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1From the Retina Service Research Laboratories, Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana; 2Alcon Research, Ltd., Fort Worth, Texas; and 3Miravant Medical Technologies, Inc., Santa Barbara, California.
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METHODS. In cynomolgus (Macaca fascicularis) and squirrel (Saimiri sciureus) monkey eyes (n = 20), matrix placement of laser photocoagulation sites elicited CNV as a component of the development of fibrovascular tissue (FVT). FVT sites received PDT according to specific drug infusion and laser light treatment parameters. FVTs and anastomoses were evaluated by fundus photography, fluorescein angiography, and histologic examination.
RESULTS. Anastomoses averaged approximately 48% of FVT sites, with greatest occurrence in the macaque. Although PDT with each photosensitizer effectively produced FVT closure, both retinal vessels and anastomoses remained patent.
CONCLUSIONS. Although PDT is effective in closing the choroidal neovascularization in FVT, this technique was ineffective in occluding anastomotic vessels and their associated tributaries within the mid- to proximal retina. Various factors (vascular diameter, composition, blood flow, orientation) may contribute to continued anastomotic patency. By convention, such vessels would typically be defined as chorioretinal anastomoses (CRAs); however, continuing studies suggest the possibility that these neovessels constitute dual-origin hybrids. Regardless of origin, viable anastomoses provide one potential mechanism for revascularization to occur after PDT and may help to explain why CRAs are considered a poor prognostic sign in patients with AMD.
This represents the first primate investigation to focus on anastomotic vessel viability as related to the prognosis for formation of a choroidal neovascular membrane (CNVM) and the efficacy of PDT. Anastomoses were evaluated as recurring components of CNV, elicited in two different primate CNV laser-trauma models, and these neovessels were evaluated for possible closure after PDT.
In this report, the term "fibrovascular tissue" (FVT) is used, rather than CNV or CNVM, to describe more accurately the combined histopathologic presence of both CNV and collagenous fibroplasia, dual components that originate from the choriocapillaris at or around the laser photocoagulation sites. FVTs may remain subretinal in their development, or they can directly infiltrate the retinal layers with intermittent neovessels that continue beyond the central FVT mass and into the proximal retina.39 These new vessels include the anastomoses examined in this investigation. Similarly, clinical detection of anastomoses can denote the physical adherence of the subretinal neovascular membrane to the neural retina and thus may contradict surgical intervention for the removal of the membrane.28
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Animals
The effects of PDT on FVT and anastomoses were evaluated in 20 eyes from young-adult cynomolgus macaque monkeys (Covance Research, Alice, TX) and from young-adult (ages, 2–4 years) squirrel monkeys (Charles River Laboratories, Houston, TX). Experiments involving the macaques were performed at the Alcon Research Laboratories (Fort Worth, TX), whereas squirrel monkey experiments were conducted at the Indiana University Laboratory Animal Resources facility (Bloomington, IN). Tissue processing and analyses for both species were performed at the Indiana University School of Medicine (Indianapolis, IN). All procedures were performed with strict adherence to the ARVO Statement for the Use of Animals and Ophthalmic and Vision Research and the guidelines for animal care and experimentation prepared by the Indiana University Institutional Animal Care and Use Committee and the Alcon Institutional Animal Care and Use Committee.
For experimental procedures, including laser photocoagulation, ocular examination, and photography, cynomolgus monkeys (weight range, 2.3–2.5 kg) received intramuscular (IM) ketamine at 10 mg/kg, along with intravenous (IV) thiopental sodium (Pentothal; Alcon Laboratories, Fort Worth, TX) at 15 to 20 mg/kg, to supplement anesthesia. For ophthalmic procedures, topical 1.0% tropicamide and 2.5% phenylephrine hydrochloride were administered to achieve maximum pupillary dilation and cycloplegia.
Squirrel monkeys (weight range, 600–950 g) received IM ketamine at 40 mg/kg and acepromazine at 2 mg/kg for anesthesia, along with IM atropine at 0.05 mg/kg, to minimize bronchial secretions. Maintenance amounts of this mixture (10%–15% of the original dose) were administered at 45-minute intervals, when necessary. For all ophthalmic procedures, topical 0.8% tropicamide and 2.5% phenylephrine hydrochloride were administered for pupillary dilation and cycloplegia.
Laser Photocoagulation
After receiving anesthesia and undergoing pupillary dilation, animals were positioned before a slit lamp (Carl Zeiss Meditec, Inc., Jena, Germany) laser-delivery system. The fundus was visualized with a Goldmann-type plano fundus contact lens (Model OGFA; Ocular Instruments, Inc., Bellevue, WA) with 2.5% hydroxypropyl methylcellulose solution as a cushioning agent.
For induction of photocoagulation sites in the cynomolgus monkeys, an argon laser (514 nm wavelength: Lambda Plus PDL II Photodynamic Laser; Coherent Inc., Santa Clara, CA) was used (390- and 455-mW power, 50-µm spot diameter, 0.1-second duration). Laser power was verified with a power meter (FieldMaster; Coherent, Inc.). In each cynomolgus eye, 16 to 20 photocoagulation sites were similarly arranged in a grid-like pattern of four rows by four to five columns within the macular region, as per criteria established by previous investigators.40 41 42 43 45
To elicit FVT development in squirrel monkey eyes, optimized laser parameters (650-mW power, 75-µm spot diameter, 0.05- or 0.1-second duration) were used with a solid-state diode laser (532-nm wavelength; OcuLight GL; Iris Medical Instruments, Inc., Mountain View, CA).39 Laser power was verified with a power meter (Radiometer Model IL1400A, with Detector Head Model SPL024F; International Light, Inc., Newburyport, MA). A typical series of nine photocoagulation sites were placed within the macula, arranged in a grid-like pattern (consisting of three horizontal rows by three vertical columns). Follow-up fundus and fluorescein angiography examinations were performed on post-FVT induction day 1 and were repeated, typically, on day 28, to confirm development of FVT.
For both species, deliberate care was taken to avoid inducing photocoagulation within the immediate vicinity of the fovea (within approximately 150 µm). Species-specific laser power settings were established and used that most reliably produced acute vapor bubbles, suggestive of Bruch’s membrane rupture. These experimental conditions also most reliably produced FVTs that emanated through the disrupted Bruch’s membrane and were capable of retinal infiltration.
Photodynamic Therapy
At 22 days after laser induction of FVTs, cynomolgus monkeys were injected intravenously with a 0.6% solution of the hydrophilic, metallotexaphyrin-class photosensitizer motexafin lutetium (Optrin; Pharmacyclics, Inc.) at a dosage of 2 mg/kg.46 47 At post-injection activation times within the optimal 10- to 45-minute time frame that previously had been determined for plateau accumulation of this PDT agent (Arbour JD, et al. IOVS 1999;40:ARVO Abstract 2111),47 laser light (732 nm, 600-mW/cm2 irradiance, 1500-µm spot diameter) was delivered transcorneally (via slit lamp adapter) by a diode laser (Visulas 732s; Carl Zeiss Meditec), developed specifically for use with this photosensitizer, to designated FVT sites at a duration-dependent light dosage of 100 J/cm2. Some FVT sites did not receive PDT treatment and were designated as drug-only control sites.
Approximately 28 days after laser induction of FVTs, squirrel monkeys were injected intravenously with the hydrophobic, pyropheophorbide-class photosensitizer indium chloride methyl pyropheophorbide (PhotoPoint; Miravant Pharmaceuticals, Inc.) at a dosage of 0.15 µmol/kg.44 48 At postinjection activation times of +70 and +72 minutes, PDT laser light (664 nm, 500 mW/cm2 irradiance, 800 µm spot diameter) was delivered transcorneally (via slit lamp adapter) to FVT sites in the column located directly nasal to the fovea at a duration-dependent light dosage of 10 J/cm2 and to FVT sites in a column located directly temporal (middle column) to the fovea at a light dosage of 20 J/cm2.48 FVT sites within the third, temporal-most column were designated as non-PDT control sites. All FVT sites for potential analyses and treatment were located within the species-appropriate portion of the macula where laser-trauma–induced CNV had been previously characterized.39
All treatment-designated central FVT sites were encompassed within the PDT beam diameter. In some instances, FVT development extended far from the central photocoagulation sites as "diffuse FVT" (consult Criswell et al.39 ), and was not included within the PDT treatment zone. These regions of extended neovascularization were not included in the histologic tissue assessment.
Ophthalmologic and Histologic Assessment
Cynomolgus monkeys underwent baseline and follow-up (day 22) slit lamp biomicroscopic and indirect ophthalmoscopic examinations. Animals were evaluated by fundus photography and fluorescein angiography (FA) at 23 days after laser induction of FVTs (i.e., 1 day after PDT), followed by euthanasia and histologic analysis of ocular tissues. For FA assessment, animals received 10% sodium fluorescein (0.14 mL/kg) administered intravenously (via the saphenous vein). Photocoagulation sites were viewed and photographed with a retinal camera and image-processing system (TRC-501A Retinal Camera and ImageNet 2000; Topcon Medical Systems, Inc., Paramus, NJ).
Squirrel monkeys underwent a baseline ophthalmologic examination 7 to 14 days before induction of laser photocoagulation sites, a follow-up examination at approximately post–laser-treatment day 28 (just before PDT treatment), and a final examination 7 days after PDT (i.e., 35 days after laser induction of FVTs), followed by euthanasia and histologic analysis of ocular tissues. This assessment included fundus photography and fluorescein angiography (FA) with a fundus camera (FK-30; Carl Zeiss, Inc.) modified (with the inclusion of a +9-D, antireflective-coated spherical lens) to image the fundus in the small diameter (
14.5 mm, axial length) eye. For early- and late-phase fluorescein angiography, 25% sodium fluorescein (0.1 mL/kg) was administered intravenously (via the saphenous vein).
Eyes from both species were enucleated immediately after euthanasia, and eyecup preparations were fixed in 4% phosphate-buffered paraformaldehyde solution (overnight at room temperature). For each eye, a single square-shaped tissue block (8–10 mm/side), containing the photocoagulation sites, optic disc, and fovea, was hand sectioned from the eyecup preparation. Tissue sections were dehydrated, embedded in paraffin, serially sectioned (6-µm thickness), and stained for light microscopy by using a regressive hematoxylin procedure (to label cell nuclei), followed by eosin counterstaining (as a cytoplasm stain and to highlight macrophages). Tissue slide sections from each laser lesion site recovered were evaluated in their entirety and photographed. Histologic tissues were assessed for the presence or absence of experimentally induced FVTs and anastomoses, as well as the relative effect of PDT on these sites. Vascular occlusion was denoted by a uniform, typically pale-pink to cream-colored vessel appearance that was devoid of blood cells, whereas patent vessels contained concentrated populations of clearly identifiable erythrocytes that were bright pink-to-reddish in appearance. Quantitative comparisons of FVT and of anastomotic development between the two primate species were performed by estimating the proportional difference between two independent samples and then determining the probability of significance by t-test (assuming n = 
).
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In this study, some of the anastomoses seemed to derive from the distally infiltrating FVT mass (Figs. 2H 2I ; small arrows), and they appeared to continue proximally to the retinal vasculature; however, this is only a qualitative observation. There were also variations in anastomotic vessel diameter that suggest possible differences in source and flow direction. Vessels that appeared to be CRAs seemed to originate from the FVT membrane and possessed smaller diameters (approximately 5 µm), characteristic of the largest-diameter vessels within the principal FVT mass.
In many other instances (Figs. 2A 2B 2C 2D 2E 2F 2G) , proximal retinal vessels within the laser photocoagulation/PDT treatment zone were noticeably larger in their vascular diameters (Figs. 2D 2E 2F ; large arrows and asterisks), measuring approximately two to five times (10–75 µm) the diameter size of normal retinal vessels (5–15 µm) that lay outside this region. Often (Figs. 2A 2B 2C 2F ; large arrows and small asterisks), from one to three primary anastomotic collaterals ("trunks") could be seen branching from these enlarged retinal vessels within the ganglion cell layer (up to several hundred micrometers away from the photocoagulation/FVT site), and they continued distally at acute angles along and through the retinal layers to reach the FVT membrane. Two or three primary trunk vessels often approached the FVT site from different (opposite) directions (large white arrows compared with small asterisks; Figs. 2A 2B 2C ) and sometimes merged into a common junction within the FVT membrane. These primary anastomotic trunks also exhibited atypically large diameters of approximately 10 to 18 µm. Smaller, but still viable, vessels approximately 5 µm in diameter (Figs. 2D 2G ; small arrows) occasionally derived from these larger vessels and from within the FVT membrane, branching tangentially at distal and proximal angles from the primary vessel and providing additional infiltration to the FVT and surrounding retinal layers. Pigmented macrophages were often observed just adjacent to these vessels (e.g., Figs. 2G 2H ; arrowheads). Up to the principal FVT membrane, neither primary nor secondary anastomotic neovessels appeared to be occluded by PDT.
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Although PDT may effectively close neovascularization in the choroid and expanding CNV membrane, this study provides evidence that anastomoses and their branches in the middle and proximal retina can remain open after PDT. Possible reasons for continued anastomotic patency may include (1) dissemination and mediation of vessel orientation (radial versus tangential) relative to the incident angle of the PDT laser beam; (2) innate differences between retinal and choroidal vessels; (3) relatively higher retinal blood flow rate that, in the proximal retina, could locally decrease photosensitizer concentration; (4) sustained arterial pressure of retinal blood vessels that could result in distension of both retinal and anastomotic trunks after PDT occlusion distally of smaller neovessels in the FVT membrane and choriocapillaris; and (5) PDT, although effective in occluding smaller diameter neovessels of choroidal origin (up to approximately 5 µm diameter), may not be effective in blocking larger diameter blood vessels (5 µm diameter and larger). Viable anastomotic vessels could contribute to the subsequent revascularization of the neovascular membrane region after PDT. Additional studies to evaluate later time points after PDT eventually will determine whether anastomoses demonstrate long-term patency and continued angiogenesis.
CRAs are considered a poor prognostic sign for laser treatment of CNV in general.10 12 Moreover, Kuhn et al.7 reported that ICG-guided photocoagulation of anastomoses in patients with AMD did not achieve effective closure. Mantel and Zografos14 have indicated that successful CRA closure by selective photocoagulation may be beneficial in the overall treatment of AMD, whereas Silva et al.16 have concluded that selective treatment of CRAs by PDT can result in stabilization or even improvement in visual acuity in most patients at 1 year after treatment.
Until recently, anastomoses have been generally considered a relatively infrequent sign of late-stage, exudative AMD, owing in part to the fact that they are difficult to visualize, particularly with FA. Wolf and Goldberg49 indicated in a previous primate investigation that CRAs did not become apparent on FA until at least 7 weeks after laser photocoagulation. Why this should happen is unclear, but it may be related to spontaneous regression of neovascularization that commences in the primate retina at approximately 5 to 8 weeks after lesion induction, thereby permitting better identification of larger anastomotic vessels. Collagenous fibroplasia surrounding the anastomotic vessels also may partially mask their presence. Moreover, anastomoses may appear as focal sites ("hot spots")3 of hyperfluorescence due to their characteristically high incidence angle of orientation through the retina, making them difficult to identify. ICGA, in conjunction with scanning laser ophthalmoscopy, can improve the analysis and localization of anastomotic "hot spots" and may provide certain technical advantages for visualizing these vessels.3 7 8 10 11 12 13 50 Optical coherence tomography (OCT) may best reveal the clinical presence of anastomoses in radial (cross-sectional) views of the retina.13
With these improved examination techniques, the clinical presence of anastomoses is now thought to be more prevalent in patients with exudative AMD than has been previously reported. CRAs have been shown to precede or to occur in tandem with more generalized neovascularization.12 29 The occurrence of CRAs is now regarded as a common feature, to even a primary manifestation, of classic CNV in exudative AMD.10 13 Kuhn et al.7 reported that one or more CRAs were visualized (on FA or ICGA) in 27% (n = 186 eyes) of 182 patients with early exudative AMD; Slakter et al.10 documented that CRAs occurred in 21% of exudative AMD eyes (n = 150), with 71% of these eyes having exhibited multiple vessels; and Axer-Siegel et al.11 indicated that anastomoses were visualized (on FA and ICGA) in conjunction with CNV in 28% of eyes (n = 205) in 153 AMD patients with occult CNV. In patients with unilateral AMD, the initial appearance of CRAs in the non-AMD-diagnosed eye now merits increased attention as a potential harbinger for contralateral neovascularization in this eye (Haddad WM, et al. IOVS 2003;44:ARVO E-Abstract 4991).
Although histologic findings in this investigation suggest the possible presence of multiple anastomoses at FVT sites, the presence of multiple anastomoses also has been discovered at FVT sites in the rat laser trauma model (Criswell et al., unpublished data, 2005). In rat, confocal microscopic analyses of FITC-albumin labeled FVT sites have revealed the presence of multiple anastomoses (commonly eight or more per site), even though only one or two anastomoses may be evident during light microscopic examination of radial tissue sections, and no anastomoses were observed during fundus or FA examinations.
Qualitative evidence in this study further suggests that anastomoses may simultaneously originate from both choroidal and retinal vascular sources and unify in and around the central FVT membrane. Some anastomoses probably represent CRAs, given that laser photocoagulation initiated neovascularization and development of FVT that originated from the choriocapillaris. However, several lines of evidence indicate that such an assumption may not apply uniformly to all identified anastomoses. In previous work, investigators have demonstrated that it is not uncommon for more than one vascular direction (choroidal to retinal versus retinal to choroidal) as well as type (artery to vein, artery to artery, vein to vein) of anastomotic permutation to evolve.11 19 32 51 52 Moreover, the larger-diameter anastomoses observed proximally may represent RCAs of retinal origin, rather than CRAs of choroidal origin. The enlarged diameters of the retinal feeder vessels and of certain proximal anastomoses suggest the possibility of a retinal arterial origin for these particular anastomoses, with colligation of these vessels and some of their smaller collateral tributaries within the FVT membrane. Human clinical evidence also suggests the possibility of dual neovascular development.4 53 Alternatively, Yannuzzi et al.3 have proposed that neovascularization can, in a subset of patients with AMD, originate as a primary manifestation of the retina vasculature (RAP) and later may evolve into an RCA with choroidal vessels.
The angiogenic potential for choroidal and retinal vascular networks to respond mutually in forming anastomoses also exists. Recent and continuing research (Hu WZ, et al. IOVS 2004;45:ARVO E-Abstract 1844) has provided evidence that laser trauma to the retinal pigment epithelium-choriocapillaris not only upregulates active growth factor expression to evoke FVT development in the choriocapillaris, but also differentially increases expression of certain precursor and active growth factors in the proximal retina, thereby increasing the possible initiation of angiogenic events within the retinal vascular layer. Clinical evidence supports this finding. Wallow et al.38 reported that in a patient with AMD, a new neovascular membrane had developed, which was supplied by the retinal vasculature, approximately 5 months after photocoagulative treatment of the original CNV membranes. Similarly, patients with early exudative AMD who were treated with laser photocoagulative therapy for vascularized retinal pigment epithelium detachments showed revascularization of the sites by anastomotic vessels of retinal origin (RCAs).7 10 14
In conclusion, this is the first primate study to examine continuing anastomotic viability in conjunction with CNV and PDT efficacy. CNV in diseases such as exudative AMD can commonly include anastomotic vessel development as an additional consequence of angiogenesis. Typical photodynamic therapy, while designed to achieve choroidal neovascular closure, may not be adequate to occlude anastomotic vessels. The continued patency of anastomoses provides one possible neovascular source for revascularization and may help to explain why the visualization of anastomoses is considered such a poor prognostic sign. Careful identification and selective highly localized treatment of anastomotic vessels by PDT or photocoagulation, in addition to standard PDT for treatment of CNVMs, may be beneficial to a patient’s long-term visual outcome. The combined use of PDT and antiangiogenic therapies may also contribute to a more favorable long-term management strategy (Haddad WM, et al. IOVS 2004;45:ARVO E-Abstract 3163).
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Acknowledgements |
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Footnotes |
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Supported in part by a grant from Alcon Research, Ltd., Fort Worth, Texas (MHC, TAC); by National Eye Institute Grant R44 EY11191, Business Innovation Research Program via Miravant Medical Technologies (TAC, MHC, RPD); by the Indiana Center for Vascular Biology and Medicine/Mr. and Mrs. Arthur R. Whale; and by an unrestricted grant from Research to Prevent Blindness, Inc., to the Department of Ophthalmology, Indiana University.
Submitted for publication December 8, 2004; revised February 7, 2005; accepted February 21, 2005.
Disclosure: M.H. Criswell, Alcon Research, Ltd. (F) and Miravant Medical Technologies, Inc. (F); T.A. Ciulla, Alcon Research, Ltd. (F) and Miravant Medical Technologies, Inc. (F); L.A. Lowseth, Alcon Research, Ltd. (E); W. Small, Miravant Medical Technologies, Inc. (E); R.P. Danis, Miravant Medical Technologies, Inc. (F); D.L. Carson, Alcon Research, Ltd. (E)
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.
Corresponding author: Mark H. Criswell, Retina Service Research Laboratories, Department of Ophthalmology, 702 Rotary Circle, Indiana University School of Medicine, Indianapolis, IN 46260; mcriswel{at}iupui.edu.
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