INJURY-INDUCED EXPRESSION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE BY GLIAL AND MICROGLIAL CELLS IN THE LEECH CENTRAL-NERVOUS-SYSTEM WITHIN MINUTES AFTER INJURY

It is known that nitric oxide (NO) is produced by injured tissues of t he mammalian central nervous system (CNS) within days of injury. The a im of the present experiments was to determine the cellular synthesis of NO in the CNS immediately after injury, using the CNS of the leech which is capable of synapse regeneration, as a step towards understand ing the role of NO in nerve repair. We report that within minutes afte r crushing the nerve cord of the leech, the region of damage stained h istochemically for NADPH diaphorase, which is indicative of nitric oxi de synthase (NOS) activity, and was immunoreactive for endothelial NOS (eNOS). On immunoblots of leech CNS extract, the same antibody detect ed a band with a relative molecular mass of 140 000, which is approxim ately the size of vertebrate eNOS. Cells expressing eNOS immunoreactiv ity as a result of injury were identified after freezing nerve cords, a procedure that produced less tissue distortion than mechanical crush ing. Immunoreactive cells included connective glia and some microglia. Calmodulin was necessary for the eNOS immunoreactivity: it was blocke d by calmodulin antagonist W7 (25 mu M), but not by similar concentrat ions of the less potent calmodulin antagonist W12. Thus in the leech C NS, in which axon and synapse regeneration is successful, an increase in NOS activity at lesions appears to be among the earliest responses to injury and may be important for repair of axons.