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, 210 (2), 489-500

Aspiration of a Cervical Spinal Contusion Injury in Preparation for Delayed Peripheral Nerve Grafting Does Not Impair Forelimb Behavior or Axon Regeneration

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Aspiration of a Cervical Spinal Contusion Injury in Preparation for Delayed Peripheral Nerve Grafting Does Not Impair Forelimb Behavior or Axon Regeneration

Harra R Sandrow et al. Exp Neurol.

Abstract

A peripheral nerve graft model was used to examine axonal growth after a unilateral cervical (C) contusion injury in adult rats and to determine if manipulation of an injury site prior to transplantation affects spontaneous behavioral recovery. After a short delay (7 d) the epicenter of a C4 contusion was exposed and aspirated without harming the cavity walls followed by apposition with one end of a pre-degenerated tibial nerve to the rostral cavity wall. After a longer delay (28 d) the aspirated cavity was treated with GDNF to promote regeneration by chronically injured neurons. In both groups forelimb and hindlimb locomotor scores decreased significantly 2 d after lesion site manipulation, but by 7 d, the forelimb score was not different from the pre-manipulation score. There was no significant difference in grid walking or grip strength scores for the affected forelimb in either group 7 d after contusion vs. 7 d after manipulation. Over 1500 brain stem and propriospinal neurons grew axons into the graft with either delay. These results demonstrate that a contusion injury site can be manipulated prior to transplantation without causing long-lasting forelimb or hindlimb behavioral deficits and that peripheral nerve grafts support axonal growth after acute or chronic contusion injury.

Figures

1
1
Histological appearance of cellular debris in contusion lesion cavity. Small densely stained cells were tightly packed in the lesion cavity forming at 7d after SCI (A), in contrast to the more loosely arranged cells in the cavity at 28d after injury (B). More spindle-shaped cells and amorphous debris were observed at the longer post injury period. Adjacent to the epicenter is spared spinal cord tissue with intact myelin to the right of dashed line in (A).
2
2
GFAP and ED1 immunocytochemistry at lesion epicenter, 7d after contusion injury. A) A thin rim of lateral white matter and dorsal and ventral medial gray matter remains at this short post injury period. The unilateral nature of the injury is obvious here. The * indicates densely packed cells that partially fill the lesion cavity which are ED1 immunoreactive (B). These comprise the majority of cells observed in Figure 1A. C) Intense staining for GFAP surrounds the lesion cavity and is spread throughout the spared gray and white matter areas of the spinal cord ipsilateral to the lesion. The astrocytic reaction contralateral to the lesion is less intensely stained, especially in the gray matter. D) Merged images of astrocyte and phagocytic cell response to contusion injury. Bar = 500 µm.
3
3
GFAP and ED1 immunocytochemistry at lesion epicenter, 28d after contusion injury. A–D are in a transverse plane and E–H are in a horizontal plane, midline to the left of image. At this long post injury period more extensive tissue damage is obvious compared to the 7d survival period. There is loss of most gray matter within the lesion epicenter and only a thin rim of spared white matter ventrally and laterally (A and E). Cellular debris that partially fills the cavity is comprised primarily of phagocytic cells (B and F) with substantial aggregation of astrocytes in gray and white matter surrounding the lesion (C and G). Merged images (D and H) indicate little migration of phagocytic cells into adjacent spinal cord tissue. Bar = 500 µm.
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4
Forelimb behavioral assessment. A) The forelimb locomotor scale (FLS) scores were comparable in the 2 groups at 2d and 7d with a significant increase in performance from 2d to 7d after contusion injury, indicative of spontaneous recovery from slight movement of all 3 joints to frequent plantar stepping. Animals in the 28d group showed further improvement beyond the 7d group. At 2d after manipulation and PN grafting to the lesion epicenter there was a significant decrease in FLS score compared to the 7d or 28d post-contusion score respectively, but by 7d post grafting animals had recovered to their pre-manipulation scores. B) Grid testing at 7d post contusion showed no significant decrease in forelimb performance in either group, with a trend towards improvement with a longer survival period. Manipulation and grafting at the epicenter did not significantly change foot placement when tested 7d later. C) Forelimb grip was not significantly impaired at any of the post-contusion intervals nor did manipulation of the epicenter lead to greater loss of function. ◇ indicates significantly greater than 2d post contusion injury; * indicates significantly less than 7d post contusion; ▲ indicates significantly less than 28d post-contusion injury.
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5
Hindlimb behavioral assessment. A) Hindlimb open field locomotor performance was not significantly different between 2d and 28d post injury. Manipulation of the lesion epicenter resulted in a significant decrease in BBB score (loss of frequency of weight support and no FL/HL coordination) that persisted through day 7 in the 2d group but not the 28d group. B) Grid testing at 7d post contusion showed no significant decrease in forelimb performance in either group, with a trend towards improvement with a longer survival period. Manipulation and grafting at the epicenter did not significantly change foot placement when tested 7d later. * indicates significantly less than 7d post contusion; ▲ indicates significantly less than 28d post-contusion injury.
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6
Peripheral nerve graft interface with injured spinal cord. Spinal cord tissue rostral to the lesion is to the right of this figure. Arrows indicate the interface of spinal cord and peripheral nerve graft that extends from the dorsal surface (top arrow) ventrally to include a portion of the ventral funiculus. Some spared tissue (*) in the epicenter below the PN graft prevented full apposition to the ventral funiculus. There was no obvious difference in the extent of apposition with a 7d or 28d delay between contusion injury and nerve grafting.
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7
Number and distribution of regenerating neurons. Delaying the PN graft for 7d or 28d had no significant effect on the number or distribution of neurons that regenerated their axon into the graft. The majority of regenerating neurons were of brain stem origin, being led by those in the reticular formation and raphe regions. While there appeared to be fewer propriospinal neurons contributing to the graft with a 28d delay, this was not significantly less than with a 7d delay because of the large variability within the groups.

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