The labelling index is not always reliable. Discrepancies between the labelling index and the mitotic rate in the rat corneal epithelium after intraperitoneal and topical administration of tritiated thymidine and colcemid

Many cell kinetic studies are based on the assumption that tritiated thymidine injected into an animal is available for incorporation into DNA for only a short time, and that it labels all cells in the S phase. The present study indicates that this is not the fact for the rat corneal epithelium. The labelling index (LI) declines considerably from the limbal area to the central cornea, while the mitotic rate is almost constant all over the corneal epithelium. The LI should therefore not be used as the only criterion in the assessment of proliferation rate.     

Circadian variations in the DNA synthesis of the rat corneal epithelium. A study using double labelling with tritiated thymidine

A prominent circadian rhythm was found in the labelling indices (LI) of the peripheral rat corneal epithelium and of the adjacent conjunctival epithelium, while almost no diurnal variation was found in the central area. Application of a double labelling technique indicated that there are rhythmic pulses of high and low influx of cells into the S phase and similar pulses of efflux of cells from the S phase. Results of the study indicate that there are different cohorts of cycling cells all over the rat corneal epithelium. Cells belonging to a rapidly proliferating cohort are observed in the peripheral cornea. There is a gradual reduction in the fraction of labelled DNA-synthesizing cells towards the centre. The considerably lower fraction of cells taking up tritiated thymidine (3H)TdR in the central cornea may be due to a higher fraction of basal cells having reached higher levels of differentiation. This may result in a shift from the salvage to the de novo pathway. The slowly proliferating cohort seems to have a prolonged S phase duration and displays practically no diurnal variation in the LI. The DNA-synthesizing cells belonging to this latter cohort probably use the salvage pathway for DNA synthesis resulting in uptake of (3H)TdR all over the cornea. The LI is thus not a reliable indicator of cell proliferation in the corneal epithelium, due both to the heterogeneity of the cell proliferation, and in particular due to the lack of labelling of the centrally located DNA-synthesizing cells. To what extent these properties may also be present in other proliferating tissues with different levels of differentiations, may be questioned.     

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.     

Biosynthesis of glycosaminoglycans by the separated tissues of the embryonic chick cornea

Corneal tissues (epithelium, endothelium, and stroma) were isolated from chick embryos at 14, 17, and 20 days of incubation and immediately labeled in vitro with d-[6-³H]glucosamine and H₂³⁵SO₄. Amount of label incorporated into each type of glycosaminoglycan or into glycopeptides was determined by specific degradative techniques, in conjunction with gel filtration chromatography. Results suggested that corneal epithelium synthesized little, if any, corneal keratan sulfates, but that corneal endothelium may have synthesized small amounts of corneal keratan sulfates. Nearly all corneal keratan sulfates were derived from the stroma. Corneal heparan sulfates appeared to be derived predominantly from corneal epithelium at later stages of development. Corneal endothelium contributed large proportions of the hyaluronic acids of the cornea. Only epithelium produced a large proportion of sulfated glycoproteins. In addition, epithelium synthesized a large proportion of a sulfated, high molecular weight polysaccharide which was resistant to treatments degrading known types of glycosaminoglycans. Each corneal tissue may not only affect corneal morphogenesis directly by contributing a unique spectrum of glycosylated proteins to the extracellular matrix, but also may regulate the extracellular matrix composition indirectly by modulating the biosynthetic activities of the other corneal tissues.     

The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye

During the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium.     

Localization of DNA-synthesizing cells and cell proliferation pattern in developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens

Localization of DNA-synthesizing cells in the developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens was investigated. DNA-synthesizing cells were scattered throughout the proventricular epithelium during all developmental stages studied. The results indicate that there is no clear proliferative zone in the proventricular epithelium of the chicken. The labeling indices (LI) of proventricular epithelial cells were measured. On the 6.5th day of incubation, the LI of glandular epithelium reached 29.5 ± 1.5%. the highest value of all the stages studied. This extremely rapid cell proliferation can be considered to be a driving force for the elongation of the proventricular glands during the following stages. Just after hatching, the LI of both the glandular and luminal (non-glandular) epithelia significantly increased from those on the 18th day of incubation. It is suggested that the rise in LI possibly reflected proventricular growth to fit in the change in the method of nourishment after hatching. In 2 week old chickens, the LI of both the glandular and luminal epithelia were reduced to approximately 1%. The active production of embryonic chicken pepsinogen in all glandular epithelial cells of the embryonic chicken revealed that proliferation and differentiation are not necessarily exclusive during the embryonic stages of proventricular development.     

Basonuclin in murine corneal and lens epithelia correlates with cellular maturation and proliferative ability

Basonuclin is a zinc finger protein with highly restricted tissue distribution. It has been found in abundance only in keratinocytes of stratified epithelia and the germ cells of the testis and ovary. We studied the expression pattern of basonuclin in relation to cellular proliferation and differentiation in murine corneal and lens epithelia, two self-renewing tissues in the eye which contain cells that proliferate throughout life. Mouse corneal and lens epithelial cells at various stages of development were labeled with BrdU for 90 min to detect cells in S phase and to establish proliferative rates. Whole eyes of mouse or rat were processed for frozen sections and cellular basonuclin was detected by either a rabbit antimouse- or a rabbit anti-human-basonuclin antibody. Basonuclin was expressed in virtually all cells in the basal layer of corneal epithelium and in the pre-equatorial lens epithelium, the respective proliferative compartments of adult corneal and lens epithelia. Basonuclin expression in corneal epithelium began at post-natal life day 4, first in a few cells and then spread to virtually all basal cells at day 20. Basonuclin was consistently absent in limbal epithelium. Lens basonuclin, which was detected earlier than that of the cornea, was confined to the pre-equatorial epithelium and was absent in equatorial cells that expressed p57KIP2, an early differentiation marker for these cells. An important distinction between corneal and lens basonuclin is that the former is predominantly nuclear whereas the latter cytoplasmic.     

Changes In the Dna Labelling Index After β Irradiation of the Mouse Epidermis

This study looked at the changes in the interfollicular DNA labelling index (LI) with time after strontium-90/yttrium-90 β irradiation of approximately 100 mm² of mouse flank skin, after a dose of 100 Gy which produces transitory moist desquamation. Within 24 hr of such a dose the LI of the irradiated area was essentionally zero (0.07 ± 0.03%), whilst those of the side area and of the control area were 15.0 ± 2.6% and 21.4 ± 2.7%, respectively. the LI of the side and the control areas then fell within 3–5 days to approximately 4% and approximately 2% respectively, whilst that of the irradiated area rose rapidly to a peak value of 30.2 ± 1.7% at 10 days post-irradiation. There was a 20% reduction in the diameter of the area with detectable radiation damage within 5 days, and this is primarily due to cell proliferation and migration from the unirradiated margins of the field. In contrast, between days 10 and 20 the major source of repopulation is probably derived from local migration and proliferation of surviving hair follicle basal cells within the irradiated field.     

A comparison of lyophilized amniotic membrane with cryopreserved amniotic membrane for the reconstruction of rabbit corneal epithelium

Many researchers have employed cryopreserved amniotic membrane (CAM) in the treatment of a severely damaged cornea, using corneal epithelial cells cultured on an amniotic membrane (AM). In this study, two Teflon rings were made for culturing the cells on the LAM and CAM, and were then used to support the AM, which is referred to in this paper as an Ahn’s AM supporter. The primary corneal epithelial cells were obtained from the limbus, using an explantation method. The corneal epithelium could be reconstructed by culturing the third-passage corneal epithelial cells on the AM. A lyophilized amniotic membrane (LAM) has a higher rate of graft take, a longer shelf life, is easier to store, and safer, due to gamma irradiation, than a CAM. The corneal epithelium reconstructed on the LAM and CAM, supported by the two-Teflon rings, was similar to normal corneal epithelium. However, the advantages of the LAM over that of the CAM make the former more useful. The reconstruction model of the corneal epithelium, using AM, is considered as a goodin vitro model for transplantation of cornel epithelium into patients with a severely damaged cornea.     

Reconstruction of a rabbit corneal epithelium on a lyophilized amniotic membrane using tilting air-liquid interface culture followed by tilting submerged culture

Herein, we reconstructed a rabbit corneal epithelium on a lyophilized amniotic membrane (LAM) using a modified version of two Teflon rings (the Ahn’s supporter). We compared the corneal epithelial cells we had differentiated in vitro using air-liquid interface (6 days, 12 days) and submerged (6 days, 12 days) cultures and followed a six-day tilting dynamic air-liquid interface culture with a six-day tilting submerged culture. We characterized the reconstructed corneal epithelium using digital photography, histological imaging, and transmission electron microscopy. The reconstructed corneal epithelium created under air-liquid interface culture exhibited a healthier basal corneal epithelial layer than that created under submerged culture. The reconstructed corneal epithelium on the LAM that was produced using the tilting dymanic culture exhibited a healthy basal layer. We therefore proposed that tilting submerged culture not only supplied nutrients from the medium to the corneal epithelial cells on the LAM, but it also removed the horny layer in the upper part of the reconstructed corneal epithelium, presumably by mimicking the effects of blinking. This study demonstrated that corneal epithelium reconstruction on a LAM using a tilting submerged culture after a tilting air-liquid interface culture may be useful not only for allogeneic or autologous transplantation, but also for in vitro toxicological test kits.     

Labelling index of human squamous cell carcinomas. Comparison of in vivo and in vitro labelling methods.

In vivo and in vitro labelling has been compared in sixteen human solid squamous cell carcinomas (ENT). The median in vivo/in vitro LI ratio was 1-2, but for two-thirds of the patients it was only 1-1, suggesting a slight LI underestimation in vitro. Two factors can possibly explain the divergences: heterogeneity from one biopsy to another in the same tumour, and lower mean grain count in the deep cell layers of the in vitro labelled tumour samples. Therefore, the fact we did not find a good agreement between in vivo and in vitro data for one-third of the tumours points out that one must be cautious in considering in vitro LI as a valid result for a given patient. However, even if the in vitro LI leads to a certain underestimate, it can provide useful data.     

The cell kinetics of the epidermis and follicular epithelium of the rat: variations with age and body site

The skin from rats of differing age was used to quantify variations in the cell kinetics of the epidermis and the follicular epithelium of different body sites. Four parameters were assessed, namely the basal cell density (BCD), the labelling index (LI), the duration of DNA synthesis (ts) and the basal cell turnover time (tT). The BCDs of the epidermis of the dorsum and the upper surface of the foot were similar in rats of 7, 14 and 52 weeks of age, but there was an indication of a progressive decline with increasing age in the BCD of the epidermis of the ear and tail. There were no age-related changes in the length of ts in any of the four body regions. The rate of cell proliferation, as indicated by the values of the LI and tT, was relatively rapid in the epidermis of the dorsum, foot and tail of rats aged 7 weeks (LI greater than 12%; tT less than 80 h). In rats aged 14 weeks this rate of proliferation was maintained in the epidermis of the dorsum. However, in the foot and tail the rate of cell proliferation was decreased (LI less than 10%; tT greater than 85 h). A fall in the rate of proliferation of the epidermis of the dorsum was only seen in 52-week-old animals. In these animals the rates of proliferation in the foot and tail were similar to those at the age of 14 weeks. In the epidermis of the ear there was no appreciable change in the rate of cell proliferation with age. The values of the cell kinetic parameters varied in the different body sites. For example, in 52-week-old animals values for tT were relatively short in the epidermis of the tail and foot and appreciably longer in the epidermis of the dorsum and ear. Considered overall, values for the cell kinetic parameters of the epidermis were comparable with those for the follicular epithelium. The only major differences between the epidermis and the follicular epithelium were in the upper surface of the foot at 7 weeks of age, and in the tail at 7 and 14 weeks of age, where the LI was higher and the tT shorter in the epidermis than in the follicular epithelium. The relevance of the observed age- and body-site-related variations in the cell kinetics of the epidermis are discussed in relation to previously described differential changes in the radiosensitivity of the skin in this strain of rat.     

Dkk2 plays an essential role in the corneal fate of the ocular surface epithelium

The Dkk family of secreted cysteine-rich proteins regulates Wnt/beta-catenin signaling by interacting with the Wnt co-receptor Lrp5/6. Here, we show that Dkk2-mediated repression of the Wnt/beta-catenin pathway is essential to promote differentiation of the corneal epithelial progenitor cells into a non-keratinizing stratified epithelium. Complete transformation of the corneal epithelium into a stratified epithelium that expresses epidermal-specific differentiation markers and develops appendages such as hair follicles is achieved in the absence of the Dkk2 gene function. We show that Dkk2 is a key regulator of the corneal versus epidermal fate of the ocular surface epithelium.     

Circadian variation in the mitotic rate of the rat corneal epithelium. Cell divisions and migration are analyzed by a mathematical model

A stathmokinetic method was used to study the diurnal variation in the mitotic rate (MR) of the rat corneal epithelium, and in the adjacent conjunctival epithelium. A prominent circadian variation in cell proliferation was observed in both epithelia, both showing almost the same pattern, which may indicate that both tissues are submitted to the same regulatory mechanisms. The average rate of cell renewal during a 24 h period indicated a mean cell renewal time of 12.3 days. This is longer than previously assumed. The MR declined toward the central cornea. Based on the above observations and the known centripetal migration of cells in the corneal epithelium, we have developed a mathematical model showing isomorphism with the renewal of the corneal epithelium.     

Epithelial Thickness and Labelling Index in Young and Old Mice1

The Labelling Index (L.I.), thickness and number of cell layers in the stratum corneum of various epithelia of groups of eight-week-old and two-year-old mice were compared. Arbitrarily selected subgroups of 5 animals were injected with 1μ Ci/gm body weight of ³H-TdR at 6 hourly intervals. Two hours after injection, specimens of skin from ear and footpad and of mucosa from tongue and palate were removed and either prepared for autoradiography or frozen for cryotomy and histological assessment. Most tissues showed a significant diurnal variation in L.I. In the older animals the L.I. was lower in all tissues except the palate (no change) and ear epidermis (higher). The thickness of the epithelium of the old animals was less in the palate but greater in the ear and footpad. The number of cell layers of the stratum corneum, when visualized by alkaline expansion, was unchanged in the footpad but significantly increased in the ears of the old mice.     

Patterns of cytokeratin and vimentin expression in the human eye

We studied the expression of the various cytokeratin (CK) polypeptides and vimentin in tissues of the human eye by applying immunocytochemical procedures using a panel of monoclonal antibodies as well as by performing biochemical analyses of microdissected tissues. Adult corneal epithelium was found to contain significant amounts of the cornea-specific CKs nos. 3 and 12 as well as CK no. 5, and several additional minor CK components. Among these last CKs, no. 19 was found to exhibit an irregular mosaic-like staining pattern in the peripheral zone of the corneal epithelium, while having a predominantly basal distribution in the limbal epithelium. Both the fetal corneal epithelium and the conjunctival epithelium were uniformly positive for CK no. 19. In the ciliary epithelium, co-expression of CKs nos. 8 and 18 and vimentin was detected, whereas in the retinal pigment epithelium, CKs nos. 8 and 18 were dominant. The present data illustrate the remarkable diversity and complexity of CK-polypeptide expression in the human eye, whose significance with respect to histogenetic and functional aspects is, as yet, only partially clear. The unusual distribution of CK no. 19 in different zones of the corneal epithelium may be related to the specific topography of corneal stem cells. The occurrence of the expression of simple-epithelium CKs in the ciliary and pigment epithelium demonstrates that, despite their neuroectodermal derivation, these are true epithelia.     

Basement membrane components in healing rabbit corneal epithelial wounds: immunofluorescence and ultrastructural studies

The nature of the substrate that supports epithelial migration in vivo is of interest, particularly with respect to mechanisms of wound healing. Immunofluorescence and electron microscopy were used to search for common substrate components in prototype rabbit corneal wounds: epithelial scrape wounds, in which the corneal or conjunctival epithelium migrated over the denuded lamina densa of the corneal basement membrane (CBM), and superficial keratectomy, in which the corneal epithelium migrated over a bare stroma without CBM. The corneal epithelium moved rapidly over the CBM or stroma to cover the defect within 2-3 d, whereas the conjunctival epithelium required 1-2 wk. In all wounds, fibronectin and fibrin/fibrinogen were deposited onto the bare surface within 8 h after wounding and persisted under the migrating epithelium until migration was complete. Bullous pemphigoid antigen (BPA), a normal component of the CBM, was removed with the epithelium upon scrape wounding and reappeared in the CBM after migration was completed. In contrast, the conjunctival epithelium had a continuous subepithelial band of BPA out to the migrating tip. Laminin, also a normal component of the CBM, was not removed in the scrape wounds, indicating that the region of least resistance to shear stress was between the BPA and laminin layers. Laminin was removed by superficial keratectomy and was not detectable under the leading edge of the migrating cells. Laminin and BPA were restored in the CBM by 2-4 wk. Type IV collagen could not be detected in normal CBM, but was conspicuously present in conjunctival basement membrane and in blood vessels. Focal bands of type IV collagen did appear in the newly synthesized CBM 2-4 wk after keratectomy. These results argue that BPA, laminin, and type IV collagen are not essential for the migration of corneal epithelium during wound healing and support the hypothesis that fibronectin and fibrin/fibrinogen are the common, perhaps the essential, components of the provisional matrix that serves as a substrate until the permanent attachment components are regenerated.     

Potential transformation of the anterior corneal epithelium of the common frog]

The possibility of transformation of the corneal anterior epithelium in the lens following its separation from the stroma was studied. The corneal epithelium was implanted into: a) empty eye orbit and b) cavity of lensless eye of the Rana temporaria tadpoles. In the eye orbit it continued, as in the normal development, to form the basal membrane. Although in the eye cavity the structures similar to lentoids arose but the specific lens proteins were shown to be asbent from them using immunofluorescence. In both the cases, thus, no transformation of the corneal epithelium in the lens was observed. The role of stroma in the stabilization of differentiation of the corneal anterior epithelium is discussed. It is suggested that the absence of increase in the mitotic activity is one of the causes of failure of the corneal epithelium transformation in the lens.     

EPITHELIAL COLLAGENS AND GLYCOSAMINOGLYCANS IN THE EMBRYONIC CORNEA : Macromolecular Order and Morphogenesis in the Basement Membrane

The embryonic corneal epithelium synthesizes both collagen and chondroitin sulfate and excretes them across the basement membrane into the subepithelial space where they assemble into a spiraling orthogonal matrix of fibrils. The assembly of collagen into fibrils is first apparent at the outer face of the basement membrane in a region of ordered chondroitin sulfate molecules. Hyaluronate, another morphogenetically important corneal macromolecule, is produced at these early stages only by the inner endothelium. These correlated biosynthetic and ultrastructural data demonstrate discrete macromolecular products of the two corneal epithelia with differing morphogenetic properties and functions.     

Corneal epithelial cell fate is maintained during repair by Notch1 signaling via the regulation of vitamin A metabolism

Integrity and preservation of a transparent cornea are essential for good vision. The corneal epithelium is stratified and nonkeratinized and is maintained and repaired by corneal stem cells. Here we demonstrate that Notch1 signaling is essential for cell fate maintenance of corneal epithelium during repair. Inducible ablation of Notch1 in the cornea combined with mechanical wounding show that Notch1-deficient corneal progenitor cells differentiate into a hyperplastic, keratinized, skin-like epithelium. This cell fate switch leads to corneal blindness and involves cell nonautonomous processes, characterized by secretion of fibroblast growth factor-2 (FGF-2) through Notch1(-/-) epithelium followed by vascularization and remodeling of the underlying stroma. Vitamin A deficiency is known to induce a similar corneal defect in humans (severe xerophthalmia). Accordingly, we found that Notch1 signaling is linked to vitamin A metabolism by regulating the expression of cellular retinol binding protein 1 (CRBP1), required to generate a pool of intracellular retinol.