(Investigative Ophthalmology and Visual Science. 1999;40:3079-3083.)
© 1999
by The Association for Research in Vision and Ophthalmology, Inc.
Light-Induced Apoptosis in the Neonatal Mouse Retina and Superior Colliculus
Kozo Sasaki1,
Hidetoshi Ino2,
Tanemichi Chiba2 and
Emiko Adachi-Usami1
From the Departments of
1 Ophthalmology and
2 Anatomy, Chiba University School of Medicine, Chiba, Japan.
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Abstract
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PURPOSE. Apoptosis occurs naturally in the rodent retina and superior colliculus
(SC) during the neonatal period. The authors used mice to demonstrate
the dependency of this apoptosis on the light stimulation and the
developmental period.
METHODS. A number of apoptotic cells were counted in the retina and SC from a
group of newborn mice reared in constant darkness (DD group), a group
reared in normal light and dark conditions (LD group), and a group
reared in constant darkness up to P7 and then transferred to normal
condition (DD-to-LD group). Terminal deoxynucleotidyl
transferase-mediated biotin-dUTP nick end labeling (TUNEL) was used for
visualization of the apoptotic cells.
RESULTS. In the LD group, apoptotic cells significantly increased in the retinal
nuclear layers, including both the outer and inner nuclear layers, the
retinal ganglion cell layer, and SC at postnatal day 1 (P1) and
postnatal day 2 (P2). The number of apoptotic cells in the ganglion
cell layer and SC reached the maximum level at P1. In contrast, in the
DD group, an increase in the number of apoptotic cells was not
observed. At P9, no significant increase in the number of apoptotic
cells was observed in the outer nuclear layer, ganglion cell layer, and
SC either in the LD, DD, or DD-to-LD groups, but the LD and DD-to-LD
groups showed a significant increase in the inner nuclear layer
compared to the DD group.
CONCLUSIONS. Apoptosis during the neonatal period in the mouse visual system is
induced by a light stimulus. This apoptosis was not induced after P7 in
the retinal ganglion cell layer and SC, even if excessive cells
survived.
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Introduction
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Irreversible damage due to degeneration of retinal visual cells
is caused by exposure to intermittent and constant strong
light.1
2
This degeneration is thought to proceed by
apoptosis.3
Rats reared in ordinary cyclic dim-light
conditions exhibit lower rhodopsin levels in photoreceptor cells than
those reared in constant darkness.3
These facts suggest
that light stimulation influences the survival of visual cells in the
retina. In addition to the apoptosis that can develop under abnormal
conditions, apoptosis also is induced in the normal developing visual
system. Studies have shown that apoptotic cells dramatically increase
after birth in mouse neonatal retina4
and rat neonatal
superior colliculus (SC).5
Generally, apoptosis during development is necessary to form
functionally normal tissue by eliminating unnecessary excessive cells.
In the nervous system, naturally occurring neuronal cell death
(apoptosis) during the neonatal period is important for the formation
of appropriate neuronal circuits by elimination of neurons with
erroneous connections.6
What kinds of stimuli distinguish
necessary neurons from unnecessary ones? In the developing visual
system, the evidence suggests that one stimulus that might is outside
light. We demonstrate here that exposure to light is necessary to
induce apoptosis in the mouse retina and SC during the neonatal period,
but this apoptosis is not induced 1 week after birth, even if excessive
neurons have not been removed.
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Materials and Methods
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Experimental Animals
We used pregnant ddY albino mice from our breeding colony. All
animals were cared for and used in accordance with the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research. The day after
mating was defined as E0 and the day of birth as P0, which corresponded
to E19. Mice were divided into three groups. Pregnant mice were
transferred to the dark room at E18, and dams and neonates were reared
in complete darkness until sacrifice (DD group). As a control, dams and
neonates were maintained on a daily cycle of 12 hours light and 12
hours darkness (LD group). The lights were on from 8 AM to 8 PM.
Illumination was provided by white fluorescent lamps (40 W, x6). The
mean distance between the lamps and cages was approximately 2 m.
Light intensity in the cages ranged from 200 to 250 lux. Dams and
neonates reared in the dark room until P7 were transferred to the
normal conditions (LD 12:12) and maintained for 2 days (DD-to-LD
group).
Preparation of Tissue Sections
Newborn mice at P0 (=E19), P1, P2, P9, P14, and P21 were overdosed
with pentobarbital and fixed by perfusing 4% paraformaldehyde in 0.1M
phosphate buffer (pH 7.4) through their hearts. The brains were cut in
half sagittally, the eyeballs were removed, and then both the brains
and the eyeballs were immersed in the 4% paraformaldehyde solution at
4°C for 2 days. E18 fetuses taken from pregnant mice anesthetized
with an overdose of pentobarbital were briefly rinsed with saline and
immersed in the fixing solution at 4°C for 2 days. After washing with
phosphate-buffered saline (PBS), tissues were immersed in 20% sucrose
in PBS overnight at 4°C and frozen with dry ice. Cross sections (10
µm) were cut by a Cryostat and stuck on glass slides coated with
0.01% poly-L-lysine. Retina sections cut
parallel along the optic axis within approximately 0.2 mm from optic
nerve were used for counting.
Terminal Deoxynucleotidyl Transferase-Mediated Biotin-dUTP Nick-End
Labeling
Sections were immersed in 0.3% Triton X-100 in 0.05 M Tris-HCl,
pH 7.5, at room temperature for 1 hour and incubated with 1.5 µg/ml
proteinase K (Boehringer Mannheim, Mannheim, Germany) for 15 minutes at
37°C. After preincubation in a terminal deoxynucleotidyl transferase
(TdT) buffer (30 mM Tris-HCl, pH 7.2, 140 mM sodium cacodylate, and 1mM
cobalt chloride) for 10 minutes, sections were incubated in a TdT
buffer containing 50 units/ml TdT (Takara, Tokyo, Japan) and 0.2 mM
biotinylated 16-dUTP (Boehringer Mannheim) in a humid atmosphere for 2
hours at 37°C. After washing with PBS, endogenous peroxidase was
inactivated by incubating in 0.3%
H2O2 and 0.1% sodium azide
for 10 minutes at room temperature. Sections were incubated with
Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) for 2
hours at room temperature and stained with 3,3'-diaminobenzidine,
H2O2 and ammonium
nickel(II) sulfate hexahydrate as substrate. Each section was
counterstained with neutral red.
Statistical Analyses
Sections including entire areas of the retina and SC at each time
point were photographed. From these data, numbers of terminal
deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling
(TUNEL)-positive cells/1000 cells were calculated. Data were analyzed
by analysis of variance (ANOVA) with Scheffé’s F
test.
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Results
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Influence of Light Stimuli on the Induction of Apoptosis in the
Neonatal Mouse Visual System
Figure 1
shows representative photographs of TUNEL-positive cells in the
neonatal retina and SC at P1. Deep dark staining of TUNEL-positive cell
nuclei was seen in the outer and inner nuclear layers (ONL and INL),
the retinal ganglion cell layer (GCL), and the SC from the LD group
(Figs. 1A
, 1C
). In the DD group, however, the number of TUNEL-positive
cells was smaller (Figs. 1B
, 1D
). We performed a quantitative analysis
for a temporal change in number of TUNEL-positive cells in the retina
and SC during the neonatal period. In the early neonatal period, when
the boundary between the ONL and INL is unclear, we did not distinguish
between these two layers in counting apoptotic cells. The nuclei of the
photoreceptor cells were included in the ONL and the nuclei of amacrine
cells, bipolar cells, horizontal cells, and Müller glia were
included in the INL. However, classification of cell types was not
performed in this experiment. Before birth (E18), few TUNEL-positive
cells were seen in the ONL and INL (0.4 ± 0.3/1000 cells) (Fig. 2A
). After birth, a small increase in the number of TUNEL-positive cells
was observed at P0 (1.1 ± 0.1). The number of apoptotic cells
clearly increased at P1 (2.8 ± 1.5, P < 0.02)
and was even greater at P2 (3.4 ± 1.4, P <
0.01). In contrast, the number of TUNEL-positive cells in the DD group
was quite low, nearly equal to the level at E18.
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Figure 1. TUNEL-positive cells in the retina (A, B) and
superior colliculus (C, D) in mice of the LD
group (A, C) and DD group (B,
D) at P1. Arrows indicate TUNEL-positive
cells showing dark nuclear staining. Note that more TUNEL-positive
cells were observed in the LD group than in the DD group. Bars, 100
µm.
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Figure 2. Developmental change in the number of TUNEL-positive cells in the
nuclear layer including the outer and inner nuclear layer
(A), retinal ganglion cell layer (B), and
superior (C) and inferior colliculi (D). Values
represent the mean ± SD; n = 5 (animal
number). *P < 0.005; **P <
0.05, relative to E18 by Scheffé’s F test.
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In the retinal GCL, ganglionic neurons with large nuclei are easily
identified. The number of apoptotic ganglion cells was small before
exposure to light at E18 (1.5 ± 1.3). After a small increase at
P0, apoptotic cells dramatically increased at P1 (17.9 ± 6.9,
P < 0.01), but then decreased at P2 (9.6 ± 4.2,
P < 0.06). The number of apoptotic ganglion cells in
the DD group did not increase from E18 to P2. At P14 and P21, there was
no apoptotic cell in the retina, in either the LD or DD group.
In the SC, the superordinate center of the retina, the temporal change
in number of neonatal apoptotic cells resembled that of retinal
ganglion cells. Although few TUNEL-positive cells were found at E18
(0.4 ± 0.5) in the LD group, a significant increase in apoptotic
cells was observed at P0 (4.4 ± 1.5, P < 0.05),
the number of apoptotic cells reached the maximum level at P1 (8.7 ± 3.4, P < 0.01), and then decreased at P2 (5.0 ± 2.2, P < 0.02). In the DD group, no significant
increase in apoptotic cells was found from E18 to P2, as in the retina.
To compare with the SC, we also analyzed the occurrence of apoptosis
during the neonatal period in the inferior colliculus. Although few
apoptotic cells were found at E18 as in the other portion of brain,
there was a significant increase in apoptotic cells at P0 in both the
LD and DD groups. This is a reasonable result because the inferior
colliculus is part of the auditory system, and breeding in the darkness
did not eliminate outside sounds.
Existence of a Critical Period for Light-Induced Apoptosis in the
Neonatal Mouse Visual System
To investigate whether the potency of light-induced apoptosis is
retained until a much later period in the development of the mouse
visual system, we performed the following experiments. Mice bred in
constant darkness until P7 were transferred and maintained under normal
conditions up to P9 (DD-to-LD group). The number of TUNEL-positive
cells was counted in the ONL, INL, GCL, and SC in P9 mice from the LD,
DD, and DD-to-LD groups (Fig. 3)
. Since the boundary between the ONL and INL is clear at this stage, we
distinguished between these two areas.
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Figure 3. TUNEL-positive cells in the LD, DD, and DD-to-LD groups at P9. A
statistically significant difference was observed only in the INL.
Values represent the mean ± SD; n = 5 (animal
number). *P < 0.005; **P <
0.05, relative to INL of the DD group by Scheffé’s
F test.
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In the ONL, TUNEL-positive cells were scarcely observed in any of the
three groups. The number of TUNEL-positive cells was at a low level in
each group. In the LD and DD-to-LD groups, the numbers in the GCL and
SC were almost at the same level. Although the DD group showed lower
values, there was no significant difference between the three groups.
These results suggest that light fails to induce apoptosis in these
areas, even if excessive cells survive by escaping from apoptosis that
occurs during the early neonatal period. In contrast, the numbers of
TUNEL-positive cells in the INL of the LD and DD-to-LD groups were
significantly higher than those in the DD group (P <
0.05 and P < 0.01, respectively). Cells in the INL
retain susceptibility to light for the induction of apoptosis. Mice
from the LD and DD groups were maintained until P14 and P21. No
apoptotic cells were found either in the retina or SC at these periods.
No morphologic change was observed in the retina and SC of DD group
mice.
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Discussion
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In the nervous system, neurons were excessively generated during
the early stages of development and were all eliminated by apoptosis,
except for those with appropriate functions in the course of
development. It is believed that the selection by apoptosis is
genetically programmed. But, we have shown here that light triggers the
induction of apoptosis in the mouse retina and SC during the early
postnatal period. This suggests some type of apoptosis depends on
outside stimuli.
The peak of apoptosis in retinal ganglion cells and neurons of the SC
from the LD group mice was at P1. To date, developmental apoptosis
observed as pyknotic cells has been reported, under normal conditions,
in the mammalian visual system. Apoptosis declined after reaching a
peak at P2 in the mouse retinal ganglion cells4
and at P0
in the rat SC.5
The patterns of temporal change of
apoptosis in the LD group were more or less consistent with these
cases. Because proliferation of these neurons terminates before birth,
their apoptosis is independent of cell cycle progression. Apoptosis was
observed in the ONL and INL during later developmental periods up to at
least P9; however, no apoptotic cells were found in any area at P14 and
P21. In addition, the population of apoptotic cells in the retina was
considerably larger than indicated by previously reported
data.4
We presume that this may be because TUNEL shows a
higher sensitivity for detection of apoptosis than light-micrographic
observation of pyknotic cells.
During the early neonatal period in rats and mice, the eyes are not
open, the retinas are immature, and the outer segment of photoreceptor
cells is not formed. So, which cells and molecules transmit information
from light stimuli to induce apoptosis during the early neonatal
period? Rhodopsin is immunohistochemically detected in immature rod
photoreceptor cells with no outer segments in rat retinas at
P3.7
Several monoclonal antibodies against rhodopsin show
immunohistochemical staining in cell bodies of immature neuroblast-like
cells positioned in the neuroblastic zone beside the pigmented
epithelium even in the P1 rat retina.8
These cells seem to
be immature photoreceptor cells. Opsin mRNA is expressed in P1 rat
retinas at low but detectable levels.9
Developmental
changes in mouse retinas may be similar to those seen in rat retinas.
This evidence suggests that rhodopsin exists in immature rod
photoreceptor cells even during the early neonatal period. We presume
that these immature photoreceptor cells may possess the light
sensitivity, even though levels of rhodopsin are low and
rhodopsin-expressing cells are much more rare in neonates than in
adults.
In rats and hamsters, the SC receives the projection from both
contralateral and ipsilateral eyes during the neonatal period. During 2
weeks after birth, most of the projection from the ipsilateral eye is
reduced and restricted to the rostromedial SC.6
10
11
12
13
This restriction is prevented by contralateral eye
enucleation10
and by injection of tetrodotoxin into the
contralateral eye to inhibit neuronal activity14
during
the neonatal period. This process is due to apoptosis of ipsilaterally
projecting retinal ganglion cells rather than to removal of
ipsilaterally projecting axons from ganglion cells with bifurcating
axons.6
These results suggest that light-induced apoptosis
occurs preferentially in ganglion cells with ipsilateral
retinocollicular projection. It is still unknown, however, why
contralateral projection always wins the game and how this competition
is regulated. Patterned visual input appears to be independent of this
phenomenon because the eyes are still not open. The influence of the
competition between these two projections on the induction of apoptosis
in the SC is also unknown.
From our results with the DD-to-LD group, we determined that a critical
period exists in the susceptibility to light for the induction of
apoptosis. In retinal ganglion cells and neurons of the SC, in
particular, light fails to induce apoptosis at P7, even when excessive
neurons destined to die survive. Mouse optic nerves enter the
SC15
several days before birth and axon branching and
synapse formation proceed around P7.16
Because
developmental change in apoptosis of the GCL and SC resemble each other
without time lag, main pathways have already formed at birth. Since the
timing of the loss of susceptibility to light-induced apoptosis is
coincident with the period of synaptic reconstruction, stimulation
through newly constructed synapses may prevent apoptosis.
Several factors related to the induction of apoptosis have been
identified recently. In the nervous system, neuronal cell death under
artificial and pathologic conditions is suppressed by antagonists of
N-methyl-D-aspartate receptors17
and inhibitors of nitric oxide synthase.18
19
The
induction mechanism of developmentally occurring neuronal cell death
has not yet been elucidated; however, similar factors also may be
included in this process.
Finally, the functional abnormality caused by a blockade of naturally
occurring apoptosis in the visual system is not clearly understood. We
suppose that DD group may show the neonatal type of retinocollicular
projection even until adulthood. Because our animal model has no neural
impairment derived from enucleation or neurotoxins, it may be used for
physiologic examination of the influences of abnormal retinocollicular
projection.
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Footnotes
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Submitted for publication December 9, 1998; revised May 6, 1999; accepted June 23, 1999.
Commercial relationships policy: N.
Corresponding author: Kozo Sasaki, Department of Ophthalmology, Naruto
Hospital, 167 Naruto, Naruto-cho, Sanbu-gun, Chiba 289-1326, Japan.
E-mail: kozosasa{at}green.ocn.ne.jp
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References
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Abstract
Introduction
