Jean R. Wrathall

Spinal cord contusion in the rat: Behavioral analysis of functional neurologic impairment☆

A graded spinal cord injury in rats was produced by dropping a 10-g weight from 2.5, 5.0, 10,0, or 17.5 cm onto the exposed dura at the T8 vertebral level. Groups of rats (N = 10) for each of these weight drop (WD) levels as well as unoperated and WD controls (0 cm) were subjected to behavioral analysis that included evaluation of simple and complex reflexes as well as spontaneous and evoked motor patterns. On the basis of this analysis, we developed a protocol for evaluating functional deficits that follow spinal cord injury in the rat. The resulting combined behavioral score, a measure of functional deficit, closely correlated with the magnitude of the mechanical injury. The protocol used for neurologic assessment was administered routinely by personnel who were easily and rapidly trained. It should therefore prove useful in detecting the effects of treatment on recovery of function in a rat model of spinal cord injury.

Spinal cord contusion in the rat: Morphometric analyses of alterations in the spinal cord ☆

Morphometric analyses were carried out on rat spinal cords which were injured by a weight drop technique. A 10-g weight was dropped 0.0, 2.5, 5.0, 7.5, 10.0, or 17.5 cm onto the dura which was exposed at the T8 vertebral level. Four weeks after injury, lesion volume, lesion length, and the dimensions of the tissue at the epicenter (lesion area, area of gray matter, and area of white matter) were measured and correlated with the height from which the weight was dropped and the results from tests of motor and sensory functional deficit. The results, based on linear regression analyses, indicated significant correlations between certain morphologic parameters (lesion volume, lesion length, and the area of gray and white matter at the epicenter) and both the height from which the weight was dropped and behavioral scores. Because the area of white matter at the epicenter is a very simple measurement which correlates well (r = 0.91) with behavioral outcome, this morphologic feature is a useful quantitative measure of the histopathologic consequences of spinal cord injury.

Temporal-spatial pattern of acute neuronal and glial loss after spinal cord contusion.

The secondary loss of neurons and glia over the first 24 h after spinal cord injury (SCI) contributes to the permanent functional deficits that are the unfortunate consequence of SCI. The progression of this acute secondary cell death in specific neuronal and glial populations has not previously been investigated in a quantitative manner. We used a well-characterized model of SCI to analyze the loss of ventral motoneurons (VMN) and ventral funicular astrocytes and oligodendrocytes at 15 min and 4, 8, and 24 h after an incomplete midthoracic contusion injury in the rat. We found that both the length of lesion and the length of spinal cord devoid of VMN increased in a time-dependent manner. The extent of VMN loss at specified distances rostral and caudal to the injury epicenter progressed symmetrically with time. Neuronal loss was accompanied by a loss of glial cells in ventral white matter that was significant at the epicenter by 4 h after injury. Oligodendrocyte loss followed the same temporal pattern as that of VMN while astrocyte loss was delayed. This information on the temporal-spatial pattern of cell loss can be used to investigate mechanisms involved in secondary injury of neurons and glia after SCI.

Spinal cord contusion in the rat: Production of graded, reproducible, injury groups ☆

A weight drop technique was used to produce a contusive injury of the spinal cord in the rat. A restricted laminectomy was carried out at T8 and the spinal column stabilized by clamps attached to the spinous processes of adjacent vertebrae. A 2.4-mm-diameter impounder was lowered onto the dura and a 10-g weight dropped 0.0, 2.5, 5.0, 7.5, 10.0, or 17.5 cm onto the impounder. The functional deficit was assessed for 4 weeks after injury and the spinal cord tissue processed for histopathologic analysis. The results indicated that groups of rats (N = 10) subjected to the weight dropped from increasing heights exhibited a graded final functional deficit as measured by scores on a modified Tarlov scale or the mean angle attained in the inclined plane test of Rivlin and Tator. Histopathologic results also indicated the production of graded lesions. Three groups of experimental animals were statistically distinguished corresponding to those with mild, moderate, or severe final functional deficit. The average functional deficit in these injury groups, produced by dropping the weight 2.5, 5.0, or 17.5 cm, respectively, was reproducible in replicate experiments. This model of spinal cord contusion in the rat may be useful in screening putative therapeutic drug regimens for subsequent clinical trials on different groups of patients with spinal cord injury.

Correlative analyses of lesion development and functional status after graded spinal cord contusive injuries in the rat

The development of both histopathological changes and functional deficits was quantitatively assessed after mild, moderate, and severe spinal cord contusive injuries. The cross-sectional area of the spinal cord at the epicenter (region of maximal damage) and the areas of hemorrhage, lesion, and remaining gray and white matter were determined from 15 min to 8 weeks after injury. From 24 h to 8 weeks after injury, functional deficits were quantified using a combined behavioral score (CBS) based on the results from a number of behavioral tests of function. Regression analysis was used to examine the correlations between the amount of residual white matter and both the severity of contusive impact and the functional deficit over time after injury. The area of hemorrhage at 15 min and 24 h was greater in the mild and moderate injury groups than after the severe contusive injury. Significant loss of gray and white matter occurred primarily between 24 h and 1 week after injury along with concomitant increases in the area of lesion. In the mild injury group the rate of lesion development appeared slower than that in the moderate and severe injury groups and significant white matter loss continued to occur between 1 and 4 weeks after injury. Behavioral tests of functional deficit were performed at 24 h and weekly thereafter. The development of stable functional deficits was observed beginning at 3 weeks after injury. There was a significant correlation between residual white matter and the degree of initial injury at 24 h after injury and all subsequent time points. However, a significant correlation between residual white matter and functional deficit, as measured by the CBS, was not observed at 24 h or 1 week but did develop by 4 weeks after injury.

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.

Distribution and time course of protein extravasation in the rat spinal cord after contusive injury

We have previously characterized a graded, spinal cord contusive injury in the rat. We have now used this reproducible model to examine vascular permeability to horseradish peroxidase (HRP) after injury. The relationship between severity of injury and distribution of protein extravasation was evaluated at 3 h after injury. After mild injury, tracer was primarily confined to central gray matter and the ventral part of the dorsal columns. After moderate injury, protein extravasation was similar to that observed after mild injury, with the exception that the central hemorrhage included pericentral while matter and occasionally extended to the pial surface. After severe injury, reaction product (RP) was more densely distributed within the central cord and peripheral white matter. The axial extent of tracer at sites proximal and distal to the impact site increased with severity of injury. At 2.0 cm from the injury, no leakage of tracer was noted after mild injury. In contrast, after moderate and severe injury limited microvascular leakage of HRP was noted. Furthermore, after severe injury, in addition to local sites of microvascular leakage, intense RP was present in the dorsal columns up to at least 2.0 cm from the injury. The time course for re-establishment of the blood-spinal cord barrier to protein was evaluated from 3 h to 14 days after moderate injury. At 3 h to 1 day, protein leakage was maximal and coincided with sites of extravasated blood components, although was consistently more extensive. By 7 days, despite resolution of the initial hemorrhage, there remained scattered evidence for protein extravasation at the injured site and at sites along the axis of the cord. The blood-spinal cord barrier to HRP was reestablished by 14 days after injury.

Amelioration of functional deficits from spinal cord trauma with systemically administered NBQX, an antagonist of non-N-methyl-D-aspartate receptors

Excitatory amino acid (EAA) receptors play a significant role in delayed neuronal death after ischemic and traumatic injury to the CNS. Recent data based on focal microinjection experiments have demonstrated that 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), a highly selective and potent antagonist of non-N-methyl-D-aspartate ionotropic EAA receptors, i.e., those preferring alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or kainate, can reduce histopathology and functional deficits after traumatic spinal cord injury (SCI). Thus, non-NMDA receptors at or near the injury site appear to be important in secondary injury processes that contribute significantly to the consequences of SCI. We have now examined the effects of systemic NBQX, using intravenous infusion, the most commonly used and temporally efficient clinical mode of drug administration. Standardized contusive SCI was produced at the T8 vertebral level in Sprague-Dawley rats. Beginning at 15 min postin-jury, NBQX was administered intravenously at 1 mg/kg/min for 30 min. Behavioral tests of hindlimb functional deficits were performed at 1 day and weekly for 1 month after SCI. Spinal cord tissue was then examined morphometrically to compare lesion size and amount of spared tissue. We found that intravenous administration of NBQX significantly reduced functional impairment after SCI. The effects included more rapid and extensive recovery of hindlimb reflexes, more rapid establishment of a reflex bladder, and a more rapid and greater degree of recovery of coordinated use of hindlimbs in open field locomotion, swimming, and maintaining position on an inclined plane. The profile of effects was similar to that seen with focal microinjection of NBQX, suggesting that even with systemic administration, the drug acts mainly at the injury site. Further, the results support a therapeutic potential for NBQX, or similar drugs that antagonize non-NMDA receptors and inhibit secondary injury processes after SCI.

Genetic approaches to neurotrauma research: opportunities and potential pitfalls of murine models.

Genetic strategies provide new ways to define the molecular cascades that regulate the responses of the mammalian nervous system to injury. Genetic interventions also provide opportunities to manipulate and control key molecular steps in these cascades, so as to modify the outcome of CNS injury. Most current genetic strategies involve the use of mice, an animal that has not heretofore been used extensively for neurotrauma research. Therefore, one purpose of the present review is to consider how mice respond to neural trauma, focusing especially on recent information that reveals important differences between mice and rats, and between different inbred strains of mice. The second aim of this review is to provide a brief introduction to the opportunities, caveats, and potential pitfalls of studies that use genetically modified animals for neurotrauma research.

Increase of interleukin-1β mRNA and protein in the spinal cord following experimental traumatic injury in the rat

Interleukin-1 beta (IL-1beta) is a major mediator of inflammation and a growth promoter for many cell types that could play an important role in the consequences of traumatic spinal cord injury (SCI). In the present study, the expression of IL-1beta and its mRNA was determined in the rat spinal cord following a standardized contusion injury. IL-1beta mRNA, measured with quantitative RT-PCR, was significantly increased in the lesion site by 1 h after SCI (35.2 +/- 5.9 vs. 9.1 +/- 2.1 pg/mg RNA, n = 3, P < 0.05) and remained significantly higher than in the normal spinal cord for at least 72 h post-injury (p.i.). IL-1beta mRNA levels in tissue immediately caudal to the lesion site did not change after the injury. IL-1beta protein levels, measured by an ELISA, were determined at the lesion site and in cerebrospinal fluid (CSF) and serum samples. IL-1beta levels in the CSF and serum were much lower than in the spinal cord. At the lesion site, IL-1beta was increased significantly by 1 h p.i., peaked at 8 h (32.3 +/- 0.1 vs. 7.6 +/- 1.9, ng/g tissue, n = 5, P < 0.05) and remained significantly higher than normal through at least 7 days p.i. These results suggest that the increased IL-1beta mRNA and protein levels are an early and local response at the lesion site that could trigger other, later, responses to traumatic SCI.