| ObjectivePrecise position,quantitatively detect the concentration of cryoprotectant in ampu-tated rabbit hind legs by Proton magnetic resonance spectroscopy(~1H-MRS)under certain conditions.Then evaluate effect of perfusion in muscle and subcutaneous tissue.To judge whether the dimethyl sulfoxide(DMSO),under the condition,achieve the effective concent-rations at the site of target.To explore a kind of method which is suitable for the best composite tissue perfusion by changing the conditions of perfusion,provide a basis for the effective application of cryoprotectants applying to cryopreservation of amputated limb.Methods1.Experiment I:Room temperature kept at 20~24?and kept solution at 37?~38?,in Normal atmospheric pressure establish standard in vitro.Prepare the physiological saline group(control groups)and 2%v/v,6%v/v,10%v/v,14%v/v,18%v/v,22%v/v,26%v/v DMSO(experimental groups)in the extractor hoods around alcohol lamp.Scanning pre-made solution one by one by the BRUKER 7.0T MRI(Magnetic Resonance Imaging).After they get structure as region of interest(ROI)locate in the middle of the three-dimensional structure.Obtaining the original spectrum of the VOI after completion of magnetic reson-ance spectrum detection.Such as access to standard spectrum through baseline correction,phase correction,parameter fitting processing,measure"peak height"and"full width half maximum(FWHM)"of dimethyl sulfoxide peaks in the standard spectrum.Calculate the area of the dimethyl sulfoxide peaks,the groups of data for review after finishing analysis,it is concluded that different concentration of samples relevant to area of the DMSO peaks.Snapping the averages into a scatter-plot and regression analysis by EXCEL and obtaining the relationship between DMSO concentration and the area under the peak.Finally establish methodology basis in measuring cryoprotectant concentrations for animal experiment.2.Experiment II:Six adult healthy New Zealand white rabbits of either gender,aged10-14 months,weighing 1.8 to 2.2kg,were enrolled in the study..All finished skin prepa-ring 24 hour and preoperative fasting water 6 hour before surgery.The rabbits were executed by air embolism through the posterior auricular vein.Amputate the skin and thigh muscle 3-5 cm away from Near side of the knee,free and protect blood vessels from harm until the surgical area be disinfected and draped.Femur and skeletal muscles were stripped to articulation coxae,joint capsule,and caput femoris was completely severed before plac-ing and fixed vein trocar in the femoral artery.The test is proceeding in rabbits'physiolo-gical temperature and blood pressure.Heparin saline was perfused into the arteria cruralis of the amputated legs until the flowing was colorless and clear.Preparing solutions of NS group,20%v/v DMSO solutions perfuse respectively into rabbit legs,lasted 40 mins.Then put rabbit hind legs one by one in the magnetic resonance imaging machine and positioning VOI,plane 2-3 cm below the knee level,in muscle tissue and subcutaneous tissue and detecting.ResultsExperiment I:In the vicinity of 2.62 PPM saline group were not detected peaks(Figure 1);All the samples can detect the corresponding wave near 2.62 ppm and each set corresponding of average area:(278.53±4.62)×10~3,(950.20±23.60)×10~3,(1481.34±21.79)×10~3,(2239.33±50.33)×10~3,(2794.35±208.12)×10~3,(3453.18±90.81)×10~3,(4130.58±295.30)×10~3,The obtained regression equation is:I=(15871·C-28.32)×10~3,(R~2=0.9992).Experiment II:The average area of subcutaneous and muscles tissue in NS group is“0”;The DMSO area of group 20%v/v is corresponding to 12.94%v/v,11.81%v/v.Conclusions1.Under certain conditions,MRS is feasible for cryoprotectants concentration detection and the concentration and peak area of dimethyl sulfoxide has highly linear correlation.2.MRS can be noninvasive,precise and quantitative detect the concentration of dimethyl sulfoxide in composite tissue. |
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