Tau pathology and neurochemical changes associated with memory dysfunction in an optimised murine model of global cerebral ischaemia - A potential model for vascular dementia?

Highlights

• This study optimised a murine model of global cerebral ischaemia.

• Cerebral ischaemia produced consistent and selective neuronal and glial cell changes in the hippocampus and the cortex.

• Cerebral ischaemia induced reduction in glutamate transporters and increase in PHF tau protein in the hippocampus.

• Reduced glutamate transporters and increased PHF tau protein are associated with memory deficits in ischaemic mice.

• This murine model may represent a preclinical model of vascular dementia.

Abstract

Cerebral ischemia is known to be a major cause of death and the later development of Alzheimer's disease and vascular dementia. However, ischemia induced cellular damage that initiates these diseases remain poorly understood. This is primarily due to lack of clinically relevant models that are highly reproducible. Here, we have optimised a murine model of global cerebral ischaemia with multiple markers to determine brain pathology, neurochemistry and correlated memory deficits in these animals. Cerebral ischaemia in mice was induced by bilateral common carotid artery occlusion. Following reperfusion, the mice were either fixed with 4% paraformaldehyde or decapitated under anaesthesia. Brains were processed for Western blotting or immunohistochemistry for glial (GLT1) and vesicular (VGLUT1, VGLUT2) glutamate transporters and paired helical filament (PHF1) tau. The PHF1 tau is the main component of neurofibrillary tangle, which is the pathological hallmark of Alzheimer's disease and vascular dementia. The novel object recognition behavioural assay was used to investigate the functional cognitive consequences in these mice. The results show consistent and selective neuronal and glial cell changes in the hippocampus and the cortex together with significant reductions in GLT1 (***P < 0.001), VGLUT1 (**P < 0.01) and VGLUT2 (***P < 0.001) expressions in the hippocampus in occluded mice as compared to sham-operated animals. These changes are associated with increased PHF1 (***P < 0.0001) protein and a significant impairment of performance (*p < 0.0006, N = 6/group) in the novel object recognition test. This model represents a useful tool for investigating cellular, biochemical and molecular mechanisms of global cerebral ischaemia and may be an ideal preclinical model for vascular dementia.

Introduction

Cerebral ischaemia, which results from stroke, cardiac arrest and cardiac surgery, is one of the most common causes of death and disability worldwide (Flynn et al., 2008; Kim and Johnston, 2011). In acute ischemic stroke a blood vessel in the brain gets occluded by thrombosis or embolism, resulting in neuronal damage and death in an area surrounding the occluded vessel (focal ischaemia) whereas global cerebral ischaemia, which is caused by cardiac arrest and cardiac surgery, encompasses wide areas of brain tissue. The pathophysiology resulting from cerebral ischaemia is a leading cause of death and adult disability and a major risk factor for later development of various neurogenerative diseases including Alzheimer's disease and vascular dementia (De la Torre, 2004a, 2004b; Hazell, 2007; Pluta et al., 2013)^, A significant proportion (60–90%) of Alzheimer's disease and vascular dementia patients exhibit cerebrovascular pathology including cerebral infarcts and ischaemic lesions leading to more rapid cognitive decline in patients diagnosed with these diseases (Kalaria, 2000). However, cerebral ischaemia induced cellular damage that initiates cerebrovascular pathology and related memory dysfunction remain poorly understood and this has mired the development of new drug treatment strategies. The possible reasons for this failure include lack of a clinically relevant model that is highly reproducible as the pathophysiology of cerebral ischaemia injury in animal models is influenced by numerous factors including the species, type of blood vessels occluded, occlusion period and reperfusion time (Hossmann, 1998).

This study aims to optimise a murine model of global cerebral ischaemia and reperfusion and uses a variety of cellular and neurochemical markers to determine the extent of neuronal and glial cell damage and changes in glutamate transporters in the hippocampus and the cortex, as assessed using immunohistochemistry (IHC). Changes in glutamate transporter proteins in the hippocampus were quantified by Western Blotting (WB). Since there is a strong correlation between cerebral ischaemia and the neurodegenerative diseases (Fujii et al., 2016; Kalaria, 2000), we have examined the expression of hyperphosphorylated tau protein in the hippocampus to determine if ischemic insult in this model results in the development of tau pathology. Hyperphosphorylated tau is known to accumulate as paired helical filament (PHF) which is the main component of neurofibrillary tangle, one of the pathological hallmarks of many neurodegenerative diseases including Alzheimer's disease and vascular dementia (Andorfer et al., 2003; Rissman et al., 2017). We have also assessed memory correlates of histologically and biochemically determined ischaemic damage in this model. Our hypothesis is that cerebral ischaemia induced cellular damage through alterations in glutamate transporters in the brain lead to neurodegenerative pathology and memory dysfunction.