| The application of eugenol as an anesthetic for fish is becoming more and more common in production practice.The research of its anesthetic mechanism has become popular for a long time.Monoamine neurotransmitters participate in a number of important physiological activities in the central nervous system.Numerous studies have shown that they are involved in the anesthetic process,but whether monoamine neurotransmitters are involved in the process of eugenol anesthesia on carps has not been reported.This study compared the anesthetic effects of eugenol nanoemulsion and emulsion on carp,and determined the most suitable eugenol formulation and optimal anesthetic concentration for anesthesia of carp.A new method for the simultaneous discovering of multiple monoamine neurotransmitters in the central nervous system of carp by HPLC-fluorescence detector was established.Changes in the content of eight monoamine neurotransmitters,such as Central Nervous System of Epinephrine(E),Norepinephrine(NE),Dopamine(DA),L-dihydroxyphenylalanine(L-DOPA),3,4-Dihydroxyphenylacetic Acid(3,4-DOPAC),5-Hydroxytryptamine(5-HT),5-Hydroxy-3-indoleacetic acid(5-HIAA)and Homovanillic Acid(HVA),in induction period,anesthesia period,deep anesthesia period and recovery period of carp under the circumstance that eugenol nanoemulsion was optimum anesthetic concentration were detected.The role of eight neurotransmitters in eugenol anesthesia and the mechanism of eugenol anesthesia was investigated.Results1.Carp weighing 0.75±0.1 kg were anesthetized with eugenol nanoemulsion and eugenol emulsion.As the concentration of eugenol increased,the anesthetic time of the carp became shorter,and the recovery time was longer.Compared to emulsions,nanoemulsions with same concentration have a shorter time for the carps to enter the anesthesia period,and the anesthetic process was smoother,the recovery time was shorter.This is a safer and more effective anesthetic for carp.The optimum anesthetic concentration for eugenol nanoemulsions was 40 mg/L for carp with a body weight of0.75±0.1 kg,while the optimal anesthetic concentration for reference drug MS-222 was 160 mg/L.2.A new method for the simultaneous determination of various monoamine neurotransmitters in the central nervous system of carp by HPLC-FLD: Eight kinds of monoamine neurotransmitters were completely separated by a gradient elution method without addition of ion-pairing reagents or derivatization of samples.The degree of separation among the substances was satisfactory and there was no tailing phenomenon.Every monoamine neurotransmitters showed a good linear relationship with the peak area in their concentration range.The correlation coefficient R2 are all greater than 0.999;the inter-day precision is in the range of 0.32%~3.82%,the intraday precision is in the range of 0.22%~4.78%.The average additional recovery rate of NE,E,L-DOPA,DA,3,4-DOPAC,5-HT,5-HIAA and HVA were 101.51%,94.71%,92.69%,101.47%,99.73%,100.35%,98.34%,and 91.47%,respectively.RSD ranged from 0.00% to 2.56%.Therefore,the method is simple,rapid,and accurate.It can be used to determine the eight monoamine transmitters in the brain of carps.3.The contents of NE in different brain tissues of carp differed significantly,and the contents were 294.29 ng/m L in the telencephalon,140.89 ng/m L in the diencephalon,111.14 ng/m L in the cerebellum,97.76 ng/m L in the midbrain,54.97 ng/m L in the medulla oblongata and 38.90 ng/m L in the spinal cord.Neuronal content was higher in brain regions with higher functions.The content of DA in each brain tissue of carps showed little difference.The content in the spinal cord was 1349.99 ng/m L,telencephalon(1048.38 ng/m L),borneol(1007.13 ng/m L),and midbrain(934.08ng/m L),diencephalon(893.11 ng/m L)and cerebellum(855.79 ng/m L).The contents of 5-HT in brain tissues of carp were less different.The contents of them were telencephalon(29.82 ng/m L),spinal cord(19.90 ng/m L),diencephalon(17.98 ng/m L),and diurnal(17.78 ng/m L),midbrain(9.82 ng/m L)and cerebellum(8.96 ng/m L),respectively.5-HIAA was detected in the midbrain(15.30 ng/m L),telencephalon(13.36 ng/m L),and spinal cord(12.37 ng/m L)of normal carps,and was not detected in other brain regions.E,L-DOPA,3,4-DOPAC and HVA were not detected in any brain regions.4.Since the carps entered the induction period,anesthesia period,deep anesthesia period,and recovery period successively,the NE of each tissue significantly decreased(P<0.01),increased significantly(P<0.01)(except spinal cord),significantly decreased(P<0.01)(except spinal cord)and then significantly increased(P<0.01)(except cerebellum)in proper order after anesthesia by eugenol.The changes of NE content in various tissues of every anesthesia period(especially the high-level center)were relatively large,and corresponded to the changes in eugenol anesthetic effect.It explained that eugenol anesthesia may work by inhibiting the function of neural pathways of carp in the NE.5.After eugenol anesthesia,the induction period of DA content in the brain,midbrain,cerebellum and medulla of carp increased significantly(P<0.01),and reached the highest level during the anesthesia(P<0.01).Decreased(P<0.01)and increased in recovery period.DA content in interbrain increased in induction period,reached the highest in anaesthesia and deep anesthesia,and decreased in recovery period;DA content in spinal cord also decreased first,and was highest in anesthesia.Then gradually decline.6.After eugenol anesthesia,the induction period of 5-HT content in each brain area of carp was extremely significantly increased(P<0.01),and the anesthetic period significantly decreased(P<0.01).The deep anesthesia period except for midbrain and Yannao was significantly more significant.Increased(P<0.01),the recovery period increased and decreased.7.After MS-222 anesthetized carp,the NE content in the telencephalon was similar to that of eugenol.The NE content in the other brain regions gradually increased during the induction period and the anesthesia period,and gradually decreased during the deep anesthesia and recovery period.The DA levels in the diencephalon,the medulla,and the spinal cord gradually decreased,while the telencephalon,midbrain,and cerebellum experienced an increase in the induction period and then gradually decreased.With the exception of diencephalon,the content of 5-HT in each brain area gradually decreased in each period.8.The eugenol anesthesia could induce the change of 5-HIAA content in some brain regions.No 5-HIAA was detected in all brain regions after MS-222 anesthesia.Conclusion: The anesthesiaof carp by eugenol may be exerted by inhibiting the function of neural pathways in the NE. |
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