The Establishment And Preliminary Study Of Eyeball And Face Combined Transplantation Model

With the development of society,traffic accidents,fallings,violent attacks and industrial accidents are increasing,and head and face injuries caused by direct or indirect effects of external violence on the head and face are also increasing.Violent injury to the forehead or eye will not only cause partial facial skin or organ defects,but might also lead to the damage of optic nerve,resulting in impaired vision and visual field,or even permanent loss of vision.Therefore,preserving the vision of these injured people and even repairing the facial defects are urgent problems to be solved.At present,the clinical development of facial transplantation has been developed well.By the end of 2016,thirty-seven facial transplantations had been successfully performed worldwide.Compared with conventional surgical repair surgery,facial transplantation surgery can help patients restore facial function and reshape appearance better.Patients can gradually restore swallowing,chewing and other functions through transplant operation.However,the development of eyeball transplantation technology is relatively slow.At present,it mainly focuses on the basic research stage.New research shows that eye transplants performed on mammals can reconstruct the blood supply of the eyeball,and some electrical activity of the retina can also be detected.However,the restoration of animal vision is hard due to the non-reproducibility of the optic nerve.The optic nerve is a special somatosensory nerve composed of the retinal ganglion cell(RGCs)axons.RGCs are located in the retina and can be divided into at least 18 different forms depending on the information they carry from the same photoreceptor.Each RGCs has a typical neuronal structure and is the only cell in the retina that can trigger action potentials.RGCs' dendrites extend across the retina to pick up signals at various levels.They pass through a long axon which clumps together to form the optic nerve to specific areas of the brain via the optic chiasm and optic pathways to produce vision.However,as a kind of nerve cell,RGCs,like other nerve cells,have a very weak regeneration ability due to its low intrinsic developmental characteristics,reduced organelle kinetics,low levels of neurotrophic factor support,and inhibitory substances produced by glial cells.Therefore,when the optic nerve is damaged,the axon of RGCs will degenerate,eventually leading to apoptosis.The number of RGCs is greatly reduced and cannot be reversed.Therefore,people pay more and more attention to the research about the repairing and regeneration mechanism of the optic axon,mainly including: 1.Neuroprotective drugs: neuroprotective drugs are used to prevent further damage to RGCs and promote its recovery.Weishaupt el al suggested that erythropoietin could protect the optic nerve and promote its regeneration,and it also could improve the visual acuity of patients with optic nerve injury by giving erythropoietin cotton thread.Lens protein,calcium channel antagonists can play a similar role.2.Neurotrophic factor: at least 20 neurotrophic factors have been found to promote the survival of RGCs and the regeneration of axons.Most of them work by altering the microenvironment in which nerve cells are located.Leibinger et al have been reported that knockout of the ciliary neurotrophic factor gene in mice can accelerate the death of the optic nerve RGCs and inhibit the regeneration of axons.Di Polo et al also reported that the application of brain-derived neurotrophic factor can significantly improve the survival of the optic nerve and promote the growth and differentiation of neural stem cells.3.Stem cell therapy: it has been reported that there are retinal stem cells in the peripheral area of the ciliary body and the retina.When the retina is damaged,these cells will proliferate and differentiate into different types of light-sensitive cells and retinal cells to play a repair role.In addition,embryonic stem cells,mesenchymal stem cells can also play a similar role.4.Peripheral nerve transplantation: by transplanting a segment of peripheral nerve to the injured area,it serves as a medium to provide support for the extension of regenerated axon.Meanwhile,schwann cells of peripheral nerve also produce a series of neurotrophic factors to promote the regeneration of axon.In recent years,due to the progress of microsurgical techniques and immunomodulation theory,as well as the new findings in the field of nerve regeneration,combined eyeball and facial transplantation become a reality gradually.In view of the highly specialized eye function and its unique anatomical structure,as well as the psychological trauma caused by facial defects and blindness,the combined eyeball and facial allograft surgery will show great attraction for the recovery,replacement and reconstruction of non-functional eyeballs in such patients.Therefore,for the short-term goal of finding new ideas to promote optic nerve regeneration and the long-term goal of making such operation being a clinical reality,it is necessary to establish a reliable animal model to test its feasibility and specific effects,as well as evaluate the survival of RGCs and the extension effect of axon.In view of this,we decided to finish the study of combined eyeball and facial transplantation by surgical design,model establishment,immunofluorescence detection: 1.Design and establishment of experimental animal model of eyeball and facial joint transplantation;2.Evaluation and discussion of experimental animal model of eyeball and facial combined transplantation.Part one:Design and establishment of experimental animal model of eyeball and facial combined transplantation.Objective:The concept of treating visual function unit as transplantation object is proposed in this study.The aim of this study is to design and establish a complete and standard surgical scheme,to establish a tissue model in vivo,and provide anatomical basis for the reconstruction of optic nerve by providing pedicled tissue in vivo.We also evaluate the feasibility and stability of the surgical scheme and model.Methods:Thirty adult healthy male Lewis rats were randomly divided into two groups: donor group(15 rats)and recipient group(15 rats).The animal model of combined eyeball and facial transplantation was established by the access of donor's eyeball and facial skin flap,the preparation of recipient's eyeball and face,and combined eyeball and facial transplantation.The operation time,blood loss,postoperative pupil size,light reflection and animal survival were evaluated.Results:(1)On the basis of udnerstanding the anatomical structure,we persistently explored and formulated specific surgical plans.With the assistance of surgical microscope,microsurgical instruments and other equipment,the obtained donor eyeball and skin flap were transplanted to the recipient's face rapidly,and blood vessels and nerves were connected successfully.(2)The operation time and blood loss of 15 groups were as follows: donor eyeball flap: operative time 1.02±0.16 hours,bleeding volume 4.01±0.21 ml;recipient eyeball preparation: operation time 0.59 ±0.07 hours,blood loss 1.35±0.12 ml;combined transplantation of the eye and face: the operation time was 1.77±0.10 hours,and the amount of bleeding was 3.32±0.19 ml.(3)The mean diameter of the pupil of the healthy side eyes of the 15 postoperative rats was 0.69±0.02 mm,with 100% direct light reflex and 0% indirect light reflex.The mean diameter of the pupil of 15 transplanted eyes was 3.06±0.17 mm,and there was 0% direct light reflex and 0% indirect light reflex.The eyeball had no light sense and no visual function for the time being.The survival rates of 15 rats on day 3,day 7,day 14 and day 28 after surgery were 93.3%,93.3%,86.7% and 80%,respectively.(4)After operation,the skin of rats was warm,ruddy and elastic;the wound secretion was few,there was no hematoma formation,there was no obvious black-purple or pale;the wound healed well and hair regenerated;the weight of rats was normal,and the whole body was in good condition.Revascularization was successful: arterial blood supply was good,venous reflux was smooth,and tissue blood supply was basically normal.Conclusion: Through the techniques of vascular reconstruction and nerve suture,a combined eyeball-facial transplantation model with visual function unit as the transplantation object was initially established.The experimental animal model had good arterial blood supply,smooth venous circulation and normal tissue blood supply.However,it has no visual function for the time being.Part Two:Evaluation and discussion of experimental animal model of eyeball and facial combined transplantation.Objective:On the basis of the model above,we evaluate the survival of RGCs in recipient rats after combined eyeball-face transplantation in order to provide histocytological and molecular biological support for the operation effects and feasibilityMethods:Sixty Lewis model rats of Eyeball-facial transplantation surgery,on the basis of using randomized grouping method,were divided into 3 groups on average: experimental group(eye facial transplantation animal model),control group(a similar facial surgery flap incision without optic neurotomy)and blank group(without any treatment such as surgery).On day 3,day 7,day 14 and day 28,samples of eyeball and optic nerve were collected for immunofluorescence staining.RGCs and optic axons were counted and compared respectively,and their gap-43 immunofluorescence staining integral optical density value(IOD)was determined.At the same time,the donor RGCs were detected to confirm whether the regenerated axons escape the graft injury and grew into the recipient optic nerve fibers.Result:(1)TUJ-1 staining and RGCs counting: RGCs of three groups were counted on day 3,day 7,day 14 and day 28: experimental group: 15.0±1.0,11.4±0.55,7.4±0.55,6.2±0.45;control Groups: 17.6 ± 0.55,17.4 ± 0.55,17.2 ± 0.84,16.4 ± 0.55;blank group: 18.2 ± 0.82,18.2 ± 0.82,17.4 ± 0.89,16.8 ± 0.84.On day 3,day 7,day 14 and day 28,the difference between the experimental group and the control group was statistically significant(P<0.05).(2)Retinal GAP-43 staining: On day 3,day 7,day 14 and day 28,the IOD values of retinal GAP-43 fluorescence intensity in the experimental group and the control group had a statistically difference(P<0.05).(3)Optic nerve GAP-43 staining: On day 3,day 7,day 14 and day 28,the difference of IOD values of optic nerve GAP-43 fluorescence intensity between the experimental group and the control group was statistically significant(P<0.05).Conclusion:On the basis of the model,further exploration was made in this study.We found that after transplantation,retinal ganglion cells decreased over time,but they still survived.This model provided an animal experimental basis for further study of visual function.