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.

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.

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.

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.

TRPV1 modulated NLRP3 inflammasome activation via calcium in experimental subarachnoid hemorrhage.

Neuroinflammation plays a key role in early brain injury (EBI) of subarachnoid hemorrhage (SAH), and NLRP3 inflammasome plays an important role in the development of neuroinflammation after SAH, but the mechanism of NLRP3 inflammasome activation after SAH is still unclear. TRPV1 is a non-selective calcium channel that is involved in the pathology of neuroinflammation, but its role in SAH has not been revealed. Our study showed that TRPV1 was significantly upregulated after SAH and was predominantly expressed in microglia/macrophages. Antagonism of TRPV1 was effective in ameliorating neurological impairment, brain edema, neuronal damage, and reducing the inflammatory response (evidenced by reducing the number of CD16/32 positive microglia/macrophages, inhibiting the expression of CD16, CD32, CD86, IL-1b, TNF-a and blocking NLRP3 inflammasome activation). However, this effect can be abolished by NLRP3 inflammasome antagonist MCC950. In vitro experiment confirmed that TRPV1 activated NLRP3 inflammasome by increasing intracellular calcium levels. In conclusion, TRPV1 mediates EBI after SAH via calcium/NLRP3, and TRPV1 is a potential therapeutic target after SAH.

Power suppression in EEG after the onset of SAH is a significant marker of early brain injury in rat models.

We analyzed the correlation between the duration of electroencephalogram (EEG) recovery and histological outcome in rats in the acute stage of subarachnoid hemorrhage (SAH) to find a new predictor of the subsequent outcome. SAH was induced in eight rats by cisternal blood injection, and the duration of cortical depolarization was measured. EEG power spectrums were given by time frequency analysis, and histology was evaluated. The appropriate frequency band and recovery percentage of EEG (defined as EEG recovery time) to predict the neuronal damage were determined from 25 patterns (5 bands × 5 recovery rates) of receiver operating characteristic (ROC) curves. Probit regression curves were depicted to evaluate the relationships between neuronal injury and duration of depolarization and EEG recovery. The optimal values of the EEG band and the EEG recovery time to predict neuronal damage were 10-15 Hz and 40%, respectively (area under the curve [AUC]: 0.97). There was a close relationship between the percentage of damaged neurons and the duration of depolarization or EEG recovery time. These results suggest that EEG recovery time, under the above frequency band and recovery rate, may be a novel marker to predict the outcome after SAH.

Alpha-Asarone Ameliorates Neurological Dysfunction of Subarachnoid Hemorrhagic Rats in Both Acute and Recovery Phases via Regulating the CaMKII-Dependent Pathways.

Early brain injury (EBI) is the leading cause of poor prognosis for patients suffering from subarachnoid hemorrhage (SAH), particularly learning and memory deficits in the repair phase. A recent report has involved calcium/calmodulin-dependent protein kinase II (CaMKII) in the pathophysiological process underlying SAH-induced EBI. Alpha-asarone (ASA), a major compound isolated from the Chinese medicinal herb Acorus tatarinowii Schott, was proven to reduce secondary brain injury by decreasing CaMKII over-phosphorylation in rats' model of intracerebral hemorrhage in our previous report. However, the effect of ASA on SAH remains unclear, and the role of CaMKII in both acute and recovery stages of SAH needs further investigation. In this work, we first established a classic SAH rat model by endovascular perforation and intraperitoneally administrated different ASA doses (10, 20, and 40 mg/kg) 2 h after successful modeling. Then, the short- and long-term neurobehavioral performances were blindly evaluated to confirm ASA's efficacy against SAH. Subsequently, we explored ASA's therapeutic mechanism in both acute and recovery stages using histopathological examination, TUNEL staining, flow cytometry, Western-blot, double-immunofluorescence staining, and transmission electron microscopy (TEM) observation. Finally, KN93, a selective CaMKII inhibitor, was applied in oxyhemoglobin-damaged HT22 cells to explore the role of CaMKII in ASA's neuroprotective effect. The results demonstrated that ASA alleviated short- and long-term neurological dysfunction, reduced mortality and seizure rate within 24 h, and prolonged 14-day survival in SAH rats. Histopathological examination showed a reduction of neuronal damage and a restoration of the hippocampal structure after ASA treatment in both acute and recovery phases of SAH. In the acute stage, the Western-blot and flow cytometer analyses showed that ASA restored E/I balance, reduced calcium overload and CaMKII phosphorylation, and inhibited mitochondrion-involved apoptosis, thus preventing neuronal damage and apoptosis underlying EBI post-SAH. In the recovery stage, the TEM observation, double-immunofluorescence staining, and Western-blot analyses indicated that ASA increased the numbers of synapses and enhanced synaptic plasticity in the ipsilateral hippocampi, probably by promoting NR2B/CaMKII interaction and activating subsequent CREB/BDNF/TrkB signaling pathways. Furthermore, KN93 notably reversed ASA's neuroprotective effect on oxyhemoglobin-damaged HT22 cells, confirming CaMKII a potential target for ASA's efficacy against SAH. Our study confirmed for the first time that ASA ameliorated the SAH rats' neurobehavioral deterioration, possibly via modulating CaMKII-involved pathways. These findings provided a promising candidate for the clinical treatment of SAH and shed light on future drug discovery against SAH.

Tanshinone IIA Alleviates Early Brain Injury after Subarachnoid Hemorrhage in Rats by Inhibiting the Activation of NF-κB/NLRP3 Inflammasome.

The abnormal activation of the nuclear factor-kappa B (NF-κB)/nod-like receptor family-pyrin domain-containing 3 (NLRP3) signaling pathway is closely related to early brain injury after subarachnoid hemorrhage (SAH). Targeting the NLRP3-inflammasome has been considered an efficient therapy for the local inflammatory response after SAH. Tanshinone IIA (Tan IIA), a major component extracted from Salvia miltiorrhiza, has been reported to have anti-inflammatory effects. The aim of this study was to investigate the effect and mechanism of Tan IIA on early brain injury after SAH. In vivo SAH injury was established by endovascular perforation technique in Sprague-Dawley rats. Limb-placement test and corner turning test were used to measure the behavior. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, hematoxylin-eosin (H&E) staining, and immunofluorescence were used to evaluate the nerve damage. Real-time RT quantitative PCR (RT-qPCR) was used to quantify the levels of inflammatory factors. Western blot was performed for the activation of the NF-κB/NLRP3 pathway. An in vitro SAH model was used to validate the conclusion. We found that the neurobehavioral impairment and cerebral edema in SAH model rats given Tan IIA were alleviated. Further study demonstrated that Tan IIA could inhibit SAH-secondary neuronal apoptosis around hematoma and alleviate brain injury. Tan IIA down-regulated the expression of interleukin-6 (IL)-6, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor (TNF)-α, and inhibited the activation of NF-κB. And the overexpression of pro-inflammatory factors NLRP3, IL-1ß, and IL-18 induced after SAH was also reversed by Tan IIA. In conclusions, Tan IIA could inhibit the NF-κB/NLRP3 inflammasome activation to protect and ameliorate SAH-followed early brain injury, and may be a preventive and therapeutic strategy against SAH.

Astrocyte-derived hepcidin aggravates neuronal iron accumulation after subarachnoid hemorrhage by decreasing neuronal ferroportin1.

Iron accumulation is one of the most essential pathological events after subarachnoid hemorrhage (SAH). Ferroportin1 (FPN1) is the only transmembrane protein responsible for exporting iron. Hepcidin, as the major regulator of FPN1, is responsible for its degradation. Our study investigated how the interaction between FPN1 and hepcidin contributes to iron accumulation after SAH. We found that iron accumulation aggravated after SAH, along with decreased FPN1 in neurons and increased hepcidin in astrocytes. After knocking down hepcidin in astrocytes, the neuronal FPN1 significantly elevated, thus attenuating iron accumulation. After SAH, p-Smad1/5 and Smad4 tended to translocate into the nucleus. Moreover, Smad4 combined more fragments of the promoter region of Hamp after OxyHb stimulation. By knocking down Smad1/5 or Smad4 in astrocytes, FPN1 level restored and iron overload attenuated, leading to alleviated neuronal cell death and improved neurological function. However, the protective role disappeared after recombinant hepcidin administration. Therefore, our study suggests that owing to the nuclear translocation of transcription factors p-Smad1/5 and Smad4, astrocyte-derived hepcidin increased significantly after SAH, leading to a decreased level of neuronal FPN1, aggravation of iron accumulation, and worse neurological outcome.

Simple procedure for assessing diffuse subarachnoid hemorrhage successfully created using filament perforation method in mice.

The murine model of subarachnoid hemorrhage (SAH) is a valuable experimental tool for investigating molecular and cellular mechanisms, and the endovascular filament perforation technique can be used to simulate prominent pathophysiological features observed after human SAH; however, current validation methods for assessing an appropriate SAH model are limited. Here, we introduce a simple procedure for selecting a mouse model of diffuse SAH. SAH was induced in 24 mice using a standard filament perforation method. After confirming survival at 24 h, SAH was scored 0-1 based on T2*-weighted images on whole-brain magnetic resonance imaging (MRI) and visual surveillance of the cisterna magna (CM) through the dura mater. The CM-based SAH grading correlated well with a reference parameter defined by extracted brain (r2 = 0.53, p < 0.0001). The receiver operating characteristic curve revealed a sensitivity of 85% and a specificity of 91% for detecting diffuse SAH, with a similar area under the curve (0.89 ± 0.06 [standard error of the mean]) as the MRI-based grading (0.72 ± 0.10, p = 0.12). Our data suggest that confirming an SAH clot in the CM is a valuable way to select a clinically relevant diffuse SAH model that can be used in future experimental studies.

Neuronal pyroptosis mediated by STAT3 in early brain injury after subarachnoid hemorrhage.

Neuroinflammation induced by early brain injury (EBI) seriously affects the prognosis of patients after subarachnoid hemorrhage (SAH). Pyroptosis can aggravate inflammatory injury by promoting the secretion of inflammatory cytokines. Meanwhile, STAT3 plays a critical role in the inflammatory response of EBI after SAH. However, whether it plays a pyroptotic role in SAH is mainly unknown. This study aimed to explore the mechanism of STAT3 in pyroptosis in EBI after SAH. C57BL/6J mice were used to establish the SAH model. Brain tissues were collected at different time points for q-RT-PCR and western blot to detect the expression level of STAT3. After intracerebroventricular injection of STAT3 inhibitor S3I-201, they were divided into sham, SAH, SAH + Vehicle, and SAH + S3I-201. Then, the SAH grade, cerebral edema content, blood-brain barrier (BBB) damage, and neurological scores of mice in each group were detected. qRT-PCR and western blot were used to detect related genes and proteins, and enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of IL-18 and IL-1ß. Immunofluorescence staining was used to observe the expression level of proteins. At the same time, S3I-201 was added to the primary neuron cells of the culture medium containing OxyHb to simulate the in vitro experiment, and the relevant indicators consistent with the in vivo experiment were detected. The expression of STAT3 was upregulated after SAH. Inhibition of STAT3 with S3I-201 attenuated neurological deficits, cerebral edema, and BBB damage after SAH. In addition, S3I-201 can also reduce the expression of pyroptosis-related inflammasomes such as GSDMD, NLRP3, Caspase 1, and AIM2 after SAH and the neurological damage caused by IL-18 and IL-1ß. Further studies have shown that STAT3 regulates pyroptosis by promoting the nuclear translocation of NF-κB p65. Our finding demonstrated that STAT3 regulates neuronal pyroptosis in EBI after SAH. Inhibition of STAT3 may be a potential target to attenuate the damage that triggers neuroinflammation after SAH.

Eriocitrin Alleviates Inflammation and Oxidative Stress in Subarachnoid Hemorrhage by Regulating DUSP14.

BACKGROUND: Inflammation and oxidative stress (OS) are major causes of aneurysmal subarachnoid hemorrhage (aSAH)-induced early brain injury (EBI). Eriocitrin (EC), a flavonoid compound, has anti-inflammatory and antioxidant actions. However, there is still no relevant studies on the role of EC in SAH. Accordingly, this research aims to clarify the anti-OS and anti-inflammatory efficacy of EC in SAH. METHOD: Rat SAH model was established in vivo and administered with Eriocitrin (25 mg/kg). In vitro, BV2 cells were exposed to oxyhemoglobin (OxyHb) for 24 hours and pretreated with Eriocitrin (1 uM/mL, 2 uM/mL, 4 uM/mL) for 30 minutes. Water maze experiments and neurological function scores were conducted to assess cognitive and motor function. TdT-mediated dUTP Nick-End Labeling (TUNEL) staining was used to detect cortical cell apoptosis. Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) were used to detect the inflammatory factors and malondialdehyde (MDA), as well as the expression of superoxide dismutase (SOD) and glutathione peroxidase (GSH-px). Western blots were used to semi quantify nuclear factor erythroid-2-related factor 2 (Nrf2), nuclear factor-κB (NF-κB), dual specificity phosphatase 14 (DUSP14) expression. RESULTS: The findings suggest that EC (25 mg/kg) reduced SAH-induced central nervous system (CNS) damage, neuronal apoptosis, inflammatory reactions and OS. Regarding a mechanistic study, EC enhanced Nrf2 and NF-κB by increasing DUSP14 activation, thereby reducing the inflammatory cytokines interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6. In addition, EC decreased MDA while markedly elevating SOD and enhancing GSH-px. Furthermore, specifically inhibiting DUSP14 expression via using protein-tyrosine-phosphatase (PTP) inhibitor IV, neutralized the protective action of EC and aggravated inflammation and OS. In vitro experiments of OxyHb-induced BV2 cells revealed that EC promoted Nrf2 while markedly suppressing NF-κB by increasing DUSP14 activation, thereby reducing the concentrations of the above inflammatory cytokines. Moreover, EC decreased MDA while evidently increasing SOD and GSH-px. CONCLUSION: In summary, this paper lays a theoretical grounding for EC treatment of SAH-induced inflammatory reactions and OS by regulating DUSP14.

GPNMB Ameliorates Neuroinflammation Via the Modulation of AMPK/NFκB Signaling Pathway After SAH in Mice.

Glycoprotein non-metastatic melanoma protein B (GPNMB) got its name from the first discovery in a cell line of non-metastatic melanoma. Later studies found that GPNMB is widely expressed in various tissues and cells of the human body, most abundant in neural tissue, epithelial tissue, bone tissue, and monocyte-macrophage system. GPNMB has been shown to have anti-inflammatory effects in a variety of neurological diseases, however, it has not been reported in subarachnoid hemorrhage (SAH). Male CD-1 mice were used and intra-arterial puncture method was applied to establish the SAH model. Exogenous recombinant GPNMB (rGPNMB) was injected intracerebroventricularly 1 h after SAH. SAH grading, brain edema and blood-brain barrier (BBB) integrity were quantified, and neurobehavioral tests were performed to evaluate the effect of GPNMB on the outcome. Dorsomorphin, the selective inhibitor on AMPK was introduced to study the downstream signaling through which the GPNMB works. Furthermore, western blot, immunofluorescence staining and ELISA were utilized to confirm the signaling. After SAH, GPNMB expression increased significantly as a result of the inflammatory response. GPNMB was expressed extensively in mouse microglia, astrocytes and neurons. The administration of rGPNMB could alleviate brain edema, restore BBB integrity and improve the neurological outcome of mice with SAH. GPNMB treatment significantly magnified the expression of p-AMPK while p-NFκB, IL-1ß, IL-6 and TNF-α were suppressed; in the meantime, the combined administration of GPNMB and AMPK inhibitor could decrease the intensity of p-AMPK and reverse the quantity of p-NFκB and the above inflammatory cytokines. GPNMB has the potential of ameliorating the brain edema and neuroinflammation, protecting the BBB and improving the neurological outcome, possibly via the AMPK/NFκB signaling pathway.

RU.521 mitigates subarachnoid hemorrhage-induced brain injury via regulating microglial polarization and neuroinflammation mediated by the cGAS/STING/NF-κB pathway.

BACKGROUND: The poor prognosis of subarachnoid hemorrhage (SAH) is often attributed to neuroinflammation. The cGAS-STING axis, a cytoplasmic pathway responsible for detecting dsDNA, plays a significant role in mediating neuroinflammation in neurological diseases. However, the effects of inhibiting cGAS with the selective small molecule inhibitor RU.521 on brain injury and the underlying mechanisms after SAH are still unclear. METHODS: The expression and microglial localization of cGAS following SAH were investigated with western blot analysis and immunofluorescent double-staining, respectively. RU.521 was administered after SAH. 2'3'-cGAMP, a second messenger converted by activated cGAS, was used to activate cGAS-STING. The assessments were carried out by adopting various techniques including neurological function scores, brain water content, blood-brain barrier permeability, western blot analysis, TUNEL staining, Nissl staining, immunofluorescence, morphological analysis, Morris water maze test, Golgi staining, CCK8, flow cytometry in the in vivo and in vitro settings. RESULTS: Following SAH, there was an observed increase in the expression levels of cGAS in rat brain tissue, with peak levels observed at 24 h post-SAH. RU.521 resulted in a reduction of brain water content and blood-brain barrier permeability, leading to an improvement in neurological deficits after SAH. RU.521 had beneficial effects on neuronal apoptosis and microglia activation, as well as improvements in microglial morphology. Additionally, RU.521 prompted a shift in microglial phenotype from M1 to M2. We also noted a decrease in the production of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6, and an increase in the level of the anti-inflammatory cytokine IL-10. Finally, RU.521 treatment was associated with improvements in cognitive function and an increase in the number of dendritic spines in the hippocampus. The therapeutic effects were mediated by the cGAS/STING/NF-κB pathway and were found to be abolished by 2'3'-cGAMP. In vitro, RU.521 significantly reduced apoptosis and neuroinflammation. CONCLUSION: The study showed that SAH leads to neuroinflammation caused by microglial activation, which contributes to early brain injury. RU.521 improved neurological outcomes and reduced neuroinflammation by regulating microglial polarization through the cGAS/STING/NF-κB pathway in early brain injury after SAH. RU.521 may be a promising candidate for the treatment of neuroinflammatory pathology after SAH. Video Abstract.

Melatonin alleviates early brain injury by inhibiting the NRF2-mediated ferroptosis pathway after subarachnoid hemorrhage.

Ferroptosis is a novel form of cell death that plays a critical role in the pathological and physiological processes of early brain injury following subarachnoid hemorrhage. Melatonin, as the most potent endogenous antioxidant, has shown strong protective effects against pathological changes following subarachnoid hemorrhage, but its impact on ferroptosis induced by subarachnoid hemorrhage remains unexplored. In our study, we established a subarachnoid hemorrhage model in male SD rats. We found that subarachnoid hemorrhage induced changes in ferroptosis-related indicators such as lipid peroxidation and iron metabolism, while intraperitoneal injection of melatonin (40 mg/kg) effectively ameliorated these changes to a certain degree. Moreover, in a subset of rats with subarachnoid hemorrhage who received pre-treatment via intravenous injection of the melatonin receptor antagonist Luzindole (1 mg/kg) and 4P-PDOT (1 mg/kg), we found that the protective effect of melatonin against subarachnoid hemorrhage includes inhibition of lipid peroxidation and reduction of iron accumulation depended on melatonin receptor 1B (MT2). Furthermore, our study demonstrated that melatonin inhibited neuronal ferroptosis by activating the NRF2 signaling pathway, as evidenced by in vivo inhibition of NRF2. In summary, melatonin acts through MT2 and activates NRF2 and downstream genes such as HO-1/NQO1 to inhibit ferroptosis in subarachnoid hemorrhage-induced neuronal injury, thereby improving neurological function in rats. These results suggest that melatonin is a promising therapeutic target for subarachnoid hemorrhage.

Single-Cell RNA Sequencing and Spatial Transcriptomics Reveal Pathogenesis of Meningeal Lymphatic Dysfunction after Experimental Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a devastating subtype of stroke with high mortality and disability rate. Meningeal lymphatic vessels (mLVs) are a newly discovered intracranial fluid transport system and are proven to drain extravasated erythrocytes from cerebrospinal fluid into deep cervical lymph nodes after SAH. However, many studies have reported that the structure and function of mLVs are injured in several central nervous system diseases. Whether SAH can cause mLVs injury and the underlying mechanism remain unclear. Herein, single-cell RNA sequencing and spatial transcriptomics are applied, along with in vivo/vitro experiments, to investigate the alteration of the cellular, molecular, and spatial pattern of mLVs after SAH. First, it is demonstrated that SAH induces mLVs impairment. Then, through bioinformatic analysis of sequencing data, it is discovered that thrombospondin 1 (THBS1) and S100A6 are strongly associated with SAH outcome. Furthermore, the THBS1-CD47 ligand-receptor pair is found to function as a key role in meningeal lymphatic endothelial cell apoptosis via regulating STAT3/Bcl-2 signaling. The results illustrate a landscape of injured mLVs after SAH for the first time and provide a potential therapeutic strategy for SAH based on mLVs protection by disrupting THBS1 and CD47 interaction.

Subarachnoid hemorrhage and axonal degeneration after C1-C2 cervical centesis in 2 horses.

Ultrasound-guided cervical centesis has gained popularity as a method for collecting cerebrospinal fluid (CSF) from standing horses. There are anecdotal reports of neck stiffness, regional swelling, sensitivity to palpation, and fever after the procedure. We report 2 horses with complications that occurred within days of C1-C2 centesis and ultimately resulted in euthanasia. Both C1-C2 centesis were performed routinely, with CSF cytologic analysis providing no evidence of blood contamination. Post-mortem examination revealed equine degenerative myeloencephalopathy as the primary disorder causing Horse 1's initial neurologic deficits, whereas Horse 2 did not have a distinct lesion explaining the horse's deficits. Both horses had evidence of subarachnoid hemorrhage at or near the centesis site with Wallerian axonal degeneration in the cranial cervical spinal cord. Although hemorrhage with associated axonal degeneration at the cervical centesis site appears to be rare, this complication of C1-C2 centesis should be considered as this technique gains popularity.

White Matter Injury: An Emerging Potential Target for Treatment after Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) refers to vascular brain injury mainly from a ruptured aneurysm, which has a high lifetime risk and imposes a substantial burden on patients, families, and society. Previous studies on SAH mainly focused on neurons in gray matter (GM). However, according to literature reports in recent years, in-depth research on the mechanism of white matter (WM) is of great significance to injury and recovery after SAH. In terms of functional recovery after SAH, all kinds of cells in the central nervous system (CNS) should be protected. In other words, it is necessary to protect not only GM but also WM, not only neurons but also glial cells and axons, and not only for the lesion itself but also for the prevention and treatment of remote damage. Clarifying the mechanism of white matter injury (WMI) and repair after SAH is of great importance. Therefore, this present review systematically summarizes the current research on WMI after SAH, which might provide therapeutic targets for treatment after SAH.