William R. Brown, PhD - US grants

Based on results of our previous research, we hypothesize that certain surface antigens on colonic epithelial cells are polarized, i.e., expressed selectively on either the apical or basolateral plasma membranes. In colonic carcinoma, this polarity is lacking, and normal apical surface antigens become expressed diffusely on surfaces of the neoplastic cells. These aberrantly expressed antigens, in contrast to those that are restricted to apices of normal epithelial cells, are exposed to the circulation and, hence, should be accessible to specific attack by immunotherapeutic agents. We have attempted to develop an animal model of aberrantly expressed surface antigens in colonic cancer. Colonic cancers were induced in mice and rats by the administration of dimethylhydrazine (DMH). Antibodies specific for apical membrane antigens of normal, mature colonic epithelial cells of the mouse and rat were used to determine whether these apical antigens are expressed diffusely on cancer cells. It was found that the cancer cells did not express antigens detected by our antisera. This outcome suggests that the colonic cancers lack differentiation antigens normally present on mature colonic epithelial cells. The present work will investigate that possibility further. We will prepare plasma membranes from cells at various stages of differentiation in rat colonic crypts. The protein and glycoprotein components of the membranes will be analyzed and compared to corresponding components of plasma membranes prepared from DMH-induced colonic cancer cells. These data will identify cells in the normal crypt that correspond in membrane composition to the colonic cancer cells. Such normal cells will be isolated and used for the preparation of heterologous antibodies to apical membrane antigens. The antibodies will then be used to determine whether the normally polarized antigens on immature colonic cells become randomly expressed on DMH-induced in situ cancers. If this occurs, the antibodies will be used in immunotherapy against transplanted and in situ colonic cancers.

DESCRIPTION (provided by applicant): This proposal is focused on studies to elucidate the mechanisms of radiation-induced cognitive dysfunction. This is an important problem because 30-50% of the patients who survive more than 18 months after whole-brain irradiation (WBI) suffer from severe cognitive deficits. We have characterized a rat model of radiation-induced cognitive dysfunction using a fractionated dose of WBI that is biologically equivalent doses typically given to brain tumor patients. During those studies, we made the novel finding that the density of the capillary bed decreased in three areas of the brain, including the hippocampus which is involved in learning and memory. This decrease occurred as early as 10 weeks post-irradiation. There was partial recovery of the capillary bed by 20 weeks, and thereafter a decline in capillary density in the irradiated rats paralleled that observed in the controls, out to one year post-irradiation. The capillary loss occurred much earlier than vascular damage previously reported for high single doses of WBI and much earlier than the appearance of cognitive deficits, which occurred 6-9 months post-irradiation, as measured in a radial-arm maze. These findings suggest that a cascade of events, beginning with capillary loss, leads eventually to cognitive dysfunction. However, the limited numbers of animals (2-6) per group and sampling times, the lack of a quantitative assessment of demyelination and glial cell damage, and the absence of dose-response data leave it uncertain whether there is a causal relationship between the capillary loss and the cognitive deficits. The specific aims of the present grant proposal should strengthen the evidence. If early damage to the blood vessels can be correlated with the late onset of cognitive deficits, it will not only have important mechanistic implications, but it would also provide an early biomarker for testing therapeutic interventions designed to ameliorate radiation-induced brain dysfunction. Such therapeutic intervention studies could potentially be performed in as little as 10 weeks.

DESCRIPTION (provided by applicant): This proposal is focused on three important public health problems: brain injury associated with cardiac surgery; leukoaraiosis (LA), a neurodegenerative disease of the white matter; and Alzheimer's disease (AD). Brain microemboli resulting from cardiac surgery occur by the millions, and cause focal ischemia that may contribute to the brain dysfunction seen in about 1/3 of these patients. We will determine whether the microemboli can be reduced by washout with increased cerebral blood flow. We will count the microemboli in the brains of dogs that have had different rates of cerebral blood flow after surgery. There will be several variables affecting blood flow in the series of dog experiments. We will also determine whether the severity of the brain injury correlates with the numbers of microemboli, by studying markers of brain injury in tissue sections: microglial activation; leukocytes; stress protein; and blood brain barrier leakage. In the CSF,we will quantitate leukocytes and the soluble brain injury markers, neuron-specific enolase and S100beta protein. LA is thought to be caused by chronic ischemia resulting from vascular pathology. In autopsy material of LA subjects (diagnosed by MRI), we will quantify the vascular density in LA lesions, normal appearing white matter, and cortex. Similar studies will be done in age-matched controls. We will also quantify the number of string vessels in those three areas. String vessels are very thin (1 micron) collagenous cords, presumably vessel remnants, with no lumen or endothelial cells. We will also quantify the number and type of cells undergoing apoptosis in the three areas. We believe that vascular pathology, with resultant ischemia, is a contributor to brain degeneration in a subset of AD patients. In the brains of AD subjects, we will quantify the vascular density in areas affected by plaques and tangles, other areas of cortex, and white matter. Similar studies will be done in controls. We will also quantify the number of string vessels in those areas. In AD subjects, we will investigate the occurrence of LA lesions (by MRI) and study two vascular pathologies: venous stenosis and occlusion caused by redundant layers of mural collagen and tortuous vessels that cause small brain cavities. Clinical impact: we expect the dog surgery experiments will lead to safer cardiac surgery, and the studies of cerebrovascular pathology in human brains from LA and AD subjects will promote a better understanding of the pathophysiolooy of these diseases and how to treat or prevent them.

This proposal is focused on a clinically important area where we can once again have an immediate impact;preventing surgery-induced microembolic injury to the brain. Air and lipid microemboli enter the circulation during both cardiac and orthopedic surgery, causing ischemia, endothelial dysfunction, and inflammation. These emboli may occlude vessels or they can be extruded through the vascular bed and irritate the endothelium, resulting in acute and chronic neurologic, renal, and splanchnic injury. Our studies show that during cardiac surgery the major source of the lipid microemboli is from the blood salvaged from the surgical field and returned to the patient. Air emboli can come from several sources, such as from cannulae or foam in the blood. In the brain, the passage of microemboli causes blood-brain barrier (BBB) breakdown, and in other vascular beds it is associated with the systemic inflammatory response syndrome. Magnetic resonance imaging (MRI) studies reveal one out of three patients may suffer a new brain lesion following cardiac procedures. In orthopedic patients, during the pressurization of the medullary canal, fat from the bone marrow is extruded into the venous circulation. The fat embolization may cause acute respiratory distress syndrome, neurologic dysfunction, or death. We will use our dog model of cardiac surgery to study brain injury from lipid and air emboli. We will investigate air emboli introduced into the cardiopulmonary bypass (CPB) circuit in two locations, in three forms: air in the venous cannula; agitated air in water in the arterial line;and agitated air in blood (protein-coated gaseous microemboli; foam) in the arterial line. We will investigate whether air emboli can be prevented during open heart surgery by flooding the operative field with argon, a low partial pressure gas. We will also determine whether knee prosthesis surgery using a state of the art reaming system, with chilling and rinse- vacuum methodology is safer than traditional bone reaming. The outcomes will include: brain edema; intravital visualization and recording of intravascular emboli;BBB leakage into the cerebrospinal fluid; emboli count in the CPB circuit, carotid artery, and jugular vein using the EDAC. Quantifier;cerebral blood flow;stereological analysis of vascular endothelial tight junctions;quantification of brain tissue hypoxia (HIF-1[unreadable]);quantification of cellular stress (Hsp70);emboli counts in the brain, lungs, and kidneys;and lipid microemboli in blood samples. We will perform MRI on six groups of dogs which simulate surgical embolic events. Histological evidence will be compared to MRI studies to evaluate the MRI resolution, specificity, and sensitivity to detect the damage from emboli. The MRI analyses will include diffusion-weighted imaging (DWI), spin echo T1, and fast spin echo T2.