Role of microglia after subarachnoid hemorrhage

Abstract

Subarachnoid hemorrhage is a devastating sequela of aneurysm rupture. Because it disproportionately affects younger patients, the population impact of hemorrhagic stroke from subarachnoid hemorrhage is substantial. Secondary brain injury is a significant contributor to morbidity after subarachnoid hemorrhage. Initial hemorrhage causes intracranial pressure elevations, disrupted cerebral perfusion pressure, global ischemia, and systemic dysfunction. These initial events are followed by two characterized timespans of secondary brain injury: the early brain injury period and the delayed cerebral ischemia period. The identification of varying microglial phenotypes across phases of secondary brain injury paired with the functions of microglia during each phase provides a basis for microglia serving a critical role in both promoting and attenuating subarachnoid hemorrhage-induced morbidity. The duality of microglial effects on outcomes following SAH is highlighted by the pleiotropic features of these cells. Here, we provide an overview of the key role of microglia in subarachnoid hemorrhage-induced secondary brain injury as both cytotoxic and restorative effectors. We first describe the ontogeny of microglial populations that respond to subarachnoid hemorrhage. We then correlate the phenotypic development of secondary brain injury after subarachnoid hemorrhage to microglial functions, synthesizing experimental data in this area.

Introduction

Subarachnoid hemorrhage (SAH) following aneurysm rupture is a debilitating neurological disease with a global prevalence of over 8 million cases.^1,2 In the United States, this translates to upwards of 30,000 cases of aneurysmal SAH annually and places a substantial burden on the healthcare system.^3,4 Despite significant practice-level improvements in the forms of novel aneurysm treatments, improved access to specialized neurocritical care, advancements in neuroimaging techniques, and use of nimodipine therapy, the morbidity and mortality of SAH remains unacceptably elevated.^{5 –8} In patients who survive, persistent neurocognitive impairments hinder psychosocial functioning in these patients, compounding challenges faced by those affected by SAH. ⁹

Early clinical and scientific investigations of SAH postulated cerebral hypoperfusion induced by vasospasm as a key driver of secondary brain injury.^{10 –12} Indeed, symptomatic vasospasm of large pial arteries, now referred to as delayed cerebral ischemia (DCI), was viewed as the main cause of patient deterioration after aneurysm rupture. ¹³ Clinical studies suggested that radiographic evidence of cerebral vasospasm may be present in up to 90% of patients with SAH. ¹⁴ A series of negative trials failed to correlate the attenuation of cerebral vasospasm with improved outcomes, paving the way for a continuous view of secondary brain injury.^{15 –19} Using a modern classification schema, delayed ischemic neurological deficit (DIND) is a focal neurological deficit or a decrease in Glasgow Coma Scale score of at least two points that lasts over one hour in the absence of any other identifiable etiologies (e.g. hydrocephalus, seizure, or fever), and occurs between days 3 and 21 after ictus.^20,21

A corollary of the negative trials targeting DCI was a shift in attention towards earlier periods of secondary injury, which galvanized a new wave of basic and translational studies. A growing body of work has implicated secondary brain injury occurring in the first 72 hours following SAH, the Early Brain Injury (EBI) period, as a major source of morbidity and mortality.^{7,22 –26} Injury during the EBI period is driven by microcirculatory dysfunction, blood-brain-barrier (BBB) disruption, neuroinflammation, oxidative cascades, cerebral edema, and various forms of cell death.^7,22 Recent studies of imaging biomarkers in patients with SAH have found the presence of EBI to independently predict the occurrence of DCI and poor patient outcomes in the chronic phase after DCI, demonstrating the relationship of these two critical periods.^7,27,28

The ongoing search for therapies to ameliorate outcomes after SAH has brought microglia to the fore of research efforts. A growing body of evidence implicates the activation and polarization of microglial cells during all phases of secondary brain injury – the acute injury period, EBI, DCI, and chronic injury– as a significant contributor to secondary brain injury, with microglia also potentially linking these periods of injury (Figure 1).^{14,29 –32} The role of microglia as effectors of neuroinflammation provides compelling support of their contributions to neuronal death and impaired neuronal communication after SAH, which can produce the long-term deficits that typify many patients surviving SAH.^{33 –35} Deleterious functions do not appear to be the sole involvement of microglia after SAH, with restorative microglial phenotypes also noted following SAH. ¹⁴

Figure 1.

Phases of secondary brain injury following aneurysm rupture. Acute aneurysm rupture activates early mediators of secondary brain injury through elevations of intracranial pressure and release of toxic blood products. Mechanisms of EBI including neuroinflammation, blood-brain-barrier disruption, microcirculatory dysfunction, cerebral edema, oxidative cascades, and neuronal death predominate the first 72 hours following hemorrhage. This is followed by DCI, which includes neuroinflammation, vasospasm, microthrombosis, autoregulatory dysfunction, and spreading depolarizations following EBI up to day 21. Chronic injury accompanies the poor long-term clinical phenotype of patients with SAH, and includes iron deposition, sustained neuroinflammation, and synaptic dysfunction.

In this review, we provide a comprehensive overview of the functions of microglia as cytotoxic and restorative mediators of secondary brain injury after SAH. First, we describe recent insights into the ontogeny of microglial populations that become involved in the development of brain injury (See Supplement 1). Next, we chronicle the development of microgliosis alongside the progression of secondary brain injury after SAH, correlating microglial activity with clinical phenotypes and outcomes. We then elaborate on cross-talk between microglia, other glial cell populations, and leukocytes to characterize the inflammatory milieu that pervades the central nervous system (CNS) after aneurysm rupture. We robustly describe the role of microglia in each phase of secondary brain injury. Finally, we include therapeutic implications of microglial functions. Our discussion is focused on preclinical data that has been derived from well-validated experimental models of SAH performed primarily in mice and rats, with clinical data provided when available.^36,37

Acute microglial activation in subarachnoid hemorrhage

Brain aneurysms become unstable following sustained hemodynamic, inflammatory, and oxidative stresses. Imbalances in the polarization state of peripheral macrophages residing in aneurysm walls (elevated M1/M2 ratio) are a major driver of aneurysm instability, and additional cellular interactions take place in aneurysms to further destabilize the diseased vessel (see Supplemental section entitled “microglial polarization” for detailed discussion of microglial states).^{38 –41} Instability promotes aneurysm growth, increasing the risk of subsequent rupture. Discrete events including coitus or strenuous exercise are reported to precipitate aneurysm rupture, but the etiology of most aneurysm ruptures remain idiopathic. ⁴² Immediately after aneurysm rupture, a series of acute events drive the first responses to SAH. These are a rapid rise in intracranial pressure (ICP) and blood extravasation into the subarachnoid spaces and ventricular system. ⁷ In addition to contributing to the classic symptoms of SAH, these events stimulate immune responses in the CNS by activating microglia.^7,43

Microglia and intracranial pressure

ICP levels are carefully regulated by a series of homeostatic mechanisms at baseline, with a normal ICP generally in the range of 7–15 mm Hg. ⁴⁴ After SAH, ICP levels climb upwards of 25 mm Hg, which causes decreases in cerebral perfusion pressure alongside global ischemic injury. ⁴⁵ The degree of ICP elevation occurring immediately at the time of ictus correlates with SAH severity. ⁴⁶ Experimental models have shown that microglia are sensitive to ICP elevations and physiologic changes that occur downstream from ICP elevations. In rodent studies of ischemic injury using middle cerebral artery occlusions, ICP elevations induced expression of the inflammatory cytokines IL-1β and IL-18 by microglia through a NOD-like receptor protein 3 (NRLP3) inflammasome-mediated pathway, possibly by promoting overproduction of reactive oxygen species (ROS). ⁴⁷ The regions of the brain that are most susceptible to pressure damage and ischemic injury from diminished CPP include the ventral cerebral cortex and hippocampus, which demonstrate more severe neuroinflammation and microglial activation during SAH in mice.^48,49 Together, these lines of evidence suggest that ICP alterations and downstream hypoperfusion may activate microglial inflammation in the acute phase of SAH.

Acute hemorrhagic products

Extravasation of hemorrhagic blood from a ruptured aneurysm is a key initiator of secondary brain injury during SAH, triggering the activation of microglia. ²⁹ Because of their strategic niche, microglia serve as sentinels and recognize the first incursions of peripheral insults into the CNS. ¹¹ Microglia express all classes of toll-like receptors (TLRs), single-pass transmembrane receptors that rapidly respond to a wide array of pathogen-associated molecular patterns (PAMPs) and initiate intracellular signaling cascades to orchestrate early innate immune responses. ⁵⁰ Toxic blood products including heme, methemoglobin, and iron products serve as agonists of TLR-4, which classically initiate intracellular pathways that culminate in inflammatory gene induction via NF-κB. ⁵¹ This results in increased transcription of IL-1β, IL-6, and TNF-α. During the acute phase of SAH and into the EBI period, activation of the transcription factor NF-κB occurs in a myeloid differentiation primary response 88 (MyD88)-mediated pathway, which gradually transitions to a TIR-domain-containing adapter-inducing interferon-β (TRIF)-mediated pathway, an intracellular adaptation that must be considered in scenarios of therapeutic targeting. ⁵² Alongside TLR-4, TLR-2 has been shown to promote induction of similar cytokines after SAH. ⁵³ TLR-2 also promotes the chemotaxis of microglia to the region of active hemorrhage and injured brain, an important early adaptation for microglia. ⁵³ Unlike TLR-4, TLR-2 signals through interferon regulatory factor 7 (IRF7) following experimental SAH. DAMPs produced intrinsically in the CNS also contribute to the activation of microglia after SAH. Neurons injured by SAH release alarmins such as high mobility box 1 protein (HMGB1), which are recognized by TLRs. ⁵⁴ Finally, Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling axes have been implicated in acute neuroinflammation after SAH, and may serve as future drug targets. ⁵⁵

Acute neuronal death

Though neuronal death develops progressively after ictus, early dying cells after SAH are seen in close proximity to regions of bleeding such as adjacent basal cisterns, and depending on aneurysm location or site of experimental SAH induction, can be observed in the somatosensory cortex, hippocampus, striatum, and basal cortex. ³³ Early activation of microglia promotes acute neuronal death, as evidence points to neuroinflammation inducing both energy dependent and energy-independent forms of cells death in this timeframe.^48,56 Metabolic imbalances that occur in consequence to dysfunctional cerebral perfusion also contribute to inflammation-induced cell death. For instance, some forms of cell death including necroptosis induce the secretion of HMGB1, which amplifies neuroinflammation and increases apoptotic and cell death-inducing mechanisms through positive feedback. ⁵⁷

Microglia in early brain injury

The EBI period immediately follows acute hemorrhage and includes the deleterious processes of diffuse blood-brain-barrier (BBB) disruption, neuroinflammation, oxidative cascades, and cerebral edema. The phenotype of EBI includes poor neurological function, accompanied by metabolic impairments, cerebral hypoxia, and imaging signs (diffuse cerebral edema, effacement of the gray-white junction, and loss of sulcal volume).^7,22 In addition to contributing to secondary brain injury during the EBI period, microglia also partake in critical neuroprotective processes during this critical period including hemoglobin scavenging via the CD163 scavenger pathway. ¹¹ Throughout the EBI period, microglial activation initiated in the acute phase of SAH is supplemented by additional activating factors, which augments their contributions to EBI. One such change occurring in this period is the congestion of meningeal lymphatics by subarachnoid blood that is being cleared, which prolongs the exposure of microglia to toxic blood breakdown products and promotes M1 polarization. ⁵⁸ The role of meningeal lymphatic drainage after SAH is essential for homeostatic function, as depletion of these pathways in mouse models leads to increased neuroinflammation and more significant neurobehavioral deficits. ⁵⁸ This change also reflects the cytotoxic M1-predominance of microglia in the EBI period. Experimental models of EBI have accounted for differences in the timing of EBI between humans and rodents. In humans, EBI encompasses the first 72 hours after aneurysm rupture, while in rodents, EBI develops within 24 hours of hemorrhage induction. ²² This important distinction facilitates timepoint selection in preclinical animal studies. ²²

Blood-brain-barrier disruption

Intracellular signaling cascades in microglia drive the production of gene products implicated in EBI. The well-described NF-κB-mediated inflammatory pathway downstream of TLR-4 terminates with the transcription of several gene products including matrix metalloproteinases (MMPs), which are calcium-dependent, zinc-containing protein cleaving enzymes. MMP-2 and MMP-9 are members of the gelatinase sub-family of proteinases, and are expressed by microglia under certain conditions. ⁵⁹ Experimental SAH models using a 24-hour timepoint identified elevations in MMP-9 but not MMP-2 after experimental SAH, honing studies of ECM degradation and BBB disruption after SAH towards MMP-9. ⁶⁰ MMP-9 regulates pathological remodeling processes by degrading extracellular matrix components including zonula occludens-1 (ZO-1), collagen IV, fibronectin, and laminin.^61,62 During the EBI period, this compromises the connections that form endothelial tight junctions, disrupts the BBB, exacerbates peripheral intrusions into the CNS, and stimulates other pathways of secondary brain injury. These MMP-9-mediated processes are responsible for early BBB disruption in EBI, with later phases of BBB disruption occurring in response to apoptotic cascades. ⁶³ Therefore, inflammatory BBB disruption during EBI is a biphasic phenomenon. ⁴⁶ The expression of MMP-9 and related endothelial damage can be modulated through inflammatory pathways alongside the direct TLR-4 pathway. TLR-4 upregulates triggering receptor expressed on myeloid cells 1 (TREM-1), which participates in positive feedback to increase the activity of MMP-9. ⁶⁴ Another important observation is that TLR-4 expressed by astrocytes does not partake in post-SAH cerebral injury. ³⁴ Therefore, BBB disruption through these signaling pathways during EBI are primarily regulated by microglia. While microglia are typically polarized towards the more neurotoxic M1 state in the EBI period, MMP-9 is also produced by restorative M2 microglia to provide controlled degradation in healing and repair after injury.^14,65 Therefore, MMP-9 can serve an important role in restorative endothelial healing at later timepoints.

Cerebral edema

A by-product of BBB disruption by metalloproteinases and other mechanisms is the generation of vasogenic edema, one of two forms of edema present after SAH.^7,66 The accumulation of high-solute extracellular fluid from plasma protein leak into the brain secondary to a diminished ability of the BBB to exclude these molecules generates osmotic forces, increasing the overall volume of water within the brain.^67,68 Microglia can worsen vasogenic edema directly through their gene products. Vascular endothelial growth factor (VEGF) is upregulated following SAH in response to diminished baseline degradation of Hypoxia-inducible factor 1 (HIF-1). ⁶⁹ VEGF induces the formation of capillary fenestrations along the cerebral vasculature and microvasculature, which is an additional source of vasogenic edema that has been targeted to attenuate EBI in preclinical studies.^70,71 Importantly, VEGF is produced by microglia polarized towards both the M1 phenotype and M2 phenotype, indicating that VEGF can be produced at times where the microglial phenotype favors cytotoxic or restorative states.^72,73 Microglia also have a role in promoting cytotoxic edema after SAH, the second major form of edema present in EBI. Cellular swelling characteristic of cytotoxic edema occurs in part due to dysfunction of Aquaporin-4 (AQP4) in astrocytes, which are the glial cells with the highest expression level of this ubiquitous water channel protein. ⁷¹ Some studies have shown that partial antibody blockade of AQP4 improves cerebral edema while complete AQP4 deficiency worsens cerebral edema. ⁷⁴ Therefore, a clear mechanism of AQP4 promoting cerebral edema during the EBI period remains elusive. Though AQP4 is expressed at high levels by astrocytes, it has been shown that microglial activation is an important driver of AQP4 disorganization. ⁷¹ Inflammatory cytokines produced by M1-polarized microglia also produce cerebral edema by promoting neuronal and glial cell toxicity that terminates in cell death through apoptosis or necroptosis.^29,75 Because of the impact microglia have on cerebral edema in EBI and the association of cerebral edema with poor patient outcomes, targeting pathways of injury causing cerebral edema are promising. Recently, You et al. demonstrated that pharmacologic modulation of microglia to promote transition from the M1 to M2 phenotype attenuated cerebral edema in rats. ⁷⁶

Microglial inflammation

Neuroinflammation encompasses the broad molecular and cellular changes within the CNS in response to insults that are orchestrated by innate immune cells. ⁷¹ In the context of EBI, neuroinflammatory changes are diverse, but can be dichotomized into microglial inflammation and non-microglial inflammation. Both forms of inflammation contribute to the cytotoxic environment after SAH that promotes neuronal and glial death, oxidative stress damage, demyelination, and excitotoxicity.^77,78

Intrinsic immune factors are a key component of microglial responses. Immune regulatory elements within microglia such as the NLRP3 inflammasome, a complex of NLRP3, ASC, and caspase-1, regulate the expression of cytokines including IL-1β, IL-18, and Gasdermin-D. ⁷⁹ The importance of the NLRP3 inflammasome in microglial inflammation is demonstrated by the absence of activated microglial morphologies when NLRP3 is deleted in preclinical models. ⁸⁰ Cytokines produced by injured or activated cells in the CNS potentiate microglial activation and induce polarization of activated microglia towards cytotoxic phenotypes. ⁸¹ The cytokines that promote cytotoxic polarization include interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). Markers of M1-polarized microglia increase rapidly throughout the first day following SAH, and remain elevated through at least day 5 after experimental SAH induction, at which point the trend of cytotoxic M1 predominance reverses to favor the restorative M2 state due to the up-regulation of M2-promoting molecules including IL-4 and TGF-β. ¹⁴ Though this transition in microglial states has been observed experimentally, it remains unclear if individual microglia change their transcriptome throughout the EBI period and beyond, if new anti-inflammatory microglia are arriving to sites of brain injury, or if there are contributions of both.^11,52

Peripheral inflammation

Microglia are implicated in communication with immune cells residing outside of the CNS that promote additional secondary brain injury. ⁸² It has been suggested that mast cells play a pivotal role in early responses to aneurysm rupture by organizing the microglial response. ⁸³ After local mast cells recognize blood-derived toxins, they rapidly degranulate and secrete tryptase, which binds to the Protease-activated receptor 2 (PAR-2) receptor on microglia, activating microglia and promoting the secretion of TNF-α, IL-1β, and IL-6. ⁸³ Microglia then communicate with additional peripheral leukocytes. Chemokines produced by microglia recruit neutrophils and monocytes from the periphery that transmigrate through the compromised BBB and enter the CNS. ⁸³ Neutrophils specifically have garnered greater attention as effectors of neuroinflammation during the EBI period based on two critical pieces of evidence. First, neutrophils have been observed entering the CNS as early as 10 minutes after experimental SAH, and may partake in the development of secondary brain injury at an early time.^84,85 Second, neutrophils recruited by microglia can subsequently stimulate microglia to adopt a more inflammatory phenotype. As part of their response against extracellular pathogens, neutrophils utilize neutrophil extracellular traps (NETs), reticular chromatin structures containing cytosolic and granular proteins assembled on nuclear products, to trap and neutralize invading pathogens. ⁸⁶ In the brain, NETs aggravate microglial inflammation and produce an inflammatory environment by promoting the cytotoxic transition of microglia.^87,88 Cortical infiltration by blood monocytes and macrophages is not observed as early as infiltration by neutrophils, reducing the role these cells may have in activating microglia. ⁸⁹ Mouse chimera models using green fluorescent protein-tagged leukocytes identified minimal infiltration of peripheral cells at a 7-day timepoint, indicating that the inflammatory impact of peripheral cells is likely confined to the earlier stages of SAH, and microglia continue to mediate the sustained inflammation observed in later stages of SAH. ⁵¹

Oxidative cascades

Oxidative compounds that promote various forms of cellular damage are derived from multiple sources during the EBI period including mitochondria of cells undergoing autophagy or apoptosis, erythrocyte breakdown products, products of activated immune cells, or by-products of neurons struggling to cope with metabolic demands.^7,90 The most direct contribution of microglia to oxidative damage is through the production of reactive oxygen species (ROS) by myeloperoxidase, which is normally reserved for pathogen elimination.^91,92 When the erythrocyte scavenging capabilities of microglia become overwhelmed by subarachnoid blood, high molecular weight compounds that induce oxidative stress response are released from partially degraded blood, which are another source of ROS. ⁹³ The most harmful blood product is free heme, which is in a ferrous state that generates free radicals through the Fenton reaction. ⁹⁴ Heme oxidase 1 (HO-1) is a critical mediator of microglial degradation of pro-oxidant heme. ⁶³ Expression of HO-1 in microglia has cytoprotective properties after SAH by facilitating the clearance of heme-derived free radicals produced during erythrophagocytosis. ⁹⁵ In animal models of ischemic stroke, HO-1 is up-regulated by M2 microglia, suggesting a potential role for M2 microglia during the predominantly cytotoxic EBI period. ⁹⁶ A final source of oxidative damage to be considered is provided by infiltrating neutrophils that can produce cytotoxic pro-oxidant granules. ⁸⁵

Blood product scavenging

The role of microglia during the EBI period is predominantly pro-inflammatory, leading researchers to suggest that therapeutic polarization of microglia from an M1 state to an M2 state using targeted agents may yield clinical benefit in patients. ⁹⁷ The deleterious role of microglia is pronounced in the context of early BBB disruption, cerebral edema, neuroinflammation, and oxidative damage, but is accompanied by protective functions of microglia that occur simultaneously. After aneurysm rupture, harmful subarachnoid blood accumulates throughout the CNS and promotes brain injury. Mechanisms for rapid clearance of this accumulating blood are therefore necessary. The primary mechanism of hemoglobin clearance is via the binding of haptoglobin, which enables internalization of haptoglobin-hemoglobin complexes via the CD163 scavenger receptor, and also impedes the participation of hemoglobin in oxidative reactions. ⁹⁸ CD163 is primarily expressed by M2-polarized microglia in the CNS, which are the cells initially tasked with neuroprotection by transferring hemoglobin-haptoglobin complexes into endosomes via clathrin-coated vesicle endocytosis for breakdown into peptides, heme, and amino acids. ⁶³ Still, the microglial CD163 hemoglobin-haptoglobin system is rapidly saturated, and must be supplemented by peripheral monocyte-derived macrophages that arrive to the hematoma and facilitate further hemoglobin breakdown using analogous mechanisms. ⁹⁴ Monocyte-derived macrophages are overall more efficacious at performing hemoglobin breakdown. ¹¹ Aside from scavenging hemoglobin from the extracellular space, CD163 is involved in anti-inflammatory signaling that favors the M2 state in microglia. ⁹⁹ Ultimately, the role of microglia in blood scavenging indicates that generalization of the EBI period as one of exclusive M1 microglial activity overlooks the diversity of microglial function after SAH and the spatial or temporal evolution of microglia in response to context-dependent signaling. Further, microglial signaling in heme scavenging provides a potential mechanism to explain the broad shift in microglial polarization states from an inflammatory one in EBI to a more restorative one at later timepoints. ¹⁴ Anti-inflammatory signaling mediated by IL-10 occurs following the binding of haptoglobin to CD163 via phosphatidylinositol-3 kinase (PI3K) and Akt, promoting further M2 polarization in microglia. This can induce changes in the expression profiles in neighboring microglia and enables a global transition in microglial phenotypes throughout the injured brain.

Additional functions

Microglia have additional functions during the EBI period that are outside of the classical pathways of EBI. White matter injury via demyelination is caused by the interaction of oligodendrocytes with neuronal axons, but is initiated by signaling molecules partially derived from activated microglia. ¹⁰⁰ Related studies have identified increased microglial activation in white matter tracts using mouse models of SAH. ¹⁰¹ Activated microglia also serve as a source of excess glutamates, which leads to glutamate excitotoxicity and downstream cellular necrosis, apoptosis, and autophagy. ¹⁰² Other forms of cell death and apoptosis are prevalent in neurons and glia during the EBI period after microglia activation due to signaling mediated by IL-1β, TNF-α, and iNOS.^7,103

Microglia in delayed cerebral ischemia

The DCI period begins at the conclusion of the EBI period and lasts up to post-hemorrhage day 21, though manifestations of DCI are most prominent in humans from days 4 through 10. ¹¹ The fundamental disturbance in DCI is a mismatch between cerebral perfusion and metabolic demands. ¹¹ This mismatch in perfusion and metabolic demands is generated by mechanisms including sustained neuroinflammation, vasospasm, microthrombosis, autoregulatory dysfunction, and spreading depolarizations. ¹⁰⁴ Of these mechanisms, microglia are most implicated in sustained neuroinflammation, vasospasm, and spreading depolarizations after SAH. Early evidence has also implicated microglia in microthrombosis since thrombin induces intracellular Ca²⁺ release in microglia, activating microglia, and microvascular clots or microhemorrhage are known to result in rapid localization in the direction of injury.^105,106 Relatedly, recent findings implicating microglia in cerebral blood flow regulation through P2RY12 signaling suggests a potential role for microglia in autoregulatory dysfunction.^107,108 Unfortunately, the two DCI mechanisms of microthrombosis and cerebral blood flow regulation have not been thoroughly studied in the context of experimental SAH. Like EBI, DCI occurs at different timepoints in animals compared to humans. There is substantial heterogeneity present among experimental timepoints used to study DCI in rodents, but the majority of studies assess outcomes between 2 and 7 days, depending on the desired endpoint (i.e., microthrombosis vs vasospasm). ¹⁰⁹

During the DCI period, the direct injury induced by ICP elevations and accumulating blood by SAH begins to wane as hemorrhagic clot is cleared from the subarachnoid space. The dampening of inflammatory signals generated by this previously cytotoxic milieu allows microglia to change their phenotype to a more restorative form representative of the M2 polarization state. ¹¹⁰ Microglial effects in DCI, however, are not entirely restorative. Observations of decreased neuronal death as far as 9 days after experimental SAH in mice following microglia depletion indicates that pathways initiated by microglia during acute SAH, the EBI period, and in DCI can continue driving secondary brain injury and neuronal death in this period.^14,52 Furthermore, evidence of interactions between microglia and cerebral vascular structures to regulate energy metabolism provide a causal pathway for microglia to promote the mismatch between perfusion and metabolic demand. ¹¹¹

Microglial phenotypic evolution

Microglia adopt a broader spectrum of expression profiles and functionalities throughout the DCI period. In the EBI period, M2 microglial phenotypes are primarily observed in the CD163-mediated haptoglobin-hemoglobin pathway, where they reduce oxidative damage and cytotoxicity. Throughout the late EBI and early DCI period, there is a broad shift towards restorative phenotypes in microglia, allowing them to perform neuroprotective functions through the expression of gene products including HO-1 and Neuroglobin.^33,63 This broad phenotypic shift occurs in the setting of an evolving milieu for activated microglia. Initial inciting factors promoting microglial activation including ICP elevations, extravasation of blood products, and some forms of brain edema are diminished, leading to decreased inflammatory signaling that promotes the cytotoxic phenotype, which allows activated microglia to adopt a restorative phenotype in response to local CNS signaling. ¹¹² Indeed, in a mouse model of SAH, the loss of M1 microglial markers were accompanied with up-regulation of the anti-inflammatory cytokines IL-4 and TGF-β. ¹¹³ One possible mechanism of the microglial phenotypic shift is through altered microglial signaling described previously. Once saturated by hemoglobin, the microglial CD163 system becomes involved in anti-inflammatory signaling, favoring expression of IL-4, IL-10, and IL-13 by M2 microglia, which can promote other microglia to adopt restorative, anti-inflammatory phenotypes through local and regional signaling. ⁸⁹ The precise temporality of phenotypic polarity shifts in DCI has begun to be elucidated. Experimental work by Zheng et al. performed in transgenic mice using an endovascular perforation model identified that at days 1 and 3, microglia expressed high levels of the cell surface markers CD16 and CD32, suggestive of pro-inflammatory M1 microglia. ¹¹³ This was followed by high expression of CD206 on days 5 and 10, suggestive of anti-inflammatory M2 microglia. Interestingly, these authors also identified morphologies and surface expression profiles representing M1 and M2 microglia simultaneously, which were coined “bipolar microglia”. This highlights the spectrum of microglial phenotypes during secondary brain injury after SAH. At an aggregate level, microglial populations in mice increase the most from days 5 to 8 after SAH, and peak between days 9 and 15.^14,114 As the profiles of microglia increasingly favor the M2 state, they begin performing neuroprotective functions such as inactivating inflammation, promoting remyelination, repairing the BBB, and facilitating neural regeneration and neurite outgrowth. ¹¹⁵ The population expansion of microglia observed in preclinical animal models has parallels in humans. Human studies performed using CSF and peripheral blood samples from patients with SAH identified low numbers of microglia immediately after SAH, which progressively increased during the DCI period. ⁷³ However, human studies have not distinguished polarization phenotypes as have animal studies, so it remains uncertain to which degree the evolution of microglial polarity in rodent models reflects events in clinical SAH.

Cerebral vasospasm

Once viewed as the primary driver of patient morbidity from SAH-induced secondary brain injury, vasospasm is considered part of a myriad of mechanisms that contribute to poor patient prognosis. Following hemolysis of hemorrhagic blood in the subarachnoid space, hemoglobin and reactive oxygen species increase the expression of endothelin-1 and reduce levels of NO, which promotes endothelial dysfunction and vasospasm. ⁷³ Microglia are involved in the development of cerebral vasospasm in a multifaceted manner, with both direct signals that promote vasospasm and downstream effects from earlier timepoints. Findings by Hanafy indicate that microglia are both necessary and sufficient to cause vasospasm in a mouse model of SAH. ⁵² Microglia produce TNF downstream of TLR-4, which then promotes RAS-related C3 botulinum toxin substrate 1 (Rac1) expression, a gene product that promotes vasoconstriction in the DCI period. ⁵² Vasoconstriction by Rac1 is compounded by the secretory activity of recruited macrophages and neutrophils, which release high levels of TNF when exposed to free hemoglobin. ¹¹⁶ Another study found that inhibition of microglial NLRP3 reduced cerebral vasospasm in mice. ⁷⁹ Analogous to the downstream signaling of NF-κB during early neuroinflammation, neuronal death induced by cerebral vasospasm occurs in a MyD88-dependent manner earlier in DCI, but a TRIF-dependent manner in late DCI. ⁵²

Human studies support the involvement of both microglia and peripheral immune cells in cerebral vasospasm. Neutrophil accumulation in the CSF following SAH correlates with the development of vasospasm, with the amount of neutrophils present identified as an independent predictor for vasospasm. ⁸⁴ Infiltration of neutrophils is facilitated by a compromised BBB, which occurs downstream of microglial inflammation during the EBI period. More prominent microglial inflammation during the EBI period may thereby promote later vasospasm by promoting more substantial BBB damage and increasing the expression of cellular adhesion molecules. Aside from targeting microglia and recruited leukocytes during the DCI period, additional therapeutic avenues to mitigate vasospasm include exogenous administration of haptoglobin to facilitate scavenging activity of microglia and recruited macrophages. ¹¹

Cortical spreading depolarizations

Cortical spreading depolarizations (CSDs) are widespread depolarizations of all cell types in the brain that diffuse through the cortex and surrounding brain tissue in a contiguous fashion, and are associated with severe neural injury and poorer clinical outcomes. ¹¹⁷ In addition, CSDs exacerbate forms of brain injury encountered at earlier timepoints following SAH such as BBB disruption, cytokine release, and cerebral edema.^118,119 An interesting hypothesis regarding the impact of CSDs on microglia was put forth by Coulibaly et al. on the basis of findings from preclinical ischemic stroke models. ³³ Briefly, a spreading calcium wave was observed to occur alongside CSDs in microglia when utilizing this model. ³³ Calcium has a pivotal role in the activation of immune cells including microglia, and thereby may induce cytotoxicity and provide a linking pathway between CSDs and microglial activation. ¹²⁰ Additionally, other glial cells are primed to adopt phenotypes that enable additional inflammation by downstream effects of CSDs. For instance, astrocytes up-regulate TLR-3 following the occurrence of CSDs, allowing astrocytes to mediate additional inflammation. ¹²¹ The present data cumulatively suggests that CSDs can induce pro-inflammatory, cytotoxic phenotypes in microglia and surrounding glial cells. Further research in this area is necessary to reconcile the inflammatory effects of CSDs in a period that is broadly characterized by anti-inflammatory microglial activity. ³³

Microglia in long-term neurobehavioral and neurocognitive outcomes

Following the conclusion of inpatient monitoring for signs of DCI, patients with a favorable clinical prognosis may be reasonably considered as candidates for discharge, having evaded the majority of windows for clinical deterioration.^122,123 Unfortunately, the majority of patients that experience aneurysm rupture demonstrate impaired cognitive function in domains including language skills, memory, motor functioning, executive functioning, and mood that spans well beyond discharge. ³² The relatively young age of patients experiencing subarachnoid hemorrhage compounds burdens placed by these impairments, particularly in the setting of individuals previously contributing to the work force. At a cellular level, this phase is typified by increased neurogenesis and increased proliferation indices as the brain attempts to heal itself via intrinsic repair processes. ³³ There is a need to clarify causes of long-term deficits in patients after SAH.

Clinical evidence has associated factors including the presence of global cerebral edema during early admission with the development of long-term neurocognitive impairments. ³² Mechanisms contributing to cerebral edema in secondary brain injury are partly driven by microglia (Figure 2). ⁷ Further, research in neurodegenerative diseases has identified sustained microglial inflammation as a causal factor in the progression of these pathologies.^124,125 Microglia-related functions may also serve as drivers of long-term neurocognitive deficits in patients after SAH, which slow recovery from the earlier phases of injury. This can occur via sustained inflammation, synaptic impairment, or long-term effects of released hemorrhagic products. ²⁹

Figure 2.

Key microglial functions following SAH and corresponding patient clinical phenotypes. In the acute injury period, ICP elevations via NLRP3 induction and hemorrhagic blood products via TLR-4 activate microglia, leading to a morphological shift from ramified to ameboid. Patients experience symptoms of SAH such as a “thunderclap” headache, nausea/vomiting, and occasional loss of consciousness. During EBI, microglia exacerbate blood-brain-barrier disruption via MMP-9 and produce VEGFs that produce capillary fenestrations and exacerbate cerebral edema. Microglia also participate in signaling with peripheral immune cells, where mast cells activate microglia, and microglia subsequently produce signals to recruit neutrophils. Microglia produce pro-inflammatory cytokines including TNF-α, IL-1β, and IFN-γ in this period, producing a predominantly cytotoxic milieu. Restorative functions of microglia in this period include heme scavenging via CD-163. During EBI, patients classically demonstrate global cerebral edema on computed tomography. In the DCI period, inflammatory signaling, especially signaling driven by TNF-α, promotes vasospasm through Rac1 in microglia. Restorative functions including repair of the blood-brain-barrier and remyelination, as well as signaling through IL-4, IL-10, and IL-13 that promote M2 polarization. Patients commonly experience large vessel vasospasm in this period, which can be observed on digital subtraction angiography and sometimes requires intervention. During the chronic injury period, microglia are stimulated by cortical iron deposits and participate in synaptic engulfment. Patients with chronic injury from SAH frequently demonstrate neurocognitive impairments.

Chronic inflammation

Microglia shift from a predominantly M1 phenotype to a predominantly M2 phenotype over time following experimental SAH, reflecting diverse functional immunologic profiles across acute hemorrhage, EBI, and DCI. ¹¹³ After the experimental DCI period, microglia increase their expression of IL-6, TLR-4, and TNF-α from days 14–28.^52,126,127 Cortical neuroinflammation with accompanying sensorimotor damage is observed at least through day 21 in rats after SAH. ¹²⁷ This line of evidence contrasts with prior views that the development of secondary brain injury ceases in this period, and patients have “recovered” from secondary brain injury. ¹²⁸ Interestingly, some have even suggested that microglial inflammation is sustained for years after SAH. ³³ Unfortunately, there is a paucity of work in chronic microglial inflammation when compared to more acute phases of secondary brain injury.

Synaptic dysfunction

Another key observation in the development cognitive impairment is the association between synapse loss in patients and severity of cognitive impairment, which was first identified in neurodegenerative diseases, but is supported by experimental SAH studies. ¹²⁹ Shi et al. demonstrated that suppression of microgliosis-mediated phagocytosis of synapses improved neurobehavioral outcomes in mice following SAH induction. ¹³⁰ Subsequent transcriptomic profiling in this study demonstrated enrichment of synapse engulfment pathway-related genes in microglia. ¹³⁰ In another study, Chang et al. demonstrated that microglial transcripts after experimental SAH favored synapse remodeling, while blood monocyte-derived macrophages had increased levels of antigen presenting gene products. ¹³¹ Evidence also implicates blood proteins involved in microthrombosis during the DCI period with microglial activity at the synapse, providing a link from mechanisms of DCI to chronic neurocognitive dysfunction. Indeed, preclinical studies indicate that fibrinogen induces dendritic spine elimination through CD11b-CD18 microglia activation. ¹³² Astrocytes activated by microglia may also generate neurocognitive impairment due to their reduced capacities to detoxify glutamate from the synaptic cleft. ⁶³ Targeting microglial functions at the synapse is a promising therapeutic strategy, but should be investigated at later timepoints in animal models. ¹³³ Additional studies into synapse loss in patients after SAH are necessary to correlate promising preclinical findings to the long-term clinical phenotype.

Iron deposition

Microglia remain active beyond the DCI period and into the chronic injury period despite an apparent withdrawal of inciting factors from SAH, suggesting sustained aggravation of these cells. Following SAH, heme and free iron derived from hemoglobin are toxic drivers of secondary brain injury. ¹³⁴ Successful sequestration of hemoglobin, first by microglia and later by recruited macrophages, is predictive of favorable outcomes in preclinical models. ¹³⁵ However, survivors of SAH demonstrate persistent cortical iron up to 6 months after SAH. ¹³⁶ In these patients, concentrations of iron depositions form a gradient that diminishes with increased distance from the brain surface, and higher levels of iron depositions were found to be associated with poorer cognitive outcomes when observed using iron-sensitive imaging. ¹³⁶ Iron depositions may continue aggravating inflammatory pathways locally. ¹³¹ Histological studies performed on patients who perished from SAH found that the highest concentration of iron was located intracellularly in macrophages, followed by intracellularly in microglia. ¹³⁶ Lower levels of iron were found in other glial cells, neurons, and in extracellular compartments. This line of evidence provides support to the interesting theory of iron deposition promoting sustained microglial inflammation and cell death through ferroptosis, but is biased by availability of samples from patients with a poor outcome after SAH.

Therapeutic considerations

Insights into the role of microglia following SAH provide a myriad of therapeutic targets at defined times that may amplify the restorative function of microglia or attenuate their cytotoxic functions to mitigate the severity of secondary brain injury, resulting in improved patient outcomes (Figure 3). As a practical matter, targeting the earliest microglial activators such as ICP elevations or the initial binding of inflammatory agonists during the acute injury period is not a feasible treatment approach given the time to presentation for patients experiencing SAH. ¹³⁷ During the EBI period, numerous therapeutic targets present themselves. First, targeting mediators of neuroinflammatory signaling axes including NF-κB, MyD88, TRIF, TLRs, and JAK/STAT may mitigate the generation of inflammatory cytokines and reduce the adoption of cytotoxic phenotypes by activated microglia. ⁹⁷ Selective targeting of cytokines, whether through direct neutralization or competitive binding, may also carry similar promise. Preventing the activation of MMP-9 may reduce BBB disruption, yielding additional downstream benefits in the form of diminished cerebral edema and peripheral infiltration. Reducing secondary injury during the EBI period likely yields benefits throughout DCI considering the link between neuroinflammation during EBI and DCI. ¹³⁸ Inducing shifts towards restorative microglial phenotypes in the EBI period, using agents such as SIRT1 agonists, may attenuate deleterious pathways of neuroinflammation. ³⁰ Iron chelation and haptoglobin have also been investigated to assist with scavenging functions after SAH. ¹³⁹ While iron toxicity may also be involved in the long-term chronic forms of injury, it is unclear if this is a source of significant inflammatory signaling or just a reflection of the initial hemorrhage severity. Similarly, the long-term effect of microglia in synaptic dysfunction must be further investigated before therapeutic targeting is piloted. Ischemic preconditioning may provide a mechanism through which microglia can be targeted prior to aneurysmal SAH, though it remains unclear how this would translate to clinical practice. ¹⁴⁰

Figure 3.

Therapeutic targeting of microglia can occur at many points. Inhibition of the transcription regulator NF-κB directly (a) or upstream via MyD88 (b) or TRIF (c) can mitigate the transcription of inflammatory gene products. Inhibition of signaling through TLRs (d) or JAK/STAT (e) provide an upstream target to minimize inflammatory activity of microglia after SAH. Binding of inflammatory cytokines (f) could provide a similar anti-inflammatory effect. Inhibition of MMP-9 can reduce the Selective polarization of microglia to an M2 phenotype may minimize the inflammatory functions of microglia while amplifying their restorative activity (h). Scavenging or binding of deposited iron (i) may reduce sustained activation of microglia.

Unfortunately, the abundance of therapeutic targets involving microglia have not translated to clinical practice. To date, no specific microglial agents are utilized in patients after SAH. In fact, the only drug routinely used that reduces patient morbidity in the period of secondary brain injury is nimodipine, which occurs through a mechanism that remains unclear but is related to the propensity for nimodipine to cross the blood-brain-barrier and attenuate neuronal excitotocity. ¹⁴¹ Prospective trials of therapeutic agents in this area are the clear next step to minimize the health care burden of SAH, and should view microglia as a leading target.

Concluding remarks

In this review, we have proposed that varying temporal and spatial microglial responses to SAH regulate secondary brain injury, which ultimately result in the clinical phenotype in these patients. Sustained investigations into the role of microglia throughout this critical period may reveal therapeutic strategies that improve the prognosis of patients that experience SAH. While the future of microglial targeting after SAH is promising, ongoing efforts must address key limitations in this area. First, timepoint selection in preclinical models must be robustly validated. While the 24-hour timepoint in rodents is widely accepted to reflect human EBI, models for DCI and chronic injury are more heterogeneous. Second, microglia-specific mice are not widely available, and more suitable animals must be developed prior to human study. Finally, findings from aneurysmal SAH are not generalizable to SAH derived from different cerebrovascular lesions or trauma, and reveal an area requiring future attention. These limitations notwithstanding, augmenting the restorative functions of microglia or inhibiting their cytotoxic functions show promise in treating secondary brain injury after SAH considering their pivotal role in multiple phases of brain injury.

Supplemental Material

sj-pdf-1-jcb-10.1177_0271678X241237070 - Supplemental material for Role of microglia after subarachnoid hemorrhage

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X241237070 for Role of microglia after subarachnoid hemorrhage by David C Lauzier and Umeshkumar Athiraman in Journal of Cerebral Blood Flow & Metabolism