Infiltration of invariant natural killer T cells occur and accelerate brain infarction in permanent ischemic stroke in mice

Invariant natural killer T (iNKT) cells are a unique subset of T cells that have been implicated in inflammation, atopy, autoimmunity, infections, and cancer. Although iNKT cells have been extensively studied over the past decade, its role in the pathogenesis of ischemic brain injury is still largely unknown. In our study, we determined whether iNKT cells infiltration occur in a mouse model of permanent cerebral ischemia. C57BL6/J male mice were treated with either alpha-galactosylceramide (α-GalCer) or vehicle control before undergoing permanent middle cerebral artery occlusion (pMCAO). α-GalCer, a glycolipid antigen, specifically activates iNKT cells by a CD1d-restricted mechanism. Using flow cytometry, 10,000 leukocytes (CD45 high cells) from the ischemic hemisphere and peripheral blood respectively were analyzed to determine the number of NK1.1+CD3+ cells at 3, 12, 24 and 48 h post-pMCAO. Cerebral infarct size, brain edema and morphological characteristics were measured at the stipulated time points by 2,3,5-triphenyltetrazolium chloride (TTC) staining, weighing, and H&E staining. The levels of IFN-γ and TNF-α in brain tissue and serum were assessed by immunohistochemistry and ELISA respectively. We found that the number of iNKT cells started increasing from 12 h (PB sample) and 24 h (ischemic hemisphere sample) respectively in the vehicle treated group. iNKT cells infiltration occurred at an earlier time-point compared in the α-GalCer treated group (T = 3H vs T = 12H in PB sample; T = 12H vs T = 24H in ischemic hemisphere sample). Brain water content at 12 h and 24 h was significantly higher in pMCAO + α-GalCer mice compared to pMCAO + vehicle mice which was in turn higher than mice that underwent sham surgery. Aggravated morphological abnormalities in HE-stained neurons and significantly increased neurons with pyknotic nuclei and cavitation in the ischemic region were observed at 24 h in the pMCAO + α-GalCer and pMCAO + vehicle groups. Cerebral infarct volume, neurological deficit Scores and brain edema were significantly increased at 24 h in the pMCAO + α-GalCer group compared to pMCAO + vehicle group. In the pMCAO + vehicle group, the serum concentrations of TNF-α and IFN-γ were increased at 12 h and 24 h post-pMCAO, and remained elevated up to 48 h. In mice treated with pMCAO + α-GalCer, TNF-α and IFN-γ were both increased at 12 h post-pMCAO, and remained elevated up to 48 h. Immunohistochemistry showed that protein expression of TNF-α and IFN-γ in brain tissues was higher in α-GalCer-treated mice. Our results demonstrate that within 48 h of focal permanent cerebral ischemia, iNKT cells infiltrate into the brain and contribute to brain infarction.