Randolph D. Glickman

Revisiting the internal absorption coefficient of the retinal pigment epithelium melanosome

Retinal pigment epithelial (RPE) cells of the vertebrate eye contain melanin packaged in structures called melanosomes. Previously, we reported that the bovine melanosome has a laser photodisruption threshold of 153.6 mJ/cm2 at 25°C and 532 nm, and an internal absorption coefficient of 2237 cm-1. Those values used an estimate of melanosome density inferred from studies of skin laser effects. We now revisit that calculation using a density value obtained from density centrifugation analysis of bovine and baboon RPE melanosomes. Stepped-density gradients (60% to 80%) of the nonionic medium, Nycodenz, were formed, and samples of melanosomes were spun on the gradients in a swinging-bucket rotor at 10,000 rpm for 60 m. Bovine melanosomes formed two populations, one at the interface between 70% and 75%, and the other between 75% and 80%, corresponding to the densities of ~1.38 and ~1.41 gm/cm3, respectively. Baboon melanosomes migrated to within the same density region. Using a density value of 1.41 gm/cm3, and assuming a water content of 52% for hydrated melanosomes, the internal absorption coefficient was calculated as 2339 cm-1. Although this calculation uses an objective density measurement, the water content remains an estimate, and the actual value in situ may differ.

Photodisruption increases the free-radical reactivity of melanosomes isolated from retinal pigment epithelium

Melanin in vivo is usually packaged in melanosomes with protein coats that restrict direct interaction of the melanin with the surrounding medium. We found that disruption of the melanosomes by exposure to a pulsed laser increased the ability of the melanin radicals to oxidize NADPH in a photochemical reaction. Retinal pigment epithelial (RPE) melanosomes were prepared from fresh bovine eyes in 0.25 M sucrose. A reaction mixture of 7 mM NADPH, approximately 7500 RPE melanosomes, and 80 mM Tris buffer, pH 7.2, was prepared in a volume of 60 (mu) l. Of the two 25-(mu) l aliquots taken from this mixture, one was pre-exposed to the 2nd-harmonic output of a Q-switched Nd:YAG laser (532 nm, 1800 10-nsec pulses at 10 Hz), and then was exposed to an Argon ion continuous wave (CW) laser (488.1 and 514.5 nm) for five minutes. The other aliquot was exposed only to the Argon laser. The CW exposure excited the melanin radicals to a reactive state that oxidized NADPH, as assayed by the loss of absorbance at 340 nm. Native melanosomes oxidized less NADPH during Ar+ laser pumping than did melanosomes pre-exposed to the YAG laser. The YAG lasers stimulatory effect on melanosomes reactivity increased as the total energy it delivered rose above 3.5 J (0.14 J/cm2/pulse X 1800 pulses), up to a maximum NADPH oxidation at about 20 J (0.2 J/cm2/pulse X 1800 pulses, beam broadened at higher pulse energy). Electron microscopic analysis of the melanosomes confirmed the progressive physical disruption of melanosomes as the YAG pulse energy increased.

Action spectrum of oxidative reactions mediated by light-activated melanin

The melanin of the retinal pigment epithelial (RPE) cells is generally thought to have a photoprotective role in the eye, yet it is excited by light to a free radical which can react with cellular components. Soluble proteins extracted from the retina are photo-oxidized by the output of a Xenon arc lamp containing UVA and visible wavelengths. The oxidative damage in this model consists of carbonyl adducts to the peptides, and is proportional to the amount of UVA present. Melanosomes isolated from bovine RPE cells and added to the retinal protein extract partly protect the proteins from photo-oxidation resulting from this broadband exposure. However, if the proteins are instead exposed to the 488 and 514.5 nm outputs of an Argon continuous wave laser, the amount of protein oxidation is markedly increased when melanosomes are present. This observation suggests that the melanin free radical is optimally excited by wavelengths in the blue-green region of the visible spectrum, and in fact the action spectrum for the photo-oxidation of NADPH by laser-excited melanin peaks between 450 and 500 nm. The present data do not distinguish between two alternative hypotheses, i.e. that the apparent action spectrum peak is due to (1) a chromophore different from the one determining the overall optical absorption of melanin, or (2) the lower efficiency of UVA photons in activating melanosomes because of their strong absorption at the solution surface. Nevertheless these data implicate melanin in the so-called blue light retinal hazard.

Pulsewidth-dependent nature of laser-induced DNA damage in RPE cells

Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.

Cytotoxicity in cultured RPE: A comparative study between continuous wave and mode-locked lasers

We have previously shown that minimum visible lesions (MVL) generated in vivo by exposures at threshold energies are not ophthalmoscopically visible at one hour, but become visible 24 hours post-exposure. Our preliminary studies toward understanding this phenomenon at the molecular level are presented. We provide threshold data relating cytotoxicity with energy for acute laser exposures in cultured retinal pigment epithelial cells. Rapid loss of cell viability is used as an initial endpoint of analysis. Comparisons of damage efficiency for continuous wave and mode-locked lasers are presented.

Evidence for excitation of fluorescence in RPE melanin by multiphoton absorption

Previously, we reported that ultrashort, near infrared (NIR) laser pulses caused more DNA breakage in cultured retinal pigment epithelial (RPE) cells than did CW, NIR laser radiation delivering a similar radiant exposure. We hypothesized that this difference was due to multiphoton absorption in an intracellular chromophore such as the RPE melanin. We investigated two-photon excitation of fluorescence in a suspension of isolated bovine RPE melanosomes exposed to a 1-KHz train of approximately 50- fsec laser pulses at 810 nm from a Ti:Sapphire laser, and compared this to the fluorescence excited by CW exposures at 406 nm from a Krypton ion laser. Fluorescence was measured with a PC-based spectrometer. The CW sources excited fluorescence with a peak at 525 nm. The fluorescence intensity depended on the irradiance of the sample, as well as the melanosome concentration. Peak fluorescence was obtained with a suspension of ~2 x 107 melanin granules/ml. The 810-nm, ultrashort pulses also excited fluorescence, but with a broader, lower-amplitude peak. The weaker fluorescence signal excited by the 810-nm ultrashort pulse laser for a given melanosome concentration, compared to 406-nm CW excitation, is possibly due to the smaller two- photon absorption cross-section. These results indicate the involvement of multiphoton absorption in DNA damage.

Hydrogen peroxide production in cultured RPE cells exposed to near infrared lasers

Hydrogen peroxide (H2O2) in situ synthesis within cultures of retinal pigment epithelial cells exposed to near-IR (NIR) continuous wave (CW) and mode-locked lasers is investigated. Any production of H2O2 as a consequence of NIR laser exposure is indicative of a multiphoton absorption event, resulting in an intracellular photochemical reaction. Comparisons of mode-locked and CW laser threshold energies required for the photo-oxidative reaction provide support for an enhancement in multiphoton absorption in tissues exposed to the high peak powers inherent in mode-locked laser beams. Due to different fluorescent staining properties our results indicate different mechanisms for oxidative stress from CW and mode- locked laser exposure.

Nonlinear optical characterization of the retina

We describe the measurements on the nonlinear absorption coefficient for the whole retina and separated components. We describe the impact of these measurements on retinal damage thresholds and damage mechanisms for various pulse regimes.