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.

Acetylcholinesterase activity as a neurotoxicity marker within the context of experimentally-simulated hyperprolinaemia: An in vitro approach.

Hyperprolinaemia is characterized by increased tissue accumulation of proline (Pro) and is known to exert serious cognitive and/or neuropsychiatric symptomatology as a direct result of Pro accumulation in the brain. The aim of this study was to explore a putative link between experimentally-simulated hyperprolinaemia and the activity of acetylcholinesterase (AChE); a crucial neurotoxicity marker. In vitro experiments were undertaken on purified eel-derived AChE, as well as on adult mouse brain homogenates, in order to examine the effect of a spectrum of Pro concentrations (3, 30, 500, and 1000 µM) on this marker. Our data showed that although Pro exerted a significant inhibitory effect on pure AChE activity, mouse brain-derived membrane-bound AChE activity was found either unaltered or significantly increased following incubation with Pro. The use of AChE activity as a neurotoxicity marker within the context of experimentally-simulated hyperprolinaemia should be considered with caution and in parallel with a number of other experimental parameters.

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.

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.

The role of cholecystokinin in the induction of aggressive behavior: a focus on the available experimental data (review).

Cholecystokinin (CCK) is a neuropeptide that is (among others) reportedly involved in the pathophysiology of psychiatric disorders. The excitatory role of CCK in negative affective emotions as well as in aversive reactions, antisocial behaviors and memories, has been indicated by numerous electrophysiological, neurochemical and behavioral methodologies on both animal models for anxiety and human studies. The current review article summarizes the existing experimental evidence with regards to the role of CCK in the induction of aggressive behavior, and: (a) synopsizes the anatomical circuits through which it could potentially mediate all types of aggressive behavior, as well as (b) highlights the potential use of these experimental evidence in the current research quest for the clinical treatment of mood and anxiety disorders.

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.

Lanthanum-induced neurotoxicity: solving the riddle of its involvement in cognitive impairment?

The conclusion of a series of recent reports highlights the significant neurotoxic effects that rare earth elements, such as lanthanum (La), can have during neurodevelopment. These findings are, herein, combined and presented using a simplified schematic overview that provides a putative solution to the riddle of La-induced cognitive impairment. Our commentary also highlights potential targets for further investigations into the mechanisms which underpin La-induced neurotoxicity, with a focus on the crucial role of the hippocampus. Within this context, a central role for the cyclic-adenosine monophosphate signalling pathway is proposed.

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.

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.

Can acetylcholinesterase activity be considered as a reliable biomarker for the assessment of cadmium-induced neurotoxicity?

Gonçalves et al. (2012) recently reported the findings of a long-awaited study on the effects of long-term dietary-induced exposure to cadmium (Cd) on the acetylcholinesterase (AChE) activity of adult rodents' brain regions. Their study can be regarded as a significant contribution to the field, as there is paucity of information on the AChE activity in brain regions following exposure to Cd. However, the Cd-induced modulation of AChE activity is an issue surrounded by controversy. We, herein, discuss and summarize the relative in vivo and in vitro experimental data, and set out to answer the straightforward question: can AChE activity be considered as a reliable biomarker for the assessment of Cd-induced neurotoxicity? At this time, we can not answer in the affirmative because of the variation in techniques used and conclusions reached. We make a plea that authors aiming to explore this potential use of brain AChE activity in the future: (a) are aware of the biases that their experimental approach might exert upon this neurochemical parameter, (b) avoid the use of anaesthesia as a mode of sacrifice and clarify its timing, (c) decide upon the use of previously-studied in vivo experimental schemes (so that they can provide comparable results), and finally, (d) identify pharmacological, biochemical and molecular approaches that are appropriate to clarify the implicated mechanism(s) through which Cd modifies AChE activity.

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.

Activation of acetylcholinesterase after U-74389G administration in a porcine model of intracerebral hemorrhage.

Spontaneous intracerebral hemorrhage (ICH) accounts for 10-15% of all strokes. Despite high incidence, morbidity and mortality, the precise pathophysiology of spontaneous ICH is not fully understood, while there is little data concerning the mechanisms that follow the primary insult of the hematoma formation. The cholinergic system, apart from its colossal importance as a neurotransmission system, seems to also play an important role in brain injury recovery. It has been recently suggested that the brain possesses a cholinergic anti-inflammatory pathway that counteracts the inflammatory responses after ICH, thereby limiting damage to the brain itself. We, herein, report the findings of our study concerning the role of acetylcholinesterase (AChE; a crucial membrane-bound enzyme involved in cholinergic neurotransmission) in a porcine model of spontaneous ICH, 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 the activation of AChE activity following U-74389G administration. The lazaroid U-74389G seems to be an established neuroprotectant and this is the first report of its supporting role in the enhancement of cholinergic response to the induction of ICH.

Short-term exposure to nickel alters the adult rat brain antioxidant status and the activities of crucial membrane-bound enzymes: neuroprotection by L-cysteine.

Nickel (Ni) is an environmental pollutant towards which human exposure can be both occupational (mainly through inhalation) and dietary (through water and food chain-induced bioaccumulation). The aim of this study was to investigate the effects of short-term Ni-administration (as NiCl(2), 13 mg/kg) on the adult rat whole brain total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), Na(+),K(+)-ATPase, and Mg(2+)-ATPase; in addition, the potential effect of the co-administration of the antioxidant L-cysteine (Cys, 7 mg/kg) on the above parameters was studied. Twenty-eight male Wistar rats were divided into four groups: A (saline-treated control), B (Ni), C (Cys), and D (Ni and Cys). All rats were treated once daily with intraperitoneal injections of the tested compounds, for 1-week. Rats were sacrificed by decapitation and the above-mentioned parameters were measured spectrophotometrically. Rats treated with Ni exhibited a significant reduction in brain TAS (-47%, p < 0.001, BvsA) that was efficiently limited by the co-administration of Cys (-4%, p > 0.05, DvsA; +83%, p < 0.001, DvsB), while Cys (group C) had no effect on TAS. The rat brain AChE activity was found significantly increased by both Ni (+30%, p < 0.001, BvsA) and Cys (+62%, p < 0.001, CvsA), while it tended to adjust to control levels by the co-administration of Ni and Cys (+13%, p < 0.001, DvsA; -13%, p < 0.001, DvsB). The activity of rat brain Na(+),K(+)-ATPase was significantly decreased by Ni-administration (-49%, p < 0.001, BvsA), while Cys supplementation could not reverse this decrease (-44%, p < 0.001, DvsA). The activity of Mg(2+)-ATPase was not affected by Ni-administration (-3%, p > 0.05, BvsA), but was significantly reduced when combined with Cys administration (-17%, p < 0.001, DvsA). The above findings suggest that Ni short-term in vivo administration causes a statistically significant decrease in the rat brain TAS and an increase in AChE activity. Both effects can be, partially or totally, reversed to control levels by Cys co-administration; Cys could thus be considered (for future applications) as a potential neuroprotective agent against chronic exposure to Ni. The activity of Na(+),K(+)-ATPase that was inhibited by Ni, could not be reversed by Cys co-administration. The matter requires further investigation in order to fully elucidate the spectrum of the neurotoxic effects of Ni.