The Efficacy of Rapamycin in Spinal Cord Injury: A Systematic Review and Meta-Analysis of preclinical studies

The Efficacy of Rapamycin in Spinal Cord Injury: A Systematic Review and Meta-Analysis of preclinical studies | 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 The Efficacy of Rapamycin in Spinal Cord Injury: A Systematic Review and Meta-Analysis of preclinical studies Hamed Zarei, Amir Azimi, Hamzah Adel Ramawad, Razieh Hajisoltani, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3948391/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 2 You are reading this latest preprint version Abstract Background Rapamycin has shown a potential role in functional and neurological recovery after neurodegenerative disease. The current study evaluates the efficacy of Rapamycin in preclinical spinal cord injury (SCI). Methods A systematic literature search was conducted in Medline, Embase, Scopus, and Web of Science databases until April 2023. Inclusion criteria were preclinical studies comparing Rapamycin treatment to a control group in animal models of SCI and reporting outcomes including locomotion, apoptosis, autophagy, inflammation, astrogliosis, neuronal counts, and signaling proteins related to the mechanistic target of Rapamycin in Akt/mTOR/p70S6K pathway. Two independent reviewers performed study screening and data extraction. For meta-analyses, a standardized mean difference (SMD) with a 95% confidence interval (CI) was calculated for each experiment and a pooled effect size was reported. The risk of bias and certainty of evidence was assessed using SYRCLE and GRADE tools, respectively. Results 18 papers were included in the study. Rapamycin significantly decreased apoptosis (TUNEL: SMD − 3.44, 95% CI -5.41 to -1.47; Caspase-3: SMD − 3.85, 95% CI -7.57 to -0.13), inflammation (TNF-α: SMD − 3.26, 95% CI -5.56 to -0.97), astrogliosis (GFAP: SMD − 0.76, 95% CI -1.28 to -0.25), and inhibited Akt/mTOR/p70S6K signaling pathway (SMD − 3.74, 95% CI -6.31 to -1.18). It increased autophagy markers (Beclin-1: SMD 1.42, 95% CI 0.51 to 2.33; LC3-II: SMD 1.09, 95% CI 0.35 to 1.82) and neuronal counts (SMD 1.18, 95% CI 0.44 to 1.91). Locomotion was not significantly influenced by the short-term effects of Rapamycin. However, treatment had significant long-term improvements in locomotion (SMD 0.74–1.54 from 1–6 weeks). Conclusion The current study indicates Rapamycin provides neuroprotection, reduces inflammation, enhances autophagy, and improves long-term locomotion in rodent SCI models. Spinal Cord Injuries Quadriplegia Hemiplegia spinal cord contusion Sirolimus Rapamycin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Spinal cord injury (SCI) is a highly disabling condition that often results in long-term damage to motor, sensory, and autonomic functions. SCI significantly impacts mobility and quality of life. Epidemiological data estimate the global incidence of traumatic SCI to be 40–80 new cases per million population annually ( 1 ). SCI is characterized by a primary injury that is followed by subsequent pathophysiological changes such as apoptosis, autophagy, ischemia, glial scarring, and oxidative stress ( 2 ). Researchers have explored numerous pharmacological agents to attenuate subsequent injuries after SCI ( 3 ). Rapamycin is a macrolide antibiotic commonly employed as an immunosuppressant to prevent organ transplant rejection ( 4 , 5 ). It inhibits the mechanistic target of Rapamycin (mTOR) pathway, which is a key regulator of cell growth, proliferation, and survival ( 6 ). Rapamycin also stimulates autophagy, an intracellular degradation process responsible for eliminating damaged organelles, harmful substances, and unneeded proteins ( 5 ). While autophagy generally protects cells, excessive or improperly regulated autophagy can also lead to cell death ( 7 ). Some studies suggested Rapamycin-induced autophagy may play a neuroprotective role in certain neurodegenerative diseases ( 8 – 11 ). Given the potential for enhanced functional and neurological recovery in SCI with Rapamycin administration, this systematic review and meta-analysis aimed to compile and evaluate the existing literature regarding the efficacy of Rapamycin treatment in SCI for the first time. Methods Study design and search strategy The protocol for this systematic review and meta-analysis was published in the PROSPERO database for systematic reviews (registration code: CRD42023439825). The present study was conducted to summarize evidence on the effectiveness of the administration of Rapamycin for SCI. Keywords related to “Rapamycin” and “Spinal cord injury” were selected from the MeSH database of Medline, and the Emtree of Embase. Recommendations from experts were also considered. The search strategy for each database included relevant tags and Boolean operators. We conducted an extensive search in Medline (via PubMed), Embase, Scopus, and Web of Science until April 3, 2023, and performed manual searches on Google and Google Scholar to ensure comprehensive coverage. The search queries for each database are presented in the supplementary materials. Selection criteria In this study, the PICO framework was applied, where the Population (P) consisted of animals with SCIs, the Intervention (I) involved Rapamycin administration, the Comparison (C) group included SCI animals receiving no treatment or vehicle, and the Outcomes (O) were motor function assessment using standard scales, sensory outcomes, and markers related to apoptosis, autophagy, inflammation, neuronal counts, and the Akt/mTOR/P70S6K signaling pathway. Exclusion criteria included review studies, non-peer-reviewed articles such as letters, commentaries, and conference abstracts, duplicate reports, studies involving animals with co-morbidities, studies without traumatic SCI induction, and in vitro studies. Other exclusions comprised studies not using Rapamycin or using combination therapy, studies without a valid control group, studies involving animals other than rats and mice, and studies not reporting relevant outcomes. Data collection Results from the systematic search were integrated into Endnote X9, and duplicate records were removed. In the initial screening, two independent researchers reviewed the titles and abstracts of all retrieved articles. Potentially relevant articles underwent a full-text review in a secondary screening. Articles meeting the inclusion criteria were selected. If full-text or essential data were missing, the corresponding author was contacted via email, with a follow-up after a week in case of no response. Data from the included articles were extracted by two independent researchers, following a checklist based on the PRISMA guidelines. Data included information about the study's first author, year of publication, characteristics of studied animals, SCI method, SCI segment and severity, Rapamycin dose, route of administration, treatment intervals with injury, repetitions of treatments, and outcome characteristics, including timing, method, and scale, mean, and standard deviations for both treated and control groups. Quality assessment The quality of the included studies was assessed using the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) risk of bias assessment tool ( 12 ). This tool evaluates the methodology and potential bias in pre-clinical studies through 10 domains. Disagreements were resolved through discussions with a third researcher when necessary. Certainty of evidence The certainty of evidence was evaluated using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework ( 13 ). Statistical analyses Statistical analyses were performed using STATA 17.0. Studies were categorized based on reported outcomes. A standardized mean difference (SMD) with a 95% confidence interval (CI) was calculated for each sample and pooled to determine an overall effect size, since the scale of reported outcomes varied in the included articles. Meta-analysis was conducted only if data were reported in at least three separate articles. A Galbraith plot was employed to detect outlier studies. If an influential outlier were detected, the data were not included in the pooled analysis. Given the clinical and methodological diversity among studies, a random effect model was chosen. To avoid heterogeneity caused by the different intervals between treatment and locomotion assessment, we stratified experiments to discriminate short-term and long-term effects of Rapamycin. Short-term effects of Rapamycin were assessed within six days of its first administration (approximately 2 half-life), while long-term effects were evaluated beyond a six-day interval from the initial dose and outcome assessment. The half-life of Rapamycin is 58–63 hours (~ 3 days) ( 12 ). Heterogeneity was assessed through visual inspection of forest plots, I 2 , and chi-square tests. Publication bias were assessed by using modified egger’s test proposed by Doleman et al ( 13 ). Results Study selection In our database search, we initially identified 2658 records. Following the elimination of duplicate entries, we were left with 1800 unique records. Subsequently, we conducted a thorough review of 89 full-text documents. Sixty-three studies did not meet our predefined eligibility criteria and were subsequently excluded. Ultimately, 18 papers were included in the study ( 3 , 5 , 6 , 14 – 28 ). Furthermore, we performed a manual search to retrieve possible missed papers. This additional search did not yield any extra articles meeting our inclusion criteria (Fig. 1 ). Study characteristics Table 1 provides an overview of the characteristics of the included studies. The animal models employed in these investigations included mice in six studies and rats in twelve studies. Three studies utilized a hemisection or transection model, twelve studies employed a contusion model, and three studies utilized a compression model to induce SCI. Sixteen studies induced thoracic and two studies induced cervical SCI. Table 1 Characteristics of the eligible studies Study Animal; Species; Gender; Weight (g) Antibiotic SCI model SCI level severity Sample size Injury to treatment (day) drug dose (mg/kg) Treatment duration Administration rout F/U (day) Chang KT, 2020 (14) Rat; SD; F; 280–300; Adult NR Transection Cervical Severe 24 0 1 daily for 1 week oral 21 Chen HC, 2013 (15) Rat; SD; F; 260–320; Adult NR Contusion Thoracic Moderate 48 0 0.5 daily for 3 days IP 28 Cordaro, 2017 (16) Mice; CD1; M; 25–30; Adult NR Compression Thoracic Severe 40 0 1 1h and 6h post-SCI IP 10 Gao K, 2015 (17) Rat; SD; M; 250–300; 8–12 Yes (type is not specified) Contusion Thoracic Severe 48 0 0.5 daily for 3 days IP 28 Ge C, 2020 (6) Rat; SD; M; 200–250; 8–12 NR Contusion Thoracic Severe 52 NR NR NR NR 28 Goldshmit, 2015 (18) Mice; C57BL/6; M; 20–30; 8–16 NR Hemisection Thoracic Severe 12 0 1 single dose IP 35 Kjell, 2014 (19) Rat; SD; F; 200–225; NR Trimethoprim Contusion Thoracic Mild and moderate 32 0 1.5 daily for 14 days oral 119 Li XG, 2019 (3) Rat; SD; M; 300–330; Adult NR Compression Thoracic Severe 32 0 1 single dose IP 1 Liu J, 2020 (20) Mice; C57; F; NR; 8 NR Contusion Cervical Moderate 32 0 1.5 daily for 12 weeks IP 84 Nottingham, 2002 (21) Rat; NR; NR; NR; NR NR Contusion Thoracic Moderate 12 0 1.5 4, 72, 144 h post-injury IP 8 Saraswat, 2018 (22) Mice; C57BL/6; F; NR; 6–8 Gentamycin Contusion Thoracic Moderate 18 0 1 daily for 3 days IP 42 Sekiguchi, 2012 (5) Mice; C57BL/6; F; NR; 10–12 NR Contusion Thoracic Mild 36 0 1 single dose IP 42 Song Y, 2014 (23) Rat; Wistar; F; 220–250; NR Ampicillin and Gentamycin Contusion Thoracic Severe 30 0 1 daily for 3 days IP 7 Tang, 2014 (25) Rat; SD; M; 200–250; NR NR Hemisection Thoracic Severe 48 0 0.5 single dose IP 90 Tateda, 2017 (26) Mice; C57BL/6; F; NR; 10–12 NR Contusion Thoracic Moderate 28 0 1 single dose IP 42 Vargova, 2021 (27) Rat; Wistar; M; 300–330; NR ampicillin Compression Thoracic Severe 23 1 5 daily for 5 days IP 7 Wang ZY, 2014 (28) Rat; SD; F; 200–220; 8 NR Contusion Thoracic Severe 20 0 5 daily for 30 days IP 30 Weng F, 2019 (24) Rat; SD; M; 220–260; 8 Gentamycin Contusion Thoracic Severe 24 0 1.8 daily for 4 weeks intrathecal 29 g: Grams; F/U: follow-up; NR: not reported; SD: Sprague-Dawley; IP: intraperitoneal. Eleven studies pertained to severe injuries, five to moderate injuries, and one to mild injuries. Remarkably, one study encompassed both mild and moderate SCI induction in distinct experiments with unique animal groups. studies used 559 animals in both arms of our study. Rapamycin treatment was initiated on the same day as the injury in almost all studies, except for one study administering it one day after injury. The studies employed a standardized dose of Rapamycin, with variations observed: 0.5 mg/kg in three studies, 1 mg/kg in eight studies, 1.5 mg/kg in three studies, 1.8 mg/kg in one study, and 5 mg/kg in two studies. Treatment duration spanned from single doses to daily doses, consistently administered for a maximum of 28 consecutive days. Notably, the administration route for Rapamycin varied, with two studies utilizing oral administration, fourteen studies utilizing intra-peritoneal delivery, one study employing intrathecal administration, and one study not specifying the administration route. Risk of bias in the studies We used the SYRCLE tool to assess the risk of bias for each of the included studies. A summary of these assessments is provided in Supplementary Materials. In terms of overall risk of bias, all studies were assessed as at high risk of bias. The justification of such judgment by the authors is the fact that none of the studies reported adequate data for sequence generation, allocation concealment, random housing, blinding of trial caregivers, and random outcome assessment. Meta-analysis of the effect of Rapamycin on spinal cord injury a) Locomotion Locomotion was assessed with BBB in rats and BMS in mice. Pooled data analysis showed that administration of Rapamycin has no significant short-term effect on locomotion following SCI (SMD = 0.78, 95% CI: -0.23 to 1.79, p = 0.079; I² = 84.71%) (Fig. 2 ). However, when we assessed the long-term effects (at least one week after the initial dose) of Rapamycin on animals at one to six weeks after injury, Rapamycin significantly improved the locomotion at all time points (0.74 ≤ SMD ≤ 1.54, p < 0.05) (Fig. 3 ). Sensitivity analysis revealed the same results when omitting studies with mild SCI. In moderate to severe SCI, Rapamycin had no significant short-term therapeutic effect (SMD = 0.94, 95% CI: -0.28 to 2.16, p = 0.130; I² = 90.76%). However, the short-term effects were robust in 1 week to 4 weeks after moderate to severe injuries (1.04 ≤ SMD ≤ 1.95, p < 0.005). b) Apoptosis and autophagy Rapamycin demonstrated a superior ability to reduce TUNEL-positive spinal cells than the control group (SMD = -3.44, 95% CI: -5.41 to -1.47, p = 0.001; I² = 75.01%). The anti-apoptotic effect of Rapamycin was confirmed in the analysis when it significantly increased BcL-2 (SMD = 6.51, 95% CI: 0.55 to 12.48, p = 0.032; I² = 97.74%) and decreased Caspase-3 (SMD = -3.85, 95% CI: -7.57 to -0.13, p = 0.043; I² = 92.67%) activity in the injured spinal cord. There was no statistical significance when studying the differences in the Bax activity (SMD = -4.96, 95% CI: -10.47 to 0.55, p = 0.078; I² = 98.33%) between Rapamycin therapy and control groups by meta-analysis following Rapamycin administration (Fig. 4 a). Autophagy biomarkers were also pooled by meta-analysis. Beclin-1 (SMD = 1.42, 95% CI: 0.51 to 2.33, p = 0.002; I² = 66.81%) and LC3-II (SMD = 1.09, 95% CI: 0.35 to 1.82, p = 0.004; I² = 11.13%) levels were clearly increased after Rapamycin treatment (Fig. 4 a). c) Inflammation Interleukin-1 (IL-1) and Tissue Necrosis Factor-α (TNF-α) were among the inflammatory outcomes of our study. Therefore, due to the insufficient number of studies, we only analyzed the effect of Rapamycin treatment on TNF-α. Pooled data analysis showed that Rapamycin could play an anti-inflammatory effect after SCI (SMD = -3.26, 95% CI: -5.56 to -0.97, p = 0.005; I² = 82.42%) (Fig. 4 b). d) GFAP and neuronal count Pooled data analysis from five studies on 63 animals demonstrated that Rapamycin treatment could significantly subside areas (indicated by GFAP) containing astrocytes in the injured animals (SMD = -0.76, 95% CI: -1.28 to -0.25, p = 0.004; I² = 37.83%) (Fig. 4 c). The analyses also showed a significantly higher number of alpha-motor (NeuN + ) neurons in the Rapamycin-treated group (SMD = 1.18, 95% CI: 0.44 to 1.91, p = 0.002; I² = 38.47%) (Fig. 4 d). e) Akt/mTOR/p70S6K signaling pathway In order to investigate the role of Rapamycin in the treatment of SCI through AKT/mTOR/p70S6K signaling pathways, we gathered data on protein levels of Akt, mTOR, and p70S6K, along with their phosphorylated forms. ‌Since we applied unrelenting criteria for pooling data in a meta-analysis, the only applied marker for meta-analysis was p-P70S6K. Pooled data analysis of 4 studies (n = 40) showed significantly lower levels of p-P70S6K within 2 weeks post-injury in the Rapamycin-treated group (SMD = -3.74, 95% CI: -6.31 to -1.18, p = 0.004; I² = 85.32%) (Fig. 4 e). Publication bias and certainty of evidence As depicted in the supplementary materials, the funnel plot asymmetry test revealed no evidence of publication bias for any of the investigated outcomes, except for locomotion at 2- and 3-weeks post-injury, and p-P70S6K, for which there are concerns regarding small study effects. The level of evidence in the assessment of the neuroprotective effects of rapamycin treatment after SCI is presented in Table 2 . Table 2 Certainty of evidence based on GRADE framework Outcome Studies (n) Effect size Risk of bias Imprecision Inconsistency (I 2 ) Indirectness Publication bias Judgment and level of evidence Short-term locomotion 6 0.80 (-0.31, 1.90) Serious Not Serious Serious Not serious Not serious Low : Rated down 2 points - Presence of serious risk of bias - Presence of serious inconsistency Long-term locomotion 1-week post-injury 11 1.54 (0.55, 2.53) Serious Not serious Serious Not serious Not serious Low : Rated down 2 points - Presence of serious risk of bias - Presence of serious inconsistency 2 weeks post-injury 5 1.17 (0.57, 1.76) Serious Not serious Serious Not serious Serious Very low : Rated down 2 points - Presence of serious risk of bias - Presence of serious inconsistency - Presence of publication bias 3 weeks post-injury 7 0.95 (0.38, 1.53) Serious Not serious Not serious Not serious Serious Low : Rated down 1 point - Presence of serious risk of bias - Presence of publication bias 4 weeks post-injury 6 1.04 (0.27, 1.81) Serious Not serious Serious Not serious Not serious Low : Rated down 2 points - Presence of serious risk of bias - Presence of serious inconsistency 5 weeks post-injury 3 1.49 (0.75, 2.22) Serious Not serious Not serious Not serious Not serious Moderate : Rated down 1 point - Presence of serious risk of bias 6 weeks post-injury 3 1.01 (0.09, 1.92) Serious Not serious Not serious Not serious Not serious Moderate : Rated down 1 point - Presence of serious risk of bias Apoptosis/Autophagy TUNEL-positive cells 3 -3.44 (-5.41, -1.47) Serious Not serious Serious Not serious Not serious Low : Rated down 2 points - Presence of serious risk of bias - Presence of serious inconsistency Bax 4 -4.96 (-10.47, 0.55) Serious Not serious Serious Not serious Not serious Low : Rated down 2 points - Presence of serious risk of bias - Presence of serious inconsistency Bcl-2 4 6.51 (0.55, 12.48) Serious Not serious Serious Not serious Not serious Low : Rated down 2 points - Presence of serious risk of bias - Presence of serious inconsistency Caspase-3 4 -3.85 (-7.57, -0.13) Serious Not serious Serious Not serious Not serious Low : Rated down 2 points - Presence of serious risk of bias - Presence of serious inconsistency Beclin-1 4 1.42 (0.51, 2.33) Serious Not serious Serious Not serious Not serious Low : Rated down 2 points - Presence of serious risk of bias - Presence of serious inconsistency LC3-II 3 1.09 (0.35, 1.82) Serious Not serious Not serious Not serious Not serious Moderate : Rated down 1 point - Presence of serious risk of bias Inflammation TNF-α 3 -3.26 (-5.56, -0.97) Serious Not serious Serious Not serious Not serious Low : Rated down 2 points - Presence of serious risk of bias - Presence of serious inconsistency Astrocytosis/Neuronal count GFAP 5 -0.76 (-1.28, -0.25) Serious Not serious Not serious Not serious Not serious Moderate : Rated down 1 point - Presence of serious risk of bias Alpha-motor neurons 4 1.18 (0.44, 1.91) Serious Not serious Not serious Not serious Not serious Moderate : Rated down 1 point - Presence of serious risk of bias Akt/mTOR/P70S6K signaling pathway p-P70S6K 4 -3.74 (-6.31, -1.18) Serious Not serious Serious Not serious Serious Very low : Rated down 2 points - Presence of serious risk of bias - Presence of serious inconsistency - Presence of publication bias GRADE: Grading of Recommendations Assessment, Development and Evaluation. Discussion Our research represents the first systematic review and meta-analysis dedicated to exploring the therapeutic potential of Rapamycin in rodent models of SCI. Our findings demonstrate significant reductions in apoptosis, inflammation, and astrogliosis, along with a substantial increase in autophagic markers. These outcomes correspond with a significant improvement in long-term locomotion recovery following Rapamycin administration in the injured animals (Fig. 5 ). Apoptosis, often referred to as programmed cell death, plays a pivotal role in the development of secondary injury after SCI ( 29 ). Mitochondria-associated cell death is among the critical mechanisms triggered by SCI ( 30 ). This process involves key proteins from the Bcl-2 family, including Bcl-2, Bax, and the executioner Caspase. When cells undergo apoptosis, Bax adheres to the outer mitochondrial membrane, forming pores through oligomerization. This pore formation allows the release of apoptogenic factors like Cytochrome C into the cytoplasm. Bcl-2, a( 31 )n anti-apoptotic protein, can inhibit apoptosis by binding to Bax, preventing the formation of these pores. In the cytoplasm, Cytochrome C contributes to the formation of the apoptosome, which activates Caspase-9. Activated Caspase-9 then transforms Pro-caspase-3 into effector Caspase-3, leading to the typical morphological changes observed in apoptotic cells ( 32 , 33 ). Our study also reveals that Rapamycin treatment results in an increase in the anti-apoptotic protein Bcl-2 and a decrease in the pro-apoptotic protein Bax. While it is important to note that individual studies examining Bax levels in the Rapamycin-treated group show a significant increase, pooled data analysis did not demonstrate a significant overall change. This discrepancy may be attributed to the limited number of studies included in the meta-analysis. Furthermore, we observed an increase in the executioner caspase, Caspase-3, suggesting that Rapamycin has complex effects on the regulation of apoptosis. Specifically, it appears that Rapamycin suppresses the mitochondrial apoptosis pathway. Additionally, our assessment of Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assays to detect apoptotic cells undergoing extensive DNA degradation during the late stages of apoptosis ( 34 ), demonstrated a decrease in the groups treated with Rapamycin, further confirming the inhibitory role of Rapamycin in the apoptotic pathway. Autophagy is the process by which cells digest their components to maintain cellular homeostasis ( 35 ). This autodigestion increases in neurons within hours after SCI, likely serving as a neuroprotective mechanism ( 36 ). The initiation of autophagy relies on the key regulatory protein Beclin-1 ( 37 ). LC3-II, a protein involved in forming autophagosomes for degradation, also reflects autophagy levels ( 38 ). Our study found that Rapamycin significantly increased Beclin-1 and LC3-II, indicating enhanced autophagic flux. Importantly, p62, which targets proteins for autophagic degradation, was lower with Rapamycin treatment in two studies ( 6 , 20 ). This decline in p62, which binds to ubiquitinated proteins and targets them for degradation via autophagy, confirms that Rapamycin truly enhances autophagic digestion rather than just accumulating autophagosomes ( 39 ). Post-trauma inflammation after SCI is characterized by increased pro-inflammatory cytokines such as TNF-α and IL-1β, attracting immune cells like macrophages and microglia, which exacerbate the inflammatory response. This inflammation contributes to secondary neuronal damage through various pathways ( 15 , 23 ). Studies have shown that Rapamycin treatment can reduce inflammation by attenuating the mTOR pathway in microglia, decreasing their activation and production of pro-inflammatory cytokines ( 40 ). Our study aligns with previous research, as it demonstrates that Rapamycin significantly lowers levels of TNF-α after injury. While an IL-1β meta-analysis was not conducted due to limited studies, the two studies included also indicated significantly lower levels of IL-1β in the Rapamycin treatment group compared to the injury control group ( 23 , 27 ). By dampening this inflammatory cascade, Rapamycin might help limit secondary damage after SCI. GFAP, an intermediate filament protein expressed predominantly in astrocytes, is commonly used to identify astrocytes and to examine astrogliosis, referring to reactive astrocyte proliferation in response to injury. Activated astrocytes secrete proinflammatory cytokines and contribute to glial scar formation and inhibit axonal regeneration ( 14 , 16 ). Studies suggest Rapamycin inhibits STAT3 signaling in astrocytes, which is known to promote reactive astrocytosis. By inhibiting STAT3 activation, Rapamycin reduces GFAP expression and other markers of astrocyte reactivity ( 41 ). Additionally, by inhibiting mTOR, Rapamycin shifts astrocyte metabolism from glycolysis to more efficient oxidative phosphorylation, which could be associated with reduced astrocyte reactivity ( 14 ). Consistent with these mechanisms, our study shows that Rapamycin reduces GFAP expression after SCI, potentially creating a more favorable environment for nerve regeneration. Alpha-motor neurons, also known as lower or skeletal motor neurons, innervate muscle fibers ( 42 ). Our study indicates that there are more surviving alpha-motor neurons in the spinal cord of the Rapamycin-treated group compared to the controls. Rapamycin likely provides neuroprotection by decreasing microglial and astrocytic activation and increasing neuronal autophagy, thereby reducing secondary damage after injury ( 14 , 15 ). Rapamycin inhibits mTOR Complex 1 (mTORC1), a key regulator of cell growth and metabolism ( 43 ). mTORC1 normally phosphorylates and activates p70S6 Kinase (p70S6K), which promotes protein synthesis and cell growth ( 15 , 44 ). Akt is a protein kinase that activates mTORC1 by phosphorylating and inhibiting TSC1/TSC2, negative regulators of mTORC1 ( 45 ). The p-Akt protein expression was assessed in four studies; two studies measured p-Akt/β -actin and two studies measured p-Akt/Akt. Therefore, data pooling did not apply to a meta-analysis. However, studies showed increased p-Akt levels within the first week of Rapamycin therapy compared to controls in SCI models, likely due to the negative feedback on PI3K/Akt signaling when mTORC1 is inhibited ( 3 , 18 , 27 ). However, one study found decreased p-Akt at 2- and 4 weeks post-injury ( 6 ). Considering Rapamycin's half-life, the early feedback-driven p-Akt upregulation may peak and normalize after prolonged treatment, leading to observable declines by 2 weeks. Our research uncovers various potential explanations for how Rapamycin contributes to enhancing motor function recovery. Firstly, we observed that Rapamycin effectively prevents apoptosis. Secondly, it reduces astrogliosis and inflammation. Thirdly, it promotes autophagy at the site of the injury. On the flip side, treatment with the mTOR inhibitor Rapamycin hampers the Akt/mTOR/p70S6K signaling pathway and decreases the expression of proteins related to myelin formation in the damaged spinal cord ( 6 ). Additionally, mTORC1 inhibition leads to the suppression of p70S6K, responsible for promoting protein synthesis and cell growth ( 15 ). Consequently, these findings have given rise to conflicting hypotheses. In alignment with these controversies, our study demonstrated a lack of significant short-term improvement and only low to moderate long-term enhancements in motor function. Our study has limitations. It's important to acknowledge that the literature has only reported a limited number of underlying pathways, and there is some degree of heterogeneity. This emphasizes the need for caution when interpreting these findings. Additionally, certain variables were too scarce to be combined, including the severity of the injury, the timing of treatment, the number and routes of administered doses, robust follow-up evaluations, and some outcomes were solely assessed through one method, such as TNF-α for inflammatory markers and p70S6K for Akt/mTOR/p70S6K signaling pathway. These considerations underscore the necessity for further experimental research in this field to ensure the generalizability of results. Another shortcoming arises from the low quality of the animal studies, all of which were judged to carry a high risk of bias. This limitation stems from the studies' failure to include some major elements outlined in CYRCLE's risk of bias tool. It is important to note that researchers cannot be faulted for this omission, as the ARRIVE guidelines for animal research do not mandate reporting on most of these items ( 46 ). Finally, there was evidence of publication bias in the assessment of locomotion at 2- and 3-weeks post-injury, as well as in p-P70S6K. This bias was predisposed by the limited number of studies for each outcome. In conclusion, Rapamycin demonstrates neuroprotective, anti-inflammatory, and pro-autophagic effects in rodent SCI models, leading to modest long-term improvements in motor function recovery versus controls. The low- to moderate-level evidence from this study emphasizes the necessity for more rigorous experimental investigations to thoroughly assess the effectiveness of Rapamycin treatment in animal models with SCI. Declarations Acknowledgments None. Author contributions All authors (HZ, RH, AZ, HAR, MY) have made substantial contributions to the conception or design of the current work; or the acquisition, analysis, or interpretation of data for the work; drafting the work, or revising it critically. All authors have provided final approval of the version to be submitted. Conception and design: MY; data extraction: HZ, RH; statistical analysis: HZ, MY; interpretation of the results: HZ, AZ, HAR; Drafting: HZ, AZ, RH; Revising the work critically: all authors; graphical abstract: HAR. Data availability statement The dataset generated and analyzed during the current study is available from the corresponding author upon reasonable request. Competing interests The authors declare no conflicts of interest. Funding/Support This research has been supported by Iran University of Medical Sciences (Grant number: 1401-4-75-25439) References Perrouin-Verbe B, Lefevre C, Kieny P, Gross R, Reiss B, Le Fort M. Spinal cord injury: A multisystem physiological impairment/dysfunction. Rev Neurol (Paris). 2021;177(5):594–605. Liu Y, Xue H, Chen H, Tang Q, Zhang Q, Liu B, et al. mTOR activation facilitates locomotor recovery in rats with spinal cord injuries: a meta-analysis. Int J Clin Exp Med. 2021;14(10):2391–404. Li XG, Du JH, Lu Y, Lin XJ. Neuroprotective effects of rapamycin on spinal cord injury in rats by increasing autophagy and Akt signaling. Neural regeneration Res. 2019;14(4):721–7. Liu Y, Yang F, Zou S, Qu L, Rapamycin. A Bacteria-Derived Immunosuppressant That Has Anti-atherosclerotic Effects and Its Clinical Application. Front Pharmacol. 2018;9:1520. Sekiguchi A, Kanno H, Ozawa H, Yamaya S, Itoi E. 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Neuroprotection in the Acute Stage Enables Functional Recovery Following Repair of Chronic Cervical Root Transection After a 3-Week Delay. Neurosurgery. 2020;87(4):823–32. Chen HC, Fong TH, Hsu PW, Chiu WT. Multifaceted effects of rapamycin on functional recovery after spinal cord injury in rats through autophagy promotion, anti-inflammation, and neuroprotection. J Surg Res. 2013;179(1):e203–10. Cordaro M, Paterniti I, Siracusa R, Impellizzeri D, Esposito E, Cuzzocrea S. KU0063794, a Dual mTORC1 and mTORC2 Inhibitor, Reduces Neural Tissue Damage and Locomotor Impairment After Spinal Cord Injury in Mice. Mol Neurobiol. 2017;54(4):2415–27. Gao K, Wang YS, Yuan YJ, Wan ZH, Yao TC, Li HH, et al. Neuroprotective effect of rapamycin on spinal cord injury via activation of the Wnt/β-catenin signaling pathway. Neural regeneration Res. 2015;10(6):951–7. Goldshmit Y, Kanner S, Zacs M, Frisca F, Pinto AR, Currie PD, et al. Rapamycin increases neuronal survival, reduces inflammation and astrocyte proliferation after spinal cord injury. Mol Cell Neurosci. 2015;68:82–91. Kjell J, Pernold K, Olson L, Abrams MB. Oral erlotinib, but not rapamycin, causes modest acceleration of bladder and hindlimb recovery from spinal cord injury in rats. Spinal Cord. 2014;52(3):186–90. Liu J, Li R, Huang Z, Lin J, Ji W, Huang Z, et al. Rapamycin Preserves Neural Tissue, Promotes Schwann Cell Myelination and Reduces Glial Scar Formation After Hemi-Contusion Spinal Cord Injury in Mice. Front Mol Neurosci. 2020;13:574041. Nottingham S, Knapp P, Springer J. FK506 treatment inhibits caspase-3 activation and promotes oligodendroglial survival following traumatic spinal cord injury. Exp Neurol. 2002;177(1):242–51. Saraswat Ohri S, Bankston AN, Mullins SA, Liu Y, Andres KR, Beare JE, et al. Blocking Autophagy in Oligodendrocytes Limits Functional Recovery after Spinal Cord Injury. J neuroscience: official J Soc Neurosci. 2018;38(26):5900–12. Song Y, Xue H, Liu TT, Liu JM, Chen D. Rapamycin plays a neuroprotective effect after spinal cord injury via anti-inflammatory effects. J Biochem Mol Toxicol. 2015;29(1):29–34. Weng F, Zhu L, Yang L, Li Y, Liu R, Fan J, et al. [Expression of B-cell lymphoma-2 protein multisite phosphorylation in autophagy after spinal cord injury in rats]. Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese. J reparative Reconstr Surg. 2019;33(5):618–27. Tang P, Hou H, Zhang L, Lan X, Mao Z, Liu D, et al. Autophagy reduces neuronal damage and promotes locomotor recovery via inhibition of apoptosis after spinal cord injury in rats. Mol Neurobiol. 2014;49(1):276–87. Tateda S, Kanno H, Ozawa H, Sekiguchi A, Yahata K, Yamaya S, et al. Rapamycin suppresses microglial activation and reduces the development of neuropathic pain after spinal cord injury. J Orthop research: official publication Orthop Res Soc. 2017;35(1):93–103. Vargova I, Machova Urdzikova L, Karova K, Smejkalova B, Sursal T, Cimermanova V et al. Involvement of mTOR Pathways in Recovery from Spinal Cord Injury by Modulation of Autophagy and Immune Response. Biomedicines. 2021;9(6). Zhang ZY, Zhang LX, Dong XQ, Yu WH, Du Q, Yang DB, et al. Comparison of the performances of copeptin and multiple biomarkers in long-term prognosis of severe traumatic brain injury. Peptides. 2014;60:13–7. Guha L, Singh N, Kumar H. Different Ways to Die: Cell Death Pathways and Their Association With Spinal Cord Injury. Neurospine. 2023;20(2):430–48. Zhang G, Zha J, Liu J, Di J. Minocycline impedes mitochondrial-dependent cell death and stabilizes expression of hypoxia inducible factor-1α in spinal cord injury. Arch Med Sci. 2019;15(2):475–83. Doleman B, Freeman SC, Lund JN, Williams JP, Sutton AJJR. Funnel plots may show asymmetry in the absence of publication bias with continuous outcomes dependent on baseline risk: presentation of a new publication bias test. 2020;11(4):522–34. He X, Sun J, Huang X. Expression of caspase-3, Bax and Bcl-2 in hippocampus of rats with diabetes and subarachnoid hemorrhage. Exp Ther Med. 2018;15(1):873–7. Hussar P. Apoptosis regulators bcl-2 and caspase-3. Encyclopedia. 2022;2(4):1624–36. Kyrylkova K, Kyryachenko S, Leid M, Kioussi C. Detection of apoptosis by TUNEL assay. Odontogenesis: Methods and Protocols. 2012:41 – 7. Tam SY, Wu VWC, Law HKW. Influence of autophagy on the efficacy of radiotherapy. Radiat Oncol. 2017;12:1–10. Luo C, Tao L. The Function and Mechanisms of Autophagy in Spinal Cord Injury. Adv Exp Med Biol. 2020;1207:649–54. Kim YC, Guan KL. mTOR: a pharmacologic target for autophagy regulation. J Clin Invest. 2015;125(1):25–32. Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol. 2004;36(12):2503–18. Liu S, Sarkar C, Dinizo M, Faden AI, Koh EY, Lipinski MM, et al. Disrupted autophagy after spinal cord injury is associated with ER stress and neuronal cell death. Cell Death Dis. 2015;6(1):e1582. Zhao JL, Wei C, Xiao X, Dong YH, Tan B, Yu J, et al. Expression of TNF–α and IL–β can be suppressed via the PPAR–γ/mTOR signaling pathway in BV–2 microglia: A potential anti–inflammation mechanism. Mol Med Rep. 2020;22(4):3559–65. Hagemann TL, Coyne S, Levin A, Wang L, Feany MB, Messing A. STAT3 Drives GFAP Accumulation and Astrocyte Pathology in a Mouse Model of Alexander Disease. Cells. 2023;12(7). Nishimura K, Ohta M, Saito M, Morita-Isogai Y, Sato H, Kuramoto E, et al. Electrophysiological and Morphological Properties of α and γ Motoneurons in the Rat Trigeminal Motor Nucleus. Front Cell Neurosci. 2018;12:9. Saxton RA, Sabatini DM. mTOR Signaling in Growth, Metabolism, and Disease. Cell. 2017;168(6):960–76. Tian J, Chang S, Ji H, Huang T, Guo H, Kang J, et al. The p70S6K/PI3K/MAPK feedback loop releases the inhibition effect of high-dose rapamycin on rat mesangial cell proliferation. Int J Immunopathol Pharmacol. 2021;35:20587384211000544. Germano CA, Clemente G, Storniolo A, Romeo MA, Ferretti E, Cirone M et al. mTORC1/ERK1/2 Interplay Regulates Protein Synthesis and Survival in Acute Myeloid Leukemia Cell Lines. Biology (Basel). 2023;12(5). Ahmadzadeh K, Dizaji SR, Yousefifard M. Lack of concordance between reporting guidelines and risk of bias assessments of preclinical studies; A call for integrated recommendations. 2023. 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(b) inflammation; (c) glial fibrillary acidic protein; (d) alpha-motor neurons; (e) p-P70S6K.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3948391/v1/f40238f4d5dbfc86fefbec0d.png"},{"id":61868737,"identity":"8959ee79-68f0-4e54-9172-37baf79fa0e1","added_by":"auto","created_at":"2024-08-06 12:39:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":75250,"visible":true,"origin":"","legend":"\u003cp\u003eA graphical abstract of the findings in the present meta-analysis\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3948391/v1/5dd3f824636d47e491346aeb.png"},{"id":61869301,"identity":"b7d87692-ab49-42e5-9ecf-f23dcdb160a4","added_by":"auto","created_at":"2024-08-06 12:47:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3350264,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3948391/v1/89a0c562-ce7b-4bb4-9a05-e4481a3463c6.pdf"},{"id":61868741,"identity":"0f58c7f1-1b0e-4559-b841-ff203af504e1","added_by":"auto","created_at":"2024-08-06 12:39:55","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2259633,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3948391/v1/7a7604030ee092b5c91bf702.docx"}],"financialInterests":"","formattedTitle":"The Efficacy of Rapamycin in Spinal Cord Injury: A Systematic Review and Meta-Analysis of preclinical studies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpinal cord injury (SCI) is a highly disabling condition that often results in long-term damage to motor, sensory, and autonomic functions. SCI significantly impacts mobility and quality of life. Epidemiological data estimate the global incidence of traumatic SCI to be 40\u0026ndash;80 new cases per million population annually (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). SCI is characterized by a primary injury that is followed by subsequent pathophysiological changes such as apoptosis, autophagy, ischemia, glial scarring, and oxidative stress (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Researchers have explored numerous pharmacological agents to attenuate subsequent injuries after SCI (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRapamycin is a macrolide antibiotic commonly employed as an immunosuppressant to prevent organ transplant rejection (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). It inhibits the mechanistic target of Rapamycin (mTOR) pathway, which is a key regulator of cell growth, proliferation, and survival (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Rapamycin also stimulates autophagy, an intracellular degradation process responsible for eliminating damaged organelles, harmful substances, and unneeded proteins (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). While autophagy generally protects cells, excessive or improperly regulated autophagy can also lead to cell death (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome studies suggested Rapamycin-induced autophagy may play a neuroprotective role in certain neurodegenerative diseases (\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Given the potential for enhanced functional and neurological recovery in SCI with Rapamycin administration, this systematic review and meta-analysis aimed to compile and evaluate the existing literature regarding the efficacy of Rapamycin treatment in SCI for the first time.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and search strategy\u003c/h2\u003e \u003cp\u003eThe protocol for this systematic review and meta-analysis was published in the PROSPERO database for systematic reviews (registration code: CRD42023439825). The present study was conducted to summarize evidence on the effectiveness of the administration of Rapamycin for SCI. Keywords related to \u0026ldquo;Rapamycin\u0026rdquo; and \u0026ldquo;Spinal cord injury\u0026rdquo; were selected from the MeSH database of Medline, and the Emtree of Embase. Recommendations from experts were also considered. The search strategy for each database included relevant tags and Boolean operators. We conducted an extensive search in Medline (via PubMed), Embase, Scopus, and Web of Science until April 3, 2023, and performed manual searches on Google and Google Scholar to ensure comprehensive coverage. The search queries for each database are presented in the supplementary materials.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSelection criteria\u003c/h2\u003e \u003cp\u003eIn this study, the PICO framework was applied, where the Population (P) consisted of animals with SCIs, the Intervention (I) involved Rapamycin administration, the Comparison (C) group included SCI animals receiving no treatment or vehicle, and the Outcomes (O) were motor function assessment using standard scales, sensory outcomes, and markers related to apoptosis, autophagy, inflammation, neuronal counts, and the Akt/mTOR/P70S6K signaling pathway. Exclusion criteria included review studies, non-peer-reviewed articles such as letters, commentaries, and conference abstracts, duplicate reports, studies involving animals with co-morbidities, studies without traumatic SCI induction, and in vitro studies. Other exclusions comprised studies not using Rapamycin or using combination therapy, studies without a valid control group, studies involving animals other than rats and mice, and studies not reporting relevant outcomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData collection\u003c/h2\u003e \u003cp\u003eResults from the systematic search were integrated into Endnote X9, and duplicate records were removed. In the initial screening, two independent researchers reviewed the titles and abstracts of all retrieved articles. Potentially relevant articles underwent a full-text review in a secondary screening. Articles meeting the inclusion criteria were selected. If full-text or essential data were missing, the corresponding author was contacted via email, with a follow-up after a week in case of no response. Data from the included articles were extracted by two independent researchers, following a checklist based on the PRISMA guidelines. Data included information about the study's first author, year of publication, characteristics of studied animals, SCI method, SCI segment and severity, Rapamycin dose, route of administration, treatment intervals with injury, repetitions of treatments, and outcome characteristics, including timing, method, and scale, mean, and standard deviations for both treated and control groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuality assessment\u003c/h2\u003e \u003cp\u003eThe quality of the included studies was assessed using the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) risk of bias assessment tool (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). This tool evaluates the methodology and potential bias in pre-clinical studies through 10 domains. Disagreements were resolved through discussions with a third researcher when necessary.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCertainty of evidence\u003c/h2\u003e \u003cp\u003eThe certainty of evidence was evaluated using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using STATA 17.0. Studies were categorized based on reported outcomes. A standardized mean difference (SMD) with a 95% confidence interval (CI) was calculated for each sample and pooled to determine an overall effect size, since the scale of reported outcomes varied in the included articles. Meta-analysis was conducted only if data were reported in at least three separate articles. A Galbraith plot was employed to detect outlier studies. If an influential outlier were detected, the data were not included in the pooled analysis. Given the clinical and methodological diversity among studies, a random effect model was chosen. To avoid heterogeneity caused by the different intervals between treatment and locomotion assessment, we stratified experiments to discriminate short-term and long-term effects of Rapamycin. Short-term effects of Rapamycin were assessed within six days of its first administration (approximately 2 half-life), while long-term effects were evaluated beyond a six-day interval from the initial dose and outcome assessment. The half-life of Rapamycin is 58\u0026ndash;63 hours (~\u0026thinsp;3 days) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHeterogeneity was assessed through visual inspection of forest plots, I\u003csup\u003e2\u003c/sup\u003e, and chi-square tests. Publication bias were assessed by using modified egger\u0026rsquo;s test proposed by Doleman et al (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStudy selection\u003c/h2\u003e \u003cp\u003eIn our database search, we initially identified 2658 records. Following the elimination of duplicate entries, we were left with 1800 unique records. Subsequently, we conducted a thorough review of 89 full-text documents. Sixty-three studies did not meet our predefined eligibility criteria and were subsequently excluded. Ultimately, 18 papers were included in the study (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Furthermore, we performed a manual search to retrieve possible missed papers. This additional search did not yield any extra articles meeting our inclusion criteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStudy characteristics\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e provides an overview of the characteristics of the included studies. The animal models employed in these investigations included mice in six studies and rats in twelve studies. Three studies utilized a hemisection or transection model, twelve studies employed a contusion model, and three studies utilized a compression model to induce SCI. Sixteen studies induced thoracic and two studies induced cervical SCI.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of the eligible studies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStudy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnimal; Species; Gender; Weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAntibiotic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSCI model\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSCI level\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eseverity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSample size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInjury to treatment (day)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003edrug dose (mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eTreatment duration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAdministration rout\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eF/U (day)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChang KT, 2020 (14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; SD; F; 280\u0026ndash;300; Adult\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTransection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCervical\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003edaily for 1 week\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eoral\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChen HC, 2013 (15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; SD; F; 260\u0026ndash;320; Adult\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003edaily for 3 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCordaro, 2017 (16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMice; CD1; M; 25\u0026ndash;30; Adult\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCompression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1h and 6h post-SCI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGao K, 2015 (17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; SD; M; 250\u0026ndash;300; 8\u0026ndash;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes (type is not specified)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003edaily for 3 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGe C, 2020 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; SD; M; 200\u0026ndash;250; 8\u0026ndash;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoldshmit, 2015 (18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMice; C57BL/6; M; 20\u0026ndash;30; 8\u0026ndash;16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHemisection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003esingle dose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKjell, 2014 (19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; SD; F; 200\u0026ndash;225; NR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrimethoprim\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMild and moderate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003edaily for 14 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eoral\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e119\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLi XG, 2019 (3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; SD; M; 300\u0026ndash;330; Adult\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCompression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003esingle dose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiu J, 2020 (20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMice; C57; F; NR; 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCervical\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003edaily for 12 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNottingham, 2002 (21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; NR; NR; NR; NR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4, 72, 144 h post-injury\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSaraswat, 2018 (22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMice; C57BL/6; F; NR; 6\u0026ndash;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGentamycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003edaily for 3 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSekiguchi, 2012 (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMice; C57BL/6; F; NR; 10\u0026ndash;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003esingle dose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSong Y, 2014 (23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; Wistar; F; 220\u0026ndash;250; NR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmpicillin and Gentamycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003edaily for 3 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTang, 2014 (25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; SD; M; 200\u0026ndash;250; NR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHemisection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003esingle dose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTateda, 2017 (26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMice; C57BL/6; F; NR; 10\u0026ndash;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003esingle dose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVargova, 2021 (27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; Wistar; M; 300\u0026ndash;330; NR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eampicillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCompression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003edaily for 5 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWang ZY, 2014 (28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; SD; F; 200\u0026ndash;220; 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003edaily for 30 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeng F, 2019 (24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat; SD; M; 220\u0026ndash;260; 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGentamycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003edaily for 4 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eintrathecal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003eg: Grams; F/U: follow-up; NR: not reported; SD: Sprague-Dawley; IP: intraperitoneal.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eEleven studies pertained to severe injuries, five to moderate injuries, and one to mild injuries. Remarkably, one study encompassed both mild and moderate SCI induction in distinct experiments with unique animal groups. studies used 559 animals in both arms of our study. Rapamycin treatment was initiated on the same day as the injury in almost all studies, except for one study administering it one day after injury. The studies employed a standardized dose of Rapamycin, with variations observed: 0.5 mg/kg in three studies, 1 mg/kg in eight studies, 1.5 mg/kg in three studies, 1.8 mg/kg in one study, and 5 mg/kg in two studies. Treatment duration spanned from single doses to daily doses, consistently administered for a maximum of 28 consecutive days. Notably, the administration route for Rapamycin varied, with two studies utilizing oral administration, fourteen studies utilizing intra-peritoneal delivery, one study employing intrathecal administration, and one study not specifying the administration route.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRisk of bias in the studies\u003c/h2\u003e \u003cp\u003eWe used the SYRCLE tool to assess the risk of bias for each of the included studies. A summary of these assessments is provided in Supplementary Materials. In terms of overall risk of bias, all studies were assessed as at high risk of bias. The justification of such judgment by the authors is the fact that none of the studies reported adequate data for sequence generation, allocation concealment, random housing, blinding of trial caregivers, and random outcome assessment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeta-analysis of the effect of Rapamycin on spinal cord injury\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ea) Locomotion\u003c/h2\u003e \u003cp\u003eLocomotion was assessed with BBB in rats and BMS in mice. Pooled data analysis showed that administration of Rapamycin has no significant short-term effect on locomotion following SCI (SMD\u0026thinsp;=\u0026thinsp;0.78, 95% CI: -0.23 to 1.79, p\u0026thinsp;=\u0026thinsp;0.079; I\u0026sup2; = 84.71%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, when we assessed the long-term effects (at least one week after the initial dose) of Rapamycin on animals at one to six weeks after injury, Rapamycin significantly improved the locomotion at all time points (0.74\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;SMD\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;1.54, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSensitivity analysis revealed the same results when omitting studies with mild SCI. In moderate to severe SCI, Rapamycin had no significant short-term therapeutic effect (SMD\u0026thinsp;=\u0026thinsp;0.94, 95% CI: -0.28 to 2.16, p\u0026thinsp;=\u0026thinsp;0.130; I\u0026sup2; = 90.76%). However, the short-term effects were robust in 1 week to 4 weeks after moderate to severe injuries (1.04\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;SMD\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;1.95, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eb) Apoptosis and autophagy\u003c/h2\u003e \u003cp\u003eRapamycin demonstrated a superior ability to reduce TUNEL-positive spinal cells than the control group (SMD = -3.44, 95% CI: -5.41 to -1.47, p\u0026thinsp;=\u0026thinsp;0.001; I\u0026sup2; = 75.01%). The anti-apoptotic effect of Rapamycin was confirmed in the analysis when it significantly increased BcL-2 (SMD\u0026thinsp;=\u0026thinsp;6.51, 95% CI: 0.55 to 12.48, p\u0026thinsp;=\u0026thinsp;0.032; I\u0026sup2; = 97.74%) and decreased Caspase-3 (SMD = -3.85, 95% CI: -7.57 to -0.13, p\u0026thinsp;=\u0026thinsp;0.043; I\u0026sup2; = 92.67%) activity in the injured spinal cord. There was no statistical significance when studying the differences in the Bax activity (SMD = -4.96, 95% CI: -10.47 to 0.55, p\u0026thinsp;=\u0026thinsp;0.078; I\u0026sup2; = 98.33%) between Rapamycin therapy and control groups by meta-analysis following Rapamycin administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAutophagy biomarkers were also pooled by meta-analysis. Beclin-1 (SMD\u0026thinsp;=\u0026thinsp;1.42, 95% CI: 0.51 to 2.33, p\u0026thinsp;=\u0026thinsp;0.002; I\u0026sup2; = 66.81%) and LC3-II (SMD\u0026thinsp;=\u0026thinsp;1.09, 95% CI: 0.35 to 1.82, p\u0026thinsp;=\u0026thinsp;0.004; I\u0026sup2; = 11.13%) levels were clearly increased after Rapamycin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ec) Inflammation\u003c/h2\u003e \u003cp\u003eInterleukin-1 (IL-1) and Tissue Necrosis Factor-α (TNF-α) were among the inflammatory outcomes of our study. Therefore, due to the insufficient number of studies, we only analyzed the effect of Rapamycin treatment on TNF-α. Pooled data analysis showed that Rapamycin could play an anti-inflammatory effect after SCI (SMD = -3.26, 95% CI: -5.56 to -0.97, p\u0026thinsp;=\u0026thinsp;0.005; I\u0026sup2; = 82.42%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ed) GFAP and neuronal count\u003c/h2\u003e \u003cp\u003ePooled data analysis from five studies on 63 animals demonstrated that Rapamycin treatment could significantly subside areas (indicated by GFAP) containing astrocytes in the injured animals (SMD = -0.76, 95% CI: -1.28 to -0.25, p\u0026thinsp;=\u0026thinsp;0.004; I\u0026sup2; = 37.83%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The analyses also showed a significantly higher number of alpha-motor (NeuN\u003csup\u003e+\u003c/sup\u003e) neurons in the Rapamycin-treated group (SMD\u0026thinsp;=\u0026thinsp;1.18, 95% CI: 0.44 to 1.91, p\u0026thinsp;=\u0026thinsp;0.002; I\u0026sup2; = 38.47%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ee) Akt/mTOR/p70S6K signaling pathway\u003c/h2\u003e \u003cp\u003eIn order to investigate the role of Rapamycin in the treatment of SCI through AKT/mTOR/p70S6K signaling pathways, we gathered data on protein levels of Akt, mTOR, and p70S6K, along with their phosphorylated forms. \u0026zwnj;Since we applied unrelenting criteria for pooling data in a meta-analysis, the only applied marker for meta-analysis was p-P70S6K. Pooled data analysis of 4 studies (n\u0026thinsp;=\u0026thinsp;40) showed significantly lower levels of p-P70S6K within 2 weeks post-injury in the Rapamycin-treated group (SMD = -3.74, 95% CI: -6.31 to -1.18, p\u0026thinsp;=\u0026thinsp;0.004; I\u0026sup2; = 85.32%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePublication bias and certainty of evidence\u003c/h2\u003e \u003cp\u003eAs depicted in the supplementary materials, the funnel plot asymmetry test revealed no evidence of publication bias for any of the investigated outcomes, except for locomotion at 2- and 3-weeks post-injury, and p-P70S6K, for which there are concerns regarding small study effects. The level of evidence in the assessment of the neuroprotective effects of rapamycin treatment after SCI is presented in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCertainty of evidence based on GRADE framework\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStudies (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEffect size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRisk of bias\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eImprecision\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInconsistency\u003c/p\u003e \u003cp\u003e(I\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIndirectness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePublication bias\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eJudgment and level of evidence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShort-term locomotion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.80 (-0.31, 1.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot Serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eLow\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 2 points\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of serious inconsistency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLong-term locomotion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1-week post-injury\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.54 (0.55, 2.53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eLow\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 2 points\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of serious inconsistency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 weeks post-injury\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.17 (0.57, 1.76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eVery low\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 2 points\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of serious inconsistency\u003c/p\u003e \u003cp\u003e- Presence of publication bias\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3 weeks post-injury\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.95 (0.38, 1.53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eLow\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 1 point\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of publication bias\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4 weeks post-injury\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.04 (0.27, 1.81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eLow\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 2 points\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of serious inconsistency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 weeks post-injury\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.49 (0.75, 2.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eModerate\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 1 point\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6 weeks post-injury\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.01 (0.09, 1.92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eModerate\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 1 point\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eApoptosis/Autophagy\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTUNEL-positive cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.44 (-5.41, -1.47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eLow\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 2 points\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of serious inconsistency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-4.96 (-10.47, 0.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eLow\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 2 points\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of serious inconsistency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBcl-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.51 (0.55, 12.48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eLow\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 2 points\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of serious inconsistency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaspase-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.85 (-7.57, -0.13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eLow\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 2 points\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of serious inconsistency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBeclin-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.42 (0.51, 2.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eLow\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 2 points\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of serious inconsistency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLC3-II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.09 (0.35, 1.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eModerate\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 1 point\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eInflammation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNF-α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.26 (-5.56, -0.97)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eLow\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 2 points\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of serious inconsistency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAstrocytosis/Neuronal count\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.76 (-1.28, -0.25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eModerate\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 1 point\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlpha-motor neurons\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.18 (0.44, 1.91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eModerate\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 1 point\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAkt/mTOR/P70S6K signaling pathway\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-P70S6K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.74 (-6.31, -1.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot serious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSerious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eVery low\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eRated down 2 points\u003c/p\u003e \u003cp\u003e- Presence of serious risk of bias\u003c/p\u003e \u003cp\u003e- Presence of serious inconsistency\u003c/p\u003e \u003cp\u003e- Presence of publication bias\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eGRADE: Grading of Recommendations Assessment, Development and Evaluation.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur research represents the first systematic review and meta-analysis dedicated to exploring the therapeutic potential of Rapamycin in rodent models of SCI. Our findings demonstrate significant reductions in apoptosis, inflammation, and astrogliosis, along with a substantial increase in autophagic markers. These outcomes correspond with a significant improvement in long-term locomotion recovery following Rapamycin administration in the injured animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eApoptosis, often referred to as programmed cell death, plays a pivotal role in the development of secondary injury after SCI (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Mitochondria-associated cell death is among the critical mechanisms triggered by SCI (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). This process involves key proteins from the Bcl-2 family, including Bcl-2, Bax, and the executioner Caspase. When cells undergo apoptosis, Bax adheres to the outer mitochondrial membrane, forming pores through oligomerization. This pore formation allows the release of apoptogenic factors like Cytochrome C into the cytoplasm. Bcl-2, a(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)n anti-apoptotic protein, can inhibit apoptosis by binding to Bax, preventing the formation of these pores. In the cytoplasm, Cytochrome C contributes to the formation of the apoptosome, which activates Caspase-9. Activated Caspase-9 then transforms Pro-caspase-3 into effector Caspase-3, leading to the typical morphological changes observed in apoptotic cells (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Our study also reveals that Rapamycin treatment results in an increase in the anti-apoptotic protein Bcl-2 and a decrease in the pro-apoptotic protein Bax. While it is important to note that individual studies examining Bax levels in the Rapamycin-treated group show a significant increase, pooled data analysis did not demonstrate a significant overall change. This discrepancy may be attributed to the limited number of studies included in the meta-analysis. Furthermore, we observed an increase in the executioner caspase, Caspase-3, suggesting that Rapamycin has complex effects on the regulation of apoptosis. Specifically, it appears that Rapamycin suppresses the mitochondrial apoptosis pathway. Additionally, our assessment of Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assays to detect apoptotic cells undergoing extensive DNA degradation during the late stages of apoptosis (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), demonstrated a decrease in the groups treated with Rapamycin, further confirming the inhibitory role of Rapamycin in the apoptotic pathway.\u003c/p\u003e \u003cp\u003eAutophagy is the process by which cells digest their components to maintain cellular homeostasis (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). This autodigestion increases in neurons within hours after SCI, likely serving as a neuroprotective mechanism (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The initiation of autophagy relies on the key regulatory protein Beclin-1 (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). LC3-II, a protein involved in forming autophagosomes for degradation, also reflects autophagy levels (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Our study found that Rapamycin significantly increased Beclin-1 and LC3-II, indicating enhanced autophagic flux. Importantly, p62, which targets proteins for autophagic degradation, was lower with Rapamycin treatment in two studies (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). This decline in p62, which binds to ubiquitinated proteins and targets them for degradation via autophagy, confirms that Rapamycin truly enhances autophagic digestion rather than just accumulating autophagosomes (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePost-trauma inflammation after SCI is characterized by increased pro-inflammatory cytokines such as TNF-α and IL-1β, attracting immune cells like macrophages and microglia, which exacerbate the inflammatory response. This inflammation contributes to secondary neuronal damage through various pathways (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Studies have shown that Rapamycin treatment can reduce inflammation by attenuating the mTOR pathway in microglia, decreasing their activation and production of pro-inflammatory cytokines (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Our study aligns with previous research, as it demonstrates that Rapamycin significantly lowers levels of TNF-α after injury. While an IL-1β meta-analysis was not conducted due to limited studies, the two studies included also indicated significantly lower levels of IL-1β in the Rapamycin treatment group compared to the injury control group (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). By dampening this inflammatory cascade, Rapamycin might help limit secondary damage after SCI.\u003c/p\u003e \u003cp\u003eGFAP, an intermediate filament protein expressed predominantly in astrocytes, is commonly used to identify astrocytes and to examine astrogliosis, referring to reactive astrocyte proliferation in response to injury. Activated astrocytes secrete proinflammatory cytokines and contribute to glial scar formation and inhibit axonal regeneration (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Studies suggest Rapamycin inhibits STAT3 signaling in astrocytes, which is known to promote reactive astrocytosis. By inhibiting STAT3 activation, Rapamycin reduces GFAP expression and other markers of astrocyte reactivity (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Additionally, by inhibiting mTOR, Rapamycin shifts astrocyte metabolism from glycolysis to more efficient oxidative phosphorylation, which could be associated with reduced astrocyte reactivity (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Consistent with these mechanisms, our study shows that Rapamycin reduces GFAP expression after SCI, potentially creating a more favorable environment for nerve regeneration.\u003c/p\u003e \u003cp\u003eAlpha-motor neurons, also known as lower or skeletal motor neurons, innervate muscle fibers (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Our study indicates that there are more surviving alpha-motor neurons in the spinal cord of the Rapamycin-treated group compared to the controls. Rapamycin likely provides neuroprotection by decreasing microglial and astrocytic activation and increasing neuronal autophagy, thereby reducing secondary damage after injury (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRapamycin inhibits mTOR Complex 1 (mTORC1), a key regulator of cell growth and metabolism (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). mTORC1 normally phosphorylates and activates p70S6 Kinase (p70S6K), which promotes protein synthesis and cell growth (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Akt is a protein kinase that activates mTORC1 by phosphorylating and inhibiting TSC1/TSC2, negative regulators of mTORC1 (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The p-Akt protein expression was assessed in four studies; two studies measured p-Akt/β -actin and two studies measured p-Akt/Akt. Therefore, data pooling did not apply to a meta-analysis. However, studies showed increased p-Akt levels within the first week of Rapamycin therapy compared to controls in SCI models, likely due to the negative feedback on PI3K/Akt signaling when mTORC1 is inhibited (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). However, one study found decreased p-Akt at 2- and 4 weeks post-injury (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Considering Rapamycin's half-life, the early feedback-driven p-Akt upregulation may peak and normalize after prolonged treatment, leading to observable declines by 2 weeks.\u003c/p\u003e \u003cp\u003eOur research uncovers various potential explanations for how Rapamycin contributes to enhancing motor function recovery. Firstly, we observed that Rapamycin effectively prevents apoptosis. Secondly, it reduces astrogliosis and inflammation. Thirdly, it promotes autophagy at the site of the injury. On the flip side, treatment with the mTOR inhibitor Rapamycin hampers the Akt/mTOR/p70S6K signaling pathway and decreases the expression of proteins related to myelin formation in the damaged spinal cord (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Additionally, mTORC1 inhibition leads to the suppression of p70S6K, responsible for promoting protein synthesis and cell growth (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Consequently, these findings have given rise to conflicting hypotheses. In alignment with these controversies, our study demonstrated a lack of significant short-term improvement and only low to moderate long-term enhancements in motor function.\u003c/p\u003e \u003cp\u003eOur study has limitations. It's important to acknowledge that the literature has only reported a limited number of underlying pathways, and there is some degree of heterogeneity. This emphasizes the need for caution when interpreting these findings. Additionally, certain variables were too scarce to be combined, including the severity of the injury, the timing of treatment, the number and routes of administered doses, robust follow-up evaluations, and some outcomes were solely assessed through one method, such as TNF-α for inflammatory markers and p70S6K for Akt/mTOR/p70S6K signaling pathway. These considerations underscore the necessity for further experimental research in this field to ensure the generalizability of results. Another shortcoming arises from the low quality of the animal studies, all of which were judged to carry a high risk of bias. This limitation stems from the studies' failure to include some major elements outlined in CYRCLE's risk of bias tool. It is important to note that researchers cannot be faulted for this omission, as the ARRIVE guidelines for animal research do not mandate reporting on most of these items (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Finally, there was evidence of publication bias in the assessment of locomotion at 2- and 3-weeks post-injury, as well as in p-P70S6K. This bias was predisposed by the limited number of studies for each outcome.\u003c/p\u003e \u003cp\u003eIn conclusion, Rapamycin demonstrates neuroprotective, anti-inflammatory, and pro-autophagic effects in rodent SCI models, leading to modest long-term improvements in motor function recovery versus controls. The low- to moderate-level evidence from this study emphasizes the necessity for more rigorous experimental investigations to thoroughly assess the effectiveness of Rapamycin treatment in animal models with SCI.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All authors (HZ, RH, AZ, HAR, MY) have made substantial contributions to the conception or design of the current work; or the acquisition, analysis, or interpretation of data for the work; drafting the work, or revising it critically. All authors have provided final approval of the version to be submitted. Conception and design: MY; data extraction: HZ, RH; statistical analysis: HZ, MY; interpretation of the results: HZ, AZ, HAR; Drafting: HZ, AZ, RH; Revising the work critically: all authors; graphical abstract: HAR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset generated and analyzed during the current study is available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding/Support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research has been supported by Iran University of Medical Sciences (Grant number: 1401-4-75-25439)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePerrouin-Verbe B, Lefevre C, Kieny P, Gross R, Reiss B, Le Fort M. 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Cell. 2017;168(6):960\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian J, Chang S, Ji H, Huang T, Guo H, Kang J, et al. The p70S6K/PI3K/MAPK feedback loop releases the inhibition effect of high-dose rapamycin on rat mesangial cell proliferation. Int J Immunopathol Pharmacol. 2021;35:20587384211000544.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGermano CA, Clemente G, Storniolo A, Romeo MA, Ferretti E, Cirone M et al. mTORC1/ERK1/2 Interplay Regulates Protein Synthesis and Survival in Acute Myeloid Leukemia Cell Lines. Biology (Basel). 2023;12(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmadzadeh K, Dizaji SR, Yousefifard M. Lack of concordance between reporting guidelines and risk of bias assessments of preclinical studies; A call for integrated recommendations. 2023.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"systematic-reviews","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sysr","sideBox":"Learn more about [Systematic Reviews](http://systematicreviewsjournal.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/sysr/default.aspx","title":"Systematic Reviews","twitterHandle":"@MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Spinal Cord Injuries, Quadriplegia, Hemiplegia, spinal cord contusion, Sirolimus, Rapamycin","lastPublishedDoi":"10.21203/rs.3.rs-3948391/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3948391/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRapamycin has shown a potential role in functional and neurological recovery after neurodegenerative disease. The current study evaluates the efficacy of Rapamycin in preclinical spinal cord injury (SCI).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA systematic literature search was conducted in Medline, Embase, Scopus, and Web of Science databases until April 2023. Inclusion criteria were preclinical studies comparing Rapamycin treatment to a control group in animal models of SCI and reporting outcomes including locomotion, apoptosis, autophagy, inflammation, astrogliosis, neuronal counts, and signaling proteins related to the mechanistic target of Rapamycin in Akt/mTOR/p70S6K pathway. Two independent reviewers performed study screening and data extraction. For meta-analyses, a standardized mean difference (SMD) with a 95% confidence interval (CI) was calculated for each experiment and a pooled effect size was reported. The risk of bias and certainty of evidence was assessed using SYRCLE and GRADE tools, respectively.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e18 papers were included in the study. Rapamycin significantly decreased apoptosis (TUNEL: SMD \u0026minus;\u0026thinsp;3.44, 95% CI -5.41 to -1.47; Caspase-3: SMD \u0026minus;\u0026thinsp;3.85, 95% CI -7.57 to -0.13), inflammation (TNF-α: SMD \u0026minus;\u0026thinsp;3.26, 95% CI -5.56 to -0.97), astrogliosis (GFAP: SMD \u0026minus;\u0026thinsp;0.76, 95% CI -1.28 to -0.25), and inhibited Akt/mTOR/p70S6K signaling pathway (SMD \u0026minus;\u0026thinsp;3.74, 95% CI -6.31 to -1.18). It increased autophagy markers (Beclin-1: SMD 1.42, 95% CI 0.51 to 2.33; LC3-II: SMD 1.09, 95% CI 0.35 to 1.82) and neuronal counts (SMD 1.18, 95% CI 0.44 to 1.91). Locomotion was not significantly influenced by the short-term effects of Rapamycin. However, treatment had significant long-term improvements in locomotion (SMD 0.74\u0026ndash;1.54 from 1\u0026ndash;6 weeks).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe current study indicates Rapamycin provides neuroprotection, reduces inflammation, enhances autophagy, and improves long-term locomotion in rodent SCI models.\u003c/p\u003e","manuscriptTitle":"The Efficacy of Rapamycin in Spinal Cord Injury: A Systematic Review and Meta-Analysis of preclinical studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-06 12:39:50","doi":"10.21203/rs.3.rs-3948391/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2024-07-12T09:44:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Systematic Reviews","date":"2024-02-10T11:59:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"systematic-reviews","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sysr","sideBox":"Learn more about [Systematic Reviews](http://systematicreviewsjournal.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/sysr/default.aspx","title":"Systematic Reviews","twitterHandle":"@MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9455f45c-634a-409b-a58c-ccc42df0f426","owner":[],"postedDate":"August 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-06T12:39:50+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-06 12:39:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3948391","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3948391","identity":"rs-3948391","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}