Download Cellular response in subretinal neovascularization induced

524
Reports
12. Tsujimura A, Shida K, Kitamura M, et al. Molecular cloning of a
murine homologue of membrane cofactor protein (CD46): preferential expression in testicular germ cells. Biochem J. 1998;330:
163-168.
13. Bora NS, Kabeer NH, Kim MC, Paryjas S, Atkinson JP, Kaplan HJ.
Expression of the complement regulatory proteins in the normal
and diseased (EAT) rat eye. In: Nussenblatt RB, Whitcup SM, Caspi
RR Gery I, eds. Advances in Ocular Immunology: Proceedings of
the 6th International Symposium on the Immunology and Immunopathology of the Eye, Bethesda, Maryland. New York:
Elsevier; 1994:83-86.
14. Ballow M, Donshik PC, Rapacz P, Maenza R, Yamase H, Muncy L.
Immune responses in monkeys to lenses from patients with con-
IOVS, February 1999, Vol. 40, No. 2
tact lens-induced giant papillary conjunctivitis. CLAOJ. 1989; 15:
64-70.
15. Cheung N-K, Walter El, Smith-Mensah WH, Ratnoff WD, Tykocinski ML, Medof ME. Decay-accelerating factor (DAF) protects
human tumor cells from complement-mediated cytotoxicity in
vitro./C/w Invest. 1988;81:1122-1128.
16. Goslings WRO, Blom DJR, DeWaard-Siebinga I, et al. Membranebound regulators of complement activation in uveal melanomas.
Invest Ophthalmol Vis Sci. 1996;37:1884-1891.
17. Bardenstein DS, LaCross G, Medof E. Surface complement regulatory proteins on choroidal melanomas [ARVO Abstracts]. Invest
Ophthalmol Vis Sci. 1996;37(3):S246. Abstract nr 1123-
Cellular Response in Subretinal
Neovascularization Induced by
bFGF-Impregnated Microspheres
prominent at 8 weeks. CD31-positive endothelial cells
were first evident at 14 days and formed neovascular
channels that were still present for up to 8 weeks. CD4and CD8-positive lymphocytes appeared in the early
stages but were few in number.
Hideya Kimura,1'5 Christine Spee,1
Taiji Sakamoto,1 David R. Hinton,2
Yuichiro Ogura,5 Yasuhiko Tabata,4
Yoshito Ikada,4 and Stephen]. Ryan1
CONCLUSIONS. SRN membranes are primarily composed of
RPE cells and vascular endothelial cells. The membrane
adheres to the retina by a gliotic band. The cellular components involved in the membrane of this model resemble
those found in SRN membranes removed from patients
with age-related macular degeneration. (Invest Ophthalmol Vis Sci. 1999;40:524-528)
determine the sequence of cellular changes
associated with a new rabbit model of subretinal neovascularization (SRN) induced by subretinal injection of basic
fibroblast growth factor (bFGF)-impregnated microspheres.
PURPOSE. TO
METHODS. bFGF-impregnated gelatin microspheres, prepared by forming a polyion complex between gelatin and
bFGF, were subretinally implanted into rabbit eyes. The
eyes were studied by immunochemistry at 3 days to 8
weeks after implantation. Antibodies to CD4, CD8, cytokeratin, CD31, glial fibrillary acidic protein (GFAP), and
RAM 11 were used.
Cytokeratin-positive retinal pigment epithelial
(RPE) cells appeared on day 3 and continued to increase in
number in the subretinal space throughout the growth of
the SRN membrane, becoming the predominant cell type.
Macrophages (RAMH-Positive) appeared early, but most
disappeared within 7 days. GFAP-positive Miiller cells
were evident early in the retina but migrated into the
subretinal space after 7 days; the gliotic adhesion they
formed between the retina and the SRN membrane was
RESULTS.
From the 'Doheny Eye Institute and the departments of Ophthalmology and Pathology, University of Southern California School of
Medicine, Los Angeles, California; and the 3 Department of Ophthalmology and Visual Sciences and ''Research Center for Biomedical
Engineering, Kyoto University, Kyoto, Japan.
Supported in part by Grants EYO1545 and EY03040 from the
National Institutes of Health, Bethesda, Maryland; and by a grant from
the Hoover Foundation, Pasadena, California. The Department of Ophthalmology is the recipient of an award from Research to Prevent
Blindness, New York, New York.
Submitted for publication February 13, 1998; revised June 25,
1998; accepted July 31, 1998.
Proprietary interest category: N.
Reprint requests: Stephen J. Ryan, Doheny Eye Institute, 1450 San
Pablo Street, Los Angeles, CA 90033-
Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017
ubretinal neovascularization (SRN) is often associated with
severe visual impairment, especially in age-related macular
Sdegeneration
(ARMD). The pathogenesis of SRN is not fully
understood; however, the new vessels of SRN are of choroidal
origin. Recent studies of surgically excised choroidal neovascular membranes (CNVMs) have provided some pathologic
information on the mechanism of CNVM formation.1"3 However, data obtained from these studies have often been limited
to late stages in the evolution of the CNVMs. Early stages of
CNVMs can only be studied in an animal model.
We have developed a new model of SRN in the rabbit
based on implanting basic fibroblast growth factor (bFGF)impregnated gelatin microspheres under the retina.4 Approximately 80% of eyes that receive bFGF-impregnated microspheres show fluorescein leakage from the CNVM 2 weeks
after implantation. These CNVMs are largely involuted within 8
weeks after microsphere implantation.
In this study we examined the time course of cellular
response into the subretinal space after initiation of the bFGF
SRN model using cell-specific antibodies. The results provide
insight into pathogenic mechanisms involved in this lesion and
demonstrate relevance to human CNVMs.
METHODS
Preparation of bFGF-impregnated Microsphere
Suspension
bFGF-impregnated microspheres were prepared by forming a
polyion complex as previously described.4 Briefly, 2.5 mg of
cross-linked gelatin microspheres were placed in 100 jixl of
distilled water containing 25 /xg of bFGF at 37°C for 1 hour,
after which 400 jal of phosphate-buffered solution (PBS; pH
IOVS, February 1999, Vol. 40, No. 2
TABLE
Reports
525
1. Time Course of Cellular Response in the Subretinal Space
Marker
Cytokeratin
CD31
RAM11
GFAP
CD4
CD8
3 Days
1 Week
2 Weeks
4 Weeks
8 Weeks
44-
Quantified by counting the number of positive cells in an average of three X40 objective fields: ± , < 1 ; +, 1-10; + + , >10.
Subretinal Implantation of bFGF-Impregnated
Microspheres
One eye each of 18 pigmented rabbits of both sexes, weighing 2.5 to 3.5 kg, was used. All procedures were in accordance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research. The animals were anesthe-
tized with a mixture (4:1) of ketamine hydrochloride (16
mg/kg) and xylazine hydrochloride (4 mg/kg), and the pupils were dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride eyedrops. The ocular surface was then
additionally anesthetized with topical instillation of 0.5%
proparacaine hydrochloride.
bFGF-impregnated microspheres were implanted under
the retina with a glass micropipette as previously described.Jf
Each eye received 50 fxl of bFGF-impregnated microsphere
suspension (2.5 jLtg of bFGF). The eyes were examined by
indirect ophthalmoscopy, photographed, and studied by fluorescein angiography weekly for up to 8 weeks.
FIGURE 1. Light micrograph of the lesion 3 days after implantation.
Many RAMll-positive cells (arrows) infiltrate under the retina-surrounding microspheres. Original magnification, X500.
FIGURE 2. Light micrograph of the lesion 2 weeks after implantation.
Note CD31-positive cells (arrows) arising from the choriocapillaris are
seen within the membrane. Original magnification, X500.
7.4) was added to prepare the 0.5% (wt/vol) microsphere
suspension for in vivo experiments. Gelatin (isoelectric point
[p/| of 4,9, molecular weight of 99,000, Nitta Gelatin, Osaka,
Japan) and human recombinant bFGF (R&D Systems, Minneapolis, MN) were used.
Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017
526
Reports
IOVS, February 1999, Vol. 40, No. 2
Burlingame, CA), and aminoethyl-carbazole (Vector) was used
to give a red color reaction. Sections were counterstained with
Mayer's modified hematoxylin.
The primary monoclonal antibodies used were cytokeratin
(mouse anti-human; Dako, Carpinteria, CA), CD4 and CD8
(mouse anti-rabbit; Spring Valley, Woodbine, MD), CD31
(mouse anti-human; Dako), glial fibrillary acidic protein (GFAP;
Sigma), and RAM11 (mouse anti-rabbit macrophage; Dako).
RESULTS
s
* * *
All experimental animals developed SRN and showed a similar
sequence of histologic and immunohistochemical findings,
which are summarized in Table 1. The eyes showed no fluorescein leakage at 3 days or 1 week. Fluorescein leakage was
visible from 2 weeks to 8 weeks after implantation; after 8
weeks the eyes showed no more leakage.
Three days after implantation, the microspheres were observed clearly under the retina. Many retinal pigment epithelial
(RPE) cells (cytokeratin-positive) and macrophages (RAM 11positive) infiltrated the sub retinal space surrounding the microspheres (Fig. 1). Adjacent Miiller cells were GFAP-positive
but did not infiltrate the subretinal space. Few CD4-positive
FIGURE 3- Light micrograph of the lesion 2 weeks after implantation.
The membrane is mainly composed of cytokeratin-positive cells. Original magnification, X500.
Immunohistochemical Examinations
Three eyes each were examined by imniunohistochemistry at
3 days and at 1 week, and four eyes each were examined by
immunohistochemistry at 2, 4, and 8 weeks after implantation.
Briefly, each eye was enucleated, and its cornea, lens, and
vitreous were removed. The eye was bisected, and the site of
the lesion was delimited by sharp cuts. One half of the lesion
was fixed in half-strength Karnovsky's solution (2.5% glutaraldehyde and 2% formaldehyde) in 0.1 M cacodylate buffer (pH
7.4). The tissues were dehydrated in a series of graded alcohols
and embedded in glycol methacrylate; 2- to 3-^m sections were
stained with periodic acid-Schiff or hematoxylin-eosin. For
immunohistochemical examinations, the other half of the tissue was embedded in optimal cutting temperature (O.C.T.)
compound (Tissue-Tek; Miles, Elkhart, IN) and frozen in liquid
nitrogen. Cryosections (8 (xtxi) were cut and stored at — 70°C.
Thawed sections were air-dried, fixed with acetone for 5 minutes, and washed with PBS (pH 7.4). The sections were
blocked for 15 minutes with 1% bovine serum albumin (Sigma,
St. Louis, MO) in PBS after blocking of endogenous peroxidase
by 0.3% hydrogen peroxide. The specimens were incubated
for 30 minutes with the primary antibody and then washed for
15 minutes with PBS. Sections were stained using the avidinbiotin complex (Vectastain ABC Elite Kit; Vector Laboratories,
Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017
FIGURE 4. Light micrograph of the lesion 4 weeks alter implantation.
GFAP-positive Miiller cells are seen traversing the retina. A GFAPpositive plaque is seen between the retina and the neovascular membrane. Original magnification, X500.
IOVS, February 1999, Vol. 40, No. 2
mononuclear cells and no CD8-positive cells were seen. No
CD31-positive endothelial cells were observed in the subretinal
space at this time.
One week after implantation, the microspheres were
mainly surrounded by cytokeratin-positive RPE cells and
RAMll-positive macropliages. GFAP-positive Miiller cells extended occasional processes into the subretinal space. A few
CD4- or CD8-positive cells were observed. CD31-positive endothelial cells were not seen in the subretinal space.
Two weeks after implantation, the microspheres were still
observed but were decreased in number. CD31-positive endothelial cells were now seen in the subretinal space and formed
neovascular channels (Fig. 2). The multilayered RPE (cytokeratin-positive) cells (Fig. 3) and endothelial cells formed a membrane-like structure. RAMll-positive macrophages were seen
but were decreased in number compared with 1 week.
Four weeks after implantation, many RPE (cytokeratinpositive) cells and some endotheiial (CD31-positive) cells were
observed in subretinal membranes. Miiller (GFAP-positive)
cells were adherent to the retinal surface of the membrane and
only rarely extended into the subretinal membrane (Fig. 4). A
few RAM11-positive macrophages were seen, but CD4- or CD8positive cells were sparse. A few microspheres were still
present in some areas.
Eight weeks after implantation, CD31-positive endothelial
cells were still seen in a membrane that was largely covered
with cytokeratin-positive RPE cells. In some areas, gliosis was
observed on the membranes.
DISCUSSION
In this study, we examined the time course of cellular infiltration into the subretinal space caused by implantation of bFGFimpregnated gelatin microspheres. The time course can be
divided into two phases: an early phase (foreign body reaction)
and a late phase (neovascularization). During the early phase,
within 1 week of implantation, microspheres were surrounded
by many RPE cells and macrophages and did not appear to be
degraded. No neovascularization was observed. It is likely that
the microspheres did not release a large amount of bFGF
during this phase because bFGF is physically immobilized in
the matrix of the cross-linked gelatin and is released as a
biologically active form only after enzymatic degradation of the
cross-linked gelatin.4 During the late phase, starting 2 weeks
after implantation, the number of microspheres decreased, and
endothelial cells migrated from the choroid forming with RPE
cells a neovascular membrane. It seems likely that the microspheres were enzymatically degraded by RPE cells or macrophages and released bFGF, resulting in proliferation of RPE
cells and choroidal endothelial cells. At 8 weeks after implantation, the neovascular membranes were covered with RPE
cells and appeared to be adherent to the retina by a gliotic
band. The microspheres were largely degraded.
Injection of microspheres alone was also associated with a
mild cellular reaction composed of macrophages and RPE cells;
however, this was not associated with neovascularization.4
The microspheres used in this study were relatively large, with
an average diameter of 40 /xm, and may have caused a foreign
body reaction. Subretinal injection itself may also result in
damage to outer segments of the photoreceptors or partial
dispersion of RPE cells that could incite RPE proliferation or a
gliotic response.
Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017
Reports
527
During the late phase, neovascularization occurs, and the
new vessels show an association with many RPE cells. Gelatin
itself enhances phagocytosis of microspheres by macrophages
through its opsonizing ability. Some microspheres may be
ingested and degraded by intracellular lysosomal enzymes, and
others may be degraded by gelatinases in the extracellular
space. RPE and endothelial cells both have been shown to be
capable of gelatinase (matrix metalloproteinase-2 and -9) secretion in vitro.5
The neovascularization may be a result of several soluble
factors. A direct or indirect effect of bFGF itself on choroidal
vessels is most likely. bFGF is a prominent promoter of endothelial cell migration and proliferation and stimulates the angiogenic process. Release of bFGF from the microspheres requires microsphere degradation that may occur by
phagocytosis by macrophages6 or RPE7 or by proteolytic digestion. A second source of angiogenic factors is from soluble
products of infiltrating macrophages6 and RPE. In particular,
migrating RPE cells have been shown to be a source of the
strongly angiogenic vascular endothelial growth factor (VEGF)
in human CNVM.3 The presence of VEGF or other secondary
angiogenic factors derived from either RPE or macrophages
will be an important focus of future study. bFGF may also
promote the development of the SRN lesion by stimulating
growth of the nonendothelial cells. bFGF is mitogenic for both
RPE8 and glial cells9 and thus may have an important role in
promoting the relatively high cellularity found in the lesions.
Therefore, although it is clear that the presence of bFGF in the
microspheres was the determinant directly associated with the
strong subretinal angiogenic response, it is possible that the
cellular or physical reactions induced by the microspheres
themselves may contribute to the stimulus.
Choroidal neovascular membranes in this model show
immunohistochemical characteristics similar to those of
CNVMs surgically excised from patients with ARMD.1"3 In
these membranes, the stromal cells are predominantly composed of RPE and vascular endothelial cells; however, small
numbers of macrophages are also seen. Most CNVMs in ARMD
include prominent extracellular matrices (ECMs) rich in collagen, laminin, and fibronectin.' CNVMs in this model appear to
be composed primarily of cellular components with less prominent ECMs. The strong proliferative response of RPE cells to
wound healing in the rabbit may explain why RPE cells are
especially prominent in the membranes in this model.10 Identification of ECM components in this model may require the
increased sensitivity of immunohistochemical methods. It is
possible that the CNVMs of ARMD contain more ECM than in
this model because the angiogenic process in ARMD is more
protracted.
GFAP-positive Miiller cells migrated into the subretinal
space after 7 days and formed a gliotic adhesion between the
retina and the SRN membrane that was most prominent at 8
weeks. The photoreceptors in the area in which microspheres
accumulated were mostly degenerated and were replaced with
proliferated Miiller cells. It seems likely that the mechanical
damage of microsphere uijection or the interference of the
normal metabolism of RPE cells by the accumulated microspheres induced the degeneration of photoreceptors. Miiller
cells tend to proliferate into the subretinal space and form
multiple layers after the degeneration of photoreceptors."
bFGF from the microspheres may also stimulate the proliferation of Miiller cells.9
528
IOVS, February 1999, Vol. 40, No. 2
Reports
We have previously demonstrated in this model a clinically relevant characteristic: CNVMs leak fluorescein and can
be detected by fluorescein angiography.4 We now show that
the immunohistochemical characteristics of cellular components involved in CNVMs of this model bear a striking resemblance to those in ARMD. Further studies of the angiogenic
responses in this model may provide some clues leading to
new therapeutic approaches to SRN.
Acknowledgments
The authors thank Thomas E. Ogclen, MD, PhD, for his helpful comments, Susan Clarke for editorial assistance, and Fernando Gallardo for
technical assistance.
4.
5.
6.
7.
8.
References
1. Grossniklaus HE, Martinez JA, Brown VB, et al. Immunohistochemical and histochemical properties of surgically excised subretinal
neovascular membranes in age-related macular degeneration. AmJ
Ophthalmol. 1992; 114:464 - 472.
2. Seregard S, Algvere PV, Berglin L. Immunohistochemical characterization of surgically removed subfoveal fibrovascular
membranes. Graefes Arch Clin Exp Ophthalmol. 1994;232:
325-329.
3. Lopez PF, Sippy BD, Lambert HM, Thach AB, Hinton DR. Transdifferentiated retinal pigment epithelial cells are immunoreactive for
vascular endothelial growth factor in surgically excised age-related
A Possible Role for p^ 1 ** 4 in
Neuronal Cell Death after Retinal
Ischemia-Reperfusion Injury
Sachiko Kuroiwa, Naomichi Katai, and
Nagahisa Yoshimura
study whether cell type-specific death occurs
in retinal ischemia-reperfusion injury and the possible
roles of pl6INK>l in the determination of cell death.
PURPOSE. TO
Retinal ischemia-reperfusion injury was induced
in rats by a ligation method. After 1 hour of ischemia and
a time of reperfusion that varied, rat eyes were enucleated. Cell death in the retina was studied by the TdT-dUTP
terminal nick-end labeling method and propidium iodide
(PI) staining. Electron microscopic observation of the retina was also performed. Immunohistochemical studies
using antibodies against syntaxin and calbindin were performed to detect amacrine cells and horizontal cells, respectively, and immunohistochemical studies using an anMETHODS.
9.
10.
11.
macular degeneration-related choroidal neovascularization. Invest
Ophthalmol Vis Sci, 1996;37:855-868.
Kimura H, Sakamoto T, Hinton DR, et al. A new model of subretinal neovascularization in the rabbit. Invest Ophthalmol Vis Sci.
1995;36:2110-2119.
Hunt RC, Fox A, al Pakalnis V, et al. Cytokines cause cultured
retinal pigment epithelial cells to secrete metalloproteinases and to
contract collagen gels. Invest Ophthalmol Vis Sci. 1993:34:31793186.
Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C.
Macrophages and angiogenesis./Leukoc Biol. 1994;55:4l0-422.
Kimura H, Ogura Y, Moritera T, Honda Y, Tabata Y, Ikada Y. In
vitro phagocytosis of polylactide microspheres by retinal pigment
epithelial cells and intracellular drug release. Curr Eye Res. 1994;
13:353-360.
Smith Thomas L, Richardson P, Parsons MA, Rennie IG, Benson M,
MacNeil S. Additive effects of extracellular matrix proteins and
platelet derived mitogens on human retinal pigment epithelial cell
proliferation and contraction. Curr Eye Res. 1996;15:739-748.
Lewis GP, Erickson PA, Guerin CJ, Anderson DH, Fisher SK. Basic
fibroblast growth factor: a potential regulator of proliferation and
intermediate filament expression in the retina. / Neurosci. 1992;
12:3968-3978.
Lopez PF, Qiong Y, Kohen L, et al. Retinal pigment epithelial
wound healing in vivo. Arch Ophthalmol. 1995; 113:14371446.
Erickson PA, Fisher SK, Anderson DH, Stern WH, Borgula GA.
Retinal detachment in the cat: the outer nuclear and outer plexiform layers. Invest Ophthalmol Vis Sci. 1983;24:927-942.
tibody against pl6 INK4 were performed to study whether
this cell cycle-related protein was expressed in dying
cells.
Most of the calbindin-positive horizontal cells in
the outer aspect of the inner nuclear layer (INL) showed
morphologic features of necrosis. In contrast, syntaxinpositive amacrine cells in the inner aspect of the INL
showed features of apoptosis. Of 320 calbindin-positive
horizontal cells, only 11 (3.4%) showed positive PI staining. Those calbindin-positive, horizontal cells were
pl6 INK4 positive. In contrast, 746 of 910 (82.0%) syntaxinpositive amacrine cells showed condensed PI staining, and
none were pl6 INK4 positive.
RESULTS.
Expression of pl6 1NK4 may regulate the fate
of retinal neurons in ischemia-reperfusion injury, and cell
type-specific death thus occurs in the retina after such
injury. (Invest Ophthalmol Vis Sci. 1999;40:528-533)
CONCLUSIONS.
n experimental retinal ischemia-reperfusion injury, neuronal
Ilayercells
mainly in the ganglion cell layer and the inner nuclear
(INL) are damaged. Previous reports have shown the
1
From the Department of Ophthalmology, Shinshu University
School of Medicine, Matsumoto, Japan.
Supported in part by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Science, Sports and Culture of the Japanese
Government (10470567 to NY).
Submitted for publication April 23, 1998; revised July 10 and
September 11, 1998; accepted October 6, 1998.
Proprietary interest category: N.
Reprint requests: Nagahisa Yoshimura, Department of Ophthalmology, Shinshu University School of Medicine, n*-*—••-"•— 2 m Q/COI
Japan.
Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017
occurrence of at least two, and possibly three, morphologically
distinct types of cell death after such injury.' Using the same
rat model, we have shown that occurrence of DNA fragmentation by TdT-dUTP terminal nick-end labeling (TUNEL) and
also by gel electrophoresis of the genomic DNA.2 Therefore,
the type of death in most of the affected cells in retinal ischemia-reperfusion injury can be classified as apoptosis. We have
also shown that aberrant expression of cell cycle-related
genes, especially c-jun and cyclin Dl, plays a role in the
process of apoptosis.2