The role of mTOR signaling pathway in spinal cord injury

The mammalian target of rapamycin (mTOR) signaling pathway plays an important role in multiple cellular functions, such as cell metabolism, proliferation and survival. Many previous studies have shown that mTOR regulates both neuroprotective and neuroregenerative functions in trauma and various diseases in the central nervous system (CNS). Recently, we reported that inhibition of mTOR using rapamycin reduces neural tissue damage and locomotor impairment after spinal cord injury (SCI) in mice. Our results demonstrated that the administration of rapamycin at four hours after injury significantly increases the activity of autophagy and reduces neuronal loss and cell death in the injured spinal cord. Furthermore, rapamycin-treated mice show significantly better locomotor function in the hindlimbs following SCI than vehicle-treated mice. These findings indicate that the inhibition of mTOR signaling using rapamycin during the acute phase of SCI produces neuroprotective effects and reduces secondary damage at lesion sites. However, the role of mTOR signaling in injured spinal cords has not yet been fully elucidated. Various functions are regulated by mTOR signaling in the CNS, and multiple pathophysiological processes occur following SCI. Here, we discuss several unresolved issues and review the evidence from related articles regarding the role and mechanisms of the mTOR signaling pathway in neuroprotection and neuroregeneration after SCI.     

Effects of nerve graft on nitric oxide synthase, NAD(P)H oxidase, and antioxidant enzymes in chronic spinal cord injury

Oxidative stress and nitrosative stress play important roles in the pathogenesis of secondary spinal cord injury. Recently, we demonstrated that peripheral nerve grafts (PNG) with acidic fibroblast growth factor (aFGF) partially restore hind limb locomotion in adult rats with completely transected spinal cords. This study investigated the protein abundances of the superoxide (O2*)-generating enzyme nicotinamide adenine dinucleotide (phosphate) oxidase (NAD(P)H oxidase; gp91phox subunit), nitric oxide synthases (NOS), antioxidant enzymes, superoxide dismutases (Cu Zn SOD, Mn SOD), catalase, and glutathione peroxidase (GPX) as well as nitrotyrosine in the spinal cord tissue 4 months after spinal cord transection in rats with and without PNG and aFGF. The protein abundances of the gp91phox subunit of NAD(P)H oxidase, Mn SOD, catalase, GPX, eNOS, and nitrotyrosine were significantly upregulated, whereas Cu Zn SOD and nNOS were unchanged in the injury group compared to the sham controls. The nerve graft with aFGF treated group showed significantly better hind limb locomotion recovery than the injury group. Although the protein abundances of gp91phox, nitrotyrosine, and Cu Zn SOD were similar in the treated group (nerve graft with aFGF) compared to the injury group, Mn SOD, GPX, catalase, and eNOS protein abundances were significantly higher, whereas nNOS was markedly lower in the treated group. We conclude that the combination of nerve graft and aFGF enhances the local antioxidant defense system after spinal cord transection in rats.     

The role of mTOR signaling pathway in spinal cord injury

The mammalian target of rapamycin (mTOR) signaling pathway plays an important role in multiple cellular functions, such as cell metabolism, proliferation and survival. Many previous studies have shown that mTOR regulates both neuroprotective and neuroregenerative functions in trauma and various diseases in the central nervous system (CNS). Recently, we reported that inhibition of mTOR using rapamycin reduces neural tissue damage and locomotor impairment after spinal cord injury (SCI) in mice. Our results demonstrated that the administration of rapamycin at four hours after injury significantly increases the activity of autophagy and reduces neuronal loss and cell death in the injured spinal cord. Furthermore, rapamycin-treated mice show significantly better locomotor function in the hindlimbs following SCI than vehicle-treated mice. These findings indicate that the inhibition of mTOR signaling using rapamycin during the acute phase of SCI produces neuroprotective effects and reduces secondary damage at lesion sites. However, the role of mTOR signaling in injured spinal cords has not yet been fully elucidated. Various functions are regulated by mTOR signaling in the CNS, and multiple pathophysiological processes occur following SCI. Here, we discuss several unresolved issues and review the evidence from related articles regarding the role and mechanisms of the mTOR signaling pathway in neuroprotection and neuroregeneration after SCI.     

Autophagy Reduces Neuronal Damage and Promotes Locomotor Recovery via Inhibition of Apoptosis After Spinal Cord Injury in Rats

Autophagy is an intracellular catabolic mechanism that maintains the balance of proteins, lipids and aging organelles. 3-Methyladenine (3-MA) is a selective inhibitor of autophagy, whereas rapamycin, an antifungal agent, is a specific inducer of autophagy, inhibiting the protein mammalian target of rapamycin. In the present study, we examined the role of autophagy, inhibited by 3-MA and enhanced by rapamycin, in a model of acute spinal cord injury in rats. We found that rapamycin could significantly increase the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 at the injury site. At the same time, the number of neurons and astrocytes with LC3 positive in the spinal cord was upregulated with time. In addition, administration of rapamycin produced an increase in the Basso, Beattie and Bresnahan scores of injured rats, indicating high recovery of locomotor function. Furthermore, expression of the proteins Bcl-2 and Bax was upregulated and downregulated, respectively. By contrast, the results for rats treated with 3-MA, which inhibits autophagy, were the opposite of those seen with the rapamycin-treated rats. These results show that induction of autophagy can produce neuroprotective effects in acute spinal cord injury in rats via inhibition of apoptosis.     

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.     

Neuroprotective effect of Scutellaria baicalensis on spinal cord injury in rats

Inflammation has been known to play an important role in the pathogenesis after spinal cord injury (SCI). Microglia are activated after injury and produce a variety of proinflammatory factors such as tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2, and reactive oxygen species leading to apoptosis of neurons and oligodendrocytes. In this study, we examined the neuroprotective effects of total ethanol extract of Scutellaria baicalensis (EESB) , after SCI. Using primary microglial cultures, EESB treatment significantly inhibited lipopolysaccharide-induced expression of such inflammatory mediators as tumor necrosis factor-α, IL-1β, IL-6, cyclooxygenase-2, and inducible nitric oxide synthase. Furthermore, reactive oxygen species and nitric oxide production were significantly attenuated by EESB treatment. For in vivo study, rats that had received a moderate spinal cord contusion injury at T9 received EESB orally at a dose of 100 mg/kg. EESB inhibited expression of proinflammatory factors and protein carbonylation and nitration after SCI. EESB also inhibited microglial activation at 4 h after injury. Furthermore, EESB significantly inhibited apoptotic cell death of neurons and oligodendrocytes and improved functional recovery after SCI. Lesion cavity and myelin loss were also reduced following EESB treatment. Thus, our data suggest that EESB significantly improve functional recovery by inhibiting inflammation and oxidative stress after injury.     

Protective effects of glucosamine-kynurenic acid after compression-induced spinal cord injury in the rat

Kynurenic acid (KYNA), a metabolite of the essential amino acid L-tryptophan, is a broad spectrum antagonist of excitatory amino acid receptors, which have also anticonvulsant and neuroprotective properties. After spinal cord injury (SCI), excitotoxicity is considered to play a significant role in the processes of secondary tissue destruction in both grey and white matter of the spinal cord. In this study, we have tested the potential therapeutic effect of glucosamine-kynurenic acid, administered after experimental compression-induced SCI in the rat. Spinal application of glucosamine-kynurenic acid continually for 24 hr after experimental SCI resulted in improved motor function recovery, beginning from the first week of evaluation and continuing until the end of the study (4 weeks). After 4 weeks?? survival, quantitative morphometric analysis of the spinal cord showed that glucosamine-kynurenic acid treatment was associated with improved tissue preservation at the lesion site. These findings indicate that spinal application of glucosaminekynurenic acid is neuroprotective and improves the outcome even when administered after spinal trauma. Our results suggest that the treatments initiated in early posttraumatic period can alleviate secondary injury and improve the final outcome 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.     

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.     

Effects of combinatorial treatment with pituitary adenylate cyclase activating peptide and human mesenchymal stem cells on spinal cord tissue repair

The aim of this study is to understand if human mesenchymal stem cells (hMSCs) and neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) have synergistic protective effect that promotes functional recovery in rats with severe spinal cord injury (SCI). To evaluate the effect of delayed combinatorial therapy of PACAP and hMSCs on spinal cord tissue repair, we used the immortalized hMSCs that retain their potential of neuronal differentiation under the stimulation of neurogenic factors and possess the properties for the production of several growth factors beneficial for neural cell survival. The results indicated that delayed treatment with PACAP and hMSCs at day 7 post SCI increased the remaining neuronal fibers in the injured spinal cord, leading to better locomotor functional recovery in SCI rats when compared to treatment only with PACAP or hMSCs. Western blotting also showed that the levels of antioxidant enzymes, Mn-superoxide dismutase (MnSOD) and peroxiredoxin-1/6 (Prx-1 and Prx-6), were increased at the lesion center 1 week after the delayed treatment with the combinatorial therapy when compared to that observed in the vehicle-treated control. Furthermore, in vitro studies showed that co-culture with hMSCs in the presence of PACAP not only increased a subpopulation of microglia expressing galectin-3, but also enhanced the ability of astrocytes to uptake extracellular glutamate. In summary, our in vivo and in vitro studies reveal that delayed transplantation of hMSCs combined with PACAP provides trophic molecules to promote neuronal cell survival, which also foster beneficial microenvironment for endogenous glia to increase their neuroprotective effect on the repair of injured spinal cord tissue.     

Intrathecal Epigallocatechin Gallate Treatment Improves Functional Recovery After Spinal Cord Injury by Upregulating the Expression of BDNF and GDNF

This study aimed to investigate the therapeutic effects of epigallocatechin-3-gallate (EGCG) administered by subarachnoid injection following spinal cord injury (SCI) in rats and to explore the underlying mechanism. Sprague–Dawley rats were randomly divided into four groups of 12 as follows: a sham group (laminectomy only); a control group; a 10 mg/kg EGCG-treated group; and a 20 mg/kg EGCG-treated group. SCI was induced in the rats using the modified weight-drop method (10 g × 4 cm) at the T10 (10th thoracic vertebral) level. EGCG (10 or 20 mg/kg) or vehicle as control was administered by subarachnoid injection at lumbar level 4 immediately after SCI. Locomotor functional recovery was assessed during the four weeks post-operation using open-field locomotor tests and inclined-plane tests. At the end of the study, the segments of spinal cord encompassing the injury site were removed for histopathological analysis. Immunohistochemical and Western blot analyses were performed to observe the expression of: the B cell CLL/lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The results showed that the EGCG-treated animals had significantly better recovery of locomotor function, less myelin loss, greater Bcl-2 expression and attenuated Bax expression. In addition, the EGCG treatment significantly increased the expression of BDNF and GDNF after SCI. These findings suggest that EGCG treatment can significantly improve locomotor recovery, and this neuroprotective effect may be related to the up-regulation of BDNF and GDNF, and the inhibition of apoptosis-related proteins. Therefore, EGCG may be a promising therapeutic agent for SCI.     

Pyrroloquinoline quinone attenuates iNOS gene expression in the injured spinal cord

Pyrroloquinoline quinone (PQQ) is a naturally occurring redox cofactor that acts as an essential nutrient, antioxidant, and redox modulator. PQQ has been demonstrated to oxidize the redox modulatory site of N-methyl-d-aspartic acid (NMDA) receptors. Such agents are known to be neuroprotective in experimental stroke models. Therefore, we examined the possible ameliorating effect of PQQ on spinal cord injury (SCI) in adult rats. Intraperitoneal administration of PQQ effectively promoted the functional recovery of SCI rats after hemi-transection, which was preceded by the attenuation of the expression of inducible nitric oxide (NO) synthase (iNOS) mRNA in the injury site. NO is involved in the secondary detrimental mechanisms and has been implicated in NMDA receptor-mediated neurotoxicity. In fact, administration of PQQ induced significantly decreased lesion size and increased axon density adjoining the lesion area. These observations suggest that PQQ protects against the secondary damage by reducing iNOS expression following primary physical injury to the spinal cord.     

bFGF通过抑制Nogo-A信号减少脊髓损伤后胶质瘢痕形成

脊髓损伤后胶质瘢痕的形成是阻碍神经恢复的关键原因之一。碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）具有良好的神经保护及促进脊髓损伤的修复作用,然而其对于胶质瘢痕的影响及其机制仍不清楚。本研究通过采用血管动脉夹（30 g）夹闭雌性SD大鼠脊髓2 min造成急性脊髓损伤模型并予以每天皮下注射bFGF（80 μg/kg）,探讨bFGF促进脊髓损伤的恢复作用是否涉及到胶质瘢痕调控和Nogo-A/NgR信号的相关机制。通过检测损伤后28 d,各组BBB评分和斜板试验,发现bFGF显著促进脊髓损伤后大鼠运动功能的恢复。HE及尼氏染色显示,bFGF处理组相对于生理盐水处理组,其神经元明显增多,空洞面积减少。同时,星形胶质细胞标记物GFAP免疫荧光结果表明,bFGF减少胶质瘢痕形成,抑制星形胶质细胞过度激活。同样,通过Western 印迹检测发现,bFGF处理后,胶质瘢痕相关蛋白（如GFAP, neurocan）以及神经突生长抑制蛋白（Nogo-A）信号通路相关蛋白质表达量下降。上述结果表明,bFGF可能通过抑制Nogo-A信号蛋白的表达,从而抑制胶质瘢痕的形成,促进脊髓损伤的恢复。此机制研究为脊髓损伤的治疗和恢复提供全新的思路和药物靶点。     

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。     

Granulocyte Colony-Stimulating Factor (G-CSF) Protects Oligpdendrocyte and Promotes Hindlimb Functional Recovery after Spinal Cord Injury in Rats

Background

Granulocyte colony-stimulating factor (G-CSF) is a protein that stimulates differentiation, proliferation, and survival of cells in the granulocytic lineage. Recently, a neuroprotective effect of G-CSF was reported in a model of cerebral infarction and we previously reported the same effect in studies of murine spinal cord injury (SCI). The aim of the present study was to elucidate the potential therapeutic effect of G-CSF for SCI in rats.

Methods

Adult female Sprague-Dawley rats were used in the present study. Contusive SCI was introduced using the Infinite Horizon Impactor (magnitude: 200 kilodyne). Recombinant human G-CSF (15.0 µg/kg) was administered by tail vein injection at 1 h after surgery and daily the next four days. The vehicle control rats received equal volumes of normal saline at the same time points.

Results

Using a contusive SCI model to examine the neuroprotective potential of G-CSF, we found that G-CSF suppressed the expression of pro-inflammatory cytokine (IL-1 beta and TNF- alpha) in mRNA and protein levels. Histological assessment with luxol fast blue staining revealed that the area of white matter spared in the injured spinal cord was significantly larger in G-CSF-treated rats. Immunohistochemical analysis showed that G-CSF promoted up-regulation of anti-apoptotic protein Bcl-Xl on oligpodendrocytes and suppressed apoptosis of oligodendrocytes after SCI. Moreover, administration of G-CSF promoted better functional recovery of hind limbs.

Conclusions

G-CSF protects oligodendrocyte from SCI-induced cell death via the suppression of inflammatory cytokines and up-regulation of anti-apoptotic protein. As a result, G-CSF attenuates white matter loss and promotes hindlimb functional recovery.     

Delayed cell cycle pathway modulation facilitates recovery after spinal cord injury

Traumatic spinal cord injury (SCI) causes tissue loss and associated neurological dysfunction through mechanical damage and secondary biochemical and physiological responses. We have previously described the pathobiological role of cell cycle pathways following rat contusion SCI by examining the effects of early intrathecal cell cycle inhibitor treatment initiation or gene knockout on secondary injury. Here, we delineate changes in cell cycle pathway activation following SCI and examine the effects of delayed (24 h) systemic administration of flavopiridol, an inhibitor of major cyclin-dependent kinases (CDKs), on functional recovery and histopathology in a rat SCI contusion model. Immunoblot analysis demonstrated a marked upregulation of cell cycle-related proteins, including pRb, cyclin D1, CDK4, E2F1 and PCNA, at various time points following SCI, along with downregulation of the endogenous CDK inhibitor p27. Treatment with flavopiridol reduced induction of cell cycle proteins and increased p27 expression in the injured spinal cord. Functional recovery was significantly improved after SCI from day 7 through day 28. Treatment significantly reduced lesion volume and the number of Iba-1⁺ microglia in the preserved tissue and increased the myelinated area of spared white matter as well as the number of CC1⁺ oligodendrocytes. Furthermore, flavopiridol attenuated expression of Iba-1 and glactin-3, associated with microglial activation and astrocytic reactivity by reduction of GFAP, NG2, and CHL1 expression. Our current study supports the role of cell cycle activation in the pathophysiology of SCI and by using a clinically relevant treatment model, provides further support for the therapeutic potential of cell cycle inhibitors in the treatment of human SCI.     

Delayed cell cycle pathway modulation facilitates recovery after spinal cord injury

Traumatic spinal cord injury (SCI) causes tissue loss and associated neurological dysfunction through mechanical damage and secondary biochemical and physiological responses. We have previously described the pathobiological role of cell cycle pathways following rat contusion SCI by examining the effects of early intrathecal cell cycle inhibitor treatment initiation or gene knockout on secondary injury. Here, we delineate changes in cell cycle pathway activation following SCI and examine the effects of delayed (24 h) systemic administration of flavopiridol, an inhibitor of major cyclin-dependent kinases (CDKs), on functional recovery and histopathology in a rat SCI contusion model. Immunoblot analysis demonstrated a marked upregulation of cell cycle-related proteins, including pRb, cyclin D1, CDK4, E2F1 and PCNA, at various time points following SCI, along with downregulation of the endogenous CDK inhibitor p27. Treatment with flavopiridol reduced induction of cell cycle proteins and increased p27 expression in the injured spinal cord. Functional recovery was significantly improved after SCI from day 7 through day 28. Treatment significantly reduced lesion volume and the number of Iba-1⁺ microglia in the preserved tissue and increased the myelinated area of spared white matter as well as the number of CC1⁺ oligodendrocytes. Furthermore, flavopiridol attenuated expression of Iba-1 and glactin-3, associated with microglial activation and astrocytic reactivity by reduction of GFAP, NG2, and CHL1 expression. Our current study supports the role of cell cycle activation in the pathophysiology of SCI and by using a clinically relevant treatment model, provides further support for the therapeutic potential of cell cycle inhibitors in the treatment of human SCI.     

Epidermal growth factor attenuates blood‐spinal cord barrier disruption via PI3K/Akt/Rac1 pathway after acute spinal cord injury

After spinal cord injury (SCI), disruption of blood–spinal cord barrier (BSCB) elicits blood cell infiltration such as neutrophils and macrophages, contributing to permanent neurological disability. Previous studies show that epidermal growth factor (EGF) produces potent neuroprotective effects in SCI models. However, little is known that whether EGF contributes to the integrity of BSCB. The present study is performed to explore the mechanism of BSCB permeability changes which are induced by EGF treatment after SCI in rats. In this study, we demonstrate that EGF administration inhibits the disruption of BSCB permeability and improves the locomotor activity in SCI model rats. Inhibition of the PI3K/Akt pathways by a specific inhibitor, LY294002, suppresses EGF‐induced Rac1 activation as well as tight junction (TJ) and adherens junction (AJ) expression. Furthermore, the protective effect of EGF on BSCB is related to the activation of Rac1 both in vivo and in vitro. Blockade of Rac1 activation with Rac1 siRNA downregulates EGF‐induced TJ and AJ proteins expression in endothelial cells. Taken together, our results indicate that EGF treatment preserves BSCB integrity and improves functional recovery after SCI via PI3K‐Akt‐Rac1 signalling pathway.     

Retinoic Acid Induced-Autophagic Flux Inhibits ER-Stress Dependent Apoptosis and Prevents Disruption of Blood-Spinal Cord Barrier after Spinal Cord Injury

Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB) which leads to infiltration of blood cells, an inflammatory response, and neuronal cell death, resulting spinal cord secondary damage. Retinoic acid (RA) has a neuroprotective effect in both ischemic brain injury and SCI, however the relationship between BSCB disruption and RA in SCI is still unclear. In this study, we demonstrated that autophagy and ER stress are involved in the protective effect of RA on the BSCB. RA attenuated BSCB permeability and decreased the loss of tight junction (TJ) molecules such as P120, β-catenin, Occludin and Claudin5 after injury in vivo as well as in Brain Microvascular Endothelial Cells (BMECs). Moreover, RA administration improved functional recovery in the rat model of SCI. RA inhibited the expression of CHOP and caspase-12 by induction of autophagic flux. However, RA had no significant effect on protein expression of GRP78 and PDI. Furthermore, combining RA with the autophagy inhibitor chloroquine (CQ) partially abolished its protective effect on the BSCB via exacerbated ER stress and subsequent loss of tight junctions. Taken together, the neuroprotective role of RA in recovery from SCI is related to prevention of of BSCB disruption via the activation of autophagic flux and the inhibition of ER stress-induced cell apoptosis. These findings lay the groundwork for future translational studies of RA for CNS diseases, especially those related to BSCB disruption.