Cytokine Profile and Glial Activation Following Brachial Plexus Roots Avulsion Injury in Mice

Cytokine Profile and Glial Activation Following Brachial Plexus Roots Avulsion Injury in Mice | 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 Cytokine Profile and Glial Activation Following Brachial Plexus Roots Avulsion Injury in Mice Ke Zhong, Yinqin Li, Ying Tang, Guangying Yu, PRINCE LAST MUDENDA ZILUNDU, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-27602/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Mar, 2021 Read the published version in Journal of Neuroimmunology → Version 1 posted You are reading this latest preprint version Abstract Background Inflammation and tissue infiltration by various immune cells play a significant role in the pathogenesis of neurons suffering the central nervous systems diseases. Although brachial plexus root avulsion (BPRA) leads to dramatic motoneurons (MNs) death and permanent loss of function, however, the crosstalk between cytokines and glial reaction in the spinal cord during injury is far beyond our knowledge. The current study is sought to investigate the alteration of specific cytokine expression patterns of BPRA injured spinal cord during an acute and subacute period. Methods Here, cytokine assay, transmission electron microscopy, and histological staining were utilized to assess the alteration of cytokine network, ultrastructure morphology, as well as glial activation and loss of MNs within two weeks, post-injury on a mouse BPRA model. Results We found that BPRA significantly changed the level of CXCL1, IL12 p70, ICAM1, IP10, MCP-5, MIP1-α and CD93. Moreover, the elevated MIP1-α and CD93 were mostly distributed in MNs of the injured, but few in healthy segments. Also, BPRA significantly induced glial reactions in the ventral horn of injured spinal segments, reflected by robustly elevated the Ionized calcium-binding adaptor molecule 1 (IBA1) and Glial fibrillary acidic protein (GFAP) positive cells. We also observed a severe progressively loss of injured MNs, with a character of both apoptosis and necrosis. Conclusion Overall, these findings suggested that the inflammatory cytokines associated with glial cell proliferation play a vital role in the pathophysiology of MNs death caused by nerve roots injury. Neurobiology of Disease cytokine profile nerve injury motoneuron degeneration mice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Motoneurons (MNs) degenerate progressively and finally die after the brachial plexus root avulsion (BPRA), an injury-causing paralysis of the target muscle groups of the upper limbs [ 1 ][ 2 ]. Neuroinflammation is one of the essential factors for triggering the neuron loss related to acute or chronic neurodegenerative disorders [ 3 ]. It has been well documented that the neuroinflammation also plays a causal role for the MNs degeneration and death associated with spinal cord injury, including avulsion injury [ 4 ][ 5 ][ 6 ][ 7 ]. It has been reported that the neuroinflammation is characteristic of the infiltration of leukocytes [ 8 ] and glial activation during the acute and subacute period after spinal cord injury [ 9 ] [ 10 ][ 11 ]. The inflammation of BPRA is attributed to that lesion created cellular debris, which activates the astrocytes and microglia, trigging the release of a wide variety of oxidative stress regulators, growth factors, and inflammatory mediators including cytokines[ 12 ]. The cytokines are proteins that coordinate the immune response throughout the CNS, recruiting phagocytic cells, like peripheral neutrophils and macrophages into the injured spinal cord [ 13 ][ 14 ], which are considered to be the first wave of infiltrating immune cells [ 15 ]. On one side, cytokines have shown a role in debris clearance, cellular remodeling, and production of pro-regenerative factors[ 16 ][ 17 ] [ 18 ][ 19 ]. For example, Several studies reported the level of anti-inflammatory cytokines, like interleukin 4 (IL-4), interleukin 10 (IL-10) and transforming growth factor-β (TGF-β), substantially enhanced after undergoing post-spinal cord injury activation by injured neurons and astrocyte[ 20 ] [ 21 ][ 22 ]. However, other studies demonstrated that either microglia or astrocytes also produce pro-inflammatory cytokines such as interleukin 1 (IL-1), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) during spinal cord injury [ 23 ][ 24 ][ 25 ], which suggest that cytokines are detrimental to CNS neurons [ 26 ] [ 27 ] and targets for the treatment of spinal cord injury [ 28 ][ 29 ][ 30 ][ 31 ]. The current study is sought to investigate how the alteration of specific cytokines expression patterns affects spinal glial activation and MNs death induced by BPRA. We observed that substantially elevated glial reactions and enhanced levels of cytokines, especially MIP-1α, which might contribute to a progressively MNs loss in the ventral horn of injured spinal segments caused by avulsion injury. Methods Animals Experiments were conducted on 76 adult male C57BL/6 mice, 8–10 weeks old, weighing 20–25 g, which were purchased from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China). The number of the animal use permit is SYXK 2017-0081. The mice were housed under a 12-hour light/dark cycle with food and water were available ad libitum. Surgical and animal care procedures were carried out under the provisions outlined in the National Health and Medical Research Council animal ethics and ARRIVE guidelines. All procedures were performed with the approval of the Animal Care and Utilization Committee of Sun Yat-sen University. Animal Model Setting The microsurgery procedure for the brachial plexus roots avulsion injury was as we described previously [ 32 ][ 33 ][ 34 ][ 35 ].In short, the mice were under anesthesia induced with ketamine/xylazine (80/20 mg/kg, i.p.) and maintained with 1% isoflurane. With each mouse in the supine position, the right side of brachial plexus was exposed, and its C5- T1 spinal nerve roots were separated under a surgical microscope (ChengHe Microsurgical Instruments Factory, Ning Bo, China). Both dorsal and ventral rootlets were pulled out using micro hemostatic forceps. All the avulsed distal parts of roots were cut away, and the success of the avulsion model confirmed under the microscope. The muscle and skin were sutured in successive layers. The mice were placed in a heated recovery chamber until they were fully awake and then returned to their cages. Tissue Preparation To assess the spinal cord cytokines profile of BPRA mice, a serial of time points [1, 2, 3, 5, 7 and 14 day post injury (dpi)], mice were anesthetized with 1% sodium pentobarbital (40 mg/kg, i.p., Sigma-Aldrich) and the cervical spinal cord was exposed by laminectomy under the surgical microscope. Subsequently, the C5 to T1 spinal segments were quickly removed and immediately divided into ipsilateral and contralateral halves. Spinal cord tissues were frozen in liquid nitrogen for subsequent protein extract. The frozen samples were homogenized in Whole-Cell Lysis buffer (KGP2100, Key GEN Biotech. Co., Ltd. Nanjing, China) containing protease inhibitor and 1 mM PMSF (P7626; Sigma-Aldrich, St. Louis, Missouri) using an electric homogenizer. The homogenates were lysed in ice for 1 h and centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatants were collected, and the protein concentrations were measured using the BCA (SL260619, Thermo Scientific; Rockford, Illinois) method and stored at − 80 °C until the time of cytokine measurement. Cytokine Assay Spinal cord samples (n = 6 mice/group for each time point) were evaluated using the 20-plex, Mouse Premixed Multi-Analyte Kit LXSASM-20 (L126405, R&D Systems, Minneapolis, MN), measured with the Luminex system (Luminex, Austin, TX), and analyzed by Bio-plex software (Bio-Rad, Hercules, CA). This panel includes: C-X-C motif chemokine ligand 1(CXCL1), tumor necrosis factor-alpha (TNF-alpha), IL-12 p70, C-C motif chemokine 2 (CCL2), IL-1beta (IL-1b), IL-2, IL-6, IL-10, IL-17A, Intercellular adhesion molecule 1(ICAM-1), C-X-C motif chemokine 10 (IP-10), regulated on activation, normal T expressed and secreted protein (RANTES), C-C motif chemokine 12(CCL12), Beta-nerve growth factor (NGFb), macrophage inflammatory protein (MIP)-1alpha (MIP1-α), interleukin (IL)-alpha (IL-1a), Stromal cell-derived factor 1(SDF-1a,), Complement component C1q receptor(CD93), C-C motif chemokine 22(CCL22). Immunofluorescence, Neutral Red Staining And Cell Counting Twenty BPRA mice were sacrificed using a lethal dose of 1% sodium pentobarbital on the 1, 3, 7, and 14 dpi, respectively (n = 5/group at each time point). They were transcardially perfused with normal saline followed by cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4). The cervical spinal cord was exposed by laminectomy under the surgical microscope. The C7-C8 spinal segments, which were defined as the region between the uppermost and the lowermost rootlets of the contralateral C7-C8 spinal nerves, were post-fixed by immersion in 4% PFA followed by overnight immersion in 30% (v/v) sucrose in PB solution at 4 °C. The transverse sections of the C7-C8 spinal cord (30 µm), were collected in 0.01M PBS. Every third section of the spinal cord was used for staining study. The immunofluorescence (IF) was evaluated under a fluorescence microscope (BX63, Olympus, Japan). The sections were rinsed three times in PBS and then treated with 0.3% Triton X-100 and 0.1% bovine serum albumin (BSA) in PBS at room temperature for 30 min. The following primary antibodies were used: mouse anti-NeuN antibody (1:500 dilution, ab104224, Abcam, UK), rabbit anti-GFAP (1:500 dilution; 80788S, CST, Danvers, Massachusetts ), rabbit anti-IBA1 (1:500 dilution; 019-19741, Wako Bioproducts, Richmond, Virginia), rabbit anti-CD93 (1:200 dilution; ab198854 Abcam, UK), and rabbit anti-MIP1-α (1:100 dilution, ab25128, Abcam, UK). Incubation was at 4 °C overnight. Then, following washing three times with PBS, the sections were incubated with respective secondary antibodies. The following antibodies were used: Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:1500, A32723, Invitrogen, USA), Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (1:1000, A32731, Invitrogen, USA). Incubation was 2 h at room temperature in the dark. After rinsing the sections three times with PBS, then incubated with Hoechst33342 (H3570, Life Technologies, USA). They were visualized under a fluorescence microscope. Staining without primary or secondary antibodies served as negative controls. In addition, the neutral red staining was performed in C7 and C8 segments to assess MNs survival as previously reported [ 36 ][ 37 ]. Briefly, Sections were stained with 1% neutral red (N4638, Sigma-Aldrich, USA) in 0.1M acetic acid (pH4.8) for 2 h followed by dehydration in ethanol. Images were captured using a digital camera attached to the microscope. Next, the quantification of cell counting on the ventral horns of the both sides spinal cord (C7-C8 segments Rexed’s lamina IX) was as described previously [ 38 ]. We computed the mean number of either IF or neural red positive cells from the total 7 ~ 8 sections obtained from each mouse. Only those neurons with both the nucleolus in the nucleus and Nissl bodies in the cytoplasm stained with neutral red we recounted under a 20 × objective lens. Two independent persons blinded to the sidedness of the groups performed cell counting, pooling of means and data analysis. Transmission Electron Microscopy Transmission electron microscopy (TEM) was used to assess motor neuron morphology in the spinal cord after BPRA at 1, 3, 7, 14 dpi (n = 5 mice/group at each time point). The mouse was perfused transcardially with 0.9% normal saline, followed by a mixture of 2% PFA and 2.5% glutaraldehyde (Sigma-Aldrich, G6257, USA) in 0.1 M PBS. Approximately 1 mm 3 of tissue per mice was dissected from the spinal cord and fixed in 2% glutaraldehyde for 2 h at 4 °C. The tissues were rinsed in 0.1 M cacodylate buffer and post-fixed with 1% osmium tetroxide for 2 h. Then, the tissue was rinsed with distilled water before undergoing dehydration in a graded ethanol series. Subsequently, the tissue was infiltrated overnight at 4 °C using a mixture of half acetone and half resin. The tissue was embedded in resin 24 h later and then cured fully as follows: 37 °C overnight, 45 °C for 12 h, and 60 °C for 24 h. After that, 70-nm sections were cut and stained with 3% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastructural changes motor neuron morphology was observed under TEM. Statistical Analysis All data were expressed as mean ± SEM (standard error of the mean). The morphological evaluation result or cytokine assay result was subjected to one-way or two-way measures ANOVA followed by a Bonferroni or Tukey post hoc test for multiple group comparisons. A value of p < 0.05 was considered statistically significant. Results BPRA substantially caused MNs loss in the ventral horn of the injured spinal cord At 1 dpi, the injured MNs started to present morphological features of both apoptosis and necrosis, reflected by slightly swollen mitochondria and condensed cytoplasm with a still visible nucleus and nucleolus. By 3–7 dpi, the cytoplasm of MNs had become hyperchromatic, the nuclear envelope was indistinct and irregular in shape, and the nucleoli and nuclei were more condensed. The cytoplasm also contained fragmented organelles, numerous, small, granular profiles and organelles are significantly reduced in the cytoplasm. Mitochondria were absent, and the cell membrane was disrupted (Fig. 1 ). Moreover, the light microscopic observation also revealed an increase in the size of the cytoplasm of injured MNs. Furthermore, the neural red positive staining cell counting of spinal ventral horn found a significant and progressive decline in the ipsilateral MNs survival rate (F (3,16) = 674.3, p < 0.0001), with only 35.34%± 0.48% ipsilateral MNs survived as compared to contralateral side at 14 dpi (Fig. 2 ). BPRA enhance the glial activation in the ventral horn of the injured spinal cord To evaluate the glial reactions that occurred in the injured spinal cord after BPRA, the expression of IBA-1 and GFAP, markers of activated microglia and astrocytes, were determined using immunoreactivity 1 to 14 dpi in both ipsilateral and contralateral side of the spinal ventral horn. Our results showed both IBA-1 average fluorescence intensity (AFI) and immunoreactivity (IR)-positive cells number of ipsilateral ventral horn gradually increased compared to the contralateral side within 1–14 days followed injury (AFI: F (3,16) = 487.7, p < 0.0001; IR cell number, F (3,16) = 249.9, p < 0.0001, Fig. 3 ). Similarly, we observed a robustly elevated GFAP expression trend in the injured ventral horn after BPRA. Compared to the contralateral side, BPRA caused an increase in GFAP AFI ( F (3,16) = 85.06, p < 0.0001) and IR cell number (F (3༌16) = 52.7, p < 0.0001) in the ipsilateral side, with the peaks at 7 dpi (Fig. 4 ). These data suggested that BPRA produced microglia and glial activation in the injured spinal cord. Discussion The present study showed that BPRA substantially caused MNs loss in the ventral horn of the injured spinal cord. The damaged MNs is morphologically characteristic of swelling and depletion of mitochondria endoplasmic reticulum, and eventual rupture of organelles, and disruption of nuclear and plasma membranes. These changes are associated with glial activation in the affected ventral horn, reflected by enhanced IBA-1 and GFAP immunoreactivity. Moreover, we also found that some essential cytokines level substantially changed during the acute and subacute phase of MNs death, especially like MIP1-α and CD93 expression increased in the inured MNs. These data strongly suggest that cytokines might have crosstalk with glial activation, which contributes to the spinal MNs loss caused by avulsion injury. Our study the changes of cytokine profile following BPRA (acute and subacute periods) in mice compared with contralateral ventral horns in the same injured spinal segments. During the first 2 weeks after BPRA, we detected significant changes in concentrations of 7 cytokines with different inflammatory and immunological activities. We found CXCL1 showed a rapidly enhanced concentration on 1 dpi then decreased to the baseline during 2–14 dpi. CXCL1, being a chemotactic substance for neutrophils, is expressed by macrophages, neutrophils, astrocytes and epithelial cells [ 40 ]. Studies in mice have shown that CXCL1 reduces the severity of multiple sclerosis and can exhibit a neuroprotective function. The concurrence of CXCR2(the receptor for CXCL1) on oligodendrocytes and induced CXCL1 on hypertrophic astrocytes in MS provides a novel mechanism for recruitment of oligodendrocytes to areas of damage, an essential prerequisite for lesion repair in this devastating condition. [ 41 ][ 42 ]. CXCL1 of might be actively involved in the regenerative processes by recruiting oligodendrocytes to the injured spinal cord on the first 1 day after BPRA. On 2 dpi, we observed a reduced concentration of IL-12 p70 and maintained a relatively low level as compared to 1 dpi. Interleukin-12 (IL-12) is a heterodimeric cytokine produced mostly by phagocytic cells in response to bacteria, bacterial products, and intracellular parasites, and to some degree by B lymphocytes. The early preference expressed in the immune response depends on the balance between IL-12, which favors Thl responses, and IL-4, which favors Th2 responses. Thus,IL-12 represents a functional bridge between the early nonspecific innate resistance and the subsequent antigen-specific adaptive immunity [ 43 ]. Pro-inflammatory cytokine IP10 peaked at 1 dpi with a second peak at 14 dpi compared to the contralateral side of the spinal cord, which is secreted by several cell types (e.g., leukocytes, macrophages and endothelial cells) in response to IFN-γ and recruits these cells to sites of infection or inflammation after binding to C-X-C motif receptor (CXCR3) [ 44 ][ 45 ]. IP10 is expressed at low levels in the spinal cord under normal conditions, but upon stimulation, the expression of this chemokine is substantially increased at sites where glial cells accumulate [ 45 ]. Previous study found the neutralization of CXCR3 significantly reduces the expression of markers of activated microglia and astrocytes as well as the levels of proinflammatory cytokines and chemokines, such as CCL2 and CXCL10 [ 45 ][ 46 ][ 47 ]. Changes in the level of pro-inflammatory cytokine MCP5 in the form of a sharp increase in its level on 1 dpi were observed in injured spinal cord and maintained a relatively high level as compared to the contralateral side till 14 dpi. The cytokine MCP5 attracts eosinophils, monocytes, and lymphocytes but not neutrophils. Previous study found sequence analyses identified HIF-1α binding sites in the promoters of MCP-5 genes and HIF-1α is involved in transcriptional induction of MCP5 in astrocytes by hypoxia [ 48 ]. We found the pro-inflammatory chemokine MIP-1 α during the first 2 weeks after BPRA with a maximum increase of 13.94 times on 1 dpi, being a macrophage inflammation protein, activates granulocytes, which leads to acute neuroinflammation [ 49 ]. Immunofluorescence staining found MIP-1 α mostly located in the injured motor neuron after BPRA. Its increase in the spinal cord tissues may be associated with disruption of the blood–brain barrier and an increased migration of macrophages and neutrophils to the site of injury. Previous studies have shown the importance of MIP-1α in demyelination in the CNS and highlight its effect by exposing MIP-1α knockout mice (MIP-1α-/-) to cuprizone and comparing pathology to wild-type mice, which found demyelination was significantly decreased in MIP-1α-/- mice (near 36% reduction) [ 50 ]. We speculate that the high expression of MIP-1α may be related to attract macrophages infiltration and demyelination of motor neurons after brachial plexus avulsion in mice. On 1 dpi, we observed a rapidly enhanced concentration of CXCL1 in the injured spinal cord. Studies in mice have shown that CXCL1 reduces the severity of multiple sclerosis and can exhibit a neuroprotective function. The concurrence of CXCR2(the receptor for CXCL1) on oligodendrocytes and induced CXCL1 on hypertrophic astrocytes in MS provides a novel mechanism for recruitment of oligodendrocytes to areas of damage, an essential prerequisite for lesion repair in this devastating condition. [ 41 ][ 42 ]. CXCL1 of might be actively involved in the regenerative processes in the CNS by recruiting oligodendrocytes to the injured spinal cord on the first 1 day after BPRA. CD93 is a type I transmembrane glycoprotein that is expressed on the surface of platelets, and myeloid, endothelial, hematopoietic stem and natural killer cells. CD93 is strongly associated with immune cell phagocytosis, which regulates innate immunity and inflammation [ 51 ]. CD93 deletion also induces phagocyte impairment on scavenging apoptotic cells[ 52 ]. Our study found CD93 IR was low in the contralateral side of spinal cord, but increased during inflammatory reaction in the ipsilateral side of spinal cord, reaching a peak at early stage of BPRA and showing distinct cytoplasmic and nuclear staining. These findings suggest that the CD93 cytoplasmic tail is mainly expressed in cell membranes yet may migrate towards the cytoplasm or nucleus with increasing inflammatory reaction. The levels of the ICAM1 gradually increased with a peak reached on 14 day. As a cell-surface glycoprotein typically expressed by both immune system cells and endothelial cells. ICAM1 is also expressed by astrocytes, though at low levels in resting, quiescent astrocytes, remarkably it is induced 12-fold following injury and subsequent astrocyte activation [ 53 ]. ICAM1 may facilitate astrocyte-lymphocyte interactions ultimately aiding recruitment of immune cells into the CNS [ 54 ]. Conclusions In our study, we evaluated the cytokine profile in spinal cord of mice with a model of BPRA in acute and subacute periods after injury. Our study also demonstrated that the MNs degeneration process, inflammatory reaction and immunological response after BPRA. A further study of a complex cytokine network imbalance after BPRA and determining its possible correlation with the severity of damage and clinical events seems important. This would provide a better understanding of the role cytokines play in the pathophysiology of the BPRA disease. Abbreviations BPRA, brachial plexus root avulsion; MNs, Motoneurons; CNS, central nervous system; TEM, transmission electron microscopy; dpi, day post injury Declarations Acknowledgements The authors acknowledge the general support provided by Yaqiong Wang for her professional assistance in the tissue process and electron microscope observation. Authors’ contributions ZK designed this study, performed the experiments and drafted the manuscript. LYQ and TY carried out extra data analysis. YGY and PRINCE participated in drafting and revised the manuscript. ZYY and XXY revised the manuscript, FR and ZLH supervised the design of the study and conceived the manuscript. All authors reviewed and approved the final version of this paper. Funding This study was supported by the National Natural Science Foundation of China, No. 81771325; the Natural Science Foundation of Guangdong Province of China, No. 2016A030310224; Young Teacher Foundation of Sun Yat-sen University, No. 59000-18841219 Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval All animal experiments were approved by the Animal Care and Use Committee of Sun Yat-sen University and the Guangdong Province Animal Care Ethics Committee. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References 10.1016/j.neuroscience.2019.01.054 Chen S, Hou Y, Zhao Z, Luo Y, Lv S, Wang Q, et al. Neuregulin-1 Accelerates Functional Motor Recovery by Improving Motoneuron Survival After Brachial Plexus Root Avulsion in Mice. Neuroscience [Internet]. IBRO; 2019;404:510–8. 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Differences in c-jun and nNOS expression levels in motoneurons following different kinds of axonal injury in adult rats. Brain Cell Biol. 2008;36:213–27. 10.1016/j.niox.2009.11.005 Wang J, Yan L, Zhao X, Wu W, Zhou LH. The diversity of nNOS gene expression in avulsion-injured spinal motoneurons among laboratory rodents. Nitric Oxide - Biol Chem [Internet]. Elsevier Inc.; 2010;22:37–42. Available from: http://dx.doi.org/10.1016/j.niox.2009.11.005 . Cheng X, Fu R, Gao M, Liu S, Li YQ, Song FH, et al. Intrathecal application of short interfering RNA knocks down c-jun expression and augments spinal motoneuron death after root avulsion in adult rats. Neuroscience. 2013;241:268–79. Mukhamedshina YO, Akhmetzyanova ER, Martynova EV, Khaiboullina SF, Galieva LR, Rizvanov AA. Systemic and local cytokine profile following spinal cord injury in rats: A multiplex analysis. Front Neurol. 2017;8:1–7. Wu X, Wittwer AJ, Carr LS, Crippes BA, DeLarco JE, Lefkowith JB. 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Krauthausen M, Saxe S, Zimmermann J, Emrich M, Heneka MT, Müller M. CXCR3 modulates glial accumulation and activation in cuprizone-induced demyelination of the central nervous system. J Neuroinflammation. 2014;11:1–12. Jiang BC, He LN, Wu XB, Shi H, Zhang WW, Zhang ZJ, et al. Promoted interaction of C/EBPα with demethylated Cxcr3 gene promoter contributes to neuropathic pain in mice. J Neurosci. 2017;37:685–700. 10.1016/j.expneurol.2014.09.019 Guan XH, Fu QC, Shi D, Bu HL, Song ZP, Xiong BR, et al. Activation of spinal chemokine receptor CXCR3 mediates bone cancer pain through an Akt-ERK crosstalk pathway in rats. Exp Neurol [Internet]. Elsevier B.V.; 2015;263:39–49. Available from: http://dx.doi.org/10.1016/j.expneurol.2014.09.019 . Mojsilovic-Petrovic J, Callaghan D, Cui H, Dean C, Stanimirovic DB, Zhang W. Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes. J Neuroinflammation. 2007;4:1–15. 10.1038/emboj.2010.256 Ren M, Guo Q, Guo L, Lenz M, Qian F, Koenen RR, et al. Polymerization of MIP-1 chemokine (CCL3 and CCL4) and clearance of MIP-1 by insulin-degrading enzyme. EMBO J [Internet]. Nature Publishing Group; 2010;29:3952–66. Available from: http://dx.doi.org/10.1038/emboj.2010.256 . McMahon EJ, Cook DN, Suzuki K, Matsushima GK. Absence of Macrophage-Inflammatory Protein-1α Delays Central Nervous System Demyelination in the Presence of an Intact Blood-Brain Barrier. J Immunol. 2001;167:2964–71. Harhausen D, Prinz V, Ziegler G, Gertz K, Endres M, Lehrach H, et al. CD93/AA4.1: A Novel Regulator of Inflammation in Murine Focal Cerebral Ischemia. J Immunol. 2010;184:6407–17. Greenlee MC, Sullivan SA, Bohlson SS. Detection and characterization of soluble CD93 released during inflammation. Inflamm Res. 2009;58:909–19. Zamanian JL, Xu L, Foo LC, Nouri N, Zhou L, Giffard RG, et al. Genomic analysis of reactive astrogliosis. J Neurosci. 2012;32:6391–410. Liddelow S, Hoyer D. Tet Operators. Curr Drug Targets [Internet]. 2016;17:156–67. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070417/pdf/CGT-16-156.pdf . Supplementary Files SupplementarymaterialsTable1.docx Cite Share Download PDF Status: Published Journal Publication published 31 Mar, 2021 Read the published version in Journal of Neuroimmunology → 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-27602","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":559564,"identity":"603a91f1-e264-416f-a593-ccd954f409ca","order_by":1,"name":"Ke Zhong","email":"","orcid":"https://orcid.org/0000-0001-8666-7102","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Zhong","suffix":""},{"id":559565,"identity":"6e753ecc-4640-4c6b-a592-bfe3aea5dfc7","order_by":2,"name":"Yinqin Li","email":"","orcid":"","institution":"Fifth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yinqin","middleName":"","lastName":"Li","suffix":""},{"id":559566,"identity":"0470683e-e81a-4d97-8433-3206d10d16ce","order_by":3,"name":"Ying Tang","email":"","orcid":"","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Tang","suffix":""},{"id":559567,"identity":"e1885721-f783-49ae-a1fb-e183f15159fe","order_by":4,"name":"Guangying Yu","email":"","orcid":"","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guangying","middleName":"","lastName":"Yu","suffix":""},{"id":559568,"identity":"7b55a842-2715-4eae-8fa9-5e0733bb3d85","order_by":5,"name":"PRINCE LAST MUDENDA ZILUNDU","email":"","orcid":"","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"PRINCE","middleName":"LAST MUDENDA","lastName":"ZILUNDU","suffix":""},{"id":559569,"identity":"1259bfa6-bd7f-44ea-9d23-d6b2187ad694","order_by":6,"name":"Yingying Zhou","email":"","orcid":"","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Zhou","suffix":""},{"id":559570,"identity":"a0dc8dcc-9065-4001-acd5-1efaf3a3e5a5","order_by":7,"name":"Xiaoying Xu","email":"","orcid":"","institution":"Sun Yat-sen University Zhongshan School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Xu","suffix":""},{"id":559571,"identity":"b3424c72-993b-49e6-935b-9ce0771d40cf","order_by":8,"name":"Rao Fu","email":"","orcid":"","institution":"School of Medicine(Shenzhen), Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rao","middleName":"","lastName":"Fu","suffix":""},{"id":559572,"identity":"22367dcc-8f2b-4c29-b534-b4a21646d211","order_by":9,"name":"Lihua Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYDACCSB+YHBAjoGBsfEAsiB+LQkGB4yBWhpI0cJwILEBSBOnRX528zGJhII76WvbDwNt+XPY3uAA88HbPAx2ebi0MM45liaRYPAsd9uZxIYDjG2HEzccYEu25mFILsalhVkixwyo5XDutgMgLQ2Hgf7iMZPmgToVG2CDakk3O/8Q5jD+b3i18EC1JJjdANrCwHaYccMBHja8WiQk0pItgFoMt90A2pLYlp448zCbseUcg2ScWuRnJB+88eHPYXmz8+kPH3z4Y23Pd7z54Y03FXY4taCCBIZmYIiAWAZEqQeDOuKVjoJRMApGwYgBACG5XrbHc3wTAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-2516-2463","institution":"","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Lihua","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2020-05-08 07:56:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-27602/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-27602/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.jneuroim.2021.577517","type":"published","date":"2021-04-01T02:43:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1081746,"identity":"5656dc1c-f4b8-4446-bf96-b4edecfe2784","added_by":"auto","created_at":"2020-05-12 18:12:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":252043,"visible":true,"origin":"","legend":"Electron microscopy (EM) photographs of motorneurons (MNs) after unilateral brachial plexus roots avulsion injury (BPRA) in mice. Representative EM microphotographs of spinal MNs at 1-14 days post-injury (dpi) of unilateral BPRA in adult mice. As compared to the MNs in the contralateral side ventral horn of injured spinal segment (a, c, e, and g), the injured MNs in the ipsilateral side show a mixed morphological feature of apoptosis and necrosis, reflected as a swollen and crenellated cellular shape and an inflated mitochondria at 1dpi (b). It was also noted that a dramatic reduction of organelles in the injured MNs cytoplasm with an irregular nuclear envelope at 3-7 dpi (d, f). Moreover, the nuclear materials demonstrated pyknotic by the 14 dpi (f). n=5 mice/group. Scale bar=5μm.N=nucleus.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27602/v1/Figure1.jpg"},{"id":1081747,"identity":"2aba3d05-f59f-4b3f-b92c-e70a945655d7","added_by":"auto","created_at":"2020-05-12 18:12:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":156720,"visible":true,"origin":"","legend":"Unilateral BPRA caused a dramatic motoneurons (MNs) loss in the ventral horn of the injured spinal cord. The spinal cord was harvested for cryostat, the 30μm section counterstained with neutral red at 1-14 days post-injury (dpi) to evaluate the MNs survival rate. Panel A is the representative microphotographs that depict the coronal section of spinal segments suffered from unilateral BPRA at 1-14 dpi (a-d). a1-d1, a2-d2 are representative images showing MNs in the contralateral (Con) and ipsilateral (Ipsi) ventral horn of the spinal cord (outlined in the rectangular area in a-d) respectively. Panel B summarizes the change of the MNs survival rate during 1-14 dpi. ****p\u003c 0.0001 vs. 1d, One-way ANOVA followed by Tukey post hoc test, n=5 mice/group. All data were presented in mean ± S.E.M. The white arrow indicates motoneurons. Scale bar=200μm (a-d) or 50 μm (a1-d1, a2-d2).","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27602/v1/Figure2.jpg"},{"id":1081748,"identity":"62bf76ee-d45a-40e1-98b5-13f8dcc50cff","added_by":"auto","created_at":"2020-05-12 18:12:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127670,"visible":true,"origin":"","legend":"Unilateral BPRA upregulated microglia expression in the ipsilateral ventral horn of the spinal cord. Upper panel a-d are representative microphotographs showing the double immunostaining of IBA-1(red colour) and NeuN (green colour) of the corresponding spinal segment. The rectangular areas focus the ipsilateral ventral horn, which was highlighted in images observed under high magnification (a1-d3) at 1-14 days post-injury (dpi). Panel e and f summarize the change of the IBA-1 relative average fluorescence intensity (AFI) and immunoreactivity (IR) positive cells in the ipsilateral ventral horn during 1-14 dpi. ****p\u003c 0.0001 vs. 1d, ####p\u003c 0.0001 vs. 3d, \u0026\u0026\u0026\u0026p\u003c 0.0001 vs. 7d, One-way ANOVA followed by Tukey post hoc test, n=5 mice/group. All data were presented in mean ± S.E.M. The white arrow indicates microglia. Scale bar=200 μm (a-d) or 50 μm (a1-d3).","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27602/v1/Figure3.jpg"},{"id":1081749,"identity":"a34139b2-eeab-42a9-ae65-7e60dc325e09","added_by":"auto","created_at":"2020-05-12 18:12:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":141805,"visible":true,"origin":"","legend":"BPRA activates astrocyte response in the ipsilateral ventral horn of the spinal cord. Upper panel a-d are representative microphotographs showing the double immunostaining of GFAP (red color) and NeuN (green color) of the corresponding spinal segment. The rectangular areas focus the ipsilateral ventral horn, which was highlighted in images observed under high magnification (a1-d3) at 1-14 days post-injury (dpi). Panel e and f summarize the change of the GFAP relative average fluorescence intensity (AFI) and immunoreactivity (IR) positive cells in the ipsilateral ventral horn during 1-14 dpi. ****p\u003c 0.0001 vs. 1d, ####p\u003c 0.0001 vs. 3d, \u0026\u0026\u0026\u0026p\u003c 0.0001 vs. 7d, One-way ANOVA followed by Tukey post hoc test, n=5 mice/group. All data were presented in mean ± S.E.M. The white arrow indicates astrocyte. Scale bar=200 μm (a-d) or 50 μm (a1-d3).","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27602/v1/Figure4.jpg"},{"id":1081750,"identity":"3286f117-f90c-4a75-93c5-717802cb6b69","added_by":"auto","created_at":"2020-05-12 18:12:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":150613,"visible":true,"origin":"","legend":"The spinal cytokines level profile during the acute and subacute phase of BPRA injury. Upper panel showing a heat map analysis of 12 cytokines profile in contralateral and ipsilateral spinal cord at 1-14 days post unilateral BPRA injury in mice. Lower panel demonstrates the concentration (pg/ml) change of 7 significantly altered cytokines caused by the injury. ****p\u003c 0.0001 vs. contralateral at each time point, ####p\u003c 0.0001 vs. 1d within the ipsilateral group, Two-way ANOVA followed by Tukey post hoc test, n=6 mice/group.","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27602/v1/Figure5.jpg"},{"id":1081751,"identity":"635a6748-ebfe-4040-9e6c-b80dfebaa9b1","added_by":"auto","created_at":"2020-05-12 18:12:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":187581,"visible":true,"origin":"","legend":"Unilateral BPRA injury elevated the expression of MIP-1α and CD93 in the spinal cord. a-d shows the representative photomicrographs of MIP-1α expression in the spinal cord after 1-14 days after avulsion injury. The rectangular areas in a-d were further magnified to demonstrate the colocalization of NeuN (green colour,a1-d1) and MIP-1α (red colour, a2-d2) positive cells in the ipsilateral ventral horn. e-h shows the representative photomicrographs of CD93 expression in the spinal cord after 1-14 days after avulsion injury. The rectangular areas in a-d were further magnified to demonstrate the colocalization of NeuN (green colour,e1-h1) and CD93 (red colour, e2-h2) positive cells in the ipsilateral ventral horn. The white arrow indicates double immunostaining cells. Scale bar=200 μm (a-h) or 50 μm (a1-h3).","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27602/v1/Figure6.jpg"},{"id":13503077,"identity":"55c665c0-8bb0-429f-9aec-29f826bc0339","added_by":"auto","created_at":"2021-09-16 23:17:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1143448,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-27602/v1/55a9d7af-d71d-422a-be7f-0c35b3717a1e.pdf"},{"id":1081745,"identity":"75b2b2f9-4384-462f-a6a7-e322a8b41a8d","added_by":"auto","created_at":"2020-05-12 18:12:17","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":21914,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialsTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-27602/v1/SupplementarymaterialsTable1.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eCytokine Profile and Glial Activation Following Brachial Plexus Roots Avulsion Injury in Mice\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eMotoneurons (MNs) degenerate progressively and finally die after the brachial plexus root avulsion (BPRA), an injury-causing paralysis of the target muscle groups of the upper limbs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e][\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Neuroinflammation is one of the essential factors for triggering the neuron loss related to acute or chronic neurodegenerative disorders [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It has been well documented that the neuroinflammation also plays a causal role for the MNs degeneration and death associated with spinal cord injury, including avulsion injury [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e][\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt has been reported that the neuroinflammation is characteristic of the infiltration of leukocytes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and glial activation during the acute and subacute period after spinal cord injury [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e][\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe inflammation of BPRA is attributed to that lesion created cellular debris, which activates the astrocytes and microglia, trigging the release of a wide variety of oxidative stress regulators, growth factors, and inflammatory mediators including cytokines[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The cytokines are proteins that coordinate the immune response throughout the CNS, recruiting phagocytic cells, like peripheral neutrophils and macrophages into the injured spinal cord [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e][\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which are considered to be the first wave of infiltrating immune cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn one side, cytokines have shown a role in debris clearance, cellular remodeling, and production of pro-regenerative factors[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e][\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e][\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For example, Several studies reported the level of anti-inflammatory cytokines, like interleukin 4 (IL-4), interleukin 10 (IL-10) and transforming growth factor-β (TGF-β), substantially enhanced after undergoing post-spinal cord injury activation by injured neurons and astrocyte[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e][\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, other studies demonstrated that either microglia or astrocytes also produce pro-inflammatory cytokines such as interleukin 1 (IL-1), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) during spinal cord injury [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e][\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e][\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which suggest that cytokines are detrimental to CNS neurons [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and targets for the treatment of spinal cord injury [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e][\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e][\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e][\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe current study is sought to investigate how the alteration of specific cytokines expression patterns affects spinal glial activation and MNs death induced by BPRA. We observed that substantially elevated glial reactions and enhanced levels of cytokines, especially MIP-1α, which might contribute to a progressively MNs loss in the ventral horn of injured spinal segments caused by avulsion injury.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eExperiments were conducted on 76 adult male C57BL/6 mice, 8\u0026ndash;10 weeks old, weighing 20\u0026ndash;25\u0026nbsp;g, which were purchased from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China). The number of the animal use permit is SYXK 2017-0081. The mice were housed under a 12-hour light/dark cycle with food and water were available ad libitum. Surgical and animal care procedures were carried out under the provisions outlined in the National Health and Medical Research Council animal ethics and ARRIVE guidelines. All procedures were performed with the approval of the Animal Care and Utilization Committee of Sun Yat-sen University.\u003c/p\u003e \u003c/div\u003e \n\u003ch2\u003eAnimal Model Setting\u003c/h2\u003e\n \u003cp\u003eThe microsurgery procedure for the brachial plexus roots avulsion injury was as we described previously [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e][\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e][\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e][\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].In short, the mice were under anesthesia induced with ketamine/xylazine (80/20\u0026nbsp;mg/kg, i.p.) and maintained with 1% isoflurane. With each mouse in the supine position, the right side of brachial plexus was exposed, and its C5- T1 spinal nerve roots were separated under a surgical microscope (ChengHe Microsurgical Instruments Factory, Ning Bo, China). Both dorsal and ventral rootlets were pulled out using micro hemostatic forceps. All the avulsed distal parts of roots were cut away, and the success of the avulsion model confirmed under the microscope. The muscle and skin were sutured in successive layers. The mice were placed in a heated recovery chamber until they were fully awake and then returned to their cages.\u003c/p\u003e \n\u003ch2\u003eTissue Preparation\u003c/h2\u003e\n \u003cp\u003eTo assess the spinal cord cytokines profile of BPRA mice, a serial of time points [1, 2, 3, 5, 7 and 14\u0026nbsp;day post injury (dpi)], mice were anesthetized with 1% sodium pentobarbital (40\u0026nbsp;mg/kg, i.p., Sigma-Aldrich) and the cervical spinal cord was exposed by laminectomy under the surgical microscope. Subsequently, the C5 to T1 spinal segments were quickly removed and immediately divided into ipsilateral and contralateral halves. Spinal cord tissues were frozen in liquid nitrogen for subsequent protein extract. The frozen samples were homogenized in Whole-Cell Lysis buffer (KGP2100, Key GEN Biotech. Co., Ltd. Nanjing, China) containing protease inhibitor and 1\u0026nbsp;mM PMSF (P7626; Sigma-Aldrich, St. Louis, Missouri) using an electric homogenizer. The homogenates were lysed in ice for 1\u0026nbsp;h and centrifuged at 12,000\u0026nbsp;rpm for 30\u0026nbsp;min at 4\u0026nbsp;\u0026deg;C. The supernatants were collected, and the protein concentrations were measured using the BCA (SL260619, Thermo Scientific; Rockford, Illinois) method and stored at \u0026minus;\u0026thinsp;80\u0026nbsp;\u0026deg;C until the time of cytokine measurement.\u003c/p\u003e \n\u003ch2\u003eCytokine Assay\u003c/h2\u003e\n \u003cp\u003eSpinal cord samples (n\u0026thinsp;=\u0026thinsp;6 mice/group for each time point) were evaluated using the 20-plex, Mouse Premixed Multi-Analyte Kit LXSASM-20 (L126405, R\u0026amp;D Systems, Minneapolis, MN), measured with the Luminex system (Luminex, Austin, TX), and analyzed by Bio-plex software (Bio-Rad, Hercules, CA). This panel includes: C-X-C motif chemokine ligand 1(CXCL1), tumor necrosis factor-alpha (TNF-alpha), IL-12 p70, C-C motif chemokine 2 (CCL2), IL-1beta (IL-1b), IL-2, IL-6, IL-10, IL-17A, Intercellular adhesion molecule 1(ICAM-1), C-X-C motif chemokine 10 (IP-10), regulated on activation, normal T expressed and secreted protein (RANTES), C-C motif chemokine 12(CCL12), Beta-nerve growth factor (NGFb), macrophage inflammatory protein (MIP)-1alpha (MIP1-α), interleukin (IL)-alpha (IL-1a), Stromal cell-derived factor 1(SDF-1a,), Complement component C1q receptor(CD93), C-C motif chemokine 22(CCL22).\u003c/p\u003e \n\u003ch2\u003eImmunofluorescence, Neutral Red Staining And Cell Counting\u003c/h2\u003e\n \u003cp\u003eTwenty BPRA mice were sacrificed using a lethal dose of 1% sodium pentobarbital on the 1, 3, 7, and 14 dpi, respectively (n\u0026thinsp;=\u0026thinsp;5/group at each time point). They were transcardially perfused with normal saline followed by cold 4% paraformaldehyde (PFA) in 0.1\u0026nbsp;M phosphate buffer (PB, pH 7.4). The cervical spinal cord was exposed by laminectomy under the surgical microscope. The C7-C8 spinal segments, which were defined as the region between the uppermost and the lowermost rootlets of the contralateral C7-C8 spinal nerves, were post-fixed by immersion in 4% PFA followed by overnight immersion in 30% (v/v) sucrose in PB solution at 4\u0026nbsp;\u0026deg;C. The transverse sections of the C7-C8 spinal cord (30\u0026nbsp;\u0026micro;m), were collected in 0.01M PBS. Every third section of the spinal cord was used for staining study.\u003c/p\u003e \u003cp\u003eThe immunofluorescence (IF) was evaluated under a fluorescence microscope (BX63, Olympus, Japan). The sections were rinsed three times in PBS and then treated with 0.3% Triton X-100 and 0.1% bovine serum albumin (BSA) in PBS at room temperature for 30\u0026nbsp;min. The following primary antibodies were used: mouse anti-NeuN antibody (1:500 dilution, ab104224, Abcam, UK), rabbit anti-GFAP (1:500 dilution; 80788S, CST, Danvers, Massachusetts ), rabbit anti-IBA1 (1:500 dilution; 019-19741, Wako Bioproducts, Richmond, Virginia), rabbit anti-CD93 (1:200 dilution; ab198854 Abcam, UK), and rabbit anti-MIP1-α (1:100 dilution, ab25128, Abcam, UK). Incubation was at 4\u0026nbsp;\u0026deg;C overnight. Then, following washing three times with PBS, the sections were incubated with respective secondary antibodies. The following antibodies were used: Goat anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:1500, A32723, Invitrogen, USA), Goat anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (1:1000, A32731, Invitrogen, USA). Incubation was 2\u0026nbsp;h at room temperature in the dark. After rinsing the sections three times with PBS, then incubated with Hoechst33342 (H3570, Life Technologies, USA). They were visualized under a fluorescence microscope. Staining without primary or secondary antibodies served as negative controls.\u003c/p\u003e \u003cp\u003eIn addition, the neutral red staining was performed in C7 and C8 segments to assess MNs survival as previously reported [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e][\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Briefly, Sections were stained with 1% neutral red (N4638, Sigma-Aldrich, USA) in 0.1M acetic acid (pH4.8) for 2\u0026nbsp;h followed by dehydration in ethanol. Images were captured using a digital camera attached to the microscope. Next, the quantification of cell counting on the ventral horns of the both sides spinal cord (C7-C8 segments Rexed\u0026rsquo;s lamina IX) was as described previously [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We computed the mean number of either IF or neural red positive cells from the total 7\u0026thinsp;~\u0026thinsp;8 sections obtained from each mouse. Only those neurons with both the nucleolus in the nucleus and Nissl bodies in the cytoplasm stained with neutral red we recounted under a 20\u0026thinsp;\u0026times;\u0026thinsp;objective lens. Two independent persons blinded to the sidedness of the groups performed cell counting, pooling of means and data analysis.\u003c/p\u003e \n\u003ch2\u003eTransmission Electron Microscopy\u003c/h2\u003e\n \u003cp\u003eTransmission electron microscopy (TEM) was used to assess motor neuron morphology in the spinal cord after BPRA at 1, 3, 7, 14 dpi (n\u0026thinsp;=\u0026thinsp;5 mice/group at each time point). The mouse was perfused transcardially with 0.9% normal saline, followed by a mixture of 2% PFA and 2.5% glutaraldehyde (Sigma-Aldrich, G6257, USA) in 0.1\u0026nbsp;M PBS. Approximately 1 mm\u003csup\u003e3\u003c/sup\u003e of tissue per mice was dissected from the spinal cord and fixed in 2% glutaraldehyde for 2\u0026nbsp;h at 4\u0026nbsp;\u0026deg;C. The tissues were rinsed in 0.1\u0026nbsp;M cacodylate buffer and post-fixed with 1% osmium tetroxide for 2\u0026nbsp;h. Then, the tissue was rinsed with distilled water before undergoing dehydration in a graded ethanol series. Subsequently, the tissue was infiltrated overnight at 4\u0026nbsp;\u0026deg;C using a mixture of half acetone and half resin. The tissue was embedded in resin 24\u0026nbsp;h later and then cured fully as follows: 37\u0026nbsp;\u0026deg;C overnight, 45\u0026nbsp;\u0026deg;C for 12\u0026nbsp;h, and 60\u0026nbsp;\u0026deg;C for 24\u0026nbsp;h. After that, 70-nm sections were cut and stained with 3% uranyl acetate for 20\u0026nbsp;min and 0.5% lead citrate for 5\u0026nbsp;min. Ultrastructural changes motor neuron morphology was observed under TEM.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of the mean). The morphological evaluation result or cytokine assay result was subjected to one-way or two-way measures ANOVA followed by a Bonferroni or Tukey post hoc test for multiple group comparisons. A value of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBPRA substantially caused MNs loss in the ventral horn of the injured spinal cord\u003c/h2\u003e \u003cp\u003eAt 1 dpi, the injured MNs started to present morphological features of both apoptosis and necrosis, reflected by slightly swollen mitochondria and condensed cytoplasm with a still visible nucleus and nucleolus. By 3\u0026ndash;7 dpi, the cytoplasm of MNs had become hyperchromatic, the nuclear envelope was indistinct and irregular in shape, and the nucleoli and nuclei were more condensed. The cytoplasm also contained fragmented organelles, numerous, small, granular profiles and organelles are significantly reduced in the cytoplasm. Mitochondria were absent, and the cell membrane was disrupted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, the light microscopic observation also revealed an increase in the size of the cytoplasm of injured MNs. Furthermore, the neural red positive staining cell counting of spinal ventral horn found a significant and progressive decline in the ipsilateral MNs survival rate (F\u003csub\u003e(3,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;674.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with only 35.34%\u0026plusmn; 0.48% ipsilateral MNs survived as compared to contralateral side at 14 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003ch2\u003eBPRA enhance the glial activation in the ventral horn of the injured spinal cord \u003c/p\u003e \u003cp\u003eTo evaluate the glial reactions that occurred in the injured spinal cord after BPRA, the expression of IBA-1 and GFAP, markers of activated microglia and astrocytes, were determined using immunoreactivity 1 to 14 dpi in both ipsilateral and contralateral side of the spinal ventral horn. Our results showed both IBA-1 average fluorescence intensity (AFI) and immunoreactivity (IR)-positive cells number of ipsilateral ventral horn gradually increased compared to the contralateral side within 1\u0026ndash;14 days followed injury (AFI: F\u003csub\u003e(3,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;487.7, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; IR cell number, F\u003csub\u003e(3,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;249.9, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, we observed a robustly elevated GFAP expression trend in the injured ventral horn after BPRA. Compared to the contralateral side, BPRA caused an increase in GFAP AFI ( F\u003csub\u003e(3,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;85.06, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and IR cell number (F\u003csub\u003e(3༌16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;52.7, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the ipsilateral side, with the peaks at 7 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These data suggested that BPRA produced microglia and glial activation in the injured spinal cord.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eThe present study showed that BPRA substantially caused MNs loss in the ventral horn of the injured spinal cord. The damaged MNs is morphologically characteristic of swelling and depletion of mitochondria endoplasmic reticulum, and eventual rupture of organelles, and disruption of nuclear and plasma membranes. These changes are associated with glial activation in the affected ventral horn, reflected by enhanced IBA-1 and GFAP immunoreactivity. Moreover, we also found that some essential cytokines level substantially changed during the acute and subacute phase of MNs death, especially like MIP1-α and CD93 expression increased in the inured MNs. These data strongly suggest that cytokines might have crosstalk with glial activation, which contributes to the spinal MNs loss caused by avulsion injury.\u003c/p\u003e \u003cp\u003eOur study the changes of cytokine profile following BPRA (acute and subacute periods) in mice compared with contralateral ventral horns in the same injured spinal segments. During the first 2 weeks after BPRA, we detected significant changes in concentrations of 7 cytokines with different inflammatory and immunological activities. We found CXCL1 showed a rapidly enhanced concentration on 1 dpi then decreased to the baseline during 2\u0026ndash;14 dpi. CXCL1, being a chemotactic substance for neutrophils, is expressed by macrophages, neutrophils, astrocytes and epithelial cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Studies in mice have shown that CXCL1 reduces the severity of multiple sclerosis and can exhibit a neuroprotective function. The concurrence of CXCR2(the receptor for CXCL1) on oligodendrocytes and induced CXCL1 on hypertrophic astrocytes in MS provides a novel mechanism for recruitment of oligodendrocytes to areas of damage, an essential prerequisite for lesion repair in this devastating condition. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e][\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. CXCL1 of might be actively involved in the regenerative processes by recruiting oligodendrocytes to the injured spinal cord on the first 1\u0026nbsp;day after BPRA.\u003c/p\u003e \u003cp\u003eOn 2 dpi, we observed a reduced concentration of IL-12 p70 and maintained a relatively low level as compared to 1 dpi. Interleukin-12 (IL-12) is a heterodimeric cytokine produced mostly by phagocytic cells in response to bacteria, bacterial products, and intracellular parasites, and to some degree by B lymphocytes. The early preference expressed in the immune response depends on the balance between IL-12, which favors Thl responses, and IL-4, which favors Th2 responses. Thus,IL-12 represents a functional bridge between the early nonspecific innate resistance and the subsequent antigen-specific adaptive immunity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePro-inflammatory cytokine IP10 peaked at 1 dpi with a second peak at 14 dpi compared to the contralateral side of the spinal cord, which is secreted by several cell types (e.g., leukocytes, macrophages and endothelial cells) in response to IFN-γ and recruits these cells to sites of infection or inflammation after binding to C-X-C motif receptor (CXCR3) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e][\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. IP10 is expressed at low levels in the spinal cord under normal conditions, but upon stimulation, the expression of this chemokine is substantially increased at sites where glial cells accumulate [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Previous study found the neutralization of CXCR3 significantly reduces the expression of markers of activated microglia and astrocytes as well as the levels of proinflammatory cytokines and chemokines, such as CCL2 and CXCL10 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e][\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e][\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChanges in the level of pro-inflammatory cytokine MCP5 in the form of a sharp increase in its level on 1 dpi were observed in injured spinal cord and maintained a relatively high level as compared to the contralateral side till 14 dpi. The cytokine MCP5 attracts eosinophils, monocytes, and lymphocytes but not neutrophils. Previous study found sequence analyses identified HIF-1α binding sites in the promoters of MCP-5 genes and HIF-1α is involved in transcriptional induction of MCP5 in astrocytes by hypoxia [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe found the pro-inflammatory chemokine MIP-1 α during the first 2 weeks after BPRA with a maximum increase of 13.94 times on 1 dpi, being a macrophage inflammation protein, activates granulocytes, which leads to acute neuroinflammation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Immunofluorescence staining found MIP-1 α mostly located in the injured motor neuron after BPRA. Its increase in the spinal cord tissues may be associated with disruption of the blood\u0026ndash;brain barrier and an increased migration of macrophages and neutrophils to the site of injury. Previous studies have shown the importance of MIP-1α in demyelination in the CNS and highlight its effect by exposing MIP-1α knockout mice (MIP-1α-/-) to cuprizone and comparing pathology to wild-type mice, which found demyelination was significantly decreased in MIP-1α-/- mice (near 36% reduction) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. We speculate that the high expression of MIP-1α may be related to attract macrophages infiltration and demyelination of motor neurons after brachial plexus avulsion in mice.\u003c/p\u003e \u003cp\u003eOn 1 dpi, we observed a rapidly enhanced concentration of CXCL1 in the injured spinal cord. Studies in mice have shown that CXCL1 reduces the severity of multiple sclerosis and can exhibit a neuroprotective function. The concurrence of CXCR2(the receptor for CXCL1) on oligodendrocytes and induced CXCL1 on hypertrophic astrocytes in MS provides a novel mechanism for recruitment of oligodendrocytes to areas of damage, an essential prerequisite for lesion repair in this devastating condition. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e][\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. CXCL1 of might be actively involved in the regenerative processes in the CNS by recruiting oligodendrocytes to the injured spinal cord on the first 1\u0026nbsp;day after BPRA.\u003c/p\u003e \u003cp\u003eCD93 is a type I transmembrane glycoprotein that is expressed on the surface of platelets, and myeloid, endothelial, hematopoietic stem and natural killer cells. CD93 is strongly associated with immune cell phagocytosis, which regulates innate immunity and inflammation [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. CD93 deletion also induces phagocyte impairment on scavenging apoptotic cells[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Our study found CD93 IR was low in the contralateral side of spinal cord, but increased during inflammatory reaction in the ipsilateral side of spinal cord, reaching a peak at early stage of BPRA and showing distinct cytoplasmic and nuclear staining. These findings suggest that the CD93 cytoplasmic tail is mainly expressed in cell membranes yet may migrate towards the cytoplasm or nucleus with increasing inflammatory reaction.\u003c/p\u003e \u003cp\u003eThe levels of the ICAM1 gradually increased with a peak reached on 14\u0026nbsp;day. As a cell-surface glycoprotein typically expressed by both immune system cells and endothelial cells. ICAM1 is also expressed by astrocytes, though at low levels in resting, quiescent astrocytes, remarkably it is induced 12-fold following injury and subsequent astrocyte activation [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. ICAM1 may facilitate astrocyte-lymphocyte interactions ultimately aiding recruitment of immune cells into the CNS [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn our study, we evaluated the cytokine profile in spinal cord of mice with a model of BPRA in acute and subacute periods after injury. Our study also demonstrated that the MNs degeneration process, inflammatory reaction and immunological response after BPRA. A further study of a complex cytokine network imbalance after BPRA and determining its possible correlation with the severity of damage and clinical events seems important. This would provide a better understanding of the role cytokines play in the pathophysiology of the BPRA disease.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eBPRA, brachial plexus root avulsion; MNs, Motoneurons; CNS, central nervous system; TEM, transmission electron microscopy; dpi, day post injury\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the general support provided by Yaqiong Wang for her professional assistance in the tissue process and electron microscope observation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZK designed this study, performed the experiments and drafted the manuscript. LYQ and TY carried out extra data analysis. YGY and PRINCE participated in drafting and revised the manuscript. ZYY and XXY revised the manuscript, FR and ZLH supervised the design of the study and conceived the manuscript. All authors reviewed and approved the final version of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China, No. 81771325; the Natural Science Foundation of Guangdong Province of China, No. 2016A030310224; Young Teacher Foundation of Sun Yat-sen University, No. 59000-18841219\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Animal Care and Use Committee of Sun Yat-sen University and the Guangdong Province Animal Care Ethics Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cdiv class=\"BibBookDOI\"\u003e10.1016/j.neuroscience.2019.01.054\u003c/div\u003e \u003cspan\u003eChen S, Hou Y, Zhao Z, Luo Y, Lv S, Wang Q, et al. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070417/pdf/CGT-16-156.pdf\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":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"cytokine profile, nerve injury, motoneuron degeneration, mice","lastPublishedDoi":"10.21203/rs.3.rs-27602/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-27602/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eInflammation and tissue infiltration by various immune cells play a significant role in the pathogenesis of neurons suffering the central nervous systems diseases. Although brachial plexus root avulsion (BPRA) leads to dramatic motoneurons (MNs) death and permanent loss of function, however, the crosstalk between cytokines and glial reaction in the spinal cord during injury is far beyond our knowledge. The current study is sought to investigate the alteration of specific cytokine expression patterns of BPRA injured spinal cord during an acute and subacute period.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eHere, cytokine assay, transmission electron microscopy, and histological staining were utilized to assess the alteration of cytokine network, ultrastructure morphology, as well as glial activation and loss of MNs within two weeks, post-injury on a mouse BPRA model.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eWe found that BPRA significantly changed the level of CXCL1, IL12 p70, ICAM1, IP10, MCP-5, MIP1-α and CD93. Moreover, the elevated MIP1-α and CD93 were mostly distributed in MNs of the injured, but few in healthy segments. Also, BPRA significantly induced glial reactions in the ventral horn of injured spinal segments, reflected by robustly elevated the Ionized calcium-binding adaptor molecule 1 (IBA1) and Glial fibrillary acidic protein (GFAP) positive cells. We also observed a severe progressively loss of injured MNs, with a character of both apoptosis and necrosis.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eOverall, these findings suggested that the inflammatory cytokines associated with glial cell proliferation play a vital role in the pathophysiology of MNs death caused by nerve roots injury.\u003c/p\u003e","manuscriptTitle":"Cytokine Profile and Glial Activation Following Brachial Plexus Roots Avulsion Injury in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-05-12 18:12:17","doi":"10.21203/rs.3.rs-27602/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":"95b13b81-eebe-4328-8974-e3efb968448b","owner":[],"postedDate":"May 12th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":98392,"name":"Neurobiology of Disease"}],"tags":[],"updatedAt":"2021-08-20T02:43:58+00:00","versionOfRecord":{"articleIdentity":"rs-27602","link":"https://doi.org/10.1016/j.jneuroim.2021.577517","journal":{"identity":"journal-of-neuroimmunology","isVorOnly":true,"title":"Journal of Neuroimmunology"},"publishedOn":"2021-04-01 02:43:58","publishedOnDateReadable":"April 1st, 2021"},"versionCreatedAt":"2020-05-12 18:12:17","video":"","vorDoi":"10.1016/j.jneuroim.2021.577517","vorDoiUrl":"https://doi.org/10.1016/j.jneuroim.2021.577517","workflowStages":[]},"version":"v1","identity":"rs-27602","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-27602","identity":"rs-27602","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}