June 2003
Volume 44, Issue 6
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Retina  |   June 2003
Noninvasive Technique for Monitoring Chorioretinal Temperature during Transpupillary Thermotherapy, with a Thermosensitive Liposome
Author Affiliations
  • Shinji Miura
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.
  • Hirokazu Nishiwaki
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.
  • Yoshiaki Ieki
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.
  • Yuya Hirata
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.
  • Junichi Kiryu
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.
  • Yoshihito Honda
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.
Investigative Ophthalmology & Visual Science June 2003, Vol.44, 2716-2721. doi:10.1167/iovs.02-1210
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      Shinji Miura, Hirokazu Nishiwaki, Yoshiaki Ieki, Yuya Hirata, Junichi Kiryu, Yoshihito Honda; Noninvasive Technique for Monitoring Chorioretinal Temperature during Transpupillary Thermotherapy, with a Thermosensitive Liposome. Invest. Ophthalmol. Vis. Sci. 2003;44(6):2716-2721. doi: 10.1167/iovs.02-1210.

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

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Abstract

purpose. To develop a technique for noninvasive and real-time monitoring of chorioretinal temperature in transpupillary thermotherapy (TTT).

method. A modified slit lamp, which was equipped with two laser wavelengths (490 nm for illumination and fluorescein excitation and 810 nm for hyperthermia), was developed for TTT and temperature monitoring. Five types of liposomes were prepared, and their phase-transition temperatures were 40°C, 46°C, 47°C, 48°C, and 52°C, respectively. Carboxyfluorescein was encapsulated in each liposome. After intravenous injection of each liposome, TTT with the modified slit lamp was performed on normal rat choroid or tissue with choroidal neovascularization (CNV). During TTT, chorioretinal temperature was monitored by observing release of fluorescein from circulating liposomes.

results. Fluorescence from liposomes was initially observed around the heated lesion immediately after TTT began and disappeared rapidly when irradiation stopped. Choroidal and retinal temperatures were monitored separately. TTT for normal retina required higher power than that for normal choroid to observe fluorescence from a 40°C, 46°C, and 47°C liposome. Retinal whitening was observed after TTT at a high-power setting. TTT for CNV required higher laser power than that for the normal choroid and retina.

conclusions. The results demonstrate the potential use of a noninvasive monitoring technique of chorioretinal temperature during TTT. The method should be useful to establish the TTT setting and achieve the optimal temperature increase in CNV.

Transpupillary thermotherapy (TTT) has recently emerged as an advancement for treating occult choroidal neovascularization (CNV) in patients who have age-related macular degeneration. 1 Initially used for the treatment of choroidal melanoma, 2 TTT is a technique in which heat is delivered to the choroid and retinal pigment epithelium (RPE) through the pupil, with a modified laser diode (usually 810 nm) delivered from the ophthalmic slit lamp. Reichel et al. 3 reported that 94% of CNV showed clinical or angiographic improvement, and vision was stabilized or improved in 75%. Miller-Rivero et al. (Miller-Rivero NE, Kaplan HJ, ARVO Abstract 932, 2000) reported that 26 of 30 eyes treated with TTT demonstrated a decrease in exudation after TTT. These earlier reports have suggested that TTT can result in closure of subfoveal CNV, with relative sparing of the overlying neurosensory retina, compared with conventional laser photocoagulation therapy. 
TTT for CNV is a subthreshold photocoagulation procedure. 4 5 A thin, white slit beam is focused in the center of the treated lesion to view any retinal changes during TTT. If retinal whitening is observed, treatment should be stopped, because the goal of treatment is to observe no retinal change in color. In contrast to conventional laser photocoagulation, in which an estimated retinal temperature increase of 42°C occurs, 6 7 the estimated retinal temperature elevation with TTT at standard clinical settings (800 mW, 60 seconds, 3.0-mm spot size) is calculated at approximately 10°C. 2 However, it is difficult to estimate the increase in chorioretinal temperature during TTT. An ophthalmoscopic end point is invisible, or just barely visible, at treatment, because clinical instrumentation for noninvasive monitoring of choroidal temperature has not been used. The thermal dosimetry techniques microthermocouple 8 and magnetic resonance imaging (MRI) 9 10 have been reported; however, at present, it appears difficult to apply these techniques for chorioretinal temperature monitoring, because the former is invasive and the latter requires specially adapted, and initially costly, devices. 
Thermosensitive liposomes encapsulating carboxyfluorescein (CF-liposomes) are vesicles of lipid bilayers. They have little fluorescein intensity in the bloodstream, because CF is entrapped into them at a high concentration. However, once CF-liposomes are warmed above their crystalline phase transition temperature (Tc), 11 which is based on the liposome’s composition, they release their contents. The release leads to dequenching of CF, and high fluorescein intensity is observed. Asrani et al., 12 13 Kiryu et al., 14 and Zeimer et al. 15 visualized choriocapillary blood flow selectively with use of a fundus camera and CF-liposome (Tc = 41°C). The authors observed CF dye front released from liposomes heated with a weak laser pulse. Temperature monitoring technique with CF-liposomes was reported by Morden et al. 16 The authors injected CF-liposome (Tc = 55°C) and measured fluorescence emission of rat liver during and after heating. They reported that fluorescence intensities correlated with the surface temperature of liver recorded with a thermographic camera. Desmettre et al. 17 18 applied this technique in ophthalmology. They evaluated retinal laser burns of rabbit fundus after intravenous injections of CF-liposomes (Tc = 55°C). 18 19 The authors also speculated about the retinal temperature increase from fluorescein intensity of laser burns. The results in these two reports correlated, in that that only one type of CF-liposome was used, with a 55°C Tc. 
We conducted this study for three purposes. First, we formulated five types of CF-liposomes that quickly release encapsulated CF at various temperatures. Second, we developed a new clinical instrument to monitor noninvasively the chorioretinal temperature during TTT. The modified slit lamp administered TTT, and we simultaneously observed fluorescein released from heated CF-liposomes. Finally, with the use of the temperature monitoring system, we compared temperature elevation during TTT in the normal choroid and retina and in that affected by CNV. 
Methods
Slit Lamp Modification for TTT
We modified a commercial slit lamp (model SL-10L; Topcon, Tokyo, Japan) for TTT (Fig. 1A) . The illumination of the slit lamp was switched to an argon laser at 490 nm (Novus 2000; Coherent, Palo Alto, CA), which made it possible to perform the treatment while visualizing and diagnosing the lesion with fluorescein angiography (FAG). In addition, a laser diode at 810 nm (F-System; Coherent) was installed as the aiming beam and to perform TTT. The zoom system allowed the spot diameter of an 810-nm laser to be continuously variable (50–500 μm). Irradiation time and power were adjusted with an attached controller. The laser spot was moved with a manipulator. Images taken by a charge-coupled device (CCD) camera (Sony, Tokyo, Japan) mounted on the slit lamp were displayed on a monitor after being amplified by a video enhancer (Seprotec, Tokyo, Japan). The images then were recorded on an S-VHS videotape (Sony). 
Measurement of Actual Laser Power
The actual laser power of the illumination laser at 490 nm and the hyperthermic laser at 810 nm were measured. The laser power analyzer (Ultima Labmaster; Coherent) was set up just in front of the rat cornea. 
CF-Liposome Preparation
As described by Ralston et al., 19 20 5,6-carboxyfluorescein (Molecular Probes, Junction City, OR), with an excitation peak of 490 nm, was purified and diluted to approximately 100 mM. Dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylglycerophosphocholine (DSPC) (all from Genzyme, Liestal, Switzerland), and myristoylpalmitoylphosphatidylcholine (MPPC; Avanti, Alabaster, AL) were used without further purification. Liposomes were prepared by using a method previously described. 15 21 Briefly, lipids (dissolved in chloroform and methanol) were dried to a thin film by rotary evaporation under vacuum. A 100-mM solution of CF was mixed with the dried lipid film, and the mixture was subjected to five freeze–thaw cycles. This process was followed by extrusion sizing in a thermobarrel extruder (Lipex Biomembranes, Vancouver, British Columbia, Canada) through a stack of two 25-mm, 0.2-μm polycarbonate membranes (Millipore, Bedford, MA) 10 times to yield large unilamellar vesicles. Free CF was removed through a resin column (Sephadex G-50; Pharmacia Biotech, Uppsala, Sweden). 
Five types of liposomes were prepared as follows: (1) 40°C liposome (Tc = 40°C), DSPC, DPPC, DPPG, and MPPC at 0:16:3:1 (M/M); (2) 46°C liposome (Tc = 46°C), DSPC, DPPC, DPPG, MPPC at 47:43:10:20 (M/M); (3) 47°C liposome (Tc = 47°C), DSPC, DPPC, DPPG, MPPC at 47:43:10:10 (M/M); (4) 48°C liposome (Tc = 48°C), DSPC, DPPC, DPPG, MPPC at 47:43:10:3 (M/M); and (5) 52°C liposome (Tc = 52°C), DSPC, DPPC, DPPG, MPPC at 16:2:2:1 (M/M). 
Release Yield Versus Suspension Temperature In Vitro
The amount of CF release was assayed by measuring fluorescence with a spectrofluorophotometer (Shimadzu, Kyoto, Japan) at 490-nm excitation and 514-nm emission. A liposome suspension of 30 μL was mixed with 3 mL of 50% human serum. A 0.1 mL of Triton X-100 (Sigma, St. Louis, MO), which disrupted the vesicles and released the entrapped CF, was added to the control samples at room temperature. The samples were heated in the water bath at different temperatures for 1 minute. The percentage of CF release was calculated by comparing the fluorescent values of heated samples with total release of CF obtained after the addition of Triton X-100 to the control sample. 
Animals and Anesthesia
Male Long-Evans (LE) rats, weighing 180 to 200 g each, were used in the study. In 13 rats, TTT was performed on normal choroid and in 6 on CNV. The animals were treated in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. They were anesthetized with intramuscular ketamine (10 mg/kg) and xylazine (4 mg/kg). Topical 1% tropicamide and 2.5% phenylephrine hydrochloride were instilled for mydriasis during laser photocoagulation and TTT. 
Induction of CNV
Dye laser irradiation (545 nm; argon dye laser model 920; Coherent) was delivered through a slit lamp (Carl Zeiss, Oberkochen, Germany) with a handheld 90-D lens (Nikon, Tokyo, Japan). A contact lens was used to retain corneal clarity throughout photocoagulation. 22 23 Laser spots were placed separately, five burns in each eye, using a setting of 100 μm in diameter, for 0.1-second duration, at 150-mW intensity. CNV was evaluated on day 14 by ophthalmoscopy, fundus photography, and conventional FAG. 
Principle of Monitoring Temperature Increase during TTT: Liposomal Temperature-Monitoring
The TTT setting selected for the present study was as follows: spot size, 0.5 mm; exposure time, 60 seconds; and power, 0 to 40 mW. TTT was delivered with the modified slit lamp through a fixed 78-D lens (Volk Optical, Mentor, OH). At the beginning of the experiment (2 minutes before TTT), liposome suspension (1.0 mL/kg) was injected into the tail vein. Immediately before TTT, the fundus image was observed, with the illuminating argon laser recorded as background fluorescence. A weak aiming beam at 810 nm was focused on the target site. Next, we started TTT with the 810-nm laser in normal choroid or laser-induced CNV. The absorbed energy of hyperthermic laser caused the tissues to warm up. When tissue temperatures reached Tc of injected liposomes, liposomes in the vasculature released their contents. Released CF was excited with the illuminating argon laser at 490 nm, and fluorescent patterns were observed on the television monitor and recorded (Fig. 1B) . When the fluorescent bolus during TTT demonstrated lobular and honeycomb patterns that Asrani et al. 12 13 had reported as choriocapillary vascular patterns, the choroidal temperature in the heated lesion was considered to be above Tc. When the bolus observed during TTT was the same pattern as the retinal capillary images in conventional FAG, the retinal temperature in the heated lesion was considered to be above Tc. After TTT on the normal fundus, two kinds of images were recorded. The first were black-and-white images recorded 1 minute after TTT to observe retinal thermal change. The others were conventional FAG images, which were obtained 5 minutes after TTT with the same system, to obtain retinal vascular images. A contact lens was used to maintain corneal clarity throughout treatment. 
Evaluation of the Minimum Power Setting for Temperature Increase in Normal Choroid and Retina and CNV
Laser power settings required to observe release of fluorescence from liposomes were compared statistically. All data are expressed as the mean ± SEM. One-way ANOVA was used to analyze the data from 40°C, 46°C, 47°C, and 48°C liposomes, with post hoc comparisons tested by the Scheffé F test. Unpaired t-test was used to compare the TTT power setting for choroid and retina after administration of the 52°C liposome. Differences were considered statistically significant at P < 0.05. 
Results
Actual Laser Power of Each Wavelength
Actual power density of the illumination laser at 490 nm was 2.9 mW/cm2 at the corneal surface. Under the recently published National Standard for Safe Use of Lasers, 24 both a retinal power density of 240 mW/cm2 and irradiation duration of 60 seconds are permissible when the eye is immobilized and the pupil is dilated. The measured intensity of the hyperthermic laser at 810 nm was equal to that indicated by the controller. 
Release Yield Versus Suspension Temperature In Vitro
The release yield was measured at different temperatures in each liposome, and the results were summarized (Fig. 2) as temperature profiles. The Tc of each liposome in vitro was at the theoretical value. The CF release rate sharply increased at each Tc. 
TTT on the Normal Choroid
Figure 3 shows the fluorescence when TTT was delivered to the normal choroid. After injection of the CF-liposome suspension, no fluorescence was observed in the choroid or retina, but retinal major vessels were slightly delineated with the illuminating argon laser (Fig. 3A) . The heated lesion was well outlined as a bright white circle during TTT (Fig. 3B) . CF dye began to spread around the heated lesion and reached a plateau immediately after the start of TTT. The video image (Fig. 3C) clearly indicated that the CF release was localized external to the neurosensory retina. That fluorescence pattern was consistent with the honeycomb reported to be the choriocapillary network. When the power of TTT was increased, both the retinal vascular and choriocapillary patterns were observed. For example, in one case, after administration of the 40°C liposome, choriocapillary fluorescence was observed with TTT at no less than 10 mW. When the power of TTT was 13 mW or more, both the lobular fluorescence pattern and retinal capillary pattern were observed (Fig. 3D) . These results indicate that 10-mW TTT produced a choroidal temperature increase above 40°C, and that a power of 13 mW was necessary to warm the retina above 40°C in this eye. When TTT was performed at a low power, no retinal color change and no abnormal fluorescence of FA were observed after TTT. In contrast, at a high-power setting, retinal whitening appeared on the overlying retina after TTT. The whitening of retina demonstrated hyperfluorescence with FAG (Fig. 3E)
TTT on the CNV
After the injection of CF liposomes, the location of CNV was determined with the illuminating argon laser before TTT, because slight fluorescent leakage was observed around CNVs (Fig. 4A) . At the start of TTT, a bright white circle that exhibited the heated lesion covered the center of the CNV lesion. Patchy fluorescence began to spread around the CNV and reached a plateau immediately after start of TTT (Fig. 4B) . It disappeared immediately after TTT ended. When fluorescence from CNV was observed, the fluorescence of vessel anastomosis was also identified. No leakage was observed with 52°C liposome at any TTT power setting. 
Minimum Power Setting for Temperature Increase in Choroid and Retina and CNVs
The minimum TTT power necessary to observe fluorescent dye release from each liposome is shown in Table 1 . To observe fluorescence from the 40°C, 46°C, or 47°C liposome, TTT for retina required a higher-power setting than that for normal choroid (40°C, P = 0.0314; 46°C, P = 0.0373; 47°C, P = 0.0497). However, the minimum power setting necessary to observe release of fluorescence from 48°C or 52°C liposome was not different between normal choroid and retina. To observe fluorescence from 40°C, 46°C, 47°C, or 48°C liposome during TTT of CNV required higher power than that for choroid (P < 0.0001) or for retina (P < 0.0001). The number of irradiated lesions on which retinal whitening was observed after TTT is also shown in Table 1
Discussion
TTT was initially reported as a hyperthermic technique for treating choroidal melanoma. Laser irradiation at 810 nm, which was minimally absorbed by ocular media, 25 was delivered to RPE and tumor melanin. The absorbed laser power produced tissue necrosis within the tumor by causing a temperature increase to approximately 65°C. The goal of therapy was to achieve a grayish-white color change in the tumor at the end of TTT. 2 Recently, TTT was applied to the treatment of CNV, as a subthreshold therapy to spare the overlying neurosensory retina. The temperature increase in TTT for CNV was calculated to be approximately 10°C, 7 which was lower than that for melanoma. In contrast to TTT for melanoma, TTT for CNV required no or barely visible color change in the treated lesion to prevent thermal coagulation from occurring in the overlying retina. However, despite the need to avoid overheating treated tissue, no clinical instrumentation for noninvasive monitoring of chorioretinal temperature has been developed. 
In the present study, we reported a technique to monitor chorioretinal temperature during TTT, the liposomal temperature-monitoring (LTM) technique. LTM consisted of applying TTT on the fundus after intravenous injections of CF-liposomes with various levels of Tc. The background fluorescein intensity before and after TTT was low, because the circulating liposomes were invisible in the angiograms due to self-quenching. However, once TTT was started, the excitation argon laser visualized brightly the bolus of CF released at the heated lesion. Temperature increase during TTT was monitored by observing the dye front from CF-liposomes circulating in the heated tissues. LTM had several advantages:
  1.  
    It demonstrated the potential to monitor small temperature increases with increased TTT power settings. We formulated five types of CF-liposomes with various Tc. Theoretically, the Tc of CF-liposome is determined with the molar ratio of phospholipids. 26 We mixed three kinds of phospholipids—DPPC (Tc = 41°C), DPPG (Tc = 40°C), and DSPC (Tc = 55°C)—to change the Tc of CF-liposomes. As the content by percentage of DSPC increased, Tc of CF-liposome increased. The most convenient and precise way to determine the Tc of the liposome was to synthesize the various phospholipids with a Tc that was the target temperature, but it was technically difficult to prepare various kinds of phospholipids with different Tc. The method used in this study made it easily possible to produce CF-liposome with Tc at arbitrary temperatures from 40°C to 55°C. Furthermore, CF-liposomes demonstrated significant release at each Tc, leading to a sharp dye front, because MPPC was contained in the lipid formulation. 27 MPPC is an acyl chain-matched lysolipid. One acyl chain defect in the MPPC introduced a slightly increased fluidity to the lipid bilayer, and MPPC containing liposomes showed slight reduction of Tc and much quicker release rate at Tc.
  2.  
    The choroidal and retinal temperatures were monitored separately, because the imaging system had high resolution; therefore, we could distinguish dye front in the choriocapillaris and retinal capillaries. The former demonstrated honeycomb lobular patterns 12 13 and the latter branch patterns similar to those obtained by conventional FA. Even when both were observed, the dye front in the retinal vasculature did not reduce the contrast of the choriocapillary dye front.
  3.  
    It is a noninvasive technique. To monitor the increase in intraocular temperature, the microthermocouple was used. Cain and Welch 8 inserted the probe into the sclera and measured laser-induced temperature increases in the rabbit eye. However, it appears difficult to use the thermocouple during TTT because it is too invasive. Other potential noninvasive approaches have included magnetic resonance imaging (MRI) and fundus reflectometry, but none has been developed. 4 Although intravenous injection of the liposome suspension was necessary, neither the phospholipids nor the CF was toxic. 14 We should to study further the potential toxicity of liposomal drugs which resolve around organs such as liver, lungs, and bone marrow.
  4.  
    The chorioretinal temperature monitoring and TTT were performed with one slit lamp, and no further instrument was used. We modified a slit lamp for TTT and LTM, and both the base model of the slit lamp and the installed lasers (810 and 490 nm) are commercially available devices.
LTM revealed some features of temperature increase in normal choroid and retina. First, the threshold retinal temperature with TTT was approximately 46°C. As we showed in the results, when TTT was performed on normal choroid, the power setting necessary to observe CF release from 46°C liposomes in the retinal vasculature could produce burns on the overlying retina. Second, retinal temperature was lower than that of the choroid when TTT was performed in a subthreshold manner (40–46°C). As shown in Table 1 , a higher power setting was needed to warm the retina than to warm the choroid. Finally, the temperature increase during TTT depended on the power used, not on the duration of heating. The chorioretinal temperature increased to a certain degree immediately after the start of TTT and that temperature can be maintained regardless of the irradiation time. Fluorescent dye front from CF-liposomes was observed and reached plateau immediately after the heating began. Little time lag existed between the choroidal temperature increase and CF release from liposomes (20–100 ms). 28 If the temperature had gradually risen along with the heating duration, CF release would have been observed only after a certain time passed. 
Our results were similar to those of previous reports of experimental models. Mainster et al. 7 29 developed theoretical models and computed the threshold temperature as 47°C. The results in the present study also showed that the threshold temperature in the retina was approximately 46°C to 47°C. It is possible that the Tc of liposomes shifted in the bloodstream. However, the accordance of fluorescence release with estimated temperature demonstrates that the shift in Tc, if any, may be negligible. Mainster et al. 7 29 also calculated the difference in absorption coefficient of retina and choroidal melanin. Because infrared wavelength laser irradiation was not absorbed in the retina, retinal heat conduction was lower than that in the choroid, which included melanin. Cain and Welch 8 reported that the retinal temperature increase occurred within 1 second and reached steady state after laser irradiation began. The consistency was an indicator of reliability of LTM. LTM was an indirect method of temperature monitoring. Because direct access to the posterior segment of the rat eye is difficult, we did not measure the retinal or choroidal temperature directly. However, these results suggest that LTM could be a potential method for accurate temperature monitoring. 
TTT for CNV required a higher power setting than that for the normal choroid. This finding suggested that it would be difficult to warm CNVs to the therapeutic temperature of 47°C. 4 In nonophthalmic models, blood flow in normal tissues considerably increases at temperatures commonly applied during hyperthermia. 30 31 In contrast, neovascular blood flow may decrease. Consequently, heat dissipation by blood flow in tumor tissues is slower than that in normal tissues during heating, and thus the temperature of the tumor increases to a higher level. CNV also has slow blood flow. 13 Nonetheless, the temperature in CNVs did not increase above that in surrounding tissues. It may have been that it was more difficult to warm CNV than normal choroid because of poor heat absorption in the RPE layer, which was the main heat absorber. It was assumed that 30% of irradiated 810-nm light was absorbed. 32 When we created high-intensity laser burns to induce neovascularization, RPE in the irradiated lesion was damaged and usually depigmented. This RPE damage would be applied to clinical CNVs, which often accompanied RPE atrophy or depigmentation from prior focal photocoagulation. 33 Another reason may be the cooling effect of choroidal blood flow. 34 35 The preserved choriocapillaris around or underlying experimental CNV would compensate for the poor heat dissipation, to slow blood flow in the CNV and maintain the temperature in the irradiated lesion. 
In conclusion, the present study indicated that LTM may have potential as a noninvasive monitoring technique of chorioretinal temperature increase during TTT. We monitored retinal and choroidal temperature separately in real time with LTM. In addition, LTM provided us with important information about thermal features of normal choroid and retina and experimental CNV. 
 
Figure 1.
 
Schematic diagram. (A) For LTM, a 490-nm argon laser (for illumination and dye excitation) and 810-nm diode laser (for aiming and TTT) were installed on a modified slit lamp. Fluorescent images of the fundus were enhanced and recorded. (B) Principle of LTM. The laser energy deposited in the tissues caused heating ( large arrow ). The CNV was heated to above the Tc of the liposome bearing the CF. The bolus of dye released from the heated tissue generated an angiogram and observed dye front.
Figure 1.
 
Schematic diagram. (A) For LTM, a 490-nm argon laser (for illumination and dye excitation) and 810-nm diode laser (for aiming and TTT) were installed on a modified slit lamp. Fluorescent images of the fundus were enhanced and recorded. (B) Principle of LTM. The laser energy deposited in the tissues caused heating ( large arrow ). The CNV was heated to above the Tc of the liposome bearing the CF. The bolus of dye released from the heated tissue generated an angiogram and observed dye front.
Figure 2.
 
Temperature profiles of the CF-liposome. The CF release rate significantly increased at the Tc of each liposome. (▪) 40°C liposome, (♦) 46°C liposome, (▴) 47°C liposome, (•) 48°C liposome, (□) 52°C liposome.
Figure 2.
 
Temperature profiles of the CF-liposome. The CF release rate significantly increased at the Tc of each liposome. (▪) 40°C liposome, (♦) 46°C liposome, (▴) 47°C liposome, (•) 48°C liposome, (□) 52°C liposome.
Figure 3.
 
TTT and LTM applied to the normal choroid. (A) Background fluorescence. Immediately after injection of CF-liposome, an illuminating argon laser slightly delineated major retinal vessels. However, no fluorescein leaked in the retinal vasculature. Arrowhead : aiming beam at 810 nm. (B) TTT at 10 mW after administration of 46°C liposome. Arrowhead : heated lesion. No release of fluorescent bolus was observed, because the temperature increase was inadequate in the choroid and retina. (C) TTT at 10 mW with 40°C liposome. Patchy fluorescein leaks were observed in the heated lesion (★), but no retinal vascular pattern was observed. (D) TTT at 15 mW with 40°C liposome. Both patchy choriocapillary fluorescence and retinal vascular fluorescence ( arrow ) were observed. (E) Retinal whitening and hyperfluorescence with conventional FA image were visible at the irradiated spot ( arrow ).
Figure 3.
 
TTT and LTM applied to the normal choroid. (A) Background fluorescence. Immediately after injection of CF-liposome, an illuminating argon laser slightly delineated major retinal vessels. However, no fluorescein leaked in the retinal vasculature. Arrowhead : aiming beam at 810 nm. (B) TTT at 10 mW after administration of 46°C liposome. Arrowhead : heated lesion. No release of fluorescent bolus was observed, because the temperature increase was inadequate in the choroid and retina. (C) TTT at 10 mW with 40°C liposome. Patchy fluorescein leaks were observed in the heated lesion (★), but no retinal vascular pattern was observed. (D) TTT at 15 mW with 40°C liposome. Both patchy choriocapillary fluorescence and retinal vascular fluorescence ( arrow ) were observed. (E) Retinal whitening and hyperfluorescence with conventional FA image were visible at the irradiated spot ( arrow ).
Figure 4.
 
TTT and LTM applied to laser-induced CNV. (A) Before TTT, an illuminating argon laser visualized the CNV lesion ( white arrow ), because of slight fluorescent leakage. (B) TTT at 15 mW after administration of 40°C liposome. After TTT began, patchy fluorescent leakage ( arrowheads ) from liposomes was observed around CNV. When CF was released from heated CNV, fluorescence was also observed in the chorioretinal anastomosis ( arrow ).
Figure 4.
 
TTT and LTM applied to laser-induced CNV. (A) Before TTT, an illuminating argon laser visualized the CNV lesion ( white arrow ), because of slight fluorescent leakage. (B) TTT at 15 mW after administration of 40°C liposome. After TTT began, patchy fluorescent leakage ( arrowheads ) from liposomes was observed around CNV. When CF was released from heated CNV, fluorescence was also observed in the chorioretinal anastomosis ( arrow ).
Table 1.
 
Minimum TTT Power Setting for the Normal Choroid, Retina, and Experimental CNVs, to Observe CF Release from Liposomes
Table 1.
 
Minimum TTT Power Setting for the Normal Choroid, Retina, and Experimental CNVs, to Observe CF Release from Liposomes
TTT Power Setting (mW) n Retinal Whitening
40°C-Liposome
 Normal choroid 10.1 ± 0.6 7 0/7
 Normal retina 13.5 ± 0.3* 8 0/8
 CNV 20.8 ± 1.5, † 6
46°C-Liposome
 Normal choroid 12.5 ± 0.6 8 0/8
 Normal retina 16.3 ± 0.8* 8 4/8
 CNV 31.0 ± 1.9, † 5
47°C-Liposome
 Normal choroid 16.4 ± 0.9 7 4/7
 Normal retina 21.4 ± 0.9* 7 7/7
 CNV 34.2 ± 2.4 6
48°C-Liposome
 Normal choroid 21.7 ± 1.1 6 6/6
 Normal retina 21.4 ± 1.4 7 7/7
 CNV 39.0 ± 1.0, † 5
52°C-Liposome
 Normal choroid 38.3 ± 1.1 6 6/6
 Normal retina 37.8 ± 1.5 7 7/7
 CNV No fluorescence observed 5
Ip, M, Kroll, A, Reichel, E. (1999) Transpupillary thermotherapy Semin Ophthalmol 14,11-18 [CrossRef] [PubMed]
Robertson, DM, Buettner, H, Bennett, SR. (1999) Transpupillary thermotherapy as primary treatment for small choroidal melanomas Arch Ophthalmol 117,1512-1519 [CrossRef] [PubMed]
Reichel, E, Berrocal, AM, Ip, M, et al (1999) Transpupillary thermotherapy of occult subfoveal choroidal neovascularization in patients with age-related macular degeneration Ophthalmology 106,1908-1914 [CrossRef] [PubMed]
Mainster, MA, Reichel, E. (2000) Transpupillary thermotherapy for age-related macular degeneration: long-pulse photocoagulation, apoptosis, and heat shock proteins Ophthalmic Surg Lasers 31,359-373 [PubMed]
Rogers, AH, Reichel, E. (2001) Transpupillary thermotherapy of subfoveal occult choroidal neovascularization Curr Opin Ophthalmol 12,212-215 [CrossRef] [PubMed]
Mainster, MA. (1999) Decreasing retinal photocoagulation damage: principles and techniques Semin Ophthalmol 14,200-209 [CrossRef] [PubMed]
Mainster, MA, Ham, WT, Jr, Delori, FC. (1983) Potential retinal hazards: instrument and environmental light sources Ophthalmology 90,927-932 [CrossRef] [PubMed]
Cain, CP, Welch, AJ. (1974) Measured and predicted laser-induced temperature increases in the rabbit fundus Invest Ophthalmol 13,60-70 [PubMed]
Stollberger, R, Ascher, PW, Huber, D, Renhart, W, Radner, H, Ebner, F. (1998) Temperature monitoring of interstitial thermal tissue coagulation using MR phase images J Magn Reson Imaging 8,188-196 [CrossRef] [PubMed]
Vogl, TJ, Weinhold, N, Mack, MG, et al (1998) Verifizierung der MR-Thermometrie mittels in vivo intralasionaler, fluoroptischer Temperaturmessung fur die laserinduzierte Thermotherapie von Lebermetastasen Rofo Fortschr Geb Rontgenstr Neuen Bildgeb Verfahr 169,182-188 [CrossRef] [PubMed]
Desmettre, T, Mordon, S, Soulie, S, Devoisselle, JM, Weisslinger, JM. (1996) Liposomes in ophthalmology: review of the literature [in French] J Fr Ophtalmol 19,716-731 [PubMed]
Asrani, S, Zou, S, D’Anna, S, Goldberg, MF, Zeimer, R. (1996) Noninvasive visualization of blood flow in the choriocapillaris of the rat Invest Ophthalmol Vis Sci 37,312-317 [PubMed]
Asrani, S, Zou, S, D’Anna, S, Phelan, A, Goldberg, M, Zeimer, R. (1996) Selective visualization of choroidal neovascular membranes Invest Ophthalmol Vis Sci 37,1642-1650 [PubMed]
Kiryu, J, Shahidi, M, Mori, MT, Ogura, Y, Asrani, S, Zeimer, R. (1994) Noninvasive visualization of the choriocapillaris and its dynamic filling Invest Ophthalmol Vis Sci 35,3724-3731 [PubMed]
Zeimer, RC, Khoobehi, B, Niesman, MR, Magin, RL. (1988) A potential method for local drug and dye delivery in the ocular vasculature Invest Ophthalmol Vis Sci 29,1179-1183 [PubMed]
Mordon, S, Desmettre, T, Devoisselle, JM. (1995) Laser-induced release of liposome-encapsulated dye to monitor tissue temperature: a preliminary in vivo study Lasers Surg Med 16,246-252 [CrossRef] [PubMed]
Desmettre, T, Devoisselle, JM, Soulie-Begu, S, Mordon, S. (1998) Diode laser-induced retinal thermal damage control with a liposome-dye system [in French] J Fr Ophtalmol 21,714-722 [PubMed]
Desmettre, TJ, Soulie-Begu, S, Devoisselle, JM, Mordon, SR. (1999) Diode laser-induced thermal damage evaluation on the retina with a liposome dye system Lasers Surg Med 24,61-68 [CrossRef] [PubMed]
Ralston, E, Blumenthal, R, Weinstein, JN, Sharrow, SO, Henkart, P. (1980) Lysophosphatidylcholine in liposomal membranes: enhanced permeability but little effect on transfer of a water-soluble fluorescent marker into human lymphocytes Biochim Biophys Acta 597,543-551 [CrossRef] [PubMed]
Ralston E, LM, Richard, D, John, N, Robert, B. (1981) Carboxyfluorescein as a probe for liposome-cell interactions effect of impurities, and purification of the dye Biochim Biophys Acta 649,1336-1337
Mayer, LD, Hope, MJ, Cullis, PR, Janoff, AS. (1985) Solute distributions and trapping efficiencies observed in freeze-thawed multilamellar vesicles Biochim Biophys Acta 817,193-196 [CrossRef] [PubMed]
Kamizuru, H, Kimura, H, Yasukawa, T, Tabata, Y, Honda, Y, Ogura, Y. () Monoclonal antibody-mediated drug targeting to choroidal neovascularization in the rat Invest Ophthalmol Vis Sci 42,2664-2672 [PubMed]
Dobi, ET, Puliafito, CA, Destro, M. (1989) A new model of experimental choroidal neovascularization in the rat Arch Ophthalmol 107,264-269 [CrossRef] [PubMed]
. Laser Institute of America. (1993) American National Standard for the Safe Use of Lasers American National Standards Institute New York.
Mainster, MA. (1986) Wavelength selection in macular photocoagulation: tissue optics, thermal effects, and laser systems Ophthalmology 93,952-058 [CrossRef] [PubMed]
Ogawa Y, TH. (1990) Targeting therapy with a drug using temperature-sensitive liposomes entrapped antitumor drug together with localized hyperthermia Jpn J Cancer Chemother 17,1127-1133
Needham, D, Dewhirst, MW. (2001) The development and testing of a new temperature-sensitive drug delivery system for the treatment of solid tumors Adv Drug Deliv Rev 53,285-305 [CrossRef] [PubMed]
Khoobehi, B, Char, CA, Peyman, GA. (1990) Assessment of laser-induced release of drugs from liposomes: an in vitro study Lasers Surg Med 10,60-65 [CrossRef] [PubMed]
Mainster, MA, White, TJ, Tips, JH, Wilson, PW. (1970) Retinal-temperature increases produced by intense light sources J Opt Soc Am 60,264-270 [CrossRef] [PubMed]
Song, CW. (1984) Effect of local hyperthermia on blood flow and microenvironment: a review Cancer Res 44,4721s-4730s [PubMed]
Bicher, HI, Hetzel, FW, Sandhu, TS, et al (1980) Effects of hyperthermia on normal and tumor microenvironment Radiology 137,523-530 [CrossRef] [PubMed]
Vogel A, BR. (1992) temperature profiles in human retina and choroid during laser coagulation with different wavelengths ranging from 514 to 810 nm Lasers Light Ophthalmol 5,9-16
Da-Pozzo, S. () Hot spots in age-related macular degeneration Ophthalmology 107,1212-1213
Parver, LM, Auker, C, Carpenter, DO. (1980) Choroidal blood flow as a heat dissipating mechanism in the macula Am J Ophthalmol 89,641-646 [CrossRef] [PubMed]
White, TJ, Mainster, MA, Tips, JH, Wilson, PW. (1970) Chorioretinal thermal behavior Bull Math Biophys 32,315-322 [CrossRef] [PubMed]
Figure 1.
 
Schematic diagram. (A) For LTM, a 490-nm argon laser (for illumination and dye excitation) and 810-nm diode laser (for aiming and TTT) were installed on a modified slit lamp. Fluorescent images of the fundus were enhanced and recorded. (B) Principle of LTM. The laser energy deposited in the tissues caused heating ( large arrow ). The CNV was heated to above the Tc of the liposome bearing the CF. The bolus of dye released from the heated tissue generated an angiogram and observed dye front.
Figure 1.
 
Schematic diagram. (A) For LTM, a 490-nm argon laser (for illumination and dye excitation) and 810-nm diode laser (for aiming and TTT) were installed on a modified slit lamp. Fluorescent images of the fundus were enhanced and recorded. (B) Principle of LTM. The laser energy deposited in the tissues caused heating ( large arrow ). The CNV was heated to above the Tc of the liposome bearing the CF. The bolus of dye released from the heated tissue generated an angiogram and observed dye front.
Figure 2.
 
Temperature profiles of the CF-liposome. The CF release rate significantly increased at the Tc of each liposome. (▪) 40°C liposome, (♦) 46°C liposome, (▴) 47°C liposome, (•) 48°C liposome, (□) 52°C liposome.
Figure 2.
 
Temperature profiles of the CF-liposome. The CF release rate significantly increased at the Tc of each liposome. (▪) 40°C liposome, (♦) 46°C liposome, (▴) 47°C liposome, (•) 48°C liposome, (□) 52°C liposome.
Figure 3.
 
TTT and LTM applied to the normal choroid. (A) Background fluorescence. Immediately after injection of CF-liposome, an illuminating argon laser slightly delineated major retinal vessels. However, no fluorescein leaked in the retinal vasculature. Arrowhead : aiming beam at 810 nm. (B) TTT at 10 mW after administration of 46°C liposome. Arrowhead : heated lesion. No release of fluorescent bolus was observed, because the temperature increase was inadequate in the choroid and retina. (C) TTT at 10 mW with 40°C liposome. Patchy fluorescein leaks were observed in the heated lesion (★), but no retinal vascular pattern was observed. (D) TTT at 15 mW with 40°C liposome. Both patchy choriocapillary fluorescence and retinal vascular fluorescence ( arrow ) were observed. (E) Retinal whitening and hyperfluorescence with conventional FA image were visible at the irradiated spot ( arrow ).
Figure 3.
 
TTT and LTM applied to the normal choroid. (A) Background fluorescence. Immediately after injection of CF-liposome, an illuminating argon laser slightly delineated major retinal vessels. However, no fluorescein leaked in the retinal vasculature. Arrowhead : aiming beam at 810 nm. (B) TTT at 10 mW after administration of 46°C liposome. Arrowhead : heated lesion. No release of fluorescent bolus was observed, because the temperature increase was inadequate in the choroid and retina. (C) TTT at 10 mW with 40°C liposome. Patchy fluorescein leaks were observed in the heated lesion (★), but no retinal vascular pattern was observed. (D) TTT at 15 mW with 40°C liposome. Both patchy choriocapillary fluorescence and retinal vascular fluorescence ( arrow ) were observed. (E) Retinal whitening and hyperfluorescence with conventional FA image were visible at the irradiated spot ( arrow ).
Figure 4.
 
TTT and LTM applied to laser-induced CNV. (A) Before TTT, an illuminating argon laser visualized the CNV lesion ( white arrow ), because of slight fluorescent leakage. (B) TTT at 15 mW after administration of 40°C liposome. After TTT began, patchy fluorescent leakage ( arrowheads ) from liposomes was observed around CNV. When CF was released from heated CNV, fluorescence was also observed in the chorioretinal anastomosis ( arrow ).
Figure 4.
 
TTT and LTM applied to laser-induced CNV. (A) Before TTT, an illuminating argon laser visualized the CNV lesion ( white arrow ), because of slight fluorescent leakage. (B) TTT at 15 mW after administration of 40°C liposome. After TTT began, patchy fluorescent leakage ( arrowheads ) from liposomes was observed around CNV. When CF was released from heated CNV, fluorescence was also observed in the chorioretinal anastomosis ( arrow ).
Table 1.
 
Minimum TTT Power Setting for the Normal Choroid, Retina, and Experimental CNVs, to Observe CF Release from Liposomes
Table 1.
 
Minimum TTT Power Setting for the Normal Choroid, Retina, and Experimental CNVs, to Observe CF Release from Liposomes
TTT Power Setting (mW) n Retinal Whitening
40°C-Liposome
 Normal choroid 10.1 ± 0.6 7 0/7
 Normal retina 13.5 ± 0.3* 8 0/8
 CNV 20.8 ± 1.5, † 6
46°C-Liposome
 Normal choroid 12.5 ± 0.6 8 0/8
 Normal retina 16.3 ± 0.8* 8 4/8
 CNV 31.0 ± 1.9, † 5
47°C-Liposome
 Normal choroid 16.4 ± 0.9 7 4/7
 Normal retina 21.4 ± 0.9* 7 7/7
 CNV 34.2 ± 2.4 6
48°C-Liposome
 Normal choroid 21.7 ± 1.1 6 6/6
 Normal retina 21.4 ± 1.4 7 7/7
 CNV 39.0 ± 1.0, † 5
52°C-Liposome
 Normal choroid 38.3 ± 1.1 6 6/6
 Normal retina 37.8 ± 1.5 7 7/7
 CNV No fluorescence observed 5
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