Studies Of Pathological Dynamics After Microvascular Injury Using Nonlinear Optical Methods
Microvascular lesions are a common feature in the aging brain and clinical evidence has correlated microvascular pathology with the development of neurodegenerative diseases such as Alzheimer's disease and dementia. Traditional animal models that replicate hemorrhagic and ischemic lesions in the brain typically affect large regions in the cortex and do not reproduce the small-scale lesions linked to neurodegeneration that likely stem from injuries to single microvessels. Due in part to this lack of smallscale injury animal models, there remains an incomplete understanding of the cellular and pathophysiological dynamics following small-scale vascular lesions, making progress on therapeutic strategies difficult. We used tightly focused femtosecond laser pulses to injure single penetrating arterioles (PA) (i.e., arterioles that plunge into the brain) in the cortex of live anesthetized rodents and used two-photon excited fluorescence (2PEF) imaging to quantify blood flow changes and to determine the time course of pathological consequences in the brain after injury. We find that after ischemic occlusion of a PA, nearby pial and penetrating arterioles do not actively compensate for the reduction of blood flow observed near the occluded blood vessel. We find that capillaries connected downstream to the clotted vessel dilate but other capillaries in the vicinity do not, suggesting that any compensatory signal that results in a physiological response travels vascularly. We ruptured individual PAs to induce microhemorrhages that resulted in extravasation of blood into the parenchyma. We find that tissue compression due to the hematoma does not collapse capillaries and cause acute ischemia. 2PEF imaging of mice expressing yellow fluorescent protein (YFP) in a subset of cortical neurons revealed no dendrite degeneration out to seven days after microhemorrhage. However, we did observe an inflammatory response by microglia/macrophages as quickly as 1.5-hrs after microhemorrhage which persisted past seven days. Lastly, we looked at spine (i.e., post-synaptic terminals on dendrites) dynamics on GFP fluorescent cortical dendrites and found a higher rate of spine loss and gain after a nearby microhemorrhage out to 14 days. This higher rate of spine turnover may help provide an understanding of the development of symptomatic dysfunction due to consequences in neuronal rewiring after a microhemorrhage. The work presented in this dissertation provides quantification of pathological consequences after both ischemic and hemorrhagic injury to a single blood vessel in the brain. We see that after a small-scale ischemic lesion, surrounding blood vessels do not elicit an active response to compensate for a lack of blood flow in the targeted blood vessel and surrounding tissue. After a hemorrhage to a single blood vessel, we do not observe any neuronal degeneration or death. These hemorrhagic lesions, however, do result in an inflammatory reaction that may lead to subtle changes in neuronal rewiring or seed the development of neurodegenerative diseases. The work presented in this dissertation can help provide new insights for the development of novel stroke therapeutics as well as provide cell specific observations about the development of pathological consequences in both ischemic and hemorrhagic lesions in the brain.
two photon imaging; hemorrhage; microvascular injury
Xu, Chunhui; Fetcho, Joseph R.
Ph. D., Biomedical Engineering
Doctor of Philosophy
dissertation or thesis