Motoneuron survival after chronic and sequential peripheral nerve injuries in the rat

Laboratory investigation

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Object

Surgical repair of peripheral nerves following chronic nerve injury is associated with poor axonal regeneration and outcome. An underlying possibility is that chronic injuries may increase motoneuron cell death. The hypothesis that substantial motoneuron death follows chronic and sequential nerve injuries was tested in adult rats in this study.

Methods

Thirty adult male Lewis rats underwent bilateral multistage surgeries. At initial surgery, Fast Blue (FB) tracer was injected at a nerve-crush injury site in the right control femoral motor nerve. The left femoral motor nerve was transected at the same level and either capped to prevent regeneration (Group 1), or repaired to allow axonal regeneration and reinnervation of the target quadriceps muscle (Group 2) (15 rats in each group). After 8 weeks in 6 rats/group, the left femoral nerve was cut and exposed to FB just proximal to prior nerve capping or repair and the rats were evaluated for FB-labeled motoneuron counts bilaterally in the spinal cord (this was considered survival after initial injury). In the remaining 9 animals/group, the left nerve was recut (sequential injury), exposed to FB, and repaired to a fresh distal saphenous nerve stump to permit axonal regeneration. Following another 6 weeks, Fluoro-Gold, a second retrograde tracer, was applied to the cut distal saphenous nerve. This allowed us to evaluate the number of motoneurons that survived (maintained FB labeling) and the number of motoneurons that survived but that also regenerated axons (double labeled with FB and Fluoro-Gold).

Results

A mean number of 350 and 392 FB-labeled motoneurons were found after 8 weeks of nerve injury on the right and the left sides, respectively. This indicated no significant cell death due to initial nerve injury alone. A similar number (mean 390) of motoneurons were counted at final end point at 14 weeks, indicating no significant cell death after sequential and chronic nerve injury. However, only 50% (mean 180) of the surviving motoneurons were double labeled, indicating that only half of the population regenerated their axons.

Conclusions

The hypothesis that significant motoneuron cell death occurs after chronic and or sequential nerve injury was rejected. Despite cell survival, only 50% of motoneurons are capable of exhibiting a regenerative response, consistent with our previous findings of reduced regeneration after chronic axotomy.

Abbreviations used in this paper: DAB = diaminobenzidine; FB = Fast Blue; FG = Fluoro-Gold.

Article Information

Address correspondence to: Rajiv Midha, M.D., M.Sc., F.R.C.S.C., Division of Neurosurgery, Department of Clinical Neurosciences, University of Calgary, Room 1195, 1403 29 Street NW, Calgary, Alberta, Canada T2N 2T9. email: rajmidha@ucalgary.ca.

Please include this information when citing this paper: published online September 18, 2009; DOI: 10.3171/2009.8.JNS09812.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Schematic diagrams of femoral motor branch surgical manipulations, in the left and right hindlimb of the rat, to examine the effect of motoneurons survival after single and sequential nerve injury. At the first surgery, FB was injected at a nerve crush injury site in the right femoral motor nerve branch to quadriceps muscle. This side served as the control (CON) side for femoral motoneuron cell counts after retrograde labeling at subsequent time points. The left side served as the experimental (EXP) side for single and sequential nerve injuries to the left femoral motor nerve branch to the quadriceps muscle. At initial surgery, the left motor nerve was severed at the same level as the right side and capped to prevent regeneration (A; the frustrated regeneration group [Group 1]) or repaired to permit regeneration (B; the regenerating group [Group 2]). After 8 weeks (in 6 rats/group), the left femoral nerve was exposed to FB just proximal to prior nerve capping or repair (C and E; Groups 1A and 2A). These rats were killed 6 days later to allow counting of FB-labeled motoneurons bilaterally in the spinal cord (survival after initial injury). In the remaining 9 animals/group (at 8 weeks), the left nerve was recut (sequential injury) and exposed to FB (same as above) after being repaired to a freshly severed distal saphenous nerve stump to permit axonal regeneration (D and F; the sequential injury groups [Groups 1B and 2B]). Following another 6 weeks, at 14 weeks after the first of 3 surgeries, FG was introduced into the distal saphenous nerve to enable counting of both surviving and regenerating femoral motoneurons.

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    Single- and double-labeling evaluation. A: Fast Blue–labeled cells were easily visualized with the ultraviolet filter as were motoneurons that were back labeled with FG. B: Since FB and FG labeling was best detected at similar fluorescence settings, and thus not clearly distinguishable from each other, we used an antibody to FG (see Methods for details) on all sections to clearly identify FG-containing cells in which the DAB reaction product can be seen as punctuate dark brown granules within the cell cytoplasm. C: For double-labeling analysis, merged images that allowed simultaneous detection of the FB label and the FG immunostaining (4 such cells are denoted by arrows and under higher magnification in the inset) were therefore evaluated and the motoneurons counted. Original magnification × 100 (A–C) and × 500 (inset).

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    A single nerve injury does not result in motoneuron cell death. The normal motoneuron pool count (~ 390) from the quadriceps motor nerve was confirmed on the control right side. Following sectioning of the quadriceps nerve on the left, and after 8 weeks, a similar number of motoneurons was back labeled on the experimental side in both the capped (Cap) (the frustrated regeneration group [Group 1A]) and motor experimental repaired (the regenerating group [Group 2A]) paradigms in the left hindlimb compared with the right side controls. Bars in the graph show mean ± SEM.

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    Chronic and sequential nerve injuries do not result in motoneuron cell death. The motoneuron pool counts, as evaluated by FB back labeling, remained virtually identical from the 8-week to the 14-week evaluation in the experimental left side (and also similar to right side controls) despite the chronicity of the injury or the sequential nature of the nerve injury. Also, note that initially frustrated regeneration (Group 1A, Cap) has no detrimental effect on survival compared with the group in which regeneration was initially enabled (Group 2A, Motor Repair). Bars in the graph show mean ± SEM.

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    Only 50% of surviving motoneurons are capable of mounting a regenerative response after prolonged axotomy. Approximately twice as many FB-labeled neurons were evident on the experimental left side in each group compared with FG and FB double-labeled neurons. The differences were highly significantly different (***p < 0.001, 2-tailed Student t-test within each group). The negative effect of prolonged axotomy on regenerative capacity is similar whether the nerves are allowed to initially regenerate (Group 2, Motor Repair) or not (Group 1, Cap). Bars in the graph show mean ± SEM.

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    A: Representative light micrographs of semithin sections from distal saphenous nerve at Week 14 (6 weeks following secondary repair in the initially capped Group 1 [upper] and the surgically repaired Group 2 [lower]). Sections from both groups exhibited a diverse population of regenerated and reasonably well-myelinated axons. Original magnification × 400. Bar = 10 μm. B: Group 2 (Motor Repair) had a larger number of myelinated axons, but the differences from Group 1 (Cap) did not achieve statistical significance. C: However, the fiber area (not shown) and myelin area were significantly greater in the motor repair group than the initially capped group. Bars represent mean ± SEM. **p < 0.01, 2-tailed Student t-test.

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