Inherited Retinal Dystrophy in the Mouse:
its Appearance in Eyes and Retinae Cultured in vitro
by
D.
R.
LUCAS1
From the Wernher Group for Research in Ophthalmological Genetics
(Medical Research Council), Royal College of Surgeons
WITH TWO PLATES
C E R T A I N strains of mice, such as C3H, exhibit a recessively inherited retinal
dystrophy. This takes the form of a degeneration and complete disappearance of
the visual cell layer; it begins on or about the 1 lth day of life and is nearly complete by the 19th day (Sorsby, Roller, Attfield, Davey, & Lucas, 1954). The process is remarkably uniform from litter to litter (Lucas & Newhouse, 1957).
In a previous paper (Lucas & Trowell, 1958) it was shown that eyes and retinae
taken from 10-day-old normal (CBA) mice could be maintained in vitro for 8
days, during which time their structure was fairly well preserved and differentiation proceeded.
In the present work, eyes and retinae from dystrophic strains of mice have
been cultured over a similar period to see if the dystrophy developed in vitro.
MATERIAL
The eyes or retinae of 25 animals from 3 litters of the C3H strain, maintained
at the M.R.C. Radiobiological Unit, Harwell, and from 2 litters of the retinal
dystrophy (Bruckner) strain maintained at the Royal College of Surgeons, were
explanted. The retinal condition is histologically identical in the two strains
and in Fi hybrids. Details of breeding experiments showing the genetic identity
of the two strains have been reported elsewhere (Lucas, 1958).
Two groups of mice were used, the one 9-10 days old (12 animals), the other
11-12 days old (13 animals). Retinae from a number of normal CBA-mice of the
stock maintained at Harwell were explanted as in vitro controls.
METHODS
The cultural and histological methods were as described in the previous paper
(Lucas & Trowell, 1958). Each experiment was performed on a group of littermates. As controls, the eyes of one mouse were fixed when the cultures were set
1
Author's address: M.R.C. Radiobiological Unit, Atomic Energy Research Establishment,
Harwell, Didcot, Berks., U.K.
[J. Embryol. exp. Morph. Vol. 6, Part 4, pp. 589-592, December 1958]
590
D. R. LUCAS—DYSTROPHIC RETINAE IN CULTURE
up ('initial' controls), and one animal was set aside to be killed at the end of the
experiment and provide in vivo controls. The eyes or retinae of the remaining
available mice were cultured for 7 days. In most experiments, cultures were also
made of normal eyes or retinae from CBA-mice as near the same age as possible.
RESULTS
In general, the cultures were equal in quality to those already reported (Lucas
& Trowell, 1958); the isolated retinae showed some loss of cells from all layers
and in the whole eyes there was usually some necrosis of the retina at the posterior pole. Retinal malformations also developed in the cultured eyes with consequent variation in the relative thickness of the different layers. These factors
made the results more difficult to interpret, but a fairly clear picture nevertheless
emerged.
Mice aged 11-12 days. The initial controls showed slight thinning of the outer
nuclear layer in the area centralis and some of the nuclei were pyknotic indicating that the dystrophy had already begun (Plate 1,fig.A). At the periphery the
outer nuclear layer was of normal thickness (Plate 1,fig.B). In all the cultured
eyes except two, which were too necrotic to interpret, the outer nuclear layer was
distinctly thinner than in the initial controls, but not so thin as in the in vivo controls (Plate 1,figs.C, D). Also, in the cultured C3H eyes the outer nuclear layer
was thinner than in cultured CBA eyes (Plate 2,fig.E).
The retinal cultures (from 2 eyes) in this group showed almost complete disappearance of the visual cell layer (Plate 2,fig.F).
Mice aged 9-10 days. In the initial controls the outer nuclear layer appeared
normal except that it contained very occasional pyknotic nuclei. In 5 of the 15
cultured eyes the'outer nuclear layer showed definite thinning as compared with
parallel cultures from normal CBA-mice. It was, however, much thicker than in
the in vivo controls. In the other 10 eyes in this group it was impossible to be sure
that any thinning had occurred because of the malformation or necrosis of the
retina referred to above.
In the 4 isolated retinae cultured in this group, the number of visual cells surviving was not consistently lower than in parallel cultures of CBA-mice.
It appeared, therefore, that the dystrophy proceeded during culture, though at
a slower rate than in vivo. It could be demonstrated more consistently when it
had already begun before culture as in the 11-12-day group.
DISCUSSION
The results show that, under the condition of these experiments, the degeneration and disappearance of the visual cell layer which normally take place between
11 and 19 days in mice of a retinal dystrophy strain are considerably delayed.
This delay is probably due to retarded differentiation. It has already been found
D. R. L U C A S — D Y S T R O P H I C R E T I N A E IN C U L T U R E
591
that the onset of degeneration in the intact animal is delayed under conditions of
malnutrition severe enough to interfere with retinal differentiation (Lucas &
Newhouse, 1957). Differentiation of the visual cells under present conditions is
undoubtedly abnormal since, though the nuclei show the changes normally
observed in vivo, the rods fail to elongate (Lucas & Trowell, 1958). It is also possible, however, that there is a humoral factor in the affected animal from the
influence of which the cultures have been removed. Wolff & Kieny (1955) have
recently demonstrated the existence of a humoral factor in extracts of Creeper
fowl embryos which retards the growth of tibial explants from both normal and
heterozygous Creeper embryos when added to the culture medium.
SUMMARY
Using an organ culture technique the eyes and retinae of mice affected by an
inherited retinal dystrophy were maintained for 7 days in vitro. The rate of the
dystrophic process was considerably retarded as compared with the living
animal.
ACKNOWLEDGEMENT
I should like to thank Dr. J. F. Loutit for making available facilities in the
M.R.C. Radiobiological Unit, Harwell, and Dr. O. A. Trowell for much advice
and encouragement.
REFERENCES
LUCAS, D. R. (1958). Retinal dystrophy strains. Mouse News Letter, 19, 43.
& NEWHOUSE, J. P. (1957). The effects of nutritional and endocrine factors on an inherited
retinal degeneration in the mouse. Arch. Ophthal. N.Y. 57, 224-35.
& TROWELL, O. A. (1958). In vitro culture of the eye and the retina of the mouse and the rat.
J. exp. Morph. 6, 178-82.
SORSBY, A., KOLLER, P. C , DAVEY, J. B., ATTFIELD, M., & LUCAS, D. R. (1954). Retinal dystrophy
in the mouse: histological and genetic aspects. / . exp. Zool. 125, 171-98.
WOLFF, E., & KIENY, MADELEINE (1957). Mise en Evidence d'une action inhibitrice de l'extrait
d'embryons de la race poules 'Courtes-pattes' Creeper sur la croissance des tibias cultiv6s in
vitro. C.R. Acad. Sci. Paris, 244,1089-91.
EXPLANATION OF PLATES
PLATE 1
FIG. A. Initial control. Area centralis of C3H mouse aged 12 days showing slight thinning of
outer nuclear layer and occasional pyknotic nuclei; fixed acid Zenker. x 400.
FIG. B. Initial control. Periphery of retina of same eye as in fig. A showing normal outer
nuclear layer (the raggedness is an artefact), x 200.
FIG. C. Periphery of retina of eye of C3H mouse aged 12 days (litter-mate of mouse from which
eye in figs. A and B originated) after 7 days in culture showing thinning of outer nuclear layer;
fixed acid Zenker. x 200.
FIG. D. In vivo control. Periphery of retina of eye of C3H mouse aged 19 days (litter-mate of
mice from which eyes in figs. A, B, and C originated) showing remnants only of the visual cell
layer; fixed acid Zenker. x 200.
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D. R. LUCAS — D Y S T R O P H I C R E T I N A E IN C U L T U R E
PLATE 2
FIG. E. In vitro control. Periphery of retina of eye of CBA-mouse aged 10 days and cultured in
same chamber as eye shown in fig. C showing normal appearance of outer nuclear layer after 7
days in culture; fixed acid Zenker. x 200.
FIG. F. Retina of C3H mouse aged 12 days after 7 days in culture, showing loss of nearly all
the visual cell layer. Cf. fig. A of which the mouse was a litter-mate; fixed formal sublimate, x 400.
FIG. G. In vitro control. Retina of CBA-mouse aged 10 days after 7 days in culture showing
amount of visual cell layer normally preserved; fixed formal sublimate, x 400.
(Manuscript received 1: iv: 58)
J. Embryol. exp. Morph.
Vol. 6, Part 4
D. R. LUCAS
Plate 1
J. Embryol. exp. Morph.
Vol. 6, Part 4
D. R. LUCAS
Plate 2