The tight junction protein occludin modulates blood-brain barrier integrity and neurological function after ischemic stroke in mice.

Blood-brain barrier (BBB) disruption contributes to brain injury and neurological impairment. Tight junctions (TJs) and cell-cell adhesion complexes develop between endothelial cells in the brain to establish and maintain the BBB. Occludin, the first transmembrane protein identified in TJs, has received intense research interest because numerous in vitro studies have suggested its importance in maintaining BBB integrity. However, its role in maintaining BBB integrity after ischemic stroke is less clear owing to the lack of in vivo evidence. This study aimed to investigate the dynamics and function of occludin across the acute and chronic phases after stroke using occludin-deficient mice. By photochemically induced thrombosis model, the expression of occludin was decreased in brain endothelial cells from ischemic lesions. The neurological function of occludin-deficient mice was continuously impaired compared to that of wild-type mice. BBB integrity evaluated by Evans blue and 0.5-kDa fluorescein in the acute phase and by 10-kDa fluorescein isothiocyanate-labeled dextran in the chronic phase was decreased to a greater extent after stroke in occludin-deficient mice. Furthermore, occludin-deficient mice showed decreased claudin-5 and neovascularization after stroke. Our study reveals that occludin plays an important role from the acute to the chronic phase after ischemic stroke in vivo.

An Older Thrombus Delays Reperfusion after Mechanical Thrombectomy for Ischemic Stroke.

BACKGROUND: Thrombosis is a dynamic process, and a thrombus undergoes physical and biochemical changes that may alter its response to reperfusion therapy. This study assessed whether thrombus age influenced reperfusion quality and outcomes after mechanical thrombectomy for cerebral embolism. METHODS: We retrospectively evaluated 185 stroke patients and thrombi that were collected during mechanical thrombectomy at three stroke centers. Thrombi were pathologically classified as fresh or older based on their granulocytes' nuclear morphology and organization. Thrombus components were quantified, and the extent of NETosis (the process of neutrophil extracellular trap formation) was assessed using the density of citrullinated histone H3-positive cells. Baseline patient characteristics, thrombus features, endovascular procedures, and functional outcomes were compared according to thrombus age. RESULTS: Fresh thrombi were acquired from 43 patients, and older thrombi were acquired from 142 patients. Older thrombi had a lower erythrocyte content (p < 0.001) and higher extent of NETosis (p = 0.006). Restricted mean survival time analysis revealed that older thrombi were associated with longer puncture-to-reperfusion times (difference: 15.6 minutes longer for older thrombi, p = 0.002). This association remained significant even after adjustment for erythrocyte content and the extent of NETosis (adjusted difference: 10.8 minutes, 95% confidence interval [CI]: 0.6-21.1 minutes, p = 0.039). Compared with fresh thrombi, older thrombi required more device passes before reperfusion (p < 0.001) and were associated with poorer functional outcomes (adjusted common odds ratio: 0.49; 95% CI: 0.24-0.99). CONCLUSION: An older thrombus delays reperfusion after mechanical thrombectomy for ischemic stroke. Adding therapies targeting thrombus maturation may improve the efficacy of mechanical thrombectomy.

MiRNA-132/212 regulates tight junction stabilization in blood-brain barrier after stroke.

MicroRNA-132/212 has been supposed as a critical gene related to the blood-brain barrier (BBB) protection after stroke, but its regulation pathway including the upstream regulator and downstream targets is still unclear. Herein, we demonstrated the cAMP response element-binding protein (CREB)-regulated transcription coactivator-1 (CRTC1) to be the upstream regulator of miRNA-132/212 using CRTC1 knockout and wild-type mice. CRTC1 deletion led to the reduction of miRNA-132/212 expression in mice brain after ischemic stroke, significantly increased infarct volume, and aggravated BBB permeability with worsening neurological deficits. Furthermore, we identified that miRNA-132 repressed Claudin-1, tight junction-associated protein-1 (TJAP-1), and RNA-binding Fox-1 (RBFox-1) by directly binding to their respective 3'-untranslated regions, which alleviated the ischemic damage by enhancing neuronal survival and BBB integrity. Moreover, the co-culture of endothelial cells with CRTC1-deficient neurons aggravated the cell vulnerability to hypoxia, also supporting the idea that miRNA-132/212 cluster is regulated by CRTC1 and acts as a crucial role in the mitigation of ischemic damage. This work is a step forward for understanding the role of miRNA-132/212 in neurovascular interaction and may be helpful for potential gene therapy of ischemic stroke.

Stroke Severity and Outcomes in Patients With Newly Diagnosed Atrial Fibrillation.

Background and Purpose: Once a stroke occurs in a patient with atrial fibrillation (AF), it is likely to be severe. Patients with newly diagnosed AF after stroke and those with known AF before stroke have different background characteristics, yet the difference in stroke severity has not been sufficiently evaluated. In the current study, we compared the stroke severity and in-hospital outcomes between these patient groups. Methods: We retrospectively analyzed a database of 196 patients with acute ischemic stroke and AF between January 2010 and October 2019. We divided the patients into two groups: patients with "newly diagnosed AF" and those with "known AF." We assessed the stroke severity using the National Institutes of Health Stroke Scale (NIHSS) score on admission and in-hospital outcomes using the modified Rankin Scale (mRS) score at discharge. Results: The proportion of newly diagnosed AF was 33% (64/196). There were no differences in age, hypertension, diabetes mellitus, and past history of heart failure between patients with newly diagnosed AF and those with known AF. Patients with newly diagnosed AF were associated with a lower proportion of male sex (male; 50 vs. 67%, p < 0.05), a lower proportion of past history of stroke (12 vs. 35%, p < 0.01), a lower CHA2DS2-VASc score (median [interquartile range]; 3 [2-4] vs. 3.5 [3-5], p < 0.01), and a lower proportion of pre-stroke oral anticoagulation (5 vs. 59%, p < 0.01). There were no differences in the NIHSS score on admission (12 [4-19] vs. 9 [3-19]) or the mRS score at discharge (3 [1-5] vs. 3 [1-5]). After adjustment for relevant covariates, newly diagnosed AF was not associated with the NIHSS score on admission [adjusted common odds ratio (OR), 0.85; 95% confidence interval (CI), 0.45-1.60] or the mRS score at discharge (adjusted common OR, 1.67; 95% CI, 0.88-3.18). After propensity score matching, newly diagnosed AF was not associated with the NIHSS score on admission (adjusted common OR, 0.91; 95% CI, 0.48-1.73) and the mRS score at discharge (adjusted common OR, 1.77; 95% CI, 0.92-3.43). Conclusion: Stroke severity and in-hospital outcomes in patients with newly diagnosed AF did not differ from those in patients with known AF after adjustment for clinically relevant factors. The importance of detection of latent AF and subsequent anticoagulation in preventing severe stroke should be further emphasized.

Functional recoveries of patients with branch atheromatous disease after rehabilitation: Comparison with other types of cerebral infarction and importance of stratification by clinical categories.

BACKGROUND: Functional recoveries after rehabilitation of patients with branch atheromatous disease (BAD) have not been well investigated, however, clinical category of cerebral infarction including BAD itself could be a potential predictive factor for functional outcome. OBJECTIVE: To describe characteristics of functional recoveries of patients with BAD through comparison with other types of cerebral infarction. METHODS: We retrospectively compared outcomes of patients with BAD (N = 222), cardioembolic cerebral infarction (CE: N = 177) and atherothrombotic cerebral infarction (AT: N = 219) by using functional independence measure (FIM) and FIM effectiveness (the proportion of potential for improvement achieved). RESULTS: Univariate analysis showed that FIM on discharge was comparable among three types of cerebral infarction, but that FIM effectiveness in patients with BAD was significantly higher than those with CE or AT. Stratified analysis revealed higher FIM effectiveness in patients with BAD compared to patients with CE or AT, if they were male, younger (≤72 years) or had supratentorial brain lesions. Multiple regression analysis demonstrated that location of the brain lesion (supratentorial vs infratentorial) and gender (male vs female) were significantly associated with FIM on discharge, and that cognitive function on admission as well as gender were significantly associated with FIM effectiveness in patients with BAD, but not in patients with CE or AT. CONCLUSIONS: Outcomes after rehabilitation of patients with BAD may be characterized by better functional improvement, especially if patients are male, relatively younger or with supratentorial lesions. The impact and the type of factors related to functional recoveries of patients with BAD may be different from other types of stroke. The present study suggested that clinical category of stroke should be taken into consideration in prediction of outcomes and planning of rehabilitation management.

Efficient optimization of high-dose formulation of novel lyophilizates for dry powder inhalation by the combination of response surface methodology and time-of-flight measurement.

Inhalation of proteins/peptides has recently received attention as various biopharmaceuticals have emerged on the market. Novel lyophilisates for dry powder inhalation (LDPIs), which are aerosolized by air impact, have been reported and LDPIs are considered an attractive option for the pulmonary administration of biopharmaceuticals. However, desirable disintegration and aerosolization properties have been unavailable in high-dose formulations, which has been a critical issue. This study aimed to investigate high-dose LDPIs and their optimization. In the present study, lysozyme (Lysoz) was used as a stable model protein and formulated with various amino acids. Furthermore, response surface methodology (RSM) and time-of-flight measurement were applied for efficient optimization. Superior disintegration and aerosolization properties were confirmed in the LDPIs with phenylalanine (Phe) and leucine (Leu). RSM results revealed that 0.5 mg/vial of Phe and 1.0 mg/vial of Leu are the optimal quantities for high-dose formulation. Based on these optimum quantities, high-dose LDPI formulations were prepared and the maximum formulable quantity of Lysoz with acceptable inhalation performance was confirmed to be 3.0 mg/vial. The results suggest that LDPI can cover the milligram-order pulmonary administration of proteins/peptides. LDPIs are expected to have biopharmaceutical applications.

Design of monoalcohol - Copolymer system for high quality silver nanowires.

Research to improve the dimensional properties of silver nanowires (Ag NWs) for transparent conductive film (TCF) applications are being carried out intensively. However, the protocol for the designed synthesis of high-quality Ag NWs is yet to be developed due to the inadequacy of knowledge on the role of parameters. Here, we attempt to elucidate the role played by the parameters and propose a monoalcohol-copolymer based system for the designed synthesis of Ag NWs superior in quality to the one synthesized using conventional ethylene glycol (EG)-polyvinylpyrrolidone (PVP) system. The key findings of the study are as follows: (1) the solubility of Ag source and the partially formed AgCl particles in monoalcohols was found to play an important role not only in the reduction to metallic Ag but also on the uniaxial growth, (2) the adsorption of capping agents on Ag NWs was carried through O and N atoms in the base molecule and their binding energies indicated that the strength is the key parameter to obtain Ag NWs with high aspect ratio, (3) the properties of nanowire could be enhanced through copolymerization of VP and base molecules that have O- and N-based ligands, and (4) the influence of copolymerization on the physical and chemical properties of the surface active agent has been theoretically and experimentally verified. Consequently, we succeeded in the development of a new technique to synthesize high yield of Ag NWs with improved aspect ratio than EG-PVP system by using benzyl alcohol as reducing solvent and N-vinylpyrrolidone/N,N-diethylaminoethyl metacrylate copolymer as a capping agent. The optical transmittance and electrical resistivity of TCFs prepared using the Ag NWs with an average diameter of 43 nm, and an average length of 13 µm were 98.6% and R: 49.1 Ω/□, respectively.