Rare corneal clones in mice suggest an age-related decrease of stem cell activity and support the limbal epithelial stem cell hypothesis

The anterior ocular surface comprises the cornea, conjunctiva and a narrow intermediate region called the limbus. It is widely accepted that the corneal epithelium is maintained by stem cells but different hypotheses propose that the stem cells that maintain the mouse corneal epithelium during normal homeostasis are located either in the basal limbal epithelium or throughout the basal corneal epithelium. There are no specific markers to help test these alternatives and new methods are required to distinguish between them. We observed that KRT5^LacZ/− transgenic mice produced rare β-galactosidase (β-gal)-positive radial stripes in the corneal epithelium. These stripes are likely to be clonal lineages of cells derived from stem cells, so they provide a lineage marker for actively proliferating stem cells. The distributions of the β-gal-positive radial stripes suggested they extended centripetally from the limbus, supporting the limbal epithelial stem cell (LESC) hypothesis. Stripe frequency declined between 15 and 30 weeks, which predicts a reduction in stem cell function with age. Pax6^+/−, KRT5^LacZ/− corneas had small patches rather than stripes, which confirms that corneal maintenance is abnormal in Pax6^+/− mice.     

Dietary restriction enhances germline stem cell maintenance

Dietary restriction (DR) increases lifespan in species ranging from yeast to primates, maintaining tissues in a youthful state and delaying reproductive senescence. However, little is known about the mechanisms by which this occurs. Here we demonstrate that, concurrent with extending lifespan, DR attenuates the age‐related decline in male germline stem cell (GSC) number in Drosophila. These data support a model whereby DR enhances maintenance of GSCs to extend the reproductive period of animals subjected to adverse nutritional conditions. This represents the first example of DR maintaining an adult stem cell pool and suggests a potential mechanism by which DR might delay aging in the tissues of higher organisms.     

Role of the cytoskeleton during injury-induced cell migration in corneal endothelium

The role of microfilaments and microtubules during injury-induced cell migration of corneal endothelial cells in situ along their natural basement membrane has been investigated using organ culture. In the noninjured tissue, actin is localized at or near the plasma membrane, whereas tubulin is observed as a delicate lattice pattern throughout the cytoplasm. Twenty-four hours after a circular freeze injury, cells surrounding the wound area extend processes into this region. Fluorescent microscopy using phallotoxins and anti-tubulin antibodies demonstrated the presence of stress fibers and microtubule reorganization within these cells. Between 24 and 48 h post-injury endothelial cells move into the wound region, and by 48 h, the injury zone is repopulated and the monolayer is becoming reestablished. When injured corneas are placed in media containing 5 x 10(-7) M cytochalasin B, endothelial cell migration occurs; but it is slow, and wound closure is not complete even by 72 h. In contrast, when tissues are cultured in the presence of 10(-8) M colchicine, cell movement is greatly reduced, complete wound closure does not occur, and endothelial cells at the wound edge fail to display extensions typical of migrating cells. Furthermore, when injured endothelia are exposed to 0.05 micrograms/ml of actinomycin D for 15 min within the first hour after injury and transferred back into culture media lacking the drug for the duration of the experiment, migration does not occur and the wound persists. These actinomycin D treated cells remain viable as shown by their ability to incorporate 3H-uridine and 3H-thymidine. Fluorescence microscopy of actinomycin D treated tissues revealed the presence of stress filaments but disorganized microtubule patterns. Interestingly, 24 h after injury, if the tissue is exposed to actinomycin D, even for periods of up to 1 h, migration is not inhibited. Our results indicate that injury-induced endothelial cell movement appears to be more dependent on microtubule than microfilament reorganization and may require a critical timing of macromolecular synthesis.     

Selection for a decrease in metastatic potential in a cell population of transplantable rat rhabdomyosarcoma RA-2

The method of individual cloning was used for checking the reduction of metastatic potential (MP) in cell population of highly malignant rhabdomyosarcoma RA-2. In the course of 15 selection cycles MP was lowered by 10-15 times. These data enabled us to consider the MP value as a quantitative character of malignant cells with low heritability (the mean value of realized heritability coefficient is equal to 0.18).     

Cell pattern in adult human corneal endothelium

A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.     

The role of corneal endothelium in macular corneal dystrophy development and recurrence

Macular corneal dystrophy(MCD) is a progressive,bilateral stromal dystrophic disease that arises from mutations in carbohydrate sulfotransferase 6(CHST6).Corneal transplantation is the ultimate therapeutic solution for MCD patients.Unfortunately,postoperative recurrence remains a significant challenge.We conducted a retrospective review of a clinical cohort comprising 102 MCD patients with 124 eyes that underwent either penetrating keratoplasty(PKP) or deep anterior lamellar keratoplasty(DALK).O...     

Scanning electron microscopic study of cell movements in the corneal endothelium of the avian embryo

Using the scanning electron microscope we have examined the appearance, in situ, of the migrating cells of the presumptive corneal endothelium in the chick embryo. The primary stroma, a collagenous layer which serves as a major substrate for the migration of the cells, was found to have many deep folds and ridges. The collagen fibrils of the stroma, as seen on its posterior surface, appear arranged more or less isotropically. Areas of orthogonal packing are small and relatively sparse. While the cells make contact with this substratum they do not seem to be guided by its topographic features. The migrating endothelial cells lack well developed ruffles, supporting the increasing prevalent idea that the pattern of cell surface activity observed in vitro during cellular locomotion, is not an absolute prerequisite for cell movement.     

Tolerance of human corneal endothelium to glycerol

As an initial step in the development of a method for corneal cryopreservation by vitrification, we attempted to establish the maximum concentration of glycerol to which human corneal endothelium could be exposed at 4 degrees C for 15 min without damage. Damage was defined as an increase in mean endothelial cell size or the inability to maintain corneal thickness for 1 week after exposure to glycerol. Using a system for long-term corneal perfusion, we perfused 24 paired human corneas with glycerol at 4 degrees C. The concentration of glycerol increased at a rate of 20% (w/v) (2.2 M) per hour until the desired maximum concentration was reached for that cornea, stabilized for 15 min, and then decreased at the same rate. The corneas were then perfused at 37 degrees C with Dulbecco's medium at a rate of 5 microliters/min under 18 mm Hg intracameral pressure for 7 days with daily measurements of corneal thickness. Endothelial morphology was examined by specular microscopy and by scanning electron microscopy. After 7 days of perfusion at 37 degrees C, there was a statistically significant direct relationship between the maximum concentration of glycerol to which the experimental eyes had been exposed and the increase in mean endothelial cell size. The mean endothelial cell size increased in corneas exposed to glycerol concentrations of 40, 50, and 60% (w/v), but did not differ significantly from baseline measurements in the corneas exposed to 30% glycerol or less. Thus, there was no detectable damage to human corneas exposed to 30% (w/v) (3.3 M) glycerol in this system. Tolerance of higher concentrations may be achieved by changes in the rates of addition and removal of glycerol or in the composition of the perfusate.     

Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate

Resveratrol, a natural phytoalexin found in grapes and red wine, increases longevity in the short-lived invertebrates Caenorhabditis elegans and Drosophila and exerts a variety of biological effects in vertebrates, including protection from ischemia and neurotoxicity. Its effects on vertebrate lifespan were not yet known. The relatively long lifespan of mice, which live at least 2.5 years, is a hurdle for life-long pharmacological trials. Here, the authors used the short-lived seasonal fish Nothobranchius furzeri with a maximum recorded lifespan of 13 weeks in captivity. Short lifespan in this species is not the result of spontaneous or targeted genetic mutations, but a natural trait correlated with the necessity to breed in an ephemeral habitat and tied with accelerated development and expression of ageing biomarkers at a cellular level. Resveratrol was added to the food starting in early adulthood and caused a dose-dependent increase of median and maximum lifespan. In addition, resveratrol delays the age-dependent decay of locomotor activity and cognitive performances and reduces the expression of neurofibrillary degeneration in the brain. These results demonstrate that food supplementation with resveratrol prolongs lifespan and retards the expression of age-dependent traits in a short-lived vertebrate.     

Symmetric and asymmetric cell division in rat corneal epithelium

Abstract. Mitotic cells in normal, mature rat corneal epithelium were examined with a light microscope on serial, semi-thick plastic sections.
Classification of mitotic figures into horizontally, obliquely or vertically positioned with reference to the epithelial basal lamina has shown that no single configuration predominates. A striking correlation between the position of the daughter cells after cytokinesis and their morphology has been observed. Horizontal cytokinetic pairs were morphologically symmetric but vertical ones were asymmetric, displaying distinct differences between daughter cells. Analysis of earlier mitotic phases has shown that the asymmetry could also be observed in vertical anaphases and telophases.
The data provide clear morphological evidence for real asymmetric (unequal) cell division in a replacing epithelium in an adult mammal. It is concluded that asymmetric cell division in the corneal epithelium coexists with, and is as frequent as symmetric (equal) cell division. Randomness of mitotic spindle positioning implies that diverse forms of cell transfer from the proliferative into the differentiative epithelial compartments must operate. Therefore, the universality of the general model of cell renewal in stratified epithelia, which assumes a strong predominance of horizontal mitoses, exclusively equal mitotic divisions and one form of cell transfer, is questioned.     

Dietary restriction mitigates the age-associated decline in mouse B cell receptor repertoire diversity

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Hypoxia protects human corneal endothelium from tertiary butyl hydroperoxide and paraquat-induced cell death in vitro

To develop new and safe approaches to protect and preserve the function of the human corneal endothelium (HCE), we have established an in vitro model of cell loss using an HCE cell line and have examined the potential for hypoxia conditioning to protect HCE from death induced by two ROS generating cytotoxins (tertiary butyl hydroperoxide [tBHP] and paraquat [PQ]). Cell death was assessed by flow cytometric analysis of Annexin-V (An) and propidium iodide (PI) double staining and oligonucleosome production. Mitochondrial membrane potential (MMP) was measured with JC-1 fluorescent dye to determine possible associations between MMP preservation and cell survival. PQ and tBHP both induced HCE cell death in a dose-dependent manner, with 48% and 32% of An(+) cells observed with 60 microM tBHP and 500 microM PQ, respectively. In addition, this level of tBHP and PQ resulted in 66% and 49% decreases in MMP, respectively. Hypoxia (0.6% +/- 0.1% oxygen) pretreatment (8 hrs) significantly reduced An(+) cells caused by 60 microM tBHP to 15%, whereas hypoxia had no effect on the decreased MMP. Hypoxia pretreatments (24 hrs) slightly reduced An(+) cells caused by 500 microM PQ to 25% and completely prevented the induced MMP reduction. Conversely, hypoxia posttreatment (24 hrs) resulted in a greater inhibition of cell death than pretreatment and yet failed to prevent the PQ-induced MMP collapse. In conclusion, these results suggest that hypoxia can sufficiently protect HCE cells against cell death caused by tBHP and PQ, although no direct link between hypoxia cell protection and MMP preservation was observed.     

Symmetric and asymmetric cell division in rat corneal epithelium

Mitotic cells in normal, mature rat corneal epithelium were examined with a light microscope on serial, semi-thick plastic sections. Classification of mitotic figures into horizontally, obliquely or vertically positioned with reference to the epithelial basal lamina has shown that no single configuration predominates. A striking correlation between the position of the daughter cells after cytokinesis and their morphology has been observed. Horizontal cytokinetic pairs were morphologically symmetric but vertical ones were asymmetric, displaying distinct differences between daughter cells. Analysis of earlier mitotic phases has shown that the asymmetry could also be observed in vertical anaphases and telophases. The data provide clear morphological evidence for real asymmetric (unequal) cell division in a replacing epithelium in an adult mammal. It is concluded that asymmetric cell division in the corneal epithelium coexists with, and is as frequent as symmetric (equal) cell division. Randomness of mitotic spindle positioning implies that diverse forms of cell transfer from the proliferative into the differentiative epithelial compartments must operate. Therefore, the universality of the general model of cell renewal in stratified epithelia, which assumes a strong predominance of horizontal mitoses, exclusively equal mitotic divisions and one form of cell transfer, is questioned.     

Effects of osmotic stress on rabbit corneal endothelium

The effects of osmotic stress on corneal endothelium were investigated by exposing rabbit corneas to anisosmotic conditions, and then perfusing the corneas with isosmotic glutathione bicarbonate Ringer solution for 4 hr at 35 degrees C. During the perfusion, endothelial function was assessed by measuring corneal thickness with a specular microscope. After perfusion, the corneas were prepared for scanning and transmission electron microscopy. Endothelial ultrastructure and function were well maintained after exposure to a wide range of osmolality (0.12-2.7 osmol/kg), but this tolerance of osmotic stress was dependent both on the duration and the temperature of exposure to the anisosmotic conditions. Exposure to an osmolality of 2.7 osmol/kg for 15 min at 23 or 37 degrees C resulted in gross damage to the endothelium when the hyperosmotic agent was sodium chloride. This damage was not the result of increased osmolality per se nor cellular shrinkage because the endothelium tolerated exposure to a sucrose solution of the same osmolality for 15 min at 37 degrees C. The detrimental effect of sodium chloride, however, was mitigated by reducing the temperature of exposure to 0 degrees C or reducing the duration of exposure to 5 min. These results suggest that endothelial cells become more tolerant of high electrolyte concentrations with reducing temperature, and this could be an important factor in the survival of the endothelium in corneal cryopreservation. The results also help to define the limits of osmotic shrinkage and swelling tolerated by endothelial cells. This will be of value in overcoming the detrimental osmotic effects associated with the addition and, in particular, the removal of cryoprotectants.