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Abstract: In a porcine liver transplant model, a brief period of oxygenated hypothermic machine perfusion (HMP) at the end of simple cold storage (SCS) has been shown to improve the viability of damaged liver grafts. To test the clinical validity of this strategy, we randomized SCS-discarded human liver grafts to either 4 hours of HMP (n = 13) or an additional 4 hours of SCS (n = 14). All livers were then warm reperfused to mimic ischemia-reperfusion injury ex vivo. The settings for HMP were: portal vein: 3 mm Hg, 300 mL/min and hepatic artery: 20 mm Hg, po 2: 300 mm Hg. Perfusion used Kidney Machine Perfusion Solution at 4°C to 8°C. During warm reperfusion, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) values were higher (P < .015) among the SCS versus HMP methods at all times. The AST slope was lower in HMP versus SCS (P = .01). The LDH slope tended to be lower for HMP versus SCS (P = .07). Morphological scores were not different between HMP and SCS. At the start of warm reperfusion, MAPK was lower in HMP versus SCS (P = .02). Endothelin-1 (EDN1) and ICAM-1 tended to be lower in HMP versus SCS (P = .1 and .07, respectively). No difference was noted in MAPK, EDN1, and ICAM-1 after 60 or 120 minutes of warm reperfusion. In conclusion, HMP down-regulated MAPK and tended to reduce EDN1 and ICAM-1 mRNA in human liver grafts. During warm reperfusion, HMP versus SCS livers showed reduced AST and LDH release but no morphological difference. Further optimization of liver HMP may require different timing/duration of perfusion and/or an higher perfusion temperature. [Copyright &y& Elsevier]

Abstract: Reduced glutathione (GSH), an important radical scavenger, has been added to various organ preservation solutions. Because GSH oxidizes into oxidized glutathione (GSSG) and only GSH has scavenging capacity, only GSH in the solution at the time of clinical use is relevant. The concentrations of GSH (GSHconc) and GSSGconc were determined in 2 static preservation solutions—University of Wisconsin (UW) and Celsior—and in 1 machine preservation solution—Kidney Preservation Solution 1 (KPS-1). We determined the half-life (T1/2) of freshly added GSH. The GSHconc in UW and KPS-1 was 0.006 ± 0.0018 mmol/L and 0.13 ± 0.30 mmol/L, respectively. The GSHconc in Celsior was 2.7 ± 0.17 mmol/L. The manufacturers of these solutions reported 3 mmol/L GSH. GSSGconc in UW, KPS-1, and Celsior was 1.58 ± 0.61 mmol/L, 1.13 ± 0.16 mmol/L, and 0.24 ± 0.01 mmol/L, respectively. T1/2 of GSH in UW, KPS-1, and Celsior was 18 days, 86 days, and 83 days, respectively. The actual GSHconc in UW and KPS-1 at the time of clinical use was substantially lower than reported by the manufacturer, owing to the relatively short T1/2 of GSH. For Celsior, the GSHconc was maintained. Therefore, addition of fresh GSH to UW and KPS-1 before clinical use is recommended. [Copyright &y& Elsevier]

Abstract: Hepatocellular carcinoma has an increasing incidence and high mortality. Treatment options are limited if the disease is not diagnosed in its early stage. The natural course of the disease is aggressive but not always predictable. Molecular profiling is a promising tool for classification in order to optimize prognosis prediction and treatment for an individual patient. In the last decade a large amount of studies has been conducted to better classify hepatocellular carcinomas. The focus of this review is on implications of molecular classification for prognosis and therapeutic decision making in HCC patients. Most studies used microarray technique for genome wide profiling, but other methods to detect genomic changes and microRNA are gaining interest. The whole genome profiling studies identified differences in affected signalling and tried to relate this to prognosis. Some common subgroups were identified, such as the proliferation cluster and the beta-catenin cluster. However, there is still little overlap between most studies. Better study design and bio-informatical analysis might help in this context. [Copyright &y& Elsevier]

Abstract: Aim: The amount of cancer cells disseminated during curative surgery for colorectal liver metastases (CRLM) may be responsible for recurrence. Haematogenous and intrahepatic cancer cell dissemination was evaluated, and its impact on cancer recurrence was assessed. Method: Twenty patients with resectable CRLM were included in a prospective study. Twelve patients underwent curative resection for 21 metastases. Ten selected metastases in eight patients were treated with radiofrequency ablation (RFA) followed by resection at the same operative session. Cancer cell dissemination was determined before, during and after surgery using ‘real time’ quantitative RT-PCR assay, based on detection and quantification of CEA and CK20 mRNA transcripts. Results: Circulating cancer cells were detected in 80% and intrahepatic cancer cells in 37% of the patients, though without impact on cancer recurrence. The amounts of disseminated cancer cells were significantly increased after surgery. This increase was similar in patients treated with and without RFA. RFA caused complete tumour destruction. Conclusion: Curative surgery for CRLM significantly increases the amount of disseminated cancer cells. Radiofrequency ablation can completely destroy selected resectable CRLM without excessive cancer cell dissemination. Neither haematogenous nor intrahepatic cancer cell dissemination were related to cancer recurrence in this small patient series. [Copyright &y& Elsevier]

Abstract: In several waterbreathing fish species, hypoxia induces a decrease in plasma free fatty acid (FFA) levels as opposed to an increase in air-breathing mammals. We hypothesised that this change is coupled to the mode of breathing. Therefore, we followed the metabolic response of cannulated air-breathing African catfish to an 8-h asphyxia period. The hematocrit and hemoglobin increased significantly upon asphyxia. However, no change was observed in the mean cellular hemoglobin concentration, indicating that more erythrocytes were brought into circulation. A continuous increase in plasma lactate concentration during asphyxia showed permanent activation of anaerobic glycolysis, pointing to a persistent oxygen shortage. Plasma glucose levels did not change, but FFA levels decreased significantly upon asphyxia with a concomitant increase in plasma noradrenaline levels. Thus, these results suggest that in the air-breathing African catfish noradrenaline mediated a decrease in plasma FFA levels similar to that in waterbreathing fish species. [Copyright &y& Elsevier]

Spontaneous action potentials were recorded longitudinally for 4–7 weeks from dissociated rat occipital cortex cells cultured on planar multi-electrode plates, during their development from isolated neurons into synaptically connected neuronal networks. Activity typically consisted of generalized bursts lasting up to several seconds, separated by variable epochs of sporadic firing at some of the active sites. These network bursts displayed discharge patterns with age-dependent firing rate profiles, and durations significantly increasing in the 3rd week in vitro and decreasing after about 1 month in vitro, when they evolved into short events with prompt onsets. These findings indicate that after about a month in vitro these cultured neuronal networks have developed a degree of excitability that allows almost instantaneous triggering of generalized discharges. Individual neurons tend to fire in specific and persistent temporal relationships to one another within these network bursts, suggesting that network connectivity maintains a core topology during its development. [Copyright &y& Elsevier]

Spontaneous bioelectric activity (SBA) taking the form of extracellularly recorded spike trains (SBA) has been quantitatively analyzed in organotypic neonatal rat visual cortex explants at different ages in vitro, and the effects investigated of both short- and long-term pharmacological suppression of glutamatergic synaptic transmission. In the presence of APV, a selective NMDA receptor blocker, 1–2- (but not 3-)week-old cultures recovered their previous SBA levels in a matter of hours, although in imitation of the acute effect of the GABAergic inhibitor picrotoxin (PTX), bursts of action potentials were abnormally short and intense. Cultures treated either overnight or chronically for 1–3 weeks with APV, the AMPA/kainate receptor blocker DNQX, or a combination of the two were found to display very different abnormalities in their firing patterns. NMDA receptor blockade for 3 weeks produced the most severe deviations from control SBA, consisting of greatly prolonged and intensified burst firing with a strong tendency to be broken up into trains of shorter spike clusters. This pattern was most closely approximated by acute GABAergic disinhibition in cultures of the same age, but this latter treatment also differed in several respects from the chronic-APV effect. In 2-week-old explants, in contrast, it was the APV+DNQX treated group which showed the most exaggerated spike bursts. Functional maturation of neocortical networks, therefore, may specifically require NMDA receptor activation (not merely a high level of neuronal firing) which initially is driven by endogenous rather than afferent evoked bioelectric activity. Putative cellular mechanisms are discussed in the context of a thorough review of the extensive but scattered literature relating activity-dependent brain development to spontaneous neuronal firing patterns. [Copyright &y& Elsevier]

Abstract: Objective: Proton magnetic resonance spectroscopy (1H MRS) is a technique to identify and quantify the composition of biofluids. Hypothermic machine perfusion (HMP) is an organ preservation method that has the potential to evaluate organ viability prior to transplantation by analyzing the composition of the perfusate. The aim of this study was to use 1H MRS to examine the perfusate during HMP of porcine livers exposed to warm ischemia (WI) and to identify potential biomarkers of liver viability. Materials and Methods: Porcine livers underwent 4 hours of HMP using kidney perfusion solution-1 (KPS-1) as perfusate after exposure to in situ WI of 0 (n = 6) or 2 hours (n = 5). Samples of the perfusate were taken at the start/end of HMP. Lactate and aspartate aminotransferase (AST) in the samples were measured biochemically as surrogates of the WI-induced damage. MRS acquisition was conducted on a 9.4 Tesla (400 MHz) high-resolution system. Results: AST increased significantly during 4 hours of HMP within groups (P < .02) and discriminated WI injury significantly from the start to the end of HMP (P < .03). 1H MRS showed significantly elevated signal intensity of lactate, alanine, and histidine during HMP within both groups (P < .02). Furthermore, alanine and histidine were significantly higher in the WI group than in the control group at the end of HMP (P = .011 and P = .038, respectively). Conclusion: AST, alanine, and histidine in HMP perfusate discriminated WI injury on porcine liver grafts and might be potential biomarkers of liver viability. [Copyright &y& Elsevier]

The clinical utility of adaptive radiotherapy (ART) for head and neck (H&N) squamous cell carcinoma (SqCC) remains poorly defined. Online daily ART (DART) promises both anatomic adaptation and planning target volume (PTV) reduction by developing a unique plan based on the anatomy at each fraction, rendering interfraction setup error negligible. In this prospective trial using CBCT-based ART, patients with H&N SCC undergoing definitive RT (+/- chemotherapy, CRT) were randomized to daily online DART with reduced PTV margins (DART arm) or standard PTV margins without ART (IGRT arm). Eligibility criteria included a diagnosis of oropharynx, larynx, or hypopharynx SqCC receiving definitive RT or CRT. All individuals received involved nodal radiotherapy (INRT) per a previous institutional study. The gross tumor volume (GTV) received 70 Gy, the primary clinical target volume (CTV) and suspicious nodes received 63 Gy, and nodes on the same level as nodal GTV or identified by an artificial intelligence (AI) algorithm received 56 Gy. The PTV margin in the DART arm was 1mm (2mm S/I), compared to 5mm in the IGRT arm. A H&N radiation oncologist adapted each DART fraction. The primary endpoint was patient-reported xerostomia at one year, requiring enrollment of 50 patients, stratified by oropharynx versus larynx/hypopharynx site. Patients completed the EORTC QLQ30/HN35, MDADI, and the Xerostomia Questionnaire (XQ) patient-reported outcome (PRO) instruments at baseline/1/3/6/12 months after treatment. Fifty patients were enrolled (26 IGRT, 24 DART). The cohort consisted of 38 oropharynx and 12 larynx/hypopharynx patients, with AJCC 7th stage I-II (n=4), III (n=10), and IVA/B (n=36). Forty-six patients (92%) were treated with CRT. The mean ipsilateral (16.0 vs. 11.5 Gy, p=0.02) parotid gland and ipsilateral (56.3 vs. 42.2 Gy, p<0.01) and contralateral (36.5 vs. 28.2 Gy, p=0.04) submandibular gland doses were significantly lower with DART. Swallowing OAR doses were not significantly different. The mean total patient in-room, recontouring, and physician at-console times were 33 (SD 5.3), 12.6 (SD 3.4), and 22 (SD 4.4) minutes per adaptive fraction, respectively. There was significantly less G2+ dermatitis in the DART arm (Grade 2+ 31% vs 8%, p=0.05) but not mucositis or dysphagia. No PRO differences were noted at one month, but at three months, trends favored reduced xerostomia with DART (XQ 43.1 vs. 31.2, p=0.2; for oropharynx patients, XQ 45 vs. 28.6, p=0.11). At a median follow-up of 7 months, one in-field, one out-of-field, and two distant recurrences were observed. Online DART for H&N SqCC may improve physician- and patient-reported acute toxicity profiles, albeit with increased resource utilization. Additional evidence and follow-up is needed to understand the potential benefits and limitations of this paradigm. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Advanced liver disease is characterized by prolonged global coagulation tests such as prothrombin time (PT). Using Model of End-stage Liver Disease (MELD) score-based allocation, many current transplant recipients show advanced end-stage liver disease with an elevated international normalized ratio (INR). The relationship between abnormalities in coagulation tests and the risk of bleeding has been recently challenged among liver disease patients. In this study we reassessed risk factors for bleeding and the clinical implications for patients who underwent orthotopic liver transplantation (OLT). Methods: We studied OLT patients between 2005 and 2011 excluding combined transplantations, retransplantations, or cases due to acute liver failure. We collected prospectively pre-OLT, during OLT, and post-OLT clinical and biochemical data to assess the risk for bleeding using linear regression models. Results: The strongest predictor of overall survival among 286 patients with a mean follow-up of 32 months was the number of blood transfusions (P = .005). The risk factor for bleeding during surgery investigated by multivariate analysis only showed the INR (P < .001) and the presence of ascites (P = .003) to independently correlate with the amount of blood transfusion. Receiver operation characteristics (ROC) analysis performed to determine the risk for massive blood transfusion (more than 6 units) revealed a cut-off value for INR ≥1.6. Appreciation of the operative field by the surgeon during the intervention as “wet” versus “dry”, amounts of blood transfusion and fresh frozen plasma, and stay in the intensive care unit (ICU) and in the hospital were all significantly different (P < .001) for patients with INR <1.6 versus INR ≥1.6. Conclusions: Bleeding during OLT affects the outcome. The risk is independently influenced by the presence of ascites (probably reflecting the degree portal hypertension) and an INR ≥1.6. To improve survival after OLT therapeutic interventions should be further explored to reduce the need for blood transfusions. [Copyright &y& Elsevier]

Abstract: Background: Livers exposed to warm ischemia (WI) are increasingly used for transplantation. The molecular mechanisms activated by WI alone (prior to procurement and transplantation) are not understood. To elucidate the pathways involved, we used microarrays to investigate the gene expression in porcine livers exposed to WI. Methods: Porcine livers (n = 6 group) were randomly subjected to WI periods of 15, 30 or 45 minutes. mRNA was extracted and gene expression determined by microarray analysis. Using bioinformatics software, we identified differentially expressed genes and related molecular pathways. We used the corresponding human annotation of the porcine microarray for the functional analysis. Results: Between 0 and 15 minutes of WI, 3530 genes were altered with a 2-log-fold change of <−0.58 or >+0.58 and P < .05. Between 0 and 30 minutes of WI, 4141 genes were differentially expressed; and between 0 and 45 minutes of WI, 2814 genes. At each time point, ∼50% of genes were up-regulated, whereas 50% were down-regulated. After pathway mapping, we found that the same pathways were induced for observed clustering of in the three WI periods: cell death, proliferation, inflammation, and metabolism pathways. Among the top genes that were up-regulated after 15 minutes of WI, the majority started to return to but did not reach baseline expression with increasing WI. A similar pattern was observed for the top suppressed genes. Conclusions: WI causes rapid changes in gene expression that affect several molecular pathways. This phenomenon seems to plateau at 15 to 30 minutes of WI. These new insights in the timing and the nature of molecular pathways induced by WI alone may help to design specific interventions to alter these changes and improve the outcome of livers from cardiac death donors. [Copyright &y& Elsevier]

Abstract: Background: Hypothermic machine perfusion (HMP) is superior to simple cold storage (SCS) for the preservation of kidney grafts. Whether HMP is superior to SCS for liver preservation is not known. Before a HMP system can be used clinically for the liver, its superiority to SCS needs to be demonstrated in an in vivo large animal transplant model. Objective: The aim was to compare outcomes after liver transplantation (LT) following preservation by SCS or HMP using technology/perfusion conditions similar to those for kidney HMP. Methods: Pig livers were perfused via the hepatic artery and portal vein for 4 hours with nonoxygenated 4°C University of Wisconsin machine perfusion solution. In the SCS group, flushed livers were stored in histidine-tryptophan-ketoglutarate solution. After preservation by SCS (n = 6) or HMP (n = 8) and LT, we assessed graft and recipient survivals, pH and lactate, hepatocellular damage [aspartate aminotransferase (AST)], Kupffer cell activation (β-galactosidase), tumor necrosis factor (TNF) α production, endothelial cell function (hyaluronic acid), and expression of Krüppel-like factor (KLF) 2 and 4, which are mediators of the flow-dependent vasoprotective endothelial phenotype. Results: No primary graft nonfunction was observed; livers recovered equally well from the postanhepatic metabolic acidosis in both groups. Pig survival was 5/6 (83%) in the SCS versus 2/8 (12.5%) in the HMP group (P = .04). Livers from both groups recovered equally well from the postanhepatic metabolic acidosis. AST in liver rinse-out samples obtained before LT were lower in the HMP than in the SCS group (P < .05). After reperfusion, AST and β-galactosidase were equally increased in both groups (P = .13 and 0.962, respectively); TNF-α and hyaluronic acid levels were higher after HMP versus SCS (P = .001 and 0.043, respectively). KLF-2 and -4 expressions were equally up-regulated after reperfusion in the SCS and HMP groups. Conclusions: In this in vivo model, liver HMP with subsequent transplantation was feasible. However, we did not demonstrate an advantage of HMP, using perfusion conditions shown to be effective for the kidney, over SCS. Despite similar immediate graft function, TNF-α generation, and endothelial cell dysfunction were more pronounced after HMP. [Copyright &y& Elsevier]

Summary: During adolescence pubertal development is said to lead to an increase in general stress sensitivity which might create a vulnerability for the emergence of psychopathology during this period. However, the empirical evidence for increasing stress sensitivity is scarce and mixed. Biological responses (salivary cortisol and alpha-amylase) were investigated during a social-evaluative stressor, the Leiden Public Speaking Task, in 295 nine to 17-year olds. Specific attention was paid to different elements of the task, that is anticipation to and delivery of the speech. Biological reactivity to the speech task increased with age and puberty, particularly during anticipation. Current findings support the idea that biological stress sensitivity increases during adolescence, at least in response to a social-evaluative situation. The increasing stress sensitivity appears related to both age and pubertal maturation, but unique contribution could not be distinguished. The importance of measuring anticipation is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Introduction and purpose: Functional proton magnetic resonance spectroscopy (MRS) can be applied to measure pharmacodynamic effects of central nervous system (CNS)-active drugs. The serotonin precursor 5-hydroxytryptophan (5-HTP), administered together with carbidopa and granisetron to improve kinetics and reduce adverse effects, acutely enhances central serotonergic neurotransmission and induces hypothalamus-pituitary-adrenal-(HPA) axis activation. We studied the hypothalamic levels of glutamate/glutamine (Glx), choline (Chol), N-acetyl-aspartate (NAA) and creatine using 7-Tesla (7T) MRS, and adrenocorticotropic hormone (ACTH) and cortisol in peripheral blood, after the administration of the 5-HTP function test in healthy volunteers. Methods: A randomized, double blind, placebo-controlled, two-way cross-over study was performed in 12 healthy males with a 7day wash-out period. After administration of the oral 5-HTP function test, ACTH and cortisol were measured over 4h and MRS scans at 7T were performed every 30min over 3h measuring Glx:Creatine, Chol:Creatine and NAA:Creatine ratios. Results: In the hypothalamus, the administration of 5-HTP had no effect on the average Glx, Chol or NAA levels over 180min but induced a significant decrease of Glx at 60min on post-hoc analysis. 5-HTP-induced significant ACTH release reaching an E max of 60.2ng/L at 80min followed by cortisol with an E max of 246.4ng/mL at 110min. Conclusions: The reduction in hypothalamic Glx levels after serotonergic stimulation is compatible with activation of excitatory neurons in this region, which is expected to cause depletion of local glutamate stores. The hypothalamic MRS-response reached its maximum prior to subsequent increases of ACTH and cortisol, which support the functional relevance of hypothalamic Glx-depletion for activation of the HPA-axis. This exploratory study shows that MRS is capable of detecting neuronal activation following functional stimulation of a targeted brain area. [Copyright &y& Elsevier]

Summary: Stress-system dysregulation is thought to increase the risk for anxiety disorders. Here we describe both hypothalamic pituitary adrenal (HPA) axis and autonomic nervous system (ANS) activity in basal non-challenging conditions and after 0.5mg dexamethasone in generalized social anxiety disorder (gSAD) patients. To ensure stress-free sampling we collected saliva and determined cortisol and alpha-amylase (sAA), the latter a relative new marker of autonomic activity. Forty-three untreated gSAD patients without comorbidity were compared with 43 age and gender matched controls in non-stressed conditions on sAA and cortisol after awakening, during the day (including late evening), and after a low dose (0.5mg) of dexamethasone. Cortisol and sAA were analyzed with mixed models. Additional analyses were done with paired t-tests. Apart from the assessments in the morning, gSAD patients had significantly higher diurnal and post-dexamethasone 1600h sAA levels. No differences between gSAD and controls in any cortisol measurements were found. In conclusion, in gSAD in basal, non-stimulated conditions and after dexamethasone, we found hyperactivity of the ANS, as measured with sAA, but not of the HPA-axis. This suggests a relative increased activity of the ANS as compared to the HPA-axis, in line with the observed hyperarousal in gSAD. [Copyright &y& Elsevier]

Calcineurin (Cn) is the target of the immunosuppressive drugs cyclosporine A (CsA), tacrolimus (Trl), and pimecrolimus (Prl). Trl and Prl are often used topically for treatment of various skin diseases. The Cn inhibitors CsA and Trl are mostly used for maintenance therapy of transplant patients. Their long-term use, however, causes a dramatic increase in skin cancer risk. By using a newly developed assay for Cn measurement in blood, we were able to demonstrate Cn activity in total skin homogenates. A significantly higher activity was found in epidermis compared to dermis. In skin cell cultures, fibroblasts showed the highest activity as compared to keratinocytes and melanocytes. Of the Cn inhibitors, Trl showed stronger inhibition than CsA and Prl (57 and 55% in fibroblast and keratinocyte cultures, respectively). Also, the lowest IC50 (the half maximal inhibitory concentration) values were found for Trl (0.5 and 1.3 nM in two different fibroblast cultures). Cn activity and its inhibition can thus be studied in dermatological samples. The effects of Cn inhibition in fibroblasts and keratinocytes may be of influence on the overall functioning of the skin immune system.Journal of Investigative Dermatology (2008) 128, 1686–1690; doi:10.1038/sj.jid.5701244; published online 17 January 2008 [ABSTRACT FROM AUTHOR]

Abstract: In recent studies we have demonstrated that arsenic (As) metabolites change the composition of neuronal cytoskeletal proteins in vivo and in vitro. To further examine the mechanism of arsenic-induced neurotoxicity with various arsenate metabolites (iAsV, MMAV and DMAV) and arsenite metabolites (iAsIII, MMAIII and DMAIII), we investigated the role of the proteolytic enzyme calpain and its involvement in the cleavage of p35 protein to p25, and also mRNA expression levels of calpain, cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase 3 beta (gsk3ß). A HeLa cell line transfected with a p35 construct (HeLa-p35) was used as a model, since all other proteins such as calpain, CDK5 and GSK3β are already present in HeLa cells as they are in neuronal cells. HeLa-p35 cells were incubated with various As metabolites and concentrations of 0, 10 and 30μM for duration of 4h. Subsequently the cells were either lysed to study their relative quantification levels of these genes or to be examined on their p35-protein expression. P35-RNA expression levels were significantly (p <0.01) increased by arsenite metabolites, while p35 protein was cleaved to p25 (and p10) after incubation with these metabolites. The cleavage of p35 is caused by calcium (Ca2+) induced activation of calpain. Inhibition of calpain activity by calpeptin prevents cleavage of p35 to p25. These results suggest that cleavage of p35 to p25 by calpain, probably As-induced Ca2+-influx, may explain the mechanism by which arsenic induces its neurotoxic effects. [Copyright &y& Elsevier]

Abstract: Neurological studies indicate that the central (CNS) and peripheral nervous system (PNS) may be affected by arsenic (As). As-exposed patients show significantly lower nerve conduction velocities (NCVs) in their peripheral nerves in comparison to healthy subjects. As may play a role in the disruption of neuroskeletal integrity, but the mechanisms by which it exerts a toxic effect on the peripheral and central nervous system are still unclear. In the present study, we examined the neurotoxic effects of various arsenic metabolites (iAsIII, iAsV, MMAV and DMAV) on two different cell lines derived from the peripheral (ST-8814) and central (SK-N-SH) nervous system. The effects of the arsenic metabolites were examined on the relative quantification levels of the cytoskeletal genes, neurofilament-light (NEFL), neurofilament-medium (NEF3), neurofilament-heavy (NEFH) and microtubule-associated protein-tau (MAPT), using real-time PCR. Our results show that iAsIII and iAsV have no significant effects on either cell lines. On the other hand, MMAV and DMAV cause significant changes in expression levels of NEF3 and NEFL genes, while the expression level of the NEFH gene is significantly increased in both cell lines. [Copyright &y& Elsevier]

Abstract: Understanding how the information is coded in large neuronal networks is one of the major challenges for neuroscience. A possible approach to investigate the information processing capabilities of the neuronal ensembles is given by the use of dissociated neuronal cultures coupled to microelectrode arrays (MEAs). Here, we describe a new strategy, based on MEAs, for studying in vitro neuronal network dynamics in interconnected sub-populations of cortical neurons. The rationale is to sub-divide the neuronal network into communicating clusters while preserving a high degree of functional connectivity within each confined sub-population, i.e. to achieve a compromise between a completely random large neuronal population and a patterned network, such as currently used with conventional MEAs. To this end, we have realized and functionally characterized a Pt microelectrode array with an integrated EPON SU-8 clustering structure, allowing to confine five relatively large yet interconnected spontaneously developing neuronal networks (i.e. thousands of cells). The clustering structure consists of five chambers of 3mm in diameter interconnected via 800μm long and 300μm wide microchannels and is integrated on the MEA of 60 thin-film Pt electrodes of 30μm diameter. Tests of the Pt microelectrodes’ stability under stimulation showed a stable behavior up to 35,000 voltage stimuli and the biocompatibility was assessed with the cultures of dissociated rat''s cortical neurons achieving cultures’ viability up to 60 days in vitro. Compared to conventional MEAs, the monitoring of spontaneous and evoked activity and the computation of the Post-Stimulus Time Histogram (PSTH) within the clusters clearly demonstrates: (i) the capability to selectively activate (through poly-synaptic pathways) specific network regions and (ii) the confinement of the network dynamics mainly in the highly connected sub-networks. [Copyright &y& Elsevier]

Changes in subunit composition of N-methyl-d-aspartate (NMDA) receptors have been reported to be affected by visual experience and may therefore form a major aspect of neuronal plasticity in the CNS during development. In contrast, putative alterations in the expression and functioning of the inhibitory GABAA receptor around eye opening have not been well defined yet. Here we describe the timing of changes in GABAA receptor subunit expression and the related synaptic functioning in the neonatal rat visual cortex and the influence of visual experience on this process.Quantitative analysis of all GABAA receptor subunit transcripts revealed a marked α3 to α1 subunit switch, in addition to a change in α4 and α5 expression. The changes were correlated with an acceleration of the decay of spontaneous inhibitory postsynaptic currents (sIPSCs). Both changes in receptor expression and synaptic functioning were initiated well before eye opening. Moreover, dark rearing could not prevent the robust upregulation of α1 or the change in sIPSC kinetics, indicating that this is not dependent of sensory (visual) input.Upon eye opening a positive correlation was observed between a faster decay of the sIPSCs and an increase in sIPSC frequency, which was absent in dark-reared animals. Thus, lack of extrinsic input to the cortex does not affect overall developmental regulation of synaptic functioning of GABAA receptors. However, we cannot exclude the possibility that visual experience is involved in proper shaping of the inhibitory network of the primary visual cortex. [Copyright &y& Elsevier]

In this study we investigated the functional implications of GABAA receptor α1 subunit deletion on dendritic arborization and spine maturation in the visual cortex. This subunit is normally strongly upregulated during early postnatal development. Our main finding is that mice lacking the GABAA receptor α1 subunit displayed an increased density of dendritic filopodia during the second and third postnatal weeks. However, there was a concomitant decreased density of mature mushroom-shaped spines, which became more pronounced in adults. In contrast, dendritic arborization was not altered in these mice. We propose that an increased efficacy of the inhibitory synaptic transmission in the α1 knock out mice may lead to an enhancement of the outgrowth of filopodia around eye opening, but to a failure in spine maturation at later stages. [Copyright &y& Elsevier]