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.

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.

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.

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.

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.

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.

Stimulator of IFN genes mediates neuroinflammatory injury by suppressing AMPK signal in experimental subarachnoid hemorrhage.

BACKGROUND: Neuroinflammation is closely associated with the poor prognosis in subarachnoid hemorrhage (SAH) patients. This study was aimed to determine the role of stimulator of IFN genes (STING), an essential regulator to innate immunity, in the context of SAH. METHODS: A total of 344 male C57BL/6 J mice were subjected to endovascular perforation to develop a model of SAH. Selective STING antagonist C-176 and STING agonist CMA were administered at 30 min or 1 h post-modeling separately. To investigate the underlying mechanism, the AMPK inhibitor compound C was administered intracerebroventricularly at 30 min before surgery. Post-SAH assessments included SAH grade, neurological test, brain water content, western blotting, RT-PCR, and immunofluorescence. Oxygenated hemoglobin was introduced into BV2 cells to establish a SAH model in vitro. RESULTS: STING was mainly distributed in microglia, and microglial STING expression was significantly increased after SAH. Administration of C-176 substantially attenuated SAH-induced brain edema and neuronal injury. More importantly, C-176 significantly alleviated both short-term and persistent neurological dysfunction after SAH. Meanwhile, STING agonist CMA remarkably exacerbated neuronal injury and deteriorated neurological impairments. Mechanically, STING activation aggravated neuroinflammation via promoting microglial activation and polarizing into M1 phenotype, evidenced by microglial morphological changes, as well as the increased level of microglial M1 markers including IL-1ß, iNOS, IL-6, TNF-α, MCP-1, and NLRP3 inflammasome, while C-176 conferred a robust anti-inflammatory effect. However, all the mentioned beneficial effects of C-176 including alleviated neuroinflammation, attenuated neuronal injury and the improved neurological function were reversed by AMPK inhibitor compound C. Meanwhile, the critical role of AMPK signal in C-176 mediated anti-inflammatory effect was also confirmed in vitro. CONCLUSION: Microglial STING yielded neuroinflammation after SAH, while pharmacologic inhibition of STING could attenuate SAH-induced inflammatory injury at least partly by activating AMPK signal. These data supported the notion that STING might be a potential therapeutic target for SAH.

RAR-Related Orphan Receptor Gamma T (RoRγt)-Related Cytokines Play a Role in Neutrophil Infiltration of the Central Nervous System After Subarachnoid Hemorrhage.

BACKGROUND: How inflammatory cells are recruited into the central nervous system is a topic of interest in a number of neurological injuries. In aneurysmal subarachnoid hemorrhage (SAH), neutrophil accumulation in the central nervous system 3 days after the hemorrhage is a critical step in the development of delayed cerebral injury (DCI). The mechanism by which neutrophils enter the central nervous system is still unclear. METHODS AND RESULTS: To identify human effectors of neutrophil recruitment, cerebrospinal fluid (CSF) samples were taken from a small, selected sample of SAH patients with external ventricular drainage devices (10 patients). Among a battery of CSF cytokines tested 3 days after SAH, five cytokines were associated with poor 90-day outcome (modified Rankin Score 3-6). A parallel study in a mouse model of mild SAH showed elevation in three cytokines in the CNS compared to sham. IL-17 and IL-2 were increased in both patients and the mouse model. IL-17 was investigated further because of its known role in neutrophil recruitment. Inhibition of RAR-Related Orphan Receptor Gamma T, the master transcription factor of IL-17, with the inverse agonist GSK805 suppressed neutrophils entry into the CNS after SAH compared to control. Using an IL-17 reporter mouse, we investigated the source of IL-17 and found that myeloid cells were a common IL-17-producing cell type in the meninges after SAH, suggesting an autocrine role for neutrophil recruitment. CONCLUSIONS: Taken together, IL-17 appears to be in important factor in the recruitment of neutrophils into the meninges after SAH and could be an important target for therapies to ameliorate DCI.

Toll-like receptor-4 pathway as a possible molecular mechanism for brain injuries after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is known as an acute catastrophic neurological disease that continues to be a serious and significant health problem worldwide. The mechanisms contributing to brain injury after SAH remain unclear despite decades of study focusing on early brain injury (EBI) and delayed brain injury (DBI). Neuroinflammation is a well-recognized consequence of SAH and may be responsible for EBI, cerebral vasospasm, and DBI. Toll-like receptors (TLRs) play a crucial role in the inflammatory response by recognizing damage-associated molecular patterns derived from the SAH. TLR4 is the most studied Toll-like receptor and is widely expressed in the central nervous system (CNS). It can be activated by the extravasated blood components in myeloid differentiation primary response-88/Toll/interleukin-1 receptor-domain-containing adapter-inducing interferon-ß (MyD88/TRIF)-dependent pathway after SAH. Transcription factors, such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK) and interferon regulatory factor (IRF), that regulate the expression of proinflammatory cytokine genes are initiated by the activation of TLR4, which cause the brain damage after SAH. TLR4 may therefore be a useful therapeutic target for overcoming EBI and DBI in post-SAH neuroinflammation, thereby improving SAH outcome. In the present review, we summarized recent findings from basic and clinical studies of SAH, with a primary focus on the biological characteristics and functions of TLR4 and discussed the mechanisms associated with TLR4 signaling pathway in EBI and DBI following SAH.

Elevated Neutrophil-Lymphocyte Ratio is Predictive of Poor Outcomes Following Aneurysmal Subarachnoid Hemorrhage.

Background Recent studies of patients with intracerebral hemorrhage suggest an association between peripheral blood neutrophil-lymphocyte ratio and neurologic deterioration. We aimed to study the prognostic utility of neutrophil-lymphocyte ratio in predicting inpatient mortality in aneurysmal subarachnoid hemorrhage. Methods We conducted a retrospective electronic medical record review of the clinical, laboratory, and radiographic data of patients with aneurysmal subarachnoid hemorrhage 18 years of age or older presenting to the neuroscience intensive care unit from January 1, 2011, to December 31, 2017. Patients with aneurysmal subarachnoid hemorrhage were divided into 2 groups (group 1, alive at discharge; group 2, deceased prior to discharge), and neutrophil-lymphocyte ratio laboratory mean values were recorded for each patient. Our primary outcome measure was inpatient mortality, and our secondary measure was incidence of pneumonia with hospitalization. Results We identified 403 patients with aneurysmal subarachnoid hemorrhage for the study. After exclusion criteria, 44 eligible patients were divided into the 2 groups (group 1, n = 32; group 2, n = 12). Mean neutrophil-lymphocyte ratio for group 1 was 11.53, and for group 2, 17.85 (P < .01). The mean neutrophil-lymphocyte ratio of those who developed pneumonia compared to those who did not was 15.28 versus 12.81, respectively (P = .39). A Kaplan-Meier plot demonstrated increased mortality among patients with a neutrophil-lymphocyte ratio equal to or greater than 12.5 compared to those with a neutrophil-lymphocyte ratio less than 12.5. Conclusions These preliminary data demonstrate that a neutrophil-lymphocyte ratio equal to or greater than 12.5 at admission predict higher inpatient mortality in patients with aneurysmal subarachnoid hemorrhage.

Innate immunity activation in the early brain injury period following subarachnoid hemorrhage.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (SAH) is a catastrophic disease with devastating consequences, including a high mortality rate and severe disabilities among survivors. Inflammation is induced following SAH, but the exact role and phenotype of innate immune cells remain poorly characterized. We investigated the inflammatory components of the early brain injury in an animal model and in SAH patients. METHOD: SAH was induced through injection of blood in the subarachnoid space of C57Bl/6 J wild-type mice. Prospective blood collections were obtained at 12 h, days 1, 2, and 7 to evaluate the systemic inflammatory consequences of SAH by flow cytometry and enzyme-linked immunosorbent-assay (ELISA). Brains were collected, enzymatically digested, or fixed to characterize infiltrating inflammatory cells and neuronal death using flow cytometry and immunofluorescence. Phenotypic evaluation was performed at day 7 using the holding time and footprint tests. We then compared the identified inflammatory proteins to the profiles obtained from the plasma of 13 human SAH patients. RESULTS: Following SAH, systemic IL-6 levels increased rapidly, whereas IL-10 levels were reduced. Neutrophils were increased both in the brain and in the blood reflecting local and peripheral inflammation following SAH. More intracerebral pro-inflammatory monocytes were found at early time points. Astrocyte and microglia activation were also increased, and mice had severe motor deficits, which were associated with an increase in the percentage of caspase-3-positive apoptotic neurons. Similarly, we found that IL-6 levels in patients were rapidly increased following SAH. ICAM-1, bFGF, IL-7, IL-12p40, and MCP-4 variations over time were different between SAH patients with good versus bad outcomes. Moreover, high levels of Flt-1 and VEGF at admission were associated with worse outcomes. CONCLUSION: SAH induces an early intracerebral infiltration and peripheral activation of innate immune cells. Furthermore, microglia and astrocytic activation are present at later time points. Our human and mouse data illustrate that SAH is a systemic inflammatory disease and that immune cells represent potential therapeutic targets to help this population of patients in need of new treatments.

Neuroinflammation and Microvascular Dysfunction After Experimental Subarachnoid Hemorrhage: Emerging Components of Early Brain Injury Related to Outcome.

Aneurysmal subarachnoid hemorrhage has a high mortality rate and, for those who survive this devastating injury, can lead to lifelong impairment. Clinical trials have demonstrated that cerebral vasospasm of larger extraparenchymal vessels is not the sole contributor to neurological outcome. Recently, the focus of intense investigation has turned to mechanisms of early brain injury that may play a larger role in outcome, including neuroinflammation and microvascular dysfunction. Extravasated blood after aneurysm rupture results in a robust inflammatory response characterized by activation of microglia, upregulation of cellular adhesion molecules, recruitment of peripheral immune cells, as well as impaired neurovascular coupling, disruption of the blood-brain barrier, and imbalances in endogenous vasodilators and vasoconstrictors. Each of these phenomena is either directly or indirectly associated with neuronal death and brain injury. Here, we review recent studies investigating these various mechanisms in experimental models of subarachnoid hemorrhage with special emphasis on neuroinflammation and its effect on microvascular dysfunction. We discuss the various therapeutic targets that have risen from these mechanistic studies and suggest the utility of a multi-targeted approach to preventing delayed injury and improving outcome after subarachnoid hemorrhage.

Autoantibodies against neuronal surface proteins in spontaneous subarachnoid and intracerebral haemorrhage.

BACKGROUND: Brain autoimmunity has been reported in patients with preceding infection of the central nervous system by herpesviridae. It has been hypothesized that neuronal damage releasing antigens might trigger secondary immune response. The objective of the study was to investigate whether brain damage due to spontaneous subarachnoid haemorrhage (SAH) or intracerebral haemorrhage (ICH) induces reactivity against neuronal surface proteins. METHODS: Patients with spontaneous SAH and ICH, who had cerebrospinal fluid (CSF) and serum sampling within 2 weeks after disease onset (baseline) and afterwards at least 10 days later (follow-up), were included. Antibodies against NMDA, GABA-B, AMPA-1/- 2 receptor, LGI1 and CASPR2 were determined by indirect immunofluorescence. RESULTS: A total of 43 SAH and 11 ICH patients aged 62 (±12) years (65% females) had simultaneous CSF/ serum sampling median 5 and 26.5 days after disease onset. At baseline, all CSF samples were collected via ventricular drainage, at follow-up 20 (37.0%) patients had CSF collection by lumbar puncture because ventricular drain had been already removed. All CSF and serum samples at baseline and follow-up tested negative for antibodies against NMDA, GABA-B, AMPA-1/- 2 receptor, LGI1 and CASPR2. CONCLUSIONS: Immunoreactivity against common neuronal surface proteins was not observed within the early disease course of spontaneous SAH and ICH.

NF-κB-Mediated Inflammation in the Pathogenesis of Intracranial Aneurysm and Subarachnoid Hemorrhage. Does Autophagy Play a Role?

The rupture of saccular intracranial aneurysms (IA) is the commonest cause of non-traumatic subarachnoid hemorrhage (SAH)—the most serious form of stroke with a high mortality rate. Aneurysm walls are usually characterized by an active inflammatory response, and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) has been identified as the main transcription factor regulating the induction of inflammation-related genes in IA lesions. This transcription factor has also been related to IA rupture and resulting SAH. We and others have shown that autophagy interacts with inflammation in many diseases, but there is no information of such interplay in IA. Moreover, NF-κB, which is a pivotal factor controlling inflammation, is regulated by autophagy-related proteins, and autophagy is regulated by NF-κB signaling. It was also shown that autophagy mediates the normal functioning of vessels, so its disturbance can be associated with vessel-related disorders. Early brain injury, delayed brain injury, and associated cerebral vasospasm are among the most serious consequences of IA rupture and are associated with impaired function of the autophagy⁻lysosomal system. Further studies on the role of the interplay between autophagy and NF-κB-mediated inflammation in IA can help to better understand IA pathogenesis and to identify IA patients with an increased SAH risk.

Fluoxetine-enhanced autophagy ameliorates early brain injury via inhibition of NLRP3 inflammasome activation following subrachnoid hemorrhage in rats.

BACKGROUND: The NLRP3 inflammasome is a multiprotein complex that regulates the innate immune inflammatory response by activating caspase-1 and subsequent IL-1ß and IL-18. Fluoxetine has been shown to have the anti-inflammatory properties in many disease models. However, the effects and mechanisms of these effects of fluoxetine in early brain injury after subarachnoid hemorrhage (SAH) have not been defined. METHODS: The SAH model was induced by an endovascular perforation in adult male Sprague-Dawley (SD) rats weighing 300-320 g. N-Ac-Tyr-Val-Ala-Asp-chloromethyl ketone (AC-YVAD-CMK) was injected intraperitoneally (5 mg/kg) 1 h after SAH. Fluoxetine was administered via intravenous route 6 h after SAH. 3-Methyladenine (3-MA) was intracerebroventricularly injected 20 min before SAH. SAH grade, neurological function, brain water content, propidium iodide (PI) staining, western blot, double immunostaining, and transmission electron microscopy were performed. RESULTS: Expression of caspase-1 increased and peaked at 24 h after SAH. Caspase activation was along with the increased necrotic cells, which occurred mainly in neurons. Necrotic cell death of microglia and astrocyte were also found. Administration of AC-YVAD-CMK, a caspase-1 inhibitor, reduced the expression of IL-1ß and IL-18 and the number of PI-positive cells, attenuated brain edema, and improved neurological function, which was also observed in fluoxetine-treated rats. Furthermore, fluoxetine treatment significantly decreased the expression of NLRP3 and cleaved caspase-1 and upregulated the expression of beclin-1, a marker for autophagy. Finally, the effects of fluoxetine in NLRP3 inflammasome activation were reversed by additional 3-MA administration. CONCLUSIONS: Together, our present study indicated that NLRP3 inflammasome and caspase-1 activation play a deleterious role in early brain injury and fluoxetine mitigates NLRP3 inflammasome and caspase-1 activation through autophagy activation after SAH, providing a potential therapeutic agent for SAH treatment.

Aneurysmal subarachnoid hemorrhage lead to systemic upregulation of IL-23/IL-17 inflammatory axis.

IL-23 and IL-17 are pro-inflammatory cytokines. IL-23 is secreted by activated macrophages and dendritic cells, while IL-17 by Th17 cells. Serum IL-23 and IL-17 are known to be elevated in numerous inflammatory diseases including neurodegenerative diseases. The role of serum IL-23 and IL-17 in aneurysmal subarachnoid hemorrhage (aSAH) has still not been investigated. The present work investigates the serum IL-23 and IL-17 levels and their association with post hemorrhagic complications and clinical outcome in patients with aSAH. METHODS: In this study, 80 patients with aSAH (Hunt and Hess grade I-V) were prospectively recruited. We enrolled 24 control patients with lumbar spinal stenosis. Peripheral venous blood was withdrawn from controls and from aSAH patients at day 1 and day 7, allowed to clot and centrifuged to obtain serum. Enzyme linked immunoassay kits were employed to quantify the serum levels of IL-23 and IL-17 by applying 50µL of serum samples. Post hemorrhagic complications and clinical outcome were documented prospectively from patient's hospital record. RESULTS: Serum IL-23 and IL-17 levels were significantly elevated in aSAH patients at day 1 and day 7 (n=80) as compared to control patients (n=24). Further analysis after dichotomy of patients who suffered from post hemorrhagic complications including cerebral vasospasm, chronic hydrocephalus, seizures, cerebral ischemia, delayed neurological deficits showed differential correlations with different post hemorrhagic complications (Table 1). Serum IL-23 and IL-17 levels did not correlate with clinical outcome. CONCLUSION: Serum IL-23 and IL-17 levels were elevated in patients with aSAH showing upregulation of IL-23/IL-17 inflammatory axis after aSAH. Serum IL-23 and IL-17 showed differential correlations with post hemorrhagic complications and no correlation with clinical outcome.

The Role of Platelet Activation and Inflammation in Early Brain Injury Following Subarachnoid Hemorrhage.

BACKGROUND: Early brain injury (EBI) following aneurysmal subarachnoid hemorrhage (SAH) is an important predictor of poor functional outcome, yet the underlying mechanism is not well understood. Animal studies suggest that platelet activation and inflammation with subsequent microthrombosis and ischemia may be a mechanism of EBI. METHODS: A prospective, hypothesis-driven study of spontaneous, SAH patients and controls was conducted. Platelet activation [thromboelastography maximum amplitude (MA)] and inflammation [C-reactive protein (CRP)] were measured serially over time during the first 72 h following SAH onset. Platelet activation and inflammatory markers were compared between controls and SAH patients with mild [Hunt-Hess (HH) 1-3] versus severe (HH 4-5) EBI. The association of these biomarkers with 3-month functional outcomes was evaluated. RESULTS: We enrolled 127 patients (106 SAH; 21 controls). Platelet activation and CRP increased incrementally with worse EBI/HH grade, and both increased over 72 h (all P < 0.01). Both were higher in severe versus mild EBI (MA 68.9 vs. 64.8 mm, P = 0.001; CRP 12.5 vs. 1.5 mg/L, P = 0.003) and compared to controls (both P < 0.003). Patients with delayed cerebral ischemia (DCI) had more platelet activation (66.6 vs. 64.9 in those without DCI, P = 0.02) within 72 h of ictus. At 3 months, death or severe disability was more likely with higher levels of platelet activation (mRS4-6 OR 1.18, 95 % CI 1.05-1.32, P = 0.007) and CRP (mRS4-6 OR 1.02, 95 % CI 1.00-1.03, P = 0.041). CONCLUSIONS: Platelet activation and inflammation occur acutely after SAH and are associated with worse EBI, DCI and poor 3-month functional outcomes. These markers may provide insight into the mechanism of EBI following SAH.

Elevated Systemic IL-6 Levels in Patients with Aneurysmal Subarachnoid Hemorrhage Is an Unspecific Marker for Post-SAH Complications.

Background: Aneurysmal subarachnoid hemorrhage (aSAH) is still a fatal and morbid disease, although bleeding aneurysms can be secured in almost all cases. Occurrence of post-SAH complications including cerebral vasospasm, delayed cerebral ischemia, hydrocephalus, epilepsy, and infections are the main determinants of clinical outcome. Hence, it is important to search for early predictors for specific post-SAH complications to treat these complications properly. Both cellular and molecular (cytokines) inflammation play a key role after aSAH during the phase of occurrence of post-SAH complications. Interleukin-6 (IL-6) is a well-known cytokine that has been extensively analyzed in cerebrospinal fluid (CSF) of patients after aSAH, but detailed studies exploring the role of systemic IL-6 in aSAH associated complications and its impact on early clinical outcome prediction are lacking. The current study aims to analyze the systemic IL-6 levels over two weeks after bleeding and its role in post-SAH complications. Methods: We recruited 80 aSAH patients prospectively who underwent peripheral venous blood withdrawal in serum gel tubes. The blood was centrifuged to harvest the serum, which was immediately frozen at -80 °C until analysis. Serum IL-6 levels were quantified using Immulite immunoassay system. Patient records including age, gender, post-SAH complications, aneurysm treatment, and clinical outcome (modified Rankin scale and Glasgow outcome scale) were retrieved to allow different subgroup analysis. Results: Serum IL-6 levels were significantly raised after aSAH compared to healthy controls over the first two weeks after hemorrhage. Serum IL-6 levels were found to be significantly elevated in aSAH patients presenting with higher Hunt and Hess grades, increasing age, and both intraventricular and intracerebral hemorrhage. Interestingly, serum IL-6 was also significantly raised in aSAH patients who developed seizures, cerebral vasospasm (CVS), and chronic hydrocephalus. IL-6 levels were sensitive to the development of infections and showed an increase in patients who developed pneumoniae. Intriguingly, we found a delayed increase in serum IL-6 in patients developing cerebral infarction. Finally, IL-6 levels were significantly higher in patients presenting with poor clinical outcome in comparison to good clinical outcome at discharge from hospital. Conclusion: Serum IL-6 levels were elevated early after aSAH and remained high over the two weeks after initial bleeding. Serum IL-6 was elevated in different aSAH associated complications, acting as a non-specific marker for post-SAH complications and an important biomarker for clinical outcome at discharge.

Lipoxin A4 Reduces Inflammation Through Formyl Peptide Receptor 2/p38 MAPK Signaling Pathway in Subarachnoid Hemorrhage Rats.

BACKGROUND AND PURPOSE: Lipoxin A4 (LXA4) has been reported to reduce inflammation in several neurological injury models. We studied the effects of LXA4 on neuroinflammation after subarachnoid hemorrhage (SAH) in a rat model. METHODS: Two hundred and thirty-eight Sprague-Dawley male rats, weight 280-320 g, were used. Exogenous LXA4 (0.3 and 1.0 nmol) were injected intracerebroventricularly at 1.5 hours after SAH. Neurological scores, brain water content, and blood-brain barrier were evaluated at 24 hours after SAH; Morris water maze and T-maze tests were examined at 21 days after SAH. The expression of endogenous LXA4 and its receptor formyl peptide receptor 2 (FPR2), as well as p38, interleukin-1ß, and interleukin-6 were studied either by ELISA or by Western blots. Neutrophil infiltration was observed by myeloperoxidase staining. FPR2 siRNA was used to knock down LXA4 receptor. RESULTS: The expression of endogenous LXA4 decreased, and the expression of FPR2 increased after SAH. Exogenous LXA4 decreased brain water content, reduced Evans blue extravasation, and improved neurological functions and improved the learning and memory ability after SAH. LXA4 reduced neutrophil infiltration and phosphorylation of p38, interleukin-1ß, and interleukin-6. These effects of LXA4 were abolished by FPR2 siRNA. CONCLUSIONS: Exogenous LXA4 inhibited inflammation by activating FPR2 and inhibiting p38 after SAH. LXA4 may serve as an alternative treatment to relieve early brain injury after SAH.