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Effects of nandrolone decanoate on respiratory
and peripheral muscles in male and female rats
ANJA BISSCHOP,1 GHISLAINE GAYAN-RAMIREZ,2 HÉLÈNE ROLLIER,2
P. N. RICHARD DEKHUIJZEN,2 RENÉ DOM,3 VERA DE BOCK,2 AND MARC DECRAMER2
lFaculty o f Physical Education and Physiotherapy , 2Respiratory Muscle Research Unit,
Laboratory o f Pneumology, and Respiratory Division , 3Department of Neuropathology,
Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Bisschop, Anja, G hislaine Gayan-Ramirez, Hélène
Rollier, P. N. R ichard D ekhuijzen, René Dom, Vera de
Bock, an d Marc D ecram er. Effects of nandrolone decano­
ate on respiratory and peripheral muscles in male and female
rats. J . Appi. Physiol. 82(4): 1112-1118, 1997.—Thirty male
and 18 female adult rats received weekly an intramuscular
injection of either saline (control; C), 1.5 mg/kg (low-dose; LD)
nandrolone decanoate or 7,5 mg/kg (high-dose; HD) nandro­
lone decanoate during 5 wk. Compared with respective C,
growth rate was stunted in male HD rats from 2 wk of
treatment on, whereas it was enhanced in female LD and HD
rats after 1 wk. Mass of all muscles studied varied proportion­
ally to body weight, except for the gastrocnemius (males:
0.49 ± 0.04 vs. C: 0.52 ± 0.03%, not significant; females;
0.17 ± 0.01 vs. C: 0.15 ± 0.01%, P < 0.05). In vitro contractile
and fatigue properties of the diaphragm remained un­
changed, except for a decrease in twitch kinetics (time to peak
tension: C, 21 ± 2; LD, 19 ± 1; FID, 19 ± 2 ms, P < 0.05;
half-relaxation time: C, 26 ± 5, LD, 25 ± 5, HD, 23 ± 3 ms,
P < 0.01). Histochemistry of the diaphragm and the gastroc­
nemius revealed a significant increase in type Ilx/b dimen­
sions. In the gastrocnemius, type I fiber dimensions also
increased. A pair-fed study, including another 24 female rats,
showed that the changes in oral food intake only partly
accounted for the observed anabolic effects.
anabolic steroids; contractile and fatigue properties; histo­
chemistry; diaphragm; gender differences; adult rats; oral
intake
might have more extensive effects on
the diaphragm compared with other skeletal muscles
because of its higher cytosol androgen-receptor density
(9) and its continuous cyclic contraction during respira­
tion. Previous studies using sedentary young rats
showed that anabolic steroids induced changes in mass
(22), histochemistry (2, 8), biochemistry, and capillary
density (9) of the diaphragm, whereas changes in
histochemistry have been disputed (22). Recently, the
effects of anabolic steroids on contractile and fatigue
properties of the diaphragm have also been examined
(22). Conversely, anabolic-androgen compounds may
have only limited effects in mature animals, as previ­
ous studies suggested (3, 14). Further study of this
issue in adult mammals is of particular interest to
patients with respiratory muscle dysfunction. Indeed,
because respiratory muscle weakness may be an impor­
tant cause of respiratory failure (27), anabolic steroid
therapy may improve diaphragmatic intrinsic strength
and endurance in patients with respiratory failure.
The present study was designed with the following
objectives: 1 ) to assess the effects of anabolic steroids on
ANABOLIC s t e r o i d s
1112
body weight, mass, and histochemistry of some respira­
tory and peripheral muscles and on in vitro contractile
and fatigue characteristics of the diaphragm in adult
postpubescent male and female rats; 2) to investigate
dose-specific effects and their interaction w ith gender;
and 3) to isolate the effects of anabolic steroids from
nutritional changes by studying pair-fed animals.
MATERIALS AND M ETHODS
Animals and Ti'eatment
Forty-two female (14 wk old, weighing 200-230 g) and 30
male (15 wk old, weighing 370-440 g) sedentary Wistar rats
were divided by gender and randomized into three groups to
receive weekly, during 5 wk, an intramuscular injection into
the left hindlimb of either 0.05 ml saline [control (C); 10
males, 14 females], 1.5 mg/kg nandrolone decanoate [low dose
(LD); 10 males, 22 females], or 7.5 mg/kg nandrolone decano­
ate [high dose (HD); 10 males, 6 females].
Nandrolone decanoate (Deca-Durabolin, 25 mg/ml; Orga­
non) was administered in an oil vehicle.
Nandrolone decanoate study. To 18 female rats (6 of each
group) and all the male rats (10 of each group), food and water
were given ad libitum. Animals were housed three per cage
and were weighed weekly. Diaphragm contractile properties
were examined in vitro, and samples of the diaphragm and
gastrocnemius were removed for histological and histochemical analyses after treatment.
Pair-fed study . The remaining 24 female W istar rats, 8
control and 16 low-dose-treated animals, were housed in
individual cages in a temperature-controlled room. Indi­
vidual food intake was measured every day at the same time,
as well as body weight. Control animals and eight low-dosetreated animals were given food and fluid ad libitum. The
other eight low-dose-treated animals [pair fed (PF)] each
received the same amount food as did controls. In addition, in
vitro diaphragm contractile properties were examined, and
samples of the diaphragm and gastrocnemius were removed
for histological and histochemical analyses after treatm ent.
Experimental Procedures
One week after the last injection, rats were anesthetized
with pentobarbital sodium (Nembutal; 55 mg/kg ip), tracheotomized, and mechanically ventilated (Harvard pump respira­
tor, South Natick, MA) with an 02-enriched gas mixture.
Contractile properties. The diaphragm was quickly re­
moved through a laparotomy and was immediately immersed
in a cooled, oxygenated Krebs solution [containing (in mM)
137 NaCl, 4 KC1, 2 CaCl2, 1 MgCl2, 1 KH2P 0 4, 12 NaHCOg,
6.5 glucose, and 0.03 d-tubocurarine chloride], A sm all rectan­
gular bundle was carefully dissected from the middle p a rt of
the lateral costal region of each hemidiaphragm, parallel to
the long axis of the fibers. Silk sutures were firmly tied to both
ends of the bundle to serve as anchoring points. Each bundle
0161-7567/97 $5.00 Copyright €> 1997 the American Physiological Society
ANABOLIC STEROIDS AND RAT DIAPHRAGM
was suspended in a water-jacketed tissue bath filled with
Krebs solution, maintained at 37°C, and continuously aer­
ated with 95% 0 2-5% C02. The central tendon side of the
bundle was anchored to a rigid support, whereas the rib
margin side was fastened to an isometric force transducer
(Maywood, Hampshire, UK) mounted to a vertical microme­
ter. Field stimulation was delivered via two large platinumstimulating electrodes placed along both sides of each bundle.
The bundles were placed at their optimal length (LQ),
defined as the length at which peak twitch force was obtained.
Stimulations were delivered through a Harvard 50-5016
stimulator (Edenbridge, Kent, UK), connected in series to a
power amplifier from power one model HS24-4.8. Pulse
duration of 0.2 ms at supramaximal voltage was used for all
stimulations. The force transducer output was amplified and
recorded on computer via analog-to-digital conversion (DT
2801-A) by using Labdat (Labdat/Anadat, RHT-InfoDat, Mon­
treal, PQ). Data analysis was performed by means of Anadat.
The following measurements were performed at L0, after a
thermoequilibration period of 15 min.
TWITCH c h a r a c t e r i s t i c s . The highest value of two succes­
sive 1-Hz stimulations was used to determine maximal twitch
tension (Pt) and its corresponding time to peak tension (TPT)
and half relaxation time (RTi/2).
MAXIMAL TETANIC TENSION (p0). Stimulation frequency was
set at 160 Hz to reach maximal force generation in rats at
37°C (24). The highest value of two successive stimulations
(train = 250 ms) was used for further analysis. In between
the two tetanic stimulations, a time interval of 2 min elapsed.
FORCE-FREQUENCY c u r v e . Each bundle was successively
stimulated (train = 250 ms) every 2 min at 25, 160, 50, 160,
80,160,120, and 160 Hz [modified after Reid and Miller (24)].
f a t i g u e p r o p e r t i e s . First, high-frequency fatigue was as­
sessed by measuring the decline in force output at 160 Hz
during the force-frequency curve. Second, low-frequency fa­
tigue was induced by a 5-min stimulation run consisting of
repeated 25-Hz stimulations of 330 ms, applied every second
[modified after Burke et al. (4)].
At the end of the in vitro experiment, length, thickness and
width of each muscle bundle were measured at L 0by means of
a caliper, and bundles were weighed after being blotted dry.
All tensions were normalized for cross-sectional area (CSÀ)
(5). Finally, the ratio of twitch to tetanic tension (Pfc/P0) was
calculated for each muscle bundle.
Muscle mass . The remaining diaphragm tissue, the para­
sternal intercostals (including sternum and chondral parts of
the ribs), the right medial scalene, and also the soleus and
gastrocnemius of the right hindlimb and the heart were
dissected, trimmed, blotted, and weighed. For the pair-fed
study, masses of the right extensor digitorum longus and
plantaris were also measured.
Histological and histochemical procedures. Material for
histochemical analysis was obtained from the same animals.
Adjacent parts of the diaphragm were used for determination
of contractile properties and for histological and histochemi­
cal analyses.
The right costal region of the diaphragm was placed at
excised length on tissue glue (Tissue-Tek, Elkhard, IN) over a
cork holder, with the fibers orientated perpendicular to the
surface of the cork. The preparations were frozen in is opentan e cooled with liquid N2. The same was done for a midbelly
section of gastrocnemius. Afterward, serial sections (10 pm),
parallel to the cork, were cut with a cryostat (—20°C). Two
sections of each muscle were taken for routine hematoxylin
and eosin staining, whereas the other serial sections were
stained for adenosinetriphosphatase (ATPase) after acid (pH
4.3 and 4.5) preincubation. A preliminary study showed that
1113
slides preincubated at this pH offered a clear separation of
the three fiber types. On the basis of their histochemical
reactions, fibers were identified as slow-twitch type I (lowmyosin ATPase, high oxidative capacity), fast-twitch type Ila
(high-myosin ATPase, high oxidative capacity), or fast-twitch
type Ilx/b (high-myosin ATPase, low oxidative capacity) (20).
It should be noted that this standard histochemical analysis
is not adapted to detect type IIx fibers, recently described in
the rat diaphragm (30). Therefore, we will refer to the type lib
fibers as type Ilx/b fibers.
Morphometrie examination was carried out with a Leitz
microscope (Wetzlar, Germany) at X25 magnification, con­
nected to a camera and digitizing board (model 2207, Numonics, Montgomeryville, PA). For each muscle, at least 250 CSAs
(determined from the number of pixels within the boundaries
of a delineated muscle fiber) and diameters (determined by
measuring the maximum diameter perpendicular to the long
axis of the cross-sectioned fiber) of individual muscle fibers
were calculated. However, because diaphragm was fixated at
excised length, diameters and CSAs were corrected for the
shortening occurring from L0i according to the formula of
Prakash et al. (21). Peripheral muscles were not corrected
because the L0 has generally been found to be between 100
and 120% of their resting length (5).
In the nandrolone decanoate study, besides 48 diaphragms,
consisting of 8 muscle preparations for each group of males
and females, also 58 gastrocnemii (males: C, 10; LD 9; HD 12;
and females: C, 10; LD, 11; HD, 6) were studied. In the
pair-fed study 22 diaphragm (C, 7; PF, 7; LD, 8) and 20
gastrocnemius samples (C, 7; PF, 5; LD, 8) were examined.
Statistical Analysis
Differences among, and interactions between, gender (malefemale) and dose (0-1.5-7.5 m g -k g '^w k “1 nandrolone de­
canoate) were assessed by using multifactorial analysis of
variance. Differences among means of combinations of levels
of the prim ary factors as drug (saline-nandrolone decanoate)
and low vs. high dose were determined by using CONTRAST
statements. Multivariate analysis of variance with repeated
measurements was used for assessment of differences as a
function of time or stimulation frequency Data derived from
two separate bundles from the same diaphragm were aver­
aged and subsequently analyzed as such. P < 0.05 was
accepted as statistically significant. In the text, Table 1, and
Figs. 1-5, values are presented as means ± SD.
RESULTS
N androlone Decanoate S tu d y
B ody a n d m uscle weights. Figure 1A shows that
starting body w eight was not different among treat­
m ent groups. In addition, treatment affected body
w eight change differently in males and females (genderdose interaction; P < 0.0001). It reduced w eight in
m ales (P < 0.01), whereas it enhanced growth in
fem ales (P < 0.0001). Males were heavier than fem ales
throughout th e entire experimental period (genderspecific effect; P < 0.0001). In male rats, low-dose and
control anim als showed a similar growth curve. In
contrast, the high-dose-treated animals showed stunted
growth from 2 w k of treatment on (P < 0.05), leading to
a final body w eigh t of 94% of both other groups (P <
0.01). In contrast, steroid-treated female rats re­
sponded after 1 w k of treatment by an enhanced growth
(P < 0.001; Fig. 1), reaching up to 120% of control at the
ANABOLIC STEROIDS AND RAT DIAPHRAGM
1114
end of therapy (P < 0.001). No differences appeared
betw een low- and high-dose-treated female animals.
Figure 1 5 presents th e body w eight change of all
groups at the end of treatm ent, illustrating th e stunted
growth in high-dose-treated m a les and the enhanced
growth in nandrolone-treated females.
B ecause final body w e ig h t w a s different among
groups, m asses of heart, respiratory, and peripheral
m uscles were expressed as a percentage of body weight
(Fig. 2). Of all exam ined m uscles, only the weight of the
gastrocnem ius w as affected differently in m ales [i.e., no
significant change (N3)] and fem ales (i.e., an increase;
P < 0.01) w ith nandrolone treatm en t (P < 0.01). The
scalenus m edius and gastrocnem ius were heavier (P <
0.0001) and the soleus and h eart were lighter (P <
0.0001) in m ales compared w ith fem ales. The weights
of the diaphragm and parasternal intercostals did not
differ between genders. Except for th e gastrocnemius, no
significant drug-specific effect w as observed in the
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Fig. 2 . Muscle masses, expressed as a percentage of body weight
(BW), of male CA; n = 30) and female { B \ n = 18) rats after 5 w k of
treatment with either saline (open bars) or nandrolone decanoate
(crosshatched bars). For the latter group, pooled values of low- and
high-dose-treated animals are presented. +Parastern, parasternal
intercostals including sternum and chondral parts o f the ribs;
gastrocn, gastrocnemius. *P < 0.05 compared with controls.
muscles investigated. Finally, no differences w ere seen
between low- and high-dose-treated animals.
-
Diaphragmatic contractile properties, t w i t c h
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FEMALES
Fig. 1. A: body w eights in m ale ( top\ n = 30) and female ( bottom ; n =
18) rats during treatm ent w ith saline ( • ) , 1.5 mg/kg nandrolone
decanoate (▼) or 7.5 mg/kg nandrolone decanoate (A). *High-dose
compared w ith 2 other groups, P < 0.001. # Controls compared with
treated animals, P < 0.001. Note th at whatever the treatment, males
were heavier than fem ales throughout treatment period (P < 0.001).
B : body w eight changes, expressed as percentage of initial, in male
and female rats after 5 wk of treatm ent w ith saline (open bars), 1.5
mg/kg nandrolone decanoate (hatched bars), or 7.5 mg/kg nandrolone
decanoate (solid bars). # P < 0,001 compared with both other groups.
*P < 0.0001 compared w ith controls.
charac­
Gender and dose interactions were not
significant. In m ales compared w ith females, P t (491 ±
114 vs. 294 ± 82 g/cm2;P < 0.0001), P Q(1,955 ± 387 vs.
1,664 ± 374 g/cm2; P < 0.05), and Pt/P 0 (0.249 ± 0.024
vs. 0.181 ± 0.019; P < 0.0001) were significantly
greater and RT^ w as significantly smaller (22 ± 2 vs.
30 ± 3 ms; P < 0.0001), whereas TPT was not different
between genders (20 ± 1 vs. 20 ± 3 ms; NS). D iaphragm
bundles contracted faster with nandrolone treatm en t
as shown by decreased TPT (C: 21 ± 2, LD: 19 ± 1, HD:
19 ± 2 ms; P < 0.05) and R T ^ (C: 26 ± 5, LD: 25 ± 5,
HD: 23 ± 3 ms; P < 0.01), whereas P t, P0, and P t/P0
showed no significant drug-specific effect. N on e of these
parameters showed significant differences b etw een lowand high-dose-treated animals.
f o r c e - f r e q u e n c y c u r v e . The effect of nandrolone on
the force-frequency curve was not different in m ales
and females. But the diaphragm of males responded
differently to increasing stimulus frequencies comt e r i s t i c s a n d p0.
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ANABOLIC STEROIDS AND RAT DIAPHRAGM
pared with females (Fig. 3; P < 0.0001, repeated
measurements). Diaphragmatic force generation, cor­
rected for CSA, was higher in males at low stimulation
frequencies (1 Hz: P < 0.0001, 25 Hz: P < 0,001, and 50
Hz: P <0.05; Fig. 3A). When expressed as a percentage
of the interposed 160-Hz stimulations (Fig. 3J3), the
force response in males was higher than in females at
all stimulation frequencies (P < 0.0001, except P < 0.01
for 120 Hz). Finally, nandrolone treatm ent did not
modify the diaphragm response to increasing stimulus
frequencies.
FATIGUE p r o p e r t i e s . During the force-frequency proto­
col, no gender and dose interaction was found for
160-Hz fatigue. But diaphragm strips of males fatigued
2.9 times more than did those of females (P < 0.0001).
Finally, nandrolone did not affect diaphragmatic 160-Hz
fatigability.
During the low-frequency fatigue run, no gender and
dose interaction was found. Diaphragm strips of both
genders fatigued to a similar degree, reaching, after 5
min of 25-Hz stimulations, 17.1 ± 4.2 and 20.7 ± 3.8%
of initial force in males and females, respectively.
Finally, nandrolone did not change diaphragmatic fa­
tigue induced by repeated 25-Hz stimulations.
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1115
Histology and histochemistry , Within one gender,
histological examination of routine hem atoxylin and
eosin-stained slides of the diaphragm and gastrocne­
m iu s showed no obvious differences between control
and treatm ent groups.
N o significant gender-dose interaction w as found for
proportions, CSAs, and diameters of types I, Ha, and
Ilx/b fibers of the diaphragm. As a consequence, sta tis­
tical analysis for drug-specific effects w as performed
gender independently. Fiber proportion w as greater for
type I (40 ± 4 vs. 37 ± 3%; P < 0.05) and w as not
significantly different for type Ha (34 ± 3 vs. 36 ± 4%;
N S) and Ilx/b fibers (27 ± 4 vs. 28 ± 5%; N S) in m ales
compared w ith females. Fiber proportion w as not influ­
enced by drug treatment. The CSAs of the three fiber
types were greater in males compared w ith females,
but this w a s only significant for type Ila (742 ± 71 vs.
623 ± 78 p m 2; P < 0.0001) and Ilx/b fibers (1,968 ± 191
vs. 1,534 ± 261 pm 2; P < 0.0001). Nandrolone treat­
m en t resulted in an increase of the CSA of type Ilx/b
fibers (C: 1,654 ± 370 vs. treated: 1,799 ± 280 pm 2; P <
0.05), w hereas that of type I (pooled values: 606 ± 84
p m 2) and type Ha fibers (pooled values: 682 ± 96 pm 2)
did not change significantly. The same gender- and
drug-specific effects were seen for the diameters of the
fiber types. Finally, the relative contribution of type
Ilx/b fibers to the total CSA of the diaphragm w as
greater in m ales compared with females (51.1 ± 4.8 vs.
48.5 ± 4.2%; P < 0.05) but did not change with
treatm ent. That of the other fiber types was not affected
by gender or dose.
In the gastrocnemius, no gender-dose interaction
w a s found for the proportions, CSAs, and diameters of
the three fiber types. The proportion of type Ila fibers
w as greater in m ales compared with fem ales (27 ± 6 vs,
21 ± 6%; P < 0.05), whereas that of type I (pooled
values: 36 ± 9%) and Ilx/b fibers (pooled values: 40 ±
9%) did not differ significantly. All fiber types were
greater in m ales compared with females (I: 2,723 ± 453
vs 2,340 ± 301 pm2, Ha: 2,617 ± 322 vs. 1,991 ± 186
p m 2, Ilx/b: 2,949 ± 376 vs. 2,207 ± 273 pm2; P <
0.0001). A s in the diaphragm, nandrolone did not
change fiber proportions but increased type Ilx/b dim en­
sions (C: 2,456 ± 566 vs. treated: 2,681 ± 446 pm2; P <
0.05). In addition, type I fiber CSA increased w ith
low-dose treatm ent in males (C: 2,738 ± 475 and HD:
2,523 ± 312 vs. LD: 3,004 ± 498 pm2, P < 0.05) as w ell
as in fem ales (C: 2,174 ± 282 vs. LD and HD pooled:
2,437 ± 2 7 5 p m 2, P < 0.05).
Pair-Fed S tu d y
40 #
Food intake . Before treatment, the food intake was
20 -i
0
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25
50
80
120
FREQUENCY [Hz)
P ig . 3. Force-frequency curve of diaphragm in maie (O; n = 29) and
fem a le ( • ; n ~ 18) rats. Diaphragm force is expressed in g/cm 2 (A)
a n d as percentage of 160-Hz stimulations [maximal tetanic tension
( P 0); B]. Dashed line, pooled SD. *P < 0.0001. #P < 0.001. @P < 0.01.
sim ilar in the three groups, averaging 113 ± 18 g/wk.
D uring treatm ent, food intake in the control group w as
stable and averaged 122 ± 37 g/wk. The food intake of
the pair-fed group followed the same course as th a t of
controls, averaging 112 ± 12 g/wk. By contrast, already
during the first week of treatment, food intake w as
significantly increased in the treated animals fed ad
libitum (133 ± 12 g) compared with both other groups
(P < 0.001). This increase further increased to 144 ± 11
ANABOLIC STEROIDS AND RAT DIAPHRAGM
1116
1.00
Similarly, no changes appeared in the force-fre­
quency curve in the three groups (Fig. 5) or in the
fatigability
of
the
diaphragm
during
the
low-frequency
0.80 fatigue run.
£
Histology and histochemistry. For both muscles stud­
CQ
ied, the muscular pattern seen on hematoxylin- and
« 0.60 eosin-stained slides was similar in pair-fed and treated
X
animals
fed
ad
libitum
and
was
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that
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UJ
X
ATPase staining revealed no significant changes in
0.20
the fiber type proportions in respect to treatm ent in the
#
muscles studied. Pooled values of all groups together
m
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were
39
±
5,
31
±
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and
30
±
4%
for
types
I,
Ha,
and
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Ilx/b,
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for
the
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the
gastroc­
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nemius,
these
values
were
41
±
4,
18
±
4
and
41
±
5%
«
C
/3
O)
for types I, Ila, and Ilx/b, respectively.
On
the
other
hand,
diaphragmatic
type
Ilx/b
fiber
Fig. 4. Muscle masses, expressed as percentage of BW, in female rats
{it = 24) after 5 wk of treatment with either saline (open bars) or dimensions increased in both treated groups, whatever
nandrolone decanoate (crosshatched bars). Latter group was pair fed.
their nutritional intake. Nevertheless, this increase
Ext.digi, extensor digitorum longns. *P < 0.05. # P < 0 .0 1 .
was only significant in treated animals fed ad libitum
(23% of control; P < 0.05) and not in pair-fed animals
g during the second week of treatment, and remained (14%; NS). Finally, diaphragm type I and Ila dimen­
sions
were
similar
in
the
three
groups,
being
520
±
49
stable afterward. Significant differences between
treated animals fed ad libitum and both other groups and 554 ± 71 pm2, respectively (pooled values).
In
the
gastrocnemius,
nandrolone
also
resulted
in
(with no differences between the latter groups) were
observed during the entire treatm ent period (P < 0.001 increased type Ilx/b fiber dimensions, reaching statisti­
cal significance in animals fed ad libitum only (18% of
to < 0.0001).
control,
P
<
0.01;
vs.
PF:
9%
of
control,
NS).
In
addition,
Body and muscle weights. Starting body weight was
similar among the three groups, averaging 208 ± 14 g. type I fiber hypertrophy was seen in the low-dose group
only
(20%
of
control;
P
<
0.01),
whereas
type
Ha
One week after the first injection, treated animals fed
ad libitum were significantly heavier than control and dimension was unchanged (pooled values: 1,948 ± 170
pair-fed groups (LD: 230 ± 16 vs. C: 209 ± 16 and PF: pm2).
208 ± 11 g; P < 0.01). This enhanced growth further DISCUSSION
increased, leading to a final body weight of 273 ± 19 g
The present data demonstrate that in healthy seden­
in low-dose-treated group vs. 223 ± 18 and 231 ± 10 g
in control and pair-fed groups, respectively (P < 0.0001). tary adult rats, 5 wk of treatm ent with 1.5 and 7.5
Except for soleus and heart weights, all muscles of mg *kg-1 *wk"“1nandrolone decanoate affected body and
pair-fed group represented a greater proportion of body muscle weights (especially gastrocnemius) positively in
weight than did controls, reaching statistical signifi­ female treated rats and negatively or not at all in male
cance in the plantaris (P < 0.05) and the extensor treated rats. In vitro contractile and fatigue characterdigitorum longus only (P < 0.01; Fig. 4). No differences
appeared between both treated groups, whatever their
100 nutritional intake.
Diaphragmatic contractile properties No statistical
80 differences in Pt and its kinetics or in P0 were found
among the three groups (Table 1), However, nandrolone
Q_
tended to decrease TPT and RT1/2) whatever the nutri­
60 «
tional intake of the animals (PF: -11 and “ 14% of C;
LU
LD: -1 5 and -15% of C, respectively; P = 0.06 vs. C).
-
-
tA
I/ ?
I
I-
L-
JZ
w
Cl
.
Table 1. Diaphragm contractile properties in C} PF}
and LD female rats
C
Pt, g/cm2
TPT, ms
RTV¿, m s
P0, g/cm 2
505 ± 5 9
20 ± 2
24 ± 4
2,235 ± 234
PF
542 ±114
19 ± 2
21 ± 2
2,369 ±365
O
cc
O
40 -
LL.
20 -
LD
419 ± 7 4
17 ± 3
20 ± 3
2,004 ± 3 9 4
Values are means ± SD. C, control; PF, pair fed; LD, low-dose
anabolic steroid treated; Pt, twitch tension; TPT, time to peak
tension; RTi/2, half relaxation time; P0) tetanic tension.
1
25
nr
~T"
50
80
120
FREQUENCY (Hz)
Fig. 5. Force-frequency curve of diaphragm in female rats. #,
Control; V, pair fed; ▼, low-dose anabolic steroid treated. Diaphragm
force is expressed as percentage of 160-Hz stimulations (P0). Dashed
line, pooled SD.
ANABOLIC STEROIDS AND RAT DIAPHRAGM
istics of rat diaphragm remained unchanged, except for
decreased twitch kinetics, because of selective type
Ilx/b fiber hypertrophy. In the gastrocnemius, type I
fiber CSA and diameter were also increased with
nandrolone treatment. Finally, although control of nu­
tritional intake is likely to be needed in anabolic steroid
studies, a pair-fed study showed th at changes in oral
food intake only partly explained the observed anabolic
changes.
It should generally be emphasized th at comparison of
results in literature is rather difficult because anabolic
effects are influenced by a series of conditions, such as
the studied species (2, 3), age (3, 14), circulating
synergistic hormones (7), muscles (3), androgen (19),
dose (2, 15, 19, 29), mode of adm inistration (17),
duration of treatment (22, 26), activity level (12, 15),
and diet (11). Indeed, these factors explain to a large
extent the inconsistencies in the literature.
In the present study, in keeping with other studies
performed on young animals (2, 19, 29), no linear
relationship within one gender between dose and effect
was found. Consequently, injection of a five times
higher dose did not produce an increased effect. Except
for the dose-dependent changes in body weight, no
systematic differences appeared in the present study
between low- and high-dose-treated animals.
In our study in adult rats, body weight increased in
females, whereas it was not affected or decreased in
males. This is similar to what has been reported in
young rats in which body weight was increased (7, 8,
19, 22) or unchanged (14,19) in females, whereas it was
not affected (7, 14, 22, 26) or stunted (14, 16) in males,
depending on the dose used. The exact mechanisms for
this gender-dependent anabolic effect is not known and
is beyond the aim of the present investigation. The
intrinsic difference in the basal level of circulating
endogenic androgens may play a role because it is
higher in sexually normal male animals compared with
females and seems to result in a smaller number of
androgen-binding sites on the muscle cytosol (6). Fur­
thermore, surpassing the physiological level of andro­
gens in males has been reported to result in 1 ) depres­
sion of the natural production of testosterone (25), 2)
downregulation of androgen-binding receptors (23), 3)
decrease in appetite (16), and 4) a metabolic conversion
to excess estradiol (15). Indeed, all these factors may
inhibit body growth. Nevertheless, the present study
does not allow us to determine the relative significance
o f these mechanisms.
Our pair-fed study illustrates th at female rats in­
creased their food intake with nandrolone treatm ent,
leading to enhanced body growth. This is in keeping
■with studies performed in prepubescent rats in which
food consumption varied with changes in body weight
(13, 16). Furthermore, in the present study no signifi­
c a n t differences in muscle weights and in diaphrag­
m a tic contractile and histochemical characteristics were
fo u n d between treated animals fed ad libitum and
pair-fed animals. These data illustrate th at the in­
creased ingestion of food alone was not fully responsible
f o r the observed anabolic effects.
1117
In prepubescent rats, muscle weights changed in
proportion to body weight (8, 19, 22). In the present
study, only the weight of the gastrocnemius failed to do
so, and, moreover, it changed gender dependently. A
graded response of different muscles to androgen admin­
istration was previously reported as being the result of
differences in the level of androgen-binding receptors
within the muscle cytosol (3, 6). Different anabolic
effects in males compared with females were observed
in young animals (7,10,22). Nevertheless, to the best of
our knowledge, a greater effect of anabolic agents on
gastrocnemius has not yet been described in rats.
In the present study, of all the contractile properties
measured, nandrolone treatment solely altered dia­
phragmatic twitch kinetics toward a fast-twitch profile,
being consistent with the observed type Ilx/b fiber
hypertrophy. In young female rats, but not in young
males, Prezant et al. (22) also found a decreased RT i/2
after testosterone treatm ent (62.5 mg*kg~1‘wk“1) but
with no concomitant changes in diaphragmatic mor­
phometry. This suggests additional mechanisms be­
sides changes in histochemistry induced by anabolic
agents, such as modification of neural and neuromuscu­
lar function (including neurotransmitter levels) (1)
and/or upregulation of the Ca2+-ATPase pump (26).
Moreover, Prezant et al. also reported enhanced dia­
phragmatic force in young female rats during short­
term (2.5-wk) testosterone treatm ent but not if tre a t­
ment was extended to 10 wk. Downregulation of muscle
cytosol androgen receptors probably explains the ob­
served discrepancies with different treatm ent dura­
tions (22). Other studies with young or adult rats
confirm in peripheral muscles our results in the dia­
phragm (8, 28).
Only few studies investigated morphological adapta­
tions of diaphragm to anabolic agents. Prezant et al.
(22) found no histochemical changes in the diaphragm
compared with pair-fed control male and female ani­
mals. In contrast, Egginton (8) reported increased type
Ha fiber proportion and dimensions compared with
female controls. In our study, a selective type Ilx/b fiber
hypertrophy was present compared with controls but
not compared with pair-fed animals. Thus it seems that
differences in morphological adaptations were observed
only when comparison was made against controls and
not against pair-fed animals.
To the best of our knowledge, histochemical alter­
ations of the gastrocnemius to anabolic steroids have
previously not been reported.
Gender-related differences in fiber composition and
morphometry, as well as in some enzymological charac­
teristics, may explain differences in contractile proper­
ties and fatigue resistance of the diaphragm between
male and female rats. Indeed, the dimensions of all
diaphragmatic fiber types, the proportion of type I
fibers, as well as the relative contribution of type Ilx/b
fibers to total CSA of the diaphragm were greater in
males compared with females. The reduced Pt3 P0, and
PL/P0 in diaphragms of female rats may additionally be
related to a greater amount of connective tissue and,
therefore, a greater series elastic component.
1118
ANABOLIC STEROIDS AND RAT DIAPHRAGM
Although the present study was performed in seden­
tary rats, our results suggest that treatment with high
therapeutic (1.5 m g-kg^-w k’ 1) or supratherapeutic
doses (7.5 m g-kg^-w k“1) of nandrolone may be of
benefit in patients (especially females) being in a
chronic state of catabolism either combined with exer­
cise reconditioning or not. Indeed, anabolic androgens
may not only act directly on the androgen receptors but
also indirectly through inhibition of the catabolic and
antianabolic action of endogeneus glucocorticoids (7,
26). Anabolic agents also may further enhance the
myotrophic effect of exercise (12), reduce fatigability,
and lead to a faster recovery as experienced by athletes,
being likely to relate to anabolic effects on the central
nervous system (18). This is supported by our findings
because nandrolone decanoate treatment did not change
fatigue resistance of the diaphragm. Whether such
effects are present in patients needs to be examined.
Finally, the administration of intermittent short bursts
rather than continuous doses of anabolic agents may be
more beneficial for muscles in terms of enhanced
muscle contractility, thereby avoiding possible down­
regulation of muscle cytosol androgen receptors.
In conclusion, this study shows that nandrolone
decanoate when given during 5 wk at high therapeutic
or supratherapeutic doses affects males and females in
a similar way except for body weight, which is en­
hanced in females and stunted in males. Diaphrag­
matic contractility and fatigability were unaltered,
except for twitch kinetics. The latter changed toward a
fast-twitch profile, consistent with a type Ilx/b hypertro­
phy Gastrocnemius weight changed disproportionally
to body weight and showed a supplemental type I fiber
hypertrophy. Finally, the anabolic steroid-induced
changes in food ingestion were only partly responsible
for the observed anabolic effects.
The authors thank N. PI ai sanee-Buts for expert technical assis­
tance and Organon Belgium for supplying us with nandrolone
decanoate,
This work was supported by a grant from the Fonds voor Ge­
neeskundig Wetenschappelijk Onderzoek.
Address for reprint requests: M. Decramer, Professor of Medicine
Respiratory Div., Univ. Hospital, Kerestraat 49, B-3000 Leuven,
Belgium.
Received 7 June 1994; accepted in final form 12 November 1996.
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