MICROGLIAL NO INDUCES DELAYED NEURONAL DEATH FOLLOWING ACUTE INJURY IN THE STRIATUM

We have established a novel injury model in the central nervous system by a stereotaxic injection of ethanol into rat striatum to induce nec rosis. With this model, we clarify a function of inducible nitric oxid e synthase (iNOS) in a healing mechanism around a necrotic lesion. A s emiquantitative reverse transcriptase-polymerase chain reaction (RT-PC R) revealed that the iNOS mRNA arose at 6 h, peaked at 24 h, and decli ned to a lower level 48 h after an intrastriatal 5-mu L ethanol inject ion. From in situ hybridization, this iNOS mRNA was expressed in the a rea surrounding the injury. By immunohistochemistry, mononuclear cells at this boundary area of necrosis were stained with anti-iNOS antibod y on the first day after the injury. These cells turned out to be reac tive microglia from the positive staining of GSA-I-B-4, ED-1 and OX-42 . Haematoxylin-eosin (HE) staining showed that neurons in this boundar y area gradually disappear up to 5 days after the injury with an incre ment of microglial cells, and this area became cavernous. Nuclei of ne urons in this area were stained positive by the terminal deoxynucleoti dyl-transferase-mediated dUTP-biotin nick end-labelling (TUNEL) assay on the first day after the injury. These TUNEL-positive neurons gradua lly disappeared toward the third day, while microglial cells increased . L-Ng-nitro-arginine methylester (L-NAME), a competitive NOS inhibito r, administration diminished the elimination of neurons by microglia i n this boundary area surrounding necrosis. Microglial NO may act as a neurotoxic agent to eliminate damaged neurons near the necrosis in the form of delayed neuronal death, and may reintegrate the neuronal circ uits with functionally intact neurons.