Effect of ischemic postconditioining on reaction of neocortex microglia after global brain ischemia in rats

Introduction. Ischemic postconditioning (IPostC) is a new concept in the brain protection strategy. Almost all researches in this area focus on the functioning and survival of neurons, while non-neuronal cells affected by IPostC remain unexplored. The aim is to study the IPostC effect on changes in microglia in the neocortex of Wistar rats after global brain ischemia during various periods of reperfusion. Materials and methods. Male Wistar rats were used as a model of a 10-minute global brain ischemia with a subsequent IPostC; the reperfusion-ischemia cycle was 15 s/15 s. In the early (2 days) and late (7 days) reperfusion periods, the number of morphologically unchanged neurons and Iba-1-positive nucleated microglyocytes in the occipital cortex was estimated. Results. It has been shown that global brain ischemia in rats leads to 25.9% (P<0.05) neuron death and an increase of 30.9% (P<0.05) in the number of Iba-1-positive microglia cells by the 2nd day of the reperfusion period in the occipital neocortex; by the 7th day of reperfusion, there was observed a neuron death significant increasing by 34.5% (P<0.05) and the number of Iba-1-positive microglia cells increasing of 65.2% (P<0.05) compared to similar indicators in sham-operated groups. The IPostC by 2 days of reperfusion was found to increase the number of unchanged neurons in the occipital region of the cerebral cortex by 18.3% (P<0.05), which is not accompanied by a significant change in the number of Iba-1-positive microglial cells; by 7 days of reperfusion the increase number of unchanged neurons was found to be 23.5% (P<0.05) in the analysed brain region , which is accompanied by a decrease in the number of Iba-1-positive microgliosis by 32.5% (P<0.05) comparing with similar indicators in groups without IPostC. Conlusions. The results of this work suggest that the cytoprotective effect of IPostC for neurons of the occipital neocortex of Wistar rats in the long-term reperfusion period is caused by blocking the infiltration of the ischemic brain region by both resident and recruited cells of the immune system.

1. Zhao H. Ischemic postconditioning as a novel avenue to protect against brain injury after stroke. J Cereb Blood Flow Metab. 2009;29:873–885. Doi: 10.1038/jcbfm.2009.13.

2. Щербак Н. С., Русакова А. Г., Галагудза М. М. и др. Морфофункциональная характеристика микроциркуляторного русла и нейронов неокортекса после ишемического посткондиционирования // Морфология. – 2016. – Т. 49, № 5. – С. 21–27. [Shcherbak NS, Rusakova AG, Galagudza MM, Yukina GYu, Barantsevich ER, Tomson VV, Shlyakhto EV. Morphofunctional Characteristics of the Microcirculatory Bed and Neurons in the Neocortex after Ischemic Postconditioning. Neurosci Behav Physi. 2016;47:841–845. (In Russ.)]. Doi: 10.1007/s11055-017-0479-y.

3. Ding ZM, Wu B, Zhang WQ, Lu XJ, Lin YC, Geng YJ, Miao YF. Neuroprotective Effects of Ischemic Preconditioning and Postconditioning on Global Brain Ischemia in Rats through the Same Effect on Inhibition of Apoptosis. Int J Mol Sci. 2012;13(5):6089–6101. Doi: 10.3390/ijms13056089.

4. Calcia MA, Bonsall DR, Bloomfield PS, Selvaraj S, Barichello T, Howes OD. Stress and neuroinflammation: a systematic review of the effects of stress on microglia and the implications for mental illness. Psychopharmacology (Berl). 2016;233(9):1637–1650. Doi: 10.1007/s00213-016-4218-9.

5. Структурная организация микроглиоцитов стриатума после транзиторной фокальной ишемии / Д. Э. Коржевский, О. В. Кирик, Е. Г. Сухорукова, Т. Д. Власов // Морфология. –2012. – Т. 141, № 2. – С. 28–32. [Korzhevskii DE, Kirik OV, Sukhorukova EG, Vlasov TD. Structural organization of microgliosis striatum after transient focal ischemia. Morfologiia. 2012;141(2):28–32. (In Russ.)].

6. Frick LR, Williams K, Pittenger C. Microglial dysregulation in psychiatric disease. Clin Dev Immunol. 2013;608654. Doi: 10.1155/2013/608654.

7. Nakajima K, Kohsaka S. Functional roles of microglia in the brain. Neurosci. Res. 1993;17:187–203. Doi: 10.1016/0168-0102(93)90047-T.

8. Boje KM, Arora PK. Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Res. 1992;587:250–256. Doi: 10.1016/0006-8993(92)91004-X.

9. Joo SP, Xie W, Xiong X, Xu B, Zhao H. Ischemic postconditioning protects against focal cerebral ischemia by inhibiting brain inflammation while attenuating peripheral lymphopenia in mice. Neuroscience. 2013;243:149–157. Doi: 10.1016/j.neuroscience.2013.03.062.

10. Ulbrich F, Goebel U, Böhringer D, Charalambous P, Lagrèze WA, Biermann J. Carbon monoxide treatment reduces microglial activation in the ischemic rat retina. Graefes Arch Clin Exp Ophthalmol. 2016;254(10):1967–1976. Doi: 10.1007/s00417-016-3435-6.

11. Esposito E, Hayakawa K, Ahn BJ, Chan SJ, Xing C, Liang AC, Kim KW, Arai K, Lo EH. Effects of ischemic postconditioning on neuronal VEGF regulation and microglial polarization in a rat model of focal cerebral ischemia. J Neurochem. 2018;146(2):160–172. Doi: 10.1111/jnc.14337.

12. Ohsawa K, Imai Y, Kanazawa H, Sasaki Y, Kohsaka S. Involvement of Iba1 in membrane ruffling and phagocytosis of macrophages/microglia. J Cell Sci. 2000;113:3073–3084.

13. Щербак Н. С., Галагудза М. М., Кузьменков А. Н. и др. Новый способ моделирования обратимой глобальной ишемии головного мозга у крыс // Бюл. эксперим. биологии и медицины. – 2011. – Т. 152, № 11. – С. 592–595. [Shcherbak NS, Galagudza MM, Kuzmenkov AN, Ovchinnikov DA, Mitrofanova LB, Barantsevich ER, Shlyakhto EV. A new rat model of reversible global cerebral ischemia. Exp Biol Med. 2012 Mar;152(5):656–658. (In Russ.)]. Doi: 10.1007/s10517-012-1600-4.

14. Сухорукова Е. Г., Кирик О. В., Коржевский Д. Э. Применение иммуногистохимического метода для выявления микроглии головного мозга в парафиновых срезах // Бюл. эксперим. биологии и медицины. – 2010. – Т. 149, № 6. – С. 709–712. [Suchorukova EG, Kirik OV, Korzhevskii DE. The use of immunohistochemical method for detection of brain microglia in paraffin sections. Bulletin of Experimental Biology and Medicine. 2010;149(6):768–770. (In Russ.)].

15. Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 1982;239:57–69. Doi: 10.1016/0006-8993(82)90833-2.

16. Pulsinelli WA, Buchan AM. The four-vessel occlusion rat model: method for complete occlusion of vertebral arteries and control of collateral circulation. Stroke. 1988;19:913–914. Doi: 10.1161/01.STR.19.7.913.

17. Davis EJ, Foster TD, Thomas WE. Cellular forms and functions of brain microglia. Brain Res. Bull. 1994;34:73–78. Doi: 10.1016/0361-9230(94)90189-918.

18. Хожай Л. И., Отеллин В. А. Реактивные изменения микроглии в неокортексе и гиппокампе у крыс после воздействия острой перинатальной гипоксии // Морфология. – 2013. – Т. 143, № 1. – С. 23–27. [Khozhai LI, Otellin VA. Reactive microglial changes in rat neocortex and hippocampus after exposure to acute perinatal hypoxia. Morphology. 2013;143(1):23–27. (In Russ.)].

19. Moon JB, Lee CH, Park CW, Cho JH, Hwang IK, Yoo KY, Choi JH, Shin HC, Won MH. Neuronal degeneration and microglial activation in the ischemic dentate gyrus of the gerbil. J Vet Med Sci. 2009;71(10):1381–1386. Doi: 10.1292/jvms.001381.

20. Gehrmann J, Bonnekoh P, Miyazawa T, Hossmann KA, Kreutzberg GW. Immunocytochemical study of an early microglial activation in ischemia. J Cereb Blood Flow Metab. 1992;12(2):257–269. Doi: 10.1038/jcbfm.1992.36.

21. Hwang IK, Yoo KY, Kim DW, Choi SY, Kang TC, Kim YS, Won MH. Ionized calcium-binding adapter molecule 1 immunoreactive cells change in the gerbil hippocampal CA1 region after ischemia/reperfusion. Neurochem Res. 2006;31(7):957–965. Doi: 10.1007/s11064-006-9101-3.

22. Lee JC, Ahn JH, Lee DH, Yan BC, Park JH, Kim IH, Cho GS, Kim YM, Lee B, Park CW, Cho JH, Lee HY, Won MH. Neuronal damage and gliosis in the somatosensory cortex induced by various durations of transient cerebral ischemia in gerbils. Brain Res. 2013;1510:78–88. Doi: 10.1016/j.brainres.2013.03.008.

23. Lee CH, Yoo KY, Choi JH, Park OK, Hwang IK, Kim SK, Kang IJ, Kim YM, Won MH. Neuronal damage is much delayed and microgliosis is more severe in the aged hippocampus induced by transient cerebral ischemia compared to the adult hippocampus. J Neurol Sci. 2010;294(1–2):1–6. Doi: 10.1016/j.jns.2010.04.014.

24. Gliem M, Mausberg AK, Lee JI, Simiantonakis I, van Rooijen N, Hartung HP, Jander S. Macrophages prevent hemorrhagic infarct transformation in murine stroke models. Ann Neurol. 2012;71(6):743–752. Doi: 10.1002/ana.23529.

25. Hovens I, Nyakas C, Schoemaker R. A novel method for evaluating microglial activation using ionized calcium-binding adaptor protein-1 staining: cell body to cell size ratio. Neuroimmunol. Neuroinflamm. 2014;1:82–88. Doi: 10.4103/2347-8659.139719.