Analysis of Corneal Response to Intra-Stromal Ring-Segment Implantation in Human Eyes. A Pathology Study J. Feito 1A, A. Astudillo 2, 1A, B.Baamonde 2, 1B, J. Alfonso 3, 2, J Merayo-Lloves 3. 1 Hospital Central de Asturias, Oviedo, Spain; A Pathology; B Ophthalmology 2 Surgery, University of Oviedo, Oviedo, Spain 3 Fundación Investigación Oftalmológica & Instituto Fernández-Vega, Oviedo, Spain INTRODUCTION Intracorneal ring (ICR, consisting of one segment) or intracorneal ring segments (ICRS, consisting of two semi-rings) refer to plastic ring (made from Poly-methyl metacrylate, PMMA) arcs inserted into the corneal stroma. ICR⁄ICRS can be used for: correction of low grades of myopia, treatment of keratoconus sometimes as an (alternative to transplantation) and for correction of residual myopia after laser treatment of severe myopia. An important advantage of ICRS is that the operation is reversible, i.e. the ring can be removed or replaced if necessary as only affects the periphery of the cornea. We want to evaluate the pathology findings observed in a patient who had an ICRS ring inserted on his cornea and who afterwards had penetrating keratoplasty. MATERIALS & METHODS Human cornea implanted with PMMA ring-segments (Ferrara ring, Ferrara & Hijos SL, Boecillo, Spain) from patient with keratoconus were analyzed for pathology studies: conventional histology and immunohistochemistry. The ICRS was placed using mechanical dissection and retired because there was a risk of corneal perforation after a temporal extrusion of the segment. Keratoplasty was performed 5 months later. The corneal button was bisected for histological and immunohistochemical study. Tissue sections were placed on formalin-fixed paraffin-embedded blocks stained with Hematoxilin-Eosin, Masson trichrome, and periodic acid-Schiff solutions and examined by light microscopy. Immunohistochemistry was performed on the sections of hemicorneal button. Tissue sections were dewaxed in xylene, rehydrated through a graded ethanol series, and washed with phosphate-buffered saline. Antigen retrieval was achieved by heat treatment in a water bath (20 minutes at 96 ºC) using a pH 9 target retrieval solution. Samples were stained using a sensitive polymer-based detection system (En Vision Plus, DakoCytomation automated immunostainer). The reference, and dilution factor of primary antibodies were as follows: vimentin (Biogenex MU074-UC, 1/200), and CD34 (Dako M7165, 1/100); to detect keratocytes; a marker of stromal cell activation CD 10 (Novocastra NCL-L-CD10-270, 1/80); a marker of marophagic phenotype CD 68 (Dako M0814, 1/2000); and a-smooth muscle actin to detect myofibroblast phenotype (Dako M851, 1/50). RESULTS We observed an atrophic epithelium in the area above the implant with a decrease in the number of layers and thinning of the few layers remaining, No irregularities or disruptions were observed in the Bowman membrane. There was also a reduction in the number of keratocites over the implant. Around the implant there was a de-orientation of the cells and hypercelularity, staining positive for SMA that mark the stromal cells as differentiated towards the myofibroblast phenotype. The stroma around the ICR segments stained more intensely with PAS than normal corneal stroma do. Also, adjacent to the implants there were stromal cells positive for CD 10 (neutral endoprotease), CD 34 (keratocites). Interestingly, there were keratocites positive for CD 68 surrounding the channel (macrophagic phenotype). These findings suggest that ring segments implants modulate the epithelial and corneal stroma with atrophy of the epithelium and adjacent stroma and changes in the phenotype of corneal keratocites around the ring segment. CONCLUSIONS Although these PMMA corneal inserts appear to be well tolerated, from the results of long-term clinical studies in humans, has been demonstarred the changes in keratocyte phenotype suggesting an active tissue repair process and physiological stress. Intra-stromal ring segments reduce the number of keratocites and epithelial cells and modulate the phenotype of keratocites with expression of markers for macrophages (CD 68), myofibroblast (SMA) and endoprotease (CD10). None of the authors have commercial interest on this study REFERENCES 1.- Histopathological Analysis of Post-Laser-Assisted In Situ Keratomileusis Corneal Ectasia With Intrastromal Ring Segments. Spirn MJ, Dawson DG, Rubinfeld RS, Burris C, Talamo J, Edelhauser HF, Grossniklaus HE. Arch Ophthalmol Nov. 2.- Histopatholocial findings after intracorneal ring segment implantation in keratoconic human corneas. Samimi S, Leger F, Touboul D, Colin J. J Cataract Refract Surg Feb. 3.- Histologic Evaluation of Corneal Stroma in Rabbits After Intrastromal Corneal Ring Implantation. Twa MD, Ruckhofer J, Kash RL, Costello M, Schanzlin DJ. Cornea Mar. D901¿? Hematoxilin-Eosin 10xSmooth Muscle Actin 20x CD 10 10x DISCUSSION The findings of epithelial atrophia and the disminution in the number of keratocytes above the implant are already well described. We haven´t observed changes in the Bowman membrane although they have been related with keratoconus. There is also evidence about a immunophenotipic change in the keratocites. The SMA staining is already described, also the reduction in the CD 34 positive keratocites. To our knowledge, this is the first report about the CD 10 and CD 68 role in the apoptotic changes tipical of this implanted corneas. Vimentin 20x PAS 10xCD 68 10x