High-resolution in vivo imaging of regimes of laser damage to the primate retina

Abstract

Purpose. To investigate fundamental mechanisms of regimes of laser induced damage to the retina and the morphological changes associated with the damage response. Methods. Varying grades of photothermal, photochemical, and photomechanical retinal laser damage were produced in eyes of eight cynomolgus monkeys. An adaptive optics confocal scanning laser ophthalmoscope and spectral domain optical coherence tomographer were combined to simultaneously collect complementary in vivo images of retinal laser damage during and following exposure. Baseline color fundus photography was performed to complement high-resolution imaging. Monkeys were perfused with 10% buffered formalin and eyes were enucleated for histological analysis. Results. Laser energies for visible retinal damage in this study were consistent with previously reported damage thresholds. Lesions were identified in OCT images that were not visible in direct ophthalmoscopic examination or fundus photos. Unique diagnostic characteristics, specific to each damage regime, were identified and associated with shape and localization of lesions to specific retinal layers. Previously undocumented retinal healing response to blue continuous wave laser exposure was recorded through a novel experimental methodology. Conclusion. This study revealed increased sensitivity of lesion detection and improved specificity to the laser of origin utilizing high-resolution imaging when compared to traditional ophthalmic imaging techniques in the retina.

Figure 1

Exposure laser setup and beam introduction into imaging system.

Figure 2

Combined AO cSLO and AO SD-OCT imaging system with wide field line scanning laser ophthalmoscope for collecting complimentary high-resolution images of retinal cross-sections and en face field of view.

Figure 3

Formation of a lesion in the retina of subject S3 immediately after a high energy retinal radiant exposure (870 mJ/cm²). The lesion (white arrows) became more reflective in the minutes following exposure and is likely associated with edema. OCT B-scans of photothermal exposure moments before (a), approximately 10 seconds after (b), and minutes later (c). Scale bar: 100 μm.

Figure 4

OCT B-scans of a multiple pulse exposure, (1,317 mJ/cm²) to the retina of subject S2 OD collected at (a) 1 hour and (b) 24 hours. (c) is an en face view of the retina photoreceptor layer of subject S3 OD after receiving a multiple pulse exposure, (874 J/cm²). Scale bar: 100 μm.

Figure 5

Picosecond lesions area grew (measured using AO SD-OCT) in size proportional to increasing energy measured (data from one subject).

Figure 6

OCT B-scans of the retina of subject S8 OD (a) 1 hour, (b) 24 hours, and (c) 7 days following moderate (325 mJ/cm²) 532 nm 40 ps exposure. White arrows in (a)–(c) denote lesion base. Within minutes following exposure, a hyperreflective thread-like structure appears and extends from the lesion site up to a blood vessel (white arrows). Damage to the RPE layer becomes more hyperreflective and distinguishable over a 24-hour period. One week later, the photoreceptor layer protrudes slightly at the exposure site. Scale bar: 100 μm.

Figure 7

Damage response to picosecond exposure (331 mJ/cm²) 1 hour following exposure. Scale bar: 100 μm.

Figure 8

(a) OCT en face images collected 1 hour after exposure of retinal area receiving 413 nm light insult (41–1,089 J/cm²). (b) Color fundus image of same retinal area shown in (a) 1 hour after exposure. (c) Color fundus image of same subject 10 days after exposure.

Figure 9

Fundus and OCT B-scans of 413 nm exposures in the retina of one subject. (a) Lesions 11–13 (474 J/cm²) were not positively identified as MVLs in color fundus photos at the 1-hour observation point. (b) At the 24-hour observation point, all three exposure sites were positively identified as a MVL. OCT B-scans collected within minutes of the initial exposure (c) reveal a hyperreflective “clumping” in the photoreceptor inner segment layer. The RPE layer shows a modest increase in reflectance with a “granular” appearance. (d) The damage surrounding the RPE and photoreceptors becomes more distinguishable 24 hours later. A wispy “threading” at the boundary of the inner nuclear layer (INL) and outer plexiform layer (OPL) for lesions 11 and 12 becomes observable. An increased reflectance of the inner retinal layers directly above the lesion site suggests an edematous reaction. Nine days later, (e) the damage appears more homogenous and extends from its base at the RPE into the connecting cilia of the photoreceptors.

Figure 10

OCT B-scan and en face fly-through of subject S6 retina after receiving 413 nm (240–1,183 J/cm²) exposures 1 hour (a)–(g), (i) and 10 days later (h).

Figure 11

Subject S6 OD histological sections of a flat mount retina nine days after exposure (1,696 J/cm²). (a) Choroid, (b) outer segment of photoreceptors, (c)-(d) junction of inner and outer segment of photoreceptors, (e) inner segment of photoreceptors, and (f) junction of inner segment and outer nuclear layer. The choroid exhibits loss of pigment as well as low numbers of macrophages, lymphocytes, and plasma cells. There is disruption of the outer segments of the photoreceptors and pigment laden macrophages (b). (c) and (d) both show the junction of inner and outer segment of photoreceptors with minimal disruption of photoreceptors and two small pigment laden macrophages. The inner segment of photoreceptors with minimal disruption of photoreceptors (linear defect is an artifact) is shown in (e). The final frame (f) is the junction of inner segment and outer nuclear layer with no detectable defect.

Figure 12

Projection view of OCT cross-sections of 413 nm exposures (24 hours) not visible in color fundus photos at the 1-hour or 24-hour time point. Retinal exposures ranged from 225 to 1,089 J/cm². Lesions eventually became visible in color fundus photos 3 days later. Note the hyperreflective regions and threading present (white arrows). Black arrow denotes thermal marker lesion.

Figure 13

Plot of lesion width versus retinal radiant exposure to 413 nm light for one subject. Measurements were collected from AO SD-OCT en face images at the RPE and photoreceptor layer at 1 hour, 24 hours, and 10 days after exposure. PR: photoreceptor layer; RPE: retinal pigment epithelial layer.