21 Feb 2020

As the bodys most energy- and oxygen-hungry organ, the brain also happens to be the most dynamic, and it's devilishly complex. How does nutrient-rich blood wend its way through the vast labyrinth of cerebral blood vessels to nourish the neurons that need it most? Two new studies describe elements of neurovascular physiology that make this feat possible. One, published in Nature on February 19 and led by Chenghua Gu at Harvard Medical School, reports that endothelial cells that line arterioles sport myriad inlets, called caveolae, which somehow control the rapid dilation of arterioles in response to neuronal stimulation. The other, published January 20 in Nature Communications and led by Martin Lauritzen of the University of Copenhagen, describes specialized sphincters that control the flow of blood from the brains arterioles into its vast capillary beds.

"The two papers, by Chow et al and Grubb et. al, significantly advance our understanding of neurovascular physiology and blood flow control," Lei Tong and Jamie Grutzendler of Yale University Medical School, wrote to Alzforum.

Neuronal activity triggers a boost in the regional supply of oxygenated blood within milliseconds. Known as neurovascular coupling, this phenomenon facilitates the coordinated activity of neural networks throughout the brain. It is also the basis of functional brain imaging. While the exact mechanisms involved remain unclear, and controversial, the conventional view is that in response to stimuli, neurons and astrocytes release factors that relax smooth muscle cells surrounding arterioles, thus dilating the vessels and upping the flow of oxygenated blood into the capillary beds connected to them (Jun 2015 news; Iadecola, 2017).However, other reports implicate the endothelial cells lining the arterioles in modulating neurovascular coupling (Chen et al., 2014; Longden et al., 2017).

In their Nature paper, first authors Brian Chow and Vicente Nuez cast arteriolar endothelial cells as key players, as opposed to mere bystanders, in neurovascular coupling. The researchers observed that, in contrast to the smooth lumen of brain capillaries, the inner surface of arterioles was ruffled, marked by numerous inlets called caveolae.

Ruffled Arterioles. Compared with the smooth surface of the capillary lumen (left, purple), the endothelial cells of arterioles (right, purple) were covered with caveolae. [Courtesy of Chow et al., Nature, 2020.]

To investigate the potential role of these caveolae in neurovascular coupling, the researchers used two-photon imaging to peer through cranial windows. They simultaneously measured neuronal activity and dilation of arterioles. Specifically, they brushed a mouses whiskers, then assessed the resulting neuronal and vascular responses in the barrel cortex, a region of the rodent somatosensory cortex. As expected, whisker brushing evoked a rapid uptick in neuronal calcium signaling in the region, then local arterioles dilated and red blood cells in downstream capillaries accelerated.

Using conditional knockout mice lacking caveolin-1, a key element of caveolae, the researchers confirmed that caveolae in arteriolar endothelial cells, but not in the smooth muscle cells encircling the vessels, were required for neurovascular coupling. They also found that arteriolar caveolae influenced neurovascular coupling independently of nitric oxidea critical vasodilatory factor.

Finally, Chow and Nuez reported that the reason arterioles have caveolae, while capillaries dont, comes down to expression of MFSD2A. Previously, Gu had reported that capillaries express high levels of this protein, which inhibits caveolae and maintains the integrity of the blood-brain barrier (Ben-Zvi et al., 2014). They found that arterioles express scant MFSD2A. When they overexpressed the protein in arterioles, caveolae largely disappeared, along with neurovascular coupling.

How do caveolae facilitate neurovascular coupling? Thats still unclear, and piecing together the signals among neurons, capillaries, and arterioles is a major focus in her lab, Gu said. She proposed that the caveolae cluster critical ion channels necessary to respond to incoming signals from capillaries, which release factors in response to neuronal stimulation. The arteriolar endothelial cells then signal to smooth muscle cells on the outside of the vessel, triggering dilation.

This proposed chain of events stands in contrast to the conventional outside-in model, which posits that vasodilatory factors released from neurons directly relax smooth muscle cells on the outside of the vessel. In our model, arteriolar endothelial cells play an active role in neurovascular coupling, Gu said. Neurovascular coupling is disrupted in aging and neurodegeneration, but whether this is a cause or consequence of disease, and how arteriolar caveolae come into play, remain to be deciphered.

In a joint comment to Alzforum, Andrew Yang and Tony Wyss-Coray of Stanford University articulated questions stimulated by these findings. Scientists have reported changes in expression of multiple genes, including caveolin-1, in brain endothelial cells with age and disease, implicating them in the diminution of neurovascular coupling (Nov 2019 news; May 2019 news).The current study provides new impetus for studying the fascinating complexity of the brain vasculature and will hopefully pave the way toward a better understanding of how this structure degenerates with age and disease, they wrote.

[The study] also raises the question whether a similar mechanism operates at the level of brain capillaries, since some recent studies have suggested that capillary dilation precedes arteriolar dilation during neurovascular coupling, noted Berislav Zlokovic of the University of Southern California in Los Angeles.

In a joint comment to Alzforum, Thomas Pfeiffer, Chanawee Hirunpattarasilp, and David Attwell of University College London made a similar point, noting that caveolin-1 expression on arterioles greatly influenced blood flow in capillaries, more than would be predicted by arteriolar dilation alone. This raises the question of whether the change of neurovascular coupling that they see on deleting caveolin-1 is not, as one might expect, occurring at the arteriole smooth muscle adjacent to the endothelial cells where the caveolae are being suppressed, but somehow instead at downstream capillary pericytes, they wrote. Attwell and others have proposed that cerebral blood flow slows in the AD brain because contractile pericytes constrict capillaries, not because smooth muscle cells encircling arterioles squeeze those vessels (Jun 2019 news).

First author Sren Grubb and colleagues addressed connections between arterioles and capillaries in their Nature Communications paper. As their name suggests, penetrating arterioles flow deep into the brain. They supply freshly oxygenated blood to numerous capillaries that branch off along the way. These capillaries, in turn, deliver oxygen and glucose to the neurons and other cells that need it. But how is this process managed in such a way that the capillaries receive an adequate, but not overwhelming, supply of arterial blood? Grubb and colleagues reported that specialized sphincters, positioned on capillaries just as they branch off of arterioles, tightly control the flow of blood into capillary beds.

The researchers used two-photon microscopy to visualize penetrating arterioles in mice that expressed a red fluorescent protein under the control of NG2 promoter, which is expressed in mural cells that surround vessels, including smooth muscle cells and pericytes. They spotted numerous pinched regions of capillaries, each surrounded by a single mural cell, at numerous branch points along penetrating arterioles. Many of these sphincters were followed by a distended region, or bulb. Out of 108 penetrating arterioles with 602 branches that they examined, 433 (72 percent) contained at least one of these sphincters. Most resided on first-order capillaries, at the proximal branch point from arterioles, and were bolstered by a structural skeleton of collagen and elastin that helped maintain the indentation.

Vascular Bottlenecks. Precapillary sphincters (boxed region) appeared at branch points between arterioles and capillaries. The indentations were surrounded by a single mural cell (red). [Courtesy of Grubb et al., Nature Communications, 2020.]

These sphincters dilated rapidly in response to whisker stimulation, then constricted for about 20 seconds before returning to their baseline diameter. During stimulation, the sphincters relative diameter increased two to three times more than did the diameter of the arteriole and capillary, and the dilation was controlled by the encircling mural cell.

These sphincters dilated rapidly in response to whisker stimulation, then constricted for about 20 seconds before returning to their baseline diameter. During stimulation, the sphincters relative diameter increased two to three times more than did the diameter of the arteriole and capillary, and the dilation was controlled by the encircling mural cell.

Based on the morphology of the mural cells and the markers they expressed, Lauritzen said the sphincter-forming cells are most likely giant, contractile pericytes, as opposed to smooth muscle cells. The sphincters shortened during dilation, and elongated during constriction. Using computational modeling, the researchers concluded that the sphincters serve as vascular bottlenecks, shielding tiny capillaries from relatively high blood pressure in arterioles, while easing in just the right amount of oxygenated blood in response to stimulation. The sphincters also ensured an even distribution of blood along the cerebrovascular tree.

Manning the Flood Gates. A precapillary sphincter widens in response to stimulation of nearby neurons. [Courtesy of Grubb et al., Nature Communications, 2020.]

Lauritzen told Alzforum that the sphincters not only ensure adequate brain perfusion and neurovascular coupling, but also protect the brain from the hearts pulse. Lauritzen believes that, as blood vessels stiffen with age, they become less adept at attenuating the rush of blood into the brain. To compensate, precapillary sphincters may clinch up, he proposed. This would lead to inadequate perfusion of the brain, and also cause microhemorrhages in backed-up arterioles. Both these problems occur with age and during neurodegeneration, he noted.

How does neuronal stimulation control the sphincters? The signaling pathway remains unclear, but Pfeiffer, Hirunpattarasilp, and Attwell commented that conceivably, pericytes or endothelial cells on capillaries downstream of the sphincter-containing vessel may sense neuronal activity and send a signal back to the sphincter to alter blood flow. It will be exciting to determine whether, in AD, constriction of the pericytes forming the sphincters has a larger effect on cerebral blood flow than constriction of contractile pericytes that are not forming sphincters, or whether the sphincter-fed vessels are relatively protected from constriction, they wrote.

Apart from preventing cerebrovascular complications related to high blood pressure, the sphincters may have a role in smooth vasomotion and the motive force for drainage of interstitial fluid from the brain, commented Roxana Carare of the University of Southampton, U.K. (Carare et al., 2020). The molecular pathways of neurovascular coupling and dysregulation may contai therapeutic targets for stroke or subarachnoid hemorrhage, Carare noted. Jessica Shugart.

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Ruffles and Sphincters Control the Spigot of Fresh Blood in the Brain - Alzforum