Retinal pigment epithelium - CiteSeerX

J. Embryol. exp. Morph. 75, 271-291 (1983) Printed in Great Britain © The Company of Biologists Limited 1983

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Retinal pigment epithelium: pattern of proliferative activity and its regulation by intraocular pressure in postnatal rats By O. G. STROEVA 1 AND I. G. PANOVA From the N. K. Koltzov Institute of Developmental Biology, USSR Academy of Sciences, Moscow

SUMMARY

The postnatal proliferative activity of retinal pigment epithelium (RPE) cells and its dependence on intraocular pressure were studied using pHjthymidine and [14C]thymidine autoradiography in normal and experimentally induced microphthalmic pigmented rats. The regulation of RPE growth by intraocular pressure was shown to involve the control of the number of binucleated cells by means of stimulation of cell entry into the S phase of the cell cycle. Binucleated cells in the rat RPE are formed by acy to kinetic mitoses between days 2 and 9. The significance of the postnatal proliferation and formation of binucleated cells in the RPE is discussed in terms of the specificity of the G2 phase for melanotropic hormone action on RPE differentiation.

INTRODUCTION

Until recently it was believed that retinal pigment epithelium (RPE) cell replication in rats is completed during intrauterine life (Puzzolo & Simone, 1979). We found that in newborn rats RPE cell proliferation resumes with a spike on the third day after birth, and a considerable proportion of RPE cells become binucleated during the first two postnatal weeks (Stroeva & Nikiphorovskaya, 1970; Marshak & Stroeva, 1973, 1974; Stroeva & Panova, 1976, 1980). By the time of eye opening the proportion of binucleated cells in the RPE central zone reaches 80 %, and then declines to 70 % in 1-year-old rats (Ts'o & Friedman, 1967; Stroeva & Brodsky, 1968; Stroeva & Nikiphorovskaya, 1970). This decline in number of binucleated cells is accompanied by tri- and tetranucleated cell formation (Fig. 1). Observation on adult mutant MSUBL rats (Stroeva & Lipgart, 1968) showed that in the RPE of microphthalmic eyes the proportion of multinucleated cells is less than that in normally sized eyes (Stroeva & Nikiphorovskaya, 1970; Marshak & Stroeva, 1974) which led us to suggest that proliferation and the occurrence of polyploidy in RPE cells are 1

Author's address: N. K. Koltzov Institute of Developmental Biology, Academy of Sciences of the USSR, 26 Vavilov Street, 117808 Moscow (B-334), USSR.

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O. G. STROEVA AND I. G. PANOVA 100'

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7 9 11 13 15 age in days

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Fig. 1. The proportion of uninucleated (filled circles), binucleated (open circles), trinucleated (filled squares), and tetranucleated (open squares) cells in the central zone of the RPE throughout the postnatal life of rats. Each point derived from cell counts on tangential sections of the RPE using ten eyes, 1000 cells per eye (averaged data from Stroeva & Nikiphorovskaya, 1970; Marshak & Stroeva, 1973; Stroeva & Panova, 1976).

controlled by intraocular pressure. The latter was shown to be a general factor controlling eye growth in the chick embryo (Coulombre, 1956). In order to test this suggestion we studied (1) the pattern of eye growth throughout life in rats; (2) the characteristics of the cycling RPE cells during thefirst2 weeks after birth (the period found to be critical for postnatal RPE differentiation), and (3) the origin of binucleated cells. Microphthalmic eyes were induced experimentally and the eye growth retardation effect on the RPE was then studied. This paper presents the results obtained. Some of our data were briefly reported earlier (Marshak, 1974; Panova & Stroeva, 1978; Stroeva & Panova, 1976,1980). The present research intends to serve as a basis for studies with more analytical methods.

Retinal pigment epithelium in postnatal rats

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MATERIALS AND METHODS

Animals Grey rats (Rattus norvegicus) with pigmented eyes randomly bred from the colony of the Institute of Developmental Biology were used in the experiments. Eye growth Rats at postnatal ages ranging from 1 day to 1 year were killed by decapitation. Following enucleation and fixation in neutral formalin+96 °ethanol+glacial acetic acid (3:1:0-3) the eyes were washed in tap water and 70° ethanol, dried on filter paper and weighed using analytical scales. Their scleral part was then cut off under the ora serrata and also weighed. For every measurement point between 10 and 45 eyes were used. Then the scleral parts of 3 to 7 eyes per agepoint were processed for histological treatment. In another experiment the scleral part of freshly enucleated eyes from 1- to 15-day-old rats cut off under the ora serrata, was incised at several places along the margin and laid flat on filter paper. The flat preparations were immersed in the fixative mixture, washed and photographed. The area of the scleral part of each eye was measured on the photo by planimetry (10 eyes for each measurement point). The gain in weight (AW/At) and in area (AS/At) of the scleral part between different ages were calculated from the equations:

AW/At = W(n) - W(n -l)/t(n) - t(n -1),

(Eq. 1)

AS/At = S(n) - S(n - l)/t(n) - t(n - 7),

(Eq. 2)

and

where W(n) and S(n) are a weight and an area of the scleral part of n-day-old rat respectively, and t(n) is the age of animals in days. Microphthalmic eyes were obtained by surgical extirpation of the lens after Coulombre & Coulombre (1964), who demonstrated the role of the lens in eye growth. Rats were subjected to surgical manipulations at the onset of the second, and at the end of the fourth postnatal days, under ether narcotization. Once the skin above the left eye and the conjunctival sac wall had been cut with a pair of scissors the lens was extirpated from the eye through a corneal linear incision, by use of a glass needle. Thereupon the skin was sutured and the animals were returned to the mother. The right intact eye of each animal was used as the control. An increase in area of individual RPE cells in normal and microphthalmic eyes was described by the nuclear concentration in the central RPE zone. A square ocular grid was projected on the slides under the light microscope at a magnification of 1000 x , and the number of nuclei per square was averaged by counting fifty such fields per eye. Nuclear concentration was calculated from the equation

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of Abercrombie (1946). In all experiments a minimum of three animals was used for each measurement point. A utoradiography For continuous labelling intact and operated rats were injected subcutaneously with [3H]thymidine (specif, activ. 9-1 Ci/mM. 1 /iCi/g) every 6 h for 19 h, and decapitated l h after the last injection. The experiments were started at 10.00a.m. For histological treatment, the eyes were processed as described above, then their scleral parts were dehydrated in serially graded ethanol and chloroform and embedded in paraffin. Serial sections, 5^m thick, were cut tangentially to the RPE in three neighbouring regions of the central (posterior) zone of the globe and from the four quadrants of the sublimbal zone referred to as peripheral. The zone between the central and peripheral ones was called equatorial (Fig. 2). The general state of microphthalmic eyes was examined from serial cross sections prepared in a plane parallel to the optic axis. Melanin was decoloured on dewaxed sections with potassium permanganate, whitened with oxalic acid and washed with tap water. Autoradiograms were prepared with a liquid emulsion (type 'M', Nil Chimphoto, Moscow), and exposed for a month at 4°C. The slides were developed in D19 developer (Schillaber, 1944) and stained over the emulsion with Carrachi haematoxylin. Proliferative activity The proportion of uninucleated and binucleated cells as well as of labelled nuclei was calculated from counts of at least 1000 cells per zone of each eye. Cell

Fig. 2. Schematic drawing of the scleral part of the eye. Open circles indicate the zones from which tangential sections were cut for cytological study of the RPE cell population: (1) peripheral zone; (2) equatorial zone, and (3) central zone.


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cycle parameters were derived graphically (Quastler & Shermann, 1959). Fourday-old rats received a single injection of [3H]thymidine, and then the fraction of labelled mitoses (FLM) was plotted as a function of time. Mitotic figures from middle prophase to telophase were scored (30-60 mitoses per eye) on tangential sections from the central RPE zone. Unless specified all nuclei with five or more silver grains over them were considered as labelled. In order to determine the duration of mitosis (IM), 4-day-old rats were injected subcutaneously with 5 jug/g of colchicine (Merk, BRD) and killed 3h later. A preliminary experiment revealed the complete blockage of metaphases in the RPE at that time. The eyes of three intact animals from the same litter provided material for the determination of the mitotic index (m), which was derived from total counts of 16 644 cells. The duration of mitosis was determined from the equation tm = (m/mc)t, (Eq. 3), where t is the effective time of colchicine action. In order to demonstrate the mitotic origin of binucleated cells, the grain density over the interphase nuclei of uni- and binucleated cells at 2 h and 27 h on the FLM curve, as well as over each of the two nuclei in 100 binucleated cells at 27 h, was obtained. The grain density was derived as the number of silver grains per nuclear area (calculated as that of an ellipse). Double labelling with [3H]thymidine and [14C]thymidine was used in order to determine the number of cell cycles between day 3 and day 9 after birth. Twentyone 3-day-old rats were each injected with a single dose of [3H]thymidine (specif, activ. 12Ci/mM; ljuCi/g). Three rats were killed 2h after the injection. The other animals were sacrificed 24, 48, 72, 96,120, and 144 h after [3H]thymidine injection (three animals per day); 2h before decapitation they were each given a single injection of [14C]thymidine (specif, activ. 54mCi/mM; 1/iCi/g). The eyes of all rats were enucleated, fixed and processed for histological treatment and autoradiography. Indices of labelled nuclei of uninucleated and binucleated cells were calculated from counting at least 1000 cells per eye on tangential sections at the central zone. All quantitative data were processed statistically.

RESULTS

Eye growth According to the weight data, the rat eye grows throughout the life of animals, whereas the growth of its scleral part has a discontinuous character (Fig. 3). The most intensive growth of the scleral part occurs from day 2 to day 5 after birth, with a maximum on day 4 (Fig. 3B). By day 5 the weight of the scleral part reaches half of that of the 1-year-old rat. The increase in weight of the scleral part was also noted between the first to third and the seventh to twelfth months of age (Fig. 3). The area of the scleral part increases continuously with a maximum rate on day 6 within the first ten postnatal days (Fig. 3A). Starting from day 5 to the

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10

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7 9 11 age in days

15

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5 7 9 age in months

12

Fig. 3. Growth of the eye and its scleral part throughout postnatal life in the rat. Each point on thefigurewas derived from measurements on 10 to 45 eyes. The total area of each scleral part was measured by planimetry from photos offlatpreparations (each point derived from measuring of 10 scleral parts). Open circles refer to the weight of the whole eye,filledcircles refer to that of the scleral part, and triangles refer to the area of the scleral part. Vertical lines are confidence intervals at 95 % significance level. In square above: (A) the gain of the scleral part area calculated from the Equation (2), and (B) the gain of the scleral part weight calculated from the Equation (1) (for details see Methods). twelfth month of age, the scleral part of the rat eye increases twofold in its weight and sixfold in its area. Proliferative activity in the rat RPE resumes during the second postnatal day. By the end of the second to third days the index of labelled nuclei in the central zone is about 20% after continuous labelling with [3H]thymidine and then


7

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Fig. 4. Indices of labelled nuclei at the different RPE zones of the intact eyes after continuous labelling with [3H]thymidine. (A) Indices of labelled nuclei in the central zone (filled circles) and at the periphery (open circles, averaged data for four quadrants), and (B) at the periphery (filled bars) and at the equatorial zones (open bars): (V), ventral; (N), nasal; (D), dorsal; and (T), temporal; each bar represents averaged data for the eyes of three animals. Vertical lines are standard error of the mean.

declines. During days 5-8, the labelling index remains at the 8 % level, then falls below 5 % by day 9, and below 1 % by the 11-15 days after birth (Fig. 4A). The average index of labelled nuclei at the RPE periphery was 7 % from the third to eighth postnatal days, and then dropped (Fig. 4A). No significant difference in the proportion of DNA-synthesizing cells was observed between different quadrants of the RPE periphery, whereas in the dorsal and temporal equatorial zones the number of labelled nuclei exceeded that in the central zone. Proliferative activity was the same in the nasal and ventral equatorial zones as in the periphery (Fig. 4B). Binucleated cells There are occasional binucleated cells (about 5 %) in the RPE central zone of newborn rats. Their proportion reaches about 50% in 5-day-old and 80% in 9-day-old rats (Fig. 1). At the periphery the 50 % level of binucleated cells was observed by day 9 and does not change later (Fig. 5). Thus the RPE zones with the highest proliferative activity are those with the maximal proportion of binucleated cells.

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5 7 age in days

Fig. 5. The proportion of uninucleated (filled circles) and binucleated (open circles) cells at the RPE periphery (averaged data for four quadrants of the globe). Each point derived from cell count on tangential sections of the RPE of one eye (1000 cells per eye). 100 -i

50 -

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20 24 27 30 33 10 h after a single r3H]thymidine injection

Fig. 6. The curve for the fraction of labelled mitoses (FLM) in the central RPE zone plotted as a function of time following a single pH]thymidine injection given to 4-day-old rats. Mitoticfiguresfrom middle prophase to telophase were scored (30 to 60 mitoses per eye) on tangential sections of the RPE. Each point represents the data for one eye. Solid line, open circles refer to the FLM at the threshold of not less than 5 grains over nucleus; dotted line,filledcircles refer to the FLM at the threshold of not less than 15 grains over nucleus.

The cell cycle and origin of binucleated cells Marshak (1974) was the first to use the cell cycle to show that binucleated cells are of mitotic origin. However she failed to present any quantitative data against the idea that binucleated cells could form by a fusion of uninucleated labelled cells. To dismiss that possibility we have repeated this experiment with 4-day-old rats using the pulse-chase method of Quastler & Shermann (1959) as well as double labelling with [3H]thymidine and [14C]thymidine. Grain density over


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labelled interphasic RPE nuclei was also determined. The results are documented in Figs 6, 7 and 8. To construct the curve of labelled mitoses (FLM) initially all mitotic figures with not less than five grains overlying them were considered as labelled. The resulting curve gives the mean value of 6-4 h for G2+I/2M, and of 25 h for the generation time (T) (measured at the 80% level of the FLM curve). The descending limb of the curve did not fall below the 50 % level which made the graphic determination of ts impossible (Fig. 6). No labelled binucleated cells were observed 2 h after [3H]thymidine injection, and 2-6 % were labelled at 27 h on the FLM curve. The population of labelled uninucleated cells was homogenous by the criterion of grain density distribution at 2h (Fig. 7, A, I), but became heterogenous by 27h (Fig. 7, A, II). Nuclei of

distribution of grain density classes

Fig. 7. Grain density distribution for interphase nuclei of uninucleated and binucleated cells in the central RPE zone on the 2nd and 27th h of the FLM curve shown in Fig. 6. Nuclear area was averaged by measuring from 50 to 70 nuclei of randomly selected RPE cells on tangential sections; grain density was termed as number of silver grains per nuclear area. Histograms (A) refer to the grain density over nuclei of uninucleated cells at 2 h (I), to that of uninucleated cells at 27 h (II), and to that of binucleated cells at 27 h (III). Histogram (B) refers to the averaged grain density over each of two nuclei of 100 randomly selected binucleated cells at 27 h. Vertical lines are confidence interval at 95 % significant level.

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some cells which had incorporated [3H]thymidine did not divide during this cycle (e.g. some cells of the fraction of the most heavily labelled nuclei). The population of the least-labelled nuclei increased enormously. These findings could not be attributed merely to cell division during a single cell cycle, and might indicate the reutilization of radioactive DNA precursors, similar to that observed for newts' eyes (Parshina & Mitashov, 1978). No reutilization effect was found when plotting the curves for labelled mitoses in the neural retina (Denham, 1967; Stroeva, 1978) and in the neuroepithelia of the iris, but it affected greatly the pattern of FLM curves for the neuroepithelia of the ciliary body (Stroeva, 1978). Possibly, it could be related to the barrier function of both pigmented epithelia. In the histogram showing grain density over the nuclei of binucleated cells at 27 h on the FLM curve (Fig. 7, A, III), the fraction of the most heavily labelled nuclei was absent, and the fraction of the least-labelled nuclei of binucleated cells

7-

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9 age in days

Fig. 8. Indices of labelled nuclei of RPE cells after double labelling with p ] thymidine and [14C]thymidine. Only nuclei of uninucleated RPE cells were initially labelled with [3H]thymidine on day 3 of age; about 50 % of them became binucleated by day 4, and the other 50 % became binucleated by postnatal day 5, and then withdrew from the cell cycle (open bars). Only nuclei of uninucleated cells were labelled with [14C]thymidine on any day (filled bars). No uninucleated cells with double label were found. Each bar derived from cell counts in the RPE central zone on tangential sections, using three animals (1000 cells per eye). Vertical lines represent the standard error of the mean.


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was less than that of uninucleated cells (Fig. 7, A, II). When comparing grain density over each of the two nuclei of binucleated cells, no statistical differences were obtained (Fig. 7B). This could not be due to cell fusion with the observed heterogeneity of labelling of the uninucleated cells at 27 h. The results obtained unambiguously demonstrate the mitotic origin of binucleated cells. To avoid the influence of a reutilization process, only labelled mitoses with not less than 15 grains over them were taken for the FLM curve reconstruction (Fig. 6). With such a correction, the graphic detection of fs at the 50 % level of FLM curve was possible. It was found to be 15 h (after subtraction of 1 h - the persistence time of [3H]thymidine in the blood). The duration of the M phase found in the experiment with colchicine (using Equation (3); see Methods) proved to be 2-3 h (m = 0-7 % ± 0-06; mc = 0-91 % ±0-15; £ = 3h). After appropriate subtraction, tGi and fci were found. Therefore, the mean T was 25 h, fe was 15-0h, tu was 2-3 h, fci was 2-5 h, and *G2 was 5-2h. The number of cell cycles in the RPE between days 3-9 was determined in a double-labelling experiment. Only nuclei of uninucleated cells were labelled in the RPE central zone at 2h following the single [3H]thymidine injection. The indices of labelled nuclei were 3-2 % in the RPE of 3-day-old rats, 5-6 % in that 50

40

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7 9 11 age in days

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15

Fig. 9. Growth of microphthalmic eyes. The growth estimation is based on the weight data for whole microphthalmic eyes obtained as the result of the lens removal at the onset of day 2 (filled squares) and at the end of day 4 (open squares), as well as for whole intact eyes (open circles) and their scleral parts (filled circles). From three to seven rats were used for each measurement point. Vertical lines represent the standard error of the mean.

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B

10 Fig. 10. (A) The left microphthalmic eye of a 15-day-old rat from which the lens had been removed surgically at the onset of day 2, and (B) the right intact eye from the same animal.

Fig. 11. A tangential section of the RPE central zone of the intact eye of a 9-day-old rat. The majority of RPE cells are binucleated ones; at the centre of the microphotograph a labelled binucleated cell is seen. Melanin grains were bleached with potassium permanganate. Sections were stained over the emulsion with Carrachi haematoxylin. Fig. 12. A tangential section from the RPE central zone of the microphthalmic eye of a 9-day-old rat (the same animal as that shown in Fig. 11). The lens was removed from the eye at the onset of the 2nd day of age. There are uninucleated cells with labelled nuclei; at the left a labelled mitosis is seen. Melanin grains were bleached with potassium permanganate. Section was stained over the emulsion with Carrachi haematoxylin.


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of 4-day-old rats, 6-2 % in the RPE of 5-day-old rats, and thereafter remained stationary (Fig. 8). In the RPE of 4-day-old rats 50% of labelled cells were uninucleated and 50 % were binucleated. On the fifth day 92-1 % of all labelled cells became binucleated. This means that the RPE cell population is heterogeneous in the duration of the Gi phase. Half the cycling cells had a Gi that considerably exceeded 5h, the mean time found in the preceding experiment. Therefore, almost all RPE uninucleated cells which had synthesized DNA at day 3 became binucleated by day 5 and withdrew from the cell cycle. New uninucleated cells enter the cell cycle during the postnatal days 4-9, as was revealed by pulse labelling with [14C]thymidine given to the rats as the second label 2 h before sacrifice. The pulse label with [14C]thymidine was 1-7 ± 0-3 % by days 4-8 and decreased to 0-7 % by day 9 (Fig. 8). Only two binucleated cells labelled with [14C]thymidine were seen in the RPE of 6- and 7-day-old rats. No uninucleated cells with two labels were found. Thus this experiment confirmed the mitotic origin of binucleated cells and showed that during this period the majority of RPE cells pass through the cell cycle only once. Experimental microphthalmia Eye growth Lens removal from the rat eye at the onset of day 2 and at the end of day 4 resulted in the same degree of microphthalmia (Figs 9 and 10). The growth pattern of the intact eyes of the operated animals did not differ from that of normal animals (cf. Fig. 3 and Fig. 9). The lens removal caused a detachment and infolding of the neural retina. As a rule the RPE of microphthalmic eyes was healthy (Figs 11 and 12). A slight increase in nuclear concentration in the RPE occurred immediately after lens removal and then progressed (Fig. 13). This g

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age in days

Fig. 13. Nuclear concentration in the RPE central zone of microphthalmic (A), and of intact (B) eyes.

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