This research project will continue studies on the molecular & cellular mechanisms of blood-brain barrier (BBB) breakdown. The BBB, which normally restricts the entry of diverse polar molecules into the brain, reflects the unique structure of the endothelial cells lining the cerebral microvessels. These cells are joined by tightly sealed junctions, & contain few pinocytotic vesicles & no fenestrae. Vasogenic brain edema, the most commonly encountered form of brain edema, features a disruption of BBB function associated with intense pinocytosis & transcytosis of blood-borne substances into the brain extracellular space, & possibly enhanced transport of other substrated (Na+, K+, CA2+, glucose, amino acids) by specific carrier-mediated transport systems in BBB. Previous studies in this laboratory established that BBB breakdown induced by cold injury is associated with a rapid biphasic incease in polyamines & their rate-limiting synthetic enzyme ornithine decarboxylase (ODC) in rat cerebral parenchyma and microvessels. This polyamine synthesis is essential for BBB breakdown and the increased transcytosis of horseradish peroxidase, as it is abolished by the ODC inhibitor Alpha-difluoromethylornithine (DFMO), and putrescine nullifies the effects of DFMO. Stimulation of ODC activity and the associated BBB breakdown induced by freeze injury apparently involves CA2+ transport and prostaglandin synthesis as both are suppressed by verapamil, dexamethasone and aspirin.
The specific aims are to study in injured cerebral microvessel endothelium: (1) the mechanisms underlying the primary (posttranslational?) and secondary (transcriptional?) regulation of ODC; (2) changes in 45Ca2+ influx and efflux, and free cytosolic CA2+ levels and their polyamine-dependence: (3) changes in rates of transcytosis and transport of 22Na+, 86Rb+, 3H-deoxyglucose, 3H-Alpha-aminoisobutyrate, and their polyamine- and Ca2+ dependence; (4) quantiative morphometry of ultrastructural and cytochemical changes in structures mediating transcytosis, and relative volumes of cytolasm, nucleus, mitochondria, endoplasmic reticulum and Golgi; (5) role of polyamines in the astrocytic response to freeze-injury (pinocytosis and lysosomal sequestration of HRP, hypertrophy, hyperplasia); (6) polyamine-dependence of focal and generalized abnormalities in EEG activity and glucose utilization developing in brain after freeze injury.
These aims will be studied in three systems: in situ in rat cerebrum after freeze and osmotic injury; in isolated cerebral microvessels; and in cultured cerebral microvessel endothelium.
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