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The human eye is relatively unexplored as a source of cells for investigating DNA damage. There have been some clinical studies, using cells from surgically removed tissues, and altered DNA bases as well as strand breaks have been measured using the comet assay. Tissues examined include corneal epithelium and endothelium, lens capsule, iris and retinal pigment epithelium. For the purpose of biomonitoring for exposure to potential mutagens in the environment, the eye-relatively unprotected as it is compared with the skin-would be a valuable object for study; non-invasive techniques exist to collect lachrymal duct cells from tears, or cells from the ocular surface by impression cytology, and these methods should be further developed and validated.

Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).

Purpose/Aim: The adult human retina has limited regenerative potential, and severe injury will result in permanent damage. Lower vertebrates handle retinal injury by activating neural stem cells (NSCs) in the ciliary marginal zone (CMZ). Müller glia-like cells expressing markers of NSCs are also present in the peripheral retina (PR) of the adult human eye, leading to the hypothesis that a CMZ-like zone might exists also in humans. In order to shed further light on this hypothesis we investigated the in vitro differentiation potential of proliferative cells isolated from the adult human PR towards a retinal phenotype.Proliferative cells were isolated from the peripheral retina of human eyes (n = 6) within 24 to 48 hours post mortem and further expanded for 2 or 3 passages before being differentiated for 1-3 weeks. Gene expression was analyzed by microarray and qRT-PCR analysis, while protein expression was identified by immunocytochemistry.A high density of cells co-staining with markers for progenitor cells and Müller glia was found in situ in the PR. Cells isolated from this region and cultured adherently showed fibrillary processes and were positive for the immature marker Nestin and the glial marker GFAP, while a few co-expressed PAX6. After 7 days of differentiation, there was a transient upregulation of early and mature photoreceptor markers, including NRL, CRX, RHO and RCVRN, as well as the Müller cell and retinal pigmented epithelium (RPE) marker CRALBP, and the early RPE marker MITF. However, the expression of all these markers dropped from Day 14 and onwards.Upon exposure of proliferating cells from the adult human PR to differentiating conditions in culture, there is a widespread change in morphology and gene expression, including the upregulation of key retinal markers. However, this upregulation is only transient and decreases after 14 days of differentiation.

Autogenous fascia lata is considered the gold standard for frontalis suspension surgery to correct severe congenital upper eyelid ptosis.This study evaluated the efficacy of a less invasive modification of the standard technique described by Crawford. A total of 85 patients with severe congenital ptosis were enrolled in this study and submitted to surgical correction by frontalis suspension using autogenous fascia lata. Among these patients, 51 had previously undergone ptosis correction using other surgical techniques. The final lid level and contour were evaluated in addition to complications, such as lagophthalmos and ptosis recurrences.Overall, the final results were evaluated as good in 71 of the cases, whereas 11 cases were graded as satisfactory and three cases as poor according to the success criteria. The average increase in eyelid height, measured as the marginal reflex distance (MRD), was 2.9 mm. The best results were obtained in the group of patients with no previous eyelid surgeries. The examples of poor results could be attributed to lagophthalmos and were all confined to the group of patients with a previously failed surgery that employed synthetic suspension materials and levator shortening procedures. No recurrences were observed during the follow-up interval, which lasted an average of 6.4 months (range = 2-59 months).This technique allows a safe surgery with an overall high rate of success. This surgery is also safe and successful in cases of congenital ptosis with associated abnormalities, such as blepharophimosis, and in cases that require secondary ptosis repair.

Purpose To examine levels of oxidative DNA base damage and expression of selected genes and proteins related to DNA damage repair in human limbal epithelium engineered ex vivo. Methods Cells were expanded from limbal tissue on cell culture‐treated inserts in medium containing fetal bovine serum, recombinant growth factors, hormones and cholera toxin (COM) and in medium with human serum as the single growth‐promoting additive (HS). Cells were analysed after two, three and four weeks in culture for DNA strand breaks and oxidized purine bases (Comet assay using the enzyme formamidopyrimidine DNA glycosylase, Fpg) and for expression of DNA repair enzymes APE1, OGG1 and Polβ by in situ hybridization (ISH) and by immunohistochemistry (IHC). Results Levels of strand breaks were substantial while levels of net Fpg‐sensitive sites (8‐oxoguanine and ring‐opened FaPy bases) were relatively low in cells engineered in COM and in HS. Both types of medium were found to support expression of base excision repair (BER) enzymes APE1, OGG1 and Polβ at the gene level. At the protein level, expression of APE1 and OGG1 was noticeable in both conditions while expression of Polβ was low. Conclusion Our findings indicate low levels of oxidative stress and/or efficient DNA purine base damage repair in human limbal epithelium engineered in a medium with human serum as the single growth‐promoting additive as well as in traditional medium with xenobiotics.

Purpose DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. Methods Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers. Results DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. Conclusion The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract.

Purpose 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. Methods 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. Results 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). Conclusions 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.

Purpose To assess the hypothesis that during corneal crosslinking (CXL) treatment, riboflavin and ultraviolet-A (UVA) may have a toxic effect on human limbal epithelial cells. Setting Center for Eye Research, Department of Ophthalmology, Oslo University Hospital Ulleval, Oslo, Norway. Design Experimental study. Methods In this vitro study, limbal biopsies from corneoscleral rims collected after corneal transplantation were treated with the following combinations: riboflavin–UVA, riboflavin only, or UVA only; a control group received no treatment. After 3 weeks of cell culture, outgrowth of epithelium from the biopsies was evaluated by measuring the area of cell expansion and the number of cell layers. The explanted biopsies were analyzed for proliferation using immunohistochemistry marker Ki-67 and for apoptosis using the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) assay. Results The mean outgrowth from the biopsies was 2.25 mm 2 ± 6.90 (SD) in the riboflavin–UVA group, 181.4 ± 94.8 mm 2 in the riboflavin-only group, 128.5 ± 129.5 mm 2 in the UVA-only group, and 176.2 ± 114.0 mm 2 in the control group. There were no statistically significant between-group differences in the number of cell layers except in the riboflavin–UVA group, in which no cells were found. Detection of apoptosis with the TUNEL-assay was found in the riboflavin–UVA group only (4/5 sections). The proliferation marker Ki-67 was positive in some sections in all groups. Conclusion Cytotoxicity and reduced cell expansion of human limbal epithelial cells occurred after riboflavin–UVA treatment in vitro, emphasizing the importance of avoiding riboflavin–UVA on the limbus during CXL. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

In addition to the ability for self-renewal and functional differentiation, neural stem/progenitor cells (NSCs) can respond to CNS injuries by targeted migration. In lower vertebrates, retinal injury is known to activate NSCs in the ciliary marginal zone (CMZ). Cells expressing markers of NSCs are also present in the ciliary body epithelium (CE) and in Müller glia in the peripheral retina (PR) of the adult human eye. However, these cells seem to be quiescent in the adult human eye and recent reports have shown that CE cells have limited properties of NSCs. In order to further clarify whether NSCs exist in the adult human eye, we tested whether NSC-like cells could be activated in eyes with proliferative vitreoretinopathy (PVR). The PR and CE were studied for NSC-associated markers in human enucleated control eyes and eyes with confirmed PVR, as well as in a mouse model of PVR. Furthermore, cells isolated from vitreous samples obtained during vitrectomies for retinal detachment were directly fixed or cultured in a stem cell-promoting medium and compared to cells cultured from the post-mortem retina and CE. In situ characterization of the normal eyes revealed robust expression of markers present in NSCs (Nestin, Sox2, Pax6) only around peripheral cysts of the proximal pars plana region and the PR, the latter population also staining for the glial marker GFAP. Although there were higher numbers of dividing cells in the CE of PVR eyes than in controls, we did not detect NSC-associated markers in the CE except around the proximal pars plana cysts. In the mice PVR eyes, Nestin activation was also found in the CE. In human PVR eyes, proliferation of both non-glial and glial cells co-staining NSC-associated markers was evident around the ora serrata region. Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR, and expressed glial - and NSC-associated markers both after direct fixation and repetitive passages. In conclusion, the adult human eye may harbor two different populations of neuroepithelial stem/progenitor cells; a non-glial population located in the proximal pars plana around peripheral cysts in addition to a population with Müller glia characteristics. Yet, we only found that the glial population was able to respond to retinal injury by targeted migration into the vitreous.

Purpose: The identification of cells with properties of retinal progenitor cells (RPCs) in the adult human ciliary margin (CM) prompted a number of studies of their proliferative and differentiation potential. One of the remaining challenges is to find a feasible method of isolating RPCs from the patient’s eye. In the human CM, only the iris pigment epithelium (IPE) is easily obtained by a minimally invasive procedure. In the light of recent studies questioning the existence of RPCs in the adult mammalian CM, we wanted to assess the potential of the adult human IPE as source of RPCs. Methods: The IPE were isolated from peripheral iridectomies during glaucoma surgery, and IPE and ciliary body (CB) epithelium were also isolated from post-mortem tissue. Cells were cultivated in sphere-promoting conditions or as monolayers. Whole-tissue samples, undifferentiated and differentiated cells were studied by immunocytochemistry, RT-PCR and transmission electron microscopy. Results: The adult human IPE, like the CB, expressed markers of RPCs such as Pax6, Sox2 and Nestin in vivo. Both sphere-promoting and monolayer cultures preserved this phenotype. However, both IPE ⁄ CB cultures expressed markers of differentiated epithelial cells such as Claudin, microphtalmia-associated transcription factor (MITF) and Cytokeratin-19. Ultrastructurally, IPE spheres displayed epithelial-like junctions and contained mature melanosomes. After induced differentiation, IPE-derived cells showed only partial neuronal differentiation expressing b-III-tubulin, Map-2 and Rhodopsin, whereas no mature glial markers were found. Conclusion: Proliferative cells with some properties of RPCs can be isolated from the adult human IPE by peripheral iridectomies. Yet, many cells retain properties of differentiated epithelial cells and lack central properties of somatic stem cells.

Explants of human retinal pigment epithelium were maintained in culture in various types of media, and examined by light and transmission electron microscopy. After one month in vitro, the central areas showed a monolayered configuration with distinct polarity and presence of ruthenium red stainable material on the apical surface. On the peripheral areas of Bruch's membrane, multilayered lesions were observed to develop and to extend from the monolayered epithelium and past the cut edge in Bruch's membrane. Cells in these lesions contained little melanin and generally lacked an apico-basal polarity. Ruthenium red staining revealed the presence of electron dense material on the apical surface of the lesions as well as in the extracellular space between cells in the various layers. Development of multilayered lesions with deposition of extracellular material are seen in various chorio-retinal disorders, including senile macular degenerations and also subsequent to laser and cryo-therapy. The findings in the present study point to the explant culture system as a valuable tool in the study of important aspects of chorio-retinal pathology.

The recently devised methods for surgical sampling of chorio-retinal tissue permits morphological evaluation of retina in various diseases. In the present report it is demonstrated, that the retinal pigment epithelium in such samples can be isolated as a viable cell population, and that the cell mass can be increased sufficiently in culture to permit biochemical as well as morphological studies. This procedure thereby expands the diagnostic potential of the sample tissue.

Retinal pigment epithelium from human eyes was maintained in organ culture, and DNA synthesis was visualized by autoradiography of cultures exposed to labelled thymidine. In Ham's F10 medium, labelling of cells was observed after 7 as well as after 14 days in vitro. At both stages the labelled cells were characteristically located adjacent to areas of epithelial damage. Cells remote from such areas did not incorporate thymidine. This pattern was observed in epithelia removed from the posterior pole as well as from the periphery of the fundus. Several types of routine media and media with human as well as foetal bovine serum (FBS) were found to support DNA synthesis in the cultured epithelium. Labelled cells were not observed in epithelium maintained in Ham's F10 without serum, or with low serum concentration. Our study demonstrates, that DNA synthesis does take place in organ cultured human retinal pigment epithelium when the appropriate culture conditions are used. Our findings indicate that induction of DNA synthesis in the cells is not promoted by an indiscriminate effect of the medium but related to the presence of epithelial damage.

Cells from samples of human choroidal tissue were collected and seeded onto isolated Bruch's membrane in vitro, and the morphology of the developing cultures was examined by light-, scanning electron- and transmission electron-microscopy. In early cultures, the cells showed a flattened, spread out appearance with short surface villi and a low degree of cellular overlapping. After 48 h, a shift towards a more elongated cell form was observed, and one week old cultures were composed of predominantly elongated cells with a pronounced degree of overlapping. In contrast, human retinal pigment epithelial cells seeded onto sheets of Bruch's membrane maintained a spread out epithelial morphology with a varying degree of overlapping throughout the culture period. The present system permits comparative studies on the behavior of choroidal and pigment epithelial cells under controlled conditions. It may serve as an in vitro model for disorders characterized by growth of these cells on Bruch's membrane in vivo.

4 × 4 mm pieces of human eye walls consisting of the sclera, the choroid and the pigment epithelium were kept in organ culture for 1 month and studied with light and transmission electron microscopy comparing the findings with normal tissue. After 1 month the choriocapillaris still had open vessel lumina lined with flattened endothelial cells containing organelles and vesicles on both inner and outer cell membranes. The endothelial cells lost their fenestrations and polarity in regard to the location of cell nuclei. Otherwise the ultrastructure was similar to that of the choriocapillaris in vitro. This study has shown that choriocapillaris can survive in organ culture for 1 month, and it permits further studies on the behaviour of the choroid under controlled conditions, and on the long-term effects of different types of trauma to the choroid in vitro.

Argon laser lesions were produced in human retinas in eyes to be enucleated because of choroidal melanomas. Lesions were subsequently isolated and maintained in vitro for one month and evaluated by light, transmission and scanning electron microscopy. In the cultured lesions ingrowing cells with a well recognizable epithelial morphology were observed to cover Bruch's membrane in the photocoagulated area, and to grow as sheets on sensory retinal tissue and on detached necrotic pigment epithelium. This growth was predominantly monolayered. In areas, however, overlapping, detachment of cells from the epithelium into the photocoagulated debris, and formation of tubulovescicular structures were observed. The present study demonstrates that lesions with a known potential for reactive changes in the retinal pigment epithelium in vivo can be isolated and cultured with a maintained potential for some of the typical reactive patterns. This opens for investigations on their genesis and on agents modifying these patterns under controlled in vitro conditions.

Samples of iris tissue were removed from human eyes with and without pseudoexfoliation syndrome and maintained in vitro for several weeks. The samples were cultured on tissue culture plastic as well as on biological basement membranes. The morphology of the cultures was examined by phase contrast-, scanning electron-, and transmission electron microscopy. Out-growth of cells with similar shapes was observed from normal iris as well as from pathological tissue on both type of substrate. In sections the outgrowing cells formed multilayered structures. In these areas the individual cells were separated by spaces containing extracellular material. The PE positive specimens revealed typical PE aggregates both within the native tissue and between the outgrown cells. Production of viable cell cultures from human eyes with pseudoexfoliation may facilitate the search for morphological and biochemical abnormalities in this syndrome.

Subretinal ingrowth of choroidal fibroblasts is of importance in several clinical settings such as age-related macular degeneration or after traumas such as laser treatment and infection. In the present investigation we describe a method for isolation and propagation of human choroidal fibroblasts in vitro. Outgrowth from the outseeded choroid was studied by means of phase contrast microscopy, scanning electron microscopy and by immunohistochemical methods. The secondary cultures were found to contain almost only fibroblasts (more than 95% of the cells), some contaminating retinal pigment cells were also found. Choroidal fibroblasts contribute to the fibrovascular subretinal scarring process, and our cell culturing system seems to be a suitable tool for studying factors regulating the behaviour of these cells in vitro.

The aim of the study was to examine for possible differences between cell cultures derived from eye with and without pseudoexfoliation. In both populations, scanning electron microscopy showed flattened epithelial cells and also spindleshaped fibroblast-like cells. The presence of these cell types was further confirmed by immunohistochemical demonstration of cytokeratin and vimentin in the cultured cells. The cells maintained in vitro showed a linear increase in uptake of leucine during a 12-h period. Within this period, the leucine recovered in the TCA precipitable fraction was considerably higher than the non-bound fraction. In cultures maintained in medium with and without L-ascorbic acid, the presence of L-ascorbic acid significantly increase the uptake of leucine into TCA precipitable material, and to a similar extent in cultures from the two populations. In conclusion, cells derived from eyes with and without pseudoexfoliation material and maintained in vitro showed similar morphology, presence of intermediate filaments, as well as uptake of leucine under various culture conditions.

Explants from the retinal pigment epithelium and the underlying choroid and sclera were dissected from human eyes and transferred to culture wells. The mechanical trauma caused by the dissection and removal of the explants, and the changes in biological milieu caused by transfer of the tissue to an in vitro system causes injury, necrosis and detachment of cells from Bruch's membrane. In the retinal pigment epithelium, cells adjacent to damaged, spherical and detaching cells and smaller cell free zones from rosettes. At the periphery of big defects, the cells spread out to cover the denuded areas of Bruch's membrane. The present work has shown that cell injury in the human retinal pigment epithelium is followed by reactive cellular changes in vitro. The result of these reactive changes are increased variation in cellular form and magnitude and in pigment concentration per unit area.

Pseudoexfoliation syndrome is a risk factor in cataract surgery because of the increased weakness of zonular apparatus and reduced pupillary dilatation. The surgical outcome of using phacoemulsification in the central zone, inducing minimal stress on the zonules, inserting a capsular tension ring in selected cases, and stretching the pupil mechanically in eyes with miotic pupils, may turn out to be uneventful in most cases. Postoperative fibrosis with subsequent shrinkage of the capsule is increased in these eyes, and these centripetal forces will further loosen the zonular fibres. Late in-the-bag intraocular lens dislocation is therefore anticipated to become a growing problem in the future. Despite the dysfunctioning of the blood-aqueous barrier in eyes with pseudoexfoliation syndrome, the frequency of postoperative inflammatory reaction is low due to the improvements made in surgical technique and equipment in recent years. Glaucoma frequently occurs in eyes with pseudoexfoliation syndrome. Compared with primary open-angle glaucoma, optic damage is more pronounced in these eyes at the time of diagnosis and response to medical therapy is poorer. Although responses to argon laser therapy and filtering surgery are roughly similar between the two types of glaucoma, there are indications that primary laser trabeculoplasty has a higher success rate in pseudoexfoliation glaucoma than in primary open-angle glaucoma.

Purpose Uveal melanoma (UM) is the most common intraocular malignancy and is fatal in approximately 50% of the cases. Cancer relapse can be seen years after treatment. Diverse mechanisms have been proposed to explain the late relapse of UM e.g cancer dormancy and the presence of cancers stem cells (CSCs). UM has the disadvantage of being difficult to culture, thus a limited number of cell lines are available. 3D sphere cultures of early passages offer a lower degree of clonal selection and perhaps a more accurate reflection of the primary tumours. The propagation of cells as spheres is believed to enrich for a CSC-like population. Methods 2D cultures were expanded in RPMI 1640 with 10% FBS, while 3D sphere cultures were expanded in hESC + MEF for 7–10 days. We compared UM primary tumours fresh frozen vs. 2D culture and 3D sphere culture of the same donors by microarray, qRT-PCR, transmission electron microscopy (TEM), immunohistochemistry (IHC) and chromatin immunoprecipitation (ChIP). Results 3D sphere cultures induce signalling pathways that enhance survival and increase oncogenic activity. They show ultrastructural resemblance to in vivo UMs. The metabolic shift seen in 3D cultures could reflect the survival patterns of pre-metastatic cancer cells. Conclusions The molecular signature of the 3D cultures reveals the presence of attractive targets for future epigenetic modulation or cancer cell vaccines.

A good understanding of the anatomical details is required to ensure optimal results during surgery of the orbit. Several indications for orbital surgery require biopsy, resection, or reconstructive procedures. The intricate relationships between the orbital septum and adjacent structures of the upper orbit can cause difficulties in interpreting the surgical anatomy of this region. The purpose of this study was to acquire further insight into the anatomy of the superior part of the orbit, with special attention paid to the orbital septum.An ex-vivo study was performed using magnetic resonance imaging (MRI) at 9.4 Tesla (isotropic resolution = 20 μm) on six human cadaver specimens to examine the superior-medial half of the orbit. To visualise the posterior layers of the upper orbit, a dissection of three of the orbits was performed prior to the MRI examination, and a flexible PVC sheet was introduced above the levator muscle.The technique enabled a visualisation of anatomically important landmarks of the anterior and posterior parts of the upper orbit at a resolution near histological levels; to the authors' knowledge, this visualisation has not been reported previously. A posterior continuation of the orbital septum, which forms a distinct anatomical structure, is revealed.The posterior aspect of the orbital septum separates the levator muscle and the orbital fat pad. Between these two structures, a surgical corridor is formed using MRI, enabling alternative access to the superior part of the orbit; this alternative access might be less invasive because the orbital septum remains undamaged.

Purpose: To evaluate the results after amnion-shielded trabeculectomy with concomitant use of mitomycin C. Methods: The study comprised patients with severely refractory glaucomas who had previously undergone two or more regular trabeculectomies with mitomycin C and, in one case, cyclodestructive cryotherapy. The patients were admitted for surgery between May 2003 and May 2004. Trabeculectomy was performed through a limbus-based incision in nine eyes of nine patients. Cryopreserved human amniotic membranes obtained from our biobank were impregnated with 0.4 mg/ml mitomycin C and washed for 2 mins in balanced salt solution. One membrane was sutured to the scleral surface and partly positioned under the scleral flap, and another was secured to subconjunctival tissue. Results: The mean preoperative intraocular pressure (IOP) was 32.2 mmHg (range 22–44 mmHg). After a follow-up of 6–18 months (mean 9.8 months), the mean IOP was 16.4 mmHg (range 11–26 mmHg). The mean number of glaucoma medications was reduced from 2.4 preoperatively to 1.4 postoperatively. There were no devastating complications. Conclusions: Amnion-shielded trabeculectomy is a procedure that should be evaluated as an option in refractory glaucoma.

Purpose p63 (isoform ΔNp63α) is a putative stemness marker for limbal epithelial stem cells (LESCs), and is essential for the proliferative potential of stem cells in stratified epithelia. The niche for LESCs is the limbus, located between the clear cornea and bulbar conjunctiva. LESCs are responsible for and able to renew and repair the corneal epithelium throughout life. In a number of disorders and after trauma, limbal stem cell deficiency (LSCD) may occur, and this can be treated by isolation and cultivation of LESCs from a healthy eye followed by transplantation to the affected eye. It is known that the percentage of p63-bright cells affect the success rate of the transplantation. Thus, we wanted to reveal whether lithium, a known Wnt signaling inducing agent, may increase the expression of p63 in human LESC cultures. Methods Small limbal biopsies (1x1-2mm) were dissected from donor corneas and cultured in a standard medium at 37 oC and 5% CO2 under different concentrations of lithium (LiCl2). The cells were cultured for 3-6 weeks and medium was changed every 2-3 days. Results The epithelial cells migrated from the limbal biopsy to form an epithelial sheet. Lithium enhanced the expression of p63 compared to controls, and this was confirmed by qRTPCR. Further studies mapping epigenetic alterations of the lithium treatment are currently conducted. Conclusion Lithium induced upregulation of p63 in LESC could be clinically important by increasing the long term success of limbal stem cell therapy.

Access to the superior mid-orbit is required for procedures on the levator muscle in the correction of upper eyelid ptosis and in surgery aimed at local lesions in this region. The purpose with this human cadaver study was to clarify the anatomical substrate for a surgical approach to the levator muscle and the upper mid-orbit structures, in which the orbital septum and the retroseptal fat pad is not harmed during surgery. Macro-anatomical dissections and histological examinations were performed on five human orbits from three formalin embalmed cadaver heads. It was found that the orbital septum extends posteriorly from its junction with the levator aponeurosis. This posterior continuation of the orbital septum encloses the superior orbital fat pad and separates this from the anterior surface of the levator muscle. In between the orbital septum and the levator, there is a dissection space that provides a minimal invasive access corridor to the structures in the upper mid-orbit.

Purpose: Storage time for donor corneas in Optisol GS is limited compared to Eye Bank Organ Culture (EBOC). We here examine the epithelium on donor corneoscleral rims after primary storage in Optisol GS and subsequent incubation in EBOC. Methods: Morphology was monitored by light and electron microscopy, expression of phenotypic and genotypic markers by immunohistochemistry and RT-PCR and changes in oxidative lipid and DNA damage by ELISA and COMET assay. Results: A prominent loss of cells was observed after storage in Optisol GS. After maintenance in EBOC, spreading apical cells were Occludin+, while the staining for E-cadherin and Connexin-43 was less intense. There were an upregulation of Occludin and a downregulation of E-cadherin and Connexin-43. Eye Bank Organ Culture was associated with an ongoing proliferative activity and a downregulation of putative progenitor/stem cell marker ABCG2 and p63. Staining for 8-OHdG and Caspase-3 did not increase, while levels of malondialdehyde and number of DNA strand breaks and oxidized bases increased. Conclusions: This dual procedure should be pursued as an option to increase the storage time and the pool of available donor corneas. The observed downregulation of markers associated with stemness during EBOC is relevant considering the potential use of donor epithelium in the treatment of ocular surface disorders. Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/onlineopen#\OnlineOpen_Terms

Purpose Lipid peroxidation content was measured in an organ culture medium after one-week storage of human donor corneas. Moreover, the effects of the medium on oxidative stress, antioxidant capacity, and the proliferation of cultured human corneal cells were studied. Methods The medium was sampled from the upper and lower halves of storage vials and from controls (n=42). Malondialdehyde (MDA) was measured by high pressure liquid chromatography (HPLC). Cultured human corneal epithelium (CRL-11515) was exposed to different medium samples and monitored for changes in MDA (enzyme-linked immunosorbent assay [ELISA]), total antioxidant capacity (antioxidant assay kit), and proliferation (Ki-67). Results A significant increase in MDA was observed in the organ culture medium in the lower level of storage vials. The addition of this fraction to cultured cells increased MDA significantly after 3 days, and the medium from both levels significantly increased MDA after 7 days. The medium from both levels significantly decreased the total antioxidant capacity of the cells but did not affect proliferative activity. Conclusions An oxidative gradient with an evident biologic effect is established in the medium in vials during organ culture of human donor corneas. Donor tissue stored at the bottom or in lower levels of such vials is exposed to a significant amount of oxidative stress.

Purpose Proliferative vitreoretinopathy (PVR) is an important cause of retinal diseases such as retinal detachment (RD). In lower vertebrates, retinal damage is known to activate neuroepithelial stem cells (NSCs) in the ciliary margin in an attempt to regenerate the neuroretina. Cells expressing some markers of NSCs are also present in the retina and the ciliary body epithelium (CB) of the adult human eye. We hypothesized that if NSCs exist in the adult human eye, they should be activated by PVR formation. Methods Cells isolated from vitreous samples (n=25) obtained during vitrectomies for RD were directly fixed or cultured in a stem cell-promoting medium, and compared to cells isolated from post mortem CB and peripheral retina (PR) using sphere-forming assay, immunohistochemistry, molecular biology and electron microscopy. Markers of NSCs were also studied in whole retinal control/PVR sections obtained from enucleations. Results Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR. These spheres stained for markers of NSCs both in vivo and after repetitive passages. Their mRNA and immunohistochemical profile resembled sphere-forming cells from the PR with only a few characteristics of CB cells. In situ characterization of the CB revealed that although there were higher numbers of dividing cells in PVR eyes than in controls, we did not detect markers of NSCs. Interestingly, markers of NSCs were evident around PR cysts with some evidence of activation following PVR formation. Conclusion A population of NSC-like cells are found in the vitreous of patients with PVR. These cells seem to be originated from the retina itself and not the CB.

In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. However, the protocol for cultivation and transplantation of HLECs differ significantly, and in most protocols growth additives such as cholera toxins, exogenous growth factors, hormones and fetal calf serum are used. In the present article, we compare for the first time human limbal epithelial cells (HLECs) cultivated on human amniotic membrane (HAM) in a complex medium (COM) including fetal bovine serum to a medium with human serum as single growth supplement (HSM), and report on our first examinations of HLECs expanded in autologous HSM and used for transplant procedures in patients with LSCD. Expanded HLECs were examined by genome-wide microarray, RT-PCR, Western blotting, and for cell viability, morphology, expression of immunohistochemical markers and colony forming efficiency. Cultivation of HLECs in HSM produced a multilayered epithelium where cells with markers associated with LESCs were detected in the basal layers. There were few transcriptional differences and comparable cell viability between cells cultivated in HSM and COM. The p63 gene associated with LESCs were expressed 3.5 fold more in HSM compared to COM, and Western blotting confirmed a stronger p63α band in HSM cultures. The cornea-specific keratin CK12 was equally found in both culture conditions, while there were significantly more CK3 positive cells in HSM. Cells in epithelial sheets on HAM remaining after transplant surgery of patients with LSCD expressed central epithelial characteristics, and dissociated cells cultured at low density on growth-arrested fibroblasts produced clones containing 21 ± 12% cells positive for p63α (n = 3). In conclusion, a culture medium without growth additives derived from animals or from animal cell cultures and with human serum as single growth supplement may serve as an equivalent replacement for the commonly used complex medium for ex vivo expansion of HLECs on HAM.

Purpose To develop simple, reproducible, animal-materials free method for cultivating limbal stem-cells and differentiating them into corneal epithelium on different human biologic materials for clinical transplantation. Methods The limbal tissues (2x2mm) were harvested from cadavers not more than 8 hours after death and proliferated in vitro on cell culture tissue plates, human amniotic membranes (HAM) or human lens capsules in medium containing human AB serum. Cell viability was tested using the MTT assay and annexin-FITC/Propidium Iodide positivity methods. Molecular gene and immunofluorescent marker studies for stemness, proliferation and differentiation were used for the analysis. Results Over a period of one year, 50 limbal tissue explants were cultivated. Emergence of cells at one edge of the explants occurred within 24 hours from culturing and formed monolayer within 14 days. Although the speed of cell growth varied among donors and types of media for growth, inadequate growth at two weeks was never recorded. The viability of the cells at 7 and 14 days of cultivation was higher than 96% except in case of HAM use where viability was below 80%. The growing cells were characterized for their positivity for stemness (P63, ABCG2), proliferation (ki67) and epithelial cell markers CK 3, 8, 12, 14, 18 and 19. Conclusion We demonstrate a simple, animal-materials free technique for generating corneal epithelium from cadavers or alternatively from autologous donors for viable cell growth on different biologic materials for transplantation. The growth of corneal epithelium on lens capsules proved to be superior compared to the other cultivation techiques.

Eye banks have procured, processed and stored donor corneas for decades. In parallel, new techniques have emerged employing allogeneic transplantation of various cells and tissues from the eye banks. This progress is a consequence of increased knowledge of stem cells, cell kinetics and immunological aspects and improved techniques for cell culturing, tissue storage and microsurgery.Review article on available transplants for treating eye diseases, based on experience with eye banking, clinical ophthalmological practice, own research and literature retrieved from PubMed, Medline and www.google.com.Treatment techniques for eye diseases, which require biological material for grafting, need efficient eye banks for continuous supply of donor material of high quality. New Norwegian legislation, based on implementation of EU Directive 2004/23/EC, demands authorization of all eye banks. The EU Directive sets high and rigorous standards for quality and safety for donation, procurement, testing, processing, storage and distribution of tissues and cells. Well-run eye banks are of great importance for modern treatment of patients suffering from eye diseases and for progress and research in ophthalmology.

Purpose A previous report has described the use of eye bank storage of cultured human limbal epithelial cells (HLECs) to provide a reliable source of tissue for treating limbal stem cell deficiency. In the present study, conventional organ culture (OC) storage and Optisol-GS (Bausch & Lomb, Irvine, CA) storage of cultured HLECs were compared. Methods Three-week HLEC cultures were either organ cultured at 31 degrees C or 23 degrees C or stored in Optisol-GS at 5 degrees C in a closed container for 1 week. Morphology was studied by light microscopy and transmission electron microscopy, and phenotypic characterization was assessed by immunohistochemistry. Apoptosis was evaluated by real-time RT-PCR microarray analysis, caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Results The ultrastructure was preserved at 23 degrees C, while storage at 31 degrees C and 5 degrees C was associated with enlarged intercellular spaces, separation of desmosomes, and detachment of epithelial cells. Cultured HLECs remained undifferentiated in all storage conditions. The expression of the antiapoptotic gene BCL2 was prominently upregulated in storage at 23 degrees C and 5 degrees C. Downregulation of BCL2A1, BIRC1, and TNF and upregulation of CARD6 in 23 degrees C and 5 degrees C storage conditions suggests a reduction in nuclear factor-kappaB activity. No significant increase in cleaved caspase-3 and TUNEL staining was observed in response to eye bank storage, and the labeling indices of cleaved caspase-3 (range, 0.0%-4.7%) and TUNEL (range, 0.0%-7.8%) were low. Conclusions These data indicate that OC storage of cultured HLECs at ambient temperature is superior to OC storage at 31 degrees C and Optisol-GS storage at 5 degrees C and that apoptosis is minimal after eye bank storage of cultured HLECs.

Purpose: To compare the histology, phenotype, ultrastructure and barrier function of cultured limbal epithelial cells using two explant culture protocols. Methods: Epithelial cells were cultured for 16 days from limbal explants, positioned with either the stromal side (stromal group) or the epithelial side (epithelial group) on intact amniotic membranes. The cultured epithelium (n = 56) was examined using light microscopy, immunohistochemistry for K3, Cx43, ABCG2 and p63 expression, Western blot analysis of ΔNp63α, transmission electron microscopy, a horseradish peroxidase (HRP) permeability assay and scanning electron microscopy. Results: The epithelial group demonstrated a significantly higher expression of p63-positive cells (85.7 ± 4.2%) than the stromal group (75.3 ± 8.9%), and Western blots showed a stronger band of ΔNp63α. K3 and ABCG2 were not detected in either group, whereas Cx43 displayed moderate immunostaining in the suprabasal layer. The number of cell layers, the desmosome number and the undulation length in the epithelial group were not significantly different from those in the stromal group. In both groups, HRP accumulated on the apical surface of the superficial cells, and scanning electron microscopy demonstrated tightly apposed superficial cells. Conclusions: Our findings indicate that limbal explants positioned epithelial side down may give rise to cultured epithelia with higher expression of p63 and ΔNp63α.

Our results indicate that OC storage of cultured HLEC at ambient temperature is superior to OC storage at 31°C and Optisol-GS storage at 5°C as the original layered structure of cultured HLEC is preserved at 23°C storage. Apoptosis is minimal following eye bank storage of cultured HLEC, though multi-gene profiling revealed interesting alterations in gene expression in cultured HLEC. Eye bank storage of cultured HLEC may provide a reliable source of tissue for treating limbal stem cell deficiency, although its feasibility for clinical use has to be evaluated further. Apoptosis Gene Expression Profiling of Ex Vivo Expanded Human Limbal Epithelial Cells Sten Raeder,1 Tor Paaske Utheim,1 Oygunn Aass Utheim,1 Savita Prabhakar,5 Yiqing Cai,2 Borghild Roald,3 Kristiane Haug,1 Anders Kvalheim,1 Edvard Berger Messelt,2 Hewen Zhang, 5 Liv Drolsum,1 Bjorn Nicolaissen,1 Torstein Lyberg,4; 1Center for Eye Research, University of Oslo, Department of Ophthalmology, Ulleval University Hospital, Norway, 2Department of Oral Biology, Faculty of Dentistry, University of Oslo, Norway, 3Department of Pathology, Ulleval University Hospital, University of Oslo, Norway. 4Center for Clinical Research, University of Oslo, Center for Eye research Ulleval University Kirkeveien, Oslo, 0407, Norway, 5SuperArray Bioscience Corporation, 7320 Executive Way, Suite 101, Frederick MD, 21704.

Aim: To investigate organ culture preservation of cultured limbal epithelial cells in order to enhance the availability of tissue-engineered epithelia that are used to treat patients with limbal stem cell deficiency. Methods: Limbal epithelial cells were cultured for 3 weeks on intact amniotic membrane fastened to a polyester membrane carrier. The cultured epithelia were stored for 1 week at 23°C in organ culture medium. The preserved epithelia were then examined using a colorimetric cell viability assay, light microscopy and immunohistochemistry. Results: The viability of the preserved epithelia was 84% (20%), and no statistically significant difference was found compared with non-preserved epithelia. In general, the cell borders were maintained, the nuclei showed no sign of degeneration, and the original layered structure was preserved. Mild intercellular oedema was occasionally observed. Expression of p63, K19 and vimentin was maintained. Conclusions: Cultured limbal epithelial cells can be preserved in organ culture medium for 1 week at room temperature, while maintaining the original layered structure and undifferentiated phenotype.

Purpose: Donor corneas are processed in eye banks and used for transplantation as a standard routine. The maximum time limit post-mortem for harvesting donor tissue varies greatly between eye banks. This study aimed to examine the corneal epithelium for structural changes post-mortem. Methods: A total of 51 corneas harvested between 14 and 163 hours post-mortem were examined using scanning electron and light microscopy. Results: Cell loss occurred through desquamation of flat superficial cells during the first days. In corneas with a post-mortem time of more than 2−3 days, large superficial cell sheets and deeper cells detached, starting centrally. Deep peripheral cells remained. The loss of the superficial cells revealed the 3-dimensional structure of the epithelium and the membrane characteristics of deeper cells. Conclusion: The longer the time post-mortem, the greater the epithelial cell loss. However, a rim of peripheral cells remained, even after 7 days. The superficial cell layer showed signs of strong lateral attachment and broke up in a sheet-like fashion. The intercellular adhesion between deeper cells and adhesion between the basal cells and the basement membrane appeared to be weak post-mortem. The cell membrane structures of the remaining cells were surprisingly well retained. The clinical implication of the study is discussed.

Our experience with the use of topical anesthesia and adjustable sutures in a one-stage operation for treatment of strabismus is discussed, and selected cases are presented. In addition to using the method in recession, we have also developed a method for using adjustable sutures in resection of muscles. We have also found the method useful in reoperations and in operations on paretic muscles.

Conjunctival tissue was removed from eyes with and without pseudo-exfoliation material during cataract surgery. Cells derived from these samples were maintained in vitro. Confluent cultures were examined by transmission electron microscopy, and non-confluent as well as confluent cultures were examined for their ability to incorporate 3H-leucine and 3H-proline into TCA-precipitated material. Cells derived from both types of samples showed deposition of extracellular matrix, and a similar incorporation of the two amino acids. It is concluded that in vitro studies allow examination of basic metabolic patterns under varying conditions, and also a search for possible differences in such functions between tissues with and without production of pseudo-exfoliation material.

The epithelium was removed from human corneas, and samples of conjunctival tissue were cultured as explants on the denuded corneal surface for 1 and 2 weeks. Cells migrating from the conjunctival explants onto the corneal surface produced a multilayer where cells on the surface generally showed a flattened appearance. The apical membrane of these cells demonstrated villi as well as microplicae. Surface projections were also detected on cells in the deeper layers of the epithelium. Neighbouring cells were connected by junctional complexes. After 2 weeks, however, a lack of intercellular junctions in some areas resulted in the formation of intraepithelial cystoid spaces. Basal cells were connected to the underlying basement membrane by hemidesmo-somes. Although transdifferentiation of the cells into a corneal epithelium was not observed within the 2 weeks, the present system provides a tool for studies on factors affecting reepithelialization of corneal epithelial defects by conjunctival cells.

In a litter of seven three-week-old English cocker spaniels, four showed persistent pupillary membrane and congenital cataract. One affected male and two affected females were kept for breeding purposes. Brother/sister mating gave birth to 14 pups. Two pups died the first day. The other 12 were euthanased at an age of one year. None of them showed any sign of eye diseases either by clinical or histological examination. The parents, one male and two females, were euthanased at the age of 20, 24 and 33 months, respectively. Clinical and histological examination of the eyes showed varying degree of loss of vision, persistent pupillary membranes, microphthalmos and cataract.