| With the development of the early diagnosis and clinical treatment technology for pregnancy, the number of nonobstetric surgeries and fetal intervention procedures had increased quickly even the new surgical methods such as fetoscopic surgery and ex-utero intrapartum treatment are invented. Most of these surgeries were performed at the second trimester of pregnancy. General anesthetic agents such as isoflurane were administrated on the mothers during operation. In order to achieved uterine quiescence and minimize the risk of miscarriage, anesthetic agents were usually maintained on high concentration about 1.5-2 minimum alveolar concentration(MAC) to relax the smooth muscle. Because most general anesthetic agents are lipophilic, which can cross the placenta easily to balance with maternal blood concentration, both mothers and fetus facilitated significant anesthetic effect. Thus the fetal brain was exposed to the agents and maybe faced to the risk of abnormal development inescapably.The second trimester of pregnancy is an important neurodevelopmental stage and also the fast-developmental stage, such as neurogenesis, neuronal migration, and corticogenesis are the major events. The fast developing brain of the fetus was sensitive to the change of environment induced by medicine. General anesthetic agents had been demonstrated that can cause neuronal injury effects. Apoptosis in neurons can be observed after anesthetic agents exposure, lead to the result of neurodegeneration and synapse loss, synaptogenesis can be delayed also. The risk of abnormal fetal brain development therefore rises dramatically. Meanwhile, this neurotoxicity effect can arise in a short term and influence for a long period.Reseachs on the fetal rodents found that the neurotoxicity of isoflurane may be performed at three aspects. First, the expression of Caspase-3 raised significantly in the hippocampus after exposure several hours later in immunohistochemical tests, which suggests that abnormal apoptosis occurrence. Second, results of behavior tests showed that anxiety-related behaviors were changed and spatial memory abilities were decreased in adulthood. Third, the count and morphology of synapses were changed significantly in the hippocampus in adulthood observed by transmission electron microscopy.Apoptosis increasing in brain in a short term is the first major adverse event of the neurotoxicity induced by general anesthetic agents. In immature brain, neurons were usually produced redundantly. Apotosis plays an important role to eliminate the superfluous and non-function cells in order to ensure the fetal brain developing normally. Interference with this process was harmful to the fetal brain. Many general anesthetic agents, including intravenous agents such as midazolam, ketamine and propofol, and the inhaled agents such as isoflurane, sevoflurane and nitrous oxide, can be proved to interfere the normal process of apotosis and subsequently influence the development of fetal brain. The adverse effects had been demonstrated that can occur in more than rodents, which can found in primates also.Antivation of α2-adrenergic receptor (α2-AR) is important for the early development of the nervous. The increase of α2-AR expression has accompanied by nerve development process from the embryonic day 12 till birth, reaching the top at embryonic day 19 and 20. Dexmedetomidine is the high selectivity agonist of α2-AR, which can promote the growth of nervous by binding α2-AR, and produce significantly neuroprotecting effect. All subtypes of α2-AR can be binded with dexmedetomidine, and the α2A-AR is the major functional subtype of neuroprection.Dexmedetomidine can rise the expression of brain-derived neurotrophic factor (BDNF) and trigger the cascades reaction of neuroprection. BDNF belongs to the neurotrophic factors family with the comprehensive role in mammals’ neuronal survival, differentiation and synaptic growth, in addition the synthesis and secretion of it depends on the activation of neuron. The BDNF can promote neuron survival, regulate the central nervous system development and synaptic plasticity, adjust the neurotransmitter release, which is an important site in nerve damage exposed to general anesthetics and the restraint of its expression can be observed.Restricted by medical ethic, there are few studies were implemented on human to explored the relation between the immature brain and the neuronal injury effect of general anesthetic agents. But this potential threat exist persistently, which may raise social problems and medical resource consumption. With the increase of these nonobstetric surgeries, more mothers and fetuses exposed to general anesthetic agents. There is an urgent need for research of alleviating these neuronal injury effects so as to meet our national policy of sound child rearing and promote the medical level of obstetrics.Our research firstly determined the effective concentration of isoflurane to provoke neuronal injury in fetal brains by administrate different concentration of agents on the mothers, combined using dexmedetomidine for different dose to evaluate its neuroprective effect and effective dose secondly. Behavior methods and transmission electron microscope were employed to examinate the effect of the neuronal injury induced by isoflurane and neuroprection by dexmedetomidine in adult. Transmission electron microscopy (TEM) and immunohistochemical method were employed to explore the reasonable mechanism. The objective of our reseach is to explore the effective method and dose of dexmedetomidine to attenuate the neuronal injury effects of fetal brain induced by isoflurane, and thus to provide theoretical evidence for clinical application.Chapterl Abnormal apotosis of fetal brain in the second trimester of pregnancy induced by exposed to isoflurane for a long durationObjectiveSimulating the general anesthesia method on nonobstetric surgeries, different concentration of isoflurane were administrated on maternal rats to expose to the fetal brain on embryonic day 14(E14). To observed the abnormal apotosis effect in the fetal brain induced by isoflurane.MethodsOn E14,50 timed-pregnancy rats were randomly assigned to 5 groups (Iso1.0, Iso1.5, Iso2.0, Oxy and Control) to receive 5 different dispositions(n=10).1.0%, 1.5% or 2.0% isoflurane in 100% oxygen were inhaled by the dams in group Iso1.0, group Iso1.5, group Iso2.0 for 4h. Single 100% oxygene was inhaled in group Oxy. Group Control without any treatments.4h after inhaling, cesarean sections were implemented to isolate the fetuses.2 fetuses were selected randomly in each dams, and the brain slices were taken at 2mm behind forehead after fixed with 4% paraformaldehyde solution. Apotosis was evaluated by Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) method. The number of apoptotic cells and the total cells number were counted by an observer blinded to the experimental protocol. The ratio between the two data above was calculated.The data of two futuses belong to the same dam were averaged as one data, so the n value of each group was 10. Measurement data were measured by (x±s), all the data were analyzed by the software SPSS 17.0. One-Way ANOVA was used to analyze the ratio in different groups, the two-two comparisons among the means were done by LSD method. A probability value of P<0.05 was considered to be statistically significant.ResultsThere was significant difference in the ratios of apoptotic cells among all groups (F=8.113, P<0.001). Comparing with group Control, the ratio in group Iso1.0, group Isol.5 and group Iso2.0 raised significantly (P<0.05) while was approximated in group Oxy(P>0.05). Comparing with group Iso2.0, the ratios in group Isol.5 is approximated(P>0.05), while group Iso1.0, group Oxy and group Control were less than it significantly (P<.05).ConclusionApotosis of the E14 fetal neuron increased significantly after 1%,1.5% and 2% isoflurane inhaled by the dam for 4h. Isoflurane may provide dose-dependent apotosis effect on the fetal brain. Single inhaling 100% oxygen for 4h did not affect apotosis.Chapter 2Neuroprective effect provided by dexmedetomidine to attenuate abnormal apotosis of fetal brain induced by isofluraneObjectiveTo evaluate the effect of combine using dexmedetomidine for the abnormal apotosis of fetal brain induced by isoflurane, and to explore the reasonable mechanism.MethodsOn E14,70 timed-pregnancy rats were randomly assigned to 7 groups (Dex5+Iso, Dex10+Iso, Dex20+Iso, NS+Iso, Dex20+Yoh+Iso, Dex20+Oxy and Control) to receive 7 different dispositions(n=10).The dams in group Dex5+Iso, group DexlO+Iso, group Dex20+Iso, group Dex20+Oxy received different dose of dexmedetomidine for 5μg/kg, 10μg/kg, 20μg/kg and 20μg/kg at two time points including 15min before and 2h in inhaling by intraperitoneal injection. Group Dex20+Yoh+Iso received the mixed agents of dexmedetomidine(20μg/kg) and yohimbine (500μg/kg) while group NS+Iso received normal saline at the same time points.1.5% isoflurane in 100% oxygene was inhaled in group Dex5+Iso, group Dex10+Iso, group Dex20+Iso, group NS+Iso, and group Dex20+Yoh+Iso for 4h while single 100% oxygene was inhaled in group Dex20+Oxy.Group Control did not receive any treatment.4h after inhaling, cesarean sections were implemented to isolate the fetuses.2 fetuses were selected randomly in each dam, and the serial brain slices were taken at 2mm behind forehead after fixed with 4% paraformaldehyde solution. Apotosis was evaluated by TUNEL method, and the number of apoptotic cells and the total cells number were counted by an observer blinded to the experimental protocol. The ratio between these two data was calculated. The expression of BDNF and Syn were examed by immunohistochemical method, and the values of mean optical density (MOD) were recorded.The data of the two futuses belong to the same dam were averaged as one data, so the n value of each group was 10. Measurement data were measured by (x±s), all the data were analyzed by the software SPSS 17.0. One-Way ANOVA was used to analyze the ratio in different groups, the two-two comparisons among the means were done by LSD method. A probability value of P<0.05 was considered to be statistically significant.ResultsThere was significant difference in the ratios of apoptotic cells among all groups (F=6.145, P<0.001).Comparing with group Control, ratios in group Dex5+Iso, group Dex10+Iso, group Dex20+Iso and group Dex20+Oxy were approximated (P>0.05), while ratios in group NS+Iso and group Dex20+Yoh+Iso raised significantly (P<0.05). Comparing with group NS+Iso, the ratios in group DexlO+Iso, group Dex20+Iso, group Dex20+Oxy and group Control were all declined significantly(P <0.05), while in group Dex5+Iso and group Dex20+Yoh+Iso were approximated (P>0.05). The ratios in group Dex5+Iso, group DexlO+Iso and group Dex20+Iso were significantly less than group Dex20+Yoh+Iso (P<0.05).There was significant difference in the MOD values of BDNF among all groups (F=6.145, P<0.001).Comparing with group Control, the values in group Dex10+Iso, group Dex20+Iso and group Dex20+Oxy were approximated (P>0.05), while values in group Dex5+Iso, group NS+Iso and group Dex20+Yoh+Iso declined significantly (P<0.05). Comparing with group NS+Iso, the values in group Dex10+Iso, group Dex20+Iso, group Dex20+Oxy and group Control were all raised significantly (P<0.05), while values in group Dex5+Iso and group Dex20+Yoh+Iso were approximated (P>0.05). The ratios in group Dex10+Iso and group Dex20+Iso were significantly more than group Dex20+Yoh+Iso (P<0.05), while the value in group Dex5+Iso was similar with group Dex20+Yoh+Iso (P>0.05).The MOD values of Syn had no significant difference among all groups(F=1.130, P=0.356).ConclusionCombined using dexmedetomidine at 10μg/kg or 20μg/kg can significantly reduce the increasing apotosis induced by isoflurane in fetal brain. Neuroprotection provoked by dexmedetomidine activating α2-AR can be reversed by yohimbine. The expression of BDNF can be raised by dexmedetomidine, and its cascade may participate in the neuroprotection. Single using dexmedetomidine did not influence apotosis and the expression of BDNF.Chapter 3 Neuroprective effect in adulthood provided by dexmedetomidine attenuated the fetal brain injury induced by isofluraneObjectiveBy behavioral tests, to evaluate whether the neuroprotective effect which provoked by dexmedetomidine can continue till adulthood in the rats exposed to isoflurane on E14. And to explore the reasonable mechanisms by anatomical and immunohistochemical methods.MethodsOn E14,50 timed-pregnancy rats were randomly assigned to 5 groups (Dex+Iso, NS+Iso, Dex+Yoh+Iso, Dex+Oxy and Control) to receive 5 different dispositions (n=10).The dams in group Dex+Iso, group Dex+Oxy received dexmedetomidine for 20μg/kg at two time points including 15min before and 2h in inhaling by intraperitoneal injection. Group Dex+Yoh+Iso received the mixed agents of dexmedetomidine(20μg/kg) and yohimbine (500μg/kg) while group NS+Iso receive normal saline at the same time points.1.5% isoflurane in 100% oxygene was inhaled in group Dex+Iso, group NS+Iso and group Dex+Yoh+Iso for 4h while single 100% oxygene was inhaled in group Dex+Oxy. Group Control did not receive any treatment.After delivery, all pups were group-housed with their dams till postnatal day 21, when they were weaned. On postnatal day 56,2 male pups belonged to each dam were randomly selected and classify by the groups their dams, to proceed behavioral test and the subsequent examination including tansmission electron microscopy (TEM) analysis and immunohistochemical tests.The pups proceeded the behavioral test as the following sequence, visual impairment exclude experiment, open-field test (OFT) and Morris water maze test (MWM). In OFT, the data of moving distance, mean speed, bowel movement times were recorded. In MWM, place navigation test were proceeded firstly on the first 4 days, and the latency time (s) to find the hidden platform from the SE start position was scored. Hidden platform search test was proceeded on day5 secondly, and the first time and the frequence of crossing the platform position were scored.After behavioral tests, one of the two pups belong to the same dam was selected to analyzed the ultrastructure of hippocampus CA1 region by TEM, Synaptic count(SC) was scored under 13500 magnified, width of synaptic cleft (WSC) and thickness of postsynaptic density(PSD) were measured under 46000 magnified, another pup was evaluated the expression of Syn and BDNF by immunohistochemical tests, and the values of MOD were recorded.The data of behavioral, anatomical and immunohistochemical tests were collected by observers blinded to the experimental protocol. The data of the two futuses belong to the same dam in behavioral tests were averaged as one data. Measurement data were measured by (x±s). all the data were analyzed by the software SPSS 17.0. Two-ways repeated measures analysis of variance was used to analyze the data in place navigation test in MWM. The comparisons among groups among the means were done by LSD method One-Way ANOVA was used to analyze the ratio in different groups. A probability value of P<0.05 was considered to be statistically significant.ResultsIn OFT, the data of moving distance and mean speed had no significant difference among all groups (F=0.339, P=0.850;F=0.393, P=0.812). There was significant difference in the data of bowel movement times among all groups (F=5.203,P=0.002). Data in group Dex+Iso was similar with group Dex+Oxy and group Control (P>0.05), while significantly less than group NS+Iso (P<0.05). Data in group Dex+Yoh+Iso was similar with group NS+Iso (P>0.05), and significantly more than group Control (P<0.05). The data in group Dex+Iso has no significant difference with group Dex+Yoh+Iso (P>0.05).In MWM, the latency time(s) in place navigation test were significantly different on different days (F=99.348, P<0.001), and reached the shortest on day4 in all groups. The two-two comparisons among the means were done by LSD method. On day3, there was no significant difference between the data in group Dex+Iso with the other groups (P>0.05). The data in group Dex+Yoh+Iso was similar with group NS+Iso (P>0.05), and significantly more than group Control (P<0.05), while the data in group Dex+Oxy was similar with group Control (P>0.05), and significantly more than group NS+Iso (P<0.05). On day4, the data in group Dex+Iso, group Dex+Oxy and group Control were approximated (P>0.05), and all significantly less than group NS+Iso (P<0.05). The data in group Dex+Yoh+Iso was significantly more than group Control (P<0.05), and similar with group NS+Iso (P>0.05). The data in Dex+Iso was significantly less than group Dex+Yoh+Iso (P<0.05). The data of first times and frequence of crossing the platform position in hidden platform search test had significant difference almost all the groups (F=3.124, P=0.024;F=4.146, P=0.006). The first time of crossing the platform position in group Dex+Iso and group Dex+Oxy were similar with group Control (P>0.05), while significantly less than group NS+Iso (P<0.05). The data in group Dex+Yoh+Iso was significantly more than group Control (P<0.05), and similar with group NS+Iso (P>0.05). The data in Dex+Iso was significantly less than group Dex+Yoh+Iso (P<0.05). The frequence of crossing the platform position in group Dex+Iso and group Dex+Oxy were similar with group Control (P>0.05), while significantly more than group NS+Iso (P<0.05). The data in group Dex+Yoh+Iso was significantly less than group Control (P<0.05) as group NS+Iso. The data in Dex+Iso was significantly more than group Dex+Yoh+Iso (P<0.05).In TEM analysis, the data of SC was significantly different among all groups (F=15.771, P<0.001). Data in group Dex+Iso and group Dex+Oxy were similar with group Control (P>0.05), while significantly more than group NS+Iso (P<0.05). The data in group Dex+Yoh+Iso was significantly less than group Control (P<0.05) as group NS+Iso. The data in Dex+Iso was significantly more than group Dex+Yoh+Iso (P<0.05). There were no significant difference in the data of WSC and PSD among all groups (F=0.119, P=0.975; F=0.638, P=0.638).In immunohistochemical tests, the MOD values of Syn was significantly different among all groups (F=4.590, P=0.003). Values in group Dex+Iso and group Dex+Oxy were similar with group Control (P>0.05), while significantly more than group NS+Iso (P<0.05). The data in group Dex+Yoh+Iso was significantly less than group Control (P<0.05) as group NS+Iso. The data in Dex+Iso was significantly more than group Dex+Yoh+Iso (P<0.05). There were no significant difference in the MOD values of BDNF among all groups(F=0.363, P=0.834).ConclusionCombined using dexmedetomidine for 20μg/kg can produce long-term effect at the male offsprings which exposed to isoflurane on E14. The abnormal changes induced by isoflurane in adulthood, including the increase of anxiety, the decrease of spatial learning and learning ability, the decrease of induced by isoflurane, the decrease of SC and the decrease of Syn expression can be attenuated by dexmedetomidine. The neuroprotective effect in spatial learning and learning ability, synapsis density and Syn expression provoked by dexmedetomidine can be reversed by yohimbine. Single using dexmedetomidine did not influence the behaviors, synaptic count and Syn expression of the male offspring in adulthood. |
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