Stroke is a common and serious condition, with few therapies. Whilst previous focus has been directed towards biochemical events within neurons, none have successfully prevented the progression of injury that occurs in the acute phase. New targeted treatments that promote recovery after stroke might be a better strategy and are desperately needed for the majority of stroke survivors. Cells comprising the neurovascular unit, including blood vessels and astrocytes, present an alternative target for supporting brain rescue and recovery in the late phase of stroke, since alteration in the unit also occurs in regions outside of the lesion. One of the major changes in the unit involves extensive morphological transition of astrocytes resulting in altered energy metabolism, decreased glutamate reuptake and recycling, and retraction of astrocyte end feed from both blood vessels and neurons. Whilst globally inhibiting transitional change in astrocytes after stroke is reported to result in further damage and functional loss, we discuss the available evidence to suggest that the transitional activation of astrocytes after stroke can be modulated for improved outcomes. In particular, we review the role of Rho-kinase (ROCK) in reactive gliosis and show that inhibiting ROCK after stroke results in reduced scar formation and improved functional recovery.

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The extent of stroke damage in patients affects the range of subsequent pathophysiological responses that influence recovery. Here we investigate the effect of lesion size on development of new blood vessels as well as inflammation and scar formation and cellular responses within the subventricular zone (SVZ) following transient focal ischemia in rats (n = 34). Endothelin-1-induced stroke resulted in neurological deficits detected between 1 and 7 days (P<0.001), but significant recovery was observed beyond this time. MCID image analysis revealed varying degrees of damage in the ipsilateral cortex and striatum with infarct volumes ranging from 0.76–77 mm3 after 14 days, where larger infarct volumes correlated with greater functional deficits up to 7 days (r = 0.53, P<0.05). Point counting of blood vessels within consistent sample regions revealed that increased vessel numbers correlated significantly with larger infarct volumes 14 days post-stroke in the core cortical infarct (r = 0.81, P<0.0001), core striatal infarct (r = 0.91, P<0.005) and surrounding border zones (r = 0.66, P<0.005; and r = 0.73, P<0.05). Cell proliferation within the SVZ also increased with infarct size (P<0.01) with a greater number of Nestin/GFAP positive cells observed extending towards the border zone in rats with larger infarcts. Lesion size correlated with both increased microglia and astrocyte activation, with severely diffuse astrocyte transition, the formation of the glial scar being more pronounced in rats with larger infarcts. Thus stroke severity affects cell proliferation within the SVZ in response to injury, which may ultimately make a further contribution to glial scar formation, an important factor to consider when developing treatment strategies that promote neurogenesis.

IntroductionDespite attempts to prevent brain injury during the hyperacute phase of stroke, most sufferers end up with significant neuronal loss and functional deficits. The use of cell-based therapies to recover the injured brain offers new hope. In the current study, we employed human neural stem cells (hNSCs) isolated from subventricular zone (SVZ), and directed their differentiation into GABAergic neurons followed by transplantation to ischemic brain.MethodsPre-differentiated GABAergic neurons, undifferentiated SVZ-hNSCs or media alone were stereotaxically transplanted into the rat brain (n=7/group) 7 days after endothelin-1 induced stroke. Neurological outcome was assessed by neurological deficit scores and the cylinder test. Transplanted cell survival, cellular phenotype and maturation were assessed using immunohistochemistry and confocal microscopy.ResultsBehavioral assessments revealed accelerated improvements in motor function 7 days post-transplant in rats treated with pre-differentiated GABAergic cells in comparison to media alone and undifferentiated hNSC treated groups. Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype, showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission. Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days, suggesting an additional trophic role of these GABAergic cells. In contrast, undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar.ConclusionOur study is the first to show enhanced exogenous repopulation of a neuronal phenotype after stroke using techniques aimed at GABAergic cell induction prior to delivery that resulted in accelerated and improved functional recovery.

Multiple methods exist to model permanent and transient ischemia under anesthesia in animals, however most human strokes occur while conscious. The use of endothelin-1 as a vasoconstrictor applied to the perivascular surface of the middle cerebral artery is one of the only methods for inducing stroke in conscious animals. Here, we describe standard operating procedures for stereotaxic placement of an ET-1 guide probe above the middle cerebral artery, induction of stroke in conscious rats, predictive outcome scoring during stroke, and neurological behavioral tests that we use to monitor transient and continuing deficits. The inclusion of long term neurological assessment is of particular importance when taking into consideration the effects of stroke on brain remodeling.