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, 27 (11), 2091-106

Characterization of a Graded Cervical Hemicontusion Spinal Cord Injury Model in Adult Male Rats

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Characterization of a Graded Cervical Hemicontusion Spinal Cord Injury Model in Adult Male Rats

Kelly A Dunham et al. J Neurotrauma.

Abstract

Most experimental models of spinal cord injury (SCI) in rodents induce damage in the thoracic cord and subsequently examine hindlimb function as an indicator of recovery. In these models, functional recovery is most attributable to white-matter preservation and is less influenced by grey-matter sparing. In contrast, most clinical cases of SCI occur at the lower cervical levels, a region in which both grey-matter and white-matter sparing contribute to functional motor recovery. Thus experimental cervical SCI models are beginning to be developed and used to assess protective and pharmacological interventions following SCI. The objective of this study was to characterize a model of graded cervical hemicontusion SCI with regard to several histological and behavioral outcome measures, including novel forelimb behavioral tasks. Using a commercially available rodent spinal cord impactor, adult male rats received hemicontusion SCI at vertebral level C5 at 100, 200, or 300 kdyn force, to produce mild, moderate, or severe injury severities. Tests of skilled and unskilled forelimb and locomotor function were employed to assess functional recovery, and spinal cord tissue was collected to assess lesion severity. Deficits in skilled and unskilled forelimb function and locomotion relating to injury severity were observed, as well as decreases in neuronal numbers, white-matter area, and white-matter gliosis. Significant correlations were observed between behavioral and histological data. Taken together, these data suggest that the forelimb functional and locomotor assessments employed here are sensitive enough to measure functional changes, and that this hemicontusion model can be used to evaluate potential protective and regenerative therapeutic strategies.

Figures

FIG. 1.
FIG. 1.
Impactor device injury parameters. Delivered contusion force (A) and measured tissue displacement (B) at the time of impact were obtained with the Infinite Horizons impactor and are presented here. Mean scores (±standard error of the mean) are reported according to group (bsignificant difference between 100 and 300 groups; csignificant difference between 100 and 200 groups; dsignificant difference between 200 and 300 groups).
FIG. 2.
FIG. 2.
Effect of injury severity on unskilled forelimb function. Unskilled forelimb function was assessed by the paw preference test. Percentages of contralateral, ipsilateral, and simultaneous paw placements are shown for days 0 (A), 7 (B), and 35 (C). Mean scores (±standard error of the mean) for contralateral paw use only at baseline, and on post-injury days 7, 14, 21, 28, and 35 are shown in D (asignificant difference between Lam and all injury groups, bsignificant difference between 100 and 300 groups, dsignificant difference between 200 and 300 groups; Lam, laminectomy control animals).
FIG. 3.
FIG. 3.
Effect of injury severity on skilled forelimb function. Skilled forelimb function was assessed by the vermicelli handling test. Images showing representative handling behaviors are shown for uninjured (A) and injured (B) animals. Mean scores (±standard error of the mean) at baseline and on post-injury days 7, 14, 21, 28, and 35 are presented in C (asignificant difference between Lam and all injury groups, bsignificant difference between 100 and 300 groups; Lam, laminectomy control animals).
FIG. 4.
FIG. 4.
Effect of injury severity on unskilled locomotion. Unskilled locomotion was assessed with the CatWalk gait analysis system. Computer representations of pawprint patterns for uninjured (A) and injured (B) animals are shown. Arrows denote the ipsilateral forepaw. Maximum pawprint area mean values (±standard error of the mean [SEM]) at baseline and on post-injury days 7, 14, 21, 28, and 35 are presented in C. Duty factor mean values (±SEM) at baseline and on post-injury days 7, 14, 21, 28, and 35 are presented in D (asignificant difference between Lam and all injury groups, bsignificant difference between 100 and 300 groups, csignificant difference between 100 and 200 groups, dsignificant difference between 200 and 300 groups, esignificant difference between Lam and 200 groups, fsignificant difference between Lam and 300 groups; Lam, laminectomy control animals).
FIG. 5.
FIG. 5.
Effect of injury severity on skilled locomotion. Skilled locomotion was assessed by the horizontal ladder test. Representation of correct paw placements, (A) forelimb slip/miss (B), and hindlimb slip/miss (C) are presented, with arrows designating the affected paw. The percentage of total rungs used (D), error score (E), and percentage of correct paw placement (F) mean scores (±standard error of the mean) at baseline and on post-injury days 7, 14, 21, 28, and 35 are presented (asignificant difference between Lam and all other groups, bsignificant difference between 100 and 300 groups, csignificant difference between 100 and 200 groups, dsignificant difference between 200 and 300 groups, esignificant difference between Lam and 200 groups, fsignificant difference between Lam and 300 groups; Lam, laminectomy control animals).
FIG. 6.
FIG. 6.
Effect of injury severity on neuron numbers in the ventral horn. Nissl substance was identified by cresyl violet histochemistry, and neurons were identified by morphology. Neurons in the ventral horn were then quantified by unbiased stereology. Representative micrographs are presented at 40× (scale bar = 400 μm in A, C, E, and G), and 100× magnification (scale bar = 200 μm in B, D, F, and H; arrows denote neurons). Neuronal loss, the presence of small infiltrating cells, and tissue disruption can be identified. Mean values (±standard error of the mean) are presented for the ipsilateral (I) and contralateral (J) spinal cord (asignificant difference between Lam and all injury groups, bsignificant difference between 100 and 300 groups; Lam, laminectomy control animals).
FIG. 7.
FIG. 7.
Effect of injury severity on white-matter area. Myelin was identified by myelin basic protein (MBP) immunohistochemistry. Representative micrographs are shown at 40× (scale bar = 400 μm in A, C, E, and G) and 100 × magnification (scale bar = 200 μm in B, D, F, and H; arrows denote myelin deposits). Disruption of intact myelin is represented by loss of staining, gross tissue disruption, and the presence of myelin deposits. Mean scores (±standard error of the mean) are presented for the ipsilateral (I) and contralateral (J) spinal cord (asignificant difference between Lam and all injury groups, bsignificant difference between 100 and 300 groups; Lam, laminectomy control animals).
FIG. 8.
FIG. 8.
Effect of injury severity on reactive gliosis in the ipsilateral spinal cord. Reactive gliosis was quantified by glial fibrillary acidic protein relative immunofluorescence (GFAPir), and representative micrographs are shown at 100× (scale bar = 200 μm in A, C, and E) and 200× magnification (scale bar = 100 μm in B, D, and F; arrows indicate GFAP-positive astrocytes). Mean scores (±standard error of the mean) are presented for gray matter GFAPir in G, and white matter GFAPir in H (asignificant difference between Lam and all injury groups, bsignificant difference between 100 and 300 groups; Lam, laminectomy control animals; Ipsi, ipsilateral; Contra, contralateral).

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