The mechanisms by which hyperglycaemia mediates its harmful effects during ischaemia have not been fully elucidated. Possible mechanistic explanations include: reduction in CBF, impaired recanalisation and increased reperfusion injury, increased lactic acidosis, enhancing excitotoxicity and increased oxidative stress and inflammation. These have mostly been investigated in animal models of stroke as only a few clinical studies have addressed the pathophysiological mechanisms associated with hyperglycaemia.
1.3.3.1 Reduced CBF
In the normal rat brain, an intraperitoneal injection of glucose to induce severe hyperglycaemia (plasma glucose: 39mmol/L) induced a 24% reduction in regional CBF (Duckrow, 1995). The reduction in CBF could not be fully explained by an increase in plasma osmolality as the injection of mannitol to induce a similar blood osmolality induced only a 10% reduction in regional CBF. In rodent models of global and focal ischaemia, moderate hyperglycaemia (blood glucose ~20mmol/L) induced a progressive reduction in CBF after the induction of ischaemia (Kawai et al., 1998). Similarly in cats, after 3 hours of focal cerebral ischaemia, moderate hyperglycaemia (plasma glucose ~20mmol/L) induced a 70% reduction in CBF after occlusion of the MCA compared with a lesser reduction of 34% in normoglycaemic animals (Wagner et al., 1992). If hyperglycaemia reduces CBF following ischaemic stroke this could lead to a significantly larger volume of hypoperfused tissue and subsequently to greater ischaemic damage and neurological deficits.
One way in which hyperglycaemia may reduce CBF is through altering the reactivity of the cerebral microcirculation. Mayhan and colleagues examined the response of rat cerebral arterioles In Vivo to various agonists (acetylcholine, histamine, NMDA and nitroglycerin) in the presence of increasing concentrations of glucose. They found that in the presence of glucose concentrations of 20 and 25mmol/L the dilation of the cerebral arterioles to the agonists acetylcholine, histamine and NMDA was inhibited but the response to nitroglycerin, a non- endothelium dependent agonist, was not (Mayhan and Patel, 1995). This indicated that acute exposure to high glucose impairs endothelium dependent (NO-mediated) vasodilation. Thus, elevated blood glucose levels may reduce CBF by impairing NO-mediated vasodilation. This is comparable to the reported effects of diabetes on the cerebrovasculature. Normally, exposure to 5% carbon dioxide (CO2) following normocapnia in healthy, non-diabetic patients causes
cerebral blood vessels to dilate, resulting in an increase in CBF. However this CO2-induced increase in CBF is markedly attenuated in diabetics, in which the
endothelial production of NO is known to be reduced (Dandona et al., 1978, Griffith et al., 1987, Lin et al.). In addition, the hyperglycaemia-mediated increase in ROS production (see below), can neutralise NO in the vessel wall which can further compromise cerebrovascular reactivity (Garg et al., 2006). Together this suggests that hyperglycaemia may reduce CBF by impairing NO production or reducing its availability.
1.3.3.2 Pre-stroke hyperglycaemia
As previously mentioned, over 50% of patients without a history of diabetes presenting with PSH are found to have diabetes or a related disorder of glucose metabolism at follow-up (3 months after stroke) (Gray et al., 2004). It is therefore highly possible that prior to their stroke, these patients had elevated blood glucose levels that could have induced morphological changes in the cerebrovasculature leading to neurovascular dysfunction. This is a condition in which the normal blood flow increases during neural activity are suppressed and is thought to be caused by the production of ROS at the level of the vascular endothelium (Moskowitz et al., 2010, Iadecola and Davisson, 2008). Morphological and functional changes might also occur in cerebral capillaries and this has been found to occur in conditions which pre-dispose to stroke such as aging, hypertension and diabetes (Bell and Ball, 1981, Tagami et al., 1990,
Junker et al., 1985, McCuskey and McCuskey, 1984). The role of such capillary changes in the aetiology and pathophysiology of stroke is uncertain at present. It is hypothesised that constriction of capillary pericytes (contractile cells that wrap around the endothelium of capillaries) during ischaemia could block the flow of blood and may explain the lack of reperfusion (no-reflow phenomenon) that occurs in some patients (Yemisci et al., 2009, Ostergaard et al., 2013). Prior to stroke, changes in capillary morphology could disturb the passage of blood through capillary beds, affecting local oxygen delivery. This could contribute to the abnormal CBF responses found in neurovascular dysfunction. During ischaemia this could be detrimental to the ischaemic penumbra where the survival of tissue depends on effective oxygen extraction to compensate for the reduced perfusion (Ostergaard et al., 2013).
1.3.3.3 Impaired recanalisation and increased reperfusion injury
Hyperglycaemia is associated with lower recanalisation rates in ischaemic stroke patients treated with rt-PA (Ribo et al., 2005). This may be caused by a decrease in plasma fibrinolytic activity and elevated plasma levels of plasminogen activator inhibitor type 1 (PAI-1), which have been reported to occur in both acute (Pandolfi et al., 2001) and chronic hyperglycaemic states (Auwerx et al., 1988, Juhan-Vague et al., 1989). Both a decrease is plasma fibrinolytic activity and increased levels of PAI-1 can reduce the thrombolytic activity of rt-PA and impair its ability to recanalise occluded blood vessels. The lower recanalization rates in stroke patients with PSH may explain the worse outcome observed in these patients as this could prevent the restoration of blood flow to the potentially viable tissue in the ischaemic penumbra causing this tissue to become incorporated into the infarct.
Evidence from animal models of transient focal cerebral ischaemia indicates that hyperglycaemia may exacerbate reperfusion injury (Nedergaard, 1987, Huang et al., 1996a, Kamada et al., 2007, Wei et al., 1997). Reperfusion injury is the tissue damage caused by the restoration of blood to previously ischaemic tissue and is believed to be caused by the generation of ROS upon restoration of CBF. Hyperglycaemia may exacerbate reperfusion injury by increasing the production of free-radicals during reperfusion (Wei et al., 1997). This could lead to microvascular damage which in turn could impair the return of blood flow to the
tissue even after recanalization. Evidence from animal stroke studies supports this hypothesis as studies have shown that the restoration of regional CBF to pre- ischaemic levels following transient focal cerebral ischaemia was impaired in hyperglycaemic but not normoglycaemic animals (Kawai et al., 1997b, Wei et al., 2003).
1.3.3.4 Lactic acidosis
During ischaemia, the reduction in CBF and the resulting lack of oxygen delivery causes a shift from aerobic to anaerobic metabolism of glucose. This leads to the production of lactic acid. The level of lactic acid production is dependent on the amount of glucose available. Therefore, elevated blood glucose levels during ischaemia leads to increased levels of lactic acid and subsequently intracellular acidosis in the brain tissue. Tissue acidosis may lead to greater ischaemic brain injury through enhancing the production of free radicals, activation of endonucleases leading to DNA fragmentation and altering gene expression or protein synthesis (Siesjo et al., 1996). Excessive lactate production may also cause the hypoperfused, yet potentially viable tissue in the ischaemic penumbra to progress to infarction. Evidence from clinical and experimental studies supports this. Parsons et al (2002) measured brain lactate levels, using magnetic resonance spectroscopy, in patients with PSH with evidence of perfusion-diffusion mismatch tissue. They found that higher blood glucose levels were associated with increased lactate production in the ischaemic brain, which in turn was associated with reduced salvage of mismatch tissue. In addition, Anderson et al reported that severe hyperglycaemia (plasma glucose: 28mmol/L) worsened tissue acidosis and mitochondrial function in the ischaemic penumbra after permanent focal cerebral ischaemia in rabbits. Infarct volume was significantly greater in hyperglycaemic animals compared to controls and this supports the hypothesis that tissue acidosis leads to the recruitment of ischemic penumbra into infarction. In the normal brain, despite the fact that lactate is considered to be potentially toxic, it is now recognised that lactate may be a valuable energy substrate for neurons in cases of high energy demand (Pellerin, 2010). Moreover, lactate metabolism is also recognised as an important substrate for ATP production during ischaemia (Pellerin and Magistretti, 1994) and therefore may be crucial for survival of the ischaemic penumbra. Furthermore, reducing brain lactate levels in patients with PSH did not influence
infarct growth (McCormick et al., 2010), indicating that brain lactic acidosis is not the only mediator of brain injury in hyperglycaemic ischaemia.
1.3.3.5 Hyperglycaemia-mediated increase in excitotoxicity
Hyperglycaemia may exacerbate the excitotoxic response after ischaemia. Following both global and focal cerebral ischaemia in rodents, the rise in extracellular glutamate levels in the ischaemic cerebral cortex was reported to be significantly greater in hyperglycaemic compared to normoglycaemic animals (Li et al., 2000b, Wei and Quast, 1998, Choi et al., 2010). Furthermore, the increase in extracellular glutamate levels correlated with an increase in ischaemic brain injury (Wei and Quast, 1998). Increased extracellular glutamate levels during ischaemia could lead to an increase in glutamate receptor stimulation which in turn may lead to significantly greater levels of intracellular Ca2+ resulting in more Ca2+ mediated cell death. The increase in Ca2+ may also
increase the activation of calcium-dependent proteolytic enzymes which can also lead to increased brain injury. In particular, an increase in the activation of the calcium-dependent cysteine protease calpain has been reported in a rat model of chronic hyperglycaemia (Smolock et al., 2011).
1.3.3.6 Increased oxidative stress and inflammation
Hyperglycaemia is linked to increased oxidative stress following ischaemia. The production of ROS during ischaemia increases in the presence of elevated glucose levels, particularly in combination with reperfusion (Tsuruta et al., 2010). Hyperglycaemia can increase the production of superoxide through the enzyme NADPH oxidase. During ischaemia/reperfusion, elevated glucose levels helps to sustain the production of NADPH, a substrate for the production of ROS, through the pentose phosphate pathway (Tang et al., 2012). The increase in superoxide production during hyperglycaemic ischaemia can disrupt the blood brain barrier leading to increased vasogenic oedema (Kamada et al., 2007).
Hyperglycaemia has also been found to have a profound effect on the inflammatory response after ischaemia. In particular, hyperglycaemia has been shown to increase several transcription pathways that increase the migration of inflammatory cells such as leukocytes into the injured brain (Li et al., 2013). The migration of circulating leukocytes into the brain is thought to amplify
inflammatory signalling pathways which in turn will enhance ischaemic damage (Wang et al., 2007). In addition, the infiltration of leukocytes into the brain following transient focal ischaemia is enhanced under hyperglycaemic ischaemia (Lin et al., 2000).