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.

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.

Steady-State Free Ca2+ in Astrocytes Is Decreased by Experience and Impacts Arteriole Tone.

Astrocytes can control basal synaptic strength and arteriole tone via their resting Ca2+ activity. However, whether resting astrocyte Ca2+ can adjust to a new steady-state level, with an impact on surrounding brain cells, remains unknown. Using two-photon Ca2+ imaging in male rat acute brain slices of the somatosensory neocortex, we found that theta burst neural activity produced an unexpected long-lasting reduction in astrocyte free Ca2+ in the soma and endfeet. The drop in intracellular Ca2+ was attenuated by antagonists targeting multiple ionotropic and metabotropic glutamate receptors, and intracellular cascades involved Ca2+ stores and nitric oxide. The reduction in astrocyte endfoot Ca2+ was coincident with an increase in arteriole tone, and both the Ca2+ drop and the tone change were prevented by an NMDA receptor antagonist. Astrocyte patch-clamp experiments verified that the glutamate receptors in question were located on astrocytes and that Ca2+ changes within astrocytes were responsible for the long-lasting change in arteriole diameter caused by theta burst neural activity. In astrocytes from animals that lived in an enriched environment, we measured a relatively lower resting Ca2+ level that occluded any further drop in Ca2+ in response to theta burst activity. These data suggest that electrically evoked patterns of neural activity or natural experience can adjust steady-state resting astrocyte Ca2+ and that the effect has an impact on basal arteriole diameter.SIGNIFICANCE STATEMENT The field of astrocyte-neuron and astrocyte-arteriole interactions is currently in a state of refinement. Experimental evidence ex vivo suggests that direct manipulation of astrocyte-free Ca2+ regulates synaptic signaling and local blood flow control; however, in vivo experiments fail to link synaptically evoked astrocyte Ca2+ transients and immediate changes to various astrocyte-mediated processes. To clarify this discrepancy, we examined a different aspect of astrocyte Ca2+: the resting, steady-state free Ca2+ of astrocytes, its modulation, and its potential role in the tonic regulation of surrounding brain cells. We found that ex vivo or in vivo neural activity induced a long-lasting reduction in resting free astrocyte Ca2+ and that this phenomenon changed arteriole tone.