Spatiotemporal Profile and Essential Role of RBM3 Expression after Spinal Cord Injury in Adult Rats

Hypoxia and other adverse conditions are usually encountered by rapidly growing cells. The RNA-binding motif protein 3 (RBM3) is induced by low temperature and hypoxia. However, its expression and function in spinal cord injury are still unclear. To investigate the certain expression and biological function in the central nervous system, we performed an acute spinal cord contusion injury (SCI) model in adult rats. Western blot analysis indicated a striking expression upregulation of RBM3 after spinal cord injury (SCI). Double immunofluorescence staining prompted that RBM3 immunoreactivity was found in astrocytes and neurons. Interestingly, RBM3 expression was increased predominantly in astrocytes. Furthermore, colocalization of RBM3 with proliferating cell nuclear antigen (PCNA) was detected in astrocytes. To further understand whether RBM3 plays a role in astrocyte proliferation, we applied lipopolysaccharide (LPS) to induce astrocyte proliferation in vitro. Western blot analysis demonstrated that RBM3 expression was positively correlated with PCNA expression following LPS stimulation. Immunofluorescence analysis showed that the expression of RBM3 was also changed following the stimulation of astrocytes with LPS, which was parallel with the data in vivo. Additionally, knocking RBM3 down with small interfering RNA (siRNA) demonstrated that RBM3 might play a significant role in the proliferation of astrocytes treated by hypoxia in vitro. These results suggest that RBM3 may be involved in the proliferation of astrocytes after SCI. To summarize, we firstly uncover the temporal and spatial expression changes of RBM3 in spinal cord injury. Our data suggest that RBM3 might be implicated in central nervous system pathophysiology after SCI.     

Expression of CAPON after Spinal Cord Injury in Rats

The adaptor protein, carboxy-terminal PDZ ligand of nNOS (CAPON), regulates the distribution of neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI) and produces the key signaling molecule nitric oxide (NO). But little is known about the role of CAPON in the pathological process of SCI. The main objective of the present study was to investigate expression of CAPON and nNOS in a spinal cord contusion model in adult rats. Real time-polymerase chain reaction (PCR) and Western blot analysis revealed that mRNA and protein for CAPON increased at 2 h after SCI and reached the peak at 8 h, gradually recovered to the baseline level at 14 days. The expression of nNOS mRNA and protein was similar to that of CAPON. During the peak expression, CAPON mRNA was found in the ventral horn, mediate zone, dorsal horn, and white matter by in situ hybridization. Immunofluorescence showed that CAPON was colocalized with nNOS in neurons, oligodendrocytes, and some astrocytes of spinal cord tissues within 5 mm from the epicenter. Interaction between CAPON and nNOS was also detected by co-immunoprecipitation. Thus, the transient expression of high levels of CAPON may provide new insight into the secondary response after SCI. Chun Cheng and Xin Li contributed equally to this work.     

Temporal and Spatial Expression of Cyclin H in Rat Spinal Cord Injury

Cyclin H regulates cell cycle transitions; it always forms trimeric cyclin-dependent protein kinases (CDK)-activating kinase (CAK) complex with CDK7 and MAT1 that phosphorylates a threonine residue in the CDK2 T loop region. However, neither the expression nor function of cyclin H in the central nervous system (CNS) injury is still clear. Therefore, we studied cyclin H in a rat spinal cord contusion model. Injury markedly increased cyclin H protein expression throughout the thoracic spinal cord but did not increase CDK7. However, double immunofluorescent staining for proliferating cell nuclear antigen (PCNA) and cell markers revealed increases of cyclin H and CDK2 in proliferating microglia and astrocytes, and the co-immunoprecipitation studies shown that the associations of cyclin H with CDK2 were enhanced evidently after injury. Our data suggest that cyclin H may play a proliferative role in spinal cord injury (SCI).     

依达拉奉对大鼠急性脊髓损伤后神经细胞凋亡的影响

目的:观察依达拉奉对大鼠急性脊髓损伤后细胞凋亡的影响,探讨脊髓保护的作用机制。方法:120只SD雄性大鼠,Allen法建立脊髓损伤模型,随机分为依达拉奉组、假手术组和对照组（均n=40）。依达拉奉组给予依达拉奉10mg·kg^-1,每日2次腹腔注射,连续6d;对照组给予等量同次数的生理盐水腹腔灌注;假手术组仅行椎板切除,不损伤脊髓,不给药。在伤后第1、7、14、28天观察各组大鼠活动情况行BBB评分,取受伤脊髓节段检测抑制羟自由基能力、caspase-3蛋白表达、原位脱氧糖核苷酸末端转移酶介导的原位末端标记法（TUNEL法）标记凋亡细胞。结果:依达拉奉组抑制羟自由基能力明显高于假手术组和对照组（P<0.05）;依达拉奉组caspase-3与对照组比各时间点的表达明显降低（P<0.05）;依达拉奉组与对照组比各时间点凋亡细胞显著减少（P<0.05）。结论:依达拉奉能减少急性脊髓损伤部位的羟自由基,下调caspase-3表达,抑制脊髓神经细胞凋亡,对继发性脊髓损伤有保护作用。     

An Upregulation of SIAH1 After Spinal Cord Injury in Adult Rats

E3 ubiquitin ligase SIAH1 is a protein associated with the onset of nontumorigenicy in revertant tumorigenic cell lines and with several apoptotic processes. However, its role in the injury of the central nervous system remains unknown. In this study, we performed acute spinal cord injury (SCI) in adult rats and investigated the protein expression and cellular localization of SIAH1 in the spinal cord. Western blot analysis revealed that SIAH1 was low expressed in normal spinal cord. It increased at 8 h after SCI, peaked at 1 day, remained for another 3 days, then declined to basal levels at 5 days after injury. Immunohistochemistry further confirmed that SIAH1 immunoactivity was expressed at low levels in gray and white matters in normal condition and increased after SCI. Double immunofluorescence staining showed that SIAH1 was coexpressed with NeuN (neuronal marker), CNPase (oligodendroglial marker), GFAP (astroglial marker), and CD11b (microglial marker) at 1 day post-injury and was also coexpressed with active caspase-3 in neurons and glial cells after injury. In addition, double immunofluorescence staining indicated that p-c-Jun NH2-kinase (JNK) coexpressed with SIAH1 in neurons and glial cells. Coimmunoprecipitation further showed that p-JNK and SIAH1 precipitated with each other in the damaged spinal cord. Taken together, these data suggest SIAH1 involvement in the injury response of the adult spinal cord of the rats.     

Gait Analysis of Adult Paraplegic Rats after Spinal Cord Repair

This study presents a novel detailed method of analysis of rat gait and uses this method to demonstrate recovery of forward locomotion patterns in adult rats made paraplegic by surgical spinal cord transection and subjected to a novel strategy for spinal cord repair. Six normal rats were compared to five animals in which the cord was transected at T8–T9, and a 5-mm segment of the spinal cord removed, and to seven animals in which, following spinal cord transection and removal of a spinal cord segment, multiple intercostal peripheral nerve bridges were implanted, rerouting pathways from white to gray matter in both directions. The implanted area was filled with fibrin glue containing acidic fibroblast growth factor. Details of the repair strategy have been published (H. Cheng, Y. Cao, and L. Olson, 1996,Science273:510–513). Gait analysis was carried out 3 and 4 months after surgery and once in the normal animals. Animals were allowed to walk across a runway with a transparent floor. Each test consisted of five trials, and each trial was videorecorded from underneath. Using frame-by-frame playback, individual footprints were then recorded regarding location and order of limb use, as well as step quality (degree of weight bearing, etc.). These data allowed measuring runway transit time, five different measures of step numbers, all possible temporal patterns of limb use, stride length, and base of support. Transected controls remained paralyzed in the hindlimbs with only occasional reflex hindlimb movements without weight bearing. Animals subjected to the full repair procedure were significantly faster than the controls, used their hindlimbs for 25–30% of the movements, and regained several of the specific limb recruitment patterns used by normal rats. Taken together, the gait analysis data demonstrate remarkable recovery of coordinated gait in the repaired animals, which was significantly better than controls for all relevant parameters, while at the same time clearly inferior to normal rats for most of the examined parameters. We conclude that normal rats use a multitude of interchangeable step sequence patterns, and that our spinal cord repair strategy leads to recovery of some of these patterns following complete spinal cord transection. These data suggest functionally relevant neuronal communication across the lesion.     

Antioxidant Therapies for Acute Spinal Cord Injury

One of the most investigated molecular mechanisms involved in the secondary pathophysiology of acute spinal cord injury (SCI) is free radical-induced, iron-catalyzed lipid peroxidation (LP) and protein oxidative/nitrative damage to spinal neurons, glia, and microvascular cells. The reactive nitrogen species peroxynitrite and its highly reactive free radicals are key initiators of LP and protein nitration in the injured spinal cord, the biochemistry, and pathophysiology of which are first of all reviewed in this article. This is followed by a presentation of the antioxidant mechanistic approaches and pharmacological compounds that have been shown to have neuroprotective properties in preclinical SCI models. Two of these, which act by inhibition of LP, are high-dose treatment with the glucocorticoid steroid methylprednisolone (MP) and the nonglucocorticoid 21-aminosteroid tirilazad, have been demonstrated in the multicenter NASCIS clinical trials to produce at least a modest improvement in neurological recovery when administered within the first 8 hours after SCI. Although these results have provided considerable validation of oxidative damage as a clinically practical neuroprotective target, there is a need for the discovery of safer and more effective antioxidant compounds for acute SCI.     

Hypothermic Treatment for Acute Spinal Cord Injury

Spinal cord injury (SCI) is a devastating condition that affects approximately 11,000 patients each year in the United States. Although a significant amount of research has been conducted to clarify the pathophysiology of SCI, there are limited therapeutic interventions that are currently available in the clinic. Moderate hypothermia has been used in a variety of experimental and clinical situations to target several neurological disorders, including traumatic brain and SCI. Recent studies using clinically relevant animal models of SCI have reported the efficacy of therapeutic hypothermia (TH) in terms of promoting long-term behavioral improvement and reducing histopathological damage. In addition, several clinical studies have demonstrated encouraging evidence for the use of TH in patients with a severe cervical spinal cord injury. Moderate hypothermia (33°C) introduced systemically by intravascular cooling strategies appears to be safe and provides some improvement of long-term recovery of function. TH remains an experimental clinical approach and randomized multicenter trials are needed to critically evaluate this potentially exciting therapeutic intervention targeting this patient population.     

Acute Hydrocephalus Following Cervical Spinal Cord Injury

We present a case of acute hydrocephalus secondary to cervical spinal cord injury in a patient with diffuse ossification of the posterior longitudinal ligament (OPLL). A 75-year-old male patient visited the emergency department with tetraparesis and spinal shock. Imaging studies showed cervical spinal cord injury with hemorrhage and diffuse OPLL from C1 to C4. We performed decompressive laminectomy and occipitocervical fusion. Two days after surgery, his mental status had deteriorated to drowsiness with dilatation of the right pupil. Findings on brain computed tomography revealed acute hydrocephalus and subarachnoid hemorrhage in the cerebellomedullary cistern, therefore, extraventricular drainage was performed immediately. Acute hydrocephalus as a complication of cervical spine trauma is rare, however, it should be considered if the patient shows deterioration of neurologic symptoms.     

The Effects of Difumarate Salt S-15176 after Spinal Cord Injury in Rats

Objective

In the present study we analyzed neuroprotective and antiapoptotic effect of the difumarate salt S-15176, as an anti-ischemic, an antioxidant and a stabilizer of mitochondrial membrane in secondary damage following spinal cord injury (SCI) in a rat model.

Methods

Three groups were performed with 30 Wistar rats; control (1), trauma (2), and a trauma+S-15176 (10 mg/kg i.p., dimethyl sulfoxide) treatment (3). SCI was performed at the thoracic level using the weight-drop technique. Spinal cord tissues were collected following intracardiac perfusion in 3rd and 7th days of posttrauma. Hematoxylin and eosin staining for histopatology, terminal deoxynucleotidyl transferase dUTP nick end labeling assay for apoptotic cells and immunohistochemistry for proapoptotic cytochrome-c, Bax and caspase 9 were performed to all groups. Functional recovery test were applied to each group in 3rd and 7th days following SCI.

Results

In trauma group, edematous regions, diffuse hemorrhage, necrosis, leukocyte infiltration and severe degeneration in motor neurons were observed prominently in gray matter. The number of apoptotic cells was significantly higher (p<0.05) than control group. In the S-15176-treated groups, apoptotic cell number in 3rd and 7th days (p<0.001), also cytochrome-c (p<0.001), Bax (p<0.001) and caspase 9 immunoreactive cells (p<0.001) were significantly decreased in number compared to trauma groups. Hemorrhage and edema in the focal areas were also noticed in gray matter of treatment groups. Results of the locomotor test were significantly increased in treatment group (p<0.05) when compared to trauma groups.

Conclusion

We suggest that difumarate salt S-15176 prevents mitochondrial pathways of apoptosis and protects spinal cord from secondary injury and helps to preserve motor function following SCI in rats.     

Activation of Matrix Metalloproteinases-9 after Photothrombotic Spinal Cord Injury Model in Rats

Objective

Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9 have been known to play an important role in secondary inflammatory reaction after spinal cord injury (SCI). The aim of this study was to investigate the expression and activity of MMP-2 and MMP-9 and to determine their relationship with disruption of endothelial blood-barrier after photochemically induced SCI in rats.

Methods

Female Sprague-Dawley rats, weighing between 250 and 300 g (aged 8 weeks) received focal spinal cord ischemia by photothrombosis using Rose Bengal. Expressions and activities of MMP-2 and MMP-9 were assessed by Western blot and gelatin zymography at various times from 6 h to 7 days. Endothelial blood-barrier integrity was assessed indirectly using spinal cord water content.

Results

Zymography and Western blot analysis demonstrated rapid up-regulation of MMP-9 protein levels in spinal cord after ischemic onset. Expressions and activities of MMP-9 showed a significant increased at 6 h after the photothrombotic ischemic event, and reached a maximum level at 24 h after the insult. By contrast, activated MMP-2 was not detected at any time point in either the experimental or the control groups. When compared with the control group, a significant increase in spinal cord water content was detected in rats at 24 h after photothrombotic SCI.

Conclusion

Early up-regulation of MMP-9 might be correlated with increased water content in the spinal cord at 24 h after SCI in rats. Results of this study suggest that MMP-9 is the key factor involved in disruption of the endothelial blood-barrier of the spinal cord and subsequent secondary damage after photothrombotic SCI in rats.     

Oligodendrocyte Fate after Spinal Cord Injury

Oligodendrocytes (OLs) are particularly susceptible to the toxicity of the acute lesion environment after spinal cord injury (SCI). They undergo both necrosis and apoptosis acutely, with apoptosis continuing at chronic time points. Loss of OLs causes demyelination and impairs axon function and survival. In parallel, a rapid and protracted OL progenitor cell proliferative response occurs, especially at the lesion borders. Proliferating and migrating OL progenitor cells differentiate into myelinating OLs, which remyelinate demyelinated axons starting at 2 weeks post-injury. The progression of OL lineage cells into mature OLs in the adult after injury recapitulates development to some degree, owing to the plethora of factors within the injury milieu. Although robust, this endogenous oligogenic response is insufficient against OL loss and demyelination. First, in this review we analyze the major spatial–temporal mechanisms of OL loss, replacement, and myelination, with the purpose of highlighting potential areas of intervention after SCI. We then discuss studies on OL protection and replacement. Growth factors have been used both to boost the endogenous progenitor response, and in conjunction with progenitor transplantation to facilitate survival and OL fate. Considerable progress has been made with embryonic stem cell-derived cells and adult neural progenitor cells. For therapies targeting oligogenesis to be successful, endogenous responses and the effects of the acute and chronic lesion environment on OL lineage cells must be understood in detail, and in relation, the optimal therapeutic window for such strategies must also be determined.     

Neurotrophic Factors Increase Axonal Growth after Spinal Cord Injury and Transplantation in the Adult Rat

The capacity of CNS neurons for axonal regrowth after injury decreases as the age of the animal at time of injury increases. After spinal cord lesions at birth, there is extensive regenerative growth into and beyond a transplant of fetal spinal cord tissue placed at the injury site. After injury in the adult, however, although host corticospinal and brainstem-spinal axons project into the transplant, their distribution is restricted to within 200 μm of the host/transplant border. The aim of this study was to determine if the administration of neurotrophic factors could increase the capacity of mature CNS neurons for regrowth after injury. Spinal cord hemisection lesions were made at cervical or thoracic levels in adult rats. Transplants of E14 fetal spinal cord tissue were placed into the lesion site. The following neurotrophic factors were administered at the site of injury and transplantation: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary-derived neurotrophic factor (CNTF), or vehicle alone. After 1–2 months survival, neuroanatomical tracing and immunocytochemical methods were used to examine the growth of host axons within the transplants. The neurotrophin administration led to increases in the extent of serotonergic, noradrenergic, and corticospinal axonal ingrowth within the transplants. The influence of the administration of the neurotrophins on the growth of injured CNS axons was not a generalized effect of growth factors per se, since the administration of CNTF had no effect on the growth of any of the descending CNS axons tested. These results indicate that in addition to influencing the survival of developing CNS and PNS neurons, neurotrophic factors are able to exert aneurotropicinfluence on injured mature CNS neurons by increasing their axonal growth within a transplant.     

Acute Inflammatory Response in Spinal Cord Following Impact Injury

Numerous factors are involved in the spread of secondary damage in spinal cord after traumatic injury, including ischemia, edema, increased excitatory amino acids, and oxidative damage to the tissue from reactive oxygen species. Neutrophils and macrophages can produce reactive oxygen species when activated and thus may contribute to the lipid peroxidation that is known to occur after spinal cord injury. This study examined the rostral–caudal distribution of neutrophils and macrophages/microglia at 4, 6, 24, and 48 h after contusion injury to the T10 spinal cord of rat (10 g weight, 50 mm drop). Neutrophils were located predominantly in necrotic regions, with a time course that peaked at 24 h as measured with assays of myeloperoxidase activity (MPO). The sharpest peak of MPO activity was localized between 4 mm rostral and caudal to the injury. Macrophages/microglia were visualized with antibodies against ED1 and OX-42. Numerous cells with a phagocytic morphology were present by 24 h, with a higher number by 48 h. These cells were predominantly located within the gray matter and dorsal funiculus white matter. The number of cells gradually declined through 6 mm rostral and caudal to the lesion. OX-42 staining also revealed reactive microglia with blunt processes, particularly at levels distant to the lesion. The number of macrophages/microglia was significantly correlated with the amount of tissue damage at each level. Treatments to decrease the inflammatory response are likely to be beneficial to recovery of function after traumatic spinal cord injury.