The Regulationl Of Propofol On The Neural Stem Cells In Hippocampal Dentate Gyrus In Developing Mice And The Underlying Mechanism

Background:Every year, millions of newborns are treated with anaesthetic agents for surgery and clinical check. However, growing and convincing preclinical evidence in rodents and primates suggest that exposure to anaesthetics in common clinical use can be neurotoxic to the developing brain, thus leading to the long-term neurological disfunction. Epidemiological study showed that children under 4 years old get increased risk of cognition disorder after repeated propofol anaesthesia. These findings have seriously questioned the safe use of general anaesthetics in obstetric and paediatric patients.General anaesthetics can be divided into inhalational anaesthetics and intravenous anaesthetics. They act by affecting NMDA and(or) GABA receptors. Previous animal research indicate that inhibiting NMDA receptor and activating GABA receptor may be toxic to the developing brain. Propofol, the typical intravenous general anaesthetic by activating GABA receptors, is widespread in obstetric and paediatric usage for little toxicity, rapid onset, quick metabolism and no accumulation. Currently, there is in dispute about the long-term effect of propofol could cause cognition disfunction, but in vivo study have found that propofol can induce apoptosis of developing neurons, repeated propofol treatment increased the number of apoptotic neurons, thus leading to cognition disorder. Up to now, it is still unclear the mechanism of the influences of propofol on the study and memory, especially its effect on the function formation of developing brain and long-term study and memory.Hippocampus is an important area related with cognition, study and memory, as well as one of places that neurogenesis continously occurs after birth in mammalians and humans. Researches showed close relation between neurogenesis in hippocampal subgranual zone and brain function development and long-term behaviors. During development, hippocampal neural stem cells maintain neurogenesis by self-proliferation. At the same time, their processes provide scaffold for the migration and mature of neurons. Factors regulating hippocampal neural stem cells might be the key target of long-term function of brain.Researches showed that cognition disfunction caused by anaesthetics neurotoxicity is i n close relation with the period of drug exposure. Neural apoptosis induced by anaesthetics usually occurs at 2 week after birth and reaches the peak at 7 day after birth. Previous resear ch showed that propofol can lead to neural apoptosis both at single dosage injection and rep eative injection. Recent study found that propofol might regulate hippocampal neurogenesis by damaging the survival and mature of hippocampal newborn neurons. In addition, propof ol significantly decreased the differentiation of neurons in hippocampal dentate gyrus in 3-month-old rats and increased the number of a-strocytes, however, in vitro study showed pro pofol at clinical dosage can promote the neural stem cells differentiate into various type of neurons. It seems that the different influence of propofol depends on the different drug dosage and the period of propofol treatment. Thus, it still needs further confirm whether propofol affect hippocampal function formation in mice at 7days after birth, when brain development is at the peak. It is unclear the relation between the effect of propofol on cognition function and the regulation of hippocampal neural stem cells in dentate gyrus. Explaining the effect and mechanism of propofol on neural stem cells in developing hippocampi will provide new knowledge of cognition disorder occured at neonates after propofol treatment, and provide a novel intervention for clinical diagnosis, treatment and prevention in obsterics and paediatrics.Methods:First of all, we injected single propofol at the dosage of 30mg/kg, 60 mg/kg or the same volume of intralipid as control in mice at 7 day after birth. By immunochemistry staining specific markers of proliferation such as BrdU, Sox2 and neural stem cells markers Nestin, BLBP, their expression were compared and analyzed to study the effect of propofol on hippocampal neural stem cells in developing dentate gyrus. In addition, GFAP and Iba1 were used to evaluate the influence of propofol on neural glial cells in hippocampal dentate gyrus. Western blot was conducted to study the expression of Sox2 for further confirm of effect of propofol on neural stem cell proliferation, and Akt, pAkt, Erk1/2, pErk1/2 were evaluated to study the mechanism of propofol on hippocampal neural stem cells.Results:1.Detect the effect of propofol on the proliferation of stem cells in P7 mice hippocampal dentate gyrus by antigen-specific cell proliferation marker Brdu and Sox2.Immunohistochemical results indicate that there were no difference in BrdU-positive cells 、Sox2-positive cells in the DG between pups treated with 30 mg/kg propofol and pups treated with vehicle. However, pups treated with60 mg/kg propofol had decreased BrdU-positive cells、Sox2-positive cells inthe DG compared to pups treated with vehicle and treated with 30 mg/kg propofol.2.Detect the effect of propofol administration during early postnatal life on mice hippocampal dentate gyrus stem cell morphology changes by early antigen- specific neural stem cell markers Nestin and BLBP.Immunofluorescence results found that pups treated with60 mg/kg propofol had decreased the number of neuron stem cells and destroy the prominences. Which indicate high dose propofol administration would damage the morphology of neuron stem cell in the mice hippocampus during early postnatal life.3.Immunofluorescence results indicate there were no difference in GFAP-positive cells in the DG between pups treated with 30 mg/kg propofol and pups treated with vehicle. However, pups treated with60 mg/kg propofol had decreased GFAP-positive cells in the DG compared to pups treated with vehicle. Besides,the immunofluorescence results of antigen- specific microglial cell marker Iba-1 indicate both of pups treated with 30 mg/kg propofol and 60 mg/kg propofol decrease the number of Iba-1 positive cells in the DG compared to pups treated with vehicle.4.Western blots revealed that Sox2 protein levels were lower in the hippocampi lysates from mice treated with 60 mg/kg propofol compared with vehicle treatment.Besides,there was significant change between mice treated with 60 mg/kg propofol and mice treated with 30 mg/kg propofol.5.Western blots revealed that the levels of pAkt and pErk were considerably lower in the hippocampi of newborn mice following propofol treatment at a dose of 30 mg/kg,and further reduction was induced at a dose of 60 mg/kg. The statistical analysis revealed that the propofol treatment(30 or 60 mg/kg) decreased the pAkt expression level by26 or 49 % compared with that of the vehicle-treated group, respectively, and propofol treatment(30 or 60 mg/kg) decreased the pERK immunoreactivity by 43 or 61 % compared with those of the vehicle treated control group.But the levels of Akt and Erk were no significant change.Conclusions:1. Propofol inhibited stem cells proliferationin the DG of newborn mice in a dose-dependent manner.2.Propofol decreased the stem cells and changed the morphology of the neural stem cells in the DG of newborn mice.3.Propofol inhibited the activation of astrocyte and microglial cells in the DG of newborn mice in a dose-dependent manner.4.Propofol inhibited the AKT and ERK pathways activation in the DG of newborn mice.