| BackgroundAnimal studies showed almost all the general anesthetics induced neurodegeneration and abnormal neurogenesis in critical period of the cerebral development which lead to changed behavior and cognitive dysfunction.Propofol played a role as a new general anesthetic by activating gamma amino acid butyric acid receptor(GABAR)or inhibiting N-methyl-D aspartate receptor(NMDAR).It was widely used in anesthesia induction,maintaining and ICU sedation for its advantage as rapid onset,quickly awake and perfect function recovery.Propofol was also found to be neurotoxic that propofol exposure in early time suppressed neurogenesis,induced neuroapoptosis,and it was reported that propofol could lead to learning and memory impairment in the later period.Anesthesia neurotoxicity in the special group of children had drawn great attention that it was extremely urgent to study its pathogenic mechanism and develop prevention measures.Cerebellum located beneath the back of the cerebrum,cover upon the pons and medulla,which accounts for most of the afterbrain.Cerebellum had lamellar structure as the brain,which was consisted of molecular layer(ML),Purkinje cell layer(PCL),and granule cell layer(GCL).Neuronal differentiation,morphogenesis and cell migration were precisely controlled and then cerebellar lamellar structure and neural circuit were formed.Cerebellar function depended on its correct neurons contact and integration of the cerebellar afferent and efferent fibers.Except for motor function,cerebellum had various non-motor functions and participated in cognition,emotion and execution process.Cerebellar nucleus received and integrated afferent message from Purkinje cells and sent efferent fibers projected to cortex,subcortex,thalamus,brainstem and hypothalamus.Through these contacts,cerebellum could regulate neurogenesis and function formation in developmental critical period of other brain regions.At the same time,cerebellar injure,especially congenital or damage in childhood,would induce neurodevelopmental disorders such as autism,attention deficit hyperactivity disorder and developmental dyslexia.Recentstudy showed that propofol could suppress neuronal activity of Purkinje cells and affect the cerebellar neural circuit,which highly suggested cerebellum as the action target of the propofol neurotoxicity.As intravenous general anesthetic,propofol caused the stress response and affected the stability of the neuroendocrine environment through the hypothalamic pituitary gonadal axis(HPA).Propofol acted on the hypothalamus immediately that lead to change of relative neural nucleus activation.What's more,disturbance of internal environment could influence the neurogenesis,synapse formation,and neural network establishment during the developmental period of the central nervous system(CNS),and that might increase the neurotoxic vulnerability of the cerebrum.Exploring the effect of propofol on cerebellum and hypothalamus to find more effect target and further study the mechanism of propofol neurotoxicity would provide new strategies and theoretical basis for the anesthesia scheme and help to prevent the neurotoxicity.MethodNeonatal mouse at postnatal day 7(P7)were treated with intraperitoneal propofol injection of 30mg/kg or 60mg/kg,and the control group received intralipid injection of the equal volume as the vehicle.To explore the effect of propofol on cerebellar development,pups were executed at P8 and cerebellum was harvested to observed the Purkinje cell,Bergmann glia,granule neurons and the morphological changes.To explore if propofol would influence the migration of granule neurons,pups received 5-Bromo-2'-deoxyuridine(BrdU)injection to mark the newborn neurons and cerebellum was harvested at P10 or P14.Distribution of the marked newborn neurons in external granule layer(EGL),molecular layer(ML)and internal granule layer(IGL)was used to evaluate migration process of the granule neurons in the cerebellum.We detected the expression of Notch signaling pathway in P8 cerebellum with western blot to further study the possible mechanism.At the same time,we explored the influence of propofol on hypothalamus.C-fos was used to find the activated area of hypothalamus.Expression of arginine vasopressin(AVP)and glucocorticoid receptor(GR)was detected to observe the stress response and change of endocrine.Activation level of microglia in hypothalamus was also observed to confirm the propofol effect.Results1?Effect of propofol on neonatal mouse cerebellar development(1)Hematoxylin-eosin(HE)staining of P8 mouse cerebellum showed there was no difference of cerebellar sagittal section area and EGL thickness in lobe IX between the vehicle and propofol-treatment group;(2)CB immunofluorescence demonstrated that compared to the vehicle-treatment group,Purkinje cell number was decreased in 60mg/kg propofol-treatment group(P<0.05)and the length of Purkinje dendrite was shortened(P<0.05).There was no difference between the vehicle and 30mg/kg group;(3)Neu N immunofluorescence showed there was no difference of the granule neurons in IGL between the vehicle and both the 30mg/kg and 60 mg/kg propofol-treatment group;(4)BLBP positive fibers was significantly decreased in both 30mg/kg and 60mg/kg propofol-treatment group compared to the vehicle-treatment group(P<0.01,P<0.01).GFAP positive fibers was decreased in 60mg/kg propofol-treatment group compared to the vehicle-treatment group(P<0.05)and the astrocyte in the white matter was increased(P<0.05);(5)Co-stain of CB and GFAP showed the contacted points between Purkinje dendrite and Bergmann fibers was significantly decreased in 60mg/kg propofol-treatment group compared to the vehicle-treatment group(P<0.01);(6)HE staining of P10 mouse cerebellum showed there was no difference of cerebellar sagittal section area in each group,while EGL thickness of lobe IX in both 30mg/kg and60mg/kg propofol-treatment group was thicker than the vehicle treated group(P<0.01,P<0.01);(7)BrdU immunofluorescence showed at P10 percentage of newborn granule cells in EGL was increased(P<0.01)in 60mg/kg propofol-treatment group compared to the vehicle-treatment group and was decreased in IGL(P<0.05).There was no difference between the vehicle and 30mg/kg propofol-treatment group.While in the P14 cerebellum there was no difference in EGL,ML and IGL between the vehicle and both 30mg/kg and60mg/kg propofol-treatment group;(8)Western blot of P8 cerebellum showed expression of Jagged1 protein was down-regulated in both 30mg/kg and 60mg/kg propofol-treatment group compared to thevehicle-treatment group(P<0.01,P<0.01).Notch1 protein was decreased(P<0.05,P<0.01)both compared to the vehicle and expression of Notch1 was lower in 60mg/kg propofol-treatment group compared to 30mg/kg(P<0.01).2?Effect of propofol on neonatal mouse hypothalamus(1)C-Fos staining showed number of activated neurons in paraventricular nucleus(PVN)was increased in both 30mg/kg and 60mg/kg propofol-treatment group(P<0.05,P<0.001)compared to the vehicle-treatment group and was higher in 60mg/kg than30mg/kg propofol-treatment group(P<0.01);(2)AVP positive neurons in PVN was significantly increased in 60mg/kg propofol-treatment group compared to the vehicle-treatment group(P<0.01),and the expression of AVP protein was up-regulated compared to both the vehicle and 30mg/kg propofol-treatment group(P<0.01,P<0.05).There was no difference between the vehicle and 30mg/kg propofol-treatment group;(3)GR positive cells in PVN was significantly increased in 60mg/kg propofol-treatment group compared to both the vehicle and 30mg/kg propofol-treatment group(P<0.001,P<0.05).Expression of GR protein was up-regulated in both 30mg/kg and60mg/kg propofol-treatment group compared to the vehicle-treatment group(P<0.05,P<0.01)and it was higher in the 60mg/kg propofol-treatment group than 30mg/kg(P<0.05);(4)Iba1 staining showed microglia in both 30mg/kg and 60mg/kg propofol-treatment group was decreased in dorsal medial hypothalamic area(DMHA)(P<0.05,P<0.01),ventral medial hypothalamic area(VMHA)(P<0.05,P<0.001)and lateral hypothalamic area(LHA)(P<0.01,P<0.01)compared to the vehicle-treatment group.ConclusionsResults above suggested that propofol exposure in early life influenced the cerebellar neurogenesis and transiently suppressed the migration of the granule neurons.By the way,propofol affected the hypothalamus,active relative neurons in PVN and lead to stress response and endocrine change. |
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