Finally, a Dye to Visualize Pericyte Function
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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
References
News Citations
- Smooth Muscle Cells, Not Pericytes, Control Brain Blood Flow
- Pericytes Don’t Go With the Flow—They Change It
Research Models Citations
Paper 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.
Further Reading
Primary Papers
- 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.
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Comments
University of Southern California, Keck School of Medicine
University of South Alabama
University of Southern California
Damisah et al. identified an incredibly useful methodology using Nissl dye NeuroTrace 500/525 to specifically label pericytes in vivo, which will more easily allow investigation of pericytes in the living brain. They should be congratulated for this outstanding effort. However, when it comes to the contractility issue, things are likely much more complicated than what has been briefly described in this paper. For instance, several recent studies have shown that pericytes contribute to the regulation of capillary diameter and blood flow, as demonstrated in vivo (Kisler et al., 2017; Mishra et al., 2016; Biesecker et al., 2016; Hall et al., 2014; Fernández-Klett et al., 2010; Dai et al., 2009) and in vitro and ex vivo (Mishra et al., 2016; Biesecker et al., 2016; Neuhaus et al., 2016; Fernández-Klett et al., 2015; Hall et al., 2014; Dai et al., 2009; Yamanishi et al., 2006; Peppiatt et al., 2006). All these studies evaluated pericyte contractility in response to different stimuli (neural, optical, pharmacological, electrophysiological), whereas Damisah et al. assessed spontaneous vasomotion of capillaries and arterioles. Vasomotion is influenced by heartbeat and respiration, and is normally less prominent in smaller vessels such as capillaries compared to arterioles, and does not necessarily inform us about cell contractility or response to stimuli.
References:
Kisler K, Nelson AR, Rege SV, Ramanathan A, Wang Y, Ahuja A, Lazic D, Tsai PS, Zhao Z, Zhou Y, Boas DA, Sakadžić S, Zlokovic BV. Pericyte degeneration leads to neurovascular uncoupling and limits oxygen supply to brain. Nat Neurosci. 2017 Mar;20(3):406-416. Epub 2017 Jan 30 PubMed.
Mishra A, Reynolds JP, Chen Y, Gourine AV, Rusakov DA, Attwell D. Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles. Nat Neurosci. 2016 Dec;19(12):1619-1627. Epub 2016 Oct 24 PubMed.
Biesecker KR, Srienc AI, Shimoda AM, Agarwal A, Bergles DE, Kofuji P, Newman EA. Glial Cell Calcium Signaling Mediates Capillary Regulation of Blood Flow in the Retina. J Neurosci. 2016 Sep 7;36(36):9435-45. PubMed.
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.
Fernández-Klett F, Offenhauser N, Dirnagl U, Priller J, Lindauer U. Pericytes in capillaries are contractile in vivo, but arterioles mediate functional hyperemia in the mouse brain. Proc Natl Acad Sci U S A. 2010 Dec 21;107(51):22290-5. Epub 2010 Dec 6 PubMed.
Dai M, Nuttall A, Yang Y, Shi X. Visualization and contractile activity of cochlear pericytes in the capillaries of the spiral ligament. Hear Res. 2009 Aug;254(1-2):100-7. Epub 2009 May 5 PubMed.
Neuhaus AA, Couch Y, Sutherland BA, Buchan AM. Novel method to study pericyte contractility and responses to ischaemia in vitro using electrical impedance. J Cereb Blood Flow Metab. 2016 Jan 1;:271678X16659495. PubMed.
Fernández-Klett F, Priller J. Diverse functions of pericytes in cerebral blood flow regulation and ischemia. J Cereb Blood Flow Metab. 2015 Jun;35(6):883-7. Epub 2015 Apr 8 PubMed.
Yamanishi S, Katsumura K, Kobayashi T, Puro DG. Extracellular lactate as a dynamic vasoactive signal in the rat retinal microvasculature. Am J Physiol Heart Circ Physiol. 2006 Mar;290(3):H925-34. Epub 2005 Nov 18 PubMed.
Peppiatt CM, Howarth C, Mobbs P, Attwell D. Bidirectional control of CNS capillary diameter by pericytes. Nature. 2006 Oct 12;443(7112):700-4. Epub 2006 Oct 1 PubMed.
Oregon Health and Science University
As shown previously for dextran-conjugated calcium green or FluoroGold injected into the blood (Hirase et al., 2004; Edwards et al., 2013), this paper shows that some capillary pericytes can be loaded with exogenously applied dye (in this case the Nissl stain NeuroTrace, which was topically applied).
As in their previous work, the authors of this paper choose to ignore the conventional definition of pericytes (made by Zimmerman in 1923, as described by Krueger and Bechmann, 2010) and define as pericytes only those cells that lack processes around the capillaries and lack smooth muscle actin labeling (see Attwell et al., 2016). As a result, they claim that NeuroTrace is a specific label for all pericytes.
However, NeuroTrace does not seem to enter the conventionally defined pericytes found on the first four branching orders of the capillary bed that have circumferential processes and express smooth muscle actin. These contractile pericytes contribute to controlling cerebral blood flow in health and disease (Peppiatt et al., 2006; Yemisci et al., 2009; Hall et al., 2014; Mishra et al., 2016; Biesecker et al., 2016; Kisler et al., 2017; Li et al. 2017; Leal-Campanari et al., 2017).
For example, the spatially isolated somata on capillaries that are labeled red by PDGFR beta-Tomato but are not labeled green by NeuroTrace in the top left panel (two cells) and bottom left panel (six cells) of Fig. 2 in this paper, which have processes extending on both sides of the capillary, would normally be defined to be pericytes—but they do not take up NeuroTrace. In this paper, however, these cells are defined not to be pericytes. Instead, the authors rather misleadingly label such spatially isolated cells as smooth muscle cells (with the curious exception of the cell with circumferential processes in Fig 4c), despite their obvious morphological difference from the contiguous rings of smooth muscle cells that wrap arterioles.
Although it is interesting that NeuroTrace enters some pericytes and not others, reinforcing the notion of pericyte heterogeneity (Hartmann et al., 2015; Nehls and Drenckhahn, 1991), the claim that NeuroTrace labels all pericytes is thus not sustainable. The paper also implies that all cells labeled by NeuroTrace must be pericytes, even if they are not associated with blood vessels. Further exhaustive labeling with other markers would be needed to make this claim.
The authors report that NeuroTrace-labeled pericytes do not show spontaneous contractile activity. While this probably reflects NeuroTrace predominantly labeling non-contractile pericytes in the middle of the capillary bed, rather than contractile pericytes located on capillaries closer to arterioles (Hall et al., 2014; movies of pericytes altering capillary diameter are available here), the obvious possibility that NeuroTrace uptake inhibits contraction also needs to be ruled out.
Finally, if it could be proven rigorously that NeuroTrace only labels pericytes that lack smooth muscle actin, and does not label the contractile pericytes that help to regulate blood flow, then it might provide a method for identifying differences in protein expression in these different types of pericyte with different functions along the capillary bed.
References:
Attwell D, Mishra A, Hall CN, O'Farrell FM, Dalkara T. What is a pericyte?. J Cereb Blood Flow Metab. 2016 Feb;36(2):451-5. Epub 2015 Oct 14 PubMed.
Biesecker KR, Srienc AI, Shimoda AM, Agarwal A, Bergles DE, Kofuji P, Newman EA. Glial Cell Calcium Signaling Mediates Capillary Regulation of Blood Flow in the Retina. J Neurosci. 2016 Sep 7;36(36):9435-45. PubMed.
Edwards IJ, Singh M, Morris S, Osborne L, Le Ruez T, Fuad M, Deuchars SA, Deuchars J. A simple method to fluorescently label pericytes in the CNS and skeletal muscle. Microvasc Res. 2013 Sep;89:164-8. Epub 2013 Jun 10 PubMed.
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.
Hartmann DA, Underly RG, Grant RI, Watson AN, Lindner V, Shih AY. Pericyte structure and distribution in the cerebral cortex revealed by high-resolution imaging of transgenic mice. Neurophotonics. 2015 Oct;2(4):041402. Epub 2015 May 27 PubMed.
Hirase H, Creso J, Singleton M, Barthó P, Buzsáki G. Two-photon imaging of brain pericytes in vivo using dextran-conjugated dyes. Glia. 2004 Apr 1;46(1):95-100. PubMed.
Kisler K, Nelson AR, Rege SV, Ramanathan A, Wang Y, Ahuja A, Lazic D, Tsai PS, Zhao Z, Zhou Y, Boas DA, Sakadžić S, Zlokovic BV. Pericyte degeneration leads to neurovascular uncoupling and limits oxygen supply to brain. Nat Neurosci. 2017 Mar;20(3):406-416. Epub 2017 Jan 30 PubMed.
Krueger M, Bechmann I. CNS pericytes: concepts, misconceptions, and a way out. Glia. 2010 Jan 1;58(1):1-10. PubMed.
Leal-Campanario R, Alarcon-Martinez L, Rieiro H, Martinez-Conde S, Alarcon-Martinez T, Zhao X, LaMee J, Popp PJ, Calhoun ME, Arribas JI, Schlegel AA, Stasi LL, Rho JM, Inge L, Otero-Millan J, Treiman DM, Macknik SL. Abnormal Capillary Vasodynamics Contribute to Ictal Neurodegeneration in Epilepsy. Sci Rep. 2017 Feb 27;7:43276. PubMed.
Li Y, Lucas-Osma AM, Black S, Bandet MV, Stephens MJ, Vavrek R, Sanelli L, Fenrich KK, Di Narzo AF, Dracheva S, Winship IR, Fouad K, Bennett DJ. Pericytes impair capillary blood flow and motor function after chronic spinal cord injury. Nat Med. 2017 May 1; PubMed.
Mishra A, Reynolds JP, Chen Y, Gourine AV, Rusakov DA, Attwell D. Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles. Nat Neurosci. 2016 Dec;19(12):1619-1627. Epub 2016 Oct 24 PubMed.
Nehls V, Drenckhahn D. Heterogeneity of microvascular pericytes for smooth muscle type alpha-actin. J Cell Biol. 1991 Apr;113(1):147-54. PubMed.
Peppiatt CM, Howarth C, Mobbs P, Attwell D. Bidirectional control of CNS capillary diameter by pericytes. Nature. 2006 Oct 12;443(7112):700-4. Epub 2006 Oct 1 PubMed.
Yemisci M, Gursoy-Ozdemir Y, Vural A, Can A, Topalkara K, Dalkara T. Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery. Nat Med. 2009 Sep;15(9):1031-7. Epub 2009 Aug 30 PubMed.
Mayo Clinic
Dr. Grutzendler’s group found that Nissl dye NeuroTrace 500/525 specifically labels brain capillary pericytes in living mice. This dye can stain Pdgfrb and NG2-expressing cells on capillaries, but not precapillary or arteriolar αSMA-expressing cells. Their finding is very interesting and provides a new strong tool to explore pericyte functions in vivo. However, it is still questionable if pericytes and smooth muscle cells can be totally distinguished by this dye due to the diversity of pericytes. It is plausible that subclasses of pericytes express αSMA. While they demonstrated that NeuroTrace-labeled capillary pericytes do not exhibit contractility, this does not necessarily mean that no “pericyte” contributes to cerebrovasculature contraction. Nonetheless, I believe this technique significantly advances pericyte research. This technique allows us to investigate the properties of capillary pericytes by distinguishing them from other vascular mural cells in living mouse models.
If follow-up studies can determine the molecular mechanism by which Nissl dye NeuroTrace 500/525 is specifically taken up by capillary pericytes, it may be possible to identify novel specific markers for this cell type. Further optimization of this method may also enable us to isolate capillary pericytes from mouse brains followed by detail characterization of these cells.
In addition, NeuroTrace labels pericytes that are not associated with vessels. Since pericytes likely migrate to sites of injury and form the core of the scar after spinal cord injury (Göritz et al., 2011), it will be interesting to investigate if NeuroTrace can detect the migrating pericytes upon brain injury.
References:
Göritz C, Dias DO, Tomilin N, Barbacid M, Shupliakov O, Frisén J. A pericyte origin of spinal cord scar tissue. Science. 2011 Jul 8;333(6039):238-42. PubMed.
Medical University of South Carolina
This is a remarkable find for pericyte research because of its specificity and ease of application to any mouse model during live imaging. It is also extremely fortuitous that it can be readily coupled with a red dye for astrocytes (SR101) and a far-red dye for arteriole elastin (Alexa633) during in vivo multiphoton studies.
It will be hard to break the stalemate on what to call a smooth muscle cell and a pericyte. This is because α-SMA expression and cell morphology (protruding cell bodies and elongated shape) historically have been used to define a subset of contractile pericytes on precapillary arterioles. α-SMA is also thought of as a verified marker of pericytes, though a number of recent studies, including work from the Grutzendler lab, have shown that it is not expressed by pericytes with classic fusiform morphology.
Semantics aside, it is important to recognize that there is a sharp transition in mural cell function where α-SMA expression ends and Neurotrace begins, i.e., when precapillary arterioles turn into capillaries. In future work on cerebral pericytes, it will be important to clarify if one is studying cells upstream or downstream of this transition point to avoid adding confusion to mural cell roles.
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