Physiological response of the retinal pigmented epithelium to 3-ns pulse laser application, in vitro and in vivo.

BACKGROUND: To treat healthy retinal pigmented epithelium (RPE) with the 3-ns retinal rejuvenation therapy (2RT) laser and to investigate the subsequent wound-healing response of these cells. METHODS: Primary rat RPE cells were treated with the 2RT laser at a range of energy settings. Treated cells were fixed up to 7 days post-irradiation and assessed for expression of proteins associated with wound-healing. For in vivo treatments, eyes of Dark Agouti rats were exposed to laser and tissues collected up to 7 days post-irradiation. Isolated wholemount RPE preparations were examined for structural and protein expression changes. RESULTS: Cultured RPE cells were ablated by 2RT laser in an energy-dependent manner. In all cases, the RPE cell layer repopulated completely within 7 days. Replenishment of RPE cells was associated with expression of the heat shock protein, Hsp27, the intermediate filament proteins, vimentin and nestin, and the cell cycle-associated protein, cyclin D1. Cellular tight junctions were lost in lased regions but re-expressed when cell replenishment was complete. In vivo, 2RT treatment gave rise to both an energy-dependent localised denudation of the RPE and the subsequent repopulation of lesion sites. Cell replenishment was associated with the increased expression of cyclin D1, vimentin and the heat shock proteins Hsp27 and αB-crystallin. CONCLUSIONS: The 2RT laser was able to target the RPE both in vitro and in vivo, causing debridement of the cells and the consequent stimulation of a wound-healing response leading to layer reformation.

Investigations into localized re-treatment of the retina with a 3-nanosecond laser.

BACKGROUND AND OBJECTIVES: Subvisual retinal lasers necessarily cause clinically invisible lesions, hence, they could intentionally or inadvertently be targeted at precisely the same or an overlapping location during repeat laser treatment. Herein, we investigated the structural integrity and cellular responses of localized re-treatment using a nanosecond laser (2RT) currently in trials for early age-related macular degeneration. MATERIALS AND METHODS: Rats were randomly assigned to one of five groups: sham, subvisual 2RT, subvisual 2RT re-treatment, visual effect 2RT, visual effect 2RT re-treatment. Re-treatment groups were lasered on days 0 and 21; single laser groups were only lasered on day 21. All rats were euthanized at day 28 and eyes were then dissected and processed for immunohistochemistry. For re-treatment, the laser was targeted at precisely the same locations on both delivery occasions. Analytical endpoints included monitoring of retinal vascular integrity overlying lesions, investigation into any potential choroidal neovascularization, assessment of the RPE, quantification of collateral injury to photoreceptors or other neuronal classes, and delineation of glial reactivity. RESULTS: Repeat laser administration to rats caused ostensibly identical retinal-RPE-choroid responses to those obtained in age-matched rats that received only a single application. Specifically, 7 days after treatment, RPE cells were re-populating lesion sites. No obvious consistent differences were evident between the single and repeat laser groups. Moreover, repeat laser caused no (measurable) additive injury to photoreceptors or other retinal neuronal classes from single laser treatment. In re-lasered animals, there was no increase in microglial activity overlying and adjacent to lesion sites relative to single lasered rats. Finally, there was no evidence of choroidal neovascularization after repeat laser treatment. CONCLUSIONS: The overall results provide a measure of confidence that re-treatment of patients with 2RT should not provide any additional risk of developing visual scotomas, choroidal neovascularizations, or inflammatory events. Indeed, the collated results indicate that the metabolic and structural disruption to the RPE-retina caused by short pulse duration laser treatment is resolved within a short time frame such that re-treatment elicits a phenotype indistinguishable from single treatment. Lasers Surg. Med. 48:602-615, 2016. © 2016 Wiley Periodicals, Inc.

Nanosecond-laser application in intermediate AMD: 12-month results of fundus appearance and macular function.

BACKGROUND: A novel, ultra-low energy nanosecond laser (retinal rejuvenation therapy) has been developed with the aim to slow progression of early age-related macular degeneration (AMD). The safety, changes in fundus characteristics and macular function in a cohort of participants with bilateral intermediate AMD are reported. DESIGN: Prospective non-randomised, pilot intervention study. PARTICIPANTS OR SAMPLES: Subjects with bilateral intermediate AMD (n = 50, aged 50-75 years). METHODS: Ultra-low energy laser pulses applied in 12 spots around the macula of one eye (0.15-0.45 mJ), using 400 µm diameter spot, 3 nanosecond pulse length, 532 nm wavelength and energy titrated to each patient. MAIN OUTCOME MEASURES: Best corrected visual acuity, drusen area and macular sensitivity (flicker perimetry) at baseline and at 3, 6 and 12 months post-laser. RESULTS: Treatment was painless with no clinically visible lesions. No participant developed choroidal neovascularization, while two with thin central retinal thickness at baseline developed atrophy at 12-month follow up. Drusen area was reduced in 44% of treated eyes and 22% of untreated fellow eyes, with changes in drusen and function not being coincident. Improvement in flicker threshold within the central 3° was observed in both the treated and untreated fellow eyes at 3 months post-laser. Of the 11 eyes at greatest risk of progression (flicker defect >15 dB), seven improved sufficiently to be taken out of this high-risk category. CONCLUSIONS: A single unilateral application of nanosecond laser to the macula produced bilateral improvements in macula appearance and function. The nanosecond retinal rejuvenation therapy laser warrants ongoing evaluation as an early intervention for AMD.

Nanosecond pulse lasers for retinal applications.

BACKGROUND AND OBJECTIVES: Thermal lasers are routinely used to treat certain retinal disorders although they cause collateral damage to photoreceptors. The current study evaluated a confined, non-conductive thermal, 3-nanosecond pulse laser in order to determine how to produce the greatest therapeutic range without causing collateral damage. Data were compared with that obtained from a standard thermal laser. MATERIALS AND METHODS: Porcine ocular explants were used; apposed neuroretina was also in place for actual laser treatment. After treatment, the retina was removed and a calcein-AM assay was used to assess retinal pigmented epithelium (RPE) cell viability in the explants. Histological methods were also employed to examine lased transverse explant sections. Three nanoseconds pulse lasers with either speckle- or gaussian-beam profile were employed in the study. Comparisons were made with a 100 milliseconds continuous wave (CW) 532 nm laser. The therapeutic energy range ratio was defined as the minimum visible effect threshold (VET) versus the minimum detectable RPE kill threshold. RESULTS: The 3-nanosecond lasers produced markedly lower minimum RPE kill threshold levels than the CW laser (e.g., 36 mJ/cm(2) for speckle-beam and 89 mJ/cm(2) for gaussian-beam profile nanosecond lasers vs. 7,958 mJ/cm(2) for CW laser). VET values were also correspondingly lower for the nanosecond lasers (130 mJ/cm(2) for 3 nanoseconds speckle-beam and 219 mJ/cm(2) for gaussian-beam profile vs. 1,0346 mJ/cm(2) for CW laser). Thus, the therapeutic range ratios obtained with the nanosecond lasers were much more favorable than that obtained by the CW laser: 3.6:1 for the speckle-beam and 2.5:1 for the gaussian-beam profile 3-nanosecond lasers versus 1.3:1 for the CW laser. CONCLUSIONS: Nanosecond lasers, particularly with a speckle-beam profile, provide a much wider therapeutic range of energies over which RPE treatment can be performed, without damage to the apposed retina, as compared with conventional CW lasers. These results may have important implications for the treatment of retinal disease.

Rapid and delayed death of cultured trabecular meshwork cells after selective laser trabeculoplasty.

BACKGROUND AND OBJECTIVE: Selective laser trabeculoplasty (SLT) is becoming increasingly employed to reduce elevated intraocular pressure in glaucoma patients. SLT is known to target the ocular trabecular meshwork (TM), but the exact response mechanisms to this treatment have not been clearly delineated. The aim of the present study, therefore, was to investigate the modes of death of cultured bovine TM cells subjected to SLT in vitro. MATERIALS AND METHODS: Bovine TM cell cultures were established, pigmented with exogenous melanin and irradiated with a Q-switched, frequency doubled, Nd:YAG laser, at different energy settings (0.05-1.0 mJ). Influences on cells were determined for up to 10 days post-treatment by trypan blue exclusion, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and by morphological assessment. Furthermore, homogeneous mixtures of pigmented and non-pigmented TM cells were irradiated to ascertain selectivity of laser effects. RESULTS: At higher energy levels (1.0, 0.75 mJ), immediate loss of cells was detected at the irradiated site. Trypan blue exclusion analysis showed that necrotic cell death subsequently occurred up to 8 hours following irradiation, peaking at 60 minutes. This was followed by delayed cell death peripheral to the irradiated area which was characteristic of apoptosis and which peaked at 2-3 days post-treatment. When mixed cultures were tested, laser treatment selectively killed pigmented cells at an energy level equivalent to the lower cell killing threshold in the initial studies (0.2 mJ) but at the higher laser energy of 0.35 mJ, all cells were non-selectively killed. CONCLUSIONS: SLT treatment killed pigmented TM cells in culture by a variety of processes (instant vaporization, rapid necrosis, delayed apoptosis), depending on the magnitude of the energy used and the distance from the center of the irradiated zone. These data may assist in the elucidation of the mechanism of action of the SLT procedure on TM cells in situ.

Retinal damage profiles and neuronal effects of laser treatment: comparison of a conventional photocoagulator and a novel 3-nanosecond pulse laser.

PURPOSE: To determine detailed effects to retinal cells and, in particular, neurons following laser photocoagulation using a conventional 532 nm Nd:YAG continuous wave (CW) laser. Furthermore, to determine whether a novel 3 ns pulse laser (retinal regeneration therapy; 2RT) could specifically ablate retinal pigment epithelium (RPE) cells without causing collateral damage to other retinal cells. METHODS: Adult Dark Agouti (DA) rats were separated into four groups: control, CW laser (12.7 J/cm(2)/pulse, 100 ms pulse duration), or 3 ns pulse 2RT laser at one of two energy settings ("High," 2RT-H, 163 mJ/cm(2)/pulse; "Low," 2RT-L, 109 mJ/cm(2)/pulse). Animals were treated and killed after 6 hours to 7 days, and retina/RPE was analyzed by histologic assessment, Western blot, polymerase chain reaction, and immunohistochemistry. RESULTS: Both lasers caused focal loss of RPE cells with no destruction of Bruch's membrane; RPE cells were present at lesion sites again within 7 days of treatments. CW and 2RT-H treatments caused extensive and moderate damage, respectively, to the outer retina. There were no obvious effects to horizontal, amacrine, or ganglion cells, as defined by immunolabeling, but an activation of PKCα within bipolar cells was noted. There was little discernible damage to any cells other than the RPE with the 2RT-L treatment. CONCLUSIONS: Conventional laser photocoagulation caused death of RPE cells with associated widespread damage to the outer retina but little influence on the inner retina. The novel 3 ns 2RT laser, however, was able to selectively kill RPE cells without causing collateral damage to photoreceptors. Potential benefits of this laser for clinical treatment of diabetic macular edema are discussed.

Glial cell and inflammatory responses to retinal laser treatment: comparison of a conventional photocoagulator and a novel, 3-nanosecond pulse laser.

PURPOSE: Retinal laser photocoagulation represents a major treatment strategy for the management of diabetic macular edema (DME). However, the thermal nature of this procedure defines that collateral tissue injury result, meaning that it cannot be used near the fovea centralis. We studied inflammatory and glial responses resulting from treatment of rats with a conventional laser and with a novel short-duration, nonthermal laser (retinal regeneration therapy [2RT]) at clinically relevant energy levels. METHODS: Pigmented Dark Agouti rats were treated with either a conventional thermal continuous wave (CW; 532-nm, 100-ms pulse duration) or a short-pulse (2RT; 532-nm, Q-switched, 3-ns pulse) laser. Settings were at visible threshold for the CW laser (12.7 J/cm(2)/pulse) and at supra- and subvisible thresholds for the 2RT laser ("high," 2RT-H, 163 mJ/cm(2)/pulse; "low," 2RT-L, 109 mJ/cm(2)/pulse). Rats were killed at various subsequent time points. Samples were processed for histology, immunohistochemistry, RT-PCR, and Western blotting. RESULTS: The CW laser caused outer retinal lesions that were associated with photoreceptor death, astrocyte and Müller cell activation, and infiltration of macrophages and neutrophils. Furthermore, inflammatory cytokines, heat shock proteins, endogenous trophic factors, and matrix metalloproteinases were induced. In comparison, all of these changes were drastically attenuated when the 2RT laser was used, particularly at the subthreshold setting. CONCLUSIONS: The conventional laser produced marked retinal damage and cellular responses consistent with an inflammatory response to thermal injury. In contrast, the 2RT laser produced negligible retinal damage and cellular responses at clinically relevant settings. These results may have important implications for the treatment of retinal disease.