| Intravenous anaesthesia is a way in which the patients are anaesthetized by mainline, lose their consciousness, rigor off their muscle, or lose ordinary respiring functions.The advantages of intravenous anaesthesia are that there is no repulsion and it can easily be used without specific anaethesia appliances. Intravenous anaesthesia is used in anaesthetic induction, anaesthetic maintenance, analgesias and pain easing after the operation. Ketamine, xylazine and midazolam are widely used for premedication, chemical restraint, and the induction and maintenance of anesthesia in small animals. The use of these three drugs as a sole anesthetic has been limited by muscle hypertonicity,myoclonus, violent recovery, convulsions, hypotension, bradycardia and respiratory depression. In the present study, we combine the three anesthetics into a compound prescription, the most appropriate prescription was determined by orthogonal experiment and orthogonal design decisive verification experiment in mice, carry out the acute toxicity, local irritation, stability test, pharmacokinetics, anesthetic effect and its effect on the immune function of the injection, providing experiment data for correct application of Shumianning injection. This study contained the follwing 6 parts.Experiment I Research on Prescription of Shumianning InjectionKetamine, xylazine and midazolam have been used extensively as small animal anesthetics for surgical procedures. Based on the results of pre-experimet, according to the facors that affect the anesthetic effect, orthogonal method was used, the concentrations of ketamine, xylazine and midazolam were selected as variable factors and table L9(34) was used to do exiperiment on the basis of loss of righting reflex in mouse. Results:The optimum prescription was:ketamine (8 g·100mL-1), xylazine (0.9 g·100mL-1) and midazolam (0.2 g·100mL-1), named as Shumianning injection. The manufacturing process is simple, which is suitable for mass production.ExperimentⅡSafety Evaluation and Long-Term Stability of Shumianning InjectionTo evaluate the safety of Shumianning injection, acute toxicity experiment was carried out in mice by up-and-down method, the results showed that ED50=0.706 mL·kg-1 body weight, LD50=2.366 mL·kg-1 body weight, TI=3.35; Injecting Shumianning into rabbits to carry out local irritation experiment, the results indicated that Shumianning had mild vein irritation and slight muscle irritation, did not cause eye irritation; To establish expiration time and suitable package for Shumianning injection, long term test was performed with 3 batches merchant injection, the results showed that the main compositions of Shumianning injection were stable, the injection looked clear, no impurity, the period of validity was 2 years at room temperature, away from light.ExperimentⅢPharmacokinetics of Shumianning in CanineTo study the pharmacokinetics of Shumianning, a reverse-phase HPLC method with UV detection is developed and validated for simultaneous determination of ketamine, xylazine and midazolam in canine plasma. Analytes are extracted from alkalinized samples into diethyl ether-methylene chloride (7:3, v/v) using single-step liquid-liquid extraction. Chromatographic separation is performed on a C18 column using a mobile phase containing acetonitrile-methanol-10mmol·L-1 sodium heptanesulfonate buffer adjusted to pH 3 with glacial acetic acid (44:10:46, v/v) at a detection wavelength of 210 nm. Shumianning was applied intravenously to 6 dogs, the concentrations of ketamine, xylazine and midazolam were measured. The concentration-time curves of ketamine, xylazine and midazolam were best described of two-compartment open model and the typical pharmacokinetics parameters of the three drugs were as follows:Ketamine Vc=1.231±0.147 L·kg-1, T1/2α=4.271±0.521 min,T1/2β=65.877±15.701min,AUC=147.331±20.54μg·min·mL-1,L(s)=0.0393±0.0055 L·min-1·kg-1;XylazineVc=0.918±0.299L·kg-1,T1/2α=4.627±2.056min,T1/2β=55.773±8.62 min, AUC=20.866±1.781μg·min·mL-1, CL(s)=0.0336±0.00289 L·min-1·kg-1; Midazolam Vc=0.339±0.055 L·kg-1, T1/2α=5.675±1.82 min, T1/2β=39.152±6.546 min, AUC=13.554±1.059μg·min·mL-1, CL(s)=0.0131±0.000975 L·min-1·kg-1. Experiment IV Anesthetic Effect of Shumianning Injection in GreyhoundIn this study the anesthetic effect of Shumianning was investigated in 9 greyhounds. The animals were injected with Shumianning (0.06 mL·kg-1Ⅳ) without premedication, to observe the time to induction, maintenance, recovery, heart rate, SpO2, respiration rate, systolic pressure, diastolic pressure, rectal temperature, effects of analgesia, sedative and muscle relaxation. The time to induction, maintenance, recovery were 33.11±3.76 sec,23±5.83 min,15±5 min, respectively; heart rate, SAP and DAP increased slowly with a peak at 5 min, then decreased steadily but remained within the normal range; rectal temperatures decreased slowly but did not vary significantly during induction and maintence of anesthesia; respiratory rate decreased rapidly at first and then increased slowly; provided good surgical anesthesia; no vomiting, nystagmus, excessive salivation.Experiment V Effect of xylazine on cell proliferation and cell cycle in mouse spleen cellsXylazine is anα2-adrenoceptor agonist extensively used in veterinary medicine. The present study is focused on the effects of xylazine with or withoutα2 antagonists (yohimbine, BRL44408, imiloxan hydrochloride and JP1302) on the proliferation and cell cycles of mouse spleen cell in culture. The results are as follows:Xylazine at doses of 0.01,0.1,0.2 and 0.4μg·mL-1 did not significantly influence the proliferation of splenocytes in cultures triggered by ConA; at concentrations ranging between 20 and 80μg·mL-1 significantly inhibited spleen cell proliferation to Con A in a dose-dependent manner. Yohimbine enhanced the inhibitory effect of xylazine(40μg·mL-1) on spleen cell proliferation, BRL 44408, imiloxan were ineffective in modulating the inhibitory effect of xylazine(40μg·mL-1); JP1302 (30,60 ng·mL-enhanced the inhibitory effect of xylazine(40μg·mL-1) on spleen cell proliferation, dose dependently. xylazine(40μg·mL-1) blocked the progression of mouse lymphocytes into S phase, enhanced the apoptosis; JP1302 enhanced the activity of cell cycle arrest of xylazine. The present results provide evidence that the inhibitory effect of xylazine on spleen cell viability probably results from activation ofα2C-adrenoceptors.Experiment VI Effect of shumianning on cell proliferation and cell cycle in mouse spleen cellsThe aim of the this study was to investigate the effects of shumianning, ketamine, xylazine and midazolam on the proliferation and cell cycles of mouse lymphocytes in culture. The results are as follows:Low concentrations of ketamine and xylazine did not significantly influence the proliferation of splenocytes in cultures triggered by ConA; at high concentrations significantly inhibited spleen cell proliferation to Con A in a dose-dependent manner. Midazolam at concentrations ranging between 0.01 and 100μg·mL-1 significantly inhibited spleen cell proliferation to ConA in a dose dependent manner. Low concentrations of shumianning (0.01-0.4μg·mL-1) did not significantly change the proliferation of splenocytes in cultures stimulated by ConA; at high concentrations (2-80μg·mL-1) significantly inhibited spleen cell proliferation to ConA, dose dependently. The in vivo administration of shumianning at doses of 0.56,2.8 and 11.2 mg·kg-1 body weight, which produced weak sedation, slight and deep anesthesia, respectively, did not significantly change the proliferation and cell cycles of splenocytes in cultures triggered by ConA. Cumulatively, these results suggest that clinically relevant concentrations of shumianning did not affect the viability of splenocytes. |
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