Release of Different Cell Mediators During Retinal Pigment Epithelium Regeneration Following Selective Retina Therapy.

Purpose: To investigate the effect of selective retina therapy (SRT) on the release of AMD-relevant cell mediators, such as matrix metalloproteinases (MMPs), VEGF, and pigment epithelium derived factor (PEDF) using different laser spot sizes and densities. Methods: Porcine RPE-choroid explants were treated with a pulsed 532 nm Nd:YAG laser using (1) large spot sizes, (2) small spot sizes with a high-density (hd) treatment, and (3) small spot sizes with a low-density (ld) treatment. Explants were cultivated in modified Ussing chambers. RPE regeneration and RPE cell death were investigated by calcein-AM staining and immunofluorescence. The MMP release was examined via zymography and immunofluorescence. VEGF and PEDF secretion was analyzed by ELISA. Results: During pigment epithelium regeneration (PER), mitosis and RPE cell migration were observed. Four days after SRT (large spot size) the content of active MMP2 increased significantly (P < 0.01). Hd treatment with small spot sizes resulted also in an increase of active MMP2 (P < 0.05). In immunofluorescence explants showed a localized expression of MMP2 within the healing lesions after irradiation. The PEDF level increased significantly (P = 0.01) after SRT with large spot sizes. VEGF secretion decreased significantly (P < 0.05) following SRT with large spot sizes and with hd treatment of small spot sizes. Conclusions: SRT induces a cytokine profile, which may improve the flux across Bruch's membrane, slows down progression of early AMD by RPE regeneration, and inhibits the formation of choroidal neovascularization. The cytokine release depends on the size and density of applied laser spots.

Power-controlled temperature guided retinal laser therapy.

Laser photocoagulation has been a treatment method for retinal diseases for decades. Recently, studies have demonstrated therapeutic benefits for subvisible effects. A treatment mode based on an automatic feedback algorithm to reliably generate subvisible and visible irradiations within a constant irradiation time is introduced. The method uses a site-individual adaptation of the laser power by monitoring the retinal temperature rise during the treatment using optoacoustics. This provides feedback to adjust the therapy laser power during the irradiation. The technique was demonstrated on rabbits in vivo using a 532-nm continuous wave Nd:YAG laser. The temperature measurement was performed with 523-nm Q-switched Nd:YLF laser pulses with 75-ns pulse duration at 1-kHz repetition rate. The beam diameter on the fundus was 200 µm for both lasers, respectively. The aim temperatures ranged from 50°C to 75°C in 11 eyes of 7 rabbits. The results showed ophthalmoscopically invisible effects below 55°C with therapy laser powers over a wide range. The standard deviation for the measured temperatures ranged from 2.1°C for an aim temperature of 50°C to 4.7°C for 75°C. The ED50 temperature value for ophthalmoscopically visible lesions in rabbits was determined as 65.3°C. The introduced method can be used for retinal irradiations with adjustable temperature elevations.

Lesion strength control by automatic temperature guided retinal photocoagulation.

Laser photocoagulation is an established treatment for a variety of retinal diseases. However, when using the same irradiation parameter, the size and strength of the lesions are unpredictable due to unknown inter- and intraindividual optical properties of the fundus layers. The aim of this work is to investigate a feedback system to generate desired lesions of preselectable strengths by automatically controlling the irradiation time. Optoacoustics were used for retinal temperature monitoring. A 532-nm continuous wave Nd:YAG laser was used for photocoagulation. A 75-ns/523-nm Q-switched Nd:YLF laser simultaneously excited temperature-dependent pressure transients, which were detected at the cornea by an ultrasonic transducer embedded in a contact lens. The temperature data were analyzed during the irradiation by a LabVIEW routine. The treatment laser was switched off automatically when the required lesion strength was achieved. Five different feedback control algorithms for different lesion sizes were developed and tested on rabbits in vivo. With a laser spot diameter of 133???m, five different lesion types with ophthalmoscopically visible diameters ranging mostly between 100 and 200???m, and different appearances were achieved by automatic exposure time control. The automatically controlled lesions were widely independent of the treatment laser power and the retinal pigmentation.

Temperature-Controlled Retinal Photocoagulation Reliably Generates Uniform Subvisible, Mild, or Moderate Lesions.

PURPOSE: Conventional retinal photocoagulation produces irregular lesions and does not allow reliable control of ophthalmoscopically invisible lesions. We applied automatically controlled retinal photocoagulation, which allows to apply uniform lesions without titration, and aimed at five different predictable lesion intensities in a study on rabbit eyes. METHODS: A conventional 532-nm photocoagulation laser was used in combination with a pulsed probe laser. They facilitated real-time fundus temperature measurements and automatic exposure time control for different predefined time/temperature dependent characteristics (TTC). We applied 225 control lesions (exposure time 200 ms) and 794 TTC lesions (5 intensities, exposure times 7-800 ms) in six rabbit eyes with variable laser power (20-66.4 mW). Starting after 2 hours, we examined fundus color and optical coherence tomographic (OCT) images over 3 months and classified lesion morphologies according to a seven-stage OCT classifier. RESULTS: Visibility rates in funduscopy (OCT) after 2 hours were 17% (68%) for TTC intensity group 1, 38% (90%) for TTC group 2 and greater than 94% (>98%) for all consecutive groups. TTC groups 1 through 4 correlated to increasing morphological lesion intensities and increasing median funduscopic and OCT diameters. Group 5 lesions were as large as, but more intense than group 4 lesions. CONCLUSIONS: Automatic, temperature controlled photocoagulation allows to apply predictable subvisible, mild, or moderate lesions without manual power titration. TRANSLATIONAL RELEVANCE: The technique will facilitate standardized, automatically controlled low and early treatment of diabetic retinopathy study (ETDRS) intensity photocoagulation independently of the treating physician, the treated eye and lesion location.

Comprehensive detection, grading, and growth behavior evaluation of subthreshold and low intensity photocoagulation lesions by optical coherence tomographic and infrared image analysis.

PURPOSE: To correlate the long-term clinical effect of photocoagulation lesions after 6 months, as measured by their retinal damage size, to exposure parameters. We used optical coherence tomographic (OCT)-based lesion classes in order to detect and assess clinically invisible and mild lesions. METHODS: In this prospective study, 488 photocoagulation lesions were imaged in 20 patients. We varied irradiation diameters (100/300 µm), exposure-times (20-200 ms), and power. Intensities were classified in OCT images after one hour, and we evaluated OCT and infrared (IR) images over six months after exposure. RESULTS: For six consecutive OCT-based lesion classes, the following parameters increased with the class: ophthalmoscopic, OCT and IR visibility rate, fundus and OCT diameter, and IR area, but not irradiation power. OCT diameters correlated with exposure-time, irradiation diameter, and OCT class. OCT classes discriminated the largest bandwidth of OCT diameters. CONCLUSION: OCT classes represent objective and valid endpoints of photocoagulation intensity even for "subthreshold" intensities. They are suitable to calculate the treated retinal area. As the area is critical for treatment efficacy, OCT classes are useful to define treatment intensity, calculate necessary lesion numbers, and universally categorize lesions in clinical studies.

Photocoagulation in rabbits: optical coherence tomographic lesion classification, wound healing reaction, and retinal temperatures.

BACKGROUND AND OBJECTIVE: The rabbit is the most common animal model to study retinal photocoagulation lesions. We present a classification of retinal lesions from rabbits, that is based on optical coherence tomographic (OCT) findings, temperature data, and OCT-follow-up data over 3 months. MATERIALS AND METHODS: Four hundred eighty-six photocoagulation lesions (modified Zeiss Visulas® 532 nm CW laser, lesion diameter 133 µm, exposure duration 200 milliseconds or variable, power variable) were analyzed from six eyes of three chinchilla gray rabbits. During the irradiation of each lesion, we used an optoacoustics-based method to measure the retinal temperature profile. Two hours, 1 week, 1 month, and 3 months after the treatment, we obtained fundus color and OCT (Spectralis®) images of each lesion. We classified the lesions according to their OCT morphology and correlated the findings to ophthalmoscopic and OCT lesion diameters, and temperatures. RESULTS: Besides an undetectable lesion class 0, we discerned subthreshold lesions that were invisible on the fundus but detectable in OCT (classes 1 and 2), very mild lesions that were partly visible on the fundus (class 3), and 3 classes of suprathreshold lesions. OCT greatest linear diameters (GLDs) were larger than ophthalmoscopic lesion diameters, both increased for increasing classes, and GLDs decreased over 3 months within each class. Mean peak end temperatures for 200 milliseconds lesions ranged from 61°C in class 2 to 80°C in class 6. CONCLUSION: The seven step rabbit lesion classifier is distinct from a previously published human lesion classifier. Threshold lesions are generated at comparable temperatures in rabbits and humans, while more intense lesions are created at lower temperatures in rabbits. The OCT lesion classifier could replace routine histology in some studies, and the presented data may be used to estimate lesion end temperatures from OCT images.

Correlation with OCT and histology of photocoagulation lesions in patients and rabbits.

PURPOSE: To examine spectral domain optical coherence tomographic (OCT) and histological images from comparable retinal photocoagulation lesions in rabbits, and to correlate these images with comparable OCT images from patients. METHODS: 508 rabbit lesions were examined by HE-stained paraffin histology. 1019 rabbit lesions versus 236 patient lesions were examined by OCT, all at the time-points 1 hr, 1 week and 4 weeks after photocoagulation. We analysed 100 µm lesions (in humans) and 133 µm lesions (in rabbits) of 200 ms exposures at powers titrated from the histological threshold up to intense damage. Lesions were matched according to morphological criteria. RESULTS: Dome-shaped layer alterations, retinal infiltration by round, pigmented cells, outer nuclear layer interruption, and eventually full thickness retinal coagulation are detectable in histology and OCT. Horizontal damage extensions are found 1½ times larger in OCT. More intense irradiation was necessary to induce comparable layer affection in rabbit OCT as in histology. Restoration of the inner retinal layers is only shown in the OCT images. Comparable primary lesions caused more pronounced OCT changes in patients than in rabbits during healing. CONCLUSIONS: Optical coherence tomographic images indicate different tissue changes than histologic images. After photocoagulation, they show wider horizontal damage diameters, but underestimate axial damage particularly during healing. Conclusions on retinal restoration should not be drawn from OCT findings alone. Retinal recovery after comparable initial lesions appears to be more complete in rabbit than in patient OCTs.

Correlation of temperature rise and optical coherence tomography characteristics in patient retinal photocoagulation.

We conducted a study to correlate the retinal temperature rise during photocoagulation to the afterward detected tissue effect in optical coherence tomography (OCT). 504 photocoagulation lesions were examined in 20 patients. The retinal temperature increase was determined in real-time during treatment based on thermoelastic tissue expansion which was probed by repetitively applied ns laser pulses. The tissue effect was examined on fundus images and OCT images of individualized lesions. We discerned seven characteristic morphological OCT lesion classes. Their validity was confirmed by increasing visibility and diameters. Mean peak temperatures at the end of irradiation ranged from approx. 60 °C to beyond 100 °C, depending on burn intensity.

Automatic temperature controlled retinal photocoagulation.

Laser coagulation is a treatment method for many retinal diseases. Due to variations in fundus pigmentation and light scattering inside the eye globe, different lesion strengths are often achieved. The aim of this work is to realize an automatic feedback algorithm to generate desired lesion strengths by controlling the retinal temperature increase with the irradiation time. Optoacoustics afford non-invasive retinal temperature monitoring during laser treatment. A 75 ns/523 nm Q-switched Nd:YLF laser was used to excite the temperature-dependent pressure amplitudes, which were detected at the cornea by an ultrasonic transducer embedded in a contact lens. A 532 nm continuous wave Nd:YAG laser served for photocoagulation. The ED50 temperatures, for which the probability of ophthalmoscopically visible lesions after one hour in vivo in rabbits was 50%, varied from 63°C for 20 ms to 49°C for 400 ms. Arrhenius parameters were extracted as ΔE=273 J mol(-1) and A=3 x 10(44) s(-1). Control algorithms for mild and strong lesions were developed, which led to average lesion diameters of 162 ± 34 µm and 189 ± 34 µm, respectively. It could be demonstrated that the sizes of the automatically controlled lesions were widely independent of the treatment laser power and the retinal pigmentation.

Real-time temperature determination during retinal photocoagulation on patients.

The induced thermal damage in retinal photocoagulation depends on the temperature increase and the time of irradiation. The temperature rise is unknown due to intraocular variations in light transmission, scattering and grade of absorption in the retinal pigment epithelium (RPE) and the choroid. Thus, in clinical practice, often stronger and deeper coagulations are applied than therapeutically needed, which can lead to extended neuroretinal damage and strong pain perception. This work focuses on an optoacoustic (OA) method to determine the temperature rise in real-time during photocoagulation by repetitively exciting thermoelastic pressure transients with nanosecond probe laser pulses, which are simultaneously applied to the treatment radiation. The temperature-dependent pressure amplitudes are non-invasively detected at the cornea with an ultrasonic transducer embedded in the contact lens. During clinical treatment, temperature courses as predicted by heat diffusion theory are observed in most cases. For laser spot diameters of 100 and 300 µm, and irradiation times of 100 and 200 ms, respectively, peak temperatures range between 70°C and 85°C for mild coagulations. The obtained data look very promising for the realization of a feedback-controlled treatment, which automatically generates preselected and reproducible coagulation strengths, unburdens the ophthalmologist from manual laser dosage, and minimizes adverse effects and pain for the patient.

Imaging thermal expansion and retinal tissue changes during photocoagulation by high speed OCT.

Visualizing retinal photocoagulation by real-time OCT measurements may considerably improve the understanding of thermally induced tissue changes and might enable a better reproducibility of the ocular laser treatment. High speed Doppler OCT with 860 frames per second imaged tissue changes in the fundus of enucleated porcine eyes during laser irradiation. Tissue motion, measured by Doppler OCT with nanometer resolution, was correlated with the temperature increase, which was measured non-invasively by optoacoustics. In enucleated eyes, the increase of the OCT signal near the retinal pigment epithelium (RPE) corresponded well to the macroscopically visible whitening of the tissue. At low irradiance, Doppler OCT revealed additionally a reversible thermal expansion of the retina. At higher irradiance additional movement due to irreversible tissue changes was observed. Measurements of the tissue expansion were also possible in vivo in a rabbit with submicrometer resolution when global tissue motion was compensated. Doppler OCT may be used for spatially resolved measurements of retinal temperature increases and thermally induced tissue changes. It can play an important role in understanding the mechanisms of photocoagulation and, eventually, lead to new strategies for retinal laser treatments.

Temperature-controlled retinal photocoagulation--a step toward automated laser treatment.

PURPOSE: Retinal laser photocoagulation carries the risk of overtreatment due to effect variation of identically applied lesions. The degree of coagulation depends on the induced temperature increase and on exposure time. We introduce temperature controlled photocoagulation (TCP), which uses optoacoustics to determine individually exposure times necessary to create reproducible lesions. METHODS: Optoacoustic temperature measurement relies on pressure waves that are excited in the retinal tissue by repetitive low-energy laser pulses. Signal amplitudes correlate with tissue temperature and are detected by a transducer in the laser contact lens. We used a continuous wave (CW) photocoagulator for treatment irradiation and superimposed probe laser pulses for simultaneous temperature measurement. Optoacoustic data of 1500 lesions (rabbit) were evaluated to develop an algorithm that controls exposure times automatically in TCP. Lesion diameters of 156 TCP lesions were compared to 156 non-controlled lesions. Histology was performed after 1 hour, and 1 and 4 weeks. RESULTS: TCP resulted in exposure times from 4 to 800 ms depending on laser power chosen. Ophthalmoscopic and histologic lesion diameters were independent of power between 14 and 200 mW. TCP lesions barely were visible with a mean diameter equal to the treatment beam (130 µm). In contrast, standard lesion diameters increased linearly and statistically significantly with power. Histology confirmed sparing of the ganglion and nerve fiber layers in TCP. CONCLUSIONS: TCP facilitates uniform retinal lesions over a wide power range. In a clinical setting, it should generate soft and reproducible lesions independently of local tissue variation and improve safety, particularly at short exposure times.