Journal of Molecular Neuroscience

, Volume 67, Issue 2, pp 217–226 | Cite as

Non-invasive Vagus Nerve Stimulation Protects Against Cerebral Ischemia/Reperfusion Injury and Promotes Microglial M2 Polarization Via Interleukin-17A Inhibition

  • Xiao-Ping Zhao
  • Yuan Zhao
  • Xiao-Ya Qin
  • Li-Yuan Wan
  • Xiao-Xuan FanEmail author


Microglia play an essential role during cerebral an ischemia/reperfusion (I/R)-related inflammatory process. Because the M2 phenotype of microglia exhibits anti-inflammation activity, it has become a promising target for anti-inflammatory therapy. Vagus nerve stimulation (VNS) reportedly has neuroprotective effects against cerebral I/R injuries via its anti-inflammatory action. The aim of this study was to investigate the ability of non-invasive VNS (nVNS) to alleviate cerebral I/R in mice by promoting microglial M2 polarization. Neurological scoring and cerebral infarct volume assessments were performed 72 h after a middle cerebral artery occlusion (MCAO)-induced stroke. M2 phenotype microglia were identified by immunohistochemistry staining using Arg-1 and Iba-1 antibodies. The protein expressions of Arg-1, IL-17A, IL-10, Bax, and Bcl-2 were detected by Western blot. Apoptotic cells were detected using TUNEL staining. According to our results, nVNS decreased infarct volume, improved neurological outcomes, reduced apoptotic neurons (TUNEL+NeuN+ cells), and promoted microglial M2 polarization as indicated by elevated Arg-1 protein expression and increased Arg-1+ cells after MCAO. Moreover, nVNS attenuated the increased levels of IL-17A protein expression after MCAO. To test the possible involvement of IL-17A in nVNS-induced neuroprotection and microglial M2 polarization, 1-μg recombinant IL-17A (rIL-17A) was intranasally administered once daily for three consecutive days after reperfusion. We found that the intranasal administration of rIL-17A nullified the nVNS-induced promotion of microglial M2 polarization. Furthermore, rIL-17A administration abolished the neuroprotective effect of nVNS. In conclusion, our study identifies microglial M2 polarization as an important mechanism underlying the nVNS-mediated neuroprotection against cerebral I/R. This effect of nVNS could be attributed to the inhibition of IL-17A expression.


Cerebral ischemia Microglia Vagus nerve stimulation IL-17A MCAO 





Arginase 1


The common carotid artery


The middle cerebral artery




Middle cerebral artery occlusion


Non-invasive VNS


The regional cerebral blood flow


The recombinant interleukin-17A


2,3,5-Triphenyltetrazolium chloride


Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling


Vagus nerve stimulation


α7 Acetylcholine receptor


Conflict of Interest

The authors declare that they have no conflict of interest.

Compliance with Ethical Standards

The experimental protocol was approved by the Ethics Committee and Animal Experimentation and was performed according to the Guidelines for Animal Experimentation of the Fourth Military Medical University. The experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80-23) revised 1996. Efforts were made to minimize animal sufferings and the number of animals used in the study.


  1. Benakis C, Garcia-Bonilla L, Iadecola C, Anrather J (2014) The role of microglia and myeloid immune cells in acute cerebral ischemia. Front Cell Neurosci 8:461. Google Scholar
  2. Benedek A, Moricz K, Juranyi Z, Gigler G, Levay G, Harsing LG Jr, Matyus P, Szenasi G, Albert M (2006) Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats. Brain Res 1116:159–165. CrossRefGoogle Scholar
  3. Bonaz B, Picq C, Sinniger V, Mayol JF, Clarencon D (2013) Vagus nerve stimulation: from epilepsy to the cholinergic anti-inflammatory pathway. Neurogastroenterol Motil 25:208–221. CrossRefGoogle Scholar
  4. del Zoppo GJ (2009) Inflammation and the neurovascular unit in the setting of focal cerebral ischemia. Neuroscience 158:972–982. CrossRefGoogle Scholar
  5. Fernandes A, Miller-Fleming L, Pais TF (2014) Microglia and inflammation: conspiracy, controversy or control? Cell Mol Life Sci 71:3969–3985.
  6. Garcia JH, Wagner S, Liu KF, Hu XJ (1995) Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation. Stroke 26:627–634 discussion 635Google Scholar
  7. Hata R, Mies G, Wiessner C, Fritze K, Hesselbarth D, Brinker G, Hossmann KA (1998) A reproducible model of middle cerebral artery occlusion in mice: hemodynamic, biochemical, and magnetic resonance imaging. J Cereb Blood Flow Metab 18:367–375.
  8. Hu X, Li P, Guo Y, Wang H, Leak RK, Chen S, Gao Y, Chen J (2012) Microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia. Stroke 43:3063–3070. CrossRefGoogle Scholar
  9. Hu X, Leak RK, Shi Y, Suenaga J, Gao Y, Zheng P, Chen J (2015) Microglial and macrophage polarization-new prospects for brain repair. Nat Rev Neurol 11:56–64. CrossRefGoogle Scholar
  10. Jiang Y, Li L, Liu B, Zhang Y, Chen Q, Li C (2014) Vagus nerve stimulation attenuates cerebral ischemia and reperfusion injury via endogenous cholinergic pathway in rat. PLoS One 9:e102342. CrossRefGoogle Scholar
  11. Jiang Y, Li L, Tan X, Liu B, Zhang Y, Li C (2015) miR-210 mediates vagus nerve stimulation-induced antioxidant stress and anti-apoptosis reactions following cerebral ischemia/reperfusion injury in rats. J Neurochem 134:173–181. CrossRefGoogle Scholar
  12. Jin Q, Cheng J, Liu Y, Wu J, Wang X, Wei S, Zhou X, Qin Z, Jia J, Zhen X (2014) Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia/macrophages and increased angiogenesis and neurogenesis following experimental stroke. Brain Behav Immun 40:131–142. CrossRefGoogle Scholar
  13. Kaczmarczyk R, Tejera D, Simon BJ, Heneka MT (2017) Microglia modulation through external vagus nerve stimulation in a murine model of Alzheimer's disease. J Neurochem 146:76–85. CrossRefGoogle Scholar
  14. Khandelwal P, Yavagal DR, Sacco RL (2016) Acute ischemic stroke intervention. J Am Coll Cardiol 67:2631–2644. CrossRefGoogle Scholar
  15. Lin Y, Zhang JC, Yao CY, Wu Y, Abdelgawad AF, Yao SL, Yuan SY (2016) Critical role of astrocytic interleukin-17 a in post-stroke survival and neuronal differentiation of neural precursor cells in adult mice. Cell Death Dis 7:e2273. CrossRefGoogle Scholar
  16. Liu AF, Zhao FB, Wang J, Lu YF, Tian J, Zhao Y, Gao Y, Hu XJ, Liu XY, Tan J, Tian YL, Shi J (2016a) Effects of vagus nerve stimulation on cognitive functioning in rats with cerebral ischemia reperfusion. J Transl Med 14:101. CrossRefGoogle Scholar
  17. Liu X, Liu J, Zhao S, Zhang H, Cai W, Cai M, Ji X, Leak RK, Gao Y, Chen J, Hu X (2016b) Interleukin-4 is essential for microglia/macrophage M2 polarization and long-term recovery after cerebral ischemia. Stroke 47:498–504. CrossRefGoogle Scholar
  18. Ma L, Pan X, Zhou F, Liu K, Wang L (2018) Hyperforin protects against acute cerebral ischemic injury through inhibition of interleukin-17A-mediated microglial activation. Brain Res 1678:254–261. CrossRefGoogle Scholar
  19. Miron VE, Boyd A, Zhao JW, Yuen TJ, Ruckh JM, Shadrach JL, van Wijngaarden P, Wagers AJ, Williams A, Franklin RJM, Ffrench-Constant C (2013) M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nat Neurosci 16:1211–1218. CrossRefGoogle Scholar
  20. Moretti R, Leger PL, Besson VC, Csaba Z, Pansiot J, Di Criscio L, Gentili A, Titomanlio L, Bonnin P, Baud O, Charriaut-Marlangue C (2016) Sildenafil, a cyclic GMP phosphodiesterase inhibitor, induces microglial modulation after focal ischemia in the neonatal mouse brain. J Neuroinflammation 13:95. CrossRefGoogle Scholar
  21. Pan J, Jin JL, Ge HM, Yin KL, Chen X, Han LJ, Chen Y, Qian L, Li XX, Xu Y (2015) Malibatol a regulates microglia M1/M2 polarization in experimental stroke in a PPARgamma-dependent manner. J Neuroinflammation 12:51. CrossRefGoogle Scholar
  22. Serhan CN, Brain SD, Buckley CD, Gilroy DW, Haslett C, O'Neill LA, Perretti M, Rossi AG, Wallace JL (2007) Resolution of inflammation: state of the art, definitions and terms. FASEB J 21:325–332. CrossRefGoogle Scholar
  23. Shin JA, Lim SM, Jeong SI, Kang JL, Park EM (2014) Noggin improves ischemic brain tissue repair and promotes alternative activation of microglia in mice. Brain Behav Immun 40:143–154. CrossRefGoogle Scholar
  24. van der Spuy WJ, Pretorius E (2012) Interrelation between inflammation, thrombosis, and neuroprotection in cerebral ischemia. Rev Neurosci 23:269–278. Google Scholar
  25. Waisman A, Hauptmann J, Regen T (2015) The role of IL-17 in CNS diseases. Acta Neuropathol 129:625–637. CrossRefGoogle Scholar
  26. Wan S, Cheng Y, Jin H, Guo D, Hua Y, Keep RF, Xi G (2016) Microglia activation and polarization after Intracerebral hemorrhage in mice: the role of protease-activated Receptor-1. Transl Stroke Res 7:478–487. CrossRefGoogle Scholar
  27. Xia CY, Zhang S, Gao Y, Wang ZZ, Chen NH (2015) Selective modulation of microglia polarization to M2 phenotype for stroke treatment. Int Immunopharmacol 25:377–382. CrossRefGoogle Scholar
  28. Xiang YX, Wang WX, Xue Z, Zhu L, Wang SB, Sun ZH (2015) Electrical stimulation of the vagus nerve protects against cerebral ischemic injury through an anti-infammatory mechanism. Neural Regen Res 10:576–582. CrossRefGoogle Scholar
  29. Yan A, Zhang T, Yang X, Shao J, Fu N, Shen F, Fu Y, Xia W (2016) Thromboxane A2 receptor antagonist SQ29548 reduces ischemic stroke-induced microglia/macrophages activation and enrichment, and ameliorates brain injury. Sci Rep 6:35885. CrossRefGoogle Scholar
  30. Yang Y, Yang LY, Orban L, Cuylear D, Thompson J, Simon B, Yang Y (2018) Non-invasive vagus nerve stimulation reduces blood-brain barrier disruption in a rat model of ischemic stroke. Brain Stimul 11:689–698.
  31. Zhang J, Wu Y, Weng Z, Zhou T, Feng T, Lin Y (2014) Glycyrrhizin protects brain against ischemia-reperfusion injury in mice through HMGB1-TLR4-IL-17A signaling pathway. Brain Res 1582:176–186. CrossRefGoogle Scholar
  32. Zhang J, Yao C, Chen J, Zhang Y, Yuan S, Lin Y (2016) Hyperforin promotes post-stroke functional recovery through interleukin (IL)-17A-mediated angiogenesis. Brain Res 1646:504–513. CrossRefGoogle Scholar
  33. Zhang Q, Lu Y, Bian H, Guo L, Zhu H (2017) Activation of the alpha7 nicotinic receptor promotes lipopolysaccharide-induced conversion of M1 microglia to M2. Am J Transl Res 9:971–985Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of NeurosurgeryAffiliated Hospital of Shaanxi University of Chinese MedicineXianyangChina
  2. 2.College of foreign languagesShaanxi University of Chinese MedicineXianyangChina
  3. 3.First Clinical Medical College of Shaanxi University of Chinese MedicineXianyangChina

Personalised recommendations