0
We're unable to sign you in at this time. Please try again in a few minutes.
Retry
We were able to sign you in, but your subscription(s) could not be found. Please try again in a few minutes.
Retry
There may be a problem with your account. Please contact the AMA Service Center to resolve this issue.
Contact the AMA Service Center:
Telephone: 1 (800) 262-2350 or 1 (312) 670-7827  *   Email: subscriptions@jamanetwork.com
Error Message ......
Access to paid content on this site is currently suspended due to excessive activity being detected from your IP address 54.242.8.162. Please contact the publisher to request reinstatement.
Laboratory Sciences |

Predilection of the Macular Region to High Incidence of Choroidal NeovascularizationAfter Intense Laser Photocoagulation in the Monkey FREE

Wei-Yong Shen, MD, PhD; Shu Yen Lee, MD; Ian Yeo, MD; Chooi-May Lai, PhD; Ranjana Mathur, MD; Donald Tan, PhD; Ian J. Constable, MD; P. Elizabeth Rakoczy, PhD
[+] Author Affiliations

From the Department of Molecular Ophthalmology, Lions Eye Institute,and Centre for Ophthalmology and Visual Science, University of Western Australia,Nedlands (Drs Shen, Lai, Constable, and Rakoczy), and Singapore Eye ResearchInstitute, National University of Singapore (Drs Lee, Yeo, Mathur, and Tan).


Arch Ophthalmol. 2004;122(3):353-360. doi:10.1001/archopht.122.3.353.
Text Size: A A A
Published online

Objective  To determine the key factors for creating a high incidence model ofchoroidal neovascularization (CNV) in the monkey.

Methods  Intense laser photocoagulation was performed in 8 eyes of 4 monkeysusing krypton red and green-yellow and Alcon frequency-doubled diode ophthalmiclasers. Eight to 13 lesions were delivered to an area between the temporalvascular arcades in each eye. Development of CNV was monitored by fluoresceinangiography at 2 and 4 weeks after laser treatment, and the results were correlatedwith histological analysis.

Results  A much higher incidence of CNV occurred in the macular region, whichrefers to an anatomic area equivalent to a mean ± SD 2.5 ± 0.4times the horizontal diameter of the optic disc in the fundus. Regardlessof the type of ophthalmic laser used, 72% of lesions developed fluoresceinleakage within the macula, compared with 12% outside the macula (P<.001). By histological analysis, 89% of lesions developed microscopicCNV within the macula vs 22% outside the macula (P<.001).

Conclusion  The macular region is predisposed to creation of laser-induced CNV inthe monkey.

Clinical Relevance  The predilection of the macular region to a high incidence of laser-inducedCNV may account for the high recurrence rate of subfoveal CNV after lasertreatment in humans.

Figures in this Article

Choroidal neovascularization (CNV), the invasion of newly formed bloodvessels from the choroid through a break in Bruch membrane, is a feature ofmany eye diseases, including age-related macular degeneration, ocular histoplasmosis,angioid streaks, high myopia, chronic uveitis, and choroidal rupture.1,2 Choroidal neovascularization developsunderneath the retina and disrupts the retinal pigment epithelium (RPE) andneurosensory retina, resulting in severe visual loss in most cases.1,2 The conventional management of CNVby laser photocoagulation has inherent drawbacks, including less dramaticeffect on subfoveal CNV, a high rate of CNV persistence and recurrence, andirreversible damage to the RPE and normal retina.35 Severalnew therapeutic strategies, including photodynamic therapy,6 viralvector–mediated gene therapy,7,8 andnew antiangiogenic drugs,9 have been introducedor are being investigated, and all of these need a reproducible and clinicallyrelated model of CNV.

Induction of CNV in animals can be achieved in 2 ways. One method isto create acute breaks in Bruch membrane traumatically9,10 orenzymatically,11 while the other is to induceCNV without immediate breaking of Bruch membrane. The disease process inducedby the latter method is slow, and the permeability of the newly formed bloodvessels is uncertain.12,13 Intenselaser photocoagulation has been used to break Bruch membrane to create CNVin the rat,14,15 rabbit,16 cat,17 and monkey.9,10 The rat and monkey models have beenthe most commonly used in eye research. Previous success rates of laser-inducedCNV of 50% to 80% have been reported in the rat.14,15 Theincidence of laser-induced CNV in the monkey, however, is only 30% to 40%,and results vary markedly.9,10

Previously, clinical and experimental investigations have overlookedthe importance of the macular region in CNV development.1,2,9,10 Ryan10 reported that 39% of laser-induced lesions developedclinical CNV (fluorescein leakage) in the macular region, with 3% in the areanasal to the optic nerve head and 0.3% in the peripheral retina of the rhesusmonkey. However, no information is available to clearly define the anatomicterritory of the macula; thus, no landmark index can be used to preciselydeliver laser lesions and achieve a high incidence of CNV in the primate model.As the retinal-choroidal circulation and macular anatomy are similar betweenhumans and primates, the information obtained from the monkey model of CNVwould be particularly valuable and crucial for assessment of new antiangiogenictherapies before clinical application. In a previous study,9 however,the low incidence of CNV in monkeys limited statistical evaluation of an antiangiogenictherapy. Considering the valuable information for clinical application andthe high cost of experimentation on monkeys, it is critical to find key factorsthat would improve the success rate of laser-induced CNV in the monkey model.In this study, we measured the anatomic size of the macular region and analyzedthe incidence of laser-induced CNV within this region, compared with thatoutside the macular region. Our results show that the macular region is predisposedto the creation of laser-induced CNV in the monkey.

ANIMAL PREPARATION AND ANESTHESIA

Eight eyes from 4 Macaca fascicularis monkeyswere used in accord with the guidelines of the Association for Research inVision and Ophthalmology on the use of animals in research and following theguidelines of the Animal Care Committee at National University of Singapore.The animals were anesthetized with ketamine hydrochloride (20 mg/kg of bodyweight), acepromazine maleate (0.25 mg/kg), and atropine sulfate (0.125 mg/kg).Pupils were dilated with 2.5% phenylephrine hydrochloride and 1% tropicamidedrops. A trained veterinarian monitored the airway, respiration, and pulseduring all procedures.

LASER PHOTOCOAGULATION

Laser photocoagulation was performed as previously described,9,10 with modifications. Three ophthalmiclasers, a krypton red (647 nm) (Coherent Radiation System, Salt Lake City,Utah), krypton green-yellow (528-568 nm) (Coherent Radiation System), andan Alcon frequency-doubled diode (532 nm) (Alcon 532 Ophthalas EyeLite Photocoagulator;Alcon, Inc, Houston, Tex), were used in this study (Table 1). With each ophthalmic laser, the levels of energy wereinitially assessed in an area away from the macula to test the ability toproduce a blister without subretinal hemorrhage. The final protocols usedwere 1.0-W power density, 50-µm spot size, and 0.2-second duration forthe krypton red; 1.5-W power density, 50-µm spot size, and 0.2-secondduration for the krypton green-yellow; and 1.5-W power density, 50-µmspot size, and 0.1-second duration for the Alcon frequency-doubled diode lasers.Eight to 13 lesions were delivered in an area between the temporal vasculararcades in each eye using a slitlamp and fundus contact laser lens.

Table Graphic Jump LocationTable 1. Development of Fluorescein Leakage After Intense Laser PhotocoagulationWith Krypton Red and Green-Yellow and Alcon Frequency-Doubled Diode (AFDD)Ophthalmic Lasers in the Macaca fascicularis Monkey
FUNDUS PHOTOGRAPHY AND FLUORESCEIN ANGIOGRAPHY

Fundus photographs were taken immediately after laser photocoagulationand at 2 and 4 weeks after laser treatment. Fundus fluorescein angiography(FFA) was performed to monitor the development of CNV at 2 and 4 weeks afterlaser treatment. Angiography was performed by intravenous injection of 10%fluorescein sodium (0.1 mL/kg of body weight). Identification of CNV was basedon fluorescein behavior during the phases of angiography from 10 seconds to10 minutes after dye injection. The status of the lesions was graded in amasked fashion by 2 examiners (W.-Y.S. and C.-M.L.) using reference angiograms.The scores were recorded as follows: 0, no hyperfluorescein staining; +, slighthyperfluorescein staining; ++, moderate fluorescein leakage; and +++, prominentfluorescein leakage or pooling. Choroidal neovascularization was characterizedby the presence of fluorescein leakage or pooling at a late phase of angiography,and only the lesions that scored 2 and higher were considered clinical CNV.9 Based on the criteria of the Macular PhotocoagulationStudy Group,18 CNV on angiography was consideredclassic when well-demarcated boundaries were discerned at an early stage ofFFA, with progressive pooling or leakage in later phases of angiography. OccultCNV was characterized by a stippled area of hyperfluorescence noted within1 to 2 minutes after dye injection, with leakage in this area in the latephase; or by areas of leakage at the level of irregular elevation of RPE inthe late phase, without well-demarcated areas of hyperfluorescence discerniblein the early phase.

HISTOLOGICAL ANALYSIS

All monkeys were humanely killed 4 weeks after laser treatment. Theeyes were enucleated and fixed in 2.5% glutaraldehyde plus 2% paraformaldehydein 0.1M phosphate-buffered saline for a minimum of 24 hours, then processedfor paraffin embedding. Paraffin sections of 5-µm thickness were cut,and serial sections were collected when the first lesion was identified. Hematoxylin-eosinstaining was performed every 4 slides. The primary goals of histological analysiswere to: (1) correlate the incidence of leaky lesions with the number of microscopicCNV lesions detected by histological analysis, (2) determine the associationof broken Bruch membrane with CNV development, and (3) orient histologicalCNV inside or outside the macular region (Table 2).

Table Graphic Jump LocationTable 2. Development of Microscopic Choroidal Neovascularization (CNV)After Intense Laser Photocoagulation With Krypton Red and Green-Yellow andAlcon Frequency-Doubled Diode (AFDD) Ophthalmic Lasers in the Macaca fascicularis Monkey

To evaluate the imbalance of CNV development between the macular andnonmacular regions, the size of the macula was initially determined by histologicalanalysis (Figure 1). The maculawas identified as a multilaminar ganglion layer, with maximum cell densityin the parafoveal retina and a gradual disappearance of ganglion cell andinner nuclear layers at the central fovea (Figure 1A). The ratio of the macula to the horizontal optic discwas measured in 4 samples from 4 monkeys. The ratio was used as an index tofurther determine the number of lesions within and outside the macular regionson angiography.

Place holder to copy figure label and caption
Figure 1.

A, The mean ± SD maculardiameter–optic disc (OD) ratio was 2.5 ± 0.4 times the horizontaldiameter of the OD in the fundus. The short arrow points to a lesion locatednext to the parafoveal retina. The macula and OD were detected in serial sections.B, The relative size of the OD to the macula in histological analysis. GCLindicates ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclearlayer; RPE, retinal pigment epithelium; and CRV, central retinal vessels.Hematoxylin-eosin staining, original magnification ×38.

Graphic Jump Location

With maximum levels of laser energy, photocoagulation using the kryptonred and green-yellow and the Alcon frequency-doubled diode lasers did notinduce obvious hemorrhage in most lesions, except for 2 testing spots showingminor hemorrhage where the lesions were delivered on small retinal vessels(data not shown). Four eyes were treated with the krypton red laser (Table 1). Of 34 lesions delivered, 5 (15%)developed fluorescein leakage on angiography. In 1 eye in which all 8 lesionswere placed outside the macular region, none showed clinical CNV by FFA (Figure 2A). In the remaining 3 eyes in which26 lesions were delivered, 5 (38%) of 13 lesions developed clinical CNV insidethe macular region, compared with none showing leakage outside the macula(Figure 2B). Using the krypton redlaser, none of the 21 lesions outside the macular region developed CNV-relatedfluorescein leakage at 2 and 4 weeks after laser treatment. Eleven lesionswere delivered into 1 eye using the krypton green-yellow laser, 3 (27%) ofwhich developed fluorescein leakage within the macular region. In contrast,none of the 8 lesions outside the macular region developed CNV-related fluoresceinleakage (Figure 2C). Using the frequency-doubleddiode laser, 33 lesions were delivered into 3 eyes, 19 (58%) of which developedCNV-related leakage (Figure 2D-F).Of 13 lesions delivered inside the macular region, all developed CNV-relatedfluorescein leakage (Figure 2D andF). Of 20 lesions that were delivered in the boundary or outside the macula,however, only 6 (30%) showed CNV-related fluorescein leakage (Figure 2D and F). In general, regardless of the type of ophthalmiclaser used, 21 (72%) of 29 lesions developed CNV-related leakage inside themacular region, compared with 6 (12%) of 49 lesions outside the macula (P<.001, unpaired t test). Inall cases, CNV-related fluorescein leakage was observed at 2 weeks and at4 weeks after laser treatment. Based on the criteria of the Macular PhotocoagulationStudy Group,18 most CNV lesions in the laser-inducedmodel showed features of classic CNV, characterized by well-demarcated boundariesdiscernible at an early stage of angiography, with progressive pooling orleakage in later phases of angiography (Figure2E and F). However, occult CNV lesions were also observed, characterizedby poorly defined areas of hyperfluorescence in the early phase and progressivepooling or leakage in later phases of angiography (white arrow, Figure 2E and F).

Place holder to copy figure label and caption
Figure 2.

Predilection of the macular regionto a high incidence of choroidal neovascularization (CNV) on fluorescein angiographyafter laser photocoagulation by the krypton red laser (A and B), krypton green-yellowlaser (C), and Alcon frequency-doubled diode laser (D, E, and F). In A-D,none of the lesions outside the macular regions developed CNV-related fluoresceinleakage. In contrast, 2, 3, 7, and 6 lesions inside the macular regions showCNV-related fluorescein leakage in B, C, D, and F, in which 6, 3, 7, and 6lesions were placed, respectively. Two lesions outside the macula (black arrows)and 1 in the boundary (curved white arrow) developed CNV-related leakage (F).Early-phase (E, 43 seconds) and late-phase (F, 7 minutes) angiograms showfluorescein leakage from well-demarcated CNV at most lesions and from poorlydemarcated CNV at 1 lesion (white arrow in E and F). Plus signs indicate thecentral fovea; circled areas, the macular region.

Graphic Jump Location

Histological analysis of 10 lesions outside the macular region in 4eyes treated by the krypton red laser showed broken Bruch membrane in 5 lesions,but all failed to develop histological CNV (Table 2 and Figure 3Aand B). Inside the macular region, however, 9 of 10 lesions showed brokenBruch membrane, and all developed microscopic CNV (Figure 3C and D). The development of CNV was initiated with disruptionof Bruch membrane, and the new vessels originated from the choriocapillariesand extended into the subretinal space, forming neovascular membrane networks.Histological analysis of the macular region in the eye treated by the kryptongreen-yellow laser was not available, because of unexpected tissue destruction.Two lesions outside the macular region showed broken Bruch membrane, but nonedeveloped microscopic CNV. Among the eyes treated by the Alcon frequency-doubleddiode laser, all 29 lesions showed broken Bruch membrane, regardless of theirgeographic location. Within the macular region, 16 (89%) of 18 lesions showedmicroscopic CNV (Figure 4A and B).In contrast, 5 (45%) of 11 lesions outside the macular region developed histologicalCNV, and most of them formed fibrous tissue. Among the few lesions showingmicroscopic CNV in the extramacular region, the number of new vessels waslimited, and most of them were embedded in firmly packed fibrous tissue (Figure 4C-F). Among some lesions, severalelongated fibroblast-like cells and pigment-laden cells enveloped the newvessels, apparently limiting their further development (Figure 4E and F). In general, regardless of the type of ophthalmiclaser used, 25 (89%) of 28 lesions developed histological CNV inside the macularregion vs 5 (22%) of 23 outside the macular region (P<.001,unpaired t test).

Place holder to copy figure label and caption
Figure 3.

Histological choroidal neovascularization(CNV) after intense laser photocoagulation treated by the krypton red laser.The figures on the right show high magnifications of the insets on the left.No CNV was detected in a lesion outside the macula in A and B, although Bdemonstrates that Bruch membrane was broken (between the 2 large arrows) andcellular infiltration (small arrows) was present. Arrows in D indicate well-formednew vessels in a CNV membrane inside the macula. GCL indicates ganglion celllayer; INL, inner nuclear layer; ONL, outer nuclear layer; and RPE, retinalpigment epithelium. Hematoxylin-eosin staining, original magnification ×94(A and C) and ×376 (B and D).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Histological choroidal neovascularization(CNV) after intense laser photocoagulation treated by the Alcon frequency-doubleddiode laser. The figures on the right show high magnifications of the insetson the left. Arrows in B indicate well-formed new vessels in a CNV membraneinside the macula. Arrows in D indicate new vessels in a CNV membrane outsidethe macula. In F, a new vessel (NV) was enveloped by several elongated fibroblast-likecells and pigment laden cells (white arrows). RPE indicates retinal pigmentepithelium. Hematoxylin-eosin staining, original magnification ×198(A and E), ×376 (B, D, and F), and ×94 (C).

Graphic Jump Location

Our results demonstrate that the macular region was predisposed to laser-inducedCNV. Fundus fluorescein angiography showed that 72% of lesions developed CNV-relatedfluorescein leakage inside the macula, compared with 12% outside the macula.Furthermore, histological analysis showed that 89% of lesions developed microscopicCNV inside the macula vs 22% outside the macula.

Previous investigations have demonstrated that the presence of fluoresceinleakage or pooling on angiography correlated with microscopic CNV, but thatnot all microscopic CNV developed fluorescein leakage on angiography.10,14,19,20 Inthe present study, 27 (35%) of 78 lesions developed fluorescein leakage onangiography, but 30 (59%) of 51 lesions on histological analysis showed microscopicCNV. This observation is similar to findings in a previous study14 ina rat model showing that 28% of lesions developed fluorescein leakage, withsubsequent histological analysis revealing microscopic CNV in 60% of lesions.In primate models, Ryan10 and Miller et al20 reported that the incidence of lesions showing fluoresceinleakage on angiography was much lower than that of microscopic CNV. In humans,Gass21 reported that, while FFA failed to showfluorescein leakage, subsequent postmortem examination demonstrated pathologicCNV development. The reason why some histological CNV membranes remained inactiveduring FFA is unclear. In the present study, most lesions inside the maculadeveloped classic CNV. However, we observed that certain lesions inside themacula developed occult CNV. By histological analysis, most CNV membranesoutside the macula contained fewer new vessels, embedded in firmly packedfibrous tissue, compared with the microscopic CNV inside the macula, and somenew vessels were enveloped by elongated fibroblast-like cells and RPE-likecells. The limited number of microscopic CNV lesions and the absence of afluid-filled space may account for the inactivation of fluorescein leakagefrom lesions outside the macula. It could be possible that the CNV lesionswithin the macular region mainly developed classic CNV, while the lesionsoutside the macular region predominantly formed occult CNV, which is difficultto interpret on angiography. In addition, the differences in endothelial cellfenestration and pericyte maturation between leaky and nonleaky new vessels—andthe local molecular mechanisms, such as the level of vascular endothelialgrowth factor that is known to initiate and maintain angiogenesis and to increasevascular permeability, may also affect the activity of lesions during angiography.15,19,20,22

Previous studies9,10 haveshown that the occurrence of hemorrhage at the time of laser photocoagulationdoes not affect CNV development. In contrast, massive subretinal hemorrhageusually leads to scar tissue formation.9,10 Becausethe intensity of laser burns varies depending on treatment protocols (powerdensity, spot size, and duration), properties and conditions of ophthalmiclasers, fundus pigmentation, and magnification factors of laser lens used,we initially delivered laser spots far away from the macular region for energytesting. The final laser protocols were designed to promote laser burns withheating blasters but without obvious hemorrhage. McAllister and colleagues23 showed that an ophthalmic laser with a green wavelengthspectrum requires at least a 1.5-W power density to reliably break Bruch membranein humans when a 50-µm spot size and a 0.1-second duration were applied.In this study, we used the most powerful laser burns to break Bruch membrane.For the krypton red laser, a 1.0-W power density was used because of the evidencethat rupture of Bruch membrane requires less power density compared with thegreen spectrum lasers.24 In the present study,of 91 lesions delivered in 8 eyes, only 2 showed minor hemorrhage in whichthe lesions hit small vessels. With the krypton red laser, rupture of Bruchmembrane occurred much more frequently inside the macular region comparedwith outside the macular region.

Based on the histological analysis, CNV only occurred in lesions inwhich Bruch membrane was broken. However, a break in Bruch membrane was notalways sufficient to induce CNV. In the laser-induced CNV model, factors affectingthe success rate of CNV may include the wavelengths and energy levels of ophthalmiclasers and the locations where lesions are delivered. Moreover, a recent study25 demonstrated age to be an independent risk factorfor severity of laser-induced CNV in rodents. With the 3 different types oflaser used, the success rate of CNV ranged from 15% to 58% in the presentstudy. Given the fact that different power densities and durations were usedfor different types of lasers, it is hard to draw any conclusions about therelative efficiency of these lasers in terms of their production of CNV, althoughit appeared that the Alcon frequency-doubled diode laser produced the highestincidence of CNV when the highest energy level was used.

The mechanisms of laser-induced CNV and the imbalance of CNV developmentbetween the macula and extramacula are unclear. It has been proposed thata balance between factors that stimulate or inhibit vessel growth controlsneovascularization.26,27 In mostnormal tissues, inhibitory factors are active and vessels remain quiescent.26 In contrast, in different pathologic states, suchas tumor growth and neovascular forms of age-related macular degeneration,neovascularization occurs because of the disruption of the balance betweenangiogenic factors and angiogenic inhibitors.2628 Recentinvestigations have shown that sole overexpression of vascular endothelialgrowth factor, one of the most potent angiogenic growth factors, is sufficientto induce ocular neovascularization.22,29 Furthermore,vascular endothelial growth factor is involved in the maturation and stabilizationof newly formed vessels.28 With a disruptionof Bruch membrane, the effect of laser photocoagulation on the developmentof CNV is often attributed to the up-regulation of angiogenic factors. Previousstudies15,3032 havedemonstrated that expression of several angiogenic factors, including vascularendothelial growth factor, basic fibroblast growth factor, and matrix metalloproteinase2, is up-regulated in activated RPE and infiltrating cells in laser-inducedCNV. Most recently, Renno et al33 demonstratedthat down-regulation of pigment epithelium–derived factor, a potentendogenous inhibitor for vascular endothelial cell proliferation and migration,also enhances laser-induced CNV. In the normal retina, pigment epithelium–derivedfactor is expressed most intensely in the outer nuclear layer (photoreceptornuclei).33 After laser treatment, however,immunostaining for the factor within the outer nuclear layer was absent ordecreased for up to 3 weeks, which seemed to parallel the up-regulation ofvascular endothelial growth factor.15,30,31,33 Inthe present study, the macular region was predisposed to the incidence oflaser-induced CNV. Anatomically and physiologically, the macula is differentfrom the extramacula by its denser distribution of photoreceptors and RPEcells and more abundant blood supply from the choroidal circulation.34,35 It is possible that these differenceslead to the imbalance between the macula and extramacula in overexpressionof angiogenic factors and in down-regulation of angiogenic inhibitors afterlaser photocoagulation. Immunostaining for angiogenic factors and angiogenicinhibitors on macular and extramacular new vessels could clarify these speculations.However, all enucleated eyes in the present study were fixed in 2.5% glutaraldehydeplus 2% paraformaldehyde for a minimum of 24 hours, which challenges the immunostainingtechniques. Our study was initially designed to increase the incidence ofCNV in the primate model for preclinical evaluation of antiangiogenic therapies,rather than to investigate the mechanisms regulating CNV formation. When freshtissue is available in the future, further investigations will be directedto elucidate the molecular mechanisms that could explain the imbalance oflaser-induced CNV between the macular and extramacular regions.

The predilection of the macular region to a high incidence of CNV isalso of clinical significance. Iatrogenic CNV is a common complication oflaser photocoagulation for treatment of subfoveal CNV.36 Previousstudies12,37,38 demonstratedthat chronic cellular processes following moderate-intensity laser treatmentresulted in gradual dissolution of Bruch membrane. Clinically, comparablelaser intensities unable to immediately break Bruch membrane induce choriocapillarybudding, with subsequent digestion of Bruch membrane and histological CNVformation.12,37,38 Thecurrent observation, the predilection of the macular region to a high incidenceof laser-induced CNV, may account for the high recurrence rate of subfovealCNV after laser treatment in humans.

Corresponding author: P. Elizabeth Rakoczy, PhD, Department of MolecularOphthalmology, Lions Eye Institute, 2 Verdun St, Nedlands, Western Australia6009 (e-mail: rakoczy@cyllene.uwa.edu.au).

Submitted for publication October 25, 2002; final revision receivedApril 23, 2003; accepted August 19, 2003.

We thank Tammy Zaknich and Benjamin Rae for their assistance in histologicalpreparations; Paul Pineda, Barathi Amutha, and Robert Ng for animal anesthesia;and Joseph Ho for fundus photography and fluorescein angiography.

Dolan  BJ Choroidal neovascularization not associated with age-related maculardegeneration. Optom Clin. 1996;555- 76
PubMed
Green  WRWilson  DJ Choroidal neovascularization. Ophthalmology. 1986;9311691176;
PubMed
Shiraga  FTakasu  IShiragami  C Treatment options in subfoveal choroidal neovascularization secondaryto age-related macular degeneration. Semin Ophthalmol. 1998;1331- 39
PubMed
Submacular Surgery Trials Pilot Study Investigators, Submacular surgery trials randomized pilot trial of laser photocoagulationversus surgery for recurrent choroidal neovascularization secondary to age-relatedmacular degeneration, I: ophthalmic outcomes submacular surgery trials pilotstudy report number 1. Am J Ophthalmol. 2000;130387- 407
PubMed
Submacular Surgey Trials Pilot Study Investigators, Submacular surgery trials randomized pilot trial of laser photocoagulationversus surgery for recurrent choroidal neovascularization secondary to age-relatedmacular degeneration, II: quality of life outcomes submacular surgery trialspilot study report number 2. Am J Ophthalmol. 2000;130408- 418
PubMed
Arnold  JBauer  BBlumenkranz  MS  et al.  Guidelines for using verteporfin (Visudyne®) in photodynamic therapyto treat choroidal neovascularization due to age-related macular degenerationand other causes. Retina. 2002;226- 18
PubMed
Lai  CCWu  WCChen  SL  et al.  Suppression of choroidal neovascularization by adeno-associated virusvector expressing angiostatin. Invest Ophthalmol Vis Sci. 2001;422401- 2407
PubMed
Lai  YKYShen  WYBrankov  MLai  CMConstable  IJRakoczy  PE Potential long-term inhibition of ocular neovascularization by recombinantadeno-associated virus-mediated secretion gene therapy. Gene Ther. 2002;9804- 813
PubMed
Shen  WYGarrett  KLWang  CG  et al.  Preclinical evaluation of a phosphorothioate oligonucleotide in theretina of rhesus monkey. Lab Invest. 2002;82167- 182
PubMed
Ryan  SJ Subretinal neovascularization: natural history of an experimental model. Arch Ophthalmol. 1982;1001804- 1809
PubMed
Ryan  SJMittl  RNMaumenee  AE Enzymatic and mechanically induced subretinal neovascularization. Albrecht Von Graefes Arch Klin Exp Ophthalmol. 1980;21521- 27
Frank  RNDas  AWeber  ML A model of subretinal neovascularization in the pigmented rat. Curr Eye Res. 1989;8239- 247
PubMed
Kimura  HSakamoto  THinton  DR  et al.  A new model of subretinal neovascularization in the rabbit. Invest Ophthalmol Vis Sci. 1995;362110- 2119
PubMed
Dobi  ETPuliafito  CADestro  M A new model of experimental choroidal neovascularization in the rat. Arch Ophthalmol. 1989;107264- 269
PubMed
Shen  WYYu  MJTBarry  CJConstable  IJRakoczy  PE Expression of cell adhesion molecules and vascular endothelial growthfactor in experimental choroidal neovascularization in the rat. Br J Ophthalmol. 1998;821063- 1071
PubMed
Van der Zypen  EFankhauser  FRaess  KEngland  C Morphologic findings in the rabbit retina following irradiation withthe free-running neodymium-YAG laser: disruption of Bruch's membrane and itseffect on the scarring process in the retina and choroid. Arch Ophthalmol. 1986;1041070- 1077
PubMed
Perry  DDRisco  JM Choroidal microvascular repair after argon laser photocoagulation. Am J Ophthalmol. 1982;93787- 793
PubMed
Macular Photocoagulation Study Group, Subfoveal neovascular lesions in age-related macular degeneration:guidelines for evaluation and treatment in the Macular Photocoagulation Study. Arch Ophthalmol. 1991;1091242- 1257
PubMed
Ohkuma  HRyan  SJ Experimental subretinal neovascularization in the monkey: permeabilityof new vessels. Arch Ophthalmol. 1983;1011102- 1110
PubMed
Miller  HMiller  BRyan  SJ Correlation of choroidal subretinal neovascularization with fluoresceinangiography. Am J Ophthalmol. 1985;99263- 271
PubMed
Gass  JDM Stereoscopic Atlas of Macular Diseases: Diagnosisand Treatment. 2nd St Louis, Mo CV Mosby Co1977;
Spilsbury  KGarrett  KLShen  WYConstable  IJRakoczy  PE Overexpression of vascular endothelial growth factor (VEGF) in theretinal pigment epithelium leads to the development of choroidal neovascularization. Am J Pathol. 2000;157135- 144
PubMed
McAllister  ILVijayasekaran  SYu  DYConstable  IJ Chorioretinal venous anastomoses: effect of different laser methodsand energy in human eyes without vein occlusion. Graefes Arch Clin Exp Ophthalmol. 1998;236174- 181
PubMed
Peyman  GALi  MYoneya  SGoldberg  MFRaichand  M Fundus photocoagulation with the argon and krypton lasers: a comparativestudy. Ophthalmic Surg. 1981;12481- 490
PubMed
Espinosa-Heidmann  DGSuner  IHernandez  EPFrazier  WDCsaky  KGCousins  SW Age as an independent risk factor for severity of experimental choroidalneovascularization. Invest Ophthalmol Vis Sci. 2002;431567- 1573
PubMed
Hanahan  DFolkman  Y Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86353- 364
PubMed
Pepper  M Manipulating angiogenesis: from basic science to the bedside. Arterioscler Thromb Vasc Biol. 1997;17605- 619
PubMed
Miller  JW Vascular endothelial growth factor and ocular neovascularization. Am J Pathol. 1997;15113- 23
PubMed
Wang  FRendahl  KGManning  WCQuiroz  DCoyne  MMiller  SS AAV-mediated expression of vascular endothelial growth factor induceschoroidal neovascularization in rat. Invest Ophthalmol Vis Sci. 2003;44781- 790
PubMed
Ishibashi  THata  YYoshikawa  HNakagawa  KSueishi  KInomata  H Expression of vascular endothelial growth factor in experimental choroidalneovascularization. Graefes Arch Clin Exp Ophthalmol. 1997;235159- 167
PubMed
Edelman  JLCastro  MR Quantitative image analysis of laser-induced choroidal neovascularizationin rat. Exp Eye Res. 2000;71523- 533
PubMed
Kvanta  AShen  WYSarman  SSeregard  SSteen  BRakoczy  E Matrix metalloproteinase (MMP) expression in experimental choroidalneovascularization. Curr Eye Res. 2000;21684- 690
PubMed
Renno  RZYoussri  AIMichaud  NGragoudas  ESMiller  JW Expression of pigment epithelium–derived factor in experimentalchoroidal neovascularization. Invest Ophthalmol Vis Sci. 2002;431574- 1580
PubMed
Bron  AJTripathi  RCTripathi  BJ Retina. Wolff's Anatomy of the Eye and Orbit 8th London, England Chapman & Hall1997;444- 488
Abdelsalam  ADel Priore  LZarbin  MA Drusen in age-related macular degeneration: pathogenesis, natural course,and laser photocoagulation–induced regression. Surv Ophthalmol. 1999;441- 29
PubMed
Lim  JI Iatrogenic choroidal neovascularization. Surv Ophthalmol. 1999;4495- 111
PubMed
Pollack  AHeriot  WJHenkind  P Cellular processes causing defects in Bruch's membrane following kryptonlaser photocoagulation. Ophthalmology. 1986;931113- 1119
PubMed
Miller  HMiller  BIshibashi  TRyan  SJ Pathogenesis of laser-induced choroidal subretinal neovascularization. Invest Ophthalmol Vis Sci. 1990;31899- 908
PubMed

Figures

Place holder to copy figure label and caption
Figure 1.

A, The mean ± SD maculardiameter–optic disc (OD) ratio was 2.5 ± 0.4 times the horizontaldiameter of the OD in the fundus. The short arrow points to a lesion locatednext to the parafoveal retina. The macula and OD were detected in serial sections.B, The relative size of the OD to the macula in histological analysis. GCLindicates ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclearlayer; RPE, retinal pigment epithelium; and CRV, central retinal vessels.Hematoxylin-eosin staining, original magnification ×38.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Predilection of the macular regionto a high incidence of choroidal neovascularization (CNV) on fluorescein angiographyafter laser photocoagulation by the krypton red laser (A and B), krypton green-yellowlaser (C), and Alcon frequency-doubled diode laser (D, E, and F). In A-D,none of the lesions outside the macular regions developed CNV-related fluoresceinleakage. In contrast, 2, 3, 7, and 6 lesions inside the macular regions showCNV-related fluorescein leakage in B, C, D, and F, in which 6, 3, 7, and 6lesions were placed, respectively. Two lesions outside the macula (black arrows)and 1 in the boundary (curved white arrow) developed CNV-related leakage (F).Early-phase (E, 43 seconds) and late-phase (F, 7 minutes) angiograms showfluorescein leakage from well-demarcated CNV at most lesions and from poorlydemarcated CNV at 1 lesion (white arrow in E and F). Plus signs indicate thecentral fovea; circled areas, the macular region.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Histological choroidal neovascularization(CNV) after intense laser photocoagulation treated by the krypton red laser.The figures on the right show high magnifications of the insets on the left.No CNV was detected in a lesion outside the macula in A and B, although Bdemonstrates that Bruch membrane was broken (between the 2 large arrows) andcellular infiltration (small arrows) was present. Arrows in D indicate well-formednew vessels in a CNV membrane inside the macula. GCL indicates ganglion celllayer; INL, inner nuclear layer; ONL, outer nuclear layer; and RPE, retinalpigment epithelium. Hematoxylin-eosin staining, original magnification ×94(A and C) and ×376 (B and D).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Histological choroidal neovascularization(CNV) after intense laser photocoagulation treated by the Alcon frequency-doubleddiode laser. The figures on the right show high magnifications of the insetson the left. Arrows in B indicate well-formed new vessels in a CNV membraneinside the macula. Arrows in D indicate new vessels in a CNV membrane outsidethe macula. In F, a new vessel (NV) was enveloped by several elongated fibroblast-likecells and pigment laden cells (white arrows). RPE indicates retinal pigmentepithelium. Hematoxylin-eosin staining, original magnification ×198(A and E), ×376 (B, D, and F), and ×94 (C).

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Development of Fluorescein Leakage After Intense Laser PhotocoagulationWith Krypton Red and Green-Yellow and Alcon Frequency-Doubled Diode (AFDD)Ophthalmic Lasers in the Macaca fascicularis Monkey
Table Graphic Jump LocationTable 2. Development of Microscopic Choroidal Neovascularization (CNV)After Intense Laser Photocoagulation With Krypton Red and Green-Yellow andAlcon Frequency-Doubled Diode (AFDD) Ophthalmic Lasers in the Macaca fascicularis Monkey

References

Dolan  BJ Choroidal neovascularization not associated with age-related maculardegeneration. Optom Clin. 1996;555- 76
PubMed
Green  WRWilson  DJ Choroidal neovascularization. Ophthalmology. 1986;9311691176;
PubMed
Shiraga  FTakasu  IShiragami  C Treatment options in subfoveal choroidal neovascularization secondaryto age-related macular degeneration. Semin Ophthalmol. 1998;1331- 39
PubMed
Submacular Surgery Trials Pilot Study Investigators, Submacular surgery trials randomized pilot trial of laser photocoagulationversus surgery for recurrent choroidal neovascularization secondary to age-relatedmacular degeneration, I: ophthalmic outcomes submacular surgery trials pilotstudy report number 1. Am J Ophthalmol. 2000;130387- 407
PubMed
Submacular Surgey Trials Pilot Study Investigators, Submacular surgery trials randomized pilot trial of laser photocoagulationversus surgery for recurrent choroidal neovascularization secondary to age-relatedmacular degeneration, II: quality of life outcomes submacular surgery trialspilot study report number 2. Am J Ophthalmol. 2000;130408- 418
PubMed
Arnold  JBauer  BBlumenkranz  MS  et al.  Guidelines for using verteporfin (Visudyne®) in photodynamic therapyto treat choroidal neovascularization due to age-related macular degenerationand other causes. Retina. 2002;226- 18
PubMed
Lai  CCWu  WCChen  SL  et al.  Suppression of choroidal neovascularization by adeno-associated virusvector expressing angiostatin. Invest Ophthalmol Vis Sci. 2001;422401- 2407
PubMed
Lai  YKYShen  WYBrankov  MLai  CMConstable  IJRakoczy  PE Potential long-term inhibition of ocular neovascularization by recombinantadeno-associated virus-mediated secretion gene therapy. Gene Ther. 2002;9804- 813
PubMed
Shen  WYGarrett  KLWang  CG  et al.  Preclinical evaluation of a phosphorothioate oligonucleotide in theretina of rhesus monkey. Lab Invest. 2002;82167- 182
PubMed
Ryan  SJ Subretinal neovascularization: natural history of an experimental model. Arch Ophthalmol. 1982;1001804- 1809
PubMed
Ryan  SJMittl  RNMaumenee  AE Enzymatic and mechanically induced subretinal neovascularization. Albrecht Von Graefes Arch Klin Exp Ophthalmol. 1980;21521- 27
Frank  RNDas  AWeber  ML A model of subretinal neovascularization in the pigmented rat. Curr Eye Res. 1989;8239- 247
PubMed
Kimura  HSakamoto  THinton  DR  et al.  A new model of subretinal neovascularization in the rabbit. Invest Ophthalmol Vis Sci. 1995;362110- 2119
PubMed
Dobi  ETPuliafito  CADestro  M A new model of experimental choroidal neovascularization in the rat. Arch Ophthalmol. 1989;107264- 269
PubMed
Shen  WYYu  MJTBarry  CJConstable  IJRakoczy  PE Expression of cell adhesion molecules and vascular endothelial growthfactor in experimental choroidal neovascularization in the rat. Br J Ophthalmol. 1998;821063- 1071
PubMed
Van der Zypen  EFankhauser  FRaess  KEngland  C Morphologic findings in the rabbit retina following irradiation withthe free-running neodymium-YAG laser: disruption of Bruch's membrane and itseffect on the scarring process in the retina and choroid. Arch Ophthalmol. 1986;1041070- 1077
PubMed
Perry  DDRisco  JM Choroidal microvascular repair after argon laser photocoagulation. Am J Ophthalmol. 1982;93787- 793
PubMed
Macular Photocoagulation Study Group, Subfoveal neovascular lesions in age-related macular degeneration:guidelines for evaluation and treatment in the Macular Photocoagulation Study. Arch Ophthalmol. 1991;1091242- 1257
PubMed
Ohkuma  HRyan  SJ Experimental subretinal neovascularization in the monkey: permeabilityof new vessels. Arch Ophthalmol. 1983;1011102- 1110
PubMed
Miller  HMiller  BRyan  SJ Correlation of choroidal subretinal neovascularization with fluoresceinangiography. Am J Ophthalmol. 1985;99263- 271
PubMed
Gass  JDM Stereoscopic Atlas of Macular Diseases: Diagnosisand Treatment. 2nd St Louis, Mo CV Mosby Co1977;
Spilsbury  KGarrett  KLShen  WYConstable  IJRakoczy  PE Overexpression of vascular endothelial growth factor (VEGF) in theretinal pigment epithelium leads to the development of choroidal neovascularization. Am J Pathol. 2000;157135- 144
PubMed
McAllister  ILVijayasekaran  SYu  DYConstable  IJ Chorioretinal venous anastomoses: effect of different laser methodsand energy in human eyes without vein occlusion. Graefes Arch Clin Exp Ophthalmol. 1998;236174- 181
PubMed
Peyman  GALi  MYoneya  SGoldberg  MFRaichand  M Fundus photocoagulation with the argon and krypton lasers: a comparativestudy. Ophthalmic Surg. 1981;12481- 490
PubMed
Espinosa-Heidmann  DGSuner  IHernandez  EPFrazier  WDCsaky  KGCousins  SW Age as an independent risk factor for severity of experimental choroidalneovascularization. Invest Ophthalmol Vis Sci. 2002;431567- 1573
PubMed
Hanahan  DFolkman  Y Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86353- 364
PubMed
Pepper  M Manipulating angiogenesis: from basic science to the bedside. Arterioscler Thromb Vasc Biol. 1997;17605- 619
PubMed
Miller  JW Vascular endothelial growth factor and ocular neovascularization. Am J Pathol. 1997;15113- 23
PubMed
Wang  FRendahl  KGManning  WCQuiroz  DCoyne  MMiller  SS AAV-mediated expression of vascular endothelial growth factor induceschoroidal neovascularization in rat. Invest Ophthalmol Vis Sci. 2003;44781- 790
PubMed
Ishibashi  THata  YYoshikawa  HNakagawa  KSueishi  KInomata  H Expression of vascular endothelial growth factor in experimental choroidalneovascularization. Graefes Arch Clin Exp Ophthalmol. 1997;235159- 167
PubMed
Edelman  JLCastro  MR Quantitative image analysis of laser-induced choroidal neovascularizationin rat. Exp Eye Res. 2000;71523- 533
PubMed
Kvanta  AShen  WYSarman  SSeregard  SSteen  BRakoczy  E Matrix metalloproteinase (MMP) expression in experimental choroidalneovascularization. Curr Eye Res. 2000;21684- 690
PubMed
Renno  RZYoussri  AIMichaud  NGragoudas  ESMiller  JW Expression of pigment epithelium–derived factor in experimentalchoroidal neovascularization. Invest Ophthalmol Vis Sci. 2002;431574- 1580
PubMed
Bron  AJTripathi  RCTripathi  BJ Retina. Wolff's Anatomy of the Eye and Orbit 8th London, England Chapman & Hall1997;444- 488
Abdelsalam  ADel Priore  LZarbin  MA Drusen in age-related macular degeneration: pathogenesis, natural course,and laser photocoagulation–induced regression. Surv Ophthalmol. 1999;441- 29
PubMed
Lim  JI Iatrogenic choroidal neovascularization. Surv Ophthalmol. 1999;4495- 111
PubMed
Pollack  AHeriot  WJHenkind  P Cellular processes causing defects in Bruch's membrane following kryptonlaser photocoagulation. Ophthalmology. 1986;931113- 1119
PubMed
Miller  HMiller  BIshibashi  TRyan  SJ Pathogenesis of laser-induced choroidal subretinal neovascularization. Invest Ophthalmol Vis Sci. 1990;31899- 908
PubMed

Correspondence

CME
Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.
NOTE:
Citing articles are presented as examples only. In non-demo SCM6 implementation, integration with CrossRef’s "Cited By" API will populate this tab (http://www.crossref.org/citedby.html).
Submit a Comment

Multimedia

Some tools below are only available to our subscribers or users with an online account.

Related Content

Customize your page view by dragging & repositioning the boxes below.

Articles Related By Topic
Related Topics