Development of white matter stroke model

Abstract

Chronic ischemia in the white matter leads to progressive degeneration of axons and/or myelin sheath surrounding them. Ischemic white matter degeneration is a frequent cause of cognitive decline in the elderly. Furthermore, ischemic changes in the white matter interact with amyloid pathology, worsening Alzheimer’s dementia-related pathological changes. Yet, it remains elusive how and why chronic ischemia leads to degeneration of myelin sheath and axonal degeneration, and as a result, there is no effective treatment to prevent ischemic white matter degeneration. The most daunting barrier to the development of effective treatment for white matter stroke is the difficulty to closely model in an experimental system the ischemic white matter degeneration that occurs in humans. The aim of my thesis research was to develop in vivo and in vitro model of ischemic/hypoxic white matter degeneration. The first part was to generate the white matter stroke animal model by combining hypertension and chronic cerebral ischemia in vivo. The second part was to establish an in vitro ischemic white matter degeneration model using cultured cerebellar slices where myelinated white matter tracts are well preserved. In the first part of the thesis, I sought to develop an animal model of ischemic white matter injury by inducing chronic cerebral hypoperfusion in animals with hypertension, which is the most significant modifiable risk factor for vascular cognitive impairment. Influence of hypertension on the development of ischemic white matter injury and cognitive dysfunction is not fully understood. I compared cognitive functions and neuropathological outcomes of chronic cerebral hypoperfusion induced by bilateral common carotid artery occlusion (BCCAO) between normotensive rats (NRs) and spontaneously hypertensive rats (SHRs). SHRs developed earlier and more severe deficits in spatial memory performance than NRs following BCCAO. Although no significant changes in the gross structure of myelinated white matter or oligodendrocyte number were noted, BCCAO resulted in subtle myelin degeneration and paranodal structural alterations at the nodes of Ranvier, regardless of hypertension. Disruption of the blood–brain barrier (BBB) was predominantly observed in the white matter of SHRs following BCCAO, implying a role of hypertension in BBB dysfunction in chronic cerebral hypoperfusion. In chronic cerebral ischemia, long-standing hypertension may aggravate impairment of BBB integrity, and the leaky BBB may in turn exacerbate dysfunction in the white matter leading to worsening of spatial cognitive performance. In the second part of the thesis study, I aimed to develop an in vitro model of ischemic white matter degeneration using cultured cerebellar slices. Cerebellar slices were obtained from postnatal day 12 mice and cultured for 12 days in vitro. At this time point, subcortical white matter axon bundles survived with exuberant myelination. Most of surviving axons from cerebellar cortices were positive with Purkinje cell marker calbindin-D28k. The cultured cerebellar slices were exposed to 2% hypoxic condition for 48 hours beginning at 10 day in vitro. Hypoxic insult resulted in white matter axons with beading appearances consisting of focal swelling and constriction. In contrast, myelin basic protein expression was preserved even at the site of severe axonal damages. Moreover, there was no significant decrease or loss of oligodendrocytes, suggesting that axonal structures are more vulnerable to hypoxia than myelin ensheathment. Electron micrographs showed accumulation of lysosome-like structures in the axoplasm after hypoxia. Consistent with the EM findings, lysosomal-associated membrane protein 1 (LAMP-1) immunoreactivity was significantly increased in the white matter in a hypoxic condition. The AMPA/kainate receptor antagonist NBQX, calpain inhibitor MDL28170, and lysosomal protease inhibitor pepstatin A, significantly attenuated hypoxia-induced beading appearances. NBQX does not prevent calpain induced alpha-spectrin proteolysis. Our slice model could be utilized to explore molecular mechanisms of ischemia-induced axon degeneration. AMPA/kainate receptor and calpain proteolysis may mediate hypoxia induced axonal degeneration in distinct pathways. Based on the results of my thesis study, I reached a conclusion that any in vivo or in vitro model for white matter ischemia developed so far could not completely represent all the components of pathological changes observed in human white matter stroke. For the time being, therefore, it would be a rational approach for the time being to find out specific pathomechanisms in each different white matter ischemia model that reflects distinctive white matter pathologies. In the future, however, more efforts should be invested to develop an animal model that more closely replicates human pathological findings in the white matter stroke, for example, by combining BBB disruption or hypoxia with cerebral hypoperfusion. To employ large animals like non-human primates or pigs might be alternative approaches to study pathomechanisms in the white matter stroke.