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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.
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