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The effects of ageing and a vitamin E-deficient diet on the lipopigment content of rat hippocampal and Purkinje neurones
Arch. Gerontol. Geriatr., 14 (1992)239-251
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4943/92/$05.00 AGG 00440
The effects of ageing and a vitamin E-deficient diet on the lipopigment content of rat hippocampal and Purkinje neurones Jonathan H. D o w s o n a, Patrizia Fattoretti b, Mary Cairns a, Nigel T. James c, Helen W i l t o n - C o x a and Carlo Bertoni-Freddari b aDepartment of Psychiatry, University of Cambridge, U.K., bl. N. R.C.A. Dipartimento di ricerche gerontologiche e geriatriche, Ancona, Italy and CDepartment of Biomedical Science, University of Sheffield, U.K. (Received 13 August 1991; accepted 27 December 1991)
Summary This study examined associations between a vitamin E-deficient diet, ageing and aspects of the morphology of neuronal lipopigment in rat hippocampal and Purkinje neurones. Groups of rats given a standard diet were killed at 6, 12, 18 and 25 months of age, while a group which had received a vitamin E-deficient diet from 1-18 months were killed at 18 months of age. Lipopigment within a neuronal cell body consists of a number of discrete regions of varying size. These were identified by fluorescence microscopy and a photograph for each individual neurone was projected onto paper, so that the outlines of the discrete regions of lipopigment could be drawn and subjected to morphometric measurements. Both ageing and vitamin E deficiency in relation to hippocampal neurones and vitamin E deficiency in relation to Purkinje neurones (in which ageing effects were not examined), were associated with a significant (< 0.05) increase in the mean total area (per rat) enclosed by the lipopigment outlines. For both vitamin E deficiency and ageing this increase was associated with both an increase in the number of relatively large discrete lipopigment regions and a decrease in the number of relatively small discrete lipopigrnent regions. The findings in relation to vitamin E deficiency could be explained by an increased rate of lipopigment formation, involving processes which also occur in ageing. Vitamin E; Lipopigment; Lipofuscin; Ageing
Introduction 'Lipopigment' consists of granules of varying size (whose maximum dimensions are usually between 1-3 ~m, Glees and Hasan, 1976) which appear yellow or brown in unstained tissue sections. It is present in many cell types and as it exhibits a striking yellow autofluorescence in unstained tissue, it can be defined as a tissue pigment Correspondence to: J.H. Dowson, Department of Psychiatry, University of Cambridge, Level E4, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, U.K.
240 which emits yellow autofluorescence when irradiated by ultraviolet or blue light (Dowson and Harris, 1981; Barden and Brizzee, 1987). The gradual accumulation of neuronal lipopigment has been considered to be one of the most consistent correlates of ageing in the mammalian nervous system, although there are considerable variations in the characteristics of lipopigment between neuronal populations (Dowson, 1989). Lipopigment is believed to contain material resulting from cell damage and/or from the normal processes of renewal of cellular constituents such as membranes, mitochondria and lysosomes. There is evidence that its formation involves the reaction of unstable 'free radicals' with such constituents in a process of lipid peroxidation (Bertoni-Freddari et al., 1984; 1987), while other processes may also be implicated, such as oxidative damage to proteins (which then undergo further reactions to form covalent crossqinks) and non-enzymic glucose-medicated protein modifications (Davies, 1987; Pongor et al, 1984). Lipopigment is usually considered as cellular debris and it has been claimed that it may be involved in a constant turnover, involving the extrusion of fragmented particles through the cell membrane (Glees and Spoerri, 1975). But it is not certain that routine removal of intraneuronal lipopigment takes place to a significant extent and opinion varies as to whether neuronal lipopigment in ageing or disease can impair cerebral function (Dowson, 1982a). Because of the variation in its properties, lipopigment should be considered as an umbrella item for a variety of related pigments (Dowson, 1982b; Dowson, 1983, Dowson et al., 1982; 1989). There is evidence that peroxidative damage is important in ageing and some disease processes such as ischaemia. Vitamin E (o~-tocopherol) appears to be an important antioxidant which is active in the inhibition and control of such damage, thus protecting unsaturated membrane lipids from free radicals by interrupting the chain of lipid peroxidation (Tappel, 1972). Various studies in mammals have examined the effects of both deficiency and supplementation of vitamin E on various biological parameters, including the characteristics of lipopigment (Porta, 1987). However, while Koistinaho et al. (1990) found that vitamin E deficiency in the rat was associated with an increase in lipopigment (as estimated by measurement of lipopigment autofluorescence) in dorsal root ganglia at 8 months of age, Davies et al. (1987), in a study of vitamin E deficiency in mice from 2-8 months (using quantitative analysis of electron micrographs), found no significant difference in the amount of lipopigment in the CA2 hippocampal neurones or in the supraoptic nucleus of the hypothalamus. Also, Katz et al. (1984) did not find increased lipopigment in rat spinal cord or inferior olivary nucleus after 17 weeks of a vitamin Edeficient diet, although an increase was reported in retinal pigment epithelium. In humans, malabsorption and vitamin E deficiency has been linked with lipopigment (ceroid) accumulation in smooth muscle and various neurological deficits (Stamp and Evans, 1987), while in rodents, a vitamin E-deficient diet has been associated with increased lipopigment in the uterus, duodenum, retinal pigment epithelium, adrenal cortical cells and extraocular muscles (Bieri and Tolliver, 1980; Robison et al., 1980; Katz et al., 1984; Herrmann et al., 1985; Katz et al., 1985; Davies et al., 1987), with a reduced cerebellar synaptic contact area (Bertoni-Freddari et al., 1984) and with electrolyte changes in brain and liver (Bertoni-Freddari et al., 1981). In humans, vitamin E supplements have been successfully used in certain deficien-
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cy states (Muller, 1986; Stamp and Evans, 1987) and may reduce the risk of atheromatous heart disease, while in rodents, vitamin E supplements have been associated with reduced lipopigment in dorsal root ganglia (Koistinaho et al., 1990), reduced lipid in brain extracts (Freund, 1979) and a protective effect in a cadmiuminduced increase in brain lipopigment (Shukla et al., 1988). The present study has examined the effects of vitamin E deficiency and ageing on neuronal lipopigment in rat hippocampal and Purkinje neurones, using a quantitative method which not only reflected the total amount of lipopigment per neurone but also examined the number of discrete regions of lipopigment in various size categories. Materials and Methods
Female Wistar rats (bred at the INRCA laboratory) were randomly selected to receive a standard laboratory diet (ad libitum), supplied by Nossan, Via E. Fermi, 8 20050 Corezzano Mi, Italy, or a vitamin E-deficient diet (ad libitum) after 1 month of age. Groups of rats which had received a standard diet were killed at 6 months (7 rats), 12 months (5 rats), 18 months (9 rats) and 25 months of age (5 rats). The group which received a vitamin E-deficient diet (9 rats) were killed at 18 months of age. Rats were killed by exposure to CO2. The components of the standard diet were yellow maize, wheat bran, wheat meat, soybean wheat, whey, oats, flax flour, dry medic, torula yeast, calcium carbonate and sodium chloride. Analysis of the diet showed carbohydrate, 41%; protein, 21%; fat (mostly unsaturated lipids), 6%; crude fibre, 7.1%; ash, 11% and unazotized extractive 52.6%. The vitamin content included vitamin E, 86.50 mg/kg and (per kg) vitamins A, 16 000 IU; D-3, 1800 IU; B-l, 8.80 mg; B-2, 18.00 rag; B-6, 8.80 mg; D-pantothenic acid, 43.47mg; K, 8.80 mg; PP, 88.00 mg; B-12, 0.088 mg; together with choline, 3680 rag; cobalt, 1.04 mg; iron, 156.5 rag; iodine, 5.21 mg; manganese, 278.26 rag; copper, 69.56 mg; zinc, 208.68 mg and methionine, 347.82 mg. This diet has proved adequate for rats; growth rates and mortality rates have been reported (Pieri et al., 1990). The vitamin E-deficient diet (from Dr Piccioni, Viale Venezia 67, 25100 Bescia, Italy) consisted of maize starch and sucrose 58%, extracted casein 18% (i.e. liposoluble vitamins had been extracted by ether), torula yeast 10%, melted filtered fat 8% (i.e. lard, from which vitamin E had been extracted), Osborne-Mendel salts 5% and a vitamin supplement lacking vitamin E. The method of extraction of vitamin E was that of Bacharach and Allchorne, (1938). Previous studies from the INRCA laboratory have reported experimental damage to cell membranes using this diet and female Wistar rats (Bertoni-Freddari et al., 1981; 1984). The brains were stored in 10% buffered formalin for up to 4 months before processing and paraffin wax-embedding. Further details of the tissue preparation have been previously reported (Dowson and Harris, 1981). Unstained 8 #m-thick sections were examined by epi-illumination with a Leitz Ortholux II microscope. The light from a mercury arc lamp passed through an exciter filter that allowed the passage of violet and blue light, and the resulting intraneuronal yellow lipopigment autofluorescence was examined with a × 100 oil-immersion objective (NA1.32) after passage through the barrier filter which transmitted above 515 nm. Lipopigment was
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defined as a pigment which exhibited yellow-green, yellow or yellow-orange autofluorescence under these conditions. In all groups of rats, pyramidal cells were examined from the stratum pyramidale of horizontal sections of the left side of the hippocampus and cells were selected from the CA3a region (Dowson, 1985). The plane of the sections was parallel to the horizontal zero plane (K6nig and Klippel, 1963) and the most rostral section of the eight adjacent sections that were examined was approximately 2 mm inferior to the superior surface of the splenium of the corpus callosum in the mid-sagittal plane. A x 63 oil-immersion objective (NA1.3) enabled each CA3a region to be divided into four adjacent fields and one hippocampal neurone was selected from each field by superimposing a 20 intersection grid, selecting an intersection at random and then identifying the neuronal body nearest to the chosen intersection. To be selected, a neuronal body had to be discrete and contain a visible nucleolus. The focus of the x 100 objective was adjusted to visualize the maximum amount of lipopigrnent in the cell body and the cell was then photographed with 400 ASA colour film. Eight adjacent sections were examined from each rat brain, involving a total of 32 hippocampal cells per rat. The observer was blind to the origin of all the sections examined. The boundary between yellow autofluorescence and green background tissue fluorescence is usually clear but, with some regions of pigment which are not in focus, there is a gradual change from yellow to green. In such cases the observer has to judge the boundary of the area corresponding to yellow pigment by the colour. Purkinje neurones were examined in those groups of rats killed at 18 months of age. (Cerebellar tissue was not available for the other groups). Purkinje neurones were examined within lobule 1 of the vermis according to the terminology of Larsell (1972). Each cerebellum was sectioned in the mid-sagittal plane and 8-tam sections from the left side were examined within 100/~m of the plane of dissection. The most caudal field in lobule I shown by the x 63 objective was the first to be examined. A graduated line was superimposed upon the row of Purkinje neurones and a position in the line was randomly selected. The Purkinje neurone body nearest to that point was selected for examination by the x 100 objective if it could be safely identified as a Purkinje cell and had a visible nucleolus. Four cells were selected, one from each of four adjacent fields from each section; eight sections per rat brain were examined which yielded a total of 32 Purkinje neurones. The focus of the x 100 objective was adjusted to visualize the maximum amount of lipopigment in the cell body and the cell was then photographed as described above. The transparencies of the photographed cell bodies were then projected onto paper keeping the magnification constant, and, for each neurone, the outlines that delineated the projected images of discrete regions of yellow lipopigment autofluorescence were drawn. The areas enclosed by each of the discrete outlines were measured automatically for each cell with a Quantimet 720 Image Analyser (Cambridge Instruments Ltd., Cambridge, U.K. ) and were automatically classified into various size categories. Also, a standard stereological technique, involving the counting of test points and surface test line intersections (Underwood, 1970), was applied to the hippocampal neurones from the rats at 6 months and 18 months of age fed with a standard diet and at 18 months of age with a vitamin E-deficient diet.
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Transparent plastic sheets bearing a 0.5-cm square test grid were randomly placed on the drawings of the outlines (boundaries) of the discrete regions of lipopigment. The number of intersections of the test lines with lipopigrnent boundaries ('cuts') were counted for each neurone section. The number of grid line intersections which were found to overlie the areas enclosed by lipopigrnent outlines ('hits') were also recorded. The distance between each test line (grid constant) was equivalent to 2.6 #m of cell dimension. The surface-to-volume (S/V) ratios for discrete regions of lipopigment, per brain, were calculated using the equation of Chalkley et al. (1949) i.e. (2 x 'cuts')/'hits' for discrete particles (Underwood, 1970) and the mean value was obtained for each group of rats. This measure reflects the mean volume of discrete regions of lipopigment, as a reduction in the mean lipopigrnent volume is accompanied by an increase in the mean surface-to-volume ratio. Statistical analyses were carried out with the SPSS/PC ÷ programme (Norusis, 1988). Comparisons between groups of rats at different ages in respect of the total number of discrete regions of lipopigrnent per rat and the total area of the outlines of discrete regions of lipopigment per rat were made with the Kruskal-Wallis oneway analysis of variance and multiple comparisons procedure (Siegel and Castellan, 1988) (Table I). F ratios were not calculated as Cochran's C-test showed significant differences in variances (P < 0.05) in respect of the group differences for the values for area (per rat) corresponding to lipopigment, both before and after square root or loge transformations of the data. Comparisons between groups of rats at different ages in respect of the mean
TABLE I The effect of ageing on the number of discrete regions of lipopigment and on the area enclosed by outlines of discrete regions of lipopigment autofluorescence in 32 hippocampal neurones for each rat. The numbers in parentheses indicate the range; n = number of rats. Age (months) 6 (n = 7)
12 (n = 5)
18 (n = 9)
25 (n = 5)
Kruskal-Wallis one-way analysis of variance
Median number (per rat) of discrete regions of lipopigmerit in 32 neurones
501 a (373-567)
710 b (640-873)
757 c (494-865)
758 a (583-905)
H = 11.13 P < 0.05 l
Median area (per rat) enclosed by lipopigment outlines in 32 neurones (t~m 2)
515 e (429-660)
1149 f (193-1210)
1729 g (920-2012)
2754 h (2280-2886)
H = 135.66 P < 0.0012
1'2In each case the hypothesis that all population medians are equal is rejected. Multiple comparisons procedure shows the following pairs of groups are significantly different at the 0.05 level: a < b, a < d , e < g , e < h.
244 n u m b e r (per rat) o f areas e n c l o s e d b y o u t l i n e s o f discrete r e g i o n s o f l i p o p i g m e n t , in v a r i o u s size categories, were m a d e with a o n e - w a y a n a l y s i s o f v a r i a n c e a n d the Scheff6 m u l t i p l e c o m p a r i s o n p r o c e d u r e ( T a b l e II). I n two size categories, s q u a r e r o o t t r a n s f o r m a t i o n s o f d a t a were r e q u i r e d to yield h o m o g e n e i t y o f v a r i a n c e as s h o w n b y C o c h r a n ' s C-test u s i n g the 0.05 level to i n d i c a t e differences. C o m p a r i s o n s b e t w e e n the v i t a m i n E-deficient g r o u p a n d the c o r r e s p o n d i n g c o n trol g r o u p ( T a b l e s III, IV) a n d c o m p a r i s o n s i n v o l v i n g the results in T a b l e VI, were m a d e with B o n f e r r o n i t-tests to allow for m u l t i p l e c o m p a r i s o n s ( G l a n t z , 1987); i.e. for x = the n u m b e r o f related t-tests, the significance level yielded b y the t-test n e e d ed to be 0.05/x to be c o n s i d e r e d s i g n i f i c a n t at the 0.05 level. T h e p o o l e d - v a r i a n c e t-test was used, u n l e s s a n F-test i n d i c a t e d s i g n i f i c a n t ( < 0.05) difference b e t w e e n the p o p u l a t i o n variances, w h e n the s e p a r a t e - v a r i a n c e t-test was s u b s t i t u t e d . Also, a t w o - w a y a n a l y s i s o f v a r i a n c e ( T a b l e V) was p e r f o r m e d for the n u m b e r o f discrete r e g i o n s o f l i p o p i g m e n t in h i p p o c a m p a l a n d P u r k i n j e n e u r o n e s in r e l a t i o n to two factors: firstly, six size categories o f discrete r e g i o n s o f l i p o p i g r n e n t a n d , s e c o n d -
TABLE II The mean number (per rat) of areas enclosed by outlines of discrete regions of lipopigment, in various size categories in a population of hippocampal neurones (32 neurones per rat) at various ages. Standard deviations are in parentheses; n = number of rats. Size categories of areas enclosed by lipopigment outlines (t~m2)
Age (months) 6 (n = 7)
12 (n = 5)
18 (n = 9)
25 (n = 5)
20- < 50
27.4 (21.8) 252.3 (71.8) 196.4a (55.5) 3.1 e (2.7) 0.4 i ( 1. I) 0
25.4 (15.5) 241.8 (79.5) 445.2 b (54.0) 11.2f (8.3) 2.0J (2.0) 0
50-<75
0
0
75- < 100
0
0
20.7 (23,8) 216.0 (97.0) 407.1 c (95.1) 38.6g (18.3) 15.7k (8.5) 2.9 (2.1) 0.1 (0.3) 0
5.0 (4.7) 135.8 (52.3) 464.8d (82.2) 61.0 h (6.5) 29.61 (3.6) 22.0 (4.9) 1.6 (1.7) 0.4 (0.9)
0- <0.5 0.5- < 1 1-<5 5 - < 10 10-<20
F ratio (One-way analysis of variance) 1.4 2.3 16,8 (P <_ 0.00009 I) 35.3 (P --- 0.000092)* 40.3 (P ~ 0.000093)*
|'2'3In each case the hypothesis that all population means are equal is rejected. In each case Scheff6 multiple comparison procedure shows the following pairs of mean values are significantly different at the 0.051evel;l:a < b,a < c,a < d ; 2 : e < g,e < h , f < g , f < h ; 3 : i < k,i < l,j < k,j < l,k < 1. P = probability of obtaining an F value at least as large as the one calculated when all population means are equal. *Square root transformation of data required to increase homogeneity of variances.
245 TABLE III The effect of vitamin E-deficient diet in rats at 18 months of age on the number of discrete regions of lipopigment and on the area enclosed by outlines of discrete regions of lipopigment in 32 hippocampal neurones and in 32 Purkinje neurones for each rat. Standard deviations are in parentheses; n = number of rats. Treatment Hippocampal neurones
Purkinje neurones
Vitamin E-deficient diet (n = 9)
Controls (standard diet) (n = 9)
Vitamin E-deficient diet (n=9)
Controls (standard diet) (n = 9)
Mean number (per rat) of discrete regions oflipopigment in 32 neurones
699 (128)
701 (160)
513 (63)
505 (53)
Mean area (per rat) enclosed by lipopigment outlines in 32 neurones (#m 2)
3069 (423)
1525 (446)*
1838 (311)
1206 (166)*
*Mean values between the two treatment groups are significantly different at the 0.05 level using the Bonferroni procedure to compensate for 4 tests on these data.
ly, a s t a n d a r d diet o r a v i t a m i n E-deficient diet. It should be n o t e d that the two largest size categories were excluded f r o m this analysis in view o f the relatively small n u m b e r s in these categories a n d the large n u m b e r o f zero values. C o c h r a n ' s C-test was used to indicate h o m o g e n e i t y o f variance, using the 0.05 level to indicate differences. F o r Purkinje neurones, all p a i r e d g r o u p s o f values for each size c a t e g o r y (i.e. c o r r e s p o n d i n g to v i t a m i n E -deficient diet a n d s t a n d a r d diet), showed h o m o g e n e i t y o f variance as defined above; h o w e v e r this was n o t the case for two size categories for h i p p o c a m p a l n e u r o n e s ( 0 - < 0 . 5 a n d 2 0 - < 50). A loge t r a n s f o r m a tion o f the d a t a for the h i p p o c a m p a l n e u r o n e s p r o d u c e d h o m o g e n e i t y in respect o f the 0 - < 0.5 c a t e g o r y b u t n o t the 2 0 - < 50 category. However, after t r a n s f o r m a t i o n , only one o f the six size categories ( 2 0 - < 50) v i o l a t e d the a s s u m p t i o n o f h o m o g e n e i t y o f variance a n d this is unlikely to invalidate the result, as the g r o u p sizes are equal (Witte, 1989). Size categories were established before the e x p e r i m e n t a n d were n o t influenced by the o u t c o m e o f the analysis.
Results The effect o f ageing o n the m e d i a n n u m b e r o f discrete regions o f l i p o p i g m e n t p e r rat a n d on the m e d i a n a r e a enclosed by l i p o p i g m e n t outlines p e r rat, in h i p p o c a m p a l
246 TABLE IV The mean number (per rat) o f areas enclosed by outlines of discrete regions of lipopigment in various size categories, in populations of hippocampal and Purkinje neurones (32 neurones per region per rat), in rats aged 18 months which had received a vitamin E -deficient diet and in control rats fed with a standard diet. Standard deviations are in parentheses; n = number of rats. Size categories of areas enclosed by lipopigment outlines (/zm 2)
0-<0.5 0.5-< 1 1- < 5 5 - < 10 10-<20 20- < 50 50- < 75 7 5 - < 100
Hippocampal neurones
Purkinje neurones
Vitamin E-deficient diet (n = 9)
Standard diet
Vitamin E-deficient diet (n = 9)
Standard diet
3.8 (2.7) 145.3 (89.2) 419.8 (51.0) 67.4 (15.5) 34.0 (7.0) 22.0 (4.5) 4.9 (2.3) 1.2 (2.2)
20.7 (23.8) 216.0 (97.0) 407.1 (95.1) 38.6* ( ! 8.3) 15.7" (8.5) 2.9* (2.1) 0. !* (0.3) 0
149.1 (38.3) 132.3 (21.4) 155.6 (31.5) 30.9 (7.8) 18.3 (5.2) 24.2 (5.2) 2.0 (1.3) 0.2 (0.4)
190.2 (32.9) 129.1 (20.8) 133.2 (20.2) 23.8 (6.2) 16.0 (4.2) 12.4" (2.8) 0.3* (0.5) 0
(n = 9)
(n = 9)
*Mean values between the two groups (vitamin E-deficient diet and standard diet) are significantly different at the 0.05 level using the Bonferroni procedure to compensate for 7 tests for each brain region.
neurones, is shown in Table I. Significant differences are found in respect to both median number per rat and median area per rat and the results suggest that the number of discrete regions show little increase above 12 months of age in contrast to the area. Therefore after 12 months the median area enclosed by a discrete lipopigment region gradually increases in size. Table II shows the effects of ageing on hippocampal neuronal lipopigment in relation to various size categories of areas enclosed by outlines of discrete regions of lipopigrnent. Analysis of variance shows that age-related increases in the numbers of discrete lipopigrnent regions are significant at the 0.05 level in three of the size categories. However, this is accompanied by age-related decreases in the two smallest categories (although these do not reach significance at the 0.05 level), which may explain why the total number of discrete regions of lipopigment per rat shows little change after 12 months of age (see Table I). The effects of the vitamin E-deficient diet and a standard diet on rats at 18 months of age are shown in Table III. While there is no significant difference in the mean number (per rat) of discrete regions of hippocampal or Purkinje neuronal lipopig-
247 TABLE V Two-way analysis of variance for the number of discrete regions of lipopigment in 32 hippocampal neurones and 32 Purkinje neurones for each of 18 rats in relation to two factors: (i) six size categories (columns) and (ii) a standard diet or a vitamin E-deficient diet (rows). Source of variance Hippocampal neurones* Between columns Between rows Interaction (rows and columns) Within groups Purkinje neurones Between columns Between rows Interaction (rows and columns) Within groups
Sum of squares
Degree of freedom
2 510 776 27 872 31 500
5 1 5
353 659
96
452 813 24 10 749
5 1 5
40 127
96
F ratio
Significance of F
136.4 7.6 18.1
_<0.0009 0.007 _<0.0009
216.7 0.1 5.1
_<0.0009 (0.81) _<0.0009
Note: 9 rats received a standard diet and 9 rats received a vitamin E-deficient diet. As there were six size categories, each of the two ANOVAs involved (9 × 2)6 = 108 values for the number of discrete regions of lipopigment. *Loge transformation of data required to increase homogeneity of variances.
m e n t b e t w e e n t h e t w o g r o u p s , v i t a m i n E d e f i c i e n c y is a s s o c i a t e d w i t h a s i g n i f i c a n t i n c r e a s e in m e a n a r e a ( p e r r a t ) e n c l o s e d b y o u t l i n e s o f d i s c r e t e r e g i o n s o f l i p o p i g m e n t in b o t h h i p p o c a m p a l a n d P u r k i n j e n e u r o n e s . T h i s s u g g e s t s t h a t v i t a m i n E defic i e n c y is a s s o c i a t e d w i t h a m e a n i n c r e a s e i n t h e size o f a d i s c r e t e r e g i o n o f lipopigrnent. Table IV examines the effect of vitamin E deficiency in relation to the v a r i o u s size c a t e g o r i e s o f a r e a s c o r r e s p o n d i n g t o d i s c r e t e r e g i o n s o f l i p o p i g r n e n t . I n
TABLE VI The effects of ageing and a vitamin E-deficient diet on morphometric properties of discrete regions of lipopigment in hippocampal neurones (32 neurones per rat) shown by a grid-counting method. Standard deviations are in parentheses; n = number of rats. Treatment
Age (months) Number of rats Mean surface-to-volume ratio for discrete regions of lipopigment (per rat)
Standard diet
Standard diet
Vitamin E-deficient diet
6 7 11.96 (1.88)
18 9 8.29 (0.99)
18 9 6.32 (0.49)
248
both neuronal populations, vitamin E deficiency is associated with a reduced number of pigment regions in the smallest size category and in the 0.5-< 1 category in hippocampal neurones (although these trends are not significant at the 0.05 level). However, in the remaining categories vitamin E deficiency is associated with an increased number of discrete pigment regions and six of the inter-group comparisons for the largest six size categories reach significance at the 0.05 level. Therefore, as with ageing in hippocampal neurones, vitamin E deficiency is associated with a decrease in the number of relatively small lipopigrnent regions and an increase in the number of relatively large lipopigment regions. The relationship between size category of discrete regions of lipopigment and vitamin E deficiency is further examined in table V, in which the two-way analysis of variance shows significant interactions between size category and vitamin E deficiency (i.e. P < 0.0009 for both hippocampal and Purkinje neurones). Thus the effect of size category on the numbers of discrete regions of lipopigment depends on whether the rats have received a vitamin E-deficient diet or a standard diet. The results of morphometric analysis by the grid counting method are shown in Table VI. These provide additional evidence (to that of Tables I and III) that ageing and vitamin E deficiency are associated with increases in the mean volume of discrete lipopigment regions. Bonferroni t-tests between values for rats at 6 and 18 months of age fed on the standard diet, and between rats at 18 months of age fed on the standard diet and those fed on the vitamin E-deficient diet, were significant at P < 0.05. Discussion
Previous comparisons have been made between ageing-related neuronal changes and those associated with a vitamin E-deficient diet; for example, both have been claimed to involve a reduction in rat cerebellar synaptic contact areas (BertoniFreddari et al., 1984), although differences in the distribution of increased lipopigment between ageing and vitamin E-deficiency have also been described (Katz et al., 1984). While the concept of vitamin E deficiency as accelerated ageing may, at best, be an over-simplification, the present study suggests that the two processes have similar effects on neuronal lipopigment, if it is assumed that the significant differences between the vitamin E-deficient and standard diet groups result from the lack of vitamin E. (This appears to be a reasonable hypothesis, although mechanisms other than direct cause and effect are possible.) The present results show that both ageing and vitamin E deficiency are associated with an increase in the mean area (per rat) enclosed by the outlines of discrete regions of lipopigrnent and that this change involves an increase in the numbers of relatively large discrete lipopigment regions, accompanied by a decrease in the number of relatively small discrete regions. Although the area within an outline of a region of yellow autofluorescence may appear filled with lipopigment, the autofluorescence may be derived from a number of lipopigment granules which can overlap when viewed under the microscope or be sufficiently close to appear fused. The present findings could reflect several processes (alone or in combination) involving changes in the density and size of lipopigment granules and changes in the rates of granule formation, of granule fusion, of granule
249 fragmentation and of extrusion of granule derivatives from the cell. (Although it has been claimed that lipopigment can be extruded from neurones, the degree of such a process is uncertain and it is not known whether any extruded lipopigment is derived from fragmentation of the larger or smaller granules.) One factor which may be relevant in vitamin E deficiency is an increase in the rate of the processes that occur in ageing, (i.e. degradation of cellular constituents such as membranes, mitochondria and lysosomes), leading to an increased rate of lipopigment formation. This would produce an increased density of lipopigment granules with increased contiguity and overlap; this may contribute to the reduction in the smaller lipopigment regions and the increase in the larger regions. Also, as the processing of lipopigment is believed to involve aggregation of lipopigment granules, accompanied by their increase in size by incorporation of cellular debris (Barden and Brizzee, 1987), an increased rate of lipopigment formation resulting from vitamin E deficiency may also be accompanied by increases in the rates of these processes, which may also contribute to the present results. Increased lipopigment formation would be expected to produce an increase in the mean total area enclosed by the lipopigment outlines, per rat, which was found in association with vitamin E deficiency (Table III). Therefore, a process of increased lipopigment formation, together with an increased rate of enlargement and aggregation of lipopigment granules, could explain the present findings in relation to vitamin E deficiency, and may reflect an increased rate of processes which occur in ageing. However, there are other explanations for the present findings; for example, vitamin E deficiency (and ageing) may reflect an impairment of the renewal processes of cellular constituents with a reduced rate of lipopigment formation. This hypothesis could agree with the present findings if this is associated with impairment of fragmentation of the relatively large lipopigment regions accompanied by a reduced rate of extrusion of the results of this process from the cell. The present study has demonstrated that a vitamin-E deficient diet can be associated with statistically significant changes (<0.05) in relation to the sizes of discrete regions of lipopigment in hippocampal and Purkinje neuronal populations in the rat. These changes are similar to those in hippocampal neurones related to ageing between 12 and 25 months. This suggests that the underlying processes initiated by vitamin E deficiency and ageing may share some common mechanisms.
Acknowledgement The authors thank Mrs A. Robson for the preparation of the manuscript.
References Barden, H. and Brizzee, K.R. (1987): The histochemistry of lipofuscin and neuromelanin. In: Lipofuscin-1987:State of the Art, pp. 339-392. Editor: I. Zs.-Nagy. Akad6miai Kiad6, Budapestand Elsevier, Amsterdam. Bacharach, A.L. and Allchorne,E. (1938):Investigationsinto the method of estimation of vitamin E: further observations of vitamin E deficiencyand implantation. Biochem.J., 32, 1298-1301.
250 Bertoni-Freddari, C., Giuti, C., Lustyik, G. and Zs.-Nagy, I. (1981): In vivo effects of vitamin E deficiency on the intracellular monovalent electrolyte concentrations in brain and liver of rat. An energy dispersive X-ray microanalytic study. Mech. Ageing Dev., 16, 169-180. Bertoni-Freddari, C., Giuli, C. and Pieri, C. (1984): Effect of chronic vitamin E deficiency on the synapses of cerebeilar glomeruli in young rats. Mech. Ageing Dev, 24, 225-232. Bertoni-Freddari, C., Meier-Ruge, W. and Ulrick, J. (1987): Aging-like alterations at the neuronal membrane of young rats fed a vitamin E-deficient diet. In: Lipofuscin-1987: State of the Art, pp. 439-440. Editor: I. Zs.-Nagy. Akad6miai Kiad6, Budapest and Elsevier Amsterdam. Bieri, J.G. and Tolliver, T.J. (1980): Lipofuscin in vitamin E deficiency and the possible role of retinol. Lipids, 15, 10-13. Chalkley, H.W., Cornfield, J. and Park, H. (1949): A method for estimating volume-surface ratios. Science, 110, 295-304. Davies, I., Davidson, Y. and Fotheringham, A.P. (1987): The effect of vitamin E deficiency on the induction of age pigment in various tissues of the mouse. Exp. Gerontol., 22, 127-137. Davies, K.J.A. (1987): Protein oxidation, protein crosslinking and proteolysis in the formation of lipofuscin; rationale and methods for the measurement of protein degradation. In: Lipofuscin-1987: State of the Art, pp. 109-133. Editor: I. Zs.-Nagy. Akad6miai Kiad6, Budapest and Elsevier, Amsterdam. Dowson, J.H. and Harris, S.J. (1981): Quantitive studies of the autofluorescence derived from neuronal iipofuscin. J. Microsc., 124, 249-258. Dowson, J.H. (1982a): Neuronal lipofuscin accumulation in ageing and Alzheimer dementia: a pathogenic mechanism? Br. J. Psychiatry, 140, 142-270. Dowson, J.H. (1982b): The evaluation of autofluorescence emission spectra derived from neuronal lipopigment. J. Microsc., 128, 261-270. Dowson, J.H., Armstrong, D., Koppang, N., Lake, B.D. and Jolly, R.D. (1982): Autofluorescence emission spectra of neuronal lipopigment in animal and human ceroidoses (ceroid-lipofuscinoses). Acta Neuropathol., 58, 152-156. Dowson, J.H. (1983): Autofluorescence emission spectra of neuronal lipopigment in a case of adult-onset ceroidosis (Kufs' disease). Acta Neuropathol. (Bed.), 59, 241-245. Dowson, J.H. (1985): Quantitative studies of the effect of aging, meclofenoxate and dihydroergotoxine in intraneuronal lipopigment accumulation in the rat. Exp. Gerontol., 20, 333-340. Dowson, J.H. (1989): Neuronal lipopigment: a marker for cognitive impairment and long-term effects of psychotropic drugs. Br. J. Psychiatry, 155, 1-11. Dowson, J.H., Wilton-Cox, H., Oldfors, A. and Sourander, P. (1989): Autofluorescence emission spectra of neuronal lipopigment in mucopolysaccharidosis (Sanfilippo's syndrome). Acta Neuropathol. (Berl.), 77, 426-429. Freund, G. (1979): The effects of chronic alchohol and vitamin E consumption on ageing pigments and learning performance in mice. Life Sci., 24, 145-152. Glantz, S.A., (1987): Primer of Biostatistics. McGraw-Hill Book Company, New York, pp. 88-90. Glees, P. and Hasan, M. (1976): Lipofuscin in neuronal aging and diseases. Norm. Pathol. Anat., 32, 1-68.
Glees, P. and Spoerri, P.E. (1975): Centrophenoxin-induced dissolution and removal of lipofuscin: an electron microscopic study. Arzneim.-Forsch., 25, 3-9. Herrmann, R.K., Robison, Jr, W.G., Bieri, J.G. and Spitznas, M. (1985): Lipofuscin accumulation in extraocular muscle of rats deficient in vitamins E and A. Graefe's Arch. Clin. Exp. Ophthalmol., 223, 272-277. Katz, M.L., Robison, Jr, W.G., Herrmann, R.K., Groome, A.B. and Bieri, J.G. (1984): Lipofuscin accumulation resulting from senescence and vitamin E deficiency; spectral properties and tissue distribution. Mech. Ageing Dev., 25, 149-159. Katz, M.L., Groome, A.B. and Robison, Jr, W.G., (1985): Localization of lipofuscin in the duodenums of vitamin E-deficient rats. J. Nutr., 115, 1355-1365. Koistinaho, J., Alho, H. and Hervonen, A. (1990): Effect of vitamin E and selenium supplement on the aging peripheral neurons of the male Sprague-Dawley rat. Mech. Ageing Dev., 51, 63-72. K6nig, J.F.R. and Klippel, R.A. (1963): The Rat Brain. Williams and Wilkins Co., Baltimore, pp. 25-139.
251 Larsell, O. (1972): The morphogenesis and adult pattern of the lobules and fissures of the cerebellum of the white rat. J. Comp. Neurol., 97, 281-356. Muller, D.P.R. (1986): Vitamin E - its role in neurological functions. Postgrad. Med. J., 62, 107-112. Norusis, M.J. (1988): The SPSS Guide to Data Analysis for SPSS/PC ÷. SPSS Inc, Chicago. Pieri, C., Recchioni, R., Moroni, F., Marcheselli, F., Falasca, M. and Piantanelli, L. (1990): Food restriction in female Wistar rats. I. Survival characteristics, membrane microviscosity and proliferative response in lymphocytes. Arch. Gerontol. Geriatr., 11, 99-108. Pongor, S., Ulrich, P.C., Bencsatti, F.A. and Cerami, A. (1984): Ageing of proteins: Isolation and identification of a fluorescent chromophore from the reaction of polypeptides with glucose. Proc. Nat. Acad. Sci. U.S.A., 81, 2684-2688. Porta, E.A. (1987): Tissue lipoperoxidation and lipofuscin accumulation as influenced by age, type of dietary fat and levels of vitamin E in rats. In: Advances in the Biosciences, Vol. 64, pp. 37-74. Editors: E.A. Totaro, P. Glees, and F.A. Pisanti. Pergamon Press, Oxford, New York. Robison Jr., W.G., Kuwabara, T. and Bieri, J.G. (1980): Deficiencies of vitamins E and A in the rat. Invest. Opthalmol. Vision Sci., 19, 1030-1037. Shukla, G.C., Hussain, T. and Chandra, S.V. (1988): Protective effect of vitamin E on cadmium-induced alterations in lipofuscin and superoxide dismutase in rat brain regions. Pharmacol. Toxicol., 63, 305-306. Siegel, S. and Castellan, Jr., W.J. (1988): Nonparametric Statistics for the Behavioural Sciences. 2nd edn., McGraw-Hill Book Company, New York, pp. 213-214. Stamp, G.W.H. and Evans, D.J. (1987): Accumulation of ceroid in smooth muscle indicates severe malabsorption and vitamin E deficiency. J. Clin. Pathoi., 40, 798-802. Tappel, A.L. (1972): Vitamin E and free radical peroxidation oflipids. Ann. N.Y. Acad. Sci., 203, 12-28. Underwood, E.E. (1970): Quantitative Stereology. Addison-Wesley Publishing Company, Reading, Massachusetts, pp. 1-38. Witte, R.S. (1989): Statistics. 3rd edn., Holt, Rinehart and Winston, Inc., New York, pp. 342-343.
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The effects of ageing and a vitamin E-deficient diet on the lipopigment content of rat hippocampal and Purkinje neurones Jonathan H. D o w s o n a, Patrizia Fattoretti b, Mary Cairns a, Nigel T. James c, Helen W i l t o n - C o x a and Carlo Bertoni-Freddari b aDepartment of Psychiatry, University of Cambridge, U.K., bl. N. R.C.A. Dipartimento di ricerche gerontologiche e geriatriche, Ancona, Italy and CDepartment of Biomedical Science, University of Sheffield, U.K. (Received 13 August 1991; accepted 27 December 1991)
Summary This study examined associations between a vitamin E-deficient diet, ageing and aspects of the morphology of neuronal lipopigment in rat hippocampal and Purkinje neurones. Groups of rats given a standard diet were killed at 6, 12, 18 and 25 months of age, while a group which had received a vitamin E-deficient diet from 1-18 months were killed at 18 months of age. Lipopigment within a neuronal cell body consists of a number of discrete regions of varying size. These were identified by fluorescence microscopy and a photograph for each individual neurone was projected onto paper, so that the outlines of the discrete regions of lipopigment could be drawn and subjected to morphometric measurements. Both ageing and vitamin E deficiency in relation to hippocampal neurones and vitamin E deficiency in relation to Purkinje neurones (in which ageing effects were not examined), were associated with a significant (< 0.05) increase in the mean total area (per rat) enclosed by the lipopigment outlines. For both vitamin E deficiency and ageing this increase was associated with both an increase in the number of relatively large discrete lipopigment regions and a decrease in the number of relatively small discrete lipopigrnent regions. The findings in relation to vitamin E deficiency could be explained by an increased rate of lipopigment formation, involving processes which also occur in ageing. Vitamin E; Lipopigment; Lipofuscin; Ageing
Introduction 'Lipopigment' consists of granules of varying size (whose maximum dimensions are usually between 1-3 ~m, Glees and Hasan, 1976) which appear yellow or brown in unstained tissue sections. It is present in many cell types and as it exhibits a striking yellow autofluorescence in unstained tissue, it can be defined as a tissue pigment Correspondence to: J.H. Dowson, Department of Psychiatry, University of Cambridge, Level E4, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, U.K.
240 which emits yellow autofluorescence when irradiated by ultraviolet or blue light (Dowson and Harris, 1981; Barden and Brizzee, 1987). The gradual accumulation of neuronal lipopigment has been considered to be one of the most consistent correlates of ageing in the mammalian nervous system, although there are considerable variations in the characteristics of lipopigment between neuronal populations (Dowson, 1989). Lipopigment is believed to contain material resulting from cell damage and/or from the normal processes of renewal of cellular constituents such as membranes, mitochondria and lysosomes. There is evidence that its formation involves the reaction of unstable 'free radicals' with such constituents in a process of lipid peroxidation (Bertoni-Freddari et al., 1984; 1987), while other processes may also be implicated, such as oxidative damage to proteins (which then undergo further reactions to form covalent crossqinks) and non-enzymic glucose-medicated protein modifications (Davies, 1987; Pongor et al, 1984). Lipopigment is usually considered as cellular debris and it has been claimed that it may be involved in a constant turnover, involving the extrusion of fragmented particles through the cell membrane (Glees and Spoerri, 1975). But it is not certain that routine removal of intraneuronal lipopigment takes place to a significant extent and opinion varies as to whether neuronal lipopigment in ageing or disease can impair cerebral function (Dowson, 1982a). Because of the variation in its properties, lipopigment should be considered as an umbrella item for a variety of related pigments (Dowson, 1982b; Dowson, 1983, Dowson et al., 1982; 1989). There is evidence that peroxidative damage is important in ageing and some disease processes such as ischaemia. Vitamin E (o~-tocopherol) appears to be an important antioxidant which is active in the inhibition and control of such damage, thus protecting unsaturated membrane lipids from free radicals by interrupting the chain of lipid peroxidation (Tappel, 1972). Various studies in mammals have examined the effects of both deficiency and supplementation of vitamin E on various biological parameters, including the characteristics of lipopigment (Porta, 1987). However, while Koistinaho et al. (1990) found that vitamin E deficiency in the rat was associated with an increase in lipopigment (as estimated by measurement of lipopigment autofluorescence) in dorsal root ganglia at 8 months of age, Davies et al. (1987), in a study of vitamin E deficiency in mice from 2-8 months (using quantitative analysis of electron micrographs), found no significant difference in the amount of lipopigment in the CA2 hippocampal neurones or in the supraoptic nucleus of the hypothalamus. Also, Katz et al. (1984) did not find increased lipopigment in rat spinal cord or inferior olivary nucleus after 17 weeks of a vitamin Edeficient diet, although an increase was reported in retinal pigment epithelium. In humans, malabsorption and vitamin E deficiency has been linked with lipopigment (ceroid) accumulation in smooth muscle and various neurological deficits (Stamp and Evans, 1987), while in rodents, a vitamin E-deficient diet has been associated with increased lipopigment in the uterus, duodenum, retinal pigment epithelium, adrenal cortical cells and extraocular muscles (Bieri and Tolliver, 1980; Robison et al., 1980; Katz et al., 1984; Herrmann et al., 1985; Katz et al., 1985; Davies et al., 1987), with a reduced cerebellar synaptic contact area (Bertoni-Freddari et al., 1984) and with electrolyte changes in brain and liver (Bertoni-Freddari et al., 1981). In humans, vitamin E supplements have been successfully used in certain deficien-
241
cy states (Muller, 1986; Stamp and Evans, 1987) and may reduce the risk of atheromatous heart disease, while in rodents, vitamin E supplements have been associated with reduced lipopigment in dorsal root ganglia (Koistinaho et al., 1990), reduced lipid in brain extracts (Freund, 1979) and a protective effect in a cadmiuminduced increase in brain lipopigment (Shukla et al., 1988). The present study has examined the effects of vitamin E deficiency and ageing on neuronal lipopigment in rat hippocampal and Purkinje neurones, using a quantitative method which not only reflected the total amount of lipopigment per neurone but also examined the number of discrete regions of lipopigment in various size categories. Materials and Methods
Female Wistar rats (bred at the INRCA laboratory) were randomly selected to receive a standard laboratory diet (ad libitum), supplied by Nossan, Via E. Fermi, 8 20050 Corezzano Mi, Italy, or a vitamin E-deficient diet (ad libitum) after 1 month of age. Groups of rats which had received a standard diet were killed at 6 months (7 rats), 12 months (5 rats), 18 months (9 rats) and 25 months of age (5 rats). The group which received a vitamin E-deficient diet (9 rats) were killed at 18 months of age. Rats were killed by exposure to CO2. The components of the standard diet were yellow maize, wheat bran, wheat meat, soybean wheat, whey, oats, flax flour, dry medic, torula yeast, calcium carbonate and sodium chloride. Analysis of the diet showed carbohydrate, 41%; protein, 21%; fat (mostly unsaturated lipids), 6%; crude fibre, 7.1%; ash, 11% and unazotized extractive 52.6%. The vitamin content included vitamin E, 86.50 mg/kg and (per kg) vitamins A, 16 000 IU; D-3, 1800 IU; B-l, 8.80 mg; B-2, 18.00 rag; B-6, 8.80 mg; D-pantothenic acid, 43.47mg; K, 8.80 mg; PP, 88.00 mg; B-12, 0.088 mg; together with choline, 3680 rag; cobalt, 1.04 mg; iron, 156.5 rag; iodine, 5.21 mg; manganese, 278.26 rag; copper, 69.56 mg; zinc, 208.68 mg and methionine, 347.82 mg. This diet has proved adequate for rats; growth rates and mortality rates have been reported (Pieri et al., 1990). The vitamin E-deficient diet (from Dr Piccioni, Viale Venezia 67, 25100 Bescia, Italy) consisted of maize starch and sucrose 58%, extracted casein 18% (i.e. liposoluble vitamins had been extracted by ether), torula yeast 10%, melted filtered fat 8% (i.e. lard, from which vitamin E had been extracted), Osborne-Mendel salts 5% and a vitamin supplement lacking vitamin E. The method of extraction of vitamin E was that of Bacharach and Allchorne, (1938). Previous studies from the INRCA laboratory have reported experimental damage to cell membranes using this diet and female Wistar rats (Bertoni-Freddari et al., 1981; 1984). The brains were stored in 10% buffered formalin for up to 4 months before processing and paraffin wax-embedding. Further details of the tissue preparation have been previously reported (Dowson and Harris, 1981). Unstained 8 #m-thick sections were examined by epi-illumination with a Leitz Ortholux II microscope. The light from a mercury arc lamp passed through an exciter filter that allowed the passage of violet and blue light, and the resulting intraneuronal yellow lipopigment autofluorescence was examined with a × 100 oil-immersion objective (NA1.32) after passage through the barrier filter which transmitted above 515 nm. Lipopigment was
242
defined as a pigment which exhibited yellow-green, yellow or yellow-orange autofluorescence under these conditions. In all groups of rats, pyramidal cells were examined from the stratum pyramidale of horizontal sections of the left side of the hippocampus and cells were selected from the CA3a region (Dowson, 1985). The plane of the sections was parallel to the horizontal zero plane (K6nig and Klippel, 1963) and the most rostral section of the eight adjacent sections that were examined was approximately 2 mm inferior to the superior surface of the splenium of the corpus callosum in the mid-sagittal plane. A x 63 oil-immersion objective (NA1.3) enabled each CA3a region to be divided into four adjacent fields and one hippocampal neurone was selected from each field by superimposing a 20 intersection grid, selecting an intersection at random and then identifying the neuronal body nearest to the chosen intersection. To be selected, a neuronal body had to be discrete and contain a visible nucleolus. The focus of the x 100 objective was adjusted to visualize the maximum amount of lipopigrnent in the cell body and the cell was then photographed with 400 ASA colour film. Eight adjacent sections were examined from each rat brain, involving a total of 32 hippocampal cells per rat. The observer was blind to the origin of all the sections examined. The boundary between yellow autofluorescence and green background tissue fluorescence is usually clear but, with some regions of pigment which are not in focus, there is a gradual change from yellow to green. In such cases the observer has to judge the boundary of the area corresponding to yellow pigment by the colour. Purkinje neurones were examined in those groups of rats killed at 18 months of age. (Cerebellar tissue was not available for the other groups). Purkinje neurones were examined within lobule 1 of the vermis according to the terminology of Larsell (1972). Each cerebellum was sectioned in the mid-sagittal plane and 8-tam sections from the left side were examined within 100/~m of the plane of dissection. The most caudal field in lobule I shown by the x 63 objective was the first to be examined. A graduated line was superimposed upon the row of Purkinje neurones and a position in the line was randomly selected. The Purkinje neurone body nearest to that point was selected for examination by the x 100 objective if it could be safely identified as a Purkinje cell and had a visible nucleolus. Four cells were selected, one from each of four adjacent fields from each section; eight sections per rat brain were examined which yielded a total of 32 Purkinje neurones. The focus of the x 100 objective was adjusted to visualize the maximum amount of lipopigment in the cell body and the cell was then photographed as described above. The transparencies of the photographed cell bodies were then projected onto paper keeping the magnification constant, and, for each neurone, the outlines that delineated the projected images of discrete regions of yellow lipopigment autofluorescence were drawn. The areas enclosed by each of the discrete outlines were measured automatically for each cell with a Quantimet 720 Image Analyser (Cambridge Instruments Ltd., Cambridge, U.K. ) and were automatically classified into various size categories. Also, a standard stereological technique, involving the counting of test points and surface test line intersections (Underwood, 1970), was applied to the hippocampal neurones from the rats at 6 months and 18 months of age fed with a standard diet and at 18 months of age with a vitamin E-deficient diet.
243
Transparent plastic sheets bearing a 0.5-cm square test grid were randomly placed on the drawings of the outlines (boundaries) of the discrete regions of lipopigment. The number of intersections of the test lines with lipopigrnent boundaries ('cuts') were counted for each neurone section. The number of grid line intersections which were found to overlie the areas enclosed by lipopigrnent outlines ('hits') were also recorded. The distance between each test line (grid constant) was equivalent to 2.6 #m of cell dimension. The surface-to-volume (S/V) ratios for discrete regions of lipopigment, per brain, were calculated using the equation of Chalkley et al. (1949) i.e. (2 x 'cuts')/'hits' for discrete particles (Underwood, 1970) and the mean value was obtained for each group of rats. This measure reflects the mean volume of discrete regions of lipopigment, as a reduction in the mean lipopigrnent volume is accompanied by an increase in the mean surface-to-volume ratio. Statistical analyses were carried out with the SPSS/PC ÷ programme (Norusis, 1988). Comparisons between groups of rats at different ages in respect of the total number of discrete regions of lipopigrnent per rat and the total area of the outlines of discrete regions of lipopigment per rat were made with the Kruskal-Wallis oneway analysis of variance and multiple comparisons procedure (Siegel and Castellan, 1988) (Table I). F ratios were not calculated as Cochran's C-test showed significant differences in variances (P < 0.05) in respect of the group differences for the values for area (per rat) corresponding to lipopigment, both before and after square root or loge transformations of the data. Comparisons between groups of rats at different ages in respect of the mean
TABLE I The effect of ageing on the number of discrete regions of lipopigment and on the area enclosed by outlines of discrete regions of lipopigment autofluorescence in 32 hippocampal neurones for each rat. The numbers in parentheses indicate the range; n = number of rats. Age (months) 6 (n = 7)
12 (n = 5)
18 (n = 9)
25 (n = 5)
Kruskal-Wallis one-way analysis of variance
Median number (per rat) of discrete regions of lipopigmerit in 32 neurones
501 a (373-567)
710 b (640-873)
757 c (494-865)
758 a (583-905)
H = 11.13 P < 0.05 l
Median area (per rat) enclosed by lipopigment outlines in 32 neurones (t~m 2)
515 e (429-660)
1149 f (193-1210)
1729 g (920-2012)
2754 h (2280-2886)
H = 135.66 P < 0.0012
1'2In each case the hypothesis that all population medians are equal is rejected. Multiple comparisons procedure shows the following pairs of groups are significantly different at the 0.05 level: a < b, a < d , e < g , e < h.
244 n u m b e r (per rat) o f areas e n c l o s e d b y o u t l i n e s o f discrete r e g i o n s o f l i p o p i g m e n t , in v a r i o u s size categories, were m a d e with a o n e - w a y a n a l y s i s o f v a r i a n c e a n d the Scheff6 m u l t i p l e c o m p a r i s o n p r o c e d u r e ( T a b l e II). I n two size categories, s q u a r e r o o t t r a n s f o r m a t i o n s o f d a t a were r e q u i r e d to yield h o m o g e n e i t y o f v a r i a n c e as s h o w n b y C o c h r a n ' s C-test u s i n g the 0.05 level to i n d i c a t e differences. C o m p a r i s o n s b e t w e e n the v i t a m i n E-deficient g r o u p a n d the c o r r e s p o n d i n g c o n trol g r o u p ( T a b l e s III, IV) a n d c o m p a r i s o n s i n v o l v i n g the results in T a b l e VI, were m a d e with B o n f e r r o n i t-tests to allow for m u l t i p l e c o m p a r i s o n s ( G l a n t z , 1987); i.e. for x = the n u m b e r o f related t-tests, the significance level yielded b y the t-test n e e d ed to be 0.05/x to be c o n s i d e r e d s i g n i f i c a n t at the 0.05 level. T h e p o o l e d - v a r i a n c e t-test was used, u n l e s s a n F-test i n d i c a t e d s i g n i f i c a n t ( < 0.05) difference b e t w e e n the p o p u l a t i o n variances, w h e n the s e p a r a t e - v a r i a n c e t-test was s u b s t i t u t e d . Also, a t w o - w a y a n a l y s i s o f v a r i a n c e ( T a b l e V) was p e r f o r m e d for the n u m b e r o f discrete r e g i o n s o f l i p o p i g m e n t in h i p p o c a m p a l a n d P u r k i n j e n e u r o n e s in r e l a t i o n to two factors: firstly, six size categories o f discrete r e g i o n s o f l i p o p i g r n e n t a n d , s e c o n d -
TABLE II The mean number (per rat) of areas enclosed by outlines of discrete regions of lipopigment, in various size categories in a population of hippocampal neurones (32 neurones per rat) at various ages. Standard deviations are in parentheses; n = number of rats. Size categories of areas enclosed by lipopigment outlines (t~m2)
Age (months) 6 (n = 7)
12 (n = 5)
18 (n = 9)
25 (n = 5)
20- < 50
27.4 (21.8) 252.3 (71.8) 196.4a (55.5) 3.1 e (2.7) 0.4 i ( 1. I) 0
25.4 (15.5) 241.8 (79.5) 445.2 b (54.0) 11.2f (8.3) 2.0J (2.0) 0
50-<75
0
0
75- < 100
0
0
20.7 (23,8) 216.0 (97.0) 407.1 c (95.1) 38.6g (18.3) 15.7k (8.5) 2.9 (2.1) 0.1 (0.3) 0
5.0 (4.7) 135.8 (52.3) 464.8d (82.2) 61.0 h (6.5) 29.61 (3.6) 22.0 (4.9) 1.6 (1.7) 0.4 (0.9)
0- <0.5 0.5- < 1 1-<5 5 - < 10 10-<20
F ratio (One-way analysis of variance) 1.4 2.3 16,8 (P <_ 0.00009 I) 35.3 (P --- 0.000092)* 40.3 (P ~ 0.000093)*
|'2'3In each case the hypothesis that all population means are equal is rejected. In each case Scheff6 multiple comparison procedure shows the following pairs of mean values are significantly different at the 0.051evel;l:a < b,a < c,a < d ; 2 : e < g,e < h , f < g , f < h ; 3 : i < k,i < l,j < k,j < l,k < 1. P = probability of obtaining an F value at least as large as the one calculated when all population means are equal. *Square root transformation of data required to increase homogeneity of variances.
245 TABLE III The effect of vitamin E-deficient diet in rats at 18 months of age on the number of discrete regions of lipopigment and on the area enclosed by outlines of discrete regions of lipopigment in 32 hippocampal neurones and in 32 Purkinje neurones for each rat. Standard deviations are in parentheses; n = number of rats. Treatment Hippocampal neurones
Purkinje neurones
Vitamin E-deficient diet (n = 9)
Controls (standard diet) (n = 9)
Vitamin E-deficient diet (n=9)
Controls (standard diet) (n = 9)
Mean number (per rat) of discrete regions oflipopigment in 32 neurones
699 (128)
701 (160)
513 (63)
505 (53)
Mean area (per rat) enclosed by lipopigment outlines in 32 neurones (#m 2)
3069 (423)
1525 (446)*
1838 (311)
1206 (166)*
*Mean values between the two treatment groups are significantly different at the 0.05 level using the Bonferroni procedure to compensate for 4 tests on these data.
ly, a s t a n d a r d diet o r a v i t a m i n E-deficient diet. It should be n o t e d that the two largest size categories were excluded f r o m this analysis in view o f the relatively small n u m b e r s in these categories a n d the large n u m b e r o f zero values. C o c h r a n ' s C-test was used to indicate h o m o g e n e i t y o f variance, using the 0.05 level to indicate differences. F o r Purkinje neurones, all p a i r e d g r o u p s o f values for each size c a t e g o r y (i.e. c o r r e s p o n d i n g to v i t a m i n E -deficient diet a n d s t a n d a r d diet), showed h o m o g e n e i t y o f variance as defined above; h o w e v e r this was n o t the case for two size categories for h i p p o c a m p a l n e u r o n e s ( 0 - < 0 . 5 a n d 2 0 - < 50). A loge t r a n s f o r m a tion o f the d a t a for the h i p p o c a m p a l n e u r o n e s p r o d u c e d h o m o g e n e i t y in respect o f the 0 - < 0.5 c a t e g o r y b u t n o t the 2 0 - < 50 category. However, after t r a n s f o r m a t i o n , only one o f the six size categories ( 2 0 - < 50) v i o l a t e d the a s s u m p t i o n o f h o m o g e n e i t y o f variance a n d this is unlikely to invalidate the result, as the g r o u p sizes are equal (Witte, 1989). Size categories were established before the e x p e r i m e n t a n d were n o t influenced by the o u t c o m e o f the analysis.
Results The effect o f ageing o n the m e d i a n n u m b e r o f discrete regions o f l i p o p i g m e n t p e r rat a n d on the m e d i a n a r e a enclosed by l i p o p i g m e n t outlines p e r rat, in h i p p o c a m p a l
246 TABLE IV The mean number (per rat) o f areas enclosed by outlines of discrete regions of lipopigment in various size categories, in populations of hippocampal and Purkinje neurones (32 neurones per region per rat), in rats aged 18 months which had received a vitamin E -deficient diet and in control rats fed with a standard diet. Standard deviations are in parentheses; n = number of rats. Size categories of areas enclosed by lipopigment outlines (/zm 2)
0-<0.5 0.5-< 1 1- < 5 5 - < 10 10-<20 20- < 50 50- < 75 7 5 - < 100
Hippocampal neurones
Purkinje neurones
Vitamin E-deficient diet (n = 9)
Standard diet
Vitamin E-deficient diet (n = 9)
Standard diet
3.8 (2.7) 145.3 (89.2) 419.8 (51.0) 67.4 (15.5) 34.0 (7.0) 22.0 (4.5) 4.9 (2.3) 1.2 (2.2)
20.7 (23.8) 216.0 (97.0) 407.1 (95.1) 38.6* ( ! 8.3) 15.7" (8.5) 2.9* (2.1) 0. !* (0.3) 0
149.1 (38.3) 132.3 (21.4) 155.6 (31.5) 30.9 (7.8) 18.3 (5.2) 24.2 (5.2) 2.0 (1.3) 0.2 (0.4)
190.2 (32.9) 129.1 (20.8) 133.2 (20.2) 23.8 (6.2) 16.0 (4.2) 12.4" (2.8) 0.3* (0.5) 0
(n = 9)
(n = 9)
*Mean values between the two groups (vitamin E-deficient diet and standard diet) are significantly different at the 0.05 level using the Bonferroni procedure to compensate for 7 tests for each brain region.
neurones, is shown in Table I. Significant differences are found in respect to both median number per rat and median area per rat and the results suggest that the number of discrete regions show little increase above 12 months of age in contrast to the area. Therefore after 12 months the median area enclosed by a discrete lipopigment region gradually increases in size. Table II shows the effects of ageing on hippocampal neuronal lipopigment in relation to various size categories of areas enclosed by outlines of discrete regions of lipopigrnent. Analysis of variance shows that age-related increases in the numbers of discrete lipopigrnent regions are significant at the 0.05 level in three of the size categories. However, this is accompanied by age-related decreases in the two smallest categories (although these do not reach significance at the 0.05 level), which may explain why the total number of discrete regions of lipopigment per rat shows little change after 12 months of age (see Table I). The effects of the vitamin E-deficient diet and a standard diet on rats at 18 months of age are shown in Table III. While there is no significant difference in the mean number (per rat) of discrete regions of hippocampal or Purkinje neuronal lipopig-
247 TABLE V Two-way analysis of variance for the number of discrete regions of lipopigment in 32 hippocampal neurones and 32 Purkinje neurones for each of 18 rats in relation to two factors: (i) six size categories (columns) and (ii) a standard diet or a vitamin E-deficient diet (rows). Source of variance Hippocampal neurones* Between columns Between rows Interaction (rows and columns) Within groups Purkinje neurones Between columns Between rows Interaction (rows and columns) Within groups
Sum of squares
Degree of freedom
2 510 776 27 872 31 500
5 1 5
353 659
96
452 813 24 10 749
5 1 5
40 127
96
F ratio
Significance of F
136.4 7.6 18.1
_<0.0009 0.007 _<0.0009
216.7 0.1 5.1
_<0.0009 (0.81) _<0.0009
Note: 9 rats received a standard diet and 9 rats received a vitamin E-deficient diet. As there were six size categories, each of the two ANOVAs involved (9 × 2)6 = 108 values for the number of discrete regions of lipopigment. *Loge transformation of data required to increase homogeneity of variances.
m e n t b e t w e e n t h e t w o g r o u p s , v i t a m i n E d e f i c i e n c y is a s s o c i a t e d w i t h a s i g n i f i c a n t i n c r e a s e in m e a n a r e a ( p e r r a t ) e n c l o s e d b y o u t l i n e s o f d i s c r e t e r e g i o n s o f l i p o p i g m e n t in b o t h h i p p o c a m p a l a n d P u r k i n j e n e u r o n e s . T h i s s u g g e s t s t h a t v i t a m i n E defic i e n c y is a s s o c i a t e d w i t h a m e a n i n c r e a s e i n t h e size o f a d i s c r e t e r e g i o n o f lipopigrnent. Table IV examines the effect of vitamin E deficiency in relation to the v a r i o u s size c a t e g o r i e s o f a r e a s c o r r e s p o n d i n g t o d i s c r e t e r e g i o n s o f l i p o p i g r n e n t . I n
TABLE VI The effects of ageing and a vitamin E-deficient diet on morphometric properties of discrete regions of lipopigment in hippocampal neurones (32 neurones per rat) shown by a grid-counting method. Standard deviations are in parentheses; n = number of rats. Treatment
Age (months) Number of rats Mean surface-to-volume ratio for discrete regions of lipopigment (per rat)
Standard diet
Standard diet
Vitamin E-deficient diet
6 7 11.96 (1.88)
18 9 8.29 (0.99)
18 9 6.32 (0.49)
248
both neuronal populations, vitamin E deficiency is associated with a reduced number of pigment regions in the smallest size category and in the 0.5-< 1 category in hippocampal neurones (although these trends are not significant at the 0.05 level). However, in the remaining categories vitamin E deficiency is associated with an increased number of discrete pigment regions and six of the inter-group comparisons for the largest six size categories reach significance at the 0.05 level. Therefore, as with ageing in hippocampal neurones, vitamin E deficiency is associated with a decrease in the number of relatively small lipopigrnent regions and an increase in the number of relatively large lipopigment regions. The relationship between size category of discrete regions of lipopigment and vitamin E deficiency is further examined in table V, in which the two-way analysis of variance shows significant interactions between size category and vitamin E deficiency (i.e. P < 0.0009 for both hippocampal and Purkinje neurones). Thus the effect of size category on the numbers of discrete regions of lipopigment depends on whether the rats have received a vitamin E-deficient diet or a standard diet. The results of morphometric analysis by the grid counting method are shown in Table VI. These provide additional evidence (to that of Tables I and III) that ageing and vitamin E deficiency are associated with increases in the mean volume of discrete lipopigment regions. Bonferroni t-tests between values for rats at 6 and 18 months of age fed on the standard diet, and between rats at 18 months of age fed on the standard diet and those fed on the vitamin E-deficient diet, were significant at P < 0.05. Discussion
Previous comparisons have been made between ageing-related neuronal changes and those associated with a vitamin E-deficient diet; for example, both have been claimed to involve a reduction in rat cerebellar synaptic contact areas (BertoniFreddari et al., 1984), although differences in the distribution of increased lipopigment between ageing and vitamin E-deficiency have also been described (Katz et al., 1984). While the concept of vitamin E deficiency as accelerated ageing may, at best, be an over-simplification, the present study suggests that the two processes have similar effects on neuronal lipopigment, if it is assumed that the significant differences between the vitamin E-deficient and standard diet groups result from the lack of vitamin E. (This appears to be a reasonable hypothesis, although mechanisms other than direct cause and effect are possible.) The present results show that both ageing and vitamin E deficiency are associated with an increase in the mean area (per rat) enclosed by the outlines of discrete regions of lipopigrnent and that this change involves an increase in the numbers of relatively large discrete lipopigment regions, accompanied by a decrease in the number of relatively small discrete regions. Although the area within an outline of a region of yellow autofluorescence may appear filled with lipopigment, the autofluorescence may be derived from a number of lipopigment granules which can overlap when viewed under the microscope or be sufficiently close to appear fused. The present findings could reflect several processes (alone or in combination) involving changes in the density and size of lipopigment granules and changes in the rates of granule formation, of granule fusion, of granule
249 fragmentation and of extrusion of granule derivatives from the cell. (Although it has been claimed that lipopigment can be extruded from neurones, the degree of such a process is uncertain and it is not known whether any extruded lipopigment is derived from fragmentation of the larger or smaller granules.) One factor which may be relevant in vitamin E deficiency is an increase in the rate of the processes that occur in ageing, (i.e. degradation of cellular constituents such as membranes, mitochondria and lysosomes), leading to an increased rate of lipopigment formation. This would produce an increased density of lipopigment granules with increased contiguity and overlap; this may contribute to the reduction in the smaller lipopigment regions and the increase in the larger regions. Also, as the processing of lipopigment is believed to involve aggregation of lipopigment granules, accompanied by their increase in size by incorporation of cellular debris (Barden and Brizzee, 1987), an increased rate of lipopigment formation resulting from vitamin E deficiency may also be accompanied by increases in the rates of these processes, which may also contribute to the present results. Increased lipopigment formation would be expected to produce an increase in the mean total area enclosed by the lipopigment outlines, per rat, which was found in association with vitamin E deficiency (Table III). Therefore, a process of increased lipopigment formation, together with an increased rate of enlargement and aggregation of lipopigment granules, could explain the present findings in relation to vitamin E deficiency, and may reflect an increased rate of processes which occur in ageing. However, there are other explanations for the present findings; for example, vitamin E deficiency (and ageing) may reflect an impairment of the renewal processes of cellular constituents with a reduced rate of lipopigment formation. This hypothesis could agree with the present findings if this is associated with impairment of fragmentation of the relatively large lipopigment regions accompanied by a reduced rate of extrusion of the results of this process from the cell. The present study has demonstrated that a vitamin-E deficient diet can be associated with statistically significant changes (<0.05) in relation to the sizes of discrete regions of lipopigment in hippocampal and Purkinje neuronal populations in the rat. These changes are similar to those in hippocampal neurones related to ageing between 12 and 25 months. This suggests that the underlying processes initiated by vitamin E deficiency and ageing may share some common mechanisms.
Acknowledgement The authors thank Mrs A. Robson for the preparation of the manuscript.
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