Muller Cell Expression of Gliol Fibrillory Acidic Protein offer Genetic ...

Muller Cell Expression of Gliol Fibrillory Acidic Protein
offer Genetic and Experimental Photoreceptor
Degeneration in the Rat Retina
Amy J. Eisenfeld, Ann H. Bunr-Milam, and P. Vijoy Sarrhy
Glial fibrillary acidic protein (GFAP) is normally found in astrocytes. In the normal rat retina at all
ages, only astrocytes stain for GFAP. This staining pattern is also found in RCS rats with inherited
retinal dystrophy younger than 38 days. Beginning on day 38, when about 61% of the photoreceptors
have degenerated, a few GFAP-positive fibers span the retina from the inner limiting membrane to
the external limiting membrane. By day 41 and at all later ages examined, the radial fibers of Muller
cells are labeled throughout the retina. To determine if the expression of GFAP in Muller cells is a
response to photoreceptor necrosis or might be a direct effect of the mutant gene, we induced
photoreceptor degeneration in normal, adult Sprague-Dawley rats by exposing them to constant light
for variable periods of time. After 3 days in constant light, there is a 20% reduction in the number
of photoreceptors and many Muller cells are positive for GFAP. Immunoblot studies confirmed that
the anti-GFAP reacted with a single protein from retina that corresponded in molecular weight and
Triton-insolubility to GFAP. The immunoblots also corroborated the results from anti-GFAP
immunostaining of control and experimental retinas. These results indicate that Muller cells express
GFAP immunoreactivity in response to experimentally as well as genetically induced photoreceptor
degeneration. Invest Ophthalmol Vis Sci 25:1321-1328, 1984
Mtiller cells are the major type of non-neuronal
cells in the vertebrate retina. Although they are
morphologically similar to radial glia and Bergmann
glial cells, unlike these cells, the Muller cells normally
do not express the glial cell-specific protein, glial
fibrillary acidic protein (GFAP). 1 " 4 However, it has
been reported that Muller cells accumulate GFAP in
response to neuronal injury 1 ' 2 and degeneration. 3 - 4
We have used antibodies to GFAP and indirect
immunofluorescence to examine the accumulation of
GFAP in Muller cells in response to genetically
induced photoreceptor degeneration in the Royal
College of Surgeons (RCS) rat. The RCS rat has an
inherited retinal dystrophy resulting in the loss of
photoreceptors between the ages of 18 and 60 days. 5
Since any alterations in Mtiller cells might be a direct
effect of the mutant gene, we also examined Muller
cells in Sprague-Dawley rats maintained in constant
From the Department of Ophthalmology, University of Washington
School of Medicine, Seattle, Washington.
Supported in part by NIH Research grant nos. EYO7O13,
EY01311, EY03523, EY03664, and EYO173O and in part by an
unrestricted grant from Research to Prevent Blindness, Inc. Dr.
Bunt-Milam is the recipient of a William and Mary Greve International
Scholar Award from Research to Prevent Blindness, Inc.
Submitted for publication: April 13, 1984.
Reprint requests: Amy J. Eisenfeld, PhD, Department of Ophthalmology
RJ-10, University of Washington, Seattle WA 98195.
light, a condition that causes loss of photoreceptors
in albino rats. 6 These models of photoreceptor degeneration
result in loss of photoreceptors with little
damage to the remainder of the retina. 6 ' 7 It was of
interest to determine if the different forms of photoreceptor
degeneration might result in any differences
in the time course of appearance of GFAP in the
Muller cells.
Animals
Materials and Methods
Pink-eyed dystrophic (RCS) rats (from breeding
pairs provided by Dr. Matthew La Vail, University of
California, San Francisco), maintained in a 12-hr
light/ 12-hr dark environment, were used as a model
for inherited photoreceptor cell degeneration. Rats of
the same ages from a congenic strain without inherited
retinal dystrophy (RCS-rdy + ) were used as controls.
All procedures involving animals were performed in
adherence to the ARVO Resolution on the Use of
Animals in Research.
Photoreceptor degeneration was experimentally induced
in 45-day-old Sprague-Dawley rats (Bellevue,
WA) by placing them in constant light (CL) for
periods of 1 day to 8 weeks. These rats were kept in
transparent cages with stainless steel wire bar covers.
In addition to 24-hr overhead room light, two lamps,
1321

1322 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / November 1984 Vol. 25
each containing two 15-watt fluorescent bulbs were
positioned 18 cm above the bottom of the cage.
These conditions resulted in an incident luminance
of approximately 200-ft candles at the floor of the
cage. The temperature in the cage was 24 ± 1 °C.
Age-matched Sprague-Dawley rats maintained in a
12-hr overhead room light/ 12-hr dark environment,
were used as controls.
All rats were enucleated under ether anesthesia
between 1:00 and 2:30 PM. After a slit was made in
the cornea, the lens and vitreous were removed and
the globe was immersed in 4% formalin in 0.13 M
phosphate buffer (pH 7.4).
Immunofluorescence
After 6 hr in fixative at room temperature, the
eyes were bisected, transferred to 30% sucrose in 0.13
M phosphate buffer and stored at 4°C overnight.
Sections were cut at a thickness of 20 nm using a
cryostat at —20°C. The sections were mounted on
chrome alum-gelatin coated slides and air-dried overnight
at room temperature. Plastic rings (0.75-cm
diameter) were mounted with fingernail polish around
the sections to form incubation wells. Each well
contained an experimental section of retina (RCS or
CL-damaged) and a control section. The sections
were treated for 10 min at room temperature with
1% goat serum and 4% bovine serum albumin (BSA)
in phosphate buffered saline (PBS) followed by overnight
incubation at 4°C in GFAP antiserum diluted
1:100 in PBS containing 0.3% Triton X-100. The
GFAP antiserum, provided by Dr. Larry Eng (Veterans
Medical Center; Palo Alto, CA), was raised in
rabbits against GFAP obtained from multiple sclerosis
plaques. 8 Control sections were treated identically
with an IgG fraction from preimmune rabbit serum.
Sections were washed twice (15 min each) with PBS
at room temperature and incubated for 30 min at
room temperature in the dark in sheep anti-rabbit
IgG-fluorescein isothiocyanate (Cappel Laboratories),
diluted 1:50 in PBS with 0.3% Triton X-100. After
two 10-min washes in phosphate buffer, the plastic
rings were removed and the sections were coverslipped
with 80% glycerol in 0.13 M phosphate buffer containing
5% n-propyl gallate. 9 The sections were examined
with a Zeiss microscope equipped for epifluorescence.
Measurement of Outer Nuclear Layer
After enucleation, eyes were stored in fixative at
4°C overnight. They were bisected vertically just
temporal to the optic nerve head. The bisected eyes
were washed for several hours in phosphate buffer
and then dehydrated through a graded series of
ethanol. The half of the eye including the optic nerve
head was embedded in plastic (Sorvall Embedding
Medium), with the cut edge of the eyecup placed flat
to allow for sectioning along the inferior-superior
plane, including the optic nerve head. In some cases
the eyes were bisected after 6 hr in fixative, and the
half without the optic nerve head was processed for
immunofluorescence, while the remaining half was
stored in fixative overnight.
Sections were cut at a thickness of 2.5 ^m on a
Sorvall JB-4 microtome and stained for 30 sec with
10% Richardson's stain. A section through the optic
nerve head from each eye was chosen and the thickness
of the outer nuclear layer was measured 250
ixm, 500 fim, and 750 /xm from the optic nerve head
in the superior and inferior hemispheres. The mean
and standard error of the mean of these six measurements
were calculated.
Preparation of Triton X-100 Insoluble Proteins
Triton-insoluble proteins were obtained from retina
according to Pruss et al. 10 Four to six retinas were
homogenized in ice-cold PBS containing the protease
inhibitors p-chloromercuribenzoate, phenyl methane
sulfonyl fluoride and o-phenanthraline, each at 1
raM concentration. The homogenate was centrifuged
at 8000 g for 10 min at 4°C. The pellet was extracted
with PBS containing 0.6 M KC1, 0.5% Triton X-100
and the protease inhibitors, and centrifuged at 8000
g for 10 min at 4°C. After a second extraction with
Triton X-100, the pellet was washed four times in
ice-cold PBS and solubilized by boiling in SDSpolyacrylamide
sample buffer." In the light damage
experiments, Sprague-Dawley rats had been exposed
to constant light for 3 or 7 days. Control animals
had been kept in 12-hr light/12-hr dark cycle. RCS
rats were 45 and 70 days old. Congenic, 45-day-old
^y" 1 " rats were used as controls.
Polyacrylamide Gel Electrophoresis and
Electroblotting to Nitrocellulose
One dimensional SDS-polyacrylamide gel electrophoresis
(PAGE) was performed according to the
procedure of Fairbanks et al" using protein standards
ranging in molecular weight from 15-94,000. Proteins
were transferred from PAGE gels to nitrocellulose
membranes (BIORAD) in a Hoefer Transphor electrophoresis
apparatus at 0.8 mV for 1 hr at room
temperature. 12 After 1 hr blocking in Tris-buffered
saline (TBS) containing 3% BSA, the blots were
treated overnight in anti-GFAP diluted in TBS and
1% BSA. After several washes, the blots were incubated
with goat anti-rabbit IgG (Cappel) for 1 hr (1:1000
dilution in TBS and 1% BSA). Following a 30-min
exposure to the peroxidase-antiperoxidase complex

No. 11 GFAP IN MULLER CELLS / Eisenfeld er al. 1023
Fig, 1. Light micrographs of normal, constant light-damaged and RCS rat retinas. A, Normal Sprague-Dawley rat retina. O, outer
segments; ON, outer nuclear layer; IN, inner nuclear layer; 1, inner plexiform layer; G, ganglion cell layer. B, Sprague-Dawley rat retina
after 3 days in constant light. C, 38-day-old RCS rat. Note the decreased thickness of the ON. The inner retina appears normal.
(1:500 in PBS and 1% BSA), the blots were stained
in Tris-saline containing 4-chloronaphthol (0.5 mg/
ml) and hydrogen peroxide (0.025%). 13
Results
Thickness of Outer Nuclear Layer
Examples of normal, CL-damaged and RCS retinas
(Figs. 1A-C) illustrate the extent of photoreceptor
degeneration. The amount of damage caused by a
3-day exposure of CL was somewhat variable from
animal to animal. The thickness of the outer nuclear
layer of a control Sprague-Dawley rat was 46.0 ± 1.0
/xm (mean ± SEM, n = 3) while for rats kept in CL
for 3 days it ranged from 22-47 nm with a mean of
37.7 ± 2.7 nm (n = 11), representing a 20% loss of
photoreceptors (Fig. IB). In the 38-day-old RCS-rdy +
rats without inherited retinal degeneration, the thickness
of the outer nuclear layer was 45.6 ± 1.0 jum (n
= 6). In 38-day-old RCS retinas with inherited retinal
degeneration, the outer nuclear layer thickness was
only 17.8 ± 0.6 ^m (n = 7), representing an average
reduction by 61% (Fig. 1C). No abnormalities were
apparent in the inner retina in either condition of
photoreceptor degeneration (Fig. IB, C).
Immunofluorescence
Sections treated with preimmune serum showed
only autofluorescence that was pale green for the
neural retina and yellow for erythrocytes (Figs. 2A>
3A). In control Sprague-Dawley rats, GFAP staining
was confined to filamentous structures in the innermost
retina, including the nerve fiber and ganglion
cell layers and encircling blood vessels (Fig. 2B).
GFAP positive cells were also abundant in the optic
nerve head. From their location and morphology,
these GFAP positive cells were interpreted as astrocytes.
In some cases, there was a light, finely particulate
staining in the outer segment layer.
After 1 day in CL, the GFAP staining did not
differ from that in control retinas. After 3 days in
CL, variable numbers of radially oriented processes
were stained, some more strongly than others. These
processes extended from the inner limiting membrane
through the inner plexiform and inner nuclear layers,
with occasional positive fibers in the outer nuclear
layer (Fig. 2C). This pattern of staining closely
matched the distribution of Muller cell processes and
appeared to represent the appearance of GFAP reactivity
in Muller cells. Staining was most intense

1324 INVESTIGATIVE OPHTHALMOLOGY & VI5UAL SCIENCE / November 1984 Vol. 25
Fig. 2, Fluorescence micrographs of control and constant light-damaged retinas treated with antibodies to GFAP. A, Control section,
exposed to constant light for 3 days and treated with preimmune serum. B, Normal retina. Only astrocytes (—•) stain for GFAP. C, Retina
exposed to constant light for 3 days. Miiller cell processes (—') express GFAP immunoreactivity. D, Retina exposed to constant light for 2
weeks. Miiller cells (—>) stain for GFAP. ON, outer nuclear layer (X576).
against the inner limiting membrane, and in the
ganglion cell and innermost inner plexiform layer.
Although the amount of photoreceptor cell loss varied
from animal to animal after 3 days in CL, every
retina examined showed Miiller cell staining. The
staining pattern remained the same at 2 weeks in CL

No. 11 GFAP IN MULLEP, CELLS / Eisenfeld er d. 1325
tig. 3. Huorescence micrographs of RCS retinas treated with antibodies to GFAP. A, Control section. A 38-day-old retina treated with
preimmune serum. B, A 25-day-old RCS retina. Only astrocytes (—») express GFAP immunoreactivity. C, A 38-day-old RCS retina. Muller
cell processes (—») first stain for GFAP at this age. D, A 6-month-old RCS retina. At this later stage of degeneration, Muller cells (—•) stain
intensely for GFAP. Accumulations of lipofuscin (•) are seen in the pigment epithelium. ON, outer nuclear layer (X576).
(Fig. ID) and at 8 wk when very few photoreceptors
could be found.
In the RCS rat, the GFAP staining was restricted
to the astrocytes until day 32, as seen in control
retinas (Figs. 2B, 3B). Beginning on day 32, an
occasional Muller fiber stained lightly for GFAP.
Muller cell processes throughout the retina were
stained consistently with anti-GFAP only after day
38 (Fig. 3C). At this time the Muller end feet and
innermost Muller radial processes stained most intensely.
As the photoreceptor degeneration progressed,
the number of GFAP positive fibers increased, and
they spanned the retina from the inner to the external
limiting membranes. At advanced stages of degeneration
(6 months; Fig. 3D), the Muller processes were
thickened and very heavily stained.
Characterization of anti-GFAP
In order to ascertain that the protein stained by
anti-GFAP was indeed GFAP, electroblot analysis
was performed on proteins from normal and degenerated
retinas. Results from the anti-GFAP electroblot
experiments are presented in Figure 4. Although

1
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No. 11 GFAP IN MULLER CELLS / Eisenfeld er ol. 1327
Fig. 4. Characterization of GFAP antibody in normal, constant light-damaged (A) and RCS (B) retinas. Lanes 1-3 are SDS-polyacrylamide
gels stained with Coomassie Blue. Lanes 4-6 are PAP stained immunoblots. Molecular weights calculated from protein standards are shown
on the left. A, Constant light-damaged retinas. Lanes (1, 4), normal retina; (2, 5) retina exposed to constant light for 7 days; (3, 6) retina
exposed to constant light for 3 days. B, RCS retinas; Lanes (1,4) normal retina; (2, 5) 40-day-old RCS retina; (3, 6) 70-day-old RCS retina.
several protein bands were seen in the Coomassie photo-oxidation and retinol-induced membranolysis. 18
shed outer segments. 1516 This leads to accumulation
References
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Blue-stained acrylamide gels, anti-GFAP stained only
one or at most two adjacent bands. Using molecular
weight markers, the size of the anti-GFAP reacting
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These unrelated mechanisms leading to photoreceptor
death might result in a different time course of GFAP
accumulation in Muller cells.
(3) Finally, there might be a direct effect on other
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of GFAP was present in both RCS-rdy + and the CL hypothetical at the present time.
control retinas. This was in accord with the immunocytochemical
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GFAP increased with progressive photoreceptor loss
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Our results provide further evidence that Muller
cells express GFAP immunoreactivity following degeneration
of apparently a single cell type, the photoreceptor.
The time course of GFAP expression here
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Discussion
wounds of the eye. 1 The different time course of
Muller cell gliosis in the RCS rat, as well as the actual
The initial appearance of GFAP immunoreactivity
significance of increased GFAP expression in astrocytes
and Muller cells in pathologic conditions 820 are
was seen in Miiller cells from CL and RCS retinas
only after substantial loss of photoreceptors. This
topics for future study. This study has shown that
loss, as determined by measurements of the outer
retinas with environmentally and genetically caused
nuclear layer, reflected a 20% decrease in CL damaged
photoreceptor degeneration may provide useful models
for the elucidation of Muller cell functions, in-
retinas and a 61% decrease in RCS rats. There are
several possible explanations for this apparent difference
in the degree of photoreceptor loss before GFAP
cluding reaction to injury. The ability to induce
accumulation of GFAP in a cell type not normally
reactivity was detected.
expressing this protein should facilitate the study of
(1) The outer nuclear layer thickness measurements
the mechanisms of reactive gliosis in other parts of
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the central nervous system.
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remaining photoreceptors was not monitored, even a
morphologically normal photoreceptor might already
Key words: Muller cells, glial fibrillary acidic protein,
photoreceptor degeneration, RCS rat, light damage
be altered functionally. Therefore, the outer nuclear
layer thickness might not be an accurate measure of
the state of degeneration. 14
(2) Although both conditions resulted ultimately
Acknowledgments
The authors wish to thank Dr. Larry Eng for the antiserum
to GFAP; Dr. Matthew La Vail for the RCS rats; Dr. J. C.
in the loss of photoreceptors, the etiologies of the two Saari for critical review of the manuscript; Mr. G. Garwin
and Ms. I. Klock for technical assistance; Mr. B. Clifton
forms of degeneration are thought to differ. In the
and Ms. D. Cannon for photographic help; and Ms. J. Seng
RCS rat, the genetic defect has been localized to the for secretarial assistance.
pigment epithelial cell, which is unable to phagocytose

1328 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / November 1984 Vol. 25
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