Mathur, Manik
(2016)
The Blood-Brain Barrier integrity and function in ischaemic injury and multiple sclerosis with role of hypothermia.
PhD thesis, University of Nottingham.
Abstract
Background
Brain injury following transient or permanent focal cerebral ischaemia matures from a multifaceted succession of pathophysiological events that progress in time and space. The key biochemical events related with these pathologies include excessive cytoskeletal remodeling, modulation of tight junction proteins, induction of oxidative stress and excessive release of pro-inflammatory cytokines. In addition, other neuropathologies such as multiple sclerosis have been shown to account for the development of these events and the consequent disruption of human blood-brain-barrier (BBB) integrity and function. However this study emphases on potentially new avenues of treatment which can protect the integrity of the blood-brain barrier and decrease post ischaemic consequences such as vasogenic oedema. Hypothermia has been hypothesised as a potent neuroprotectant and its putative effects have been assessed in the present research work.
Objectives
This thesis focuses on the putative role of hypothermia during intra- and post-ischaemic stroke mimicked by oxygen-glucose deprivation and followed by reperfusion. The impact of hypothermia has been studied on various inflammatory pathways such as pro-inflammatory cytokine levels, rate of apoptosis, extent of cytoskeleton remodeling and integrity and function of the BBB. In addition, we have also focused on the neuroinflammatory effects of multiple sclerosis (MS) on the BBB integrity.
Method
To mimic ischaemic insult, human brain microvascular endothelial cells, human astrocytes and human pericytes were exposed to oxygen-glucose deprivation (OGD) for 4 and 20 hours in the absence or presence of hypothermia (intra-ischaemic episode) and 4 hours of OGD followed by 20 hours of reperfusion in the presence of hypothermia (post-ischaemic episode) and cells left untreated were served as controls. Similarly, to assess BBB properties, all three cells were cultured simultaneously to establish an in vitro model of human BBB before exposing them to the experimental conditions. The integrity and function of triple cultures were measured via transendothelial electrical resistance (TEER) and paracellular flux via permeability markers such as sodium fluorescein (NaF) and Evan’s blue-labelled albumin (EBA). Various inflammatory inhibitors were also used during the insult to assess the distortion of the barrier. Furthermore, the triple cultures were exposed to multiple sclerosis patients’ serum mainly to study the neuroinflammatory effects. Moreover, with help of specific enzyme-linked immunosorbent assay-based kits pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-1β were measured during intra- and post-ischaemic episodes in the absence or presence of moderate hypothermia. Lastly, to evaluate the status of oxidative stress during acute ischaemic insult, the levels of superoxide anion generation and NADPH oxidase activities were measured.
Results
This study shows that moderate hypothermia significantly decreases pro-inflammatory cytokine levels when introduced during intra- and post-ischaemic episodes. Additionally, moderate hypothermia also maintains the BBB integrity and function during intra-and post-ischaemic scenarios. As evidenced by significant increase in resistance across the barrier and by concurrent decrease paracellular flux values. In addition, moderate hypothermia manages to reduce the oxidative stress impact during intra- and post-ischaemic episodes as documented by a decrease in NADPH oxidase and superoxide anion levels. Furthermore, the outlined study shows that MS patients’ serum is fortified with various inflammatory components such as pro-inflammatory cytokines, increase rate of apoptosis and resulting in destruction of the BBB architecture.
Conclusions
In this study, we have found moderate hypothermia to be a robust neuroprotectant in during intra- and post-ischaemic episodes of ischaemic injury mimicked by oxygen-glucose deprivation alone or followed by reperfusion. If adequately and appropriately maintained, moderate hypothermia may be an effective therapeutic option with minimal adverse effects. Furthermore, MS associated BBB destruction shows a new conduit for future research.
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