The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane

PURPOSE: To determine the feasibility of using human amniotic membrane (AM) as a substrate for culturing oral epithelial cells and to investigate the possibility of using autologous cultivated oral epithelial cells in ocular surface reconstruction. METHODS: An ocular surface injury was created in one eye of each of eight adult albino rabbits by a lamellar keratectomy, and a conjunctival excision was performed, including and extending 5 mm outside the limbus. Oral mucosal biopsy specimens were obtained from these eight adult albino rabbits and cultivated for 3 weeks on a denuded AM carrier. The cultivated epithelium was examined by electron microscopy (EM) and immunohistochemically labeled for several keratins. At 3 to 4 weeks after the ocular surface injury, the conjunctivalized corneal surfaces of the eight rabbits were surgically reconstructed by transplanting the autologous cultivated oral epithelial cells on the AM carrier. RESULTS: The cultivated oral epithelial sheet had four to five layers of stratified, well-differentiated cells. EM revealed that the epithelial cells were very similar in appearance to those of normal corneal epithelium, had numerous desmosomal junctions, and were attached to a basement membrane with hemidesmosomes. Immunohistochemistry confirmed the presence of the keratin pair 4 and 13 and keratin-3 in the cultivated oral epithelial cells. Corneas that were grafted with the cultivated oral epithelial cells on an AM carrier were clear and were all epithelialized 10 days after surgery. CONCLUSIONS: Cultures of oral epithelial cells can be generated to confluence on AM expanded ex vivo from biopsy-derived oral mucosal tissue. Autologous transplantation was performed with these cultivated oral epithelial cells onto the ocular surfaces of keratectomized rabbit eyes. Autologous transplantation of cultivated oral epithelium is a feasible method for ocular surface reconstruction. The long-term outcome of such transplantation is not yet clear, and its feasibility in clinical use should be evaluated further.     

UV absorption in human oral mucosal epithelial sheets for ocular surface reconstruction

BACKGROUND: Ocular surface reconstruction with autologous oral mucosal epithelium has attracted attention as a novel treatment strategy that avoids allograft rejection. OBJECTIVES: To evaluate the absorption of ultraviolet (UV) A or B irradiation by human oral mucosal epithelium cultured on human amniotic membrane. METHODS: Human oral mucosal and limbal epithelial cells were co-cultured on amniotic membrane with inactivated 3T3 fibroblasts. The cell sheets were also subjected to UV-A (365 nm) or UV-B (302 nm) irradiation at energy levels ranging from 50 to 800 microW/cm2, and the UV absorption rate was measured with a UV irradiation meter. RESULTS: Cultured oral mucosal epithelium had a structure with 3-5 layers of cells, consistent with the histological features of cultured corneal limbal epithelium after 4 weeks. The decrease in UV-A absorption of cultivated oral mucosal epithelium ranged from 25 to 36% of that for cultured corneal epithelium. The increase in UV-B absorption by cultured oral mucosal epithelium between 200 and 800 microW/cm2 was approximately 145% of that for cultured corneal limbal epithelium. CONCLUSION: Our data demonstrated that cultured oral mucosal epithelium has low UV-A and high UV-B absorption capacity as compared with those of cultured corneal epithelium, suggesting that oral mucosal epithelium can compensate for UV absorption of corneal epithelium.     

Attempted oral immunization with chlamydial lipopolysaccharide subunit vaccine

The effects of oral immunization with a recombinant vaccine expressing chlamydial lipopolysaccharide (LPS) on subsequent ocular challenge with Chlamydia trachomatis were studied in cynomolgus monkeys. Groups of four or five monkeys were given an oral vaccine containing 5 X 10(8) parent or recombinant Escherichia coli on days 0, 14, and 35 and were challenged with either 2 X 10(3) or 5 X 10(3) inclusion forming units of viable purified elementary bodies on day 42. On clinical and microbiologic grounds, oral immunization failed to protect monkeys against subsequent ocular challenge. Antichlamydial IgG or IgA antibodies were not induced by oral vaccination, and the antibody response following ocular challenge was similar in vaccinated and nonvaccinated animals. Paradoxically, however, while nonvaccinated control animals developed antibodies against chlamydial LPS detectable by immunoblotting after chlamydial challenge, the LPS vaccinated animals did not. This study demonstrates that the oral recombinant vaccine expressing chlamydial LPS was ineffective in protecting against chlamydial eye infection and strongly suggests that chlamydial LPS may not be an important antigen for protective immunity against chlamydia.     

Primary vitrectomy for combined rhegmatogenous retinal detachment and choroidal detachment with or without oral corticosteroids: a pilot study

PURPOSE: The occurrence of choroidal detachment (CD) in eyes with primary rhegmatogenous retinal detachment (RRD) is relatively uncommon (2%-4.5%). Recent reports suggest that primary vitrectomy yields better anatomic success than scleral buckling. However, for these inflamed eyes with low intraocular pressure, the influence of preoperative oral steroids on reattachment rates has not been elucidated yet. METHODS: Twenty eyes with combined RRD and CD that underwent primary vitrectomy were randomized to receive oral steroids (for 1 week) or no oral steroids before surgery. RESULTS: Preoperative clinical data such as mean age, lens status, Snellen visual acuity, duration of macular detachment, CD (size and extent), and retinal detachment characteristics (e.g., extent, number of retinal breaks, atrophic or tractional retinal break, size of retinal break, and location of retinal break) were similarly distributed in both groups. Single-operation anatomic success was 81.8% (9/11) among those patients who received preoperative oral steroids and was 66.7% (6/9) among those who did not receive preoperative oral steroids. After reoperation, anatomic success was 100% in both groups. The mean follow-up was 20.1 months. CONCLUSION: The results suggest that administration of oral steroids before primary vitrectomy in eyes with combined RRD and CD improves reattachment rates.     

Treatment of adult gonococcal keratoconjunctivitis with oral norfloxacin

We evaluated the efficacy of oral norfloxacin in 15 patients with culture-proven gonococcal eye disease caused by Neisseria gonorrhoeae. The first seven patients received 1,200 mg of oral norfloxacin for three consecutive days. The other eight patients were each treated with a single oral dose of 1,200 mg of norfloxacin. All control cultures were negative, and there was no progression of the corneal lesions after treatment was initiated. No adverse effects were observed. The results of this study suggested that a single dose of oral norfloxacin may be a valuable alternative to the currently recommended treatment regimens for gonococcal eye disease because it combines high efficacy and low toxicity with low cost and excellent patient compliance.     

The use of autologous serum in the development of corneal and oral epithelial equivalents in patients with Stevens-Johnson syndrome

PURPOSE: To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS). METHODS: AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry. RESULTS: BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy. CONCLUSIONS: AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.     

Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface

PURPOSE: Autologous stem cell transplantation for total limbal stem cell deficiency is immunologically preferable, to avoid allograft rejection. This study was undertaken to investigate the possibility of a novel tissue engineering approach for ocular surface reconstruction, using autologous oral mucosal epithelial stem cells expanded ex vivo on temperature-responsive cell culture surfaces. METHODS: Rabbit oral mucosal epithelial cells cultured on temperature-responsive culture surfaces with mitomycin-C-treated 3T3 feeder cells for 2 weeks produced confluent epithelial cell sheets. Putative progenitor cell populations were estimated by colony-forming assays. Autologous transplantation of these cell sheets to surgically manipulated eyes was performed, and ocular surface reconstruction and cell phenotypic modulation were examined. RESULTS: All cultured oral epithelial cells were nonenzymatically harvested as transplantable intact cell sheets by reducing culture temperature to 20 degrees C. Oral epithelial cells were stratified in three to five cell layers more similar to corneal epithelium than to oral mucosal epithelium. Colony-forming assays and immunofluorescence for p63, beta1-integrin, and connexin 43 indicated retention of viable stem and/or progenitor cell populations in cell sheets. Autologous transplantation to rabbit corneal surfaces successfully reconstructed the corneal surface, with restoration of transparency. Four weeks after transplantation, epithelial stratification was similar to that in the corneal epithelium, although the keratin expression profile retained characteristics of the oral mucosal epithelium. CONCLUSIONS: Cell sheet harvest technology enables fabrication of viable, transplantable, tissue-engineered epithelial cell sheets that retain putative progenitor cells from autologous oral mucosal epithelial cells. Promising clinical capabilities for autologous tissue-engineered epithelial cell sheets for ocular surface reconstruction are indicated.     

Oral acyclovir (Zovirax) in herpes simplex dendritic corneal ulceration.

Sixty patients with simple dendritic corneal ulceration were randomly assigned to double blind treatment with either acyclovir tablets (400 mg) or acyclovir ophthalmic ointment administered five times daily. There was no significant difference in the proportions of patients healed in either treatment group (88.9% on oral acyclovir and 96.6% on acyclovir ointment). The median healing time was five days in both groups. No systemic or significant local side effects were noted in either treatment group. Trough levels of acyclovir in the tear fluid of those who received the oral preparation were within or above the range of mean in-vitro ID50 levels for herpes simplex virus type 1. We conclude that oral administration of acyclovir (400 mg, five times daily) may be an effective alternative to topical therapy in selected patients.     

A comparison of local and systemic acyclovir in the management of herpetic disciform keratitis.

Forty-three patients with active herpetic disciform keratitis were entered into an open study to compare the efficacy of oral acyclovir (400 mg) with acyclovir ophthalmic ointment (3%) to inhibit viral replication during treatment with 0.05% prednisolone eye drops. All patients, regardless of the mode of therapy, were treated five times a day until they were healed. The mean time to heal in the oral group was 25.9 days and in the topical group was 25.3 days. Resolution of lacrimation was significantly faster in the oral group (12.1 days versus 27.6 days). The patients on tablets also showed a greater improvement in visual acuity. No statistically significant differences were found between the two groups in the incidence of recurrences over a three-year post-treatment period. It is concluded that oral acyclovir treatment is an effective alternative to ophthalmic ointment in the management of herpetic disciform keratitis.     

Oral acyclovir for herpes zoster ophthalmicus.

BACKGROUND: Reports on the natural history of herpes zoster ophthalmicus stress its high morbidity related to vicious scars on eyelids, ocular complications, and post-herpetic neuralgia. Early treatment with oral acyclovir is effective, but the optimal duration of treatment has not been defined. METHODS: The authors performed a bicentric, prospective, randomized, double-masked study of 86 patients with acute herpes zoster ophthalmicus, within 72 hours of skin eruption, who received oral acyclovir (800 mg 5 times daily), either for 7 days (plus 7 days oral placebo) or for 14 days. All patients concomitantly received ophthalmic 3% acyclovir ointment; follow-up was at least 6 months. RESULTS: Statistical analyses of subjective symptoms, skin lesions, and ocular complications showed no significant differences between the groups, suggesting that a 7-day course of treatment was sufficient. Drug tolerance was good. Pooled data from both groups corroborated earlier reports that prompt treatment with oral acyclovir reduces the severity of the skin eruption, the incidence and severity of late ocular manifestations, and the intensity of postherpetic neuralgia. At 6 months, late ocular inflammatory complications were seen in 29.1% of our 86 patients, versus 50% to 71% of untreated patients described by others. Only 13% of our patients experienced post-herpetic neuralgia, which in no case required the use of analgesics. CONCLUSION: The authors believe it is not useful to prolong treatment with 800 mg of oral acyclovir 5 times daily for more than 7 days in herpes zoster ophthalmicus. This study confirms the efficacy of oral acyclovir not only against skin lesions and ocular complications, but also against postherpetic neuralgia in herpes zoster ophthalmicus.     

Cataract surgery under topical anesthesia with oral anticoagulants

BACKGROUND: Approximately 14 % of cataract surgery patients receive blood-thinning agents. In a prospective study, the influence of oral anticoagulants on intraoperative and postoperative hemorrhages in patients undergoing cataract surgery in topical anesthesia was investigated. PATIENTS AND METHODS: 128 patients presenting for cataract surgery under oral anticoagulation were included. The mean preoperative prothrombin time was 39 +/- 18 %. Most patients (81 %) continued their oral anticoagulation (prothrombin time 34 +/- 13 %). All surgeries were performed in topical anesthesia. RESULTS: In 9 patients (7 %) an ocular hemorrhagic event was observed. These were not sight-threatening and resorbed spontaneously within a few days. Only one patient (0.8 %) had a slight hemorrhage in the anterior chamber. There were no differences (P > 0.05) between patients with or without hemorrhagic complications in the postoperative visual acuity, the intraocular pressure, the prothrombin time or the discontinuation of oral anticoagulants. CONCLUSIONS: Cataract surgery in topical anesthesia under oral anticoagulation did not increase the risk of sight-threatening hemorrhages. The continuation of oral anticoagulation seems particularly indicated for ambulatory cataract surgery.     

Visual outcomes of treated periocular capillary haemangiomas in childhood: a 10‐year review

Purpose: To report our experiences of treating periocular capillary haemangioma with particular emphasis on visual outcomes. Methods: Patients who underwent treatment for periocular capillary haemangioma were identified, and a chart review was performed. Results: There were seventeen patients in the study. The main indications for treatment were increased size of lesion with threat of amblyopia and partial or full pupil occlusion. Seven patients required more than one type of treatment. Fourteen patients had intralesional steroid treatment and six patients had oral steroids. Intralesional steroids gave variable results with minimal side‐effects, oral steroids were very effective but 4 out of 6 patients developed side‐effects. The average pretreatment astigmatism was 2.4D and post treatment this had reduced to 1.3D. Final visual outcomes were available for 15 patients, 13 had visual acuity of 6/7.5 or better, two were amblyopic with vision of 6/9‐2 and 6/18. Conclusion: In this study, intralesional steroids were used for localized lesions and oral steroids for diffuse haemangiomas with orbital involvement. Treatment especially with oral steroids was found to halt the proliferative phase and assist early haemangioma regression reducing ocular sequelae.     

Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders

BACKGROUND/AIMS: To determine outcomes of transplants of cultivated autologous oral epithelial cells in patients with severe ocular surface disorders. METHODS: The eyes (n = 6) of four patients with Stevens-Johnson syndrome (three eyes) or chemical burns (three eyes) were studied. Autologous oral epithelial cells, grown for 2-3 weeks on a denuded amniotic membrane carrier in the presence of 3T3 fibroblasts, were air lifted. The resultant sheet was transplanted onto the damaged eye, and acceptance of the sheet by the corneal surface was confirmed 48 hours after surgery. The success of ocular surface reconstruction, graft survival, changes in visual acuity, and postoperative complications were assessed and the quality of the cultivated oral epithelial sheet was evaluated histologically. RESULTS: At 48 hours after transplant, the entire corneal surface of all six eyes was free of epithelial defects indicating complete survival of the transplanted oral epithelium. Visual acuity was improved in all eyes. During follow up (mean 13.8 (SD 2.9) months), the corneal surface remained stable, although all eyes manifested mild peripheral neovascularisation. CONCLUSIONS: Autologous oral epithelial cells grown on denuded amniotic membrane can be transplanted to treat severe ocular surface disorders.     

Resolution of macular edema after systemic treatment with furosemide

We report two cases of macular edema treated with the oral administration of furosemide. The first case presented here was a 78-year-old male patient with visual disturbance of the left eye. He had been taking an oral agent for diabetes and had chronic renal failure for 7 years. From 10 days prior to the visit, he had visual disturbance of the left eye accompanied by systemic edema. There were no specific findings in the anterior segment, but sub-retinal fluid was observed in the left fundus. Macular edema was observed on fluorescein angiography and optical coherence tomography; therefore, the oral administration of furosemide was initiated. After seven days, the sub-retinal fluid disappeared. The second case was a 43-year-old female patient with visual disturbance of the left eye who had been taking hypoglycemic agents for diabetes for 13 years. There were no specific findings in the anterior segment, but flame-shaped retinal hemorrhages were scattered over both posterior poles, neovascularization was observed in the left eye, and, of particular note, sub-retinal fluid was detected in the macula of the left eye. Macular edema was also observed on fluorescein angiography and optical coherence tomography, and oral administration of furosemide was initiated. After 3 weeks, the macular edema had significantly decreased.     

The effect of oral contraceptives on colour vision in diabetic women.

The results of previous studies of the effect of oral contraceptives on visual function have produced discrepancies and have lacked specific restraints on the control and experimental groups. We compared 14 diabetics with normal acuity who were taking oral contraceptives with equivalent diabetic and control groups and used the Pickford-Nicholson anomaloscope and the Farnsworth-Munsell 100-Hue test. Diabetics using oral contraceptives showed a definite trend to increases in tritanomaly and extended red-green matching ranges. When these losses were classified by degree of defect a significantly higher incidence of major defects was found among diabetics on "the pill". Partial correlations for colour variables, duration of diabetes and duration of contraceptive therapy revealed a compounding effect. While subjects using oral contraceptives demonstrated trends toward the extreme scores, these effects were found only in one out of every four users.     

Transplantable cultivated mucosal epithelial sheet for ocular surface reconstruction

Ocular surface reconstruction by tissue engineering using somatic stem cells is a second-generation therapeutic modality. In view of future treatment of bilaterally affected, severe ocular surface disorders, two types of transplantable cultivated mucosal epithelial sheets can be used for reconstruction. One is an allogeneic corneal epithelial stem cell sheet, and the other is an autologous oral mucosal epithelial cell sheet. We first investigated the feasibility of amniotic membrane as an epithelial carrier, and found that denuded amniotic membrane was the most appropriate substrate for this purpose. Thus, cultivated corneal epithelial stem cell sheets were created by co-culturing with 3T3 fibroblast and air-lifting on amniotic membrane. These epithelial sheets demonstrated positive keratin 3 and 12 specific to in vivo corneal epithelium, light junction related proteins and proliferative activity. The transplanted allogeneic human corneal epithelial sheets existed successfully on the corneal surface, and were quite effective in achieving ocular surface stability in severe ocular surface disorders. A few cases, however, developed immunological reactions or opportunistic infections, etc. Secondly, we established transplantable autologous cultivated oral mucosal epithelial sheets in rabbits. The in vitro oral mucosal epithelial sheets showed histological characteristics similar to those of in vivo corneal epithelial sheets; for example, positive keratin 3 expression. Based on the fact that, the transplanted autologous oral mucosal epithelial sheets resembled corneal epithelium and that we achieved the recovery of corneal transparency in rabbits, we propose that cultivated oral mucosal epithelium may become the substitute for corneal epithelium in ocular surface reconstruction.     

自体口腔黏膜上皮细胞体外培养异体角膜移植治疗眼表重度烧伤

目的观察自体口腔粘膜上皮细胞体外培养联合异体角膜基质治疗重度眼表烧伤临床效果。方法随机将重度眼表烧伤患者分为两组,移植组行自体口腔粘膜上皮细胞联合异体角膜移植术,对照组为异体角膜移植术,分别在术后1 d、7 d、30 d、90 d、120 d观察角膜荧光染色、植片水肿及角膜新生血管情况。结果在各观察点角膜荧光染色得分对照组均高于移植组;在第7天、30天时其水肿程度好于对照组;术后30 d、90 d、120 d新生血管数量和范围对照组均高于移植组。结论对于严重的双眼化学烧伤者,自体口腔粘膜上皮细胞联合异体角膜板层移植具有较好疗效,为挽救眼球,争取二次复明手术提供了一个新的可行性方法。     

和血明目片联合激光光凝治疗Eales病的疗效观察

探讨和血明目片联合激光光凝治疗视网膜静脉周围炎（Eales病）的临床效果。 方法：将30例42眼早期视网膜静脉周围炎患者随机分为2组,对照组行视网膜病变区光凝治疗,治疗组行视网膜病变区光凝治疗外,给予和血明目片口服3mo。对照组和治疗组中查出病因者,给予相关对症治疗,随访1mo~3a。 结果：对照组有效率为80%,治疗组有效率为95.5%,经统计学分析,两组有统计学差异（P＜0.05）。 结论：视网膜静脉周围炎患者,行病变区光凝并给予和血明目片口服,效果显著     

口服阿昔洛韦联合玻璃酸钠及氟米龙眼液治疗带状疱疹性角膜炎

目的：探讨口服阿昔洛韦联合玻璃酸钠及氟米龙眼液滴眼治疗带状疱疹性角膜炎疗效。

方法：将本院收治的40例眼部带状疱疹患者分为两组,观察组20例,对照组20例。观察组应用阿昔洛韦200mg口服,5次/d,玻璃酸钠眼液点眼,4次/d,当发生角膜基质炎、盘状角膜炎或者角膜内皮炎时使用1g/L氟米龙眼液点眼,4次/d; 对照组给予1.5g/L更昔洛韦眼用凝胶滴眼4次/d。随访3～4wk,观察两组患者的疗效。

结果：观察组在患者局部疼痛及畏光缓解时间、角膜损伤愈合时间与对照组比较,观察组均优于对照组,差异有统计学意义(P<0.05); 两组有效率比较,差异无统计学意义(P=0.633)。

结论：口服阿昔洛韦联合玻璃酸钠及氟米龙眼液治疗带状疱疹性角膜炎疗效满意。