Enhancing Th17 cells drainage through meningeal lymphatic vessels alleviate neuroinflammation after subarachnoid hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe cerebrovascular disorder primarily caused by the rupture of aneurysm, which results in a high mortality rate and consequently imposes a significant burden on society. The occurrence of SAH initiates an immune response that further exacerbates brain damage. The acute inflammatory reaction subsequent to SAH plays a crucial role in determining the prognosis. Th17 cells, a subset of T cells, are related to the brain injury following SAH, and it is unclear how Th17 cells are cleared in the brain. Meningeal lymphatic vessels are a newly discovered intracranial fluid transport system that has been shown to drain large molecules and immune cells to deep cervical lymph nodes. There is limited understanding of the role of the meningeal lymphatic system in SAH. The objective of this research is to explore the impact and underlying mechanism of drainage Th17 cells by meningeal lymphatics on SAH. METHODS: Treatments to manipulate meningeal lymphatic function and the CCR7-CCL21 pathway were administered, including laser ablation, injection of VEGF-C geneknockout, and protein injection. Mouse behavior was assessed using the balance beam experiment and the modified Garcia scoring system. Flow cytometry, enzyme-linked immunosorbent assays (ELISA), and immunofluorescence staining were used to study the impact of meningeal lymphatic on SAH drainage. Select patients with unruptured and ruptured aneurysms in our hospital as the control group and the SAH group, with 7 cases in each group. Peripheral blood and cerebrospinal fluid (CSF) samples were assessed by ELISA and flow cytometry. RESULTS: Mice with SAH showed substantial behavioral abnormalities and brain damage in which immune cells accumulated in the brain. Laser ablation of the meningeal lymphatic system or knockout of the CCR7 gene leads to Th17 cell aggregation in the meninges, resulting in a decreased neurological function score and increased levels of inflammatory factors. Injection of VEGF-C or CCL21 protein promotes Th17 cell drainage to lymph nodes, an increased neurological function score, and decreased levels of inflammatory factors. Clinical blood and CSF results showed that inflammatory factors in SAH group were significantly increased. The number of Th17 cells in the SAH group was significantly higher than the control group. Clinical results confirmed Th17 cells aggravated the level of neuroinflammation after SAH. CONCLUSION: This study shows that improving the drainage of Th17 cells by meningeal lymphatics via the CCR7-CCL21 pathway can reduce brain damage and improve behavior in the SAH mouse model. This could lead to new treatment options for SAH.

Identification of key genes and immune infiltration in peripheral blood biomarker analysis of delayed cerebral ischemia: Valproic acid as a potential therapeutic drug.

BACKGROUND: Delayed cerebral ischemia (DCI) is a common and serious complication of subarachnoid hemorrhage (SAH). Its pathogenesis is not fully understood. Here, we developed a predictive model based on peripheral blood biomarkers and validated the model using several bioinformatic multi-analysis methods. METHODS: Six datasets were obtained from the GEO database. Characteristic genes were screened using weighted correlation network analysis (WGCNA) and differentially expressed genes. Three machine learning algorithms, elastic networks-LASSO, support vector machines (SVM-RFE) and random forests (RF), were also used to construct diagnostic prediction models for key genes. To further evaluate the performance and predictive value of the diagnostic models, nomogram model were constructed, and the clinical value of the models was assessed using Decision Curve Analysis (DCA), Area Under the Check Curve (AUC), Clinical Impact Curve (CIC), and validated in the mouse single-cell RNA-seq dataset. Mendelian randomization(MR) analysis explored the causal relationship between SAH and stroke, and the intermediate influencing factors. We validated this by retrospectively analyzing the qPCR levels of the most relevant genes in SAH and SAH-DCI patients. This experiment demonstrated a statistically significant difference between SAH and SAH-DCI and normal group controls. Finally, potential small molecule compounds interacting with the selected features were screened from the Comparative Toxicogenomics Database (CTD). RESULTS: The fGSEA results showed that activation of Toll-like receptor signaling and leukocyte transendothelial cell migration pathways were positively correlated with the DCI phenotype, whereas cytokine signaling pathways and natural killer cell-mediated cytotoxicity were negatively correlated. Consensus feature selection of DEG genes using WGCNA and three machine learning algorithms resulted in the identification of six genes (SPOCK2, TRRAP, CIB1, BCL11B, PDZD8 and LAT), which were used to predict DCI diagnosis with high accuracy. Three external datasets and the mouse single-cell dataset showed high accuracy of the diagnostic model, in addition to high performance and predictive value of the diagnostic model in DCA and CIC. MR analysis looked at stroke after SAH independent of SAH, but associated with multiple intermediate factors including Hypertensive diseases, Total triglycerides levels in medium HDL and Platelet count. qPCR confirmed that significant differences in DCI signature genes were observed between the SAH and SAH-DCI groups. Finally, valproic acid became a potential therapeutic agent for DCI based on the results of target prediction and molecular docking of the characterized genes. CONCLUSION: This diagnostic model can identify SAH patients at high risk for DCI and may provide potential mechanisms and therapeutic targets for DCI. Valproic acid may be an important future drug for the treatment of DCI.

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.

Predictive effects of admission white blood cell counts and hounsfield unit values on delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.

OBJECTIVE: Neuroinflammatory response is deemed the primary pathogenesis of delayed cerebral ischemia (DCI) caused by aneurysmal subarachnoid hemorrhage (aSAH). Both white blood cell (WBC) count and Hounsfield Unit (HU) are gradually considered can reflect inflammation in DCI. This study aims to identify the relationship between WBC count and HU value and investigate the effects of both indicators in predicting DCI after aSAH. METHODS: We enrolled 109 patients with aSAH admitted within 24 h of onset in our study. A multivariate logistic regression analysis was used to evaluate the admission WBC count, HU value, and combined WBC-HU associated with DCI. The receiver operating characteristic curve and area under the curve (AUC) were used to determine thresholds and detect the predictive ability of these predictors. These indicators were also compared with the established inflammation markers. RESULTS: Thirty-six (33%) patients developed DCI. Both WBC count and HU value were strongly associated with the admission glucose level (ρ = .303, p = .001; ρ = .273, p = .004), World Federation of Neurosurgical Societies grade (ρ = .452, p < .001; ρ = .578; p < .001), Hunt-Hess grade (ρ = .450, p < .001; ρ = .510, p < .001), and modified Fisher scale score (ρ = .357, p < .001; ρ = .330, p < .001). After controlling these public variables, WBC count (ρ = .300, p = .002) positively correlated with HU value. An early elevated WBC (odds ratio [OR] 1.449, 95% confidence interval [CI]: 1.183-1.774, p < .001) count and HU value (OR 1.304, 95%CI: 1.149-1.479, p < .001) could independently predict the occurrence of DCI. However, only these patients with both WBC count and HU value exceeding the cut-off points (OR 36.89, 95%CI: 5.606-242.78, p < .001) were strongly correlated with DCI. Compared with a single WBC count (AUC 0.811, 95%CI: 0.729-0.892, p < .001) or HU value (AUC 0.869, 95%CI: 0.803-0.936, p < .001), the combined WBC-HU (AUC 0.898, 95%CI: 0.839-0.957, p < .001) demonstrated a better ability to predict the occurrence of DCI. Inspiringly, the prediction performance of these indicators outperformed the established inflammatory markers. CONCLUSION: An early elevated WBC count and HU value could independently predict DCI occurrence between 4 and 30 days after aSAH. Furthermore, WBC count was positively correlated with HU value, and the combined WBC-HU demonstrated a superior prediction ability for DCI development compared with the individual indicator.

TREM1 Regulates Neuroinflammatory Injury by Modulate Proinflammatory Subtype Transition of Microglia and Formation of Neutrophil Extracellular Traps via Interaction With SYK in Experimental Subarachnoid Hemorrhage.

Neuroinflammation is a key process in the pathogenesis of subarachnoid hemorrhage (SAH) and contributes to poor outcome in patients. The purpose of this study is to explore the effect of triggering receptor expressed on myeloid cells 1 (TREM1) in the SAH, as well as its potential mechanism. In our study, plasma levels of soluble TREM1 was increased significantly after SAH and correlated to SAH severity and serum C-reactiveprotein. TREM1 inhibitory peptide LP17 alleviated the neurological deficits, attenuated brain water content, and reduced neuronal damage after SAH. Meanwhile, TREM1 inhibitory peptide decreased neuroinflammation (evidenced by the decreased levels of markers including IL-6, IL-1ß, TNF-α) by attenuating proinflammatory subtype transition of microglia (evidenced by the decreased levels of markers including CD68, CD16, CD86) and decreasing the formation of neutrophil extracellular traps (evidenced by the decreased levels of markers including CitH3, MPO, and NE). Further mechanistic study identified that TREM1 can activate downstream proinflammatory pathways through interacting with spleen tyrosine kinase (SYK). In conclusion, inhibition of TREM1 alleviates neuroinflammation by attenuating proinflammatory subtype transition of microglia and decreasing the formation of neutrophil extracellular traps through interacting with SYK after SAH. TREM1 may be a a promising therapeutic target for SAH.

A20 Establishes Negative Feedback With TRAF6/NF-κB and Attenuates Early Brain Injury After Experimental Subarachnoid Hemorrhage.

Nuclear factor (NF)-κB-ty -50mediated neuroinflammation plays a crucial role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). As an important negative feedback regulator of NF-κB, A20 is essential for inflammatory homeostasis. Herein, we tested the hypothesis that A20 attenuates EBI by establishing NF-κB-associated negative feedback after experimental SAH. In vivo and in vitro models of SAH were established. TPCA-1 and lentivirus were used for NF-κB inhibition and A20 silencing/overexpression, respectively. Cellular localization of A20 in the brain was determined via immunofluorescence. Western blotting and enzyme-linked immunosorbent assays were applied to observe the expression of members of the A20/tumor necrosis factor receptor-associated factor 6 (TRAF6)/NF-κB pathway and inflammatory cytokines (IL-6, IL-1ß, TNF-α). Evans blue staining, TUNEL staining, Nissl staining, brain water content, and modified Garcia score were performed to evaluate the neuroprotective effect of A20. A20 expression by astrocytes, microglia, and neurons was increased at 24 h after SAH. A20 and inflammatory cytokine levels were decreased while TRAF6 expression was elevated after NF-κB inhibition. TRAF6, NF-κB, and inflammatory cytokine levels were increased after A20 silencing but suppressed with A20 overexpression. Also, Bcl-2, Bax, MMP-9, ZO-1 protein levels; Evans blue, TUNEL, and Nissl staining; brain water content; and modified Garcia score showed that A20 exerted a neuroprotective effect after SAH. A20 expression was regulated by NF-κB. In turn, increased A20 expression inhibited TRAF6 and NF-κB to reduce the subsequent inflammatory response. Our data also suggest that negative feedback regulation mechanism of the A20/TRAF6/NF-κB pathway and the neuroprotective role of A20 to attenuate EBI after SAH.

Admission Neutrophil to Lymphocyte Ratio for Predicting Outcome in Subarachnoid Hemorrhage.

PURPOSE: We sought to evaluate the relationship between admission neutrophil-to-lymphocyte ratio (NLR) and functional outcome in aneurysmal subarachnoid hemorrhage (aSAH) patients. MATERIAL AND METHODS: Consecutive patients with aSAH were treated at two tertiary stroke centers during a five-year period. Functional outcome was defined as discharge modified Rankin score dichotomized at scores 0-2 (good) vs. 3-6 (poor). RESULTS: 474 aSAH patients were evaluated with a mean NLR 8.6 (SD 8.3). In multivariable logistic regression analysis, poor functional outcome was independently associated with higher NLR, older age, poorer clinical status on admission, prehospital statin use, and vasospasm. Increasing NLR analyzed as a continuous variable was independently associated with higher odds of poor functional outcome (OR 1.03, 95%CI 1.00-1.07, p=0.05) after adjustment for potential confounders. When dichotomized using ROC curve analysis, a threshold NLR value of greater than 6.48 was independently associated with higher odds of poor functional outcome (OR 1.71, 95%CI 1.07-2.74, p=0.03) after adjustment for potential confounders. CONCLUSIONS: Higher admission NLR is an independent predictor for poor functional outcome at discharge in aSAH patients. The evaluation of anti-inflammatory targets in the future may allow for improved functional outcome after aSAH.

Systemic Inflammation Response Index and Systemic Immune-inflammation Index for Predicting the Prognosis of Patients with Aneurysmal Subarachnoid Hemorrhage.

OBJECTIVES: Inflammatory response plays a pivotal role in the progress of aneurysmal subarachnoid hemorrhage (aSAH). As novel inflammatory markers, systemic inflammation response index (SIRI) and systemic immune-inflammation (SII) index could reflect clinical outcomes of patients with various diseases. The aim of this study was to ascertain whether initial SIRI and SII index were associated with prognosis of aSAH patients. METHODS: A total of 680 patients with aSAH were enrolled. Their prognosis was evaluated with modified Rankin Scale (mRS) at 3 months, and unfavorable clinical outcome was defined as mRS score of 3-6. Receiver operating characteristic (ROC) curve analysis was performed to identify cutoff values of SIRI and SII index for predicting clinical outcomes. Univariate and multivariate regression analyses were performed to explore relationships of SIRI and SII index with prognosis of patients. RESULTS: Optimal cutoff values of SIRI and SII index to discriminate between favorable and unfavorable clinical outcomes were 3.2 × 109/L and 960 × 109/L, respectively (P < 0.001 and 0.004, respectively). In multivariate analysis, SIRI value ≥ 3.2 × 109/L (odds ratio [OR]: 1.82, 95% CI: 1.46-3.24; P = 0.021) and SII index value ≥ 960 × 109/L (OR: 1.68, 95% CI: 1.24-2.74; P = 0.040) were independent predicting factors for poor prognosis after aSAH. CONCLUSIONS: SIRI and SII index values are associated with clinical outcomes of patients with aSAH. Elevated SIRI and SII index could be independent predicting factors for a poor prognosis after aSAH.

5-Lipoxygenase inhibition reduces inflammation and neuronal apoptosis via AKT signaling after subarachnoid hemorrhage in rats.

Early brain injury (EBI) is a major contributor to the high mortality and morbidity after subarachnoid hemorrhage (SAH). Inflammatory responses and neuronal apoptosis are important causes of EBI. Because 5- lipoxygenase (5-LOX) is known to be involved various central nervous system diseases, we investigated the effects of 5-LOX inhibition during EBI after SAH. Zileuton and LY294002 were used to inhibit expression of 5-LOX and Akt, respectively. We found that 5-LOX expression was significantly increased in the cytoplasm of cortical neurons after SAH and was accompanied by upregulated expression of the inflammatory factors LTB4, TNF-α, IL-1ß and IL-6; upregulation of the pro-apoptotic factor Bax; downregulation of the anti-apoptotic factor Bcl-2; and an increased apoptosis rate. Gastric Zileuton administration significantly suppressed all of those effects and improved neurological function. Zileuton also upregulated activated (phosphorylated) AKT levels, and these beneficial effects of Zileuton were abolished by intracerebroventricular infusion of the PI3K inhibitor LY294002. Taken together, these findings indicate that 5-LOX mediates pro-inflammatory and pro-apoptotic effects that contribute to EBI after SAH and that those effects are suppressed by activation of PI3K/Akt signaling. This suggests targeting 5-LOX may be an effective approach to treating EBI after SAH.

Interleukin 6 and Aneurysmal Subarachnoid Hemorrhage. A Narrative Review.

Interleukin 6 (IL-6) is a prominent proinflammatory cytokine. Neuroinflammation in general, and IL-6 signaling in particular, appear to play a major role in the pathobiology and pathophysiology of aneurysm formation and aneurysmal subarachnoid hemorrhage (SAH). Most importantly, elevated IL-6 CSF (rather than serum) levels appear to correlate with delayed cerebral ischemia (DCI, "vasospasm") and secondary ("vasospastic") infarctions. IL-6 CSF levels may also reflect other forms of injury to the brain following SAH, i.e., early brain damage and septic complications of SAH and aneurysm treatment. This would explain why many researchers have found an association between IL-6 levels and patient outcomes. These findings clearly suggest CSF IL-6 as a candidate biomarker in SAH patients. However, at this point, discrepant findings in variable study settings, as well as timing and other issues, e.g., defining proper clinical endpoints (i.e., secondary clinical deterioration vs. angiographic vasospasm vs. secondary vasospastic infarct) do not allow for its routine use. It is also tempting to speculate about potential therapeutic measures targeting elevated IL-6 CSF levels and neuroinflammation in SAH patients. Corticosteroids and anti-platelet drugs are indeed used in many SAH cases (not necessarily with the intention to interfere with detrimental inflammatory signaling), however, no convincing benefit has been demonstrated yet. The lack of a robust clinical perspective against the background of a relatively large body of data linking IL-6 and neuroinflammation with the pathophysiology of SAH is somewhat disappointing. One underlying reason might be that most relevant studies only report correlative data. The specific molecular pathways behind elevated IL-6 levels in SAH patients and their various interactions still remain to be delineated. We are optimistic that future research in this field will result in a better understanding of the role of neuroinflammation in the pathophysiology of SAH, which in turn, will translate into the identification of suitable biomarkers and even potential therapeutic targets.

Role of Adaptor Protein Myeloid Differentiation 88 (MyD88) in Post-Subarachnoid Hemorrhage Inflammation: A Systematic Review.

Myeloid differentiation 88 (MyD88) is a well-established inflammatory adaptor protein. It is one of the essential downstream proteins of the toll-like receptor 4 (TLR4) signaling pathway. TLRs are pattern recognition receptors that are usually activated by the damage-associated molecular pattern molecules (DAMPs). Sterile inflammation is triggered by the endogenous DAMPs released in response to global cerebral ischemia and from extravasated blood after subarachnoid hemorrhage (SAH). In this review, we highlight the importance of the neuroinflammatory role of the MyD88 in the SAH. We also explore a few possible pharmacological agents that can be used to decrease SAH-associated neuroinflammation by modulating the MyD88 dependent functions. Pharmacological agents such as flavonoids, melatonin, fluoxetine, pentoxifylline and progesterone have been investigated experimentally to reduce the SAH-associated inflammation. Inhibition of the MyD88 not only reduces the expression of pro-inflammatory cytokines, but also potentially inhibits other processes that can augment the SAH associated inflammation. Further investigations are required to translate these findings in the clinical setting.

In a model of SAH-induced neurogenic fever, BAT thermogenesis is mediated by erythrocytes and blocked by agonism of adenosine A1 receptors.

Neurogenic fever (NF) after subarachnoid hemorrhage (SAH) is a major cause of morbidity that is associated with poor outcomes and prolonged stay in the neurointensive care unit (NICU). Though SAH is a much more common cause of fever than sepsis in the NICU, it is often a diagnosis of exclusion, requiring significant effort to rule out an infectious source. NF does not respond to standard anti-pyretic medications such as COX inhibitors, and lack of good medical therapy has led to the introduction of external cooling systems that have their own associated problems. In a rodent model of SAH, we measured the effects of injecting whole blood, blood plasma, or erythrocytes on the sympathetic nerve activity to brown adipose tissue and on febrile thermogenesis. We demonstrate that following SAH the acute activation of brown adipose tissue leading to NF, is not dependent on PGE2, that subarachnoid space injection of whole blood or erythrocytes, but not plasma alone, is sufficient to trigger brown adipose tissue thermogenesis, and that activation of adenosine A1 receptors in the CNS can block the brown adipose tissue thermogenic component contributing to NF after SAH. These findings point to a distinct thermogenic mechanism for generating NF, compared to those due to infectious causes, and will hopefully lead to new therapies.

Interleukin 6 in cerebrospinal fluid is a biomarker for delayed cerebral ischemia (DCI) related infarctions after aneurysmal subarachnoid hemorrhage.

Interleukin 6 (IL-6) is a prominent proinflammatory cytokine and has been discussed as a potential biomarker for delayed cerebral ischemia (DCI) following aneurysmal subarachnoid hemorrhage. In the present study we have analyzed the time course of serum and cerebrospinal fluid (CSF) IL-6 levels in 82 patients with severe aneurysmal subarachnoid hemorrhage (SAH) requiring external ventricular drains in correlation to angiographic vasospasm, delayed cerebral ischemia, secondary infarctions and other clinical parameters. We observed much higher daily mean IL-6 levels (but also large interindividual variations) in the CSF than the serum of the patients with a peak between days 4 and 14 including a maximum on day 5 after SAH. Individual CSF peak levels correlated significantly with DCI (mean day 4-14 peak, DCI: 26,291 ± 24,159 pg/ml vs. no DCI: 16,184 ± 13,163 pg/ml; P = 0.023). Importantly, CSF IL-6 levels differed significantly between cases with DCI and infarctions and patients with DCI and no infarction (mean day 4-14 peak, DCI with infarction: 37,209 ± 26,951 pg/ml vs. DCI, no infarction: 15,123 ± 11,239 pg/ml; P = 0.003), while findings in the latter patient group were similar to cases with no vasospasm (mean day 4-14 peak, DCI, no infarction: 15,123 ± 11,239 vs. no DCI: 15,840 ± 12,979; P = 0.873). Together, these data support a potential role for elevated CSF IL-6 levels as a biomarker for DCI with infarction rather than for DCI in general. This fits well with a growing body of evidence linking neuroinflammation to ischemia and infarction, but (together with the large interindividual variations observed) limits the diagnostic usefulness of CSF IL-6 levels in SAH patients.

The role of immune inflammation in aneurysmal subarachnoid hemorrhage.

Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating disease, which mainly caused by the rupture of an intracranial aneurysm. Clinical trials have demonstrated that cerebral vasospasm (CVS) is not the sole contributor to delayed cerebral ischemia (DCI) and poor outcomes in patients with aSAH. Currently, accumulating evidence suggests that early brain injury (EBI), which occurs within 72 h after the onset of aSAH, lays the foundation for subsequent pathophysiological changes and poor outcomes of patients. The pathological mechanisms of EBI mainly include increased intracranial pressure, oxidative stress, neuroinflammation, blood-brain barrier (BBB) disruption, cerebral edema and cell death. Among them, the brain immune inflammatory responses involve a variety of immune cells and active substances, which play an important role in EBI after aSAH and may be related to DCI and long-term outcomes. Thus, attention should be paid to strategies targeting cerebral immune inflammatory responses. In this review, we discuss the role of immune inflammatory responses in the occurrence and development of aSAH, as well as some inflammatory biomarkers related to CVS, DCI, and aSAH outcomes. In addition, we also summarize the potential therapeutic drugs that target cerebral immune inflammatory responses for patients with aSAH in current research.

Respuesta inmune locorregional y sistémica en hemorragia subaracnoidea aneurismática aguda / Locoregional and systemic immune response in acute aneurysmal subarachnoid hemorrhage

La hemorragia subaracnoidea aneurismática (HSAa) aguda es una enfermedad que afecta a todas las edades, pero fundamentalmente a mujeres jóvenes en torno a los 50 años. Su impacto individual, familiar y sanitario continúa siendo aún inaceptablemente elevado. Esto se debe, en parte, al conocimiento parcial de los mecanismos injuriantes y reparadores que se desencadenan una vez que el aneurisma se rompe y la sangre arterial se vuelca al espacio subaracnoideo y/o ventricular. La respuesta inmune locorregional y sistémica tiene, potencialmente, un rol protagónico como uno de los principales mecanismos en juego desde los primeros minutos (injuria precoz). Su posible rol como puente o enlace hacia la injuria diferida (vasoespasmo-isquemia) también ha sido postulado. La respuesta innata ha recibido mayor atención (investigación) a la fecha. Sin embargo, la respuesta inmune adquirida también ha captado el interés neurocientífico en los últimos años. No menos importante es la interacción neuro-sistémica que caracteriza a esta enfermedad como una entidad clínica con un impacto multiorgánico precoz. En la presente tesis exploramos tanto la respuesta inmune innata como adquirida. Hicimos énfasis en aquellos efectores celulares más importantes y lo complementamos con el análisis de las citoquinas relacionadas y aquellas variables clínicas de interés tales como severidad del sangrado, vasoespasmo, mortalidad y con la técnica seleccionada para el tratamiento del saco aneurismático. Los resultados encontrados son originales, en algunos casos, mientras que otros corroboran hechos ya conocidos, típicos de la enfermedad. Entre los mismos destacamos que la muestra de pacientes enrolados padeció una HSAa aguda grave tanto desde el punto de vista clínico (pobre grado clínico) como tomográfico (abundante sangre volcada al espacio subaracnoideo). En estas condiciones, observamos una hiperleucocitosis con un aumento de los neutrófilos con un mayor estado de activación, particularmente a nivel del LCR. Concomitantemente aumentaron los monocitos totales y sus subpoblaciones a nivel de la sangre periférica. Por otra parte, tanto las células dendríticas como Natural Killers disminuyeron a nivel de la sangre periférica. Particularmente interesante e intrigante resultó ser la objetivación del predominio en LCR de la subpoblación NK CD56brigth CD16-. Con respecto al análisis de los linfocitos y subpoblaciones, observamos un descenso relativo de los mismos a nivel de la sangre periférica, pero no a nivel del LCR. Sin duda alguna, entre los hallazgos originales más atractivos desde un punto de vista patogénico, se encuentran los referentes a las variaciones de las subpoblaciones de células T CD4+ y CD8+ y su mayor estado de activación tanto a nivel de la sangre periférica como del LCR. Pero, además, detectamos un disbalance proinflamatorio del eje Th17/Treg (aumento del cociente) tanto a nivel de la sangre periférica como del LCR. Concomitantemente, la IL-17A aumentó en ambos compartimentos y su incremento a nivel de la sangre periférica en la etapa precoz se asoció al desarrollo ulterior de vasoespasmo y mayor mortalidad. Las restantes citoquinas analizadas (IL-2, IL-4, IL-6, IL-10, TNFα, INFγ) también se incrementaron significativamente tanto a nivel de la sangre periférica como del LCR, pero su incremento no se asoció estadísticamente con ninguna de las variables clínicas de interés mencionadas. Con respecto al posible impacto de la estrategia terapéutica seleccionada para el tratamiento del saco aneurismático sobre la respuesta inmune precoz y diferida, encontramos resultados potencialmente opuestos en las subpoblaciones Th1/Th2 a nivel del LCR, pero sin una asociación estadísticamente significativa con el perfil de citoquinas secretadas a dicho nivel. En suma, hemos demostrado, al igual que diversos investigadores alrededor del mundo, que la respuesta inmune innata tiene un papel protagónico en esta patología. Además, con el estudio del estado de activación hemos jerarquizado el rol de la respuesta inmune adaptativa CD4+ y CD8+. Postulamos al disbalance proinflamatorio del eje Th17/Treg como un potencial jugador patogénico clave y proponemos a la IL-17A como un prometedor biomarcador precoz de mayor morbimortalidad. Sin duda alguna, nuevas estrategias de investigación, experimental y clínica, podrán eventualmente, confirmar nuestros alentadores resultados preliminares comentados

Activation of adenosine A3 receptor reduces early brain injury by alleviating neuroinflammation after subarachnoid hemorrhage in elderly rats.

The incidence of subarachnoid hemorrhage (SAH) and hazard ratio of death increase with age. Overactivation of microglia contributes to brain damage. This study aimed to investigate the effects of A3 adenosine receptors (A3R) activation on neurofunction and microglial phenotype polarization in the context of SAH in aged rats. The A3R agonist (CI-IB-MECA) and antagonist (MRS1523) were used in the SAH model. Microglia were cultured to mimic SAH in the presence or absence of CI-IB-MECA and/or siRNA for A3R. The neurofunction and status of the microglial phenotype were evaluated. The P38 inhibitor SB202190 and the STAT6 inhibitor AS1517499 were used to explore the signaling pathway. The results showed that SAH induced microglia to polarize to the M(LPS) phenotype both in vivo and in vitro. CI-IB-MECA distinctly skewed microglia towards the M(IL-4) phenotype and ameliorated neurological dysfunction, along with the downregulation of inflammatory cytokines. Knockdown of A3R or inhibition of P38 and/or STAT6 weakened the effects of CI-IB-MECA on microglial phenotypic shifting. Collectively, our findings suggest that activation of A3R exerted anti-inflammatory and neuroprotective effects by regulating microglial phenotype polarization through P38/STAT6 pathway and indicated that A3R agonists may be a promising therapeutic options for the treatment of brain injury after SAH.

Mast Cell Promotes the Development of Intracranial Aneurysm Rupture.

BACKGROUND AND PURPOSE: Inflammation has emerged as a key component of the pathophysiology of intracranial aneurysms. Mast cells have been detected in human intracranial aneurysm tissues, and their presence was associated with intramural microhemorrhage and wall degeneration. We hypothesized that mast cells play a critical role in the development of aneurysmal rupture, and that mast cells can be used as a therapeutic target for the prevention of aneurysm rupture. METHODS: Intracranial aneurysms were induced in adult mice using a combination of induced systemic hypertension and a single injection of elastase into the cerebrospinal fluid. Aneurysm formation and rupture were assessed over 3 weeks. Roles of mast cells were assessed using a mast cell stabilizer (cromolyn), a mast cell activator (C48/80), and mice that are genetically lacking mature mast cells (KitW-sh/W-sh mice). RESULTS: Pharmacological stabilization of mast cells with cromolyn markedly decreased the rupture rate of aneurysms (80% versus 19%, n=10 versus n =16) without affecting the aneurysm formation. The activation of mast cells with C48/80 significantly increased the rupture rate of aneurysms (25% versus 100%, n=4 versus n=5) without affecting the overall rate of aneurysm formation. Furthermore, the genetic deficiency of mast cells significantly prevented aneurysm rupture (80% versus 25%, n=10 versus n=8, wild-type versus KitW-sh/W-sh mice). CONCLUSIONS: These results suggest that mast cells play a key role in promoting aneurysm rupture but not formation. Stabilizers of mast cells may have a potential therapeutic value in preventing intracranial aneurysm rupture in patients.

Inhibition of EZH2 (Enhancer of Zeste Homolog 2) Attenuates Neuroinflammation via H3k27me3/SOCS3/TRAF6/NF-κB (Trimethylation of Histone 3 Lysine 27/Suppressor of Cytokine Signaling 3/Tumor Necrosis Factor Receptor Family 6/Nuclear Factor-κB) in a Rat Model of Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: Neuroinflammation has been proven to play an important role in the pathogenesis of early brain injury after subarachnoid hemorrhage (SAH). EZH2 (enhancer of zeste homolog 2)-mediated H3K27Me3 (trimethylation of histone 3 lysine 27) has been recognized to play a critical role in multiple inflammatory diseases. However, there is still a lack of evidence to address the effect of EZH2 on the immune response of SAH. Therefore, the aim of this study was to determine the role of EZH2 in SAH-induced neuroinflammation and explore the effect of EZH2 inhibition with its specific inhibitor EPZ6438. METHODS: The endovascular perforation method was performed on rats to induce subarachnoid hemorrhage. EPZ6438, a specific EZH2 inhibitor, was administered intraperitoneally at 1 hour after SAH. SOCS3 (Suppressor of cytokine signaling 3) siRNA and H3K27me3 CRISPR were administered intracerebroventricularly at 48 hours before SAH to explore potential mechanisms. The SAH grade, short-term and long-term neurobehavioral tests, immunofluorescence staining, and western blots were performed after SAH. RESULTS: The expression of EZH2 and H3K27me3 peaked at 24 hours after SAH. In addition, inhibition of EZH2 with EPZ6438 significantly improved neurological deficits both in short-term and long-term outcome studies. Moreover, EPZ6438 treatment significantly decreased the levels of EZH2, H3K27Me3, pathway-related proteins TRAF6 (TNF [tumor necrosis factor] receptor family 6), NF-κB (nuclear factor-κB) p65, proinflammatory cytokines TNF-α, IL (interleukin)-6, IL-1ß, but increased the expression levels of SOCS3 and anti-inflammatory cytokine IL-10. Furthermore, administration of SOCS3 siRNA and H3k27me3-activating CRISPR partly abolished the neuroprotective effect of EPZ6438, which indicated that the neuroprotective effect of EPZ6438 acted, at least partly, through activation of SOCS3. CONCLUSIONS: In summary, the inhibition of EZH2 by EPZ6438 attenuated neuroinflammation via H3K27me3/SOCS3/TRAF6/NF-κB signaling pathway after SAH in rats. By targeting EZH2, this study may provide an innovative method to ameliorate early brain injury after SAH.

Fingolimod administration improves neurological functions of mice with subarachnoid hemorrhage.

PURPOSE: To investigate the brain protective effects of fingolimod on inflammatory response of SAH mice. METHODS: We utilized an endovascular mouse perforation model of SAH. Mice were divided into three groups: sham group, SAH group and SAH + Fingolimod group. Mice received either saline or fingolimod (1 mg/kg) intraperitoneally 2 h after sham surgery or SAH. The modified neurological severity score (mNSS) and Morris water maze were respectively used to evaluate the influence of nerve function. Evens blue (EB) extravasation was used to detect the permeability of blood-brain barrier, and water content in brain tissue was also detected. Flow cytometry, ELISA kits and western blotting were used to detect inflammatory factors in brain tissue. RESULTS: The results showed that compared with SAH group, after treatment, the delay time of locating the hidden platform was shorter. The mNSS results showed that fingolimod improved the behavior of SAH mice. In addition, fingolimod could reduce the water content in brain. Flow cytometry results showed that after 3 d of treatment, fingolimod significantly increased Treg cells and down-regulated NK cells. Western blotting results showed fingolimod inhibited the expression of inflammatory cytokines in brain tissue. ELISA kit results showed that fingolimod could down-regulate IL-6 and TNF-α and up-regulate IL-10 and TGF-ß1 in serum. CONCLUSIONS: Fingolimod could regulate the inflammatory response to alleviate SAH-induced brain damage and promote neurological recovery, which provides a new therapeutic strategy for SAH treatment.

Targeting CCL20 inhibits subarachnoid hemorrhage-related neuroinflammation in mice.

Recent evidence suggests that CC chemokine ligand 20 (CCL20) is upregulated after subarachnoid hemorrhage (SAH). Here, we investigated the functions of CCL20 in SAH injury and its underlying mechanisms of action. We found that CCL20 is upregulated in an SAH mouse model and in cultured primary microglia and neurons. CCL20-neutralizing antibody alleviated SAH-induced neurological deficits, decreased brain water content and neuronal apoptosis, and repressed microglial activation. We observed increased levels of CCL20, CC chemokine receptor 6 (CCR6), interleukin 1 beta (IL-1ß), and tumor necrosis factor alpha (TNF-α), as well as of microglial activation in microglia treated with oxyhemoglobin (OxyHb). CCL20 or CCR6 knockdown reversed the effects of OxyHb on microglia. Conditioned medium from OxyHb-treated microglia induced neuronal apoptosis, while the percentage of apoptotic neurons in the conditioned medium from microglia transfected with CCL20 siRNA or CCR6 siRNA was decreased. We observed no decrease in OxyHb-induced apoptosis in CCL20-knockdown neurons. Conditioned medium from OxyHb-treated neurons led to microglial activation and induced CCR6, IL-1ß and TNF-α expression, while CCL20 knockdown in neurons or CCR6 knockdown in microglia reversed those effects. Our results thus suggest CCL20 may be targeted to elicit therapeutic benefits after SAH injury.