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Background: Breastmilk is widely regarded as the healthiest choice for both infants and mothers due to its numerous advantages over formula, such as higher concentrations of essential nutrients and antibodies, easier digestion, and superior taste. The World Health Organization International Code of Marketing of Breast-milk Substitutes was adopted over 40 years ago to mitigate the effects of infant formula marketing on a woman's decision to breastfeed. Yet, the commercial formula milk industry has continued to market their products aggressively and through an increasing variety of social media channels. This study examines the impact of formula milk marketing on specific components of the health system to understand how systems that are built to support and sustain breastfeeding may have been captured and repurposed by industry practices., Methods: As part of A Multi-country Study of Early Infant Feeding Decisions, this qualitative study was conducted in two representative cities in China, Beijing and Jinan, in 2020. The participants were recruited through convenience sampling and snowballing sampling methods. Data were collected through 40 in-depth interviews and 10 focus group discussions with health professionals, pregnant women, and formula milk marketing executives. A thematic analytical method was used to analyze the main themes and sub-themes that detail the characteristics of formula milk marketing and its impact on the health system in China., Results: Our results identified the most common practices used by the formula milk industry to reach health workers as well as mothers and their families through health systems. Such practices include (1) provider/facility-based marketing to sponsor academic activities (e.g., seminars, conferences, and research projects) and promote specialized infant formula that targets health care providers and facilities, which influence the health consultations and recommendations provided by health professionals to mothers and families; and (2) receiver-based marketing that targets Chinese women and increases marketing exposure through lectures provided by maternity schools at health facilities during the antenatal period and health promotion activities organized by formula milk companies outside of facilities during the postnatal care period., Conclusions: Formula milk marketing aims to influence the practices and services within the health system in China, both directly through health professionals and facilities, and also indirectly by targeting mothers. These marketing strategies have impacted the perceptions of providers on formula milk, and strategies targeting consumers have aimed to further influence and undermine mothers' intentions to continue breastfeeding. Stricter regulation of industry marketing practices is needed to create a supportive environment for mothers to breastfeed., Competing Interests: Declarations. Ethics approval and consent to participate: All methods were carried out in accordance with relevant guidelines and regulations. The study received ethical approval from the ethics committee of Capital Institute of Pediatrics, Beijing, China (No. SHERLL2020002). Written informed consent was obtained from all subjects. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Hypothesis: The objective of this study is to obtain comprehensive morphometric measurements of the incomplete partition type II (IP-II) cochlea to provide a better understanding of intracochlear anatomy and important considerations for electrode selection and insertion., Background: IP-II is the most common bony inner ear malformation that often requires cochlear implantation. Currently, there is significant controversy on electrode selection due to a lack of research that can provide reliable, high-resolution measurements., Methods: Three-dimensional reconstructions of the cochlea were made from hematoxylin and eosin-stained slides from 11 archival human temporal bones from 8 adult IP-II patients (one paired) and 2 fetuses. Detailed measurements of the angular and linear length of the spiral ganglion neurons and cochlear duct at the modiolar and lateral wall of the scala tympani as well as cross-sectional areas and vertical height measurements of the scala tympani at 90-degree intervals were measured., Results: The spiral ganglia neurons terminated at 540.5 ± 45.4 degrees, which corresponded to the beginning of the interscalar septal defect. The corresponding Rosenthal's canal length was 12.75 ± 0.82 mm, and the lateral wall length was 23.95 ± 1.04. The average cochlear duct length was 32.44 mm ± 1.58 mm, corresponding to an average angular distance of 951.6 ± 80 degrees. The modiolar height demonstrated less variation within the scala tympani but was significantly smaller at 0 and 90 degrees compared with the normal cochlea. The lateral wall height was also significantly smaller at 0, 180, and 540 degrees. There was a drastic decrease in lateral wall height at 540 degrees to 0.4 mm, which is smaller than the apical dimension of many electrodes., Conclusion: This is the first study to provide detailed morphometric measurements of the IP-II cochlea including spiral ganglion neuron length and scala tympani height. These measurements directly relate to electrode selection for cochlear implantation., Competing Interests: The authors disclose no conflicts of interest., (Copyright © 2024, Otology & Neurotology, Inc.)

Background: Preadolescents, who are in a transitional phase of development, may experience higher exposure to heterosexual interactions while facing higher risks regarding misinformation in sexual knowledge and unsafe engagement in sexual activities. There is a deficiency in the availability of qualified educators and age-appropriate teaching materials for sexuality education in China., Methods: We implemented an animation-based comprehensive sexuality education package among preadolescents aged 9 to 12 years from eight schools in Anhui, China. The first round of intervention included 1,835 participants, lasting 2 months from September to November 2020. A total of 374 participants, accounting for 52% of the intervention group, received a second round of intervention in September 2021. Participants completed immediate follow-up assessment and 1-year follow-up assessment to assess changes in their sexual knowledge, attitudes, and other outcomes. Propensity score matching and difference-in-difference analysis were performed to determine the short- and long-term impacts., Results: Significant improvements were observed for both sexual knowledge and sexual attitudes in the immediate follow-up. There was no significant effect on pornography-seeking behavior or awareness of experiencing sexual abuse. After 1 year, the effect was sustained for sexual knowledge, but slightly declined for sexual attitudes. The second intervention significantly improved sexual knowledge; however, no significant change in sexual attitudes, pornography-seeking behavior, or awareness of experiencing sexual abuse was observed., Conclusions: Our comprehensive sexuality education package was effective in improving sexual knowledge both immediately and 1 year after the intervention. Repeated intervention can be an effective strategy for promoting preadolescent health development regarding comprehensive sexuality education., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

This study examined whether maternal depression is related to Early Childhood Developmental (ECD) delay among children by quantifying the mediating contribution of responsive caregiving. We used data from 1235 children (Children's mean age = 50.4 months; 582 girls, 653 boys, 93.9% were Han), selected through convenience sampling, in 2021. 4.7% of children had ECD delay, 34.3% of mothers had depression. Children with depressed mothers were less likely to receive responsive caregiving (OR 4.35, 95% CI 2.60-7.27), and those who did not receive responsive caregiving were more likely to experience ECD delay (OR 3.89, 95% CI 1.89-8.02). Responsive caregiving partly mediated the relationship between maternal depression and ECD. Early intervention for children with depressed mothers is worthy of further investigation., (© 2024. The Author(s).)

Hypothesis: Transforming growth factor beta-1 (TGFβ-1) and connective tissue growth factor (CTGF) are upregulated in the implanted human cochlea., Background: Cochlear implantation can lead to insertion trauma and intracochlear new tissue formation, which can detrimentally affect implant performance. TGFβ-1 and CTGF are profibrotic proteins implicated in various pathologic conditions, but little is known about their role in the cochlea. The present study aimed to characterize the expression of these proteins in the human implanted cochlea., Methods: Archival human temporal bones (HTB) acquired from 12 patients with previous CI and histopathological evidence of new tissue formation as well as surgical samples of human intracochlear scar tissue surrounding the explanted CI were used in this study. Histopathologic analysis of fibrosis and osteoneogenesis was conducted using H&E. Protein expression was characterized using immunofluorescence. RNA expression from surgical specimens of fibrotic tissue surrounding the CI was quantified using qRT-PCR., Results: TGFβ-1 and CTGF protein expressions were upregulated in the areas of fibrosis and osteoneogenesis surrounding the CI HTB. Similarly, surgical samples demonstrated upregulation of protein and mRNA expression of TGFβ-1 and mild upregulation of CTGF compared with control. TGFβ-1 was expressed diffusely within the fibrous capsule, whereas CTGF was expressed in the thickened portion toward the modiolus and the fibrosis-osteoneogensis junction., Conclusion: To our knowledge, this is the first study to demonstrate increased expression of TGFβ-1 and CTGF in the human implanted cochlea and may provide better understanding of the mechanism behind this pathogenic process to better develop future mitigating interventions., Competing Interests: The authors disclose no conflicts of interest., (Copyright © 2024, Otology & Neurotology, Inc.)

Background: The relationship between multimorbidity (i.e. ≥ 2 chronic conditions) and incontinence (i.e. urinary and/or faecal incontinence) is underexplored. This study investigated the association between multimorbidity and incident incontinence in Chinese adults aged ≥50 years., Methods: Data from the 2011 and 2015 waves of the China Health and Retirement Longitudinal Study were used. The association between 12 chronic conditions, multimorbidity and new-onset incontinence was analysed using weighted logistic regression models. Mediation analysis was conducted to explore the potential mediators (self-reported health, subjective memory, depressive symptoms, disability, cognitive function, handgrip strength, mobility limitations, medications and frailty status) between multimorbidity and incontinence., Findings: Among 9,986 individuals aged ≥50 years who were continent at baseline, 5.3% (n = 521) were newly incontinent 4 years later (incident cases). The risk of incident incontinence increased with physical multimorbidity (OR 2.04, 95% CI 1.62-2.57). Compared to no chronic condition, having 1, 2, 3 and ≥ 4 chronic conditions were associated with incident incontinence with OR (95% CI): 1.41 (1.01-1.97), 1.74 (1.24-2.44), 2.82 (1.93-4.12) and 3.99 (2.29-6.95), respectively. The association between multimorbidity and incontinence was mediated by self-reported health (41.2%), medications (26.6%), mobility limitations (20.9%), depressive symptoms (12.8%), disability (11.6%), subjective memory (8.7%) and frailty status (8.3%)., Conclusion: This longitudinal study found that physical multimorbidity and specific chronic conditions may increase the risk of new-onset incontinence among Chinese adults aged ≥50 years. Self-reported health, medications and mobility limitations seemed to be important intermediate conditions between multimorbidity and incident incontinence., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Geriatrics Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose of Review: Although pre-exposure prophylaxis (PrEP) is effective for reducing risk of HIV transmission, stigma persists as a barrier to HIV prevention. Digital technologies present opportunities to access hard-to-reach populations and increase the efficiency of established interventions. This review examines current digital interventions addressing stigma to improve PrEP-related outcomes., Recent Findings: Digital technologies are increasingly used for HIV prevention and include a wide range of formats. Recent interventions focused on stigma and PrEP tend to engage mobile phone-related technology and focus on younger populations with particular attention to men who have sex with men and transgender women. Digital interventions that address stigma are promising for improving PrEP-related outcomes. No single technology currently demonstrates consistent superiority. Limited access to PrEP and heightened stigma in under-resourced countries present challenges for interventions supporting diverse communities. Further research should examine how digital interventions can reduce stigma beyond the individual level to enhance PrEP use and explore opportunities to improve and integrate approaches to stigma measurement., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Progress in reducing maternal and neonatal mortality, particularly in low-income and middle-income countries (LMICs) and regions, is insufficient to achieve the Sustainable Developmental Goals by 2030. High-quality postnatal care (PNC) for mothers and neonates is crucial for mothers and babies, yet it remains the most neglected intervention on the continuum of maternal and child care. We aimed to estimate the associations between observable factors and high-quality maternal and neonatal PNC in pooled and country-specific analyses., Methods: In this cross-sectional study, we used the most recent (2015-2022) Demographic and Health Surveys from 23 countries across Africa (n = 14), Southeast Asia (n = 3), Eastern Mediterranean (n = 2), Europe (n = 2), Americas (n = 1), and Western Pacific (n = 1). Women who, within the last 5 years, were aged 15-49 years at their last live birth that had delivered a singleton child were included. We identified eleven PNC behaviours recommended by the World Health Organization (WHO) to measure PNC quality, and applied thresholds to create binary outcomes for quality maternal and neonatal PNC. 15 factors were included in our analysis to assess their association with high-quality PNC for mothers and neonates in a series of single-adjusted and mutually adjusted logistic regression models, both in pooled and country-specific analysis. We also conducted two sets of subgroup analyses for place of residence and maternal age at last birth, and two sets of supplementary analyses to test the robustness of the results., Findings: Among 172,526 women and their most recent child, 41.42% (40.93-41.91) received quality maternal PNC while 42.34% (41.86-42.83) received quality neonatal PNC. In the pooled analysis, we found that the factors showing the strongest associations with quality maternal PNC were delivery by skilled birth attendants (SBAs) (OR: 4.92; 95% CI: 4.32-5.59), four or more antenatal care (ANC) visits (OR: 1.69, 1.58-1.81), and institutional delivery (OR: 1.61; 1.46-1.78). Consistent results were found for all factors of quality newborn care (e.g., delivery by SBA: OR, 4.25; 3.75-4.81; four or more ANC visits: OR, 1.83; 1.70-1.96) except institutional delivery. The association between these leading factors and PNC quality were broadly consistent across countries. Subgroup analyses and sensitivity analyses showed generally consistent results., Interpretation: Our study demonstrated that institutional delivery and frequent ANC visits had the strongest positive associations with quality PNC for both mothers and neonates. Our findings highlight that improvements to the quality of maternal and neonatal PNC in the LMICs we assessed are urgently needed to achieve ambitious maternal, newborn, and child health goals., Funding: China National Natural Science Foundation., Competing Interests: We declare no competing interests., (© 2023 The Author(s).)

Background: Implementation of Kangaroo Mother Care (KMC) in resource-limited areas of China may face unique barriers, such as a lack of resources, geographic location and more traditional culture among others. This qualitative study analyses the facilitators and barriers to implementing KMC in county-level health facilities in resource-limited areas of China for the promotion of KMC on a larger scale., Methods: Participants from 4 of the 18 pilot counties where early essential newborn care was implemented through the Safe Neonatal Project and 4 control counties not enrolled in Safe Neonatal Project were selected using purposive sampling. A total of 155 participants were interviewed, including stakeholders of the Safe Neonatal Project such as national maternal health experts, relevant government officials and medical staff. Thematic analysis was used to process and analyse the interview content in order to summarise the facilitators and barriers to implementing KMC., Results: KMC was accepted in the pilot areas but still faced certain challenges due to institutional regulation, resource provision and the perceptions of health staff, postpartum mothers and their families as well as COVID-19 prevention and control regulations. The facilitators identified were government officials and medical staff acceptance and the incorporation of KMC into routine clinical care. The barriers identified were a lack of dedicated funding and other resources, the present scope of health insurance and KMC cost-sharing mechanism, providers' knowledge and practical abilities, parental awareness, postpartum discomfort, fathers' inadequate involvement, and the impact from COVID-19., Conclusion: The Safe Neonatal Project pilot experience indicated the feasibility of implementing KMC in more areas of China. Optimising institutional regulations, providing necessary supporting resources and enhancing education and training may help to refine the implementation and scale-up of KMC practice in China., (© 2023. The Author(s).)

Background: Trilateral South‒South cooperation is envisioned as an equal and empowering partnership model but still faces certain challenges. This study addresses whether and how trilateral South‒South cooperation can transform traditional development assistance for health (DAH) and explores the opportunities and challenges of trilateral South‒South cooperation for transforming future DAH, in the theme of "the emerging development partner's DAH transformation facilitated by a multilateral organization"., Methods: We evaluate a maternal, newborn, and child health (MNCH) project involving the Democratic Republic of Congo (DRC), the United Nations Children's Fund (UNICEF), and China (hereinafter referred to as the "DRC-UNICEF-China project"). We analyze data from project documents and seventeen semi-structured interviews using a pragmatic analytical framework based on the DAH program logic model and the OECD's trilateral cooperation framework., Results: Evidence from the DRC-UNICEF-China MNCH project suggests that trilateral South‒South cooperation facilitated by a multilateral organization can provide transformative opportunities for emerging development partners' DAH to generate and deliver context-based, demand-oriented solutions, harmonize rules and procedures, institutionalize mutual learning and knowledge sharing, and increase the visibility of emerging development partners as sources for South‒South development experience transfer. However, the project revealed some challenges, including the neglect of key stakeholders in the complex governance structure, the high transaction costs needed to ensure transparency, and the harm local absence of the emerging development partner poses to long-term DAH engagement., Conclusions: This study echoes some of the findings in trilateral SSC literature that claim power structures and philanthropic, normative justification for health equity are often juxtaposed in trilateral SSC partnerships. The opportunities offered by the DRC-UNICEF-China project align with China's cognitive learning process for strengthening international engagement and global image building. However, challenges may arise as a result of complex governance structures and the entrustment of facilitating partners, which can threaten the effectiveness of trilateral cooperation. We call for strengthening the beneficiary partner's ownership at all levels, engaging the emerging development partner to better understand the beneficiary partner's local context(s) and needs, and ensuring available resources to support programmatic activities and long-term partnerships for the health and well-being of the beneficiaries., (© 2023. The Author(s).)

Purpose: A previous immunofluorescent study suggests that, in collapsing glomerulopathy, most hyperplastic podocytes that stained positively for a progenitor cell marker CD133 are derived from CD133 + parietal epithelial cells. In pathology practice, not all renal biopsies with collapsing glomerulopathy show the typical morphologic features for this entity, which include florid podocyte hyperplasia, collapsing glomerular capillary loops, and cystic tubular dilation. This study was made to determine if CD133 staining using an immunohistochemical method can be used to confirm hyperplastic podocytes and identify extensive acute tubular injury in collapsing glomerulopathy., Methods: Twenty-one collapsing glomerulopathy biopsies were stained for CD133 and compared with 15 biopsies with focal segmental glomerulosclerosis, not otherwise specified (FSGS)., Results: All patients with collapsing glomerulopathy were of African American descent with prominent renal failure and nephrotic range proteinuria. In contrast, the FSGS group consisted of patients from a variety of ethnic backgrounds with nephrotic range proteinuria but relatively low serum creatinine. The striking finding was that all collapsing glomerulopathy cases showed positive CD133 staining in the clusters of hyperplastic podocytes. There was significantly higher CD133-positive staining rate for hyperplastic podocytes (38%) in the glomeruli of the collapsing glomerulopathy group when compared to small clusters of hyperplastic podocytes in the FSGS group (8%). In addition, when compared to the relatively weak CD133 staining in the proximal tubules of the FSGS group, the proximal tubules of the collapsing glomerulopathy group all showed diffuse and strong CD133 staining as a feature of severe acute tubular injury, which corresponded to the high serum creatinine levels in these patients., Conclusion: Our data indicate that the combination of the distinctive mosaic CD133 staining in hyperplastic podocytes and the diffuse tubular CD133 staining is helpful in supporting a diagnosis of collapsing glomerulopathy., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The high lipid content of the brain, coupled with its heavy oxygen dependence and relatively weak antioxidant system, makes it highly susceptible to oxidative DNA damage that contributes to neurodegeneration. This study is aimed at identifying specific ROS-responsive miRNAs that modulate the expression and activity of the DNA repair proteins in human astrocytes, which could serve as potential biomarkers and lead to the development of targeted therapeutic strategies for neurological diseases. Oxidative DNA damage was established after treatment of human astrocytes with 10μM sodium dichromate for 16 h. Comet assay analysis indicated a significant increase in oxidized guanine lesions. RT-qPCR and ELISA assays confirmed that sodium dichromate reduced the mRNA and protein expression levels of the human base-excision repair enzyme, 8-deoxyguanosine DNA glycosylase 1 (hOGG1). Small RNAseq data were generated on an Ion Torrent™ system and the differentially expressed miRNAs were identified using Partek Flow® software. The biologically significant miRNAs were selected using miRNet 2.0. Oxidative-stress-induced DNA damage was associated with a significant decrease in miRNA expression: 231 downregulated miRNAs and 2 upregulated miRNAs (p < 0.05; >2-fold). In addition to identifying multiple miRNA-mRNA pairs involved in DNA repair processes, this study uncovered a novel miRNA-mRNA pair interaction: miR-1248:OGG1. Inhibition of miR-1248 via the transfection of its inhibitor restored the expression levels of hOGG1. Therefore, targeting the identified microRNA candidates could ameliorate the nuclear DNA damage caused by the brain's exposure to mutagens, reduce the incidence and improve the treatment of cancer and neurodegenerative disorders.

Tobacco exposure and human papillomavirus (HPV) infection are among the main risk factors for the development of head and neck squamous cell carcinoma (HNSCC). Interestingly, recent studies show that tumors from HPV positive (HPV+) smokers and non-smokers have similar mutational profiles, which suggests that HPV could prevent mutation induction or accumulation in the intermediate risk group composed of HPV+ smokers. Hence, we tested this observation by analyzing the effects of 4-Nitroquinoline N-oxide (4NQO), a mutagen and smoking mimetic, in NOK (normal oral keratinocytes), NOK E6.E7 (NOK cells transfected with E6.E7 oncogenes of HPV), HPV+ and HPV negative (HPV-) HNSCC cells. Oxidative DNA damage, γH2AX foci formation, DNA repair protein activation, cell cycle phase analysis, apoptotic cell death, cell viability and clonogenic cell survival were analyzed after 4NQO treatment in NOK, NOK E6.E7 , HPV+ and HPV- HNSCC cells. 4NQO increased oxidative base damage and γH2AX foci formation in NOK E6.E7 , HPV+ and HPV- HNSCC cells. Phosphorylation of homologous recombination (HR) repair proteins was higher in NOK E6.E7 and HPV+ HNSCC cells compared to NOK and HPV- HNSCC cells respectively. HPV+ and HPV- HNSCC cells showed differential activation of cell cycle regulatory proteins, increased apoptosis, and decreased cell viability upon 4NQO-induced DNA damage. Taken together, 4NQO (a smoking mimetic), induced higher activation of HR repair in HPV+ HNSCC cells compared to HPV- HNSCC cells. This may allow for increased mutational resistance and help explain why HPV+ smokers have a worse prognosis than HPV+ non-smokers., (Published by Elsevier Ltd.)

Background: Proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) is a glomerular disease defined by non-organized glomerular deposits of heavy and light chain-restricted immunoglobulin and is rarely reported in children., Methods: We characterized a series of nine pediatric patients from two academic centers with biopsy-proven PGNMID and additionally describe two patients with monotypic IgG in the setting of IgM deposition., Results: Each patient presented with hematuria and/or proteinuria; however, only five had elevated serum creatinine. Prodromal or concurrent infection was identified in six patients, low C3 in five, and alternate complement pathway gene variants in two. No monoclonal serum proteins were identified in five tested patients. Seven patients had monotypic deposits composed of IgG3-λ, two showed IgG3-κ, and one each IgG1 and IgG3 with lambda dominance in the setting of IgM deposition. The glomerular pattern was predominantly mesangial proliferative or membranoproliferative glomerulonephritis (MPGN). Treatment and outcomes were variable; four patients have recent PGNMID diagnoses and therefore minimal follow up, one had relatively stable kidney function for over a decade, and six experienced kidney failure, with four receiving transplants. Recurrent deposits of the same isotype were identified in five of six transplanted kidneys, corresponding to three of four transplanted patients. One of these patients developed PGNMID recurrences in three separate kidney allografts over a 20-year disease course., Conclusions: Our study emphasizes the need for upfront IgG subclass investigation in pediatric mesangial or MPGN with IgG deposition and monotypic or biased light-chain staining. Furthermore, this pediatric experience suggests expanded pathogenic considerations in PGNMID. Graphical abstract.

Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies with a median survival time of about 4 months. Currently, there is no effective treatment, and the development of new therapies is an important and urgent issue for ATC patients. YM155 is a small molecule that was identified as the top candidate in a high-throughput screen of small molecule inhibitors performed against a panel of ATC cell lines by the National Cancer Institute. However, there were no follow-up studies investigating YM155 in ATC. Here, we determined the effects of YM155 on ATC and human primary benign thyroid cell (PBTC) survival with alamarBlue assay. Our data show that YM155 inhibited proliferation of ATC cell lines while sparing normal thyroid cells, suggesting a high therapeutic window. YM155-induced DNA damage was detected by measuring phosphorylation of γ-H2AX as a marker for DNA double-strand breaks. The formamidopyrimidine-DNA glycosylase (FPG)-modified alkaline comet assay in conjunction with reactive oxygen species (ROS) assay and glutathione (GSH)/glutathione (GSSG) assay suggests that YM155-mediated oxidative stress contributes to DNA damage. In addition, we provide evidence that YM155 causes cell cycle arrest in S phase and in the G2/M transition and causes apoptosis, as seen with flow cytometry. In this study, we show for the first time the multiple effects of YM155 in ATC cells, furthering a potential therapeutic approach for ATC.

White matter damage is an important contributor to long-term neurological deficit after stroke. Our previous study has shown that inhibition of CD147 ameliorates acute ischemic stroke in mice. In this study, we aimed to investigate whether inhibition of CD147 promotes white matter repair and long-term functional recovery after ischemic stroke.Male adult C57BL/6 mice were subjected to transient (1-h) middle cerebral artery occlusion (tMCAO). Anti-CD147 function-blocking antibody (αCD147) was injected intravenously once daily for 3 days beginning 4 h after onset of ischemia. Sensorimotor and cognitive functions were evaluated up to 28 days after stroke. We found that αCD147 treatment not only prevented neuronal and oligodendrocyte cell death in the acute phase, but also profoundly protected white matter integrity and reduced brain atrophy and tissue loss in the late phase, leading to improved sensorimotor and cognitive functions for at least 28 days after stroke. Mechanistically, we found that αCD147 treatment increased the number of proliferating NG2(+)/PDGFRα(+) oligodendrocyte precursor cells (OPCs) and newly generated mature APC(+)/Sox10(+) oligodendrocytes after stroke, possibly through upregulation of SDF-1/CXCR4 axis in OPCs. In conclusion, inhibition of CD147 promotes long-term functional recovery after stroke, at least in part, by enhancing oligodendrogenesis and white matter repair., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Phosphoinositide 3-kinase γ (PI3Kγ) expressed in immune cells is linked to neuroinflammation in several neurological diseases. However, the expression and role of PI3Kγ in preclinical traumatic brain injury (TBI) have not been investigated. In WT mice, we found that TBI induced rapid and extensive expression of PI3Kγ in neurons within the perilesional cortex and the ipsilateral hippocampal subfields (CA1, CA3), which peaked between 1 and 3 days and declined significantly 7 days after TBI. Intriguingly, the induction of neuronal PI3Kγ in these subregions of the brain spatiotemporally coincided with both the TBI-induced activation of the neuronal ER stress pathway (p-eIF2α, ATF4, and CHOP) and neuronal cell death (marked by TUNEL-positive neurons) 3 days after TBI. Further, we show that the absence of PI3Kγ in knockout mice profoundly reduced the TBI-induced activation of the ER stress pathway and neuronal cell death. White matter disruption is a better predictor of long-term clinical outcomes than focal lesion size. We show that PI3Kγ deficiency not only reduced brain tissue loss but also alleviated white matter injury (determined by axonal injury and demyelination) up to 28 days after TBI. Importantly, PI3Kγ-knockout mice exhibited greater functional recovery including forepaw use, sensorimotor balance and coordination, and spatial learning and memory up to 28 days after TBI. These results unveil a previously unappreciated role for neuronal PI3Kγ in the regulation of ER stress associated with neuronal cell death, white matter damage, and long-term functional impairment after TBI.

Glioblastoma (GBM) has a poor prognosis despite intensive treatment with surgery and chemoradiotherapy. Previous studies using dose-escalated radiotherapy have demonstrated improved survival; however, increased rates of radionecrosis have limited its use. Development of radiosensitizers could improve patient outcome. In the present study, we report the use of sodium sulfide (Na 2 S), a hydrogen sulfide (H 2 S) donor, to selectively kill GBM cells (T98G and U87) while sparing normal human cerebral microvascular endothelial cells (hCMEC/D3). Na 2 S also decreased mitochondrial respiration, increased oxidative stress and induced γH2AX foci and oxidative base damage in GBM cells. Since Na 2 S did not significantly alter T98G capacity to perform non-homologous end-joining or base excision repair, it is possible that GBM cell killing could be attributed to increased damage induction due to enhanced reactive oxygen species production. Interestingly, Na 2 S enhanced mitochondrial respiration, produced a more reducing environment and did not induce high levels of DNA damage in hCMEC/D3. Taken together, this data suggests involvement of mitochondrial respiration in Na 2 S toxicity in GBM cells. The fact that survival of LN-18 GBM cells lacking mitochondrial DNA (ρ 0 ) was not altered by Na 2 S whereas the survival of LN-18 ρ + cells was compromised supports this conclusion. When cells were treated with Na 2 S and photon or proton radiation, GBM cell killing was enhanced, which opens the possibility of H 2 S being a radiosensitizer. Therefore, this study provides the first evidence that H 2 S donors could be used in GBM therapy to potentiate radiation-induced killing., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Genetic and pharmacological inhibition of the PI3Kγ (phosphoinositide 3-kinase-γ) exerts anti-inflammatory and protective effects in a number of inflammatory and autoimmune diseases. SHRs (spontaneously hypertensive rats) subjected to embolic middle cerebral occlusion were treated with AS605240 (30 mg/kg) at 2 or 4 hours, tPA (tissue-type plasminogen activator; 10 mg/kg) at 2 or 6 hours, or AS605240 at 4 hours plus tPA at 6 hours. Infarct volume, brain hemorrhage, neurological function, microvascular thrombosis, and cerebral microvessel patency were examined. We found that treatment with AS605240 alone at 2 hours or the combination treatment with AS605240 at 4 hours and tPA at 6 hours significantly reduced infarct volume and neurological deficits at 3 days after stroke compared with ischemic rats treated with saline, AS605240 alone at 4 hours, and tPA alone at 6 hours. Moreover, the combination treatment effectively prevented the delayed tPA-induced cerebral hemorrhage. These protective effects are associated with reduced disruption of the blood-brain barrier, reduced downstream microvascular thrombosis, and improved microvascular patency by AS605240. Inhibition of the NF-κB (nuclear transcription factor-κB)-dependent MMP (matrix metalloproteinase)-9 and PAI-1 (plasminogen activator inhibitor-1) in the ischemic brain endothelium may underlie the neurovascular protective effect of AS605240. In addition, the combination treatment significantly reduced circulating platelet P-selectin expression and platelet-leukocyte aggregation compared with ischemic rats treated with saline or tPA alone at 6 hours. In conclusion, inhibition of PI3Kγ with AS605240 reduces delayed tPA-induced intracerebral hemorrhage and improves microvascular patency, which likely contributes to neuroprotective effect of the combination treatment.

The original version of this Article presented incorrect information for citation 50. This has now been corrected in both the PDF and HTML versions of the Article. Petrov, M. S. Predicting the severity of acute pancreatitis: choose the right horse before hitching the cart. Dig. Dis. Sci. 56, 3402-3404 (2011).Parikh, R. et al. Understanding and using sensitivity, specificity and predictive values Indian J. Ophthalmol. 56, 45-50 (2008).

Background: Hospital discharge codes are increasingly used in gastroenterology research, but their accuracy in the setting of acute pancreatitis (AP) and chronic pancreatitis (CP), one of the most frequent digestive diseases, has never been assessed systematically. The aim was to conduct a systematic literature review and determine accuracy of diagnostic codes for AP and CP, as well as the effect of covariates., Methods: Three databases (Pubmed, EMBASE and Scopus) were searched by two independent reviewers for relevant studies that used International Classification of Disease (ICD) codes. Summary estimates of sensitivity, specificity and positive predictive value were obtained from bivariate random-effects regression models. Sensitivity and subgroup analyses according to recurrence of AP and age of the study population were performed., Results: A total of 24 cohorts encompassing 18,106 patients were included. The pooled estimates of sensitivity and specificity of ICD codes for AP were 0.85 and 0.96, respectively. The pooled estimates of sensitivity and specificity of ICD codes for CP were 0.75 and 0.94, respectively. The positive predictive value of ICD codes was 0.71 for either AP or CP. It increased to 0.78 when applied to incident episode of AP only. The positive predictive value decreased to 0.68 when the ICD codes were applied to paediatric patients., Conclusion: Nearly three out of ten patients are misidentified as having either AP or CP with the indiscriminate use of ICD codes. Limiting the use of ICD codes to adult patients with incident episode of AP may improve identification of patients with pancreatitis in administrative databases.

Background and Purpose: Taurine (2-aminoethansulfolic amino acid) exerts neuroprotective actions in experimental stroke. Here, we investigated the effect of taurine in combination with delayed tPA (tissue-type plasminogen activator) on embolic stroke., Methods: Rats subjected to embolic middle cerebral artery occlusion were treated with taurine (50 mg/kg) at 4 hours in combination with tPA (10 mg/kg) at 6 hours. Control groups consisted of ischemic rats treated with either taurine (50 mg/kg) or saline at 4 hours or tPA (10 mg/kg) alone at 2 or 6 hours after middle cerebral artery occlusion., Results: We found that combination treatment with taurine and tPA robustly reduced infarct volume and neurological deficits 3 days after stroke, whereas treatment with taurine alone had a less-significant protective effect. tPA alone at 6 hours had no effects on infarct volume but instead induced intracerebral hemorrhage. The combination treatment with taurine prevented tPA-associated hemorrhage and reduced intravascular deposition of fibrin/fibrinogen and platelets in downstream microvessels and hence improved microvascular patency. These protective effects are associated with profound inhibition of CD147 (cluster of differentiation 147)-dependent MMP-9 (matrix metalloproteinase-9) pathway in ischemic brain endothelium by taurine. Notably, targeted inhibition of CD147 by intracerebroventricular injection of the rat CD147 siRNA profoundly inhibited ischemia-induced and tPA-enhanced MMP-9 activity in ischemic brain endothelium and blocked tPA-induced cerebral hemorrhage. Finally, the combination treatment with taurine and tPA improved long-term outcome at least 45 days after stroke compared with saline-treated group., Conclusions: Our results suggest that taurine in combination with tPA may be a clinically feasible approach toward future attempts at combination stroke therapy., (© 2018 American Heart Association, Inc.)

Purpose: Cell migration/invasion assays are widely used in commercial drug discovery screening. 3D printing enables the creation of diverse geometric restrictive barrier designs for use in cell motility studies, permitting on-demand assays. Here, the utility of 3D printed cell exclusion spacers (CES) was validated as a cell motility assay., Methods: A novel CES fit was fabricated using 3D printing and customized to the size and contour of 12 cell culture plates including 6 well plates of basal human brain vascular endothelial (D3) cell migration cells compared with 6 well plates with D3 cells challenged with 1uM cytochalasin D (Cyto-D), an F-actin anti-motility drug. Control and Cyto-D treated cells were monitored over 3 days under optical microscopy., Results: Day 3 cell migration distance for untreated D3 cells was 1515.943μm ± 10.346μm compared to 356.909μm ± 38.562μm for the Cyt-D treated D3 cells (p < 0.0001). By day 3, untreated D3 cells reached confluency and completely filled the original voided spacer regions, while the Cyt-D treated D3 cells remained significantly less motile., Conclusions: Cell migration distances were significantly reduced by Cyto-D, supporting the use of 3D printing for cell exclusion assays. 3D printed CES have great potential for studying cell motility, migration/invasion, and complex multi-cell interactions.

The role of platelets in the development of thrombosis and abrupt closure after angioplasty is well recognized. However, the direct impact of platelets on neointima formation after arterial injury remains undetermined. Herein, we show that neointima formation after carotid artery wire injury reduces markedly in CD40 -/- apolipoprotein E-deficient (apoE -/- ) mice but only slightly in CD40 ligand -/- apoE -/- mice, compared with apoE -/- mice. Wild-type and CD40-deficient platelets were isolated from blood of apoE -/- and CD40 -/- apoE -/- mice, respectively. The i.v. injection of thrombin-activated platelets into CD40 -/- apoE -/- mice was performed every 5 days, starting at 2 days before wire injury. Injection of wild-type platelets promoted neointima formation, which was associated with increased inflammation by stimulating leukocyte recruitment via up-regulation of circulating platelet surface P-selectin expression and the formation of platelet-leukocyte aggregates. It was also associated with further promoting the luminal deposition of platelet-derived regulated on activation normal T cell expressed and secreted/chemokine (C-C motif) ligand 5 and expression of monocyte chemoattractant protein-1 and vascular cell adhesion molecule 1 in wire-injured carotid arteries. Remarkably, all these inflammatory actions by activated platelets were abrogated by lack of CD40 on injected platelets. Moreover, injection of wild-type platelets inhibited endothelial recovery in wire-injured carotid arteries, but this effect was also abrogated by lack of CD40 on injected platelets. Results suggest that platelet CD40 plays a pivotal role in neointima formation after arterial injury and might represent an attractive target to prevent restenosis after vascular interventions., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Inflammation and thrombosis currently are recognized as critical contributors to the pathogenesis of ischemic stroke. CD147 (cluster of differentiation 147), also known as extracellular matrix metalloproteinase inducer, can function as a key mediator of inflammatory and immune responses. CD147 expression is increased in the brain after cerebral ischemia, but its role in the pathogenesis of ischemic stroke remains unknown. In this study, we show that CD147 acts as a key player in ischemic stroke by driving thrombotic and inflammatory responses., Methods: Focal cerebral ischemia was induced in C57BL/6 mice by a 60-minute transient middle cerebral artery occlusion. Animals were treated with anti-CD147 function-blocking antibody (αCD147) or isotype control antibody. Blood-brain barrier permeability, thrombus formation, and microvascular patency were assessed 24 hours after ischemia. Infarct size, neurological deficits, and inflammatory cells invaded in the brain were assessed 72 hours after ischemia., Results: CD147 expression was rapidly increased in ischemic brain endothelium after transient middle cerebral artery occlusion. Inhibition of CD147 reduced infarct size and improved functional outcome on day 3 after transient middle cerebral artery occlusion. The neuroprotective effects were associated with (1) prevented blood-brain barrier damage, (2) decreased intravascular fibrin and platelet deposition, which in turn reduced thrombosis and increased cerebral perfusion, and (3) reduced brain inflammatory cell infiltration. The underlying mechanism may include reduced NF-κB (nuclear factor κB) activation, MMP-9 (matrix metalloproteinase-9) activity, and PAI-1 (plasminogen activator inhibitor-1) expression in brain microvascular endothelial cells., Conclusions: Inhibition of CD147 ameliorates acute ischemic stroke by reducing thromboinflammation. CD147 might represent a novel and promising therapeutic target for ischemic stroke and possibly other thromboinflammatory disorders., (© 2017 American Heart Association, Inc.)

Background: There is a lack of robust estimates of the worldwide incidence and mortality of acute pancreatitis, chronic pancreatitis, pancreatic cysts, and pancreatic cancer in the general population. Our aim was to quantitate and compare the incidence and mortality of major pancreatic diseases in high-quality population-based cohort studies., Methods: Three databases (PubMed, Embase, and Scopus) were searched independently by two reviewers. Data from eligible studies were subject to meta-analysis to obtain global estimates. A number of prespecified subgroup analyses and meta-regression analyses were also done., Findings: 48 population-based cohort studies (35 on pancreatic cancer, ten on acute pancreatitis, three on chronic pancreatitis, and none on pancreatic cysts) were identified, with a total study population of 296 million individuals and 119 000 patients with pancreatic diseases. Global estimates of incidence and mortality were 8·14 cases (95% CI 6·63-9·98) per 100 000 person-years and 6·92 deaths (95% CI 3·72-12·89) per 100 000 person-years for pancreatic cancer, 33·74 cases (95% CI 23·33-48·81) per 100 000 person-years and 1·60 deaths (95% CI 0·85-1·58) per 100 000 person-years for acute pancreatitis, and 9·62 cases (95% CI 7·86-11·78) per 100 000 person-years and 0·09 deaths (95% CI 0·02-0·47) per 100 000 person-years for chronic pancreatitis. Subgroup analysis based on the WHO regions showed that the incidences of both pancreatic cancer and acute pancreatitis, and mortality from pancreatic cancer, were significantly higher in the American region than in the European and Western Pacific regions, while the incidence of chronic pancreatitis was significantly higher in the European region than in the American region. Mortality from pancreatic cancer was lowest in the Southeast Asian region. The incidence of chronic pancreatitis was twice as high in men as in women, although there was no difference between sexes for pancreatic cancer or acute pancreatitis., Interpretation: Globally, acute pancreatitis is the most common pancreatic disease whilst pancreatic cancer is the most lethal. However, their burden is not equal across the globe. The epidemiological estimates reported in this study could inform future high-quality studies., Funding: None., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Abnormal glucose metabolism is present in almost 40% of patients after acute pancreatitis, but its pathophysiology has been poorly investigated. Pancreatic hormone derangements have been sparingly studied to date, and their relationship with abnormal glucose metabolism is largely unknown. The aim was to investigate the associations between pancreatic hormones and glucose metabolism after acute pancreatitis, including the effect of potential confounders. This was a cross-sectional study of 83 adult patients after acute pancreatitis. Fasting venous blood was collected from all patients and used for analysis of insulin, glucagon, pancreatic polypeptide, amylin, somatostatin, C-peptide, glucose, and hemoglobin A1c. Statistical analyses were conducted using the modified Poisson regression, multivariable linear regression, and Spearman's correlation. Age, sex, body mass index, recurrence of acute pancreatitis, duration from first attack, severity, and etiology were adjusted for. Increased insulin was significantly associated with abnormal glucose metabolism after acute pancreatitis, in both unadjusted (P = 0.038) and adjusted (P = 0.001) analyses. Patients with abnormal glucose metabolism also had significantly decreased pancreatic polypeptide (P = 0.001) and increased amylin (P = 0.047) in adjusted analyses. Somatostatin, C-peptide, and glucagon were not changed significantly in both unadjusted and adjusted analyses. Increased insulin resistance and reduced insulin clearance may be important components of hyperinsulinemic compensation in patients after acute pancreatitis. Increased amylin and reduced pancreatic polypeptide fasting levels characterize impaired glucose homeostasis. Clinical studies investigating islet-cell hormonal responses to mixed-nutrient meal testing and euglycemic-hyperinsulinemic clamps are now warranted for further insights into the role of pancreatic hormones in glucose metabolism derangements secondary to pancreatic diseases., (Copyright © 2016 the American Physiological Society.)

Background & Aim: Acute pancreatitis (AP) and chronic pancreatitis (CP) traditionally have been thought to be distinct diseases, but there is evidence that AP can progress to CP. Little is known about the mechanisms of pancreatitis progression. We performed a meta-analysis to quantify the frequency of transition of AP to CP and identify risk factors for progression., Methods: We searched PubMed, Scopus, and Embase for studies of patients with AP who developed CP, published from 1966 through November 2014. Pooled prevalence and 95% confidence intervals (CIs) were calculated for these outcomes, and sensitivity, subgroup, and meta-regression analyses were conducted., Results: We analyzed 14 studies, which included a total of 8492 patients. The pooled prevalence of recurrent AP was 22% (95% CI, 18%-26%), and the pooled prevalence of CP was 10% (95% CI, 6%-15%). Sensitivity analyses yielded a pooled prevalence of CP of 10% (95% CI, 4%-19%) and 36% (95% CI, 20%-53%) in patients after the first occurrence and recurrent AP, respectively. Subgroup analyses found alcohol use and smoking to be the largest risk factors for the development of CP, with pooled prevalence values of 65% (95% CI, 48%-56%) and 61% (95% CI, 47%-73%), respectively. Meta-regression analysis found that men were more likely than women to transition from AP to CP., Conclusions: Ten percent of patients with a first episode of AP and 36% of patients with recurrent AP develop CP; the risk is higher among smokers, alcoholics, and men. Prospective clinical studies are needed to study pancreatitis progression., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

This study is to evaluate the effects of Simvastatin on left ventricular hypertrophy and left ventricular function in patients with essential hypertension. Untreated or noncompliance with drug treatment patients with simple essential hypertension were treated with a therapy on the basis of using Telmisartan to decrease blood pressure (BP). There were 237 patients who had essential hypertension combined with left ventricular hypertrophy as diagnosed by echocardiography, taken after their BPs were decreased to meet the values of the standard normal. Among them, there were only 41 out of the original 237 patients, 17.3%, who had simple essential hypertension combined with left ventricular hypertrophy without any other co-existing disease. They were the patients selected for this study. All patients were randomly, indiscriminately divided into two groups: one was the control group (Group T), treated with the Telmisartan-based monotherapy; the other was the target group (Group TS), treated with the Telmisartan-based plus simvastatin therapy. The changes of left ventricular hypertrophy and left ventricular function were rediagnosed by echocardiography after 1 year. The results we obtained from this study were as follows: (i) The average BPs at the beginning of the study, of simple essential hypertension combined with left ventricular hypertrophy, were high levels (systolic blood pressure (SBP) 189.21 ± 19.91 mm Hg, diastolic blood pressure 101.40 ± 16.92 mm Hg). (ii) The Telmisartan-based plus simvastatin therapy was significantly effective in lowering the SBP (128.26 ± 9.33 mm Hg vs. 139.22 ± 16.34 mm Hg). (iii) After the 1-year treatment, the parameters of left ventricular hypertrophy in both groups were improved. Compared to group T, there were no differences in the characteristics of the subjects, including interventricular septum, left ventricular mass, left ventricular mass index, ejection fraction, left atrium inner diameter at baseline. The patients' interventricular septum (Group TS 10.30 ± 1.80 mm vs. Group T 10.99 ± 1.68 mm, P < .05), LVM (Group TS 177.43 ± 65.40 g vs. Group T 181.28 ± 65.09 g, P < .05), and LVMI (Group TS 100.97 ± 37.33 g/m(2) vs. Group T 106.54 ± 27.95 g/m(2), P < .05), all dropped more prominently (P < .05) in group TS; the ejection fraction rose more remarkably in group TS (Group TS: 57.50 ± 16.41% to 65.43 ± 11.60%, P < .01 while showing no change in Group T); the left ventricular hypertrophy reversed more significantly and the left ventricular systolic function improved more. (iv) The left atrium inner diameter of Group TS decreased (P < .01), the ratio of E/A, which indicates the left ventricular diastolic function, continued to drop further, showing no change to the trend of left ventricular diastolic function declination. Patients who have hypertension with left ventricular hypertrophy usually suffer other accompanying diseases at the same time. Telmisartan-based plus Simvastatin treatment can significantly reduce SBP, reverse left ventricular hypertrophy, improve the left ventricular systolic function, but it has no effect on reversing the left ventricular diastolic function. This experiment indicated that Simvastatin can reverse left ventricular hypertrophy and improve left systolic function.

Transport systems frequently include auxiliary proteins that perform subfunctions within the transporter protein complex. Two such proteins found in Gram-negative bacteria are the Membrane Fusion Proteins (MFPs) and the Outer Membrane Auxiliary (OMA) proteins. We here demonstrate that OMAs present in alpha-proteobacteria (but not in other bacterial types) contain a long alpha-helical region that is homologous to corresponding regions in the MFPs. The results suggest that during their evolution, OMAs, specifically from alpha-proteobacteria, exchanged their own alpha-helical domain for one derived from an MFP. The structural and functional implications of these findings are discussed.

K+ and Cl- homeostasis have been implicated in cell volume regulation and apoptosis. We addressed the hypothesis that K+ and Cl- efflux may contribute to apoptotic cell shrinkage and apoptotic death in cultured cortical neurons. CLC-2 and CLC-3 chloride channels were detected in cultured cortical neurons. The Cl- channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the outwardly rectifying Cl- current, prevented apoptotic cell shrinkage, and mildly attenuated cell death induced by staurosporine, C2-ceramide, or serum deprivation. Cl- channel blockers, however, at concentrations that prevented cell shrinkage had no significant effects on caspase activation and/or DNA fragmentation. Cell death in the presence of a Cl- channel blocker was still sensitive to blockade by the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk). Electron microscopy revealed that, although DIDS prevented apoptotic cell shrinkage, certain apoptotic ultrastructural alterations still took place in injured neurons. On the other hand, the K+ channel blocker tetraethylammonium (TEA), clofilium, or the caspase inhibitor z-VAD-fmk prevented cell shrinkage as well as caspase activation and/or DNA damage, and showed stronger neuroprotection against apoptotic alterations and cell death. The results indicate that neurons may undergo apoptotic process without cell shrinkage and imply distinct roles for Cl- and K+ homeostasis in regulating different apoptotic events., (Copyright 2004 Springer-Verlag)

The Na+, K+-ATPase (Na+, K+-pump) plays critical roles in maintaining ion homeostasis. Blocking the Na+, K+-pump may lead to apoptosis. By contrast, whether an apoptotic insult may affect the Na+, K+-pump activity is largely undefined. In cultured cortical neurons, the Na+, K+-pump activity measured as a membrane current Ipump was time-dependently suppressed by apoptotic insults including serum deprivation, staurosporine, and C2-ceramide, concomitant with depletion of intracellular ATP and production of reactive oxygen species. Signifying a putative relationship among these events, Ipump was highly sensitive to changes in ATP and reactive oxygen species levels. Moreover, the apoptosis-associated Na+, K+-pump failure and serum deprivation-induced neuronal death were antagonized by pyruvate and succinate in ATP- and reactive-oxygen-species-dependent manners. We suggest that failure of the Na+, K+-pump as a result of a combination of energy deficiency and production of reactive oxygen species is a common event in the apoptotic cascade; preserving the pump activity provides a neuroprotective strategy in certain pathological conditions.

K(+) channel blockers such as 4-aminopyridine (4-AP) can be toxic to neurons; the cellular mechanism underlying the toxicity, however, is obscure. In cultured mouse cortical neurons, we tested the hypothesis that the toxic effect of 4-AP might result from inhibiting the Na(+),K(+)-ATPase (Na(+),K(+)-pump) and thereafter induction of a hybrid death of concomitant apoptosis and necrosis. The Na(+),K(+)-pump activity, monitored as whole-cell membrane currents, was markedly blocked by 4-AP in concentration- and voltage-dependent manners in low millimolar ranges. At similar concentrations, 4-AP induced a neuronal death sensitive to attenuation by the caspase inhibitor Z-VAD-FMK (Z-Val-Ala-Asp(OMe)-fluoromethyl ketone) or Ca(2+) chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Electron microscopy confirmed hybrid ultrastructural features of coexisting apoptotic and necrotic components in same cells. We suggest that 4-AP is a potent antagonist of the Na(+),K(+)-ATPase and an inducer of the hybrid death of central neurons.

Dysfunction of the Na(+),K(+)-ATPase (Na(+),K(+)-pump), due to reduced energy supply or increased endogenous ouabain-like inhibitors, likely occurs under pathological conditions in the central nervous system. In cultured mouse cortical neurons, we examined the hypothesis that a mild non-toxic inhibition of the Na(+),K(+)-ATPase could synergistically sensitize the vulnerability of neurons to normally non-lethal apoptotic signals. Ouabain at a low concentration of 0.1 microM slightly lessened the Na(+),K(+)-pump activity measured as an ouabain-sensitive current, yet did not affect K(+) homeostasis and viability of cortical neurons. Co-exposure to 0.1 microM ouabain plus non-lethal C(2)-ceramide (5 microM) or beta-amyloid 1-42 (5 microM), however, induced marked intracellular K(+) loss, caspase-3 cleavage, DNA laddering, and synergistically triggered neuronal death. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) predominantly blocked the caspase activation and neuronal death. These results suggest that slight impairment of Na(+),K(+)-pump activity may amplify the disruption of K(+) homeostasis in the presence of a non-lethal apoptotic insult, leading to activation of apoptotic cascade and substantial neuronal injury.

Energy deficiency and dysfunction of the Na+, K+-ATPase are common consequences of many pathological insults. The nature and mechanism of cell injury induced by impaired Na+, K+-ATPase, however, are not well defined. We used cultured cortical neurons to examine the hypothesis that blocking the Na+, K+-ATPase induces apoptosis by depleting cellular K+ and, concurrently, induces necrotic injury in the same cells by increasing intracellular Ca2+ and Na+. The Na+, K+-ATPase inhibitor ouabain induced concentration-dependent neuronal death. Ouabain triggered transient neuronal cell swelling followed by cell shrinkage, accompanied by intracellular Ca2+ and Na+ increase, K+ decrease, cytochrome c release, caspase-3 activation, and DNA laddering. Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and necrosis in individual cells. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) blocked >50% of ouabain-induced neuronal death. Potassium channel blockers or high K+ medium, but not Ca2+ channel blockade, prevented cytochrome c release, caspase activation, and DNA damage. Blocking of K+, Ca2+, or Na+ channels or high K+ medium each attenuated the ouabain-induced cell death; combined inhibition of K+ channels and Ca2+ or Na+ channels resulted in additional protection. Moreover, coapplication of Z-VAD-FMK and nifedipine produced virtually complete neuroprotection. These results suggest that the neuronal death associated with Na+, K+-pump failure consists of concurrent apoptotic and necrotic components, mediated by intracellular depletion of K+ and accumulation of Ca2+ and Na+, respectively. The ouabain-induced hybrid death may represent a distinct form of cell death related to the brain injury of inadequate energy supply and disrupted ion homeostasis.

Activation of ionotropic glutamate receptors can induce neuronal apoptosis in vitro and in vivo. We showed previously that activation of the N-methyl-D-aspartic acid (NMDA) subtype of glutamate receptors in a low Ca(2+) and low Na(+) condition induced apoptotic neuronal death, and that the K(+) efflux via NMDA receptor channels was likely a key event in NMDA-induced apoptosis. Since non-NMDA receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and kainate receptors, are also permeable to K(+), we tested the hypothesis that stimulating K(+) efflux via non-NMDA receptor channels could induce apoptosis in cultured cortical neurons. Using a Ca(2+)-free and Na(+)-free external solution, application of kainate revealed outward membrane currents carried by K(+) efflux. In a low Ca(2+)/low Na(+) medium, a 5-h exposure to 50-500 microM AMPA in the presence of the NMDA receptor antagonist MK801 induced dose-dependent neuronal death 24 h after the onset of the insult, accompanied by intracellular K(+) reduction and caspase-3 activation. The AMPA-induced cell death was attenuated by the caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) and by the protein synthesis inhibitor cycloheximide. Reducing K(+) efflux by raising extracellular K(+) concentration from 5 to 25 mM attenuated AMPA-triggered cell death, the Ca(2+) channel antagonist nifedipine showed no effect on the AMPA toxicity. Kainate induced similar neuronal death sensitive to attenuation by Z-VAD-FMK or elevated extracellular K(+).We suggest that the non-NMDA receptor-mediated K(+) efflux may participate in apoptotic process and that blocking excessive K(+) efflux mediated by NMDA and non-NMDA receptors may selectively prevent neuronal apoptosis under certain pathological conditions.

Inhibition of catechol catechol-O-methyltransferase (COMT) in the brains of subjects treated with L-DOPA (L-3,4-dihydroxylphenylalanine) and an aromatic amino acid decarboxylase (AADC) inhibitor is suggested to cause an increase of L-DOPA, which might lead to oxidative damage through enhanced formation of free radicals. To investigate this hypothesis, the acute effects of two doses of the systemically administered COMT inhibitors entacapone (peripheral) and tolcapone (peripheral and central) on the extracellular formation of hydroxyl radicals in vivo following treatment with L-DOPA and the AADC inhibitor carbidopa were examined. The formation of extracellular hydroxyl radicals were determined by the measurement of 2,3-dihydroxybenzoic acid (2,3-DHBA), a reaction product of hydroxyl radicals with sodium salicylate, using microdialysis in the striatum of anesthetised rats. The COMT inhibitors were administered together with 50 mg/kg i.p. carbidopa as 5% gum arabic suspensions intraperitoneally (i.p.) at doses of 0, 1.0, and 10 mg/kg body weight to a total of 36 male HAN-Wistars rats. L-DOPA was injected i.p. 40 min after drugs of interest. Microdialysis samples were collected every 20 min for 400 min at a perfusion rate of 1 microl/min. Systemically administered 10 mg/kg tolcapone, but not entacapone, induced an increase in hydroxyl radical formation in the striatum of anesthetised rats following treatment with L-DOPA/carbidopa. The increase in hydroxyl radical formation was reflected by higher extracellular concentrations of the hydroxylate product of salicylate, 2,3-DHBA, peaking at 192% of baseline at the end of the observation period. Similar results were also found using the AUC (area under the curve) value estimated for the observation period. We conclude that the increase in hydroxyl radical formation is likely to result from an increased rate of monoamine oxidase-mediated and non-enzymatic (autoxidation) dopamine metabolism following increased central availability caused by reduction in COMT-mediated metabolism. We cannot, however, exclude the possibility that hydroxyl radicals are produced by tolcapone as a result of uncoupling mitochondrial oxidative phosphorylation.

Tetraethylammonium (TEA), the quaternary ammonium ion and nonselective K(+) channel blocker, is protective against neuronal apoptosis. We now tested two TEA analogs, tetrapentylammonium (TPeA) and tetrahexylammonium (THA), for their effects on apoptotic neuronal death and for their pharmacological profiles on membrane currents in cultured mouse cortical neurons. TPeA and THA (0.1-1.0 microM) attenuated staurosporine-induced caspase-3 activation and neuronal apoptosis. TPeA and THA blocked the outward delayed rectifier K(+) (I(K)) current in concentration-dependent manners with IC(50) values of 2.7 and 1.9 microM, respectively. I(K) was blocked by TPeA in a use-dependent manner, whereas THA blocked I(K) regardless of activation state of the channel. TPeA at 1 microM inhibited the high voltage-activated (HVA) Ca(2+) current and the A-type K(+) current (I(A)). TPeA (1-10 microM) also blocked the fast inactivating Na(+) current. The ligand-gated N-methyl-D-aspartate (NMDA) receptor current was not affected by up to 20 microM TPeA. THA at 1 microM showed inhibitory effects on I(A), HVA Ca(2+), and Na(+) currents. THA (10 microM) suppressed NMDA currents. The data suggest that, as K(+) channel blockers and apoptosis antagonists, TPeA and THA are much more potent than TEA; however, they have nonspecific actions on several voltage-gated or ligand-gated channels.

We have investigated the acute effects of systemic administration of Tinuvin 123 on nigro-striatal dopaminergic neurons in the C57Bl/6 mouse. Tinuvin 123 was administered subcutaneously (s.c.) twice, 16 h apart, at doses of 0, 2, 20 or 200 mg/kg body weight to a total of 48 male C57Bl/6 mice (12 animals/group). Seven days following the last dose the animals were decapitated and the brains removed. No deaths occurred during the study. There were no differences between the mean body weights of any of the experimental groups prior to or following Tinuvin 123 treatment. Animals treated s.c. with 2 mg/kg Tinuvin 123 exhibited no changes in striatal dopamine or metabolite concentrations compared with vehicle-treated animals. Higher doses of Tinuvin 123 (20 and 200 mg/kg) resulted in a moderate loss of striatal dopamine (31 and 38%) but concentrations of the dopamine metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid and the neurotransmitters serotonin, aspartate, gamma aminobutyric acid and glutamate were unchanged. The total number of tyrosine hydroxylase-immunoreactive neurons in the entire substantia nigra were equivalent in the vehicle- and Tinuvin 123-treated animals at all doses, thus no neuronal loss was demonstrated. In conclusion, this study demonstrates no evidence that systemic administered Tinuvin 123 induces dopaminergic neurotoxicity in C57Bl/6 mice.

1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo), a neurotoxin structurally similar to the dopaminergic neurotoxin MPTP, may be formed in humans treated with chloral hydrate or exposed to trichloroethylene, a widely used industrial solvent. Systemically administered TaClo (0.4 mg/kg, i.p.) induced an immediate and transient release of dopamine (DA) and serotonin (5-HT) measured using microdialysis. However, only 5-HT was increased significantly (area under the curve, AUC, for the 1-2 h-period following TaClo administration: 400% compared with the respective control value; 2-3 h-period: 326%). This was followed by a progressive increase in hydroxyl radical formation reflected by higher extracellular concentrations of the hydroxylate product of salicylic acid, 2,3-dihydroxybenzoic acid (AUC for the 1-2 h period following TaClo administration: 182% compared with the respective control value; 2-3 h period: 190%). In contrast, extracellular glutamate and GABA were increased 2-3 h post-injection by 64 and 51%, respectively. These data suggest that TaClo stimulates the generation of hydroxyl free radicals via an acute release of 5-HT and perhaps DA.