Tuberculous pleural effusion-induced Arg-1+ macrophage polarization contributes to lung cancer progression via autophagy signaling.

BACKGROUND: The association between tuberculous fibrosis and lung cancer development has been reported by some epidemiological and experimental studies; however, its underlying mechanisms remain unclear, and the role of macrophage (MФ) polarization in cancer progression is unknown. The aim of the present study was to investigate the role of M2 Arg-1+ MФ in tuberculous pleurisy-assisted tumorigenicity in vitro and in vivo. METHODS: The interactions between tuberculous pleural effusion (TPE)-induced M2 Arg-1+ MФ and A549 lung cancer cells were evaluated. A murine model injected with cancer cells 2 weeks after Mycobacterium bovis bacillus Calmette-Guérin pleural infection was used to validate the involvement of tuberculous fibrosis to tumor invasion. RESULTS: Increased CXCL9 and CXCL10 levels of TPE induced M2 Arg-1+ MФ polarization of murine bone marrow-derived MФ. TPE-induced M2 Arg-1+ MФ polarization facilitated lung cancer proliferation via autophagy signaling and E-cadherin signaling in vitro. An inhibitor of arginase-1 targeting M2 Arg-1+ MФ both in vitro and in vivo significantly reduced tuberculous fibrosis-induced metastatic potential of lung cancer and decreased autophagy signaling and E-cadherin expression. CONCLUSION: Tuberculous pleural fibrosis induces M2 Arg-1+ polarization, and M2 Arg-1+ MФ contribute to lung cancer metastasis via autophagy and E-cadherin signaling. Therefore, M2 Arg-1+ tumor associated MФ may be a novel therapeutic target for tuberculous fibrosis-induced lung cancer progression.

Genome-wide association study identifies novel susceptibilities to adult moyamoya disease.

Genome-wide association study has limited to discover single-nucleotide polymorphisms (SNPs) in several ethnicities. Here, we investigated an initial GWAS to identify genetic modifiers predicting with adult moyamoya disease (MMD) in Koreans. GWAS was performed in 216 patients with MMD and 296 controls using the large-scale Asian-specific Axiom Precision Medicine Research Array. A subsequent fine-mapping analysis was conducted to assess the causal variants associated with adult MMD. A total of 489,966 out of 802,688 SNPs were subjected to quality control analysis. Twenty-one SNPs reached a genome-wide significance threshold (p = 5 × 10-8) after pruning linkage disequilibrium (r2 < 0.8) and mis-clustered SNPs. Among these variants, the 17q25.3 region including TBC1D16, CCDC40, GAA, RNF213, and ENDOV genes was broadly associated with MMD (p = 3.1 × 10-20 to 4.2 × 10-8). Mutations in RNF213 including rs8082521 (Q1133K), rs10782008 (V1195M), rs9913636 (E1272Q), rs8074015 (D1331G), and rs9674961 (S2334N) showed a genome-wide significance (1.9 × 10-8 < p < 4.3 × 10-12) and were also replicated in the East-Asian populations. In subsequent analysis, RNF213 mutations were validated in a fine-mapping outcome (log10BF > 7). Most of the loci associated with MMD including 17q25.3 regions were detected with a statistical power greater than 80%. This study identifies several novel and known variations predicting adult MMD in Koreans. These findings may good biomarkers to evaluate MMD susceptibility and its clinical outcomes.

Oxiracetam alleviates anti-inflammatory activity and ameliorates cognitive impairment in the early phase of traumatic brain injury.

BACKGROUND: We aimed to investigate the effects of oxiracetam on cognitive impairment in the early phase of traumatic brain injury (TBI), for which no specific treatment is currently available. METHODS: The in vitro study used a cell injury controller to damage SH-SY5Y cells and evaluate the effect of oxiracetam at a dosage of 100 nM. The in vivo study used a stereotaxic impactor to induce a TBI model in C57BL/6 J mice and analyzed immunohistochemical changes and cognitive function after an intraperitoneal injection of oxiracetam (30 mg/kg/day) for 5 days. The number of mice used in this study was 60. They were divided into three groups (sham, TBI, and TBI with oxiracetam treatment) (20 mice in each group). RESULTS: The in vitro study showed that oxiracetam treatment resulted in increased superoxide dismutase (SOD)1 and SOD2 mRNA expression. The mRNA and protein expression of COX-2, NLRP3, caspase-1, and interleukin (IL)-1 ß were decreased after oxiracetam treatment, along with decreases in intracellular reactive oxygen species production and apoptotic effects. TBI mice treated with oxiracetam exhibited the loss of fewer cortical damaged lesions, less brain edema, and fewer Fluoro-Jade B (FJB)-positive and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)-positive cells compared to those without oxiracetam treatment. The mRNA and protein expression of COX-2, NLRP3, caspase-1, and IL-1ß were decreased significantly after oxiracetam treatment. These inflammation-related markers, which colocalized with Iba-1-positive or GFAP-positive cells after TBI, were also decreased after oxiracetam treatment. TBI mice treated with oxiracetam had a smaller decrease in preference and more latency time than those not treated with oxiracetam, suggesting the amelioration of impaired cognitive impairment. CONCLUSIONS: Oxiracetam may be helpful in restoring cognitive impairment by ameliorating neuroinflammation in the early phase of TBI.

Human embryonic stem cell-derived cerebral organoids for treatment of mild traumatic brain injury in a mouse model.

OBJECTIVE: There are no effective treatments for relieving neuronal dysfunction after mild traumatic brain injury (TBI). Here, we evaluated therapeutic efficacy of human embryonic stem cell-derived cerebral organoids (hCOs) in a mild TBI model, in terms of repair of damaged cortical regions, neurogenesis, and improved cognitive function. METHODS: Male C57BL/6 J mice were randomly divided into sham-operated, mild TBI, and mild TBI with hCO groups. hCOs cultured at 8 weeks were used for transplantation. Mice were sacrificed at 7 and 14 days after transplantation followed by immunofluorescence staining, cytokine profile microarray, and novel object recognition test. RESULTS: 8W-hCOs transplantation significantly reduced neuronal cell death, recovered microvessel density, and promoted neurogenesis in the ipsilateral subventricular zone and dentate gyrus of hippocampus after mild TBI. In addition, increased angiogenesis into the engrafted hCOs was observed. Microarray results of hCOs revealed neuronal differentiation potential and higher expression of early brain development proteins associated with neurogenesis, angiogenesis and extracellular matrix remodeling. Ultimately, 8W-hCO transplantation resulted in reconstruction of damaged cortex and improvement in cognitive function after mild TBI. CONCLUSION: hCO transplantation may be feasible for treating mild TBI-related neuronal dysfunction via reconstruction of damaged cortex and neurogenesis in the hippocampus.

Extracellular Mitochondrial Dysfunction in Cerebrospinal Fluid of Patients with Delayed Cerebral Ischemia after Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: Mitochondrial dysfunction is related to brain ischemic injury and neural cell death. However, little is known about the association between mitochondrial dysfunction of cerebrospinal fluid (CSF) and delayed cerebral ischemia (DCI) following subarachnoid hemorrhage (SAH). The objective of this study was to investigate whether extracellular CSF mitochondria might serve as a potential biomarker for DCI. METHODS: CSF samples were serially collected at 1, 3, and 5 days following SAH in 33 patients (DCI, n = 12; and non-DCI, n = 21) who underwent coil embolization. To monitor mitochondrial membrane potentials, JC-1 dye was used. The ratio (red/green) of JC-1 was considered as an indicator of intact mitochondrial membrane potential. Flow cytometry was done to analyze extracellular mitochondria particles and their possible cellular origins. RESULTS: DCI patients had lower JC-1 red/green ratios than non-DCI patients at 1 day (3.35 [3.20-3.75] vs. 3.70 [3.40-3.95] in non-DCI) and 3 days (4.65 [4.45-5.00] vs. 5.10 [4.65-5.30] in non-DCI) after SAH. At 5 days after SAH, JC-1 red/green ratio was significantly lower in DCI than that in non-DCI (3.05 [2.90-3.35] vs. 4.20 [4.10-4.50]; p < 0.01) patients. DCI patients had a higher percentage of vWF-positive mitochondria (40.10% [38.25%-44.90%] vs. 30.20% [25.70%-36.68%]) and a lower percentage of GLAST-positive mitochondria particles (26.85% [17.10%-30.00%] vs. 31.60% [26.70%-35.00%]) than non-DCI patients. However, there was no significant difference in CD45-positive (p = 0.369) or CD41/61-positive mitochondrial particles (p = 0.155) between the two groups of patients. CONCLUSIONS: Mitochondrial membrane potential could be a marker of DCI. JC-1 ratios seemed to be able to predict future DCI onset. Further studies are needed to determine detailed mechanisms of extracellular mitochondria-mediated cell-to-cell signals in DCI.

Characterization of the TCR ß Chain CDR3 Repertoire in Subarachnoid Hemorrhage Patients with Delayed Cerebral Ischemia.

Little is known of the adaptive immune response to subarachnoid hemorrhage (SAH). This study was the first to investigate whether T cell receptor (TCR) immune repertoire may provide a better understanding of T cell immunology in delayed cerebral ischemia (DCI). We serially collected peripheral blood in five SAH patients with DCI. High-throughput sequencing was used to analyze the TCR ß chain (TCRB) complimentary determining regions (CDR) 3 repertoire. We evaluated the compositions and variations of the repertoire between admission and the DCI period, for severe DCI and non-severe DCI patients. Clonality did not differ significantly between admission and DCI. Severe DCI patients had significantly lower clonality than non-severe DCI patients (p value = 0.019). A read frequency of 0.005% ≤ - < 0.05% dominated the clonal expansion in non-severe DCI patients. Regarding repertoire diversity, severe DCI had a higher diversity score on admission than non-severe DCI. The CDR3 lengths were similar between admission and DCI. Among 728 annotated V-J gene pairs, we found that the relative frequencies of two V-J pairs were different at the occurrence of DCI than at admission, with T cells increasing by over 15%. TCRB CDR3 repertoires may serve as biomarkers to identify severe DCI patients.

Prolyl hydroxylase inhibition protects against murine MC903-induced skin inflammation by downregulating TSLP.

Previously, we reported an anti-inflammatory effect of mTORC1 in a mouse model of type 2 skin inflammation. TSLP, one of the epithelial cell-derived cytokines, was upregulated by Raptor deficiency or rapamycin treatment, which was inhibited by dimethyloxalylglycine (DMOG). However, it remains unclear how DMOG regulates TSLP expression and type 2 skin inflammation. In this study, we investigated the protective effect of DMOG on MC903 (calcipotriol)-induced type 2 skin inflammation. Morphological and immunological changes were assessed by H-E staining, flow cytometry and RT-qPCR. DMOG treatment attenuated MC903-induced skin inflammation in a T cell-independent manner. The anti-inflammatory effect of DMOG was accompanied by downregulation of TSLP and IL-33, and supplementation with recombinant TSLP and IL-33 abolished the effect of DMOG. MC903 increased ROS levels in skin tissue, which was prevented by DMOG. Furthermore, the ROS scavenger N-acetylcysteine (NAC) downregulated TSLP and ameliorated MC903-induced skin inflammation, as did DMOG. Finally, the effect of DMOG on ROS and TSLP was reduced by HIF knockdown. These results suggest that DMOG downregulates TSLP and ROS through the HIF pathway, which reduces MC903-induced skin inflammation.

Longitudinal Genome-Wide Association Study of Cognitive Impairment after Subarachnoid Hemorrhage.

OBJECTIVES: The occurrence of cognitive deficits after subarachnoid hemorrhage (SAH) is highly possible, leading to vascular dementia. We performed a novel longitudinal genome-wide association study (GWAS) to identify genetic modifications associated with cognitive impairment following SAH in a long-term prospective cohort study. MATERIALS AND METHODS: This GWAS involved 153 patients with SAH sharing 5,971,372 markers after high-throughput imputation. Genome-wide Cox proportional hazard regression testing was performed to estimate the hazard ratio (HR) and 95% confidence interval (CI). Subsequently, a weighted polygenetic risk score (wPRS) was determined, based on GWAS-driven loci and risk stratification. RESULTS: Cognitive impairment was observed in 65 patients (42.5%) during a mean follow-up of 37.7 ± 12.4 months. Five genome-wide signals, including rs138753053 (PDCD6IP-LOC101928135, HR = 28.33, p = 3.4 × 10-8), rs56823384 (LINC00499, HR = 12.47, p = 2.8 × 10-9), rs145397166 (CASC15, HR = 11.16, p = 1.7 × 10-8), rs10503670 (LPL-SLC18A1, HR = 2.88, p = 4.0 × 10-8), and rs76507772 (IRS2, HR = 5.99, p = 3.5 × 10-8), were significantly associated with cognitive impairment following SAH. In addition, the well-constructed wPRS containing five markers showed nominal ability to predict cognitive impairment (AUROC = 0.745, 95% CI: 0.667-0.824). Tertile stratification showed a higher effectiveness in predicting cognitive impairment, especially in those with haptoglobin 2-1 (HR = 44.59, 95% CI: 8.61-231.08). CONCLUSIONS: Our study revealed novel susceptible loci for cognitive impairment, longitudinally measured in patients with SAH. The clinical utility of these loci will be evaluated in further follow-up studies.

Therapeutic Effect of Donepezil on Neuroinflammation and Cognitive Impairment after Moderate Traumatic Brain Injury.

BACKGROUND: Despite the important clinical issue of cognitive impairment after moderate traumatic brain injury (TBI), there is currently no suitable treatment. Here, we used in vitro and in vivo models to investigate the effect of Donepezil-an acetylcholinesterase (AChE) inhibitor-on cognitive impairment in the acute period following injury, while focusing on neuroinflammation and autophagy- and mitophagy-related markers. METHODS: The purpose of the in vitro study was to investigate potential neuroprotective effects in TBI-induced cells after donepezil treatment, and the in vivo study, the purpose was to investigate therapeutic effects on cognitive impairment in the acute period after injury by analyzing neuroinflammation and autophagy- and mitophagy-related markers. The in vitro TBI model involved injuring SH-SY5Y cells using a cell-injury controller and then investigating the effect of donepezil at a concentration of 80 µM. The in vivo TBI model was made using a stereotaxic impactor for male C57BL/6J mice. Immuno-histochemical markers and cognitive functions were compared after 7 days of donepezil treatment (1 mg/kg/day). Mice were divided into four groups: sham operation with saline treatment, sham operation with donepezil treatment, TBI with saline treatment, and TBI with donepezil treatment (18 mice in each group). Donepezil treatment was administered within 4 h post-TBI. RESULTS: In vitro, donepezil was found to lead to increased cell viability and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), along with decreased reactive oxygen species (ROS), lactate-dehydrogenase (LDH), 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA)-positive cells, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. The mRNA and protein expressions of neuroinflammation (Cyclooxygenase-2, COX-2; NOD-like receptor protein 3, NLRP3; Caspase-1; and Interleukin-1 beta, IL-1ß), as well as autophagy- and mitophagy-related markers (death-associated protein kinase 1, DAPK1; PTEN-induced kinase 1, PINK1; BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like, BNIP3L; Beclin-1, BECN1; BCL2-associated X protein, BAX; microtubule-associated protein 1A/1B-light chain 3B (LC3B); Sequestosome-1; and p62) were all found to decrease after donepezil treatment. The in vivo study also showed that donepezil treatment resulted in decreased levels of cortical tissue losses and brain swelling in TBI compared to the TBI group without donepezil treatment. Donepezil treatment was also shown to decrease the mRNA and Western blotting expressions of all markers, and especially COX-2 and BNIP3L, which showed the most significant decreases. Moreover, TBI mice showed an decreased escape latency, increased alteration rate, and improved preference index, altogether pointing to better cognitive performance after donepezil treatment. CONCLUSIONS: Donepezil treatment may be beneficial in improving cognitive impairment in the early phase of moderate traumatic brain injury by ameliorating neuroinflammation, as well as autophagy and mitophagy.

Modeling of the brain-lung axis using organoids in traumatic brain injury: an updated review.

Clinical outcome after traumatic brain injury (TBI) is closely associated conditions of other organs, especially lungs as well as degree of brain injury. Even if there is no direct lung damage, severe brain injury can enhance sympathetic tones on blood vessels and vascular resistance, resulting in neurogenic pulmonary edema. Conversely, lung damage can worsen brain damage by dysregulating immunity. These findings suggest the importance of brain-lung axis interactions in TBI. However, little research has been conducted on the topic. An advanced disease model using stem cell technology may be an alternative for investigating the brain and lungs simultaneously but separately, as they can be potential candidates for improving the clinical outcomes of TBI.In this review, we describe the importance of brain-lung axis interactions in TBI by focusing on the concepts and reproducibility of brain and lung organoids in vitro. We also summarize recent research using pluripotent stem cell-derived brain organoids and their preclinical applications in various brain disease conditions and explore how they mimic the brain-lung axis. Reviewing the current status and discussing the limitations and potential perspectives in organoid research may offer a better understanding of pathophysiological interactions between the brain and lung after TBI.

Bone-Marrow-Derived Mesenchymal Stem Cells Attenuate Behavioral and Cognitive Dysfunction after Subarachnoid Hemorrhage via HMGB1-RAGE Axis Mediation.

We evaluated the therapeutic effects of bone-marrow-derived mesenchymal stem cells (BMSCs) on behavioral and cognitive function in a mouse model of mild subarachnoid hemorrhage (SAH) and explored the underlying mechanisms in conjunction with the HMGB1-RAGE axis. The SAH models were generated in a total of 126 male C57BL/6J mice via endovascular perforation and evaluated 24 h and 72 h after the intravenous administration of BMSCs (3 × 105 cells). The BMSCs were administered once, at 3 h, or twice, at 3 h and 48 h after the model induction. The therapeutic effects of the BMSCs were compared to those of the saline administration. Compared to saline-treated SAH-model mice, at 3 h, the mice with mild SAH treated with the BMSCs showed significant improvements in their neurological scores and cerebral edema. The administration of the BMSCs decreased the mRNA expression of HMGB1, RAGE, TLR4, and MyD88, as well as the protein expression of HMGB1 and phosphorylated NF-kB p65. Furthermore, the numbers of slips per walking time, impairments in short-term memory, and the recognition of novel objects were improved. There was some improvement in inflammatory-marker levels and cognitive function according to the BMSCs' administration times, but no large differences were seen. The administration of BMSCs improved behavioral and cognitive dysfunction by ameliorating HMGB1-RAGE axis-mediated neuroinflammation after SAH.

WITHDRAWN: Effect of Therapeutic Hypothermia on Cognitive Impairment in a Mouse Model of Ischemia-Reperfusion Injury.

Ahead of Print article withdrawn by publisher.

Therapeutic effect of a hydrogel-based neural stem cell delivery sheet for mild traumatic brain injury.

OBJECTIVE: There are no effective clinically applicable treatments for neuronal dysfunction after mild traumatic brain injury (TBI). Here, we evaluated the therapeutic effect of a new delivery method of mouse neural stem cell (mNSC) spheroids using a hydrogel, in terms of improvement in damaged cortical lesions and cognitive impairment after mild TBI. METHODS: mNSCs were isolated from the subventricular zone and subgranular zone by a hydrogel-based culture system. GFP-transduced mNSCs were generated into spheroids and wrapped into a sheet for transplantation. Male C57BL/6J mice were randomly divided into four groups: sham operation, TBI, TBI with mNSC spheroids, and TBI with mNSC spheroid sheet transplantation covering the damaged cortex. Histopathological and immunohistochemical features and cognitive function were evaluated 7, 14, and 28 days after transplantation following TBI. RESULTS: Hydrogel-based culture systems and mNSC isolation were successfully established from the adult mice. Essential transcription factors for NSCs, such as SOX2, PAX6, Olig2, nestin, and doublecortin (DCX), were highly expressed in the mNSCs. A transplanted hydrogel-based mNSC spheroid sheet showed good engraftment and survival ability, differentiated into TUJ1-positive neurons, promoted angiogenesis, and reduced neuronal degeneration. Also, TBI mice treated with mNSC spheroid sheet transplantation exhibited a significantly increased preference for a new object, suggesting improved cognitive function compared to the mNSC spheroids or no treatment groups. CONCLUSION: Transplantation with a hydrogel-based mNSC spheroid sheet showed engraftment, migration, and stability of delivered cells in a hostile microenvironment after TBI, resulting in improved cognitive function via reconstruction of the damaged cortex. STATEMENT OF SIGNIFICANCE: This study presents the therapeutic effect of a new delivery method of mouse neural stem cells spheroids using a hydrogel, in terms of improvement in damaged cortical lesions and cognitive impairment after traumatic brain injury. Collagen/fibrin hydrogel allowed long-term survival and migratory ability of NSCs spheroids. Furthermore, transplanted hydrogel-based mNSCs spheroids sheet showed good engraftment, migration, and stability of delivered cells in a hostile microenvironment, resulting in reconstruction of the damaged cortex and improved cognitive function after TBI. Therefore, we suggest that a hydrogel-based mNSCs spheroids sheet could help to improve cognitive impairment after TBI.

Novel Genome-Wide Interactions Mediated via BOLL and EDNRA Polymorphisms in Intracranial Aneurysm.

OBJECTIVE: The association between boule (BOLL) and endothelin receptor type A (EDNRA) loci and intracranial aneurysm (IA) formation has been reported via genome-wide association studies. We sought to identify genome-wide interactions involving BOLL and EDNRA loci for IA in a Korean adult cohort. METHODS: Genome-wide pairwise interaction analyses of BOLL and EDNRA involving 250 patients with IA and 296 controls were performed using the additive effect model after adjusting for confounding factors. RESULTS: Among 512575 single-nucleotide polymorphisms (SNPs), 23 and 11 common SNPs suggested a genome-wide interaction threshold (p<1.25×10-8) involving rs700651 (BOLL) and rs6841581 (EDNRA). Rather than singe SNP effect of BOLL or EDNRA on IA development, they showed a synergistic effect on IA formation via multifactorial pair-wise interactions. The rs1105980 of PTCH1 gene showed the most significant interaction with rs700651 (natural log-transformed odds ratio [lnOR], 1.53; p=6.41×10-11). The rs74585958 of RYK gene interacted strongly with rs6841581 (lnOR, -19.91; p=1.64×10-9). Although, there was no direct interaction between BOLL and EDNRA variants, two EDNRA-interacting gene variants of TNIK (rs11925024 and rs1231) and FTO (rs9302654), and one BOLL-interacting METTL4 gene variant (rs549315) exhibited marginal interaction with BOLL gene. CONCLUSION: BOLL or EDNRA may have a synergistic effect on IA formation via multifactorial pair-wise interactions.

Updated Trans-Ethnic Meta-Analysis of Associations between Inflammation-Related Genes and Intracranial Aneurysm.

OBJECTIVE: We performed an expanded multi-ethnic meta-analysis to identify associations between inflammation-related loci with intracranial aneurysm (IA) susceptibility. This meta-analysis possesses increased statistical power as it is based on the most data ever evaluated. METHODS: We searched and reviewed relevant literature through electronic search engines up to August 2022. Overall estimates were calculated under the fixed- or random-effect models using pooled odds ratio (OR) and 95% confidence intervals (CIs). Subgroup analyses were performed according to ethnicity. RESULTS: Our meta-analysis enrolled 15 studies and involved 3070 patients and 5528 controls including European, Asian, Hispanic, and mixed ethnic populations. Of 17 inflammation-related variants, the rs1800796 locus (interleukin [IL]-6) showed the most significant genome-wide association with IA in East-Asian populations, including 1276 IA patients and 1322 controls (OR, 0.65; 95% CI, 0.56-0.75; p=3.24×10-9) under a fixed-effect model. However, this association was not observed in the European population (OR, 1.09; 95% CI, 0.80-1.47; p=0.5929). Three other variants, rs16944 (IL-1ß), rs2195940 (IL-12B), and rs1800629 (tumor necrosis factor-α) showed a statistically nominal association with IA in both the overall, as well as East-Asian populations (0.01

Autophagy and mitophagy-related extracellular mitochondrial dysfunction of cerebrospinal fluid cells in patients with hemorrhagic moyamoya disease.

We aimed to investigate whether mitochondrial dysfunction in extracellular cerebrospinal fluid (CSF), which is associated with autophagy and mitophagy, might be involved in neurological outcomes in adult patients with hemorrhagic moyamoya disease (MMD) whose pathogenesis related to poor outcomes is not well-known. CSF samples were collected from 43 adult MMD patients and analyzed according to outcomes at 3 months. Fluorescence-activated cell sorter analysis (FACS) and the JC-1 red/green ratio were used to assess mitochondrial cells and intact mitochondrial membrane potential (MMP). We performed quantitative real-time polymerase chain reaction and Western blotting analyses of autophagy and mitophagy-related markers, including HIF1α, ATG5, pBECN1, BECN1, BAX, BNIP3L, DAPK1, and PINK1. Finally, FACS analysis with specific fluorescence-conjugated antibodies was performed to evaluate the potential cellular origin of CSF mitochondrial cells. Twenty-seven females (62.8%) with a mean age of 47.4 ± 9.7 years were included in the study. Among 43 patients with hemorrhagic MMD, 23 (53.5%) had poor outcomes. The difference in MMP was evident between the two groups (2.4 ± 0.2 in patients with poor outcome vs. 3.5 ± 0.4 in patients with good outcome; p = 0.02). A significantly higher expression (2-ΔCt) of HIF1α, ATG5, DAPK1 followed by BAX and BNIP3L mRNA and protein was also observed in poor-outcome patients compared to those with good outcomes. Higher percentage of vWF-positive mitochondria, suggesting endothelial cell origins, was observed in patients with good outcome compared with those with poor outcome (25.0 ± 1.4% in patients with good outcome vs. 17.5 ± 1.5% in those with poor outcome; p < 0.01). We observed the association between increased mitochondrial dysfunction concomitant with autophagy and mitophagy in CSF cells and neurological outcomes in adult patients with hemorrhagic MMD. Further prospective multicenter studies are needed to determine whether it has a diagnostic value for risk prediction.

Identification of Differentially-Methylated Genes and Pathways in Patients with Delayed Cerebral Ischemia Following Subarachnoid Hemorrhage.

OBJECTIVE: We reported the differentially methylated genes in patients with subarachnoid hemorrhage (SAH) using bioinformatics analyses to explore the biological characteristics of the development of delayed cerebral ischemia (DCI). METHODS: DNA methylation profiles obtained from 40 SAH patients from an epigenome-wide association study were analyzed. Functional enrichment analysis, protein-protein interaction (PPI) network, and module analyses were carried out. RESULTS: A total of 13 patients (32.5%) experienced DCI during the follow-up. In total, we categorized the genes into the two groups of hypermethylation (n=910) and hypomethylation (n=870). The hypermethylated genes referred to biological processes of organic cyclic compound biosynthesis, nucleobase-containing compound biosynthesis, heterocycle biosynthesis, aromatic compound biosynthesis and cellular nitrogen compound biosynthesis. The hypomethylated genes referred to biological processes of carbohydrate metabolism, the regulation of cell size, and the detection of a stimulus, and molecular functions of amylase activity, and hydrolase activity. Based on PPI network and module analysis, three hypermethylation modules were mainly associated with antigen-processing, Golgi-to-ER retrograde transport, and G alpha (i) signaling events, and two hypomethylation modules were associated with post-translational protein phosphorylation and the regulation of natural killer cell chemotaxis. VHL, KIF3A, KIFAP3, RACGAP1, and OPRM1 were identified as hub genes for hypermethylation, and ALB and IL5 as hub genes for hypomethylation. CONCLUSION: This study provided novel insights into DCI pathogenesis following SAH. Differently methylated hub genes can be useful biomarkers for the accurate DCI diagnosis.

The Therapeutic Role of Nanoparticle Shape in Traumatic Brain Injury : An in vitro Comparative Study.

OBJECTIVE: To perform a comparative analysis of therapeutic effects associated with two different shapes of ceria nanoparticles, ceria nanorods (Ceria NRs) and ceria nanospheres (Ceria NSs), in an in vitro model of traumatic brain injury (TBI). METHODS: In vitro TBI was induced using six-well confluent plates by manually scratching with a sterile pipette tip in a 6×6-square grid. The cells were then incubated and classified into cells with scratch injury without nanoparticles and cells with scratch injury, which were treated separately with 1.16 mM of Ceria NSs and Ceria NRs. Antioxidant activities and anti-inflammatory effects were analyzed. RESULTS: Ceria NRs and Ceria NSs significantly reduced the level of reactive oxygen species compared with the control group of SH-SY5Y cells treated with Dulbecco's phosphate-buffered saline. The mRNA expression of superoxide dismutases was also reduced in nanoparticle-treated SH-SY5Y cells, but apparently the degree of mRNA expression decrease was not dependent on the nanoparticle shape. Exposure to ceria nanoparticles also decreased the cyclooxygenase-2 expression, especially prominent in Ceria NR-treated group than that in Ceria NS-treated group. CONCLUSION: Ceria nanoparticles exhibit antioxidant and anti-inflammatory effects in TBI models in vitro. Ceria NRs had better antiinflammatory effect than Ceria NSs, but showed similar antioxidant activity.

Recent Stem Cell Research on Hemorrhagic Stroke : An Update.

Although technological advances and clinical studies on stem cells have been increasingly reported in stroke, research targeting hemorrhagic stroke is still lacking compared to that targeting ischemic stroke. Studies on hemorrhagic stroke are also being conducted, mainly in the USA and China. However, little research has been conducted in Korea. In reality, stem cell research or treatment is unfamiliar to many domestic neurosurgeons. Nevertheless, given the increased interest in regenerative medicine and the increase of life expectancy, attention should be paid to this topic. In this paper, we summarized pre-clinical rodent studies and clinical trials using stem cells for hemorrhagic stroke. In addition, we discussed results of domestic investigations and future perspectives on stem cell research for a better understanding.

Bioinformatics Analysis of Autophagy and Mitophagy Markers Associated with Delayed Cerebral Ischemia Following Subarachnoid Hemorrhage.

OBJECTIVE: To evaluate the interactions among differentially expressed autophagy and mitophagy markers in subarachnoid hemorrhage (SAH) patients with delayed cerebral ischemia (DCI). METHODS: The expression data of autophagy and mitophagy-related makers in the cerebrospinal fluid (CSF) cells was analyzed by real-time reverse transcription-polymerase chain reaction and Western blotting. The markers included death-associated protein kinase (DAPK)-1, BCL2 interacting protein 3 like (BNIP3L), Bcl-1 antagonist X, phosphatase and tensin homolog-induced kinase (PINK), Unc-51 like autophagy activating kinase 1, nuclear dot protein 52, and p62. In silico functional analyses including gene ontology enrichment and the protein-protein interaction network were performed. RESULTS: A total of 56 SAH patients were included and 22 (38.6%) of them experienced DCI. The DCI patients had significantly increased mRNA levels of DAPK1, BNIP3L, and PINK1, and increased expression of BECN1 compared to the non-DCI patients. The most enriched biological process was the positive regulation of autophagy, followed by the response to mitochondrial depolarization. The molecular functions ubiquitin-like protein ligase binding and ubiquitin-protein ligase binding were enriched. In the cluster of cellular components, Lewy bodies and the phagophore assembly site were enriched. BECN1 was the most connected gene among the differentially expressed markers related to autophagy and mitophagy in the development of DCI. CONCLUSION: Our study may provide novel insight into mitochondrial dysfunction in DCI pathogenesis.