Effects of aging and food restriction on the trigeminal ganglion: A morphometric study

Mechanisms of Ageing and Development, 65 (1992) 111- 125

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Elsevier Scientific Publishers Ireland Ltd.

EFFECTS OF AGING AND FOOD RESTRICTION ON THE TRIGEMINAL GANGLION: A M O R P H O M E T R I C STUDY

M.A. BIEDENBACH a, D.N. K A L U a and D.C. HERBERT b aDepartments of Physiology and bCellular/Structural Biology, University of Texas Health Science Center, San Antonio, Texas 78284 (USA) (Received November 30th, 1991)

SUMMARY

A quantitative morphometric study of the rat trigeminal ganglion was conducted to determine the changes that occur with aging. All measurements were tracked from young to old age in two rat groups simultaneously. One group was fed ad libitum, the other was maintained on restricted food intake from 6 weeks on. Immunocytochemical and radioimmunoassay techniques were used to study the neuron group that produces the peptide, C G R P and to compare it with the CGRP-negative neuron group. We observed that in the trigeminal ganglion, soma diameters and nucleus diameters of all neurons, whether C G R P positive or negative, increased modestly with age; so did total ganglion weight. Food restriction delayed, but did not prevent the increases in neuron diameters. No significant changes occurred as a function of age in the total number of neurons per ganglion, the ratio of CGRP positive to CGRP negative neurons and ganglion content of CGRP. Food restriction did not affect the parameters that remained constant with age. These findings are in contrast to the marked inhibitory effect of food restriction on age-related increase in thyroid calcitonin, a hormone that is encoded by the same gene as CGRP.

Key words: Trigeminal ganglion; CGRP; Neuron numbers; Neuron size; Fisher 344 male rat; Aging and food restriction INTRODUCTION

It is now well established that food restriction without malnutrition delays aging and prolongs the life span of rats. Although its mechanism of action is unknown, Correspondence to: D.N. Kalu, Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78284-7756, USA 0047-6374/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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food restriction prevents or delays the onset of a wide variety of age-related alterations in physiologic activities and disease processes [1-4]. One of the systems markedly influenced by food restriction is the calcitonin endocrine system [5,6]. Several investigators [9,10] have reported that thyroidal and circulating calcitonin levels increase progressively with aging in rats and we have demonstrated that this increase is drastically reduced by food restriction [5,6,9]. The calcitonin gene is of particular interest because it encodes the precursor RNA for the mRNAs for both calcitonin and calcitonin gene related peptide (CGRP) [10,11]. As a result of alternative post-transcriptional RNA processing, the mRNA containing the calcitonin sequence is expressed almost exclusively in the thyroid C-cells whereas the m R N A containing the sequence for CGRP is expressed almost exclusively in neural tissues [11]. Among the neural tissues with high concentration of CGRP is the trigeminal ganglion [10,11]. CGRP has also been located in all the dorsal root ganglia and in several sensory ganglia associated with the vagal nerve [121. From ganglion cell bodies CGRP is transported to the distal and central ends of their axons and at the central ends is assumed to act, like other neuropeptides, as transmitter or modulator of neural transmission. At distal ends where these sensory axons reach many target tissues in visceral organs, around blood vessels, in skin and muscle [12,13], CGRP may exert trophic or other effects. The preferential occurrence of CGRP in axons of smaller diameter and its frequent co-existence with substance P are thought to indicate important roles in nociception and other small-axon-associated functions, such as numerous visceral functions. In this study, the trigeminal ganglion was used as a model for studying the effects of diet and aging on a neuropeptide, CGRP, whose close genetic relative, calcitonin, is markedly influenced by aging and food restriction. Studies concerned with the effects of aging on neuron numbers in various brain regions often yield conflicting results; either a decline or no change in neuron numbers is reported as in a recent review [14]. The main problem is that neuron populations are vast and for practical purposes only small samples in restricted brain volumes can be studied. However, borders of specific neural structures are usually not clear at the microscopic level, so that accurate delimitation in different animals of a tissue volume in which neurons are to be enumerated, may be impossible. Hence in the literature neuron numbers tend to be reported as neuron density, i.e., the number of neurons per unit area in a histological section, without knowing whether the originally studied tissue volume remained constant, increased or decreased. Only a few aging studies on cerebral cortex were able to track at least one dimension of the 3-dimensional volume, namely the perpendicular distance from the pia mater to the subcortical white matter [15-17]. In the trigeminal ganglion however, a quantitative morphometric study with accurate enumeration of all neurons is possible. Right and left ganglia are located in the base of the skull on either side of the mid line in isolation from other neural

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horizontal plane with the microtome set at 16 ~m thickness. Individual ganglia yielded 82-95 sections. Rabbit anti-human C G R P antiserum that crossreacts 100% with rat C G R P was obtained from Peninsula Labs, California. The antiserum was diluted 1:1250 in a 0.01-M phosphate buffer (pH 7.4) containing saline and incubated with the tissue sections overnight at room temperature. The sections were then treated with the reagents in an ABC kit (Vector Laboratories) following the protocol supplied by Vector Laboratories which is similar to that published by Hsu et al. [18]. The staining reaction was visualized with the chromogen diaminobenzidine (DAB) using a solution containing 0.05% DAB and 0.01% H202. Specificity of the primary antiserum was determined from control studies as outlined by Sternberger [19]. No immunostaining was observed when the primary antiserum was either absorbed with its homologous antigen or replaced by normal rabbit serum. Hematoxylin and eosin served as counterstain for the CGRP-negative neurons. An age-related increase in ganglion wet weight became apparent at the end of the 24-month period, when the question arose, whether this was accompanied by an agerelated change in water content and dry weight. We obtained some ganglia from rats used for other studies and determined the percentage of dry weight per ganglion in 6 A and 6 R ganglia aged 6 months, in 6 A and 6 R ganglia aged 24 months and in 2 R ganglia aged 32 months. After dissecting out the ganglia, they were weighed immediately, transferred to a vacuum oven at 60°C, left overnight and weighed the next day several times at 4-h intervals to constant weight. The final weight was expressed as a percent of the wet weight.

Morphometric analysis The following procedures were carried out on each histologically processed ganglion. At low power (40x, w. 4x obj.) of the light microscope, the outline of the neuron-containing area was traced in every second of the serial sections and the total neuron-containing area per ganglion computed. For analysis of individual neurons, three sections per ganglion were selected from along the dorsoventral axis, at 25%, 50% and 75% depth; e.g., in a ganglion with 88 serial sections, all neurons containing nucleoli were traced and analyzed in sections 22, 44, 66. These three sections contained usually between 4 and 5% of a ganglion's total neuron containing area and for each neuron the circumferences of the soma and nucleus were traced. Tracing was done with the aid of a drawing tube (500x, with 50x oil objective) and a roll of paper with numbered squares. A square was slightly smaller than the microscope field and was superimposed on the microscope field through the drawing tube. This drawing arrangement permitted systematic covering of the whole section, square by square and helped to ensure that each neuron in the section was included, but not traced more than once. Tracings of C G R P positive cells were marked to permit separate analysis of C G R P positive neurons (brownish cytoplasm), and C G R P negative neurons (bluish cytoplasm). These cell tracings were entered into data files of a digital computer (Minc-11, Digital Equipment Corporation) by re-tracing them

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from the paper roll via a graphic digitizer (Summagraphics Bit Pad One) as we have done previously with other structures [20]. Each file contained the sample of one ganglion, approximately 2300-3700 neuron tracings. The analysis programs computed the total number of neurons per ganglion, % of CGRP positive and negative neurons, cross sectional area and diameter of each neuron and its nucleus and the circularity indices (or form factors) [20] of neurons and nuclei. To determine whether the neuron counts in Table I needed correction for double counting of split cells [21], a few hundred nucleoli and their nuclei were traced at 1000 magnification (50x oil immersion objective) in 6- and 24-months ganglia and their mean diameters computed. The correction formula [21] is N = nt/(t + d), where n = raw cell count, N = corrected cell count, d = diameter in micrometers of the structure actually counted, in our case the nucleoli, t = section thickness in micrometers. Generally, nucleolus diameter increased with nucleus diameter. Mean nucleolar diameter in 6-month-A rats was 3.0 ttm for CGRP negative cells and 2.7 #m for CGRP positive cells; in 24-month-A rats, it was 3.2 #m for CGRP negative cells and 3.0 #m for CGRP positive cells. At 24 months of age there was no significant difference in nucleolar diameter between A and R ganglia. Inserting the measured nucleolar diameters into the formula yielded respective correction factors of 0.84, 0.86, 0.83, 0.84, respectively. However, other studies indicate that any correction needed, is less than that obtained from the formula [21,22]. In rat pelvic ganglia [22] correction factors were determined in two ways, empirically and with the formula. The former yielded 0.88, but the formula 0.71. This would probably bring the "empirical correction factors in our study to 0.95-1.0; hence no correction was applied in Table I. Overall, all ganglia were processed and analyzed identically so that any observed changes are due solely to differences in age or diet.

CGRP radioimmunoassay The left and right trigeminal ganglia were dissected out, pooled and homogenized in 2 M acetic acid with a Brinkmann polytron homogenizer (Brinkmann Instruments, Westbury, N.Y.). The homogenate was boiled for 5 min, cooled and spun in a refrigerated centrifuge. The supernatant was aliquoted in 200 #1 samples and lyophilized and stored until required for analysis. For CGRP radioimmunoassay, the samples were reconstituted in 200 #1 2 M acetic acid. Unless boiled for 5 min as described above, the trigeminal extract would not easily resolubilize in 2 M acetic acid. Synthetic human CGRP standards or test samples at three different dilutions were incubated overnight in phosphate buffer, (pH 7.5), with rabbit anti human CGRP antiserum that crossreacts with rat CGRP. The antiserum (RB 2035) was kindly provided by Dr. Gkonos (VA American Lake, Tacoma, WA) and the final dilution used in the assay was 1:50000. Radioiodine-labeled human CGRP ([125I]hCGRP; 10 000 counts/min) was added with the first antibody and incubated overnight, following which IgGsorb was used to precipitate antibody bound [~25I]hCGRP. Immunoprecipitated radioactivity was counted in a Micromedic

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gamma counter for 3 min. Test samples and standards were corrected for nonspecific tracer binding determined from assay tubes that were treated similarly, except that no CGRP antibody was added.

Statistics Statistical analysis of data in Table I and Fig. 1 was done using analysis of variance. RESULTS

Wet weights and peptide content In Fig. 1 data from the proliferative thyroidal calcitonin-cells are compared with data from the presumed postmitotic trigeminal ganglion neurons. Thyroid gland weight and thyroid calcitonin (Figs. 1A,B) increased markedly with age, but such increases were almost absent in food restricted animals. Equivalent data from the trigeminal ganglion (Figs. 1C,D) show that trigeminal C G R P content, in contrast to the thyroid, did not change significantly with age, nor did food restriction have a consistent effect on its level. Ganglion weight did increase but to a lesser degree than

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Age (months) Fig. 1. Comparison of thyroid gland (A,B; modified from Fig. 2 of ref. 17, with permission) with the trigeminal ganglion (C,D) as a function of age and diet. (A), weight of thyroid gland; at 24 months the weight of R glands is 65% that of A glands. (B), calcitonin (CT) content of thyroid gland; at 24 months CT content in R glands is 23% that in A glands. (C), weight of trigeminal ganglion; at 24 months the weight of R ganglia is 92% that of A ganglia. (D), CGRP content of ganglia. Each data point: mean

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thyroid weight; mean wet weight of the A ganglia increased from 20.9 mg to 26 mg between 6 and 24 months of age, a 25% increase (in contrast to a 65% increase in thyroid weight). Food restriction kept ganglion weight slightly lower at each age point but between 6 and 24 months there was nevertheless a similar percentage

TABLE I EFFECTS OF A G I N G A N D F O O D RESTRICTION ON THE NUMBERS A N D SIZES OF TRIGEMINAL GANGLION NEURONS

Parameters

Diet

Age 6 months

12 months

24 months

68 153 ± 1345 64568 ± 3657

66 065 ± 3989 68 887 ± 4 0 9 3

71 078 ± 2380 67 822 ± 2018

Total number of neurons

A R

% of CGRP positive neurons

A R

25 ± 4.0 22 ± 1.0

23 ± 3.5 21 ± 1.0

27 ± 2 . 1 28 ± 0.5

Diameter (t~m) of CGRPpositive neurons

A % R

20.5 ± 0.35 100% 20.2 ± 0.40 100%

20.7 ± 0.35 101% 19.3 ± 0.10 96%

21.7 ± 0.32* 106% 21.4 ± 0* 106%

Diameter ~ m ) of CGRPnegative neurons

A % R

22.8 ± 0.29 100% 22.7 ± 0.50 100%

24.5 ± 0.1 108% 22.6 ± 0.1 99%

25.3 ± 0.2* 110% 24.2 ± 0.3* 107%

Diameter (#m) of CGRPpositive nuclei

A % R

10.1 ± 0 100% 10.1 ± 0.05 100%

10.2 ± 0.05 101% 9.5 ± 0.10 94%

10.8 ± 0.38* 107% 10.8 ± 0.05* 107%

Diameter (/~m) of CGRPnegative nuclei

A % R

10.8 ± 0.18 100% 10.9 ± 0.07 100%

11.2 ± 0.04 104% 10.6 ± 0.05 97%

12.0 ± 0.38* 111% 12.0 ± 0.15" I 10%

C.I. of CGRP-positive neurons

A R

0.89 ± 0 0.89 ± 0

0.89 ± 0.005 0.89 ± 0.005

0.89 ± 0.003 0.89 ± 0

C.I. of CGRP-positive nuclei

A R

0.93 ± 0.003 0.92 ± 0

0.92 ± 0 0.92 ± 0.005

0.92 ± 0.003 0.93 ± 0

All numbers represent mean value per ganglion ± S.E.M. N = 3; at each age 3 A and 3 R ganglia were analyzed. A = Ad lib±turn fed; R = food restricted; C.I. = circularity index or form factor. Effects of aging can be seen by reading from left to right, effects of food restriction by comparing A and R data. Aging changes in diameters are also shown in % with 6 month values = 100%. *Significant age related trend between 6 and 24 months, P < 0.025.

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increase in weight of the R as of the A ganglia. The percent dry weight of the ganglia remained constant in all age and dietary groups: 30.9°/,, and 31.1°/,, in 6- and 24month-old A rats and 31.4%, 31.4% and 30.5% in 6-, 24- and 32-month-old R rats, respectively. This finding indicates that the ratio of water to solid substances in the trigeminal ganglion remained constant during an age-related increase in the wet weight of the ganglia in both dietary groups. Neuron numbers and sizes Results of the morphometric analysis are shown in Table 1 as a function of age (6, 12, 24 months) and diet (A,R). Some preliminary results were reported previously [23,24]. The total number of neurons per ganglion did not change significantly with age or food restriction. Approximately 1/4 of neurons per ganglion were C G R P positive and this ratio of C G R P positive to C G R P negative neurons also remained constant as a function of age and diet. Mean soma diameter of C G R P positive neurons is smaller than that of C G R P negative ones in all age and diet groups. However, in both these neuron groups there was an age related increase in soma size which in A ganglia was detectable already at 12 months but in R ganglia only at 24 months. Between 6 and 24 months the mean soma diameter increased 10% (22.8 #m

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to 25.3 #m) for the CGRP negative neurons and 6% (20.5 #m to 21.7 #m) for the CGRP positive neurons. Converting these soma diameters into soma volumes (with the formula for a sphere, a reasonable approximation for Cl's of 0.89), adjusting for the ratio of CGRP positive to CGRP negative neurons and summing the total number of neurons/ganglion, yielded an age related increase in the summed neuron volume per ganglion of approximately 35%. Comparison with the total ganglion wet weight increase of 25% indicates that as function of age the neuron portion (1/3 of the ganglion at 6 months) undergoes a greater percentage increase than the axon portion. In both neuron groups, nucleus diameter showed with age a similar or slightly larger percent increase as soma diameter (Table I). Again, in the R ganglia, the increase in nucleus diameter was not yet detectable at 12 months, but by 24 months the percent increase and even the absolute value was as in the A ganglia. However, by 24 months the absolute values of soma diameter remained slightly smaller in R than in A ganglia, as if food restriction provided some protection against soma increase, but not against nucleus increase. Circularity indices (CI) did not change with age or diet. Neuron shape being rather consistently round to oval in CGRP positive and negative neurons, yielded Cl's of 0.89-0.90 in all groups. The Cl's of the nuclei were also constant with age and diet and at 0.92-0.93 somewhat more spherical than somas. 20 --

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Soma Diameter, p.m Fig. 3. Frequency distributions of soma diameters of the two main neuron groups, CGRP and nonCGRP. This figure is from data from a 6-month A ganglion, but is representative of all ages and of both diets. CGRP neurons, n = 790 = 100%; non-CGRP neurons, n = 2376 = 100%.

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Frequency distributions of soma and nucleus diameters The neuron populations represented by mean diameter in Table I, are displayed in more detail in Fig. 2 and as frequency distribution histograms in Figs. 3-5. A typical microscopic field is shown in Fig. 2. After scanning many microscopic sections of young and old ganglia, several impressions are obtained: neurons of all sizes, as well as CGRP positive and negative neurons, are intermingled and distributed throughout the ganglion. In any one ganglion, CGRP staining intensity varied among neurons, ranging from intense staining of the cytoplasm to various degrees of weaker staining. This pattern appeared similar in young, old, A and R ganglia. The investigator cannot tell which section is from old or young ganglia. The relatively small age related diameter increases could be detected only by careful measurements. Figure 3 shows the typical size distributions of the CGRP positive and CGRP negative neuron groups in any one ganglion. The CGRP positive neurons always have a narrower diameter range and a greater component of small neurons, than the CGRP negative neurons, but are not restricted solely to small neurons. CGRP occurs in some fairly large neurons. Only the small percentage of neurons > 40 #m never seems to contain CGRP. The age-related increases in the diameters of the CGRP positive and C G R P negative neurons are shown in Fig. 4. Typically, these diameter increases, were associated with little change in the range of diameters; by

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24 months the number of neurons in the small diameter bins simply decreased and those in the larger diameter bins, beyond the mean diameter, increased. The effect of age on frequency distributions of nucleus diameters is shown in Fig. 5 for C G R P positive (A) and C G R P negative (B) neurons. Like soma diameters, nucleus diameters of C G R P positive neurons tend to be smaller than those of C G R P negative neurons. Between 6 and 24 months of age, nucleus diameters of both neuron groups increased 7-11% in a manner similar to soma diameters. Plotting nucleus diameters versus soma diameters showed that nucleus size tended to increase with soma size, in all age and diet groups. However, in the smaller neurons, nucleus diameter constitutes a larger percent of soma diameter than in the larger neurons; approximately 60% in the smallest neurons, 38% in the largest neurons and 48-50% in neurons of mean diameter. In old A ganglia (where nucleus and soma diameters increased) these percentages were not significantly different from those in young A ganglia. However, in 24-month R ganglia (where soma diameter increases were somewhat less than in A ganglia) the percentages were slightly higher, e.g., mean values for A ganglia 49-509/0, versus 47-48% for A ganglia. These percentage increases, although not significant, were consistent and present in both C G R P positive and negative neurons.

122 DISCUSSION The small size of the trigeminal ganglion and its separate location from other neural structures made it possible to conduct this rigorous quantitative study as a function of age and diet. Within the age range investigated, neuron number per ganglion, ratio of CGRP positive to CGRP negative neurons and ganglion content of CGRP remained constant. Food restriction did not affect these parameters that remained constant with age. In contrast, in the thyroid where there are dramatic age related increases in gland weight, calcitonin content and C-cell proliferation, all these increases are almost completely prevented by food restriction [5,6]. Clearly, aging changes in the trigeminal ganglion do not mirror those in the thyroid gland. No neuron proliferation occurred and neurons that produce CGRP (encoded by the same gene as calcitonin [10,11]) act like other ganglion neurons, not like calcitonin producing thyroidal C-cells. A constant number of ganglion neurons with aging may not be surprising considering neurons are generally considered postmitotic. However, a quantitative study on lumbar dorsal root ganglia of Wistar rats reported a steady age-related increase in the number of ganglion neurons, although it was not determined in the study whether the new neurons were in fact due to neuron proliferation or derived from small undifferentiated cells in the ganglia [25]. Although neuron number and CGRP content remained constant, the data revealed that in the trigeminal ganglion several structural parameters (soma size, nucleus size, whole ganglion wet weight) increased with age. The comparatively small magnitudes of age-related diameter increases and of food restriction effects were not apparent on simply viewing microscope slides. The fact that all neuron sizes as well as peptide positive and negative neurons were intermingled may contribute to the difficulty in detecting small morphological changes. Possibly in the ganglia of still older rats, the observed diameter increases may progress and additional morphological changes may develop. For example, in a study on the visual cortex [16] no age-related changes in cortical thickness and neuron density were detectable in old A rats, but in much older rats, where life span had been extended by food restriction, decreases in cortical thickness and neuron density were observed. A survey of the literature shows that age-related structural changes in the nervous system do not occur uniformly throughout the brain [17,26-28]. Depending on the method of investigation, on the species, age and brain regions investigated, findings have ranged from no age-related change to age-related decline (in neuron number, size, dendritic extent, synapses) and, much less frequently, to age-related increase (of somas, dendritic trees, synapses) [16,17,27-32]. Whether age-related size increases are indeed infrequent or need careful quantitative measurement, remains to be seen. That aging changes are not uniform throughout the brain is not surprising, considering the regional differences in the biochemical properties of neurons, in their transmitter systems and in their hierarchical location within different neural circuits.

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The age related increase in soma and nucleus size of both CGRP positive and CGRP negative neurons indicates that the increase is independent of CGRP producing mechanisms, since the peptide content did not increase progressively with aging. Soma size increases have been observed in several other neural systems. In the olfactory bulb [29,30] a continuous expansion of mitral cell dendrites that receive synapses from olfactory axons, occurred between young and old age. This dendritesynapse enlargement was preceded (and probably triggered) by an increase in the numbers of the peripherally located olfactory epithelial cells which emit the olfactory axons. In old age, when some loss of mitral cells occurred, the dendrite expansion of the remaining cells became more pronounced. Such events are not likely to explain the size increase of trigeminal ganglion cells, since these cells do not receive synapses from peripheral axons. Expansions of soma, nucleus or dendrites have also been observed concomitant with loss of neighboring neurons and were considered compensatory responses [28,31]. Neural degenerative events are usually accompanied by an increase in size and number of glia cells, assumed to constitute a reactive gliosis [28,33]. Since in the trigeminal ganglion neuron number and peptide content remained constant, it is unlikely that the soma size increase was a compensatory response to neuron loss. It is, of course, possible that the soma size increase occurred in response to age related biochemical changes that were not yet accompanied by structural correlates. Finally, it has been reported that the gene for neurofilaments encodes neuron size and that an increase in the expression of this gene might lead to neuron (and axon) enlargement [34,35]. We do not yet know whether this mechanism could account for the size increase in the trigeminal ganglion and whether the initial increase is in the nucleus and triggers a proportionate increase in the soma (cytoplasm). Analysis of data at additional age points might reveal whether soma or nucleus increase occurs first. Calculations referred to in Results indicated that the size increase of the individual neurons could account for a large part of the whole ganglion weight increase, but not for all. Other elements in the ganglion may contribute to the weight increase, e.g., axons, Schwann cells, glia cells. Food restriction counteracted to a remarkable degree the age-related weight increase and cell proliferation in the thyroid, but in the trigeminal ganglion its effects were small. Although in the ganglion food restriction affected selectively elements that changed with age, the increase in nucleus size was not prevented, only delayed; by 24 months of age nucleus size in A and R ganglia was not significantly different. The increase in soma size was similarly delayed, but a small, beneficial effect remained at 24 months. The effect of food restriction on ganglion wet weight was similar to the effect on soma diameters. While food restriction markedly prolongs life span [4,36], comparison of thyroid and trigeminal ganglion data suggests that the effects of food restriction occur in proportion to the magnitude of the particular aging changes. Another interpretation is that different kinds of aging changes are differentially sensitive to the action of food restriction, e.g., cell proliferation in the thyroid

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being highly sensitive; soma size and whole weight increase in the trigeminal ganglion being mildly sensitive and still other changes being nearly insensitive [37]. ACKNOWLEDGEMENTS

This study was supported by NIA grant AG-01188. The animals for this study were obtained from the Core of the Program Project under the direction of Dr. B.P. Yu. Myra Garza Zapata and Martha Castilleja provided technical assistance with tissue processing. Dr. Alex McMahan provided assistance with statistical analysis. Dr. A.C. Brown (Oregon Health Sciences University) developed the computer programs used for the morphometric analysis. REFERENCES 1 G. Fernandes, E.J. Yunis, R.A. Good, Suppression of adenocarcinoma by the immunological consequences of caloric restriction. Nature, 263 (1976) 504-506. 2 E.J. Masoro, B.P. Yu, H.A. Bertrand and F.T. Lynd, Nutritional probe of the aging process. Fed. Proc., 39 (1980) 3178-3182. 3 R. Weindruch and R.L. Walford, Dietary restriction in mice beginning at 1 year of age: effect of lifespan and spontaneous cancer incidence. Science, 215 (1981) 1415-1418. 4 B.P. Yu, E.J. Masoro, I. Murata, H.A. Bertrand and F.T. Lynd, Lifespan study of SPF Fischer 344 male rats fed ad libitum or restricted diets: Longevity, growth, lean body mass and disease. J. GerontoL, 37 (1982) 130-141. 5 D.N. Kalu, R. Cockerham, B.P. Yu and B.A. Ross, A lifelong dietary modulation of calcitonin levels in rats. Endocrinology, 113 (1983) 2010-2016. 6 D.N. Kalu, D.C. Herbert, R.R. Hardin, B.P. Yu, G. Kaplan and J.W. Jacobs, Mechanisms of dietary modulation of calcitonin levels in Fischer rats. £ GerontoL (BioL Sci.), 43 (1988) B125-BI31. 7 T.C. Peng, C.W. Cooper and S.C. Garner, Thyroid and blood thyrocalcitonin concentrations and C-cell abundance in two strains of rats at different ages. Pro. Soc. Exp. BioL Meal, 153 (1976) 268. 8 B.A. Roos, C.W. Cooper, A.L. Frelinger and L.J. Deftos, Acute and chronic fluctuations of immunoreactive biologically active plasma calcitonin in the rat. Endocrinology, 103 (1978) 2180. 9 D.N. Kalu, E.I. Masoro, B.P. Yu, R.R. Hardin and B.W. Hollis, Modulation of age-related hyperparathyroidism and senile bone loss in Fischer rats by soy protein and food restriction. Endocrinology, 122 (1988) 1847-1855. 10 M.G. Rosenfeld, S.G. Amara and R.E. Evans, Alternative RNA processing: Determining neuronal phenotype. Science, 225 (1984) 1315-1320. 11 M.G. Rosenfeld, J.-J. Mermod, S.G. Amara, L.W. Swanson, P.E. Sawchenko, J. Rivier, W.W. Vale and R.M. Evans, Production of a novel neuropeptide encoded by the calcitonin gene via tissuespecific RNA processing. Nature, 304 (1983) 129-135. 12 G. Terenghi, J.M. Polak, J. Rodrigo, P.K. Mulderry and S.R. Bloom, Calcitonin gene-related peptide-immunoreactive nerves in the tongue, epiglottis and pharynx of the rat: occurrence, distribution and origin. Brain Res., 365 (1986) 1-14. 13 E.C. Goodman and L.L. lversen, Calcitonin gene-related peptide: novel neuropeptide. Life Sci., 38 (1986) 2169-2178. 14 A. Peters, Aging in monkey cerebral cortex. In A. Peters and E.G. Jones (eds.), Cerebral Cortex, Vol. 9, Plenum Press, New York, 1991, pp. 485-510. 15 G. Henderson, B.E. Tomlinson and P.H. Gibson, Cell counts in human cerebral cortex in normal adults throughout life using an image analysing computer. £ NeuroL Sci., 46 (1980) 113-136. 16 A. Peters, K.M. Harriman and C.D. West, The effect of increased longevity, produced by dietary

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