Demographic profile and clinical characteristics of patients presenting with acute ocular burns

Purpose: To study and compare the demographic and clinical profile of acute ocular burns (AOB) in children and adults. Methods: This retrospective case series included 271 children (338 eyes) and 1300 adults (1809 eyes) who presented to two tertiary eye care centers within one month of sustaining AOB. Data regarding demographics, causative agents, severity of injury, visual acuity (VA), and treatment were collected and analyzed. Results: Males were more commonly affected particularly among adults (81% versus 64%, P < 0.00001). Among children, 79% sustained domestic injuries, whereas 59% of adults had work?place injuries (P < 0.0001). Most cases were due to alkali (38%) and acids (22%). Edible lime (chuna, 32%), superglue (14%), and firecrackers (12%) in children, and chuna (7%), insecticides, lye, superglue (6% each), toilet cleaner (4%) and battery acid (3%) in adults, were the main causative agents. The percentage of cases with Dua grade IV?VI was greater in children (16% versus 9%; P = 0.0001). Amniotic membrane grafting and/or tarsorrhaphy were needed in 36% and 14% of affected eyes in children and adults, respectively (P < 0.00001). The median presenting VA was logMAR 0.5 in children and logMAR 0.3 in adults (P = 0.0001), which improved significantly with treatment in both groups (P < 0.0001), but the final VA in eyes with Dua grade IV?VI burns was poorer in children (logMAR 1.3 versus logMAR 0.8, P = 0.04). Conclusion: The findings clearly delineate the at?risk groups, causative agents, clinical severity, and treatment outcomes of AOB. Increased awareness and data?driven targeted preventive strategies are needed to reduce the avoidable ocular morbidity in AOB

Dry eye disease in head and neck cancer patients undergoing radiotherapy

Purpose: To study the incidence of dry eye disease (DED) in head and neck cancer (HNC) patients undergoing external beam radiotherapy (EBRT), to find a correlation between tumor location and total radiation dose with DED, and to report various radiotherapy (RT) induced acute toxic effects on ocular and adnexal structures. Methods: A prospective cohort study was conducted at a tertiary eye?care center on 90 patients of HNC undergoing EBRT from March 2021 to May 2022. All underwent a thorough clinical history and complete ophthalmological examination including an ocular surface disease index (OSDI) questionnaire, visual acuity, anterior segment, angle and posterior segment examination, dry eye workup including the Schirmer test, tear meniscus height, tear break?up time, corneal fluorescein staining and grading, and meibography by auto?refractometer and its scoring at each visit. Patients were evaluated before the start of RT and then at 1 week, 4 weeks, and 12 weeks post?RT. Radiation records of all patients were noted. Data were analyzed using percentage and Microsoft Excel. Results: Of the 90 patients, 66 were male and 24 female (M: F ratio of 2.75) with a median age of 52.5 years (range 24 to 80 years). The most common HNC was the carcinoma oral cavity and lip. Most patients received a total radiation dose between 46 to 55 Gy. DED developed in 48 (53.3%) patients. The incidence of DED increased with the increase in total radiation dose (r = 0.987). DED was also found to be correlated with tumor location (r = 0.983). Conclusion: The incidence of DED positively correlated with the total radiation dose and tumor location.

E-PIX: An enhanced grading for acute ocular burns

An enhanced online and manual grading system, based on the I’s and E’s, for acute ocular chemical injuries is being proposed. E-PIX is designed to be an online/manual grading system that includes all the parameters that adversely affect the outcome of acute chemical injuries. The importance of addressing the I’s and E’s in chemical burns cannot be underestimated. These include the need for the documentation and management of epithelial defect (E), intraocular pressure (P) (IOP), ischemia (scleral) (I), and exposure (X) (acronym - E-PIX). Epithelial defect includes that involving the limbus (L), along with conjunctival (C), corneal (K), and tarsal (T). These additional parameters are graded and represented as an annotation along with the limbal grade providing a comprehensive grading for the injury. A manual entry sheet and a freely accessible online grade generator are a part of the system. The proposed enhanced grading offers a final annotation that provides a clear understanding of all factors that can lead to vision-threatening complications ensuring their assessment and hence subsequently their addressal to improve outcomes, if abnormal. The prognostication continues to be based on the grade of limbal involvement. The additional annotations impact prognosis and outcome if not addressed. Including the laterality of injury provides, in addition, a futuristic understanding of available options. The grade generator retains the flexibility to be dynamic with changes reflecting upon the healing process in the acute stage. The proposed system aims to provide primary and tertiary caregivers alike with a uniform grading system.

Modified Supportive Simple Limbal Epithelial Transplantation (M-SLET): A surgical technique modified for limbal stem cell deficiency

This study aimed to develop and modify the surgical technique of simple limbal epithelial transplantation in patients with limbal stem cell deficiency to provide support to epithelial explants during the post-operative period. This is a case series of five eyes of five patients who underwent modified supportive simple limbal epithelial transplantation (M-SLET) surgery. The health and stability of the ocular surface were assessed based on clinical slit lamp examination; they were the main outcome measures. All patients had a stable ocular surface and healed epithelium during all the follow-up visits. The M-SLET technique provides additional support to limbal epithelial explants, adhering to the cornea, thus creating a stable epithelial surface. This is particularly important when there is a risk of explants being dislodged by eye rubbing.

Ocular manifestations in Kindler syndrome

We aimed describe the chronic ocular sequelae of Kindler syndrome. All cases of Kindler syndrome with ocular involvement that presented to a tertiary eye care center were included. Three cases of Kindler syndrome with ocular changes were reviewed. Case 1 (10 years, female) had recurrent epithelial breakdown with severe dry eye and corneal opacity secondary to keratitis. Case 2 (28 years, male) had symblepharon , ocular surface keratinization , and severe dry eye. Case 3 (16 years , female ) had partial limbal stem cell deficiency with dry eye. All cases were treated with topical lubricants, short course of low?potency steroids and immuno?modulators. Attention must be paid to the eye in addition to the oro?an?genital mucosa to avoid longterm ocular sequelae

Feasibility of corneal epithelial transplantation with polyethylene glycol hydrogel membrane as a carrier for limbal stem cell deficiency / 中华实验眼科杂志

Objective:To investigate whether polyethylene glycol hydrogel films (PHFs) can be used as a carrier for the expansion of corneal epithelial cells (CECs) in vitro and whether PHFs can be used in the treatment of limbal stem cell deficiency (LSCD). Methods:Sebacoyl chloride, dihydroxyl PCL and glycerol ethoxylate were used to synthesize PHFs.The thickness, transmittance and mechanical tensile properties of PHFs were measured.Four clean-grade New Zealand white rabbits were selected to culture primary limbal epithelial cells.The expression of keratin marker AE1/AE3 and stem cell marker p63 in the cultured cells were observed under a fluorescence microscope.The cells were divided into negative control group cultured with common cell culture solution, positive control group cultured with cell culture solution containing 100 μmol/L H 2O 2, and PHFs+ CECs group lined with PHFs cultured with common cell culture solution for 24 hours.The proliferation and apoptosis of cells in the three groups were observed by MTT and TUNEL staining, respectively.Fifteen clean-grade New Zealand white rabbits were divided into control group, PHFs group and PHFs+ CECs group by random number table method, with 5 rabbits in each group.LSCD model was constructed in the three groups.The control group was not given any treatment after modeling.In PHFs group, empty PHFs were placed on the corneal surface of rabbits.In PHFs+ CECs group, tissue-engineered grafts constructed with CECs after passage implanted on PHFs were placed on the corneal surface of rabbits.The corneal defect area of rabbits was detected and scored by fluorescein sodium staining.The histological characteristics of rabbits corneal epithelium was observed by hematoxylin-eosin staining.The use and care of animals complied with Guide for the Care and Use of Laboratory Animals by the U. S.National Research Council.The experimental protocol was approved by the Research and Clinical Trial Ethics Committee of The First Affiliated Hospital of Harbin Medical University (No.2021006). Results:The synthetic PHFs were with a thickness ≤150 μm, a tensile strength about 6 MPa, and a transmittance over than 99% in the range of 400-700 nm.Most of the cells from primary culture of limbal tissue were positive for AE1/AE3 and p63.MTT test results showed that the A490 value of PHFs+ CECs group, negative control group and positive control group was 0.59±0.01, 0.65±0.07 and 0.06±0.04, respectively, showing a statistically significant overall difference ( F=12.25, P<0.05). The A490 values of PHFs+ CECs group and negative control group were significantly higher than that of positive control group, and the differences were statistically significant (both at P<0.05). TUNEL test results showed that there was a significant difference in the TUNEL-positive cell rate among the three groups ( F=13.45, P<0.05), and the rates of TUNEL-positive cells in PHFs+ CECs group and negative control group were significantly lower than that in positive control group (both at P<0.05). Fluorescein sodium staining results showed that with the extension of postoperative period, the corneal fluorescein sodium staining score of the three groups decreased, which decreased successively in control group, PHFs group and PHFs+ CECs group.Hematoxylin-eosin staining showed fewer irregularly shaped corneal epithelial cells in the control group, and sparse single layer of corneal epithelial cells in some areas of the PHFs group.In PHFs+ CECs group, the corneal epithelium coverage was the largest, and the cell layers increased to 3-5, and the cells were with regular morphology and in close arrangement. Conclusions:PHFs have enough toughness, high transmittance and can expand corneal epithelium in vitro.PHFs are suitable for corneal epithelial transplantation and can promote the repair of corneal epithelium in rabbit model of LSCD.

Regulation of limbal stem cells viawnt signaling in the treatment of limbal stem cell deficiency / 中国组织工程研究

BACKGROUND: Wnt signaling pathway plays an important role in the regulation of stem cells, but its regulatory effect on limbal stem cells is not well defined. OBJECTIVE: To investigate the regulation of limbal stem cells via Wnt signaling pathway and its function in the treatment of limbal stem cell deficiency. METHODS: Rat limbal segments were digested with Dispase and trypsin/EDTA and then single limbal stem cells were seeded in 3D-Matrigel. In experimental group LiCl (500 μmol/L) was added to the culture system. Control group received no LiCl. The expression levels of p63α, CK12, CEBPδ and Ki67 in limbal stem cells were detected by qRT-PCR on the 7th day of culture. The expression level of β-catenin in limbal stem cells was detected by immunofluorescence staining. The rat model of limbal stem cell deficiency was made by alkali burn method. Rats in treatment group were treated with Wnt-activated limbal stem cells by subconjunctival injection. Rats in control group were treated with PBS. Rats were checked by slit lamp every day. On the 4th day after treatment, the immunofluorescence staining and hematoxylin-eosin staining were applied to evaluate the repair of limbus. RESULTS AND CONCLUSION: (1) The limbal stem cells aggregated in 3D-Matrigel. β-Catenin was negative in the control group. β-Catenin was found in the cytoplasm and nucleus of the limbal stem cells in the experimental group. (2) qRT-PCR results showed there was no significant difference in the levels of p63α, CK12 or CEBPδ between control and experimental groups (P > 0.05). The Ki67 level in the experimental group was significantly higher than that in the control group (P < 0.05). (3) The rat models of limbal stem cell deficiency were established. The degree of corneal opacity in the treatment group was significantly lower than that in the control group (P < 0.05). Hematoxylin-eosin staining results observed that the corneal epithelial cells in the treatment group were neatly arranged, the cell size was uniform, and repair was good. Immunofluorescence staining showed that β-catenin was found in the cytoplasm and nucleus of most corneal epithelial cells in the treatment group. While β-catenin was only weakly positive in the control group, and was invisible in the cytoplasm or nucleus. (4) These results indicate that activation of Wnt signaling pathway in limbal stem cells enhances their proliferation and keeps them in an undifferentiated self-renewal state. Wnt-activated limbal stem cells can promote the regeneration of corneal epithelium and reduce the degree of corneal opacity in the treatment of limbal stem cell deficiency. The regulation of limbal stem cells via Wnt signaling pathway is expected to provide new ideas for the treatment of limbal stem cell deficiency.

Contact Lens Induced Limbal Stem Cell Deficiency: Clinical Features in Korean Patients

PURPOSE: To describe the clinical features of Korean patients with contact lens-induced limbal stem cell deficiency (CL-LSCD).METHODS: Medical records of 22 patients who were diagnosed with CL-LSCD between 2014 and 2019 were reviewed retrospectively. Outcome measures included demographics, clinical presentation, treatment, clinical course, and pattern of contact lens (CL) wear.RESULTS: Forty-two eyes of 22 patients were found to have typical changes associated with CL-LSCD. Twenty (91%) patients were women and mean age was 36 ± 12 years. All patients had myopia with mean spherical equivalent of −7.52 ± 3.2 diopter. Twenty (91%) patients had bilateral disease and the location of limbal involvement was diffuse in 20 eyes (47.6%) and partial in 22 eyes (52.4%, superior in 20 eyes and inferior in 2 eyes). Fourteen (63.6%) patients complained of decreased visual acuity. Average period of CL wear was 14 ± 9 years. Four patients used cosmetic colored CLs and four patients had a history of overnight CL wear. All 12 patients who completed follow-up (28 ± 42 weeks) showed improvement in visual acuity and ocular surface condition after cessation of CL wear and medical treatment. Of them, five (42%) patients showed full recovery while seven (58%) showed partial recovery.CONCLUSIONS: If a patient with a history of CL wear for an extended period of time presents with decreased visual acuity, practitioners should perform detailed examinations with suspicion of CL-LSCD, including fluorescein staining. CL-LSCD is usually reversible and close follow-up with conservative treatment is recommended as the initial treatment option.

Experimental study on decompensation model of rabbit corneal limbal stem cells induced by alkali burn / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To explore a new method to induce the animal model of rabbit partial limbal stem cell deficiency(LSCD).<p>METHODS: LSCD was induced through corneal alkali burn, C57 mice and New Zealand rabbits were used to establish the animal models. Corneal alkali burn manipulation was accomplished in experimental animals under general anesthesia combined with surface anesthesia in the operated eye. Specifically, mice(<i>n</i>=30)were used to induce complete LSCD model. In brief, the filter paper(diameter of 3mm)that immersed in 1mol/L potassium hydroxide solution was placed on the central corneal surface of the left eye for 30s, followed by washing with saline. In addition, rabbits(<i>n</i>=19)were utilized to establish the partial LSCD model. Briefly, the nictitating membrane(third eyelid)was resected, and the filter paper(diameter of 5mm)that immersed in 1mol/L potassium hydroxide solution was placed on the superior temporal peripheral corneal surface of the left for 30s, followed by washing with saline. After surgery, the model eyes were treated with 0.5% Levofloxacin Hydrochloride Eye Drops four times a day. In addition, the slit-lamp microscope was adopted for observation and photo-taking before burn, as well as at 1, 2, 4wk and 2mo after burn; meanwhile, complications such as corneal ulcer and perforation were recorded. 2mo after surgery, the corneal goblet cell distribution was detected with impression cytology, and the severity of LSCD was classified according to slit-lamp microscopic findings and corneal impression cytology. The animals were sacrificed 2mo after surgery, cornea and conjunctiva sections were made to observe angiogenesis and goblet cell distribution in cornea. Animals died accidentally were not counted into the total number, and the successful induction rates of complete LSCD and partial LSCD models were compared.<p>RESULTS: Six out of the 30 mice died accidentally, while 2 developed corneal perforation after burn, and the remaining 22 had developed complete LSCD only, yielding the successful induction rate of 92%. 2mo after burn, extensive angiogenesis distribution in the superficial and deep corneal stromal layers could be observed, and pathological sections revealed corneal angiogenesis. Seven out of the 19 rabbits died accidentally, while the remaining 12 had various degrees of LSCD only(partial LSCD, average involving 1.17±0.39 quadrants). Additionally, no corneal perforation was observed, and the successful induction rate was 100%. The result of Fisher's exact test <i>P</i> value is 0.543, without statistical difference. No goblet cells were observed in the normal corneal region, while goblet cells were observed in the LSCD region, with the average density of 58.60±12.58 cell/HP.<p>CONCLUSION: Central corneal alkali burn can induce complete LSCD; however, some animals will experience failure in model induction due to corneal ulcer and perforation, LSCD is generally serious and may be combined with angiogenesis in deep cornea. Alkali burn in superior temporal cornea can induce partial LSCD, which may be combined with relatively minor corneal lesion, and the corneal angiogenesis is located in the superficial layer.

Primary limbal stem cell transplantation in the surgical management of extensive ocular surface squamous neoplasia involving the limbus

Purpose: To determine the role of primary limbal stem cell transplantation (LSCT) in managing extensive ocular surface squamous neoplasia (OSSN) with more than 3 quadrants of limbal involvement to prevent manifestations of limbal stem cell deficiency. Methods: A multi-center, comparative, and interventional clinical study of cases and historical controls was done. Patients with a limbal mass suggestive of OSSN involving more than 3 quadrants of limbus (> 9 clock hours) and no local, regional or systemic metastasis, who underwent primary surgical excision with or without adjuvant chemotherapy and had a minimum follow-up of 12 months were included in this study. Two historical controls underwent tumor excision with only amniotic membrane grafting. Three cases underwent tumor excision along with LSCT in the form of conjunctival limbal autograft (CLAG) or simple limbal epithelial transplant (SLET). Results: None of the eyes had any recurrences of OSSN during the follow-up period. Cases with primary LSCT with tumor excision showed better ocular surface stability in the long term. Conclusion: Concomitant LSCT either in the form of CLAG or SLET appears to provide better long-term outcomes and is advocated while excision of extensive OSSN involving the limbus.

Results of simple limbal epithelial transplantation in unilateral ocular surface burn

Purpose: This study aimed to report the long-term outcomes of autologous Simple Limbal Epithelial Transplantation (SLET) performed for unilateral limbal stem cell deficiency (LSCD) following chemical burn at a tertiary eye center in North India. Methods: This was a single-center prospective interventional case series of patients who developed unilateral LSCD after suffering from ocular surface burns and who underwent SLET between October 2012 and May 2016 with a follow-up period of at least 6 months. The primary outcome measure was restoration of a completely epithelized, stable, and avascular corneal surface. The secondary outcome measure was percentage of eyes, which reported visual gain. Results: The study included 30 eyes of 30 patients, 18 adults and 12 children, at a median follow-up of 1.1 years (range: 6 months to 3.5 years), 21 of 30 eyes (70%; 95% confidence interval, 53.6%–86.2%) maintained successful outcome. Visual acuity gain was seen in 71.4% of successful cases. The clinical factors associated with failure were identified as acid injury, severe symblepharon at the time of presentation, and SLET combined with penetrating keratoplasty (PK). Conclusion: Autologous SLET is an effective limbal cell transplantation technique for the treatment of unilateral LSCD. It is especially beneficial for centers where cell cultivation laboratory is unavailable. Presence of severe symblepharon, which requires PK peroperatively , has poor outcome.

Research progress of ocular surface reconstruction with ex vivo cultured tissue engineered cell sheet / 中华实验眼科杂志

Limbal stem cells (LSCs),the source of corneal epithelial cells,play an important role in the ocular surface. In recent years, with the development of somatic stem cell application and tissue engineering, biomaterials and cell culture technology,progress has been made on the basic researches and clinical applications of ocular surface reconstruction with ex vivo cultured limbal epithelial and oral mucosal epithelial cell sheet transplantation. However, there are several issues, including the successful clinical outcomes for ocular surface reconstruction,and the in vivo tracking of donor stem cells,remained indefinitive. This article introduced and compared recent advancements of tissue engineering techniques ex vivo cultured autologous or allogeneic limbal,oral mucosal epithelial cells in ocular surface reconstruction,so as to provide a useful direction for the future research of ocular surface reconstruction.

A Two Case of 360 Degree Keratolimbal Allograft

PURPOSE: To report the clinical outcomes of 360-degree keratolimbal allograft in 2 patients. CASE SUMMARY: An 83-year-old female who had uncontrolled Mooren's ulcer invading 360 degrees of the limbus with corneal opacity received a 360-degree keratolimbal allograft (KLAL). Another 63-year-old female who had central corneal opacity and corneal neovascularization due to severe limbal cell deficiency with chemical injury received a 360-degree KLAL. During the average 17.5 months of follow-up, both eyes were tectonically maintained without severe graft rejection. CONCLUSIONS: A 360-degree KLAL may be an effective tectonic procedure for corneal opacity caused by limbal stem cell deficiency. Herein, we report 2 cases of successfully performed 360-degree KLAL with a literature review.

Transforming ocular surface stem cell research into successful clinical practice.

It has only been a quarter of a century since the discovery of adult stem cells at the human corneo-scleral limbus. These limbal stem cells are responsible for generating a constant and unending supply of corneal epithelial cells throughout life, thus maintaining a stable and uniformly refractive corneal surface. Establishing this hitherto unknown association between ocular surface disease and limbal dysfunction helped usher in therapeutic approaches that successfully addressed blinding conditions such as ocular burns, which were previously considered incurable. Subsequent advances in ocular surface biology through basic science research have translated into innovations that have made the surgical technique of limbal stem cell transplantation simpler and more predictable. This review recapitulates the basic biology of the limbus and the rationale and principles of limbal stem cell transplantation in ocular surface disease. An evidence-based algorithm is presented, which is tailored to clinical considerations such as laterality of affl iction, severity of limbal damage and concurrent need for other procedures. Additionally, novel fi ndings in the form of factors infl uencing the survival and function of limbal stem cells aft er transplantation and the possibility of substituting limbal cells with epithelial stem cells of other lineages is also discussed. Finally this review focuses on the future directions in which both basic science and clinical research in this fi eld is headed.

Establishment of assessment of rabbit model of total limbal stem cell deficiency / 中华实验眼科杂志

Background The deficiency of limbus stem cells will cause various kinds of the disorder of eye surface,in serious condition,it will lead to corneal conjunctivalization,chronic inflammation,lasting deficiency of corneal epithelium and corneal opacity accompanying long-term visual loss.Objective The aim of this study was to investigate how to establish and evaluate the successful model of total limbal stem cell deficiency (LSCD).Methods Twenty Japanese white rabbits aged 3-5 months were selected.The limbus corneal epithelium was removed from 1 mm inner to 2 mm outer of the limbus 100-150 μm in deep,and the total central epithelium and shallow stroma were simultaneously removed in the left eyes using scalpel.The experimental eyes were examined by slitlamp 2,3,4,5 weeks after operation,and the inflammatory score was performed according to corneal opacity,corneal neovascularization and corneal flurescein dye using international universal rating criteria of LSCD model.Cornea tissue was obtained in various time points mentioned above to examine the structural change and goblet cells.The expression of cytokeratin 3(CK3) in the cornea was detected using indirect immuno-fluorescence staining.Fisher Exact Probability method was used to compare the successful rate of models in different time points.Results The successful rate of models rate was 12.5％,62.5％,81.3％ and 87.5％,respectively 2,3,4,5 weeks after operation,showing a significant difference between the 3-week group and 2-week group (P =0.009).However,no significant differences were found in the model successful rate between the 3-week group and the 4-week group,the 4-week group and the 5-week group (P =0.465,0.200).There was a little difference between the 3-week group and the 5-week group(P=0.049).The mean time of achieving successful model was(3.21 ±0.80) weeks.The result of hematoxylin & eosin staining showed the edema of cornea stroma,a mass of inflammatory cells infiltration and new vessels.Goblet cells could be found by Periodic acid-Schiff staining 5 weeks after operation.Expression of CK3 was absent in the corneas of successful model eyes.Conclusions The LSCD model can be successfully established by cutting off the epithelium of limbus and central cornea,and the average time of model formation is about 3.2 weeks.

Short-term outcome of Boston Type 1 keratoprosthesis for bilateral limbal stem cell deficiency.

This study reports the short-term functional and anatomical outcome of Boston Type 1 keratoprosthesis (Boston Kpro) implantation for bilateral limbal stem cell deficiency (LCSD). Retrospective analysis was done on eight eyes of eight patients who underwent Boston Kpro implantation between July 2009 and October 2009. The best corrected visual acuity (BCVA) and slit-lamp biomicroscopy findings were assessed at 1, 3 and 6 months postoperatively. All eight eyes retained the prosthesis. BCVA of 20/40 or better was achieved in 8, 6, and 5 eyes at 1, 3, and 6 months, respectively, postoperatively. One patient each developed epithelial defect, sterile stromal melt and fungal keratitis in the late postoperative period associated with antecedent loss of the soft contact lens from the eye. Boston Kpro has good short-term visual and anatomical outcome in patients with bilateral LSCD, provided compliance with postoperative care can be ensured.

Amniotic Membrane Transplanted in Conjunctiva as a Mesenchymal Stem Cells Carrier for Limbal Stem Cell Deficiency

PURPOSE: To report the use of amniotic membrane (AM) as a mesenchymal stem cell (MSC) carrier in cornea perforation with severe limbal stem cell deficiency due to a chemical burn on both eyes. CASE SUMMARY: The patient in the present study is a 43-year-old man with a history of bilateral alkaline chemical injury six years previously. The patient suffered from total limbal stem cell deficiency (LSCD) in the injured eyes and underwent numerous operations including autologous conjunctival flap, AM graft, penetrating keratoplasty, keratolimbal autograft and ex vivo expanded limbal allogrft. However, the patient's ocular condition deteriorated due to persistent LSCD, which led to corneal perforation resulting in the need for an urgent therapeutic operation. In the patient's past ocular history, Ahmed valve implantation was performed for uncontrolled IOP. Revision of the Ahmed tube due to exposure and conjunctival erosion was performed again via placement of an AM over the exposed tube one year previous to presentation to our hospital. The authors performed a transplanted AM graft at the cornea perforation using sub-conjunctival AM believed to contain MSCs. Six months postoperatively, the ocular surface was well mainatained and the graft remained healthy. Transplanted AM showed morphological and immunohistochemical findings similar to those of MSCs. CONCLUSIONS: Results from the present study confirm that AM transplanted into the conjunctiva is a useful carrier of bone marrow-derived MSCs in severe ocular surface diseases.

Culture & characterisation of limbal epithelial cells & oral mucosal cells.

Background & objectives: Sources of autologous tissue that can functionally replace the corneal epithelium have been considered as an alternative to allogenous limbal transplants for limbal stem cells deficiency (LSCD). The aim of the present study was to compare the characterization of oral mucosa with limbal epithelial cells by markers using reverse transcriptase polymerase chain reaction (RT-PCR). Methods: Experiments were performed using oral tissue (n=6) obtained from patients who underwent oral mucosal graft for LSCD. Confluent cultures of limbus and oral mucosa epithelial cells were characterized by the pututative stem cell markers using RT-PCR. The morphological characteristics of cultivated epithelial cells were analyzed by haematoxylin and eosin staining and phase contrast microscopy. Results: Confluent sheets of epithelial cells were seen at the end of 14th day resembling the morphological features of limbal epithelia. RT–PCR analysis showed that cultured oral epithelial cells expressed markers such as ABCG2, p63, delta Np63, isoforms of p63, Keratin 3 (K3), membrane protein – Mucin (MUC 1, 4 and 16) and Antimicrobial Peptide - AMP (Human β Defensin – hBD 1, 2 and 3). Interpretation & conclusions: Oral epithelial cultures have morphological features resembling corneal and limbal epithelial cells by expressing similar marker genes. Thus, feasibility of clinical use of oral epithelial cells need be evaluated for allogenous limbal transplants.

Transplantation of in vivo Cultivated Limbal Corneal Epithelial Cells with Total Limbal Stem Cell Deficiency

PURPOSE: To treat limbal stem cell deficiency, we investigated the epithelial characteristics in rabbits with total limbal stem cell deficiency (LSCD) after reconstruction with autologous limbal corneal epithelial cells in vivo expanded on amniotic membrane. METHODS: The right eyes of 10 rabbits were rendered total LSCD, as verified by impression cytology. In the left eyes of 10 rabbits, in vivo limbal corneal epithelial cells culture was achieved. In group A (n=4), we evaluated the characteristics of the cultured limbal corneal epithelial cells and in group B (n=6) we evaluated the characteristics of the transplanted limbal corneal epithelial cells at postoperative 1, 2, and 4 weeks. RESULTS: Successful epithelial growth was observed on amniotic membrane in all eyes of group A. Transplanted epithelial cells were well remained in five eyes of group B. The epithelial cells of group A and B were confirmed with corneal specificity by immunohistochemical staining (AK-2, AE-5). CONCLUSIONS: The corneal surface with unilateral total LSCD can be successfully reconstructed by transplantation with autologous limbal corneal epithelial cells in vivo expanded on amniotic membrane.