Analysis of different expression RNA binding protein genes in mouse microglia cell from the brains of mice 72 h after subarachnoid hemorrhage or sham operation.

BACKGROUND: The prognosis of brain injury caused by subarachnoid hemorrhage (SAH) is poor. Previous studies showed that abnormal function of RBPs might be involved in brain injury, neuroinflammation and further affect microglia homeostasis. However, no studies have systematically analyzed the genome-wide abnormal expression of RBPs genes in microglia during SAH. METHODS: RNA-seq data of microglia from the SAH mouse group (SAH) and control sham-operated mouse group (sham) were downloaded from the GEO database in GSE167957, including four samples from the sham group and four samples from the SAH group for subsequent analysis.Utilizing GO and KEGG functional enrichment analyses, we conducted a comprehensive study of differentially expressed genes (DEGs), alternative splicing patterns, and co-expression networks to gain deeper insights into the differential expression of RNA-binding proteins (RBPs) and differential alternative splicing events (ASEs) between the SAH (subarachnoid hemorrhage) and sham groups. This analysis aimed to elucidate the potential mechanisms underlying the aberrant expression of RBPs in microglia during brain injury caused by SAH. RESULTS: ASEs and co-expression analyses of differentially expressed RBPs and differential ASEs were carried out in microglia in terms of gene expression. GO and KEGG functional enrichment analysis showed that aberrantly expressed RBPs such as Mcm7, Mtdh, SRSF3, and Hnrnpa2b1 may affect and regulate downstream Csnk1d, Uckl1 and other protein phosphorylation-related genes by alterative splicing. CONCLUSION: RBPs were aberrantly expressed in microglia during the development of brain injury secondary to SAH, regulating alterative splicing of downstream genes and influencing the progression of SAH brain injury in this study. This implies that RBPs are important for the identification of new therapeutic targets for brain injury after SAH.

Injection of Collagen Binding Domain-Brain Derived Neurotrophic Factor Promotes SIRT1 Expression: Improving Neuroinflammation in Experimental Subarachnoid Hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe cerebrovascular disease, often leading to neuroinflammation and neuronal damage. Activation of the Nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is closely associated with post-SAH neuroinflammation, while activation of Nicotinamide Adenine Dinucleotide (NAD)-dependent deacetylase sirtuin-1 (SIRT1) has neuroprotective effects. This study aimed to investigate the impact of injectable Collagen Binding Domain-Brain Derived Neurotrophic Factor (CBD-BDNF) on neuroinflammation and neuronal damage following SAH. METHODS: After establishing the SAH model, experimental animals were divided into three groups: sham surgery group (Sham), SAH group, and SAH+neuroregenerative scaffold (CBD-BDNF treatment) group. Behavioral performance was evaluated using neurofunctional deficit, beam balance, and Y-maze tests. Expression of inflammatory factors and essential proteins was quantitatively analyzed using Enzyme-Linked Immunosorbent Assay (ELISA) kits and immunoblotting. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) staining was used to assess cell apoptosis. To further investigate the mechanism of action of CBD-BDNF on SIRT1, the model animals were treated with EX527 (SIRT1 inhibitor) for comparative studies. RESULTS: Neurological deficit tests, CBD-BDNF improves functional outcomes after SAH. Compared to the SAH group, the SAH+neuroregenerative scaffold group showed significantly increased expression of SIRT1 protein and significantly decreased expression of NLRP3, Apoptosis-associated speck-like protein containing a CARD (ASC), and c-caspase-1. The inflammatory cytokines Interleukin-1 beta (IL-1ß), IL-6, and IL-18 levels also significantly decreased in the SAH+neuroregenerative scaffold group. Additionally, animals in the SAH+neuroregenerative scaffold group showed better neurofunctional recovery in neurofunctional deficit and beam balance tests. The number of apoptotic cells significantly decreased in the SAH+neuroregenerative scaffold group compared to the SAH group. However, when SIRT1 was inhibited with EX527, the aforementioned neuroprotective effects were reversed, indicating the involvement of CBD-BDNF through SIRT1 activation. CONCLUSION: This study demonstrates that injectable CBD-BDNF can significantly alleviate neuroinflammation and neuronal damage resulting from SAH by blocking NLRP3 inflammasome activation and promoting SIRT1 expression. These findings provide a new therapeutic strategy for neuroprotection after SAH and reveal the mechanism of action of CBD-BDNF as a potential therapeutic agent. Future research will further explore the long-term efficacy and safety of CBD-BDNF.

Lactylation of histone by BRD4 regulates astrocyte polarization after experimental subarachnoid hemorrhage.

Under subarachnoid hemorrhage (SAH) conditions, astrocytes undergo a marked intensification of glycolytic activity, resulting in the generation of substantial amounts of lactate to maintain the energy demand for neurons and other brain cells. Lactate has garnered increasing attention in recent years because of its emerging role in critical biological processes such as inflammation regulation and neuroprotection, particularly through its histone lactylation. Bromodomain-containing protein 4 (BRD4) plays a crucial role in maintaining neural development and promoting memory formation in the central nervous system. Nonetheless, the function and regulatory mechanism of BRD4 and histone lactylation in astrocytes following SAH remain elusive. Our findings indicate that BRD4, a crucial epigenetic regulator, plays a definitive role in histone lactylation. Both in vitro and in vivo, these results demonstrated that targeted silencing of BRD4 in astrocytes can significantly reduce H4K8la lactylation, thereby aggravating the A1 polarization of astrocytes and ultimately affecting the recovery of neural function and prognosis in mice after SAH. In summary, BRD4 plays a pivotal role in modulating astrocyte polarization following SAH via histone lactylation. Targeting this mechanism might offer an efficient therapeutic strategy for SAH.

Microglia aggravate white matter injury via C3/C3aR pathway after experimental subarachnoid hemorrhage.

The activation of glial cells is intimately associated with the pathophysiology of neuroinflammation and white matter injury (WMI) during both acute and chronic phases following subarachnoid hemorrhage (SAH). The complement C3a receptor (C3aR) has a dual role in modulating inflammation and contributes to neurodevelopment, neuroplasticity, and neurodegeneration. However, its impact on WMI in the context of SAH remains unclear. In this study, 175 male C57BL/6J mice underwent SAH through endovascular perforation. Oxyhemoglobin (oxy-Hb) was employed to simulate SAH in vitro. A suite of techniques, including immunohistochemistry, transcriptomic sequencing, and a range of molecular biotechnologies, were utilized to evaluate the activation of the C3-C3aR pathway on microglial polarization and WMI. Results revealed that post-SAH abnormal activation of microglia was accompanied by upregulation of complement C3 and C3aR. The inhibition of C3aR decreased abnormal microglial activation, attenuated neuroinflammation, and ameliorated WMI and cognitive deficits following SAH. RNA-Seq indicated that C3aR inhibition downregulated several immune and inflammatory pathways and mitigated cellular injury by reducing p53-induced death domain protein 1 (Pidd1) and Protein kinase RNA-like ER kinase (Perk) expression, two factors mainly function in sensing and responding to cellular stress and endoplasmic reticulum (ER) stress. The deleterious effects of the C3-C3aR axis in the context of SAH may be related to endoplasmic reticulum (ER) stress-dependent cellular injury and inflammasome formation. Agonists of Perk can exacerbate the cellular injury and neuroinflammation, which was attenuated by C3aR inhibition after SAH. Additionally, intranasal administration of C3a during the subacute phase of SAH was found to decrease astrocyte reactivity and alleviate cognitive deficits post-SAH. This research deepens our understanding of the complex pathophysiology of WMI following SAH and underscores the therapeutic potential of C3a treatment in promoting white matter repair and enhancing functional recovery prognosis. These insights pave the way for future clinical application of C3a-based therapies, promising significant benefits in the treatment of SAH and its related complications.

Protective Effects of Adropin in Experimental Subarachnoid Hemorrhage.

PURPOSE: We aimed to investigate early effects of exogenously administered adropin (AD) on neurological function, endothelial nitric oxide synthase (eNOS) expression, nitrite/nitrate levels, oxidative stress, and apoptosis in subarachnoid hemorrhage (SAH). METHODS: Following intracerebroventricular AD administration (10 µg/5 µl at a rate of 1 µl/min) SAH model was carried out in Sprague-Dawley rats by injection of autologous blood into the prechiasmatic cistern. The effects of AD were assessed 24 h following SAH. The modified Garcia score was employed to evaluate functional insufficiencies. Adropin and caspase-3 proteins were measured by ELISA, while nitrite/nitrate levels, total antioxidant capacity (TAC) and reactive oxygen/nitrogen species (ROS/RNS) were assayed by standard kits. eNOS expression and apoptotic neurons were detected by immunohistochemical analysis. RESULTS: The SAH group performed notably lower on the modified Garcia score compared to sham and SAH + AD groups. Adropin administration increased brain eNOS expression, nitrite/nitrate and AD levels compared to SHAM and SAH groups. SAH produced enhanced ROS/RNS generation and reduced antioxidant capacity in the brain. Adropin boosted brain TAC and diminished ROS/RNS production in SAH rats and no considerable change amongst SHAM and SAH + AD groups were detected. Apoptotic cells were notably increased in intensity and number after SAH and were reduced by AD administration. CONCLUSIONS: Adropin increases eNOS expression and reduces neurobehavioral deficits, oxidative stress, and apoptotic cell death in SAH model. Presented results indicate that AD provides protection in early brain injury associated with SAH.

PARP-1 dependent cell death pathway (Parthanatos) mediates early brain injury after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a neurological condition with high mortality and poor prognosis, and there are currently no effective therapeutic drugs available. Poly (ADP-ribose) polymerase 1 (PARP-1) dependent cell death pathway-parthanatos is closely associated with stroke. We investigated improvements in neurological function, oxidative stress, blood-brain barrier and parthanatos-related protein expression in rats with SAH after intraperitoneal administration of PARP-1 inhibitor (AG14361). Our study found that the expression of parthanatos-related proteins was significantly increased after SAH. Immunofluorescence staining showed increased expression of apoptosis-inducing factor (AIF) in the nucleus after SAH. Administration of PARP-1 inhibitor significantly reduced malondialdehyde (MDA) level and the expression of parthanatos-related proteins. Immunofluorescence staining showed that PARP-1 inhibitor reduced the expression of 8-hydroxy-2' -deoxyguanosine (8-OHdG) and thus reduced oxidative stress. Moreover, PARP-1 inhibitor could inhibit inflammation-associated proteins level and neuronal apoptosis, protect the blood-brain barrier and significantly improve neurological function after SAH. These results suggest that PARP-1 inhibitor can significantly improve SAH, and the underlying mechanism may be through inhibiting parthanatos pathway.

BCAT1 alleviates early brain injury by inhibiting ferroptosis through PI3K/AKT/mTOR/GPX4 pathway after subarachnoid hemorrhage.

Regulation of the redox system by branched-chain amino acid transferase 1 (BCAT1) is of great significance in the occurrence and development of diseases, but the relationship between BCAT1 and subarachnoid hemorrhage (SAH) is still unknown. Ferroptosis, featured by iron-dependent lipid peroxidation accompanied by the depletion of glutathione peroxidase 4 (GPX4), has been implicated in the pathological process of early brain injury after subarachnoid hemorrhage. This study established SAH model by endovascular perforation and adding oxyhemoglobin (Hb) to HT22 cells and delved into the mechanism of BCAT1 in SAH-induced ferroptotic neuronal cell death. It was found that SAH-induced neuronal ferroptosis could be inhibited by BCAT1 overexpression (OE) in rats and HT22 cells, and BCAT1 OE alleviated neurological deficits and cognitive dysfunction in rats after SAH. In addition, the effect of BCAT1 could be reversed by the Ly294002, a specific inhibitor of the PI3K pathway. In summary, our present study indicated that BCAT1 OE alleviated early brain injury EBI after SAH by inhibiting neuron ferroptosis via activation of PI3K/AKT/mTOR pathway and the elevation of GPX4. These results suggested that BCAT1 was a promising therapeutic target for subarachnoid hemorrhage.

GPR30 alleviated subarachnoid hemorrhage-induced blood-brain barrier dysfunction by activating the PI3K/Akt and Nrf2/HO-1 pathways.

The blood-brain barrier (BBB) plays a critical role in the development and outcome of subarachnoid hemorrhage (SAH). This study focuses on the potential mechanism by which G-protein-coupled estrogen receptor 30 (GPR30) affects the BBB after SAH. A rat SAH model was established using an intravascular perforation approach. G1 (GPR30 agonist) was administered to investigate the mechanism of BBB damage after SAH. Brain water content, Western blotting, Evans blue leakage, and immunofluorescence staining were performed. Brain microvascular endothelial cells were induced by hemin to establish SAH model in vitro. By adding LY294002 [a phosphatidylinositol 3-kinase (PI3K) blocker] and zinc protoporphyrin IX (ZnPP IX) [a heme oxygenase 1 (HO-1) antagonist], the mechanism of improving BBB integrity through the activation of GPR30 was studied. In vivo, GPR30 activation improved BBB disruption, as evidenced by decreased cerebral edema, downregulated albumin expression, and reduced extravasation of Evans blue and IgG after G1 administration in SAH rats. Moreover, SAH downregulated the levels of tight junction (TJ) proteins, whereas treatment with G1 reversed the effect of SAH. The protective effect of G1 on BBB integrity in vitro was consistent with that in vivo, as evidenced by G1 reducing the impact of hemin on transendothelial electrical resistance (TEER) value, dextran diffusivity, and TJ protein levels in brain microvascular endothelial cells. In addition, G1 activated the PI3K/ protein kinase B (Akt) and nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 pathways both in vivo and in vitro. Furthermore, the administration of LY294002 and ZnPP IX partially reversed the protective effect of G1 on BBB integrity in hemin-stimulated cells. We demonstrated that the activation of GPR30, at least partly through the PI3K/Akt and Nrf2/HO-1 pathways, alleviated BBB damage both in vivo and in vitro. This study introduced a novel therapeutic approach for protecting the BBB after SAH.NEW & NOTEWORTHY The PI3K/Akt and Nrf2/HO-1 pathways might be potential mechanisms by which GPR30 protected the integrity of the BBB in SAH models. Therefore, treatment of SAH with GPR30 activator might be a promising therapeutic strategy.

Validation of a simplified model for subarachnoid hemorrhage in mice.

BACKGROUND: Subarachnoid hemorrhage (SAH) represents a severe injury to the brain and is associated with a high mortality (40%). Several experimental SAH models are described in the literature requiring specialized equipment and a high degree of surgical expertise. Our goal was to validate a simplified, cost-effective model to permit future studies of SAH. METHODS: SAH was induced by injection of homologous blood into the cisterna magna. Perfusion-fixation then perfusion of gelatinous India ink was performed. Brains and brainstems were collected and imaged for analysis of cerebral vasospasm. Triphenyl tetrazolium chloride (TTC) staining was used to analyze brain tissue cell death 24 hours following stroke. A composite neuroscore was utilized to assess SAH-related neurologic deficits. RESULTS: Anterior cerebral artery and basilary artery diameters were significantly reduced at 24 hours post SAH induction. Middle cerebral artery diameter was also reduced; however, the results were not significant. TTC staining showed no infarcted tissue. Neuroscores were significantly lower in the SAH mice, indicating the presence of functional deficits. CONCLUSIONS: This simplified model of SAH elicits pathological changes consistent with those described for more complex models in the literature. Therefore, it can be used in future preclinical studies examining the pathophysiology of SAH and novel treatment options.

Oroxin A alleviates early brain injury after subarachnoid hemorrhage by regulating ferroptosis and neuroinflammation.

BACKGROUND: Subarachnoid hemorrhage (SAH), a severe subtype of stroke, is characterized by notably high mortality and morbidity, largely due to the lack of effective therapeutic options. Although the neuroprotective potential of PPARg and Nrf2 has been recognized, investigative efforts into oroxin A (OA), remain limited in preclinical studies. METHODS: SAH was modeled in vivo through filament perforation in male C57BL/6 mice and in vitro by exposing HT22 cells to hemin to induce neuronal damage. Following the administration of OA, a series of methods were employed to assess neurological behaviors, brain water content, neuronal damage, cell ferroptosis, and the extent of neuroinflammation. RESULTS: The findings indicated that OA treatment markedly improved survival rates, enhanced neurological functions, mitigated neuronal death and brain edema, and attenuated the inflammatory response. These effects of OA were linked to the suppression of microglial activation. Moreover, OA administration was found to diminish ferroptosis in neuronal cells, a critical factor in early brain injury (EBI) following SAH. Further mechanistic investigations uncovered that OA facilitated the translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) from the cytoplasm to the nucleus, thereby activating the Nrf2/GPX4 pathway. Importantly, OA also upregulated the expression of FSP1, suggesting a significant and parallel protective effect against ferroptosis in EBI following SAH in synergy with GPX4. CONCLUSION: In summary, this research indicated that the PPARg activator OA augmented the neurological results in rodent models and diminished neuronal death. This neuroprotection was achieved primarily by suppressing neuronal ferroptosis. The underlying mechanism was associated with the alleviation of cellular death through the Nrf2/GPX4 and FSP1/CoQ10 pathways.

FGF21 attenuates neuroinflammation following subarachnoid hemorrhage through promoting mitophagy and inhibiting the cGAS-STING pathway.

BACKGROUND: Subarachnoid hemorrhage (SAH) represents a form of cerebrovascular event characterized by a notable mortality and morbidity rate. Fibroblast growth factor 21 (FGF21), a versatile hormone predominantly synthesized by the hepatic tissue, has emerged as a promising neuroprotective agent. Nevertheless, the precise impacts and underlying mechanisms of FGF21 in the context of SAH remain enigmatic. METHODS: To elucidate the role of FGF21 in inhibiting the microglial cGAS-STING pathway and providing protection against SAH-induced cerebral injury, a series of cellular and molecular techniques, including western blot analysis, real-time polymerase chain reaction, immunohistochemistry, RNA sequencing, and behavioral assays, were employed. RESULTS: Administration of recombinant fibroblast growth factor 21 (rFGF21) effectively mitigated neural apoptosis, improved cerebral edema, and attenuated neurological impairments post-SAH. Transcriptomic analysis revealed that SAH triggered the upregulation of numerous genes linked to innate immunity, particularly those involved in the type I interferon (IFN-I) pathway and microglial function, which were notably suppressed upon adjunctive rFGF21 treatment. Mechanistically, rFGF21 intervention facilitated mitophagy in an AMP-activated protein kinase (AMPK)-dependent manner, thereby preventing mitochondrial DNA (mtDNA) release into the cytoplasm and dampening the activation of the DNA-sensing cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. Conditional knockout of STING in microglia markedly ameliorated the inflammatory response and mitigated secondary brain injuries post-SAH. CONCLUSION: Our results present the initial evidence that FGF21 confers a protective effect against neuroinflammation-associated brain damage subsequent to SAH. Mechanistically, we have elucidated a novel pathway by which FGF21 exerts this neuroprotection through inhibition of the cGAS-STING signaling cascade.

Establishment of a novel protocol for assessing the severity of subarachnoid hemorrhage in circle Willis perforation mouse model.

The Circle of Willis perforation (cWp) mouse model is a key tool in subarachnoid hemorrhage (SAH) research; however, inconsistent bleeding volumes can challenge experimental reliability. To address this issue, we introduced the ROB Scoring System, a novel protocol integrating Rotarod Tests (RT), Open-field Tests (OT) video analysis, and daily Body Weight Loss (BWL) monitoring to precisely categorize SAH severity. Forty C57BL/6 mice underwent cWp SAH induction, categorized by ROB into severity subgroups (severe, moderate, mild). Validation compared ROB trends in subgroups, and ROB outcomes with autopsy results on postoperative days three and seven for acute and sub-acute evaluations. Mortality rates were analyzed via the survival log-rank test, revealing a significant difference among SAH subgroups (P < 0.05). Strong correlations between ROB grades and autopsy findings underscored its precision. Notably, the severe group exhibited 100% mortality within 4 days post SAH onset. Single parameters (RT, OT, BWL) were insufficient for distinguishing SAH severity levels. The ROB score represents a significant advancement, offering an objective method for precise categorization and addressing inherent bleeding variations in the cWp SAH model. This standardized protocol enhances the reliability and effectiveness of the SAH translational research, providing a valuable tool for future investigations into this critical area.

p97 inhibits integrated stress response-induced neuronal apoptosis after subarachnoid hemorrhage in mice by enhancing proteasome function.

Neuronal apoptosis is a common pathological change in early brain injury after subarachnoid hemorrhage (SAH), and it is closely associated with neurological deficits. According to previous research, p97 exhibits a remarkable anti-cardiomyocyte apoptosis effect. p97 is a critical molecule in the growth and development of the nervous system. However, it remains unknown whether p97 can exert an anti-neuronal apoptosis effect in SAH. In the present study, we examined the role of p97 in neuronal apoptosis induced after SAH and investigated the underlying mechanism. We established an in vivo SAH mice model and overexpressed the p97 protein through transfection of the mouse cerebral cortex. We analyzed the protective effect of p97 on neurons and evaluated short-term and long-term neurobehavior in mice after SAH. p97 was found to be significantly downregulated in the cerebral cortex of the affected side in mice after SAH. The site showing reduced p97 expression also exhibited a high level of neuronal apoptosis. Adeno-associated virus-mediated overexpression of p97 significantly reduced the extent of neuronal apoptosis, improved early and long-term neurological function, and repaired the neuronal damage in the long term. These neuroprotective effects were accompanied by enhanced proteasome function and inhibition of the integrated stress response (ISR) apoptotic pathway involving eIF2α/CHOP. The administration of the p97 inhibitor NMS-873 induced a contradictory effect. Subsequently, we observed that inhibiting the function of the proteasome with the proteasome inhibitor PS-341 blocked the anti-neuronal apoptosis effect of p97 and enhanced the activation of the ISR apoptotic pathway. However, the detrimental effects of NMS-873 and PS-341 in mice with SAH were mitigated by the administration of the ISR inhibitor ISRIB. These results suggest that p97 can promote neuronal survival and improve neurological function in mice after SAH. The anti-neuronal apoptosis effect of p97 is achieved by enhancing proteasome function and inhibiting the overactivation of the ISR apoptotic pathway.

Galectin-3 modulates microglial activation and neuroinflammation in early brain injury after subarachnoid hemorrhage.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (SAH) is a devastating acute cerebrovascular event with high mortality and permanent disability rates. Higher galectin-3 levels on days 1-3 have been shown to predict the development of delayed cerebral infarction or adverse outcomes after SAH. Recent single-cell analysis of microglial transcriptomic diversity in SAH revealed that galectin could influence the development and course of neuroinflammation after SAH. METHODS: This study aimed to investigate the role and mechanism of galectin-3 in SAH and to determine whether galectin-3 inhibition prevents early brain injury by reducing microglia polarization using a mouse model of SAH and oxyhemoglobin-treated activation of mouse BV2 cells in vitro. RESULTS: We found that the expression of galectin-3 began to increase 12 h after SAH and continued to increase up to 72 h. Importantly, TD139-inhibited galectin-3 expression reduced the release of inflammatory factors in microglial cells. In the experimental SAH model, TD139 treatment alleviated neuroinflammatory damage after SAH and improved defects in neurological functions. Furthermore, we demonstrated that galectin-3 inhibition affected the activation and M1 polarization of microglial cells after SAH. TD139 treatment inhibited the expression of TLR4, p-NF-κB p65, and NF-κB p65 in microglia activated by oxyhemoglobin as well as eliminated the increased expression and phosphorylation of JAK2 and STAT3. CONCLUSION: These findings suggest that regulating microglia polarization by galectin-3 after SAH to improve neuroinflammation may be a potential therapeutic target.

Neuron-targeted liposomal coenzyme Q10 attenuates neuronal ferroptosis after subarachnoid hemorrhage by activating the ferroptosis suppressor protein 1/coenzyme Q10 system.

Subarachnoid hemorrhage (SAH) is primarily attributed to the rupture of intracranial aneurysms and is associated with a high incidence of disability and mortality. SAH disrupts the blood‒brain barrier, leading to the release of iron ions from blood within the subarachnoid space, subsequently inducing neuronal ferroptosis. A recently discovered protein, known as ferroptosis suppressor protein 1 (FSP1), exerts anti-ferroptotic effects by facilitating the conversion of oxidative coenzyme Q 10 (CoQ10) to its reduced form, which effectively scavenges reactive oxygen radicals and mitigates iron-induced ferroptosis. In our investigation, we observed an increase in FSP1 levels following SAH. However, the depletion of CoQ10 caused by SAH hindered the biological function of FSP1. Therefore, we created neuron-targeted liposomal CoQ10 by introducing the neuron-targeting peptide Tet1 onto the surface of liposomal CoQ10. Our objective was to determine whether this formulation could activate the FSP1 system and subsequently inhibit neuronal ferroptosis. Our findings revealed that neuron-targeted liposomal CoQ10 effectively localized to neurons at the lesion site after SAH. Furthermore, it facilitated the upregulation of FSP1, reduced the accumulation of malondialdehyde and reactive oxygen species, inhibited neuronal ferroptosis, and exerted neuroprotective effects both in vitro and in vivo. Our study provides evidence that supplementation with CoQ10 can effectively activate the FSP1 system. Additionally, we developed a neuron-targeted liposomal CoQ10 formulation that can be selectively delivered to neurons at the site of SAH. This innovative approach represents a promising therapeutic strategy for neuronal ferroptosis following SAH. STATEMENT OF SIGNIFICANCE: Subarachnoid hemorrhage (SAH) is primarily attributed to the rupture of intracranial aneurysms and is associated with a high incidence of disability and mortality. Ferroptosis suppressor protein 1 (FSP1), exerts anti-ferroptotic effects by facilitating the conversion of oxidative coenzyme Q 10 (CoQ10) to its reduced form, which effectively scavenges reactive oxygen radicals and mitigates iron-induced ferroptosis. In our investigation, we observed an increase in FSP1 levels following SAH. However, the depletion of CoQ10 caused by SAH hindered the biological function of FSP1. Therefore, we created neuron-targeted liposomal CoQ10. We find that it effectively localized to neurons at the lesion site after SAH and activated the FSP1/CoQ10 system. This innovative approach represents a promising therapeutic strategy for neuronal ferroptosis following SAH and other central nervous system diseases characterized by disruption of the blood-brain barrier.

LAMC1 attenuates neuronal apoptosis via FAK/PI3K/AKT signaling pathway after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The poor prognosis in patients with subarachnoid hemorrhage (SAH) is often attributed to neuronal apoptosis. Recent evidence suggests that Laminin subunit gamma 1 (LAMC1) is essential for cell survival and proliferation. However, the effects of LAMC1 on early brain injury after SAH and the underlying mechanisms are unknown. The current study aimed to reveal the anti-neuronal apoptotic effect and the potential mechanism of LAMC1 in the rat and in the in vitro SAH models. METHODS: The SAH model of Sprague-Dawley rats was established by endovascular perforation. Recombinant LAMC1 (rLAMC1) was administered intranasally 30 min after modeling. LAMC1 small interfering RNA (LAMC1 siRNA), focal adhesion kinase (FAK)-specific inhibitor Y15 and PI3K-specific inhibitor LY294002 were administered before SAH modeling to explore the neuroprotection mechanism of rLAMC1. HT22 cells were cultured and stimulated by oxyhemoglobin to establish an in vitro model of SAH. Subsequently, SAH grades, neurobehavioral tests, brain water content, blood-brain barrier permeability, western blotting, immunofluorescence, TUNEL, and Fluoro-Jade C staining were performed. RESULTS: The expression of endogenous LAMC1 was markedly decreased after SAH, both in vitro and in vivo. rLAMC1 significantly reduced the brain water content and blood-brain barrier permeability, improved short- and long-term neurobehavior, and decreased neuronal apoptosis. Furthermore, rLAMC1 treatment significantly increased the expression of p-FAK, p-PI3K, p-AKT, Bcl-XL, and Bcl-2 and decreased the expression of Bax and cleaved caspase -3. Conversely, knockdown of endogenous LAMC1 aggravated the neurological impairment, suppressed the expression of Bcl-XL and Bcl-2, and upregulated the expression of Bax and cleaved caspase-3. Additionally, the administration of Y15 and LY294002 abolished the protective roles of rLAMC1. In vitro, rLAMC1 significantly reduced neuronal apoptosis, and the protective effects were also abolished by Y15 and LY294002. CONCLUSION: Exogenous LAMC1 treatment improved neurological deficits after SAH in rats, and attenuated neuronal apoptosis in both in vitro and in vivo SAH models, at least partially through the FAK/PI3K/AKT pathway.

Subarachnoid Hemorrhage Depletes Calcitonin Gene-Related Peptide Levels of Trigeminal Neurons in Rat Dura Mater.

Subarachnoid hemorrhage (SAH) remains a major cause of cerebrovascular morbidity, eliciting severe headaches and vasospasms that have been shown to inversely correlate with vasodilator calcitonin gene-related peptide (CGRP) levels. Although dura mater trigeminal afferents are an important source of intracranial CGRP, little is known about the effects of SAH on these neurons in preclinical models. The present study evaluated changes in CGRP levels and expression in trigeminal primary afferents innervating the dura mater 72 h after experimentally induced SAH in adult rats. SAH, eliciting marked damage revealed by neurological examination, significantly reduced the density of CGRP-immunoreactive nerve fibers both in the dura mater and the trigeminal caudal nucleus in the medulla but did not affect the total dural nerve fiber density. SAH attenuated ex vivo dural CGRP release by ~40% and in the trigeminal ganglion, reduced both CGRP mRNA levels and the number of highly CGRP-immunoreactive cell bodies. In summary, we provide novel complementary evidence that SAH negatively affects the integrity of the CGRP-expressing rat trigeminal neurons. Reduced CGRP levels suggest likely impaired meningeal neurovascular functions contributing to SAH complications. Further studies are to be performed to reveal the importance of impaired CGRP synthesis and its consequences in central sensory processing.

Changes in arterial myocyte excitability induced by subarachnoid hemorrhage in a rat model.

Aneurismal subarachnoid hemorrhage (aSAH) is a neurovascular disease produced by the rupture of the cerebral arteries and the extravasation of blood to the subarachnoid space and is accompanied by severe comorbidities. Secondarily associated vasospasm is one of the main side effects after hydrocephalus and possible rebleeding. Here, we analyze the alterations in function in the arteries of a rat model of SAH. For this, autologous blood was injected into the cisterna magna. We performed electrophysiological, microfluorimetric, and molecular biology experiments at different times after SAH to determine the functional and molecular changes induced by the hemorrhage. Our results confirmed that in SAH animals, arterial myocytes were depolarized on days 5 and 7, had higher [Ca2+]i on baseline, peaks and plateaus, and were more excitable at low levels of depolarization on day 7, than in the control and sham animals. Microarray analysis showed that, on day 7, the sets of genes related to voltage-dependent Ca2+ channels and K+ dynamics in SAH animals decreased, while the voltage-independent Ca2+ dynamics genes were over-represented. In conclusion, after SAH, several mechanisms involved in arterial reactivity were altered in our animal model, suggesting that there is no unique cause of vasospasm and alterations in several signaling pathways are involved in its development.

Role of microglia after subarachnoid hemorrhage.

Subarachnoid hemorrhage is a devastating sequela of aneurysm rupture. Because it disproportionately affects younger patients, the population impact of hemorrhagic stroke from subarachnoid hemorrhage is substantial. Secondary brain injury is a significant contributor to morbidity after subarachnoid hemorrhage. Initial hemorrhage causes intracranial pressure elevations, disrupted cerebral perfusion pressure, global ischemia, and systemic dysfunction. These initial events are followed by two characterized timespans of secondary brain injury: the early brain injury period and the delayed cerebral ischemia period. The identification of varying microglial phenotypes across phases of secondary brain injury paired with the functions of microglia during each phase provides a basis for microglia serving a critical role in both promoting and attenuating subarachnoid hemorrhage-induced morbidity. The duality of microglial effects on outcomes following SAH is highlighted by the pleiotropic features of these cells. Here, we provide an overview of the key role of microglia in subarachnoid hemorrhage-induced secondary brain injury as both cytotoxic and restorative effectors. We first describe the ontogeny of microglial populations that respond to subarachnoid hemorrhage. We then correlate the phenotypic development of secondary brain injury after subarachnoid hemorrhage to microglial functions, synthesizing experimental data in this area.