Role of N-formyl peptide receptor 2 in germinal matrix hemorrhage: an intrinsic review of a hematoma resolving pathway

Abstract

Germinal matrix hemorrhage is one of the leading causes of morbidity, mortality, and acquired infantile hydrocephalus in preterm infants in the United States, with little progress made in its clinical management. Blood clots have been shown to elicit secondary brain injury after germinal matrix hemorrhage, by disrupting normal cerebrospinal fluid circulation and absorption after germinal matrix hemorrhage causing post-hemorrhagic hydrocephalus development. Current evidence suggests that rapid hematoma resolution is necessary to improve neurological outcomes after hemorrhagic stroke. Various articles have demonstrated the beneficial effects of stimulating the polarization of microglia cells into the M2 phenotype, as it has been suggested that they play an essential role in the rapid phagocytosis of the blood clot after hemorrhagic models of stroke. N-formyl peptide receptor 2 (FPR2), a G-protein-coupled receptor, has been shown to be neuroprotective after stroke. FPR2 activation has been associated with the upregulation of phagocytic macrophage clearance, yet its mechanism has not been fully explored. Recent literature suggests that FPR2 may play a role in the stimulation of scavenger receptor CD36. Scavenger receptor CD36 plays a vital role in microglia phagocytic blood clot clearance after germinal matrix hemorrhage. FPR2 has been shown to phosphorylate extracellular-signal-regulated kinase 1/2 (ERK1/2), which then promotes the transcription of the dual-specificity protein phosphatase 1 (DUSP1) gene. In this review, we present an intrinsic outline of the main components involved in FPR2 stimulation and hematoma resolution after germinal matrix hemorrhage.

Introduction

Germinal matrix hemorrhage (GMH) occurs in approximately 3.5 per 1,000 live births per year and is the leading cause of mortality and morbidity in premature/low-weight infants in the United States (Heron et al., 2010; Koschnitzky et al., 2018). The germinal matrix is a highly vascularized region that is the site of neuronal and glial proliferation, which is extremely important in the development of the neonatal central nervous system (CNS) (Segado-Arenas et al., 2018). Due to the fragility of these blood vessels within this structure, changes in hemodynamic and cardiorespiratory instability cause these vessels to spontaneously rupture, leading to GMH (Ballabh, 2010, 2014). It has been reported that 30–75% of patients that survive GMH develop long-term neurocognitive consequences, such as cerebral palsy, learning impairments, and post-hemorrhagic hydrocephalus (Sherlock et al., 2005; Luu et al., 2009; Ramagiri et al., 2022). Additionally, 42% of preterm patients with GMH develop cerebral palsy, 15.6% of children have a visual deficiency, and 7.8% have a hearing impairment (Gilard et al., 2018; Cizmeci et al., 2020). It should be noted that although males were met with more severe grades of intraventricular hemorrhage compared to female patients, there were no statistically significant gender disparities in the severity of the hemorrhage and neurodevelopmental outcomes (Matijevic et al., 2019). Clinically there are no therapeutics that target the acute stage of GMH. The blood clot after GMH has been identified as the causative factor leading to debilitating consequences such as post-hemorrhagic hydrocephalus, periventricular leukomalacia, brain atrophy, and cerebral palsy (Ballabh, 2010, 2014; Sadegh et al., 2023). Previous preclinical studies in GMH have shown the beneficial effects of the quick resolution of the hematoma through endogenous microglial phagocytic pathways, which resulted in improved neurological outcomes (Flores et al., 2016; Liu et al., 2021). Therefore, a safe and non-invasive therapeutic that directly targets the hematoma after GMH is necessary. The hematoma-resolving actions of FPR2 stimulation after GMH and a complete signaling pathway have been recently elucidated (Flores et al., 2023). In this review article, we further look into the multiple endogenous components involved after the stimulation of FPR2. A deeper understanding of FPR2 may provide more evidence for its potential use as a therapeutic target for treating GMH in the early stages.

Retrieval Strategy

A computer-based online search of the PubMed database was performed to retrieve articles published up to May 1, 2023. A combination of the following text words (MeSH terms) was used to maximize search specificity and sensitivity: “germinal matrix hemorrhage”; “microglia polarization”; “hematoma resolution”; “M1 Microglia”; “M2 microglia”; “phagocytosis”; “post-hemorrhagic hydrocephalus”; “n-formyl peptide receptors”; “cerebrospinal fluid”, “peroxisome proliferator-activated receptor gamma”, “cluster of differentiation”, “blood clot”, “stroke” and “hemorrhage.” Search results were screened by the title and abstract, and only studies exploring the relationship between hematoma resolution and germinal matrix hemorrhage were included to investigate the effects of FPR2 on resolving the hematoma after hemorrhage. No language or study type restrictions were applied. Phagocytic mechanisms in other cell types outside of microglia/macrophages were excluded.

Germinal Matrix Hemorrhage Pathophysiology

GMH is defined as the rupture of immature blood vessels within the subependymal (or periventricular) germinal matrix, where the debilitating consequences include the formation of post-hemorrhagic hydrocephalus (Ballabh, 2010; Heron et al., 2010). It has been suggested that germinal matrix/intraventricular hemorrhage occurs due to hemodynamic and cardiorespiratory instability in premature/low-birth-weight infants, causing abrupt fluctuations in cerebral blood flow in the fragile germinal matrix and resulting in spontaneous bleeding (Ballabh, 2014). The blood after hemorrhage invades the ventricles, allowing the blood to mix with the cerebrospinal fluid (CSF) and circulate towards the subarachnoid space. It is suggested that once the blood invades the ventricles, a blood clot is formed, obstructing the cerebral aqueduct or formania of Luschka and Magendi or by microthrombi obstructing small CSF outflow passages in the subarachnoid space (Strahle et al., 2012). The rupture of the blood vessels after GMH causes hematoma formation, leading to erythrocyte lysis releasing hemoglobin, which is further broken down into heme and Iron (Figure 1). The release of iron into the surrounding tissue from metabolized hemoglobin consequently causes iron overload which has been associated with CSF overproduction and PHH development (Klebe et al., 2017). Preclinical models of GMH-IVH have demonstrated that hemoglobin-metabolized products were found in the CSF and were key contributors to the development of ventriculomegaly (Strahle et al., 2012; Klebe et al., 2017).

Figure 1.

Systemic overview of the GMH.

The rupture of blood vessels in the germinal matrix space leads to the formation of hematoma and the release of thrombin into the CNS. After hemorrhage, the blood clot undergoes erythrocyte lysis releasing hemoglobin, which is metabolized into heme and iron. The by-products of the blood clot after erythrocyte lysis contribute to an increase in CSF production, inflammation, cytotoxicity, oxidative stress, and a decrease in CSF circulation leading to long-term neurological consequences. Created using Microsoft Power Point version 2304. CSF: Cerebrospinal fluid; GMH: germinal matrix hemorrhage.

Models for the Induction of Germinal Matrix Hemorrhage

To understand the complications associated with GMH and to explore novel therapeutic options, the following models have been utilized: Dogs (Goddard et al., 1980), sheep (Reynolds et al., 1979), lambs (Wheeler et al., 1979), rats (Lekic et al., 2011; Ramagiri et al., 2022), mice (Segado-Arenas et al., 2018), rabbits (Dohare et al., 2016), or pigs (Mayfrank et al., 1997). The most commonly used animal is rodents (mice and rats) due to the feasibility, greater understanding of rodent neurodevelopment, and behavioral consequences, which allow for better comparisons with humans (Bockhorst et al., 2008; Semple et al., 2013; Tartaglione et al., 2016). It is well known that Day 6 rats (P6) are equivalent to 35 weeks of gestation in humans, and P0 mice are similar to 22–24 gestational weeks in humans (Clancy et al., 2007; Hagberg et al., 2002). Furthermore, the most commonly used animal models of GMH are lesion-induced models such as intraventricular administration of autologous blood or collagenase in rodents and intraperitoneal administration of glycerol in rabbits (Fischer et al., 1986; Lekic et al., 2011; Tao et al., 2016). The advantage of using lesion-induced models is that the hemorrhage’s time and location are controlled. The disadvantages of these models are that they do not mimic a spontaneous rupture of blood vessels within the germinal matrix. However, models which mimic a spontaneous rupture of blood vessels are limited in utility as the spontaneous development of GMH is extremely low and unreliable (Lorenzo et al., 1982).

Glycerol administration causes an increase in osmolarity and intravascular dehydration, which leads to a decrease in intracranial pressure leading to the rupture of blood vessels within the germinal matrix (Georgiadis et al., 2008; Ballabh, 2014). The advantage of intraventricular administration of glycerol is the observation of a large amount of extracellular hemoglobin found in the periventricular white matter, which is also exhibited in patients (Ley et al., 2016). Disadvantages of glycerol-induced GMH are the impact the toxic effect of glycerol on organs and that glycerol can produce bleeds outside of the germinal matrix, such as subarachnoid, subdural, deep white substance or cortical basal ganglia hemorrhage (Lekic et al., 2015).

Autologous blood administration uses maternal blood or blood from other neonates to be injected at the site of the germinal matrix (Aquilina et al., 2011; Lee et al., 2018). The advantages of this model are that it does not rely on exogenous proteins and mimics the natural coagulation and inflammatory pathways associated with the exposure of hemoglobin to the surrounding tissue (Sansing et al., 2011; Krafft et al., 2012). The negative drawback of this model is that it does not mimic the rupture of the blood vessels in the germinal matrix (Dawes et al., 2016).

Collagenase-induced GMH uses the administration of bacterial collagenase, which contains protease proteins that lyses the extracellular matrix causing the rupture of the fragile blood vessels in the germinal matrix (Krafft et al., 2012). The advantages of this model are that it produces consistent hemorrhages that mimic grade 3/4, consistent ventricular dilatation, and behavioral deficits associated with severe hemorrhages (Lekic et al., 2011). Disadvantages of this model involve the possible exaggerated inflammatory response due to the collagenase and may induce cerebral ischemic injury, which has not yet been evaluated in neonates only in adult models using collagenase (MacLellan et al., 2008; Krafft et al., 2012).

Microglia Role after Germinal Matrix Hemorrhage

Resident macrophages and peripheral microglia play a pivotal role in CNS development and homeostasis in the neonatal brain (Pierre et al., 2017). Microglia have been shown to have various functions based on environmental stimuli and can conform to two distinct phenotypes: activated pro-inflammatory phenotype (M1), which has been associated with neurological injury, or alternatively-activated anti-inflammatory phenotype (M2), which has been associated with wound healing (Kanazawa et al., 2017; Pierre et al., 2017). It was thought that M1 microglia cells become terminal and die, yet evidence suggests that M1 microglia can undergo phenotype switching from M1 to M2 (Hashimoto et al., 2013; Yona et al., 2013; Orihuela et al., 2016). The invading hematoma causes mechanical pressure on glia and neuronal cells, resulting in apoptosis and cytotoxicity, which invokes the activation of the inflammatory response (Strahle et al., 2012; Abrantes De Lacerda Almeida et al., 2019). Activated microglia have been shown to recruit hematogenous phagocytes to the site of injury, which engulf the blood clot and damage surrounding tissues (Alshareef et al., 2022). M1, microglia GMH, has been shown to contribute to secondary brain injury through the mediation of pro-inflammatory cytokines. The inhibition of M1 microglia after GMH leads to improved neurological function and inflammation inhibition (Xiao et al., 2021). M1 phenotype surface markers commonly investigated are CD14, CD16, CD32, CD40, CD86, and MHCII (Jurga et al., 2020).

At 24 hours, M2 markers were found at the ischemic core, suggesting that microglia cells are alternatively activated and promote tissue repair (Perego et al., 2011). Common surface markers to investigate M2 phenotype are CD163 and CD206 (Liu et al., 2021; Flores et al., 2023). Notably, CD163, a hemoglobin scavenger receptor, is a macrophage-specific protein and represents macrophage switching of the M1 microglia cells into the M2 phenotype and is used to identify macrophages from the periphery (Porcheray et al., 2005; Daftarian et al., 2020). It was recently demonstrated that CD163 protein expression was significantly decreased after GMH, yet its expression was significantly increased at 72 hours and remained elevated at seven days post-ictus (Liu et al., 2021). This data suggests that peripheral monocytes play a role in the polarization of microglia/macrophages in the CNS. However, microglia M2 polarization post-GMH has been understudied, but our research group has shown that this phenotype plays a significant role in phagocytosis in GMH (Flores et al., 2016; Xiao et al., 2021; Xu et al., 2022). M2 microglia can be categorized into three subtypes: M2a, M2b, and M2c. Both M2b and M2c have been shown to play a role in phagocytosis and the removal of tissue debris, while M2a plays a role in cell regeneration (Roszer, 2015). Recent studies in GMH have shown therapeutics that shift microglia/macrophages into the M2 phenotype, enhancing phagocytic blood clot clearance, attenuating short and long-term neurological deficits, and reducing PHH (Xiao et al., 2021; Alshareef et al., 2022; Xu et al., 2022; Flores et al., 2023). Furthermore, M2 microglia play a prominent role in regulating iron as the cell type can contain large intracellular labile iron pools, which effectively take up and spontaneously release iron at low concentrations away from the site of injury to avoid iron overload (Flores et al., 2023) which plays a significant role in CSF production and hydrocephalus formation (Corna et al., 2010). Therefore, we must harness novel mechanisms that upregulate M2 microglia cells for the quick removal of the hematoma.

N-Formyl Peptide Receptor 2 and Its Actions on CD36 after Germinal Matrix Hemorrhage

N-formyl peptide receptors (FPR) 1, 2, and 3 belong to a family of G-protein-coupled receptors that are expressed on microglia, endothelial cells, microglia/macrophages, and astrocytes in the central nervous system. FPR1 plays a significant role in neutrophil oxidative burst and activating the innate immune system to promote inflammatory pathways. FPR3 participates in the allergic reaction process and dendritic cell maturation (Senchenkova et al., 2019). Stimulation of the n-formyl peptide receptor 2 (FPR2) has been shown to be neuroprotective in models of stroke (Ding et al., 2020; Flores et al., 2023). FPR2 agonism has been shown to modulate the innate immune response to resolve injury through the increase in macrophage phagocytosis (Senchenkova et al., 2019). Additionally, FPR2 activation has also been found to polarize microglia into the M2 phenotype (Yuan et al., 2022), which is primarily responsible for mediating wound healing and enhancing macrophage phagocytosis of blood clots in GMH (Flores et al., 2023). These findings suggest that FPR2 may play a significant role in immunomodulation that provides protection against multiple maladies. FPR2 has only been recently studied in the CNS and was shown to be neuroprotective through anti-inflammatory mechanisms in adult stroke models (Vital et al., 2016; Ding et al., 2020). Most of the investigated mechanisms of FPR2 have focused on the inhibition of prothrombotic activity and MAPK/P38 signaling pathway after stroke (Vital et al., 2016; Senchenkova et al., 2019; Ding et al., 2020). A recent study indicated that the upregulation of FPR2 leads to improved neurobehavior and the increased expression of scavenger receptor CD36 after GMH (Flores et al., 2023).

Currently, many of the studies conducted on phagocytic hematoma clearance are based on adult hemorrhagic stroke models. Previous work in intracerebral hemorrhage (ICH) has shown that scavenger receptor CD36 plays a significant role in the phagocytosis of blood clots, and its upregulation was shown to be neuroprotective (Li et al., 2021). Scavenger receptor CD36, a trans-membrane glycoprotein, is located on the cell surface of several cell types, including monocytes, astrocytes, endothelial cells, and microglia. CD36 modulation has been connected to various pathways such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and nuclear factor-erythroid 2 p45-related factor. Such pathways have been shown to mediate microglia/macrophage polarization to the M2 phenotype, which is responsible for microglia phagocytic actions (Liu et al., 2022a, b). PPAR-γ, a nuclear hormone receptor, stimulation has been reported to directly induce the expression of CD36 through the increase of CD36 transcription (Zhao et al., 2007). However, M2’s role in germinal matrix hemorrhage has been greatly understudied; further characterization of microglia subtypes and function needs to be conducted in the neonatal CNS. It was previously demonstrated that PPAR-γ receptor activation upregulated CD36, which increased M2-positive cells and enhanced hematoma resolution. Recently, the link between FPR2 and CD36 was established, as FPR2 agonism leads to an increase in CD36 protein expression (Flores et al., 2023).

Annexin A1 and Its Actions on FPR2 after Germinal Matrix Hemorrhage

Annexin A1, a glucocorticoid-regulated protein, belongs to a family of Ca²⁺ -dependent phospholipid-binding proteins, and it has been found that endogenous AnxA1 is commonly expressed on glial cells in the CNS of adult human and rodent brains, more specifically microglia/macrophages (McArthur et al., 2010). FPR2 agonist Annexin A1 has been shown to have therapeutic effects in preclinical stroke models. Previous studies have found that endogenous AnxA1 increased in conjugation with an influx of neutrophils at the site of injury. Specifically, AnxA1 is released from neutrophil cytosolic granules to the cell surface, interacting with FPRs in an autocrine/paracrine fashion (Gavins, 2010). Various publications have demonstrated the protective anti-inflammatory characteristics of AnxA1 treatment in adult stroke models (Thygesen et al., 2019; Ding et al., 2020). Furthermore, AnxA1 has been attributed to play a key role in the microglia phagocytosis of various molecules (Purvis et al., 2019). For these very reasons, AnxA1 was used to assess the neuroprotective effects of FPR2 on hematoma resolution. It was recently elucidated that AnxA1 significantly increased hematoma resolution and improved neurobehavior. Additionally, the enhancement of hematoma resolution led to improved long-term neurobehavior and reduced the development of post-hemorrhagic hydrocephalus in AnxA1-treated animals. Because AnxA1 stimulation of FPR2 may act on various other cell types, global inhibition microglia cells with liposomal clodronate was used to determine if the actions of FPR2 on hematoma resolutions were elicited by microglia cells. Here it was demonstrated that AnxA1-treated animal groups did not significantly reduce hematoma volume in the presence of FPR2 KO CRISPR, suggesting that AnxA1 hematoma resolving characteristics were mediated through microglia cells (Flores et al., 2023). Thus, it is clinically relevant to use AnxA1 as a treatment to upregulate the neuroprotective effects of FPR2, as we would be targeting an endogenous mechanism.

FPR2 Phagocytic Signaling Pathway: ERK1/2/DUSP1/CD36

FPR2 stimulation has been associated with the phosphorylation of extracellular-signal-regulated kinase 1/2 (ERK1/2) (Ansari et al., 2021). Extracellular signal-regulation kinase 1/2, members of the mitogen-activated protein kinase family, have been noted as playing a pivotal role in many pathophysiologies such as stroke, Alzheimer’s disease, and traumatic brain injury (Sun and Nan, 2017; Khezri et al., 2023). Various experimental articles have identified ERK1/2 as a potent effector of neuronal death and neuroinflammation (Sun and Nan, 2017). Additionally, the upregulation of ERK1/2 has been shown to activate and polarize microglia into the M1 phenotype, whereas its inhibition leads to the phenotypic shift of microglia from M1 to M2 (Zhao et al., 2020). In GMH, ERK1/2 inactivation through its phosphorylation was demonstrated to be beneficial in the polarization of Microglia from M1 to M2 (Flores et al., 2023). The phosphorylation of ERK1/2 has also been demonstrated to promote the activation of dual-specificity protein phosphatase 1 (DUSP1) and its gene transcription (Finelli et al., 2013). DUSP1, a member of the threonine-tyrosine dual-specificity phosphatase family, is neuroprotective in neurological disorders (Taylor et al., 2013; Zhang et al., 2022). Its activation resulted in beneficial outcomes after stroke (Zhang et al., 2022; Flores et al., 2023). Furthermore, DUSP1 has been attributed to promoting Microglia polarization towards the M2 phenotype (Wang et al., 2021). It was reported that there was a significant increase in DUSP1 protein expression in FPR2 agonist-treated groups after GMH, signifying that DUSP1 plays a significant role in the FPR2 signaling pathway and in the GMH pathophysiology (Flores et al., 2023). Current literature suggests that DUSP1 may act on the CD36 receptor, thereby playing a role in the upregulation of hematoma clot clearance (Cattaneo et al., 2013). This manuscript was the first to link the connection between DUSP1 and CD36, which played an important role in FPR2-mediated hematoma resolution after GMH. More notably, FPR2 knockdown and inhibition, through the use of FPR2 CRISPR and FPR2 inhibitor, significantly decreased ERK1/2, DUSP1, and CD36 protein expression and attenuated FPR2 induced hematoma resolution at 72 hours after GMH (Flores et al., 2023).

The Potential of FPR2 Used as a Therapeutic Target

Currently, the only treatment option for GMH targets post-hemorrhagic hydrocephalus, where the clinical management relies on inserting shunts that drain excess CSF from the ventricles into the peritoneum. However, this procedure can lead to postsurgical complications, which include infection, shunt obstruction, seizures, over/under drainage, and shunt replacement, causing a socioeconomic burden (Woernle et al., 2013). Thus far, no clinical trials target the blood clot after GMH, yet clinical trials in adults with hemorrhagic stroke give us insight into the potential approaches of directly removing the blood clot. So far, clinical trials in adult hemorrhagic stroke showed no beneficial effect in the direct removal of the blood clot (Hanley et al., 2019). As a result, combinational treatments involving direct removal and delivery of therapeutics, such as anti-inflammatory agents targeting secondary brain injury, have been executed in adult hemorrhagic stroke trials. This suggests that an endogenous mechanism that targets the removal of the blood clot and promotes wound healing may be more beneficial than solely removing the blood clot (Sembill et al., 2018). The recent study on FPR2 elucidated a novel endogenous mechanism that directly resolves the primary causative factor of secondary brain injury after GMH. Here we proposed a complete mechanism of action of FPR2 stimulation after GMH. FPR2 stimulation resulted in the quick removal of the blood clot after GMH, improving behavior and brain morphology outcomes. Additionally, FPR2 agonism increased M2 microglia cells suggesting that the actions of FPR2 on hematoma resolution are mediated through microglia phagocytosis. To further confirm that FPR2 acts on microglia, the global inhibition of microglia in the CNS leads to the attenuation of FPR2 agonism-induced hematoma resolution (Flores et al., 2023). The Overall findings provide pertinent information on viable therapeutic targets that not only attenuate long-term PHH development after GMH but also for a therapeutic approach for adult hemorrhagic stroke.

Summary/Conclusion

Significant progress is currently being made in elucidating mechanisms of hematoma resolution in germinal matrix hemorrhage. Our research group has identified a novel role for FPR2 in neonatal germinal matrix hemorrhage pathophysiology. It was found that FPR2 played a significant role in enhancing hematoma resolution, thus improving overall outcomes in the short and long term. Additionally, FPR2 actions are mediated through the polarization of microglia into the M2 phenotype, which plays a primary role in the phagocytosis of red blood cells. We then investigated a novel signaling pathway, where FPR2 agonism resulted in the upregulation of FPR2, p-ERK(1/2), DUSP1, and CD36. Figure 2 provides a detailed schematic of the FPR2 signaling pathway. Lastly, pharmacological and gene knock-down of FPR2 resulted in the decreased expression of this signaling pathway. Although we are shedding light on a new mechanism of action, further studies need to be conducted to investigate the overall actions of FPR2, such as the role that this receptor has on microglia subtypes, cell types such as neutrophils after GMH.

Figure 2.

FPR2 as a therapeutic target for GMH.

Spontaneous rupture of blood vessels within the germinal matrix space leads to blood clot formation. The blood clot leads to the obstruction of the cerebral aqueduct or formania of Luschka and Magendi or by microthrombi obstructing small CSF outflow passages in the subarachnoid space leading to decreased circulation and absorption of the CSF dynamics. Disruption of the CSF dynamics causes hydrocephalus formation, leading to long-term neurological deficits. In the proposed mechanism of FPR2, AnxA1 binds and stimulates FPR2 causing the phosphorylation of ERK1/2. The phosphorylation of ERK1/2 stimulates DUSP1 and increases its gene transcription. DUSP1’s stimulation then leads to the activation of CD36, promoting M2 phenotype polarization and phagocytosis of the blood clot. This quick resolution of the hematoma results in improved neurological outcomes after GMH. Created using Microsoft Power Point version 2304. AnxA1: Annexin A1; CD36: cluster of differentiation 36; CSF: cerebrospinal fluid; DUSP1: dual specificity phosphatase 1; ERK: extracellular signal-regulated kinase; FPR2: N-formyl peptide receptor 2; GMH: germinal matrix hemorrhage; M2: microglia M2 phenotype.

Future Directions

The recent study on FPR2 in GMH was limited in that it primarily focused on hematoma resolution, microglia cells, short- and long-term outcomes, and one signaling pathway after FPR2 agonism. Although both male and female rodents were used for this study, further investigation needs to be conducted to investigate hormone differences between males and females and how they may affect the pathophysiology of GMH and treatment mechanisms. In this current study, animals were treated only at the 1-hour time point after GMH; thus delayed treatment regimens need to be assessed to investigate the time window of AnxA1 treatment. Additionally, other FPR2 agonists need to be assessed to compare if they stimulate the exact mechanisms of action that AnxA1 acts on. As previously mentioned, M2 microglia have various subtypes which were not assessed in the primary study and need to be conducted to characterize the mechanism of action of microglia cells fully. Lastly, to investigate the role of FPR2 on other cell types, such as astrocytes and neutrophils, that also have the capacity to transform into phagocytes (Flores et al., 2023).