Identification of blood exosomal metabolomic profiling for high-altitude cerebral edema.

High-altitude cerebral edema (HACE) is a severe neurological condition that can occur at high altitudes. It is characterized by the accumulation of fluid in the brain, leading to a range of symptoms, including severe headache, confusion, loss of coordination, and even coma and death. Exosomes play a crucial role in intercellular communication, and their contents have been found to change in various diseases. This study analyzed the metabolomic characteristics of blood exosomes from HACE patients compared to those from healthy controls (HCs) with the aim of identifying specific metabolites or metabolic pathways associated with the development of HACE conditions. A total of 21 HACE patients and 21 healthy controls were recruited for this study. Comprehensive metabolomic profiling of the serum exosome samples was conducted using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC‒MS/MS). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed to identify the metabolic pathways affected in HACE patients. Twenty-six metabolites, including ( +)-camphoric acid, choline, adenosine, adenosine 5'-monophosphate, deoxyguanosine 5'-monophosphate, guanosine, and hypoxanthine-9-ß-D-arabinofuranoside, among others, exhibited significant changes in expression in HACE patients compared to HCs. Additionally, these differentially abundant metabolites were confirmed to be potential biomarkers for HACE. KEGG pathway enrichment analysis revealed several pathways that significantly affect energy metabolism regulation (such as purine metabolism, thermogenesis, and nucleotide metabolism), estrogen-related pathways (the estrogen signaling pathway, GnRH signaling pathway, and GnRH pathway), cyclic nucleotide signaling pathways (the cGMP-PKG signaling pathway and cAMP signaling pathway), and hormone synthesis and secretion pathways (renin secretion, parathyroid hormone synthesis, secretion and action, and aldosterone synthesis and secretion). In patients with HACE, adenosine, guanosine, and hypoxanthine-9-ß-D-arabinofuranoside were negatively correlated with height. Deoxyguanosine 5'-monophosphate is negatively correlated with weight and BMI. Additionally, LPE (18:2/0:0) and pregnanetriol were positively correlated with age. This study identified potential biomarkers for HACE and provided valuable insights into the underlying metabolic mechanisms of this disease. These findings may lead to potential targets for early diagnosis and therapeutic intervention in HACE patients.

Ac2-26 activated the AKT1/GSK3ß pathway to reduce cerebral neurons pyroptosis and improve cerebral function in rats after cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) results in brain injury, which is primarily caused by inflammation. Ac2-26 protects against ischemic or hemorrhage brain injury. The present study was to explore the effect and mechanism of Ac2-26 on brain injury in CPB rats. METHODS: Forty-eight rats were randomized into sham, CPB, Ac, Ac/AKT1, Ac/GSK3ßi and Ac/AKT1/GSK3ßa groups. Rats in sham group only received anesthesia and in the other groups received standard CPB surgery. Rats in the sham and CPB groups received saline, and rats in the Ac, Ac/AKT1, Ac/GSK3ßi and Ac/AKT1/GSK3ßa groups received Ac2-26 immediately after CPB. Rats in the Ac/AKT1, Ac/GSK3ßi and Ac/AKT1/GSK3ßa groups were injected with shRNA, inhibitor and agonist of GSK3ß respectively. The neurological function score, brain edema and histological score were evaluated. The neuronal survival and hippocampal pyroptosis were assessed. The cytokines, activity of NF-κB, S100 calcium-binding protein ß(S100ß) and neuron-specific enolase (NSE), and oxidative were tested. The NLRP3, cleaved-caspase-1 and cleaved-gadermin D (GSDMD) in the brain were also detected. RESULTS: Compared to the sham group, all indicators were aggravated in rats that underwent CPB. Compared to the CPB group, Ac2-26 significantly improved neurological scores and brain edema and ameliorated pathological injury. Ac2-26 reduced the local and systemic inflammation, oxidative stress response and promoted neuronal survival. Ac2-26 reduced hippocampal pyroptosis and decreased pyroptotic proteins in brain tissue. The protection of Ac2-26 was notably lessened by shRNA and inhibitor of GSK3ß. The agonist of GSK3ß recovered the protection of Ac2-26 in presence of shRNA. CONCLUSIONS: Ac2-26 significantly improved neurological function, reduced brain injury via regulating inflammation, oxidative stress response and pyroptosis after CPB. The protective effect of Ac2-26 primarily depended on AKT1/ GSK3ß pathway.

Endothelial EGLN3-PKM2 signaling induces the formation of acute astrocytic barrier to alleviate immune cell infiltration after subarachnoid hemorrhage.

BACKGROUND: Most subarachnoid hemorrhage (SAH) patients have no obvious hematoma lesions but exhibit blood-brain barrier dysfunction and vasogenic brain edema. However, there is a few days between blood‒brain barrier dysfunction and vasogenic brain edema. The present study sought to investigate whether this phenomenon is caused by endothelial injury induced by the acute astrocytic barrier, also known as the glial limitans. METHODS: Bioinformatics analyses of human endothelial cells and astrocytes under hypoxia were performed based on the GEO database. Wild-type, EGLN3 and PKM2 conditional knock-in mice were used to confirm glial limitan formation after SAH. Then, the effect of endothelial EGLN3-PKM2 signaling on temporal and spatial changes in glial limitans was evaluated in both in vivo and in vitro models of SAH. RESULTS: The data indicate that in the acute phase after SAH, astrocytes can form a temporary protective barrier, the glia limitans, around blood vessels that helps maintain barrier function and improve neurological prognosis. Molecular docking studies have shown that endothelial cells and astrocytes can promote glial limitans-based protection against early brain injury through EGLN3/PKM2 signaling and further activation of the PKC/ERK/MAPK signaling pathway in astrocytes after SAH. CONCLUSION: Improving the ability to maintain glial limitans may be a new therapeutic strategy for improving the prognosis of SAH patients.

Inhibition of TREM-1 alleviates neuroinflammation by modulating microglial polarization via SYK/p38MAPK signaling pathway after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI), as a major public health problem, is characterized by high incidence rate, disability rate, and mortality rate. Neuroinflammation plays a crucial role in the pathogenesis of TBI. Triggering receptor expressed on myeloid cells-1 (TREM-1) is recognized as an amplifier of the inflammation in diseases of the central nervous system (CNS). However, the function of TREM-1 remains unclear post-TBI. This study aimed to investigate the function of TREM-1 in neuroinflammation induced by TBI. METHODS: Brain water content (BWC), modified neurological severity score (mNSS), and Morris Water Maze (MWM) were measured to evaluate the effect of TREM-1 inhibition on nervous system function and outcome after TBI. TREM-1 expression in vivo was evaluated by Western blotting. The cellular localization of TREM-1 in the damaged region was observed via immunofluorescence staining. We also conducted Western blotting to examine expression of SYK, p-SYK and other downstream proteins. RESULTS: We found that inhibition of TREM-1 reduced brain edema, decreased mNSS and improved neurobehavioral outcomes after TBI. It was further determined that TREM-1 was expressed on microglia and modulated subtype transition of microglia. Inhibition of TREM-1 alleviated neuroinflammation, which was associated with SYK/p38MAPK signaling pathway. CONCLUSIONS: These findings suggest that TREM-1 can be a potential clinical therapeutic target for alleviating neuroinflammation after TBI.

Activating transcription factor 6 alleviates secondary brain injury by increasing cystathionine γ-lyase expression in a rat model of intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) comprises primary and secondary injuries, the latter of which induces increased inflammation and apoptosis and is more severe. Activating transcription factor 6 (ATF6) is a type-II transmembrane protein in the endoplasmic reticulum (ER). ATF6 target genes could improve ER homeostasis, which contributes to cryoprotection. Hence, we predict that ATF6 will have a protective effect on brain tissue after ICH. METHOD: The ICH rat model was generated through autologous blood injection into the right basal ganglia, the expression of ATF6 after ICH was determined by WB and IF. The expression of ATF6 was effectively controlled by means of intervention, and a series of measures was used to detect cell death, neuroinflammation, brain edema, blood-brain barrier and other indicators after ICH. Finally, the effects on long-term neural function of rats were measured by behavioral means. RESULT: ATF6 was significantly increased in the ICH-induced brain tissues. Further, ATF6 was found to modulate the expression of cystathionine γ-lyase (CTH) after ICH. Upregulation of ATF6 attenuated neuronal apoptosis and inflammation in ICH rats, along with mitigation of ICH-induced brain edema, blood-brain barrier deterioration, and cognitive behavior defects. Conversely, ATF6 genetic knockdown induced effects counter to those aforementioned. CONCLUSIONS: This study thereby emphasizes the crucial role of ATF6 in secondary brain injury in response to ICH, indicating that ATF6 upregulation may potentially ameliorate ICH-induced secondary brain injury. Consequently, ATF6 could serve as a promising therapeutic target to alleviate clinical ICH-induced secondary brain injuries.

Mas receptor activation facilitates innate hematoma resolution and neurological recovery after hemorrhagic stroke in mice.

BACKGROUND: Intracerebral hemorrhage (ICH) is a devastating neurological disease causing severe sensorimotor dysfunction and cognitive decline, yet there is no effective treatment strategy to alleviate outcomes of these patients. The Mas axis-mediated neuroprotection is involved in the pathology of various neurological diseases, however, the role of the Mas receptor in the setting of ICH remains to be elucidated. METHODS: C57BL/6 mice were used to establish the ICH model by injection of collagenase into mice striatum. The Mas receptor agonist AVE0991 was administered intranasally (0.9 mg/kg) after ICH. Using a combination of behavioral tests, Western blots, immunofluorescence staining, hematoma volume, brain edema, quantitative-PCR, TUNEL staining, Fluoro-Jade C staining, Nissl staining, and pharmacological methods, we examined the impact of intranasal application of AVE0991 on hematoma absorption and neurological outcomes following ICH and investigated the underlying mechanism. RESULTS: Mas receptor was found to be significantly expressed in activated microglia/macrophages, and the peak expression of Mas receptor in microglia/macrophages was observed at approximately 3-5 days, followed by a subsequent decline. Activation of Mas by AVE0991 post-treatment promoted hematoma absorption, reduced brain edema, and improved both short- and long-term neurological functions in ICH mice. Moreover, AVE0991 treatment effectively attenuated neuronal apoptosis, inhibited neutrophil infiltration, and reduced the release of inflammatory cytokines in perihematomal areas after ICH. Mechanistically, AVE0991 post-treatment significantly promoted the transformation of microglia/macrophages towards an anti-inflammatory, phagocytic, and reparative phenotype, and this functional phenotypic transition of microglia/macrophages by Mas activation was abolished by both Mas inhibitor A779 and Nrf2 inhibitor ML385. Furthermore, hematoma clearance and neuroprotective effects of AVE0991 treatment were reversed after microglia depletion in ICH. CONCLUSIONS: Mas activation can promote hematoma absorption, ameliorate neurological deficits, alleviate neuron apoptosis, reduced neuroinflammation, and regulate the function and phenotype of microglia/macrophages via Akt/Nrf2 signaling pathway after ICH. Thus, intranasal application of Mas agonist ACE0991 may provide promising strategy for clinical treatment of ICH patients.

Roles of mechanosensitive ion channel PIEZO1 in the pathogenesis of brain injury after experimental intracerebral hemorrhage.

Secondary brain injury after intracerebral hemorrhage (ICH) is the main cause of poor prognosis in ICH patients, but the underlying mechanisms remain less known. The involvement of Piezo1 in brain injury after ICH was studied in a mouse model of ICH. ICH was established by injecting autologous arterial blood into the basal ganglia in mice. After vehicle, Piezo1 blocker, GsMTx4, Piezo1 activator, Yoda-1, or together with mannitol (tail vein injection) was injected into the left lateral ventricle of mouse brain, Piezo1 level and the roles of Piezo1 in neuronal injury, brain edema, and neurological dysfunctions after ICH were determined by the various indicated methods. Piezo1 protein level in neurons was significantly upregulated 24 h after ICH in vivo (human and mice). Piezo1 protein level was also dramatically upregulated in HT22 cells (a murine neuron cell line) cultured in vitro 24 h after hemin treatment as an in vitro ICH model. GsMTx4 treatment or together with mannitol significantly downregulated Piezo1 and AQP4 levels, markedly increased Bcl2 level, maintained more neurons alive, considerably restored brain blood flow, remarkably relieved brain edema, substantially decreased serum IL-6 level, and almost fully reversed the neurological dysfunctions at ICH 24 h group mice. In contrast, Yoda-1 treatment achieved the opposite effects. In conclusion, Piezo1 plays a crucial role in the pathogenesis of brain injury after ICH and may be a target for clinical treatment of ICH.

The NLRP3 inhibitor, OLT1177 attenuates brain injury in experimental intracerebral hemorrhage.

BACKGROUND AND PURPOSE: It has been reported activation of NLRP3 inflammasome after intracerebral hemorrhage (ICH) ictus exacerbates neuroinflammation and brain injury. We hypothesized that inhibition of NLRP3 by OLT1177 (dapansutrile), a novel NLRP3 inflammasome inhibitor, could reduce brain edema and attenuate brain injury in experimental ICH. METHODS: ICH was induced by injection of autologous blood into basal ganglia in mice models. Sixty-three C57Bl/6 male mice were randomly grouped into the sham, vehicle, OLT1177 (Dapansutrile, 200 mg/kg intraperitoneally) and treated for consecutive three days, starting from 1 h after ICH surgery. Behavioral test, brain edema, brain water content, blood-brain barrier integrity and vascular permeability, cell apoptosis, and NLRP3 and its downstream protein levels were measured. RESULTS: OLT1177 significantly reduced cerebral edema after ICH and contributed to the attenuation of neurological deficits. OLT1177 could preserve blood-brain barrier integrity and lessen vascular leakage. In addition, OLT1177 preserved microglia morphological shift and significantly inhibited the activation of caspase-1 and release of IL-1ß. We also found that OLT1177 can protect against neuronal loss in the affected hemisphere. CONCLUSIONS: OLT1177 (dapansutrile) could significantly attenuate the brain edema after ICH and effectively alleviate the neurological deficit. This result suggests that the novel NLRP3 inhibitor, OLT1177, might serve as a promising candidate for the treatment of ICH.

Soluble PD-L1 reprograms blood monocytes to prevent cerebral edema and facilitate recovery after ischemic stroke.

Acute cerebral ischemia triggers a profound inflammatory response. While macrophages polarized to an M2-like phenotype clear debris and facilitate tissue repair, aberrant or prolonged macrophage activation is counterproductive to recovery. The inhibitory immune checkpoint Programmed Cell Death Protein 1 (PD-1) is upregulated on macrophage precursors (monocytes) in the blood after acute cerebrovascular injury. To investigate the therapeutic potential of PD-1 activation, we immunophenotyped circulating monocytes from patients and found that PD-1 expression was upregulated in the acute period after stroke. Murine studies using a temporary middle cerebral artery (MCA) occlusion (MCAO) model showed that intraperitoneal administration of soluble Programmed Death Ligand-1 (sPD-L1) significantly decreased brain edema and improved overall survival. Mice receiving sPD-L1 also had higher performance scores short-term, and more closely resembled sham animals on assessments of long-term functional recovery. These clinical and radiographic benefits were abrogated in global and myeloid-specific PD-1 knockout animals, confirming PD-1+ monocytes as the therapeutic target of sPD-L1. Single-cell RNA sequencing revealed that treatment skewed monocyte maturation to a non-classical Ly6Clo, CD43hi, PD-L1+ phenotype. These data support peripheral activation of PD-1 on inflammatory monocytes as a therapeutic strategy to treat neuroinflammation after acute ischemic stroke.

Transplantation of Neural Stem Cells-Overexpressed Ku70 Improves Neurological Deficits in a Mice Model of Cerebral Ischemia Stroke.

Cerebral ischemic stroke is a cerebrovascular disease, which is related to DNA damage. Many researches have shown that Ku70 is a key regulator for DNA damage. Here, we aimed to explore Ku70 roles in cerebral ischemic stroke and its potential molecular mechanism. In our study, neural stem cells (NSCs) were induced by oxygen-glucose deprivation/reoxygenation (OGD/R) for constructing cerebral ischemic stroke cell model. CCK8 assay, Brdu/GFP staining, flow cytometry and TUNEL staining were performed to examine cell proliferation, cell cycle and apoptosis, respectively. Relative mRNA and protein levels were detected by quantitative real-time PCR and western blot analysis, respectively. Ku70 positive cells were examined by immunofluorescence staining. Comet assay was employed to determine DNA damage. Animal experiments were performed to assess the effect of transplanting NSCs and Ku70-overexpressed NSCs on neurological deficits, infarct volume, brain edema and blood‒brain barrier (BBB) integrity in middle cerebral artery occlusion (MCAO) model. Our data found that Ku70 expression was decreased in NSCs after OGD/R. Overexpression of Ku70 reduced DNA damage and apoptosis of OGD/R-induced NSCs. Knockdown of Ku70 promoted the activity of ATM/p53. Moreover, KU60019 (ATM-specific inhibitor) reversed the promoting effects of Ku70 silencing on DNA damage and apoptosis in OGD/R-induced NSCs. In animal experiments, transplantation of NSCs-overexpressed Ku70 enhanced cell survival, improved motor function, reduced infarct volume, relieved brain edema and alleviated BBB dysfunction in MCAO mice models. In conclusion, Ku70 overexpression repressed the DNA damage and apoptosis in OGD/R-induced NSCs by regulating ATM/p53 pathway, and transplantation of NSCs-overexpressed Ku70 played neuroprotective effects in MCAO mice models.

Lessons on brain edema in HE: from cellular to animal models and clinical studies.

Brain edema is considered as a common feature associated with hepatic encephalopathy (HE). However, its central role as cause or consequence of HE and its implication in the development of the neurological alterations linked to HE are still under debate. It is now well accepted that type A and type C HE are biologically and clinically different, leading to different manifestations of brain edema. As a result, the findings on brain edema/swelling in type C HE are variable and sometimes controversial. In the light of the changing natural history of liver disease, better description of the clinical trajectory of cirrhosis and understanding of molecular mechanisms of HE, and the role of brain edema as a central component in the pathogenesis of HE is revisited in the current review. Furthermore, this review highlights the main techniques to measure brain edema and their advantages/disadvantages together with an in-depth description of the main ex-vivo/in-vivo findings using cell cultures, animal models and humans with HE. These findings are instrumental in elucidating the role of brain edema in HE and also in designing new multimodal studies by performing in-vivo combined with ex-vivo experiments for a better characterization of brain edema longitudinally and of its role in HE, especially in type C HE where water content changes are small.

Matrix Metalloproteinase-9 inhibitors as therapeutic drugs for traumatic brain injury.

Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality among young adults and the elderly. In the United States, TBI is responsible for around 30 percent of all injuries brought on by injuries in general. Vasogenic cerebral edema due to blood-brain barrier (BBB) dysfunction and the associated elevation of intracranial pressure (ICP) are some of the major causes of secondary injuries following traumatic brain injury. Matrix metalloproteinase-9 (MMP-9) is a therapeutic target for being an enzyme that degrades the proteins that make up a part of the microvascular basal lamina as well as inter-endothelial tight junctions of the blood-brain barrier. MMP-9-mediated BBB dysfunctions and the compromise of the BBB is a major pathway that leads the development of vasogenic cerebral edema, elevation of ICP, poor cerebral perfusion and brain herniation following traumatic brain injury. That makes MMP-9 an effective therapeutic target and endogenous or exogenous MMP-9 inhibitors as therapeutic drugs for preventing secondary brain damage after traumatic brain injury. Although our understanding of the mechanisms that underlie the primary and secondary stages of damage following a TBI has significantly improved in recent years, such information has not yet resulted in the successful development of novel pharmacological treatment options for traumatic brain injury. Recent pre-clinical and/or clinical studies have demonstrated that there are several compounds with specific or non-specific MMP-9 inhibitory properties either directly binding and inhibiting MMP-9 or by indirectly inhibiting MMP-9, with potential as therapeutic agents for traumatic brain injury. This article reviews the efficacy of several such medications and potential agents that include endogenous and exogeneous compounds that are at various levels of research and development. MMP-9-based therapeutic drug development has enormous potential in the pharmacological treatment of cerebral edema and/or neuronal injury resulting from traumatic brain injury.

Inhibition of EphA4 reduces vasogenic edema after experimental stroke in mice by protecting the blood-brain barrier integrity.

Cerebral vasogenic edema, a severe complication of ischemic stroke, aggravates neurological deficits. However, therapeutics to reduce cerebral edema still represent a significant unmet medical need. Brain microvascular endothelial cells (BMECs), vital for maintaining the blood-brain barrier (BBB), represent the first defense barrier for vasogenic edema. Here, we analyzed the proteomic profiles of the cultured mouse BMECs during oxygen-glucose deprivation and reperfusion (OGD/R). Besides the extensively altered cytoskeletal proteins, ephrin type-A receptor 4 (EphA4) expressions and its activated phosphorylated form p-EphA4 were significantly increased. Blocking EphA4 using EphA4-Fc, a specific and well-tolerated inhibitor shown in our ongoing human phase I trial, effectively reduced OGD/R-induced BMECs contraction and tight junction damage. EphA4-Fc did not protect OGD/R-induced neuronal and astrocytic death. However, administration of EphA4-Fc, before or after the onset of transient middle cerebral artery occlusion (tMCAO), reduced brain edema by about 50%, leading to improved neurological function recovery. The BBB permeability test also confirmed that cerebral BBB integrity was well maintained in tMCAO brains treated with EphA4-Fc. Therefore, EphA4 was critical in signaling BMECs-mediated BBB breakdown and vasogenic edema during cerebral ischemia. EphA4-Fc is promising for the treatment of clinical post-stroke edema.

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.

(S)-roscovitine, a CDK inhibitor, decreases cerebral edema and modulates AQP4 and α1-syntrophin interaction on a pre-clinical model of acute ischemic stroke.

Cerebral edema is one of the deadliest complications of ischemic stroke for which there is currently no pharmaceutical treatment. Aquaporin-4 (AQP4), a water-channel polarized at the astrocyte endfoot, is known to be highly implicated in cerebral edema. We previously showed in randomized studies that (S)-roscovitine, a cyclin-dependent kinase inhibitor, reduced cerebral edema 48 h after induction of focal transient ischemia, but its mechanisms of action were unclear. In our recent blind randomized study, we confirmed that (S)-roscovitine was able to reduce cerebral edema by 65% at 24 h post-stroke (t test, p = .006). Immunofluorescence analysis of AQP4 distribution in astrocytes revealed that (S)-roscovitine decreased the non-perivascular pool of AQP4 by 53% and drastically increased AQP4 clusters in astrocyte perivascular end-feet (671%, t test p = .005) compared to vehicle. Non-perivascular and clustered AQP4 compartments were negatively correlated (R = -0.78; p < .0001), suggesting a communicating vessels effect between the two compartments. α1-syntrophin, AQP4 anchoring protein, was colocalized with AQP4 in astrocyte endfeet, and this colocalization was maintained in ischemic area as observed on confocal microscopy. Moreover, (S)-roscovitine increased AQP4/α1-syntrophin interaction (40%, MW p = .0083) as quantified by proximity ligation assay. The quantified interaction was negatively correlated with brain edema in both treated and placebo groups (R = -.57; p = .0074). We showed for the first time, that a kinase inhibitor modulated AQP4/α1-syntrophin interaction, and was implicated in the reduction of cerebral edema. These findings suggest that (S)-roscovitine may hold promise as a potential treatment for cerebral edema in ischemic stroke and as modulator of AQP4 function in other neurological diseases.

Cranial venous-outflow obstruction promotes neuroinflammation via ADAM17/solTNF-α/NF-κB pathway following experimental TBI.

Traumatic brain injury (TBI) is a global public health problem. As an important cause of secondary injury, cerebrovascular reaction can cause secondary bleeding, venous sinus thrombosis, and malignant brain swelling. Recent clinical studies have confirmed that intracranial venous return disorder is closely related to the prognosis of patients, yet the specific molecular mechanism involved in this process is still unclear. This study used an acute subdural hematoma (ASDH) model with cranial venous outflow obstruction (CVO) to explore how CVO aggravates the pathological process after TBI, especially for inflammation and tissue damage. The results suggest that intracranial venous return disorder exacerbates neurological deficits and brain edema in rats with ASDH by aggravating the destruction of endothelial cell tight junctions (TJs) proteins and promoting the expression of inflammatory factors, the activation of microglia and expression of recombinant A disintegrin and metalloprotease 17 (ADAM17) as well as the secretion of solTNF-α, a soluble form of tumor necrosis factor-alpha (TNFα), which in turn increase IκB-α ((inhibitor of the transcription factor nuclear factor-κB) and NF-κB p65. Our study revealed a molecular basis of how CVO aggravates inflammation and tissue damage.

Potentiating glymphatic drainage minimizes post-traumatic cerebral oedema.

Cerebral oedema is associated with morbidity and mortality after traumatic brain injury (TBI)1. Noradrenaline levels are increased after TBI2-4, and the amplitude of the increase in noradrenaline predicts both the extent of injury5 and the likelihood of mortality6. Glymphatic impairment is both a feature of and a contributor to brain injury7,8, but its relationship with the injury-associated surge in noradrenaline is unclear. Here we report that acute post-traumatic oedema results from a suppression of glymphatic and lymphatic fluid flow that occurs in response to excessive systemic release of noradrenaline. This post-TBI adrenergic storm was associated with reduced contractility of cervical lymphatic vessels, consistent with diminished return of glymphatic and lymphatic fluid to the systemic circulation. Accordingly, pan-adrenergic receptor inhibition normalized central venous pressure and partly restored glymphatic and cervical lymphatic flow in a mouse model of TBI, and these actions led to substantially reduced brain oedema and improved functional outcomes. Furthermore, post-traumatic inhibition of adrenergic signalling boosted lymphatic export of cellular debris from the traumatic lesion, substantially reducing secondary inflammation and accumulation of phosphorylated tau. These observations suggest that targeting the noradrenergic control of central glymphatic flow may offer a therapeutic approach for treating acute TBI.

Potential mechanism of TMEM2/CD44 in endoplasmic reticulum stress­induced neuronal apoptosis in a rat model of traumatic brain injury.

Traumatic brain injury (TBI) can lead to the disruption of endoplasmic reticulum (ER) homeostasis in neurons and induce ER stress. Transmembrane protein 2 (TMEM2) may regulate ER stress through the p38/ERK signaling pathway, independent of the classic unfolded protein response (UPR) pathway. The present study examined the expression of TMEM2 following TBI in a rat model, in an aim to determine whether the mitogen­activated protein kinase (MAPK) signaling pathway is controlled by TMEM2/CD44 to mitigate secondary brain injury. For this purpose, 89 Sprague­Dawley rats were used to establish the model of TBI, and TMEM2 siRNA was used to silence TMEM2. Western blot analysis, immunofluorescence, TUNEL assay and Fluoro­Jade C staining, the wet­dry method and behavioral scoring were used for analyses. The results revealed that TMEM2 was activated following TBI in rats. The silencing of TMEM2 resulted in a significant increase in the levels of p38 and ERK (components of MAPK signaling), while brain edema, neuronal apoptosis and degeneration were significantly aggravated. TBI increased TMEM2/CD44­aggravated brain edema and neurological impairment, possibly by regulating ERK and p38 signaling. TMEM2/CD44 may thus be a target for the prevention and control of TBI.

Revisiting the role of the complement system in intracerebral hemorrhage and therapeutic prospects.

Intracerebral hemorrhage (ICH) is a stroke subtype characterized by non-traumatic rupture of blood vessels in the brain, resulting in blood pooling in the brain parenchyma. Despite its lower incidence than ischemic stroke, ICH remains a significant contributor to stroke-related mortality, and most survivors experience poor outcomes that significantly impact their quality of life. ICH has been accompanied by various complex pathological damage, including mechanical damage of brain tissue, hematoma mass effect, and then leads to inflammatory response, thrombin activation, erythrocyte lysis, excitatory amino acid toxicity, complement activation, and other pathological changes. Accumulating evidence has demonstrated that activation of complement cascade occurs in the early stage of brain injury, and the excessive complement activation after ICH will affect the occurrence of secondary brain injury (SBI) through multiple complex pathological processes, aggravating brain edema, and pathological brain injury. Therefore, the review summarized the pathological mechanisms of brain injury after ICH, specifically the complement role in ICH, and its related pathological mechanisms, to comprehensively understand the specific mechanism of different complements at different stages after ICH. Furthermore, we systematically reviewed the current state of complement-targeted therapies for ICH, providing a reference and basis for future clinical transformation of complement-targeted therapy for ICH.

Cation flux through SUR1-TRPM4 and NCX1 in astrocyte endfeet induces water influx through AQP4 and brain swelling after ischemic stroke.

Brain swelling causes morbidity and mortality in various brain injuries and diseases but lacks effective treatments. Brain swelling is linked to the influx of water into perivascular astrocytes through channels called aquaporins. Water accumulation in astrocytes increases their volume, which contributes to brain swelling. Using a mouse model of severe ischemic stroke, we identified a potentially targetable mechanism that promoted the cell surface localization of aquaporin 4 (AQP4) in perivascular astrocytic endfeet, which completely ensheathe the brain's capillaries. Cerebral ischemia increased the abundance of the heteromeric cation channel SUR1-TRPM4 and of the Na+/Ca2+ exchanger NCX1 in the endfeet of perivascular astrocytes. The influx of Na+ through SUR1-TRPM4 induced Ca2+ transport into cells through NCX1 operating in reverse mode, thus raising the intra-endfoot concentration of Ca2+. This increase in Ca2+ stimulated calmodulin-dependent translocation of AQP4 to the plasma membrane and water influx, which led to cellular edema and brain swelling. Pharmacological inhibition or astrocyte-specific deletion of SUR1-TRPM4 or NCX1 reduced brain swelling and improved neurological function in mice to a similar extent as an AQP4 inhibitor and was independent of infarct size. Thus, channels in astrocyte endfeet could be targeted to reduce postischemic brain swelling in stroke patients.