Overlapping Distribution of Osteopontin and Calcium in the Ischemic Core of Rat Brain after Transient Focal Ischemia

Osteopontin (OPN), an adhesive glycoprotein, has recently been proposed to act as an opsonin that facilitates phagocytosis of neuronal debris by macrophages in the ischemic brain. The present study was designed to elucidate the process whereby OPN binds to neuronal cell debris in a rat model of ischemic stroke. Significant co-localization of the OPN protein and calcium deposits in the ischemic core were observed by combining alizarin red staining and OPN immunohistochemistry. In addition, electron microscopy (EM) using the osmium/potassium dichromate method revealed that electron-dense precipitates, typical of calcium deposits, were localized mainly along the periphery of putative degenerating neurites. This topical pattern of calcium precipitates resembled the distribution of OPN as detected by immunogold-silver EM. Combining immunogold-silver EM and electron probe microanalysis further demonstrated that the OPN protein was localized at the periphery of cell debris or degenerating neurites, corresponding with locally higher concentrations of calcium and phosphorus, and that the relative magnitude of OPN accumulation was comparable to that of calcium and phosphorus. These data suggest that calcium precipitation provides a matrix for the binding of the OPN protein within the debris or degenerating neurites induced by ischemic injury. Therefore, OPN binding to calcium deposits may be involved in phagocytosis of such debris, and may participate in the regulation of ectopic calcification in the ischemic brain.