| Nerve growth factor (NGF) is one of the identified and best known neurotrophic factors. It has been showed to be able to promote the growth , differentiation and regulate the survival and synaptogenesis of Central and peripheral neurons. In retinal pathologic conditions,NGF not only protect neurons and prevent the visual neurons' apoptosis. but also ehance their survival ability against pathological environment. However, the studies on NGF topical administration to eye such as subconjunctival injection and intravitreal injection indicated that there are insurmountable difficults. which greatly limits its widely clinical application. There need more novel .scientific administration methods and preparation so as to fit for clinical use.Abjective:To abtain a novel NGF medical preparation, we introduce rm2.5s- P NGF into collagen, an inartifificial and biodegradable material, and make it into control-released drug membrane, implant it in subconjunctiva of the experimental animals.Evaluate whether NGF is able to be released from the drug membranes and whether NGF penetrates the wall of rabbit eyesball into intraocular tissures . The intravitreal pharmacokinetics of NGF were observed systematically,either. This provide a theoretical and experimental evidence for clinic applicationMethods:The study was divided into two steps to perform.A: The preparation of uni lateral NGF-col lagen control -re I easedmembrane:weigh out fresh bovine tendon tissure 20g, after cutting up, wash it thoroughly and sterilize for 5 minutes with scour-chlorhexidine . and then use tri-distilkd water to wash 8 times. Following that , pour down the breef tendon tissure into a triturator and triturate it repeatedly ..After that, fall down triturated tendon on the filer gauze to syringe and degrease. weigh out pepsin 66mg. dissolve it in the new confected 0. 1M hydrochloric acid (HC1) solution 100ml and then add the triturated breef tendon into them, add water in solution and dilute to 1000ml, mix round the solution into equality. incubate for 72 hour at 30 constant tempreture .During the 72 hour .mix round the solution at intervals of 5 hour .Romove the solution into eppendorf tubes, symmetrically arrange tubes in a high-speed centrifuge ,centrifugate at 7000 rpm for 15 minutes at 4 XT. After that, Take the supernatant from centrifugal solution, abandon remains. Use 4M sodium hydroxide solution to regulate pH value of the supernatant to 10-11, stop reaction of the pepsin in the supernatant. The supernatant now become collagen gel. Store the collagen gel in a refrigeratoiy overnight at 4. Then add the collagen gel into supersaturated sodium chloride solution and salt out twice to get rid of resolvable impurity. After salting our the collagen gel ,then get the solidified collagen, enclose the seperated collagen tissure in a dialytic bag through a filler. The diarytic bag was dialysed for 4 day in 5000ml tri-distilled water. Use silver nitrate to test the content of chloric ion in tri-distrilled water until there is not white precipitation occurrence in water. At that time, take out the collagen tissure from the dialytic bag and add NGF powdery reagent in the collagen tissure ,then mix them with 2% acetic acid 18ml and add water to 360ml , incubate 2 hours. Fill the mixture in a mould ,and take shape after store , reconstitute and freeze it out in a freeze-dry machine for 72 hours. Once the NGF-collagen membrane has been finished,the following step is to unilaterally process the membrane: weigh out 240mg poly-lactic-acid (PLA), dissolve it in 12ml chloroform and pour PL A solution on a sterile glass-groove,incubate 20 minutes with that place the shaped spongyNGF-collagen membrane on the PL A film. After the membrane has dried, divide averagely the membrane into 54 equal shares, therefore every piece contains 100 u g NQF with dimension of 4mm XIOmm. Thus, the unilaterally control-released delivery membrane s of NGF have been made. At last.keep them at 4'C in a refrigeratory for use.B. Studies on the intravitreal pharmacokinetics of NG...
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