MFGE8 mitigates brain injury in a rat model of SAH by maintaining vascular endothelial integrity via TIGß5/PI3K/CXCL12 signaling.

Leaked blood components, injured endothelial cells, local inflammatory response and vasospasm may converge to promote microthrombosis following subarachnoid hemorrhage (SAH). Previously, we showed that the milk fat globule-epidermal growth factor 8 (MFGE8) can mitigate SAH-induced microthrombosis. This present study was aimed to explore the molecular pathway participated in MFGE8-dependent protection on vascular endothelium. Immunofluorescence, immunoblot and behavioral tests were used to determine the molecular partner and signaling pathway mediating the effect of MFGE8 in vascular endothelium in rats with experimental SAH and controls, together with the applications of RNA silencing and pharmacological intervention methods. Relative to control, recombinant human MFGE8 (rhMFGE8) treatment increased 5-bromo-2'-deoxyuridine (BrdU) labeled new endothelial cells, reduced TUNUL-positive endothelial cells and elevated the expression of phosphatidylinositol 3-kinase (PI3K) and chemokine (C-X-C motif) ligand 12 (CXCL12), in the brains of SAH rats. These effects were reversed by MFGE8 RNA silencing, as well as following cilengitide and wortmannin intervention. These results suggest that MFGE8 promotes endothelial regeneration and mitigates endothelial DNA damage through the activation of the TIGß5/PI3K/CXCL12 signaling pathway.

Inhibition of mast cell tryptase attenuates neuroinflammation via PAR-2/p38/NFκB pathway following asphyxial cardiac arrest in rats.

BACKGROUND: Cardiac arrest survivors suffer from neurological dysfunction including cognitive impairment. Cerebral mast cells, the key regulators of neuroinflammation contribute to neuroinflammation-associated cognitive dysfunction. Mast cell tryptase was demonstrated to have a proinflammatory effect on microglia via the activation of microglial protease-activated receptor-2 (PAR-2). This study investigated the potential anti-neuroinflammatory effect of mast cell tryptase inhibition and the underlying mechanism of PAR-2/p-p38/NFκB signaling following asphyxia-induced cardiac arrest in rats. METHODS: Adult male Sprague-Dawley rats resuscitated from 10 min of asphyxia-induced cardiac arrest were randomized to four separate experiments including time-course, short-term outcomes, long-term outcomes and mechanism studies. The effect of mast cell tryptase inhibition on asphyxial cardiac arrest outcomes was examined after intranasal administration of selective mast cell tryptase inhibitor (APC366; 50 µg/rat or 150 µg/rat). AC55541 (selective PAR-2 activator; 30 µg/rat) and SB203580 (selective p38 inhibitor; 300 µg/rat) were used for intervention. Short-term neurocognitive functions were evaluated using the neurological deficit score, number of seizures, adhesive tape removal test, and T-maze test, while long-term cognitive functions were evaluated using the Morris water maze test. Hippocampal neuronal degeneration was evaluated by Fluoro-Jade C staining. RESULTS: Mast cell tryptase and PAR-2 were dramatically increased in the brain following asphyxia-induced cardiac arrest. The inhibition of mast cell tryptase by APC366 improved both short- and long-term neurological outcomes in resuscitated rats. Such behavioral benefits were associated with reduced expressions of PAR-2, p-p38, NFκB, TNF-α, and IL-6 in the brain as well as less hippocampal neuronal degeneration. The anti-neuroinflammatory effect of APC366 was abolished by AC55541, which when used alone, indeed further exacerbated neuroinflammation, hippocampal neuronal degeneration, and neurologic deficits following cardiac arrest. The deleterious effects aggregated by AC55541 were minimized by p38 inhibitor. CONCLUSIONS: The inhibition of mast cell tryptase attenuated neuroinflammation, led to less hippocampal neuronal death and improved neurological deficits following cardiac arrest. This effect was at least partly mediated via inhibiting the PAR-2/p-p38/NFκB signaling pathway. Thus, mast cell tryptase might be a novel therapeutic target in the management of neurological impairment following cardiac arrest.

17-Allylamino-Demethoxygeldanamycin Ameliorate Microthrombosis Via HSP90/RIP3/NLRP3 Pathway After Subarachnoid Hemorrhage in Rats.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe and emergent cerebrovascular disease, the prognosis of which usually very poor. Microthrombi formation highlighted with inflammation occurs early after SAH. As the main cause of DCI, microthrombosis associated with the prognosis of SAH. The aim of this study was to show HSP90 inhibitor 17-AAG effect on microthrombosis after SAH in rats. METHODS: Ninety-five SD rats were used for the experiment. For time course study, the rats were randomly divided into five groups: sham group and SAH group with different time point (1d, 2d, 3d, 5d). Endovascular perforation method was conducted for SAH model. Neurological score, SAH grade, and mortality were measured after SAH. The samples of the left hemisphere brain were collected. The expression of HSP90 was detected by Western blot. The microthrombosis after SAH in rats' brain was detected by immunohistochemistry. For mechanism study, rats were randomly divided into three groups: sham, SAH + vehicle, and SAH +17-AAG (n = 6/group). 17-AAG was given by intraperitoneal injection (80 mg/kg) 1 h after SAH. Neurological function were measured at 24 h after SAH. The expression of RIP3, NLRP3, ASC, and IL-1ß was measured by Western blot. Microthrombosis was detected by immunohistochemistry. RESULTS: Our results showed that the HSP90 protein level increased and peaked at 2 days after SAH. Microthrombosis caused by SAH was increased in 1 day and peaked at 2 days after SAH. Administration HSP90 specific inhibitor 17-AAG reduced expression of RIP3, NLRP3, ASC, and IL-1ß, reduced microthrombosis after SAH, and improved neurobehavior when compared to vehicle group. CONCLUSIONS: 17-AAG can ameliorate microthrombosis via HSP90/RIP3/NLRP3 pathway and improve neurobehavior after SAH.

Recombinant Human Milk Fat Globule-Epidermal Growth Factor 8 Attenuates Microthrombosis after Subarachnoid Hemorrhage in Rats.

BACKGROUND: Microthrombosis after subarachnoid hemorrhage has an adverse effect on prognosis. Milk fat globule-epidermal growth factor 8 promotes phagocytosis of phagocytic cells and may reduce microthrombosis. This study investigated the effects of recombinant human milk fat globule-epidermal growth factor 8 on microthrombosis and neurological function after subarachnoid hemorrhage. METHODS: Rats subarachnoid hemorrhage model was induced by intravascular puncture method. Western blot was performed to measure the expression of endogenous milk fat globule-epidermal growth factor 8 after subarachnoid hemorrhage. Microthrombosis was quantified by microthrombi count using immunohistochemistry and immunofluorescence. The neuroprotective effect of recombinant human milk fat globule-epidermal growth factor 8 administration was evaluated by modified Garcia score, beam balance, Rotarod test, and Morris water maze. RESULTS: Endogenous milk fat globule-epidermal growth factor 8 protein level increased after subarachnoid hemorrhage. Microthrombosis was significantly increased in subarachnoid hemorrhage rats brain, while recombinant human milk fat globule-epidermal growth factor 8 dramatically reduced microthrombosis as well as improve short- and long- term neurobehavior after subarachnoid hemorrhage. CONCLUSIONS: Recombinant human milk fat globule-epidermal growth factor 8 reduces microthrombosis and improves neurological function after subarachnoid hemorrhage, which may be an effective strategy for treating subarachnoid hemorrhage.

Activation of retinoid X receptor by bexarotene attenuates neuroinflammation via PPARγ/SIRT6/FoxO3a pathway after subarachnoid hemorrhage in rats.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a life-threatening subtype of stroke with high mortality and disabilities. Retinoid X receptor (RXR) has been shown to be neuroprotective against ischemia/reperfusion injury. This study aimed to investigate the effects of the selective RXR agonist bexarotene on neuroinflammation in a rat model of SAH. METHODS: Two hundred male Sprague-Dawley rats were used. The endovascular perforation induced SAH. Bexarotene was administered intraperitoneally at 1 h after SAH induction. To investigate the underlying mechanism, the selective RXR antagonist UVI3003 and RXR siRNA or SIRT6 inhibitor OSS128167 was administered via intracerebroventricular 1 h before SAH induction. Post-SAH assessments including SAH grade, neurological score, brain water content, Western blot, and immunofluorescence were performed. RESULTS: The endogenous RXR and sirtuin 6 (SIRT6) protein levels were increased after SAH. Bexarotene treatment significantly reduced brain edema and improved the short-/long-term neurological deficit after SAH. Mechanistically, bexarotene increased the levels of PPARγ and SIRT6; decreased the expression of phosphorylated FoxO3a (p-FoxO3a), IL-6, IL-1ß, and TNF-a; and inhibited the microglia activation and neutrophils infiltration at 24 h after SAH. Either UVI3003, OSS128167, or RXR siRNA abolished the neuroprotective effects of bexarotene and its regulation on protein levels of PPARγ/SIRT6/p-FoxO3a after SAH. CONCLUSIONS: The activation of RXR by bexarotene attenuated neuroinflammation and improved neurological deficits after SAH. The anti-neuroinflammatory effect was at least partially through regulating PPARγ/SIRT6/FoxO3a pathway. Bexarotene may be a promising therapeutic strategy in the management of SAH patients.

Maresin 1 Activates LGR6 to Alleviate Neuroinflammation via the CREB/JMJD3/IRF4 Pathway in a Rat Model of Subarachnoid Hemorrhage.

Neuroinflammation is an early event of brain injury after subarachnoid hemorrhage (SAH). Whether the macrophage mediators in resolving inflammation 1 (MaR1) is involved in SAH pathogenesis is unknown. In this study, 205 male Sprague-Dawley rats were subjected to SAH via endovascular perforation in the experimental and control groups. MaR1 was dosed intranasally at 1 h after SAH, with LGR6 siRNA and KG-501, GSK-J4 administered to determine the signaling pathway. Neurobehavioral, histological and biochemical data were obtained from the animal groups with designated treatments. The results showed: (i) The leucine-rich repeat containing G protein-coupled receptor 6 (LGR6) was decreased after SAH and reached to the lowest level at 24 h after SAH. Jumonji d3 (JMJD3) protein levels tended to increase and peaked at 24 h after SAH. LGR6 and JMJD3 expression were co-localized with microglia. (ii) MaR1 administration mitigated short-term neurological deficits, brain edema and long-term neurobehavioral performance after SAH, and attenuated microglial activation and neutrophil infiltration. (iii) Knockdown of LGR6, inhibition of CREB phosphorylation or JMJD3 activity abolished the anti-neuroinflammatory effect of MaR1 on the expression of CREB, CBP, JMJD3, IRF4, IRF5, IL-1ß, IL-6 and IL-10, thus prevented microglial activation and neutrophil infiltration. Together, the results show that MaR1 can activate LGR6 and affect CREB/JMJD3/IRF4 signaling to attenuate neuroinflammation after SAH, pointing to a potential pharmacological utility in this disorder.

MFGE8 promotes adult hippocampal neurogenesis in rats following experimental subarachnoid hemorrhage via modifying the integrin ß3/Akt signaling pathway.

Subarachnoid hemorrhage (SAH) is one of the most severe type of cerebral strokes, which can cause multiple cellular changes in the brain leading to neuronal injury and neurological deficits. Specifically, SAH can impair adult neurogenesis in the hippocampal dentate gyrus, thus may affecting poststroke neurological and cognitive recovery. Here, we identified a non-canonical role of milk fat globule epidermal growth factor 8 (MFGE8) in rat brain after experimental SAH, involving a stimulation on adult hippocampal neurogenesis(AHN). Experimental SAH was induced in Sprague-Dawley rats via endovascular perforation, with the in vivo effect of MFGE8 evaluated via the application of recombinant human MFGE8 (rhMFGE8) along with pharmacological interventions, as determined by hemorrhagic grading, neurobehavioral test, and histological and biochemical analyses of neurogenesis related markers. Results: Levels of the endogenous hippocampal MFGE8 protein, integrin-ß3 and protein kinase B (p-Akt) were elevated in the SAH relative to control groups, while that of hippocalcin (HPCA) and cyclin D1 showed the opposite change. Intraventricular rhMGFE8 infusion reversed the decrease in doublecortin (DCX) immature neurons in the DG after SAH, along with improved the short/long term neurobehavioral scores. rhMGFE8 treatment elevated the levels of phosphatidylinositol 3-kinase (PI3K), p-Akt, mammalian target of rapamycin (mTOR), CyclinD1, HPCA and DCX in hippocampal lysates, but not that of integrin ß3 and Akt, at 24 hr after SAH. Treatment of integrin ß3 siRNA, the PI3K selective inhibitor ly294002 or Akt selective inhibitor MK2206 abolished the effects of rhMGFE8 after SAH. In conclusion, MFGE8 is upregulated in the hippocampus in adult rats with reduced granule cell genesis. rhMFGE8 administration can rescue this impaired adult neurogenesis and improve neurobehavioral recovery. Mechanistically, the effect of MFGE8 on hippocampal adult neurogenesis is mediated by the activation of integrin ß3/Akt pathway. These findings suggest that exogenous MFGE8 may be of potential therapeutic value in SAH management. Graphical abstract and proposed pathway of rhMFGE8 administration attenuate hippocampal injury by improving neurogenesis in SAH models. SAH caused hippocampal injury and neurogenesis interruption. Administered exogenous MFGE8, recombinant human MFGE8(rhMFGE8), could ameliorate hippocampal injury and improve neurological functions after SAH. Mechanistically, MFGE8 bind to the receptor integrin ß3, which activated the PI3K/Akt pathway to increase the mTOR expression, and further promote the expression of cyclin D1, HPCA and DCX. rhMFGE8 could attenuated hippocampal injury by improving neurogenesis after SAH, however, know down integrin ß3 or pharmacological inhibited PI3K/Akt by ly294002 or MK2206 reversed the neuro-protective effect of rhMFGE8.

Exosome-Encapsulated microRNA-140-5p Alleviates Neuronal Injury Following Subarachnoid Hemorrhage by Regulating IGFBP5-Mediated PI3K/AKT Signaling Pathway.

Recent literature has highlighted the therapeutic implication of exosomes (Exos) released by adipose tissue-originated stromal cells (ADSCs) in regenerative medicine. Herein, the current study sought to examine the potential protective effects of ADSC-Exos on neuronal injury following subarachnoid hemorrhage (SAH) by delivering miR-140-5p. Firstly, isolated primary neurons were co-cultured together with well-identified ADSC-Exos. TDP-43-treated neurons were subsequently treated with PKH67-ADSC-Exos and Cy3-miR-140-5p to assess whether ADSC-Exos could transmit miR-140-5p to the recipient neurons to affect their behaviors. Moreover, a luciferase assay was carried out to identify the presumable binding of miR-140-5p to IGFBP5. IGFBP5 rescue experimentation was also performed to testify whether IGFBP5 conferred the impact of miR-140-5p on neuronal damage. The role of PI3K/AKT signaling pathway was further analyzed with the application of its inhibitor miltefosine. Lastly, SAH rat models were developed for in vivo validation. It was found that ADSC-Exos conferred protection against TDP-43-caused neuronal injury by augmenting viability and suppressing cell apoptosis. In addition, miR-140-5p was transmitted from ADSC-Exos to neurons and post-transcriptionally downregulated the expression of IGFBP5. As a result, by means of suppressing IGFBP5 and activating the PI3K/AKT signaling pathway, miR-140-5p from ADSC-Exos induced a neuroprotective effect. Furthermore, in vivo findings substantiated the aforementioned protective role of ADSC-Exos-miR-140-5p, contributing to protection against SAH-caused neurological dysfunction. Collectively, our findings indicated that ADSC-Exos-miR-140-5p could inhibit TDP-43-induced neuronal injury and attenuate neurological dysfunction of SAH rats by inhibiting IGFBP5 and activating the PI3K/Akt signaling pathway.

Elevation of CSF Sortilin Following Subarachnoid Hemorrhage in Patients and Experimental Model Rats.

Subarachnoid hemorrhage (SAH) can cause acute neuronal injury and chronic neurocognitive deficits; biomarkers reflecting its associated neuronal injury are of potential prognostic value. Sortilin, a member of the vacuolar protein sorting 10p (Vps10p) family, is enriched in neurons and is likely involved in neurodegenerative diseases. Here, we explored sortilin in the cerebrospinal fluid (CSF) as a potential biomarker for early neuronal injury after SAH. Sortilin levels in the CSF of SAH patients (n = 11) and controls (n = 6) were analyzed by immunoblot. SAH rats surviving 3-72 h (h) were evaluated neurologically, with their brain and CSF samples examined histologically and biochemically. Sortilin protein ~100 kDa was detected in the CSF from SAH patients only, with its levels correlated to Hunt-Hess scale. Rats in the SAH groups showed poorer Garcia score and beam balancing capability than sham controls. Sortilin ~100 kDa was detectable in the CSF of the SAH, but not sham, animals. Levels of sortilin ~100 kDa and fragments ~40 kDa in cortical lysates were elevated in the SAH relative to control rats. Levels of cortical glial fibrillary acidic protein (GFAP) were also elevated in the SAH rats. In immunohistochemistry, the pattern of sortilin labeling in the brain was largely comparable between the SAH and control rats, whereas an increased astrocytic GFAP immunolabeling was evident in the former. Together, these results suggest that SAH can cause an early and remarkable rise of sortilin products in CSF, likely reflecting neuronal change. Sortilin could be further explored as a potential biomarker in some brain disorders.

Activation of Melanocortin 1 Receptor Attenuates Early Brain Injury in a Rat Model of Subarachnoid Hemorrhage viathe Suppression of Neuroinflammation through AMPK/TBK1/NF-κB Pathway in Rats.

Neuroinflammation plays a vital role in early brain injury (EBI) following subarachnoid hemorrhage (SAH). The hypothesis of this study was that activation of melanocortin 1 receptor (MC1R) with BMS-470539 attenuates EBI by suppression of neuroinflammation after SAH. We utilized BMS-470539, MSG-606, and MRT-68601 to verify the neuroprotective effects of MC1R. We evaluated brain water content, short-term and long-term neurobehavior after SAH. Western blotting and immunofluorescence staining were utilized to assess the changes of protein levels. The results of western blotting suggested that the expressions of MC1R, phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK), and phosphorylated-TANK binding kinase 1 (p-TBK1) were increased and reached their peak points at 24 h following SAH. Moreover, BMS-470539 treatment notably attenuated neurological deficits caused by SAH, and also notably improved long-term spatial learning and memory abilities after SAH. The underlying mechanisms of the neuroprotection of BMS-470539 involved the suppression of microglia activation, promotion of CD206+ microglia transformation and reduction of neutrophil infiltration by increasing the levels of p-AMPK and p-TBK1 while decreasing the levels of NF-κB, IL-1ß, and TNFα. The neuroprotective effects of BMS-470539 were significantly abolished by MSG-606 and MRT-68601. The activation of MC1R with BMS-470539 notably attenuates EBI after SAH by suppression of microglial activation and neutrophil infiltration via the AMPK/TBK1/NF-κB signaling pathway.

Hydrogen Inhalation Attenuates Oxidative Stress Related Endothelial Cells Injury After Subarachnoid Hemorrhage in Rats.

Background: Subarachnoid hemorrhage (SAH) is a devastating cerebrovascular disease with poor clinical outcome. Nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome serves a key role in inflammatory response, which may lead to endothelial cell injury and blood-brain barrier (BBB) disruption. Hydrogen (H2) is considered a neuroprotective antioxidant. This study was set out to explore whether hydrogen inhalation protects against SAH induced endothelial cell injury, BBB disruption, microthrombosis and vasospasm in rats. Methods: One hundred eighty-two male SD rats were used for the study. SAH was induced by endovascular perforation. H2 at a concentration of 3.3% was inhaled beginning at 0.5 h after SAH for duration of 30, 60 or 120 min, followed by single administration or once daily administration for 3 days. The temporal expression of NLRP3 and ASC in the brain was determined, with the effect of hydrogen inhalation evaluated. In addition, brain water content, oxidative stress markers, inflammasome, apoptotic markers, microthrombosis, and vasospasm were evaluated at 24 or 72 h after SAH. Results: The expression of NLRP3 and ASC were upregulated after SAH associated with elevated expression of MDA, 8-OHdG, 4-HNE, HO-1, TLR4/NF-κB, inflammatory and apoptotic makers. Hydrogen inhalation reduced the expression of these inflammatory and apoptotic makers in the vessels, brain edema, microthrombi formation, and vasospasm in rats with SAH relative to control. Hydrogen inhalation also improved short-term and long-term neurological recovery after SAH. Conclusion: Hydrogen inhalation can ameliorate oxidative stress related endothelial cells injury in the brain and improve neurobehavioral outcomes in rats following SAH. Mechanistically, the above beneficial effects might be related to, at least in part, the inhibition of activation of ROS/NLRP3 axis.

Neurogenesis changes and the fate of progenitor cells after subarachnoid hemorrhage in rats.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a devastating cerebrovascular disease that leads to poor outcomes. Neurogenesis, an essential recovery mechanism after brain injury, has not been fully elucidated after SAH. METHODS: A total of 122 SD rats were used in this study. For experiment one, the rats were randomly divided into six groups: sham and SAH with different time points (1,3,5,7,14 days) (n = 12/group). An endovascular perforation method was conducted for SAH model. Rats were injected with 5-Bromo-2'-deoxyuridine (BrdU, 50 mg/kg) 24 h before euthanasia at different time points after SAH. The BrdU labeled cells were detected by immunohistochemistry; Doublecortin (DCX) and glial fibrillary acidic protein (GFAP) were measured by western blot and immunohistochemistry. For experiment two, rats were randomly divided into five groups: sham and SAH with different time points (1, 2, 4, 8 weeks) (n = 6/group). Rats received BrdU (50 mg/kg) once daily for 7 days after the induction of SAH. Double immunofluorescence staining was used to verify proliferation, differentiation and migration of progenitor cells. Rotarod test and water maze used to test the neurobehavioral recovery. RESULTS: Our results showed that BrdU positive cells in hippocampus changed overtime after SAH. BrdU positive cells decreased as early as 1 day reaching lowest levels at 3 days after SAH, after which it gradually recovered. Similar change patterns were observed with DCX, which was reversed with GFAP. In addition, BrdU did not co-localize with cleaved caspase-3. The BrdU positive cells mainly differentiated into immature neurons for short-term fate, whereas they differentiated into mature neurons for long-term fate but not astrocytes, which facilitated neurobehavioral recovery after SAH. CONCLUSION: Neurogenesis in the hippocampus changes overtime after SAH. The neuronal progenitor cells may play an essential role in the neurobehavioral recovery after brain injury induced by SAH, since short-term progenitors helped with the recovery of immature neurons in the hippocampus, whereas long-term progenitors differentiated into mature neurons.

The MC4 receptor agonist RO27-3225 inhibits NLRP1-dependent neuronal pyroptosis via the ASK1/JNK/p38 MAPK pathway in a mouse model of intracerebral haemorrhage.

BACKGROUND AND PURPOSE: Inflammasome-mediated pyroptosis is an important neuronal cell death mechanism. Previous studies reported that activation of melanocortin MC4 receptor exerted neuroprotection in several neurological diseases. Here, we have investigated the role of MC4 receptor activation with RO27-3225 in suppressing neuronal pyroptosis after experimental intracerebral haemorrhage (ICH) and the underlying mechanism. EXPERIMENTAL APPROACH: One hundred and sixty-nine male CD1 mice were used. ICH was induced by injection of bacterial collagenase into the right-side basal ganglia. RO27-3225, a selective agonist of MC4 receptor, was injected intraperitoneally at 1 hr after ICH. To elucidate the underlying mechanism, we used the specific MC4 receptor antagonist HS024 and NQDI-1, a specific inhibitor of the apoptosis signalling-regulating kinase 1 (ASK1). Neurological tests, Western blot, Fluoro-Jade C, TUNEL, and immunofluorescence staining were conducted. KEY RESULTS: Expression of MC4 receptor and the NOD-like receptor family, pyrin domain containing 1 (NLRP1) inflammasome in brain were increased after ICH. RO27-3225 treatment decreased neuronal pyroptosis and neurobehavioural deficits at 24 and 72 hr after ICH. RO27-3225 reduced the expression of p-ASK1, p-JNK, p-p38 MAPK, NLRP1 inflammasome, cleaved caspase-1, and IL-1ß after ICH. HS024 pretreatment prevented the effects of RO27-3225. Similar to RO27-3225, NQDI-1 alone improved neurological functions and down-regulated ASK1/JNK/p38MAPK expression after ICH. CONCLUSIONS AND IMPLICATIONS: RO27-3225 suppressed NLRP1-dependent neuronal pyroptosis and improved neurological function, possibly mediated by activation of MC4 receptor and inhibition of ASK1/JNK/p38 MAPK signalling pathways, after experimental ICH in mice. The MC4 receptor may be a promising therapeutic target for the management of ICH.

Activation of GPR30 with G1 attenuates neuronal apoptosis via src/EGFR/stat3 signaling pathway after subarachnoid hemorrhage in male rats.

BACKGROUND: Neuron apoptosis plays a vital role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). Previous studies showed that the activation of G protein-coupled receptor 30 (GPR30) with GPR30 agonist G1 was anti-apoptotic after experimental trauma brain injury and global cerebral ischemia in male rats or mice. However, the role of GPR30 activation with G1 has not been clarified in SAH. The aim of this study was to investigate the anti-apoptotic effect of GPR30 activation and the underlying mechanism of src/EGFR/stat3 signaling pathway in a male rat model of SAH. METHODS: A total of 215 male rats and 18 female rats were used. SAH was induced by intravascular perforation. G1 was administrated intravenously 1 h after SAH. For mechanism study, the GPR30 antagonist G15 or epidermal growth factor receptor (EGFR) antagonist AG1478 was administrated intravenously 1 h before SAH, small interfering ribonucleic acid (siRNA) for GPR30 and EGFR were administered intracerebroventricularly 48 h before SAH. Post-SAH assessments included SAH Grade, neurological deficits, western blot, terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) staining, Fluoro-Jade C (FJC) staining, Nissl staining and immunofluorescence. RESULTS: The expression of endogenous GPR30 in male rats was increased at 3 h and peaked at 24 h after SAH, which mainly co-localized with neurons, but there was no significant increase in intact female rats at 24 h after SAH. The G1 post-treatment significantly reduced the short-term and long-term neurological deficit as well as neuronal apoptosis in male rats, but it did not significantly improve the short-term outcome of intact female rats. Mechanistic studies indicated that G15 or GPR30 siRNA and AG1478 or EGFR siRNA reversed the anti-neuronal apoptosis effects of G1 and its effects on protein expressions of src/EGFR/stat3 signaling pathway. CONCLUSION: G1 reduced EBI through attenuating neuronal apoptosis after SAH in male rats, partly via activating src/EGFR/stat3/signaling pathway. G1 may provide a promising therapeutic strategy for SAH patients.

LRP1 activation attenuates white matter injury by modulating microglial polarization through Shc1/PI3K/Akt pathway after subarachnoid hemorrhage in rats.

White matter injury (WMI) is associated with motor deficits and cognitive dysfunctions in subarachnoid hemorrhage (SAH) patients. Therapeutic strategy targeting WMI would likely improve the neurological outcomes after SAH. Low-density lipoprotein receptor-related protein-1 (LRP1), a scavenger receptor of apolipoprotein E (apoE), is able to modulate microglia polarization towards anti-inflammatory M2 phenotypes during inflammatory and oxidative insult. In the present study, we investigated the effects of LRP1 activation on WMI and underlying mechanisms of M2 microglial polarization in a rat model of SAH. Two hundred and seventeen male Sprague Dawley rats (weight 280-330 g) were used. SAH was induced by endovascular perforation. LPR1 ligand, apoE-mimic peptide COG1410 was administered intraperitoneally. Microglial depletion kit liposomal clodronate (CLP), LPR1 siRNA or PI3K inhibitor were administered intracerebroventricularly. Post-SAH assessments included neurobehavioral tests, brain water content, immunohistochemistry, Golgi staining, western blot and co-immunoprecipitation. SAH induced WMI shown as the accumulation of amyloid precursor protein and neurofilament heavy polypeptide as well as myelin loss. Microglial depletion by CLP significantly suppressed WMI after SAH. COG1410 reduced brain water content, increased the anti-inflammatory M2 microglial phenotypes, attenuated WMI and improved neurological function after SAH. LRP1 was bound with endogenous apoE and intracellular adaptor protein Shc1. The benefits of COG1410 were reversed by LPR1 siRNA or PI3K inhibitor. LRP1 activation attenuated WMI and improved neurological function by modulating M2 microglial polarization at least in part through Shc1/PI3K/Akt signaling in a rat model of SAH. The apoE-mimic peptide COG1410 may serve as a promising treatment in the management of SAH patients.

Milk Fat Globule-Epidermal Growth Factor-8 Pretreatment Attenuates Apoptosis and Inflammation via the Integrin-ß3 Pathway after Surgical Brain Injury in Rats.

Iatrogenic brain injury inevitably occurs in neurosurgical operations, leading to brain edema, ischemia, intracranial hematoma, and other postoperative complications, eventually worsening neurological outcomes of patients. If apoptotic cells are not rapidly eliminated by phagocytic engulfment, they may communicate with surrounding cells to undergo secondary necrosis and releasing toxic signals. Recent studies have shown that milk fat globule-epidermal growth factor-8 (MFGE8), which promotes phagocytosis and inhibits inflammation, is an endogenous protective factor in response to brain infarction, Alzheimer's disease, subarachnoid hemorrhage, and prion disease. In the present study, we sought to investigate the different effects of both pretreated and posttreated recombinant milk fat globule-epidermal growth factor-8 (rhMFGE8) for the surgical brain injury (SBI) rat model and potential involvement of its receptor integrin ß3 for apoptosis and neuroinflammation after SBI. One hundred and sixty-seven male rats were employed in the preset study. Experiment 1 was performed to evaluate neurological scores and MFGE8, cleaved caspase-3 (CC3), and interleukine-1 beta (IL-1ß) levels at 3, 24, and 120 h after SBI. Experiment 2 was performed to evaluate the effects of rhMFGE8 pretreatment (10 min before SBI) and rhMFGE8 posttreatment (6 h after SBI) on brain edema at 24 and 72 h after SBI. Experiment 3 was performed to evaluate the potential anti-apoptotic and anti-inflammatory effects of rhMFGE8 pretreatment and posttreatment. Experiment 4 sought to investigate the involvement of the integrin-ß3 signal in the effects of MFGE8 pretreatment. Our data showed rhMFGE8 pretreatment alleviated neurological deficits and decreased brain water content and apoptotic cells in the SBI model, which exhibited neurological dysfunction, apoptosis, and inflammation. Meanwhile, MFGE8 siRNA, which inhibited endogenous MFGE8 expression, significantly increased IL-1ß, TUNEL positive cells, and CC3. Furthermore, knockdown of its receptor integrin ß3 by siRNA abolished the effects of rhMFGE8 in the SBI model. In conclusion, we found that rhMFGE8 pretreatment effectively alleviated neurological deficits and decreased brain water content and apoptotic cells in the SBI model through the MFGE8/integrin-ß3 pathway, and treatment time was an important factor in achieving curative effects. Therefore, MFGE8 pretreatment may serve as a promising therapeutic strategy for SBI patients.

Subarachnoid hemorrhage enhances the expression of TDP-43 in the brain of experimental rats and human subjects.

The transactive response DNA-binding protein of 43 (TDP-43) may be involved in neurodegenerative disease and in the response to brain injury; however, alterations in the expression of TDP-43 following subarachnoid hemorrhage (SAH) require further investigation. The present study reported a notable elevation in the expression of TDP-43 within the cerebrospinal fluid (CSF) of patients with aneurysmal SAH and increased brain expression of TDP-43 in a rat model of SAH. The TDP-43 protein and a derivative migrated at 43 and 24 kDa, respectively, as observed via the immunoblotting of concentrated CSF samples obtained from patients with SAH; no signal was detected in the CSF from healthy controls. SAH in rats was induced by intravascular suture puncture. The expression levels of TDP-43 in rat cortical lysates following SAH were increased at 0.5 h, peaked at 48 h and remained significantly elevated at 72 h post-injury, compared with sham controls. TDP-43 immunolabeling indication localization within neurons, astrocytes and microglia in the experimental rats. Collectively, the findings of the present study indicated the early involvement of TDP-43 in the brain in response to SAH, and that expression levels of TDP-43 in the CSF may serve as a prognostic biomarker among patients with this condition.

Inhibition of Heat Shock Protein 90 by 17-AAG Reduces Inflammation via P2X7 Receptor/NLRP3 Inflammasome Pathway and Increases Neurogenesis After Subarachnoid Hemorrhage in Mice.

Subarachnoid hemorrhage (SAH) is a life-threatening cerebrovascular disease that usually has a poor prognosis. Heat shock proteins (HSPs) have been implicated in the mechanisms of SAH-associated damage, including increased inflammation and reduced neurogenesis. The aim of this study was to investigate the effects of HSP90 inhibition on inflammation and neurogenesis in a mouse model of experimental SAH induced by endovascular surgery. Western blotting showed HSP90 levels to be decreased, while neurogenesis, evaluated by 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry, was decreased in the hippocampuses of SAH mice. SAH also induced pro-inflammatory factors such as interleukin-1ß (IL-1ß), capase-1 and the NLRP3 inflammasome. However, intraperitoneal administration of the specific HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) reduced the levels of HSP90, NLRP3, ASC, caspase-1 and IL-1ß, while increasing the levels of brain-derived neurotrophic factor and doublecortin (DCX), as well as the number of BrdU-positive cells in SAH mice. In addition, 17-AGG improved short- and long-term neurobehavioral outcomes. The neuroprotective and anti-inflammatory effects of 17-AGG were reversed by recombinant HSP90 (rHSP90); this detrimental effect of HSP90 was inhibited by the specific P2X7 receptor (P2X7R) inhibitor A438079, indicating that SAH-induced inflammation and inhibition of neurogenesis were likely mediated by HSP90 and the P2X7R/NLRP3 inflammasome pathway. HSP90 inhibition by 17-AAG may be a promising therapeutic strategy for the treatment of SAH.