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Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury
Published on: September 4, 2013
High-dose dexamethasone regulates microglial polarization via the GR/JAK1/STAT3 signaling pathway after traumatic
Mengshi Yang1, Miao Bai2, Yuan Zhuang1
1Beijing Tiantan Hospital, Capital Medical University, Beijing, China.
Abstract:
JOURNAL/nrgr/04.03/01300535-202509000-00023/figure1/v/2024-11-05T132919Z/r/image-tiff Although microglial polarization and neuroinflammation are crucial cellular responses after traumatic brain injury, the fundamental regulatory and functional mechanisms remain insufficiently understood. As potent anti-inflammatory agents, the use of glucocorticoids in traumatic brain injury is still controversial, and their regulatory effects on microglial polarization are not yet known. In the present study, we sought to determine whether exacerbation of traumatic brain injury caused by high-dose dexamethasone is related to its regulatory effects on microglial polarization and its mechanisms of action. In vitro cultured BV2 cells and primary microglia and a controlled cortical impact mouse model were used to investigate the effects of dexamethasone on microglial polarization. Lipopolysaccharide, dexamethasone, RU486 (a glucocorticoid receptor antagonist), and ruxolitinib (a Janus kinase 1 antagonist) were administered. RNA-sequencing data obtained from a C57BL/6 mouse model of traumatic brain injury were used to identify potential targets of dexamethasone. The Morris water maze, quantitative reverse transcription-polymerase chain reaction, western blotting, immunofluorescence and confocal microscopy analysis, and TUNEL, Nissl, and Golgi staining were performed to investigate our hypothesis. High-throughput sequencing results showed that arginase 1, a marker of M2 microglia, was significantly downregulated in the dexamethasone group compared with the traumatic brain injury group at 3 days post-traumatic brain injury. Thus dexamethasone inhibited M1 and M2 microglia, with a more pronounced inhibitory effect on M2 microglia in vitro and in vivo . Glucocorticoid receptor plays an indispensable role in microglial polarization after dexamethasone treatment following traumatic brain injury. Additionally, glucocorticoid receptor activation increased the number of apoptotic cells and neuronal death, and also decreased the density of dendritic spines. A possible downstream receptor signaling mechanism is the GR/JAK1/STAT3 pathway. Overactivation of glucocorticoid receptor by high-dose dexamethasone reduced the expression of M2 microglia, which plays an anti-inflammatory role. In contrast, inhibiting the activation of glucocorticoid receptor reduced the number of apoptotic glia and neurons and decreased the loss of dendritic spines after traumatic brain injury. Dexamethasone may exert its neurotoxic effects by inhibiting M2 microglia through the GR/JAK1/STAT3 signaling pathway.
Insights
High-dose dexamethasone exacerbates traumatic brain injury by inhibiting anti-inflammatory M2 microglia via the glucocorticoid receptor/Janus kinase 1/signal transducer and activator of transcription 3 pathway. Inhibiting this pathway may protect against neuronal damage.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Microglial polarization and neuroinflammation are key responses to traumatic brain injury (TBI), but regulatory mechanisms are unclear.
- The role of glucocorticoids, like dexamethasone, in TBI is controversial, with their effects on microglial polarization unknown.
Purpose of the Study:
- To investigate if high-dose dexamethasone exacerbates TBI by altering microglial polarization.
- To elucidate the underlying mechanisms of dexamethasone's effects on microglial polarization and TBI outcomes.
Main Methods:
- In vitro (BV2 cells, primary microglia) and in vivo (controlled cortical impact mouse model) studies.
- Administration of lipopolysaccharide, dexamethasone, glucocorticoid receptor antagonist (RU486), and Janus kinase 1 antagonist (ruxolitinib).
- RNA-sequencing, Morris water maze, qPCR, Western blotting, immunofluorescence, and histological analyses (TUNEL, Nissl, Golgi staining).
Main Results:
- Dexamethasone significantly downregulated arginase 1 (M2 microglia marker) in TBI mice.
- Dexamethasone inhibited both M1 and M2 microglia, with a stronger inhibitory effect on M2 microglia.
- Glucocorticoid receptor activation increased apoptosis and neuronal death, and decreased dendritic spine density.
- The GR/JAK1/STAT3 pathway was identified as a potential downstream signaling mechanism.
Conclusions:
- High-dose dexamethasone impairs TBI recovery by inhibiting anti-inflammatory M2 microglia through the GR/JAK1/STAT3 pathway.
- Blocking glucocorticoid receptor activation reduced glial and neuronal apoptosis and preserved dendritic spine density.
- Dexamethasone's neurotoxic effects in TBI may stem from M2 microglia inhibition via this pathway.
