Inhibition of LAR attenuates neuroinflammation through RhoA/IRS-1/Akt signaling pathway after intracerebral hemorrhage in mice.

Leukocyte common antigen-related phosphatase (LAR) is widely expressed in the central nervous system and is known to regulate a variety of processes including cell growth, differentiation, and inflammation. However, little is currently known about LAR signaling mediated neuroinflammation after intracerebral hemorrhage (ICH). The objective of this study was to investigate the role of LAR in ICH using autologous blood injection-induced ICH mouse model. Expression of endogenous proteins, brain edema and neurological function after ICH were evaluated. Extracellular LAR peptide (ELP), an inhibitor of LAR, was administered to ICH mice and outcomes were evaluated. LAR activating-CRISPR or IRS inhibitor NT-157 was administered to elucidate the mechanism. The results showed that expressions of LAR, its endogenous agonist chondroitin sulfate proteoglycans (CSPGs) including neurocan and brevican, and downstream factor RhoA increased after ICH. Administration of ELP reduced brain edema, improved neurological function, and decreased microglia activation after ICH. ELP decreased RhoA and phosphorylated serine-IRS1, increased phosphorylated tyrosine-IRS1 and p-Akt, and attenuated neuroinflammation after ICH, which was reversed by LAR activating-CRISPR or NT-157. In conclusion, this study demonstrated that LAR contributed to neuroinflammation after ICH via RhoA/IRS-1 pathway, and ELP may be a potential therapeutic strategy to attenuate LAR mediated neuroinflammation after ICH.

Metformin Preserves VE-Cadherin in Choroid Plexus and Attenuates Hydrocephalus via VEGF/VEGFR2/p-Src in an Intraventricular Hemorrhage Rat Model.

Hydrocephalus induced by intraventricular hemorrhage (IVH) is associated with unfavorable prognosis. The increased permeability of choroid plexus and breakdown of the blood-brain barrier (BBB) was reported as a prominent mechanism of IVH-induced hydrocephalus, and vascular endothelial-cadherin (VE-cadherin) was demonstrated to be relevant. Metformin was reported to protect endothelial junction and preserve permeability widely; however, its role in hydrocephalus remains unclear. In this study, the decreased expression of VE-cadherin in the choroid plexus, accompanied with ventricle dilation, was investigated in an IVH rat model induced by intraventricular injection of autologous blood. Metformin treatment ameliorated hydrocephalus and upregulated VE-cadherin expression in choroid plexus meanwhile. We then observed that the internalization of VE-cadherin caused by the activation of vascular endothelial growth factor (VEGF) signaling after IVH was related to the occurrence of hydrocephalus, whereas it can be reversed by metformin treatment. Restraining VEGF signaling by antagonizing VEGFR2 or inhibiting Src phosphorylation increased the expression of VE-cadherin and decreased the severity of hydrocephalus after IVH. Our study demonstrated that the internalization of VE-cadherin via the activation of VEGF signaling may contribute to IVH-induced hydrocephalus, and metformin may be a potential protector via suppressing this pathway.

TLR7 (Toll-Like Receptor 7) Facilitates Heme Scavenging Through the BTK (Bruton Tyrosine Kinase)-CRT (Calreticulin)-LRP1 (Low-Density Lipoprotein Receptor-Related Protein-1)-Hx (Hemopexin) Pathway in Murine Intracerebral Hemorrhage.

Background and Purpose- Heme and iron are considered to be key factors responsible for secondary insults after intracerebral hemorrhage (ICH). Our previous study showed that LRP1 (low-density lipoprotein receptor-related protein-1)-Hx (hemopexin) facilitates removal of heme. The TLR7 (Toll-like receptor 7)-BTK (Bruton tyrosine kinase)-CRT (calreticulin) pathway regulates the expression of LRP1-Hx. This study is designed to clarify whether TLR7 activation facilitates heme scavenging and to establish the potential role of the BTK-CRT-LRP1-Hx signaling pathway in the pathophysiology of ICH. Methods- ICH was induced by stereotactic, intrastriatal injection of type VII collagenase. Mice received TLR7 agonist (imiquimod) via intraperitoneal injection after ICH induction. TLR7 inhibitor (ODN2088), BTK inhibitor (LFM-A13), and CRT agonist (thapsigargin) were given in different groups to further evaluate the underlying pathway. Mice were randomly divided into sham, ICH+vehicle (normal saline), ICH+Imiquimod (2.5, 5, and 10 µg/g), ICH+ODN2088, ICH+LFM-A13, ICH+thapsigargin, and ICH+ODN2088+thapsigargin. Imiquimod was administered twice daily starting at 6 hours after ICH; ODN2088 was administered by intracerebroventricular injection at 30 minutes, and LFM-A13 or thapsigargin was administered by intraperitoneal injection at 3 hours after ICH induction. Neurological scores, cognitive abilities, as well as brain edema, blood-brain barrier permeability, hemoglobin level, brain expression of TLR7/BTK/CRT/LRP1/Hx were analyzed. Results- Low dosage imiquimod significantly attenuated hematoma volume, brain edema, BBB permeability, and neurological deficits after ICH. Imiquimod also increased protein expressions of TLR7, BTK, CRT, LRP1, and Hx; ODN2088 reduced TLR7, BTK, CRT, LRP1, and Hx expressions. Conclusions- TLR7 plays an important role in heme scavenging after ICH by modulating the BTK-CRT-LRP1-Hx pathway. TLR7 may offer protective effects by promoting heme resolution and reduction of brain edema after ICH.

Association of Renal Dysfunction With Remote Diffusion-Weighted Imaging Lesions and Total Burden of Cerebral Small Vessel Disease in Patients With Primary Intracerebral Hemorrhage.

Objective: Remote diffusion-weighted imaging (DWI) lesions (R-DWIL) found in intracerebral hemorrhage (ICH) patients are considered as an additional marker of cerebral small vessel disease (cSVD). This study aimed to investigate the association of renal dysfunction and R-DWIL, as well as the total burden of cSVD on magnetic resonance imaging among patients with primary ICH. Methods: One hundred and twenty-six consecutive patients were prospectively enrolled. R-DWIL on DWI, as well as other imaging markers of cSVD, including lacunes, white matter lesions, cerebral microbleeds, and enlarged perivascular spaces were rated using validated scales. Renal dysfunction was evaluated either by reduced estimated glomerular filtration rate (eGFR) or the presence of proteinuria or increased cystatin C. Results: After adjustments for potential confounders by logistic regression, impaired eGFR [odds ratio (OR) 6.00, 95% confidence interval (CI) 1.73-20.78], proteinuria (OR 3.07, 95% CI 1.25-7.54) and increased cystatin C (OR 2.73, 95% CI 1.11-6.72) were correlated with presence of R-DWIL. A similar association was also found between cystatin C levels (OR 3.16, 95% CI 1.39-7.19), proteinuria (OR 2.79, 95% CI 1.34-5.83) and the comprehensive cSVD burden. Conclusions: Renal dysfunction are associated with the presence of R-DWIL, and total burden of cSVD in patients with primary ICH.

Congenital anomaly of the posterior arch of the atlas: a rare risk factor for posterior circulation stroke.

We report an extremely rare case involving a posterior arch anomaly of the atlas, causing a vertebral artery dissection (VAD) induced posterior circulation stroke. A 16-year-old girl was admitted to hospital because of new onset dizziness. VAD related multiple infarction in the posterior circulation was revealed. The congenital posterior arch anomaly of the atlas, along with instability of the atlantoaxial joint, were discovered accidentally during follow-up. This is the first case of ischemic stroke related to atlantal posterior arch aplasia and atlantoaxial instability. Although rare, it reminds us that investigation of the craniovertebral junction should be considered when stroke occurs in young patients.

Recombinant Gas6 augments Axl and facilitates immune restoration in an intracerebral hemorrhage mouse model.

Axl, a tyrosine kinase receptor, was recently identified as an essential component regulating innate immune response. Suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 are potent Axl-inducible negative inflammatory regulators. This study investigated the role of Axl signaling pathway in immune restoration in an autologous blood-injection mouse model of intracerebral hemorrhage. Recombinant growth arrest-specific 6 (Gas6) and R428 were administrated as specific agonist and antagonist. In vivo knockdown of Axl or suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 by siRNA was applied. After intracerebral hemorrhage, the expression of endogenous Axl, soluble Axl, and Gas6 was increased, whereas the expression of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 was inhibited. Recombinant growth arrest-specific 6 administration alleviated brain edema and improved neurobehavioral performances. Moreover, enhanced Axl phosphorylation with cleavage of soluble Axl (sAxl), and an upregulation of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 were observed. In vivo knockdown of Axl and R428 administration both abolished the effect of recombinant growth arrest-specific 6 on brain edema and also decreased the expression suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3. In vivo knockdown of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 aggravated cytokine releasing despite of recombinant growth arrest-specific 6. In conclusion, Axl plays essential role in immune restoration after intracerebral hemorrhage. And recombinant growth arrest-specific 6 attenuated brain injury after intracerebral hemorrhage, probably by enhancing Axl phosphorylation and production of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3.

Different risk factors for poor outcome between patients with positive and negative susceptibility vessel sign.

PURPOSE: The absence of the susceptibility vessel sign (negative SVS) on gradient-recalled echo or susceptibility-weighted imaging (SWI) in thrombolytic therapy has not been well studied. Since positive and negative SVS may have different components, we aimed to investigate the difference in risk factors for clinical outcome between patients with positive and negative SVS. METHODS: We retrospectively examined clinical and imaging data from 85 consecutive patients with acute ischemic stroke with middle cerebral artery occlusion who underwent SWI before intravenous thrombolysis (IVT). We then examined the predictors of negative SVS and the risk factors for a poor outcome (defined as modified Rankin Scale score ≥3) 3 months after IVT in subgroup analysis. RESULTS: Multivariate regression analysis indicated that previous antiplatelet use (OR 0.076; 95% CI 0.007 to 0.847; p=0.036) and shorter time from onset to treatment (OR 1.051; 95% CI 1.003 to 1.102; p=0.037) were inversely associated with poor outcome in patients with negative SVS, while higher baseline National Institutes of Health Stroke Scale (NIHSS) score was associated with poor outcome in patients with positive SVS (OR 1.222; 95% CI 1.084 to 1.377; p=0.001). CONCLUSIONS: The risk factors for clinical outcome after IVT in patients with negative SVS may differ from those with positive SVS.

Hyperbaric Oxygen Preconditioning Attenuates Hemorrhagic Transformation Through Reactive Oxygen Species/Thioredoxin-Interacting Protein/Nod-Like Receptor Protein 3 Pathway in Hyperglycemic Middle Cerebral Artery Occlusion Rats.

OBJECTIVES: To clarify whether hyperbaric oxygen preconditioning can attenuate hyperglycemia-enhanced hemorrhagic transformation and to establish a role for Nod-like receptor protein 3 inflammasome in the pathophysiology of hemorrhagic transformation. DESIGN: Controlled prospective animal study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats weighing 260-280 g. INTERVENTIONS: Rats received 1-hour-long hyperbaric oxygen preconditioning for five consecutive days. Hyperglycemic middle cerebral artery occlusion model was induced at 24 hours after the last hyperbaric oxygen exposure. Reactive oxygen species scavenger (N-acetyl-L-cysteine), thioredoxin-interacting protein small interfering RNA, and Nod-like receptor protein 3 small interfering RNA were given in different groups separately to verify the possible pathway. MEASUREMENTS AND MAIN RESULTS: Rats were randomly divided into sham, middle cerebral artery occlusion, middle cerebral artery occlusion + dextrose, middle cerebral artery occlusion + dextrose + normobaric oxygen preconditioning, middle cerebral artery occlusion + dextrose + hyperbaric oxygen, middle cerebral artery occlusion + dextrose + hyperbaric oxygen + N-acetyl-L-cysteine, middle cerebral artery occlusion + dextrose + hyperbaric oxygen + control small interfering RNA, middle cerebral artery occlusion + dextrose + hyperbaric oxygen + thioredoxin-interacting protein small interfering RNA, and middle cerebral artery occlusion + dextrose + hyperbaric oxygen + Nod-like receptor protein 3 small interfering RNA groups. Hyperglycemia was induced by administration of 50% dextrose (6 mL/kg) intraperitoneally 30 minutes before middle cerebral artery occlusion. Control small interfering RNA/thioredoxin-interacting protein small interfering RNA or Nod-like receptor protein 3 small interfering RNA (500 pmol/5 µL) were injected intracerebroventricularly 72 hours before middle cerebral artery occlusion for intervention. The neurologic scores, infarction and hemorrhage volumes, the expression of Nod-like receptor protein 3, and its downstream targets were analyzed. Hyperbaric oxygen preconditioning decreased both infarction and hemorrhage volumes and improved neurobehavioral function. In addition, hyperbaric oxygen preconditioning provided additional protective effects in hemorrhagic transformation, which was independent of infarction volume. The benefits of hyperbaric oxygen preconditioning on hyperglycemic middle cerebral artery occlusion rats were reversed after blocking the reactive oxygen species/thioredoxin-interacting protein/Nod-like receptor protein 3 pathway. CONCLUSIONS: Nod-like receptor protein 3 inflammasome played an important role in hyperglycemia-enhanced hemorrhagic transformation. Hyperbaric oxygen preconditioning attenuated hemorrhagic transformation through reactive oxygen species/thioredoxin-interacting protein/Nod-like receptor protein 3 pathway.