Spinal Cord Injury Causes Prominent Tau Pathology Associated with Brain Post-Injury Sequela

Spinal Cord Injury Causes Prominent Tau Pathology Associated with Brain Post-Injury Sequela | 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 Spinal Cord Injury Causes Prominent Tau Pathology Associated with Brain Post-Injury Sequela Elnaz Nakhjiri, Shaqayeq Roqanian, Hamid Soltani Zangbar, Manuchehr Seyedi Vafaee, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-932182/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Spinal cord injury (SCI) can lead to neurological impairment with significant functional and cognitive deficits. It is obvious that SCI causes focal neurodegeneration that gradually expands to the other cord areas. On the other hand, it is clear that traumatic brain injuries result in tau protein pathology and profound neurodegeneration. Tau is a microtubule-associated protein, which is highly expressed in neurons, and its abnormalities result in neuronal cell death. Moreover, it is clear that tau pathology spreads in various brain areas upon trauma. Therefore, we herein examined tau pathology in the spinal cord as well as brain samples at various time-points in severe SCI mouse models. We examined the effects of severe SCI on locomotor function, spatial memory, and anxiety/risk-taking behavior. We found a gradual increased tau pathology in the spinal cord as well as brain areas; confirmed by immunostaining and immunoblotting. Moreover, we studied the brain samples with electron microscopy and observed disrupted mitochondria and microtubule structure upon SCI. SCI caused motor dysfunction, memory impairment, and abnormal risk-taking behavior. Importantly, pathogenic cis P-tau elimination with systemic administration of respective monoclonal antibody restored SCI-related pathological and functional consequences. Thus, our finding suggests that SCI results in profound tauopathy, which spreads to brain areas, reflecting brain dysfunction. Moreover, tau immunotherapy with anti- cis P-tau antibody could suppress the pathogenic outcomes in the SCI mouse models, which would have profound clinical implications in the SCI patients. Neurobiology of Disease Spinal cord injury Tau pathology Spreading Brain destruction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The global prevalence of spinal cord injury (SCI) is approximately 500000 people each year due to motor vehicle crashes, falls, violence, and sporting accidents [ 1 , 2 ], and can result in substantial neurological impairment along with significant emotional and psychological distress [ 3 ]. SCI can interrupt nerve impulses conduction, resulting in neurological dysfunction [ 4 ]. Primary injury affecting the spinal cord has been shown to promptly disrupt cell membranes, myelin and axons destruction within the longitudinal tracts. Moreover, the SCI damages microvessels, resulting in destructive secondary injury by releasing different harmful factors [ 5 ]. Different cellular and molecular mechanisms in the secondary injury process may lead to comprehensive neurodegeneration [ 6 ]. Such cascades as active biological processes offer a chance of treating SCI by selective inhibitors. SCI is also able to change systemic immune functions, whereby affecting the brain [ 7 ]. Moreover, the released agents may reach to the brain via cerebrospinal fluid (CSF). The brain abnormalities, caused by SCI, are due to afferent and efferent routes modifications. However, there are remarkable neuropathological hallmarks triggered by SCI, such as decreased number of cortical neurons [ 4 , 8 – 12 ]. Moreover, there is cognitive impairment in 60% of the SCI population [ 4 , 10 – 12 ]. Despite extensive considerations, it remains uncertain how SCI results in the brain abnormalities. It is clear that tau protein abnormality is a major pathological hallmark upon traumatic brain injury (TBI) [ 13 ]. Tau is a microtubule-associated protein that promotes microtubules (MTs) formation and stabilization [ 14 , 15 ]. Tau is commonly subjected to hyperphosphorylation in tauopathies on Ser/Thr residues, which in turn impairs the function of the MT as well as changes in protein integrity, resulting in its aggregation and tangle formation [ 16 , 17 ]; particularly in chronic traumatic encephalopathy (CTE) [ 18 – 23 ], and Alzheimer’s disease (AD) [ 16 , 17 ]. It is clear that phosphorylated tau at Thr231 exists in the two distinct cis and trans conformation in which that cis pThr231-tau ( cis P-tau) conformer is extremely neurotoxic and early driver of tauopathy process upon TBI. Peptidyl-prolyl cis / trans isomerase (Pin1) suppresses the tau pathology development as well as neurodegeneration in AD by changing the phosphorylated tau at Thr231-Pro motif from neurotoxic cis to the physiological trans conformation [ 24 – 32 ]. Moreover, tauopathy process could be blocked by cis P-tau monoclonal antibody ( cis mAb); both in vitro and in vivo [ 33 , 34 ]. Also, pathogenic cis P-tau shows a prion nature and spreads in brain areas as well as CSF in tauopathy mouse models [ 33 – 37 ]. A few studies have investigated total tau and P-tau concentrations in CSF, serum, and spinal cord tissue in patients and experimental animals with SCI. However, it remains to be fully understood the molecular mechanism of tau pathology in SCI. Also, the causative link between SCI and brain dysfunction remains elusive thus far. Thus, we herein examined tau pathology process in severe SCI (sSCI) mouse models; as proposed previously [ 38 ]. We induced sSCI and investigated various pathogenic tau species formation, and neurodegeneration in both cord and brain tissues at different time points to examine whether SCI injury can lead to the brain pathology. Materials And Methods Animals and study design. Male Balb/c mice (2–3 months old) weighting 22–26 g, were obtained and housed in clear plastic cages, under controlled temperature and humidity in a 12 h light/dark cycle with free access to water and food. All animals were allowed to acclimate to their new surroundings for 1 week before they undergo any experimental procedures. All protocols were approved by the ethics committee of Tabriz University of Medical Sciences (approval No. IR.TBZMED.VCR.REC.1398.067). All experiments were performed according to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH Publications). -54 adult male mice were randomly divided into six groups ( n =-9): Sham group (laminectomy surgery without compression injury), sSCI (48h), sSCI (2W), and sSCI (1M) groups (severe compression injury at 8th thoracic segment ‘T8’ of the spinal cord and were sacrificed 48 h, 2 weeks, and 1 months after the SCI), sSCI (2M) + IgG group (severe compression injury at T8 and received IgG after the SCI for 2 months), and sSCI (2M) + cis mAb group (severe compression injury at T8 and received cis mAb after the SCI for 2 months). Laminectomy and calibrated forceps model of spinal cord compression. All procedures were performed under sterile conditions. Mice were anesthetized with 4% isoflurane, and a laminectomy was done at T7-9 to expose the T8 segment of the spinal cord, without any damage to the dura. Pairs of forceps were applied for laterally compressing the spinal cord to the corresponding thickness (0.25 mm) for 15 sec [ 39 , 40 ]. Sham group received laminectomy and forceps placement around the spinal cord, without a compression. Muscles and skin were stitched after forceps removal followed by administration of saline solution for rehydration (1 ml), buprenorphine (0.05 mg/kg) to alleviate pain and ciprofloxacin (5 mg/kg) to treat/prevent bladder infection, all subcutaneously (SC) two timed a day for 3 days. Animals were monitored in a temperature‐controlled room until recovery and then transferred to their separate cage. Bladders of SCI mice were manually expressed two times a day until the establishing of the urinary reflex. Locomotor analysis. Motor function was assessed in mice to ensure that an effective SCI or a successful laminectomy was done. Hind-limb function in groups was tested by the Basso mouse scale (BMS) one day after injury in the open-field (OF) [ 41 ]. In brief, animals were individually placed in the OF chamber (22.5 × 22.5 cm) and allowed to freely explore for 5 min. Two evaluators independently gave each animal a score of 0 to 9, with a score of 0 indicating a complete defect in locomotor function and a score of 9 indicating no locomotor deficits. To determine the characteristics of cis P-tau, AT8 P-tau, and AT100 P-tau induction upon SCI, mice in the sSCI (48h) and sSCI (2W) groups were anesthetized with intraperitoneal (IP) injection of ketamine (60 mg/kg) and xylazine (10 mg/kg) 48 h and 2 weeks after the SCI, and the spinal cord tissue samples were collected for immunoblotting and immunofluorescence staining analyses. Antibody treatment of mice. To evaluate the efficacy of cis mAb in treating SCI, we examined whether cis mAb could affect intracellular P-tau in sSCI (2M) + cis mAb, and sSCI (2M) + IgG groups. Undergoing sSCI, male mice were randomly treated with mouse cis mAb or mouse IgG. Animals received 1 dose of cis mAb/IgG IP pre-treatment (200 µg/per mouse) 3 days before the injury, single IP post-injury treatment (20 µg in 5 µl) 15 min after SCI, then IP post-treatment (200 µg) every 4 days for 2 weeks, followed by 200 µg weekly for the rest of the two month treatment (33). Transmission electron microscopy (TEM). The ultrastructural assessment was carried out using the TEM method. The brain and spinal cord specimens, from sham and SCI mouse models treated with either control IgG or cis mAb, were cut into pieces of 2 × 2 mm. Briefly, the cells were fixed in glutaraldehyde 2.5%, buffered at 0.1 M phosphate (pH 7.4), osmium tetroxide 1% was used for post-fixation, and finally embedding was performed using resin. Ultrathin sections around 60–90 nanometer were cut and taken on a copper grid and stained with a mixture of uranyl acetate and lead citrate and examined under a ZIESS electron microscope (EM902A), and then viewed by a Leo 906 (Leo, Germany) transmission electron microscope. Immunoblotting analysis. For immunoblotting, spinal cord and the brain samples were homogenized in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP 40, 0.1% SDS, 0.5% Na-deoxycholate, 50 mM NaF) containing proteinase and phosphatase inhibitors and then mixed with the SDS sample buffer and loaded onto a gel after boiling. The proteins were resolved by polyacrylamide gel electrophoresis 12% and transferred to a PVDF membrane. Then the membranes were blocked with 2% milk in TBST (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween 20) for 1 h. Next, the membranes were incubated with primary antibodies overnight at 4ºC. Then, the immunoblots were incubated with HRP-conjugated secondary antibody in 2% milk in TBST. The signals were detected using chemiluminescence reagent (Perkin Elmer, San Jose, CA). The membranes were washed 6 times with TBST after each step. Immunoblotting results were quantified with imageJ. The band of interests were normalized against actin. Immunostaining analysis. Mice were deeply anesthetized and perfused through the left ventricle with 10% neutral buffered formalin. The spinal cord and brain were removed immediately, and post-fixed in 10% neutral buffered formalin overnight. A 1.5 cm segment of spinal cord centered on the injury site and brain were embedded into paraffin, and then cut into 8 µm increments by a microtome. Sections were dewaxed, and then dehydrated in a serial dilution of ethanol. A 5% ammonium chloride solution (Merck, 101145) were used to quench autofluorescence. The sections were placed in a steamer (0.01 M) Sodium citrate (Sigma-Aldrich, S4641) for 20 minutes for antigen enhancement. Then, the slides were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, T8532) for 15 min, and blocked with 10% anti-goat serum for one hour. Next, the slides were incubated with following primary antibodies overnight: cis P-tau mAb (gift from KP. Lu), AT8 P-tau (MN1020), and AT100 P-tau (MN1060). The samples were incubated with the anti-mouse secondary antibodies (Alexa Fluor 488 or 594) at 37°C for one hour. The nucli were stained with Dapi (Invitrogen, D1306), and the images were visualized by a fluorescent microscope (BX71; Olympus equipped with DP72 digital camera). Twenty ROIs (The 350×450 µm 2 dimension representative view) from the cortices of the mouse brains were randomly selected and quantified for each group. The immunofluorescence intensity were quantified with ImageJ ( n = 3/per group). Open-field locomotion. On days 1, 30, and 60 post-injury, hind-limbs functions and spontaneous locomotor activity were evaluated in the OF test. We used the BMS and the related subscale to score locomotor performance. To determination of spontaneous locomotor activity, as mice freely explore the OF chamber for 5 min, a computer-based video-tracking system (Noldus Ethovision) recorded total traveled distance and speed. The OF test is not only used to measure motor activity, but also to measure other factors such as anxiety. Rodents usually spend more time in contact with the walls of the chamber than in the center area (thigmotaxis) [ 42 , 43 ]. On day 60 after injury, the mouse freely explored the maze for 5 min, and the tracking software recorded movement. Mice that spend most of their time in the unprotected central area of the maze indicate anxiolytic-like behaviors [ 42 , 43 ]. Elevated plus-maze (EPM) test. The EPM task is an experiment model to evaluate anxiety/risk-taking behavior; which examines cortical circuits [ 44 – 49 ]. The test is performed on a plus-shaped apparatus which is raised 50 cm above the ground, with two opposite open (aversive, without walls) and two opposite closed (safe, 15 cm high walls) arms (30 × 5 cm), and a central square. A mouse is placed on the central square of the apparatus, facing an open arm, and explore through the maze for 5 min. The number of open arm entries and total time exploring open arms were recorded (Noldus Ethovision). The maze was thoroughly cleaned after each trial to avoid any odorant confounding the test. Mice with normal levels of anxiety/risk-taking behavior enter the open arms less often and spend less time in the open arms. Entering the open arms is being addressed to abnormal risk-taking and anxiety behaviour associated with a cognitive decline [ 33 , 50 ]. Y-maze spontaneous alternation test. The Y-maze test is used for the evaluation of spatial working memory or to quantify cognitive deficits in rodents. The Y-maze consists of three identical black arms (30 cm long, 6 cm wide with 15 cm high walls) mounted in the shape of the letter ‘Y’ at a 120° angle from each other. Rodents usually show a tendency to explore a new arm rather than exploring the arms previously visited. One arm (arms A–C) was randomly selected as the ‘start’ arm, and the mouse is placed at the end of it and allowed to move through each arm. The number of arm entries (when all four paws enter the arm) was recorded for 10 min. Alternation is consecutive entries into each arm without any repeats. The alternation percentage is calculated by using the following formula: (Number of alternations / Number of arm entries) × 100. A mouse with intact working memory scored significantly ˃ 50% [ 51 – 53 ]. At the end of the second month, and after the behavioral tests, mice were anesthetized by ketamine-xylazine IP injection. For electron microscopy, immunoblotting, and immunofluorescence staining analyses, samples were collected from the brain and spinal cord tissues. Statistical analysis. All normally distributed data were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s HSD multiple comparisons post hoc test to compare the groups mean differences. All data were analyzed with SPSS software version 22, and were reported as the means ± standard deviation (SD) with P < 0.05 considered significant. Results Compression severe SCI induced tau pathology in the cord neurons. We used SCI mouse models to study tau pathology triggered by the injury by performing immunoblotting and immunofluorescence staining on the spinal cord tissues. Severe SCI acutely and persistently induced pathogenic cis P-tau 48 h after the injury, and remained at high levels over 2 weeks. Robust cis P-tau signal (1.128 ± 0.03875) and (1.319 ± 0.06179) were detected in the cords at 48 hours and two weeks after injury (Fig. 1 a, P < 0.0001, P = 0.0035). Immunoblotting also showed the progressive increase in AT8 P-tau [ F (2, 4) = 122.8, P = 0.0056], (early tangle), and AT100 P-tau [ F (2, 4) = 255.8, P = 0.0034], (late tangle) in the cord tissues upon trauma in a timely manner (Fig. 1 b-e). It seems that the SCI engendered tau pathology in the cord tissue. Immunotherapy with anti cis mAb improved tau pathology consequences triggered by sSCI. It is clear that cis P-tau is associated with neurodegenerative outcomes and progression of tauopathy triggered by TBI. As mentioned before, we showed that cis P-tau was induced 48 h of sSCI, and thus, there could be a relationship between cis P-tau and pathological changes after injury. To understand the brain complications triggered by SCI, and specially, the effectiveness of cis mAb on neurodegenerative pathologies triggered by sSCI, we treated the animal models with either cis mAb or control IgG isotype for 2 months. We have shown that pathogenic cis P-tau spread to various brain areas after sSCI, resulting in brain tau pathogenicity. However, that cis mAb treatment effectively suppressed tau pathology (Fig. 2 ); consistent with previous findings [ 33 , 34 , 54 – 57 ]. Immunofluorescence staining of sSCI mouse brains showed profound cis P-tau signals one month after injury (11.70 ± 2.078, P = 0.0191) (Fig. 2 a-b). Immunoblotting assay also confirmed the presence of profound amount of cis P-tau (1.296 ± 0.068, P = 0.0228), AT8 P-tau (1.386 ± 0.08493, P = 0.0102), and AT100 P-tau (1.242 ± 0.088, P = 0.0132) in the cortices of sSCI mice while there was no significant amount of the P-tau epitopes in sham animals (0.4958 ± 0.076, 0.247 ± 0.050, 0.334 ± 0.040, Fig. 2 c-f). Interestingly, while immunotherapy with control IgG did not suppress cis P-tau formation in the cortices of the sSCI animals compared to the sham groups (27.94 ± 3.554, P = 0.0234), cis mAb administration effectively stopped tau pathology and cistauosis in the immunostained sSCI mouse’s brains (1.611 ± 0.4818, P = 0.7424, Fig. 2 a-b). Similarly, the efficacy of cis mAb administration to hinder cis P-tau (0.3967 ± 0.1611), AT8 P-tau (0.4870 ± 0.02438), and AT100 P-tau formation (0.5332 ± 0.07576) was shown in the immunoblots of sSCI mouse brains; while IgG treatment did not change the amount of tau epitopes formation (Fig. 2 c-f). It seems that SCI could induce tau pathology in both cord and brain areas and cis mAb immunotherapy of sSCI animals can efficiently impediment the pathogenic P-tau formation in the cortices of mouse brains. SCI caused motor function impairment, improved by cis mAb. Open-field test was performed on days 1, 30, and 60 of the sSCI to examine spontaneous locomotor activity, and hind-limbs functions using the BMS scores [ 41 ]. On every three days of the experiment, mice from both sSCI (1M) and sSCI (2M) + IgG groups displayed significant reduction in total traveled distance ( P < 0.001) during the test, compared with sham mice. The reduction in total traveled distance was paralleled by reduction in walking speed in sSCI (1M) and sSCI (2M) + IgG mice ( P < 0.001) versus sham mice. Importantly, on days 30, and 60 after injury, we observed significant improvement of spontaneous locomotor activity in cis mAb-treated sSCI group ( P < 0.001) compared with sSCI and IgG-treated sSCI groups (Fig. 4 a, b). All mice indicated almost complete loss of motor function one day after injury (BMS score of 1). Hind-limb functional recovery, using the BMS score, significantly improved in cis mAb-treated mice compared with IgG-treated animals ( P < 0.001) by day 60 (Fig. 4 c). Therefore, cis P-tau elimination improved the recovery of spontaneous locomotor activity as well as hind-limb function in sSCI mice. SCI caused cognitive dysfunction, restored by cis mAb. The Y-maze spontaneous alternation test was performed to measure spatial working memory. Sham group displayed functional working memory with ~ 65% spontaneous alteration. Severe SCI significantly reduced percentage of spontaneous alteration ( P < 0.001) compared with the sham group. SCI mice treated with cis mAb showed an increased percentages of spontaneous alteration compared with the IgG-treated sSCI animals ( P = 0.002 or P < 0.01) (Fig. 5 a). To examine the anxiolytic properties of cis mAb, we performed open-field and elevated-plus maze tests 60 days after SCI. The sham animals frequently entered the center portion of the maze while the sSCI mouse treated with IgG spent limited time in the center of the maze in the open-field test. These findings demonstrated of high levels of thigmotaxis (tendency to stay close the walls), indicating an increased anxiety-like behavior. We observed that cis mAb-treated sSCI mouse spent more time in the center area compared to the IgG-treated sSCI animals (Fig. 5 b). In the EPM test, sham animals explored significantly less time in the open arms than IgG-treated sSCI ( P = 0.009 or P < 0.01) mice. Moreover, cis mAb-treated group, similar to the sham group, spent significantly less time in the open arms ( P < 0.001) compared with IgG-treated sSCI groups (Fig. 5 c). SCI (2M) + IgG mice displayed more total open arm entries than the sham and cis mAb-treated sSCI mice (Fig. 5 d). As a result, all IgG-treated sSCI mice, exploring the two open arms, showed anxiety/risk-taking behavior by contrast, cis mAb-treated mice displayed minimal anxious behavior. Our data confirm this viewpoint that, rodents prefer protected areas such as the nest. Thus, cis mAb not only eliminates cis P-tau and cistauosis , but also restores behavioral deficits triggered by SCI. Discussion It is clear that SCI impairs brain function and results in cognitive decline in the patients. However, the molecular mechanisms by which SCI affects the brain has remained elusive thus far. SCI can reorganize the cerebral cortex and thalamus by anterograde and retrograde [ 60 ]. For example, the thalamic nucleus can send cortical efferents to the hippocampal compartment [ 61 ]. Moreover, injured neurons in spinal cord produce cysteine-cysteine chemokine ligand 21, which activates microglias at distant spinal cord sections as well as thalamus [ 62 , 63 ]. SCI also alters systemic immune functions [ 64 ]; affecting the brain. It is possible that neuronal cell injury or death releases intracellular MT binding proteins into the extracellular space, whereby the proteins travel to the brain through the CSF [ 65 , 66 ]. Several studies have shown that tau level increases in the CSF during acute TBI [ 67 ] as well as SCI [ 68 ]. Despite extensive considerations, the underlying molecular mechanisms mediating brain pathology due to the SCI remain elusive [ 69 ]. It has been shown that single severe TBI (ssTBI) or repetitive mild TBI (rmTBI) induce pathogenic cis P-tau in axons few hours following injury, leading to neurodegeneration. Cis P-tau has a prion nature and spreads to various brain areas [ 33 , 35 ]. Notably, cis mAb treatment can eliminate pathogenic cis P-tau and restore axonal pathologies, such as MT defects, organelle transport and long-term potentiation (LTP), and prevent developing many short- and long-term pathological and functional consequences after ssTBI or rmTBI [ 33 – 35 , 59 ]. We herein explored tau pathologies triggered by SCI in order to determine and treat brain complications, including axonal pathology, functional deficits, and cognitive impairment; as proposed previously [ 38 ]. We found that cis P-tau is the initial and important mediator of neurodegeneration and functional defects following SCI in the mouse model. Moreover, we evaluated the cis mAb therapy effects on pathological and functional brain complications following injury. We observed: (1) Severe SCI had a robust and persistent effect on cis P-tau induction, and led to histological changes in the spinal cord and brain tissues; (2) Severe SCI caused locomotor impairment, cognitive deficits and anxiety-like behaviors; (3) Treating SCI mice with cis mAb effectively prevented the development of extensive tauopathy, improved histopathological consequences, restored motor and cognitive function. Importantly, our histological results showed that cis P-tau level at the site of injury was significantly increased, and spread from the spinal cord to the brain within 2 months of the injury. These findings indicate that cis P-tau induction is essential to develop several pathological and functional outcomes following SCI. Conclusion We herein examined the relationship between SCI and tau pathology in the mouse models. We have shown that pathogenic tau induced focally in the spinal cord tissue early after a severe compression SCI, which spread to the brain areas; likely through CSF, and induced prominent tauopathy, resulting in motor dysfunction. Taken these together, we propose cis P-tau as a reliable biomarker to evaluate the SCI pathologic outcomes. Additional studies in pre-clinical SCI models and patients with different degrees of SCI are needed to determine the value of cis P‐tau as a biomarker. The biomarkers may propose novel SCI therapeutic targets as well as treatment approaches. In summary, our results suggest that pathogenic cis P-tau is a tauopathy driver in SCI, which is a potential diagnosis and therapeutic target for immunotherapy. Abbreviations SCI, Spinal cord injury; CSF, Cerebrospinal Fluid; TBI, Traumatic Brain Injury; MTs, Microtubules; CTE, Chronic Traumatic Encephalography; cis P-tau, cis pThr231-tau; AD, Alzheimer’s disease; Pin1, Peptidyl-prolyl cis / trans isomerase; Cis mAb, cis P-tau monoclonal antibody; sSCI, Severe SCI; NIH, National Institute of Health; SC, Subcutaneously; BMS, the Basso Mouse Scale; OF, Open-field; IP, intraperitoneal; TEM, Transmission Electron Microscopy; EPM, Elevated plus-maze ; ROI, Region Of Interest; ANOVA, Analysis of Variance; SD, Standard Deviation; ssTBI, Single Severe TBI; rmTBI, Repetitive Mild TBI; LTP, long-term potentiation. Declarations Support or grant information: The grant of this study was provided by Neurosciences Research Center of Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran; Council for Stem Cell Sciences and Technologies, Tehran, Iran (Grant number: 11/35721); and grant #1397-A-5443 from Royan Institute for Stem Cell Biology & Technology, Tehran, Iran. Funding Information. The grant of this study was provided by Neurosciences Research Center of Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran; Council for Stem Cell Sciences and Technologies, Tehran, Iran (Grant number: 11/35721); and grant #1397-A-5443 from Royan Institute for Stem Cell Biology & Technology, Tehran, Iran. Conflict of Interest. The authors have no conflicts of interest to declare that are relevant to the content of this article. Ethics approval. This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Tabriz University of Medical Sciences (Code of ethics: IR.TBZMED.VCR.REC.1398.067). Consent to participate. Not applicable. Consent for publication. All the authors approve content of the manuscript and agree with Molecular Neurobiology publication policies. Availability of data and material. The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors' contributions. All authors contributed to the study conception and design. Elnaz Nakhjiri expressed the idea of the article and performed the literature search. The first draft of the manuscript was written by Elnaz Nakhjiri, Koorosh Shahpasand and Parviz Shahabi. All authors edited the manuscript and approved the final manuscript. References Ackery A, Tator C, Krassioukov A (2004) A global perspective on spinal cord injury epidemiology. 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Exp Neurol 246:35–43. https://linkinghub.elsevier.com/retrieve/pii/S0014488612000337 Supplementary Files Graphical.png Graphical Abstract Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-932182","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":53930591,"identity":"2b84e501-6e54-47c7-916a-d8965168fa96","order_by":0,"name":"Elnaz Nakhjiri","email":"","orcid":"","institution":"Tabriz Medical University: Tabriz University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elnaz","middleName":"","lastName":"Nakhjiri","suffix":""},{"id":53930592,"identity":"eac5e429-3c2a-4047-a8dc-865daa1d2fb0","order_by":1,"name":"Shaqayeq Roqanian","email":"","orcid":"","institution":"Royan Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shaqayeq","middleName":"","lastName":"Roqanian","suffix":""},{"id":53930593,"identity":"bd99add6-0e34-4a86-880d-2077de271fa8","order_by":2,"name":"Hamid Soltani Zangbar","email":"","orcid":"","institution":"Tabriz Medical University: Tabriz University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hamid","middleName":"Soltani","lastName":"Zangbar","suffix":""},{"id":53930594,"identity":"a3c8f917-1769-4ff9-ab2f-b1a47bdea16d","order_by":3,"name":"Manuchehr Seyedi Vafaee","email":"","orcid":"","institution":"SDU: Syddansk Universitet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Manuchehr","middleName":"Seyedi","lastName":"Vafaee","suffix":""},{"id":53930595,"identity":"ff1676ed-49b7-4be1-a145-a3a906842150","order_by":4,"name":"Daryoush Mohammadnejad","email":"","orcid":"","institution":"Tabriz Medical University: Tabriz University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Daryoush","middleName":"","lastName":"Mohammadnejad","suffix":""},{"id":53930596,"identity":"2b5c6d45-b669-4f06-a00b-ee0189ed9c74","order_by":5,"name":"Shahin Ahmadian","email":"","orcid":"","institution":"Tehran University: University of Tehran","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shahin","middleName":"","lastName":"Ahmadian","suffix":""},{"id":53930597,"identity":"7c443204-8b8d-47fb-8181-dee12bd15dd6","order_by":6,"name":"Selva Zamanzadeh","email":"","orcid":"","institution":"Azad University: Islamic Azad University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Selva","middleName":"","lastName":"Zamanzadeh","suffix":""},{"id":53930598,"identity":"6d31e84b-9ad3-442e-ad00-d0ec8d037e91","order_by":7,"name":"Ehsan Ehsani","email":"","orcid":"","institution":"Azad University: Islamic Azad University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ehsan","middleName":"","lastName":"Ehsani","suffix":""},{"id":53930599,"identity":"70ca1a9f-f11a-4991-b2b0-5d587c637c93","order_by":8,"name":"Parviz Shahabi","email":"","orcid":"","institution":"Tabriz Medical University: Tabriz University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Parviz","middleName":"","lastName":"Shahabi","suffix":""},{"id":53930600,"identity":"dbe30ea5-8ca3-40b0-98a4-6aaa8c2f3ffc","order_by":9,"name":"koorosh Shahpasand","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYDACCQY2KCux8cEHIMXGTryW5MOGM0BamInXkpYmzQOiCWmRn9387HFBTZ28OXuOmbTNr23yfMwMjB8+5uDWYnDnmLnxjGOHDXf2vDG2zu27bdjGzMAsOXMbHi0SCWbSPGwHGDfcyDG8ndtzmxGohY2ZF48W+Rnp36R5/tXZA7UYSFv23LYnqIXhBtALvG3MiRtupCVJM/y4nUhQi8GNnDLpmX2HkzeceXzYsLfhdnIbM2MzXr8AHbZNuuBbne2G48Co/PHntu389uaDHz7icxgDckQwtoHJBvzqUbQw/CGoeBSMglEwCkYgAACetFLIubBGwAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2153-0564","institution":"Royan","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"koorosh","middleName":"","lastName":"Shahpasand","suffix":""}],"badges":[],"createdAt":"2021-09-23 20:46:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-932182/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-932182/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":14071983,"identity":"3e84d8a7-32c2-4d25-86b1-4b2022146a67","added_by":"auto","created_at":"2021-09-28 16:21:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":807576,"visible":true,"origin":"","legend":"Severe SCI had a robust and persistent effect on tau pathogenicity induction in the cord. Mice were subjected to SCI by calibrated forceps. a, The sham and sSCI mouse cords (48h, and 2w after injury), were stained with anti cis P-tau antibody followed by immunofluorescence staining. Cis, green; DNA, blue; Scale bar, 100 µm; n=3. b-e, Immunoblots were stained with cis P-tau, AT8 P-tau, and AT100 P-tau antibodies, followed by quantification analysis . Protein bands were quantified with ImageJ software and were normalized against actin. Data statistically analyzed by one-way ANOVA by Tukey’s post-hoc test (mean ± SD.). *P \u003c 0.05, **P \u003c 0.01. h, hours; w, weeks.","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-932182/v1/e466c659c2d7c3fa5b66818c.png"},{"id":14071988,"identity":"50518f70-b5cf-47b9-ac01-362aa2be78fe","added_by":"auto","created_at":"2021-09-28 16:21:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1376908,"visible":true,"origin":"","legend":"Treating sSCI mice with cis mAb blocked cistauosis and tau pathology in the brain. a,b, Severe SCI mice were treated with cis mAb or control IgG for 2 months. The cortices of sham and sSCI mouse brains were stained with cis P-tau followed by immunofluorescence intensity quantification. Cis P-tau, red; Dapi, blue; Scale bar, 100 µm; n=3. c-f, Immunoblots were stained with cis P-tau, AT8 P-tau, AT100 P-tau, and actin antibodies, followed by quantification analysis. The protein bands were quantified with ImageJ and were normalized against actin, n=3. Data statistically analyzed by one-way ANOVA by Tukey’s post-hoc test (mean ± SD.). *P \u003c 0.05, **P \u003c 0.01. M, month; cis, cis mAb; ns: not significant.","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-932182/v1/3b67822ff3c75272f9a46932.png"},{"id":14071985,"identity":"243a74a5-a775-47c7-a4bc-356c5de13fae","added_by":"auto","created_at":"2021-09-28 16:21:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2316087,"visible":true,"origin":"","legend":"Treating sSCI mice with cis mAb suppressed the spinal cord and the brain ultrastructure destruction. a, b, Electron micrographs of spinal cord and brain sSCI mouse models; either treated or untreated with cis p-tau mAb. Axonal MTs (blue open arrows) and MIs (red filled arrows). Scale bars, 100 nm. M, month; MT, microtubule; MI, mitochondria.","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-932182/v1/90d7af1f7c097970c7c33abf.png"},{"id":14072195,"identity":"3a08253f-8c8f-4628-9a0e-3365ec05be54","added_by":"auto","created_at":"2021-09-28 16:24:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":369551,"visible":true,"origin":"","legend":"Cis P-tau elimination in sSCI mice improved motor function. a-c, sSCI mice were subjected to open-field test, and spontaneous locomotor activity (a, b), as well as hind-limb locomotor functions, using the BMS score (c). Data are expressed as mean ± SD. ***P \u003c 0.001 versus sham, ###P \u003c 0.001 vs sSCI (2M) + cis mAb. M, month; D, day; BMS, Basso mouse scale.","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-932182/v1/d3aeee6935aaa5b924da4bac.png"},{"id":14072460,"identity":"d69e3628-3c10-4932-8030-393e0ef19b55","added_by":"auto","created_at":"2021-09-28 16:27:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":712605,"visible":true,"origin":"","legend":"Cis P-tau Immunotherapy restores cognitive dysfunction in SCI mice. a, After 2 months treatment with IgG or cis mAb, sSCI mice were subjected to the Y-maze spontaneous alteration test to assess spatial working memory. b, sSCI mice were subjected to the OF test to assess anxiety-like behavior, and the path taken by a mouse over 5 min was recorded. c, d, sSCI mice were subjected to the EPM test to assess anxiety/risk-taking behavior, and time spent in the open arms and open arm entries were measured. Data are expressed as mean ± SD. ***P \u003c 0.001 vs sham, **P \u003c 0.01 vs sham, ###P \u003c 0.001 vs sSCI (2M) + cis mAb, ##P \u003c 0.01 vs sSCI (2M) + cis mAb. M, month; OAT, open arm time; OAE, open arm entry.","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-932182/v1/096ea43d80aeeb5520d97afd.png"},{"id":18264521,"identity":"8a197706-f29a-4005-aedf-d6988c086e93","added_by":"auto","created_at":"2022-02-16 04:08:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4085625,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-932182/v1/94316131-8aa1-4450-be2d-ed293b296d48.pdf"},{"id":14071984,"identity":"09a6eecd-4188-4c89-a3c8-a9233a4ec26d","added_by":"auto","created_at":"2021-09-28 16:21:37","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":825684,"visible":true,"origin":"","legend":"Graphical Abstract","description":"","filename":"Graphical.png","url":"https://assets-eu.researchsquare.com/files/rs-932182/v1/c3924cba77df5c4765fa8cc4.png"}],"financialInterests":"","formattedTitle":"Spinal Cord Injury Causes Prominent Tau Pathology Associated with Brain Post-Injury Sequela","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe global prevalence of spinal cord injury (SCI) is approximately 500000 people each year due to motor vehicle crashes, falls, violence, and sporting accidents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and can result in substantial neurological impairment along with significant emotional and psychological distress [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSCI can interrupt nerve impulses conduction, resulting in neurological dysfunction [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Primary injury affecting the spinal cord has been shown to promptly disrupt cell membranes, myelin and axons destruction within the longitudinal tracts. Moreover, the SCI damages microvessels, resulting in destructive secondary injury by releasing different harmful factors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Different cellular and molecular mechanisms in the secondary injury process may lead to comprehensive neurodegeneration [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Such cascades as active biological processes offer a chance of treating SCI by selective inhibitors.\u003c/p\u003e \u003cp\u003eSCI is also able to change systemic immune functions, whereby affecting the brain [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, the released agents may reach to the brain via cerebrospinal fluid (CSF). The brain abnormalities, caused by SCI, are due to afferent and efferent routes modifications. However, there are remarkable neuropathological hallmarks triggered by SCI, such as decreased number of cortical neurons [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, there is cognitive impairment in 60% of the SCI population [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite extensive considerations, it remains uncertain how SCI results in the brain abnormalities. It is clear that tau protein abnormality is a major pathological hallmark upon traumatic brain injury (TBI) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Tau is a microtubule-associated protein that promotes microtubules (MTs) formation and stabilization [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Tau is commonly subjected to hyperphosphorylation in tauopathies on Ser/Thr residues, which in turn impairs the function of the MT as well as changes in protein integrity, resulting in its aggregation and tangle formation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]; particularly in chronic traumatic encephalopathy (CTE) [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and Alzheimer\u0026rsquo;s disease (AD) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is clear that phosphorylated tau at Thr231 exists in the two distinct \u003cem\u003ecis\u003c/em\u003e and \u003cem\u003etrans\u003c/em\u003e conformation in which that \u003cem\u003ecis\u003c/em\u003e pThr231-tau (\u003cem\u003ecis\u003c/em\u003e P-tau) conformer is extremely neurotoxic and early driver of tauopathy process upon TBI. Peptidyl-prolyl \u003cem\u003ecis\u003c/em\u003e/\u003cem\u003etrans\u003c/em\u003e isomerase (Pin1) suppresses the tau pathology development as well as neurodegeneration in AD by changing the phosphorylated tau at Thr231-Pro motif from neurotoxic \u003cem\u003ecis\u003c/em\u003e to the physiological \u003cem\u003etrans\u003c/em\u003e conformation [\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28 CR29 CR30 CR31\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Moreover, tauopathy process could be blocked by \u003cem\u003ecis\u003c/em\u003e P-tau monoclonal antibody (\u003cem\u003ecis\u003c/em\u003e mAb); both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Also, pathogenic \u003cem\u003ecis\u003c/em\u003e P-tau shows a prion nature and spreads in brain areas as well as CSF in tauopathy mouse models [\u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA few studies have investigated total tau and P-tau concentrations in CSF, serum, and spinal cord tissue in patients and experimental animals with SCI. However, it remains to be fully understood the molecular mechanism of tau pathology in SCI. Also, the causative link between SCI and brain dysfunction remains elusive thus far. Thus, we herein examined tau pathology process in severe SCI (sSCI) mouse models; as proposed previously [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We induced sSCI and investigated various pathogenic tau species formation, and neurodegeneration in both cord and brain tissues at different time points to examine whether SCI injury can lead to the brain pathology.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cb\u003eAnimals and study design.\u003c/b\u003e Male Balb/c mice (2\u0026ndash;3 months old) weighting 22\u0026ndash;26 g, were obtained and housed in clear plastic cages, under controlled temperature and humidity in a 12 h light/dark cycle with free access to water and food. All animals were allowed to acclimate to their new surroundings for 1 week before they undergo any experimental procedures. All protocols were approved by the ethics committee of Tabriz University of Medical Sciences (approval No. IR.TBZMED.VCR.REC.1398.067). All experiments were performed according to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH Publications).\u003c/p\u003e \u003cp\u003e-54 adult male mice were randomly divided into six groups (\u003cem\u003en\u003c/em\u003e=-9): Sham group (laminectomy surgery without compression injury), sSCI (48h), sSCI (2W), and sSCI (1M) groups (severe compression injury at 8th thoracic segment \u0026lsquo;T8\u0026rsquo; of the spinal cord and were sacrificed 48 h, 2 weeks, and 1 months after the SCI), sSCI (2M)\u0026thinsp;+\u0026thinsp;IgG group (severe compression injury at T8 and received IgG after the SCI for 2 months), and sSCI (2M)\u0026thinsp;+\u0026thinsp;\u003cem\u003ecis\u003c/em\u003e mAb group (severe compression injury at T8 and received \u003cem\u003ecis\u003c/em\u003e mAb after the SCI for 2 months).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLaminectomy and calibrated forceps model of spinal cord compression.\u003c/b\u003e All procedures were performed under sterile conditions. Mice were anesthetized with 4% isoflurane, and a laminectomy was done at T7-9 to expose the T8 segment of the spinal cord, without any damage to the dura. Pairs of forceps were applied for laterally compressing the spinal cord to the corresponding thickness (0.25 mm) for 15 sec [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Sham group received laminectomy and forceps placement around the spinal cord, without a compression. Muscles and skin were stitched after forceps removal followed by administration of saline solution for rehydration (1 ml), buprenorphine (0.05 mg/kg) to alleviate pain and ciprofloxacin (5 mg/kg) to treat/prevent bladder infection, all subcutaneously (SC) two timed a day for 3 days. Animals were monitored in a temperature‐controlled room until recovery and then transferred to their separate cage. Bladders of SCI mice were manually expressed two times a day until the establishing of the urinary reflex.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLocomotor analysis.\u003c/b\u003e Motor function was assessed in mice to ensure that an effective SCI or a successful laminectomy was done. Hind-limb function in groups was tested by the Basso mouse scale (BMS) one day after injury in the open-field (OF) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In brief, animals were individually placed in the OF chamber (22.5 \u0026times; 22.5 cm) and allowed to freely explore for 5 min. Two evaluators independently gave each animal a score of 0 to 9, with a score of 0 indicating a complete defect in locomotor function and a score of 9 indicating no locomotor deficits.\u003c/p\u003e \u003cp\u003eTo determine the characteristics of \u003cem\u003ecis\u003c/em\u003e P-tau, AT8 P-tau, and AT100 P-tau induction upon SCI, mice in the sSCI (48h) and sSCI (2W) groups were anesthetized with intraperitoneal (IP) injection of ketamine (60 mg/kg) and xylazine (10 mg/kg) 48 h and 2 weeks after the SCI, and the spinal cord tissue samples were collected for immunoblotting and immunofluorescence staining analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibody treatment of mice.\u003c/b\u003e To evaluate the efficacy of \u003cem\u003ecis\u003c/em\u003e mAb in treating SCI, we examined whether \u003cem\u003ecis\u003c/em\u003e mAb could affect intracellular P-tau in sSCI (2M)\u0026thinsp;+\u0026thinsp;\u003cem\u003ecis\u003c/em\u003e mAb, and sSCI (2M)\u0026thinsp;+\u0026thinsp;IgG groups. Undergoing sSCI, male mice were randomly treated with mouse \u003cem\u003ecis\u003c/em\u003e mAb or mouse IgG. Animals received 1 dose of \u003cem\u003ecis\u003c/em\u003e mAb/IgG IP pre-treatment (200 \u0026micro;g/per mouse) 3 days before the injury, single IP post-injury treatment (20 \u0026micro;g in 5 \u0026micro;l) 15 min after SCI, then IP post-treatment (200 \u0026micro;g) every 4 days for 2 weeks, followed by 200 \u0026micro;g weekly for the rest of the two month treatment (33).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransmission electron microscopy (TEM).\u003c/b\u003e The ultrastructural assessment was carried out using the TEM method. The brain and spinal cord specimens, from sham and SCI mouse models treated with either control IgG or \u003cem\u003ecis\u003c/em\u003e mAb, were cut into pieces of 2 \u0026times; 2 mm. Briefly, the cells were fixed in glutaraldehyde 2.5%, buffered at 0.1 M phosphate (pH 7.4), osmium tetroxide 1% was used for post-fixation, and finally embedding was performed using resin. Ultrathin sections around 60\u0026ndash;90 nanometer were cut and taken on a copper grid and stained with a mixture of uranyl acetate and lead citrate and examined under a ZIESS electron microscope (EM902A), and then viewed by a Leo 906 (Leo, Germany) transmission electron microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoblotting analysis.\u003c/b\u003e For immunoblotting, spinal cord and the brain samples were homogenized in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP 40, 0.1% SDS, 0.5% Na-deoxycholate, 50 mM NaF) containing proteinase and phosphatase inhibitors and then mixed with the SDS sample buffer and loaded onto a gel after boiling. The proteins were resolved by polyacrylamide gel electrophoresis 12% and transferred to a PVDF membrane. Then the membranes were blocked with 2% milk in TBST (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween 20) for 1 h. Next, the membranes were incubated with primary antibodies overnight at 4\u0026ordm;C. Then, the immunoblots were incubated with HRP-conjugated secondary antibody in 2% milk in TBST. The signals were detected using chemiluminescence reagent (Perkin Elmer, San Jose, CA). The membranes were washed 6 times with TBST after each step. Immunoblotting results were quantified with imageJ. The band of interests were normalized against actin.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunostaining analysis.\u003c/b\u003e Mice were deeply anesthetized and perfused through the left ventricle with 10% neutral buffered formalin. The spinal cord and brain were removed immediately, and post-fixed in 10% neutral buffered formalin overnight. A 1.5 cm segment of spinal cord centered on the injury site and brain were embedded into paraffin, and then cut into 8 \u0026micro;m increments by a microtome.\u003c/p\u003e \u003cp\u003eSections were dewaxed, and then dehydrated in a serial dilution of ethanol. A 5% ammonium chloride solution (Merck, 101145) were used to quench autofluorescence. The sections were placed in a steamer (0.01 M) Sodium citrate (Sigma-Aldrich, S4641) for 20 minutes for antigen enhancement. Then, the slides were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, T8532) for 15 min, and blocked with 10% anti-goat serum for one hour. Next, the slides were incubated with following primary antibodies overnight: \u003cem\u003ecis\u003c/em\u003e P-tau mAb (gift from KP. Lu), AT8 P-tau (MN1020), and AT100 P-tau (MN1060). The samples were incubated with the anti-mouse secondary antibodies (Alexa Fluor 488 or 594) at 37\u0026deg;C for one hour. The nucli were stained with Dapi (Invitrogen, D1306), and the images were visualized by a fluorescent microscope (BX71; Olympus equipped with DP72 digital camera).\u003c/p\u003e \u003cp\u003eTwenty ROIs (The 350\u0026times;450 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e dimension representative view) from the cortices of the mouse brains were randomly selected and quantified for each group. The immunofluorescence intensity were quantified with ImageJ (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3/per group).\u003c/p\u003e \u003cp\u003e \u003cb\u003eOpen-field locomotion.\u003c/b\u003e On days 1, 30, and 60 post-injury, hind-limbs functions and spontaneous locomotor activity were evaluated in the OF test. We used the BMS and the related subscale to score locomotor performance. To determination of spontaneous locomotor activity, as mice freely explore the OF chamber for 5 min, a computer-based video-tracking system (Noldus Ethovision) recorded total traveled distance and speed. The OF test is not only used to measure motor activity, but also to measure other factors such as anxiety. Rodents usually spend more time in contact with the walls of the chamber than in the center area (thigmotaxis) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. On day 60 after injury, the mouse freely explored the maze for 5 min, and the tracking software recorded movement. Mice that spend most of their time in the unprotected central area of the maze indicate anxiolytic-like behaviors [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eElevated plus-maze (EPM) test.\u003c/b\u003e The EPM task is an experiment model to evaluate anxiety/risk-taking behavior; which examines cortical circuits [\u003cspan additionalcitationids=\"CR45 CR46 CR47 CR48\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The test is performed on a plus-shaped apparatus which is raised 50 cm above the ground, with two opposite open (aversive, without walls) and two opposite closed (safe, 15 cm high walls) arms (30 \u0026times; 5 cm), and a central square. A mouse is placed on the central square of the apparatus, facing an open arm, and explore through the maze for 5 min. The number of open arm entries and total time exploring open arms were recorded (Noldus Ethovision). The maze was thoroughly cleaned after each trial to avoid any odorant confounding the test. Mice with normal levels of anxiety/risk-taking behavior enter the open arms less often and spend less time in the open arms. Entering the open arms is being addressed to abnormal risk-taking and anxiety behaviour associated with a cognitive decline [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eY-maze spontaneous alternation test.\u003c/b\u003e The Y-maze test is used for the evaluation of spatial working memory or to quantify cognitive deficits in rodents. The Y-maze consists of three identical black arms (30 cm long, 6 cm wide with 15 cm high walls) mounted in the shape of the letter \u0026lsquo;Y\u0026rsquo; at a 120\u0026deg; angle from each other. Rodents usually show a tendency to explore a new arm rather than exploring the arms previously visited. One arm (arms A\u0026ndash;C) was randomly selected as the \u0026lsquo;start\u0026rsquo; arm, and the mouse is placed at the end of it and allowed to move through each arm. The number of arm entries (when all four paws enter the arm) was recorded for 10 min. Alternation is consecutive entries into each arm without any repeats. The alternation percentage is calculated by using the following formula: (Number of alternations / Number of arm entries) \u0026times; 100. A mouse with intact working memory scored significantly ˃ 50% [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAt the end of the second month, and after the behavioral tests, mice were anesthetized by ketamine-xylazine IP injection. For electron microscopy, immunoblotting, and immunofluorescence staining analyses, samples were collected from the brain and spinal cord tissues.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e All normally distributed data were evaluated using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s HSD multiple comparisons post hoc test to compare the groups mean differences. All data were analyzed with SPSS software version 22, and were reported as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCompression severe SCI induced tau pathology in the cord neurons.\u003c/b\u003e We used SCI mouse models to study tau pathology triggered by the injury by performing immunoblotting and immunofluorescence staining on the spinal cord tissues. Severe SCI acutely and persistently induced pathogenic \u003cem\u003ecis\u003c/em\u003e P-tau 48 h after the injury, and remained at high levels over 2 weeks. Robust \u003cem\u003ecis\u003c/em\u003e P-tau signal (1.128\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03875) and (1.319\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06179) were detected in the cords at 48 hours and two weeks after injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0035). Immunoblotting also showed the progressive increase in AT8 P-tau [\u003cem\u003eF\u003c/em\u003e (2, 4)\u0026thinsp;=\u0026thinsp;122.8, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0056], (early tangle), and AT100 P-tau [\u003cem\u003eF\u003c/em\u003e (2, 4)\u0026thinsp;=\u0026thinsp;255.8, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0034], (late tangle) in the cord tissues upon trauma in a timely manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-e). It seems that the SCI engendered tau pathology in the cord tissue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunotherapy with anti\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ecis\u003c/span\u003e \u003cb\u003emAb improved tau pathology consequences triggered by sSCI.\u003c/b\u003e It is clear that \u003cem\u003ecis\u003c/em\u003e P-tau is associated with neurodegenerative outcomes and progression of tauopathy triggered by TBI. As mentioned before, we showed that \u003cem\u003ecis\u003c/em\u003e P-tau was induced 48 h of sSCI, and thus, there could be a relationship between \u003cem\u003ecis\u003c/em\u003e P-tau and pathological changes after injury. To understand the brain complications triggered by SCI, and specially, the effectiveness of \u003cem\u003ecis\u003c/em\u003e mAb on neurodegenerative pathologies triggered by sSCI, we treated the animal models with either \u003cem\u003ecis\u003c/em\u003e mAb or control IgG isotype for 2 months. We have shown that pathogenic \u003cem\u003ecis\u003c/em\u003e P-tau spread to various brain areas after sSCI, resulting in brain tau pathogenicity. However, that \u003cem\u003ecis\u003c/em\u003e mAb treatment effectively suppressed tau pathology (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e); consistent with previous findings [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR55 CR56\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence staining of sSCI mouse brains showed profound \u003cem\u003ecis\u003c/em\u003e P-tau signals one month after injury (11.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.078, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0191) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b). Immunoblotting assay also confirmed the presence of profound amount of \u003cem\u003ecis\u003c/em\u003e P-tau (1.296\u0026thinsp;\u0026plusmn;\u0026thinsp;0.068, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0228), AT8 P-tau (1.386\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08493, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0102), and AT100 P-tau (1.242\u0026thinsp;\u0026plusmn;\u0026thinsp;0.088, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0132) in the cortices of sSCI mice while there was no significant amount of the P-tau epitopes in sham animals (0.4958\u0026thinsp;\u0026plusmn;\u0026thinsp;0.076, 0.247\u0026thinsp;\u0026plusmn;\u0026thinsp;0.050, 0.334\u0026thinsp;\u0026plusmn;\u0026thinsp;0.040, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-f). Interestingly, while immunotherapy with control IgG did not suppress \u003cem\u003ecis\u003c/em\u003e P-tau formation in the cortices of the sSCI animals compared to the sham groups (27.94\u0026thinsp;\u0026plusmn;\u0026thinsp;3.554, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0234), \u003cem\u003ecis\u003c/em\u003e mAb administration effectively stopped tau pathology and \u003cem\u003ecistauosis\u003c/em\u003e in the immunostained sSCI mouse\u0026rsquo;s brains (1.611\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4818, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.7424, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b). Similarly, the efficacy of \u003cem\u003ecis\u003c/em\u003e mAb administration to hinder \u003cem\u003ecis\u003c/em\u003e P-tau (0.3967\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1611), AT8 P-tau (0.4870\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02438), and AT100 P-tau formation (0.5332\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07576) was shown in the immunoblots of sSCI mouse brains; while IgG treatment did not change the amount of tau epitopes formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-f).\u003c/p\u003e \u003cp\u003eIt seems that SCI could induce tau pathology in both cord and brain areas and \u003cem\u003ecis\u003c/em\u003e mAb immunotherapy of sSCI animals can efficiently impediment the pathogenic P-tau formation in the cortices of mouse brains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSCI caused motor function impairment, improved by\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ecis\u003c/span\u003e \u003cb\u003emAb.\u003c/b\u003e Open-field test was performed on days 1, 30, and 60 of the sSCI to examine spontaneous locomotor activity, and hind-limbs functions using the BMS scores [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. On every three days of the experiment, mice from both sSCI (1M) and sSCI (2M)\u0026thinsp;+\u0026thinsp;IgG groups displayed significant reduction in total traveled distance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) during the test, compared with sham mice. The reduction in total traveled distance was paralleled by reduction in walking speed in sSCI (1M) and sSCI (2M)\u0026thinsp;+\u0026thinsp;IgG mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) versus sham mice. Importantly, on days 30, and 60 after injury, we observed significant improvement of spontaneous locomotor activity in \u003cem\u003ecis\u003c/em\u003e mAb-treated sSCI group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared with sSCI and IgG-treated sSCI groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003eAll mice indicated almost complete loss of motor function one day after injury (BMS score of 1). Hind-limb functional recovery, using the BMS score, significantly improved in \u003cem\u003ecis\u003c/em\u003e mAb-treated mice compared with IgG-treated animals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) by day 60 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Therefore, \u003cem\u003ecis\u003c/em\u003e P-tau elimination improved the recovery of spontaneous locomotor activity as well as hind-limb function in sSCI mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSCI caused cognitive dysfunction, restored by\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ecis\u003c/span\u003e \u003cb\u003emAb.\u003c/b\u003e The Y-maze spontaneous alternation test was performed to measure spatial working memory. Sham group displayed functional working memory with ~\u0026thinsp;65% spontaneous alteration. Severe SCI significantly reduced percentage of spontaneous alteration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared with the sham group. SCI mice treated with \u003cem\u003ecis\u003c/em\u003e mAb showed an increased percentages of spontaneous alteration compared with the IgG-treated sSCI animals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eTo examine the anxiolytic properties of \u003cem\u003ecis\u003c/em\u003e mAb, we performed open-field and elevated-plus maze tests 60 days after SCI. The sham animals frequently entered the center portion of the maze while the sSCI mouse treated with IgG spent limited time in the center of the maze in the open-field test. These findings demonstrated of high levels of thigmotaxis (tendency to stay close the walls), indicating an increased anxiety-like behavior. We observed that \u003cem\u003ecis\u003c/em\u003e mAb-treated sSCI mouse spent more time in the center area compared to the IgG-treated sSCI animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In the EPM test, sham animals explored significantly less time in the open arms than IgG-treated sSCI (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) mice. Moreover, \u003cem\u003ecis\u003c/em\u003e mAb-treated group, similar to the sham group, spent significantly less time in the open arms (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared with IgG-treated sSCI groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). SCI (2M)\u0026thinsp;+\u0026thinsp;IgG mice displayed more total open arm entries than the sham and \u003cem\u003ecis\u003c/em\u003e mAb-treated sSCI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). As a result, all IgG-treated sSCI mice, exploring the two open arms, showed anxiety/risk-taking behavior by contrast, \u003cem\u003ecis\u003c/em\u003e mAb-treated mice displayed minimal anxious behavior. Our data confirm this viewpoint that, rodents prefer protected areas such as the nest. Thus, \u003cem\u003ecis\u003c/em\u003e mAb not only eliminates \u003cem\u003ecis\u003c/em\u003e P-tau and \u003cem\u003ecistauosis\u003c/em\u003e, but also restores behavioral deficits triggered by SCI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt is clear that SCI impairs brain function and results in cognitive decline in the patients. However, the molecular mechanisms by which SCI affects the brain has remained elusive thus far. SCI can reorganize the cerebral cortex and thalamus by anterograde and retrograde [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. For example, the thalamic nucleus can send cortical efferents to the hippocampal compartment [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Moreover, injured neurons in spinal cord produce cysteine-cysteine chemokine ligand 21, which activates microglias at distant spinal cord sections as well as thalamus [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. SCI also alters systemic immune functions [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]; affecting the brain. It is possible that neuronal cell injury or death releases intracellular MT binding proteins into the extracellular space, whereby the proteins travel to the brain through the CSF [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Several studies have shown that tau level increases in the CSF during acute TBI [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] as well as SCI [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Despite extensive considerations, the underlying molecular mechanisms mediating brain pathology due to the SCI remain elusive [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt has been shown that single severe TBI (ssTBI) or repetitive mild TBI (rmTBI) induce pathogenic \u003cem\u003ecis\u003c/em\u003e P-tau in axons few hours following injury, leading to neurodegeneration. \u003cem\u003eCis\u003c/em\u003e P-tau has a prion nature and spreads to various brain areas [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Notably, \u003cem\u003ecis\u003c/em\u003e mAb treatment can eliminate pathogenic \u003cem\u003ecis\u003c/em\u003e P-tau and restore axonal pathologies, such as MT defects, organelle transport and long-term potentiation (LTP), and prevent developing many short- and long-term pathological and functional consequences after ssTBI or rmTBI [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe herein explored tau pathologies triggered by SCI in order to determine and treat brain complications, including axonal pathology, functional deficits, and cognitive impairment; as proposed previously [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We found that \u003cem\u003ecis\u003c/em\u003e P-tau is the initial and important mediator of neurodegeneration and functional defects following SCI in the mouse model. Moreover, we evaluated the \u003cem\u003ecis\u003c/em\u003e mAb therapy effects on pathological and functional brain complications following injury. We observed: (1) Severe SCI had a robust and persistent effect on \u003cem\u003ecis\u003c/em\u003e P-tau induction, and led to histological changes in the spinal cord and brain tissues; (2) Severe SCI caused locomotor impairment, cognitive deficits and anxiety-like behaviors; (3) Treating SCI mice with \u003cem\u003ecis\u003c/em\u003e mAb effectively prevented the development of extensive tauopathy, improved histopathological consequences, restored motor and cognitive function. Importantly, our histological results showed that \u003cem\u003ecis\u003c/em\u003e P-tau level at the site of injury was significantly increased, and spread from the spinal cord to the brain within 2 months of the injury. These findings indicate that \u003cem\u003ecis\u003c/em\u003e P-tau induction is essential to develop several pathological and functional outcomes following SCI.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe herein examined the relationship between SCI and tau pathology in the mouse models. We have shown that pathogenic tau induced focally in the spinal cord tissue early after a severe compression SCI, which spread to the brain areas; likely through CSF, and induced prominent tauopathy, resulting in motor dysfunction. Taken these together, we propose \u003cem\u003ecis\u003c/em\u003e P-tau as a reliable biomarker to evaluate the SCI pathologic outcomes.\u003c/p\u003e \u003cp\u003eAdditional studies in pre-clinical SCI models and patients with different degrees of SCI are needed to determine the value of \u003cem\u003ecis\u003c/em\u003e P‐tau as a biomarker. The biomarkers may propose novel SCI therapeutic targets as well as treatment approaches. In summary, our results suggest that pathogenic \u003cem\u003ecis\u003c/em\u003e P-tau is a tauopathy driver in SCI, which is a potential diagnosis and therapeutic target for immunotherapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSCI, Spinal cord injury; CSF, Cerebrospinal Fluid; TBI, Traumatic Brain Injury; MTs, Microtubules;\u0026nbsp;CTE, Chronic Traumatic Encephalography;\u0026nbsp;\u003cem\u003ecis\u003c/em\u003e P-tau,\u003cem\u003e\u0026nbsp;cis\u003c/em\u003e pThr231-tau;\u0026nbsp;AD, Alzheimer\u0026rsquo;s disease;\u0026nbsp;Pin1, Peptidyl-prolyl \u003cem\u003ecis\u003c/em\u003e/\u003cem\u003etrans\u003c/em\u003e isomerase;\u0026nbsp;\u003cem\u003eCis\u003c/em\u003e mAb,\u003cem\u003e\u0026nbsp;cis\u003c/em\u003e P-tau monoclonal antibody; sSCI, Severe SCI;\u0026nbsp;NIH, National Institute of Health;\u0026nbsp;SC, Subcutaneously; BMS, the Basso Mouse Scale; OF, Open-field;\u0026nbsp;IP, intraperitoneal; TEM, Transmission Electron Microscopy;\u0026nbsp;EPM, Elevated plus-maze\u003cstrong\u003e;\u0026nbsp;\u003c/strong\u003eROI, Region Of Interest; ANOVA, Analysis of Variance; SD, Standard Deviation; ssTBI, Single Severe TBI; rmTBI, Repetitive Mild TBI; LTP, long-term potentiation.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupport or grant information:\u0026nbsp;\u003c/strong\u003eThe grant of this study was provided by Neurosciences Research Center of Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran; Council for Stem Cell Sciences and Technologies, Tehran, Iran (Grant number: 11/35721); and grant #1397-A-5443 from Royan Institute for Stem Cell Biology \u0026amp; Technology, Tehran, Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Information.\u0026nbsp;\u003c/strong\u003eThe grant of this study was provided by Neurosciences Research Center of Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran; Council for Stem Cell Sciences and Technologies, Tehran, Iran (Grant number: 11/35721); and grant #1397-A-5443 from Royan Institute for Stem Cell Biology \u0026amp; Technology, Tehran, Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest.\u0026nbsp;\u003c/strong\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval.\u003c/strong\u003e This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Tabriz University of Medical Sciences (Code of ethics: IR.TBZMED.VCR.REC.1398.067).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate.\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication.\u003c/strong\u003e All the authors approve content of the manuscript and agree with \u003cem\u003eMolecular Neurobiology\u003c/em\u003e publication policies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material.\u003c/strong\u003e The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions.\u003c/strong\u003e All authors contributed to the study conception and design. Elnaz Nakhjiri expressed the idea of the article and performed the literature search. The first draft of the manuscript was written by Elnaz Nakhjiri, Koorosh Shahpasand and Parviz Shahabi. All authors edited the manuscript and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAckery A, Tator C, Krassioukov A (2004) A global perspective on spinal cord injury epidemiology. J Neurotrauma 21(10):1355\u0026ndash;1370\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan den Berg MEL, Castellote JM, Mahillo-Fernandez I, de Pedro-Cuesta J (2010) Incidence of Spinal Cord Injury Worldwide: A Systematic Review. 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Exp Neurol 246:35\u0026ndash;43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://linkinghub.elsevier.com/retrieve/pii/S0014488612000337\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spinal cord injury, Tau pathology, Spreading, Brain destruction ","lastPublishedDoi":"10.21203/rs.3.rs-932182/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-932182/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpinal cord injury (SCI) can lead to neurological impairment with significant functional and cognitive deficits. It is obvious that SCI causes focal neurodegeneration that gradually expands to the other cord areas. On the other hand, it is clear that traumatic brain injuries result in tau protein pathology and profound neurodegeneration. Tau is a microtubule-associated protein, which is highly expressed in neurons, and its abnormalities result in neuronal cell death. Moreover, it is clear that tau pathology spreads in various brain areas upon trauma. Therefore, we herein examined tau pathology in the spinal cord as well as brain samples at various time-points in severe SCI mouse models. We examined the effects of severe SCI on locomotor function, spatial memory, and anxiety/risk-taking behavior. We found a gradual increased tau pathology in the spinal cord as well as brain areas; confirmed by immunostaining and immunoblotting. Moreover, we studied the brain samples with electron microscopy and observed disrupted mitochondria and microtubule structure upon SCI. SCI caused motor dysfunction, memory impairment, and abnormal risk-taking behavior. Importantly, pathogenic \u003cem\u003ecis\u003c/em\u003e P-tau elimination with systemic administration of respective monoclonal antibody restored SCI-related pathological and functional consequences. Thus, our finding suggests that SCI results in profound tauopathy, which spreads to brain areas, reflecting brain dysfunction. Moreover, tau immunotherapy with anti-\u003cem\u003ecis\u003c/em\u003e P-tau antibody could suppress the pathogenic outcomes in the SCI mouse models, which would have profound clinical implications in the SCI patients.\u003c/p\u003e","manuscriptTitle":"Spinal Cord Injury Causes Prominent Tau Pathology Associated with Brain Post-Injury Sequela","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-09-28 16:21:35","doi":"10.21203/rs.3.rs-932182/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":"8a8aa784-85ad-4596-b905-cac858814b38","owner":[],"postedDate":"September 28th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":7505513,"name":"Neurobiology of Disease"}],"tags":[],"updatedAt":"2022-02-16T04:08:33+00:00","versionOfRecord":[],"versionCreatedAt":"2021-09-28 16:21:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-932182","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-932182","identity":"rs-932182","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}