Effects Of Ketamine On Autophagy And Apoptosis Of Fetal Rat Nerve Cells And Ability Of Learning And Memory In Young Offspring Rat

Anesthesia is one of the necessary measures in animals’ clinical diagnosis and treatment of animals.The clinical anesthesia research mainly focuses on the anesthetic effects.In safety evaluation,more emphasis is placed on the safety effect of drug does while ignore the toxic effects on the narcotic itself.Mutiple narcotics can pass through the blood-brain barrier and placental barrier,while exerting anesthetic action,narcotic also has other effects.Such as,the damage to nerve cells and the decline of learning and memory ability have become popular issues in anesthesia research.As a narcotic analgesics commonly used clinically,ketamine is currently the commonly used drug in veterinary clinics,such as caesarean section.However,its complex neurotoxic effects were still unclear.Pregnancy is a crucial period for the growth and development of embryos,vulnerable to external stimuli.If narcotic is given at this time,it is bound to cause harm to the embryo.Based on this,it has real needs and practical significance to study the toxic side effects of ketamine on the nervous system.This study can also help exploring the action mechanism,providing data basis for the rational application and guiding clinical rational drug use.In this experiment,ketamine was injected into rats on the 19 th day of pregnancy.We detected protein levels of autophagy and apoptosis related genes such as LC3,C-Caspase-3(Cleaved-Caspase-3),Beclin-1,Atg5,Bax,Bcl-2,Atg4 and p62.At the same time,ketamine was used in vitro cultured PC12 to detect protein levels of autophagy and apoptosis related proteins.Also,we observed changes of autophagic flow by using mRFP-EGFP-LC3 dual-fluorescent autophagy indicator system.To investigate the long-term effects of ketamine on young offspring,behavioral tests were performed on days 25-30 after the birth of the young offspring.The levels of CaMKII,p-CaMKII,CaMKIV,p-CaMKIV,ERK,p-ERK,PKA,CREB,p-CREB,p-PKA,and BDNF were measured to reveal the mechanism of neurotoxicity of ketamine on the brain.The results found:(1)Ketamine significantly up-regulated the protein expressions of C-Caspase-3,Beclin1,Bax,Atg5 and LC3-II in fetal rat hippocampus,while downregulating the expressions of Bcl-2,Atg4,P62,and LC3-I.Indicated Ketamine cause autophagy and apoptosis in rat hippocampus.(2)Ketamine significantly down-regulated the ability of T-AOC and up-regulate the contents of ROS and MDA,suggesting that ketamine cause oxidative stress in fetal rathippocampus.(3)The experiments in vitro were consistent with experiments in vivo,and ketamine could induce autophagy in PC12 in a dose-dependent manner.(4)Autophagy inhibitor Nac can alleviate the occurrence of autophagy in nerve cells caused by ketamine,suggesting Nac can be used to antagonize the side effects of ketamine in the future.(5)Ketamine can lead to a decrease in the number of neurons in the hippocampal CA1 and CA3 regions of the offspring,and it can lead to a decrease in the number of neuronal synaptic spines.(6)Ketamine can affect the spatial and conditional learning and memory ability of offspring.(7)Ketamine can significantly reduce the protein levels of ERK,p-ERK,PKA,p-PKA,p-CREB and BDNF in hippocampus of offspring,and increase the protein level of CREB.(8)The PKA inhibitor H89 and ERK inhibitor SCH772984 were used in PC12 cells.The results showed ketamine significantly decreased the protein levels of ERK,p-ERK,PKA,p-PKA,CREB,p-CREB,and BDNF.The inhibition of ERK or PKA alone did not result in a change in the protein level of CREB or p-CREB,but resulting in a significant decrease in the content of p-CREB when ERK and PKA inhibited combined.These results showed,autophagy and apoptosis happened in ketamine treated fetal rat hippocampal and PC12.Moreover,the apoptosis was regulated by autophagy.Ketamine induced the accumulation of ROS in fetal rat hippocampal and PC12,which may the direct cause of autophagy and apoptosis.The neurotoxicity caused by ketamine during pregnancy affects the learning and memory ability of offspring rats in terms of space and conditions.This decline in learning and memory may be due to the reducing neuronal and synaptic spine density by ketamine.At the molecular level,ketamine may affect the learning and memory ability of offspring by inhibiting the CREB pathway.In addition,in ketamine exposed PC12 cells,ERK and PKA have greetings effects on the regulation of CREB.It is recommended that the use of ketamine should be reduced or avoided in the future for pregnant,caesarean animals and newborns who need surgery.