Understanding The Mechanisms Leading To Microvascular Dysfunction And Poor Cerebral Perfusion In Stroke

INTRODUCTION

Thrombosis Research Group

The Thrombosis Group at the Heart Research Institute (HRI) conducts experimental and clinical research into atherothrombotic disease. This research aims to establish the mechanisms that lead to platelet hyperactivity and pathological blood clot formation in healthy individuals. This experimental foundation can then serve as a starting point for the development of new approaches in treating and preventing devastating diseases like stroke and myocardial infarction. A current, and vital, focus of research within the Thrombosis Group is on the mechanisms leading to microvascular dysfunction and poor cerebral perfusion in stroke (Heart Research Institute, 2019).

Ischaemic Stroke and Reperfusion Injury

Acute ischaemic stroke is caused by a thrombotic or embolic arterial occlusion in the brain that decreases local blood flow resulting ischaemic cell injury and, eventually, cell death (Dirnagl, Iadecola and Moskowitz, 1999). A cerebral infarct consists of a core region, in which functional impairment of the cell has progressed to irreversible injury characterised by morphologic change, and a surrounding penumbra, in which (intermittent) functional impairment of the cell has not progressed and is thus reversible (Heiss, 2000). As hypoperfusion endures, penumbral tissue is recruited into the core, growing the region of inevitable necrosis (Baron, 1999). The basis of acute ischaemic stroke therapy is to salvage reversibly injured tissue through early reperfusion. The mainstay of medical treatment is intravenous administration of a thrombolytic agent, which has a successful recanalization rate of almost half (Rha and Saver, 2007).

Recanalization does not invariably progress to complete resolution of reversible injury; several pathologic sequelae can result from ischaemia and/or subsequent reperfusion, causing further local injury and possibly remote organ damage. One such phenomenon, called microvascular obstruction (MVO) or no-reflow, occurs in the parenchymal tissue surrounding the occlusion as a result of ischamia/reperfusion (I/R) injury (Kloner, King and Harrington, 2018). Despite resumed patency in the affected vessel, reperfusion may be limited in micro vessels due to ischaemic swelling and post-ischaemic inflammatory reactions, the latter of which may circulate to other regions of the body (Yuan et al., 2017). Another, very serious, complication following restoration of blood flow (either spontaneously or by thrombolysis) to vasculature with an ischaemia- or reperfusion injury-induced increase in endothelial permeability can result in haemorrhage (Álvarez-Sabín et al., 2013). The mechanisms that underly these phenomena are not fully understood and thus development of therapeutic and preventative treatments is somewhat limited (Albers et al., 2011).

RESULTS

Laboratory Approaches to Microvascular Obstruction

The present understanding of the mechanisms underlying microvascular dysfunction in stroke has been elucidated by a range of traditional and novel techniques. To understand the role of the haemodynamic disturbances caused by thrombi on neutrophil recruitment, an experiment conducted at the HRI utilised a novel model of the thrombo-pathologic vasculature. Under a confocal microscope, isolated platelets were passed through a microfluidic channel embedded with either a 10m- or 40m-tall post. The platelets were then removed, an agonist was (variably) added, and finally neutrophils were passed through the channel.

A single experimental run in not sufficient to draw meaningful conclusions from. However, the overall trends from this experiment are well reflected by the data above. It is clear that a larger post size corresponds with a greater amount of neutrophil recruitment, in states of stimulated and unstimulated platelet-posts. The increase in neutrophils between the 10m and 40m posts may indicate that, independent of platelet activation, larger flow disturbances cause greater neutrophil recruitment. The increase in neutrophils between the unstimulated and stimulated platelet-posts may confirm that platelets play a role in neutrophil recruitment and trafficking.

In order to quantify fibrin formation and platelet deposition on ischaemia/reperfusion-injured epithelium, another experiment conducted at the HRI utilised in vivo models and fluorescent microscopy. Using mice, a sham (surgery without ischaemia/reperfusion) and a disease (surgery and ischaemia/reperfusion) model were prepared and injected with fibrin, platelet, and endothelial injury (Annexin V) labels. After the sham and disease procedures, the mice were sacrificed, and the ischaemic tissue was imaged. The relationship between endothelial injury and fibrin formation/platelet deposition was analysed using specialised software.

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It is clear from these results that greater endothelial injury is associated with higher levels of fibrin formation and platelet deposition. There is a trend in the ischaemia/reperfusion model that shows that increasing levels of platelet deposition correspond with increasing levels of endothelial injury. There is no obvious trend in either of the sham model measures nor the levels of fibrin formation in the ischaemia/reperfusion model. A larger data set could confirm or explicate these findings.

Findings from Specific Laboratory Techniques

The first experiment described, in which thrombi size and neutrophil recruitment were correlated, involves a relatively novel technique. Microfluidic channels have been previously used to model the microvascular environment in stroke, leading to a greater understanding of a variety of pathologic mechanisms including: the relationship between endothelial cells and inflammatory cascades (Sfriso et al., 2018) and the neutrophil-based pathogenesis of renal ischemia-reperfusion injury (Chaturvedi et al., 2013). However, the microfluidic channels embedded with posts were created especially by HRI researchers for this project and thus other research has not utilised the same model yet.

The second experiment described, in which endothelial injury and fibrin and platelet levels were correlated, involves a relatively modern and widely used technique. Related research conducted at the HRI utilised these experimental processes in the discovery that neutrophil-platelet macroaggregates formed in the gut, which are bound by platelet membrane fragments, cause a distinct thrombotic pathology in the lungs (Yuan et al., 2017). Two other important findings to come from research based on vivo models imaged using fluorescent microscopy are that: protein aggregates accumulate in neurons during ischaemia and contribute to cell death (Hu et al., 2001); and matrix metalloproteinases degrade tight junction proteins resulting in BBB disruption and thus oedema after reperfusion (Rosenberg and Yang, 2007).

CONCLUSIONS

Hypothesised Mechanisms of Microvascular Obstruction

Ischaemia/reperfusion injury manifests as both structural (including endothelial swelling and intravascular, blood-component obstruction) and functional (including extravascular-driven vessel compression) changes (Granger and Kvietys, 2017). Intravascular obstruction seems to occur due to a combination of factors, important among which are endothelial cell swelling due to the sequelae of changes in ionic homeostasis and capillary plugging by blood components (Bai and Lyden, 2015). The central pathologic event underlying cytotoxic oedema (ischaemic swelling) appears to be hypoxic ATP depletion, which impairs the action of Na+/K+-ATPase pumps leading to intracellular sodium accumulation (Song and Yu, 2013). The mechanisms underlying capillary plugging likely involves inflammatory stimulation and aggregation of leukocytes (mainly neutrophils) as well as platelets and fibrin (Danton and Dietrich, 2003). Reperfusion of ischaemic tissue activates or releases various inflammatory mediators, including complement proteins, cytokines (e.g. IL-1) and reactive oxygen species, and exposes the injury-revealed extracellular matrix components (e.g. tissue factor, collagen) to blood flow (Burrows et al., 2016; Nieswandt, Pleines and Bender, 2011). Respectively, this leads to an increase the expression of adhesion molecules on endothelial cells (ICAM-1, P-selectin, etc.), allowing for leukocyte recruitment, and activation of platelets and fibrin formation, ultimately resulting in a leukocyte-platelet plug (Yilmaz and Granger, 2009; Thomas et al., 1993).

Therapeutic Targets

A number of potential targets exist to develop therapies for the treatment of microvascular obstruction due to cerebral ischaemia/reperfusion injury. Agents to block leukocyte receptors to prevent endothelial interaction have shown early experiment success (Smith et al., 2015; Stoll and Nieswandt, 2019). Inhibition of platelet activity in the I/R injured tissue has had success and failure – agents to block platelet activation have not shown much promise (Kleinschnitz et al., 2007), while agents to block platelet adhesion have had early success (Chen et al., 2018). Agents to inhibit complement activation and antioxidant therapy have been proposed with initial studies showing varying results (Eltzschig and Collard, 2004).

Despite significant progress, there is still not a complete picture of the mechanisms behind microvascular obstruction and ischaemia/reperfusion injury at large. Particularly important areas of the pathogenesis yet to be understood include the inflammatory cascade that leads to leukocyte recruitment and the multifactorial process that creates blood brain barrier dysfunction.