The proposed project aims to explore the use of neuronal progenitor cells as a possible source of exogenous neurons to be used for replacement of loss neurons following cerebral ischemia. In addition, behavioral measures will be used to evaluate if the implanted cells can reverse the cognitive deficits seen after ischemic stroke. Stroke is a major health problem in the United States, it is the third leading cause of death. In addition to the physical and emotional suffering associated with the disease, treatment of stroke victims consumes about 40 billion dollars annually, mainly because of nursing home admissions. Among doctors and the public, strokes have long been considered catastrophic events for which little can be done. Now this pessimistic assumption is being challenged with the use of anti-thrombolytic agents in the acute period following the onset of stroke symptoms. Neural transplantation is evolving as a stroke remedy even after the critical period has passed. The successful use of transplantation in Parkinson's disease has spurred the use of this approach to the treatment of stroke. Results of transplantation in human stroke victims are still pending. The barriers to wide spread use of fetal cell for transplantation are formidable, beginning with availability to ethical considerations. Therefore, efforts to develop a reliable alternative source of cells are crucial if transplantation is to develop as a treatment strategy. The objectives of this project are: (1) to examine the chronic survival and differentiation of the immortalized neuronal cell line, RN33B, following transplantation into the adult rat hippocampus after varying degree of cerebral ischemia and (2) explore the efficacy of the RN33B cell line in ameliorating spatial learning deficits induced by cerebral ischemia. In phase 1 of the study, animals will be subjected to mild, moderate and severe cerebral ischemia. Ischemia will be induced by occlusion of both carotid arteries accompanied by hypotension (2-VO). After a ten-day recovery period, animals will receive unilateral transplantation of RN33B cells into the hippocampus. Eight weeks post transplantation, animals will be sacrificed and the brains removed for histological analyses. Transplanted cells will be detected in vivo using antibodies against beta-galactosidase. Measures of survival and differentiation will include: stereological count of beta-galactosidase immunoreactive cells located in the hippocampus and characterization of the degree of morphological differentiation. In phase II, one ischemic group will be used for follow-up behavioral studies. A new set of animals will undergo the ischemic surgery and transplantation procedure. Animals will be assessed for the recovery of behavioral deficits using the water maze acquisition and open field tests. Behavioral testing will be performed prior to and eight weeks post transplantation of RN33B cells.
