Research Reveals Gender-Specific Epigenetic Regulatory Mechanisms in Neuroinflammation, Injury, and Recovery

Background

Ischemic stroke is one of the leading causes of death and disability worldwide. Due to limited understanding of its linked disease mechanisms, current treatments for alleviating acute ischemic brain injury are very limited. Recent studies have shown that epigenetic mechanisms, such as histone lysine acetylation/deacetylation, are involved in ischemia-induced neuronal injury and death. However, another common epigenetic mechanism - lysine methylation/demethylation - has not been thoroughly investigated in cerebral ischemia. In particular, the impact of gender on post-stroke outcomes necessitates the consideration of gender factors in research. This study aims to elucidate these molecular mechanisms through the recently developed internal carotid artery occlusion (ICAO) mouse model.

Source

This paper was written by Mydhili Radhakrishnan, Vincy Vijay, B. Supraja Acharya, Papia Basuthakur, Shashikant Patel, Kalyani Soren, Arvind Kumar, Sumana Chakravarty, and others from the Indian Institute of Chemical Technology (IICT) and the Centre for Cellular and Molecular Biology (CCMB). The paper was accepted in December 2023 and published in the journal Neuromolecular Medicine in 2024.

Research Process

Experimental Design

The study used 7-month-old male and female CD1 mice, divided into experimental and sham surgery groups. The experimental group underwent internal carotid artery occlusion (ICAO) to induce ischemia, while the sham surgery group did not. Specific experimental steps included:

1. Internal Carotid Artery Occlusion Surgery: After anesthesia, the left internal carotid artery was occluded for 90 minutes, followed by reperfusion.

2. Blood Flow Perfusion Imaging: Laser Doppler imaging technique was used to measure blood flow perfusion rates before occlusion, after occlusion, and after reperfusion.

3. Behavioral Tests: Including Neurological Deficit Score (NDS), grip strength measurement, rotarod test, and open field test, to assess behavioral function recovery at various time points.

4. Tissue Collection and Fixation: Mice were humanely euthanized at different time points, and brain tissues were collected for molecular biology studies such as quantitative PCR (qPCR), Western Blot, and Chromatin Immunoprecipitation (ChIP-qPCR).

5. Mitochondrial Enzyme Activity Assay: 2,3,5-Triphenyltetrazolium chloride (TTC) staining was used to evaluate mitochondrial enzyme activity in the ischemic area.

6. Histological Staining: Hematoxylin and Eosin (H&E) staining was performed to observe cellular damage.

Data Analysis

Data were analyzed for statistical significance using two-way ANOVA, with P<0.05 considered statistically significant.

Key Findings

1. Blood Flow Perfusion: Occlusion led to a significant decrease in blood flow perfusion rates in the left common carotid artery (ROI 1) and internal carotid artery (ROI 2), but recovered after reperfusion.

2. Behavioral Tests: Female mice showed faster neurological function recovery at various time points compared to male mice, particularly with significantly reduced neurological deficit scores after 1 day. Grip strength measurements showed recovery in female mice after 3 days and in male mice after 5 days. Rotarod test results showed complete recovery in female mice after 7 days, while male mice did not fully recover.

3. Mitochondrial Enzyme Activity: Ischemia-induced damage was significant in the striatum of both sexes, but significant mitochondrial dysfunction was observed only in the hippocampal region of female mice.

4. H&E Staining: Large areas of vacuolization were observed in striatal tissue, indicating significant tissue damage.

5. Inflammatory Factor Expression: After 1 day, HIF-1α, TNF-α, IL-1β, and NLRP3 were significantly upregulated in male mice, while in female mice, these factors were significantly upregulated after 6 hours and gradually returned to baseline levels thereafter.

6. Epigenetic Regulatory Mechanisms: After 1 day, H3K9me2 sites in the striatum of male mice were significantly reduced, while in female mice, H3K9me2 was significantly reduced after 6 hours, and KDM4B/JMJD2B was upregulated.

Conclusion

This study, using the ICAO model, reveals for the first time the important role of H3K9me2 in gender-specific neuroinflammatory responses. The study shows that female mice recover faster than male mice, possibly due to early inflammatory responses. Gender-specific epigenetic modifications play a key role in ischemic brain injury and recovery processes, with H3K9me2 potentially becoming a new therapeutic target, and gender being an important variable.

Research Highlights

1. In-depth study of the internal carotid artery occlusion (ICAO) model, revealing gender-specific mechanisms of neuroinflammation and recovery.
2. Proposal of H3K9me2 as a potential new therapeutic target for stroke-induced neuronal injury and recovery.
3. This study provides a theoretical basis for incorporating gender factors in future stroke treatment research.

Impact and Significance

This study reveals the importance of gender differences in ischemic stroke, providing new perspectives and therapeutic targets for future disease treatment research, with significant scientific and application value. By gaining a deeper understanding of the molecular and cellular mechanisms in different genders after stroke, gender-specific treatment methods can be further developed to improve the prognosis of stroke patients.