| Flap graft technique is an important technology of plastic surgery field,and is widely used in orthopedics,microsurgery,oral and maxillofacial surgery,ophthalmology and other professional fields.But flap graft often takes a long time period of ischemia and hypoxia,and the ischemia-reperfusion will inevitably causes certain damage to tissue,and affect the survival of flap graft.The necrosis of flap will inevitably bring patients with irreversible physical damage and economic loss.Spasmodic contraction of blood vessels in the process of clinical island flap transplantation is a common cause of poor results of the transplanted tissues part survived,and the ischemia-reperfusion injury caused by the recanalization of blood vessels in free flap tissue,also seriously affect the flap survival.At present,it is think that formation of oxygen free radicals during reperfusion cause lipid peroxidation and membrane increased permeability,tissue edema and intracellular calcium overload,which cause skin flap.And,the studies in this field still remain in one or more vascular active drugs application,to remove oxygen free radical,to improve the membrane permeability,and to inhibit calcium inside,and even to reduce inflammatory cells infiltration,etc.However,the studies are empty about which the target accumulation to the flap of certain drugs to play the role of more obvious improvement,and what’s the difference between traditional method of administration and targeted therapy.Magnetic liquid is a kind of solid and liquid mixed two-phase fluid containing nano magnetic particles,which is widely used in cell separation,magnetic heat therapy,magnetic control endovascular magnetic microspheres embolism,magnetic drug targeting therapy and so on.Especially in magnetic drug targeting therapy,magnetic liquid as the drug carrier,can carry drugs positioning mobile to accumulate them in the body,and increase the concentration of the drug target place,reduce drug toxicity and side effects to the normal tissue,under the action of outside magnetic field.In recent years,some scholars abroad have applied magnetics drug to targeting therapy of patients in clinical trials,and the results and datum were suggested that the solutions were safe and successful.Current researches in this field are mainly concentrated in tumor treatment,and in the field of plastic surgery,especially in the flap,few researches are reported.The alprostadil,also as known as prostaglandins E,a kind of naturel prostaglandins(PG),is an oxidation product of two high r-linoleic fat acid,a polyunsaturated fatty acid.Endogenous prostaglandin E widely distributes in the body,but most are quickly fired after playing a local biology role.The alprostadil as a kind of efficient biological active substances,has obviously dilated blood vessels,improved the spasmodic contraction of blood vessels,but also has inhibited platelet aggregation,reduced thromboxane A2,blood viscosity and erythrocyte aggregation to improve microcirculation.And it also can prevent arthrosis lipid plaque formation and improve nerve damage,via lowering endothelin(ET),increasing the expression of endothelial nitric oxide synthase,nitric oxide(NO)in serum.The magnetic alprostadillipid microspheres made of the alprostadilcoating at about 0.2 microns of lipid microsphere and magnetic fluids.On and under the action of magnetic field,the magnetic alprostadillipid microsphere can deliver drugs targeted to the lesion site,so as to achieve targeted treatment of disease.Compared to the traditional method of administration,it can effectively prevent the adverse drug reactions,and have the features of "targeting sustainability,safety and high efficiency",etc.However,there are no studiesabouttargeted accumulating to flap of the magnetic alprostadillipid microsphere,and the role of improving spasmodic contraction of blood vessel,reducing ischemia-reperfusion injury of the flap and increasing the survival rate of flap,at present.Objective:The aims are to product the magnetic alprostadil lipid microsphere,the establishment of ischemia-reperfusion injury model of rat’s abdomen flap,and to test the level of lipid peroxides,free radicals and capillaries generation before and after ischemia reperfusion.Study the influences of the magnetic alprostadil lipid microspheretomalondialdehyde(MDA),nitric oxide(NO)and the microvascular generation when targeting treatment of ischemia-reperfusion injury of the rat’s abdomen flap.Discuss the effect of this new method of administration,targeted therapy of the magnetic alprostadil lipid microsphere to rat flap,to the survival rate of flap.Provide new train of thought and theory basis for clinical prevention and treatment of flap of ischemia-reperfusion injury,improving the survival rate of flap transplant.Methods: 1.The magnetic alprostadil lipid microsphere preparation and response to magnetic field Use the emulsification heat curing technology: Dissolve the bovine serumalbumin in distilled water solution of alprostadil,add magnetic liquid,and then mixed them with castor oiltoultrasonic emulsification.Then stir the mix of emulsion and heating castor oil,add ether to degrease,centrifuge,abandon the oil,then precipitate and rinse,and finally displace them in distilled water,freeze drying to the powder of the magnetic alprostadil lipid microsphere.Add the magnetic alprostadil lipid microsphere to 0.1% saline according to the concentration ratio of 1:10,and ultrasonic disperse them,observe the microspheres dispersion under inverted microscope;At the same time,drop the suspension liquid one drop on the slide,and put a rubidium iron boron magnet with 3000 gauss on the other slide,observe the gathering and movement of the microspheres.2.To establish ischemia-reperfusion injury model of a rat’s abdomen flap: The female SD rats were generally anesthetized,and design rat’s abdomen island flap according to the methods of Manson.Raised the subcutaneous intima of the size of 3 cm x 6 cm island flap layer with the abdominal shallow vessels.Block the blood flow of abdominal shallow vessels with microvascular clamps to cause skin flap of ischemia,and then reperfusion after 5 hours,finally cause ischemia-reperfusion injury model.3.The experimental groups 30 SD rats were randomly divided into three groups: experimental group,control group,and the blank group,10 rats in each group.Block the blood flow of abdominal shallow vessels with microvascular clamps to cause skin flap of ischemia,and then reperfusion after 5 hours,finally cause ischemia-reperfusion injury model.At the same time,the magnetic alprostadil lipid microspheres were injected in the experimental group rats by the contralateral femoral artery intubation,and physiological saline was injected in control group rats.Blood did not be blocked and no drugs were injected in blank group rats,after preparation of the normal skin flap.Experimental group rats’ flaps were coverage with rubidium iron boron magnet outside.The flaps were suture in situ in the experimental group and control group rats after reperfusion two hours,and in the blank group rats after preparation seven hours.4.Flap blood flow measurement: Measurement the flap blood flow before ischemia,at 1,2,5 hours after ischemia and 5 min,30 min,2 hours after reperfusion,with laser Doppler blood flow meter respectively.5.The content of malondialdehyde(MDA)and nitric oxide(NO)of flaps: The MDA and NO were two of the important indices for flap ischemia-reperfusion injury.In this study,the content of MDA and NO were measured respectively by the barbituric acid method and Griess Reagent method,in flap edge organizations of control group and experiment group in 0.5 hour and 2 hours after reperfusion,the blank group in 2 h after flap preparation.6.Testing Vascular endothelial growth factor(VEGF): Flap tissues specimen were made into slices,and detect the expression of VEGF by using immunohistochemically method and immunofluorescence method.7.The average capillary number and diameter of detection: Flap tissue samples were made into slices,and stained by routine HE method to detect capillary number and diameter on average.8.Determination of the flap survival rate: Test the flap design area and the flap survival area using coordinate sticker 7 days after Flap were sutured,and calculate the flap survival rate.9.Statistical methods: Statistical analysis was conducted by applying SPSS17.0 statistical software.Experimental results were showed with mean ±standard deviation,comparison between groups used single factor analysis of variance,and P < 0.05 is statistical significant.Results: 1.The skin flap blood flow measurement: compared with the blank group,flap blood flows gradually reduced with ischemia period lengthen.The flap blood flow 5 hour after ischemic was about 30% of the microcirculation blood flow before ischemia;Blood flow volume gradually increased after reperfusion(P < 0.05),and the experimental group flap blood volume were significantly more than it in the control group and blank group(P < 0.05).2.The content of MDA:There were no significant statistical differences in MDA content in the three groups at the moment when flap was made,(P > 0.05).Compared with the blank group,MDA content was not significant increase in the experimental group 0.5 hour after reperfusion,while it was significantly increased in control group(P < 0.05);2 hours after reperfusion,MDA content was increased in the experimental group and the control group,and it was significantly higher in the control group than that of the experimental group(P < 0.05).3.The content of NO: There were no significant statistical differences in NO content in the three groups at the moment when flap was made,(P > 0.05).0.5 hour and 2 hours after reperfusion,NO content was decreased in the experimental group and the control group compared with the blank group,and it was significantly higher in the experimental group than that of the control group(P < 0.05).4.The expression of VEGF and capillaries numbers: by VEGF immunohistochemistry and immunofluorescence staining and HE staining,a large number of new capillaries were observed and the average number of capillaries increased in the experimental group rat’s abdominal flap.The expression of VEGF also was more than that of the control group(P < 0.05);Compared to the control group,capillary number decreased,the expression of VEGF decreased significantly in blank group(P < 0.05).5.The survival rate of skin flap and flap observations: the flap survival rate of the experimental group after 7 days was significantly higher than the control group,the flap survival rate was 81.6 ±7.4%(that of control group was 42.1 ± 5.6%,and the blank group was 100%),and the difference was statistically significant(P < 0.05).Only did proximal tissues survive in the control group,most of the rest is dark brown,dry,scab skin and inelastic,hairless.When touching,the temperature was obviously lower than normal skin;5-syringe needle pierced it without bleeding.Survival area of experimental group flap was significantly greater than the control group,and most of flap grew well,flap edema was lighter,color and texture were normal.Hair growth and normal skin temperature were observed,and bright red blood flew out when 5-syringe needle Pierced it;only was the remote flap dark brown,it was shown ductile,inelastic,no hemorrhage,necrosis.Blank group flap color was reddish,and normal temperature and texture,visible hair growth,no necrosis was observed.Conclusion: 1.After ischemia,flap blood flow decreased,reperfusion could restore blood flow.Ischemia reperfusion could reduce the NO synthesis and VEGF expression,and could increase MDA level and the average number of capillaries,while flap survival rate was significantly reduced in 7 days.2.Targeted therapy of alprostadil can improve the survival rate of flap,by increasing the NO content and decreasing MDA level,reducing the oxygen free radicals and lipid peroxide,and improving the skin microcirculation,relieving skin flap ischemia-reperfusion injury,and increasing new blood vessels numbers. |
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