S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Medicine (의학과) Theses (Master's Degree_의학과)
The effect of hypothermia on the NLRP3 inflammasome expression in a rat model of focal cerebral ischemia
백서 국소뇌허혈 모델에서 저체온요법이 NLRP3 인플라마좀 발현에 미치는 영향
- 의과대학 의학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 : 의학과, 2015. 2. 윤병우.
- Background: Hypothermia is generally known to reduce brain injury following cerebral ischemia by various mechanisms including anti-inflammatory effects. However, the exact anti-inflammatory mechanism of hypothermia is not well known. Recently, several evidence have suggested that NLRP3 inflammasome is involved in the pathogenesis of sterile inflammatory response by processing caspase-1 and Interleukin (IL)-1β to an active stage following cerebral ischemia. The association between hypothermia and inflammasome in the cerebral ischemia animal model is not reported. So we hypothesized that hypothermia can attenuate the expression of NLRP3 inflammasome in a rat model of focal cerebral ischemia.
Methods and Results: For in vitro study, BV-2 cells (immortalized mouse microglial cell line) were divided by two groups, control and oxygen-glucose deprivation groups. Each groups were re-divided by different temperatures, normothermia (37 °C) and hypothermia (33 °C). Hypothermia was maintained for 3 h in the beginning of study. Western blotting with antibodies to IL-1β, IL-18 and NLRP3 inflammasome complexes including NLRP3 protein, ASC, and caspase-1 and immunohistochemical staining with caspase-1 antibody were done. Western blotting showed increased expression levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 after OGD group and reduced significantly by hypothermia. Caspase-1 expressed in the cytosol of BV-2 cell in all groups and showed relatively decreased expression in OGD with hypothermia group compared with OGD with normothermia group.
For in vivo study, transient middle cerebral artery occlusion (MCAO) model was induced in male Sprague–Dawley (SD) rats using the suture occlusion technique. Sixteen SD rats underwent left MCA occlusion for 2 h followed by reperfusion for 22 h. For the hypothermia, rats were cooled by alcohol spraying and evaporating methods within 15 min at the occlusion of MCA. During 2 h with occlusion period, rectal temperature was maintained with 33 °C (hypothermia group) or 37 °C (normothermia group). Rats were sacrificed at 24 h after ischemia. Nissl staining to evaluate the infarct volume showed reduced infarct volume in hypothermia group compared with the normothermia group (normothermia, 248 ± 52 mm3
hypothermia, 128 ± 27 mm3
n=8 per group). To analyze the effects of hypothermia on the expression of IL-1β, and NLRP3 proteins in ischemic brain tissue, we measured IL-1β, and NLRP3 levels in ischemic or contralateral non-ischemic brain tissues. Compared with contralateral non-ischemic hemisphere, Western blot with IL-1β and NLRP3 antibodies showed significantly increased expression levels in ischemic hemisphere (p<0.001 in normothermia groups
p<0.05 in hypothermia groups). IL-1β and NLRP3 proteins were significantly decreased in ischemic brain tissue with hypothermia than ischemic brain tissue with normothermia (p<0.05 by Western blot with IL-1β antibody
p<0.001 with NLRP3 antibody).
Conclusion: Our study revealed that NLRP3 inflammasome is overexpressed in the BV-2 microglial cells with OGD models, and hypothermia attenuates the NLRP3 inflammasome expression and infarct volume in a rat model of focal cerebral ischemia.