Intranasal Administration of Insulin Reduces Chronic Behavioral Abnormality and Neuronal Apoptosis Induced by General Anesthesia in Neonatal Mice.
ABSTRACT: Children, after multiple exposures to general anesthesia, appear to be at an increased risk of developing learning disabilities. Almost all general anesthetics-including sevoflurane, which is commonly used for children-are potentially neurotoxic to the developing brain. Anesthesia exposure during development might also be associated with behavioral deficiencies later in life. To date, there is no treatment to prevent anesthesia-induced neurotoxicity and behavioral changes. In this study, we anesthetized 7-day-old neonatal mice with sevoflurane for 3 h per day for three consecutive days and found that the anesthesia led to mild behavioral abnormalities later in life that were detectable by using the novel object recognition test, Morris water maze, and fear conditioning test. Biochemical and immunohistochemical studies indicate that anesthesia induced a decrease in brain levels of postsynaptic density 95 (PSD95), a postsynaptic marker, and marked activation of neuronal apoptosis in neonatal mice. Importantly, insulin administered through intranasal delivery prior to anesthesia was found to prevent the anesthesia-induced long-term behavioral abnormalities, reduction of PSD95, and activation of neuronal apoptosis. These findings suggest that intranasal insulin administration could be an effective approach to prevent the increased risk of neurotoxicity and chronic damage caused by anesthesia in the developing brain.
Project description:Children with multiple exposures to anesthesia and surgery may have an increased risk of developing cognitive impairment. Sevoflurane, a commonly used anesthetic in children, has been reported to decrease levels of postsynaptic density 95 protein. However, the upstream mechanisms and downstream consequences of the sevoflurane-induced reduction in postsynaptic density 95 protein levels remains largely unknown. We therefore set out to assess whether sevoflurane acts on ubiquitination-proteasome pathway to facilitate postsynaptic density 95 protein degradation.Six-day-old wild-type mice received anesthesia with 3% sevoflurane 2?h daily for 3 days starting on postnatal day 6. We determined the effects of the sevoflurane anesthesia on mRNA, protein and ubiquitinated levels of postsynaptic density 95 protein in neurons, and synaptosomes and hippocampus of young mice. Cognitive function in the mice was determined at postnatal day 31 by using a Morris water maze. Proteasome inhibitor MG132 and E3 ligase mouse double mutant 2 homolog inhibitor Nutlin-3 were used for the interaction studies.The sevoflurane anesthesia decreased protein, but not mRNA, levels of postsynaptic density 95, and reduced ubiquitinated postsynaptic density 95 protein levels in neurons, synaptosomes, and hippocampus of young mice. Both MG132 and Nutlin-3 blocked these sevoflurane-induced effects. Sevoflurane promoted the interaction of mouse double mutant 2 homolog and postsynaptic density 95 protein in neurons. Finally, MG132 and Nutlin-3 ameliorated the sevoflurane-induced cognitive impairment in the mice.These data suggest that sevoflurane acts on the ubiquitination-proteasome pathway to facilitate postsynaptic density 95 protein degradation, which then decreases postsynaptic density 95 protein levels, leading to cognitive impairment in young mice. These studies would further promote the mechanistic investigation of anesthesia neurotoxicity in the developing brain.
Project description:Recent studies have suggested that children undergoing surgery under anesthesia could be at an increased risk for the development of learning disabilities, but whether anesthetics contribute to this learning disability is unclear. Therefore, the authors set out to assess the effects of sevoflurane, the most commonly used inhalation anesthetic, on caspase activation, apoptosis, beta-amyloid protein levels, and neuroinflammation in the brain tissues of neonatal naïve and Alzheimer disease (AD) transgenic mice.Six-day-old naïve and AD transgenic (B6.Cg-Tg[amyloid precursor protein swe, PSEN1dE9]85Dbo/J) mice were treated with sevoflurane. The mice were killed at the end of the anesthesia, and the brain tissues were harvested and then subjected to Western blot, immunocytochemistry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction.Herein, the authors show for the first time that sevoflurane anesthesia induced caspase activation and apoptosis, altered amyloid precursor protein processing, and increased beta-amyloid protein levels in the brain tissues of neonatal mice. Furthermore, sevoflurane anesthesia led to a greater degree of neurotoxicity in the brain tissues of the AD transgenic mice when compared with naïve mice and increased tumor necrosis factor-alpha levels in the brain tissues of only the AD transgenic mice. Finally, inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate attenuated sevoflurane-induced caspase-3 activation and beta-amyloid protein accumulation in vivo.These results suggest that sevoflurane may induce neurotoxicity in neonatal mice. AD transgenic mice could be more vulnerable to such neurotoxicity. These findings should promote more studies to determine the potential neurotoxicity of anesthesia in animals and humans, especially in children.
Project description:Elderly individuals are at increased risk of cognitive decline after anesthesia. General anesthesia is believed to be a risk factor for Alzheimer's disease (AD). At present, there is no treatment that can prevent anesthesia-induced postoperative cognitive dysfunction. Here, we treated mice with daily intranasal administration of insulin (1.75 U/day) for one week before anesthesia induced by intraperitoneal injection of propofol and maintained by inhalation of sevoflurane for 1 hr. We found that the insulin treatment prevented anesthesia-induced deficit in spatial learning and memory, as measured by Morris water maze task during 1-5 days after exposure to anesthesia. The insulin treatment also attenuated anesthesia-induced hyperphosphorylation of tau and promoted the expression of synaptic proteins and insulin signaling in the brain. These findings show a therapeutic potential of intranasal administration of insulin before surgery to reduce the risk of anesthesia-induced cognitive decline and AD.
Project description:BACKGROUND:Anesthesia may induce neurotoxicity and neurocognitive impairment in young mice. However, the underlying mechanism remains largely to be determined. Meanwhile, autophagy is involved in brain development and contributes to neurodegenerative diseases. We, therefore, set out to determine the effects of sevoflurane on autophagy in the hippocampus of young mice and on cognitive function in the mice. METHODS:Six day-old mice received 3% sevoflurane, for two hours daily, on postnatal days (P) 6, 7 and 8. We then decapitated the mice and harvested the hippocampus of the young mice at P8. The level of LC3, the ratio of LC3-II to LC3-I, and SQSTM1/p62 level associated with the autophagy in the hippocampus of the mice were assessed by using Western blotting. We used different groups of mice for behavioral testing via the Morris Water Maze from P31 to P37. RESULTS:The anesthetic sevoflurane increased the level of LC3-II and ratio of LC3-II/LC3-I, decreased the p62 level in the hippocampus of the young mice, and induced cognitive impairment in the mice. 3-Methyladenine, the inhibitor of autophagy, attenuated the activation of autophagy and ameliorated the cognitive impairment induced by sevoflurane in the young mice. CONCLUSION:These data showed that sevoflurane anesthesia might induce cognitive impairment in the young mice via activation of autophagy in the hippocampus of the young mice. These findings from the proof of concept studies have established a system and suggest the role of autophagy in anesthesia neurotoxicity and cognitive impairment in the young mice, pending further investigation.
Project description:The highly organized laminar structure of the mammalian brain is dependent on successful neuronal migration, and migration deficits can cause lissencephaly and behavioral and cognitive defects. Here, we investigated the contribution of neuronal migration dysregulation to anesthesia-induced neurotoxicity in the fetal brain. Pregnant C57BL/6 mice at embryonic day 14.5 received 2.5% sevoflurane daily for two days. Cortical neuron migration and axon lengths were evaluated using GFP immunostaining. Morris water maze tests were performed to assess the effects of sevoflurane exposure on spatial memory in offspring. We found that sevoflurane exposure decreased axon length and caused cognitive defects in young mice. RNA sequencing revealed that these defects were associated with reduced neuro-oncological ventral antigen 2 (Nova2) expression. In utero electroporation experiments using Nova2 shRNA recapitulated this finding. Nova2 shRNA inhibited neuronal migration and decreased axon lengths. Finally, we found that Netrin-1/Deleted in Colorectal Cancer (Dcc) proteins acted downstream of Nova2 to suppresses neuronal migration. These findings describe a novel mechanism by which prenatal anesthesia exposure affects embryonic neural development and postnatal behavior.
Project description:Sevoflurane is one of the most commonly used volatile anaesthetics for children, but the safety of prolonged or repeated clinical use of sevoflurane in infants or children is controversial. Here, we investigated the effects of sevoflurane on rats in early life and the time scale of those effects. Our behavioral results indicated that repeated short-term exposure of new-born rats to sevoflurane caused learning and memory impairment, while a single exposure of rats to sevoflurane was relatively safe. Further mechanistic investigation revealed that repeated sevoflurane exposure impaired long-term potentiation (LTP), downregulated the expression of certain synaptogenesis-related proteins (GluR1, PSD95) and upregulated proteins related to endoplasmic reticulum (ER) stress in the hippocampus. An ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), reversed the changes in the levels of synaptic plasticity proteins. Our results provide new evidence for the clinical concerns regarding repeated sevoflurane anesthesia.
Project description:Sevoflurane is a volatile anesthetic that has been widely used in general anesthesia, yet its safety in pediatric use is a public concern. This study sought to evaluate whether prolonged exposure of infant monkeys to a clinically relevant concentration of sevoflurane is associated with any adverse effects on the developing brain. Infant monkeys were exposed to 2.5% sevoflurane for 9 h, and frontal cortical tissues were harvested for DNA microarray, lipidomics, Luminex protein, and histological assays. DNA microarray analysis showed that sevoflurane exposure resulted in a broad identification of differentially expressed genes (DEGs) in the monkey brain. In general, these genes were associated with nervous system development, function, and neural cell viability. Notably, a number of DEGs were closely related to lipid metabolism. Lipidomic analysis demonstrated that critical lipid components, (eg, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol) were significantly downregulated by prolonged exposure of sevoflurane. Luminex protein analysis indicated abnormal levels of cytokines in sevoflurane-exposed brains. Consistently, Fluoro-Jade C staining revealed more degenerating neurons after sevoflurane exposure. These data demonstrate that a clinically relevant concentration of sevoflurane (2.5%) is capable of inducing and maintaining an effective surgical plane of anesthesia in the developing nonhuman primate and that a prolonged exposure of 9 h resulted in profound changes in gene expression, cytokine levels, lipid metabolism, and subsequently, neuronal damage. Generally, sevoflurane-induced neuronal damage was also associated with changes in lipid content, composition, or both; and specific lipid changes could provide insights into the molecular mechanism(s) underlying anesthetic-induced neurotoxicity and may be sensitive biomarkers for the early detection of anesthetic-induced neuronal damage.
Project description:General anesthesia increases the risk for cognitive impairment post operation, especially in the elderly and vulnerable individuals. Recent animal studies on the impact of anesthesia on postoperative cognitive impairment have provided some valuable insights, but much remains to be understood. Here, by using mice of various ages and conditions, we found that anesthesia with propofol and sevoflurane caused significant deficits in spatial learning and memory, as tested using Morris Water Maze (MWM) 2-6 days after anesthesia exposure, in aged (17-18 months old) wild-type (WT) mice and in adult (7-8 months old) 3xTg-AD mice (a triple transgenic mouse model of Alzheimer's disease (AD)), but not in adult WT mice. Anesthesia resulted in long-term neurobehavioral changes in the fear conditioning task carried out 65 days after exposure to anesthesia in 3xTg-AD mice. Importantly, daily intranasal administration of insulin (1.75 U/mouse/day) for only 3 days prior to anesthesia completely prevented the anesthesia-induced deficits in spatial learning and memory and the long-term neurobehavioral changes tested 65 days after exposure to anesthesia in 3xTg-AD mice. These results indicate that aging and AD-like brain pathology increase the vulnerability to cognitive impairment after anesthesia and that intranasal treatment with insulin can prevent anesthesia-induced cognitive impairment.
Project description:Results of animal studies have raised a significant concern that commonly used general anesthetics may induce neurotoxicity in children. It may be difficult to resolve this concern with human studies because randomizing children only for testing anesthetic toxicity may not be feasible. We randomized 6-day old male Cynomolgus monkeys to receive or not to receive sevoflurane anesthesia at surgical plane for 5 h. Sevoflurane is the most commonly used general anesthetic in children in the U.S.A. Here, we showed that sevoflurane anesthesia did not affect the behavior evaluated by holding cage method when the monkeys were 3 and 7 months old. However, there was an age-dependent decrease in the frequency of stress events and environmental exploration behavior during the test. Sevoflurane also did not affect the learning and memory of the monkeys when they were assessed from the age of 7 months. Finally, sevoflurane did not affect the expression of multiple neuron-specific proteins in the hippocampus and cerebral cortex of 10-month old monkeys after all behavioral and cognitive tests were completed. These results suggest that exposure of neonatal monkey to sevoflurane may not affect cognition, behavior and neuronal structures in childhood, indicating the safety of sevoflurane anesthesia in children.
Project description:Sevoflurane is widely used in pediatric anesthesia and former studies showed that it causes neurodegeneration in the developing brain. The present study was carried out to investigate the effects of sevoflurane on neurogenesis, neurodegeneration and behavior.We administered 5-bromodeoxyuridine, an S-phase marker, before, during, and after 4 h of sevoflurane given to rats on postnatal day 7 to assess dentate gyrus progenitor proliferation and Fluoro-Jade staining for degeneration. Spatial reference memory was tested 2 and 6 weeks after anesthesia.Sevoflurane decreased progenitor proliferation and increased cell death until at least 4 days after anesthesia. Spatial reference memory was not affected at 2 weeks but was affected at 6 weeks after sevoflurane administration.Sevoflurane reduces neurogenesis and increases the death of progenitor cells in developing brain. This might mediate the late-onset neurocognitive outcome after sevoflurane application.