Cytoskeletal and activity-related changes in spinal motoneurons after root avulsion

Abstract

Compelling evidence shows that after root avulsion motoneurons attempt to survive and regenerate before dying. In order to study these mechanisms, unilateral avulsion of L4-L5 spinal roots was performed in adult rats, and the ventral spinal cords were studied from 3 to 28 days post-operation (dpo). Electrophysiological results indicated complete denervation of L4-L5 muscles in the injured limb without functional effects on the contralateral hindlimb. Estimated cell counts showed progressive loss of avulsed motoneurons; at 28 dpo, the number of surviving motoneurons averaged 13% (+/-3), 22% (+/-0.9), and 29% (+/-0.7) in samples stained with cresyl violet, immunostained for choline acetyltransferase (ChAT) and for SMI32, respectively. Regarding glial reactivity, we found an early microglial response, with the highest increase of isolectin B4 (IB4) and Iba1 expression at 3 days, and a slower and progressive response of astrocytes, shown by increasing expression of glial fibrillary acidic protein (GFAP) during the 28 days studied. Accumulation of phosphorylated (RT97+) and non-phosphorylated (SMI32+) neurofilaments was observed in the soma of axotomized motoneurons from 3 dpo. RT97 expression was present in approximately 80% (+/-3.7) of surviving motoneurons at 7 dpo, and it was related to the regenerative response of motoneurons, as revealed by decreased ChAT expression and co-localization with GAP43. Intense SMI32 immunostaining was observed during the first week after avulsion, appearing in 37% (+/-0.9) of surviving motoneurons, but it was not associated with GAP43 expression. Calcitonin gene-related peptide (CGRP) expression in motoneurons was markedly reduced from the second week after avulsion. This study presents a detailed description of motoneuron reaction after root avulsion, which suggests an early time-window during the first 2 weeks for attempts to repair the injury and promote motoneuron survival and regeneration.