Changes in Morphology and Optical Properties of

Changes in Morphology and Optical Properties of Sclera due to Hyperosmotic agent Raiyan T. Zaman, Henry G. Rylander III, Narasimhan Rajaram, Tianyi Wang, Nitin Asokan, James W. Tunnell, Ashley J. Welch Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas 78712

ABSTRACT 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.

Keywords: hyperosmotic agents, optical clearing, optical properties, reduced scattering coefficient, absorption coefficient, diffuse optical spectroscopy, linear-probe, sclera.

Acknowledgements: This research has been supported by the National Science Foundation. Spatial thanks to Kathryn Starr at the Animal Resource Center (ARC) who helped Raiyan with the anesthetic part of the animal experiment.

1. INTRODUCTION Hyperosmotic agents i.e. 100% anhydrous glycerol, substantially increase the penetration depth of light in skin [1−6]. The increase in light penetration depth is accomplished by reducing light scattering in the tissue [4, 6]. Thus, the primary and the secondary goals of this study were to investigate the change in morphology and optical properties of sclera due to 100% anhydrous glycerol. A potential benefit of the optical clearing technique is the improvement of laser therapeutic technique that relies on sufficient light penetration to a target embedded in sub-sclera. Combining optical clearing with laser radiation could reduce the laser fluences required for a therapeutic effect to treat ocular diseases.

Optical Interactions with Tissue and Cells XX, edited by Steven L. Jacques, E. Duco Jansen, William P. Roach, Proc. of SPIE Vol. 7175, 71750D · © 2009 SPIE · CCC code: 1605-7422/09/$18 · doi: 10.1117/12.809701

Proc. of SPIE Vol. 7175 71750D-1

In the study we performed our experiments on in vivo sclera of 8 rabbits and 3 pigs. For the primary goal, a camera attached to a slit lamp was used to capture the morphological changes occurred to the sclera during dehydration cycle or after glycerol application. These morphological changes were reversed when the sclera was hydrated with 0.9% saline. For the secondary goal, we used a diffuse optical spectroscopy (DOS) system with a linear fiber arrangement to measure reflectance from the sclera during the dehydration and hydration cycles. 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. The changes in optical properties were consistent with the morphological changes observed in sclera during these cycles.

2. METHODOLOGY 2.1. Animal preparation Female SPF Dutch Belted (n=8) rabbits weighing 4 to 5 lbs and female miniature pigs (n=3) were used to identify the changes in morphology and optical properties of sclera after using hyperosmotic agent. All experimental procedures were conducted according to protocols approved by the Animal Care Committee of The University of Texas at Austin. Prior to the experiment, the rabbits were anesthetized with glycopyrrolate 0.02mg/kg intramuscularly (IM) followed by 5mg/kg xylazine and 40mg/kg ketamine hydrochloride (Rompun-Ketaset) in the proportions: 60% of 20 mg/ml Rompun to 40% of 100 mg/ml Ketaset by volume. Anesthesia was maintained with isoflurane (1.5−3%) inhalant during the experiment. Isoflurane was supplemented by acepromazine (0.5−1.0 mg/kg) administered intramuscularly as needed for individual rabbits demonstrating a high intolerance to isoflurane. One hour prior to termination of isoflurane, buprenorphine 0.01−0.05 mg/kg was administered IM for pain management. A mixture of Telazol (50 mg/mL) and xylazine (50 mg/mL) were administered IM (4.4 mg Telazol/kg with 2.2 mg xylazine/kg) to the pigs as a pre-anesthetic. They were also maintained on isoflorane gas anesthesia for the entire experiment. The pigs were given 2−4 mg/kg Carprofen IM one hour before the end of the experiment for pain management. In all experiments, the rabbits and pigs were positioned in the ventral recumbent position. Oxygen saturation and pulse, heart and respiration rate were continuously monitored throughout the study. Animals were kept warm throughout the experimental procedures with warm water blankets. 2.2. In vivo eye model In this study we were interested in three layers of the in vivo eye model (i) conjunctive, (ii) sclera, and (iii) choroid. The conjunctiva is the thin clear outermost membrane that covers the sclera [7, 8]. The sclera is just beneath the conjunctiva, called the white of the eye, which is opaque due to hydration. Collagen accounts for 90% of the dry weight of all mammalian sclera. In addition, some fine blood vessel arcades exist on the surface of the sclera. The thickness of the sclera varies from 1000 µm at the posterior pole to 300 µm just behind the rectus muscle insertions. It is continuous with the dura mater and the cornea, and provides an attachment for the extraocular muscle insertions. The choroid lies between the retina and sclera and consists of four layers (i) Haller's layer (outermost layer), (ii) Sattler's layer, (iii) Choriocapillaris, and (iv) Bruch's membrane (innermost layer). Haller’s and Sattler's layers consist of large and medium diameter blood vessels, respectively; Choriocapillaris layer has capillaries [9]. Melanin, a darkly colored chromophore that occurs throughout the choroid layer helps limit uncontrolled reflection within the eye and increases vision. Figure 1a illustrates the diffusion model for multi layers eye tissue with optimal penetration depth. In our model, we assumed three different cases (i) at baseline (no glycerol application) the light scattered from the collimated beam undergoes multiple reflections and propagations; (ii) after glycerol application, the sclera started to clear and increases light penetration depth leads to a higher absorption at the choroid layer (iii) after 8 minutes sclera started to dehydrate and sclera became less transparent which leads to backscattering and internal reflection. In another word, the remitted light at the surface of sclera emerges to the probe. A cross section with a transmission electron microscope (TEM) through sclera and choroid near the rectus muscle of a rabbit eye is shown in Figure 1b.


(a)

(b)

Remitted Conjunctiva Scler

Sclera Reflected Absorbed

Choroid Choroid Choriocapillar Bruch’s membrane Retinal pigment epithelium

Choriocapillaries Retinal pigment epithelium

Figure 1: Eye model used to calculated the optical properties of sclera (b) micrograph of a rabbit eye with TEM

2.3. Surgical procedure A wire eye speculum was placed in the eye to hold the eye lead at a fixed position. The position of the eye was stabilized with a suture placed in the limbus. We introduced glycerol to the sclera close to limbus where the sclera is only 300 µm thick to get an optimal penetration depth of diffuse light based on our probe geometry. Glycerol was delivered to sclera in 2 methods (i) injecting through conjunctiva (using a hypodermic needle 27G ½), (ii) direct topical application after 0.3 cm incision at conjunctiva. The glycerol was injected very slowly due to the high viscosity of the 100% anhydrous glycerol. 2.4. Photography of sclera treated with glycerol and saline An EOS digital SLR camera (Digital Rebel XT Canon Japan) attached to a slit lamp (Topcon SL-6E) to capture the morphological changes of the sclera. The camera shutter was triggered by digital single-lens reflex (DSLR) software. A photograph was taken of the in vivo sclera at baseline (pre glycerol application), immediately after glycerol application (0 minute), and every 2 minutes thereafter up to 26 minutes and after hydrating sclera with 0.9% saline. 2.5. Diffuse Optical Spectroscopy (DOS) instrument The system we used to collect the diffuse reflectance is described in detail by Zaman et al. [10]. Figure 2 illustrates the DOS system consisting of three main components: (i) light source (LS-1 Tungsten Halogen lamp, Ocean Optics), (ii) optical fiber probe (custom built), and (iii) spectrometer (USB4000, Ocean Optics). The tungsten halogen lamp was connected to the optical probe through a SubMiniature version A (SMA) connector. The sample was illuminated with one optical fiber (the source fiber), and the reflected light was collected with a separate optical fiber (the detector fiber), 370 µm from the source fiber. The collector fiber was coupled to the spectrometer, and the reflectance spectrum was collected over a wavelength range of 425−700 nm. Each reflectance spectrum was collected in a fraction of a second. Reflectance spectrum from the sclera was colleted at baseline, immediate after glycerol injection to sclera, then every two minutes up to 18 minutes. Then sclera was hydrated with 0.9% saline and reflectance spectrum was collected immediate after saline application to sclera, then every 2 minutes upto 12 minutes. Prior to spectral analysis, recorded signals were corrected for system response. We subtracted the detector dark or static current and normalized the sample reflectance by the reflectance of a Lambertian reflector (Spectralon, Labsphere Inc.; 40% reflector).


USB 4000 Spectrometer SMA connectct

Hdloqen [drop

SMA connect

USB curFtor Computer stordqe

4- Source

Detector } 370 pm

Collection Fiber Bundle

W,ite Light Exc

*

icri Fiber

To sclera

Figure 2. Schematic illustration of the instrumentation setup for DOS system with sclera

2.6. Tissue Phantoms—Lookup Table (LUT) We used a Lookup table (LUT)-based inverse algorithm to determine the optical properties of sclera. The development of the LUT-based model has been described in detail previously [11]. The optical properties are determined from steadystate diffuse spectra that are valid for linear fiber-based probe geometries with close source-detector separations of 370 µm. The LUT is based solely on experimental measurements of known optical properties (calibration standards)—tissuesimulating phantoms. These phantoms were made with polystyrene microspheres (diameter = 1µm; Poly-sciences) and 10% India ink (Salis International) dissolved in water to simulate scattering and absorption, respectively. We used Mie theory to calculate reduced scattering coefficient (µs′) of the tissue phantoms and measured absorption coefficient (µa) of a various concentration of India ink solution using DOS system. A 6 × 10 matrix consists of 60 tissue phantoms with varying scattering [µs′(λ) = 0.25−4 mm-1] and absorption parameters [µa (λ) = 0−6.41 mm-1], based on previously reported values for tissue [12]. These 60 phantoms covered the entire range of the LUT matrix. The probe was placed in contact with the surface of the tissue phantoms, and white light spectra from the phantoms were shown in Figure 3.

0.6 U (

U a)

2

3

21 :

Figure 3. Resulting LUT [R(µs′, µa)] for tissue phantom made from polystyrene bead with various concentration of India ink (10% concentration).


3. RESULTS 3.1. Sclera: change in morphology The results from the primary goal of this study are shown in Figure 4. These photographs showed the morphological changes which occurred to sclera due to a hyperosmotic agent i.e. 100% anhydrous glycerol. The initial clearing of sclera started 3 minutes after injecting glycerol. The sclera became completely transparent at 8 minutes and stayed clear for 10−15 minutes. The clear sclera became less transparent over next 11 minutes and completely opaque once we applied 0.9% saline to hydrate 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 topically applied directly to sclera after the incision at the conjunctiva, immediately the sclera became transparent. (a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

1k

Figure 4. Sclera after injecting glycerol at (a) 0 minutes (b) 8 minutes (c) 26 minutes, sclera became less transparent (d) completely opaque after topical application of 0.9% saline. Sclera after direct application of glycerol after 0.3 cm incision at conjunctiva (e) 0 minutes (f) 6 minutes (g) 8 minutes (h) completely opaque after topical application of 0.9% saline.

3.2. Sclera: change in optical properties For the secondary goal, optical properties of sclera were measured before and after dehydration of the tissue with glycerol. The optical properties were also measured after hydrating the tissue with 0.9% saline. All data were compared at baseline. The change in optical properties of sclera depends on how scattering and absorption changes due to 100% anhydrous glycerol. Thus, the result section is divided into two main sections (i) scattering coefficient (ii) absorption coefficient. At baseline, the optical properties are generated due to the light interaction between sclera and air. However, once the hyperosmotic agent is introduced to the sclera the light penetrating depth increases and optical properties are measured from choroid. The error bars for all optical properties are calculated by using standard deviation. 3.2.1 Scattering coefficient The probability of scattering per infinitesimal path length in the sclera is defined with scattering coefficient (µs). In this study we calculated reduced scattering coefficient, µs′, based on the measured µs and g (average expected cosine of angle scattering). The µs′ decreased for both pigs and rabbits sclera immediately after glycerol injection (Figure 5a, 5b). The maximum reduction of 54%−57% in scattering occurred at 4 and 8 minutes for pig’s and rabbit’s sclera, respectively and


was followed by a gradual increase. After hydrating the tissue the scattering increased linearly with respect to time and return to baseline 12 minutes after the saline application.

3.5

(b)

Dehydration

R educed Scattering C oefficient (mm -1 )

R educed Scattering C oefficient (mm -1 )

(a) Hydration

3.0 2.5 2.0 1.5 1.0 0.5 0.0 base

0

4

8

12

16

0

4

8

12

3.5

Dehydration

Hydration

3.0 2.5 2.0 1.5 1.0 0.5 0.0 base

0

Observation Time (Minutes)

4

8

12

16

0

4

8

12

Observation Time (Minutes)

Figure 5. µs′ of (a) rabbit (n=8) and (b) pig (n=3) sclera at baseline, after dehydration by injecting 100% anhydrous glycerol and hydration with 0.9%. All values (rabbit and pig studies combined) are not statistically significant (p