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Background: Perihematomal edema (PHE) after post-intracerebral hemorrhage (ICH) has complex pathophysiological mechanisms that are poorly understood. The complicated immune response in the post-ICH brain constitutes a crucial component of PHE pathophysiology. In this study, we aimed to characterize the transcriptional profiles of immune cell populations in human PHE tissue and explore the microscopic differences between different types of immune cells., Methods: 9 patients with basal ganglia intracerebral hemorrhage (hematoma volume 50-100 ml) were enrolled in this study. A multi-stage profile was developed, comprising Group1 (n = 3, 0-6 h post-ICH, G1), Group2 (n = 3, 6-24 h post-ICH, G2), and Group3 (n = 3, 24-48 h post-ICH, G3). A minimal quantity of edematous tissue surrounding the hematoma was preserved during hematoma evacuation. Single cell RNA sequencing (scRNA-seq) was used to map immune cell populations within comprehensively resected PHE samples collected from patients at different stages after ICH., Results: We established, for the first time, a comprehensive landscape of diverse immune cell populations in human PHE tissue at a single-cell level. Our study identified 12 microglia subsets and 5 neutrophil subsets in human PHE tissue. What's more, we discovered that the secreted phosphoprotein-1 (SPP1) pathway served as the basis for self-communication between microglia subclusters during the progression of PHE. Additionally, we traced the trajectory branches of different neutrophil subtypes. Finally, we also demonstrated that microglia-produced osteopontin (OPN) could regulate the immune environment in PHE tissue by interacting with CD44-positive cells., Conclusions: As a result of our research, we have gained valuable insight into the immune-microenvironment within PHE tissue, which could potentially be used to develop novel treatment modalities for ICH., (© 2024. The Author(s).)

Background: Germinal matrix hemorrhage (GMH) is defined by the rupture of immature blood vessels in the germinal matrix, where subsequent hemorrhage enters the subependymal zone and the cerebral lateral ventricles. The consequent blood clot has been identified as the causative factor of secondary brain injury, which triggers a series of complex parallel and sequential harmful mechanisms, including neuroinflammation. The orphan G-protein-coupled receptor 40 (GPR40), a free fatty acid (FFA) receptor 1, has been shown to exert anti-inflammatory effects when activated and improved outcomes in animal models of stroke. We aimed to investigate the anti-inflammatory effects of GPR40 and its underlying mechanisms after GMH., Methods: GMH model was induced in 7-day-old rat pups by an intraparenchymal injection of bacterial collagenase. GPR40 agonist, GW9508, was administered intranasally 1 h, 25 h, and 49 h after GMH induction. CRISPR targeting GPR40, PAK4, and KDM6B were administered through intracerebroventricular injection 48 h before GMH induction. Neurologic scores, microglia polarization, and brain morphology were evaluated by negative geotaxis, right reflex, rotarod test, foot fault test, Morris water maze, immunofluorescence staining, Western blots, and nissl staining respectfully., Results: The results demonstrated that GW9508 improved neurological and morphological outcomes after GMH in the short (24 h, 48 h, 72h) and long-term (days 21-27). However, the neuroprotective effects of treatment were abolished by GW1100, a selective GPR40 antagonist. GW9508 treatment increased populations of M2 microglia and decreased M1 microglia in periventricular areas 24 h after GMH induction. GW9508 upregulated the phosphorylation of PAK4, CREB, and protein level of KDM6B, CD206, IL-10, which was also met with the downregulation of inflammatory markers IL-1β and TNF-α. The mechanism study demonstrated that the knockdown of GPR40, PAK4, and KDM6B reversed the neuroprotective effects brought on by GW9508. This evidence suggests that GPR40/PAK4/CREB/KDM6B signaling pathway in microglia plays a role in the attenuation of neuroinflammation after GMH., Conclusions: In conclusion, the present study demonstrates that the activation of GPR40 attenuated GMH-induced neuroinflammation through the activation of the PAK4/CREB/KDM6B signaling pathway, and M2 microglia may be a major mediator of this effect. Thus, GPR40 may serve as a potential target in the reduction of the inflammatory response following GMH, thereby improving neurological outcomes in the short- and long-term., (© 2021. The Author(s).)

Background: Intracerebral hemorrhage (ICH) has a high morbidity and mortality. The peripheral immune system and cross-talk between peripheral blood and brain have been implicated in the ICH immune response. Thus, we delineated the gene networks associated with human ICH in the peripheral blood transcriptome. We also compared the differentially expressed genes in blood following ICH to a prior human study of perihematomal brain tissue., Methods: We performed peripheral blood whole-transcriptome analysis of ICH and matched vascular risk factor control subjects (n = 66). Gene co-expression network analysis identified groups of co-expressed genes (modules) associated with ICH and their most interconnected genes (hubs). Mixed-effects regression identified differentially expressed genes in ICH compared to controls., Results: Of seven ICH-associated modules, six were enriched with cell-specific genes: one neutrophil module, one neutrophil plus monocyte module, one T cell module, one Natural Killer cell module, and two erythroblast modules. The neutrophil/monocyte modules were enriched in inflammatory/immune pathways; the T cell module in T cell receptor signaling genes; and the Natural Killer cell module in genes regulating alternative splicing, epigenetic, and post-translational modifications. One erythroblast module was enriched in autophagy pathways implicated in experimental ICH, and NRF2 signaling implicated in hematoma clearance. Many hub genes or module members, such as IARS, mTOR, S1PR1, LCK, FYN, SKAP1, ITK, AMBRA1, NLRC4, IL6R, IL17RA, GAB2, MXD1, PIK3CD, NUMB, MAPK14, DDX24, EVL, TDP1, ATG3, WDFY3, GSK3B, STAT3, STX3, CSF3R, PIP4K2A, ANXA3, DGAT2, LRP10, FLOT2, ANK1, CR1, SLC4A1, and DYSF, have been implicated in neuroinflammation, cell death, transcriptional regulation, and some as experimental ICH therapeutic targets. Gene-level analysis revealed 1225 genes (FDR p < 0.05, fold-change > |1.2|) have altered expression in ICH in peripheral blood. There was significant overlap of the 1225 genes with dysregulated genes in human perihematomal brain tissue (p = 7 × 10 -3 ). Overlapping genes were enriched for neutrophil-specific genes (p = 6.4 × 10 -08 ) involved in interleukin, neuroinflammation, apoptosis, and PPAR signaling., Conclusions: This study delineates key processes underlying ICH pathophysiology, complements experimental ICH findings, and the hub genes significantly expand the list of novel ICH therapeutic targets. The overlap between blood and brain gene responses underscores the importance of examining blood-brain interactions in human ICH.

Background: Inflammation following intracerebral hemorrhage (ICH) significantly contributes to secondary brain damage and poor outcomes. Prostaglandin E2 (PGE2) is known to modulate neuroinflammatory responses and is upregulated in response to brain injury as a result of changes in inducible cyclooxygenase 2 (COX-2) and the membrane-bound type of PGE synthase. Inhibition of COX-2 activity has been reported to attenuate ICH-induced brain injury; however, the clinical utility of such drugs is limited due to the potential for severe side effects. Therefore, it is now important to search for downstream targets capable of preferentially modulating PGE2 signaling, and the four E prostanoid receptors, EP1-4, which are the main targets of PGE2, remain a viable therapeutic option. We have previously shown that EP1 receptor deletion aggravates ICH-induced brain injury and impairs functional recovery, thus the current study aimed to elaborate on these results by including a pharmacologic approach targeting the EP1 receptor., Results: Chronic post-treatment with the selective EP1 receptor antagonist, SC-51089, increased lesion volume by 30.1 ± 14.5% (p < 0.05) and treatment with the EP1 agonist, 17-pt-PGE2, improved neuromuscular functional recovery on grip strength (p < 0.01) and hanging wire (p < 0.05) behavioral testing. To begin identifying the mechanisms involved in EP1-mediated neuroprotection after ICH, histology was performed to assess ferric iron content, neuroinflammation, leukocyte transendothelial migratory potential, and peripheral neutrophil and immunoglobulin infiltration. Following ICH, mice treated with the antagonist displayed increased ferric iron (p < 0.05) and cortical microgliosis (p < 0.05), whereas treatment with the agonist decreased cortical (p < 0.01) and striatal (p < 0.001) astrogliosis, leukocyte transendothelial migratory potential (p < 0.01), neutrophil infiltration (p < 0.05), and blood brain barrier breakdown (p < 0.05)., Conclusions: In agreement with our previous results, selective antagonism of the EP1 receptor aggravated ICH-induced brain injury. Furthermore, EP1 receptor agonism improved anatomical outcomes and functional recovery. Thus, the present data continues to reinforce a putative role for EP1 as a new and more selective therapeutic target for the treatment of ICH that could reduce the side effects associated with COX-2 inhibition while still exploiting the beneficial effects.

Background: Mammalian target of rapamycin (mTOR), a serine/threonine kinase, regulates many processes, including cell growth and the immune response. mTOR is also dysregulated in several neurological diseases, such as traumatic brain injury (TBI), stroke, and neurodegenerative disease. However, the role of mTOR in intracerebral hemorrhage (ICH) remains unexplored. The aims of our study were to determine whether inhibiting mTOR signaling could affect the outcome after ICH and to investigate the possible underlying mechanism., Methods: A rat ICH model was induced by intracerebral injection of collagenase IV into the striatum, and mTOR activation was inhibited by administration of rapamycin. mTOR signaling activation was determined by western blotting. Neurobehavioral deficit after ICH was determined by a set of modified Neurological Severity Scores (mNSS). The levels of CD4+CD25+Foxp3+ regulatory T cells (Tregs) and cytokines were examined using flow cytometry and ELISA, respectively., Results: Our results demonstrated thatmTOR signaling was activated 30 minutes and returned to its basal level 1 day after ICH. Increased p-mTOR, which mean that mTOR signaling was activated, was predominantly located around the hematoma. Rapamycin treatment significantly improved the neurobehavioral deficit after ICH, increased the number of Tregs, increased levels of interleukin-10 and transforming growth factor-β and reduced interferon-γ both in peripheral blood and brain., Conclusions: Our study suggests that mTOR improves ICH outcome and modulates immune response after ICH.

Background: Microglia/macrophages are known to play important roles in initiating brain inflammation after spontaneous intracerebral hemorrhage (ICH). T cell immunoglobulin and mucin domain-3 (Tim-3) have been proven to play a critical part in several inflammatory diseases through regulation of both adaptive and innate immune responses. Tim-3 can be expressed by microglia/macrophages and regulates their function in the innate immune response. However, the effect of Tim-3 on inflammatory responses following ICH is unclear., Methods: In this study, we investigated Tim-3 expression, the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and brain water content in peri-hematomal brain tissue at 12 hours and at 1, 3, 5, and 7 days post-ICH in wild type (WT) ICH and Tim-3-/- ICH mice. The numbers of Tim-3 positive cells,astrocytes, neutrophils and microglia/macrophages were detected using immunofluorescence staining. Cytokines were measured by ELISA. Double immunofluorescence labeling was performed to identify the cellular source of Tim-3 expression. Mouse neurological deficit scores were assessed through animal behavior., Results: Expression of Tim-3 increased early in mouse peri-hematomal brain tissue after autologous blood injection, peaked at day 1, and was positively correlated with the concentrations of TNF-α, IL-1β, and brain water content. Tim-3 was predominantly expressed in microglia/macrophages. Compared with WT mice, Tim-3-/- mice had reduced ICH-induced brain inflammation with decreased TNF-α and IL-1β, cerebral edema and neurological deficit scores. Moreover, Tim-/- inhibited activation of microglia/macrophages. The number of activated microglia/macrophages in Tim-3-/- ICH mice was much lower than that in WT ICH mice., Conclusions: Our findings demonstrate that Tim-3 plays an important role in brain inflammation after ICH, and may be a potential treatment target.

Background: Intracerebral hemorrhage (ICH) is a devastating stroke subtype characterized by a prominent neuroinflammatory response. Antagonism of pro-inflammatory cytokines by specific antibodies represents a compelling therapeutic strategy to improve neurological outcome in patients after ICH. To test this hypothesis, the tumor necrosis factor alpha (TNF-α) antibody CNTO5048 was administered to mice after ICH induction, and histological and functional endpoints were assessed., Methods: Using 10 to 12-week-old C57BL/6J male mice, ICH was induced by collagenase injection into the left basal ganglia. Brain TNF-α concentration, microglia activation/macrophage recruitment, hematoma volume, cerebral edema, and rotorod latency were assessed in mice treated with the TNF-α antibody, CNTO5048, or vehicle., Results: After ICH induction, mice treated with CNTO5048 demonstrated reduction in microglial activation/macrophage recruitment compared to vehicle-treated animals, as assessed by unbiased stereology (P = 0.049). This reduction in F4/80-positive cells was associated with a reduction in cleaved caspase-3 (P = 0.046) and cerebral edema (P = 0.026) despite similar hematoma volumes, when compared to mice treated with vehicle control. Treatment with CNTO5048 after ICH induction was associated with a reduction in functional deficit when compared to mice treated with vehicle control, as assessed by rotorod latencies (P = 0.024)., Conclusions: Post-injury treatment with the TNF-α antibody CNTO5048 results in less neuroinflammation and improved functional outcomes in a murine model of ICH.

Intracerebral hemorrhage (ICH) is a common type of fatal stroke, accounting for about 15% to 20% of all strokes. Hemorrhagic strokes are associated with high mortality and morbidity, and increasing evidence shows that innate immune responses and inflammatory injury play a critical role in ICH-induced neurological deficits. However, the signaling pathways involved in ICH-induced inflammatory responses remain elusive. Toll-like receptor 4 (TLR4) belongs to a large family of pattern recognition receptors that play a key role in innate immunity and inflammatory responses. In this review, we summarize recent findings concerning the involvement of TLR4 signaling in ICH-induced inflammation and brain injury. We discuss the key mechanisms associated with TLR4 signaling in ICH and explore the potential for therapeutic intervention by targeting TLR4 signaling.

Background: Intracerebral hemorrhage (ICH) is a devastating disease that carries a 30 day mortality of approximately 45%. Only 20% of survivors return to independent function at 6 months. The role of inflammation in the pathophysiology of ICH is increasingly recognized. Several clinical studies have demonstrated an association between inflammatory markers and outcomes after ICH; however the relationship between serum biomarkers and functional outcomes amongst survivors has not been previously evaluated. Activation of the inflammatory response as measured by change in peripheral leukocyte count was examined and assessment of mortality and functional outcomes after ICH was determined., Findings: Patients with spontaneous ICH admitted to a tertiary care center between January 2005 and April 2010 were included. The change in leukocyte count was measured as the difference between the maximum leukocyte count in the first 72 hours and the leukocyte count on admission. Mortality was the primary outcome. Secondary outcomes were mortality at 1 year, discharge disposition and the modified Barthel index (MBI) at 3 months compared to pre-admission MBI. 423 cases were included. The in-hospital mortality was 30.4%. The change in leukocyte count predicted worse discharge disposition (OR = 1.258, p = 0.009). The change in leukocyte count was also significantly correlated with a decline in the MBI at 3 months. These relationships remained even after removal of all patients with evidence of infection., Conclusions: Greater changes in leukocyte count over the first 72 hours after admission predicted both worse short term and long term functional outcomes after ICH.

Background: Anti-Aβ immunotherapy is a promising approach to the prevention and treatment of Alzheimer's disease (AD) currently in clinical trials. There is extensive evidence, both in mice and humans that a significant adverse event is the occurrence of microhemorrhages. Also, vasogenic edema was reported in phase 2 of a passive immunization clinical trial. In order to overcome these vascular adverse effects it is critical that we understand the mechanism(s) by which they occur., Methods: We have examined the matrix metalloproteinase (MMP) protein degradation system in two previously published anti-Aβ immunotherapy studies. The first was a passive immunization study in which we examined 22 month old APPSw mice that had received anti-Aβ antibodies for 1, 2 or 3 months. The second is an active vaccination study in which we examined 16 month old APPSw/NOS2-/- mice treated with Aβ vaccination for 4 months., Results: There is a significant activation of the MMP2 and MMP9 proteinase degradation systems by anti-Aβ immunotherapy, regardless of whether this is delivered through active vaccination or passive immunization. We have characterized this activation by gene expression, protein expression and zymography assessment of MMP activity., Conclusions: Since the MMP2 and MMP9 systems are heavily implicated in the pathophysiology of intracerbral hemorrhage, these data may provide a potential mechanism of microhemorrhage due to immunotherapy. Increased activity of the MMP system, therefore, is likely to be a major factor in increased microhemorrhage occurrence.