Unraveling neurotoxicity discrepancies: comparative in vitro and In vivo analysis of colistin and polymyxin B and the underlying mechanisms

Unraveling neurotoxicity discrepancies: comparative in vitro and In vivo analysis of colistin and polymyxin B and the underlying mechanisms | 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 Unraveling neurotoxicity discrepancies: comparative in vitro and In vivo analysis of colistin and polymyxin B and the underlying mechanisms Rui Yang, Debiao Xiang, Fang Yuan, Yuan Yang, Pengkai Wang, Bing Xu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3322528/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2024 Read the published version in Molecular Neurobiology → Version 1 posted 5 You are reading this latest preprint version Abstract Background : Polymyxins, including colistin and polymyxin B, are a final resort against Gram-negative bacterial infections. However, its clinical application is restricted due to concerns related to neurotoxicity. Despite the similar antibacterial spectrum and mode of action shared between colistin and polymyxin B, there is still a lack of definitive evidence to support the idea that their neurotoxicity profiles are identical. Purpose : To comprehensively compare the neurotoxicity between colistin and polymyxin B both in vivo and in vitro and establish a theoretical foundation to guide the rational use of polymyxins within clinical settings. Methods : In vitro experiments simulated nerve damage by exposing N2a and RSC96 cells to colistin and polymyxin B. The evaluation of nerve injury included assessments of cell viability and apoptosis. To discern the variance in the mechanisms of nerve injury between colistin and polymyxin B, oxidative stress levels were examined, such as SOD, CAT, GSH, and MDA. In in vivo experiments, a rat nerve injury model was created through intraventricular injections of colistin and polymyxin B, respectively. The impact of these drugs on brain injury in rats, particularly within the hippocampus and medulla oblongata, was measured using HE and Nissl staining. The potential influence of polymyxins on the ferroptosis pathway was evaluated by assessing the levels of LPO and Fe 2+ and the degree of mitochondrial impairment. Results : At equivalent doses, colistin demonstrated a reduced level of neurotoxicity compared to polymyxin B, both in vitro and in vivo . In vitro experiments revealed greater cell viability and a lower apoptosis rate after colistin treatment than after polymyxin B treatment. This variance in outcomes could be attributed to the comparatively lower levels of oxidative stress associated with colistin administration . In a rat model, nerve injury resulted in observable damage to both the hippocampus and the medulla oblongata. A comprehensive assessment of the extent of damage in the CA1 to CA4 regions of the hippocampus, the nucleus of the solitary tract, and the hypoglossal nucleus of the medulla oblongata underscored that the neurotoxic effects of colistin remained milder compared to those elicited by polymyxin B. Even when evaluated at equivalent multiples of clinically recommended doses, colistin exhibited lower neurotoxicity in vivo than polymyxin B. For the first time, this study demonstrated the role of ferroptosis in polymyxin B-induced nerve damage. The activation levels observed within the ferroptosis pathway due to polymyxin B exceeded those triggered by colistin. Conclusions : Colistin exhibited a marked reduction in neurotoxicity compared to polymyxin B, evident in both the equivalent and clinically recommended doses. These findings suggest that, from the perspective of neurotoxicity, colistin presents a more favorable option for clinical use. Polymyxin B Colistin Neurotoxicity Comparison Ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 INTRODUCTION With the emergence of multidrug-resistant (MDR) bacterial infections and the slow pace of new antibiotic development, polymyxins have emerged as the "last line of defense" against Gram-negative MDR bacteria [ 1 ]. Colistin and polymyxin B are the primary representatives of polymyxins used in clinical settings [ 2 ]. Although colistin and polymyxin B share a quasi-identical structure, except d-phenylalanine being substituted by d-leucine [ 3 ], this subtle alteration in structure leads to a spectrum of divergent pharmacokinetic (PK) attributes and toxic reactions [ 3 ]. Clinical investigations and in vitro analyses have underscored the relative reduction in nephrotoxicity of polymyxin B compared to colistin [ 4 , 5 ]. Consequently, polymyxin B gains preference in patients with compromised renal function. Beyond nephrotoxicity, neurotoxicity is critical, mainly when these drugs are administered intraventricularly or intrathecally for central nervous system (CNS) infections. A previous study involving such administration reported a 28% incidence of neurotoxicity for polymyxins [ 6 ]. Current approaches to mitigate polymyxin-induced neurotoxicity often involve dose reduction [ 7 , 8 ], but this strategy could compromise therapeutic efficacy [ 9 ]. Although specific studies have identified neuroprotective agents capable of improving polymyxin neurotoxicity, many of these investigations are confined to cellular or animal experiments, distant from clinical implementation [ 10 – 13 ]. Patients with CNS infections often contend with concurrent CNS damage [ 14 ]. Thus, the inclination leans toward using polymyxins with less neurotoxicity. However, whether there are substantial neurotoxicity discrepancies among various polymyxins remains uncharted territory. Previous research has suggested an association between polymyxin neurotoxicity and the initiation of apoptosis through oxidative stress [ 15 ]. Reactive oxygen species (ROS), generated during normal cellular metabolism, are typically counteracted by intracellular antioxidant proteins such as Superoxide Dismutase (SOD), catalase (CAT), and glutathione (GSH) [ 16 ]. Polymyxins, however, can induce an excessive ROS surge that exceeds the neutralization capacity of the antioxidant protein, thus inflicting oxidative stress-induced damage [ 15 ]. This elevated ROS production may also trigger mitochondrial dysfunction [ 17 ]. Experiments in mice administered colistin (15 mg/kg/day intravenous injection for 7 consecutive days) showed significant pathological alterations in the ultrastructure of brain mitochondria, including swollen or degenerated cristae and even complete loss of cristae. Furthermore, alterations in mitochondrial membrane permeability, membrane potential, and succinic dehydrogenase activity were observed. Oxidative stress-induced factors accelerate cytochrome C (Cyt c) release from damaged mitochondria, activating a cascade of caspases, a group of cysteine proteases. In our previous investigations, both colistin and polymyxin B were found to increase cyt c release while simultaneously inhibiting the NRF2 antioxidant pathway [ 11 ]. Existing evidence implies a similarity in the neurotoxic mechanisms of colistin and polymyxin B [ 15 , 18 ]. Although previous studies have revealed substantial distinctions in the impact of polymyxin B and colistin on the viability of renal cells HEK293 and NRK-52E [ 5 ], the degree of variation in the toxicity of the two drugs on nerve cells remains unexplored. The primary objective of this study was to perform a comparative analysis of the neurotoxicity profiles of colistin and polymyxin B, employing both in vitro and in vivo approaches. Additionally, the study aimed to unravel the underlying mechanisms responsible for these observed differences. In the in vivo phase, a rat nerve injury model was established by localized brain injections. The divergence in neurotoxic effects between colistin and polymyxin B was evaluated by pathological slices. Meanwhile, the in vitro experiment investigated how colistin and polymyxin B affect the apoptosis and ferroptosis pathways within N2a and RSC96 cells. 2 MATERIALS AND METHODS 2.1 Materials For in vitro experiments, colistin and polymyxin B were obtained from the National Institutes for Food and Drug Control in Beijing, China. In the in vivo phase, colistin injections were supplied by Shanghai Xinya Pharmaceutical Co., Ltd. (Shanghai, China). Polymyxin B injections were obtained from Shanghai Shangyao's First Biochemical Pharmaceutical Co., Ltd. (Shanghai, China). N2a cells, RSC96 cells, MEM medium, DMEM medium, and fetal bovine serum were acquired from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The CCK-8 reagent was obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China), and the ROS assay kit was sourced from Beyotime Biotechnology (Shanghai, China). The antibodies were purchased from Abcam (Cambridge, UK). 2.2 Cell culture N2a cells and RSC96 cells were cultured in MEM medium and DMEM medium, respectively, which supplemented with 10% (v/v) FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin (Hyclone, Los Angeles, America), at 37℃ in a 5% CO 2 atmosphere. 2.3 Animals Female Sprague-Dawley (SD) rats, with body weights of 220 g to 250 g, were chosen as subjects for this study. The rats underwent an initial three-day adaptation period, after which they were randomly assigned to different groups (n = 6). Individual rats were housed in separate cages and had unrestricted access to food and water. They were allowed to adjust to their environment, adjusting to factors such as temperature, humidity, light cycles, and ventilation conditions. All animal experiments were examined and approved by the Ethics Committee of the Hunan Experimental Animal Center (Hunan Drug Safety Evaluation Research Center), approval number: IACUC-157023. 2.4 Cell viability assay To elucidate the neurotoxic effects of polymyxin B and colistin on N2a and RSC96 cells, the appropriate experimental concentrations of polymyxin B and colistin in N2a and RSC96 cells were determined. Cell viability was detected by a CCK-8 detection kit. N2a and RSC96 cells were inoculated in 96-well plates (5×10 3 cells/well) and treated with polymyxin B (50, 100, 200, 400, 500, 600, 800, 1000, 1200 µM) and colistin (50, 100, 400, 500, 600, 800, 1000, 1200 µM) for 24 h. After exposure, the medium was removed and replaced with a medium containing 10% CCK-8. Incubation was performed for 2 h, after which the absorbance was measured at 405 nm. 2.5 Cell apoptosis The annexin V-FITC apoptosis detection kit (Beyotime, Shanghai, China) was used to detect the apoptosis rate. N2a and RSC96 cells were seeded in 100 mm cell culture dishes (5×10 6 cells/well). N2a cells were treated with colistin or polymyxin B (150, 300, 600 µM), while RSC96 cells were treated with colistin or polymyxin B (300, 600, 900 µM) at 37°C for 24 h. After treatment, N2a and RSC96 cells were harvested with 0.25% trypsin, washed twice with PBS, and resuspended in 195 µL Annexin V-FITC binding buffer. The resuspended cells were incubated with 5 µL Annexin V-FITC and 10 µL propidium iodide in the dark for 20 min. Analysis was performed using a DxFLEX flow cytometer (Beckman Coulter, Los Angeles, US). 2.6 Assessment of cellular ROS levels The Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China) was used to detect the ROS level in cells. First, N2a and RSC96 cells were seeded in 100 mm cell culture dishes (5×10 6 cells/well). N2a cells were then treated with colistin (150, 300, 600 µM) and polymyxin B (150, 300, 600 µM) at 37 o C for 24 h, and RSC96 cells were treated, respectively, with colistin (300, 600, 900 µM) and polymyxin B (300, 600, 900 µM) at 37 o C for 24 h. After treatment, cells were treated with 0.25% trypsin, washed with PBS 3 times, then treated with DCFH-DA basic culture medium containing 10 µM for 20 min at 37 o C, and finally analyzed by DxFLEX flow cytometry (Beckman Coulter in Los Angeles, US). 2.7 Evaluation of oxidative stress levels An oxidative stress test kit (Nanjing Jiancheng, Nanjing, China) was used to detect SOD, CAT, GSH, and MDA activity levels. N2a and RSC96 cells were seeded in 100 mm cell culture plates and treated according to the method recommended by the manufacturer. After treatment, N2a and RSC96 cells were collected with a scraper in a centrifuge tube and centrifuged at 800 g for 5 min. After the supernatant was removed, PBS resuscitated the cells. The cells were then ultrasonicated at 250 W for 10 min on ice, after which the cell lysates were centrifuged at 20,000 g for 10 min at 4°C. The supernatant was collected and assayed for SOD, CAT, GSH, and MDA activities. Protein concentrations were quantified using the BCA protein assay kit (Boster, Wuhan, China). 2.8 Western blot analysis Cells were collected with a scraper, then transferred to a centrifuge tube and centrifuged at 800 g for 5 min at 20℃. After discarding the supernatant, PBS was added to resuspend the cells and centrifuged at 800 g for 5 min at 20℃. The supernatant was discarded, and the appropriate amount of PBS was added. Cells were broken in ice using an ultrasonic cell grinder (Scientz Biotechnology, Ningbo, China) at 300 W power for 10 min. The sample was centrifuged, and the supernatant was taken for Western blotting analysis. 10% sodium dodecyl sulfate-PAGE gel was used for electrophoresis, and the protein was transferred to the PVDF membrane (Applied Technology Company, Beijing, China). The membrane was blocked with 5% (w/w) skimmed milk powder in TBST for 2 h, washed with TBST 3 times, and incubated overnight with rabbit polyclonal antibody at 4℃. 2.9 Assessment of cellular lipid peroxide levels The lipid peroxide (LPO) level was detected using an LPO detection kit (Nanjing Jiancheng, Nanjing, China). N2a and RSC96 cells were inoculated in 100 mm cell culture dishes and treated according to the method recommended by the manufacturer. After treatment, N2a and RSC96 cells were collected with a scraper and centrifuged at 800 g for 5 min. After the supernatant was removed, cells were resuspended with PBS. Subsequently, a 250 W ultrasound was applied for 10 min. The resulting cell suspension was then utilized to assess the LPO level. A BCA protein detection kit (Boster, Wuhan, China) was used to quantify the protein concentration. 2.11 Evaluation of cellular iron concentration The concentration of ferrous ions was detected by a cell ferrous colorimetric assay kit (Elabscience Biotechnology, Wuhan, China). First, drug-treated cells were collected with a cell scraper, and the number of cells in each group was obtained using a cell counter. The lysate contained in the kit was used to lyse the cell for 10 min on ice and then centrifuged at 15,000 g for 10 min. The concentration of ferrous ions in the supernatant was detected at 593 nm. 2.12 Mitochondrial morphology The structure of mitochondria was evaluated using the Mito Tracker Red CMXRos fluorescence indicator (Beyotime, Shanghai, China). Cells were inoculated in 12-well plates (2×10 5 cells/well), and cell slides were placed in each well. Cells were treated with different concentrations of polymyxin B and colistin at 37℃ for 6 h. Cells were incubated in MEM containing 100 nM Mito Tracker Red CMXRos for 30 min. The slides were cleaned with PBS, supplemented with an anti-fading mounting medium, covered, and observed through a laser scanning confocal microscope (FV3000, Olympus, Tokyo, Japan). 2.13 Evaluation of the mitochondrial membrane potential Δψm was detected with the fluorescent indicator JC-1 (Beyotime, Shanghai, China). Cells were treated with different concentrations of polymyxin B and colistin at 37°C for 24 h and then incubated in MEM containing 10 µM JC-1 at 37°C for 30 min. The cells were then washed with PBS. An anti-Fade patch containing DAPI was added to the cell slide, covered, and observed with a laser scanning confocal microscope (FV3000, Olympus, Tokyo, Japan). The fluorescence change from red to green indicates the loss of memory. 2.14 Intraventricular injection of polymyxin B and colistin in rats After a three-day adaptive feeding, the rats were randomly divided into control, sham, and administration groups. To prevent infections, a single intraperitoneal injection of ceftriaxone at 210 mg/kg was administered to rats 30 min before the surgical procedure. After continuous anesthesia induction using isoflurane, rats were fixed on a stereotactic instrument (KOPF, Florida, US). An incision was made longitudinally on the scalp, approximately 15 mm long. The periosteum was scraped, completely exposing the anterior fontanelle. The precise positioning involved moving the positioning needle 0.8 mm posteriorly and 1.5 mm laterally from the origin of the anterior fontanelle, with a penetration depth of 3.5 mm. Subsequently, a hole was drilled in the skull, and a guide tube was inserted, which was then affixed to the skull using a small amount of dental cement. After this surgical procedure, the rats were given another three days to adjust to their surroundings. Following this period, actual drug administration was conducted. Medical solutions, 20 µL, were administered to the lateral ventricle using a microinjection pump at a controlled rate of 4 µL/min. Administration doses were translated into equivalent doses for adults: the low dose (750 U/day of colistin and 300 U/day of polymyxin B) was determined based on the adult dose, the medium dose (1,875 U/day of colistin and 750 U/day of polymyxin B) increased to 2.5 times the adult dose, and the high dose (4,687.5 U/day of colistin and 1,875 U/day of polymyxin B) increased to 6.25 times the adult dose. Dosage was administered once daily over five days. After the administration on day 5, the rats were euthanized with an overdose of anesthetics. As for the sham group, an equivalent volume of vehicle solution was administered in parallel. 2.15 Statistical analysis Data are presented as mean ± standard deviation for a sample size. Analysis was conducted using GraphPad Prism software (GraphPad Software, USA), and mean values were compared using one-way analysis of variance (ANOVA). A significance threshold of P ≤ 0.05 indicates statistical significance, while P ≤ 0.01 is considered highly significant. 3 RESULTS 3.1 Effects of colistin and polymyxin B on N2a and RSC96 cell viability N2a and RSC96 cells were subjected to drug treatment for 24 h. As drug doses escalated, the viability of both cell types exhibited a dose-dependent decrease (Fig. 1 ). The IC50 values for colistin and polymyxin B in N2a cells were determined to be 183 µM and 127 µM, respectively. In RSC96 cells, the IC50 values for colistin and polymyxin B were 810 µM and 579 µM, respectively (Table 1 ). Polymyxin B demonstrated a greater degree of neurotoxicity compared to colistin. Table 1 In vitro toxicity of colistin and polymyxin B in different cells IC 50 (µM) 1 Cells Colistin Polymyxin B N2a 183.17 ± 17.10 127.40 ± 18.30 RSC96 810.13 ± 15.45 579.03 ± 42.52 1 Data shown are means ± SDs. IC50 (drug concentration resulting in 50% of maximal reduction in cell viability) Figure 2. Apoptosis induced by colistin and polymyxin B. (A) Western blot staining of Cyt c, Caspase-3, and Caspase-9.Data are presented as mean ± SD for n = 3; (B) The apoptosis rate were analyzed by flow cytometry. Data are presented as mean ± SD for n = 3. 3.2 Difference in colistin and polymyxin B-induced apoptosis in vitro The apoptosis rate detected by flow cytometry and the expression level of apoptotic proteins (Cytc, Caspase-3, Caspase-9) detected by Western blot were used to compare the ability of polymyxin B and colistin-induced neuronal apoptosis. Cytc and its downstream Caspase-3 and Caspase-9 can promote apoptosis. In both N2a and RSC96 cells, Cytc, Caspase-3, and Caspase-9 expression levels in the polymyxin B group were higher than those of colistin at the same dose (150 µM, 300 µM, 600 µM, 900 µM) (P < 0.05; P < 0.01; P < 0.0001; Fig. 1 A). Polymyxin B induced a higher apoptosis rate in N2a and RSC96 cells at the same dose (P < 0.005; P < 0.0001; Fig. 1 B). Furthermore, flow cytometry showed that almost all apoptotic cells were in the late stage of apoptosis. Figure 3. Oxidative stress was induced by colistin and polymyxin B. (A) SOD, GSH, CAT and MDA levels were measured by ELISA; Data are presented as mean ± SD for n = 3; (B)The ROS level were analyzed by flow cytometry. Data are presented as mean ± SD for n = 3. 3.3 Oxidative stress induced by colistin and polymyxin B in N2a and RSC96 cells At the same dose, N2a and RSC96 cells were induced by colistin and polymyxin B, and changes in SOD, CAT, GSH, MDA, and ROS were detected to assess the toxicity of the two drugs on nerve cells. In both N2a and RSC96 cells, upon exposure to identical doses of the two drugs (150 µM, 300 µM, 600 µM, 900 µM), the levels of GSH, SOD, and CAT were consistently lower in the polymyxin B group compared to the colistin group. The MDA content in the polymyxin B group was higher than in the colistin group (P < 0.05; P < 0.01; P < 0.005; P < 0.0001; Fig. 3A ). Furthermore, flow cytometry showed that polymyxin B could induce a higher level of ROS than Colistin at the same dose (P < 0.0001; Fig. 3B ). The morphology of mitochondria in the control group was linear under a confocal laser microscope ( Fig. 4A ). With the aggravation of injury, the morphology of the mitochondria changed from linear to punctate. Almost all mitochondria were punctate after cells were treated with high doses of colistin and polymyxin B ( Fig. 4A ). The mitochondrial membrane potential of the polymyxin B group was lower than that of the colistin group at the same dose(P < 0.01; P < 0.0001; Fig. 4B ). Figure 4. Mitochondrial dysfunction was induced by colistin and polymyxin B. (A) Mitochondrial morphologies were assessed by confocal microscopy imaging after staining of cells with the fluorescent indicator Mito-Tracker; (B) The mitochondrial membrane potential (MMP) of cells. The mitochondrial membrane potential (MMP) was measured by using the JC-1 dye. The JC-1 aggregate form, indicating the normal MMP function, is presented in red. The JC-1 monomeric form, indicating disrupted MMP, is shown in green. Data are presented as mean ± SD for n = 3. 3.4 Difference of neurotoxicity between colistin and polymyxin B in vivo The extent of nerve injury resulting from varying doses was evaluated using HE and Nissl staining. Upon HE staining of brain tissue, neurons within the hippocampal CA1-CA4 regions of both the control and the sham groups showed organized arrangement, regular morphology, and minimal intercellular gaps ( Fig. 5 ). At administration, no substantial damage was observed when the recommended clinical dose of colistin was used ( Fig. 5 ). Minor damage was apparent in the CA1 and CA4 areas, while significant damage was evident in the CA2 and CA3 areas after administering colistin 2.5 times the clinical dose ( Fig. 5 ). Elevating the dose to 6.25 times the clinical standard resulted in severe damage in all CA1-CA4 areas, characterized by an almost complete absence of neurons ( Fig. 5 ). Regarding polymyxin B, minor damage was observed in the CA1 and CA4 areas, and substantial damage was observed in the CA2 and CA3 regions at the recommended clinical dose ( Fig. 5 ). When polymyxin B was administered 2.5 times the clinical dose, except for CA4, CA1-CA3 areas exhibited an almost complete neuronal absence ( Fig. 5 ). The impact of polymyxins on the administered area was so severe that a significant number of cells were not adequately preserved by the fixative and consequently lost when administered 6.25 times the clinical dose ( Fig. 5 ). In the contralateral hippocampus, the CA1-CA4 region of the colistin group did not show apparent lesions. However, after treatment with 1 times the clinical dose of both colistin and polymyxin B, the intercellular spaces within the CA1-CA4 region of the polymyxin B group grew markedly ( Fig. 6A ). Furthermore, in the polymyxin B group receiving a dose equivalent to 1 times the clinical dose, distinct neurons within the CA3 region showed evident shrinkage ( Fig. 6A ). Within the colistin group, the intercellular spaces within the CA2-CA3 area were enlarged and only a few neurons experienced shrinkage. Neuronal cells maintained a regular arrangement even at 2.5 times the clinical dose ( Fig. 6A ). In contrast, within the polymyxin B group, a substantial number of neurons in the CA1-CA3 area shrank, leading to a significant increase in intercellular space, resulting in the loss of the original cellular arrangement. This effect was even more pronounced, and numerous cells within the CA4 area exhibited shrinkage 2.5 times the clinical dose ( Fig. 6A ). At 6.25 times the clinical dose, no regions displaying healthy characteristics were observed within the polymyxin B group ( Fig. 6A ). In addition, the solitary nucleus in medulla oblongata was also damaged by polymyxins. With the increase of the dose of polymyxins, neurons in the solitary nucleus tract were shrinkage and the inter-cellular gaps between cells was notably larger. Polymyxin B exhibits more damage than colistin at an equivalent dose ( Fig. 7 ). The results of Nissl staining showed that more neurons shifted compared to the colistin group, and the number of Nissl bodies decreased in the polymyxin B group at the same dose ( Fig. 6B ). Figure 5. Hippocampal damage was induced by colistin and polymyxin B at the administration side in rats. Figure 6. Hippocampal damage was induced by colistin and polymyxin B at the contralateral hippocampus in rats. (A) HE staining of the hippocampal CA1-CA4 regions; (B) Nissl staining of the hippocampal CA1-CA4 regions, Data are presented as mean ± SD for n = 3. Figure 7. Medulla oblongata was induced by colistin and polymyxin B Figure 8. Ferroptosis was induced by colistin and polymyxin B. (A) Fe 2+ and LPO levels were measured by ELISA; (B)Western blot staining of GPX4, xCT, and ACSL4 for cells, Data are presented as mean ± SD for n = 3. 3.5 Effects of colistin and polymyxin B on ferroptosis in N2a and RSC96 cells Ferroptosis is closely related to oxidative stress, as mentioned above. The concentration of iron ions, the level of ROS, and the content of LPO increased ( Fig. 8A ). At the same time, the GSH content decreased, and the expression of ferroptosis-related proteins changed when ferroptosis was activated. Intracellular iron concentration, ROS level, and LPO level increased in a dose-dependent manner. After cells were treated with colistin and polymyxin, iron concentration, ROS level, and LPO level in the polymyxin B group were higher than in the colistin group. In contrast, the GSH content in the polymyxin B group was lower than in the colistin group. Glutathione peroxidase 4 (GPX4) could inhibit ferroptosis, but GPX4 expression in cells treated with colistin and polymyxin B decreased dose-dependently. The expression level of GPX4 in the polymyxin B group was lower than in the colistin group at the same dose (P < 0.05; P < 0.01; P < 0.005; Fig. 8B ). Amino acid transport system xc- (xCT) plays a pivotal role in counteracting ferroptosis by facilitating the increase of intracellular GSH synthesis through the transportation of cystine into cells. decreased dose-dependently after colistin and polymyxin B treatment, and xCT expression in the polymyxin B group was lower than in the colistin group at the same dose (P < 0. 05; Fig. 8B ). The long-chain family member-4 of Acyl-CoA synthetase (ACSL4) can promote the oxidation of polyunsaturated fatty acids to produce lipid peroxides, which can induce iron death. ACSL4 expression increased dose-dependently after cells were treated with colistin and polymyxin B, and ACSL4 expression in the polymyxin B group was higher than in the colistin group at the same dose (P < 0.05; P < 0.005; Fig. 8B ). Lipid hydroperoxide is one of the products of ferroptosis. The intracellular LPO and Fe 2+ content increased in a dose-dependent manner after treatment with colistin and polymyxin B, and the LPO and Fe 2+ content in the polymyxin B group was higher than in the colistin group at the same dose (P < 0.05; P < 0.01; P < 0.005; P < 0.0001; Fig. 8A ). The mitochondria will be damaged when ferroptosis is activated. Our results showed that the mitochondrial membrane potential decreased after colistin and polymyxin B treatment, and the mitochondrial phantom potential in the polymyxin B group was lower than that of the colistin group at the same dose. In addition to the mitochondrial membrane potential, we observed the morphology of mitochondria by laser confocal microscope and electron microscope. At the same dose, mitochondrial punctate patterns were evident when examined using a confocal laser microscope. 4 Discussion Several notable distinctions have emerged between polymyxin B and colistin in recent years, including PK/PD indices and nephrotoxicity [ 3 , 19 – 21 ]. However, this study marks the first comparison of neurotoxicity between these two drugs. Our findings indicate that polymyxin B exhibits more pronounced neurotoxicity than colistin at an equivalent dose, both in vivo and in vitro . We also attempted to elucidate the mechanisms behind these differences in neurotoxicity, highlighting the potential role of varying degrees of damage induced by oxidative stress as a significant factor. In particular, this study also for the first time presents the association between the neurotoxicity of polymyxins and ferroptosis. This study found that colistin and polymyxin B induce oxidative stress within cells, consistent with previous research findings [ 15 ]. Following colistin and polymyxin B treatment, there was substantial depletion of reductive molecules (CAT, SOD, and GSH) within the cells, coupled with the generation of oxidative products (ROS, MDA) on a large scale. This dynamic led to a disruption in the cellular redox equilibrium. Oxidative stress-induced damage to DNA and the endoplasmic reticulum subsequently triggered up-regulation in the expression of key factors such as Caspase-3, Caspase-9, and Cytc. This, in turn, led to mitochondrial impairment and an increased rate of apoptosis [ 22 – 24 ]. In particular, the potency of polymyxin B in inducing the effects mentioned above was significantly more pronounced compared to colistin at equivalent doses. We observed that N2a cells sustained more severe damage than RSC96 cells when subjected to the same dose. This suggests central nervous cells might exhibit higher colistin and polymyxin B sensitivity than peripheral nerve cells. Peripheral nerve cells have significantly higher regenerative capabilities than central nerve cells, which could explain the increased susceptibility of central nerve cells to more severe injuries [ 25 – 27 ]. Reports concerning the neurotoxicity of polymyxins predominantly involve intravenous administration regimens. In particular, the neurotoxic effects of polymyxins tend to manifest in the periphery and subsequently recover after a period of drug discontinuation. This pattern might be attributed to the challenge of facilitating intravenous administration through the blood-brain barrier and the relatively robust recovery potential of peripheral nerve cells after injury [ 7 , 8 , 28 – 30 ]. Although intracerebroventricular injection could increase drug exposure within the CNS, it is imperative to remain vigilant about neurotoxicity due to the limited regenerative capacity of cells within the CNS. In recent studies, ferroptosis, an emerging form of programmed cell death, has been associated with oxidative stress [ 31 , 32 ]. However, the involvement of ferroptosis in polymyxin-induced neurotoxicity remains unknown. In this study, we observed that both drugs activated ferroptosis, and polymyxin B demonstrated a more potent effect than colistin. Specifically, polymyxin B caused a higher generation of Fe 2+ compared to colistin. This Fe 2+ contributes to the Fenton reaction, leading to the production of lipid peroxides. Polymyxin B induced elevated expression of ACSL4, resulting in larger quantities of LPO. Compared to the colistin group, the polymyxin B group exhibited lower levels of GPX4 and xCT expression, making LPO clearance more difficult. This robust induction of ferroptosis by polymyxin B is probably a critical factor contributing to its more severe neurotoxic effects than colistin. We established a dose-to-toxicity relationship to initially discern the disparities in neurotoxicity between the two drugs in an in vivo setting. We observed a consistent pattern in which the areas most susceptible to damage were initially CA2 and CA3, with increasing dose concentrations resulting in the involvement of the CA1 and CA4 regions. This implies that CA2 and CA3 exhibit a greater vulnerability to injury than CA1 and CA4. Although the exact function of the CA4 region remains unclear, lesions in CA1, CA2, and CA3 are related to clinical symptoms. Lesions in the CA2 region are correlated with an abnormal cognitive state, which can contribute to hallucinations [ 33 ]. Furthermore, lesions in the CA1 and CA3 regions of the hippocampus are associated with memory deficits and epilepsy. This connection underscores how patients administered polymyxins can experience temporary memory loss and epilepsy due to the resulting damage to these regions [ 34 , 35 ]. Furthermore, our study demonstrated that colistin exhibited lower levels of damage than polymyxin B when administered at the exact multiple of the clinical dose. This observation suggests colistin could be safer than polymyxin B in intraventricular injection. Of particular importance, the pathological sections highlighted a stark contrast in the severity of the damage between the hippocampus on the administration region and the contralateral hippocampus. This discrepancy could be attributed to the possibility of excessively elevated local concentrations on the administration side. Significant damage was evident even at the lowest doses of colistin (750 U/d) and polymyxin B (300 U/d). In particular, polymyxin B induced more severe damage on the administration side than colistin. It should be noted that the minimum concentrations utilized in this study for polymyxin B and colistin were 15 U/µL and 37.5 U/µL, respectively. These concentrations closely approximate those employed in clinical settings (polymyxin B at 10 U/µL, colistin at 25 U/µL for 5 mL injections). This observation strongly implies the need to carefully consider injuries from the administration side when intraventricular injection of polymyxins is used in clinical scenarios. 5 Conclusions The study revealed a more significant neurotoxic impact of polymyxin B in vitro and in vivo than colistin. This increased neurotoxicity could be attributed to elevated levels of damage induced by oxidative stress. The neurotoxicity of polymyxins is attributed not only to the apoptosis pathway but also to the ferroptosis pathway. Compared to polymyxin B, colistin demonstrated a safer profile in terms of neurotoxicity. Declarations Ethics approval : All animal experiments were examined and approved by the Ethics Committee of the Hunan Experimental Animal Center (Hunan Drug Safety Evaluation Research Center), approval number: IACUC-157023. Consent to participate : Not applicable Consent for publication : All authors consent to the publication of this manuscript. Availability of data and materials : Data will be made available on request. Competing interests : The authors declare that they have no conflict of interest. Funding : This work was supported by the Hunan Provincial Key Laboratory of Anti-Resistance Microbial Drugs (No. 2023TP1013), “Changsha Anti-Infective Drugs Engineering Technology Research Center, China” [No. kq1801120], “Changsha Municipal Natural Science Foundation, China”[No. kq2208463, No. kq2208464] and “Hunan Provincial Science and Technology Department Foundation,China” [No. 2016SK4008, No. 2020SK52901]. Authors' contributions : Rui Yang: Investigation, experimenting, and Writing-Original Draft. Debiao Xiang: Conceptualization, Project administration, Validation. Fang Yuan: Visualization, Data curation. Pengkai Wang and Yuan Yang: Data curation. Bing Xu: Formal analysis and Visualization. Xin Li: Funding acquisition, Project administration, Resources, Conceptualization, and Supervision. Acknowledgments : The Hunan Provincial Key Laboratory of Anti-Resistance Microbial Drugs (No. 2023TP1013), “Changsha Anti-Infective Drugs Engineering Technology Research Center, China” [No. kq1801120], “Changsha Municipal Natural Science Foundation, China”[No. kq2208463, No. kq2208464] and “Hunan Provincial Science and Technology Department Foundation,China” [No. 2016SK4008, No. 2020SK52901]. References Nang SC, Azad M, Velkov T, Zhou QT, Li J (2021) Rescuing the Last-Line Polymyxins: Achievements and Challenges. Pharmacol Rev 73(2):679–728 Vaara M (2010) Polymyxins and their novel derivatives. Curr Opin Microbiol 13(5):574–581 Cai Y, Lee W, Kwa AL (2015) Polymyxin B versus colistin: an update. Expert Rev Anti Infect Ther 13(12):1481–1497 Aggarwal R, Dewan A (2018) Comparison of nephrotoxicity of Colistin with Polymyxin B administered in currently recommended doses: a prospective study. Ann Clin Microbiol Antimicrob 17(1):15 Phe K, Lee Y, McDaneld PM et al (2014) In vitro assessment and multicenter cohort study of comparative nephrotoxicity rates associated with colistimethate versus polymyxin B therapy. Antimicrob Agents Chemother 58(5):2740–2746 Falagas ME, Bliziotis IA, Tam VH (2007) Intraventricular or intrathecal use of polymyxins in patients with Gram-negative meningitis: a systematic review of the available evidence. Int J Antimicrob Agents 29(1):9–25 Myint T, Evans ME, Burgess DR, Greenberg RN (2016) Respiratory Muscle Paralysis Associated With Colistin, Polymyxin B, and Muscle Relaxants Drugs: A Case Report. J Investig Med High Impact Case Rep 4(1):2324709616638362 Camargo C, Narula T, Jackson DA, Padro T, Freeman WD (2021) Colistin neurotoxicity mimicking Guillain-Barré syndrome in a patient with cystic fibrosis: case report and review. Oxf Med Case Reports 2021(9):omab080 Tsuji BT, Pogue JM, Zavascki AP et al (2019) International Consensus Guidelines for the Optimal Use of the Polymyxins: Endorsed by the American College of Clinical Pharmacy (ACCP), European Society of Clinical Microbiology and Infectious Diseases (ESCMID), Infectious Diseases Society of America (IDSA), International Society for Anti-infective Pharmacology (ISAP), Society of Critical Care Medicine (SCCM), and Society of Infectious Diseases Pharmacists (SIDP). Pharmacotherapy 39(1):10–39 Dai C, Tang S, Deng S et al (2015) Lycopene attenuates colistin-induced nephrotoxicity in mice via activation of the Nrf2/HO-1 pathway. Antimicrob Agents Chemother 59(1):579–585 Xiong L, Xiang D, Yuan F et al (2023) Piceatannol-3'-O-β-D-glucopyranoside attenuates colistin-induced neurotoxicity by suppressing oxidative stress via the NRF2/HO-1 pathway. Biomed Pharmacother 161:114419 Dai C, Xiong J, Wang Y, Shen J, Velkov T, Xiao X (2020) Nerve Growth Factor Confers Neuroprotection against Colistin-Induced Peripheral Neurotoxicity. ACS Infect Dis 6(6):1451–1459 Liu Y, Dai C, Gao R, Li J (2013) Ascorbic acid protects against colistin sulfate-induced neurotoxicity in PC12 cells. Toxicol Mech Methods 23(8):584–590 Giovane RA, Lavender PD (2018) Central Nervous System Infections. Prim Care 45(3):505–518 Dai C, Xiao X, Li J et al (2019) Molecular Mechanisms of Neurotoxicity Induced by Polymyxins and Chemoprevention. ACS Chem Neurosci 10(1):120–131 Sies H (2015) Oxidative stress: a concept in redox biology and medicine. Redox Biol 4:180–183 Dai C, Tang S, Biao X, Xiao X, Chen C, Li J (2019) Colistin-induced peripheral neurotoxicity involves mitochondrial dysfunction and oxidative stress in mice. Mol Biol Rep 46(2):1963–1972 Velkov T, Dai C, Ciccotosto GD, Cappai R, Hoyer D, Li J (2018) Polymyxins for CNS infections: Pharmacology and neurotoxicity. Pharmacol Ther 181:85–90 Nation RL, Velkov T, Li J (2014) Colistin and polymyxin B: peas in a pod, or chalk and cheese. Clin Infect Dis 59(1):88–94 Kassamali Z, Danziger L (2015) To B or not to B, that is the question: is it time to replace colistin with polymyxin B. Pharmacotherapy 35(1):17–21 Kwa A, Kasiakou SK, Tam VH, Falagas ME (2007) Polymyxin B: similarities to and differences from colistin (polymyxin E). Expert Rev Anti Infect Ther 5(5):811–821 Radi E, Formichi P, Battisti C, Federico A (2014) Apoptosis and oxidative stress in neurodegenerative diseases. J Alzheimers Dis 42(Suppl 3):S125–S152 Manucha W, Vallés PG (2012) Apoptosis modulated by oxidative stress and inflammation during obstructive nephropathy. Inflamm Allergy Drug Targets 11(4):303–312 Zhu C, Zhang C, Cui X, Wu J, Cui Z, Shen X (2021) Trichosanthin inhibits cervical cancer by regulating oxidative stress-induced apoptosis. Bioengineered 12(1):2779–2790 Brosius Lutz A, Lucas TA, Carson GA et al (2022) An RNA-sequencing transcriptome of the rodent Schwann cell response to peripheral nerve injury. J Neuroinflammation 19(1):105 Mahar M, Cavalli V (2018) Intrinsic mechanisms of neuronal axon regeneration. Nat Rev Neurosci 19(6):323–337 Feng R, Muraleedharan Saraswathy V, Mokalled MH, Cavalli V (2023) Self-renewing macrophages in dorsal root ganglia contribute to promote nerve regeneration. Proc Natl Acad Sci U S A 120(7):e2215906120 Ni M, Meng X, Wang L, Zhao Y, Yu M, Shi S (2020) Polymyxin B-induced rhabdomyolysis: A case report. Med (Baltim) 99(43):e22924 Weinstein L, Doan TL, Smith MA (2009) Neurotoxicity in patients treated with intravenous polymyxin B: Two case reports. Am J Health Syst Pharm 66(4):345–347 Wunsch H, Moitra VK, Patel M, Dzierba AL (2012) Polymyxin use associated with respiratory arrest. Chest 141(2):515–517 Ren JX, Li C, Yan XL, Qu Y, Yang Y, Guo ZN (2021) Crosstalk between Oxidative Stress and Ferroptosis/Oxytosis in Ischemic Stroke: Possible Targets and Molecular Mechanisms. Oxid Med Cell Longev. 2021: 6643382 Chen GH, Song CC, Pantopoulos K, Wei XL, Zheng H, Luo Z (2022) Mitochondrial oxidative stress mediated Fe-induced ferroptosis via the NRF2-ARE pathway. Free Radic Biol Med 180:95–107 Zhao F, Behnisch T (2023) The Enigmatic CA2: Exploring the Understudied Region of the Hippocampus and Its Involvement in Parkinson's Disease. Biomedicines. 11(7) Lee I, Jerman TS, Kesner RP (2005) Disruption of delayed memory for a sequence of spatial locations following CA1- or CA3-lesions of the dorsal hippocampus. Neurobiol Learn Mem 84(2):138–147 Wheal HV (1989) Function of synapses in the CA1 region of the hippocampus: their contribution to the generation or control of epileptiform activity. Comp Biochem Physiol A Comp Physiol 93(1):211–220 Cite Share Download PDF Status: Published Journal Publication published 28 Oct, 2024 Read the published version in Molecular Neurobiology → Version 1 posted Reviewers agreed at journal 05 Dec, 2023 Reviewers invited by journal 26 Sep, 2023 Editor invited by journal 22 Sep, 2023 Editor assigned by journal 07 Sep, 2023 First submitted to journal 05 Sep, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3322528","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":231470946,"identity":"00351e84-4451-4d3d-8cfc-2f2c5538bdc1","order_by":0,"name":"Rui Yang","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Yang","suffix":""},{"id":231470947,"identity":"5aacc1e6-c03b-4fc7-adb7-95488fa8c540","order_by":1,"name":"Debiao Xiang","email":"","orcid":"","institution":"The Third Hospital of Changsha","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Debiao","middleName":"","lastName":"Xiang","suffix":""},{"id":231470948,"identity":"1117fb7a-6fbc-4ec6-8c26-d8e55a724e1d","order_by":2,"name":"Fang Yuan","email":"","orcid":"","institution":"The Third Hospital of Changsha","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Yuan","suffix":""},{"id":231470949,"identity":"d145e2bf-9fce-454a-b963-8044588480be","order_by":3,"name":"Yuan Yang","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Yang","suffix":""},{"id":231470950,"identity":"08c3d72b-1d7c-468e-b772-f21acb8a1f14","order_by":4,"name":"Pengkai Wang","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pengkai","middleName":"","lastName":"Wang","suffix":""},{"id":231470951,"identity":"92ca545b-a9d0-41da-945e-111cc909e058","order_by":5,"name":"Bing Xu","email":"","orcid":"","institution":"The Third Hospital of Changsha","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Xu","suffix":""},{"id":231470952,"identity":"a11b10de-fbfe-4136-885d-1f36438e280e","order_by":6,"name":"Xin Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYDACZiB+YGADYhqQoCXBII0ULSCQwHCYBC0Gx3kPMCQUnJeX7z+8+TNvG4M8v9gB/Fokm/kSgA67bbjhRlqZNFCL4czZCfi18DPzmP8AakkwkOAxYwZqAbHxa2Fj5jEAKjuXIN9/xvgzUVr4IVoOJDAcyDGQJkqLZDNYSzLYL5JzzkkQ9ovB+TMGDB/+2IFD7MObMht5fmkCWlAAEy+bBAnKQYDxxx8SdYyCUTAKRsGIAAB1AjoB/xpnBAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-8866-6848","institution":"The Third Hospital of Changsha","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-09-04 00:53:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3322528/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3322528/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-024-04577-8","type":"published","date":"2024-10-28T16:20:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":43005698,"identity":"689b41a0-9e31-497d-81a7-cc0dff593d49","added_by":"auto","created_at":"2023-09-12 14:06:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":112725,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of colistin and polymyxin B on cell viability of N2a and RSC96 cells.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3322528/v1/1277a9f0cd405ca08510b3e0.png"},{"id":43005699,"identity":"5cc6bf97-580b-4af3-a110-35e5ded1d1ab","added_by":"auto","created_at":"2023-09-12 14:06:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2559478,"visible":true,"origin":"","legend":"\u003cp\u003eApoptosis induced by colistin and polymyxin B. (A) Western blot staining of Cyt c, Caspase-3, and Caspase-9.Data are presented as mean ± SD for n = 3; (B) The apoptosis rate were analyzed by flow cytometry. Data are presented as mean ± SD for n = 3.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3322528/v1/bf5ca03fa8ee876d8b62d5a7.png"},{"id":43007726,"identity":"654d60c5-aaa4-4ce9-b3db-91b2c568c870","added_by":"auto","created_at":"2023-09-12 14:14:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":750182,"visible":true,"origin":"","legend":"\u003cp\u003eOxidative stress was induced by colistin and polymyxin B. (A) SOD, GSH, CAT and MDA levels were measured by ELISA; Data are presented as mean ± SD for n = 3; (B)The ROS level were analyzed by flow cytometry. Data are presented as mean ± SD for n = 3.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3322528/v1/7c284d4201bcc530ae7c1c10.png"},{"id":43007727,"identity":"f2d9aaf6-03e7-400d-ab56-7a3700a416c1","added_by":"auto","created_at":"2023-09-12 14:14:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11574126,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondrial dysfunction was induced by colistin and polymyxin B. (A) Mitochondrial morphologies were assessed by confocal microscopy imaging after staining of cells with the fluorescent indicator Mito-Tracker; (B) The mitochondrial membrane potential (MMP) of cells. The mitochondrial membrane potential (MMP) was measured by using the JC-1 dye. The JC-1 aggregate form, indicating the normal MMP function, is presented in red. The JC-1 monomeric form, indicating disrupted MMP, is shown in green. Data are presented as mean ± SD for n = 3.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3322528/v1/770e71500e6616144d23cd5d.png"},{"id":43005705,"identity":"915c1c78-d7bd-4405-a905-aa13585a6027","added_by":"auto","created_at":"2023-09-12 14:06:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37258427,"visible":true,"origin":"","legend":"\u003cp\u003eHippocampal damage was induced by colistin and polymyxin B at the administration side in rats.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3322528/v1/88c36221beb13bb76af4408f.png"},{"id":43005713,"identity":"6ab07664-bc84-441d-876e-cc248c439a48","added_by":"auto","created_at":"2023-09-12 14:06:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":66633684,"visible":true,"origin":"","legend":"\u003cp\u003eHippocampal damage was induced by colistin and polymyxin B at the contralateral hippocampus in rats. (A) HE staining of the hippocampal CA1-CA4 regions; (B) Nissl staining of the hippocampal CA1-CA4 regions, Data are presented as mean ± SD for n = 3.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3322528/v1/748d98ec5cd60b92786e15ab.png"},{"id":43005704,"identity":"20f60adb-1034-4e6d-8451-6bce4ce1b208","added_by":"auto","created_at":"2023-09-12 14:06:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":23942744,"visible":true,"origin":"","legend":"\u003cp\u003eMedulla oblongata was induced by colistin and polymyxin B\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3322528/v1/343348a7017e9b684e2be4b9.png"},{"id":43005701,"identity":"41d1a94e-eeeb-46b3-8f24-4e9986629efd","added_by":"auto","created_at":"2023-09-12 14:06:38","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1483517,"visible":true,"origin":"","legend":"\u003cp\u003eFerroptosis was induced by colistin and polymyxin B. (A) Fe\u003csup\u003e2+\u003c/sup\u003e and LPO levels were measured by ELISA; (B)Western blot staining of GPX4, xCT, and ACSL4 for cells, Data are presented as mean ± SD for n = 3.\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-3322528/v1/e63fcbca7af2fb4d3105a5dd.png"},{"id":68207212,"identity":"becdead5-9132-4421-8045-6b64ca5d856b","added_by":"auto","created_at":"2024-11-04 16:35:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":189471289,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3322528/v1/675eed4f-c300-4184-8e20-4d78458dcb2e.pdf"}],"financialInterests":"","formattedTitle":"Unraveling neurotoxicity discrepancies: comparative in vitro and In vivo analysis of colistin and polymyxin B and the underlying mechanisms","fulltext":[{"header":"1 INTRODUCTION","content":"\u003cp\u003eWith the emergence of multidrug-resistant (MDR) bacterial infections and the slow pace of new antibiotic development, polymyxins have emerged as the \"last line of defense\" against Gram-negative MDR bacteria [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Colistin and polymyxin B are the primary representatives of polymyxins used in clinical settings [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although colistin and polymyxin B share a quasi-identical structure, except d-phenylalanine being substituted by d-leucine [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], this subtle alteration in structure leads to a spectrum of divergent pharmacokinetic (PK) attributes and toxic reactions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Clinical investigations and \u003cem\u003ein vitro\u003c/em\u003e analyses have underscored the relative reduction in nephrotoxicity of polymyxin B compared to colistin [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Consequently, polymyxin B gains preference in patients with compromised renal function. Beyond nephrotoxicity, neurotoxicity is critical, mainly when these drugs are administered intraventricularly or intrathecally for central nervous system (CNS) infections. A previous study involving such administration reported a 28% incidence of neurotoxicity for polymyxins [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrent approaches to mitigate polymyxin-induced neurotoxicity often involve dose reduction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], but this strategy could compromise therapeutic efficacy [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Although specific studies have identified neuroprotective agents capable of improving polymyxin neurotoxicity, many of these investigations are confined to cellular or animal experiments, distant from clinical implementation [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Patients with CNS infections often contend with concurrent CNS damage [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thus, the inclination leans toward using polymyxins with less neurotoxicity. However, whether there are substantial neurotoxicity discrepancies among various polymyxins remains uncharted territory.\u003c/p\u003e \u003cp\u003ePrevious research has suggested an association between polymyxin neurotoxicity and the initiation of apoptosis through oxidative stress [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Reactive oxygen species (ROS), generated during normal cellular metabolism, are typically counteracted by intracellular antioxidant proteins such as Superoxide Dismutase (SOD), catalase (CAT), and glutathione (GSH) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Polymyxins, however, can induce an excessive ROS surge that exceeds the neutralization capacity of the antioxidant protein, thus inflicting oxidative stress-induced damage [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This elevated ROS production may also trigger mitochondrial dysfunction [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Experiments in mice administered colistin (15 mg/kg/day intravenous injection for 7 consecutive days) showed significant pathological alterations in the ultrastructure of brain mitochondria, including swollen or degenerated cristae and even complete loss of cristae. Furthermore, alterations in mitochondrial membrane permeability, membrane potential, and succinic dehydrogenase activity were observed. Oxidative stress-induced factors accelerate cytochrome C (Cyt c) release from damaged mitochondria, activating a cascade of caspases, a group of cysteine proteases. In our previous investigations, both colistin and polymyxin B were found to increase cyt c release while simultaneously inhibiting the NRF2 antioxidant pathway [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Existing evidence implies a similarity in the neurotoxic mechanisms of colistin and polymyxin B [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Although previous studies have revealed substantial distinctions in the impact of polymyxin B and colistin on the viability of renal cells HEK293 and NRK-52E [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], the degree of variation in the toxicity of the two drugs on nerve cells remains unexplored.\u003c/p\u003e \u003cp\u003eThe primary objective of this study was to perform a comparative analysis of the neurotoxicity profiles of colistin and polymyxin B, employing both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e approaches. Additionally, the study aimed to unravel the underlying mechanisms responsible for these observed differences. In the \u003cem\u003ein vivo\u003c/em\u003e phase, a rat nerve injury model was established by localized brain injections. The divergence in neurotoxic effects between colistin and polymyxin B was evaluated by pathological slices. Meanwhile, the \u003cem\u003ein vitro\u003c/em\u003e experiment investigated how colistin and polymyxin B affect the apoptosis and ferroptosis pathways within N2a and RSC96 cells.\u003c/p\u003e"},{"header":"2 MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e experiments, colistin and polymyxin B were obtained from the National Institutes for Food and Drug Control in Beijing, China. In the \u003cem\u003ein vivo\u003c/em\u003e phase, colistin injections were supplied by Shanghai Xinya Pharmaceutical Co., Ltd. (Shanghai, China). Polymyxin B injections were obtained from Shanghai Shangyao's First Biochemical Pharmaceutical Co., Ltd. (Shanghai, China). N2a cells, RSC96 cells, MEM medium, DMEM medium, and fetal bovine serum were acquired from Procell Life Science \u0026amp; Technology Co., Ltd. (Wuhan, China). The CCK-8 reagent was obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China), and the ROS assay kit was sourced from Beyotime Biotechnology (Shanghai, China). The antibodies were purchased from Abcam (Cambridge, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell culture\u003c/h2\u003e \u003cp\u003eN2a cells and RSC96 cells were cultured in MEM medium and DMEM medium, respectively, which supplemented with 10% (v/v) FBS, 100 units/mL penicillin, and 100 \u0026micro;g/mL streptomycin (Hyclone, Los Angeles, America), at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Animals\u003c/h2\u003e \u003cp\u003eFemale Sprague-Dawley (SD) rats, with body weights of 220 g to 250 g, were chosen as subjects for this study. The rats underwent an initial three-day adaptation period, after which they were randomly assigned to different groups (n\u0026thinsp;=\u0026thinsp;6). Individual rats were housed in separate cages and had unrestricted access to food and water. They were allowed to adjust to their environment, adjusting to factors such as temperature, humidity, light cycles, and ventilation conditions. All animal experiments were examined and approved by the Ethics Committee of the Hunan Experimental Animal Center (Hunan Drug Safety Evaluation Research Center), approval number: IACUC-157023.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell viability assay\u003c/h2\u003e \u003cp\u003eTo elucidate the neurotoxic effects of polymyxin B and colistin on N2a and RSC96 cells, the appropriate experimental concentrations of polymyxin B and colistin in N2a and RSC96 cells were determined. Cell viability was detected by a CCK-8 detection kit. N2a and RSC96 cells were inoculated in 96-well plates (5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well) and treated with polymyxin B (50, 100, 200, 400, 500, 600, 800, 1000, 1200 \u0026micro;M) and colistin (50, 100, 400, 500, 600, 800, 1000, 1200 \u0026micro;M) for 24 h. After exposure, the medium was removed and replaced with a medium containing 10% CCK-8. Incubation was performed for 2 h, after which the absorbance was measured at 405 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell apoptosis\u003c/h2\u003e \u003cp\u003eThe annexin V-FITC apoptosis detection kit (Beyotime, Shanghai, China) was used to detect the apoptosis rate. N2a and RSC96 cells were seeded in 100 mm cell culture dishes (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/well). N2a cells were treated with colistin or polymyxin B (150, 300, 600 \u0026micro;M), while RSC96 cells were treated with colistin or polymyxin B (300, 600, 900 \u0026micro;M) at 37\u0026deg;C for 24 h. After treatment, N2a and RSC96 cells were harvested with 0.25% trypsin, washed twice with PBS, and resuspended in 195 \u0026micro;L Annexin V-FITC binding buffer. The resuspended cells were incubated with 5 \u0026micro;L Annexin V-FITC and 10 \u0026micro;L propidium iodide in the dark for 20 min. Analysis was performed using a DxFLEX flow cytometer (Beckman Coulter, Los Angeles, US).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Assessment of cellular ROS levels\u003c/h2\u003e \u003cp\u003eThe Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China) was used to detect the ROS level in cells. First, N2a and RSC96 cells were seeded in 100 mm cell culture dishes (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/well). N2a cells were then treated with colistin (150, 300, 600 \u0026micro;M) and polymyxin B (150, 300, 600 \u0026micro;M) at 37\u003csup\u003eo\u003c/sup\u003eC for 24 h, and RSC96 cells were treated, respectively, with colistin (300, 600, 900 \u0026micro;M) and polymyxin B (300, 600, 900 \u0026micro;M) at 37\u003csup\u003eo\u003c/sup\u003eC for 24 h. After treatment, cells were treated with 0.25% trypsin, washed with PBS 3 times, then treated with DCFH-DA basic culture medium containing 10 \u0026micro;M for 20 min at 37\u003csup\u003eo\u003c/sup\u003eC, and finally analyzed by DxFLEX flow cytometry (Beckman Coulter in Los Angeles, US).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Evaluation of oxidative stress levels\u003c/h2\u003e \u003cp\u003eAn oxidative stress test kit (Nanjing Jiancheng, Nanjing, China) was used to detect SOD, CAT, GSH, and MDA activity levels. N2a and RSC96 cells were seeded in 100 mm cell culture plates and treated according to the method recommended by the manufacturer. After treatment, N2a and RSC96 cells were collected with a scraper in a centrifuge tube and centrifuged at 800 \u003cem\u003eg\u003c/em\u003e for 5 min. After the supernatant was removed, PBS resuscitated the cells. The cells were then ultrasonicated at 250 W for 10 min on ice, after which the cell lysates were centrifuged at 20,000 \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C. The supernatant was collected and assayed for SOD, CAT, GSH, and MDA activities. Protein concentrations were quantified using the BCA protein assay kit (Boster, Wuhan, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Western blot analysis\u003c/h2\u003e \u003cp\u003eCells were collected with a scraper, then transferred to a centrifuge tube and centrifuged at 800 \u003cem\u003eg\u003c/em\u003e for 5 min at 20℃. After discarding the supernatant, PBS was added to resuspend the cells and centrifuged at 800 \u003cem\u003eg\u003c/em\u003e for 5 min at 20℃. The supernatant was discarded, and the appropriate amount of PBS was added. Cells were broken in ice using an ultrasonic cell grinder (Scientz Biotechnology, Ningbo, China) at 300 W power for 10 min. The sample was centrifuged, and the supernatant was taken for Western blotting analysis. 10% sodium dodecyl sulfate-PAGE gel was used for electrophoresis, and the protein was transferred to the PVDF membrane (Applied Technology Company, Beijing, China). The membrane was blocked with 5% (w/w) skimmed milk powder in TBST for 2 h, washed with TBST 3 times, and incubated overnight with rabbit polyclonal antibody at 4℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Assessment of cellular lipid peroxide levels\u003c/h2\u003e \u003cp\u003eThe lipid peroxide (LPO) level was detected using an LPO detection kit (Nanjing Jiancheng, Nanjing, China). N2a and RSC96 cells were inoculated in 100 mm cell culture dishes and treated according to the method recommended by the manufacturer. After treatment, N2a and RSC96 cells were collected with a scraper and centrifuged at 800 \u003cem\u003eg\u003c/em\u003e for 5 min. After the supernatant was removed, cells were resuspended with PBS. Subsequently, a 250 W ultrasound was applied for 10 min. The resulting cell suspension was then utilized to assess the LPO level. A BCA protein detection kit (Boster, Wuhan, China) was used to quantify the protein concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Evaluation of cellular iron concentration\u003c/h2\u003e \u003cp\u003eThe concentration of ferrous ions was detected by a cell ferrous colorimetric assay kit (Elabscience Biotechnology, Wuhan, China). First, drug-treated cells were collected with a cell scraper, and the number of cells in each group was obtained using a cell counter. The lysate contained in the kit was used to lyse the cell for 10 min on ice and then centrifuged at 15,000\u003cem\u003eg\u003c/em\u003e for 10 min. The concentration of ferrous ions in the supernatant was detected at 593 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Mitochondrial morphology\u003c/h2\u003e \u003cp\u003eThe structure of mitochondria was evaluated using the Mito Tracker Red CMXRos fluorescence indicator (Beyotime, Shanghai, China). Cells were inoculated in 12-well plates (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well), and cell slides were placed in each well. Cells were treated with different concentrations of polymyxin B and colistin at 37℃ for 6 h. Cells were incubated in MEM containing 100 nM Mito Tracker Red CMXRos for 30 min. The slides were cleaned with PBS, supplemented with an anti-fading mounting medium, covered, and observed through a laser scanning confocal microscope (FV3000, Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Evaluation of the mitochondrial membrane potential\u003c/h2\u003e \u003cp\u003eΔψm was detected with the fluorescent indicator JC-1 (Beyotime, Shanghai, China). Cells were treated with different concentrations of polymyxin B and colistin at 37\u0026deg;C for 24 h and then incubated in MEM containing 10 \u0026micro;M JC-1 at 37\u0026deg;C for 30 min. The cells were then washed with PBS. An anti-Fade patch containing DAPI was added to the cell slide, covered, and observed with a laser scanning confocal microscope (FV3000, Olympus, Tokyo, Japan). The fluorescence change from red to green indicates the loss of memory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Intraventricular injection of polymyxin B and colistin in rats\u003c/h2\u003e \u003cp\u003eAfter a three-day adaptive feeding, the rats were randomly divided into control, sham, and administration groups. To prevent infections, a single intraperitoneal injection of ceftriaxone at 210 mg/kg was administered to rats 30 min before the surgical procedure. After continuous anesthesia induction using isoflurane, rats were fixed on a stereotactic instrument (KOPF, Florida, US). An incision was made longitudinally on the scalp, approximately 15 mm long. The periosteum was scraped, completely exposing the anterior fontanelle. The precise positioning involved moving the positioning needle 0.8 mm posteriorly and 1.5 mm laterally from the origin of the anterior fontanelle, with a penetration depth of 3.5 mm. Subsequently, a hole was drilled in the skull, and a guide tube was inserted, which was then affixed to the skull using a small amount of dental cement. After this surgical procedure, the rats were given another three days to adjust to their surroundings. Following this period, actual drug administration was conducted. Medical solutions, 20 \u0026micro;L, were administered to the lateral ventricle using a microinjection pump at a controlled rate of 4 \u0026micro;L/min.\u003c/p\u003e \u003cp\u003eAdministration doses were translated into equivalent doses for adults: the low dose (750 U/day of colistin and 300 U/day of polymyxin B) was determined based on the adult dose, the medium dose (1,875 U/day of colistin and 750 U/day of polymyxin B) increased to 2.5 times the adult dose, and the high dose (4,687.5 U/day of colistin and 1,875 U/day of polymyxin B) increased to 6.25 times the adult dose. Dosage was administered once daily over five days. After the administration on day 5, the rats were euthanized with an overdose of anesthetics. As for the sham group, an equivalent volume of vehicle solution was administered in parallel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Statistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation for a sample size. Analysis was conducted using GraphPad Prism software (GraphPad Software, USA), and mean values were compared using one-way analysis of variance (ANOVA). A significance threshold of P\u0026thinsp;\u0026le;\u0026thinsp;0.05 indicates statistical significance, while P\u0026thinsp;\u0026le;\u0026thinsp;0.01 is considered highly significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 RESULTS","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effects of colistin and polymyxin B on N2a and RSC96 cell viability\u003c/h2\u003e \u003cp\u003eN2a and RSC96 cells were subjected to drug treatment for 24 h. As drug doses escalated, the viability of both cell types exhibited a dose-dependent decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The IC50 values for colistin and polymyxin B in N2a cells were determined to be 183 \u0026micro;M and 127 \u0026micro;M, respectively. In RSC96 cells, the IC50 values for colistin and polymyxin B were 810 \u0026micro;M and 579 \u0026micro;M, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Polymyxin B demonstrated a greater degree of neurotoxicity compared to colistin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e toxicity of colistin and polymyxin B in different cells\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e(\u0026micro;M)\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColistin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolymyxin B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN2a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e183.17\u0026thinsp;\u0026plusmn;\u0026thinsp;17.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e127.40\u0026thinsp;\u0026plusmn;\u0026thinsp;18.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRSC96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e810.13\u0026thinsp;\u0026plusmn;\u0026thinsp;15.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e579.03\u0026thinsp;\u0026plusmn;\u0026thinsp;42.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e1\u003c/sup\u003e Data shown are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs. IC50 (drug concentration resulting in 50% of maximal reduction in cell viability)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2.\u003c/b\u003e Apoptosis induced by colistin and polymyxin B. (A) Western blot staining of Cyt c, Caspase-3, and Caspase-9.Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for n\u0026thinsp;=\u0026thinsp;3; (B) The apoptosis rate were analyzed by flow cytometry. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for n\u0026thinsp;=\u0026thinsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Difference in colistin and polymyxin B-induced apoptosis \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe apoptosis rate detected by flow cytometry and the expression level of apoptotic proteins (Cytc, Caspase-3, Caspase-9) detected by Western blot were used to compare the ability of polymyxin B and colistin-induced neuronal apoptosis. Cytc and its downstream Caspase-3 and Caspase-9 can promote apoptosis. In both N2a and RSC96 cells, Cytc, Caspase-3, and Caspase-9 expression levels in the polymyxin B group were higher than those of colistin at the same dose (150 \u0026micro;M, 300 \u0026micro;M, 600 \u0026micro;M, 900 \u0026micro;M) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Polymyxin B induced a higher apoptosis rate in N2a and RSC96 cells at the same dose (P\u0026thinsp;\u0026lt;\u0026thinsp;0.005; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, flow cytometry showed that almost all apoptotic cells were in the late stage of apoptosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 3.\u003c/b\u003e Oxidative stress was induced by colistin and polymyxin B. (A) SOD, GSH, CAT and MDA levels were measured by ELISA; Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for n\u0026thinsp;=\u0026thinsp;3; (B)The ROS level were analyzed by flow cytometry. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for n\u0026thinsp;=\u0026thinsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Oxidative stress induced by colistin and polymyxin B in N2a and RSC96 cells\u003c/h2\u003e \u003cp\u003eAt the same dose, N2a and RSC96 cells were induced by colistin and polymyxin B, and changes in SOD, CAT, GSH, MDA, and ROS were detected to assess the toxicity of the two drugs on nerve cells. In both N2a and RSC96 cells, upon exposure to identical doses of the two drugs (150 \u0026micro;M, 300 \u0026micro;M, 600 \u0026micro;M, 900 \u0026micro;M), the levels of GSH, SOD, and CAT were consistently lower in the polymyxin B group compared to the colistin group. The MDA content in the polymyxin B group was higher than in the colistin group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; P\u0026thinsp;\u0026lt;\u0026thinsp;0.005; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; \u003cb\u003eFig.\u0026nbsp;3A\u003c/b\u003e). Furthermore, flow cytometry showed that polymyxin B could induce a higher level of ROS than Colistin at the same dose (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; \u003cb\u003eFig.\u0026nbsp;3B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe morphology of mitochondria in the control group was linear under a confocal laser microscope (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e). With the aggravation of injury, the morphology of the mitochondria changed from linear to punctate. Almost all mitochondria were punctate after cells were treated with high doses of colistin and polymyxin B (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e). The mitochondrial membrane potential of the polymyxin B group was lower than that of the colistin group at the same dose(P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; \u003cb\u003eFig.\u0026nbsp;4B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 4.\u003c/b\u003e Mitochondrial dysfunction was induced by colistin and polymyxin B. (A) Mitochondrial morphologies were assessed by confocal microscopy imaging after staining of cells with the fluorescent indicator Mito-Tracker; (B) The mitochondrial membrane potential (MMP) of cells. The mitochondrial membrane potential (MMP) was measured by using the JC-1 dye. The JC-1 aggregate form, indicating the normal MMP function, is presented in red. The JC-1 monomeric form, indicating disrupted MMP, is shown in green. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for n\u0026thinsp;=\u0026thinsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Difference of neurotoxicity between colistin and polymyxin B \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe extent of nerve injury resulting from varying doses was evaluated using HE and Nissl staining. Upon HE staining of brain tissue, neurons within the hippocampal CA1-CA4 regions of both the control and the sham groups showed organized arrangement, regular morphology, and minimal intercellular gaps (\u003cb\u003eFig.\u0026nbsp;5\u003c/b\u003e). At administration, no substantial damage was observed when the recommended clinical dose of colistin was used (\u003cb\u003eFig.\u0026nbsp;5\u003c/b\u003e). Minor damage was apparent in the CA1 and CA4 areas, while significant damage was evident in the CA2 and CA3 areas after administering colistin 2.5 times the clinical dose (\u003cb\u003eFig.\u0026nbsp;5\u003c/b\u003e). Elevating the dose to 6.25 times the clinical standard resulted in severe damage in all CA1-CA4 areas, characterized by an almost complete absence of neurons (\u003cb\u003eFig.\u0026nbsp;5\u003c/b\u003e). Regarding polymyxin B, minor damage was observed in the CA1 and CA4 areas, and substantial damage was observed in the CA2 and CA3 regions at the recommended clinical dose (\u003cb\u003eFig.\u0026nbsp;5\u003c/b\u003e). When polymyxin B was administered 2.5 times the clinical dose, except for CA4, CA1-CA3 areas exhibited an almost complete neuronal absence (\u003cb\u003eFig.\u0026nbsp;5\u003c/b\u003e). The impact of polymyxins on the administered area was so severe that a significant number of cells were not adequately preserved by the fixative and consequently lost when administered 6.25 times the clinical dose (\u003cb\u003eFig.\u0026nbsp;5\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn the contralateral hippocampus, the CA1-CA4 region of the colistin group did not show apparent lesions. However, after treatment with 1 times the clinical dose of both colistin and polymyxin B, the intercellular spaces within the CA1-CA4 region of the polymyxin B group grew markedly (\u003cb\u003eFig.\u0026nbsp;6A\u003c/b\u003e). Furthermore, in the polymyxin B group receiving a dose equivalent to 1 times the clinical dose, distinct neurons within the CA3 region showed evident shrinkage (\u003cb\u003eFig.\u0026nbsp;6A\u003c/b\u003e). Within the colistin group, the intercellular spaces within the CA2-CA3 area were enlarged and only a few neurons experienced shrinkage. Neuronal cells maintained a regular arrangement even at 2.5 times the clinical dose (\u003cb\u003eFig.\u0026nbsp;6A\u003c/b\u003e). In contrast, within the polymyxin B group, a substantial number of neurons in the CA1-CA3 area shrank, leading to a significant increase in intercellular space, resulting in the loss of the original cellular arrangement. This effect was even more pronounced, and numerous cells within the CA4 area exhibited shrinkage 2.5 times the clinical dose (\u003cb\u003eFig.\u0026nbsp;6A\u003c/b\u003e). At 6.25 times the clinical dose, no regions displaying healthy characteristics were observed within the polymyxin B group (\u003cb\u003eFig.\u0026nbsp;6A\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn addition, the solitary nucleus in medulla oblongata was also damaged by polymyxins. With the increase of the dose of polymyxins, neurons in the solitary nucleus tract were shrinkage and the inter-cellular gaps between cells was notably larger. Polymyxin B exhibits more damage than colistin at an equivalent dose (\u003cb\u003eFig.\u0026nbsp;7\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe results of Nissl staining showed that more neurons shifted compared to the colistin group, and the number of Nissl bodies decreased in the polymyxin B group at the same dose (\u003cb\u003eFig.\u0026nbsp;6B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 5.\u003c/b\u003e Hippocampal damage was induced by colistin and polymyxin B at the administration side in rats.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 6.\u003c/b\u003e Hippocampal damage was induced by colistin and polymyxin B at the contralateral hippocampus in rats. (A) HE staining of the hippocampal CA1-CA4 regions; (B) Nissl staining of the hippocampal CA1-CA4 regions, Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for n\u0026thinsp;=\u0026thinsp;3.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 7.\u003c/b\u003e Medulla oblongata was induced by colistin and polymyxin B\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 8.\u003c/b\u003e Ferroptosis was induced by colistin and polymyxin B. (A) Fe\u003csup\u003e2+\u003c/sup\u003e and LPO levels were measured by ELISA; (B)Western blot staining of GPX4, xCT, and ACSL4 for cells, Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for n\u0026thinsp;=\u0026thinsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effects of colistin and polymyxin B on ferroptosis in N2a and RSC96 cells\u003c/h2\u003e \u003cp\u003eFerroptosis is closely related to oxidative stress, as mentioned above. The concentration of iron ions, the level of ROS, and the content of LPO increased (\u003cb\u003eFig.\u0026nbsp;8A\u003c/b\u003e). At the same time, the GSH content decreased, and the expression of ferroptosis-related proteins changed when ferroptosis was activated. Intracellular iron concentration, ROS level, and LPO level increased in a dose-dependent manner. After cells were treated with colistin and polymyxin, iron concentration, ROS level, and LPO level in the polymyxin B group were higher than in the colistin group. In contrast, the GSH content in the polymyxin B group was lower than in the colistin group. Glutathione peroxidase 4 (GPX4) could inhibit ferroptosis, but GPX4 expression in cells treated with colistin and polymyxin B decreased dose-dependently. The expression level of GPX4 in the polymyxin B group was lower than in the colistin group at the same dose (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; P\u0026thinsp;\u0026lt;\u0026thinsp;0.005; \u003cb\u003eFig.\u0026nbsp;8B\u003c/b\u003e). Amino acid transport system xc- (xCT) plays a pivotal role in counteracting ferroptosis by facilitating the increase of intracellular GSH synthesis through the transportation of cystine into cells. decreased dose-dependently after colistin and polymyxin B treatment, and xCT expression in the polymyxin B group was lower than in the colistin group at the same dose (P\u0026thinsp;\u0026lt;\u0026thinsp;0. 05; \u003cb\u003eFig.\u0026nbsp;8B\u003c/b\u003e). The long-chain family member-4 of Acyl-CoA synthetase (ACSL4) can promote the oxidation of polyunsaturated fatty acids to produce lipid peroxides, which can induce iron death. ACSL4 expression increased dose-dependently after cells were treated with colistin and polymyxin B, and ACSL4 expression in the polymyxin B group was higher than in the colistin group at the same dose (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; P\u0026thinsp;\u0026lt;\u0026thinsp;0.005; \u003cb\u003eFig.\u0026nbsp;8B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eLipid hydroperoxide is one of the products of ferroptosis. The intracellular LPO and Fe\u003csup\u003e2+\u003c/sup\u003e content increased in a dose-dependent manner after treatment with colistin and polymyxin B, and the LPO and Fe\u003csup\u003e2+\u003c/sup\u003econtent in the polymyxin B group was higher than in the colistin group at the same dose (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; P\u0026thinsp;\u0026lt;\u0026thinsp;0.005; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; \u003cb\u003eFig.\u0026nbsp;8A\u003c/b\u003e). The mitochondria will be damaged when ferroptosis is activated. Our results showed that the mitochondrial membrane potential decreased after colistin and polymyxin B treatment, and the mitochondrial phantom potential in the polymyxin B group was lower than that of the colistin group at the same dose. In addition to the mitochondrial membrane potential, we observed the morphology of mitochondria by laser confocal microscope and electron microscope. At the same dose, mitochondrial punctate patterns were evident when examined using a confocal laser microscope.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eSeveral notable distinctions have emerged between polymyxin B and colistin in recent years, including PK/PD indices and nephrotoxicity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, this study marks the first comparison of neurotoxicity between these two drugs. Our findings indicate that polymyxin B exhibits more pronounced neurotoxicity than colistin at an equivalent dose, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. We also attempted to elucidate the mechanisms behind these differences in neurotoxicity, highlighting the potential role of varying degrees of damage induced by oxidative stress as a significant factor. In particular, this study also for the first time presents the association between the neurotoxicity of polymyxins and ferroptosis.\u003c/p\u003e \u003cp\u003eThis study found that colistin and polymyxin B induce oxidative stress within cells, consistent with previous research findings [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Following colistin and polymyxin B treatment, there was substantial depletion of reductive molecules (CAT, SOD, and GSH) within the cells, coupled with the generation of oxidative products (ROS, MDA) on a large scale. This dynamic led to a disruption in the cellular redox equilibrium. Oxidative stress-induced damage to DNA and the endoplasmic reticulum subsequently triggered up-regulation in the expression of key factors such as Caspase-3, Caspase-9, and Cytc. This, in turn, led to mitochondrial impairment and an increased rate of apoptosis [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In particular, the potency of polymyxin B in inducing the effects mentioned above was significantly more pronounced compared to colistin at equivalent doses.\u003c/p\u003e \u003cp\u003eWe observed that N2a cells sustained more severe damage than RSC96 cells when subjected to the same dose. This suggests central nervous cells might exhibit higher colistin and polymyxin B sensitivity than peripheral nerve cells. Peripheral nerve cells have significantly higher regenerative capabilities than central nerve cells, which could explain the increased susceptibility of central nerve cells to more severe injuries [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Reports concerning the neurotoxicity of polymyxins predominantly involve intravenous administration regimens. In particular, the neurotoxic effects of polymyxins tend to manifest in the periphery and subsequently recover after a period of drug discontinuation. This pattern might be attributed to the challenge of facilitating intravenous administration through the blood-brain barrier and the relatively robust recovery potential of peripheral nerve cells after injury [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although intracerebroventricular injection could increase drug exposure within the CNS, it is imperative to remain vigilant about neurotoxicity due to the limited regenerative capacity of cells within the CNS.\u003c/p\u003e \u003cp\u003eIn recent studies, ferroptosis, an emerging form of programmed cell death, has been associated with oxidative stress [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, the involvement of ferroptosis in polymyxin-induced neurotoxicity remains unknown. In this study, we observed that both drugs activated ferroptosis, and polymyxin B demonstrated a more potent effect than colistin. Specifically, polymyxin B caused a higher generation of Fe\u003csup\u003e2+\u003c/sup\u003e compared to colistin. This Fe\u003csup\u003e2+\u003c/sup\u003e contributes to the Fenton reaction, leading to the production of lipid peroxides. Polymyxin B induced elevated expression of ACSL4, resulting in larger quantities of LPO. Compared to the colistin group, the polymyxin B group exhibited lower levels of GPX4 and xCT expression, making LPO clearance more difficult. This robust induction of ferroptosis by polymyxin B is probably a critical factor contributing to its more severe neurotoxic effects than colistin.\u003c/p\u003e \u003cp\u003eWe established a dose-to-toxicity relationship to initially discern the disparities in neurotoxicity between the two drugs in an \u003cem\u003ein vivo\u003c/em\u003e setting. We observed a consistent pattern in which the areas most susceptible to damage were initially CA2 and CA3, with increasing dose concentrations resulting in the involvement of the CA1 and CA4 regions. This implies that CA2 and CA3 exhibit a greater vulnerability to injury than CA1 and CA4. Although the exact function of the CA4 region remains unclear, lesions in CA1, CA2, and CA3 are related to clinical symptoms. Lesions in the CA2 region are correlated with an abnormal cognitive state, which can contribute to hallucinations [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Furthermore, lesions in the CA1 and CA3 regions of the hippocampus are associated with memory deficits and epilepsy. This connection underscores how patients administered polymyxins can experience temporary memory loss and epilepsy due to the resulting damage to these regions [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, our study demonstrated that colistin exhibited lower levels of damage than polymyxin B when administered at the exact multiple of the clinical dose. This observation suggests colistin could be safer than polymyxin B in intraventricular injection.\u003c/p\u003e \u003cp\u003eOf particular importance, the pathological sections highlighted a stark contrast in the severity of the damage between the hippocampus on the administration region and the contralateral hippocampus. This discrepancy could be attributed to the possibility of excessively elevated local concentrations on the administration side. Significant damage was evident even at the lowest doses of colistin (750 U/d) and polymyxin B (300 U/d). In particular, polymyxin B induced more severe damage on the administration side than colistin. It should be noted that the minimum concentrations utilized in this study for polymyxin B and colistin were 15 U/\u0026micro;L and 37.5 U/\u0026micro;L, respectively. These concentrations closely approximate those employed in clinical settings (polymyxin B at 10 U/\u0026micro;L, colistin at 25 U/\u0026micro;L for 5 mL injections). This observation strongly implies the need to carefully consider injuries from the administration side when intraventricular injection of polymyxins is used in clinical scenarios.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThe study revealed a more significant neurotoxic impact of polymyxin B \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e than colistin. This increased neurotoxicity could be attributed to elevated levels of damage induced by oxidative stress. The neurotoxicity of polymyxins is attributed not only to the apoptosis pathway but also to the ferroptosis pathway. Compared to polymyxin B, colistin demonstrated a safer profile in terms of neurotoxicity.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e: All animal experiments were examined and approved by the Ethics Committee of the Hunan Experimental Animal Center (Hunan Drug Safety Evaluation Research Center), approval number: IACUC-157023.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: All authors consent to the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e: Data will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work was supported by the Hunan Provincial Key Laboratory of Anti-Resistance Microbial Drugs (No. 2023TP1013),\u0026nbsp;\u0026ldquo;Changsha Anti-Infective Drugs Engineering Technology Research Center, China\u0026rdquo;\u0026nbsp;[No. kq1801120],\u0026nbsp;\u0026ldquo;Changsha Municipal Natural Science Foundation, China\u0026rdquo;[No. kq2208463, No. kq2208464] and\u0026nbsp;\u0026ldquo;Hunan Provincial Science and Technology Department Foundation,China\u0026rdquo;\u0026nbsp;[No. 2016SK4008, \u0026nbsp;No. 2020SK52901].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e: Rui Yang: Investigation, experimenting, and Writing-Original Draft. Debiao Xiang: Conceptualization, Project administration, Validation. Fang Yuan: Visualization, Data curation. Pengkai Wang and Yuan Yang: Data curation. Bing Xu: Formal analysis and Visualization. Xin Li: Funding acquisition, Project administration, Resources, Conceptualization, and Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e: The Hunan Provincial Key Laboratory of Anti-Resistance Microbial Drugs (No. 2023TP1013),\u0026nbsp;\u0026ldquo;Changsha Anti-Infective Drugs Engineering Technology Research Center, China\u0026rdquo;\u0026nbsp;[No. kq1801120],\u0026nbsp;\u0026ldquo;Changsha Municipal Natural Science Foundation, China\u0026rdquo;[No. kq2208463, No. kq2208464] and\u0026nbsp;\u0026ldquo;Hunan Provincial Science and Technology Department Foundation,China\u0026rdquo;\u0026nbsp;[No. 2016SK4008, \u0026nbsp;No. 2020SK52901].\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNang SC, Azad M, Velkov T, Zhou QT, Li J (2021) Rescuing the Last-Line Polymyxins: Achievements and Challenges. 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Chest 141(2):515\u0026ndash;517\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen JX, Li C, Yan XL, Qu Y, Yang Y, Guo ZN (2021) Crosstalk between Oxidative Stress and Ferroptosis/Oxytosis in Ischemic Stroke: Possible Targets and Molecular Mechanisms. Oxid Med Cell Longev. 2021: 6643382\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen GH, Song CC, Pantopoulos K, Wei XL, Zheng H, Luo Z (2022) Mitochondrial oxidative stress mediated Fe-induced ferroptosis via the NRF2-ARE pathway. Free Radic Biol Med 180:95\u0026ndash;107\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao F, Behnisch T (2023) The Enigmatic CA2: Exploring the Understudied Region of the Hippocampus and Its Involvement in Parkinson's Disease. Biomedicines. 11(7)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee I, Jerman TS, Kesner RP (2005) Disruption of delayed memory for a sequence of spatial locations following CA1- or CA3-lesions of the dorsal hippocampus. Neurobiol Learn Mem 84(2):138\u0026ndash;147\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWheal HV (1989) Function of synapses in the CA1 region of the hippocampus: their contribution to the generation or control of epileptiform activity. Comp Biochem Physiol A Comp Physiol 93(1):211\u0026ndash;220\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Polymyxin B, Colistin, Neurotoxicity, Comparison, Ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-3322528/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3322528/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/em\u003e: Polymyxins, including colistin and polymyxin B, are a final resort against Gram-negative bacterial infections. However, its clinical application is restricted due to concerns related to neurotoxicity. Despite the similar antibacterial spectrum and mode of action shared between colistin and polymyxin B, there is still a lack of definitive evidence to support the idea that their neurotoxicity profiles are identical.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/em\u003e: To comprehensively compare the neurotoxicity between colistin and polymyxin B both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e and establish a theoretical foundation to guide the rational use of polymyxins within clinical settings.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/em\u003e: \u003cem\u003eIn vitro\u003c/em\u003e experiments simulated nerve damage by exposing N2a and RSC96 cells to colistin and polymyxin B. The evaluation of nerve injury included assessments of cell viability and apoptosis. To discern the variance in the mechanisms of nerve injury between colistin and polymyxin B, oxidative stress levels were examined, such as SOD, CAT, GSH, and MDA. In \u003cem\u003ein vivo\u003c/em\u003e experiments, a rat nerve injury model was created through intraventricular injections of colistin and polymyxin B, respectively. The impact of these drugs on brain injury in rats, particularly within the hippocampus and medulla oblongata, was measured using HE and Nissl staining. The potential influence of polymyxins on the ferroptosis pathway was evaluated by assessing the levels of LPO and Fe\u003csup\u003e2+\u003c/sup\u003e and the degree of mitochondrial impairment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/em\u003e: At equivalent doses, colistin demonstrated a reduced level of neurotoxicity compared to polymyxin B, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. \u003cem\u003eIn vitro\u003c/em\u003e experiments revealed greater cell viability and a lower apoptosis rate after colistin treatment than after polymyxin B treatment. This variance in outcomes could be attributed to the comparatively lower levels of oxidative stress associated with colistin administration\u003cem\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/em\u003eIn a rat model, nerve injury resulted in observable damage to both the hippocampus and the medulla oblongata. A comprehensive assessment of the extent of damage in the CA1 to CA4 regions of the hippocampus, the nucleus of the solitary tract, and the hypoglossal nucleus of the medulla oblongata underscored that the neurotoxic effects of colistin remained milder compared to those elicited by polymyxin B. Even when evaluated at equivalent multiples of clinically recommended doses, colistin exhibited lower neurotoxicity \u003cem\u003ein vivo\u003c/em\u003e than polymyxin B. For the first time, this study demonstrated the role of ferroptosis in polymyxin B-induced nerve damage. The activation levels observed within the ferroptosis pathway due to polymyxin B exceeded those triggered by colistin.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/em\u003e: Colistin exhibited a marked reduction in neurotoxicity compared to polymyxin B, evident in both the equivalent and clinically recommended doses. These findings suggest that, from the perspective of neurotoxicity, colistin presents a more favorable option for clinical use.\u003c/p\u003e","manuscriptTitle":"Unraveling neurotoxicity discrepancies: comparative in vitro and In vivo analysis of colistin and polymyxin B and the underlying mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-12 14:06:33","doi":"10.21203/rs.3.rs-3322528/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-12-05T15:02:46+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-09-27T00:37:17+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Molecular Neurobiology","date":"2023-09-22T20:29:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-09-07T07:03:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2023-09-05T08:25:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"22a1aad1-d475-44fa-8d86-6686a0af2fc8","owner":[],"postedDate":"September 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-04T16:25:21+00:00","versionOfRecord":{"articleIdentity":"rs-3322528","link":"https://doi.org/10.1007/s12035-024-04577-8","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2024-10-28 16:20:06","publishedOnDateReadable":"October 28th, 2024"},"versionCreatedAt":"2023-09-12 14:06:33","video":"","vorDoi":"10.1007/s12035-024-04577-8","vorDoiUrl":"https://doi.org/10.1007/s12035-024-04577-8","workflowStages":[]},"version":"v1","identity":"rs-3322528","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3322528","identity":"rs-3322528","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}