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Abstract
Purpose
To compare histopathological and apoptotic changes of ophthalmoscopically similar subthreshold laser burns made by a low power-long duration (LD) and a high power-short duration (SD) subthreshold laser treatment.
Methods
Ophthalmoscopically invisible subthreshold laser burns with a 3.0 mm spot size were made using an 810 nm diode laser on the rabbit retina. Lasers were applied for 60 seconds in the LD group, and 1 second in the SD group. Laser power was adjusted to achieve ophthalmoscopically invisible burns just below the threshold. The rabbits were sacrificed at 6, 12, 24, and 72 hours, 1, 2, and 4 weeks after laser treatment. The eyes were processed for light microscopic examination using hematoxylin and eosin (H&E), toluidine blue, and TdT-dUTP terminal nick-end labeling (TUNEL) staining. Eyes were also processed for electron microscopic examination.
Results
The changes in the retina were different between the two groups. The LD group showed abundant TUNEL positive cells in all the retinal layers at 6 hours after laser treatment, and distinct histological changes in the outer nuclear layer. Conversely, in the SD group, apoptosis did not occur and histological alteration in the outer nuclear layer was minimal.
References
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Figure 1.
Fundus photograph of marker laser burns: Single laser (*) was applied as a superior and inferior marker for the long duration subthreshold laser treatment (LD) and two juxtaposed lasers (**) for the short duration subthreshold laser treatment (SD). Subthres-hold lasers of 3.0 mm in spot size had been applied for 60 seconds in the LD group, and for 1 second for SD group within the marker burns.
Figure 2.
H&E stained histological findings of the long duration (LD, A and C) and short duration (SD, B and D) subthreshold laser group. ×400. (A) LD at 12 hours, (B) SD at 12 hours, (C) LD at 4 weeks, and (D) SD at 4 weeks after the laser treatment. Retina became swollen soon after the subthreshold laser treatment in both groups. Vacuole formation in the cytoplasm of inner nuclear layer is more evident in the LD group (A and B). Altered photoreceptor inner and outer segments, and intracytoplasmic vacuolization in the retinal pigment epithelium are noted. Choroid did not show any remarkable alteration except for mild stromal edema. Retinal thickness has decreased with time in the both group, while reduction of cellularity was more marked in the LD group.
Figure 3.
TUNEL staining of the long duration (LD, A and C) and short duration (SD, B and D) subthreshold laser group, ×400. (A) LD at 6 hours, (B) SD at 6 hours, (C) LD at 24 hours, and (D) SD at 24 hours after the laser treatment. Numerous TUNEL-positive cells stained brown are observed in GCL, INL and ONL at 6 hours after long duration subthreshold laser treatment. They decreased rapidly with time. GCL=ganglion cell layer; INL=inner nuclear layer; ONL=outer nuclear layer.
Figure 4.
Transmission electron micrograph of LD group. (A) Six hours after the laser. Focal eruption of retinal pigment epithelium with severe cytoplasmic alteration is observed. (Original magnification ×2,000; bar indicates 2 µm) (B) At 6 hours. Damaged ONL with pyknosis and altered cytoplasm of the cells are noted. (×2,500; bar, 2 µm) (C) At 12 hours. Vacuolizations in the cytoplasm of retinal pigment epithelial cell and disrupted photo-receptor outer segments are observed. Several phagosomes dislodged from the outer segment are scattered in the cytoplasm. (×2,500; bar, 2 µm) (D) One week after LD laser. Damaged photoreceptor inner and outer segments are closely associated with retinal pigment epithelial cells that had lost their apical microvillus. Vacuoles are still present in the RPE cytoplasm. (×2,500; bar indicates 2 µm)
Figure 5.
Transmission electron micrograph of SD group. (A) Six hours after the laser. Apical microvilli were destroyed and numerous vacuoles are observed in the cytoplasm of the RPE. (original magnification ×6,500; bar indicates 1 µm) (B) At 6 hours. (×3,000; bar, 2 µm) Pyknosis and abnormal cytoplasm with heterochromatin condensation is observed but is less significant than LD group. (C) At 12 hours. Deformed erythrocyte and obstructed capillary lumen beneath the well preserved Bruch membrane is seen. Photoreceptor outer segments are disrupted and several phagosomes engulfed in the cytoplasm of the retinal pigment epithelium are noted. (×3,000; bar, 2 µm) (D) At 1 week. The number of photoreceptor outer segment decreased. Note the altered apical microvilli and vacuoles in the cytoplasm of retinal pigment epithelial cell. (×2,500; bar, 2 µm)
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