Imran Ahmed Bhutto

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. Anat Rec 264:63–71, 2001.

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 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.

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 changes in Otsuka Long-Evans Tokushima Fatty rats (spontaneously diabetic rat) - Possibility of a new experimental model for diabetic retinopathy

PURPOSE To study retinal changes in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a spontaneously diabetic rat, and evaluate it as a possible new diabetic retinopathy model. METHODS We examined the retinas of OLETF rats and Long-Evans Tokushima Otsuka (LETO) rats as controls under both a transmission electron microscope (TEM) and a scanning electron microscope (SEM). RESULTS We detected higher blood sugar level in the OLETF rats than in the LETO rats beginning at 5 months of age. The inner nuclear layers of the retina decreased from 3-4 rows to 2 rows, whereas the photoreceptor cell nuclei decreased from 8 rows to 3-6 rows. Retinal pigment epithelial cells decreased in height, and basal infoldings were poorly developed. Retinal capillary basement membranes were significantly thicker in the OLETF rats than in the LETO rats, and endothelial cell damage was observed. The SEM of vascular corrosion casts of OLETF rats showed tortuosity, microaneurysms, and loop formations. CONCLUSIONS The development of spontaneous hyperglycemia in OLETF rats was associated with alterations in retina ultrastructure. Changes were similar to those seen in diabetic retinopathy, but did not include either hemorrhages or exudates. The OLETF rat may be a useful animal model for the study of ocular complications in humans.

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

&NA; 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.

Retinal and choroidal vasculature in rats with spontaneous diabetes type 2 treated with the angiotensin-converting enzyme inhibitor cilazapril: Corrosion cast and electron-microscopic study

The long-term effects of the angiotensin-converting enzyme (ACE) inhibitor cilazapril on retinal and choroidal circulation in rats with spontaneous diabetes type 2 were assessed by corrosion casts, scanning electron microscopy (SEM) and transmission electron microscopy. One group of 20 male Otsuka Long-Evans Tokushima Fatty (OLETF) rats was treated with 10 mg/kg/day of cilazapril from 4 to 64 weeks of age, and 20 other OLETF rats received no treatment. A third group, 20 male Long-Evans Tokushima Otsuka (LETO) rats, served as age-matched controls. At regular intervals, the rats were weighed, and their blood glucose was measured. Before the experiment, their systolic blood pressure and total cholesterol level were determined. At 64 weeks of age, the OLETF rats weighed significantly less than the cilazapril-treated OLETF and the LETO rats (p < 0.0001). At the same age, 100% of the untreated OLETF rats had bilateral cataracts, while the lens was clear and no fundus abnormality was detected in the cilazapril-treated OLETF rats and the LETO rats. Cilazapril lowered systolic blood pressure to a nearly normal level, significantly prevented the increase in blood sugar and inhibited the increase in serum cholesterol in the OLETF rats throughout the treatment. In the 64-week-old OLETF rats without treatment, corrosion cast and SEM revealed diabetic retinal and choroidal vascular changes: tortuosity of the vessels, variations in caliber, narrowing of arteries, arterio-arteriolar anastomoses and hairpin loop formation in precapillary arterioles, sparse collecting venules in the choroid and marked capillary changes such as caliber irregularity, narrowing, tortuosity, loop formation and decreased capillaries, outpouching and microaneurysms. In the cilazapril-treated OLETF rats, these changes were markedly decreased to the level seen in the LETO rats, in which the retinal and choroidal blood vessels had a definite and fairly constant pattern and the capillaries were more regularly and densely arranged and had a remarkably uniform caliber. Our results show that the long-term administration of cilazapril before or from the initial onset significantly prevented the increase in blood sugar and inhibited the increases in serum cholesterol in OLETF rats throughout the treatment, lowered systolic arterial pressure to a nearly normal level and prevented diabetic ocular complications. The effects of cilazapril on the diabetic retinal and choroidal vasculature are described for the first time. SEM of corrosion casts is a valuable and easy technique for showing precisely and three-dimensionally the effects of some drugs on the vasculature.

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.

Retinal Vascular Changes during Aging in Wistar Kyoto Rats

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.

Natural Course of Experimental Choroidal Neovascularization: Three-Dimensional Study with Corrosion Cast and Scanning Electron Microscope

Objectives: The details of the morphological features of choroidal neovascularization (CNV) remain unclear. The purpose of this study was to establish a CNV rat model and study the natural course of CNV using vascular casts and a scanning electron microscope (SEM). Methods: Focal laser photocoagulation (argon green 50 mW, 0.04 s, 200 µm) was applied to Brown Norway pigmented rats. Choroidal vascular casts were prepared 1 and 3 days, 1 and 2 weeks, and 1, 3 and 6 months after laser photocoagulation. The choroidal casts were examined with a SEM. Results: One day after photocoagulation, corrosion casts and SEM revealed complete defects of the choriocapillaris at the laser shot sites. One week after photocoagulation, small vascular buds originating from the damaged choriocapillaris were observed. Two weeks after photocoagulation, newly formed CNV originating from an individual laser burn was observed. One to three months after photocoagulation, these new vessels were connected to each other to form CNV networks. Six months later, some thin and atrophic vessels were observed in the CNV network. Conclusions: We succeeded in making fine corrosion casts of CNV formed by photocoagulation in pigmented rats and in demonstrating the details of CNV formation and regression. It is hoped that the results of this study will contribute to the development of a drug therapy for CNV and to the interpretation of diagnostic imaging of CNV in humans.