H. Heath

Ultrastructural alterations during the development of retinopathy in sucrose-fed and streptozotocin-diabetic rats

Abstract Normal and streptozotocin-diabetic rats were maintained for 6–11 months on diets in which the sole source of carbohydrate was either 68% corn starch or sucrose. Perpendicular cross-sections of 15 capillaries from the retinal outer plexiform layer from each animal were examined qualitatively and quantitatively. The retinopathy produced by sucrose feeding to normal animals although identical by light microscopy to that produced by streptozotocin was not so ultrastructurally. Ramifications of the basement membrane, nodules and deposition of collagen-like material within the basement membrane were observed but no uniform thickening took place. Similar pathological changes were present in the diabetic capillaries except that a significant increase in the thickness of the basement membrane occurred. This thickening was not related to either blood glucose levels or the severity of the retinopathy indicating that the development of retinopathy is not related to basement membrane thickening, but rather to the deposition of material within the membrane.

The effects of an aldose reductase inhibitor upon the sorbitol pathway, fructose-1-phosphate and lactate in the retina and nerve of streptozotocin-diabetic rats

UNLABELLED To investigate the aetiology of complications secondary to experimental diabetes in the rat, the concentrations of glucose, sorbitol, fructose, fructose-1-phosphate, lactate and inositol were measured in the retina and sciatic nerve of streptozotocin-diabetic and normal rats treated for 22 days with ICI 105552 (1-(3,4-dichlorobenzyl)-3-methyl-1,2-dihydro-2-oxoquinol-4-ylacetic acid, sodium salt; 50 mg/kg body wt daily), an inhibitor of aldose reductase (E.C. 1.1.1.21) the first enzyme of the sorbitol pathway. In the diabetic nerve, where accumulation of sorbitol may be pathogenic, treatment with ICI 105552 reduced the accumulations of sorbitol (70%), fructose (47%) and lactate (34%) without affecting glucose, fructose-1-phosphate or inositol. In the nerves of controls, the inhibitor reduced both sorbitol (23%) and fructose (20%) levels without other effects. In the diabetic retina where accumulation of sorbitol or lactate might be pathogenic, treatment with ICI 105552 had no statistically significant effect upon the concentrations of glucose, sorbitol, fructose, fructose-1-phosphate or inositol. However, the inhibitor reduced the concentration of lactate to below the non-diabetic level. In the retinas of controls, dosage with ICI 105552 reduced the concentration of sorbitol by 36% without other effects. THE RESULTS (1) demonstrated that ICI 105552 was a potent inhibitor of aldose reductase in sciatic nerve and suggested that a proportion of nerve lactate in diabetes could result from sorbitol pathway activity; (2) implied that flux through the sorbitol pathway, eventually to form lactate, increases in diabetic retina; and (3) indicated, by the drugs reduction of retinal lactate concentration, that inhibitors of aldose reductase might be of potential use in diabetic retinopathy.

The active transport of ascorbic acid by the rat retina

The uptake of l -[I- 14 C]ascorbic acid by the rat retina in vitro has been investigated. A study of the effects of anaerobiosis, fluoride, iodoacetate, dinitrophenol, ouabain and variations in glucose and sodium ion concentrations showed that ascorbic acid was transported into the retina by an energy-dependent, sodium-sensitive process. 80% of the required energy could be derived from anaerobic glycolysis. This transport was inhibited by corticosteroids and enhanced by acetazolamide. l -[I- 14 C]Dehydroascorbic acid entered the rat retina in vitro by a process of passive diffusion.

Identification of fructose as the retinopathic agent associated with the ingestion of sucrose-rich diets in the rat☆

In order to determine whether the fructose moiety of sucrose or the lack of some factor essential for the integrity of the microvascular system was responsible for the development of sucrose retinopathy in the rat, a series of diets containing possible sources of such a factor and/or fructose was tested over a 6-mo period. Examination of the isolated rat retinal vascular systems showed conclusively that fructose was the dietary microangiopathic agent associated with sucrose-induced retinopathy. The microvascular lesions produced were similar to those found in diabetic rats maintained over the same period. Cross-sectional studies of the retinas revealed that microvascular lesions preceded the associated degeneration of neural tissue rather than vice versa since the majority of rats with retinopathy showed no signs of neural damage. Sucrose feeding was found to produce a significant elevation (p < 0.001) in blood fructose concentration and a slight increase, albeit not significant (p < 0.01), in retinal fructose-1-phosphate (F1P) levels. The results are discussed in relation to the changes in retinal sorbitol, fructose, FIP, and lactate metabolism found in diabetes.

The effects of long-term treatment of streptozotocin-diabetic rats with an aldose reductase inhibitor

To test the possible involvement of the sorbitol pathway in the pathogenesis of retinopathy in long-term experimentally-diabetic rats, streptozotocin-diabetic and normal rats were dosed orally (50 mg/kg body weight daily) for up to 373 days with an aldose reductase inhibitor (ICI 105552) or a placebo. Long-term treatment with ICI 105552 (1,(3,4-dichlorobenzyl)-3-methyl-1,2-dihydro-2-oxoquinol-4-ylaceti c acid; sodium salt) markedly reduced the normal accumulations of sorbitol and fructose in the sciatic nerves (86 and 69% reductions) and seminal vesicles with coagulating glands (75 and 49% reductions). Thus, by these criteria, the inhibitor was as effective after several months of dosing as after three weeks. There was no evidence that treatment with this aldose reductase inhibitor had any protective effect against the development of pathological changes in the retina and kidney of these rats.

The separation of bound ascorbic acid from rat adrenals by gel filtration.

Abstract A new method is described for the study of bound ascorbic acid. Extracts of adrenals from rats injected previously with l -[I-14C]ascorbic acid were separated by gel filtration on Sephadex G-50 columns into eight fractions. The first six fractions to be eluted were destroyed by prior digestion of the adrenal homogenate with proteolytic enzymes and they are thought to represent protein or polypeptide-bound forms of ascorbic acid. 24 h after the administration of the labelled vitamin 41.5% of the total counts were found in these fractions. The two low molecular weight fractions consisted of ascorbic acid and dehydroascorbic acid.

Inhibition of Alcohol Dehydrogenase by Chloroquine

CHLOROQUINE is an anti-malarial drug which is also used in the treatment of rheumatoid arthritis. It has been shown to inhibit various thiol-containing enzymes such as succinic dehydrogenase1, glutamic dehydrogenase2 and the α-ketoglutarate oxidizing system3. The prolonged administration of large doses of chloroquine causes the development of an irreversible retinopathy4. Chloroquine accumulates in the retinal pigment epithelium5 and it also inhibits NADH-monodehydroascorbic acid transhydrogenase, a thiol-containing enzyme present in retinal microsomes6. Alcohol dehydrogenase is an important enzyme in the visual cycle, being involved in the retinol–retinal conversion, and in this communication it has been established that this thiol-containing enzyme is competitively inhibited by chloroquine.

Chemical changes in human diabetic, cataractous and β,β′-iminodipropionitrile-treated rat lens capsules*

Thickening of vascular basement membrane has been shown to occur in diabetes by many researchers. Lens capsule contains a collagen-like protein and has chemical and immunological properties similar to those of other basement membranes. Significant increases in the hydroxyproline content of both the anterior and posterior human capsule were found to occur in diabetes. No significant change occurred in the posterior capsule from human cataractous lenses, but the content of hydroxyproline in the anterior capsule was raised significantly. Treatment of rats with β,β′-iminodipropionitrile causes a retinal angiopathy with increased PAS positivity. The hydroxyproline content of the lens capsules of these treated rats was increased whereas the hexosamine content was decreased. No changes were detected in the sialic acid content.

Effect of β,β′-iminodipropionitrile and related compounds on the electroretinogram and the retinal vascular system of the rat

The subcutaneous injection of 200 mg/100 g body weight of β,β′-iminodipropionitrile to rats causes the development of a progressive retinal angiopathy. The course of these changes was followed by the histological examination of the retinal vascular system and the determination of the electroretinogram, after the administration of this compound. It was concluded that β,β′-iminodipropiontrile does not immediately inhibit any energy-providing processes of the retina since no changes were observed in the electroretinogram for the first 2 days after dosage. A decrease of b-wave amplitude but no change of the a-wave occurred on the third day. The positive components of the b-wave were non-existent by the fourth day and the remaining electroretinal response was extinct by the eighth day. PAS-positive deposits were observable in the retinal arterioles on the fifth day and the angiopathy, consisting of endothelial hypercellularity, PAS-positive deposition, acellular capillaries and micro-aneurysms, became progressively more severe over the next 16 days. Similar changes were not caused by iminodiacetonitrile, β,β′-oxydipropionitrile or 2,2′-iminodiethanol and the toxicity would appear to be specifically associated with the structure of β,β′-iminodipropionitrile.

Changes in the intraocular pressure and the electroretinogram in the β,β′-iminodipropionitrile-treated rabbit

The development of retinal microangiopathy and central nervous system disturbances commences in animals 3 days after the administration of β,β′-Iminodipropionitrile. The intraocular pressure of rabbits decreases significantly within 24 hr following the subcutaneous injection of 75–125 mg IDPN/100 g body weight. At the high dose level, this change was permanent and preceded alterations in the ERG, which first occurred on the third day.