This project has a dual focus. On the one hand our analysis will attempt to identify those pathological changes in the brain that may account for the observed behavioral changes in cognitive function while on the other hand it will also attempt to identify the pattern of development of small vessel disease. To this end one hemisphere will be immersion fixed in 4% paraformaldehyde and then cut into serial sections. These will be stained with standard Nissl and myelin procedures as well as with H&E and surveyed for changes in blood vessels, for microinfarctions and for demyelination - all changes observed in our pilot work. In addition other series will be processed with immunocytochemical methods using antibodies to serum proteins to identify areas of vascular leakage and Glial Fibrillary Acidic Protein to identify areas of minute necrosis. Since our pilot observations suggest that these obvious changes are probable not sufficient to explain the widespread behavioral changes, the other hemisphere of the brain will be flash frozen and cut on a cryostat into 15 micron thick serial sections. These sections will be processed using ligand binding methods to ascertain if there are changes in neurotransmitter specific afferents and/or their associated receptors which may be especially vulnerable to mild ischemia that may occur before or independent of the microinfarctions. Since the thickening of small vessel walls may result in inadequate dilation during the course of normal daily vicissitudes of blood pressure, the territory supplied by these vessels may be inadequately perfused. In the cerebral cortex the most likely areas to be effected are the superficial cortical layers, namely I and II. Indeed studies in spontaneously hypertensive rats have shown preferential changes in the apical dendritic tree in layers I and II of retrosplenial cortex. This analysis suggests that the most likely afferent systems to be effected would be the cholinergic, dopaminergic and noradrenergic systems that terminate preferentially in layer I. To determine if these afferent system are uniquely affected sections will be processed using radioactively labeled ligands that bind to the neurotransmitter specific uptake sites located on axon terminals. This method provides a quantitative techniques for comparing afferent density in different layers of cortex in the same animals and also allows between animal comparisons to be made. In addition we will also use radioactively labeled ligands that bind to specific neurotransmitter receptor subtypes to determine, quantitatively, if there is a loss of receptors in these layers. While many is a regression of dendrites of specific neuronal populations, then this method should detect loss of post-synaptic receptors associated with these neuronal populations. Since the major infracortical receptor systems are the excitatory amino acids and the GABAergic interneurons both of these receptors will be assessed as well as the major subtypes of the ACH, DA and NE systems.
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