Niclosamide as a Neuroprotective and Anti-Inflammatory Agent: A Potential Therapeutic Approach for Spinal Cord Injury

Niclosamide as a Neuroprotective and Anti-Inflammatory Agent: A Potential Therapeutic Approach for Spinal Cord Injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Niclosamide as a Neuroprotective and Anti-Inflammatory Agent: A Potential Therapeutic Approach for Spinal Cord Injury Ali Darabniya, Sara Shahriari, Reza Fazeli, Razie Mohammad Jafari, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7609682/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Spinal cord injury (SCI) is a life-altering condition of the central nervous system resulting in permanent motor, sensory, and functional deficits. The pathophysiology involved includes: primary neuronal necrosis, secondary inflammation, oxidative stress, apoptosis, and impaired neurogenesis, which collectively inhibit tissue repair. Despite efforts, there is currently no effective therapy that offers radial spinal cord regeneration, which highlights our need for pharmacological agents with anti-inflammatory and neuroprotective properties. Niclosamide is an FDA-approved anti-parasitic agent that has received increasing attention recently due to its ability to modulate important signaling pathways such as STAT3, NF-κB, MAPK/ERK, and mTOR. We therefore sought to investigate the therapeutic potential of niclosamide in a rat model of SCI. Animals were given a single intraperitoneal injection of niclosamide (2, 5, or 10 mg/kg) immediately after the clip-induced injury. Functional recovery was assessed by the BBB and Tail-Flick tests for 28 days. The animals that had received 10 mg/kg niclosamide exhibited significant improvement in locomotor and sensory outcomes compared to intact SCI animals. MRI imaging performed on day 7 showed a decrease in lesion size and greater preservation of the surrounding tissue in niclosamide-treated animals. Histopathological assessment on day 28 showed less inflammation and hemorrhage; less vacuolization of the neuron cell bodies; and a decrease in cyst formation. ELISA testing indicated significantly lower levels of IL-6, IL-1β, and TNF-α, and western blot analysis indicated low phosphorylation of ERK1/2, p38 MAPK, STAT3, and mTOR, demonstrating inhibition of inflammatory and apoptotic signaling pathways following injury. These findings collectively indicate that niclosamide has neuroprotective effects across multiple targets and leads to improvements in functional recovery following SCI. These results support niclosamide as a promising candidate for spinal cord repair. STAT3 NF-κB MAPK/ERK mTOR TNF-α Neuropathic pain Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Spinal cord injury (SCI) is a severe and debilitating neurological condition resulting in permanent motor and sensory deficits, profoundly impacting patients’ quality of life. The complex pathophysiology of SCI involves both primary mechanical injury and secondary processes, including inflammation, oxidative stress, apoptotic cell death, and glial scar formation, all contributing to poor functional recovery and limited neuroregeneration (Ahuja et al., 2017; Kwon et al., 2004). Despite advances in research, no effective curative treatment exists, and current therapeutic strategies mainly focus on mitigating secondary damage and neuroinflammation. Therefore, identifying pharmacological agents capable of reducing neuroinflammation and limiting secondary injury is of high clinical relevance (Palanisamy et al., 2023). Niclosamide, a long-approved FDA anthelmintic agent, is primarily used against tapeworm infections by disrupting mitochondrial oxidative phosphorylation (Chen et al., 2017). However, recent studies have highlighted its anti-inflammatory, antioxidant, and neuroprotective effects in various disease models (Milani et al., 2024). Niclosamide exerts these effects mainly through the suppression of key intracellular inflammatory pathways, notably STAT3 and NF-κB signaling, which play major roles in immune activation, cytokine production, and apoptosis following central nervous system injuries (Huanget al., 2015; Thatikonda et al., 2020; Lu et al., 2025; Wu et al., 2013). Another crucial pathological mechanism following SCI involves the aberrant activation of mitogen-activated protein kinase (MAPK) pathways, particularly p38 MAPK and ERK1/2. Hyperactivation of p38 MAPK and p-ERK1/2 has been associated with increased inflammatory responses, glial activation, and neuronal apoptosis after SCI (Huang et al., 2024; Gwak et al., 2009). Thus, pharmacological agents capable of inhibiting these pathways may reduce secondary tissue damage and support neuroprotection. Additionally, dysregulation of the mTOR (mammalian target of rapamycin) pathway, particularly overactivation of mTORC1, contributes to enhanced inflammation, oxidative stress, and impaired autophagy after SCI (Darabniya, 2025; Panwar et al., 2023). Niclosamide has been recognized as an effective inhibitor of mTORC1, capable of alleviating inflammatory cascades and supporting neuronal survival (Fonseca et al., 2012). To date, no experimental study has evaluated the effects of niclosamide on these inflammatory pathways in a spinal cord injury model. This study aims to investigate the therapeutic potential of niclosamide in a rat model of SCI, focusing on its effects on functional recovery, neuroinflammation, and modulation of STAT3, p38 MAPK, p-ERK1/2, and mTOR pathways. Uncovering its molecular mechanisms may introduce new therapeutic strategies for spinal cord injury management. 2. Materials and Methods 2.1.Animals This study was conducted on 48 male Sprague–Dawley rats (250–300 g, n = 8 per group) obtained from an accredited animal research facility. The animals were housed under standard laboratory conditions (23 ± 2°C, 50 ± 5% humidity, 12-hour light/dark cycle) and had ad libitum access to food and water. To minimize stress, animals were acclimatized for one week before the experimental procedures 2.2.Drugs Niclosamide was obtained from Behrood Atrak Pharmaceutical Company (Arak, Iran), while Buprenorphine was sourced from Sigma (St. Louis, Missouri, USA). Ketamine and Xylazine were purchased from Alfasan (Woerden, Netherlands). All drugs were dissolved in normal saline (0.9%) and administered intraperitoneally at the time of surgery at a dosage volume of 1 ml/kg. 2.3.Induction of Spinal Cord Injury (SCI) Rats were anesthetized with intraperitoneal ketamine (87.7mg/kg) and xylazine (12.3 mg/kg). A T8 laminectomy was performed to expose the spinal cord, followed by a moderate contusion injury using an aneurysm clip (110 g, 60 sec), a widely accepted model that replicates secondary damage caused by post-traumatic inflammation and apoptosis. After injury, the muscles and skin were sutured, and the animals were transferred to individual recovery cages for post-operative monitoring. Manual bladder expression was performed twice daily until spontaneous urination resumed (Afshari et al., 2018; Aghili et al., 2024). (as shown in Fig. 1 ) Spinal cord injury was induced on day 0, followed by a single intraperitoneal injection of Niclosamide. Behavioral assessments were conducted on specified days, an MRI was performed on day 7, and tissue samples were collected on day 28 for histological and molecular analyses. 2.4.Post-Surgical Care To prevent infection and manage post-operative pain, the following treatments were administered: • Cefazolin (25 mg/kg, IP) – A broad-spectrum antibiotic, administered once daily for three days (Flecknell et al., 2017). • Buprenorphine (0.05 mg/kg, SC) – An opioid analgesic, administered twice daily for three days (Albus et al., 2012). The general health of the animals, including body weight, hydration, and signs of distress, was monitored daily throughout the study. 2.5.Experimental Groups Rats were randomly assigned to six experimental groups (n = 8 per group): 1. Control (SCI + Saline) – Received normal saline (0.9%) intraperitoneally. 2. Sham (Laminectomy Only) – Underwent laminectomy without spinal cord injury. 3. Dimethyl sulfoxide (DMSO) Control (SCI + Vehicle) – Received DMSO + saline (0.9%) intraperitoneally to control for potential solvent effects (Blevins et al., 2002). 4. Niclosamide 2 mg/kg – Administered intraperitoneally immediately after SCI. 5. Niclosamide 5 mg/kg – Administered intraperitoneally immediately after SCI. 6. Niclosamide 10 mg/kg – Administered intraperitoneally immediately after SCI. Niclosamide was first dissolved in DMSO and then diluted with normal saline (0.9%) to prepare the final injection solution (Bhanushali et al., 2022). The DMSO control group received the same volume of solvent without the drug to assess potential vehicle effects. 2.6.MRI Acquisition Magnetic resonance imaging (MRI) was performed on day 7 post-injury to evaluate structural alterations in the spinal cord. Three additional rats were used exclusively for imaging purposes and were not part of the primary experimental groups used for behavioral, histological, or molecular assessments. This design was intended to eliminate any potential confounding effects of anesthesia or imaging procedures on the main outcome measures. Animals were anesthetized via intraperitoneal injection of ketamine (87.7 mg/kg) and xylazine (12.3 mg/kg) prior to scanning. MRI was conducted using a clinical 1.5 Tesla scanner (Siemens Avanto 1.5T, Siemens Healthcare, Erlangen, Germany) equipped with a dedicated knee coil, allowing for high-resolution imaging of the rat thoracolumbar spinal cord. T2-weighted sagittal images were acquired to assess lesion extent, edema, and tissue integrity. Imaging parameters were optimized for small animal scanning and included repetition time (TR), echo time (TE), slice thickness, and field of view (FOV) settings tailored to enhance visualization of spinal cord architecture. Following MRI acquisition, the animals were euthanized according to the experimental protocol and were not used for any subsequent procedures. 2.7.Behavioral Assessments 2.7.1. Body Weight Monitoring The body weight of each rat was recorded one day before surgery and on postoperative days 7, 14, 21, and 28 to assess general health status and recovery progression. Measurements were performed using a digital scale with an accuracy of ± 0.1 g. Drug dosages, including niclosamide (10 mg/kg), were calculated based on the individual body weight before administration. 2.7.2.BBB Locomotor Scoring Locomotor recovery was assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, a widely validated method for evaluating functional recovery in SCI models. This scoring system assesses hind limb movement, joint coordination, weight support, and stepping ability. Rats were placed in an open-field arena (90 × 90 × 20 cm) for 10 minutes, and two independent blinded observers assigned BBB scores based on locomotor performance. The scoring system ranges from 0 (complete paralysis) to 21 (normal locomotion): • Scores 0–7: Minimal movement, slight joint flexion, and no weight support. • Scores 8–13: Some weight support with uncoordinated stepping but no consistent limb coordination. • Scores 14–21: Near-normal stepping with coordinated limb movements and improved gait patterns. BBB scoring was conducted on days 0, 3, 5, 7, 14, 21, and 28 post-injury, providing a comprehensive evaluation of locomotor recovery over time. A score below 10 indicated severe functional impairment, while a score above 15 suggested significant recovery (Basso et al., 1995). 2.7.3 Neuropathic pain assessment Tail-Flick Test (Thermal Nociception) The tail-flick test was used to assess thermal pain sensitivity and spinal reflex function. The test was conducted on days 7, 14, 21, and 28 post-SCI. In this test, the rat’s tail was exposed to a focused infrared heat source, and the latency to withdraw the tail was recorded. A prolonged withdrawal time indicates reduced pain sensitivity, which may be due to SCI-induced sensory impairment. Conversely, a significantly shorter withdrawal time suggests thermal hyperalgesia, indicating heightened pain sensitivity. To minimize variability, each rat was tested three times at 5-minute intervals, and the average withdrawal latency was calculated. The maximum cutoff time was set to 10 seconds to prevent tissue damage (Kim et al., 2013). 2.8.Histopathological scoring For histopathological analysis, the injury epicenter of the spinal cord was collected from each animal at day 28 post-injury and fixed in 4% paraformaldehyde for 72 hours. Tissues were then processed for paraffin embedding, and transverse sections (5 µm) were prepared. Sections were deparaffinized at 70°C for 20 minutes, immersed in xylene for 3 minutes, rehydrated through a graded ethanol series, and stained with hematoxylin (Sigma-Aldrich, USA) for 15 minutes. After rinsing in tap water, eosin staining was performed for 2 minutes. Slides were dehydrated in 90%, 96%, and 100% ethanol (2 minutes each), cleared in xylene, and mounted with mounting medium (Merck, Germany). Histological evaluations were performed by a blinded pathologist using a calibrated grid via Axiovision software (Zeiss, Germany) under ×100 magnification. The grid was centered on the lesion site, and a 2 mm area rostral and caudal to the injury was analyzed. Three fields spaced 100 µm apart were assessed per section. A semi-quantitative scoring system was used to assess three parameters: ( 1 ) inflammatory cell infiltration, ( 2 ) neuronal vacuolation, and ( 3 ) parenchymal hemorrhage. Each parameter was scored from 0 to 3 (0 = none, 1 = mild, 2 = moderate, 3 = severe), with a total maximum score of 9 (Qiao et al., 2019). 2.9.Molecular Analysis 2.9.1.ELISA (Enzyme-Linked Immunosorbent Assay) To evaluate the levels of key inflammatory cytokines in the spinal cord, an enzyme-linked immunosorbent assay (ELISA) was performed for IL-6, IL-1β, and TNF-α. Frozen tissue samples were first homogenized thoroughly using a mechanical homogenizer to achieve a uniform mixture. The resulting homogenates were centrifuged at 4,000 rpm for 15 minutes at 4°C to remove cellular debris, and the clear supernatant was collected for analysis. Cytokine levels were quantified using commercially available ELISA kits, following the manufacturers’ protocols: • IL-6 (Cat No: CSB-E04640r,cusabio,USA) • IL-1β (Cat No: RLB00, R&D, USA) • TNF-α (Cat No: RTA00, R&D,USA) Absorbance was measured at 450 nm using a microplate reader. For each cytokine, a standard curve was plotted from known concentrations, and sample concentrations were interpolated using the curve’s linear regression equation. The measured cytokine concentrations were statistically analyzed to compare inflammatory responses among different experimental groups (Darabniya et al., 2025). 2.9.2.Western blot analysis Western blot analysis was conducted according to standard protocols with slight modifications optimized for this study. Briefly, frozen spinal cord tissue samples were homogenized in RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails. Homogenates were centrifuged at 14,000 rpm for 20 minutes at 4°C to collect the supernatant. Total protein concentration was measured using the Bradford Protein Assay Kit (DB0017, DNAbioTech, Iran) following the manufacturer’s instructions. Equal amounts of protein (20 µg per sample) were mixed with an equal volume of 2X Laemmli sample buffer, boiled at 95°C for 5 minutes, and separated on 10% SDS-PAGE gels. The proteins were transferred onto 0.2 µm PVDF membranes (Cat No: 162–0177, Bio-Rad Laboratories, CA, USA) using a semi-dry transfer system. Membranes were blocked with 5% BSA (Cat No: A-7888, Sigma Aldrich, MO, USA) in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature and then incubated overnight at 4°C with the following primary antibodies: • p38 MAPK (1:1000, Cat No: ab308333, Abcam) • Phospho-p38 MAPK (p-p38) (1:1000, Cat No: a50384, antibodies) • Phospho-mTOR (Ser2448) (1:1000, Cat No: 2971S, Cellsignal) • STAT3 (1:1000, Cat No: ab68153, Abcam) • Phosphor-STAT3 (Phospho Y705) (0.5 µg/ml, Cat No: ab171358, Abcam) • Phospho-ERK1/2 (1:5000, Cat No: ab76299, Abcam) • β-actin (1:2500, Cat No: ab8227, Abcam) as an internal loading control After three washes with TBST, membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:10,000, Cat No: ab6721, Abcam) for 1 hour at room temperature. Protein bands were visualized using an Enhanced Chemiluminescence (ECL) detection system and imaged using a chemiluminescent imaging system. Densitometric analysis was performed using ImageJ software (NIH, USA). Protein expression levels were normalized to β-actin, and relative expression levels were calculated by dividing the area under the curve (AUC) of each target protein by that of the corresponding β-actin band. The results were then statistically compared among experimental groups (Darabniya et al., 2025). 2.10.Statistical Analysis All data were expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using Stata software version 17 (StataCorp LLC, College Station, TX, USA). The normality of data distribution was assessed using the Shapiro–Wilk test. Depending on the study design, one-way or two-way ANOVA was applied, followed by Tukey’s post hoc test for multiple group comparisons. For behavioral assessments over time (e.g., BBB and tail-flick tests), repeated measures ANOVA was used. A p-value less than 0.05 was considered statistically significant. Graphs were generated using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA). 2.11.Statement of ethics This study was conducted in strict accordance with both institutional and national ethical standards for animal research. All experimental procedures complied with the ethical principles of the Declaration of Helsinki (World Medical Association, 2013) and followed the guidelines of the International Association for the Study of Pain (International Association for the Study of Pain.Guidelines for the Use of Animals in Research, 1983) for the humane use of animals in scientific research. The experimental protocol was reviewed and approved by the Ethics Committee of Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1403.419). Additionally, the principal investigator was certified in “Ethical Principles and Techniques for Working with Laboratory Animals,” a program officially accredited by the Iranian Ministry of Health and conducted by Tehran University of Medical Sciences. 3. Results 3.1.MRI Findings To assess the structural integrity of the spinal cord and determine the extent of injury, T2-weighted sagittal MRI imaging was performed on day 7 post-injury. Representative images from each group are shown in Fig. 2 . In the SCI (control) group (Fig. 2 -A), a pronounced hyperintense signal was observed at the injury epicenter, indicative of severe edema, tissue disruption, and potential cystic cavitation. These features reflect extensive inflammatory infiltration and structural damage to the spinal cord. In contrast, the niclosamide-treated group (10 mg/kg, Fig. 2 -B) showed markedly reduced hyperintensity, suggesting less edema and better tissue preservation. The spinal cord architecture appeared more intact, implying that niclosamide administration led to attenuated secondary injury and enhanced neuroprotection. In the sham-operated group (Fig. 2 -C), spinal cord morphology and signal intensity were normal, confirming the absence of trauma and the validity of the surgical control. These imaging results are consistent with the behavioral outcomes (significantly improved BBB locomotor scores and tail flick latency in the niclosamide-treated group), histopathological findings (lower injury scores), and molecular analyses (reduced pro-inflammatory cytokines and modulation of key signaling pathways including p38, mTOR, STAT3, and ERK1/2). Collectively, the data support the neuroprotective and anti-inflammatory effects of niclosamide, particularly at the 10 mg/kg dose, in mitigating spinal cord damage and promoting functional recovery. 3.2. Behavioral Outcomes Following SCI and Niclosamide Treatment 3.2.1. Niclosamide Preserves Body Weight After Spinal Cord Injury (Protective metabolic effect) To monitor the general health status and recovery progress, body weight was measured on days 0 (baseline), 7, 14, 21, and 28 post-injury. As shown in Fig. 3 , rats in the SCI (Control) and DMSO groups exhibited a progressive and significant decline in body weight throughout the study period, reflecting systemic effects of injury and inflammation. In contrast, treatment with Niclosamide resulted in a dose-dependent attenuation of weight loss. Notably, the 10 mg/kg Niclosamide group showed a significant improvement in weight retention from day 14 onward compared to the SCI group (****p < 0.0001), suggesting a potential protective or systemic stabilizing effect. The sham-operated animals maintained a stable body weight throughout the experiment. 3.2.2. Niclosamide Enhances Locomotor Recovery in a Dose-Dependent Manner (Functional restoration assessed by BBB scoring) Locomotor function was evaluated using the BBB (Basso, Beattie, Bresnahan) scoring system on days 0, 1, 3, 5, 7, 14, 21, and 28. As illustrated in Fig. 4 , animals in the SCI and DMSO groups demonstrated poor motor recovery with consistently low BBB scores across the time points. In contrast, Niclosamide-treated groups exhibited a marked and dose-dependent improvement in hindlimb motor function. Particularly, the 10 mg/kg group showed significantly higher BBB scores starting from day 5 and maintained improved recovery through day 28 compared to SCI controls (***p < 0.001, ****p < 0.0001). This finding indicates enhanced functional restoration in Niclosamide-treated rats. Sham animals preserved normal motor activity with a consistent score of 21 throughout the study. 3.2.3. Niclosamide Attenuates Thermal Hyperalgesia in SCI Rats (Improved sensory function assessed by Tail Flick Test) The tail flick test was performed to assess thermal nociceptive thresholds. As shown in Fig. 5 , SCI and DMSO groups exhibited a peak in latency on day 7, followed by a decline indicating the development of thermal hyperalgesia over time. In contrast, rats treated with Niclosamide maintained higher tail flick latencies in a dose-dependent manner. The 10 mg/kg group showed significantly prolonged latencies at days 14, 21, and 28 compared to the SCI group (***p < 0.001, ****p < 0.0001), suggesting reduced hyperalgesia and better sensory recovery. Sham animals exhibited stable baseline latency throughout the experimental period. 3.3. Histopathological Assessment Histological evaluation of spinal cord tissue at day 28 post-injury revealed significant differences among experimental groups in terms of tissue integrity and pathological scoring. In the SCI (control) and DMSO-treated groups, histopathological damage was pronounced, as evidenced by higher cumulative scores reflecting severe inflammatory cell infiltration, axonal vacuolation, hemorrhage, and cyst formation. The average histopathological score in these groups was approximately 2.8 to 3, indicating substantial structural damage. In contrast, treatment with niclosamide led to a dose-dependent reduction in tissue damage. Animals receiving 2 mg/kg of niclosamide showed a moderate decrease in histological injury scores compared to SCI and DMSO groups, while 5 mg/kg provided further reduction. Notably, the group treated with 10 mg/kg niclosamide exhibited a marked improvement in tissue morphology, with significantly lower scores (mean score < 1.0), approaching near-normal histological features. Statistical analysis confirmed that this reduction was highly significant (****p < 0.0001) when compared with both the SCI and vehicle groups. (as shown in Fig. 6 ) These findings suggest that a single intraperitoneal administration of niclosamide immediately after SCI provides robust neuroprotective effects, as evidenced by a substantial reduction in the hallmarks of secondary tissue damage. The data support the hypothesis that niclosamide attenuates inflammatory and degenerative processes in the injured spinal cord and may promote structural preservation in a dose-dependent manner.(as shown in Fig. 7 ) 3.4. Molecular Results: Effect of Niclosamide on Inflammatory Cytokines and Signaling Pathways To investigate the molecular mechanisms underlying the observed neuroprotective effects of niclosamide, levels of key inflammatory cytokines and intracellular signaling proteins were assessed in spinal cord tissues on day 28 post-injury, specifically in the group treated with 10 mg/kg niclosamide, compared to SCI and sham groups. 3.4.1. ELISA Results: Niclosamide Attenuates Inflammatory Cytokine Expression To investigate the anti-inflammatory effects of niclosamide following spinal cord injury (SCI), ELISA analysis was performed to quantify the levels of key pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 in spinal cord tissues on day 28 post-injury (Fig. 8 ). TNF-α (Fig. 8 -A): The SCI group exhibited a significant increase in TNF-α levels compared to the sham group (p < 0.001). Treatment with niclosamide (10 mg/kg) resulted in a significant reduction in TNF-α levels compared to the SCI group (p < 0.01), indicating suppression of acute inflammatory response. IL-1β (Fig. 8 -B): Similarly, IL-1β levels were markedly elevated in the SCI group relative to sham (p < 0.001). Niclosamide treatment significantly reduced IL-1β expression compared to the SCI group (p < 0.01), supporting its role in modulating neuroinflammation. IL-6 (Fig. 8 -C): The SCI group showed a significant elevation in IL-6 levels versus sham (p < 0.001). Niclosamide administration significantly decreased IL-6 levels compared to SCI (p < 0.01), although the values remained higher than the sham group (p < 0.05). These findings confirm the anti-inflammatory potential of niclosamide in the SCI model through effective downregulation of proinflammatory cytokines. 3.4.2 Western Blot Results: To investigate the molecular mechanisms underlying the neuroprotective effects of niclosamide, we evaluated the expression of key signaling proteins involved in inflammation and neuroregeneration using Western blotting. Proteins assessed included phosphorylated and total forms of ERK1/2, MAPK, STAT3, and mTOR. Densitometric quantification of protein bands was normalized to β-actin and is shown in Fig. 9 . Following spinal cord injury (SCI), expression levels of P-ERK1/2, P-MAPK/MAPK, P-mTOR, and P-STAT3/STAT3 were significantly increased compared to the sham group (p < 0.01 to p < 0.0001), indicating activation of these pathways in response to injury. Treatment with a single dose of niclosamide (10 mg/kg) markedly attenuated these increases across all four markers: • P-ERK1/2/β-actin expression (Fig. 9 -A) was significantly reduced in the niclosamide group compared to SCI (p < 0.01). • P-MAPK/MAPK ratio (Fig. 9 -B) was also significantly lower in niclosamide-treated rats (p < 0.01). • A significant decrease in P-mTOR/β-actin was observed in the niclosamide group (Fig. 9 -C, p < 0.01). • Notably, P-STAT3/STAT3 expression (Fig. 9 -D) was also significantly downregulated by niclosamide compared to untreated SCI animals (p < 0.0001). Representative Western blot bands for each protein are presented in Fig. 9 -E, showing a clear visual reduction in phosphorylation levels following niclosamide administration. These results suggest that niclosamide suppresses injury-induced activation of MAPK/ERK, mTOR, and STAT3 pathways, supporting its anti-inflammatory and neuroprotective role in the SCI model. 4. Discussion In the present study, we demonstrate that a single intraperitoneal dose of niclosamide significantly attenuated spinal cord injury (SCI)–induced pathological and functional deficits in a rat model. While niclosamide is traditionally recognized as an anti-helminthic agent, our data contribute to an expanding body of evidence suggesting that this compound possesses potent anti-inflammatory and neuroprotective properties that could be repurposed for central nervous system (CNS) disorders (Apolloni et al., 2023; Milani et al., 2024). A key strength of our study is the comprehensive evaluation encompassing behavioral, molecular, MRI, and histopathological endpoints, allowing for a multidimensional assessment of therapeutic efficacy. The significant improvements in BBB scores, tail-flick latency, and preservation of body weight in niclosamide-treated animals underscore its capacity to mitigate both motor and sensory impairments following SCI. While previous studies have shown similar functional benefits with anti-inflammatory agents such as methylprednisolone, the fact that niclosamide achieved meaningful recovery with a single post-injury dose is remarkable and clinically promising (Evaniew et al., 2015). From a mechanistic standpoint, our results indicate that niclosamide effectively suppresses SCI-induced upregulation of proinflammatory cytokines (IL‑6, IL‑1β, TNF‑α), as demonstrated by ELISA assays. More importantly, Western blot analysis revealed significant downregulation of phosphorylated ERK1/2, p38 MAPK, STAT3, and mTOR, suggesting that niclosamide interferes with multiple converging signaling cascades known to mediate neuroinflammation and cell death (Sekulovski et al., 2020; Singh et al., 2024). Importantly, NF‑κB is a master transcription factor driving expression of key inflammatory mediators such as TNF‑α and IL‑1β, and its sustained activation in the injured spinal cord contributes to chronic neuroinflammation and tissue damage. Inhibition of NF‑κB signaling has been shown to reduce the expression of proinflammatory cytokines and preserve spinal cord tissue integrity (Brambilla et al., 2005). Although we did not directly assess NF‑κB activation, the reduction in cytokine levels in the niclosamide-treated group supports potential suppression of this pathway. Interestingly, while the ERK1/2 pathway is implicated in neuronal survival and regeneration, sustained activation in the late phase of SCI (as in day 28 of our model) may reflect ongoing inflammatory responses and glial scar formation rather than regenerative processes (Yu et al., 2005). Thus, the downregulation of p‑ERK1/2 observed here may represent a beneficial suppression of chronic inflammatory signaling rather than inhibition of neurogenesis (Li et al., 2021). STAT3 activation plays dual roles in promoting astrocyte reactivity and scarring while also contributing to limited regeneration (Herrmann et al., 2008). Our findings that niclosamide suppresses STAT3 phosphorylation point toward a net anti-inflammatory and anti-gliotic effect. The modulation of mTOR signaling by niclosamide also merits attention. mTORC1 is a critical regulator of cellular metabolism and autophagy, and its inhibition has been shown to promote neuroprotection in various CNS injury models, including SCI, by enhancing autophagy markers and reducing neuronal death (Ding et al., 2022; Kanno et al., 2012). In our study, reduced phosphorylation of mTOR suggests that niclosamide may restore metabolic homeostasis and prevent apoptosis in the injured spinal cord, consistent with these prior preclinical observations. Together, these data suggest that niclosamide acts as a multi-targeted therapeutic, modulating multiple pathways—NF‑κB, MAPK/ERK, STAT3, mTOR—that are central to the pathophysiology of SCI. To our knowledge, this is the first study evaluating niclosamide in a spinal cord injury model. The robust efficacy noted with a single dose, combined with its safety profile, underscores its translational potential. Nevertheless, certain limitations warrant acknowledgement. We assessed only one dose level for molecular analyses, and long-term outcomes beyond 28 days remain unknown. Moreover, while Western blotting provided mechanistic insights, further studies employing immunofluorescence, neuronal tracing, or single-cell transcriptomics could offer a deeper understanding of cell-specific effects. Additionally, whether niclosamide’s benefits extend to chronic SCI models or other injury paradigms requires further investigation. 5. Conclusion This study demonstrates, for the first time, that a single intraperitoneal dose of niclosamide (10 mg/kg) administered immediately after spinal cord injury exerts significant neuroprotective and anti-inflammatory effects in a rat contusion model. Niclosamide markedly improved locomotor and sensory function, reduced histopathological damage, attenuated proinflammatory cytokine expression, and suppressed the activation of key signaling pathways involved in secondary injury, including STAT3, mTOR, and MAPK/ERK. These results highlight the potential of niclosamide as a promising therapeutic agent for SCI, capable of modulating both inflammatory and regenerative mechanisms. Given its FDA-approved status and favorable safety profile, niclosamide may be a viable candidate for repurposing in the treatment of spinal cord injury. However, further investigations are warranted to explore its long-term efficacy, optimal dosing strategies, and underlying cellular mechanisms in both acute and chronic models. Declarations Acknowledgments The authors would like to express their sincere gratitude to Dr. Masoud Fazeli, radiologist and faculty member at Arak University of Medical Sciences, for generously providing access to MRI facilities and technical support. We also acknowledge Mr. Shamseddin Mashayekhi, CEO of Behrood Atrak Pharmaceutical Company, for his supportive role in facilitating this research. Special thanks to Dr. Shahrzad Jalilpour Rezaei for her assistance in editing several figures used in the manuscript. Author Contributions Ali Darabniya led the study, performed the spinal cord injury surgeries, developed the scientific framework, conducted all experimental procedures, analyzed the data, and drafted the initial version of the manuscript. Ahmad Reza Dehpour was responsible for the overall study design and conceptual development, supervised the research process, and contributed to the critical revision of the manuscript. Sara Shahriari and Reza Fazeli contributed to the execution of experiments, data collection, and data analysis, and both reviewed the initial draft and provided constructive feedback. Seyed Mohammad Tavangar performed histopathological assessments, contributed to the pathology section of the manuscript, and participated in reviewing the draft and giving constructive comments. Razieh Mohammadi Jafari assisted in laboratory procedures and animal care, and also contributed to reviewing and improving the early version of the manuscript. All authors reviewed and approved the final version of the manuscript. Funding This study was financially supported by the Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran (Grant No 1403-3-101-74387) Data availability The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Consent to Participate Not applicable. 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Brambilla R, Bracchi-Ricard V, Hu WH, Frydel B, et al. Inhibition of astroglial nuclear factor kappaB reduces inflammation and improves functional recovery after spinal cord injury. J Exp Med. 2005;202(1):145–56. doi: 10.1084/jem.20041918 . Yu CG, Yezierski RP. Activation of the ERK1/2 signaling cascade by excitotoxic spinal cord injury. Brain Res Mol Brain Res. 2005;138(2):244–55. doi: 10.1016/j.molbrainres.2005.04.013 . Li J, Jia Z, Zhang Q, et al. Inhibition of ERK1/2 phosphorylation attenuates spinal cord injury induced astrocyte activation and inflammation through negatively regulating aquaporin-4 in rats. Brain Res Bull. 2021;170:162–173. doi: 10.1016/j.brainresbull.2021.02.014 . Herrmann JE, Imura T, Song B, et al. STAT3 is a critical regulator of astrogliosis and scar formation after spinal cord injury. J Neurosci. 2008;28(28):7231–43. doi: 10.1523/JNEUROSCI.1709-08.2008 . Ding Y, Chen Q. mTOR pathway: A potential therapeutic target for spinal cord injury. Biomed Pharmacother. 2022;145:112430. doi: 10.1016/j.biopha.2021.112430 . Kanno H, Ozawa H, Sekiguchi A, et al. The role of mTOR signaling pathway in spinal cord injury. Cell Cycle. 2012;11(17):31759. doi: 10.4161/cc.21262 . Epub 2012 Aug 16. Additional Declarations No competing interests reported. Supplementary Files Bactin.jpg pErk12.jpg STAT3.jpg MAPK.jpg PhosphomTOR.jpg PSTAT3.jpg PMAPK.jpg VideoS1Niclo10Day281.Mp4.mp4 VideoS2Niclo10Day282.Mp4.mp4 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":708273,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic overview of the experimental design.\u003c/p\u003e\n\u003cp\u003eSpinal cord injury was induced on day 0, followed by a single intraperitoneal injection of Niclosamide. Behavioral assessments were conducted on specified days, an MRI was performed on day 7, and tissue samples were collected on day 28 for histological and molecular analyses.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/1c7bf3cb569fae18a81e7a07.png"},{"id":92739169,"identity":"fd5ea5c1-0bec-4766-9776-7308c4e85f9c","added_by":"auto","created_at":"2025-10-03 17:02:17","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":269152,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative sagittal T2-weighted MRI images of rat spinal cords obtained on day 7 post-injury.\u003c/p\u003e\n\u003cp\u003e(A) The SCI group (control) shows a prominent hyperintense cavity at the injury site, indicating extensive edema and tissue disruption. (B) The niclosamide-treated group (10 mg/kg) exhibits reduced lesion size and better preservation of spinal cord structure compared to the control.(C) The sham-operated group demonstrates intact spinal cord morphology with no evidence of lesion or edema.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/cf725fe989dd52be22611445.jpeg"},{"id":92737644,"identity":"112d383b-e753-4751-9c82-7953b1fa9b72","added_by":"auto","created_at":"2025-10-03 16:46:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":174837,"visible":true,"origin":"","legend":"\u003cp\u003eBody weight changes in rats following spinal cord injury (SCI) and treatment with Niclosamide. Weight was recorded on days 0, 7, 14, 21, and 28. Niclosamide-treated groups showed a dose-dependent attenuation of weight loss compared to the SCI and DMSO groups. Data are presented as mean ± SEM. Statistical significance vs. SCI group: ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/53140643b2098e379cc46a68.png"},{"id":92737642,"identity":"33df143e-2dad-4025-8833-40b27de7e6a4","added_by":"auto","created_at":"2025-10-03 16:46:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":214433,"visible":true,"origin":"","legend":"\u003cp\u003eBBB locomotor scores of rats over 28 days post-injury. Niclosamide improved locomotor recovery in a dose-dependent manner, with the 10 mg/kg group showing significant improvements starting from day 5. Sham group maintained normal function. Data are mean ± SEM. Significance vs. SCI group: **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/be30a51139db9efcd84f5000.png"},{"id":92739172,"identity":"fdb4d7d0-fefc-4680-b560-4c2fbdab21d7","added_by":"auto","created_at":"2025-10-03 17:02:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":180265,"visible":true,"origin":"","legend":"\u003cp\u003eTail flick latency measurements in seconds, used to assess thermal nociception following SCI. Niclosamide-treated animals demonstrated prolonged latencies, indicating reduced hyperalgesia compared to SCI and DMSO groups. Data are mean ± SEM.\u003c/p\u003e\n\u003cp\u003e***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 vs. SCI.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/58f0bf7340c80a85954e289c.png"},{"id":92739171,"identity":"0d14ddb2-1335-4095-ad58-1d74daea9475","added_by":"auto","created_at":"2025-10-03 17:02:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59778,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological scoring of spinal cord tissue at day 28 post-injury.\u003c/p\u003e\n\u003cp\u003eHistological evaluation was performed using hematoxylin and eosin (H\u0026amp;E) staining to assess key pathological features including axonal vacuolation, inflammatory cell infiltration, hemorrhage, and cyst formation. The SCI and DMSO groups exhibited significantly higher scores, indicating severe tissue damage. In contrast, niclosamide-treated groups, particularly the 10 mg/kg dose, showed marked reductions in histopathological scores, suggesting neuroprotective and anti-inflammatory effects. Data are presented as mean ± SEM.\u003c/p\u003e\n\u003cp\u003eStatistical significance: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ***p \u0026lt; 0.0001 vs. SCI group.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/3dd687704997b5a22bd8582a.png"},{"id":92737662,"identity":"87fdada6-7042-4876-ba60-c44478f18a7c","added_by":"auto","created_at":"2025-10-03 16:46:18","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":464990,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological evaluation of spinal cord tissue on day 28 post-injury using H\u0026amp;E staining.\u003c/p\u003e\n\u003cp\u003eA₁, A₂: Spinal cord tissue from the SCI group (control) showing severe tissue disorganization, necrosis, and cavitation. A₁ captured at low magnification (×100) and A₂ at high magnification (×400).\u003c/p\u003e\n\u003cp\u003eB₁, B₂: Sections from the niclosamide-treated group (10 mg/kg) demonstrating improved tissue integrity, reduced vacuolation, and decreased cellular degeneration. B₁ at ×100, B₂ at ×400.\u003c/p\u003e\n\u003cp\u003eC₁, C₂: Samples from the sham-operated group, showing normal spinal cord architecture with intact neurons and preserved tissue structure. C₁ at ×100, C₂ at ×400.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/43fde628e0f59cf22a3c74f9.jpeg"},{"id":92737630,"identity":"a59cee6f-13da-4c0b-8045-903cf98df295","added_by":"auto","created_at":"2025-10-03 16:46:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":267923,"visible":true,"origin":"","legend":"\u003cp\u003eNiclosamide attenuates the expression of pro-inflammatory cytokines in spinal cord tissue following SCI.\u003c/p\u003e\n\u003cp\u003eQuantitative ELISA analysis of (A) TNF-α, (B) IL-1β, and (C) IL-6 levels in spinal cord homogenates at day 28 post-injury. The SCI group exhibited significantly elevated cytokine levels compared to the sham group. Treatment with niclosamide (10 mg/kg) significantly reduced the levels of all three cytokines. Data are presented as mean ± SD (n=8 per group). Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/a1e6226b0da0bd065013f792.png"},{"id":92737664,"identity":"496f80fc-f81a-48a7-ac97-4bd54d36489b","added_by":"auto","created_at":"2025-10-03 16:46:18","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":618260,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot analysis of key signaling proteins in spinal cord tissue.\u003c/p\u003e\n\u003cp\u003e(A–D) Densitometric quantification of the relative expression of P-ERK1/2, P-MAPK, P-mTOR, and P-STAT3 in the sham, SCI, and niclosamide (10 mg/kg) groups.\u003c/p\u003e\n\u003cp\u003e(E) Representative immunoblots of phosphorylated and total protein levels. β-actin was used as a loading control. Data are presented as mean ± SD; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/3cdeab17ef867f65b8b1e3e7.png"},{"id":94473965,"identity":"2c59fd0a-a30a-46b1-be3f-6dbe39108ddb","added_by":"auto","created_at":"2025-10-27 15:46:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4075940,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/42dc2541-baa2-4f15-9984-cb67269e0b4d.pdf"},{"id":92737643,"identity":"49468cb3-9e0f-4b8c-839d-9a121b16186b","added_by":"auto","created_at":"2025-10-03 16:46:18","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":156985,"visible":true,"origin":"","legend":"","description":"","filename":"Bactin.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/4e98f603baf2644ba8f96f29.jpg"},{"id":92737633,"identity":"e50436e7-6c56-49ed-a4c1-72fab5a50757","added_by":"auto","created_at":"2025-10-03 16:46:17","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":174568,"visible":true,"origin":"","legend":"","description":"","filename":"pErk12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/9e9df41e4d9d69a328548c53.jpg"},{"id":92737648,"identity":"a3ce7d16-8d90-4b18-8bd6-74caa8083852","added_by":"auto","created_at":"2025-10-03 16:46:18","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":169391,"visible":true,"origin":"","legend":"","description":"","filename":"STAT3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/320561c841468054368990c4.jpg"},{"id":92738671,"identity":"c0b6a726-010a-4356-9ed4-99eefeb71ff4","added_by":"auto","created_at":"2025-10-03 16:54:18","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":164123,"visible":true,"origin":"","legend":"","description":"","filename":"MAPK.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/3995aeff5dc75fda636855c8.jpg"},{"id":92737636,"identity":"531af766-e8ca-48c7-809c-70de79a7f31d","added_by":"auto","created_at":"2025-10-03 16:46:17","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":162408,"visible":true,"origin":"","legend":"","description":"","filename":"PhosphomTOR.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/575d5a6acf30ddab85b90030.jpg"},{"id":92737638,"identity":"68cf768d-3f1a-4c46-a53c-df2b64ad38e8","added_by":"auto","created_at":"2025-10-03 16:46:17","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":184465,"visible":true,"origin":"","legend":"","description":"","filename":"PSTAT3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/a4d0328a9d780749d3a7c613.jpg"},{"id":92737678,"identity":"c209c7aa-1e49-4166-99f9-d5315f1ce7c6","added_by":"auto","created_at":"2025-10-03 16:46:19","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":170837,"visible":true,"origin":"","legend":"","description":"","filename":"PMAPK.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/eb9ffe7e8180eaf4adebc588.jpg"},{"id":92737693,"identity":"c0994d6e-1465-4af8-9cb3-1237bab37b93","added_by":"auto","created_at":"2025-10-03 16:46:19","extension":"mp4","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15666579,"visible":true,"origin":"","legend":"","description":"","filename":"VideoS1Niclo10Day281.Mp4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/50b576f5b3b625cd9180e579.mp4"},{"id":92737645,"identity":"f61a7966-2ace-4476-90f2-fee211dc2471","added_by":"auto","created_at":"2025-10-03 16:46:18","extension":"mp4","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":8083884,"visible":true,"origin":"","legend":"","description":"","filename":"VideoS2Niclo10Day282.Mp4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7609682/v1/6ac583aa60299bab2f07a870.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"Niclosamide as a Neuroprotective and Anti-Inflammatory Agent: A Potential Therapeutic Approach for Spinal Cord Injury","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSpinal cord injury (SCI) is a severe and debilitating neurological condition resulting in permanent motor and sensory deficits, profoundly impacting patients\u0026rsquo; quality of life. The complex pathophysiology of SCI involves both primary mechanical injury and secondary processes, including inflammation, oxidative stress, apoptotic cell death, and glial scar formation, all contributing to poor functional recovery and limited neuroregeneration (Ahuja et al., 2017; Kwon et al., 2004).\u003c/p\u003e\u003cp\u003eDespite advances in research, no effective curative treatment exists, and current therapeutic strategies mainly focus on mitigating secondary damage and neuroinflammation. Therefore, identifying pharmacological agents capable of reducing neuroinflammation and limiting secondary injury is of high clinical relevance (Palanisamy et al., 2023).\u003c/p\u003e\u003cp\u003eNiclosamide, a long-approved FDA anthelmintic agent, is primarily used against tapeworm infections by disrupting mitochondrial oxidative phosphorylation (Chen et al., 2017). However, recent studies have highlighted its anti-inflammatory, antioxidant, and neuroprotective effects in various disease models (Milani et al., 2024). Niclosamide exerts these effects mainly through the suppression of key intracellular inflammatory pathways, notably STAT3 and NF-κB signaling, which play major roles in immune activation, cytokine production, and apoptosis following central nervous system injuries (Huanget al., 2015; Thatikonda et al., 2020; Lu et al., 2025; Wu et al., 2013).\u003c/p\u003e\u003cp\u003eAnother crucial pathological mechanism following SCI involves the aberrant activation of mitogen-activated protein kinase (MAPK) pathways, particularly p38 MAPK and ERK1/2. Hyperactivation of p38 MAPK and p-ERK1/2 has been associated with increased inflammatory responses, glial activation, and neuronal apoptosis after SCI (Huang et al., 2024; Gwak et al., 2009). Thus, pharmacological agents capable of inhibiting these pathways may reduce secondary tissue damage and support neuroprotection.\u003c/p\u003e\u003cp\u003eAdditionally, dysregulation of the mTOR (mammalian target of rapamycin) pathway, particularly overactivation of mTORC1, contributes to enhanced inflammation, oxidative stress, and impaired autophagy after SCI (Darabniya, 2025; Panwar et al., 2023). Niclosamide has been recognized as an effective inhibitor of mTORC1, capable of alleviating inflammatory cascades and supporting neuronal survival (Fonseca et al., 2012).\u003c/p\u003e\u003cp\u003eTo date, no experimental study has evaluated the effects of niclosamide on these inflammatory pathways in a spinal cord injury model. This study aims to investigate the therapeutic potential of niclosamide in a rat model of SCI, focusing on its effects on functional recovery, neuroinflammation, and modulation of STAT3, p38 MAPK, p-ERK1/2, and mTOR pathways. Uncovering its molecular mechanisms may introduce new therapeutic strategies for spinal cord injury management.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1.Animals\u003c/h2\u003e\n \u003cp\u003eThis study was conducted on 48 male Sprague\u0026ndash;Dawley rats (250\u0026ndash;300 g, n\u0026thinsp;=\u0026thinsp;8 per group) obtained from an accredited animal research facility. The animals were housed under standard laboratory conditions (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5% humidity, 12-hour light/dark cycle) and had ad libitum access to food and water. To minimize stress, animals were acclimatized for one week before the experimental procedures\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2.Drugs\u003c/h2\u003e\n \u003cp\u003eNiclosamide was obtained from Behrood Atrak Pharmaceutical Company (Arak, Iran), while Buprenorphine was sourced from Sigma (St. Louis, Missouri, USA). Ketamine and Xylazine were purchased from Alfasan (Woerden, Netherlands). All drugs were dissolved in normal saline (0.9%) and administered intraperitoneally at the time of surgery at a dosage volume of 1 ml/kg.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3.Induction of Spinal Cord Injury (SCI)\u003c/h2\u003e\n \u003cp\u003eRats were anesthetized with intraperitoneal ketamine (87.7mg/kg) and xylazine (12.3 mg/kg). A T8 laminectomy was performed to expose the spinal cord, followed by a moderate contusion injury using an aneurysm clip (110 g, 60 sec), a widely accepted model that replicates secondary damage caused by post-traumatic inflammation and apoptosis. After injury, the muscles and skin were sutured, and the animals were transferred to individual recovery cages for post-operative monitoring. Manual bladder expression was performed twice daily until spontaneous urination resumed (Afshari et al., 2018; Aghili et al., 2024). (as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n \u003cp\u003eSpinal cord injury was induced on day 0, followed by a single intraperitoneal injection of Niclosamide. Behavioral assessments were conducted on specified days, an MRI was performed on day 7, and tissue samples were collected on day 28 for histological and molecular analyses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4.Post-Surgical Care\u003c/h2\u003e\n \u003cp\u003eTo prevent infection and manage post-operative pain, the following treatments were administered:\u003c/p\u003e\n \u003cp\u003e\u0026bull; Cefazolin (25 mg/kg, IP) \u0026ndash; A broad-spectrum antibiotic, administered once daily for three days (Flecknell et al., 2017).\u003c/p\u003e\n \u003cp\u003e\u0026bull; Buprenorphine (0.05 mg/kg, SC) \u0026ndash; An opioid analgesic, administered twice daily for three days (Albus et al., 2012).\u003c/p\u003e\n \u003cp\u003eThe general health of the animals, including body weight, hydration, and signs of distress, was monitored daily throughout the study.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5.Experimental Groups\u003c/h2\u003e\n \u003cp\u003eRats were randomly assigned to six experimental groups (n\u0026thinsp;=\u0026thinsp;8 per group):\u003c/p\u003e\n \u003cp\u003e\u003cspan\u003e1. Control (SCI\u0026thinsp;+\u0026thinsp;Saline) \u0026ndash; Received normal saline (0.9%) intraperitoneally.\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan\u003e2. Sham (Laminectomy Only) \u0026ndash; Underwent laminectomy without spinal cord injury.\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan\u003e3. Dimethyl sulfoxide (DMSO) Control (SCI\u0026thinsp;+\u0026thinsp;Vehicle) \u0026ndash; Received DMSO\u0026thinsp;+\u0026thinsp;saline (0.9%) intraperitoneally to control for potential solvent effects (Blevins et al., 2002).\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan\u003e4. Niclosamide 2 mg/kg \u0026ndash; Administered intraperitoneally immediately after SCI.\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan\u003e5. Niclosamide 5 mg/kg \u0026ndash; Administered intraperitoneally immediately after SCI.\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan\u003e6. Niclosamide 10 mg/kg \u0026ndash; Administered intraperitoneally immediately after SCI.\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eNiclosamide was first dissolved in DMSO and then diluted with normal saline (0.9%) to prepare the final injection solution (Bhanushali et al., 2022).\u003c/p\u003e\n \u003cp\u003eThe DMSO control group received the same volume of solvent without the drug to assess potential vehicle effects.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6.MRI Acquisition\u003c/h2\u003e\n \u003cp\u003eMagnetic resonance imaging (MRI) was performed on day 7 post-injury to evaluate structural alterations in the spinal cord. Three additional rats were used exclusively for imaging purposes and were not part of the primary experimental groups used for behavioral, histological, or molecular assessments. This design was intended to eliminate any potential confounding effects of anesthesia or imaging procedures on the main outcome measures.\u003c/p\u003e\n \u003cp\u003eAnimals were anesthetized via intraperitoneal injection of ketamine (87.7 mg/kg) and xylazine (12.3 mg/kg) prior to scanning. MRI was conducted using a clinical 1.5 Tesla scanner (Siemens Avanto 1.5T, Siemens Healthcare, Erlangen, Germany) equipped with a dedicated knee coil, allowing for high-resolution imaging of the rat thoracolumbar spinal cord.\u003c/p\u003e\n \u003cp\u003eT2-weighted sagittal images were acquired to assess lesion extent, edema, and tissue integrity. Imaging parameters were optimized for small animal scanning and included repetition time (TR), echo time (TE), slice thickness, and field of view (FOV) settings tailored to enhance visualization of spinal cord architecture.\u003c/p\u003e\n \u003cp\u003eFollowing MRI acquisition, the animals were euthanized according to the experimental protocol and were not used for any subsequent procedures.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7.Behavioral Assessments\u003c/h2\u003e\n \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\n \u003ch2\u003e2.7.1. Body Weight Monitoring\u003c/h2\u003e\n \u003cp\u003eThe body weight of each rat was recorded one day before surgery and on postoperative days 7, 14, 21, and 28 to assess general health status and recovery progression. Measurements were performed using a digital scale with an accuracy of \u0026plusmn;\u0026thinsp;0.1 g. Drug dosages, including niclosamide (10 mg/kg), were calculated based on the individual body weight before administration.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n \u003ch2\u003e2.7.2.BBB Locomotor Scoring\u003c/h2\u003e\n \u003cp\u003eLocomotor recovery was assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, a widely validated method for evaluating functional recovery in SCI models. This scoring system assesses hind limb movement, joint coordination, weight support, and stepping ability.\u003c/p\u003e\n \u003cp\u003eRats were placed in an open-field arena (90 \u0026times; 90 \u0026times; 20 cm) for 10 minutes, and two independent blinded observers assigned BBB scores based on locomotor performance. The scoring system ranges from 0 (complete paralysis) to 21 (normal locomotion):\u003c/p\u003e\n \u003cp\u003e\u0026bull; Scores 0\u0026ndash;7: Minimal movement, slight joint flexion, and no weight support.\u003c/p\u003e\n \u003cp\u003e\u0026bull; Scores 8\u0026ndash;13: Some weight support with uncoordinated stepping but no consistent limb coordination.\u003c/p\u003e\n \u003cp\u003e\u0026bull; Scores 14\u0026ndash;21: Near-normal stepping with coordinated limb movements and improved gait patterns.\u003c/p\u003e\n \u003cp\u003eBBB scoring was conducted on days 0, 3, 5, 7, 14, 21, and 28 post-injury, providing a comprehensive evaluation of locomotor recovery over time. A score below 10 indicated severe functional impairment, while a score above 15 suggested significant recovery (Basso et al., 1995).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003e2.7.3 Neuropathic pain assessment\u003c/h2\u003e\n \u003cp\u003eTail-Flick Test (Thermal Nociception)\u003c/p\u003e\n \u003cp\u003eThe tail-flick test was used to assess thermal pain sensitivity and spinal reflex function. The test was conducted on days 7, 14, 21, and 28 post-SCI.\u003c/p\u003e\n \u003cp\u003eIn this test, the rat\u0026rsquo;s tail was exposed to a focused infrared heat source, and the latency to withdraw the tail was recorded. A prolonged withdrawal time indicates reduced pain sensitivity, which may be due to SCI-induced sensory impairment. Conversely, a significantly shorter withdrawal time suggests thermal hyperalgesia, indicating heightened pain sensitivity.\u003c/p\u003e\n \u003cp\u003eTo minimize variability, each rat was tested three times at 5-minute intervals, and the average withdrawal latency was calculated. The maximum cutoff time was set to 10 seconds to prevent tissue damage (Kim et al., 2013).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8.Histopathological scoring\u003c/h2\u003e\n \u003cp\u003eFor histopathological analysis, the injury epicenter of the spinal cord was collected from each animal at day 28 post-injury and fixed in 4% paraformaldehyde for 72 hours. Tissues were then processed for paraffin embedding, and transverse sections (5 \u0026micro;m) were prepared. Sections were deparaffinized at 70\u0026deg;C for 20 minutes, immersed in xylene for 3 minutes, rehydrated through a graded ethanol series, and stained with hematoxylin (Sigma-Aldrich, USA) for 15 minutes. After rinsing in tap water, eosin staining was performed for 2 minutes. Slides were dehydrated in 90%, 96%, and 100% ethanol (2 minutes each), cleared in xylene, and mounted with mounting medium (Merck, Germany).\u003c/p\u003e\n \u003cp\u003eHistological evaluations were performed by a blinded pathologist using a calibrated grid via Axiovision software (Zeiss, Germany) under \u0026times;100 magnification. The grid was centered on the lesion site, and a 2 mm area rostral and caudal to the injury was analyzed. Three fields spaced 100 \u0026micro;m apart were assessed per section.\u003c/p\u003e\n \u003cp\u003eA semi-quantitative scoring system was used to assess three parameters: (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) inflammatory cell infiltration, (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) neuronal vacuolation, and (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e) parenchymal hemorrhage. Each parameter was scored from 0 to 3 (0\u0026thinsp;=\u0026thinsp;none, 1\u0026thinsp;=\u0026thinsp;mild, 2\u0026thinsp;=\u0026thinsp;moderate, 3\u0026thinsp;=\u0026thinsp;severe), with a total maximum score of 9 (Qiao et al., 2019).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9.Molecular Analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e2.9.1.ELISA (Enzyme-Linked Immunosorbent Assay)\u003c/h2\u003e\n \u003cp\u003eTo evaluate the levels of key inflammatory cytokines in the spinal cord, an enzyme-linked immunosorbent assay (ELISA) was performed for IL-6, IL-1\u0026beta;, and TNF-\u0026alpha;. Frozen tissue samples were first homogenized thoroughly using a mechanical homogenizer to achieve a uniform mixture. The resulting homogenates were centrifuged at 4,000 rpm for 15 minutes at 4\u0026deg;C to remove cellular debris, and the clear supernatant was collected for analysis.\u003c/p\u003e\n \u003cp\u003eCytokine levels were quantified using commercially available ELISA kits, following the manufacturers\u0026rsquo; protocols:\u003c/p\u003e\n \u003cp\u003e\u0026bull; IL-6 (Cat No: CSB-E04640r,cusabio,USA)\u003c/p\u003e\n \u003cp\u003e\u0026bull; IL-1\u0026beta; (Cat No: RLB00, R\u0026amp;D, USA)\u003c/p\u003e\n \u003cp\u003e\u0026bull; TNF-\u0026alpha; (Cat No: RTA00, R\u0026amp;D,USA)\u003c/p\u003e\n \u003cp\u003eAbsorbance was measured at 450 nm using a microplate reader. For each cytokine, a standard curve was plotted from known concentrations, and sample concentrations were interpolated using the curve\u0026rsquo;s linear regression equation. The measured cytokine concentrations were statistically analyzed to compare inflammatory responses among different experimental groups (Darabniya et al., 2025).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003e2.9.2.Western blot analysis\u003c/h2\u003e\n \u003cp\u003eWestern blot analysis was conducted according to standard protocols with slight modifications optimized for this study. Briefly, frozen spinal cord tissue samples were homogenized in RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails. Homogenates were centrifuged at 14,000 rpm for 20 minutes at 4\u0026deg;C to collect the supernatant. Total protein concentration was measured using the Bradford Protein Assay Kit (DB0017, DNAbioTech, Iran) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n \u003cp\u003eEqual amounts of protein (20 \u0026micro;g per sample) were mixed with an equal volume of 2X Laemmli sample buffer, boiled at 95\u0026deg;C for 5 minutes, and separated on 10% SDS-PAGE gels. The proteins were transferred onto 0.2 \u0026micro;m PVDF membranes (Cat No: 162\u0026ndash;0177, Bio-Rad Laboratories, CA, USA) using a semi-dry transfer system.\u003c/p\u003e\n \u003cp\u003eMembranes were blocked with 5% BSA (Cat No: A-7888, Sigma Aldrich, MO, USA) in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature and then incubated overnight at 4\u0026deg;C with the following primary antibodies:\u003c/p\u003e\n \u003cp\u003e\u0026bull; p38 MAPK (1:1000, Cat No: ab308333, Abcam)\u003c/p\u003e\n \u003cp\u003e\u0026bull; Phospho-p38 MAPK (p-p38) (1:1000, Cat No: a50384, antibodies)\u003c/p\u003e\n \u003cp\u003e\u0026bull; Phospho-mTOR (Ser2448) (1:1000, Cat No: 2971S, Cellsignal)\u003c/p\u003e\n \u003cp\u003e\u0026bull; STAT3 (1:1000, Cat No: ab68153, Abcam)\u003c/p\u003e\n \u003cp\u003e\u0026bull; Phosphor-STAT3 (Phospho Y705) (0.5 \u0026micro;g/ml, Cat No: ab171358, Abcam)\u003c/p\u003e\n \u003cp\u003e\u0026bull; Phospho-ERK1/2 (1:5000, Cat No: ab76299, Abcam)\u003c/p\u003e\n \u003cp\u003e\u0026bull; \u0026beta;-actin (1:2500, Cat No: ab8227, Abcam) as an internal loading control\u003c/p\u003e\n \u003cp\u003eAfter three washes with TBST, membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:10,000, Cat No: ab6721, Abcam) for 1 hour at room temperature.\u003c/p\u003e\n \u003cp\u003eProtein bands were visualized using an Enhanced Chemiluminescence (ECL) detection system and imaged using a chemiluminescent imaging system. Densitometric analysis was performed using ImageJ software (NIH, USA). Protein expression levels were normalized to \u0026beta;-actin, and relative expression levels were calculated by dividing the area under the curve (AUC) of each target protein by that of the corresponding \u0026beta;-actin band. The results were then statistically compared among experimental groups (Darabniya et al., 2025).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10.Statistical Analysis\u003c/h2\u003e\n \u003cp\u003eAll data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses were performed using Stata software version 17 (StataCorp LLC, College Station, TX, USA). The normality of data distribution was assessed using the Shapiro\u0026ndash;Wilk test. Depending on the study design, one-way or two-way ANOVA was applied, followed by Tukey\u0026rsquo;s post hoc test for multiple group comparisons. For behavioral assessments over time (e.g., BBB and tail-flick tests), repeated measures ANOVA was used. A p-value less than 0.05 was considered statistically significant.\u003c/p\u003e\n \u003cp\u003eGraphs were generated using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11.Statement of ethics\u003c/h2\u003e\n \u003cp\u003eThis study was conducted in strict accordance with both institutional and national ethical standards for animal research. All experimental procedures complied with the ethical principles of the Declaration of Helsinki (World Medical Association, 2013) and followed the guidelines of the International Association for the Study of Pain (International Association for the Study of Pain.Guidelines for the Use of Animals in Research, 1983) for the humane use of animals in scientific research. The experimental protocol was reviewed and approved by the Ethics Committee of Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1403.419). Additionally, the principal investigator was certified in \u0026ldquo;Ethical Principles and Techniques for Working with Laboratory Animals,\u0026rdquo; a program officially accredited by the Iranian Ministry of Health and conducted by Tehran University of Medical Sciences.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1.MRI Findings\u003c/h2\u003e\n \u003cp\u003eTo assess the structural integrity of the spinal cord and determine the extent of injury, T2-weighted sagittal MRI imaging was performed on day 7 post-injury. Representative images from each group are shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eIn the SCI (control) group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e-A), a pronounced hyperintense signal was observed at the injury epicenter, indicative of severe edema, tissue disruption, and potential cystic cavitation. These features reflect extensive inflammatory infiltration and structural damage to the spinal cord.\u003c/p\u003e\n \u003cp\u003eIn contrast, the niclosamide-treated group (10 mg/kg, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e-B) showed markedly reduced hyperintensity, suggesting less edema and better tissue preservation. The spinal cord architecture appeared more intact, implying that niclosamide administration led to attenuated secondary injury and enhanced neuroprotection.\u003c/p\u003e\n \u003cp\u003eIn the sham-operated group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e-C), spinal cord morphology and signal intensity were normal, confirming the absence of trauma and the validity of the surgical control.\u003c/p\u003e\n \u003cp\u003eThese imaging results are consistent with the behavioral outcomes (significantly improved BBB locomotor scores and tail flick latency in the niclosamide-treated group), histopathological findings (lower injury scores), and molecular analyses (reduced pro-inflammatory cytokines and modulation of key signaling pathways including p38, mTOR, STAT3, and ERK1/2). Collectively, the data support the neuroprotective and anti-inflammatory effects of niclosamide, particularly at the 10 mg/kg dose, in mitigating spinal cord damage and promoting functional recovery.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Behavioral Outcomes Following SCI and Niclosamide Treatment\u003c/h2\u003e\n \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.1. Niclosamide Preserves Body Weight After Spinal Cord Injury\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003e(Protective metabolic effect)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo monitor the general health status and recovery progress, body weight was measured on days 0 (baseline), 7, 14, 21, and 28 post-injury. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, rats in the SCI (Control) and DMSO groups exhibited a progressive and significant decline in body weight throughout the study period, reflecting systemic effects of injury and inflammation. In contrast, treatment with Niclosamide resulted in a dose-dependent attenuation of weight loss. Notably, the 10 mg/kg Niclosamide group showed a significant improvement in weight retention from day 14 onward compared to the SCI group (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), suggesting a potential protective or systemic stabilizing effect. The sham-operated animals maintained a stable body weight throughout the experiment.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.2. Niclosamide Enhances Locomotor Recovery in a Dose-Dependent Manner\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003e(Functional restoration assessed by BBB scoring)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eLocomotor function was evaluated using the BBB (Basso, Beattie, Bresnahan) scoring system on days 0, 1, 3, 5, 7, 14, 21, and 28. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, animals in the SCI and DMSO groups demonstrated poor motor recovery with consistently low BBB scores across the time points. In contrast, Niclosamide-treated groups exhibited a marked and dose-dependent improvement in hindlimb motor function. Particularly, the 10 mg/kg group showed significantly higher BBB scores starting from day 5 and maintained improved recovery through day 28 compared to SCI controls (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). This finding indicates enhanced functional restoration in Niclosamide-treated rats. Sham animals preserved normal motor activity with a consistent score of 21 throughout the study.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.3. Niclosamide Attenuates Thermal Hyperalgesia in SCI Rats\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003e(Improved sensory function assessed by Tail Flick Test)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe tail flick test was performed to assess thermal nociceptive thresholds. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, SCI and DMSO groups exhibited a peak in latency on day 7, followed by a decline indicating the development of thermal hyperalgesia over time. In contrast, rats treated with Niclosamide maintained higher tail flick latencies in a dose-dependent manner. The 10 mg/kg group showed significantly prolonged latencies at days 14, 21, and 28 compared to the SCI group (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), suggesting reduced hyperalgesia and better sensory recovery. Sham animals exhibited stable baseline latency throughout the experimental period.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Histopathological Assessment\u003c/h2\u003e\n \u003cp\u003eHistological evaluation of spinal cord tissue at day 28 post-injury revealed significant differences among experimental groups in terms of tissue integrity and pathological scoring. In the SCI (control) and DMSO-treated groups, histopathological damage was pronounced, as evidenced by higher cumulative scores reflecting severe inflammatory cell infiltration, axonal vacuolation, hemorrhage, and cyst formation. The average histopathological score in these groups was approximately 2.8 to 3, indicating substantial structural damage.\u003c/p\u003e\n \u003cp\u003eIn contrast, treatment with niclosamide led to a dose-dependent reduction in tissue damage. Animals receiving 2 mg/kg of niclosamide showed a moderate decrease in histological injury scores compared to SCI and DMSO groups, while 5 mg/kg provided further reduction. Notably, the group treated with 10 mg/kg niclosamide exhibited a marked improvement in tissue morphology, with significantly lower scores (mean score\u0026thinsp;\u0026lt;\u0026thinsp;1.0), approaching near-normal histological features. Statistical analysis confirmed that this reduction was highly significant (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) when compared with both the SCI and vehicle groups. (as shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e\n \u003cp\u003eThese findings suggest that a single intraperitoneal administration of niclosamide immediately after SCI provides robust neuroprotective effects, as evidenced by a substantial reduction in the hallmarks of secondary tissue damage. The data support the hypothesis that niclosamide attenuates inflammatory and degenerative processes in the injured spinal cord and may promote structural preservation in a dose-dependent manner.(as shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Molecular Results: Effect of Niclosamide on Inflammatory Cytokines and Signaling Pathways\u003c/h2\u003e\n \u003cp\u003eTo investigate the molecular mechanisms underlying the observed neuroprotective effects of niclosamide, levels of key inflammatory cytokines and intracellular signaling proteins were assessed in spinal cord tissues on day 28 post-injury, specifically in the group treated with 10 mg/kg niclosamide, compared to SCI and sham groups.\u003c/p\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.1. ELISA Results: Niclosamide Attenuates Inflammatory Cytokine Expression\u003c/h2\u003e\n \u003cp\u003eTo investigate the anti-inflammatory effects of niclosamide following spinal cord injury (SCI), ELISA analysis was performed to quantify the levels of key pro-inflammatory cytokines including TNF-\u0026alpha;, IL-1\u0026beta;, and IL-6 in spinal cord tissues on day 28 post-injury (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eTNF-\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e-A):\u003c/p\u003e\n \u003cp\u003eThe SCI group exhibited a significant increase in TNF-\u0026alpha; levels compared to the sham group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Treatment with niclosamide (10 mg/kg) resulted in a significant reduction in TNF-\u0026alpha; levels compared to the SCI group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating suppression of acute inflammatory response.\u003c/p\u003e\n \u003cp\u003eIL-1\u0026beta; (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e-B):\u003c/p\u003e\n \u003cp\u003eSimilarly, IL-1\u0026beta; levels were markedly elevated in the SCI group relative to sham (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Niclosamide treatment significantly reduced IL-1\u0026beta; expression compared to the SCI group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), supporting its role in modulating neuroinflammation.\u003c/p\u003e\n \u003cp\u003eIL-6 (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e-C):\u003c/p\u003e\n \u003cp\u003eThe SCI group showed a significant elevation in IL-6 levels versus sham (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Niclosamide administration significantly decreased IL-6 levels compared to SCI (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), although the values remained higher than the sham group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eThese findings confirm the anti-inflammatory potential of niclosamide in the SCI model through effective downregulation of proinflammatory cytokines.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.2 Western Blot Results:\u003c/h2\u003e\n \u003cp\u003eTo investigate the molecular mechanisms underlying the neuroprotective effects of niclosamide, we evaluated the expression of key signaling proteins involved in inflammation and neuroregeneration using Western blotting. Proteins assessed included phosphorylated and total forms of ERK1/2, MAPK, STAT3, and mTOR. Densitometric quantification of protein bands was normalized to \u0026beta;-actin and is shown in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eFollowing spinal cord injury (SCI), expression levels of P-ERK1/2, P-MAPK/MAPK, P-mTOR, and P-STAT3/STAT3 were significantly increased compared to the sham group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 to p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating activation of these pathways in response to injury. Treatment with a single dose of niclosamide (10 mg/kg) markedly attenuated these increases across all four markers:\u003c/p\u003e\n \u003cp\u003e\u0026bull; P-ERK1/2/\u0026beta;-actin expression (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e-A) was significantly reduced in the niclosamide group compared to SCI (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n \u003cp\u003e\u0026bull; P-MAPK/MAPK ratio (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e-B) was also significantly lower in niclosamide-treated rats (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n \u003cp\u003e\u0026bull; A significant decrease in P-mTOR/\u0026beta;-actin was observed in the niclosamide group (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e-C, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n \u003cp\u003e\u0026bull; Notably, P-STAT3/STAT3 expression (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e-D) was also significantly downregulated by niclosamide compared to untreated SCI animals (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\n \u003cp\u003eRepresentative Western blot bands for each protein are presented in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e-E, showing a clear visual reduction in phosphorylation levels following niclosamide administration.\u003c/p\u003e\n \u003cp\u003eThese results suggest that niclosamide suppresses injury-induced activation of MAPK/ERK, mTOR, and STAT3 pathways, supporting its anti-inflammatory and neuroprotective role in the SCI model.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the present study, we demonstrate that a single intraperitoneal dose of niclosamide significantly attenuated spinal cord injury (SCI)\u0026ndash;induced pathological and functional deficits in a rat model. While niclosamide is traditionally recognized as an anti-helminthic agent, our data contribute to an expanding body of evidence suggesting that this compound possesses potent anti-inflammatory and neuroprotective properties that could be repurposed for central nervous system (CNS) disorders (Apolloni et al., 2023; Milani et al., 2024).\u003c/p\u003e\u003cp\u003eA key strength of our study is the comprehensive evaluation encompassing behavioral, molecular, MRI, and histopathological endpoints, allowing for a multidimensional assessment of therapeutic efficacy. The significant improvements in BBB scores, tail-flick latency, and preservation of body weight in niclosamide-treated animals underscore its capacity to mitigate both motor and sensory impairments following SCI. While previous studies have shown similar functional benefits with anti-inflammatory agents such as methylprednisolone, the fact that niclosamide achieved meaningful recovery with a single post-injury dose is remarkable and clinically promising (Evaniew et al., 2015).\u003c/p\u003e\u003cp\u003eFrom a mechanistic standpoint, our results indicate that niclosamide effectively suppresses SCI-induced upregulation of proinflammatory cytokines (IL‑6, IL‑1β, TNF‑α), as demonstrated by ELISA assays. More importantly, Western blot analysis revealed significant downregulation of phosphorylated ERK1/2, p38 MAPK, STAT3, and mTOR, suggesting that niclosamide interferes with multiple converging signaling cascades known to mediate neuroinflammation and cell death (Sekulovski et al., 2020; Singh et al., 2024).\u003c/p\u003e\u003cp\u003eImportantly, NF‑κB is a master transcription factor driving expression of key inflammatory mediators such as TNF‑α and IL‑1β, and its sustained activation in the injured spinal cord contributes to chronic neuroinflammation and tissue damage. Inhibition of NF‑κB signaling has been shown to reduce the expression of proinflammatory cytokines and preserve spinal cord tissue integrity (Brambilla et al., 2005). Although we did not directly assess NF‑κB activation, the reduction in cytokine levels in the niclosamide-treated group supports potential suppression of this pathway.\u003c/p\u003e\u003cp\u003eInterestingly, while the ERK1/2 pathway is implicated in neuronal survival and regeneration, sustained activation in the late phase of SCI (as in day 28 of our model) may reflect ongoing inflammatory responses and glial scar formation rather than regenerative processes (Yu et al., 2005). Thus, the downregulation of p‑ERK1/2 observed here may represent a beneficial suppression of chronic inflammatory signaling rather than inhibition of neurogenesis (Li et al., 2021). STAT3 activation plays dual roles in promoting astrocyte reactivity and scarring while also contributing to limited regeneration (Herrmann et al., 2008). Our findings that niclosamide suppresses STAT3 phosphorylation point toward a net anti-inflammatory and anti-gliotic effect.\u003c/p\u003e\u003cp\u003eThe modulation of mTOR signaling by niclosamide also merits attention. mTORC1 is a critical regulator of cellular metabolism and autophagy, and its inhibition has been shown to promote neuroprotection in various CNS injury models, including SCI, by enhancing autophagy markers and reducing neuronal death (Ding et al., 2022; Kanno et al., 2012). In our study, reduced phosphorylation of mTOR suggests that niclosamide may restore metabolic homeostasis and prevent apoptosis in the injured spinal cord, consistent with these prior preclinical observations.\u003c/p\u003e\u003cp\u003eTogether, these data suggest that niclosamide acts as a multi-targeted therapeutic, modulating multiple pathways\u0026mdash;NF‑κB, MAPK/ERK, STAT3, mTOR\u0026mdash;that are central to the pathophysiology of SCI. To our knowledge, this is the first study evaluating niclosamide in a spinal cord injury model. The robust efficacy noted with a single dose, combined with its safety profile, underscores its translational potential.\u003c/p\u003e\u003cp\u003eNevertheless, certain limitations warrant acknowledgement. We assessed only one dose level for molecular analyses, and long-term outcomes beyond 28 days remain unknown.\u003c/p\u003e\u003cp\u003eMoreover, while Western blotting provided mechanistic insights, further studies employing immunofluorescence, neuronal tracing, or single-cell transcriptomics could offer a deeper understanding of cell-specific effects. Additionally, whether niclosamide\u0026rsquo;s benefits extend to chronic SCI models or other injury paradigms requires further investigation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrates, for the first time, that a single intraperitoneal dose of niclosamide (10 mg/kg) administered immediately after spinal cord injury exerts significant neuroprotective and anti-inflammatory effects in a rat contusion model. Niclosamide markedly improved locomotor and sensory function, reduced histopathological damage, attenuated proinflammatory cytokine expression, and suppressed the activation of key signaling pathways involved in secondary injury, including STAT3, mTOR, and MAPK/ERK.\u003c/p\u003e\u003cp\u003eThese results highlight the potential of niclosamide as a promising therapeutic agent for SCI, capable of modulating both inflammatory and regenerative mechanisms. Given its FDA-approved status and favorable safety profile, niclosamide may be a viable candidate for repurposing in the treatment of spinal cord injury. However, further investigations are warranted to explore its long-term efficacy, optimal dosing strategies, and underlying cellular mechanisms in both acute and chronic models.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their sincere gratitude to Dr. Masoud Fazeli, radiologist and faculty member at Arak University of Medical Sciences, for generously providing access to MRI facilities and technical support.\u003c/p\u003e\n\u003cp\u003eWe also acknowledge Mr. Shamseddin Mashayekhi, CEO of Behrood Atrak Pharmaceutical Company, for his supportive role in facilitating this research.\u003c/p\u003e\n\u003cp\u003eSpecial thanks to Dr. Shahrzad Jalilpour Rezaei for her assistance in editing several figures used in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAli Darabniya led the study, performed the spinal cord injury surgeries, developed the scientific framework, conducted all experimental procedures, analyzed the data, and drafted the initial version of the manuscript.\u003c/p\u003e\n\u003cp\u003eAhmad Reza Dehpour was responsible for the overall study design and conceptual development, supervised the research process, and contributed to the critical revision of the manuscript.\u003c/p\u003e\n\u003cp\u003eSara Shahriari and Reza Fazeli contributed to the execution of experiments, data collection, and data analysis, and both reviewed the initial draft and provided constructive feedback.\u003c/p\u003e\n\u003cp\u003eSeyed Mohammad Tavangar performed histopathological assessments, contributed to the pathology section of the manuscript, and participated in reviewing the draft and giving constructive comments.\u003c/p\u003e\n\u003cp\u003eRazieh Mohammadi Jafari assisted in laboratory procedures and animal care, and also contributed to reviewing and improving the early version of the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran (Grant No 1403-3-101-74387)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. No human participants were involved in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhuja CS, Nori S, Tetreault L, et al. Traumatic Spinal Cord Injury-Repair and Regeneration. Neurosurgery. 2017;80(3S):S9-S22. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/neuros/nyw080\u003c/span\u003e\u003cspan address=\"10.1093/neuros/nyw080\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKwon BK, Tetzlaff W, Grauer JN, et al. Pathophysiology and pharmacologic treatment of acute spinal cord injury. 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Epub 2012 Aug 16.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"STAT3, NF-κB, MAPK/ERK, mTOR, TNF-α, Neuropathic pain","lastPublishedDoi":"10.21203/rs.3.rs-7609682/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7609682/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpinal cord injury (SCI) is a life-altering condition of the central nervous system resulting in permanent motor, sensory, and functional deficits. The pathophysiology involved includes: primary neuronal necrosis, secondary inflammation, oxidative stress, apoptosis, and impaired neurogenesis, which collectively inhibit tissue repair. Despite efforts, there is currently no effective therapy that offers radial spinal cord regeneration, which highlights our need for pharmacological agents with anti-inflammatory and neuroprotective properties.\u003c/p\u003e\u003cp\u003eNiclosamide is an FDA-approved anti-parasitic agent that has received increasing attention recently due to its ability to modulate important signaling pathways such as STAT3, NF-κB, MAPK/ERK, and mTOR. We therefore sought to investigate the therapeutic potential of niclosamide in a rat model of SCI. Animals were given a single intraperitoneal injection of niclosamide (2, 5, or 10 mg/kg) immediately after the clip-induced injury. Functional recovery was assessed by the BBB and Tail-Flick tests for 28 days. The animals that had received 10 mg/kg niclosamide exhibited significant improvement in locomotor and sensory outcomes compared to intact SCI animals.\u003c/p\u003e\u003cp\u003eMRI imaging performed on day 7 showed a decrease in lesion size and greater preservation of the surrounding tissue in niclosamide-treated animals. Histopathological assessment on day 28 showed less inflammation and hemorrhage; less vacuolization of the neuron cell bodies; and a decrease in cyst formation. ELISA testing indicated significantly lower levels of IL-6, IL-1β, and TNF-α, and western blot analysis indicated low phosphorylation of ERK1/2, p38 MAPK, STAT3, and mTOR, demonstrating inhibition of inflammatory and apoptotic signaling pathways following injury.\u003c/p\u003e\u003cp\u003eThese findings collectively indicate that niclosamide has neuroprotective effects across multiple targets and leads to improvements in functional recovery following SCI. These results support niclosamide as a promising candidate for spinal cord repair.\u003c/p\u003e","manuscriptTitle":"Niclosamide as a Neuroprotective and Anti-Inflammatory Agent: A Potential Therapeutic Approach for Spinal Cord Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 16:46:11","doi":"10.21203/rs.3.rs-7609682/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2846c1ba-bed1-4929-93e4-ca639dd8a053","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-12T10:25:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-03 16:46:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7609682","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7609682","identity":"rs-7609682","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}