Denise Hileeto

Noninvasive imaging of the early effect of sodium iodate toxicity in a rat model of outer retina degeneration with spectral domain optical coherence tomography

Abstract. An ultrahigh resolution spectral domain optical coherence tomography (SD-OCT) system is used to observe for the first time in vivo the early effect of sodium iodate (NaIO3) toxicity on retinal morphology. Retinal degeneration is induced in rats via tail vein injection of NaIO3 and structural changes in the outer retina are assessed longitudinally at baseline and 1, 2, 3, 6, 8, and 10 h, and 12 post drug administration with OCT, H&E histology, and IgG immunochemistry. Disruption of the structural integrity and changes in the optical reflectivity of the photoreceptor inner (IS) and outer segment (OS) layers are observed as early as 1 h post NaIO3 injection. A new layer is observed in the OCT tomograms to form between the retinal pigmented epithelium and the photoreceptors OS a few hours post NaIO3 injection. The dynamics and the low optical reflectivity of this layer, as well as cell swelling and disruption of the blood-retina barrier observed in the histological and immunohistochemistry cross-sections suggest that the layer corresponds to temporary fluid accumulation in the retina. Results from this study demonstrate the effectiveness of OCT technology for monitoring dynamic changes in the retinal morphology and provide better understanding of the early stages of outer retina degeneration induced by NaIO3 toxicity.

Phenylboronic acid modified mucoadhesive nanoparticle drug carriers facilitate weekly treatment of experimentally-induced dry eye syndrome

Topical formulations, commonly applied for treatment of anterior eye diseases, require frequent administration due to rapid clearance from the ocular surface, typically through the lacrimal drainage system or through over-spillage onto the lids. We report on a mucoadhesive nanoparticle drug delivery system that may be used to prolong the precorneal residence time of encapsulated drugs. The nanoparticles were formed from self-assembly of block copolymers composed of poly(d, l-lactide) and Dextran. The enhanced mucoadhesion properties were achieved by surface functionalizing the nanoparticles with phenylboronic acid. The nanoparticles encapsulated up to 12 wt.% of Cyclosporine A (CycA) and sustained the release for up to five days at a clinically relevant dose, which led us to explore the therapeutic efficacy of the formulation with reduced administration frequency. By administering CycA-loaded nanoparticles to dry eye-induced mice once a week, inflammatory infiltrates were eliminated and the ocular surface completely recovered. The same once a week dosage of the nanoparticles also showed no signs of physical irritation or inflammatory responses in acute (1 week) and chronic (12 weeks) studies in healthy rabbit eyes. These findings indicate that the nanoparticles may significantly reduce the frequency of administration for effective treatment of anterior eye diseases without causing ocular irritation.

Prolonged Ocular Retention of Mucoadhesive Nanoparticle Eye Drop Formulation Enables Treatment of Eye Diseases Using Significantly Reduced Dosage

Eye diseases, such as dry eye syndrome, are commonly treated with eye drop formulations. However, eye drop formulations require frequent dosing with high drug concentrations due to poor ocular surface retention, which leads to poor patient compliance and high risks of side effects. We developed a mucoadhesive nanoparticle eye drop delivery platform to prolong the ocular retention of topical drugs, thus enabling treatment of eye diseases using reduced dosage. Using fluorescent imaging on rabbit eyes, we showed ocular retention of the fluorescent dye delivered through these nanoparticles beyond 24 h while free dyes were mostly cleared from the ocular surface within 3 h after administration. Utilizing the prolonged retention of the nanoparticles, we demonstrated effective treatment of experimentally induced dry eye in mice by delivering cyclosporin A (CsA) bound to this delivery system. The once a week dosing of 0.005 to 0.01% CsA in NP eye drop formulation demonstrated both the elimination of the inflammation signs and the recovery of ocular surface goblet cells after a month. Thrice daily administration of RESTASIS on mice only showed elimination without recovering the ocular surface goblet cells. The mucoadhesive nanoparticle eye drop platform demonstrated prolonged ocular surface retention and effective treatment of dry eye conditions with up to 50- to 100-fold reduction in overall dosage of CsA compared to RESTASIS, which may significantly reduce side effects and, by extending the interdosing interval, improve patient compliance.

Development of a curved, stratified, in vitro model to assess ocular biocompatibility.

Purpose To further improve in vitro models of the cornea, this study focused on the creation of a three-dimensional, stratified, curved epithelium; and the subsequent characterization and evaluation of its suitability as a model for biocompatibility testing. Methods Immortalized human corneal epithelial cells were grown to confluency on curved cellulose filters for seven days, and were then differentiated and stratified using an air-liquid interface for seven days before testing. Varying concentrations of a commercial ophthalmic solution containing benzalkonium chloride (BAK), a known cytotoxic agent, and two relevant ocular surfactants were tested on the model. A whole balafilcon A lens soaked in phosphate buffered saline (BA PBS) was also used to assess biocompatibility and verify the validity of the model. Viability assays as well as flow cytometry were performed on the cells to investigate changes in cell death and integrin expression. Results The reconstructed curved corneal epithelium was composed of 3–5 layers of cells. Increasing concentrations of BAK showed dose-dependent decreased cell viability and increased integrin expression and cell death. No significant change in viability was observed in the presence of the surfactants. As expected, the BA PBS combination appeared to be very biocompatible with no adverse change in cell viability or integrin expression. Conclusions The stratified, curved, epithelial model proved to be sensitive to distinct changes in cytotoxicity and is suitable for continued assessment for biocompatibility testing of contact lenses. Our results showed that flow cytometry can provide a quantitative measure of the cell response to biomaterials or cytotoxic compounds for both the supernatant and adherent cell populations. As a specifically designed in vitro model of the corneal epithelium, this quantitative model for biocompatibility at the ocular surface may help improve our understanding of cell-material interactions and reduce the use of animal testing.

In Vivo Imaging and Morphometry of the Human Pre-Descemet's Layer and Endothelium With Ultrahigh-Resolution Optical Coherence Tomography.

PURPOSE To visualize in vivo and quantify the thickness of the posterior corneal layers: the acellular pre-Descemets layer (PDL), Descemets membrane (DM), and endothelium (END) in healthy subjects, using ultrahigh-resolution optical coherence tomography (UHR-OCT). METHODS A research-grade, 800-nm UHR-OCT system with 0.95-μm axial resolution in corneal tissue was used to image in vivo the posterior cornea in healthy subjects. The system offers approximately 98 dB sensitivity for 680 μW optical power incident on the cornea and 34,000 A-scans/s image acquisition rate. This study comprised 20 healthy subjects, aged 20 to 60 years. The thickness of the PDL, DM, and END layers was measured both with a custom, automatic segmentation algorithm and manually. RESULTS The boundaries and structure of the posterior corneal layers were clearly visible in the UHR-OCT images. The average thickness was measured to be 6.6 ± 1.4 μm (PDL), 10.4 ± 2.9 μm (DM), and 4.8 ± 0.4 μm (END), which agrees well with published data from ex vivo studies. Both the END and DM thickness showed minor spatial variations, whereas the PDL showed up to 2× thickness change for different locations on the same cross-sectional corneal image or over the entire imaged region of the cornea. CONCLUSIONS Our data indicate that all three layers of the posterior cornea can be clearly visualized in vivo and their thicknesses measured precisely with UHR-OCT. Although the PDL thickness showed large spatial variations, the thickness of the DM and END layers was consistent over the entire imaged region of the cornea.

Sub-micrometer axial resolution OCT for in-vivo imaging of the cellular structure of healthy and keratoconic human corneas

Corneal degenerative conditions such as keratoconus (KC) cause progressive damage to the anterior corneal tissue and eventually severely compromise visual acuity. The ability to visualize corneal tissue damage in-vivo at cellular or sub-cellular level at different stages of development of KC and other corneal diseases, can aid the early diagnostics as well as the development of more effective treatment approaches for various corneal pathologies, including keratoconus. Here, we present the optical design of an optical coherence tomography system that can achieve 0.95 µm axial resolution in biological tissue and provide test results for the systems spatial resolution and sensitivity. Corneal images acquired in-vivo with this system from healthy and keratoconic human subjects reveal the cellular and sub-cellular structure of the corneal epithelium, as well as the normal and abnormal structure of the Bowmans membrane and the anterior corneal stroma.

In-vivo imaging of the palisades of Vogt and the limbal crypts with sub-micrometer axial resolution optical coherence tomography

A research-grade OCT system was used to image in-vivo and without contact with the tissue, the cellular structure and microvasculature of the healthy human corneo-scleral limbus. The OCT system provided 0.95 µm axial and 4 µm (2 µm) lateral resolution in biological tissue depending on the magnification of the imaging objective. Cross-sectional OCT images acquired tangentially from the inferior limbus showed reflective, loop-like features that correspond to the fibrous folds of the palisades of Vogt (POV). The high OCT resolution allowed for visualization of individual cells inside the limbal crypts, capillaries extending from the inside of the POVs fibrous folds and connecting to a lateral grid of micro-vessels located in the connective tissue directly below the POV, as well as reflections from individual red blood cells inside the capillaries. Difference in the reflective properties of the POV was observed among subjects of various pigmentation levels of the POV. Morphological features observed in the high resolution OCT images correlated well with histology. The ability to visualize the limbal morphology and microvasculature in-vivo at cellular level can aid the diagnostics and treatment of limbal stem cell dysfunction and dystrophies.

Prevalence of vision loss among hospital in-patients; a risk factor for falls?

Despite poor vision being a risk factor for falls, current hospital policies and practices often do not include a vision assessment at patient admission or in the hospitals incident reporting system when a fall occurs. Our purpose was to document the prevalence of vision loss in hospital general medicine units to increase awareness of poor vision as a potential risk factor for falls that occur within the hospital, and inform future preventative practice.

High speed UHR-OCT for in-vivo volumetric imaging of the palisades of Vogt and the cellular structure of the limbal crypts in the healthy and pathological human corneo-scleral limbus (Conference Presentation)

Limbal stem cell dysfunction (LSCD) causes morphological and physiological changes in the limbus that result in decreased vision, photophobia, tearing, chronic inflammation and hyperemia, recurrent episodes of pain, and blindness in severe cases. Currently, clinical in-vivo imaging of the palisaded of Vogt (POV) and the cellular structure of the limbal crypts in the human corneo-scleral limbus is accomplished by in-vivo confocal microscopy (IVCM). However, IVCM requires physical contact with the limbal tissue that can cause pain and inflammation. In this study, we used a novel high speed, ultra-high resolution optical coherence tomography (UHR-OCT) system to generate volumetric, cellular resolution image of the healthy and pathological human corneo-scleral limbus. The UHR-OCT system has a compact fiber-optic design. A femtosecond laser with 790 nm central wavelength and ~150 nm spectral bandwidth (at 3dB) was used to achieve ~1.4 µm axial resolution in biological tissue. The UHR-OCT system also utilizes a high resolution spectrometer (Cobra, Wasatch Photonics) connected to a novel line scan camera with a tall pixel design, 2048 pixel array and a maximum readout rate of 250 kHz. The system’s SNR was 96 dB at 100 µm away from the zero delay line, with ~10 dB roll-off over 1.5 mm scanning range for ~800 µm power of the imaging beam. Volumetric images of the POV and the cellular structure of the limbal crypts were acquired in-vivo and without contact with the limbal tissue from healthy and LSCD and subjects. This study was approved by the University of Waterloo Research Ethics Committee.

In-vivo volumetric imaging of the cellular structure of healthy and pathological human cornea with high-speed UHR-OCT (Conference Presentation)

Degenerative conditions such as keratoconus and Fuch’s dystrophy can alter over time the cellular structure of the human corneal epithelium and endothelium respectively. A high-speed UHR-OCT system, capable of generating volumetric images of the cellular structure of the human cornea was built. The UHR-OCT system has a compact fiber-optic design that utilizes a commercial femtolaser with the central wavelength of 790 nm and 3dB spectral bandwidth of 150 nm to achieve ~ 1.4 µm axial resolution in corneal tissue. The optical design of the OCT imaging probe ensured ~2 µm OCT lateral resolution in corneal tissue. At the detection end of the UHR-OCT system, a high-resolution spectrometer (Cobra, Wasatch Photonics) is interfaced with a novel line scan camera. The camera has a tall pixel design, 2048 pixel array and a maximum readout rate of 250 kHz. The system’s SNR was 96 dB at 100 µm away from the zero delay line, with a 10 dB roll-off over 1.5 mm scanning range for ~800 µm power of the imaging beam incident on the corneal surface. Volumetric images of healthy and pathological corneas were acquired in-vivo from healthy volunteers and subjects with keratoconus and Fuch’s dystrophy and the images were compared with typical histological images. This study was approved by the University of Waterloo Research Ethics Committee.