A microphysiological model of the neurovascular unit capable of demonstrating neurovascular coupling

SUMMARY
The brain does not contain any significant energy stores but relies on blood flow to supply its metabolic needs,
needs which vary both by region and over time. Neurovascular coupling (NVC) refers to the coordinated activity
of multiple cell types within the brain to respond to spatially and temporally varying levels of neural activity (and
associated metabolic needs) by dynamically modulating vessel lumen diameter and thereby redirecting cerebral
blood flow to regions of greatest need. Dysfunctional NVC is closely associated with the cognitive decline seen
in many diseases, and thus a better understanding of both the mechanisms of healthy NVC in humans as well
as approaches to rescue impaired NVC in a diseased state could yield crucial information regarding potential
therapies to aid in the recovery of cognitive ability. Current human microphysiological models of the
cerebrovasculature and surrounding environment (the “neurovascular unit” or NVU) are unable to model NVC
because 1) they lack the contractile mural cells needed to constrict or dilate the vessel and 2) the ability for cells
in culture to transduce the relevant signals has not been established. To overcome this critical gap in NVU
model functionality, we will develop the first engineered NVU capable of demonstrating any aspect of NVC.
While there are many mechanisms involved, we choose to model the well-established glutamate-NMDA-nNOS-
NO pathway that occurs at cerebral parenchymal arterioles and is thought to contribute to a substantial portion
of NVC response. In this pathway, glutamate released from active neurons stimulates N-methyl-D-aspartate
(NMDA) receptors in interneurons, causing an increase in intracellular Ca2+ and activating the Ca2+-dependent
enzyme neuronal nitric oxide synthase (nNOS), resulting in release of NO that can act directly on smooth muscle
cells (SMCs) as a vasodilator. In Aim 1, we focus on the “actuators”: the SMCs. We will conduct studies both
with SMCs alone and in co-culture with endothelial cells (ECs) in a coaxial configuration on the wall of an
engineered microvessel, and demonstrate appropriate vasoconstriction or vasodilation in response to vasoactive
agents. Aim 2 focuses on producing a population of iPSC-derived nNOS+ interneurons and validating their
ability to transduce glutamate signaling into NO release, first in 2D culture and then in a tubular volume
surrounding the lumen of our 3D culture model. Finally, in Aim 3, we demonstrate optogenetic stimulation of
iPSC-derived glutamatergic neurons and measure resulting release of glutamate, first in 2D culture and then in
3D. Subsequently, we incorporate the other stages of our model: the nNOS+ interneurons (transducing released
glutamate into NO) and the SMCs (responding to secreted NO by relaxing and causing vasodilation). Successful
completion of all three Aims will result in a human NVU model in which optogenetic stimulation of neurons results
in vasodilation of a nearby engineered microvessel. Such a model would be a first (but crucial) step towards an
in vitro human model of NVC in health and disease, enabling future identification of therapeutic targets and
screening for drug candidates to rescue dysfunctional NVC and restore impaired cognition.

Grant Number: 5R21MH136576-02
NIH Institute/Center: NIH
Principal Investigator: Leon Bellan