Hyperacute Neuropathological Findings after Proton Beam Radiosurgery of the Rat Hippocampus

Abstract

To study the hyperacute histological and immunohistochemical effects of stereotactic proton beam irradiation of the rat hippocampus. Nine rats underwent proton beam radiosurgery of one hippocampus with nominal doses of cobalt-2, -12, and -60 Gray equivalents (n = 3 each). Control animals (n = 3) were not irradiated. Animals were killed 5 hours after irradiation and brain sections were stained for Nissl, silver degeneration, deoxyribonucleic acid (DNA) fragmentation (DNAF), and the activated form of two mitogen-activated protein kinases (MAPKs), phospho-Erk1/2 (P-Erk1/2) and p38. Stained cells in the hippocampus expressing DNAF and/or P-Erk1/2 were counted. Confocal microscopy with double immunofluorescent staining was used to examine cellular colocalization of DNAF and P-Erk1/2. Both DNAF and P-Erk1/2 showed quantitative dose-dependent increases in staining in the targeted hippocampus compared with the contralateral side and controls. This finding was restricted to the subgranular proliferative zone of the hippocampus. Both markers also were up-regulated on the contralateral side when compared with controls in a dose-dependent fashion. Simultaneous staining for DNAF and P-Erk1/2 was found in fewer than half of all cells. p38 was unchanged compared with controls. Although Nissl staining appeared normal, silver stain confirmed dose-dependent cellular degeneration. DNAF, a marker of cell death, was present in rat hippocampi within 5 hours of delivery of cobalt-2 Gray equivalents stereotactically focused irradiation, suggesting that even low-dose radiosurgery has hyperacute neurotoxic effects. Activated mitogen-activated protein kinase was incompletely colocalized with DNAF, suggesting that activation of this cascade is neither necessary nor sufficient to initiate acute cell death after irradiation.