The Cytoarchitecture of Normal Mouse Lens Epithelium ' NANCY S . RAFFERTY Department of Anatomy, Northwestern University Medical and Dental Schools, Chicago, Illinois 60611 ABSTRACT The lens epithelium of 30 gm male albino CFl mice was characterized by determining the area dimensions and the mitotic rate of the total and of the several different regions of whole-mount preparations. A ring of nonpigmented iris cells is found to adhere to the outer surface of the lens cuticle, which serves to delineate an inner central zone from an outer peripheral zone of the epithelium. A high number of dividing cells in the peripheral area, especially immediately adjacent to the meridional rows, but including the area overlain by the iridial fringe of cells, identifies this wide region as the proliferative zone. The mitotic rate, furthermore, undergoes marked diurnal variation, rising in the late evening through the early morning hours and diminishing during the late morning and afternoon hours. The relative large size of the rabbit, rat, and frog lens has encouraged their use in a whole-mount technique, developed by Howard ('52). In this technique, the entire fixed epithelium covering the anterior surface of the lens, along with the overlying acellular cuticle, is removed €rom the lens fiber mass and flattened Ionto microscope slides. A variety of cytological, histochemical, biochemical and autoradiographic studies, appropriate to investigations on cell population kinetics or cataractogenesis, can thus be performed on the total cell population comprising this tissue. Owing perhaps to its small size, the mouse lens has not been favored as material for such studies, except by a few investigators (Hanna, '65; von Sallmann, '57; von Sallmann et al., '57; Voaden and Leeson, '70; Weinstock and Stewart, '61). Mouse lens is reported to be susceptible to x-ray radiation cataract (von Sallmann, '57; and von Sallmann, et al., '57) and to drug-induced temporary opacities (Weinstock and Stewart, '61). Injury-induced cataract in the mouse is presently under investigation by the author. In the present report, the feasibility of whole-mount preparations of the mouse lens epithelium is studied and the cytoarchitecture of the lens epithelium described. The normal mitotic rate and diurnal variations in the rate, furthermore, form a standard baseANAT. REC., 173: 225-228. line for comparison with changes induced by injury. It was found that the mouse lens is well suited to whole-mount studies and, due to its small size, has the advantage that the total preparation can be scanned with a 1000-fold magnification (e.g., for mitotic counts) within a two-hour period. MATERIALS AND METHODS This study was done during the summer months. Carworth Farms (CFl) albino male mice, weighing between 28 and 30 gm were used. The mice were killed by cervical dislocation, and the eyes immediately removed and placed into 3 :1 ethanol : acetic acid for 12 to 24 hours. The eyes were rinsed and stored in 70% ethanol. The fixed lens was removed through a slit in the eye and placed in Delafields hematoxylin for six to seven seconds to facilitate visualization and removal of the epithelium. Removal of the epithelium was accomplished in tap water under the dissecting microscope ( X 25), as described previously for the frog lens (Rafferty, '67). However, the shape of the fixed mouse lens is so nearly round that the anterior and posterior surfaces are difficult to distinReceived Sept. 28, '71. Accepted Jan. 24, '72. 1 This investigation was supported by Public Health Service Research grant 7 R01 EY00698-01 from the National Eye Institute. 225 226 NANCY S. RAFFERTY guish. It was observed, however, that the non-pigmented iris leaves a fringe of cells at its attachment to the anterior surface of the lens, and this serves as a marker of the anterior surface. The detached epithelium was transferred with its cuticle down to a glass slide and four or five radial cuts were made to flatten it. After air drying the preparations were restained in hematoxylin for 15 minutes, dehydrated, and cover slips mounted. The diurnal variation in mitotic rate of the epithelium was determined in mice killed at four hour intervals, beginning at 2 PM, using three mice per interval. The number of mitoses and their position with respect to the adhering iridial fringe, were counted in the entire lens epithelium. The total area of each epithelium to the beginning of the meridional rows, the area inside the iridial fringe (central zone), and the area inside, including the iridial fringe area, were found from the diameters measured under 25 magnifications with a B & L stage micrometer (Lines A, B and C in fig. lb, respectively). The area outside the iridial fringe (peripheral area), and the area underlying the iridial fringe were calculated by appropriate subtractions of the above measured figures. These calculated areas were compared with areas mzasured directly with a Gelman planimeter on camera lucida drawings of two mouse lenses. OBSERVATIONS Figure l a illustrates a typical whole mount preparation of mouse lens epithelium. The adhering iridial fringe is seen as a darkened ring, separating a central from a peripheral area (but, of course, the iris cells are separated from the lens cells by the lens cuticle). The lens cells of both areas are polygonal and regular. The density of the cell population appears to increase from the central to the peripheral areas. Near the periphery the epithelial cells align themselves into orderly meridional rows (fig. 2 ) , and as they approach the lens bow region their elongation and differentiation into lens fibers takes place. The equatorial diameter of the intact fixed lens is approximately 2.3 mm, and the polar diameter, 2.1 mm. The average diameter of the 33 flattened epithelia is 2.90 mm, and the average surface area is 6.59 -+. 0.05 mm'. The central zone within the circular attachment of the iris has an average diameter of 1.65 mm and a surface area of 2.17 5 0.12 mm'. The iridial fringe area averages 0.43 mm'. By subtraction, the peripheral area outside of the iridial fringe is 3.99 mm2. These dimensions are summarized in figure lb. Fig. l a Whole-mount preparation of mouse lens epithelium showing adhering iridial fringe (IF), separating a central zone ( C Z ) from a peripheral area which includes the proliferative zone (PZ), and which gives way to the meridional rows ( M R ) and lens bow (LB). X 2#5. Fig. l b Line drawing of the epithelium shown in l a with summary figures of area dimensions (mm2) of different regions. 227 NORMAL MOUSE LENS EPITHELIUM Fig. 2 Portion of whole-mount preparation of mouse lens epithelium showing meridional rows ((top) and mitoses (arrows) in the proliferative zone. x 250. These calculated areas agree within 11 % with direct planimeter measurements d camera lucida outlines: for example, total area, planimeter measurement is 5.89 mm2,compared with 6.59 mm', calculated area; central area, 2.19 mm2, com- pared with 2.17 mm'; and peripheral area, 3.70 mm2, compared with 3.99 mm'. The planimeter measurements are the more accurate because they exclude the wedgeshaped spaces at the periphery which result from the radial cuts necessary for flattening the preparations. Areas calculated from diameter measurements include these spaces and the area of the peripheral region especially is increased by the latter method. The average number of mitoses found in each region of the epithelium at each time interval is presented in table 1. If the mitoses are converted into the number per mm2, the highest number corresponds to the peripheral area. This area includes the normal proliferative zone of vertebrate lenses. Although dividing cells are found throughout the peripheral area, the greatest concentration appears just anterior to the meridional rows. Some mitoses are observed a few cells anterior to the rows (fig. 2). The total numbers of mitoses counted in epithelia obtained at four hour intervals (table 1 ) point to a mitotic peak in the late evening and early morning hours, and a decline in mitotic rate in the late morning and afternoon hours. DISCUSSION The normal lens epithelium of male albino CF1 mice weighing 30 gm is shown to have a high rate of cell division which fluctuates diurnally, rising in the late evening and early morning hours and diminishing in the daytime hours. This diurnal variation agrees with that de- TABLE 1 Average number of mitoses in three regions of mouse k n s epithelium Time of sacrifice Number of eyes Peripheral area Central area Area underlying iridial fringe Total epithelium 6 5 5 6 6 5 226.0225.4 238.42 10.2 283.22 16.1 279.02 15.3 358.8 2 31.5 302.0 f36.4 22.0 2 2.6 12.8f3.1 24.4 f6.3 27.8 2 6.5 3 7 . 6 2 5.7 15.223.2 10.5 2 3.1 10.620.8 15.0?2.2 17.024.7 36.0 6.8 12.0-C3.4 258.5 2 30.2 261.8-C 10.6 322.6" 14.2 323.8 C 19.5 432.5-C31.7 329.2 2 39.2 281.8 23.8 17.3 323.0 2 Pm 6 pm 10 pm 2 am 6 am 10 am Average number of mitoses Average area (33 epithelia) Average number mitosis/mmz 3.99 mm2 70.6 2.17 mm2 10.9 * 0.43 mm2 40.2 6.59 mm2 49.0 228 NANCY S. RAFFERTY scribed by von Sallmann ('52) in the lens epithelium of chinchilla rabbits; it does not agree with the findings in Swiss mouse lens by Voaden and Leeson ('70), who found high levels of mitotic activity in the early morning ( 7 AM) and low levels in the late evening (10 P M ) . The discrepancy may result from their use of female mice, of a different strain, or the fact that their data did not include the intervals between 10 PM and 7 AM, when the mitotic activity may have shown the upswing described in the present report. In any case, if the total number of mitoses from the six intervals studied in this report are averaged, a value of 323 mitoses per lens epithelium is obtained. This value coincides with the midday mitotic averages, which is fortuitous in planning future experiments in which diurnal variation may be a critical factor. The number of mitoses reported in the total lens epithelium is considerably higher than that found by von Sallmann et al. ('57) in Swiss strain mice: 323 compared with 42 mitoses, respectively. This differ'ence probably results from the exclusion by the latter authors of both early prophases and late telophases, which in this experiment are included, and from other differences in their counting techniques (e.g., their counts were made under lower magnification). Miki ('61) pointed out that both body weight and the season of the year, among other factors, influence the mitotic rate in the lens epithelium of rats: the rate increases as the body weight increases, and peaks during the summer season. These factors were appreciated by Hanna ('65) who carefully studied the patterns of DNA, RNA, and protein synthesis in the developing lens of Swiss mice. He describes the 180-day or older mouse lens as having a proliferative zone confined to a narrow band just anterior to the lens equator, opposite the ciliary processes. This mature condition evolves from a broader proliferative zone involving most of the epithelium of very young mice, and which with aging contracts to a restricted band. In the epithelia of 30 gm mice (approximately eight weeks of age) studied in the present report, the proliferative zone appears wide with a somewhat higher concentration of mitoses close to the meridional rows. The area underlying the iridial fringe should perhaps be included in the proliferative zone since it has a higher concentration of mitoses than the adjacent central area. The adhering iridial fringe provides a convenient marker between adjacent areas of the lens epithelium which otherwise has a remarkably uniform appearance, though a different physiological activity. The mitotic rate in the whole epithelium and in the different areas delineated by the iris cells, and the diurnal rhythmicity in mitoses described in this report form a normal baseline against which changes induced by injury may be compared. LITERATURE CITED Hanna, C. 1965 Changes in DNA, RNA, and protein synthesis in the developing lens. Invest. Ophthal., 4: 480-491. Howard, A. 1952 Whole mounts of rabbit lens epithelium for cytological study. Stain Tech., 27: 313-315. Miki, T. 1961 Fluctuation in mitosis count of lens epithelium; influence of age, season, and adrenal hormones. Acta SOC.Ophthal. Japan, 65: 2207-2224. Rafferty, N. S. 1967 Proliferative response in experimentally injured frog lens epithelium: autoradiographic evidence for movement of DNA synthesis towards the injury. J. Morph., 121: 295-310. von Sallmann, L. 1952 Experimental studies on early lens changes after roentgen irridiation. 111. Effect of x-radiation on mitotic activity and nuclear fragmentation of lens epithelium in normal and cysteine-treated rabbits. Arch. Ophthal., 47: 305-320. 1957 The lens epithelium in the pathogenesis of cataract. Am. J. Ophthal., 44: 159-1 70. von Sallmann, L., L. Caravaggio, C. M. Munoz and A. Drungis 1957 Species differences in the radiosensitivity of the lens. Am. J. Ophthal., 4 3 : 693-704. Voaden, M. J., and S. J. Leeson 1970 A chalone in the mammalian lens. 1. Effect of bilateral adrenalectomy on the mitotic activity of the adult mouse lens. Exp. Eye Res., 9: 57-66. Weinstock, M., and H. C. Stewart 1961 Occurrence in rodents of reversible drug-induced oaacities of the lens. Brit. J. Ophthal., 45: 468214.