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1 3. LITERATURE REVIEW 3. A Influence of environmental factors, demographics and genes on retinal vascular morphology Residing in regions with higher air pollution concentrations and experiencing daily increases in air pollution were each associated with narrower retinal arteriolar diameters in older individuals; (Adar et.al.,2010) findings which support the hypothesis that important vascular phenomena are associated with small increases in short or long-term air pollution exposures, even at current exposure levels, and further corroborate reported associations between air pollution and the development and exacerbation of clinical cardiovascular disease. Smoking has been found related to larger retinal arterial and venous diameters in three large population studies on vascular abnormalities(Ikram et al.,2004;Kleinet al.,2003).Such a tendency was also found in a recent Danish Twin Study(Taarnhoj et al.,2006). The underlying mechanism may be that high carbon monoxide levels in smokers cause decreased oxygen delivery to the retina, resulting in retinal hypoxia, which causes vessel dilation. Even though sporadically reported in certain studies,there are yet no conclusive or unequivocal data on the association between retinal vessel calibres and alcohol consumption, or with estrogen replacement therapy in women, or with the use of ocular betablockers, or the use of systemic antihypertensive medications. Because of various systemic, environmental and genetic effects, it is challenging to define uniform reference values that can be applied across populations even though it has already been shown that separate reference values are needed for children, (Mitchell et al.,2007) . The results of a 2008 study on Danish Twins (Taarnhoj et al.,2006) suggests that genetic factors determine much of the variation in retinal vessel calibres. Theresesrchers quoted that there is a large variation intortuosity of retinal 2 arteries in healthy subjects and the predominant determinants are genetic, accounting for 82% of the observed variations.A genome-wide linkage study of the Beaver Dam population established that retinal artery and vein diameters were linked to multiple gene loci in regions associated with hypertension, endothelial dysfunction, and vasculogenesis. (Xing et al.,2006). Generalized retinal arterial narrowing was associated with present or past blood pressure and increasing age; with the arterial diameters decreasing by 2.3 to 4.8 micrometer per decade while the mean retinal arterial diameters were higher in women than in men across all age groups (Wong & Michelle,2004). Unique biometric technology based retinal recognition is now being deployed in commercial identification applications through retinal scanning devices. Two famous studies have confirmed the uniqueness of the blood vessel pattern found in the human retina. The first one was published by Dr Carleton Simon and Dr Isodore Goldstein (Simon & Goldstein 1935), and describes how every retina contains a unique blood vessel pattern. In another paper, they even suggested usage of photographs of these patterns as a means of personal identification. The second study was conducted during the 1950s by Dr Paul Towerwho discovered that - even among identical twins - the blood vessel patterns of the retina are unique and different(Tower,1950).It is likely that genetic, systemic and environmental factors proactively interact to contribute to variations in retinal artery and veins and assessing these specific factors in new studies may allow for a greater understanding, prevention and treatment of complex vascular diseases in humans in the future. 3 3B. Retinal vascular signs: Systemic associations and health implications The retinal vasculature is the only microcirculation that can be visualized in vivo in humans and analysis of the retinal vessels can provide potentially valuable disease biomarkers.Retinal microvascular abnormalities, like generalized / focal arteriolar narrowing, branching pattern, tortuoisity, arteriovenous nicking and retinopathy reflect cumulative vascular damage from aging, hypertension, diabetes, inflammations, infections and other systemic phenomenon. These abnormalities can be observed in 2-15% of non symptomatic general population and are often the first indicators of underlying pathology(Pose et al.,2005). Generalized arteriolar narrowing and arteriovenous nicking appear to be irreversible long-term markers of hypertension, related to current as well as past blood pressure levels. There is an association between retinal microvascular abnormalities and stroke. Retinal vasculature is a useful risk indicator for impending cerebrovascular diseases. The retina and brain are highly metabolically active tissues with increased demands on metabolic substrates via specialized vascular networks. Embryologically, the human retina is an extension of diencephalon, and both these organs share a similar vascularization pattern during development ( Lutty et al. 2002). There is a close anatomical relationship between the macrovascular and microvascular blood supply to the brain and retina, and both networks share similar vascular regulatory processes (Delaey & Van de Voorde, 2000). Assessment of cerebral and retinal vasculature is important in determining an individual’s risk of cerebrovascular diseases, such as vascular dementia (Yoshikawa et al. 2003) and stroke. The severity of retinal characteristics is proportional to atherosclerosis, ischemic heart disease and cardiovascular mortality. Increasing age and arterial hypertension are associated with reductions in retinal vascular caliber and the extent of narrowing 4 has been recognized as a major sign of end-organ damage. Retinal vessel tortuosity has been linked to unexplained retinal haemorrhages and endocrinopathies. Pituitary hormone insufficiencies show specific ocular fundus characteristics with isolated tortuosity of retinal veins that may facilitate early diagnosis and treatment. The association between retinal vascular abnormalities, midline brain lesions and hormone deficiencies has been well described in septo-optic dysplasia syndrome(Hellstro¨m et al.,1999).Patientswith hydrocephalus have significantly straighter retinal arteries and fewer vessel branching points (Kaufman & Alm,2003).Preterm birth affects the retinal vascular system functionally as well as structurally, resulting in lower threshold for the development of vascular disease along with an increased casual blood pressure in these subjects; suggesting that birth status has effects on retinal vasculature that persist up to adult life.Increased retinal vein tortuosity is caused by increase in vascular transmural pressurewhile diameters are more sensitive than (Andersson&Hellstorm,2009).People with tortuosity Growth at lower Hormone pressures insufficiencies, regardless of hormonal treatment, have a significantly lower number of vascular branching points.Impaired retinal perfusion occurs in cases with cerebral malaria; filling defects (ghost vessels) and mottling of blood (dark columns)being evidence of hypoxia and ischemia(White&Lewallen,2009).It has been suggested that hepatocyte growth factor and blood cell count, particularly platelets are associated with retinal vessel diameter and may be important in the pathogenesis of micro vascular disease(Chiea et al.,2010).During Sickle Cell retinopathy, a remodeling of the retinal vascular bed occurs; the tortuosity can change or a part of the vascular tree can disappear. Retinal vessels can become blocked with emboli, cholesterol or calcium plaques, that travel with the blood flow and become lodged at sites of narrowing. The risk of occlusion is proportional to the number of arterial-venous 5 overcrossings and branching degree of vessel arcades. Retinal vascular occlusive disorder collectively constitutes one of the major causes of blindness.Retinopathy that includes unique vessel abnormalities, appearance of narrowed and blocked blood vessels, neovascularization and patterns of retinal whitening suggests impaired perfusion that has diagnostic value in patients and its presence and severity can predict the length of coma and risk of death. Retinal blood vessel appearance is an important indicator for physical activity level, fasting and dehydration, diabetes, hypertension and arteriosclerosis and is useful to access the severity and progression of these conditions (Heitmar et al.,2008; Wong et al.,2005). Retinal vascular net and particularly the bifurcation points are specific anatomical landmarks for diagnostic and invasive procedures. There is clear evidence regarding involvement of aging, hypertension, arteriosclerosis, inflammation, endothelial dysfunction, smoking, and blood lipids in retinal vascular characteristics, even to the extent that retinal vessel abnormalities may precede health events like arterial hypertension, stroke and coronary heart disease(Yoshikawa et al.,2003). Wider retinal venous calibre has been reported to be independently associated with progression of diabetic retinopathy, diabetic macular edema, and incidence of proliferative diabetic retinopathy Generalized arterial narrowing was significantly associated with 10-year cardiovascular death in the famous Beaver Dam Study(Wong &McIntosh,2005), showing a relationship between retinal arterial narrowing and various diseases and disease markers; therefore,an association would be expected between increased mortality and retinal arterial narrowing. Cerebral and retinal microcirculations share similaranatomical, embryological, and physical characteristics. There is evidence that small-vessel disease is related to both clinical and sub-clinical stroke but the underlying mechanisms are not fully understood.Retinal imaging has revealed relatively 6 narrow retinal venules in patients with early Alzheimer Disease.White matter lesions, which are caused by small vessel abnormalities, were associated with increased risk of clinical stroke. However, the clinical utility of using vascular calibers in cardiovascular or cerebrovascular risk prediction and in disease management requires further evaluation. In a conclusive note, we can say that because of its unique architecture and dictated by its function; both, the diseases of the eye, as well as diseases that affect the circulation and the brain can manifest themselves in the human retina. These include ocular diseases like macular degeneration and glaucoma, the first and third most important causes of blindness in the developed world. Also, a number of systemic diseases can affect the retina. These include complications of diabetic retinopathy from diabetes, the second most common cause of blindness in the developed world, hypertensive retinopathy from cardiovascular disease, and multiple sclerosis. Thus, while on the one hand, retina is vulnerable to organ-specific and systemic diseases; on the other hand, retinal imaging allows diseases of the eye proper, as well as complications of diabetes, hypertension and other cardiovascular diseases to be timely detected, diagnosed and managed. 7 3C. Imaging based visualization of blood vessels in the retina: History and current status 3C.1History of Retinal Imaging Somewhat paradoxically, the same optical properties of the human eye that allow image formationalso prevent the direct inspection of retina. The red reflex, a blurred reflection of the retina makes the pupil appear red if light is shined into the eye at the appropriate angle, was known for centuries and this red reflex prevents the visibility of retina in imaging. Therefore, special techniques are needed to obtain a focused image of the retina. The French physician Jean Mery made the first attempt to image the retina in a cat by demonstrating that if a live cat is immersed in water, its retinal vessels are visible from the outside. Quite logically, the impracticality of such an approach for humans lead to the invention of the principles of the ophthalmoscope in 1823 by a Czech scientist Jan Evangelista Purkyně (Purkinje) and its reinvention in 1845 by Charles Babbage (Abramoff et al. ,2010) . Here,it is imperative to note that Babbage alsooriginated the concept of a programmable computer and thus the link between computationand retinal imaging is not a new one. Finally, the ophthalmoscope was reinvented againby von Helmholtz in 1851(Abramoff et al. ,2010) . Thus, inspection and evaluation of the retinabecame routine for ophthalmologists, and in 1853,the first images of the human retina werepublished by the Dutch ophthalmologist 'Van Trigt' while earlier sketches by Purkyněprovided drawings of his own retinal vasculature . (Fig.7 a) Considering the prevalence of infectious diseases at the time and because the ophthalmoscoperequired the physician to come quite close to the face of the patient, it was preferably attractive to image the eye photographically. Thus, the first useful photographic images of the human retina, showing bloodvessels, were obtained in 1891 by the German ophthalmologist Gerloff. In 1910, Gullstrand 8 developed the fundus camera, a concept still used to image the retina today .He received the Nobel Prize for this invention (Abramoff et al.,2010).Because of its safety and cost effectiveness at documenting retinal abnormalities, fundus imaging has till date remained the primary method of retinal imaging.The second most important development in eye imaging was the invention of fluorescein angiography where a fundus camera with additional narrow band filters is used to image a fluorescent dyeinjected into the bloodstream. It still remains widely used because it allows an understanding of the functional state of the retinal circulation. Concerns about its patient safety and long term cost-effectiveness are leading it to be slowly but surely replaced by tomographic imagingmethods for its primary applications, namely image-guided treatment of macular edema andthe “wet form” of macular degeneration. One of the major limitations of fundus photography is that it obtains a 2-D representation of the 3-D semi-transparent retinal tissues projected onto the imaging plane.The initial approach, as first described by Allen in 1964 was to depict the 3-D shape of the retina in stereo fundus photography, where multi-angle images of the retina are combined by the human observer into a 3-D shape. Subsequently, using the confocal aperture to obtain multiple images of the retina at different confocal depths, "Confocal scanning laser ophthalmoscopy" was developed, yielding estimates of 3-D shape. Tomographic imaging of the human retina became commonplace with the development of femtosecond lasers ,super-luminescent diodes and the application of optical coherence tomography to retinal imaging , which allows truly 3-D optical sectioning of the retina. 9 3C.2. Current Status of Retinal Imaging During the past 160 years, retinal imaging has developed remarkably and is a now a mainstay of the clinical care and management of patients with retinal as well as systemic diseases. Fundus photography is now widely used for population-based, large scale detection of diabeticretinopathy, glaucoma, and age-related macular degeneration. Optical coherencetomography (OCT) and fluorescein angiography are widely used in the clinical diagnosis andmanagement of patients with diabetic retinopathy, macular degeneration, and inflammatory retinal diseases. Also, OCT is widely used in preparation for and follow-up in vitreo-retinalsurgery. The following modalities/techniques all belong to the broad category of fundus imaging: .Fundus photography including the red-free photography . Color fundus . Stereo fundus photography . Hyperspectral imaging . Scanning laser ophthalmoscopy (SLO) . Adaptive optics SLO . Fluorescein angiography and indocyanine angiography—image intensities represent the amounts of emitted photons from the fluorescein or indocyanine green fluorophore that was injected into the subject’s circulation.(Fig.7 b) Retinal angiography allows the physician to analyze intraretinal circulation and to define the interrelationships between various layers of the retina, Retinal Pigment Epithelial complex(RPE), and choroid. Angiography is not only documentary and supportive of clinical differential diagnosis but also enables opthalmologists to 10 make reasoned therapeutic decisions, particularly in the treatment of Age Related Macular Degeneration and diabetic maculopathy. Angiography allows to confirm the diagnosis and retain a "hard copy" for analysis throughout the course of the disease. Accurate analysis and meaningful diagnostic conclusions result from the recognition of several important phenomena that occur during the course of an angiographic study and a standard protocol should be followed when interpreting a fluorescein angiogram. Sodium fluorescein is a fluorescent dye compound which can be administered intravenously or orally. It adheres to leucocytes of blood and when stimulated by the "exciting" light of the fundus camera, it emits yellow-green light, which is captured either with film or with digital imaging. In traditional fluorescein angiography, 5 ml of 10% sodium fluorescein is injected into the antecubital vein of forearm in a 3- to 7-second bolus. Mild, moderate, and severe reactions to the injection of sodium fluorescein have sporadically been described and should be watched out for. The dye flows to the heart and lungs and back through the heart before entering the postequatorial choroidal circulation via the short posterior ciliary arteries. This initiates the transit phase of the angiogram. The transit phase (10-30 seconds post injection) records the first passage of dye as it flows through the ocular blood vessels and into the ocular tissue. The earliest stage of dye filling is called the prearterial phase when the dyediffuses throughout the choroid and into the choriocapillaris, where it appears as "choroidal flush" or "irregular background fluorescence." Cilioretinal arteries fill with dye concurrently with choroid. A second later, the arterial phase begins as the retinal arteries fill. During the venous stage, veins completely fill with dye and the arteries begin to empty. The arteriovenous phase shows laminar flow in the venous system which refers to the pattern of filling in the outer region of the blood column, within the major retinal veins. This causes the vessel to appear more fluorescent at its margin 11 than at the center. Most notable in the arteriovenous phase are fine vascular details of the macular arterioles, the foveal avascular zone, and the diffuse background choroid.The recirculation phase photographs provide a less distinct picture of retinal vascular pattern due to staining of retinal and choroidal vasculature and the diffusion of dye through retinal tissues. In general, the first complete circulation of fluorescein through the eye reveals the most distinct picture of retinal pathology from which the terms hypofluorescence and hyperfluorescence are evaluated. Pathologic lesions can be defined as hypofluorescent if they appear less brilliant on angiography than normal background fluorescence of the fundus, and hyperfluorescent if they appear brighter than background structures. Hypofluorescent lesions are associated with decreased fluorescence due to blocking or obscuration of the fluorescent pattern, as with sub retinal blood or scar tissue. Alternatively, hypofluorescence is the result of poor filling of an area of retinal vasculature. For example, in capillary nonperfusion associated with diabetic retinopathy or retinal vein occlusion. Breakdown of the blood—inner retina barrier and blood—outer retina barrier may be diagnostic in a variety of disease states. Leakage from a breach in tight junctions of retinal vessel endothelium is reflective of damage to the vessel wall and a corresponding loss of physiologic balance in the osmotic gradient between the intra- and extravascular tissue compartments. Defects in the RPE barrier are seen as leakage points or focal areas of hyperfluorescence and imply that fluid from the choriocapillaris and choroid have entered the subretinal space. Choroidal vessels in an abnormal location like in the subretinal space anterior to the RPE cause dye to leak from choroidal intravascular space, causing relative hyperfluorescence. Abnormal vessels having non-tight junction endothelial cells , such as the neovascular blood vessels associated with diabetes or venous occlusion leak dye or hyperfluorescence into the vitreous cavity. 12 3C.3. Digital imaging of the ocular fundus In the past years, retinal image analysis became a popular research field, with three main factors explaining this trend: Firstly,the retina is the only location where blood vessels can be visualizednon-invasively in vivo; Secondly, retinal images can be produced and distributed with low timeand financial costs; Thirdly, retinal vessels are strong indicators for the presence of diseases such as diabetic retinopathy,retinal vessel occlusion ,glaucoma and arterial hypertension.The use of retinal image analysis offers new techniques to analyze different aspects of retinal vascular topography, including retinal vascular widths, geometrical attributes at vessel bifurcations and vessel tracking. Being predominantly automated and objective, these techniques offer the opportunity to study and to identify retinal microvascular abnormalities as markers of metabolic and systemic disorders.Digital technology has revolutionized the way in which we can acquire, store, and share retinal images, and it gives us considerably very many possibilities for quantitative assessment of fundus and retinal vascular pathological changes. The major advantage of digital imaging in clinical ophthalmology is its ability to conveniently enhance and adjust photographs in order to visualize the smallest of pathologic changes in the ocular fundus. A fundus camera is a specialized lowpower microscope with an attached camera which illuminates the fundus of the eye(Fig.7c) .Light is projected through a set of filters and lenses on to an annular mirror, creating a ring of illumination through the dilated pupil to form an image at the film plane or the array sensor. The first step in digital imaging is digital image capture, which is the electronic recording of light information and its conversion to a set of numbers which are stored in a related image file. The advantage of digital photography is immediate feedback control of image focus and luminosity, 13 enabling immediate adjustment of exposure settings. Magnification of the eye under fundus photography is very complex, and increases from the centre out towards because the eyes are ball shaped. Sharpness in a photograph is a visual phenomenon that is difficult to quantify and an image is described as sharp when the borders defining the blood vessels are distinct and clear. Magnification differs between eyes because of existing differences in the refraction, axial length,distance and angle of the camera and refers to the number of specific points (pixels) of picture information that are contained in an image file. High-resolution images contain a high number of pixels and can exhibit greater image detail. The clarity of fundus photography is related to the focus of the camera, the clarity of the patient’s tear film,anterior chamber, lens and vitreous, since illuminating and imaging rays pass twice through these structures. Dry or swollen corneas, cataracts, and vitreous haemorrhages are conditions that can blur the image. The sharpness of a final fundus photograph is also compromised by limitations in the specific camera’s optical system(Tyler et al.,2003).Vessel measurements are reported in absolute numbers or points and by setting a mean vertical disc diameter to 1.8 mm, the measured distance in pixels in a digital image can be converted to mm. However, this method is potentially prone to bias, since individuals have different sized optic discs, refraction and axial length. But to get around this magnification issue would be far too comprehensive for large population studies and researchers within the field have accepted that which there is no good solution at the moment for the magnification problem. 14 Primitivefunduscopy with candle.First image of human retina drawn by Van Trigt ,1853 Early drawing of retinal vasculature including outlines of ONH and fovea published byPurkyne in 1823 Modern Fundus camera Automated vessel analysis Figure. 7 a. Retinal imaging: "Then and now" 15 The characteristic phases in a normal fluorescein angiogram.A and B represent preinjection photographs; C– E are the transit (early) phase of the angiogram; and F–I are the recirculation ("mid" and "late") phase. Figure.7b.Fluorescein Angiography Entry of fluorescein into the choroidal and retinal circulations. (Source:Kanski;200Ed.) 16 Figure.7.c. Working of the modern Fundus camera: A fundus camera is a specialized low-power microscope with an attached camera. (Source: Google images:fundus camera) 17 3C.4 Computer assisted methods for analysis of retinal blood vessels In the context of clinical research, methods for automatically analyzing retinal images hold high relevance as they offer the potential to examine a large number of images with time and cost savings and provide more objective measurements than current observer-driven techniques. Historically, researches on retinal vescular morphology can be divided into three eras: the visual impression era, the quantitative era, and the digital imaging era. The first era of visual impression was basic and yielded limited data based on subjective and qualitative observations.Terminologies of the time wereretinal arterial 'narrowing' in hypertensive retinopathy described by Gowers (Gowers ,1876) and arteriovenous ''nicking as a sign of hypertensive retinopathy described by Marcus Gunn (Gunn ,1898).The 1970s witnessed a movement into the quantitative era, thanks to the introduction of the wide-angle fundus camera.A new era of modern digital imaging began in the 1990s and like the rest of the imaging world today, fundus photography also participated in the digital revolution. Recent advances in digital image analysis have allowed objective, fast, and accurate semi-automated quantitative measurements of retinal vessels, particularly vessel calibers (Hubbard et al.,1999).The advantages of these methods include the ability to quickly and easily measure retinal vessel diameters of large study populations and the ability to follow them over prolonged periods of time. Another advantage is the ability to use statistics in order to adjust for important systemic and environmentalfactors when analyzing associations and correlations with ocular and systemic conditions.Completely automatic fundus photographic vessel measurement programs that are quicker and highly accurate have recently been presented, but have not yet been unequivocally tested in large population studies and of the many 18 detection methods that are available, the results arenot always satisfactory. Methods giving a sensitivity of 92% with a specificity of 91% are regarded acceptable. In some applications, vessels are the main interest of the image (Perdersenet al.,2000), while in other cases, they merely act to obstructthe observed area(Waldock et al.,1998) . Blood vessels are also used as landmarks in registration methods (Lloret et al.,2000 ;Zana &Klein,1999). Methods for detecting retinal blood vessels generally fall into one of three categories (Hoover et al.,2000). . -Kernel-based -Classifier-based -Tracking-based Kernel-based methods convolute the image with a kernel based on a predefined model; Tracking methods use a model to track the vessels, starting at given points; Classifier-based methods use a two-step approach starting with a segmentation step (often by employing one of the mentioned kernel-based methods) and next the regions are classified according to many features which allows the incorporation oflarge-scale properties, but only after an initial segmentation step. Numerous algorithms have been presented in the last two decades for the segmentation of retinal blood vessels. Many of them are based on the assumption that the intensity profile of retinal vessels is Gaussian-like. Directional Gaussian filters,as well as their first or second order derivatives,have been widely applied in order to enhance the retinal blood vessels. Many other filters for highlighting the vessels have been proposed like morphological filters with linear structuring elements,modified top-hat, tramline algorithms, line detectors and 19 wavelets(Rossant et al.,2011). In all cases, proper detection is crucial and till date, due to the unique properties of each acquisition technique; a single generally acknowledged vessel detection algorithm does not exist. Current methods of retinal vessel measurements are subject to a considerable amount of subjective human input and a fair amount of inter and intra-grader variability exists in retinal vessel measurements. Accurate quantification of changes in vessels like calibre and tortuosity or branching patterns is difficult to automate fully because of large variations in image type, size and quality and in practice, measurements are frequently obtained using semi-automated computer-assisted methods, which can be both laborious and open to user bias. . 20 3C.5 Fundus photography and some prominent retinal vascular population based researches The field of quantitative fundus photographic vessel studies is extensive and photographic assessment of retina, specially its arteries and veins has demonstrated statistically significant effects of systemic variables, environmental conditions, and genetic factors. Some major studies, covering fundus image based retinal analysis of populations, including a total of 34480 subjects are enumerated here. a. The Atherosclerosis Risk in Communities Study (ARIC): a prospective cohort study of 15792 persons, from US communities, initiated in 1987 through 1989 which investigated cardiovascular disease and associated risk factors among middle-aged people.(Wong &McIntosh,2005;Wong&Michelle,2004) b. The Blue Mountains Eye Study (BMES):an Australian population based cohort research of common eye diseases that included 3355 participants in 1992-94, where mydriatic 30 degree fundus photos were used to examine microvascular characteristics and their association with ocular and systemic diseases.(Wong &McIntosh,2005;Wong&Michelle,2004) c. The Beaver Dam Eye Study: a study of 4926 persons aged 43-84 years living in Beaver Dam, Wisconsin. Baseline examinations were performed in 1988-1990 in order to investigate relationships between microvascular characters and ocular and systemic diseases.(Wong &McIntosh,2005;Wong&Michelle,2004) d. The Cardiovascular Health Study (CHS): a prospective cohort study initiated in 1989-1990 in 4 communities living in USA during 1997-98 . Fundus photos of 2405 people, aged 69-97 years were taken to study etiology and risk prediction of 21 coronary heart disease and stroke among older people (Wong &McIntosh,2005;Wong&Michelle,2004). e. The Wisconsin Epidemiologic Study of Diabetic Retinopathy (XVIII and XIX) :this population based research included 996 persons diagnosed with diabetes at age younger than 30 years who were taking insulin, and 225 controls without diabetes, all from South Central Wisconsin during 1980-82.This study examined epidemiologic and clinical features of diabetic retinopathy, retinal vein and artery diameters and other ocular characteristics(Wong &McIntosh,2005). f. The Rotterdam Study: a population-based cohort study on presence of chronic diseases in the elderly; including 7983 persons, from a district near the city of Rotterdam aged over 55 years in 1990-1993(Wong &McIntosh,2005). Summary of the aforementioned research findings: These above mentioned large scale population based studies (a-f) elucidate the following: There is evidence suggesting that retinal arterial narrowing is associated with increasingage, elevated blood pressure (past, current, and future), increasing degrees of macular diabetic oedema, increasing severity of diabetic retinopathy, increased risk of Coronary Heart Disease and increased mortality from Coronary Heart Disease and stroke. Generalized arterial narrowing seems to be related to chronically elevated blood pressure levels although which event precedes the other still remains to be shown.Also, evidence suggests that arterial narrowing may precede the development of hypertension. Lower mean Arterio Venous Ratio(AVR) indirectly shares certain risk factors with atherosclerosis.Lower mean AVR is associated with increased risk of diabetes mellitus and stroke, although this association is mostly driven by venous dilation rather than arterial narrowing; reflecting the importance of analyzingarterial and venous diameters separately.