| Background:Sevoflurane is a wide-used inhaled anesthetic, especially in pediatric surgeries with its good controllability, fast acting and low irritation to airway. However, some recent researches have proved that all common-used inhaled anesthetics will lead to any extents of neurotoxicity. It mainly affects on learning and memorizing function, particularly in children and the elders.Children exposed to inhaled anesthetics during the development period, they are likely to have some changes in behaviors and mental symptoms as they’re growing up. Meanwhile, the postoperative cognitive dysfunction (POCD), is somehow related to the inhaled anesthetics. A clinical review shows that anesthesia and surgeries for children less than 4 years old tend to decrease their ability to read, write and study mathematics. Also animal studies display that brain cells are to damage undergoing a long time, high dosage of sevoflurane exposure. Even more, it may result in a long-range neural cognitive dysfunction.In the latest statistics, there’re approximately 6 million children undergoing surgeries and anesthesia only in US, including 1.5 million baby patients. Therefore, to prevent neurotoxicity in developing brain from inhaled anesthetics has been concerned by many doctors and scientists in the recent years. So far, since how anesthetics work remains incompletely demonstrated, most of the researches still stay on animal level. Plus, it’s difficult to carry out on human patients. So we still stay in the very first stage of figuring the prevention and cure for central neurotoxicity followed by anesthetics. And most vivo hormones or drugs are used but show no exact effects.Thyroid hormone (TH), is one of the most important hormones for humans and animals when they’re growing up. It plays an important role in brain developing by inactivating mTOR signal transduction pathway, influencing gene transcription and protein translation, regulating proliferation of neurons and improving the cognitive function like learning and memorizing. Even, TH in natal period is the key for brain development. Anesthetics and its metabolic products may affect on thyroid gland and then interferes the secretion of TH, which is likely to induce neurotoxicity. In view of this, the purpose of this study is to discover the potential mechanism of how to prevent and cure sevoflurane-induced neurotoxicity in the perspective of cognitive dysfunction, by observing the changes of cognitive function, hippocampal neurons, PSD-95, NR2A/NR2B and cellular apoptosis in sevoflurane-exposed natal rats which are loaded with TH. And also, this may provide evidence for clinical use.Objective:To observe the influence to cognitive function and emotion in young rats who have exposed to sevoflurane in natal period by open field test (OPT), sugar preference test (SPT), elevated plus maze test (EPMT) and water maze test (WMT). To observe how TH influences the density of apoptosis in hippocampus in sevoflurane-exposed natal rats by TUNEL. Western blot is used for the expression of NR2A, NR2B, PSD-95 in hippocampus. BrdU remarking technique is for observing the cellular proliferation in DG area in adolescent rats. Golgi staining is for observing the hippocampal neurons in adults. Combine all the results to discuss whether T3 can lessen the neurotoxicity induced by sevoflurane in natal rats during anesthesia and its mechanism.Methods:5 female virgin SD rats,180-220 g, labor after mating with 2 male SD rats.7 days after,7-day offspring (P7) are divided into 3 groups (NS, SEV, T3) randomly for each mother rats. The number of male and female offsprings are equal in each group. 5ml/kg NS is injected intraperitoneally for group NS.5ml/kg NS is injected intraperitoneally for group SEV 0.5 h before exposure to sevoflurane.100 ug/kg T3 is injected intraperitoneally for group T30.5 h before exposure to sevoflurane. Injections for all groups are required every 24 h in 3 days. Group SEV and T3 rats are put into a crystal cube through which air containing sevoflurane flows at 1 L/min for 6h after first administration. And 1 L/min air for 6h in the same equipment for group NS. The whole cube will be place on a warmer to keep rats temperature around 37-38 degree centigrade. Monitor breathing and skin color during the anesthesia.3 of each group are selected randomly for brain paraffined. Brain paraffin section will then be used for counting the density of TUNEL (+) cells in DG and SVZ area. On the other hand,5 of each group will be administrated BrdU 50 mg/kg for 7 days right at the day finishing anesthesia. Then brain paraffin sections will be done when the administration is over. Ratio of the BrdU/DCX (++) from BrdU (+) and of the BrdU/GFAP (++) from BrdU (+) are counted through immoflurorescence. For the rest of the offsprings, they’re required to run behavior tests starting from P25. Each of the groups is separated into 2 parts. First part of the offsprings is for WMT and EPMT while the second part is for SPT and OFT. For the first part,13 of each group is about to run WMT for 3 days and begins to run EPMT a day after accomplishing WMT. For the second one, all rats are about to run OFT for 3 days (P25) and 3-day SPT starts form P28. When all behavior tests are done,5 of each group are randomly picked to make brain paraffin section for testing the density of CA1 and CA3 area in hippocampus by Nissl Staining. Another 3 of each group get killed and rapidly removed the brains for Golgi staining to observe the number of dendritic branches, total length and the distribution of dendrites of CA1 and CA3 pyramidal cells. Every 5 from each group (1 per mother rat) will get their brain removed onto the ice and hippocampus is rapidly separated for testing the expression of NR2A, NR2B and PSD-95 by western blot.Results:1. All rats were stable, no respiratory depression or other complications during the anesthesia. In the 9 days after administration, there’s no statistical significance of the weights among all rats (P> 0.05). There were one from group T3 and one from group SEV that showed low weight, who had been eliminated from the experiment.2.6h after anesthesia, compared to group NS and T3, TUNEL(+) cells in DG and SVZ area apparently increased in group SEV (P< 0.01, P< 0.05). There’s no significant difference about TUNEL (+) cells in DG and SVZ area between group NS and T3 (P> 0.05). On P15 day, ratio of the number of BrdU/GFAP (++) and BrdU (+) in group SEV was apparently lower than the one in group NS and T3 (P< 0.05) while there was no significant difference between group T3 and NS. And there showed the ratio of the number of BrdU/DCX (++) and BrdU (+) in group SEV was apparently lower than NS (P< 0.05), But no significant difference between SEV and T3 (P> 0.05). It tells us sevoflurane will decrease the proliferation of neurons in natal rats, whereas T3 can lessen the effect sevoflurane brings.3. In P25-30, group SEV was considered that their ability to learn and memorizing decreased, with anxiety to some extent. While there was no significant difference of learning and memorizing between group T3 and NS. Plus, the change of emotion in group T3 seemed lightened compared to group SEV. Details:the escaping latency in group SEV was significantly longer than group T3 and NS (P< 0.05, P< 0.01) whereas there was no significant difference between T3 and SEV; in SPT, SP(sugar preference) in group SEV was much lower than group NS and T3 (P< 0.01, P< 0.05) whereas there was no significant difference between NS and T3; during P25-27, in OFT, time of staying in the central area in group SEV increased (P< 0.05), but group T3 was decreased than SEV; as P30, in EPM, there was no significant difference about the time of staying in the open arms among 3 groups (P> 0.05). All of these behavior science tests suggest natal rats undergone sevoflurane anesthesia are likely to acquire impairment of learning and memorizing, with some anxious emotion changes as usual in adolescent period. And the use of T3 may improve the impairment sevoflurane leads to.4. In P30, the density of neural cells in CA3 area in group NS and T3 was obviously higher than group SEV (P< 0.01) while there was no significant difference between T3 and NS. Total length of CA3 pyramidal cells in hippocampus from group SEV and T3 was significantly shorter than group NS (P< 0.001, P< 0.05). The number of dendritic branches of pyramidal cells from group SEV was lower than group NS (P< 0.01) while there was no significant difference between group T3 and NS. For the pyramidal cells in CA1, total length of dendrites of group SEV was significantly shorter than group NS (P< 0.05), while there was no significant difference between group T3 and NS (P>0.05). For the number of dendritic branches of pyramidal cells, there was no statistical significance among 3 groups(P>0.05). Statistics tells that sevoflurane may damage the development of hippocampus, which results in any levels of eccyliosis to pyramidal cells both in CA1 and CA3 area. However, the use of T3 may lessen this effect.5. In PI5, group SEV showed lower expression of PSD-95, NR2A and NR2B (P< 0.05) and the expression of these protein in group T3 was higher than group SEV. Results implicates that sevoflurane may disarrange the expression of NMDA receptor’s subunits in natal rats, which results in a lower expression of PSD-95, leading to abnormal development in neural synapses.T3 could reduce the interference of sevoflurane on the development stage of brain nerve growth related protein.Conclusion:Sevoflurane anesthesia may induce impairment in developing brain in natal rats, such as increase of neural apoptosis in hippocampus. It also stunts the cellular proliferation in DG area of hippocampus, which mainly stunts the astrocyte and newborn immature neuron. That may results in the decrease of CA3 and eccyliosis for pyramidal cells in CA1 and CA3 when the rats have grown up, which even leads to abnormality in behavior, acting mild depression-like emotion change and impairment of learning and memorizing. During the anesthesia, administration of T3 may alleviate the apoptosis, relieve the lack of CA3 neurons in hippocampus and improve the eccyliosis of pyramidal neurons. The mechanism is assumed that T3 may ameliorate the metabolism of brain cells during anesthesia, which decreases the apoptosis and influence to NR2A/NR2B expression and increase the expression of PSD-95 to improve the growth of hippocampal cells. |
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