Hypophysitis related to immune checkpoint inhibitors: An intriguing adverse event with many faces.

INTRODUCTION: The incorporation of immune checkpoint inhibitors in the oncologists' arsenal is a milestone in cancer therapeutics, though not being devoid of toxicities.Areas covered: The present review provides a comprehensive and up-to-date overview of the immune-related hypophysitis with focus on the elusive biological background, the wide spectrum of the epidemiological profile, the varying clinical aspects, and the diagnostic and therapeutic challenges.Expert opinion: Historically considered distinctive of anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) monoclonal antibodies (mAbs), the immune-related hypophysitis is increasingly correlated with the anti-programmed cell-death (PD) protein 1 (PD-1)/anti-PD ligand 1 (PD-L1) mAbs. The distinct phenotype of hypophysitis related to anti-PD1/anti-PD-L1 mAbs is highlighted with focus on the immune-related isolated adrenocorticotropic (ACTH) deficiency. The immune-related central diabetes insipidus is discussed as a rare aspect of anti-CTL-A4 mAbs-induced hypophysitis, recently related to anti-PD1/anti-PD-L1 mAbs as well. The present review builds on existing literature concerning immune-related hypophysitis underscoring the pending issues still to be addressed, including (i) pathogenesis; (ii) correlation with preexisting autoimmunity; (iii) predictive value; (iv) utility of high-dose glucocorticoids; and (v) establishment of evidence-based diagnostic and therapeutic protocols. Increased awareness and constant vigilance are advocated as cornerstone of a multidisciplinary approach to ensure optimal patients' care.

Effects of gestational thiamine-deprivation and/or exposure to ethanol on crucial offspring rat brain enzyme activities.

OBJECTIVE: The fetal alcohol spectrum disorder (FASD) is a group of clinical conditions associated with the in utero exposure to ethanol (EtOH). We have recently examined the effects of a moderate maternal exposure to EtOH on crucial brain enzyme activities in offspring rats, and discussed the translational challenges arising when attempting to simulate any of the clinical conditions associated with FASD. MATERIALS AND METHODS: In this current study, we: (i) address the need for a more consistent and reliable in vivo experimental platform that could simulate milder cases of FASD complicated by simultaneous thiamine-deprivation during gestation and (ii) explore the effects of such a moderate maternal exposure pattern to EtOH and a thiamine-deficient diet (TDD) on crucial enzyme activities in the offspring rat brains. RESULTS: We demonstrate a significant decrease in the newborn and 21-day-old offspring body and brain weight due to maternal dietary thiamine-deprivation, as well as evidence of crucial brain enzyme activity alterations that in some cases are present in the offspring rat brains long after birth (and the end of the maternal exposure to both EtOH and TDD). CONCLUSIONS: Our findings provide a preliminary characterization of important neurochemical effects due to maternal exposure to EtOH and TDD during gestation that might affect the offspring rat neurodevelopment, and that characterization should be further explored in a brain region-specific manner level as well as through the parallel examination of changes in the offspring rat brain lipid composition.

How Far Are We from Prescribing Fasting as Anticancer Medicine?

(1) Background: the present review provides a comprehensive and up-to date overview of the potential exploitation of fasting as an anticancer strategy. The rationale for this concept is that fasting elicits a differential stress response in the setting of unfavorable conditions, empowering the survival of normal cells, while killing cancer cells. (2) Methods: the present narrative review presents the basic aspects of the hormonal, molecular, and cellular response to fasting, focusing on the interrelationship of fasting with oxidative stress. It also presents nonclinical and clinical evidence concerning the implementation of fasting as adjuvant to chemotherapy, highlighting current challenges and future perspectives. (3) Results: there is ample nonclinical evidence indicating that fasting can mitigate the toxicity of chemotherapy and/or increase the efficacy of chemotherapy. The relevant clinical research is encouraging, albeit still in its infancy. The path forward for implementing fasting in oncology is a personalized approach, entailing counteraction of current challenges, including: (i) patient selection; (ii) fasting patterns; (iii) timeline of fasting and refeeding; (iv) validation of biomarkers for assessment of fasting; and (v) establishment of protocols for patients' monitoring. (4) Conclusion: prescribing fasting as anticancer medicine may not be far away if large randomized clinical trials consolidate its safety and efficacy.

"Extracellular matrix remodelling in the liver of rats subjected to dietary choline deprivation and/or thioacetamide administration".

Choline deprivation is a recognized experimental approach to nonalcoholic steatohepatitis, while thioacetamide (TAA)-induced liver fibrosis resembles alcoholic liver fibrogenesis. In order to elucidate the effect of TAA on liver extracellular matrix composition under choline deprivation due to choline-deficient diet (CDD) administration, we evaluated the transcriptional and immunohistochemical (IHC) pattern of major hepatic matrix metalloproteinases (namely, MMP-2, -9) and their tissue inhibitors (TIMP-1, -2) in adult male albino Wistar rats at 30, 60 and 90 days. In the CDD+TAA group, IHC showed an early progressive increase in MMP-2 expression, while MMP-9 initially exhibited a significant increase followed by a gradual decrease; TIMP-1 and TIMP-2 IHC expressions showed gradual increase throughout the experiment. The MMPs-TIMPs regulation at the transcriptional level was found to be increased in all groups throughout the experiment. The increased MMP-2/TIMP-2 and suppressed MMP-9/TIMP-1 ratios in IHC and in real-time polymerase chain reaction (RT-PCR) seemed to correlate with the degree of liver fibrosis. These results support the important role of MMPs and TIMPs in controlling the hepatic pathogenesis and shed more light on the recently described experimental approach to liver disease (steatohepatitis) under the impact of two insults (TAA and CDD).

Immunohistochemical determination of the extracellular matrix modulation in a rat model of choline-deprived myocardium: the effects of carnitine.

Choline has been identified as an essential nutrient with crucial role in many vital biological functions. Recent studies have demonstrated that heart dysfunction can develop in the setting of choline deprivation even in the absence of underlying heart disease. Matrix metalloproteinases (MMPs) are responsible for extracellular matrix degradation, and the dysregulation of MMP-2 and MMP-9 has been involved in the pathogenesis of various cardiovascular disorders. The aim of the study was to investigate the role of MMPs and their inhibitors (TIMPs), in the pathogenesis of choline deficiency-induced cardiomyopathy, and the way they are affected by carnitine supplementation. Male Wistar Albino adult rats were divided into four groups and received standard or choline-deficient diet with or without L-carnitine in drinking water (0.15% w/v) for 1 month. Heart tissue immunohistochemistry for MMP-2, MMP-9, TIMP-1, and TIMP-2 was performed. Choline deficiency was associated with suppressed immunohistochemical expression of MMP-2 and an increased expression of TIMP-2 compared to control, while it had no impact on TIMP-1. MMP-9 expression was decreased without, however, reaching statistical significance. Carnitine did not affect MMP-2, MMP-9, TIMP-1 or TIMP-2 expression. The pattern of TIMP and MMP modulation observed in a choline deficiency setting appears to promote fibrosis. Carnitine, although shown to suppress fibrosis, does not seem to affect MMP-2, MMP-9, TIMP-1 or TIMP-2 expression. Further studies will be required to identify the mechanism underlying the beneficial effects of carnitine.

Exposure to ethanol during neurodevelopment modifies crucial offspring rat brain enzyme activities in a region-specific manner.

The experimental simulation of conditions falling within "the fetal alcohol spectrum disorder" (FASD) requires the maternal exposure to ethanol (EtOH) during crucial neurodevelopmental periods; EtOH has been linked to a number of neurotoxic effects on the fetus, which are dependent upon the extent and the magnitude of the maternal exposure to EtOH and for which very little is known with regard to the exact mechanism(s) involved. The current study has examined the effects of moderate maternal exposure to EtOH (10 % v/v in the drinking water) throughout gestation, or gestation and lactation, on crucial 21-day-old offspring Wistar rat brain parameters, such as the activities of acetylcholinesterase (AChE) and two adenosine triphosphatases (Na(+),K(+)-ATPase and Mg(2+)-ATPase), in major offspring CNS regions (frontal cortex, hippocampus, hypothalamus, cerebellum and pons). The implemented experimental setting has provided a comparative view of the neurotoxic effects of maternal exposure to EtOH between gestation alone and a wider exposure timeframe that better covers the human third trimester-matching CNS neurodevelopment period (gestation and lactation), and has revealed a CNS region-specific susceptibility of the examined crucial neurochemical parameters to the EtOH exposure schemes attempted. Amongst these parameters, of particular importance is the recorded extensive stimulation of Na(+),K(+)-ATPase in the frontal cortex of the EtOH-exposed offspring that seems to be a result of the deleterious effect of EtOH during gestation. Although this stimulation could be inversely related to the observed inhibition of AChE in the same CNS region, its dependency upon the EtOH-induced modulation of other systems of neurotransmission cannot be excluded and must be further clarified in future experimental attempts aiming to simulate and to shed more light on the milder forms of the FASD-related pathophysiology.

Neuronal tumour necrosis factor-α and interleukin-1ß expression in a porcine model of intracerebral haemorrhage: Modulation by U-74389G.

Tumour necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) are important mediators of intracerebral haemorrhage (ICH) inflammatory response. Lazaroids, established antioxidants and neuroprotectants, have been studied in several brain pathologies. The present study was designed to investigate: a) TNF-α and IL-1ß changes, in neurons and b) U-74389G effects, 4 and 24h after haematoma induction in a porcine model of intracerebral haemorrhage. In twenty male landrace pigs (swines) aged 135-150 days old, autologous whole blood was injected around the right basal ganglia territory; in ten of the pigs the lazaroid compound U-74389G was administered. Brain TNF-α and IL-1ß immunopositive neurons were determined by immunoarray techniques at 4 and 24h timepoints. After the haematoma induction the number of TNF-α immunopositive neurons ipsilateral to the haematoma was significantly higher compared to the contralateral site at 4h (p<0.0005), while U-74389G significantly reduced the number of TNF-α immunopositive neurons, ipsilateral to the haematoma, at 4h (p=0.002); at 24h, TNF-α immunopositive neurons were found significantly lower in the control group ipsilateral to the haematoma in comparison to 4h timepoint(p<0.0005). The number of IL-1ß immunopositive neurons at 4h after the hematoma induction was significantly higher ipsilateral to the haematoma site (p<0.0005). U-74389G had no statistical significant effect. TNF-α and IL-1ß, increase in neurons, 4h after the haematoma induction, ipsilateral to the haematoma site. The administration of the antioxidant compound U-74389G, results in early (at 4h) decrease of TNF-α immunopositive neurons but shows no statistical significant effect to IL-1ß immunopossitive neurons.

Inhibition of Na(+),K(+)-ATPase in the hypothalamus, pons and cerebellum of the offspring rat due to experimentally-induced maternal hypothyroidism.

Neurodevelopment is known to be particularly susceptible to thyroid hormone insufficiency and can result in extensive structural and functional deficits within the central nervous system (CNS), subsequently leading to the establishment of cognitive impairment and neuropsychiatric symptomatology. The current study evaluated the effects of gestational and/or lactational maternal exposure to propylthiouracil (PTU)-induced hypothyroidism (as a suggestive multilevel experimental approach to the study of hypothyroidism-induced changes that has been developed and characterized by the authors) on crucial brain enzyme activities of 21-day-old Wistar rat offspring in a CNS region-specific manner. The activities of acetylcholinesterase (AChE), Na(+),K(+)-ATPase and Mg(2+)-ATPase in the offspring hypothalamus, cerebellum and pons were assessed. The study demonstrated that maternal exposure to PTU (0.05% w/v in the drinking water) during the critical periods of neurodevelopment can result in an inhibition of hypothalamic, pontine and cerebellar Na(+),K(+)-ATPase; a major marker of neuronal excitability and metabolic energy production as well as an important regulator of important systems of neurotransmission. On the other hand, no significant changes in the activities of the herein offspring CNS regions' AChE and Mg(2+)-ATPase were recorded. The observed Na(+),K(+)-ATPase inhibition: (i) is region-specific (and non-detectable in whole brain homogenetes), (ii) could constitute a central event in the pathophysiology of clinically-relevant hypothyroidism-associated developmental neurotoxicity, (iii) occurs under all examined experimental schemes, and (iv) certainly deserves further clarification at a molecular and histopathological level. As these findings are analyzed and compared to the available literature, they also underline the need for the adoption and further study of Na(+),K(+)-ATPase activity as a consistent neurochemical marker within the context of a systematic comparative study of existing (and novel) simulation approaches to congenital and early age hypothyroidism.

Experimentally-induced maternal hypothyroidism alters crucial enzyme activities in the frontal cortex and hippocampus of the offspring rat.

Thyroid hormone insufficiency during neurodevelopment can result into significant structural and functional changes within the developing central nervous system (CNS), and is associated with the establishment of serious cognitive impairment and neuropsychiatric symptomatology. The aim of the present study was to shed more light on the effects of gestational and/or lactational maternal exposure to propylthiouracil (PTU)-induced hypothyroidism as a multilevel experimental approach to the study of hypothyroidism-induced changes on crucial brain enzyme activities of 21-day-old Wistar rat offspring in a brain region-specific manner. This experimental approach has been recently developed and characterized by the authors based on neurochemical analyses performed on newborn and 21-day-old rat offspring whole brain homogenates; as a continuum to this effort, the current study focused on two CNS regions of major significance for cognitive development: the frontal cortex and the hippocampus. Maternal exposure to PTU in the drinking water during gestation and/or lactation resulted into changes in the activities of acetylcholinesterase and two important adenosinetriphosphatases (Na(+),K(+)- and Mg(2+)-ATPase), that seemed to take place in a CNS-region-specific manner and that were dependent upon the PTU-exposure timeframe followed. As these findings are analyzed and compared to the available literature, they: (i) highlight the variability involved in the changes of the aforementioned enzymatic parameters in the studied CNS regions (attributed to both the different neuroanatomical composition and the thyroid-hormone-dependent neurodevelopmental growth/differentiation patterns of the latter), (ii) reveal important information with regards to the neurochemical mechanisms that could be involved in the way clinical hypothyroidism could affect optimal neurodevelopment and, ultimately, cognitive function, as well as (iii) underline the need for the adoption of more consistent approaches towards the experimental simulation of congenital and early-age-occurring hypothyroidism.

Effects of experimentally-induced maternal hypothyroidism on crucial offspring rat brain enzyme activities.

Hypothyroidism is known to exert significant structural and functional changes to the developing central nervous system, and can lead to the establishment of serious mental retardation and neurological problems. The aim of the present study was to shed more light on the effects of gestational and/or lactational maternal exposure to propylthiouracil-induced experimental hypothyroidism on crucial brain enzyme activities of Wistar rat offspring, at two time-points of their lives: at birth (day-1) and at 21 days of age (end of lactation). Under all studied experimental conditions, offspring brain acetylcholinesterase (AChE) activity was found to be significantly decreased due to maternal hypothyroidism, in contrast to the two studied adenosinetriphosphatase (Na(+),K(+)-ATPase and Mg(2+)-ATPase) activities that were only found to be significantly altered right after birth (increased and decreased, respectively, following an exposure to gestational maternal hypothyroidism) and were restored to control levels by the end of lactation. As our findings regarding the pattern of effects that maternal hypothyroidism has on the above-mentioned crucial offspring brain enzyme activities are compared to those reported in the literature, several differences are revealed that could be attributed to both the mode of the experimental simulation approach followed as well as to the time-frames examined. These findings could provide the basis for a debate on the need of a more consistent experimental approach to hypothyroidism during neurodevelopment as well as for a further evaluation of the herein presented and discussed neurochemical (and, ultimately, neurodevelopmental) effects of experimentally-induced maternal hypothyroidism, in a brain region-specific manner.

Postnuclear supernatants of rat brain regions as substrates for the in vitro assessment of cadmium-induced neurotoxicity on acetylcholinesterase activity.

Acetylcholinesterase (AChE) activity is thought to be a major neurotoxicity biomarker. Considering the recently highlighted controversy over the use of AChE activity as a biomarker for the neurotoxicity induced by cadmium (Cd; a major environmental contaminant), we have evaluated the in vitro effects of different concentrations of Cd on AChE activity in postnuclear supernatants of brain regions of newborn, 21-day-old, and adult male Wistar rats. Our findings demonstrate that Cd is a consistent inhibitor of AChE activity at concentrations higher than 10(-3) M as well as that, at a concentration of 10(-2) M, Cd induces an almost absolute inhibition of this crucial enzyme in the examined postnuclear supernatants. These findings confirm previous in vitro experiments of ours, but are not in full agreement with the available in vivo findings; in fact, they underline that this in vitro approach to Cd-induced neurotoxicity does not produce the distinctive brain region-specific responses in terms of AChE activity that we have recently observed in vivo. Our study does not support the use of AChE activity as a biomarker for the assessment of Cd-induced neurotoxicity in rat brain-derived postnuclear supernatants, at least under the examined in vitro experimental conditions.

Carnitine modulates crucial myocardial adenosine triphosphatases and acetylcholinesterase enzyme activities in choline-deprived rats.

Choline is an essential nutrient, and choline deficiency has been associated with cardiovascular morbidity. Choline is also the precursor of acetylcholine (cholinergic component of the heart's autonomic nervous system), whose levels are regulated by acetylcholinesterase (AChE). Cardiac contraction-relaxation cycles depend on ion gradients established by pumps like the adenosine triphosphatases (ATPases) Na(+)/K(+)-ATPase and Mg(2+)-ATPase. This study aimed to investigate the impact of dietary choline deprivation on the activity of rat myocardial AChE (cholinergic marker), Na(+)/K(+)-ATPase, and Mg(2+)-ATPase, and the possible effects of carnitine supplementation (carnitine, structurally relevant to choline, is used as an adjunct in treating cardiac diseases). Adult male albino Wistar rats were distributed among 4 groups, and were fed a standard or choline-deficient diet for one month with or without carnitine in their drinking water (0.15% w/v). The enzyme activities were determined spectrophotometrically in the myocardium homogenate. Choline deficiency seems to affect the activity of the aforementioned parameters, but only the combination of choline deprivation and carnitine supplementation increased myocardial Na(+)/K(+)-ATPase activity along with a concomitant decrease in the activities of Mg(2+)-ATPase and AChE. The results suggest that carnitine, in the setting of choline deficiency, modulates cholinergic myocardial neurotransmission and the ATPase activity in favour of cardiac work efficiency.

Developmental neurotoxicity of cadmium on enzyme activities of crucial offspring rat brain regions.

Cadmium (Cd) is an environmental contaminant known to exert significant neurotoxic effects on both humans and experimental animals. The aim of this study was to shed more light on the effects of gestational (in utero) and lactational maternal exposure to Cd (50 ppm of Cd as Cd-chloride in the drinking water) on crucial brain enzyme activities in important rat offspring brain regions (frontal cortex, hippocampus, hypothalamus, pons and cerebellum). Our study provides a brain region-specific view of the changes in the activities of three crucial brain enzymes as a result of the developmental neurotoxicity of Cd. Maternal exposure to Cd during both gestation and lactation results into significant changes in the activities of acetylcholinesterase and Na(+),K(+)-ATPase in the frontal cortex and the cerebellum of the offspring rats, as well as in a significant increase in the hippocampal Mg(2+)-ATPase activity. These brain-region-specific findings underline the need for further research in the field of Cd-induced developmental neurotoxicity. Deeper understanding of the mechanisms underlying the neurodevelopmental deficits taking place due to in utero and early age exposure to Cd could shed more light on the causes of its well-established cognitive implications.

Gestational exposure to cadmium alters crucial offspring rat brain enzyme activities: the role of cadmium-free lactation.

The present study aimed to shed more light on the effects of gestational (in utero) exposure to cadmium (Cd) on crucial brain enzyme activities of Wistar rat offspring, as well as to assess the potential protective/restorative role that a Cd-free lactation might have on these effects. In contrast to earlier findings of ours regarding the pattern of effects that adult-onset exposure to Cd has on brain AChE, Na(+),K(+)- and Mg(2+)-ATPase activities, as well as in contrast to similar experimental approaches implementing the sacrificing mode of anaesthesia, in utero exposure to Cd-chloride results in increased AChE and Na(+),K(+)-ATPase activities in the newborn rat brain homogenates that were ameliorated through a Cd-free lactation (as assessed in the brain of 21-day-old offspring). Mg(2+)-ATPase activity was not found to be significantly modified under the examined experimental conditions. These findings could provide the basis for a further evaluation of the herein discussed neurotoxic effects of in utero exposure to Cd, in a brain region-specific manner.

CD3(-)CD16(-)CD56(bright) immunoregulatory NK cells are increased in the tumor microenvironment and inversely correlate with advanced stages in patients with papillary thyroid cancer.

BACKGROUND: The innate immune system is the first line of defense and plays a key role in thyroid cancer development. The role of the tumor-infiltrating natural killer (NK) cells is becoming increasingly important in research and potential cancer therapies. NK cell subpopulations, CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright), demonstrate a significant role in the tumor immuno-surveillance process. METHODS: We investigated the distribution of CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright) NK subpopulations in tissue and blood samples from patients with papillary thyroid cancer (PTC) and nodular goiter (NG). Twenty-eight patients with PTC, 13 patients with NG, and 50 healthy donors were included in the study. Tissue and blood samples from all patients and blood samples from healthy donors were analyzed for CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright) NK cells by flow cytometry. RESULTS: A significant predominance of CD3(-)CD16(+)CD56(dim) cells compared to CD3(-)CD16(-)CD56(bright) NK cells was found in blood samples in all groups (p<0.0001 in PTC, NG, and healthy donors). Increased infiltration by CD3(-)CD16(-)CD56(bright) NK cells was observed in thyroid tissue of patients with PTC, as compared to CD3(-)CD16(+)CD56(dim) NK cells (p=0.046), while CD3(-)CD16(+)CD56(dim) NK cells demonstrated a higher infiltration of NG tissues. CD3(-)CD16(+)CD56(dim) NK cell tissue infiltration positively correlated with advanced stages of PTC. In contrast, the CD3(-)CD16(-)CD56(bright) NK cell population was negatively associated with tumor stage in patients with PTC. CONCLUSION: CD3(-)CD16(-)CD56(bright) NK cell infiltration seems to be associated with PTC progression. These findings contribute to a better understanding of the immune response in PTC and may lead to novel immunotherapeutic approaches in these patients.

Heart dysfunction induced by choline-deficiency in adult rats: the protective role of L-carnitine.

Choline is a B vitamin co-factor and its deficiency seems to impair heart function. Carnitine, a chemical analog of choline, has been used as adjunct in the management of cardiac diseases. The study investigates the effects of choline deficiency on myocardial performance in adult rats and the possible modifications after carnitine administration. Wistar Albino rats (n=24), about 3 months old, were randomized into four groups fed with: (a) standard diet (control-CA), (b) choline deficient diet (CDD), (c) standard diet and carnitine in drinking water 0.15% w/v (CARN) and (d) choline deficient diet and carnitine (CDD+CARN). After four weeks of treatment, we assessed cardiac function under isometric conditions using the Langendorff preparations [Left Ventricular Developed Pressure (LVDP-mmHg), positive and negative first derivative of LVDP were evaluated], measured serum homocysteine and brain natriuretic peptide (BNP) levels and performed histopathology analyses. In the CDD group a compromised myocardium contractility compared to control (P=0.01), as assessed by LVDP, was noted along with a significantly impaired diastolic left ventricular function, as assessed by (-) dp/dt (P=0.02) that were prevented by carnitine. Systolic force, assessed by (+) dp/dt, showed no statistical difference between groups. A significant increase in serum BNP concentration was found in the CDD group (P<0.004) which was attenuated by carnitine (P<0.05), whereas homocysteine presented contradictory results (higher in the CDD+CARN group). Heart histopathology revealed a lymphocytic infiltration of myocardium and valves in the CDD group that was reduced by carnitine. In conclusion, choline deficiency in adult rats impairs heart performance; carnitine acts against these changes.

Experimentally-induced Wernicke's encephalopathy modifies crucial rat brain parameters: the importance of Na+, K+ -ATPase and a potentially neuroprotective role for antioxidant supplementation.

Wernicke's encephalopathy (WE) is a serious neuropsychiatric syndrome caused by chronic alcoholism and thiamine (T) deficiency. Our aim was to shed more light on the pathophysiology of WE, by introducing a modified in vivo experimental model of WE and by focusing on changes provoked in the total antioxidant status (TAS) and three crucial brain enzyme activities in adult rats. Rats were placed on ethanol (EtOH) consumption (20 % v/v) for a total of 5 weeks. By the end of the third week, rats were fed a T-deficient diet (TDD) and were treated with pyrithiamine (PT; 0.25 mg/kg) for the remaining 2 weeks. Following the induction of WE symptomatology, rats were treated with three consecutive (every 8 h) injections of saline or T (100 mg/kg) and were sacrificed. Brain homogenates were generated and used for spectrophotometrical evaluation of TAS and enzymatic activities. Additionally, in vitro experiments were conducted on brain homogenates or pure enzymes incubated with T or neuromodulatory antioxidants. Pre-exposure to EtOH provided a successful protocol modification that did not affect the expected time of WE symptomatology onset. Administration of T ameliorated this symptomatology. WE provoked oxidative stress that was partially limited by T administration, while T itself also caused oxidative stress to a smaller extent. Brain acetylcholinesterase (AChE) was found inhibited by WE and was further inhibited by T administration. In vitro experiments demonstrated a potential neuroprotective role for L-carnitine (Carn). Brain sodium-potassium adenosine triphosphatase (Na(+),K(+)-ATPase) activity was found increased in WE and was reduced to control levels by in vivo T administration; this increase was also evident in groups exposed to PT or to TDD, but not to EtOH. In vitro experiments demonstrated a potential neuroprotective role for this Na(+),K(+)-ATPase stimulation through T or L-cysteine (Cys) administration. Brain magnesium adenosine triphosphatase (Mg(2+)-ATPase) activity was found decreased by prolonged exposure to EtOH, but was not affected by the experimental induction of WE. Our data suggest that T administration inhibits AChE, which is also found inhibited in WE. Moreover, increased brain Na(+),K(+)-ATPase activity could be a marker of T deficiency in WE, while combined T and antioxidant co-supplementation of Cys and/or Carn could be neuroprotective in terms of restoring the examined crucial brain enzyme activities to control levels.

Modulation of crucial adenosinetriphosphatase activities due to U-74389G administration in a porcine model of intracerebral hemorrhage.

Spontaneous intracerebral hemorrhage (ICH) represents a partially-understood cerebrovascular disease of high incidence, morbidity and mortality. We, herein, report the findings of our study concerning the role of two important adenosinetriphosphatases (ATPases) in a porcine model of spontaneous ICH that we have recently developed (by following recent references as well as previously-established models and techniques), with a focus on the first 4 and 24 h following the lesion's induction, in combination with a study of the effectiveness of the lazaroid antioxidant U-74389G administration. Our study demonstrates that the examined ICH model does not cause a decrease in Na(+),K(+)-ATPase activity (the levels of which are responsible for a very large part of neuronal energy expenditure) in the perihematomal basal ganglia territory, nor a change in the activity of Mg(2+)-ATPase. This is the first report focusing on these crucial ATPases in the experimental setting of ICH and differs from the majority of the findings concerning the behavior of these (crucial for central nervous system cell survival) enzymes under stroke-related ischemic conditions. The administration of U-74389G (an established antioxidant) in this ICH model revealed an injury specific type of behavior, that could be considered as neuroprotective provided that one considers that Na(+),K(+)- and Mg(2+)-ATPase inhibition might in this case diminish the local ATP consumption.

Large-scale bone mineral histomorphometry-report of a simplified technique.

AIMS: The aim of this study was the development of a simplified technique for bone mineral histomorphology on large undecalcified bone samples. Established techniques, such as undecalcified bone thin sectioning, ultrathin grinding, surface-stained block grinding and micro-computerized tomography (CT), are expensive, time-consuming and put very high demands on equipment, safety standards, personnel and laboratory facilities. METHODS AND RESULTS: The method is based on the surface-stained block-grinding principle; however, its novelty lies in the selection of user-friendly, safe and low-cost materials, equipment and digitization techniques. We describe in detail the relevant steps, as well as many practical tips for their successful implementation: accurate bone cutting in thin sections with a customized arrangement on a commercial bandsaw, defatting with sodium hypochlorite, embedding in epoxy resin blocks at room temperature, silicon carbide paper grinding, von Kossa staining, flatbed scanner digitization and image processing. CONCLUSION: We believe that the proposed methodology could contribute to the expansion of the study of bone tissue, as it enables the rapid examination of bone specimens on a large scale with minimal laboratory requirements and consumables costs.