Cornea - ARVO

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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaperformed using qPCR.Results: Gene expression of SP and NK-1 R in human cornealfibroblasts was verified in the primary cultures. The presence of SPwas also confirmed at the protein level using immunocytochemistry.Western blot and immunocytochemistry showed that the full lengthisoform of NK-1 R is the dominant isoform. Anti-Fas treatmentinduced apoptosis in the cells, and SP augmented this response, asseen by an increase in activated apoptotic enzymes. Treating cellswith SP also lead to a decreased amount of phosphorylated/activatedAkt. The effects of SP were reduced with a specific NK-1 Rantagonist.Conclusions: SP is produced by human corneal fibroblasts. Anti-Fastreatment induces apoptosis in these cells and SP augments thisresponse through a NK-1 R specific pathway, by reducingphosphorylation of Akt. This suggests that SP may play a role in theapoptosis occurring in corneal wound healing.Commercial Relationships: Peter Boman, None; Sandrine LeRoux, None; Patrik Danielson, NoneSupport: Swedish Society of Medicine, Cronqvist Foundation, KMAFoundation, Ögonfonden FoundationProgram Number: 5228 Poster Board Number: C0147Presentation Time: 2:45 PM - 4:30 PMTGF-β1 modulates the functional expression of the NK-1receptor in human corneal fibroblastsSandrine LE ROUX, Peter Boman, Patrik Danielson. Anatomy, Dept.of Integrative Medical Biology, Anatomy, Umeå University, Umeå,Sweden.Purpose: Transforming growth factor beta 1 (TGF-β1) is a cytokineinvolved in a variety of processes, such as differentiation offibroblasts into myofibroblasts. TGF-β1 has also been shown to delaythe internalization of the Neurokinin-1 receptor (NK-1 R) after itsactivation by its ligand, the neuropeptide substance P (SP).NK-1 R comprises two naturally occurring variants, a full-length anda truncated form, triggering different cellular responses. SP has beenshown to be involved in important events in the cornea essential forthe proper organization of collagen fibers and the transparency of thestroma. An impaired signaling through NK-1 R could thus disturb theextracellular matrix and impact the vision quality.We hypothesize that TGF-β1 modulates the expression pattern andfunction of the NK-1 R in human corneal fibroblasts. The purpose ofthis study was to test that hypothesis.Methods: Cultures of primary corneal fibroblasts were set-up withcells derived from healthy corneas, obtained from donatedtransplantation graft leftovers. Immunocytochemistry for the TGF-βreceptor type I (TGF-βRI) and NK-1 R was performed. Geneexpression was assessed with qPCR. Western blot was used toconfirm the gene expression results at the protein level.Results: Expression of TGF-βRI was confirmed on corneal stromalderived fibroblasts. Treating the fibroblasts with TGF-β1significantly reduced the expression of the NK-1 R gene.Furthermore, immunocytochemistry for NK-1 R showed that the fulllengthversion of the receptor is reduced after treatment by TGF-β1(figure).Conclusions: TGF-β1 downregulates the gene expression of NK-1 Rin human corneal fibroblasts, and the results also suggest that itdisturbs the signaling pathways triggered by the receptor by reducingthe full-length version of it, which might explain the delay ininternalization after activation by SP seen with TGF-β1 treatment.Immunostaining of human corneal fibroblasts for the inner loop(green; c, f) and the C-terminal (red; b, e) of the NK-1 receptor.Merged pictures in a (of b and c) and d (of e and f), with DAPI addedfor demonstrating nuclei (bluish). The inner loop is found in both thefull-length and the truncated isoform, whereas the C-terminal is onlypresent in the full-length receptor. Untreated cells (a-c) showoverlapping immunostainings, whereas cells treated with TGF-β1 (df)show reduced staining for the full-length isoform.Commercial Relationships: Sandrine LE ROUX, None; PeterBoman, None; Patrik Danielson, NoneSupport: Swedish Society of Medicine; Cronqvist Foundation; KMAFoundation; Ögonfonden FoundationProgram Number: 5229 Poster Board Number: C0148Presentation Time: 2:45 PM - 4:30 PMA Long-Lasting Scar in Murine CorneaAndrew Hertsenberg, Sayan Basu, Yiqin Du, James L. Funderburgh.Ophthalmology, University of Pittsburgh School of Medicine,Pittsburgh, PA.Purpose: Penetrating keratoplasty is a successful therapy for cornealscarring but, due do graft failure and declining donor tissueavailability, new treatment methods for corneal scarring and haze areunder investigation. Animal models of long-term scarring areimportant tools to assess these new approaches. The purpose of thisstudy is to evaluate a penetrating murine corneal injury model forlong-term changes in stromal transparency and stromal connectivetissue that replicate those typical of human corneal scarring.Methods: A wound was created by surgical removal of a 1 mm fullthickness button from C57B6 mouse corneas. Corneas were imagedby optical coherence tomography (OCT) 48 hr after wounding andweekly for 2 months. Average pixel intensity for each OCT stack wasmeasured using ImageJ. After 2 months, eyes were enucleated andfixed. Corneal collagen was examined with single harmonicgenerated (SHG) images by 2-photon microscopy. Corneas weredigested in chondroitinase ABC and stained as whole mounts withantibodies to collagen III, biglycan, chondroitin-4-sulfate, anddecorin. Birefringence was analyzed by viewing the mounted corneasthrough a cross polarizing filters.Results: Two months after wounding, polarized light microscopyrevealed a regular, patterned collagen birefringence in unwoundedcorneas, but a markedly irregular pattern in wounded area. 2-photonimages confirmed the loss of collagen organization in the scar.Immunohistochemistry revealed localized concentrations of collagenIII and biglycan at the site of the wound. OCT images documentedcorneal thickening and a distinct increase in light scatter intensity inthe wound region persisting at least 2 months after wounding.Conclusions: Previous studies showed the transient appearance offibroblasts and myofibroblasts in penetrating murine corneal wounds,however, presence and persistence of non-transparent scar tissue has©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneanot been documented. This study demonstrated light scatter, haze,disruption in collagen organization, and accumulation of extracellularmatrix components typical of human corneal scars to persist in thecentral cornea for at least 2 months after wounding. The mouse is anideal animal model due to its low cost, genetic manipulability, andrelative homology to humans. As such, a mouse model that closelyresembles corneal scarring would expedite the search for analternative treatment to corneal haze in humans.Commercial Relationships: Andrew Hertsenberg, None; SayanBasu, None; Yiqin Du, None; James L. Funderburgh, NoneSupport: NIH Grants EY016415, P30-EY008098, Eye & EarFoundation of Pittsburgh, Research to Prevent BlindnessProgram Number: 5230 Poster Board Number: C0149Presentation Time: 2:45 PM - 4:30 PMAUTOLOGOUS PLATELET RICH PLASMA THERAPY INRESISTANT CORNEAL ULCERSYakup Aksoy 1 , Uzeyir Erdem 2 . 1 Ophthalmology, Gülhane MilitaryMedical Academy Haydarpasa Education Hospital, istanbul, Turkey;2 Ophthalmology, Gülhane Military Medical Faculty, Ankara, Turkey.Purpose: To assess the results and the application procedures of theplatelet rich plasma therapy which is a novel option in resistantcorneal ulcers?Methods: We performed limited debridement after harvestingadequate samples from the margin of the lesions for culture and HSVPCR in nine eyes of the eight corneal ulcer sterile patients who had ahistory of resistant corneal ulcer one or more year inconclusive trialsof various therapies. After the debridement all eyes administeredautologous platelet rich plasma (8 drops/day).Results: Corneal ulcers of all cases were healed in about 4,3(±2,3)weeks. Visual acuity improvement was 4,1(±2,4) lines. In one case,epithelial defect relapsed after six months but, healed in 3 weeksagain using the same protocol. we also applied wet PTK in 4 eyesafter the treatment period.Conclusions: We found that autologous platelet rich plasma therapyis effective in resistant corneal ulcers as an adjunctive therapy.Further investigations should be done in wider and different caseseries to prove its benefits in resistant corneal ulcers and also indifferent clinical applications.Commercial Relationships: Yakup Aksoy, None; Uzeyir Erdem,NoneProgram Number: 5231 Poster Board Number: C0150Presentation Time: 2:45 PM - 4:30 PMInitial Evidence that Immune Cell Infiltrates are CandidateSources of Post-Surface Ablation Stromal RougheningDaniel J. Gibson, Gregory S. Schultz. Obstetrics and Gynecology,University of Florida, Gainesville, FL.Purpose: To investigate potential cellular sources of previouslyobserved post-surgical roughening of the stroma in surface ablatedcorneas.Methods: New Zealand White rabbits were anesthetized andreceived bi-lateral 6.0mm x 125µm deep excimer phototherapeutickeratectomy wounds. Rabbits were euthanized daily to generate atime line of wound healing up until re-epithelialization. Immediatelypost-euthanization, the corneas were excised and fixed overnight. Thefixed tissues were paraffin embedded, sectioned, and stained withH&E. The histological time course was grossly analyzed by lightmicroscopy, with an emphasis on finding non-epithelial cells enteringthe wounded stromal surface.Results: Based on nuclear morphology, there were many neutrophilspresent in the anterior stroma with the highest concentration being inregions which were not yet re-epithelialized. Several of theneutrophils were observed in proximity with stromal fibroblasts.Several cellular masses were observed entering the wounded stromaahead of the migrating epithelium with nuclei shaped like neutrophilsor another class of granulocyte. The cells were either still round andon the surface or beginning to flatten and enter the stroma. In at leastone sample, a neutrophil was present in front of an epithelial frontwhich had entered the stroma.Conclusions: Neutrophils are in the right place and at the right timeto be considered as the source of post-surgical, biologically induced,stromal roughening. Hypothetically, the proteolytic activity of theseinfiltrating cells and the tissue displaced by them as they migrate intothe stroma creates “tunnels” which can subsequently be entered bythe migrating epithelium. Additionally, the crudely apparent cell-cellinteraction between the neutrophils and fibroblasts may have a role inactivation of the fibroblasts. Given that topical steroids have beenreported to be reduce haze and that they are potent neutralizers ofneutrophil activities, it may be through a combination of reducedstromal roughening and reduced fibroblast interactions. Additionalwork is needed to validate this proposed mechanism of action, but theobservations reported herein strongly suggest that the neutrophils areat least responsible for biologically induced roughening of the stromafollowing surface ablation surgery.Commercial Relationships: Daniel J. Gibson, None; Gregory S.Schultz, NoneSupport: NEI R01-EY05587Program Number: 5232 Poster Board Number: C0151Presentation Time: 2:45 PM - 4:30 PMOutcomes of Traumatic Dehiscence of Penetrating Keratoplastyversus Severe Open-Globe InjuryTherese Peron, Maria A. Woodward, Roni M. Shtein. Department ofOphthalmology, University of Michigan, Ann Arbor, MI.Purpose: To compare outcomes of patients with traumaticdehiscence of penetrating keratoplasty (PK) to those with severe openglobe (OG) injuries.Methods: A retrospective review of 160 consecutive patients withsurgical repair of traumatic open globe injuries at a single center from2006 to 2012 was conducted. Of those, 36 patients underwent repairof dehisced PK, 16 of which had adequate data to calculate theOcular Trauma Score (OTS). OTS was calculated for the remainingnon-PK open globes, resulting in 34 eyes with OTS of similarseverity. Ophthalmic and demographic factors were analyzed using t-tests, Pearson correlation coefficients, chi squared tests, and Fisher’sexact test.Results: There was a trend of patients with dehisced PK being olderthan the OTS-matched patients with OG injuries (60 vs.48; p=0.06).In the overall cohort, older age correlated weakly with lower OTS (r=-0.4, p=0.008). There was no correlation between sex and OTS(p=0.20). Associated lens injury at the time of initial trauma wasmore common in the PK group (p=0.03), but was not significantlyassociated with poor OTS (p=0.18). The extent of the ocularlaceration was not significantly different between the groups(p=0.22). Of the 40 patients with postoperative follow-up, 10required subsequent enucleation - 1 from the PK group and 9 fromthe OG group (p=0.12). Of the remaining 30 patients, 67% requiredsurgery beyond the initial repair. Additional surgeries were similarlylikely in both groups (p=1.00). Two patients developedendophthalmitis, 1 from each group. Patients with dehisced PKrequired subsequent cornea surgery more often than those with OGinjuries (p=0.0025), but had similar rates for subsequent retina,glaucoma, and lens surgeries (p=0.12, p=0.55, p=0.23, respectively).The mean final visual acuity of the PK group was 20/400 versus20/100 in the OG group, but this was not statistically significant©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
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Cornea - ARVO

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