Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery

Abstract

Here we show that ischemia induces sustained contraction of pericytes on microvessels in the intact mouse brain. Pericytes remain contracted despite successful reopening of the middle cerebral artery after 2 h of ischemia. Pericyte contraction causes capillary constriction and obstructs erythrocyte flow. Suppression of oxidative-nitrative stress relieves pericyte contraction, reduces erythrocyte entrapment and restores microvascular patency; hence, tissue survival improves. In contrast, peroxynitrite application causes pericyte contraction. We also show that the microvessel wall is the major source of oxygen and nitrogen radicals causing ischemia and reperfusion–induced microvascular dysfunction. These findings point to a major but previously not recognized pathophysiological mechanism; ischemia and reperfusion-induced injury to pericytes may impair microcirculatory reflow and negatively affect survival by limiting substrate and drug delivery to tissue already under metabolic stress, despite recanalization of an occluded artery. Agents that can restore pericyte dysfunction and microvascular patency may increase the success of thrombolytic and neuroprotective treatments.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Ischemia and reperfusion induces segmental narrowing of capillaries due to sustained contraction of pericytes.
Figure 2: Ischemic pericytes contract and hence constrict the adjoining capillary.
Figure 3: Suppression of oxidative-nitrative stress during reperfusion relieves pericyte contraction and restores microvascular patency.
Figure 4: Ischemia- or peroxynitrite-induced pericyte contraction colocalize with 3-nitrotyrosine immunolabeling.
Figure 5: Despite successful recirculation, capillaries in the MCA territory were filled with trapped erythrocytes.
Figure 6: Effects of ischemia and peroxynitrite on microvessels visualized with FITC-dextran and monitored through a cranial window.

References

  1. 1

    Juttler, E., Kohrmann, M. & Schellinger, P.D. Therapy for early reperfusion after stroke. Nat. Clin. Pract. Cardiovasc. Med. 3, 656–663 (2006).

    CAS  Article  Google Scholar 

  2. 2

    Molina, C.A. & Saver, J.L. Extending reperfusion therapy for acute ischemic stroke: emerging pharmacological, mechanical, and imaging strategies. Stroke 36, 2311–2320 (2005).

    Article  Google Scholar 

  3. 3

    Lo, E.H., Dalkara, T. & Moskowitz, M.A. Mechanisms, challenges and opportunities in stroke. Nat. Rev. Neurosci. 4, 399–415 (2003).

    CAS  Article  Google Scholar 

  4. 4

    Abbott, N.J., Ronnback, L. & Hansson, E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat. Rev. Neurosci. 7, 41–53 (2006).

    CAS  Article  Google Scholar 

  5. 5

    Little, J.R., Kerr, F.W. & Sundt, T.M. Jr. Microcirculatory obstruction in focal cerebral ischemia. Relationship to neuronal alterations. Mayo Clin. Proc. 50, 264–270 (1975).

    CAS  PubMed  Google Scholar 

  6. 6

    del Zoppo, G.J., Schmid-Schonbein, G.W., Mori, E., Copeland, B.R. & Chang, C.M. Polymorphonuclear leukocytes occlude capillaries following middle cerebral artery occlusion and reperfusion in baboons. Stroke 22, 1276–1283 (1991).

    CAS  Article  Google Scholar 

  7. 7

    Little, J.R., Kerr, F.W.L. & Thoralf, M.S. Microcirculatory obstruction in focal cerebral ischemia: an electron microscopic investigation in monkeys. Stroke 7, 25–30 (1976).

    Article  Google Scholar 

  8. 8

    Hallenbeck, J.M. et al. Polymorphonuclear leukocyte accumulation in brain regions with low blood flow during the early postischemic period. Stroke 17, 246–253 (1986).

    CAS  Article  Google Scholar 

  9. 9

    del Zoppo, G.J. & Mabuchi, T. Cerebral microvessel responses to focal ischemia. J. Cereb. Blood Flow Metab. 23, 879–894 (2003).

    Article  Google Scholar 

  10. 10

    Garcia, J.H., Liu, K.F., Yoshida, Y., Chen, S. & Lian, J. Brain microvessels: factors altering their patency after the occlusion of a middle cerebral artery (Wistar rat). Am. J. Pathol. 145, 728–740 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Ohtake, M., Morino, S., Kaidoh, T. & Inoue, T. Three-dimensional structural changes in cerebral microvessels after transient focal cerebral ischemia in rats: scanning electron microscopic study of corrosion casts. Neuropathology 24, 219–227 (2004).

    Article  Google Scholar 

  12. 12

    Peppiatt, C.M., Howarth, C., Mobbs, P. & Attwell, D. Bidirectional control of CNS capillary diameter by pericytes. Nature 443, 700–704 (2006).

    CAS  Article  Google Scholar 

  13. 13

    Chan, P.H. Role of oxidants in ischemic brain damage. Stroke 27, 1124–1129 (1996).

    CAS  Article  Google Scholar 

  14. 14

    Chong, Z.Z., Li, F. & Maiese, K. Oxidative stress in the brain: novel cellular targets that govern survival during neurodegenerative disease. Prog. Neurobiol. 75, 207–246 (2005).

    CAS  Article  Google Scholar 

  15. 15

    Heo, J.H., Han, S.W. & Lee, S.K. Free radicals as triggers of brain edema formation after stroke. Free Radic. Biol. Med. 39, 51–70 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Gürsoy-Ozdemir, Y., Bolay, H., Saribas, O. & Dalkara, T. Role of endothelial nitric oxide generation and peroxynitrite formation in reperfusion injury after focal cerebral ischemia. Stroke 31, 1974–1980 (2000).

    Article  Google Scholar 

  17. 17

    Gürsoy-Ozdemir, Y., Can, A. & Dalkara, T. Reperfusion-induced oxidative/nitrative injury to neurovascular unit after focal cerebral ischemia. Stroke 35, 1449–1453 (2004).

    Article  Google Scholar 

  18. 18

    Gibson, C.L., Coughlan, T.C. & Murphy, S.P. Glial nitric oxide and ischemia. Glia 50, 417–426 (2005).

    Article  Google Scholar 

  19. 19

    Iadecola, C., Zhang, F., Casey, R., Nagayama, M. & Ross, M.E. Delayed reduction of ischemic brain injury and neurological deficits in mice lacking the inducible nitric oxide synthase gene. J. Neurosci. 17, 9157–9164 (1997).

    CAS  Article  Google Scholar 

  20. 20

    Newell, D.W., Barth, A., Papermaster, V. & Malouf, A.T. Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures. J. Neurosci. 15, 7702–7711 (1995).

    CAS  Article  Google Scholar 

  21. 21

    Liu, S., Connor, J., Peterson, S., Shuttleworth, C.W. & Liu, K.J. Direct visualization of trapped erythrocytes in rat brain after focal ischemia and reperfusion. J. Cereb. Blood Flow Metab. 22, 1222–1230 (2002).

    Article  Google Scholar 

  22. 22

    Wei, E.P., Kontos, H.A. & Beckman, J.S. Mechanisms of cerebral vasodilation by superoxide, hydrogen peroxide, and peroxynitrite. Am. J. Physiol. 271, H1262–H1266 (1996).

    CAS  Article  Google Scholar 

  23. 23

    Hossmann, K.A. Pathophysiology and therapy of experimental stroke. Cell. Mol. Neurobiol. 26, 1057–1084 (2006).

    Article  Google Scholar 

  24. 24

    Li, P.A. et al. Capillary patency after transient middle cerebral artery occlusion of 2 h duration. Neurosci. Lett. 253, 191–194 (1998).

    CAS  Article  Google Scholar 

  25. 25

    Allt, G. & Lawrenson, J.G. Pericytes: cell biology and pathology. Cells Tissues Organs 169, 1–11 (2001).

    CAS  Article  Google Scholar 

  26. 26

    Balabanov, R. & Dore-Duffy, P. Role of the CNS microvascular pericyte in the blood-brain barrier. J. Neurosci. Res. 53, 637–644 (1998).

    CAS  Article  Google Scholar 

  27. 27

    Bandopadhyay, R. et al. Contractile proteins in pericytes at the blood-brain and blood-retinal barriers. J. Neurocytol. 30, 35–44 (2001).

    CAS  Article  Google Scholar 

  28. 28

    Kamouchi, M. et al. Hydrogen peroxide–induced Ca2+ responses in CNS pericytes. Neurosci. Lett. 416, 12–16 (2007).

    CAS  Article  Google Scholar 

  29. 29

    Chrissobolis, S. & Sobey, C.G. Recent evidence for an involvement of rho-kinase in cerebral vascular disease. Stroke 37, 2174–2180 (2006).

    CAS  Article  Google Scholar 

  30. 30

    Rees, D.D., Palmer, R.M., Schulz, R., Hodson, H.F. & Moncada, S. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br. J. Pharmacol. 101, 746–752 (1990).

    CAS  Article  Google Scholar 

  31. 31

    Huang, Z. et al. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 265, 1883–1885 (1994).

    CAS  Article  Google Scholar 

  32. 32

    Faraci, F.M. Reactive oxygen species: influence on cerebral vascular tone. J. Appl. Physiol. 100, 739–743 (2006).

    CAS  Article  Google Scholar 

  33. 33

    Pacher, P., Beckman, J.S. & Liaudet, L. Nitric oxide and peroxynitrite in health and disease. Physiol. Rev. 87, 315–424 (2007).

    CAS  Article  Google Scholar 

  34. 34

    Ames, A., III, Wright, R.L., Kowada, M., Thurston, J.M. & Majno, G. Cerebral ischemia. II. The no-reflow phenomenon. Am. J. Pathol. 52, 437–453 (1968).

    PubMed  PubMed Central  Google Scholar 

  35. 35

    Fischer, E.G. Impaired perfusion following cerebrovascular stasis. A review. Arch. Neurol. 29, 361–366 (1973).

    CAS  Article  Google Scholar 

  36. 36

    Crowell, R.M. & Olsson, Y. Impaired microvascular filling after focal cerebral ischemia in the monkey. Modification by treatment. Neurology 22, 500–504 (1972).

    CAS  Article  Google Scholar 

  37. 37

    Ritter, L.S., Orozco, J.A., Coull, B.M., McDonagh, P.F. & Rosenblum, W.I. Leukocyte accumulation and hemodynamic changes in the cerebral microcirculation during early reperfusion after stroke. Stroke 31, 1153–1161 (2000).

    CAS  Article  Google Scholar 

  38. 38

    Anwar, M., Buchweitz-Milton, E. & Weiss, H.R. Effect of prazosin on microvascular perfusion during middle cerebral artery ligation in the rat. Circ. Res. 63, 27–34 (1988).

    CAS  Article  Google Scholar 

  39. 39

    Folbergrová, J., Zhao, Q., Katsura, K. & Siesjo, B.K. N-tert-butyl-α-phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia. Proc. Natl. Acad. Sci. USA 92, 5057–5061 (1995).

    Article  Google Scholar 

  40. 40

    Belayev, L. et al. Albumin therapy of transient focal cerebral ischemia: in vivo analysis of dynamic microvascular responses. Stroke 33, 1077–1084 (2002).

    Article  Google Scholar 

  41. 41

    Verdouw, P.D., Jennewein, H.M., Heiligers, J., Duncker, D.J. & Saxena, P.R. Redistribution of carotid artery blood flow by 5-HT: effects of the 5–HT2 receptor antagonists ketanserin and Wal 1307. Eur. J. Pharmacol. 102, 499–509 (1984).

    CAS  Article  Google Scholar 

  42. 42

    Hudetz, A.G. Blood flow in the cerebral capillary network: a review emphasizing observations with intravital microscopy. Microcirculation 4, 233–252 (1997).

    CAS  Article  Google Scholar 

  43. 43

    Ozerdem, U., Grako, K.A., Dahlin-Huppe, K., Monosov, E. & Stallcup, W.B. NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis. Dev. Dyn. 222, 218–227 (2001).

    CAS  Article  Google Scholar 

  44. 44

    Wu, D.M., Kawamura, H., Sakagami, K., Kobayashi, M. & Puro, D.G. Cholinergic regulation of pericyte-containing retinal microvessels. Am. J. Physiol. Heart Circ. Physiol. 284, H2083–H2090 (2003).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by The Turkish Academy of Sciences (T.D. and Y.G.-O.), Hacettepe University Research Fund 0401105001 (T.D.), Scientific and Technical Research Council of Turkey 104S254 (Y.G.-O.), Ankara University Biotechnology Institute 2001K120240 (A.C.) and Brain Research Association (M.Y.). We are grateful to M.A. Moskowitz for his support and comments. Part of this study was presented at the Society For Neuroscience 37th Annual Meeting in San Diego, California, 2007.

Author information

Affiliations

Authors

Contributions

M.Y. performed the in vivo experiments and histology studies and contributed to the in vitro studies, design of the experiments, data analyses and preparation of the figures; Y.G.-O. conducted and performed the intravital microscopy experiments and in vitro studies and contributed to the histology studies, design of the experiments and preparation of the figures; A.V. contributed to the intravital microscopy experiments and in vitro studies, performed image analyses and contributed to the preparation of the figures; A.C. conducted the confocal and DIC microscopy studies and prepared the figures; K.T. contributed to the in vivo experiments and performed the in vivo experiments with knockout mice; T.D. designed and supervised the project, contributed to the data analyses and wrote the manuscript.

Corresponding author

Correspondence to Turgay Dalkara.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Data and Supplementary Methods (PDF 1850 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yemisci, M., Gursoy-Ozdemir, Y., Vural, A. et al. Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery. Nat Med 15, 1031–1037 (2009). https://doi.org/10.1038/nm.2022

Download citation

Further reading