Experimental Study Of The Evolution Of Amniotic Membrane After Allogeneic Cultivated Limbal Epithelial Transplantation

PurposeUse of different methods to observe human amniotic membrane changes aftertransplantation of allograft limbal stem cell patch which human amniotic membrane as acarrier, provide a reference for this way of clinical treatments.Method1. Building and evaluation of ocular alkali burn animal model:1) model building:select35healthy New Zealand white rabbits eyes, right eye as the model eye, left eye forthe normal control eyes, cut off the third eyelid, put filter paper soaked with1mol/LNaOHsolution affixed to the limbus for30s, at the same time drop50μl1%NaOH solution in theconjunctiva, a large number of physiological saline solution wash lasts for5minutes,coated with0.3%ofloxacin eye ointment in conjunctival sac qn, regularly slit lampobservation and photography.2) model evaluation: to observe the model of ocular surfacestability underwent slit lamp and score, RT-OCT examination, in vivo confocal microscopyexamination, impression cytology observation, histological examination, Schirmer test.2. Implement transplantation surgery use patch of the amniotic membrane as a carriercultured limbal stem cell and have a dynamic follow-up observation of amniotic change after operation:1) according to the clinical score standard selected6-9points model21eyes for the surgery, postoperative suture eyelid fissure.2) after operation1d,7d,0.5,1,2,3months, slit lamp observation; after surgery1,2,3months, RT-OCT, in vivo laserconfocal microscopy, different time points were taken76(cured group3, the failure group3)cornea histopathological HE staining and observed under the microscope camera.ResultFirst, the evaluation of ocular alkali burn model1.5months±10d after the model, according to the chemical injury clinical score,6-9points,28eyes of35(80%),4eyes <6points,1eye>10points,1eye corneal perforation,1eye endophthalmitis, and ultimately atrophy of eyeball. The slit lamp examination revealedthe cornea and conjunctivitis is stabilized, corneal opacity, a large number of new vesselsinto the corneal superficial stroma, iris is unclear, sodium fluorescein were negative forstaining the cornea and conjunctiva; RT-OCT examination revealed corneal thickening, thescan is difficult to penetrate the cornea, the visible shadow of the corneal stromaneovascularization; in vivo confocal microscopy examination showed epithelial cellsarranged in disorder, inflammatory cell infiltration, but not normal epithelial cells structure,a large number of new blood vessels in the corneal superficial stroma, red blood cell flowin the tube, nerve tissue severely damaged, stromal scarring, fiber derangement,inflammatory cell infiltration, it is difficult to scan to the endothelial cell layer; impressioncytology to check corneal and conjunctival goblet cells see corneal epithelial cells withirregular shape, find goblet cells in the corneal epithelium, the number of goblet cells inconjunctival epithelium less than normal; corneal tissue HE staining cortical epitheliumcells derangement, new blood vessels in the corneal superficial stroma, see red blood cellswithin the lumen, thickening of the matrix, fiber derangement, inflammatory cellinfiltration, deep matrix structure is still normal, no significant changes in the endothelialsurface; Schirmer test results showed that the model tears reduced fluid secretion, thedifference between the control eyes statistically significant, P <0.05.Second, changes after transplantation of amniotic membrane patch 1. The results is divided into two categories: cure and failure groups,after1monthpostoperative,21eyes of cured12, failed9,2and3months later2and1eyes failed. Stableocular surface of the cured group, the cornea is still transparent, the iris is still clear, and noneovascularization or limbal margin only2mm a small number of neovascularization;failure group ocular surface neovascularizated again, corneal opacity, iris is unclear.Different results of operations, different changes of the amniotic membrane.2. Slit-lamp observation: after operation1d, amnion exist, amniotic transparency ispoor, pale, iris is unclear, sodium fluorescein staining was negative,7d amniotic thinnerthan previous, transparency slightly improved,0.5months, the amniotic no dissolved off.Cure group,1months, amniotic membrane yet transparent, thickness, iris is clear, haveno neovascularization;2months, the amniotic vaguely exist, not find the typical amnion, itis difficult to to determine whether amniotic membrane;3months, ocular surface stabilityof the corneal epithelium, transparency, no obvious neovascularization at the limbus, cracksidentify unclear whether amniotic exist,sodium fluorescein staining was negative.Failure group,1months after operation, new blood vessels grow into the periphery ofthe cornea,≤1/2quadrant, less transparent, below epithelial to see a frosted glass-like graycloudy, like amniotic exist;2months, neovascularization at the periphery of thecornea,>1/2quadrant, the amniotic membrane is unclear, frosted glass-like structuredisappeared subcutaneously in the local area on the cornea;3months, ocularneovascularization again, the whole cornea visible neovascularization, amniotic membraneis unclear, and sodium fluorescein staining was negative.3.RT-OCT examination: cure group,1months after operation, amniotic membraneperformance continuous reflective enhanced arc structure under epithelial layer, welldefined with epithelium and stroma layer, thickness is about36±5.4μm;2months stillsubcutaneous continuous reflective enhanced arc structure visible on the cornea, thethickness is25±3.4μm;3months amnion-like reflective enhanced arc structure is still exist,a thickness of15±1.9μm.Failure group,1months after operation amniotic exist, the performance of continuous reflective enhanced arc structure under epithelial layer, thickness about38±5.8μm, thebelow corneal stromal edema;2month, amniotic layer is thinner than previous, yetcontinuous, approximately27±3.7μm, under amniotic membrane of new blood vesselsvisible shadow;3months membrane-like structure is not continuous, the shadow ofneovascularization increased, amniotic membrane thickness of about9±1.7μm. Differenttime points between the two groups,3months after operation, the difference was statisticallysignificant (P <0.05).4. In vivo laser confocal microscopy:1month after operation, the cure group observedto the bottom of the epithelium showing homogeneous, reflecting enhanced cell-freestructure, surrounded by a little of inflammatory cells infiltration;2months, reflective of theamnion gradually weakened, around the occasional inflammatory cell infiltration, noneovascularization;3months, can still be observed showing homogeneous, reflectingenhanced cell-free structure, with no inflammatory cell infiltration, no neovascularization,the endothelial cell layer can be observed;The bottom of the amnion epithelium showing homogeneous, reflecting enhancedcell-free structure and large number of inflammatory cells was observed1month later inthe failure group; Neovascularization can be seen after2months, inflammatory cellsinfiltration;3months later, We just found the reflective enhanced cell-free amnioticmembrane structure in some place, surrounded by added neovasculars and increasedinflammatory cell, the endothelial cell layer couldn’t be observed.5. Corneal tissue HE staining: amniotic membrane structure were integrated,and thejunction between the corneal stroma and the amniotic membrane were close-knited in thecure group1month later; the junction between the corneal stroma and the amnioticmembrane were looser than formerly2month later.with a little inflammatory cells could beseen.after3months, the whole amniotic membrane thinner than before, and the structureof the amniotic membrane was integrated, the junction between the corneal stroma and theamniotic membrane were close-knited,and below the amniotic membrane, corneal stromastructure arranged in neat rows. Amniotic membrane structure were integrated,and the junction between the cornealstroma and the amniotic membrane were loosen in the failure group1month later; and inthe same time, large number of inflammatory cells and neovascularizations could be seen;After2months, amniotic membrane became thinner than before, the structure is stillcontinuous, inflammatory cells and newborn blood vessels could be seen obvious,,Corneal shallow matrix scared;After3months, the amniotic membranes were thinner thanever,and the structure no longer integrated, inflammatory cells、scared tissues and newbornblood vessels could be seen severe clear.Conclusion1. Alkaline solution burns the New Zealand White rabbits limbal cornea caused by thecircular filter paper soaked in1%NaOH,and eye drops low consistence lye simultaneouslycan successfully established the New Zealand white rabbit ocular alkali burn model ofcorneal limbal stem cell deficiency2. Changes of amniotic membrane conditions after transplantation are closely relatedto the circumstances of surrounded tissues.If the surgery was effective. the amnioticmembrane as a stable carrier could be existe in the corneal stroma for a long time；if theoperation fails, the amniotic membrane would disappeared gradually by the inflammatorycells and neovascularization.