Acta Ophthalmologica 2013
Histology and immunochemistry
evaluation of autologous
translocation of retinal pigment
epithelium-choroid graft in
porcine eyes
Ivan Fernandez-Bueno,1,2 Enrique Rodriguez de la Rua,1
Denise Hileeto,1 Maria Luisa Parrado,1,2 Marta RegueiroPurriños,3 Anna Sala-Puigdollers,1 Girish K. Srivastava,1,2,4
Jose Manuel Gonzalo-Orden3 and Jose Carlos Pastor1,2
1
Universitary Institute of Applied Ophthalmobiology (IOBA), University of
Valladolid, Valladolid, Spain
2
Networking Research Center on Bioengineering, Biomaterials and Nanomedicine
(CIBER-BBN), Valladolid, Spain
3
Institute of Biomedicine (IBIOMED), University of León, León, Spain
4
Castilla and Léon Regenerative Medicine and Cell Therapy Network Centre, Spain
ABSTRACT.
Purpose: To evaluate structure and cellular functionality of retinal pigment epithelium
Introduction
(RPE)-choroid grafts after autologous translocation in porcine eyes.
Methods: Retinal pigment epithelium-choroid grafts were obtained from the nasal midperiphery donor site and translocated to the central area in 12 pigs (12 eyes). Grafts
were placed under the central retina through a retinotomy. Ophthalmoscopic and pathological evaluations were performed immediately (n = 1) and at 15 (n = 3) and 30
(n = 3) days after surgery. Untranslocated nasal RPE-choroid grafts were obtained at
time of surgery and used as controls. Specimens were evaluated by standard histology
and by immunochemical studies of RPE65, CRALBP and GFAP.
Results: Five animals were lost to follow-up owing to surgery or anaesthesia complications. Ophthalmoscopic examination revealed that the grafts remained in place at all
time-points studied. Fifteen and thirty days postsurgery, some areas of the transplanted
RPE maintained a monolayered structure. Retinal pigment epithelium cells were firmly
attached to Bruch’s membrane and predominantly preserved polarity and pigment distribution. However, RPE65, CRALBP and GFAP patterns of expression and distribution
were diminished and modified during follow-up. Ophthalmoscopic retinal detachment
and proliferative vitreoretinopathy (PVR), confirmed by microscopic evaluation, complicated all cases at 30 days of follow-up.
Conclusion: Autologous RPE-choroid grafts survived up to 30 days in porcine eyes.
Histological and immunochemical evaluation revealed preserved transplanted RPE cells
morphology accompanied by alterations in the immunoreactivity expression of functional proteins, and development of significant PVR. The data presented in this manuscript provide insights into the fate, viability and cellular functionality of the
transplanted RPE-choroid graft, serving as foundation for further knowledge and
improvement of this technique.
Age-related macular degeneration
(AMD) is a common retinal degenerative disease and an important cause of
blindness (Congdon et al. 2004), especially among the elderly in developed
countries. There are two basic clinical
forms of AMD: wet (or exudative)
and dry (or atrophic). Wet forms of
the disease have been treated in recent
years with different therapies, now
mainly by intravitreal injections of
anti-angiogenic factors (Gragoudas
et al. 2004; Rosenfeld et al. 2006).
Also, there is some evidence supporting the use of antioxidants and nutritional supplements in patients with
mild forms of AMD to reduce the
progression of the disease (AREDS
2001). However, there is currently no
cure for either the advanced stages of
dry AMD or for severe cases of wet
AMD associated with submacular
retinal pigment epithelium (RPE)
atrophy, with or without subretinal
haemorrhages or RPE tears.
Replacement of the diseased RPE
cells is considered an attractive possibility for cases of AMD not suitable to be
managed with the standard treatments
Key words: age-related macular degeneration – autologous RPE-choroid graft – experimental
animal models – proliferative vitreoretinopathy – retinal pigment epithelium
Acta Ophthalmol. 2013: 91: e125–e132
ª 2012 The Authors
Acta Ophthalmologica ª 2012 Acta Ophthalmologica Scandinavica Foundation
doi: 10.1111/aos.12001
e125
Acta Ophthalmologica 2013
(Binder et al. 2007). Different cell therapies are under investigation and some
have shown promising results (Limb
et al. 2006). In that sense, autologous
translocation of the choroid and RPE
has been used to replace the damaged
RPE in selected cases of advanced
AMD. Various modifications of this
surgical technique were developed during the last decade with different results
in AMD patients (Binder et al. 2002,
2007; Stanga et al. 2002; van Meurs &
Van Den Biesen 2003; MacLaren et al.
2005, 2007; Joussen et al. 2006, 2007;
Maaijwee et al. 2007a, 2008; Heussen
et al. 2008; Ma et al. 2009). However,
there are little data regarding the histopathological findings after this transplant, because of the obvious difficulties
in obtaining samples from the eyes of
living patients. Thus, in vitro and in vivo
histopathological findings will add
additional information to this field of
study.
In this work, we performed RPEchoroid autologous transplantation in
pig eyes closely following the technique described by van Meurs & Van
Den Biesen (2003). Our goal was to
expand the histological and immunochemical
information
previously
reported about the structure and functionality of the RPE cells after transplantation (Maaijwee et al. 2007b).
The second aim was to assess the feasibility of using autologous transplantation
in
pig
eyes
as
an
experimentation model for translational studies with possible human
applications.
Material and Methods
Animals
The use of animals in this study was
in accordance with the recommendations of the Association for Research
in Vision and Ophthalmology (ARVO)
and approved by the Ethics Committee of the University of León, Spain.
Surgery was performed on one eye of
12 young female domestic pigs
(8 ± 2 weeks). The other eye served
as the nonsurgical control.
Surgical procedure
Twenty-four hours before and after surgery, the food intake of the animals was
restricted while maintaining free access
to water. Animals were premedicated
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and anaesthetized as described by Perez
de Prado et al. (2009). Body temperature was maintained between 36.5 and
37.5C with the aid of an under-table
heating device. Analgesia was induced
with a bolus of buprenorphine
(0.02 mg ⁄ kg, intramuscularly, Buprex;
Schering-Plough,
Kenilworth,
NJ,
USA). During surgery, the muscle relaxant atracurium (4 lg ⁄ kg ⁄ min, intravenously, Tracrium; Glaxo Wellcome,
Burgos, Spain) was perfused to facilitate eye manipulation. Prophylactic
antibiotic use was with amoxicillin ⁄ clavulanic acid (20 mg ⁄ kg, intramuscularly; Ratiopharm, Madrid,
Spain) in a single preintervention
injection. The conjunctival sac was
washed with povidone-iodine solution
(5%, Betadine; Meda Manufacturing, Bordeaux, France), and pupils
were dilated by topical tropicamide
(0.5%,
Colircusi
Tropicamida;
Alcon, Barcelona, Spain).
Autologous translocation of the
choroid and RPE was carried out closely following a technique described
by van Meurs & Van Den Biesen
(2003). Briefly, a standard 20G pars
plana lensectomy and a pars plana
vitrectomy were performed using an
Accurus Vitrectomy System (Alcon,
Ft. Worth, TX, USA). After the
induction of a posterior hyaloid vitreous detachment, the central retina was
detached by the subretinal injection of
Ringer’s lactate solution through a
retinotomy performed with a 39G
angled rigid microinjection cannula
(Synergetics Inc.,; Paris, France).
Recipient RPE and Bruch’s membrane
were left intact. Afterwards, demarcation of a rectangular area of
2 · 2 mm, using an endolaser probe,
at the donor nasal midperiphery of
the same eye was performed. Using a
bimanual technique with vitreous forceps and scissors, a full-thickness graft
of neuroretina, RPE and choroid was
carefully separated from the sclera.
The graft was held from the choroidal
side and the neuroretina was removed.
The graft, then consisting of the RPE
and choroid, was translocated to the
recipient bed in the central area using
a special forceps for this manoeuver
(DORC, Zuidland, the Netherlands).
Care was taken to avoid any damage
or rolling of the graft as the choroid
side was placed against the native
RPE of the graft bed. To achieve this
objective, a bubble of perfluorooctane
(ARCOTANE
C8F18;
Prohosa,
Madrid, Spain) was injected. Then,
after fluid ⁄ air exchange, the eye was
filled up with 1000 Cst silicone oil
(AJL Ophthalmics, Vitoria, Spain).
Untranslocated RPE-choroid fullthickness grafts were obtained at time
of surgery from the nasal midperiphery
of the same eye and used as controls.
Postoperative analgesia was maintained with buprenorphine (0.02 mg ⁄
kg ⁄ 8 hr, intramuscularly; Buprex;
Schering-Plough). Antibiotic treatment,
amoxicillin ⁄ clavulanic
acid
(500 mg ⁄ 12 hr, orally), was administered for 5 days after surgery.
Follow-up
A veterinarian followed the animals
every day to rule out painful complications. Indirect ophthalmoscopy was
conducted by an ophthalmologist
before and immediately after the operation and again before enucleation.
As described below (Results section),
five of the animals were lost to followup. After a follow-up of 15 (n = 3) or
30 days (n = 3), the animals were
anaesthetized as described and killed
by intravenous injection of an overdose
of sodium pentobarbital (100 mg ⁄ kg,
Eutanax; Fatro Iberica, Barcelona,
Spain). Furthermore, to determine if
the complex surgical procedure
induced neuroretinal and ⁄ or RPE
damage, one animal was killed immediately after surgery and the eye processed (n = 1). Afterwards, the eyes
were enucleated and prepared as
described below.
Tissue processing
The ocular globes were sectioned into
two halves along the horizontal axis.
For light microscopy, ocular globes
were fixed for 24 hr in 10% formalin,
embedded in paraffin and processed
for sectioning (3 lm). Multiple parallel serial sections at different levels
were taken from both halves. Some
sections were stained with haematoxylin and eosin (H&E) and periodic
Schiff reagent (PAS). These were
examined microscopically until the
exact site of the transplanted RPE
was identified.
For immunohistochemical studies,
the paraffin-embedded sections were
pretreated for antigen retrieval with
citrate buffer (pH 6) for 15 min at
Acta Ophthalmologica 2013
100C. After rinsing in water for
5 min, specimens were incubated for
5 min in 3% hydrogen peroxide to
eliminate
endogenous
peroxidase
activity. After another 5-min water
rinse followed by three changes of
phosphate-buffered saline (PBS, pH
7.4; Invitrogen Ltd., Paisley, UK), the
sections were blocked with 0.5%
Triton X-100 in PBS, 10% normal
goat serum and 1% BSA (all SigmaAldrich, St. Louis, MO, USA) for
1 hr at room temperature.
To evaluate possible modifications
in structure and functionality of the
RPE, the sections were incubated
overnight at 4C with monoclonal
mouse anti-human retinal pigment
epithelium-specific 65 kDa protein
(RPE65, 1:50; Novus Biologicals
LTD, Cambridge, UK) in 0.5%
Triton X-100 in PBS. Other sections
were incubated with monoclonal
mouse anti-human [B2] cellular retinaldehyde-binding protein (CRALBP,
1:1000; Abcam plc., Cambridge, UK).
To study glial cells and the development of reactive gliosis, sections were
incubated with anti-CRALBP as
described above or polyclonal rabbit
anti-cow glial fibrillary acidic protein
(GFAP, undiluted; DakoCytomation
Inc., Carpinteria, CA, USA) for
30 min at room temperature.
After incubation with primary antibodies, the sections were washed three
times in PBS and exposed for 2 hr at
room temperature to a 1:200 dilution
of goat anti-rabbit Alexa Fluor488,
goat anti-mouse Alexa Fluor488 or
goat anti-mouse Alexa Fluor568
(IgG(H+L); Molecular Probes, Eugene,
CA, USA). Cellular nuclei were stained
with 4¢,6-diamino-2-phenylindole dihydrochloride (DAPI, 10 lg ⁄ ml; Invitrogen, Eugene, OR, USA) for 10 min at
(A)
room temperature. Slides were then
coverslipped with 1:1 PBS ⁄ glycerol.
Substitution of the primary antibody with PBS and secondary antibody omission was included as
negative controls. Microscopy was
performed with a Leica DM4000B
light microscope (Leica Microsystems
GmbH, Wetzlar, Germany) equipped
for epifluorescence. Images were captured with a Leica DFC490 camera
and processed with the appropriate
software (Leica Application Suite,
Version 2.8.1; Leica Microsystems
GmbH). TIFF images were enhanced
using adobe photoshop software (Version 10.0.1 for Macintosh; Adobe Systems Inc., San Jose, CA, USA).
Comparative studies based on the
expression of the immunohistochemical markers were carried out on
images acquired at the same levels of
exposure, intensity and gain.
Results
Surgery
The first four surgical attempts could
not be finished because of surgical or
anaesthesia
complications
that
included massive choroidal haemorrhage in the first two cases, malignant
hyperthermia in the third and a complete iatrogenic retinal detachment in
the fourth case. The following eight
cases were surgically successful; however, one animal (number 6) had to be
killed after 7 days because of acute
postsurgical endophthalmitis. The eye
was not included for the final pathological analysis. Thus, pathological
evaluation was made on the surgical
control taken immediately after surgery
(n = 1), and on eyes taken 15 (n = 3)
and 30 (n = 3) days after surgery.
(B)
Ophthalmoscopic
examination
revealed that the grafts remained in
place at both follow-up time-points. At
15 days, the neuroretina was attached
in all cases, and no clinical evidence of
PVR was observed. However, in the
three animals followed for 30 days, the
retina was detached in each. These eyes
also showed ophthalmoscopic findings
of severe proliferative vitreoretinopathy (PVR) with funnel shape and evident epiretinal bands.
Light microscopy
Untranslocated RPE-choroid samples
revealed adequately preserved RPE
and Bruch’s membrane morphology
after surgical procedure for the preparation of the grafts (Fig. 1A). There
was no histological evidence of damage at the neural retina or recipient
RPE in the eye obtained immediately
after surgery. The site from where the
RPE-choroid graft was excised
showed architectural disorganization
and no significant fibrosis of the
remaining choroid at both 15 and
30 days after surgery (Fig. 1B).
Except for one animal, all of the
RPE-choroid grafts were adequately
placed and oriented in the central bed
of the recipient. In one case (animal
number 10, at 15 days), the graft was
placed rolled over so that the transplanted RPE was against the native
RPE. The donor graft showed various
changes in different areas at 15 and
30 days after surgery. Some graft
regions demonstrated only small clusters of pigmented cells with abnormal
pigment distribution, loss of cellular
polarity and no distinguishable
Bruch’s membrane (Fig. 1C). However, in other regions (Fig. 2A–C), the
graft transplant site contained RPE
(C)
Fig. 1. Histology of untranslocated retinal pigment epithelium (RPE)-choroid sample (A); and donor site (B) and transplanted graft at 30 days
(C). RPE cells showed an adequate preservation of its morphology with intact Bruch’s membrane after surgical procedure for the preparation of
the graft (A, PAS). The remaining choroid at the site of the donor RPE-choroid graft was disorganized and presented no significant fibrosis
(B, H&E). Graft RPE (C, PAS) demonstrating some areas with disorganized clusters of pigmented cells with abnormal pigment distribution, loss
of polarity and absent Bruch’s membrane at 30 days. Scale bars: 20 lm.
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Acta Ophthalmologica 2013
(A)
(B)
(C)
Fig. 2. Histology of the transplanted grafts at 30 days. Transplanted graft showing double-layered retinal pigment epithelium (RPE; A–C). The
native choroid vasculature beneath the graft appeared viable, open and perfused (A and B). Graft RPE cells, firmly attached to Bruch’s membrane (B, arrows), maintained a monolayer that had a normal appearance just above the native RPE. Some RPE cells from the graft seemed to
be firmly attached to the native ones (B, asterisks). In these regions, none of the remaining donor choroid was apparent. Both graft and native
RPE layers were firmly attached to one another and demonstrated predominantly preserved polarity (C, oil immersion, arrows) and some pigment
distribution (C, oil immersion, asterisks). A-C: PAS staining. Scale bars: 20 lm.
cells that were firmly attached to the
Bruch’s membrane (Fig. 2B, arrows)
and to the native RPE (Fig. 2B,
asterisks), with no evidence of the
donor choroid (Fig. 2A). In these
areas, the transplanted RPE maintained a monolayered structure with
normal appearance, predominantly
preserved polarity (Fig. 2C, arrows)
and some pigment distribution
(Fig. 2C, asterisks), located just above
the native Bruch’s membrane and
RPE layer. Compared with untranslocated grafts, cellular content of pigment was decreased both in recipient
and transplanted RPE. In other areas
of the graft (data not shown), the
transplanted RPE layer was firmly
attached to the native RPE with
some residual choroid tissue still present in between. In all samples, the
native choroidal vasculature beneath
the graft contained erythrocytes and
other blood cells and thus appeared
viable,
open
and
perfused
(Fig. 2A,B). Bridging vessels between
the recipient and the graft were not
observed.
At 15 days after transplantation,
the neuroretinal area overlying the
graft site revealed intraretinal modifications, such as reactive gliosis, formation of subretinal membranes, and
neuronal loss and disorganization;
however, the neuroretina remained
attached at this time-point (Fig. 3A).
The retina surrounding the graft
showed preserved inner and outer retinal architecture. By 30 days after surgery, the neuroretina was detached
except in the peripheral regions of the
globe, and there were also vast areas
of advanced PVR (Fig. 3B) showing
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(A)
(B)
Fig. 3. Histology of the neuroretina at the graft site. Intraretinal modifications were apparent
at 15 days (A). These modifications included reactive gliosis with the formation of subretinal
membranes (A, arrows), neuronal loss and cellular disorganization. At 30 days (B), the central
neuroretina was totally detached and disorganized. Epiretinal membranes can be appreciated
(B, arrows; asterisk indicates the optic nerve). A & B: H&E staining. Scale bars: 20 lm (A) and
50 lm (B).
epiretinal membranes and intraretinal
changes.
Immunochemistry
Retinal pigment epithelium (RPE65 and
CRALBP)
In sections from untranslocated RPEchoroid grafts (Fig. 4A), RPE65 was
abundantly distributed in the apical
cytoplasm, extending almost to the
perinuclear cytoplasm of typical looking RPE cells. Comparable RPE65
immunoreactivity expression and distribution patterns were observed
15 days after surgery (data not
shown). However, 30 days after surgery (Fig. 4B), RPE65 in the native
midperipheral RPE had shifted to the
basal portion of the cells, with immunoreactivity expression levels similar to
that of the controls (Fig. 4A). In the
native RPE near the recipient site
(Fig. 4C), RPE65 demonstrated focal
weak
positivity
(Fig. 4C,
inset,
arrow),
which
corresponded
to
marked reduction in its expression.
However, at the graft site (Fig. 4D),
this protein was present in both the
donor and the recipient RPE monolayers. There was no clearly defined
pattern of distribution inside the cells.
Furthermore, the protein expression
level appeared to be higher than in
the native RPE surrounding the recipient site (Fig. 4C).
Untranslocated grafts (Fig. 5A)
revealed
CRALBP
distribution
throughout the cytoplasm of the RPE
cells. In the transplanted site 15 days
after surgery (Fig. 5B), CRALBP was
still present in the RPE cells of both
the donor and recipient layers, but the
level of this protein immunoreactivity
expression was considerably reduced.
However, by 30 days, CRALBP was
no longer detectable in donor or
recipient RPE (Fig. 5C).
Acta Ophthalmologica 2013
was still attached, Müller cell CRALBP
labeled extensions formed a continuous
layer located between the photoreceptor outer segments and the RPE. These
extensions appeared to be connected to
each other, forming subretinal membranes; these included some cellular
nuclei (Fig. 6A). At 30 days, CRALBP
expression was reduced in the external
Neural retina (CRALBP and GFAP)
The sample obtained immediately
after surgery revealed the presence of
CRALBP in the cytoplasm of Müller
cells, mainly at the neuroretinal external layers. GFAP was present at the
inner limiting membrane and nerve
fibre layer. However, at 15 days after
transplantation, when the neuroretina
(A)
(B)
neuroretinal layers, mainly in areas
revealing a disorganized neuroretina
(Fig. 6B). At both study times, GFAP
expression was increased. At 30 days,
the GFAP-positive intermediate filaments appeared to occupy the entire
cytoplasm of the Müller cells in these
gliotic areas (Fig. 6C).
(C)
(D)
Fig. 4. Distribution of RPE65 in untranslocated sample and transplanted graft areas at 30 days. In untranslocated grafts (A), RPE65 (red) was
abundantly distributed in the apical cytoplasm, extending almost to the perinuclear cytoplasm of typical looking retinal pigment epithelium
(RPE) cells. In the midperipheral RPE of operated eyes at 30 days (B), RPE65 was distributed more basally but at approximately the same immunoreactivity expression level as controls (A). In the native RPE near the recipient site at 30 days (C), the RPE65 immunoreactivity was reduced (inset,
arrow). In the graft site at 30 days (D), RPE65 was present in both RPE monolayers, donor (arrows) and recipient bed-site (asterisks), with no
clearly defined pattern of distribution inside cells. Protein expression level appeared to be higher than the nearest RPE unilayer (C). DAPI dye
(blue). Scale bars: 20 lm.
(A)
(B)
(C)
Fig. 5. Distribution of CRALBP in untranslocated sample and transplanted grafts at 15 and 30 days. CRALBP (green) in untranslocated grafts
(A) was present throughout the cytoplasm of the retinal pigment epithelium (RPE) cells. In the grafts 15 days after surgery (B), CRALBP expression was reduced, but this protein was still present in both donor and recipient bed-site RPE cells. Thirty days after surgery (C), CRALBP was
no longer detectable in donor or recipient RPEs. In this sample, green channel stimulation revealed autofluorescent blood cells inside choroidal
vessels (asterisk). DAPI dye (blue). Scale bars: 20 lm.
(A)
(B)
(C)
Fig. 6. Neuroretinal distribution of CRALBP and GFAP near transplanted grafts at 15 and ⁄ or 30 days post-transplantation. At 15 days after
surgery (A), Müller cell extensions expressing CRALBP (green) formed a continuous layer, located between photoreceptor outer segments and
CRALBP-positive retinal pigment epithelium (RPE; arrows). At 30 days (B), all of the retinas were detached, and there was reduced expression
of CRALBP in the external disorganized neuroretinal layers. At 30 days (C), GFAP expression (green) was increased within the cytoplasm of the
Müller cells. The GFAP-positive intermediate filaments reached the external layers of the neuroretina. DAPI (blue). Scale bars: 50 lm (A) and
20 lm (B&C).
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Acta Ophthalmologica 2013
Discussion
Autologous translocation of the choroid and RPE is one of the few
surgical techniques already employed
for selected cases of human AMD.
Some clinical series have demonstrated that this procedure is associated with a high risk of potential
complications, but clinical benefits in
a number of patients have also been
reported (Stanga et al. 2002; Binder
et al. 2007; Maaijwee et al. 2007a).
There is not much histopathology
information about the survival and
functionality of transplanted cells in
patients because of the difficulties of
accessing chorioretinal specimens in
humans. Furthermore, there are only
a few reports on the pathological findings in animals after RPE transplantation (Maaijwee et al. 2007b; Cong
et al. 2008; Hu et al. 2008). Maaijwee
et al. (2007b) focused on the revascularization of the RPE-choroid graft in
pigs, and also provided useful information regarding the morphology of
the transplanted cells. However, they
did not study the potential functionality of those cells. Cong et al. (2008)
described a novel rabbit model for
studying RPE transplantation, but the
obvious differences between rabbit
and human retina limit its usefulness
with regard to similar procedures in
humans. Hu et al. (2008) had reported
good survival and function of the
transplant in a partial-thickness RPEchoroid graft in rabbits. They suggest
that the transplantation of a partialthickness graft provides better results
than the full-thickness one. However,
these results need to be confirmed in
animals with holangiotic retinas. For
these reasons, we used a porcine
model to expand the existing knowledge regarding the survival, morphology and cellular function of the RPE.
A successfully established animal
model could then serve as a control
group to compare new RPE transplantation procedures and methodologies that might then be applied to
human eyes.
The porcine eye was selected
because the neuroretina and particularly the choroid are, in many ways,
similar to that of humans (De Schaepdrijver et al. 1989; Simoens et al.
1992; Garcia-Layana et al. 1997;
Hendrickson & Hicks 2002). Furthermore, the comparable length and size
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of the globe allowed the use of standard human vitrectomy instruments
for the surgical procedure. Because of
the above-mentioned characteristics,
pigs are more appropriate than other
experimental animals such as rabbits,
rodents, etc. However, modifications
of the technique developed by van
Meurs & Van Den Biesen (2003), as
obtaining the donor graft from the
nasal midperiphery instead of the 12
o’clock position, had to be performed
owing to eyes location and bone conformation of the porcine skull. Furthermore, as documented by others,
the present study shows that the surgical learning curve of the procedure is
prolonged and is associated with a
high rate of complications. The porcine intraocular vascular system has a
high tendency to bleed (Maaijwee
et al. 2007b), and the posterior vitreous attachment of the retina makes it
difficult to achieve complete removal
of the cortical vitreous. Probably for
these reasons, the major problem with
this model is the development of
PVR, as also observed by Maaijwee
et al. (2007b). Furthermore, RPE cell
migration during surgery could contribute to the development of this
postsurgical complication (Charteris
1995). As noted by Maaijwee et al.
(2007b), this model will not be useful
unless the PVR rate and the surgical
complications can be reduced. Finally,
another pitfall of this model is that in
young pigs, the RPE is healthy. For
the porcine model to be most useful,
strategies must be developed to create
damage in the RPE that is similar to
human AMD. To achieve this,
debridement of the RPE combined
with the injection of subretinal mitomycin C has been reported (Del Priore
et al. 1996). However, the mechanism
of RPE damage in that model is very
different from that of AMD, so new
alternatives need to be developed.
A remarkable finding of this study
is the maintenance of the transplanted
RPE layer during the 30 postsurgical
days of this experiment, but with
apparent alteration and possible
reduction in the functionality of the
RPE cells, based in immunohistochemical staining of RPE65 and
CRALBP. The polarity of the RPE
cells was well preserved, with some
pigment
distribution,
and
they
remained firmly attached to the
Bruch’s membrane. However, immu-
nohistochemical evaluation suggested
a progressive dysfunction of these
cells. The expression of RPE65, which
converts all-trans retinol to 11-cis
retinal, was diminished at 30 days and
redistributed from the apical cytoplasm to a more even distribution
throughout the cytoplasm of the RPE
cells. Additionally, the expression of
CRALBP, a retinoid-binding protein
important in vitamin A metabolism,
was not detectable at 30 days.
Although
histological
evaluation
showed that the RPE cells predominantly maintained polarity, the modifications at the molecular level may
indicate that they were beginning to
lose that polarity. This would lead to
improper processing and release of
retinoids, as discussed by Carlson &
Bok (1992, 1999). Consistent with
these findings, a clinical series showed
graft survival but with progressive
reduction of visual function (MacLaren et al. 2005). Other studies, however, suggested that the RPE
functionality could be restored (van
Meurs et al. 2004; Joussen et al. 2006,
2007). In this context, it is not clear if
the changes in the expression of
RPE65 and CRALBP represent a progressive decline in the function of the
RPE cells, or these changes represent
a temporary alteration in their function with a possible partial or complete regain of their original state at a
later point in time. Further studies
would be necessary to clarify this
issue.
Neovascularization of the transplanted graft observed by Maaijwee
et al. (2007b) could not be confirmed
in our studies because the used stainings were not suitable to accurately
and reliably asses the vasculature. In
this study, debridement of the RPE
has not been performed because revascularization of the graft in porcine
eyes is not improved with this
manoeuver (Maaijwee et al. 2007b).
Even more, PAS staining revealed that
the native Bruch’s membrane was still
present beneath the graft at 30 days.
However, Maaijwee et al. (2007b)
described the degeneration of the
intact native Bruch’s membrane
1 week and at 3 months after surgery.
The reason for the differences between
these two studies could be owing to
the size of the transplanted graft.
Maaijwee et al. (2007b) performed
translocation of 9 mm2 grafts whereas
Acta Ophthalmologica 2013
we transplanted 2 mm2 pieces to closely
reproduce the technique described in
humans (van Meurs & Van Den
Biesen 2003; Maaijwee et al. 2007a;
MacLaren et al. 2007). In that sense,
small graft size may allow maintenance of Bruch’s membrane. Nevertheless, it could not influence PVR
development in this model.
No appreciable histological modifications in retinal tissue were observed
immediately after surgery. However,
we cannot discount the presence of
early, undetected molecular changes
associated with retinal damage.
Intraretinal reactive gliosis and neuronal
cell disorganization were present
15 days after transplantation. These
intraretinal modifications were similar
to findings described retinectomy specimens in patients with intraretinal
PVR (Pastor et al. 2006; Charteris
et al. 2007). The neuroretinal distribution of CRALBP and GFAP confirmed the observations made on
H&E and PAS stainings. They
showed subretinal membranes growing from Müller cell extensions. These
Müller cell processes interfered with
the contacts between photoreceptor
outer segments and the RPE. Additionally, the extensions plus the
intraretinal modifications observed at
15 days could have contributed to the
posterior retinal detachment and PVR
development present at 30 days after
surgery. The reduction of CRALBP
expression in the neuroretinal external
layers at that time also revealed a
reduction of Müller cells functionality.
In summary, this study showed survival of autologous porcine RPEchoroid grafts, with preservation of
polarity and some remaining pigment
distribution, in both donor and recipient RPE layers, up to 30 days of
follow-up.
Nevertheless,
immuno
chemical evaluation revealed alterations in functional proteins of the
transplanted cells. Further improvement of the model is necessary to
control the development of PVR.
Over time, graft dysfunction and PVR
development could occur simultaneously and their interaction could
constitute an intricate mutually related
pathophysiological dependency. Thus,
it is difficult to determine if the graft
dysfunction causes the development of
PVR, or the trauma of the surgery
with subsequent PVR development
prevents the graft from functioning
properly. However, the data presented
in this manuscript provide additional
insights into the fate, viability and cellular functionality of the transplanted
RPE-choroid graft, serving as foundation for further knowledge and
improvement of this technique.
Acknowledgements
Work supported by grants from Cajas
de ahorros de Castilla y León and
Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y
León to JC Pastor and from National
Plan of I+D+I 2008–2011 and
ISCIII-Subdirección General de Evaluación y Fomento de la Investigación
(PS09 ⁄ 00938) (MICNN) cofinanced
by FEDER funds to GK Srivastava.
CIBER-BBN is an initiative funded
by the VI National R&D&I Plan
2008–2011, Iniciativa Ingenio 2010,
Consolider Program and CIBER
Actions and financed by the Instituto
de Salud Carlos III with assistance
from the European Regional Development Fund. I Fernandez-Bueno and
D Hileeto were supported by Junta de
Castilla y León and Ministerio de
Educación y Ciencia (PTA-2003-0100986), respectively. Presented in part
at the 40th Annual Scientific Meeting
of the American College of Veterinary
Ophthalmologists (ACVO) 2009; at
the European College of Veterinary
Ophthalmologists (ECVO) Conference
2010; and at the XIV Congreso de la
Sociedad Española de Retina y Vı́treo
(SERV) 2010.
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Received on December 2nd, 2011.
Accepted on August 19th, 2012.
Correspondence:
Ivan Fernandez-Bueno, BVSc, PhD
Universitary Institute of Applied Ophthalmobiology (IOBA)
University of Valladolid
Paseo de Belén 17
47011 Valladolid
Spain
Tel: + 34 983 184753
Fax: + 34 983 184762
Email: ifernandezb@ioba.med.uva.es