Corneal lymphangiogenesis facilitates ocular surface inflammation and cell trafficking in dry eye disease.

PURPOSE: While the normal cornea has limited innervation by the lymphatic system, chronic immune-inflammatory disorders such as dry eye (DE) can induce lymphangiogenesis in the ocular surface. Using a conditional knock-down murine model, Lyve-1Cre;VEGFR2flox mice, this study investigated the role of lymphangiogenesis in the pathophysiology of DE. METHODS: DE was induced in both wild type (WT) B6 and Lyve-1Cre;VEGFR2flox mice. Tissue immunostaining and volumetric gross measurements were used to assess changes in the ocular surface, skin, and lymph nodes (LNs). The expression of lymphangiogenic factors (TNF-α, IL-6/-8/-12/-17, VEGF-C/-D, IFN-γ, VEGFR-2/-3, Lyve-1, and podoplanin) and the frequency of immune cells (CD4, CD11b, and CD207) on the ocular surface and lacrimal glands were quantified by real-time polymerase chain reaction and flow cytometry. RESULTS: Compared to WT mice, there were fewer lymphatic vessels and a reduction in lymphangiogenic markers in the ocular surface and skin of Lyve-1Cre;VEGFR2flox mice. After DE induction, mRNA levels of TNF-α, IL-8, and IFN-γ were significantly reduced in Lyve-1Cre;VEGFR2flox mice compared to WT mice (p < .01). Surprisingly, the LNs from Lyve-1Cre;VEGFR2flox mice with DE were significantly smaller and populated by fewer dendritic cells and effector T cells than those from WT mice (p < .001). Furthermore, immunostaining showed corneal nerves in the DE-induced Lyve-1Cre;VEGFR2flox mice were notably intact like in the naïve condition. CONCLUSIONS: Inhibition of lymphangiogenesis in the cornea effectively attenuates not only the inflammatory response including trafficking of immune cells but also preserves corneal nerves under desiccating stress. Corneal lymphangiogenesis might be a contributing factor in deterioration on the ocular surface homeostasis.

Efficient cultivation conditions for human limbal epithelial cells.

To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

Short-term Efficacy of Topical Immunosuppressive Agents on the Survival of Cultivated Allo-Conjunctival Equivalents.

PURPOSE: To investigate the short-term efficacy of topical immunosuppressive agents on the survival of cultivated allo-conjunctival equivalents. METHODS: Twenty-five eyes of New Zealand white rabbits were included. Temporal conjunctivae were trephined to a diameter of 7.5 mm, and then cultured allo-conjunctival epithelial cells on amniotic membrane were transplanted onto them. Various immunosuppressants including steroid, cyclosporine, and rapamycin were applied topically four times a day for a week. Epithelial defects and graft edema were graded daily. Numbers of inflammatory cells were measured in H&E. PKH26 and cytokeratin 4 and 7 were immunostained. RESULTS: Earlier epithelialization was observed in 1% steroid-treated eyes and defects persisted significantly in 0.5% CsA applied eyes. In histology, PKH26 positive cells considered as donor cells were only found in 1% steroid or 0.01% rapamycin applied eyes. 1% steroid- or 0.01% rapamycin-applied eyes both showed positive staining for keratin-4 and -7. Inflammatory cells were less found in 1% steroid or 0.01% rapamycin treated eyes. CONCLUSIONS: Topical steroid or rapamycin can help to suppress acute inflammation and enhance the acute survival of transplanted conjunctival cells.

Experience of Comamonas acidovorans keratitis with delayed onset and treatment response in immunocompromised cornea.

PURPOSE: To report 2 cases of Comamonas acidovorans keratitis in immunocompromised cornea. METHODS: A complete review of the medical records of the two cases of Comamonas acidovorans keratitis. RESULTS: We found some similarities in clinical courses of two cases. Both of them showed development of keratitis during the management with corticosteroids, delayed onset, slow response to antibiotics, and relatively less affected corneal epithelium. CONCLUSIONS: Comamonas acidovorans is known as a less virulent organism. However it can cause an indolent infection that responds slowly even to adequate antibiotics therapy in immunocompromised corneas.

Clinical efficacy of amniotic membrane transplantation in the treatment of various ocular surface diseases.

PURPOSE: To investigate the efficacy of permanent amniotic membrane transplantation (AMT) for the treatment of various ocular surface diseases. METHODS: The medical records of 62 eyes from 58 patients who had undergone permanent AMT were reviewed. The amniotic patches were grafted for the treatment of neurotrophic ulcers (n=15), inflammatory corneal ulcers (n=15), scleral ulcers (n=11), painful bullous keratopathy (n=8) and pterygium as an adjuvant to a conjunctival autograft (n=13). Cryo-preserved or freeze-dried amniotic membrane (AM) were used. The overall success rate, the interval to epithelialization, pain-subsiding time, and complications were evaluated. The pain relief and the full epithelialization interval in the bullous keratopathy patients given the cryo-preserved AM were compared with those given the freeze-dried AM. RESULTS: The success rate in the patients with neurotrophic ulcer, inflammatory corneal ulcer, scleral ulcer and bullous keratopathy were 93.3%, 66.7%, 92.9% and 100%, respectively. A conjunctival autograft with AMT showed a 100% success rate without recurrence. The time to re-epithelialization was 24.4+/-24.2, 20.4+/-5.8, and 16.9+/-7.0 days in patients with neurotrophic, inflammatory and scleral ulcers, respectively (p=0.431). The pain relief interval in the cryo-preserved and freeze-dried AM group was 17.7 and 23.3 days, and the re-epithelialization interval was 29 and 22 days, respectively, which was insignificant. CONCLUSIONS: AMT has a high success in the treatment of neurotrophic, scleral ulcer, bullous keratopathy and pterygium with a low rate of complications but presented only partial success in the treatment of inflammatory corneal ulcers. The clinical efficacy of AMT was not related to the methods used to preserve the AM.

Efficient cryopreservative conditions for cultivated limbal and conjunctival epithelial cells.

PURPOSE: To examine the effects of cryopreservation on the viability of cultivated corneal limbal and conjunctival epithelial cells and to evaluate the optimal conditions for cryopreservation. METHODS: The cultivated human limbal epithelial cells (HLECs) were stored in media including 20%, 50%, and 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) at -196 degrees C for 1 week. The cultivated rabbit conjunctival epithelial cells were stored in 10%, 20%, and 50% FBS with 10% glycerol or DMSO as a cryoprotectant at -196 degrees C for 1 week. After thawing, cell viability was assessed using the trypan blue vital staining and 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay. Immunofluorescent staining was performed with cytokeratin 3/12 antibody. Colony-forming efficiency (CFE) was evaluated 2 weeks after culture. RESULTS: HLECs cryopreserved with 50% FBS showed the highest cell viability, whereas those with 20% FBS revealed the lowest survival rate (87.1% +/- 0.8% and 79.8% +/- 4.01%, respectively; P = 0.030). CFE of HLECs was 2.13 +/- 1.35%, 2.31 +/- 2.23%, and 1.94 +/- 0.72% in cells with 20%, 50%, and 90% FBS, respectively (P > 0.05). For conjunctival epithelial cells, the cell viability was the highest with 50% FBS and 10% glycerol (95.0% +/- 4.27%), and the lowest survival rate was observed in the condition of 10% FBS and 10% DMSO (80.0% +/- 5.49%). CFE of cryopreserved conjunctival epithelial cells was 14.1% +/- 1.9% in cells with 20% FBS and glycerol and 13.5% +/- 2.0% in those with 20% FBS and DMSO (P > 0.05). HLECs expressed CK3/12 after cryopreservation in all conditions examined. CONCLUSIONS: The best results were yielded by 50% FBS for cell viability in HLECs. Glycerol seems to be superior to DMSO in cell viability of the rabbit conjunctival epithelium after cryopreservation.

Long-term outcome in ocular intractable surface disease with Seoul-type keratoprosthesis.

PURPOSE: To evaluate the long-term clinical efficacy of the Seoul-type keratoprosthesis (S-KPro). METHODS: S-KPros were implanted into 4 unsighted and 5 sighted eyes in 9 patients: 6 patients were diagnosed with Stevens-Johnson syndrome, 2 had chemical burns, and 1 suffered from ocular pemphigoid. The preoperative visual acuity ranged from light perception to hand motion. The average follow-up period was 62.8 months. We evaluated several clinical factors, including visual acuity, visual field, number of additional grafting procedures, number of capsulotomy procedures, and the interval between retinal detachment and skirt exposure. RESULTS: The S-KPro showed anatomic success for an average of 62.8 months in 66.7% of the eyes. The average visual acuity preservation time was 31.6 months. Localized glaucomatous visual field defect was not found in any of the sighted patients; however, diffuse visual field constriction was observed after long-term follow-up. Average time of skirt exposure and mean number of additional grafting procedures were 12.9 months and 2.44, respectively. Retinal detachments were developed in all the patients at a mean time interval of 2 months after S-KPro exchange. CONCLUSIONS: S-KPro achieved visual rehabilitation for an average of 31.6 months with long-term anatomic stability in patients with severe intractable ocular surface disease.

Biocompatibility of nanocomposites used for artificial conjunctiva: in vivo experiments.

PURPOSE: To evaluate the biocompatibility of nanocomposites used for artificial conjunctiva. METHODS: Fifty New Zealand white rabbits were used for the experiments. Nanocomposites of poly -caprolactone (PCL) and of PCL coated with polyvinyl alcohol (PCL+PVA), polyvinyl pyrrolidone (PCL+PVP), or chitosan (PCL+C), and amniotic membrane (AM) as a control, were cut into small disks with a diameter of 3.5 mm. The disks were inserted underneath the conjunctiva to measure their inflammation-inducing properties. To investigate epithelial adhesion and goblet cell differentiation, the disks were transplanted after round conjunctival excision. Cultivated conjunctival epithelial cells on nanocomposite were then transplanted onto the abdomen of Balb/c athymic mice. The number of inflammatory cells and the density of goblet cells were measured using hematoxylin and eosin, periodic-acid-Schiff, and immunohistochemistry after 2 weeks. RESULTS: The number of inflammatory cells found inside of the inserts was as follows: 21 +/- 4.9 for controls, 21 +/- 15.1 for PCL, 49.6 +/- 26.0 for PCL+PVP, 40.2 +/- 17.1 for PCL+C, and 13.8 +/- 3.9 for PCL+PVA. In PCL+PVA, the accumulation of inflammatory cells was significantly lower than in the controls (p < 0.01, Mann-Whitney U). The reepithelialization of conjunctival cells was accomplished in more than 75% of all disks except for the PCL+C. In addition, we found the differentiation of goblet cells in the following order from greatest to least: amniotic membrane, PCL, and PCL+PVP. CONCLUSIONS: Nanocomposites of PCL were biocompatible in rabbit conjunctiva, suggesting that PCL may be considered as a candidate for use in the development of artificial conjunctiva.

Isolation of putative corneal epithelial stem cells from cultured limbal tissue.

PURPOSE: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue. METHODS: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2 U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation. RESULTS: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3 x 10(4) cell/ml and 8.06 x 10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21 x 10(6) cell/ml with a trypsin/EDTA treatment (p < 0.05). CFE was 9.67 +/- 2.13% and 6.63 +/- 2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61 +/- 0.42% and 5.21 +/- 4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67 +/- 2.24% and 1.17 +/- 6.13%, respectively (p < 0.05). CONCLUSIONS: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.

The side population cells in the rabbit limbus sensitively increased in response to the central cornea wounding.

PURPOSE: Side population (SP) cells are known to reside in the limbus as putative corneal epithelial stem cells. This study was performed to demonstrate the presence and the characteristics of SP cells in the rabbit limbal epithelium and explore their sensitivity in response to the central cornea wounding. METHODS: To sort out the SP cells, freshly isolated rabbit limbal and central corneal epithelial cells were subjected to Hoechst 33342 dye efflux assay. For characterization of the sorted SP cells, RT-PCR analysis, semi-dry three-dimensional (3-D) cell culture, and transplantation in nude mice were performed. To explore wound sensitivity of the limbal SP cells, the rabbit central cornea was wounded by direct contact of a 6-mm paper disk soaked with 1 N NaOH, and changes in the population size of the SP cells and the colony-forming efficiency (CFE) was monitored on days 1, 2, and 5 after wounding. RESULTS: The SP cells were present in the rabbit limbal epithelium with an incidence of 0.73% +/- 0.14% (n = 8) and were smaller in cell size than the major population (MP) cells, quiescent in the cell cycle, and in the undifferentiated state. The SP cells were able to regenerate the cornea-like structure with basal enrichment of p63-positive cells by in vitro 3-D culture and in vivo transplantation, all of which were best achieved by the whole population (WP) of cells comprising SP and MP cells. After central cornea wounding, this rare population of the limbal SP cells increased in size fivefold on day 1 and then decreased on day 2. The transient increase in the SP cells was subsequently followed by the propagation of an increase in CFE in the limbal MP cells on day 2 and then in the corneal MP cells on day 5. In the hematopoietic colony-forming assay, the limbal SP cells gave approximately eightfold higher CFU than the limbal MP cells. CONCLUSIONS: The SP cells identified in the rabbit limbus are an undifferentiated and noncycling rare epithelial cell population, which sensitively respond to the central cornea wounding by their transient increase in the population size.

Adhesion complex in cultivated limbal epithelium on amniotic membrane after in vivo transplantation.

PURPOSE: To investigate adhesion complex formation in cultivated human limbal epithelium after transplantation into the limbal deficient model. METHODS: Cultivated epithelium on amniotic membrane was transplanted into limbal deficient rabbits. The transplanted rabbits and the controls were sacrificed at 1, 2, 3, and 4 weeks. The adhesion complex was examined by electron microscopy and immunohistochemistry. RESULTS: Morphologically identifiable hemidesmosomes appeared at 1 week, and matured adhesion complex was found at 3 weeks. Collagen VII was partly stained after transplantation. The mean numbers of hemidesmosomes/2.25 microm were 2.3 +/- 0.9, 2.5 +/- 0.5, 5.2 +/- 1.0, and 4.0 +/- 0.9 at 1, 2, 3, and 4 weeks, and all they were smaller than those in the control, respectively (p < 0.05). It reached 137.4% of the density of hemidesmosomes in human cornea at 3 weeks. The average depths of anchoring fibril were 0.10 +/- 0.03, 0.27 +/- 0.06, 0.45 +/- 0.06, and 0.46 +/- 0.12 microm at 1, 2, 3, and 4 weeks, reaching 75.0% of that in the human cornea after 3 weeks, although they were shallower than that of the control, respectively (p < 0.05). CONCLUSIONS: Assembly of adhesion complex in cultivated epithelium transplanted in limbal deficient rabbit might recover to the level of that in the human after 3 weeks, although it was delayed compared with that in normal wound healing of the rabbit.

Change of proliferation rate of corneal epithelium in the rabbit with orthokeratology lens.

OBJECTIVE: To investigate the cell proliferation rate of normal corneal epithelium with extended orthokeratology lens (OKL) wear in comparison with extended rigid gas-permeable (RGP) lens wear. METHODS: Twenty-four rabbits were fitted unilaterally with either an OKL or an RGP lens, and the other eye was used as a control. They were injected with 5-bromo-2-deoxyuridine (BrdU) 24 h prior to being sacrificed. The rabbits were sacrificed at 1, 3, 7 and 14 days after lens fitting. The cornea from the superior limbus to the center was taken at 1.0-mm intervals. The BrdU-labeled cells were counted in medium power fields (x200) in each sample using light microscopy. RESULTS: The number of BrdU-labeled cells in the RGP lens group initially increased, but the number decreased in the corneal center and superior limbus by 32 and 8%, respectively, after 14 days. There was no statistically significant change. However, the number of BrdU-labeled cells in the OKL group decreased after 3 days, and the number of BrdU-labeled cells was reduced in the center and superior limbus by 63 and 8%, respectively, after 14 days. The change in proliferation in the corneal center in the OKL-wearing rabbits was statistically significant compared to the control (p < 0.05). CONCLUSIONS: Wearing an OKL had a greater effect on the change of the proliferation pattern in the epithelium than wearing an RGP lens, which suggests that the OKL might be less physiologic than the RGP lens is.

Optimization of contact lens fitting in keratectasia patients after laser in situ keratomileusis.

PURPOSE: To evaluate the clinical outcomes of contact lens fitting in eyes with keratectasia after laser in situ keratomileusis (LASIK) to optimize the performance of the contact lenses. SETTING: Contact Lens Clinic, Seoul National University Hospital, Seoul, Korea. METHODS: Six eyes of 4 patients with keratectasia were examined. Keratectasia was diagnosed based on abnormal topographic findings, progressive myopic changes, and uncorrected visual acuity with glasses. A multicurve lens or reverse-geometry lens (RGL) was selected based on the anterior elevation map. The base curve radius and peripheral parameters were determined based on the axial map and the evaluation of the fluorescein pattern of the lens. The contact lens fitting characteristics and visual acuity were evaluated. Patients were interviewed about their level of comfort, daily contact lens wear time, and glare. RESULTS: Multicurve lenses and RGLs were fitted in 4 eyes and 2 eyes, respectively. The base curve radius of the multicurve lenses ranged from 7.2 to 7.5 mm; the alignment curve radius of RGLs was 7.6 mm in both cases. The radii of peripheral curves were customized to enhance tear interchange. Visual acuity improved to 20/30 or better in all eyes. All patients were completely satisfied with the comfort of the fitting. The mean daily wearing time was 12.7 hours. One patient reported tolerable glare in the eye after being fitted with an RGL. CONCLUSION: Proper contact lens fitting based on topographic data and slitlamp evaluation allowed good visual rehabilitation and comfortable extended daily wear in patients with keratectasia after LASIK.

Clinical performance and fitting characteristics with a multicurve lens for keratoconus.

PURPOSE: To evaluate the clinical outcome and fitting characteristics with a multicurve lens for keratoconus in a Korean population. METHODS: Sixty eyes with keratoconus were fit with a multicurve lens based on topographic index and fluorescein pattern on slitlamp biomicroscopy. Three-point touch was applied in 59 eyes, and apical clearance was adopted in an eye with a persistent epithelial defect. The mean follow-up was 11.4 months. Visual acuity, comfort, glare, daily contact lens-wearing time, and the changes in topographic indices after fitting were evaluated. The relationship between base curve radius (BCR) and topographic indices were analyzed along with the relationship between BCR and peripheral curve radius. RESULTS: A total of 71.6% of the eyes achieved a visual acuity of 20/30 or better. Flatter BCR was related to better visual acuity. Ninety-four percent of the patients tolerated the fitting, and 85% of them reported complete comfort. The mean daily wearing time was 11.9 hours. Four eyes reported glare. The change of Sim K during follow-up was not statistically significant. Prefitting Sim Kmax and average Sim K were significantly related to the BCR. Mean differences between the BCR and first peripheral curve radius were 1.06 +/- 0.21 mm with a BCR less than 6.00 mm and 0.81 +/- 0.2 mm with a BCR greater than 6.00 mm. These differences were statistically significant. CONCLUSIONS: Customization with the multicurve lens enabled patients with keratoconus to wear the contact lens comfortably and seemed not to aggravate the progression of keratoconus. Sim Kmax and the relationship between BCR and first peripheral curve radius seem to be useful information to optimize performance of the multicurve lens.

Seoul-type keratoprosthesis: preliminary results of the first 7 human cases.

OBJECTIVE: To evaluate the clinical efficacy of a newly designed Seoul-type keratoprosthesis (S-KPro). METHODS: The S-KPro, which consists of a polymethyl methacrylate optic, a skirt (polyurethane or polypropylene), and polypropylene haptics, was developed and implanted into 2 unsighted and 5 sighted eyes of 7 patients. One patient had a chemical burn, another had an ocular pemphigoid, and the remainder were diagnosed as having Stevens-Johnson syndrome. The preoperative visual acuities ranged from light perception to hand motions. The average follow-up time was 25.6 months. MAIN OUTCOME MEASURES: We evaluated anatomical stability, visual acuity, retinal status, and the visual field. RESULTS: At the last follow-up visit, the S-KPro was well placed in 6 patients. The best-corrected visual acuities of the sighted patients ranged from 20/100 to 20/60 in the affected eye. One patient each experienced retinal detachment or endophthalmitis. Partial extrusion was found in the patient with glaucoma. A retroprosthetic membrane was detected in 1 patient and was treated with an Nd:YAG laser. No glaucomatous visual field defects were found in any of the sighted patients. CONCLUSIONS: Anatomical success was achieved in 6 of 7 eyes. In 3 of the 5 sighted eyes, the S-KPro could rehabilitate corneal blindness not correctable with keratoplasty.