Defining compartmentalized stem cell populations with distinct cell division dynamics in the ocular surface epithelium.

Adult tissues contain label-retaining cells (LRCs), which are relatively slow-cycling and considered to represent a property of tissue stem cells (SCs). In the ocular surface epithelium, LRCs are present in the limbus and conjunctival fornix; however, the character of these LRCs remains unclear, owing to lack of appropriate molecular markers. Using three CreER transgenic mouse lines, we demonstrate that the ocular surface epithelium accommodates spatially distinct populations with different cell division dynamics. In the limbus, long-lived Slc1a3CreER-labeled SCs either migrate centripetally toward the central cornea or slowly expand their clones laterally within the limbal region. In the central cornea, non-LRCs labeled with Dlx1CreER and K14CreER behave as short-lived progenitor cells. The conjunctival epithelium in the bulbar, fornix and palpebral compartment is regenerated by regionally unique SC populations. Severe damage to the cornea leads to the cancellation of SC compartments and conjunctivalization, whereas milder limbal injury induces a rapid increase of laterally expanding clones in the limbus. Taken together, our work defines compartmentalized multiple SC/progenitor populations of the mouse eye in homeostasis and their behavioral changes in response to injury.

Posttranscriptional regulation of MMP-9 by HuR contributes to IL-1ß-induced pterygium fibroblast migration and invasion.

Inflammation is considered to be critical in the pterygium progression and recurrence. However, the underlying molecular mechanism is not well understood. Herein, we investigated the potential role of RNA binding protein human antigen R (HuR) responsible for the impact of inflammation on pterygium development. The expression of HuR and matrix metallopeptidase-9 (MMP-9) in pterygium and normal conjunctiva was detected with immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The influence of interleukin-1ß (IL-1ß) on HuR expression and cellular distribution was determined with western blot and immunofluorescence. The pterygium fibroblast (PTF) migration was determined with scratch wound healing assay and Transwell migration assay. MMP-9 production was determined with qRT-PCR and gelatin zymography. The interaction between HuR and MMP-9 was investigated with RNP immunoprecipitation (IP) followed by RT-PCR and messenger RNA (mRNA) stability analysis. HuR and MMP-9 expression are elevated in pterygium, especially progressive pterygium compared with normal conjunctiva. IL-1ß could increase the expression and nucleus-cytoplasm shuttle of HuR in cultured PTFs. HuR mediated the stimulatory effect of IL-1ß on PTF migration and MMP-9 production. HuR bound to MMP-9 mRNA and in turn increased it stability. Our results suggest that posttranscriptional regulation of MMP-9 via stabilizing mRNA by HuR might contribute to the stimulatory effect of inflammatory factor IL-1ß on pterygium progression. These findings shed light on the pathogenesis of pterygium and provide a promising target for adjuvant treatment of pterygium.

Expression of Pluripotency Markers, SOX2 and OCT4, in Pterygium Development.

Pterygium is a common ocular surface disease characterized by the abnormal epithelial proliferation, matrix remodeling, vascularization and the migration of the lesion. Although the etiology of pterygium is elusive, recent studies have focused on the role of limbal stem cells (LSCs) damage and effects of UVB. This study aimed to determine the expression levels of pluripotent markers of SOX2 and OCT4 in primary pterygium and normal conjunctiva. Using real time polymerase chain reaction (PCR), the SOX2 and OCT4 expressions were compared in primary pterygium and normal conjunctiva. This study assessed the correlation between SOX2 mRNA expression and OCT4 mRNA expression, as well as the association between the clinicopathological indices and both gene expression levels. The relative mRNA expression levels of OCT4 genes in primary pterygium were significantly reduced compared to the normal conjunctiva tissues. The association between OCT4 gene expression and the clinicopathological indices reported significant laterality (P = .004) and marginal growth activity indices (P = .063). The univariate correlation between the SOX2 and OCT4 expressions was statistically significant (P = .001). The present study emphasized the downregulation of pluripotent marker OCT4 genes in the pterygium. It is speculated that these results may predict a new avenue for exploring the role of stem cell deficiency in the development of pterygium.

A wide temporal window for conjunctival papillae development ensures the formation of a complete sclerotic ring.

BACKGROUND: The conjunctival papillae are epithelial thickenings of the conjunctiva that are required for the induction of underlying bones (the scleral ossicles). These transient papillae develop and become inductively active over an extended temporal period (HH 30-36, 6.5-10 dpf). While their inductive capacity was discovered in the mid-1900s, little is known about their development. RESULTS: Through a series of timed surgical ablations followed by in situ hybridization for Bmp2, we show that the ring of conjunctival papillae is not altered if the conjunctival epithelium is ablated either prior to or shortly after papillae induction (i.e., HH 29-30, 6.5-7 dpf). A conjunctival papilla ablated at or prior to HH 34 (8 dpf), when the complete ring is present, regenerates and quickly becomes inductively active, inducing an underlying scleral condensation with only a slight delay. This regenerative capacity extends until HH 35.5, a full 36 hours beyond the normal timeline of papillae induction. As such, the period of epithelial competency for papilla induction is longer than previously identified. CONCLUSIONS: Papilla regeneration is a mechanism that ensures the formation of a complete sclerotic ring and provides another level of redundancy for the induction of a complete sclerotic ring during the normal inductive period. Developmental Dynamics 246:381-391, 2017. © 2017 Wiley Periodicals, Inc.

Gene expression analysis during the induction and patterning of the conjunctival papillae in the chick embryonic eye.

The induction and patterning of the conjunctival papillae (i.e. epithelial thickenings of the conjunctiva required for the induction of the underlying, neural crest-derived scleral ossicles) is complex. It takes place over a period of two days and follows a defined spatiotemporal sequence. In this study, we investigated the spatial and temporal expression pattern of four genes over seven morphological stages of development of these papillae. We show that ß-catenin is expressed during the pre-patterning of the epithelium prior to papilla induction and second that ß-catenin, Ednrb and Inhba are expressed during the induction and patterning of the conjunctival papillae. Furthermore, we identified two genes, ß-catenin and Prox1, that may be involved in the induction of the underlying scleral bones. These data provide an excellent baseline for future studies, setting the stage for functional studies aimed at examining the role of these genes in the patterning of the scleral ossicle system. This study also outlines the similarities between the conjunctival papillae and other placodes and may provide insights into the evolution and development of the conjunctival papillae.

TGFß signaling inhibits goblet cell differentiation via SPDEF in conjunctival epithelium.

The ocular surface epithelia, including the stratified but non-keratinized corneal, limbal and conjunctival epithelium, in concert with the epidermal keratinized eyelid epithelium, function together to maintain eye health and vision. Abnormalities in cellular proliferation or differentiation in any of these surface epithelia are central in the pathogenesis of many ocular surface disorders. Goblet cells are important secretory cell components of various epithelia, including the conjunctiva; however, mechanisms that regulate goblet cell differentiation in the conjunctiva are not well understood. Herein, we report that conditional deletion of transforming growth factor ß receptor II (Tgfbr2) in keratin 14-positive stratified epithelia causes ocular surface epithelial hyperplasia and conjunctival goblet cell expansion that invaginates into the subconjunctival stroma in the mouse eye. We found that, in the absence of an external phenotype, the ocular surface epithelium develops properly, but young mice displayed conjunctival goblet cell expansion, demonstrating that TGFß signaling is required for normal restriction of goblet cells within the conjunctiva. We observed increased expression of SAM-pointed domain containing ETS transcription factor (SPDEF) in stratified conjunctival epithelial cells in Tgfbr2 cKO mice, suggesting that TGFß restricted goblet cell differentiation directly by repressing Spdef transcription. Gain of function of Spdef in keratin 14-positive epithelia resulted in the ectopic formation of goblet cells in the eyelid and peripheral cornea in adult mice. We found that Smad3 bound two distinct sites on the Spdef promoter and that treatment of keratin 14-positive cells with TGFß inhibited SPDEF activation, thereby identifying a novel mechanistic role for TGFß in regulating goblet cell differentiation.

Development, alteration and real time dynamics of conjunctiva-associated lymphoid tissue.

PURPOSE: Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model. METHODS: Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy. RESULTS: Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min. CONCLUSIONS: CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.

Discontinuous LYVE-1 expression in corneal limbal lymphatics: dual function as microvalves and immunological hot spots.

LYVE-1(+) corneal lymphatics contribute to drainage and immunity. LYVE-1 is widely accepted as the most reliable lymphatic marker because of its continuous expression in lymphatic endothelium. LYVE-1 expression in corneal lymphatics has not been examined. In this study, we report intact CD31(+) corneal lymphatic capillary endothelial cells that do not express LYVE-1. The number of LYVE-1(-) gaps initially increased until 8 wk of age but was significantly reduced in aged mice. C57BL/6 mice showed a notably higher number of the LYVE-1(-)/CD31(+) lymphatic regions than BALB/c mice, which suggests a genetic predisposition for this histological feature. The LYVE-1(-) lymphatic gaps expressed podoplanin and VE-cadherin but not αSMA or FOXC2. Interestingly, the number of LYVE-1(-) gaps in FGF-2, but not VEGF-A, implanted corneas was significantly lower than in untreated corneas. Over 70% of the CD45(+) leukocytes were found in the proximity of the LYVE-1(-) gaps. Using a novel in vivo imaging technique for visualization of leukocyte migration into and out of corneal stroma, we showed reentry of extravasated leukocytes from angiogenic vessels into newly grown corneal lymphatics. This process was inhibited by VE-cadherin blockade. To date, existence of lymphatic valves in cornea is unknown. Electron microscopy showed overlapping lymphatic endothelial ends, reminiscent of microvalves in corneal lymphatics. This work introduces a novel corneal endothelial lymphatic phenotype that lacks LYVE-1. LYVE-1(-) lymphatic endothelium could serve as microvalves, supporting unidirectional flow, as well as immunological hot spots that facilitate reentry of stromal macropahges.

Conditional disruption of mouse Klf5 results in defective eyelids with malformed meibomian glands, abnormal cornea and loss of conjunctival goblet cells.

Members of the Krüppel-like family of transcription factors regulate diverse developmental processes in various organs. Previously, we have demonstrated the role of Klf4 in the mouse ocular surface. Herein, we determined the role of the structurally related Klf5, using Klf5-conditional null (Klf5CN) mice derived by mating Klf5-LoxP and Le-Cre mice. Klf5 mRNA was detected as early as embryonic day 12 (E12) in the cornea, conjunctiva and eyelids, wherein its expression increased during development. Though the embryonic eye morphogenesis was unaltered in the Klf5CN mice, postnatal maturation was defective, resulting in smaller eyes with swollen eyelids that failed to separate properly. Klf5CN palpebral epidermis was hyperplastic with 7-9 layers of keratinocytes, compared with 2-3 in the wild type (WT). Klf5CN eyelid hair follicles and sebaceous glands were significantly enlarged, and the meibomian glands malformed. Klf5CN lacrimal glands displayed increased vasculature and large number of infiltrating cells. Klf5CN corneas were translucent, thicker with defective epithelial basement membrane and hypercellular stroma. Klf5CN conjunctiva lacked goblet cells, demonstrating that Klf5 is required for conjunctival goblet cell development. The number of Ki67-positive mitotic cells was more than doubled, consistent with the increased number of Klf5CN ocular surface epithelial cells. Co-ablation of Klf4 and Klf5 resulted in a more severe ocular surface phenotype compared with Klf4CN or Klf5CN, demonstrating that Klf4 and Klf5 share few if any, redundant functions. Thus, Klf5CN mice provide a useful model for investigating ocular surface pathologies involving meibomian gland dysfunction, blepharitis, corneal or conjunctival defects.

Mouse conjunctival forniceal gene expression during postnatal development and its regulation by Kruppel-like factor 4.

PURPOSE: To identify the changes in postnatal mouse conjunctival forniceal gene expression and their regulation by Klf4 during the eye-opening stage when the goblet cells first appear. METHODS: Laser microdissection (LMD) was used to collect conjunctival forniceal cells from postnatal (PN) day 9, PN14 and PN20 wild-type (WT), and PN14 Klf4-conditional null (Klf4CN) mice, in which goblet cells are absent, developing, present, and missing, respectively. Microarrays were used to compare gene expression among these groups. Expression of selected genes was validated by quantitative RT-PCR, and spatiotemporal expression was assessed by in situ hybridization. RESULTS: This study identified 668, 251, 1160, and 139 transcripts that were increased and 492, 377, 1419, and 57 transcripts that were decreased between PN9 and PN14, PN14 and PN20, PN9 and PN20, and PN14 WT and Klf4CN conjunctiva, respectively. Transcripts encoding transcription factors Spdef, FoxA1, and FoxA3 that regulate goblet cell development in other mucosal epithelia, and epithelium-specific Ets (ESE) transcription factor family members were increased during conjunctival development. Components of pathways related to the mesenchymal-epithelial transition, glycoprotein biosynthesis, mucosal immunity, signaling, and endocytic and neural regulation were increased during conjunctival development. Conjunctival Klf4 target genes differed significantly from the previously identified corneal Klf4 target genes, implying tissue-dependent regulatory targets for Klf4. CONCLUSIONS: The changes in gene expression accompanying mouse conjunctival development were identified, and the role of Klf4 in this process was determined. This study provides new probes for examining conjunctival development and function and reveals that the gene regulatory network necessary for goblet cell development is conserved across different mucosal epithelia.

The effect of autologous serum eye drops on the conjunctivalization over exposed porous polyethylene orbital implant (Medpor(®)) in the rabbit model.

PURPOSE: To assess the efficacy of autologous serum eye drops (AS) in enhancing conjunctivalization over a scleral patch graft on exposed porous polyethylene orbital implant (Medpor(®)). METHODS: Eighteen rabbits were underwent evisceration, implantation of Medpor(®) and homologous scleral patch graft. The conjunctival edge was sutured leaving 5 mm-diameter circular defect. In group A, 20% AS were applied 8 times a day and only balanced salt solution was applied in group B. Digital anterior segment photography was obtained 1, 3, 7, 10, 14, and 21 days after surgery and analyzed with Image Pro Plus(®) software. RESULTS: In the early postoperative period, the average conjunctival growth rate (mm(2)/day) was faster in group A, but failed to show statistical significance (3.79 vs. 3.03, p = 0.26 in 1~3days, 2.39 vs. 1.80, p = 0.59 in 4- days, 0.03 vs. -0.02, p = 0.94 in 7-10 days, Mann-Whitney test). The complete healing rate was higher (67% vs. 56%, p = 0.5, Fisher's exact test) and the mean elapsed time for complete healing (days) was shorter in group A (17.3 vs. 18.2, p = 0.83, Mann-Whitney test), but did not show a statistically significant difference. CONCLUSION: In a rabbit model, 20% AS did not appear to facilitate the healing of small conjunctival defects of exposed porous orbital implant.

Effect of hypothyroidism on postnatal conjunctival development in rats.

AIM: To study the conjunctival development in hypothyroid genetically epilepsy-prone rats (GEPRs) with serum T3 and T4 significantly lower than in normal rats. METHODS: A structural, ultrastructural and histochemical study on the conjunctival epithelium of GEPRs and of control Sprague-Dawley (SD) rats before and after eyelid opening, with particular regard to goblet cell differentiation. RESULTS: From birth to day 12, no goblet cells were demonstrated on the conjunctival surface of both strains, so that the epithelium was formed only by a cuboidal basal layer and by a superficial layer of roundish or flattened cells. On day 16, after the eyelid opening, Alcian blue (AB)-positive goblet cells filled with homogeneous granules were demonstrated isolated, in GEPRs, or clustered, in SD rats, in both the fornices and palpebral conjunctiva. The epithelium showed a basal layer and many layers of flattened cells and was taller in SD rats (8-10 layers) than in GEPRs (6-7 layers). At 3 months, the epithelium in SD rats was higher with generally clustered goblet cells, whilst in GEPRs goblet cells were both isolated or clustered. In both strains, the goblet cells showed a marked AB/periodic acid-Schiff positivity all over the conjunctival surface and were filled with granules of different density. In both strains, goblet cells were absent at birth and their appearance, as AB-positive cells, was concomitant with eyelid opening. CONCLUSIONS: Hypothyroid rats showed a conjunctival development different than that of normothyroid rats for both epithelial and goblet cells. It appears that thyroid hormone imbalance may influence conjunctival development.

Proliferative and migratory aptitude in pterygium.

Pterygium is a chronic fibrovascular overgrowth on the corneal surface and is often associated with inflammation, astigmatism and obstructed vision. The common treatment is surgical removal but post-operative recurrences with more aggressive behavior are common. However, there is a controversy in the pathogenesis of primary pterygium between limbal stem cell failure versus proliferation. In this study, we explore the proliferative and migratory aptitude in pterygium by characterizing the growth and migration pattern of pterygial cells in the head (on the cornea), the neck (over the focal limbus), and the body (on the conjunctiva) epithelia of 12 full-length primary pterygia. Immunofluorescence and quantification analyses showed a spatial expression pattern of markers for stem cells, cell growth, and matrix metalloproteinases. Beside the basal epithelia in all three regions, p63α(strong) cells were located in suprabasal layers in head, weak in the body and absent in neck. Pertinent cell proliferation in head than body epithelia was revealed by its higher colony-forming efficiency. ATP-binding cassette transporter glycoprotein family member-2 and cytokeratin-15 were found mainly in the body basal epithelia, similar to that in normal conjunctiva. Much fewer proliferating stem-like cells in the neck region supported the limbal failure as a cause of pterygium formation. Pax6, matrix metalloproteinase-2 and -9 were more expressed in the head than in the other two regions. Our results indicate the importance of pterygium head in tissue growth and invasion and its likely involvement in post-operation recurrence.

FGF-regulated BMP signaling is required for eyelid closure and to specify conjunctival epithelial cell fate.

There are conflicting reports about whether BMP signaling is required for eyelid closure during fetal development. This question was addressed using mice deficient in BMP or TGFbeta signaling in prospective eyelid and conjunctival epithelial cells. Genes encoding two type I BMP receptors, the type II TGFbeta receptor, two BMP- or two TGFbeta-activated R-Smads or the co-Smad Smad4 were deleted from the ocular surface ectoderm using Cre recombinase. Only mice with deletion of components of the BMP pathway had an 'eyelid open at birth' phenotype. Mice lacking Fgf10 or Fgfr2 also have open eyelids at birth. To better understand the pathways that regulate BMP expression and function during eyelid development, we localized BMPs and BMP signaling intermediates in Fgfr2 and Smad4 conditional knockout (CKO) mice. We found that Fgfr2 was required for the expression of Bmp4, the normal distribution of Shh signaling and for preserving the differentiation of the conjunctival epithelium. FGF signaling also promoted the expression of the Wnt antagonist Sfrp1 and suppressed Wnt signaling in the prospective eyelid epithelial cells, independently of BMP function. Transcripts encoding Foxc1 and Foxc2, which were previously shown to be necessary for eyelid closure, were not detectable in Smad4(CKO) animals. c-Jun, another key regulator of eyelid closure, was present and phosphorylated in eyelid periderm cells at the time of fusion, but failed to translocate to the nucleus in the absence of BMP function. Smad4(CKO) mice also showed premature differentiation of the conjunctival epithelium, conjunctival hyperplasia and the acquisition of epidermal characteristics, including formation of an ectopic row of hair follicles in place of the Meibomian glands. A second row of eyelashes is a feature of human lymphedema-distichiasis syndrome, which is associated with mutations in FOXC2.

Ex vivo growth of rabbit bulbar, fornix and palpebral conjunctival epithelia in a serum-free and feeder layer-free culture system.

This study was conducted to explore the feasibility of culturing conjunctiva epithelial cells in serum-free and feeder layer-free culture system with regard to the cell morphology and immunocytochemistry of the rabbit bulbar, fornix and palpebral conjunctiva epithelia. The results showed that epithelium cells from all the three conjunctiva regions can be cultured in a serum-free and feeder layer-free environment. We obtained highest epithelial growth from fornix region with minimum invasion of fibroblast cells compared to other area. All cultured cells were stained positive for cytokeratin 19 and MUC5AC and negative for cytokeratin 3. These findings suggested that fornix was a better source of cells for the development of tissue engineered conjunctiva for future clinical application.

Conjunctival expansion using a subtenon's silicone implant in New Zealand white rabbits.

PURPOSE: In the field of ophthalmology, the conjunctival autograft is a useful therapeutic material in many cases, but the small size of the autograft is a disadvantage. Therefore, we evaluated the feasibility of taking an expanded sample of conjunctival tissue using a subtenon's silicone implant. MATERIALS AND METHODS: We included a total of nine rabbits; eight rabbits were operative cases, and one was a control. A portion of conjunctival tissue from the control rabbit, which did not undergo surgery, was dissected and examined to determine whether it was histologically different from the experimental group. The surgical procedure was performed on eight rabbits via a subtenon's insertion of a silicone sponge in the left superior-temporal portion; after surgery, we dropped antibiotics into the eyes. We sacrificed a pair of rabbits every three days (on days 3, 6, 9, and 12) after surgery, removed the expanded conjunctival tissues with the silicone sponge implants, and measured their sizes. RESULTS: The mean size of the expanded conjunctival tissues was 194.4 mm2. On the third day, we were able to harvest a 223.56 mm2 section of conjunctival tissue, which was the most expanded sample of tissue in the study. On the twelfth day, we removed a 160.38 mm2 section of conjunctival tissue, which was the least expanded sample of tissue. Statistically, there were no significant differences in the mean dimensions of the expanded conjunctival tissues for each time period. Microscopic examinations showed no histological differences between the expanded conjunctival tissues and the normal conjunctival tissues. CONCLUSION: The results reveal that this procedure is a useful method to expand the conjunctiva for grafting and transplantation.

Future design of a new keratoprosthesis. Physical and biological analysis of polymeric substrates for epithelial cell growth.

One of the main issues in the development of new biocolonizable materials is to understand the influence of the synthetic material on the biological response in terms of cellular adhesion, proliferation, and differentiation. In this study, we characterized different polymeric materials (with different hydrophobicity/hydrophilicity ratios and electrical charges) using dynamic-mechanical analysis, equilibrium water content, and surface energy. Cell adhesion, viability, morphology, and proliferation studies were conducted with these materials using a conjunctival epithelial cell line (IOBA-NHC). The biological data regarding physicochemical parameters of the materials were also correlated. When conjunctival epithelial cells were grown on poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymers, P(EA-co-HEA), samples with up to 20% hydrophilic groups on their polymeric chain showed adhesion, viability, and proliferation, although these three factors decreased as the hydrophilic group content increased. The poly(ethyl acrylate-co-methacrylic acid) 90/10 copolymer, P(EA-co-MAAc) 90/10, showed better results than poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymers and were even better than tissue control polystyrene (TCPS). This feature is explained by the presence of electrical charges on the surface of the poly(ethyl acrylate-co-methacrylic acid) 90/10 copolymer. The fact that the ionic groups are configured in domains structured in nanophases as happens in this copolymer improves cell adhesion even further.

Conjunctival Expansion Using a Subtenon's Silicone Implant in New Zealand White Rabbits

PURPOSE: In the field of ophthalmology, the conjunctival autograft is a useful therapeutic material in many cases, but the small size of the autograft is a disadvantage. Therefore, we evaluated the feasibility of taking an expanded sample of conjunctival tissue using a subtenon's silicone implant. MATERIALS AND METHODS: We included a total of nine rabbits; eight rabbits were operative cases, and one was a control. A portion of conjunctival tissue from the control rabbit, which did not undergo surgery, was dissected and examined to determine whether it was histologically different from the experimental group. The surgical procedure was performed on eight rabbits via a subtenon's insertion of a silicone sponge in the left superior-temporal portion; after surgery, we dropped antibiotics into the eyes. We sacrificed a pair of rabbits every three days (on days 3, 6, 9, and 12) after surgery, removed the expanded conjunctival tissues with the silicone sponge implants, and measured their sizes. RESULTS: The mean size of the expanded conjunctival tissues was 194.4mm(2). On the third day, we were able to harvest a 223.56mm(2) section of conjunctival tissue, which was the most expanded sample of tissue in the study. On the twelfth day, we removed a 160.38mm(2) section of conjunctival tissue, which was the least expanded sample of tissue. Statistically, there were no significant differences in the mean dimensions of the expanded conjunctival tissues for each time period. Microscopic examinations showed no histological differences between the expanded conjunctival tissues and the normal conjunctival tissues. CONCLUSION: The results reveal that this procedure is a useful method to expand the conjunctiva for grafting and transplantation.

The structure of the human semilunar plica at different stages of its development--a morphological and morphometric study.

In this study development of the semilunar plica was examined histologycally by making sections through the eyes of eleven foetuses at different stages of gestation, two newborns and an old man. We found that in the early stages of its development the semilunar fold covered a bigger part of the orbit and later did not keep up with the growth of the eyeball and the lids. In its development three different kinds of germinal glands could be seen in the semilunar plica. Beside poorly differentiated buds of the surface epithelium which can be classified as rudiments of the nictitating or Harderian gland, serous glands were detected which could be beginnings of Krause's glands. Additionally, a new kind of plica gland was identified in which ductular structures, with an onion skin appearance, could be discriminated from mucous acini. The surface of the plica developed slowly by an increase in the layers of its epithelium as well as by the maturation of the epithelial cells from a two-layered cuboidal to a multi-layered cylindrical epithelium. In general the palpebral side of the plica consisted of a higher number of epithelial layers and more goblet cells than its bulbar side. Moreover, the surface of the palpebral side appeared more irregular and enlarged by numerous pleatings of its epithelium. The initially loose mesenchymal connective tissue was soon condensed by firstly an increase of the number of cells and later by an increase in the fibre density. In the tight collagenous connective tissue no elastic or reticular fibres or cartilaginous structures could be found. Very dense vascularization of the plica semilunaris was seen early in development. The blood vessels rising from the root of the plica divided themselves into a central and a subepithelial vascular net. Later on, some of them showed an enlarged lumen and were covered by a thin layer of muscle cells. A few unmyelinated neurofibres were found next to blood vessels and glands. Muscle cells could not be detected. A very dense concentration of leukocytes in the plica even in early intrauterine development was very striking and could be observed in this kind for the first time. These were mostly lymphoplasmocytic elements but granulocytes and macrophages could also be seen around the blood vessels and in the subepithelial area where they were arranged as follicles. Some of these cells even passed through the epithelium and could be found in the conjunctival fissure. The discovery of dense infiltration with both specific and non-specific immune cells, abundant vascularization and secretory structures (goblet cells and surface enlargement) in the semilunar plica suggest that it plays an important role as a specialized organ in human eye protection. Its anatomical position at the medial border of the eye supports this theory. Its origin from the third eyelid of the mammals could be recognized to a certain extent.

Proliferation and apoptosis in the epithelium of the developing human cornea and conjunctiva.

To determine the distribution of proliferating and apoptotic cells in the human cornea during prenatal and early postnatal development, we examined sections of the bulbar conjunctiva, the limbus as well as the central and peripheral cornea between 11 weeks of gestation and 6 months after birth. The objective was to localize dividing cells by proliferating cell nuclear antigen-like immunoreactivity (PCNA-LI) and apoptotic cells by terminal transferase-mediated nick-end labeling (TUNEL). Before the 17th gestational week, PCNA-LI was absent in all 4 regions examined, indicating negligible cell proliferation during early development. After 20 weeks, strong PCNA-labeling was observed in all regions examined suggestive of high proliferative activity not only in the limbus and the bulbar conjunctiva, but also in the central and peripheral cornea. This rise in proliferative activity was followed by a steady decline: after 28 weeks, anti-PCNA staining gradually disappeared in the central and peripheral cornea, so that, at 6 months after birth, it was confined to the limbus and the bulbar conjunctiva, resembling the picture described for the adult cornea. TUNEL-positive cells were virtually absent in all 4 regions examined before the 38th gestational week. Apoptotic cells only started to appear at 38 weeks; at this stage, they were confined to the bulbar conjunctival epithelium. At 6 months after birth, TUNEL-positive cells were observed in the bulbar conjunctival epithelium and the entire cornea; the limbus, however remained devoid of apoptotic cells throughout the entire prenatal and early postnatal period. The present study for the first time localizes proliferating and apoptotic cells in the epithelium of the developing human cornea. Three stages of development can be distinguished: Minimal proliferation (until 17th week), vigorous proliferation over the entire cornea including the limbus and the bulbar conjunctiva (until 28th week) and gradual decrease in proliferative activity (after 28th week) accompanied by the appearance of apoptotic cells.