Temporal and spatial patterns of expression of laminin, chondroitin sulphate proteoglycan and HNK-1 immunoreactivity during regeneration in the goldfish optic nerve

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Abstract
Current views suggest that the extracellular environment is critically important for successful axonal regeneration in the CNS. The goldfish optic nerve readily regenerates, indicating the presence of an environment that supports regeneration. An analysis of changes that occur during regeneration, in this model may help identify those molecules that contribute to a favourable environment for axonal regrowth. We examined the distribution and expression of two extracellular matrix molecules, laminin and chondroitin sulphate proteoglycan, and a carbohydrate epitope shared by a family of adhesion molecules (HNK-1), using immunocytochemical detection in sections from the normal adult goldfish optic nerve and in nerves from one hour to five months following optic nerve crush. We also usedin vitro preparations to determine if neurites in retinal explants could express these same molecules. The linear distributions of laminin and chondroitin sulphate proteoglycan immunoreactivity in control optic nerves are co-extensive with the glia limitans, suggesting both are expressed by non-neuronal components surrounding the axon fascicles. Between one and three weeks postoperatively when axons elongate and reach their target, laminin and chondroitin sulphate proteoglycan immunoreactivity increases around the crush site and distally. At six weeks postoperatively the pattern of immunoreactivity has returned to normal. While the temporal pattern of changes in immunoreactivity is similar, the spatial pattern of these two extracellular proteins in the regenerating nerve differs. Chondroitin sulphate proteoglycan immunoreactivity is organized in discrete columns associated with regenerating axons while laminin immunoreactivity is more diffusely distributed. Examination of retinal explants reveals growing neurites express chondroitin sulphate proteoglycan but not laminin. Our results suggest that laminin is only associated with non-neuronal cells, while chondroitin sulphate proteoglycan is associated with axons as well as non-neuronal cells. HNK-1 immunoreactivity is co-extensive with both the glia limitans and axon fascicles and is more extensively distributed in the intact nerve than either laminin or chondroitin sulphate proteoglycan immunoreactivity. In contrast to laminin and chondroitin sulphate proteoglycan, HNK-1 immunoreactivity is substantially decreased at the crush site within one week following optic nerve crush. HNK-1 immunoreactivity reappears through the crush site during the next several weeks, although non-immunoreactive regions, co-extensive with areas predominantly containing non-neuronal cells, persist both proximal and distal to the crush, up to six weeks postoperatively. The pattern suggests that HNK-1 epitope expression by these non-neuronal cells is decreased during axonal regeneration. Our results show that each of these molecules is constitutively expressed with a unique distribution in the normal goldfish optic nerve and each exhibits different patterns of change during regeneration. It thus appears that each may contribute to modifications of the environment that supports axonal regeneration. Both neurons and non-neuronal cells contribute to these changes.