Brain injury and repair after Intracerebral hemorrhage: The role of microglia and brain-infiltrating macrophages

1. Introduction

Intracerebral hemorrhage (ICH), defined as spontaneous extravasation of blood into the brain parenchyma, is the second most common form of stroke, accounting for 10–20% of stroke cases worldwide [1, 2]. ICH is the deadliest subtype of stroke, with a very high mortality rate of approximately 40% at one month post the initial ictus. The mortality rate of ICH has not changed over the past two decades [3], despite recent advances in preclinical research. Also, only around 26% of ICH survivors are able to achieve functional independence at one year after symptom onset [4] further indicating the devastating nature of the disease. Currently, the worldwide incidence of ICH is 2 million cases per year [3], with approximately 120,000 cases per year in the United States [5–7]. Importantly, the worldwide incidence and hospital admissions of ICH have increased by ~47% over the last 20 years [1] and 18% in the past ten years [8], respectively. Also, the incidence is estimated to double by 2050 [9] due to increasing life expectancy, population aging and the widespread use of anticoagulants [10].

Based on the pathogenesis of the disease, ICH is classified as primary or secondary ICH. Primary ICH results from the spontaneous rupture of blood vessels at the bifurcation of small arteries mostly due to hypertension or amyloid angiopathy [2] whereas cerebral bleeding that occurs as a consequence of brain tumors, and ischemic stroke is regarded as secondary ICH. The risk factors of ICH include, but are not limited to, hypertension, amyloid angiopathy and advanced age. Of note, around 80% of ICH patients present with hypertension. Cerebral amyloid angiopathy is observed in approximately 20% of ICH patients and is found mostly in the elderly [1, 11]. Other risk factors are smoking, diabetes, alcohol abuse, drug abuse, ethnicity, genetic factors, decreased LDL cholesterol, and anticoagulant and thrombolytic agents [11].

ICH results in severe brain damage which is, in general, categorized into primary brain injury and secondary brain injury [12]. The primary injury is the direct insult to the brain due to the blood vessel rupture and the subsequent hematoma formation [2] and is mainly attributed to the mass effect of the hematoma. Primary injury often leads to the mechanical disruption of brain tissue surrounding the hematoma and subsequent elevation of intracranial pressure (ICP) resulting in brain herniation, based on the size and location of the hematoma [1]. The hematoma is not a static entity due to continued bleeding and in ~30% of ICH patients, significant hematoma expansion is observed which causes an exacerbation of ICP [13] and subsequent decline of neurological functions. The continued cerebral bleeding occurs in 14–20% of ICH patients and the brain regions that are highly vulnerable to hypertensive ICH include the basal ganglia, cerebellum, brainstem, and cortex.

Secondary brain injury (SBI) includes a cascade of cellular and molecular changes in the brain that are initiated by primary brain injury, the release of blood components from the hematoma, and innate immune response characterized by glial activation and infiltration of blood-borne immune cells into the brain [12]. The extravasated blood components such as hemoglobin, hemin, thrombin and iron (Fe) contribute to secondary brain damage [14–16]. Of note, secondary brain injury contributes to further destruction of brain tissue apart from the initial damage due to the cerebral bleeding, and can result in severe neurological deficits and sometimes delayed fatality [17, 18]. ICH patients often exhibit progressive deterioration suggesting a critical role of secondary damage in the pathophysiology of ICH [16, 19]. The key mechanisms of secondary brain injury include neuroinflammation [20], oxidative stress [21] and neurotoxicity induced by blood components [22], which often results in blood- brain barrier break down [23], development of cerebral edema [13], hematoma expansion [13] and severe neurological deterioration or death.

Currently, there is no effective treatment for ICH and the clinical management of ICH is largely restricted to supportive care and surgery for selected patients [4]. Of note, surgical intervention to remove the hematoma has not been shown to provide a significant increase in patient survival, or improve neurological recovery in surviving patients [19]. Therefore, it is pivotal to find other avenues that can improve the prognosis of ICH patients. Along these lines, delineating the molecular mechanisms of secondary brain injury, which is responsible for both acute as well as long term neurological outcomes after ICH is critical. Further, secondary brain injury persists for a prolonged period and hence, it could provide a wider window for therapeutic intervention after ICH. While blood components being the key molecular mediators of secondary brain damage, the major cellular contributor of secondary brain injury after ICH is microglia, the inflammatory cells of the CNS (central nervous system). This review focuses on the role of microglia and brain-infiltrating monocyte-derived macrophages in secondary brain injury and brain repair after ICH. Also, the review discusses the potential of targeting microglia and brain-infiltrating macrophages for therapeutic intervention after ICH. The clinical and preclinical aspects that we discuss throughout this manuscript are related to ICH that occurs in adults, but not in infants and neonatal ICH differs in epidemiology, pathophysiology and clinical management from that in adults. Hence, the concepts described in this article may not fit for ICH in infants.

2. Microglia/macrophage activation and ICH

In a non-injured adult brain, microglia is critical for maintaining brain homeostasis, remodeling synapses and constantly surveying the brain for damaged or dead neurons and foreign bodies [24]. Microglia are the resident immune cells of the CNS and the first-responders of brain injury. Neuroinflammation, characterized by microglial activation, infiltration of leukocytes and subsequent release of cytokines, chemokines and various neurotoxic molecules, is a key determinant of secondary brain injury after ICH [25].

Hemorrhagic injury to brain results in prominent microglial activation in preclinical models of ICH characterized by enlarged cell body with thick and short cell processes and increased expression of microglia or macrophage-specific molecular markers such as Iba1 or cd11b in comparison to non-activated or resting microglia observed in an un-injured or healthy adult brain [26]. Microglial activation was mostly observed in the brain region that encircles the hematoma, called the perihematomal brain region and upon hemorrhagic brain injury microglial/macrophage activation was observed at 1 hour and 4 hours post-ICH in the collagenase injection model [27] and the blood injection model of ICH [28], respectively. In the collagenase injection model, peak microglia/ macrophage activation was observed at day 3 and day 5 post-ICH and there was an 8-fold increase in the number of activated microglia or macrophages on day 3 and day 5 post-ICH in comparison to experimental control [4]. The increase in the number of microglia or macrophages after ICH could be due to the injury-induced proliferation of microglia or migration of microglia to the injury site or brain infiltration of blood-borne macrophages (macrophages). Macrophages have been found to migrate into the injury site within 12 hours in a preclinical model of ICH [29]. Also, activated microglia/macrophages in different brain regions have different morphologic characteristics, indicating different functions [30]. Microglial/macrophage activation persists for 4 weeks in preclinical models. Notably, microglial activation also occurs after ICH in human patients, with brain invasion of leukocytes (neutrophils, macrophages) [31]. Activated microglia and macrophages share several immune cell markers in common [32]. Therefore, it is difficult to differentiate activated microglia from macrophages by commonly used techniques like immunohistochemistry and hence, they are often grouped as microglia/macrophages when discussing their functional roles following a brain injury [33].

Although the precise functional role of activated microglia is yet to be defined, activated microglia are regarded as the critical cellular regulators of neuroinflammation after ICH based on their ability to release cytokines, chemokines, prostaglandins, proteases, ferrous iron, glutamate, and reactive oxygen species [34–36]. The cytokines, in turn, cause further activation of microglia, resulting in a self-propagating inflammatory cascade leading to neuronal dysfunction and cell death [37, 38]. Furthermore, activated microglia also modulate neuroinflammation by recruiting and activating blood-derived macrophages [39–42]. And several preclinical studies focused on assessing the microglia or macrophage activation in the ipsilateral brain region as a measure of neuroinflammatory response post-ICH. Post-injury administration of a tripeptide MIF (macrophage/microglial inhibitory factor, tuftsin fragment 1–3, Thr-Lys-Pro) in a collagenaseinjection mouse model of ICH resulted in decreased microglial/macrophage activation, hemorrhagic lesion size, neurodegeneration, cerebral edema, and acute neurological deficits post-ICH [43]. Further, tuftsin attenuated thrombin-induced release of IL-1-β and TNF-α from rat microglia and intracerebral infusion of tuftsin attenuated cerebral edema in a blood-injection model of ICH [44]. Also, atorvastatin-mediated attenuation of cerebral edema and neuronal death on day 3 post-ICH in a pre-clinical model was associated with the reduction in microglia or macrophage activation [45]. Furthermore, minocycline, a tetracycline derivative and an inhibitor of microglia/macrophage activation attenuated cerebral edema and improved neurological outcomes after ICH [46]. Apart from cytokines, chemokines are also critical regulators of neuroinflammation and CXCR4 (C-X-C chemokine receptor type 4) antagonist, CX807-mediated neuroprotection in a preclinical model of ICH was associated with a profound reduction in microglia/macrophage activation [47]. Altogether, microglial/macrophage activation correlates with blood brain-barrier damage, brain swelling/edema, hematoma expansion, peri-hematomal cell death, neurological deterioration, and poor functional recovery after ICH [45, 48–55] and reduction in microglia/macrophage activation in the acute phase of ICH is associated with improved functional recovery in preclinical models.

Microglia/macrophages are activated by various inflammatory stimuli following ICH that include blood components such as thrombin, hemoglobin, heme, iron and plasma proteins such as fibrinogen [4]. Necrotic neurons release many cellular contents, such as ATP, neurotransmitters, heat shock proteins and high-mobility group protein B1 (HMGB1), that also activate microglia following ICH [33]. The inflammatory stimuli hemin (a hemoglobin degradation product that accumulates in the brain after hemorrhage) and fibrinogen act through Toll-like receptors that are found predominantly on the surface of microglia and macrophages [56–59] and potentiate microglia and macrophage activation [57]. Exogenous hemin treatment increased the expression of TLR4, and proinflammatory cytokines, such as TNF-α, IL-1β and IL-6 in cultured microglia [57]. Consistently, TLR4 expression is found to be up-regulated after ICH [60] and contributes to an inflammatory response through the nuclear factor-κB (NFκB) signaling pathway [33]. TLR4 knockout animals exhibited decreased microglial activation, brain infiltration of neutrophils and monocytes, and improved functional recovery at 3 days post-ICH [60]. Furthermore, the administration of a TLR4 antagonist and anti-TLR4 antibody reduced brain water content, neurological deficit scores, proinflammatory cytokine levels, and brain infiltration of monocytes/macrophages after ICH [57, 61] suggesting a major role of TLR-4 in acute neuroinflammation following ICH. Also, recent clinical studies have shown that increased expression of TLR4 in monocytes is associated with poor functional outcome and enhanced residual lesion volume in ICH patients [62]. Apart from TLR-4, recent studies document NLRP3 inflammasome as a potent regulator of neuroinflammation, and proinflammatory cytokines secretion after ICH [63, 64]. TLR4-NFkB signaling can cause augmented expression of NLRP3 and pro-IL-1 β [64]. Activation of NLRP3 by inflammatory stimuli results in the oligomerization of NLRP3 with an adaptor protein ASC and the inactive form of the enzyme caspase-1 leading to caspase-1 activation and subsequent generation of biologically active proinflammatory cytokines, IL-1β and IL-18 from its precursors. Notably, expression of NLRP3, caspase-1, and IL-1β is up regulated in microglia after collagenase-induced ICH and heme activates NLRP3 inflammasome [63]. In addition, recent studies report increased levels of complement 3 (C3) in the ipsilateral brain area in a preclinical model of ICH and genetic ablation of C3 attenuated ICH-induced IL-1β production suggesting a role of NLRP3 signaling pathway in complement-induced neuroinflammation after ICH [65]. Altogether, NLRP3 activation and NFκB upregulation play a critical role in ICH-induced inflammatory response. In addition, the inhibition of microglia/macrophage inflammatory pathways in preclinical models of ICH has resulted in a reduction in the brain infiltration of leukocytes (neutrophils, macrophages) [61, 66] implicating that microglia recruits other immune cell types to the injury site. To this end, microglia release CXCL2 a chemokine that attracts neutrophils. It has been shown that neutrophils also contribute to inflammation following ICH and its brain recruitment exacerbated the secondary brain injury [33]. Neutrophils may damage the brain by producing reactive oxygen species, releasing pro-inflammatory proteases and thereby modulating blood–brain-barrier permeability[67].

ICH results in profound neuronal death in the peri-hematomal and hematomal brain region contributing to patient morbidity and mortality. Consistent with the role of microglial/macrophage activation in inducing cell death [68–70], neuronal death in the ipsilateral brain region after ICH spatially and temporarily correlated with microglia/macrophage activation [71]. Types of cell death observed after ICH are necrosis, apoptosis, pyroptosis, and autophagy [72–76]. NFκB, a transcription factor is a key regulator of proinflammatory cytokines, such as TNF-α and IL-1-β in various pathological conditions including ICH [57] that can cause direct or indirect neurotoxicity after a brain injury. NFκB transcriptional activity correlated with perilesional cell death after ICH in rats [48] and IL-1β and TNF-α antagonists attenuated apoptotic cell death in experimental ICH [77]. Also, activation of NFκB is positively related to the progression of apoptotic cell death in patients with ICH [78]. In addition, NLRP3-induced pyroptosis, documented in experimental models of ICH, may play a significant role in brain damage after ICH [79] because apart from resulting in brain cell loss, pyroptosis is associated with the release of proinflammatory mediators that can further damage the brain. Given the role of cell death in neurological deficits after ICH, further studies are warranted determining the occurrence of pyroptosis in human ICH and delineating further the precise molecular mechanisms of inflammation-induced cell death in preclinical models.

3. Conclusions and future directions

Overall, microglial/macrophage activation plays a detrimental role in the acute phase of ICH. However, preclinical studies have mostly employed male young subjects and hence further studies are warranted testing the efficacy of inhibiting microglia/macrophage activation in female subjects. Moreover, preclinical studies involving aged animals are also required, given the prevalence of ICH in the elderly population. To date, a few studies have documented altered microglial/ macrophage activation in aged animals after ICH implicating the need to further explore the therapeutic efficacy of targeting microglial or macrophage activation in improving neurological outcomes. Also, the development of novel transgenic mouse models using Cre-lox technology could help in functionally differentiating microglia from macrophages and that may result in the identification of microglia or macrophage- specific molecular targets for therapeutic intervention after ICH. In addition, how acute inhibition of microglia/macrophage activation modulates long- term neurobehavioral outcomes after ICH requires investigation. Further, activated microglia and macrophage are less susceptible to blood-induced toxicity in comparison to neurons making them an efficient target for therapeutic intervention after ICH warranting further studies.