Collaterals and stroke reperfusion: Too few leads to too much.

Adequate reperfusion after ischemic stroke is a major determinant of functional outcome yet remains unpredictable and insufficient for most survivors. In this issue of Neuron, Binder et al.1 identify leptomeningeal collaterals (LMCs) in mice and human patients as a key factor in regulating reperfusion and hemorrhagic transformation following stroke.

Optical opening of the blood-brain barrier for targeted and ultra-sparse viral infection of cells in mouse cortex.

Adeno-associated viruses (AAVs) are used in a wide array of experimental situations for driving expression of biosensors, recombinases, and opto-/chemo-genetic actuators in the brain. However, conventional approaches for minimally invasive, spatially precise, and ultra-sparse AAV-mediated transduction of cells during imaging experiments have remained a significant challenge. Here, we show that intravenous injection of commercially available AAVs at different doses, combined with laser-based perforation of cortical capillaries through a cranial widow, allows for ultra-sparse, titratable, and micron-level precision for delivery of viral vectors with relatively little inflammation or tissue damage. Further, we show the utility of this approach for eliciting sparse expression of GCaMP6, channelrhodopsin, or fluorescent reporters in neurons and astrocytes within specific functional domains in normal and stroke-damaged cortex. This technique represents a facile approach for targeted delivery of viral vectors that should assist in the study of cell types and circuits in the cortex.

Sex and interferon gamma signaling regulate microglia migration in the adult mouse cortex in vivo.

Although microglia possess the unique ability to migrate, whether mobility is evident in all microglia, is sex dependent, and what molecular mechanisms drive this, is not well understood in the adult brain. Using longitudinal in vivo two-photon imaging of sparsely labeled microglia, we find a relatively small population of microglia (~5%) are mobile under normal conditions. Following injury (microbleed), the fraction of mobile microglia increased in a sex-dependent manner, with male microglia migrating significantly greater distances toward the microbleed relative to their female counterparts. To understand the signaling pathways involved, we interrogated the role of interferon gamma (IFNγ). Our data show that in male mice, stimulating microglia with IFNγ promotes migration whereas inhibiting IFNγ receptor 1 signaling inhibits them. By contrast, female microglia were generally unaffected by these manipulations. These findings highlight the diversity of microglia migratory responses to injury, its dependence on sex and the signaling mechanisms that modulate this behavior.

Behavioral and Neural Activity-Dependent Recanalization of Plugged Capillaries in the Brain of Adult and Aged Mice.

The capillaries of the brain, owing to their small diameter and low perfusion pressure, are vulnerable to interruptions in blood flow. These tiny occlusions can have outsized consequences on angioarchitecture and brain function; especially when exacerbated by disease states or accumulate with aging. A distinctive feature of the brain's microvasculature is the ability for active neurons to recruit local blood flow. The coupling of neural activity to blood flow could play an important role in recanalizing obstructed capillaries. To investigate this idea, we experimentally induced capillary obstructions in mice by injecting fluorescent microspheres and then manipulated neural activity levels though behavioral or pharmacologic approaches. We show that engaging adult and aged mice with 12 h exposure to an enriched environment (group housing, novel objects, exercise wheels) was sufficient to significantly reduce the density of obstructed capillaries throughout the forebrain. In order to more directly manipulate neural activity, we pharmacologically suppressed or increased neuronal activity in the somatosensory cortex. When we suppressed cortical activity, recanalization was impaired given the density of obstructed capillaries was significantly increased. Conversely, increasing cortical activity improved capillary recanalization. Since systemic cardiovascular factors (changes in heart rate, blood pressure) could explain these effects on recanalization, we demonstrate that unilateral manipulations of neural activity through whisker trimming or injection of muscimol, still had significant and hemisphere specific effects on recanalization, even in mice exposed to enrichment where cardiovascular effects would be evident in both hemispheres. In summary, our studies reveal that neural activity bi-directionally regulates the recanalization of obstructed capillaries. Further, we show that stimulating brain activity through behavioral engagement (i.e., environmental enrichment) can promote vascular health throughout the lifespan.

Microvascular basis of cognitive impairment in type 1 diabetes.

The complex computations of the brain require a constant supply of blood flow to meet its immense metabolic needs. Perturbations in blood supply, even in the smallest vascular networks, can have a profound effect on neuronal function and cognition. Type 1 diabetes is a prevalent and insidious metabolic disorder that progressively and heterogeneously disrupts vascular signalling and function in the brain. As a result, it is associated with an array of adverse vascular changes such as impaired regulation of vascular tone, pathological neovascularization and vasoregression, capillary plugging and blood brain barrier disruption. In this review, we highlight the link between microvascular dysfunction and cognitive impairment that is commonly associated with type 1 diabetes, with the aim of synthesizing current knowledge in this field.

Longitudinal functional imaging of VIP interneurons reveals sup-population specific effects of stroke that are rescued with chemogenetic therapy.

Stroke profoundly disrupts cortical excitability which impedes recovery, but how it affects the function of specific inhibitory interneurons, or subpopulations therein, is poorly understood. Interneurons expressing vasoactive intestinal peptide (VIP) represent an intriguing stroke target because they can regulate cortical excitability through disinhibition. Here we chemogenetically augmented VIP interneuron excitability in a murine model of photothrombotic stroke and show that it enhances somatosensory responses and improves recovery of paw function. Using longitudinal calcium imaging, we discovered that stroke primarily disrupts the fidelity (fraction of responsive trials) and predictability of sensory responses within a subset of highly active VIP neurons. Partial recovery of responses occurred largely within these active neurons and was not accompanied by the recruitment of minimally active neurons. Importantly, chemogenetic stimulation preserved sensory response fidelity and predictability in highly active neurons. These findings provide a new depth of understanding into how stroke and prospective therapies (chemogenetics), can influence subpopulations of inhibitory interneurons.

Invasion of phagocytic Galectin 3 expressing macrophages in the diabetic brain disrupts vascular repair.

The cellular events that dictate the repair of damaged vessels in the brain, especially in those with vascular risk factors such as diabetes, is poorly understood. Here, we dissected the role of resident microglia and infiltrative macrophages in determining the repair of ruptured cerebral microvessels. Using in vivo time-lapse imaging, gene expression analysis, and immunohistochemistry, we identified a unique population of phagocytic Galectin 3 (Gal3) expressing macrophages, distinct from resident microglia, which infiltrated and aggregated at the site of injury in diabetic mice and were associated with the elimination of microvessels. Depletion of these infiltrative macrophages in diabetic mice attenuated phagocytic activity and prevented the loss of blood vessels after injury. These findings highlight a previously unknown role for infiltrative Gal3 expressing macrophages in promoting vessel elimination after brain injury and provide impetus for future studies to determine whether depleting these cells can facilitate vascular repair in at risk populations.

Susceptibility to capillary plugging can predict brain region specific vessel loss with aging.

Vessel loss in the aging brain is commonly reported, yet important questions remain concerning whether there are regional vulnerabilities and what mechanisms could account for these regional differences, if they exist. Here we imaged and quantified vessel length, tortuosity and width in 15 brain regions in young adult and aged mice. Our data indicate that vessel loss was most pronounced in white matter followed by cortical, then subcortical grey matter regions, while some regions (visual cortex, amygdala, thalamus) showed no decline with aging. Regions supplied by the anterior cerebral artery were more vulnerable to loss than those supplied by middle or posterior cerebral arteries. Vessel width and tortuosity generally increased with age but neither reliably predicted regional vessel loss. Since capillaries are naturally prone to plugging and prolonged obstructions often lead to vessel pruning, we hypothesized that regional susceptibilities to plugging could help predict vessel loss. By mapping the distribution of microsphere-induced capillary obstructions, we discovered that regions with a higher density of persistent obstructions were more likely to show vessel loss with aging and vice versa. These findings indicate that age-related vessel loss is region specific and can be explained, at least partially, by regional susceptibilities to capillary plugging.

Pannexin 1 Regulates Network Ensembles and Dendritic Spine Development in Cortical Neurons.

Dendritic spines are the postsynaptic targets of excitatory synaptic inputs that undergo extensive proliferation and maturation during the first postnatal month in mice. However, our understanding of the molecular mechanisms that regulate spines during this critical period is limited. Previous work has shown that pannexin 1 (Panx1) regulates neurite growth and synaptic plasticity. We therefore investigated the impact of global Panx1 KO on spontaneous cortical neuron activity using Ca2+ imaging and in silico network analysis. Panx1 KO increased both the number and size of spontaneous co-active cortical neuron network ensembles. To understand the basis for these findings, we investigated Panx1 expression in postnatal synaptosome preparations from early postnatal mouse cortex. Between 2 and 4 postnatal weeks, we observed a precipitous drop in cortical synaptosome protein levels of Panx1, suggesting it regulates synapse proliferation and/or maturation. At the same time points, we observed significant enrichment of the excitatory postsynaptic density proteins PSD-95, GluA1, and GluN2a in cortical synaptosomes from global Panx1 knock-out mice. Ex vivo analysis of pyramidal neuron structure in somatosensory cortex revealed a consistent increase in dendritic spine densities in both male and female Panx1 KO mice. Similar findings were observed in an excitatory neuron-specific Panx1 KO line (Emx1-Cre driven; Panx1 cKOE) and in primary Panx1 KO cortical neurons cultured in vitro. Altogether, our study suggests that Panx1 negatively regulates cortical dendritic spine development.

Suppressing Interferon-γ Stimulates Microglial Responses and Repair of Microbleeds in the Diabetic Brain.

Microcirculatory damage is a common complication for those with vascular risk factors, such as diabetes. To resolve vascular insults, the brain's immune cells (microglia) must rapidly envelop the site of injury. Currently, it is unknown whether Type 1 diabetes, a condition associated with chronic immune system dysfunction, alters microglial responses to damage and what mechanisms are responsible. Using in vivo two-photon microscopy in adult male mice, we show that microglial envelopment of laser-induced cerebral microbleeds is diminished in a hyperglycemic mouse model of Type 1 diabetes, which could not be fully rescued with chronic insulin treatment. Microglia were important for vessel repair because reduced microglial accumulation in diabetic mice or near-complete depletion in healthy controls was associated with greater secondary leakage of the damaged vessel. Broadly suppressing inflammation with dexamethasone in diabetic mice but not healthy controls, significantly enhanced microglial responses to microbleeds and attenuated secondary vessel leakage. These enhancements were associated with changes in IFN-γ signaling because dexamethasone suppressed abnormally high levels of IFN-γ protein levels in brain and blood serum of diabetic mice. Further, blocking IFN-γ in diabetic mice with neutralizing antibodies restored normal microglial chemotaxic responses and purinoceptor P2ry12 gene expression, as well as mitigated secondary leakage. These results suggest that abnormal IFN-γ signaling disrupts microglial function in the diabetic brain, and that immunotherapies targeting IFN-γ can stimulate microglial repair of damaged vessels.SIGNIFICANCE STATEMENT Although Type 1 diabetes is an established risk factor for vascular complications, such as microbleeds, and is known to hinder wound healing in the body, no study has examined how diabetes impacts the brain's innate immune reparative response (involving cells called microglia) to vascular injury. Here we show that microglial responses to brain microbleeds were diminished in diabetic animals, which also exacerbated secondary leakage from damaged vessels. These impairments were related to abnormally high levels of the proinflammatory cytokine IFN-γ because reducing IFN-γ with immunosuppressant drugs or blocking antibodies helped restore normal microglial responses and repair of damaged vessels. These data highlight the use of IFN-γ modulating therapeutics to enhance vascular repair in at-risk populations.

VEGF signaling regulates the fate of obstructed capillaries in mouse cortex.

Cortical capillaries are prone to obstruction, which over time, could have a major impact on brain angioarchitecture and function. The mechanisms that govern the removal of these obstructions and what long-term fate awaits obstructed capillaries, remains a mystery. We estimate that ~0.12% of mouse cortical capillaries are obstructed each day (lasting >20 min), preferentially in superficial layers and lower order branches. Tracking natural or microsphere-induced obstructions revealed that 75-80% of capillaries recanalized within 24 hr. Remarkably, 30% of all obstructed capillaries were pruned by 21 days, including some that had regained flow. Pruning involved regression of endothelial cells, which was not compensated for by sprouting. Using this information, we predicted capillary loss with aging that closely matched experimental estimates. Genetic knockdown or inhibition of VEGF-R2 signaling was a critical factor in promoting capillary recanalization and minimizing subsequent pruning. Our studies reveal the incidence, mechanism and long-term outcome of capillary obstructions which can also explain age-related capillary rarefaction.

Optogenetic rewiring of thalamocortical circuits to restore function in the stroke injured brain.

To regain sensorimotor functions after stroke, surviving neural circuits must reorganize and form new connections. Although the thalamus is critical for processing and relaying sensory information to the cortex, little is known about how stroke affects the structure and function of these connections, or whether a therapeutic approach targeting these circuits can improve recovery. Here we reveal with in vivo calcium imaging that stroke in somatosensory cortex dampens the excitability of surviving thalamocortical circuits. Given this deficit, we hypothesized that chronic transcranial window optogenetic stimulation of thalamocortical axons could facilitate recovery. Using two-photon imaging, we show that optogenetic stimulation promotes the formation of new and stable thalamocortical synaptic boutons, without impacting axon branch dynamics. Stimulation also enhances the recovery of somatosensory cortical circuit function and forepaw sensorimotor abilities. These results demonstrate that an optogenetic approach can rewire thalamocortical circuits and restore function in the damaged brain.

Illuminating the Effects of Stroke on the Diabetic Brain: Insights From Imaging Neural and Vascular Networks in Experimental Animal Models.

Type 1 diabetes is known to cause circulatory problems in the eyes, heart, and limbs, and the brain is no exception. Because of the insidious effects of diabetes on brain circulation, patients with diabetes are two to four times more likely to have an ischemic stroke and are less likely to regain functions that are lost. To provide a more mechanistic understanding of this clinically significant problem, imaging studies have focused on how stroke affects neural and vascular networks in experimental models of type 1 diabetes. The emerging picture is that diabetes leads to maladaptive changes in the cerebrovascular system that ultimately limit neuronal rewiring and recovery of functions after stroke. At the cellular and systems level, diabetes is associated with abnormal cerebral blood flow in surviving brain regions and greater disruption of the blood-brain barrier. The abnormal vascular responses to stroke can be partly attributed to aberrant vascular endothelial growth factor (VEGF) signaling because genetic or pharmacological inhibition of VEGF signaling can mitigate vascular dysfunction and improve stroke recovery in diabetic animals. These experimental studies offer new insights and strategies for optimizing stroke recovery in diabetic populations.

Ultrastructural analysis of blood-brain barrier breakdown in the peri-infarct zone in young adult and aged mice.

Following ischemia, the blood-brain barrier is compromised in the peri-infarct zone leading to secondary injury and dysfunction that can limit recovery. Currently, it is uncertain what structural changes could account for blood-brain barrier permeability, particularly with aging. Here we examined the ultrastructure of early and delayed changes (3 versus 72 h) to the blood-brain barrier in young adult and aged mice (3-4 versus 18 months) subjected to photothrombotic stroke. At both time points and ages, permeability was associated with a striking increase in endothelial caveolae and vacuoles. Tight junctions were generally intact although small spaces were detected in a few cases. In young mice, ischemia led to a significant increase in pericyte process area and vessel coverage whereas these changes were attenuated with aging. Stroke led to an expansion of the basement membrane region that peaked at 3 h and partially recovered by 72 h in both age groups. Astrocyte endfeet and their mitochondria were severely swollen at both times points and ages. Our results suggest that blood-brain barrier permeability in young and aged animals is mediated by transcellular pathways (caveolae/vacuoles), rather than tight junction loss. Further, our data indicate that the effects of ischemia on pericytes and basement membrane are affected by aging.

Delayed inhibition of VEGF signaling after stroke attenuates blood-brain barrier breakdown and improves functional recovery in a comorbidity-dependent manner.

Diabetes is a common comorbidity in stroke patients and a strong predictor of poor functional outcome. To provide a more mechanistic understanding of this clinically relevant problem, we focused on how diabetes affects blood-brain barrier (BBB) function after stroke. Because the BBB can be compromised for days after stroke and thus further exacerbate ischemic injury, manipulating its function presents a unique opportunity for enhancing stroke recovery long after the window for thrombolytics has passed. Using a mouse model of Type 1 diabetes, we discovered that ischemic stroke leads to an abnormal and persistent increase in vascular endothelial growth factor receptor 2 (VEGF-R2) expression in peri-infarct vascular networks. Correlating with this, BBB permeability was markedly increased in diabetic mice, which could not be prevented with insulin treatment after stroke. Imaging of capillary ultrastructure revealed that BBB permeability was associated with an increase in endothelial transcytosis rather than a loss of tight junctions. Pharmacological inhibition (initiated 2.5 d after stroke) or vascular-specific knockdown of VEGF-R2 after stroke attenuated BBB permeability, loss of synaptic structure in peri-infarct regions, and improved recovery of forepaw function. However, the beneficial effects of VEGF-R2 inhibition on stroke recovery were restricted to diabetic mice and appeared to worsen BBB permeability in nondiabetic mice. Collectively, these results suggest that aberrant VEGF signaling and BBB dysfunction after stroke plays a crucial role in limiting functional recovery in an experimental model of diabetes. Furthermore, our data highlight the need to develop more personalized stroke treatments for a heterogeneous clinical population.

VEGF can protect against blood brain barrier dysfunction, dendritic spine loss and spatial memory impairment in an experimental model of diabetes.

Clinical and experimental studies have shown a clear link between diabetes, vascular dysfunction and cognitive impairment. However, the molecular underpinnings of this association remain unclear. Since vascular endothelial growth factor (VEGF) signaling is important for maintaining vascular integrity and function, we hypothesized that vascular and cognitive impairment in the diabetic brain could be related to a deficiency in VEGF signaling. Here we show that chronic hyperglycemia (~8weeks) in a mouse model of type 1 diabetes leads to a selective reduction in the expression of VEGF and its cognate receptor (VEGF-R2) in the hippocampus. Correlating with this, diabetic mice showed selective deficits in spatial memory in the Morris water maze, increased vessel area, width and permeability in the dentate gyrus/CA1 region of the hippocampus and reduced spine densities in CA1 neurons. Chronic low dose infusion of VEGF in diabetic mice was sufficient to restore VEGF signaling, protect them from memory deficits, as well as vascular and synaptic abnormalities in the hippocampus. These findings suggest that a hippocampal specific reduction in VEGF signaling and resultant vascular/neuronal defects may underlie early manifestations of cognitive impairment commonly associated with diabetes. Furthermore, restoring VEGF signaling may be a useful strategy for preserving hippocampal-related brain circuitry in degenerative vascular diseases.

Induction of ischemic stroke in awake freely moving mice reveals that isoflurane anesthesia can mask the benefits of a neuroprotection therapy.

Anesthetics such as isoflurane are commonly used to sedate experimental animals during the induction of stroke. Since these agents are known to modulate synaptic excitability, inflammation and blood flow, they could hinder the development and discovery of new neuroprotection therapies. To address this issue, we developed a protocol for inducing photothrombotic occlusion of cerebral vessels in fully conscious mice and tested two potential neuroprotectant drugs (a GluN2B or α4ß2 nicotinic receptor antagonist). Our data show in vehicle treated mice that just 20 min of exposure to isoflurane during stroke induction can significantly reduce ischemic cortical damage relative to mice that were awake during stroke. When comparing potential stroke therapies, none provided any level of neuroprotection if the stroke was induced with anesthesia. However, if mice were fully conscious during stroke, the α4ß2 nicotinic receptor antagonist reduced ischemic damage by 23% relative to vehicle treated controls, whereas the GluN2B antagonist had no significant effect. These results suggest that isoflurane anesthesia can occlude the benefits of certain stroke treatments and warrant caution when using anesthetics for pre-clinical testing of neuroprotective agents.

Diabetes augments in vivo microvascular blood flow dynamics after stroke.

Stroke usually affects people with underlying medical conditions. In particular, diabetics are significantly more likely to have a stroke and the prognosis for recovery is poor. Because diabetes is associated with degenerative changes in the vasculature of many organs, we sought to determine how hyperglycemia affects blood flow dynamics after an ischemic stroke. Longitudinal in vivo two-photon imaging was used to track microvessels before and after photothrombotic stroke in a diabetic mouse model. Chronic hyperglycemia exacerbated acute (3-7 d) ischemia-induced increases in blood flow velocity, vessel lumen diameter, and red blood cell flux in peri-infarct regions. These changes in blood flow dynamics were most evident in superficial blood vessels within 500 µm from the infarct, rather than deeper or more distant cortical regions. Long-term imaging of diabetic mice not subjected to stroke indicated that these acute stroke-related changes in vascular function could not be attributed to complications from hyperglycemia alone. Treating diabetic mice with insulin immediately after stroke resulted in less severe alterations in blood flow within the first 7 d of recovery, but had more variable results at later time points. Analysis of microvessel branching patterns revealed that stroke led to a pruning of microvessels in peri-infarct cortex, with very few instances of sprouting. These results indicate that chronic hyperglycemia significantly affects the vascular response to ischemic stroke and that insulin only partially mitigates these changes. The combination of these acute and chronic alterations in blood flow dynamics could underlie diabetes-related deficits in cortical plasticity and stroke recovery.

Stroke induces long-lasting deficits in the temporal fidelity of sensory processing in the somatosensory cortex.

Recovery from stroke is rarely complete as humans and experimental animals typically show lingering deficits in sensory function. One explanation for limited recovery could be that rewired cortical networks do not process sensory stimuli with the same temporal precision as they normally would. To examine how well peri-infarct and more distant cortical networks process successive vibro-tactile stimulations of the affected forepaw (a measure of temporal fidelity), we imaged cortical depolarizations with millisecond temporal resolution using voltage-sensitive dyes. In control mice, paired forepaw stimulations (ranging from 50 to 200 milliseconds apart) induced temporally distinct depolarizations in primary forelimb somatosensory (FLS1) cortex, and to a lesser extent in secondary FLS (FLS2) cortex. For mice imaged 3 months after stroke, the first forepaw stimulus reliably evoked a strong depolarization in the surviving region of FLS1 and FLS2 cortex. However, depolarizations to subsequent forepaw stimuli were significantly reduced or completely absent (for stimuli ≤100 milliseconds apart) in the FLS1 cortex, whereas FLS2 responses were relatively unaffected. Our data reveal that stroke induces long-lasting impairments in how well the rewired FLS1 cortex processes temporal aspects of sensory stimuli. Future therapies directed at enhancing the temporal fidelity of cortical circuits may be necessary for achieving full recovery of sensory functions.