Studies on cerebral ischemia
During stroke, hypoxia, hypoglycemia and acidosis transiently appear after the reduction of cerebral blood flow. These metabolic insults induce neuronal loss that result from two distinct death mechanisms - necrosis and apoptosis. Necrotic neuronal loss begins within minutes, yet neuronal function returns to the surviving neurons. The recovery of neuronal function can be transient; many neurons succumb to apoptosis approximately 3-4 days following reperfusion. We have demonstrated that a thirty minute acidification of intracellular pH from 7.3 to 6.5 induced both necrotic and apoptotic loss of neurons in cultured hippocampal slices. Necrosis and apoptosis were distinguished temporally, morphologically, and pharmacologically. Necrosis was observed eight hours following acidosis and apoptosis after sixteen hours. Apoptotic neuronal loss was accompanied by DNA fragmentation and was blocked by inhibitors of protein and RNA synthesis, ectopic expression of the anti-apoptotic gene bcl-2 [An inhibitor of caspases, proteases that mediate apoptosis, was also effective]. Necrotic neuronal loss was unaffected by these treatments. Hypothermia, a treatment known to attenuate apoptosis following ischemic injury, blocked both acidosis-induced necrosis and apoptosis. In contrast to the thirty minute acidosis treatment that caused both apoptosis and necrosis, a fifteen minute treatment produced only apoptosis. These results indicate that acidosis is neurotoxic in vitro and suggests that acidosis contributes to both necrotic and apoptotic neuronal loss following cerebral ischemia.
We have developed a method to place slices of adult hippocampus into long-term culture. Exposure of adult cultured slices to brief hypoxia/hypoglycemia induces selective and delayed loss of CA1 neurons. Experimental parameters that confound in vivo studies are avoided such as variations in cerebral microcirculation, brain temperature, and trauma due to survival surgery. In contrast to other in vitro models, neuronal loss following H/H is selective and delayed. The adult slice culture model will be a valuable tool to determine the molecular mechanism of ischemic injury as well to test potential neuroprotective therapies.
Caspase activation is believed to be necessary for apoptosis. Specific caspase inhibitors have been developed that block apoptosis. Experiments in the first specific aim will examine when caspases are activated following hypoxia and determine the necessity of caspase activation for apoptosis.
Figure 1. Delayed and CA1 specific neuronal loss induced by 10 minutes of hypoxia/hypoglycemia in slice cultures isolated from adult rats. Slice cultures received 10' H/H (left) or N/N (right). Neuronal loss was assayed with PI staining at 1 day intervals for 8 days after the insult. False color mages of PI epifluorescence were obtained using an intensified CCD camera (9X magnification).
Selected Publications
Moskowitz, S. I., Basu, S. B., and Bergold, P. J. (2001). Chronic and cyclical neuronal loss in hippocampal slice cultures following transient inhibition of the type 1 isoform of superoxide dismutase. Brain Res. 913, 207-219.
Xiang, Z.-M., Yuan, M., Hassan, G. W., Gampel, M. and Bergold, P. J. (2004). Lactate-induced excitotoxicity in hippocampal slice cultures. Exp. Neurol. 186, 70-77.
Hassen, G. W., Tian, D., Ding, D. and Bergold, P. J. (2004). A new model of ischemic preconditioning using young adult hippocampal slice cultures. Brain Res. Prot. 13, 135-143.
Cracco, J. B., Serrano, P., Moskowitz, S. I., Bergold, P. J., and Sacktor, T.C. (2005). Protein synthesis-dependent LTP in the isolated dendrites of CA1 pyramidal cells. Hippocampus 5, 551-556.
Cao, H., Kass, I., Cottrell, J. E., and Bergold, P. J. (2005). Pre-ischemic or Post-ischemic administration of lidocaine or thiopental attenuates ischemic injury in rat Hippocampal Slice Cultures. Anesthes. Analges. 101, 1163-1169.
Gilbert, E., and Bergold, P. J. (2005). Oxidation of 14C-labeled substrates by hippocampal Slice Cultures. Brain Res. Prot. 5, 135-141.
Gilbert, E., Tang, H. M., Ludvig, N., and Bergold, P. J. (2006). Lactate suppresses neural firing and glucose metabolism in hippocampal neurons. Brain Res. 1117, 213-223.
Tian, D., Dimitreeva, R. I., Doris, P. C., Sacktor, T. C., and Bergold, P. J. (2006). PKM regulation of Na/K ATPase: A potential neuroprotective mechanism of ischemic preconditioning. Submitted.
List of Publications (Pub Med)
Personnel
Anna Yermakova, Ph.D., Postdoctoral Fellow
Service Functions
Reviewer for various scientific journals.