Reduced Photoreceptor Damage after Photodynamic Therapy through Blockade of Nitric Oxide Synthase in a Model of Choroidal Neovascularization

Abstract

purpose. To investigate the role of nitric oxide synthase (NOS) in photoreceptor degeneration associated with photodynamic therapy (PDT) in a laser-induced model of choroidal neovascularization (CNV).

methods. PDT was performed in monkey and Brown Norway rats with laser-induced CNV. L-NAME, a NOS inhibitor, or saline was injected intraperitoneally in rats with CNV. An NO donor, or saline, was injected intravitreously into normal rats. Photoreceptor apoptosis was evaluated by TUNEL and electron microscopy. NOS, ED-1, and cleaved-caspase-3 (c-casp-3) expression were determined by immunohistochemistry. CNV lesions were examined by fluorescence angiography and choroidal flat mount.

results. TUNEL and electron microscopy showed photoreceptor apoptosis after PDT. In rats, there were significantly more TUNEL-positive cells in the photoreceptors 24 hours after PDT, whereas in the CNV lesions there were more TUNEL-positive cells 6 hours after PDT. C-casp-3 was detected in the CNV lesions but not in the photoreceptors after PDT. There was no difference in the numbers of ED-1–positive macrophages before and after PDT. However, inducible NOS (iNOS) was increased after PDT in macrophages. Intravitreous injection of the NO donor without PDT also induced substantial photoreceptor apoptosis. L-NAME–treated animals had significantly fewer TUNEL-positive cells in the photoreceptors than saline-treated animals after PDT (P < 0.05). There were no differences in CNV size and leakage between L-NAME– and saline-treated groups.

conclusions. iNOS expression in macrophages contributes to PDT-induced photoreceptor degeneration. NOS inhibition reduces PDT-induced photoreceptor degeneration without compromising the treatment effect of PDT in an experimental model of CNV.

Photodynamic therapy (PDT) using the photosensitizing dye verteporfin has been demonstrated in large clinical trials to be an effective and safe treatment for subfoveal choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) ^{1 2 3} and pathologic myopia. ⁴ However, even with PDT, treated patients still generally lose vision over time. ⁵ PDT preserves retinal tissue compared with traditional laser photocoagulation ⁶ and transpupillary thermotherapy, ⁷ but preclinical studies of animal models have shown that limited damage is still inflicted on the retina in rat ^{8 9} and monkey. ^{10 11 12 13 14} Given that most patients need repeated treatments, there is reason for concern about cumulative retinal damage. ¹² In addition, the mechanism of PDT-induced damage is likely complex. Singlet oxygen, which has a short lifespan, is thought to cause direct cell damage in a localized fashion after PDT. However, PDT may also cause indirect damage to the tissue. For example, inflammatory cells, such as neutrophils and monocytes or macrophages, are activated by PDT, and they may be responsible for releasing other factors that cause further damage to the tissue. ¹⁵ Therefore, it is important to understand the cellular events after PDT and the relations between this and photoreceptor degeneration. 

Macrophages play important roles in inflammation and angiogenesis, such as occurs during experimental and clinical CNV. ^{16 17 18} In animal models, bone marrow–derived macrophages infiltrate the retina after laser induction of CNV, ¹⁷ and the depletion of macrophages decreases CNV size. ¹⁶ CNV size reduction after macrophage depletion is also associated with decreased VEGF protein levels in the choroid. ¹⁶ However, it is unknown what role macrophages play in the retina and CNV after PDT. 

Nitric oxide (NO) is generated by the oxidation of arginine, a reaction catalyzed by the enzyme nitric oxide synthase (NOS). ¹⁹ It is an important signaling molecule that mediates a variety of essential physiological processes, including neurotransmission, vasodilation, and host cell defense. ²⁰ The main signaling pathway of NO is activation of soluble guanylyl cyclase, formation of cGMP, and concomitant protein phosphorylation. ^{20 21} NOS has three isoforms, endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). eNOS and nNOS are constitutively expressed proteins. Although small amounts of NO mediate physiological functions, excess NO can mediate tissue injury. NO is known to influence cell viability in a variety of models. Its effect may be proapoptotic or antiapoptotic, depending on the biological environment—the presence or absence of stimulatory or inhibitory cosignals. ²¹ In the retina, most evidence is that NO is toxic to photoreceptors. ^{22 23 24}  

In this study, we used animal models to demonstrate photoreceptor apoptosis after PDT. Furthermore, we investigated the role of NOS in PDT-induced photoreceptor degeneration. 

Methods

Animals

All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines established by the Animal Care Committee of the Massachusetts Eye and Ear Infirmary. Cynomolgus monkeys (supplied by Covance Research Products, Alice, TX) were anesthetized as described previously. ^{10 11 12 13} Brown Norway (BN; supplied by Charles River Laboratories, Inc., Wilmington, MA) rats 3 to 5 month of age were anesthetized with 0.2 mL to 0.3 mL of a 50:50 mixture of 100 mg/mL ketamine and 20 mg/mL xylazine. Pupils were dilated with 5.0% phenylephrine and 0.8% tropicamide. 

Induction of CNV and PDT for Monkey

All monkey sections were obtained from specimens in previous studies in our laboratory. The experimental protocol that followed included CNV using high-intensity argon laser, as previously described, ^{10 11 12 13} with fluorescein angiography to detect CNV formation. Two weeks after laser induction of CNV, PDT was performed as previously described. ^{10 11 12 13} Briefly, verteporfin for injection (Visudyne; Novartis, Basel, Switzerland) was infused as an intravenous bolus (6 mg/m²) over 15 seconds, followed by a flush with 3 mL saline. Five to 100 minutes after the end of intravenous infusion of verteporfin, PDT was performed using 689-nm light at fluences of 50 J/cm² delivered over 2 minutes 47 seconds (for paraffin sections and TUNEL staining) or 150 J/cm² delivered over 4 minutes 9 seconds (for electron microscopy) using a diode laser (Coherent Medical Laser, Palo Alto, CA) and a slit lamp adaptor. The spot size was 1250 μm on the cornea surface. Eight CNV lesions from seven monkeys that underwent PDT irradiation 15 to 20 minutes after verteporfin infusion were included in this study. Twenty-four hours after PDT, eyes were enucleated and fixed in 10% formalin or modified Karnovsky fixative (pH 7.4) at 4°C overnight for TUNEL (two lesions from one monkey) or electron microscopy (six lesions from six monkeys). 

Induction of CNV and PDT for Rats

CNV was induced with a 532-nm laser (Oculight GLx; Iridex, Mountain View, CA) in rats, as previously described. ⁸ Six laser spots (150 mW, 100 μm, 100 ms) were placed in each eye of the rat using a slit lamp delivery system and a coverglass as a contact lens. Production of a bubble at the time of laser confirmed the rupture of the Bruch membrane. If significant hemorrhages occurred and obscured the view of the retina, the eye was excluded. 

PDT was performed as previously described for rats. ⁸ Two weeks after laser injury, 6 mg/m² verteporfin was injected into the tail vein as a bolus. Laser light of 689 nm was administered using a diode laser delivered through a slit lamp adaptor 12 to 20 minutes after verteporfin injection. The laser spot size was set at 800 μm and was confirmed with a micrometer. The amount of irradiance delivered was 600 mW/cm² for 42 seconds. The dose of verteporfin was decided in a preliminary study in which 3 mg/m², 6 mg/m², and 12 mg/m² verteporfin was injected; 6 mg/m² verteporfin caused moderate photoreceptor apoptosis (data not shown). 

Treatment Groups of Rats

PDT was performed 2 weeks after laser induction of CNV in rats. Each group consisted of five rats killed at 6 and 24 hours after PDT. Five rats with only laser-induced CNV were used as control. Other animals were treated with N-nitro-L-arginine methyl ester (L-NAME; 60 mg/kg) or vehicle control (saline) intraperitoneally 1 hour before PDT and once a day after PDT for 6 days. Five rats from each group were killed at 6 hours, 24 hours, and 7 days after PDT. 

Evaluation of CNV Size and Leakage in Rats

Five rats treated with L-NAME before PDT and five control rats treated with saline and PDT were used for evaluation of CNV size and leakage. After 0.2 mL of 2% fluorescein sodium was injected intraperitoneally, fluorescein angiography was performed 1 day before and 1 and 6 days after PDT with the use of a digital fundus camera (TCR50IA; Topcon, Paramus, NJ). A choroidal neovascular membrane was defined as closed after treatment if there was no leakage from the treated membrane compared with baseline. ^{8 25} Angiograms were graded by two masked readers. 

Seven days after PDT, CNV lesion size was measured with choroidal flat mount in accordance with previously reported methods that had been slightly modified. ²⁶ Briefly, rats were anesthetized and perfused through the left ventricle with 20 mL PBS, followed by 20 mL of 5 mg/mL fluorescein-labeled dextran (FITC-dextran; MWt, 2 × 10⁶; Sigma, St. Louis, MO) in 1% gelatin. Eyes were enucleated and fixed in 4% paraformaldehyde for 3 hours. The anterior segment and retina were removed from the eyecup. Four to 6 relaxing radial incisions were made, and the remaining RPE-choroid-sclera complex was flat mounted (Vectashield Mounting Medium; Vector Laboratories, Burlingame, CA) and coverslipped. Pictures of the choroidal flat mounts were taken under a microscope (Leica Microsystems, Wetzlar, Germany). Software (Openlab; Improvision, Boston, MA) was used by two masked investigators to measure the magnitude of the hyperfluorescent areas corresponding to the CNV lesions. The average size of one CNV lesion in each animal was determined. 

Intravitreous Injection of NO Donor in Normal Rat Eyes

After rats were anesthetized and pupils were dilated, sodium nitroprusside (SNP), an NO donor, was dissolved in PBS at a concentration of 1 mM. SNP or vehicle control was injected in the right eye of each rat (n = 4). Five microliters of SNP or vehicle was injected intravitreously in normal eyes using a 32-gauge needle attached to a Hamilton syringe through the sclera 1 mm posterior to the limbus. ^{23 27} Rats were killed 24 hours after intravitreous injection. Eyes were enucleated, and sections were prepared for TUNEL staining. 

Immunohistochemistry

Immunohistochemistry was performed as previously reported. ^{27 28 29} Eyes were fixed in 4% paraformaldehyde at 4°C overnight. The anterior segment and the lens were removed, and the remaining eyecup was cryoprotected with 20% sucrose in 0.1 M PBS (pH, 7.4; 0.15 M NaCl). Eyecups were then embedded in optimum cutting temperature (OCT) compound. Sections were cut at 10 μm with a cryostat (Leica Microsystems). Only sections that were cut in the middle of the CNV lesions were included. Five sections from each CNV lesion were selected to stain for the antibodies listed in Table 1 . Sections were incubated with blocking buffer (PBS containing 10% goat serum, 0.5% gelatin, 3% BSA, and 0.2% Tween 20) for 1 hour and were incubated with primary antibodies as listed in Table 1 . As a control, the slides were incubated without any primary antibody. Sections were then incubated with fluorescence-conjugated secondary antibody: goat anti–mouse IgG Alexa Fluor 488 (Molecular Probes, Carlsbad, CA) or goat anti–rabbit IgG cy5 (Zymed Laboratories, San Francisco, CA). Sections were mounted with mounting medium with DAPI (Vectashield Mounting Medium; Vector Laboratories). Pictures were taken under a microscope (Leica Microsystems). 

TUNEL

TUNEL staining was performed according to the manufacturer’s protocol (ApopTag Fluorescein In Situ Apoptosis Detection Kit; Chemicon, Temecula, CA), as previously described. ^{27 29} Briefly, cryosections of rat eyecups and deparaffinized sections of monkey eyecups were washed with PBS twice and incubated with TdT enzyme at 37°C for 1 hour. Sections were washed 3 times in PBS for 1 minute and incubated with anti–digoxigenin conjugate (FITC) in a humidified chamber for 30 minutes at room temperature, followed by three rinses with 0.1 M PBS. Sections were mounted with mounting medium (Vectashield Mounting Medium; Vector Laboratories) with DAPI. 

Six and 24 hours after PDT, TUNEL-positive cells in the ONL overlying CNV lesions of rat eyes without PDT were counted. Only sections that were cut through the middle of the CNV lesions were included. Three sections from each CNV lesion were stained for TUNEL. An area of 340 μm × 240 μm with the CNV lesion in the middle was selected. TUNEL-positive cells in the ONL were counted by two masked investigators and normalized by the area of the ONL. The average number of TUNEL-positive cells in three sections from each lesion was calculated, and the average of all the lesions in one animal was calculated for statistical analysis. The area of ONL was measured (OpenLab; Improvision). 

For SNP-injected normal eyes, six sections near or across the optic nerve, vertical to the cornea plane, 200 μm apart from each other, were stained by TUNEL. Retina pictures approximately 3 disc diameters from the optic nerve were taken. 

Electron Microscopy

Rat and monkey eyes were enucleated 24 hours after PDT and were processed for electron microscopy, as previously described. ^{13 30 31} The eyes were fixed in modified Karnovsky fixative (pH, 7.4) for 20 minutes, and the anterior segment was removed. Eyecups were fixed overnight at 4°C and transferred to 0.1 M cacodylate buffer (pH, 7). Then eyecups were postfixed in 2% veronal acetate buffer osmium tetroxide, dehydrated in ethanol and water, and embedded in Epon. Ultrathin sections were cut from blocks, mounted on copper grids, stained with uranyl acetate in methanol and Sato lead stain, and examined with a transmission electron microscope (CM 10; Phillips, Eindhoven, The Netherlands). 

Statistical Analysis

Paired Student’s t-test was used to compare between different groups. Statistical significance was set at P < 0.05. 

Results

Photoreceptor Degeneration after PDT in the Monkey

We have previously reported that photoreceptor apoptosis occurs after PDT in rat (Young TA, et al. IOVS 2003;44:ARVO E-Abstract 3932; Matsubara A, et al. IOVS 2006;47: ARVO E-Abstract 907). In this study, TUNEL-positive cells were detected in the ONL of monkey retina overlying CNV 24 hours after PDT (Fig. 1 , arrows). Retina overlying the CNV lesion but not exposed to light irradiation showed no TUNEL-positive cells. Electron microscopic examination showed apoptotic photoreceptors (Fig. 1 , asterisks), with cellular shrinkage and chromatin condensation 24 hours after PDT. This suggests that PDT causes some photoreceptor degeneration in the retina overlying the CNV. 

Photoreceptor Degeneration after PDT in the Rat

To further investigate the mechanism of PDT-induced photoreceptor degeneration, PDT was performed in the rat CNV model. TUNEL and electron microscopy were used to detect apoptosis after PDT (Fig. 2) . Before PDT, TUNEL-positive cells were not seen in the CNV or the photoreceptors. At 6 and 24 hours after PDT, TUNEL-positive cells were detected in the photoreceptor and in CNV lesions. However, the time course of TUNEL-positive cells differed between the photoreceptors and the cells in CNV lesions. The number of TUNEL-positive cells in CNV was greater 6 hours after PDT than 24 hours after it; in the photoreceptors, the number was greater at 24 hours. Most of the TUNEL-positive photoreceptors were distributed close to the original laser injury. Electron microscopy confirmed the presence of apoptotic photoreceptors (Fig. 2 , white asterisks), with findings similar to those seen in the monkey model, with cellular shrinkage, chromatin condensation, and apoptotic body formation (Fig. 2 , arrowheads). There were also some migrating cells (Fig. 2 , black asterisks) containing pigment at the marginal area of CNV. 

Cleaved Caspase-3 (c-casp-3) Detected in CNV But Not in the Photoreceptors after PDT

Because caspases play an important role in apoptosis, we examined the expression of the activated form of caspase 3 (c-casp-3) in rat CNV after PDT using immunohistochemistry (Fig. 3) . Before PDT, there was no expression of c-casp-3 in the CNV lesion or the retina. Six and 24 hours after PDT, c-casp-3–positive cells were detected in the CNV but not in the retina, suggesting that the mechanism for apoptosis in the CNV and the photoreceptor may be different. 

Lack of Macrophage Recruitment after PDT

Macrophages have been detected in human ¹⁸ and experimental CNV. ^{16 17} We examined the number of macrophages in the CNV before and after PDT using immunohistochemical staining of ED-1, a macrophage marker, and found that most of the macrophages localized in the laser-injured retina, which was close to the TUNEL-positive photoreceptors. There was no change in the number of macrophages in the laser-injured retina 6 or 24 hours after PDT (Fig. 4) , suggesting that no additional macrophages were recruited to the CNV within 24 hours of PDT. 

Increased iNOS in the CNV after PDT, and Macrophages as Cellular Sources for iNOS

It has been reported that NO may cause photoreceptor apoptosis ^{22 23} and that macrophages can express NOS. ³² iNOS was increased in the CNV after PDT and was colocalized with ED-1–positive cells by double staining of ED-1 and iNOS (Fig. 5) . eNOS and nNOS were not increased after PDT, according to immunohistochemistry findings (Fig. 5) . 

Induction of Photoreceptor Apoptosis in Rats after Intravenous Injection of NO Donor

SNP, an NO donor, or PBS was injected intravitreously in rats without PDT. In the control eyes injected with PBS, no TUNEL-positive cells were detected in the retina. Twenty-four hours after intravitreous injection of SNP, numerous TUNEL-positive cells were noted in the ONL (Fig. 6) . No TUNEL-positive cells were detected in other layers of the retina or in the choroid, suggesting that NO is more toxic to photoreceptors than other cells in the retina and choroids, such as endothelial cells. 

Reduced Photoreceptor Apoptosis after PDT By L-NAME

To study whether increased iNOS had any biological effect, L-NAME (60 mg/kg), a NOS inhibitor, was injected intraperitoneally 1 hour before PDT. L-NAME reduced the number of TUNEL-positive cells in the photoreceptor layer after PDT (Fig. 7) . Numbers of positive cells in the photoreceptor layer at 6 and 24 hours after PDT were 371 ± 74/mm² and 1002 ± 401/mm², respectively. For L-NAME–treated groups, the numbers decreased to 83 ± 37/mm² and 59 ± 9/mm². At both time points, the differences were significant (P < 0.05). 

Treatment Effect of PDT Uncompromised by NOS Inhibition

To investigate whether NOS inhibition affected the original PDT treatment effect for CNV, we injected animals with L-NAME, performed masked reading of graded fluorescein angiography, and made choroidal flat mounts perfused with high molecular–weight FITC–dextran to demonstrate the leakage and extent of CNV after PDT. With the use of fluorescein angiography, CNV was closed 1 day after PDT in saline- and L-NAME–treated groups (Fig. 8) . In the early phase, hypofluorescence was observed in the treated CNV area. In the late phase, hyperfluorescence was predominant at the periphery of the treatment zone. In choroidal flat mounts made 7 days after PDT (Fig. 9) , the average area of FITC–dextran perfused new vessels in PDT–treated lesions were much smaller than CNV lesions without PDT, but there was no difference between L-NAME– and saline-treated groups after PDT. These data suggest that the treatment effect of PDT for CNV is not compromised by NOS inhibition. 

Discussion

The goal of our study was to better understand the mechanisms of retinal damage caused by PDT and to explore how to suppress PDT-induced photoreceptor degeneration. We demonstrated by electron microscopy and TUNEL that PDT caused photoreceptor apoptosis. The time course of TUNEL-positive cell in the ONL was different from that in the CNV lesion, and caspase activation was not detected in the photoreceptors by immunohistochemistry. ED1-positive macrophages were already detected before PDT, and the number of ED1-positive cells did not change after PDT. However, PDT caused the upregulation of iNOS in macrophages, and SNP, a NOS donor, induced photoreceptor apoptosis. Furthermore, L-NAME, a NOS inhibitor, reduced PDT-induced photoreceptor apoptosis without compromising the effectiveness of PDT for closing the CNV. Hence, we showed for the first time that PDT causes photoreceptor apoptosis through NOS activation in macrophages and that NOS inhibition significantly suppresses PDT-induced photoreceptor degeneration. 

Our group and others ^{8 9 10 11 12 13 14} have reported that despite its significant therapeutic effects, PDT causes photoreceptor degeneration. Previous reports were based primarily on morphologic evidence. In this study, we have demonstrated PDT-induced photoreceptor degeneration by two different approaches, electron microscopy and TUNEL. The activation of caspase-3 was detected in CNV lesions but not in the photoreceptors after PDT, and intravitreous injection in a NO donor also does not activate caspase-3 in the ONL (data not shown). Photoreceptor apoptosis occurs in various retinal diseases, including retinitis pigmentosa, ³³ retinal detachment, ³⁴ and macular degeneration. ³⁵ Generally, the activation of caspases, a family of cysteine proteases, is a central event in apoptosis. ³⁶ However, photoreceptor apoptosis may also occur in a caspase-independent manner under different conditions. ^{24 30 37} For example, light-induced photoreceptor apoptosis in mouse is caspase independent. ²⁴ In these mice, nNOS expression is increased after light injury, whereas inhibition of nNOS and guanylate cyclase, a downstream effector of NO, reduces photoreceptor apoptosis. In an in vitro study, oxidative stress also induces caspase-independent retinal cell apoptosis. ³⁷ All this suggests that photoreceptor apoptosis after PDT may be caspase independent. After PDT, photoreceptor apoptosis and cell apoptosis in CNV lesions appear to follow different pathways, with photoreceptors using a caspase-independent pathway and cells in the CNV lesion using a caspase-dependent pathway. This important distinction may provide the opportunity to selectively block the apoptosis of photoreceptors and to protect them after PDT. 

Macrophages play important roles in inflammation and angiogenesis. They recognize and respond directly to injury-related stimuli by releasing potent proinflammatory and proangiogenic cytokines. Macrophages express NOS after stimulation by inflammatory factors such as IFN-γ and thereby produce NO. ³² They are also one of the cellular components of human ¹⁸ and experimental CNV. ^{16 17} One of the reported mechanisms for PDT-induced tumor cell death is the activation of inflammatory cells. ¹⁵ Marked increases of tumor NOS and NO level were detected after PDT, ³⁸ and NOS inhibition reduces PDT-mediated tumor destruction. ³⁹ We noticed that after PDT, most of the TUNEL-positive photoreceptors were adjacent to the original laser injury lesion (Fig. 2) , which is also where macrophages tend to accumulate. Some of the major upstream factors of iNOS expression in macrophages are inflammatory agents. ³² Recent clinical studies have shown that PDT combined with the anti-inflammation therapy intravitreal triamcinolone is more effective than PDT monotherapy for CNV secondary to AMD. ⁴⁰ Taken together, these data suggest that PDT-induced inflammation in the CNV lesion might activate macrophages to produce iNOS, which results in excessive NO release and subsequently in photoreceptor apoptosis. A major mechanism for cell injury produced by NO in vivo is due to its diffusion-limited reaction with superoxide (O₂ ^–) to form peroxynitrite (ONOO^–). ⁴¹ Peroxynitrite is a potent, harmful oxidant that can diffuse through the cell membrane and react with intracellular molecules. ²⁰ Activated macrophages can also produce superoxide. In this study, we demonstrated that iNOS was increased in macrophages and that NOS inhibition reduced photoreceptor apoptosis. Because oxygen is necessary for photodynamic reaction, anti-inflammatory treatment may be an alternative strategy for the suppression of PDT-induced photoreceptor degeneration. 

NO has been shown to be proangiogenic in the retina and choroid, and the blockade of NOS leads to a significant reduction of CNV. ^{42 43} In our study, a single injection of NOS inhibitor did not change the CNV closure rate demonstrated by angiography 1 day after PDT or CNV size measured on flat mount 1 week after PDT. There are two possible reasons for this. First, the effects of NOS inhibition on CNV growth after laser injury and on endothelial cell survival after PDT might not have been similar. Second, the amount of NOS inhibitor was different from that used in previous studies. ^{42 43} Other investigators used either daily administration of NOS inhibitor for 2 weeks or NOS knockout mice. Our results suggest that neuroprotective agents could be used as adjunct treatment of PDT to protect photoreceptors without undermining the desired therapeutic effect of CNV closure. This strategy may lead to better treatment outcomes after PDT for CNV. 

In conclusion, in an experimental model of CNV, NOS inhibition reduced PDT-induced photoreceptor degeneration without compromising the beneficial treatment effect of PDT. Adjunctive NOS inhibition may have neuroprotective benefits in reducing photoreceptor injury after PDT. 

 Supported by the Neovascular Research Fund, National Eye Institute Core Grant P30 EY014104, National Institutes of Health Grant AI050775, Alcon Award, Massachusetts Lions Foundation, and Research to Prevent Blindness.

 Submitted for publication August 18, 2006; revised October 27, 2006; accepted March 19, 2007.

 Disclosure: H. She, None; T. Nakazawa, None; A. Matsubara, None; T. Hisatomi, None; T.A. Young, None; N. Michaud, None; E. Connolly, None; A. Hafezi-Moghadam, None; E.S. Gragoudas, None; J.W. Miller, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Primary Antibodies Used in This Study

Antigen	Host	Working Dilution	Manufacturer
c-casp-3	Rabbit	1:50	Cell Signaling (Danvers, MA)
ED-1	Mouse	1:100	Serotec (Oxford, UK)
eNOS	Rabbit	1:100	Affinity BioReagents (Golden, CO)
iNOS	Rabbit	1:50	BD Biosciences (Franklin Lakes, NJ)
nNOS	Rabbit	1:50	Chemicon (Temecula, CA)

Figure 1.

 

Photoreceptor apoptosis after PDT in monkey retina. CNV was first induced by laser. Twenty-four hours after PDT, TUNEL-positive cells could be detected in the ONL (arrows), whereas in the retina not exposed to PDT irradiation in the same eye, no TUNEL-positive cells were detected. Electron microscopy (EM) image shows apoptotic photoreceptors (asterisks) with cellular shrinkage and chromatin condensation after PDT. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer.

Figure 2.

 

Apoptosis of photoreceptors after PDT shown by electron microscopy (EM) and TUNEL in rats. Before PDT, there were no TUNEL-positive cells in the CNV lesions or the photoreceptors. At 6 and 24 hours after PDT, TUNEL-positive cells were detected in photoreceptors (arrows) and CNV lesions. The bar chart shows the quantification of TUNEL-positive cells in the photoreceptor layer after PDT. n = 5 animals per group. *P < 0.05; paired t-test. Values represent mean ± SEM. Twenty-four hours after PDT, apoptotic photoreceptors (white asterisks) are observed in the ONL with cellular shrinkage, chromatin condensation, and apoptotic body formation (arrowheads). Some migrating cells (black asterisks) with pigment at the marginal area of CNV are also observed. INL, inner nuclear layer; ONL, outer nuclear layer; CNV, choroidal neovascularization.

Figure 3.

 

Immunohistology of c-casp-3. Before PDT, there was no c-casp-3 signal in laser-induced CNV lesions or in the retina. At 6 hours and 24 hours after PDT, cells stained positive for c-casp-3 in the CNV (arrows), but no signal was detected in the retina. Scale bar, 50 μm. INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 4.

 

Immunohistochemistry staining of ED-1, a macrophage marker, shows no difference in macrophage (arrow) distribution in the retina over CNV lesions before and after PDT. There was no difference between the groups. n = 5 animals per group. Values represent mean ± SEM. Scale bar, 50 μm. INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 5.

 

Immunohistochemistry of iNOS, eNOS, and nNOS and double staining of iNOS and ED1. iNOS signal (arrows) was increased in the CNV lesion after PDT, especially in the laser-injured retina overlying the CNV lesions, where macrophages were distributed. iNOS was colocalized with ED-1–positive macrophages. eNOS and nNOS staining did not undergo obvious changes after PDT. Scale bars, 50 μm. INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 6.

 

TUNEL staining of normal rat eye injected intravitreously with SNP or PBS. In the control eyes injected with PBS, no TUNEL-positive cell was detected in the retina. Twenty-four hours after intravitreous injection of SNP, an NO donor, numerous TUNEL-positive cells were observed in the ONL, but there were no signals in the INL or GCL. Scale bar, 50 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 7.

 

TUNEL shows that the NOS inhibitor, L-NAME, can reduce TUNEL-positive cells in the photoreceptor layer after PDT. One hour before PDT, a NOS inhibitor, L-NAME (60 mg/kg) or saline was injected intraperitoneally in BN rats. At 6 hours and 24 hours after PDT, significantly fewer TUNEL-positive cells (arrows) were observed in the photoreceptor layer in L-NAME–treated groups compared with control. n = 5 animals per group. *P < 0.05; paired t-test. Values represent mean ± SEM. Scale bar, 50 μm. INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 8.

 

Fluorescein angiography shows no difference between saline-treated (5 rats, 10 eyes) and L-NAME–treated (5 rats, 9 eyes) groups after PDT, with CNV lesions angiographically closed 1 day after PDT in both groups. In the early phase, hypofluorescence developed in the CNV area (arrows). In the late phase, hyperfluorescence developed predominantly at the periphery of the treatment zone.

Figure 9.

 

Choroidal flat mount shows no difference between saline- and L-NAME–treated groups 1 week after PDT. Smaller areas of FITC–dextran-perfused new vessels can be seen (arrows) in PDT-treated CNV lesions, in contrast to CNV lesions without any treatment. Values represent mean ± SEM. Scale bar, 100 μm.