A Quantitative Immunohistochemical Study of the Cellular Response to Crush Injury in Optic Nerve

Computer-enhanced immunohistochemical techniques were applied to quantify the cellular response of rat optic nerve to crush injury to attain the spatial and temporal detail necessary for resolving trends in the process that may be affected by experimental therapy. Changes in the major cell populations of the optic nerve during 6 weeks following crush injury were analyzed using antibodies against β-tubulin (TuJ1), neurofilament (8-152), glial fibrillary acidic protein (GFAP), galactocerebrosides (GalC), ED-1, and von Willebrand factor to detect the presence of axons, astrocytes, oligodendrocytes, macrophages, and endothelial cells. The markers for axons, astrocytes, oligodendrocytes, and endothelial cells decreased their activity and remained depressed in the area of direct mechanical injury throughout 6 weeks following crush injury. A spatial peak in the axonal makers TuJ1 and 8-152 at the proximal boundary of the injury at both 2 and 3 weeks is consistent with the idea that the injury zone is a barrier obstructing distal elongation of sprouted neurites. Reactive astrocytes were GFAP-positive in the nerve segments proximal and distal to the injury with marker density above 1.5 times normal at 2 and 3 weeks. GFAP appeared to be absent in the site of injury even at 6 weeks postinjury, as shown by both immunohistochemistry and Western blot analysis. ED-1-positive macrophages were the most numerous population in the injury site from 1 through 6 weeks with a peak value measured at 2 weeks. The optical density of ED-1 activity in the distal and proximal nerve segments was less than 20% the density in the injury at all measured time points.