Periodic Report Summary 1 - NEUVASCHEMIA (Neurovascular coupling in stroke - the brain microvasculature as a target for prevention of ischemic brain damage) Project objectives:The overall aim of the project was to reveal the molecular mechanisms underlying dysfunctional neurovascular coupling after stroke, and identify novel targets for treatment aimed at normalising brain blood flow in stroke patients. Rationale Every three minutes someone dies from a stroke, which occurs when the blood supply to a part of or the entire brain is disrupted, typically due to a brain haemorrhage or blood clot or a cardiac arrest. In Europe, stroke is the second most common cause of death and the leading cause of long-term disabilities (European Heart Network 2008), leaving many sufferers dependent others to care for them. With an ageing European population the human and economic costs of stroke are likely to increase significantly in the future. For decades, most stroke research has focused on protection of neurons from ischemic damage by blockade of neuronal cell death mechanisms, but without clinical success. However, this approach overlooks the fact that without a properly regulated blood circulation in the brain in the days following a stroke, any attempt of neuroprotection will have low chances of success. To develop better stroke therapies, we must understand how the brain vasculature and its interaction with neurons and glia cells, termed the neurovascular unit, is altered after a stroke. This was emphasised by the National Heart, Lung and Blood Institute Working Group on Cerebrovascular Biology and Disease: ‘enhanced basic knowledge about the pathobiology of the vascular component of the neurovascular unit would promote translational approaches leading to novel stroke treatment strategies’ (Iadecola et al., 2006). Professor Edvinsson’s lab has shown that arteries on the brain surface (pial arteries) upregulate certain vasoconstrictor receptors after stroke, leading to decreased cerebral blood flow and worsened outcome. However, pial arteries contribute only half the vascular resistance in the brain. The other half lies in their branches penetrating into the brain, called brain parenchymal arterioles, which are enveloped by astrocytic processes called endfeet. When a brain area is busy, e.g with cognitive tasks, the neuronal activity is ‘sensed’ by local astrocytes that via their endfeet induce increased blood flow in local parenchymal arterioles to meet the area’s metabolic need. This is termed neurovascular coupling. It has been demonstrated that neurovascular coupling can be impaired in stroke patients, even months after the stroke, but the underlying mechanisms are not understood.Research objectivesThe objectives of the project was to answer the following four questions:1) Is neurovascular coupling impaired following a global ischemic stroke in mice?2) Are intracellular Ca2+ signals in the neurovascular unit altered after global ischemic stroke?3) Do brain parenchymal arterioles upregulate vasoconstrictor receptors after global ischemic stroke?4) Can dysfunctional neurovascular coupling after stroke be prevented by inhibition of the extracellular regulated kinase-1/2 (ERK1/2) pathway?Work performed since the beginning of the project:1) With the aim of answering objective 1 and 2, neurovascular coupling was investigated in acute brain slices from sham-operated rats and rats with induced global cerebral ischemia. By means of a unique technique developed in Mark Nelson's laboratory (outgoing host), brain slices loaded with the calcium indicator Fluo-4 were stimulated with electrical field stimulation to engage neurovascular coupling, and calcium signals in astrocytic endfeet as well as diameter changes of parenchymal arterioles in response to the neuronal stimulation were measured and quantified.2) With the aim of answering objective 3, pilot experiments were performed investigating the putative upregulation of G-protein coupled vasoconstrictor receptors in isolated, pressurized parenchymal arterioles from rats with global cerebral ischemia compared to sham-operated rats. However, since no profound differences in the contractile properties of the ischmeic arterioles were observed, the fellow decided to continue her investigations focusing on changes in the dilatory function of inward rectifyer potassium channels in the parenchymal arterioles. The function of these channels were investigated in isolated, pressurized parenchymal arterioles as well as in acute brain slices.Since the research related to objective 4 was planned for the second year of the proejct, these investigations will not be performed, due to early termination of the project.Main results achieved so far:1) In acute brain slices, the fellow demonstrated that the dilation of parenchymal arterioles in response to neuronal stimulation (that is the process of neurovascular coupling) is completely abrogated in rats subjected to global cerebral ischemia. However, when the calcium signals in astrocytic endfeet that mediates the neurovascular coupling process were invetsigated these signals turned out to be unaltered in the ischemic rats. This suggets that the deficit giving rise to the abrogated neurovascular coupling is to be found in the parenchymal arterioles, rather than in the neuronal and astrocytic components of the neurovascular unit. 2) When investigating the dilatory function of inward rectifying potassium channels in parenchymal arterioles by stimualting isolated arterioles and arterioles in acute brain slices with small increases in the extracellular potassium concentration, the fellow demonstrated that the function of these channesl is markedly reduced after global cerberal ischemia. This explains the inability of the parenchymal arterioles to dilate in response to neuronal stimulation, that is the abrogated neurovascular coupling.Expected final results and their potential impact and use:Since the project will be terminated early (November 2013) the results described above are the final results of the project.The project and the achieved results will have impact at two levels:A) via the qualifications gained by the fellow during her stay at the outgoing institution. Towards the end of her stay at the outgoing institution, the fellow was offered a research position in a larger Danish Pharmaceutical company, which she accepted. The job offer was highly motivated by the qualifications obtained by the fellow at the outgoing host institution and the secondary competences obtained as a Marie Curie International Outgoing Fellow. The fellow will now implement these new scientific and secondary competencies for the benefit of European pharmaceutical industry.B) via the obtained research results. The research conduced during the project has recealed a new aspect of the pathophysiology following incidences of global cerebral ischemia. The fellow has demonstrated that the process of neurovascular coupling, which ensures sufficient blood supply to active brain areas under normal circumstances, is completely abrogated after global cerebral ischemia. This has potentially devastating consequences in terms of worsening of the ischemic brain damage following for example a cardiac arrest. Moreover, the research conducted by the fellow has uncovered one of the important molecular mechanisms underlying the abrogation of neurovacular coupling after global cerebral ischemia, namely the reduced function of inward rectifying potassium channels in the smooth muscle layer of the cerebral parenchymal arterioles penetrating and supplying the brain tissue. This deepened understanding of the molecular mechanism behind ischemic abrogation of neurovascular coupling may lead to development of novel therapeutic strategies targeting the cerebrovascular pathophysiology following cardiac arrest.