January 2006
Volume 47, Issue 1
Free
Retina  |   January 2006
Effect of Intravitreal Injection of Ranibizumab in Combination with Verteporfin PDT on Normal Primate Retina and Choroid
Author Affiliations
  • Ivana K. Kim
    From the Angiogenesis and Laser Laboratory, Retina Research Institute, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts;
  • Deeba Husain
    Department of Ophthalmology, Boston University School of Medicine, Boston, Massachusetts;
  • Norman Michaud
    From the Angiogenesis and Laser Laboratory, Retina Research Institute, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts;
  • Edward Connolly
    From the Angiogenesis and Laser Laboratory, Retina Research Institute, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts;
  • Anne Marie Lane
    From the Angiogenesis and Laser Laboratory, Retina Research Institute, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts;
  • Khayyam Durrani
    From the Angiogenesis and Laser Laboratory, Retina Research Institute, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts;
  • Ali Hafezi-Moghadam
    From the Angiogenesis and Laser Laboratory, Retina Research Institute, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts;
  • Evangelos S. Gragoudas
    From the Angiogenesis and Laser Laboratory, Retina Research Institute, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts;
  • Charles A. O’Neill
    BioMarin Pharmaceuticals, Novato, California; and the
  • Joseph C. Beyer
    Department of Safety Assessment, Genentech, Inc., South San Francisco, California.
  • Joan W. Miller
    From the Angiogenesis and Laser Laboratory, Retina Research Institute, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts;
Investigative Ophthalmology & Visual Science January 2006, Vol.47, 357-363. doi:10.1167/iovs.04-0087
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      Ivana K. Kim, Deeba Husain, Norman Michaud, Edward Connolly, Anne Marie Lane, Khayyam Durrani, Ali Hafezi-Moghadam, Evangelos S. Gragoudas, Charles A. O’Neill, Joseph C. Beyer, Joan W. Miller; Effect of Intravitreal Injection of Ranibizumab in Combination with Verteporfin PDT on Normal Primate Retina and Choroid. Invest. Ophthalmol. Vis. Sci. 2006;47(1):357-363. doi: 10.1167/iovs.04-0087.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To evaluate the effect of intravitreal injection of a monoclonal antibody fragment (ranibizumab, also known as rhuFab V2 and Lucentis; Genentech, S. San Francisco, CA) directed against vascular endothelial growth factor (VEGF) in combination with verteporfin photodynamic therapy (PDT) on normal primate retina and choroid.

methods. Eight cynomolgus monkeys were treated with intravitreal ranibizumab in one eye and placebo in the other, alternating with verteporfin PDT in both eyes on a weekly basis for 6 to 7 weeks. Treatment effects were evaluated by color fundus photography, fluorescein angiography, and light and electron microscopy.

results. Over the course of the study, increasing retinal pigment epithelial changes, with corresponding window defects, developed in both eyes of all animals on fluorescein angiography over the course of the study. No complications attributable to the intravitreal injection of ranibizumab were observed. Histologic analysis revealed a similar reduction in choriocapillaris density in the irradiated area of eyes treated with PDT alone compared with those that received combination treatment.

conclusions. In this limited study of normal monkey eyes, no severe adverse effects from the combination of intravitreal ranibizumab and verteporfin PDT were demonstrated compared with PDT alone.

Despite the use of photodynamic therapy (PDT), choroidal neovascularization (CNV) continues to cause severe vision loss in patients with AMD (AMD). Although PDT with verteporfin has been shown to reduce the risk of visual loss in patients with subfoveal CNV, the incidence of visual loss is still substantial. Results from the Verteporfin in Photodynamic Therapy trial showed that 29% of patients who received PDT for occult subfoveal CNV lost six or more lines of vision after 2 years, and 55% of these patients lost three or more lines of vision. 1 Additional limitations to PDT include the necessity of multiple treatments with the potential for cumulative damage to retinal structures and the existence of lesions not amenable to treatment. 
The discovery of the role of vascular endothelial growth factor (VEGF) in ocular angiogenesis has identified this molecule as an important therapeutic target for neovascular conditions such as exudative AMD. VEGF expression has been demonstrated in surgically excised choroidal neovascular membranes from patients with AMD 2 3 and in laser-induced animal models of CNV (Husain D, IOVS 1997;36:ARVO Abstract S501). 4 Furthermore, overexpression of VEGF in the RPE cells of rats and sustained delivery of VEGF in the subretinal space of primates induce CNV. 5 6 An anti-VEGF aptamer (pegaptanib sodium) has been approved for the treatment of neovascular AMD, and other agents for inhibiting VEGF are currently under investigation. In a primate model, the antigen-binding fragment of a recombinant humanized monoclonal antibody against VEGF (ranibizumab) has been found to prevent experimental CNV and to decrease leakage from formed CNV. 7 Clinical trials with this agent have shown promising early results (http://www.gene.com/gene/news/pressreleases/display.do?method=detail&id=8727). 
Combination therapy with an anti-VEGF agent such as ranibizumab in addition to PDT has the potential to provide improved visual outcomes for patients with neovascular AMD. In preliminary animal studies, this combination appears to cause greater reduction in angiographic leakage from experimental CNV than does PDT alone. 8 However, with improved efficacy, there is a potential risk of increased toxicity. 
Light and electron microscopy in both animal and human studies has demonstrated occlusion of normal choriocapillaris after PDT treatment. 9 10 Reperfusion of the choriocapillaris occurs during the 4 to 7 weeks after PDT, with reduplication of basement membrane suggestive of recanalization. 11 As VEGF may stimulate the migration and proliferation of endothelial cells involved in recanalization, inhibition of this growth factor could impair the regenerative process necessary for optimal visual function after PDT. 
In this study we investigated the effect of intravitreally administered ranibizumab in combination with verteporfin PDT on normal retina and choroid in the cynomolgus monkey. 
Materials and Methods
Animals
Eight cynomolgus monkeys (Macaca fascicularis), aged 2 to 4 years, obtained from Covance Biomedical Products, Inc. (Alice, TX), were used in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Permission allowing for the bilateral treatments was obtained from the Animal Care Committee at the Massachusetts Eye and Ear Infirmary. The monkeys were anesthetized for all procedures with intramuscular injections of ketamine hydrochloride (20 mg/kg), acepromazine maleate (0.25 mg/kg), and atropine sulfate (0.125mg/kg). Supplemental anesthesia of ketamine (5–6 mg/kg) was administered intramuscularly as needed. In addition, proparacaine hydrochloride 0.5% was used topically. Enucleation was performed after intravenous administration of ketamine (20–25 mg/kg). Animals were killed with a pentobarbital sodium veterinary euthanasia solution (J. A. Webster, Inc., Sterling, MA) administered intravenously. 
Ranibizumab
Recombinant humanized antigen-binding fragment of a monoclonal antibody against human endothelial growth factor (ranibizumab) was obtained from Genentech, Inc. (South San Francisco, CA). Ranibizumab was stored in lyophilized form at 2 °C to 8°C. The drug was reconstituted on the day of injection to a concentration of 25 mg/mL with vehicle supplied by the manufacturer. The reconstituted ranibizumab was then diluted to a concentration of either 10 or 40 mg/mL with sterile water. The final concentration of the drug that was to be injected in the eye was confirmed by spectral analysis. A volume of 50 μL of the drug solution was withdrawn with a needle with a 5-μm filter on a 1-mL tuberculin (TB) syringe. For placebo injections, 50 μL of vehicle was withdrawn in a 1-mL TB syringe. 
Intravitreal Injection
After the animal was anesthetized, a drop of proparacaine followed by a drop of povidone iodine 5% solution was placed in the conjunctival sac of each eye. A self-retaining eye speculum was placed in the eye. Intravitreal injections of 50 μL per eye were performed 2 mm behind the limbus in the temporal quadrant with a 30-gauge needle. Bacitracin ointment was placed in the eye after the injection. 
Photography
Fundus color photography was performed with a fundus camera (model 50VT; Topcon America Corp., Paramus, NJ) and 35-mm film. Photographs were taken of each eye and included photographs of the posterior pole and two midperipheral fields (temporal and nasal). Fluorescein angiography was performed on the Imagenet Digital Angiography System (Topcon America Corp.). Red-free photographs of both eyes were taken followed by fluorescein angiography using 0.1 mL/kg of 10% sodium fluorescein (Akorn, Inc., Abita Springs, LA) injected intravenously. After the fluorescein injection, a rapid series of photographs of the posterior pole were taken of the right eye followed by the posterior pole of the left before 1 minute, and then at approximately 1 to 2, and 5 minutes. Between 2 and 5 minutes, two midperipheral fields (temporal and nasal) were taken of each eye. Baseline photographs and fluorescein angiograms were performed before the experiments were started and then weekly before and after each PDT or injection. 
Photodynamic Therapy
Verteporfin for injection (Visudyne; Novartis, Basel, Switzerland) was purchased from the manufacturer or agent of the manufacturer (QLT Inc., Vancouver, British Columbia, Canada). The dye was handled, reconstituted, and stored based on the manufacturer’s guidelines. Reconstituted verteporfin was protected from light at all times and used within 4 hours of its reconstitution. The volume required to achieve a dose of 6 mg/m2 was withdrawn with a syringe from the vial and diluted with 5% dextrose in water (D5W) for a total injection volume of 10 mL. Verteporfin was administered intravenously using a syringe pump over 10 minutes followed by a flush of D5W. Fifteen minutes after the start of intravenous infusion of verteporfin, the retina was irradiated with 689-nm light at 600 mW/cm2 and 100 J/cm2 using a diode laser and laser link apparatus (Coherent, Inc.. Palo Alto, CA). The second eye was treated within 5 minutes of the first eye, so that treatments were completed within 20 minutes of the start of verteporfin infusion. 
Treatment Groups
Group I.
Four animals initially received 500 μg ranibizumab intravitreally in one eye and 50 μL vehicle in the other. For subsequent injections, 2000 μg ranibizumab was given in the combination treatment eye and vehicle in the control eye every 2 weeks, for four injections. One week after each injection, both eyes underwent verteporfin PDT for three treatments. The animals were followed for 49 days (2 weeks after the last PDT). 
Group II.
Four animals initially received verteporfin PDT in both eyes followed 1 week later by 500 μg of intravitreal ranibizumab in one eye and 50 μL vehicle in the other. Verteporfin PDT was repeated on both eyes every 2 weeks for three treatments. One week after the second and third PDT, 2000 μg ranibizumab (combination treatment eye) or 50 μL vehicle (control eye) was injected intravitreally for three injections. The animals were observed for 42 days (2 weeks after the last PDT). 
Safety Evaluation and Outcomes
Each animal was examined at the slit lamp and with indirect ophthalmoscopy, to identify and record inflammation and other toxic effects every week before any treatment was performed on the eye. Anterior chamber and vitreous cells were graded using a 2-mm slit lamp beam at high magnification, with grading based on the American Academy of Ophthalmology scheme. 12  
Ophthalmic Evaluation and Analysis
The fundus photographs and fluorescein angiograms were evaluated by two masked and experienced examiners (ESG, JWM). 
Histopathological Analysis
The globes were carefully removed from each animal and dissected clean of orbital tissue. The globes were then rinsed in saline and placed in modified Karnovsky’s fixative consisting of 2% glutaraldehyde and 2.5% formaldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C. Within 10 minutes, each globe was opened and the anterior segment removed. The posterior pole was placed in fixative overnight and then changed to buffer (0.1 M cacodylate) until processed for routine light and electron microscopy. 
Each eye was prepared for light and electron microscopy by sectioning into blocks that contained the areas of interest. Tissue was postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer for 2 hours at room temperature then dehydrated in a series of ethanols, infiltrated with propylene oxide and Epon, and embedded in Epon. Blocks were cut into 1-μm sections and stained with 0.5% toluidine blue in borate buffer. For electron microscopy, thin sections were stained with saturated uranyl acetate in methanol and Sato’s lead stain. 
Choriocapillaris Quantitation
Light microscopic images were captured with a five-color digital camera (Q-color; Olympus, Lake Success, NY) mounted on a microscope (Leica, Deerfield, IL) at 40× objective magnification. Image-analysis software (AnalySIS; Soft Imaging Systems) software was used for computer-controlled capture and subsequent analysis after calibration with a stage micrometer. Various measures including number of capillary lumens, luminal area, and luminal perimeter were evaluated. The parameter determined to be the most reliable in repeated counts of control sections was the total of all capillary lengths in the axis parallel to Bruch’s membrane expressed as a percentage of the length of Bruch’s. At least two areas along two different nerve-fovea planes were counted, for four to eight areas per eye. Statistical analysis was performed with repeated-measures analysis of variance (ANOVA). 
Results
Color Photographs
Serial fundus photography revealed whitening of the outer retinal layers in the area of the PDT spot, which was most prominent the first day after the initial PDT. This whitening faded over the following 1 to 2 weeks. Retinal whitening was not observed in the previously treated areas with subsequent PDT procedures. With successive treatments, increased pigment mottling was noted in the treated areas (Fig. 1) . No new retinal abnormalities were noted on photographs taken after intravitreal injections of ranibizumab or vehicle. 
In two animals from group II, a cotton wool spot with a small amount of retinal hemorrhage was noted within the PDT spot in the right eye after the first treatment (Fig. 2)and resolved within 2 weeks. Although this small area of retinal ischemia was noted only in the right eye of each animal, it was observed before the first ranibizumab injection and thus was considered an effect of PDT rather than ranibizumab. 
Fluorescein Angiography
The fluorescein angiographic findings in the animals that received combination ranibizumab and PDT treatments were consistent with those after PDT alone. Both eyes in all the animals demonstrated early hypofluorescence in the area of the laser spot, with late leakage in the same area on angiograms taken the day after PDT treatment. With successive PDT treatments, the amount of late leakage was less pronounced. In some eyes, a rim of more intense, late leakage appeared when a portion of the laser spot was applied over a previously untreated area. In addition, there appeared to be RPE changes in the central macula from multiple PDT applications, as evidenced by window defects on the angiograms from the later part of the follow-up period (Fig. 3) . Some animals exhibited a small central area of late staining in both the control and ranibizumab eyes. 
An effect on the retinal vasculature was observed in two animals from group II. A small area of capillary nonperfusion was noted in the right eye of both these animals on day 1 (1 day after first PDT). This area was within the laser spot and was associated with retinal vessel staining in one of the animals. Color photographs from that day revealed a cotton wool spot in the same area and a small amount of retinal hemorrhage in both animals. Although this finding occurred only in the right eye of each animal, it was observed before the first injection of ranibizumab and thus must have been related to PDT. The area of nonperfusion in both animals became less prominent in subsequent angiograms but did not completely resolve by the end of the study period (Fig. 2)
Slit Lamp Examination
All eyes treated with ranibizumab exhibited anterior chamber cells within 24 hours of injection. As shown in Tables 1this inflammatory reaction was most prominent after the first injection and resolved within 1 to 2 weeks. No significant vitreous inflammatory reaction was noted in any eye. 
Histologic Analysis
Histologic evaluation was performed on both eyes of two animals from group I and two animals in group II. Significant retinal pigment epithelial changes were observed in the irradiated area of all animals studied. Proliferation of the RPE with areas of multilayering was observed within the area of PDT. Macrophages were noted in the subretinal space. There was variable shortening of photoreceptor outer segments and some pyknosis in the outer nuclear layer. In two animals there were small focal areas of absent photoreceptor layer. The inner retina appeared intact in all cases. Gross distortion of the retina and blood in the subretinal space was noted in one eye of one animal. There was no evidence of subretinal hemorrhage in this eye on color photographs and fluorescein angiography performed just before enucleation. Thus, these histologic findings were probably induced at the time of enucleation and not a direct result of the treatment regimen. 
All examined eyes had a reduction in the density of choriocapillaris in the irradiated area compared with the untreated choroid. In addition, the vessels in the choriocapillaris appeared further removed from Bruch’s membrane than in untreated areas (Fig. 4) . Changes in choriocapillaris density were quantitated by calculating the total capillary luminal length expressed as a fraction of the length of Bruch’s membrane. Sections from the irradiated areas of seven eyes treated with PDT alone and seven eyes treated with PDT+ranibizumab were analyzed in addition to nonirradiated areas from three eyes treated with PDT alone and three eyes which received the combination treatment. There was a significant reduction in choriocapillaris density in the irradiated areas compared with the nonirradiated areas in both groups (Fig. 5) . There was a trend toward slightly reduced density in the irradiated area of combination treatment eyes versus that in eyes treated with PDT alone. However, this difference did not reach statistical significance. These findings indicate that PDT serves as the primary factor in reduction of choriocapillaris density and that the addition of ranibizumab does not appear to have significant detrimental effects. 
Electron Microscopy
Transmission electron microscopy demonstrated generally normal retinal architecture, but the choroid and RPE appeared partially modified in both PDT+ranibizumab and PDT-alone eyes. In both eyes, the choriocapillaris was perfused, but vessels were fewer in number, were further from Bruch’s membrane, and showed evidence of duplication of the basal lamina. The capillary fenestrations appeared normal; however, the nuclear regions extended into the lumen. Cell processes were present between the choriocapillaris and RPE, including some containing pigment, presumably processes of pigment cells of the choroid. Basal lamina duplication was occurred with all capillaries examined. The laminae were variable in density and were rarely complete around the cell. These layers appear different from the original capillary basal lamina, which composed the outer portion of Bruch’s membrane. Vesicles and dense deposits were noted within Bruch’s membrane. The RPE appeared to be a continuous epithelium, occasionally multilayered, but with minimal basal infoldings. Duplication of the RPE basal lamina, was also observed, with acellular pockets occasionally found between the laminae (Fig. 6) . Fewer vessels were seen in the area examined from the PDT+ranibizumab eye compared with the PDT alone eye. In addition, more cell processes were found between Bruch’s membrane and the new capillaries in the combination treatment eye. This may have been a sampling artifact concerning the location of the section within the treated area. 
Discussion
The results of this study suggest that the effects of combined treatment with verteporfin PDT and intravitreal ranibizumab on normal primate retina and choroid are similar to those seen with PDT alone. Findings on fundus photography and fluorescein angiography were similar between eyes that received the combination therapy and eyes that had PDT alone. The pattern of deep retinal whitening that appeared 24 hours after the initial PDT application and not after subsequent PDT procedures followed by late pigmentary changes in the treatment area is consistent with previous studies on repeated PDT in the primate. 13 Late fluorescein leakage in the irradiated area 24 hours after PDT, which decreased in intensity and duration with subsequent treatments has also been reported. No effects on the retinal vasculature associated with intravitreal ranibizumab injections were observed. However, two instances of mild retinal capillary nonperfusion after PDT were noted in this study. Retinal vascular damage after PDT with higher drug doses and a higher fluence of 150 J/cm2 has been reported in both animal and clinical studies. 9 14 The findings in this report suggest that mild retinal damage is also possible at a fluence of 100 J/cm2, further justifying the use of the lower value of 50 J/cm2 in clinical practice. 
The histopathologic findings in this investigation were compared to those from a previous analysis of the effects of repeated verteporfin PDT on normal retina and choroid of monkey eyes. 13 In the previous experiments, PDT was applied every 2 weeks for three treatments, and retinas were examined 3 and 6 weeks later. Laser light doses used in that study were the same as those used in this study, and verteporfin doses included 6 mg/m2, as was used in the present study. Retina and choroid from both sets of eyes were similar, with changes mostly localized to the outer retina and choriocapillaris. The photoreceptors showed some disarray of outer segments. There was the suggestion of some mild reduction in the thickness of the outer nuclear layer, although quantitative analysis was not performed to see whether there was a reduction in the population of outer layer nuclei. A reduced vessel density was noted in the choriocapillaris, and the RPE contained areas of multilayering, along with pigmented macrophages in the subretinal space. In comparison to this previous study of repeated verteporfin PDT alone, eyes treated with combination ranibizumab and PDT in our study revealed no significant differences by qualitative histologic analysis. 
Transmission electron microscopy on representative eyes from this series confirmed the previously noted finding of basement membrane reduplication in the choriocapillaris. 11 13 Studies on regeneration of rat and rabbit skeletal muscle fibers and capillaries after ischemic or cold injury have indicated that the basal lamina serves as a scaffold for proliferation and migration of endothelial cells during capillary reconstruction. 15 These experiments also revealed multilayering of basal lamina in cases of repeated injury, with the number of layers roughly corresponding to the number of insults. Observations of choriocapillaris regeneration in the rabbit after sodium iodate–induced atrophy have indicated that a process of recanalization occurs, involving sprouting from choroidal venules and remnant choriocapillaris and the production of new endothelial cells. 16 Newly formed endothelial tubes from all sources formed in association with remnants of preexisting basement membrane resulting in areas of redundant or duplicated basement membrane. Thus, the finding of basement membrane reduplication in PDT studies indicates that choriocapillaris recanalization is important in the response to PDT. 
It has been previously shown that at the dye and light doses required for CNV closure, choriocapillaris occlusion, RPE necrosis, and some pyknosis of the outer nuclear layer occurs. 9 The recovery phase involves RPE repopulation of the treated area as well as recanalization of the choriocapillaris. 11 Although VEGF may play a role in this process, we were unable to detect a dramatic adverse effect of VEGF inhibition on choriocapillaris recovery after PDT in this limited study. The results of our quantitative analysis confirm that PDT induces a measurable reduction in choriocapillaris density. However, the addition of ranibizumab was not shown to cause further loss of choriocapillaris vessels in this limited number of animals. Calculations were performed to determine the number of eyes necessary to detect with power of 0.80 and 95% confidence, a statistically significant difference in choriocapillaris density in eyes treated with ranibizumab+PDT versus PDT alone, of the magnitude suggested by our data. Given the small difference, at least 35 eyes per treatment group would be necessary, requiring 35 more primates to be killed. We feel that such a study would not be justified. 
The safety of combination therapy suggested by this preclinical study is likely to hold true in the clinical setting, because this experimental protocol was designed to increase the potential of discovering any adverse effects of PDT combined with ranibizumab therapy. The PDT applications and intravitreal injections in this study were repeated at 2-week intervals, which is more frequent than would be applied in a clinical setting. In addition, a fluence of 100 J/cm2 was used in this investigation, compared with the 50 J/cm2 currently used for treating patients. Both the high frequency of PDT and higher fluence would be expected to induce more cumulative damage to choriocapillaris and RPE than would occur with standard treatment parameters. In clinical application, combination treatment with PDT and intravitreal ranibizumab may reduce the number of PDT applications necessary to close CNV and/or increase the interval between treatments. Such an effect on current treatment strategies would theoretically allow for less cumulative damage to normal RPE and choriocapillaris and provide greater time for recovery between PDT applications. 
This study was designed to demonstrate any severe, clinically significant, adverse effect on normal choroidal vasculature that may arise from the combination of PDT and intravitreal ranibizumab. Our data do not demonstrate any such effects. Given the limited number of animal studied, subtle effects on choriocapillaris density cannot be definitively excluded. Furthermore, the choriocapillaris recovery from injury in these young, normal monkey eyes might be more robust than in aged eyes with macular degeneration. However, the high frequency of PDT (every 2 weeks instead of every 12 weeks as usual in clinical practice) and the lack of subretinal fluid or hemorrhage in these normal eyes would tend to increase the severity of injury from PDT. Results of this limited study suggest that combination treatment with PDT and intravitreal ranibizumab appears to have no additional severe detrimental effects on normal choroid and retina compared to PDT alone. The visual impact of such combination therapy will be demonstrated by ongoing clinical trials. 
 
Figure 1.
 
(A) Baseline fundus photographs of one animal in group I. (B) Fundus appearance of same animal 2 weeks after initial PDT. The right eye had received two intravitreal injections of ranibizumab and the left eye had received injections of vehicle only. (C) Fundus appearance of same animal at the end of the study period, 2 weeks after third PDT, and 1 week after the fourth ranibizumab injection. Corresponding fluorescein angiograms shown in Figure 3 .
Figure 1.
 
(A) Baseline fundus photographs of one animal in group I. (B) Fundus appearance of same animal 2 weeks after initial PDT. The right eye had received two intravitreal injections of ranibizumab and the left eye had received injections of vehicle only. (C) Fundus appearance of same animal at the end of the study period, 2 weeks after third PDT, and 1 week after the fourth ranibizumab injection. Corresponding fluorescein angiograms shown in Figure 3 .
Figure 2.
 
(A) Right eye of an animal from group II 1 week after initial PDT and before ranibizumab injection, demonstrating an area of retinal ischemia on color photographs with associated capillary nonperfusion occurring in the early phase of angiography. (B) The same animal 5 weeks later (2 weeks after third PDT, 1 week after the third ranibizumab injection), with some persistent capillary nonperfusion observed in the early frame and late staining.
Figure 2.
 
(A) Right eye of an animal from group II 1 week after initial PDT and before ranibizumab injection, demonstrating an area of retinal ischemia on color photographs with associated capillary nonperfusion occurring in the early phase of angiography. (B) The same animal 5 weeks later (2 weeks after third PDT, 1 week after the third ranibizumab injection), with some persistent capillary nonperfusion observed in the early frame and late staining.
Figure 3.
 
(A, B) Early- and late-phase fluorescein angiograms of a group I animal 2 weeks after initial PDT, demonstrating some persistent late leakage in the irradiated area. (A) Right eye 1 week after the second ranibizumab injection. (B) Left eye 1 week after the second placebo injection. (C, D) Early- and late-phase angiograms of the same animal 2 weeks after the third PDT and 1 week after the fourth set of injections. Window defects and blockage due to pigmentary changes were seen, but no retinal vascular effects were noted.
Figure 3.
 
(A, B) Early- and late-phase fluorescein angiograms of a group I animal 2 weeks after initial PDT, demonstrating some persistent late leakage in the irradiated area. (A) Right eye 1 week after the second ranibizumab injection. (B) Left eye 1 week after the second placebo injection. (C, D) Early- and late-phase angiograms of the same animal 2 weeks after the third PDT and 1 week after the fourth set of injections. Window defects and blockage due to pigmentary changes were seen, but no retinal vascular effects were noted.
Table 1.
 
Anterior Chamber Inflammatory Reaction
Table 1.
 
Anterior Chamber Inflammatory Reaction
Day Combination PDT Alone
Intervention AC Cells Intervention AC Cells
Group I
0 Ranibizumab Vehicle
1 1–4+ 0
7 PDT 0 PDT 0–0.5+
8 0 0
14 Ranibizumab 0 Vehicle 0
15 1–3+ 0
21 PDT 0 PDT 0
22 0 0
28 Ranibizumab 0 Vehicle 0
29 1–2+ 0
35 PDT 0 PDT 0
36 0 0
42 Ranibizumab 0 Vehicle 0
43 1–3+ 0
49 0 0
Group II
0 PDT PDT
1 0 0
7 Ranibizumab 0 Vehicle 0
8 2–3+ 0
14 PDT 0–1+ PDT 0
15 0–1+ 0
21 Ranibizumab 0 Vehicle 0
22 1–3+ 0
28 PDT 0–1+ PDT 0
29 0–1+ 0
35 Ranibizumab 0 Vehicle 0
36 1–2+ 0
42 0 0
Figure 4.
 
Photomicrographs of eyes from an animal treated three times with PDT on normal choroid. (A, C) Right eye treated with PDT plus ranibizumab; (B, D) left eye treated with PDT only. Similar results were seen in eyes from both groups. (A, B) Outer segments were shortened, the recovering RPE was lightly pigmented, and there were numerous heavily pigmented macrophages present in the subretinal space. Areas of multilayering of the repopulated RPE were also observed. There appeared to be a reduction of the number of vessels in the choriocapillaris (arrowheads) and some were displaced away from Bruch’s membrane (arrow). (C, D) Sections taken from areas outside the irradiated zone, representing normal choriocapillaris architecture. Magnification, ×100.
Figure 4.
 
Photomicrographs of eyes from an animal treated three times with PDT on normal choroid. (A, C) Right eye treated with PDT plus ranibizumab; (B, D) left eye treated with PDT only. Similar results were seen in eyes from both groups. (A, B) Outer segments were shortened, the recovering RPE was lightly pigmented, and there were numerous heavily pigmented macrophages present in the subretinal space. Areas of multilayering of the repopulated RPE were also observed. There appeared to be a reduction of the number of vessels in the choriocapillaris (arrowheads) and some were displaced away from Bruch’s membrane (arrow). (C, D) Sections taken from areas outside the irradiated zone, representing normal choriocapillaris architecture. Magnification, ×100.
Figure 5.
 
Comparison of choriocapillaris densities after various treatment conditions. (A) Densities expressed as total luminal length relative to overlying Bruch’s membrane. (B) Plot demonstrating range of values for each treatment category.
Figure 5.
 
Comparison of choriocapillaris densities after various treatment conditions. (A) Densities expressed as total luminal length relative to overlying Bruch’s membrane. (B) Plot demonstrating range of values for each treatment category.
Figure 6.
 
Transmission electron micrograph of the region of Bruch’s membrane (large arrow) from an area of retina treated three times with PDT. This region was perfused by a capillary (C) and was covered by proliferating RPE (R). The capillary was separated from the original basal lamina in Bruch’s membrane and showed incomplete, duplicated basal laminae (arrowheads) away from a complete layer. The RPE also showed duplicated basal lamina (small arrows) in primitive infoldings. Bar, 1 μm.
Figure 6.
 
Transmission electron micrograph of the region of Bruch’s membrane (large arrow) from an area of retina treated three times with PDT. This region was perfused by a capillary (C) and was covered by proliferating RPE (R). The capillary was separated from the original basal lamina in Bruch’s membrane and showed incomplete, duplicated basal laminae (arrowheads) away from a complete layer. The RPE also showed duplicated basal lamina (small arrows) in primitive infoldings. Bar, 1 μm.
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Figure 1.
 
(A) Baseline fundus photographs of one animal in group I. (B) Fundus appearance of same animal 2 weeks after initial PDT. The right eye had received two intravitreal injections of ranibizumab and the left eye had received injections of vehicle only. (C) Fundus appearance of same animal at the end of the study period, 2 weeks after third PDT, and 1 week after the fourth ranibizumab injection. Corresponding fluorescein angiograms shown in Figure 3 .
Figure 1.
 
(A) Baseline fundus photographs of one animal in group I. (B) Fundus appearance of same animal 2 weeks after initial PDT. The right eye had received two intravitreal injections of ranibizumab and the left eye had received injections of vehicle only. (C) Fundus appearance of same animal at the end of the study period, 2 weeks after third PDT, and 1 week after the fourth ranibizumab injection. Corresponding fluorescein angiograms shown in Figure 3 .
Figure 2.
 
(A) Right eye of an animal from group II 1 week after initial PDT and before ranibizumab injection, demonstrating an area of retinal ischemia on color photographs with associated capillary nonperfusion occurring in the early phase of angiography. (B) The same animal 5 weeks later (2 weeks after third PDT, 1 week after the third ranibizumab injection), with some persistent capillary nonperfusion observed in the early frame and late staining.
Figure 2.
 
(A) Right eye of an animal from group II 1 week after initial PDT and before ranibizumab injection, demonstrating an area of retinal ischemia on color photographs with associated capillary nonperfusion occurring in the early phase of angiography. (B) The same animal 5 weeks later (2 weeks after third PDT, 1 week after the third ranibizumab injection), with some persistent capillary nonperfusion observed in the early frame and late staining.
Figure 3.
 
(A, B) Early- and late-phase fluorescein angiograms of a group I animal 2 weeks after initial PDT, demonstrating some persistent late leakage in the irradiated area. (A) Right eye 1 week after the second ranibizumab injection. (B) Left eye 1 week after the second placebo injection. (C, D) Early- and late-phase angiograms of the same animal 2 weeks after the third PDT and 1 week after the fourth set of injections. Window defects and blockage due to pigmentary changes were seen, but no retinal vascular effects were noted.
Figure 3.
 
(A, B) Early- and late-phase fluorescein angiograms of a group I animal 2 weeks after initial PDT, demonstrating some persistent late leakage in the irradiated area. (A) Right eye 1 week after the second ranibizumab injection. (B) Left eye 1 week after the second placebo injection. (C, D) Early- and late-phase angiograms of the same animal 2 weeks after the third PDT and 1 week after the fourth set of injections. Window defects and blockage due to pigmentary changes were seen, but no retinal vascular effects were noted.
Figure 4.
 
Photomicrographs of eyes from an animal treated three times with PDT on normal choroid. (A, C) Right eye treated with PDT plus ranibizumab; (B, D) left eye treated with PDT only. Similar results were seen in eyes from both groups. (A, B) Outer segments were shortened, the recovering RPE was lightly pigmented, and there were numerous heavily pigmented macrophages present in the subretinal space. Areas of multilayering of the repopulated RPE were also observed. There appeared to be a reduction of the number of vessels in the choriocapillaris (arrowheads) and some were displaced away from Bruch’s membrane (arrow). (C, D) Sections taken from areas outside the irradiated zone, representing normal choriocapillaris architecture. Magnification, ×100.
Figure 4.
 
Photomicrographs of eyes from an animal treated three times with PDT on normal choroid. (A, C) Right eye treated with PDT plus ranibizumab; (B, D) left eye treated with PDT only. Similar results were seen in eyes from both groups. (A, B) Outer segments were shortened, the recovering RPE was lightly pigmented, and there were numerous heavily pigmented macrophages present in the subretinal space. Areas of multilayering of the repopulated RPE were also observed. There appeared to be a reduction of the number of vessels in the choriocapillaris (arrowheads) and some were displaced away from Bruch’s membrane (arrow). (C, D) Sections taken from areas outside the irradiated zone, representing normal choriocapillaris architecture. Magnification, ×100.
Figure 5.
 
Comparison of choriocapillaris densities after various treatment conditions. (A) Densities expressed as total luminal length relative to overlying Bruch’s membrane. (B) Plot demonstrating range of values for each treatment category.
Figure 5.
 
Comparison of choriocapillaris densities after various treatment conditions. (A) Densities expressed as total luminal length relative to overlying Bruch’s membrane. (B) Plot demonstrating range of values for each treatment category.
Figure 6.
 
Transmission electron micrograph of the region of Bruch’s membrane (large arrow) from an area of retina treated three times with PDT. This region was perfused by a capillary (C) and was covered by proliferating RPE (R). The capillary was separated from the original basal lamina in Bruch’s membrane and showed incomplete, duplicated basal laminae (arrowheads) away from a complete layer. The RPE also showed duplicated basal lamina (small arrows) in primitive infoldings. Bar, 1 μm.
Figure 6.
 
Transmission electron micrograph of the region of Bruch’s membrane (large arrow) from an area of retina treated three times with PDT. This region was perfused by a capillary (C) and was covered by proliferating RPE (R). The capillary was separated from the original basal lamina in Bruch’s membrane and showed incomplete, duplicated basal laminae (arrowheads) away from a complete layer. The RPE also showed duplicated basal lamina (small arrows) in primitive infoldings. Bar, 1 μm.
Table 1.
 
Anterior Chamber Inflammatory Reaction
Table 1.
 
Anterior Chamber Inflammatory Reaction
Day Combination PDT Alone
Intervention AC Cells Intervention AC Cells
Group I
0 Ranibizumab Vehicle
1 1–4+ 0
7 PDT 0 PDT 0–0.5+
8 0 0
14 Ranibizumab 0 Vehicle 0
15 1–3+ 0
21 PDT 0 PDT 0
22 0 0
28 Ranibizumab 0 Vehicle 0
29 1–2+ 0
35 PDT 0 PDT 0
36 0 0
42 Ranibizumab 0 Vehicle 0
43 1–3+ 0
49 0 0
Group II
0 PDT PDT
1 0 0
7 Ranibizumab 0 Vehicle 0
8 2–3+ 0
14 PDT 0–1+ PDT 0
15 0–1+ 0
21 Ranibizumab 0 Vehicle 0
22 1–3+ 0
28 PDT 0–1+ PDT 0
29 0–1+ 0
35 Ranibizumab 0 Vehicle 0
36 1–2+ 0
42 0 0
×
×

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