Rolf Bjerknes

KINETICS OF CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure.

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G2 phase DNA content were measured by flow cytometry of isolated basal cells, and the [3H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined.

THE STATHMOKINETIC METHOD IN VIVO TIME‐RESPONSE WITH SPECIAL REFERENCE TO CIRCADIAN VARIATIONS IN EPIDERMAL CELL PROLIFERATION IN THE HAIRLESS MOUSE

Groups of hairless mice were injected i.p. with a stathmokinetic dose of 0·15 mg colcemid at seven different times of the day and animals killed 0, 15 and 30 min, 1, 2, 3 and 4 hr after the injection. The proportion of cells in metaphase and ana/telophase was determined in histological sections.

Cell renewal of the rat corneal epithelium: A method to compare corresponding corneal areas from individual animals

Abstract A stathmokinetic method to study the mitotic rate in the corneal epithelium of the rat is described and applied. Horizontal and vertical diameter sections were used. In order to analyze the mitotic rate in the various parts of the corneal epithelium, each vision field (objective 100, eye piece 12.5), comprising 182 μm basement membrane, was regarded as a separate unit. Since the number of vision fields acros the cornea varies from specimen to specimen, we constructed a mathematical method to correlate corresponding corneal areas from different eyes. Using this method it is shown that the mitotic rate is almost equal all over the cornea, with no definite reduction in the central areas. There is no area of significantly high proliferation rate, either in the limbal area or in the adjacent conjunctiva.

Migration of cells in the rat corneal epithelium

Abstract The migration of cells in the rat corneal epithelium was studied using continuous labelling with tritiated thymidine [3H]TdR, during a 24 h period. Most mitoses resulted in 2 new basal cells. Cells leaving the basal cell layer moved vertically to the surface in all areas of the corneal epithelium. The first labelled cells reached the surface 3 days after the first injection of [3H]TdR. No stream of cells from the conjunctiva to the cornea in the limbal areas was observed, and no centripetal migration of epithelial cells in the cornea could be observed. After a cell has lost its attachment to the basement membrane, it is committed to be exfoliated in a few days without undergoing mitosis. Thus the slow centripetal migration of epithelial cells and the exchange of centrally located cells, as indicated by clinical findings and experimental studies, can only be explained by migration of basal cells.

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

SummaryA 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.

Cell kinetics during healing of corneal epithelial wounds

Abstract. After removing a circular area of the central corneal epithelium of the rat eye, the labelling indices and the mitotic rates were measured at various times after wounding, both in the cornea and in the adjacent conjunctival epithelium. The proliferative response was most marked in the corneal epithelium adjacent to the wound, but there was also a definite response in the epithelium covering the denuded areas, and in the conjunctival epithelium. The study demonstrated that the conjunctiva itself plays a role in the healing of a central corneal epithelial wound. The similarities in the cellular response may indicate that both epithelia are under the influence of the same growth‐suppressing factors (chalones), and must be looked upon as a unit. However, no support was found for the theory that the limbal area serves as a generative organ for the corneal epithelium.

Cell kinetics of conjunctival and corneal epithelium during regeneration of different-sized corneal epithelial defects.

Abstract. Rats with small (diam. 1.7 mm), medium sized (diam. 35 mm) or large (diam. 5.5 mm) corneal epithelial erosions in one eye were killed 1,2 or 4 days after the injury. The proliferative response was evaluated by measuring the labelling index and the mitotic rate in the corneal epithelium and in the adjacent conjunctiva. The small erosions triggered a proliferative response in the cornea only with the maximum response occurring midperipherally. The medium sized erosions induced a higher and more extensive response in the cornea and also a slight increase of the labelling index in the limbal area. The large erosions induced an even more pronounced response in the peripheral cornea and an increase both of the labelling index and the mitotic rate well beyond the limbal part of the conjunctiva. It is concluded that the magnitude and the extent both of the conjunctival and the corneal regenerative response to a corneal abrasion is correlated to the size of the corneal defect. Temporary reduction in the conjunctival epithelial cell number shows that both cells in the limbal and the extralimbal conjunctiva migrate centri‐petally during healing of large corneal wounds. It is suggested that the stem cell theory should be modified. The limbal area is probably an area in which conjunctival epithelial cells or conjunctiva‐derivated cells transform or differentiate to corneal epithelial cells.

The early cell kinetic response during healing of corneal epithelial wounds.

The labeling index and the mitotic rate were measured at 4-hour intervals during the first 24 hours after a central abrasion had been made in the corneal epithelium of six groups of four rats each. The proliferative response was noted in the conjunctival, the limbal, and the corneal epithelium. After 24 hours, the density of epithelial cells was equal throughout the corneal epithelium, but there was only half the normal number of cells. Physiological mechanisms seem strongly to regulate the total number of cells per square unit throughout the healing corneal epithelium, but the nature of these mechanisms is unknown.

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

Abstract. 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.