Deciphering the mechanobiology of microglia in traumatic brain injury with advanced microsystems

Advanced microsystems in traumatic brain injury research: Traumatic brain injury (TBI) results from a mechanical insult to the brain, leading to neuronal and axonal damage and subsequently causing a secondary injury. Within minutes of TBI, a neuroinflammatory response is triggered, driven by intricate molecular and cellular inflammatory processes. The temporal progression of TBI events has been investigated using animal models, human surgical and post-mortem tissue samples, and the analysis of cerebrospinal fluid and plasma from TBI patients. However, the complex interplay between injured brain cells remains poorly understood. TBI induces immediate cell death at the site of impact (primary injury), which is dependent on the severity and depth of the injury. Damaged cells release damage-associated molecular patterns, which signal to resident and infiltrating immune cells via pattern recognition receptors. At the injury site, astrocytes, microglia, and damaged neurons secrete cytokines and chemokines, which activate microglia and astrocytes, and recruit peripheral immune cells that penetrate the compromised blood–brain barrier during the acute post-traumatic period (Nespoli et al., 2024). Consequently, TBI is a mechanobiological disorder characterized not only by the dysregulation of neuronal cells but also by a complex interplay of signaling pathways among brain cells.

Microglia, along with infiltrated peripheral macrophages and T cells, play a pivotal role in delayed secondary injury following TBI. As the principal immune effectors in the brain, microglia are believed to adopt a reactive pro-inflammatory phenotype in neurotrauma, which can persist for weeks or even years. Within minutes of TBI, microglia initiate the brain’s innate immune response, serving as the first line of defense due to their rapid and localized reaction to injury. The process of microglial activation following TBI is intricate and multifaceted, necessitating advanced microsystems to standardize culture conditions for more precise analysis. Traditionally, microglia have been categorized dichotomously; however, recent advancements incorporating modern techniques of epigenetic, transcriptomic, metabolomic, and proteomic data suggest a multidimensional spectrum of coexisting states (Paolicelli et al., 2022). Understanding the mechanical activation of microglial cells is essential for delineating the molecular pathways involved in stress propagation through brain tissues, which is fundamental for developing effective pharmacological treatments with clinical applicability. On one hand, the use of in vivo models closely replicates the pathophysiology of TBI but does not allow for the deciphering of the complexity of cellular interactions operating in brain tissue. On the other hand, current in vitro injury models, however, often fail to replicate the mechanisms of primary and secondary damage accurately and focus on neurons leaving out glial cells. Therefore, there is a critical need to develop advanced in vitro models that recapitulate the complex cellular interactions among the principal brain cell types (microglia, astrocytes, neurons, and oligodendrocytes). These models should standardize culture conditions, transition from 2D to 3D brain organoids, and incorporate the application of mechanical stresses analogous to those observed in TBI events using cutting-edge biomolecular techniques (Figure 1). Such advancements are pivotal for enhancing our understanding of cellular responses to neurotrauma and addressing existing knowledge gaps.

Figure 1.

Multidisciplinary strategies for studying TBI mechanisms.

Multicellular in vitro models represent a valuable tool for replicating the complex cellular interactions observed within brain tissues, essential for studying the physiopathology of TBI. However, the integration of advanced in vitro models employing microsystems, such as microprinted neuronal networks, microfluidic chips, soft hydrogels with controlled rigidity, or 3D organoids, allows for the exploration of the influence of the biochemical and mechanical environment on brain cells under precisely controlled conditions. These cutting-edge in vitro models can be further enhanced by a diverse range of read-out techniques, including mechanobiology, physical and biochemical interactions, proteomics, metabolomics, transcriptomics, and gene expression analysis, as well as electrophysiology and mechanical assays under optogenetic control. The combination of these techniques offers unprecedented insights into the intricate dynamics of brain tissue responses. Created with BioRender.com. TBI: Traumatic brain injury.

Mechanical activation and mechanotransduction in microglial cells: TBI presents a multifaceted challenge due to the diverse nature of mechanical trauma and the intricate composition of brain tissue. To simplify the modeling and understand the ramifications of a mechanical injury, it is imperative to consider mechanical stretch injury as a prominent force involved in the etiology of TBI. Such injuries directly impact and impair neurons, astrocytes, and endothelial cells. However, the specific detrimental effects of stretch injury on microglia remain elusive.

To explore the response of microglial cells to mechanical trauma, we subjected them to single uniaxial deformation resulting in approximately 20% stretch (Procès et al., 2024), closely mirroring the effects of a cerebral concussion. We observed distinct morphological changes in microglial cells following injury, with minimal alterations observed in their cytokine and chemokine profiles. Our findings elucidate that a single instance of stretching induces a unique phenotype, emphasizing the pronounced mechanosensitivity of microglia. Stretched microglial cells exhibit distinct characteristics, including elevated levels of Iba1 protein, a denser actin cytoskeleton, and enhanced migratory persistence. These adaptations in microglial cell migration patterns and cytoskeletal dynamics following mechanical injury align with previous findings implicating the integrin/FAK pathway in microglial reactivity to stretch (Hemphill et al., 2011).

Current advancements in mechanobiology underscore the profound impact of the mechanical environment on gene expression, mediated by the stress and strain exerted on the nucleus. Mechanical trauma has been shown to directly influence chromatin condensation and can lead to double-strand DNA breaks (Procès et al., 2024). Utilizing 2D protein micropatterns, tailored 3D biomaterials, or microfluidic devices to replicate the mechanical properties of the cell microenvironment and apply mechanical forces (Figure 1) offers significant potential in elucidating the impact of the mechanical environment on gene expression in microglial cells.

Insight into synaptic stripping following brain injury: In the aftermath of axonal injury associated with TBI, microglial cells engage in a phenomenon termed “synaptic stripping”, wherein synapses are removed or stripped off from the affected cell body. This process preferentially targets excitotoxic glutamatergic nerve terminals over inhibitory glycinergic and GABAergic nerve terminals, suggesting a reparative effect. Functional major histocompatibility complex class I proteins are implicated in orchestrating this stripping process, specifically preserving inhibitory influences on injured motoneurons. Notably, studies in β2-Microglobulin knockout mice demonstrated a marked reduction in synaptic terminals on the surface of affected neurons. Depletion of microglia post-injury resulted in enhanced functional recovery, while microglia depletion during the insult exacerbated neuronal loss (Rice et al., 2015). These results align with our recent findings (Procès et al., 2024) highlighting the role of microglia in acute response on neuronal networks following TBI.

We combined stretching assays on microglia and neuronal networks cultured in multicompartment microfluidic devices to demonstrate that mechanically-activated microglial cells exhibit synaptic stripping activities when introduced into a healthy neuronal network. Mechanical activation of microglial cells via stretching specifically targeted and removed synaptophysin and PSD-95 from pre- and post-synaptic sites, respectively. This selective protein removal was not observed in microglia activated by lipopolysaccharide. Using advanced microsystems to simulate TBI events, we demonstrated that healthy microglia protect injured neurons, whereas mechanically activated microglia modulates the connectivity of a healthy neuronal network. Notably, the presence of stretch-activated microglia resulted in a significant reduction in synaptophysin levels, a phenomenon not observed with healthy microglia (Wang et al., 2022). Further research integrating in vitro electrophysiological measurements, such as multielectrode arrays, in advanced microsystems of multicellular cultures (e.g., brain-on-a-chip; microfluidic devices) could help decipher whether synaptic stripping is beneficial or detrimental to neuronal connectivity, and thus neurotransmission.

Unveiling mechanisms of microglial activation: The intricate molecular mechanisms governing the mechanical activation of microglia remain partially elucidated. Our findings suggest that microglial cells are sensitive to mechanical forces, presumably mediated by mechanosensitive receptors (Procès et al., 2024). Building upon our previous investigations demonstrating the mechanosensitivity of astrocytes, we propose a shared mechanism involving mechanosensitive channels across glial cells within the central nervous system (Lantoine et al., 2021). Mechanosensing at the cellular level primarily hinges on mechanically-gated channels integrated with cell membrane lipid bilayers. Among these, Piezo1 emerges as a pivotal player, implicated in various chronic inflammatory pathologies, acting as both a sensor and an effector of cellular and tissue mechanical deformations.

Recent studies underscore Piezo1’s role in transducing mechanical forces into intracellular inflammatory signals, potentially exacerbating inflammation and brain injury. Moreover, Piezo1 senses fluctuations in mechanical stress within the local environment and modulates the progression of chronic inflammation. Notably, Toll-like receptor 4 signaling via Piezo1 enhances macrophage-mediated responses during bacterial infection. Upon lipopolysaccharide stimulation, macrophages assemble the Piezo1-Toll-like receptor 4 complex, remodel F-actin organization, and enhance phagocytosis, mitochondrion-phagosomal ROS production, and bacterial clearance through calcium signal-induced activation of the CaMKII-Mst1/2-Rac axis (Geng et al., 2021). Subsequent investigations into Piezo1 channel activation in microglial cells demonstrate its downregulatory effect on the pro-inflammatory function of microglial cells, especially the production of tumor necrosis factor-α and interleukin-6, by initiating intracellular Ca²⁺ signaling to inhibit the nuclear factor kappa B inflammatory signaling pathway (Malko et al., 2023). Piezo1 serves as a mechanosensory channel through which microglia sense amyloid-β fibril stiffness, regulating microglial response to amyloid-β plaques and limiting Alzheimer’s disease-like pathology in Alzheimer’s disease mice, thus highlighting a protective role of microglial mechanobiology in Alzheimer’s disease pathogenesis (Hu et al., 2023). These findings underscore Piezo1 channel as a previously unrecognized mechanism regulating microglial cell function, offering a promising avenue for targeting this molecular mechanism to alleviate neuroinflammation and associated pathologies in the central nervous system.

This insight holds relevance in the context of TBI, where significant remodeling of the actin cytoskeleton and upregulation of Iba1 was observed in microglial following mechanical loading. We postulate that Piezo1 intricately mediates mechanotransduction processes during cellular deformation, potentially exacerbating tissue inflammation. Substantial evidence supports the involvement of Piezo1 in the development and progression of chronic inflammatory diseases.

Future investigations should delve into the involvement of Piezo1 in microglial cell mechanoresponse to short mechanical loading using well-designed microsystems (Figure 1). To fully understand the precise role of Piezo1 in the mechanical activation of microglial cells, further experiments using agonists and antagonists and cyclic stretches for chronic traumatisms are required. We emphasize that modulating these pathways can enhance neuroregeneration following brain injuries.

Concluding remarks and perspectives: TBI presents a multifaceted pathology encompassing both biochemical and biophysical dimensions. Historically, research has predominantly emphasized the biochemical aspects, and the burgeoning field of mechanobiology has significantly enriched TBI investigations. This expansion has introduced new avenues of inquiry, particularly concerning the mechanosensitivity of microglia. Microglia, like other cell types, possess an exceptional ability to perceive and react to mechanical cues, dynamically adjusting their function and morphology in response to environmental changes (Procès et al., 2022). This mechanosensing capability likely underpins microglial discrimination between diverse cellular densities and tissue stiffness levels, contributing to region-specific morphological and functional specialization across different brain areas, both in health and disease.

Despite these insights, the underlying mechanisms governing microglial activation in response to mechanical stimuli remain incompletely understood. Within the brain, microglial cells play a pivotal role in adapting to varying environmental cues by detecting and responding to alterations in extracellular metabolite levels. They also coordinate cellular interactions during spinal cord repair in mice, suggesting a significant role in modulating astrocyte gene expression and phenotype during the formation of the glial scar around the injury site. Following TBI, cells undergo significant metabolic shifts that fuel neuroinflammation. However, questions persist regarding microglial metabolism post-mechanical injury, particularly concerning the impact on mitochondrial machinery and alterations in metabolic pathway engagement. It is imperative to explore these metabolic shifts’ ramifications on neuroinflammation resolution.

Investigating the impact of mechanical cues on microglial behavior by replicating the intricate physico-chemical properties of their microenvironment — such as viscoelastic properties, corrugated patterns (Luciano et al., 2021), or gradients of chemoattractant (Figure 1) — holds tremendous potential for advancing our understanding of these cells’ adaptability to diverse stimuli. Cutting-edge microsystems are crucial for these investigations, providing the precise control needed to replicate the complex mechanical environments microglia encounter in vivo. Furthermore, optogenetics emerges as a promising tool for unraveling the complexities of microglial behavior in TBI events, offering unprecedented control and precision in studying their responses to light-mediated stimulation (Figure 1). This technology opens new avenues for therapeutic interventions and treatments, potentially revolutionizing our approach to mitigating the impacts of TBI.

SG acknowledges funding from FEDER Prostem Research Project, No. 1510614 (Wallonia DG06), the F.R.S.-FNRS Epiforce Project, No. T.0092.21, the F.R.S.-FNRS CellSqueezer Project, No. J.0061.23, the F.R.S.-FNRS Optopattern Project, No. U.NO26.22 and the Interreg MAT(T)ISSE Project, which is financially supported by Interreg France-Wallonie-Vlaanderen (Fonds Européen de Développement Régional, FEDER-ERDF), Programme Wallon d’Investissement Région Wallone pour les instruments d’imagerie (INSTIMAG UMONS #1910169). AP acknowledges postdoctoral support from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (AdG grant agreement no. 834317, Fueling Transport, PI Frédéric Saudou).