Sami Harik, MD - US grants

The goals of this proposal are to investigate the basic cellular mechanisms that underlie the complex functions of the brain microcirculation, and how this circulation is controlled to meet the fastidious requirements of the brain. The endothelial cells of brain microvessels stand at the interface between the systemic circulation and nervous tissue, and have a vital role in maintaining a stable environment for neuronal function. To accomplish this, the brain capillary endothelium is endowed with unique features, such as tight intercellular junctions, and a variety of transporters for essential lipid-insoluble molecules like glucose and amino acids. Many gaps remain in our knowledge of the basic cellular mechanisms underlying the complex functions of the brain's blood vessels. The proposed experiments will employ complementary biochemical, pharmacological, and ultrastructural techniques to study the brain microcirculation in intact tissue, and in preparations enriched with isolated microvessels. The experiments will address: (1) Cellular mechanisms by which adenosine and its analogues exert their affects on the cerebral circulation. Adenosine receptors and the adenosine transporter will be investigated in isolated cerebral microvessels by ligand binding techniques. (2) The presence of receptors for putative peptide neurotransmitters which may regulate the cerebral circulation: angiotensin II, vasopressin, cholecystokin, and vasoactive intestinal polypeptide. (3) Ultrastructural investigation of heterogeneities in the distribution of the glucose transporter and sodium, potassium-ATPase in different brain regions, and within the microvascular unit, by immunocytochemical methods. (4) Isolation and biochemical characterization of the basal lamina of brain microvessels. (5) Biochemical determination and possible ultrastructural localization of carbonic anhydrase in cerebral microvessels. A better understanding of how the brain microcirculation functions may be a prerequisite for appreciating the pathophysiology of the cerebral circulation. This research may provide scientific bases for rational therapy of cerebral vascular disorders, metabolic encephalopathies and blood-brain barrier dysfunction.

The aim of the proposed experiments is to continue the study of the biological role of endogenous cerebral norepinephrine in the control of brain oxidative metabolism and its local vascular perfusion and permeability with particular attention to the provision of glucose as the major substrate for brain metabolism. The physiological mechanisms underlying this influence of norepinephrine and their significance will be investigated. The models chosen for these studies include effects of chemical lesion of the nucleus locus ceruleus in the rat. The locus ceruleus is the site of origin of most, if not all, the noradrenergic innervation of the cerebral cortex. The effects of ablation of the locus ceruleus will be studied using a variety of physiological in vivo methodologies and concomitantly by biochemical and pharmacological in vitro and in vivo techniques. Physiological techniques measure cerebral blood flow, the extraction of glucose by the brain, monitoring the redox state of cytochrome oxidase and local blood volume by dual wavelength reflection spectrophotometry, and the ability of the vascular endothelium to exclude large molecules (such as albumin) from entering the brain. The results obtained from these experiments will be correlated with quantitative biochemical analyses of catecholamine neurotransmitters, the enzymes that synthesize them and their metabolites and of compounds involved in intermediary metabolism. Since the locus ceruleus, in awake animals, is ordinarily activated during stressful conditions, experiments will explore the abnormal responses of the norepinephrine-depleted cerebral cortex during such stressful conditions, that are usually associated with increased metabolic demands such as immobilization stress and status epilepticus. The mechanisms underlying the influence of norepinephrine on cerebral physiology will be investigated vis-a-vis the density of noradrenergic receptors in the cerebral cortex and the pharmacological effect of various noradrenergic receptor agonists and antagonists on these functions.

Our aim is to continue the study of the biological role of endogenous cerebral norepinephrine in the control of brain metabolism and its local microvascular perfusion and capillary permeability. The physiological mechanisms underlying this influence of norepinephrine on brain function and their significance in abnormal pathophysiological conditions will be investigated. The results is previously obtained from this laboratory provide ample evidence for such a role, especially when cerebral metabolic requirements are increased, and during pathophysiological conditions such as hypertension, epilepsy, and brain ischemia. One of the paradigms chosen for these studies include the unilateral chemical lesion of the nucleus ceruleus in the rat. The locus ceruleus is the site of origin of most of the noradrenergic innervation of the cerebral cortex and hippocampus. Effects of locus ceruleus lesion will be studied using a combination of physiological, biochemical, pharmacological, and morphological methods in vitro and in vivo. Physiological studies will measure cerebral blood flow and capillary permeability, and will determine whether the noradrenergic innervation of the cerebral circulation plays a role in capillary recruitment and in maintaining the integrity of the blood-brain barrier. Functions of the blood-brain barrier will be assessed by its ability to exclude large molecules such as albumin from entering the brain, and by the active transport of water and electrolytes from blood-to-brain and vice versa. Biochemical and morphological methods will be used to study the response of noradrenergic receptors to the ablation of the putative noradrenergic innervation of cerebral blood vessels, and the mechanisms by which the brain compensates for chronic noradrenergic denervation after locus ceruleus lesion to restitute function. These latter experiments will shed light on the nature of factors that are responsible for the recovery of function, and will provide basic information regarding the phenomena of plasticity, regeneration, and sprouting in the adult mammalian brain.