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q 2002 American Society for Photobiology 0031-8655/02 $5.0010.00

Photochemistry and Photobiology, 2002, 75(2): 149–158

Laser Targeted Photo-occlusion of Rat Choroidal NeovascularizationWithout Collateral Damage†¶

Hirokazu Nishiwaki1,2, Ran Zeimer*‡2, Morton F. Goldberg2, Salvatore A. D’Anna2, Stanley A. Vinores2

and Rhonda Grebe2

1Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Kyoto University, Japanand 2Johns Hopkins University School of Medicine, Wilmer Ophthalmological Institute, Baltimore, MD

Received 14 June 2001; accepted 14 November 2001

ABSTRACT

Laser targeted photo-occlusion (LTO) is a novel methodbeing developed to treat choroidal neovascular mem-branes (CNV) in age-related and other macular degen-erations. A photosensitive agent, encapsulated in heat-sensitive liposomes, is administered intravenously. A lowpower laser warms the targeted tissue and releases a bo-lus of photosensitizer. The photosensitizer is activated af-ter it clears from the normal choriocapillaris but notfrom the CNV. Forty-five experimental CNV were in-duced in seven rats. Five weeks after LTO, complete oc-clusion was observed by laser targeted angiography(LTA) in 76% of treated CNV, and partial occlusion wasfound in the remaining 24%. The tissues outside the CNVbut within the area treated by LTO showed no flow al-teration and no dye leakage. All untreated CNV werepatent on LTA at 5 weeks. Light microscopy and electronmicroscopy confirmed the results in treated and controllesions. Moreover, treated areas next to lesions showednormal photoreceptors, retinal pigment epithelium(RPE), Bruch’s membrane and choriocapillaris. Theseresults indicate that LTO may improve current photo-dynamic therapy by alleviating the need for repeatedtreatments and by avoiding the long-term risks associ-

†Presented in part at the 1999 meeting of the Association for Re-search in Ophthalmology, Fort Lauderdale, FL.

¶Posted on the web site on November 28, 2001.*To whom correspondence should be addressed at: Johns Hopkins

University School of Medicine, Wilmer Ophthalmological Insti-tute, Wilmer/Woods Bldg. 355, 600 N. Wolfe Street, Baltimore,MD 21287-9131, USA. Fax: 410-614-2895; e-mail: rzei-mer@jhmi.edu

‡Dr. Zeimer is entitled to sales royalties from PhotoVision Phar-maceuticals, which is developing products related to the researchdescribed in this paper. In addition, he serves as a consultant tothe company. The terms of this arrangement have been reviewedand approved by the Johns Hopkins University in accordance withits conflict of interest policies. None of the other authors has afinancial interest.

Abbreviations: AlPcS4, aluminum phthalocyanine tetrasulfonate;AMD, age-related macular degeneration; CCD, charge-coupleddetector; CF, carboxyfluorescein; CNV, choroidal neovasculiza-tion; LTA, laser targeted angiography; LTO, laser targeted photo-occlusion; PDT, photodynamic therapy; RPE, retinal pigment ep-ithelium.

ated with damage to the RPE and occlusion of normalchoriocapillaries.

INTRODUCTION

Choroidal neovascularization (CNV) accounts for the ma-jority of legally blind eyes with age-related macular degen-eration (AMD) (1). The efficacy of the current treatment forCNV, based on thermal photocoagulation, is very limited asit is applicable only to a minority of cases and the recurrencerate is high, and it causes massive and permanent damageto the adjacent neurosensory retina, retinal pigment epithe-lium (RPE), Bruch’s membranes and normal choroidal ves-sels. A therapy for occult and classic CNV that is effectiveand causes minimal collateral damage is needed.

Photodynamic therapy (PDT) is being investigated andutilized as a therapy that may be better than thermal pho-tocoagulation. It consists of intravenously injecting a pho-tosensitive agent and exposing it to the appropriate wave-length of light in order to generate free radicals and singletoxygen that supposedly cause damage, resulting in vaso-oc-clusion. PDT following systemic injection of a photosensi-tizer has been investigated in animals for the occlusion ofCNV (2–7). These studies have shown that occlusion can beachieved by chemical means, and the results have led toclinical trials. A TAP study (8) has indicated that at 24months following treatment, the visual acuity benefit of ver-teporfin therapy was clearly demonstrated when the area ofclassic CNV occupied 50% or more of the area of the lesion.Another report has shown that cessation of fluorescein leak-age from classic CNV could be achieved for at least 1 to 4weeks, and the rate and severity of ocular or systemic ad-verse events were not increased by multiple applications (9).However, the success was very modest, as cessation of fluo-rescein leakage from classic CNV for at least 1 to 4 weekswas achieved in only 6.5% of patients, and progression ofCNV beyond the area identified before retreatment was not-ed in 48 to 90% of the eyes (9). The limited success isbecause of the necessity of keeping both the photosensitizerand light doses low enough to avoid collateral damage insurrounding normal tissues. This damage is caused by theundesirable presence of the photosensitizer in the normalretinal and choroidal vasculatures, the RPE and the subreti-nal space commonly accompanying CNV and in the RPE.

150 Hirokazu Nishiwaki et al.

Figure 1. Schematic explanation of LTO principle. A: CNV under the RPE and all normal blood vessels are perfused with liposomes. B:A releasing (argon laser) beam warms tissues that absorb the light efficiently. C: A bolus of dye is released from the liposomes in vesselsthat have been warmed to 418C. D: The bolus clears quickly from the normal choroidal vessels because of their rapid flow. E: The bolusclears from the choriocapillaris and remains only in the CNV, because of its slow flow. F: A second beam (diode laser) activates thephotosensitizer and causes damage only in the CNV.

Upon activation, the photosensitizer damages the CNV, butit also affects the normal tissues in which it is present.

We have developed a method, laser targeted drug deliv-ery, to deliver drugs that can be applied to target a photo-sensitizer specifically to CNV. The method consists of en-capsulating a drug in heat-sensitive liposomes, injecting theliposomes intravenously and releasing their contents at thesite of choice by noninvasively warming up the targeted tis-sue momentarily with a low power laser pulse directedthrough the pupil (10). Laser targeted delivery has been suc-cessfully applied to deliver an angiographic dye locally spe-cifically within the retinal vasculature (11,12), the choroidalvasculature (13,14) and CNV(15), i.e. laser targeted angi-ography (LTA). By replacing the dye with a photosensitizer,one can target PDT to specific tissues. This new method,laser targeted photo-occlusion (LTO), has been used suc-cessfully experimentally to occlude normal choriocapillariswithout damaging the RPE and retina (16). The goal of thepresent study was to evaluate, with electron microscopy,light microscopy and LTA, whether LTO is capable of oc-cluding CNV in a rat model while sparing adjacent normaltissues.

MATERIALS AND METHODSPrinciple of LTO. The principle of the application of LTO to thetreatment of CNV is illustrated in Fig. 1. A low power laser beamirradiates the CNV and the surrounding tissues at a wavelengthstrongly absorbed by blood in the CNV and choriocapillaris as wellas by pigments in the RPE and choroid. The absorbed energy causesthe tissues to warm up. On the other hand, the flow of blood and

the presence of nonabsorbing tissues, such as the nonvascular por-tion of the retina, tend to cool the tissues. The RPE, choriocapillarisand CNV are warmed up most efficiently because they are strongabsorbers. As a consequence, these tissues are the first to reach thetemperature necessary to melt the liposomes (418C). This causes therelease of their content (Fig. 1). After 200 ms, the low power laserused for release is turned off, and the tissues cool with sufficientrapidity to stop further release of photosensitizer from the liposomes.We have shown that the bolus first clears from large choroidal ves-sels and then from the normal choriocapillaries, but remains longerin the CNV because of its slow blood flow (15). At this point, asecond source of light, with a wavelength appropriate for excitingthe photosensitizer, is used to irradiate the targeted area, therebycausing damage, primarily to the CNV. The short-duration bolusdelivery (200 ms) also ensures that only a minimal amount of pho-tosensitizer leaks into the surrounding tissues, such as the RPE,where it might be retained.

Aluminum phthalocyanine tetrasulfonate (AlPcS4) was used as aphotosensitizer because its wavelength for sensitization (675 nm)assures penetration through blood, it is water soluble and thus canbe encapsulated efficiently, it has one of the highest light absorptioncoefficients among the various photosensitizers, and it is clearedrapidly from the body, thereby minimizing skin photosensitivity. Li-posomes containing AlPcS4 were formed according to the procedureof Hope et al. (17).

In order to perform LTA, a second formulation of liposomes wasprepared for encapsulation of 6-carboxyfluorescein (CF) (MolecularProbes, Junction City, OR).

Instrumentation. As shown in Fig. 2, a fundus camera (Zeiss,Oberkochen, Germany) was modified to provide video angiogramsand to deliver one laser beam to release the content of the liposomesand a second one to activate the photosensitizer. The first laser wasalso used to illuminate the fundus. The output of the charge-coupleddetector (CCD) camera was fed into a video image enhancer (OpticalElectronics, Tucson, AZ) and recorded on magnetic tapes with a

Photochemistry and Photobiology, 2002, 75(2) 151

Figure 2. Schematic description of the instrumentation. A funduscamera (Zeiss) was modified to provide video angiograms—to de-liver the two laser beams used to release the content of the lipo-somes, initiate the photosensitizer activation, and use the first laserto illuminate the fundus. The video imaging was achieved by opti-cally coupling [3] a CCD (charge-coupled device) video camera(Texas Instruments, Dallas, TX) [1] to the fundus conjugate planenormally occupied by photographic film. A filter [2] placed in frontof the CCD blocked light other than fluorescence. An argon laser(Coherent Radiation) was used for the release of the liposomes’content. Its output, filtered to deliver only at 488 nm, was coupledinto a fiber-optic cable. This cable was attached to a fiber-opticsplitter that divided the light into two separate fiber-optic cables.One of these cables was coupled by a lens [7] to the conjugate imageplane of the fundus usually occupied by the observer’s eyepiece.The other fiber-optic cable was coupled by a lens [8] to the locationordinarily occupied by the flash lamp and was used to illuminatethe fundus and generate video angiograms. This resulted in a 208circle illuminated with 8 mW in the rat eye. A diode laser emittingat 675 nm (Spectra Diode) was used to photosensitize AlPcS4. Thelaser beam was focused on a third fiber by an optical coupler. Theoutput of the fiber was collimated by a lens [6] and diverted towardthe camera by a dichroic mirror reflecting in red [4], to irradiate a1200 mm circle with a power of 3 mW for a duration of 500 ms,yielding 0.13 J/cm2 for each release and activation.

high-frequency video recorder (Beta Cam; Sony, Tokyo, Japan). Lat-er, the tape was played back, and video sequences were digitizedwith a frame grabber for subsequent analysis (Epix, Northbrook, IL).

An argon laser (Coherent Radiation, Palo Alto, CA) was used forthe release of the liposomes’ content. Its output, filtered to deliveronly at 488 nm, was used to warm the fundus minimally and causethe release of the photosensitizer or of the fluorescein. The powerof the laser was increased gradually until the temperature in thetissue was sufficient to cause release from liposomes, observed as abright fluorescent bolus. Typically, this was achieved with a powerof 16 mW applied in a 600 mm spot for a duration of 200 ms. Adiode laser emitting at 675 nm (Spectra Diode Labs, San Jose, CA)was used to photosensitize AlPcS4. The delivery of the laser beamswas controlled by shutters that were activated by outputs of an in-terface board located in a personal computer. The activation of thedifferent shutters was synchronized by a dedicated program that usedLabView (National Instruments, Austin, TX) to generate a virtualinstrument.

Animals and procedures. Animals were used in accordance withthe ARVO Statement for the Use of Animals in Ophthalmic andVision Research, and the protocol was approved by The Johns Hop-kins University Animal Care and Use Committee. Seven Long-Evans black-hooded, pigmented rats (weighing 250 to 350 g) wereanesthetized, for all procedures, with 50 mg/kg ketamine and 10 mg/kg xylazine, administered intramuscularly. The pupils were dilatedwith tropicamide (1%), phenylephrine hydrochloride (10%) and at-ropine sulfate (1%). The animals were sacrificed 5 weeks after LTOwith an overdose of pentobarbital sodium solution.

Induction of experimental CNV. CNV was induced by kryptonlaser burns as described by Dobi et al. (18). The laser was set at aspot size of 100 mm and to an exposure duration of 0.1 s. The powerwas raised until a bubble was seen or until it reached 100 mW.Eight to 10 lesions were created in each eye between the majorretinal vessels.

Identification of CNV and assessment of dye clearance. LTA wasperformed from 14 to 21 days after the creation of the lesions. Weused LTA, not traditional fluorescein angiography, to detect CNV,because our previous studies showed that LTA is more sensitive thanfluorescein angiography in detecting occult CNV (15). The digitizedimages were analyzed with image processing software (National In-stitute of Health public domain software). The fluorescence decayin CNV and in the adjacent normal choriocapillaris was assessed bymeasuring the pixel density (256 steps) within a 15 pixel diametercircle (corresponding to 50 mm in the retina) as a function of time.The bolus was considered to have cleared when the fluorescenceintensity of the measured area decayed to one-tenth of its peak value.The comparison between the clearance time of the normal chorio-capillaris and the CNV allowed the determination, for each lesion,of the delay needed to insure substantial washout in the normalchoriocapillaries.

Procedure for LTO. CF liposome formulation was first injectedat the time of treatment at a CF dose of 4 mg/kg. Ten minutes afterinjection, LTA was performed to confirm the presence of CNV, setthe power necessary for release and estimate the clearance rate ofthe normal choriocapillaris. This was followed by a second liposomeformulation of AlPcS4 at a dose of 2.5 mg/kg. LTO was performedby activating the sensitizing radiation after the release of the pho-tosensitizer from the liposomes. The time delay between the releaseand the activation of the photosensitizer was determined experimen-tally in each area of CNV according to the clearance rate. The treat-ment consisted of a single sequence of 20 consecutive releases andactivations. Each activation lasted 0.5 s at a power density of 270mW/cm2, thus yielding a cumulative energy density of 2.7 J/cm2.

Forty-five areas of CNV were identified in seven rats. Thirty-fourwere selected for LTO treatment, and 11 (one or two per rat) similarareas of CNV were chosen for controls.

Assessment of treatment. The lesions were observed with LTA at35 days after treatment. In addition, red-free images of the treatedarea were obtained before and after treatment. The CNV was con-sidered completely occluded if there was no flow in the lesion andif no increased hyperfluorescence was observed after repeating LTAthree to five times. This ensured that leaky lesions that led to ac-cumulation of dye after multiple releases were not considered oc-cluded. Repeated LTA was very useful in distinguishing betweendye accumulation in CNV and scar tissue. Scar tissue often showedhyperfluorescence, but it did not increase as LTA was repeated. LTAwas also used to assess the flow around the lesion and determine ifit was affected by treatment.

Histologic examination. The eyes were enucleated and placed im-mediately in 2% paraformaldehyde and 2% glutaraldehyde in a 0.1M phosphate buffer, pH 7.4. Several small needle punctures weremade around the limbal area to allow fixative to enter the orbit. Theeyes remained in the fixative solution overnight. They were trans-ferred to a 0.1 M phosphate buffer, washed two times for 10 mineach and then dissected for electron microscopy. The eyes were cutinto trapezoid-shaped pieces to fit into 14 3 5 3 6 mm thick molds,and the locations of all treated areas within the tissue blocks wererecorded. The specimens were then postfixed in 1% OsO4 in 0.1 Mphosphate buffer, washed in 0.1 M phosphate buffer, dehydrated ina graded series of ethanols, stained en bloc with 1% uranyl acetatein absolute ethanol and embedded in LX112 resin (Ladd, Burlington,VT). Polymerization was carried out at 608C for 36 h. One-micronsections were cut on a Sorvall MT2 microtome and stained with 1%toluidine blue and 1% sodium borate in water. Each specimen wasstep sectioned in 50 mm increments until the thermal lesion used toinduce CNV was located. The semithin sections were examined andphotographed on a Zeiss Axioskop microscope and camera system.Photographs were taken in the lesion area and its immediate vicinity.The specimens were then trimmed around the lesion into a pyramidshape (;2.5 to 2.8 mm wide 3 2.5 to 3 mm long) for thin sectioning.Because the thermal lesions were generally 200 to 300 mm in di-ameter, they also included adjacent areas of normal tissue. Thin

152 Hirokazu Nishiwaki et al.

Figure 3. Laser-targeted angiography of a treated lesion. This lesion is treated lesion 8 (Table 1). Top: 2 weeks after laser photocoagulationto induce CNV. Next page: 5 weeks after LTO. The circle indicates the laser spot used to release the content of the liposomes and generatea bolus. The arrowheads point to normal choriocapillaris lobules that clear within 600 to 1200 ms. The CNV is very slow in clearing andremains highlighted at 1800 ms. The arrows point to anastomoses that carry the bolus from the choroid to retinal veins.

sections (70 to 90 nm thick) were cut on a Reichert-Jung Ultracutmicrotome using a 3 mm Diatome diamond knife. Step sections wereprepared for each lesion. Four to five grids were made from eacharea under investigation for electron microscopy. Ten, twenty or fiftymicrons were trimmed away and subsequent semithin and thin sec-tions were prepared. The sections were stained with an aqueoussolution of 2% uranyl acetate, poststained with lead citrate and ex-amined on a JOEL JEM 100CX II electron microscope operating atan accelerating voltage of 60 kV. Photographs were obtained in amosaic pattern in order to include the thermal lesion and the adjacentregions.

A pathologist, masked as to the identity of the specimens and themicrographs, identified and marked the boundaries of the laser-in-duced lesion. Within these boundaries, the pathologist marked (1)thrombosed vessels; (2) patent vessels containing red blood cells;and (3) empty vessels containing no cells. The pathologist also ex-amined the areas adjacent to the lesion to determine if damage waspresent. This could not be assessed within the region of the lesionbecause the laser burn used to induce CNV destroyed the tissue. Adifferent investigator tabulated the data.

RESULTSDelay between release and activation

The bolus released during LTA was washed out from thenormal choriocapillaris within 0.7 to 1.1 s (average 0.9 s).

This time was used to delay the diode-activation of the pho-tosensitizer after its argon-induced release from the lipo-somes.

Evaluation by LTA and red free images

At 5 weeks after treatment, complete vascular occlusion wasobserved by LTA in 26 (76%) of 34 lesions, and partialocclusion was found in the remaining 8 (24%) lesions. Inall the 11 control (untreated) lesions, CNV was perfused at5 weeks.

The areas adjacent to the lesion, but within the field ofdiode laser irradiation, were examined for RPE changes bycomparing red-free images and LTA before and at the endof the follow-up period. In 74% (n 5 25) of these areas, nochanges were noticeable. In the remaining 26% (n 5 9),slight discoloration of RPE was observed in the red-free im-ages. No window defect could be observed using LTA.

The same surrounding areas were examined for bloodflow changes on LTA. These areas showed no flow distur-bance, and no dye was found to leak and accumulate after

Photochemistry and Photobiology, 2002, 75(2) 153

Figure 3. Continued.

LTA bolus delivery. Figure 3 illustrates LTA findings in anarea of CNV treated successfully by LTO. LTA before treat-ment shows three patterns of dye clearance after the releaseof the bolus. In the area adjacent to the lesion, the bolushighlights lobular patterns (indicated by the arrowheads) thatfill and clear within 600 to 1200 ms. In contrast, an irregularpattern surrounds the center of the lesion. The fluorescencein this pattern decays much slower and is still present 1800ms after bolus delivery. The third pattern, indicated by thearrows, consists of a laminar flow of dye that progresses,from the center of the lesion, along a small chorio-retinalanastomosis and into a large retinal vessel with flow towardthe disk. LTA at the end of follow-up shows normal lobularpatterns around the lesion and an irregular pattern at the siteof the lesion. However, in contrast to the pretreatment situ-ation, both patterns decay at the same rate, leaving no fluo-rescence at 1800 ms. The pattern of linear flow is still pre-sent after treatment. Figure 3 indicates that there may be anadditional small vessel draining into the large retinal vessel.

At 5 weeks, all the 11 lesions that were not treated byLTO were found to be patent by LTA. Figure 4 illustratesthe fluorescent patterns in an untreated CNV. LTA delineatesan irregular pattern at the lesion before and after treatment.

This pattern evolves slowly and is still visible after 1200 ms.Two linear flow patterns are clearly visible at baseline andare present but faint at follow-up.

Histological examination

Untreated CNV. Table 1 summarizes the results. Of the fouruntreated lesions, three showed more than one lumen openby light microscopy and electron microscopy, and no occlu-sions. The remaining lesion showed open vessels by electronmicroscopy, which were accompanied by spontaneously oc-cluded ones.

Figure 5, obtained from untreated lesion 2 in Table 1,illustrates the findings. Three control electron micrographs,obtained from two different areas, indicate the presence ofblood vessels (marked by black arrows) in scar tissue locatedabove (i.e. internal to) Bruch’s membrane (empty arrow).The vessels are seen clearly within the scar. The vesselspossess a clear lumen within which intact blood cells arepresent.

Treated CNV. In treated CNV (Table 1) six of the eightlesions showed no CNV by light microscopy. Lesion 1 hadsome occluded vessels in the area of CNV, but also more

154 Hirokazu Nishiwaki et al.

Figure 4. Laser targeted angiography of an untreated lesion. This lesion is untreated lesion 2 (Table 1). Top: 2 weeks after laser photoco-agulation to induce CNV. Next page: 5 weeks later. The circle indicates the laser spot used to release the content of the liposomes andgenerate a bolus. The arrowhead points to normal choriocapillaris lobule that clears earlier (600 to 1200 ms) than the CNV, which remainshighlighted after 1800 ms. The arrow points to anastomoses that carry the bolus from the choroid to retinal veins.

than one open lumen in this area. Lesion 7 showed numerousopen vessels. Six of the eight lesions showed no CNV oroccluded CNV by electron microscopy. The combination oflight microscopy and electron microscopy indicates that fivelesions were totally occluded, two were partially occludedand one was not occluded (lesion 7).

In Fig. 6, obtained from treated lesion 8 in Table 1, thecomposite illustrates that the findings are not limited to arestricted area. The figure is provided to give a sense of thenature of the occlusive effect of LTO. The scar, which orig-inally contained CNV, according to LTA (Fig. 3), is delin-eated by the arrowheads. In this entire region, there is nodiscernable vascular lumen or residue thereof.

Area adjacent to treated CNV. All the histological sec-tions obtained at the periphery of the lesion and examinedby electron microscopy showed normal photoreceptors, nor-mal RPE, intact Bruch’s membrane and perfused, normal-looking choriocapillaris. Figure 7, taken from two regions oftreated lesion 8 demonstrates normal-looking photorecep-tors, RPE with well-defined organelles (black arrowhead),

intact Bruch’s membrane (empty arrow) and healthy-look-ing, perfused choriocapillaris (filled arrow).

DISCUSSION

The results of a recent clinical trial of PDT (9) indicate thatit can provide a better treatment than standard thermal laserphotocoagulation in particular circumstances. Beneficial out-comes with respect to visual acuity and contrast sensitivitynoted at the month 12 examination were sustained to themonth 24 examination (8). Although the results are encour-aging, they indicate that the treatment success is limited.Leakage activity reappeared 4 to 12 weeks after retreatment,but it seemed to be reduced after multiple treatments (9).For patients with follow-up after the last retreatment, 48 and90% of the eyes with classic CNV progressed beyond theinitial area after infusion of 6 mg/m2 verteporfin and irra-diation of 100 J/cm2 and 50 to 100 J/cm2, respectively (9).

Some limitations of the present PDT can be explainedfrom studies in monkeys with the same photosensitizer. A

Photochemistry and Photobiology, 2002, 75(2) 155

Figure 4. Continued.

dose of 0.375 mg/kg, followed 20 to 50 min later by irra-diation of 150 J/cm2 led, at best, to the envelopment of theCNV by proliferative RPE cells (19). Lesions considered bythe authors to have been adequately treated showed lack ofleakage, by fluorescein angiography, at 4 weeks. However,as pointed out by the authors, the lesion contained open ves-sels, illustrated histologically (fig. 4 of Husain et al. (19)).In other words, lack of fluorescein leakage does not ensurethat the CNV is not perfused. It is likely that fluoresceinleakage is not apparent when the CNV is newly envelopedby RPE cells. The example shown by the investigators toillustrate the success of their treatment would be considered,in our study, to be a failure, because more than one openvessel is present in the lesion. The regimen adopted by theinvestigators could not be intensified because the dose nec-essary to occlude CNV, when applied to the normal retinacaused damage: occasional pyknosis in the inner retina, milddisarray of photoreceptors, abnormal vessel walls in the cho-riocapillaris and pigment-laden cells in the subretinal space,with phagocytic activity (19). Therefore, to prevent damage,a lower energy density of 50 to 100 J/cm2 was adopted forthe clinical trials (9). However, this chosen dose is one-thirdto two-thirds of a dose that does not cause occlusion in an-

imals. The fact that the dose of the photosensitizer or thelight or both needs to be limited to avoid collateral damageis primarily because of the lack of targeting specifically toCNV. The current challenge of PDT is thus to improve thetargeting, specifically to increase the ratio between the con-centration in the CNV and the adjacent normal tissues. LTOis capable of increasing this ratio, and thus the aim of thepresent work was to assess its efficacy by in vivo observa-tions followed by histologic evaluation.

A bolus of dye released locally by LTA in the choroidwas shown to perfuse the CNV and the choriocapillaris andto clear more rapidly from the normal choriocapillaris thanfrom the CNV (15). Because of this difference in rate ofclearance, only the CNV contains dye after a short time de-lay. This is illustrated in our previous work (15) and in Figs.3 and 4 of this article, in which only the abnormal net ofvessels remains highlighted after 1800 ms. LTA was alsoperformed by releasing the bolus in choroidal arteries up-stream from the lesion (between the optic disk and the le-sion). The abnormal pattern of vessels was observed again,thus demonstrating that these vessels are fed by the choroidalcirculation. These observations led us to conclude that an

156 Hirokazu Nishiwaki et al.

Table 1. Light microscopy and electron microscopy of treated andcontrol CNV. The X indicates the presence of the feature in thecolumn. The shaded areas indicate vessel closure or lack of vesselfor the tested lesions and vessel patency for the untreated lesions

Figure 5. Electron microscopy of anuntreated lesion. This lesion is un-treated lesion 2 (Table 1 and Fig. 4),which was prepared for histologyshortly after LTA shown in Fig. 4b.Black arrows indicate patent vesselswith red blood cells. They are locatedabove (internal to) Bruch’s mem-brane (indicated by the white arrow)and within the scar generated by laserphotocoagulation to induce CNV.

irregular pattern of vessels with slow flow is characteristicof CNV.

Our model of CNV was created by an intense burn thatpartially destroys the retina and leads to its atrophy in andabove the scar. This process has been used by others tocreate anastomoses between the choroidal and retinal vas-culatures. The presence of anastomoses in our model is il-lustrated clearly by the flow pattern delineated by LTA. Asshown in Figs. 3 and 4, some choroidal vessels filled anddrained rapidly into the retinal circulation. These vesselstended to have a linear pattern different from the lacy patternof CNV. Because of the rapid clearance of dye of such anas-tomoses, they were not damaged by LTO and remained pre-sent throughout the follow-up period, as illustrated in Figs.3 and 4.

We chose to assess the treatment by LTA and histologyat 5 weeks after treatment. This relatively long-term follow-up was chosen for a better comparison with conventionalPDT, which shows recurrence after 4 weeks. Any improved

treatment needs to be capable of reducing this long-termrecurrence, and a better performance within days after treat-ment would not necessarily be a sign that the new therapyis advantageous. On the other hand, our long-term follow-up was limited to 5 weeks, because the literature indicatedthat the CNV model is transient and limited to such a period.Moreover, this was the time period chosen by (19) in theirexperimental evaluation of PDT.

LTA showed that at 5 weeks, 76% of the treated CNVwere completely occluded, and the remaining 24% were par-tially occluded. In contrast, all the control untreated CNVwere found patent by LTA. This is a major improvementover the results of conventional PDT, which, as mentionedabove, typically leads to temporary lack of leakage, whichrecurs within a similar follow-up period. The effect of PDTwith verteporfin showed that, at 4 weeks, patent CNV ves-sels remain in lesions considered successfully treated (seefig. 4 of Husain et al. (19)).

The fact that LTA revealed patent CNV at 5 weeks in thecontrol lesions is not in contradiction with the observationby others that, on conventional angiography, leakage grad-ually disappears. LTA is unique in its capability to assessperfusion regardless of leakage, whereas conventional an-giography provides information on leakage rather than onpatency. This unique capability of LTA has been demon-strated in comparison with fluorescein angiography (15) andis further validated by the histology of control lesions dis-cussed subsequently.

Electron microscopy confirmed the LTA assessment ofpatent CNV in four out of five untreated lesions and partiallyconfirmed it in the fifth lesion. Electron microscopy alsoindicated that LTA correctly identifies the closure of vesselsin a majority of instances: six of eight lesions deemed closedby LTA either had, on examination by electron microscopy,no visible CNV, or the CNV was damaged and occluded byultrastructural criteria. Moreover, as seen in Fig. 6, LTO cancause such severe damage to the blood vessels that theydisappear. From the degree of disruption of the lumen, alsoseen in other treated lesions, it seems that the damage islikely to be irreversible. Finally, electron microscopy con-firmed the LTA assessment that choriocapillaries in areasadjacent to the CNV and exposed to excitation during LTOremain patent.

Although there was good agreement between LTA and

Photochemistry and Photobiology, 2002, 75(2) 157

Figure 6. Electron microscopy of a treated lesion. This lesion istreated lesion 8 (Table 1 and Fig. 3), which was prepared for his-tology shortly after LTA shown in Fig. 3. A composite was createdto show most of the scar seen in Fig. 3 by LTA to harbor CNVprior to treatment. Vessels or remains of vessels are absent in thescar indicated by the arrowheads.

Figure 7. Electron microscopy of a region immediately adjacent toa treated and occluded CNV. These sections were taken from treatedlesion 8 (Table 1 and Fig. 3) at a location less than 50 mm from thescar. This region was within the area treated by LTO. Note theintegrity of the RPE including the nucleus (arrowheads) and Bruch’smembrane (white arrow) and the well-perfused choriocapillaris(black arrow).

electron microscopy, the comparison was complicated by thepresence of choroido-retinal anastomoses. Because, as men-tioned above, LTO does not occlude such anastomoses, his-tological sections used to assess LTO inevitably includedsome open vessels, whether they were treated or not. Weattempted to eliminate this confounding factor by requiringthe presence of more than one open vessel per lesion toclassify it as containing perfused CNV. This cutoff is some-what arbitrary because more than one anastomosis couldhave been present in the section.

The results illustrated in Fig. 3 showed that LTO can oc-clude CNV even when it is covered by RPE cells and scartissue. This is very encouraging because it indicates thatLTO might be efficient in treating occult CNV, which is alsolocated under the RPE and surrounded by deposits and scartissue.

Electron microscopy confirmed the preservation of adja-cent tissues that are essential to long-term normalcy of visualfunction. The RPE was found normal in treated areas im-mediately adjacent to the thermal injury used to create theCNV. The preservation of the RPE may be particularly im-portant in AMD, because it is already abnormal and likelyto be more sensitive to additional insult. The intact natureof Bruch’s membrane after LTO is also important in AMD,because breaks are believed to be associated with prolifer-ation of new vessels into the subretinal space, possibly lead-ing to recurrence. The posttreatment integrity of the chorio-capillaris, illustrated by electron microscopy, is an importantstep toward enhancing the efficacy of PDT. It is possiblethat the closure of the choriocapillaris by conventional PDT,even if temporary, plays a role in the recurrence of CNVbecause ischemia may induce new vessel proliferation,thereby adding to preexisting proliferative signals in AMD.

There does not seem to be any inherent obstacle towardthe clinical application of LTA and LTO. As far as toxicityis concerned, liposomes have already been injected intrave-nously in clinical practice to carry a variety of drugs (20).They are used in clinical PDT for AMD (9), and pilot studiesof our formulation have demonstrated safety in animals (21).

The clinical procedures required for LTO would not bemarkedly different from those currently used for PDT. Oncethe CNV is identified through conventional angiography or,preferably, by LTA, the photosensitizer–liposome formula-tion will be infused and LTO will be performed. If experi-ence shows that clearance of the normal choriocapillarisvaries greatly among individuals, the treatment will be pre-ceded by a 2 s LTA sequence from which an automaticalgorithm will determine the delay to be used during theLTO sequence.

In summary, this study indicates that LTO has the advan-tages of PDT while avoiding or minimizing many of its cur-rent limitations. LTO may prove to be a more efficient andsafer PDT that may permit the treatment of both occult andclassic CNV (subfoveal, juxtafoveal or extrafoveal) and mayopen the way for early intervention before significant visionloss occurs.

Acknowledgements Supported by Research Grants EY 07768, EY10017 and Core Grant EY 1765 from the National Institutes ofHealth, Bethesda, MD, the Lew R. Wasserman Merit Award

158 Hirokazu Nishiwaki et al.

(S.A.V), an Alcon Research Institute Award (R.Z), gifts from Wil-liam H. Young and Mr. and Mrs. Harold W. McGraw Jr. (R.Z.), andan unrestricted research grant from Research to Prevent Blindness,Inc., New York (M.F.G.).

REFERENCES

1. Ferris, F. L., S. L. Fine and L. A. Hyman (1984) Age-relatedmacular degeneration and blindness due to neovascular macu-lopathy. Arch. Ophthalmol. 102, 1640–1642.

2. Miller, H. and B. Miller (1993) Photodynamic therapy of sub-retinal neovascularization in the monkey eye. Arch. Ophthalmol.111, 855–860.

3. Kliman, G. H., C. A. Puliafito, D. Stern, S. Borirakchanyavatand W. A. Gregory (1994) Phthalocyanine photodynamic ther-apy: new strategy for closure of choroidal neovascularization.Lasers Surg. Med. 15, 2–10.

4. Kliman, G. H., C. A. Puliafito, G. A. Grossman and W. A.Gregory (1994) Retinal and choroidal vessel closure usingphthalocyanine photodynamic therapy. Lasers Surg. Med. 15,11–18.

5. Miller, J. W., A. W. Walsh and M. Kramer (1995) Photodynam-ic therapy of experimental choroidal neovascularization usinglipoprotein-delivered benzoporphyrin. Arch. Ophthalmol. 113,810–818.

6. Schmidt-Erfurth, U., T. Hasan, E. Gragoudas, N. Michaud, T.J. Flotte and R. Birngruber (1994) Vascular targeting in pho-todynamic occlusion of subretinal vessels. Ophthalmology 101,1953–1961.

7. Reinke, M., C. Canakis, D. Husain, N. Michaud, T. J. Flotte,E. S. Gragoudas and J. W. Miller (2000) Verteporfin photo-dynamic therapy retreatment of normal retina and choroid inthe cynomolgus monkey. Ophthalmology 106, 1915–1923.

8. Treatment of Age-related Macular Degeneration with Photody-namic Therapy (TAP) Study Group (2000) Photodynamic ther-apy of subfoveal choroidal neovascularization in age-relatedmacular degeneration with verteporfin: one-year results of 2 ran-domized clinical trails—TAP report 1. Arch. Ophthalmol. 117,1329–1345.

9. Schmidt-Erfurth, U., J. W. Miller, M. Sickenberg, H. Laqua, I.Barbazetto, E. S. Gragoudas, L. Zografos, B. Piguet, C. J. Pour-naras, G. Donati, A. M. Lane, R. Birngruber, H. van den Berg,H. A. Strong, U. Manjuris, T. Gray, M. Fsadni, and N. M. Bres-sler (1999) Photodynamic therapy with verteporfin for choroidalneovascularization caused by age-related macular degeneration:

results of retreatments in a phase 1 and 2 study. Arch. Ophthal-mol. 117, 1177–1187.

10. Zeimer, R., B. Khoobehi, M. R. Niesman and R. L. Magin(1988) A potential method for local drug and dye delivery inthe ocular vasculature. IOVS (Investig. Ophthalmol. Vis. Sci.)29, 1179–1183.

11. Zeimer, R., T. Guran, M. Shahidi and M. T. Mori (1990) Vi-sualization of the retinal microvasculature by targeted dye de-livery. IOVS (Investig. Ophthalmol. Vis. Sci.) 31, 1459–1465.

12. Guran, T., R. Zeimer, M. Shahidi and M. Mori (1990) Quanti-tative analysis of retinal hemodynamics using targeted dye de-livery. IOVS (Investig. Ophthalmol. Vis. Sci.) 31, 2300–2306.

13. Kiryu, J., M. Shahidi, M. Mori, Y. Ogura, S. Asrani and R.Zeimer (1994) Noninvasive visualization of the choriocapillarisand its dynamic filling. IOVS (Investig. Ophthalmol. Vis. Sci.)35, 3724–3732.

14. Asrani, S., S. Zou, S. D’Anna, M. F. Goldberg and R. Zeimer(1996) Noninvasive visualization of the blood flow in the cho-riocapillaris of the normal rat. IOVS (Investig. Ophthalmol. Vis.Sci.) 37, 312–317.

15. Asrani, S., S. Zou, S. D’Anna, A. Phelan, M. F. Goldberg andR. Zeimer (1996) Selective visualization of choroidal neovas-cular membranes. IOVS (Investig. Ophthalmol. Vis. Sci.) 37,1642–1650.

16. Asrani, S., S. Zou, S. D’Anna, G. A. Lutty, S. A. Vinores, M. F.Goldberg and R. Zeimer (1997) Feasibility of laser-targetedphoto occlusion of the choriocapillary layer in rats. IOVS (In-vestig. Ophthalmol. Vis. Sci.) 38, 2702–2710.

17. Hope, M. J., M. B. Bally, G. Webb and P. R. Cullis (1985)Production of large unilamellar vesicles by a rapid extrusionprocedure. Characterization of size distribution, trapped volumeand ability to maintain a membrane potential. Biochim. Biophys.Acta 812, 55–65.

18. Dobi, E. T., C. A. Puliafito and M. Destro (1989) A new modelof experimental choroidal neovascularization in the rat. Arch.Ophthalmol. 107, 264–269.

19. Husain, D., M. Kramer, A. G. Kenny, N. F. T. J. Michaud, E.S. Gragoudas and J. W. Miller (1999) Effects of photodynamictherapy using verteporfin on experimental choroidal neovascu-larization and normal retina and choroid up to 7 weeks aftertreatment. IOVS (Investig. Ophthalmol. Vis. Sci.) 40, 2322–2331.

20. Lasic, D. D. and D. Papahadjopoulos (1995) Liposomes revis-ited. Science 267, 1275–1276.

21. Asrani, S., S. D’Anna, H. Alkan-Onyuksel, W. Wang, D. Good-man and R. Zeimer (1995) Systemic toxicology and laser safetyof laser targeted angiography with heat sensitive liposomes. J.Ocul. Pharmacol. Ther. 11, 575–584.