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

Article metrics

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 optionsAccess 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).

  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).

  3. 3

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

  4. 4

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

  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).

  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).

  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).

  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).

  9. 9

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

  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).

  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).

  12. 12

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

  13. 13

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

  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).

  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).

  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).

  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).

  18. 18

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

  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).

  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).

  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).

  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).

  23. 23

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

  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).

  25. 25

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

  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).

  27. 27

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

  28. 28

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

  29. 29

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

  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).

  31. 31

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

  32. 32

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

  33. 33

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

  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).

  35. 35

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

  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).

  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).

  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).

  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).

  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).

  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).

  42. 42

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

  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).

  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).

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

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.

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

Further reading