Reactive gliosis (RG), whose hallmark is increased immunoreactivity (IR) for GFAP, is induced by CNS injury including seizures. Cholinolytic seizures produce a marked increase in GFAP-IR in piriform cortex within 1 hr of seizure, however, the extracellular signals initiating RG are largely unknown. Our working hypothesis is that signals such as acidic pH initiate RG as measured by increased astrocytic GFAP in response to CNS injury or seizure. To investigate the potential signals for RG, we developed an in vitro system ideally suited to this purpose a non-reactive, """"""""mature"""""""" astrocyte culture. Decreasing the pH of such cultures to 5.8-6.0 provokes a rapid increase in GFAP-IR within hours. This increase is diminished by Ca++ channel blockers. The time course of this reactivity mimics that of our in vivo animal model. We propose to test three specific hypotheses using an in vitro culture system in parallel with an in vivo animal model. Hypothesis A: In seizures, hyperactive neurons increase glycolytic flux resulting in the production of lactic acid and decreased extracellular pH. The decreased pH stimulates RG both in vivo and in vitro.
Aim 1. Correlate lactic acid levels /extracellular pH during cholinolytic seizures in vivo with increases in GFAP-IR. 2. Investigate the effects of acidic pH on GFAP-IR in cultured non- reactive, """"""""mature' astrocytes. 3. Determine if infusion of lactic acid into a focal brain area increases GFAP in normal animals. A second experiment will determine if infusion of buffers into the brain (piriform cortex) of animals experiencing cholinolytic seizures prevents increased GFAP. Hypothesis B: GFAP-IR increases as the result of a cascade of signals initiated by reduced pH.
Aim 4. Investigate the effect of acidic pH on Cab translocation in cultured, """"""""mature' astrocytes. Hypothesis C: GFAP responds in a biphasic manner. First, there is a transient increase in IR that is independent of de novo protein synthesis. Second, there is synthesis of GFAP mRNA and protein; this process may persist for weeks. 5 Determine if acidic pH causes posttranslational modifications (e.g., phosphorylation) of GFAP or structural changes in filaments in vivo and in vitro. a.
Aim 6. Investigate the expression of immediate early genes (IEGs), GFAP mRNA and GFAP protein during induced seizures, and, in parallel, in acid-treated mature"""""""" astrocyte cultures. The significance of this proposal is that it will allow us to characterize the signals involved in RG and thus, provide us with a pharmacological strategy to modify the degree and the time course of the RG response to injury. This would be important since reactive astrocytes form a """"""""glial scar which has long been thought to a form barrier to regenerating CNS axons.
| Lee, Y B; Du, S; Rhim, H et al. (2000) Rapid increase in immunoreactivity to GFAP in astrocytes in vitro induced by acidic pH is mediated by calcium influx and calpain I. Brain Res 864:220-9 |
| Du, S; Rubin, A; Klepper, S et al. (1999) Calcium influx and activation of calpain I mediate acute reactive gliosis in injured spinal cord. Exp Neurol 157:96-105 |
| Kim, Y C; Kim, S R; Markelonis, G J et al. (1998) Ginsenosides Rb1 and Rg3 protect cultured rat cortical cells from glutamate-induced neurodegeneration. J Neurosci Res 53:426-32 |
