MCT1-Mediated Endothelial Cell Lactate Shuttle as a Target for Promoting Axon Regeneration after Spinal Cord Injury

MCT1-Mediated Endothelial Cell Lactate Shuttle as a Target for Promoting Axon Regeneration after Spinal Cord Injury | 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 MCT1-Mediated Endothelial Cell Lactate Shuttle as a Target for Promoting Axon Regeneration after Spinal Cord Injury Chaoran Shi, Jiaqi Xu, Yinghe Ding, Feifei Yuan, Fengzhang Zhu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4079758/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 Vascular damage following spinal cord injury (SCI) precipitates ischemia and hypoxia at the injury site, leading to profound metabolic disturbances. The implications of these metabolic disorders on neural tissue remodeling and functional recovery remain poorly understood. Our study elucidates the consequences of the hypoxic environment induced by SCI, which significantly enhances glycolysis and lactate production at the injury's epicenter. And our findings revealed a marked decrease in the expression of Monocarboxylate Transporter 1 (MCT1), a crucial transporter facilitating lactate delivery to neurons and consequently supporting their energy metabolism, within vascular endothelial cells emerging after SCI. This decrease disrupts lactate transport to neurons, resulting in metabolic imbalances that impede axonal regeneration. Remarkably, our research demonstrates that targeted delivery of adeno-associated virus (AAV) injections to restore MCT1 expression in endothelial cells (ECs) promotes axonal regeneration and functional recovery in SCI mouse models. These findings reveal a previously unrecognized connection between lactate shuttling from ECs to neurons after SCI and neural functional recovery. Highlighting a novel metabolic pathway for intervention, our study opens new therapeutic avenues for the treatment of spinal cord injuries, suggesting that targeting lactate transport mechanisms may offer significant benefits in SCI recovery. Spinal cord injury endothelial cell lactate shuttle neuron metabolism axon regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Spinal Cord Injury (SCI) poses a formidable challenge in contemporary medicine, characterized by its substantial contribution to premature mortality and enduring disability. Over the span from 1990 to 2019, a notable escalation has been observed globally in the incidence, prevalence, and years lived with disability attributable to SCI ( 1 ). The pathophysiology of SCI encompasses a multifaceted process, wherein an initial mechanical insult precipitates a cascade of secondary damage ( 2 ). The pathophysiology of SCI encompasses a multifaceted process, wherein an initial mechanical insult precipitates a cascade of secondary damage ( 3 )., alongside the compromise of vasculature integrity, evidenced by the disruption of endothelial cell tight junctions. Such disturbances lead to the breakdown of the blood-spinal cord barrier (BSCB), augmenting vascular permeability ( 4 , 5 ). Subsequently, the injury site is subjected to sustained ischemia, perpetuated by ongoing edema ( 6 ), vessel thrombosis, vasospasm, and a disruption of homeostatic equilibrium ( 7 ). This pathophysiological milieu fosters metabolic dysfunction among cells at the injury epicenter, situated in an ischemic and hypoxic environment ( 8 – 10 ). The aberrant alteration of metabolic products post-SCI and their impact on nerve regeneration remains an area of unresolved inquiry ( 11 ). It underscores the imperative of managing energy metabolism within the injury epicenter as a pivotal aspect of SCI recovery. Endothelial cells (ECs) play a key role in regulating selective transport and metabolic exchange between the blood and the spinal cord as a vital component of the BSCB ( 12 ). Post-SCI, ECs undergo regeneration at the site of injury; however, their functional attributes are markedly diminished relative to their healthy counterparts, spanning diminished barrier functionality to compromised substance transport ( 13 , 14 ). Increasingly, lactate is recognized not merely as a metabolic byproduct but as a crucial energy substrate and signaling molecule that fosters axon regeneration post-SCI ( 15 , 16 ), This revelation posits lactate homeostasis as a critical determinant in SCI recovery. In the central nervous system, lactate can transport between highly glycolytic cells to adjacent cells, a phenomenon known as lactate shuttle ( 17 ). Many studies have confirmed the involvement of the astrocyte-neuron lactate shuttle (ANLS) in memory formation, the generation of neuropathic pain, and the further exacerbation of neurodegenerative diseases. ( 18 – 21 ). This shuttle mechanism suggests that astrocytes respond to lactate-mediated neuronal activity by increasing their aerobic glycolysis levels. However, it remains unclear whether a lactate shuttle exists between spinal cord ECs and neurons, and the mechanism by lactate mediates neuronal metabolism after SCI. Monocarboxylic transporters (MCTs) play a significant role in the lactate shuttle, which may promote intracellular and intercellular lactate shuttle in the central nervous system ( 22 ). There is research indicating that ECs predominantly express Monocarboxylic transporter1 (MCT1) in the spinal cord ( 23 ), indicating ECs may regulate axon regeneration by transporting lactate via MCT1. However, the lack of evidence regarding how MCT1 is expressed in ECs after SCI and its functional implications in this context requires further investigation. In this study, metabolomics analysis revealed elevated lactate levels post-SCI. Bioinformatics analysis subsequently indicated a reduced expression of MCT1, a lactate transporter, in ECs following injury. Furthermore, in vitro co-culture experiments employing a lactate sensor substantiated the presence of a lactate shuttle between ECs and neurons. Leveraging adenovirus mediated MCT1 overexpression through in situ injection in a mouse model, we delineated the pivotal role of endothelial cell MCT1 in modulating energy metabolism, crucial for the regeneration of endothelial cells and axons post-SCI. This insight heralds a novel metabolic therapeutic strategy for SCI rehabilitation. 2. Materials and Methods 2.1 Cell Lines Mouse brain endothelial cells (bEnd.3 cells, CL-0598, Procell) were maintained with culture media containing 10% FBS ( FBS-CP500, NEWZERUM), 1% 100* Penicillin-Streptomycin Solution (PB180120, Procell) in DMEM (10-013-CVRV, CORNING) at 37℃ in a 5% CO2-humidified incubator. Cell line was obtained directly from Pricella, with no additional cell authentication performed. Mouse nerve cells (CATH.a cells, HTX2209, ATCC)were maintained in CATH.a cell-specific medium (CM-0665, Procell) at 37℃ in a 5% CO2-humidified incubator. Cell line was obtained directly from ATCC, with no additional cell authentication performed. 2.2 Cell treatment The bEnd.3 or CATH.a were seeded in six-well plates at a density of 10^5 cells/ml. We exposed bEnd.3 or CATH.a with 10mM exogenous L-lactate (L6402, sigma) or 5 mM α-cyano-4-hydroxycinnamic acid (α-CHCA) (S8612, Selleck) for 1 day before being examined.. For the contact co-culture experiments, we used Ibidi 2-well inserts (81176, ibidi) were installed into 20mm confocal culture dish (801001, NEST) with sterile tweezers and gently pushed with a gloved fingertip. Pretreated bEnd.3 or CATH.a with Laconic infection were seeded into opposite chambers at a density of 1 × 10 5 cells/cm 2 and incubated overnight. The culture insert was then removed with sterile tweezers and the well topped up with 1 ml of DMEM media. After 48-hour incubation, cells were fixed with freshly prepared 4% PFA (AWI0056b, Abiowell) for 30 minutes at room temperature. After fixing, cells were subjected to imaging using a Zeiss confocal laser scanning microscope LSM 9. 2.3 Lentiviral vector construction and transfection The construct for Laconic (Plasmid #44238; San Martin et al, 2013) was obtained from Addgene. Seed well-conditioned target cells at a density of 5 × 10 4 cells/well. Plate in a 24-well plate, and after cell counting, add 500 µL of DMEM culture medium to each well. After 24 hours, replace with DMEM medium containing 1 µg/mL Polybrene-plus, choosing an MOI (Multiplicity of Infection) value of 40, and add 20 µL of 1 × 10 8 TU/mL viral particles. After 24 hours, replace with fresh culture medium. 72 hours post viral infection, observe under a fluorescence microscope to evaluate the efficiency of lentiviral infection in the target cells. 2.4 Adeno-associated virus construction and transfection Adeno-associated virus used in this study was produced at the Obio Technology (Shanghai, China). The following AAV plasmid was used and detailed sequence information is available as detailed or upon request: pAAV-SIc16a1-FLAG-mCherry (Obio Technology). Injection volumes, coordinates and experimental purpose are described below. 2.5 Mice Adult male or female C57BL/6 mice (8–10 weeks old, weight 20-25g) were used in this study. The animals were purchased from Hunan SJA Laboratory Animal Company Limited and were kept in a pathogen-free animal facility at Central South University, following standard purification procedures. They were housed in the laboratory animal unit, with a 12-hour day/night cycle, and were provided ad libitum access to food and water. All mice are housed in a laboratory animal facility with an ambient temperature of 22–24°C and relative humidity of 60–80%. Manual bladder voiding and all other animal care was performed 2 to 4 times daily throughout the entire experiment. 2.6 SCI models The Animal Care and Use Committee of Central South University, Changsha, China, approved all animal protocols and experimental procedures. Perform routine anesthesia, skin preparation, disinfection, and draping. Make an incision centered on the T10 spinous process, sequentially cutting through the skin, subcutaneous tissue, muscles, and lamina to expose the spinal cord. Severe crush SCI were made after laminectomy of a single vertebra by using No. 5 Dumont forceps (Fine Science Tools, Foster City, CA) without spacers and with a tip width of 0.5mm to completely compress the entire spinal cord laterally from both sides for 5 second. Irrigate the wound, then close the incision in layers, cover with a dressing, and secure it. Postoperatively, mice are housed individually with standard feed and water available ad libitum. Administer penicillin 20,000 units bi-daily via intramuscular injection for postoperative infection prevention for 3 days. Manually express the bladder 2 to 4 times daily to assist with urination. 2.7 Spinal injections for AAV To overexpress MCT1 in endothelial cells, partial laminectomy at the T9 spinal level was performed two weeks prior to spinal cord injury, using 30-33G Hamilton needles to bilaterally inject pAAV-Slc16a1-FLAG-mCherry2 (0.5 µl per injection) at two depths of 0.8 mm and 0.4 mm below the dorsal surface, spaced 1 mm apart, with each injection maintained for 5 minutes. Post-injection, the needle was left in the spinal tissue for 15–20 minutes. During the injection process, care was taken to keep the mice warm. The needle was then slowly withdrawn. The skin was sutured and the wound disinfected. For postoperative pain relief, mice were administered carprofen via intramuscular injection daily for 3 days. Animals were perfused 28 days after AAV injection. 2.8 Perfusions At different time points post-SCI, 1% sodium pentobarbital (50 mg/kg) is administered intraperitoneally for deep anesthesia in mice. Rapid thoracotomy is performed to expose the heart, and a size 12 medical intravenous catheter is inserted into the left ventricle up to the origin of the ascending aorta and secured. The right atrium is incised, followed by sequential perfusion with pre-warmed heparinized saline and 10% formalin solution, maintaining the 10% formalin perfusion for approximately one hour. The specimens are then stored in a 4°C refrigerator. 2.9 Immunofluorescence For immunofluorescence of tissue, frozen sections of 16-µm thickness from the spinal cord containing the lesion site were harvested at different time points post-SCI (or sham group) and cut along the sagittal plane. The frozen sections were rewarmed at room temperature for 15 minutes and rinsed three times with a PBS solution for 10 minutes each time. The immunohistochemical pen was used to circle the spinal cord sections on the slides. The sections were then permeabilized with 100µL of PBS solution containing 0.1% Tween 20 and 0.3% Triton-X 100 for 30 minutes and blocked with 5% BSA in PBS for an additional 30 minutes. After blocking, the sections were incubated with a group of primary antibodies including F4/80 (Abcam, ab6640, 1:400), TUJ-1 (CST, 5568, 1:400), GFAP (Proteintech, 16825-1-AP, 1:800), MCT1 (Proteintech, 20139-1-AP, 1:200), CD31(R&D systems, inc., fab3628G, and 1:200), overnight at 4℃. After being rinsed five times for 10 minutes each with PBS containing 0.1% Tween 20, the sections were incubated for one hour at room temperature with species-appropriate secondary antibodies conjugated with Alexa Fluor 594 (Abcam, 1:400) or Alexa Fluor 488 (Abcam, 1:400). Subsequently, the sections were rinsed with PBS containing 0.1% Tween 20 before being mounted on slides and covered with DAPI (Genetex). For immunofluorescence of cells, we place sterile 24-well slides into a 24-well plate. The bEnd.3 or CATH.a, post trypsinization, are seeded onto the slides at a density of 5 × 10 4 cells/well, with 0.5 ml of complete culture medium added per well. The culture plate is then incubated in a 37°C, 5% CO 2 incubator. Once cells reach 85% confluency, the medium is aspirated, and old culture medium is washed off with PBS along the walls of the well. Each well is then fixed with 500 µl of 4% paraformaldehyde for 12 minutes, followed by a PBS wash to remove the paraformaldehyde. Each well is treated with 200 µl of 0.1% Triton-X100 in PBST for 20 minutes. After aspiration of the PBST, the wells are blocked with 3% BSA in PBS solution for 30 minutes. The blocking solution is then aspirated, and primary antibodies including CD31, MCT1, and TUJ are added and incubated overnight at 4°C. The next day, primary antibodies are aspirated, and the wells are washed three times with PBS for 5 minutes each. Corresponding secondary antibodies are then added and incubated at room temperature for 1.5 hours. After aspiration of the secondary antibodies, the wells are washed three times with PBS for 5 minutes each. A drop of DAPI solution containing an anti-fade agent is then added to the slide, and the cell slide is picked up with forceps and inverted onto the slide. The sections were analyzed under a fluorescence microscope or confocal microscope (Zeiss). To validate antibody specificity and distinguish genuine target staining from the background, secondary antibody-only controls were employed. The ImageJ software was used for quantitative analysis of the images, while the Imaris 9.0 software was used for 3D reconstruction. 2.10 Western Blotting We utilize a BCA Protein Assay Kit for protein concentration normalization. After adjusting protein samples to a protein to loading buffer ratio of 4:1, we denature the proteins in a 95°C metal bath for 10 minutes, followed by storage at -20°C for future use. During electrophoresis, samples are loaded into the gel wells. The voltage is set to 90V for the first 30 minutes, then adjusted to 120V until the protein bands are fully separated. Subsequently, PVDF membranes are activated with anhydrous ethanol and carefully placed on the SDS-PAGE gel, ensuring no bubbles are formed. The PVDF membrane and SDS gel are sandwiched between sponges and filter paper and electrotransferred in an ice-water mixture at 300mA for 90 minutes. The PVDF membrane is then blocked with 5% non-fat milk in TBST solution on a shaker for 1.5 hours. Afterward, the membrane is washed three times with TBST, each for 5 minutes. The corresponding bands are cut, and primary antibodies are added for incubation at 4°C overnight. The next day, the primary antibodies are aspirated, and the membrane is washed three times with TBST for 10 minutes each. Corresponding Western blot secondary antibodies are then added and incubated at room temperature on a shaker for 1 hour. After aspiration of the secondary antibodies, the membrane is washed three times with TBST for 10 minutes each. Finally, using the chemiluminescence reagent (ShareBio, SB-WB001), the immunoreactive bands were visualized with a ChemiDoc XRS Plus luminescent image analyzer (Bio Rad, England). The image analysis was performed using ImageJ software, and the relative expression levels of the target proteins to β-actin were used for statistical comparison. 2.11 Electrophysiology Capture the mice to be tested and anesthetize with 0.3% sodium pentobarbital. Shave, disinfect the animal, and securely fixate it in a stereotaxic apparatus, maintaining body temperature with a heating pad. Perform a craniotomy to expose the M1 area of the motor cortex. Insert stimulation electrodes, guided by the stereotaxic device, to a depth of 700–1000 mm from the brain surface, targeting corticospinal neurons in the sensorimotor cortex. Position recording electrodes to penetrate the contralateral thigh’s sciatic nerve distal end to record muscle action potentials induced by electrical stimulation. Amplify and record bioelectrical signals using the BL-420F Biological Function Experiment System. Stimulation parameters are as follows: stimulation type: micro-voltage, mode: single pulse; delay 100 ms; frequency 100 Hz, stimulation intensity: 14V. 2.12 BMS score evaluation, LSS swimming The recovery of hindlimb motor function in SCI (Spinal Cord Injury) mice is evaluated using the Basso Mouse Scale (BMS) scoring system ( 24 ). Assessments are conducted pre-injury and on days 1, 3, 7, 14, and 28 post-injury. The BMS score ranges from 0 to 9, with 0 indicating complete paralysis and 9 indicating normal hindlimb motor function. Before each scoring session, mice are placed on the testing platform in advance to acclimate to the environment. Each mouse is observed for a duration of 4 minutes. Mice underwent three consecutive days of swimming training in a 5 x 15 cm water tank, moving from one side to the other. Surgery was performed after the completion of training. On postoperative day 28, mice were placed in the same water tank and observed for 30 seconds. The Louisville Swim Scale (LSS) was used to evaluate the swimming abilities of the mice, including assessments of hindlimb movement, hindlimb alternation, forelimb reliance, trunk stability, and body angle ( 25 ). All experiments were conducted with the mice being randomly assigned by two trained observers blinded to the group allocations. Both observers simultaneously observed and recorded the mice's BMS scores, LSS scores. The final score was determined as the average of the scores given by the two observers. 2.13 Cell Lateral Migration Assay Firstly, use Mark pen to draw lines on the backside of a six-well plate to determine the scratch positions. Seed bEnd.3 cells in the six-well plate, with approximately 200,000 cells per well. Allow the cells to grow and fully cover the surface of the six-well plate, then replace the medium with serum-free culture medium for 6 hours to induce starvation. Use a 200µl pipette tip, held perpendicular to the cell surface, to draw lines according to the positions marked by the marker pen. Subsequently, wash away the detached cells with PBS, followed by replacing the culture medium with fresh serum-free medium. Incubate the cells in a constant-temperature CO2 incubator at 37°C for 12 hours. Use an optical microscope to observe cell migration at 0 and 24 hours at the scratch site and calculate the migration distance using Image J software. 2.14 Tube formation assay When bEnd.3 cells reach approximately 85% confluence, the serum-containing medium is replaced with serum-free medium to induce cell starvation for 5 hours. Subsequently, after removing the medium and digesting with trypsin, cells are centrifuged at 1000 rpm for 5 minutes. The experimental and control groups of bEnd.3 cells are adjusted to a concentration of 80,000 cells/ml using serum-containing culture medium based on cell counting. Pre-cooled pipette tips are used to evenly coat a 96-well plate with Matrigel matrix gel at 100 µl per well, and the plate is then incubated in a constant-temperature incubator for 30 minutes. Once the matrix gel solidifies, 500 µl of cell suspension is added to each well of a 24-well plate and incubated in a constant-temperature incubator for 6 hours. The formation of capillary-like structures by the cells is observed using an optical microscope, and Image J software is employed for quantitative analysis of the tube formation. 2.15 Bioinformatic analysis of the single-cell transcriptomic dataset The single-cell RNA sequencing data of the contusive mouse spinal cord injury (GSE162610) and the information on the corresponding annotation were retrieved from the GEO database [doi : 10.1084/jem.20210040. ]. Data processing and analysis were performed using the R package "Seurat" [doi : 10.1038/nbt.3192. ]. The genes expressed in less than 10 cells were excluded. The gene expression matrix was normalized and scaled. We selected the top 20 principal components by performing PCA based on 3,000 variable genes. The FindNeighbors and FindClusters functions were used to cluster cells on a shared-nearest-neighbor graph. We visualized the expression level of interested genes using violin plots. The single-nucleus RNA sequencing data of the human adult spinal cord from seven donors (GSE190442) were also obtained and underwent consistent analysis workflow. [doi : 10.1016/j.neuron.2023.01.007. ]. 2.16 Statistical analysis The statistical analysis of the results was performed using GraphPad Prism (version 7.0, USA). All data were reported as mean ± standard deviation (SD). Normality was determined using the Shapiro-Wilk test. For group number = 2, the homogeneity of variances was tested using the F-test. When the data followed a normal distribution and homogeneity of variance, an unpaired t test was conducted for the statistical analysis. For group number > 2, the homogeneity of variances was tested using the Brown-Forsythe test. When the data followed a normal distribution and homogeneity of variance, an ordinary one-way ANOVA and Tukey's multiple comparisons test were performed for the statistical analysis. Two-way ANOVA was utilized for data with two variables, grouping and time. All differences among and between groups were considered statistically significant at p < 0.05. In the figures, ns denotes p ≥ 0.05, ∗ denotes p < 0.05, ∗∗ denotes p < 0.01, ∗∗∗ denotes p < 0.001, and ∗∗∗∗ denotes p < 0.0001. 3. Results 3.1 Enhanced glycolysis metabolism after spinal cord injury Existing literature has demonstrated that glycolysis plays a crucial role in the survival and regeneration of neurons in Drosophila ( 15 , 25 ). However, its role in mice after SCI remains to be investigated. We obtained in situ spinal cord tissue samples from mice after 7 days post-SCI. Targeted metabolomics sequencing was performed to compare changes in glycolysis-related metabolites before and after SCI. The heatmap revealed significant enhancement of metabolites such as lactate, D-glucose-6-phosphate, β-D-fructose-6-phosphate, and adenosine monophosphate post-injury (Figs. 1 A, B). Additionally, KEGG analysis indicated that the metabolites increased after SCI were mainly glycolysis pathway-related metabolites (Fig. 1 C). Concurrently, volcano plots were used to identify the most significantly altered differential metabolites post-injury, revealing a noticeable increase in glycolysis-related metabolites such as lactate, D-glucose-6-phosphate, and β-D-fructose-6-phosphate (Fig. 1 D). Therefore, we identified lactate, a core molecule of glycolysis, as an important functional metabolite post- SCI, providing a direction for subsequent experiments. 3.2 Endothelium-derived lactate feeds nerve cells Endothelial cells are critical in regulating metabolic exchange between blood and spinal cord parenchyma, forming a crucial component of the neurovascular unit alongside neurons ( 12 , 26 ). Given their ability to produce and release a significant amount of lactate, and considering their physical proximity to neurons, we investigated the role of ECs lactate in neuron biology. To examine whether neurons utilize lactate derived from ECs, we employed Laconic, a Förster Resonance Energy Transfer (FRET)-based quantitative intracellular lactate sensor ( 27 ). We infected mouse neuron cells (CATH.a) with a lentivirus expressing Laconic and cultured them in the presence of a glycolysis inhibitor (2-Deoxy-D-glucose, 8 mM) to reduce the production of basal glycolytic lactate. As expected, an increase in lactate concentration from 10 mM to 100 mM led to a concentration-dependent increase in the YFP/CFP fluorescence ratio (Figs. 2 A and B). To investigate whether lactate derived from endothelial cells is taken up by neurons, we co-cultured bEnd.3 and CATH.a in a two-well cell culture dish (Fig. 2 C). Bend.3 and Laconic-infected CATH.a were seeded in separate wells of the culture dish. Twenty-four hours after cell seeding and confirming cell attached, the culture insert was removed to allow cell migration (Fig. 2 D). After a further 24-hour examination, we observed a significant increase in the YFP/CFP fluorescence ratio in CATH.a cells that were in direct contact with the ECs, but no increase in CATH.a cells that had no ECs contact (Figs. 2 E-G). Taken together, these data suggest that lactate from ECs can enter neuron cells, and direct contact between ECs and neurons promotes this process. 3.3 Deficiency of MCT1 in ECs after SCI Elevated lactate levels in the epicenter of injury mice following SCI may be associated with lactate transport mechanisms deficiency due to ECs damage. Therefore, we investigated the altered transport proteins in spinal cord vascular endothelial cells post- SCI. Lactate transport is primarily mediated by monocarboxylate transport proteins (MCTs, also known as Slc16a family) ( 28 ). Through analysis of existing databases from human adult spinal cord ( 29 ), we found that Monocarboxylate Transporter 1,4,5,7,8 (SLC16a1, SLC16a2, SLC16a4) is highly expressed in adult ECs (Fig. 3 A), and MCT1 is predominantly expressed in ECs within the spinal cord (Fig. 3 B), we have focused on the role of MCT1 in ECs-to-Neuron lactate shuttling. Through analysis of single-cell data from mice with spinal cord injury ( 30 ), we found that MCT1 expression is significantly reduced post-SCI (Fig. 3 C). Literature indicates that MCT1, a transmembrane transporter monocarboxylate, plays a crucial role for transmembrane transport of lactate, pyruvate, and ketone bodies ( 31 ), which is an essential protein for maintaining lactate homeostasis in the spinal cord. Additionally, we revealed the spatiotemporal changes of MCT1 post-SCI using immunofluorescence. Additionally, immunofluorescence confirmed angiogenesis at the epicenter of injury 7 days post- SCI, with the newly formed blood vessels appearing dilated and malformed at 14 and 28 days post- SCI, suggesting partial loss of function of these vessels after SCI (Fig. 3 D). Furthermore, our study revealed a decrease in MCT1 expression in the blood vessels formed via angiogenesis at the injury center of the spinal cord (Fig. 3 E). Confocal microscopy observations in SCI mice also confirmed the lack of MCT1 expression in the ECs at the injury center (Fig. 3 F-H). These findings indicate that endothelial cell-specific expression of MCT1. Newly formed blood vessels display morphological abnormalities, accompanied by decreased MCT1 expression after SCI. 3.4 Deficiency of MCT1 in ECs inhibits angiogenesis and glycolysis production To investigate the impact of MCT1 on the biological functions of ECs, we conducted tube formation experiments and transverse migration experiments (scratch assays). Compared to the Control group, after lactate treatment, there were no significant changes in the number and branching length of endothelial cell tubes. However, downregulation of MCT1 expression by adding the MCT1-specific inhibitor α-cyano-4-hydroxycinnamic acid (α-CHCA) resulted in a reduction in the number and branching length of cell tubes. Additionally, the addition of lactate did not significantly improve tube formation in ECs after MCT1 downregulation (Fig. 4 A, B). In comparison to the Control group, lateral migration of endothelial cells treated with lactate showed no significant change, while inhibiting MCT1 expression significantly reduced the lateral migration distance of ECs (Fig. 4 C, D). Simultaneously, we verified the expression of key enzymes related to glycolysis, Hexokinase 1 (HK1), Phosphofructokinase 1 (PFKM), and Pyruvate kinase 1 (PKM1), after lactate treatment. WB experiments indicated increased expression of glycolytic enzymes in ECs after lactate treatment. However, the addition of α-CHCA resulted in a decrease in the expression of glycolytic enzymes in endothelial cells, and the simultaneous addition of lactate and α-CHCA did not improve the inhibited glycolysis situation (Fig. 4 E, F). These results suggest that MCT1 is necessary for lactate transport through ECs. Reduction of MCT1 significantly inhibits tube formation and migration ability of ECs. Moreover, impaired lactate transport also leads to a reduction in glycolysis levels in ECs. 3.5 Neurons Utilize Lactate to Enhance Glycolysis and Promote Axonal Regeneration In order to ascertain that neurons can enhance glycolysis by utilizing lactate delivered by ECs, we co-cultured bEnd.3 and CATH.a cells infected with Laconic lentivirus in Ibidi chambers. Lactate or α-CHCA was added to the bEnd.3 side chamber, and after a 24-hour treatment, the media was exchanged before allowing extensive contact between the two cell types. Experimental results revealed that CATH.a cells in contact with bEnd.3 exhibited an increased CFP/YFP ratio upon lactate treatment, as demonstrated by laser confocal microscopy (Fig. 5 A, B). Conversely, α-CHCA treatment led to a decrease in the CFP/YFP ratio, indicating reduced lactate entry from bEnd.3 into CATH.a. Subsequently, we isolated the cytoplasm and axons of CATH.a cells, simulating axon fragmentation after spinal cord injury by severing distal axons. Experimental findings indicated that lactate treatment of CATH.a cells promoted axon regeneration. However, axon regeneration was inhibited when the glycolysis inhibitor 2-Deoxy-D-glucose (2-DG) was used. Simultaneous treatment with lactate and 2-DG restored the inhibitory capacity of axon regeneration. Additionally, WB results demonstrated enhanced glycolytic capability in CATH.a cells after lactate treatment, while 2-DG treatment suppressed glycolytic capacity (Fig. 5 E, F). These findings suggest that ECs can transport lactate to neurons, and neurons can enhance their regenerative capacity by utilizing lactate to increase glycolysis. 3.6 Spinal Cord ECs overexpression of MCT1 Rescues Impaired SCI Mice Neurogenesis To validate the association between decreased MCT1 in ECs and axon regeneration in SCI mice, we induced overexpression of MCT1 in spinal cord ECs using a spinal cord-specific AAV-MCT1. After successful injection of AAV-MCT1, the expression of MCT1 in the ECs at the injury epicenter of SCI mice was restored. (Fig. 6 A-D). We also confirmed the specific expression of AAV-MCT1, as immunofluorescence experiments demonstrated its absence in astrocytes, neurons, and macrophages (Fig. 6 E, F). We then assessed whether the restored MCT1 could improve axonal regeneration in SCI mice. AAV-MCT1 injection improved the neurite regrowth besides the lesion core and increased the amount of TUJ positive signal in spinal cord sections compared to the Control groups. (Fig. 6 G, H). These results provide direct in vivo evidence demonstrating that spinal cord endothelial cell MCT1 can modulate lactate shuttle and axonal regeneration in SCI mice. 3.7 Spinal Cord Vasculature Overexpression of MCT1 Rescues Impaired SCI Mice Functional Recovery Following AAV-MCT1 injection, electrophysiological tests revealed a significant increase in the amplitude of SCI mouse hind limb motor-evoked potentials (MEP) at 28 days post-injection (dpi) (Fig. 7 A, B). Basso Mouse Scale (BMS) scores assessing hind limb motor function indicated improved recovery in AAV-MCT1-treated SCI mice starting from 7 dpi. By the end of 28 dpi, the hind limb motor function in treated SCI mice showed a marked improvement (Fig. 7 C). Swimming tests demonstrated that mice in the AAV-MCT1 group exhibited more stable trunk movements, fewer tilted body angles, and less drooping tails, resulting in a significantly elevated LSS swimming score (Fig. 7 D, E). Moreover, analysis of the H&E-stained slices of the SCI mouse bladder indicated a significant increase in the thickness of the detrusor muscle layer in the neurogenic bladder following treatment with AAV-MCT1, implying the restoration of nerve innervation in the bladder (Fig. 7 F, G). Overall, our research findings confirm a positive therapeutic effect of restoring MCT1 expression in spinal cord ECs on functional recovery in SCI mice. 4. Discussion The results of this study demonstrate that endothelium-derived lactate can be uptaken and utilized by neurons for energy generation and axonal regeneration, which is crucial for maintaining normal neuron function and axon regeneration. After SCI, there is abnormal lactate metabolism at the injury epicenter, where ECs destruction leads to obstruction of lactate shuttle. Additionally, we identified MCT1 as the endothelial cell-specific lactate transporter. By upregulating the expression of MCT1 in ECs, we restored lactate transport function, enhanced neuronal glycolysis, promoting axon regeneration and neurological function recovery after SCI. It highlights that MCT1 serves as a novel therapeutic target for SCI, providing a new direction after SCI treatment from a metabolic perspective. Previous studies have shown that in traumatic SCI, the primary injury damages cells and triggers complex secondary injury cascades ( 2 ). Sustained microvascular thrombosis and spasms at the injury epicenter cause further neurons death. Our study, from a metabolic perspective, investigates whether the loss of microvascular function after spinal cord injury leads to metabolic changes in the epicenter of injury. Energy metabolomic analysis of mice after SCI reveals an elevation in glycolysis-related metabolites, with lactate being a significantly increased metabolite post-injury. Existing literature suggests that endothelial cells can maintain central nervous system energy metabolism by transporting lactate, thereby regulating neuronal functional stability ( 32 ). Meanwhile, enhancing glycolysis in glial cells can promote axon regeneration at the epicenter of injury ( 15 ), and there is also literature suggesting that increasing the neuronal intrinsic glycolytic capacity can inhibit neuronal apoptosis ( 33 ). Therefore, focusing on metabolic changes at the site of injury after SCI may provide new research directions for studying neurological function recovery post-SCI. We and others have previously shown that restoring cellular energy metabolism after SCI can promote axon regeneration and functional recovery ( 34 , 35 ). Lactate had been widely accepted as a metabolic waste product; however, recent studies have shown that lactate is a crucial fuel in energy metabolism ( 36 ). In the central nerve system, lactate serves as a vital source of energy, although its contribution to the tricarboxylic acid (TCA) cycle compared to glucose remains a contentious issue ( 37 ). The roles of lactate in delivery of oxidative and gluconeogenic substrates as well as in cell signaling is termed the lactate shuttle ( 38 ). Research on lactate shuttle initially focused on describing and analyzing the whole organ lactate shuttle mechanisms ( 39 , 40 ). However, cellular lactate shuttle pathways have garnered increasing attention in recent years. Two recent studies have indicated that pericytes and macrophages can fulfill their energy requirements by receiving lactate from endothelial cells. Endothelial-derived lactate is absorbed by pericytes to maintain blood-brain barrier stability ( 41 ), while lactate produced by endothelial cells induces macrophage polarization to promote muscle regeneration following ischemic injury ( 42 ).Restoring cellular glycolysis is a crucial pathway for promoting axonal regeneration and functional recovery after spinal cord injury ( 43 ). Additionally, Furthermore, endothelial cells can also utilize lactate as a driving force, along with high rates of glycolysis, to promote angiogenesis ( 44 ). Our study aims to elucidate the biological functions of lactate. We found that endothelial cells can enhance their glycolytic capacity and tube-forming migration ability by utilizing exogenous lactate. Additionally, we discovered that neurons rely on endothelium-derived lactate to provide energy. This is crucial for studying lactate shuttle and functional recovery of neurovascular after SCI, as a lack of endothelium-derived lactate can lead to impediments in axon regeneration and diminished neurological recovery. Endothelium-derived lactate is present at high concentrations locally, it becomes an easily accessible fuel source around blood vessels ( 45 ). Neurons and ECs are typically in direct contact throughout the entire neurovascular network, endothelium‐derived lactate may be the main source of energy supply for neurons. Although neurons are closely associated with ECs, lactate cannot be transferred directly through physical contact; there are lactate-related transport proteins mediating lactate shuttle between cells ( 46 ). Our data suggest that MCT1, a lactate transporter protein, is specifically expressed in endothelial cells within the spinal cord, playing a crucial role in the extracellular secretion of lactate by these cells. Neurons uptake lactate into the cell for energy utilization through their specific transport proteins. While we demonstrate the role of lactate in endothelial cells through MCT1-mediated extracellular lactate secretion, the specific MCT members responsible for lactate uptake in neurons, their efficiency in lactate uptake, and their specific functions in vivo still require further investigation. After SCI, insufficient axon regeneration often leads to poor recovery, representing one of the most pressing challenges in SCI treatment. Developing successful regenerative strategies to reconnect axons within the central nervous system is paramount for spinal cord injury research. It is widely believed that inadequate intrinsic neuronal growth capacity, a deficient growth environment, and the absence of neurotrophic factors are the main reasons for hindered axonal regeneration ( 47 , 48 ). Currently, while there is considerable research on axonal regeneration in adults ( 49 – 51 ), the translation of these findings into clinically applicable therapeutic methods is severely limited. Metabolic reprogramming emerges as a novel strategy to stimulate axonal regeneration in the central nervous system ( 52 , 53 ). Previous studies have shown that lactate serves as an energy source for neurons ( 54 ), and local application of lactate to the injured spinal cord can promote corticospinal tract axonal regeneration ( 15 ).Our research further reveals an unexpected connection between endothelial cell-neuron lactate shuttling after SCI and neural functional recovery, offering a novel metabolic target for spinal cord injury treatment. We found that impaired lactate shuttling due to the lack of MCT1 in endothelial cells at the epicenter of injury impedes neuronal energy metabolism, resulting in weakened axonal regeneration capacity. Restoring MCT1 expression in endothelial cells at the epicenter of injury significantly promotes axon regeneration and functional recovery. While we did not specifically study whether other cells transport lactate to neurons after SCI, besides endothelial cells, other cells may also contribute. For instance, it has been reported that astrocytes secrete lactate to fuel neuronal mitochondria in traumatic brain injury ( 55 ). Interestingly, lactate not only serves as a metabolic substrate but also functions as a signaling molecule to modulate neuronal function ( 54 , 56 , 57 ). Whether this also promotes axonal regeneration warrants further investigation. 5. Conclusion In summary, our study demonstrates that lactate produced by endothelial cells is utilized by adjacent neurons for energy metabolism and axonal regeneration. The impaired lactate metabolism observed in mice following spinal cord injury is attributed to the lack of MCT1 in endothelial cells at the epicenter of injury. Restoring MCT1 expression at the lesion site in adult mammals promotes significant axonal regeneration and functional recovery Declarations Ethics approval and consent to participate All research protocols were approved by the Animal Ethics Committee of Central South University. Animal care and use during our experiment were conducted under the guidelines of the Administration Committee of Affairs Concerning Experimental Animals in Hunan Province, China. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that there is no conflict of interest regarding the publication of this paper. Funding This work was supported by the Key Program of the National Natural Science Foundation of China (No. 82030071), the National Natural Science Foundation of China (No. 82202722, No. 81874004), the Science and Technology Major Project of Changsha (NO. kh2103008), the Science Foundation of Xiangya Hospital for Young Scholar (Grant No. 2021q18), the Natural Science Foundation of Changsha city (Grant Nos. kq2202378), and Graduate students of Central South University independently explore innovative projects (2022ZZTS094). Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Authorship contribution statement Liyuan Jiang, Jianzhong Hu: Conceptualized and designed the project. Chaoran Shi, Jiaqi Xu: Performed major experiments and analyzed data. Yinghe Ding: Performed bioinformatic analysis and data analysis. Feifei Yuan, Fengzhang Zhu: Performed animal experiments and data validation. Hongbin Lu, Chunyue Duan, Tianding Wu Provided consultation, review & editing, and supervision. All authors had approved the manuscript. Acknowledgements The authors would like to thank Hui Xie and other staff from the Movement System Injury and Repair Research Center, Xiangya Hospital, Central South University, Changsha, China. References Global regional. national burden of spinal cord injury, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet Neurol. 2023;22(11):1026–47. Ahuja CS, Wilson JR, Nori S, Kotter MRN, Druschel C, Curt A, et al. Traumatic spinal cord injury. Nat Rev Dis Primers. 2017;3:17018. Grossman SD, Rosenberg LJ, Wrathall JR. Temporal-spatial pattern of acute neuronal and glial loss after spinal cord contusion. Exp Neurol. 2001;168(2):273–82. Mautes AE, Weinzierl MR, Donovan F, Noble LJ. 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Na(+) /K(+) -ATPase coupled to endothelin receptor type B stimulates peripheral nerve regeneration via lactate signalling. Eur J Neurosci. 2017;46(5):2096–107. Additional Declarations No competing interests reported. Supplementary Files Graphicabstract.jpg FulluncroppedBlotsimageFigure4HK1.tif FulluncroppedBlotsimageFigure4PFKM.jpg FulluncroppedBlotsimageFigure4PKM1.jpg FulluncroppedBlotsimageFigure4actin.jpg FulluncroppedBlotsimageFigure5HK1.jpg FulluncroppedBlotsimageFigure5PKM1.jpg FulluncroppedBlotsimageFigure5actin.jpg FulluncroppedBlotsimageFigure5pfkm.jpg 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4079758","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":279019236,"identity":"acd2c744-4cea-4d22-a61f-d16dde94d753","order_by":0,"name":"Chaoran Shi","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Chaoran","middleName":"","lastName":"Shi","suffix":""},{"id":279019237,"identity":"615714a2-daa9-4a95-a310-1f4f086e191d","order_by":1,"name":"Jiaqi Xu","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Xu","suffix":""},{"id":279019238,"identity":"9f6be41b-7640-4831-bc45-9fc14907062a","order_by":2,"name":"Yinghe Ding","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Yinghe","middleName":"","lastName":"Ding","suffix":""},{"id":279019239,"identity":"78da3577-3b74-4007-b1d1-89dcb9a6892c","order_by":3,"name":"Feifei Yuan","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Feifei","middleName":"","lastName":"Yuan","suffix":""},{"id":279019240,"identity":"849944c8-1bd4-4e2d-b850-06d8b58020e1","order_by":4,"name":"Fengzhang Zhu","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Fengzhang","middleName":"","lastName":"Zhu","suffix":""},{"id":279019241,"identity":"230ac48c-80b1-4335-b477-771d95094d81","order_by":5,"name":"Tianding Wu","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Tianding","middleName":"","lastName":"Wu","suffix":""},{"id":279019242,"identity":"22786924-fc39-488a-8ebb-79d252326f3a","order_by":6,"name":"Chunyue Duan","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Chunyue","middleName":"","lastName":"Duan","suffix":""},{"id":279019243,"identity":"c0d55d29-3884-4784-9e72-2bd53b015860","order_by":7,"name":"Hongbin Lu","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Hongbin","middleName":"","lastName":"Lu","suffix":""},{"id":279019244,"identity":"0d0277f1-29d6-48d3-893e-6a7ecc70214b","order_by":8,"name":"Jianzhong Hu","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Jianzhong","middleName":"","lastName":"Hu","suffix":""},{"id":279019245,"identity":"9ff51627-60e7-451b-a285-3765fa6f2d25","order_by":9,"name":"Liyuan Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYBACxmYQWSAB5VZAKAkcqpG0GMDUnCFCCwQYwAxoI0ILczvzs4dfDCwSNxw/e0yad56dvcEB5oO3eRjs8nA7jM3cWMZAInHDmbxkY95tyYkbDrAlW/MwJBfj8YuZtARIy4Ecw8e82w4kGBzgMZPmYTiQ2IBTC/s3iJbzbwwO8845AHQY/zcCWnjMJD+AtNwA2dJwgHHDAR42QlrKpIGBbDzzxhtjwznHkhNnHmYztpxjkIxTi2H/8W2SPyrqZPvO55hJvKmxs+c73vzwxpsKO9xagBLMPAwMjiAFTDwgIWYQYYBDPRDIgxz3g4HBngHKGAWjYBSMglGAAQDymFN2apxpTwAAAABJRU5ErkJggg==","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":true,"prefix":"","firstName":"Liyuan","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2024-03-12 03:35:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4079758/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4079758/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52765343,"identity":"30bbce8c-d5aa-4762-b5d0-0c54e8454818","added_by":"auto","created_at":"2024-03-15 13:21:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4531344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrecision-targeted metabolomics analysis identified the most affected functional metabolites after SCI. \u003c/strong\u003e(A) KEGG analysis of 32 metabolites with heatmap to examine the metabolic pathways with significant changes in energy metabolism after SCI. (B) Comparative analysis of 32 energy metabolism metabolites in spinal cord tissues with volcano plotting to identify five glycolysis-related functional metabolites (red points) that were mostly changed after SCI (|log2FoldChange|\u0026gt;0.1 and P-value \u0026lt; 0.01). (C) Heatmap overview of the most significantly differential metabolites in the epicenter of injury tissue that were clearly changed after SCI. (D) Violin plot shown the expressional levels of four glycolysis metabolism related metabolites that were mostly alternated after SCI. (n = 3 for each group, * *p \u0026lt; 0.01, ∗∗∗∗ p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/a06cddac8c7ee54c6ac90264.jpg"},{"id":52765356,"identity":"426fa2ed-1b30-46f5-b9d3-beee5a27ea4a","added_by":"auto","created_at":"2024-03-15 13:21:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8182261,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndothelium-derived lactate feeds nerve cells. \u003c/strong\u003e(A) Representative images showing CFP (blue) and YFP (yellow) fluorescence in control and lactate (100 mM)‐treated CATH.a. (scale bar = 10μm). (B) Quantification of the CFP/YFP fluorescence ratio of laconic (lactate sensor) showing concentration‐dependent lactate uptake in CATH.a. (n = 6 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM). (C) Representative images for the lactate uptake assay with 2 well chamber. Cells were seeded on a glass bottom dish. bEnd.3 and adenoviral laconic‐infected HBVPs were separated by 2 well chamber. After attachment, the 2 well chamber was removed for cell migration. (D) Schematic illustration for the lactate uptake assay after chamber removal. At 24 h after chamber removal, the fluorescence of CFP and YFP were observed in the middle of the wells (yellow area). (E) Representative confocal image showing CFP (blue) and YFP (yellow), Light field (white) fluorescence from bEnd.3 and laconic‐infected CATH.a on a 2 well chamber. (scale bar = 100μm). (F) Localized enlargements showing CFP (blue) and YFP (yellow), Light field (white) fluorescence from bEnd.3 and laconic‐infected CATH.a on a 2 well chamber. The upper panel shows CATH.a with endothelial contact and the lower panel shows CATH.a without endothelial contact (scale bar = 10μm). (G) Quantification of the CFP/YFP fluorescence ratio of laconic in CATH.a with endothelial contact (EC‐NEU contact) or CATH.a alone (NEU only) (n = 8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student’s t test, ∗∗** p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/440563c7292dfcc77065bc13.jpg"},{"id":52765349,"identity":"443c95e0-6482-48ee-890a-e4b35caa0c14","added_by":"auto","created_at":"2024-03-15 13:21:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7822891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatial and temporal expression of MCT1 after SCI.\u003c/strong\u003e (A) The expression level of MCTs in endothelial cells, in single-cell dataset GSE190442. (B) The expression level of SLC16a1 (MCT1) in different cell types, in single-cell dataset GSE162610. (C) The expression level of SLC16a1 in different time point post injury, in single-cell dataset GSE162610. (D) Representative fluorescence images showing the expression of MCT1 in the spinal cord of the sham and 7, 14, 28 days post injury (dpi) groups (red: MCT1, green: CD31, blue: DAPI, scale bar: 200μm). (E) Quantification of the MCT1 area /CD31 area intensity in the central and margin regions of 7, 14, 28 days post injury groups in (D) (n = 8, mean ± SD, unpaired t-test, ****p \u0026lt; 0.0001). (F) Representative fluorescence images showing the colocalization of CD31 with MCT1 in the spinal cord at 14dpi (red: MCT1, green: CD31, blue: DAPI, scale bar: 200μm). (G) Localized enlargements of fluorescence confocal images from (F). The upper panel shows the margin region of injury site and the lower panel shows the central region of injury site (red: MCT1, green: CD31, blue: DAPI, scale bar: 10μm). (H) Quantification of the Colocalization of MCT1 cells with CD31 cells in the central and margin regions in (E) (n = 3, mean ± SD, unpaired t-test, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/d4e4a43d701267a5bc946843.jpg"},{"id":52765352,"identity":"e5964bc6-98e5-4a98-a08d-ce55501573c1","added_by":"auto","created_at":"2024-03-15 13:21:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4448967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of ECs MCT1 Expression Leads to Decreased Biological and Metabolic Functions.\u003c/strong\u003e (A) Representative images showing the tube-forming ability of bEnd.3 Cells under lactate or α-CHCA treatment in four groups (scale bar = 100μm). (B) Quantification of tube-forming ability in (A). (n = 6, mean ± SD, one-way ANOVA, *p \u0026lt; 0.05, ****p \u0026lt; 0.0001). (C) Representative images showing the lateral migration ability of bEnd.3 Cells under lactate or α-CHCA treatment in four groups (scale bar = 100μm). (D) Quantification of tube-forming ability in (C) (n = 3, mean ± SD, one-way ANOVA, ns not significant, ****p \u0026lt; 0.0001). (E) Western blotting analysis of the levels of HK1, PFKM, PKM1 and β-actin in bEnd.3 Cells with different treatments. (F) Quantification of the relative expression of HK1, PFKM, PKM1 and β-actin in (E) (n = 3, mean ± SD, one-way ANOVA, ns not significant, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/c76dc0562294a91e3b0f68c4.jpg"},{"id":52765358,"identity":"21c66311-943e-462c-978e-eaa07cd2ceb7","added_by":"auto","created_at":"2024-03-15 13:21:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8388181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeurons Utilize Lactate to Enhance Glycolysis and Promote Axon Regeneration.\u003c/strong\u003e (A) Representative confocal image showing CFP (blue) and YFP (yellow), Light field (white) fluorescence from lactate or α-CHCA treated bEnd.3 and laconic‐infected CATH.a on a 2 well chamber. (scale bar = 100μm). (B) Quantification of the CFP/YFP fluorescence ratio of laconic in CATH.a with bEnd.3 under lactate treatment or under treatment (n = 8, mean ± SD, unpaired t-test, ****p \u0026lt; 0.0001). (C) Representative immunofluorescent images of the regenerative axons under lactate or 2-DG treatment in four groups (green: Tuj1, blue: DAPI, scale bar = 100μm). (D) Quantification of total axon length in (C) (n = 8, mean ± SD, one-way ANOVA, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001). (E) Western blotting analysis of the levels of HK1, PFKM, PKM1 and β-actin in CATH.a Cells with different treatments. (F) Quantification of the relative expression of HK1, PFKM, PKM1 and β-actin in (E) (n = 3, mean ± SD, one-way ANOVA, ns not significant, ns not significant, *p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/b560a9a3d8c713612aa3a830.jpg"},{"id":52765907,"identity":"8270b727-e8a1-40b7-8ae1-415809e9255a","added_by":"auto","created_at":"2024-03-15 13:29:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":15130359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpinal Cord ECs Overexpression of MCT1 Rescues Impaired Neurogenesis in SCI Mice.\u003c/strong\u003e (A) Representative fluorescence images showing the colocalization of CD31, MCT1 with MCT1-AAV in the injured spinal cord at 14dpi, in the AAV-MCT1 treated group (red: MCT1, green: CD31, blue: DAPI, scale bar: 200μm). And Localized enlargements of immunofluorescence staining. The upper panel shows the margin region of the injury site, and the lower panel shows the central region of the injury site (white: MCT1, red: AAV-MCT1, green: CD31, blue: DAPI, scale bar: 20μm). (B) Representative fluorescence images showing the colocalization of CD31, MCT1 with MCT1-AAV in the injured spinal cord at 14dpi, in the control group (red: MCT1, green: CD31, blue: DAPI, scale bar: 200μm). (C) Quantification of the Colocalization of MCT1 cells with CD31 cells in the central and margin regions in (A) (n = 3, mean ± SD, unpaired t-test, ns not significant). (D) Quantification of the MCT1 area/ CD31 area in the central region of the injury site from (A, B) (n = 3, mean ± SD, unpaired t-test, ns not significant). (E) Representative immunofluorescence images of GFAP, TUJ, F4/80 (green), and AAV (red) in the injury site (scale bar: 50μm). (F) Quantification of the percentage of AAV+ cells in GFAP+, TUJ+, and F4/80+ cells in (F) (n = 6, mean ± SD, one-way ANOVA, ****p \u0026lt; 0.0001). (G) Representative immunofluorescent stains of TUJ1 images of the spinal cord at 28 dpi in each group. (scale bar: 200μm). (H) Quantification of TUJ1 positive signals in different areas roster to the epicenter of control, and AAV-MCT1 groups in (H) (n = 6, mean ± SD, unpaired t-test, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/5428ae9798e07009cd1a8aad.jpg"},{"id":52765346,"identity":"48c57a57-bf78-452a-9f4e-36f9dad406af","added_by":"auto","created_at":"2024-03-15 13:21:03","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4342143,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMCT1 treated enhanced tissue repair and functional recovery after spinal cord injury. \u003c/strong\u003e(A) Representative images of hindlimb motor evoked potentials (MEPs) in the sham, control, and treatment groups at 28 dpi. (B) Quantification of the amplitude of hindlimb in (A) (n=6, mean ± SD, one-way ANOVA, ∗∗p \u0026lt; 0.01). (C) Basso Mouse Scale (BMS) scores over time post-injury in the sham, control, and treatment groups (n=6, mean ± SD, two-way ANOVA, Tukey's multiple comparisons, ns not significant, ∗ p \u0026lt; 0.05, ∗∗ p \u0026lt; 0.01, ∗∗∗ p \u0026lt; 0.001, ∗∗∗∗ p \u0026lt; 0.0001). (D) Representative images of swimming test at 28 dpi in the sham, control, and treatment groups. (E) Quantification of the swimming test in (D) using the Louisville Swim Scale (LSS) swim score (n=6, mean ± SD, one-way ANOVA, ∗∗ p \u0026lt; 0.01). (F) Representative images of hematoxylin and eosin staining (H\u0026amp;E) of the bladder (scale bar=200μm) and bladder macroscopic photo at 28 dpi in the sham, control, and treatment groups. (G) Quantification of the detrusor muscle thickness in (F) (n=6, mean ± SD, one-way ANOVA, Tukey's multiple comparisons, ∗ p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/42b68d7c152d71e9b3b3ccec.jpg"},{"id":52849125,"identity":"4f3d8d67-2fcd-4428-a8c5-9799aad7b7d9","added_by":"auto","created_at":"2024-03-17 21:07:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1873858,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/2742132c-ff96-48c9-944c-340b4209ff7d.pdf"},{"id":52765347,"identity":"a49fe3f7-82ff-4aa1-bea6-ea4eaa10b976","added_by":"auto","created_at":"2024-03-15 13:21:03","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4890990,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/81c24b2b729d42b0123b3167.jpg"},{"id":52765342,"identity":"ab3e9add-c8f5-4f96-80a5-79d1076380c6","added_by":"auto","created_at":"2024-03-15 13:21:03","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2311720,"visible":true,"origin":"","legend":"","description":"","filename":"FulluncroppedBlotsimageFigure4HK1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/4574726c5e4640d9c879956c.tif"},{"id":52765906,"identity":"4c47dd8c-1685-4cbd-84f1-687fdbb6ced1","added_by":"auto","created_at":"2024-03-15 13:29:04","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":64300,"visible":true,"origin":"","legend":"","description":"","filename":"FulluncroppedBlotsimageFigure4PFKM.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/f97c753d14fe3bdf29ec8249.jpg"},{"id":52765351,"identity":"d6ceb8c4-41c2-447f-99f9-3ac030f1f3e4","added_by":"auto","created_at":"2024-03-15 13:21:04","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":91109,"visible":true,"origin":"","legend":"","description":"","filename":"FulluncroppedBlotsimageFigure4PKM1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/e10516d984b000dc86e7e9ed.jpg"},{"id":52765344,"identity":"c5a4b439-a577-45c7-8d6a-3231ecd49705","added_by":"auto","created_at":"2024-03-15 13:21:03","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":49540,"visible":true,"origin":"","legend":"","description":"","filename":"FulluncroppedBlotsimageFigure4actin.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/3223971bcbe6f03f25a4f081.jpg"},{"id":52765353,"identity":"cd8fc641-81c3-4575-b55c-8a1c7b129009","added_by":"auto","created_at":"2024-03-15 13:21:04","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":74525,"visible":true,"origin":"","legend":"","description":"","filename":"FulluncroppedBlotsimageFigure5HK1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/a2b6dfae60cba03a30d44072.jpg"},{"id":52765905,"identity":"b5671a16-6b40-424c-b141-cc862c46fa5f","added_by":"auto","created_at":"2024-03-15 13:29:04","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":56041,"visible":true,"origin":"","legend":"","description":"","filename":"FulluncroppedBlotsimageFigure5PKM1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/9bf5a369e2c9d43a53b57012.jpg"},{"id":52765355,"identity":"463c808f-6624-4135-932d-d8d492f838e4","added_by":"auto","created_at":"2024-03-15 13:21:05","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":68603,"visible":true,"origin":"","legend":"","description":"","filename":"FulluncroppedBlotsimageFigure5actin.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/362225f3f96f3a9b3412e691.jpg"},{"id":52765904,"identity":"6c19ed5b-1aa1-427f-80e4-e4c1bf2e6f07","added_by":"auto","created_at":"2024-03-15 13:29:04","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":71485,"visible":true,"origin":"","legend":"","description":"","filename":"FulluncroppedBlotsimageFigure5pfkm.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4079758/v1/9697d204576aed77f402e494.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"MCT1-Mediated Endothelial Cell Lactate Shuttle as a Target for Promoting Axon Regeneration after Spinal Cord Injury","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSpinal Cord Injury (SCI) poses a formidable challenge in contemporary medicine, characterized by its substantial contribution to premature mortality and enduring disability. Over the span from 1990 to 2019, a notable escalation has been observed globally in the incidence, prevalence, and years lived with disability attributable to SCI (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The pathophysiology of SCI encompasses a multifaceted process, wherein an initial mechanical insult precipitates a cascade of secondary damage (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The pathophysiology of SCI encompasses a multifaceted process, wherein an initial mechanical insult precipitates a cascade of secondary damage (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)., alongside the compromise of vasculature integrity, evidenced by the disruption of endothelial cell tight junctions. Such disturbances lead to the breakdown of the blood-spinal cord barrier (BSCB), augmenting vascular permeability (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSubsequently, the injury site is subjected to sustained ischemia, perpetuated by ongoing edema (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), vessel thrombosis, vasospasm, and a disruption of homeostatic equilibrium (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). This pathophysiological milieu fosters metabolic dysfunction among cells at the injury epicenter, situated in an ischemic and hypoxic environment (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The aberrant alteration of metabolic products post-SCI and their impact on nerve regeneration remains an area of unresolved inquiry (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). It underscores the imperative of managing energy metabolism within the injury epicenter as a pivotal aspect of SCI recovery.\u003c/p\u003e \u003cp\u003eEndothelial cells (ECs) play a key role in regulating selective transport and metabolic exchange between the blood and the spinal cord as a vital component of the BSCB (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Post-SCI, ECs undergo regeneration at the site of injury; however, their functional attributes are markedly diminished relative to their healthy counterparts, spanning diminished barrier functionality to compromised substance transport (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Increasingly, lactate is recognized not merely as a metabolic byproduct but as a crucial energy substrate and signaling molecule that fosters axon regeneration post-SCI (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), This revelation posits lactate homeostasis as a critical determinant in SCI recovery. In the central nervous system, lactate can transport between highly glycolytic cells to adjacent cells, a phenomenon known as lactate shuttle (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Many studies have confirmed the involvement of the astrocyte-neuron lactate shuttle (ANLS) in memory formation, the generation of neuropathic pain, and the further exacerbation of neurodegenerative diseases. (\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). This shuttle mechanism suggests that astrocytes respond to lactate-mediated neuronal activity by increasing their aerobic glycolysis levels. However, it remains unclear whether a lactate shuttle exists between spinal cord ECs and neurons, and the mechanism by lactate mediates neuronal metabolism after SCI.\u003c/p\u003e \u003cp\u003eMonocarboxylic transporters (MCTs) play a significant role in the lactate shuttle, which may promote intracellular and intercellular lactate shuttle in the central nervous system (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). There is research indicating that ECs predominantly express Monocarboxylic transporter1 (MCT1) in the spinal cord (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), indicating ECs may regulate axon regeneration by transporting lactate via MCT1. However, the lack of evidence regarding how MCT1 is expressed in ECs after SCI and its functional implications in this context requires further investigation.\u003c/p\u003e \u003cp\u003eIn this study, metabolomics analysis revealed elevated lactate levels post-SCI. Bioinformatics analysis subsequently indicated a reduced expression of MCT1, a lactate transporter, in ECs following injury. Furthermore, in vitro co-culture experiments employing a lactate sensor substantiated the presence of a lactate shuttle between ECs and neurons. Leveraging adenovirus mediated MCT1 overexpression through in situ injection in a mouse model, we delineated the pivotal role of endothelial cell MCT1 in modulating energy metabolism, crucial for the regeneration of endothelial cells and axons post-SCI. This insight heralds a novel metabolic therapeutic strategy for SCI rehabilitation.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell Lines\u003c/h2\u003e \u003cp\u003eMouse brain endothelial cells (bEnd.3 cells, CL-0598, Procell) were maintained with culture media containing 10% FBS ( FBS-CP500, NEWZERUM), 1% 100* Penicillin-Streptomycin Solution (PB180120, Procell) in DMEM (10-013-CVRV, CORNING) at 37℃ in a 5% CO2-humidified incubator. Cell line was obtained directly from Pricella, with no additional cell authentication performed. Mouse nerve cells (CATH.a cells, HTX2209, ATCC)were maintained in CATH.a cell-specific medium (CM-0665, Procell) at 37℃ in a 5% CO2-humidified incubator. Cell line was obtained directly from ATCC, with no additional cell authentication performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell treatment\u003c/h2\u003e \u003cp\u003eThe bEnd.3 or CATH.a were seeded in six-well plates at a density of 10^5 cells/ml. We exposed bEnd.3 or CATH.a with 10mM exogenous L-lactate (L6402, sigma) or 5 mM α-cyano-4-hydroxycinnamic acid (α-CHCA) (S8612, Selleck) for 1 day before being examined..\u003c/p\u003e \u003cp\u003eFor the contact co-culture experiments, we used Ibidi 2-well inserts (81176, ibidi) were installed into 20mm confocal culture dish (801001, NEST) with sterile tweezers and gently pushed with a gloved fingertip. Pretreated bEnd.3 or CATH.a with Laconic infection were seeded into opposite chambers at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e and incubated overnight. The culture insert was then removed with sterile tweezers and the well topped up with 1 ml of DMEM media. After 48-hour incubation, cells were fixed with freshly prepared 4% PFA (AWI0056b, Abiowell) for 30 minutes at room temperature. After fixing, cells were subjected to imaging using a Zeiss confocal laser scanning microscope LSM 9.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Lentiviral vector construction and transfection\u003c/h2\u003e \u003cp\u003eThe construct for Laconic (Plasmid #44238; San Martin et al, 2013) was obtained from Addgene. Seed well-conditioned target cells at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well. Plate in a 24-well plate, and after cell counting, add 500 \u0026micro;L of DMEM culture medium to each well. After 24 hours, replace with DMEM medium containing 1 \u0026micro;g/mL Polybrene-plus, choosing an MOI (Multiplicity of Infection) value of 40, and add 20 \u0026micro;L of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e TU/mL viral particles. After 24 hours, replace with fresh culture medium. 72 hours post viral infection, observe under a fluorescence microscope to evaluate the efficiency of lentiviral infection in the target cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Adeno-associated virus construction and transfection\u003c/h2\u003e \u003cp\u003eAdeno-associated virus used in this study was produced at the Obio Technology (Shanghai, China). The following AAV plasmid was used and detailed sequence information is available as detailed or upon request: pAAV-SIc16a1-FLAG-mCherry (Obio Technology). Injection volumes, coordinates and experimental purpose are described below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Mice\u003c/h2\u003e \u003cp\u003eAdult male or female C57BL/6 mice (8\u0026ndash;10 weeks old, weight 20-25g) were used in this study. The animals were purchased from Hunan SJA Laboratory Animal Company Limited and were kept in a pathogen-free animal facility at Central South University, following standard purification procedures. They were housed in the laboratory animal unit, with a 12-hour day/night cycle, and were provided ad libitum access to food and water. All mice are housed in a laboratory animal facility with an ambient temperature of 22\u0026ndash;24\u0026deg;C and relative humidity of 60\u0026ndash;80%. Manual bladder voiding and all other animal care was performed 2 to 4 times daily throughout the entire experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 SCI models\u003c/h2\u003e \u003cp\u003e The Animal Care and Use Committee of Central South University, Changsha, China, approved all animal protocols and experimental procedures. Perform routine anesthesia, skin preparation, disinfection, and draping. Make an incision centered on the T10 spinous process, sequentially cutting through the skin, subcutaneous tissue, muscles, and lamina to expose the spinal cord. Severe crush SCI were made after laminectomy of a single vertebra by using No. 5 Dumont forceps (Fine Science Tools, Foster City, CA) without spacers and with a tip width of 0.5mm to completely compress the entire spinal cord laterally from both sides for 5 second. Irrigate the wound, then close the incision in layers, cover with a dressing, and secure it. Postoperatively, mice are housed individually with standard feed and water available ad libitum. Administer penicillin 20,000 units bi-daily via intramuscular injection for postoperative infection prevention for 3 days. Manually express the bladder 2 to 4 times daily to assist with urination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Spinal injections for AAV\u003c/h2\u003e \u003cp\u003eTo overexpress MCT1 in endothelial cells, partial laminectomy at the T9 spinal level was performed two weeks prior to spinal cord injury, using 30-33G Hamilton needles to bilaterally inject pAAV-Slc16a1-FLAG-mCherry2 (0.5 \u0026micro;l per injection) at two depths of 0.8 mm and 0.4 mm below the dorsal surface, spaced 1 mm apart, with each injection maintained for 5 minutes. Post-injection, the needle was left in the spinal tissue for 15\u0026ndash;20 minutes. During the injection process, care was taken to keep the mice warm. The needle was then slowly withdrawn. The skin was sutured and the wound disinfected. For postoperative pain relief, mice were administered carprofen via intramuscular injection daily for 3 days. Animals were perfused 28 days after AAV injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Perfusions\u003c/h2\u003e \u003cp\u003eAt different time points post-SCI, 1% sodium pentobarbital (50 mg/kg) is administered intraperitoneally for deep anesthesia in mice. Rapid thoracotomy is performed to expose the heart, and a size 12 medical intravenous catheter is inserted into the left ventricle up to the origin of the ascending aorta and secured. The right atrium is incised, followed by sequential perfusion with pre-warmed heparinized saline and 10% formalin solution, maintaining the 10% formalin perfusion for approximately one hour. The specimens are then stored in a 4\u0026deg;C refrigerator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunofluorescence\u003c/h2\u003e \u003cp\u003eFor immunofluorescence of tissue, frozen sections of 16-\u0026micro;m thickness from the spinal cord containing the lesion site were harvested at different time points post-SCI (or sham group) and cut along the sagittal plane. The frozen sections were rewarmed at room temperature for 15 minutes and rinsed three times with a PBS solution for 10 minutes each time. The immunohistochemical pen was used to circle the spinal cord sections on the slides. The sections were then permeabilized with 100\u0026micro;L of PBS solution containing 0.1% Tween 20 and 0.3% Triton-X 100 for 30 minutes and blocked with 5% BSA in PBS for an additional 30 minutes. After blocking, the sections were incubated with a group of primary antibodies including F4/80 (Abcam, ab6640, 1:400), TUJ-1 (CST, 5568, 1:400), GFAP (Proteintech, 16825-1-AP, 1:800), MCT1 (Proteintech, 20139-1-AP, 1:200), CD31(R\u0026amp;D systems, inc., fab3628G, and 1:200), overnight at 4℃. After being rinsed five times for 10 minutes each with PBS containing 0.1% Tween 20, the sections were incubated for one hour at room temperature with species-appropriate secondary antibodies conjugated with Alexa Fluor 594 (Abcam, 1:400) or Alexa Fluor 488 (Abcam, 1:400). Subsequently, the sections were rinsed with PBS containing 0.1% Tween 20 before being mounted on slides and covered with DAPI (Genetex).\u003c/p\u003e \u003cp\u003eFor immunofluorescence of cells, we place sterile 24-well slides into a 24-well plate. The bEnd.3 or CATH.a, post trypsinization, are seeded onto the slides at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well, with 0.5 ml of complete culture medium added per well. The culture plate is then incubated in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Once cells reach 85% confluency, the medium is aspirated, and old culture medium is washed off with PBS along the walls of the well. Each well is then fixed with 500 \u0026micro;l of 4% paraformaldehyde for 12 minutes, followed by a PBS wash to remove the paraformaldehyde. Each well is treated with 200 \u0026micro;l of 0.1% Triton-X100 in PBST for 20 minutes. After aspiration of the PBST, the wells are blocked with 3% BSA in PBS solution for 30 minutes. The blocking solution is then aspirated, and primary antibodies including CD31, MCT1, and TUJ are added and incubated overnight at 4\u0026deg;C. The next day, primary antibodies are aspirated, and the wells are washed three times with PBS for 5 minutes each. Corresponding secondary antibodies are then added and incubated at room temperature for 1.5 hours. After aspiration of the secondary antibodies, the wells are washed three times with PBS for 5 minutes each. A drop of DAPI solution containing an anti-fade agent is then added to the slide, and the cell slide is picked up with forceps and inverted onto the slide. The sections were analyzed under a fluorescence microscope or confocal microscope (Zeiss). To validate antibody specificity and distinguish genuine target staining from the background, secondary antibody-only controls were employed. The ImageJ software was used for quantitative analysis of the images, while the Imaris 9.0 software was used for 3D reconstruction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western Blotting\u003c/h2\u003e \u003cp\u003eWe utilize a BCA Protein Assay Kit for protein concentration normalization. After adjusting protein samples to a protein to loading buffer ratio of 4:1, we denature the proteins in a 95\u0026deg;C metal bath for 10 minutes, followed by storage at -20\u0026deg;C for future use. During electrophoresis, samples are loaded into the gel wells. The voltage is set to 90V for the first 30 minutes, then adjusted to 120V until the protein bands are fully separated. Subsequently, PVDF membranes are activated with anhydrous ethanol and carefully placed on the SDS-PAGE gel, ensuring no bubbles are formed. The PVDF membrane and SDS gel are sandwiched between sponges and filter paper and electrotransferred in an ice-water mixture at 300mA for 90 minutes. The PVDF membrane is then blocked with 5% non-fat milk in TBST solution on a shaker for 1.5 hours. Afterward, the membrane is washed three times with TBST, each for 5 minutes. The corresponding bands are cut, and primary antibodies are added for incubation at 4\u0026deg;C overnight. The next day, the primary antibodies are aspirated, and the membrane is washed three times with TBST for 10 minutes each. Corresponding Western blot secondary antibodies are then added and incubated at room temperature on a shaker for 1 hour. After aspiration of the secondary antibodies, the membrane is washed three times with TBST for 10 minutes each. Finally, using the chemiluminescence reagent (ShareBio, SB-WB001), the immunoreactive bands were visualized with a ChemiDoc XRS Plus luminescent image analyzer (Bio Rad, England). The image analysis was performed using ImageJ software, and the relative expression levels of the target proteins to β-actin were used for statistical comparison.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Electrophysiology\u003c/h2\u003e \u003cp\u003eCapture the mice to be tested and anesthetize with 0.3% sodium pentobarbital. Shave, disinfect the animal, and securely fixate it in a stereotaxic apparatus, maintaining body temperature with a heating pad. Perform a craniotomy to expose the M1 area of the motor cortex. Insert stimulation electrodes, guided by the stereotaxic device, to a depth of 700\u0026ndash;1000 mm from the brain surface, targeting corticospinal neurons in the sensorimotor cortex. Position recording electrodes to penetrate the contralateral thigh\u0026rsquo;s sciatic nerve distal end to record muscle action potentials induced by electrical stimulation. Amplify and record bioelectrical signals using the BL-420F Biological Function Experiment System. Stimulation parameters are as follows: stimulation type: micro-voltage, mode: single pulse; delay 100 ms; frequency 100 Hz, stimulation intensity: 14V.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 BMS score evaluation, LSS swimming\u003c/h2\u003e \u003cp\u003eThe recovery of hindlimb motor function in SCI (Spinal Cord Injury) mice is evaluated using the Basso Mouse Scale (BMS) scoring system (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Assessments are conducted pre-injury and on days 1, 3, 7, 14, and 28 post-injury. The BMS score ranges from 0 to 9, with 0 indicating complete paralysis and 9 indicating normal hindlimb motor function. Before each scoring session, mice are placed on the testing platform in advance to acclimate to the environment. Each mouse is observed for a duration of 4 minutes.\u003c/p\u003e \u003cp\u003eMice underwent three consecutive days of swimming training in a 5 x 15 cm water tank, moving from one side to the other. Surgery was performed after the completion of training. On postoperative day 28, mice were placed in the same water tank and observed for 30 seconds. The Louisville Swim Scale (LSS) was used to evaluate the swimming abilities of the mice, including assessments of hindlimb movement, hindlimb alternation, forelimb reliance, trunk stability, and body angle (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll experiments were conducted with the mice being randomly assigned by two trained observers blinded to the group allocations. Both observers simultaneously observed and recorded the mice's BMS scores, LSS scores. The final score was determined as the average of the scores given by the two observers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Cell Lateral Migration Assay\u003c/h2\u003e \u003cp\u003eFirstly, use Mark pen to draw lines on the backside of a six-well plate to determine the scratch positions. Seed bEnd.3 cells in the six-well plate, with approximately 200,000 cells per well. Allow the cells to grow and fully cover the surface of the six-well plate, then replace the medium with serum-free culture medium for 6 hours to induce starvation. Use a 200\u0026micro;l pipette tip, held perpendicular to the cell surface, to draw lines according to the positions marked by the marker pen. Subsequently, wash away the detached cells with PBS, followed by replacing the culture medium with fresh serum-free medium. Incubate the cells in a constant-temperature CO2 incubator at 37\u0026deg;C for 12 hours. Use an optical microscope to observe cell migration at 0 and 24 hours at the scratch site and calculate the migration distance using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Tube formation assay\u003c/h2\u003e \u003cp\u003eWhen bEnd.3 cells reach approximately 85% confluence, the serum-containing medium is replaced with serum-free medium to induce cell starvation for 5 hours. Subsequently, after removing the medium and digesting with trypsin, cells are centrifuged at 1000 rpm for 5 minutes. The experimental and control groups of bEnd.3 cells are adjusted to a concentration of 80,000 cells/ml using serum-containing culture medium based on cell counting. Pre-cooled pipette tips are used to evenly coat a 96-well plate with Matrigel matrix gel at 100 \u0026micro;l per well, and the plate is then incubated in a constant-temperature incubator for 30 minutes. Once the matrix gel solidifies, 500 \u0026micro;l of cell suspension is added to each well of a 24-well plate and incubated in a constant-temperature incubator for 6 hours. The formation of capillary-like structures by the cells is observed using an optical microscope, and Image J software is employed for quantitative analysis of the tube formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Bioinformatic analysis of the single-cell transcriptomic dataset\u003c/h2\u003e \u003cp\u003eThe single-cell RNA sequencing data of the contusive mouse spinal cord injury (GSE162610) and the information on the corresponding annotation were retrieved from the GEO database \u003cb\u003e[doi\u003c/b\u003e: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1084/jem.20210040.\u003c/span\u003e\u003cspan address=\"10.1084/jem.20210040.\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cb\u003e].\u003c/b\u003e Data processing and analysis were performed using the R package \"Seurat\" \u003cb\u003e[doi\u003c/b\u003e: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nbt.3192.\u003c/span\u003e\u003cspan address=\"10.1038/nbt.3192.\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cb\u003e].\u003c/b\u003e The genes expressed in less than 10 cells were excluded. The gene expression matrix was normalized and scaled. We selected the top 20 principal components by performing PCA based on 3,000 variable genes. The FindNeighbors and FindClusters functions were used to cluster cells on a shared-nearest-neighbor graph. We visualized the expression level of interested genes using violin plots. The single-nucleus RNA sequencing data of the human adult spinal cord from seven donors (GSE190442) were also obtained and underwent consistent analysis workflow. \u003cb\u003e[doi\u003c/b\u003e: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neuron.2023.01.007.\u003c/span\u003e\u003cspan address=\"10.1016/j.neuron.2023.01.007.\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cb\u003e].\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis of the results was performed using GraphPad Prism (version 7.0, USA). All data were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Normality was determined using the Shapiro-Wilk test. For group number\u0026thinsp;=\u0026thinsp;2, the homogeneity of variances was tested using the F-test. When the data followed a normal distribution and homogeneity of variance, an unpaired t test was conducted for the statistical analysis. For group number\u0026thinsp;\u0026gt;\u0026thinsp;2, the homogeneity of variances was tested using the Brown-Forsythe test. When the data followed a normal distribution and homogeneity of variance, an ordinary one-way ANOVA and Tukey's multiple comparisons test were performed for the statistical analysis. Two-way ANOVA was utilized for data with two variables, grouping and time.\u003c/p\u003e \u003cp\u003eAll differences among and between groups were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. In the figures, ns denotes p\u0026thinsp;\u0026ge;\u0026thinsp;0.05, \u0026lowast; denotes p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u0026lowast;\u0026lowast; denotes p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u0026lowast;\u0026lowast;\u0026lowast; denotes p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and \u0026lowast;\u0026lowast;\u0026lowast;\u0026lowast; denotes p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Enhanced glycolysis metabolism after spinal cord injury\u003c/h2\u003e \u003cp\u003eExisting literature has demonstrated that glycolysis plays a crucial role in the survival and regeneration of neurons in Drosophila (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, its role in mice after SCI remains to be investigated. We obtained in situ spinal cord tissue samples from mice after 7 days post-SCI. Targeted metabolomics sequencing was performed to compare changes in glycolysis-related metabolites before and after SCI. The heatmap revealed significant enhancement of metabolites such as lactate, D-glucose-6-phosphate, β-D-fructose-6-phosphate, and adenosine monophosphate post-injury (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Additionally, KEGG analysis indicated that the metabolites increased after SCI were mainly glycolysis pathway-related metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Concurrently, volcano plots were used to identify the most significantly altered differential metabolites post-injury, revealing a noticeable increase in glycolysis-related metabolites such as lactate, D-glucose-6-phosphate, and β-D-fructose-6-phosphate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Therefore, we identified lactate, a core molecule of glycolysis, as an important functional metabolite post- SCI, providing a direction for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Endothelium-derived lactate feeds nerve cells\u003c/h2\u003e \u003cp\u003eEndothelial cells are critical in regulating metabolic exchange between blood and spinal cord parenchyma, forming a crucial component of the neurovascular unit alongside neurons (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Given their ability to produce and release a significant amount of lactate, and considering their physical proximity to neurons, we investigated the role of ECs lactate in neuron biology. To examine whether neurons utilize lactate derived from ECs, we employed Laconic, a F\u0026ouml;rster Resonance Energy Transfer (FRET)-based quantitative intracellular lactate sensor (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). We infected mouse neuron cells (CATH.a) with a lentivirus expressing Laconic and cultured them in the presence of a glycolysis inhibitor (2-Deoxy-D-glucose, 8 mM) to reduce the production of basal glycolytic lactate. As expected, an increase in lactate concentration from 10 mM to 100 mM led to a concentration-dependent increase in the YFP/CFP fluorescence ratio (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). To investigate whether lactate derived from endothelial cells is taken up by neurons, we co-cultured bEnd.3 and CATH.a in a two-well cell culture dish (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Bend.3 and Laconic-infected CATH.a were seeded in separate wells of the culture dish. Twenty-four hours after cell seeding and confirming cell attached, the culture insert was removed to allow cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). After a further 24-hour examination, we observed a significant increase in the YFP/CFP fluorescence ratio in CATH.a cells that were in direct contact with the ECs, but no increase in CATH.a cells that had no ECs contact (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G). Taken together, these data suggest that lactate from ECs can enter neuron cells, and direct contact between ECs and neurons promotes this process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Deficiency of MCT1 in ECs after SCI\u003c/h2\u003e \u003cp\u003eElevated lactate levels in the epicenter of injury mice following SCI may be associated with lactate transport mechanisms deficiency due to ECs damage. Therefore, we investigated the altered transport proteins in spinal cord vascular endothelial cells post- SCI. Lactate transport is primarily mediated by monocarboxylate transport proteins (MCTs, also known as Slc16a family) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Through analysis of existing databases from human adult spinal cord (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), we found that Monocarboxylate Transporter 1,4,5,7,8 (SLC16a1, SLC16a2, SLC16a4) is highly expressed in adult ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), and MCT1 is predominantly expressed in ECs within the spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), we have focused on the role of MCT1 in ECs-to-Neuron lactate shuttling. Through analysis of single-cell data from mice with spinal cord injury (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), we found that MCT1 expression is significantly reduced post-SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Literature indicates that MCT1, a transmembrane transporter monocarboxylate, plays a crucial role for transmembrane transport of lactate, pyruvate, and ketone bodies (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), which is an essential protein for maintaining lactate homeostasis in the spinal cord. Additionally, we revealed the spatiotemporal changes of MCT1 post-SCI using immunofluorescence. Additionally, immunofluorescence confirmed angiogenesis at the epicenter of injury 7 days post- SCI, with the newly formed blood vessels appearing dilated and malformed at 14 and 28 days post- SCI, suggesting partial loss of function of these vessels after SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Furthermore, our study revealed a decrease in MCT1 expression in the blood vessels formed via angiogenesis at the injury center of the spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Confocal microscopy observations in SCI mice also confirmed the lack of MCT1 expression in the ECs at the injury center (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-H). These findings indicate that endothelial cell-specific expression of MCT1. Newly formed blood vessels display morphological abnormalities, accompanied by decreased MCT1 expression after SCI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Deficiency of MCT1 in ECs inhibits angiogenesis and glycolysis production\u003c/h2\u003e \u003cp\u003eTo investigate the impact of MCT1 on the biological functions of ECs, we conducted tube formation experiments and transverse migration experiments (scratch assays). Compared to the Control group, after lactate treatment, there were no significant changes in the number and branching length of endothelial cell tubes. However, downregulation of MCT1 expression by adding the MCT1-specific inhibitor α-cyano-4-hydroxycinnamic acid (α-CHCA) resulted in a reduction in the number and branching length of cell tubes. Additionally, the addition of lactate did not significantly improve tube formation in ECs after MCT1 downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). In comparison to the Control group, lateral migration of endothelial cells treated with lactate showed no significant change, while inhibiting MCT1 expression significantly reduced the lateral migration distance of ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Simultaneously, we verified the expression of key enzymes related to glycolysis, Hexokinase 1 (HK1), Phosphofructokinase 1 (PFKM), and Pyruvate kinase 1 (PKM1), after lactate treatment. WB experiments indicated increased expression of glycolytic enzymes in ECs after lactate treatment. However, the addition of α-CHCA resulted in a decrease in the expression of glycolytic enzymes in endothelial cells, and the simultaneous addition of lactate and α-CHCA did not improve the inhibited glycolysis situation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). These results suggest that MCT1 is necessary for lactate transport through ECs. Reduction of MCT1 significantly inhibits tube formation and migration ability of ECs. Moreover, impaired lactate transport also leads to a reduction in glycolysis levels in ECs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Neurons Utilize Lactate to Enhance Glycolysis and Promote Axonal Regeneration\u003c/h2\u003e \u003cp\u003eIn order to ascertain that neurons can enhance glycolysis by utilizing lactate delivered by ECs, we co-cultured bEnd.3 and CATH.a cells infected with Laconic lentivirus in Ibidi chambers. Lactate or α-CHCA was added to the bEnd.3 side chamber, and after a 24-hour treatment, the media was exchanged before allowing extensive contact between the two cell types. Experimental results revealed that CATH.a cells in contact with bEnd.3 exhibited an increased CFP/YFP ratio upon lactate treatment, as demonstrated by laser confocal microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Conversely, α-CHCA treatment led to a decrease in the CFP/YFP ratio, indicating reduced lactate entry from bEnd.3 into CATH.a. Subsequently, we isolated the cytoplasm and axons of CATH.a cells, simulating axon fragmentation after spinal cord injury by severing distal axons. Experimental findings indicated that lactate treatment of CATH.a cells promoted axon regeneration. However, axon regeneration was inhibited when the glycolysis inhibitor 2-Deoxy-D-glucose (2-DG) was used. Simultaneous treatment with lactate and 2-DG restored the inhibitory capacity of axon regeneration. Additionally, WB results demonstrated enhanced glycolytic capability in CATH.a cells after lactate treatment, while 2-DG treatment suppressed glycolytic capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). These findings suggest that ECs can transport lactate to neurons, and neurons can enhance their regenerative capacity by utilizing lactate to increase glycolysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Spinal Cord ECs overexpression of MCT1 Rescues Impaired SCI Mice Neurogenesis\u003c/h2\u003e \u003cp\u003eTo validate the association between decreased MCT1 in ECs and axon regeneration in SCI mice, we induced overexpression of MCT1 in spinal cord ECs using a spinal cord-specific AAV-MCT1. After successful injection of AAV-MCT1, the expression of MCT1 in the ECs at the injury epicenter of SCI mice was restored. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). We also confirmed the specific expression of AAV-MCT1, as immunofluorescence experiments demonstrated its absence in astrocytes, neurons, and macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). We then assessed whether the restored MCT1 could improve axonal regeneration in SCI mice. AAV-MCT1 injection improved the neurite regrowth besides the lesion core and increased the amount of TUJ positive signal in spinal cord sections compared to the Control groups. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, H). These results provide direct in vivo evidence demonstrating that spinal cord endothelial cell MCT1 can modulate lactate shuttle and axonal regeneration in SCI mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Spinal Cord Vasculature Overexpression of MCT1 Rescues Impaired SCI Mice Functional Recovery\u003c/h2\u003e \u003cp\u003eFollowing AAV-MCT1 injection, electrophysiological tests revealed a significant increase in the amplitude of SCI mouse hind limb motor-evoked potentials (MEP) at 28 days post-injection (dpi) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). Basso Mouse Scale (BMS) scores assessing hind limb motor function indicated improved recovery in AAV-MCT1-treated SCI mice starting from 7 dpi. By the end of 28 dpi, the hind limb motor function in treated SCI mice showed a marked improvement (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Swimming tests demonstrated that mice in the AAV-MCT1 group exhibited more stable trunk movements, fewer tilted body angles, and less drooping tails, resulting in a significantly elevated LSS swimming score (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, E). Moreover, analysis of the H\u0026amp;E-stained slices of the SCI mouse bladder indicated a significant increase in the thickness of the detrusor muscle layer in the neurogenic bladder following treatment with AAV-MCT1, implying the restoration of nerve innervation in the bladder (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, G). Overall, our research findings confirm a positive therapeutic effect of restoring MCT1 expression in spinal cord ECs on functional recovery in SCI mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe results of this study demonstrate that endothelium-derived lactate can be uptaken and utilized by neurons for energy generation and axonal regeneration, which is crucial for maintaining normal neuron function and axon regeneration. After SCI, there is abnormal lactate metabolism at the injury epicenter, where ECs destruction leads to obstruction of lactate shuttle. Additionally, we identified MCT1 as the endothelial cell-specific lactate transporter. By upregulating the expression of MCT1 in ECs, we restored lactate transport function, enhanced neuronal glycolysis, promoting axon regeneration and neurological function recovery after SCI. It highlights that MCT1 serves as a novel therapeutic target for SCI, providing a new direction after SCI treatment from a metabolic perspective.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that in traumatic SCI, the primary injury damages cells and triggers complex secondary injury cascades (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Sustained microvascular thrombosis and spasms at the injury epicenter cause further neurons death. Our study, from a metabolic perspective, investigates whether the loss of microvascular function after spinal cord injury leads to metabolic changes in the epicenter of injury. Energy metabolomic analysis of mice after SCI reveals an elevation in glycolysis-related metabolites, with lactate being a significantly increased metabolite post-injury. Existing literature suggests that endothelial cells can maintain central nervous system energy metabolism by transporting lactate, thereby regulating neuronal functional stability (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Meanwhile, enhancing glycolysis in glial cells can promote axon regeneration at the epicenter of injury (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), and there is also literature suggesting that increasing the neuronal intrinsic glycolytic capacity can inhibit neuronal apoptosis (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Therefore, focusing on metabolic changes at the site of injury after SCI may provide new research directions for studying neurological function recovery post-SCI.\u003c/p\u003e \u003cp\u003eWe and others have previously shown that restoring cellular energy metabolism after SCI can promote axon regeneration and functional recovery (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Lactate had been widely accepted as a metabolic waste product; however, recent studies have shown that lactate is a crucial fuel in energy metabolism (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In the central nerve system, lactate serves as a vital source of energy, although its contribution to the tricarboxylic acid (TCA) cycle compared to glucose remains a contentious issue (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The roles of lactate in delivery of oxidative and gluconeogenic substrates as well as in cell signaling is termed the lactate shuttle (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Research on lactate shuttle initially focused on describing and analyzing the whole organ lactate shuttle mechanisms (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). However, cellular lactate shuttle pathways have garnered increasing attention in recent years. Two recent studies have indicated that pericytes and macrophages can fulfill their energy requirements by receiving lactate from endothelial cells. Endothelial-derived lactate is absorbed by pericytes to maintain blood-brain barrier stability (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), while lactate produced by endothelial cells induces macrophage polarization to promote muscle regeneration following ischemic injury (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).Restoring cellular glycolysis is a crucial pathway for promoting axonal regeneration and functional recovery after spinal cord injury (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Additionally, Furthermore, endothelial cells can also utilize lactate as a driving force, along with high rates of glycolysis, to promote angiogenesis (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Our study aims to elucidate the biological functions of lactate. We found that endothelial cells can enhance their glycolytic capacity and tube-forming migration ability by utilizing exogenous lactate. Additionally, we discovered that neurons rely on endothelium-derived lactate to provide energy. This is crucial for studying lactate shuttle and functional recovery of neurovascular after SCI, as a lack of endothelium-derived lactate can lead to impediments in axon regeneration and diminished neurological recovery.\u003c/p\u003e \u003cp\u003eEndothelium-derived lactate is present at high concentrations locally, it becomes an easily accessible fuel source around blood vessels (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Neurons and ECs are typically in direct contact throughout the entire neurovascular network, endothelium‐derived lactate may be the main source of energy supply for neurons. Although neurons are closely associated with ECs, lactate cannot be transferred directly through physical contact; there are lactate-related transport proteins mediating lactate shuttle between cells (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Our data suggest that MCT1, a lactate transporter protein, is specifically expressed in endothelial cells within the spinal cord, playing a crucial role in the extracellular secretion of lactate by these cells. Neurons uptake lactate into the cell for energy utilization through their specific transport proteins. While we demonstrate the role of lactate in endothelial cells through MCT1-mediated extracellular lactate secretion, the specific MCT members responsible for lactate uptake in neurons, their efficiency in lactate uptake, and their specific functions in vivo still require further investigation.\u003c/p\u003e \u003cp\u003eAfter SCI, insufficient axon regeneration often leads to poor recovery, representing one of the most pressing challenges in SCI treatment. Developing successful regenerative strategies to reconnect axons within the central nervous system is paramount for spinal cord injury research. It is widely believed that inadequate intrinsic neuronal growth capacity, a deficient growth environment, and the absence of neurotrophic factors are the main reasons for hindered axonal regeneration (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Currently, while there is considerable research on axonal regeneration in adults (\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), the translation of these findings into clinically applicable therapeutic methods is severely limited. Metabolic reprogramming emerges as a novel strategy to stimulate axonal regeneration in the central nervous system (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Previous studies have shown that lactate serves as an energy source for neurons (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), and local application of lactate to the injured spinal cord can promote corticospinal tract axonal regeneration (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).Our research further reveals an unexpected connection between endothelial cell-neuron lactate shuttling after SCI and neural functional recovery, offering a novel metabolic target for spinal cord injury treatment. We found that impaired lactate shuttling due to the lack of MCT1 in endothelial cells at the epicenter of injury impedes neuronal energy metabolism, resulting in weakened axonal regeneration capacity. Restoring MCT1 expression in endothelial cells at the epicenter of injury significantly promotes axon regeneration and functional recovery. While we did not specifically study whether other cells transport lactate to neurons after SCI, besides endothelial cells, other cells may also contribute. For instance, it has been reported that astrocytes secrete lactate to fuel neuronal mitochondria in traumatic brain injury (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Interestingly, lactate not only serves as a metabolic substrate but also functions as a signaling molecule to modulate neuronal function (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Whether this also promotes axonal regeneration warrants further investigation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, our study demonstrates that lactate produced by endothelial cells is utilized by adjacent neurons for energy metabolism and axonal regeneration. The impaired lactate metabolism observed in mice following spinal cord injury is attributed to the lack of MCT1 in endothelial cells at the epicenter of injury. Restoring MCT1 expression at the lesion site in adult mammals promotes significant axonal regeneration and functional recovery\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll research protocols were approved by the Animal Ethics Committee of Central South University. Animal care and use during our experiment were conducted under the guidelines of the Administration Committee of Affairs Concerning Experimental Animals in Hunan Province, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest regarding the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Key Program of the National Natural Science Foundation of China (No. 82030071), the National Natural Science Foundation of China (No. 82202722, No. 81874004), the Science and Technology Major Project of Changsha (NO. kh2103008), the Science Foundation of Xiangya Hospital for Young Scholar (Grant No. 2021q18), the Natural Science Foundation of Changsha city (Grant Nos. kq2202378), and Graduate students of Central South University independently explore innovative projects (2022ZZTS094).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiyuan Jiang, Jianzhong Hu: Conceptualized and designed the project. Chaoran Shi, Jiaqi Xu: Performed major experiments and analyzed data. Yinghe Ding: Performed bioinformatic analysis and data analysis. Feifei Yuan, Fengzhang Zhu: Performed animal experiments and data validation. Hongbin Lu, Chunyue Duan, Tianding Wu Provided consultation, review \u0026amp; editing, and supervision. All authors had approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Hui Xie and other staff from the Movement System Injury and Repair Research Center, Xiangya Hospital, Central South University, Changsha, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGlobal regional. national burden of spinal cord injury, 1990\u0026ndash;2019: a systematic analysis for the Global Burden of Disease Study 2019. 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Eur J Neurosci. 2017;46(5):2096\u0026ndash;107.\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":"Spinal cord injury, endothelial cell, lactate shuttle, neuron metabolism, axon regeneration","lastPublishedDoi":"10.21203/rs.3.rs-4079758/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4079758/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVascular damage following spinal cord injury (SCI) precipitates ischemia and hypoxia at the injury site, leading to profound metabolic disturbances. The implications of these metabolic disorders on neural tissue remodeling and functional recovery remain poorly understood. Our study elucidates the consequences of the hypoxic environment induced by SCI, which significantly enhances glycolysis and lactate production at the injury's epicenter. And our findings revealed a marked decrease in the expression of Monocarboxylate Transporter 1 (MCT1), a crucial transporter facilitating lactate delivery to neurons and consequently supporting their energy metabolism, within vascular endothelial cells emerging after SCI. This decrease disrupts lactate transport to neurons, resulting in metabolic imbalances that impede axonal regeneration. Remarkably, our research demonstrates that targeted delivery of adeno-associated virus (AAV) injections to restore MCT1 expression in endothelial cells (ECs) promotes axonal regeneration and functional recovery in SCI mouse models. These findings reveal a previously unrecognized connection between lactate shuttling from ECs to neurons after SCI and neural functional recovery. Highlighting a novel metabolic pathway for intervention, our study opens new therapeutic avenues for the treatment of spinal cord injuries, suggesting that targeting lactate transport mechanisms may offer significant benefits in SCI recovery.\u003c/p\u003e","manuscriptTitle":"MCT1-Mediated Endothelial Cell Lactate Shuttle as a Target for Promoting Axon Regeneration after Spinal Cord Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-15 13:20:58","doi":"10.21203/rs.3.rs-4079758/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":"07adc81f-62c7-44f8-aa76-a49cbd1bc266","owner":[],"postedDate":"March 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-17T20:59:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-15 13:20:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4079758","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4079758","identity":"rs-4079758","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}