Biomarkers in Spinal Cord Injury: from Prognosis to Treatment

Spinal cord injury (SCI) is considered an incurable condition, having a heterogenous recovery and uncertain prognosis. Therefore, a reliable prediction of the improvement in the acute phase could benefit patients. Physicians are unanimous in insisting that at the initial damage of the spinal cord (SC), the patient should be carefully evaluated in order to help selecting an appropriate neuroprotective treatment. However, currently, neurologic impairment after SCI is measured and classified by functional examination. The identification of prognostic biomarkers of SCI would help to designate SC injured patients and correlate to diagnosis and correct treatment. Some proteins have already been identified as good potential biomarkers of central nervous system injury, both in cerebrospinal fluid (CSF) and blood serum. However, the problem for using them as biomarkers is the way they should be collected, as acquiring CSF through a lumbar puncture is significantly invasive. Remarkably, microRNAs (miRNAs) have emerged as interesting biomarker candidates because of their stability in biological fluids and their tissue specificity. Several miRNAs have been identified to have their expressions altered in SCI in many animal models, making them promising candidates as biomarkers after SCI. Moreover, there are yet no effective therapies for SCI. It is already known that altered lysophospholipids (LPs) signaling are involved in the biology of disorders, such as inflammation. Reports have demonstrated that LPs when locally distributed can regulate SCI repair and key secondary injury processes such as apoptosis and inflammation, and so could become in the future new therapeutic approaches for treating SCI.     

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.     

The relationships among the severity of spinal cord injury,motor and somatosensory evoked potentials and spinal cord blood flow

To characterize the changes in axonal function in the motor and somatosensory tracts of the cord after spinal cord injury (SCI) and to correlate these changes with spinal cord blood flow (SCBF), the relationships among the severity of SCI, motor and somatosensory evoked potentials (MEPs and SSEPs) and SCBF were examined. Fifteen rats received a 1.5 g (n = 5), 20 g (n = 5) or 56 g (n = 5) clip compression injury of the cord at C8. SCBF at the injury site was measured by the hydrogen clearance technique 35 min before and 30 min after SCI. Concomitantly MEPs from the cord at T10 (MEP-C) and from the sciatic nerve (MEP-N) and SSEPs were recorded.A linear relationship (r = −0.89, P < 0.002) was found between the severity of SCI and the reduction in SCBF at the injury site. Linear discriminant analysis revealed that both the MEP (P < 0.0001) and SSEP (P < 0.003) were significantly related to the severity of SCI. Furthermore, the amplitude of the MEP (r = 0.65, P < 0.0001) and SSEP (r = 0.58, P < 0.0011) was significantly correlated with the posttraumatic SCBF. Multiple regression revealed that both the severity of cord injury and the degree of posttraumatic ischemia were significantly related to axonal dysfunction after SCI. While the MEP was more sensitive to injury than the SSEP, the SSEP more accurately distinguished between mild and moderate severities of cord injury.Axonal conduction in the motor and somatosensory tracts of the cord was significantly correlated with the reduction in posttraumatic SCBF and, therefore, these data provide quantitative evidence linking posttraumatic ischemia to axonal dysfunction following acute cord injury. Furthermore, this study validates the hypothesis that the combined recording of MEPs and SSEPs is an accurate technique to assess the physiological integrity of the cord after injury.     

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.¹ Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5° (animals rotated at 22.5°), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya''s staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.     

Chondroitinase ABC treatment and the phenotype of neural progenitor cells isolated from injured rat spinal cord

The aim of the present study was to investigate whether enzyme chondroitinase ABC (ChABC) treatment influences the phenotype of neural progenitor cells (NPCs) derived from injured rat spinal cord. Adult as well as fetal spinal cords contain a pool of endogenous neural progenitors cells, which play a key role in the neuroregenerative processes following spinal cord injury (SCI) and hold particular promise for therapeutic approaches in CNS injury or neurodegenerative disorders. In our study we used in vitro model to demonstrate the differentiation potential of NPCs isolated from adult rat spinal cord after SCI, treated with ChABC. The intrathecal delivery of ChABC (10 U/ml) was performed at day 1 and 2 after SCI. The present findings indicate that the impact of SCI resulted in a decrease of all NPCs phenotypes and the ChABC treatment, on the contrary, caused an opposite effect.     

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。     

Effects of retrograde gene transfer of brain-derived neurotrophic factor in the rostral spinal cord of a compression model in rat

Recovery after spinal cord injury (SCI) is rare in humans and experimental animals. Following SCI in adults, changes in gene expression and the regulation of these genes are associated with the pathological development of the injury. High levels of brain-derived neurotrophic factor (BDNF) in the injury area during the post-injury period contribute to enhanced neuroprotection and axonal regeneration. Intervention at the level of gene regulation has the potential to promote SCI repair. In this study, the injection of adenovirus-mediated BDNF in the lesion area (rostral spinal cord) up-regulated the expression of BDNF in the injury zone of a compression model in rat, thereby protecting neurons and enhancing behavioral function.     

Mesenchymal Stem Cells from Rat Bone Marrow Downregulate Caspase-3-mediated Apoptotic Pathway After Spinal Cord Injury in Rats

Mesenchymal stem cells have been intensively studied for their potential use in reparative strategies for neurodegenerative diseases and traumatic injuries. We used mesenchymal stem cells (rMSC) from rat bone marrow to evaluate the therapeutic potential after spinal cord injury (SCI). Immunohistochemistry confirmed a large number of apoptotic neurons and oligodendrocytes in caudal segments 2 mm away from the lesion site. Expression of caspase-3 on both neurons and oligodendrocytes after SCI was significantly downregulated by rMSC. Caspase-3 downregulation by rMSC involves increased expression of FLIP and XIAP in the cytosol and inhibition of PARP cleavage in the nucleus. Animals treated with rMSC had higher Basso, Beattie, Bresnahan (BBB) locomotor scoring and better recovery of hind limb sensitivity. Treatment with rMSC had a positive effect on behavioral outcome and histopathological assessment after SCI. The ability of rMSC to incorporate into the spinal cord, differentiate and to improve locomotor recovery hold promise for a potential cure after SCI. Special issue in honor of Naren Banik.     

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。     

Tauroursodeoxycholic acid alleviates secondary injury in the spinal cord via up-regulation of CIBZ gene

Spinal cord injury (SCI) is generally divided into primary and secondary injuries, and apoptosis is an important event of the secondary injury. As an endogenous bile acid and recognized endoplasmic reticulum (ER) stress inhibitor, tauroursodeoxycholic acid (TUDCA) administration has been reported to have a potentially therapeutic effect on neurodegenerative diseases, but its real mechanism is still unclear. In this study, we evaluated whether TUDCA could alleviate traumatic damage of the spinal cord and improve locomotion function in a mouse model of SCI. Traumatic SCI mice were intraperitoneally injected with TUDCA, and the effects were evaluated based on motor function assessment, histopathology, apoptosis detection, qRT-PCR, and western blot at different time periods. TUDCA administration can improve motor function and reduce secondary injury and lesion area after SCI. Furthermore, the apoptotic ratios were significantly reduced; Grp78, Erdj4, and CHOP were attenuated by the treatment. Unexpectedly, the levels of CIBZ, a novel therapeutic target for SCI, were specifically up-regulated. Taken together, it is suggested that TUDCA effectively suppressed ER stress through targeted up-regulation of CIBZ. This study also provides a new strategy for relieving secondary damage by inhibiting apoptosis in the early treatment of spinal cord injury.     

Glial and axonal regeneration following spinal cord injury

Spinal cord injury (SCI) has been regarded clinically as an irreversible damage caused by tissue contusion due to a blunt external force. Past research had focused on the analysis of the pathogenesis of secondary injury that extends from the injury epicenter to the periphery, as well as tissue damage and neural cell death associated with secondary injury. Recent studies, however, have proven that neural stem (progenitor) cells are also present in the brain and spinal cord of adult mammals including humans. Analyses using spinal cord injury models have also demonstrated active dynamics of cells expressing several stem cell markers, and methods aiming at functional reconstruction by promoting the potential self-regeneration capacity of the spinal cord are being explored. Furthermore, reconstruction of the neural circuit requires not only replenishment or regeneration of neural cells but also regeneration of axons. Analysis of the tissue microenvironment after spinal cord injury and research aiming to remove axonal regeneration inhibitors have also made progress. SCI is one of the simplest central nervous injuries, but its pathogenesis is associated with diverse factors, and further studies are required to elucidate these complex interactions in order to achieve spinal cord regeneration and functional reconstruction.     

Extremely low-frequency electromagnetic fields: A possible non-invasive therapeutic tool for spinal cord injury rehabilitation

Traumatic insults to the spinal cord induce both immediate mechanical damage and subsequent tissue degeneration. The latter involves a range of events namely cellular disturbance, homeostatic imbalance, ionic and neurotransmitters derangement that ultimately result in loss of sensorimotor functions. The targets for improving function after spinal cord injury (SCI) are mainly directed toward limiting these secondary injury events. Extremely low-frequency electromagnetic field (ELF-EMF) is a possible non-invasive therapeutic intervention for SCI rehabilitation which has the potential to constrain the secondary injury-induced events. In the present review, we discuss the effects of ELF-EMF on experimental and clinical SCI as well as on biological system.     

Long-Term Changes in Spinal Cord Evoked Potentials After Compression Spinal Cord Injury in the Rat

SUMMARY 1. After traumatic spinal cord injury (SCI), histological and neurological consequences are developing for several days and even weeks. However, little is known about the dynamics of changes in spinal axonal conductivity. The aim of this study was to record and compare repeated spinal cord evoked potentials (SCEP) after SCI in the rat during a 4 weeks’ interval. These recordings were used: (i) for studying the dynamics of functional changes in spinal axons after SCI, and (ii) to define the value of SCEP as an independent outcome parameter in SCI studies.2. We have used two pairs of chronically implanted epidural electrodes for stimulation/recording. The electrodes were placed below and above the site of injury, respectively. Animals with implanted electrodes underwent spinal cord compression injury induced by epidural balloon inflation at Th8–Th9 level. There were five experimental groups of animals, including one control group (sham-operated, no injury), and four injury groups (different degrees of SCI).3. After SCI, SCEP waveform was either significantly reduced or completely lost. Partial recovery of SCEPs was observed in all groups. The onset and extent of recovery clearly correlated with the severity of injury.There was good correlation between quantitated SCEP variables and the volumes of the compressing balloon. However, sensitivity of electropohysiological parameters was inferior compared to neurological and morphometric outcomes.4. Our study shows for the first time, that the dynamics of axonal recovery depends on the degree of injury. After mild injury, recovery of signal is rapid. However, after severe injury, axonal conductivity can re-appear after as long as 2 weeks postinjury.In conclusion, SCEPs can be used as an independent parameter of outcome after SCI, but in general, the sensitivity of electrophysiological data were worse than standard morphological and neurological evaluations.     

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.     

Increased expression of nitric oxide synthase interacting protein (NOSIP) following traumatic spinal cord injury in rats

Previous studies indicated that nitric oxide (NO) is involved in secondary damage of spinal cord injury (SCI), which worsens the primary physical injury to the central nervous systems. Recently, nitric oxide synthase interacting protein (NOSIP) has been identified to interact with neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase by inhibiting the NO production. However, its expression and function after a central nervous system injury remains unclear. In this study, we examined the expression and cellular localization of NOSIP in the spinal cord of an adult rat. Western blot analysis indicated that NOSIP protein levels increased at day1 post-injury and peaked at day 14. Double immunofluorescence staining showed that NOSIP was primarily expressed in neurons and glial cells in the intact spinal cord. Interestingly, this study also showed that the expression of NOSIP significantly increased in astrocytes after injury. Furthermore, injury-induced expression of NOSIP was co-expressed with proliferating cell nuclear antigen (PCNA) positive astrocytes after injury. We also showed the NOSIP was co-localized with nNOS in gray matter and white matter after SCI. All these data taken together suggested that NOSIP may play an important roles in astrogliogenesis after a spinal cord injury.