Cognitive Deficits In Offspring Rats Exposed To Prenatal Maternal Seizure And Neuroprotective Effect Of Astaxanthin

Epilepsy is one of the oldest neurological conditions known to humankind. Up till now, it is still a very common neurological disease second only to stroke. Maternal epilepsy is the commonest neurological disease in pregnancy with substantial effects on maternal and fetal health. Prenatal maternal seizures have been found to be associated with an increased risk of mental retardation in the infants and might lead to cognitive deficits in them later in life. Howerer, its pathophysiologic mechanisms are not fully understood and it is difficult for researchers to reach a reliable conclusion due to influences of disturbing factors such as teratogenic effects of antiepileptic medication, genetic predisposition and socio-economical factors. Compared to human studies, animal studies can eliminate these disturbing factors and therefore have an advantage in exploring the effects of maternal seizures on neurodevelopment of offspring.The developing brain is highly vulnerable to different insults, and prenatal maternal convulsions can increase the risk for hypoxia-ischemia and acidosis in utero, which results in irreversible damages of the fetus. Hypoxia has been demonstrated to induce oxidative stress in adult and fetal tissues. Studies have shown that oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, such as amyotrophic lateralizing sclerosis, Parkinson’s disease and epilepsy. Furthermore, oxidative stress has been reported to have association with fetal brain damage induced by preeclampsia, chronic intrauterine hypoxia, intrauterine growth restriction and intrauterine inflammation. Fetal oxidative stress might be involved in adult brain damage and cognitive deficits induced by maternal epilepsy. This mechanism appears to provide new targets for fetal protection during prenatal maternal seizures but has not been involved in previous researches. Astaxanthin(ATX), a natural carotenoid, widely distributes in algae, crustaceans, salmonoids and many other such organisms. Previous researches confirmed that ATX has diverse bio-activities such as anti-oxidant, anti-inflammatory and anti-apoptotic properties. Additionally, the neuroprotective effects of ATX have attracted considerable attention based on its ability to cross the brain-blood barrier. However, to our knowledge, the neuroprotective effects of ATX on epilepsy have not been investigated.In this study, we used chronic amygdale-kindled rat to evaluate neuronal damage and cognitive impairment of offspring induced by maternal epilepsy throughout pregnancy and confirm the effect of oxidative stress on fetal brain damage. Meanwhile, after determining the neuroprotective effects of ATX on amygdale-kindled rats, we investigated whether ATX can ameliorate rats brain damage induced by prenatal maternal epilepsy and explore its potential mechanisms. Therefore, the present study was aimed to provide new targets for preventing fetal brain damage induced by prenatal maternal seizures. PartⅠ Cognitive deficits in offspring rats exposed to prenatal maternal seizure induced by amygdala kindlingObjective: To observe neuronal damage and cognitive impairment of offspring induced by maternal seizure throughout pregnancy, and to determine the effect of oxidative stress on fetal brain damage by the detection of oxidative stress parameters in hippocampus and pathological changes of placenta.Methods: Adult female Spraque-Dawley(SD) rats were randomly divided into three groups: control group, kindling group and sham group. Electrodes were implanted into the rats of kindling group and sham group, but amygdala kindling procedure was performed only in kindling group. The rats were kindled once by electric stimulation of the amygdale each day. All the rats were allowed to mate after one week’s fully kindling. The pregnant rats in kindling group received electric stimulation every 48 hr. Dams were allowed to deliver naturally. Effects of maternal seizure on the number of offspring, the survival rate of pups and body weight of pups were observed.Hematoxylin-Eosin(HE) staining was used to visualize changes of maternal placenta. Nissal’s staining was used to detect hippocampal morphology of P0 and P84 offspring. We used Morris water maze(MWM) test to assess the cognitive function of adult offspring. Oxidative stress parameters, malondialdehyde(MDA) and glutathione(GSH), in the hippocampus of P0 pups were examined by spectrophotometry.Results: 1. Amygdala kindling pregnant rat model was performed successfully. Eepileptic seizure was not found to have association with pregancy. 2. The survival rate of pups in kindling group was much greater than that in the control group(P<0.01). No significant difference was found between kindling group and the control group(P>0.05) in the number of pups per litter and pups birth-weight. There was no difference between control and sham groups(P>0.05). 3. Findings from HE staining are as follows: Placental tissues from control and sham groups were normal while the histologic abnormalities of placentas from kindling group were characterized by thickening of the villus vascular walls, luminal stenosis, trophoblasts hyperplasia, abnormalities of trophoblasts with nuclear pyknosis and karyorrhexis and accumulation of inflammatory lymphocytes in labyrinthine zone. 4. Nissl staining showed that neurons in hippocampus of P0 and P84 offspring from control and sham groups were normal while neuronal damages were obvious in hippocampus of P0 and P84 offspring from kindling groups, and the damages in P0 pups were severe with a marked loss of neuron, shrinkage of cells and nuclear pyknosis and karyorrhexis. 5. The level of GSH of kindling group was significantly lower compared to that of control group(P < 0.01) and the level of MDA was much higher than that of control group(P < 0.01). There was no difference between control and sham groups(P > 0.05). 6. In the Morris water maze, all rats showed a progressive decline in the escape latency with training. From the 3rd day to the 5th day, Rats in the kindling group exhibited significantly prolonged escape latency as compared to those in the control group(P<0.01). In the probe trial, the kindling group spent significantly less time in the target quadrant than the control group(P<0.01),and the number of crossing the platform obviously decreased as compared to the control group(P<0.01). In addition, there was no difference between control and sham groups(P >0.05).Conclusions : The results in this study demonstrated that prenatal maternal seizures induced obvious pathological damages of hippocampus and cognitive impairment of offspring. Hypoxia-ischemia of placenta and fetal oxidative stress might be contributing factors in this process. Part Ⅱ The neuroprotective effect of astaxanthin on amygdale-kindled ratsObjective: to evaluate the protective effect of Astaxanthin(ATX) on neuronal damage of hippocampus induced by seizures and to explore the underlying mechanisms including anti-oxidant and anti-apoptotic effects.Methods: Adult male Spraque-Dawley(SD) rats were randomly divided into four groups: control group, ATX group, kindling group and kindling+ATX group. Electrodes were implanted into all animals, but amygdala kindling procedure was performed in kindling and kindling+ATX groups. Electric stimulation was delivered to the rats in kindling group once each day for 15 days. ATX(30mg/kg/d) was injected intraperitoneally at 1hr before each electric stimulation. The mean seizure stage and the after discharge duration(ADD) in the course of kindling were detected. Nissal’s staining was used to detect the hippocampal neuron of rats. Oxidative stress parameters(MDA and GSH) in the hippocampus of rats were examined by spectrophotometry.Results: 1. There were significant differences in seizure stage between the kindling and kindling+ATX groups from the 12 th day to the 15 th day(P<0.05). Meanwhile, pretreatment with ATX markedly decreased the after discharge duration(ADD) compared with the kindling group from the 8th day to the 15 th day(P<0.05). No seizure activities were observed in the control and ATX groups. 2. Nissl staining showed that: neurons in hippocampus of control group were normal while in the kindled rats, neuron loss was obvious with shrunken plasma body and pyknotic nuclei. Treatment with ATX markedly ameliorated those neuronal damages. The hippocampus neurons of ATX group showed no significant change as compared to those of the control group. 3. Compared to the control group, the level of MDA in the kindling group was significantly higher(P<0.01), and the level of glutathione(GSH) was significantly lower(P<0.01). However, Treatment with ATX led to a significant decrease in the level of MDA(P<0.01) and a noticeable increase in GSH level(P<0.05) compared to the kindling group. Nevertheless, compared to the control group, in the kindling+ATX group the oxidative stress level was still higher(P < 0.05). No significant difference was found between the control and ATX per se groups(P > 0.05).Conclusions:The results in this part demonstrated that ATX can protect hippocampus neuron from being damaged by seizures and it is a safe drug. The neuroprotective mechanisms of ATX included attenuating the oxidative damage and partly ameliorating seizure. Therefore, these results provided valid evidence for the further study of ATX in prenatal maternal seizure. Part Ⅲ The neuroprotective effect of astaxanthin on newborn rats exposed to prenatal maternal seizureObjective: This study was aimed to investigate the protective effect of ATX on neuronal damage of new pups induced by maternal seizure throughout pregnancy. We also explore the underlying mechanisms by evaluating whether ATX can inhibit fetal oxidative stress, ameliorate pathological damages of placenta and influence CREB-BDNF pathway in this process.Methods: Adult female Spraque-Dawley(SD) rats were randomly divided into four groups: control group, ATX group, kindling group and kindling+ATX group. Electrodes were implanted into all animals, but amygdala kindling procedure was performed in kindling and kindling+ATX groups. The rats were kindled once by electric stimulation of the amygdale each day. All the rats were allowed to mate after one week’s fully kindling. ATX(30mg/kg/d) was injected intraperitoneally during pregnancy(GD1-GD20). The pregnant rats in kindling and kindling+ATX groups received electric stimulation every 48 hr. Dams were allowed to deliver naturally. Effects of maternal seizure on the mortality and body weight of the postnatal rats were observed. HE staining was used to visualize changes of maternal placenta. Using Nissal’s staining was used to detect hippocampal morphology of P0 pups. MDA and GSH in the hippocampus of P0 pups were examined by spectrophotometry. The expression of CERB and BDNF were determined using Western blot and real-time fluorescence quantitative polymerase chain reaction(PCR).Results: 1.The survival rate of pups in kindling group was much lower than that in the control group(P<0.01), but prenatal ATX treatment led to a significant increase(P<0.05). There was no difference in survival rate of pups between control and ATX groups(P > 0.05). The number of pups per litter and pups birth-weight in all groups showed no significant difference(P>0.05). 2. Findings from HE staining are as follows: Placental tissues from control were normal while the histologic abnormalities of placentas from kindling group were characterized by thickening of the villus vascular walls, luminal stenosis, trophoblasts hyperplasia, abnormalities of trophoblasts with nuclear pyknosis and karyorrhexis and accumulation of inflammatory lymphocytes in labyrinthine zone. But this situation got better when treated with ATX. No significant difference in the placental pathology was found between ATX only study group and the control group. 3. Nissl staining showed that hippocampal neurons of P0 pups in control group were normal. The damages in kindling group were severe, but prenatal ATX treatment markedly ameliorated those neuronal damages. The hippocampus neurons of ATX group showed no significant change compared to the control group. 4. In P0 pups of kindling group, the level of MDA was markedly higher than that in the control group(P<0.01), and the level of GSH was significantly lower than that in the control group(P<0.01). However, prenatal ATX treatment led to a significant decrease in MDA concentration(P<0.01) and a noticeable increase in GSH concentration(P<0.05) compared to the kindling group. ATX per se caused no change as compared to the control group(P>0.05). 5. Western blot analysis showed that: CREB and BDNF in the hippocampus of P0 pups were significantly lower in the kindling group as compared to the control group(P<0.01),but prenatal ATX administration led to significant increase in both CREB and BDNF levels(P<0.01)as compared to the kindling group.ATX per se caused no change comparing to the control group(P>0.05).6.real-time PCR showed that the levels of CREB and BDNF m RNA in the hippocampus of P0 pups were significantly declined in kindling group as compared to the control group(P<0.01).However,prenatal treatment with ATX led to a significant increase in CREB and BDNF m RNA levels as compared to the kindling group(P<0.01 for CREB,P<0.05 for BDNF).ATX per se caused no change as compared to the control group(P>0.05).Conclusions:These findings indicated a neuroprotective effect of ATX on offspring exposed to maternal seizures in utero and suggest that antioxidative effect, placental protection and activation of CREB-BDNF pathway are involved in this process. Part Ⅳ The protective effect of astaxanthin on cognitive deficits in adult offspring exposed to prenatal maternal seizureObjective: This study was aimed to evaluate the effect of prenatal ATX treatment on cognitive impairment and pathological damages of hippocampus in adult offspring induced by maternal seizure throughout pregnancy. We also explore the underlying mechanisms by investigating whether ATX can long-term influence CREB-BDNF pathway in this process.Methods: Adult female Spraque-Dawley(SD) rats were randomly divided into four groups: control group, ATX group, kindling group and kindling+ATX group. Electrodes were implanted into all animals, but amygdala kindling procedure was performed in kindling and kindling+ATX groups. The rats were kindled once by electric stimulation of the amygdale each day. All the rats were allowed to mate after one week’s fully kindling. ATX(30mg/kg/d) was injected intraperitoneally during pregnancy(GD1-GD20). The pregnant rats in kindling and kindling+ATX groups received electric stimulation every 48 hr. Dams were allowed to deliver naturally. The offspring were allowed to grow to 12 weeks of age(adulthood). Using Nissal’s staining and transmission electron microscope, we investigated hippocampal morphology of adult offspring. Morris water maze test was used to assess the cognitive function of adult offspring. The expression of CERB and BDNF of hippocampus were determined using Western blot and real-time fluorescence quantitative polymerase chain reaction(PCR).Results: 1. In the Morris water maze, all rats showed a progressive decline in the escape latency with training. From the 3rd day to the 5th day, rats in the kindling group exhibited significantly prolonged escape latency as compared to the control group(P<0.01). However, the poor performance was rescued by prenatal treatment with ATX(P<0.05 on the 3rd day, P<0.01 on the the 4th day and the 5th day). In the probe trial, the kindling group spent significantly less time in the target quadrant than the control group(P<0.01), and the number of crossing the platform obviously decreased as compared to the control group(P<0.01). However, prenatal treatment with ATX markedly improved time in the target quadrant and the number of crossing the platform(P<0.01). In addition, there was no difference between control and sham groups(P>0.05). 2. Nissl staining showed that hippocampal neurons of adult offspring in control group were normal. The damages in kindling group were obvious, but not severe and those neuronal damages were ameliorated by prenatal ATX treatment. The hippocampus neurons of ATX group showed no significant change as compared to the control group. 3. Hippocampal CA1 area of adult offspring under electron microscope: there were normal neurons and synapses in control and ATX groups. In kindling group,ultrastructure of neurons and synapses represent obvious damage, but they were improved in kindling+ATX group.4.Western blot analysis showed that CREB and BDNF in the hippocampus of adult offspring were significantly lower in the kindling group as compared to the control group(P<0.01), but prenatal ATX administration led to significant increase in both CREB and BDNF levels(P<0.01). ATX per se caused no change compared to the control group(P>0.05). 5. real-time PCR showed that the levels of CREB and BDNF m RNA in the hippocampus of adult offspring declined significantly in kindling group as compared to the control group(P<0.01), but prenatal treatment with ATX led to a significant increase in CREB and BDNF m RNA levels(P<0.01). ATX per se caused no change compared to the control group(P>0.05).Conclusions:These findings demonstrated that prenatal treatment with ATX can ameliorate the cognitive impairment and pathological damages of hippocampus in adult offspring induced by maternal seizures in utero, and its protective effects were partly due to long-term up-regulation of CREB-BDNF pathways. Our results further supported the idea that ATX might serve as a novel, clinically useful nutrient for preventing long-term brain damage of offspring induced by maternal seizures.