1. eduardoandreghetti@uol.com.br Scanning Electron Microscopy in Rabbit Corneas with Intrastromal Corneal Segment(ICS) Eduardo Andreghetti(1) Maria Rosa Bet Moraes Silva(2) Maria Aparecida Domingues(3) Victor Andrigheti Coronado Antunes(4) 1,4-Instituto de Oftalmologia de Assis & Botucatu Medical School(UNESP) 2,3-Botucatu Medical School(UNESP) -The authors have no financial interest in this study

2. eduardoandreghetti@uol.com.br I – Purposes To evaluate by scanning electron microscopy rabbit corneas implanted with ICS uncoated and coated with chondroitin sulfate, concerning with the epithelium, stroma, Descemet membrane and endothelium To evaluate by scanning electron microscopy rabbit corneas implanted with ICS uncoated and coated with chondroitin sulfate, concerning with the epithelium, stroma, Descemet membrane and endothelium

3. eduardoandreghetti@uol.com.br II – Methods  30 albino adult Norfolk rabbits  Proceeding from: Central Vivarium Botucatu Medical School -UNESP  Aproved by Ethical Comission for Animal Research Botucatu Medical School – UNESP (Botucatu, SP –Brazil)  Group G1 – ICS PMMA Classic (Grupo G2 – ICS PMMA+ Chondroitin Sulfate coating (Right Eye)  G1 + G2 Left Eyes – Control Group  Surgery Technic: Oliveira CS et al, 2004 (01)  Posoperatory drug:Ofloxacino 0.3% eyedrops each 6 hours – 20 days  ICS:thickness 125 micra, curvature 150º, apex diameter 5mm, two holes at each tip, pyramidal section with constant base line pyramidal section with constant base line 01) Oliveira CS, Moreira H, Wahab AS, Godoy G. Análise de nova técnica para o implante de anel de Ferrara no ceratocone. Arq Bras Oftalmol 2004;67(3):509-17.

4. eduardoandreghetti@uol.com.br II – Methods  Enucleation - both eyes at 60 º P.O. day  Division of corneas in two halves  Fixation in glutaraldeid 2,5% buffered phosfate 0,1 M pH 7.3, for at least 3 hours  Washing in deionized water  Pos-fixation in osmium tetroxide 0,5%  Dehidratation in crescent series of ethanol (from 7,5 to 100%)  Crtical Point with liquid CO2 ( CPD 020 / Pfeiffer – Vacuum Gmb-H-Lietchtenstein)  Corneal tissue assemblage in “stubs”  Metalization with 15 nm of gold (MED 010 / Balzers- Union-Liechtenstein) Scanning Eletron microscopes used:  QUANTA 200 FEI – Netherlands  100/515 PHILLIPS - Netherlands

5. eduardoandreghetti@uol.com.br III - Results Epithelium – Control Eye - OS Corneal crossection –Epithelium (left), endothelium (right) Epithelial surface

6. eduardoandreghetti@uol.com.br  Over ICS G2 – OD epithelium Sfoliation  p=0.81 III - Results G 1 - OD  epithelium membrane Rupture  p=0.83

7. eduardoandreghetti@uol.com.br 12 x 72 x G1 - OD Intense compactation of stromal lamellae anterior to ICS p=0.87 III - Results  Stroma - OS  Normal compactation of lamellae

8. eduardoandreghetti@uol.com.br  Normal Endothelium - OS III - Results Endothelium gaps at ICS way G1 - OD P=0.79 Descemet membrane G1 - OD Preserved in both groups

9. eduardoandreghetti@uol.com.br III - Results Endothelium Edema Endothelium edema at the border of ICS way G2 - OD P=0.78 Endothelium folds At the edge area of the ICSI way there were many endothelial folds(p=0.81) G2 - OD

10. eduardoandreghetti@uol.com.br ICS III - Results Inner view of ICS pushing corneal inner layers forward into anterior chamber –G2 OD ICS compacting stromal lamellae and It´s pyramidal Section – G2 - OD Microgaps along ICS polimeralstructure

11. eduardoandreghetti@uol.com.br IV- Comment  Epithelial sfoliation, compactness of stromal lamellae, mechanical folding of endothelium with different intensities and gaps between endothelium cells by disrupture of intercels bridges were the most important alterations in this study and were present in both G1 and G2 groups (with no diferent significance).  ICS scanning microstructure showed a great amount of gaps that may have some clinical importance

12. eduardoandreghetti@uol.com.br V - CONCLUSION  There are indications that leads to necessity of mechanical improve on the ICSs studied as the indications that could be studied concerning to standard surgical technic used in this study.  The ICS in both ( uncoated and coated) experimental groups had a very similar behavior concerning the studied parameters