Establishment of a novel protocol for assessing the severity of subarachnoid hemorrhage in circle Willis perforation mouse model.

The Circle of Willis perforation (cWp) mouse model is a key tool in subarachnoid hemorrhage (SAH) research; however, inconsistent bleeding volumes can challenge experimental reliability. To address this issue, we introduced the ROB Scoring System, a novel protocol integrating Rotarod Tests (RT), Open-field Tests (OT) video analysis, and daily Body Weight Loss (BWL) monitoring to precisely categorize SAH severity. Forty C57BL/6 mice underwent cWp SAH induction, categorized by ROB into severity subgroups (severe, moderate, mild). Validation compared ROB trends in subgroups, and ROB outcomes with autopsy results on postoperative days three and seven for acute and sub-acute evaluations. Mortality rates were analyzed via the survival log-rank test, revealing a significant difference among SAH subgroups (P < 0.05). Strong correlations between ROB grades and autopsy findings underscored its precision. Notably, the severe group exhibited 100% mortality within 4 days post SAH onset. Single parameters (RT, OT, BWL) were insufficient for distinguishing SAH severity levels. The ROB score represents a significant advancement, offering an objective method for precise categorization and addressing inherent bleeding variations in the cWp SAH model. This standardized protocol enhances the reliability and effectiveness of the SAH translational research, providing a valuable tool for future investigations into this critical area.

Tobacco smoke condensate-induced senescence in endothelial cells was ameliorated by colchicine treatment via suppression of NF-κB and MAPKs P38 and ERK pathways activation.

Smoking is the major cause of cardiovascular diseases and cancer. It induces oxidative stress, leading to DNA damage and cellular senescence. Senescent cells increase the expression and release of pro-inflammatory molecules and matrix metalloproteinase, which are known to play a vital role in the initiation and progression of cardiovascular diseases and metastasis in cancer. The current study investigated the smoking induced cellular senescence and employed colchicine that blocked senescence in endothelial cells exposed to tobacco smoke condensate. Colchicine prevented oxidative stress and DNA damage in tobacco smoke-condensate-treated endothelial cells. Colchicin reduced ß-gal activity, improved Lamin B1, and attenuated cell growth arrest markers P21 and P53. Colchicine also ameliorated the expression of SASP factors and inhibited the activation of NF-kB and MAPKs P38 and ERK. In summary, colchicine inhibited tobacco smoke condensate-induced senescence in endothelial cells by blocking the activation of NF-kB and MAPKs P38 and ERK.

CLR (C-Reactive Protein to Lymphocyte Ratio) Served as a Promising Predictive Biomarker for Cerebral Vasospasm in Aneurysmal Subarachnoid Hemorrhage (aSAH): A Retrospective Cohort Study.

Background: Subarachnoid hemorrhage is a devastating disease. Even after state-of-the-art treatment patients suffer from complications, including cerebral vasospasm (CVS), delayed cerebral ischemia (DCI), and chronic hydrocephalus (CH) following aneurysmal subarachnoid hemorrhage (aSAH). The aim of our study is to identify the predictive value of the C-reactive protein to lymphocyte ratio (CLR) for neurological functional outcome and complications after aSAH. Methods: We retrospectively analyzed a total of 166 aSAH patients who met the inclusion criteria enrolled in our study. Multivariate logistic regression analyses were performed to evaluate the independent risk factors. The predictive value of different models was compared by calculating the areas under the receiver operating characteristic (ROC) curve. Results: On-admission levels of CLR in patients with poor outcomes (6 months mRS 3-6), CVS, DCI, and CH were significantly higher than those in patients with good outcomes (6 months mRS 0-2), non-CVS, non-DCI, and non-CH. Multivariate logistic regression analysis revealed that admission CLR was independently associated with CVS (OR [95% CI] 2.116 [1.507-2.971]; p < 0.001), and DCI (OR [95% CI] 1.594 [1.220-2.084]; p = 0.001). In ROC analysis, the area under the curve (AUC) of CLR for poor outcomes (6 months mRS 3-6), CVS, DCI, and CH prediction were (AUC [95% CI] 0.639 [0.555-0.724]; p = 0.002), (AUC [95% CI] 0.834 [0.767-0.901]; p < 0.001), (AUC [95% CI] 0.679 [0.581-0.777]; p < 0.001), and (AUC [95% CI] 0.628 [0.543-0.713]; p = 0.005) revealing that admission CLR had a favorable predictive value for CVS after aSAH. The sensitivity and specificity of admission CLR for CVS prediction were 77.1% and 75.4%. On-admission CLR of 0.757 mg × 10-6 was identified as the best cutoff threshold to discriminate between CVS and non-CVS (CVS: CLR < 0.757 mg × 10-6 11/100 [11.0%] vs. CLR ≥ 0.757 mg × 10-6 37/66 [56.1%]; p < 0.001). Conclusions: High levels of on-admission CLR serve as an independent risk factor for CVS and DCI after aSAH. Admission CLR is an easy-to-quantify laboratory parameter that efficiently predicts the CVS after aSAH, which can provide some guidance for clinicians to evaluate for possible progression and treatment strategies in patients with aSAH.

Current Mouse Models of Intracranial Aneurysms: Analysis of Pharmacological Agents Used to Induce Aneurysms and Their Impact on Translational Research.

Intracranial aneurysms (IAs) are rare vascular lesions that are more frequently found in women. The pathophysiology behind the formation and growth of IAs is complex. Hence, to date, no single pharmacological option exists to treat them. Animal models, especially mouse models, represent a valuable tool to explore such complex scientific questions. Genetic modification in a mouse model of IAs, including deletion or overexpression of a particular gene, provides an excellent means for examining basic mechanisms behind disease pathophysiology and developing novel pharmacological approaches. All existing animal models need some pharmacological treatments, surgical interventions, or both to develop IAs, which is different from the spontaneous and natural development of aneurysms under the influence of the classical risk factors. The benefit of such animal models is the development of IAs in a limited time. However, clinical translation of the results is often challenging because of the artificial course of IA development and growth. Here, we summarize the continuous improvement in mouse models of IAs. Moreover, we discuss the pros and cons of existing mouse models of IAs and highlight the main translational roadblocks and how to improve them to increase the success of translational IA research.

Colchicine prevents oxidative stress-induced endothelial cell senescence via blocking NF-κB and MAPKs: implications in vascular diseases.

BACKGROUND: Smoking, alcohol abuse, and hypertension are - among others, potential risk factors for cardiovascular diseases. These risk factors generate oxidative stress and cause oxidative stress-induced DNA damage, resulting in cellular senescence and senescence-associated secretory phenotype (SASP). The SASP factors in feed-forward response exacerbate inflammation and cause tissue remodeling, resulting in atherosclerotic plaque formation and rupture. RESULTS: Colchicine inhibited ROS generation and mitigated oxidative stress-induced DNA damage. It dampened oxidative stress-induced endothelial cell senescence and improved the expression of DNA repair protein KU80 and aging marker Lamin B1. The drug attenuated the expression of senescence marker P21 at mRNA and protein levels. The pathway analysis showed that colchicine inhibited NF-κB and MAPKs pathways and subdued mTOR activation. Colchicine also attenuated mRNA expression of interleukin (IL)-1ß, IL-6, IL-8, MCP-1, ICAM-1, and E-selectin. Furthermore, colchicine reduced the mRNA and protein expression of matrix metalloproteinase (MMP-2). CONCLUSION: In summary, colchicine blocked oxidative stress-induced senescence and SASP by inhibiting the activation of NF-κB and MAPKs pathways.

mTOR Inhibitor Rapalink-1 Prevents Ethanol-Induced Senescence in Endothelial Cells.

The cardiovascular risk factors, including smoking, ethanol, and oxidative stress, can induce cellular senescence. The senescent cells increase the expression and release of pro-inflammatory molecules and matrix metalloproteinase (MMPs). These pro-inflammatory molecules and MMPs promote the infiltration and accumulation of inflammatory cells in the vascular tissue, exacerbating vascular tissue inflammation. MMPs damage vascular tissue by degenerating the extracellular matrix. Consequently, these cellular and molecular events promote the initiation and progression of cardiovascular diseases. We used Rapalink-1, an mTOR inhibitor, to block ethanol-induced senescence. Rapalink-1 inhibited oxidative-stress-induced DNA damage and senescence in endothelial cells exposed to ethanol. It attenuated the relative protein expression of senescence marker P21 and improved the relative protein expression of DNA repair protein KU70 and aging marker Lamin B1. It inhibited the activation of NF-κB, MAPKs (P38 and ERK), and mTOR pathway proteins (mTOR, 4EBP-1, and S6). Moreover, Rapalink-1 suppressed ethanol-induced mRNA expression of ICAM-1, E-selectin, MCP-1, IL-8, MMP-2, and TIMP-2. Rapalink-1 also reduced the relative protein expression of MMP-2. In summary, Rapalink-1 prevented senescence, inhibited pro-inflammatory pathway activation, and ameliorated pro-inflammatory molecule expression and MMP-2.

The Role of NF-κB in Intracranial Aneurysm Pathogenesis: A Systematic Review.

Intracranial aneurysms (IAs) are abnormal dilations of the cerebral vessels, which pose a persistent threat of cerebral hemorrhage. Inflammation is known to contribute to IA development. The nuclear factor "kappa-light-chain-enhancer" of activated B-cells (NF-κB) is the major driver of inflammation. It increases the expression of inflammatory markers and matrix metalloproteinases (MMPs), which contribute heavily to the pathogenesis of IAs. NF-κB activation has been linked to IA rupture and resulting subarachnoid hemorrhage. Moreover, NF-κB activation can result in endothelial dysfunction, smooth muscle cell phenotypic switching, and infiltration of inflammatory cells in the arterial wall, which subsequently leads to the initiation and progression of IAs and consequently results in rupture. After a systematic search, abstract screening, and full-text screening, 30 research articles were included in the review. In this systematic review, we summarized the scientific literature reporting findings on NF-κB's role in the pathogenesis of IAs. In conclusion, the activation of the NF-κB pathway was associated with IA formation, progression, and rupture.

Colchicine Protects against Ethanol-Induced Senescence and Senescence-Associated Secretory Phenotype in Endothelial Cells.

Inflammaging is a potential risk factor for cardiovascular diseases. It results in the development of thrombosis and atherosclerosis. The accumulation of senescent cells in vessels causes vascular inflammaging and contributes to plaque formation and rupture. In addition to being an acquired risk factor for cardiovascular diseases, ethanol can induce inflammation and senescence, both of which have been implicated in cardiovascular diseases. In the current study, we used colchicine to abate the cellular damaging effects of ethanol on endothelial cells. Colchicine prevented senescence and averted oxidative stress in endothelial cells exposed to ethanol. It lowered the relative protein expression of aging and senescence marker P21 and restored expression of the DNA repair proteins KU70/KU80. Colchicine inhibited the activation of nuclear factor kappa B (NFκ-B) and mitogen activated protein kinases (MAPKs) in ethanol-treated endothelial cells. It reduced ethanol-induced senescence-associated secretory phenotype. In summary, we show that colchicine ameliorated the ethanol-caused molecular events, resulting in attenuated senescence and senescence-associated secretory phenotype in endothelial cells.

TGF-ß Activated Kinase 1 (TAK1) Is Activated in Microglia After Experimental Epilepsy and Contributes to Epileptogenesis.

Increasing evidence suggests that inflammation promotes epileptogenesis. TAK1 is a central enzyme in the upstream pathway of NF-κB and is known to play a central role in promoting neuroinflammation in neurodegenerative diseases. Here, we investigated the cellular role of TAK1 in experimental epilepsy. C57Bl6 and transgenic mice with inducible and microglia-specific deletion of Tak1 (Cx3cr1CreER:Tak1fl/fl) were subjected to the unilateral intracortical kainate mouse model of temporal lobe epilepsy (TLE). Immunohistochemical staining was performed to quantify different cell populations. The epileptic activity was monitored by continuous telemetric electroencephalogram (EEG) recordings over a period of 4 weeks. The results show that TAK1 was activated predominantly in microglia at an early stage of kainate-induced epileptogenesis. Tak1 deletion in microglia resulted in reduced hippocampal reactive microgliosis and a significant decrease in chronic epileptic activity. Overall, our data suggest that TAK1-dependent microglial activation contributes to the pathogenesis of chronic epilepsy.

Doxycycline Attenuated Ethanol-Induced Inflammaging in Endothelial Cells: Implications in Alcohol-Mediated Vascular Diseases.

Excess alcohol consumption is a potential risk factor for cardiovascular diseases and is linked to accelerated aging. Drug discovery to reduce toxic cellular events of alcohol is required. Here, we investigated the effects of ethanol on human umbilical vein endothelial cells (HUVECs) and explored if doxycycline attenuates ethanol-mediated molecular events in endothelial cells. Initially, a drug screening using a panel of 170 drugs was performed, and doxycycline was selected for further experiments. HUVECs were treated with different concentrations (300 mM and 400 mM) of ethanol with or without doxycycline (10 µg/mL). Telomere length was quantified as telomere to single-copy gene (T/S) ratio. Telomere length and the mRNA expression were quantified by qRT-PCR, and protein level was analyzed by Western blot (WB). Ethanol treatment accelerated cellular aging, and doxycycline treatment recovered telomere length. Pathway analysis showed that doxycycline inhibited mTOR and NFκ-B activation. Doxycycline restored the expression of aging-associated proteins, including lamin b1 and DNA repair proteins KU70 and KU80. Doxycycline reduced senescence and senescence-associated secretory phenotype (SASP) in ethanol-treated HUVECs. In conclusion, we report that ethanol-induced inflammation and aging in HUVECs were ameliorated by doxycycline.

SARS-CoV-2 infection increases risk of intracranial hemorrhage.

Introduction: SARS-CoV-2 virus infection causes a dysbalanced and severe inflammatory response, including hypercytokinemia and immunodepression. Systemic inflammation triggered by a viral infection can potentially cause vascular damage, which may lead to cardiovascular and neurovascular events. Research question: The aim was to investigate whether CNS complications are related to COVID-19. Materials and methods: We examined 21 patients suffering from stroke and intracranial hemorrhage (ICH) and 9 (43%) of them were male. We compared relative frequencies using Fisher's exact test. As we had few observations and many variables, we used principal component analysis (PCA) to reduce data dimensionality. We trained a linear support vector machine (SVM) on the first two PCs of the laboratory data to predict COVID-19. Results: Patients suffering from stroke had either hypertension or SARS-CoV-2 infection, but seldom both (OR = 0.05, p = 0.0075). The presence of SARS-CoV-2 infection was strongly associated with the logarithm of CRP (p = 1.4e-07) and with D-DIMER (p = 1.6e-05) and moderately with PT (p = 0.0024). SARS-CoV-2 infection was not related to any other factor. CRP, D-DIMER, PT, and INR were all related to each other (R 2 ranging from 0.19 to 0.52, p ranging from 0.012 to < 0.0001). The first two PCs covered 96% of the variance in the four variables. Using them, perfect linear discrimination between patients suffering from COVID-19 and other patients could be achieved. Discussion and conclusion: SARS-CoV-2 infection causes systemic inflammation, which is suggested as a predictor of the severe course of ICH. SARS-CoV-2 infection is an additional risk factor for vascular complications.

Meta-review on Perforation Model of Subarachnoid Hemorrhage in Mice: Filament Material as a Possible Moderator of Mortality.

Robust preclinical models are inevitable for researchers to unravel pathomechanisms of subarachnoidal hemorrhage (SAH). For the mouse perforation model of SAH, the goal of this meta-review was the determination of variances in mortality, SAH severity grade, and vasospasm, and their experimental moderators, as many researchers are facing with incomparable results. We searched on the databases PubMed, Embase, and Web of Science for articles describing in vivo experiments using the SAH perforation mouse model and measuring mortality, SAH grade, and/or vasospasm. After screening, 42 articles (total of 1964 mice) were included into systematic review and meta-analysis. Certain model characteristics were insufficiently reported, e.g., perforation location (not reported in six articles), filament (material (n = 15) and tip texture (n = 25)), mouse age (n = 14), and weight (n = 10). Used injective anesthetics and location of perforation showed large variation. In a random-effects meta-analysis, the overall animal mortality following SAH was 21.3% [95% CI: 17.5%, 25.7%] and increased with longer observational periods. Filament material significantly correlated with animal mortality (p = 0.024) after exclusion of hyperacute studies (time after SAH induction < 24 h). Reported mean SAH grade was 10.7 [9.6, 11.7] on the scale of Sugawara (J Neurosci Methods 167:327-34, 2008). Furthermore, mean diameter of large cerebral arteries after SAH was reduced by 27.6% compared to sham-operated non-SAH mice. Uniforming standards of experimental procedures and their reporting are indispensable to increase overall comparability.

Intracranial Aneurysm Rupture after SARS-CoV2 Infection: Case Report and Review of Literature.

BACKGROUND: SARS-CoV virus infection results in a dysbalanced and severe inflammatory response with hypercytokinemia and immunodepression. Viral infection triggers systemic inflammation and the virus itself can potentially cause vascular damage, including blood-brain barrier (BBB) disruption and alterations in the coagulation system, which may result in cardiovascular and neurovascular events. Here, we review the literature and present a case of COVID-19 infection leading to an aneurysmal subarachnoid haemorrhage (aSAH). CASE DESCRIPTION: A 61-year-old woman presented with dyspnea, cough, and fever. She had a history of hypertension and was overweight with a body mass-index of 34. There was no history of subarachnoid hemorrhage in the family. Due to low oxygen saturation (89%) she was admitted into ICU. A chest CT showed a typical picture of COVID-19 pneumonia. The PCR-based test of an oropharyngeal swab was COVID-19-positive. In addition to oxygen support she was prescribed with favipiravir and hydroxychloroquine. She experienced a sudden headache and lost consciousness on the second day. Computer tomography (CT) with CT-angiography revealed a subarachnoid haemorrhage in the basal cisterns from a ruptured anterior communicating artery aneurysm. The aneurysm was clipped microsurgically through a left-sided standard pterional approach and the patient was admitted again to the intensive care unit for further intensive medical treatment. Post-operatively, the patient showed slight motor dysphasia. No other neurological deficits. CONCLUSION: Systemic inflammation and ventilator support-associated blood pressure fluctuations may trigger aneurysmal subarachnoid haemorrhage secondary to COVID-19 infection. COVID-19 infection could be considered as one of the possible risk factors leading to instability and rupture of intracranial aneurysm.

Testing the Stability of Drug Resistance on Cryopreserved, Gene-Engineered Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) have emerged as a powerful tool for in vitro modelling of diseases with broad application in drug development or toxicology testing. These assays usually require large quantities of hiPSC, which can entail long-term storage via cryopreservation of the same cell charges. However, it is essential that cryopreservation does not oppose durable changes on the cells. In this project, we characterize one parameter of functionality of one that is well established in the field, in a different research context, an applied hiPSC line (iPS11), namely their resistance to a medium size library of chemo interventions (>160 drugs). We demonstrate that cells, before and after cryopreservation, do not change their relative overall drug response phenotypes, as defined by identification of the top 20 interventions causing dose-dependent reduction of cell growth. Importantly, also frozen cells that are exogenously enforced for stable overexpression of oncogenes myelocytomatosis (cMYC) or tumor protein 53 mutation (TP53R175H), respectively, are not changed in their relative top 20 drugs response compared to their non-frozen counterparts. Taken together, our results support iPSCs as a reliable in vitro platform for in vitro pharmacology, further raising hopes that this technology supports biomarker-associated drug development. Given the general debate on ethical and economic problems associated with the reproducibly crisis in biomedicine, our results may be of interest to a wider audience beyond stem cell research.

A computational guided, functional validation of a novel therapeutic antibody proposes Notch signaling as a clinical relevant and druggable target in glioma.

The Notch signaling network determines stemness in various tissues and targeting signaling activity in malignant brain cancers by gamma-secretase inhibitors (GSI) has shown promising preclinical success. However, the clinical translation remains challenging due to severe toxicity side effects and emergence of therapy resistance. Better anti-Notch directed therapies, specifically directed against the tumor promoting Notch receptor 1 signaling framework, and biomarkers predicting response to such therapy are of highest clinical need. We assessed multiple patient datasets to probe the clinical relevance Notch1 activation and possible differential distribution amongst molecular subtypes in brain cancers. We functionally assessed the biological effects of the first-in-human tested blocking antibody against Notch1 receptor (brontictuzumab, BRON) in a collection of glioma stem-like cell (GSC) models and compared its effects to genetic Notch1 inhibition as well as classical pharmacological Notch inhibitor treatment using gamma-secretase inhibitor MRK003. We also assess effects on Wingless (WNT) stem cell signaling activation, which includes the interrogation of genetic WNT inhibition models. Our computed transcriptional Notch pathway activation score is upregulated in neural stem cells, as compared to astrocytes; as well as in GSCs, as compared to differentiated glioblastoma cells. Moreover, the Notch signature is clinical predictive in our glioblastoma patient discovery and validation cohort. Notch signature is significantly increased in tumors with mutant IDH1 genome and tumors without 1p and 19q co-deletion. In GSCs with elevated Notch1 expression, BRON treatment blocks transcription of Notch pathway target genes Hes1/Hey1, significantly reduced the amount of cleaved Notch1 receptor protein and caused significantly impairment of cellular invasion. Benchmarking this phenotype to those observed with genetic Notch1 inhibition in corresponding cell models did result in higher reduction of cell invasion under chemotherapy. BRON treatment caused signs of upregulation of Wingless (WNT) stem cell signaling activity, and vice versa, blockage of WNT signaling caused induction of Notch target gene expression in our models. We extend the list of evidences that elevated Notch signal expression is a biomarker signature declaring stem cell prevalence and useful for predicting negative clinical course in glioblastoma. By using functional assays, we validated a first in man tested Notch1 receptor specific antibody as a promising drug candidate in the context of neuro oncology and propose biomarker panel to predict resistance and therapy success of this treatment option. We note that the observed phenotype seems only in part due to Notch1 blockage and the drug candidate leads to activation of off target signals. Further studies addressing a possible emergence of therapy resistance due to WNT activation need to be conducted. We further validated our 3D disease modeling technology to be of benefit for drug development projects.

Robot technology identifies a Parkinsonian therapeutics repurpose to target stem cells of glioblastoma.

Aim: Glioblastoma is a heterogeneous lethal disease, regulated by a stem-cell hierarchy and the neurotransmitter microenvironment. The identification of chemotherapies targeting individual cancer stem cells is a clinical need. Methodology: A robotic workstation was programmed to perform a drug concentration to cell-growth analysis on an in vitro model of glioblastoma stem cells (GSCs). Mode-of-action analysis of the selected top substance was performed with manual repetition assays and acquisition of further parameters. Results: We identified 22 therapeutic potential substances. Three suggested a repurpose potential of neurotransmitter signal-modulating agents to target GSCs, out of which the Parkinson's therapeutic trihexyphenidyl was most effective. Manual repetition assays and initial mode of action characterization revealed suppression of cell proliferation, cell cycle and survival. Conclusion: Anti-neurotransmitter signaling directed therapy has potential to target GSCs. We established a drug testing facility that is able to define a mid-scale chemo responsome of in vitro cancer models, possibly also suitable for other cell systems.

Collective cell migration of thyroid carcinoma cells: a beneficial ability to override unfavourable substrates.

PURPOSE: Tumor cell invasion and metastasis are life threatening events. Invasive tumor cells tend to migrate as collective sheets. In the present in vitro study we aimed to (i) assess whether collective tumor cells gain benefits in their migratory potential compared to single cells and (ii) to identify its putative underlying molecular mechanisms. METHODS: The migratory potential of single and collective carcinoma cells was assessed using video time lapse microscopy and cell migration assays in the absence and presence of seven potential gap junction inhibitors or the Rac1 inhibitor Z62954982. The perturbation of gap junctions was assessed using a dye diffusion assay. In addition, LDH-based cytotoxicity and RT-PCR-based expression analyses were performed. RESULTS: Whereas single breast, cervix and thyroid carcinoma cells were virtually immobile on unfavourable plastic surfaces, we found that they gained pronounced migratory capacities as collectives under comparable conditions. Thyroid carcinoma cells, that were studied in more detail, were found to express specific subsets of connexins and to form active gap junctions as revealed by dye diffusion analysis. Although all potential gap junction blockers suppressed intercellular dye diffusion in at least one of the cell lines tested, only two of them were found to inhibit collective cell migration and none of them to inhibit single cell migration. In the presence of the Rac1 inhibitor Z62954982 collective migration, but not single cell migration, was found to be reduced up to 20 %. CONCLUSIONS: Our data indicate that collective migration enables tumor cells to cross otherwise unfavourable substrate areas. This capacity seems to be independent of intercellular communication via gap junctions, whereas Rac1-dependent intracellular signalling seems to be essential.

Experimental febrile seizures impair interastrocytic gap junction coupling in juvenile mice.

Prolonged and focal febrile seizures (FSs) have been associated with the development of temporal lobe epilepsy (TLE), although the underlying mechanism and the contribution of predisposing risk factors are still poorly understood. Using a kainate model of TLE, we previously provided strong evidence that interruption of astrocyte gap junction-mediated intercellular communication represents a crucial event in epileptogenesis. To elucidate this aspect further, we induced seizures in immature mice by hyperthermia (HT) to study the consequences of FSs on the hippocampal astrocytic network. Changes in interastrocytic coupling were assessed by tracer diffusion studies in acute slices from mice 5 days after experimental FS induction. The results reveal that HT-induced FSs cause a pronounced reduction of astrocyte gap junctional coupling in the hippocampus by more than 50%. Western blot analysis indicated that reduced connexin43 protein expression and/or changes in the phosphorylation status account for this astrocyte dysfunction. Remarkably, uncoupling occurred in the absence of neuronal death and reactive gliosis. These data provide a mechanistic link between FSs and the subsequent development of TLE and further strengthen the emerging view that astrocytes have a central role in the pathogenesis of this disorder. © 2016 Wiley Periodicals, Inc.

Pharmacogenetic prediction of individual variability in drug response based on CYP2D6, CYP2C9 and CYP2C19 genetic polymorphisms.

Interindividual variability in drug response depends on a number of genetic and environmental factors. The metabolic enzymes are well known for their contribution to this variability due to drug-drug interactions and genetic polymorphisms. The phase I drug metabolism is highly dependent upon the cytochrome P450 mono-oxygenases (CYP) and their genetic polymorphism leads to the variable internal drug exposures. The highly polymorphic CYP2C9, CYP2C19 and CYP2D6 isozymes are responsible for metabolizing a large portion of routinely prescribed drugs and contribute significantly to adverse drug reactions and therapeutic failures. In this review, two attractive and easily implementable approaches are highlighted to recommend drug doses ensuring similar internal exposures in the face of these polymorphisms. The first approach relies on subpopulation-based dose recommendations that consider the original population dose as an average of the doses recommended in genetically polymorphic subpopulations. By using bioequivalence principles and assuming linear gene-dose effect, dose recommendations can be made for different metabolic phenotypes. The second approach relates area under the curve to two characteristic parameters; the contribution ratio (CR), computes for the contribution of the metabolic enzyme and the fractional activity (FA), considers the impact of the genetic polymorphism. This approach provides valid and error free internal drug exposure predictions and can take into consideration genetic polymorphisms and drug interactions and/ or both simultaneously. Despite certain advantages and limitations, both approaches provide a good initial frame-work for devising models to predict internal exposure and individualize drug therapy, one of the promises from human genome project.

Mechanisms underlying the osteo- and adipo-differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are considered a promising cell source for regenerative medicine, because they have the potential to differentiate into a variety of lineages among which the mesoderm-derived lineages such adipo- or osteogenesis are investigated best. Human MSCs can be harvested in reasonable to large amounts from several parts of the patient's body and due to this possible autologous origin, allorecognition can be avoided. In addition, even in allogenic origin-derived donor cells, hMSCs generate a local immunosuppressive microenvironment, causing only a weak immune reaction. There is an increasing need for bone replacement in patients from all ages, due to a variety of reasons such as a new recreational behavior in young adults or age-related diseases. Adipogenic differentiation is another interesting lineage, because fat tissue is considered to be a major factor triggering atherosclerosis that ultimately leads to cardiovascular diseases, the main cause of death in industrialized countries. However, understanding the differentiation process in detail is obligatory to achieve a tight control of the process for future clinical applications to avoid undesired side effects. In this review, the current findings for adipo- and osteo-differentiation are summarized together with a brief statement on first clinical trials.