(Investigative Ophthalmology and Visual Science. 2000;41:552-560.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Expression of Ciliary Neurotrophic Factor Activated by Retinal Müller Cells in Eyes with NMDA- and Kainic Acid–Induced Neuronal Death
Megumi Honjo1,
Hidenobu Tanihara2,
Noriaki Kido1,
Masaru Inatani1,
Kazushiro Okazaki1 and
Yoshihito Honda1
1 From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan and
2 Department of Ophthalmology, Tenri Hospital, Tenri, Japan.
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Abstract
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PURPOSE. To elucidate the role of retinal Müller cells in
N-methyl-D-aspartate (NMDA)- or kainic acid
(KA)-induced retinal damage.
METHODS. In experimental eyes, NMDA or KA was injected into the vitreous of rat
eyes. Immunohistochemistry and western blot analysis were conducted to
elucidate expression and localization of glial fibrillary acidic
protein (GFAP) and ciliary neurotrophic factor (CNTF). In addition, the
neuroprotective effects of CNTF were calculated by counting cells in
the ganglion cell layer (GCL) and by measuring the thickness of the
various retinal layers.
RESULTS. Morphometric analysis of retinal damage in NMDA- and KA-injected eyes
showed significant cell loss in the GCL and thinning of the inner
plexiform layer (IPL) of the retina, but not of other retinal layers.
Immunohistochemistry demonstrated disappearance and/or decrease in
immunoreactivities of calbindin- and calretinin- positive cells and
their neurites and upregulated expression of both GFAP and CNTF in
experimental eyes. Western blot analysis showed an increase in protein
expression for CNTF in retinas of experimental eyes. Confocal images
and sequential localization demonstrated colocalization of CNTF and
GFAP in the inner retinal layer and possibly in Müller cells. In
addition, pretreatment with CNTF (1 µg) before the intravitreal
injection of NMDA (or KA) demonstrated that CNTF has neuroprotective
effects against NMDA- or KA-induced neuronal death in the retina.
CONCLUSIONS. These studies revealed the upregulated expression of CNTF and GFAP in
Müller cells in response to NMDA- and KA-induced neuronal death,
suggesting that production of CNTF in Müller cells may be a part
of the endogenous neuroprotective system in the
retina.
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Introduction
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Upregulated expression of neuroprotective molecules plays an
important role in fending off various insults to neural
tissues.1
2
3
4
Ciliary neurotrophic factor (CNTF) is a
representative neuroprotective molecule that was initially isolated
from the eye,5
and upregulated expression of CNTF has been
reported in eyes subjected to mechanical injury,6
7
light
exposure,8
and ischemia.9
Because
intravitreal injection of this neurotrophic factor protects retinal
neurons from various insults such as axotomy,10
ischemia,11
inherited retinal degeneration and light
induced retinal degeneration,12
upregulation of CNTF has
been hypothesized to play an important role in the endogenous
neuroprotective system.6
7
8
9
13
On the other hand, changes
in the expression and distribution of glial fibrillary acidic protein
(GFAP) in retinal glial components have been reported in eyes with
various diseases and/or injuries, including glaucoma,14
retinal detachment,15
light-induced damage,16
retinal degeneration,17
mechanical injury18
and ischemia.19
Because upregulation of GFAP occurs in
response to a number of conditions, as noted above, this may represent
a responsive mechanism elicited by stress or injury in the retina.
Because the intraocular origin of CNTF expression is hypothesized to be
glial cells, astrocytes, and Müller cells, the number of
GFAP-positive cells may reflect the number of CNTF-expressing
cells.6
7
It is well known that glutamate receptor–related neurotoxicity can be
induced by N-methyl-D-aspartate (NMDA) and
kainic acid (KA),20
21
22
and herein, we report upregulated
expression of CNTF in retinal Müller cells in response to NMDA-
or KA-induced retinal damage.
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Materials and Methods
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Animals
Experiments were performed on adult male Sprague–Dawley rats
(200–300 g). All animals were killed by an intraperitoneal overdose
injection of pentobarbital. Animals were given water and food ad
libitum. All studies were conducted in accordance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research.
Animal Models of NMDA- and KA-Induced Neuronal Death
NMDA and KA were obtained from Sigma (St. Louis, MO), and rat CNTF
was obtained from Genzyme (Cambridge, MA). Animal models were made in a
manner similar to that described by Morizane et al.23
Briefly, rats were anesthetized with an intraperitoneal injection of 50
mg/kg of sodium pentobarbital. The pupil was dilated with phenylephrine
hydrochloride and tropicamide drops, and a single dose of 5 µl of 40
mM NMDA (total amount, 200 nmol) or 5 µl of 1 mM KA (total amount, 5
nmol) was injected into the vitreous space. A microsyringe with a
30-gauge needle was inserted 2 mm behind the limbus at the
superotemporal quadrant and directed toward the optic nerve. When the
tip of the needle reached the midvitreous, the injection was
administered in a single, swift action. All procedures were performed
under microscopy. In this series, rats that received only an injection
of 5 µl of 0.1 M phosphate-buffered saline (PBS) served as controls.
Morphometric Analysis
Morphometric analysis was carried out in a manner as described
previously.23
Briefly, 7 days after NMDA or KA injection,
animals were killed by an intraperitoneal overdose injection of
pentobarbital, and the eyes were enucleated. Eyes were immersed
overnight at 4°C in fixative solution containing 2.5% glutaraldehyde
and 2% formalin in 0.1 M phosphate buffer (pH 7.4), followed by
fixation at 4°C in 10% formalin in 0.1 M phosphate buffer (pH 7.4)
for at least 24 hours, followed by dehydration and paraffin embedding.
Transverse sections of the rat retinas, 3-µm thick, were made through
the optic disc. The sections then were stained with hematoxylin
and eosin and subjected to morphometric analysis. The extent of NMDA-
or KA-induced retinal neuronal death was quantified by counts of cells
in the ganglion cell layer (GCL) and the thickness of the various
retinal layers, such as the inner plexiform layer (IPL), inner nuclear
layer (INL), outer plexiform layer (OPL), and outer nuclear layer (ONL)
at a distance of 1.0 to 1.5 mm from the optic disc. Units for numerical
values were cell number per millimeter for GCL cells and per micrometer
for thickness of retinal layers. Data from three sections were averaged
for each eye and compiled. The results were expressed as the means ± SE. All data were analyzed by analysis of variance (ANOVA), and
appropriate group comparisons between PBS-injected control eyes and
experimental eyes were performed.
Antibodies
The following specific antibodies were used: rabbit polyclonal
antibody to human GFAP (Dako Japan, Kyoto, Japan); mouse monoclonal
antibody to rat CNTF (Boehringer Mannheim, Mannheim, Germany); mouse
monoclonal anti-calbindin D antibody (Sigma Chemical Co.); rabbit
polyclonal antibody to calretinin (Chemicon International, Temecula,
CA), biotinylated anti-rat CNTF antibody (R&D Systems Inc.,
Minneapolis, MN). Appropriate secondary antibodies labeled with
dichlorotriazinylamino fluorescein or indocarbocyanin dyes Cy3 were
obtained from Chemicon International.
Immunohistochemical Procedures
Adult rats were perfusion-fixed with 4% paraformaldehyde/PBS
before enucleation. Subsequently, the enucleated eyes were further
fixed for 2 hours at 4°C in 4% paraformaldehyde/0.1 M PBS, washed
for 5 minutes in PBS, then gently shaken overnight at 4°C in 15%
sucrose/0.1 M PBS, embedded in Tissue-Tek (Miles, Inc., Elkhart, IN),
and frozen in liquid nitrogen. Sections (10 µm) were cut on a
cryostat and collected onto silanized slides (Dako Japan), and
air-dried.
The samples were incubated successively in methanol at -20°C for 20
minutes, in 5% skim milk in PBS ("blocking solution") for 30
minutes, and in a solution of antibodies to CNTF (1:50 dilution in
blocking solution) (Boehringer Mannheim), GFAP (1:100 dilution in
blocking solution), calbindin-D (1:500 dilution in blocking solution),
calretinin (1:500 dilution in blocking solution), for 60 minutes at
room temperature. They were then treated for 30 minutes with secondary
antibodies (Cy3-conjugated anti-mouse IgG or anti-rabbit IgG) diluted
1:200. For double-label immunostaining, the same procedures were
repeated. Sections were mounted in 90% glycerol/10% PBS. The
secondary antibodies used for double-staining experiments were labeled
with Cy3 for CNTF and fluorescein isothiocyanate (FITC) for GFAP. As
for immunohistochemistry for CNTF and GFAP, sequential sections were
stained with each antibody as well as double-stained sections.
Fluorescence was visualized under an epifluorescence microscope (Zeiss
Axioplan, Oberkochen, Germany) and with a confocal laser scanning
microscope (Bio-Rad Laboratories, Hercules, CA). To display results
from double-labeled sections simultaneously, confocal images were
color-coded and superimposed. After immunostaining of the 10-µm-thick
transverse sections of rat retinas, immunoreactive cells at a
determined layer were counted at a distance of 1.0 to 1.5 mm from the
optic disc. Units for numerical values were cell number per millimeter.
Data were obtained from at least six eyes, and the results are shown as
the means ± SE. P values and statistical significance
were calculated by ANOVA between PBS-injected control eyes and
experimental eyes.
Western Blot Analysis
For western blot analysis, 7 days after NMDA or KA injection,
animals were killed by an intraperitoneal overdose injection of
pentobarbital, and the eyes were enucleated. The animals that received
PBS injection served as controls. The retinal tissues were isolated
with fine forceps and Vannas scissors under a dissecting microscope.
Then samples were lysed in sample buffer containing 2% sodium
dodecylsulfate (SDS) and were subjected to SDS-polyacrylamide gel
electrophoresis (PAGE) using a polyacrylamide gradient gel (15%–25%)
(Daiichi Pure Chemicals, Tokyo, Japan). Each sample was containing 50
µg protein. The concentration of each sample was measured by Bio-Rad
DC protein assay (Bio-Rad Laboratories). After electrophoresis, the
proteins were electrophoretically transferred onto polyvinylidene
difluoride membranes (Millipore Co., Bedford, MA). After blocking the
membrane with 5% skim milk and 0.002% Tween 20 in Tris-buffered
saline (TBS; pH 7.4) at 4°C for 16 hours, the membrane was incubated
in a 1:500 dilution of biotinylated anti-rat CNTF antibody (R&D Systems
Inc.) for 2 hours at room temperature and then washed with TBS five
times for 5 minutes. The membrane was then incubated with ABC solution
(ABC Elite kit; Vector, Burlingame, CA). After washing the membrane
with TBS for 5 minutes five times, the blotted protein bands were
stained with dimethylaminoazobenzene (DAB) solution. Optical
densities of the labeled bands were measured by a Power Macintosh G3
computer (Apple Computer, Cupertino, CA) and NIH Image 1.59.
Pretreatment with CNTF
A single injection of 1 µl CNTF at two concentrations (0.1 or 1
µg/µl) was administered into the vitreous space using a 30-gauge
needle in the same manner as described above. Two days later, a single
injection of 5 µl of 40 mM NMDA (200 nmol) or 5 µl of 1 mM KA (5
nmol) was administered into the vitreous space in a single dose. One
week later the animals were killed, and the eyes were prepared for
histologic examination. Morphometric analyses and immunohistochemical
studies were conducted as described above. The control animals received
injection of 1 µl of vehicle (PBS) 2 days before NMDA or KA injection
and were killed 1 week later in the same manner.
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Results
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Morphometric Analysis of NMDA- and KA-Induced Retinal Damage
In eyes that underwent intravitreal injection of NMDA (200 nmol),
cell loss in the GCL and thinning of the IPL were observed. Statistical
analysis of results obtained from the morphometric studies showed
significant differences between experimental and control eyes in cell
counts in the GCL and thickness of the IPL (P < 0.0001
and P < 0.0001, ANOVA), but not for other layers; that
is, INL, OPL, and ONL (Fig. 1A
). Furthermore, similar analyses of eyes that received intravitreal
injections of KA (5 nmol) showed statistically significant differences
between experimental and control eyes in cell counts in the GCL and
thickness of the IPL (P < 0.0001 and P < 0.0001, ANOVA), but not for other layers (Fig. 1B)
.
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Figure 1. Morphometric analysis of glutamate receptor–mediated retinal damage on
the seventh posttreatment day. In NMDA (200 nmol)-injected eyes
(A) and kainic acid (5 nmol)-injected eyes (B),
the degree of NMDA-induced retinal neuronal death was quantified by
cell counts (cell number/mm) of cells present in the ganglion cell
layer (GCL) and the thickness (µm) of retinal layers such as the
inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform
layer (OPL) and outer nuclear layer (ONL) at a distance of 1.0 to 1.5
mm from the optic disc. Units for numerical values were cell number per
millimeter for GCL cells and per micrometer for thickness of retinal
layers. Data from three sections were averaged for each eye. The
results are shown as the means ± SE (n = 10).
P values and statistical significance were calculated by
ANOVA between PBS-injected control eyes and experimental eyes.
Morphometric analysis showed statistically significant cell loss in GCL
cells (P < 0.0001) and thinning of IPL thickness
(P < 0.0001). NS, not significant.
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Immunohistochemical Studies for Calcium-Binding Proteins
In PBS-injected (control) eyes, immunohistochemical studies
demonstrated that calretinin immunoreactivities were seen in cells
located in the GCL and INL as well as in axons of the nerve fiber layer
(NFL) and IPL. Furthermore, three calretinin-positive layers were
observed within the IPL (Fig. 2A
). This result seemed to be in agreement with previously reported
data.24
In NMDA-injected eyes, there was a significant
loss of GCL cells and partial cell components in the INL, and more
intense staining of cells in the INL and small granulelike
immunostaining in the IPL were seen (Fig. 2B) . In experimental eyes
using intravitreal injection of KA, no calretinin-positive bands were
observed, although small granulelike immunostaining was seen in the IPL
(Fig. 2D)
. In control eyes, immunoreactivities of calbindin, another
calcium-binding protein and cell type marker for amacrine and
horizontal cells,24
25
26
27
were faint in cells at the
vitreous side of the INL and much more conspicuous in cells of the
outer layer of the INL (Fig. 2F) . This result seemed to be in good
accordance with previous reports.24
25
26
27
One week after
intravitreal injection of NMDA, numerous calbindin-positive cells in
the inner side of the INL (possibly amacrine cells) showed
disappearance and/or decrease in immunoreactivities although
quantification of the cells was difficult because of the faint
immunoreactivities (Fig. 2G)
. In contrast, the population of
calbindin-positive cells in the outer side of the INL (possibly
horizontal cells) in experimental eyes seemed to be same as in control
eyes. A similar observation was confirmed by experiments using
intravitreal injection of KA (Fig. 2I)
.
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Figure 2. Immunohistochemistry for calcium-binding proteins in retina.
(A through E) Light microscopic photographs show
representative immunohistochemistry for calretinin. In PBS-injected
control eyes (A), calretinin immunoreactivities were seen in
cells in the GCL and INL, in addition to their neurites. Three
calretinin-positive layers were observed in the IPL of control eyes. In
NMDA (200 nmol)-injected eyes (B), at the first
posttreatment week, significant loss of GCL cells and partial cell
components in the INL were found, and calretinin-immunoreactive small
granulelike immunostaining was observed. On the other hand, in KA (5
nmol)-injected eyes (D), no calretinin-positive bands were
observed although small granulelike immnunostaining was found in the
IPL (D). With pretreatment of CNTF, loss of small
granulelike immunostaining was recovered (C). This
observation was confirmed by experiments using intravitreal injection
of KA (D, E). (F through J)
Light microscopic photographs showed representative
immunohistochemistry for calbindin. In PBS-injected control eyes
(F), immunoreactivities of calbindin were found in cells of
the INL. In NMDA (200 nmol)-injected eyes (G), at the first
posttreatment week, the number of calbindin-positive cells in the inner
side of the INL decreased, but the population of calbindin-positive
cells in the outer side of the INL (possibly horizontal cells) in
experimental eyes seemed to be same as in control eyes. This
observation was confirmed by experiments using intravitreal injection
of KA (I). Pretreatment with CNTF showed no significant
protective effect against decreasing the number of calbindin-positive
cells (H, J). NFL, nerve fiber layer; GCL,
ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear
layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Bar, 50
µm.
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Immunohistochemical Studies for GFAP and CNTF
In PBS-injected (control) eyes, immunohistochemical studies
using polyclonal antibodies against GFAP showed immunoreactivity in
some of the longitudinal cells embedded in the nerve fiber layer, which
might be astrocytes, but no immunoreactivity was found in the outer
layer of the retina (Fig. 3A
). In contrast, in the experimental eyes that had received an
intravitreal injection of NMDA, faint radial staining was seen at early
stage (3 days after NMDA injection) (Fig. 3B)
, and strong
immunoreactivity against GFAP was found in the inner layer (NFL and
GCL), and radial staining was seen throughout the retina at later stage
(7 days after NMDA injection) (Fig. 3C)
. This was confirmed in other
experimental eyes that had undergone intravitreal injection of KA
(Figs. 3D
3E)
.
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Figure 3. Immunohistochemistry for glial fibrillary acidic protein (GFAP) and
ciliary neurotrophic factor (CNTF). Upregulated immunoreactivities of
GFAP were shown in both NMDA- and KA-injected eyes.
Arrowheads indicate the NFL. (A through
E) Light microscopic photographs showed representative
immunohistochemistry for GFAP. (A) PBS-injected control
eyes; (B) 3 days after NMDA (200 nmol) injection;
(C) 7 days after NMDA (200 nmol) injection; (D) 3
days after KA (5 nmol) injection; (E) 7 days after KA (5
nmol) injection. (F through J) Light
microscopic photographs showed representative immunohistochemistry for
CNTF. Upregulated immunoreactivities of CNTF were shown in both NMDA-
and KA-injected eyes. (F) PBS-injected control eyes;
(G) 3 days after NMDA (200 nmol) injection; (H) 7
days after NMDA (200 nmol) injection; (I) 3 days after KA (5
nmol) injection; (J) 7 days after KA (5 nmol) injection.
Bar, 50 µm.
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Additional immunohistochemical studies were conducted in an
attempt to elucidate the expression of CNTF in rat retinas. In control
eyes, no CNTF immunoreactivity was detected, save for faint
immunostaining in the NFL (Fig. 3F)
. However, as early as 3
days after treatment with NMDA, CNTF immunoreactivity became detectable
in the NFL and individual cells in the INL. Faint labeling also was
detectable in radial processes of Müller cells (Fig. 3G)
. At a
later stage as late as 7 days after NMDA injection, more conspicuous
CNTF immunoreactivities were observed in the NFL and cells in the INL;
there also was less conspicuous radial staining throughout the retina
(Fig. 3H)
. A greater intensity of immunostaining was observed in the
region of the cell nucleus compared with the cytoplasm. This was
confirmed in other experimental eyes that had undergone intravitreal
injection of KA (Figs. 3I
3J)
. Alteration in immunoreactivities for
CNTF was more drastic in KA-injected eyes than in NMDA-injected eyes.
Immunohistochemical study of sequential sections as well as confocal
microscopic observation of the double-stained sections by GFAP and CNTF
revealed colocalization of the two proteins in the radial staining
pattern (Fig. 4)
. We quantitated cell counts of CNTF-positive cells associated with
immunostaining for both GFAP and CNTF in radial staining. The mean cell
count (±SE) of CNTF-positive cells was 3.3 ± 1.8, 48.3 ±
4.5, and 44.1 ± 3.5 (number/mm), respectively, in PBS-injected
control eyes, NMDA-injected eyes, and KA-injected eyes at 7 days after
NMDA or KA injection (Table 1)
. Statistical analysis showed a significant increase in the
mean cell count (± SE) of CNTF-positive cells during the observation
period (up to 1 week) in both NMDA- and KA-injected eyes
(P < 0.0001 for NMDA treatment and
P < 0.0001 for KA treatment, ANOVA) (Table 1)
.
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Figure 4. Sequential localization and colocalization of GFAP and CNTF.
Immunohistochemistry of sequential sections of KA-injected eyes showed
sequential localization of CNTF (A) and GFAP (B).
Also, confocal images were shown in the right-most panel.
The CNTF signals (red; Cy3) coincide with the GFAP ones
(green; FITC) (C). (A)
Arrowheads indicate CNTF-positive radial staining
throughout the retina. (B) Arrow indicates
GFAP-positive Müller cell and radial staining. (C)
Large arrowheads point the colocalization of CNTF and
GFAP in radial staining. Bar, 50 µm.
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Western Blot Analysis
To identify and quantify protein expression of CNTF, we carried
out a western blot analysis. Western blot analysis showed a band of
approximately 23 kDa, which corresponds to the reported molecular size
of rat CNTF,9
28
in experiments using anti-rat CNTF
antibody (Fig. 5)
. Densitometric analysis of the positive bands of expected length
showed that, in experimental eyes with NMDA injection (Fig. 5A)
, the
optical density was 2.13 ± 0.38-fold that of the control eyes
(1.00 ± 0.13) (P = 0.0082, ANOVA). Also, similar
experiments showed that, in experimental eyes with KA injection (Fig. 5B)
, the optical density was 7.51 ± 0.98-fold that of the
PBS-injected (control) eyes (1.00 ± 0.24) (P =
0.0004, ANOVA).
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Figure 5. Western blot analysis for ciliary neurotrophic factor (CNTF). Western
blot analysis for CNTF showed upregulated expression of CNTF proteins
in NMDA (200 nmol)-injected eyes (n = 3)
(A) and KA (5 nmol)-injected eyes (n =
3) (B). Lanes 1 to 3:
PBS-injected eyes as controls; lanes 4 to
6: NMDA- or KA-injected eyes. A single band of
approximately 23 kDa was detected (arrowhead).
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Morphometric Analysis for Neuroprotective Effects of CNTF
In an effort to elucidate neuroprotective effects of CNTF
against NMDA- or KA-induced retinal damage, we conducted
morphometric analyses after intravitreal injection of NMDA and KA (Fig. 6)
. Two days after injection of 1 µl (0.1 or 1 µg/µl) recombinant
rat CNTF (or 1 µl of PBS as a negative control) into the vitreous, 5
µl of NMDA (200 nmol) or KA (5 nmol) was injected. With CNTF or PBS
pretreatment, no inflammatory findings were observed on histologic
examination. Also, in both NMDA- and KA-injected eyes, cell loss in the
GCL and thinning of the IPL appeared to be inhibited.
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Figure 6. Light micrographs of retinal sections of eyes with glutamate
receptor-mediated retinal damage and those pretreated with CNTF.
Light microscopic photographs show representative retinal
tissues in control and experimental eyes after intravitreal injection
of NMDA (200 nmol) and KA (5 nmol). (A) PBS-injected eyes as
controls; (B) NMDA-injected eyes without pretreatment;
(C) NMDA-injected eyes after pretreatment with CNTF (1 µl
of 0.1 µg/µl); (D) NMDA-injected eyes after pretreatment
with CNTF (1 µl of 1 µg/µl); (E) PBS-injected eyes as
controls; (F) KA-injected eyes without pretreatment;
(G) KA-injected eyes after pretreatment with CNTF (1 µl of
0.1 µg/µl); (H) KA-injected eyes after pretreatment with
CNTF (1 µl of 1 µg/µl). Bar, 50 µm.
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In NMDA-injected eyes, the mean number (±SE) of GCL cells was
25.9 ± 0.9, 25.8 ± 1.6, and 40.1 ± 3.9, respectively,
in PBS-treated eyes (control), CNTF (0.1 µg)-treated eyes, and CNTF
(1 µg)-treated eyes (PBS versus CNTF 0.1 µg, P =
0.9681; PBS versus CNTF 1 µg, P < 0.0001, ANOVA)
(Table 2)
. The mean thickness (±SE) of the IPL was 18.8 ± 1.4, 18.7 ± 1.2, and 25.1 ± 1.6, respectively, in PBS-treated eyes, CNTF
(0.1 µg)-treated eyes, and CNTF (1 µg)-treated eyes (PBS versus
CNTF 0.1 µg, P = 0.9622; PBS versus CNTF 1 µg,
P = 0.0073, ANOVA) (Table 2)
. Also, in KA-injected
eyes, the mean number (±SE) of GCL cells was 26.4 ± 1.6,
28.5 ± 5.9, and 30.6 ± 3.5, respectively, in PBS-treated
eyes (control), CNTF (0.1 µg)-treated eyes, and CNTF (1 µg)-treated
eyes (PBS versus CNTF 0.1 µg, P = 0.6191 and PBS
versus CNTF 1 µg, P = 0.3720, ANOVA) (Table 2)
. The mean thickness (±SE) of IPL was 9.0 ± 1.2,
9.8 ± 2.3, and 16.9 ± 2.2, respectively, in PBS-treated
eyes (control), CNTF (0.1 µg)-treated eyes and CNTF (1 µg)-treated
eyes (PBS versus CNTF 0.1 µg, P = 0.9084 and PBS
versus CNTF 1 µg, P = 0.0485, ANOVA) (Table 2)
.
To show a neuroprotective effect of CNTF pretreatment,
immunohistochemical studies for calbindin/calretinin were performed in
NMDA (200 nmol)- or KA (5nmol)-injected eyes after CNTF (1 µg)
pretreatment (Figs. 2C
2E
2H
2J)
. As for calretinin
immunoreactivity, the same staining pattern was observed in eyes with
PBS pretreatment before NMDA or KA injection (negative controls) as
shown in NMDA- or KA-injected eyes without any pretreatment. In eyes
with CNTF pretreatment, the small granulelike immunostaining against
calretinin seen in the IPL was preserved compared to the control (Fig. 2C) . As for calbindin immunoreactivity, decrease of the number of
calbindin-positive cells in the inner side of the INL (possibly
amacrine cells) appeared to be inhibited in the experimental eyes,
although quantification of the cells was difficult because of the faint
immunoreactivities (Fig. 2H)
. In contrast, the population of
calbindin-positive cells in the outer side of the INL (possibly
horizontal cells) in experimental eyes seemed to be same as in control
eyes. A similar observation was confirmed by experiments using
intravitreal injection of KA (Figs. 2E
2J)
.
|
Discussion
|
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In several common ocular diseases, including glaucoma, ischemia,
and optic nerve damage, high levels of glutamate in the vitreous have
been reported.29
30
31
Because glutamate is not only a
neurotransmitter in retina, but also a neurotoxic excitatory amino acid
against neuronal cells, prolonged high levels of glutamate have been
hypothesized to result in serious damage to the
retina.32
33
Glutamate receptor–related neuronal death
can be explained by an influx of calcium ion, nitric oxide synthesis
and subsequent free radical formation, depletion of ATP and various
enzymatic reactions of calcium-dependent enzymes.34
It is
well known that glutamate receptor–related neurotoxicity can be
induced by NMDA and KA.20
21
22
In the study described
herein, we investigated alterations in the expression of CNTF in NMDA-
or KA-injected eyes and also the efficacy of CNTF in experimental eyes.
Our study showed upregulation of GFAP in NMDA- and KA-injected rat
eyes, suggesting that glial components throughout the retina may be
part of the retinal response against NMDA- and KA-induced neuronal cell
death. Because responsive upregulation of GFAP as well as CNTF in
retinal glial cells was elicited within a short incubation period (as
early as 3–7 days) in our study, NMDA or KA treatments may have their
effect directly on retinal glial cells. However, we were unable to draw
any conclusion on this point because we cannot deny the possibility
that these events may follow the early events of neurodegenerative
processes even before cell loss of retinal neuronal cells. Astrocytes
are ubiquitous in the NFL and in the GCL, and Müller cells extend
all the way from the external to the internal limiting
membrane.35
36
Our immunohistochemical studies on control
sections demonstrated faint expression of GFAP in the NFL and GCL, but
none in outer layers, which is in agreement with previous
reports.14
15
16
17
18
19
After the intravitreal injection of NMDA
and KA, conspicuous immunolabeling for GFAP was observed in a radial
pattern between the NFL and external limiting membrane in addition to
longitudinal immunoreactivity in the NFL and GCL. Thus, the major cell
types responsible for the upregulation of GFAP in eyes with
experimental eyes are thought to be retinal Müller cells. Changes
in the expression and distribution of GFAP in retinal glial components
have been reported in eyes with various diseases and/or injuries, such
as retinal detachment,15
laser-induced
injury,16
retinal degeneration,17
and
mechanical injury.18
Because upregulation of GFAP occurs
in response to a number of conditions, as described above, it may
represent a nonspecific neuroprotective mechanism in response to stress
or injury in the retina. In our previous report,14
upregulation of GFAP was identified in monkey eyes with experimental
glaucoma and our immunohistochemical studies demonstrated that the
cells responsible are most probably both astrocytes and Müller
cells. Because glaucoma is one of the most common ocular diseases
related to elevated levels of glutamate in the vitreous
body,37
it is possible that it may be caused by abnormal
release and/or uptake of glutamate, secondary to intraocular
pressure–related primary neural damage. This also may be the case in
eyes with ischemia and optic nerve damage, both of which result in
elevation in intravitreal glutamate
concentrations.29
30
31
38
Thus, our results show that the
treatments with NMDA or KA elicit activation of retinal glial
components, whether by a direct or an indirect mechanism.
Immunohistochemical studies against calcium-binding proteins
demonstrated that, in experimental eyes, GCL cells and partial cell
components in the INL showed disappearance and/or decrease in
immunoreactivities. This result seems to be in good accordance with the
cell loss of these layers, as shown in morphometric analysis. On the
other hand, we were unable to detect a significant change in the
population of the calbindin-positive cells in the outer side of the
INL (possibly horizontal cells) in experimental eyes in comparison with
control eyes. Although some investigation suggested that horizontal
cells possess sensitivity to KA,21
22
38
39
40
Morgan et
al.22
41
have reported that, with low (6 nmol) and
intermediate (60 nmol) doses of KA, the retinal lesions were confined
to the IPL and amacrine cells, and horizontal cells were substantially
intact. Our study was conducted under experimental conditions using an
experimental condition with 5 nmol of KA in which only inner retinal
layers were affected. The used dose of KA in this study was comparable
to "low dose" of the previous report by Morgan et al., and the
result of our study also seemed to be in good accordance with their
result.
Our immunohistochemical studies showed upregulated expression of CNTF
in retinal Müller cells, as well as in astrocytes of the NFL and
GCL. Confocal images of double staining and sequential localization
demonstrated colocalization of GFAP and CNTF in radial processes,
indicating that activated glial components express this neurotrophic
factor throughout the structure of the retina. Similar upregulation of
CNTF has been reported in eyes with mechanical injury, light-induced
retinal damage, and ischemia.6
7
8
9
Also, we observed that
CNTF might have a preferential disposition to the nucleus rather than
the cytoplasm. This result is in good accordance with CNTF
immunoreactivity in adult rodent central nervous system and rat retina
after pressure-induced ischemia.9
28
The nuclear
localization of CNTF may be important in defining the function of these
CNTF-positive cells and suggests the possibility that CNTF might
interact with cellular components directly to affect gene
transcription. Also, faint to moderate expression of CNTF was observed
in cells (possibly astrocytes) in the NFL and GCL, which corresponds to
GFAP expression. Responsive upregulated expression of CNTF and GFAP in
Müller cells may take part in a series of events of NMDA- and
KA-induced neurodegeneration. Because CNTF activates transcriptional
expression of GFAP in glial cells,42
the secondary
upregulation of GFAP expression in Müller cells may be caused by
the primary release of the CNTF. Also, it is possible that alteration
in distribution of the expression may result in neuroprotective effects
in a larger area.
In conclusion, our studies have shown that upregulated expression of
CNTF and GFAP in retinal Müller cells occurs in response to NMDA-
and KA-induced neuronal death. Thus, in addition to astrocytes,
activation of retinal Müller cells may play a role in the
intrinsic neuroprotective system of retina.
|
Footnotes
|
|---|
Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, and from the Ministry of Health and Welfare, Japan.
Submitted for publication March 12, 1999; revised August 26, 1999; accepted September 23, 1999.
Commercial relationships policy: N.
Corresponding author: Hidenobu Tanihara, Department of Ophthalmology, Tenri Hospital, Mishima-cho 200, Tenri, 632-0015, Japan. tanihara{at}pearl.ocn.ne.jp
|
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Top
Abstract
Introduction
