Purpose: To examine the conjunctiva and cornea of riboflavin-deficient rats with scanning (SEM) and transmission (TEM) electron microscopes. Materials and Methods: Three-week-old Wistar Kyoto rats were fed a riboflavin-deficient diet (0.05 mg riboflavin/100 g) for 3 months. As a recovery experiment, rats which had been on a riboflavin-deficient diet for 3 months were given water with 1 mg riboflavin/300 ml for 2 months. The conjunctiva and the cornea were examined with SEM and TEM. Results: The serum riboflavin level was significantly lower in the riboflavin-deficient group than in the controls. In rats on a riboflavin-deficient diet for 3 months, SEM showed decreased microvilli and microplicae in the superficial epithelium of the conjunctiva and a decrease in the number of goblet cells. The cornea showed many dark cells and a marked decrease of microvilli and microplicae. In the riboflavin-deficient rats, TEM of the conjunctiva showed a decrease of microvilli and microplicae in the most superficial epithelial cells, a decrease in the layers of the epithelium and a marked decrease in the number of goblet cells, while the cornea had decreased microvilli and microplicae in the superficial epithelium, dark wing cells, loss of the basement membrane and hemidesmosomes of basal cells, cell debris and degenerative stroma cells and deposits of dense bodies in the subepithelial layer of the stroma. In rats recovered from riboflavin deficiency, the conjunctiva and cornea showed no abnormalities. Discussion: Riboflavin plays a role in the development and maintenance of the surface structures of epithelial cells. Riboflavin may also be necessary for the development and maintenance of goblet cells. Conclusion: Riboflavin is essential for maintaining the structure and function of the ocular surface.

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.

Purpose: The ultrastructure of the optic nerves in magnesium (Mg)-deficient rats was studied to elucidate the function of Mg. Methods: After delivery, mother Wistar Kyoto rats were fed a low-Mg diet containing 0.1 mg Mg/100 g diet with all other nutrients and distilled and deionized water. Infant rats were suckled by their mothers for 21 days and then fed the same Mg-deficient diet. Control mother rats were fed commercial rat pellets containing 24 mg Mg/100 g diet and all other nutrients. The optic nerves were examined by electron microscopy at 6 weeks of age. Results: In the Mg-deficient rats, serum Mg levels were significantly lower and calcium levels higher than in the control rats. Ultrastructural findings were: significantly fewer myelinated axons and significantly thinner myelin sheaths in the Mg-deficient rats than in the control and pair-fed controls, and more numerous unmyelinated axons. There were multifocal areas of destruction and necrosis in the optic nerve of Mg-deficient rats. Conclusions: This study suggests that the optic nerve needs Mg for the development and maintenance of its cell structure.

Purpose: We studied the cornea and conjunctiva of Zn-deficient rats with an electron microscope and time-of-flight secondary ion mass spectrometry (TOF-SIMS) to elucidate the role of trace elements in the cornea and conjunctiva. Materials and Methods: Twenty-one-day-old Wistar Kyoto rats were fed a Zn-deficient diet and deionized water for 7 weeks and then killed. The control rats were fed a Zn-deficient diet and deionized water supplemented with 3 mg Zn/100 ml. After 7 weeks on the deficient diet, another group of rats was given drinking water containing 3 mg Zn/100 ml and the usual diet containing 4.7 mg Zn/100 g for 8 weeks for recovery. The cornea and conjunctiva were examined by electron microscopy and TOF-SIMS. Results: Microvilli and microplicae in the most superficial layer of the epithelium of the Zn-deficient rat conjunctiva and cornea were prominently reduced, and dark cells were significantly increased. The numbers of goblet cells were decreased in the conjunctiva of the Zn-deficient group. Zn, Ca and Al ions were significantly fewer, but K, Fe, Cl and S ions were significantly more numerous in the Zn-deficient group than in the control group. In the cornea of the Zn-deficient group, there was significantly more Cl but less Ca and vitamin C than in the controls. Discussion: Zn deficiency may interfere with protein, nucleic acid and collagen synthesis through the reduction of Zn-containing enzymes. Myosin-like substance, actin filaments and tonofibrils are important structural components for microvilli and microplicae in the epithelium. Maldevelopment of these structural components may be related to disturbed activities of Zn-containing enzymes in protein and collagen synthesis because of Zn deficiency. In addition, Zn deficiency caused changes in the levels of Zn and other trace elements such as Ca, Al, S, Fe, and Cl and vitamin C. Conclusion: Zn deficiency resulted in poorly developed microvilli and microplicae on the ocular surface tissues, reduced the number of goblet cells and changed the quantity of trace elements and vitamin C.

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