Roger C. Moretz

Preclinical changes in weight of scrapie-infected mice as a function of scrapie agent-mouse strain combination.

Several inbred strains of mice were injected with different scrapie agents and their total body weight was monitored throughout the incubation period. As a control, mice were injected with normal mouse brain homogenate. For most combinations of scrapie agent and mouse strain, weights during the preclinical phase were similar to or lower than the average weight of controls. For some combinations there was a significant increase in weight (compared to controls) during the latter part of the preclinical phase of disease. The effect was dependent on both agent and mouse strain, i.e., in some cases a mouse strain showed the increase with one scrapie agent but not another and some scrapie agents caused the increase in one inbred strain of mouse but not in another strain. The increase in weight was due to accumulations of fat rather than a generalized increase in weight of various organs. With one mouse strain (SJL), there was increased vacuolation seen in the hypothalamus of mice injected with scrapie agents that showed the increase in weight compared to the lesion intensity with an agent which did not cause the weight increase.

Increased blood-brain barrier permeability in scrapie-infected mice.

The present investigation was designed to study the ultrastructural integrity of the blood-brain barrier (BBB) in the cerebral microvasculature of scrapie-infected mice showing clinical illness. Cerebral microvessels from either IM, VM, or C57BL/6J mice, terminally affected with various strains of scrapie agent showed a focal leakage of horseradish peroxidase (HRP) in all agent-strain and mouse-strain combinations. This leakage was most pronounced in and near the primary site of agent inoculation, but was also observed in microvessels scattered throughout the brain. Cytochemical studies also revealed a redistribution of plasmalemma-bound alkaline phosphatase in the endothelial cells. In control mice, the enzymatic activity was mainly concentrated in the luminal plasmalemma, while in the scrapie-infected mice the activity also appeared in the abluminal side in the majority of microvessels. Our observations are evidence that the BBB of the mouse is altered in some way by the scrapie agent. Such an alteration may have important implications for human disease, since the scrapie agent is related to the group of “slow” viral infections, including kuru and Creutzfeldt-Jakob disease. Scrapie may also serve as an important model for the study of senile dementia of the Alzheimer type (SDAT).

Genetic Control of Scrapie: Incubation Period and Plaque Formation in I Mice

The host component of control of scrapie incubation period in the mouse is manifested largely through the action of the Sinc gene. Only one mouse strain (VM) has been found that is p7p7 (prolonged incubation for ME7 agent) and two other strains have been derived from VM. All other strains, designated s7s7, have a short incubation for ME7. In the present study, the I strain was shown to fulfil the criteria that are characteristic of mouse strains with the p7 allele of Sinc: a comparatively long incubation period for ME7 and a short incubation period for 22A, the incubation period for F1 hybrid mice (s7s7 X p7p7) either fell between the incubation periods for the parental strains (with ME7) or were longer than either parent (with 139A and 22A), amyloid plaques occurred following injection of ME7 and 87V but not after 22A or 139A, lesion profiles for four scrapie strains were similar in I mice and p7p7 mouse strains, and injection of 87V led to disease in less than 300 days. Finally, allelism tests using F1 hybrid mice (I X a p7p7 mouse strain) and progeny of backcrosses between these F1 mice and I mice failed to reveal the segregation of additional major genes affecting scrapie incubation period.

Nutritional Taurine Deficiency and Feline Pregnancy and Outcome

The high concentrations of taurine and the function of taurine in excitable tissues have been the subject of speculation for many years. In recent years it has become possible to examine the functions of taurine further by perturbing the concentration in tissues; in the cat by dietary restriction and in rodents by administering the taurine transport inhibitor guanidinoethane sulfonate. Both of these strategies result in reduced tissue taurine concentrations and retinal degeneration. There has been additional interest in the possible functions of taurine in the developing nervous system because taurine concentrations are especially high, taurine generally being the free amino acid present is the greatest concentration in late fetal and early postnatal brain (4). We have explored the role of taurine in development by mating and breeding female cats that had been acclimated to synthetic diets containing various amounts of taurine for long periods of time (more than 6 months). It soon became clear that although taurine-deficient females apparently came into estrus normally and generally appeared to conceive normally, they experienced difficulty in successfully completing pregnancies (5). Reproductive losses included fetal resorption, abortion, stillbirth, and low birthweights (mean birth weight 70 g) of live kittens at term (Fig. 1). Live kittens had a poor survival rate, and grew slowly. Females fed the same diet supplemented with 0.05% taurine did not experience such difficulties (mean birthweight 104 g). Table 1 details the outcome of pregnancies to date.

Abnormal visual acuity and retinal morphology in rhesus monkeys fed a taurine-free diet during the first three postnatal months.

Over ten years ago, taurine was identified as an essential nutrient for cats (7). Without a dietary source of this amino acid, such as their natural taurine-rich diet of meat and fish, both kittens and adult cats undergo a decline in plasma and tissue taurine levels, reduced electroretinogram amplitudes with prolonged peak latencies, and progressive retinal degeneration which eventually results in blindness (8,24,26). As Dr. Sturman describes elsewhere in this volume, taurine deprivation of pregnant cats and their kittens also produces more widespread nervous system disturbances, including abnormal development of the cerebellum and visual cortex (19,27). Although cats appear to be unique in the severity of the effects produced by dietary taurine deprivation, it is now clear that they are not the only species affected. Recent studies of both human children and infant monkeys show that changes in retinal structure and function are associated with taurine deprivation.

A Simple Screening Procedure for Evaluating Central Nervous System Tissue Sections Showing Structural and Cytochemical Alterations of the Blood-Brain Barrier

A simple method for rapidly screening and evaluating many areas of central nervous system tissue before and after flat embedding in Beem capsules is described. This method uses light microscopy to select regions surrounding needle track injuries of brain tissue for subsequent fine structural and enzyme cytochemical analysis of the blood-brain barrier. The mouse cerebral cortex was sectioned with a tissue chopper at 40-50 micrometers and reacted with diaminobenzidine to demonstrate the presence of exogenous horseradish peroxidase near an injured central nervous system site. Following the enzyme reaction, both osmicated and unosmicated tissue slices were processed for routine electron microscopy, infiltrated with unpolymerized resin, and evaluated on glass slides by light microscopy prior to flat embedding and polymerization. Numerous tissue specimens can be screened in this way for maximum information per tissue slice, and extra tissue samples can be polymerized on the glass slides and conveniently stored for future sectioning.