Immunohistochemical localization of low density lipoprotein receptor-related protein 1 and alpha(2)-Macroglobulin in retinal and choroidal tissue of proliferative retinopathies.

The immunolocalization of the low density lipoprotein receptor-related protein 1 (LRP1) and its ligand alpha 2-Macroglobulin (alpha(2)M) was examined in tissues from human donor eyes of normal, diabetic and sickle cell disease subjects. Streptavidin alkaline phosphatase immunohistochemistry was performed with a mouse anti-human LRP1 and rabbit anti-human alpha(2)M antibodies. Retinal and choroidal blood vessels were labeled with mouse anti-human CD34 antibody in adjacent tissue sections. Mean scores for immunostaining from the pathological and control eyes were statistically compared. LRP1 immunoreactivity was very weak to negative in the neural retina of normal subjects except in scattered astrocytes. LRP1 expression in diabetic eyes was detected in the internal limiting membrane (ILM), astrocytes, inner photoreceptor matrix, choriocapillaris and choroidal stroma. The ligand alpha(2)M, however, was limited mainly to blood vessel walls, some areas of the inner nuclear layer (INL), photoreceptors, RPE-Bruch's membrane-choriocapillaris complex, intercapillary septa, and choroidal stroma. In sickle cell eyes, avascular and vascular retina as well as choroidal neovascularization (CNV) were analyzed. In avascular areas, LRP1 immunoreactivity was in innermost retina (presumably ILM, astrocytes, and Muller cells) and INL as well as RPE-Bruch's membrane-choriocapillaris complex and choroidal stroma. alpha(2)M was very weak in avascular peripheral retina compared to vascularized areas and limited to stroma in choroid. In contrast, in areas with CNV, LRP1 immunoreactivity was significantly decreased in overlying retina and in RPE-Bruch's membrane and choroidal stroma compared to the controls, while alpha(2)M was elevated in RPE-Bruch's membrane near CNV compared to normal areas in sickle cell choroid. The mean scores revealed that LRP1 and alpha(2)M in neural retina were significantly elevated in astrocytes and ILM in diabetic eyes (p < or = 0.05), whereas in sickle cell eyes scores were elevated in ILM and INL (p < or = 0.05). In addition, alpha(2)M immunoreactivity was in photoreceptors in both ischemic retinopathies. In choroid, the patterns of LRP1 and alpha(2)M expression were different and not coincident. This is the first demonstration of the presence of LRP1 and alpha(2)M in human proliferative retinopathies. Elevated LRP1 expression in sickle cell neural retina and diabetic inner retina and choroid suggests that LRP1 plays an important role in ischemic neovascular diseases.

Localisation of SDF-1 and its receptor CXCR4 in retina and choroid of aged human eyes and in eyes with age related macular degeneration.

AIM: To examine the immunolocalisation of stromal cell derived factor 1 (SDF-1) and its receptor CXCR4 in aged control human donor eyes and eyes with age related macular degeneration (AMD). METHODS: Postmortem eyes from eight aged control donors (mean age 79.8 years) and from 12 donors with AMD (mean age 83.9 years) were cryopreserved and sectioned through the macular region. SDF-1 and CXCR4 were localised using streptavidin alkaline phosphatase immunohistochemistry and then sections were bleached. Three independent masked observers scored the immunohistochemical reaction product. RESULTS: In aged control retinas, SDF-1 immunoreactivity was most intense in inner photoreceptor matrix (IPM). CXCR4 showed a similar pattern of immunostaining, but was more prominent in inner segments of photoreceptors. In aged control and AMD choroid, SDF-1 and CXCR4 localisations were most prominent in retinal pigment epithelial (RPE) cells and choroidal stroma. However, the intensity for SDF-1 was significantly reduced in RPE (p < 0.0001) and choroidal stroma (p < 0.05) in late AMD eyes. SDF-1 and CXCR4 immunoreactivities were weak or nearly absent in disciform scars with choroidal neovascularisation (CNV). Circulating cells, presumably leucocytes, were most intensely positive for CXCR4. CONCLUSIONS: These results show that changes in distribution and relative levels of SDF-1/CXCR4 were not evident in early AMD. This suggests that SDF-1/CXCR4 may not contribute to the formation of CNV in AMD, in that CXCR4+ cells were not incorporated into neovascularisation. However, the examples of CNV studied were within disciform scars, so the authors cannot comment on the role of SDF-1/CXCR4 in the early stages of CNV formation.

Impaired expression of thrombospondin-1 in eyes with age related macular degeneration.

AIMS: This study investigated the expression and localisation of thrombospondin-1 (TSP-1), a known anti-angiogenic extracellular matrix protein, in normal aged control human eyes and eyes with age related macular degeneration (AMD). METHODS: Immunohistochemical analysis with mouse anti-human TSP-1 antibody and mouse anti-human CD 34 antibody, as a blood vessel marker, was performed on frozen sections from macular and peripheral blocks of aged control donor eyes (n = 12; mean age 78.8 years), and eyes with AMD (n = 12; mean age 83.9 years). Pigment in retinal pigment epithelium (RPE) and choroidal melanocytes was bleached. Three independent observers scored the immunohistochemical reaction product. RESULTS: In the macular region, TSP-1 expression was observed intensely in Bruch's membrane and weakly in RPE basement membrane, choriocapillaris, and the wall of large choroidal blood vessels in the aged control eyes. In eyes with AMD, TSP-1 immunoreactivity was significantly lower in all structures except RPE basement membrane (p<0.01). There was significantly lower TSP-1 in the far periphery than the equator and submacular regions in all eyes. TSP-1 immunoreactivity was low in choroidal neovascularisation (CNV), but it was high and diffuse in adjacent scar tissue. CONCLUSION: These findings suggest that decreased TSP-1 in Bruch's membrane and choroidal vessels during AMD may permit the formation of CNV.

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.

Corrosion cast demonstration of choroidal vasculature in normal Wistar Kyoto rat.

No previous attempt has been made so to demonstrate the details of the choroid vascularization in the rat. This paper describes the choroidal vascular pattern of the normal Wistar Kyoto rat using the corrosion cast method. 6-month-old normal Wistar Kyoto rats were used. Vascular casts prepared by our previously described technique were observed with a Hitachi S-2360N scanning electron microscope. Corrosion casts of the entire choroidal vasculature showed that the ophthalmic artery branched into two main ciliary arteries: the nasal and temporal arteries, which extend forward to form the iridociliary circle. Further the inferior ciliary artery arises from the temporal ciliary artery. In the posterior eye segment these arteries form four to seven branches supplying the adjacent choriocapillaris. There are different arrangements of choriocapillaris. The larger veins formed four vortex veins, one in each quadrant, draining blood from the anterior region of the choroid or iris. Venous blood from the central region, peripapillary choroid and sometimes optic nerve head is drained by smaller veins, which run more or less directly into the posterior ciliary veins. In conclusion corrosion casts and SEM can show the details of the choroidal vascular architecture. These three-dimensional (3-D) findings of choroidal vessels of the rat are very useful for the study of choroid vascular abnormalities in various pathological conditions.

Microvascular architecture of the rat choroid: corrosion cast study.

This study presents the details of the microvasculature of the rat choroid visualized by scanning electron microscopy of vascular corrosion casts. Wistar Kyoto rats were anesthetized with intraperitoneal sodium pentobarbital. The vascular system was perfused with heparinized saline, and Mercox resin was injected into the cannulated carotid arteries. After polymerization of the resin, the vascular casts were macerated with potassium hydroxide, washed with water, and freeze dried. The casts were examined with a Hitachi S-2360N scanning electron microscope (SEM). Corrosion casts of the entire choroidal vasculature revealed that the two long posterior ciliary arteries supplied the entire uveal vasculature. In the posterior choroid, these arteries formed five to seven branches on each side supplying the adjacent choriocapillaris. No interarterial or arteriovenous anastomoses were seen. The choriocapillaris appeared as a nonhomogeneous and nonlobular monolayer capillary network, consisting of dense honeycomb and irregular patterns. There are two distinct venous systems in the rat choroid. The venous blood from the central region, peripapillary choroid, and sometimes the optic nerve head drain into the posterior ciliary vein. The venous blood from the iris, ciliary body, anterior choroid, and half of the posterior choroid drain into the vortex veins. Corrosion casts and the SEM have shown details of the choroidal vascular architecture. These three-dimensional observations indicate that the rat choroidal vasculature has different features from those of humans and other primates. Despite these interspecies differences, the establishment of a thorough baseline concept of choroidal vasculature should permit additional studies of the choroidal pathology and enable the proper interpretation of results from rat experimental models for extrapolation to humans.

Retinal vascular changes in rats with inherited hypercholesterolemia--corrosion cast demonstration.

PURPOSE: To demonstrate specific hypercholesterolemic changes in the retinal vascular architecture. METHODS: Corrosion casts of 12- to 18-month-old rats with inherited hypercholesterolemia (RICO) and of control Wistar Kyoto (WKy) rats were examined with a scanning electron microscope (SEM). The diameters of the retinal arteries, veins and capillaries were measured in photographs with a caliber micrometer. The capillary branches were counted in the micrographs with the use of Adobe Photoshop. The retinal capillaries were examined by transmission electron microscopy (TEM). RESULTS: SEM examination of the vascular casts of 15-month-old RICO rats showed slight tortuosity of large vessels at the posterior pole of the retina. The precapillary arterioles branching from the major artery were longer and straighter than normal. Retinal capillary changes such as caliber irregularity and narrowing in the capillary network were more severe in 18-month-old RICO rats. The most prominent finding was marked straightening of the capillaries in the inner and outer layers of the capillary network, which looked like fine strings. Intercapillary spaces became wider, and finally capillaries looked scattered. The diameter of the retinal capillaries lumen in RICO rats was significantly narrower than that in WKy rats (p < 0.0001). The capillary branches were fewer in 18-month-old RICO rats than that in 18-month-old WKy rats (p < 0.0001). Neither local stenosis or obstruction in the arterioles and venules nor any arteriovenous crossing defect was seen in young and old RICO rats. Transmission electron microscopy of 16-month-old RICO rat retinas revealed that the capillaries in the inner and outer plexiform layers contained scarce cytoplasmic components, vacuoles in endothelial cells and basement membranes of irregular thickness. Capillary pericytes were swollen and irregular in shape, contained vacuolated mitochondria and scarce cytoplasmic components. CONCLUSIONS: These findings indicate that the retinal capillary changes are probably related to hypercholesterolemia.

Retinal vascular architecture is maintained in retinal degeneration: corrosion cast and electron microscope study.

PURPOSE: To demonstrate the changes of the retinal vascular architecture in the diffusely degenerated thin retina. METHODS: Three-week-old weanling Wistar Kyoto rats were divided randomly into two groups. One group (n = 20) was fed a vitamin E-deficient solid diet and the other group (n = 20) was fed a solid rat chow diet. Rats were maintained on their respective diets for 14 months and then killed for scanning electron microscopy of vascular corrosion casts, light and electron microscopy and biochemical determinations. RESULTS: The serum level of vitamin E in the E-deficient rats was 1.0 +/- 0.49 microg/ml, while that in the rats fed a normal diet was 13.7 +/- 1.0 microg/ml (Student's t-test, p = 0.0001). In vitamin E-deficient rats, light microscopy showed degenerated retinas only half as thick as normal. Corrosion casts and scanning electron microscopy revealed that the retinal capillaries of the entire retina were decreased in number and scattered with localised narrowing, calibre irregularity and frequent loop formation. In the posterior pole of the retina, some capillaries clustered into small tortuous knots. However, the two-layered architecture of the capillary network in the retina was maintained. The differences in calibre of retinal capillaries between the vitamin E-deficient and normal rats were statistically significant (p < 0.0001). No remarkable abnormal changes were observed in the large retinal vessels other than arterial calibre differences (p < 0.022). No arteriovenous shunts, crossing defects or microaneurysms were seen. Transmission electron microscopy revealed complete disappearance of the photoreceptor outer and inner segments and nuclei. The retinal pigment epithelium contained lipofuscin granules and retinal capillaries with narrow lumens. The capillary endothelial cells were thickened and had scarce cytoplasmic components with vacuoles and irregularly thickened basement membranes. The capillary pericytes had vacuoles. No abnormalities were seen in the control normal rats. CONCLUSION: These findings indicate that the decrease in retinal capillaries in vitamin E-deficient rats is secondary to retinal degeneration. It was assumed that the morphological changes in the capillary network reflected structural damage to the retinal vascular cells caused by free radicals and lipid peroxides generated by oxidation. However, even in such severe degeneration the retinal vascular architecture, including the main artery and vein and two-layer capillary networks, was maintained. This is may be because of the basic anatomical arrangement of the blood vessels.

Retinal capillary changes in Otsuka Long-Evans Tokushima fatty rats (spontaneously diabetic strain). Electron-microscopic study.

The Otsuka Long-Evans Tokushima fatty (OLETF) rat is a spontaneously diabetic strain with polyuria, polydipsia and mild obesity. The pathological features of OLETF rats closely resemble those of patients with non-insulin-dependent diabetes mellitus. The purpose of this study is to investigate the retinal capillary changes in the OLETF rat and to confirm the valuability of the OLETF rat as the model of diabetic retinal disease. One-month-old male OLETF rats and age- and sex-matched Long-Evans Tokushima Otsuka (LETO) controls were supplied by Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan). Body weight and blood sugar levels were measured monthly. Their eyes were enucleated 14 months after birth. Ultrathin sections were made and examined with a transmission electron microscope. According to their location, two kinds of retinal capillaries were differentiated: those in the nerve fiber layer (NFL) and those in the outer plexiform layer (OPL). The image of each capillary was transferred to a computed image analyzer, and basement membrane thickness and the ratio of the pericyte area to total capillary cross-section area were determined. Corrosion casts of retinal vessels were made and examined with scanning electron microscopy (SEM). OLETF rats gained more weight than LETO rats from the beginning, and the difference increased gradually with age. The blood sugar level of OLETF rats was higher than that of LETO rats after 5 months of age. In the retinal capillaries of 14-month-old OLETF rats, basement membranes were significantly thicker (OLETF: 209 +/- 51 nm in NFL, 132 +/- 23 nm in OPL; LETO: 118 +/- 28 nm in NFL, 79 +/- 14 nm in OPL), and the ratio of pericyte area to the capillary cross-section area was significantly lower than that of the controls (OLETF: 0.131 +/- 0.92 in NFL, 0.111 +/- 0.102 in OPL; LETO: 0.288 +/- 0.142 in NFL, 0.198 +/- 0.136 in OPL). The endothelial cell cytoplasm had degenerated. SEM examination of the vascular corrosion cast of a 14-month-old OLETF rat showed caliber irregularity, narrowing, tortuosity and loop formations of capillaries. The morphological changes in the retinal capillaries of OLETF rats were similar to those seen in diabetic patients. The OLETF rat may be a useful animal model for the study of ocular diabetic complications in humans.

Vascular architecture of degenerated retina in WBN/Kob rats: corrosion cast and electron microscopic study.

The purpose of the present study is to determine the changes of vascular architecture in the degenerated retina. We used mainly corrosion casts of the retinal vasculature and scanning electron microscopy to obtain a wide three-dimensional view. WBN/Kob rats (a spontaneously diabetic strain) were used because their outer retinas degenerate and become very thin with age. In 15-month-old rats, localized constriction and irregular caliber of the capillaries were evident in the vascular casts. Two layers of capillary network in the retina were maintained, but the capillaries were decreased in number. Numerous loop formations were present in the superficial capillary networks. Neither microaneurysms nor arteriovenous shunts were seen in young and old rats. Transmission electron microscopy revealed that capillary pericytes in the inner and outer plexiform layers had thickened basement membranes and that endothelial cells had many vesicles in their cytoplasm. Thus the retinal capillary changes in WBN/Kob rats are nondiabetic but due to hereditary retinal degeneration, although the systemic and pancreatic abnormalities in this rat strain are diabetic. Even when the retina becomes very thin, two layer capillary networks remain.

Effects of cilazapril on the retinal vessels in spontaneously hypertensive rats: corrosion cast and scanning electron microscopic study.

The effects of the long-term oral angiotensin-converting enzyme inhibitor, cilazapril, on retinal circulation in stroke-prone spontaneously hypertensive (SHR-SP) rats were assessed by scanning electron microscopy (SEM), corrosion casts and transmission electron microscopy (TEM). Two groups of 20 male SHR-SP rats were compared. One group was treated with 10 mg/kg/day of cilazapril from 4 to 40 weeks of age, and the other group received no treatment. A third group of male Wistar-Kyoto (WKY) rats served as age-matched controls. At regular intervals the rats were weighed, and their systolic blood pressure was measured. Cilazapril normalized systolic arterial pressure to 121+/-2.7 mm Hg (SD) in the treated SHR-SP rats. There was no significant difference in body weight between the two groups of SHR-SP. In the 40-week-old SHR-SP rats without treatment corrosion cast and SEM revealed hypertensive retinal vascular changes. In the 40-week-old SHR-SP rats treated with cilazapril, these changes were markedly decreased to the level seen in WKY rats. The differences in caliber of retinal capillaries between the treated SHR-SP and untreated SHR-SP rats were statistically significant (p<.0001). TEM in the cilazapril-treated SHR-SP rats revealed intact basement membranes (0.29+/-0.057 microm) of the endothelial cells and pericytes, but in the untreated SHR-SP rats the basement membrane was thickened (0.51+/-0.123 microm) (p<.0001) and the pericytes damaged. Our results show that the long-term administration of cilazapril decreased systolic arterial pressure to a nearly normal level and prevented hypertensive retinal vascular changes, probably by improving endothelial function. The effects of cilazapril on the retinal vasculature are described for the first time. SEM of corrosion casts is a valuable technique for showing the effects of some drugs on the vasculature easily, precisely and three-dimensionally.

Vascular changes in retinas of spontaneously hypertensive rats demonstrated by corrosion casts.

The changes in the capillary network of the retina in spontaneously hypertensive rats (SHR) were demonstrated by scanning and transmission electron microscopy. Corrosion casts with scanning electron microscopy provided a three-dimensional view of the microvasculature of the rat retina and could detect the conspicuous features of the retinal vasculature in SHR. In general, when hypertension lasted for 6-7 months in SHR, tortuosity of the retinal vessels was noted, and later the SHR retina showed increased tortuosity and generalized narrowing of the vessels, localized constriction of the veins, arteriovenous crossing defects, and marked capillary changes, such as caliber irregularity, narrowing, bead-like capillaries, loop formation and localized obliteration. At a later stage, when the blood pressure had been sustained for a long time, there was marked capillary constriction which first affected the superficial capillary network, then scattered capillary network constriction and finally capillary dropout. Transmission electron microscopy revealed narrow capillary lumina and thin endothelium with scarce cytoplasmic components and damaged pericytes. These morphological changes in the capillary network were probably due to structural damage to the endothelial cells, facilitated possibly by compression of the precapillary arterioles. The severity of these changes was usually proportional to the degree and duration of hypertension. These findings indicate that the retinal capillary changes are probably related to hypertension.

Histologic study of living response to artificially synthesized hydroxyapatite implant: 1-year follow-up.

We implanted artificially synthesized hydroxyapatite spheres into the orbits of 13 rabbits after enucleation. The spheres were removed 1, 2, 3, 6, and 12 months after implantation and examined by light and scanning electron microscopy. Tissue breakdown and exposure of the artificially synthesized hydroxyapatite implants were not observed. Month after month, fibrovascular tissues gradually invaded the pores of the artificially synthesized hydroxyapatite spheres deeper and deeper. Although the hydroxyapatite we used was completely artificially synthesized, we observed a mild foreign-body reaction around the artificially synthesized hydroxyapatite spheres. After 12 months, however, relief of the foreign-body reaction had occurred. Hydroxyapatite spheres for orbital implants after enucleation without scleral enveloping are appropriate.

[A study of living response to artificially synthesized hydroxyapatite implant in the rabbit orbit].

We implanted artificially synthesized hydroxyapatite (a-HA) spheres into the orbits of sixteen rabbits after enucleation. The spheres were removed 1, 2, 3, and 6 months after implantation and examined by light and scanning electron microscopy. Tissue breakdown and exposure of a-HA implants were not observed in any case. As time passed, fibrovascular tissue gradually invaded the pores of the HA spheres deeper and deeper. Although the HA used was completely artificially, we observed a mild foreign body reaction around the a-HA sphere. HA spheres are appropriate for orbital implants after enucleation without scleral enveloping.

Corrosion cast demonstration of retinal vasculature of normal Wistar-Kyoto rats.

Plastic corrosion casts of the rat retinal vasculature were studied by scanning electron microscopy. This technique demonstrated the entire retinal vasculature of the rat. The retinal blood vessels supplying the rat's retina have a definite and fairly constant pattern. At the disk there are usually six main artery and vein branches that run symmetrically towards the periphery. Veins are wider and more tortuous. The characteristic arrangement of endothelial cell nuclear indentations clearly differentiates arteries from veins. Retinal arteries have side-arm and dichotomous branchings. The number of vessel branches is greater on the nasal side than on the temporal side of the retina. The vein-over-artery crossing phenomenon is more frequent than the artery-over-vein. Retinal capillaries appear tortuous and are arranged cylindrically in two layers. The superficial network of capillaries comes essentially from arterioles, while deep layer capillary networks are more regularly and densely arranged and are mainly connected with venules. The inner and the outer capillary networks have interconnections, vertical runs and short vascular bridges. Within the retina there are regional variations in capillary pattern and distribution. More regular, dense and rich capillary networks are observed in the peripheral area than at the posterior pole area. No arteriovenous shunts were seen. The study of such plastic casts makes possible a more accurate assessment of some aspects of vascular abnormalities. These findings will be helpful in further investigations of retinal vascular abnormalities.

Retinal vascular changes during aging in Wistar Kyoto rats. Application of corrosion cast and scanning electron microscopy.

Retinal vasculature changes during aging in normal Wistar Kyoto rats maintained to longevity without known diseases were studied by corrosion casts and scanning electron microscopy (SEM). Six-month-old rat retina showed regular running and arrangement of retinal blood vessels. In 18-month-old rats, a definite morphological change was moderate tortuosity of the vessels, especially at the posterior pole of the retina. In 24-month-old rats, the retinal arteries and veins were very tortuous and narrow, and their calibers were irregular. In some animals occlusion of the veins and localized constriction of the arteries and veins were evident in the vascular casts. Neither arteriovenous crossing defects nor microaneurysms were seen. Retinal capillaries showed marked changes: localized narrowing, constriction, bead-like features, caliber irregularity and occlusion. The corrosion cast method with SEM can demonstrate the details of the capillary networks in the rat retina. The findings demonstrated appear to be related to aging.