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Effect of an antioxidant compound (2-mercaptoethanol) on the nerve terminals of the aging small intestine
Experimental Gerontology, Vol. 25, pp. 135-140, 1990 Printed in the USA. All rights reserved.
0531-5565/90 $3.00 + .00 Copyright t~ 1990 Pergamon Press plc
EFFECT OF AN ANTIOXIDANT COMPOUND (2-MERCAPTOETHANOL) ON THE NERVE TERMINALS OF THE AGING SMALL INTESTINE
ERZSI~BET FEHI~R a n d L . PI~NZES1 First Department of Anatomy and ~Gerontology Center, Semmelweis University Medical School, Budapest, Hungary
- - The effect of 2-mercaptoethanol (2-ME) treatment on the nerve elements of the small intestine has been investigated in old mice. In control animals only a few synapses and a very small number of vesicles were found in the nerve terminals. Some of the nerve fibers were observed to be degenerating. After drinking 2-ME daily for 17 months, both the number of synapses and vesicles within the nerve terminals increased. No degenerated fibers were observed. It is assumed that 2-ME has a beneficial influence on the peripheral intestinal nervous system increasing the number of synapses and the vesicle population in the nerve terminals. Abstract
Key Words: 2-mercaptoethanol, autonomic nerves, aging small intestine, ultrastructure
INTRODUCTION A LARGEnumber of morphological and biochemical changes are observed in the aging central nervous system (Sheffer, 1973); however, only a few findings are available concerning the peripheral nervous system. Primary morphological changes that are age-related occur in the myelin sheaths (Terry et al., 1964). It has been shown that free radicals damage cells by initiating lipid peroxidation of the membrane lipids (Wills, 1969; Kellogg and Fridovich, 1977; Halliwell, 1981). Peroxidation of membrane lipids leads to cell necrosis and death. This oxidative damage is mainly due to activated oxygen species such as the superoxide radical, hydroxy radicals and singtet oxygen. 2-mercaptoethanol is one of the most efficient of the antioxidant compounds evaluated so far (Harman, 1986). On the other hand, the role of the biological antioxidants, such as; vitamin E, should not be omitted. Comprehensive studies provide ample evidence to support its action as a potent antioxidant (Tappel, 1970; Tappel et al., 1973; Harman, 1978; Porta et al., 1981). It has been known for some time that these antioxidants have a positive effect on the life span of
Correspondence to: E. Feh~r, First Department of Anatomy, Semmelweis University Medical School, Budapest, IX, Ttizolt6 u. 58, P.O. Box, 95, Hungary, H-1450. (Received 6 December 1988; Accepted 9 May 1989)
135
136
E FEH~R and L. PI~NZES
FiG. 1. A part of the myentericplexus of an old animal after 2-ME treatment. Arrow points to a synapse on the surface of the nerve cell body (NC). Bar scale = 1 p,m. laboratory animals (Harman, 1961; Makinodan and Albright, 1979a; 1979b; Heindrick et al., 1980; Kikuchi et al., 1982; Heidrick et al., 1984). Their effects are thought to include the trapping of free radicals and a scavenging capacity. Using conventional transmission electron microscopy, we observed the effects of 2-ME on the ultrastructure of nerve terminals in the aging small intestine. This investigation is also an attempt to identify the changes in the number of synapses and vesicles of the nerve terminals after antioxidant treatment. MATERIALS AND METHODS The experiments were performed on male CBA/Ca inbred mice (Lati/G6d6116, Hungary) of 25 to 34 g body weight. The animals used were 2.5 and 22 months of age. All the young and
FIG.2. A part of the myentericplexus of the younganimal. Arrow shows the varicose nerveterminal containingmainly large granular vesicles close to the nerve cell body (NC). Bar scale = I Ixm.
EFFECT OF AN ANTIOXIDANT ON NERVE FIBERS
137
FIG. 3. Arrow points to the varicose nerve terminal containing small clear and large granulated vesicles in the tunica mucosa of the young animal. Bar scale = 1 p,m. half of the old animals served as controls. The experimental animals were treated with 8 p,g/animal 2-mercaptoethanol (Merk-Schuchardt, FRG) in drinking water for a period of almost 17 months from 5.5 months of age onwards. The animals were fed a pelleted laboratory chow ad libitum. After treatment with 2-ME for 17 months, the animals were killed and a piece of the small intestine was fixed in a combination of 2.5% paraformaldehyde and 2.5% glutaraldehyde. Postosmication was carried out for 1 h in 1% osmium tetroxide and the tissue was then embedded in Durcupan. Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined with a Tesla BS 500 electron microscope. For statistical analysis the number of synapses were counted in areas of 300 i~m2 of myenteric and submucous plexuses from each animal and was calculated for 100 ixm2 tissue area as the average for each group. The number of vesicles were also counted within 50 nerve profiles.
F[o. 4. After 2-ME treatment the varicose nerve terminals contain a large population of vesicles (arrows). Bar scale = 1 p,m.
138
E. FEHC:Rand L. PI~NZES
Fie. 5. A part of the myenteric plexus of an old animal. Arrows show degenerated nerve fibers close to the nerve cell body (NC). Dotted arrow points to the nerve fibers without a vesicular population. Bar scale = 1 p,m.
RESULTS Varicose nerve fibers can be observed in all layers of the intestinal wall, both within the submucous and myenteric ganglia and in the muscle layers as well as in the tunica mucosa -up to less than 1 p~m distance below the epithelial lining of the mucosa. The neural processes have their usual relation to the Schwann cells. In large part they are completely embedded with connecting mesaxons, while others are only lightly impressed into the Schwann cell processes. Several nerve fibers were in close apposition to the muscle cells with a space of 100 to 200 nm between the plasma membranes. No distinct membrane thickenings or specializations were observed even in the closest situation. Some of the nerve terminals could be observed in synapses on the surface of nerve cells (Fig. 1) or with other nerve processes. A large population
FIG. 6. A bundle of nerve terminals in the submucous plexus of an old control animal. Note that the nerve terminals contain only microtubules and some mitochondria. Bar scale = 1 la,m.
EFFECT OF AN ANT1OXIDANT ON NERVE FIBERS
139
TABLE I . THE EFFECT OF 2-MERCAPTOETHANOL ON THE QUANTITY OF SYNAPSES AND VESICLES IN NERVE TERMINALS
Animals Y o u n g (n = 3) (3 m o n t h old) Old c o n t r o l ( n = 4) (24 m o n t h old) 2 - M E treated (n = 5) (24 m o n t h old)
Number of Synapses/ 100 ixm2 Areas
Number of vesicles within 50 nerve fibers
18 + 2.5
1720 ± 62
5 ± 1.8
130 ± 18
12 -
2.2
780 - 46
of vesicles could be found within the varicose nerve terminal fibers of the young (Figs. 2 and 3) and of 2-ME treated animals (Fig. 4). Most of the nerve processes contained mainly large granulated vesicles of 80 to 120 nm in diameter with small clear vesicles of 30 to 50 nm in diameter. In the old animals only a few synapses and a very small number of vesicles were found in the nerve terminals (Figs. 5 and 6). The number of nerve terminals was also decreased and they contained only microtubules and neurofilaments without vesicles. Some of the nerve fibers were observed to be degenerating (Fig. 5). After 2-ME treatment, the number of synapses and the number of vesicles within the nerve terminals increased as shown in Table 1.
DISCUSSION Electron microscopic studies of nerve fibers in the gut have shown that many nerve fibers contain a large number of vesicles as described previously (Gabella, 1972; Fehrr and Vajda, 1974). The total number of nerve fibers in the tunica mucosa, tela submucosa and in the inner circular muscle layer was 38/100 ixm2 (100%) in the young; 32/100 ixm2 (84%) in the treated, senile animals and only 12/100 p,m2 (32%) in the senile animals without treatment. The total number of nerve fibers in the myenteric plexus was 71/100 ixm2 (100%) in the young, 67/100 ixm2 (93%) in the treated, senile animals and only 27/100 p,m2 (37.5%) in the senile animals without treatment. We conclude that administration of 2-ME causes marked changes in the number of fibers with an increase of synapses and vesicles in the nerve terminals (Table 1). In our recent study (Prnzes et al., 1988) using long-term experiments, it was shown that 2-ME has an overall impact on age-related metabolic changes of CBA/Ca inbred mice. It was also demonstrated that significant increases occur in the levels of some of the polyunsaturated fatty acids in the lipid fractions of the liver and brain. We regard these changes as favorable because it is known that there is an apparent increase in lipid saturation levels as aging advances (Prnzes et al., 1988). Unfortunately, there is little data regarding this effect on the aging nerve tissue elments. Nevertheless, Malone and Szoke (1982) have found corresponding changes in the molecular composition of myelin, with changes in structural stability as well. They found that the cholesterol:phospholipid ratio of myelin subfractions increases with age and that this may influence membrane stability. On the other hand, the authors observed a major incremental change in the ratio of unsaturated: saturated long chain fatty acids. Malone and Szoke (1982) concluded that these changes may lead to an increased fluidity and decreased stability with age
140
E. FEHI~Rand L. PI~NZES
in m y e l i n . T h e s e c h a n g e s , h o w e v e r , r e f e r to rats o f o n l y 3, 13, a n d 19 m o n t h s o f age, thus t h e r e is n o c o n v i n c i n g d a t a o n w h e t h e r this t r e n d c o n t i n u e s as the a n i m a l s r e a c h o l d age.
REFERENCES FEHI~,R, E. and VAJDA, J. Degeneration analysis of the extrinsic nerve elements of the small intestine. Acta Anat. 87, 97-109, 1974. GABELLA, G. Fine Structure of the myenteric plexus in the guinea-pig ileum. J. Anat. 111, 69-97, 1972. HALLIWELL, B. The biological effects of the snperoxide radical and its products. Bull. Eur. Physiopath. Resp. 17, (Suppl) 21-28, 1981. HARMAN, D. Prolongation of the normal lifespan and inhibition of spontaneous cancer by antioxidants. J. Geront. 16, 247-254, 1961. HARMAN, D. Free radical theory of aging: Nutritional complications. Age 1, 145-152, 1978. HARMAN, D. Free radical theory of ageing: Role of free radicals in the organization and evolution of life, ageing and disease processes. In; Free Radicals, Ageing, and Degenerative Diseases, Johnson, J.E., Jr., Walford, R., Harman, D., and Miguel, I. (Editors), pp. 3-49, Alan R. Liss, New York, 1986. HEIDRICK, M.L., ALBRIGHT, J.W., and MAKINODAN, T. Restoration of impaired immune functions in ageing animals. IV. Action of 2-mercaptoethanol in enhancing age-reduced immune responsiveness. Mech. Ageing Dev. 13, 367-378, 1980. HEIDRICK, M.L., HENDRICKS, L.C., and COOK, D.E. Effect of dietary 2-mercaptoethanol on the life span, immune system, immuno incidence and lipid peroxidation damage in spleen lymphocytes of ageing BC3F mice. Mech. Ageing Dev. 27, 341-358, 1984. KELLOGG, E.W., III and FRIDOVICH, I. Liposome oxidation and erythrocyte lysis by enzymatically generated superoxide and hydrogen peroxide. J. Biol. Chem. 252, 6721-6728, 1977. KIKUCHI, Y., HIRAMOTO, R.N., and GHANTA, V.K. Kitogen response of peripheral blood and splenic lymphocytes and effect of 2-mercaptoethanol in tumor-bearing mice. Cancer lmmunol. Immunother. 12, 225-230, 1982. MALONE, M.J. and SZOKE, M.C. Neurochemical studies in ageing brain. I. Structural changes in myelin lipids. J. Gerontol. 37, 262-267, 1982. MAKINODAN, T. and ALBRIGHT, J.W. Restoration of impaired immune functions in ageing animals. II. Effect of Mercaptoethanol in enhancing the reduced primary antibody responsiveness in vitro. Mech. Ageing Dev. 10, 325-340, 1979a. MAKINODAN, T. and ALBRIGHT, J.W. Restoration of impaired immune functions in ageing animals: III. Effect of mercaptoethanol in enhancing the reduced primary antibody responsiveness in vitro. Mech. Ageing Dev. 11, 1-8, 1979b. PI~NZES, L., NOBLE, R.C., BEREGI, E., IMRE, S., IZSAK, J., and RI~GIUS, O. Effect of 2-mercaptoethanol on some metabolic indices of the ageing CBA/Ca mice. Mech. Ageing Dev. 45, 75-92, 1988. PORTA, E.A., KEOPUHIWA, L., JOUN, N.S., and NI'ITA, T. Effects of the type of dietary fat at two levels of vitamin E in Wistar male rats during development and aging. III. Biochemical and morphometric parameters of the liver. Mech. Ageing Dev. 15, 297-335, 1981. SHEFFER, F. F. The absolute number of neurons and thickness of cerebral cortex during ageing, senile and vascular dementia and Pick's Alzheimer's diseases. Neurosci. Behav. Physiol. 33, 319-324, 1973. TAPPEL, A.L. Biological antioxidant protection against lipid peroxidation damage. Am. J. Clin. Nutr. 23, 1137-1139, 1970. TAPPEL, A.L., FLETCHER, G., and DEAMER, D. Effect of antioxidants and nutrients on lipid peroxidation fluroescent products and ageing parameters in the mouse. J. Geront. 28, 415-424, 1973. TERRY, R.D., GONATAS, N.K., and WEISS, M. Ultrastructural studies in Alzheimer's presenile dementia. Am. J. Path. 44, 269-297, 1964. WILLS, E.D. Lipid peroxid formation in microsomes. General considerations. Biochem. J. 113, 315-324, 1969.
0531-5565/90 $3.00 + .00 Copyright t~ 1990 Pergamon Press plc
EFFECT OF AN ANTIOXIDANT COMPOUND (2-MERCAPTOETHANOL) ON THE NERVE TERMINALS OF THE AGING SMALL INTESTINE
ERZSI~BET FEHI~R a n d L . PI~NZES1 First Department of Anatomy and ~Gerontology Center, Semmelweis University Medical School, Budapest, Hungary
- - The effect of 2-mercaptoethanol (2-ME) treatment on the nerve elements of the small intestine has been investigated in old mice. In control animals only a few synapses and a very small number of vesicles were found in the nerve terminals. Some of the nerve fibers were observed to be degenerating. After drinking 2-ME daily for 17 months, both the number of synapses and vesicles within the nerve terminals increased. No degenerated fibers were observed. It is assumed that 2-ME has a beneficial influence on the peripheral intestinal nervous system increasing the number of synapses and the vesicle population in the nerve terminals. Abstract
Key Words: 2-mercaptoethanol, autonomic nerves, aging small intestine, ultrastructure
INTRODUCTION A LARGEnumber of morphological and biochemical changes are observed in the aging central nervous system (Sheffer, 1973); however, only a few findings are available concerning the peripheral nervous system. Primary morphological changes that are age-related occur in the myelin sheaths (Terry et al., 1964). It has been shown that free radicals damage cells by initiating lipid peroxidation of the membrane lipids (Wills, 1969; Kellogg and Fridovich, 1977; Halliwell, 1981). Peroxidation of membrane lipids leads to cell necrosis and death. This oxidative damage is mainly due to activated oxygen species such as the superoxide radical, hydroxy radicals and singtet oxygen. 2-mercaptoethanol is one of the most efficient of the antioxidant compounds evaluated so far (Harman, 1986). On the other hand, the role of the biological antioxidants, such as; vitamin E, should not be omitted. Comprehensive studies provide ample evidence to support its action as a potent antioxidant (Tappel, 1970; Tappel et al., 1973; Harman, 1978; Porta et al., 1981). It has been known for some time that these antioxidants have a positive effect on the life span of
Correspondence to: E. Feh~r, First Department of Anatomy, Semmelweis University Medical School, Budapest, IX, Ttizolt6 u. 58, P.O. Box, 95, Hungary, H-1450. (Received 6 December 1988; Accepted 9 May 1989)
135
136
E FEH~R and L. PI~NZES
FiG. 1. A part of the myentericplexus of an old animal after 2-ME treatment. Arrow points to a synapse on the surface of the nerve cell body (NC). Bar scale = 1 p,m. laboratory animals (Harman, 1961; Makinodan and Albright, 1979a; 1979b; Heindrick et al., 1980; Kikuchi et al., 1982; Heidrick et al., 1984). Their effects are thought to include the trapping of free radicals and a scavenging capacity. Using conventional transmission electron microscopy, we observed the effects of 2-ME on the ultrastructure of nerve terminals in the aging small intestine. This investigation is also an attempt to identify the changes in the number of synapses and vesicles of the nerve terminals after antioxidant treatment. MATERIALS AND METHODS The experiments were performed on male CBA/Ca inbred mice (Lati/G6d6116, Hungary) of 25 to 34 g body weight. The animals used were 2.5 and 22 months of age. All the young and
FIG.2. A part of the myentericplexus of the younganimal. Arrow shows the varicose nerveterminal containingmainly large granular vesicles close to the nerve cell body (NC). Bar scale = I Ixm.
EFFECT OF AN ANTIOXIDANT ON NERVE FIBERS
137
FIG. 3. Arrow points to the varicose nerve terminal containing small clear and large granulated vesicles in the tunica mucosa of the young animal. Bar scale = 1 p,m. half of the old animals served as controls. The experimental animals were treated with 8 p,g/animal 2-mercaptoethanol (Merk-Schuchardt, FRG) in drinking water for a period of almost 17 months from 5.5 months of age onwards. The animals were fed a pelleted laboratory chow ad libitum. After treatment with 2-ME for 17 months, the animals were killed and a piece of the small intestine was fixed in a combination of 2.5% paraformaldehyde and 2.5% glutaraldehyde. Postosmication was carried out for 1 h in 1% osmium tetroxide and the tissue was then embedded in Durcupan. Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined with a Tesla BS 500 electron microscope. For statistical analysis the number of synapses were counted in areas of 300 i~m2 of myenteric and submucous plexuses from each animal and was calculated for 100 ixm2 tissue area as the average for each group. The number of vesicles were also counted within 50 nerve profiles.
F[o. 4. After 2-ME treatment the varicose nerve terminals contain a large population of vesicles (arrows). Bar scale = 1 p,m.
138
E. FEHC:Rand L. PI~NZES
Fie. 5. A part of the myenteric plexus of an old animal. Arrows show degenerated nerve fibers close to the nerve cell body (NC). Dotted arrow points to the nerve fibers without a vesicular population. Bar scale = 1 p,m.
RESULTS Varicose nerve fibers can be observed in all layers of the intestinal wall, both within the submucous and myenteric ganglia and in the muscle layers as well as in the tunica mucosa -up to less than 1 p~m distance below the epithelial lining of the mucosa. The neural processes have their usual relation to the Schwann cells. In large part they are completely embedded with connecting mesaxons, while others are only lightly impressed into the Schwann cell processes. Several nerve fibers were in close apposition to the muscle cells with a space of 100 to 200 nm between the plasma membranes. No distinct membrane thickenings or specializations were observed even in the closest situation. Some of the nerve terminals could be observed in synapses on the surface of nerve cells (Fig. 1) or with other nerve processes. A large population
FIG. 6. A bundle of nerve terminals in the submucous plexus of an old control animal. Note that the nerve terminals contain only microtubules and some mitochondria. Bar scale = 1 la,m.
EFFECT OF AN ANT1OXIDANT ON NERVE FIBERS
139
TABLE I . THE EFFECT OF 2-MERCAPTOETHANOL ON THE QUANTITY OF SYNAPSES AND VESICLES IN NERVE TERMINALS
Animals Y o u n g (n = 3) (3 m o n t h old) Old c o n t r o l ( n = 4) (24 m o n t h old) 2 - M E treated (n = 5) (24 m o n t h old)
Number of Synapses/ 100 ixm2 Areas
Number of vesicles within 50 nerve fibers
18 + 2.5
1720 ± 62
5 ± 1.8
130 ± 18
12 -
2.2
780 - 46
of vesicles could be found within the varicose nerve terminal fibers of the young (Figs. 2 and 3) and of 2-ME treated animals (Fig. 4). Most of the nerve processes contained mainly large granulated vesicles of 80 to 120 nm in diameter with small clear vesicles of 30 to 50 nm in diameter. In the old animals only a few synapses and a very small number of vesicles were found in the nerve terminals (Figs. 5 and 6). The number of nerve terminals was also decreased and they contained only microtubules and neurofilaments without vesicles. Some of the nerve fibers were observed to be degenerating (Fig. 5). After 2-ME treatment, the number of synapses and the number of vesicles within the nerve terminals increased as shown in Table 1.
DISCUSSION Electron microscopic studies of nerve fibers in the gut have shown that many nerve fibers contain a large number of vesicles as described previously (Gabella, 1972; Fehrr and Vajda, 1974). The total number of nerve fibers in the tunica mucosa, tela submucosa and in the inner circular muscle layer was 38/100 ixm2 (100%) in the young; 32/100 ixm2 (84%) in the treated, senile animals and only 12/100 p,m2 (32%) in the senile animals without treatment. The total number of nerve fibers in the myenteric plexus was 71/100 ixm2 (100%) in the young, 67/100 ixm2 (93%) in the treated, senile animals and only 27/100 p,m2 (37.5%) in the senile animals without treatment. We conclude that administration of 2-ME causes marked changes in the number of fibers with an increase of synapses and vesicles in the nerve terminals (Table 1). In our recent study (Prnzes et al., 1988) using long-term experiments, it was shown that 2-ME has an overall impact on age-related metabolic changes of CBA/Ca inbred mice. It was also demonstrated that significant increases occur in the levels of some of the polyunsaturated fatty acids in the lipid fractions of the liver and brain. We regard these changes as favorable because it is known that there is an apparent increase in lipid saturation levels as aging advances (Prnzes et al., 1988). Unfortunately, there is little data regarding this effect on the aging nerve tissue elments. Nevertheless, Malone and Szoke (1982) have found corresponding changes in the molecular composition of myelin, with changes in structural stability as well. They found that the cholesterol:phospholipid ratio of myelin subfractions increases with age and that this may influence membrane stability. On the other hand, the authors observed a major incremental change in the ratio of unsaturated: saturated long chain fatty acids. Malone and Szoke (1982) concluded that these changes may lead to an increased fluidity and decreased stability with age
140
E. FEHI~Rand L. PI~NZES
in m y e l i n . T h e s e c h a n g e s , h o w e v e r , r e f e r to rats o f o n l y 3, 13, a n d 19 m o n t h s o f age, thus t h e r e is n o c o n v i n c i n g d a t a o n w h e t h e r this t r e n d c o n t i n u e s as the a n i m a l s r e a c h o l d age.
REFERENCES FEHI~,R, E. and VAJDA, J. Degeneration analysis of the extrinsic nerve elements of the small intestine. Acta Anat. 87, 97-109, 1974. GABELLA, G. Fine Structure of the myenteric plexus in the guinea-pig ileum. J. Anat. 111, 69-97, 1972. HALLIWELL, B. The biological effects of the snperoxide radical and its products. Bull. Eur. Physiopath. Resp. 17, (Suppl) 21-28, 1981. HARMAN, D. Prolongation of the normal lifespan and inhibition of spontaneous cancer by antioxidants. J. Geront. 16, 247-254, 1961. HARMAN, D. Free radical theory of aging: Nutritional complications. Age 1, 145-152, 1978. HARMAN, D. Free radical theory of ageing: Role of free radicals in the organization and evolution of life, ageing and disease processes. In; Free Radicals, Ageing, and Degenerative Diseases, Johnson, J.E., Jr., Walford, R., Harman, D., and Miguel, I. (Editors), pp. 3-49, Alan R. Liss, New York, 1986. HEIDRICK, M.L., ALBRIGHT, J.W., and MAKINODAN, T. Restoration of impaired immune functions in ageing animals. IV. Action of 2-mercaptoethanol in enhancing age-reduced immune responsiveness. Mech. Ageing Dev. 13, 367-378, 1980. HEIDRICK, M.L., HENDRICKS, L.C., and COOK, D.E. Effect of dietary 2-mercaptoethanol on the life span, immune system, immuno incidence and lipid peroxidation damage in spleen lymphocytes of ageing BC3F mice. Mech. Ageing Dev. 27, 341-358, 1984. KELLOGG, E.W., III and FRIDOVICH, I. Liposome oxidation and erythrocyte lysis by enzymatically generated superoxide and hydrogen peroxide. J. Biol. Chem. 252, 6721-6728, 1977. KIKUCHI, Y., HIRAMOTO, R.N., and GHANTA, V.K. Kitogen response of peripheral blood and splenic lymphocytes and effect of 2-mercaptoethanol in tumor-bearing mice. Cancer lmmunol. Immunother. 12, 225-230, 1982. MALONE, M.J. and SZOKE, M.C. Neurochemical studies in ageing brain. I. Structural changes in myelin lipids. J. Gerontol. 37, 262-267, 1982. MAKINODAN, T. and ALBRIGHT, J.W. Restoration of impaired immune functions in ageing animals. II. Effect of Mercaptoethanol in enhancing the reduced primary antibody responsiveness in vitro. Mech. Ageing Dev. 10, 325-340, 1979a. MAKINODAN, T. and ALBRIGHT, J.W. Restoration of impaired immune functions in ageing animals: III. Effect of mercaptoethanol in enhancing the reduced primary antibody responsiveness in vitro. Mech. Ageing Dev. 11, 1-8, 1979b. PI~NZES, L., NOBLE, R.C., BEREGI, E., IMRE, S., IZSAK, J., and RI~GIUS, O. Effect of 2-mercaptoethanol on some metabolic indices of the ageing CBA/Ca mice. Mech. Ageing Dev. 45, 75-92, 1988. PORTA, E.A., KEOPUHIWA, L., JOUN, N.S., and NI'ITA, T. Effects of the type of dietary fat at two levels of vitamin E in Wistar male rats during development and aging. III. Biochemical and morphometric parameters of the liver. Mech. Ageing Dev. 15, 297-335, 1981. SHEFFER, F. F. The absolute number of neurons and thickness of cerebral cortex during ageing, senile and vascular dementia and Pick's Alzheimer's diseases. Neurosci. Behav. Physiol. 33, 319-324, 1973. TAPPEL, A.L. Biological antioxidant protection against lipid peroxidation damage. Am. J. Clin. Nutr. 23, 1137-1139, 1970. TAPPEL, A.L., FLETCHER, G., and DEAMER, D. Effect of antioxidants and nutrients on lipid peroxidation fluroescent products and ageing parameters in the mouse. J. Geront. 28, 415-424, 1973. TERRY, R.D., GONATAS, N.K., and WEISS, M. Ultrastructural studies in Alzheimer's presenile dementia. Am. J. Path. 44, 269-297, 1964. WILLS, E.D. Lipid peroxid formation in microsomes. General considerations. Biochem. J. 113, 315-324, 1969.