Download Lysosomes and melanin granules of the retinal pigment

312 Reports
17. Dystcr-Aas, H. K.: A comparison between
the aqueous flare inducing effect of a «melanocyte stimulating hormone and a minimal trauma, Acta Ophthalmol. 43: 30, 1965.
18. Dystcr-Aas, H. K., and Krakau, C. E. T.: A
photo-electric instrument for measuring the
aqueous flare in the intact eye, Ophthalmologica 14C: 48, 1963.
Lysosomes and melanin granules of the
retinal pigment epithelium in a mouse
model of the Chediak-Higashi syndrome.
W. GERALD ROBISON, JR., TOICIIIRO KUWA1JARA, AND DAVID G. COGAN.
The origin of giant granules in the retinal pigment epithelium of the beige mouse was investigated with electron microscopy and ultrastructural
histochemistry. These granules were found to contain melanin and acid phosphatase. Apparently
they arise from fusions of primary lysosomes with
melanin granules which are already enlarged, from
multiple fusions among melanosomes. Therefore,
the giant granules are not primary lysosomes, nor
are they simply enlarged melanin granules as
suspected from light microscopic studies. A deficiency of primary lysosomes in the pigment epithelium results, suggesting a defect in intracellular
digestion similar to that found in the leukocytes
of Chediak-Higashi patients and several animal
models. Affected humans probably have defective
digestion in their retinal pigment epithelium also;
ivhich could impair the renewal process for rod
outer segments. Thus, Chediak-Higashi patients
may show an increased susceptibility to light
damage due not only to hypopigmentation, but
to defective intracellular digestion, as well.
The Chediak-Higashi syndrome is a rare human
disorder of recessive inheritance which results in
decreased oculocutaneous pigmentation, photophobia, nystagmus, and recurrent pyogenic infections.1 The increased susceptibility to infection
is attributed mainly to a block in intracellular
digestion, though neutropenia and lowered cheniotaxis of leukocytes are contributing factors.-- •"•
Phagocytic leukocytes contain abnormally large
granules which represent multiply fused lysosomes.1 Cells with such enlarged lysosomes fail
to degranulate properly and are unable to carry
out normal digestion.:l- r> The pigment epithelium
also contains enlarged granules, but these were
reported to be abnormal melanin deposits,11 and
their possible relation to lysosomes has not been
investigated.
The beige mutant of the C57 black mouse is
a good animal model for the Chediak-Higashi
syndrome."- ' We have studied its retinal pigment epithelium using electron microscopy and
Investigative Ophthalmology
April 1975
acid phosphatase histochemistry to determine the
relations of the abnormally large granules to
melanin granules and to lysosomes.
Materials and methods. Eyes were obtained
from three black mice (C57BL/6 N1H- +/+) and
three beige mutants (C57BL/6 MM- bg/bg)
ages 25 to 33 days. The central retina and
choroid were excised in fixative and carefully
sliced with a sharp razor blade into pieces that
were 200 to 400 cm wide. These tissue sections
were fixed for 180 minutes at 4° C. in 2.5 per
cent glutaraldehyde and 6 per cent sucrose
buffered to pH 7.2 with 50 mM sodium cacodylate. Some of the tissue sections were processed
directly for ultrastructural studies. Others were
washed overnight in 50 mM sodium cacodylatc
(pH 7.2) with 8 per cent sucrose at 4° C ,
rinsed in 200 mM Tris maleate at pH 5 for 60
minutes, and then were incubated at 37° C.
lor 60 minutes in one of the following media
for acid phosphatase histochemistry.
The incubation medium for the demonstration of
acid phosphatase contained 5 ml. HJO, 5 ml. 50 mM
MnCI-, 10 ml. 200 mM Tris maleale (pH 5.0),
10 ml. 25 mM cytidine S'-monophosphate, 3 Cm.
sucrose, and 20 nil. 0.2 per cent lead nitrate.s- !)
Control tissues were treated by one of the
following methods: (1) heated at 60° C. for 30
minutes prior to incubation; (2) incubated in
medium with 10 mM sodium fluoride added;
(3) incubated in medium without cytidine 5 monophosphate or other substrate; and (4) left
unincubated and not postfixed.
Following incubation the tissues were rinsed
briefly in 200 mM Tris maleate (pH 5.0) at 37°
C. and then in 120 mM cacodylate buffer (pH
7.2) at room temperature. They were postfixed
and embedded for electron microscopy. Only the
first 100 Mm ol each piece was sectioned in order
to avoid artifacts related to limited penetration
by the histochemical media. Sections were examined and photographed with a JEM 10013 electron
microscope, unstained or after uranyl acetate and
lead citrate staining.
Results.
Comparative morphology. The retinal pigment
epithelium of the beige mouse contains giant
granules of irregular shapes which may approximate the sizes of nuclei (Fig. 1). These granules
may be as large as 3 /mi wide and 11 /mi long.
They are especially conspicuous for their high
density (light and electron opacity). They contain material which is structurally identifiable as
melanin, but they differ from typical melanin
granules in that their content is not homogeneous,
and their position in the cell is usually central rather
than apical. The smaller (melanin) granules which
are present abundantly in the apical portions of
normal retinal pigment epithelium are absent.
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Reports 313
Fig. 1. Retinal pigment epithelium and choroid of a beige mouse which is a mutant of the
C57 mouse, and an animal model for the Chediak-Higashi syndrome. Compare the giant
intracellular granules with the melanin granules of the C57 black mouse, as seen in the
inset of the same magnification. (xl2,300.)
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314
Investigative Ophthalmology
April 1975
Reports
Fig. 2. Retinal pigment epithelium of a beige mouse incubated for the demonstration of
acid phosphatase activity. Reacted lysosomes (L) and unreacted phagosomes (Ph) are
prominent. A giant granule is seen at left. (xl4,500.)
The giant granules of the beige mouse often
exhibit melanin in different stages of aggregation
within the same granule. Some granules appear
to be in the process of fusing, and many have
concentric rings of varied densities, suggesting
that additions to their size were acquired in discrete steps. They also contain material of low
density in their peripheral regions which does
not resemble melanin.
The giant granules do not have the structure
of primary lysosomes, nor are they similar to any
typical cell organelle.
The primary lysosomes in the retinal pigment
epithelium of the beige mouse are decreased in
number, even though their size range is normal
(0.2 Mm to 0.9 /im). The phagosomes (0.6 Mm
to 2 Mm) and secondary lysosomes {0.3 /ira to
2 /tin) are also normal in size, but they are
more abundant than normal and a greater proportion of them is localized basally in the pigment epithelial cells. The nuclei are morphologically normal with dimensions of approximately 3.6
Mm by 6 Mm. There appears to be no change in
the number or morphology of mitochondria.
The melanocyte in the choroid of the beige
mouse has giant granules also (Fig. 1). These
are somewhat more regular in shape than those
of the pigment epithelium, and may be as large
as 2 /im by 4 Mm.
The C57 black mouse has typical melanin
granules (Fig. 1, inset). Some of the granules
in the retinal pigment epithelium are spherical,
with diameters near 1 Mm, but most are oblong
and may be as large as 0.9 Mm on their short
axis and 1.6 Mm on their long axis. The choroid
of the black mouse has melanin granules which
are mainly spheroid and have diameters up to
0.7 jum.
EM histochemistry. Acid phosphatase, a common lysosomal enzyme, served as a marker for
distinguishing lysosomes from organelles of similar structure. The pigment epithelium of both
black and beige mice incubated for the demonstration of acid phosphatase activity (Figs. 2 and
3) showed significant amounts of lead precipitate
in the primary and secondary lysosomes, and in
the smooth endoplasmic reticulum adjacent to the
Golgi. The melanin granules of the pigment epithelium in C57 black mice often showed small
amounts of precipitate, perhaps just above background levels. Some of the giant granules of the
beige mice exhibited a similar low level of reactivity also; however, many of these giant granules (Chediak-Higashi granules) exhibited high
levels, equivalent to those found in lysosomes
(Fig. 4). Such was never observed in the melanin
granules of the black mice, even with extended
incubation times.
We found in beige mice many lysosome-like
bodies which appeared to have. fused with the
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membranes of the giant granules (Fig. 4). In
such instances the contents of the granules, at the
periphery, and the contents of the "fused bodies"
exhibited acid phosphatase activity. These enzyme-rich bodies which fused with the giant
granules appear to be primary lysosonies. Their
fusion with the giant granules shows an affinity normally reserved to reaction with phagosomes.
Beige mice showed many bodies which had
the appearance of secondary lysosomes, but
showed no lead precipitate (Fig. 3). Often these
were located basally. They were interpreted to
be phagosomes of long standing which had received insufficient acid phosphatase for digestion
of their contents. In contrast, essentially all
structurally distinct secondary lysosomes in the
C57 black mouse demonstrated acid phosphatase
activity.
No lead precipitate was observed in control
tissues incubated without substrate. Some of the
heat-treated tissues and those incubated with
the addition of sodium fluoride showed a little
precipitate, which appeared to be nonspecific.
This was believed to result from incomplete
inhibition or spontaneous precipitation, and was
considered to be insignificant.
Discussion. The retinal pigment epithelium of
the beige mouse contains giant granules which
are highly variable in size and shape. Many of
them exhibit acid phosphatase activity as well
as structural evidence of melanin content. Our
observations suggest that their enzyme activity
derives from fusion of primary lysosomes with
enlarged melanin granules. Such fusions appear
to be common, and may lead to the observed
deficiency in the number of primary lysosomes
in the retinal pigment epithelium of the beige
mutant. The digestion of engulfed outer segment
membranes of the photoreceptor cells may be
affected adversely. Such is consistent with our
observation that there is an apparent accumulation of phagosomes in the retinal pigment epithelium of the beige mutant. We observed no
extracellular accumulation of discarded rod outer
segments.
Greatly enlarged cytoplasmic granules (inclusion bodies) and impaired intracelluJar digestion
in leukocytes are said to be pathognomonic for
the Chediak-Higashi syndrome in humans,1"'1 and
for the beige mutant in mice.7 Granulocytes
and other phagocytic cells appear to engulf bacteria normally, but the contact of digestive enzymes with ingested material is greatly delayed
or absent.3* s Apparently, the large cytoplasmic
granules derive from multiple fusions of primary
lysosomes, and the sequestering of their enzymes
into one large lysosome (the Chediak-Higashi
granule).4 Such bizarre lysosomes seldom fuse
with phagosomes and, therefore, do not form
Reports 315
Fig. 3. Retinal pigment epithelium of a beige
mouse showing a secondary lysosome (above)
which reacted positively for acid phosphatase activity, and a phagosome (below) which reacted
negatively. (x23,000.)
secondary lysosomes in which substrate and enzymes are brought together.
The giant granules of the retinal pigment epithelium cannot be classified as primary lysosomes
since they contain melanin as well as acid phosphatase. They would fit the definition of secondary
lysosomes better, even though there is no evidence as yet that their contents are digested. They
probably originate by fusions among developing
melanin granules,0 followed by fusions of melanin
granules with primary lysosomes.
Such disturbances in the mechanism of intracellular digestion appear to be related to the
pathogenesis of the Chediak-Higashi syndrome.
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316 Reports
Investigative Ophthalmology
April 1975
Fig. 4. Retinal pigment epithelium of a beige mouse reacted for acid phosphatase activity.
Chediak-Higashi granules as well as primary lysosomes are reacted positively. Often lysosomes
(arrows) appear to fuse with the granules. (x37,000.)
Probably the retinal pigment epithelium of
Chediak-Higashi patients, like that of the beige
mouse, contains giant granules with melanin and
acid phosphatase instead of typical pigment
granules and normal numbers of primary lysosomes. Defective intracellular digestion in the
pigment epithelium is suggested, and this may be
accompanied by a delayed renewal of rod outer
segments and perhaps by an increased susceptibility of the patient to irreversible light damage.
Preliminary evidence to be reported subsequently suggests an abnormal susceptibility of the
beige mouse to photic damage. Probably defective
digestion of rod outer segments is an important
factor, but hypopigmentation must be contributory. The ocular tissues of beige mice and
Chediak-Higashi patients that would normally
provide protection from photons are lightly pigmented. Unlike normal tissues they concentrate
what may be a typical amount of melanin in a
few giant granules rather than disperse it into
many small granules. The net effect is lighter
pigmentation and less filtering capacity.
We thank Dr. Sheldon M. Wolff of the Laboratory of Clinical Investigation, National Institute
of Allergy and Infectious Diseases, and Dr. Francis
J. Judge, Dr. Carl T. Hansen, and Mr. Richard
L. Pierson of the Veterinary Resources Branch of
the Division of Research Services of the National
Institutes of Health, Bethesda, Md., for advice,
cooperation, and the animals obtained. We acknowledge the technical assistance of Mrs. Sandra
Dubinsky.
From the Laboratory of Vision Research, National Eye Institute, National Institutes of Health,
United States Department of Health, Education,
and Welfare, Bethesda, Md. 20014. Submitted for
publication Oct. 25, 1974. Reprint requests: Dr.
W. G. Robison, Jr., Bldg. 6-211, National Eye
Institute, National Institutes of Health, Bethesda,
Md. 20014.
Key words: pigment epithelium, Chediak-Higashi
syndrome, lysosome, retina, acid phosphatase,
melanin, EM histochemistry, beige mouse.
REFERENCES
1. Blume, R. S., and Wolff, S. M.: The ChediakHigashi syndrome: studies in four patients and
a review of the literature, Medicine 51: 247,
1972.
2. Wolff, S. M., Dale, D. C , Clark, R. A., et al.:
The Chediak-Higashi syndrome: studies of
host defenses, Ann. Intern. Med. 76: 293,
1972.
3. Root, R. K., Rosenthal, A. S., and Balestra,
D. J.: Abnormal bactericidal, metabolic, and
lysosomal functions of Chediak-Higashi syndrome leukocytes, J. Clin. Invest. 51: 649,
1972.
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4. Davis, W. C , Spicer, S. S., Greene, W. B.,
et al.: Ultrastructure of bone marrow granulocytes in normal mink and mink with the
homolog of the Chediak-Higashi trait of humans. I. Origin of the abnormal granules present in the neutrophils of mink with the
C-HS trait, Lab. Invest. 24: 303, 1971.
5. Prieur, D. J., Davis, W. C , and Padgett,
G. A.: Defective function of renal lysosomes
in mice with the Chediak-Higashi syndrome,
Am. J. Pathol. 07: 227, 1972.
6. Lutzncr, M. A., and Lowrie, C. T.: Ultrastructure of the development of the normal
black and giant beige melanin granules in the
mouse, in Pigmentation, Its Genesis and Biological Control, Riley, V., editor. Proceedings
of the Seventh International Pigment Cell
Conference. New York, 1972, AppletonCentury-Crofts.
7. Oliver, C , and Essner, E.: Distribution of
anomalous lysosomes in the beige mouse: a
homolog of Chediak-Higashi syndrome, J.
Histochem. Cytochem. 21: 218, 1973.
8. Barka, T., and Anderson, P. J.: Histochemical
methods for acid phosphatase using hexazonium pararosanilin as coupler, J. Histochem.
Cytochem. 10: 741, 1962.
9. NovikofF, A. B.: Lysosomes in the physiology
and pathology of cells: contributions of staining methods, in: Ciba Foundation Symposium
on Lysosomes, deReuck, A. V. S., and Cameron, M. P., editors. Boston, 1963, Little,
Brown and Company, p. 36.
Closely spaced saccades. A.
KAREN A.
TERRY BAHILL,
BAHILL, MICHAEL R.
CLARK,
AND LAWRENCE STARK.
The relationships between saccadic velocity,
duration, and magnitude have been used to prove
the normalcy of saccades with intersaccadic intervals of less than 200 ms. Pairs of normal saccades with small intersaccadic intervals will have
the second saccade larger or smaller and going in
the same or the opposite direction than the first
saccade. These normal saccades may be horizontal, vertical, or oblique.
Saccades are the fast, staccato eye movements
characteristically exhibited by people who are
reading or looking about a scene. These saccades
are usually spaced about 200 ms. apart; however,
Fig. 1 shows normal saccades with smaller intersaccadic intervals. The important and consistent
relationships between duration, maximum saccadic velocity, and saccadic amplitude, called the
main sequence1 and shown in Fig. 2, are used to
prove the normalcy of contiguous saccades.
Young and Stark11 realized that most saccades
are separated by about 200 ms. so they modeled
the saccadic eye movement system as a sample
data system with a sampling interval of 200 ms.
317
Many experimenters have observed infrequent
saccadic pairs with smaller intersaccadic intervals.
There are reports of saccades with intersaccadic
intervals as small as 160 ms.,'1 120 ms.,4 100 ms.,r>
75 ms.,'1 70 ms.,7 50 ms.,s 40 ms.,9 10 ms.,10 and
0 ms. M " i;i To elicit closely spaced saccades, some
experimenters'- '-• i:i used pulse-step stimuli—for
example, the target could jump five degrees right,
pause for 50 ms., then jump another three degrees
right. Other experimenters recorded more natural
saccades during reading,9 fixation,* or in response
to a step change in target position.•'<• 10 A variety
of other effects can evoke closely spaced saccades:
for instance, fatigue/' flickering illumination,r>
voluntary pauses,7 and abrupt decelerations of
a moving target.11 In all of these reports, the
second saccade was usually smaller, and in the
same direction as the first saccade. One reporter11
emphasized that the second saccades of his pairs
were abnormal, while the others made no mention
of normalcy. Our present findings indicate that no
refractory period is necessary in order for the
subsequent saccadic eye movement to be normal.
Methods. The infrared photodiode method of
eye position measurement1' - was used to record
the saccades of this report. The photodiodes were
mounted on a pair of spectacle frames worn by
the subject. The subject's head was stabilized
with a head rest and a bite bar covered with
dental impression compound. Saccades as small
as three minutes of arc have been measured with
this equipment.1 The bandwidth of the complete
system, including photodiodes, direct current,
(DC) amplifiers, computerized velocity algorithm,
computerized slow-down plotting routine, and the
X-Y plotter was in excess of 1,000 Hz. The data
of Fig. 3 are for five normal unfatigued subjects.
The saccades of Fig. 1 were made while tracking
a spot of light that jumped periodically, with a
frequency of 0.33 Hz., between various predetermined pairs of points. The oblique saccadic
eye movements of Fig. 4 were made while repetitively saccading between two continuously observable targets. We have also recorded closely spaced
saccades using electro-oculography (EOG). Corneal reflection,1- (;- *• ° EOC,3- 10- i a . i;t suction contact lens,"1 photodiode,11 and psychophysical7 techniques have been used by others to record closely
spaced saccades.
Results. Fig. 1 shows naturally occurring, normal,
human saccades with successively smaller intersaccadic intervals (ISI). ISI is defined as the time
between saccadic eye movements; more specifically, the time between the end of the first saccade,
until the beginning of the next saccade. This
definition of ISI is illustrated in the idealized
drawing of closely spaced saccades near the
vertical axis of Fig. 3. We have recorded many
saccadic pairs with small intersaccadic intervals;
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