| Objective: To investigate the mechanism underlying disorders of cortical development (DCDs), We developed an animal model of DCDs induced by Carmustine and explored the optimal dose and methodology of established animal model of DCDs.Methods:1.The establishment and assessment of rat model of DCDs induced by Carmustine: Pregnant Sprague-Dawley rats were given intraperitoneal injection of carmustine with different doses on embryonic day 17. The rats in model one were given injection of carmustine with 5mg/kg. The rats in model two were given injection of carmustine with 10mg/kg. The rats in model three were given injection of carmustine with 15mg/kg. The rats in model four were given injection of carmustine with 20mg/kg. BCNU exposure in utero produces histologic alterations suggestive of DCDs in rat offspring. The control group were untreated.The following aspects were observed in all groups. We calculated the survival rate of rat offspring in each group and observed their general state on birth and the postnatal daily behaviors including consciousness, viability and epileptic seizure.The body weight reflecting developmental condition of rat offspring were measured at P0, P7, P21, P56 and P84. we adopted Morris-water-maze experiment to test the study and memory capacity of rat offspring in each group and used Kainic acid inducing seizure to compare their seizure threshold and mortality induced by seizure. After measureing the wet weight of brains and observing their appearance, we examined the changes of brain pathology in cerebral cortex and hippocampus to confirm the typies of DCDs at P21 and P84.we assessed that we could or not establish the animal model of DCDs induced by Carmustine successfully by above aspects.we also judged the best drug dose of successful model and evaluated the credibility of methodology in the animal model of DCDs.2.Detection of related neurotransmitters: To explore the seizure mechanism underlying DCDs, immunohistochemistry method was used to detect the neuronal distribution of neuropeptide Y and Calretinin in brains of rat offspring of optimal dose model.Results:1.The survival rate of rat offspring in models group and control group: The survival rates of rat offspring in control group, model one, model two, model three and model four were 100%, 100%, 93.1%, 83.3% and 0% respectively.The all rat offspring in model four were death at P0.2.The results of general ethology observation: The observation results of rat offspring in model one were similar with those of control group. No abnormity existed in model two at the process of growth and development. The rat offspring in model three were poor than control group on birth, followed by less activities, dullness and disturbance of intelligence in daily life. No obviously seizure was observed in all model groups.3.Condition of growth and development in rat offspring: The rat offspring in control group and model one were found to have no difference in body weight (P>0.05). The mean weight of rat offspring in model two was reduced slightly at P0 (P<0.05), but the difference of weight was disappear when they were adult. The rat offspring in model three indicated an overall reduction in mean weight at every time point, the phenomenon was significantly with age (P<0.05).4.Morris-water-maze experiment: The rat offspring in model one did not showed any disparity with control group on the escape latency and times of crossing platform (P>0.05).The escape latency of rat offspring in model two was longer than that of control group at first day and second day, but the difference was disappear at third day and fourth day, the times of crossing platform of rat offspring in model two was equal with that of control group (P>0.05). In the training period escape latency of rat offspring in model three was always longer than that of control group (P<0.05). After removing platform on fifth day, the times of crossing platform of rat offspring in model three was not so many as that of control group (P<0.05).5.Kainic acid inducing seizure experiment: There was no difference between model one and control group on latent period and time duration of kainic acid inducing seizure and mortality induced by seizure of rat offspring (P>0.05). The latent period of kainic acid inducing seizure was shortened in model two (P<0.05), but the time of epileptic state and the mortality induced by seizure of rat offspring in model two were as same as that of control group (P>0.05).The rat offspring in model three had a significantly lower seizure threshold and longer time of epileptic state than those of control group, the mortality induced by seizure of rat offspring was obviously higher than that of control group (P<0.05).6.Comparison of cerebral appearance and brain weight and pathological examination of brains: The disparation of cerebral appearance and brain weight were unexist between model one and control group, there was no pathologic change in model one. Rat offspring in model two have no significant change in weight and construction of brain, the incidence rate of DCDs was 41.67%. Reduced weight and abnormal construction of brain were displaied in model three, the difference was increased with age. The incidence rate of DCDs was 100%. Typical pathological features included that model rats showed a thin cortical plate and disorder of layer structure, nerve cells in cerebral cortex were absence and dysplasia, distinct clusters of neuronal elements that represent heterotopias emerged in cerebral cortex and hippocampus. These morphologic features of DCDs found in this model were shared with cortical dysplasia and neuronal heterotopias in humans.7.Detection of neurotransmitters: The results demonstrated that neuronal numbers of neuropeptide Y were decreased obviously in cerebral cortex and hippocampus of rat offspring in model three (P<0.05). The neuronal architecture and expression of calretinin were abnormal in rat offspring in model three.Conclusions:1.The method that pregnant SD rats are given intraperitoneal injection of carmustine with 15mg/kg on embryonic day 17 can establish an ideal model of DCDs in offspring, But carmustine treatment with 5mg/kg or 10mg/kg or 20mg/kg to pregnant rats can not establish successfully the model of DCDs in offspring.2.Rat offspring in the model show poor growth and development, abnormal behaviors, disorders of study and memory, lower threshold of epileptic seizure and have typically pathologic changes of DCDs, these commendably reveal the features of ethology and pathology in humans.3.The neuronic abnormity of neuropeptide Y and calretinin in the brain of model rat offspring provides theoretical clew for the mechanism of epileptic seizure induced by DCDs.4.DCDs of rat offspring in the model can simulate cortical dysplasia, neuronal heterotopia that is similar in humans. The rat offspring in our model have higher survival rate than that of rat offspring in mode that carmustine treatment with 20mg/kg of pregnant rats on embryonic day 15 in abroad. The model has better repeatability and being a new, pragmatic animal model of DCDs.
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