Long-term effects of a lumbosacral ventral root avulsion injury on axotomized motor neurons and avulsed ventral roots in a non-human primate model of cauda equina injury

Abstract

Here, we have translated from the rat to the non-human primate a unilateral lumbosacral injury as a model for cauda equina injury. In this morphological study, we have investigated retrograde effects of a unilateral L6-S2 ventral root avulsion (VRA) injury as well as the long-term effects of Wallerian degeneration on avulsed ventral roots at 6-10 months post-operatively in four adult male rhesus monkeys. Immunohistochemistry for choline acetyl transferase and glial fibrillary acidic protein demonstrated a significant loss of the majority of the axotomized motoneurons in the affected L6-S2 segments and signs of an associated astrocytic glial response within the ventral horn of the L6 and S1 spinal cord segments. Quantitative analysis of the avulsed ventral roots showed that they exhibited normal size and were populated by a normal number of myelinated axons. However, the myelinated axons in the avulsed ventral roots were markedly smaller in caliber compared to the fibers of the intact contralateral ventral roots, which served as controls. Ultrastructural studies confirmed the presence of small myelinated axons and a population of unmyelinated axons within the avulsed roots. In addition, collagen fibers were readily identified within the endoneurium of the avulsed roots. In summary, a lumbosacral VRA injury resulted in retrograde motoneuron loss and astrocytic glial activation in the ventral horn. Surprisingly, the Wallerian degeneration of motor axons in the avulsed ventral roots was followed by a repopulation of the avulsed roots by small myelinated and unmyelinated fibers. We speculate that the small axons may represent sprouting or axonal regeneration by primary afferents or autonomic fibers.

Keywords: ChAT; GFAP; PBS; VRA; Wallerian degeneration; choline acetyltransferase; electron microscopy; glial fibrillary acidic protein; phosphate-buffered saline; rhesus macaque; spinal cord; ventral root avulsion.

Figure 1

Surgical display of left sided L6, L7, S1, and S2 ventral roots after a hemi-laminectomy and dural opening (A). The identification of the ventral roots were based on vertebral landmarks and characteristic differences in caliber for individual lumbosacral ventral roots. Sutures have been attached to each ventral root to assist with the identification and presentation. The L6-S2 ventral roots have been avulsed from the surface of the spinal cord (B). Note fine rootlets at the tip of the avulsed ventral root. Transverse sections of the L6, L7, S1, and S2 ventral roots harvested from the intact contralateral side at the end of experiment (C). Note the characteristic large size of the L6 and L7 ventral roots compared to the much smaller S1 and S2 ventral roots. Also note that ventral roots exhibit a very thin epineurium and that regions of densely packed myelinated fibers are separated by streaks of connective tissues.

Figure 2

Representative photomicrographs of the ventral horn of the L6 Intact (A) and L6 Avulsed (B) sides as well as the S1 Intact (C) and S1 Avulsed (D) sides in a Rhesus monkey at 6 months after a unilateral L6-S2 VRA injury. The sections have been processed for ChAT immunoreactivity. Note that the avulsed side appears more sparsely populated by cholinergic motor neurons.

Figure 3

Graph demonstrating the number of motor neurons in a 2 mm spinal cord slice for the L6-S2 segments. The left and right ends of each line represent the motor neuron count for the intact and avulsed sides, respectively. The black, blue, brown, and green color codes for the lines each represent a different subject. Note a significantly decreased number of motor neurons in all segments with * indicating p < 0.05 and ** indicating p < 0.01.

Figure 4

Representative photomicrographs of the ventral horn of the L7 Intact (A) and L7 Avulsed (B) as well as the S1 Intact (C) and S1 Avulsed (D) sides in a Rhesus monkey at 6 months after a unilateral L6-S2 VRA injury. The sections have been processed for GFAP immunoreactivity, which is readily identified bilaterally.

Figure 5

Graph demonstrating immunoreactivity for GFAP in the ventral horn (A) and dorsal horn (B) of the L6-S2 segments. The left and right ends of each line represent the number of pixels corresponding to GFAP labeling for the intact and avulsed sides, respectively. The black, blue, brown, and green color codes for the lines each represent a different subject. Note a significantly increased GFAP immunoreactivity in the ventral horn of the L6 and S1 segments with * indicating p < 0.05 and ** indicating p < 0.01.

Figure 6

Photomicrographs of representative plastic embedded and toluidine blue stained L6-S2 ventral roots at 6 months after a unilateral L6-S2 VRA injury. The intact L6, L7, S1, and S2 ventral roots are presented in A, C, E, and G, respectively. The avulsed L6, L7, S1, and S2 are presented in B, B, F, and H, respectively. Note the presence of a large number of small myelinated axons in the avulsed ventral roots.

Figure 7

Graphs demonstrating ventral root cross-section area (upper graph) and number of ventral root axons (lower graph) in intact and avulsed L6-S2 ventral roots at 6–8.5 months after a unilateral L6-S2 VRA injury. The left and right ends of each line represent the values for the intact and avulsed roots, respectively. The black, blue, brown, and green color codes for the lines each represent a different subject.

Figure 7

Graphs demonstrating ventral root cross-section area (upper graph) and number of ventral root axons (lower graph) in intact and avulsed L6-S2 ventral roots at 6–8.5 months after a unilateral L6-S2 VRA injury. The left and right ends of each line represent the values for the intact and avulsed roots, respectively. The black, blue, brown, and green color codes for the lines each represent a different subject.

Figure 8

Graph demonstrating the size distribution for the cross sectional fiber area in intact (upper graph) and avulsed (lower graph) L6-S2 ventral roots at 6–8.5 months after a unilateral VRA injury. Note the predominance of small myelinated fibers in the avulsed ventral roots with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001.

Figure 9

A: Electron micrograph of avulsed ventral root demonstrating several small and medium caliber myelinated axons (ax), a Schwann cell (SC), and unmyelinated axons, each indicated by an asterisk. B: Electron micrograph demonstrating a Schwann cell (SC) in close association with a small myelinated axon (ax). The endoneurium exhibits numerous collagen fibers (see insert).