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Updated: Aug 20, 2026
Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
[Role of microglial pyroptosis in hypoxic-ischemic brain damage]
Lan-Lan Tan1, Mei Li, Chen-Xi Feng
1Department of Neonatology, Children's Hospital of Soochow University, Suzhou, Jiangsu 215025, China. xing_feng66@suda.edu.cn.
Objective:
To investigate the role of microglial pyroptosis in hypoxic-ischemic brain damage.
Methods:
An oxygen-glucose deprivation/reoxygenation (OGD/R) model of rat microglial cells were cultured in vitro. Western blot was used to measure the expression of the pyroptosis-related proteins caspase-1, interleukin-1β (IL-1β), and N-terminal gasdermin D (GSDMD-N) at 0, 1, 3, 6, 12, and 24 hours after OGD/R. After the microglial cells were transfected with lentivirus-mediated silenced gasdermin D (GSDMD), immunofluorescence assay and Western blot were used to measure the transfection rate of GSDMD. Microglial cell lines were divided into three groups: normal control, negative control, and LV-sh_GSDMD (lentivirus-mediated GSDMD silencing). CCK-8 assay and LDH kit were used to observe the effect of GSDMD silencing on the viability and toxicity of microglial cells at 24 hours after OGD/R. Western blot was used to observe the effect of GSDMD silencing on the levels of caspase-1, GSDMD-N, and IL-1β in the microglial cells at 24 hours after OGD/R.
Results:
The expression levels of the pyroptosis-related proteins caspase-1, GSDMD-N, and IL-1β in microglial cells were upregulated since 0 hour after OGD/R and reached the peak levels at 24 hours. A microglial cell model of lentivirus-mediated GSDMD silencing was successfully constructed. At 24 hours after OGD/R, compared with the normal control group, the GSDMD silencing group had a significant increase in the cell viability and a significant reduction in the cytotoxicity (P<0.05), as well as significant reductions in the protein expression levels of caspase-1, GSDMD-N, and IL-1β in microglial cells (P<0.05).
Conclusions:
Lentivirus silencing of the key substrate protein for pyroptosis GSDMD can alleviate hypoxic-ischemic brain damage, suggesting that microglial pyroptosis aggravates hypoxic-ischemic brain damage.
Insights
Microglial pyroptosis, a cell death process, exacerbates hypoxic-ischemic brain damage. Silencing gasdermin D (GSDMD) in microglia reduces cell death and alleviates brain injury.
Area of Science:
- Neuroscience
- Cell Biology
- Pathology
Background:
- Hypoxic-ischemic (HI) brain damage is a significant cause of neurological dysfunction.
- Microglia play a critical role in neuroinflammation and brain injury.
- Pyroptosis, a programmed inflammatory cell death, has been implicated in various neurological disorders.
Purpose of the Study:
- To investigate the role of microglial pyroptosis in hypoxic-ischemic brain damage.
- To determine the effect of silencing gasdermin D (GSDMD), a key pyroptosis protein, on microglial cells under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions.
Main Methods:
- Established an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model using rat microglial cells.
- Utilized Western blot to measure pyroptosis-related proteins (caspase-1, IL-1β, GSDMD-N).
- Employed lentivirus-mediated GSDMD silencing and assessed its impact on cell viability, cytotoxicity, and protein expression.
Main Results:
- OGD/R upregulated caspase-1, GSDMD-N, and IL-1β expression in microglial cells, peaking at 24 hours.
- Successful construction of a lentivirus-mediated GSDMD-silenced microglial cell model.
- GSDMD silencing significantly increased cell viability and reduced cytotoxicity, along with decreased levels of pyroptosis markers post-OGD/R.
Conclusions:
- Microglial pyroptosis, mediated by GSDMD, contributes to hypoxic-ischemic brain damage.
- Silencing GSDMD in microglia alleviates cell death and reduces the severity of hypoxic-ischemic brain injury.
- Targeting microglial pyroptosis presents a potential therapeutic strategy for hypoxic-ischemic brain damage.
