The Animal Model Of Malformation Of Cortical Development Induced By Methylazoxymethanol Acetate

Background: Malformation of cortical development(MCD)is a blanket terms,it contains a broad spectrum diseases attribute to the changes in genetic and environmental condition which can interference the normal process of nerve cells proliferation,migration or differentiation.The clinical manifestation of patients with MCD is varying,ranging from early,severe neurological impairment to epilepsy or unexpected deficits which should be detectable by screening.The rapid evolution of molecular biology,genetics and imaging not only has enriched the knowledge about the normal development of the cerebral cortex but also expanded its abnormal types.A large number of clinical and basic research data is accumulating and many hypotheses are putting forward.Due to the limitations in clinical and pathologic specimen research,appropriate animal model always plays an important role in verifying the clinical and basic research observations and makes great contribution to translational medicine.The offspring of the prenatal rats which were accepted methylazoxymethanol acetate(MAM)treatment with pathological,molecular and behavioral changes which is similar to patients with MCD is considered promising and popular.Glucocorticoid receptor(GR)and mineralocorticoid receptor(MR)are members of the nuclear receptor superfamily,they can regulate cell proliferation,differentiation and apoptosis by regulating gene transcription,their corporate ligand is glucocorticoid,they play an important role in maintaining the function of Hypothalamic-Pituitary-Adrenal axis(HPA),it has been confirmed that a variety of diseases are associated with their abnormality.Epilepsy is one of the common diseases in nervous system,the occurrence of epilepsy is closely related to HPA axis,MCD is an important cause of epilepsy,especially refractory epilepsy,the curative effect of glucocorticoid which plays a role when it combines with its receptor is poor,abnormality in receptor may be possible cause.In view of the important role of GR and MR,we hypothesize that GR and MR may be involved in the formation and epileptogenesis of MCD.Objective: To establish a new animal model of MCD which is induced by MAM,investigate the relationship between the extent of histopathology and the age as well as the epilepsy seizure susceptibility and the effect of epilepsy seizure on the MCD histopathology,then investigate the roles of GR and MR in the formation and epileptogenesis of MCD.Methods: Pregant Sprague-Dawley rats received 2 MAM intraperitonesl on embryonic day 15 to establish the MCD model,12 hours apart between the two operations.The control group was treated with normal saline.Observe daily activities everyday and analysis the EEG of the next generation rats on postnatal day 90.Thioine-Giemsa staining was applied to observe the histological changes in the cortex and hippocampus of the P0,7,14,28,60,90 rats;immunofluorescence staining of Neu N was used to reveal the distribution of neuron in the cortex and hippocampus.Pilocarine was administrated to establish epilepsy animal model and investigate the effect of epilepsy seizure on the existing pathological morphology.Real Time-PCR(RT-PCR)and Western Blot were used to detect the m RNA expression and protein expression of GR and MR in the 2 month old rats,cerebral cortex respectively.Results: The MAM group rats are dull and curtail their activities.The MAM group rats,size of cerebral cortex are lesser than normal counterpart,their superior and inferior colliculi are not covered by the occipital cortex completely.The MAM group rats,brain weight were lighter than the age matched control group.The histopathological and immunofluorescence staining results reveal that the MAM group rats,cerebral cortex decrease and the laminar organization disrupt;there are heterotopic neurons,hypertrophic neurons and proliferation nodules in the hippocampal and cortex;the pathology exacerbate with age.The MAM group rats,seizure susceptibility elevates,a low-does pilocarine can induce them appearing generalized tonic-clonic seizure(GTCS),namely ?-? grade seizure,even status epilepticus,following mildly spontaneous behavioural seizures once in a while.The hypertrophic/dysmorphic pyramidal neurons are observed in the MAM-PILO group rats by Thioine-Giemsa staining.The degree of malformation of cortical development in the MAM-PILO group rats is more serious than the MAM group rats.RT-PCR showed the MAM group rats,relative m RNA expression of GR are significantly lower than the normal counterpart(p<0.05),but the relative m RNA expression of MR don,t show significant difference.Western Blot result is in accordance with the RT-PCR,it showed the MAM group rats,relative protein expression of GR are significantly lower than the normal counterpart(p<0.05),but the protein expression of MR don,t show significant difference.Conclusion: The rat model of MCD can be established by 2 MAM intraperitoneal on embryonic day 15,its pathological features resemble the patient with MCD and its pathology exacerbate with age,it may be a suitable model for MCD.The MAM group rats,seizure susceptibility elevates,epilepsy seizure can aggravate the pathology of MCD.Combining utilization MAM and pilocarine could be a good approach to establish animal model of epilepsy patient who is based on MCD.The downregulation of GR or the inbalance between GR and MR may be connected with the formation of pathological morphology and elevatory seizure susceptibility in the MAM group rats.