In Vivo Two‐photon Fluorescence Microscopy Reveals Disturbed Cerebral Capillary Blood Flow and Increased Susceptibility to Ischemic Insults in Diabetic Mice

Abstract

Aims Diabetes mellitus increases the risk of stroke but the mechanisms are unclear. The present study tested the hypothesis that diabetes mellitus disturbs the brain microcirculation and increases the susceptibility to cerebral damage in a middle cerebral artery occlusion (MCAO) model of ischemia. Methods Diabetes was induced by streptozocin in mice expressing green fluorescent protein in endothelial cells (Tie2-GFP mice). Four weeks later, they were subjected to transient (20 min) MCAO. In vivo blood flow was measured by two-photon laser scanning microscopy (TPLSM) in cerebral arteries, veins and capillaries. Results There was a significant decrease in red blood cell (RBC) velocity in capillaries in diabetic mice as assessed by TPLSM, yet the regional cerebral blood flow, as assessed by laser-Doppler flowmetry, was maintained. Brain capillary flow developed turbulence after MCAO only in diabetic mice. These mice sustained increased neurological deficits after MCAO which were accompanied by an exaggerated degradation of tight junction proteins and blunted CaMKII phosphorylation in cerebral tissues indicating disruption of the blood brain barrier and disturbed cognitive potential. Conclusion diabetic mice are more susceptible to disturbances of cerebral capillary blood flow which may predispose them to neurovascular defects following ischemia.

Impact of Metabolic Diseases on Cerebral Circulation: Structural and Functional Consequences

“…It was also reported that STZ-mediated changes in cerebral perfusion and metabolism are region specific (261). A two-photon fluorescence microscopy study reported disturbed cerebral capillary blood flow in STZ-induced T1D mice increases the susceptibility to ischemic insults (123). Another group reported similar findings and specifically showed that diabetes-mediated changes in CBF are associated with impaired recovery after stroke (209).…”

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

“…Recent in vivo imaging studies have shown that both T1D and T2D exacerbate the acute reduction in regional blood flow and velocity of blood flow in peri-infarct arteries and microvessels after MCA occlusion ( Fig. 2A-E and I) (35,36). These acute deficits in blood flow are evident in the first few hours after stroke and may be linked to an impaired recruitment of collateral cerebral blood flow (36) or a downregulation of clotbusting proteins, such as tissue plasminogen activator, in cerebral capillaries (37).…”

“…In animal models of T1D and T2D, several studies have shown a correlation between increased BBB disruption after stroke and an exaggerated loss of TJC protein expression in peri-infarct regions. However, direct visual evidence of tight junction disruption with electron microscopy were missing in these studies (8), which relied on Western blots or fluorescence microscopy (35). This caveat is significant because recent studies that combined electron microscopy with fluorescence imaging of TJC proteins in regions with confirmed BBB disruption noted that tight junction ultrastructure is generally unaltered or, at worst, contain small gaps at these sites (48,49).…”

“…*P < 0.05, **P < 0.01, ***P < 0.001 vs. Con; #P < 0.05, ##P < 0.01, ###P < 0.001. Panels A-E modified with permission from Huang et al (35). F-H: Diabetic animals also showed abnormal patterns of blood flow in the days to weeks after stroke.…”

Multiparametric Imaging of Organ System Interfaces

“…Longitudinal in vivo two-photon microscopy investigating the role of fractalkine and its receptor CX3CR1 in leukocytes after ischemia has revealed that CX3CR1 deficiency induced an early protective inflammatory environment 80 . Intravital two-photon microscopy has also revealed that diabetes disturbs cerebral capillary blood flow after stroke 81 . Recent in vivo two-photon calcium imaging suggested that microglia are vital to the response after stroke, in that they protect against brain injury by stabilizing neuronal networks.…”