Regulation of cerebral blood flow is tightly controlled by changes in intracerebral arteriole diameter. To study these changes in real time, we have continuously developed innovative ex vivo imaging techniques of the mouse brain microcirculation. With ex vivo pressurized arterioles (diameter ~15 µm) isolated from different brain regions, e.g. cortex, amygdala, hippocampus, we can measure real-time constrictions and relaxations as well as changes in membrane potential in response to vasoactive agents. Furthermore, we are able to combine genetic expression of Ca2+ indicator in endothelial or smooth muscle cells with confocal microscopy to analyze intracellular Ca2+ signaling.

Pericytes, the last line of cerebral blood flow regulation: The last decade has seen a dramatic upsurge in research on pericytes, the mural cells that wrap around capillary endothelial cells, with a focus on their role regulating cerebral blood flow in both health and disease. Because of technical challenges, several questions regarding their reactivity to blood pressure and the contractile pathways in place have been left unanswered. Through our pioneering work on capillaries-to-arteriole signaling, we have extended our ability to image brain microcirculation to the pericyte level not only in vivo, but also ex vivo.

GCaMP6f fluorescence reflecting cytosolic Ca²⁺ concentration increases and spontaneous Ca²⁺ signals in transitional pericytes in an ex vivo capillary-parenchymal arteriole preparation pressurized at 20 mmHg (left side) and 40 mmHg (right side).

Local cerebral blood flow is precisely regulated by neurovascular coupling to ensure that neuronal metabolic demands are satisfied. Vasodilation of parenchymal arterioles is thought to mediate a large part of the response due to their ability to dilate rapidly in response to endogenous substances. In a seminal study conducted with Dr. Longden (presently at the University of Maryland) in Dr. Mark Nelson’s lab at the University of Vermont, we provided unequivocal evidence that brain capillaries act as a sensory web to translate neural activity into blood flow. We demonstrated a central role for capillary endothelial cells in sensing neural activity and communicating it to upstream arterioles in the form of an electrical vasodilatory signal. We found that this signal is initiated by extracellular potassium—a byproduct of neural activity—which activates capillary endothelial cell KIR2.1 channels to produce a rapidly propagating retrograde hyperpolarization that causes upstream arteriolar dilation, increasing blood flow into the capillary bed. To enable these experiments, we developed a novel ex vivo pressurized capillary-parenchymal arteriole (CaPA) preparation that allows the study of conducted electrical signaling from the capillaries to the upstream arteriole. Computational modelling with our collaborator Dr. Tsoukias further validated our model.

Capillary stimulation with PGE2 1 μM evokes upstream arterial dilation in ex vivo CaPA preparation.

Using the aforementioned approaches in mouse brain microcirculation allows us to use transgenic mouse models for different conditions such cerebral small vessel disease, Alzheimer’s disease, hypertension and more recently, autism spectrum disorders, to investigate the pathomechanisms at play. In particular, our long-term transatlantic collaboration with Dr. Anne Joutel (Paris, France) has focused on the CADASIL syndrome, a hereditary form of small vessel disease caused by mutations in the NOTCH3 receptor. Cerebral small vessel disease is responsible for more than 25% of ischemic strokes and is the leading cause of age-related cognitive decline and dementia after Alzheimer’s disease. At the capillary level, we have found that CADASIL is associated with reduced synthesis of the phospholipid PIP₂, which prevents the Kir2.1 channel-initiated capillary-to-arteriole electrical signaling that supports vasodilatory responses during functional hyperemia (see Neurovascular coupling above). We further showed that systemic injection of exogenous PIP₂ is sufficient to rescue this deficit in CADASIL mice, restoring adequate cerebral blood flow in response to neuronal activation (patent number 62/823,378; “Methods to promote cerebral blood flow in the brain”). We are actively carrying on this finding to develop a chronic PIP₂-based treatment and measure the impact of neurovascular deficits on cognitive functions.

Exogenous application of PIP₂ fully restores upstream arterial dilation in response to capillary stimulation with 10 mM K⁺ in CADASIL mice.