A Rat Model of Second Impact Syndrome in Diffuse Traumatic Brain Injury: Evidence of Facial Hypersensitivity, Neurological and Other Behavioral Alterations, and Immunological and Histopathological Tissue Changes

A Rat Model of Second Impact Syndrome in Diffuse Traumatic Brain Injury: Evidence of Facial Hypersensitivity, Neurological and Other Behavioral Alterations, and Immunological and Histopathological Tissue Changes | 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 A Rat Model of Second Impact Syndrome in Diffuse Traumatic Brain Injury: Evidence of Facial Hypersensitivity, Neurological and Other Behavioral Alterations, and Immunological and Histopathological Tissue Changes Leonardo de Macedo Filho, Ana Carolina Aragao, Vito Thayson dos Santos, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7273712/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Traumatic brain injury (TBI) can lead to chronic complications, including headache and facial hypersensitivity. Second-impact syndrome (SIS), occurring when a second TBI precedes recovery from an initial injury, often causes severe long-term symptoms. Neuroinflammation is implicated, but the comprehensive pathophysiology of SIS remains unclear. This study characterized neurological/behavioral outcomes, facial nociception, plasma inflammatory markers, and central nervous system (CNS) histopathology in rat models of SIS and single mild TBI. Female Wistar rats (n = 6/group) were assigned to naïve control, sham surgery, single impact (TBI), or double impact (SIS; intervals: 1h, 24h, 3d, 7d, 14d). Mild TBI was induced using a modified Marmarou weight-drop model. Assessments (facial mechanical sensitivity via von Frey, Neurological Severity Scale (NSS), Neurobehavioral Scale (NBS)) were performed pre-injury and on days 1,3,5,7 post-injury. Plasma IL-6 and TNF-α were measured by ELISA. Histopathology (H&E) assessed CNS inflammation. All impact groups developed transient facial mechanical allodynia and acute neurobehavioral deficits (impaired NSS/NBS at day 1). SIS groups, particularly with longer intervals (≥ 24h), exhibited more prolonged facial allodynia and significant neurological deficits compared to single TBI at specific timepoints. Plasma cytokines showed minimal changes. Histopathology revealed significantly elevated CNS inflammation in both TBI and SIS groups versus controls, with SIS groups (intervals 24h-14d) showing robust responses. Repeated TBI, especially with longer recovery intervals between injuries, results in more severe and prolonged facial allodynia, neurological deficits, and CNS neuroinflammation than a single mild TBI. These findings illuminate SIS pathophysiology and underscore the critical need for protection post-concussion. Traumatic brain injury Second-impact syndrome Facial pain Neurobehavioral Neuroinflammation Rat model Figures Figure 1 Figure 2 Figure 3 Introduction Traumatic brain injury (TBI) is an insult to the brain caused by an external force, leading to a range of neurological impairments and long-term consequences (Ng and Lee 2019). Second impact syndrome (SIS) refers to a second head injury that occurs before full recovery from an initial concussion (Tator 2013; McLendon et al. 2016; Romero-Reyes and Uyanik 2014). SIS often results in both acute and chronic exacerbation of symptoms. For example, patients may develop post-traumatic headache (PTH) that resembles migraine and is frequently accompanied by facial mechanical allodynia (Lucas et al. 2014; Xiong et al. 2013; Mustafa et al. 2017). SIS also stimulates neuroinflammatory and oxidative-stress processes that can worsen brain damage and impair neurobehavioral recovery (de Macedo Filho et al. 2024; Alam et al. 2020; Freire et al. 2023). Previous rodent studies have modeled single versus repeated TBI to investigate migraine-like hypersensitivity, facial allodynia, and underlying immunological and histopathological changes (Woodcock and Morganti-Kossmann 2013; Mørch et al. 2007; Hu 1990; Bree and Levy 2018; Tanaka and Zhang 2022; Studlack et al. 2018; Ou et al. 2022; Mustafa et al. 2016). However, a direct comparison of neurological, behavioral, immunological, and histological outcomes between single-TBI and SIS models has not been reported. Therefore, we designed this study to compare the post-injury effects of one versus two head impacts in rats. We measured facial nociception, comprehensive neurological and behavioral function (NSS/NBS), plasma cytokine levels (IL-6, TNF-α), and CNS histopathology in a rat SIS model, and compared these findings to a single-TBI model and control animals. Materials and Methods Animals and Experimental Groups Female Wistar rats (mean ± SD body weight, 194.0 ± 16.5 g) were obtained from the Experimental Biology Center (NUBEX) at the University of Fortaleza. All protocols were approved by the University of Fortaleza Animal Ethics Committee (protocol #6442130818) and followed Brazilian guidelines for animal research. Rats were randomly allocated to eight groups (n=6 per group; Table 1): (1) Naïve controls (no anesthesia or surgery); (2) Sham controls (anesthesia and scalp incision without impact); (3) TBI (1-impact) (anesthesia + one weight-drop impact); and (4–8) SIS (2-impacts) groups with two identical impacts separated by 1 hour, 24 hours, 3 days, 7 days, or 14 days, respectively. Behavioral and neurological tests were performed 1 day before the first procedure (baseline) and on post-operative days 1, 3, 5, and 7 (denoted PO1–PO7). The interval between the first and second impacts was set according to group (as above). After the final assessment on PO7, animals were euthanized for blood collection and brain histology. All assessments were conducted by investigators blinded to group assignment. Traumatic Brain Injury Model A modified Marmarou impact acceleration model was used [19]. Rats were anesthetized with ketamine (70 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and placed prone on a foam bed. A midline scalp incision (~20 mm) was made, and a 10-mm diameter, 3-mm thick stainless-steel disc was affixed to the skull midline (sagittal suture) between bregma and lambda using cyanoacrylate glue. Once the adhesive set, a 450-g weight was dropped from a height of 1 m through a Plexiglas tube onto the disc, causing a mild diffuse brain injury (Fig. 1). The mortality rate was 0% across all groups. After impact, the scalp was sutured, and animals were allowed to recover under observation. Behavioral and Neurological Assessments Facial Mechanical Sensitivity: Facial nociception was evaluated using an electronic von Frey apparatus (Insight Equipamentos, Brazil). Rats were tested in their home cages while awake. At baseline (1 day pre-injury) and on PO1, PO3, PO5, and PO7, von Frey filaments of increasing force were applied by a blind evaluator (V.T.d.S.) to three sites on the vibrissal pad. The withdrawal threshold at each site was defined as the minimal force eliciting a head-withdrawal or scratching response in ≥3 of 5 trials. The final threshold for each animal was the mean of the three sites. Forces above 15 g were not applied to avoid triggering head extension responses. Neurological Severity Scale (NSS) and Neurobehavioral Scale (NBS): At the same timepoints (baseline, PO1, PO3, PO5, PO7), overall neurological and behavioral function was scored. The NSS [20] is a composite scale (0–28; 0 = normal, ≥20 = severe deficit) assessing motor/sensory reflexes and coordination. The NBS [20] evaluates general behavior across five domains (including exploration, proprioception, and response to novel objects) with a total score of 0–20 (20 = normal). Higher NSS scores indicate worse deficits, while higher NBS scores indicate better function. A single blinded evaluator (D.A.) performed all assessments. Cytokine Assays Blood samples were collected from anesthetized rats on PO1 and PO7 (via cardiac puncture) immediately before euthanasia. (Naïve controls were bled only on PO7.) Plasma was isolated by centrifugation. Levels of IL-6 and TNF-α in plasma were measured using DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions [21]. Assays were read by two independent blinded investigators (R.C.L.J., L.M.N.), and cytokine concentrations were calculated from standard curves. Results are expressed as pg per 100 µL of plasma [22]. Histology Immediately after blood collection, animals were perfused with 10% buffered formalin. Brains were extracted, post-fixed in formalin, and coronally sectioned (5 µm) using a microtome (Leica, Germany). Sections were stained with hematoxylin-eosin and analyzed under light microscopy using CaseViewer software (3DHistech, Hungary). Two blinded observers (J.M., D.O.) performed semi-quantitative scoring of pathology in various CNS regions. We scored congestion, vasogenic edema, cytotoxic edema, neuronal necrosis (in basal ganglia, cerebellum, and cortex), and inflammatory cell presence. Each parameter was rated on a scale from 0 (none) to 4 (severe) as previously described [23]. Statistical Analysis All data were tested for normality (Shapiro–Wilk); ~31% of measures were non-normal. Therefore, nonparametric statistics were used. Two-group comparisons (e.g. impact vs control at a given time) were performed with two-tailed Mann–Whitney U tests [24,25]. For outcomes involving multiple groups or repeated measures (e.g. NSS, NBS, histology), we used Kruskal–Wallis tests. Histological scores from the two raters were averaged. Each impact group (with its interval) was compared to the naïve and sham control groups at each timepoint (Pre-op, PO1, PO3, PO5, PO7) using Mann–Whitney tests. Significance was set at p < 0.05. Data are reported as mean ± standard error. Results Facial Mechanical Sensitivity All experimental groups receiving either single (TBI) or double (SIS) impacts demonstrated significantly reduced mechanical response thresholds to facial stimulation compared to naïve controls (Table 2, Figure 2A). However, post-hoc analysis revealed specific temporal patterns in these hypersensitivity responses. Notably, the SIS group at 14 days post-injury (14D) exhibited statistically significant mechanical hypersensitivity at both PO3 (p = 0.0461) and PO5 (p = 0.0223) timepoints relative to naïve controls. Similarly, the 24-hour post-impact SIS group (24H) showed significant threshold reduction at PO5 (p = 0.0316). When compared to sham controls, we observed robust mechanical hypersensitivity at PO1 in multiple experimental groups: single-impact TBI (p = 0.005), as well as SIS groups at 1 hour (p = 0.0084), 24 hours (p = 0.0304), 3 days (p = 0.0449), and 7 days (p = 0.0304) post-injury. This hypersensitivity pattern persisted at PO3 in SIS groups at 1 hour (p = 0.0219), 3 days (p = 0.0318), and 14 days (p = 0.0097). The PO5 timepoint showed significant threshold reductions in single-impact TBI (p = 0.0325) and SIS groups at 24 hours (p = 0.0219) and 14 days (p = 0.0065). Importantly, no significant differences were observed between experimental and sham groups at the PO7 timepoint. Neurological and Behavioral Assessments At the PO1 time point, Neurological Severity Scale (NSS) scores were significantly elevated, and Neurobehavioral Scale (NBS) scores were reduced across all impact groups compared to naïve controls (Figure 2B and Table 3). NSS increases reached statistical significance in the single-impact group (p = 0.0048) and all double-impact cohorts—1-hour (1H; p = 0.0013), 24-hour (24H; p = 0.0014), 3-day (3D; p = 0.0013), 7-day (7D; p = 0.0014), and 14-day (14D; p = 0.0014) intervals. Similarly, NBS reductions were observed in the single-impact group (p = 0.0048) and all double-impact groups (1H: p = 0.0045; 24H: p= 0.0013; 3D: p= 0.0014; 7D: p = 0.0014; 14D: p = 0.0013). Relative to sham controls, NSS scores were elevated in double-impact groups with 1H (p = 0.0293), 7D (p = 0.0423), and 14D (p = 0.0500) intervals at PO1. NBS scores were also reduced in double-impact groups with 3D (p = 0.0300) and 14D (p = 0.0421) intervals compared to sham controls. No statistically significant differences in NSS or NBS scores were observed between impact groups and controls (naïve or sham) at subsequent time points (PO3, PO5, PO7). Inflammatory Mediators and Histopathology Plasma IL-6 and TNF-α levels (Fig. 2C and Table 4) showed no differences between any impact group and naïve controls at either timepoint. However, comparisons to sham controls revealed some changes: the 2-impacts (14d) group had lower TNF-α (p = 0.0221), and the 2-impacts (24h), (3d), and (7d) groups had higher IL-6 (p = 0.0463, 0.0065, and 0.0318, respectively). Histologically, injured brains showed increased inflammation and damage. Compared to naïve controls, the 1-impact group (p = 0.0314) and all SIS groups (24h–14d) had significantly higher inflammation scores (Fig. 2D, Fig. 3, Table 5). Specifically, inflammation was elevated in 2-impacts (24h) (p = 0.00228), 2-impacts (3d) (p = 0.00228), 2-impacts (7d) (p = 0.00118), and 2-impacts (14d) (p = 0.00231) relative to naïve (the 3d and 14d p-values were corrected from earlier errors). Relative to sham, significant inflammation increases were seen in 2-impacts (3d) (p = 0.0117), (7d) (p = 0.00320), and (14d) (p = 0.00797). No significant edema or necrosis differences were detected in other regions. Overall, these findings indicate that SIS produced a robust CNS inflammatory response, especially with longer inter-injury intervals. Discussion Our study reveals that rats subjected to repeated mild TBIs (SIS model) exhibit pronounced facial hypersensitivity, neurological deficits, and brain inflammation, compared to single-TBI or uninjured controls. Facial mechanical hypersensitivity developed rapidly after injury. All impact groups showed reduced withdrawal thresholds, reflecting facial allodynia, but the effect was strongest when two impacts occurred within short intervals. For example, the 14-day SIS group had significantly lower thresholds on day 3–5 post-injury, and even the 24-hour SIS group was sensitized by day 5. These results align with prior rodent studies where TBI induced migraine-like hypersensitivity and trigeminal sensitization (Mørch et al. 2007; Hu 1990; Bree and Levy 2018; Tanaka and Zhang 2022; Wattiez et al. 2021). Clinical reports also note that second injuries can precipitate severe headache early on (Minen et al. 2016; Kontos et al. 2013; Theeler et al. 2013). The mechanisms likely involve peripheral and central sensitization of trigeminal pathways and inflammation of meningeal structures (Mustafa et al. 2017; Mørch et al. 2007; Hu 1990; Bree and Levy 2018; Tanaka and Zhang 2022). For instance, Mustafa et al. (2017) showed that neurotransmitter changes (e.g., serotonin) and enhanced trigeminal excitability underlie post-traumatic headache in rodents. Our data extend this by showing that an additional impact can worsen and prolong facial allodynia. Notably, even a 1-hour interval between impacts produced acute hypersensitivity (day 1), suggesting that very short intervals fall within the SIS window (McLendon et al. 2016). Longer intervals (several days) also caused persistent allodynia, indicating that the brain remains vulnerable for at least 2 weeks after an injury. Neurological and behavioral deficits were also observed acutely. Immediately after injury (PO1), all impact groups scored worse than controls on the NSS and NBS. This reflects the immediate sensorimotor and cognitive disturbances that follow concussive brain trauma. Some SIS groups showed borderline significance versus sham at day 1 (e.g., higher NSS in 1h, 7d, 14d SIS; lower NBS in 3d, 14d SIS), implying that multiple injuries can slightly exacerbate acute deficits. These findings are consistent with clinical observations: a second concussion before recovery can trigger rapid decline, brain swelling, and serious disability (McLendon et al. 2016; Zeidan and Johnson 2011). The present deficits mirror reports that repetitive injuries prolong recovery and worsen outcomes (Sagher 2013; McKee and Daneshvar 2015; Kamins and Giza 2016; Rodney et al. 2018). However, by days 3–7 post-injury, performance largely recovered to control levels, consistent with the transient nature of many acute concussion symptoms. Longer-term impacts (e.g., chronic traumatic encephalopathy) were beyond this short 7-day study. Inflammatory markers showed modest changes. IL-6 and TNF-α are known to have complex roles after TBI (Woodcock and Morganti-Kossmann 2013; Rodney et al. 2018; Ziebell and Morganti-Kossmann 2010; Amanollahi et al. 2023). In our SIS model, groups with two impacts had higher IL-6 levels (24h, 3d, 7d) compared to sham, supporting the idea that repeated injury augments the IL-6 response (Engelhardt et al. 2020; Patterson and Holahan 2012; Parkin et al. 2019). IL-6 can be neuroprotective or neurotoxic depending on context (Ooi et al. 2022). Conversely, TNF-α was decreased in the 14d SIS group relative to sham, which might reflect a shift toward resolving inflammation or a neuroprotective phase (Ferreira et al. 2014; Galgano et al. 2017). These dynamic changes suggest that timing and context determine cytokine effects. Similar variability has been reported in other TBI studies (Ling et al. 2004; Choi et al. 1994; Finamor et al. 2023), highlighting the need for detailed time-course analyses. Histopathological changes were pronounced in SIS rats. We observed neuronal necrosis and edema in multiple CNS areas, along with increased inflammatory cells and vascular congestion, especially in SIS groups with longer intervals. TBI is known to cause cortical and subcortical cell death and inflammation (Meymandi et al. 2018; McKee and Daneshvar 2015). Our data suggest that repetitive injury exacerbates these pathologies: rats receiving two impacts beyond 24 hours apart showed more widespread edema and damage than those with a single impact. This is consistent with the concept that SIS leads to compounded neuropathology. Pro-inflammatory cytokines likely attracted immune cells and worsened edema (Ziebell and Morganti-Kossmann 2010). Thus, repeated concussions produce additive brain injury in this model, in line with other reports of cumulative TBI damage. Study Limitations: The findings of our study should be interpreted with several caveats. Behavioral scoring is inherently subjective, though we minimized bias with validated scales and blinded observers. Our histological analysis was semi-quantitative, lacking cell-specific markers (e.g., for microglia or apoptotic neurons), which could refine interpretation. Only female rats were studied, so sex differences in SIS responses remain unknown. We also limited observations to 7 days post-injury; longer-term follow-up is needed to assess persistent hypersensitivity or neurodegeneration. Future work should include more detailed immunohistochemistry, both sexes, and extended timepoints to capture chronic effects of SIS. Conclusion In conclusion, this study provides a comprehensive comparison of single versus repeated mild TBI in rats. We show that a second impact (SIS) results in more severe facial hypersensitivity, acute neurological deficits, and CNS inflammation than a single injury, particularly when the second impact occurs more than one day later. These results improve our understanding of SIS pathophysiology and underscore the importance of strict return-to-play guidelines after a concussion. By delineating these changes, our work lays the groundwork for future studies on the mechanisms and treatment of SIS. The findings support clinical observations that SIS can produce more severe and longer-lasting deficits than an isolated concussion, and they may inform development of protective and therapeutic strategies for repetitive brain injury. Declarations Conflict of Interest: The authors declare no conflicts of interest. Funding: This research received no external funding. Author Contribution LdMF: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft, Writing – review & editing, Supervision.ACA, VTdS, DA: Conceptualization, Methodology, Writing – original draft.LP: Formal analysis.RCLJ, LMN: Investigation, Formal analysis.JM, DO: Investigation.GM, ESG, HW, DB, NS: Writing – review & editing.BS, GH: Conceptualization, Supervision, Writing – review & editing.AR: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft, Writing – review & editing, Supervision. Acknowledgement This research received the Natus Resident/Fellow Award for Traumatic Brain Injury at the CNS 2024 Annual Meeting. We thank the Nucleo de Pesquisa Experimental da UNIFOR (NUBEX) at the Universidade de Fortaleza for research support and animal care facilities. References Abe K, Hashimoto Y, Yatsushiro S et al (2013) Simultaneous immunoassay analysis of plasma IL-6 and TNF-α on a microchip. 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JAMA 315(10):1014–1025. https://doi.org/10.1001/jama.2016.1203 Zeidan F, Johnson SK (2011) Second Impact Syndrome. In: Kreutzer JS, DeLuca J, Caplan B (eds) Encyclopedia of Clinical Neuropsychology. Springer, New York, NY. https://doi.org/10.1007/978-0-387-79948-3_572 Ziebell JM, Morganti-Kossmann MC (2010) Involvement of pro- and anti-inflammatory cytokines and chemokines in the pathophysiology of traumatic brain injury. Neurotherapeutics 7(1):22–30. https://doi.org/10.1016/j.nurt.2009.10.016 Tables Table 1 to 5 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table15.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Naive\u003c/p\u003e\n\u003cp\u003egroup: \u003cstrong\u003eA)\u003c/strong\u003e neuronal death in cerebral cortex (44.3x); \u003cstrong\u003eB)\u003c/strong\u003e neuronal death in cerebellum (37.2x). Sham group; \u003cstrong\u003eC)\u003c/strong\u003e neuronal death in cerebellum (23.6x); \u003cstrong\u003eD-F)\u003c/strong\u003e signs of congestion and edema (14.8x); \u003cstrong\u003eG)\u003c/strong\u003e neuronal death in cerebral cortex (53.1x); \u003cstrong\u003eH)\u003c/strong\u003e congestion of choroid plexus (6.0x). One impact group; \u003cstrong\u003eI)\u003c/strong\u003e neuronal death in cerebral cortex (53.1x). Two impacts at an interval of 1 hour: \u003cstrong\u003eJ)\u003c/strong\u003e neuronal death in cerebellum (27.0x). Two impacts at\u003c/p\u003e\n\u003cp\u003ean interval of 24 hours; \u003cstrong\u003eK)\u003c/strong\u003e signs of congestion and edema in cerebral cortex (36.8x). Two impacts at an interval of 3 days: \u003cstrong\u003eL-M)\u003c/strong\u003e neuronal death in cerebral cortex (44.3x). Two impacts at an interval of 7 days: \u003cstrong\u003eN)\u003c/strong\u003e neuronal death in cerebral cortex (36.9x); \u003cstrong\u003eO)\u003c/strong\u003e neuronal death in hippocampus (40.9x); \u003cstrong\u003eP)\u003c/strong\u003e signs of congestion and edema in choroid plexus and basal ganglia.\u003c/p\u003e\n\u003cp\u003e(53.1x); \u003cstrong\u003eQ-R)\u003c/strong\u003e neuronal death in cerebral cortex (44.3x). Two impacts at an interval of 14 days: \u003cstrong\u003eS)\u003c/strong\u003e edema in cerebral cortex (44.3x).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7273712/v1/be08ea602e11ce4714338a5d.jpeg"},{"id":96965411,"identity":"935e5315-08c8-4952-9666-8b2ed32874d0","added_by":"auto","created_at":"2025-11-28 06:24:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1428145,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7273712/v1/cef65f56-faee-4ad3-8d9c-4bd6d9874f95.pdf"},{"id":89567335,"identity":"e047bfa9-ae4b-4bca-8f82-b54f5715c716","added_by":"auto","created_at":"2025-08-21 11:11:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26935,"visible":true,"origin":"","legend":"","description":"","filename":"Table15.docx","url":"https://assets-eu.researchsquare.com/files/rs-7273712/v1/5fbc2a733d3d37b19e39d0b8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Rat Model of Second Impact Syndrome in Diffuse Traumatic Brain Injury: Evidence of Facial Hypersensitivity, Neurological and Other Behavioral Alterations, and Immunological and Histopathological Tissue Changes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTraumatic brain injury (TBI) is an insult to the brain caused by an external force, leading to a range of neurological impairments and long-term consequences (Ng and Lee 2019). Second impact syndrome (SIS) refers to a second head injury that occurs before full recovery from an initial concussion (Tator 2013; McLendon et al. 2016; Romero-Reyes and Uyanik 2014). SIS often results in both acute and chronic exacerbation of symptoms. For example, patients may develop post-traumatic headache (PTH) that resembles migraine and is frequently accompanied by facial mechanical allodynia (Lucas et al. 2014; Xiong et al. 2013; Mustafa et al. 2017). SIS also stimulates neuroinflammatory and oxidative-stress processes that can worsen brain damage and impair neurobehavioral recovery (de Macedo Filho et al. 2024; Alam et al. 2020; Freire et al. 2023).\u003c/p\u003e\u003cp\u003ePrevious rodent studies have modeled single versus repeated TBI to investigate migraine-like hypersensitivity, facial allodynia, and underlying immunological and histopathological changes (Woodcock and Morganti-Kossmann 2013; M\u0026oslash;rch et al. 2007; Hu 1990; Bree and Levy 2018; Tanaka and Zhang 2022; Studlack et al. 2018; Ou et al. 2022; Mustafa et al. 2016). However, a direct comparison of neurological, behavioral, immunological, and histological outcomes between single-TBI and SIS models has not been reported. Therefore, we designed this study to compare the post-injury effects of one versus two head impacts in rats. We measured facial nociception, comprehensive neurological and behavioral function (NSS/NBS), plasma cytokine levels (IL-6, TNF-α), and CNS histopathology in a rat SIS model, and compared these findings to a single-TBI model and control animals.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals and Experimental Groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale Wistar rats (mean \u0026plusmn; SD body weight, 194.0 \u0026plusmn; 16.5 g) were obtained from the Experimental Biology Center (NUBEX) at the University of Fortaleza. All protocols were approved by the University of Fortaleza Animal Ethics Committee (protocol #6442130818) and followed Brazilian guidelines for animal research. Rats were randomly allocated to eight groups (n=6 per group; Table 1): (1) \u003cstrong\u003eNa\u0026iuml;ve controls\u003c/strong\u003e (no anesthesia or surgery); (2) \u003cstrong\u003eSham controls\u003c/strong\u003e (anesthesia and scalp incision without impact); (3) \u003cstrong\u003eTBI (1-impact)\u003c/strong\u003e (anesthesia + one weight-drop impact); and (4\u0026ndash;8) \u003cstrong\u003eSIS (2-impacts)\u003c/strong\u003e groups with two identical impacts separated by 1 hour, 24 hours, 3 days, 7 days, or 14 days, respectively. Behavioral and neurological tests were performed 1 day before the first procedure (baseline) and on post-operative days 1, 3, 5, and 7 (denoted PO1\u0026ndash;PO7). The interval between the first and second impacts was set according to group (as above). After the final assessment on PO7, animals were euthanized for blood collection and brain histology. All assessments were conducted by investigators blinded to group assignment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTraumatic Brain Injury Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA modified Marmarou impact acceleration model was used [19]. Rats were anesthetized with ketamine (70 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and placed prone on a foam bed. A midline scalp incision (~20 mm) was made, and a 10-mm diameter, 3-mm thick stainless-steel disc was affixed to the skull midline (sagittal suture) between bregma and lambda using cyanoacrylate glue. Once the adhesive set, a 450-g weight was dropped from a height of 1 m through a Plexiglas tube onto the disc, causing a mild diffuse brain injury (Fig. 1). The mortality rate was 0% across all groups. After impact, the scalp was sutured, and animals were allowed to recover under observation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioral and Neurological Assessments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFacial Mechanical Sensitivity:\u003c/strong\u003e Facial nociception was evaluated using an electronic von Frey apparatus (Insight Equipamentos, Brazil). Rats were tested in their home cages while awake. At baseline (1 day pre-injury) and on PO1, PO3, PO5, and PO7, von Frey filaments of increasing force were applied by a blind evaluator (V.T.d.S.) to three sites on the vibrissal pad. The withdrawal threshold at each site was defined as the minimal force eliciting a head-withdrawal or scratching response in \u0026ge;3 of 5 trials. The final threshold for each animal was the mean of the three sites. Forces above 15 g were not applied to avoid triggering head extension responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeurological Severity Scale (NSS) and Neurobehavioral Scale (NBS):\u003c/strong\u003e At the same timepoints (baseline, PO1, PO3, PO5, PO7), overall neurological and behavioral function was scored. The NSS [20] is a composite scale (0\u0026ndash;28; 0 = normal, \u0026ge;20 = severe deficit) assessing motor/sensory reflexes and coordination. The NBS [20] evaluates general behavior across five domains (including exploration, proprioception, and response to novel objects) with a total score of 0\u0026ndash;20 (20 = normal). Higher NSS scores indicate worse deficits, while higher NBS scores indicate better function. A single blinded evaluator (D.A.) performed all assessments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were collected from anesthetized rats on PO1 and PO7 (via cardiac puncture) immediately before euthanasia. (Na\u0026iuml;ve controls were bled only on PO7.) Plasma was isolated by centrifugation. Levels of IL-6 and TNF-\u0026alpha; in plasma were measured using DuoSet ELISA kits (R\u0026amp;D Systems, Minneapolis, MN, USA) following the manufacturer\u0026rsquo;s instructions [21]. Assays were read by two independent blinded investigators (R.C.L.J., L.M.N.), and cytokine concentrations were calculated from standard curves. Results are expressed as pg per 100 \u0026micro;L of plasma [22].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmediately after blood collection, animals were perfused with 10% buffered formalin. Brains were extracted, post-fixed in formalin, and coronally sectioned (5 \u0026micro;m) using a microtome (Leica, Germany). Sections were stained with hematoxylin-eosin and analyzed under light microscopy using CaseViewer software (3DHistech, Hungary). Two blinded observers (J.M., D.O.) performed semi-quantitative scoring of pathology in various CNS regions. We scored congestion, vasogenic edema, cytotoxic edema, neuronal necrosis (in basal ganglia, cerebellum, and cortex), and inflammatory cell presence. Each parameter was rated on a scale from 0 (none) to 4 (severe) as previously described [23].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were tested for normality (Shapiro\u0026ndash;Wilk); ~31% of measures were non-normal. Therefore, nonparametric statistics were used. Two-group comparisons (e.g. impact vs control at a given time) were performed with two-tailed Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e tests [24,25]. For outcomes involving multiple groups or repeated measures (e.g. NSS, NBS, histology), we used Kruskal\u0026ndash;Wallis tests. Histological scores from the two raters were averaged. Each impact group (with its interval) was compared to the na\u0026iuml;ve and sham control groups at each timepoint (Pre-op, PO1, PO3, PO5, PO7) using Mann\u0026ndash;Whitney tests. Significance was set at p \u0026lt; 0.05. Data are reported as mean \u0026plusmn; standard error.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eFacial Mechanical Sensitivity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental groups receiving either single (TBI) or double (SIS) impacts demonstrated significantly reduced mechanical response thresholds to facial stimulation compared to na\u0026iuml;ve controls (Table 2, Figure 2A). However, post-hoc analysis revealed specific temporal patterns in these hypersensitivity responses.\u003c/p\u003e\n\u003cp\u003eNotably, the SIS group at 14 days post-injury (14D) exhibited statistically significant mechanical hypersensitivity at both PO3 (p = 0.0461) and PO5 (p = 0.0223) timepoints relative to na\u0026iuml;ve controls. Similarly, the 24-hour post-impact SIS group (24H) showed significant threshold reduction at PO5 (p = 0.0316).\u003c/p\u003e\n\u003cp\u003eWhen compared to sham controls, we observed robust mechanical hypersensitivity at PO1 in multiple experimental groups: single-impact TBI (p = 0.005), as well as SIS groups at 1 hour (p = 0.0084), 24 hours (p = 0.0304), 3 days (p = 0.0449), and 7 days (p = 0.0304) post-injury. This hypersensitivity pattern persisted at PO3 in SIS groups at 1 hour (p = 0.0219), 3 days (p = 0.0318), and 14 days (p = 0.0097). The PO5 timepoint showed significant threshold reductions in single-impact TBI (p = 0.0325) and SIS groups at 24 hours (p = 0.0219) and 14 days (p = 0.0065). Importantly, no significant differences were observed between experimental and sham groups at the PO7 timepoint.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeurological and Behavioral Assessments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the PO1 time point, Neurological Severity Scale (NSS) scores were significantly elevated, and Neurobehavioral Scale (NBS) scores were reduced across all impact groups compared to na\u0026iuml;ve controls (Figure 2B and Table 3). NSS increases reached statistical significance in the single-impact group (p = 0.0048) and all double-impact cohorts\u0026mdash;1-hour (1H; p = 0.0013), 24-hour (24H; p = 0.0014), 3-day (3D; p = 0.0013), 7-day (7D; p = 0.0014), and 14-day (14D; p = 0.0014) intervals. Similarly, NBS reductions were observed in the single-impact group (p = 0.0048) and all double-impact groups (1H: p = 0.0045; 24H: p= 0.0013; 3D: p= 0.0014; 7D: p = 0.0014; 14D: p = 0.0013).\u003c/p\u003e\n\u003cp\u003eRelative to sham controls, NSS scores were elevated in double-impact groups with 1H (p = 0.0293), 7D (p = 0.0423), and 14D (p = 0.0500) intervals at PO1. NBS scores were also reduced in double-impact groups with 3D (p = 0.0300) and 14D (p = 0.0421) intervals compared to sham controls.\u003c/p\u003e\n\u003cp\u003eNo statistically significant differences in NSS or NBS scores were observed between impact groups and controls (na\u0026iuml;ve or sham) at subsequent time points (PO3, PO5, PO7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInflammatory Mediators and Histopathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasma IL-6 and TNF-\u0026alpha; levels (Fig. 2C and Table 4) showed no differences between any impact group and na\u0026iuml;ve controls at either timepoint. However, comparisons to sham controls revealed some changes: the 2-impacts (14d) group had lower TNF-\u0026alpha; (p = 0.0221), and the 2-impacts (24h), (3d), and (7d) groups had higher IL-6 (p = 0.0463, 0.0065, and 0.0318, respectively).\u003c/p\u003e\n\u003cp\u003eHistologically, injured brains showed increased inflammation and damage. Compared to na\u0026iuml;ve controls, the 1-impact group (p = 0.0314) and all SIS groups (24h\u0026ndash;14d) had significantly higher inflammation scores (Fig. 2D, Fig. 3, Table 5). Specifically, inflammation was elevated in 2-impacts (24h) (p = 0.00228), 2-impacts (3d) (p = 0.00228), 2-impacts (7d) (p = 0.00118), and 2-impacts (14d) (p = 0.00231) relative to na\u0026iuml;ve (the 3d and 14d p-values were corrected from earlier errors). Relative to sham, significant inflammation increases were seen in 2-impacts (3d) (p = 0.0117), (7d) (p = 0.00320), and (14d) (p = 0.00797). No significant edema or necrosis differences were detected in other regions. Overall, these findings indicate that SIS produced a robust CNS inflammatory response, especially with longer inter-injury intervals.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study reveals that rats subjected to repeated mild TBIs (SIS model) exhibit pronounced facial hypersensitivity, neurological deficits, and brain inflammation, compared to single-TBI or uninjured controls. Facial mechanical hypersensitivity developed rapidly after injury. All impact groups showed reduced withdrawal thresholds, reflecting facial allodynia, but the effect was strongest when two impacts occurred within short intervals. For example, the 14-day SIS group had significantly lower thresholds on day 3\u0026ndash;5 post-injury, and even the 24-hour SIS group was sensitized by day 5. These results align with prior rodent studies where TBI induced migraine-like hypersensitivity and trigeminal sensitization (M\u0026oslash;rch et al. 2007; Hu 1990; Bree and Levy 2018; Tanaka and Zhang 2022; Wattiez et al. 2021). Clinical reports also note that second injuries can precipitate severe headache early on (Minen et al. 2016; Kontos et al. 2013; Theeler et al. 2013). The mechanisms likely involve peripheral and central sensitization of trigeminal pathways and inflammation of meningeal structures (Mustafa et al. 2017; M\u0026oslash;rch et al. 2007; Hu 1990; Bree and Levy 2018; Tanaka and Zhang 2022). For instance, Mustafa et al. (2017) showed that neurotransmitter changes (e.g., serotonin) and enhanced trigeminal excitability underlie post-traumatic headache in rodents. Our data extend this by showing that an additional impact can worsen and prolong facial allodynia. Notably, even a 1-hour interval between impacts produced acute hypersensitivity (day 1), suggesting that very short intervals fall within the SIS window (McLendon et al. 2016). Longer intervals (several days) also caused persistent allodynia, indicating that the brain remains vulnerable for at least 2 weeks after an injury.\u003c/p\u003e\u003cp\u003eNeurological and behavioral deficits were also observed acutely. Immediately after injury (PO1), all impact groups scored worse than controls on the NSS and NBS. This reflects the immediate sensorimotor and cognitive disturbances that follow concussive brain trauma. Some SIS groups showed borderline significance versus sham at day 1 (e.g., higher NSS in 1h, 7d, 14d SIS; lower NBS in 3d, 14d SIS), implying that multiple injuries can slightly exacerbate acute deficits. These findings are consistent with clinical observations: a second concussion before recovery can trigger rapid decline, brain swelling, and serious disability (McLendon et al. 2016; Zeidan and Johnson 2011). The present deficits mirror reports that repetitive injuries prolong recovery and worsen outcomes (Sagher 2013; McKee and Daneshvar 2015; Kamins and Giza 2016; Rodney et al. 2018). However, by days 3\u0026ndash;7 post-injury, performance largely recovered to control levels, consistent with the transient nature of many acute concussion symptoms. Longer-term impacts (e.g., chronic traumatic encephalopathy) were beyond this short 7-day study.\u003c/p\u003e\u003cp\u003eInflammatory markers showed modest changes. IL-6 and TNF-α are known to have complex roles after TBI (Woodcock and Morganti-Kossmann 2013; Rodney et al. 2018; Ziebell and Morganti-Kossmann 2010; Amanollahi et al. 2023). In our SIS model, groups with two impacts had higher IL-6 levels (24h, 3d, 7d) compared to sham, supporting the idea that repeated injury augments the IL-6 response (Engelhardt et al. 2020; Patterson and Holahan 2012; Parkin et al. 2019). IL-6 can be neuroprotective or neurotoxic depending on context (Ooi et al. 2022). Conversely, TNF-α was decreased in the 14d SIS group relative to sham, which might reflect a shift toward resolving inflammation or a neuroprotective phase (Ferreira et al. 2014; Galgano et al. 2017). These dynamic changes suggest that timing and context determine cytokine effects. Similar variability has been reported in other TBI studies (Ling et al. 2004; Choi et al. 1994; Finamor et al. 2023), highlighting the need for detailed time-course analyses.\u003c/p\u003e\u003cp\u003eHistopathological changes were pronounced in SIS rats. We observed neuronal necrosis and edema in multiple CNS areas, along with increased inflammatory cells and vascular congestion, especially in SIS groups with longer intervals. TBI is known to cause cortical and subcortical cell death and inflammation (Meymandi et al. 2018; McKee and Daneshvar 2015). Our data suggest that repetitive injury exacerbates these pathologies: rats receiving two impacts beyond 24 hours apart showed more widespread edema and damage than those with a single impact. This is consistent with the concept that SIS leads to compounded neuropathology. Pro-inflammatory cytokines likely attracted immune cells and worsened edema (Ziebell and Morganti-Kossmann 2010). Thus, repeated concussions produce additive brain injury in this model, in line with other reports of cumulative TBI damage.\u003c/p\u003e\u003cp\u003eStudy Limitations: The findings of our study should be interpreted with several caveats. Behavioral scoring is inherently subjective, though we minimized bias with validated scales and blinded observers. Our histological analysis was semi-quantitative, lacking cell-specific markers (e.g., for microglia or apoptotic neurons), which could refine interpretation. Only female rats were studied, so sex differences in SIS responses remain unknown. We also limited observations to 7 days post-injury; longer-term follow-up is needed to assess persistent hypersensitivity or neurodegeneration. Future work should include more detailed immunohistochemistry, both sexes, and extended timepoints to capture chronic effects of SIS.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study provides a comprehensive comparison of single versus repeated mild TBI in rats. We show that a second impact (SIS) results in more severe facial hypersensitivity, acute neurological deficits, and CNS inflammation than a single injury, particularly when the second impact occurs more than one day later. These results improve our understanding of SIS pathophysiology and underscore the importance of strict return-to-play guidelines after a concussion. By delineating these changes, our work lays the groundwork for future studies on the mechanisms and treatment of SIS. The findings support clinical observations that SIS can produce more severe and longer-lasting deficits than an isolated concussion, and they may inform development of protective and therapeutic strategies for repetitive brain injury.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest:\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis research received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLdMF: Conceptualization, Methodology, Investigation, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Supervision.ACA, VTdS, DA: Conceptualization, Methodology, Writing \u0026ndash; original draft.LP: Formal analysis.RCLJ, LMN: Investigation, Formal analysis.JM, DO: Investigation.GM, ESG, HW, DB, NS: Writing \u0026ndash; review \u0026amp; editing.BS, GH: Conceptualization, Supervision, Writing \u0026ndash; review \u0026amp; editing.AR: Conceptualization, Methodology, Investigation, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research received the Natus Resident/Fellow Award for Traumatic Brain Injury at the CNS 2024 Annual Meeting. 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Neurotherapeutics 7(1):22\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.nurt.2009.10.016\u003c/span\u003e\u003cspan address=\"10.1016/j.nurt.2009.10.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Traumatic brain injury, Second-impact syndrome, Facial pain, Neurobehavioral, Neuroinflammation, Rat model","lastPublishedDoi":"10.21203/rs.3.rs-7273712/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7273712/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTraumatic brain injury (TBI) can lead to chronic complications, including headache and facial hypersensitivity. Second-impact syndrome (SIS), occurring when a second TBI precedes recovery from an initial injury, often causes severe long-term symptoms. Neuroinflammation is implicated, but the comprehensive pathophysiology of SIS remains unclear. This study characterized neurological/behavioral outcomes, facial nociception, plasma inflammatory markers, and central nervous system (CNS) histopathology in rat models of SIS and single mild TBI. Female Wistar rats (n\u0026thinsp;=\u0026thinsp;6/group) were assigned to na\u0026iuml;ve control, sham surgery, single impact (TBI), or double impact (SIS; intervals: 1h, 24h, 3d, 7d, 14d). Mild TBI was induced using a modified Marmarou weight-drop model. Assessments (facial mechanical sensitivity via von Frey, Neurological Severity Scale (NSS), Neurobehavioral Scale (NBS)) were performed pre-injury and on days 1,3,5,7 post-injury. Plasma IL-6 and TNF-α were measured by ELISA. Histopathology (H\u0026amp;E) assessed CNS inflammation. All impact groups developed transient facial mechanical allodynia and acute neurobehavioral deficits (impaired NSS/NBS at day 1). SIS groups, particularly with longer intervals (\u0026ge;\u0026thinsp;24h), exhibited more prolonged facial allodynia and significant neurological deficits compared to single TBI at specific timepoints. Plasma cytokines showed minimal changes. Histopathology revealed significantly elevated CNS inflammation in both TBI and SIS groups versus controls, with SIS groups (intervals 24h-14d) showing robust responses. Repeated TBI, especially with longer recovery intervals between injuries, results in more severe and prolonged facial allodynia, neurological deficits, and CNS neuroinflammation than a single mild TBI. These findings illuminate SIS pathophysiology and underscore the critical need for protection post-concussion.\u003c/p\u003e","manuscriptTitle":"A Rat Model of Second Impact Syndrome in Diffuse Traumatic Brain Injury: Evidence of Facial Hypersensitivity, Neurological and Other Behavioral Alterations, and Immunological and Histopathological Tissue Changes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 11:11:12","doi":"10.21203/rs.3.rs-7273712/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1cdbb74-4c01-405f-8a78-47c323b1b042","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-28T06:24:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-21 11:11:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7273712","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7273712","identity":"rs-7273712","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}