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Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation
Published on: February 16, 2018
Expression of proteinase-activated receptors in mouse microglial cells
Stephanie Balcaitis1, Yiheng Xie, Jonathan R Weinstein
1Department of Neurosurgery, University of Washington, Seattle, 98195, USA.
Abstract:
Microglia are the resident immune cells of the CNS: they are activated rapidly by CNS damage and perform the function of tissue macrophages. The first steps during microglial activation are currently under intense study, and it is widely believed that substances released from damaged brain tissue can trigger this process. We recently reported that the blood coagulation factor thrombin, which enters the CNS during breakdown of the blood-brain barrier, activates microglial cells. The cellular effects of thrombin and trypsin-like proteases are mediated by proteinase-activated receptors (PARs). Based on our prior data we hypothesized that microglial cells express these receptors. Using RT-PCR and flow cytometry, we report that primary mouse microglial cells, as well as the murine microglial cell lines BV-2 and N9, indeed express PARs, albeit at different levels. Demonstrating multiple PARs on microglia may enhance the attractiveness of PARs as therapeutic targets in neuroinflammatory disorders.
Insights
Microglia, the brain's immune cells, are activated by substances like thrombin. This study confirms microglia express proteinase-activated receptors (PARs), suggesting PARs as potential therapeutic targets for neuroinflammation.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia are the primary immune cells in the central nervous system (CNS), acting as tissue macrophages.
- Microglial activation is a rapid response to CNS damage, triggered by substances released from injured brain tissue.
- Thrombin, a blood coagulation factor entering the CNS during blood-brain barrier breakdown, has been shown to activate microglia.
Purpose of the Study:
- To investigate the expression of proteinase-activated receptors (PARs) on microglial cells.
- To determine if PARs mediate the effects of thrombin and other trypsin-like proteases on microglia.
- To explore the therapeutic potential of targeting PARs in neuroinflammatory disorders.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) was used to detect PAR gene expression.
- Flow cytometry was employed to confirm PAR protein expression on microglial cells.
- Primary mouse microglial cells and murine microglial cell lines (BV-2, N9) were utilized.
Main Results:
- Primary mouse microglia and the BV-2 and N9 cell lines express multiple PARs.
- The expression levels of PARs varied among the different microglial cell types studied.
- This confirms that microglia possess the molecular machinery to respond to PAR-activating proteases.
Conclusions:
- Microglial cells express proteinase-activated receptors (PARs).
- The presence of PARs on microglia supports their role in sensing CNS damage and inflammation.
- PARs represent promising therapeutic targets for treating neuroinflammatory conditions.
