The first clinical application of the laser

Abstract

In 1960, the established method of photocoagulation was the xenon arc photocoagulator. The ruby laser, invented that year by Maiman, had very different characteristics: a single short pulse, deep red wavelength, and a highly collimated beam. An experimental ruby laser photocoagulator was built, tested on rabbits, and successfully used to photocoagulate the retina of a patient in 1961. This was the first clinical application of the laser.

After the invention of the ruby laser by Maiman in 1960,1 there was considerable interest in possible medical applications. In 1961, Zaret et al. reported on experimental laser photocoagulation of rabbits.2 Investigations of the pathological effects on skin were conducted by Goldman et al., and reported in 1963.3

At that time, photocoagulation of the retina using a xenon arc source was an established procedure, following the pioneering work of Meyer-Schwicke- rath.4 Treatment required a 1000-W xenon arc lamp, and an optical delivery system that was impressive in its design, size, and articulation. The patient was typically seated in a recliner chair, and the eyepiece of the delivery system was brought to the patient. Exposure times were approximately one second, and were frequently a source of discomfort.

The ruby laser also required a large power supply plus a powerful flash lamp. The laser output was about 1 msec in duration, so that the laser light itself could not be used for aiming. Furthermore, the energy output from the first ruby lasers was only about one quarter of the energy that was required by the xenon arc lamp to produce a photocoagulation on the human retina. However, the short duration of the laser output more than compensated for the low energy level, as was soon demonstrated in experiments on rabbits.

Aiming the laser beam required a different approach from that used in the xenon arc instrument, since the ruby laser emitted a single pulse, followed by a significant pause for recharging the bank of capacitors. Aiming the beam into the eye was accomplished by using a unique property of the laser: the light output emerges from a laser rod in a direction precisely perpendicular to the mirror end surface of the rod. Aiming was achieved by collimating white light from a point source so that the beam was perpendicular to the end surface of the laser. A portion of the collimated white light was directed into the eye using the mirror of an indirect ophthalmoscope. The laser output followed the same path through the pupil and to the retina.

Experiments on rabbits were carried out at the research laboratory of American Optical Co. by Charles J. Campbell, MD. They served to define the range of power output that would produce appropriate photocoagulations. When the instrument and the aiming system were ready for clinical application, the instrument, with its power supply and large bank of capacitors, was transported to the Harkness Eye Institute at Columbia University. Dr. Campbell treated the first patient on November 22nd, 1961. Other participants in the project were Charles J. Koester, Elias Snitzer, Stephen M. MacNeille, Vonda Curtis, and M. Catherine Rittler.

The New York Times and Wall Street Journal reported the event during the following weeks, and the first presentation was at a meeting of the Optical Society of America in March 1962.5 At the same meeting, Zaret et al. reported ruby laser photocoagulation experiments on rabbits.6

The first human subject had a diagnosis of advanced angiomatosis retinae that had destroyed virtually all vision in the affected eye. Xenon arc photoco-

Fig. 1. The patient’s fundus photograph taken immediately after the laser exposures. Two laser photocoagulations can be seen on or near blood vessels just above the center of the photograph.

agulation had been proposed to destroy the abnormal vessels, but because of the patient’s youth, that procedure would have required general anesthesia. The ruby laser held the promise of a more readily tolerated therapeutic approach, even by a young patient. Therefore, ruby laser photocoagulation was explored for the first human patient, even though the wavelength of the ruby laser (0.694 µm) was not optimum for absorption by the blood and blood vessels.

Figure 1 is the fundus photograph taken immediately after the laser exposure. An area of the retina was selected, away from the pathology, in order to determine the power required for retinal coagulations. Normal vessels were selected because they posed a lesser risk of fragility. The areas of pathology were then subjected to photocoagulation intensities, based on the calibration exposures.

Figure 2 is the same area as Figure 1, photographed six days after exposure, showing scars and pigmentation in the treated areas, with excellent localized coagulation. However, the blood vessels appear to be intact. Even though the treatment could be performed comfortably, it was concluded that the single pulse ruby laser exposures were not effective in destroying blood vessels. These and other early results were reported at the American Academy of Ophthalmology and Otolaryngology in 19637 and in the Archives of Ophthalmology in 1964.8

A water-cooled, rapidly pulsed (60 Hz) ruby laser photocoagulator was later developed by American Optical Co. Campbell and Rittler reported that the appearance of retinal lesions was virtually identical to lesions produced with an argon laser.9 An indirect ophthalmoscope was used for viewing the fundus and aiming the beam. Absorption of the laser light by blood or blood vessels was limited. Photophobia from the ruby laser light was not marked.

Fig. 2. The same area as in Figure 1, photographed six days after laser exposure.

Ross et al. developed a ruby laser pulsed at 80 Hz.10 This laser, effectively a continuous source, was combined with an adaptation of the Goldmann threemirror contact lens and provided a precise aiming system for photocoagulation.11

Thus, as the characteristics of the ruby laser were developed and improved for the purpose of photocoagulation, the aiming systems were adapted to provide optimum viewing of the fundus and aiming of the laser beam.

Conclusions

The first laser photocoagulator demonstrated the feasibility of the laser as a light source for clinical photocoagulation. The ruby photocoagulator also identified the limitations of a very short pulse, deep red wavelength light source. These limitations have since been removed by improved systems that employ ruby and other laser sources.

References

1.Maiman TH: Stimulated optical radiation in ruby. Nature 187:493-494, 1960

2.Zaret MM, Breinin GM, Schmidt H, Ripps H, Siegel IM: Ocular lesions produced by an optical maser (Laser). Science 134:1525-1526, 1961

3.Goldman L, Blaney DJ, Kindel DJ, Richfield D, Frank EK: Pathology of the effect of the laser beam on the skin. Nature 197:912-914, 1963

4.Meyer-Schwickerath G: Light coagulation. St Louis, MO: CV Mosby 1960

5.Koester CJ, Snitzer E, Campbell CJ, Rittler MC: Experimental laser retina coagulator. J Opt Soc Am 52:607, 1962

6.Zaret MM, Ripps H, Siegel IM, Breinin GM: Biomedical experimentation with optical masers. J Opt Soc Am 52:607, 1962

7.Campbell CJ, Rittler MC, Koester CJ: The optical maser as a retinal coagulator: an evaluation. Trans Am Acad Ophthalmol Otol 67:58-67, 1963

8.Noyori KS, Campbell CJ, Rittler MC, Koester CJ: The characteristics of experimental laser coagulations of the retina. Arch Ophthalmol 72:254-263, 1964

9.Campbell CJ, Rittler MC: Effects of lasers on the eye. New York Academy of Science: Laser Conference, February 1970, p 36. 1970

10.Röss D et al: Pumping new life into ruby lasers. Electronics 39:115-118, 1966

11.Fankhauser F, Lotmar W: Photocoagulation though the Goldmann contact glass. Ophthalmology 77:320-330, 1967

Introduction

Retinal photocoagulation has been performed for more than 30 years and its value in various macular diseases has been established. Typically, the power of the laser is adjusted to produce white or gray retinal lesion depending on the depth of the coagulation. Heat conduction from the irradiated retinal pigment epithelium (RPE) into the retina may lead to irreversible thermal denaturation of the outer and inner segments.1-3 However, the benefit of retinal laser treatment has traditionally been attributed to the destruction of retinal tissue. So, the threshold energy required to effect most treatments remains unknown. Heat conduction from the RPE into the retina in a typical laser lesion leads to irreversible thermal denaturation of the outer and inner seg- ments.1-3 The exact biological mechanism of how such damage produces a therapeutic effect is poorly understood. However, for a variety of retinal diseases, which are thought to be associated with a degradation of the RPE, it might be sufficient to selectively damage the malfunctioning RPE, but to spare the overlying photoreceptors in order to avoid scotoma, which is especially useful within the macula. If the damaged RPE is rejuvenated in the healing process, due to migration and proliferation of the adjoining RPE, minimal, destructive, selective RPE treatment might be optimal. Various aspects of this new selective RPE treatment will be discussed in the following paragraphs and will be evaluated with respect to conventional laser photocoagulation.

Healing response after conventional laser photocoagulation

The most common explanation for the beneficial effect of photocoagulation in diabetic retinopathy is the destruction of oxygen consuming photoreceptors.4 Another theory suggests that the beneficial effect results from the restoration of a new RPE barrier and the subsequent production of a variety of growth factors.5-8 While the exact interaction mechanisms of the different growth factors is not well understood, the vascular endothelium growth factor (VEGF) seems to play a major role in regulation of the neovascularization associated with ischemic retina.9,10

In the treatment of diabetic macular edema, the beneficial effect is thought to be mediated by restoration of a new RPE barrier.11 A similar effect can be postulated in the treatment of drusen. Drusen are located within Bruch’s membrane or beneath the RPE, and often disappear after photocoagulation of the surrounding tissues. The value of prophylactic treatment of drusen is now being studied by several investigators.

Based on the fact that drusen are located deep in the retina, there is no clear rationale routinely to include the neural retina in photocoagulation. For instance, in central serous retinopathy (CSR), the rationale of therapy is the photocoagulation and subsequent formation of a new RPE barrier, replacing the old diseased RPE cells. Destruction of the photoreceptors would only appear to be an unwanted side-effect of such a disease, and the same may be true in the treatment of macular edema.

Following laser photocoagulation, the targeted tissue undergoes a healing process which is inde-

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pendent of the laser used. Typically, the tissue in the laser target area will be replaced by proliferating glial tissue originating from the surrounding retina and choroid. In addition, RPE cells contribute a significant part to this healing process,15 and the outer blood retinal barrier is reformed after about seven days.16 In animal experiments, it has been shown that the RPE may respond in several ways after injury. Adjacent RPE cells may spread out and the defect will be filled by hypertrophy of the neighboring RPE cells.17,18 Another mechanism is cell division of the RPE cells. This has been shown after photocoagulation in rabbits,19 in monkeys after retinal detachment,19 and in rabbits after surgi- cally-induced RPE defects.20 Glaser et al.21 have shown that RPE cells produce inhibitors for neovascularization, suggesting that these cells may play a role in regulation of the neovascularization process. In addition, Boulton et al.22 found a significant change in growth factors in the vitreous after panretinal photocoagulation. Yoshimura et al.8 showed that photocoagulated RPE cells produce inhibitors of endothelial cells. The molecular and immunological characteristics of these inhibitors correlate with TGF-ß2. Regenerating RPE cells are known to produce more TGF-ß2 compared to normal RPE cells.23

Based on the experimental findings elucidated above, photocoagulation and stimulation of the RPE layer alone might theoretically be enough to mediate a biological response in the treatment of retinal diseases, without destroying the photoreceptors. That is, rather than targeting the sensory retina, we should consider the very important role of the RPE cells. It may be that these, rather than the sensory retina, are of primary importance when producing an effect from photocoaguation.

Temperature and histology after laser photocoagulation

When laser light strikes the retina, a high percentage of the energy is absorbed in the RPE layer, and this percentage is wavelength-dependent. With green light, about 50% of the laser light is absorbed by the RPE.24 The typical exposure time of conventional argon laser treatment is 100 msec or more. While the laser energy is being absorbed, it is converted to heat which diffuses out of the absorbing RPE layer in three dimensions; i.e., heat is conducted posteriorly into the choroid and anteriorly in the direction of the neural retina. Heat is also transferred horizontally, along the RPE cells and retinal layers. Heat diffuses out of an absorbing structure at a speed of roughly 1 µsec per µm (1 µs = 10-6 s). Hence, a laser exposure of 100 ms results in considerable heat conduction. The spatial and temporal temperature distribution can be calculated by mathematical models, and can be verified experimentally.25,26 Figure 1 shows the calculated temperature profile inside the RPE and inside the neural retina. It is obvious that

J. Roider et al.

Temperature [°C]

Retina

Fig. 1. Computed spatial temperature profile in the RPE and retina after exposure to a 100 ms argon (514 nm) laser pulse with various exposure times (50 and 100 ms, 1 s) and various threshold powers (spot size 110 µm). There is only a small temperature gradient between the RPE and the neural retina.

only a small temperature difference exists between the RPE and neural retina after a 100-ms exposure. This difference is about 18% from the RPE to 5 µm into the retina. Therefore, lowering the laser power, while keeping the exposure time constant, will not protect the retina from thermal damage. Instead, the exposure duration must be reduced.

Figure 2 shows the histology of the retina of an argon laser exposure with 20 mW and 100 ms on a 110-µm spot diameter. The power was chosen so that the lesion was not clinically visible, but it was detectable by fluorescein angiography. Despite the subtle nature of the lesion, the photoreceptors have been irreversibly damaged. Over time, the damaged photoreceptors will be replaced by scar tissue. This drop out of photoreceptors, after even mild photocoagulation very close to the damage threshold, is obvious.

Concept of selective retinal pigment epithelium treatment

Sparing of adjacent structures is only possible if the laser pulse duration is matched with the target’s physical characteristics. The pulse duration needed to spare the neural retina can be estimated by the thermal relaxation time or the time interval required for the heat to diffuse out of an absorbing tissue.25,27,28 If we consider that the size of an RPE cell is about 10 µm, high temperatures can be confined to the RPE cell itself, if the exposure time is of the order of a few microseconds rather than the customary millisecond settings.

Figure 3 shows the spatial temperature profile as calculated after a argon laser exposure of 1 µsec. The difference in the temperature profile compared

Fig. 2. Light micrograph of an ophthalmoscopically non-vis- ible laser lesion obtained 14 days after exposure to a 100 ms argon laser pulse (power: 20 mW, spot diameter: 110 µm). A significant dropout and damage of photoreceptors is seen. Magnification 500 ×.

to Figure 1 is obvious. It is conceivable that most of the heat is concentrated within the RPE at the end of the laser pulse, and the temperature elevation inside the retina can be minimized. Hot spots do occur, but these are situated around single melanin granules. Since no more laser energy is delivered at the end of the laser pulse, the temperature profile quickly smoothes out and leads to only a very low temperature increase at the retina. This effect is especially pronounced if repetitive laser pulses are applied in such a manner that the following laser pulse is applied only after the retinal tissue has had sufficient time to completely cool down to baseline. Thus, the pulse repetition rate has to be matched to the irradiation in order to achieve high temperatures inside the RPE and low temperatures at the adjoining photoreceptors.

If the melanosome temperatures become high enough, thermal denaturation of cell proteins close to the granules, as well as coagulation of the whole cell, can take place. However, the temperatures required for denaturation in the microsecond time domain are still unknown. If the vaporization temperature around the melanin granules is exceeded, i.e., about 140°C, microbubble formation occurs.29 The expansion and collapse of the these microbubbles can cause thermomechnical disruption in the RPE cells.25 Figure 4 shows the histological effect after exposure to a chain of 500 repetitive 5-µs laser exposures. It is obvious that the RPE is heavily damaged, but most of the photoreceptors have been spared. Similar histological effects have been shown after repetitive 200-nsec laser pulses with a Nd: YAG (532 nm) laser.30

RPE defects after selective RPE treatment18,31 may be covered by a new population of RPE cells. Cells close to and far away from the treatment site react, and a new RPE barrier is quickly restored. This effect on the RPE is not different from the reaction of the RPE after conventional laser photocoagulation.2,15 Since the RPE cells do not divide during their lifetime, apart from special circumstances, therapeutical laser effects could be explained by direct or indirect stimuli originating from RPE alterations.

µm

Fig. 3. Computed spatial temperature profile in the RPE and retina after exposure to a 1 µs argon (514 nm) laser pulse (2 µJ, 110 µm). A significant temperature gradient between the RPE and retina is conceivable. The hottest spots are conceivable around single melanin granules, which are absorbers of the laser light inside the RPE.

Fig. 4. Transmission electron micrograph of an ophthalmoscopically non-visible laser lesion obtained two hours days after exposure to 500 repetitive 5-µs argon laser pulses (3 µJ, 110 µm, 500 Hz). Most of the photoreceptors have been spared and the outer segments look normal. Magnification 3000 ×.

Therapeutical effects after selective retinal pigment epithelium treatment

A salient question is whether, and in which diseases, selective RPE treatment leads to a positive therapeutic effect. In a first clinical pilot study, we focused on three pathological conditions: diabetic macular edema, central serous retinopathy, and drusen in age related macular degeneration (AMD). Treatment was performed either using a chain of repetitive laser pulses with a frequency-doubled Nd:YLF laser (wavelength 527 nm, pulse duration 1.7 µs, 100 and 500 Hz repetition rate, 30 and 100 pulses and a retinal spot diameter of 160 µm) or a pulsed Nd:YAG laser (wavelength 532 nm, pulse duration 800 ns, 500 Hz repetition rate, 100 pulses and a retinal spot diameter of 200 µm).

Figure 5 shows the appearance of the fundus of a patient who had been treated for drusen with a

Fig. 5. Fundus photograph in a patient: (A) before, and (B) 12 months after laser treatment for drusen. A significant reduction in the number of drusen is visible.

Nd:YAG laser. After test exposures, selective RPE spots were placed in a horseshoe pattern around the fovea. Drusen slowly began to disappear after three months.32

In another study, 12 patients with diabetic maculopathy (group 1), ten with soft drusen (group 2), and four with central serous retinopathy (CSR) (group

3) were treated and followed up for one year.32 Treatment was performed using an Nd:YLF laser. Laser energy was based on the visibility of test lesions on fluorescein angiography (50-130 µJ). Patients were examined at various times by ophthalmoscopy, fluoresceinand ICG-angiography, as well as by infrared imaging. After six months, hard exudates disappeared in six of nine patients in group 1, and leakage had disappeared in six of 12 diabetic patients. In group 2, there were less drusen in seven of ten patients. In group 3, serous detachment had disappeared in three of four cases. Visual acuity was stable in all cases.

Selectivity of retinal pigment epithelium treatment (sparing the retina)

In a first pilot study, test laser lesions were used to investigate whether sparing of the retina was possible in the human retina.33 In order to investigate whether selective RPE effects were really selective from a microperimetry point of view, microperimetry was performed directly on top of laser lesions during a follow-up period of up to one year. A repetitive pulsed Nd:YLF laser was applied in 17 patients, using pulse energies of from 20-130 µJ. To establish the necessary energy, test exposures were performed in the inferior macular region (see, for example, Fig. 6A). Of 179 test lesions, 73 were followed at various times by performing microperimetry directly on top of the laser lesions. For testing, laser lesion threshold stimuli were determined before laser exposure. The threshold sensitivity values were defined as the minimal contrast at which a response was obtained. This threshold value was used to evaluate the test lesions during the follow-up period.

Probit analysis showed that all the test lesions were at the threshold of RPE disruption and that none of the laser effects were visible by means of ophthalmoscopy during photocoagulation, and were only detectable by fluorescein angiography. After exposure with 500 pulses, retinal defects could be detected in up to 73% of patients (100 µJ) after the first day. Most of these defects were no longer detectable after three months. After exposure with 100

r

*

V

pulses, no defects could be detected with 70 and 100 mJ after one day, and the neural retina remained undamaged during the follow-up period. Table 1 summarizes the results of relative defects after selective RPE treatment.33

Figure 6A shows the fluorescein angiogram of a patient in whom laser effects have been applied to the part below the macula. This patient was scheduled for treatment for soft drusen. Figure 6B shows the corresponding infrared image with the threshold stimuli recognized by the patient superimposed. No retinal defects could be detected after selective RPE treatment with pulse energies below 100 µJ and 100 or 500 pulses.

Our findings are in contrast to microperimetry findings after conventional laser photocoagulation, which is associated with thermal damage to the outer and inner nuclear layer and replacement by scar

tissue.2,3,15,31,34 It is not surprising that such lesions lead to absolute scotomas on microperimetry. Figure 7 shows an example of such a patient treated with conventional laser techniques for diabetic macular edema and proliferative diabetic retinopathy. Absolute scotomas can be seen over each argon laser exposure. If such lesions are located temporal to the fovea, the patient may have problems with reading, despite good visual acuity.

Autofluorescent behavior after conventional and selective laser treatment (imaging the retinal pigment epithelium)

Photoreceptors degenerate as soon as they are devoid of RPE cells for an extended period. The question of whether RPE cells regenerate is crucial for

retinal sparing laser treatment.

The RPE contains high amounts of age-accumu- lated lipofuscein, which is also a strong fluorophore. Lipofuscin can be divided into ten subcomponents and is considered to be a storage granule within the pigment epithelium.35 The excitation spectrum of fundus autofluorescence has a broad band between 450 and 540 nm, with a maximum around 500 nm. The emission spectrum of lipofuscin ranges around 500780 nm, with a maximum at 620 nm.35 In selective RPE treatment, most of the energy is absorbed in the RPE, and thus a change of its autofluorescence due to the thermal effects of lipofuscein can be expected. Since changes in the RPE occur both during and after laser treatment in the healing phase, monitoring its autofluorescence might be a helpful tool both for verifying as well as characterizing the laser effects.

After mild, barely visible continuous wave photocoagulation (100-200 ms, 100 µm, power equivalent to a barely visible laser burn), there is a change in autofluorescent behavior over a period of one year, which is much longer than we would have expected from animal experiments. Autofluorescence has been observed by retina angiography (HRA) in the fluorescence mode, using excitation and barrier filters, as used during fluorescein angiography, excitation wavelength 488 nm, without using fluorescein. This was examined in 13 patients, in whom focal diabetic macular edema had been treated by conventional laser parameters, as described above. In 12 of the 13 patients treated by conventional focal laser photocoagulation, the laser spots appeared as hypofluorescent areas one hour after treatment. The autofluorescent behavior of different laser exposures did not vary between patients, but did change dramatically with time. Figure 8A shows fluorescent behavior one hour after focal laser treatment. One month after focal laser treatment, all laser spots examined appeared as hyperfluorescent areas (Fig. 8B). This situation remained unchanged for three months. After six months, most of the spots (94%) were highly hyperfluorescent. Some had a mixed form with a central white hyperfluorescent island

surrounded by a dark hypofluorescent area. After a period of more than 12 months, most of the laser spots once again appeared hypofluorescent (85%) (see Fig. 8C), while 15% showed a mixed appearance. The changes in autofluorescent behavior were significant between one hour and one month after treatment (P = 0.0001), and also between six months and one year (P = 0.001). These findings show that changes within the RPE cells occur after laser treatment over a long period of time.

These autofluorescence findings can partially be explained by the histological results of animal experiments. After one hour, autofluorescence photographs show that all cells are damaged and that the irradiated area appears to be hypofluorescent. These findings show that all fluorophores are completely destroyed. After laser treatment, the cell debris from damaged RPE cells and photoreceptors is phagocitized by RPE cells sliding in from the surrounding area or by macrophages originating from the choriocapillaris.18 Abundant phagosomes can be found within these cells during this highly active period. These storage granules accumulate within the cells and could be responsible for the hyperfluorescent signal. In animal experiments, the damaged irradiated area is repopulated by RPE cells or RPEderived cells. After conventional laser photocoagulation, all photoreceptors are irreversibly destroyed and scarring can be seen. Lipofuscein is a storage granule. Several observations have confirmed that most of the material accumulating in RPE lipofuscin granules in AMD originates from phagocytosis of photoreceptor material.36,37 When the RPE is destroyed, the newly formed RPE does not accumulate the same amount of lipofuscin as seen in animals reared in dim light.38 This would suggest that no storage granules develop. This may explain why the irradiated area appears to be hypofluorescent after one year, when scarring has occurred. In the meantime, transition from a highly active period to a quiet period occurs. One year after conventional laser treatment, the RPE has obviously obtained a different status of viability compared to the initial one.

Future studies will show whether the RPE is capable of completely regenerating after selective treatment. Figure 9 shows the autofluorescent image one hour (Fig. 9B) and one year (Fig. 9C) after selective RPE treatment in a 36-year-old patient. Images of the laser spots between the two large vessels one year after selective RPE treatment are very similar to the autofluorescence image of the surrounding area, suggesting the complete recovery from damage after selective RPE treatment.

Monitoring retinal pigment epithelium damage for dosimetry by means of autofluorescence

Selective RPE treatment requires a refinement of laser protocol, since it is a problem that RPE laser lesions are not clinically visible after placement. This is in contrast to conventional retinal photocoagulation where the laser spots can be seen as a whitening, since the coagulated retina scatters the illumination light of the slit lamp in all directions. Fluorescein angiography is even more sen-sitive in detecting coagulated areas, compared to conventional observation or photography. In fact, after conventional cw laser exposure, fluorescein angiography is twice as sensitive as ordinary black and white or color fundus photographs.36

In selective RPE treatment, the heat is confined to the RPE, which shows that, on its own, RPE damage cannot be observed by white light illumination. However, in animal experiments, using repetitive µs or 200 nsec laser exposures,18,30 fluorescein angiography shows RPE damage quite well, by indirectly demarking a tight junction defect in the treated RPE areas. However, noninvasive dosimetry control is needed. Preferably, this should serve directly during treatment as an on-line method of control.

In order to monitor the acute changes in autofluorescence during selective RPE treatment, the laser beam itself was simultaneously used for fluorescence excitation, since its wavelength of 527 nm is well within the excitation spectrum of lipofuscein. The

fluorescent light originating from the fundus was detected by a photodiode coupled to the slit lamp. A dichroic beam splitter was mounted in the pathway of the laser beam within the slit lamp, as shown in Figure 10. The excitation spectrum of the fundus autofluorescence and the wavelength of the laser is described on the previous page first paragraph and the laser wavelength a few lines ahead. Figure 11 shows its spectral transmission. The photodiode current was recorded by a PC and was promptly integrated over each laser pulse applied.

By means of this set-up, the fluorescence signal is detected during selective RPE treatment. The fluorescence intensity was strong enough for a photodiode instead of a more sensitive photomultiplier to be used. In the four patients treated for diabetic macular edema, decay of autofluorescence over the laser pulses applied was detected at each laser spot.

Figure 12A shows the typical time course of the signal for different pulse energies of single spots in one patient. Time and spectral integrated fluorescence was normalized to the fluorescence of the first pulse. For all pulse energies, fluorescent intensity decreases with an increasing number of pulses. In order to quantify fluorescent decay, a monoexponential decay according to I(n) ~ e-n/τ, was fitted to the data; I(n) represents the fluorescent intensity after n pulses within the chain, τ is the decay constant.

Figure 12B shows the mean decay constants and their statistical variations at various pulse energies in four different patients. For 50 and 70 µJ, two spots per patient were applied (test exposures), while many more spots were applied and averaged in Figure 12B at 100 and 130 µJ (treatment pulses). In two patients, the decay constant slightly decreased with pulse energy, however, this was not statistically relevant. In the other two patients, strong variations with pulse energy were found.

In order to use the decay of the autofluorescence as an on-line dosimetry criterium, a threshold value has to be defined. Thus, the decay constant has to be significantly different with radiant exposures below and above RPE damage. This could be proved

Fig. 11. A: Transmission spectrum of the beam splitter, as used with the slit lamp to separate the fluorescent light (B) from the laser beam. (The excitation and emission spectra is taken from Delori et al.35)

in vitro using porcine RPE samples, in which a high correlation between autofluorescence and cell damage, demarked with a viability stain, was found. Clinically, correspondence has to be found with respect to fluorescein leakage in angiography. In the four patients, threshold energy for RPE disruption was between 70 and 130 µJ. However, the decay constants from 50-130 µJ do not differ significantly,

Fig. 12. A: Autofluorescent decay during laser irradiation with a chain of 93 repetitive laser pulses at a rate of 500 Hz, obtained from patient A. The fluorescent intensity decreases on average for all pulses, however strong variations between pulses were detected. B: Autofluorescent decay constants (see text) of four patients treated for diabetic macular edema with various pulse energies. Error bars indicate 1 SD.

the first clinical results presented here show high fluctuations of the decay constant, and so far no clearcut threshold. We are reporting the first results. It should be taken into account that micromovement of the fundus during irradiation, as well as the nonuniform lipofuscin distribution across the RPE, influence autoflourescence. Furthermore, fluorescein angiography and a change in autofluorescence signify very different events, which are not necessarily correlated. With fluorescein angiography, a break-up of tight junctions between the RPE cells can be detected. After laser photocoagulation, this happens if the cell is totally destroyed as shown by histology after laser treatment.18,31 The decay of RPE autofluorescence may reflect the thermal alteration of lipofuscin, or at least, the temperature dependence of its fluorescence. However, up to now, it is unclear whether the on-line detection of autofluorescence can be used for dosimetry in order to monitor RPE damage during laser treatment.

Imaging of RPE autofluorescence after treatment by retinal angiography in the autofluorescent mode (Heidelberg Engineering, Heidelberg, HRA) typically showed a faint hypofluorescent spot at the site of exposure ten minutes after treatment. This hypofluorescent spot became more pronounced one hour after treatment (Fig. 13B), as discussed above. In conclusion, two-dimensional imaging of autofluorescence one hour after laser exposure has been proved to be reliable in detecting RPE damage, as long as the signal is not weakened by fluid originating from retinal edema.

Conclusions

Both experimental and initial clinical results are encouraging and support the concept of selective RPE treatment, which seems to be effective for certain diseases, while being less harmful to visual func-

tion. From a theoretical point of view, this technique may require different strategies in order to achieve efficacy; i.e., more RPE changes may be required compared to conventional laser photocoagulation. Macrophages, which are one of the cells that also react and migrate after continuous wave photocoagulation, are less likely to be as stimulated after selective RPE treatment compared to customary treatment.18 It may be that macrophages play an important role in the resolution of certain retinal diseases.

These pilot studies on selective RPE treatment, as well as studies on subthreshold diode laser burns,39,40 suggest that actual blanching of the retina, as for example that recommended by early treatment diabetic retinopathy (ETDR) studies, is not necessary. Despite the undebatable advantages of laser photocoagulation with current laser techniques, local defects cannot be avoided with these techniques. As shown above, the use of selective RPE treatment can avoid the formation of local scotomas. Another advantage of solely RPE-related laser effects is that retreatment may be easier to implement while still achieving a therapeutic effect.

Acknowledgements

The authors wish to thank Georg Schüle, Carsten Framme, Hann Elsner and Norman Michaud for their collaboration and contribution to the joint project on selective RPE treatment.

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