Microglia and Macrophages in Central Nervous System Homeostasis and Disease Progression: Guardians and Executioners

Abstract

Microglia and macrophages are critical immune cells within the central nervous system (CNS), with distinct roles in development, homeostasis, and disease. Once viewed as passive bystanders, these cells are now recognized for their dynamic phenotypic plasticity, which enables them to respond to a wide range of physiological and pathological stimuli. During homeostasis, microglia and CNS-resident macrophages actively participate in synaptic pruning, neuronal support, myelin regulation, and immune surveillance, contributing to CNS integrity. However, under pathological conditions, these cells can adopt neurotoxic phenotypes, exacerbating neuroinflammation, oxidative stress, and neuronal damage in diseases such as Alzheimer’s, Parkinson’s, multiple sclerosis, and glioblastoma. This review synthesizes emerging insights into the molecular, epigenetic, and metabolic mechanisms that govern the behavior of microglia and macrophages, highlighting their developmental origins, niche-specific programming, and interactions with other CNS cells. We also explore novel therapeutic strategies aimed at modulating these immune cells to restore CNS homeostasis, including nanotechnology-based approaches for selective targeting, reprogramming, and imaging. Understanding the complex roles of microglia and macrophages in both health and disease is crucial for the development of precise therapies targeting neuroimmune interfaces. Continued advances in single-cell technologies and nanomedicine are paving the way for future therapeutic interventions in neurological disorders.

1. Introduction

The central nervous system (CNS) was once thought to be immunologically privileged and isolated from classical immune surveillance; the central nervous system (CNS) is now understood as a dynamic immunological environment governed mainly by its intrinsic immune cells—microglia and CNS-resident macrophages [1,2]. These cells are increasingly recognized as central regulators of neural development, synaptic plasticity, injury response, and neurodegeneration. Their ability to sense, interpret, and react to environmental cues makes them pivotal in maintaining CNS homeostasis and places them at the forefront of numerous neuropathological processes [3,4]. Microglia, originating from yolk sac progenitors during early embryogenesis, are the primary immune effector cells within the CNS parenchyma [5,6]. In contrast, CNS-associated macrophages (CAMs), derived from peripheral hematopoietic precursors, reside in distinct anatomical niches such as the meninges, perivascular spaces, and choroid plexus [7,8]. Both cell types display a high degree of plasticity, adapting their phenotype and function in response to physiological or pathological stimuli. Advances in single-cell RNA sequencing, spatial transcriptomics, and epigenomic profiling have revealed a previously unappreciated level of cellular heterogeneity and context-specific functionality across the CNS immune landscape [6,9,10].

Under physiological conditions, microglia contribute to neurogenesis, myelination, synaptic pruning, and the clearance of apoptotic cells and debris. Their role as ‘housekeepers’ of the CNS is crucial for neuronal health and circuit refinement [8,11]. However, these cells may undergo maladaptive activation during neurodegenerative diseases, traumatic injuries, and infections. They can shift toward pro-inflammatory and neurotoxic phenotypes, contributing to chronic neuroinflammation, oxidative stress, and progressive neuronal damage. This dichotomous behavior—oscillating between neuroprotection and neurotoxicity—has led to the characterization of microglia and macrophages as both guardians and executioners of the CNS [12,13,14]. Given their central role in health and disease, microglia and macrophages are increasingly being targeted as potential therapeutic agents in neurological conditions, including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and glioblastoma. Yet, clinical translation remains challenging due to their phenotypic plasticity, context dependency, and difficulty selectively targeting disease-promoting subpopulations without disrupting beneficial homeostatic functions [15,16].

A total of 169 peer-reviewed articles, published across leading scientific journals, were critically evaluated and thoroughly analyzed to ensure comprehensive coverage of the current advancements, emerging trends, and ongoing challenges in the field. The selected literature spans foundational theoretical studies, experimental breakthroughs, and translational applications, providing a multidisciplinary perspective that supports the in-depth synthesis and interpretation presented throughout this review. This review consolidates emerging advances in the molecular, epigenetic, and metabolic pathways that regulate the function and plasticity of microglia and macrophages within the central nervous system (CNS). Emphasis is placed on their developmental ontogeny, region-specific specialization, and dynamic crosstalk with neuronal and non-neuronal cell types. We further evaluate innovative therapeutic strategies aimed at modulating these innate immune populations to re-establish CNS homeostasis. In particular, we highlight nanotechnology-enabled platforms for selective cellular targeting, phenotypic reprogramming, and real-time in vivo imaging. A comprehensive understanding of microglial and macrophage behavior in both physiological and pathological contexts is essential for the rational design of next-generation neuroimmune therapeutics. Advances in single-cell multi-omics, high-resolution spatial transcriptomics, and precision nanomedicine are anticipated to revolutionize therapeutic paradigms for a broad spectrum of neurological disorders.

2. Origin and Developmental Ontogeny of CNS-Resident Immune Cells

The central nervous system (CNS) harbors a heterogeneous population of resident immune cells, among which microglia and border-associated macrophages (BAMs) represent distinct lineages with unique developmental trajectories and functional specializations. Microglia, the parenchymal macrophages of the CNS, originate from primitive myeloid progenitors in the yolk sac during early embryogenesis [6,17,18]. This early colonization precedes the establishment of definitive hematopoiesis and is independent of bone marrow-derived monocytes. Lineage-tracing studies have demonstrated that these yolk sac-derived progenitors, characterized by the expression of the transcription factors PU.1 and IRF8, migrate into the neuroepithelium via the developing vasculature and proliferate to populate the entire CNS. Once established, microglia undergo a tightly regulated maturation process mediated by signals from the local CNS microenvironment. Among these, the transforming growth factor-β (TGF-β) is pivotal in maintaining microglial identity and function [18,19]. TGF-β, in concert with colony-stimulating factor 1 (CSF1), ensures the expression of microglia-specific genes while suppressing the expression of genes associated with peripheral macrophage lineages. This molecular programming is critical for sustaining homeostatic functions such as synaptic pruning and neuronal support and mounting appropriate responses during CNS injury and disease [20,21].

In contrast, BAMs—including perivascular, meningeal, and choroid plexus macrophages—display a dual ontogeny. While a significant fraction arises from the same yolk sac progenitors as microglia, additional populations are replenished by bone marrow-derived monocytes under steady-state and inflammatory conditions. This dichotomy underscores the specialized roles of BAMs in surveilling the CNS borders and mediating immune responses at interfaces with the peripheral circulation [6,17]. Although they share specific molecular markers with microglia, BAMs exhibit distinct transcriptional profiles that reflect their unique anatomical niches and functional imperatives. Recent single-cell transcriptomic analyses have refined our understanding of CNS-resident immune cell ontogeny by revealing subtle differences in gene expression patterns delineating distinct microglial subpopulations. These studies indicate that, even within the ostensibly homogenous microglial compartment, region-specific phenotypes emerge that are likely shaped by local neuronal and vascular cues [22,23]. Such heterogeneity is particularly interesting given its implications for differential responses to pathological stimuli, including neurodegenerative processes and acute inflammatory challenges.

The developmental divergence of microglia and BAMs has significant implications for CNS homeostasis and disease progression (Figure 1,

Table 1. Developmental ontogeny, molecular signatures, and functional specialization of CNS-Resident immune cells.

Cell Type	Developmental Origin	Key Molecular/Transcriptional Features	Functional Role/Localization
Microglia [28]	Derived from primitive yolk sac progenitors during early embryogenesis	High expression of transcription factors such as PU.1 and IRF8; maturation is critically dependent on TGF-β and CSF1 signaling	Reside within the CNS parenchyma; engage in immune surveillance, synaptic pruning, and support of neuronal homeostasis
Perivascular Macrophages [6]	Dual origin: a subset from yolk sac progenitors and another from bone marrow-derived monocytes	Exhibit transcriptional profiles distinct from parenchymal microglia, reflecting adaptations to their vascular niche	Located in perivascular spaces, they play essential roles in maintaining vascular homeostasis and mediating immune cell trafficking
Meningeal Macrophages [6]	Dual origin: contributions from both yolk sac-derived progenitors and bone marrow-derived cells	Display unique molecular markers that differentiate them from microglia and other BAMs; specific gene expression patterns	They rely on the meninges, contribute to immune surveillance at the CNS borders, and help preserve barrier integrity
Choroid Plexus Macrophages [15]	Dual origin: originating from yolk sac progenitors and replenished by bone marrow-derived monocytes	Possess niche-specific transcriptional profiles that reflect their adaptation to the choroid plexus microenvironment	Localized within the choroid plexus, regulate cerebrospinal fluid composition by modulating immune cell entry

). Microglia, as the primary resident immune cells, are essential for the developmental sculpting of neural circuits and, as key responders in pathological contexts, modulating neuroinflammation and contributing to tissue repair. In contrast, the strategic positioning of BAMs at CNS interfaces suggests they may play critical roles in regulating immune cell trafficking and bridging communication between the CNS and systemic circulation [24,25]. Understanding these ontogenic distinctions is fundamental for deciphering the complex interplay between CNS immunity and neurodegenerative disorders. In summary, CNS-resident immune cells’ origin and developmental trajectories reflect an intricate interplay between embryonic programming and local environmental cues, establishing a foundation for homeostatic regulation and disease pathogenesis. This duality of origin highlights the evolutionary adaptation of the CNS immune system and offers avenues for targeted therapeutic interventions in CNS disorders [26,27].

3. Roles in CNS Homeostasis

Microglia and CNS-associated macrophages play multifaceted roles in maintaining central nervous system (CNS) homeostasis through highly regulated molecular mechanisms, integral from early development to aging [29,30]. These roles include synaptic pruning, neuronal support, regulation of myelination, and continuous immune surveillance of the CNS microenvironment. Recent insights from high-resolution transcriptomics and in vivo imaging have revealed that microglia are not merely passive immune sentinels but active modulators of neural circuitry and plasticity [29,30]. During neurodevelopment and adulthood, microglia contribute to the refinement of neural networks through synaptic pruning—an activity-dependent process that removes weak or redundant synapses. This function is essential for proper neuroplasticity, learning, and memory. The complement cascade, particularly the C1q and C3 proteins, tags synapses for elimination, while microglia express complement receptor 3 (CR3) to phagocytose these synaptic elements. Dysregulation in this pathway has been linked to synaptic loss in Alzheimer’s disease and schizophrenia, suggesting that maintaining its critical balance is fundamental to healthy brain function [31,32].

Beyond pruning, microglia and CNS macrophages support neuronal viability by secreting neurotrophic factors such as brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1), and transforming growth factor-beta (TGF-β). These molecules promote neuronal survival, synaptogenesis, and repair following injury. Moreover, microglial-derived BDNF modulates synaptic plasticity through TrkB signaling, influencing long-term potentiation (LTP) and memory consolidation [33,34]. This trophic support’s spatial and temporal control underscores their importance in homeostatic and reparative processes. Microglia interact closely with oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes to regulate myelin dynamics. They contribute to developmental myelination and remyelination following demyelinating injuries. Depending on their activation state, microglia-derived cytokines and extracellular vesicles containing miRNAs can promote or inhibit oligodendrocyte differentiation. Moreover, clearance of myelin debris by microglia is a prerequisite for efficient remyelination, highlighting their dual role in debris removal and regenerative signaling [35,36].

In the steady state, microglia adopt a ramified morphology optimized for surveying the CNS parenchyma. This homeostatic surveillance is regulated by a unique repertoire of receptors termed the “sense.” These include pattern recognition receptors (e.g., TREM2, TLRs), purinergic receptors (e.g., P2RY12), and scavenger receptors (e.g., CD36) [37,38]. Through these receptors, microglia detect changes in the extracellular milieu—such as ATP release from dying cells or accumulation of misfolded proteins—and initiate appropriate responses, including phagocytosis, cytokine release, or metabolic adaptation [29,30]. Collectively, the intricate roles of microglia and CNS macrophages in synaptic pruning, trophic support, myelin regulation, and immune surveillance underscore their indispensable function in preserving CNS integrity. Dysfunctions in any of these homeostatic roles can initiate or exacerbate neuropathological conditions. Understanding these mechanisms at the molecular and cellular levels, as highlighted in the recent works of Paolicelli et al. and Bennett et al., provides critical insight into targeting microglial dynamics in neurodegenerative and neurodevelopmental disorders [39,40,41]. The mechanistic interplay between cortical spreading depolarizations (CCHs) and the purinergic system involves a complex bidirectional modulation where extracellular ATP release during CCHs activates purinergic P2 receptors on neurons and glial cells, triggering intracellular calcium signaling cascades and inflammatory responses. Concurrently, activation of P1 adenosine receptors mediates neuroprotective feedback by modulating neurotransmitter release and vascular tone. This dynamic interaction influences neuronal excitability, glial activation, and blood–brain barrier integrity, thereby shaping the pathophysiological outcomes of CCHs. Unraveling these mechanisms is crucial for targeting purinergic signaling to modulate CCH-related neurological disorders effectively [42,43]. Purinergic receptors are classified primarily into P1 and P2 families based on their endogenous ligands and signaling mechanisms. P1 receptors are G protein-coupled receptors activated by adenosine, mediating mainly inhibitory and neuroprotective effects through the modulation of cAMP pathways. In contrast, P2 receptors respond to extracellular nucleotides such as ATP and are subdivided into ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptors function as ligand-gated ion channels, facilitating rapid cation influx, while P2Y receptors are G protein-coupled, triggering diverse intracellular signaling cascades. For example, the activation of P2X7 receptors—a specific subtype of P2X—has been implicated in pro-inflammatory responses and cell death, distinct from broader P2X receptor family effects. Clarifying receptor subtype-specific roles is essential for precise therapeutic targeting [44,45,46].

Recent research highlights the diverse roles of purinergic signaling in various physiological and pathological processes. Purinergic P2 receptors are implicated in homeostasis, metabolism, and nociception, offering potential therapeutic targets for multiple diseases [47]. In autism spectrum disorder, mesenchymal stem cells may modulate neuroinflammation through purinergic activity, particularly adenosine signaling [48]. Purinergic signaling is also hypothesized to be involved in the cytokine storm associated with monkeypox infections, suggesting potential therapeutic interventions through receptor modulation [49]. Furthermore, purinergic signaling plays a crucial role in controlling the generation and release of extracellular vesicles, particularly in immune and cancer cells, mediating long-distance cellular communication [44]. These findings collectively underscore the importance of purinergic signaling in various biological processes and its potential as a therapeutic target in multiple disorders.

4. Dysregulation and Disease Progression

Although microglia and CNS-associated macrophages (CAMs) are essential for maintaining CNS homeostasis, their dysregulation under pathological conditions can significantly contribute to the onset and progression of neurological diseases [50,51]. Chronic activation, maladaptive phenotypic changes, and aberrant immune responses orchestrated by these cells can promote neuroinflammation, neurodegeneration, and neural dysfunction. The duality of microglial and macrophage functions—ranging from neuroprotective to neurotoxic—underscores their critical role in determining disease outcomes across various neurodegenerative, neuroinflammatory, and neurovascular disorders [52,53].

4.1. Neurodegenerative Diseases: Alzheimer’s, Parkinson’s, and Beyond

In neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), microglia and CAMs undergo significant changes in their phenotypic states [54,55]. In AD, for example, microglia display a pro-inflammatory, neurotoxic phenotype in response to amyloid-β plaques and tau tangles. This neuroinflammatory response, driven by the activation of innate immune receptors like TLR4, the NLRP3 inflammasome, and the complement system, worsens neuronal damage and synaptic loss [56,57]. Though they initially promote amyloid clearance, prolonged activation of microglia results in chronic inflammation, oxidative stress, and the release of neurotoxic substances, creating a vicious cycle of neuronal degeneration. Similarly, in Parkinson’s disease (PD), microglia are activated in response to the accumulation of α-synuclein aggregates, which contribute to the death of dopaminergic neurons in the substantia nigra. This inflammatory environment is characterized by elevated levels of cytokines (e.g., TNF-α IL-1β) and reactive oxygen species (ROS) and accelerates neurodegeneration and threatens neuronal survival. The failure of microglial clearance mechanisms further worsens the accumulation of toxic protein aggregates, indicating that restoring microglial homeostasis could be a valuable therapeutic approach [58,59,60]. In Huntington’s disease, microglia are involved in the neuroinflammatory responses to the buildup of the mutant huntingtin protein, activating both microglial and peripheral immune cells. CAMs also play a critical role in these diseases, especially in promoting blood–brain barrier disruption and allowing the infiltration of peripheral immune cells, which may contribute to disease progression [60,61].

4.2. Neuroinflammatory Disorders: Multiple Sclerosis and Autoimmunity

In autoimmune diseases such as multiple sclerosis (MS), the roles of microglia and CAMs take on a more pronounced immunopathological character [62,63]. In MS, microglia are activated in response to the disruption of the blood–brain barrier, leading to the infiltration of autoreactive T cells and the onset of demyelination [36,64]. Microglial activation in MS is typified by an increase in pro-inflammatory cytokines (e.g., IL-6, IL-12) and matrix metalloproteinases, contributing to the degradation of myelin and oligodendrocyte injury. Furthermore, microglial-derived ROS and nitric oxide can directly damage axonal structures, impairing neuronal communication and promoting disability progression [65,66]. CAMs, particularly those in the perivascular space, also play key roles in MS by orchestrating the recruitment of peripheral immune cells into the CNS and modulating the neuroinflammatory microenvironment. The interplay between microglia and T cells is crucial for the exacerbation of the disease, and therapies targeting this interaction are being explored in clinical trials [67,68].

4.3. Trauma and Stroke: Inflammatory Activation and Tissue Repair

In acute CNS injuries, such as traumatic brain injury (TBI) and ischemic stroke, microglia and CAMs play essential roles in both the response to injury and the subsequent tissue repair process [69,70]. Following TBI or stroke, microglia and CAMs rapidly activate, responding to the influx of damage-associated molecular patterns (DAMPs) and initiating an inflammatory cascade. In the early stages of injury, their activation is neuroprotective, promoting the clearance of debris and supporting tissue repair [71]. However, prolonged activation can lead to chronic neuroinflammation, gliosis, and exacerbated neuronal damage. During ischemia, microglia are implicated in the neuroinflammatory response to the disruption of the BBB, promoting the recruitment of neutrophils and macrophages to the site of injury [72,73]. The microglial response can have dual effects: while acute activation aids tissue repair, prolonged activation can contribute to secondary brain damage by releasing pro-inflammatory mediators and exacerbating oxidative stress [74,75].

4.4. Aging and Neurodegenerative Progression

Aging is associated with a gradual shift in the functional states of microglia and CAMs, resulting in a chronic low-grade neuroinflammatory environment known as inflammation [76,77]. In the aging brain, microglia display signs of cellular senescence, including altered gene expression, diminished phagocytic capacity, and the accumulation of dysfunctional mitochondria. This senescent microglial state is linked to cognitive decline and increased vulnerability to neurodegenerative diseases [78,79]. Moreover, aging leads to a decline in the regenerative capacity of the CNS, partly due to impaired neurogenesis and neurorestorative functions of microglia. CAMs, too, change with age, showing an increased propensity for pro-inflammatory activation. This shift contributes to the acceleration of age-related neurodegenerative diseases and cognitive decline [80].

4.5. Cancer and the Tumor Microenvironment

Emerging evidence highlights the role of microglia and CAMs in the tumor microenvironment (TME), particularly in gliomas and other CNS cancers. Microglia and CAMs are actively recruited to tumors, where they can exhibit both tumor-promoting and tumor-suppressive activities depending on their polarization state [81,82,83]. Tumor-associated microglia (TAMs) often adopt an M2-like phenotype that supports tumor growth by promoting angiogenesis, immune suppression, and extracellular matrix remodeling. However, microglial responses to tumor-related signals are complex, and specific microglial subpopulations may also help suppress tumorigenesis through immune surveillance and the release of anti-tumoral cytokines [84,85,86]. CAMs contribute to the immune landscape of CNS tumors by influencing the infiltration of peripheral immune cells, promoting tumor cell invasion, and modulating the blood–brain barrier. These interactions create an immune-tolerant environment, which can hinder the efficacy of immunotherapies [87].

4.6. The Neuroimmune Interface in the CNS

The neuroimmune interface in the CNS has garnered significant attention in recent years due to its critical role in modulating the pathophysiology of mood disorders, notably depression and stress-related conditions. Central to this interface are microglia, the resident macrophages of the brain, and peripheral macrophages, which may infiltrate the CNS under certain pathological conditions [88,89]. Both cell types contribute to neuroinflammatory processes that have been implicated in the etiology and progression of depressive disorders. Microglia, serving as the primary immune surveillance cells in the CNS, dynamically respond to environmental cues through a complex interplay of receptor-mediated signaling pathways [88,89]. Under homeostatic conditions, microglia support neural plasticity and synaptic remodeling, contributing to the maintenance of neuronal circuits. However, in response to chronic stress or injury, these cells undergo phenotypic changes, adopting a pro-inflammatory profile characterized by the upregulation of cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6. This pro-inflammatory cascade has been proposed as a mechanistic link to the cytokine hypothesis of depression, whereby sustained microglial activation disrupts synaptic function and neurogenesis, ultimately leading to behavioral deficits associated with depressive states [90].

Recent research has underscored the dual role of microglia in neuroprotection and neurotoxicity. On the one hand, microglial activation can promote the clearance of cellular debris and support reparative processes; on the other hand, excessive or chronic activation may result in a maladaptive inflammatory milieu that exacerbates neuronal dysfunction [90]. This dichotomy is further complicated by the phenotypic heterogeneity of microglia, which can adopt either a classically activated (M1) pro-inflammatory state or an alternatively activated (M2) anti-inflammatory state [91]. Shifts in this balance, particularly under conditions of prolonged stress, are thought to precipitate neuroinflammatory responses that impair neuroplasticity and contribute to the persistence of depressive symptoms. Moreover, peripheral macrophages, which are functionally analogous to microglia, can be recruited to the CNS when the integrity of the blood–brain barrier is compromised—a phenomenon often observed in chronic stress and systemic inflammation [92,93]. These infiltrating macrophages can amplify the local inflammatory response, further perturbing neuronal homeostasis. The interaction between resident microglia and peripheral macrophages appears to be pivotal in shaping the inflammatory landscape of the brain. For instance, recent studies have demonstrated that stress-induced peripheral inflammation can prime microglia, making them more susceptible to subsequent stimuli, thereby establishing a feed-forward loop that perpetuates the inflammatory state [92,93].

The implications of these findings extend to potential therapeutic interventions. Modulating microglial activation states and limiting the recruitment of peripheral macrophages into the CNS are emerging strategies in treating depression. Pharmacological agents that target specific signaling pathways—such as those inhibiting pro-inflammatory cytokine production or enhancing anti-inflammatory responses—have shown promise in preclinical models [94,95]. Additionally, novel approaches aimed at restoring the balance between the M1 and M2 phenotypes may hold therapeutic potential, as they could mitigate the deleterious effects of chronic neuroinflammation without compromising the essential homeostatic functions of these immune cells [96,97]. In summary, the intricate interplay between microglia and peripheral macrophages plays a pivotal role in the neuroinflammatory processes that underlie depression and stress-related disorders. Continued elucidation of these mechanisms is critical for developing targeted interventions aimed at modulating neuroimmune responses, thereby offering new avenues for treating mood disorders [96,97].

5. Nanotechnology-Based Modulation of Microglia and Macrophages

Microglia and macrophages constitute the primary immune sentinels of the CNS, executing dual roles in preserving homeostasis and propagating neuroinflammation [98,99,100,101]. These dynamic and plastic immune cells orchestrate neurodevelopmental synaptic pruning, phagocytosis of cellular debris, and immune surveillance under physiological conditions [29,102]. However, in response to pathological cues, such as Aβ aggregates, α-synuclein fibrils, or traumatic injury, they undergo phenotypic reprogramming toward neurotoxic, pro-inflammatory states (M1-like), contributing to the pathogenesis of neurodegenerative diseases such as AD, PD, and MS [103]. Recent advances in nanotechnology offer unprecedented avenues to investigate and therapeutically manipulate microglial/macrophage function with spatial and temporal precision [104,105].

Nanotechnology-based approaches enable selective targeting, reprogramming, and imaging of CNS-resident microglia and infiltrating macrophages via engineered nanoparticles (NPs), exosomes, and nanocarriers (Table 2). These nanodevices can be designed to traverse the BBB, exploit microglial surface receptor expression (e.g., TREM2, CD11b, CX3CR1), and modulate intracellular signaling pathways such as NF-κB, STAT1/6, and mTOR, thereby influencing inflammatory responses, phagocytic capacity, and metabolic polarization [106,107]. Functionalized polymeric nanoparticles, such as PEG-PLA, PLGA, or dendrimer-based systems, have been employed for the targeted delivery of anti-inflammatory agents (e.g., minocycline, dexamethasone), siRNAs, and gene-editing tools (e.g., CRISPR-Cas9) directly to activated microglia [108,109]. For instance, nanocarriers encapsulating siRNA against pro-inflammatory mediators like TNF-α or IL-1β have demonstrated the capacity to attenuate glial-mediated neurotoxicity in murine models of AD. Additionally, exosome-mimetic nanovesicles derived from neural stem cells or M2-polarized macrophages offer biocompatible vehicles for transferring therapeutic cargoes, including miRNAs and neurotrophic factors, to reprogram microglial activity toward neuroprotective phenotypes [110,111,112].

Engineered nanomaterials can be designed to cross the blood–brain barrier and selectively target microglia, enabling in situ immunomodulation [113]. These nanodevices can influence microglial phenotypes, shifting them between pro-inflammatory (M1) and anti-inflammatory (M2) states to address various CNS pathologies [113,114]. Microglia-targeting nanotherapeutics can be engineered by conjugating specific receptor-targeting ligands, leveraging microglial phagocytic properties, and incorporating therapeutic agents to modulate activation pathways [115]. Such approaches may help resolve multiple pathological determinants of neurodegenerative diseases and guide microglial phenotypes toward a more neuroprotective state [115]. Nanomedicines targeting specific disease-associated cells in the CNS, including microglia, neurons, and astrocytes, show potential to enhance therapeutic efficacy while reducing side effects [116]. Nanoparticles can be engineered to cross the blood–brain barrier and selectively target microglia, enabling therapeutic manipulation of their function [117]. These nanodevices can modulate microglial states, influencing inflammatory responses and phagocytic capacity in various CNS conditions [117,118]. Nanomaterials also allow for macrophage visualization and metabolic reprogramming, potentially customizing their phenotype for therapeutic purposes [117]. Furthermore, nanotheranostics show promise in overcoming the blood–brain barrier, addressing a significant challenge in treating CNS diseases [119]. These nanotechnology-based approaches offer unprecedented precision in investigating and manipulating microglial and macrophage function, paving the way for novel therapeutic interventions in neurodegenerative disorders, autoimmune diseases, and other CNS pathologies [117,118].

As depicted in the schematic of a pathological central nervous system environment (Figure 2), nanoparticles introduced into the brain parenchyma can significantly contribute to neuroinflammation by interacting with resident immune cells. The figure illustrates that nanoparticles are one of the factors leading to the activation of microglia, transitioning them from a homeostatic state to a reactive phenotype [98,99,100,101]. Once activated, these microglia become a significant source of inflammatory mediators within the brain microenvironment [98,120]. Specifically, the diagram indicates the release of potent pro-inflammatory cytokines, including Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), and Tumor Necrosis Factor-alpha (TNF-β), as well as the chemokine Monocyte Chemoattractant Protein-1 (MCP-1). These molecules are known to propagate inflammatory cascades, potentially recruiting other immune cells and directly impacting neuronal function and viability. Additionally, the figure notes the involvement of Transforming Growth Factor-beta (TGF-β), a cytokine with complex roles that can be both pro- and anti-inflammatory depending on the context [98,99,100,101]. This nanoparticle-induced inflammatory milieu, driven by activated microglia, is shown to be a key contributor, alongside oxidative stress and Aβ plaque pathology, to the ultimate outcomes of neuronal damage and dementia illustrated in Figure 2.

In parallel, magnetic and plasmonic nanoparticles offer real-time tracking and theranostic capabilities, enabling the in vivo visualization of microglial migration, phagocytosis, and response to inflammatory insults [98,99,100,101]. For example, superparamagnetic iron oxide nanoparticles (SPIONs) allow MRI-based mapping of microglial infiltration in demyelinating lesions. At the same time, gold nanorods and nanoshells can facilitate photothermal modulation of immune responses within the CNS parenchyma [121,122]. Furthermore, nanotechnology interfaces seamlessly with optogenetic and photodynamic strategies to modulate microglial behavior via light-responsive nanomaterials. Such approaches permit localized activation or silencing of microglial function with subcellular resolution, offering a non-invasive strategy to influence neuroimmune interactions in real time [123,124]. Nanotechnology provides a mechanistic platform for dissecting the cellular and molecular underpinnings of microglial and macrophage biology in CNS homeostasis and disease and paves the way for precision neuroimmunotherapy. Future studies integrating nanotechnology with single-cell transcriptomics, spatial proteomics, and systems neurobiology will further refine our capacity to harness these innate immune cells for neurorestorative purposes [125,126,127].

Table 2. Nanotechnology strategies targeting microglia and macrophages in CNS homeostasis and disease.

Nanotechnology Approach	Targeted Cell Type	Mechanism of Action	Therapeutic Application
Polymeric Nanoparticles (e.g., PLGA, PEG-PLA) [128]	Microglia	Deliver anti-inflammatory agents (e.g., minocycline, dexamethasone) to modulate activation states	Attenuation of neuroinflammation in neurodegenerative diseases
siRNA-Loaded Nanocarriers [129]	Microglia	Gene silencing of pro-inflammatory cytokines (e.g., TNF-α, IL-1β)	Reduction in neurotoxic responses in Alzheimer’s disease models
Exosome-Mimetic Nanovesicles [130]	Microglia and Macrophages	Transfer of therapeutic cargoes (e.g., miRNAs, neurotrophic factors) to reprogram immune cell phenotypes	Promotion of neuroprotection and repair mechanisms
Superparamagnetic Iron Oxide Nanoparticles (SPIONs) [131,132]	Microglia	Enable magnetic resonance imaging (MRI) tracking of microglial migration and activation	Visualization of inflammatory processes in CNS disorders
Gold Nanorods/Nanoshells [133,134]	Microglia	Facilitate photothermal modulation of immune responses	Targeted modulation of neuroinflammation
Light-Responsive Nanomaterials [135]	Microglia	Integration with optogenetic tools to control activation states via light stimulation	Non-invasive modulation of microglial behavior

Table 2. Nanotechnology strategies targeting microglia and macrophages in CNS homeostasis and disease.

Nanotechnology Approach	Targeted Cell Type	Mechanism of Action	Therapeutic Application
Polymeric Nanoparticles (e.g., PLGA, PEG-PLA) [128]	Microglia	Deliver anti-inflammatory agents (e.g., minocycline, dexamethasone) to modulate activation states	Attenuation of neuroinflammation in neurodegenerative diseases
siRNA-Loaded Nanocarriers [129]	Microglia	Gene silencing of pro-inflammatory cytokines (e.g., TNF-α, IL-1β)	Reduction in neurotoxic responses in Alzheimer’s disease models
Exosome-Mimetic Nanovesicles [130]	Microglia and Macrophages	Transfer of therapeutic cargoes (e.g., miRNAs, neurotrophic factors) to reprogram immune cell phenotypes	Promotion of neuroprotection and repair mechanisms
Superparamagnetic Iron Oxide Nanoparticles (SPIONs) [131,132]	Microglia	Enable magnetic resonance imaging (MRI) tracking of microglial migration and activation	Visualization of inflammatory processes in CNS disorders
Gold Nanorods/Nanoshells [133,134]	Microglia	Facilitate photothermal modulation of immune responses	Targeted modulation of neuroinflammation
Light-Responsive Nanomaterials [135]	Microglia	Integration with optogenetic tools to control activation states via light stimulation	Non-invasive modulation of microglial behavior

6. Mechanobiology Techniques for Intracellular Probing

It is now widely recognized that most cellular functions are dependent on the mechanical properties of both the microenvironment and the cell itself [136,137]. Mechanobiology investigates how microglia and macrophages perceive and react to mechanical cues and forces, and how these mechanical signals are transduced into biochemical responses. While significant efforts over the past four decades have elucidated cellular mechanics at the whole-cell scale, recent technological advancements have enabled the exploration of intracellular mechanotransduction—specifically, how mechanical forces are conveyed within microglia and macrophages at the subcellular level [136,137]. The application and measurement of forces inside these cells facilitate the investigation of key questions, particularly in domains such as cell migration and division. These biological processes involve substantial deformation and structural rearrangement of intracellular components, which can now be examined by force application or real-time force monitoring [136,137]. For example, migration of microglia and macrophages often entails nuclear deformation as they navigate through confined microenvironments. Similarly, in the context of cellular energetics and metabolism, the critical importance of mechanical properties is becoming increasingly acknowledged. Intracellular mechanical forces are necessary for the transport, fission, and fusion of mitochondria, all of which are vital for mitochondrial function and energy generation. Beyond the nucleus and mitochondria, other organelles involved in the secretory pathway—including the endoplasmic reticulum (ER), Golgi apparatus, and endosomes—also demonstrate mechanosensitivity. Since intracellular trafficking governs protein and lipid synthesis, sorting, export, and import, the mechanosensitivity of endomembranes is indispensable for cellular mechanical response pathways [138,139].

Therefore, the investigation of the mechanical characteristics of subcellular structures, including organelles and the cytoplasm, has emerged as a major area of scientific inquiry. Although numerous reviews have analyzed techniques related to whole cells and tissues, relatively few have focused on advanced methodologies suitable for probing the interior of microglia and macrophages at high spatial and temporal resolution [138,139]. This section emphasizes such methodologies. Enhancing spatio-temporal precision enables the capture of subtle or short-lived phenomena [140,141]. For instance, in cancer research, variations in biophysical parameters like intracellular viscosity have been linked to elevated metastatic potential. Additionally, refined measurement capabilities are crucial for improving computational models of biological processes.

Two key distinctions must be made here. First, there is a difference between techniques that function as force generators versus those that serve as force sensors. To comprehensively assess the mechanical properties of an intracellular organelle, it is essential to apply forces and to evaluate its rheological attributes. Some of the experimental techniques described herein function as force generators, force sensors, or both. The second distinction is between techniques that act exclusively at the intracellular level versus those operating at the transcellular or extracellular levels [142,143]. For example, force generators that operate exclusively intracellularly can apply spatially localized forces, as in the case of optical tweezers, which are capable of inducing localized deformation of an organelle at the micrometer scale. In contrast, transcellular or extracellular methods are typically broader and less spatially resolved. This review focuses solely on the former—techniques capable of localized mechanical manipulation. It should be further emphasized that each technique possesses distinct temporal characteristics, allowing forces to be applied or recorded over varying durations, at specific frequencies, and in either reversible or irreversible modes [144,145].

In this section, we present six state-of-the-art experimental techniques optimized for assessing the mechanical properties of subcellular components in microglia and macrophages. These methods, arranged from force generators to mechanical sensors, include optogenetics, engineered microglial/macrophage-based nanorobots, optical tweezers (OT), Brillouin microscopy, molecular tension probes, and magnetic particles. We highlight these particular methodologies because, unlike earlier approaches, they offer high spatio-temporal accuracy, enabling precise interrogation of specific organelles without inadvertently altering adjacent cellular structures [146,147]. Conventional techniques for manipulating cells and their organelles include cell stretching systems, parallel microplate rheometers, microfluidic devices, and atomic force microscope (AFM) cantilevers. An alternative approach involves culturing cells on patterned adhesive surfaces, which induces varying degrees of cellular stretching and, consequently, organelle deformation. Similarly, the mechanical behavior of intracellular systems can be modulated by culturing cells on substrates with different stiffness levels or through osmotic challenges that alter cell volume [148,149,150]. On the sensing front, AFM has been widely regarded as the gold standard for mechanical property measurements, though its use is generally limited to the cell surface and topography. Micropipette aspiration can also be employed to evaluate global viscoelastic properties of living microglia or macrophages, as well as their organelles. Moreover, force generation within the intracellular environment can be achieved through techniques like photoablation, which uses lasers to sever cellular structures, including cytoskeletal filaments such as actin within cells [148,149,150]. Chromophore-assisted laser inactivation (CALI) is another approach that allows for localized and temporally controlled inactivation of specific proteins; when applied to cytoskeletal proteins or motor proteins, it can effectively modulate intracellular forces. Nevertheless, as these methods primarily lead to the relaxation of intracellular or intercellular tension rather than the direct application of controllable forces—and as force estimation from these experiments necessitates complex modeling—we do not elaborate on them further. Interested readers are referred to recent in-depth reviews on these techniques [148,151,152]. We propose that the immediate future of intracellular mechanics in microglia and macrophages lies in refining and applying the six experimental strategies presented in this review. This discussion is not intended to be exhaustive regarding available methodologies, nor are the organelles discussed meant to be comprehensive examples. Each technique is illustrated using select case studies to demonstrate its potential and application.

6.1. Optogenetics and Its Application in Mechanotransduction of Intracellular Organelles

In this section, we explore the emerging role of optogenetics—a sophisticated experimental strategy—in elucidating the mechanosensitivity of intracellular organelles. We present a molecular-level explanation of the technique, highlight a recent application targeting the endoplasmic reticulum (ER) in live-cell systems, and underscore its numerous methodological advantages [153,154]. Optogenetics enables the spatially and temporally precise regulation of protein expression or localization within live cells through light exposure. This is made possible by two critical molecular components: a photosensitive protein that undergoes conformational change upon illumination, and a second, functionally relevant protein that interacts with the former. Notable examples include Cry2/CIB1 and iLID/SspB systems [153,154]. The photosensitive protein can be engineered to localize to specific organelles, thereby acting as a spatial anchor. By selectively illuminating defined regions within the cell, researchers can achieve precise subcellular control over protein activity. Key physical parameters that influence the response include the illuminated region’s area, illumination duration and intensity, and the local concentration of optogenetic proteins. Activation is typically rapid—within sub-second timescales—and remains effective for several minutes after cessation of light exposure [155,156,157].

A recent study exemplified the utility of optogenetics in demonstrating the ER’s mechanosensitivity. This involved the development of LIMER (Light-Inducible ER-Specific Mechanostimulator), which directs mechanical force to the ER via light-induced recruitment of a modified kinesin-1 motor protein [153,158,159]. Blue light (470 nm) illumination prompted localized ER contraction. It was hypothesized that this mechanical perturbation activated mechanosensitive ion channels in the ER membrane, facilitating calcium ion (Ca²⁺) release—an essential process in cellular signaling (Figure 3) [160,161]. GCaMP6, a genetically encoded Ca²⁺ indicator, was utilized for its robust expression, low cytotoxicity, and spatial specificity. To pinpoint the responsible ion channels—TRPV1 and PKD2—the researchers applied the TRPV1 inhibitor SB-366791 and employed PKD2 knockdown strategies, both of which led to significantly diminished Ca²⁺ flux. The study illustrates multiple strengths of optogenetics: it allows for spatially confined activation through defined regions of interest (ROIs) and temporally controlled stimulation over seconds [162,163]. The technique is minimally invasive, applicable in vivo, and biologically orthogonal, meaning it does not interfere with unrelated cellular processes. However, limitations include the technical complexity of generating stable cell lines that express the optogenetic components and potential issues with light penetration in optically heterogeneous tissues, such as the brain, where lipid-water interfaces cause scattering and refraction, thereby impairing spatial resolution. Nevertheless, optogenetics remains among the most precise and non-invasive approaches currently available for applying mechanical stimuli to intracellular structures.

6.2. Nanorobots

A parallel approach to probing intracellular mechanics involves the utilization of nanorobots, an emerging class of abiotic micro- and nanoscale machines that have garnered significant attention in the biomedical literature, particularly for applications in targeted drug delivery, biosensing, and theranostics [164,165]. Despite their prominence in these domains, their role in interrogating intracellular mechanical environments remains surprisingly underexplored, with notable exceptions such as certain magnetic micro- and nanoprobes (Figure 3) [164,165]. Nanorobots typically incorporate propulsion mechanisms driven by magnetic fields, chemical reactions, ultrasound, or light stimuli and exhibit diverse morphologies tailored for specific biomedical functions. For instance, the Magnetically Propelled Nanomotor (MPN) represents a widely studied class of helical nanorobots composed of biocompatible materials such as iron oxide, nickel, or polymeric composites, and navigated via a rotating magnetic field. These MPNs, typically ranging from 100 nm to a few micrometers in length, can achieve speeds of up to tens of micrometers per second, depending on the field strength and medium viscosity. While initially designed for intracellular cargo delivery (e.g., transfection agents or gene therapy vectors), MPNs could be calibrated to function as active micromechanical probes, for instance, by applying force against a bead held in optical tweezers (OT), thereby enabling real-time measurement of viscoelastic properties within the cytoplasm [166,167,168].

Another intriguing subclass of nanorobots incorporates photothermal nanomaterials, which convert near-infrared (NIR) light into localized heat, producing asymmetric thermophoretic propulsion. These light-activated nanorobots, fabricated from gold nanorods, carbon-based structures, or Janus particles, often adopt intricate morphologies such as spherical, urchin-like, or flower-like architectures [166,169,170]. Their sizes typically range from 50 to 500 nm, and they can be precisely activated and steered using spatiotemporally modulated laser fields. Nonetheless, as with magnetic particles, these systems frequently exhibit linear or unidirectional motion toward or away from external gradients, thus lacking adaptive navigational capabilities in complex intracellular landscapes [171,172,173]. Recent advances aim to overcome these limitations through the incorporation of smart materials and feedback control algorithms, enabling more sophisticated behaviors such as swarming, shape transformation, or multi-modal propulsion switching [171,174,175]. Fabrication strategies for such nanorobots include top-down approaches like electron-beam lithography and 3D nanoprinting, as well as bottom-up methods involving self-assembly, templating, and bioconjugation with synthetic or biomimetic components. Although these technologies remain in the early translational stages, they offer substantial promise for mechanobiological interrogation of intracellular environments. Importantly, unlike optogenetics—where light modulates genetically encoded actuators—these nanorobots represent entirely abiotic systems governed by external physical fields, marking a significant step toward non-genetic and autonomous intracellular manipulation [172,176,177].

7. Conclusions

In conclusion, microglia and macrophages are integral players in the central nervous system (CNS), not only serving as the primary immune sentinels but also actively contributing to homeostatic processes such as synaptic pruning, neuronal support, and myelin regulation. Their development and differentiation, stemming from distinct ontogenetic origins, underline their specialized functions and roles in maintaining CNS integrity. Despite their essential functions, both microglia and macrophages exhibit phenotypic plasticity, which, under pathological conditions, can shift them toward neurotoxic states, exacerbating diseases such as Alzheimer’s, Parkinson’s, multiple sclerosis, and glioblastoma. These shifts emphasize the complex duality of their roles—simultaneously acting as protectors and effectors in disease progression. Recent advancements in single-cell transcriptomics, spatial omics, and in vivo imaging have provided deeper insights into their molecular mechanisms, revealing a nuanced understanding of how these cells modulate neuroinflammation, tissue repair, and immune surveillance. Moreover, the evolving field of nanotechnology offers promising therapeutic avenues for reprogramming or modulating microglial and macrophage phenotypes, enabling targeted interventions to restore homeostasis and halt disease progression. However, challenges remain in fully understanding the intricate molecular and epigenetic controls governing their behavior, as well as the potential risks of manipulating their activation states. Future research will need to balance the therapeutic potential of microglia and macrophage modulation with the preservation of their critical roles in CNS development and maintenance. The integration of cutting-edge technologies, such as optogenetics and nanorobotics, holds immense promise in dissecting cellular mechanobiology at unprecedented resolutions, potentially offering new approaches for diagnosing and treating CNS disorders. In sum, microglia and macrophages represent a dynamic and essential arm of the immune system in the CNS, and therapeutic strategies aimed at their precise modulation could significantly impact the treatment of neurodegenerative, inflammatory, and even neoplastic CNS conditions. As we continue to unravel the complexity of these cells, their manipulation stands to redefine how we approach neurological diseases, offering hope for more effective and targeted interventions in the future.