Effect Of Mannitol On Blood-brain Barrier Penetration And Pharmacokinetics Of Cerebrospinal Fluid Of Vancomycin In Rabbits

Part 1 Effects of intravenous mannitol on the opening of blood-brain barrier in rabbitsObjective:To verify whether intravenous mannitol can promote the opening of blood-brain barrier.Methods:The current study verified whether intravenous mannitol can promote the opening of blood-brain barrier.There were 12 general grade healthy rabbits,randomly divided into 2 groups?mannitol group,control group?,6 rabbits in each group.After the animals were anesthetized,2%evans blue(3 m L·kg^-1)was injected intravenously.After 10 minutes,the mannitol group was intravenously injected with 20%mannitol(4.90 g·kg^-1,0.1 m L·s^-1);the control group was injected with an equal volume of 0.9%sodium chloride injection.After 1 hour of circulation,cardiac perfusion was performed,and the brain was decapitated to observe the blue staining of brain tissue.The wet weight of the brain tissue was recorded,evans blue was extracted with formamide,and the absorbance value was determined by spectrophotometer at620 nm.The content of evans blue was calculated according to the standard curve,the results of the two groups were all expressed as evans blue content(?g·g^-1)per gram of brain tissue,and the data were statistically processed using SPSS 21.0 software.Results:The standard curve equation of Evens blue is Y=0.007X-0.0002?R²=0.9998?,and the linear relationship is good in the concentration range of2.5-100?g·m L^-1.Compared with the control group,the brain blue staining of mannitol group is more obvious,and the content of evans blue is also significantly different.Conclusions:Intravenous mannitol can promote the opening of blood-brain barrier.Part 2 Establishment of HPLC method for determination of vancomycin concentration in cerebrospinal fluid of rabbitsObjective:High performance liquid chromatography method?HPLC?was established to determine the concentration of vancomycin in rabbit cerebrospinal fluid.Methods:A Diamonsil C₁₈column was used,with the mobile phase of methanol-0.05 mol·L^-1 potassium dihydrogen phosphate buffer?adjusted to p H 3.2 with phosphoric acid??17:83,V/V?,at the detection wavelength of 236nm.The flow rate was 1.0 m L·min^-1,and the column temperature was set at30?.Norvancomycin was used as an internal standard.Rabbit cerebrospinal fluid samples were precipitated with 4%perchloric acid solution.The specificity,linearity,detection limit and quantitative limit,extraction recovery rate and method recovery rate,intraday precision and daytime precision,room temperature placement stability after sample treatment,low temperature preservation stability and repeated freeze-thaw stability were investigated respectively.Results:Endogenous substances in rabbit cerebrospinal fluid do not interfere with the determination of vancomycin and internal standard.The vancomycin and internal standard peaks were well-formed and completely separated,and the retention time was 14.3 min and 11.2 min,respectively.The standard curve equation of vancomycin is Y=0.536X+0.020?R²=0.9996?,and the linear range is 0.25-10?g·m L^-1.The recovery rate of vancomycin is93.80%-101.2%.The recovery rate of internal standard norvancomycin is?98.09±0.04?%,and the method recovery rate of vancomycin is90.98%-99.70%.The RSD values for both intraday and daytime precision are less than 15%.CSF samples are stable when placed 8 h at room temperature after processed,stored at-40?f or 7 days and under freeze-thaw cycles.Conclusions:This study established an HPLC method for measuring the concentration of vancomycin in the cerebrospinal fluid of rabbits.This method has the advantages of simple operation,rapid analysis and good specificity.It is suitable for the determination of vancomycin concentration in the cerebrospinal fluid of domestic rabbits,and lays a methodological foundation for the subsequent study of the effect of mannitol on the vancomycin pharmacokinetics in cerebrospinal fluid.Part 3 Effect of mannitol on blood-brain barrier penetration and pharmacokinetics of cerebrospinal fluid of vancomycinObjective:To investigate the effects of different doses of mannitol intravenously on vancomycin blood-brain barrier permeability and cerebrospinal fluid pharmacokinetics.Methods:There were 24 general grade healthy rabbits,randomly divided into 3 groups?low dose group,high dose group,control group?,8 rabbits in each group.The cerebrospinal fluid was extracted from cerebellar medulla oblongata cistern at each time point after administration and was treated by precipitation of protein in 4%perchloric acid solution Vancomycin concentration was determined in cerebrospinal fluid by HPLC.DAS 2.1.1software was used to fit the cerebrospinal fluid drug-time curve,calculate the pharmacokinetic parameters,and SPSS 21.0 software were used for statistical processing.Results:The main pharmacokinetics parameters of vancomycin in low dose mannitol group,high dose mannitol group and control group are as?7.28±4.67?h;T_maxare?2.21±0.80?h,?1.88±0.84?h and?2.50±0.76?h;C_maxCompared with the control group,the vancomycin cerebrospinal fluid concentration and main pharmacokinetic parameters in the low-dose mannitol of vancomycin in the high-dose group increase by 28.92%,15.38%,and57.46%,respectively?P<0.05?.And the drug concentrations in the cerebrospinal fluid increase by 81.25%,65.48%and 33.09%?P<0.05?at 1,2,3 h time points,respectively.Conclusions:Intravenous administration of high-dose mannitol promotes the ability of vancomycin to penetrate the blood-brain barrier in rabbits and increases the concentration of vancomycin in the CNS.