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Age-related changes in brain and spinal cord lipids of the male garden lizard
Arch. Gerontol. Geriatr., 13 (1991)43-50
43
© 1991 Elsevier Science Publishers B.V. 0167-4943/91/$03.50
A G G 00381
Age-related changes in brain and spinal cord lipids of the male garden lizard P.V. M a n i b a b u a a n d B . K . P a t n a i k b a Department of Zoology, Vikram Deb College, Jeypore (K)-764001, Orissa, India, and ~ Department of Zoology, Berhampur University, Berhampur-760007, Orissa, India (Received 25 March 1990; revised version received 16 November 1990; accepted 22 November 1990)
Summary The lipid composition (cholesterol, phospholipid triglyceride and fatty acids) of brain and spinal cord of the male garden lizard was traced during maturation and ageing. The total brain cholesterol content of both the tissues showed a significant rise during maturation, remaining almost constant thereafter. The free cholesterol content of the brain increased between middle and old age and that of the spinal cord increased during maturation. The esterified cholesterol content of the brain increased during maturation, the same parameter being not age-dependent in the spinal cord. While in the brain the phospholipid content increased during maturation followed by a decrease in old age, it showed a reverse trend in the spinal cord. The level of triglyceride in the brain declined during maturation followed by an increase in old age with no appreciable age change in the spinal cord. A comparison between young and old age-groups revealed a decrease in fatty acid content of the brain. A similar trend of decline was observed during maturation in the spinal cord. The pattern of age changes in the lipid profile of nervous tissue of the lizard, a n o n - m a m m a l i a n vertebrate, almost conform to the pattern observed in a majority of mammals. Lipid composition; Lizard brain; Lizard spinal cord
Introduction Basic molecular-biological research in the central nervous system (CNS) is essential for a variety of reasons. Findings on age-dependent parameters in the CNS might help in selecting the true indicators of physiological ageing and in distinguishing the changes resulting from normal physiological and pathological conditions. Finally on the basis of such investigations a pharmacological intervention in the ageing brain could be possible (Iwangoff et al., 1979; Meier-Ruge et al., 1980). Correspondence should be addressed to B.K. Patnaik, Department of Zoology, Berhampur University, Berhampur-760007, Orissa, India.
44
Moreover it has been suggested that the primary shifts in the CNS determine the dynamics of the most important functions of the organism during ageing (Frolkis and Bezrukov, 1979; Frolkis, 1982). The lipid content of the nervous tissue of mammals (Mcllwain and Bachelard, 1971) and reptiles (Khalil and Abdel-Messeih, 1962) is high. The amount of myelin in the CNS increases with growth and maturation of animals (Sabri and Davidson, 1977). Myelination of nerve fibres is accompanied by an accumulation of hpids during maturation (Himwich, 1973) and ageing (Jacks et al., 1989). Age changes in lipid components a n d / o r in association of lipids and proteins might affect the structure and function of neuronal and glial cell membranes (Strehler, 1977) as envisaged in the membrane hypothesis of ageing (Zs.-Nagy, 1979). An accumulation of the age pigment, lipofuscin, a product of lipid peroxidation is well documented (Tappel, 1970). Therefore, changes in the metabolism of lipids and their role as activators and restrainers of enzyme regulation may have considerable bearing on the ageing brain (Masoro, 1977). Information of age changes in mammalian brain lipids is far from complete (Masoro, 1976). From a comparative point of view it is also necessary to verify the situation in non-mammalian vertebrates in search of a common mechanism of the ageing process. The present study concerns age changes in some lipid components of the brain and spinal cord of a short-lived species of reptile, the garden lizard Calotes versicolor. It is known for mammals that the three major levels of CNS function are the spinal cord level, the lower brain level and the cortical level (Guyton, 1986). Function at the cortical level is very much limited in reptiles (Goldby and Gamble, 1957). Therefore, the distinctions in lipid profile of the brain and spinal cord of the ageing garden lizard might reflect functional differences between the two main regions of the CNS.
Materials and Methods
Male garden lizards of three different age-groups (young, less than 1 year old; middle-aged, 1 year old; and old, aged 2 - 4 years) as described in our earlier publications (Patnaik and Behera, 1981; Sahu and Patnaik, 1989) were used as the experimental animals. They were maintained in the laboratory at room temperature (28 _+ 2 o C) on a diet of goat liver on alternate days in proportion to their size, and tap water was provided ad libitum. The period of collection of the lizards from Berhampur localities was confined to the non-breeding phase, i.e., from September to April. Separate individuals were used for the estimation of different parameters. To maintain homogeneity, the estimation of a parameter in all the three age groups was completed at a stretch within a period of 1 month.
Preparation of tissue for analysis The lizards were sacrificed with a blow on the head. The brain and spinal cord were dissected out. The spinal cord covered few centimeters extending from the brain stem upto and including the brachial enlargement. Both tissues were quickly
45 w a s h e d in ice cold distilled water, d r i e d b e t w e e n the folds of a W h a t m a n No. 1 filter p a p e r , weighed in a m o n o p a n b a l a n c e a n d i m m e d i a t e l y used for further analysis. T r i g l y c e r i d e s were e s t i m a t e d with triolein (Centron, B o m b a y ) a s s t a n d a r d b y H a n t z s c h r e a c t i o n as d e s c r i b e d b y Varley et al. (1980). F r e e fatty acids ( F F A ) were e s t i m a t e d as d e s c r i b e d b y D u n c o m b e (1963) a n d S c h m i d t et al. (1971) using stearic a c i d (Sigma) as the s t a n d a r d . P h o s p h o l i p i d s were d e t e r m i n e d following the m e t h o d of Y o u n g b u r g a n d Y o u n g b u r g (1930) as d e s c r i b e d b y Oser (1965), the t o t a l cholesterol as p e r C a r r a n d D r e k t e r (1956) a n d the free cholesterol a c c o r d i n g to H u a n g et al. (1961) with cholesterol (Sigma) as the s t a n d a r d . T h e values of esterified cholesterol were derived from the difference b e t w e e n total a n d free cholesterol contents.
Results
Cholesterol T h e free cholesterol c o n t e n t of the b r a i n d i d not show a n y significant c h a n g e b e t w e e n the y o u n g a n d m i d d l e - a g e d groups b u t i n c r e a s e d from the m i d d l e - a g e d to the o l d age groups. O n the o t h e r h a n d the esterified cholesterol i n c r e a s e d d u r i n g the p e r i o d of m a t u r a t i o n a n d r e m a i n e d a l m o s t c o n s t a n t thereafter. W h i l e the free cholesterol c o n t e n t of the spinal cord i n c r e a s e d b e t w e e n the y o u n g a n d m i d d l e - a g e d g r o u p s only, the ester±fled cholesterol c o n t e n t was n o t a g e - d e p e n dent. T h e total cholesterol c o n t e n t (free + ester±lied) of b r a i n a n d spinal c o r d i n c r e a s e d significantly d u r i n g m a t u r a t i o n , i.e., f r o m y o u n g to m i d d l e - a g e d a n d r e m a i n e d c o n s t a n t thereafter ( T a b l e I).
Phospholipid T h e p h o s p h o l i p i d c o n t e n t of the b r a i n increased b e t w e e n the y o u n g a n d m i d d l e aged groups a n d d e c r e a s e d thereafter, b u t in the spinal c o r d a reverse t r e n d was
TABLE I Age-related changes in cholesterol contents of brain and spinal cord of the male garden lizard Age group Young (Y) P Middle-age (MA) P Old(O)
Free cholesterol *
Esterified cholesterol *
Total cholesterol *
Brain
Spinal cord
Brain
Spinal cord
Brain
Spinal cord
15.30+ 0.49 (12) NS 15.68±0.58 (9) < 0.05 18.85+0.99 (14)
30.88+ 1.45 (12) < 0.001 40.87±2.06 (10) NS 40.39±3.48 (7)
3.75 + 0.35 (10) < 0.01 6.14±0.75 (8) NS 4.98±0.38 (12)
12.01± 1.08 (12) NS 12.98±2.13 (8) NS 13.19±1.51 (8)
18.59± 0.42 (12) < 0.001 22.25±0.81 (11) NS 23.51±1.00 (14)
43.66± 2.00 (12) < 0.001 61.78±2.00 (8) NS 56.65±1.37 (6)
* Values are rag/g, wet weight, mean 5: SEM. Number in parentheses indicates number of animals. P values refer to comparison between subsequent age groups. NS, not significant at probability level of 0.05.
46 TABLE II Age-related changes in phospholipid, total cholesterol/phospholipid ratio and phospholipid ratio in brain and spinal cord of the male garden lizard Age group
Young (Y) P Middle-aged (MA) P Old(O)
**
free cholesterol/
Phospholipid *
Total cholesterol/ phospholipid ratio * *
Free cholesterol/ phospholipid ratio * *
Brain
Spinal cord
Brain
Spinal cord
Brain
Spinal cord
4:68 ± 0.27 (13) < 0.01 5.83 ± 0.26 (12) < 0.001 4.26±0.19 (16)
30.72 + 2.11 (9) < 0.001 18.21± 1.62 (9) < 0.05 23.19± 1.40 (8)
3.97
1.42
3.26
1.00
4.34
3.39
2.68
2.24
5.51
2.44
4.42
1.74
* Values are mg phosphorus/g, wet weight, mean ± SEM. Calculations are based on mean values as mentioned in this table and Table I.
observed, i.e., the parameter decreased from young to middle-aged and increased thereafter (Table II).
Cholesterol / phospholipid ratio The total cholesterol/phospholipid ratio of the brain showed a trend to increase with age. On the other hand the parameter in the spinal cord increased during maturation followed by a decrease. The free cholesterol/phospholipid ratio of the brain decreased during maturation but increased between middle and old age. The same parameter increased in the spinal cord during maturation followed by a decrease from the middle-age to the old age groups (Table II).
TABLE I11 Age-related changes in triglyceride and free fatty acid contents of brain and spinal cord of the male garden lizard Age group
Triglycerides Brain
Spinal cord
Brain
Spinal cord
Young (Y)
19.45 + 0.64 (7) < 0.02 17.10 + 0.54 (11) < 0.02 19.34 _+0.51 (8)
18.31 + 1.01 (8) NS 19.96 + 0.68 (10) NS 20.16 _+0.94 (9)
18.59 + 1.97 (8) NS 14.26 + 1.71 (6) NS 11.52 + 1.42 (9) < 0.02
30.99 + 2.12 (8) < 0.05 22.28 + 2.76 (12) NS 24.12 _+2.84 (10)
P Middle-aged (MA) P Old (O) P (between Y and O)
Free fatty acids
Values are m g / g , wet weight, mean_+SEM. Number in parentheses indicates number of animals, P values refer to comparison between subsequent age groups. NS, not significant at probability level of 0.05.
47
Triglycerides The triglyceride content of the brain decreased during maturation followed by an increase between middle-aged and old age groups. In the spinal cord the same parameter was not age-dependent (Table III).
Free fatty acids While the free fatty acid content in the brain did not show any significant change between subsequent age groups, a comparison between young and old revealed a significant decline. In the spinal cord the parameter decreased between the young and middle-aged groups and remained constant thereafter (Table III).
Discussion
Ageing organisms exhibit a progressive inability to utilize lipids. Both the processes, i.e., the synthesis and degradation are reduced in old age but the later event is much slower than the former, the net result being accumulation of lipid with age (Story et al., 1976). Our earlier observation indicated that the total lipid content of the brain of the male garden lizard increased with age (Patnaik and Jena, 1972). Since the lipase activity of the brain declined with age in the male garden lizard, the accumulation of lipid could be partly due to reduced rate of utilization. However, age changes in lipid fractions may not show a trend similar to that of total lipid. Moreover, age trends are likely to differ between brain and spinal cord because of a difference in the lipid composition of grey and white matter. Myelination during which lipids increase, is a progressive process which does not occur simultaneously in various parts of the nervous system. The completion of myelination coincides with the establishment of function (Best and Taylor, 1961). Myelination is completed earher in the spinal cord than in anterior parts of the brain. Concomitantly the adult level of lipid composition is also reached earlier in the spinal cord than in brain regions (Timiras, 1972). The higher values of lipid composition are also reached earlier in the spinal cord than in brain regions (Timiras, 1972). The higher values of lipid components in the spinal cord at corresponding ages and the confinement of an increasing trend to maturation (young to middle-aged) in a majority of cases barring phospholipid, suggest the completion of myelination during that period. Similar observations were made in mammalian brain (Rajalakshmi and Nakhari, 1975). Myelin is rich in cholesterol and phospholipids. Increase in myelin lipids is a major contributor to the increase in whole brain/spinal cord lipids (Timiras, 1972). Our results indicate a significant rise in total cholesterol content of brain and spinal cord of the garden lizard during maturation followed by no significant change thereafter. This result partially agrees with the report that the cholesterol content of mouse brain homogenate increased with advancing age (Sun and Samorajski, 1972).
48 Age changes in the phospholipid content of nervous tissue were found to be inconsistent. While an age-related trend to increase was observed in rat brain (Sun and Samorajski, 1972; Mattieu et al., 1975), a reverse trend was reported for the human brain and the spinal cord (Burger, 1957). Again the individual fractions of phospholipids were found to show different age-related trends (Horrocks, 1973). Our results in age changes in the phospholipid content of the brain during maturation is in agreement with the observation in rat brain. The opposite age-related trends in brain and spinal cord also suggest that changes in phospholipid fraction are dependent on different degrees of functional changes occurring during ageing. An increase in the cholesterol/phospholipid ratio and the free cholesterol/ phospholipid ratio during ageing has been implicated with an increase in microviscocity of cell membrane (Rivnay et al., 1978), a parameter related to the permeability of membrane, information flow across the plasma membrane, growth control of cells and cellular recognition. The cholesterol/phospholipid ratio in brain myelin of rat increases with advancing age (Malone and Szoke, 1982). Our results in the garden lizard indicate a trend of an age-related rise in total cholesterol/ phospholipid ratio in brain during maturation and ageing and in spinal cord during maturation. A rise in the free cholesterol/phospholipid ratio was observed during ageing (middle-aged to old) of the brain and maturation of the spinal cord. Age-related alterations in membrane properties such as an increase in the cholesterol/phospholipid ratio might result in an increase in rigidity leading to derangement in cellular functions as envisaged in the membrane hypothesis of ageing (Zs.-Nagy, 1979). However, it has also been theorized that the rate of development of the membrane alterations may depend on the genetically determined species specific chemical composition of the cell membranes as well as on the efficiency of the eventual protecting mechanism (Zs.-Nagy, 1978). Triglycerides, the neutral lipid are considered as metabolic storage compounds with several physiological functions (Overturf and Dryer, 1969). Age-related changes in triglyceride content of mammalian tissues are inconsistent. While in some tissues (arteries/blood) the parameter increased with age (Carlson et al., 1968), in others (brain microsome/serum) it declined (Hawcroft and Martin, 1974; Kessler and Rawlins, 1983). The hepatic triglyceride content of F-344 rats did not change with age (Hashimoto et al., 1981). It appears that while an accumulation of cholesterol and phospholipid contributes significantly to an increase in total lipid content of brain during maturation, an increase in triglyceride content between middle and old age is a major contributor of lipid accumulation during ageing. On the other hand while the cholesterol content of the spinal cord assumes greater significance during maturation, the phospholipid is predominant in advanced age (middle-aged to old) and the triglyceride content shows no significant age change. The free fatty acid content of a tissue is dependent on the degree of its biosynthesis, the lipolytic activity and the extent to which it undergoes oxidation. In rat brain the ability to biosynthesize fatty acids decreases with age as is evident from the decrease in the activity of synthetase (E1-Hassan et al., 1981). A decline in fatty acid content of the brain between young
49 a n d o l d a n d t h a t o f t h e s p i n a l c o r d b e t w e e n y o u n g a n d m i d d l e - a g e d lizards suggest a d e c l i n e in rate o f s y n t h e s i s d u r i n g ageing. T a k e n t o g e t h e r the p a t t e r n o f a g e - r e l a t e d c h a n g e s in lipid c o m p o s i t i o n o f the b r a i n a n d s p i n a l c o r d o f the m a l e g a r d e n lizard, a n o n - m a m m a l i a n v e r t e b r a t e , d o e s n o t s h o w a m a r k e d d e p a r t u r e f r o m t h a t o b s e r v e d in a m a j o r i t y o f m a m m a l s . T h i s o b s e r v a t i o n a l o n g w i t h the f i n d i n g s o n o t h e r p a r a m e t e r s r e p o r t e d e a r l i e r ( P a t n a i k , 1981) m a y t e n t a t i v e l y s u p p o r t t h e c o n c e p t o f a c o m m o n m e c h a n i s m o f a g e i n g p r o c e s s ( e s ) in v e r t e b r a t e s .
Acknowledgements T h e a u t h o r s t h a n k the a u t h o r i t i e s o f B e r h a m p u r U n i v e r s i t y for p r o v i d i n g l a b o r a t o r y facilities for the w o r k a n d the G o v e r n m e n t of O r i s s a for g r a n t i n g s t u d y l e a v e to M a n i b a b u for c o n d u c t i n g the e x p e r i m e n t a l w o r k .
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50 Khalil, F. and Abdel-Messeih, G. (1962): Tissue constituents of reptiles in relation to their mode of life, II. Lipid content. Comp. Biochem. Physiol., 6, 171-174. Malone, M.J. and Szoke, M.C. (1982): Neurochemical studies in ageing brain, I. Structural changes in myelin lipids. J. Gerontol., 37, 262-267. Masoro, E.J. (1976): Lipids and lipid metabolism as a function of aging. In: Annual Review of Experimental Aging Research. Vol. I, pp. 199-215. Editors: M.F. Elias, E.F Eleftheriou and P.K. Elias, EAR Inc., Bar Harbor, ME. Masoro, E.J. (1977): Lipids and lipid metabolism. Annu. Rev. Physiol., 39, 301-321. Mattieu, J.M., Wielmer, S. and Herschkowitz, N. (1975): Biochemical changes in mouse brain during myelination. Brain Res., 55, 391-402. Mcllwain, H. and Bachelard, H.S. (1971): Biochemistry and the Central Nervous System. p. 308-367. Churchill Livingstone, Edinburgh. Meier-Ruge, W., Gygax, P. and Wierusperger, N. (1980): A synoptic view of pathological and experimental pharmacology in gerontological brain research. In: Psychopharmacology of Aging, pp. 65-97. Editors: C. Eisdorfer and W.E. Fann, Spectrum, New York. Oser, B. (1965): Hawk's Physiological Chemistry, 14th edn., p. 1060. McGraw Hill, New York. Overturf, M. and Dryer, R.L. (1969): Experiments in the biochemistry of animal lipids. In: Experiments in Physiology and Biochemistry, Vol. 2, pp. 89-164. Editor: G.A. Kerkut, Academic Press, London. Patnaik, B.K. (1981): Growth: reptiles. In: Comparative Animal Nutrition, Vol. 4, pp. 139-178. Editor: M. Rechcigl Jr., Karger, Basel. Patnaik, B.K. and Behera, H.N. (1981): Age determination in the tropical agamid garden lizard, Calotes versicolor (Daudin) based on bone histology. Exp. Gerontol., 16, 295-307. Patnaik, B.K. and Jena, R.N. (1972): Age changes in the brain of garden lizard, Calotes versicolor, I. Change in brainweight/bodyweight ratio, water content, total lipid, phospholipid. Exp. Gerontoi., 7, 281-285. Rajalakshmi, R. and Nakhari, H.L. (1975): Effects of nutritional deficiencies on spinal cord lipids in rats. Indian J. Biochem. Biophys., 13 (Suppl.), 25. Rivnay, B., Globerson, A. and Shinitzky, M. (1978): Viscosity of lymphocyte plasma membrane in aging mice and its possible relation to serum cholesterol. Mech. Ageing Dev., 10, 71-79. Sabri, M.I. and Davidson, A.N. (1977): The synthesis of myelin in developing rat brain. J. Neurochem., 30, 309-314. Sahu, N. and Patnaik, B.K. (1989): Age related changes in the response of hepatic succinic dehydrogenase activity to thyroxine and thiourea in lizards. Gerontology, 34, 179-183. Schmidt, F.H., Stork, H. and Dahi, K.V. (1971): Lipase photometric assay. In: Methods of Enzymatic Analysis, Vol. 2, p. 819. Editor: H.U. Bergmeyer, Academic Press, New York. Story, J.A., Tepper, S.A. and Kritchevsky, D. (1976): Age related changes in the lipid metabolism of Fisher 344 rats. Lipids, 11,623-627. Strehler, B.L. (1977): Time, Cells and Ageing, 2nd edn., p. 267. Academic Press, New York. Sun, G.Y. and Samorajski, T. (1972): Age changes in lipid composition of whole homogenates and isolated myelin fraction of mouse brain. J. Gerontol., 27, 10-17. Tappel, A.I. (1970): Biological antioxidants protection against peroxidation damage. Am. J. Clin. Nutr., 23, 1137-1139. Timiras, P.S. (1972): Developmental Physiology and Aging, p. 129. Macmillan, New York. Varley, H., Gowenlock, A.H. and Bell, M. (1980): Practical Clinical Chemistry, Vol. 1, 5th edn., pp. 671-677. The White Friars Press, Tonbridge. Zs.-Nagy, I. (1978): A membrane hypothesis of aging. J. Theor. Biol., 75, 189-195. Zs.-Nagy, I. (1979): The role of membrane structure and function in cellular aging: a review. Mech. Ageing Dev.., 9, 237-246.
43
© 1991 Elsevier Science Publishers B.V. 0167-4943/91/$03.50
A G G 00381
Age-related changes in brain and spinal cord lipids of the male garden lizard P.V. M a n i b a b u a a n d B . K . P a t n a i k b a Department of Zoology, Vikram Deb College, Jeypore (K)-764001, Orissa, India, and ~ Department of Zoology, Berhampur University, Berhampur-760007, Orissa, India (Received 25 March 1990; revised version received 16 November 1990; accepted 22 November 1990)
Summary The lipid composition (cholesterol, phospholipid triglyceride and fatty acids) of brain and spinal cord of the male garden lizard was traced during maturation and ageing. The total brain cholesterol content of both the tissues showed a significant rise during maturation, remaining almost constant thereafter. The free cholesterol content of the brain increased between middle and old age and that of the spinal cord increased during maturation. The esterified cholesterol content of the brain increased during maturation, the same parameter being not age-dependent in the spinal cord. While in the brain the phospholipid content increased during maturation followed by a decrease in old age, it showed a reverse trend in the spinal cord. The level of triglyceride in the brain declined during maturation followed by an increase in old age with no appreciable age change in the spinal cord. A comparison between young and old age-groups revealed a decrease in fatty acid content of the brain. A similar trend of decline was observed during maturation in the spinal cord. The pattern of age changes in the lipid profile of nervous tissue of the lizard, a n o n - m a m m a l i a n vertebrate, almost conform to the pattern observed in a majority of mammals. Lipid composition; Lizard brain; Lizard spinal cord
Introduction Basic molecular-biological research in the central nervous system (CNS) is essential for a variety of reasons. Findings on age-dependent parameters in the CNS might help in selecting the true indicators of physiological ageing and in distinguishing the changes resulting from normal physiological and pathological conditions. Finally on the basis of such investigations a pharmacological intervention in the ageing brain could be possible (Iwangoff et al., 1979; Meier-Ruge et al., 1980). Correspondence should be addressed to B.K. Patnaik, Department of Zoology, Berhampur University, Berhampur-760007, Orissa, India.
44
Moreover it has been suggested that the primary shifts in the CNS determine the dynamics of the most important functions of the organism during ageing (Frolkis and Bezrukov, 1979; Frolkis, 1982). The lipid content of the nervous tissue of mammals (Mcllwain and Bachelard, 1971) and reptiles (Khalil and Abdel-Messeih, 1962) is high. The amount of myelin in the CNS increases with growth and maturation of animals (Sabri and Davidson, 1977). Myelination of nerve fibres is accompanied by an accumulation of hpids during maturation (Himwich, 1973) and ageing (Jacks et al., 1989). Age changes in lipid components a n d / o r in association of lipids and proteins might affect the structure and function of neuronal and glial cell membranes (Strehler, 1977) as envisaged in the membrane hypothesis of ageing (Zs.-Nagy, 1979). An accumulation of the age pigment, lipofuscin, a product of lipid peroxidation is well documented (Tappel, 1970). Therefore, changes in the metabolism of lipids and their role as activators and restrainers of enzyme regulation may have considerable bearing on the ageing brain (Masoro, 1977). Information of age changes in mammalian brain lipids is far from complete (Masoro, 1976). From a comparative point of view it is also necessary to verify the situation in non-mammalian vertebrates in search of a common mechanism of the ageing process. The present study concerns age changes in some lipid components of the brain and spinal cord of a short-lived species of reptile, the garden lizard Calotes versicolor. It is known for mammals that the three major levels of CNS function are the spinal cord level, the lower brain level and the cortical level (Guyton, 1986). Function at the cortical level is very much limited in reptiles (Goldby and Gamble, 1957). Therefore, the distinctions in lipid profile of the brain and spinal cord of the ageing garden lizard might reflect functional differences between the two main regions of the CNS.
Materials and Methods
Male garden lizards of three different age-groups (young, less than 1 year old; middle-aged, 1 year old; and old, aged 2 - 4 years) as described in our earlier publications (Patnaik and Behera, 1981; Sahu and Patnaik, 1989) were used as the experimental animals. They were maintained in the laboratory at room temperature (28 _+ 2 o C) on a diet of goat liver on alternate days in proportion to their size, and tap water was provided ad libitum. The period of collection of the lizards from Berhampur localities was confined to the non-breeding phase, i.e., from September to April. Separate individuals were used for the estimation of different parameters. To maintain homogeneity, the estimation of a parameter in all the three age groups was completed at a stretch within a period of 1 month.
Preparation of tissue for analysis The lizards were sacrificed with a blow on the head. The brain and spinal cord were dissected out. The spinal cord covered few centimeters extending from the brain stem upto and including the brachial enlargement. Both tissues were quickly
45 w a s h e d in ice cold distilled water, d r i e d b e t w e e n the folds of a W h a t m a n No. 1 filter p a p e r , weighed in a m o n o p a n b a l a n c e a n d i m m e d i a t e l y used for further analysis. T r i g l y c e r i d e s were e s t i m a t e d with triolein (Centron, B o m b a y ) a s s t a n d a r d b y H a n t z s c h r e a c t i o n as d e s c r i b e d b y Varley et al. (1980). F r e e fatty acids ( F F A ) were e s t i m a t e d as d e s c r i b e d b y D u n c o m b e (1963) a n d S c h m i d t et al. (1971) using stearic a c i d (Sigma) as the s t a n d a r d . P h o s p h o l i p i d s were d e t e r m i n e d following the m e t h o d of Y o u n g b u r g a n d Y o u n g b u r g (1930) as d e s c r i b e d b y Oser (1965), the t o t a l cholesterol as p e r C a r r a n d D r e k t e r (1956) a n d the free cholesterol a c c o r d i n g to H u a n g et al. (1961) with cholesterol (Sigma) as the s t a n d a r d . T h e values of esterified cholesterol were derived from the difference b e t w e e n total a n d free cholesterol contents.
Results
Cholesterol T h e free cholesterol c o n t e n t of the b r a i n d i d not show a n y significant c h a n g e b e t w e e n the y o u n g a n d m i d d l e - a g e d groups b u t i n c r e a s e d from the m i d d l e - a g e d to the o l d age groups. O n the o t h e r h a n d the esterified cholesterol i n c r e a s e d d u r i n g the p e r i o d of m a t u r a t i o n a n d r e m a i n e d a l m o s t c o n s t a n t thereafter. W h i l e the free cholesterol c o n t e n t of the spinal cord i n c r e a s e d b e t w e e n the y o u n g a n d m i d d l e - a g e d g r o u p s only, the ester±fled cholesterol c o n t e n t was n o t a g e - d e p e n dent. T h e total cholesterol c o n t e n t (free + ester±lied) of b r a i n a n d spinal c o r d i n c r e a s e d significantly d u r i n g m a t u r a t i o n , i.e., f r o m y o u n g to m i d d l e - a g e d a n d r e m a i n e d c o n s t a n t thereafter ( T a b l e I).
Phospholipid T h e p h o s p h o l i p i d c o n t e n t of the b r a i n increased b e t w e e n the y o u n g a n d m i d d l e aged groups a n d d e c r e a s e d thereafter, b u t in the spinal c o r d a reverse t r e n d was
TABLE I Age-related changes in cholesterol contents of brain and spinal cord of the male garden lizard Age group Young (Y) P Middle-age (MA) P Old(O)
Free cholesterol *
Esterified cholesterol *
Total cholesterol *
Brain
Spinal cord
Brain
Spinal cord
Brain
Spinal cord
15.30+ 0.49 (12) NS 15.68±0.58 (9) < 0.05 18.85+0.99 (14)
30.88+ 1.45 (12) < 0.001 40.87±2.06 (10) NS 40.39±3.48 (7)
3.75 + 0.35 (10) < 0.01 6.14±0.75 (8) NS 4.98±0.38 (12)
12.01± 1.08 (12) NS 12.98±2.13 (8) NS 13.19±1.51 (8)
18.59± 0.42 (12) < 0.001 22.25±0.81 (11) NS 23.51±1.00 (14)
43.66± 2.00 (12) < 0.001 61.78±2.00 (8) NS 56.65±1.37 (6)
* Values are rag/g, wet weight, mean 5: SEM. Number in parentheses indicates number of animals. P values refer to comparison between subsequent age groups. NS, not significant at probability level of 0.05.
46 TABLE II Age-related changes in phospholipid, total cholesterol/phospholipid ratio and phospholipid ratio in brain and spinal cord of the male garden lizard Age group
Young (Y) P Middle-aged (MA) P Old(O)
**
free cholesterol/
Phospholipid *
Total cholesterol/ phospholipid ratio * *
Free cholesterol/ phospholipid ratio * *
Brain
Spinal cord
Brain
Spinal cord
Brain
Spinal cord
4:68 ± 0.27 (13) < 0.01 5.83 ± 0.26 (12) < 0.001 4.26±0.19 (16)
30.72 + 2.11 (9) < 0.001 18.21± 1.62 (9) < 0.05 23.19± 1.40 (8)
3.97
1.42
3.26
1.00
4.34
3.39
2.68
2.24
5.51
2.44
4.42
1.74
* Values are mg phosphorus/g, wet weight, mean ± SEM. Calculations are based on mean values as mentioned in this table and Table I.
observed, i.e., the parameter decreased from young to middle-aged and increased thereafter (Table II).
Cholesterol / phospholipid ratio The total cholesterol/phospholipid ratio of the brain showed a trend to increase with age. On the other hand the parameter in the spinal cord increased during maturation followed by a decrease. The free cholesterol/phospholipid ratio of the brain decreased during maturation but increased between middle and old age. The same parameter increased in the spinal cord during maturation followed by a decrease from the middle-age to the old age groups (Table II).
TABLE I11 Age-related changes in triglyceride and free fatty acid contents of brain and spinal cord of the male garden lizard Age group
Triglycerides Brain
Spinal cord
Brain
Spinal cord
Young (Y)
19.45 + 0.64 (7) < 0.02 17.10 + 0.54 (11) < 0.02 19.34 _+0.51 (8)
18.31 + 1.01 (8) NS 19.96 + 0.68 (10) NS 20.16 _+0.94 (9)
18.59 + 1.97 (8) NS 14.26 + 1.71 (6) NS 11.52 + 1.42 (9) < 0.02
30.99 + 2.12 (8) < 0.05 22.28 + 2.76 (12) NS 24.12 _+2.84 (10)
P Middle-aged (MA) P Old (O) P (between Y and O)
Free fatty acids
Values are m g / g , wet weight, mean_+SEM. Number in parentheses indicates number of animals, P values refer to comparison between subsequent age groups. NS, not significant at probability level of 0.05.
47
Triglycerides The triglyceride content of the brain decreased during maturation followed by an increase between middle-aged and old age groups. In the spinal cord the same parameter was not age-dependent (Table III).
Free fatty acids While the free fatty acid content in the brain did not show any significant change between subsequent age groups, a comparison between young and old revealed a significant decline. In the spinal cord the parameter decreased between the young and middle-aged groups and remained constant thereafter (Table III).
Discussion
Ageing organisms exhibit a progressive inability to utilize lipids. Both the processes, i.e., the synthesis and degradation are reduced in old age but the later event is much slower than the former, the net result being accumulation of lipid with age (Story et al., 1976). Our earlier observation indicated that the total lipid content of the brain of the male garden lizard increased with age (Patnaik and Jena, 1972). Since the lipase activity of the brain declined with age in the male garden lizard, the accumulation of lipid could be partly due to reduced rate of utilization. However, age changes in lipid fractions may not show a trend similar to that of total lipid. Moreover, age trends are likely to differ between brain and spinal cord because of a difference in the lipid composition of grey and white matter. Myelination during which lipids increase, is a progressive process which does not occur simultaneously in various parts of the nervous system. The completion of myelination coincides with the establishment of function (Best and Taylor, 1961). Myelination is completed earher in the spinal cord than in anterior parts of the brain. Concomitantly the adult level of lipid composition is also reached earlier in the spinal cord than in brain regions (Timiras, 1972). The higher values of lipid composition are also reached earlier in the spinal cord than in brain regions (Timiras, 1972). The higher values of lipid components in the spinal cord at corresponding ages and the confinement of an increasing trend to maturation (young to middle-aged) in a majority of cases barring phospholipid, suggest the completion of myelination during that period. Similar observations were made in mammalian brain (Rajalakshmi and Nakhari, 1975). Myelin is rich in cholesterol and phospholipids. Increase in myelin lipids is a major contributor to the increase in whole brain/spinal cord lipids (Timiras, 1972). Our results indicate a significant rise in total cholesterol content of brain and spinal cord of the garden lizard during maturation followed by no significant change thereafter. This result partially agrees with the report that the cholesterol content of mouse brain homogenate increased with advancing age (Sun and Samorajski, 1972).
48 Age changes in the phospholipid content of nervous tissue were found to be inconsistent. While an age-related trend to increase was observed in rat brain (Sun and Samorajski, 1972; Mattieu et al., 1975), a reverse trend was reported for the human brain and the spinal cord (Burger, 1957). Again the individual fractions of phospholipids were found to show different age-related trends (Horrocks, 1973). Our results in age changes in the phospholipid content of the brain during maturation is in agreement with the observation in rat brain. The opposite age-related trends in brain and spinal cord also suggest that changes in phospholipid fraction are dependent on different degrees of functional changes occurring during ageing. An increase in the cholesterol/phospholipid ratio and the free cholesterol/ phospholipid ratio during ageing has been implicated with an increase in microviscocity of cell membrane (Rivnay et al., 1978), a parameter related to the permeability of membrane, information flow across the plasma membrane, growth control of cells and cellular recognition. The cholesterol/phospholipid ratio in brain myelin of rat increases with advancing age (Malone and Szoke, 1982). Our results in the garden lizard indicate a trend of an age-related rise in total cholesterol/ phospholipid ratio in brain during maturation and ageing and in spinal cord during maturation. A rise in the free cholesterol/phospholipid ratio was observed during ageing (middle-aged to old) of the brain and maturation of the spinal cord. Age-related alterations in membrane properties such as an increase in the cholesterol/phospholipid ratio might result in an increase in rigidity leading to derangement in cellular functions as envisaged in the membrane hypothesis of ageing (Zs.-Nagy, 1979). However, it has also been theorized that the rate of development of the membrane alterations may depend on the genetically determined species specific chemical composition of the cell membranes as well as on the efficiency of the eventual protecting mechanism (Zs.-Nagy, 1978). Triglycerides, the neutral lipid are considered as metabolic storage compounds with several physiological functions (Overturf and Dryer, 1969). Age-related changes in triglyceride content of mammalian tissues are inconsistent. While in some tissues (arteries/blood) the parameter increased with age (Carlson et al., 1968), in others (brain microsome/serum) it declined (Hawcroft and Martin, 1974; Kessler and Rawlins, 1983). The hepatic triglyceride content of F-344 rats did not change with age (Hashimoto et al., 1981). It appears that while an accumulation of cholesterol and phospholipid contributes significantly to an increase in total lipid content of brain during maturation, an increase in triglyceride content between middle and old age is a major contributor of lipid accumulation during ageing. On the other hand while the cholesterol content of the spinal cord assumes greater significance during maturation, the phospholipid is predominant in advanced age (middle-aged to old) and the triglyceride content shows no significant age change. The free fatty acid content of a tissue is dependent on the degree of its biosynthesis, the lipolytic activity and the extent to which it undergoes oxidation. In rat brain the ability to biosynthesize fatty acids decreases with age as is evident from the decrease in the activity of synthetase (E1-Hassan et al., 1981). A decline in fatty acid content of the brain between young
49 a n d o l d a n d t h a t o f t h e s p i n a l c o r d b e t w e e n y o u n g a n d m i d d l e - a g e d lizards suggest a d e c l i n e in rate o f s y n t h e s i s d u r i n g ageing. T a k e n t o g e t h e r the p a t t e r n o f a g e - r e l a t e d c h a n g e s in lipid c o m p o s i t i o n o f the b r a i n a n d s p i n a l c o r d o f the m a l e g a r d e n lizard, a n o n - m a m m a l i a n v e r t e b r a t e , d o e s n o t s h o w a m a r k e d d e p a r t u r e f r o m t h a t o b s e r v e d in a m a j o r i t y o f m a m m a l s . T h i s o b s e r v a t i o n a l o n g w i t h the f i n d i n g s o n o t h e r p a r a m e t e r s r e p o r t e d e a r l i e r ( P a t n a i k , 1981) m a y t e n t a t i v e l y s u p p o r t t h e c o n c e p t o f a c o m m o n m e c h a n i s m o f a g e i n g p r o c e s s ( e s ) in v e r t e b r a t e s .
Acknowledgements T h e a u t h o r s t h a n k the a u t h o r i t i e s o f B e r h a m p u r U n i v e r s i t y for p r o v i d i n g l a b o r a t o r y facilities for the w o r k a n d the G o v e r n m e n t of O r i s s a for g r a n t i n g s t u d y l e a v e to M a n i b a b u for c o n d u c t i n g the e x p e r i m e n t a l w o r k .
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