In the treatment of various retinal pigment epithelium (RPE) related retinal diseases, selective retina therapy (SRT) is highly demanded, as SRT intends to selectively damage the RPE while sparing the neurosensory retina (NSR) and the choroid. A gentle method for removing diseased host RPE cells is still missing regarding RPE stem cell therapy. Cell therapeutics for age-related macular degeneration are often implanted regardless of host RPE status in the target zone, which may result in RPE multilayering. Here, we study a novel laser for selective large-area RPE removal without damaging the surrounding tissue prior to RPE implantation to promote subretinal integration. Therefore, pigmented rabbit eyes were exposed to laser pulses of 8 μs in duration (wavelength, 532 nm; top-hat beam profile, 223 × 223 μm2). Postirradiation retinal changes were assessed with color fundus photography, fluorescein angiography, indocyanine green angiography, and optical coherence tomography (OCT). Here we present the histological outcome of four animals after laser treatment. Following euthanization, the eyes of the animals were processed for histology, sectioned in 5 μm paraffin sections and stained with hematoxylin and eosin. Particular emphasis was given to an OCT vs light microscopy comparison. Our results reveal that RPE can be removed selectively using laser pulses of 8 µs duration in the green spectral range without damaging the NSR. Therefore, this regime proves to be applicable in the sense of SRT.
The treatment of retinal pigment epithelium (RPE) related diseases, utilizing minimally invasive laser techniques like selective retina therapy (SRT) is highly demanded. However, due to the strong inter- and intraindividual variability of RPE absorption, as well as optical transmission, laser microsurgery requires reliable real-time feedback-controlled dosimetry (RFD) to prevent unwanted retinal overexposure. The formation of microbubbles around the strong absorbing melanosomes inside RPE cells, has been identified as the leading mechanism of RPE cell damage during low microsecond laser pulse exposure. Their formation and collapse cause measurable optoacoustic (OA) transients. In the presented experiment OA transients are compared to fringe-washouts in simultaneously recorded optical coherence tomography (OCT) M-scans directly following RPE laser irradiation. Ex-vivo porcine RPE-choroid-sclera explants were exposed to laser pulses of 8, 12, 16 and 20 μs duration and pulse energies ranging from 15 to 100 μJ (wavelength: 532 nm, exposure area: 120 × 120 μm2). Simultaneously, time-resolved OCT M-scans were recorded (central wavelength: 840 nm, scan rate: 77 kHz). Post irradiation, RPE cell damage was quantified using a calcein-AM viability assay and correlated with OA transients and fringe-washouts in OCT M-scans. The results show that the detection of fringe-washouts in OCT M-scans linearly scales with OA transients and correctly identifies the destruction of RPE cells. Furthermore, the findings indicate that the optical detection is more sensitive for SRT dosimetry than OA, because OCT reacts to fast dynamic changes of the scattering structure, which possibly are related to minute cell collapses yet indiscernible by OA transients.
Selective retina therapy (SRT) is currently used in clinical studies to treat several chorioretinal diseases. For SRT a laser pulse duration of 1,7 μs is currently used. At this pulse duration the retinal pigment epithelium (RPE) cells are destroyed by transient microbubbles without damaging the neuronal retina. So far it is unclear whether slightly longer laser pulses are still acting thermomechanically or whether thermal effects show responsible for cell damage close above damage threshold. In order to investigate the damage threshold increase with pulse duration, a novel laser with adjustable pulse duration in the range of 2-20 μs was used to investigate RPE damage on ex-vivo porcine RPE explants. The specimen were fixed in an eye model and were exposed to laser pulse energies ranging from 15-150 μJ with a top hat square of 120×120μm2, exhibiting a spatial intensity modulation factor of 1,3. Viability tests using binary evaluation result in threshold values with peak radiant exposures of 233 mJ/cm2 and 389 mJ/cm2 for 2 μs and 20 μs laser durations, respectively. An almost logarithmic increase of the threshold radiant exposure over pulse duration was found.
Selective retina therapy (SRT) is a short pulse (μs-regime) alternative to conventional laser photocoagulation (LPC) for treatment of retinal diseases. LPC leads to collateral damage of retinal layers adjacent to the retinal pigment epithelium (RPE), including healthy, non-regenerative photoreceptors due to the high thermal load, whereas in SRT, RPE cells are destroyed by microbubbles without damaging the neuronal retina. A novel experimental SRT laser operating at 532 nm wavelength can deliver 2 – 20 μs pulse sequences. Its tight integration into an upgraded diagnostic SPECTRALIS system combines beam control for treatment planning with real-time optical coherence tomography (OCT) overexposure protection of the photoreceptors. This “Spectralis Centaurus” system, was built and preliminary tested on porcine ex-vivo samples, reaching an unprecedented accuracy with unique planning and follow-up capabilities for upcoming clinical cellular level micro-surgery. The combination of OCT with SRT selectively limits cell death to the RPE by precisely controlling energy deposition while optically monitoring tissue response.
Ocular optical coherence tomography at the wavelengths ranges of 850 and 1060 nm have been integrated with a confocal scanning laser ophthalmoscope eye-tracker as a clinical commercial-class system. Collinear optics enables an exact overlap of the different channels to produce precisely overlapping depth-scans for evaluating the similarities and differences between the wavelengths to extract additional physiologic information. A reliable segmentation algorithm utilizing Graphcuts has been implemented and applied to automatically extract retinal and choroidal shape in cross-sections and volumes. The device has been tested in normals and pathologies including a cross-sectional and longitudinal study of myopia progress and control with a duplicate instrument in Asian children.
Selective retina therapy and optical coherence tomography have been combined to monitor laser-tissue interaction in real-time. An ex-vivo study of porcine eyes unveils mechanisms that enable automated and accurate dose-control during laser-therapy.
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