Umbilical Cord Blood Stem Cell Mediated Downregulation of Fas Improves Functional Recovery of Rats after Spinal Cord Injury

Human umbilical cord blood stem cells (hUCB), due to their primitive nature and ability to develop into nonhematopoietic cells of various tissue lineages, represent a potentially useful source for cell-based therapies after spinal cord injury (SCI). To evaluate their therapeutic potential, hUCB were stereotactically transplanted into the injury epicenter, one week after SCI in rats. Our results show the presence of a substantial number of surviving hUCB in the injured spinal cord up to five weeks after transplantation. Three weeks after SCI, apoptotic cells were found especially in the dorsal white matter and gray matter, which are positive for both neuron and oligodendrocyte markers. Expression of Fas on both neurons and oligodendrocytes was efficiently downregulated by hUCB. This ultimately resulted in downregulation of caspase-3 extrinsic pathway proteins involving increased expression of FLIP, XIAP and inhibition of PARP cleavage. In hUCB-treated rats, the PI3K/Akt pathway was also involved in antiapoptotic actions. Further, structural integrity of the cytoskeletal proteins α-tubulin, MAP2A&2B and NF-200 has been preserved in hUCB treatments. The behavioral scores of hind limbs of hUCB-treated rats improved significantly than those of the injured group, showing functional recovery. Taken together, our results indicate that hUCB-mediated downregulation of Fas and caspases leads to functional recovery of hind limbs of rats after SCI.     

FGF1 improves functional recovery through inducing PRDX1 to regulate autophagy and anti‐ROS after spinal cord injury

Fibroblast growth factor 1 (FGF1) is thought to exert protective and regenerative effects on neurons following spinal cord injury (SCI), although the mechanism of these effects is not well understood. The use of FGF1 as a therapeutic agent is limited by its lack of physicochemical stability and its limited capacity to cross the blood‐spinal cord barrier. Here, we demonstrated that overexpression of FGF1 in spinal cord following SCI significantly reduced tissue loss, protected neurons in the ventricornu, ameliorated pathological morphology of the lesion, dramatically improved tissue recovery via neuroprotection, and promoted axonal regeneration and remyelination both in vivo and in vivo. In addition, the autophagy and the expression levels of PRDX1 (an antioxidant protein) were induced by AAV‐FGF1 in PC12 cells after H₂O₂ treatment. Furthermore, the autophagy levels were not changed in PRDX1‐suppressing cells that were treated by AAV‐FGF1. Taken together, these results suggest that FGF1 improves functional recovery mainly through inducing PRDX1 expression to increase autophagy and anti‐ROS activity after SCI.     

Electro-acupuncture upregulates CGRP expression after rat spinal cord transection

Calcitonin gene-related peptide (CGRP) plays a variety of important roles within the nervous system. Increasing CGRP expression could improve the survival of injured neurons and prevent neuronal loss. In this study, we first evaluated in vitro the neuroprotective function of CGRP on mechanically injured cerebellar granule neurons (CGNs) of rats. We then verified this result through exogenous administration of CGRP in a spinal cord transected completely in rats. Finally, we investigated the effect of electro-acupuncture (EA) on CGRP expression following the spinal cord transected completely in rats. We found that EA can improve CGRP expression, and exogenous CGRP may promote the survival of injured neurons, both in vivo and in vitro. Our results suggest that CGRP may be a specific neuropeptide expressed in GV-EA treatment of spinal cord injuries (SCI), and that CGRP may play a neuroprotective role in survival of neurons injured mechanically.     

Spatiotemporal Patterns of SSeCKS Expression After Rat Spinal Cord Injury

Src suppressed C kinase substrate (SSeCKS) was identified as a PKC substrate/PKC-binding protein, which plays a role in mitogenic regulatory activity and has a function in the control of cell signaling and cytoskeletal arrangement. However its distribution and function in the central nervous system (CNS) lesion remain unclear. In this study, we mainly investigated the mRNA and protein expression and cellular localization of SSeCKS during spinal cord injury (SCI). Real-time PCR and Western blot analysis revealed that SSeCKS was present in normal whole spinal cord. It gradually increased, reached a peak at 3 days for its mRNA level and 5 days for its protein level after SCI, and then declined during the following days. In ventral horn, the expression of SSeCKS underwent a temporal pattern that was similar with the whole spinal cord in both mRNA and protein level. However, in dorsal horn, the mRNA and protein for SSeCKS expression were significantly increased at 1 day for its mRNA level and 3 days for its protein level, and then gradually declined to the baseline level, ultimately up-regulated again from 7 to 14 days. The protein expression of SSeCKS was further analysed by immunohistochemistry. The positively stained areas for SSeCKS changed with the similar pattern to that of protein expression detected by immunoblotting analysis. Double immunofluorescence staining showed that SSeCKS immunoreactivity (IR) was found in neurons, astrocytes, oligodendrocytes of spinal cord tissues within 5 mm from the lesion site. Importantly, injury-induced expression of SSeCKS was co-labeled by active caspase-3 (apoptotic marker), Tau-1 (the marker for pathological oligodendrocyte) and β-1,4-galactosyltransferase 1 (GalT). All the results suggested that SSeCKS might play important roles in spinal cord pathophysiology and further research is needed to have a good understanding of its function and mechanism. Feng Xiao and Min Fei contributed equally to this work.     

Temporal expression profile of CXC chemokines in serum of patients with spinal cord injury

Chemokines, a subclass of cytokine superfamily have both pro-inflammatory and migratory role and serve as chemoattractant of immune cells during the inflammatory responses ensuing spinal cord injury (SCI). The chemokines, especially CXCL-1, CXCL-9, CXCL-10 and CXCL-12 contribute significant part in the inflammatory secondary damage of SCI. Inhibiting chemokine’s activity and thereby the secondary damage cascades has been suggested as a chemokine-targeted therapeutic approach to SCI. To optimize the inhibition of secondary injury through targeted chemokine therapy, accurate knowledge about the temporal profile of these cytokines following SCI is required. Hence, the present study was planned to determine the serum levels of CXCL-1, CXCL-9, CXCL-10 and CXCL-12 at 3–6 h, 7 and 28 days and 3 m after SCI in male and female SCI patients (n = 78) and compare with age- and sex-matched patients with non-spinal cord injuries (NSCI, n = 70) and healthy volunteers (n = 100). ANOVA with Tukey post hoc analysis was used to determine the differences between the groups. The data from the present study show that the serum level of CXCL-1, CXCL-9 and CXCL-10 peaked on day 7 post-SCI and then declined to the control level. In contrast, significantly elevated level of CXCL-12 persisted for 28 days post SCI. In addition, post-SCI expression of CXCL-12 was found to be sex-dependent. Male SCI patients expressed significantly higher CXCL-12 when compared to control and SCI female. We did not observe any change in chemokines level of NSCI. Further, the age of the patients did not influence chemokines expression after SCI. These observations along with SCI-induced CSF-chemokine level should contribute to the identification of selective and temporal chemokine targeted therapy after SCI.     

Tamoxifen attenuates inflammatory-mediated damage and improves functional outcome after spinal cord injury in rats

Tamoxifen has been found to be neuroprotective in both transient and permanent experimental ischemic stroke. However, it remains unknown whether this agent shows a similar beneficial effect after spinal cord injury (SCI), and what are its underlying mechanisms. In this study, we investigated the efficacy of tamoxifen treatment in attenuating SCI-induced pathology. Blood–spinal cord barrier (BSCB) permeability, tissue edema formation, microglial activation, neuronal cell death and myelin loss were determined in rats subjected to spinal cord contusion. The results showed that tamoxifen, administered at 30 min post-injury, significantly decreased interleukin-1β (IL-1β) production induced by microglial activation, alleviated the amount of Evans blue leakage and edema formation. In addition, tamoxifen treatment clearly reduced the number of apoptotic neurons post-SCI. The myelin loss and the increase in production of myelin-associated axonal growth inhibitors were also found to be significantly attenuated at day 3 post-injury. Furthermore, rats treated with tamoxifen scored much higher on the locomotor rating scale after SCI than did vehicle-treated rats, suggesting improved functional outcome after SCI. Together, these results demonstrate that tamoxifen provides neuroprotective effects for treatment of SCI-related pathology and disability, and is therefore a potential neuroprotectant for human spinal cord injury therapy.     

Expression of Sam68 Associates with Neuronal Apoptosis and Reactive Astrocytes After Spinal Cord Injury

Src-associated in mitosis (Sam68; 68 kDa) is a novel RNA-binding protein that belongs to the signal transduction and activation of RNA family involved in various biological processes. However, the expression and roles of Sam68 in the central nervous system remain unknown. In the present study, we performed a spinal cord injury (SCI) model in adult rats and found a significant increase of Sam68 protein levels in this model, which reached a peak at day 3 and then gradually returned to normal levels at day 14 after SCI. We use immunohistochemistry analysis revealing a widespread distribution of Sam68 in the spinal cord. In addition, double-immunofluorescence staining showed that Sam68 immunoreactivity was found predominantly in neurons and astrocytes. Moreover, colocalization of Sam68/active caspase-3 has been respectively detected in neuronal nuclei, and colocalization of Sam68/PCNA has been detected in glial fibrillary acidic protein. In vitro, we found that depletion of Sam68 by short interfering RNA inhibits neuronal apoptosis and astrocyte proliferation and decreases cyclin D1 protein levels. In conclusion, this is the first study to find the Sam68 expression in SCI. Our results suggest that Sam68 might be illustrated in the apoptosis of neurons and proliferation of astrocytes after SCI. This research will provide new drug targets for clinical treatment of SCI.     

Gramine promotes functional recovery after spinal cord injury via ameliorating microglia activation

In recent years, a large number of studies have reported that neuroinflammation aggravates the occurrence of secondary injury after spinal cord injury. Gramine (GM), a natural indole alkaloid, possesses various pharmacological properties; however, the anti-inflammation property remains unclear. In our study, Gramine was investigated in vitro and in vivo to explore the neuroprotection effects. In vitro experiment, our results suggest that Gramine treatment can inhibit release of pro-inflammatory mediators. Moreover, Gramine prevented apoptosis of PC12 cells which was caused by activated HAPI microglia, and the inflammatory secretion ability of microglia was inhibited by Gramine through NF-κB pathway. The in vivo experiment is that 80 mg/kg Gramine was injected orthotopically to rats after spinal cord injury (SCI). Behavioural and histological analyses demonstrated that Gramine treatment may alleviate microglia activation and then boost recovery of motor function after SCI. Overall, our research has demonstrated that Gramine exerts suppressed microglia activation and promotes motor functional recovery after SCI through NF-κB pathway, which may put forward the prospect of clinical treatment of inflammation-related central nervous diseases.     

Expression of SGTA correlates with neuronal apoptosis and reactive gliosis after spinal cord injury

Small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA) is a novel TPR-containing protein involved in various biological processes. However, the expression and roles of SGTA in the central nervous system remain unknown. We have produced an acute spinal cord injury (SCI) model in adult rats and found that SGTA protein levels first significantly increase, reach a peak at day 3 and then gradually return to normal level at day 14 after SCI. These changes are striking in neurons, astrocytes and microglia. Additionally, colocalization of SGTA/active caspase-3 has been detected in neurons and colocalization of SGTA/proliferating cell nuclear antigen has been detected in astrocytes and microglial. In vitro, SGTA depletion by short interfering RNA inhibits astrocyte proliferation and decreases cyclinA and cyclinD1 protein levels. SGTA knockdown also reduces neuronal apoptosis. We speculate that SGTA is involved in biochemical and physiological responses after SCI.     

Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation

Spinal cord injury (SCI) is a severe neurological disease; however, few drugs have been proved to treat SCI effectively. Neuroinflammation is the major pathogenesis of SCI secondary injury and considered to be the therapeutic target of SCI. Salidroside (Sal) has been reported to exert anti‐inflammatory effects in airway, adipose and myocardial tissue; however, the role of Sal in SCI therapeutics has not been clarified. In this study, we showed that Sal could improve the functional recovery of spinal cord in rats as revealed by increased BBB locomotor rating scale, angle of incline, and decreased cavity of spinal cord injury and apoptosis of neurons in vivo. Immunofluorescence double staining of microglia marker and M1/M2 marker demonstrated that Sal could suppress M1 microglia polarization and activate M2 microglia polarization in vivo. To verify how Sal exerts its effects on microglia polarization and neuron protection, we performed the mechanism study in vitro in microglia cell line BV‐2 and neuron cell line PC12. The results showed that Sal prevents apoptosis of PC12 cells in coculture with LPS‐induced M1 BV‐2 microglia, also the inflammatory secretion phenotype of M1 BV‐2 microglia was suppressed by Sal, and further studies demonstrated that autophagic flux regulation through AMPK/mTOR pathway was involved in Sal regulated microglia polarization after SCI. Overall, our study illustrated that Sal could promote spinal cord injury functional recovery in rats, and the mechanism may relate to its microglia polarization modulation through AMPK‐/mTOR‐mediated autophagic flux stimulation.     

Spinal cord injury causes sustained disruption of the blood-testis barrier in the rat

There is a high incidence of infertility in males following traumatic spinal cord injury (SCI). Quality of semen is frequently poor in these patients, but the pathophysiological mechanism(s) causing this are not known. Blood-testis barrier (BTB) integrity following SCI has not previously been examined. The objective of this study was to characterize the effects of spinal contusion injury on the BTB in the rat. 63 adult, male Sprague Dawley rats received SCI (n = 28), laminectomy only (n = 7) or served as uninjured, age-matched controls (n = 28). Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), BTB permeability to the vascular contrast agent gadopentate dimeglumine (Gd) was assessed at either 72 hours-, or 10 months post-SCI. DCE-MRI data revealed that BTB permeability to Gd was greater than controls at both 72 h and 10 mo post-SCI. Histological evaluation of testis tissue showed increased BTB permeability to immunoglobulin G at both 72 hours- and 10 months post-SCI, compared to age-matched sham-operated and uninjured controls. Tight junctional integrity within the seminiferous epithelium was assessed; at 72 hours post-SCI, decreased expression of the tight junction protein occludin was observed. Presence of inflammation in the testes was also examined. High expression of the proinflammatory cytokine interleukin-1 beta was detected in testis tissue. CD68⁺ immune cell infiltrate and mast cells were also detected within the seminiferous epithelium of both acute and chronic SCI groups but not in controls. In addition, extensive germ cell apoptosis was observed at 72 h post-SCI. Based on these results, we conclude that SCI is followed by compromised BTB integrity by as early as 72 hours post-injury in rats and is accompanied by a substantial immune response within the testis. Furthermore, our results indicate that the BTB remains compromised and testis immune cell infiltration persists for months after the initial injury.     

大鼠脊髓损伤后表皮生长因子受体在脊髓的表达特点

目的研究大鼠脊髓损伤(spinal cord injury,SCI)后表皮生长因子受体(epidermal growth factor re-ceptor,EGFR)在脊髓的表达特点及意义。方法健康成年雄性SD大鼠,随机分为4组(每组10只):假手术组,SCI术后3 d、7 d和14 d组。应用Basso Beattie Bresnahan(BBB)评分观察大鼠行为学改变;逆转录-聚合酶链反应(RT-PCR)检测损伤段脊髓组织中EGFR mRNA表达水平;免疫组织化学方法观察损伤段脊髓灰质中EGFR蛋白表达情况;并对EGFRmRNA及蛋白表达情况与BBB评分进行相关性分析。结果行为学观察发现大鼠脊髓损伤后下肢神经功能逐步恢复;RT-PCR结果显示EGFR mRNA在假手术组大鼠脊髓中微量表达,SCI术后3 d表达显著升高,随后趋于下降,14 d时仍高于假手术组(P<0.01);免疫组织化学染色显示损伤段脊髓灰质中EGFR阳性细胞数在损伤后3 d显著高于假手术组(P<0.01),随后趋于下降,但14 d时仍高于假手术组(P<0.01);EGFR mRNA及蛋白的表达均与BBB评分呈显著负相关(r=-0.956,P<0.05;r=-0.966,P<0.05)。结论EGFR在大鼠脊髓损伤后具有时相分布特点,且与动物行为呈负相关,提示其表达可能阻碍损伤后的神经功能恢复。     

Electro-acupuncture upregulates CGRP expression after rat spinal cord transection

Calcitonin gene-related peptide (CGRP) plays a variety of important roles within the nervous system. Increasing CGRP expression could improve the survival of injured neurons and prevent neuronal loss. In this study, we first evaluated in vitro the neuroprotective function of CGRP on mechanically injured cerebellar granule neurons (CGNs) of rats. We then verified this result through exogenous administration of CGRP in a spinal cord transected completely in rats. Finally, we investigated the effect of electro-acupuncture (EA) on CGRP expression following the spinal cord transected completely in rats. We found that EA can improve CGRP expression, and exogenous CGRP may promote the survival of injured neurons, both in vivo and in vitro. Our results suggest that CGRP may be a specific neuropeptide expressed in GV-EA treatment of spinal cord injuries (SCI), and that CGRP may play a neuroprotective role in survival of neurons injured mechanically.     

Up-regulation of TRAF2 Suppresses Neuronal Apoptosis after Rat Spinal Cord Injury

Tumor necrosis factor receptor-associated factor 2 (TRAF2) for signal transduction of the cell death receptor is well established. However, the role of TRAF2 in spinal cord injury (SCI) remains unclear. In this study, we detected the dynamic change patterns of TRAF2 expression using an acute spinal cord contusion (SCC) model in adult rats. Western blot analysis and immunohistochemistry identified significant upregulation of TRAF2 after SCI. Double-immunofluorescent staining demonstrated that the upregulated TRAF2 was found predominantly in neurons. Moreover, colocalization of TRAF2 with active caspase-3/-8 was detected in NeuN-positive cells. In vitro, we analyzed the association of TRAF2 with active caspase-3/8 on PC12 cells by western blot analysis, which paralleled the in vivo data. Knockdown ofTRAF2 with siRNA demonstrated its probable anti-apoptotic role in the process of neuronal apoptosis after SCI. To summarize, we have revealed for the first time the temporal and spatial expression profile of TRAF2 in SCI. Our data suggest that upregulation of TRAF2 triggered by trauma plays an important role in suppressing neuronal apoptosis after SCI.     

Spinal neuropeptide expression and neuropathic behavior in the acute and chronic phases after spinal cord injury: Effects of progesterone administration

Patients with spinal cord injury (SCI) develop chronic pain that severely compromises their quality of life. We have previously reported that progesterone (PG), a neuroprotective steroid, could offer a promising therapeutic strategy for neuropathic pain. In the present study, we explored temporal changes in the expression of the neuropeptides galanin and tyrosine (NPY) and their receptors (GalR1 and GalR2; Y1R and Y2R, respectively) in the injured spinal cord and evaluated the impact of PG administration on both neuropeptide systems and neuropathic behavior. Male rats were subjected to spinal cord hemisection at T13 level, received daily subcutaneous injections of PG or vehicle, and were evaluated for signs of mechanical and thermal allodynia. Real time PCR was used to determine relative mRNA levels of neuropeptides and receptors, both in the acute (1 day) and chronic (28 days) phases after injury. A significant increase in Y1R and Y2R expression, as well as a significant downregulation in GalR2 mRNA levels, was observed 1 day after SCI. Interestingly, PG early treatment prevented Y1R upregulation and resulted in lower NPY, Y2R and GalR1 mRNA levels. In the chronic phase, injured rats showed well-established mechanical and cold allodynia and significant increases in galanin, NPY, GalR1 and Y1R mRNAs, while maintaining reduced GalR2 expression. Animals receiving PG treatment showed basal expression levels of galanin, NPY, GalR1 and Y1R, and reduced Y2R mRNA levels. Also, and in line with previously published observations, PG-treated animals did not develop mechanical allodynia and showed reduced sensitivity to cold stimulation. Altogether, we show that SCI leads to considerable changes in the spinal expression of galanin, NPY and their associated receptors, and that early and sustained PG administration prevents them. Moreover, our data suggest the participation of galaninergic and NPYergic systems in the plastic changes associated with SCI-induced neuropathic pain, and further supports the therapeutic potential of PG- or neuropeptide-based therapies to prevent and/or treat chronic pain after central injuries.     

LIN28 Expression in Rat Spinal Cord After Injury

LIN28, an RNA-binding protein, is known to be involved in the regulation of many cellular processes, such as embryonic stem cell proliferation, cell fate succession, developmental timing, and oncogenesis. However, its expression and function in central nervous system still unclear. In this study, we performed an acute spinal cord contusion injury (SCI) model in adult rats and investigated the dynamic changes of LIN28 expression in spinal cord. Western blot and immunohistochemistry analysis revealed that LIN28 was present in normal spinal cord. It gradually increased, reached a peak at 3 day, and then nearly declined to the basal level at 14 days after SCI. Double immunofluorescence staining showed that LIN28 immunoreactivity was found in neurons, astrocytes and a handful of microglia. Interestingly, LIN28 expression was increased predominantly in astrocytes but not in neurons. Moreover, the colocalization of LIN28 and proliferating cell nuclear antigen was detected after injury. Western blot showed that LIN28 participated in lipopolysaccharide (LPS) induced astrocytes inflammatory responses by NF-κB signaling pathway. These results suggested that LIN28 may be involved in the pathologic process of SCI, and further research is needed to have a good understanding of its function and mechanism.