On the Indeterminates of Glaucoma:the Controversy of Arterial Blood Pressure and Retinal Perfusion

Glaucoma is a chronic eye disease characterized by thinning of the retina, death of ganglion cells, and progressive loss of vision, eventually leading to blindness. The prevalence of glaucoma is estimated at 1-3% of those over 40 years old. With a constantly aging population, this number is expected to increase significantly over the next 10 years. Even with treatment, about 15% of people with glaucoma currently develop residual vision or tunnel vision and eventually become blind or partially sighted. The mechanisms behind ganglion cell death are poorly understood. Elevated eye pressure is the main risk factor for glaucoma, but treatment in the form of medication, laser, or surgery can only slow the decline, not stop it. In addition, high intraocular pressure is neither necessary nor sufficient for the development of glaucoma, indicating the existence of other unknown risk factors. It has been established that the death of ganglion cells results in a decreased oxygen demand and a concomitant decrease in blood flow. However, there is also a hypothesis that reduced or unstable blood supply is not only a consequence, but also a cause of glaucoma. This is known as the ‘chicken-egg’ dilemma in glaucoma. It is supported by the observation that the risk of developing glaucoma is higher in people with very low blood pressure (sometimes even as a result of overtreatment of high blood pressure).This dissertation is an attempt to methodically examine whether blood pressure can be linked to changes in the retina that could suggest susceptibility to glaucoma. For this purpose, we analyze epidemiological data from the Groningen Longitudinal Glaucoma Study, we use advanced imaging techniques to model the microcirculation, and we describe its relationship with the neural structure and oxygen consumption of the retina. We provide evidence leaning towards the existence of a vascular component, likely pertinent to glaucoma

Retinal and choroidal oximetry using snapshot multispectral imaging

The principal aim of the research was to validate and develop a non-invasive multispectral imaging technique of measuring oxygen saturation in retinal and choroidal vasculature. The Image replicating imaging spectrometer (IRIS) snapshot multispectral fundus camera used for retinal imaging, and image analysis protocols used to perform oximetry are described. In vitro and in vivo validation of oximetry techniques was performed. For in vitro validation an artificial eye containing blood at varying oxygenation was used. For in vivo validation the pig was used as an animal model. The calculated oxygen saturation was compared to blood gas analysis (gold standard) results and was found to be in close agreement. Retinal oximetry was performed on healthy human subjects. The average oxygen saturation value (± SD) for retinal arterioles and venules were 96.08% ± 1.9% and 68.04% ± 2.1%, respectively. The application of retinal oximetry technique was explored by conducting human hypoxia trials, in which the effect of acute mild hypoxia on retinal oxygenation and autoregulation was assessed on healthy human subjects. Hypoxic exposure resulted in a decline in both retinal arterial and venous saturation, as well as a significant increase in retinal vessel calibre, suggesting an autoregulatory response. This thesis also explored the possibility of exploiting fundus reflections to measure the choroidal oxygenation non-invasively. Fundus reflection intensity at two wavelengths, 780 nm and 800 nm (oxygen sensitive and isosbestic) were used to calculate intensity ratio (R), which is directly proportional to the blood oxygenation. A pilot study on 10 healthy humans was conducted. Fundus reflection was recorded at room air (normoxia) and 15% inspired oxygen (mild hypoxia). A significant reduction (P < 0.001) in intensity ratio was observed during hypoxia

Studying fatty acid content in red blood cell membranes in diabetic retinopathy of type 2 diabetes patients

Introduction. Studying pathogenetic pathways that are formed under conditions of hyperglycaemia and trigger further metabolic changes resulting in diabetes complications over time are of special interest both in theoretical and practical medicine. Membrane of the red blood cell that stays in blood constantly and relatively durable is an obvious participant of all metabolic changes and cannot but reflect changes in metabolism. Objective: to study content of fatty acids (FAs) in red blood cell membranes of patients with diabetic retinopathy (DR) with type 2 diabetes (T2D). Materials and methods. The study enrolled 73 subjects maximally matched by age and gender: 50 patients with established long-lasting T2D complicated with DR and 23 patients in the control group. All the biochemical tests were performed at the certified laboratory under the standard methods. Gas chromatography was applied to study FA spectrum. Statistical analysis was performed in IBM SPSS Statistics 23. Results and discussion. The key difference between the lipid metabolism of patients with complicated long-lasting T2D and healthy subjects was a significant difference in FA content redistribution in the red blood cell membranes represented by increased “saturation”. The content of saturated FAs (SAFAs)in patients with DR was higher than in the CG: 1.5-fold for palmitic (С:16), 2-fold for myristic(С:14), pentadecanoic (С:15) and margaric (С:17) (Р &lt; 0.05). The content of saturated stearic (С:18) FA was not changed significantly.The content of unsaturated FAs(USFAs) have changed multidirectionally: the content of linoleic (С18:2) and arachidonic (С20:4) decreased 1.5-1.7-fold, respectively. The content of linolenic (С18:3) increased 2-fold, and content of oleic FA (С18:1) was not changed significantly. Conclusion. Study of the fluctuations of FA levels in red blood cell membranesplays animportant role in the development of insulin resistance, T2D and its microvascular complicationsthat can be applied in creating treatment and prevention strategies ofDRin type 2 diabetes patients

The Retinal Microvasculature in Secondary Progressive Multiple Sclerosis

In light of new data regarding pathology of multiple sclerosis (MS), more research is needed into the vascular aspects of the disease. Demyelination caused by inflammation is historically thought of as the main cause of disability in the disease. Recent studies, however, have suggested that MS is in fact a spectrum of overlapping phenotypes consisting of inflammation, oxidative damage and hypoperfusion. The microvasculature plays an important role in all of these pathogenic processes and its dysfunction may therefore be of crucial importance to the development and progression of the disease. This thesis focuses on investigating the microvasculature of the retina as a surrogate for the brain by assessing the vascular structure, blood flow dynamics and oxygen transfer of the retinal blood vessels in secondary progressive multiple sclerosis (SPMS). Studying the retinal microvasculature using a multimodal imaging approach has allowed us to develop a more detailed understanding of blood flow in MS and to identify new imaging markers for trials into neuroprotective drugs in MS. The work done in this thesis demonstrated; i) a higher rate of retinal microvascular abnormalities in MS which progresses with disease severity, ii) evidence of retinal vascular remodelling in SPMS and iii) changes in blood velocity and flow in the retina in SPMS. These observations pave the way for future investigations into the mechanisms of vascular alterations and vascular dysfunction in MS, and provide a set of imaging markers to further explore other cerebrovascular diseases through the retina

Phototherapeutic Devices for the Treatment of Diabetic Retinopathy

Diabetic retinopathy is a microvascular disease of the retina and a leading cause of vision loss worldwide. In the non-proliferative phase, diabetes-induced degradation of the retinal blood supply leads to edema and progressive tissue hypoxia. In response, the retinal tissue expresses proangiogenic growth factors (e.g. vascular endothelial growth factor), which drive proliferation of aberrant blood vessels within the eye. These poorly formed vessels leak fluid and blood cells into the eye and grow into the vitreous, which puts traction on the retina and leads to detachment. Given the hypoxic etiology, retinal oxygen tension and metabolism have received considerable attention. Dark-adapted conditions drive the retina to a significantly lower oxygen tension compared to light- adapted conditions as rod cells consume more energy in order to boost sensitivity. While tolerable in the healthy retina, it has been hypothesized that increased nightly metabolism overwhelms the compromised oxygen supply in the diabetic retina, leading to hypoxia and pathological vascular endothelial growth factor expression. This thesis develops ocular devices that shine light onto the retina to modulate rod metabolism, reducing oxygen demand and mitigating nightly hypoxia. The phototherapeutic effect is characterized through mathematical modeling of retinal metabolism and in vivo testing. Implantable phototherapy devices are designed, fabricated, and evaluated. This thesis also develops overnight phototherapeutic contact lenses utilizing radioluminescence, chemiluminescence, and electroluminescence approaches. Phototherapy holds promise as a non-invasive, preventative therapy for the treatment of hypoxic retinal diseases such as diabetic retinopathy.</p

Retinal Blood Flow and Markers of Vascular Inflammation and Endothelial Dysfunction in Type 2 Diabetes

Abnormal leukocyte adhesion (i.e. leukostasis) to retinal vascular endothelial cells occurs in early diabetes. The processes of leukostasis have been clearly demonstrated in the vascular endothelium of patients with diabetes. In non-proliferative DR, clinical outcomes are manifested by excessive permeability from inflammatory progression leading to inner blood retinal barrier disruption, endothelial cell damage and widespread capillary nonperfusion. Diabetes promotes vascular leakage in DR by upregulation of adhesion molecules. Moreover, many of the pathological changes in NPDR are related to abnormalities in retinal blood flow. Studies have shown that specific circulating markers of inflammatory activity and endothelial dysfunction are associated with clinical signs of diabetic retinopathy. However, few have found an association between circulating levels of inflammatory and endothelial dysfunctional markers and abnormal retinal hemodynamics in patients with non-proliferative DR. The specific aims of this thesis are as follows: (Chapter 3)To correlate baseline levels of inflammatory and endothelial dysfunction markers and 1) baseline retinal arteriolar hemodynamics and 2) any disturbance in retinal hemodynamics over 6-month time in terms of vessel diameter, blood velocity, maximum-to-minimum velocity ratio and volumetric flow. In Chapter 4: To correlate circulating levels of inflammatory and endothelial dysfunction markers and 1) baseline vascular reactivity and 2) any disturbance in vascular reactivity after 6-month time in terms of vessel diameter, blood velocity, maximum-to-minimum velocity ratio and volumetric flow in patients with increasing non-proliferative diabetic retinopathy (NPDR) severity. Methods for Chapter 3: Diabetes subjects were stratified into either mild-to-moderate (Group 2) or moderate-to-severe (Group 3) NPDR based on their retinopathy status. Age-matched non-diabetics were recruited as controls (Group 1). Forearm blood sample was collected to determine baseline levels of inflammatory and endothelial dysfunctional markers. At visit 1, baseline retinal hemodynamics was acquired using Canon Laser Blood Flowmeter. Patients returned for a visit 2 (6 month follow-up visit) and retinal hemodynamics was reassessed. Baseline levels of inflammatory and endothelial dysfunctional markers compared between groups and correlated with both baseline and change in retinal hemodynamic parameters over 6-month time. For Chapter 4: Diabetes subjects were stratified into either mild-to-moderate NPDR or moderate-to-severe NPDR based on their retinopathy status. Age-matched non-diabetics were recruited as controls. At visit 1, forearm blood sample was collected to determine levels of inflammatory and endothelial dysfunctional markers and baseline vascular reactivity response was acquired. Retinal blood flow data was acquired while subjects breathed air. Retinal blood flow measurements were then acquired after exposure to isocapnic hyperoxic stimuli. At visit 2 (6 month follow-up), retinal vascular reactivity was reassessed. Baseline levels of inflammatory and endothelial dysfunctional markers compared between groups and correlated with both magnitude of baseline and change in vascular reactivity in terms of retinal hemodynamics. Results of Chapter 3: Maximum-to-minimum velocity ratio (max: min) was found to be significantly elevated in the group 3 compared to group 1 at baseline (0.72 vs. 0.49, after Bonferroni correction P<0.01). Both sICAM-1 and sE-selectin were significantly elevated as a function of group (ANOVA p=0.02 and p=0.04). A post hoc Bonferroni test showed that Group 3 had significantly higher in both sICAM-1 and sE-selectin levels compared to Group 1 (234.0 vs. 151.5 ng/ml, P=0.02 and 53.4 vs. 27.6 ng/ml, P<0.01, respectively). Hemoglobin A1c was significantly elevated across the groups (ANOVA p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher hemoglobin A1c level compared to Group 1 (7.9 vs. 5.6 % , P<0.01). There were no significant associations found between baseline markers of inflammation and baseline retinal hemodynamics across all groups. The Δ velocity was correlated with the baseline sICAM-1 (r=0.42, p=0.02) and A1c levels (r=0.37, p=0.04) in patients with NPDR. After adjustment for all other variables (A1c, hsCRP and vWF), Δ velocity, sICAM-1 and A1c were found not to be reliable predictors of baseline retinal hemodynamics. For Chapter 4: There were no significant differences in magnitude of retinal vascular reactivity in hemodynamic parameters between groups at visit 1 or visit 2. Over 6 months time, compliance was found to be significantly reduced in patients of Group 3 compared to Group 2 (-0.4 vs. 0.1, t-test p<0.01). Both sICAM-1 and sE-selectin were significantly elevated as a function of group (ANOVA p=0.02 and p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher in both sICAM-1 and sE-selectin levels compared to Group 1 (243.4 vs. 157.3ngml, P<0.01 and 57.0 vs. 29.3 ng/ml, P<0.01, respectively). Hemoglobin A1c was significantly elevated across the groups (ANOVA p<0.01). A post hoc Bonferroni test showed that Group 3 had significantly higher hemoglobin A1c level compared to Group 1 (8.8 vs. 5.6 % , P<0.01). Baseline VR in blood velocity weakly correlates with sE-selectin (r=0.31, p=0.04) across all groups while sVCAM-1 was associated with VR in terms of blood flow (r=-0.62, p<0.01) in patients with mild-to-moderate NPDR. The ∆ blood flow after 6 months was found to be weakly associated with sE-selectin (r=0.46, p=0.03) across all groups. Finally, the ∆ blood velocity after 6 month time was found to be moderately correlated with baseline vWF Ag level (r=-0.78, p=0.02). Multiple regression analysis found that vascular inflammatory and endothelial function markers had weak predictive power for Δ hemodynamic parameters. Conclusions Chapter 3: We found weak associations between circulating markers and baseline or the disturbance in retinal hemodynamics after 6 months time. Overall, we found both an increase in rigidity of the arteriolar circulation and elevated inflammatory adhesion markers (sICAM-1 and sE-selectin) within the same population sample. Change in velocity over the follow-up period was correlated with sICAM-1 and A1c levels in patients with NPDR but the level of association was such that neither sICAM-1 nor A1c proved to reliably predict retinal hemodynamics. Finally, in Chapter 4 we demonstrated two important characteristics in early NPDR; 1) a disturbance in vascular reactivity in terms of compliance and 2) an increase in systemic markers of inflammation were found in patients with NPDR. Although systemic markers of vascular inflammation and endothelial dysfunction are not predictive of hemodynamic parameters, our study found moderate associations between baseline and disturbances in VR after 6 months time. Therefore, there is evidence that inflammation and vascular function may be related with respect to their development in NPDR

Human retinal oximetry using hyperspectral imaging

The aim of the work reported in this thesis was to investigate the possibility of measuring human retinal oxygen saturation using hyperspectral imaging. A direct non-invasive quantitative mapping of retinal oxygen saturation is enabled by hyperspectral imaging whereby the absorption spectra of oxygenated and deoxygenated haemoglobin are recorded and analysed. Implementation of spectral retinal imaging thus requires ophthalmic instrumentation capable of efficiently recording the requisite spectral data cube. For this purpose, a spectral retinal imager was developed for the first time by integrating a liquid crystal tuneable filter into the illumination system of a conventional fundus camera to enable the recording of narrow-band spectral images in time sequence from 400nm to 700nm. Postprocessing algorithms were developed to enable accurate exploitation of spectral retinal images and overcome the confounding problems associated with this technique due to the erratic eye motion and illumination variation. Several algorithms were developed to provide semi-quantitative and quantitative oxygen saturation measurements. Accurate quantitative measurements necessitated an optical model of light propagation into the retina that takes into account the absorption and scattering of light by red blood cells. To validate the oxygen saturation measurements and algorithms, a model eye was constructed and measurements were compared with gold-standard measurements obtained by a Co-Oximeter. The accuracy of the oxygen saturation measurements was (3.31%± 2.19) for oxygenated blood samples. Clinical trials from healthy and diseased subjects were analysed and oxygen saturation measurements were compared to establish a merit of certain retinal diseases. Oxygen saturation measurements were in agreement with clinician expectations in both veins (48%±9) and arteries (96%±5). We also present in this thesis the development of novel clinical instrument based on IRIS to perform retinal oximetry.Al-baath University, Syri

Oxygen Transporter and Generator Devices to Treat Diabetic Retinopathy

In recent years, Micro-Electrical Mechanical Systems (MEMS) have opened new areas of the human body to non-pharmacological treatment. Miniaturized implants have started to appear in volume or power constrained areas, such as the eye and the heart. In particular, the eye benefits from miniaturization, as it is very sensitive to pressure and volumetric changes, which can affect eyesight and blood flow. Diabetic retinopathy is the worldwide leading cause of blindness among working age adults. As the numbers of diabetics increases, so does the number of retinopathies. By 2030, 191 million people are expected to be affected by the disease. As a patient’s retinopathy progresses, the chronic hyperglycemia from diabetes causes permanent changes to the vasculature; vessels become leaky and occluded, tissue becomes hypoxic due to this ischemia and begins to release vascular endothelial growth factor (VEGF) to promote angiogenesis. Currently, treatments exist only for severe non-proliferative or proliferative DR, and rely on blocking VEGF (vascular endothelial growth factor) or panretinal laser photocoagulation to reduce retinal metabolic demand. VEGF antagonists are expensive; costing up to $164k per quality life adjusted year and must be administered by intravitreal injections monthly. Laser photocoagulation also requires retreatment and is known to reduce peripheral vision—up to 20% of the peripheral retina is ablated. Another treatment approach may be to supply oxygen. Oxygen is a strong vasoconstrictor and suppresses the hypoxic signaling that leads to release of VEGF. These two effects reduce the plasma volume leaked into tissue, which in turn reduces edema, and may help prevent ischemic related cell death. Literature supports this assertion. A study of nasally inspired oxygen in patients with macular edema showed a reduction of edema and improvement of visual acuity following 3 months of treatment. Another study on rabbits with an induced ischemia demonstrated that intravitreal oxygenation maintained the retina to a near healthy condition. In this thesis, two devices, the oxytransporter and oxygenerator, that treat diabetic retinopathy are designed and tested. The former shuttles oxygen from areas of high concentration to the ischemic retina. The latter generates oxygen by electrolysis. This thesis is grounded on a computational model of oxygen consumption in the retina. To estimate the oxygen consumption, the model accounts for the anatomical distribution of tissue and vasculature in the retina. Previous models in literature averaged over the effects in the inner retina. The model estimates that the devices must supply 0.25nmol/s of oxygen to the human macula with an oxygen tension dependent on the degree of ischemia. A nanoporous filler material was developed and integrated into the oxytransporter to allow this device to operate in the high humidity environment of the eye. The material is capable of withstanding an environment with water vapor 1.4 times the bulk saturation pressure. Theory behind the material was tested and compared to simulation. Benchtop testing over a month demonstrated the stability of the device in conditions similar to the eye. This oxytransporter was implanted in rabbits and the diffusor, or output membrane, reached the favorable mark of 100mmHg in the vitreous humor from atmospheric oxygen alone. This is estimated to be sufficient to treat a mild to moderate ischemia in humans. The oxygenerator is powered from a coil up to 3cm away, and can provide 0.25nmol/s continuously with an oxygen tension of up to 300mmHg for a human sized diffusor. A steady state test demonstrated the capability of maintaining the oxygen tension in the device by modulating the input power. The device is replenished through osmosis from the vitreous humor, and can absorb moisture at a rate comparable to the required oxygen consumption. One week implantation in vivo in rabbits demonstrated that the oxygen tension exceeded 200mmHg at the diffusor, which is estimated to be sufficient to treat severe ischemia. Future work should involve a study of the long term effects of oxygen in an ischemic animal model.</p