Retinal microvascular structure and function in patients with risk factors of atherosclerosis and coronary artery disease.

OBJECTIVE: Retinal microvascular signs are markers of cardiovascular disease risk. There are limited data, on relationships between retinal microvascular signs and retinal microvascular endothelial function. We sought to determine the relationship of retinal vascular signs with retinal microvascular endothelial function in patients with or at high risk of coronary artery disease. METHODS: Participants with atherosclerosis risk factors and coronary disease (n=258; mean age 57±11 years) were recruited to have static and dynamic retinal vascular assessment. Retinal arteriolar dilatation in response to flicker light (FI-RAD) was measured using the Digital Vessel Analyser and expressed as percentage increase over baseline diameter. Static retinal photographs were acquired utilising a digital fundus camera for measurement of central retinal artery and vein equivalent (CRAE and CRVE), arteriovenous nicking (AVN) and focal arteriolar narrowing (FAN). RESULTS: Intra-class correlation coefficient was 0.82 for flicker-light induced retinal arteriolar dilatation. There were modest associations in retinal vascular measurements between eyes. For each 10 µm decrease in retinal arteriolar diameter, the absolute increase in FI-RAD was 0.28% (95% CI 0.11, 0.45; p=0.002) independent of age, gender and atherosclerosis risk factors. AVN and FAN were associated with attenuated FI-RAD (ß=-0.67%; 95% CI -1.20, -0.15; p=0.012) and (ß=-0.83%; 95% CI -1.44, -0.23; p=0.007) respectively after adjustment for age and gender. CONCLUSION: Assessment of retinal microvascular endothelial function is reproducible and correlated with retinal microvascular structure and signs, independent of atherosclerosis risk factors. Assessment of retinal vascular structure and function may provide insights into atherosclerotic disease.

Loss of caveolin-1 causes blood-retinal barrier breakdown, venous enlargement, and mural cell alteration.

Blood-retinal barrier (BRB) breakdown and related vascular changes are implicated in several ocular diseases. The molecules and mechanisms regulating BRB integrity and pathophysiology are not fully elucidated. Caveolin-1 (Cav-1) ablation results in loss of caveolae and microvascular pathologies, but the role of Cav-1 in the retina is largely unknown. We examined BRB integrity and vasculature in Cav-1 knockout mice and found a significant increase in BRB permeability, compared with wild-type controls, with branch veins being frequent sites of breakdown. Vascular hyperpermeability occurred without apparent alteration in junctional proteins. Such hyperpermeability was not rescued by inhibiting eNOS activity. Veins of Cav-1 knockout retinas exhibited additional pathological features, including i) eNOS-independent enlargement, ii) altered expression of mural cell markers (eg, down-regulation of NG2 and up-regulation of αSMA), and iii) dramatic alterations in mural cell phenotype near the optic nerve head. We observed a significant NO-dependent increase in retinal artery diameter in Cav-1 knockout mice, suggesting that Cav-1 plays a role in autoregulation of resistance vessels in the retina. These findings implicate Cav-1 in maintaining BRB integrity in retinal vasculature and suggest a previously undefined role in the retinal venous system and associated mural cells. Our results are relevant to clinically significant retinal disorders with vascular pathologies, including diabetic retinopathy, uveoretinitis, and primary open-angle glaucoma.

In vitro induction of protein complexes between bevacizumab, VEGF-A¹65 and heparin: explanation for deposits observed on endothelial veins in monkey eyes.

PURPOSE: By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A(165) and heparin. METHODS: Cynomolgus monkeys were intravitreally injected with 1.25 mg bevacizumab. The eyes were enucleated between 1 and 14 days after injection and investigated by electron microscopy and immunohistochemistry. Human umbilical vein endothelial cells (HUVEC) were incubated with bevacizumab, VEGF-A(165) and heparin at different concentrations. Treatments with ranibizumab served as control. Bevacizumab and ranibizumab were detected immunohistochemically using Cy-3 or immunogold labelled antibodies. RESULTS: Treated animals showed bevacizumab locally at high concentration within retinal blood vessels. Electron-dense deposits inside retinal vessels and between erythrocytes were detected in three out of four treated monkeys. In vitro, many globular aggregates heavily stained with anti-human IgG were only observed with equimolar amounts (240 nM) of bevacizumab and VEGF-A(165) and 0.2 U/ml heparin and not after ranibizumab treatment. The immunogold labelling specifically localised ultrastructurally the complexes formed between bevacizumab, VEGF-A(165) and heparin at the surfaces of HUVEC cells. CONCLUSIONS: Heparin promotes bevacizumab immune complex deposition on to endothelial cells. Our in vitro results could explain the presence of deposits observed on endothelial veins in monkey eyes intravitreally injected with bevacizumab.

In vitro noncontact intravascular femtosecond laser surgery in models of branch retinal vein occlusion.

PURPOSE: To investigate intravenous femtosecond laser surgery in models of branch retinal vein occlusion. MATERIALS AND METHODS: Non-amplified near infrared femtosecond laser was used to ablate polyamide sutures and human hairs inserted into the vascular lumina of porcine retinal veins in vitro. Specimens were subjected to multiphoton laser scanning microscopy and electron microscopy. RESULTS: Regular laser cuts within sutures and hairs were detected with laser microscopy and electron microscopy. Neither laser microscopy nor histology revealed collateral damage of the vascular wall. CONCLUSIONS: Non-amplified femtosecond lasers may allow precise atraumatic non-contact intravenous retinal surgery controlled by high-resolution imaging of the target.

Retinal vascular changes and systemic diseases: corrosion cast demonstration.

The purpose of the present study is to clarify the specific retinal vascular changes in rat models of single spontaneous or hereditary systemic diseases. We used Wistar Kyoto (WKy) rats 6 months of age as normal controls, 24-month-old Wistar Kyoto rats in studies of aging, 16-month-old spontaneously hypertensive rats (SHR), 18-month-old rats with inherited hypercholesterolemia (RICO) for arteriosclerosis, and 18-month-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats for diabetes mellitus. Retinal vascular corrosion casts were made and observed with a scanning electron microscope. The retinal vessels were also examined with a transmission electron microscope. Specific changes in the retinal vessels were: narrowing and thin thread-like capillaries in aging; capillary tortuosity, irregularity and narrowing in hypertension; straightening in hypercholesterolemia; and loop formation and microaneurysms in diabetes mellitus. These specific changes in the retinal vessels in each systemic disease can be clearly and easily revealed in a three-dimensional fashion by corrosion casts followed by scanning electron microscopy.

Scanning electron microscopic study of the posterior ciliary veins in domestic ungulates.

Vascular corrosion casts of 12 equine, 15 bovine and 50 porcine eyes were studied scanning electron microscopically for the presence of posterior ciliary veins. These veins drain a postequatorial segment of the choroid and emerge near the posterior bulbar pole. They complement the four vorticose veins that emerge near or at the equatorial zone of the eyeball and the slender choroidoretinal veins that drain the peridiscal area of the choroid. Posterior ciliary veins were observed in all equine and bovine eyes examined. In these species they presented a large variation in size, number and position. In contrast, posterior ciliary veins were only present in two porcine eyes where they were represented by one and two vessels, respectively. The morphology and variability of these vessels is illustrated and their nomenclature and functional significance are discussed.

Microvasculature of the rat optic nerve head.

PURPOSE: To describe the arterial blood supply, capillary bed, and venous drainage of the rat optic nerve head. METHODS: Ocular microvascular castings from 6 Wistar rats were prepared by injection of epoxy resin through the common carotid arteries. After polymerization, tissues were digested with 6 M KOH, and the castings washed, dried, and coated for scanning electron microscopy. RESULTS: Immediately posterior to the globe, the ophthalmic artery trifurcates into the central retinal artery and two posterior ciliary arteries. The central retinal artery directly provides capillaries to the nerve fiber layer and only contributes to capillary beds in the neck of the nerve head. The remainder is supplied by branches of the posterior ciliary arteries that are analogous to the primate circle of Zinn-Haller. Arterioles arising from these branches supply the capillaries of the transitional, or laminar, region of the optic nerve head. These capillaries are continuous with those of the neck and retrobulbar optic nerve head. All optic nerve head capillaries drain into the central retinal vein and veins of the optic nerve sheath. A flat choroidal sinus communicates with the central retinal vein, the choriocapillaris, and with large veins of the optic nerve sheath. CONCLUSIONS: The microvasculature of the rat optic nerve head bears several similarities to that of the primate, with a centripetal blood supply from posterior ciliary arteries and drainage into the central retinal and optic nerve sheath veins. Association of nerve sheath veins with the choroid represents an important difference from the primate.

Retinopathy in diabetic (KKA gamma) mice: diabetic microvascular changes to the retina in KKA gamma mice revealed by light and electron microscopy.

Pericytic changes in the retinal vessels of diabetic (KKA gamma) and control (C57BL) mice were studied by light and electron microscopy. An improved histochemical technique for alkaline phosphatase was used in the light microscopic study. In the control mice, a continuous pathway was identified extending from the retinal arterioles, via the superficial and deep retinal capillaries, to the retinal venules. The deep retinal capillaries formed networks and were localized within the deeper retinal layers; the retinal arterioles, superficial capillaries, and venules were present in the nerve fiber layer. Examination of KKA gamma mice, aged 16 to 28 weeks, revealed engorgement of the arterioles, hypertrophy of the pericytes (which contained numerous actin filaments) within the superficial retinal capillaries, and narrowing of the deep retinal capillaries. These microvascular changes indicate retinal hyperperfusion, local hypertension of the superficial retinal capillaries, adaptive hyperfunctional changes in the pericytes of these capillaries, and ischemia of the deep retinal capillaries. The pericytic changes observed in the diabetic capillaries contrasted sharply with previous reports; an explanation for this variance is suggested.

Microvasculature of the human optic nerve.

PURPOSE: Methyl methacrylate vascular corrosion casting techniques were used to examine the normal anterior optic nerve microvasculature in 18 human eye bank eyes. METHODS: Selective cannulation of the central retinal artery, the short posterior ciliary arteries, or both, allowed the methyl methacrylate to be injected into the anterior optic nerve circulation. Preflushing with tissue plasminogen activator greatly enhanced the filling of the fine microvasculature by dissolving the intraluminal clots. RESULTS: The superficial nerve fiber layer of the optic nerve received its primary blood supply from the central retinal artery. In 11 of 13 eyes injected with methyl methacrylate through the short posterior ciliary arteries, there was a perineural, circular arterial anastomosis (circle of Zinn-Haller) at the scleral level. Branches from this circle penetrated the optic nerve to supply the prelaminar and laminar regions and the peripapillary choroid. In the two eyes without this arterial circle, direct branches from the short posterior ciliary arteries supplied the anterior optic nerve. The venous drainage of the anterior optic nerve was almost entirely through the central retinal vein and its tributaries. CONCLUSIONS: This study demonstrates that the main arterial vascular supply to the anterior optic nerve is from the short posterior ciliary arteries. The contribution of the peripapillary choroid to the anterior optic nerve is minimal in comparison to the direct contribution from the short posterior ciliary arteries.

Optimal conditions required for the creation of an iatrogenic chorioretinal venous anastomosis in the dog using argon green laser photocoagulation.

We have previously reported iatrogenic retinal to choroidal vein anastomosis, developed as a potential method of by-passing the site of obstruction to venous outflow in retinal venous occlusion in dogs (1), and in rats (2). In order to minimise tissue damage to the retina and choroid and increase the rate of success in these experiments, we investigated in the dog model the factors that would promote an anastomosis and compare the effects of three different power levels. A small spot size (50 microns) argon green laser beam of 514 nm at power levels of 0.5w, 1.5w and 2.5w were used. Spaced serial sections from each lesion were examined by light and by transmission electron microscopy. Morphometric measurements of the corresponding retinal pigment epithelium (RPE)/glial scar was computed using IBMPC digitising pad and sigma scan software and the extent of tissue damage at the different power levels assessed. At the lowest power level of 0.5w the damage to the retina was mild and there was an absence of anastomosis formation. At the 1.5w power level an anastomosis formed in 40% of the lesions. At the highest power level of 2.5w a 71% rate of success was obtained however, the damage to the retina tended to be severe. The results of this study also indicate that disruption of Bruch's membrane and vein rupture at the time of irradiation are essential for anastomosis formation, which may be further enhanced by necrotic tissue, retinal pigment epithelial and glial scar formation and inflammation. These findings are useful in establishing optimal conditions for the creation of a chorioretinal venous anastomosis, for consideration in human trials.

Stereological estimation of Weibel-Palade bodies in the retinal vasculature of normal and diabetic dogs.

The absolute volume of Weibel-Palade (WP) bodies, the storage organelles of von Willebrand factor (vWF), was estimated by a stereological method in a known volume of central retina from normal and 5-year diabetic dogs. The results showed that the volume of WP bodies present in the endothelium of the retinal vasculature varies with blood vessel type and in diabetes. In both diabetic and normal dogs the endothelium of the retinal veins contained a higher volume of WP bodies than that of the retinal arteries. In dogs which had been diabetic for a duration of 5 years the volume of WP bodies present in the endothelium of retinal veins was significantly greater than in the endothelium of veins from the control animals. However, there was no significant difference in the volume of WP bodies present in the endothelium of retinal arteries or capillaries between the two groups of animals.

Significance of Bruch's membrane in the creation of iatrogenic chorioretinal venous anastomosis.

Iatrogenic retinal to choroidal vein anastomoses, as a method of bypassing retinal venous occlusions has been reported in dogs in which Bruch's membrane is poorly formed. In order to determine whether chorioretinal venous anastomoses can be induced in an animal with a Bruch's membrane that is well developed as in humans, pigmented rats were chosen. A high intensity, small spot size argon green laser beam of 514 nm was used to induce the anastomosis. Three out of 5 rat eyes developed a retinal vein to choroidal vein anastomosis. The success rate of iatrogenic retinal to choroidal vein anastomoses in the rat was comparable to that obtained in the dog. This study suggests that Bruch's membrane was not significant in the creation of an iatrogenic chorioretinal venous anastomosis.

[Recanalization of venule following experimental retinal branch vein occlusion].

Retinal branch vein occlusion was experimentally produced in monkey (macaca irus) by dye laser photocoagulation. Venous dilatation, retinal edema and punctate hemorrhages were the ophthalmoscopic changes immediately after the occurrence of venous occlusion by photocoagulation. Fluorescein angiography revealed delay of retinal circulation and dye leakage from these venules. Fluorescein angiography on the 2nd day revealed caliber irregularities in the venules distal to the photocoagulated points. Histopathologically, thrombus formation and partial defect of the endothelial cells were observed in the venules in the areas which showed recovery with immature endothelial cells on the 4th day. Young endothelial cells proliferating along the endothelial basement membrane, which remained around the thrombi, and recanalization were observed on the 7th day. These results reveals a process of recanalization in the thrombogenous retinal venous occlusion.

Optic nerve head microvasculature of the rabbit eye.

Vascular luminal castings of rabbit eyes were microdissected and studied with scanning electron microscopy to elucidate the three-dimensional angioarchitecture of the optic nerve head. Using sequential microdissection, an incomplete arterial circle was identified as terminal branches of two to three short posterior ciliary arteries around the optic nerve head. Several recurrent branches from the arterial circle form a pial arterial network. This pial system supplies the optic nerve head microvasculature and receives numerous venules from them. The only large vessel to enter the optic nerve is a central retinal artery that has few branches within the optic nerve and provides several branches at the surface of the optic disc. Moderately numerous vessels connect the retinal and ciliary vascular layers within the optic nerve head. Few arterioles to the optic nerve head arise from the choroid; however, there are a small number of capillary and numerous venous connections between them. These results indicate that the principal blood supply of the rabbit optic nerve head is derived from the short posterior ciliary arteries by the arterial circle. The retinal arteries contribute to the surface vasculature of the optic nerve head. The pial system also plays a significant role in both supply and drainage of the rabbit optic nerve head.

[A scanning electron microscopic study of experimental endothelial injury and regeneration following photochemically induced thrombosis in rat retinal venules].

The natural course of retinal venous obstruction and of endothelial injury and regeneration following photochemically induced thrombosis in Wistar-Kyoto rats was studied by scanning electron microscopy utilizing the freeze-splitting method. The eyes were examined ophthalmoscopically and histologically at 30 min, 6, 24 h, 2, 3, 4 and 5 days after the insult. At 30 minutes after the irradiation, the vascular lumen of retinal venule was completely occluded with a large amount of fibrous materials considered to be fibrin mesh-work and a mass of deformed erythrocytes. By 6 hours after the irradiation, the fibrous materials had decreased and deformed erythrocytes had also decreased in number. On the other hand, multi-layered platelets with pseudopods aggregated over the irradiated area. Similar platelet aggregation was also seen at 24 hours after the irradiation, however, fibrous materials and deformed erythrocytes were rarely observed. These aggregates persisted for up to 2 days after the irradiation, when exposure of the subendothelium was also observed. By 3 days after the irradiation, there had been no platelet aggregates and the injured area was reendothelialized. But the endothelial surface was rugged and had many crater-like pores. In addition, some of these endothelial cells protruded or were elevated toward the lumen. Endothelial abnormalities such as rugged surface and crater-like pores were also found at 5 days after the irradiation. These findings suggested that rapid thrombolysis was caused by activation of the local fibrinolytic system, that the injured area was rapidly reendothelialized within about 3 days but that mild endothelial damage still existed for more than 5 days.

Experimental retinal vascular occlusion. III. An ultrastructural study of simultaneous occlusion of central retinal vein and artery.

The retinal changes following the simultaneous occlusion of retinal vessels were examined at an ultrastructural level. The preservation of endothelial cells that prevented leakage of blood and horseradish peroxidase tracer material in the surrounding retinal tissues explained the lack of hemorrhages observed clinically. Mueller cell death resulted in severe structural disorganization of the retina, with disruption of the inner limiting membrane, involvement of the outer retinal layers, and fleurette formation. The retinal macrocysts were derived from ischemic infarction.

Scanning electron microscopy in retinal research.

The literature on scanning electron microscopy (SEM) pertaining to the retina has been surveyed and described. The first two papers on SEM and retina appeared in 1969. Most of the earlier studies concentrated on descriptions [by SEM alone, or with light microscopy (LM) and transmission electron microscopy (TEM)] of the appearance of various retinae and retinal cells [fish, newt, primates, rodents and rabbits, bullfrog]; or embryology of the chick retina. Two papers dealt with retinal disease. In all there were 25 papers in SEM/retinal research before and in 1974. Since 1975 there have been 111 papers which have used SEM in retinal research in very many different fields and in diverse ways, viz: the morphology of the retina, specific retinal cells, and the retinal vessels; embryology; labelling of retinal cells with surface markers; naturally occurring and experimentally-induced retinal disease; the effects of light, including lasers and photocoagulation, and other radiation on the retina, and phagocytosis and the retinal pigment epithelium (RPE). There have also been a number of papers concerned with techniques for SEM, applicable to retinal research, and several review articles. SEM has proven to be a very valuable adjunct to TEM in retinal research and it has become an accepted and almost routine way of extending and enhancing reports on retinal research. Properly used, SEM provides a laconic way of presenting information that might otherwise require many serial sections and long explanations.

Retinal phlebitis with chorioretinal emboli.

Emboli to the eye may cause retinal vascular occlusive disease with a wide range of clinicopathologic manifestations including arteriolar occlusion, retinal ischemia and infarction, and retinal neovascularization. Clinical observations of a progressive obliterative arteriolitis in patients with systemic embolic disease have led to the speculation that retinal vasculitis may be a feature of ocular embolic disease. A postmortem examination of the enucleated eyes of two elderly female patients disclosed gross and histopathologic features of retinal periphlebitis associated with many chorioretinal calcific emboli. These patients also had premortem and postmortem manifestations of systemic thromboembolic disease originating from the heart and great vessels. One patient had a progressive decrease in visual acuity, paracentral scotoma, and midperipheral perivascular sheathing. These findings suggest that ocular embolism may sometimes be a factor in the development of retinal phlebitis.