Ischemia, hypoxia and stroke are potential complications of invasive medical procedures. Exposure to focal brain ischemia may occur during neurosurgical resection, during carotid endarterectomy, and during cardiac bypass or total circulatory arrest procedures. Magnetic resonance imaging, for example, demonstrates new ischemic lesions in 58% of patients with valvular disease undergoing cardiopulmonary bypass. The consequences of Central Nervous System (CNS) deficits as a result of ischemic or hypoxic insult are so severe as to warrant investigation of novel methods of minimizing damage. Our long-range goal is to prevent CNS damage by the introduction of intracellular nucleic acids (DNA and mRNA), which encode neuroprotective proteins. This program of studies will test the hypothesis that delivery of a neuroprotective agent before a near-lethal stress can protect neurons. We will investigate Heat Shock Protein 70 (HSP70) as a candidate neuroprotective gene. This member of the Heat Shock Protein (HSP) family has been shown to protect against ischemic cell death in a variety of tissues and species. An essential step in determining the potential clinical usefulness of this strategy is to evaluate the uptake, anatomic distribution and cellular localization of mRNA and DNA introduced into the cerebrospinal fluid. We have transfected CNS neurons with mRNA or DNA encoding GFP, luciferase and HSP70 in vitro, and in vivo in the ventricular system of rats. We have demonstrated widespread distribution, uptake, and expression in vivo. Expression is seen in a large fraction of cells, which include neurons, both ipsilateral and contralateral to the site of CSF infusion, and throughout coronal sections along the longitudinal axis of the rat brain. Initial stereology analysis confirms transfection to deep cell layers. Neither gross neurological impairment nor histological evidence for inflammatory response have been seen. We are now ready to begin studies to determine whether intraventricularly introduced HSP70 can protect hippocampal pyramidal cells from global ischemia-induced cell death. We will investigate neuroprotection in a transient four-vessel global occlusion model. As a part of this project we will evaluate: Timing of transfection relative to the exposure of hypoxia, dose-response of introduced mRNA or DNA, immunohistochemical quantification of the HSP70 protein, and cellular toxicity including apoptosis. Behavioral assessments will include sensory motor and vestibulomotor function. Injury limited to CA1 in the ischemia-sensitive hippocampus after transient global occlusion will be assessed by measurement of learning and memory function using the Morris water maze, also well established in the laboratory of one of the Consultants for this application.
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| Anderson, Dua M; Hall, Leon L; Ayyalapu, Anitha R et al. (2003) Stability of mRNA/cationic lipid lipoplexes in human and rat cerebrospinal fluid: methods and evidence for nonviral mRNA gene delivery to the central nervous system. Hum Gene Ther 14:191-202 |
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