Microglia, the brain’s resident immune cells, play a crucial role in maintaining cerebral homeostasis. As the central nervous system’s first line of defense, they rapidly respond to changes in the brain’s microenvironment. Recent advances in single-cell RNA sequencing (scRNA-seq) have unveiled the complex functional diversity of microglia, shedding light on their involvement in various neurological disorders.
Microglia are pivotal in regulating neuroimmune processes and mediating neuroinflammation. They are implicated in a spectrum of neurological disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis, brain tumors, and stroke. Upon inflammatory stimuli, microglia undergo rapid phenotypic and functional transformations, migrating to injury sites and orchestrating neuroinflammation through diverse immune mechanisms.
Microglia in Ischemic Stroke: A Closer Look
Ischemic stroke is a leading cause of permanent neurological impairment and death globally, with limited therapeutic options. Following cerebral ischemia-reperfusion injury, microglia are rapidly activated, initiating an early immune response. Traditionally, microglia have been classified into proinflammatory (M1) and anti-inflammatory (M2) phenotypes. However, recent studies challenge this binary classification, highlighting the complexity of microglial subpopulations and their distinct roles post-injury.
Activated microglia release proinflammatory cytokines that exacerbate neuronal death and disrupt the blood-brain barrier. They also secrete complement components, recognize stressed neurons for removal, and phagocytose damaged cells. Importantly, microglia exhibit immune-metabolic flexibility, expressing genes involved in diverse metabolic pathways and displaying distinct metabolic signatures under different inflammatory conditions.
Revealing Microglial Heterogeneity
The advent of scRNA-seq has expanded our understanding of microglial heterogeneity, uncovering disease-specific subpopulations such as disease-associated microglia (DAM) in AD. In a recent study, researchers used scRNA-seq to characterize markers of major brain cell types, providing a comprehensive view of the molecular mechanisms underlying cerebral ischemia-reperfusion injury.
Through distinct gene expression patterns, ischemic stroke-associated microglia (ISAM) were identified, revealing their transcriptional dynamics and heterogeneity during post-stroke neuroinflammation.
The study identified stroke-associated microglial subpopulations characterized by signature genes, immune functions, metabolic states, and regulatory microenvironments. These findings offer new insights into the pathogenesis of ischemic stroke and highlight potential therapeutic strategies targeting these microglial populations.
Methodological Insights: Animal Models and scRNA-Seq
Researchers utilized healthy male Sprague-Dawley rats to model cerebral ischemia-reperfusion. The middle cerebral artery occlusion/reperfusion (MCAO/R) model was established, and the success of the cerebral ischemia model was assessed using the modified Zea-Longa neurological deficit score.
Single-cell suspensions were prepared for scRNA-seq, capturing individual cells and extracting RNA for gene expression profiling. Quality control, clustering, and dimensionality reduction analyses were conducted using advanced bioinformatics tools, revealing 14 major cell clusters, including microglia, astrocytes, and endothelial cells.
Understanding Microglial Subclusters
Microglial heterogeneity was further explored at the single-cell level, categorizing them into seven distinct subclusters. These subclusters exhibited unique molecular characteristics and functions, with some emerging specifically following cerebral ischemia-reperfusion injury.
Notably, the MG5 subcluster predominated during acute cerebral ischemia-reperfusion injury, characterized by high expression of genes associated with neuroinflammation, tissue repair, and immune response.
Functional characterization of microglial subclusters revealed distinct roles, with some involved in proinflammatory signaling pathways and others in tissue repair and regeneration. These findings underscore the complexity of microglial responses and their potential as therapeutic targets.
Metabolic Reprogramming and Microglial Function
Ischemic stroke induces metabolic reprogramming in microglia, influencing their functional states. Following cerebral ischemia-reperfusion injury, microglia engage in lipid and amino acid metabolism, crucial for membrane remodeling and inflammatory processes.
Different microglial subsets exhibit heterogeneous metabolic profiles, reflecting their adaptive responses to ischemic damage. Under homeostatic conditions, microglia rely on the TCA cycle and oxidative phosphorylation (OXPHOS) for energy production. Post-ischemic activation shifts microglia toward glycolysis, enabling rapid ATP production to sustain inflammatory responses.
These metabolic changes activate downstream inflammatory and oxidative stress signaling pathways, further driving the alteration of microglial functions.
Targeting microglial metabolic pathways, particularly those involving succinate dehydrogenase (SDH) and its downstream effectors, may offer promising therapeutic strategies for mitigating post-stroke neuronal damage and preventing neurodegenerative disorders.
Implications and Future Directions
This study highlights the dynamic transformation of microglial phenotypes following cerebral ischemia-reperfusion injury, emphasizing the emergence of ischemic stroke-associated microglia. The findings reveal the remarkable plasticity of microglia in response to injury and underscore the importance of targeting specific microglial subpopulations to modulate neuroinflammation and promote tissue repair.
While the study provides valuable insights, it also acknowledges limitations, including species differences and the focus on a single time point post-injury. Future research should explore temporal dynamics and intercellular communication mechanisms to fully understand microglial biology and develop precise therapeutic strategies for ischemic stroke.
Overall, the study advances our understanding of microglial diversity and their roles in brain disorders, offering new avenues for therapeutic intervention and highlighting the potential of scRNA-seq in uncovering cellular heterogeneity and function.
