Histological Effect and Protein Expression in Subthreshold TranspupillaryThermotherapy in Rabbit Eyes

Objective  To investigate the histological effect of subthreshold transpupillarythermotherapy (TTT) on the retina.

Methods  We performed TTT in normal pigmented rabbit eyes using an 810-nm diodelaser with spot size of 1.2 mm, power of 50 mW, and varying durations of 15,30, or 60 seconds. Four weeks later, fluorescein angiography was performed,and the enucleated eyes were examined by means of electron microscopy andimmunohistochemical staining.

Results  Funduscopy immediately and at 4 weeks showed no discernable changesat TTT sites, and fluorescein angiography at 4 weeks showed no abnormalities.However, electron microscopy showed photoreceptor and retinal pigment epitheliumcell disruption, changes more prominent with longer durations of treatment.Immunohistochemical staining was positive for heat shock protein 60, heatshock protein 70, tumor necrosis factor α, and vascular cell adhesionmolecule 1 in the photoreceptors and retinal pigment epithelium at TTT sites.Untreated control eyes showed no staining.

Conclusions  Despite the absence of changes evident by funduscopy and fluoresceinangiography, TTT resulted in dose-dependent histological changes in photoreceptorsand retinal pigment epithelium. The induction of heat shock proteins, cytokines,and cell adhesion molecules may play a role in the tissue response to subthresholdTTT.

Clinical Relevance  Unrecognized damage to the retina and retinal pigment epithelium maycontribute to visual loss in eyes that undergo subthreshold TTT.

Subthreshold transpupillary thermotherapy (TTT) using the 810-nm diodelaser represents a method for delivering low-energy, large-spot-size hyperthermiato the retina and choroid. Recently, TTT has been used with some success toreduce exudation associated with subfoveal choroidal neovascularization (CNV)in age-related macular degeneration.^1,2 Ithas been estimated that the temperature elevation one can expect with subthresholdTTT is 4°C to 10°C.^3,4 However,the exact temperature elevation achieved with such therapy, the influenceof various clinical variables on treatment, and dose-dependent histopathologicalchanges that occur with subthreshold TTT are not known. Furthermore, althoughsome researchers have suggested that heat shock protein (HSP) and apoptosismay play a role in the tissue response to TTT,^4,5 themechanism of action of TTT in eyes with CNV remains largely unknown.

The purpose of this study was to examine the histological effect ofTTT in an experimental animal model. We performed subthreshold TTT in normalpigmented rabbit eyes and examined dose-dependent histopathological changesby means of light and electron microscopy. In addition, immunohistochemicalstaining was performed to investigate expression of HSP60, HSP70, tumor necrosisfactor α (TNF-α), and vascular cell adhesion molecule 1 (VCAM-1).

We used rabbits weighing approximately 2 kg with normal pigmented eyes.All animals were housed and experiments were conducted in accordance withthe Association for Research in Vision and Ophthalmology Statement on theUse of Animals in Ophthalmic and Vision Research. General anesthesia was inducedby intramuscular injection of 30 mg/kg ketamine hydrochloride and 6 mg/kgxylazine hydrochloride. Pupils were dilated with 0.5% tropicamide and 0.5%phenylephrine eye drops.

We performed TTT using an 810-nm diode laser (OcuLight SLx; IRIS MedicalInstruments, Inc, Mountain View, Calif) fitted with a TTT slitlamp adapter(SLALS; IRIS Medical Instruments Inc). In all laser applications, we useda Goldmann laser–coated contact lens (−67 diopters) with a magnificationfactor of approximately 1.247 in rabbits with 810-nm radiation calculatedusing values from Murphy and Howland⁶ (MartinA. Mainster, PhD, MD, e-mail communication, May 27, 2002). First, we investigatedthe power required to create a funduscopically visible laser burn (thresholdapplication). Using a spot size of 1.2 mm and a power setting of 75 mW orgreater, a 60-second TTT application produced whitening of the retina. However,at a power of 50 mW with the same spot size and duration, no funduscopicallyvisible change to the retina was observed (subthreshold application). We confirmedthis subthreshold power setting in several different animals. Because 50 mWwas the lowest power setting available on the laser system used, durationof treatment was changed to vary the total laser energy dose in the subthresholdapplications in this study.

Before TTT, 2 strong photocoagulative burns spaced slightly apart werecreated using the diode laser (size, 50 mm; power, 800 mW; duration, 0.2 seconds)in the posterior pole to serve as markers for use in subsequent histopathologicalprocessing. This was followed by 3 subthreshold applications (3 spots) ofTTT placed in a slightly overlapping fashion between the 2 marker burns. Lasersettings were as follows: diameter of 1.2 mm; power of 50 mW; and durationof 15, 30, or 60 seconds (with a fluence of 71, 143, or 286 J/cm²,respectively). Color fundus photographs were taken immediately and at 4 weeksafter treatment. Fluorescein angiography was performed at 4 weeks after treatmentusing a 1-mL injection of 10% fluorescein via the marginal ear vein. The animalswere then killed, and the eyes were immediately removed for histopathologicalprocessing.

Freshly enucleated eyes were fixed in 2.5% glutaraldehyde and 2% formaldehydefor 48 hours. Sections of the posterior pole were stained with toluidine blueand examined by means of light microscopy. Samples postfixed in 1% bufferedosmium tetroxide and stained with uranyl were examined by means of transmissionelectron microscopy (JEM-1010; JEOL, Ltd, Tokyo, Japan). For immunohistochemistry,sections were blocked and incubated with primary antibody against HSP60, HSP70,VCAM-1 (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif), or TNF-α(Techne Corp, Minneapolis, Minn). Sections were then washed, incubated withsecondary antibody using fluorescein isothiocyanate–conjugated anti–goatIgG (Vector Laboratories Inc, Burlingame, Calif), and viewed with a fluorescencemicroscope (Model BX50; Olympus Corp, Tokyo). Normal untreated eyes used forcontrol samples were prepared in a similar manner for light and electron microscopyand for immunohistochemical staining.

Ophthalmoscopically, no visible change to the retina was observed forall durations of treatment, immediately or at 4 weeks after TTT application(Figure 1A and B), with the exceptionof 1 eye with relatively heavy fundus pigmentation that underwent TTT fora duration of 60 seconds. Development of some pigmentary changes was observedby 4 weeks after TTT, and this eye was not included in the histopathologicalexamination results. Regardless, fluorescein angiography performed at 4 weeksshowed no hypofluorescence or hyperfluorescence in the area of TTT applicationin this eye or in any other eye (Figure 1C).

In comparison with control eyes (Figure2A), disruption of the normal configuration of photoreceptor outersegments was observed at 4 weeks after TTT application, even with the shortestduration of treatment of 15 seconds (Figure2B). This disruption was increasingly more pronounced at 30 and60 seconds of treatment (Figure 2Cand D), with observation of fewer cell nuclei and overall thinning of theouter nuclear layer. In addition, with progressive TTT duration, decreaseddensity of nuclei in the outer nuclear layer and greater pigmentation in theretinal pigment epithelium (RPE) were noted. No changes were observed in theinner layers of the retina or in the choroid for any duration of treatment.

At low magnification, overall thinning of the photoreceptor layer withdisruption of the outer and inner segments was observed, even at the shortestTTT duration of 15 seconds compared with controls (Figure 3A and B). With 60 seconds of treatment, the loss and disruptionof photoreceptors were more pronounced (Figure3C). Higher magnification showed vacuolization and distention ofphotoreceptor outer segments, with disruption of the lamellar structures ofphotoreceptors and apical microvilli of RPE cells at 15 (Figure 3D) and 30 seconds (not shown) of TTT duration. The normalbasal infoldings of RPE cells appeared to be preserved. No changes were observedin Bruch's membrane or the anterior choroid for any duration of treatment.

Results of tests for all antibodies were negative in all untreated controls(Figure 4A, D, F, and H). Stainingfor HSP60 was observed in the RPE and the inner segments of photoreceptorsin eyes that received 30 seconds of TTT (Figure 4B), with the addition of staining for HSP60 observed inthe photoreceptor outer segments at 60 seconds of treatment (Figure 4C). Staining for HSP70, TNF-α, and VCAM-1 was alsoobserved in the RPE and the photoreceptor outer and inner segments for 30and 60 seconds of TTT, with the staining appearing more pronounced for 60seconds of TTT (Figure 4E, G, andI; data for 30 seconds of TTT are not shown). No staining for HSP60, HSP70,TNF-α, or VCAM-1 was observed in the inner layers of the retina, theouter nuclear layer, or the choroid for any duration of treatment.

Despite reports that TTT leads to decreased exudation in patients withCNV in age-related macular degeneration,^1,2 themechanism of action of this therapy has yet to be delineated. Because littleor no color change or burn is observed in the retina immediately after thesubthreshold treatment involved in TTT, it is clear that the tissue effectsdiffer from those of conventional laser photocoagulation. In addition, itis presumed that the higher wavelength of the infrared laser used (810 nm)would result in deeper tissue penetration and, consequently, less damage tothe sensory retina when compared with photocoagulation using the argon orthe dye laser. Thus, it has been suggested that TTT may be an ideal treatmentfor lesions involving the center of the fovea.

The present study shows that, even in the absence of funduscopic orangiographic evidence of alterations to the fundus, the outer retina and RPEare affected histologically by subthreshold TTT. Dose-dependent disruptionof photoreceptor outer segments with loss of the outer nuclear layer was observedby means of light and electron microscopy. Furthermore, dose-dependent vacuolizationof photoreceptor outer segments and RPE cells and disruption of RPE apicalmicrovilli were also observed by means of electron microscopy. No adverseeffect on the choroid was noted at the subthreshold energy levels used. Becausenormal pigmented rabbit eyes were used in these experiments, the results obtainedcannot be directly extrapolated to the clinical setting in which TTT is usedto treat a CNV lesion under the retina. Such lesions are usually in associationwith subretinal fluid, and this may serve to insulate the retina to some degreeagainst heat absorption during TTT application. However, in eyes with CNVin which there is little associated subretinal fluid, our findings certainlyhighlight the need to consider decreasing power settings.

Previous histological studies of TTT have involved threshold settingsto photocoagulate choroidal tumors.^7-9 However,1 study examined subthreshold TTT to the normal macula in a human eye withchoroidal melanoma that subsequently underwent enucleation.¹⁰ Thatstudy reported abnormal cytopolasmic lipofuscin and melanofuscin granulesin RPE cells and disruption of photoreceptor outer segments, with no changesobserved in the choroid. Aside from the RPE granules, these findings are inagreement with what we observed in rabbit eyes.

Interpretation of our results must be tempered by differences in therefractive error and structure of rabbit vs human eyes. The irradiance (power/area)achieved using the same power, spot size, and contact lens will be lower inrabbit eyes than in human eyes.^6,11,12 Forexample, using the Goldmann lens and 810-nm radiation, the magnification factoris 1.247 in rabbits but only 1.08 in human eyes.¹³ Thisresults in an approximately 33% higher irradiance in human eyes than in rabbiteyes. Therefore, one would expect the tissue effects observed in rabbits tobe more pronounced in human eyes using identical laser settings.

The mechanism of action in the treatment of CNV using TTT is currentlyunclear. The expression of HSPs are known to be induced by heat and otherpathologic stresses,^14-17 andrecent studies have suggested that HSPs may play a major role in the effectof TTT.^4,5 Heat shock proteinsare believed to act as molecular chaperones, theoretically allowing cellsto adapt to unfavorable changes in their environment.^18-22 Heatshock proteins are also known to induce apoptosis, and therefore this mayalso contribute to the cellular alterations observed after TTT.^4,5,23 Weexamined the expression of HSP60 and HSP70 by immunohistochemistry after TTTin rabbit eyes and found both proteins to have a dose-dependent expressionin the RPE and outer retina. In contrast, Desmettre and colleagues⁵ reported the presence of HSP70 expression 24 hoursafter TTT in rabbits in choroidal cells, including capillary endothelial cells,but not in the retina. Expression of other proteins was not examined in thatstudy. The fact that we assessed protein expression at 4 weeks after TTT mayaccount for the difference in results between the study by Desmettre et aland our study. Expression of HSP has been shown to be induced within severalhours after hyperthermia and to revert to control levels within 48 hours.^14,15 Expression of HSP60 and HSP70 at4 weeks after TTT may therefore be the result of molecular interactions otherthan those induced by transient hyperthermia.

Indeed, HSPs have also been implicated in the down-regulation of inflammatorycytokines such as TNF-α, interleukin 1, and interleukin 6, and someof these same inflammatory cytokines have been shown to enhance HSP expressionin an apparent cross-regulatory function.^24-30 Inaddition, HSP60 and HSP70 have each been shown to induce expression of intracellularadhesion molecule 1 and VCAM-1,^31,32 andone might speculate that the long-term clinical effects of TTT are relatedto such adhesion molecule expression in the vessels of CNV lesions. In thepresent study, dose-dependent expression of TNF-α and VCAM-1 were alsoobserved by means of immunohistochemical staining in the RPE and photoreceptors4 weeks after TTT. Although these data suggest that the tissue effects ofTTT involve the late expression of HSPs, TNF-α, and VCAM-1 in normalrabbit eyes, further experiments are clearly required to determine how suchprotein expression might lead to decreased exudation in eyes with CNV.

Subthreshold TTT in normal pigmented rabbit eyes produced alterationsto the photoreceptor outer segments and RPE cells when assessed at 4 weeksafter treatment. These alterations were accompanied by expression of HSP60,HSP70, TNF-α, and VCAM-1 within the outer retina and RPE. All changeswere dose dependent and suggest that expression of HSPs, cytokines, and celladhesion molecules may contribute to a delayed tissue response to subthresholdTTT.

Correspondence: Annabelle A. Okada, MD, Department of Ophthalmology,Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611,Japan (aokada@po.iijnet.or.jp)

Submitted for publication October 22, 2002; final revision receivedJanuary 23, 2004; accepted March 26, 2004.

The authors thank Martin A. Mainster, PhD, MD, for his generous adviceregarding lens image magnification in the rabbit eye, and also Minoru Fukudaand Nobuko Takahashi for their technical assistance.