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Immunocytochemical Characterization of Macular Hole Opercula
Eric Ezra, FRCS, FRCOphth;
Robert N. Fariss, PhD;
Daniel E. Possin, BS;
William G. Aylward, FRCS, FRCOphth;
Zdenek J. Gregor, FRCS, FRCOphth;
Philip J. Luthert, PhD;
Ann H. Milam, PhD
Arch Ophthalmol. 2001;119:223-231.
ABSTRACT
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Objectives To immunocytochemically characterize the neural and glial elements of
idiopathic full-thickness macular hole (FTMH) opercula excised during vitrectomy,
and to correlate them with the outcome of surgery.
Methods Opercula were collected from eyes undergoing vitrectomy for stage 3
FTMH and processed for transmission electron microscopy, light epifluorescence,
and laser scanning confocal microscopy. Glia were identified using anti–glial
fibrillary acid protein (GFAP), antivimentin, and anti–cellular retinaldehyde
binding protein antibodies. Anti–phosphodiesterase gamma and antirhodopsin
were used for cone and rod photoreceptors, and anticytokeratin was used for
retinal pigment epithelium. The findings were correlated with the clinical
data before and after surgery. For statistical analysis, data were combined
with those of a previous study by the authors of 18 opercula.
Results Opercula from 12 consecutive eyes of 12 patients were studied. In all
opercula, GFAP, vimentin, and cellular retinaldehyde binding protein–positive
glia were present. Six (50%) of 12 opercula contained more than 5 photoreceptors
with somata and internal photoreceptor fibres, but lacking outer segments,
demonstrating strong immunoreactivity to anti-phosphodiesterase gamma without
antirhodopsin reactivity consistent with cones. Further, 2 (17%) of 12 opercula
showed few cones (1-5 cones), and 4 (33%) of 12 contained only glia. Clinicopathologic
correlation of the 30 opercula from the 2 studies showed that eyes with opercula
containing more than 5 photoreceptors were associated with a worse anatomical
closure rate after initial surgery, compared with those with fewer than 5
photoreceptors (P = .004). Once closure had been
achieved with reoperation, median postoperative vision was similar in both
groups (20/40 and 20/60, respectively).
Conclusions A spectrum of opercula occur in FTMH ranging from those containing only
glia to those containing numerous cones. The extent of foveal neuroretinal
tissue loss may affect the outcome of surgery.
INTRODUCTION
THE PATHOPHYSIOLOGICAL mechanisms leading to the formation of idiopathic
full-thickness macular holes (FTMH) have been a subject of much debate recently.
In their original description of the clinical staging of FTMH, Gass,1-2 and Johnson and Gass3
postulated that they resulted from tangential vitreofoveal traction. This
was caused by focal contraction of the prefoveolar vitreous cortex, leading
to progression from foveolar detachment (stages 1a and 1b), to early FTMH
(stage 2), to a mature FTMH with vitreofoveal separation with or without an
operculum (stage 3), and finally, to a hole with a complete posterior vitreous
detachment (stage 4). Other mechanisms have also been proposed, including
anteroposterior vitreofoveal traction, mechanical traction resulting from
fluid movements in the premacular bursa, and degeneration of a foveal cyst
with operculation of the anterior cyst wall.4
Recent clinical studies reporting the efficacy of the surgical treatment
of FTMH have shown a significant anatomical and functional benefit in the
majority of eyes undergoing vitrectomy.5-15
These studies have also shown that although an anatomical closure rate of
70% to 100% can be achieved with surgery, the visual results may be variable,
with some eyes achieving near normal visual acuity following anatomical closure,
and others showing little or no visual improvement after closure. In a reappraisal
of the mechanisms leading to FTMH formation, Gass16
suggested that in the majority of holes, tangential vitreofoveal traction
resulted in a foveal dehiscence without significant loss of foveal tissue,
thereby explaining the marked visual improvement observed in some eyes after
surgery. Recent clinicopathological studies17-18
on opercula excised during vitrectomy for stage 3 holes, which were performed
to determine whether or not they contain neural tissue, have shown that 2
types occur in association with FTMH: those containing only glial tissue and
those containing glial and neuroretinal tissue. Opercula containing only glial
tissue (frequently with segments of internal limiting membrane) have been
termed "pseudo-opercula" by some authors,17
who have postulated that they represent reactive glial tissue adherent to
the separated posterior vitreous cortex in the region of the hole. Others
have postulated that they may represent glial tissue avulsed from the inner-retinal
lamellae at the fovea.18 Opercula containing
neuroretinal tissue have been termed "true opercula"; some of which have been
thought to arise from full-thickness foveal tears.18
In our previous study18 of 18 opercula using
transmission electron microscopy, 39% of opercula contained axons, pedicles,
and somata consistent with cones, suggesting that these were produced by avulsion
of foveal retina rather than a purely reactive gliosis in response to a foveal
dehiscence. Sixty-one percent contained only glia and internal limiting membrane,
consistent with avulsed inner-retinal tissue. Opercula containing photoreceptors
were associated with a worse postoperative anatomical closure rate after initial
surgery. Further ultrastructural and clinicopathological data on FTMH opercula
has been sparse because of the difficulties associated with the collection
and processing of these minute specimens.
The objectives of this study were to further characterize the cellular
components of opercula identified by previous ultrastructural studies using
immunocytochemical methods, and to provide further clinicopathological correlation.
PATIENTS AND METHODS
Opercula from patients undergoing vitreous surgery for stage 3 FTMH
were collected for immunocytochemical analysis. A stage 3 FTMH was defined,
according to Gass' definition,1-3,16
as a lesion with vitreomacular separation detectable on slitlamp examination
by the presence of a free operculum suspended on the separated posterior vitreous
cortex, anterior to the preretinal plane. Thus, only eyes with free opercula
visible preoperatively were included. Preoperative details of all patients
were recorded, and included age, sex, duration of symptoms, previous ocular
and medical history, best-corrected Snellen visual acuity, and slitlamp and
fundus examinations with a 78-diopter lens and Goldmann fundus contact lens.
The size of the hole was measured preoperatively from red-free photographs
and fundus fluorescein angiogram. All opercula included in this study were
identified preoperatively as free-floating preretinal opacities suspended
on the posterior aspect of the vitreous cortex anterior to a stage 3 hole.
Visual outcome was defined as the postoperative refracted Snellen visual acuity
at 6 months.
The operative procedure used in all eyes has been described elsewhere.13 All operations were performed by 1 surgeon (Z.J.G.).
Following a 3-port pars plana vitrectomy, the posterior vitreous cortex was
separated from the retinal surface by aspiration using the vitreous cutter.
In all cases, the operculum remained attached to the posterior vitreous cortex
following its elevation from the retinal surface and was identified while
suspended on the detached vitreous cortex in the vitreous cavity. The vitreous
cortex temporal to the operculum was then partially excised, and a pair of
noncrushing, cupped, intraocular foreign body forceps was used to grasp the
operculum. The vitreous cortex adjacent to the forceps was then excised with
the cutter under coaxial illumination to allow removal of the forceps from
the eye. Thus, all opercula were collected using an atraumatic technique prior
to any manipulation or peeling of membranes on the surface of the retina.
Specimens were then carefully removed from the forceps and immediately fixed
and processed for immunocytochemical analysis.
The retinal surface was then examined with a membrane pick to identify
any epiretinal membranes. Membrane peeling was performed only when membranes
could be identified. In no case was an attempt made to remove the internal
limiting membrane. Following examination of the retinal periphery, air-fluid
exchange was performed with aspiration of subretinal fluid using a 34-gauge
cannula. The hole was then dried under air for 10 minutes, and an air-perfluoropropane
(14%-16% C3F8) exchange was performed to complete the
procedure. Reoperations after failed initial surgery were carried out using
a standardized technique involving rigourous epiretinal membrane, internal
limiting membrane dissection, and C3F8 gas tamponade,
as has been described elsewhere.19
TRANSMISSION ELECTRON MICROSCOPIC IMMUNOCYTOCHEMISTRY
For this technique, each specimen was fixed immediately in a solution
of 4% paraformaldehyde for 20 minutes and then manipulated into small (approximately
2 x 2 x 2 mm) blocks of set 10% agarose gel to allow subsequent
handling of the specimen. The blocks containing the specimens were then rinsed
in 3 changes of 0.1-mol/L sodium phosphate buffer (pH, 7.4). Subsequent dehydration
was achieved by transfer of the agarose block through ascending alcohols (5-minute
immersions in 50%, 70%, and 90% ethanol and 4 changes of 100% ethanol), followed
by embedding in London Resin white resin (LR White; Ted Pella Inc, Redding,
Calif) and overnight curing at 60°C. Semithin (1 µm) and ultrathin
(100 nm) sections were then cut using glass and diamond knives, respectively,
with an ultramicrotome. The opercula were step sectioned, taking 10 to 20
cuts at 20-µm intervals.
Ultrathin sections were then placed on copper grids and processed for
transmission electron microscopic (TEM) immunocytochemistry by the silver
enhancement method for immunogold labeling of electron microscopic (EM) sections,
using a modification of a previously described technique.20
Following initial incubation at 20°C for 30 minutes with a blocking buffer
(pH, 8.2), containing tris buffered saline (TBS), bovine serum albumin fraction
V (BSA) and normal blocking serum, grids were incubated in primary antibody
in BSA-TBS overnight at 4°C. This was followed by 3 washes in BSA-TBS
with NaCl for 10 minutes. Grids were then incubated with 5 nm colloidal gold
secondary antibody in BSA-TBS with NaCl (1:10 dilution) for 2 hours at 20°C
and washed 3 times (for 5,10, and 15 mins) in BSA-TBS with NaCl. The grids
were then silver enhanced using the IntenSETM M Silver Enhancement Kit (Amersham
Life Sciences Inc, Arlington Heights, Ill). The procedure produces easily
viewed particles 20 to 40 nm in diameter from the 1-to-5–nm colloidal
gold-labeled TEM sections. Following silver enhancement, the grids were stained
with uranyl acetate and lead citrate before viewing with a TEM. Specimens
from the previous study using TEM18 were reviewed.
In those specimens in which photoreceptors had been identified, the grids
were reexamined and photoreceptors counted through the stepped sections for
each specimen.
COLOCALIZATION STUDIES WITH LIGHT EPIFLUORESCENCE AND LASER SCANNING
CONFOCAL MICROSCOPY
In view of their small size, for this technique, whole opercula were
processed without sectioning. Opercula were immediately fixed in 4% paraformaldehyde,
followed by rinsing in 4 changes (3 for 10 minutes; 1 for 60 minutes) of chilled
phosphate-buffered saline (PBS) containing 0.5% BSA, 0.1% NaN3,
and Triton x-100 (0.1%) at a pH of 7.3. This was followed by incubation with
blocking buffer for 4 hours at 20°C, primary antibodies diluted in PBS-azide
at 4°C overnight and 4 further washes in PBS in the same maner as the
first washes. Specimens were then placed in fluorescent secondary antibody
diluted in PBS overnight at 4°C. Finally, after 4 further washes in PBS,
specimens were mounted in 5% N-propyl gallate in
glycerol (to retard photobleaching) and coverslipped. Immunofluorescence was
analyzed using epifluorescence and confocal microscopy.
ANTIBODIES AND NUCLEAR LABEL
The following primary antibodies were used: rabbit polyclonal anti–phosphodiesterase
gamma (PDEG), which was generated against peptide amino acids 73 through 87
at a dilution of 1:1000 for cones and rods21;
mouse monoclonal antirhodopsin (Rho 4D2) at a dilution of 1:20 for rods; rabbit
polyclonal antiglial fibrillary acid protein (anti-GFAP) and mouse monoclonal
anti-GFAP (Dako Corp, Carpinteria, Calif), at dilutions of 1:200 and 1:20,
respectively; mouse monoclonal antivimentin (Clone V9; Dako Corp), at a dilution
of 1:200 for activated glial cells; mouse polyclonal anticellular retinaldehyde
binding protein (clone Immunoglobulin 83), at a dilution of 1:200 for Muller
glia and retinal pigment epithelium cells; and mouse monoclonal anti-cytokeratin
(Clone Cam-52; Becton and Dickinson, Sparks, Md), at a dilution of 1:200,
for RPE cells.
For double labeling studies, 2 primary antibodies raised in different
species were used, as were the 2 secondary antibodies, which were labeled
with fluorescein isothiocyanate (FITC) or indocarbocyanine (Cy3) (Jackson
Immuno-Research Laboratories Inc, West Grove, Pa). As a pannuclear label in
some preparations, 4',6'-diamidino-2-phenylindole (DAPI; Molecular
Probes, Eugene, Ore) was used at 2 µg/mL. Sections were photographed
as single, double, or triple exposures for epifluorescence microscopy. For
confocal microscopy, "Z series" scans were compiled of the entire specimen,
at 1-µm increments per scan, in each fluorescence channel. The images
from each channel were then superimposed using the NIH Image software version
1.6 (National Institutes of Health, Bethesda, Md) to produce composite double-labeled
or triple-labeled images. The composite "Z series" image of the entire specimen
therefore enabled individual cells to be counted.
CONTROLS
Control sections of healthy adult macular and foveal retina were processed
as above for immunofluorescence and TEM and used as positive controls. These
were also used for negative (no primary antibody) controls for immunofluorescence
microscopy, as opercula were used as whole mounts without sectioning and no
sections of opercula were available for controls. Ultrathin sections of opercula
were used as negative controls for TEM immunocytochemistry.
STATISTICAL ANALYSIS
For data analysis, the Fisher exact or Mantel-Haenszel tests were used
for comparison of proportions; the 2-tailed t test
for comparison of means; and the Pearson product moment correlation for strength
of association between interval data.
RESULTS
Twelve opercula from 12 eyes of 12 patients undergoing surgery for stage
3 FTMH were collected and processed for immunocytochemical characterization.
Baseline data analysis revealed that there was a mean age of 66.2 years (range,
61-75 years), a subject sample with 1 (8%) of 12 patients being men, a mean
symptom duration of 4.9 months (range, 1-9 months), a mean preoperative hole
diameter of 410 µm (range, 310-500 µm), a median preoperative
visual acuity of 20/120 (range, 20/60-20/200). Ten opercula were processed
for immunofluorescence by light epifluorescence and laser scanning confocal
microscopy, and 2 for TEM immunocytochemistry.
In the immunofluorescence study (Figure
1, Figure 2, and Figure 3), 9 were double labeled with anti-PDEG
and antirhodopsin antibodies. Of these, 2 of 9 cases showed no reactivity
to anti-PDEG (cases 1 and 2). Another 2 of 9 (cases 3 and 4) showed that between
1 and 5 cells had positive reactivity to anti-PDEG (Figure 1A, case 3). Two of nine cases (cases 5 and 6) showed that
5 to 100 cells were reactive to anti-PDEG (Figure 3A and 3B), and 3 of 9 cases (cases 7, 8, and 9) had more
than 100 cells positive for PDEG antibodies (Figure 1B and Figure 2).
All 9 cases showed negative reactivity to antirhodopsin. One further operculum
(case 10) was double labeled with anti-PDEG and anti-GFAP and showed positive
reactivity to both antibodies (Figure 3C).
Two opercula (cases 11 and 12) were step sectioned and processed for TEM immunocytochemistry.
Both showed negative reactivity to anti-PDEG, antirhodopsin and Cam-52, and
positive reactivity to anti-GFAP, anti–cellular retinaldehyde binding
protein and antivimentin.
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Figure 1. Epifluorescence micrographs of
2 opercula containing cones with positive labeling to anti–phosphodiesterase
gamma (PDEG) or fluorescein isothiocyanate (green) and negative labeling to
antirhodopsin or indocarbocyanine (red). Nuclei are labeled with 4'6'-diamidino-2-phenylindole
(DAPI) (blue). The anti-PDEG antibody labels the peptide amino acids 73 through
87, which is identical in the rod and cone isoforms of PDEG and therefore
labels both rod and cones. In the healthy retina, PDEG is localized throughout
the cone outer segments and to the cytoplasm of the cone inner segments, somata,
and axons.21 In rods, PDEG is localized to the
outer segments only.21 Rhodopsin, meanwhile,
is localized to the outer segments of rods only. In retinal detachment, PDEG
expression declines in both cones and rods,21
while rhodopsin becomes redistributed to the plasma membrane of the entire
rod. Thus, in the opercula described here, positive PDEG labeling (green)
confirms the presence of photoreceptors, and negative rhodopsin labeling (red)
confirms the photoreceptors to be cones rather than rods. Note that while
cones are labeled positively with both anti-PDEG and DAPI (nuclei) (blue),
numerous other cells are present that only label positively for nuclear DAPI.
A, Case 3. An operculum (original magnification x1000) measuring 67
x 65 µm. Note that only 2 cones are present showing positive anti-PDEG
reactivity in their somata (arrows) and axons (arrowheads). B, Case 7. An
operculum (original magnification x600) measuring 120 x 80 µm
showing numerous cones and no rods. The patient initially had a stage 2 pericentric
("can opener") full-thickness macular hole at initial visit, which, by the
time of admission for surgery, had evolved into a fully operculated stage
3 hole.
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Figure 2. Pseudocolor laser scanning confocal
micrographs. A, Positive control healthy human fovea (original magnification
x300). The section is through the foveal pit and is double labeled with
anti–phosphodiesterase gamma (PDEG) (red) and antirhodopsin (green).
Note that in the healthy fovea, PDEG is localized throughout the cone photoreceptors
from outer segments to the Henle layer (asterisk) and in rod outer segments,
while rhodopsin is localized in the outer segments of rods that are absent
at the foveal center. Cone photoreceptors thus appear red (PDEG-positive and
rhodopsin-negative), rod outer segments appear yellow (PDEG-positive and rhodopsin-negative),
and rod cell bodies and axons do not fluoresce at all (PDEG-negative and rhodopsin-negative).
The vitreoretinal interface is also indicated (arrows). B, Positive control
healthy human macula (original magnification x1200). The section is
through the juxtafoveal area and is single labeled only with anti-PDEG (red),
showing PDEG localization in cones throughout the outer segments (os), cone
cell bodies (cc) in the outer nuclear layer (on) and Henle layer (H), and
in rod os. Rod cell bodies appear as negative images (rc). C, Case 8. Operculum
(original magnification x1000) measuring 95 x 100 µm, double
labeled with anti-PDEG (red) and antirhodopsin (green), showing 286 cones
(PDEG-positive and rhodopsin-negative).
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Figure 3. Pseudocolor laser scanning confocal
micrographs (stereopairs). A, Case 5. Operculum with numerous cones (original
magnification x1000) measuring 100 x 130 µm triple labeled
with anti–phosphodiesterase gamma (PDEG) (red), antirhodopsin (green),
and 4',6'-diamidino-2-phenylindole (DAPI) for nuclei (blue). A
total of 46 cones can be seen with positive reactivity to anti-PDEG (red)
only and nuclear DAPI (blue). The nuclei (PDEG-positive and rhodopsin-positive)
appear pink, while cone axons (PDEG-positive) appear red. Also seen is a population
of cells with positive DAPI nuclear labeling (blue) and negative labeling
to both anti-PDEG and antirhodopsin. B, Case 6. Operculum (original magnification
x800) measuring 140 x 100 µm with a total of 41 cones. C,
Case 10. Operculum (original magnification x1000) measuring 95 x
60 µm triple labeled with anti-PDEG (red), anti–glial fibrillary
acid protein (GFAP) (green), and DAPI (blue) for nuclei. A total of 14 cones
are seen with positive reactivity to anti-PDEG and DAPI. Two adjacent glia
with typical long processes are seen with strong reactivity to anti-GFAP (green).
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The presence of cells with positive reactivity to anti-PDEG and negative
to antirhodopsin in opercula was indicative of cone photoreceptors as rods
that have shed their outer segments. In the setting of a detachment of the
photoreceptor layer from the RPE, cells would be expected to show positive
reactivity to both anti-PDEG and antirhodopsin in the plasma membrane surrounding
their somata.21 In all specimens, cone photoreceptors
were lacking in inner and outer segments but had preserved axons, often with
intact pedicles. In the 6 of 9 opercula that contained more than 5 cones,
the cones were distributed in a random fashion without discernable retinal
architecture, consistent with tissue undergoing gliosis.
Overall, 4 (33%) of 12 opercula contained only glia, while 8 (67%) of
12 contained cones in addition to glia. Of these 8 opercula, 2 (17%) contained
very few cones (<5), and 6 (50%) contained more than 5 cones. Of the 6
with more than 5 cones, 3 (25%) contained a moderate number of cones (range,
5-50 cones), and 3 (25%) contained more than 50 cones. All opercula were negative
for rod photoreceptors, and no correlation was evident between either the
duration of symptoms or the size of the operculum and the presence of cones
in the operculum. Similarly, there was no correlation between preoperative
hole diameter and the presence of cones in the opercula.
Clinically, 8 (75%) of 12 eyes had successful anatomical closure after
the first operation. In the group with opercula containing more than 5 cones,
3 (50%) of 6 were closed after the first surgery, compared with 5 (84%) of
6 in the group with fewer than 5 cones. In the cases in which surgery had
failed, reoperation was undertaken in 2 of 3 eyes in the group with more than
5 cones and 1 of 1 in the group with opercula containing fewer than 5 cones,
with all 3 reoperations resulting in successful anatomical closure. The remaining
patient in the first group declined reoperation. Analysis of the postoperative
visual acuity following successful closure showed an overall median visual
acuity of 20/60 (range, 20/20-20/200), with both groups showing median postoperative
acuities of 20/60. Neither the initial anatomical result nor the final visual
acuity after successful closure seemed to be affected by the overall size
of the operculum or the number of cones within the operculum.
In 1 patient (case 7, Figure 1B),
a pericentric ("can-opener") stage 2 FTMH was noted at the initial visit,
which subsequently progressed to a stage 3 FTMH with vitreofoveal separation
and a free, mobile operculum by the time she was admitted for surgery. The
operculum contained abundant cones.
For further statistical analysis, the data were combined with those
of our previously published study.18 This allowed
statistical analysis on a total of 30 opercula from 30 eyes of 30 patients,
with a mean age of 68 years (range, 60-84 years), a mean duration of symptoms
of 4.6 months (range, 1-9 months), and a subject population in which 8 (27%)
of 30 patients were male . Of the 30 opercula, 15 (50%) contained photoreceptors,
and 15 (50%) contained only glia. Overall, 17 (57%) of 30 had few (2/30 had
<5 cones) or no cones (15/30 contained glia only) and 13 (43%) of 30 had
more than 5 cones. Anatomical closure was achieved after the first operations
in 18 (60%) of 30 patients and in 27 (90%) of 30 after reoperations, with
the remaining 3 patients declining a second operation. Although the primary
closure rate was significantly better (P = .004)
in cases with opercula containing few or no photoreceptors (14/17 [82%]) compared
with those with opercula containing photoreceptors (4/13 [30%]), the closure
rate after reoperation was similar (16/17 [94%]; 11/13 [85%]) in both groups.
There was no correlation between anatomical closure and preoperative hole
size (mean, 465 µm; range, 310-585 µm) (P
= .10). There was also no correlation between preoperative hole size and the
presence of cones within opercula.
Analysis of visual outcome showed that overall, the median visual acuity
improved from 20/120 preoperatively, to 20/60 in the 27 of 30 cases in which
successful anatomical closure was achieved. There was a trend for better visual
acuity results in the 17 cases with opercula containing fewer than 5 cones
(preoperative median, 20/120; postoperative median, 20/40), compared with
the 13 cases with opercula containing more than 5 cones (preoperative median,
20/120; postoperative median, 20/60), which did not reach statistical significance
(P = .24). Although a worse preoperative visual acuity
was associated with a worse final acuity after hole closure (P<.001), other preoperative factors such as age, duration of symptoms
before initial surgery, and sex did not affect either anatomical or visual
outcome. There were also no significant baseline differences in age, sex,
duration, and preoperative visual acuity between the 2 groups of opercula,
and no correlation between the duration of symptoms before initial surgery
and the number of cone photoreceptors in opercula.
COMMENT
Two studies have previously reported on the ultrastructure of macular
hole opercula. Madreperla et al17 reported on
2 opercula studied by TEM, both of which contained only fibroglial cells and
no significant neuroretinal elements. They suggested that these "pseudo-opercula"
were formed by the aggregation of epiretinal glia as part of an attempted
healing response around the hole. In a more recent study,18
we reported on the ultrastructure of 18 opercula studied by TEM, showing 61%
to contain glia only and 39% to contain glia and neuroretinal tissue, including
cone nuclei, axons, and pedicles forming synaptic complexes typical of the
external plexiform layer.
The current study describes techniques for processing opercula for immunocytochemistry
using TEM, epifluorescence, and confocal microscopy. Confocal microscopy allowed
the examination of whole-mounted opercula, which, due to their size and thickness,
are ideally suited for this method. This affords much additional information
in terms of 3-dimensional structure and qualitative and quantitative analysis
of various cellular populations within opercula. Transmission electron microscopic
immunocytochemistry was used as a complementary technique to allow the study
of immunoreactivity to multiple antibodies in any one specimen, as epifluorescence
and confocal microscopy only allowed the study of 2 antibodies on any whole-mounted
operculum.
Confocal microscopy of whole-mounted opercula proved to be an extremely
sensitive technique, allowing the identification of very small numbers of
photoreceptors (<5) within opercula in 2 specimens, which may not have
been possible with serial-section EM examination. This probably explains the
higher incidence of opercula with any photoreceptors in this study (67%) compared
with our previous study with EM (39%).18 The
incidence of photoreceptor-rich opercula (>5) in the current study was 50%.
Thus, for the detection of photoreceptors or other neural elements, confocal
epifluorescence seems to be a more sensitive method compared with EM, which
necessitates a step-sectioning method.
Experimental work has shown that in healthy attached retina, PDEG is
localized throughout the cone outer segments and the cytoplasm of the cone
inner segments, somata, and axons.21 In rods,
PDEG is localized to the outer segments only.21
Rhodopsin is localized to the outer segments of rods. In detached retina,
PDEG expression declines in both cones and rods,21
while rhodopsin becomes redistributed to the plasma membrane of the entire
rod.22 Thus, in the opercula described here,
positive PDEG labeling confirms the presence of photoreceptors, and negative
rhodopsin labeling confirms the photoreceptors to be cones rather than rods,
which is consistent with central foveal tissue.
Although we cannot exclude the possibility that opercula with very few
or no cones represent opercula in which photoreceptors have degenerated with
time, both the current study and our previous data18
showed no correlation between the presence of photoreceptors and the duration
of symptoms. This suggests that the distinction between photoreceptor-rich
opercula and opercula with few or no photoreceptors is a real one. On the
other hand, it is likely that the number of cones demonstrated in any operculum
by immunocytochemical labeling represents only a proportion of those originally
avulsed, with a significant number having undergone degeneration in the operculum.
The numerous cells in opercula that show positive DAPI nuclear labelling but
negative reactivity for both anti-PDEG and antirhodopsin represent glial cells,
which constitute the majority cell population within opercula.
The paucity of photoreceptor inner and outer segments in cones found
in opercula during this and the previous study18
is not unexpected since separation of the retina from the RPE in a chronic
retinal detachment leads to marked degeneration of outer segments within 2
to 4 weeks,23-24 with slower inner
segment degeneration after months. In the vast majority of eyes with stage
3 FTMH at the time of vitrectomy, any foveal cones present in the operculum
would have been separated from the RPE, first during the initial foveal detachment
phase (stage 1) and then during the time between progression from stage 2
to stage 3 FTMH. This occurs during a period of weeks or months. In photoreceptor-rich
opercula, the lack of healthy retinal architecture, rounding of the edges
of the opercula, the paucity of inner and outer segments, and the marked glial
response are consistent with gliotic, chronically detached retinal tissue
rather than retinal tissue avulsed from the edges of the hole at the time
of surgery.
The findings described in this study provide further evidence that a
significant proportion of macular holes arise from avulsion of foveal neural
tissue rather than from a pure foveal dehiscence without tissue loss or avulsion
of only superficial inner-retinal glial tissue. The variation in the quantity
of photoreceptors reflects a variable degree of foveal tissue loss during
hole formation. The presence of numerous cones and the lack of rods in opercula
is consistent with tissue from the central fovea, with the abundance of cone
nuclei in some cases suggesting that these operculum contains tissue from
deeper within the fovea.
The current study also supports some of Gass's concepts regarding healthy
foveal architecture and the mechanism of FTMH formation. In his most recent
hypothesis25 based on the interpretation of
previous studies of Hogan and Alvarado26 and
Yamada,27 he emphasizes the presence of an inverted
"cone" of Müller cells at the central foveola. These cells may provide
important anatomical support for the healthy fovea, without which the underlying
retinal tissue would be susceptible to disruption.They may also act as the
primary site of xanthophyl storage within the healthy fovea and play an primary
role in FTMH formation. In this process, migration and proliferation of the
Müller cells (an aging process) into the prefoveolar vitreous cortex
causes contraction of the vitreous cortex and further disruption of the Müller
cell "cone." This in turn results in a full-thickness foveal defect, centrifugal
retraction of photoreceptors, and formation of a prehole opacity, which represents
an avulsed segment of the Müller cell cone.
These hypotheses are consistent with the findings of our current and
previous studies.17-18 The spectrum
of tissue found in opercula reflect the degree of tissue avulsion from the
Müller cell cone. At one end of the spectrum are the majority of opercula
(accounting for approximately 80% of all lesions) formed by avulsion of tissue
from around the superficial base of the Müller cell cone. This base consists
of glial elements (found in 100% of opercula17-18)
and internal limiting membrane (found in 61%-100% of opercula17-18)
with or without occasional cone photoreceptors from the more superficial layers
of the neuroretina at the fovea. In these specimens, it is impossible to distinguish
histologically between glia originating from beneath (ie, from superficial
inner retina) or superficial to the internal limiting membrane (ie, epiretinal).
It is likely that in the majority, both inner-retinal and epiretinal glia
are avulsed within the operculum. The presence of cone pedicles and second-order
neurites in some opercula would be consistent with tissue avulsed from more
superficial peripheral inner neuroretina (further than 350 µm from the
center of the foveola) around the periphery of the base of the Müller
cell cone. The fact that the majority of opercula are less than 350 µm
in diameter is a result of contraction that is owing to glial remodeling after
avulsion. In these cases, avulsion of the whole or part of the Müller
cell cone results in disruption of the central foveola, an umbo dehiscence
and subsequent centrifugal retraction of foveolar tissue without significant
neuroretinal tissue loss.
At the other end of the spectrum are opercula rich in photoreceptor
elements, which result from avulsion of neuroretinal tissue from deeper within
the fovea, around the truncated apex of the Müller cell cone. In some
cases, a small number of foveolar cones may also be avulsed. In any case,
these would only represent a small proportion of the total 2500 cones in the
healthy fovea,26 even accounting for photoreceptor
degeneration within the free operculum with time. In these cases, neuroretinal
foveal tissue loss occurs, followed by further retraction and enlargement
of the foveal defect. Such cases probably only account for only about 20%
of all FTMH.
The histological findings, therefore, suggest that the majority of opercula
are in fact "true" opercula, containing epiretinal glia, internal limiting
membrane, and glia from the superficial base of the Müller cell cone,
with or without occasional photoreceptor elements. They also suggest that
a more useful distinction would be between "glial" and "photoreceptor-rich"
opercula rather than between "true" and "pseudo" opercula.
The worse anatomical results after surgery in cases with opercula containing
photoreceptor tissue could be accounted for by (1) a larger central foveal
neuroretinal defect before surgery and/or (2) a more pronounced glial response
occurring around the edges of the hole as a result of greater tissue disruption
at the fovea. Both factors would make the hole edges less likely to reappose
and heal after surgery. This is also supported by previous histological studies28-30 on postmortem eyes with
successfully treated FTMH showing variable residual retinal defects of between
16 µm and 250 µm, which may reflect a variable amount of foveal
tissue loss. Although the studies of Funata et al28
and Madreperla et al29 showed effective reapposition
of the hole edges, consistent with minimal or no foveal tissue loss, the study
by Rosa et al30 showed a much larger residual
foveal defect of 250 µm in a healed macular hole after surgery, consistent
with significant loss of central foveal tissue.
The overall primary surgical closure rate of 64% in the combined series
of 30 eyes is lower than those reported by other series but is comparable
with the 69% closure rate reported for stage 3 and stage 4 holes by Freeman
et al15 in their prospective randomized study.
The eyes included in the our 2 studies may represent one end of the spectrum
of stage 3 holes, in which greater foveal tissue loss and more pronounced
glial proliferation have occurred, resulting in a worse prognosis following
surgery. Once anatomical closure has been achieved after reoperation, the
lack of correlation between the presence of cones in opercula and a worse
visual outcome is likely to reflect the small number of foveal cones avulsed
in even the most photoreceptor-rich operculum. Even accounting for photoreceptor
degeneration within the operculum before surgery, this number would still
represent only a very small portion of the foveal cone population of 2500.
In terms of visual function, the loss of such a small number of cones is probably
of less consequence compared with the improvement of photoreceptor function
in the rest of the macula following successful reapposition of photoreceptors
to the RPE with resolution of the fluid cuff. In any case, these clinicopatholological
correlates should be interpreted with caution in view of the sample sizes
of the studies, which do not permit detailed statistical analysis. Ideally,
larger studies should be conducted to allow adequate stratification and analysis
of the different types of opercula and their clinical outcomes.
Although the current study provides further insight into the spectrum
of tissue avulsed from the central foveola, the primary mechanism initiating
vitreofoveal traction in the early stages of hole formation remains unclear.
Gass16, 25 has proposed that glial
proliferation and migration forms in the Müller cell cone into the prefoveal
vitreous cortex (demonstrated histologically in prefoveal vitreous removed
during surgery for impending holes31-32),
results in prefoveal vitreous contraction, and is the primary initiating mechanism.
Others33 have suggested that different factors
such as mechanical forces transmitted to the foveola in the presence of a
partial vitreomacular separation with residual foveal tethering (as seen on
optical coherence tomography33) may be the primary
mechanisms. Thus the glial activity around the foveola, as in opercula,17-18 and epiretinal membranes34
found at the edges of FTMHs might represent a secondary healing response.17 Although in vitro and in vivo data suggest that glial
membranes may contract as a result of cellular migration35
rather than cellular myofibroblastic contraction,36-38
it remains uncertain whether or not such forces are sufficient per se, or
can contribute significantly to FTMH. Indeed, as much as 20% of apparently
healthy asymptomatic eyes may show epiretinal glial membranes at postmortem
examination without any evidence of epimacular traction.39-40
The nebulous epiretinal membranes observed in intraoperatively in some cases
of FTMH41 may not necessarily be associated
with significant traction.
Interestingly, case 7, observed to progress from a pericentric stage
2 lesion to a to stage 3 lesion, was associated with the formation of a photoreceptor-rich
operculum. This suggests that significant foveal tissue may be avulsed in
some cases, and that in such opercula, avulsion of a segment of glial membrane
overlying an "occult" hole leads to avulsion of the adherent underlying foveal
cones. In fact, previous clinical data have suggested that pericentric stage
2 holes are associated with a worse prognosis42
and this may reflect the larger neuroretinal tissue defect in these cases.
In summary, our data suggest that although some FTMHs are associated
with minimal or no foveal tissue loss (ie, begin as an umbo dehiscence as
described by Gass16, 25) others are
associated with more extensive central tissue avulsion including neuroretinal
tissue with cone photoreceptors, from the fovea. The correlation between the
spectrum of histological disease and variation in clinical outcome following
surgery requires further elucidation with studies of greater numbers of opercula
to allow more detailed statistical analysis.
AUTHOR INFORMATION
Accepted for publication November 3, 2000.
This article was supported by grant 301 from the Guide Dogs for the
Blind Association, London, England; grant 311 from the Stringer Bequest to
the Special Trustees of Moorfields Eye Hospital, London, England (Drs Ezra,
Gregor, and Aylward); grant EY0-1311 from the National Institutes of Health,
Bethesda, Md (Dr Milam); the Foundation Fighting Blindness, Hunt Valley, Md
(Dr Milam); and the Paul and Evania Bell Mackall Foundation Trust, New York
(Dr Milam).
The authors would like to thank Paulette Brunner from the W. M. Keck
Centre for Advanced Studies of Neural Signaling, University of Washington,
Seattle, and Glen MacDonald of the Virginia Merrill Hearing Research Center
and the Center on Human Development and Disability, University of Washington,
Seattle, for their help and advice on confocal image analysis. We also thank
professors A. C. Bird, MD, FRCS, FRCOphth, and W. R. Lee, FRCPath, FRCOphth,
for their contributions to the discussion. The authors also thank B. K. Fung,
PhD, from the Department of Ophthalmology, Jules Stein Institute, University
of California Los Angeles, who provided the anti-PDEG antibody; R. S. Molday,
PhD, from the Department of Biochemistry and Molecular Biology, University
of British Columbia, Vancouver, who provided the antirhodopsin (Rho 4D2);
and J. C. Saari, PhD, from the Department of Ophthalmology, University of
Washington, Seattle, who provided the anticellular retinaldehyde binding protein
(Immunoglobulin 83).
Corresponding author: Eric Ezra, FRCS, FRCOphth, Vitreoretinal Unit,
Moorfields Eye Hospital, City Road, London EC1V 2PD, England (e-mail: ericezra{at}hotmail.com).
From the Vitreoretinal Unit, Moorfields Eye Hospital, London (Drs Ezra,
Aylward, and Gregor); the Department of Pathology, Institute of Ophthalmology,
London, England (Drs Ezra and Luthert); and the Department of Ophthalmology,
University of Washington School of Medicine, Seattle (Dr Fariss, Mr Possin,
and Dr Milam); and the Scheie Eye Institute, University of Pennsylvania, Philadelphia
(Dr Milam).
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