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.     

Sustained Calpain Inhibition Improves Locomotor Function and Tissue Sparing Following Contusive Spinal Cord Injury

Following contusive spinal cord injury (SCI), calpain activity is dramatically increased and remains elevated for days to weeks. Although calpain inhibition has previously been demonstrated to be neuroprotective following spinal cord injury, most studies administered the calpain inhibitor at a single time point. We hypothesized that sustained calpain inhibition would improve functional and pathological outcomes, as compared to the results obtained with a single postinjury administration of the calpain inhibitor. Contusion SCI was produced in female Long-Evans rats using the Infinite Horizon spinal cord injury impactor at the 200 kdyn force setting. Open-field locomotor function was evaluated until 6 weeks postinjury. Histological assessment of lesion volume and tissue sparing was performed at 6 weeks after SCI. Calpain inhibitor MDL28170 administered as a single postinjury i.v. bolus (20 mg/kg) or as a daily i.p. dose (1 mg/kg) improved locomotor function, but did not increase tissue sparing. Combined i.v. and daily i.p. MDL28170 administration resulted in significant improvement in both functional and pathological outcome measures, supporting the calpain theory of SCI proposed by Dr. Banik and colleagues. Special issue in honor of Naren Banik.     

Changes in NMDA receptor subunit expression in response to contusive spinal cord injury

Differential assembly of N-methyl-D-aspartate (NMDA) receptor subunits determines their functional characteristics. Using in situ hybridization, we found a selective increase of the subunits NR1 and NR2A mRNA at 24 h in ventral motor neurons (VMN) caudal to a standardized spinal cord contusion injury (SCI). Other neuronal cell populations and VMN rostral to the injury site appeared unaffected. Significant up-regulation of NR2A mRNA also was seen 1 month after SCI in thoracic and lumbar VMN. The selective effects on VMN caudal to the injury site suggest that the loss of descending innervation leads to increased NMDA receptor subunit expression in these cells after SCI, which may alter their responses to glutamate. In contrast, protein levels determined by western blot analysis show decreased levels of NR2A 1 month after SCI in whole thoracic segments of spinal cord that included the injury sites. No effects of injury were seen on subunit levels in cervical or lumbar segments. Taken together with our previous study showing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit down-regulation after injury, our data suggest that glutamate receptor composition is significantly altered after SCI. These changes need to be taken into account to properly understand the function of, and potential pharmacotherapy for, the chronically injured spinal cord.     

Omega-3 fatty acids and neurological injury

Studies with omega-3 polyunsaturated fatty acids (PUFA) have shown that these compounds have therapeutic potential in several indications in neurology and psychiatry. Acute spinal cord injury (SCI) is an event with devastating consequences, and no satisfactory treatment is available at present. The pathogenetic mechanisms associated with SCI include excitotoxicity, increased oxidation and inflammation. We review here our recent studies, which suggest that omega-3 PUFA have significant neuroprotective potential in spinal cord trauma. In a first study, we administered an intravenous bolus of alpha-linolenic acid (LNA) or docosahexaenoic acid (DHA) 30 min after spinal cord hemisection injury in adult rats. The omega-3 PUFA led to increased neuronal and glial survival, and a significantly improved neurological outcome. In subsequent studies, we tested DHA in a more severe compression model of SCI. We also explored a combined acute and chronic treatment regime using DHA. Saline or DHA was administered intravenously 30 min after compression of the spinal cord. After injury, the saline group received a standard control diet, whereas DHA-injected animals received either a control or a DHA-enriched diet for 6 weeks following injury. We assessed locomotor recovery and analysed markers for cell survival and axonal damage, and we also investigated the effects of the treatment on the inflammatory reaction and the oxidative stress that follow SCI. We showed that the acute DHA treatment is neuroprotective after compression SCI, even if the treatment is delayed up to an hour after injury. The DHA injection led to an increased neuronal and glial cell survival, and the effect of the DHA injection was amplified by addition of DHA to the diet. Rats treated with a DHA injection and a DHA-enriched diet performed significantly better at 6 weeks in terms of neurological outcome. The analysis of the tissue after DHA administration showed that the fatty acid significantly reduced lipid peroxidation, protein oxidation and RNA/DNA oxidation, and the induction of COX-2. Parallel studies in a facial nerve injury model in mice also showed pro-regenerative effects of chronic dietary administration of DHA after nerve lesion. These observations suggest that treatment with omega-3 PUFA could represent a promising therapeutic approach in the management of neurological injury.     

Involvement of acidic fibroblast growth factor in spinal cord injury repair processes revealed by a proteomics approach

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01-0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.     

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.     

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.     

P2X7 receptor inhibition improves recovery after spinal cord injury

Secondary injury exacerbates the extent of spinal cord insults, yet the mechanistic basis of this phenomenon has largely been unexplored. Here we report that broad regions of the peritraumatic zone are characterized by a sustained process of pathologic, high ATP release. Spinal cord neurons expressed P2X7 purine receptors (P2X7R), and exposure to ATP led to high-frequency spiking, irreversible increases in cytosolic calcium and cell death. To assess the potential effect of P2X7R blockade in ameliorating acute spinal cord injury (SCI), we delivered P2X7R antagonists OxATP or PPADS to rats after acute impact injury. We found that both OxATP and PPADS significantly improved functional recovery and diminished cell death in the peritraumatic zone. These observations demonstrate that SCI is associated with prolonged purinergic receptor activation, which results in excitotoxicity-based neuronal degeneration. P2X7R antagonists inhibit this process, reducing both the histological extent and functional sequelae of acute SCI.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cells Mesenchymal stem cells in the treatment of spinal cord injuries:A review

With technological advances in basic research,the intricate mechanism of secondary delayed spinal cord injury(SCI)continues to unravel at a rapid pace.However,despite our deeper understanding of the molecular changes occurring after initial insult to the spinal cord,the cure for paralysis remains elusive.Current treatment of SCI is limited to early administration of high dose steroids to mitigate the harmful effect of cord edema that occurs after SCI and to reduce the cascade of secondary delayed SCI.R ecent evident-based clinical studies have cast doubt on the clinical benefit of steroids in SCI and intense focus on stem cell-based therapy has yielded some encouraging results.An array of mesenchymal stem cells(MSCs)from various sources with novel and promising strategies are being developed to improve function after SCI.In this review,we briefly discuss the pathophysiology of spinal cord injuries and characteristics and the potential sources of MSCs that can be used in the treatment of SCI.We will discuss the progress of MSCs application in research,focusing on the neuroprotective properties of MSCs.Finally,we will discuss the results from preclinical and clinical trials involving stem cell-based therapy in SCI.     

Systemic bisperoxovanadium activates Akt/mTOR, reduces autophagy, and enhances recovery following cervical spinal cord injury

Secondary damage following primary spinal cord injury extends pathology beyond the site of initial trauma, and effective management is imperative for maximizing anatomical and functional recovery. Bisperoxovanadium compounds have proven neuroprotective effects in several central nervous system injury/disease models, however, no mechanism has been linked to such neuroprotection from bisperoxovanadium treatment following spinal trauma. The goal of this study was to assess acute bisperoxovanadium treatment effects on neuroprotection and functional recovery following cervical unilateral contusive spinal cord injury, and investigate a potential mechanism of the compound's action. Two experimental groups of rats were established to 1) assess twice-daily 7 day treatment of the compound, potassium bisperoxo (picolinato) vanadium, on long-term recovery of skilled forelimb activity using a novel food manipulation test, and neuroprotection 6 weeks following injury and 2) elucidate an acute mechanistic link for the action of the drug post-injury. Immunofluorescence and Western blotting were performed to assess cellular signaling 1 day following SCI, and histochemistry and forelimb functional analysis were utilized to assess neuroprotection and recovery 6 weeks after injury. Bisperoxovanadium promoted significant neuroprotection through reduced motorneuron death, increased tissue sparing, and minimized cavity formation in rats. Enhanced forelimb functional ability during a treat-eating assessment was also observed. Additionally, bisperoxovanadium significantly enhanced downstream Akt and mammalian target of rapamycin signaling and reduced autophagic activity, suggesting inhibition of the phosphatase and tensin homologue deleted on chromosome ten as a potential mechanism of bisperoxovanadium action following traumatic spinal cord injury. Overall, this study demonstrates the efficacy of a clinically applicable pharmacological therapy for rapid initiation of neuroprotection post-spinal cord injury, and sheds light on the signaling involved in its action.     

Minocycline neuroprotects, reduces microgliosis, and inhibits caspase protease expression early after spinal cord injury

Minocycline, a clinically used tetracycline for over 40 years, crosses the blood-brain barrier and prevents caspase up-regulation. It reduces apoptosis in mouse models of Huntington's disease and familial amyotrophic lateral sclerosis (ALS) and is in clinical trial for sporadic ALS. Because apoptosis also occurs after brain and spinal cord (SCI) injury, its prevention may be useful in improving recovery. We analyzed minocycline's neuroprotective effects over 28 days following contusion SCI and found significant functional recovery compared to tetracycline. Histology, immunocytochemistry, and image analysis indicated statistically significant tissue sparing, reduced apoptosis and microgliosis, and less activated caspase-3 and substrate cleavage. Since our original report in abstract form, others have published both positive and negative effects of minocycline in various rodent models of SCI and with various routes of administration. We have since found decreased tumor necrosis factor-alpha, as well as caspase-3 mRNA expression, as possible mechanisms of action for minocycline's ameliorative action. These results support reports that modulating apoptosis, caspases, and microglia provide promising therapeutic targets for prevention and/or limiting the degree of functional loss after CNS trauma. Minocycline, and more potent chemically synthesized tetracyclines, may find a place in the therapeutic arsenal to promote recovery early after SCI in humans.     

NK1 Receptor Blockade Is Ineffective in Improving Outcome following a Balloon Compression Model of Spinal Cord Injury

The neuropeptide substance P (SP) is a well-known mediator of neurogenic inflammation following a variety of CNS disorders. Indeed, inhibition of SP through antagonism of its receptor, the tachykinin NK1 receptor, has been shown to be beneficial following both traumatic brain injury and stroke. Such studies demonstrated that administration of an NK1 receptor antagonist reduced blood-brain-barrier permeability, edema development and improved functional outcome. Furthermore, our recent studies have demonstrated a potential role for SP in mediating neurogenic inflammation following traumatic spinal cord injury (SCI). Accordingly, the present study investigates whether inhibition of SP may similarly play a neuroprotective role following traumatic SCI. A closed balloon compression injury was induced at T10 in New Zealand White rabbits. At 30 minutes post-injury an NK1 receptor antagonist was administered intravenously. Animals were thereafter assessed for blood spinal cord barrier (BSCB) permeability, spinal water content (edema), intrathecal pressure (ITP), and histological and functional outcome from 5 hours to 2 weeks post-SCI. Administration of an NK1 receptor antagonist was not effective in reducing BSCB permeability, edema, ITP, or functional deficits following SCI. We conclude that SP mediated neurogenic inflammation does not seem to play a major role in BSCB disruption, edema development and consequential tissue damage seen in acute traumatic SCI. Rather it is likely that the severe primary insult and subsequent hemorrhage may be the key contributing factors to ongoing SCI injury.     

药物及靶向治疗在脊髓损伤中的治疗新进展

脊髓损伤(spinalcordinjury,SCI)是一种严重的损伤,它对患者的影响是相当持久的,SCI治疗的难点主要是由于损伤后脊髓中的微环境不利于神经细胞的再生、轴突的生长和新突触的形成,从而影响了脊髓组织的修复。现在SCI治疗的策略就是要改善损伤脊髓微环境,减少不利因素,从而促进脊髓结构修复和功能重建。本研究综述近年来逐渐发展起来的药物及靶向治疗方法,为SCI的新治疗提供参考依据,真正提高患者的生活质量。     

Oncostatin M Reduces Lesion Size and Promotes Functional Recovery and Neurite Outgrowth After Spinal Cord Injury

The family of interleukin (IL)-6 like cytokines plays an important role in the neuroinflammatory response to injury by regulating both neural as well as immune responses. Here, we show that expression of the IL-6 family member oncostatin M (OSM) and its receptor is upregulated after spinal cord injury (SCI). To reveal the relevance of increased OSM signaling in the pathophysiology of SCI, OSM was applied locally after spinal cord hemisection in mice. OSM treatment significantly improved locomotor recovery after mild and severe SCI. Improved recovery in OSM-treated mice was associated with a reduced lesion size. OSM significantly diminished astrogliosis and immune cell infiltration. Thus, OSM limits secondary damage after CNS trauma. In vitro viability assays demonstrated that OSM protects primary neurons in culture from cell death, suggesting that the underlying mechanism involves direct neuroprotective effects of OSM. Furthermore, OSM dose-dependently promoted neurite outgrowth in cultured neurons, indicating that the cytokine plays an additional role in CNS repair. Indeed, our in vivo experiments demonstrate that OSM treatment increases plasticity of serotonergic fibers after SCI. Together, our data show that OSM is produced at the lesion site, where it protects the CNS from further damage and promotes recovery.     

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.     

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.     

KU0063794, a Dual mTORC1 and mTORC2 Inhibitor,Reduces Neural Tissue Damage and Locomotor Impairment After Spinal Cord Injury in Mice

Autophagy is an intracellular catabolic mechanism for the degradation of cytoplasmic constituents in the autophagosomal–lysosomal pathway. This mechanism plays an important role in homeostasis and it is defective in certain diseases. Preceding studies have revealed that autophagy is developing as an important moderator of pathological responses associated to spinal cord injury (SCI) and plays a crucial role in secondary injury initiating a progressive degeneration of the spinal cord. Thus, based on this evidence in this study, we used two different selective inhibitors of mTOR activity to explore the functional role of autophagy in an in vivo model of SCI as well as to determine whether the autophagic process is involved in spinal cord tissue damage. We treated animals with a novel synthetic inhibitor temsirolimus and with a dual mTORC1 and mTORC2 inhibitor KU0063794 matched all with the well-known inhibitor of mTOR the rapamycin. Our results demonstrated that mTOR inhibitors could regulate the neuroinflammation associated to SCI and the results that we obtained evidently demonstrated that rapamycin and temsirolimus significantly diminished the expression of iNOS, COX2, GFAP, and re-established nNOS levels, but the administration of KU0063794 is able to blunt the neuroinflammation better than rapamycin and temsirolimus. In addition, neuronal loss and cell mortality in the spinal cord after injury were considerably reduced in the KU0063794-treated mice. Accordingly, taken together our results denote that the administration of KU0063794 produced a neuroprotective function at the lesion site following SCI, representing a novel therapeutic approach after SCI.     

Acute leptin treatment enhances functional recovery after spinal cord injury

Background

Spinal cord injury is a major cause of long-term disability and has no current clinically accepted treatment. Leptin, an adipocyte-derived hormone, is best known as a regulator of food intake and energy expenditure. Interestingly, several studies have demonstrated that leptin has significant effects on proliferation and cell survival in different neuropathologies. Here, we sought to evaluate the role of leptin after spinal cord injury.

Findings

Based on its proposed neuroprotective role, we have evaluated the effects of a single, acute intraparenchymal injection of leptin in a clinically relevant animal model of spinal cord injury. As determined by quantitative Real Time-PCR, endogenous leptin and the long isoform of the leptin receptor genes show time-dependent variations in their expression in the healthy and injured adult spinal cord. Immunohistochemical analysis of post-injury tissue showed the long isoform of the leptin receptor expression in oligodendrocytes and, to a lesser extent, in astrocytes, microglia/macrophages and neurons. Moreover, leptin administered after spinal cord injury increased the expression of neuroprotective genes, reduced caspase-3 activity and decreased the expression of pro-inflammatory molecules. In addition, histological analysis performed at the completion of the study showed that leptin treatment reduced microglial reactivity and increased caudal myelin preservation, but it did not modulate astroglial reactivity. Consequently, leptin improved the recovery of sensory and locomotor functioning.

Conclusions

Our data suggest that leptin has a prominent neuroprotective and anti-inflammatory role in spinal cord damage and highlights leptin as a promising therapeutic agent.     

Real-time quantitative PCR analysis of temporal-spatial alterations in gene expression after spinal cord contusion

Rat spinal cord contusion injury models the histopathology associated with much clinical spinal cord injury (SCI). Studies on altered gene expression after SCI in these models may identify therapeutic targets for reducing secondary injury after the initial trauma and/or enhancing recovery processes. However, complex spatial and temporal alterations after injury could complicate interpretation of changes in gene expression. To test this hypothesis, we selected six genes and studied their temporal and spatial patterns of expression at 1 h, 1, 3 and 7 days after a standardized spinal cord contusion produced by a weight drop device (10 g x 25 mm at T8). Real-time RT-PCR using TaqMan probes was employed to quantify mRNA for proteolipid protein, glyceraldehyde-3-phosphate dehydrogenase, glial fibrillary acidic protein, nestin, and the GluR2 and NR1 subunits of glutamate receptors. We found widely different temporal and spatial patterns of altered gene expression after SCI, including instances of opposing up- and down-regulation at different locations in tissue immediately adjacent to the injury site. We conclude that greater use of the reliable and extremely sensitive technique of quantitative real-time PCR for regional tissue analysis is important for understanding the altered gene expression that occurs after CNS trauma.     

应用cDNA微阵列技术筛选大鼠脊髓损伤修复相关基因

脊髓损伤是一类常见的、高致残率的中枢神经系统疾病,由于多种复杂因素影响其损伤后的修复过程,损伤脊髓的再生能力非常有限。本研究采用cDNA微阵列技术筛选大鼠脊髓损伤后出现的差异表达基因。实验组动物在T8-T9进行脊髓全横断手术,对照组动物只打开椎板;4．5d后取脊髓进行RNA提取并在反转录过程中进行Cy3／Cy5标记,然后与预制的、带有4041条特异性探针的芯片进行杂交。Cy5／Cy3信号比值≥2．0视为脊髓损伤后出现差异表达的基因。通过筛选,我们得到了65个上调表达基因（21个已知基因,30个已知EST和14个未知基因）和79个下调基因（20个已知基因,42个已知EST和17个未知基因）。进一步通过半定量RT-PCR对其中的5个上调已知基因（Timpl,Tagln,Vim,Fc gamma receptor,Ctss）和三个下调已知基因（stearyl-CoA desaturase,F2,Ensa）的表达情况进行了验证,结果显示与芯片结果一致。这些基因可能在脊髓损伤后的修复过程中起一定的作用,对其深入研究将有助于揭示脊髓损伤修复的分子机制。