| Isoflurane is one of the most commonly used inhalational anesthetics. It was discovered in 1982 and has been used for nearly twenty years. It can be used in surgery patients of all ages including children. But in 1999, it was firstly found that antagonist of NMDA receptor could lead to a wide range of neuronal apoptosis in developmental rats. Since isolfurane can also partly block NMDA receptors, the problem of whether isoflurane anesthesia could induce developmental neurotoxicity in human beings caused extensive attention in anesthesiologists, neurologists and patients.Current experimental results showed that clinical-relevant concentration and time of isoflurane exposure in developmental period could increase apoptotic neurons in central nervous system including cortex, hippocampus, thalamus and spinal cord in rats. It was found that isoflurane anesthesia could disturb development of synapse; growth rate of dendrites, numbers and morphology of neurites changed after isoflurane exposure in vitro and vivo. Researchers also found that isoflurane anesthesia in developmental period lead to long-term behavioral changes. However, there are still many controversial results in current researches, particularly in the mechanism of isoflurane-induced neurotoxicity and long-term structural and behavioral changes after isoflurane exposure in developmental period. In addition, we lack drugs that can effectively prevent or eliminate these neurotoxicity effects at present.In this study, we aimed to confirm the developmental neurotoxicity and investigate the possible mechanism through observing the neuronal apoptosis in cortex and hippocampus, long-term neuronal density and learning and memory ability, cortical and hippocampal mRNA expression of pro-inflammatory cytokines and related signaling pathway in rats 7 days after birth after exposure to clinical-relevant concentration of isoflurane. Based on these results, we then observed the protective effect of minocycline, a common tetracycline antibiotic, on isoflurane-induced developmental neurotoxicity through in vitro and in vivo study. These studies are expected to provide theoretical basis for the safe use of isoflurane in clinic.1,To observe the effect of isoflurane exposure on neuronal apoptosis in cortex and hippocampus and long-term neuronal density changes of developmental rats.2,To investigate the effect of isoflurane exposure on cortical and hippocampal mRNA expression of pro-inflammatory cytokines and related signaling pathway of developmental rats.3,To determine the effect of isoflurane anesthesia on long-term learning and memory ability of development period rats.4,To observe the protective effect of minocycline on isoflurane-induced developmental neurotoxicity in vitro and in vivo.1,Calculation of apoptotic neurons and neuronal density Seven-day-old Sprague-Dawley (SD) rats were randomly assigned to received 6h of 1.5% isoflurane exposure in 35℃(anesthesia group), or room air (control group). Arterial blood gas was measured and active caspase-3 positive neurons in rats cortical and hippocampus were detected with immunohistochemistry after 6h of exposure in each group. The cortical neuron density was assessed in brain slice stained with NeuN 53 days after exposure. Apoptotic neurons and neuronal density between two groups was compared.2,Investigate the expressions of pro-inflammatory cytokines mRNA and IκBαprotein in dynamic Seven-day-old SD rats were randomly assigned into isoflurane group (group A) and control group(group C).Rats in group A were exposed to 1.5% isoflurane for 6 hours, while group C to air. mRNA of IL-1β,IL-6 and TNF-αin cortex and hippocampus were assessed by real-time PCR at the time point of 0,2,4,6 hours during the isoflurane exposure and 4,6, 12,24 hours after the exposure. Total proteins were extracted and IκBαprotein was measured by Western blot at the time point of 2,4,6 hours.3,Long-term learning and memory ability detected with Morris Water Maze Seven-day-old SD rats were randomly assigned into isoflurane group (group A) and control group (group C). Rats in group A were exposed to 1.5% isoflurane for 6 hours, while group C to air. After the isoflurane exposure, rats were returned to continue feeding after the birth 60 d. Learning and memory ability was tested by Morris Water Maze test.4,Protective role of minocycline on primary cultured neurons Cortical neurons of newborn SD rats were cultured for 5 days with the density of 1 X 105 and then divided into four groups, control group (group C), anesthesia group (group A), minocycline at 1μmol/L group(group M1) and minocycline at 10μmol/L group (group M2). Neurons in group C were treated with room air in 37℃for 6 h, while group A with 1.5% isoflurane for 6 h. Neurons in group M1 and M2 were pretreated with minocycline at 1 and 10μmol/L for ten minutes and then treated at the same time with group A.Morphological changes of neurons were observed with phase contrast microscope, neuron viability was evaluated with MTT assay and apoptotic neurons were detected with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).5,Protective role of minocycline on 7-day-old rats Seven-day-old SD rats were randomly divided into three groups, control group (group C), anesthesia group (group A) and minocycline group (group M).Rats in group C were treated with room air in 35℃for 6 h, while group A with 1.5% isoflurane for 6h. Rats in group M were intraperitoneal injected with minocycline at 45mg/kg for 12h and then treated at the same time with group A. mRNA of IL-1β, IL-6 and TNF-αin hippocampus and cortex were assessed by real-time PCR at the time point of 0,2,4,6 hours during the isoflurane exposure.Total proteins were extracted and IκBαprotein was measured by Western blot at the time point of 4 hours after isoflurane exposure. Active caspase-3 positive neurons in rats cortical and hippocampus were detected with immunohistochemistry after 6h of exposure in each group.1,Calculation of apoptotic neurons and neuronal density No difference was found in arterial blood gas analysis between two groups. Active caspase-3 positive neurons increased significantly in cortex after 6h of 1.5% isoflurane exposure in group A. While, there was no significant difference of apoptotic neurons in hippocampus between two groups.The neuron density was also similar in cortex in two groups.2,Expressions of pro-inflammatory cytokines mRNA and IκBαprotein in dynamic Hypoxia was not observed during the isoflurane exposure. Copmared with group C, level of IL-1βand TNF-αmRNA in hippocampus began to elevate at 2 hours reached their peak at 4 and 6 hours during the exposure and went back to basal level at 12 and 24 hours after the exposure. Level of IL-6 mRNA was 2 and 1.7 folds at the point of 4 and 6 hours during the exposure and reached the basal level at 6 hour after the exposure. Similar results were found in cortex.Expression of IκB protein was decreased after the isoflurane exposure.3,Morris Water Maze Latency of searching platform in rats of two groups was decreased with the training days. But no statistical difference was observed in swimming speed, latency of searching platform, searching time in target quadrant and times of platform crossing between two groups.4,Protection of minocycline on primary cultured neurons In group A, neuron morphology changed obviously, neurons swelling appeared and so on. Neurons in group M2 were similar with group C, while group M1 with group A. Compared with group C, cell viability in group A and M1 decreased (P<0.05).Apoptotic neurons in group A and M1 were more than those in group C and M2 (P<0.05).5,Protection of minocycline on 7-day-old rats Apoptosis neurons in group M were significantly less than that in group A (P<0.05).By contrast with group A, the expressions of mRNA of IL-1β,IL-6 and TNF-a in group M were significantly decreased (P<0.05). IκBαprotein in group M was increased when compared with group A (P<0.05).1,Isoflurane exposure for 6 h led to increased cortical neuron apoptosis, but no significant reduction of spatial learning and memory ability and neuron density were observed.2,The expression of proinflammatorial cytokines elevated in the hippocampus of immature rats after the 6 hours of isoflurane exposure, and may be related with activation of NF-κB/IκB signal pathway.3,Minocycline protect the primary cultured rat cortical neurons and seven-day-old rats from isoflurane-induced developmental neurotoxicity through anti-apoptosis and anti-inflammatory mechnisms. |
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