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Exposure of mouse neuroblastoma cells in culture to low pH for 8 days caused a sixfold increase in cell lipofuscin pigment. Pigment content was measured as the percent of cells having clumps of acid phosphatase-staining material. Hydergine, between 0.1 and 3 microgram/ml, caused a concentration-related decrease in pigment content. Hydergine also stimulated neurite formation in cells in regular pH medium and was slightly toxic to cells in low pH medium. These results support the use of neuroblastoma cells as an in vitro model for age pigment studies.

Previous studies in our laboratory demonstrated an age-related increase in the brain-reactive antibodies in sera of aging mice. The present paper deals with the study of brain-reactive antibodies in sera of non-human primates (Macaca nemestrina) of three age groups (4, 10 and 20 years) by an indirect immunofluorescence method. The results indicated a progressive increase in the serum levels of these antibodies with advancing age. Since all three age groups examined demonstrated brain-reactive antibodies in sera, the age of onset of these antibodies could not be determined in these animals. It has been suggested that the formation of these antibodies could be evidence of autoimmune reactions which might play a significant role in neuronal degeneration during aging.

Previous studies have demonstrated that underfeeding in both premature and young mature animals may extend the life span as well as preserve the functions of the immune system. The effects of caloric restriction for a period of 12 months on different organs as well as the formation of brain-reactive antibodies in young mature mice (3 months) were tested. These animals showed a significantly lower weight of the total body and various organs including the brain, spleen, adrenals and kidneys. The brain weight/body weight ratio, on the other hand, was significantly higher in these mice. Sera in the dietary animals were mostly negative while those of control animals of the same age and sex had high levels. The present study supports the earlier observations that controlled dietary restriction is able to slow down the age-related deterioration of the immune system. The inhibition of brain-reactive antibody formation in these animals might also be related to a delayed onset of autoimmune disorders.

The effects of centrophenoxine on the retinal pigment epithelium (RPE) of 17 month old female mice have been studied. Animals were injected subcutaneously for 3 months (60 injections) with the drug (0.1 mg/g of body wt) daily in 0.1 M phosphate buffered saline at pH 7.0. The morphological changes in the pigment layers of the retina of both eyes were studied by light and electron microscopy and the lipofuscin pigment was demonstrated by its autofluorescence and ultrastructural characteristics. There was a significant reduction of the lipofuscin pigment in the treated animals, but the melanin pigment remained unchanged. The lipofuscin granules also appeared less osmiophilic and showed a greater preponderance of membranes and vacuoles. Although the precise mechanism of action of the drug is not clear, an increased protective function of the pigment epithelium by the drug has been suggested.

The cerebellar cortices in 4, 10 and 20 year of Macaca nemestrina have been examined for the number of Purkinje (P) and granule cells and the deposition of lipofuscin in P cells in relation to aging. Lipofuscin distribution significantly increased with in the P cells in these animals. The number of P cells was significantly reduced, while there were no changes in the number of granule cells. It appears from this and other studies that the Purkinje cells are more prone to aging changes than the granule cells of the cerebellum both in lipofuscin formation and cell loss. Although the precise functional significance of these changes in P cells is not clear, their vulnerability may be related to changes in motor function in old age.

The extent to which immune processes contribute to senescence-related neurological/behavioral impairment was examined using an adoptive transfer procedure. C57BL/6 mice aged 22 to 24 months showed impaired ability for acquisition of an active avoidance response when compared with younger mice aged 3 months. An immunofluorescence assay of the sera of these mice indicated that only sera from the senescent mice reacted with brain antigen. When tested three months following irradiation and receipt of bone marrow/spleen cell suspensions from senescent mice, young mice showed senescence-like serum-brain reactivity and declines in their abilities to acquire learning. Young control mice receiving cell suspensions from age-matched donors showed no evidence of serum-brain reactivity or learning deficits, suggesting that impaired learning was related to acquisition of aged immunity and not a nonspecific effect of the transfer procedure. These findings indicate that immune processes may be involved in the etiology of senescence-related neurological/behavioral dysfunctions.

Previous studies in our laboratory demonstrated that brain-reactive antibodies are present in the serum of old mice and these are separated from the brain antigen by the blood-brain barrier. In this paper the specificity of these antibodies to neuronal antigen has been studied using sera from old mice with or without absorption by the homogenates of different organs and also by direct treatment with sections of those organs. The results indicate that these brain-reactive antibodies found in sera of old mice are specific to neuronal antigen in the central nervous system.

Previous studies have demonstrated an increase in brain-reactive antibodies (BRA) in sera of various mammals as a function of age. The present study attempts to obtain a better understanding of the mode of formation of BRA by using various immunologic manipulations in young and old C57BL/6 mice. The mean BRA levels were significantly higher in older mice than in the young ones. Mixtures of bone marrow cells and spleen cells from old donors were able to adoptively transfer high BRA levels in young irradiated mice and this was not altered by pretreatment of the cells with anti-Thy-1.2 or anti-Ig serum plus complement. Transfer of young cells to old irradiated hosts did not lower the high BRA levels in the hosts. However, when the old hosts were also thymectomized and grafted with neonatal thymuses, BRA levels were significantly lowered. The studies tend to indicate that thymic involution or loss of thymic function in aging plays an important role in BRA formation in sera of aged mice.

The possible role of damaged neurons as an antigenic stimulant in the formation of brain reactive antibodies (BRA) has been studied. When neurons of the ventral horn of the spinal cord were damaged by axonal injury, the damaged cells showed no evidence of antigen-antibody reaction within 12 weeks, nor could BRA be demonstrated in the blood during that time in young mice. The possible migration of radio-labeled gamma-globulin across the blood-brain-barrier (BBB) has also been investigated in young mice. Only a very low rate of migration has been observed across the BBB and this might account for the scattered loss of nerve cells in the brains of old animals.

Senescent mice showed attenuation of habituation in locomotor activity and marked deficit in the motor performance requiring limb coordination. Also, the binding of 3H-diazepam to the crude synaptosomal fraction prepared from cerebral cortex of the senescent mice was significantly altered. The binding maximum (Bmax) was significantly greater in the aged (23-26 months) mice, but no change was found in the apparent dissociation constant (Kd). Senescent related changes appear to include neurochemical alteration of the endogenous benzodiazepine system (increased receptor sites without any change in receptor affinity) and, perhaps, related physiological deficits in the limb coordination and/or strength.

Previous studies in our and other laboratories indicated that there is a reduction in the neuronal lipofuscin in old rodents after several weeks of treatment with centrophenoxine. The present study investigates whether this chemical can prevent pigment formation if given early in life before the onset of pigmentogenesis. The study shows that the drug did not stop lipofuscin formation in 1 month old mice. But there was a consistent decrease in the pigment in the neurons of cerebral cortex and hippocampus of the treated animals compared to the age-matched controls. The degree of reduction was largely dependent on the duration of the treatment and a significant diminution was noted after treatment for five months or more.

Previous studies have indicated an increase in brain-reactive antibodies (BRA) in sera of aging mammals and an autoimmune disorder underlying senescence has been suggested. Since New Zealand Black (NZB) mice have a shorter lifespan and greater propensity for autoimmune diseases than C57BL/6 mice, various age groups from both strains of mice were investigated for simultaneous occurrence of BRA serum titer and deficits in learning. NZB mice exhibited a marked learning deficit as well as higher BRA levels at all ages. C57BL/6 mice showed increased BRA and a learning deficit only at advanced ages. The findings of "precocious" BRA titers along with marked learning deficits, both occurring at young ages in NZB mice and both similar to defects seen in the normal mice at senescence and in patients with senile-dementia, suggest that NZB mice may serve as a useful animal model of pre-senile dementia.

Previous studies have shown increased life span and decreased lipofuscin deposition in brain structures when both premature and young adult animals are calorically restricted of an otherwise nutritionally adequate diet. Three-months-old C57BL/6 mice were subjected to 12 months of caloric restriction (2 g/day), and subsequently exposed to a radial maze learning paradigm. Mice in the diet restriction group showed faster learning and higher asymptotic performance on the radial maze task, as well as lower lipofuscin deposition in the neurons of hippocampus and frontal cortex relative to control mice fed ad libitum. The results suggest that dietary restriction has effects on radial maze learning, and this improved behavioral performance was associated with significant reduction in lipofuscin pigment deposition in the neurons of hippocampus and frontal cortex.