Effect of prolonged physical training on the histochemically demonstrable catecholamines in the...

Journal of the Autonomic Nervous System, 10 (1984) 181-191 181 Elsevier

JAN 00340

Effect of prolonged physical training on the histochemically demonstrable catecholamines in the

sympathetic neurons, the adrenal gland and extra-adrenal catecholamine storing cells of the rat

Hannu Alho 1, Jari Koist inaho 1, Vuokko Kovanen 2, Harri Suominen 2 and Antt i Hervonen *

I Department of Biomedical Sciences, University of Tampere, Box 607, SF - 33101 Tampere and 2 Department of Health Sciences, University ofJyvi~skyl~ SF- 40100 Jyviiskylii (Finland)

(Received October 28th, 1983) (Revised version received February 7th, 1984)

(Accepted February 9th, 1984)

K e y words." physical training - sympathetic ganglia - catecholamine storing cells - heart innervation - microfluorimetry - aging

Abstract

The effect of daily physical training for 24 months on the sympathetic neurons, adrenal gland, extra-adrenal catecholamine storing cells and on the heart was investigated in rats. The tissue catecholamine fluorescence intensity was determined by microfluorimetric quantitation of catecholamines. The maximal and final body weights were significantly lower in trained animals. The trained rats showed promi- nent increase of heart weight relative to body weight, while the adrenals did not enlarge. The adrenergic nerve fiber density of the heart and the fluorescence intensity of the terminal axons were significantly increased. There were no changes in the fluorescence intensity of the perikarya of the sympathetic neurons and the amount of extra-adrenal catecholamine storing cells after physical exercise. The volume of the superior cervical ganglion was doubled and the neuronal perikarya were enlarged in trained animals. The prolonged physical training throughout the

* Present address: Laboratory of Neurosciences, Gerontology Research Center, Baltimore City Hospitals, Baltimore, MD 21224, U.S.A.

Correspondence: H. Alho, Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere, Finland.

0165-1838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

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life span of the rat gave new information about the reactions of the sympathetic nervous system to physical exercise.

Introduction

There have been several studies of the effects of physical training and stress on the heart and adrenal glands in laboratory animals [3,5,8,9,13,14,18,20,21,24,25,30]. In most studies, comparatively short training has been used, and only a few reports on training during several months are available [8,9,20,24].

Life-long daily exercise has been suggested to exert favourable effects on cardio- vascular functions. While the physiological parameters like blood pressure and heart rate can be determined with accuracy, the basic events taking place at the cellular level during prolonged training are unknown. Furthermore, the possible effect of increasing age on the final outcome of the exercise has not been considered in any of the previous studies. The present project was planned to describe the morphological, physiological and biochemical changes due to prolonged submaximal exercise in the locomotor, neuroendocrine and nervous systems. In this paper, the results concern- ing the sympathoadrenal system will be reported, and the other results will be published elsewhere.

Materials and methods

Initially 3-week-old male Wistar rats (24 rats, weighing about 50 g) were used. The animals were divided into two groups. The experimental group (12 rats) was trained to run on a treadmill with an electric fence at the end of the apparatus. The rest of the animals served as controls. The running program lasted for two years. The following training schedule was used: during the first week the experimental rats were trained to run on the apparatus for 10-40 min a day at a speed of 10-18 m/ra in . Within two weeks the training time and the speed of the treadmill were gradually increased to 60 min at the speed of 20m/min. After 2 months, the speed was increased to 22 m / m i n , and after 4 months to 25 m/min . During the last 5 months, the speed was decreased to 20 m/min . This training program was carried out for 5 days a week during two years. The animals had food and drinking water ad libitum. The rats were maintained in a standard laboratory facility with a constant l ight-dark cycle (dark 18.00-06.00 h), constant temperature (20 + 1 °C) and humid- ity (40 _+ 5%).

After the experimental period, the animals were anaesthetized with ether, and the adrenal glands, hypogastric ganglia (HGG), superior cervical ganglia (SCG) and the heart were prepared for study. The adrenals and the hearts were cleaned and weighed. To study the extra-adrenal chromaffin tissue, the abdominal para-aortic and interrenal connective tissues were prepared for analysis. The specimens were immersed in propane cooled with liquid nitrogen, and the formaldehyde-induced

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fluorescence (FIF) method for the histochemical demonstration of catecholamines [12] was used. This method combined with microfluorimetry has proved to be sufficiently sensitive for use in the quantification of monoamines both in noradrenergic nerve terminals [29] and in sympathetic ganglion cells [2].

The specimens were freeze-dried and then exposed to paraformaldehyde vapor for 60 min at 80°C, after which the specimens were embedded in paraffin under vacuum. The excitation light source was a stabilized HBO 100 mercury lamp (Osram). The lamp housing contained 2 mm KG1 and 4 mm BG 38 filters. The specimens were excited with the 405 nm HG-peak obtained through the filter set D (BG 3, KP 425, TK 455 and K 460) of the Ploemopak II epi-illuminator. The second barrier filter (Veril B60 band interference filter, Leitz) was set at 485 nm for catecholamines. For the measurement of the FIF intensity, Leitz MPV 2 microspec- trofluorimeter was used. The diameter of the circular measuring field for FIF intensity measurement was 7.5 #m in sympathetic ganglia, and 4.5 /~m in adrenal medullary (AM) cells and in the sympathetic nerves of the left auricle. The FIF intensity was measured starting at a random point from 90 individual nerve fibers of the auricle, and from 180 ceils of SCG, H G G and AM at 4 different levels from each animal. The quantitation of the FIF intensity of catecholamines was performed according to the method of Alho et al. [2]. The specimens of para-aortic and interrenal connective tissue were sectioned serially and the number of paraganglia was counted. Every tenth section of each ganglion was photographed through a fluorescence microscope. Each section (66 x magnification) was coded, and under "bl ind" conditions, employed to measure the minor and the major radius of the object in that section. From these measurements, the total ganglion volume was estimated as described by Konigsmark [19]. The cell sizes of adrenergic neurons were measured with a micrometric analysis from the fluorescence photographs. The diameters of the neurons with a visible nucleus were measured in two directions perpendicular to each other. The cell size was measured from 200 cells at 4 different levels (distance 100 ~tm) in the middle part of a ganglion. The density of the neurons was counted from fluorescence photographs. The number of neurons with visible nucleus were counted from standard area (10,000/~m 2) at 4 different levels in the middle part of a ganglion. The density of the sympathetic nerves of the left auricle of the heart was estimated from color slides from a standard area with constant magnification ( × 62.5). The slides were divided into 150 squares, and the number of fluorescent nerve fibers in each square was counted. The number of fibers was counted from 10-15 slides in each animal. All the data were analyzed statistically by using two-tailed Student's t-test.

Results

At the beginning of the experiment, some of the rats refused to run and tried to jump to the brim of the treadmill or lie on the electric fence. During the first 2 or 3 weeks the rats were weary after the training period, but during the next month the rats became less affected by the exercise. Three of the trained animals died during

184

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Fig. 2. Fluorescence photomicrograph of sections from left auricular tissue. Formaldehyde treatment is used to demonstrate adrenergic nerves. The green-fluorescent sympathetic nerve fibers and the varicosities are well seen. A: trained animals. B: control animals. Note that the fluorescence intensity and the density of the nerve fibers is increased in trained animals, x 120.

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T A B L E II

T H E E F F E C T O F P R O L O N G E D R U N N I N G O N T H E F I F I N T E N S I T Y IN S U P E R I O R C E R V I -

C A L G A N G L I O N (SCG) , H Y P O G A S T R I C G A N G L I O N ( t t G G ) , A D R E N A L M E D U L L A ( A M )

A N D S Y M P A T H E T I C N E R V E F I B E R S (SN) 1N T H E L E F T A U R I C L E

The data represent the mean + S.D. The fluorescence was measured f rom 18(1 cells a n d f rom 90 nerve fibers at 4 different levels in each specimen,

n Fluorescence intensity

S ( ' G H G G A M SN

Trained 5 74.8 _+ 7,8 40.3 +_ 4.1 523.8 :± 53.9 38.9 + 4.9

Controls 5 72.4 +_- 5.7 3 7 7 +_ 3.8 495.5 ~ 80.3 29.3 + 2,3 *

• P < 0.01, two-tailed Student's t-test.

the first 12 months, while all controls survived the first year, and during the last year 4 control and 2 trained animals died (Fig. 1). Seven experimental animals survived through the whole experiment, but 2 of them had to stop running 1 3 weeks before finishing the training program.

There were no differences between the trained rats and the control ones in the initial body weight (BW). The final BW was significantly lower (P < 0.01) and the maximal BW was very significantly lower ( P < 0.001 ) in the trained animals (Table l). There was no significant difference in the heart weight (HW), but the H W / B W ratio was significantly larger in the trained animals (Table l). The weight of the adrenal glands and also the ratio adrenal we ight /BW were not significantly changed after exercise.

The cytoplasmic fluorescence intensity (arbitrary mean values of sympathetic neurons in the superior cervical ganglion (SCG), in the hypogastric ganglion (HGG) and in the adrenal medullary (AM) cells is presented in Table II. There was no significant difference in the fluorescence intensity of AM, HGG and SCG between the two groups. The mean fluorescence intensity of sympathetic nerve fibers in the left auricle was increased from 29.3 + 2.3 in control animals to 38.9 _+ 4.9 (P < 0.01)

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Fig. 3. The effect of training on the heart weight (A), fluorescence intensity of nerve fibers in the left auricle (B), and nerve fiber density of left auricle (C>. White columns = controls (mean +_ S.D.): two-tailed Student's t-test; **P < 0.01+ ***P < 0.001.

187

T A B L E 1II

T H E E F F E C T O F P R O L O N G E D R U N N I N G ON A D R E N E R G I C C E L L SIZE, C E L L D E N S I T Y

A N D G A N G L I O N V O L U M E O F SCG

The data represent mean + S.D.; n = n u m b e r of ganglia.

n Cell size Cell density Ganglion volume (/~m) ce l l s / /~m 2 × 104 / tm 3 × 106

Trained 4 33.02 _+ 0.4 5.8 _+ 0.3 2011 + 241.5

Con t ro l s 4 31.6 _+ 0.6 * 6.3 + 0.3 1105 _+ 121.5 *

• P < 0.01, two-ta i led S tudent ' s t-test.

in the trained ones (Table II). In the left auricle, the prolonged running caused an increase of the density of fluorescent nerve fibers from 0.24 + 0.03 to 0.48 + 0.02 (P < 0.001) fibers per 1400/tm 2 (Fig. 3). This increase was also seen in photographs (Fig. 2). The effects of running on the heart are summarized in Fig. 3.

The superior cervical ganglion (SCG) was larger in the experimental animals than

Fig. 4. Fluorescence micrographs of representative sections of superior cervical ganglion. A: trained animals. B: control animals . × 65.

188

in the untrained ones. The volume of SCG was 1105 × 106 /tm 3 in the control animals, and 2011 × 106/~m 3 (P < 0.01) in the trained ones (Table III, Fig. 4). The cell size of the principal neurons in SCG was 33.2/xm in trained animals and 31.6 /~m in control animals (P<0 .01) (Table III). The cell density of sympathetic neurons in SCG was smaller in the trained animals (Table III).

In abdominal para-aortic and interrenal connective tissue the amount and the distribution of the paraganglia was not statistically changed. The number of small intensely fluorescent (SIF) cells of the SCG did not change in the trained animals.

Discussion

The present investigation demonstrated that prolonged physical exercise throughout the life span of the rat can elicit the enlargement of superior cervical ganglion and the increase of sympathetic innervation in the left auricle of the heart. The absolute heart weight did not increase but the heart weight/body weight ratio increased in trained animals as shown earlier [14,24,30]. The adrenal weight did not increase, which differed from earlier results [14,24,25].

Many investigations have been made on the effects of physiological training on the cardiovascular system and adrenal glands of laboratory rats [5,8,9,13,14, 18,20,21,24,25]. The results have varied depending on the training program used. The final and the maximal BW were lower in the trained animals, which is in accordance with earlier results [14,20,21,28,29]. Leon and Bloor [20] have found that intermittent training, also used by De Schryver et al. [9] (running at the speed of 12.5 m / m i n for 90 min three times a week) is not heavy enough to induce cardiac hypertrophy in rats. Running at the speed of 37 m/ra in for 80 rain [24] or swimming one hour daily 5 days per week for 3 months [21] has caused cardiac hypertrophy. The training used in this investigation (25 m/rain, one hour daily 5 days per week) did not increase the absolute heart weight, but the H W / B W ratio was significantly higher in trained animals. The H W / B W ratio may better reflect the capacity of the heart than the absolute heart weight.

The fluorescence intensity was increased in sympathetic nerve terminals in the left auricle after training. The increase of fluorescence is due to the increase of catecholamine contents in terminals [29]. De Schryver et al. [8,9] have reported significant decrease in biochemicaUy measured heart catecholamine concentration after comparatively light training. After swimming there was no reduction of cardiac noradrenaline concentration [21], and after prolonged running a tendency to an increase of the NA concentration [24]. The increase of CA concentration and nerve fiber density observed in the left auricle of the heart may be due to the increased and prolonged muscular work of the heart. It is possible that the moderate hypertrophy induced by prolonged physical training is accompanied by trophic stimulation of the growth and arborization of the terminal adrenergic network. The increase of NA concentration in axons may reflect adjustment to higher storage levels due to repeated bursts of stimulation. The increased density of the terminal adrenergic network may be functionally important. During normal aging, a gradual diminution

189

of the vascular innervation takes place [1,23], sympathetic ganglia lose fluorescent neurons [16,26], and the turnover slows down [28]. The increase of terminal arborization may compensate for these changes and maintain a proper stimulation level during the exercise.

Many investigations have shown that the catecholamine release of the adrenals increases during heavy physical training [3,5,13,18]. Ostman and Sj6strand [24,25] have shown that prolonged exercise (4 months) increases the adrenal weight and biochemically measured total adrenaline amount, while the catecholamine con- centration in adrenal glands remains unchanged. This is probably mainly due to an increase of the adrenal cortical tissue as a result of general adaptation to stress caused by training. In the present investigation, the prolonged running did not cause any changes either in the weight or in the CA concentration of the adrenals. It is likely that the prolonged training used here was not a stress to the rats, at least in the latter part of the experiment. The adrenomedullary cells, though stimulated during the exercise, were capable of synthetizing the required catecholamine without any permanent changes in their function. The kind of exercise used in this study does not seem to exceed the normal functional capacity of adrenal glands.

The extra-adrenal catecholamine storing cells (paraganglia and SIF cells) of the rat degenerate during the first postnatal weeks [7], but their amount increases again in aging animals [27]. The prolonged exercise in this study did not have any measurable effects on amounts or distribution of the extra-adrenal catecholamine storing cells.

It is known that cold stress can increase the NA concentration in the sympathetic ganglion [6]. In the present study, the CA concentration in the sympathetic ganglia was unchanged. This can be caused by an adaptation process, or the training program used was not hard enough to increase the preganglionic stimulus and the NA concentration.

One of the most surprising results was the growth of the SCG. The volume of the SCG was almost doubled and the size of principal neurons was increased in the trained animals. The enlargement of ganglion volume and of neuronal perikarya due to exercise is a new observation. As a phenomenon it can be compared with the effects of the nerve growth factor (NGF) on the cells of SCG [4,15,22]. The trophic effect cannot be due to the heart only, because several other organs are also innervated by the SCG. It is more probable that the prolonged and repeated stimulation itself causes this until now unknown expansion of the cell bodies, and that it is also accompanied by an increased density of the peripheral axonal net.

While the expansion of the cell bodies explains only partly the growth of the ganglia, there must also be an increase in the cell counts or in the inter-neuronal space. The ganglion size was increased, and the cell density was unchanged in the trained animals. So the total number of neurons must be larger in trained animals. In the sympathetic ganglia of the young rat, a few poorly differentiated neuroblasts can be found which still have mitotic capability [10,17]. In the normal Wistar rat, there is a decrease in the total number of the neurons of the SCG from 2 days until adulthood [15]. The major decrease in the number of these cells occurs between the 5th and the 30th postnatal days, and the normal loss of cells during this period is

190

approximately 30%. This loss can be prevented by adminis t ra t ion of nerve growth factor, and the total volume of SCH can be doubled [15,22]. The t raining program in

the present investigation began at the age of 3 weeks, and probably the physical exercise somehow prevented the normal loss of neurons during the maturat ion,

mediated probably by the N G F from the target organ. The effect of glucocorticoids on the sympathetic neurons might also explain the observed enlargement of SCG. It is likely that in the beginning of the experiment the t raining was a stress to the rats,

and the adrenals released glucocorticoids. In the newborn rat, t reatment with hydrocort isone increased the cellular volume and CA fluorescence in sympathetic

ganglia [11]. Probably the volume of the ganglion is also enlarged after corticoid treatment.

In conclusion, prolonged physical t ra ining lasting throughout the life span of the

rat gave new informat ion about the reactions of the sympathetic nervous system to physical exercise. The enlargement of superior cervical ganglia due to exercise is a

new observation, and, as a phenomenon , comparable to the effects of nerve growth factor a n d / o r hydrocortisone. There was no clear evidence that the exercise was a stress to the animals. The training had compensatory effects on the age-related changes of the cardiovascular system

Acknowledgements

The authors wish to thank Mrs. Tuovi Nyk~nen and Mrs. Bertta Salmi from the Univers i ty of Jyv~skyl~i for taking care of the daily runn ing program of the rats. This study was supported by the Emil Aal tonen Founda t ion , Finland.

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