Rebamipide suppresses TNF-α production and macrophage infiltration in the conjunctiva.

PURPOSE: To evaluate the anti-inflammatory effect of rebamipide during corneal epithelial wound healing using a mouse wound repair model. METHODS: A 2-mm circular disc of the central cornea was demarcated in the right eye of C57BL/6 mice and the epithelium removed. Rebamipide 2% eyedrop was instilled onto the wounded eye 5 times a day (n = 26). Phosphate-buffered saline (PBS) was used in the control group (n = 26). Corneal and conjunctival IL-1ß and TNF-α levels were measured at 6 h and 24 h postinjury by ELISA. The wounded area was evaluated by fluorescein staining at 24 h postinjury. Macrophage infiltration was assessed immunohistochemically, and TNF-α secretion from macrophages was examined in vitro. RESULTS: Conjunctival IL-1ß and corneal IL-1ß levels were not significantly different between PBS-treated and rebamipide-treated groups. However, conjunctival TNF-α level was significantly lower in the rebamipide-treated group compared with the PBS-treated group. Macrophage migration into the conjunctiva, but not into the cornea, was suppressed by rebamipide treatment. In addition, TNF-α secretion from cultured macrophages was suppressed by rebamipide in a concentration-dependent manner. Rebamipide treatment significantly accelerated corneal epithelial wound healing at 24 h postinjury. CONCLUSIONS: In a mouse corneal epithelial wound model, rebamipide suppressed TNF-α secretion and macrophage infiltration in the conjunctiva, which might have contributed to accelerated corneal epithelial wound healing in the first 24 h following injury.

In vivo challenging of polymyxins and levofloxacin eye drop against multidrug-resistant Pseudomonas aeruginosa keratitis.

The purposes of this study were to establish a rabbit multidrug-resistant Pseudomonas aeruginosa (MDRP) keratitis model, and test the efficacy of levofloxacin, colistin methanesulfate (CL-M), colistin sulfate (CL-S) and polymyxin B (PL-B) against MDRP infection. In a rabbit eye, making a 2-mm circular corneal excision, and MDRP strain #601 or representative P. aeruginosa strain IID1210 were instilled into the corneal concavity. IID1210 was used to confirm this model developed P. aeruginosa keratitis. After MDRP keratitis developed, we treated the eyes with levofloxacin, CL-M, CL-S or PL-B eye drops. The infected eyes were evaluated by clinical score, histopathological examination and viable bacterial count (CFU). Rabbits developed MDRP keratitis reproducibly after instilled the bacteria into the corneal lesion. MDRP produced severe keratitis similarly with IID1210, as shown by slit lamp examination and clinical score. In MDRP keratitis models, clinical scores and viable bacterial counts were significantly lower in levofloxacin- and CL-M-treated groups compared with PBS-treated group, but the magnitudes of reduction were not remarkable. However, clinical scores were dramatically lowered in CL-S- and PL-B-treated groups compared with PBS-treated group. CL-S- and PL-B-treated group were kept corneal translucency and little influx of polymorphonuclear neutrophils in histopathological examination. In addition, both CL-S- and PL-B-treated groups were not detected viable bacteria in infected cornea. Using our MDRP keratitis model, we showed that topical levofloxacin and CL-M are not adequately effective, while CL-S and PL-B are efficacious in controlling MDRP keratitis. Especially, PL-B, which is commercially available eye drop, might be most effective against MDRP.

Corneal Perforation Associated with Lacrimal Canaliculitis: A Case Series.

Purpose: To report seven eyes of six patients diagnosed with corneal perforation and lacrimal canaliculitis in a single facility. Methods: Clinical records of patients with corneal perforation accompanied by lacrimal canaliculitis seen by the authors were reviewed. Results: Six patients (7 eyes) with corneal perforation accompanied by lacrimal canaliculitis were identified. All patients were female, and all were treated with topical antibiotics while five were receiving topical corticosteroids. Two patients had a history of dacryocystitis and three had systemic immune diseases. The corneal lesions did not respond to topical antibiotics but were effectively treated by removal of concretions in the lacrimal canaliculi and lacrimal duct drainage together with conjunctival autograft or corneal transplantation. Conclusion: Lacrimal canaliculitis is a risk factor for corneal perforation. When corneal perforation does not respond to antibiotics, lacrimal canaliculitis should be considered.

Corticosteroid eye drop instillation aggravates the development of Acanthamoeba keratitis in rabbit corneas inoculated with Acanthamoeba and bacteria.

The role of topical corticosteroids in management of Acanthamoeba keratitis (AK) remains controversial. Using a rabbit AK model, we investigated whether corticosteroid use is a risk factor of AK. Acanthamoeba (1 × 105/ml) was incubated with two densities of P. aeruginosa (PA; high-PA: 1 × 108/ml, low-PA: 3 × 105/ml) before corneal inoculation. Rabbit corneas were inoculated with Acanthamoeba alone or Acanthamoeba plus PA and administered levofloxacin and betamethasone sodium phosphate (BSP) eye drops for 5 or 7 days. Infected rabbit eyes were evaluated for clinical score and Acanthamoeba by histological examination. Acanthamoeba alone and BSP treatment did not produce keratitis. Corneas inoculated with Acanthamoeba plus low-PA treated immediately with levofloxacin and BSP remained clear with few infiltrates. Corneas inoculated with Acanthamoeba plus low-PA treated with levofloxacin immediately and BSP 12 h later developed severe keratitis. Corneas inoculated with Acanthamoeba plus high-PA treated immediately with levofloxacin and BSP also developed severe keratitis. Acanthamoebae were detected by PAS staining in corneas inoculated with Acanthamoeba plus high-PA treated with levofloxacin and BSP. Topical corticosteroids have the potential to aggravate AK when cornea is infected by Acanthamoeba with a critical number of bacteria or when corticosteroids are given after infection has established by Acanthamoeba with small number of bacteria.

Corneal lymphangiogenesis ameliorates corneal inflammation and edema in late stage of bacterial keratitis.

Lymphatic vessels play a crucial role in systemic immune response and regulation of tissue fluid homeostasis. Corneal lymphangiogenesis in bacterial keratitis has not been studied. In this study, we investigated the mechanism and the role of corneal lymphangiogenesis in a murine bacterial keratitis model using Pseudomonas aeruginosa. We first demonstrated that corneal lymphangiogenesis was enhanced mainly in the late stage of bacterial keratitis, contrary to corneal angiogenesis that started earlier. Corresponding to the delayed lymphangiogenesis, expression of the pro-lymphangiogenic factors VEGF-C and VEGFR-3 increased in the late stage of bacterial keratitis. We further found that F4/80 and CD11b positive macrophages played an essential role in corneal lymphangiogenesis. Notably, macrophages were specifically involved in corneal lymphangiogenesis in the late stage of bacterial keratitis. Finally, we demonstrated the beneficial role of corneal lymphangiogenesis in ameliorating the clinical course of bacterial keratitis. Our study showed that bacterial activity was not directly involved in the late stage of keratitis, while corneal lymphangiogenesis reduced corneal edema and clinical manifestation in the late stage of bacterial keratitis. These findings suggest that the process of lymphangiogenesis in bacterial keratitis ameliorates corneal inflammation and edema in the late stage of bacterial keratitis.

Corneal deposits associated with topical tosufloxacin following penetrating keratoplasty: a case report.

PURPOSE: To report the adverse event of topical tosufloxacin administered after penetrating keratoplasty in one patient. CASE REPORT: A 60-year-old female was referred to our hospital for treatment of vision loss due to corneal opacification, etiology was unknown. Slit lamp examination showed dense opacification in corneal stroma. Penetrating keratoplasty was performed on her left eye. She was treated with topical applications of 1.5% levofloxacin, 0.5% cefmenoxime, 0.1% betamethasone, 0.1% hyaluronate sodium, and 3% aciclovir after penetrating keratoplasty. Delayed epithelialization of the donor graft was observed at day 4 post-transplantation. Because of the concern that levofloxacin induced corneal epithelialization delay, 1.5% levofloxacin was changed to 0.3% tosufloxacin. At day 6 post-transplantation, deposits on the epithelial defect of the donor graft were observed. Tosufloxacin was suspected to be the cause of deposits, and tosufloxacin eye drop was discontinued. The deposits decreased gradually and completely disappeared by 5 months post-transplantation. CONCLUSION: Topical tosufloxacin treatment has the risk of precipitation on compromised corneas such as corneal grafts with epithelial defect after penetrating keratoplasty. After discontinuation of therapy, the deposit may resolve spontaneously without surgical removal.

Number of Bacteria and Time of Coincubation With Bacteria Required for the Development of Acanthamoeba Keratitis.

PURPOSE: We hypothesized that bacteria may be a factor contributing to the development of Acanthamoeba keratitis (AK). We investigated interactions between Acanthamoeba and Pseudomonas aeruginosa for the development of keratitis in rabbit corneas. METHODS: Acanthamoeba castellanii (ATCC50492) and P. aeruginosa (PAO-1) were used. Two densities of P. aeruginosa (high, 1 × 10/mL; low, 3 × 10/mL) and 2 durations of coincubation (long, 6 h; short, 2 h) of Acanthamoeba with 1 × 10/mL of P. aeruginosa were tested. Acanthamoeba alone or Acanthamoeba coincubated with P. aeruginosa was inoculated into rabbit corneas. After inoculation, levofloxacin (LVFX) eye drops were administered. The clinical score of the cornea was evaluated after inoculation. RESULTS: Acanthamoeba alone did not produce keratitis during a 5-day observation period. Rabbit corneas inoculated with Acanthamoeba coincubated with low-density P. aeruginosa followed by topical LVFX were clear with few infiltrates. Corneas inoculated with Acanthamoeba coincubated with high-density P. aeruginosa followed by LVFX treatment developed severe keratitis, and clinical scores were significantly higher compared with high-density P. aeruginosa alone followed by LVFX treatment (scores 7, 9.6, 8.5 vs. 3, 3.5, 3.25 on days 1-3, all P < 0.01). The long (6 h) coincubation time of Acanthamoeba with high-density P. aeruginosa resulted in more severe keratitis compared with short (2 h) coincubation (scores, 9.7, 12.7, 12.1, 9.8, 8.7 vs. 7, 9.6, 8.5, 6.9, 5.6 on days 1-5, all P < 0.01). CONCLUSIONS: These results suggest that the presence of bacteria is essential and a critical number of bacteria is required for the development of AK. The time of coexistence with bacteria may be an important determinant of the severity of AK.

Conjunctival flora in patients with human immunodeficiency virus infection.

PURPOSE: To compare the conjunctival flora of human immunodeficiency virus (HIV)-positive and HIV-negative patients. Also, to assess the prophylactic effect of oral clarithromycin against Mycobacterium avium complex on the conjunctival flora of HIV-positive patients. METHODS: Ninety-four eyes of 47 HIV-positive patients and 122 eyes of 61 control patients were examined. All participants had a detailed anterior segment examination, including conjunctival cultures and laboratory blood tests. Culture results for different organisms were evaluated by chi-square analysis between the groups. The effect of systemic antibiotic treatment on the conjunctival flora of patients with HIV infection was evaluated by chi-square analysis. RESULTS: Bacterial organisms in the conjunctival sac were detected in four out of 28 (14.3%) eyes of HIV-positive patients treated with systemic clarithromycin and in 32 out of 66 (48.5%) eyes of HIV-positive patients without systemic clarithromycin treatment (p < 0.01). The CD4-positive T-cell counts in these groups were 158/microl and 416/microl, respectively (p < 0.01). Bacterial organisms were also detected in 46 of 122 (37.7%) control eyes. No difference was observed in the types and proportions of organisms isolated from the conjunctiva between HIV-positive patients without systemic clarithromycin treatment and controls. CONCLUSION: There was no difference between the conjunctival flora of HIV-negative and HIV-positive patients. Systemic clarithromycin treatment decreased the conjunctival flora of HIV patients, including those who had a CD4 count that was less than 50/microl.

Investigation of the Role of Bacteria in the Development of Acanthamoeba Keratitis.

PURPOSE: Recently, much interest has been shown in bacteria extracted from Acanthamoeba strains isolated from patients with Acanthamoeba keratitis (AK). We hypothesized that the bacteria in Acanthamoeba strains may be a contributing factor in the development of AK. To prove this hypothesis, we investigated the involvement of bacteria harbored by Acanthamoeba in causing progressive ocular infection in rabbit corneas. METHODS: One Acanthamoeba strain (T4 genotype) that harbored bacteria was isolated from a patient with AK. The Acanthamoeba strain pretreated or not pretreated with levofloxacin (LVFX) was inoculated into rabbit corneas. We also tested the effect of LVFX eye drops on keratitis induced by the Acanthamoeba strain. The infected rabbit eyes were evaluated for clinical scores, Acanthamoeba 18S rDNA and bacterial 16S rDNA numbers were analyzed by the real-time polymerase chain reaction, and the presence of Acanthamoeba was analyzed by histological examination. RESULTS: Inoculation of nonpretreated Acanthamoeba resulted in severe keratitis. In contrast, inoculation of LVFX-pretreated Acanthamoeba did not induce keratitis (mean clinical score, 17.3 vs. 2.3; P < 0.05). Rabbit corneas inoculated with nonpretreated Acanthamoeba followed by topical LVFX therapy developed severe keratitis. In corneas inoculated with nonpretreated Acanthamoeba followed by LVFX therapy, the number of Acanthamoeba 18S rDNA copies was significantly higher than in other groups (P < 0.05), whereas the bacterial 16S rDNA gene was undetectable. Acanthamoeba cysts were detected by Fungiflora Y staining only in corneas inoculated with nonpretreated Acanthamoeba followed by LVFX therapy. CONCLUSIONS: These results suggest that the presence of bacteria in Acanthamoeba may be required for the development of AK.

[Corneal endothelial cell changes in triple procedure].

PURPOSE: A retrospective study was performed to examine time-related changes in the corneal endothelium in eyes that underwent a triple procedure consisting of penetrating keratoplasty, extracapsular excision, and intraocular lens (IOL) insertion. MATERIALS AND METHODS: We studied 39 eyes from 36 patients who underwent a triple procedure associated with corneal transplantation at Tokyo Medical University between February 1989 and May 1996. The ages of the patients at the time of operation ranged from 61 to 91 years (mean, 70.9 +/- 10.8 years). The postoperative follow-up period ranged from 13.6 to 107.5 months (mean, 53.4 +/- 27.1 months). The following were evaluated: 1) cure rate of transparency, 2) complications, 3) time-related changes of endothelial cell density during 7 years after operation, and 4) comparison of changes in endothelial cell density associated with various factors such as primary disease, graft, IOL, and rejection. RESULT: The following results were obtained. 1. The cure rate of transparency of the graft at the last examination(a mean of 53.4 +/- 27.1 months after surgery) was 82.1%. 2. The incidence of rejection at the last examination was 35.9%. 3. The mean endothelial cell density in the graft was 2945.8 cells/mm2 before operation, and decreased to 1806.7 cells/mm2 one year after operation, and further to 1161.1 cells/mm2 five years after operation, showing a reduction of 60.6% compared to the mean before operation. 4. The annual rate of endothelial cell reduction in the same patient was approximately 25% the first year after operation and gradually lessened from the second year, reaching a stable level of 6% after the third year. 5. The punching method(done from the epithelial side), diameter of optical zone of IOL (6 mm or less), and rejection contributed to the reduction of endothelial cell density by one year after the operation. CONCLUSION: The triple procedure is considered to be a technique that may be chosen actively.

Depiction of cavity formation in Terrien marginal degeneration by anterior segment optical coherence tomography.

PURPOSE: To evaluate detailed morphologic changes of Terrien marginal degeneration (TMD) using anterior segment optical coherence tomography (AS-OCT). METHODS: Two patients with TMD were examined by 1310-nm wavelength swept source optical coherence tomography, corneal topography, and slit-lamp microscopy. RESULTS: AS-OCT depicted corneal thinning with intact epithelial layer in the peripheral cornea of both patients. Moreover, AS-OCT images of both patients clearly showed cavity formation surrounded and enclosed by intact epithelial and endothelial layers in the thinned area. CONCLUSION: We demonstrate here the existence of cavity formation in the peripheral cornea with TMD. This observation suggests a possibility that corneal thinning in TMD is associated with cavity formation in the corneal stroma. AS-OCT is useful for the observation of detailed morphologic changes of diseased cornea.

Non-Descemet's stripping automated endothelial keratoplasty for bullous keratopathy secondary to iridoschisis.

PURPOSE: To report a case of bullous keratopathy secondary to iridoschisis treated by non-Descemet's stripping automated endothelial keratoplasty (nDSAEK). CASE REPORT: A 79-year-old woman was referred to our hospital with loss of vision in the left eye. Slit lamp examination of her left eye showed a shallow anterior chamber with cataract and schisis in the inferior quadrant of iris stroma. Bullous keratopathy secondary to iridoschisis was diagnosed. Cataract surgery with iridectomy succeeded to deepen the anterior chamber and remove the floating iris leaf, although corneal edema remained. Four days later, nDSAEK was performed, which resolved corneal edema and restored visual acuity. CONCLUSION: The two-step surgery of cataract surgery plus iridectomy followed by nDSAEK may be an effective strategy for treating bullous keratopathy secondary to iridoschisis.