A study of low-nutrient diets used for aging studies in the rat.

The effects of diets of low caloric value on rats used in aging studies were investigated. Groups of 85 Sprague-Dawley rats were fed ad libitum from 3 months of age on three different diets containing 8 or 10 Megajoule (MJ) of metabolizable energy and 80 or 100 g of crude protein/kg. Body weights, food consumption, and morphological and biochemical parameters were monitored throughout life. Kidneys were examined histologically. Rats given the diet with highest energy and protein ate less food, attained greater weights, and had larger abdominal fat deposits than those on the lower energy diets. They had a raised proteinuria, and nearly half developed glomerulosclerosis and tubulo-interstitial damage by 26 months. There was no significant difference in mortality between the groups, and no other serious abnormalities were observed. It is concluded that rats can be maintained into old age with no signs of nutritional inadequacy on diets with lower energy and protein contents than those in general use.

Hematologic and biochemical profiles of selectively bred WHHL rabbits.

In Watanabe heritable hyperlipidemic (WHHL) rabbits, hypercholesterolemia and hypertriglyceridemia develop from birth, because of a deficiency of low-density lipoprotein receptors, and are followed by a consequent early development of aortic atherosclerosis. This closely resembles human familial hypercholesterolemia. Starting in 1984, we have developed a closed colony by breeding two male and two female homozygous WHHL rabbits, obtained from Japan (Dr. Watanabe, Kobe University). In our facility, the application of a selective breeding program, strictly based on mating parents that both have high serum lipid concentrations, has produced markedly elevated cholesterol (701 +/- 172 mg/dl, mean +/- SD) and triglyceride (780 +/- 325 mg/dl) concentrations in weaning rabbits. Clinical chemical analysis revealed no kidney or liver function abnormalities even in animals with extremely high lipid concentrations, and hematologic profiles were very similar in WHHL and age-matched New Zealand White rabbits, with the exception of platelet count, which was significantly higher in WHHL rabbits. Platelet aggregation induced by collagen and platelet-activating factor was significantly reduced in WHHL rabbits, whereas thrombin and prothrombin times appeared normal when compared with those in New Zealand White rabbits.

Aortic response to relaxing agents in Watanabe heritable hyperlipidemic (WHHL) rabbits of different age.

Serum and aortic tissue cholesterol levels in parallel with aortic relaxation to endothelium-dependent and independent drugs were determined in Watanabe heritable hyperlipidemic (WHHL) rabbits in comparison with New Zealand (N.Z.) normocholesterolemic rabbits, aged 4-14 months. Serum cholesterol was elevated (626 +/- 99 mg/100 ml) in 4-6-month-old WHHL rabbits and significantly lower in 12-14-month-old animals (344 +/- 51 mg/100 ml). Cholesterol infiltration in thoracic aorta was high in young WHHL compared with N.Z. rabbits (0.88 +/- 0.3 mg/100 mg fresh tissue vs. 0.08 +/- 0.003 mg/100 mg, respectively) and it did not vary with age. In N.Z. rabbits, serum and aortic cholesterol levels were low from 4 to 14 months of age. The aortic relaxation to acetylcholine (0.03-3 microM) on EC50 noradrenaline precontracted rings was similar in 4-6-month-old WHHL and N.Z. rabbits of the same age. In WHHL rabbits, the relaxation to acetylcholine was significantly reduced in 7-11- (-35% at maximum) and in 12-14-month-old rabbits (-40% at maximum). In N.Z. rabbits the response to acetylcholine was not modified in the 3 age groups. The relaxation to ATP (30 microM to 3 mM) was reduced by age both in N.Z. and in WHHL rabbits, but in 12-14-month-old WHHL rabbits the maximal relaxing response was significantly more elevated than in age-matched N.Z. rabbits (50.1 +/- 2.5% vs. 35.1 +/- 3.2%, respectively). The aortic relaxation to NaNO2 (10 microM to 3 mM) was reduced by age both in N.Z. and in WHHL rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium-dependent relaxation, cholesterol content and high energy metabolite balance in Watanabe hyperlipemic rabbit aorta.

Functional and metabolic parameters of thoracic aorta from Watanabe heritable hyperlipemic (WHHL) rabbits (aged 11-14 months) were investigated in vitro. The aortic preparations, normally responsive to noradrenaline, showed a diminished response to the endothelium-dependent agent, acetylcholine, in comparison with control preparations from age-matched New Zealand rabbits (maximal relaxation: 33 +/- 4% in WHHL vs. 52 +/- 2% in controls: P less than 0.005). ATP relaxant effect (only partially endothelium-dependent) was unimpaired in WHHL aorta, and it was much higher than in controls (maximal response: 63 +/- 6% vs. 37 +/- 3%, respectively; P less than 0.005). The response to NaNO2, an endothelium-independent relaxant, was unchanged in WHHL aortas. Acetylcholine-induced response was found to be inversely related to the degree of total cholesterol infiltration in aorta (r = -0.62, P less than 0.05). No correlation was observed between either total serum cholesterol or triglycerides and ACh-induced response. Furthermore, the concentration of adenine nucleotides and nucleosides in the aortic tissue of WHHL rabbits was lower than in controls, indicating a loss of energy balance. The results indicate a functional damage induced by genetic hyperlipidemia on endothelium-dependent relaxation and an impairment of energy-rich phosphate metabolism of the aortic wall. The relationship between functional and metabolic parameters is not yet clarified.

General toxicity and peripheral nerve alterations induced by chronic vincristine treatment in the rabbit.

The effects of five 0.3 mg/kg intravenous administrations of vincristine (VCR) at weekly intervals were studied in the rabbit. Body weight gain was impaired starting from the first injection, while gross signs of motor paralysis and hair loss initiated from the third week. At the end of the observation period blood analysis revealed normocytic normochromic anemia, elevated serum creatine kinase, and low serum alkaline phosphatase, whereas all the tested parameters related to liver and kidney functions where within normal limits. The decreased number of red blood cells was the consequence of a complete, although reversible, blockade of staminal hematopoietic activity. Two important indexes of peripheral nerve function were clearly altered at the end of the treatment: (i) the sciatic nerve conduction velocity in vitro was 27% reduced and (ii) the latency between sciatic nerve stimulation and extensor digitorum longus (EDL) twitch in vivo was 34% prolonged. The usefulness of the rabbit as an animal model to study side-effects of VCR treatment is discussed.

Development of diabetic neuropathy in the C57BL/Ks (db/db) mouse and its treatment with gangliosides.

We studied the development of diabetic neuropathy and its treatment with gangliosides using the C57BL/Ks mouse. The results of axonal morphometry showed the presence of a progressive axonal atrophy which was maximal at 180 days of age. To 400 days of age there was no longer any significant difference, perhaps due to aging processes. Nerve conduction velocity changed significantly from the early days of life. Thirty-day treatment with gangliosides significantly improved nerve conduction velocity and axonal morphometry at 180 and 280 days of life. No effect was observed with treatments at 30 or 60 days. It was previously shown that the early phase of the C57BL/Ks mouse neuropathy was reversed by insulin, whereas the late phase (180 days) was not. We showed elsewhere that at 180 days of age in the C57BL/Ks mouse there was a drastic decrease in slow transport of AChE (G1 and G2 molecular forms) indicating a shift in neuronal metabolism and suggesting that the disease was then more intrinsically neuronal. Using the suggestion of Robertson and Sima (Diabetes 29: 60-67, 1980) we label the first phase of the neuropathy "metabolic" (treatable with insulin) and the second phase "neuronal" (treatable with gangliosides). This "neuronal" phase could be related to the degenerative stage of human diabetic neuropathy.

Functional and morphological abnormalities induced by ouabain intoxication of the rabbit retina.

Several researchers have recently used an intravitreal ouabain injection to induce a suitable model of experimental retinopathy and optic neuropathy in various animals. Ouabain administration into the vitreous body of rabbit causes an irreversible degeneration of the retinal layers and consequently of the optic nerve. The degeneration is proportional to the amount of injected drug. Electroretinographic recordings (ERG) show that these structural abnormalities are related to an inhibition of the electric retinal activity as the dose-dependent reduction of ERG waves amplitude has shown. Moreover, ERG and visual evoked responses (VER) measured at the same time evidence that the intravitreal injection of 1.7 nmol ouabain may block the impulse conduction along the optic nerve. This can be proved by the fact that 90 min after an ouabain injection VER disappears, while ERG is only partially reduced. These results are correlated with both morphological observation and autoradiographic studies on 3H-ouabain distribution in different retinal layers.

Monosialoganglioside (GM1) treatment of ouabain-induced retinopathy in the rabbit.

The mammalian retina is markedly influenced by cardiac glycosides. When nanomolar concentrations of ouabain are intravitreously injected into the rabbit, the visually evoked response completely disappears within 90 min, while scotopic ERG recordings show a remarkably decreased amplitude of the principal waves. When 33 nmol/kg monosialoganglioside are injected intravenously 30 min before topical intoxication, this functional impairment is significantly reduced. The electroretinographic response shows a limited amplitude reduction, while the cortical potential never disappears completely. Histological observations of intoxicated retinas show that a degenerative process begins in photoreceptor outer segment 24 h after the intravitreal ouabain injection. Presently, this process involves both the outer and inner nuclear layers and, finally, the ganglion cell layer. Comparing the intoxicated treated and untreated retinas, no difference is found in the degenerative pattern of the two groups. Autoradiographic studies are also reported to correlate the protective effect of monoganglioside (GM1) on this toxic retinopathy with its preferential accumulation in different retinal tissues.