Injury-induced nitric oxide signaling in the leech central nervous system
Abstract
Axons of the leech Hirudo medicinalis regenerate following injury by crush or transection. We have used this system to study the early cellular and molecular events that occur following injury, in order to understand the remarkable regenerative capacity of the leech CNS. We demonstrate that injury to the leech CNS results in endothelial nitric oxide Synthase (eNOS)-like immunoreactivity in the glial and microglial cells at the injured site and that this injury-induced eNOS activity guides microglial migration and leads to accumulation. In particular, application of the nitric oxide (NO) donor, SPNO, significantly reduces the velocity of migrating microglia and prevents their accumulation. Application of a NOS inhibitor does not affect the speed of the migrating microglia, and allows the microglia to migrate through the lesion, thus preventing accumulation at the injury site. These data suggest that in the absence of NO, the microglia continue to migrate, but fail to accumulate at the injury site. We have directly measured the NO produced by the eNOS-like protein at the injury site with a novel NO-selective microelectrode. These experiments indicate that following injury, there is a rapid efflux of NO followed by a slow lingering component. Injury-induced NO appears to activate soluble guanylate cyclase (sGC) present in the leech CNS. Injury to the leech CNS induces specific NO-dependent cGMP-like immunoreactivity at the lesion site that occurs in a time-dependent manner following injury. Inhibition of sGC activity with [1H-[1,2,4] oxadizole [4,3-a] quinoxalin-1-one] (ODQ) blocks this cGMP-like immunoreactivity at the lesion site. Injury-induced NO-dependent cGMP-like immuunoreactivity shares a similar distribution with accumulated microglia at the lesion site. We suggest that the injury-induced NO exerts its effect on the migrating microglia through cGMP and hypothesize that the injury-induced NO-dependent cGMP serves as a directional cue for the migrating microglia.
Degree
Ph.D.
Advisors
Sahley, Purdue University.
Subject Area
Neurology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.