Sommaire L’oxygène fait partie intégrante de l’environnement et ceci jusqu’au fonctionnement même des structures cellulaires qui composent le corps humain. Deux systèmes sont intimement liés à la distribution de l’oxygène, ce sont les systèmes cardiovasculaire et respiratoire. La transparence du système optique de l’œil peut être exploitée afin de mesurer de façon non invasive la concentration en oxyhémoglobine du sang qui alimente le système nerveux central. L’oxygénation capillaire de l’œil a été mesurée par spectro-réflectométrie dans deux régions de l’œil: d’une part les capillaires de la zone du nerf optique qui représentent principalement la circulation rétinienne; d’autre part, les capillaires du limbe cornéen. Cinq sujets volontaires, non fumeurs, sains, âgés de 20 à 45 ans et cinq sujets volontaires, fumeurs, sains, âgés de 20 à 40 ans ont participé à cette étude. Tous ces sujets ont été exposés à des conditions d’hyper et d’hypo oxygénation. Une séance d’expérimentations était composée d’un enregistrement continu de 360 secondes. Durant la première étape de 60 secondes, le sujet respirait de l’air ambiant. Durant une deuxième étape de 180 secondes, le sujet était exposé soit à une condition d’hyper (60% O2) soit, à une condition d’hypo oxygénation (15% O2), tandis que les 120 dernières secondes de la séance de mesure permettait d’exposer le sujet, une fois de plus à l’air ambiant. Le rythme cardiaque et les changements d’oxygénation artérielle au niveau du doigt étaient mesurés pendant ce temps vec le sphygmo-oxymètre. Les variations du taux d’oxyhémoglobine du sang au niveau capillaire de l’œil (nerf optique ou sclérotique) étaient toujours en corrélation directe avec les variations du taux d’oxyhémoglobine artériel. Toutefois, les capillaires du nerf optique offrent plus de précision pour les mesures d’oxygénation, relativement aux mesures d’oxygénation du sang contenu dans les capillaires de la sclérotique. La précision de la mesure de la concentration d’oxyhémoglobine obtenue dans cette étude par spectro-réflectométrie de l’œil, en fait un instrument utile au diagnostic d’une grande partie des pathologies pulmonaires ou oculaires. / Abstract Oxygen is an integral part of the environment. It has a major role to play in the cellular structures of the human body. The two systems, which are closely related to the distribution of oxygen within the body are the cardiovascular and the respiratory systems. The transparency of the eye can be used as a way to non-invasively measure the concentration of oxyhemoglobin in the blood which feeds the central nervous system. The capillary oxygenation of the eye was measured using multichannel spectro-reflectometry technology. Two areas of the eye were studied: the capillaries of the optical nerve which represent retinal circulation as well as the capillaries of the eye at the corneal limbus. Five healthy non-smoking volunteers aged 20-45 years and five healthy smoking volunteers aged 20-40 years took part in this study. All subjects were exposed to hypo and hyper oxygenation conditions. The experimental session was composed of a continuous recording of 360 seconds. In the first 60 seconds, volunteers breathed ambient air, followed by a second phase of 180 seconds during which the subjects were either exposed to a condition of hyper (60% O2) or hypo oxygenation (15% O2). The final phase of the experimental session was an exposure to ambient air. Cardiac rhythm and arterial oxygenation were measured at the tip of the finger with a sphygmo-oxymeter. Clearly, variations in the oxyhemoglobin blood concentration in the capillaries of the optic nerve or the sclerotic are always directly correlated with variations in the arterial oxyhemoglobin levels. However, the optic nerve capillaries offer more precision for measurements of oxygenation as compared to the sclerotic capillaries. In this study, accurate information has been provided on the oxyhemoglobin concentration using spectro-reflectometry of the eye. This technology could become a tool in the diagnosis of various pulmonary or ocular pathologies.

Purpose: To determine the vascular reactivity (VR) capacity and visual function (VF) response to potent vasoconstrictor and vasodilatory provocations of retinal arterioles in healthy subjects. Methods: One hyperoxic hypocapnic and two graded hypoxic hypercapnic stimuli were administered. VR in response to gas provocation was assessed using the Canon Laser Blood Flowmeter. VF was assessed using high and low contrast ETDRS logMAR charts, Medmont C-100, and H.R.R. Pseudoisochromatic Plates. Results: Flow reduced by 23% (p=0.0001) during hyperoxic hypocapnia and increased by 18% (p=0.0129) during hypoxic hypercapnia. During hyperoxic hypocapnia, high contrast VA improved by -0.026 (p=0.0372). During hypoxic hypercapnia, high and low contrast VA were reduced (+0.033, p=0.0110; +0.025, p=0.0058, respectively). Colour vision was unaffected. Conclusions: The retinal arterioles demonstrated a greater capacity for vasoconstriction than vasodilation in response to the stimuli used in our study. Hyperoxic hypocapnia improved high contrast VA while hypoxic hypercapnia reduced high and low contrast VA.

Purpose: To determine the vascular reactivity (VR) capacity and visual function (VF) response to potent vasoconstrictor and vasodilatory provocations of retinal arterioles in healthy subjects. Methods: One hyperoxic hypocapnic and two graded hypoxic hypercapnic stimuli were administered. VR in response to gas provocation was assessed using the Canon Laser Blood Flowmeter. VF was assessed using high and low contrast ETDRS logMAR charts, Medmont C-100, and H.R.R. Pseudoisochromatic Plates. Results: Flow reduced by 23% (p=0.0001) during hyperoxic hypocapnia and increased by 18% (p=0.0129) during hypoxic hypercapnia. During hyperoxic hypocapnia, high contrast VA improved by -0.026 (p=0.0372). During hypoxic hypercapnia, high and low contrast VA were reduced (+0.033, p=0.0110; +0.025, p=0.0058, respectively). Colour vision was unaffected. Conclusions: The retinal arterioles demonstrated a greater capacity for vasoconstriction than vasodilation in response to the stimuli used in our study. Hyperoxic hypocapnia improved high contrast VA while hypoxic hypercapnia reduced high and low contrast VA.

Current anti-VEGF biologics, such as bevacizumab and VEGF trap, have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, their toxicity profile, including proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this PhD thesis was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of biologics were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup, “sticky-traps”, is composed of VEGF trap linked to a sequence of hydrophobic amino acids, with affinity for heparin sulfate proteoglycans of the ECM. The second subgroup, “lassos”, is composed of a C-terminus positioned form of VEGF trap linked to single-chain variable domain antibodies specific for either HER2 (HER2/V lasso) or fibronectin extra domain B (EDB; EDB/V lasso), expressed on breast cancer cell surfaces or in the vascular bed of solid tumours, respectively. ii Using a novel transgenic method, piggyBac transposons, biologics were expressed in transgenic cancer cell lines in a doxycycline inducible manner. They were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the sticky-traps adhered to the ECM and the HER2/V lasso inhibited the proliferation of HER2 positive cancer cell lines. Sticky-traps as well as lassos were able to inhibit or delay tumour growth of A-673, Pc-3, SKOV-3 and HT-29 xenografts. In contrast to soluble VEGF trap, sticky-traps were retained at the tumour site and were undetectable in the circulation. Moreover, sticky-traps, in contrast to VEGF trap, did not delay wound healing and regression of trachea blood vessels. Furthermore, transgenic studies indicated that HER2/V lasso is more effective compared to anti-HER2 Ab and VEGF trap used alone or in combination. These novel classes of antiangiogenic molecules could be advantageous in a clinical setting. Using the principles established in my PhD thesis work, similar dual function biologics can be designed for inhibition of other molecules with disease relevance.

Two novel personal calibration procedures that do not require active user participation are presented. These procedures, in conjunction with a state-of-the-art remote eye-gaze tracking (REGT) technology, allow estimation of the angle between the optical and visual axes (angle kappa) automatically without explicit/active user involvement. The first algorithm for the binocular estimation of angle kappa (BEAK) is based on the assumption that at each time instant both eyes look at the same point on a surface with a known geometry (e.g., a computer monitor). The sensitivity of the BEAK procedure to the geometry of the observation surface and to the noise in the estimates of the optical axis is studied both analytically and in computer simulations. Experimental results with 4 adult subjects suggest that with the current REGT technology angle kappa can be estimated with an RMS error of 0.5°. The second personal calibration algorithm (‘calibrate and validate’, CaVa) adopts a probabilistic approach to the estimation of angle kappa in infants. Even though the presentation of visual stimuli at known positions is part of the procedure, the CaVa algorithm does not require/assume continuous fixation on the presented targets. If an infant attends to roughly half of the presented targets, angle kappa can be estimated accurately and with high confidence. In experiments with five babies, the average difference between repeated measurements of angle kappa was 0.04 ± 0.31°. The second part of the thesis describes two methods for automated measurement of eye misalignment in adults and infants. These methods are based on the user-calibration-free (UCF) technology presented in the first part of the thesis. The first method is based on the clinical Hirschberg test. It is shown that the UCF-REGT technology can improve significantly the accuracy of the Hirschberg test by enabling the estimation of subject-specific parameters (the Hirschberg ratio and angle kappa) in infants. The maximum error in the estimation of the horizontal and vertical components of eye misalignment in five orthotropic infants was shown to be less than 1°, which is significantly better than the accuracy of a standard clinical Hirschberg test. Finally, a novel Eye-Tracker Based Test (ETBT) for the estimation of the maximum (manifest + latent) angle of deviation is presented. ETBT is based on the UCF-REGT system. ETBT allows free head movements and does not require continuous fixation on specific targets. Experiments with 22 adult subjects demonstrated a good agreement of 0.7 ± 1.7° between ETBT and the gold-standard clinical procedure—the altenate prism and cover test. A pilot study with 5 orthotropic infants and one infant with strabismus demonstrated that the ETBT can be used in infants.

Current anti-VEGF biologics, such as bevacizumab and VEGF trap, have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, their toxicity profile, including proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this PhD thesis was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of biologics were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup, “sticky-traps”, is composed of VEGF trap linked to a sequence of hydrophobic amino acids, with affinity for heparin sulfate proteoglycans of the ECM. The second subgroup, “lassos”, is composed of a C-terminus positioned form of VEGF trap linked to single-chain variable domain antibodies specific for either HER2 (HER2/V lasso) or fibronectin extra domain B (EDB; EDB/V lasso), expressed on breast cancer cell surfaces or in the vascular bed of solid tumours, respectively. ii Using a novel transgenic method, piggyBac transposons, biologics were expressed in transgenic cancer cell lines in a doxycycline inducible manner. They were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the sticky-traps adhered to the ECM and the HER2/V lasso inhibited the proliferation of HER2 positive cancer cell lines. Sticky-traps as well as lassos were able to inhibit or delay tumour growth of A-673, Pc-3, SKOV-3 and HT-29 xenografts. In contrast to soluble VEGF trap, sticky-traps were retained at the tumour site and were undetectable in the circulation. Moreover, sticky-traps, in contrast to VEGF trap, did not delay wound healing and regression of trachea blood vessels. Furthermore, transgenic studies indicated that HER2/V lasso is more effective compared to anti-HER2 Ab and VEGF trap used alone or in combination. These novel classes of antiangiogenic molecules could be advantageous in a clinical setting. Using the principles established in my PhD thesis work, similar dual function biologics can be designed for inhibition of other molecules with disease relevance.

Two novel personal calibration procedures that do not require active user participation are presented. These procedures, in conjunction with a state-of-the-art remote eye-gaze tracking (REGT) technology, allow estimation of the angle between the optical and visual axes (angle kappa) automatically without explicit/active user involvement. The first algorithm for the binocular estimation of angle kappa (BEAK) is based on the assumption that at each time instant both eyes look at the same point on a surface with a known geometry (e.g., a computer monitor). The sensitivity of the BEAK procedure to the geometry of the observation surface and to the noise in the estimates of the optical axis is studied both analytically and in computer simulations. Experimental results with 4 adult subjects suggest that with the current REGT technology angle kappa can be estimated with an RMS error of 0.5°. The second personal calibration algorithm (‘calibrate and validate’, CaVa) adopts a probabilistic approach to the estimation of angle kappa in infants. Even though the presentation of visual stimuli at known positions is part of the procedure, the CaVa algorithm does not require/assume continuous fixation on the presented targets. If an infant attends to roughly half of the presented targets, angle kappa can be estimated accurately and with high confidence. In experiments with five babies, the average difference between repeated measurements of angle kappa was 0.04 ± 0.31°. The second part of the thesis describes two methods for automated measurement of eye misalignment in adults and infants. These methods are based on the user-calibration-free (UCF) technology presented in the first part of the thesis. The first method is based on the clinical Hirschberg test. It is shown that the UCF-REGT technology can improve significantly the accuracy of the Hirschberg test by enabling the estimation of subject-specific parameters (the Hirschberg ratio and angle kappa) in infants. The maximum error in the estimation of the horizontal and vertical components of eye misalignment in five orthotropic infants was shown to be less than 1°, which is significantly better than the accuracy of a standard clinical Hirschberg test. Finally, a novel Eye-Tracker Based Test (ETBT) for the estimation of the maximum (manifest + latent) angle of deviation is presented. ETBT is based on the UCF-REGT system. ETBT allows free head movements and does not require continuous fixation on specific targets. Experiments with 22 adult subjects demonstrated a good agreement of 0.7 ± 1.7° between ETBT and the gold-standard clinical procedure—the altenate prism and cover test. A pilot study with 5 orthotropic infants and one infant with strabismus demonstrated that the ETBT can be used in infants.

Sommaire L’oxygène fait partie intégrante de l’environnement et ceci jusqu’au fonctionnement même des structures cellulaires qui composent le corps humain. Deux systèmes sont intimement liés à la distribution de l’oxygène, ce sont les systèmes cardiovasculaire et respiratoire. La transparence du système optique de l’œil peut être exploitée afin de mesurer de façon non invasive la concentration en oxyhémoglobine du sang qui alimente le système nerveux central. L’oxygénation capillaire de l’œil a été mesurée par spectro-réflectométrie dans deux régions de l’œil: d’une part les capillaires de la zone du nerf optique qui représentent principalement la circulation rétinienne; d’autre part, les capillaires du limbe cornéen. Cinq sujets volontaires, non fumeurs, sains, âgés de 20 à 45 ans et cinq sujets volontaires, fumeurs, sains, âgés de 20 à 40 ans ont participé à cette étude. Tous ces sujets ont été exposés à des conditions d’hyper et d’hypo oxygénation. Une séance d’expérimentations était composée d’un enregistrement continu de 360 secondes. Durant la première étape de 60 secondes, le sujet respirait de l’air ambiant. Durant une deuxième étape de 180 secondes, le sujet était exposé soit à une condition d’hyper (60% O2) soit, à une condition d’hypo oxygénation (15% O2), tandis que les 120 dernières secondes de la séance de mesure permettait d’exposer le sujet, une fois de plus à l’air ambiant. Le rythme cardiaque et les changements d’oxygénation artérielle au niveau du doigt étaient mesurés pendant ce temps vec le sphygmo-oxymètre. Les variations du taux d’oxyhémoglobine du sang au niveau capillaire de l’œil (nerf optique ou sclérotique) étaient toujours en corrélation directe avec les variations du taux d’oxyhémoglobine artériel. Toutefois, les capillaires du nerf optique offrent plus de précision pour les mesures d’oxygénation, relativement aux mesures d’oxygénation du sang contenu dans les capillaires de la sclérotique. La précision de la mesure de la concentration d’oxyhémoglobine obtenue dans cette étude par spectro-réflectométrie de l’œil, en fait un instrument utile au diagnostic d’une grande partie des pathologies pulmonaires ou oculaires. / Abstract Oxygen is an integral part of the environment. It has a major role to play in the cellular structures of the human body. The two systems, which are closely related to the distribution of oxygen within the body are the cardiovascular and the respiratory systems. The transparency of the eye can be used as a way to non-invasively measure the concentration of oxyhemoglobin in the blood which feeds the central nervous system. The capillary oxygenation of the eye was measured using multichannel spectro-reflectometry technology. Two areas of the eye were studied: the capillaries of the optical nerve which represent retinal circulation as well as the capillaries of the eye at the corneal limbus. Five healthy non-smoking volunteers aged 20-45 years and five healthy smoking volunteers aged 20-40 years took part in this study. All subjects were exposed to hypo and hyper oxygenation conditions. The experimental session was composed of a continuous recording of 360 seconds. In the first 60 seconds, volunteers breathed ambient air, followed by a second phase of 180 seconds during which the subjects were either exposed to a condition of hyper (60% O2) or hypo oxygenation (15% O2). The final phase of the experimental session was an exposure to ambient air. Cardiac rhythm and arterial oxygenation were measured at the tip of the finger with a sphygmo-oxymeter. Clearly, variations in the oxyhemoglobin blood concentration in the capillaries of the optic nerve or the sclerotic are always directly correlated with variations in the arterial oxyhemoglobin levels. However, the optic nerve capillaries offer more precision for measurements of oxygenation as compared to the sclerotic capillaries. In this study, accurate information has been provided on the oxyhemoglobin concentration using spectro-reflectometry of the eye. This technology could become a tool in the diagnosis of various pulmonary or ocular pathologies.

No description available.

Master of Science / Department of Clinical Sciences / Amy J. Rankin / Objective – To measure blood aqueous-barrier breakdown following aqueocentesis using various needle sizes and to monitor the intraocular pressure (IOP) response. Animals – 24 healthy, adult dogs received treatment (24 treated eyes, 24 contralateral eyes); 3 dogs were untreated controls (6 control eyes). Procedures – Dogs receiving treatment were divided into 3 equal groups (25-, 27-, or 30- gauge needle aqueocentesis). In each dog the treated eye was determined randomly, the contralateral eye was untreated. Dogs that did not have aqueocentesis performed in either eye were used as controls. Aqueocentesis at the lateral limbus was performed under sedation and topical anesthesia. Anterior chamber fluorophotometry was performed before and after aqueocentesis on day 1. On days 2-5 sedation and fluorophotometry were repeated. Intraocular pressure was measured with a rebound tonometer at multiple time points. Results – Aqueocentesis resulted in blood-aqueous barrier breakdown in all treated eyes with barrier reestablishment present by day 5 detected by fluorophotometry. On day 2 the contralateral untreated eyes of all groups also showed statistically significant increased fluorescence (P < 0.05) following treatment of the opposite eye, but these values were not statistically significantly greater than untreated controls. In treated eyes there was no statistical difference in fluorescein concentration or IOP between 27- and 30-gauge needles. Use of the 25- gauge needle resulted in a statistically significant increase in anterior chamber fluorescence on days 3 and 5. It also caused a statistically significant increase in IOP 20 minutes following aqueocentesis as compared to the 27- and 30-gauge needles. Aside from this transient ocular hypertension, rapid resolution of ocular hypotony following aqueocentesis was observed in all treatment groups. Conclusions and Clinical Relevance – Aqueocentesis using a 25-gauge needle resulted in a greater degree of blood-aqueous barrier breakdown and a brief state of intraocular hypertension following paracentesis. Use of a 27- or 30-gauge needle is recommended for aqueous paracentesis. A consensual ocular reaction appeared to occur in dogs following unilateral traumatic blood-aqueous barrier breakdown and may be of clinical significance. Statistical significance was limited in this study due to high variability and large standard deviations.