Therapeutic Benefits of Mesenchymal Stromal Cells in a Rat Model of Hemoglobin-Induced Hypertensive Intracerebral Hemorrhage

  • Ding, Rui (Department of Neurosurgery, Jingmen No. 1 People's Hospital) ;
  • Lin, Chunnan (Department of Neurosurgery, Maoming People's Hospital) ;
  • Wei, ShanShan (Department of Hematology, Jingmen No. 1 People's Hospital) ;
  • Zhang, Naichong (Department of Neurosurgery, Maoming People's Hospital) ;
  • Tang, Liangang (Department of Neurosurgery, Maoming People's Hospital) ;
  • Lin, Yumao (Department of Neurosurgery, Maoming People's Hospital) ;
  • Chen, Zhijun (Department of Neurosurgery, Jingmen No. 1 People's Hospital) ;
  • Xie, Teng (Department of Neurosurgery, Jingmen No. 1 People's Hospital) ;
  • Chen, XiaoWei (Department of Neurosurgery, Jingmen No. 1 People's Hospital) ;
  • Feng, Yu (Department of Neurosurgery, Jingmen No. 1 People's Hospital) ;
  • Wu, LiHua (Department of Neurosurgery, Jingmen No. 1 People's Hospital)
  • Received : 2016.10.25
  • Accepted : 2017.01.09
  • Published : 2017.02.28


Previous studies have shown that bone marrow mesenchymal stromal cell (MSC) transplantation significantly improves the recovery of neurological function in a rat model of intracerebral hemorrhage. Potential repair mechanisms involve anti-inflammation, anti-apoptosis and angiogenesis. However, few studies have focused on the effects of MSCs on inducible nitric oxide synthase (iNOS) expression and subsequent peroxynitrite formation after hypertensive intracerebral hemorrhage (HICH). In this study, MSCs were transplanted intracerebrally into rats 6 hours after HICH. The modified neurological severity score and the modified limb placing test were used to measure behavioral outcomes. Blood-brain barrier disruption and neuronal loss were measured by zonula occludens-1 (ZO-1) and neuronal nucleus (NeuN) expression, respectively. Concomitant edema formation was evaluated by H&E staining and brain water content. The effect of MSCs treatment on neuroinflammation was analyzed by immunohistochemical analysis or polymerase chain reaction of CD68, Iba1, iNOS expression and subsequent peroxynitrite formation, and by an enzyme-linked immunosorbent assay of pro-inflammatory factors (IL-$1{\beta}$ and TNF-${\alpha}$). The MSCs-treated HICH group showed better performance on behavioral scores and lower brain water content compared to controls. Moreover, the MSC injection increased NeuN and ZO-1 expression measured by immunochemistry/immunofluorescence. Furthermore, MSCs reduced not only levels of CD68, Iba1 and pro-inflammatory factors, but it also inhibited iNOS expression and peroxynitrite formation in perihematomal regions. The results suggest that intracerebral administration of MSCs accelerates neurological function recovery in HICH rats. This may result from the ability of MSCs to suppress inflammation, at least in part, by inhibiting iNOS expression and subsequent peroxynitrite formation.


hypertensive intracerebral hemorrhage;iNOS;mesenchymal stromal cells;$ONOO^-$


  1. Aronowski, J., and Hall, C.E. (2005). New horizons for primary intracerebral hemorrhage treatment: experience from preclinical studies. Neurol Res. 27, 268-279.
  2. Balami, J.S., and Buchan, A.M. (2012). Complications of intracerebral haemorrhage. Lancet Neurol. 11, 101-118.
  3. Beckman, J.S., and Koppenol, W.H. (1996). Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271, C1424-1437.
  4. Bhasin, R.R., Xi, G., Hua, Y., Keep, R.F., and Hoff, J.T. (2002). Experimental intracerebral hemorrhage: effect of lysed erythrocytes on brain edema and blood-brain barrier permeability. Acta Neurochir. Suppl. 81, 249-251.
  5. Candelise, L., Gattinoni, M., Bersano, A., Micieli, G., Sterzi, R., and Morabito, A. (2007). Stroke-unit care for acute stroke patients: an observational follow-up study. Lancet 369, 299-305.
  6. Chen, M., Li, X., Zhang, X., He, X., Lai, L., Liu, Y., Zhu, G., Li, W., Li, H., Fang, Q., Wang, Z., and Duan, C. (2015). The inhibitory effect of mesenchymal stem cell on blood-brain barrier disruption following intracerebral hemorrhage in rats: contribution of TSG-6. J. Neuroinflammation 12, 61.
  7. Di Nicola, M., Carlo-Stella, C., Magni, M., Milanesi, M., Longoni, P.D., Matteucci, P., Grisanti, S., and Gianni, A.M. (2002). Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 99, 3838-3843.
  8. Ding, R., Chen, Y., Yang, S., Deng, X., Fu, Z., Feng, L., Cai, Y., Du M, Zhou, Y., and Tang, Y. (2014). Blood-brain barrier disruption induced by hemoglobin in vivo: Involvement of up-regulation of nitric oxide synthase and peroxynitrite formation. Brain Res. 1571, 25-38.
  9. Ding, R., Feng, L., He, L., Chen, Y., Wen, P., Fu, Z., Lin, C., Yang, S., Deng, X., Zeng, J., and Sun, G. (2015). Peroxynitrite decomposition catalyst prevents matrix metalloproteinase-9 activation and neurovascular injury after hemoglobin injection into the caudate nucleus of rats. Neuroscience 297, 182-193.
  10. Fatar, M., Stroick, M., Griebe, M., Marwedel, I., Kern, S., Bieback, K., Giesel, F.L., Zechmann, C., Kreisel, S., Vollmar, F., et al., (2008). Lipoaspirate-derived adult mesenchymal stem cells improve functional outcome during intracerebral hemorrhage by proliferation of endogenous progenitor cells stem cells in intracerebral hemorrhages. Neurosci Lett. 443, 174-178.
  11. Gong, C., Hoff, J.T., and Keep, R.F. (2000). Acute inflammatory reaction following experimental intracerebral hemorrhage in rat. Brain Res. 871, 57-65.
  12. Hermann, A., Gastl, R., Liebau, S., Popa, M.O., Fiedler, J., Boehm, B.O., Maisel, M., Lerche, H., Schwarz, J., Brenner, R., et al. (2004). Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells. J. Cell Sci. 117, 4411-4422.
  13. Hirabayashi, H., Takizawa, S., Fukuyama, N., Nakazawa, H., and Shinohara, Y. (2000). Nitrotyrosine generation via inducible nitric oxide synthase in vascular wall in focal ischemia-reperfusion. Brain Res. 852, 319-325.
  14. Hua, Y., Schallert, T., Keep, R.F., Wu, J., Hoff, J.T., and Xi, G. (2002). Behavioral tests after intracerebral hemorrhage in the rat. Stroke 33, 2478-2484.
  15. Huang, F.P., Xi, G., Keep, R.F., Hua, Y., Nemoianu, A., and Hoff, J.T. (2002). Brain edema after experimental intracerebral hemorrhage: role of hemoglobin degradation products. J. Neurosurg. 96, 287-93.
  16. Jiang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D., Ortiz-Gonzalez, X.R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., et al. (2002). Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41-49.
  17. Jin, S.Z., Meng, X.W., Sun, X., Han, M.Z., Liu, B.R., Wang, X.H., and Pei, F.H. (2011). Hepatocyte growth factor promotes liver regeneration induced by transfusion of bone marrow mononuclear cells in a murine acute liver failure model. J. Hepatobiliary Pancreat. Sci. 18, 397-405.
  18. Jung, K.H., Chu, K., Jeong, S.W., Han, S.Y., Lee, S.T., Kim, J.Y., Kim, M., and Roh, J.K. (2004). HMG-CoA reductase inhibitor, atorvastatin, promotes sensorimotor recovery, suppressing acute inflammatory reaction after experimental intracerebral hemorrhage. Stroke 35, 1744-9.
  19. Katsu, M., Niizuma, K., Yoshioka, H., Okami, N., Sakata, H., and Chan, P.H. (2010). Hemoglobin-induced oxidative stress contributes to matrix metalloproteinase activation and blood-brain barrier dysfunction in vivo. J. Cereb. Blood Flow Metab. 30, 1939-1950.
  20. Khan, M., Dhammu, T.S., Sakakima, H., Shunmugavel, A., Gilg, A.G., Singh, A.K., and Singh, I. (2012). The inhibitory effect of Snitrosoglutathione on blood-brain barrier disruption and peroxynitrite formation in a rat model of experimental stroke. J. Neurochem. 123 Suppl 2, 86-97.
  21. Kim, D.W., Im, S.H., Kim, J.Y., Kim, D.E., Oh, G.T., and Jeong, S.W. (2009). Decreased brain edema after collagenase-induced intracerebral hemorrhage in mice lacking the inducible nitric oxide synthase gene. Laboratory investigation. J. Neurosurg. 111, 995-1000.
  22. Liang, H., Yin, Y., Lin, T., Guan, D., Ma, B., Li, C., Wang, Y., and Zhang, X. (2013). Transplantation of bone marrow stromal cells enhances nerve regeneration of the corticospinal tract and improves recovery of neurological functions in a collagenase-induced rat model of intracerebral hemorrhage. Mol. Cells 36, 17-24.
  23. Liao, W., Zhong, J., Yu, J., Xie, J., Liu, Y., Du L, Yang, S., Liu, P., Xu, J., Wang, J., Han, Z., and Han, Z.C. (2009). Therapeutic benefit of human umbilical cord derived mesenchymal stromal cells in intracerebral hemorrhage rat: implications of anti-inflammation and angiogenesis. Cell. Physiol. Biochem. 24, 307-316.
  24. Masada, T., Hua, Y., Xi, G., Yang, G.Y., Hoff, J.T., and Keep, R.F. (2001). Attenuation of intracerebral hemorrhage and thrombininduced brain edema by overexpression of interleukin-1 receptor antagonist. J. Neurosurg. 95, 680-686.
  25. Meirelles, L.S., Fontes, A.M., Covas, D.T., and Caplan, A.I. (2009). Mechanisms involved in the therapeutic properties of mesenchymal stem cells. Cytokine Growth Factor Rev. 20, 419-427.
  26. Moncada, S., and Bolanos, J.P. (2006). Nitric oxide, cell bioenergetics and neurodegeneration. J. Neurochem. 97, 1676-1689.
  27. Murphy, T.H., and Corbett, D. (2009). Plasticity during stroke recovery: from synapse to behaviour. Nat. Rev. Neurosci. 10, 861-872.
  28. Nagai, A., Kim, W.K., Lee, H.J., Jeong, H.S., Kim, K.S., Hong, S.H., Park, I.H., and Kim, S.U. (2007). Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow. PLoS One 2, e1272.
  29. Nogawa, S., Forster, C., Zhang, F., Nagayama, M., Ross, M.E., and Iadecola, C. (1998). Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia. Proc. Natl. Acad. Sci. USA 95, 10966-10971.
  30. Ocarino, N.M., Bozzi, A., Pereira, R.D., Breyner, N.M., Silva, V.L., Castanheira, P., Goes, A.M., and Serakides, R., (2008). Behavior of mesenchymal stem cells stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) in osteogenic and non osteogenic cultures. Biocell 32, 175-183.
  31. Pacher, P., Beckman, J.S., and Liaudet, L. (2007). Nitric oxide and peroxynitrite in health and disease. Physiol. Rev. 87, 315-424.
  32. Pannu, R., and Singh, I., (2006). Pharmacological strategies for the regulation of inducible nitric oxide synthase: neurodegenerative versus neuroprotective mechanisms. Neurochem. Int. 49, 170-182.
  33. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147.
  34. Qureshi, A.I., Mendelow, A.D., and Hanley, D.F. (2009). Intracerebral haemorrhage. Lancet 373, 1632-1644.
  35. Seyfried, D.M., Han, Y., Yang, D., Ding, J., Shen, L.H., Savant-Bhonsale, S., and Chopp, M. (2010). Localization of bone marrow stromal cells to the injury site after intracerebral hemorrhage in rats. J. Neurosurg. 112, 329-335.
  36. Steinberg, G.K., Kondziolka, D., Wechsler, L.R., Lunsford, L.D., Coburn, M.L., Billigen, J.B., Kim, A.S., Johnson, J.N., Bates, D., King, B., et al. (2016). Clinical outcomes of transplanted modified bone marrow-derived mesenchymal stem cells in stroke: a phase 1/2a study. Stroke 47, 1817-1824.
  37. Suofu, Y., Clark, J., Broderick, J., Wagner, K.R., Tomsick, T., Sa, Y., and Lu, A. (2010). Peroxynitrite decomposition catalyst prevents matrix metalloproteinase activation and neurovascular injury after prolonged cerebral ischemia in rats. J. Neurochem. 115, 1266-1276.
  38. Tejima, E., Zhao, B.Q., Tsuji, K., Rosell, A., van Leyen, K., Gonzalez, R.G., Montaner, J., Wang, X., and Lo, E.H., (2007). Astrocytic induction of matrix metalloproteinase-9 and edema in brain hemorrhage. J. Cereb. Blood Flow Metab. 27, 460-468.
  39. Vaquero, J., Otero, L., Bonilla, C., Aguayo, C., Rico, M.A., Rodriguez, A., and Zurita, M. (2013). Cell therapy with bone marrow stromal cells after intracerebral hemorrhage: impact of platelet-rich plasma scaffolds. Cytotherapy 15, 33-43.
  40. Virag, L., Szabo, E., Gergely, P., and Szabo, C. (2003). Peroxynitriteinduced cytotoxicity: mechanism and opportunities for intervention. Toxicol. Lett. 140-141, 113-124.
  41. Wang, J., and Dore, S. (2008). Heme oxygenase 2 deficiency increases brain swelling and inflammation after intracerebral hemorrhage. Neuroscience 155, 1133-1141.
  42. Wang, J., Fields, J., Zhao, C., Langer, J., Thimmulappa, R.K., Kensler, T.W., Yamamoto, M., Biswal, S., and Dore, S. (2007). Role of Nrf2 in protection against intracerebral hemorrhage injury in mice. Free Radic. Biol. Med. 43, 408-414.
  43. Wang, S.P., Wang, Z.H., Peng, D.Y., Li, S.M., Wang, H., and Wang, X.H. (2012). Therapeutic effect of mesenchymal stem cells in rats with intracerebral hemorrhage: reduced apoptosis and enhanced neuroprotection. Mol. Med. Rep. 6, 848-854.
  44. Wu, B., Ma, Q., Suzuki, H., Chen, C., Liu, W., Tang, J., and Zhang, J. (2011). Recombinant osteopontin attenuates brain injury after intracerebral hemorrhage in mice. Neurocrit. Care 14, 109-117.
  45. Xi, G., Keep, R.F., and Hoff, J.T. (1998). Erythrocytes and delayed brain edema formation following intracerebral hemorrhage in rats. J. Neurosurg. 89, 991-6.
  46. Xi, G., Hua, Y., Bhasin, R.R., Ennis, S.R., Keep, R.F., and Hoff, J.T. (2001). Mechanisms of edema formation after intracerebral hemorrhage: effects of extravasated red blood cells on blood flow and blood-brain barrier integrity. Stroke 32, 2932-2938.
  47. Yan, K., Zhang, R., Sun, C., Chen, L., Li, P., Liu, Y., Peng, L., Sun, H., Qin, K., Chen, F., et al. (2013). Bone marrow-derived mesenchymal stem cells maintain the resting phenotype of microglia and inhibit microglial activation. PLoS One 8, e84116.
  48. Yang, S., Chen, Y., Deng, X., Jiang, W., Li, B., Fu, Z., Du, M., and Ding, R. (2013). Hemoglobin-induced nitric oxide synthase overexpression and nitric oxide production contribute to blood-brain barrier disruption in the rat. J. Mol. Neurosci. 51, 352-363.
  49. Zhang, L., Schallert, T., Zhang, Z.G., Jiang, Q., Arniego, P., Li, Q., Lu, M., and Chopp, M. (2002). A test for detecting long-term sensorimotor dysfunction in the mouse after focal cerebral ischemia. J. Neurosci. Methods 117, 207-214.
  50. Zhang, R., Liu, Y., Yan, K., Chen, L., Chen, X.R., Li, P., Chen, F.F., and Jiang, X.D. (2013). Anti-inflammatory and immunomodulatory mechanisms of mesenchymal stem cell transplantation in experimental traumatic brain injury. J. Neuroinflammation 10, 106.
  51. Zhao, X., Zhang, Y., Strong, R., Zhang, J., Grotta, J.C., and Aronowski, J. (2007). Distinct patterns of intracerebral hemorrhageinduced alterations in NF-kappaB subunit, iNOS, and COX-2 expression. J. Neurochem. 101, 652-663.