5-fluorouracil standard and 5-fluorouracil injection solution ( 25 mg / ml ) were procured from shanghai xudong haipu pharmaceutical co. , ltd . high pressure liquid chromatography ( hplc ) grade methanol and ultra - pure water were procured from j.t . lyophilized and co sterilized bio - amniotic membranes with size of 4  5 mm and free of hepatitis b , hepatitis c , syphilis , human immunodeficiency virus ( hiv ) , and other pathogens were procured from jiangxi rui - ji medical devices co. ltd . after prefreezing them for 10 h at 0c to 56c , they were lyophilized at 10c to 56c till their water content reached 1 - 2% . healthy adult new zealand white rabbits ( 2 - 3 kg , 30 males and 30 females ) were procured from jinan experimental animal center and kept under normal conditions . 5-fluorouracil was analyzed on diamonsil c18 column ( 5 m , 4.6  250 mm ) using methanol - water ( 2:98 ) as mobile phase at flow rate of 1.0 ml / min and column temperature of 30c , and monitored by absorption at 266 nm . 5-fluorouracil standard was first dissolved in methanol and then prepared as 100 g / ml methanol - water ( 2:98 ) solution . twenty bio - amniotic membranes were immersed in 2 ml of 25 mg / ml 5-fluorouracil solution for 5 or 10 min , respectively , transferred into 1 ml mobile phase , and vortexed for 2 min . 5-fluorouracil was identified by comparing its retention time to that of the standard , and its amount per bio - amniotic membrane was calculated by comparing peak areas of supernatant and standard . male and female rabbits were randomly assigned into three groups with 20 in each group . rabbits in group a , b , and c were implemented trabeculectomy and intraoperatively transplanted rehydrated bio - amniotic membranes that had been immersed in 5-fluorouracil solution for 5 or 10 min , or in saline for 5 min , respectively . two hours prior to the operation , the operative eye of each rabbit was examined by electroretinogram ( erg ) . in detail , the body hair in the central part was removed using depilatory and the pupil was fully zoomed by alternately dropping 1% atropine and 10% phenylephrine eye drops for three times . rabbits were adapted to dark environment for 40 min and injected 3% pentobarbital sodium solution ( 1 ml / kg ) from the ear vein . under general anesthesia , the experimental eyelid was fully opened and placed right in the front of full - field stimulating ball . then the corneal contact electrode , reference electrode , and grounding electrode were placed on the corneal surface , surface skin of center forehead , and surface skin of middle ear edge , respectively . the parameters of electrophysiological diagnostic equipment were set as 13.7 cd / m for flash intensity of stimulating light , 2 s for flash interval , 0.1 - 75 hz for passband to detect b - wave , and five times for magnifying recording waveform . the results were automatically processed , analyzed , and printed by a computer and the amplitudes ( aa , ba ) of b - waves were recorded . after examination , the operation was performed under general anesthesia by intramuscularly injecting 50 mg / kg ketamine hydrochoride and 10 mg / kg chlorpromazine . after washing with sterile saline , the operative eye was routinely disinfected with iodine disinfectant and covered with sterile towels . conjunctival flap based on the fornix was created at the superior temporal quadrant 5 - 7 mm behind the corneoscleral edge of eyeball . conjunctiva and tenon 's capsule were bluntly separated to 1 mm in cornea transparent zone and a sclera flap with size of about 3  4 mm and thickness of l/2 of the sclera was created . deep scleral tissues equivalent to 1  2 mm trabecular along with iris root were excised and a preimmersed bio - amniotic membrane was placed underneath the sclera flap with epithelium upward . the scleral flap was then sutured one stitch each at the two corners and the bulbar conjunctiva was sutured one to two stitches with 10 - 0 nylon needles to make sure that both tenon 's capsule and bulbar conjunctiva reached watertight state . after the operation , 10,000 units of gentamicin and 1 mg of dexamethasone were immediately injected underneath the bulbar conjunctiva and dexamethasone / neomycin and 2.5 mg / ml chloramphenicol eye drops were postoperatively applied three times a day . the dispersion degree , bubble wall thickness and color , degree of vascular filling , neovascularization , and bleb height were observed . filtering blebs at late stage ( 2 weeks after operation ) were divided into four types based on their appearance and function as proposed by krofeld . type iii blebs are scar - like ones with blood - filling , slightly raised conjunctival hyperemia and adhesive sub - conjunctival tissues , and vessel - rich scleral surface . type iv blebs are capped with partial dome - shaped bulge , and have cystic hyperplasia and a dense ball fascia cavity . the former two types are functioning and the latter two types are nonfunctioning [ table 1 ] . bleb formation of rabbits in different groups before sacrifice baseline iop was defined as iop before being prepared as hypertension model . postoperative iop was defined as iop observed with schiotz at 14 , 21 , and 28 days postoperation [ table 2 ] . intraocular pressure of rabbits before and after operation at different times at 7 , 14 , 21 , and 28 days postoperation , rabbits in each group were randomly selected . after subjecting them to erg their eyeballs were immediately enucleated , and their retinas within 4 mm range of optic disc were observed by transmission electron microscopy ( tem ) . data were expressed as mean  standard deviation and analyzed using multi - factor analysis of variance for completely randomized design in spss 12.0 software . pairwise comparisons among groups were analyzed using least significant difference ( lsd ) t - test . 5-fluorouracil standard and 5-fluorouracil injection solution ( 25 mg / ml ) were procured from shanghai xudong haipu pharmaceutical co. , ltd . high pressure liquid chromatography ( hplc ) grade methanol and ultra - pure water were procured from j.t . lyophilized and co sterilized bio - amniotic membranes with size of 4  5 mm and free of hepatitis b , hepatitis c , syphilis , human immunodeficiency virus ( hiv ) , and other pathogens were procured from jiangxi rui - ji medical devices co. ltd . after prefreezing them for 10 h at 0c to 56c , they were lyophilized at 10c to 56c till their water content reached 1 - 2% . healthy adult new zealand white rabbits ( 2 - 3 kg , 30 males and 30 females ) were procured from jinan experimental animal center and kept under normal conditions . 5-fluorouracil was analyzed on diamonsil c18 column ( 5 m , 4.6  250 mm ) using methanol - water ( 2:98 ) as mobile phase at flow rate of 1.0 ml / min and column temperature of 30c , and monitored by absorption at 266 nm . 5-fluorouracil standard was first dissolved in methanol and then prepared as 100 g / ml methanol - water ( 2:98 ) solution . twenty bio - amniotic membranes were immersed in 2 ml of 25 mg / ml 5-fluorouracil solution for 5 or 10 min , respectively , transferred into 1 ml mobile phase , and vortexed for 2 min . 5-fluorouracil was identified by comparing its retention time to that of the standard , and its amount per bio - amniotic membrane was calculated by comparing peak areas of supernatant and standard . male and female rabbits were randomly assigned into three groups with 20 in each group . rabbits in group a , b , and c were implemented trabeculectomy and intraoperatively transplanted rehydrated bio - amniotic membranes that had been immersed in 5-fluorouracil solution for 5 or 10 min , or in saline for 5 min , respectively . two hours prior to the operation , the operative eye of each rabbit was examined by electroretinogram ( erg ) . in detail , the body hair in the central part was removed using depilatory and the pupil was fully zoomed by alternately dropping 1% atropine and 10% phenylephrine eye drops for three times . rabbits were adapted to dark environment for 40 min and injected 3% pentobarbital sodium solution ( 1 ml / kg ) from the ear vein . under general anesthesia , the experimental eyelid was fully opened and placed right in the front of full - field stimulating ball . then the corneal contact electrode , reference electrode , and grounding electrode were placed on the corneal surface , surface skin of center forehead , and surface skin of middle ear edge , respectively . the parameters of electrophysiological diagnostic equipment were set as 13.7 cd / m for flash intensity of stimulating light , 2 s for flash interval , 0.1 - 75 hz for passband to detect b - wave , and five times for magnifying recording waveform . the results were automatically processed , analyzed , and printed by a computer and the amplitudes ( aa , ba ) of b - waves were recorded . after examination , the operation was performed under general anesthesia by intramuscularly injecting 50 mg / kg ketamine hydrochoride and 10 mg / kg chlorpromazine . after washing with sterile saline , the operative eye was routinely disinfected with iodine disinfectant and covered with sterile towels . conjunctival flap based on the fornix was created at the superior temporal quadrant 5 - 7 mm behind the corneoscleral edge of eyeball . conjunctiva and tenon 's capsule were bluntly separated to 1 mm in cornea transparent zone and a sclera flap with size of about 3  4 mm and thickness of l/2 of the sclera was created . deep scleral tissues equivalent to 1  2 mm trabecular along with iris root were excised and a preimmersed bio - amniotic membrane was placed underneath the sclera flap with epithelium upward . the scleral flap was then sutured one stitch each at the two corners and the bulbar conjunctiva was sutured one to two stitches with 10 - 0 nylon needles to make sure that both tenon 's capsule and bulbar conjunctiva reached watertight state . after the operation , 10,000 units of gentamicin and 1 mg of dexamethasone were immediately injected underneath the bulbar conjunctiva and dexamethasone / neomycin and 2.5 mg / ml chloramphenicol eye drops were postoperatively applied three times a day . the dispersion degree , bubble wall thickness and color , degree of vascular filling , neovascularization , and bleb height were observed . filtering blebs at late stage ( 2 weeks after operation ) were divided into four types based on their appearance and function as proposed by krofeld . type iii blebs are scar - like ones with blood - filling , slightly raised conjunctival hyperemia and adhesive sub - conjunctival tissues , and vessel - rich scleral surface . type iv blebs are capped with partial dome - shaped bulge , and have cystic hyperplasia and a dense ball fascia cavity . the former two types are functioning and the latter two types are nonfunctioning [ table 1 ] . postoperative iop was defined as iop observed with schiotz at 14 , 21 , and 28 days postoperation [ table 2 ] . at 7 , 14 , 21 , and 28 days postoperation , rabbits in each group were randomly selected . after subjecting them to erg their eyeballs were immediately enucleated , and their retinas within 4 mm range of optic disc were observed by transmission electron microscopy ( tem ) . data were expressed as mean  standard deviation and analyzed using multi - factor analysis of variance for completely randomized design in spss 12.0 software . pairwise comparisons among groups were analyzed using least significant difference ( lsd ) t - test . we first measured the amount of 5-fluorouracil per bio - amniotic membrane after being immersed in 25 mg / ml 5-fluorouracil solution by hplc . 1 shows the representative peaks of the standard and samples immersed for 5 and 10 min , respectively . table 3 lists the variation of hplc analysis of 5-fluorouracil and table 4 lists the calculated 5-fluorouracil amount absorbed by bio - amniotic membranes . as shown , the absorbed 5-fluorouracil amount per bio - amniotic membrane was 59 - 76 g . ( c ) sample immersed for 10 min amount of 5-fluorouracil absorbed by bio - amniotic membranes egr b - wave latency and amplitude of rabbits in different groups ( v , n=20 ) table 4 shows the a - wave and b - wave latencies and amplitudes of egr of rabbits in different groups . as shown , there were no significant differences in a - wave and b - wave latencies and amplitudes among the three groups ( p = 0.165 and p = 0.052 , respectively ) . 2 and 3 shows the tem images of retinal ganglion cells of rabbits in different groups . as shown , the nuclei of retinal ganglion cells of rabbits in all three groups were normal . ( a ) showing clear microvilli structure and intact and smooth glass membrane ( 5000 ) . ( b ) showing clear microvilli structure and intact and smooth glass membrane ( 7500 ) . ( c ) showing clear microvilli structure and intact and smooth glass membrane ( 7500 ) . ( e ) showing neatly arranged retinal outer segment membrane disc , but abnormal structures ( 7500 ) . ( f ) showing neatly arranged , intact retinal outer segment membrane disc ( 5000 ) in this study , we confirmed with hplc that each bio - amniotic membrane ( 4  5 mm ) could absorb 59.004 and 75.828 g of 5-fluorouracil after remaining immersed in 25 mg / ml 5-fluorouracil solution for 5 and 10 min , respectively . we further implanted the 5-fu containing amniotic membrane under the scleral flap of filtration channel of rabbit eyes in trabeculectomy to observe postoperative iop changes and bleb morphology , and to preliminarily assess the effect of the surgical approach on rabbit eyes with focuses on retinal function and morphology using erg and electron microscopy . the experiment indicated that application of this surgical approach in rabbit eyes could promote the formation of a well - functioning bleb and maintain a good and relatively low iop within a certain time , while having no significant effect on retinal structure and function . inhibiting fibroblast proliferation , angiogenesis and collagen synthesis within 14 days of surgery is the key to control postoperative scar formation at early stage . its in vivo metabolite 2-deoxy-5-fluoro - uridin can block conversion of deoxyuridine to deoxythymidine by inhibiting thymidylate synthase and consequently inhibit dna synthesis , eventually causing cell death , especially those in s phase . moreover , 5-fluorouracil has been shown to inhibit fibroblast activity and increase the success rate of filtration surgery . in glaucoma treatment , it is primarily applied by frequent subconjunctival injection . however , the drug hardly directly impacts the filtration pathway , and high local drug concentration beneath conjunctiva is toxic to the corneal and conjunctiva . bio - amniotic membranes prepared from fresh amniotic membranes using lyophilizing techniques and co sterilization are transparent . studies on their biological ultrastructures found that they are rich in collagen fibers and reticular fibers arranged in woven status with mesh gap about 0.5 - 15 m and could absorb a large amount of drugs with size less than their porous size . because collagen degradation is slow , the absorbed drugs can be gradually released over an extended period to maintain local drug concentration at certain level for a long period . thus , bio - amniotic membrane is a good , semi - quantitative , membrane - controlled drug delivery system . borhani reported that the highest toxic 5-fluorouracil concentration is 0.25 mg / ml for retina and mannis reported that the highest toxic 5-fluorouracil concentration is 1 - 10 mg / ml for corneal endothelium . in this study , each bio - amniotic membrane can absorb maximum 75.828 g of 5-fluorouracil , far below its toxic concentration to eye . we also found that supplementing 5-fluorouracil to bio - amniotic membrane had no significant effects on retinal structure and function . because our sample size is small , its efficacy and safety need further in - depth , repeated investigation using a large sample size . in addition , we used 5-fu instead of mitomycin c to preliminarily explore the safety of the method because the latter has 100-fold stronger antifibroblast proliferation function . in short ,
background : to observe the impact of application of bio - amniotic membrane immersed in 5-fluorouracil solution in trabeculectomy on the retina in a rabbit model.materials and methods : healthy white new zealand rabbits were randomly assigned into three groups with 20 in each group . 
 bio - amniotic membranes of 4  5 mm immersed in either physiological saline / water for 10 min , or 25 mg / ml 5-fluorouracil solution for 5 and 10 min , respectively , were applied on rabbit eyes during trabeculectomy . at 7 , 14 , 21 , and 28 days of postoperation , five rabbits from each group were examined with electroretinogram ( erg ) . after being examined for eye pressure and bleb morphology , 
 rabbits were sacrificed by air embolism and their retinas were collected and examined by transmission electron microscopy ( tem ) . 
 in addition , 5-fluorouracil amount in bio - amniotic membranes was measured using high - performance liquid chromatography.results:each bio - amniotic membrane could absorb 59.004 g and 75.828 g 5-fluorouracil after being immersed in 5-fluorouracil solution for 5 and 10 min , respectively . 
 application of these bio - amniotic membranes in trabeculectomy could promote the formation of well - functioning bleb and maintain intraocular pressure , although it had no effect on retina structures as examined with erg and tem.conclusion:application of 5-fu soaked bio - amniotic membrane in rabbit eye trabeculectomy is effective and safe .