Sevoflurane Post-Conditioning Ameliorates Neuronal Deficits and Axon Demyelination After Neonatal Hypoxic Ischemic Brain Injury: Role of Microglia/Macrophage


Microglia/macrophages have been identified to be highly polarized after ischemia. Interestingly, the polarization of these microglia/macrophages varies immensely under differing disease conditions. Post-conditioning using sevoflurane, a volatile anesthetic, could provide long-term neuroprotection to neonatal rats after hypoxic-ischemic brain injury (HIBI). Thus, the current study aimed at investigating the effects of sevoflurane post-conditioning (SPC) on microglia/macrophage polarization after HIBI induction in neonatal rats. Additionally, we aimed at identifying the underpinning mechanisms specifically related to autophagy and lysosomal protease enzyme, cathepsin B. To develop a HIBI model, 7-day-old Sprague–Dawley rats underwent left common carotid artery ligation followed by 2 h of hypoxia. The role of microglia/macrophages in the neuroprotection conferred by SPC was examined by left-side intra-cerebroventricular injection with adenovirus vector carrying catB-GFP or rapamycin. The number of interleukin (IL)-1β+ cells, cathepsin B+ cells, light chain 3B positive (LC3B+) cells among ionized calcium binding adaptor molecule 1(Iba1+)cells to investigate microglia polarization, neuronal apoptosis to assess neuronal death in the acute phase were tested at 24 h after HIBI. Behavioral tests including suspension test, Morris water maze tests were performed to investigate the long-term effects of SPC, at 21 to 34 days post HIBI. Nissl staining and myelin basic protein (MBP) immunostaining to assess the long-term neuronal and myelin damage were performed at 34 days after HIBI. Based on the obtained results post HIBI, we observed the cells that were positive for IL-1β, cathepsin B, and LC3B among Iba1 positive cell population in the hippocampus were significantly decreased after SPC treatment. SPC significantly attenuated the HIBI-induced increase in neuronal apoptosis, improved long-term cognitive function, and attenuated HI-induced decrease of Nissl-positive cells and MBP expression. However, these trends were reversed by injection of adenovirus vector carrying catB-GFP and rapamycin. SPC attenuated microglia polarization towards neurotoxic phenotypes, alleviates neuronal death and axon demyelination after HIBI in neonatal rats by regulating microglia autophagy and cathepsin B expression, and therefore provided long-term cognitive, learning and memory protection.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8



Hypoxic-ischemic brain injury


Hypoxia ischemia


Sevoflurane post-conditioning


Cornu ammonis


Morris water maze


Dentate gyrus


Central nervous system


Induced nitrogen monoxide synthase




Ionized calcium binding adaptor molecule


Myelin basic protein


  1. Anderson MF, Blomstrand F, Blomstrand C, Eriksson PS, Nilsson M (2003) Astrocytes and stroke: networking for survival? Neurochem Res 28(2):293–305

    CAS  Article  Google Scholar 

  2. Bhalala US, Koehler RC, Kannan S (2015) Neuroinflammation and neuroimmune dysregulation after acute hypoxic-ischemic injury of developing brain. Front Pediatr 14(2):144

    Google Scholar 

  3. Canu N, Tufi R, Serafino AL, Amadoro G, Ciotti MT, Calissano P (2005) Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells. J Neurochem 92(5):1228–1242

    CAS  Article  Google Scholar 

  4. Dang DD, Saiyin H, Yu Q, Liang WM (2018) Effects of sevoflurane preconditioning on microglia/macrophage dynamics and phagocytosis profile against cerebral ischemia in rats. CNS Neurosci Ther 24(6):564–571

    CAS  Article  Google Scholar 

  5. Donat CK, Scott G, Gentleman SM, Sastre M (2017) Microglial activation in traumatic brain injury. Front Aging Neurosci 9:208

    Article  Google Scholar 

  6. Gao Y, Wang Z, He W, Ma W, Ni X (2019) Mild hypothermia protects neurons against oxygen glucose deprivation via poly (ADP-ribose) signaling. J Matern Fetal Neonatal Med 32(10):1633–1639

    CAS  Article  Google Scholar 

  7. Gluckman PD, Wyatt JS, Azzopardi D, Ballard R, Edwards AD, Ferriero DM, Polin RA, Robertson CM, Thoresen M, Whitelaw A, Gunn AJ (2005) Selective head cooling with mild systemic hypothermia after neonatal encephalopathy: multicentre randomised trial. Lancet 365(9460):663–670

    Article  Google Scholar 

  8. Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T, Fitzgerald K, Latz E, Moore K, Golenbock D (2008) The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat Immunol 9(8):857–865

    CAS  Article  Google Scholar 

  9. Huang J, Zhang L, Qu Y, Zhou Y, Zhu J, Li Y, Zhu T, Zhao F, Tang J, Dezhi Mu (2018) Histone acetylation of oligodendrocytes protects against white matter injury induced by inflammation and hypoxia-ischemia through activation of BDNF-TrkB signaling pathway in neonatal rats. Brain Res 1688:33–46

    CAS  Article  Google Scholar 

  10. Koo E, Sheldon RA, Lee BS, Vexler ZS, Ferriero DM (2017) Effects of therapeutic hypothermia on white matter injury from murine neonatal hypoxia-ischemia. Pediatr Res 82(3):518–526

    CAS  Article  Google Scholar 

  11. Kreutzberg GW (1996) Microglia: a sensor for pathological events in the CNS. Trends Neurosci 19(8):312–318

    CAS  Article  Google Scholar 

  12. Menzies FM, Henriquez FL, Alexander J, Roberts CW (2011) Selective inhibition and augmentation of alternative macrophage activation by progesterone. Immunology 134(3):281–291

    CAS  Article  Google Scholar 

  13. Mizushima N (2007) Autophagy: process and function. Genes Dev 21(22):2861–2873

    CAS  Article  Google Scholar 

  14. Neher JJ, Neniskyte U, Zhao JW, Bal-Price A, Tolkovsky AM, Brown GC (2011) Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death. J Immunol 186(8):4973–4983

    CAS  Article  Google Scholar 

  15. Ni J, Wu Z, Peterts C, Yamamoto K, Qing H, Nakanishi H (2015) The critical role of proteolytic relay through cathepsins B and E in the phenotypic change of microglia/macrophage. J Neurosci 35(36):12488–12501

    CAS  Article  Google Scholar 

  16. Papazian O (2018) Neonatal hypoxic-ischemic encephalopathy. Medicina (B Aires) 78(Suppl 2):36–41

    Google Scholar 

  17. Tsuchiya K, Kohda Y, Yoshida M, Zhao L, Ueno T, Yamashita J (1999) Postictal blockade of ischemic hippocampal neuronal death in primates using selective cathepsin inhibitors. Exp Neurol 155(2):187–194

    CAS  Article  Google Scholar 

  18. Vannucci RC, Connor JR, Mauger DT, Palmer C, Smith MB, Towfighi J, Vannucci SJ (1999) Rat model of perinatal hypoxic-ischemic brain damage. J Neurosci Res 55(2):158–163

    CAS  Article  Google Scholar 

  19. Wang G, Shi Y, Jiang X, Leak RK, Hu X, Wu Y, Pu H, Li W, Tang B, Wang Y, Gao Y, Zheng P, Bennett M, Chen J (2015) HDAC inhibition prevents white matter injury by modulating microglia/macrophage polarization through the GSK3β/PTEN/Akt axis. Proc Natl Acad Sci USA 112(9):2853–2858

    CAS  Article  Google Scholar 

  20. Wang S, Xue H, Xu Y, Niu J, Zhao P (2019) Sevoflurane postconditioning inhibits autophagy through activation of the extracellular signal-regulated kinase cascade, alleviating hypoxic-ischemic brain injury in neonatal rats. Neurochem Res 44(2):347–356

    CAS  Article  Google Scholar 

  21. Wendt W, Schulten R, Stichel CC, Lübbert H (2009) Intra- versus extracellular effects of microglia-derived cysteine proteases in a conditioned medium transfer model. J Neurochem 110(6):1931–1941

    CAS  Article  Google Scholar 

  22. Wu Z, Sun L, Hashioka S, Yu S, Schwab C, Okada R, Hayashi Y, McGeer P, Nakanishi H (2013) Differential pathways for interleukin-1β production activated by chromogranin A and amyloid β in microglia. Neurobiol Aging 34(12):2715–2725

    CAS  Article  Google Scholar 

  23. Xue H, Xu Y, Wang S, Wu ZY, Li XY, Zhang YH, Niu JY, Gao QS, Zhao P (2019) Sevoflurane post-conditioning alleviates neonatal rat hypoxic-ischemic cerebral injury via Ezh2-regulated autophagy. Drug Des Devel Ther 13:1691–1706

    CAS  Article  Google Scholar 

  24. Xu Y, Tian Y, Tian Y, Li X, Zhao P (2016) Autophagy activation involved in hypoxic-ischemic brain injury induces cognitive and memory impairment in neonatal rats. J Neurochem 139(5):795–805

    CAS  Article  Google Scholar 

  25. Yu X, Zhang F, Shi J (2019) Effect of sevoflurane treatment on microglia activation, NF-kB and MAPK activities. Immunobiology 224(5):638–644

    CAS  Article  Google Scholar 

  26. Zhao P, Ji G, Xue H, Yu W, Zhao X, Ding M, Yang Y, Zuo Z (2014) Isoflurane postconditioning improved long-term neurological outcome possibly via inhibiting the mitochondrial permeability transition pore in neonatal rats after brain hypoxia-ischemia. Neuroscience 280:193–203

    CAS  Article  Google Scholar 

  27. Zhou XY, Luo Y, Zhu YM, Liu ZH, Kent TA, Rong JG, Li W, Qiao SG, Li M, Ni Y, Ishidoh K, Zhang HL (2017) Inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membrane in ischemic astrocytes. Cell Death Dis 8(2):e2618

    CAS  Article  Google Scholar 

  28. Zhu YM, Gao X, Ni Y, Li W, Kent TA, Qiao SG, Wang C, Xu XX, Zhang HL (2017) Sevoflurane postconditioning attenuates reactive astrogliosis and glial scar formation after ischemia-reperfusion brain injury. Neuroscience 356:125–141

    CAS  Article  Google Scholar 

  29. Ziemka-Nalecz M, Jaworska J, Zalewska T (2017) Insights into the neuroinflammatory responses after neonatal hypoxia-ischemia. J Neuropathol Exp Neurol 76(8):644–654

    CAS  Article  Google Scholar 

Download references


Thanks to Prof. Lili Wang’s suggestion and help in completing the experiments.


This research is supported by grant from the National Natural Science Foundation of China (Nos. 81671311, 81870838), Liaoning Province Distinguished Professor Support Program (No. XLYC1802096) and Outstanding Scientific Fund of Shengjing Hospital (No. 201708), and Colleges and Universities Basic Research Project Fund of Liaoning Province (No. LQNK201709).

Author information




H.X. did the experiment and wrote the paper; Q.G. and J.N. contributed in statistical analysis; Y.Z., Z.W. and C.L. did the experiment; Ping Zhao designed the experiment and offered the fund.

Corresponding author

Correspondence to Ping Zhao.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

Animal experiments were all performed according to the recommendations of the National Institutes of Health Guidelines for the Care and Use of Laboratory animals. Protocols described below were all approved by the Animal Review Board of Shengjing Hospital, China Medical University. This article does not contain any studies with human participants performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Xue, H., Zhang, YH., Gao, QS. et al. Sevoflurane Post-Conditioning Ameliorates Neuronal Deficits and Axon Demyelination After Neonatal Hypoxic Ischemic Brain Injury: Role of Microglia/Macrophage. Cell Mol Neurobiol (2020).

Download citation


  • Sevoflurane post-conditioning
  • Hypoxic-ischemic brain injury
  • Neonatal rat
  • Microglia/macrophage
  • Cathepsin B