Investigating the effects of thrombin on SIM-A9 microglial cell line: relevance to haemorrhagic stroke

Background:
Microglia promote neuroinflammation following haemorrhagic stroke, when activated in their proinflammatory states. Thrombin activates microglia after haemorrhagic stroke, inducing pro-inflammatory signalling pathways and cytokines production (e.g. IL-1b). Thrombin activates the NLRP3 inflammasome, while inflammasome-dependent release of HMGB1 following thrombin stimulation has not been studied.

Aims:
To investigate the effects of thrombin on the induction of pro-inflammatory phenotypes in microglia and the expression of NLRP3 and HMGB1 in microglia.

Method:
Spontaneously immortalised microglia (SIM-A9) were exposed to thrombin (10, 20 and 40 U/ml), LPS (1mg/ ml, positive control), or vehicle controls for 24 hours. Pro-inflammatory populations, NLRP3 and HMGB1 expression, MAPK and NF-jB activation, as well as IL-1b released were then examined using flow cytometry, RTqPCR, immunoblotting and ELISA, respectively.

Results/Conclusions:
Increased pro-inflammatory populations were recorded in all groups, including the untreated ones (Figure 1), while expression of NLRP3 and HMGB1 was statistically similar before and after thrombin stimulation (n ¼ 3, p>0.05). Preliminary results detected activated MAPK signalling in both thrombintreated and untreated groups (n ¼ 1), and no detectable IL-1b levels. These preliminary results showed that thrombin does not induce pro-inflammatory microglial polarisation or NLRP3 and HMGB1 expression versus vehicle controls. We recommend that pre-existing pro-inflammatory states of SIM-A9 cells are investigated for future studies using SIM-A9s for assessing microglia as protagonists for neuroinflammation.