Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaVickery E. Trinkaus-Randall, Albert Lee, Kelsey Derricks, MatthewNugent. Ophthalmology L904, Boston University Sch of Med,Boston, MA.Purpose: Damage to corneal nerves can originate from traumaincluding surgery, infection and chemical injury. Previously wedemonstrated that injured neuronal and epithelial cells release ATPand induce a rapid response that is detected by imaging Ca2+mobilization. Our immediate goal was to understand how neuronalepithelialsignaling was altered under environmental stress.Methods: Cell cultures and organ cultures were used to determinecell communication.To perform the experiments we used a combination of a confocal(Carl Zeiss inverted LSM 700 and 510) and a multiphoton/confocalmicroscope system (Carl Zeiss upright LSM 710 NLO). Ca2+mobilization was monitored in cell-cultures and in corneas fromThy1-GFP-rats. To analyze the response to stimuli we developed acustom MATLAB software script to analyze calcium flux movies.The script can individually identify cells, normalize each calciumsignal value to the cell’s individual background, and elucidate themagnitude of response, and clusters of coordinated cells.Results: In intact corneas from Thy1-GFP-rats, the fluorescentsensory neurons were detected passing through the basal lamina andthe thin apical processes entered the basal epithelium labeled with aCa2+ indicator dye. In cultured cells the ATP stimulated responsegenerated by epithelial wound medium generated a rapid wave thatwas inhibited using Apyrase, a nucleotidase. In contrast media frominjured neuronal cells elicited both the rapid wave and a secondslower wave that was present in multiple cell clusters.Communication between cells was delineated with custom MATLABcell clustering analysis. The neuronal wound media was dissectedusing ionotropic (N-methyl-D-aspartate (NMDA) receptor inhibitorsand Apyrase where the latter abrogated the first peak but left theoscillatory glutamatergic response, while NMDA inhibitorsdiminished only the secondary response. Factors released by neuronalcells facilitated directed epithelial cell migration. When cells weresubjected to neuronal media under environmental stress there was adecrease in Ca2+ mobilization and cell clusters. In addition there wasa change in localization of an NMDA receptor (NR1).Conclusions: These results suggest that two separate modes of Ca2+mobilization mediate cell communication and provide insight intomechanisms that corneal nerves and epithelia signal to each other inresponse to injury.Commercial Relationships: Vickery E. Trinkaus-Randall, None;Albert Lee, None; Kelsey Derricks, None; Matthew Nugent, NoneSupport: NIH EY06000, Mass Lions Eye Research Fund, NewEngland Corneal Transplant FundProgram Number: 5978Presentation Time: 11:30 AM - 11:45 AMCiliary neurotrophic Factor accelerates corneal NerveRegeneration in a Murine ModelRudolf F. Guthoff, Maria Reichard, Marine Hovakimyan, OliverStachs. Ophthalmology, University of Rostock, Rostock, Germany.Purpose: The aim of this study was the in vivo examination of themouse subbasal nerve fibre plexus (SBP) during regenerationprocess. Particularly, investigations addressed the regenerationcapabilities of the injured SBP, and the influence of local ciliaryneurotrophic factor (CNTF) application on the regeneration process.We have created a simple yet effective and well-tolerated injurymodel designed to influence a distinct corneal nerve area.Methods: Twelve-week-old BALB/c mice were included in thisstudy. A circular incision through corneal epithelium and anteriorstroma was generated with a custom-made guided trephine system tocut the nerves in SBP. The surgery was performed unilaterally.Animals were subdivided in 2 groups, one of them becoming CNTFeye drops 3 times daily. In vivo confocal laser scanning microscopy(Heidelberg Retina Tomograph/Rostock Cornea Module) was used tocharacterize the SBP before and up to 8 weeks after surgery. Nervefibre density (NFD) was determined with the semi-automatic nervetracing program NeuronJ.Results: NFD was reduced in both groups 24 hours after circularcorneal incision. The regeneration of subbasal nerve fibres was basedon sprouting out of stromal nerves within the cut and on regrowth ofsubbasal nerves over the scar from outside the cut. The statisticallysignificant NFD reduction was observed for up to 3 weekspostoperatively in both groups, with and without CNTF, whencompared to intact controls. The neuroprotective influence of CNTFeye drops was observed as early as 1 week after injury. At that timepoint the CNTF group displayed 38.39 % of baseline NFD, comparedwith only 13.02% in the group without CNTF.The difference between both groups remained significant for up to 4weeks postoperatively.Eight week postoperatively NFD displayed 92.49% and 88.65% ofbaseline in the groups with and without CNTF-treatment,respectively.Conclusions: Uneventful healing of SBP could be demonstrated aftersurgical dissection in BALB/c murine corneas. The regenerationprocess was significantly accelerated by topical application of CNTF.Commercial Relationships: Rudolf F. Guthoff, None; MariaReichard, None; Marine Hovakimyan, None; Oliver Stachs, NoneProgram Number: 5979Presentation Time: 11:45 AM - 12:00 PMThe Mechanotransducers YAP and TAZ are Modulated bySubstratum Compliance in Corneal Stromal Cells and duringCorneal Wound HealingChristopher J. Murphy 1, 2 , Sara M. Thomasy 1 , Vijaykrishna K.Raghuanthan 1 , Christopher M. Reilly 3 , Paul Russell 1 . 1 Surgical andRadiological Sciences, School of Veterinary Medicine, University ofCalifornia, Davis, Davis, CA; 2 Ophthalmology & Vision Science,School of Medicine, University of California, Davis, Davis, CA;3 Pathology, Immunology & Microbiology, School of VeterinaryMedicine, University of California, Davis, Davis, CA.Purpose: Biophysical cues profoundly influence corneal cellularbehavior including processes integral to corneal wound healing suchas migration and proliferation. Mechanotransduction or thetranslation of external biomechanical cues into internal biochemicalevents remains poorly understood in corneal cells. Yes-associatedprotein (YAP) and transcriptional coactivator with PDZ-bindingmotif (TAZ) have been identified as cellular mechanotransducers.Thus, the purposes of this study were 1) to determine the effects ofsubstratum stiffness on expression of YAP/TAZ in human cornealfibroblasts (HCF) and myofibroblasts and 2) to determine the effectsof corneal wounding on expression and localization of YAP/TAZ.Methods: Primary HCFs were cultured on polyacrylamide substratesof differing stiffness (10 or 85 kPa) or on tissue culture plastic (TCP;>1GPa) in serum-containing media with 0 or 10 ng/ml TGFβ-1.Expression of YAP, TAZ and α-smooth muscle actin (αSMA) toconfirm fibroblast to myofibroblast transition was determined byqPCR. Rabbits underwent epithelial debridement only or incombination with phototherapeuic keratectomy (PTK; 6 mmdiameter, 100 μm deep) on the right eye (OD); the left eye (OS)served as a control. Rabbits were euthanized 6 days later and theglobes were removed and fixed in formalin. Immunohistochemistryfor YAP/TAZ was performed.Results: In the presence of 10 ng/ml TGFβ-1, TAZ expression was©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.