Veterinary Ophthalmology (2009) 12, 3, 158–169
Blackwell Publishing Inc
Functional and structural assessment of retinal sheet allograft
transplantation in feline hereditary retinal degeneration
Magdalene J. Seiler,*a Robert B. Aramant,†a Mathias W. Seeliger,‡ Ragnheidur Bragadottir,§
Melissa Mahoney¶ and Kristina Narfstrom**
*Departments of Ophthalmology, and Cell and Neurobiology, Keck School of Medicine, University of South California, Los Angeles, CA, USA ; †Department of
Anatomical Sciences and Neurobiology, University of Louisville, Louisville, KY, USA; ‡Ocular Neurodegeneration Research Group, Institute for Ophthalmic
Research, Tuebingen, Germany; §Department of Ophthalmology, Ulleval University Hospital, Oslo, Norway; ¶Chemical Engineering, University of Colorado,
Boulder, CO, USA; **Departments of Veterinary Medicine and Surgery, and Ophthalmology, University of Missouri-Columbia, MO, USA
Address communications to:
K. Narfstrom
Tel.: +573 882 2095
Fax: +573 884 5444
e-mail: narfstromk@missouri.edu
a
Present address: Anatomy and
Neurobiology, Reeve-Irvine
Research Center, University of
California, Irvine, CA, USA.
Abbreviations: GCL, ganglion
cell layer; IPL, inner plexiform
layer; NBL, neuroblastic layer;
INL, inner nuclear layer;
OPL, outer plexiform layer;
ONL, outer nuclear layer;
RPE, retinal pigment epithelium;
Rhod, rhodopsin; Glusynth,
glutamine synthetase; DAPI,
4,6-diamidino-2-phenylindole;
PKC, protein kinase C; Syn,
synaptophysin; Calb, Calbindin;
Parv, parvalbumin; H, host;
T, transplant.
Abstract
Purpose To investigate whether sheets of fetal retinal allografts can integrate into the
dystrophic Abyssinian cat retina with progressive rod cone degeneration.
Methods Fetal retinal sheets (cat gestational day 42), incubated with BDNF
microspheres, were transplanted to the subretinal space of four cats at an early disease
stage. Cats were studied by fundus examinations, bilateral full-field flash ERGs, and
indocyanine green and fluorescein angiograms up to 4 months following surgery. E42
donor and transplanted eyes were analyzed by histology and immunohistochemistry for
retinal markers.
Results Funduscopy and angiography showed good integration of the transplants in two
of four cats, including extension of host blood vessels into the transplant and some
scarring in the host. In these two, transplants were found in the subretinal space with
laminated areas, with photoreceptor outer segments in normal contacts with the host
retinal pigment epithelium. In some areas, transplants appeared to be well-integrated
within the host neural retina. Neither of these two cats showed functional improvement
in ERGs. In the other two cats, only remnants of donor tissue were left. Transplants
stained for all investigated cellular markers. No PKC immunoreactivity was detected in
the fetal donor retina at E42, but developed in the 4-month-old grafts.
Conclusions Fetal sheet transplants can integrate well within a degenerating cat retina and
develop good lamination of photoreceptors. Functional improvement was not
demonstrated by ERG in cats with well-laminated grafts. Transplants need to be further
evaluated in cat host retinas with a more advanced retinal degeneration using longer
follow-up times.
Key Words: Abyssinian cat, fetal sheets, fluorescein angiography, ICG,
immunohistochemistry, retinal degeneration, retinal transplantation, scanning laser
ophthalmoscope
INTRO DUC TIO N
Retinal degenerative diseases such as age-related macular
degeneration1,2 and retinitis pigmentosa (RP)3 affect a
considerable part of the human population and lead to
irreversible vision loss. Such diseases primarily affect the
photoreceptors and/or retinal pigment epithelium (RPE)
while leaving the inner retina functional for some time.4–7 If
the diseased cells can be replaced and the new cells can make
appropriate connections with the functional part of the host
retina, a degenerated retina might be repaired and eyesight
restored.
Animal models of retinal degeneration have been used
by researchers to elucidate physiological and biochemical
changes associated with these diseases.5,8–10 Retinal transplantation using fetal retinal sheets and a custom-made
implantation tool appear to be promising for replacing dying
photoreceptor cells.11–15
© 2009 American College of Veterinary Ophthalmologists
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BDNF has been shown to enhance retinal ganglion cell
axonal outgrowth and survival16–18 and thereby promoting
cellular connectivity and also has a protective effect on photoreceptors19,20 through Müller glial cells.21 Its secretion is
activity-dependent.22 It has been demonstrated that BDNF
microsphere treatment enhances the functional effect of
retinal transplants.23
Using a well-established large animal model for human
recessive RP24 we wanted to investigate whether intact sheets
of feline fetal retina, treated with BDNF microspheres
integrate into the dystrophic Abyssinian cat retina and if the
transplanted tissue develops normally in vivo. This cat model
has been used previously for transplantation of small pieces
of 3–5-day-old kitten retina to the subretinal space.25 These
transplants developed rosettes. This cat model was later
also used for transplantation of full-thickness retinal sheets
derived from kittens26 using a different instrument and
procedure than the one presented in this article. In another
study, the same model was used for transplantation of dissociated retinal progenitor cells.27
MAT ERIALS AN D M ETH OD S
with Hibernate E medium (BrainBits, Springfield, IL) with
B-27 supplements (Invitrogen, Carlsbad, CA), the microspheres
were resuspended in Hibernate E medium at a concentration of
5 mg/mL.
Donor tissue
Neural retinas were dissected from cat fetuses of gestational
day 42. The gestation of cats is 63 days. The fetuses were
euthanized by an overdose of medetodimine and ketamine
intraperitoneally. After enucleation of the eyes, a circumferential cut was made in the pars plana, and anterior eye
segments were removed. The neural retina was then dissected
free from RPE/choroid and vitreal blood vessels, and cut
into pieces (1.7 × 3 mm). The pieces were flattened in a
small amount of medium in a Petri dish and then incubated
with a small drop (50–100 μL) of BDNF treated microspheres
in Hibernate E medium with B-27 for 3 h on ice or overnight
at 4 °C. The correct orientation of the tissue could easily be
recognized in the dissection microscope by looking at the
ganglion cell axons. Immediately before transplantation,
the tissue was taken up into the nozzle of a custom-made
implantation instrument that has been used previously in
human clinical trials30,31 (review32).
Experimental animals
All experimental procedures were in accordance with the
Association for Research in Vision and Ophthalmology
statement for the Use of animals in Ophthalmologic and
Vision Research, and were approved by Animal Care and
Use committee of the University of Missouri. Pregnant
cats were obtained from LibertyResearch Inc., Waverly,
NY. Recipients were four Abyssinian cats, age 1–1.5 years
(cat #1–4), born and maintained at the University of
Missouri, affected with an early stage of the hereditary
retinal degeneration.24,28
BDNF microspheres
BDNF microspheres (average diameter 1 ± 0.5 μm) were
prepared using a previously described methodology.29 A
release curve of the microspheres for BDNF has been
published.23 First, 300 mg of (50 : 50) PLGA (Mn = 54 100
Birmingham Polymers) was dissolved in 2 mL of methylene
chloride. On ice, 100 μL of a solution containing 500 μg
BDNF was added to the dissolved PLGA. After sonication,
4 mL of aqueous 1% polyvinyl alcohol (PVA, Mw = 25 000,
Polysciences) was mixed into this emulsion. To achieve
microsphere formation, this double-emulsion was poured
into a beaker with 100 mL of aqueous 0.3% PVA and stirred
for 3 h. Microspheres were collected by centrifugation at
3130 × g for 10 min and washed three times with de-ionized
water before they were frozen in liquid nitrogen and then
lyophilized for 24 h. Microspheres were stored at 4 °C
until use.
To make the microspheres positively charged to adhere to
tissue, a suspension of 5 mg BDNF microspheres was
incubated for 1 h in 10 μg/mL poly-dl-lysine in PBS immediately before transplantation. After washing three times
Transplantation procedure
After induction of medetodimine and ketamine anesthesia
(medetomidine 0.1 mg/kg IM, Domitor, Novartis, Pfizer Animal
Health, Exton, PA, and ketamine 5 mg/kg IM, Ketaset III,
Fort Dodge Animal Health, Fort Dodge, IA) and dilation of
the right pupil with atropine (Isopto-Atropin 0.5% Alcon,
Fort Worth, TX) the eye was prepared for aseptic surgery as
previously described.26 In short, the eye was stabilized with
4/0 silk stay sutures and a lateral canthotomy was performed.
The sclera was exposed temporally with a radial incision
through conjunctiva and Tenon’s capsule. Two sclerotomies
were performed, 6–7 mm from the limbus, approximately
5 mm apart, using a 20G MVR blade (Alcon Surgical) and
care taken not to incise the intrascleral venous plexus by
avoiding visible veins. Intrascleral ports were found not to be
needed because of the rigid structure of the cat sclera. A
magnifying lens (Machemer flat lens, Ocular Instruments,
Bellevue, WA) was placed on the cornea for intraocular
viewing through a binocular operating microscope (Zeiss,
Berlin). A partial vitrectomy, encompassing approximately
30% of the vitreous, was made in the region overlying the
transplantation area. A retinotomy was performed two disc
diameters superior nasal to the optic nerve head and a retinal
bleb was formed through infusion of BSS with a glass micropipette (drawn from a borosilicate glass tube; TW 150-4,
World Precision Instruments Inc., Sarasota, FL) attached to a
1.0-mL syringe with silicone tubing. The small retinotomy was
enlarged with a fan shaped movement of the micropipette tip.
Using a custom-made instrument for delivery, a sheet of the
transplant was placed in the subretinal space by a standard
vitreoretinal approach (ab interno). The details of the surgery
with this instrument in rats and humans have been previously
© 2009 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 12, 158–169
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SEILER ET AL.
Table 1. Antibodies for cell type characterization
Primary antibodies
MAP-1 A
(microtubule-associated protein 1 A)
Synaptophysin
Parvalbumin
Glutamine synthetase
Protein kinase C (PKC)
Rhodopsin
Recoverin
Calbindin
Calretinin
Secondary antibodies
Rhodamine RedXTM anti-mouse IgG
Alexa Fluor 488 anti-rabbit IgG
Rhodamine RedXTM anti-rabbit IgG
Species Specific for
Dilution
Supplier
Mouse
1 : 2000
Amersham, Pittsburgh, PA
Mouse
Mouse
Mouse
Rabbit
Rabbit
Rabbit
Rabbit
Rabbit
Goat
Goat
Neuronal cell processes, ganglion cell
dendrites
Synaptic layers
Amacrine cells + horizontal cells
Glial cells
Rod bipolar cells; cone outer segments
Rod photoreceptors
Rod + cone photoreceptors; cone bipolar cells
Horizontal + amacrine cells; cones
Amacrine and ganglion cells
Mouse antibodies
Rabbit antibodies
Rabbit antibodies
described.33 Only one eye received a transplant, the contralateral
eye served as a control.
1 : 5000
1 : 500
1 : 1000
1 : 200
1 : 1000
1 : 500
1 : 2000
1 : 2000
1 : 200
@
Sigma, St. Louis, MO
!
Transduction Labs, Lexington, KY
Oxford Biomed. Products, Oxford, MI
Plantner62
McGinnis;63 Dizhoor64
Calbiochem, San Diego, CA
Chemicon, Temecula, CA
Molecular Probes, Eugene, OR
tissues and layers because of their reflection and transmission
characteristics, in particular the degree of absorption by
melanin pigment.35
Post-operative examination
Cats were studied post-operatively on a daily basis for the first
week, then once weekly till they were euthanized. Complete
ophthalmic exams were performed including indirect
ophthalmoscopy and slit-lamp biomicroscopy after pupillary
dilation. Starting one month post-surgery, then every month
until euthanasia, simultaneous bilateral full-field flash elctroretinograms (ERGs) were performed using a computerized
ERG (ERG System Tor, Global Eye Program, Rejmyre,
Sweden) with full-field white light stimulation. ERGs were
recorded, using similar anesthesia protocol as for surgery,
after the cats had been dark-adapted for a minimum of 1.5 h.
A jet corneal contact lens served as the active electrode, and
subdermal needle electrodes, inserted approximately 10 mm
temporal to the temporal canthus of the eye and at the
ipsilateral occipital crest, served as the reference and ground
electrodes, respectively. Scotopic ERG responses were recorded
by stimulation using white light between –6 and 0.6 log units
of standard flash (SF) (SF = 1cd.s/m2) in increments of
0.3–0.5 log units, by removing neutral density filters from
the light path. After 10 min of light adaptation at 30 cd/m2,
photopic stimulation was performed at 5.1 Hz, followed by
flicker recordings at 30 and 50 Hz. The photopic ERGs
were recorded at a light stimulus of intensity 0.0 log cd.s/m2,
which is equivalent to 1 cd.s/m2. Four months following
surgery, the engraftment site morphology was further
examined using scanning laser ophthalmoscopy (HRA I SLO,
Heidelberg Engineering, Dossenheim, Germany). The SLO
unit was modified for use in animals.34 The vascular status of
the retina and the graft was assessed with fluorescence
angiography (FA) using fluorescein dye and a blue (488 nm)
laser stimulus. The status of the choroidal circulation was
visualized by ICG angiography using indocyanine green dye
and an infrared (795 nm) laser stimulus. The comparison of
images taken with the different wavelengths together with
the two different dyes, provides information about specific
Tissue processing
Four months after surgery, cats were euthanized according
to veterinary standards by IV injection of euthanasia solution
(Beuthanasia-d-Special, Shering Plough Animal Health,
Omaha, NE), their eyes harvested immediately and fixed in
4% paraformaldehyde in 0.1 m sodium-phosphate buffer. Donor,
contralateral and transplanted eyes were infiltrated with 30%
sucrose, embedded in Tissue Tek OCT compound (Sakura
Finetek, Torrance, CA) and cut on a Leica cryostat (10 μm
sections). Sections were stained with H&E, and with
antibodies for retinal markers (recoverin, rhodopsin, PKC,
MAP 1 A, calbindin, calretinin, synaptophysin, glutamine
synthetase) (see Table 1). Sections were blocked with 20%
goat serum at room temperature before incubation with
primary antibodies overnight at 4 °C. After washing thrice
with phosphate-buffered saline (PBS), sections were incubated
in the appropriate fluorescent secondary antibodies for 1 h
at room temperature. After another PBS wash, sections were
coverslipped with 4,6-diamidino-2-phenylindole (DAPI)containing mounting medium (Vector Laboratories, Burlingame CA). Fluorescent images were taken on a Nikon FXA
microscope, and deconvoluted using Autoquant deconvolution
software (Autodeblur software 9.2 and 9.3). Confocal images
were scanned on a Zeiss LSM 510 confocal microscope (Carl
Zeiss, Germany). Scans at different focus depths were combined
into one projection image.
R E S U LT S
Clinical examination
In one of four cats (cat #4), the grafting procedure resulted
in blood in the vitreous cavity the day after surgery. After a
few days a secondary cataract developed, which made visualization of the fundus impossible. The other three cats had a
normal post-operative appearance of the eyes. The subretinal
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Figure 1. Scanning-laser ophthalmoscope (SLO) imaging. Images were obtained from the nasal part of the central tapetal fundus during fluorescence
angiography (a–c), native infrared imaging (d), and during indocyanine green angiography (ICGA, e –f ). (a) Autofluorescence image before injection
of dye, (b) early phase, and (c) later phase of FA depicting mainly the retinal circulation. (d) Native infrared image of the transplant. (e) ICGA image
allowing to visualize both the choroidal and the retinal circulation. (f ) Detail of (e) showing also minor supporting vessels and roundish pigmented
structures presumably resembling rosettes.
bleb was observed to become flat within 2–3 days following
surgery. In two of the four cats, there was subretinal hemorrhage
in the bleb area, which resolved within 3–4 weeks after surgery.
SLO imaging
Four months after surgery, the graft appeared well-integrated
and connected to the host’s vascular system (e.g. Fig. 1d, native
infrared). To explore this in depth, a FA and indocyanine green
angiography (ICGA) were performed (Fig. 1).
In FA, the autofluorescence image before injection of dye
(Fig. 1a) revealed dark coloration at the site of the graft as
the light path is blocked by the extra tissue, but also in a few
surrounding regions presumably because of manipulations
during surgery. Interestingly, these sites did not show
alterations in choroidal filling (Fig. 1b) during the early phase
of FA. In the later phases of FA, a number of vessels supporting
the graft became obvious (Fig. 1c), but no leakage was apparent.
The graft was surrounded by a number of well-demarcated
hyperfluorescent areas probably reflecting window defects.
The ICGA indicates that the major choroidal vessels were
not disturbed by the graft or the surgical procedure (Fig. 1e).
The extra tissue of the graft led to a shadowing of the choroidal
structures below the transplant area (Fig. 1e). The magnified
view of the transplant area (Fig. 1f ) reveals details of the
vascular connection but also the rosette-like structures
further described in the histological analysis below.
Elctroretinogram
For the two cats with well-laminated transplants (cats #1
and 2), there were no differences in waveforms, response
amplitudes or implicit times between transplanted and
contralateral eyes, 1 month following surgery (Fig. 2) and in
the follow-up studies up to 4 months after transplantation.
The same was found for cat #3. Although there were
slight increases in ERG amplitudes in the treated eye for
cat #4 post-operatively, there was a great variability between
eyes and recordings, especially for this cat. For details in
regards to maximum a- and b-wave responses obtained for
the four treated cats in scotopic conditions see Table 2.
Histology
Examples of the histology of fetal cat retina are shown in
Fig. 3. At the time of transplantation, the donor tissue
contained a ganglion cell and an outer neuroblastic layer.
Strong staining of cones was observed close to the outer
limiting membrane (Fig. 3a,b). Staining for calretinin, a
marker for amacrine cells, showed labeled cells in the
ganglion cell layer and scattered cells in the neuroblastic
layer (Fig. 3c). Calbindin, a marker for horizontal and
amacrine cells, stained few cells in the ganglion cell layer and
migrating large cells with processes (indicating developing
horizontal cells) as well as smaller developing amacrine cells
(Fig. 3d). No immunoreactivity for protein kinase C, a
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SEILER ET AL.
Figure 2. Representative ERG responses from transplanted cat, 4 months after surgery. Scotopic responses were initially obtained after 1.5 h of
dark adaptation followed by photopic responses after 10 min of light adaptation using 30 cd/m2 of background light. Upper left: rod responses
at –3.0 log cd.s/m2, and lower left: mixed rod cone responses at 0.6 log cd.s/m2. Upper right: cone responses at 0.0 log cd.s/m2, lower right: cone and
inner retinal responses using 30 Hz flickering light stimulation. Upper tracing in each set of recordings is from the right transplanted eye and lower
tracing is from the left untreated eye.
Table 2. ERG a- and b-wave amplitudes (in μV) and implicit times (in ms) in cat 1–4 before surgery and at 1 and 4 months, respectively, after surgery
using high white light intensity (4 cd.s/m2) stimulation in the scotopic state
Pre-operative
OD
OS
Post-operative 1 month
Post-operative 4 months
OD
OD
OS
OS
Cat/ERG parameters
b
a
b
a
b
a
b
a
b
a
b
a
1/Amplitude
Implicit time
2/Amplitude
Implicit time
3/Amplitude
Implicit time
4/Amplitude
Implicit time
597
66
666
59
777
61
409
63
180
10
111
9
314
9
118
9
596
66
648
59
712
57
493
62
174
10
111
10
296
9
118
9
527
64
638
54
562
64
444
59
152
9
104
10
208
10
118
9
596
64
611
62
652
64
555
59
180
9
97
9
250
9
125
8
577
68
650
59
520
64
520
57
150
9
103
9
208
10
125
9
576
65
636
59
666
64
402
59
164
9
95
9
263
10
90
9
marker for rod bipolar cells, was found at this early stage
(data not shown).
At 4 months after transplantation, in two (cats #3 and 4) of
the four cats, Bruch’s membrane and choroid had been
damaged; and only remnants of donor tissue were found in
the subretinal space, indicating rejection (data not shown).
In the other two cats (cats #1 and 2), intact laminated transplants were found in the subretinal space. The host retina
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Figure 3. Fetal cat retina at E45. (a) Double labeled
image for the nuclear marker DAPI (blue) and
recoverin (red), a marker for photoreceptors and
cone bipolar cells. (b) shows a confocal image.
Developing cones at the outer limiting membrane
are strongly stained, faint label of developing cone
bipolar cells. (c) Staining of scattered cells in the
neuroblastic layer and in the ganglion cell layer for
calretinin (red) (amacrine cell marker). (d) Staining
for calbindin (red) (marker for horizontal and
amacrine cells). Bar = 20 μm. (a) (c),
(d) Deconvoluted images (Autoqant Autodeblur
software).
contained approximately seven rows of photoreceptors in
the outer nuclear layer outside the transplant area, similar to
the numbers found in the untreated eye (Fig. 4). A good
apposition between transplant photoreceptor outer segments
and the host RPE was observed in the laminated areas (Fig. 5).
Within the transplant area, most host photoreceptors had
disappeared, and the host inner retina was traumatically
disrupted and gliotic (Fig. 5). Recipient and transplant
photoreceptors looked differently in H&E staining (Fig. 5).
Figure 5(c,d) show enlargements of the transplant–host interface.
Transplants stained for all investigated cellular markers. The
antibody for glutamine synthetase, labeled radial processes
of glial Müller cells in the host retina (Fig. 6a) and in the
transplant Fig. 6(b). Photoreceptor outer segments stained
for rhodopsin, both in the host retina (Fig. 6a) and in the
transplant (Fig. 6b). The antibody for synaptophysin (a
synaptic marker) strongly stained the outer and inner
plexiform layer in the host retina (Fig. 7a) and in the transplant
(Fig. 7b). The antibody for PKC labeled rod bipolar cells in
Figure 4. Histology of contralateral nonoperated eye, staining with
H&E. In the central retina, approximately 7 rows of nuclei are seen in
the outer nuclear layer at this early stage of rod/come degeneration
(1–1.5 years). Bar = 100 μm.
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SEILER ET AL.
Figure 5. H&E stained images of transplant of cat
1. Approximate transplant-host border in the outer
nuclear layer is indicated by arrows in A, B, and C.
Small white dashes indicate the transplant-host
interface in C and D. Question marks in C and D
indicate areas where it is unclear which cells belong
to transplant or host retina. (a,b) Low power
composite images of serial sections. The
detachment of the outer segments from RPE and
choroid on the right side of (a) and (b) is a sectioning
artifact. The transplant outer nuclear layer is facing
the host RPE. Most of the host photoreceptors have
degenerated in the transplant area. The transplant
appears to have folded on itself with photoreceptor
rosettes toward the host retina. It is difficult to
determine the exact transplant-host border towards
the host retina. Box in B) indicates area of
enlargement in (c). Bar = 100 μm. (c) Enlargement
of (b), showing the transition from host to
transplant outer nuclear layer. Bar = 50 μm.
(d) Further enlargement of transplant-host
interface. Bar = 20 μm.
host retina (Fig. 7a) and in the transplant (Fig. 7b–d). In the
laminated transplant areas, rod bipolar cells appeared to be
organized similar to the host retina. More disorganization
was seen where the transplant had folded on itself. In the
transition area between transplant and host, crossing of PKC
immunoreactive processes and merging of synaptic layers
could be observed (Fig. 7b,c). Figure 8 shows the distribution
of parvalbumin (a marker for horizontal and AII amacrine
cells) and calbindin (a marker for cones, horizontal cells and
another amacrine cell population). Horizontal cells were
double-labeled by calbindin and parvalbumin, both in host
and transplant. Immunoreactivity (and density) of transplant
cones for calbindin appeared to be somewhat lower than in
the host retina (Fig. 8c–e).
DISC USSIO N
The purpose of this study was to test whether sheets of fetal
cat retina transplanted to a cat model of retinal degeneration,
can develop lamination and integrate with the host retina,
using an instrument and procedure that has provided good
results in rodents models11,15,36 and is currently being used
in clinical trials in human patients.30,31,37
The Abyssinian cat is a well-established model for retinal
degeneration.24 The model has been carefully characterized
and clinical, morphological and electrophysiological similarities
to human RP have been shown.38
In an earlier study that used the Abyssinian mutant for
retinal transplantation, dissociated neuro-retinal cells were
administered subretinally using a one-port sclerotomy
procedure, without vitrectomy.25 These transplants survived
for at least 6 months, but severe rosette formation was
invariably observed. Seiler and Aramant,11 and Ghosh et al.,39
showed that rosette formation is reduced by transplanting
whole sheets of minimally traumatized neuroretina in rat
and rabbit, respectively. Bragadottir and Narfstrom26 developed
a technique, using a two-port sclerotomy and vitrectomy,
for transplanting larger neuro-retinal donor tissue into the
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Figure 6. Double labeling for glial marker
glutamine synthetase (red) and photoreceptor
marker rhodopsin (green), with nuclear DAPI
counterstain (blue). Confocal images. (a) transplant
area (transplant of cat 1), composite of 8 confocal
images. Arrow points to transplant-host border (also
demarcated with white dashed line). The transplant
has developed an outer nuclear layer facing the host
RPE (RPE cannot be seen). Photoreceptor outer
segments stain green. It is unclear whether the
rosettes close to the inner nuclear layer of the host
retina belong to the host or the transplant.
Bar = 100 μm. (b) host retina outside transplant.
Bar = 20 μm.
Figure 7. Double labeling for synaptic marker
synaptophysin (red) and rod bipolar cell marker
PKC (green), with DAPI nuclear counterstain
(blue). (a) transplant of cat 1, overview
(deconvoluted image). Arrows indicate approximate
transplant-host border in outer nuclear layer, white
dashed lines indicate interface between transplant
and host. Question marks indicate areas where it is
unclear which cells belong to transplant or host
retina. White dots indicate a hollow space within
the transplant where it has folded on itself.
Bipolar cells are well-stained in the laminated
area of the transplant, but less stained in the more
disorganized areas. Intense red line indicates strong
synaptophysin stain of outer plexiform layer of the
host retina and also in laminated area of transplant;
loss of synaptophysin staining in host outer
plexiform layer overlying transplant. (b–d) Confocal
images (cat #2). (b) host retina outside transplant
(confocal image). (c) Edge of transplant. Arrow
points to transplant-host border. (d) Enlargement of
(c). Bar = 500 μm (a); = 20 μm (b,c,d).
subretinal space of cat eyes. Further development of that
technique was performed in the present study by utilizing a
custom made instrument for atraumatic delivery of oriented
pieces of neuroretinal tissue.15,40
The surgical success rate in the present small number of
cats transplanted was comparably low with only two of four
recipients showing well-laminated transplants with good
apposition between the transplant photoreceptors and the
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SEILER ET AL.
Figure 8. Double labeling of transplant area for
parvalbumin (red) and calbindin (green). Both
antibodies stain similar cell populations of amacrine
and horizontal cells. However, A-II amacrine cells
only stain for parvalbumin; and cones only stain for
calbindin. Strong double-staining of the outer
plexiform layer in transplant and host. (a, b) Low
power overview images. (a) Calbindin, (b)
Parvalbumin Bar = 200 μm. (c) Double-labeled
confocal image, overview (composite of 7 confocal
images). Note the lower number of calbindinimmunoreactive cones in the transplant. Horizontal
cells appear to be larger in the host retina.
Bar = 100 μm. (d) Further enlargement of C.
Bar = 20 μm. White dashed line indicates
approximate transplant-host border at edge of
graft: Bar = 20 μm.
RPE. One reason for this is that posterior segment surgery is
technically difficult to perform in domestic cats,26 with deep
orbital sockets and little space to manipulate the eye. Furthermore, there is easily bleeding from the ciliary plexus and the
choroidal tissue, with a tendency to occur upon the initial
sclerotomy procedures, and also at other times during surgery
when instruments are entered or withdrawn from the intraocular
surgery site.26 The Teflon nozzles for the instrument (the
inserter) used in the present study were developed for
humans and were a bit too large for the smaller cat eye.
Apparently, the donor tissue had folded back on itself
which explains the thickness of the transplants in some areas
and the disorganization of its inner layers towards the host
neural retina.
We have shown that intact fetal sheet transplants can integrate
well within a degenerating cat retina and partially develop good
lamination. In certain areas, transplant photoreceptors had
developed outer segments in contact with host RPE. Transplant
photoreceptors in the laminated areas expressed rhodopsin
and synaptophysin in the correct cellular compartments. All
this together indicates the potential function of transplant
photoreceptors. Graft–host transition in the outer nuclear
layer could be observed in two of the transplanted retinas.
Some rosette formation and disorganized inner retinal layers
could be seen in the transplant area as well. Trauma to the
tissue during the surgery may have promoted formation of
these rosettes. This finding was corroborated with breaks in
the RPE layer including Bruch’s membrane observed by SLO
angiography and morphology in the present study.
Although BDNF was used for improving the transplant
success,23 the effects could not be evaluated in this study,
because of the lack of positive controls and the early stage of
the retinal degeneration.
At the time of sacrifice, the recipient retina still contained
about seven rows of photoreceptors. Host photoreceptors
had degenerated in the transplant area, probably because of
the increased distance from the RPE. Although the donor
tissue had not been prelabeled, transplant photoreceptors
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r e t i n a l s h e e t t r a n s p l a n t i n f e l i n e r e t i n a l d e g e n e r a t i o n 167
could be clearly distinguished from host photoreceptors in
the transplant–host interface (Fig. 5).
Both transplants contained laminated areas with good
apposition of photoreceptors to host RPE, in addition to
rosetted areas. It appeared that the edges of the donor tissue
had folded up during or after the insertion and formed
rosettes. Transplant photoreceptors appeared to have fully
matured in the laminated areas, as judged by both rhodopsin
and synaptophysin staining. Other retinal markers used
showed good expression. PKC-expressing rod bipolar cells
had developed in the transplants, showing relatively normal
morphology in laminated areas, but appeared to be disorganized
in rosetted areas. Similar development of retinal marker
expression has been shown for fetal sheet transplants in
rodent models of retinal degeneration.11,36,40,41
The donor tissue in the current study was fetal (E42), about
25 day younger in development than the older postnatal
tissues used in the other studies.25,26 Although microglial cells
can be identified as early as E12 in the rat42 and E10 to E11.5
in the mouse,43 fetal retinal tissue may be less immunogenic
because it contains less microglia than older tissue.42,44 In
mice, microglia density peaked between E17.5 and E18.5,
but decreased at P0, and increased later postnatally43 whereas in
the rat, microglia density continuously increased until P7.42 In
developing human fetal retina, microglia was associated with
developing blood vessels.44 Since the fetal retina used for
transplantation did not yet contain retinal blood vessels, and
vitreous blood vessels were dissected away, microglial cell
density was probably low.
The subretinal space into which the transplants are placed
has been shown to be an ‘immune privileged’ site, similar to
the anterior chamber of the eye and the central nervous
system.45–47 This means that allografts of neonatal retina,
but also other foreign antigens, do not elicit a classic immune
response. If there is a later challenge to the immune system
such as a skin graft, rejection of allogeneic fetal transplants
can occur by delayed hypersensitivity,48 and examples of
allograft rejection have been described.49–53
Ma and Streilein54 transplanted retinal cell aggregates
(disrupted tissue) of neonatal retina to the anterior chamber and
into the subretinal space of allogeneic or syngeneic recipient
mice. At 35 days post-transplantation, allogeneic subretinal
transplants contained an increased number of microglial
cells expressing major histocompatibility complex class I and
class II antigens which induce sensitization of the recipients
and serve as targets of immune rejection.54,55 This was
confirmed in another study56 that hypothesized that some
modification of the immune system prevented the allografts
from being rejected.
Fetal microglia may have a weak capacity to present antigens
which may contribute to the prolonged survival of the allograft.
Gregerson et al. demonstrated that CD45+ microglia have a
weak antigen presenting activity and might not be strong
enough to activate T-cells.57,58
In contrast to previous studies in this cat model,25,26 the
transplants in two cats (#1 and 2) observed in the current study
appeared to be well-‘integrated’ with the recipient host retina.
Crossing of bipolar processes was sometimes observed (see
Fig. 7). The presence of bridging fibers has been shown to
depend on the absence of an outer nuclear layer.59 Apparently
the large size of the transplants contributed to a rapid
degeneration of the host outer nuclear layer over the transplant which may have enhanced the transplant ‘integration’.
Although clinical and morphological studies suggested good
neuroretinal integration, functional improvement could not
be demonstrated by ERG in the two cats with successful
transplants. One reason for this is probably the short followup time for the transplanted cats in relation to the progression
of the disease process.24 Follow-up time need to be at least
12–18 months to evaluate marked electrophysiologic differences
(Narfstrom, unpublished observation, 2008). In the present
study the cats were still young and in a comparably early
stage of disease. The progression of disease is slow and the
remaining host photoreceptors function for a long period of
time in both eyes. Furthermore, the trauma of surgery
causes an up-regulation of positive growth regulating factors
in the transplanted eye masking the differences between the
operated eye and the control eye even further.60,61
In summary, this study shows that it is possible to perform
retinal transplantation in cat with a slowly progressive humanlike retinal degenerative disease. Well-laminated transplants
can develop under good surgical conditions. Further studies are
needed to evaluate host–transplant integration and functional
effects using longer follow-up times, preferably transplanted in
cat host retinas with a more advanced retinal degeneration.
A C K N O W LE D G M E N T S
The authors thank Lynette Caro, Leilani Castaner, and Ginny
Dodam at the University of Missouri, Columbia, and Zhenhai
Chen at the Doheny Eye Institute, Los Angeles, for technical
and surgical assistance. Supported by: Foundation Fighting
Blindness; Private Funds, Foundation for Retinal Research,
Fletcher Jones Foundation, NIH EY03040, Lincy Foundation
National Cancer Institute, and German Research Council grant
DFG See837/4-1. M. J. Seiler and R. B. Aramant have a proprietary interest in the implantation instrument and procedure.
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