(G-257) Isolation of Cerebral Microvascular Networks to Investigate Capillary-Mediated Neurovascular Coupling

Blood flow regulation is crucial for the normal functioning of the brain and the delivery of oxygen and nutrients to active neurons. Neurovascular Coupling (NVC) is a mechanism that links neural activity to changes in cerebral blood flow, a process that can be impaired in various neurodegenerative disorders such as dementia and Alzheimer’s. It is well established that neuronal activity releases signals that dilate arterioles, increasing blood flow to the active region of the brain. Recent findings suggest that in addition to the well-established signaling between neurons and feeding arterioles, direct communication also exists between neurons and the smallest blood vessels, the capillaries. To further investigate this novel mode of neurovascular communication, we plan to adapt a recently developed capillary-arteriolar ex-vivo preparation (Rosehart et al. J Vis Exp, 2019) and develop a protocol for isolating extensive capillary networks attached to feeding parenchymal arterioles. Our goal is to examine the electrical properties of the capillary network and elucidate the microvascular network communication and coordination in response to local neuronal activity.

For our experiments, we utilize C57BL6 mice, 2 - 3 months of age, (wild-type, WT) and mice with a germline knockout of the Kir2.1 gene -that encodes for the inward rectifying K⁺ channel-, (Kir-KO). Transgenic (Tg) mice are generated by crossing mice that express CRE recombinase under an endothelial-specific promoter (Cadherin 5), with Kir2.1 floxed Tg mice (exon 1 of the Kir2.1 gene is flanked by loxP sites). We confirm the presence of LoxP and Cre recombinase by genotyping the offspring through Polymerase Chain Reaction (PCR) to confirm their genetic constitution.

For brain isolation, we euthanize the mice through CO₂ inhalation followed by the removal of the skin and hair over the skull. An incision with the tips of dissection scissors from the occiput region to the frontal region of the head will be performed. The brain is then dissected and placed in a Calcium-free aCSF solution until further use. Under a high magnification dissecting microscope, the middle cerebral artery is traced (Fig. 1A) and pial arteries are dissected along with parenchymal tissue containing capillary networks.  Removed tissue is carefully dissociated using micro forceps and placed on the surface of the dissecting solution where the vascular network is isolated and stretched by surface tension.  The stretched network is picked up by a coverslip and transferred to a perfusion chamber for electrophysiology and fluorescence microscopy studies. The procedure is optimized to produced long capillary networks attached to their feeding arteriole (Fig.1B).

The proposed ex-vivo preparation will provide an invaluable tool in elucidating the contribution of capillary dynamics and signaling in blood flow regulation in health and in disease. Recent findings suggest that neurovascular communication and blood flow control is compromised in Alzheimer’s and in Small Vessel Diseases of the Brain (SVD). Compromised capillary-mediated signaling has also been suggested to contribute to perfusion deficits. We are primarily interested in the electrical properties of the capillary network including signaling dynamics along capillaries and communication with the upstream parenchymal arterioles. We will investigate the ability of electrical signals to regulate capillary diameter, their propagation characteristics within the capillary network and the biophysical determinants of their conduction towards the upstream feeding arterioles.  

Our preliminary efforts suggest that long capillary networks can be isolated and stretched in a perfusion chamber allowing electrophysiological characterization using sharp electrodes or fluorescence microscopy. Furthermore, our previous theoretical studies have identified the Kir channel as a key molecular player in mediating electrical signaling in microvascular networks.  The use of Kir-KO mice will enable us to explore the specific contributions of Kir 2.1 channel in neurovascular coupling and test our previous theoretical predictions about its role in electrical propagation along the network (Moshkforoush et al. PNAS, 2020).

In summary, an ex vivo capillary-arteriolar preparation serves as a suitable model for investigating electrical properties of the capillary network and its role in neurovascular coupling. The preparation will provide insights for the understanding of blood flow control in the brain in health and in neurovascular disorders.

The work is supported by the National Institute of Neurological Disorders and Stroke, and the National Institute of Aging of the NIH under award number R01NS119971.

Rosehart, A. C. et al., (2019). Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study. Journal of visualized experiments : JoVE, (154), 10.3791/60676. https://doi.org/10.3791/60676

Moshkforoush , A et al., (2020, June 19). The capillary Kir channel as sensor and amplifier of neuronal signals: Modeling insights on K+-mediated neurovascular communication.” PNAS. https://www.pnas.org/doi/10.1073/pnas.2000151117