Changes in structure and function of cultured cerebral arterioles : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
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[College Station, Tex.] :
[Texas A&M University System Health Science Center],
[2010]
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| Abstract: | ABSTRACT: Delayed cerebral vasospasm is a major cause of morbidity and mortality following subarachnoid hemorrhage. Recent evidence has suggested the involvement of the microcirculation in the development of cerebral ischemia associated with vasospasm. We sought, therefore, to describe the in vivo homeostatic environment of cerebral (pial) arterioles, and then develop two ex vivo organ culture systems with which to investigate the effects of serum and clotted blood on arteriolar structure and function. In our initial investigations into the in vivo environment of pial arterioles, which are embedded within the pia mater and contact brain parenchyma, we hypothesized that axial and circumferential perivascular tethering would limit circumferential wall stretch and stress. Using a cannulated microvessel preparation, the mechanics of pial arterioles were quantified in vitro at low (1.10) or normal (1.24) levels of axial stretch as well as with or without circumferential perivascular tethering. Neither circumferential stretch nor stress differed significantly with changes in axial stretch, but myogenic responsiveness was enhanced slightly at the lower level of stretch. Circumferential tethering caused arteriolar geometry to change from a circular cross section at physiological pressure to an elliptical one at non-physiological pressures. We calculated that the observed levels of ellipticity could cause a modest decrease in volumetric blood flow or a more pronounced alteration in shear stress around the circumference of the arteriole. An elliptical cross-section may thus increase vascular resistance or promote inward remodeling at non-physiological pressures. This characterization of effects of perivascular tethering of pial arterioles may help explain perturbations of cerebral blood flow under some pathological conditions as well as the propensity of the cerebral circulation to remodel in response to dramatic changes in pressure. We next developed an ex vivo organ culture system to study the changes in cerebral arteriolar structure and function that might take place after subarachnoid hemorrhage. Cerebral or skeletal muscle arterioles were cultured in a pressure myograph system in cell culture media. Contractile and dilatory responses, as well as passive diameter and wall thickness, were measured after 0, 24, 72, and 120 hours of culture. Control culture conditions maintained cerebral arteriolar contractility for 5 days with only a moderate loss of the myogenic response. The passive pressure-diameter relationship, the circumferential stretch-stress relationship, and arteriolar wall thickness were not altered with culture. In contrast, skeletal muscle arterioles demonstrated a more rapid loss of contracility, a reduction in passive diameter, and a dramatic decrease in compliance. Culture with a high concentration of serum, which contains many factors implicated in the development of cerebral vasospasm, did not elicit remodeling of either cerebral or gastrocnemius arterioles. In summary, these results suggest an inherent, organ-specific difference in the control of arteriolar remodeling of cerebral and skeletal muscle arterioles. We further concluded that the pressure myograph-based culture protocol is an appropriate technique with which to study the vascular remodeling that might contribute to cerebral ischemia following subarachnoid hemorrhage. Because serum and clotted blood contain many pro-angiogenic factors, we developed a second organ culture system to investigate the potential effects of a subarachnoid clot on neovessel formation from cerebral arterioles. As in the previous study, arterioles from the gastrocnemius were used as a comparison. Arterioles were embedded in a soft collagen gel made with media containing 1, 5, 10, or 20% rabbit serum and imaged at days 0, 2, 4, and 6 of culture. In cerebral arterioles, we found that endothelial cell migration, network formation, angiogenic sprouting, and adventitial collagen production all increased with serum concentration. In contrast, the cells that migrated from gastrocnemius arterioles did not form branched networks and very few angiogenic sprouts were seen, suggesting another inherent, organ-specific difference in the control of neovascularization between cerebral and skeletal muscle arterioles. In the last study, we used both organ culture systems to investigate the effects of clotted arterial blood on cerebral arterioles. Although the exposure of cannulated arterioles to clotted blood caused mild vasoconstriction, prolonged exposure decreased arteriolar contractility and increased wall cross-sectional area, but did not cause any vasospasm-like remodeling. Culture of collagen-embedded arterioles with clotted blood enhanced cell migration and angiogenic sprouting, but decreased the numbers of network-forming cells migrating from the basilar artery. To identify the factor(s) produced by clotted blood responsible for the changes seen in our culture systems, we cultured non-heparinized blood for up to 5 days and used heparin affinity chromatography to identify proteins that were released in a delayed manner as the clot degraded. We discovered a ~70 kDa protein that was released into the serum in large quantities on days 1 to 3 of culture; this protein was identified as histidine rich glycoprotein (HRG) by N-terminal amino acid sequencing and western blotting. Cannulated arterioles were found to respond biphasically to HRG by dilating at low concentrations (50 to 100 nM) and constricting at higher concentrations (0.5 to 2 [mu]M). Arterioles cultured with HRG (100 nM) displayed reduced contractility and increased wall cross-sectional area, much like those cultured with clotted blood. In collagen-embedded arterioles, however, HRG decreased numbers of angiogenic sprouts, but did not significantly affect other indices of neovascularization. In summary, we have described the in vivo mechanical environment of cerebral arterioles on the pial surface and have used this data to develop a culture system that maintains cerebral arteriolar contractility for up to 5 days without causing vascular remodeling. A second organ culture system was also designed to investigate angiogenesis and vasculogenesis in intact arterioles; using these two systems, we documented fundamental differences in remodeling and neovascularization between cerebral and skeletal muscle arterioles. Finally, we used the ex vivo culture systems to describe the effects of clotted arterial blood, as well HRG, a degradation product of the ageing blood clot, on cerebral arterioles. While our experiments were not successful in replicating vasospasm in culture, we have gained valuable insight into the possible effects of clotted blood on cerebral arterioles. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Office of Research and Graduate Studies of The Texas A&M University System Health Science Center in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2010." Approved as to style and content by: Jay D. Humphrey, Emily Wilson, Gregory J. Bix, Jonathan A. Friedman, Travis W. Hein, Harris Granger. |
| Physical Description: | xvi, 192 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 175-176). |