STREAMING OF LABELED CELLS IN THE CONJUNCTIVAL EPITHELIUM

This study examines epithelial cell streaming and turnover in normal r at bulbar conjunctiva. Twenty seven male adult random-bred Hebrew rats weighing between 250-300 g, were injected i.p. with [H-3]-thymidine. Three rats were killed at various times, thereafter from 1 h to 28 day s. The enucleated eyes were fixed in formalin, cut into 5 mu m thick s ections, dipped into liquid emulsion, exposed for three weeks and stai ned with haematoxylin and eosin. Conjunctival epithelium was scanned f rom the limbus and outward, using an ocular micrometer grid with 10x10 divisions. In each consecutive field the grid was positioned along th e basement membrane which was. defined as the x-axis. The y-axis exten ded from the basement membrane outward. The x,y coordinate of each nuc leus with three grains or more and its grain content were recorded alo ng the entire epithelium. Conjunctival epithelium is divided into two cell kinetic compartments: a progenitor (P), along the basal and supra basal layer, in which cells proliferate, and a non proliferating Q-co mpartment, in the layers above. One hour after labelling most of the l abelled cells were in the basal and supra basal layers. From then onwa rd labelled cells streamed along both axes. Their x-velocity was 10.5/-2.4 mu m/day and the y-velocity 9.3+/-5.4 mu m/day. Cells are elimin ated at the epithelial surface which is the outer Q-compartment bounda ry. Basal cell turnover was estimated from grain count dilution curves . The time it takes for the grains in a cell to reach half of their in itial value was 8.3 days. It is closely related to the cell's generati on time. The present study demonstrates that conjunctival epithelium i n the rat streams along two axes, x, and y: 1 The x-axis extends along the basal layer, from the limbus and outward. 2 The y-axis extends fr om the basal layer into the layers above it. Cells first stream along the x-direction and then turn y-ward. Since cells are ultimately exfol iated from the conjunctival surface, and since the conjunctiva maintai ns steady state, we propose that stem cells located in the limbus gene rate transitional cells that stream along the two axes. Macroscopicall y the limbus is circular, and the stem cells are situated around the c ornea. Each stem cell and its streaming progeny can be viewed as a con junctival epithelial unit. We propose that conjunctival and corneal ep ithelium, are the descendants of an uncommitted stem cell that generat es two differentiation pathways, a corneal and a conjunctival. Relativ ely little is known on conjunctival cell turnover. The conjunctiva is covered with stratified columnar epithelium with goblet cells. In albi no rabbits conjunctival epithelial healing was studied following n-hep tanol and trephined conjunctival wounding (Geggel et al. 1984). Within one day a single layered leading edge started migrating toward the wo und centre. By the third day conjunctival epithelium covered the entir e wound and started forming additional layers. Goblet cells appeared a t the wound periphery after one week and within 3 weeks penetrated the entire epithelium. Conjunctival epithelial wound healing resembles th at of the cornea (Elgjo 1969, Hanna & O'Brien 1960). The present study applied two methods for estimating cell turnover; cell kinetics and c ell kinematics. The first estimates the rate of cell proliferation, an d cell kinematics studies cell displacement (Zajicek 1986, Zajicek et al. 1988). Cell displacement is closely linked with cell proliferation . In squamous epithelia, e.g. epidermis (Potten 1981) or esophagus (Le blond et al. 1965, Marques-Pereira & Leblond 1965), only basal cells p roliferate. Of the two daughter cells formed during a mitosis, one rep laces the dividing ancestor and remains in the basal layer, while the other is displaced into the layers above. In order to make room for th e newly formed cell, all cells above are displaced outward by one cell width. Thus each basal cell division is associated with the displacem ent of the cell column located above it by one cell location. The natu re of cell displacement is still obscure. Since cells are neither pull ed nor pushed in a mechanical sense, they simply stream, in the same w ay as a log floating in a river, that is neither pushed nor pulled. We described cell streaming in the liver (Zajicek et al. 1988), salivary glands (Schwartz-Arad et al 1988; Zajicek et al. 1985, Zajicek et al. 1989), adrenal cortex (Zajicek et al. 1986), and pancreas (Zajicek et al. 1990!. The present study demonstrates that conjunctival epitheliu m streams from the limbus and outward.