| PrefaceVacuum Freeze - drying Preservation ( VFDP) of cornea is developed on the base of cryopreservation and drying preservation. The freeze - drying corneas lost its water by freezing and sublimating under vacuum pressure. Evaluating of the activity of rabbit corneal endothelial cell enzyme through enzyme histochemistry and electron microscope histochemical technics to determine how much the VFDP influences the ability of rabbit cornea endothelial cell.Materials and methods1. Experimental instrumentsFreeze - drying machinery: ZLG - 0. 05, produced by Northeast universityDimethylsulfoxide(DMSO) , ATP, levamisole, NBT, ect. Sigma Chemical CO. P. O USA20% Human serum Album ( HSA) : Shanghai RAAS Blood Products CO. LtdSimple controlled freezer; made by ourselves2. Animal and group:18 albino rabbits, 2. 0 - 3. Okg. Divide 36 eyes into threegroups. They are freeze - drying group, and two control groups including fresh group and cryopreservation group.3. Basic operation:3.1 Making up protective agent;Consist of 5% and 7. 5% (v/v) DMSO respectively and 20% HSA3. 2 Preparation the rabbit corneas;The corneas were removed with a rim of sclera as astraumatially as possible, taking care not to rub the endothelium.3.3 Rehydrative media;1640 media and 20% rabbit serum.3.4 Cryopreservation of cornea;Incubated excised corneas in each of two cryoprotective agents for 20 min; procedure freezing at the rate of 1. 0C/min to -30C, then accelerated to 10t/min until - 80C. Dip into the liquid nitrogen.3.5 VFDP of cornea;Prepreparation and freezing same as cryopreservation. Freeze drying in the vacuum freeze - drying machine. Packaging by puffing nitrogen ; stored in room temperature or refrigerator.3. 6 Thaw of cryopreserved cornea3.7 Rehydration of freeze - drying cornea4. Light enzyme histochemistry4.1 Na+ -K + -ATPase :Fixation in formalin; washed in PBS; incubated in the corresponding reaction media for enzyme activity; postdeposed and observed by LM.4.2 SDH:Incubated at first; fixation in formalin; washed; and observed byLM.5. Electron histochemical technicsSections were postfixed in 1%OsO4; dehydrated and embedded in epoxy resin; light stained with Uranyl acetate and lead citrate; observed under a transmission electron microscope (TEM).6. Statistical analysisObtain the Integrated Optical Density Average (IOD) of cornea histochemical stain by Metamorph stage collecting system and then analyzed by SPSS11.0 software.ResultsFor all three groups, the ATPase and SDH enzyme histochemistry dyeing appears positive reaction under the LM. The IOD of freeze -drying group is lower than that of cryopreservation group and fresh group. The difference is significant (P <0. 01). Linear precipitates of ATP enzyme grains on the cell membrane can be observed under the TEM.SDH electron histochemical stain had no result, because of the lost of endothelium.DiscussionCorneal VFDP is set up on the base of cryopreservation and drying store. Freeze - drying cornea cannot be oxidized and contaminated, it can be stored in room temperature or refrigerator; transported conveniently. This preservation is defined that the water of cornea is freezed and then sublimated in the drying chamber under vacuum pres-sure. Before use, rehydrating the freeze - drying cornea to recover its physical state as possible.There are several enzymes in the corneal endothelial cell. Some of them have high activity, SDH, ALP, ATPase, etc. Measuring the activity of rabbit corneal endothelial cell enzymes through enzyme his-tochemistry and electron microscope histochemical technics can reflex the ability of rabbit cornea endothelial cell.In the present study, the ATPase and SDH enzyme histochemistry dyeing of all the three groups appears positive reaction. It appears the endothelial cells of three groups viable. The average integrated optical density (IOD) of freeze - drying group is lower than that of cryopr-eservation group and fresh g... |
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