The Melecular Mechanis Of Ketamine Effect On Early Embryo

Objective:Ketamine is a nonspecific antagonist of N-methyl-aspartate (NMDA) receptor, which is commonly used in clinical anesthesia and is a new popular drug around the world. There are many researches about the abuse of ketamine on early embryonic development. It is belived that ketamine works as a NMDA receptor antagonist. NMDA receptor, a kind of ligand gating of Ca2+ highly transparent ion channels, plays an important role in the development of the nervous system, neuron survival and synaptic plasticity. Since the melecular mechanism of ketamine effect on early embryo remains unknown, interventions to avoid birth defects caused by ketamine abuse are still not efficacious. Our research will be helpful to understand the mechanisms of the drug toxic effect, evaluate the potential risks in clinical anesthesia and provide inteventions for birth defects caused by drug abuse.Materials and methods:Embryo in situ hybridization, embryonic microinjection, RT-PCR, dual luciferase assay, molecular cloning, plasmid construction, cell culture and transfection, electrophoretic mobility shift assay.Results:1. Ketamine exposure in embryo caused a series of developmental defects such as pigment cells reduction, bulky bowel, intestinal neuron loss, eyes dysplasia and craniofacial cartilage shrink or disappear.2. Ketamine can inhibit the expression of Zic5 gene in the neural crest area.3. Ketamine decreased the expression of downstream genes in Notch signaling pathway.4. NICD, Notch intracellular domain, can obviously enhance zic5 gene expression.5. Ten plasmids were constructed with different fragments of Zic5 gene promoter regions. Luciferase assay showed NICD enhanced-200/-29 with a higher promoter activity than-186/-29. It was estimated that the binding site of Zic5 gene promoter with NICD was in-186/-200.6. Electrophoresis mobility shift assay showed that Zic5-200/-186 probe can bond with the total protein of SY5Y cell transfected with NICD.Conclusion:1. Ketamine causes development defects by interfering with the embryo neural crest development.2. Zic5 is the molecular targets of ketamine on early embryonic neural crest.3. NICD can up regulate the gene expression and promoter activity of Zic5 through combination with-200/-186.4. Notch signaling pathway plays a significant role in ketamine interference on neural crest development.