C-fos expression in the spinal cord of rats exhibiting allodynia following contusive spinal cord injury

Contusive spinal cord injury (SCI) may result in central neuropathic pain marked by allodynia-like features in the dermatomes close to the level of injury. The aim of this study was to compare the laminar distribution of activated neurons (as determined by c-fos immediate early gene expression) in the spinal cord immediately above the level of a SCI in rats with or without allodynia-like features. Non-noxious mechanical stimulation was applied to half the animals in the dermatomes corresponding to the level of injury prior to perfusion. Stimulation resulted in a significant increase in c-fos labelling in all laminae of the spinal dorsal horn in the segment immediately above the level of injury only in allodynic animals. Animals that had allodynia also demonstrated a significant increase in the level of c-fos labelling in lamina III, IV and V of the dorsal horn without stimulation. Thus, allodynia following SCI is associated with significant increases in basal and evoked c-fos expression (“neuronal activity”) in response to non-noxious mechanical stimulation. The data also suggest that allodynia-like behaviour following SCI cannot be accounted for solely by changes occurring at a spinal level.     

Cell proliferation and replacement following contusive spinal cord injury

After spinal cord injury (SCI), about 50% of the oligodendrocytes and astrocytes in the residual white matter at the injury site are lost by 24 h. However, chronically after SCI, the density of oligodendrocytes is normal. Previous studies have shown that the adult rat spinal cord contains a pool of proliferating glial progenitors whose progeny could help restore cell density after injury. To study proliferation in response to injury, we performed SCI on adult female rats at the T8 level, using a standardized contusion model. Animals received bromodeoxyuridine (BrdU) injections during the first week after SCI, and were perfused within 2 h for acute studies, and at 6 weeks for chronic studies. The tissue was analyzed using immunohistochemical detection of BrdU and cell marker antigens. We demonstrate that cell proliferation in the residual white matter is increased at 1-7 days after SCI, peaking on day 3. Dividing cells include oligodendrocytes, astrocytes, microglia/macrophages, and a high proportion of NG2(+) glial precursors. By 6 weeks, some cells that had been labeled 2-4 days after SCI were still present. Double immunohistochemistry showed that while very few of these cells expressed NG2 or the microglia/macrophage marker OX42, about 50% expressed CC1 or glial fibrillary acidic protein (GFAP), markers of mature oligodendrocytes and astrocytes, respectively. The post-injury environment represented by residual white matter is thus permissive to the differentiation of glial precursors. Cells that are stimulated to divide during the first week after SCI develop chronically into mature phenotypes that replace macroglia lost after injury.     

Responses of spinal neurones to cutaneous and dorsal root stimuli in rats with mechanical allodynia after contusive spinal cord injury

The firing of neurones in spinal segments adjacent to a contusive T13 spinal cord injury was characterised in anaesthetised rats. Three groups of rats were examined: (1) allodynic spinally injured, (2) non-allodynic spinally injured and (3) normal, uninjured. Spinal cord field potentials evoked by electrical dorsal root stimulation and the responses of 207 dorsal horn neurones to mechanical stimuli applied to the skin were studied. Within the lesioned spinal segment few active neurones were encountered and field potentials were absent. Depolarising field potentials recorded rostral to the lesion were reduced in both allodynic and non-allodynic animals compared to uninjured controls, while those recorded in caudal segments were enhanced in allodynic animals. Neuronal recordings revealed that allodynia was associated with exaggerated responses, including afterdischarges, to innocuous and noxious mechanical stimuli in a proportion of wide dynamic range, but not low threshold, neurones. These changes were observed both rostral and caudal to the site of injury. The results suggest that an increased responsiveness of some dorsal horn neurones in segments neighbouring a contusive spinal cord injury may contribute to the expression of mechanical allodynia. It is proposed that a relative lack of inhibition underlies altered cell responses.     

Neuroprotective effect of apigenin in rats after contusive spinal cord injury

Apigenin, a common plant flavonoid, has been extensively studied and showed a wide variety of beneficial effects. The aim of this study was to determine the therapeutic efficacy of starting apigenin treatment 3 day after spinal cord injury (SCI) in rat and to investigate the underlying mechanism. SCI was induced using the modified weight-drop method in Sprague–Dawley rats. The SCI animals were randomly assigned to five groups: sham control group, SCI model group, the methylprednisolone sodium succinate (MPSS) group, the 10 mg/kg apigenin treatment group and the 20 mg/kg apigenin treatment group. First, neuronal function after SCI was evaluated with Basso Beattie Bresnahan locomotor rating scale (BBB) and the result showed that injured animals treated with apigenin showed a significant increase in BBB scores. To explore the underlying mechanism, antioxidative effect of apigenin was assessed by measuring malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities after SCI. Apigenin treatment reversed the decrease of SOD and GSH-Px activity, and the increase of MDA level caused by SCI, suggesting its antioxidative role in response to the injury. In addition, apigenin treatment decreased serum interleukin-1β, tumor necrosis factor-α and intercellular adhesion molecule-1 release after SCI, suggesting an anti-inflammatory effect of apigenin. Finally, apigenin treatment affected the expression level of apoptosis-related gene Bax, Bcl-2 and caspase-3, which indicated its antiapoptosis role after SCI. Our data suggest that apigenin significantly promotes the recovery of rat neuronal function after SCI, which is associated with its antioxidative, anti-inflammatory and antiapoptotic properties.     

Temporal progressive antigen expression in radial glia after contusive spinal cord injury in adult rats

In the development of the CNS, radial glial cells are among the first cells derived from neuroepithelial cells. Recent studies have reported that radial glia possess properties of neural stem cells. We analyzed the antigen expression and distribution of radial glia after spinal cord injury (SCI). Sprague-Dawley rats had a laminectomy at Th11-12, and spinal cord contusion was created by compression with 30 g of force for 10 min. In the injury group, rats were examined at 24 h and 1, 4, and 12 weeks after injury. Frozen sections of 20-microm thickness were prepared from regions 5 and 10 mm rostral and caudal to the injury epicenter. Immunohistochemical staining was performed using antibodies to 3CB2 (a specific marker for radial glia), nestin, and glial fibrillary acidic protein (GFAP). At 1 week after injury, radial glia that bound anti-3CB2 MAb had spread throughout the white matter from below the pial surface. From 4 weeks after injury, 3CB2 expression was also observed in the gray matter around the central canal, and was especially strong around the ependymal cells and around blood vessels. In double-immunohistochemical assays for 3CB2 and GFAP or 3CB2 and nestin, coexpression was observed in subpial structures that extended into the white matter as arborizing processes and around blood vessels in the gray matter. The present study demonstrated the emergence of radial glia after SCI in adult mammals. Radial glia derived from subpial astrocytes most likely play an important role in neural repair and regeneration after SCI.     

Regional heterogeneity in astrocyte responses following contusive spinal cord injury in mice

Astrocytes and their precursors respond to spinal cord injury (SCI) by proliferating, migrating, and altering phenotype. This contributes to glial scar formation at the lesion border and gliosis in spared gray and white matter. The present study was undertaken to evaluate astrocyte changes over time and determine when and where interventions might be targeted to alter the astrocyte response. Bromodeoxyuridine (BrdU) was administered to mice 3 days after SCI, and cells expressing BrdU and the astrocyte marker, glial fibrillary acidic protein (GFAP), were counted at 3, 7, and 49 days post‐injury (DPI). BrdU‐labeled cells accumulated at the lesion border by 7 DPI and approximately half of these expressed GFAP. In spared white matter, the total number of BrdU+ cells decreased, while the percentage of BrdU+ cells expressing GFAP increased at 49 DPI. Phenotypic changes were examined using the progenitor marker nestin, the radial glial marker, brain lipid binding protein (BLBP), and GFAP. Nestin was upregulated by 3 DPI and declined between 7 and 49 DPI in all regions, and GFAP increased and remained above naïve levels at all timepoints. BLBP increased early and remained high along the lesion border and spared white matter, but was expressed transiently by cells lining the central canal and in a unique population of small cells found within the lesion and in gray matter rostral and caudal to the border. The results demonstrate that the astrocyte response to SCI is regionally heterogeneous, and suggests astrocyte populations that could be targeted by interventions. J. Comp. Neurol. 518:1370–1390, 2010. © 2009 Wiley‐Liss, Inc.     

Behavioral assessment of functional deficit in rats with contusive spinal cord injury

We have previously reported a method for assessing functional deficits in rats after contusive spinal cord injury in which a Combined Behavioral Score (CBS) is calculated that is indicative of the overall percent deficit (Exp. Neurol., 88: 123-134, 1985). The test battery used includes several neurologic tests as well as the Motor Score in which use of the hindlimbs in locomotion is graded. In this report we present correction criteria in order to reduce potential interlaboratory variability in assessing functional deficit by the CBS. Groups of rats were subjected to contusive injury using a weight drop technique. The calculated CBS at 4 weeks was compared to that obtained if the Motor Score was over- or underestimated by 1 grade. The results indicated a considerable effect on the calculated CBS, especially when the Motor Score was underestimated in mildly injured animals. Behavioral test data were examined in terms of the distribution of the responses to the various other behavioral tests in relation to the Motor Score. The results were used to develop a set of correction criteria that minimized the effects on the CBS of subjective errors in the Motor Score.     

Spinal AMPA receptor inhibition attenuates mechanical allodynia and neuronal hyperexcitability following spinal cord injury in rats

In this study, we examined whether a competitive AMPA receptor antagonist, NBQX, attenuates mechanical allodynia and hyperexcitability of spinal neurons in remote, caudal regions in persistent central neuropathic pain following spinal cord injury in rats. Spinal cord injury was produced by unilateral T13 transverse spinal hemisection, from dorsal to ventral, in male Sprague Dawley rats (200-250 g). Mechanical thresholds were measured behaviorally, and the excitability of wide-dynamic-range (WDR) dorsal horn neurons in the lumbar cord (L4-L5) was measured to assess central neuropathicpain. On postoperation day (POD) 28 after spinalhemisection, mechanical thresholds were significantly decreased in both injured (ipsilateral) and noninjured (contralateral) hindpaws compared with preinjury and sham control, respectively (P < 0.05). Intrathecal administration of NBQX (0.25, 0.5, 1 mM) significantly reversed the decreased mechanical thresholds in both hindpaws, dose dependently (P < 0.05). The excitability of WDR neurons was significantly enhanced on both sides of the lumbar dorsal horn 28 days following spinal hemisection (P < 0.05). The hyperexcitability of WDR neurons was attenuated by topical administration of NBQX (0.125, 0.25, 0.5, 1 mM), dose dependently (P < 0.05). Regression analysis indicated that at least three molecules of NBQX bond per receptor complex, and are needed to achieve inhibition of WDR hyperexcitability. In conclusion, our study demonstrates that the AMPA receptor plays an important role in behaviors related to the maintenance of central neuropathic pain below the level of spinal cord injury.     

The effects of ciliary neurotrophic factor on neurological function and glial activity following contusive spinal cord injury in the rats

Ciliary neurotrophic factor (CNTF) has been implicated in the pathophysiology of injury to the central nervous system. The rapid increase in CNTF production following spinal cord injury (SCI) in rats is thought to serve a role in the neuronal survival and functional recovery. In this study, 40 SD rats were divided into four groups: sham-operated group, saline-treated group, 5- and 10-microg CNTF group. Saline and CNTF were given through lumbar intrathecal catheter for 10 days after T10 segment of spinal cord were injured by modified Allen contusion method. Animals were behaviorally tested for 6 weeks using the Basso, Beattie, Bresnahan locomotor rating scale and inclined plane test. At the end of 6 week, rubrospinal neurons of five rats in each group were labeled by retrograde transport of the horseradish peroxidase (HRP) from the lesion site, and then the labeled red nucleus neuron (RN) numbers were counted. Additional rats were histologically assessed for tissue sparing and neuronal loss and reactive gliosis at the injury site and adjacent areas. Rats treated with CNTF regained greater improvements in hindlimb function than controls. The amount of spared tissue was significantly higher in CNTF-treated animals than in controls. After CNTF treatment, the number of HRP-labeled RN neurons were significantly increased. Astrocytes and microglia reactivity was more pronounced in CNTF-treated animals than in controls. These results indicate that intrathecal infusion of exogenous CNTF following SCI may significantly reduce tissue damage and protect the rubrospinal descending tracks and enhances functional recovery, and may also induce more gliosis.     

A biochemical investigation of spinal cord after contusive injury

A closed spinal cord contusive injury system which is easily and reproducibly inflicted is described. At various times after injury, homogenates from individual segments along the cord were evaluated for total protein, lipid soluble galactose, (galactolipid), ceramide, 2′,3′-cyclic nucleotide 3′-phosphohydrolase, and myelin. Homogenates and myelin were also evaluated by SDS-polyacrylamide gel electrophoresis. In addition, the tissues were evaluated by light and electron microscopy. The results indicated that there was little change, in comparison with normal spinal cord tissue, in any biochemical components studied as long as 48 h after contusive injury. This was especially evident when the data were normalized on the basis of protein and/or galactolipid per tissue slice. This was in marked contrast to the progressive tissue necrosis and unraveling of the myelin sheath evident by light and electron microscopic examination of the injury site. The data suggest that at short times after injury, the architecture of the myelin sheath is disorganized yet the biochemical building blocks are still present.     

Increase in nerve growth factor-like immunoreactivity and decrease in choline acetyltransferase following contusive spinal cord injury

We have previously described a graded spinal cord injury model in the rat. Mild contusive injury results in an initially severe functional deficit that is attenuated over time to reveal the mild chronic deficits that characterize this injury. In this study, we have shown that mild contusive injury also results in a significant decrease in choline acetyltransferase (ChAT) activity during the first week after injury. At 1 week ChAT activity is maximally reduced at the site of the contusion and is also significantly lowered throughout the spinal cord. ChAT activity then rebounds during the following 3 weeks, partially at the injury site where there is considerable loss of gray and white matter, and completely in rostral and caudal cord segments. The rebound in ChAT activity is temporally associated with the partial recovery of function. Further, the changes in ChAT activity after injury are mirrored by changes in nerve growth factor-like immunoreactivity (NGF-LI) as determined by a specific two-site ELISA. NGF-LI increases significantly after injury, reaching a maximum at 7 days after contusion and at the injury site. However, levels of NGF-LI are also significantly increased throughout the spinal cord. NGF-LI then decreases at 2 and 4 weeks as ChAT activity rebounds. Further experiments will be needed to examine the possibility of a role for NGF in promoting the recovery of function after spinal cord injury.     

Diagnostic accuracy of evoked potentials for functional impairment after contusive spinal cord injury in adult rats

Iatrogenic spinal cord injury (SCI) is a cause of potentially debilitating post-operative neurologic complications. Currently, intra-operative neurophysiological monitoring (IONM) via somatosensory evoked potentials and motor-evoked potentials is used to detect and prevent impending SCI. However, no empirically validated interventions exist to halt the progression of iatrogenic SCI once it is detected. This is in part due to the lack of a suitable translational model that mimics the circumstances surrounding iatrogenic SCI detected via IONM. Here, we evaluate a model of simulated contusive iatrogenic SCI detected via IONM in adult female Sprague-Dawley rats. We show that transient losses of somatosensory evoked potentials responses are 88.24% sensitive (95% confidence interval [CI] 63.53–98.20) and 80% specific (95% CI 51.91–95.43) for significant functional impairment following simulated iatrogenic SCI. Similarly, we show that transient losses in motor-evoked potentials responses are 70.83% sensitive (95% CI 48.91–87.33) and 100% specific (95% CI 62.91–100.00) for significant functional impairment following simulated iatrogenic SCI. These results indicate that our model is a suitable replica of the circumstances surrounding clinical iatrogenic SCI.     

Pain with no gain: allodynia following neural stem cell transplantation in spinal cord injury

Transplantation of neural stem cells (NSCs) in the injured spinal cord has been shown to improve functional outcome; however, recent evidence has demonstrated forelimb allodynia following transplantation of embryonic NSCs. The aim of this study was to investigate whether transplantation of murine C17.2 NSCs alone or transfected with glial-derived neurotrophic factor (C17.2/GDNF) would induce allodynia in transplanted spinal cord-injured animals. One week after a T8-level spinal cord injury (SCI), C17.2, C17.2/GDNF or normal saline was injected at the injury site. Locomotor function and sensory recovery to thermal and mechanical stimuli were then measured. Spinal cords were processed immunohistochemically at the injury/transplantation site for characterization of NSC survival and differentiation; and at the cervicothoracic level for calcitonin gene-related peptide (CGRP), a neuropeptide expressed in dorsal horn nocioceptive neurons, and growth-associated protein-43 (GAP43), a marker of neuronal sprouting. Locomotor function was not significantly improved following NSC transplantation at any time (P >0.05). Significant forelimb thermal and mechanical allodynia were observed following transplantation with both NSC populations (P <0.05). The C17.2 and C17.2/GDNF NSCs survived and differentiated into a predominately astrocytic population. Calcitonin gene-related peptide and GAP43 immunoreactivity significantly increased and co-localized in cervicothoracic dorsal horn laminae I-III following C17.2 and C17.2/GDNF transplantation. This study demonstrated that murine C17.2 NSCs differentiated primarily into astrocytes when transplanted into the injured spinal cord, and resulted in thermal and mechanical forelimb allodynia. Sprouting of nocioceptive afferents occurred rostral to the injury/transplantation site only in allodynic animals, suggesting a principal role in this aberrant pain state. Further, a difference in the degree of allodynia was noted between C17.2- and C17.2/GDNF transplant-treated groups; this difference correlated with the level of CGRP/GAP43 immunoreactivity and sprouting observed in the cervicothoracic dorsal horns. Both allodynia- and CGRP/GAP43-positive afferent sprouting were less in the C17.2/GDNF group compared to the C17.2 group, suggesting a possible protective or analgesic effect of GDNF on post-injury neuropathic pain.     

An inexpensive apparatus for producing graded spinal cord contusive injury in the rat

We recently described an experimental model of spinal cord contusive injury in the rat in which a modified weight-drop apparatus is used to generate reproducible mild, moderate, or severe final functional deficit. We now report several inexpensive alternatives for portions of the original apparatus which allow the production of contusive injuries without the necessity of an expensive commercial stereotaxic frame or custom-machined clamp holders.