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

Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2022
Thesis identifier
  • T16304
Person Identifier (Local)
  • 202052119
Qualification Level
Qualification Name
Department, School or Faculty
  • Haemorrhagic stroke accounts for approximately 20% of the total stroke cases annually and is characterised by high mortality rates. The activated pro-inflammatory microglia are protagonists in the development of neuroinflammation following stroke. Thrombin has been shown to be one of the blood products driving microglial activation, neurotoxic reactions, contributing to neuroinflammation. Activation of microglia by thrombin induces pro-inflammatory signalling pathways and production of cytokines, such as IL-1β. Thrombin can also influence the activation of the NLRP3 inflammasome, while inflammasome-dependent release of HMGB1 following thrombin stimulation has not been studied. The present study, focused on investigating the effects of thrombin on the induction of microglial pro-inflammatory phenotypes, as well as the expression of NLRP3 and HMGB1, using the novel spontaneously immortalised microglial cell line (SIM-A9). The cells were cultured and exposed to thrombin (10, 20 and 40 U/ml), as well as LPS (1µg/ml, as positive control). Pro-inflammatory populations, NLRP3 and HMGB1 expression, MAPK and NF-κB activations, as well as IL-1β released were them examined using flow cytometry, RT-qPCR, immunoblotting and ELISA respectively. The results obtained showed no thrombin-induced effect on the induction of proinflammatory phenotypes in SIM-A9, as the levels of pro-inflammatory populations remained high in all groups, including the untreated ones. Similarly, the expression of the NLRP3 and HMGB1 remained relatively stable before and after thrombin stimulation. The western blot analysis revealed an activated signal of the MAPK pathway in both treated and untreated cells (n=1), while the ELISA analysis revealed that there were no detectable levels of IL-1β following the stimulation with thrombin. The results suggest that thrombin does not affect neither the phenotypical profile of the SIM-A9, or the expression of the NLRP3 and HMGB1. However, the results from the FACS analysis and the western blots, suggest that in the pro-inflammatory phenotypes could pre-exist in the specific microglial line. This could be an important limitation for the experiments conducted and it should be considered for future studies.
Advisor / supervisor
  • Carswell, Hilary
Resource Type
Date Created
  • 2021