Μιχαήλ Κουτσιλιέρης, Καθηγητής-Διευθυντής Εργαστηρίου Φυσιολογίας, Ιατρική, ΕΚΠΑ
Χάρις Λιάπη, Αναπληρώτρια Καθηγήτρια, Ιατρική, ΕΚΠΑ
Σταμάτιος Θεοχάρης, Αναπληρωτής Καθηγητής, Ιατρική, ΕΚΠΑ
Στυλιανός Τσακίρης , τ. Αναπληρωτής Καθηγητής, Ιατρική, ΕΚΠΑ
Παναγούλα Αγγελογιάννη , Αναπληρώτρια Καθηγήτρια, Ιατρική, ΕΚΠΑ
Αθανάσιος Αρμακόλας, Επίκουρος Καθηγητής, Ιατρική, ΕΚΠΑ
ΑναστάσιοςΦιλίππου , Επίκουρος Καθηγητής, Ιατρική, ΕΚΠΑ
Background: As it is known, the activities of brain enzymes, acetylcholinesterase (AChE) and two adenosine triphosphatases (Na+, K+-ATPase and Mg2+-ATPase) are critical parameters for the assessment of cholinergic function, the synaptic excitability and the control of protein synthesis rate and cell growth, respectively. The activities of these enzymes are subject to variations from the action of free radicals and vary considerably between different brain regions during the experimental induction of metabolic disorders that affect (among others), and the CNS. The purpose of this study is: i) to investigate the effects of mild in vivo exposure to ethanol (EtOH) (10% v/v in drinking water) in pregnant rats, on the activities of AChE, (Na+, K+)-ATPase and Mg2+-ATPase in whole brain and brain region (frontal cortex, hippocampus, hypothalamus, and cerebellum) homogenates of both newborn and 21-day-old offspring rats, during gestation, and throughout the duration of both gestation and lactation, and ii) to investigate the effects of mild in vivo administration of EtOH and/or thiamine deficient diet (TDD) to pregnant rats during gestation, on the activities of the aforementioned enzymes in whole brain homogenates, in addition to the liver histopathological examination, of both newborn and 21-day-old offspring rats.
Materials and Methods: For the in vivo experiments of the present study (two different experiments were performed), female and male adult AlbinoWistar were used. Food and water were provided ad libitum. In the 1st experimental phase, each male rat was placed with two female rats in each case, in order mating to be achieved. Following that (as assessed through the examination for the presence of an ejaculatory plug in the vagina), males were removed and females were divided into three groups: (i) Control (receiving tap water during both gestation and lactation), (ii) EtOH(G) (receiving 10% v/v of EtOH in the drinking water during gestation), and (iii) EtOH(G+L) (receiving 10% v/v of EtOH in the drinking water during gestation and lactation). At the end of lactation period, the 21-day-old rat offsprings were weighted, sacrificed by decapitation and their brains were rapidly removed, providing tissue samples for the corresponding subgroups Control, EtOH(G) and EtOH(G+L). Apart from those whole brains, other offspring rats were used in order for the following brain regions to be isolated and pooled into amounts sufficient for the undertaking of the below-mentioned neurochemical determinations in their homogenates: frontal cortex, hippocampus, hypothalamus, cerebellum and pons. In the 2nd experimental phase, after mating confirmed (same way as described acove), males were removed and females were divided into four groups: (i) Control (receiving tap water and normal diet during both gestation and lactation), (ii) EtOH (receiving normal diet and 10% v/v of EtOH in the drinking water during gestation), (iii) TDD (receiving special T deficient diet and tap water during gestation), and iv) TDD+EtOH (receiving 10% v/v of EtOH in the drinking water and special thiamine deficient diet during gestation). At birth, sufficient amount of newborn rats from each group were weighted, sacrificed by decapitation and their brains were rapidly removed, providing tissue samples for the Control1, EtOH1, TDD1 και EtOH1+TDD1 subgroups, respectively. The rest newborn rats from each group were left to grow until the end of the lactation period and in the same time the dams of all groups were receiving tap water and normal diet (same as the Control group). At the end of the lactation period sufficient amount of 21-day-old ofspring rats from each group were also weighted, sacrificed by decapitation and their brains were rapidly removed, providing tissue samples for the Control21, EtOH21, TDD21 και EtOH21+TDD21 subgroups, respectively. It should be also noted that all newborn and offspring rat groups from both experimental phases consisted of rats from both sexes. Tissue from whole brain and brain regions from the 1st experimental phase, as well as, whole brain tissue and liver tissue from the 2nd experimental phase, were rapidly removed, washed with isotonic buffer and weighted. In addition, liver tissue were immersed in formalin and were stained with haematoxylin-eosin stain. The obtained rat brain tissue was homogenized and the activities of AChE, (Νa+,Κ+)- ATPase and Mg2+-AΤΡase were assessed in all subgroups.
Results: In the 1st experimental phase, exposure to EtOH during gestation has resulted in a statistically-significant inhibition of AChE in the 21-day-old offspring whole brain and frontal cortex of the EtOH(G) subgroup, as compared to the corresponding Control activities. The frontal cortex of the offspring of the EtOH(G+L) subgroup has also demonstrated a statistically-significant decrease in AChE activity when compared to the respective Control values. The 21-day-old offspring frontal cortex Νa+,Κ+-ΑΤΡase activities have also been particularly affected by the undertaken tratments: the homogenates of both EtOH(G) and EtOH(G+L) subgroups revealed an extensive (and statistically-significant) increase as compared to Control21 subgroup. Interestingly, the offspring whole brain, hippocampal and hypothalamic Νa+,Κ+-ΑΤΡase activities were not affected by the undertaken maternal exposure to ethanol, while the EtOH(G) cerebellar and pontine Νa+,Κ+-ΑΤΡase activities demonstrated statistically-significant changes when compared to Control21 activities. Finally, offspring Mg2+-ATPase was found to be stimulated to a statistically-significant level (as compared to that of the Control21 subgroup) in the hippocampal samples of both EtOH-exposed subgroups and unaffected in the whole brain, the frontal cortex and the pons. The EtOH(G+L) hypothalamic Mg2+-ATPase activity was found increased as compared to Control21, while an increase of this enzyme’s activity was also recorded in the EtOH(G+L) cerebellar samples as compared to the EtOH(G) subgroup. In the 2nd experimental phase, TDD treatment during gestation has resulted in a statistically-significant stimulation of AChE activity in the newborn whole brain as compared to the respective Control1 activity. In addition, the 21-day-old offspring whole brain of the EtOH21 subgroup has resulted in a statistically-significant increase of the AChE activity as compared to the respective Control21 activity. Instead, at the subgroup TDD21+EtOH21 which ethanol and thiamine deficient diet were co-administated, a statistically-significant inhibition of the AChE activity of the 21-day-old whole brain was presented as compared to the respective Control21 activity. Same response was resulted in the whole brain AChE activity of the TDD21+EtOH21 subgroup as compared to the TDD21 subgroup. The newborn whole brain Νa+,Κ+-ΑΤΡase activities have also been particularly affected by the undertaken treatments: the homogenates of both EtOH1 and ΤDD1+EtOH1 subgroups revealed a statistically-significant increase in the Νa+,Κ+-ΑΤΡase activity, as compared to Control1 subgroup. Instead, the newborn whole brain Νa+,Κ+-ΑΤΡase activity of the TDD1 subgroup was found decreased, as compared as compared to Control1 subgroup. Interestingly, the newborn whole brain Νa+,Κ+-ΑΤΡase activity of the ΤDD1+EtOH1 subgroup revealed an extensive increase as compared to TDD1 subgroup. In addition, the 21-year-old offspring whole brain Νa+,Κ+-ΑΤΡase activity of the TDD21+EtOH21 subgroup was presented statistically-significant enhanced as compared to Control21 subgroup. Finally, the whole brain Mg2+-ATPase activities of both newborn and 21-day-old offspring rats were found unaffected under all examined subgroups. After histopathological examination of all the examined subgroups, in the 2nd experimental phase, no significant alterations were observed in both newborn and 21-day-old rat livers as compared to the respective Control subgroups.
Conclusions: The experimental design of the 1st experimental phase on the present study, compares the neurotoxic effects of maternal exposure to EtOH in a broader time frame (pregnancy and lactation), which in humans corresponds to neurodevelopmental CNS period during the third trimester of pregnancy, and also reveales the vulnereability of critical neurochemical parameters by specific brain region, through the different EtOH exposed schemed that were implemented. Among the considered enzymatic parameters of particular importance is the demonstrated extensive stimulation of Na+, K+-ATPase in the frontal cortex of the 21-day-old offspring rats, which is likely due to the reduced degree of dendrites branching in this specific brain region due to maternal exposure in EtOH. In contrast, the inhibition of cerebellar Na+, K+-ATPase activity of the 21-day-offspring rats, may indicate the existence of a region-specific alteration in the activity of this enzyme, which may be due to different histology and function of the examined brain regions, during the delicate period of neuronal development. Finally, the significant increament that was observed in the hippocampal Mg2+-ATPase activity of the 21-day-old offspring rats, is a finding that reported for the first time in the relative literature, since very little is known on the effects of maternal exposure to EtOH in the brain Mg2+-ATPase activity of the offsprings (existing findings are mainly related to the effects of EtOH exposure in the adulthood period). This enhancement in 21-day-old offspring hippocampal Mg2+-ATPase activity can be translated: (i) as an indicator for this specific brain region, regarding the neurotoxicity induced by FASD, and / or (ii) a cellular responce of this specific brain region to overcome the structural neurodevelopmental delay from EtOH exposure, by increasing the rate of protein synthesis and cell growth. Despite the aforementioned interesting findings, further research is needed in order to simulate and elucidate the pathophysiology of milder forms of FASD. In the 2nd experimental phase of the present study, a finding of particular interest is the observed increase in the newborn rats whole brain AChE activity of TDD1 subgroup, compared to the Control1 subgroup, which may be due to one or more of the following mechanisms: i) the reduction of physiological inhibition of the AChE activity due to the ability of the T to act as an in vitro AChE inhibitor and to block the transmission of nerve impulses in vivo, ii) the affect of TD-state on the cholinergic system (and therefore the activity of AChE), may be induced by the complicated and largely unspecified synergistic interactions between different brain neurotransmitter systems, and iii) the observed stimulation in the AChE activity of the TDD1 subgroup, may be due to a possible response of the cholinergic system to compensate for the reduced levels of neurotransmission due to possible ROS accumulation in the brain (as a result of cellular alterations in the TD-state), during prenatal exposure to TD. The observed recovery on the 21-day-old offspring whole brain AChE activity of the TDD21 subgroup, may be due the sufficient amount of T uptake through lactation that restores the normal cholinergic function. In the co-administration subgroups, the observed reduction in the 21-day-old offspring whole brain AChE activity of the TDD21+EtOH21 subgroup, as compared to the Control21 subgroup, may be due to a reduction of the cerebral TPP levels because of the synergistic neurotoxic effects of EtOH exposure in combination to TD-state. Furthermore, the significant reduction also observed in the 21-day-old offspring whole brain AChE activity of the TDD21+EtOH21 subgroup, as compared to the TDD21 subgroup, demonstrates that the neurotoxic effects of co-administration are more pronounced, than those which can cause the TD-state itself. Regarding to the Na+, K+-ATPase activity findings, the observed decrease in the newborn whole brain TDD1 subgroup, as compared Control1 subgroup, is probably due to the decreased ATP synthesis resulting from the impaired catabolism of neuronal cells due to reduced TPP brain levels in TD-state. Instead, the observed increase in the newborn whole brain Na+, K+-ATPase activity of EtOH1 subgroup, compared to the Control1 subgroup, is probably related to the excessive glutamate release, that can cause neuronal damage, due to induced hyperstimulation. Furthermore, the observed increase in whole brain Na+, K+-ATPase activities of EtOH and TDD co-administration subgroups, in both newborns and 21-day-old offsprings, as compared to the respective Control subgroups, is possibly due to: the mitochondrial dysfunction, the induced energy loss, the oxidative stress because of ROS formation, the disturbances in cell proliferation and cell migration, and the induced cell death, at the delicate period of neuronal development. The extensive increase observed in the newborn whole brain Na+, K+-ATPase activity of TDD1+EtOH1 subgroup, compared to the TDD1 subgroup, may can be explained from the fact that the neurotoxic effects of EtOH exposure are much more severe than those caused by the TD-state, resulting to the observed enhancement of the newborn whole brain Na+, K+-ATPase activity of TDD1+EtOH1 subgroup. The whole brain Mg2+-ATPase activity was not revealed any significant changes in both newborn and 21-day-old offspring examined subgroups. Finally, after histopathological examination of all examined subgroups, we can assume that the in utero mild EtOH exposure did not cause any significant alterations in the histopathological picture of the liver, in both newborn and 21-day-old offspring rats.