program number: 1978 poster board number: d959 1:45 pm - 3

Copyright 2011 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

268 Corneal Wound Healing I

Monday, May 2, 2011, 1:45 PM - 3:30 PM

Hall B/C Poster Session

Program #/Board # Range: 1976-2030/D957-D1011 Track 1: Physiology and Pathology

Track 2: Repair, Regeneration, and Restoration

Track 3: Imaging and Other Methods

Organizing Section: Cornea

Contributing Section(s): Retinal Cell Biology

Program Number: 1976 Poster Board Number: D957

Presentation Time: 1:45 PM - 3:30 PM

Increase in Leukocyte Elastase Inhibitor during Wound Healing in Bovine

Corneal Endothelial Cells Silvia Chifflet

1, Cristian Justet

1, Frances Evans

1, Alicia Torriglia

2.

1Bioquimica,

Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay; 2U872

Eq. 17, INSERM, Paris, France.

Purpose: Proteases and protease inhibitors interplay are crucial events during

wound healing. Here we study the modifications in LEI (Leukocyte Elastase

Inhibitor) expression during wound healing in bovine corneal endothelial cells

(BCEC), its dependence on the modality of healing (actin cable or lamellipoidial crawling) and on some well-recognized healing response initiators, such as the

calcium and the reactive oxygen species (ROS) waves.

Methods: Wounds in BCEC monolayers were performed with a syringe needle, to

remove, or a silicon-coated wire, to maintain the underlying ECM. Previously we

have shown that BCEC heals by actin cable in the absence of ECM and by

laemllipodial crawling in its presence. LEI expression was determined by IIF and

Western Blot. The calcium wave (Fluo-4 staining) was inhibited by cyclopiazonic acid (CPA)/EDTA and the ROS wave by N-acetylcysteine (NAC).

Results: After incisional wounding in BCEC, there is a progressive rise in LEI at

the wound border, that propagates several rows of cells towards the center of the

monolayer, reaches its maximum at approximately 4 h and decreases as soon as the

two margins re-establish contact. The LEI increase is larger under conditions of

ECM preservation. The inhibition of the calcium or the ROS waves does not

modify the LEI increase.

Conclusions: The increase in LEI expression suggests a role for this enzyme in the healing response. Moreover, the larger increase in lamellar zones may indicate a

participation of the protein in cytoskeletal dynamics.

Commercial Relationships: Silvia Chifflet, None; Cristian Justet,

None; Frances Evans, None; Alicia Torriglia, None

Support: ECOS, CSIC, PEDECIBA



Efficacy Of Plasma Rich In Growth Factors In The Wound Healing Of

Corneal Epithelium In Rabbits Juan A. Duran

1, Jaime Etxebarria

2,3, Raquel Hernáez-Moya

2, María-Celia

Morales4, Vanesa Freire

4, Noelia Andollo

2.

1Instituto de Oftalmologia, Basque

Country University, Vizcaya, Spain; 2Cell Biology and Histology, Basque Country

University, Leioa, Spain; 3Ophthalmology, Hospital de Cruces, Barakaldo, Spain;

4I+D+i, Instituto Clínico-Quirúrgico de Oftalmología, Bilbao, Spain.

Purpose: To evaluate the efficacy of Plasma Rich in Growth Factors (PRGF) treatment on the wound healing process in mechanically produced corneal ulcers.

Methods: Eight millilitres of blood from 24 New Zealand rabbits were obtained by

venipuncture. PRGF was obtained and conserved as previously described. An

epithelial debridement of 9 mm of diameter was done in 1 eye from each of the 24

rabbits. Four groups of 6 rabbits were treated topically 4 times daily with: 1) non-

diluted, 2) 50% diluted, 3) 20% diluted PRGF, or 4) unpreserved tear substitute

(control). The healing of the ulcers was measured by its diameter, and the degree of

infiltration and neovascularization. The healing was supervised every day up to the complete healing of the wounds as seen with fluorescein staining. All rabbits were

treated under the ARVO statements on the Care and Use of Animals in Vision

Research.

Results: The results show that PRGF accelerated the corneal wound healing

process. Although the time of corneal wound healing was reduced when treated

with 20 and 50% PRGF, there were not significant differences with respect to

control treatment. However, when we used the non-diluted concentration the wound healing was faster and it showed statistically significant differences. In all

cases we could confirm the normal histology of the healed epithelium.

Conclusions: Treatment with PRGF can be an efficient way to provide essential

components to the ocular surface and to improve the reepithelization of corneal

defects when compared to treatment with pharmaceutical tear substitutes.

Commercial Relationships: Juan A. Duran, None; Jaime Etxebarria,

None; Raquel Hernáez-Moya, None; María-Celia Morales, None; Vanesa

Freire, None; Noelia Andollo, None Support: None



Difluprednate 0.05% following Corneal Transplant Surgery (CTS) in Infants

and Children Anna Djougarian

1, Gerald W. Zaidman

2.

1New York Medical College, Valhalla,

NY; 2Ophthalmology, Westchester Medical Center, Valhalla, NY.

Purpose: To investigate the efficacy and complications of difluprednate in

pediatric patients following CTS compared to other topical corticosteroids.

Methods: A chart review was conducted of pediatric patients 16 years or younger

operated on from 2000-2010. Eyes were separated into 2 groups: Group A was

treated with difluprednate 0.05% and Group B was treated with other topical

corticosteroids - prednisolone acetate 1%, fluorometholone 0.25%, sulfacetamide sodium 10%/prednisolone acetate 0.2%, tobramycin 0.3%/dexamethasone 0.1%,

rimexolone 1%, neomycin sulfate equivalent to neomycin 3.5 mg/polymyxin B

sulfates 10,000 units/dexamethasone 0.1% and loteprednol etabonate 0.5%.

Complications were determined by the occurrence of glaucoma and cataracts.

Efficacy was measured by the prevention of transplant rejection.

Results: Group A had 15 eyes of 13 children, between 3 months and 11 years old.

Children were followed for 1 to 15 months. 9 eyes had controlled glaucoma from

treatment with topical corticosteroids prior to difluprednate; the remaining 6 were treated only with difluprednate and of these, 3 eyes had glaucoma prior to

difluprednate while 3 did not. In the 9 eyes with glaucoma, switching to

difluprednate led to normal pressures in 6 eyes, with persistently elevated pressures

in 3 eyes. In the other 6 eyes treated with difluprednate, use of difluprednate led to

medically controlled glaucoma in 3 eyes, all of which already had elevated

intraocular pressure (IOP) before treatment. The other 3 eyes remained normal.

None needed glaucoma surgery. 1 eye required cataract surgery. 2 grafts failed. Group B had 35 eyes of 28 children, between 3 months to 18 years old. Follow-up

ranged from 1 month to 10 years. 32 (91.4%) of the 35 eyes had glaucoma

following treatment with topical corticosteroids, 3 (8.6%) did not. Of the 32 eyes

with glaucoma, 16 (50%) had elevated IOP prior to surgery while 16 (50%) did not.

The 3 eyes that did not have glaucoma following surgery had normal IOP prior to

treatment with topical corticosteroids. 1 (2.9%) of the 35 eyes needed a glaucoma

drainage implant. 12 (34.3%) required cataract surgery. 10 (28.6%) grafts failed.

Conclusions: Patients treated with difluprednate (Group A) had less problems with IOP, fewer cataract surgeries and less graft failures than Group B. This suggests

that difluprednate may have less complications and may be more effective than

other topical corticosteroids. However, the follow-up time in Group A was

significantly shorter than Group B and some of these complications take time to

occur.

Commercial Relationships: Anna Djougarian, None; Gerald W. Zaidman,

None Support: None



Slug Is Upregulated During Wound Healing And Downregulates Tp63 In

Corneal Epithelial Cells Keiichi Aomatsu

1A,1B, Tokuzo Arao

1B, Kosuke Abe

1A, Koji Sugioka

1A, Aya

Kodama1A

, Hiroshi Mishima2, Kazuto Nishio

1B, Yoshikazu Shimomura

1A.

AOphthalmology,

BGenome Biology,

1Kinki Univ Faculty of Med, Osaka-Sayama,

Japan; 2Ophthalmology, Nara Hospital Kinki Univ Faculty of Med, Ikoma, Japan.

Purpose: Involvement of epithelial mesenchymal transition (EMT) in corneal

wound healing remains largely unclear. The purpose of study is to gain the insight

into EMT and corneal wound healing.

Methods: Slug expression in murine corneal tissues was evaluated using

fluorescence staining. Snail and Slug were retrovirally and stably introduced into

SV40-immortalized human corneal epithelial cells (HCECs). All cells were

cultured in DMEM/F12 medium supplemented with 15% fetal bovine serum and gentamicin at 37

oC with 5% CO2. Real-time PCR was carried out using Thermal

Cycler Dice ® RealTime System. GAPDH was used as an internal control to

normalize and compare each sample. In western blot analysis, antibodies used in

this study were as follows: anti-snail, slug, E-cadherin, vimentin, beta-actin, N-

cadherin. Rhodamine-conjugated Fhalloidin, E-cadherin and vimentin antibody and

4‟,6-diamidino-2-phenylindole (DAPI) were used for fluorescent cellular staining.

Results: Slug was overexpressed in corneal epithelial cells during wound healing in vivo, while Snail was not upregulated. Therefore, we hypothesized that Slug

plays key roles during wound healing. Overexpression of Snail or Slug induced the

cellular morphology and cadherin switch in HCECs, demonstrating that these

transcription factors were able to mediate clear EMT in HCECs. Cellular

proliferation was suppressed in these transfectant. Regarding a stemness of corneal

epithelial cells, overexpression of Snail or Slug mediated the downregulation of

TP63. The result suggests that Snail or Slug expression may be involve in the

stemness of corneal epithelial cells during wound healing. Conclusions: We found that slug is upregulated during corneal wound healing and

the expression lead to downregulate the TP63 in corneal epithelial cells. Our

findings provide novel insight into EMT, corneal wound healing and the stemness

of corneal epithelial cells.

Commercial Relationships: Keiichi Aomatsu, None; Tokuzo Arao,

None; Kosuke Abe, None; Koji Sugioka, None; Aya Kodama, None; Hiroshi

Mishima, None; Kazuto Nishio, None; Yoshikazu Shimomura, None


Support: None



Initial Evidence for an Epithelial-to-Mesenchymal Transition Basis For

Corneal Scarring Daniel J. Gibson, Gregory S. Schultz. Biochemistry & Molecular Biology,

University of Florida, Gainesville, FL.

Purpose: To investigate the source of cells which form a light scattering scar

following a stroma penetrating wound to the cornea.

Methods: Twelve rabbits received bilateral central 6.0 mm diameter, 125 μm deep

PTK wounds. Just prior to euthanization, each eye was anesthetized, the pupils

dilated, and the wound was photographed. Two rabbits were euthanized at 30 min, 1 day, 2 days, 5 days, 7 days, and 10 days post-wounding and an 8.0 mm diameter

punch encompassing the wound was collected, fixed, paraffin embedded and

sectioned. One slide per eye was stained with H&E for general histological

analysis. Immunohistochemical staining for α-smooth muscle actin (α-SMA) was

used to identify myofibroblasts and staining for tenascin-C (TNC) was used to

identify loci of EMT.

Results: Corneal haze became apparent in the day 5 eyes and began as a ring of

haze at the wound margin with occasional points of haze in the body of the wound. The haze then spread from the points of nucleation. Both the patterns of α-SMA

and TNC staining mirrored the spreading pattern seen with the haze. General

histological analysis revealed that during re-epithelialization: 1) an occasional

epithelial cell was found to have partially invaded the wounded stroma, 2) that the

remaining stromal fibroblasts were still apparent, 3) there was a significant number

of neutrophils which had invaded the stroma and were associating with the stromal

fibroblasts. At day 5 the entire wound volume was filled with epithelial-like cells. At the basal layer, the nuclei remained flattened and in places, a weak association

between the basement stroma and the nascent epithelium was evidenced by blister-

like formations with what appears to be a flattened nuclei at each the basal and

apical surfaces. The distribution of this “blistering” mirrored the haze and IHC

staining. Finally, the wound volume had been reconstituted with stroma-like tissue

and the epithelium is restored to its typically thickness.

Conclusions: The spread of corneal haze and its molecular markers mirrors the

pattern of re-epithelialization whereby both start at the wound margin and migrate towards the wound‟s center. The co-localization of markers which also mirror the

same spreading pattern for haze and EMT further strengthen the evidentiary basis

underlying the theory that the light scattering regenerated stromal tissue is derived

from the epithelium, not the stromal fibroblasts.

Commercial Relationships: Daniel J. Gibson, None; Gregory S. Schultz, None

Support: NEI T32

Program Number: 1981 Poster Board Number: D962 Presentation Time: 1:45 PM - 3:30 PM

Vitamin A Palmitate Prevents Ocular Surface From Damages Induced By

Antiglaucomatous Eyedrops: A Randomized Control Trial Tingting Qian

1, Jiaxu Hong

2, JianJiang Xu

3.

1Ophthalmology, Eye & Ear, Nose,

Throat Hospital, Shanghai, China; 2Ophthalmology, Eye & Ear, Nose, Throat

Hospital, Shanghai, China; 3Ophthalmology, Eye & ENT Hospital of Fudan Univ,

Shanghai, China.

Purpose: To determine whether vitamin A palmitate can prevent ocular surface from damages induced by prolonged use of topical antiglaucomatous eyedrops.

Methods: Sixty eyes of 30 New Zealand white rabbits were randomized to 1 of 5

treatment groups: Vitamin A palmitate 1% QD (group 1), timolol maleate 0.5%

BID (group 2), brimonidne 0.2% BID (group 3), combined Vitamin A palmitate

1% QD with timolol maleate 0.5% BID (group 4), or combined Vitamin A 1% QD

with brimonidne 0.2% BID (group 5) for 30 days. Tear break-up times and basal

Schirmer's tests were measured and the conjunctival changes were determined by

impression cytology. Corneal damage was evaluated by scanning electron microscopy.

Results: Rabbits in groups 2 and 3 showed statistically significant fewer normal

tear break-up time and basal Schirmer's tests than before (P<0.05). Also, in the

conjunctival impression cytology, the number of global in the epithelium in groups

2 and 3 was significantly lower than before (P<0.05). According to Nelson's

method, specimens of groups 2 and 3 were graded and scored to 1, while groups 1,

4 and 5 were all scored to 0. Corneal damage in groups 2 and 3 assessed with scanning electron microscopy showed loss of microvilli, increasing number of

epithelial holes, cell peeling, loss of hexagonal shape and retraction of cell borders.

Ocular surface seemed not to change obviously in group 1, 4 and 5.

Conclusions: 1-month treatment with glaucoma medications containing

preservatives without vitamin A resulted in corneal and conjunctival damage. The

long-term use of glaucoma medications with vitamin A might help preserve ocular

health.

Commercial Relationships: Tingting Qian, None; Jiaxu Hong, None; JianJiang

Xu, None

Support: Ministry of Health Grant (2010-2013); Shanghai Municipality Grant

(10XD1401100); Fudan University Grant (2009-2011).



Topical Treatment With Neuroprotectin D1 Increases Corneal Nerve

Regeneration After Experimental Surgery Maria S. Cortina

1, Jiucheng He

2, Tiffany Russ

2, Nicolas G. Bazan

2, Haydee E.

Bazan2.

1Ophthalmology, University of Illinois Eye and Ear Infirmary, Chicago,

IL; 2Ophthalmology & Neuroscience Ctr, LSU Health Sciences Center, New

Orleans, LA.

Purpose: Treatment with pigment epithelial derived factor (PEDF) in association

with docosahexaenoic acid (DHA) after lamellar keratectomy increases the

regeneration of corneal nerves. We have also shown that corneal sensation returns

to normal levels in treated animals at 8 weeks after surgery (Cortina et al, IOVS,

2010). However the mechanism by which PEDF and DHA exert their effect in

corneal nerve regeneration is not known. We hypothesize that this effect is due to

the synthesis of neuroprotectin D1 (NPD1) from DHA, which increases in corneas treated with PEDF+DHA. The purpose of this study is to define if topical NPD1

treatment increases the regeneration of corneal nerves after surgery.

Methods: An 8 mm corneal stromal dissection was performed in the left eyes of

adult New Zealand rabbits. The treatment group received topical NPD1 (100ng)

application three times a day for 6 weeks. The control group received vehicle drops

(2μl of ethanol in 50μl of PBS). Corneal sensitivity was assessed weekly with a

Cochet-Bonnet aesthesiometer. Rabbits were sacrificed at 8 weeks and corneas

were processed for immunohistochemistry. Corneal nerves were stained with βIII tubulin. The βIII tubulin-positive area at the subepithelial nerve plexus was

calculated and compared to the total area using an image analysis program.

Results: Subepithelial corneal nerve area in the NPD1-treated group was increased

over two fold compared to the vehicle-treated group (13.08 +/- 2.0 vs 5.85 +/- 1.2).

This difference was statistically significant with a p value of 0.006. Six weeks after

surgery there was a 67% recovery of corneal sensitivity in the NPD1-treated

animals, while in the vehicle-treated group there was only a 30% recovery of sensitivity.

Conclusions: Topical NPD1 treatment promotes functional regeneration of

damaged corneal nerves after experimental surgery. This lipid mediator could be a

novel therapeutic agent for the treatment of neurotrophic keratitis and dry eye that

develops as a result of corneal nerve damage.

Commercial Relationships: Maria S. Cortina, None; Jiucheng He,

None; Tiffany Russ, None; Nicolas G. Bazan, None; Haydee E. Bazan, None

Support: This work is supported by grant R01 EY 019465



Safety Profile of Stromal Hydration of Clear Corneal Incisions with

Cefuroxime Mariya Moosajee

1,2, Dhani Tracey-White

1, Richard P. Harbottle

1, Veronica

Ferguson2.

1Molecular Medicine, Imperial College London, London, United

Kingdom; 2Imperial College Healthcare NHS Trust, Western Eye Hospital,

London, United Kingdom.

Purpose: Sutureless clear corneal incisions for phacoemulsification are widely

used, but it has been suggested that an increase in post-operative endophthalmitis

may be due to the ingression of ocular surface fluid including bacteria through the

http://files.abstractsonline.com/CTRL/ec/0/6be/9f6/3a9/4cb/381/f82/9d8/697/c5d/0f/g3804_1.JPG

http://files.abstractsonline.com/CTRL/ec/0/6be/9f6/3a9/4cb/381/f82/9d8/697/c5d/0f/g3804_2.JPG


wound. To improve the self-sealing status of corneal incisions, stromal hydration of

the wound can be performed with balanced salt solution (BSS) with variable

persistence. Stromal hydration together with intracameral cefuroxime (1mg/0.1ml)

has reduced endophthalmitis rates following cataract surgery. This study aims to investigate the safety profile of stromal hydration of clear corneal incisions with

cefuroxime. Therapeutic levels of antibiotic sequestered at the wound may further

reduce rates of post-operative endophthalmitis.

Methods: MF-1 mice had clear corneal incisions placed in each eye. Stromal

hydration was performed with either 5 μl of 10 mg/ml cefuroxime, cefuroxime-

texas red conjugate, or BSS (each group n=30). Corneas were harvested at 1 hour,

day 1, and weeks 1, 2 and 6 post-operatively. Confocal microscopy was used to

monitor presence of cefuroxime-texas red conjugate. Corneal morphology and histology was examined by light microscopy, and apoptosis was detected by

TUNEL assay to evaluate the safety profile of this technique.

Results: Cefuroxime stromal hydration did not alter corneal morphology; there was

no evidence of corneal scarring or vascularisation. Corneal histology and levels of

apoptosis were minimal and comparable in the BSS and cefuroxime stromal

hydration groups up to 6 weeks post-operatively. Confocal microscopy detects

cefuroxime-texas red up to 48 hours surrounding the corneal wound.

Conclusions: Stromal hydration of clear corneal incisions with cefuroxime is safe in mouse corneas. A reservoir of cefuroxime at the wound can act as a barrier of

defence against infection. This technique has the potential to reduce rates of post-

operative endophthalmitis if applied to phacoemulsification or other ocular surgery

involving similar corneal incisions.

Commercial Relationships: Mariya Moosajee, None; Dhani Tracey-White,

None; Richard P. Harbottle, None; Veronica Ferguson, None

Support: Eli Webster Fund



Heme Oxygenase (HO)-2 Deficiency Leads To The Development Of Epithelial

Defects In The Injured Cornea Lars Bellner

1A, Michael W. Dunn

1B, Michal L. Schwartzman

1A.

APharmacology,

BOphthalmology,

1New York Medical College, Valhalla, NY.

Purpose: HO-2 is highly expressed in the corneal epithelium and is a component of

the HO system that represents an intrinsic cytoprotective and anti-inflammatory system. We have shown that epithelial injury in HO-2 null mice leads to chronic

inflammatory complications including ulceration and neovascularization (Seta et al,

2006) and further that knockdown of HO-2 in human corneal epithelial cells

resulted in a significant reduction in healing rate following injury due to inhibition

of migration (Halilovic et al 2010). We evaluate the effect of HO-2 deletion and

biliverdin application on the development of corneal defects.

Methods: HO-2 null mice were treated with biliverdin (100 µl of 100 µM ip once daily; and topical (10 µl of 100 µM), or its vehicle (HBSS, pH 7.4) one hour prior

to injury and t.i.d. thereafter. The corneal epithelium was removed using an Alger

Brush in anesthetized mice. Re-epithelialization was assessed at days 2, 4 and 7 by

fluorescein staining. Infiltrating neutrophils were detected through GR1-staining of

histological sections. TUNEL and DHE staining was used to show apoptotic

changes and oxidative stress, respectively. QPCR was used to detect the mRNA

levels of NADPH oxidases 2 and 4, and MMP- 2 and -9.

Results: After epithelial removal, non-penetrating ulcerations developed in less than 25% of injured eyes of WT mice, compared to 90% of injured eyes of HO-2

null mice. In HO-2 null mice the average area of the defect was 31.7%±4.9% and

32.3%±3.2% (n=8-10), at days 4 and 7 after injury, respectively. biliverdin-

treatment significantly accelerated wound closure when compared with vehicle-

treated group reducing the size of the ulceration, 17.5%±2.6% and 13.8%±3.3%

(n=8-10), at days 4 and 7, respectively. Biliverdin-treatment also caused a drop in

the number of inflammatory cells, lessened oxidative stress as shown by a 4-fold

reduction of DHE intensity of histological slides at day 4 after injury, and NADPH-oxidases -2 and -4 mRNA levels were reduced by 50% at day 7. TUNEL staining

showed a significant reduction in the number of dead cells per histological section.

Conclusions: The demonstration that the inflammatory responses, including the

infiltration of inflammatory cells and the oxidative stress are exaggerated in HO-2

knockout mice, together with the development of epithelial defects strongly

supports the notion that the HO system is critical for controlling the inflammatory

and repair response. Biliverdin/bilirubin, potent intrinsic antioxidants and anti-inflammatory molecules, may offer a novel strategy to treat corneal conditions.

Commercial Relationships: Lars Bellner, None; Michael W. Dunn,

None; Michal L. Schwartzman, None

Support: NIH grant EY06513 (MLS)



LAU-0901 Protects Deep Corneal Injuries Affected By PAF in an in vivo

Model Azucena H. Kakazu, Jiucheng He, Tiffany C. Russ, Haydee E. Bazan.

Ophthalmology/Neuroscience Center, LSU Health Sciences Center, New Orleans,

LA.

Purpose: To evaluate the effect of a selective PAF receptor antagonist LAU-0901

(SCP-09001) on an in vivo mouse model of corneal epithelial and stroma injury.

Methods: C57BL/6 mice were injured by scraping the corneal epithelium and

anterior stroma (about 25 %) using an Algerbrush II under a surgical microscope.

Mice were divided into three groups: to the first group, PAF (10 µg/ml, 2x 5 µl)

was applied topically; to the second group, LAU-0901 (30 mg/Kg) was injected i.p. after PAF application; the third group received only vehicle as control. This

treatment took place once a day. Additional mice received no injury or treatment.

Mice were sacrificed at 1, 2, and 7 days after injury. The corneas were processed

for histopathology and immunofluorescence with antibodies for metalloproteinase-

9 (MMP-9), fibronectin (FN) and α-smooth muscle actin (α-SMA).

Results: One day after injury there was a 50 % delay in epithelial wound closure in

presence of PAF compared to vehicle treated animals. The delay was also

significant 2 days after the injury. LAU-0901 completely blocked the PAF effect. Treatment with PAF for 2 days increased MMP-9 and decreased FN expression in

the epithelium and stroma; consequently, fewer myofibroblasts migrated to the

wounded area. By 7 days, MMP-9 was still upregulated and FN was

downregulated. All these changes were inhibited when the animals were treated

with LAU-0901.

Conclusions: PAF is a strong inflammatory mediator and a potent activator of

MMP-9 that promotes FN degradation, thereby delaying epithelial wound healing

and migration of myofibroblasts from the limbal area. The inhibition of PAF action by LAU-0901 could be important in the treatment of corneal injuries that

compromise the stroma.

Commercial Relationships: Azucena H. Kakazu, None; Jiucheng He,

None; Tiffany C. Russ, None; Haydee E. Bazan, None

Support: NIH grant EY 004928



Matrix Metalloproteinase 14 Overexpression Reduces Corneal Scarring Pierre R. Fournie

1,2, Stéphane Galiacy

2, Massoudi Dawiyat

2, Angélique Erraud

2,

Isabelle Raymond-Letron2,3

, Fabienne Rolling4, François Malecaze

1,2.

1Ophthalmology, Purpan Hospital, Toulouse, France;

2INSERM U563, Toulouse,

France; 3Département de Pathologie, Ecole Nationale Vétérinaire, Toulouse,

France; 4INSERM UMR U649, Nantes, France.

Purpose: Corneal wound healing is an everyday preoccupation for

ophthalmologists. Corneal transparency depends on the scarring quality after a traumatic corneal wound, but also after refractive corneal surgery. Cicatrisation and

fibrosis formation involve epithelial/fibroblast interactions via paracrin signals

inducing extracellular matrix (ECM) remodeling. The major event is fibroblast

activation and differentiation into myofibroblasts. These cells have a key role in the

fibrotic response. They acquire contractile properties, and synthesize a new ECM,

mainly composed of type III collagen. This scar tissue is less organized than the

regular stroma, thus explaining corneal opacity. ECM remodeling is a critical step which aims to digest the excess of ECM by proteolysis of type III collagen.

MMP14 is a membrane-bound fibrillar collagenase from the Matrix

Metalloproteinase family. We hypothesized that its overexpression in the corneal

stroma during wound repair will increase ECM remodeling and thus prevent

collagen deposition in the scar tissue.

Methods: We developed an adeno-associated virus-based vector expressing murine

MMP14 under the control of the CMV promoter. We evaluated MMP14

overexpression after viral transfection in a murine model of corneal wound healing. We characterized several parameters: clinical observation, histology, and wound

healing markers.

Results: We demonstrated that a single and simple direct injection of recombinant

adeno-associated virus-based vector expressing murine MMP14 can modulate gene

expression of murine stromal keratocytes. We observed that MMP14

overexpression reduced corneal opacity. Our preliminary results showed a

decreased in edema and corneal scar formation, associated with a decreased

expression of alpha smooth actin and type III collagen. Conclusions: These results represent proof of concept that gene transfer of

MMP14 can reduce scar formation, which could have therapeutic applications after

corneal trauma.

Commercial Relationships: Pierre R. Fournie, None; Stéphane Galiacy,

None; Massoudi Dawiyat, None; Angélique Erraud, None; Isabelle Raymond-

Letron, None; Fabienne Rolling, None; François Malecaze, None

Support: Fondation de l‟Avenir (study ET7-474), la Fondation de France (grants „Berthe Fouassier‟: 2008002176, 2009002318 and 2009002320), the Laboratoires

Pierre Fabre and the INSERM



The Efficiency Of Autocytokine Therapy In The Treatment Of The Corneal

Damages. (Experimental Study) Olga I. Krivosheina, Igor V. Zapuskalov, Nadezda Levchenco, Julia Khoroshikh.

Ophthalmology, Siberian State Medical Univ, Tomsk, Russian Federation. Purpose: To study efficiency of the introstromal introductions of the autologic

mononuclear blood cells in treatment of the induced damage of a cornea.

Methods: A series of experiments on 20 rabbits of breed the Chinchilla. Corneal

epithelium was removed out totally and mechanical damage of stroma and

endothelium was executed. The animals of the 1st group besides antibacterial and


metabolic remedies was injected autological mononuclear blood cells into the

stroma. The cells allocated by the method fractionating centrifugation on a density

gradient. In the 2nd animal group traditional pharmacotherapy was given. The

general duration of experiment was 21 days. During experiment external examination, biomicroscopy, fluorescent test, photoregistration were spent. The

material fence was made for 3, 7, 14, 21 days of the experiment.

Results: For the 3d day of experiment corneal epithelium of the 1st group was

presented by 5-6 layers of flat cells, in the stroma was observed diffuse

mononuclear infiltration, bunches of collagen fibers settled disordered down. In the

2nd group corneal epithelium was presented by hydropical dystrophic

epitheliocites. The stroma was edematous, collagenic fibers were friable located. At

the limbus was found out dense polymorphic nuclear infiltration. Corneal epithelium was desquamated. At the 7

th day, in animals of the 1

st group, the course

of collagen fibers in corneal stroma became ordered. In limbic area was found out

diffuse congestion of mononuclear cells and the individual expanded vessels of.

The endothelium remained on all extent. In animals of the 2nd

group, the nidal fibrin

deposits was applying on the cornea, diffuse lymphocytic infiltration and

neovascularisation were observed in the stroma. At the 14th

day at animals of the

1st group corneal epithelium had a normal structure. The stroma contained strictly

located collagenic fibres. In the 2nd group in back 1/3 own substance of the cornea was homogeneous destruction of the corneal stroma. At the 21

st day of the

experimant, the corneal epithelium in animals of the 1st group had the normal

structure. In the 2nd group the Boumen‟s membrane was a little thickened. At the

2/3 parts was marked active formation of collagenic fibres, in back 1/3 of stromal

layers - the hypostasis and homogenization remained.

Conclusions: The intrastromal introduction of the autologic mononuclear cells at

cases of cornea damage stimulates reparative regeneration, promoting fast healing and thus interfering scarring and neovascularisation of the cornea.

Commercial Relationships: Olga I. Krivosheina, None; Igor V. Zapuskalov,

None; Nadezda Levchenco, None; Julia Khoroshikh, None

Support: None



Inhibition of PRK-induced Corneal Haze By Trichostatin-A Involves

Epigenetic Modification Ashish Tandon

1,2, Rangan Gupta

1,2, Ajay Sharma

1,2, Yasaman J. Hemmat

1,2, Rajiv

R. Mohan1,2

. 1Mason Eye Institute, University of Missouri, Columbia, MO;

2Harry

S. Truman Veterans Administration Hospital, Columbia, MO.

Purpose: Recently we reported significant inhibition of photorefractive

keratectomy (PRK)-induced haze in rabbit cornea by Trichostatin-A (TSA), a

histone deacetylase inhibitor. This study investigated whether the anti-fibrotic

effects of TSA on cornea are due to epigenetic modifications of TGFβ. Methods: Human corneal fibroblasts (HSF) and New Zealand White rabbits (2.5-

3.0kg) were used. Fibrosis in HSF was induced with TGFβ (1ng/ml) using serum-

free conditions and rabbit cornea with -9.0 diopter PRK with an excimer laser.

Doses of TSA showing significant inhibition of corneal fibrosis were used for in

vivo and in vitro experiments. Slit lamp biomicroscopy, biochemical assays

(TUNEL, trypan blue, histone acetyltransferase, histone deacetylase), real-time

PCR, western blotting, and immunocytochemistry techniques were used to examine

epigenetic modifications. Results: TGFβ1 induced phenotypic changes, increased extracellular matrix

synthesis and assembly of actin filaments (αSMA, fibronectin and phalloidin; 8-

10±2.1 fold; p<0.01-0.001), and TSA significantly inhibited the levels of tested

proteins and mRNA in HSF (46-83%; p<0.001) and rabbit corneal sections (73%;

p<0.001). HSF treated with TGFβ1 reduced histone H3 acetylation whereas TSA

treatment to TGFβ1-stimulated HSF showed dose-dependent restoration of

acetylated histone H3 levels. Quantification studies are underway.

Conclusions: This study suggests that epigenetic regulation plays an important role in corneal fibrosis. Understanding the molecular hierarchy of events with respect to

reactivation of transcription and reversal of histone modification will be critical for

understanding and developing mechanism-based anti-fibrotic treatments for corneal

scarring.

Commercial Relationships: Ashish Tandon, None; Rangan Gupta, None; Ajay

Sharma, None; Yasaman J. Hemmat, None; Rajiv R. Mohan, None

Support: VA Merit 1I01BX000357-01 (RRM), NEI RO1EY17294 (RRM) and Unrestricted Research to Prevent Blindness



The Structural Role Of Pax6 Over-expression In The Formation Of

Microcorneas Erin P. Dooley

1A, Craig Boote

1A, Natalie Dora

2, John D. West

2, Christina S.

Kamma-Lorger1A

, Andrew J. Quantock1A

, Keith M. Meek1B

. ASchool of Optometry

and Vision Sciences, BOptometry & Vision Sciences,

1Cardiff University, Cardiff,

United Kingdom; 2Reproductive and Developmental Sciences, University of

Edinburgh, Edinburgh, United Kingdom.

Purpose: To better understand the structural role of Pax6 over-expression in

hemizygous PAX77+/-

transgenic mice (carrying 5-6 copies of the human PAX6

gene) in the formation of microcorneas.

Methods: Corneas of CBA-PAX77+/-

mice (hemizygous PAX77+/-

mice on a

CBA/Ca genetic background) and wild-type (CBA/Ca) mice were dissected,

preserved in buffered paraformaldehyde and examined by wide angle X-ray

scattering at station I02 at the Diamond Light Source (Didcot, UK). The corneas were scanned using a 200 µm x 200 µm beam at a scanning interval of 0.25 mm

with a 10 second exposure time. X-ray scatter patterns were recorded at each

position, and subsequent data analysis quantified the preferential orientation and

distribution of stromal collagen fibrils across the cornea from limbus to limbus.

Results: PAX77+/-

corneas were smaller than the wild-type strain. Differences in

the preferential alignment and distribution of stromal collagen fibrils were evident

between the PAX77+/-

and wild-type strains. Preliminary data showed that in

PAX77+/-

corneas collagen lamellae were oriented in a unilateral inferior/superior direction throughout, unlike wild-type corneas which showed a more annular

arrangement of lamellae.

Conclusions: Pax6 over-expression in PAX77+/-

corneas is associated with changes

in collagen lamella arrangement, which may be related to the microcornea

phenotype.

Commercial Relationships: Erin P. Dooley, None; Craig Boote, None; Natalie

Dora, None; John D. West, None; Christina S. Kamma-Lorger, None; Andrew

J. Quantock, None; Keith M. Meek, None Support: MRC Grant G0600755



Severe Airbag Related Ocular Alkali Injury Shawn S. Barnes

1, William Wong, Jr.

2, John C. Affeldt

3.

1University of Hawaii

School of Medicine, Honolulu, HI; 2Hawaii Vision Clinic, Inc., Honolulu, HI;

3Ophthalmology, Loma Linda University School of Medicine, San Juan Capistrano,

CA.

Purpose: The instantaneous (50msec) inflation of motor vehicle accident (MVA)

triggered airbags is achieved through the explosive ignition of sodium azide.

Reaction byproducts include powdered metallic oxides, sodium bicarbonate and the

potent alkali sodium hydroxide (lye). This occasionally released alkali is

responsible for ~20% of all airbag related eye injuries, with the majority manifested

as rapidly healing corneal abrasions or mild anterior segment alkali burns. We

report for the first time a severe (Pfister-Koski classification scheme) bilateral airbag related ocular alkali injury.

Methods: Interventional case report

Results: A 47 yo male with no previous ocular history was involved in a rural

setting MVA, sustaining multiple life-threating injuries. Transport logistics and

prioritized injury care resulted in no ocular lavage until 7 hours post injury. Initial

eye exam revealed a VA of LP OU, opaque de-epithelialized corneas OU, diffuse

limbal opacification OU, and retained chemical particulates. Six months after injury, BCVA was 20/200 OD and 20/80 OS. Slit-lamp exam revealed diffusely

scarred and vascularized corneas OU, with symblepharon OD and inferior sulcus

contraction OS. The patient is currently awaiting bilateral limbal stem cell and

corneal transplantation.

Conclusions: As demonstrated in this case, airbags despite their life saving intent

can also be associated with serious ocular injuries including severe alkali burns.

This case emphasises the importance of ER guide lined immediate ocular lavage

following all airbag deployment MVA's, and also suggests the addition of particulate clearing forniceal sweeps to those

Commercial Relationships: Shawn S. Barnes, None; William Wong, Jr.,

None; John C. Affeldt, None

Support: None



Effect Of Plasma Rich In Growth Factors (PRGF) On Corneal Epithelial

Wound Healing Eduardo Anitua

1, Francisco Muruzabal

1, Maria De la Fuente

1, Jesús Merayo

2,

Gorka Orive1.

1Eduardo Anitua Foundation, Vitoria, Spain;

2Instituto

Oftalmologico Fernandez-Vega, Oviedo, Spain.

Purpose: To evaluate the effects of PRGF on corneal epithelial cell proliferation

and wound healing.

Methods: Blood from healthy donors was collected, centrifuged and, Plasma Rich

in Growth Factors (PRGF) was drawn off avoiding the buffy coat. Human corneal epithelial cells (HCE) transfected with SV40 adenivirus were cultured. The

proliferation assays was carried out using the cyquant assay after the treatment of

cells with PRGF. In order to quantify the wound closure potential of epithelial cells

after the treatment of PRGF, cells were plated in culture inserts placed on a 24-well

plate at high density. After removing the inserts, cells were treated with PRGF and

the wound healing area was photographed each 4 hours until complete closure.

Results: Proliferation rate of epithelial cells stimulated with PRGF increased

significantly when compared with PRGF non-treated group. Results from the in vitro wound healing study revealed that experimental area of 8.1 mm

2 was totally

closed by the epithelial cells (100% of the damage area) in 24 hours after treatment

with PRGF, while the non-treated control group was only partially closed (35% of

total area).


Conclusions: These results suggest that PRGF could accelerate corneal epithelial

wound healing.

Commercial Relationships: Eduardo Anitua, Pioneer of PRGF technology (P);

Francisco Muruzabal, Scientist of BTI (E); Maria De la Fuente, Scientist of BTI (E); Jesús Merayo, None; Gorka Orive, Scientist of BTI (E)

Support: None



Influence Of Early Amniotic Membrane And Mesenchymal Stem Cell

Transplantation On The Clinical Outcome After Severe Corneal Burn In Rats Andreas Heidt, Philipp Eberwein, Daniel Böhringer, Johannes Schwartzkopff,

Yvonne Kern, Thomas Reinhard. University-eye-hospital Freiburg, Freiburg, Germany.

Purpose: The Purpose of this study was to investigate the influence of amniotic

membrane transplantation (AM) with our without mesenchymal stem cell (MSC)

transplantation on corneal woundhealing after severe corneal burn.

Methods: MSC were isolated from 3 weeks old female Lewis rats and cultured

until passage 4. Then, they were analyzed for their marker profile by flow

cytometry and used for experiments. Those were as follows: 12 weeks old rats were

anaesthetized and a corneal burn was done in one eye using 0.1N NaOH for 1 min. After intense rinsing with water the rats were divided into 4 treatment groups: 1

st

group no intervention, 2nd

group lid suture, 3rd

group double layer of AM sutured

onto the cornea + lid suture, 4th

group 1 layer of AM covered with MSC plus a

second layer of AM on top of the first sutured onto the cornea + lid suture. The

ocular surface was evaluated every 3-4 days for 4 weeks. Each week, rats of all

groups were euthanized and corneal mRNA expression of acute inflammatory and

angiogenic cytokines was analyzed. Results: In ocular surface evaluation, rats in group 2, 3 and 4 showed a

significantly faster epithelialisation (p<0,001) and less corneal opacity than group 1

(p<0,05). There was no difference between group 2-4 concerning these parameters.

Corneal vaskularisation did not significantly differ between group 1- 4. mRNA

expression of the MMP-2 and TSP1- gene peaked 2 weeks after the injury in group

1 while the treated groups showed lower or no up-regulation. IL-6 showed a strong

up-regulation after 1 week in group 2-4, while up-regulation in the control group

(group 1) occurred after 3 weeks. Concerning VEGF-expression, there was slight up-regulation throughout the follow-up in all 4 groups with no significant

differences.

Conclusions: Treatment groups showed significantly better clinical outcome than

the control (group 1). Interestingly, there was no significant difference between the

treatment groups regarding our clinical outcome measures. MSC or AM

transplantation did not further improve the clinical outcome in comparison to lid

suture alone. These results correlated with the regulation of the gene expression. Future studies using AM and MSC in the acute phase after corneal burn may either

use later time points after injury or repeated transplantation of AM +/- MSC in

order to check for more beneficial effects of these treatments.

Commercial Relationships: Andreas Heidt, None; Philipp Eberwein,

None; Daniel Böhringer, None; Johannes Schwartzkopff, None; Yvonne Kern,

None; Thomas Reinhard, None

Support: None


Comparison of Corneal Healing After Exposure to the Blistering Agents

Nitrogen Mustard and UVB Iris Po

1, Andrea S. DeSantis

1, Rita A. Hahn

1, Donald R. Gerecke

1, Jeffrey D.

Laskin2, Marion K. Gordon

1.

1Pharmacology and Toxicology, Rutgers University,

Piscataway, NJ; 2Environmental and Occupational Medicine, UMDNJ, Robert

Wood Johnson Medical School, Piscataway, NJ.

Purpose: Nitrogen mustard (NM) and ultraviolet B light (UVB) are vesicants that induce microbullae in the cornea. Severe exposures result in the loss of epithelial-

stromal integrity. Physicians treat mustard wounds under the assumption that they

heal more slowly than equivalent injuries with epithelial-stromal (or epidermal-

dermal) separations. We set out to experimentally determine whether or not this

presumption is true.

Methods: Rabbit corneas in air lifted organ cultures (Gordon et al., J. Ocul

Pharmacol. Ther. 26: 407-419, 2010) were exposed to different intensities of UVB and different exposures of NM. UVB exposures were 100, 400, 800, 1200 and 1600

mJ/cm2. For NM, corneas were exposed to 100 nmoles of the vesicant for 30, 60 or

120 min. Composites of overlapping micrographs of H&E stained sections

covering the entire diameter of cornea were analyzed to determine what conditions

resulted in equivalent phenotypes at 24 hr post exposure. Corneas with equivalent

24 hr injuries were allowed to heal for 1-10 days. The timing of re-epithelialization

was determined and the appearance and removal of provisional matrix components

SPARC and hevin were assessed by immunofluorescence. Results: At 24 hours post exposure, a nearly equivalent epithelial-stromal

separation was observed with 2000mJ/cm2 UVB (61.7% separation) and with a 60

min exposure to 100 nmol NM (58.3% separation). The UVB-exposed corneas re-

epithelialized within 5 days, while the NM-exposed cornea took 7 days to re-

epithelialize. Immunofluorescence analysis showed that provisional wound matrix

components HEVIN and SPARC persisted at least 1 day longer in the NM-exposed

corneas than in UVB-exposed corneas.

Conclusions: These data experimentally verify that mustard-induced corneal

injuries heal more slowly than equivalent UVB injuries. Both re-epithelialization and removal of provisional matrix components require longer times in NM-exposed

corneas.

Commercial Relationships: Iris Po, None; Andrea S. DeSantis, None; Rita A.

Hahn, None; Donald R. Gerecke, None; Jeffrey D. Laskin, None; Marion K.

Gordon, None

Support: EY009056; AR055073; ES005022



Evaluation Of Ultra-thin Biocellulose Membranes For Ophthalmological Use Anna Dobias

1A, Anna-Katharina Salz

1B, Nina Harmening

1A, Constance Ace

2, Peter

Walter1A

, Gabriele Thumann1B,1A

. ADepartment of Ophthalmology,

BIZKF Aachen,

1RWTH Aachen University, Aachen, Germany;

2Research and Development, Xylos

Corporation, Langhorne, PA.

Purpose: Bioengineering of ophthalmic structures requires a biocompatible

support for cell growth and proliferation. Here we have examined the

biocompatibility of biocellulose membranes and whether these membranes support cell proliferation.

Methods: Cell viability, proliferation and morphology were assessed for ARPE-19

cells cultured on biocellulose membranes. Biocompatibility was examined by

implanting 33 biocellulose membranes subconjunctivally. At 1, 3 and 5 weeks,

eyes were enucleated and areas containing the implants were fixed in formalin,

embedded in paraffin, sectioned and stained with H&E. Histological sections were

analyzed for the presence of membrane degradation and inflammation. Results: On biocellulose membranes 100% of the cells were viable with only a rare

dead cell observed; however cell proliferation was lower than for cells cultured on

plastic. On biocellulose membranes cells appeared more compact and less spread

than cells cultured on a plastic substratum. Biocellulose membranes implanted

subconjunctivally demonstrated excellent biocompatibility similar to that observed

with amniotic membranes, with only mild inflammation observed during the first

week post implantation. Histological examination revealed no degradation of the

membranes during a 5 weeks follow up. A few lymphocytes and giant cells were observed indicating a very minor foreign body reaction. Placed on the cornea the

biocellulose membranes adapted perfectly to the convex corneal shape without

folding. The membranes could be sutured (Vicryl-7) to the limbus without tearing

when tensile force was applied.

Conclusions: The results show that the biocellulose biomaterial demonstrates very

good biocompatibility as proven by the lack of inflammatory reactions at the site of

implantation. The membranes are not toxic since cells remained viable and proliferated for a period of 3 weeks; however proliferation was slower than on

plastic. Biocellulose membranes exhibit excellent deformability and satisfactory

transparency, when applied to the corneal surface. Therefore, biocellulose

membranes are a promising biomaterial for use as a repair matrix for corneal

applications such as in corneal ulcers, for subconjunctival implants for the repair

and protection of ophthalmic tissues and as a cell transfer sheet for the

transplantation of cells where control of initial propagation rates is desirable.

Commercial Relationships: Anna Dobias, Xylos Corporation, USA (F); Anna-

Katharina Salz, None; Nina Harmening, None; Constance Ace, None; Peter

Walter, None; Gabriele Thumann, None

Support: Xylos Corporation USA



2-Arachidonoylglycerol (2-AG) Induces Corneal Epithelial Cell Migration via

Cannabinoid CB1 Receptors Shimin Li

1A, Sherry Hu

1B, Douglas McHugh

1B, Alex Straiker

1B, Joseph A.

Bonanno1A

. ASchool of Optometry,

BPsychological and Brain Sciences,

1Indiana

University, Bloomington, IN.

Purpose: Cannabinoids are a group of ~70 compounds present in Cannabis plants.

Best known for their psychoactive properties, they also possess immunosuppressive

and anti-inflammatory properties, and have potential as therapeutic agents in

alleviating such conditions as pain, nausea, diabetes, septic shock, rheumatoid

arthritis, chronic hepatitis, and intraocular pressure in glaucoma. Some cannabinoids such as Δ

9-THC are known to act through classical cannabinoid

receptors (CB1, CB2) and potentially other putative receptors (e.g. GPR55,

GPR119, GPR92 and GPR18). CB1 receptors are known to be present in the eye,

but the distribution of the others remains poorly studied. Furthermore, a

constellation of proteins involved in the produce of the endogenous cannabinoids,

anandamide (AEA) and 2-arachidonoylglycerol (2-AG), have been identified. In

the present study, we examined the presence of five candidate cannabinoid

receptors as well as 10 cannabinoid-related proteins in bovine anterior eyes and assessed 2-AG-induced corneal epithelial cell migration to provide insight for the

medicinal potential of cannabinoids, particularly for wound healing during corneal

injury.

Methods: Bovine eyes were dissected to collect corneal epithelium and

endothelium, trabecular meshwork, and retina tissue. Primary corneal epithelial


cells were obtained via dispase digestion. The expression of a panel of cannabinoid

receptors (CB1, CB2 and several GPRs), enzymes for biosynthesis of AEA and 2-

AG (NAPE-PLD, ABHDs, FAAH, NAAA, DGLα/β and MGL) and cannabinoid

receptor interacting protein (CRIP1a) were examined by RT-PCR. CB1 protein was also examined by western blotting. 2-AG-induced epithelial cell migration was

examined using a Boyden Chamber assay with Diff-Quik staining.

Results: mRNA for CB1, GPR18, ABHD4, ABHD12, MGL and CRIP1a was

detectable in the bovine corneal epithelium. CB1 protein was also found in this

tissue, while CB2 was not detectable. 2-AG-induced corneal epithelial cell

migration in a concentration-dependent manner with the peak at 100nM, which was

able to enhance the migration by 6-fold when compared to the control.

Furthermore, 2-AG induced-migration was blocked by a CB1 antagonist SR141716. Conclusions: Our results demonstrate the presence of several additional

components of the endocannabinoid signaling system in mammalian anterior eyes.

Corneal epithelial cell migration is mediated via activation of cannabinoid receptor

CB1 by 2-AG. This event may alter the cytokine profile and promote cornea wound

healing.

Commercial Relationships: Shimin Li, None; Sherry Hu, None; Douglas

McHugh, None; Alex Straiker, None; Joseph A. Bonanno, None

Support: NIH Grant EY08834



Corneal But Not Neutrophil Ho-2 Expression Contributes To Corneal Healing Giuseppina Marrazzo

1A, Lars Bellner

1A, Adna Halilovic

1A, Michael W. Dunn

1B,

Michal L. Schwartzman1A

. APharmacology,

BOphthalmology,

1New York Medical

College, Valhalla, NY.

Purpose: Heme oxygenase (HO) is the rate-limiting enzyme in heme degradation. Our previous studies demonstrated that the HO system, in particular, the

constitutive HO-2 is critical for a self resolving inflammatory and repair response.

Epithelial injury in HO-2 null mice leads to impaired healing and chronic

inflammation in the cornea. This study was undertaken to examine the possible

relationship between HO-2 and the recruitment of inflammatory cells.

Methods: Re-epithelialization and neutrophil recruitment were assessed in wild

type (WT) and HO-2 knockout (KO) mice treated with Gr-1 monoclonal antibody

to deplete peripheral neutrophils. Re-epithelialization was measured by fluorescein staining. The number of inflammatory cells was visualized using H&E staining.

Adhesion assay was performed using aortic endothelial cells (mAEC) from WT and

KO mice and neutrophils isolated from WT mice. HO-1 and HO-2 expression was

assessed by real-time PCR.

Results: Wound closure in intact WT mice was 60% higher than in neutrophil-

depleted WT mice and 85% higher in intact KO mice than neutrophil-depleted KO

mice. H&E staining confirmed the lack of inflammatory cells in the cornea in neutrophil-depleted mice and showed that the number of neutrophils is markedly

increased in corneas from HO-2 KO mice as compared to WT mice. WT

neutrophils adhered more to mAEC KO cells than to WT. Real time PCR showed a

significant difference in the HO-1expression between neutrophils purified from

intact WT and neutrophil-depleted WT mice; no significant changes were observed

in HO-1 expression in neutrophils of KO mice intact or neutrophil-depleted.

Conclusions: The HO-2 null mice showed an exaggerated corneal inflammation

associated with increased number of inflammatory cells compared to WT. Our results demonstrated that the elevated number of neutrophils recruited to the injured

tissues in HO-2 KO mice are not mainly responsible for the delay in healing, on the

contrary, the lack of neutrophils, through depletion, increase significantly the delay

in wound healing in both WT and KO.

Commercial Relationships: Giuseppina Marrazzo, None; Lars Bellner,

None; Adna Halilovic, None; Michael W. Dunn, None; Michal L.

Schwartzman, None

Support: Supported in part by NIH grant EY06513 (MLS) and the international PhD program in Neuropharmacology, University of Catania, Medical School

(GM).



Ocular Side Effects Of Inhibitors Of The Epidermal Growth Factor Receptor:

Report Of 4 Cases Nicolas Molina, Raoul A. Saint-Jean, Josep Torras, Merce Morral, Maite Sainz de la Maza. Hospital Clinic of Barcelona, Barcelona, Spain.

Purpose: To describe the ocular effects associated with the administration of

epidermal growth factor (EGF) receptor inhibitors, Panitumumab and Erlotinib.

Methods: Noncomparative interventional case series included 8 eyes of 4 patients

in treatment with EGF receptor inhibitors, 3 with Erlotinib for end-stage lung

carcinoma and 1 with Panitumumab for end-stage colorectal cancer.

Results: Multiple epithelial defects were observed in 8 eyes, corneal melting and

thinning were observed in 3 eyes of 2 patients, 2 eyes of 1 patient presented with lower lid ectropion, and corneal perforation in 2 eyes of 2 patients, both requiring a

penetrating keratoplasty.

Conclusions: Severe ocular side effects, including perforated corneal ulcers, may

be associated with the use of the EGF inhibitors panitumab and erlotinib.

Commercial Relationships: Nicolas Molina, None; Raoul A. Saint-Jean,

None; Josep Torras, None; Merce Morral, None; Maite Sainz de la Maza, None

Support: None


Effectiveness of Heated Hematic Derivatives (Autologous Serum & Plasma

Rich in Growth Factors) in Corneal Epithelial Wound Healing Claudia Nunez

1, Jesus Merayo-Lloves

1, Guilherme Ferrara

1, Ignacio Alcalde

1,

Manuel Chacon1, Carlos Alonso-Ron

1, Eduardo Anitua

2.

1Fundacion de

Investigacion Oftalmologica, Instituto Oftalmologico Fernandez-Vega, Oviedo,

Spain; 2Research / Development, Fundación Anitua. BTI, Vitoria-Gasteiz, Spain.

Purpose: To study and compare the effectiveness of heated autologous hematic derivatives in corneal epithelium recovery after experimental trauma in a cell

culture model.

Methods: A wound was practiced in cultures of SV40-immortalized human

corneal epithelial cells grown until confluence and incubated in different hematic

derivatives as culture media: plasma, activated plasma, Plasma Rich in Growth

Factors (PRGF), heated-activated-plasma and heated-PRGF (50ºC, 20 minutes).

Those media were obtained from blood processing taken from the same donor.

There were also two negative controls: DMEM/F12 and PBS. Photographs of wounded areas were taken each 12 hours and analyzed with a cuantitative software.

Velocity of closure and time of healing were measured.

Results: All hematic derivatives increase wound healing and the closure of the

epithelial wound, compared with the negative controls. The higest rate of repare

was obtained at the PRGF group. Interestingly, the rate of healing and the velocity

of recovery of epithelial wound was increased in all hematic derivatives by

exposing media to heat. This findings could be related to the denaturation of heat-sensible proteins present in the hematic derivatives.

Conclusions: Topical application of heated hematic derivatives increase the in

vitro epithelial wound healing compared to non heated. These results could be

transfer to clinical studies in order to evaluate the putative application for epithelial

wound healing therapy.

Commercial Relationships: Claudia Nunez, None; Jesus Merayo-Lloves,

None; Guilherme Ferrara, None; Ignacio Alcalde, None; Manuel Chacon,

None; Carlos Alonso-Ron, None; Eduardo Anitua, Inventor (E) Support: in part by Fundacion Mª Cristina Masaveu Peterson and CajAstur



http://files.abstractsonline.com/CTRL/26/e/f69/e66/efd/407/a8e/42c/f1c/1b5/9d1/f3/g5866_2.JPG

http://files.abstractsonline.com/CTRL/26/e/f69/e66/efd/407/a8e/42c/f1c/1b5/9d1/f3/g5866_4.JPG


Development Of Stereotactic Methods To Target The Ophthalmic Branch Of

The Trigeminal Ganglion Natalia Karagianni, John Pena, Mark I. Rosenblatt. Margaret M. Dyson Vision

Research Institute, Weill Cornell Medical College, New York, NY. Purpose: The ophthalmic branch of the trigeminal ganglion supplies sensory

nerves to the cornea. Our aim is to develop a stereotactic approach to reproducibly

apply agents to the trigeminal ganglia that can modulate corneal neurobiology.

Methods: Six to 8 week-old male C57BL/6 mice were deeply anesthetized and

immobilized within a neurosurgical stereotactic frame. Following removal of the

superficial dermal layer and bone, a 35g microinjection needle was passed to

previously published stereotactic coordinates for the trigeminal ganglia. India ink

dye was microinjected through the needle. Following injection, the animal was sacrificed and the true localization of the injected ink was compared to the expected

location of the injection. Initial comparative data was used to refine the stereotactic

coordinates corresponding to the ophthalmic branch of the trigeminal ganglion and

the reproducibility of these revised co-ordinates evaluated in littermates of the

initially examined animal.

Results: Using the published coordinates on 4 mice from 4 different litters to inject

the India Ink to their V1 nerve, only in 1 out of the 4 mice (25%) the injected ink

was located on the V1 nerve. In the other 3 mice the true V1 location was found to differ from the injected ink location by an average of +0.08mm on the x axis, -

0.5mm on the y axis, and -0.36mm on the z axis. After revising the coordinates

according to the true V1 location and using them to inject the India ink to the V1

nerve of each mouse‟s littermates, in 3 out of the 4 litters (75%) the injected ink

was located at the true V1 location.

Conclusions: Establishment of a reproducible system of microinjection at the

ophthalmic branch of the trigeminal nerve was achieved. This system can be used to apply agents such as nerve growth factors, analgesics, or gene vectors at the cell

bodies corresponding to the nerves providing sensation of the cornea. The system

may improve our ability to study basic and translational aspects of corneal

neurobiology.

Commercial Relationships: Natalia Karagianni, None; John Pena, None; Mark

I. Rosenblatt, None

Support: RPB Career Development Award and NIH Grant R01EY018594


Combine Use Of Platelet Gel And Autolougus Serum Eye Drop In The

Management Of Non-Healing Corneal Ulcer: in vivi confocal microscopy

study Marco Marenco, Isabella Mariani, Magda Gharbyia, Stefano Lupo, Serena

Fragiotta, Giancarlo Ferrazza, Enzo Maria Vingolo. Ophalmology, Sapienza

University of Roma, Rome, Italy. Purpose: to report the use of combined Platlet-gel and autologous serum eye drop

in the treatment of persistent corneal ulcers and analyze the effect on corneal tissue

by the mean of in vivo confocal microscopy .

Methods: 11 patients with persistent corneal ulcers not responsive to antibiotic

treatment were treated with combined therapy of platelet-gel and autologous serum

eyedrops.

Best Corrected Visual Acuity, complete eye examination, anterior segment

photograph with fluorescein stain and in vivo confocal microscopy were performed and analyzed in all patients for a minimum follow up of 3 months. Preparation: 10

ml of platelet rich plasma (PRP) were obtained from 16 ml of autologous peripheral

blood into two REGEN ™ RTHT tubes after centrifugation 8 minutes at 1500xg.

It‟s possible to variate PRP volume transferring in another tube then draw off 2 ml

supernatant after centrifugation and homogenize by inversion of the tube. In this

way the platelet number can reach twice or three times greater than peripheral

values. Platelet gel was performed adding PRP 5 ml of autologous serum plus 2 ml

of Ca-gluconate. All patients received topical treatment with antibiotic therapy associated with gel 3 times daily for 3 days followed by topical instillation of

autologous serum eye drops 4 times daily until corneal ulcer closure.

Results: in all cases relief of symptoms was obtained and after 20 days closure of

corneal ulcer with complete re-epithelialization occurred in all 11 patients.

Confocal microscopy performed at resolution of revealed regeneration of nerve

plexus with flocculation in all cases.

Conclusions: The presence of plateles together with plasma improve tissues regeneration. Platelet-gel and autologous serum have been used in the treatment of

several corneal disorders. Both of them are simple, non invasive and well tolerated

and provide a good support for corneal healing in persistent corneal ulcer.

Commercial Relationships: Marco Marenco, None; Isabella Mariani,

None; Magda Gharbyia, None; Stefano Lupo, None; Serena Fragiotta,

None; Giancarlo Ferrazza, None; Enzo Maria Vingolo, None

Support: None


Impact Of Storage Time On Cryopreserved Amniotic Membrane Henning Thomasen

1, Mikk Pauklin

2, Bernhard Noelle

3, Gerd Geerling

4, Jan

Vetter5, Philipp Steven

6, Klaus-Peter Steuhl

1, Daniel Meller

1.

1Department of

Ophthalmology, University Hospital Essen, Essen, Germany; 2Department of

Ophthalmology, University Tartu, Tartu, Estonia; 3Department of Ophthalmology,

University of Kiel, Kiel, Germany; 4Ophthalmology, University of Wurzburg,

Wurzburg, Germany; 5Department of Ophthalmology, University of Mainz, Mainz,

Germany; 6Ophthalmology, University of Luebeck, Luebeck, Germany.

Purpose: Cryopreserved amniotic membrane (AM) is widely used in

ophthalmology because of its anti-angiogenic, anti-inflammatory and wound

healing promoting capabilities. A common method to conserve the tissue is the

storage in cell culture medium containing 50% glycerol at -80°C. The aim of this

study was to examine the influence of storage time on the sterility as well as the

histological and biological properties of cryopreserved AM.

Methods: Amniotic membrane from different donors which was stored in cell

culture medium containing 50% glycerol for different time periods, on average 4 months (group 1), 15 months (group 2) and 24 months (group 3), at -80°C was

analysed. Samples of the tissue and cryo-medium were examined for bacterial and

fungal contamination. Tissue samples were incubated in 0.5ml/cm2 serum-free

medium at 37°C. The medium was changed after 1, 2, and 3 days. The proteins

released by AM were TCA-precipitated and the proteins TIMP-1 and IL-1ra were

analyzed using Western blotting and semi quantified by means of image analysis.

Integrity of the amniotic epithelium and the basement membrane components

collagen IV, collagen VII, laminin, laminin 5 and fibronectin were examined by haematoxylin eosin stain and immunohistochemistry in cryosections of AM.

Results: None of the examined samples showed bacterial or fungal contamination.

The soluble proteins TIMP-1 and IL-1ra were found in all samples of medium

incubated for all time periods. The examined proteins were detectable after one-day

incubation but the staining signal diminished significantly in the second and third

wash after 48h and 72h. Differences in the intensity of the Western Blot signal

between the three particular groups were statistically not significant. The epithelia of all samples were intact. The basement membranes of all samples showed a

similar distribution of collagen IV, collagen VII, laminin, laminin 5 and

fibronectin.

Conclusions: Long-term storage of amniotic membrane in cell culture media with

50% glycerol does not significantly impair sterility, histology or biological

properties of AM.

Commercial Relationships: Henning Thomasen, None; Mikk Pauklin,

None; Bernhard Noelle, None; Gerd Geerling, None; Jan Vetter, None; Philipp

Steven, None; Klaus-Peter Steuhl, None; Daniel Meller, None

Support: None



Plasma Rich In Growth Factors (PRGF) Stimulate Scarness Wound Healing

In The Stromal Cells Of The Ocular Surface Tissues Gorka Orive

1, Eduardo Anitua

1, Francisco Muruzabal

1, Maria De la Fuente

1,

Jesús Merayo2.

1Biotechnology Institute, Vitoria, Spain;

2Instituto Oftalmologico

Fernandez-Vega, Oviedo, Spain.

Purpose: Plasma rich in growth factors (PRGF) technology is an autologous

platelet-enriched plasma obtained from patient‟s own blood, which after activation

with calcium chloride allows the release of a pool of biologically active proteins

that influence and promote a range of biological process including cell recruitment,

growth and differentiation. Since ocular surface wound healing is mediated by

different growth factors, we decided to explore the potential of PRGF technology in stimulating the biological processes related with fibroblast-induced tissue repair.

Furthermore, the anti-fibrotic properties of PRGF were also studied.

Methods: Blood from healthy donors was collected, centrifuged and, whole plasma

column (PRGF) was drawn off avoiding the buffy coat. Primary human cells

including keratocytes and conjunctival fibroblasts were used to perform the “in

vitro” investigations. The potential of PRGF in promoting wound healing was

evaluated by means of a proliferation and migration assays. Fibroblast cells were

induced to myofibroblast differentiation after the treatment with 2.5 ng/ml of TGF-β1. The capability of PRGF to prevent and inhibit TGF-β1-induced differentiation

was evaluated.

Results: Results show that this autologous approach enhances significantly

proliferation and migration of both keratocytes and conjunctival fibroblasts. In

addition, PRGF prevents and inhibits TGF-β1-induced myofibroblast

differentiation.

Conclusions: These results suggest that PRGF can reduce scarring while stimulate wound healing in ocular surface.

Commercial Relationships: Gorka Orive, Scientist of BTI (E); Eduardo

Anitua, Pioneer of the PRGF technology (P); Francisco Muruzabal, Scientist of

BTI (E); Maria De la Fuente, Scientist of BTI (E); Jesús Merayo, None

Support: None



Differential Effects of Vascular Endothelial Growth Factor on Existing and

Regenerating Corneal Nerves In Vivo Shima Fukuoka, Natalia Karagianni, Mark I. Rosenblatt. Margaret M. Dyson

Vision Research Institute, Weill Cornell Medical College, New York, NY.

Purpose: Vascular endothelial growth factor (VEGF) can mediate nerve growth in

addition to its well-described effects on angiogenesis. We evaluated the effects of


VEGF on existing and regenerating corneal nerves in thy1-YFP mice using a

corneal micropocket assay.

Methods: Sucralfate/hydron pellets were impregnated with VEGF or vehicle

(negative control). A partial thickness corneal stromal micropocket was made in the cornea of an anesthetized thy-1-YFP mouse, and the pellets implanted midway

between the central corneal and limbus. For the analysis of VEGF effects on

corneal nerve regeneration, mice received a superficial injury via debridement of

the epithelium and underlying corneal nerve plexus one day after pellet

implantation. Mice were sacrificed 4 days after surgery and nerves imaged via

fluorescence microscopy at the site of pellet implantation and at a site 180 degrees

away from the pellet. Images were analyzed using neuron analysis software

(Neurolucida) to quantify axonal density and morphology. Stimulation of angiogenesis was monitored via slit lamp examination.

Results: In the absence of concomitant nerve injury, Mice receiving VEGF pellets

demonstrated minimal neurogenesis at the site of pellet implantation and no

changes in nerve density at sites away from the pellet. However, when a

concomitant superficial nerve injury was applied with the pellets, a significant

increase in nerve regeneration was measured at the site of the pellet as well as at

sites distant from the pellet in mice receiving VEGF. No angiogenesis was

observed with the doses of VEGF used in these experiments. Conclusions: Sub-angiogenic concentrations of VEGF appeared to have minimal

effects on pre-existing innervation of the cornea, but did significantly stimulate

corneal nerve regeneration following injury to the corneal sub-basal neuronal

plexus. These data suggest a specific role for VEGF in corneal neurobiology and in

possible use of VEGF in treating corneal nerve injury.

Commercial Relationships: Shima Fukuoka, None; Natalia Karagianni,

None; Mark I. Rosenblatt, None Support: RPB Career Development Award and NIH Grant R01EY018594



Amelioration Of Ultraviolet-induced Photokeratitis Treated With Astaxanthin

Eye Drops In Mice Anton Lennikov

1A, Nobuyoshi Kitaichi

1A, Risa Fukase

1A, Miyuki Murata

1A, Kousuke

Noda1A

, Shigeaki Ohno1B

, Ryo Ando1A

, Susumu Ishida1A

. ADepartment of

Ophthalmology, BDepartment of Ocular Inflammation and Immunology,

1Hokkaido

University, Sapporo, Japan.

Purpose: Astaxanthin (AST), a red carotenoid found in marine animals and

vegetables, has potential clinical applications due to its higher antioxidant activity

than β-carotene and α-tocopherol. Acute ultraviolet (UV) exposure causes

photokeratitis and induces various inflammatory changes in the cornea. In the

present study, we examined whether topical administration of AST has therapeutic

effects on UV-photokeratitis in mice. Methods: Six- to 8-week-old C57BL/6 male mice were used. C57BL/6 male mice

were administered AST in instillation form at the concentrations of 1 mg/ml, 0.1

mg/ml, and 0.01 mg/ml to right eyes, whereas left eyes were instilled with vehicle

alone. After the instillation, the mice were irradiated with UVB at the dose of 400

mJ/cm2 under anesthesia. Eyeballs were collected 24 hours after irradiation, and

corneal damage was evaluated by TUNEL. As an in vitro study, NIH-3T3 cells

irradiated with UVB were cultured with or without AST. Cytotoxicity was

quantified with LDH assay. Results: UVB irradiation caused disruption of the corneal basement membrane and

UVB irradiation caused disruption of the corneal basement membrane and thinning

of the corneal epithelium; however, the epithelium was well preserved after

irradiation in AST-treated corneas. The corneal epithelium thickness was

35.75±1.7, 29.75±1.7 and 8.5±2.8 μm in mice treated with 1, 0.1 and 0.01 mg/ml of

AST, respectively. The mean corneal epithelial thickness was 4.75±4.6 μm in

untreated eyes after irradiation. Non-irradiated corneal epithelium was 38.25±2.5

μm thick. Apoptotic cells were counted as 2.75±3.7, 2.25±2.8, 19.0±3.2, and 23.0±5.3 in eyes treated with 1, 0.1, 0.01, and 0 mg/ml of AST, respectively.

Significantly fewer apoptotic cells were observed in AST-treated UV-irradiated

corneas than controls (p<0.01). In vitro study showed less cytotoxicity in AST-

treated cells after UVB-irradiation. The percentages of mean cytotoxicity after

irradiation were 23.0±5.3%, 59.25±5.3%, 77.75±7.6 %, and 86.75±4.3% in wells

added 1, 0.1, 0.01, and 0 mg of AST, respectively.

Conclusions: These results suggest that AST has the protective effect against UVB damage in vivo and in vitro. AST might be a promising naturally-derived material

protecting ocular surface from the toxicity of ultraviolet.

Commercial Relationships: Anton Lennikov, None; Nobuyoshi Kitaichi,

None; Risa Fukase, None; Miyuki Murata, None; Kousuke Noda,

None; Shigeaki Ohno, None; Ryo Ando, None; Susumu Ishida, None

Support: Mishima Memorial Foundation Grant



Vegf Promotes Trigeminal Neuron Growth Through Multiple VEGF Receptor

Types Zan Pan, Aihong Liu, Natalia Karagianni, Mark I. Rosenblatt. Margaret M. Dyson

Vision Research Institute, Weill Cornell Medical College, New York, NY.

Purpose: Our previous data demonstrated that vascular endothelial growth factor

(VEGF) can regulate the growth of corneal nerves. We sought to identify the

VEGF receptor sub-types (VEGFR1, VEGFR2, or neuropilin-1(NPR1)) which

mediate the potent effect of VEGF on isolated trigeminal neurons in vitro. Methods: Thy1-YFP mice were sacrificed and trigeminal ganglia (TG) isolated

following craniotomy. Isolated neurons were isolated from TG by limited

enzymatic treatment with papain and collagenase/dispase followed by Percoll

gradient centrifugation. Neurons were seeded on poly-D-lysine coated culture

plates and incubated in Neurobasal A media containing 1% B27 supplement. Two

hours after plating, neurons were treated for 1 hour with either VEGFR2 kinase

inhibitors (SU1498 and Ki8751), specific neutralizing antibodies for VEGFR1,

VEGFR2 or NPR1 or random IgG. Subsequently, 50ng/ml VEGF was added to each pre-treated well to evaluate for VEGF-dependent neuronal growth. Neurons

were imaged by fluorescence microscopy at multiple time points. Images were

analyzed using quantitative neuronal tracing software (Neurolucida).

Results: Primary cultured trigeminal neurons extended dendrites 24 hr after

plating. By 72 hours, 50 ng/ml VEGF increased dendrite numbers, branches, length

and tortuosity by 3-fold. SU1498 (5uM) or Ki 8751 (5nM) completely suppressed

dendrite elongation in the presence of VEGF. Neutralizing antibodies for VEGFR1,

VEGFR2, and NPR1 inhibited dendrite growth in a dose-dependent manner with nearly complete inhibition achieved by 10ug/ml anti-VEGFR1, 2.5 ng/ml anti-

VEGFR2, and 2ug/ml anti-NPR1. Random IgG did not affect dendrite growth.

Conclusions: VEGF promotion of trigeminal neuron growth is mediated through

multiple receptors, including VEGFR receptors type 1, 2 and NPR 1. VEGF

represents a potential drug target to promote restoration of corneal innervation

following corneal injury. Further study is needed to identify opportunities to

optimize VEGF-mediated neurogenesis while minimizing its possible angiogenic effects.

Commercial Relationships: Zan Pan, None; Aihong Liu, None; Natalia

Karagianni, None; Mark I. Rosenblatt, None

Support: RPB Career Development Award and NIH Grant R01EY018594



Intracameral Bevacizumab Reduces Deep Stromal Neovascularization And

Improves Corneal Graft Clarity Vikram S. Brar, Monali Sakhalkar, S Balaiya, KV Chalam. Ophthalmology,

University of Florida, Jacksonville, FL.

Purpose: To demonstrate the efficacy of intracameral bevacizumab in treating deep

corneal neovascularization secondary to graft failure.

Methods: We report a case of a 41 year-old male who presented with corneal graft

failure demonstrating deep stromal neovascularization and central corneal opacity.

He had a history of keratoconus for which he had undergone three prior penetrating keratoplasties in the affected eye. His best corrected visual acuity was 20/400. The

pupil could not be visualized. Clinical improvement did not occur despite therapy

with 40mg oral prednisone, hourly prednisolone acetate 1% eye drops, and a

subconjunctival injection of bevacizumab (1.25mg). Subsequently, he was given 2

intracameral injections of bevacizumab (1.25mg) spaced 4 weeks apart. Topical

prednisolone acetate was continued. Slit lamp photography and aqueous samples

were obtained prior to and following intracameral injections.

Aqueous samples were analyzed by immunobead assay using Luminex 100 IS fluoroanalyzer to detect concentration of vascular endothelial growth factor

(VEGF).

Results: There was marked reduction in the corneal stromal neovascularization and

central opacification following two intracameral injections of bevacizumab. The

resultant clarity allowed the pupil and iris to be visualized and the patient‟s vision

improved to 20/200. Pre-injection VEGF level was 7.912pg/mL, which increased to

9.308pg/mL prior to the second injection.

Conclusions: We report successful regression of deep stromal corneal neovascularization with improvement in graft clarity, following intracameral

injections of bevacizumab. Clinical improvement in this case was not related to

aqueous concentrations of VEGF which remained normal despite anti-VEGF

therapy, suggesting an alternative mechanism of action in this case.

Commercial Relationships: Vikram S. Brar, None; Monali Sakhalkar, None; S.

Balaiya, None; KV Chalam, None

Support: None



Proteomic Analysis of Corneal Tissue Following Metallic Injury Shari A. Seidman

1A, Natasha Johnson

1A, Toral P. Parikh

1A, Eric Geron

1A, Sanjoy K.

Bhattacharya1B

. AOphthalmology,

BBascom Palmer Eye Institute,

1University of

Miami/ Bascom Palmer Eye Institute, Miami, FL.

Purpose: To determine whether metallic object penetration and length of exposure

time results in predictable changes in corneal protein profile. To determine whether penetration by metallic objects of different compositions (copper, iron and lead)

results in different protein changes for the bovine and porcine eyes.

Methods: Enucleated bovine (n=30) and porcine (n=30) were used for exposure to

copper nails, iron pins and lead pellets. Eyes were subjected to Fluorescein staining

to confirm corneal epithelial integrity. Laemellar dissection techniques were


employed to isolate layers of the cornea for exposure. Excised cornea was

subjected to protein extraction. Protein amount was determined by

spectrophotometry and protein profile was determined using SDS-PAGE analysis.

Results: Metallic object penetration resulted in lower protein extractability from corneal tissue compared to controls. Increased depth of injury also negatively

affected protein extractability compared to controls. The changes in protein profiles

were different for different metals (copper, iron and lead). Protein profile changes

were different for penetration to different depths and dependent on several other

factors in a complex manner.

Conclusions: Exposure to metallic objects results predictable changes in corneal

protein profile. The different duration and composition of metallic object shows

differences in protein profile. The depth and duration of exposure results in reproducible changes suggesting

severity of exposure can be predicted from corneal

protein profile changes.

Commercial Relationships: Shari A. Seidman, None; Natasha Johnson,

None; Toral P. Parikh, None; Eric Geron, None; Sanjoy K. Bhattacharya, None

Support: RPB Career Development Award (SKB), unrestricted funds from RPB to

University of Miami.; DOD Grant W81XWH-09-1-0674 (Project 2.2); NIH core

grant P30-EY14801


Lipidomic Analyses of Corneal Tissue following Alkali Exposure Ashley M. Crane, Andrew D. Coggin, Bryan R. Cam, Byron L. Lam, Sanjoy K.

Bhattacharya. Bascom Palmer Eye Institute, University of Miami Miller School of

Medicine, Miami, FL.

Purpose: To determine whether corneal exposure to alkaline chemicals results in

predictable lipid profile changes. To determine whether lipid profile changes correlate to specific alkali strength and exposure duration.

Methods: Enucleated bovine (n= 40), porcine (n= 40), and cadaver human eyes

(n= 6) were procured from Just Meats, Chillicothe, Ohio and Lions Eye Bank

Florida respectively and exposed to varying concentrations of sodium and

ammonium hydroxide. The corneas were excised and the corneal lipids were

extracted via the Bligh and Dyer lipid extraction method. The extracted lipid

solutions were analyzed with thin layer chromatography (TLC). Lipid standards

were purchased and run simultaneously on the TLC to assess for presence of specific lipid classes. Mass spectrometry is pending to determine exact lipid species

present in corneal tissue. This research adhered to ARVO statement for use of

animals in research and Helsinki Declaration respectively.

Results: TLC analysis revealed that alkali exposure duration indeed alters the lipid

profile. The lipid solution extracted from bovine eyes that were exposed to 11

molar sodium hydroxide revealed significant profile changes that manifested at the

30-minute time point. A new spot on the TLC plate appeared at the 30-minute time point that was not present in the previous 30-second and 12-minute time points.

This spot remained for the following 60-minute, 8-hour, and 24-hour time points.

Further mass spectrometry will be used to determine the exact composition of this

novel spot.

Conclusions: Alkali exposure duration does indeed modify the corneal lipid

profile.

Commercial Relationships: Ashley M. Crane, None; Andrew D. Coggin,

None; Bryan R. Cam, None; Byron L. Lam, None; Sanjoy K. Bhattacharya, None

Support: DOD grant W81XWH-09-1-0674 (Project 2.2); NIH core grant P30-

EY14801



Study Of The Therapeutic Action Of 0,1% Riboflavin/ultraviole Tradiation

On The Experimental Ey Burn In Rabbits Marcello C. Barboza

1, Sergio Felberg

1, Guilherme Barboza

1, Paulo Dantas

1, Elcio

Sato2.

1Ophthalmology, Santa Casa de Sao Paulo, Sao Paulo, Brazil;

2Ophthalmology, Unifesp, Sao Paulo, Brazil.

Purpose: To evaluate the effect of riboflavin/ultraviolet radiation (collagen

crosslinking) after ocular alkali burn in rabbits

Methods: Ten rabbits had the right corneal limbal structure burned with NaOH 4N

and were divided in two groups. Control group was treated with clinical therapy

and Case Group was treated with clinical therapy plus riboflavin/ultraviolet radiation (collagen crosslinking) after one hour.

Clinical parameters were evaluate at 1, 7, 15, 30 days. All animals were sacrificed

after 30 days and the corneas were evaluate by a pathologist.

Results: In the preliminary study, two corneas in the Case group and one cornea in

the Control group were analyzed until now. There was no difference in clinical

parameters. The histopathology exam showed more organized collagen fibers and

more bridges linking collagens fibers in case group than control group.

Conclusions: Pilot study suggests that anatomical changes seem to occur in the collagen fibers after riboflavin/ultraviolet light (collagen crosslinking) in corneas

with acute alkali ocular burn.

Commercial Relationships: Marcello C. Barboza, None; Sergio Felberg,

None; Guilherme Barboza, None; Paulo Dantas, None; Elcio Sato, None Support: None



Ten-year results of Phototherapeutic Keratectomy on Recurrent Corneal

Erosions Belquiz A. Nassaralla

1A, Joao J. Nassaralla, Jr.

1B.

ACataract Cornea & Refractive

Surgery, BRetina and Vitreous,

1Instituto de Olhos de Goiania, Goiania, Brazil.

Purpose: To determine the ten-year visual results and outcome of excimer laser

phototherapeutic keratectomy (PTK) for recurrent corneal erosions.

Methods: A retrospective chart review of 26 eyes of 23 patients with recurrent

corneal erosions treated by PTK from 1996 to 2000 was performed. All eyes had

failed to respond to conventional therapy. Data regarding the preoperative and

postoperative best-corrected visual acuity (BCVA), spherical equivalent (SE),

symptomatic relief, incidence of recurrence, and complications arising from the

laser treatment were analyzed. The mean duration of symptoms prior to PTK was 18 months (range, 8 to 36 months). The corneal epithelium was debrided, and laser

ablation was performed to a depth of 5 micron with an ablation zone of 7 to 9 mm,

using the Technolas 217C Plano Scan excimer laser. Mean postoperative follow-up

was 12 years (range, 10 to 14 years).

Results: At the last follow-up visit, 15 eyes (57.69%) were free of symptoms. Five

eyes (19.2%) had occasional mild symptoms of irritation and photophobia upon

awakening. Recurrence of painful corneal erosions occurred in six eyes (23.07%),

which required a PTK retreatment. Twenty four eyes had a preserved or improved BCVA, while 2 eyes showed deterioration of 1 line on Snellen testing. Eleven eyes

had no change in SE; the rest had a change of less than +/-0.75 diopters. There

were no major complications during the follow-up period.

Conclusions: Excimer PTK is a safe and effective procedure for relieving

symptoms and improving visual acuity in patients with recurrent corneal erosions

refractory to conventional treatment.

Commercial Relationships: Belquiz A. Nassaralla, None; Joao J. Nassaralla,

Jr., None

Support: None

Clinical Trial: http: //www.clinicaltrials.gov, NCT01251692



Tgfβ Concentration And Corneal Wound Healing

http://files.abstractsonline.com/CTRL/11/b/50f/92b/fdf/44c/68b/7c3/8ee/346/7ac/91/g1546_1.JPG

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Audrey M. Bernstein1, Rajiv R. Mohan

2, Jonathan C. Tovey

3, Nadia Pelaez

1,

Lingyan Wang1.

1Ophthalmology, Mount Sinai School of Medicine, New York,

NY; 2Mason Eye Institute, University of Missouri-Columbia, Columbia, MO;

3Ophthalmology, University of Missouri, Columbia, MO.

Purpose: We have investigated the role of TGFβ concentration in fibroblast

migration and corneal wound healing as a means to enhance stromal repopulation

and improve corneal stromal wound healing after LASIK.

Methods: Human corneal fibroblasts (HCFs) were cultured on collagen in

supplemented serum-free media (SSFM). Cell migration was assessed by a scratch-

wound assay and results were quantified using T-Scratch software.

Immunocytochemistry was used to evaluate SMAD 2/3 localization and α-SMA

expression. Active TGFβ was quantified by co-culturing HCFs with TMLC cells containing the PAI-1 promoter linked to the luciferase gene. Wounding in vivo was

performed by creating a corneal flap of 8mm in diameter and 200μm in depth with

a FlapMaker Disposable Microkeratome in New Zealand White rabbits (2.5-3.0kg).

At 0 and 24 hours, corneas were excised and embedded for histological

examination to measure wound closure and α-SMA expression. The contralateral

eyes served as unoperated controls.

Results: We demonstrated that endogenous levels of active TGFβ1 (0.01ng/ml)

stimulated cell migration in vitro since neutralizing antibody to TGFβ1 inhibited cell migration by 3.6-fold compared to IgG control, which had no effect. Addition

of 1.0 ng/ml TGFβ1 (100-fold higher than endogenous levels) also reduced cell

migration by 3.2-fold, and promoted fibrotic markers: SMAD 2/3 nuclear

translocalization and myofibroblast differentiation. Similarly after corneal

wounding in rabbits in vivo, application of 0.01ng/ml TGFβ1 significantly

increased the rate of wound closure with reduced α-SMA expression compared to

1.0 ng/ml TGFβ1. Conclusions: These in vitro and in vivo data suggest that low levels of TGFβ1

(0.01ng/ml) promote cell migration and wound closure with a reduced fibrotic

response. Thus controlling but not eliminating TGFβ may be a key to promoting

regenerative healing in the cornea.

Commercial Relationships: Audrey M. Bernstein, None; Rajiv R. Mohan,

None; Jonathan C. Tovey, None; Nadia Pelaez, None; Lingyan Wang, None

Support: (AMB) NIH/NEI EY017030 and the Research to Prevent Blindness and

(RRM) NIH/NEI EY017294



Optimization Of in vitro Conditions Of TGFβ And PDGF Modulation Of

V+A+D+ Myofibroblast Development From Precursor Cells Vivek Singh, Flavia L. Barbosa, Vandana Agrawal, Steven E. Wilson. Cole Eye

Institute, Cleveland Clinic, Cleveland, OH.

Purpose: To test the hypotheses that: (a) development of mature vimentin+/alpha-smooth muscle actin+/desmin+ (V+A+D+) myofibroblasts from corneal stromal or

bone marrow-derived precursor cells are regulated by the coordinated, sequential

action of TGFβ and PDGF; (b) myofibroblast development in vitro follows a

similar developmental pathway of marker expression as it does in vivo.

Methods: Corneal keratocyte was isolated as per Yoshida et.al. (2005) protocol

from the fresh eyes of swiss Webster mice (Pel freeze). The corneal fibroblast

finally obtained was cultured in serum free media ( augmented DMEM/F12) or low

serum RSF media at different doses of TGFβ (0.1 to 2.0 ng/mL) with and w/o mice PDGF (2.0ng/mL) to determine optimal doses and time that promote transition of

the mouse corneal fibroblast to V+A+D+ myofibroblasts. At each time point (2 to

15 days), with each growth factor concentration, we determined the mean % of

vimentin+, SMA+ and desmin+ cells (all from single IHC).

Results: See Fig 1 & 2

Conclusions: We have established the optimal concentration and time required to

convert the corneal fibroblast to myofibroblast (V+A-D- to V+A+D- to

V+A+D+).Our results suggest that dose of 0.5ng/ml and 1.0ng/mL of TGFβ along with 2.0ng/mL PDGF in DMEM/F12 serum free media are the optimal

concentrations to study the above transformation in sequential manner to V+A+D+

myofibroblast ( Fig-1 & 2). The present results will help in better understanding of

the biology of myofibroblast development and will lead to prevent haze after PTK

and other surface ablation procedures, perhaps through the modulation of cytokine

effects on the myofibroblasts that underlie haze.

Commercial Relationships: Vivek Singh, None; Flavia L. Barbosa,

None; Vandana Agrawal, None; Steven E. Wilson, None

Support: Supported by EY10056 and EY15638 from National Eye Institute,

National Institutes of Health, Bethesda, Maryland and Research to Prevent

Blindness, New York, NY.



Dependence Of Tgfβ-induced Corneal Fibrosis On Trpv1 Activation Yuanquan Yang, Hua Yang, Zheng Wang, Fan Zhang, Peter S. Reinach. Biological

Sciences, SUNY College of Optometry, New York, NY.

Purpose: Previously we reported functional expression of transient receptor

potential cation channel V1 (TRPV1) in cultured human corneal fibroblasts. As

TRPV1 activation by severe injury in mice contributes to inappropriate healing, we

determined if the presence of TRPV1 is essential for TGFβ to induce myofibroblast

transdifferentiation.

Methods: Fresh human cadaver corneas were obtained from New York Upstate Transplant Service. Stromal fibroblasts were isolated and cultured following a

described method. Western blot analysis probed for α-smooth muscle actin (SMA)

and TRPV1 protein expression. Immunocytochemistry stained for α-SMA

expression. ELISA evaluated the effects of capsaicin (CAP), a TRPV1 selective

agonist, on IL-6 release.

Results: TGF-β1 (1ng/ml) treatment for 48 h upregulated TRPV1 protein

expression. Preincubation with capsazepine (CPZ, 5 µM), a selective TRPV1 antagonist, significantly reduced TGF-β1 induced α-SMA protein expression. After

24 h exposure to CAP (50 µM), IL-6 maximally rose 2.5-fold above the control

level, which was blocked by either CPZ (5 µM) or SB-203580 (10 µM), a p38

mitogen-activated protein kinase (MAPK) inhibitor.

Conclusions: TRPV1 blockade delays and reduces TGF-β1-induced

transdifferentiation of human corneal fibroblasts. TRPV1 activation induces IL-6

release through p38 MAPK pathway activation, which may also contribute to

fibrosis. Commercial Relationships: Yuanquan Yang, None; Hua Yang, None; Zheng

Wang, None; Fan Zhang, None; Peter S. Reinach, None

Support: EY04795, W81XWH-09-2-0162



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Cyclosporin A Suppresses Tgfβ1-induced Collagen Typei/iv Expression In

Both Primary And Cell Line Of Conjunctival Fibroblasts: A Possible

Implication In Modulation Of Fibrosis Alessandra Micera

1,2, Bijorn Omar Balzamino

1,2, Loredana Mastrella

2, Paolo

Lauretti2, Stefano Bonini

2.

1Lab. Ophthalmology, IRCCS-GB Bietti Eye

Foundation, Rome, Italy; 2CIR, Lab Ophthalmology, University Campus Bio-

Medico, Rome, Italy.

Purpose: To explore in vitro the influence of Cyclosporin A (CsA), a powerful

immunosuppressive agent, on fibrosis in term of proliferation, differentiation, ECM

metabolism and contraction of primary (from n=3/biopsies and from ScienCell Res.

Lab, Carlsbad, CA) cultures of conjunctival fibroblasts. CsA effects were compared

with those of TGFβ1. Methods: Both fibroblasts (FBs) and in vitro induced myofibroblasts (myoFBs)

were used for CsA (Restasis, Allergan Inc., Irvine, CA) at scalar doses (50, 200,

400 and 800 ng/mL). Cells were also exposed to 10ng/mL TGFβ1, 800ng/mL CsA

either alone or combined. Sister untreated cells were used as control. After 24

hours, cell viability and proliferation were assessed. Extracellular metabolism was

investigated by measuring collagen typeI/IV (csELISA) and MMP9/TIMP1

(zymography, ssELISA) protein expression. Extracellular matrix contraction was

monitored according to the 3D gel assay. Statistical analysis was assessed by parametric ANOVA-Tukey Kramer post hoc assays.

Results: Increasing CsA doses did not influence either proliferation nor αSMA

expression in both FBs and myoFBs. CsA did not modulate significantly collagen

typeI/IV nor MMP9 expression, while TGFβ1 exposure resulted in a drastic

increase of collagen typeI/IV in both cell types (p<.05). A trend toward a MMP9

increase was detected upon CsA exposure while a trend to a decrease was observed

for TIMP1 (p>.05). Addition of 800ng/mL CsA to 10ng/mL TGFβ1 abolished this typeI/IV increase in both FBs and myoFBs (p<.05). Merely to MMP9/TIMP1,

addition of 800ng/mL CsA to 10ng/mL TGFβ1 resulted in a suppression of TGFβ1-

induced MMP9/TIMP1 expression in both cell types. At low doses, CsA triggered

FB mediated contraction of a 3D gel with no effects at higher doses, as compared

to those of TGFβ1.

Conclusions: CsA eye drops are therapeutically used for severe allergic

conjunctivitis, but the mechanism of action is actually unknown. Herein, CsA

appears to modulate profibrogenic effects induced by TGFβ1, a well-known factor involved in FBs/myoFBs activation, suggesting new potential mechanisms of CsA

in ocular tissue remodeling/fibrosis.

Commercial Relationships: Alessandra Micera, None; Bijorn Omar

Balzamino, None; Loredana Mastrella, None; Paolo Lauretti, None; Stefano

Bonini, None

Support: None


Vimentin is a Novel Target for Corneal Fibrosis Paola Bargagna-Mohan

1,2, Riya R. Paranthan

2, Keiko Nakayama

3, Keiichi T.

Nakayama4, Royce Mohan

1,2.

1Neuroscience, University of Connecticut Health

Center, Farmington, CT; 2Ophthalmology and Visual Science, University of

Kentucky, Lexington, KY; 3Division of Developmental Genetics, Tohoku

University Graduate School of Medicine, Sendai, Miyagi, Japan; 4Department of

Molecular and Cellular Biology, Kyushu University, Fukuoka, Japan. Purpose: Fibrosis is an enigmatic consequence of many ocular blinding diseases

and it especially has devastating effects on the refractive property of the transparent

cornea. The goal of this study was to validate that vimentin is a key target for

corneal fibrosis.

Methods: We used the corneal model of combined alkali burn with epithelial

scrape injury to elicit corneal fibrosis. Mice were also subjected to the vimentin

inhibitor withaferin A (WFA; 2 mg/kg/day; intrapertoneal injection) for 7-14 days.

Results: Injured wild type mice sustain critical hallmarks of corneal fibrosis that concede from conjunctivalization, neovascularization, inflammatory infiltration and

myofibroblast activation, which become progressively insidious by 14 days post

injury. Injured corneas of vimentin deficient (Vim KO) mice have delayed

myofibroblast activation, downregulated TGF-beta expression by 7 days post

injury, and by day 14 show decreased CD11b-positive infiltrates and recover

expression of the abundant corneal crystallin tissue transketolase. We show that

wild type mice do not recover injury-downregulated expression of the critical cell cycle inhibitor p27

kip1 and maintain the conjunctival phenotype with globlet cell

expression, whereas, Vim KO mice engage a regenerative repair mechanism that is

evident by day 7 with re-expression of p27kip1

that is mainated through day 14

when corneal clarity is fully recovered. This in vivo mechanism of epithelial cell

growth arrest mediated by genetic deficiency of vimentin is mimicked by treatment

of wild-type mice with WFA in vivo that also causes recovery of p27kip1

expression

due to potent downregulation of its cognate ubiquitin conjugating E3 ligase Skp2.

Moreover, exploiting WFA to cause conditional knockout of vimentin in vitro, we show that G2/M cell cycle arrest exerted by WFA is completely abrogated in

cultured cells from mice deficient in p27kip1

or Skp2.

Conclusions: These findings for the first time illuminate a previously unrecognized

important role for vimentin that acts as a central target for corneal fibrosis resulting

from its broad expression in stromal, inflammatory, vascular and limbal

compartments of the diseased cornea. We additionally demonstrate that this type III

intermediate filament is druggable in vivo and afford WFA as a drug lead for

therapeutic development.

Commercial Relationships: Paola Bargagna-Mohan, patent pending (P); Riya

R. Paranthan, None; Keiko Nakayama, None; Keiichi T. Nakayama, None; Royce Mohan, patent pending (P)

Support: NIH Grant R01 EY0167821; Research to Prevent Blindness Challenge

Grant; Figh for Sight Foundation; Kentucky Science and Technology Corp.

COMMFUND-718-RFP-006; Kentucky Science and Engineering Fo



Proteomic Analysis Of Murine Corneas During Nerve Regeneration Lisette Yco, Abed Namavari, Shweta V. Chaudhary, Joy Sarkar, Sandeep Jain. Ophthalmology and Visual Sciences, University of Illinois Eye and Ear Infirmary,

Chicago, IL.

Purpose: To identify proteins expressed during nerve regeneration following

lamellar flap surgery in murine corneas.

Methods: Hinged lamellar corneal flap was fashioned surgically in Thy1-YFP

neurofluorescent mice (n=8). Corneas were harvested at 4 weeks and the flaps were

separated from the bed. Two-dimensional electrophoresis was performed according

to the carrier ampholine method of isoelectric focusing. Gels were scanned with a laser densitometer. The images were analyzed using Progenesis Same Spots

software. Spot integrated density above background was expressed as a percentage

of total density above background of all spots measured. Protein spots with >1.7

fold difference (p<0.05) were punched out and MALDI Mass spectrometric

analysis was performed on the digest using Applied Biosystems Voyager DE Pro

mass spectrometer in the linear mode.

Results: Eighteen protein spots showed >1.7 fold difference. Of these spots Calmodulin-like protein 3 showed the highest difference in expression (14.5 fold).

Other proteins identified were Transgelin-2, Annexin A1, Annexin A2 and

cytokeratins. The expression of calmodulin-like protein 3 was confirmed by qPCR.

Conclusions: Calcium sensor proteins like Calmodulin are abundantly expressed

during corneal nerve regeneration. Calmodulin may play a role in during corneal

nerve regeneration by regulating calcium signaling dependent secretion of

neurotrophins.

Commercial Relationships: Lisette Yco, None; Abed Namavari, None; Shweta

V. Chaudhary, None; Joy Sarkar, None; Sandeep Jain, None

Support: NEI Grant K08EY018874



Prevalence and Distribution of Hemangiogenesis and Lymphangiogenesis in

Pathologic Corneas Makambo Tshionyi, Elizabeth Shay, Elisa Lunde, Amy Lin, Kyu-Yeon Han, Sandeep Jain, Jin-Hong Chang, Dimitri Azar. Ophthalmology and Visual Sciences,

University of Illinois College of Medicine, Chicago, IL.

Purpose: We characterized the prevalence and distribution of hemangiogenesis

(HA) and lymphangiogenesis (LA) in human corneal specimens exhibiting 13

underlying pathologies.

Methods: Human corneal specimens collectively exhibiting Acanthamoeba

keratitis, bullous keratopathy, chronic keratitis, corneal scarring, Fuchs‟ dystrophy,

fungal keratitis, graft failure, graft rejection, herpes simplex virus keratitis, inflammatory pannus, keratoconus, pterygium, and ulcerative keratitis with

perforation were obtained from consenting subjects (n=2 or n=3 for each

pathology; total sample size, n=35). The pathologic specimens were stained with

hematoxylin and Eosin (H&E) to determine the presence or absence of (i) corneal

neovascularization, as well as (ii) comparatively superficial or deep stromal

distribution of neovascularization (NV). Immunohistochemical staining was then

performed to differentiate the prevalence and distribution of HA (positive for

CD31) from that of LA (positive for lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)).

Results: The double negative (CD31+/LYVE-1+) phenotype, indicating the

absence of NV, was exhibited by 21 specimens (60%). The mixed phenotype

(CD31+/LYVE-1-), indicating the presence of HA and the absence of LA, was

exhibited by 12 specimens (34%). The double-positive (CD31+/LYVE-1+)

phenotype, indicating the presence of both HA and LA, was exhibited by two

specimens (6%). Notably, the CD31+/LYVE-1+ mixed phenotype, indicating the presence of LA and the absence of HA, was not detected amongst the specimens.

Comparatively deep stromal NV exhibited in a 4: 3 ratio to superficial stromal NV.

Conclusions: The double-negative phenotype was more prevalent in corneal

specimens exhibiting non-inflammatory pathologies; the ratio of the double-

negative phenotype to the combined neovascular phenotypes (i.e., CD31+ or

LYVE-1+) was 3: 2. Among the neovascular phenotypes, HA was seven times

more prevalent than was LA. Specimens exhibiting LA presented only with the

double-positive phenotype. NV was predominantly detected in the deep stromal, rather than the superficial stromal, tissue layer. Our results suggest potential

therapeutic utility of targeting anti-neovascular therapies specifically to corneal

HA, LA, or mixed pathology.


Commercial Relationships: Makambo Tshionyi, None; Elizabeth Shay,

None; Elisa Lunde, None; Amy Lin, None; Kyu-Yeon Han, None; Sandeep

Jain, None; Jin-Hong Chang, None; Dimitri Azar, None

Support: EY14048 (JHC), EY10101 (DTA), EY01792 (DTA), and an unrestricted grant from Research to Prevent Blindness, New York, NY.



A Mouse Model of Limbal Stem Cell Deficiency Induced by Topical

Medication of Benzalkonium Chloride Zhirong Lin, Tong Zhou, Huan He, Xiaochen Liu, Hui He, Zuguo Liu.

Ophthalmology, Eye Institute of Xiamen University, Xiamen City, Fujian Province,

China. Purpose: To develop a mouse model of limbal stem cell deficieny induced by

topical administration of benzalkonium chloride and investigate the possible

mechanisms.

Methods: Benzalkonium Chloride (BAC) at concentrations ranging from 0% to

0.5% was applied to the mouse ocular surface for 28 days. Scoring of length of new

vessels (NV), corneal inflammatory index and fluorescent staining test were

performed to evaluate the toxic effects of BAC on the ocular surface. Global

specimens were collected on day (D) 28 and labeled with a series of antibodies including cytokeratin 12 (K12), cytokeratin 19 (K19), connexin 43 (Cx43), P63,

ABCG2, EGF-receptor, β-catenin, CD4, cytokeratin 10 (K10), Ki67. In situ

TUNEL assay and transmission electron microscopy (TEM) were also applied.

TKE2 cells was cultured for MTT assay.

Results: BAC at concentration of 0.5% four times per day successfully induced

typical manifestations of limbal stem cell deficiency, including corneal

neovascularization, severe stromal inflammation, and diffuse epithelial staining. Conjunctiva-specdific K19 and Cx43 was positive in the corneal surface after BAC

treatment. The stem cell associated markers, P63, ABCG2, EGF-receptor, and β-

catenin were absent in the limbal basal epithelium. CD4 was present in the corneal

stroma. K10 emerged in the corneal and conjunctival surface. TUNEL assay

revealed extensive apoptosis in the entire cornea and limbal zone. TEM showed the

irregular basement membrane and the loss of stem cell-specific ultrastructure at the

limbus. MTT revealed the apparent decrease of proliferative capacity in TKE2 cells

after BAC treatment. Conclusions: Topical administration of 0.5% BAC at high frequency in mouse

induces changes resembling that of limbal stem cell deficiency in humans, and

thus, represents a novel model of limbal stem cell deficiency.

Commercial Relationships: Zhirong Lin, None; Tong Zhou, None; Huan He,

None; Xiaochen Liu, None; Hui He, None; Zuguo Liu, None

Support: the National Natural Science Foundation of China (NSFC, No.

30872810, 30910270), the Ministry of Science and Technology of China (863 Program, No. 2006AA02A131)



Injury Induced Expression Of Chloride Channels Contributes To Corneal

Wound Electric Currents Min Zhao

1A, Brian Reid

1B, Lin Cao

2, Tsung-Yu Chen

1C, Xiaodong Zhang

1A.

ADermatology and Ophthalmology,

BDermatology & Ophthalmology,

CNeurology,

1University of California Davis, Davis, CA;

2Dermatology & Ophthalmology, UC

Davis & University of Aberdeen, Davis, CA.

Purpose: The corneal epithelium has multiple functions including mechanical

protection from pathogens and refraction of light onto the lens and retina. The

integrity of the epithelial surface is necessary for normal vision. Fortunately most

corneal epithelial wounds are repaired promptly. Wounding the corneal epithelium,

and many other epithelia, results in production of large electric currents and fields

at the wound which stimulate epithelial cell division and migration into the wound

bed to initiate and promote healing. These endogenous wound currents appears to be actively-regulated and we have shown that enhancing or inhibiting the wound

currents pharmacologically can increase or decrease wound healing, respectively.

Using ion-selective microelectrodes, we have shown that chloride is the major ion

generating the wound electric currents, with lesser contributions by calcium and

sodium. The purpose of this study was to identify the ion channels and pumps

responsible. By defining the electrophysiological properties of the cornea, we will

be able to understand the mechanisms of endogenous wound electric current generation, and offer new approaches to manage corneal injury and/or disease.

Methods: We examined the expression and distribution of Cl- channels in cornea

wounds using quantitative PCR, Western blot and immunohistochemistry. Patch

clamp with flash induced photorelease of calcium and ion selective probe were

used to determine the Cl- currents in corneal epithelial cells. Using a vibrating

probe system we measured cornea wound electric currents in ex vivo eyes in the

presence of various ion channel blockers.

Results: Wounding induced significant changes in expression profiles of Cl- channels. We found Ca

2+ dependent Cl- currents in corneal epithelial cells. Broad-

spectrum chloride channel blocker DIDS (4,4′-diisothiocyanatostilbene-2,2′-

disulfonic acid disodium salt hydrate) significantly reduced (almost halved) cornea

wound current (P<0.003). Fluoxetine hydrochloride, which blocks calcium-

dependent chloride current, reduced wound current slightly. Interestingly,

mefloquine hydrochloride reversed the small outward current normally seen in

unwounded cornea to produce an inward current (P<0.03). Injury to the cornea

caused significant re-distribution and increased expression of ion channels near the

wound. Conclusions: Injury induced changes in expression profile of Cl- channels in

corneal epithelium contribute to cornea wound electric currents.

Commercial Relationships: Min Zhao, None; Brian Reid, None; Lin Cao,

None; Tsung-Yu Chen, None; Xiaodong Zhang, None

Support: NIH 1R01EY019101. Also 1R01GM065447, CIRM RB1-01417, NSF

MCB-0951199



Betacellulin as a Potential Therapeutic Agent in Corneal Epithelial Wound

Healing Joanne L. Peterson, Brian P. Ceresa. Cell Biology, University of Oklahoma HSC,

Oklahoma City, OK.

Purpose: Experimentally it has been shown that epidermal growth factor (EGF)

and its receptor (EGFR/ErbB1) are necessary and sufficient for promoting the

cellular events associated with corneal epithelial wound healing. However, the

clinical use of EGF has mixed results and there is no clear evidence that exogenous EGF can enhance wound healing of the corneal epithelium in patients. In an

attempt to resolve the disparity between experimental and clinical data, the role of

other EGFR ligands should be explored. Preliminary data from our lab, and others,

indicated that betacellulin (BTC), an EGF-like ligand, induced corneal wound

healing more efficaciously that EGF and therefore may be a potential therapeutic

agent. The purpose of this study is to determine the molecular mechanisms that

contribute to BTC‟s enhanced efficacy. Methods: Porcine corneas were superficially wounded (epithelial layer) and treated

with BTC, EGF, or media to assess qualitative and quantitative differences in

ligand-mediated epithelial wound healing. Radiolabeled BTC (125

I-BTC) was used

with immortalized and primary corneal epithelial cell cultures to determine ligand

binding properties of BTC.

Results: After 48 hours, the epithelial thickness of the wounded area in eyes

treated with BTC is approximately 1.5-fold greater than those treated with EGF and

2-fold greater than media alone. The binding affinity of BTC in both immortalized and primary cells is consistent with published data and the number of available

binding sites in immortalized cells is greater than those in primary cells. Unlabeled

ligands BTC and EGF, but not neuregulin 4, were able to compete for 125

I-BTC

binding to receptors. Additionally, BTC binding is less sensitive to acid

dissociation than EGF binding.

Conclusions: BTC mediates corneal epithelial wound healing at a greater rate than

EGF. The binding properties of BTC are a likely mechanism for why BTC is a more efficacious ligand. Our data demonstrate that BTC binds with a higher

affinity than EGF. Further, binding of BTC to receptors is less sensitive to changes

in pH, indicating the ligand: receptor complex would stay intact longer. A third

possibility is that BTC binds to an additional population of receptors that are either

more prevalent or intrinsically more efficacious in mediating corneal epithelial

wound healing.

Commercial Relationships: Joanne L. Peterson, None; Brian P. Ceresa, None

Support: NIH Grant Number P20 RR017703, NIGMS 1R01GM092874-01A1



Inflammatory Response And Extracellular Matrix Remodeling During Wound

Healing In Embryonic Corneas James W. Spurlin, III, Peter Y. Lwigale. Biochemistry and Cell Biology, Rice

University, Houston, TX.

Purpose: Despite increasing evidence of the regenerative potential of embryonic

tissue, very little is known about wound healing in embryonic cornea. In this study we examine the inflammatory response and regeneration of the corneal

extracellular matrix in wounded embryonic corneas.

Methods: We have developed a novel method of accessing and manipulating chick

embryos at late stages of development enabling us to wound corneas and observe

the healing process over time. A single wound traversing the epithelium and

anterior stroma was created in the central cornea of one eye in each embryo at

embryonic day (E7). Wounded and control corneas were collected between E7-E18 and analyzed for various aspects of the wound healing cascade observed in adult

corneas.

Results: We observed widening of the epithelial wound within 16 hours post-

wounding. There was no significant difference in cellular responses to

inflammatory signals such as cytokine-induced apoptosis and activated cell

proliferation between wounded and control corneas. Transdifferentiation of repair

fibroblasts specified by smooth muscle actin, was observed briefly between E8-

E10, and are absent in healed embryonic corneas. Surprisingly, regeneration of the cornea epithelium was not complete until E16. Extracellular matrix molecules such

as fibronectin was up-regulated whereas collagen 1 expression was down-regulated

in the stroma at the wound site, but returned to normal as the epithelium

regenerated. Expression of keratan sulfate proteoglycan remained the unchanged

during the healing process. By E18, majority of the wounded corneas were fully


regenerated and were similar in transparency as unwounded controls.

Conclusions: These data suggest that there is a reduced inflammatory response and

minimal ECM reconstruction during tissue regeneration in wounded embryonic

corneas, allowing for scar-free wound healing. Commercial Relationships: James W. Spurlin, III, None; Peter Y. Lwigale,

None

Support: NIH EY018050



Effects Of Loss Of TRPV4 On The Inflammation And Scarring Of An Alkali-

burned Cornea In Mice Yuka Okada

1A, Peter S. Reinach

2, Kumi Shirai

1A, Ai Kitano

1A, Masayasu

Miyajima1B

, Shizuya Saika1A

. AOphthalmology,

BLaboratory Animal Center,

1Wakayama Medical University, Wakayama, Japan;

2Biological Sciences, SUNY

College of Optometry, New York, NY.

Purpose: We reported that in mice transient receptor potential vanilloid 1 (TRPV1)

and transient receptor potential ankrin 1 (TRPA1) gene ablation markedly

improved the corneal wound healing response to alkali burning (ARVO, 2009,

2010). Currently, we determined if transient receptor potential vanilloid 4 (TRPV4)

gene ablation also affects inflammation and scarring severity during wound healing of alkali-burned mice corneas.

Methods: 1) Immunohistochemistry probed for TRPV4 protein expression in mice

corneas . 2) Three microliters of 1 N NaOH were applied under general anesthesia

to the right eye of 6-8 week old TRPV4(-/-)(KO) (n=44) or TRPV4(+/+) (WT)

(n=44) mice to produce an ocular surface alkali burn. The eyes were processed for

histology/immunohistochemistry or real time RT-PCR.

Results: 1) TRPV4 protein expression was detected in the corneal epithelium. 2) Stroma of the KO healing corneas were more transparent as compared with those of

WT mice at 5 to 20 days post-alkali burn. Hematoxylin-eosin histological staining

indicated more marked inflammation in the thickened stroma in the corneas of WT

mice. Immunohistochemical and real time RT-PCR examinations showed less α-

smooth muscle actin, F4/80, myeloperoxidase, TGFβ1, Collagen1a1 and MCP-1

expression in the cornea of KO mice after alkali burn.

Conclusions: TRPV4 is a calcium-permeable channel activated by extracellular

hypotonicity, polyunsaturated fatty acids, phorbol esters, and heat. To the best of our knowledge, this report shows for the first time that in TRPV4 KO mice, the

corneal wound healing response to an alkali burn has an improved outcome since

there is less inflammation and scarring than in the WT counterpart.

Commercial Relationships: Yuka Okada, None; Peter S. Reinach, None; Kumi

Shirai, None; Ai Kitano, None; Masayasu Miyajima, None; Shizuya Saika,

None

Support: Education, Science, Sports and Culture of Japan C21592241



Management of Visually Significant Corneal Lacerations in the Pediatric

Population Adrian W. Jachens

1, Gerald W. Zaidman

2.

1Ophthalmology, New York Medical

College, New York, NY; 2Ophthalmology, Westchester Medical Center, Valhalla,

NY.

Purpose: To analyze the management options in pediatric corneal lacerations in order to improve long-term visual results.

Methods: We performed a retrospective chart review of all pediatric patients with

corneal lacerations treated from 1999- 2010. We analyzed initial management, time

from referral to repair, need for secondary surgery, complications and final visual

result.

Results: Seven patients were identified ranging from 20 months to 9 years of age.

Follow up ranged from 3 to 89 months. All patients had primary closure of their

wound within 24 hours of the trauma. The time between referral and secondary surgery ranged from nine to twenty two weeks with an average of 14.4 weeks. All

patients had secondary surgeries with all having a penetrating keratoplasty (PKP),

and five having a cataract extraction. All cataract extractions had acrylic posterior

chamber intra-ocular lens (IOL) implantation. One patient was aphakic and had a

secondary sulcus-fixated lens implant performed in conjunction with the PKP. Five

patients also needed an iridoplasty; one needed an anterior vitrectomy. Significant

complications included medically controlled glaucoma and non compliance. No patients required glaucoma surgery and there have been no graft rejections. Four

patients, ages five to nine, had vision better or equal to 20/60 with the time between

injury and secondary surgery ranging from nine to twenty weeks with an average of

14 weeks. One patient, age three, had 20/200 vision with twelve weeks between

injury and secondary surgery. One patient, age two, had hand motion vision with

twenty-two weeks between injury and secondary surgery, mostly due to poor

compliance. Finally, one patient is too early in his clinical course to determine their

outcome. Conclusions: We found that time between original injury and secondary surgery

did not correlate with final visual acuity. Glaucoma occurred in a majority of the

patients but it was well controlled and did not affect final vision. We found that the

most important predictors of a good visual outcome were older age at time of injury

and compliance to treatment by parent and patient.

Commercial Relationships: Adrian W. Jachens, None; Gerald W. Zaidman,

None

Support: None


Novel Laser-Activated Biological Glue for Sealing Corneal Wounds Alan G. Fong

1, Hyung Cho

1, Tofik Ali

2, Swathi Reddy

1, Barbara Soltz

3, Robert

Soltz3, Anurag Shrivastava

1, Roy S. Chuck

1.

1Department of Ophthalmology, Albert

Einstein College Of Medicine, Montefiore Medical Center, Bronx, NY; 2Department of Ophthalmology, University of Rochester Medical Center, Flaum

Eye Institute, Rochester, NY; 3Conversion Energy Enterprises, Spring Valley, NY.

Purpose: To evaluate the effectiveness of a novel laser-activated collagen-flavin bioglue for sealing corneal incisions by measuring bursting pressures after wound

closure.

Methods: A 2.85mm keratome blade was used to create a non-self-sealing central

perpendicular corneal wound in 15 cadaver calf eyes. Each incision was performed

by the same surgeon. After drying the corneal wound of residual aqueous humor,

the bioglue solder was applied in a strip completely covering the incision. A diode-

pumped solid state portable blue light laser (457nm +/-5nm at 450mW)

(Conversion Energy Enterprises, Spring Valley, NY) was then used to activate the solder for 3 minutes, resulting in cross-linking to tissue. Wound stability was

subsequently tested using a stepwise infusion of basic salt solution at 15mL/hr, and

pressure changes were monitored via digital manometer. Bursting pressure was

recorded, defined as the first sign of leakage of fluid from the corneal wound.

Results: A total of 15 fresh calf cadaver eyes were used, but only 13 eyes with

bursting pressures below 1000mmHg (the maximum limit for digital manometer)

were included for analysis. The mean corrected bursting pressure (bursting minus baseline intraocular pressure) after wound closure with the solder was

251.15mmHg (SD 87.53).

Conclusions: The laser-activated collagen-flavin solder was effective in sealing

central full-thickness corneal wounds in cadaver calf eyes.

Commercial Relationships: Alan G. Fong, None; Hyung Cho, None; Tofik Ali,

None; Swathi Reddy, None; Barbara Soltz, Conversion Energy Enterprises (P, I,

E); Robert Soltz, Conversion Energy Enterprises (E, I, P); Anurag Shrivastava,

None; Roy S. Chuck, None Support: None



Analysis of Thrombin Activity and Cyr61 Regulation in a Rat Cornea Organ

Culture System Emily A. Andreae

1A, Debra J. Warejcka

1A, Sally S. Twining

1B.

ABiochemistry,

BBiochemistry, Ophthalmology,

1Medical College of Wisconsin, Milwaukee, WI.

Purpose: Thrombin stimulation increases Cyr61 (CCN1) gene and protein

expression in cultured corneal cells. Cyr61 is an extracellular matrix-associated

protein that promotes migration and proliferation of various cell types and

stimulates angiogenesis. Our goal is to determine whether prothrombin is activated

and whether thrombin upregulates Cyr61 synthesis and stimulates Cyr61 cleavage

in a rat cornea organ culture system.

Methods: Corneas were removed from Sprague-Dawley rats and placed in organ

culture. The rat lens was used as a base to maintain corneal shape. Epithelial scrape wounds were made in one group of corneas prior to organ culture. Serum-free

medium was added drop-wise to the corneal surface every 24 hours. In a set of

wounded and unwounded corneas, serum-free medium spiked with hirudin, a

specific inhibitor of thrombin, was added to the corneal surface. Conditioned

medium was collected for all experiments and the proteins separated by SDS-

PAGE, transblotted, and probed for Cyr61 and antithrombin III.

Results: Prothrombin activation to thrombin was observed in response to

wounding through detection of a two-fold increase in levels of antithrombin III-thrombin complexes. Cyr61 protein was also increased approximately three-fold in

the wounded corneas. The rate of extracellular Cyr61 degradation appeared to

increase in response to injury. Hirudin inhibited the increase in Cyr61 secretion and

proteolytic degradation in wounded corneas which indicates that thrombin is

involved in these effects.

Conclusions: Prothrombin is activated to thrombin upon corneal injury, and

thrombin upregulates Cyr61 protein expression in response to corneal injury. These results support the involvement of thrombin generated upon corneal injury in the

regulation of synthesis and processing of proteins such as Cyr61.

Commercial Relationships: Emily A. Andreae, None; Debra J. Warejcka,

None; Sally S. Twining, None

Support: RO1-EY12731



Ocular Safety Evaluation of Newly Developed Cyclosporine in Cationic

Emulsion in an in vitro Corneal Wound Healing Model and in an Acute in vivo

Rabbit Model Hong Liang

1,2, Christophe Baudouin

1,2, Phillipe Daull

3, Luisa Riancho

2, Jean-

Sébastien Garrigue3, Françoise Brignole-Baudouin

2,4.

1Ophthalmology-Hosp Paris,


Chino Des Quinze-Vights, Paris, France; 2INSERM, UMR_S968, UPMC

University, Vision Institute, Paris, France; 3Novagali Pharma, Evry, France;

4Paris

Descartes University, Faculty of Pharmaceutical and Biological Sciences,

Toxicology Department, Paris, France. Purpose: Topical preparation of cyclosporine (CsA) has been developed to treat

dry eye while inducing side effects, such as allergy and irritation. The present study

aims at evaluating the cytotoxicity profile of newly developed CsA in cationic

emulsion (CEm) (a) in an in vitro corneal would healing model using human

corneal epithelial (HCE) cells (b) and in an acute in vivo rabbit model.

Methods: Four CsA formulations were tested: 1) 0.05%CsA-0.005% cetalkonium

chloride (CKC)-CEm, 2) 0.05%CsA-0.02% benzalkonium chloride (BAC)-CE, 3)

0.05%CsA-oil solvent excipient (Oi), commercial 0.05%CsA-Em (Restasis®

). Phosphate buffered saline (PBS) was used as control. (a) A wound was created by

scratching through a confluent HCE cell layer. Cytotoxicity, cell migration and

proliferation were analyzed at 2, 24 and 72hours after a 30 min exposure to

solutions diluted to 1/10; (b) The eye drops were applied to rabbit eyes 15 times at

5-min intervals, and the ocular surface structures were examined using slit-lamp

and corneal in vivo confocal microscopy (IVCM).

Results: (a) The in vitro study showed that CsA-BAC delayed corneal healing

process by damaging dramatically the remaining HCE cells. The other solutions maintained a normal healing rate with a particular behavior for CsA-CKC that

improves the healing process when compared to control. (b) CsA associated to

BAC showed the highest toxicity by clinical observations and IVCM, inducing

redness, chemosis with damaged corneal epithelium and inflammatory infiltration.

CsA-Oi and CsA-Em induced moderate inflammatory cell infiltration around the

conjunctiva-associated lymphoid tissue structures, and CsA-CKC showed the

lowest toxicity with patterns similar to those of the control. Conclusions: The combination of these in vitro and in vivo models evaluated the

tolerance/cytotoxicity and the dynamic wound healing potential of CsA in different

formulations. The CsA in cationic emulsion appeared to offer the best tolerance

without delaying HCE healing. It will be of great interest to protect dry eye surface

and improve long-term tolerance of CsA topical treatments.

Commercial Relationships: Hong Liang, The study was supported by an

unrestricted grant from Novagali Pharma and Quinze-Vingts Hospital. (I);

Christophe Baudouin, Consultant for Novagali Pharma (I); Phillipe Daull, Novagali Pharma Employee (E); Luisa Riancho, None; Jean-Sébastien Garrigue,

Employee of Novagali Pharma (E); Françoise Brignole-Baudouin, None

Support: unrestricted grant from INSERM



Topical Cyclosporine Effect On Corneal Nerve Regeneration Shweta V. Chaudhary, Abed Namavari, Joy Sarkar, Lisette Yco, May Bakir, Sandeep Jain. Ophthalmology, University of Illinois Chicago, Chicago, IL.

Purpose: To analyze the effect of topical Cyclosporine on corneal nerve

regeneration after lamellar flap surgery in murine corneas.

Methods: A 2.0 mm lamellar corneal flap was created in Thy-1 YFP murine

neurofluorescent corneas (n=20) using a preset 50 um diamond blade for initial

incision and a Grieshaber UltraVit spatula for lamellar dissection. Mice were

divided into study group and control group with n=10 in each group. Study group

received Cyclosporine 0.1% eye drops twice a day and control group received Optive eye drops, twice daily for 6 weeks each. Using Stereolumar microscope,

sequential in vivo images were taken at baseline, 2 weeks, 4 weeks and 6 weeks.

Maximum intensity projection of z-stacks were used to calculate nerve fiber length

density, neurite extension and corneal T-cell infiltration. Neurite extension was

calculated as percent increase in nerve fiber density between weeks 6 and week 2,

using Neurolucida software. YFP+ T-cells were enumerated in the cornea.

Results: In the control group, mean nerve fiber length density at 2 weeks and 6

weeks was 15.151mm/mm2 and 21.123 mm/mm2, respectively, while in study group mean nerve fiber length density at 2 weeks and 6 weeks was 12.95 mm/mm2

and 16.734 mm/mm2, respectively. Neurite extension increased by 41.04% (+/-

0.14) over a period of 4 weeks in control group and by 28.27% (+/- 0.07) in the

study group. Average number of YFP+ T-cell infiltrates observed in the control

group at 2 weeks and 6 weeks were 8 and 9, respectively and1 and 1 in the study

group. Neurite extension and T-cell infiltration was significantly lower in the study

group (p<0.05). Conclusions: Cyclosporine inhibits inflammatory cell infiltration as well as retards

the nerve regeneration in uncomplicated lamellar flaps. Mild T-cell infiltration may

be facilitative towards nerve regeneration.

Commercial Relationships: Shweta V. Chaudhary, None; Abed Namavari,

None; Joy Sarkar, None; Lisette Yco, None; May Bakir, None; Sandeep Jain,

None

Support: NEI Grant K08EY018874


A Chemical Screen to Identify Pharmacological Inhibitors of Corneal Fibrosis Gabriel M. Gordon, M. Elizabeth Fini. USC Institute for Genetic Medicine, Keck

School of Medicine of USC, Los Angeles, CA.

Purpose: Corneal repair occurs on a spectrum from regenerative to fibrotic, the

former restoring corneal clarity, the latter causing scarring and obstructed vision.

We are developing a method to efficiently screen chemical libraries, using

transcriptional activity of surrogate marker genes for fibrosis as the read-out in a cell-based assay performed in high content format. This study examined use of the

α-smooth muscle actin (SMA) gene.

Methods: A surrogate reporter construct was created utilizing 1 kb of the SMA

gene promoter to drive expression of the firefly luciferase gene (a gift of Dr.

Michael Keogh). Test cells were co-transfected with this construct along with a

renilla luciferase gene driven by the constitutively active CMV promoter, which

serves as a readout for cell viability. We used primary rabbit keratocytes (PRK) or

an immortalized human keratocyte cell line (HTK; a gift of Dr. Jamie Jester). Transfected cells were treated for 24 hours with TGF-β2 (T2) to activate the

program of fibrotic gene expression. A subset of cells were co-treated with 7

different test compounds. The firefly to renilla expression ratio (F/R ratio) was

calculated to identify compounds that block SMA gene activity, while also

maintaining cell viability. SMA gene and protein expression were also assessed by

RT-PCR and immunocytochemistry (ICC).

Results: T2 treatment increased the F/R ratio by 2.7 fold in HTKs. Co-treatment

with anisomycin (ANI), lovastatin (LOV), iodotubercidin (IODO), and SB203580 (SB) significantly reduced the F/R ratio by 0.1, 1.4, 0.6, and 1.1 fold respectively.

Similarly, T2 treatment of PRKs resulted in a 4.5 fold increase in the F/R ratio and

treatment with ANI, LOV, IODO, and SB reduced the ratio to 1.7, 1, 0.9, and 1.1

fold respectively. RT-PCR analysis showed T2 treatment increased SMA mRNA

by 10 fold in HTKs and 69 fold in PRKs. ANI treatment reduced this to 1.3 fold in

HTKs and 3.2 fold in PRKs while SB treatment reduced expression to 2.6 fold in

HTKs and 17.7 fold in PRKs. ICC staining of both HTKs and PRKs showed that ANI and SB both significantly reduced SMA protein levels. We will show how

combining this assay with a second assay utilizing the T2 gene as surrogate

(reported last year), provides a more comprehensive picture of a compound‟s anti-

fibrotic effect.

Conclusions: We have developed a high throughput screening method for

identifying inhibitors of corneal fibrosis, and we have identified two lead

compounds. Applying this screening model to chemical libraries with thousands of

compounds will uncover more potential inhibitors of corneal scarring, which can then be further assessed in a clinical setting.

Commercial Relationships: Gabriel M. Gordon, None; M. Elizabeth Fini, None

Support: NIH Grant EY09828, NIH grant P30-EY003040



Effect Of Topical Administration Of Slpi And Fp-mc, Inhibitors Of Nuclear

Factor K B, In Rats With Corneal Alkali Burns, 7 Days Post Injury Juan P. Salica

1, Eduardo Chuluyan

2, Juan E. Gallo

1, Paulo Maffia

2, Diego

Guerrieri2, Andrés Rodriguez

1.

1Ophthalmology, Universidad Austral, Pilar,

Argentina; 2Pharmacology, Universidad Nacional de Buenos Aires, Capital

Federal, Argentina.

Purpose: To evaluate the efficacy of topical administration of SLPI (Secretory

Leucocyte Protease Inhibitor) and FP-MC (Fusion Protein Cementoin-SLPI ) in

rats with alkali injured corneas after 7 days. Results at day 3 had shown that FP-

MC had antiangiogenic and antiinflammatory properties. Methods: An alkali injury was performed on the right cornea of 18 rats under

general anesthesia, using a 3 mm diameter filter paper containing 1 mol/L sodium

hydroxide during 40 seconds. Animals were divided into three groups (n=6 each)

and received SLPI (20 µg), FP-MC (2 µg) or vehicle. Treatment was topically

administered four times a day for up to 7 days. Thereafter, animals were sacrificed

and eyes were extracted and processed using H & E. Histological examination was

masked and separately performed by two investigators. The variables studied in a

40x field in the center plus a 40x field in the periphery of the corneas were 1) number of PMN cells, 2) total number of cells in stroma. Extension and depth of

neovessels was also analysed in these fields. Results were compared between

groups using T-test.

Results: The mean of PMN cells was 3.3 in FP-MC treated group, 69.5 in SLPI,

340.3 in Buffer and 0.4 in normal corneas (PF-MC p<0,001 compared to Buffer

and SLPI groups). The total number of cells in stroma showed a mean of 101 in FP-

MC treated animals, 256 in SLPI, 574 in Buffer and 85 in normal corneas, respectively. The difference between PF-MC rats and other groups was statistically

significant (p<0.05).

Topographycally, from periphery to the center, FP-MC rats only disclosed

neovessels in the first 40X field in 18.7% of cases while in SLPI and buffer groups

neovessels reached out the forth and sixth fields in 4.1% and 14.6% of cases,

respectively. There was a direct relationship between severity of inflammation and

depth of neovascularization. FP-MC showed neovessels only in the superficial third

of the corneas in 18.7% of cases, SLPI in 77%, and Buffer in 100% of cases. In SLPI and buffer groups neovessels reached the deepest third of the cornea in 12.5%

and 37.5%, respectively.

Conclusions: FP-MC, a novel protein, showed antiinflammatory and

antiangiogenic properties at 7 days of treatment. Further research will be carried

out to determine the usefulness of this agent in ophthalmology.


Commercial Relationships: Juan P. Salica, None; Eduardo Chuluyan,

None; Juan E. Gallo, None; Paulo Maffia, None; Diego Guerrieri,

None; Andrés Rodriguez, None

Support: None



Does Rabbit Bone Marrow -derived Cells Implantation Contribute To Corneal

Epithelial Repair? Benedicte Tougeron Brousseau

1, Julie Gueudry

1, Marek Lamacz

2, Jean-Pierre

Vannier2, Marc Muraine

1.

1Ophthalmology, University Hospital of Rouen, Rouen,

France; 2Difema-Merci Laboratory, Medical School University, Rouen, France.

Purpose: Bone marrow-derived stem cells were reported to differentiate in epithelial cells in vitro and in vivo. Amniotic membrane is a good basement

membrane for cultivating limbal corneal epithelial cells. We explore the potential

of adherent bone marrow-derived stem cells to provide a therapy for limbal stem

cells insufficiency after transfer on human amniotic membrane and we analyze

their outcome in vivo.

Methods: Rabbit adherent bone marrow stem cells (mesenchymal stem cell, MSC)

were expanded and transferred on denuded amniotic membrane (MA). To trace

stem cells, we infected them with a retroviral vector encoding the Green Fluorescent Protein (GFP). Optimal conditions for expansion of rabbit adherent

bone marrow cells were determined. A confluent culture of MSC was established

on MA. Bone marrow cells on MA were analysed by HES coloration and epithelial

differentiation were analysed by immunohistochemistry. MA were autologous

transplanted onto rabbit eyes injured by a lamellar keratectomy extending 3 mm

outside the limbus, performed one month ago. After one month, animals were

sacrificed. Dissected corneas were analysing with HES coloration and fluorescent microscopy.

Results: After transplantion on the rabbit eyes with limbal stem cells insufficiency,

GFP fluorescence was detect in the cornea. The examination with fluorescent

microscopy has showed a presence of MSC labelled with GFP in the cornea

stroma. Nevertheless, their was no amelioration of the ocular surface disorder in

microscopic analysis. Moreover, majority of MSC were lysed after 3 weeks of the

transplantation.

Conclusions: Autologous transplantion of MSC is feasible by using amniotic membrane as a basement membrane. The transplantation MSC does not seem to

ameliorate the ocular surface disorder in our model probably due to their lysis. It

would be interesting to research the reasons of this lyse and evaluate the

possibilities of gene transfer in MSC for therapeutic strategies.

Commercial Relationships: Benedicte Tougeron Brousseau, None; Julie

Gueudry, None; Marek Lamacz, None; Jean-Pierre Vannier, None; Marc

Muraine, None Support: None

program number: 1978 poster board number: d959 1:45 pm - 3

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