Light scattering in the normally white sclera prevents diagnostic imaging or delivery of a focused laser beam to a target in the underlying choroid layer. In this study, we examine optical clearing of the sclera and changes in blood flow resulting from the application of glycerol to the sclera of rabbits. Recovery dynamics are monitored after the application of saline. The speed of clearing for injection delivery is compared to the direct application of glycerol through an incision in the conjunctiva. Although, the same volume of glycerol was applied, the sclera cleared much faster (5 to 10 s) with the topical application of glycerol compared to the injection method (3 min). In addition, the direct topical application of glycerol spreads over a larger area in the sclera than the latter method. A diffuse optical spectroscopy system provided spectral analysis of the remitted light every two minutes during clearing and rehydration. Comparison of measurements to those obtained from phantoms with various absorption and scattering properties provided estimates of the absorption coefficient and reduced scattering coefficient of rabbit eye tissue.
The primary and the secondary goals of this study were to investigate the change in morphology and optical properties of
sclera due to a hyperosmotic agent i.e. 100% anhydrous glycerol. We performed our experiments in vivo on the sclera of
8 rabbits and 3 miniature pigs. All the animals were under anesthetic for the entire experiment according to an approved
protocol. The position of the eye was stabilized with a suture placed in the limbus. Glycerol was delivered to sclera in 2
methods (i) injection (using a hypodermic needle 27G ½), (ii) direct application after 0.3 cm incision at conjunctiva. A
camera attached to a slit lamp was used to capture the morphological changes of the sclera. For the secondary goal we
used a diffuse optical spectroscopy (DOS) system with a linear fiber arrangement to measure reflectance from the sclera
before and after application of glycerol. The probe source-detector separation was set to 370 μm for optimal penetration
depth. We fit the measured diffuse reflectance to a Lookup Table (LUT)-based inverse model specific to our probe
geometry to determine the scattering and absorption properties of the sclera. This method estimated the size and density
of scatterers, absorbers-blood volume fraction, melanin concentration, oxygen saturation, and blood vessel size. The
results illustrated that the initial clearing of sclera started 3 minutes after injecting glycerol to sclera. The sclera became
completely transparent at 8 minutes and stayed clear for 10-15 minutes. During this time the choroid layer was visible
through sclera. The clear sclera became less transparent over next 11 minutes and became completely opaque once we
applied 0.9% saline to hydrate the sclera. These dehydration and hydration cycles were repeated 4 times for each eye and
the results were consistent for all animal models. When glycerol was applied directly to sclera after the incision at the
conjunctiva, the sclera became transparent instantaneously. For the secondary goal, the changes in optical properties of
sclera were monitored during the dehydration and hydration cycles. The reduced scattering coefficient decreased when
glycerol was injected and it further reduced with direct application. The scattering increased after re-hydration. We also
measured the blood volume fraction, melanin concentration, oxygen saturation, and blood vessels diameter to calculate
absorption coefficient with the DOS system. This study provided a novel way to identify morphological changes of
sclera in addition to measuring changes in optical properties due to hyper osmotic agent. The changes in optical
properties were consistent with the morphological changes in sclera during the dehydration and hydration cycles.
A series of experiments were conducted in vivo on porcine skin to determine the ED50 damage thresholds for 1214 nm continuous wave laser irradiation. These results provide new information for refinement of Maximum Permissible Exposure (MPE). The study employed exposure durations of 1 sec, 3 sec, and 10 seconds with nominal spot diameters of 6 mm, 8 mm and 10 mm and as a function of laser power. The effect of each irradiation was evaluated acutely, one hour after exposure, and 24 hours post exposure. Probit analysis was conducted to estimate the dose for 50% probability of laser-induced damage (ED50); Damage was defined as persistent redness at the site of irradiation for the pig skin after 24 hours. The results indicated that Maximum Permissible Exposure (MPE) limits should be lowered for the laser beam diameters larger than 6 mm.
Optical changes in skin blood flow due to the presence of glycerol were measured from a two-dimensional map of blood
flow in skin blood vessels with a dynamic imaging technique using laser speckle. In this study a dorsal skin-flap window
was implanted on the hamster skin with and without a hyper-osmotic agent i.e. glycerol. The hyper-osmotic drug was
delivered to the skin through the open dermal end of the window model. A two-dimensional map of blood flow in skin
blood vessels were obtained with very high spatial and temporal resolution by imaging the speckle pattern with a CCD
camera. Preliminary studies demonstrated that hyper-osmotic agents such as glycerol not only make tissue temporarily
translucent, but also reduce blood flow. The blood perfusion was measured every 3 minutes up to 36-60 minutes after
diffusion of anhydrous glycerol. Small capillaries blood flow reduced significantly within 3-9 minutes. Perfusion rate in
lager blood vessels i.e. all arteries and some veins decreased (speckle contrasts increased from 0.0115 to 0.384) over
time. However, the blood flow in some veins reduced significantly in 36 minutes. After 24 hours the blood perfusion
further reduced in capillaries. However, the blood flow increased in larger blood vessels in 24 hours compared to an hour
after application of glycerol. For further investigation the speckle contrast measurement were verified with color
Doppler optical coherence tomography.
Conference Committee Involvement (1)
Optics and Ionizing Radiation
3 February 2020 | San Francisco, California, United States
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