PEDF-34 attenuates neurological deficit and suppresses astrocyte-dependent neuroinflammation by modulating astrocyte polarization via 67LR/JNK/STAT1 signaling pathway after subarachnoid hemorrhage in rats.

BACKGROUND: Reactive astrocytes participate in various pathophysiology after subarachnoid hemorrhage (SAH), including neuroinflammation, glymphatic-lymphatic system dysfunction, brain edema, BBB disruption, and cell death. Astrocytes transform into two new reactive phenotypes with changed morphology, altered gene expression, and secretion profiles, termed detrimental A1 and beneficial A2. This study investigates the effect of 67LR activation by PEDF-34, a PEDF peptide, on neuroinflammation and astrocyte polarization after the experimental SAH. METHODS: A total of 318 male adult Sprague-Dawley rats were used in experiments in vivo, of which 272 rats were subjected to the endovascular perforation model of SAH and 46 rats underwent sham surgery. 67LR agonist (PEDF-34) was administrated intranasally 1 h after SAH. 67LR-specific inhibitor (NSC-47924) and STAT1 transcriptional activator (2-NP) were injected intracerebroventricularly 48 h before SAH. Short- and long-term neurological tests, brain water content, immunostaining, Nissl staining, western blot, and ELISA assay were performed. In experiments in vitro, primary astrocyte culture with hemoglobin (Hb) stimulation was used to mimic SAH. The expression of the PEDF-34/67LR signaling pathway and neuro-inflammatory cytokines were assessed using Western blot, ELISA, and immunohistochemistry assays both in vivo and in vitro. RESULTS: Endogenous PEDF and 67LR expressions were significantly reduced at 6 h after SAH. 67LR was expressed in astrocytes and neurons. Intranasal administration of PEDF-34 significantly reduced brain water content, pro-inflammatory cytokines, and short-term and long-term neurological deficits after SAH. The ratio of p-JNK/JNK and p-STAT1/STAT1 and the expression of CFB and C3 (A1 astrocytes marker), significantly decreased after PEDF-34 treatment, along with fewer expression of TNF-α and IL-1ß at 24 h after SAH. However, 2-NP (STAT1 transcriptional activator) and NSC-47924 (67LR inhibitor) reversed the protective effects of PEDF-34 in vivo and in vitro by promoting A1 astrocyte polarization with increased inflammatory cytokines. CONCLUSION: PEDF-34 activated 67LR, attenuating neuroinflammation and inhibiting astrocyte A1 polarization partly via the JNK/STAT1 pathway, suggesting that PEDF-34 might be a potential treatment for SAH patients.

UDP-Glucose/P2Y14 Receptor Signaling Exacerbates Neuronal Apoptosis After Subarachnoid Hemorrhage in Rats.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe subtype of stroke with poor outcomes. Abnormal glucose metabolism often occurs after SAH, but the strict control of blood glucose levels is not always beneficial. This study aimed to investigate the contribution of uridine diphosphate glucose (UDP-G), an intermediate of glucose/glycogen metabolism, and its receptor P2Y14 (P2Y purinoceptor 14) to SAH pathology and explored the potential targeted treatments in rats. METHODS: A total of 218 Sprague-Dawley male rats were used. SAH was induced by endovascular perforation. Brain expressions of P2Y14, uridine diphosphate glucose (UDP-G), and its converting enzyme UGP2 (UDP-G pyrophosphorylase-2) were evaluated. Exogenous UDP-G or selective P2Y14 inhibitor was administered intranasally at 1 hour after SAH to explore their potential effects. Intranasal Ugp2 or P2ry14 siRNA was delivered 24 hours before SAH for mechanistic evaluation. Primary neuron culture and hemoglobin stimulation were used as in vitro model of SAH. Post-SAH evaluation included liquid chromatography-mass spectrometry measurement of brain endogenous UDP-G level, neurobehavioral assessments, Western blotting, immunohistochemistry, TUNEL staining, and Nissl staining. RESULTS: There was an acute elevation of endogenous brain UDP-G and UGP2 after SAH, and P2Y14 was expressed in neurons. Although P2Y14 inhibitor decreased neurological dysfunction, neuronal apoptosis, and proapoptotic molecules, exogenous UDP-G exacerbated these outcomes at 24 hours after SAH. Early inhibition of P2Y14 preserved long-term neuronal survival in the hippocampus, amygdala, and cortex with improved neurocognition and depressive-like behavior. In addition, in vivo knockdown of Ugp2- and P2ry14-reduced neurological deficits and proapoptotic molecules at 24 hours after SAH, and furthermore in vitro knockdown of P2ry14-reduced apoptosis in hemoglobin stimulated primary neuron. CONCLUSIONS: These findings suggest a detrimental role of brain UDP-G/P2Y14 signaling in SAH, as a part of glucose metabolic pathology at the tissue level. P2Y14 inhibitor 4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid hydrochloride may serve as a potential therapeutic target in treating patients with SAH.

Recombinant Slit2 suppresses neuroinflammation and Cdc42-mediated brain infiltration of peripheral immune cells via Robo1-srGAP1 pathway in a rat model of germinal matrix hemorrhage.

BACKGROUND: Germinal matrix hemorrhage (GMH) is a devastating neonatal stroke, in which neuroinflammation is a critical pathological contributor. Slit2, a secreted extracellular matrix protein, plays a repulsive role in axon guidance and leukocyte chemotaxis via the roundabout1 (Robo1) receptor. This study aimed to explore effects of recombinant Slit2 on neuroinflammation and the underlying mechanism in a rat model of GMH. METHODS: GMH was induced by stereotactically infusing 0.3 U of bacterial collagenase into the germinal matrix of 7-day-old Sprague Dawley rats. Recombinant Slit2 or its vehicle was administered intranasally at 1 h after GMH and daily for 3 consecutive days. A decoy receptor recombinant Robo1 was co-administered with recombinant Slit2 after GMH. Slit2 siRNA, srGAP1 siRNA or the scrambled sequences were administered intracerebroventricularly 24 h before GMH. Neurobehavior, brain water content, Western blotting, immunofluorescence staining and Cdc42 activity assays were performed. RESULTS: The endogenous brain Slit2 and Robo1 expressions were increased after GMH. Robo1 was expressed on neuron, astrocytes and infiltrated peripheral immune cells in the brain. Endogenous Slit2 knockdown by Slit2 siRNA exacerbated brain edema and neurological deficits following GMH. Recombinant Slit2 (rSlit2) reduced neurological deficits, proinflammatory cytokines, intercellular adhesion molecules, peripheral immune cell markers, neuronal apoptosis and Cdc42 activity in the brain tissue after GMH. The anti-neuroinflammation effects were reversed by recombinant Robo1 co-administration or srGAP1 siRNA. CONCLUSIONS: Recombinant Slit2 reduced neuroinflammation and neuron apoptosis after GMH. Its anti-neuroinflammation effects by suppressing onCdc42-mediated brain peripheral immune cells infiltration was at least in part via Robo1-srGAP1 pathway. These results imply that recombinant Slit2 may have potentials as a therapeutic option for neonatal brain injuries.

Recruitment of regulatory T cells with rCCL17 promotes M2 microglia/macrophage polarization through TGFß/TGFßR/Smad2/3 pathway in a mouse model of intracerebral hemorrhage.

AIMS: Intracerebral hemorrhage (ICH) is a severe neurological condition with high mortality and morbidity. Microglia activation and peripheral inflammatory cells infiltration play an important role in ICH prognosis. Previous studies demonstrated that regulatory T cells (Tregs) ameliorated neuroinflammation following experimental ICH. However, the molecular mechanism underlying such effects of Tregs remains unclear. The objective was to examine how Tregs recruitment induced by recombinant CC chemokine ligand 17 (rCCL17) influences microglia/macrophage polarization in an intrastriatal autologous blood injection ICH animal model, and to determine if TGFß/TGFß-R/Smad2/3 pathway was involved. METHODS: 380 adult CD1 mice (male, eight weeks old) were subjected to sham surgery or autologous blood injection induced ICH. A CD25-specific mouse antibody or isotype control mAb was injected intraventricular (i.c.v) 48 h prior to ICH induction to deplete Tregs. rCCL17, a CC chemokine receptor 4 (CCR4) ligand, was delivered intranasally at 1 h post-ICH. SB431542, a specific inhibitor of TGF-ß was administered intraperitoneally 1 h before ICH induction. Following the ICH, neurobehavioral testing, brain edema, hematoma volume, hemoglobin content, western blotting, double immunofluorescence labeling, and immunohistochemistry were performed. RESULTS: Endogenous expressions of CCL17, Tregs marker Foxp3, and the number of Tregs in perihematomal region increased following ICH. Tregs depletion with a CD25 antibody aggravated neurological deficits and brain edema, increased inflammatory cytokines, neutrophil infiltration, oxidative stress, and reduced the rate of hematoma resolution in ICH mice. rCCL17 treatment increased the number of Tregs in the brain, ameliorated neurological deficits and brain edema after ICH, and promoted microglia/macrophage polarization toward M2 phenotype which was reversed with CD25 antibody. Moreover, rCCL17 increased the expressions of brain TGF-ß/phosphorylated-Smad2/3 which was abrogated with the selective TGFß inhibitor SB431542. CONCLUSIONS: rCCL17-mediated Tregs recruitment may be a potential therapeutic strategy to promote M2 microglia/macrophages polarization and alleviate early brain injury following ICH.

Inhibition of lysophosphatidic acid receptor 1 relieves PMN recruitment in CNS via LPA1/TSP1/CXCR2 pathway and alleviates disruption on blood-brain barrier following intracerebral haemorrhage in mice.

BACKGROUD: The frequencies of morbidity and impairment associated with spontaneous intracerebral haemorrhage (ICH) are comparatively high. Blood-brain barrier (BBB) integrity was compromised due to subsequent brain injury induced by ICH, which is crucial for a poor prognosis. Polymorphonuclear leukocyte (PMN) strongly modulate the disruption of BBB in the central nervous system (CNS). The lysophosphatidic acid receptor 1 (LPA1) mediated thrombospondin-1 (TSP1) regulation in astrocytes, which induce macrophage inflammatory protein 2(MIP2) secretion. MIP2 enhance PMN recruitment through CXC chemokine type 2 (CXCR2) activation. The purpose of this study was to investigate whether the LPA1-mediated inhibition of PMN recruitment and BBB protection after ICH is regulated by TSP1 and CXCR2 networks. METHODS: ICH induction was performed in CD1 mice using collagenase administration. AM966, a targeted LPA1 antagonist, was orally administered 1 and 12 h following ICH. further identify possible LPA1-mediated BBB protection mechanisms, we intracerebroventricularly (ICV) administered a CXCR2 ligand MIP2, as well as TSP1 CRISPR activation (ACT) with AM966. Consequently, we performed neurobehavioral, brain water content (BWC), Evans blue staining (EBS), immunofluorescence (IF), and western blot (WB) analyses. RESULTS: After ICH, astrocytes showed signs of LPA1, which peaked after 24 h, while PMN\ displayed evidence of CXCR2. The AM966-mediated LPA1 suppression relieved PMN recruitment, diminished brain oedema, demonstrated extravasation (as evidenced by EBS), protected BBB integrity, and enhanced neurologic activity following ICH. AM966 treatment strongly reduced TSP1, CXCR2, Occludin, and Claudin-5 expressions and PMN recruitment following ICH, and their expressions were restored by MIP2 and TSP1 CRISPR (ACT). CONCLUSIONS: This study shows that LAP1 suppression reduced PMN recruitment after ICH in mice via TSP1/CXCR2 signalling, which minimized BBB disruption and improved the CNS's neurobehavioral functioning. Hence, LPA1 is a strong candidate for therapy to reduce PMN recruitment and offer protection of BBB integrity after ICH.

Inhibition of LAR attenuates neuroinflammation through RhoA/IRS-1/Akt signaling pathway after intracerebral hemorrhage in mice.

Leukocyte common antigen-related phosphatase (LAR) is widely expressed in the central nervous system and is known to regulate a variety of processes including cell growth, differentiation, and inflammation. However, little is currently known about LAR signaling mediated neuroinflammation after intracerebral hemorrhage (ICH). The objective of this study was to investigate the role of LAR in ICH using autologous blood injection-induced ICH mouse model. Expression of endogenous proteins, brain edema and neurological function after ICH were evaluated. Extracellular LAR peptide (ELP), an inhibitor of LAR, was administered to ICH mice and outcomes were evaluated. LAR activating-CRISPR or IRS inhibitor NT-157 was administered to elucidate the mechanism. The results showed that expressions of LAR, its endogenous agonist chondroitin sulfate proteoglycans (CSPGs) including neurocan and brevican, and downstream factor RhoA increased after ICH. Administration of ELP reduced brain edema, improved neurological function, and decreased microglia activation after ICH. ELP decreased RhoA and phosphorylated serine-IRS1, increased phosphorylated tyrosine-IRS1 and p-Akt, and attenuated neuroinflammation after ICH, which was reversed by LAR activating-CRISPR or NT-157. In conclusion, this study demonstrated that LAR contributed to neuroinflammation after ICH via RhoA/IRS-1 pathway, and ELP may be a potential therapeutic strategy to attenuate LAR mediated neuroinflammation after ICH.

Annexin A1 upregulates hematoma resolution via the FPR2/p-ERK(1/2)/DUSP1/CD36 signaling pathway after germinal matrix hemorrhage.

Germinal matrix hemorrhage (GMH) is one of the leading causes of morbidity and mortality in preterm infants in the United States, with little progress made in its clinical management. Blood clots disrupting normal cerebrospinal fluid circulation and absorption after germinal matrix hemorrhage are key contributors towards post-hemorrhagic hydrocephalus development. n-formyl peptide receptor 2 (FPR2), a G-protein-coupled receptor, has been associated with the activation of p-ERK1/2, which in turn promotes the transcription of the DUSP1 gene, which may play a role in CD36 signaling. CD36 scavenger, a transmembrane glycoprotein, plays an essential role in microglia phagocytic blood clot clearance after GMH. FPR2's role in blood clot clearance after hemorrhagic stroke is unknown. We hypothesize that FPR2 activation by FPR2 agonist Annexin A1 (AnxA1) will enhance hematoma resolution via the upregulation of the CD36 signaling pathway, thereby improving short- and long-term neurological outcomes. Bacterial collagenase (0.3 U) was infused intraparenchymally into the right hemispheric ganglionic eminence in P7 rat pups to induce GMH. AnxA1 and FPR2 Inhibitor (Boc2) were given at 1-h post-GMH via intranasal administration. FPR2 CRISPR was given 48-h prior to GMH induction. Short-term neurological deficits were assessed using negative geotaxis test. Hematoma volume was assessed using hemoglobin assay. Protein expression was assessed using western blots. Long-term neurocognitive deficits and motor coordination were assessed using Morris water maze, rotarod, and foot fault tests. We have demonstrated that AnxA1 treatment enhances hematoma resolution and improved short and long-term outcomes. Lastly, FPR2 agonist AnxA1 treatment resulted in the upregulation of the FPR2/p-ERK(1/2)/DUSP1/CD36 signaling pathway.

Kynurenine/Aryl Hydrocarbon Receptor Modulates Mitochondria-Mediated Oxidative Stress and Neuronal Apoptosis in Experimental Intracerebral Hemorrhage.

Aims: Oxidative stress and neuronal apoptosis play crucial roles in the pathological processes of secondary injury after intracerebral hemorrhage (ICH). Aryl hydrocarbon receptor (AHR), together with its endogenous ligand kynurenine, is known to mediate free radical accumulation and neuronal excitotoxicity in central nervous systems. Herein, we investigate the pathological roles of kynurenine/AHR after ICH. Results: Endogenous AHR knockout alleviated reactive oxygen species accumulation and neuronal apoptosis in ipsilateral hemisphere at 48 h after ICH in mice. The ICH insult resulted in an increase of total and nucleus AHR protein levels and AHR transcriptional activity. Inhibition of AHR provided both short- and long- term neurological benefits by attenuating mitochondria-mediated oxidative stress and neuronal apoptosis after ICH in mice. RhoA-Bax signaling activated mitochondrial death pathway and participated in deleterious actions of AHR. Finally, we reported that exogenous kynurenine aggravated AHR activation and mediated the brain mentioned earlier. Male animals were used in the experiments. Innovation: We show for the first time that kynurenine/AHR mediates mitochondria death and free radical accumulation, at least partially via the RhoA/Bax signaling pathway. Pharmacological antagonists of AHR and kynurenine may ameliorate neurobehavioral function and improve the prognosis of patients with ICH. Conclusion: Kynurenine/AHR may serve as a potential therapeutic target to attenuate mitochondria-mediated oxidative stress and neuronal cells impairment in patients with ICH. Antioxid. Redox Signal. 37, 1111-1129.

Inhibition of Aryl Hydrocarbon Receptor Attenuates Hyperglycemia-Induced Hematoma Expansion in an Intracerebral Hemorrhage Mouse Model.

Background Hyperglycemia is associated with greater hematoma expansion (HE) and worse clinical prognosis after intracerebral hemorrhage (ICH). However, the clinical benefits of intensive glucose normalization remain controversial, and there are no approved therapies for reducing HE. The aryl hydrocarbon receptor (AHR) has been shown to participate in hyperglycemia-induced blood-brain barrier (BBB) dysfunction and brain injury after stroke. Herein, we investigated the role of AHR in hyperglycemia-induced HE in a male mouse model of ICH. Methods and Results CD1 mice (n=387) were used in this study. Mice were subjected to ICH by collagenase injection. Fifty percent dextrose was injected intraperitoneally 3 hours after ICH. AHR knockout clustered regularly interspaced short palindromic repeat was administered intracerebroventricularly to evaluate the role of AHR after ICH. A selective AHR inhibitor, 6,2',4'-trimethoxyflavone, was administered intraperitoneally 2 hours or 6 hours after ICH for outcome study. To evaluate the effect of AHR on HE, 3-methylcholanthrene, an AHR agonist, was injected intraperitoneally 2 hours after ICH. The results showed hyperglycemic ICH upregulated AHR accompanied by greater HE. AHR inhibition provided neurological benefits by restricting HE and preserving BBB function after hyperglycemic ICH. In vivo knockdown of AHR further limited HE and enhanced the BBB integrity. Hyperglycemia directly activated AHR as a physiological stimulus in vivo. The thrombospondin-1/transforming growth factor-ß/vascular endothelial growth factor axis partly participated in AHR signaling after ICH, which inhibited the expressions of BBB-related proteins, ZO-1 and Claudin-5. Conclusions AHR may serve as a potential therapeutic target to attenuate hyperglycemia-induced hematoma expansion and to preserve the BBB in patients with ICH.

Activation of Frizzled-7 attenuates blood-brain barrier disruption through Dvl/ß-catenin/WISP1 signaling pathway after intracerebral hemorrhage in mice.

BACKGROUND: Destruction of blood-brain barrier (BBB) ​​is one of the main mechanisms of secondary brain injury following intracerebral hemorrhage (ICH). Frizzled-7 is a key protein expressed on the surface of endothelial cells that controls vascular permeability through the Wnt-canonical pathway involving WNT1-inducible signaling pathway protein 1 (WISPI). This study aimed to investigate the role of Frizzled-7 signaling in BBB preservation after ICH in mice. METHODS: Adult CD1 mice were subjected to sham surgery or collagenase-induced ICH. Frizzled-7 activation or knockdown was performed by administration of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) by intracerebroventricular injection at 48 h before ICH induction. WISP1 activation or WISP1 knockdown was performed to evaluate the underlying signaling pathway. Post-ICH assessments included neurobehavior, brain edema, BBB permeability, hemoglobin level, western blot and immunofluorescence. RESULTS: The brain expressions of Frizzled-7 and WISP1 significantly increased post-ICH. Frizzled-7 was expressed in endothelial cells, astrocytes, and neurons after ICH. Activation of Frizzled-7 significantly improved neurological function, reduced brain water content and attenuated BBB permeability to large molecular weight substances after ICH. Whereas, knockdown of Frizzled-7 worsened neurological function and brain edema after ICH. Activation of Frizzled-7 significantly increased the expressions of Dvl, ß-Catenin, WISP1, VE-Cadherin, Claudin-5, ZO-1 and reduced the expression of phospho-ß-Catenin. WISP1 knockdown abolished the effects of Frizzled-7 activation on the expressions of VE-Cadherin, Claudin-5 and ZO-1 at 24 h after ICH. CONCLUSIONS: Frizzled-7 activation potentially attenuated BBB permeability and improved neurological deficits after ICH through Dvl​​/ß-Catenin/WISP1 pathway. Frizzled-7 may be a potential target for the development of ICH therapeutic drugs.

Neurokinin Receptor 1 (NK1R) Antagonist Aprepitant Enhances Hematoma Clearance by Regulating Microglial Polarization via PKC/p38MAPK/NFκB Pathway After Experimental Intracerebral Hemorrhage in Mice.

Hematoma clearance is an important therapeutic target to improve outcome following intracerebral hemorrhage (ICH). Recent studies showed that Neurokinin receptor-1 (NK1R) inhibition exerts protective effects in various neurological disease models, but its role in ICH has not been explored. The objective of this study was to investigate the role of NK1R and its relation to hematoma clearance after ICH using an autologous blood injection mouse model. A total of 332 adult male CD1 mice were used. We found that the expression levels of NK1R and its endogenous ligand, substance P (SP), were significantly upregulated after ICH. Intraperitoneal administration of the NK1R selective antagonist, Aprepitant, significantly improved neurobehavior, reduced hematoma volume and hemoglobin levels after ICH, and promoted microglia polarization towards M2 phenotype. Aprepitant decreased phosphorylated PKC, p38MAPK, and NFκB p65, and downregulated M1 markers while upregulating M2 markers after ICH. Intracerebroventricular administration of the NK1R agonist, GR73632 or PKC agonist, phorbol 12-myristate 13-acetate (PMA) reversed the effects of Aprepitant. To demonstrate the upstream mediator of NK1R activation, we performed thrombin injection and found that it increased SP. Inhibiting thrombin suppressed SP and decreased M1 markers while increasing M2 microglia polarization. Thus, NK1R inhibition promoted hematoma clearance after ICH by increasing M2 microglial polarization via downregulating PKC/p38MAPK/NFκB signaling pathway, and thrombin may be a key upstream mediator of NK1R activation. Therapeutic interventions inhibiting NK1R signaling may be a new target for the treatment of ICH.

TREM (Triggering Receptor Expressed on Myeloid Cells)-1 Inhibition Attenuates Neuroinflammation via PKC (Protein Kinase C) δ/CARD9 (Caspase Recruitment Domain Family Member 9) Signaling Pathway After Intracerebral Hemorrhage in Mice.

Background and Purpose: Intracerebral hemorrhage (ICH) is a devastating subtype of stroke with high mortality and disability. Inflammatory response promotes secondary brain injury after ICH. TREM (triggering receptor expressed on myeloid cells)-1 is a key regulator of inflammation. The aim of this study was to evaluate the role of TREM-1 in neuroinflammatory response after ICH in mice. Methods: CD1 mice (n=275) were used in this study. Mice were subjected to ICH by autologous blood injection. TREM-1 knockout CRISPR was administered intracerebroventricularly to evaluate the role of TREM-1 after ICH. A selective TREM-1 inhibitor, LP17, was administered intranasally 2 hours after ICH. To elucidate TREM-1 signaling pathway, CARD9 (caspase recruitment domain family member 9) activation CRISPR was administered with LP17 and TREM-1 activating anti-mouse TREM-1 monoclonal antibody (mAb) was administered with Rottlerin, a specific PKC (protein kinase C) δ inhibitor. Lastly, to evaluate the role of HMGB1 (high-mobility group box 1) in TREM-1 mediated microglia activation, glycyrrhizin, an inhibitor of HMBG1 was administered with TREM-1 activating mAb. Neurobehavioral test, brain water content, Western blot, immunofluorescence staining, and coimmunoprecipitation was performed. Results: TREM-1 knockout reduced ICH-induced neurobehavioral deficits and neuroinflammatory response. The temporal expression of HMGB1, TREM-1, PKC δ, and CARD9 increased after ICH. TREM-1 was expressed on microglia. Intranasal administration of LP17 significantly decreased brain edema and improved neurobehavioral outcomes at 24 and 72 hours after ICH. LP17 promoted M2 microglia polarization and reduced proinflammatory cytokines after ICH, which was reversed with CARD9 activation CRISPR. TREM-1 mAb increased neurobehavior deficits, proinflammatory cytokines, and reduced M2 microglia after ICH, which was reversed with Rottlerin. HMBG1 interaction with TREM-1 increased after ICH, and glycyrrhizin reduced neuroinflammation and promoted M2 microglia which was reversed with TREM-1 mAb. Conclusions: This study demonstrated that TREM-1 enhanced neuroinflammation by modulating microglia polarization after ICH, and this regulation was partly mediated via PKC δ/CARD9 signaling pathway and increased HMGB1 activation of TREM-1.

Autophagy & Phagocytosis in Neurological Disorders and their Possible Cross-talk.

Autophagy and phagocytosis are two important endogenous lysosomal dependent clearing systems in the organism. In some neurological disorders, excessive autophagy or dysfunctional phagocytosis has been shown to contribute to brain injury. Recent studies have revealed that there are underlying interactions between these two processes. However, different studies show inconsistent results for the contribution of autophagy to the phagocytic process in diverse phagocytes and relatively little is known about the link between them especially in the brain. It is critical to understand the role that autophagy plays in phagocytic process in order to promote the clearance of endogenous and exogenous detrimental materials. In this review, we highlight the studies focusing on phagocytosis and autophagy occurring in the brain and summarizing the possible regulatory roles of autophagy in the process of phagocytosis. Balancing the roles of autophagy and phagocytosis may be a promising therapeutic strategy for the treatment of some neurological diseases in the future.

Recombinant CCL17-dependent CCR4 activation alleviates neuroinflammation and neuronal apoptosis through the PI3K/AKT/Foxo1 signaling pathway after ICH in mice.

BACKGROUND: Intracerebral hemorrhage (ICH), a devastating subtype of stroke, is associated with high mortality and morbidity. Neuroinflammation is an important factor leading to ICH-induced neurological injuries. C-C Chemokine Receptor 4 (CCR4) plays an important role in enhancing hematoma clearance after ICH. However, it is unclear whether CCR4 activation can ameliorate neuroinflammation and apoptosis of neurons following ICH. The aim of the present study was to examine the effects of recombinant CCL17 (rCCL17)-dependent CCR4 activation on neuroinflammation and neuronal apoptosis in an intrastriatal autologous blood injection ICH model, and to determine whether the PI3K/AKT/Foxo1 signaling pathway was involved. METHODS: Two hundred twenty-six adult (8-week-old) male CD1 mice were randomly assigned to sham and ICH surgery groups. An intrastriatal autologous blood injection ICH model was used. rCCL17, a CCR4 ligand, was delivered by intranasal administration at 1 h, 3 h, and 6 h post-ICH. CCL17 antibody was administrated by intraventricular injection at 1 h post-ICH. C021, a specific inhibitor of CCR4 and GDC0068, an AKT inhibitor were delivered intraperitoneally 1 h prior to ICH induction. Brain edema, neurobehavioral assessments, western blotting, Fluoro-Jade C staining, terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunofluorescence staining were conducted. RESULTS: Endogenous expression of CCL17 and CCR4 were increased following ICH, peaking at 5 days post-induction. CCR4 was found to co-localize with microglia, neurons, and astrocytes. rCCL17 treatment decreased brain water content, attenuated short- and long-term neurological deficits, deceased activation of microglia/macrophages and infiltration of neutrophils, and inhibited neuronal apoptosis in the perihematomal region post-ICH. Moreover, rCCL17 treatment post-ICH significantly increased the expression of CCR4, PI3K, phosphorylated AKT, and Bcl-2, while Foxo1, IL-1ß, TNF-α, and Bax expression were decreased. The neuroprotective effects of rCCL17 were reversed with the administration of C021 or GDC0068. CONCLUSIONS: rCCL17-dependent CCR4 activation ameliorated neurological deficits, reduced brain edema, and ameliorated neuroinflammation and neuronal apoptosis, at least in part, through the PI3K/AKT/Foxo1 signaling pathway after ICH. Thus, activation of CCR4 may provide a promising therapeutic approach for the early management of ICH.

Inhibition of lysophosphatidic acid receptor 1 attenuates neuroinflammation via PGE2/EP2/NOX2 signalling and improves the outcome of intracerebral haemorrhage in mice.

Lysophosphatidic acid receptor 1 (LPA1) plays a critical role in proinflammatory processes in the central nervous system by modulating microglia activation. The aim of this study was to explore the anti-inflammatory effects and neurological function improvement of LPA1 inhibition after intracerebral haemorrhage (ICH) in mice and to determine whether prostaglandin E2 (PGE2), E-type prostaglandin receptor 2 (EP2), and NADPH oxidase 2 (NOX2) signalling are involved in LPA1-mediated neuroinflammation. ICH was induced in CD1 mice by autologous whole blood injection. AM966, a selective LPA1 antagonist, was administered by oral gavage 1 h and 12 h after ICH. The LPA1 endogenous ligand, LPA was administered to verify the effect of LPA1 activation. To elucidate potential inflammatory mechanisms of LPA1, the selective EP2 activator butaprost was administered by intracerebroventricular injection with either AM966 or LPA1 CRISPR knockout (KO). Water content of the brain, neurobehavior, immunofluorescence staining, and western blot were performed. After ICH, EP2 was expressed in microglia whereas LPA1 was expressed in microglia, neurons, and astrocytes, which peaked after 24 h. AM966 inhibition of LPA1 improved neurologic function, reduced brain oedema, and suppressed perihematomal inflammatory cells after ICH. LPA administration aggravated neurological deficits after ICH. AM966 treatment and LPA1 CRISPR KO both decreased the expressions of PGE2, EP2, NOX2, NF-κB, TNF-α, IL-6, and IL-1ß expressions after ICH, which was reversed by butaprost. This study demonstrated that inhibition of LPA1 attenuated neuroinflammation caused by ICH via PGE2/EP2/NOX2 signalling pathway in mice, which consequently improved neurobehavioral functions and alleviated brain oedema. LPA1 may be a promising therapeutic target to attenuate ICH-induced secondary brain injury.

Recombinant CCL17 Enhances Hematoma Resolution and Activation of CCR4/ERK/Nrf2/CD163 Signaling Pathway After Intracerebral Hemorrhage in Mice.

Hematoma is a crucial factor leading to poor prognosis after intracerebral hemorrhage (ICH). Promoting microglial phagocytosis to enhance hematoma resolution may be an important therapeutic target for recovery after ICH. C-C chemokine receptor 4 (CCR4) is important for regulating immune balance in the central nervous system. However, whether CCR4 activation can attenuate hematoma after ICH remains unknown. We aimed to evaluate whether CCL17 (a specific ligand of CCR4) treatment can promote hematoma resolution through CCR4/ERK/Nrf2/CD163 pathway after ICH. A total of 261 adult male CD1 mice were used. Mice were subjected to intrastriatal injection of autologous blood to induce ICH and randomly assigned to receive recombinant CCL17 (rCCL17) or vehicle which was administered intranasally at 1 h after ICH. To elucidate the underlying mechanism, C021, a selective inhibitor of CCR4 and ML385 and a selective inhibitor of Nrf2 were administered 1 h prior to ICH induction. Clustered regularly interspaced short palindromic repeats (CRISPR) knockout for CD163 was administered by intracerebroventricular injection at 48 h before ICH. Brain edema, short- and long-term neurobehavior evaluation, hematoma volume, hemoglobin content, western blot, and immunofluorescence staining were performed. Endogenous CCL17, CCR4, and CD163 expression increased and peaked at 72 h after ICH. CCR4 was expressed by microglia. CCR4 activation with rCCL17 significantly improved neurobehavioral scores and reduced hematoma volume and brain edema compared with vehicle. Moreover, rCCL17 treatment significantly promoted phosphorylation of ERK1/2, increased the expression Nrf2, and upregulated CD163 expression after ICH. The protective effects of rCCL17 were abolished by administration of C021, ML385, and CD163 CRISPR knockout. This study demonstrated that CCR4 activation with rCCL17 promoted hematoma resolution by increasing CD163 expression and CCR4/ERK/Nrf2 pathway activation after ICH, thereby reducing brain edema and improving neurological function. Overall, our study suggests that CCR4 activation may be a potential therapeutic strategy to attenuate hematoma in early brain injury after ICH.

The Activation of Phosphatidylserine/CD36/TGF-ß1 Pathway prior to Surgical Brain Injury Attenuates Neuroinflammation in Rats.

Neuroinflammation plays an important pathological role in experimental surgical brain injury (SBI). Apoptotic associated with phosphatidylserine (PS) externalization promotes anti-inflammatory mediator TGF-ß1 release. In the present study, we investigated the anti-neuroinflammation effect of PS liposome or isoflurane pretreatment via PS/CD36/TGF-ß1 signaling in a rat model of SBI. A total of 120 male Sprague-Dawley rats (weighing 280-330 gms) were used. SBI was induced by partial right frontal lobe corticotomy. Intranasal PS liposome or isoflurane inhalation was administered prior to SBI induction. CD36 small interfering RNA (siRNA) was administered intracerebroventricularly. Recombinant Annexin V protein (rAnnexin V) was delivered intranasally. Post-SBI assessments included neurological tests, brain water content, Western blot, and immunohistochemistry. Endogenous CD36 protein levels but not TGF-ß1 was significantly increased within peri-resection brain tissues over 72 h after SBI. SBI rats were associated with increased brain water content surrounding corticotomy and neurological deficits. PS liposome pretreatment significantly reduced brain water content and improved some neurological deficits at 24 hours and 72 hours after SBI. PS liposome increased CD36 and TGF-ß1 protein levels, but decreased IL-1ß and TNFα protein levels in peri-resection brain tissues at 24 hours after SBI. CD36 siRNA or rAnnexin V partially countered the protective effect of PS liposome. Isoflurane pretreatment produced similar antineuroinflammation and neurological benefits in SBI rats partially by upregulating CD36/Lyn/TGF-ß1 signaling. Collectively, our findings suggest that the activation of PS/CD36/TGF-ß1 pathway by PS liposome or isoflurane prior to SBI could attenuate neuroinflammation and improve neurological outcomes in rats. PS liposome or isoflurane pretreatment may serve as an effective preventive strategy to minimize the brain injury caused by neurosurgical procedures in patients.

Orexin A alleviates neuroinflammation via OXR2/CaMKKß/AMPK signaling pathway after ICH in mice.

BACKGROUND: Orexins are two neuropeptides (orexin A, OXA; orexin B, OXB) secreted mainly from the lateral hypothalamus, which exert a wide range of physiological effects by activating two types of receptors (orexin receptor 1, OXR1; orexin receptor 2, OXR2). OXA has equal affinity for OXR1 and OXR2, whereas OXB binds preferentially to OXR2. OXA rapidly crosses the blood-brain barrier by simple diffusion. Many studies have reported OXA's protective effect on neurological diseases via regulating inflammatory response which is also a fundamental pathological process in intracerebral hemorrhage (ICH). However, neuroprotective mechanisms of OXA have not been explored in ICH. METHODS: ICH models were established using stereotactic injection of autologous arterial blood into the right basal ganglia of male CD-1 mice. Exogenous OXA was administered intranasally; CaMKKß inhibitor (STO-609), OXR1 antagonist (SB-334867), and OXR2 antagonist (JNJ-10397049) were administered intraperitoneally. Neurobehavioral tests, hematoma volume, and brain water content were evaluated after ICH. Western blot and ELISA were utilized to evaluate downstream mechanisms. RESULTS: OXA, OXR1, and OXR2 were expressed moderately in microglia and astrocytes and abundantly in neurons. Expression of OXA decreased whereas OXR1 and OXR2 increased after ICH. OXA treatment significantly improved not only short-term but also long-term neurofunctional outcomes and reduced brain edema in ipsilateral hemisphere. OXA administration upregulated p-CaMKKß, p-AMPK, and anti-inflammatory cytokines while downregulated p-NFκB and pro-inflammatory cytokines after ICH; this effect was reversed by STO-609 or JNJ-10397049 but not SB-334867. CONCLUSIONS: OXA improved neurofunctional outcomes and mitigated brain edema after ICH, possibly through alleviating neuroinflammation via OXR2/CaMKKß/AMPK pathway.