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Effects of controlled dietary restriction on brainreactive antibodies in sera of aging mice
Mechanisms of Ageing and Development, 18 (1982) 97-102
97
EFFECTS OF CONTROLLED DIETARY RESTRICTION ON BRAINREACTIVE ANTIBODIES IN SERA OF AGING MICE
KALIDAS NANDY* Geriatric Research, Education and Clinical Center, Veterans Administration Hospital, Bedford, Massachusetts, and Department o f Anatomy and Neurology, Boston University School of Medicine, Boston, Massachusetts (U.S.A.)
(Received September 15, 1981)
SUMMARY 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.
INTRODUCTION
Previous studies in our laboratory demonstrated the presence of brain-reactive antibodies (BRA) in sera of aging mice [1, 2]. These antibodies begin to appear around 6 - 9 months of age and thereafter increase progressively as a function of age [3]. The agerelated increase of the antibodies has also been observed in the sera of non-human primates (Macaca nemestrina) of different ages (4, 10 and 20 years) as well as of humans ranging in age from 40 to 80 years [4, 5]. BRA specifically react with the brain tissue, although some cross-reaction with thymic tissue was also noted [6]. The blood-brain
*Address for correspondence: Veterans Administration Hospital, 200 Springs Road, Bedford, MA 01730, U.S.A. 0047-6374/82/0000-0000/$02.75
© Elsevier Sequoia/Printed in The Netherlands
98 barrier appeared to play an important role in separating the circulating antibodies from the brain antigen, and disruption of the barrier resulted in antigen-antibody reaction and evidence of morphological damage in the surrounding cells [7, 8]. Several studies indicated that nutritional manipulation, underfeeding or undernutrition can significantly prolong ultimate survival in rats, mice, fish, rotifer [ 9 - 1 6 ] . Although a dramatic extension of the maximum lifespan could be achieved by underfeeding or protein restriction in the post-weaning period of the animals, a significant increase was also observed in mature animals [ 1 7 - 2 0 ] . Such a life-prolonging effect was associated with a reduction of the age-related changes in the immune system and an enhanced immune response to tumors [ 2 1 , 2 2 ] . This paper deals with the effects of prolonged dietary restriction on the formation of BRA in sera of aging mice following maturity.
MATERIALSAND METHODS Twenty-five 3-month-old female C57BL/6 mice were fed 2.5 g of Purina mouse chow (daily average intake was 5 g). The same number of animals of the same age and sex, but fed ad libitum, were used as controls. The dietary regime was carried out for 12 months and supplemental vitamins A, B complex, C and D and minerals were added in the drinking water each week for both groups. Animals were housed in a special temperature- and humidity-controlled environmental chamber with 12 h light and dark cycles and their body weights were measured initially and each week. At the end of 12 months, ten experimental animals and ten control animals were sacrificed and the remaining ones were kept to study the effects of more prolonged dietary regime. Blood was drawn from the chest cavity of the animals under anesthesia and sera were separated by centrifugation following coagulation. A number of organs including liver, brain, kidneys (both), adrenals (both) and spleen were dissected out and weighed and the brain weight/body weight ratio was calculated for each animal. BRA were tested by an indirect immunofluorescence method by treating serial frozen sections (10/~m) of the brain of the animal with its own serum prior to incubation with fluorescein-tabelled anti-mouse gamma-globulin [1]. A bright greenish fluorescence was consistently noted in the regions indicating antigen-antibody reaction. The number of pyramidal cells in the frontal cortex and hippocampus showing positive reaction were counted under a fluorescence microscope. Alternate serial sections of the brain were stained by cresyl violet and the total number of cells were counted in ten fields in each of ten sections through the same areas under the microscope. The number of cells with positive antigen-antibody reaction were also counted for each mouse and the percentage of the total cells showing specific reactions were calculated. Sera from experimental animals were tested by the same method with the brain sections from the control mice and vice versa. Control tests were carried out by treating anti-mouse gamma-globulin with normal mouse serum prior to incubation and also treating brain sections with fluoresceinlabeled normal rabbit globulin. All statistical analyses of the data from both groups were done by t-test.
99 TABLE I WEIGHTS OF WHOLE BODY AND DIFFERENT ORGANS IN RESTRICTED DIET AND CONTROL GROUPS OF C57BL/6 MICE
Total body Brain Brain weight/body weight Liver Spleen Kidneys (both) Adrenals (both)
Restricted diet
Controls
18.66 0.4139 0.0221 1.1134 0.0363 0.2677 0.0079
26.460 0.4390 0.0165 1.0893 0.0737 0.3059 0.0123
± 1.67 ± 0.0187 ± 0.0019 ± 0.1334 ± 0.0079 ± 0.0173 ± 0.0015
-+ 2.740 ± 0.0214 ± 0.0019 ± 0.0957 ± 0.0144 ± 0.0249 ± 0.0017
The numbers represent mean weights in g ± S.D. Significance levels were determined by t-test and differences between the two groups were significant (p < 0.05) in all cases except the liver.
RESULTS Ten animals fed on calorically restricted diet and another ten on ad libitum diet were examined in this experiment. Animals were weighed initially (average 16 g), weekly, as well as prior to sacrifice. The weights o f different organs (brain, liver, spleen, both kidneys and both adrenals) and the brain weight/body weight ratios were calculated. The mean b o d y weight o f the control and experimental groups were 26.46 + 2.74 and 18.66 + 1.67 g, respectively, and the difference was statistically significant (p < 0.05). All the organs studied with the exception o f the liver showed a significantly lower weight (p < 0.05), while the brain weight/body weight ratio was significantly ( p < 0.05) higher in the dietary animals (Table I). Sagittal sections o f the brain were tested against the serum from the same mouse for a n t i g e n - a n t i b o d y reaction and the positive reaction was primarily indicated by a greenish fluorescence in the cells. Primarily, the neurons o f the frontal cortex and hippocampus were studied and the reaction Was mostly located in the cell wall or the nucleus. The percentage o f the reacting cells relative to the total number o f cells was analyzed in those two .areas of the brain. While sera o f control animals consistently demonstrated a high level of BRA, those from the experimental animals were negative (Table II). The difference in the levels o f BRA in sera o f the two groups was highly significant ( p < 0.001). Sera of control mice were also tested against the brain sections o f dietary mice and vice versa and no significant difference in the reactivity was noted.
DISCUSSION McCay et al. [9] first demonstrated that the growth period in rats on dietary restriction could be delayed to 7 6 6 - 9 1 1 days with marked increase o f the maximum life span. The same authors later observed that the dietary animals were less susceptible to
100 TABLE II BRAIN-REACTIVE ANTIBODIES IN SERA OF CONTROL AND DIETARY MICE Restricted diet
Control
Animal
Pyramidal cells
Hippocampal cells
Animal
Pyramidal cells
Hippocampal cells
1 2 3 4 5 6 7 8 9 10
0 0 0 9.24 0 1.32 0.73 0 0 0
0 0 0 1.49 0 0.65 0.55 0 0 0
1 2 3 4 5 6 7 8 9 10
50.38 44.00 45.28 52.10 49.76 43.14 32.14 39.02 46.56 30.92
51.19 46.00 35.00 58.00 41.81 60.80 44.97 34.29 49.00 43.06
Mean
1.12
0.26
Mean -+ S.D.
44.8582 -+8.58696
46.412 -+8.70924
The values represent percentage of reacting cells by indirect immunofluorescence when sera from mice of restricted diet and control groups were treated with brain sections. Significance level was determined by t-test and the differences between two graphs were highly significant for both types of ceils (p < 0.001). lung diseases and spontaneous tumors [10, 11 ]. A significant increase in the longevity in young mature mice (3 months) by dietary restriction was also noted by Barrows and Roeder [23] and Nolen [17]. Ross [19, 24] confirmed the previous studies and further noted that the dietary restriction was more effective early in life and less so in later ages. Ross and Bras [25] observed that both low protein/caloric ratio diet under ad libitum conditions and restricted isocaloric diet reduced the risk of spontaneous tumors, whereas protein over-nutrition had a reverse effect. Gerbase-DeLima et al. [12, 13] reported that the dietary restriction beginning after weaning in C57BL/6 mice had immunosuppressive effects by all parameters and that these mice responded better immunologically than controls in mid-life. These authors concluded that the immune system matured less rapidly and stayed younger in these animals longer than the controls. Fernandes et al. [26, 27] studied the effects o f dietary restriction on NZB mice who have a shorter lifespan and are more susceptible to development o f autoimmune diseases. The lifespan of these animals was significantly prolonged with a more vigorous immune system and delayed onset of hemolytic anemia [28]. In this study, young adult mice (3 months) on the calorically restricted diet for 12 months exhibited a significantly lower b o d y weight and appeared more healthy and active. Out of 25 animals on dietary regime only two died as compared with five mice in the control group during the same period. While the brain weight was significantly lower in this group, the brain weight/body weight ratio was significantly increased. This observation might be o f importance since a direct correlation between the brain weight/body weight ratio and the maximum life span has been earlier reported in mammals [29]. This
101 increased ratio in the dietary group also correlates with the longer survivorship of the animals. All organs studied except the liver showed a reduction in weights in the dietary animals, although the reason for this change is not clear in this study. An age-related increase in the serum levels of BRA was observed in different mammals including humans, and a significant role of these in the neuronal degeneration in aging has been suggested [2, 4, 5, 3 0 - 3 2 ] . The underlying immunological mechanism of the antibody formation is not clearly understood. Makinodan [33] reported that the decline of the immune system may occur in normal individuals with aging and this was also associated with increased incidence of autoimmune disorders. Autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, chronic thyroiditis, amyloidosis and autoimmune hemolytic anemia tend to increase with age in humans [34]. The formation of BRA could be another evidence of autoimmune disorder in older animals [5]. It appears that dietary maniplalation was capable of influencing the body in a variety of ways and slowing the age-related changes in the immune system. In this study, formation of BRA was almost completely inhibited in most dietary animals, while control animals exhibited high levels in their sera. Previous studies in our laboratory indicated that BRA began to develop in these animals around 9 months of age and thereafter increased progressively as a function of age [3]. If the formation o f BRA is considered as evidence of immunologic dysfunction or autoimmune disorder, its virtual absence in 15month-old dietary animals might be indicative of either a sustained immunologic vigor and/or a delayed onset of autoimmune disorders.
ACKNOWLEDGMENT This work was supported by the Research Fund of Veterans Administration and National Institute o f Health N S - 1 2 9 6 4 .
REFERENCES 1 J. Thxeatt, K. Nandy and R. Fritz, Brain-reactive antibodies in serum of old mice. J. Gerontol., 26 (1971) 316-323. 2 K. Nandy, Immune reaction in aging brain and senile dementia. In K. Nandy and 1. Sherwin (eds.), The Aging Brain and Senile Dementia, Plenum Press, New York, 1977, pp. 181-196. 3 K. Nandy, Brain-reactive antibodies in mouse serum as a function of age. J. Gerontol., 27 (1972) 173-177. 4 K. Nandy, Brain-reactive antibodies in sera of aging non-human primates. Mech. Ageing Dev., 16 (1981) 141-147. 5 K. Nandy, Senile dementia: an immune hypothesis. In J. A. Mortimer and L. M. Schuman (eds.), The Epidemiology of Dementia, Oxford University Press, New York, 1981. 6 K. Nandy, R. B. Fritz and J. Threatt, Specificity of brain reactive antibodies in serum of old mice. J. Gerontol., 30 (1975) 269-274. 7 K. Nandy, Neuronal degeneration in aging and after experimental injury. Exp. Gerontol., 7 (1972) 303-311. 8 K. Nandy, Significance of brain-reactive antibodies in serum of aged mice. J. Gerontol., 30 (1975) 412-416.
102 9 C. M. McCay, M. F. Crowell and L. A. Maynard, The effect of retarded growth upon the length of the lifespan and upon the ultimate body size. J. Nutr., 10 (1935) 63-79. 10 C. M. McCay, L. A. Maynard, G. Sperling and L. L. Barnes, Retarded growth, lifespan, ultimate body size and age changes in the albino rat after feeding diets restricted in calories. J. Nutr., 18 (1939) 1 - 3 . 11 C. M. McCay, G. Sperling and L. L. Barnes, Growth, aging, chronic diseases in rats. Arch. Biochem., 2 (1943) 469-479. 12 M. Gerbase-DeLima, R. K. Liu, K. E. Cheney, R. Mickey and R. L. Walford, Immune function and survival in a long-lived mouse strain subjected to undernutrition. Gerontologia, 21 (1975) 1 8 4 202. 13 M. Gerbase-DeLima, P. Meredith and R. L. Walford, Age-related changes, including synergy and suppression, in the mixed lymphocyte reaction in long-lived mice. Fed. Proc., 34 (1975) 159-161. 14 A. Comfort, Mutation, autoimmunity and aging. Lancet, ii (1963) 138-140. 15 M. H. Ross, Nutrition and longevity in experimental animals. In M. Winick (ed.), Nutrition and Aging, Wiley, New York, 1976, pp. 4 3 - 5 7 . 16 M. H. Ross, E. Lustbader and G. Bras, Dietary practices and growth responses as predictors of longevity. Nature, 262 (1976) 548-553. 17 G. A. Nolen, Effects of various restricted dietary regimens on the growth, health and longevity of albino rats. J. Nutr., 102 (1972) 1477-1494. 18 D. S. Miller and P. R. Payne, Longevity and protein intake. Exp. Gerontol., 3 (1968) 231-234. 19 M. H. Ross, Length of life and caloric intake. Am. J. Clin. Nutr., 25 (1972) 834-838. 20 C. H. Barrows, Jr. and G. Kokkonen, Protein synthesis, development, growth and life span. Growth, 39 (1975) 525-533. 21 R. G. Bell and L. A. Hazell, Influence of dietary protein restriction on immune competence: I. Effect on the capacity of ceils from lymphoid organs to induce graft vs host reactions. J. Exp. Med., 141 (1975) 127-137. 22 D. G. Jose and R. A. Good, Quantitative effect of nutritional proteins and caloric deficiency upon immune responses to tumors in mice. Cancer Res., 33 (1973) 807-812. 23 C. 1t. Barrows Jr. and L. M. Roeder, The effect of reduced dietary intake on enzymatic activities and life span of rats. J. Gerontol., 20 (1965) 69-71. 24 M. H. Ross, Length of life and nutrition in the rat. J. Nutr., 25 (1961) 197-210. 25 M. H. Ross and G. Bras, Influence of protein under and overnutrition on spontaneous tumor prevalence in the rat. J. Nutr., 103 (1973) 944-963. 26 G. E. Fernandes, J. Yunis and R. A. Good, Influence of diet on survival of mice. Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 1279-1283. 27 G. E. Fernandes, J. Yunis and R. A. Good, Influence of protein restriction on immune functions in NZB mice. J. lmmunol., 116 (1976) 782-790. 28 G. Fernandes, P. Friend and E. J. Yunis, Influence of calorie restriction on autoimmune disease. Fed. Proc., 36 (1977) 1313. 29 G. A. Sacher, Maturation and longevity in relation to cranial capacity in hominid evolution. In R. Tuttle (ed.), Primates: Functional Morphology and Evolution, Mouton, The Hague, 1975, pp. 417-441. 30 K. Nandy, Brain-reactive antibodies in aging and senile dementia. In R. D. Terry, R. Katzman and K. L. Bick (eds.), Alzheimer's Disease--Senile Dementia and Related Disorders. Raven Press, New York, 1978. 31 J. Chaffee, M. Nassef and R. Stephen, Cytotoxic autoantibody to the brain. In K. Nandy (ed.), Senile Dementia: A Biomedical Approach, Elsevier/North Holland, New York, 1978, pp. 61-72. 32 C. R. Ingram, K. J. Phegan and H. T. Bluementhal, Significance of an aging-linked neuron binding gamma-globulin fraction of human sera. Z Gerontol., 29 (1974) 20-72. 33 T. Makinodan, Immunobiology of aging. J. Am. Geriatr. Soc., 24 (1976) 249-252. 34 P. R. J. Burch, An lnquiry Concerning Growth, Disease and Aging, Oliver and Boyd, Edinburgh, 1968.
97
EFFECTS OF CONTROLLED DIETARY RESTRICTION ON BRAINREACTIVE ANTIBODIES IN SERA OF AGING MICE
KALIDAS NANDY* Geriatric Research, Education and Clinical Center, Veterans Administration Hospital, Bedford, Massachusetts, and Department o f Anatomy and Neurology, Boston University School of Medicine, Boston, Massachusetts (U.S.A.)
(Received September 15, 1981)
SUMMARY 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.
INTRODUCTION
Previous studies in our laboratory demonstrated the presence of brain-reactive antibodies (BRA) in sera of aging mice [1, 2]. These antibodies begin to appear around 6 - 9 months of age and thereafter increase progressively as a function of age [3]. The agerelated increase of the antibodies has also been observed in the sera of non-human primates (Macaca nemestrina) of different ages (4, 10 and 20 years) as well as of humans ranging in age from 40 to 80 years [4, 5]. BRA specifically react with the brain tissue, although some cross-reaction with thymic tissue was also noted [6]. The blood-brain
*Address for correspondence: Veterans Administration Hospital, 200 Springs Road, Bedford, MA 01730, U.S.A. 0047-6374/82/0000-0000/$02.75
© Elsevier Sequoia/Printed in The Netherlands
98 barrier appeared to play an important role in separating the circulating antibodies from the brain antigen, and disruption of the barrier resulted in antigen-antibody reaction and evidence of morphological damage in the surrounding cells [7, 8]. Several studies indicated that nutritional manipulation, underfeeding or undernutrition can significantly prolong ultimate survival in rats, mice, fish, rotifer [ 9 - 1 6 ] . Although a dramatic extension of the maximum lifespan could be achieved by underfeeding or protein restriction in the post-weaning period of the animals, a significant increase was also observed in mature animals [ 1 7 - 2 0 ] . Such a life-prolonging effect was associated with a reduction of the age-related changes in the immune system and an enhanced immune response to tumors [ 2 1 , 2 2 ] . This paper deals with the effects of prolonged dietary restriction on the formation of BRA in sera of aging mice following maturity.
MATERIALSAND METHODS Twenty-five 3-month-old female C57BL/6 mice were fed 2.5 g of Purina mouse chow (daily average intake was 5 g). The same number of animals of the same age and sex, but fed ad libitum, were used as controls. The dietary regime was carried out for 12 months and supplemental vitamins A, B complex, C and D and minerals were added in the drinking water each week for both groups. Animals were housed in a special temperature- and humidity-controlled environmental chamber with 12 h light and dark cycles and their body weights were measured initially and each week. At the end of 12 months, ten experimental animals and ten control animals were sacrificed and the remaining ones were kept to study the effects of more prolonged dietary regime. Blood was drawn from the chest cavity of the animals under anesthesia and sera were separated by centrifugation following coagulation. A number of organs including liver, brain, kidneys (both), adrenals (both) and spleen were dissected out and weighed and the brain weight/body weight ratio was calculated for each animal. BRA were tested by an indirect immunofluorescence method by treating serial frozen sections (10/~m) of the brain of the animal with its own serum prior to incubation with fluorescein-tabelled anti-mouse gamma-globulin [1]. A bright greenish fluorescence was consistently noted in the regions indicating antigen-antibody reaction. The number of pyramidal cells in the frontal cortex and hippocampus showing positive reaction were counted under a fluorescence microscope. Alternate serial sections of the brain were stained by cresyl violet and the total number of cells were counted in ten fields in each of ten sections through the same areas under the microscope. The number of cells with positive antigen-antibody reaction were also counted for each mouse and the percentage of the total cells showing specific reactions were calculated. Sera from experimental animals were tested by the same method with the brain sections from the control mice and vice versa. Control tests were carried out by treating anti-mouse gamma-globulin with normal mouse serum prior to incubation and also treating brain sections with fluoresceinlabeled normal rabbit globulin. All statistical analyses of the data from both groups were done by t-test.
99 TABLE I WEIGHTS OF WHOLE BODY AND DIFFERENT ORGANS IN RESTRICTED DIET AND CONTROL GROUPS OF C57BL/6 MICE
Total body Brain Brain weight/body weight Liver Spleen Kidneys (both) Adrenals (both)
Restricted diet
Controls
18.66 0.4139 0.0221 1.1134 0.0363 0.2677 0.0079
26.460 0.4390 0.0165 1.0893 0.0737 0.3059 0.0123
± 1.67 ± 0.0187 ± 0.0019 ± 0.1334 ± 0.0079 ± 0.0173 ± 0.0015
-+ 2.740 ± 0.0214 ± 0.0019 ± 0.0957 ± 0.0144 ± 0.0249 ± 0.0017
The numbers represent mean weights in g ± S.D. Significance levels were determined by t-test and differences between the two groups were significant (p < 0.05) in all cases except the liver.
RESULTS Ten animals fed on calorically restricted diet and another ten on ad libitum diet were examined in this experiment. Animals were weighed initially (average 16 g), weekly, as well as prior to sacrifice. The weights o f different organs (brain, liver, spleen, both kidneys and both adrenals) and the brain weight/body weight ratios were calculated. The mean b o d y weight o f the control and experimental groups were 26.46 + 2.74 and 18.66 + 1.67 g, respectively, and the difference was statistically significant (p < 0.05). All the organs studied with the exception o f the liver showed a significantly lower weight (p < 0.05), while the brain weight/body weight ratio was significantly ( p < 0.05) higher in the dietary animals (Table I). Sagittal sections o f the brain were tested against the serum from the same mouse for a n t i g e n - a n t i b o d y reaction and the positive reaction was primarily indicated by a greenish fluorescence in the cells. Primarily, the neurons o f the frontal cortex and hippocampus were studied and the reaction Was mostly located in the cell wall or the nucleus. The percentage o f the reacting cells relative to the total number o f cells was analyzed in those two .areas of the brain. While sera o f control animals consistently demonstrated a high level of BRA, those from the experimental animals were negative (Table II). The difference in the levels o f BRA in sera o f the two groups was highly significant ( p < 0.001). Sera of control mice were also tested against the brain sections o f dietary mice and vice versa and no significant difference in the reactivity was noted.
DISCUSSION McCay et al. [9] first demonstrated that the growth period in rats on dietary restriction could be delayed to 7 6 6 - 9 1 1 days with marked increase o f the maximum life span. The same authors later observed that the dietary animals were less susceptible to
100 TABLE II BRAIN-REACTIVE ANTIBODIES IN SERA OF CONTROL AND DIETARY MICE Restricted diet
Control
Animal
Pyramidal cells
Hippocampal cells
Animal
Pyramidal cells
Hippocampal cells
1 2 3 4 5 6 7 8 9 10
0 0 0 9.24 0 1.32 0.73 0 0 0
0 0 0 1.49 0 0.65 0.55 0 0 0
1 2 3 4 5 6 7 8 9 10
50.38 44.00 45.28 52.10 49.76 43.14 32.14 39.02 46.56 30.92
51.19 46.00 35.00 58.00 41.81 60.80 44.97 34.29 49.00 43.06
Mean
1.12
0.26
Mean -+ S.D.
44.8582 -+8.58696
46.412 -+8.70924
The values represent percentage of reacting cells by indirect immunofluorescence when sera from mice of restricted diet and control groups were treated with brain sections. Significance level was determined by t-test and the differences between two graphs were highly significant for both types of ceils (p < 0.001). lung diseases and spontaneous tumors [10, 11 ]. A significant increase in the longevity in young mature mice (3 months) by dietary restriction was also noted by Barrows and Roeder [23] and Nolen [17]. Ross [19, 24] confirmed the previous studies and further noted that the dietary restriction was more effective early in life and less so in later ages. Ross and Bras [25] observed that both low protein/caloric ratio diet under ad libitum conditions and restricted isocaloric diet reduced the risk of spontaneous tumors, whereas protein over-nutrition had a reverse effect. Gerbase-DeLima et al. [12, 13] reported that the dietary restriction beginning after weaning in C57BL/6 mice had immunosuppressive effects by all parameters and that these mice responded better immunologically than controls in mid-life. These authors concluded that the immune system matured less rapidly and stayed younger in these animals longer than the controls. Fernandes et al. [26, 27] studied the effects o f dietary restriction on NZB mice who have a shorter lifespan and are more susceptible to development o f autoimmune diseases. The lifespan of these animals was significantly prolonged with a more vigorous immune system and delayed onset of hemolytic anemia [28]. In this study, young adult mice (3 months) on the calorically restricted diet for 12 months exhibited a significantly lower b o d y weight and appeared more healthy and active. Out of 25 animals on dietary regime only two died as compared with five mice in the control group during the same period. While the brain weight was significantly lower in this group, the brain weight/body weight ratio was significantly increased. This observation might be o f importance since a direct correlation between the brain weight/body weight ratio and the maximum life span has been earlier reported in mammals [29]. This
101 increased ratio in the dietary group also correlates with the longer survivorship of the animals. All organs studied except the liver showed a reduction in weights in the dietary animals, although the reason for this change is not clear in this study. An age-related increase in the serum levels of BRA was observed in different mammals including humans, and a significant role of these in the neuronal degeneration in aging has been suggested [2, 4, 5, 3 0 - 3 2 ] . The underlying immunological mechanism of the antibody formation is not clearly understood. Makinodan [33] reported that the decline of the immune system may occur in normal individuals with aging and this was also associated with increased incidence of autoimmune disorders. Autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, chronic thyroiditis, amyloidosis and autoimmune hemolytic anemia tend to increase with age in humans [34]. The formation of BRA could be another evidence of autoimmune disorder in older animals [5]. It appears that dietary maniplalation was capable of influencing the body in a variety of ways and slowing the age-related changes in the immune system. In this study, formation of BRA was almost completely inhibited in most dietary animals, while control animals exhibited high levels in their sera. Previous studies in our laboratory indicated that BRA began to develop in these animals around 9 months of age and thereafter increased progressively as a function of age [3]. If the formation o f BRA is considered as evidence of immunologic dysfunction or autoimmune disorder, its virtual absence in 15month-old dietary animals might be indicative of either a sustained immunologic vigor and/or a delayed onset of autoimmune disorders.
ACKNOWLEDGMENT This work was supported by the Research Fund of Veterans Administration and National Institute o f Health N S - 1 2 9 6 4 .
REFERENCES 1 J. Thxeatt, K. Nandy and R. Fritz, Brain-reactive antibodies in serum of old mice. J. Gerontol., 26 (1971) 316-323. 2 K. Nandy, Immune reaction in aging brain and senile dementia. In K. Nandy and 1. Sherwin (eds.), The Aging Brain and Senile Dementia, Plenum Press, New York, 1977, pp. 181-196. 3 K. Nandy, Brain-reactive antibodies in mouse serum as a function of age. J. Gerontol., 27 (1972) 173-177. 4 K. Nandy, Brain-reactive antibodies in sera of aging non-human primates. Mech. Ageing Dev., 16 (1981) 141-147. 5 K. Nandy, Senile dementia: an immune hypothesis. In J. A. Mortimer and L. M. Schuman (eds.), The Epidemiology of Dementia, Oxford University Press, New York, 1981. 6 K. Nandy, R. B. Fritz and J. Threatt, Specificity of brain reactive antibodies in serum of old mice. J. Gerontol., 30 (1975) 269-274. 7 K. Nandy, Neuronal degeneration in aging and after experimental injury. Exp. Gerontol., 7 (1972) 303-311. 8 K. Nandy, Significance of brain-reactive antibodies in serum of aged mice. J. Gerontol., 30 (1975) 412-416.
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