Pericytes—dainty little cells that decorate the brain’s smallest blood vessels— are drawing growing research interest, and controversy. For one, whether these cells help control cerebral blood flow has been a contentious issue, with different groups reporting conflicting data. Part of the disagreement stems from how pericytes are defined; with no specific marker for these cells, researchers may not all be studying the same populations. Now, in the May 15 Nature Neuroscience, researchers led by Jaime Grutzendler at Yale School of Medicine, New Haven, Connecticut, debut a new tool that may help resolve the question. The researchers identified a dye that selectively labels perivascular cells in capillary beds of live mice, while eschewing adjacent smooth muscle cells that surround larger arterioles. The labeled cells possess the round cell bodies and long spidery arms characteristic of pericytes, but do not express smooth muscle actin or demonstrate any ability to squeeze blood vessels, the authors report. “This is the first clear-cut evidence that these cells form a unique cell population that is distinct from anything immediately adjacent to them,” Grutzendler told Alzforum. He believes the dye could be useful for in vivo studies of vascular development and disease processes, as well as for isolating this cell population via fluorescence-activated cell sorting (FACS) for detailed transcriptomic and proteomic studies.
Others hailed the discovery, calling it an important advance. “A reliable, robust method of labeling pericytes was both necessary and urgent. The present method should allow for more in-depth investigations of the role of pericytes in different neurologic diseases,” Roxana Carare at the University of Southampton, U.K., wrote to Alzforum.
The dye might help researchers reach consensus on what defines pericytes, others stressed. “Having a resource like this will bring clarity to the field. Now there’s an opportunity for different labs to use this dye and see how it holds up against their definition of pericytes,” Jeffrey Iliff at Oregon Health and Science University, Portland, told Alzforum. However, Takahisa Kanekiyo at the Mayo Clinic in Jacksonville, Florida, noted that it remains possible the dye labels only a subpopulation of pericytes. He said further studies will be needed to fully settle the debate over whether any pericyte population contributes to brain blood flow (see comment below).
Two Distinct Populations. The putative pericyte dye (green) labels round cells on capillaries, but not mural cells expressing α-actin (red) on larger vessels (left; vessels blue). An abrupt transition point divides the two cell types (right). [Courtesy of Damisah et al., Nature Neuroscience.]
The issue has divided vascular researchers. Grutzendler and colleagues previously reported that only ring-like smooth muscle cells, which express α-actin and ensheathe larger precapillaries and arterioles, pinch and relax blood vessels (see Jun 2015 news). Other groups disagree. David Attwell and colleagues at University College London, claim that pericytes regulate blood flow in health and disease, while researchers led by Berislav Zlokovic at the University of Southern California, Los Angeles, recently reported that depletion of pericytes in mice attenuated increases in brain blood flow evoked by neural activity (see Hall et al., 2014; Feb 2017 news). A lack of agreement over what size vessels constitute capillaries versus precapillaries or arterioles confuses the debate further. Compounding the issue, the commonly used markers for pericytes PDGFRβ and NG2 also light up vascular smooth muscle cells, making it difficult to specifically identify or manipulate pericytes.
The discovery of a dye that appears to distinguish pericytes came about by accident. Joint first authors Eyiyemisi Damisah and Robert Hill were studying neuronal labels, including the green fluorescent dye NeuroTrace 500/525. This commercially available dye is typically used to identify neurons in fixed brain sections since it binds to Nissl bodies, granules of rough endoplasmic reticulum found in neuronal soma. However, in living mouse brains NeuroTrace 500/525 behaved quite differently, the authors found. Instead of neurons, it lit up only the spidery cells clinging to the smallest blood vessels. The label penetrated up to 400 microns into mouse cortex, and lasted there for two to three days. The authors saw the same staining pattern whether they applied the dye directly to the cortex through a cranial window, or injected it into the parenchyma. They saw no signs of cell toxicity.
Were these labeled cells pericytes? To investigate, the authors applied NeuroTrace to the cortices of transgenic mice that carried a fluorescent marker in all mural cells, including pericytes and smooth muscle cells. The dye lit up all of the round cells lining the capillary bed, but none of the ring-like cells surrounding larger vessels. In a separate transgenic mouse that expressed a red fluorescent marker for α-actin, NeuroTrace labeled only those mural cells without actin, and the demarcation between the two cell types appeared stark (see images above). The authors used high-resolution confocal imaging and electron microscopy to confirm that NeuroTrace 500/525-labeled cells were surrounded on all sides by basal lamina, a key characteristic of pericytes.
The data suggest that pericytes and vascular smooth muscle cells represent two distinct cell populations with no overlap, Grutzendler said. He sees no evidence for the existence of transitional cells that possess characteristics of both types. Some researchers had proposed such transitional cells sheath terminal arterioles and precapillaries.
While many commenters found these data compelling, they noted the findings do not yet fully settle the question of what pericytes are. For this, researchers need to trace the embryonic origin of both the mural cells that take up the dye and those that do not, said Costantino Iadecola at Weill Cornell Medical College, New York. Some studies have suggested that pericytes and vascular smooth muscle cells have distinct origins, and NeuroTrace could help test that idea. Iadecola also recommended using single-cell transcriptomics to identify molecular markers unique to pericytes, and to see if these label the cells that take up NeuroTrace. “Only after we do that will we be able to definitively distinguish between these cell types,” he said.
Christer Betsholtz at Uppsala University, Sweden, has been analyzing mural cell expression profiles with single-cell approaches. “I am not surprised the authors find that pericytes and vascular smooth muscle cells differ substantially. This fits very well with our own unpublished data,” he wrote to Alzforum.
Regardless of the precise dividing line between mural cell types, researchers agree that NeuroTrace 500/525 labels a specific population of capillary pericytes. What kind of studies could it facilitate? To take a first stab, Grutzendler and colleagues used the dye to examine the behavior of labeled cells and their associated vessels in awake and anesthetized mice, using just three mice per group. They found that vessels decorated with the labeled cells did not contract or relax during time-lapse imaging, suggesting that these pericytes did not control blood flow. Supporting this, in a mouse model of ischemia, only vessels surrounded by mural cells expressing α-actin tightened or collapsed, while those sporting labeled pericytes did not change diameter. However, Zlokovic and colleagues were unconvinced, noting that capillaries normally exhibit little spontaneous contraction or expansion, but may still change size in response to stimuli (see comment below).
Some studies suggest that pericyte loss during aging contributes to neurodegeneration. The authors saw no evidence of age-related pericyte loss in wild-type mice with NeuroTrace, however. Elderly and young mice had similar numbers and densities of labeled cells in the brain. In 5xFAD mice that model Alzheimer’s disease pathology, vessels containing NeuroTrace-positive cells did not accumulate amyloid deposits, but larger arterioles did. While these findings are preliminary, the dye may eventually help researchers determine whether pericyte loss is a common feature of aging and AD, researchers said.
Commenters noted some limitations of NeuroTrace. Because it leaches out of tissues during staining, it is only useful for live imaging. In addition, the dye only penetrates the upper regions of the cortex. “There is a great diversity in brain vasculature beyond what we see in the cortex, and in all likelihood, there will be a similar diversity in pericyte taxonomy and function throughout the brain,” Iliff noted.
At the same time, researchers were excited by some of the implications of the finding. The selective uptake of NeuroTrace by capillary pericytes suggest that these cells possess a unique molecular transporter that might shed light on their function, many scientists observed. Iliff speculated that the existence of such a transporter might eventually allow researchers to develop a PET ligand for labeling pericytes in human brain. Grutzendler and colleagues are trying to identify the dye’s transporter and are exploring its use in different species.—Madolyn Bowman Rogers
- Smooth Muscle Cells, Not Pericytes, Control Brain Blood Flow
- Pericytes Don’t Go With the Flow—They Change It
Research Models Citations
- Hall CN, Reynell C, Gesslein B, Hamilton NB, Mishra A, Sutherland BA, O'Farrell FM, Buchan AM, Lauritzen M, Attwell D. Capillary pericytes regulate cerebral blood flow in health and disease. Nature. 2014 Apr 3;508(7494):55-60. Epub 2014 Mar 26 PubMed.
- Damisah EC, Hill RA, Tong L, Murray KN, Grutzendler J. A fluoro-Nissl dye identifies pericytes as distinct vascular mural cells during in vivo brain imaging. Nat Neurosci. 2017 Jul;20(7):1023-1032. Epub 2017 May 15 PubMed.