Limbal Area Research Articles

Evaluation of the Tightness of Contact between Limbal Sclera Tunnel and Tube following Ahmed Glaucoma Valve Implantation

To investigate whether the tightness of contact between the tube and the limbal sclera tunnel can be evaluated with high-magnification anterior segment optical coherence tomography (OCT) imaging following Ahmed glaucoma valve implantation. Tightness between the tube and the limbal sclera tunnel was investigated with the CAM-L cornea lens adapter of the Optovue Fourier-domain OCT (RTVue-OCT) for 21 uncomplicated Ahmed glaucoma valves implanted in 20 eyes of 19 patients with glaucoma. Nineteen valves were implanted 4 to 124 months earlier (late postoperative cases) and 2 valves 1 day prior to the imaging (early postoperative cases). All valves were introduced into the anterior chamber via a limbal sclera tunnel. The limbal intratunnel part of the tube was successfully imaged in all but 2 cases where an additional full-thickness sclera patch was used. In 14 cases, the contact was tight without tube compression. In 5 cases, the tube was partially compressed but remained open in the limbal sclera tunnel, and redilated behind the limbus. No case with loose contact or peritubular filtration was seen. The posterior run of the tube was successfully imaged in all 19 cases without a full-thickness sclera patch. High-magnification imaging with the CAM- L anterior segment adapter of the RTVue-OCT allows detailed examination of the limbal insertion area of tubes in both the early and late postoperative periods. Therefore this method may potentially be applied for detection of complications related to tube insertion after glaucoma drainage device surgery.

Development of a microfabricated artificial limbus with micropockets for cell delivery to the cornea

The aim of this study was to develop a synthetic alternative to the human corneal limbus for use initially as an ex vivo model in which to study corneal stem cell function within a niche environment and ultimately to develop an implantable limbus for future clinical use. Microstereolithography was used for the fabrication of polyethylene glycol diacrylate (PEGDA) based rings on a macroscopic (1.2 cm) scale containing unique microfeatures (pockets) which were then modified with fibronectin to promote cell adhesion. These rings were designed to mimic the limbal area of the eye containing structures of the approximate size and shape of the stem cell microenvironments found in the palisades of Vogt. The attachment of rabbit limbal fibroblasts and rabbit limbal epithelial cells to the PEGDA rings was increased by pretreating the microfabricated structures with biotinylated fibronectin. Cell outgrowth from fibronectin coated microfabricated structures was 50% greater than from rings without structures or fibronectin coating. The cell loaded rings were then placed on an ex vivo wounded cornea model and the outgrowth of cells to form a multilayered epithelium was observed. We suggest this is a new approach to investigating limbal stem cells niches and the first steps towards a new approach for corneal regeneration.

Investigation of Trace Elements in Pterygium Tissue

Purpose: In this study, we compare the concentrations of the essential trace elements chromium (Cr), copper (Cu), selenium (Se), manganese (Mn), boron (B) and zinc (Zn) in both pterygium and normal conjunctiva and investigate the role they play in the development of pterygium.Methods: Included in the study were 38 patients with pterygium and 38 control patients in matching age groups who had been operated on for strabismus or cataracts and in whom conjunctiva samples were collected from the nasal limbus area. All conjunctiva samples were kept at −80 °C until the performance of the biochemical investigations. The B, Cr, Mn, Cu, Zn and Se levels in the samples were then measured. The levels of all tissue trace elements were determined by using Agilent 7500a Inductively Coupled Plasma-Mass Spectrometer (Agilent Technologies, Santa Clara, CA).Results: Cr, Mn, Zn and Se levels are significantly lower in the study group as compared to those of the control group (p < 0.001 for all four values). Regarding the level of B, there was no significant difference between the groups. The Cu levels of almost all subjects in the control group and all subjects in the pterygium group were under the detection limit.Conclusions: These findings indicate that remarkable differences in Mn, Zn, Se and Cr levels exist in pterygium tissues. Further investigation of electrolyte composition of the conjunctiva is needed to understand the genesis and developmental mechanism of pterygium.

Der Melanozyt und das Auge: eine Übersicht unter besonderer Berücksichtigung der Kornea

Hintergrund: Verschiedene Arbeiten insbesondere aus der Dermatologie haben in den letzten Jahren deutlich werden lassen, dass der Melanozyt ein „Multitalent“ ist, dessen Bedeutung über die reine UV-Protektion hinausgeht. Neben (neuro-)humoralen und immunologischen Funktionen könnten Melanozyten auch eine Rolle als Fänger von freien Radikalen sowie bei der Druckperzeption spielen und so unter Umständen Teil des zu vermutenden „intraokularen Drucksensors“ sein. Es wird gemeinhin angenommen, dass es in der Kornea unter physiologischen Bedingungen keine Melanozyten gibt. Eine systematische Untersuchung zu diesem Sachverhalt anhand einer größeren Zahl von Proben hat es unseres Wissens bisher aber noch nicht gegeben. Methodik: Es wurden insgesamt 103 korneale Gewebsproben (ganze Augen, Hornhautexplantate mit verschiedenen Erkrankungen, korneosklerale Spendersegmente) sowie 13 Pterygien (korneale Anteile) mit dem Melanozyten-spezifischen monoklonalen Antikörper Melan A immunhistochemisch untersucht. Ergebnisse: In gesunder Kornea finden sich Melanozyten regelhaft im Bereich des Limbus, um sich dann zentralwärts in ca. 1 mm Limbusabstand zu verlieren. Das Epithel der mittelperipheren und zentralen Hornhaut ist Melanozyten-frei. Unter pathologischen Bedingungen (Dystrophien, Narben, Ulzera u. a.) kommt es ausnahmsweise zu einer aberranten melanozytären Besiedelung der mittelperipheren, aber nur sehr selten der zentralen Hornhaut. In mehr als der Hälfte der Pterygien sind Melanozyten nachweisbar. Schlussfolgerungen: Melanozyten können unter bestimmten, pathologischen Bedingungen zentralere Bereiche des Hornhautepithels besiedeln, sodass die wenigen, in der Literatur beschriebenen „kornealen Melanome“ theoretisch „vor Ort“ entstanden sein könnten. Die Hornhaut besitzt offenbar Mechanismen, um die Invasion von Melanozyten trotz der zentripetalen Epithelmigration zu verhindern. Hypothetischerweise geschieht dieses durch einen „CoMeSuF“ („Corneal Melanocyte Suppression Factor“), dessen Identifizierung Aufgabe der kommenden Jahre sein soll. Dieses vor allem auch deshalb, weil ein „CoMeSuF“ potenziell zur Behandlung melanozytärer Proliferationen (Melanome) herangezogen werden könnte.

Immunolocalization of Different Collagens in the Cornea of Human Fetal Eyes: A Developmental Approach

Purpose: To study the corneal development in the human fetal eye with particular emphasis on the epithelial basement membrane and Bowman’s layer. Thus, immunohistochemical markers supposed to stain this region were employed.Material and Methods: 19 formalin-fixed fetal eyes and a 16-day-old newborn’s cornea without any obvious irregularities of the anterior segment were investigated. The age of the fetal eyes ranged from 11 to 38 week of gestation (WoG). The eyes (including the corneal thickness) were measured and, in addition to routine hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) stains, immunohistochemical labeling with antibodies to collagen IV, V, IX, and XVII was performed.Results: Analysis of the H&E stains revealed that measurements of corneal thickness correlated well with corneal development as a basic indicator for maturation. In a more detailed immunohistochemical analysis, collagen IV was expressed in the epithelial basement membrane (BM) of the cornea, conjunctiva, and Descemet’s membrane in fetal eyes up to the age of 23 WoG. In fetal eyes older than 23 WoG, staining was confined to the limbal area only. With the antibody against collagen V, the corneal stroma and the BM were intensely stained. Bowman’s layer (first detected at 17 WoG by light microscopy) was not labeled. Anti-collagen IX labeled predominantly the conjunctival and corneal epithelium. With anti-collagen XVII, the BM of the cornea and conjunctiva was stained in all fetal eyes, whereas intracellular expression in the epithelium increased with age.Conclusion: Our results indicate maturation-associated variations of collagen expression in the human cornea. Measurements of the corneal thickness may serve as an additional parameter to narrow down the developmental age with possible implications for pediatric pathology and forensic issues.

The “Replacement Hypothesis”

Previous studies have shown that the stem cells of corneal epithelia are located at the limbal basal layer. Limbal stem cells are believed to be the source of corneal epithelial cell proliferation and differentiation. This study tested the replacement hypothesis, which suggests that corneal stem cell origin epithelia may be replaced by limbal stem cell origin epithelia after 2 weeks of age in mice. The cytokeratin 12 expression pattern in the cornea was examined using K12 IRES-Cre and Cre reporter mice. Before 2 weeks of age, K12 expression in corneal epithelia showed a mosaic pattern. After 2 weeks of age, centripetal K12 IRES-Cre expression gradually elongated from the limbal area. Around 12 weeks of age, the mosaic expression pattern disappeared from the center of the cornea. Temporal and spatial observations of K12 IRES-Cre expression patterns suggested that the mosaic pattern cells proliferated and amassed at the same position from day 15.5 of the embryonic stage at the latest. Therefore, these cells were considered corneal stem cell origin epithelia. In contrast, centripetal pattern cell populations were considered limbal stem cell origin epithelia because they originated from the limbal area and moved to the center of the cornea. These observations suggest that corneal stem cell origin epithelia are replaced by limbal stem cell origin epithelia after 2 weeks of age in mice.

Aquaporins 6-12 in the human eye

Aquaporins (AQPs) are widely expressed and have diverse distribution patterns in the eye. AQPs 0-5 have been localized at the cellular level in human eyes. We investigated the presence of the more recently discovered AQPs 6-12 in the human eye. RT-PCR was performed on fresh tissue from two human eyes divided into the cornea, corneal limbus, ciliary body and iris, lens, choroid, optic nerve, retina and sclera. Each structure was examined to detect the mRNA of AQPs 6-12. Twenty-one human eyes were examined using immunohistochemical and immunofluorescence techniques to determine the topographical localization of AQPs 6-12. mRNA transcripts of AQP7, AQP9 and AQP11 were found in the ciliary body, corneo-limbal tissue, optic nerve, retina and sclera. AQP9 and AQP11 mRNA was also detected in the choroid. No mRNA of AQP6, AQP8, AQP10 or AQP12 was detected. Anti-AQP7 immunolabelling was detected in the corneal epithelium, corneal endothelium, trabecular meshwork endothelium, ciliary epithelia, lens epithelium, the inner and outer limiting membrane of the retina, the retinal pigment epithelium and the capillary endothelium of all parts of the eye. AQP9 immunolabelling was detected in the nonpigmented ciliary epithelium and retinal ganglion cells. AQP11 immunolabelling was detected in the corneo-limbal epithelium, nonpigmented ciliary epithelium and inner limiting membrane of the retina. Selective expression of AQP7, AQP9 and AQP11 was found within various structures of the human eye. The detection of these aquaporins in the eye implies a role that may be related not only to water transport but also to the transport of glycerol, lactate and ammonia, with importance for metabolism, especially in the retina.

Evaluation of intraocular pressure elevation in a modified laser-induced glaucoma rat model

The main drawbacks of currently described pressure induced glaucoma animal models are, that intraocular pressure (IOP) either rises slowly, leading to a heterogeneous onset of glaucoma in the treated animals or that IOP normalizes before significant damage occurs, necessitating re-treatment. Furthermore, a variable magnitude of IOP increase often results when particles are introduced into the anterior chamber. In order to develop a simple and reproducible rat glaucoma model with sustained IOP elevation after a single treatment we induced occlusion of the chamber angle by anterior chamber paracentesis and subsequent laser coagulation of the limbal area with 35, 40 or 45 laser burns. Right eyes served as controls. IOP was measured three times weekly using TonoLab rebound tonometry in awake animals. After four weeks, retinal tissue was harvested and processed for whole mount preparation. The number of prelabeled, fluorogold-positive retinal ganglion cells (RGCs) was analyzed under a fluorescence microscope. The eyes were further analyzed histologically. Results are expressed as means and standard deviation. Amplitude and duration of the IOP elevation increased with the number of laser burns. Two weeks after 35, 40 or 45 translimbal laser burns the IOP difference between treated and control eye was 7.5 ± 5, 14 ± 8 or 19 ± 9 mmHg, respectively; the RGC density/mm2 28 days after treatment was 1488 ± 238 for control eyes (n = 31) and 1514 ± 287 (n = 10), 955 ± 378 (n = 10) or 447 ± 350 (n = 11) for the respective laser groups. Mean IOP of all control eyes over the observation period was 12.4 ± 0.8 mmHg. The chamber angle showed pigment accumulation in the trabecular meshwork of all laser groups and confluent peripheral anterior synechia after 40 and 45 laser burns. Histologic examination of the retina revealed increasing glia activation in a pressure dependant manner. In this study, >91% of laser treated rats developed secondary glaucoma with sustained IOP elevation for at least 2 weeks. The amount of IOP elevation and RGC loss correspond with the number of laser burns applied. This relatively high success rate after a single procedure may constitutes an advantage over established glaucoma models, as this decreases the risk of complications (e.g. corneal decompensation, intraocular bleeding or inflammation) and, thus, improves the outcome.

Clinical transplantation ofex vivoexpanded autologous limbal epithelial cells using a culture medium with human serum as single supplement: a retrospective case series

Presently, our clinic is the only centre in Scandinavia that offers patients with corneal surface pathology including limbal stem cell deficiency (LSCD) transplantation of ex vivo expanded limbal epithelial cells (LECs). We here present clinical data of the first nine patients with LSCD who were transplanted with autologous LECs expanded in medium completely free of any animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), and with autologous human serum as the only growth supplement. We conducted a noncomparative retrospective study of patients with LSCD at our centre between 2009 and 2011. The diagnosis was based on history and clinical signs. A biopsy was taken from healthy limbus, and the epithelium was expanded on amniotic membrane (AM) in medium containing autologous serum and subsequently transplanted to the affected eye. Successful outcome was defined as relief of pain and photophobia and/or improved best corrected visual acuity (BCVA) and/or reestablishment of a stable corneal epithelium and regression of corneal vascularization. Five of the nine transplanted patients (55.6%) had an improvement in either subjective symptoms or objective findings (11- to 28-month follow-up). Our clinical study shows that patients with LSCD can be treated successfully with transplantation of LECs expanded ex vivo in a medium with autologous serum as the only growth supplement. The use of this novel culture system, which is devoid of animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), reduces the risks of inter-species disease transmission and host immune responses to xenogenic proteins, both obvious advantages for the patient.

Predicting estimates of oxygen transmissibility for scleral lenses

Although scleral contact lenses are prescribed with increasing frequency, little is known about their long-term effects on ocular physiology. The main goal of this paper is to predict values of oxygen transmissibility of scleral lens systems by applying the concept of resistors in series to parameters characteristic of current scleral lenses. A second aim is to find the maximal lens and post-lens tear layer thickness combinations above which hypoxia-induced corneal swelling would be found. Theoretical calculations were used to predict the oxygen transmissibility of scleral lens systems, considering several material permeabilities (Dks 100-170), varying lens thicknesses (250-500 μm), the known tear permeability (Dk of 80) and expected post-lens tear layer thicknesses (100-400 μm). The Holden-Mertz Dk/t criteria of 24 Fatt units for the central cornea and the Harvitt-Bonanno criteria of 35 Fatt units for the limbal area were used as reference points. Our calculations of oxygen transmissibility, with varying tear layer and lens thicknesses, ranged from 10 to 36.7 at the scleral lens centers and from 17.4 to 62.6 at the peripheries. Our calculations of maximum central lens thicknesses show a practical range of 250-495 μm, in conjunction with a post-lens tear layer thickness of 100-250 μm. Our computations show that most modern scleral lenses, with recommended fitting techniques, should lead to some level of hypoxia-induced corneal swelling. Recommendations are made to minimize hypoxia-induced corneal swelling: highest Dk available (>150) lens with a maximal central thickness of 250 μm and fitted with a clearance that does not exceed 200 μm.

Identification of foreign bodies on the ocular surface after uneventful intravitreal injections

Editor, Intravitreal injections with anti-Vvascular endothelial growth factor (VEGF) for various retinal diseases have become very common intraocular procedures worldwide. Known but rare complications to intravitreal injections are endophthalmitis, uveitis, an increase in intraocular pressure, retinal detachment and retinal tear (Day et al. 2011; Meyer et al. 2011). In addition, approximately 10% of the patients experience a self-limiting postinjection discomfort (Rodrigues et al. 2011). Although this surface discomfort is not sight threatening, it may affect the willingness of the patients to continue the treatment, in particular because of its repetitive nature. Here, we describe a potential cause of discomfort owing to foreign bodies after intravitreal injections of 0.05 ml ranibizumab (Lucentis; Genentech, South San Francisco, CA, USA), because we found small plastic particles on the ocular surface of patients during four procedures. All procedures were performed on different patients, at different dates and by different surgeons. To our knowledge, this has not been reported earlier. All patients had previously received uncomplicated intravitreal injections and they had all been done in the same manner. Injections are at our department routinely given under sterile conditions in an operating room with the patient in supine position without the use of a surgical microscope. Disinfection of the eyelids was carried out with topical 5% povidone-iodine and topical anaesthesia (0.5% tetracaine and 0.4% oxybuprocain). Handling and preparation of the 0.05 ml ranibizumab was performed under sterile conditions and according to the manufactures instructions. The medication was used before the expiration date. We used the package supplied 1-ml syringe, 18-gauge filter-needle [BD – BD Blunt Fill Needle - Filter 18G 1½ 1.2 × 40 mm (5 mikr.m.) REF305211] and the 30-gauge injection needle (BD Microlance 30G 0.3 × 13 mm REF 304000). 0.05 ml of ranibizumab was sucked up from the capped vial into the filter-needle (18G). The filter-needle was then exchanged with the injection needle (30G), which was attached on to the syringe. A sterile 40 × 40 centimetre surgical drape with a prefabricated ocular opening was placed over the eye, and an eye speculum was used to fixate the lashes. During the intravitreal injections, small plastic foreign bodies on the ocular surface were immediately observed after the intravitreal injection was completed, but before the eye speculum was removed. The foreign bodies were flat, transparent, purple in colour and with varying shape and a maximum size of 1 × 0.5 mm (Fig. 1A). The foreign bodies were removed from the limbus area and inferior fornix using a cotton tip (Fig. 1A). After removal of the particles, the procedure was completed with removal of the eye speculum and surgical drape, without further complications. No postinjection discomfort was reported. Cotton tip with the plastic particles (arrows) removed from the ocular surface (A). Superficial defect (arrow) in the plastic of 18-gauge filter-needle (B). The plastic foreign bodies, probably originated from the surface of plastic part of the filter-needle, were small, and superficial defects were found (Fig. 1B). These defects might have been caused by the manual attachment and removal of syringe from the filter-needle during preparation of the 0.05 ml ranibizumab. The particles may have been transferred to the ocular surface by sticking to the sterile surgical gloves. This is not the first time that plastic particles have been reported to shed from syringes (Cant et al. 1988) and silicone oil microdroplets originating from syringes have been reported intraocularly after anti-VEGF treatment (Kocabora et al. 2010). If the particles had not been removed they had most likely caused foreign body sensation and might have lead to infection or corneal epithelial damage. We suggest that care should be taken to observe for potential foreign bodies even though the intravitreal injection is given under sterile condition, and patients with continuous ocular discomfort after intravitreal injections should be evaluated for possible foreign bodies.

Trabeculectomy pearls of wisdom; mitomycin-soaked pledget ‘necklace’ suture

Sir, We read with interest the correspondence from Lindfield and co-authors about a method of placing a mitomycin-soaked sponge so that it would not get lost.1 We have published on this subject, which we agree is of importance.2 It seems likely that one reason why surgeons are reluctant to place sponges in a larger area, 6–10 mm posterior to the limbus, where they are most likely to cause aqueous drain away from the limbal area, and to lead to more diffuse blebs,3 is because the surgeons fear they will not be able to retrieve the sponge. Our technique avoids taking these risks by using color-tailed sponges (Codman Surgical Patties, available from Codman, Raynham, MA, USA) for antimetabolite application. We like our technique because it does not require an extra step, it is safe and inexpensive, and there is no possibility of the suture becoming detached from the sponge. It is surprising to us that the idea of using a technique to prevent loss of mitomycin-soaked sponges in trabeculectomy does not seem to have caught on. There is no down side, and there are many advantages.

In vivo microstructural and microvascular imaging of the human corneo-scleral limbus using optical coherence tomography

The corneo-scleral limbus contains several biological components, which are important constituents for understanding, diagnosing and managing several ocular pathologies, such as glaucoma and corneal abnormalities. An anterior segment optical coherence tomography (AS-OCT) system integrated with optical microangiography (OMAG) is used in this study to non-invasively visualize the three-dimensional microstructural and microvascular properties of the limbal region. Advantages include first the ability to correct optical distortion of microstructural images enabling quantification of relationships in the anterior chamber angle. Second, microvascular images enable the visualization of the microcirculation in the limbal area without the use of exogenous contrast agents. Third, by combining the microstructural and microvascular information, the aqueous outflow pathway can be identified. The proposed AS-OCT can serve as a useful tool for ophthalmological research to determine normal and pathologic changes in the outflow system. As a clinical tool it has the potential to detect early aqueous outflow system abnormalities that lead to the pressure elevation in glaucoma. Recent surgical innovations and their implementations also rely on an assessment of outflow system structure and function, which can be revealed by AS-OCT.

Der Melanozyt und das Auge: eine Übersicht unter besonderer Berücksichtigung der Kornea

Various publications especially from the field of dermatology have indicated in the recent years that the melanocyte is a "multitalent" with--besides UV-protection--(neuro-)humoral and immunological functions. Moreover, the melanocyte could play a role as a scavenger of free radicals or in pressure perception, so that it could even perhaps be part of the "intraocular pressure sensor". It is generally assumed that the cornea is devoid of melanocytes under physiological conditions. However, to the best of our knowledge a systematic investigation with a larger quantity of specimens has not been performed thus far. 103 corneal specimens (whole eyes, corneal explants with different corneal diseases, corneoscleral donor buttons) and 13 pterygia (corneal part) were studied immunohistochemically using the monoclonal antibody Melan A which is specific for melanocytes. In healthy cornea melanocytes are found in the limbal area. In the corneal periphery, up to 1 mm distant from the limbus, the melanocytes disappear so that the mid-peripheral and the central epithelium of the cornea are devoid of melanocytes. Under pathological conditions (dystrophies, scars, ulcers) there is only exceptionally an invasion of melanocytes into the mid-peripheral corneal epithelium. The central epithelium almost always remains free of melanocytes even in various corneal diseases. In more than 50% of the pterygia melanocytes can be found in the epithelium. Under certain, pathological conditions melanocytes can settle in more central regions of the corneal epithelium. Thus, the very few "corneal melanomas" described in the literature could have theoretically developed within the cornea itself (and not within the limbus). Obviously, the cornea possesses mechanisms to inhibit centripetal migration of melanocytes perhaps via a (still hypothetic) "corneal melanocyte suppression factor" ("CoMeSuF"). To identify this factor will be the task for the coming years. If this factor is really existent it could possibly serve as a therapy for melanocytic proliferations (melanomas).

Isolation and Characterization of Multi-Potent Stem Cells from Human Orbital Fat Tissues

Loss of corneal epithelial cells results in visual problems. Stem cells isolated from the limbal area of the ocular surface are able to replenish lost corneal epithelial cells. However, destruction of the healthy limbus tissue is inevitable. Theoretically, orbital fat should be an excellent source to isolate stem cells for regenerating ocular tissues as the orbital connective tissues share the same embryonic origin with the ocular proper in early organogenesis. The aim of this study is to isolate stem cells from the human orbital fat and to explore their differentiation potentials into epithelial cells. It was found that spindle-shaped, fibroblast-like cells with extensive proliferation potentials could be isolated from orbital fat tissues. These orbital fat-derived stem cells (OFSCs) possessed multi-lineage differentiation potential to become osteoblasts, chondrocytes, and adipocytes. Upon mix-culture with corneal epithelial cells, OFSCs changed their morphology to round, polygonal epithelial-like cells. Loss of CD105 expression and increased expression of epithelial cell markers, including epithelial-specific antigen and zonal occludin-1, were found upon mix-culture with corneal epithelial cells. Moreover, corneal epithelial differentiation was evidenced by expression of cytokeratin -19 and cytokeratin -3 after mix-culture with corneal epithelial cells, whereas human adipose-derived stem cells from subcutaneous fat were unable to differentiate into corneal epithelial cells under the same induction condition. We further found that direct contact with corneal epithelial cells was essential for OFSCs to commit to corneal epithelial cells. Taken together, orbital fat tissues are a novel source for multi-potent stem cells that possess the potential to differentiate into corneal epithelial lineage. OFSCs are therefore a potential candidate for cell therapy and tissue engineering of corneal epithelium.

Investigation of K14/K5 as a Stem Cell Marker in the Limbal Region of the Bovine Cornea

BackgroundIdentification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. Here we report on the limitations of K14/K5 as a limbal stem cell marker.Methodology/Principal FindingsK14/K5 expression was measured by immunohistochemistry, Western blotting and Real time PCR and compared between bovine epithelial cells in the limbus and central cornea. A functional study was also included to investigate changes in K5/14 expression within cultured limbal epithelial cells undergoing forced differentiation. K14 expression (or its partner K5) was detected in quiescent epithelial cells from both the limbal area and central cornea. K14 was localized predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Western blotting revealed K14 expression in both limbus and central cornea (higher levels in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus. Following forced differentiation in culture the limbal epithelial cells revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in a known differentiation marker.Conclusions/SignificanceK14 and its partner K5 are limited not only to the limbus but also to the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific in situ. Furthermore K14/K5 expression levels were not lowered (in fact they increased) within a limbal epithelial cell culture undergoing forced differentiation suggesting K14/K5 is an unreliable maker for undifferentiated cells ex vivo.

Influence and mechanism of He-Ne laser on scar formation of filtration canal after trabeculectomy in rabbit.

To investigate the influence of He-Ne laser on connective tissue growth factor (CTGF) expression and collagen formation of fibroblast in filtration site after trabeculectomy in rabbit, and to discuss the mechanism for preventing scar formation with He-Ne laser in vivo. The upper nasal limbus area next to the upper rectus muscle in right eyes received 10 minutes He-Ne laser irradiation (200mW/cm(2)) every day for three days, the left eyes served as control. Twenty-four hours after the last irradiation, both eyes of the rabbits were took trabeculectomy surgery. The expressions of CTGF in the filtration area were tested on the 7(th), 14(th) and 28(th) day after surgery and collagen density was tested on the 14(th) and 28(th) day after surgery. Each of the time point had 7 rabbits. The expression of CTGF was lower than that of the control group's on the 7(th) and 14(th) day after trabeculectomy surgery (P=0.01, P=0.005). When examined on the 14(th) and 28(th) day, the collagen density of irradiation group were significantly lower than that of the control group's (P=0.013, P=0.01). Pretreating the filtration area with 200mW/cm(2) He-Ne laser may be helpful in preventing scar formation after trabeculectomy in rabbit, possibly due to downregulation of the expression of CTGF and collagen synthesis in fibroblasts. He-Ne laser may be developed into a new scar preventing method in filtration surgery.

In vivo laser scanning confocal microscopy of vernal keratoconjunctivitis

To demonstrate the morphological characteristics of vernal keratoconjunctivitis (VKC) by using in vivo laser scanning confocal microscope (LSCM). Descriptive analysis of VKC by LSCM performed in EENT Hospital of Fudan University. 26 patients diagnosed as VKC and 26 age-matched healthy controls were enrolled in the study. The HRTII/RCM was used to examine bulbar conjunctiva, tarsal conjunctiva, superior limbus and inferior limbus. The density of Langerhans cells and inflammatory cells in tarsal and bulbar conjunctiva were calculated. The morphology of Vogt Palisades was analysed and normal rate of Vogt Palisades at superior and inferior limbus was calculated, respectively. Infiltration of a larger number of Langerhans cells and inflammatory cells could be identified in both bulbar conjunctiva and tarsal conjunctiva compared with normal subjects. Furthermore, the density of Langerhans cells in tarsal conjunctiva was significantly higher in tarsal form and mix form than that in bulbar form (P = 0.048). Normal Vogt Palisades were visible only in 15 and 23 eyes at superior and inferior limbus respectively. Abnormal Vogt Palisades were characterized by atrophy or absence of stromal papillae and disappearance of bright basal cells, accompanied with the infiltration of more Langerhans cells. At superior limbal area, bulbar form had the highest abnormal rate, followed by mix form, both were significantly higher than tarsal form (P = 0.005). The in vivo morphological characteristics of conjunctiva and limbus in VKC included the infiltration of Langerhans cells and inflammatory cells in epithelium and stroma, and the destruction of Vogt Palisades.

Mapping the entire human corneal nerve architecture

We developed an approach to generate a three-dimensional map that facilitates the assessment of epithelial nerve density in different corneal areas to define aging and gender influence on human corneal nerve architecture. Twenty-eight fresh human eyes from 14 donors of different ages were studied. Corneal nerves were stained and consecutive images acquired with a fluorescence microscope, recorded at the same plane, and merged for viewing the complete epithelial and stromal nerve architecture. After whole mount examination, the same cornea was also used for transection. Stromal nerves entered the cornea in a radial pattern, subsequently dividing into smaller branches. Some branches connected at the center of the stroma, but most penetrated upward into the epithelium. No differences were observed between nerve densities in the four corneal quadrants. Epithelial innervation in the limbal and most of the peripheral area was supplied by a superficial network surrounding the limbal area. Central epithelial nerves were supplied by branches of the stromal nerve network. Epithelial nerve density and terminal numbers were higher in the center of the cornea, rather than the periphery. There were no differences in epithelial nerve density between genders, but there was a progressive nerve density reduction concomitant with aging, mainly in eye samples of donors 70-years of age and older. The modified technique of tissue preparation used for this study allowed for observation of new nerve structure features and, for the first time, provided a complete view of the human corneal nerve architecture. Our study reveals that aging decreases the number of central epithelial nerve terminals, and increases the presence of irregular anomalies beneath the basal layer.

The influence of He-Ne laser on scar formation after trabeculectomy in rabbits.

To investigate the influence of He-Ne lasers on scar formation in the filtration canal after trabeculectomy in a rabbit model, as well as to explore the mechanisms for preventing scar formation when using He-Ne lasers in vivo. Experiment 1: Four groups were established (four eyes in each group). In 12 eyes, the upper nasal limbus area next to the upper rectus muscle received 10 minutes of He-Ne laser irradiation (100, 150, 200mW/cm(2); 60, 90, 120J/cm(2)) every day for three days. Four eyes served as controls. Twenty-four hours after the final irradiation, the rabbits were sacrificed and the irradiated tissue was excised, fixed with paraformaldehyde and tested for proliferating cell nuclear antigen (PCNA), connective tissue growth factor (CTGF) and apoptosis (TUNEL). Experiment 2: Forty-two rabbits were randomly divided into two groups and standard trabeculectomy was performed in the right eyes either after 200mW/cm(2) He-Ne laser irradiation or not in the filtration area. The expression of PCNA and CTGF, apoptosis and collagen density in the filtration area were tested on the 7(th), 14(th) and 28(th) day after surgery. Experiment 1: There were no more PCNA and CTGF positive cells in the He-Ne irradiation group than in the control group. No apoptotic cells were found in either group. Experiment 2: The expression of PCNA and CTGF was lower in the He-Ne irradiation group than in the control group on the 7(th) and 14(th) day after trabeculectomy surgery (P<0.05); no apoptotic cells were detected in either group. Collagen density was significantly lower in the He-Ne irradiation group than in the control group on the 14(th) and 28(th) day after surgery (P<0.05). Pretreating the filtration area with 200mW/cm(2) (120J/cm(2)) of He-Ne laser irradiation may be helpful in preventing scar formation after trabeculectomy, possibly due to the downregulation of the expression of PCNA, CTGF and collagen synthesis in fibroblasts.