Download The potential usefulness to research of retina obtained by

January 1988
Vol. 29/1
Investigative Ophthalmology
& Visual Science
Articles
The Potential Usefulness to Research of Retina
Obtained by Biopsy
Alan C. Bird,* Debora D. Forber,t^: Alan E. Kreiger,j-^ Bradley R. Srraarsma,t:{: and Dean
Retinal biopsy has been performed on normal rabbits and dogs. It was shown that retinal samples
could be obtained by internal and external routes in rabbits, but in dogs inability to achieve adequate
vitrectomy precluded useful retinal biopsy by the internal route. A single external biopsy specimen of 3
mm diameter was more than adequate to undertake standard histopathological examination, immunocytochemical experiments and determination of cyclic nucleotide levels. The quality of the micrographs, immunocytochemical labelling of rhodopsin and phosphodiesterase, and cyclic nucleotide
analyses were similar to those obtained with retinas from freshly enucleated eyes. The surgical
exercise was well tolerated by most eyes and does not preclude serial biopsies being undertaken. It is
concluded that retinal biopsy provides material of sufficient quantity and quality to satisfy many
laboratory needs in retinal research. Invest Ophthalmol Vis Sci 29:2-11,1988
The search for the pathogeneses of retinal diseases
has been hampered by the shortage of tissue available
for laboratory studies. This has been particularly
damaging in the study of degenerative and genetically
determined disorders. Despite major efforts, only a
limited amount of material has been obtained from
patients after death; moreover, in many instances this
material has given only limited information because
either the time interval between death and enucleation resulted in considerable autolysis, or the advanced state of disease allowed the conclusion that
cell death had occurred but prevented analysis of the
cause of metabolic failure. Correlation between the
functional characteristics of the diseased retina and
the laboratory findings has been hampered further by
the prolonged time interval between the last clinical
examination and death of the patient, since few if any
of the common inherited retinal d'rorders are associated with a shortened life expectancy.
From the *Department of Clinical Ophthalmology, Institute of
Ophthalmology, Moorfields Eye Hospital, London, the Departments of tOphthalmology and §Anatomy and the JJules Stein Eye
Institute, UCLA School of Medicine, Los Angeles, California.
Supported by National Eye Institute Grants EY-OO331,
EY-02651, and EY-00444, an International Research Scholar
Award from Research to Prevent Blindness, Inc. and a Center
Grant from the National Retinitis Pigmentosa Foundation, Fighting Blindness, Baltimore, Maryland. Dr. Bok is Dolly Green Professor of Ophthalmology.
Submitted for publication: April 24, 1986; accepted August 20,
1987.
Reprint requests: Dr. Dean Bok, Jules Stein Eye Institute, UCLA
School of Medicine, Los Angeles, CA 90024.
Retinal biopsy has been considered as a possible
solution to this problem. Such an exercise might be
justified with fully informed consent if it could be
shown that search for therapy for retinal dystrophies
was unlikely to be successful without biopsy and that
various objectives could be met. First it must be
shown that the eye would not be put at great risk by
the operation of the biopsy. Surgical techniques have
been devised in rabbits and primates which appear to
minimize this risk1"4; recently this work has been extended to humans.5"9 It has been shown that hypotensive anesthesia and hyperventilation prevent choroidal bleeding.56 Vitrectomy has been undertaken
by some workers to reduce the risk of postoperative
retinal detachment due to proliferative vitreo-retinal
disease although others have found this unnecessary.
Furthermore, good wound closure prevents postoperative ocular hypotony and incarceration of ocular tissues. In some instances the tissue has been obtained
by an internal approach, while in others an external
approach has been used. Development of these surgical techniques has allowed successful removal of large
intraocular tumors.8
Second, biopsy would be unhelpful if laboratory
expertise was inadequate for useful information to be
obtained from the specimen. Laboratory techniques
have now advanced sufficiently such that the activity
of various enzymes and the concentration of many
proteins and lipids can be assessed on small quantities of tissue. Immunocytochemistry and in situ hybridization studies can be performed with great precision. In addition, a limited number of metabolic
attributes can be tested in organ support systems, and
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RETINAL'DIOPSY/Birderd.
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retinal pigment epithelium has now been successfully
cultured in many centers. However, most laboratory
work on animal retinas has been undertaken on relatively large quantities of material removed under
controlled conditions from freshly enucleated eyes,
which contrasts with the limited volume of tissue
which could be removed by biopsy.
Finally, it is clear that damage occurring at the time
of surgery should not render the specimen unusable
in the laboratory. The prolonged period of high illumination required by the procedure could cause
pathological changes due to heating, drying or phototoxicity. Good preservation of retinal morphology in
biopsy specimens has been demonstrated by several
authors,6'7'9 but no other attributes of retinal function
have been tested.
In order to address this last potential problem, a
series of experiments have been undertaken to assess
the suitability of biopsy material obtained for laboratory analysis. Tissue was collected from normal rabbits and dogs and analyzed using biochemical and
histological techniques.
Materials and Methods
Surgical Technique
Internal biopsy: Vitrectomy was performed initially using an Ocutome Model 800A (Berkeley Bioengineering, San Leandro, CA), with the cutting
probe, infusion port and light pipe inserted through
paralimbal sclerotomy sites. Internal biopsy was performed with a 1 mm trephine which was hollow
throughout its length so that axial suction could be
applied to the tissue being cut. The trephine was inserted through an enlarged paralimbal sclerotomy
and the retinal sample was obtained under direct vision through the operating microscope by rotating
the trephine with slight pressure against the inner
surface of the wall of the eyeball. Light suction was
applied to the tissue throughout the cutting. The tissue was recovered by washing the retinal disc from
the trephine using balanced salt solution.
External biopsy: After opening the conjunctiva,
sutures were passed under the rectus muscles to allow
the globe to be manipulated. A full thickness 5 mm
scleral flap with a stepped edge was reflected, exposing the outer surface of the choroid. If possible, the
material was obtained using a 3 mm trephine with
axial suction; if that failed, the sample was excised
with scissors. After each procedure any prolapsed vitreous was abscised and the sclera was repaired with
8-0 interrupted sutures. The tissue obtained was
placed in a petri dish in balanced salt solution, the
retina separated from the choroid, and processed immediately.
Large samples obtained by the external route were
divided into two or four pieces for the various analyses while the small internal biopsies were used intact.
The retinal samples obtained by the external route
were restricted to the equatorial fundus by the limitations imposed by exposure. In order to avoid damage
to the lens, more posterior tissue from within the
central 20 degrees was obtained by the internal route.
All procedures were in keeping with the ARVO Resolution on the Use of Animals in Research.
Preparation of Rabbit Anti-bovine Opsin Antibodies
Rod outer segments were isolated from 40 fresh
bovine eyes according to the method of Papermaster
and Dreyer.10 Opsin was separated from other proteins on a 4 mm sodium dodecyl sulfate polyacrylamine gel (SDS-PAGE) according to the method of
Laemmli11 and the position of the opsin band determined by scanning the gel at 280 nm. The region was
excised from the gel and protein was extracted and
further purified by hydroxyapatite chromatography
according to previously published methods.12 Subsequent SDS-PAGE analysis and silver staining of the
gel showed no contaminants other than opsin multimers. One hundred micrograms of purified opsin in
complete Freund's adjuvant was injected intradermally into each of two rabbits on a weekly basis for 6
weeks. Animals were bled from the marginal ear vein
on the fifth, sixth, and seventh weeks. The presence
and specificity of opsin antibodies were determined
by labelled antibody staining of nitrocellulose replicas
(Western blots) obtained by electrophoretic transfer
of solubilized outer segment proteins from SDS
gels.13 Staining of outer segment proteins was limited
to opsin and its multimers. Specific antiopsin IgG
was affinity-purified from immune serum by coupling purified bovine opsin to cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals,
Piscataway, NJ) following instructions provided by
the manufacturer, except that NaCl was omitted
from the buffer that was used to bind the antigen.
After the serum sample was loaded onto the affinity
column and washed to remove unbound proteins,
specific IgG was eluted with 4 M MgC^ in phosphate
buffered saline (PBS; 10 mM sodium phosphate in
0.9% NaCl). One milliliter fractions were eluted into
5 ml volumes of PBS in order to achieve rapid dilution of the MgCl2. IgG was dialyzed against PBS and
concentrated prior to use.
Preparation of Rabbit Anti-Bovine Cyclic Guanosine
Monophosphate Phosphodiesterase
(cGMP-PDE) Antibodies
Rod outer segments were isolated from 500 frozen-thawed bovine retinas (Hormel, Austin, MN)
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / January 1988
and homogenized in 10 mM MOPS, 30 mM NaCl,
60 mM KC1, 2 mM MgCl2, 0.1 mM PMSF and 1
mM DTT, pH 7.5 (buffer A). The suspension was
centrifuged at 40,000 g for 30 min and the supernatant was discarded. The pellet was resuspended in 5
mM Tris, 0.5 mM MgCl2, 0.1 mM PMSF, 1 mM
DTT, Ph 7.5 and bleached on ice for 10 min. After
centrifugation at 40,000 g for 30 min, the resulting
pellet was re-extracted three more times as before,
and the four supernatants were combined and centrifuged at 40,000 g for 3 hr. The clear supernatant was
applied to a DEAE 50-A Sephadex column and
eluted with a 0-1 M NaCl gradient in 20 mM MOPS,
1.5 mM MgCl2, 0.1 mM PMSF, and 1 mM DTT, pH
7.5. Fractions (0.5 ml) were collected and aliquots
were assayed for cGMP-PDE activity as described
previously.14 Fractions containing enzyme activity
were pooled, dialyzed against buffer A, concentrated
to 1 ml and subjected to HPLC on a TSK-3000 column. cGMP-PDE was eluted with buffer A and the
fractions containing activity were pooled and concentrated. Subsequent SDS-PAGE and Coomassie
blue staining of the gel showed mainly the typical
84-88 kDa doublet corresponding to cGMP-PDE together with three very minor contaminants (120, 95,
and 67 kDa proteins). From this enriched preparation 750 jug of cGMP-PDE in complete Freund's adjuvant were injected intradermally into two rabbits.
The animals received a 500 tig boost after 2 weeks
and 200 tig boosts every month thereafter. They were
bled from the marginal ear vein on the fifth week and
thereafter every 2 weeks.
Polyclonal antibodies were affinity-purified from
the immune serum following the procedure described
by Talian et al15 with minor modifications. One percent pigskin gelatin and 2% BSA in PBS was used as
the blocking buffer for the nitrocellulose sheets and
incubation in the buffer was carried out for 4 hr at
37°C. Excised nitrocellulose strips containing the
electrophoretically transferred cGMP-PDE were incubated overnight at 37°C in immune serum diluted
1:15. The bound antibodies were eluted and concentrated to 0.3 OD (1 cm light path) for immunocytochemical staining. Specificity of the affinity-purified
antibody for cGMP-PDE was determined by Western
blot analysis.13 Bound antibody was visualized by
horseradish peroxidase bound to goat anti-rabbit IgG.
Development of the colored reaction product was
carried out with 0.05% 4-chloronaphthol and 0.01%
H2O2 in PBS.
Analysis of Tissue
Histology and routine electron microscopy: Following biopsy, the specimens were fixed at 21°C by immersion for 1 hr in a mixture of 2.5% glutaraldehyde
Vol. 29
and 2% formaldehyde buffered at pH 7.4 with 0.1 M
sodium phosphate. They werefixedadditionally for 1
hr in 1% osmium tetroxide, dehydrated in a graded
ethanol series (50%-100%), further treated with propylene oxide and embedded in Araldite 502 (Ciba
Products Co., Summit, NJ). Silver-gold sections were
cut with a diamond knife and stained with lead and
uranium salts.
Immunocytochemistry: Specimens for immunocytochemistry were fixed in mixed aldehydes as described above and then stored for 5 days at 4°C in
phosphate-buffered 4% formaldehyde. Thereafter,
they were dehydrated at room temperature in a
graded series of dimethyl formamide (50%-100%)
and embedded in Lowicryl K4M (Polysciences, Warrington, PA). The Lowicryl was polymerized with ultraviolet light for 24 hr at 4°C and additionally for 48
hr at room temperature. After washing in 0.1 M
phosphate buffer (pH 7.4) and blocking with 4% bovine serum albumin (BSA) in phosphate buffer, sections supported by Formvar and carbon on copper
grids were reacted for 1 hr with affinity-purified rabbit polyclonal IgG antibodies directed against bovine
opsin or bovine light-activated cGMP-PDE. The
concentration of affinity-purified primary antibody
was 0.1 OD (A280, 1 cm light path) for opsin antibodies and 0.3 OD for cGMP-PDE antibodies. In each
case, the antibodies were diluted in 0.1 M phosphate
buffer containing 1% BSA. After washing in 0.1 M
phosphate buffer, the sections were washed in saline
(0.9%) containing 20 mM Tris and then incubated in
goat anti-rabbit IgG antibodies adsorbed to 10 nm
colloidal gold (Janssen Life Sciences Products, Piscataway, NJ). The colloidal gold-labelled antibodies
were diluted 1:49 with Tris saline and incubation was
for 1 hr. The sections were washed in phosphate
buffer, fixed for 20 min in phosphate-buffered 2%
glutaraldehyde, washed with distilled water and dried
before viewing in the electron microscope.
Cyclic nucleotides: Each biopsy sample was homogenized in 200 fA of 0.1 N HC1. Two 10/zl aliquots
were separated for the determination of protein by
the method of Lowry et al.16 The rest of the homogenate was boiled for 1 min, centrifuged at 4000 g for
10 min, and the supernatant fraction was removed.
Cyclic GMP and cAMP were measured by radioimmunoassay as described by Farber and Lolley.17 For
cGMP determinations, two 30 tA aliquots of each
supernatant were diluted to 100 til with 50 mM sodium acetate, pH 6.2 and were acetylated with 5 n\ of
a mixture of acetic anhydride: triethylamine (1:2)
prior to assay. For cAMP measurements, two 20 til
aliquots were used. The dilution buffer in this case
was 50 mM sodium acetate, pH 6.2 containing 21
mM CaCl2; acetylation was done in the same way.
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RETINAL DIOP5Y / Bird er ol.
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The succinyltyrosine [I125] methyl ester derivatives of
cyclic GMP and cyclic AMP and the antibodies
against these cyclic nucleotides were purchased from
New England Nuclear (Boston, MA). After completion of the radioimmunoassay and counting of the
samples, nonspecific binding was subtracted, total
cpm/bound in the standards were plotted against increasing femtomoles of standard added and the cyclic
nucleotide concentration in the tissue samples was
read from the linear plot. The results were expressed
as picomoles of cyclic nucleotide per milligram protein.
Results
Surgical Results
In each of two rabbits external biopsy was undertaken in one eye and internal biopsy on the other as a
non-survival experiment. No particular surgical difficulties were encountered and good samples of retina
and pigment epithelium were obtained by each
method. Six additional rabbits were allowed to survive following unilateral internal biopsy of three and
unilateral external biopsy of the remaining three.
There were few external signs of inflammation 1
week after surgery and the ocular media remained
clear. After 3 weeks the wounds appeared clean by
ophthalmoscopy, and the retina elsewhere remained
flat in all but one, in which there was limited detachment around the internal biopsy site. Two animals
with flat retinas were sacrificed at this stage and four
were observed for a further 6 months. At this time the
appearance of the wounds was unchanged (Fig. 1)
except for one in which complete retinal detachment
developed in the animal that had previously had limited detachment. In addition, one animal had limited
posterior lens opacities.
In two dogs internal biopsy was attempted in one
eye using identical techniques; it was found that the
nature of the vitreous precluded adequate vitrectomy
and the specimens obtained were unsatisfactory. In
both instances the dog was allowed to survive the
operation. Uveitis developed and complete retinal
detachment followed within 7 days. The dogs were
sacrificed at that time. In two additional dogs, external biopsy was performed. Apart from bleeding from
the sclera no surgical difficulties were encountered
during the exposure. It proved impossible to obtain
adequate samples with a trephine and tissue was removed using scissors. The retina remained flat and
the ocular media clear during the 4 weeks following
the procedure. External biopsy was undertaken in the
fellow eye of each dog prior to sacrifice.
The tissue obtained was relatively easy to handle,
although in dogs the vitreous adherent to the inner
Fig. J. Photograph of a rabbit fundus 6 months after internal
biopsy showing a round atrophic lesion (arrow) near the visual
streak (VS). There is some hyperpigmentation at the level of the
retinal pigment epithelium arround the lesion. Magnification X4.3.
retina made it difficult to free the samples from surgical instruments.
Histopathology
The morphology of the biopsy material both from
dogs and rabbits was well-preserved apart from minor
changes in the intra- and interdiscal spaces (Fig. 2).
After surgery, examination confirmed that the retina
was flat on either side of the biopsy site (Fig. 3a, d)
with the exception of the animal with detachment.
The inner surface at one internal biopsy site of a
rabbit in which the choroid had not been disturbed
was covered with a monolayer of retinal pigment epithelial cells (Fig. 3c), and with fibrous tissue at the
other sites. On either side of the lesion there was a 1
mm "die back" zone (Fig. 3a, d) with loss of receptors
on its central edge and absence of outer segments
more peripherally. Beyond the margins of this zone
the receptors appeared quite normal (Fig. 3a, b, d).
Immunocytochemistry
Ultrathin sections of Lowicryl K4M-embedded
biopsies were readily stained by the two-stage colloidal gold method. Localization of two antigens,
namely opsin, an intrinsic membrane protein and
cGMP-PDE, a soluble enzyme, were detected in tissue sections of both species under study (Figs. 4, 5).
Rod outer segments were heavily labelled with opsin
antibody and significant binding was evident in inner
segments (Fig. 4a), the sites of opsin synthesis and
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1988
Vol. 29
pteiai
i,
Fig. 2. Light and electron microscopy of rabbit (a, b) and dog (c, d) biopsies. Apart from some distortion of the inter- and intradiscal spaces,
the retinal morphology shows good preservation of structure. Rabbit retina removed by internal biopsy retained some pigment epithelium
(short arrows). Dog retina removed by the external route lacked pigment epithelium and contained adherent red blood cells (long arrows).
Magnification Figures 2a and 2c X770; Figure 2b X44.000; Figure 2d XI 3,750.
transport.18"20 Cyclic GMP-PDE, an important enzyme in phototransduction whose activity is depressed in Irish Setter dogs and rd mice with inherited
retinal degeneration,21'22 was also readily detectable
in rod outer segments (Figs. 4b, 5a). The number of
copies per cell for cGMP-PDE is smaller than opsin
by about ten-fold,23 hence the labelling density for
this protein was considerably less than that for opsin.
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RETINAL BIOPSY / Dird er ol.
1
9k
Cyclic Nucleotides
Although the protein content of the rabbit or dog
biopsies varied from one another, the levels of cGMP
or cAMP expressed per mg of protein were quite reproducible in the different specimens, particularly for
the type of surgical procedure used (Table I). Rabbit
specimens I, 2 and 3 were external biopsies whereas
Fig. 3. Light and electron microscopy of rabbit (a, b, c) and
dog (d) biopsy sites and adjacent retina following internal
biopsy in the rabbit and external biopsy in the dog. The rabbit biopsy site corresponds to
the tissue sample shown in Figure 2a. Retinal pigment epithelial cells (RPE) were seen to
have proliferated and covered
the biopsy site in the rabbit (c).
The cells had a reduced number
of melanin granules and a profusion of apical microvilli.
Some of the proliferating cells
appeared to be budding off
from the monolayer, although
no further evidence of their
proliferation was seen elsewhere in the retina. Both rabbit
(a) and dog (d) retinal sections
show atrophy of the photoreceptor cell outer sements within
I mm of the edge of the biopsy
(short arrows). Within this dieback zone, there were atrophic
neurones with proliferating
glial elements (long arrows).
Magnification Figures 3a and
3d XI20; Figure 3b X470; Figure 3c X5200.
samples 4, 5 and 6 were obtained internally. The
levels of cGMP were in the same range for all of the
rabbit samples, although cAMP levels were considerably lower in the external biopsies. Only external
biopsies were obtained from dogs and the levels of
cGMP and cAMP measured were in agreement with
those reported in the Hterature.for normal dog retinas
of different species.22'24
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / January 1988
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Fig. 4. Electron microscope immunocytochemistry of dog rods showing the junctinal zone between the inner and outer segment joined by
the connecting cilium (C). Goat anti-rabbit antibodies adsorbed to colloidal gold particles were used to demonstrate binding sites for rabbit
antibodies to opsin (a) and light-activated cGMP-PDE (b). Labelling of the rod outer segments (OS) is in accordance with the relative density
of antigen as measured by biochemical means. Labelling of newly synthesized opsin in transit from inner segment (IS) sites of origin is also
evident in Figure 4a. Magnification X27.500.
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RETINAL DIOP5Y / Dird er ol.
No. 1
OS
Fig. 5. Electron microscopy of the junction between the inner and outer segments of rabbit rods showing colloidal gold labelling of
cGMP-PDE over the outer segment (OS) discs (a). Control in which nonimmune primary antibodies were substituted for immune antibodies
is shown in b. Magnification X27,500.
Discussion
Our experience shows that it is possible to obtain
samples of retina from the rabbit and dog without
destroying the integrity of the sample. The only technique which failed was the internal approach in the
dog eye due to the difficulty in achieving an adequate
vitrectomy. The surgical difficulties were somewhat
greater in the dog than in the rabbit because of bleeding during exposure of the choroid and adherent
qualities of the vitreous. Hypotensive anesthesia and
controlled hyperventilation, neither of which were
available to us, would have minimized the bleeding
tendency.
Tissue loss in the eye due to the surgical procedure
comprised the sample of retina removed and a 1 mm
zone around the biopsy site. Such loss of peripheral
retina would not be expected to cause appreciable
visual loss and there were no detectable behavioral
changes in the animals. Apart from a small area
around the biopsy site, the retina appeared normal
after biopsy such that the exercise would not prejudice the usefulness of subsequent biopsies. In all but
one eye in this series it would have been possible to
Table 1. Protein and cyclic nucleotide content of
rabbit and dog biopsies
Animal
Rabbit
Specimen
1
2
3
4
5
6
Dog
1
2
3
4
Protein
(Mg)
Cyclic
GMP*
Cyclic
AMP*
72
62
20.0
20.8
30.5
31.8
20.1
25.5
36.0
41.0
63.0
76.1
74.5
76.2
15.6
14,0
14.0
13.7
26.3
is
20
58
61
79
15S
72
94
24.2
23.6
25.2
* Picomoles per mg protein. Values represent the mean of two aliquots
from each sample; each measurement was done in duplicate.
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10
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1988
undertake a second or more biopsies from one eye or
alternately from the other eye, since the fellow eye
had recovered sufficiently from surgery. Clearly the
procedure is not totally innocuous, as shown by the
detachment in one rabbit after internal biopsy. However, there is sufficient evidence from this study as
well as from previous work to suggest that biopsies
can be taken without high risk of destroying the eye.
There is little doubt that the risk would vary from one
species to another. The observation that the procedure is relatively safe in one species does not allow the
conclusion that it would be safe in others.
This experience shows that the specimens taken
were more than adequate in size for laboratory examination. Three of the four quarters of a 3 mm sample
of retina would allow histological examination, multiple immunocytochemical experiments and analysis
of cyclic nucleotides, A remaining sample would be
available for additional analysis such as in situ hybridization or could be kept in reserve if one sample
proved inadequate. Alternatively, it is possible that a
slightly smaller specimen might be obtained.
It is also shown that the removal of a retinal biopsy
under normal surgical conditions does not prejudice
the usefulness of the sample for research. The appearance of the tissue by electron microscopy showed little artifact, and the distribution and concentration of
antigen label was in accord with that found in retina
obtained by other means. Two analyses of each specimen were undertaken to determine the levels of each
cyclic nucleotide. Interestingly, the results from the
rabbit biopsies indicated some variation in cGMP
levels among the different samples independent of
the type of surgery performed, whereas cAMP levels
seemed to be considerably lower in the external biopsies than in the internal ones. This may be related to
the relative distribution of each cyclic nucleotide in
the rabbit retina. cGMP is known to be concentrated
mainly in the outer segments of rod photoreceptors,25
which are present in abundance throughout the rabbit and dog retina, whereas cAMP is enriched in the
cone visual cells most of which are located in the
central retina and in the cells of the inner retina.2627
Furthermore, most vertebrate retinas, including that
of the rabbit, are thicker in the posterior or central
region than in the periphery. Thus, samples obtained
by the internal procedure had more cellular components enriched in cAMP than did the external biopsies.
The potential usefulness of this biopsy technique is
not limited to the study or diagnosis of human retinal
disease. When animals are in short supply, as is the
case with some breeds of dog with inherited retinal
degeneration, biopsy could be used to good advantage in these cases as well as in humans. Although
Vol. 29
biopsy would not be useful in the documentation of
regional distribution of change at a given point in the
evolution of the disease, it would allow serial documentation of degeneration in the equatorial retina
without having to sacrifice the animal when retinal
material is needed.
With the current level of expertise it has been possible to identify fundamental metabolic abnormalities
in animals with early onset, genetically determined
retinal dystrophies. While progressively more information is becoming available on human retinal degeneration, the irregular and unpredictable supply of
material which is frequently not ideally suited for
laboratory analysis will not allow advances in the understanding of human disease at the same rate as has
occurred in animals. That laboratory investigative
techniques may reveal important new information
concerning the pathogenesis of retinal dystrophies in
man was illustrated recently by the demonstration of
abnormal incorporation of sugars by receptors of
normal morphology derived from a patient with retinitis pigmentosa.28 It is only by biopsy that retina
could ever be obtained prospectively from patients
with inherited retinal receptor disease. Such an exercise might not be justified before clinical and laboratory sciences have provided sufficient information to
formulate hypotheses concerning the pathogenesis of
human retinal dystrophies. When such a time comes
it is important that the laboratory techniques will
have been adapted to make maximum use of the
small quantities of tissue likely to be available from
biopsy.
Key words: retinal biopsy, photoreceptors, pigment epithelium, retinal glia, retinitis pigmentosa, immunocytochemistry, cyclic nucleotides
Acknowledgments
The authors gratefully acknowledge the technical assistance of Caryl Lightfoot, Marcia Lloyd, Alice Van Dyke,
and Walter Miyamasu. We thank Dr. Brian Matsumoto for
preparation of the opsin antibodies.
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