Targeted downregulation of FIS1 in excitatory neurons within the spinal dorsal horn alleviates neuropathic pain through the mitigation of mitochondrial fragmentation

Targeted downregulation of FIS1 in excitatory neurons within the spinal dorsal horn alleviates neuropathic pain through the mitigation of mitochondrial fragmentation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Targeted downregulation of FIS1 in excitatory neurons within the spinal dorsal horn alleviates neuropathic pain through the mitigation of mitochondrial fragmentation Chang-Lei Zhu, Shu-Jiao Li, Zhi-Peng Lin, Zi-Wei Ni, Ke Tian, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5916414/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Neuropathic pain has been shown to induce abnormal mitochondrial fission in neurons, yet the analgesic potential of inhibiting this process remains unclear. Our previous studies demonstrated that targeted regulation of the dynamin-related protein (DRP1) can alleviate neuropathic pain; however, the downstream molecular signaling mechanisms remain to be elucidated. Methods: To investigate the role of mitochondrial dynamics in neuropathic pain, we utilized C57BL/6J mice, GAD2-Cre mice, and vGluT2-Cre mice. Mitochondrial network changes in pain states were assessed using GAD2-MITO and vGluT2-MITO transgenic mouse models combined with MiNA analysis. Pain thresholds and the expression levels of various molecules in the spinal dorsal horn (SDH) were evaluated through behavioral tests, immunofluorescence, and Western blotting. Mitochondrial morphology and function in pain conditions were examined using electron microscopy, mitochondrial membrane potential, reactive oxygen species, and adenosine triphosphate assays. The effects of antioxidant analgesics epigallocatechin gallate (EGCG) and Cinnamic Acid on molecular changes in the SDH during pain states were also investigated. Results: We observed that mitochondrial networks in both excitatory and inhibitory neurons of the SDH were disrupted in spared nerve injury mice, as evidenced by GAD2-MITO and vGluT2-MITO transgenic mouse models. Specifically, down-regulating FIS1 in excitatory neurons, but not in inhibitory neurons, within the SDH elicited analgesic effects, as evidenced by experiments conducted with vGluT2-Cre and GAD2-Cre mouse models. Additionally, epigallocatechin gallate (EGCG), which effectively down-regulates FIS1 in the SDH, concurrently inhibited SNI-induced neuropathic pain. These findings suggest that reducing mitochondrial fragmentation by down-regulating FIS1 in SDH excitatory neurons can alleviate neuropathic pain. Neuropathic pain Spinal dorsal horn Excitatory neurons Mitochondrial fission protein 1 Mitochondrial fragmentation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Neuropathic pain has a prevalence of approximately 10% and is on the rise, becoming one of the most common and severe chronic clinical symptoms [ 1 , 2 ]. It is a type of pain triggered or caused by primary damage or dysfunction of the nervous system. Due to the incomplete understanding of the pathogenesis of neuropathic pain, its clinical treatment remains a significant challenge [ 1 – 3 ]. Currently, the treatment options for neuropathic pain are limited, mainly including opioids and antioxidants. Therefore, in-depth exploration of the pathogenesis of neuropathic pain is necessary and may provide new targets for the clinical treatment of neuropathic pain. The spinal dorsal horn (SDH), as the primary center for pain information integration, is a key breakthrough point in the study of the mechanism of neuropathic pain [ 4 – 6 ]. As the primary gateway for the ascending conduction of peripheral pain stimuli, the SDH is crucial for the integration of pain signals and central sensitization [ 7 ]. In neuropathic pain, both excitatory and inhibitory neurons in the spinal dorsal horn exhibit functional abnormalities [ 8 ]. Inhibitory neurons in the spinal dorsal horn are mainly distributed in the superficial layer, while excitatory neurons are widely distributed throughout the spinal dorsal horn [ 9 ]. There is a close relationship between neuropathic pain and mitochondria. In various animal models of pain, mitochondrial homeostasis imbalance can be observed in the central nervous system neurons, ultimately leading to the occurrence and development of neuropathic pain [ 10 – 13 ]. Mitochondrial dysfunction and excessive fragmentation can induce the pathological production of reactive oxygen species and play a significant role in the state of neuropathic pain [ 14 ]. Improving mitochondrial function and morphology can relieve neuropathic pain [ 15 ]. The changes in mitochondrial structure and function are mainly mediated by mitochondrial fission and fusion [ 16 ]. Mitochondrial fission and fusion determine the fate of mitochondria [ 14 ]. Mitochondrial fission protein 1 (FIS1) is an important molecule in mitochondrial fission [ 17 ]. FIS1 is widely distributed on the outer membrane of mitochondria, and its main function is to recruit free DRP1 in the cytoplasm and mediate the peripheral fission of mitochondria [ 18 , 19 ]. Therefore, this study investigated the effects of upregulation and downregulation of FIS1 in the SDH of SNI mice on their gait and pain behavior. We found that upregulation of FIS1 in the SDH of normal mice could cause spontaneous pain behavior, while inhibition of FIS1 in the SDH of SNI mice could alleviate neuropathic pain. By constructing transgenic mice with excitatory and inhibitory neuron specificity, we discovered that downregulation of FIS1 in inhibitory neurons of the SDH of SNI mice could exert analgesic effects and improve the morphology and function of mitochondria in the SDH. Additionally, we screened two FIS1-related clinical analgesic drugs, epigallocatechin gallate and cinnamic acid. Among them, epigallocatechin gallate could alleviate neuropathic pain by inhibiting FIS1, further confirming that inhibition of FIS1 expression can relieve neuropathic pain. This study will promote in-depth research on the mitochondrial mechanism of neuropathic pain and is expected to provide new molecular targets and clinical drugs for the analgesic treatment of neuropathic pain. 2. Methods 2.1 Animals Adult (6–8 w) male wild-type C57BL/6J, vGluT2-ires Cre, GAD2-ires Cre, vGluT2-Mito-GFP, and GAD2-Mito-GFP mice were used in this study. Among them, C57BL/6 J mice were purchased from the Animal Center of the Fourth Military Medical University in Xi 'an, China. All mice were placed in 12 hours of light (8 am)/darkness (8 PM). Male mice were used throughout the study to further control for differences caused by gender differences in the animals. All animal testing protocols are based on procedures approved by the Animal Ethics and Welfare Committee of the Air Force Medical University Institutional Animal Care and Use Committee (IACUC-20190107) and follow institutional guidelines for the use of laboratory animals. All experiments were conducted by double-blind method. vGluT2-ires Cre and GAD2-IRES Cre mice purchased from Jackson's laboratory displayed internal ribosome entry sites and CRE recombinases at the 3’utr of vesicular glutamate transporter site 2 (vGluT 2) and glutamate decarboxylase site 2 (GAD2). Therefore, endogenous vGluT2 and GAD2 promoter/enhancer elements target glutamatergic and GABAergic populations in the central nervous system represented by vGluT2 and GAD2 positive neurons for Cre expression [ 20 , 21 ]. When vGluT2-ires-Cre and GAD2-ires-Cre mice were infected with viral vectors containing double floxed inverse open reading frames (DIO), Including rAAV-CMV-DIO- (EGFP-U6) -SHRNA (scramble) -Wpre-HGH poly A (Fis1 NC); rAAV-CMV-DIO- (EGFP-U6) -SHRNA (Fis1) -Wpre-HGH poly A (KD), will achieve knockdown (KD) regulation in the corresponding neurons. GFP-MITO label adhesive mice were produced by GemPharmatech Co, Ltd (Nanjing, China) and attached to a MITO-Tag box for the CAG-LSL-GFP-MITO label. A construct containing CAG-loxP-STOP-loxP-Kozak-GFP-MITO-TGA-pA targets Rosa26 of mouse Gt (ROSA) 26s. When GFP-MiTO-labeled mice were hybridized with vGluT2-ires-Cre and GAD2-ires-Cre drivers, the resulting mouse lines, called vGluT2-MITO-GFP and GAD2-MITO-GFP, exhibited bright MITO-GFP fluorescence. It is specifically localized in all glutamatergic and GABAergic central nervous mitochondria expressed by vGluT2 and GAD2 [ 20 , 21 ]. 2.2 Surgical procedures for SNI models The mice were anesthetized with pentobarbital (50 mg/kg, i.p) and an incision of about 1.5 cm was made at the upper margin of the right hind limb. The muscle is then gently separated to reveal the main sciatic nerve and its 3 branches, which are directly separated. Nerve injury (SNI) is induced by ligation and severing of 2–4 mm segments of the common peroneal and tibial nerves. The sciatic nerve and its branches were only exposed in control mice [ 22 ]. After surgery, all mice were monitored for infection. 2.3 Behavioral detection The experiment time was 09:00–12:00 in the morning. To minimize possible transfer effects and potential visual or olfactory effects, the mice were moved to a behavioral laboratory ahead of time and acclimated to the environment for at least 30 minutes. After each test, the surface of the instrument was cleaned with 75% alcohol to avoid odor interference from the previous mouse. 2.3.1. Mechanical hypersensitivity Mechanical hypersensitivity behavior was evaluated within two weeks after SNI. von Frey wire (No.37450-275, Aesthesio, Italy) was used to evaluate the foot regression threshold of mice in response to mechanical stimulation [ 23 ]. Hold each von Frey wire perpendicular to the side of the back claw for about 5 seconds. Stimulate the right hind foot (5 minutes apart). The mechanical threshold (in grams) is defined as the first fiber that causes at least 3 foot retractions in 5 tests. A series of calibrated von Frey wires were used to measure PWT before (day − 1) and after (SNI) days 1, 3, 5, 7, 10, 14. 2.3.2. Hyperalgesia thermalis The latency of the response to the hot plate was measured before (baseline level) and after SNI to indicate hyperthermic algesia [ 24 ]. The mice were placed on a hot plate (LS-6B, Jinan, China) and the temperature was adjusted to 54 ± 0.5◦C. Record the time of the first nociceptive response (licking, recoiling, or jumping) of the operative side paw and immediately remove the animal from the hot plate. To avoid injury to the Java, the maximum detection time was maintained at 25 seconds. 2.3.3. Open field test(OFT) Anxiety-like emotions of mice were detected by OFT experiment, which indirectly reflected pain response [ 25 , 26 ]. The experimental device was a 50 × 50 cm plexiglass square box with a wall 50 cm high. The mice were placed in the center of the plexiglass box and were free to explore the whole box for 15 minutes. 2.3.4. Elevated plus maze (EPM) EPM test was used to detect anxiety-like emotions in mice, which indirectly reflected pain response [ 27 ]. The elevated Cross maze was made of plexiglass and consists of two open arms (35 x 7 cm) and two closed arms (35 x 7 cm), the latter surrounded by walls 15 cm high. The device was raised 50 centimeters above the ground. Each mouse was placed in the center of the device and allowed to move freely for five minutes. Anxiety-like behavior was determined by measuring the position preferences of mice with open arms for 5 minutes. 2.3.5. Rotarod test Rotary rod test was used to detect the ability of mouse feet to grasp the ground and indirectly reflect the pain response [ 28 ]. The effect of pain on exercise endurance by responding to it during rod time. The mice were placed on a rotating rod fatigue instrument (BYZ-007, Dongguan, China) and started to rotate the rotating rod. Starting at 0 rpm and gradually increasing to 40 rpm, the acceleration time and measurement time are both 5 minutes. The stay time of the mice on the rotating rod was observed. 2.3.6. Catwalk analysis Gait analysis was used to detect pain response in mice [ 29 , 30 ]. Gait analysis was performed using the Catwalk XT system (Noldus, Netherlands), which has proven to be a very reliable method for measuring pain-related behaviors. The mice were placed at the open end of an enclosed glass platform and walked autonomously on a glass floor, during which a high-speed camera under the device captured images of each paw and transmitted the data to gait analysis software (Catwalk XT, version 10.6; Noldus). In this study, three parameters were identified that could be used to evaluate neuropathic pain-related dynamic behavior: (1) the duration of contact with the glass plate by the standing paw; (2) the maximum paw contact area; (3) The area of the complete paw print when walking. 2.4. Stereotactic viral vector injection Viral vectors used in this study include: pAAV-CMV-Fis1-3xFLAG-EF1a-EGFP-tWPA(OE), pAAV-CMV-MCS-EF1a-EGFP-tWPA(OENC), pAAV-U6-shRNA (Fis1)-CMV-EGFP-WPRE (KD), pAAV-U6-shRNA (NC2)-CMV-EGFP-WPRE (KDNC), rAAV-CMV-DIO-(EGFP-U6)-SHRNA (scramble)-Wpre-HGH poly A (KDNC), rAAV-CMV-DIO-(EGFP-U6)-SHRNA (Fis1)-WPRE-HGH pA (KD), all supplied by OBiO (China). Mice were sedated with pentobarbital sodium (50 mg/kg, i.p) prior to placement in a stereotaxator (RWD, China). Target spinal position by palpation. The paraspinous muscle above the L4/5 vertebra was excised and the right vertebra was partially excised for injection. The injection volume was 400 nL and the injection speed was 40 nL/min. To inject the virus into the SDH, the stereolocator was moved 500 nm to the right side of the spinal cord, with a final tip depth of 300 nm. The needle stays in the spinal cord for another 10 minutes and is then removed. The wound was sutured intermittently in layers, and iodine disinfectant was applied to the closed wound. 2.5. Tissue preparation and immunofluorescence analysis The mice were anesthetized with pentobarbital sodium (100 mg/kg, i.p), transorally infused with 50 mL 0.01 M phosphate buffered saline (PBS, pH 7.4), and then fixed with 4% paraformaldehyde (pH 7.4). The spinal cords of mice were then collected and soaked in 0.1M PBS containing 30% sucrose at 4℃. In low temperature thermostat (Leica CM1800; Heidelberg, Germany). Sections were incubated at room temperature for 30 min in a blocking PBS buffer containing 0.3% Triton X-100 and 0.05% sodium azide and 10% bovine serum albumin. After the primary antibody was incubated at 4°C overnight, the secondary antibody was incubated (Table 1 ), and DAPI (1:1000, #BMD0063, Abbkine, CN) was added and placed at room temperature for 12 minutes [ 31 ]. All images were taken under the LEICA laser confocal microscope STELLARIS5 (Germany). Table 1 Antibodies used for Western blot or Immunofluoresence. Antibody Source Vendor Dilution Cat.# WB/IF β- actin Mouse Abclonal 1:5000 AC004 WB c-FOS Rabbit Cell Signaling Technology 1: 1000 2250T IF COX Ⅳ Rabbit Cell Signaling Technology 1: 1000 4850T WB DRP1 Rabbit Cell Signaling Technology 1: 1000 8570T WB FIS1 Rabbit Cell Signaling Technology 1: 250 and 1༚1000 32523T IF/WB GAD2 Mouse Santa Cruz Animal Health 1: 250 sc-365180 IF MFF Rabbit Cell Signaling Technology 1: 1000 84580T WB NeuN Rabbit Abcam 1: 250 ab177487 IF OPA1 Rabbit Cell Signaling Technology 1: 1000 67589T WB PDHA1 Mouse Abcam 1: 1000 ab168379 WB SDHB Mouse Abcam 1: 1000 ab175255 WB TFAM Rabbit Cell Signaling Technology 1: 1000 8076T WB VDAC Rabbit Abcam 1: 250 ab15895 IF vGluT2 Mouse Abcam 1: 250 ab79157 IF Alexa 488 Rabbit Abbkine 1: 500 A23220 IF Alexa 594 Mouse Abbkine 1: 500 A23420 IF Alexa 594 Rabbit Abbkine 1: 500 A23620 IF Alexa 649 Rabbit Abbkine 1: 500 A23610 IF HRP Rabbit Abbkine 1:5000 A21020 WB HRP Mouse Abbkine 1:5000 A21010 WB 2.6. Western blot Proteins were isolated from homogenate tissue using modified RIPA buffers with protease and phosphatase inhibitors (Thermo Fisher Scientific). Assay Reagent Kit was used to determine protein concentration. The protein was isolated by 12.5% SDS-PAGE and transferred to 4.5um PVDF membrane, then blocked in TBST with 5% skim milk and incubated at room temperature for 2 hours. The primary antibody was incubated at 4°C overnight and the secondary antibody HRP was incubated (Table 1 ). Fusion FX EDGE chemiluminescence imaging scanning film was used, and ImageJ was used for gray value analysis [ 32 ]. 2.7. Transmission electron microscope (TEM) After anesthesia, mice were injected 0.01 M PBS, 4% formaldehyde and 0.1% glutaraldehyde PBS successively. Spinal cord tissue was placed in 4% glutaraldehyde solution for tissue fixation. After fixation, each spinal cord tissue was washed twice with 0.1 M PBS. It was then washed with 1% osmic acid (TED PELLA, Inc.No.18451). Dye for 2 hours, then dehydrate with 50%, 70%, 90% ethanol and 100% acetone, respectively, and then soak with acetone: embedding agent (Embed 812, DDSA, NMA, DMP-30) at room temperature 1:1 for 2 hours. Finally, the tissue was removed from the embedding agent and stored overnight at room temperature. On the second day, the tissue was placed in a special electron microscope plate, incubated at 60◦C for 48 hours, and 70 nm thick slices were prepared using an ultra-thin secting mechanism. The sections were placed on a copper rack and stained with lead nitrate and uranyl acetate for 10 minutes, respectively. TEM (JEM 1400, Olympus, Japan) was used to record images. The mitochondrial morphological data were analyzed by imagej based image analysis method. Finally, 16 continuous tissue sections were selected for TEM analysis, and slices with a thickness of about 1500 nm were obtained. The size of each mitochondria was obtained by drawing an electron microscope sagittal picture of the SDH mitochondria. By using ImageJ's hand-drawn selection, the contours of mitochondria and internal vacuoles are carefully drawn. The number of mitochondria is the total number of mitochondria in each image, and then the image area is calculated according to the scale carried by the image, and the density, circumference and circle rate of mitochondria are calculated. The same method was used to calculate the area, circumference, and density of mitochondrial vacuoles [ 33 ]. 2.8. Mitochondrial network analysis (MiNA) Mitochondrial Network Analysis (MiNA) uses the FiJi distribution, freely available on the ImageJ platform, and combines open source tools into a simple macro toolset. Spinal cord sections of vGluT2-Mito-GFP and GAD2-Mito-GFP mice were photographed under laser scanning confocal microscopy (LEICA, STELLARIS5). Mitochondrial network analysis was performed using the MiNA plug-in in ImageJ [ 20 , 34 ]. 2.9. Mitochondrial extraction Mice were anesthetized with pentobarbital sodium (100 mg/kg, i.p) and SDH was rapidly removed. Purification of mitochondria using a mitochondrial extraction kit (SM0020, Solarbio, CN) : First, the tissue was cut as small as possible with scissors, then broken with a pre-cooled tissue analyzer (QIAGEN). Centrifuge 1000 g at 4°C twice for 5 minutes, then 12000 g for 10 minutes. The supernatant was discarded and the mitochondrial precipitation was placed on ice by re-suspension with Store Buffer. Finally, mitochondrial proteins were quantified by BCA method. 2.10. Mitochondrial membrane Potential Detection (MMP) Mitochondrial membrane potential (JC-1) was detected using an enhanced mitochondrial membrane potential assay kit (Beyotime, C2003S, CN). Detection with fluorescent enzyme marker: After purified mitochondria were mixed with the working liquid, the enzyme marker was directly used to scan, and the excitation wavelength was 485 nm and the emission wavelength was 590 nm to detect the fluorescence intensity of mitochondrial JC-1 [ 35 ]. 2.11. ROS assay Mitochondrial reactive oxygen Species ROS assay kit (Bestbio, BB-460913, CN) was used to detect ROS levels in purified mitochondria. The fluorescence intensity of purified mitochondria was measured with an enzyme labeler at excitation wavelength of 485 nm and emission wavelength of 590 nm [ 36 ]. 2.12. ATP detection Tissue ATP levels were measured using an enhanced ATP test kit (Beyotime, S0027, China) according to manufacturer's instructions. The luminescence was measured at 560 nm using an enzyme labeler, a standard curve was generated and used to calculate ATP concentration. Histamin was quantified by BCA [ 37 ]. 2.13 Network pharmacological analysis Database: Comparative Toxicogenomics Database ( https://ctdbase.org/ ), MitoCarta3.0 dataset ( https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways ), the STRING database ( https://cn.string-db.org/ ), GSEA database ( https://www.gsea-msigdb.org/gsea/index.jsp ). Drawing: microscopic letter ( https://www.bioinformatics.com.cn/ ), Chiplot ( https://www.chiplot.online/ ). Chronic Pain, Cinnamic Acid and Epigallocatechin Gallate were selected as keywords to predict the target genes with Comparative Toxicogenomics Database. The PPI network interaction was analyzed by using STRING database. GO and KEGG path enrichment analysis was performed for key targets using DAVID database and Weisheng platform. Weisheng and Chiplot were used for image rendering. A total of 16 targets were associated with keywords. After intersection with mouse mitochondrial gene set, the intersection targets were input into STRING database for protein interaction analysis, and free targets were removed. A total of 16 target genes related to mitochondria were obtained and identified as target genes. Mitochondrial Fission was searched for in the GSEA database, and Cinnamic Acid, Chronic Pain, Epigallocatechin Gallate and Epigallocatechin gallate were selected. Finally, DRP1 and FIS1 were the key proteins. 2.14 Intraperitoneal drug injection SC-9 [ 38 ] (TargetMol, T83057, USA; Cinnamic Acid (MCE, HY-N0610A, USA: 30 mg/kg) [ 39 ], and Epigallocatechin Gallate [ 40 ] (MCE, HY-13653, USA: 20 mg/kg) were intraperitoneally administrated to SNI mice on days 7, 10, and 14 post-SNI. Mechanical pain was assessed using von Frey filaments, while thermal pain was evaluated using a hot plate test, both conducted 4 hours after administration. Statistical analysis was performed as described in section 4 . 2.15 Statistical analysis The statistical comparison methods of each research method were detailed in the legend. Quantitative data were presented as mean ± S.E.M. For statistical analysis, unpaired t- tests were employed for single comparisons, while two-way ANOVA was utilized for multiple comparisons. All analyses were conducted using SPSS 26.0 software and GraphPadPrism 9.0. A P < 0.05 was considered statistically significant. 3. Results 3.1 Spared nerve injury (SNI)-induced neuropathic pain upregulated the expression levels of DRP1 and FIS1 in the ipsilateral SDH and activated SDH neurons. The spared nerve injury (SNI) neuropathic pain model was established by separating the right common peroneal nerve (CPN), sural nerve (SN) and tibial nerve (TN) of mice and ligating the CPN and TN (Fig. 1 A). The results of the von Frey mechanical pain threshold test (Fig. 1 B) showed that the paw withdrawal threshold of mice decreased from 1.0 g to 0.02 g on the first day after SNI ( P < 0.0001) and remained so until the 14th day after surgery ( P < 0.0001), while no such change was observed in the Sham group, indicating that SNI caused mechanical allodynia in mice. The results of the hot plate test (Fig. 1 C) showed that the latency of mice decreased from 10 s to 6.5 s on the first day after SNI ( P < 0.0001) and remained so until the 14th day after surgery, while no such change was observed in the Sham group, indicating that SNI caused thermal hyperalgesia in mice. The open field test (OFT) and elevated plus maze (EPM) experiments provided indicators of anxiety-like behavior in animals. The OFT results showed (Fig. 1 D and E) that the distance and time spent in the central area by the SNI group mice were significantly lower than those of the Sham group ( P < 0.01). The EPM results indicated (Fig. 1 F and G) that the number of entries into the open arms, the time spent in the open arms, and the distance traveled in the open arms of the SNI group mice were all significantly lower than those of the Sham group ( P < 0.05). If animals exhibit hyperalgesia, it would also be manifested as abnormal results in the rotarod test. The rotarod test results (Fig. 1 H) showed that the time spent on the rod by the SNI group mice was significantly shorter than that of the Sham group ( P < 0.0001). The Catwalk gait analysis provided fine behavioral indicators of the mice. The Catwalk gait analysis results (Fig. 1 I and J) indicated that the standing time of the right hind limb, the maximum contact area of the right hind paw upon landing, and the footprint area of the right hind paw in the SNI group were all significantly reduced compared with those in the Sham group ( P < 0.001). Further, 14 days after SNI or Sham, the spinal dorsal horn (SDH) tissue on the stimulated side was collected for Western blot analysis of 8 mitochondrial-related molecules (Fig. 1 K). The results showed (Fig. 1 L and M) that there was no statistically significant difference between the two groups in optic atrophy protein 1 (OPA1), mitochondrial fission factor (MFF), pyruvate dehydrogenase alpha (PDHA1), succinate dehydrogenase complex B subunit (SDHB), and mitochondrial transcription factor A (TFAM). However, the expression of mitochondrial fission-related proteins dynamin-related protein 1 (DRP1) and mitochondrial fission protein 1 (FIS1) was significantly increased in the SNI group compared with the Sham group ( P < 0.01), while the expression of complex IV (COX IV), a key molecule in the mitochondrial electron transport chain, was significantly decreased in the SNI group compared with the Sham group ( P < 0.01). Further, immunofluorescence staining of nuclear phosphoprotein FOS was used to detect neuronal activation (Fig. 1 N). The FOS staining (green) results showed (Fig. 1 O) that the number of FOS-positive cells in the dorsal horn of the stimulated side of the spinal cord in the SNI group was significantly increased compared with the Sham group ( P < 0.0001). Previous papers published by our research group have confirmed that SNI causes an increase in the expression of mitochondrial fission protein DRP1 in the spinal cord dorsal horn [ 41 ]. Therefore, this study focused on another important mitochondrial fission protein, FIS1, for research. FIS1 immunofluorescence staining (green) experiments were used to verify the Western blot detection results (Fig. 1 P). The results showed (Fig. 1 Q) that the relative fluorescence expression area and relative fluorescence intensity of FIS1 in the dorsal horn of the stimulated side of the spinal cord in the SNI group were significantly increased compared with the Sham group ( P < 0.01). The above results suggested that there was neuronal activation in SDH of SNI mice, and the expressions of DRP1 and FIS1 were both increase, while the complex IV of the mitochondrial respiratory chain was reduced, indicating mitochondrial respiratory dysfunction. 3.2 DRP1-FIS1 contact inhibitor SC-9 could alleviate mechanical allodynia and thermal hyperalgesia. Next, we will examined whether FIS1 affects neuropathic pain. Previous studies have shown that DRP1, which is located in the cytoplasm, translocates to the outer mitochondrial membrane and binds to FIS1 to initiate mitochondrial fission. Our previous published work has confirmed that targeting upregulation of DRP1 at the spinal cord level produces analgesic effects [ 41 ]. Recently, it was reported that Anti-inflammatory agent 49 (SC-9) is a contact inhibitor of DRP1 and FIS1, which can inhibit the interaction between DRP1 and FIS1 without affecting the physiological function of DRP1 [ 38 ]. Therefore, we intraperitoneally injected SC-9 into SNI mice to detect the changes in mechanical allodynia and thermal hyperalgesia when the FIS1-dependent initiation stage of mitochondrial fission is inhibited. We performed SNI surgery on C57BL/6J mice and administered SC-9 (50 mg/kg) intraperitoneally on the 7th, 10th, and 14th days after the operation. Behavioral tests were conducted 4 hours after each injection (Fig. 2 A). The results showed that on the 7th, 10th, and 14th days after SNI, the mechanical threshold of mice in the SC-9 injection group was significantly increased compared with that of the control group (Fig. 2 B) ( P < 0.01), and the thermal pain latency was significantly increased (Fig. 2 C) ( P < 0.01). After the last behavioral test, the spinal dorsal horn proteins of the two groups of mice were extracted for Western blot analysis (Fig. 2 D). The results showed that SC-9 inhibited the expression of FIS1 in SDH after SNI, but did not affect the expression of DRP1 and MFF [ 14 ] (Fig. 2 E) ( P < 0.0001). The above results indicated that the DRP1-FIS1 contact inhibitor SC-9 significantly alleviates mechanical allodynia and thermal hyperalgesia caused by SNI. 3.3 Upregulating FIS1 in SDH neurons induced spontaneous pain in normal mice, while downregulating FIS1 alleviated pain in SNI mice. The previous results indicated that the inhibitor of FIS1 exerted an analgesic effect in the system. Therefore, we conducted two experiments: one was to up-regulate the expression of FIS1 in SDH neurons of normal mice, and the other was to down-regulate the expression of FIS1 in SDH neurons of SNI mice. Then, we observed the pain behavior of the mice. Firstly, we injected the Fis1 overexpression (OE) adeno-associated virus (AAV) targeting neurons into the unilateral (right) SDH of C57BL/6J mice, with the empty virus as the control. Behavioral observations were performed on days 1, 3, 5, 7, 10, and 14 after 14 days of injection (when the virus began to exert a stable effect), and spinal cord tissue was collected after the last behavioral test (Fig. 3 A). The results of the von Frey mechanical pain threshold test (Fig. 3 B) showed that the withdrawal threshold of the right hind paw of the OE treatment group was significantly lower than that of the empty virus group from the 1st day of observation ( P < 0.05), and it continuously decreased from 1.0 g to 0.02 g over the next 14 days ( P < 0.0001). The results of the hot plate test (Fig. 3 C) showed that the latency of the OE treatment group was significantly lower than that of the empty virus group from the 1st day of observation ( P < 0.01), and it continuously decreased to 5 s over the next 14 days ( P < 0.0001). Although the OFT results (Fig. 3 D and E) and Catwalk gait analysis results (Fig. 3 I and J) showed no significant differences between the OE group and the control group, the EPM results (Fig. 3 F and G) indicated that the number of entries into the open arms, the time spent in the open arms, and the distance traveled in the open arms of the OE treatment group were significantly lower than those of the control group ( P < 0.01). The results of the rotarod test (Fig. 3 H) showed that the time spent on the rotarod by the OE treatment group was significantly shorter than that of the control group ( P < 0.01). Further, Western blot analysis was performed on the dorsal horn tissue of the stimulated side (right side) of the spinal cord (Fig. 3 L and K). The results showed that the expression of FIS1 in the OE group was significantly increased compared to the control group ( P < 0.05), confirming the reliability of the Fis1 OE AAV. Meanwhile, the expression level of DRP1 in the OE treatment group was significantly lower than that in the control group ( P < 0.05), while the expression levels of the other six molecules (OPA1, PDHA1, MFF, SDHB, TFAM, and COX IV) showed no significant differences between the two groups. To further down-regulate the expression of FIS1 in SDH neurons of SNI mice. We injected Fis1 knockdown (KD) AAV into the unilateral (right) SDH of mice by spinal cord injection, with empty virus as the control (Fig. 3 M). The experimental process was consistent with OE group. The results of the von Frey mechanical pain threshold test (Fig. 3 N) showed that the withdrawal threshold of the right hind paw of SNI mice in the KD treatment group increased from 0.16 g to 0.5 g on the 1st day compared with the empty virus group (P < 0.05), and this increase persisted for 14 days. The results of the hot plate test (Fig. 3 O) showed that the latency of SNI mice in the KD treatment group increased from 6 s to 9 s on the 1st day compared with the empty virus group ( P < 0.001), and this increase persisted for 14 days. The results of the OFT (Fig. 3 P and Q) showed that the distance and time of activity in the central area of SNI mice in the KD treatment group were significantly increased compared with the control group ( P < 0.05). The results of the EPM (Fig. 3 R and S) showed that the time spent in the open arms of SNI mice in the KD treatment group was significantly increased compared with the control group ( P < 0.01). The results of the Catwalk gait analysis (Fig. 3 T and U) showed that the standing time of the right hind limb, the maximum contact area of the right hind paw, and the area of the right hind paw print of SNI mice in the KD treatment group were significantly increased compared with the control group ( P < 0.05). The results of the rotarod test (Fig. 3 V) showed that the time on the rod of SNI mice in the KD treatment group was significantly prolonged compared with the control group ( P < 0.01). Subsequently, the SDH of the stimulated side was sampled for Western blot detection (Fig. 3 W). The results showed (Fig. 3 X) that the expression of FIS1 in the KD group was significantly lower than that in the control group ( P < 0.01), confirming the reliability of Fis1 KD AAV. Meanwhile, the expression levels of DRP1 and PDHA1 in the KD treatment group of SNI mice were significantly increased compared with the control group ( P < 0.01), while the expression level of SDHB was significantly decreased, and there was no difference in the expression of OPA1, MFF, TFAM and COX IV molecules. The above results suggested that targeted up-regulation of FIS1 expression in SDH neurons causes spontaneous pain in normal mice, while targeted down-regulation alleviated the pain response caused by SNI. 3.4 Both excitatory and inhibitory neurons within the SDH of SNI mice activated and the FIS1 level was elevated in both types of neurons. Previous studies have shown that the SDH contains both excitatory and inhibitory neurons, with inhibitory neurons mainly located in layers I-III, while excitatory neurons are distributed throughout the SDH [ 7 ]. Our results suggested that regulation of FIS1 levels in SDH neurons caused changes in mechanical and thermal hyperalgesia in normal mice. To distinguish the neuronal types in the SDH of normal mice that respond to FIS1 expression after SNI, we first performed double immunofluorescence staining with the neuronal activation marker FOS (green) and vesicular glutamate transporter site 2 (vGluT 2) / glutamate decarboxylase site 2 (GAD2) (red) (Fig. 4 A and B). The results of the double immunofluorescence staining (Fig. 4 C) showed that the number of FOS-positive cells co-localized with GAD2 in the SDH after SNI was significantly higher than that in the Sham group; similarly, the number of FOS-positive cells co-localized with vGluT2 in the SDH after SNI was also significantly higher than that in the Sham group ( P < 0.0001). Subsequently, we used the same method to perform double immunofluorescence staining for FIS1 (green) and GAD2/vGluT2 (red). Statistical analysis was performed using the commonly used Manders' Colocalization Coefficients (MCC) in immunofluorescence. M1 represents the ratio of the co-labeled fluorescence area of the two proteins to the fluorescence area of a single channel protein. Here, it indicates the ratio of the co-labeled fluorescence area of FIS1 and GAD2 or vGluT2 to the single-labeled fluorescence area of GAD2 or vGluT2. The results showed (Fig. 4 D-F) that the M1 values related to GAD2 and vGluT2 in the SDH after SNI were significantly increased compared to the Sham group ( P < 0.0001). The above results indicated that both excitatory and inhibitory neurons in the SDH were activated after SNI, and FIS1 was expressed in both types of neurons. Therefore, the results of this part of the study could not yet answer which type of neuron FIS1 exerts its pain regulatory effect on. 3.5 Mitochondrial networks in both excitatory and inhibitory neurons of the SDH were disrupted in SNI mice, as demonstrated through the use of specifically constructed GAD2-MITO and vGluT2-MITO transgenic mouse models. Therefore, we designed the following experiment. We first constructed GAD2-MITO and vGluT2-MITO transgenic mice that could respectively express green fluorescent protein on the outer mitochondrial membrane of excitatory and inhibitory neurons (Fig. 5 A). When GFP-MITO labeled mice were crossed with vGluT2-ires-Cre and GAD2-ires-Cre driver factors, the offsprings were called vGluT2-MITO-GFP and GAD2-MITO-GFP, showing bright MITO-GFP fluorescence, specifically localized in all vGluT2 and GAD2 expressing central nervous system mitochondria of glutamatergic and GABAergic neurons neurons [ 20 ]. Then, we performed voltage-dependent anion channel (VDAC) immunofluorescence staining on the SDH of normal GAD2-MITO and vGluT2-MITO mice. The co-localization analysis of confocal fluorescence images showed that VDAC (purple) completely overlapped with mitochondria (green) in both types of mice, indicating that the green fluorescence in GAD2-MITO and vGluT2-MITO mice indeed labeled the mitochondria in excitatory and inhibitory neurons (Fig. 5 B). Subsequently, SNI models were established using GAD2-MITO and vGluT2-MITO mice. On the 14th day after surgery, the spinal cords were taken for NeuN fluorescence staining (a neuronal marker, red), and confocal images of the SDH region were taken. The mitochondrial network analysis (MiNA) plugin in ImageJ software was used to analyze the mitochondrial network of SDH. The analysis indicators included four: the total length of mitochondrial imprints, the length of the mitochondrial network, the length of the mitochondrial network branches, and the number of mitochondrial network branch points. Figure 5 C and D showed the original confocal images and the ImageJ processed MiNA images of GAD2-MITO and vGluT2-MITO mice 14 days after SNI, respectively. The statistical results (Fig. 5 E) showed that the four indicators of inhibitory neurons (GAD2 + neurons) in GAD2-MITO mice were significantly lower than those in the Sham group ( P < 0.0001) 14 days after SNI. At the same time, the four indicators of non-inhibitory neurons (non-GAD2 + neurons) were also significantly lower than those in the Sham group ( P < 0.05). The statistical results (Fig. 5 E) showed that three of the four indicators of excitatory neurons (vGluT2 + neurons) in vGluT2-MITO mice were significantly lower than those in the Sham group (all mitochondrial imprint lengths, mitochondrial network lengths, and the number of mitochondrial network branch points) ( P < 0.0001) 14 days after SNI, while the length of mitochondrial network branches showed no significant difference. At the same time, three of the four indicators of non-excitatory neurons (non-vGluT2 + neurons) were significantly lower than those in the Sham group (all mitochondrial imprint lengths, mitochondrial network lengths, and the number of mitochondrial network branch points) ( P < 0.0001), while the length of mitochondrial network branches showed no significant difference. In addition, we performed FIS1 (red) immunofluorescence staining (Fig. 5 F and G). The results showed (Fig. 5 H) that the expression level of FIS1 in inhibitory neurons (GAD2 + neurons) in the SDH of GAD2-MITO mice after SNI was significantly increased compared with the Sham group ( P < 0.0001); similarly, the expression level of FIS1 in excitatory neurons (vGluT2 + neurons) in the SDH of vGluT2-MITO mice after SNI was significantly increased compared with the Sham group ( P < 0.0001). These results suggested that the mitochondrial networks in both excitatory and inhibitory neurons in the SDH was weakened after SNI, accompanied by an increase in FIS1 expression. Therefore, the results of this part of the study suggested that the pain regulatory effect of FIS1 may be achieved through both excitatory and inhibitory neurons. However, this was puzzling because the states of these two types of neurons are completely opposite in spinal cord sensitization in neuropathic pain. According to the spinal cord gate control theory, the sensitization of excitatory neurons and the disinhibition of inhibitory neurons lead to central sensitization in neuropathic pain. Therefore, we next precisely regulated FIS1 in the two types of neurons to try to identify the target cells of FIS1 in neuropathic pain. 3.6 Down-regulating FIS1 specifically in excitatory neurons, but not inhibitory neurons, of spinal dorsal horn could exert analgesic effects, as demonstrated using vGluT2-Cre mice and GAD2-Cre mice. We conducted behavioral and molecular biological experiments using vGluT2-Cre mice and GAD2-Cre mice combined with targeted injection of Fis1 RNA interference (KD) recombination adeno-associated virus (rAAV) carrying the DIO element. We first targeted the injection of Fis1 RNA interference (KD) rAAV into vGluT2-Cre mice to down-regulate FIS1 in the excitatory neurons of the right side of the SDH in mice, with empty virus as the control. At 14 days, the viral expression reached a stable level, at which point unilateral (right side) SNI modeling was performed, and then behavioral observations of mice were conducted on days 1, 3, 5, 7, 10, and 14 after SNI (Catwalk and rotarod experiments were only conducted on day 14), and the spinal cord was taken for double immunofluorescence staining of FIS1 and vGluT2 after the last behavioral observation (Fig. 6 A). The results of the von Frey mechanical pain threshold test (Fig. 6 B) showed that the right hind paw withdrawal threshold of SNI mice in the SDH excitatory neuron Fis1 KD treatment group increased from 0.01g to 0.4g on day 3 compared with the empty virus group ( P < 0.05), and this effect persisted until day 14. The results of the hot plate test (Fig. 6 C) showed that the latency of SNI mice in the Fis1 KD treatment group on day 10 increased from 5s to 7s compared with the empty virus group ( P < 0.001), and this effect persisted until day 14. The results of the OFT (Fig. 6 D and E) showed that there was no significant difference in the distance and time of activity in the central area between the Fis1 KD treatment group and the control group. The results of the EPM (Fig. 6 F and G) showed that there was no significant difference in the time spent in the open arms between the Fis1 KD treatment group and the control group. The results of the Catwalk gait analysis (Fig. 6 H and I) showed that the standing time of the right hind limb, the maximum contact area of the right hind paw, and the area of the right hind paw print of SNI mice in the Fis1 KD treatment group on day 14 were significantly increased compared with the control group ( P < 0.05). The results of the rotarod test (Fig. 6 J) showed that the time on the rod of SNI mice in the Fis1 KD treatment group was significantly longer than that of the control group ( P < 0.05). The results of dual immunofluorescence staining for FIS1 (green) and vGluT2 (red) showed (Fig. 6 K and L) that the M1 value of double-labeled FIS1 and vGluT2 in the SDH of the SNI group treated with Fis1 KD was significantly lower than that of the empty virus group ( P < 0.0001). The above research process was then repeated in GAD2-Cre mice (Fig. 6 M). The results of the von Frey mechanical pain threshold test (Fig. 6 N) showed that the withdrawal threshold of the right hind paw of SNI mice in the SDH inhibitory neuron Fis1 KD treatment group was not significantly different from that of the empty virus group. The results of the hot plate test (Fig. 6 O) showed that the latency of SNI mice in the Fis1 KD treatment group was not significantly different from that of the empty virus group. The results of the OFT (Fig. 6 P and Q) showed that the distance and time of activity in the central area of SNI mice in the SDH excitatory neuron Fis1 KD treatment group were not significantly different from those of the control group. The results of the EPM (Fig. 6 R and S) showed that the time spent in the open arm of SNI mice in the Fis1 KD treatment group was not significantly different from that of the control group. The results of the rotarod test (Fig. 6 T) showed that the time on the rod of SNI mice in the SDH inhibitory neuron Fis1 KD treatment group was not significantly different from that of the control group. The results of the Catwalk gait analysis (Fig. 6 U and V) showed that the standing time of the right hind limb, the maximum contact area of the right hind paw, and the area of the right hind paw print of SNI mice in the Fis1 KD treatment group were not significantly different from those of the control group. The results of double immunofluorescence staining for FIS1 (green) and GAD2 (red) (Fig. 6 W and X) showed that the M1 value of double-labeled FIS1 and GAD2 in the SDH of the SNI group treated with Fis1 KD was significantly lower than that of the empty virus group ( P < 0.0001). The above results indicated that down-regulating FIS1 in excitatory neurons in the SDH could significantly exert analgesic effect in SNI mice, but it could not alleviate the anxiety-like behavior. On the contrary, down-regulating FIS1 in SDH inhibitory neurons had no effect on the pain response and anxiety-like behavior after SNI. Thus, we finally discovered that the target cells for FIS1 analgesia were the excitatory neurons in the spinal dorsal horn. 3.7 Targeted downregulation of FIS1 in the SDH of excitatory neurons in SNI mice mitigated mitochondrial dysfunction. Previous research results have shown that neuropathic pain can lead to a decrease in adenosine triphosphate (ATP) production and mitochondrial membrane potential (MMP) levels, and an increase in reactive oxygen species (ROS) generation [ 42 – 44 ]. The results of the earlier part of this study showed that SNI causes a decrease in the expression of COX IV of the mitochondrial electron transport chain in the SDH tissue of mice and damage to the mitochondrial network structure within neurons. Therefore, we hypothesized that the mechanism of FIS1 downregulation for analgesia is related to mitochondrial function. To verify this hypothesis, we conducted five groups of experiments. In the first group, C57BL/6J mice were subjected to sham treatment and SNI model groups. On the 14th day after surgery, the dorsal horn of the stimulated side of the spinal cord was taken, and then mitochondria were purified. The experimental results indicated that compared with the sham group, the SNI group had a decreased mitochondrial MMP level in SDH ( P < 0.01) (Fig. 7 A), reduced ATP production ( P < 0.05) (Fig. 7 B), and increased ROS generation ( P < 0.05) (Fig. 7 C). In the second group, C57BL/6J mice were given intraspinal injection of AAV-Fis1-OE into the spinal dorsal horn. On the 28th day after injection, the dorsal horn of the stimulated side of the spinal cord was taken, and then mitochondria were purified. The relative levels of ATP, MMP, and ROS were detected. The results showed that the Fis1 OE treatment group had a lower MMP level ( P < 0.05) (Fig. 7 D) and increased ROS generation ( P < 0.05) (Fig. 7 F) compared with the empty virus group (Fis1-NC), but there was no significant difference in ATP levels between the two groups (Fig. 7 E). In the third group, Fis1-KD AAV was injected into the SDH of normal mice, with empty virus as the control. Fourteen days after virus injection, SNI modeling was performed. Fourteen days after SNI, the dorsal horn of the stimulated side of the spinal cord was taken, and then mitochondria were purified to detect the relative levels of ATP, MMP, and ROS. The results showed that the MMP level in the SNI mice group treated with Fis1-KD was higher than that in the control group (Fis1-NC) ( P < 0.05) (Fig. 7 G), ATP production increased (Fig. 7 H) ( P < 0.05), and ROS production decreased ( P < 0.0001) (Fig. 7 I). In the fourth group, Fis1-KD rAAV virus was injected into the SDH of GAD2-Cre mice, with empty virus as the control. The results showed that the ROS production in the GAD2-Cre SNI mice treated with Fis1-KD was lower than that in the control group (Fis1-NC) ( P < 0.05) (Fig. 7 L), while there was no significant difference in MMP (Fig. 7 J) and ATP (Fig. 7 K) levels. The fifth group experiment was similar to the fourth group, except that the animals were changed to vGluT2-Cre mice. The results showed that the ATP production in the vGluT2-Cre SNI mice treated with Fis1-KD was higher than that in the control group (Fis1-NC) ( P < 0.01) (Fig. 7 N), and ROS production decreased ( P < 0.05) (Fig. 7 O), while there was no significant difference in MMP (Fig. 7 M) levels between the two groups. The above results indicated that SNI and targeted upregulation of FIS1 in SDH both caused mitochondrial dysfunction; targeted downregulation of FIS1 in excitatory neurons of SNI mice SDH alleviated mitochondrial dysfunction. 3.8 Targeted down-regulation of FIS1 in the SDH decreased the mitochondrial vacuoles and mitochondrial fragmentation induced by SNI. Previous studies have found that morphological changes in mitochondria, such as swollen cristae, increased vacuoles, and abnormal fission and fusion, are associated with various neurological disorders, such as Alzheimer's disease and Parkinson's disease [ 1 , 2 , 45 – 47 ]. To observe the morphological changes in mitochondria after SDH-targeted regulation of Fis1 in common mice alleviates neuropathic pain, we used transmission electron microscopy to observe and analyze seven indicators of mitochondria in SDH: the number, perimeter, area, circularity, and the number, perimeter, and area of vacuoles within mitochondria. The above electron microscopy detection techniques have been reported in our previous studies [ 41 ]. We conducted three groups of experiments. In the first group, we established the Sham treatment and SNI model groups in C57BL/6J mice and took the dorsal horn of the stimulated side of the spinal cord for electron microscopy observation on the 14th day after surgery. The transmission electron microscopy (TEM) results showed (Fig. 8 A) that in the SNI group, the number of SDH mitochondria was increased ( P < 0.0001), the perimeter was decreased ( P < 0.0001), the area was decreased ( P < 0.0001), and the circularity was increased (P < 0.0001) compared with the Sham group (Fig. 8 B). At the same time, the number of vacuoles within mitochondria was increased ( P < 0.0001), the perimeter of vacuoles was increased ( P < 0.05), and the area was increased ( P < 0.05) (Fig. 8 C). The second group was given intraspinal injection of AAV-Fis1-OE into the SDH of C57BL/6J mice. After 28 days, the dorsal horn of the stimulated side of the spinal cord was taken for electron microscopy observation. The results of transmission electron microscopy showed (Fig. 8 D) that compared with the empty virus group, the Fis1-OE treated group had an increased number of mitochondria ( P < 0.0001), decreased perimeter ( P < 0.0001), decreased area ( P < 0.0001), and increased circularity ( P < 0.0001) (Fig. 8 E), and the number of vacuoles within mitochondria increased ( P < 0.01), the perimeter increased ( P < 0.0001), and the area increased ( P < 0.0001) (Fig. 8 F). The third group was given intraspinal injection of AAV-Fis1-KD into the SDH of C57BL/6J mice. Fourteen days later, SNI modeling was performed. Fourteen days after SNI, the dorsal horn of the stimulated side of the spinal cord was taken for electron microscopy observation. The results of transmission electron microscopy showed (Fig. 8 G) that compared with the control group, the SNI mice treated with Fis1-KD had a decreased number of mitochondria ( P < 0.0001), increased perimeter ( P < 0.0001), increased area ( P < 0.0001), and decreased circularity ( P < 0.0001) (Fig. 8 H), and the number of vacuoles within mitochondria decreased ( P < 0.05), the perimeter decreased ( P < 0.0001), and the area decreased ( P < 0.0001) (Fig. 8 I). The above results indicated that both SNI and targeted upregulation of FIS1 in SDH caused mitochondrial fragmentation, while targeted downregulation of FIS1 in SDH neurons of SNI mice reduced mitochondrial fragmentation. 3.9 EGCG, which was capable of down-regulating FIS1 in the SDH, could simultaneously inhibit SNI-induced neuropathic pain. Previous experiments have shown that targeting and down-regulating the level of Fis1 can exert analgesic effects by correcting mitochondrial fragmentation. Therefore, we proposed the next scientific hypothesis that analgesic drugs may exert their analgesic effects by down-regulating the level of FIS1. To verify this hypothesis, we selected two analgesic drugs, epigallocatechin gallate (EGCG) and cinnamic acid (CA), for the following experiments. EGCG is the most effective active component of tea polyphenols [ 40 ]. CA is an organic acid isolated from cinnamon bark [ 48 ]. They are well-known antioxidant drugs and have analgesic effects [ 39 , 49 ]. Firstly, we used network pharmacology methods to analyze the possible molecular mechanisms by which EGCG and CA exert analgesic effects. By inputting the keywords "Chronic Pain", "Cinnamic Acid", and "Epigallocatechin Gallate" into the human Comparative Toxicogenomics Database, we obtained 7806, 36, and 2393 related genes respectively. Taking the intersection, we obtained 16 genes, including BAX, BCL2, CASP3, CAT, CYP2E1, CYP3A4, DNM1L, FIS1, HMGCR, MMP2, MMP9, NOS3, PPARG, SLC2A4, TNF, etc. (Fig. 9 A). The above results suggested that the 16 genes, including DNM1L (i.e., Drp1) and FIS1, may be the molecular targets through which EGCG and CA exert their analgesic effects. Then, STRING database was used for protein-protein interaction networks (PPI) network interaction analysis, and one free gene (CAT) was excluded (Fig. 9 B). Subsequently, the database for annotation, visualization and integrated discovery (DAVID) database and microbiomics platform were utilized to conduct gene ontology (GO) analysis on the remaining 15 genes. The results of gene ontology biological process (GO-BP) analysis indicated that these 15 genes were mainly enriched in biological processes such as "cellular response to chemical stress", "oxidative stress response", and "alteration of mitochondrial morphology and function" (Fig. 9 C). The results of gene ontology cellular component (GO-CC) analysis showed that these 15 genes were mainly enriched in cellular components such as "mitochondrial outer membrane", "organelle outer membrane", and "lipid raft" (Fig. 9 D). The results of gene ontology molecular function (GO-MF) analysis revealed that these 15 genes were mainly enriched in molecular functions such as "oxidoreductase activity", "BH domain binding", and "protein phosphatase binding" (Fig. 9 E). Then, the keywords "mouse" and "mitochondrial fission" were input into the gene set enrichment analysis (GSEA) database, and 23 disease-related mitochondrial fission genes were obtained (Aurka, Bnip3, Dcn, Ddhd1, Ddhd2, Drp1, Fis1, Igtp, Irgm1, Irgm2, Kdr, Marchf5, Mcu, Mff, Mief1, Mief2, Mul1, Pgam5, Pink1, Rala, Ralbp1, Spire1, Vps35). The intersection of the above 15 genes and the 23 genes yielded 2 genes, namely Fis1 and Drp1 (Fig. 9 F). Based on the above bioinformatics analysis, we reached a preliminary conclusion that EGCG and CA might exert analgesic effects through the Fis1 or Drp1 molecular pathways. Next, we conducted behavioral verification for this. We performed SNI surgery on common C57BL/6J mice. On the 7th, 10th, and 14th days after the operation, EGCG (50 mg/kg) and CA (50 mg/kg) were intraperitoneally injected (normal saline was used as the control), and behavioral tests were conducted 4 hours after each injection. After the last test, the dorsal horn of the spinal cord was taken and proteins were extracted for Western Blot detection (Fig. 10 A). The results of the von Frey test showed that the withdrawal threshold of the right hind paw in the EGCG group increased from 0.02 g to 0.3 g on the 7th day after SNI compared with the control group ( P < 0.05), and this effect was maintained until the 14th day. However, there was no significant difference in the CA group compared with the control group (Fig. 10 B). The results of the hot plate test showed that the latency in the EGCG group increased from 5 s to 7 s on the 7th day after SNI compared with the control group ( P < 0.0001), and this effect was maintained until the 14th day. However, there was no significant difference in the CA group compared with the control group (Fig. 10 C). The Western blot results showed that the EGCG group (Fig. 10 D and E) could significantly reverse the increased expression of DRP1 ( P < 0.0001) and FIS1 ( P < 0.05) in the spinal dorsal horn caused by SNI, without affecting the expression level of MFF. Meanwhile, the CA group had no effect on the expression levels of the above proteins in the spinal dorsal horn after SNI (Fig. 10 F and G). These results suggested that EGCG, but not CA, could inhibit the expression of FIS1. At the same time, EGCG, but not CA, could exert an analgesic effect against SNI. This preliminarily validated our hypothesis that drugs capable of down-regulating the expression level of FIS1 in the spinal dorsal horn could also inhibit SNI-induced neuropathic pain. 4. Discussion This study demonstrated three key findings : first, mitochondrial networks in both excitatory and inhibitory neurons of the SDH were disrupted in spared nerve injury (SNI) mice, as demonstrated through the use of specifically constructed GAD2-MITO and vGluT2-MITO transgenic mouse models; second, selectively down-regulating FIS1 specifically in excitatory neurons, but not inhibitory neurons, of spinal dorsal horn could exert analgesic effects, as demonstrated using vGluT2-Cre mice and GAD2-Cre mice; third, epigallocatechin gallate (EGCG), which is capable of down-regulating FIS1 in the spinal dorsal horn, concurrently inhibited SNI-induced neuropathic pain. Our research findings suggest that FIS1 may represent a novel molecular target for the treatment of pain. This study supported that FIS1-mediated mitochondrial morphology and function alterations in the SDH could regulate neuropathic pain. A large number of literatures have indicated that mitochondrial dysfunction plays a significant role in the pathogenesis of neuropathic pain, with the main mechanism being the abnormal DRP1-mitochondrial fission-ROS cycle in the spinal dorsal horn [ 3 , 50 ]. Previous studies have shown that in neuropathic pain models, mitochondrial fragmentation and a reduction in the average planar area of mitochondria occur in the spinal dorsal horn, along with mitochondrial dysfunction [ 41 ], and enhancing mitochondrial function in the spinal dorsal horn can alleviate pain. Our research group has previously confirmed these conclusions and found that the DRP1 in the spinal dorsal horn increases, and regulating DRP1 in the spinal dorsal horn can exert analgesic effects and improve mitochondrial function [ 41 ]. However, previous work has not distinguished the types of neurons in the spinal dorsal horn regarding the above mechanisms. Our research results also found mitochondrial fragmentation and dysfunction in the spinal dorsal horn, and improving mitochondrial function in excitatory neurons in the spinal dorsal horn could alleviate neuropathic pain. Moreover, our study demonstrated that mitochondrial dysfunction in the spinal dorsal horn is caused by FIS1-mediated mitochondrial fragmentation. The mitochondrial fission protein FIS1 mediates the assembly of the mitochondrial fission complex and participates in peripheral mitochondrial fission, being an important factor in the process of mitochondrial fusion and fission [ 14 ]. Previous studies have found that inhibiting Fis1 can reduce mitochondrial fission in rat retinal endothelial cells [ 51 ]. We obtained the same mitochondrial morphological results by down-regulating FIS1 in the spinal dorsal horn of the SNI model and further proved that down-regulating FIS1 can exert analgesic effects by reducing ROS levels, increasing MMP and ATP content. Based on the current research results, it can be considered that targeting the regulation of FIS1 expression in SDH can affect mitochondrial morphology and function in response to neuropathic pain stress. This study showed that the morphological dysfunction of mitochondria in excitatory neurons in dorsal horn of spinal cord caused neuropathic pain. Interneurons in SDH can be divided into two categories: excitatory neurons and inhibitory neurons. Inhibitory neurons mainly exist in layers I-III, while excitatory neurons are almost distributed throughout SDH [ 7 ]. Previous studies have shown that both excitatory and inhibitory interneurons in the dorsal horn play an important role in neuropathic pain [ 52 ]. Consistent with previous studies, we found that both excitatory and inhibitory neurons were activated in neuropathic pain models with increased intracellular mitochondrial fission and reduced mitochondrial function. In addition, we found increased intracellular FIS1 expression in both. Considering that improving the function of spinal dorsal horn mitochondria can alleviate neuropathic pain [ 53 ], there are significant differences in somatosensory response and plasticity of SDH excitatory and inhibitory neurons [ 8 ]. We down-regulated FIS1 in excitatory nerves and inhibitory neurons of spinal dorsal horn in neuropathic pain mice. Interestingly, we only found that down-regulating FIS1 in excitatory neurons of SNI model alleviated mechanical pain and thermal pain, and effectively decreased mitochondrial fragmentation in excitatory neurons and enhanced mitochondrial function in spinal dorsal horn. Although downregulating FIS1 in SDH inhibitory neurons of SNI model decreased ROS levels, we found that had no effect on relieving pain. This may be related to the fact that peripheral nerve injury mainly activates excitatory neurons in the I-III layer of the dorsal horn of the spinal cord, and the excitatory neurons in the SDH in the pain model are more sensitive to thermal stimulation than the inhibitory neurons [ 7 , 54 ]. It has been reported that epigallocatechin gallate and cinnamic acid can be used for pain treatment. Epigallocatechin gallate is mainly used for postoperative pain treatment. Cinnamic acid is mainly used for cancer pain [ 39 , 49 ]. The application of these two drugs in neuropathic pain has not been reported. Epigallocatechin-3-gallate (EGCG), the main bioactive component of theocatechin, has been reported to rescue mitochondrial function and may improve cell function by inhibiting mitochondrial reactive oxygen species mediated iron death [ 40 , 55 ]. In addition, EGCG reduced DRP1 and FIS1 expression, reduced mitochondrial fragmentation, and improved mitochondrial dynamic function in subarachnoid hemorrhage models [ 55 ]. The main pathogenesis of neuropathic pain is the increase of ROS and mitochondrial fragmentation in the dorsal horn of the spinal cord, and the mechanism of EGCG is similar. Fortunately, after intrabitoneal injection of EGCG into the SNI model, we observed that EGCG had the same analgesic effect on the neuropathic pain model, and EGCG reduced the expression of FIS1 in the spinal dorsal horn of the SNI model. Therefore, we believed that the analgesic effect of EGCG may be mediated by reducing the expression of FIS1, thereby reducing mitochondrial fragmentation, and thus improving mitochondrial function. Cinnamic acid is a phenolic polyphenol. It has been reported in previous studies that it can play an anti-inflammatory and antioxidant role by reducing oxidative stress and inhibiting over-activated immune response [ 56 , 57 ]. It can also have an analgesic effect on oxaliplatin induced cold atopic pain by inhibiting spinal cord pain transmission [ 39 ]. Unfortunately, cinnamic acid was not able to relieve neuropathic pain in our SNI model, which may be related to the fact that the analgesic target of cinnamic acid is spinal wide dynamic range neurons rather than vGluT2 neurons, but we do not rule out the effectiveness of CA in other types of pain models. In summary, our present findings supported that mitochondria at the spinal cord level are sensitive to neuropathic pain, that SDH-targeted up-regulation of FIS1 can cause pain response, and that SDH-targeted down-regulation of mitotic protein FIS1 in excitatory neurons could alleviate neuropathic pain induced by SNI models. This study will promote the in-depth study of the mitochondrial mechanism of neuropathic pain, and is expected to provide new molecular targets and clinical drugs for the analgesic treatment of neuropathic pain. Although the current study results can demonstrate the key role of FIS1 in analgesia, there are still many shortcomings. After Fis1 knockout in GAD2-Cre mice, mitochondrial function was also impaired, indicating that Fis1 knockout also has an effect on the mitochondria of inhibitory neurons, which is worthy of further study. Secondly, due to the problem of experimental conditions, we cannot isolate the two types of neurons for mitochondrial function detection at present, and can only detect these two types of neurons as a whole. TEM analysis is an intuitive method to observe the morphological changes of mitochondria. We performed electron microscopic analysis after SNI and Fis1 over-expression and Fis1 knockout, but Fis1 knockout in Cre mice was not analyzed under electron microscope because we could not distinguish neuron types under electron microscope, which is what we need to improve in the future. 5. Conclusion In this study, neuropathic pain after nerve injury was associated with morphological abnormalities and dysfunction of spinal dorsal horn mitochondria in mice. The expression of FIS1 increases in spinal dorsal horn in pain state. Targeted down-regulation of FIS1 expression in spinal dorsal horn excitatory neurons of neuropathic pain mice can alleviate pain by reducing mitochondrial fragmentation and improving mitochondrial function. Our findings suggest that FIS1 may be a novel molecular target for the treatment of pain. Abbreviations AAV Adeno-associated virus ATP Adenosine triphosphate CA Cinnamic Acid CPN Common peroneal nerve COX Ⅳ Cytochrome c oxidase DRP1 Dynamin-related protein 1 EGCG Epigallocatechin gallate EPM Elevated plus maze FIS1 Fission protein 1 GABA Gamma-aminobutyric acid GAD2 Glutamic acid decarboxylase 2 KD Knockdown MFF Mitochondrial fission factor MiNA Mitochondrial network analysis MITO Mitochondria MMP Mitochondrial membrane potential OE Overexpression OFT Open field test OPA1 Optic atrophy 1 PBS Phosphate buffer saline PDHA1 Pyruvate dehydrogenase e1 subunit alpha 1 PWT Paw-withdrawal threshold rAAV Recombination adeno-associated virus ROS Reactive oxygen species SC-9 Anti-inflammatory agent 49 SDH Spinal dorsal horn SDHB Succinate dehydrogenase b SN Sural nerve SNI Spared nerve injury TEM Transmission electron microscope TFAM Mitochondrial transcription factor A TN Tibial nerve VDAC Voltage-dependent anion channel vGluT2 Vesicular glutamate transporter 2 Declarations Acknowledgments This work was supported by the following funds and foundations: Military Medicine Advancement Program, Air Force Military Medical University, 2020SWAQ04, Ya-Yun Wang; Shaanxi Innovation Capability Support Program, 2023-CX-PT-33, Ya-Yun Wang; Natural Science Basic Research Program of Shaanxi Province, 2024JC-ZDXM-60, Yan-Ling Yang; Xijing Hospital Clinical New Technology, 2023XJSY27, Yan-Ling Yang; Natural Science Basic Research Program of Shaanxi Province, 2022JQ-820, Fei-Fei Wu; National Natural Science Foundation of China, 82201627, Fei-Fei Wu. Authorship Western blot and immunofluorescence staining were performed by Chang-Lei Zhu and Shu-Jiao Li. Chang-Lei Zhu, Shu-Jiao Li and Ke Tian performed stereotactic surgery and behavioral tests. Yun-Qiang Huang, Fei-Fei Wu, Shuai Zhang and Hui Liu prepare animals. Chang-Lei Zhu, Zi-Wei Ni, Jing-Jing Tie, Zhi-Peng Lin, You-Sheng Wu, Fei Tian and Nan-Nan Liu analyze the data. Ya-Yun Wang and Yan-Ling Yang designed the study. Chang-Lei Zhu and Kun-Long Zhang wrote the draft. The electron microscope analysis was carried out by Zhi-Peng Lin and Xue-Yin Pu. Drawing and layout by Zhi-Peng Lin and Yu-Lu Xia. Ya-Yun Wang and Yan-Ling Yang supervised the experiments and revised the manuscript. All the authors read and approved the final manuscript. Declaration of interest The authors have declared that there is no conflict of interest. Data availability Data will be made available on request. References Finnerup NB, Kuner R, Jensen TS (2021) Neuropathic Pain: From Mechanisms to Treatment. 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Supplementary Files graphicalabstract.jpg Graphical abstract This study demonstrated that in neuropathic pain mice, the expression level of mitochondrial fission protein 1 (FIS1) in the excitatory neurons of the spinal dorsal horn significantly increases, accompanied by mitochondrial fragmentation, decreased mitochondrial membrane potential and adenosine triphosphate (ATP) content, and elevated reactive oxygen species (ROS) levels in the neurons. Targeted down-regulation of FIS1 in the excitatory neurons of the spinal dorsal horn alleviates neuropathic pain by correcting mitochondrial fragmentation, increasing mitochondrial membrane potential and ATP levels, and reducing ROS levels. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5916414","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":409280638,"identity":"fe47efc0-b320-4568-94aa-634a4c84e1f9","order_by":0,"name":"Chang-Lei Zhu","email":"","orcid":"","institution":"National Demonstration Center for Experimental Preclinical Medicine Education Air Force Medical University (Fourth Military Medical 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06:08:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5916414/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5916414/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75177376,"identity":"2c9c10a6-94bc-48fb-bde0-69695050db75","added_by":"auto","created_at":"2025-01-31 15:33:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":319507,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSNI-induced neuropathic pain increased DRP1 and FIS1 level in the ipsilateral SDH and activated SDH neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) SNI model construction diagram.\u003c/p\u003e\n\u003cp\u003e(B, C) von Frey mechanical threshold and hot plate reaction latency in C57BL/6J mice (n = 6 animals per group).\u003c/p\u003e\n\u003cp\u003e(D-G) Open field experiment and elevated cross maze experiment and trajectory analysis.\u003c/p\u003e\n\u003cp\u003e(H) Rotarod test analysis.\u003c/p\u003e\n\u003cp\u003e(I, J) Catwalk gait analysis and parameter analysis, including standing time, maximum paw contact area and complete paw print area.\u003c/p\u003e\n\u003cp\u003e(K) SDH sampling diagram.\u003c/p\u003e\n\u003cp\u003e(L, M) Western blot results and analysis (n = 3 animals for each group).\u003c/p\u003e\n\u003cp\u003e(N, O) SDH FOS immunofluorescence staining and analysis (Bar = 100 μm or 30 μm, n = 3 animals for each group, 10 slices were selected).\u003c/p\u003e\n\u003cp\u003e(P, Q) SDH FIS1 immunofluorescence staining and analysis (Bar = 100 μm or 30 μm, n = 3 animals per group, 6 slices were selected).\u003c/p\u003e\n\u003cp\u003eThe above data were analyzed with Unpaired t test. Data are presented as mean ± S.E.M. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/81f205ff67ac3584cc14c950.jpg"},{"id":75177371,"identity":"6d683b43-bf93-45ef-b17e-c607ab5346d0","added_by":"auto","created_at":"2025-01-31 15:33:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":631413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDRP1-FIS1 contact inhibitor SC-9 could alleviate mechanical allodynia and thermal hyperalgesia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The chemical structure of SC-9 and the behavioral test diagram after administration.\u003c/p\u003e\n\u003cp\u003e(B, C) Detection of von Frey mechanical threshold and hot plate latency after administration of SNI mice (n = 6 animals per group).\u003c/p\u003e\n\u003cp\u003e(D, E) Western blot analysis of SNI administration group and control group (n = 3 animals in each group).\u003c/p\u003e\n\u003cp\u003eData were analyzed using Unpaired t test and Two way ANOVA. Data are presented as mean ± S.E.M. **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/e75a502afe64837b3adb4448.jpg"},{"id":75177391,"identity":"41e37818-50a5-4b06-8740-4eb9a00611ef","added_by":"auto","created_at":"2025-01-31 15:33:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":368907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpregulating FIS1 in SDH neurons induced spontaneous pain in normal mice, while downregulating FIS1 alleviated pain in SNI mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) C57BL/6J mouse targeted injection Fis1 OE AAV and behavioral test diagram.\u003c/p\u003e\n\u003cp\u003e(B, C) The mechanical threshold of von Frey and the incubation period of hot plate reaction in OE mice (n = 6 animals per group).\u003c/p\u003e\n\u003cp\u003e(D-G) Open field experiment and elevated cross maze experiment and trajectory analysis.\u003c/p\u003e\n\u003cp\u003e(H) Rotarod test analysis.\u003c/p\u003e\n\u003cp\u003e(I, J) Catwalk gait analysis and parameter analysis, including standing time, maximum paw contact area and complete paw print area.\u003c/p\u003e\n\u003cp\u003e(K, L) Western blot results and analysis (n = 3 animals for each group).\u003c/p\u003e\n\u003cp\u003e(M) SNI model and behavioral test diagram were constructed after targeted injection of Fis1 KD AAV in C57BL/6J mice.\u003c/p\u003e\n\u003cp\u003e(N, O) von Frey mechanical threshold and hot plate reaction latency were detected in KD mice (n = 6 animals in NC group, n = 5 animals in KD group).\u003c/p\u003e\n\u003cp\u003e(P-S) Open field experiment and elevated cross maze experiment and trajectory analysis.\u003c/p\u003e\n\u003cp\u003e(T) Rotating rod fatigue test analysis.\u003c/p\u003e\n\u003cp\u003e(U, V) Catwalk gait analysis and parameter analysis, including standing time, maximum paw contact area and complete paw print area.\u003c/p\u003e\n\u003cp\u003e(W, X) Western blot results and analysis (n = 3 animals per group).\u003c/p\u003e\n\u003cp\u003eThe above data were analyzed with Unpaired t test. Data are presented as mean ± S.E.M. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/975f1d763bfa34edee425f2d.jpg"},{"id":75177373,"identity":"939f846c-003e-410f-8c82-b019648b9230","added_by":"auto","created_at":"2025-01-31 15:33:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":667993,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBoth excitatory and inhibitory neurons within the SDH of SNI mice activated and the FIS1 level was elevated in both types of neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) C57BL/6J mice constructed SNI were co-labeled with FOS and GAD2 (Bar = 100 μm or 20 μm, n = 5 for each group).\u003c/p\u003e\n\u003cp\u003e(B) Co-labeling of FOS and vGluT2 after SNI construction in C57BL/6 mice (Bar = 100 μm or 20 μm, n = 5 in each group).\u003c/p\u003e\n\u003cp\u003e(C) Percentage of colocalized neurons in all FOS activations.\u003c/p\u003e\n\u003cp\u003e(D) Co-labeling staining of FIS1 with GAD2 after SNI construction in C57BL/6J mice (Bar = 100 μm or 20 μm, n = 5 for each group).\u003c/p\u003e\n\u003cp\u003e(E) Co-labeling of FIS1 and vGluT2 after SNI construction in C57BL/6J mice (Bar = 100 μm or 20 μm, n = 5 for each group).\u003c/p\u003e\n\u003cp\u003e(F) The proportion of colocalization to GAD2 or vGluT2.\u003c/p\u003e\n\u003cp\u003eThe above data were analyzed using Two ANOVA methods. Data are presented as mean ± S.E.M. ****\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/3fd840bfbb22a13bc11206cc.jpg"},{"id":75177374,"identity":"93d18f50-98ad-4554-a748-5b692b4afae4","added_by":"auto","created_at":"2025-01-31 15:33:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":747523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial networks in both excitatory and inhibitory neurons of the SDH were disrupted in SNI mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Construction strategy of GAD2-MITO and vGluT2-MITO transgenic mice.\u003c/p\u003e\n\u003cp\u003e(B) MITO and VDAC colocalization analysis under 3D layer scan (Bar = 30 μm).\u003c/p\u003e\n\u003cp\u003e(C) MiNA of NeuN-labeled mature neurons and non-neurons (Bar = 30 μm or 10 μm, n = 3 in each group) in 3D layer scan after SNI in GAD2-MITO mice.\u003c/p\u003e\n\u003cp\u003e(D) MiNA of NEUN-labeled mature neurons and non-neurons in vGluT2-MITO mice under 3D layer scan after SNI (Bar = 30 μm or 10 μm, n = 3 in each group).\u003c/p\u003e\n\u003cp\u003e(E) MiNA statistical analysis of neuronal cell bodies and cell-body connections.\u003c/p\u003e\n\u003cp\u003e(F) Co-localization analysis of MITO and FIS1 in GAD2-MITO mice after 3D layer scan after SNI (Bar = 30μm, n = 3 per group).\u003c/p\u003e\n\u003cp\u003e(G) Co-localization analysis of MITO and FIS1 in vGluT2-MITO mice under 3D layer scan after SNI (Bar = 30 μm, n = 3 per group).\u003c/p\u003e\n\u003cp\u003e(H) The proportion of FIS1 and MITO co-localization in MITO.\u003c/p\u003e\n\u003cp\u003eThe above data were analyzed using Two ANOVA methods. Data are presented as mean ± S.E.M. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/eb274f40d1a462a1e7855199.jpg"},{"id":75177380,"identity":"fe126f87-9360-4c87-a065-443228303655","added_by":"auto","created_at":"2025-01-31 15:33:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":412610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDown-regulating FIS1 in excitatory neurons but not inhibitory neurons of spinal dorsal horn could exert analgesic effects.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) vGluT2-Cre mouse SDH targeted injection Fis1 KD rAAV and behavioral test diagram.\u003c/p\u003e\n\u003cp\u003e(B, C) Detection of von Frey mechanical threshold and hot plate reaction latency in KD mice (n = 3 in NC group and n = 4 in KD group).\u003c/p\u003e\n\u003cp\u003e(D-G) Open field experiment and elevated cross maze experiment and trajectory analysis.\u003c/p\u003e\n\u003cp\u003e(H, I) Catwalk gait analysis and parameter analysis, including standing time, maximum paw contact area and complete paw print area.\u003c/p\u003e\n\u003cp\u003e(J) Rotarod test analysis.\u003c/p\u003e\n\u003cp\u003e(K, L) co-localization analysis of Fis1 KD with vGluT2 in vGluT2-Cre mice (Bar = 100 μm or 20 μm, n = 3 in each group).\u003c/p\u003e\n\u003cp\u003e(M) GAD2-Cre mice SDH targeted injection Fis1 KD rAAV and behavioral test diagram.\u003c/p\u003e\n\u003cp\u003e(N, O) von Frey mechanical threshold and hot plate latency detection in KD mice (n = 5).\u003c/p\u003e\n\u003cp\u003e(P-S) Open field experiment and elevated cross maze experiment and trajectory analysis.\u003c/p\u003e\n\u003cp\u003e(T) Rotarod test analysis.\u003c/p\u003e\n\u003cp\u003e(U, V) Catwalk gait analysis and parameter analysis, including standing time, maximum paw contact area and complete paw print area.\u003c/p\u003e\n\u003cp\u003e(W, X) Co-localization analysis of Fis1 KD with GAD2 in GAD2-CRE mice (Bar = 100 μm or 20 μm, n = 3 per group).\u003c/p\u003e\n\u003cp\u003eThe above data were analyzed with Unpaired t test. Data are presented as mean ± S.E.M. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/653d9f1b7b930331cc3ffcb8.jpg"},{"id":75177418,"identity":"48f6049a-c8b6-46ca-a8ef-7ae17fcf01ee","added_by":"auto","created_at":"2025-01-31 15:33:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2385859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeted downregulation of FIS1 in the SDH of excitatory neurons in SNI mice mitigated mitochondrial dysfunction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) The levels of MMP, ATP and ROS in SNI mice SDH were detected (n = 3).\u003c/p\u003e\n\u003cp\u003e(D-F) Detection of MMP, ATP and ROS levels in C57 BL/6 mice after SDH targeted up-regulation of FIS1 (n = 3).\u003c/p\u003e\n\u003cp\u003e(G-I) Levels of MMP, ATP and ROS in C57 BL/6J mice after SDH targeted down-regulation of FIS1 (n = 3).\u003c/p\u003e\n\u003cp\u003e(J-L) Levels of MMP, ATP and ROS were detected after SDH targeted down-regulation of FIS1 in vGluT2-Cre mice (n = 3).\u003c/p\u003e\n\u003cp\u003e(M-O) Detection of MMP, ATP and ROS levels after SDH targeted down-regulation of FIS1 in GAD2-Cre mice (n = 3).\u003c/p\u003e\n\u003cp\u003eThe above data were analyzed with Unpaired t test. Data are presented as mean ± S.E.M. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/4f2121612473d1cead8bb112.jpg"},{"id":75177415,"identity":"542840a1-8235-4429-a1f2-21ca6d6104a8","added_by":"auto","created_at":"2025-01-31 15:33:19","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":470228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeted down-regulation of FIS1 in the SDH decreased the mitochondrial vacuoles and mitochondrial fragmentation induced by SNI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Electron microscopic images of SDH mitochondria in SNI mice (Bar = 1 μm or 500 nm, n = 3).\u003c/p\u003e\n\u003cp\u003e(B) Mitochondrial morphological analysis (267 mitochondria in Sham group and 572 in SNI group).\u003c/p\u003e\n\u003cp\u003e(C) Morphological analysis of mitochondrial vacuoles (46 vacuoles in Sham group and 341 vacuoles in SNI group).\u003c/p\u003e\n\u003cp\u003e(D) Electron microscopic images of SDH mitochondria in Fis1 OE mice (Bar = 1 μm or 500 nm, n = 3).\u003c/p\u003e\n\u003cp\u003e(E) Mitochondrial morphological analysis (235 mitochondria in the OENC group and 495 in the OE group).\u003c/p\u003e\n\u003cp\u003e(F) Morphological analysis of mitochondrial vacuoles (65 vacuoles in OENC group and 196 in OE group).\u003c/p\u003e\n\u003cp\u003e(G) Electron microscopic images of SDH mitochondria in Fis1 KD mice (Bar = 1 μm or 500 nm, n = 3).\u003c/p\u003e\n\u003cp\u003e(H) Mitochondrial morphological analysis (531 mitochondria in KDNC group and 271 mitochondria in KD group).\u003c/p\u003e\n\u003cp\u003e(I) Morphological analysis of mitochondria vacuoles (279 vacuoles in KDNC group and 122 vacuoles in KD group).\u003c/p\u003e\n\u003cp\u003eThe above data were analyzed with Unpaired t test. Data are presented as mean ± S.E.M. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/75f05ed5369bcf4a4f113895.jpg"},{"id":75177395,"identity":"403e2f5b-15ff-4c50-9110-5af6bd0bc64b","added_by":"auto","created_at":"2025-01-31 15:33:18","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":187118,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNetwork pharmacological analysis suggested that epigallocatechin gallate and cinnamic acid may exert analgesic effect by inhibiting FIS1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Wayne diagram of the intersection of chronic pain, cinnamic acid and epigallocatechin gallate genes.\u003c/p\u003e\n\u003cp\u003e(B) Protein-protein interaction maps between 16 key genes.\u003c/p\u003e\n\u003cp\u003e(C) Bioprocess enrichment map of intersection genes; (D) Cell component enrichment analysis diagram of intersection genes.\u003c/p\u003e\n\u003cp\u003e(E) Molecular functional enrichment map of intersection genes.\u003c/p\u003e\n\u003cp\u003e(F) Wayne diagram of intersection genes and mitochondrial division gene sets.\u003c/p\u003e","description":"","filename":"image10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/30d4c61ffc97cafe15ae69c1.jpg"},{"id":75177423,"identity":"79d3da44-94e9-4079-913e-27f195179edd","added_by":"auto","created_at":"2025-01-31 15:33:20","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1181924,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEpigallocatechin gallate could relieve neuropathic pain by inhibiting FIS1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The chemical structure formula of EGCG and CA and the behavioral test diagram after administration.\u003c/p\u003e\n\u003cp\u003e(B, C) Detection of von Frey mechanical threshold and hot plate reaction latency after administration of SNI mice (n = 5 per group).\u003c/p\u003e\n\u003cp\u003e(D, E) Western blot results and analysis of CA group and control group (n = 3 in each group).\u003c/p\u003e\n\u003cp\u003e(F, G) Western blot analysis of EGCG group and control group (n = 3 in each group).\u003c/p\u003e\n\u003cp\u003eData were analyzed using Unpaired t test and Two way ANOVA. Data are presented as mean ± S.E.M. *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/dfc9ed0fa7332de0b47aa694.jpg"},{"id":75433685,"identity":"28166526-90f6-4f97-888b-0dc667b2975c","added_by":"auto","created_at":"2025-02-04 14:02:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9563747,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/d5933339-eed8-46c0-bef1-2b4179476ec3.pdf"},{"id":75177372,"identity":"e7866e10-8b27-4d1c-9127-6d5e7598c477","added_by":"auto","created_at":"2025-01-31 15:33:15","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":138085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study demonstrated that in neuropathic pain mice, the expression level of mitochondrial fission protein 1 (FIS1) in the excitatory neurons of the spinal dorsal horn significantly increases, accompanied by mitochondrial fragmentation, decreased mitochondrial membrane potential and adenosine triphosphate (ATP) content, and elevated reactive oxygen species (ROS) levels in the neurons. Targeted down-regulation of FIS1 in the excitatory neurons of the spinal dorsal horn alleviates neuropathic pain by correcting mitochondrial fragmentation, increasing mitochondrial membrane potential and ATP levels, and reducing ROS levels.\u003c/p\u003e","description":"","filename":"graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5916414/v1/d7376339ea5b52f4358585bd.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeted downregulation of FIS1 in excitatory neurons within the spinal dorsal horn alleviates neuropathic pain through the mitigation of mitochondrial fragmentation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNeuropathic pain has a prevalence of approximately 10% and is on the rise, becoming one of the most common and severe chronic clinical symptoms [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is a type of pain triggered or caused by primary damage or dysfunction of the nervous system. Due to the incomplete understanding of the pathogenesis of neuropathic pain, its clinical treatment remains a significant challenge [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Currently, the treatment options for neuropathic pain are limited, mainly including opioids and antioxidants. Therefore, in-depth exploration of the pathogenesis of neuropathic pain is necessary and may provide new targets for the clinical treatment of neuropathic pain.\u003c/p\u003e \u003cp\u003eThe spinal dorsal horn (SDH), as the primary center for pain information integration, is a key breakthrough point in the study of the mechanism of neuropathic pain [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. As the primary gateway for the ascending conduction of peripheral pain stimuli, the SDH is crucial for the integration of pain signals and central sensitization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In neuropathic pain, both excitatory and inhibitory neurons in the spinal dorsal horn exhibit functional abnormalities [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Inhibitory neurons in the spinal dorsal horn are mainly distributed in the superficial layer, while excitatory neurons are widely distributed throughout the spinal dorsal horn [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere is a close relationship between neuropathic pain and mitochondria. In various animal models of pain, mitochondrial homeostasis imbalance can be observed in the central nervous system neurons, ultimately leading to the occurrence and development of neuropathic pain [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Mitochondrial dysfunction and excessive fragmentation can induce the pathological production of reactive oxygen species and play a significant role in the state of neuropathic pain [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Improving mitochondrial function and morphology can relieve neuropathic pain [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The changes in mitochondrial structure and function are mainly mediated by mitochondrial fission and fusion [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Mitochondrial fission and fusion determine the fate of mitochondria [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Mitochondrial fission protein 1 (FIS1) is an important molecule in mitochondrial fission [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. FIS1 is widely distributed on the outer membrane of mitochondria, and its main function is to recruit free DRP1 in the cytoplasm and mediate the peripheral fission of mitochondria [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, this study investigated the effects of upregulation and downregulation of FIS1 in the SDH of SNI mice on their gait and pain behavior. We found that upregulation of FIS1 in the SDH of normal mice could cause spontaneous pain behavior, while inhibition of FIS1 in the SDH of SNI mice could alleviate neuropathic pain. By constructing transgenic mice with excitatory and inhibitory neuron specificity, we discovered that downregulation of FIS1 in inhibitory neurons of the SDH of SNI mice could exert analgesic effects and improve the morphology and function of mitochondria in the SDH. Additionally, we screened two FIS1-related clinical analgesic drugs, epigallocatechin gallate and cinnamic acid. Among them, epigallocatechin gallate could alleviate neuropathic pain by inhibiting FIS1, further confirming that inhibition of FIS1 expression can relieve neuropathic pain. This study will promote in-depth research on the mitochondrial mechanism of neuropathic pain and is expected to provide new molecular targets and clinical drugs for the analgesic treatment of neuropathic pain.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eAdult (6\u0026ndash;8 w) male wild-type C57BL/6J, vGluT2-ires Cre, GAD2-ires Cre, vGluT2-Mito-GFP, and GAD2-Mito-GFP mice were used in this study. Among them, C57BL/6 J mice were purchased from the Animal Center of the Fourth Military Medical University in Xi 'an, China. All mice were placed in 12 hours of light (8 am)/darkness (8 PM). Male mice were used throughout the study to further control for differences caused by gender differences in the animals. All animal testing protocols are based on procedures approved by the Animal Ethics and Welfare Committee of the Air Force Medical University Institutional Animal Care and Use Committee (IACUC-20190107) and follow institutional guidelines for the use of laboratory animals. All experiments were conducted by double-blind method.\u003c/p\u003e \u003cp\u003evGluT2-ires Cre and GAD2-IRES Cre mice purchased from Jackson's laboratory displayed internal ribosome entry sites and CRE recombinases at the 3\u0026rsquo;utr of vesicular glutamate transporter site 2 (vGluT 2) and glutamate decarboxylase site 2 (GAD2). Therefore, endogenous vGluT2 and GAD2 promoter/enhancer elements target glutamatergic and GABAergic populations in the central nervous system represented by vGluT2 and GAD2 positive neurons for Cre expression [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. When vGluT2-ires-Cre and GAD2-ires-Cre mice were infected with viral vectors containing double floxed inverse open reading frames (DIO), Including rAAV-CMV-DIO- (EGFP-U6) -SHRNA (scramble) -Wpre-HGH poly A (Fis1 NC); rAAV-CMV-DIO- (EGFP-U6) -SHRNA (Fis1) -Wpre-HGH poly A (KD), will achieve knockdown (KD) regulation in the corresponding neurons.\u003c/p\u003e \u003cp\u003eGFP-MITO label adhesive mice were produced by GemPharmatech Co, Ltd (Nanjing, China) and attached to a MITO-Tag box for the CAG-LSL-GFP-MITO label. A construct containing CAG-loxP-STOP-loxP-Kozak-GFP-MITO-TGA-pA targets Rosa26 of mouse Gt (ROSA) 26s. When GFP-MiTO-labeled mice were hybridized with vGluT2-ires-Cre and GAD2-ires-Cre drivers, the resulting mouse lines, called vGluT2-MITO-GFP and GAD2-MITO-GFP, exhibited bright MITO-GFP fluorescence. It is specifically localized in all glutamatergic and GABAergic central nervous mitochondria expressed by vGluT2 and GAD2 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Surgical procedures for SNI models\u003c/h2\u003e \u003cp\u003eThe mice were anesthetized with pentobarbital (50 mg/kg, i.p) and an incision of about 1.5 cm was made at the upper margin of the right hind limb. The muscle is then gently separated to reveal the main sciatic nerve and its 3 branches, which are directly separated. Nerve injury (SNI) is induced by ligation and severing of 2\u0026ndash;4 mm segments of the common peroneal and tibial nerves. The sciatic nerve and its branches were only exposed in control mice [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. After surgery, all mice were monitored for infection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Behavioral detection\u003c/h2\u003e \u003cp\u003eThe experiment time was 09:00\u0026ndash;12:00 in the morning. To minimize possible transfer effects and potential visual or olfactory effects, the mice were moved to a behavioral laboratory ahead of time and acclimated to the environment for at least 30 minutes. After each test, the surface of the instrument was cleaned with 75% alcohol to avoid odor interference from the previous mouse.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003e2.3.1. Mechanical hypersensitivity\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eMechanical hypersensitivity behavior was evaluated within two weeks after SNI. von Frey wire (No.37450-275, Aesthesio, Italy) was used to evaluate the foot regression threshold of mice in response to mechanical stimulation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Hold each von Frey wire perpendicular to the side of the back claw for about 5 seconds. Stimulate the right hind foot (5 minutes apart). The mechanical threshold (in grams) is defined as the first fiber that causes at least 3 foot retractions in 5 tests. A series of calibrated von Frey wires were used to measure PWT before (day \u0026minus;\u0026thinsp;1) and after (SNI) days 1, 3, 5, 7, 10, 14.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003e2.3.2. Hyperalgesia thermalis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe latency of the response to the hot plate was measured before (baseline level) and after SNI to indicate hyperthermic algesia [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The mice were placed on a hot plate (LS-6B, Jinan, China) and the temperature was adjusted to 54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5◦C. Record the time of the first nociceptive response (licking, recoiling, or jumping) of the operative side paw and immediately remove the animal from the hot plate. To avoid injury to the Java, the maximum detection time was maintained at 25 seconds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Open field test(OFT)\u003c/h2\u003e \u003cp\u003eAnxiety-like emotions of mice were detected by OFT experiment, which indirectly reflected pain response [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The experimental device was a 50 \u0026times; 50 cm plexiglass square box with a wall 50 cm high. The mice were placed in the center of the plexiglass box and were free to explore the whole box for 15 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003e2.3.4.\u003c/em\u003e Elevated plus maze \u003cem\u003e(EPM)\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eEPM test was used to detect anxiety-like emotions in mice, which indirectly reflected pain response [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The elevated Cross maze was made of plexiglass and consists of two open arms (35 x 7 cm) and two closed arms (35 x 7 cm), the latter surrounded by walls 15 cm high. The device was raised 50 centimeters above the ground. Each mouse was placed in the center of the device and allowed to move freely for five minutes. Anxiety-like behavior was determined by measuring the position preferences of mice with open arms for 5 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003e2.3.5. Rotarod test\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eRotary rod test was used to detect the ability of mouse feet to grasp the ground and indirectly reflect the pain response [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The effect of pain on exercise endurance by responding to it during rod time. The mice were placed on a rotating rod fatigue instrument (BYZ-007, Dongguan, China) and started to rotate the rotating rod. Starting at 0 rpm and gradually increasing to 40 rpm, the acceleration time and measurement time are both 5 minutes. The stay time of the mice on the rotating rod was observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003e2.3.6. Catwalk analysis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eGait analysis was used to detect pain response in mice [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Gait analysis was performed using the Catwalk XT system (Noldus, Netherlands), which has proven to be a very reliable method for measuring pain-related behaviors. The mice were placed at the open end of an enclosed glass platform and walked autonomously on a glass floor, during which a high-speed camera under the device captured images of each paw and transmitted the data to gait analysis software (Catwalk XT, version 10.6; Noldus). In this study, three parameters were identified that could be used to evaluate neuropathic pain-related dynamic behavior: (1) the duration of contact with the glass plate by the standing paw; (2) the maximum paw contact area; (3) The area of the complete paw print when walking.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.4. Stereotactic viral vector injection\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eViral vectors used in this study include: pAAV-CMV-Fis1-3xFLAG-EF1a-EGFP-tWPA(OE), pAAV-CMV-MCS-EF1a-EGFP-tWPA(OENC), pAAV-U6-shRNA (Fis1)-CMV-EGFP-WPRE (KD), pAAV-U6-shRNA (NC2)-CMV-EGFP-WPRE (KDNC), rAAV-CMV-DIO-(EGFP-U6)-SHRNA (scramble)-Wpre-HGH poly A (KDNC), rAAV-CMV-DIO-(EGFP-U6)-SHRNA (Fis1)-WPRE-HGH pA (KD), all supplied by OBiO (China). Mice were sedated with pentobarbital sodium (50 mg/kg, i.p) prior to placement in a stereotaxator (RWD, China). Target spinal position by palpation. The paraspinous muscle above the L4/5 vertebra was excised and the right vertebra was partially excised for injection. The injection volume was 400 nL and the injection speed was 40 nL/min. To inject the virus into the SDH, the stereolocator was moved 500 nm to the right side of the spinal cord, with a final tip depth of 300 nm. The needle stays in the spinal cord for another 10 minutes and is then removed. The wound was sutured intermittently in layers, and iodine disinfectant was applied to the closed wound.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.5. Tissue preparation and immunofluorescence analysis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe mice were anesthetized with pentobarbital sodium (100 mg/kg, i.p), transorally infused with 50 mL 0.01 M phosphate buffered saline (PBS, pH 7.4), and then fixed with 4% paraformaldehyde (pH 7.4). The spinal cords of mice were then collected and soaked in 0.1M PBS containing 30% sucrose at 4℃. In low temperature thermostat (Leica CM1800; Heidelberg, Germany). Sections were incubated at room temperature for 30 min in a blocking PBS buffer containing 0.3% Triton X-100 and 0.05% sodium azide and 10% bovine serum albumin. After the primary antibody was incubated at 4\u0026deg;C overnight, the secondary antibody was incubated (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and DAPI (1:1000, #BMD0063, Abbkine, CN) was added and placed at room temperature for 12 minutes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. All images were taken under the LEICA laser confocal microscope STELLARIS5 (Germany).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibodies used for Western blot or Immunofluoresence.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVendor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCat.#\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWB/IF\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ- actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAC004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec-FOS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2250T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOX Ⅳ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4850T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDRP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8570T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFIS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 250 and 1༚1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32523T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIF/WB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSanta Cruz Animal Health\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003esc-365180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMFF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e84580T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeuN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab177487\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOPA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67589T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDHA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab168379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSDHB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab175255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTFAM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8076T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVDAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab15895\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003evGluT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab79157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlexa 488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbbkine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA23220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlexa 594\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbbkine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA23420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlexa 594\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbbkine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA23620\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlexa 649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbbkine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1: 500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA23610\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHRP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbbkine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA21020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHRP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbbkine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA21010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.6. Western blot\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eProteins were isolated from homogenate tissue using modified RIPA buffers with protease and phosphatase inhibitors (Thermo Fisher Scientific). Assay Reagent Kit was used to determine protein concentration. The protein was isolated by 12.5% SDS-PAGE and transferred to 4.5um PVDF membrane, then blocked in TBST with 5% skim milk and incubated at room temperature for 2 hours. The primary antibody was incubated at 4\u0026deg;C overnight and the secondary antibody HRP was incubated (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Fusion FX EDGE chemiluminescence imaging scanning film was used, and ImageJ was used for gray value analysis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.7. Transmission electron microscope (TEM)\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAfter anesthesia, mice were injected 0.01 M PBS, 4% formaldehyde and 0.1% glutaraldehyde PBS successively. Spinal cord tissue was placed in 4% glutaraldehyde solution for tissue fixation. After fixation, each spinal cord tissue was washed twice with 0.1 M PBS. It was then washed with 1% osmic acid (TED PELLA, Inc.No.18451). Dye for 2 hours, then dehydrate with 50%, 70%, 90% ethanol and 100% acetone, respectively, and then soak with acetone: embedding agent (Embed 812, DDSA, NMA, DMP-30) at room temperature 1:1 for 2 hours. Finally, the tissue was removed from the embedding agent and stored overnight at room temperature. On the second day, the tissue was placed in a special electron microscope plate, incubated at 60◦C for 48 hours, and 70 nm thick slices were prepared using an ultra-thin secting mechanism. The sections were placed on a copper rack and stained with lead nitrate and uranyl acetate for 10 minutes, respectively. TEM (JEM 1400, Olympus, Japan) was used to record images. The mitochondrial morphological data were analyzed by imagej based image analysis method. Finally, 16 continuous tissue sections were selected for TEM analysis, and slices with a thickness of about 1500 nm were obtained. The size of each mitochondria was obtained by drawing an electron microscope sagittal picture of the SDH mitochondria. By using ImageJ's hand-drawn selection, the contours of mitochondria and internal vacuoles are carefully drawn. The number of mitochondria is the total number of mitochondria in each image, and then the image area is calculated according to the scale carried by the image, and the density, circumference and circle rate of mitochondria are calculated. The same method was used to calculate the area, circumference, and density of mitochondrial vacuoles [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.8. Mitochondrial network analysis (MiNA)\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eMitochondrial Network Analysis (MiNA) uses the FiJi distribution, freely available on the ImageJ platform, and combines open source tools into a simple macro toolset. Spinal cord sections of vGluT2-Mito-GFP and GAD2-Mito-GFP mice were photographed under laser scanning confocal microscopy (LEICA, STELLARIS5). Mitochondrial network analysis was performed using the MiNA plug-in in ImageJ [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.9. Mitochondrial extraction\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eMice were anesthetized with pentobarbital sodium (100 mg/kg, i.p) and SDH was rapidly removed. Purification of mitochondria using a mitochondrial extraction kit (SM0020, Solarbio, CN) : First, the tissue was cut as small as possible with scissors, then broken with a pre-cooled tissue analyzer (QIAGEN). Centrifuge 1000 g at 4\u0026deg;C twice for 5 minutes, then 12000 g for 10 minutes. The supernatant was discarded and the mitochondrial precipitation was placed on ice by re-suspension with Store Buffer. Finally, mitochondrial proteins were quantified by BCA method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.10. Mitochondrial membrane Potential Detection (MMP)\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eMitochondrial membrane potential (JC-1) was detected using an enhanced mitochondrial membrane potential assay kit (Beyotime, C2003S, CN). Detection with fluorescent enzyme marker: After purified mitochondria were mixed with the working liquid, the enzyme marker was directly used to scan, and the excitation wavelength was 485 nm and the emission wavelength was 590 nm to detect the fluorescence intensity of mitochondrial JC-1 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.11. ROS assay\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eMitochondrial reactive oxygen Species ROS assay kit (Bestbio, BB-460913, CN) was used to detect ROS levels in purified mitochondria. The fluorescence intensity of purified mitochondria was measured with an enzyme labeler at excitation wavelength of 485 nm and emission wavelength of 590 nm [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.12. ATP detection\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTissue ATP levels were measured using an enhanced ATP test kit (Beyotime, S0027, China) according to manufacturer's instructions. The luminescence was measured at 560 nm using an enzyme labeler, a standard curve was generated and used to calculate ATP concentration. Histamin was quantified by BCA [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.13 Network pharmacological analysis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eDatabase: Comparative Toxicogenomics Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ctdbase.org/\u003c/span\u003e\u003cspan address=\"https://ctdbase.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), MitoCarta3.0 dataset (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways\u003c/span\u003e\u003cspan address=\"https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), GSEA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gsea-msigdb.org/gsea/index.jsp\u003c/span\u003e\u003cspan address=\"https://www.gsea-msigdb.org/gsea/index.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Drawing: microscopic letter (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Chiplot (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chiplot.online/\u003c/span\u003e\u003cspan address=\"https://www.chiplot.online/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChronic Pain, Cinnamic Acid and Epigallocatechin Gallate were selected as keywords to predict the target genes with Comparative Toxicogenomics Database. The PPI network interaction was analyzed by using STRING database. GO and KEGG path enrichment analysis was performed for key targets using DAVID database and Weisheng platform. Weisheng and Chiplot were used for image rendering. A total of 16 targets were associated with keywords. After intersection with mouse mitochondrial gene set, the intersection targets were input into STRING database for protein interaction analysis, and free targets were removed. A total of 16 target genes related to mitochondria were obtained and identified as target genes. Mitochondrial Fission was searched for in the GSEA database, and Cinnamic Acid, Chronic Pain, Epigallocatechin Gallate and Epigallocatechin gallate were selected. Finally, DRP1 and FIS1 were the key proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.14 Intraperitoneal drug injection\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eSC-9 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] (TargetMol, T83057, USA; Cinnamic Acid (MCE, HY-N0610A, USA: 30 mg/kg) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and Epigallocatechin Gallate [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] (MCE, HY-13653, USA: 20 mg/kg) were intraperitoneally administrated to SNI mice on days 7, 10, and 14 post-SNI. Mechanical pain was assessed using von Frey filaments, while thermal pain was evaluated using a hot plate test, both conducted 4 hours after administration. Statistical analysis was performed as described in section \u003cspan refid=\"Sec26\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.15 Statistical analysis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe statistical comparison methods of each research method were detailed in the legend. Quantitative data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. For statistical analysis, unpaired \u003cem\u003et-\u003c/em\u003etests were employed for single comparisons, while two-way ANOVA was utilized for multiple comparisons. All analyses were conducted using SPSS 26.0 software and GraphPadPrism 9.0. A \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003e3.1 Spared nerve injury (SNI)-induced neuropathic pain upregulated the expression levels of DRP1 and FIS1 in the ipsilateral SDH and activated SDH neurons.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe spared nerve injury (SNI) neuropathic pain model was established by separating the right common peroneal nerve (CPN), sural nerve (SN) and tibial nerve (TN) of mice and ligating the CPN and TN (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results of the von Frey mechanical pain threshold test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) showed that the paw withdrawal threshold of mice decreased from 1.0 g to 0.02 g on the first day after SNI (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and remained so until the 14th day after surgery (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while no such change was observed in the Sham group, indicating that SNI caused mechanical allodynia in mice. The results of the hot plate test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) showed that the latency of mice decreased from 10 s to 6.5 s on the first day after SNI (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and remained so until the 14th day after surgery, while no such change was observed in the Sham group, indicating that SNI caused thermal hyperalgesia in mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe open field test (OFT) and elevated plus maze (EPM) experiments provided indicators of anxiety-like behavior in animals. The OFT results showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and E) that the distance and time spent in the central area by the SNI group mice were significantly lower than those of the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The EPM results indicated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and G) that the number of entries into the open arms, the time spent in the open arms, and the distance traveled in the open arms of the SNI group mice were all significantly lower than those of the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eIf animals exhibit hyperalgesia, it would also be manifested as abnormal results in the rotarod test. The rotarod test results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) showed that the time spent on the rod by the SNI group mice was significantly shorter than that of the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The Catwalk gait analysis provided fine behavioral indicators of the mice. The Catwalk gait analysis results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI and J) indicated that the standing time of the right hind limb, the maximum contact area of the right hind paw upon landing, and the footprint area of the right hind paw in the SNI group were all significantly reduced compared with those in the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eFurther, 14 days after SNI or Sham, the spinal dorsal horn (SDH) tissue on the stimulated side was collected for Western blot analysis of 8 mitochondrial-related molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). The results showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL and M) that there was no statistically significant difference between the two groups in optic atrophy protein 1 (OPA1), mitochondrial fission factor (MFF), pyruvate dehydrogenase alpha (PDHA1), succinate dehydrogenase complex B subunit (SDHB), and mitochondrial transcription factor A (TFAM). However, the expression of mitochondrial fission-related proteins dynamin-related protein 1 (DRP1) and mitochondrial fission protein 1 (FIS1) was significantly increased in the SNI group compared with the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the expression of complex IV (COX IV), a key molecule in the mitochondrial electron transport chain, was significantly decreased in the SNI group compared with the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eFurther, immunofluorescence staining of nuclear phosphoprotein FOS was used to detect neuronal activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN). The FOS staining (green) results showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO) that the number of FOS-positive cells in the dorsal horn of the stimulated side of the spinal cord in the SNI group was significantly increased compared with the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003ePrevious papers published by our research group have confirmed that SNI causes an increase in the expression of mitochondrial fission protein DRP1 in the spinal cord dorsal horn [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, this study focused on another important mitochondrial fission protein, FIS1, for research. FIS1 immunofluorescence staining (green) experiments were used to verify the Western blot detection results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eP). The results showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ) that the relative fluorescence expression area and relative fluorescence intensity of FIS1 in the dorsal horn of the stimulated side of the spinal cord in the SNI group were significantly increased compared with the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eThe above results suggested that there was neuronal activation in SDH of SNI mice, and the expressions of DRP1 and FIS1 were both increase, while the complex IV of the mitochondrial respiratory chain was reduced, indicating mitochondrial respiratory dysfunction.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.2 DRP1-FIS1 contact inhibitor SC-9 could alleviate mechanical allodynia and thermal hyperalgesia.\u003c/h2\u003e \u003cp\u003eNext, we will examined whether FIS1 affects neuropathic pain. Previous studies have shown that DRP1, which is located in the cytoplasm, translocates to the outer mitochondrial membrane and binds to FIS1 to initiate mitochondrial fission. Our previous published work has confirmed that targeting upregulation of DRP1 at the spinal cord level produces analgesic effects [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Recently, it was reported that Anti-inflammatory agent 49 (SC-9) is a contact inhibitor of DRP1 and FIS1, which can inhibit the interaction between DRP1 and FIS1 without affecting the physiological function of DRP1 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, we intraperitoneally injected SC-9 into SNI mice to detect the changes in mechanical allodynia and thermal hyperalgesia when the FIS1-dependent initiation stage of mitochondrial fission is inhibited.\u003c/p\u003e \u003cp\u003eWe performed SNI surgery on C57BL/6J mice and administered SC-9 (50 mg/kg) intraperitoneally on the 7th, 10th, and 14th days after the operation. Behavioral tests were conducted 4 hours after each injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The results showed that on the 7th, 10th, and 14th days after SNI, the mechanical threshold of mice in the SC-9 injection group was significantly increased compared with that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the thermal pain latency was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter the last behavioral test, the spinal dorsal horn proteins of the two groups of mice were extracted for Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The results showed that SC-9 inhibited the expression of FIS1 in SDH after SNI, but did not affect the expression of DRP1 and MFF [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eThe above results indicated that the DRP1-FIS1 contact inhibitor SC-9 significantly alleviates mechanical allodynia and thermal hyperalgesia caused by SNI.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Upregulating FIS1 in SDH neurons induced spontaneous pain in normal mice, while downregulating FIS1 alleviated pain in SNI mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe previous results indicated that the inhibitor of FIS1 exerted an analgesic effect in the system. Therefore, we conducted two experiments: one was to up-regulate the expression of FIS1 in SDH neurons of normal mice, and the other was to down-regulate the expression of FIS1 in SDH neurons of SNI mice. Then, we observed the pain behavior of the mice.\u003c/p\u003e \u003cp\u003eFirstly, we injected the Fis1 overexpression (OE) adeno-associated virus (AAV) targeting neurons into the unilateral (right) SDH of C57BL/6J mice, with the empty virus as the control. Behavioral observations were performed on days 1, 3, 5, 7, 10, and 14 after 14 days of injection (when the virus began to exert a stable effect), and spinal cord tissue was collected after the last behavioral test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of the von Frey mechanical pain threshold test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) showed that the withdrawal threshold of the right hind paw of the OE treatment group was significantly lower than that of the empty virus group from the 1st day of observation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and it continuously decreased from 1.0 g to 0.02 g over the next 14 days (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The results of the hot plate test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) showed that the latency of the OE treatment group was significantly lower than that of the empty virus group from the 1st day of observation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and it continuously decreased to 5 s over the next 14 days (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eAlthough the OFT results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and E) and Catwalk gait analysis results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI and J) showed no significant differences between the OE group and the control group, the EPM results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and G) indicated that the number of entries into the open arms, the time spent in the open arms, and the distance traveled in the open arms of the OE treatment group were significantly lower than those of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The results of the rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) showed that the time spent on the rotarod by the OE treatment group was significantly shorter than that of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eFurther, Western blot analysis was performed on the dorsal horn tissue of the stimulated side (right side) of the spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL and K). The results showed that the expression of FIS1 in the OE group was significantly increased compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), confirming the reliability of the Fis1 OE AAV. Meanwhile, the expression level of DRP1 in the OE treatment group was significantly lower than that in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the expression levels of the other six molecules (OPA1, PDHA1, MFF, SDHB, TFAM, and COX IV) showed no significant differences between the two groups.\u003c/p\u003e \u003cp\u003eTo further down-regulate the expression of FIS1 in SDH neurons of SNI mice. We injected Fis1 knockdown (KD) AAV into the unilateral (right) SDH of mice by spinal cord injection, with empty virus as the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). The experimental process was consistent with OE group.\u003c/p\u003e \u003cp\u003eThe results of the von Frey mechanical pain threshold test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN) showed that the withdrawal threshold of the right hind paw of SNI mice in the KD treatment group increased from 0.16 g to 0.5 g on the 1st day compared with the empty virus group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and this increase persisted for 14 days. The results of the hot plate test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO) showed that the latency of SNI mice in the KD treatment group increased from 6 s to 9 s on the 1st day compared with the empty virus group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and this increase persisted for 14 days.\u003c/p\u003e \u003cp\u003eThe results of the OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP and Q) showed that the distance and time of activity in the central area of SNI mice in the KD treatment group were significantly increased compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results of the EPM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eR and S) showed that the time spent in the open arms of SNI mice in the KD treatment group was significantly increased compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eThe results of the Catwalk gait analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eT and U) showed that the standing time of the right hind limb, the maximum contact area of the right hind paw, and the area of the right hind paw print of SNI mice in the KD treatment group were significantly increased compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe results of the rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eV) showed that the time on the rod of SNI mice in the KD treatment group was significantly prolonged compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eSubsequently, the SDH of the stimulated side was sampled for Western blot detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eW). The results showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eX) that the expression of FIS1 in the KD group was significantly lower than that in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), confirming the reliability of Fis1 KD AAV. Meanwhile, the expression levels of DRP1 and PDHA1 in the KD treatment group of SNI mice were significantly increased compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the expression level of SDHB was significantly decreased, and there was no difference in the expression of OPA1, MFF, TFAM and COX IV molecules.\u003c/p\u003e \u003cp\u003eThe above results suggested that targeted up-regulation of FIS1 expression in SDH neurons causes spontaneous pain in normal mice, while targeted down-regulation alleviated the pain response caused by SNI.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 Both excitatory and inhibitory neurons within the SDH of SNI mice activated and the FIS1 level was elevated in both types of neurons.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have shown that the SDH contains both excitatory and inhibitory neurons, with inhibitory neurons mainly located in layers I-III, while excitatory neurons are distributed throughout the SDH [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Our results suggested that regulation of FIS1 levels in SDH neurons caused changes in mechanical and thermal hyperalgesia in normal mice. To distinguish the neuronal types in the SDH of normal mice that respond to FIS1 expression after SNI, we first performed double immunofluorescence staining with the neuronal activation marker FOS (green) and vesicular glutamate transporter site 2 (vGluT 2) / glutamate decarboxylase site 2 (GAD2) (red) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). The results of the double immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) showed that the number of FOS-positive cells co-localized with GAD2 in the SDH after SNI was significantly higher than that in the Sham group; similarly, the number of FOS-positive cells co-localized with vGluT2 in the SDH after SNI was also significantly higher than that in the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we used the same method to perform double immunofluorescence staining for FIS1 (green) and GAD2/vGluT2 (red). Statistical analysis was performed using the commonly used Manders' Colocalization Coefficients (MCC) in immunofluorescence. M1 represents the ratio of the co-labeled fluorescence area of the two proteins to the fluorescence area of a single channel protein. Here, it indicates the ratio of the co-labeled fluorescence area of FIS1 and GAD2 or vGluT2 to the single-labeled fluorescence area of GAD2 or vGluT2. The results showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F) that the M1 values related to GAD2 and vGluT2 in the SDH after SNI were significantly increased compared to the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eThe above results indicated that both excitatory and inhibitory neurons in the SDH were activated after SNI, and FIS1 was expressed in both types of neurons. Therefore, the results of this part of the study could not yet answer which type of neuron FIS1 exerts its pain regulatory effect on.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 Mitochondrial networks in both excitatory and inhibitory neurons of the SDH were disrupted in SNI mice, as demonstrated through the use of specifically constructed GAD2-MITO and vGluT2-MITO transgenic mouse models.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTherefore, we designed the following experiment. We first constructed GAD2-MITO and vGluT2-MITO transgenic mice that could respectively express green fluorescent protein on the outer mitochondrial membrane of excitatory and inhibitory neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). When GFP-MITO labeled mice were crossed with vGluT2-ires-Cre and GAD2-ires-Cre driver factors, the offsprings were called vGluT2-MITO-GFP and GAD2-MITO-GFP, showing bright MITO-GFP fluorescence, specifically localized in all vGluT2 and GAD2 expressing central nervous system mitochondria of glutamatergic and GABAergic neurons neurons [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, we performed voltage-dependent anion channel (VDAC) immunofluorescence staining on the SDH of normal GAD2-MITO and vGluT2-MITO mice. The co-localization analysis of confocal fluorescence images showed that VDAC (purple) completely overlapped with mitochondria (green) in both types of mice, indicating that the green fluorescence in GAD2-MITO and vGluT2-MITO mice indeed labeled the mitochondria in excitatory and inhibitory neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eSubsequently, SNI models were established using GAD2-MITO and vGluT2-MITO mice. On the 14th day after surgery, the spinal cords were taken for NeuN fluorescence staining (a neuronal marker, red), and confocal images of the SDH region were taken. The mitochondrial network analysis (MiNA) plugin in ImageJ software was used to analyze the mitochondrial network of SDH. The analysis indicators included four: the total length of mitochondrial imprints, the length of the mitochondrial network, the length of the mitochondrial network branches, and the number of mitochondrial network branch points. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D showed the original confocal images and the ImageJ processed MiNA images of GAD2-MITO and vGluT2-MITO mice 14 days after SNI, respectively.\u003c/p\u003e \u003cp\u003eThe statistical results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) showed that the four indicators of inhibitory neurons (GAD2\u003csup\u003e+\u003c/sup\u003e neurons) in GAD2-MITO mice were significantly lower than those in the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) 14 days after SNI. At the same time, the four indicators of non-inhibitory neurons (non-GAD2\u003csup\u003e+\u003c/sup\u003e neurons) were also significantly lower than those in the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe statistical results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) showed that three of the four indicators of excitatory neurons (vGluT2\u003csup\u003e+\u003c/sup\u003e neurons) in vGluT2-MITO mice were significantly lower than those in the Sham group (all mitochondrial imprint lengths, mitochondrial network lengths, and the number of mitochondrial network branch points) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) 14 days after SNI, while the length of mitochondrial network branches showed no significant difference. At the same time, three of the four indicators of non-excitatory neurons (non-vGluT2\u003csup\u003e+\u003c/sup\u003e neurons) were significantly lower than those in the Sham group (all mitochondrial imprint lengths, mitochondrial network lengths, and the number of mitochondrial network branch points) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while the length of mitochondrial network branches showed no significant difference.\u003c/p\u003e \u003cp\u003eIn addition, we performed FIS1 (red) immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and G). The results showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH) that the expression level of FIS1 in inhibitory neurons (GAD2\u003csup\u003e+\u003c/sup\u003e neurons) in the SDH of GAD2-MITO mice after SNI was significantly increased compared with the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001); similarly, the expression level of FIS1 in excitatory neurons (vGluT2\u003csup\u003e+\u003c/sup\u003e neurons) in the SDH of vGluT2-MITO mice after SNI was significantly increased compared with the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eThese results suggested that the mitochondrial networks in both excitatory and inhibitory neurons in the SDH was weakened after SNI, accompanied by an increase in FIS1 expression. Therefore, the results of this part of the study suggested that the pain regulatory effect of FIS1 may be achieved through both excitatory and inhibitory neurons. However, this was puzzling because the states of these two types of neurons are completely opposite in spinal cord sensitization in neuropathic pain. According to the spinal cord gate control theory, the sensitization of excitatory neurons and the disinhibition of inhibitory neurons lead to central sensitization in neuropathic pain. Therefore, we next precisely regulated FIS1 in the two types of neurons to try to identify the target cells of FIS1 in neuropathic pain.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6 Down-regulating FIS1 specifically in excitatory neurons, but not inhibitory neurons, of spinal dorsal horn could exert analgesic effects, as demonstrated using vGluT2-Cre mice and GAD2-Cre mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe conducted behavioral and molecular biological experiments using vGluT2-Cre mice and GAD2-Cre mice combined with targeted injection of Fis1 RNA interference (KD) recombination adeno-associated virus (rAAV) carrying the DIO element.\u003c/p\u003e \u003cp\u003eWe first targeted the injection of Fis1 RNA interference (KD) rAAV into vGluT2-Cre mice to down-regulate FIS1 in the excitatory neurons of the right side of the SDH in mice, with empty virus as the control. At 14 days, the viral expression reached a stable level, at which point unilateral (right side) SNI modeling was performed, and then behavioral observations of mice were conducted on days 1, 3, 5, 7, 10, and 14 after SNI (Catwalk and rotarod experiments were only conducted on day 14), and the spinal cord was taken for double immunofluorescence staining of FIS1 and vGluT2 after the last behavioral observation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of the von Frey mechanical pain threshold test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) showed that the right hind paw withdrawal threshold of SNI mice in the SDH excitatory neuron Fis1 KD treatment group increased from 0.01g to 0.4g on day 3 compared with the empty virus group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and this effect persisted until day 14. The results of the hot plate test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) showed that the latency of SNI mice in the Fis1 KD treatment group on day 10 increased from 5s to 7s compared with the empty virus group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and this effect persisted until day 14. The results of the OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and E) showed that there was no significant difference in the distance and time of activity in the central area between the Fis1 KD treatment group and the control group. The results of the EPM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF and G) showed that there was no significant difference in the time spent in the open arms between the Fis1 KD treatment group and the control group. The results of the Catwalk gait analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH and I) showed that the standing time of the right hind limb, the maximum contact area of the right hind paw, and the area of the right hind paw print of SNI mice in the Fis1 KD treatment group on day 14 were significantly increased compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results of the rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ) showed that the time on the rod of SNI mice in the Fis1 KD treatment group was significantly longer than that of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe results of dual immunofluorescence staining for FIS1 (green) and vGluT2 (red) showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK and L) that the M1 value of double-labeled FIS1 and vGluT2 in the SDH of the SNI group treated with Fis1 KD was significantly lower than that of the empty virus group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eThe above research process was then repeated in GAD2-Cre mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). The results of the von Frey mechanical pain threshold test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN) showed that the withdrawal threshold of the right hind paw of SNI mice in the SDH inhibitory neuron Fis1 KD treatment group was not significantly different from that of the empty virus group. The results of the hot plate test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO) showed that the latency of SNI mice in the Fis1 KD treatment group was not significantly different from that of the empty virus group. The results of the OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eP and Q) showed that the distance and time of activity in the central area of SNI mice in the SDH excitatory neuron Fis1 KD treatment group were not significantly different from those of the control group. The results of the EPM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eR and S) showed that the time spent in the open arm of SNI mice in the Fis1 KD treatment group was not significantly different from that of the control group. The results of the rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eT) showed that the time on the rod of SNI mice in the SDH inhibitory neuron Fis1 KD treatment group was not significantly different from that of the control group. The results of the Catwalk gait analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eU and V) showed that the standing time of the right hind limb, the maximum contact area of the right hind paw, and the area of the right hind paw print of SNI mice in the Fis1 KD treatment group were not significantly different from those of the control group.\u003c/p\u003e \u003cp\u003eThe results of double immunofluorescence staining for FIS1 (green) and GAD2 (red) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eW and X) showed that the M1 value of double-labeled FIS1 and GAD2 in the SDH of the SNI group treated with Fis1 KD was significantly lower than that of the empty virus group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eThe above results indicated that down-regulating FIS1 in excitatory neurons in the SDH could significantly exert analgesic effect in SNI mice, but it could not alleviate the anxiety-like behavior. On the contrary, down-regulating FIS1 in SDH inhibitory neurons had no effect on the pain response and anxiety-like behavior after SNI. Thus, we finally discovered that the target cells for FIS1 analgesia were the excitatory neurons in the spinal dorsal horn.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.7 Targeted downregulation of FIS1 in the SDH of excitatory neurons in SNI mice mitigated mitochondrial dysfunction.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious research results have shown that neuropathic pain can lead to a decrease in adenosine triphosphate (ATP) production and mitochondrial membrane potential (MMP) levels, and an increase in reactive oxygen species (ROS) generation [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The results of the earlier part of this study showed that SNI causes a decrease in the expression of COX IV of the mitochondrial electron transport chain in the SDH tissue of mice and damage to the mitochondrial network structure within neurons. Therefore, we hypothesized that the mechanism of FIS1 downregulation for analgesia is related to mitochondrial function. To verify this hypothesis, we conducted five groups of experiments.\u003c/p\u003e \u003cp\u003eIn the first group, C57BL/6J mice were subjected to sham treatment and SNI model groups. On the 14th day after surgery, the dorsal horn of the stimulated side of the spinal cord was taken, and then mitochondria were purified. The experimental results indicated that compared with the sham group, the SNI group had a decreased mitochondrial MMP level in SDH (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), reduced ATP production (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), and increased ROS generation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the second group, C57BL/6J mice were given intraspinal injection of AAV-Fis1-OE into the spinal dorsal horn. On the 28th day after injection, the dorsal horn of the stimulated side of the spinal cord was taken, and then mitochondria were purified. The relative levels of ATP, MMP, and ROS were detected. The results showed that the Fis1 OE treatment group had a lower MMP level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) and increased ROS generation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF) compared with the empty virus group (Fis1-NC), but there was no significant difference in ATP levels between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eIn the third group, Fis1-KD AAV was injected into the SDH of normal mice, with empty virus as the control. Fourteen days after virus injection, SNI modeling was performed. Fourteen days after SNI, the dorsal horn of the stimulated side of the spinal cord was taken, and then mitochondria were purified to detect the relative levels of ATP, MMP, and ROS. The results showed that the MMP level in the SNI mice group treated with Fis1-KD was higher than that in the control group (Fis1-NC) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG), ATP production increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and ROS production decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eIn the fourth group, Fis1-KD rAAV virus was injected into the SDH of GAD2-Cre mice, with empty virus as the control. The results showed that the ROS production in the GAD2-Cre SNI mice treated with Fis1-KD was lower than that in the control group (Fis1-NC) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL), while there was no significant difference in MMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ) and ATP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK) levels.\u003c/p\u003e \u003cp\u003eThe fifth group experiment was similar to the fourth group, except that the animals were changed to vGluT2-Cre mice. The results showed that the ATP production in the vGluT2-Cre SNI mice treated with Fis1-KD was higher than that in the control group (Fis1-NC) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eN), and ROS production decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eO), while there was no significant difference in MMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eM) levels between the two groups.\u003c/p\u003e \u003cp\u003eThe above results indicated that SNI and targeted upregulation of FIS1 in SDH both caused mitochondrial dysfunction; targeted downregulation of FIS1 in excitatory neurons of SNI mice SDH alleviated mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.8 Targeted down-regulation of FIS1 in the SDH decreased the mitochondrial vacuoles and mitochondrial fragmentation induced by SNI.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have found that morphological changes in mitochondria, such as swollen cristae, increased vacuoles, and abnormal fission and fusion, are associated with various neurological disorders, such as Alzheimer's disease and Parkinson's disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. To observe the morphological changes in mitochondria after SDH-targeted regulation of Fis1 in common mice alleviates neuropathic pain, we used transmission electron microscopy to observe and analyze seven indicators of mitochondria in SDH: the number, perimeter, area, circularity, and the number, perimeter, and area of vacuoles within mitochondria. The above electron microscopy detection techniques have been reported in our previous studies [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. We conducted three groups of experiments.\u003c/p\u003e \u003cp\u003eIn the first group, we established the Sham treatment and SNI model groups in C57BL/6J mice and took the dorsal horn of the stimulated side of the spinal cord for electron microscopy observation on the 14th day after surgery. The transmission electron microscopy (TEM) results showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) that in the SNI group, the number of SDH mitochondria was increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), the perimeter was decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), the area was decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and the circularity was increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared with the Sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). At the same time, the number of vacuoles within mitochondria was increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), the perimeter of vacuoles was increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the area was increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe second group was given intraspinal injection of AAV-Fis1-OE into the SDH of C57BL/6J mice. After 28 days, the dorsal horn of the stimulated side of the spinal cord was taken for electron microscopy observation. The results of transmission electron microscopy showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD) that compared with the empty virus group, the Fis1-OE treated group had an increased number of mitochondria (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), decreased perimeter (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), decreased area (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and increased circularity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE), and the number of vacuoles within mitochondria increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), the perimeter increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and the area increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eThe third group was given intraspinal injection of AAV-Fis1-KD into the SDH of C57BL/6J mice. Fourteen days later, SNI modeling was performed. Fourteen days after SNI, the dorsal horn of the stimulated side of the spinal cord was taken for electron microscopy observation. The results of transmission electron microscopy showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG) that compared with the control group, the SNI mice treated with Fis1-KD had a decreased number of mitochondria (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), increased perimeter (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), increased area (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and decreased circularity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH), and the number of vacuoles within mitochondria decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the perimeter decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and the area decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eThe above results indicated that both SNI and targeted upregulation of FIS1 in SDH caused mitochondrial fragmentation, while targeted downregulation of FIS1 in SDH neurons of SNI mice reduced mitochondrial fragmentation.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.9 EGCG, which was capable of down-regulating FIS1 in the SDH, could simultaneously inhibit SNI-induced neuropathic pain.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious experiments have shown that targeting and down-regulating the level of Fis1 can exert analgesic effects by correcting mitochondrial fragmentation. Therefore, we proposed the next scientific hypothesis that analgesic drugs may exert their analgesic effects by down-regulating the level of FIS1. To verify this hypothesis, we selected two analgesic drugs, epigallocatechin gallate (EGCG) and cinnamic acid (CA), for the following experiments.\u003c/p\u003e \u003cp\u003eEGCG is the most effective active component of tea polyphenols [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. CA is an organic acid isolated from cinnamon bark [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. They are well-known antioxidant drugs and have analgesic effects [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFirstly, we used network pharmacology methods to analyze the possible molecular mechanisms by which EGCG and CA exert analgesic effects. By inputting the keywords \"Chronic Pain\", \"Cinnamic Acid\", and \"Epigallocatechin Gallate\" into the human Comparative Toxicogenomics Database, we obtained 7806, 36, and 2393 related genes respectively. Taking the intersection, we obtained 16 genes, including BAX, BCL2, CASP3, CAT, CYP2E1, CYP3A4, DNM1L, FIS1, HMGCR, MMP2, MMP9, NOS3, PPARG, SLC2A4, TNF, etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). The above results suggested that the 16 genes, including DNM1L (i.e., Drp1) and FIS1, may be the molecular targets through which EGCG and CA exert their analgesic effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, STRING database was used for protein-protein interaction networks (PPI) network interaction analysis, and one free gene (CAT) was excluded (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Subsequently, the database for annotation, visualization and integrated discovery (DAVID) database and microbiomics platform were utilized to conduct gene ontology (GO) analysis on the remaining 15 genes. The results of gene ontology biological process (GO-BP) analysis indicated that these 15 genes were mainly enriched in biological processes such as \"cellular response to chemical stress\", \"oxidative stress response\", and \"alteration of mitochondrial morphology and function\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). The results of gene ontology cellular component (GO-CC) analysis showed that these 15 genes were mainly enriched in cellular components such as \"mitochondrial outer membrane\", \"organelle outer membrane\", and \"lipid raft\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). The results of gene ontology molecular function (GO-MF) analysis revealed that these 15 genes were mainly enriched in molecular functions such as \"oxidoreductase activity\", \"BH domain binding\", and \"protein phosphatase binding\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). Then, the keywords \"mouse\" and \"mitochondrial fission\" were input into the gene set enrichment analysis (GSEA) database, and 23 disease-related mitochondrial fission genes were obtained (Aurka, Bnip3, Dcn, Ddhd1, Ddhd2, Drp1, Fis1, Igtp, Irgm1, Irgm2, Kdr, Marchf5, Mcu, Mff, Mief1, Mief2, Mul1, Pgam5, Pink1, Rala, Ralbp1, Spire1, Vps35). The intersection of the above 15 genes and the 23 genes yielded 2 genes, namely Fis1 and Drp1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eBased on the above bioinformatics analysis, we reached a preliminary conclusion that EGCG and CA might exert analgesic effects through the Fis1 or Drp1 molecular pathways. Next, we conducted behavioral verification for this.\u003c/p\u003e \u003cp\u003eWe performed SNI surgery on common C57BL/6J mice. On the 7th, 10th, and 14th days after the operation, EGCG (50 mg/kg) and CA (50 mg/kg) were intraperitoneally injected (normal saline was used as the control), and behavioral tests were conducted 4 hours after each injection. After the last test, the dorsal horn of the spinal cord was taken and proteins were extracted for Western Blot detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of the von Frey test showed that the withdrawal threshold of the right hind paw in the EGCG group increased from 0.02 g to 0.3 g on the 7th day after SNI compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and this effect was maintained until the 14th day. However, there was no significant difference in the CA group compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe results of the hot plate test showed that the latency in the EGCG group increased from 5 s to 7 s on the 7th day after SNI compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and this effect was maintained until the 14th day. However, there was no significant difference in the CA group compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe Western blot results showed that the EGCG group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD and E) could significantly reverse the increased expression of DRP1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and FIS1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the spinal dorsal horn caused by SNI, without affecting the expression level of MFF. Meanwhile, the CA group had no effect on the expression levels of the above proteins in the spinal dorsal horn after SNI (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eF and G).\u003c/p\u003e \u003cp\u003eThese results suggested that EGCG, but not CA, could inhibit the expression of FIS1. At the same time, EGCG, but not CA, could exert an analgesic effect against SNI. This preliminarily validated our hypothesis that drugs capable of down-regulating the expression level of FIS1 in the spinal dorsal horn could also inhibit SNI-induced neuropathic pain.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study demonstrated three key findings : first, mitochondrial networks in both excitatory and inhibitory neurons of the SDH were disrupted in spared nerve injury (SNI) mice, as demonstrated through the use of specifically constructed GAD2-MITO and vGluT2-MITO transgenic mouse models; second, selectively down-regulating FIS1 specifically in excitatory neurons, but not inhibitory neurons, of spinal dorsal horn could exert analgesic effects, as demonstrated using vGluT2-Cre mice and GAD2-Cre mice; third, epigallocatechin gallate (EGCG), which is capable of down-regulating FIS1 in the spinal dorsal horn, concurrently inhibited SNI-induced neuropathic pain. Our research findings suggest that FIS1 may represent a novel molecular target for the treatment of pain.\u003c/p\u003e \u003cp\u003eThis study supported that FIS1-mediated mitochondrial morphology and function alterations in the SDH could regulate neuropathic pain. A large number of literatures have indicated that mitochondrial dysfunction plays a significant role in the pathogenesis of neuropathic pain, with the main mechanism being the abnormal DRP1-mitochondrial fission-ROS cycle in the spinal dorsal horn [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Previous studies have shown that in neuropathic pain models, mitochondrial fragmentation and a reduction in the average planar area of mitochondria occur in the spinal dorsal horn, along with mitochondrial dysfunction [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and enhancing mitochondrial function in the spinal dorsal horn can alleviate pain. Our research group has previously confirmed these conclusions and found that the DRP1 in the spinal dorsal horn increases, and regulating DRP1 in the spinal dorsal horn can exert analgesic effects and improve mitochondrial function [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, previous work has not distinguished the types of neurons in the spinal dorsal horn regarding the above mechanisms. Our research results also found mitochondrial fragmentation and dysfunction in the spinal dorsal horn, and improving mitochondrial function in excitatory neurons in the spinal dorsal horn could alleviate neuropathic pain. Moreover, our study demonstrated that mitochondrial dysfunction in the spinal dorsal horn is caused by FIS1-mediated mitochondrial fragmentation. The mitochondrial fission protein FIS1 mediates the assembly of the mitochondrial fission complex and participates in peripheral mitochondrial fission, being an important factor in the process of mitochondrial fusion and fission [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Previous studies have found that inhibiting Fis1 can reduce mitochondrial fission in rat retinal endothelial cells [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. We obtained the same mitochondrial morphological results by down-regulating FIS1 in the spinal dorsal horn of the SNI model and further proved that down-regulating FIS1 can exert analgesic effects by reducing ROS levels, increasing MMP and ATP content. Based on the current research results, it can be considered that targeting the regulation of FIS1 expression in SDH can affect mitochondrial morphology and function in response to neuropathic pain stress.\u003c/p\u003e \u003cp\u003eThis study showed that the morphological dysfunction of mitochondria in excitatory neurons in dorsal horn of spinal cord caused neuropathic pain. Interneurons in SDH can be divided into two categories: excitatory neurons and inhibitory neurons. Inhibitory neurons mainly exist in layers I-III, while excitatory neurons are almost distributed throughout SDH [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previous studies have shown that both excitatory and inhibitory interneurons in the dorsal horn play an important role in neuropathic pain [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Consistent with previous studies, we found that both excitatory and inhibitory neurons were activated in neuropathic pain models with increased intracellular mitochondrial fission and reduced mitochondrial function. In addition, we found increased intracellular FIS1 expression in both. Considering that improving the function of spinal dorsal horn mitochondria can alleviate neuropathic pain [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], there are significant differences in somatosensory response and plasticity of SDH excitatory and inhibitory neurons [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. We down-regulated FIS1 in excitatory nerves and inhibitory neurons of spinal dorsal horn in neuropathic pain mice. Interestingly, we only found that down-regulating FIS1 in excitatory neurons of SNI model alleviated mechanical pain and thermal pain, and effectively decreased mitochondrial fragmentation in excitatory neurons and enhanced mitochondrial function in spinal dorsal horn. Although downregulating FIS1 in SDH inhibitory neurons of SNI model decreased ROS levels, we found that had no effect on relieving pain. This may be related to the fact that peripheral nerve injury mainly activates excitatory neurons in the I-III layer of the dorsal horn of the spinal cord, and the excitatory neurons in the SDH in the pain model are more sensitive to thermal stimulation than the inhibitory neurons [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt has been reported that epigallocatechin gallate and cinnamic acid can be used for pain treatment. Epigallocatechin gallate is mainly used for postoperative pain treatment. Cinnamic acid is mainly used for cancer pain [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The application of these two drugs in neuropathic pain has not been reported. Epigallocatechin-3-gallate (EGCG), the main bioactive component of theocatechin, has been reported to rescue mitochondrial function and may improve cell function by inhibiting mitochondrial reactive oxygen species mediated iron death [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In addition, EGCG reduced DRP1 and FIS1 expression, reduced mitochondrial fragmentation, and improved mitochondrial dynamic function in subarachnoid hemorrhage models [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The main pathogenesis of neuropathic pain is the increase of ROS and mitochondrial fragmentation in the dorsal horn of the spinal cord, and the mechanism of EGCG is similar. Fortunately, after intrabitoneal injection of EGCG into the SNI model, we observed that EGCG had the same analgesic effect on the neuropathic pain model, and EGCG reduced the expression of FIS1 in the spinal dorsal horn of the SNI model. Therefore, we believed that the analgesic effect of EGCG may be mediated by reducing the expression of FIS1, thereby reducing mitochondrial fragmentation, and thus improving mitochondrial function. Cinnamic acid is a phenolic polyphenol. It has been reported in previous studies that it can play an anti-inflammatory and antioxidant role by reducing oxidative stress and inhibiting over-activated immune response [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. It can also have an analgesic effect on oxaliplatin induced cold atopic pain by inhibiting spinal cord pain transmission [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Unfortunately, cinnamic acid was not able to relieve neuropathic pain in our SNI model, which may be related to the fact that the analgesic target of cinnamic acid is spinal wide dynamic range neurons rather than vGluT2 neurons, but we do not rule out the effectiveness of CA in other types of pain models.\u003c/p\u003e \u003cp\u003eIn summary, our present findings supported that mitochondria at the spinal cord level are sensitive to neuropathic pain, that SDH-targeted up-regulation of FIS1 can cause pain response, and that SDH-targeted down-regulation of mitotic protein FIS1 in excitatory neurons could alleviate neuropathic pain induced by SNI models. This study will promote the in-depth study of the mitochondrial mechanism of neuropathic pain, and is expected to provide new molecular targets and clinical drugs for the analgesic treatment of neuropathic pain.\u003c/p\u003e \u003cp\u003eAlthough the current study results can demonstrate the key role of FIS1 in analgesia, there are still many shortcomings. After Fis1 knockout in GAD2-Cre mice, mitochondrial function was also impaired, indicating that Fis1 knockout also has an effect on the mitochondria of inhibitory neurons, which is worthy of further study. Secondly, due to the problem of experimental conditions, we cannot isolate the two types of neurons for mitochondrial function detection at present, and can only detect these two types of neurons as a whole. TEM analysis is an intuitive method to observe the morphological changes of mitochondria. We performed electron microscopic analysis after SNI and Fis1 over-expression and Fis1 knockout, but Fis1 knockout in Cre mice was not analyzed under electron microscope because we could not distinguish neuron types under electron microscope, which is what we need to improve in the future.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, neuropathic pain after nerve injury was associated with morphological abnormalities and dysfunction of spinal dorsal horn mitochondria in mice. The expression of FIS1 increases in spinal dorsal horn in pain state. Targeted down-regulation of FIS1 expression in spinal dorsal horn excitatory neurons of neuropathic pain mice can alleviate pain by reducing mitochondrial fragmentation and improving mitochondrial function. Our findings suggest that FIS1 may be a novel molecular target for the treatment of pain.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"470\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAAV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAdeno-associated virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eATP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAdenosine triphosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCinnamic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCPN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCommon peroneal nerve\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCOX Ⅳ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCytochrome c oxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDRP1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDynamin-related protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEGCG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eEpigallocatechin gallate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEPM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eElevated plus maze\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFIS1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eFission protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGABA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGamma-aminobutyric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGAD2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGlutamic acid decarboxylase 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eKD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eKnockdown\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMFF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMitochondrial fission factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMiNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMitochondrial network analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMITO\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMitochondria\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMMP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMitochondrial membrane potential\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eOE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOverexpression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eOFT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpen field test\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eOPA1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOptic atrophy 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePBS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePhosphate buffer saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePDHA1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePyruvate dehydrogenase e1 subunit alpha 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePWT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePaw-withdrawal threshold\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003erAAV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRecombination adeno-associated virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eROS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eReactive oxygen species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSC-9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAnti-inflammatory agent 49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSDH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSpinal dorsal horn\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSDHB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSuccinate dehydrogenase b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSural nerve\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSNI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSpared nerve injury\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTransmission electron microscope\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTFAM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMitochondrial transcription factor A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTibial nerve\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eVDAC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eVoltage-dependent anion channel\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003evGluT2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eVesicular glutamate transporter 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the following funds and foundations: Military Medicine Advancement Program, Air Force Military Medical University, 2020SWAQ04, Ya-Yun Wang; Shaanxi Innovation Capability Support Program, 2023-CX-PT-33, Ya-Yun Wang; Natural Science Basic Research Program of Shaanxi Province, 2024JC-ZDXM-60, Yan-Ling Yang; Xijing Hospital Clinical New Technology, 2023XJSY27, Yan-Ling Yang; Natural Science Basic Research Program of Shaanxi Province, 2022JQ-820, Fei-Fei Wu; National Natural Science Foundation of China, 82201627, Fei-Fei Wu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot and immunofluorescence staining were performed by Chang-Lei Zhu and Shu-Jiao Li. Chang-Lei Zhu, Shu-Jiao Li and Ke Tian performed stereotactic surgery and behavioral tests. Yun-Qiang Huang, Fei-Fei Wu, Shuai Zhang and Hui Liu prepare animals. Chang-Lei Zhu, Zi-Wei Ni, Jing-Jing Tie, Zhi-Peng Lin, You-Sheng Wu, Fei Tian and Nan-Nan Liu analyze the data. Ya-Yun Wang and Yan-Ling Yang designed the study. Chang-Lei Zhu and Kun-Long Zhang wrote the draft. The electron microscope analysis was carried out by Zhi-Peng Lin and Xue-Yin Pu. Drawing and layout by Zhi-Peng Lin and Yu-Lu Xia. Ya-Yun Wang and Yan-Ling Yang supervised the experiments and revised the manuscript. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFinnerup NB, Kuner R, Jensen TS (2021) Neuropathic Pain: From Mechanisms to Treatment. Physiol Rev 101:259\u0026ndash;301\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaron R, Binder A, Wasner G (2010) Neuropathic pain: diagnosis, pathophysiological mechanisms, and treatment. Lancet Neurol 9:807\u0026ndash;819\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai CQ, Guo Y, Chu XY (2020) Neuropathic Pain: the Dysfunction of Drp1, Mitochondria, and ROS Homeostasis. 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Phytochemistry 206:113532\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Neuropathic pain, Spinal dorsal horn, Excitatory neurons, Mitochondrial fission protein 1, Mitochondrial fragmentation","lastPublishedDoi":"10.21203/rs.3.rs-5916414/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5916414/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Neuropathic pain has been shown to induce abnormal mitochondrial fission in neurons, yet the analgesic potential of inhibiting this process remains unclear. Our previous studies demonstrated that targeted regulation of the dynamin-related protein (DRP1) can alleviate neuropathic pain; however, the downstream molecular signaling mechanisms remain to be elucidated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e To investigate the role of mitochondrial dynamics in neuropathic pain, we utilized C57BL/6J mice, GAD2-Cre mice, and vGluT2-Cre mice. Mitochondrial network changes in pain states were assessed using GAD2-MITO and vGluT2-MITO transgenic mouse models combined with MiNA analysis. Pain thresholds and the expression levels of various molecules in the spinal dorsal horn (SDH) were evaluated through behavioral tests, immunofluorescence, and Western blotting. Mitochondrial morphology and function in pain conditions were examined using electron microscopy, mitochondrial membrane potential, reactive oxygen species, and adenosine triphosphate assays. The effects of antioxidant analgesics epigallocatechin gallate (EGCG) and Cinnamic Acid on molecular changes in the SDH during pain states were also investigated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e We observed that mitochondrial networks in both excitatory and inhibitory neurons of the SDH were disrupted in spared nerve injury mice, as evidenced by GAD2-MITO and vGluT2-MITO transgenic mouse models. Specifically, down-regulating FIS1 in excitatory neurons, but not in inhibitory neurons, within the SDH elicited analgesic effects, as evidenced by experiments conducted with vGluT2-Cre and GAD2-Cre mouse models. Additionally, epigallocatechin gallate (EGCG), which effectively down-regulates FIS1 in the SDH, concurrently inhibited SNI-induced neuropathic pain. These findings suggest that reducing mitochondrial fragmentation by down-regulating FIS1 in SDH excitatory neurons can alleviate neuropathic pain.\u003c/p\u003e","manuscriptTitle":"Targeted downregulation of FIS1 in excitatory neurons within the spinal dorsal horn alleviates neuropathic pain through the mitigation of mitochondrial fragmentation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-31 15:33:10","doi":"10.21203/rs.3.rs-5916414/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43472571-3931-4cdf-a35a-270803914191","owner":[],"postedDate":"January 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-04T13:53:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-31 15:33:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5916414","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5916414","identity":"rs-5916414","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}