Egypt. J. Exp. Biol. (Zool.), 8(2): 273 – 285 (2012)
© The Egyptian Soc iet y of Experi m ent al Biology
RESEARCH ARTICLE
N a bila I . E l- D e so uk i*
Am a l I. E l- R e f aiy* *
D a lia F. A. Ab o u- Za id*
Am ir a A. Abd e l - K a de r*
H IST O L O GIC AL , H IST O C H E MIC AL , AN D I M MU N O H IST OC H E M IC AL S T U DIES OF
T H E C AR D IAC MU SCL E O F T H E AL B IN O R AT U N D ER IM MO B IL IZ AT IO N
ST RESS AND T HE C UR AT IVE R OL E OF D IAZ EP AM
ABSTRACT:
The present study is planned to study the effect
of immobilizati on stress on the cardiac muscle
of adult male albino rats and the curati ve role of
diazepam. The study was carried out on 80
albino rats; the animals were divided into eight
groups: group 1 served as control rats; group 2
unstressed rats injected intraperitoneally daily
with 0.1 mg/kg b.w. diazepam for 30 days;
groups 3, 4, and 5 served as immobilized
stressed-rats for 30 days (by restricti ng
movement for 2 hr daily at different durations
5, 15, and 30 days, respectively); groups 6, 7,
and 8 stressed rats treated daily with 0.1 mg/kg
b.w. diazepam for 30 days. The histological
study of the cardiac myofi bres of the stressedrats revealed disorganizati on of the muscle
fi bres, vacuolation of the sarcoplasm, pyknosis
of the nuclei, congestion and dilatation of blood
vessels in endomysium. The thickness of
collagen fibres gradually increased and became
compact dense in the stressed rats till 30 days,
and they were more obvious around the blood
vessels. The histochemical study demonstrated
a marked reduction in the glycogen and protein
contents of the cardiac muscle, and was timedependent. The immunohistochemical study
revealed that the rats stressed for different
durations manif ested the disappearance of the
cytoskeletal desmin protein filaments at
intercalated discs and Z-lines of the cardiac
myofi bres. Treatment wit h diazepam for 30
days to stressed rats demonstrated markedly
improvements of the architecture of cardiac
myofi bres and restoration of the two main
chemical components; glycogen and protein
contents. Also, restoration of desmin in the
cardiac myofibres was elucidated. The results
indicated that diazepam is recommended to be
used as a curative drug to improve the
disturbances in the cardiomyocytes and
myofi brils assembly caused under the effect of
stress.
KEY WORDS :
Cardiac muscle, Desmin, Diazepam, Histochemistry,
Histology, Immunohistochemistry, Rat, Stress
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CORRE SPONDE NCE:
Nabil a Ibrahim El-Desouki
Departm ent of Zoology, Faculty of Science,
Tanta University, Egypt.
E-m ail: nabil adesoky@ yahoo.com
Am al I. E l- R ef a iy* *
D al ia F. A. Ab o u- Z a id*
Am ir a A. Abd e l - K ad er *
*Dept. of Zoology, Faculty of Science, Tanta
Uni versity
**Dept.
of
Biol ogical
&
Environm ental
Science, Faculty of H om e Economic, Al-Azhar
Uni v.
ART ICLE CODE: 35.01.12
INTRODU CTION:
Stress is an aversi ve stimulus, which
perturbs the physiologi cal homeostasis, and
its impact is refl ect ed on a vari ety of
bi ologi cal systems. According to t he t asks in
the li fe, peopl e can see t he degree of stress,
more tasks-more stress. Stress responses
can be categori zed as specific or nonspecifi c.
Specific str essors are typicall y short-t erm
such as a sudden increase i n environmental
temperature. Anim als typicall y react to
specif ic str essors by tr ying to combat the
stressor.
How ever,
in
Long-term
or
nonspecific str ess, animal s t ake m easures to
adapt to the stressor rather than deali ng wit h
it dir ectly (Pacak and Palkovits, 2001).
It has been postulated that str ess is
involved in pathogenesi s of vari ety of
diseases
like depression and
anxiety.
Immobilizati on (r estr aint) str ess has been
commonly used as acute and chronic str ess
inducers in rats (Bharihoke et al., 2000; Iwa
et al., 2006; K ulkarni and Juvekar, 2008).
Exposure of striated muscl e ti ssue to
intensi ve and prolonged compressi on may
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274
Egypt. J. Exp. Biol. (Zool.), 8(2): 273 – 285 (2012)
pathologically alter it s microstr ucture and
lead t o loss of muscle fibres cross-striati on
and infiltr ation of inflammator y cell (Bosboom
et al., 2001;Appell et al., 2004), and al so
effected on immune system (Brehe and W ay,
2008).
Under
imm obili zation
str ess,
the
histological changes were eluci dated in
mammary gl ands (Soliman,
2006) and
induced ult rastr uctural changes i n the colonic
mucosa (El-Deri eny and Mousa, 2006).
Moreover, Erosy et al. (2008) documented
that the stomach epitheli um of W ister albi no
rat under stress showed ulceration in som e
areas, dil atation and degenerati on in gastric
glandular cells and prominent congesti on of
the
capillari es.
Besi des,
degen erated
epit heli um and severe vascular congesti on
were observed in the ileum and colon aft er
the exposure to acut e water-avoidance stress
for 2 hr dail y and chronic str ess for 5 days.
Gabry et al. (2011) also dem onstr ated many
histopathological changes in the gastric
mucosa of rats under immobili zati on str ess
for different durations.
El- Ref aiy (2010) documented that the
immobil izati on str ess of rats for 2 hr daily for
different durations (5, 15 and 30 days)
caused marked reduction of sperm atogenesis,
deg en erati on
of
germ inal
epit heli um,
thickening
of
basement
m embrane,
haemorr hage and marked increm ent of
collagen fi bres. Mazroa and Asker (2010)
found
ultrastructural
changes
in
zona
fasciculate cell s of rat adrenal cort ex aft er
exposure to high am bient tem perature str ess.
El-Desouki et al. (2011) el ucidated the
abnorm alities in ultrastr ucture of the three
adrenal cortical zonal cell s of r at under 2 hr
of immobili zation stress daily for 30 days.
W hen an anim al fi rst encounters a
stressor, the neurogenic system is activat ed.
The neurogenic system is composed of the
sympathetic postgangli onic neurons and
adrenal m edull ary ti ssue (Joels et al., 2007).
Activati on of the neurogenic system leads to
marked increases i n blood pressure m uscle
tone, nerve sensitivity, blood sugar and
respirati on. This i s brought about by secreti on
of the neurogenic amines, epinephrine and
norepinephrine. Also, when the hypothalam icpituit ary-adrenal cortical system is activated
by str ess (Ulrich-Lai et al., 2006), the
hypothal amus
produces
corticotrophinreleasing fact or, which in turn stimulat es the
pituit ary
to
release
adrenocorticotr opic
hormone
(ACTH).
In
most
mammals,
secretion of ACTH under str ess causes the
cells of the adrenal cortical ti ssue to
proli ferate and to secrete corticosteroi ds
especially cort isol fr om zona fascicul ata
(Armari o et al., 2008).
Benzodiazepines
(BDZs),
such
as
diazepam, are widely used pri maril y as
anxiolytic, muscle relaxant,
sedative /
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hypnoti c and anti convulsant drugs. BDZs
reduce stress responses by acti ng on highaffinit y receptor sites present in the central
nervous system; these specifi c bindi ng sites
on gama-aminobutyric aci d (GABA)-gated
chlori de channels are call ed GABA-r eceptorchloride-complex (Engel
et al. ,
2007).
Nevertheless, besides t he central receptors
described f or BDZs, peri pheral-type binding
sites have also been identifi ed for them in
hum an stomach, small int estine, col on, liver,
lung,
thyroid
gland,
pancreas,
breast,
prostate,
ovary
and
in
mitochondri al
m embranes (Bribes et al., 2004).
In fact, diazepam treatm ent has been
found to reverse the effect of different
stressf ul stimuli (Lane et al., 1982), to
suppress noise induced gastric hypomotili ty
(G ue et al. , 1987), to im prove the histol ogical
and histochemical alt erati ons of testes of
immobili zed rat (El-Refai y, 2010), to recover
both the histological changes in gastri c
mucosa (G abry et al., 2011) and the
ultr astr uctural alterations induced by str ess in
rat adrenal cort ex (El-Desouki et al., 2011).
The aim of this work is to study the
ef fect of immobilizati on str ess on the
histological, hi stochemical and on desmin of
the cardiac m yocytes of mal e al bino rats and
the curati ve role of diazepam.
M ATERIAL AND M ETHO DS:
Animals:
Ei ghty adult male albino rats, each
wei ghing 100 ± 5 g, were used in t he pr esent
work. The anim als were kept under the sam e
natural
envi ronm ent al
condition
of
temperature and photoperi od with fr ee food
and water. All procedures and treatm ents
were in accordance with the protocol of
National Anim al Care and Use Commi tt ee and
Guideli nes for the care and use of
experim ental animals.
Immobilization stress:
Rat s were exposed to str ess for 2 hr daily
between 10:00 and 12:00 a.m . The anim als
were individually pl aced in wire stainl ess
m esh restr ainers (5×7×12 cm in dim ension)
as descri bed by Soli man (2006). This
procedure effecti vely restricted movem ent of
the animals. Contr ol group were housed in
norm al stainless cages (20 × 25 × 35) Cm.
Treatment:
Stressed rats were dail y injected
intr aperi toneally for 30 days with the
therapeuti c dose of diazepam which is 0.1
mg/kg b.w. according to Paget and Barnes
(1964). Diazepam was received fr om Amoun
Pharm aceuti cal Industries Co. Cair o, Egypt.
Experimental design:
The rats were divi ded into eight groups,
10 rat s each. Group1: rats received no
tr eatm ent and served as contr ol; group 2:
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275
El-Desouki et al., The Cardiac Muscle of the Albino Rat under Immobilization Stress and the Curative Role of Diazepam
RES ULTS :
Effect of stress on cortis ol horm on e:
Table 1. Effect of stress on the level of c ortis ol
Group
Control
Cortisol hormone
Ug/dl
1.35
Stressed rats
Stress for
5 days
Stress for
15 days
Stress for
30 days
1.53
4.5
4.03
Relation between stress and level of
cortisol
level of hormon
(Ug l dl)
unstressed
rats
injected
dail y
intr aperitoneall y with a dose of diazepam
0.1mg/kg b.w. f or 30 days; groups 3, 4, and
5: rats expos ed to str ess daily for 5, 15, and
30 days, respectively; groups 6, 7, and 8 rats
injected
daily
i ntr aperitoneally
wit h
a
diazepam dose of 0.1 mg/kg b.w. for 30 days
aft er 5, 15, and 30 days, respectively of
applying the str ess.
At the end of the experim ent, the rats
were sacrificed then the blood sera were
collected to m easure the l evel of cortisol.
Serum cortisol was det ermined by using a
radioimmunoassay kit (Biochemical, Costa
Mesa, CA, USA) and the val ues were
expressed as Ug cortisol /dl serum (Ulrich-Lai
et al., 2006).
The cardi ac muscl e specimens fr om the
left ventricles of the contr ol and treated
animals were fi xed in 10% neutr al formali n,
then dehydrated through alcohols, cleared in
xyl ene and then embedded in paraffi n wax,
then cut t o obtain sections of 5 m t hickness
which were used for:
1) Histological study:
a- Haem atoxylin and eosin; H&E (Bancroft
and Steven, 1996).
b- Other secti ons stained with azan to
dem onstr ate the coll agen fi bres (Humason,
1972).
2) For histochemical studies,
aP eri odic acid Schiff 's reagent
(Pearse, 1963) was used t o dem onstrate
glycogen content.
b) Bromophenol blue st ain (BPB) w as
used to detect the total protei n contents
(Mazia et al., 1953).
3) For immunohistochemical study:
P araffin sections of cardiac muscle and
monoclonal anti body (RD301) against desmin
were used. Clone, RD301 is a mouse
monoclonal IgG 2b antibody and reacts
exclusively with desmin and is expressed in
smooth, cardi ac, and striated m uscle cells,
and was obtained from (Therm o Fisher
Scientific Industries). Avi din bi oti nylated
horseradish
peroxi dase
com plex
ABC)
techni que is appli ed as detecti on reagent .
Colour reaction w as developed by usi ng
diamino–benzidine ( DAB) and gave a brown
colour to desmin. Haematoxylin was used for
counterstaining (Hsu et al., 1981).
5
4
3
2
1
0
0
10
20
30
40
(Tim e/day)
Fig 1. The c orrelation between stress and level of
c ortis ol hormone
Histological observations:
Haematoxyli n and eosin (H&E):
In control rats group, the myocardium is
striated and arr anged in a l inear array that
branches and anatomizes in a specific patt ern
givi ng the appearance of a sheet . The cardiac
muscl e fi bres are joined toget her by
intercalated discs. They contai n acidophil ic
cytopl asm with oval centr all y located nucl ei.
The cardi ac muscle fibres are separat ed by
deli cate layer of connecti ve ti ssue wit h well
evidenced m yocardi al blood capillari es (Fig.
2a). No changes were dem onstr ated in
cardiomyocytes aft er tr eatm ent wit h diazepam
only for 30 days (Fig. 2b).
Fig. 2. Longitudinal s ection of the c ardiac muscle
showing normal br anc hed striat ed c ardiac
myoc yt e with intact interc alat ed discs (arr ows),
ac idophilic s arc oplas m and c entrally-loc ated
nuclei (N): a) a c ontrol rat, b) a r at treated with
diazepam only f or 30 days. H&E,
Bar = 6.25
µm.
T he
c or t is ol
h or m on e
v al u es
wer e
m e as ur ed in t h e b lo od s er a of r ats . T h e v al u e
was 1. 35 U g/ d l in t he c on tr ol r ats . Af t er 5 d ays of
s tr es s , t h e h or m on e l e v el s in t h e
b lo od s er a
The rats group str essed for 5 days
wer e i nc r e as ed f r om 1. 3 5 U g/ d l t o 1. 53 U g/ dl .
revealed
vari ety of histological changes; pale
T he incr em ent of t he hor m on e le ve ls c on t in u ed
acidophilic cytoplasm of many cardiac
af t er 15 d a ys of s tr es s wher e it r eac hed 4. 5 35
myocyt es and perinucl ear vacuolation were
Ug/ d l t h en p os t 3 0 d ays of s tr ess , th e c ort is ol
seen. These changes w ere accom panied wit h
le v el s r eac hed 4. 03 U g/ d l (T abl e 1 & Fig. 1).
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Egypt. J. Exp. Biol. (Zool.), 8(2): 273 – 285 (2012)
nuclear peri pheralization and pyknosis. Also,
congestion of myocardi al blood vessels w as
dem onstr ated (Fig. 3a). Treatm ent wit h
diazepam at a dose of 0.1mg/kg b.w. for 30
days of the stressed rats exhibit ed an
im provem ent in myocardial cell s and almost
appeared like normal (Fig. 3b).
Fig. 3. Longitudinal section of the cardiac muscle of :
a) a r at stressed f or 5 days showing
disarr angem ent of cardiac myocyte, c ytoplasm ic
vac uol at ion, deg eneration of muscle fibres (thic k
arr ows), pyknot ic nuc lei (thin arr ows), dilatation
and c ongestion of blood vess els (BV), b)
treat ment of stress ed rat for 5 days with
diazepam
f or
30
days
showing
normal
hist ological appearance of c ardiac myof ibres and
normal nuc lei (N). H&E, Bar = 6.25 µ m.
The rat s group stressed for 15 days
reveal ed more obvious alt erati ons, where
most
of
the
cardiac
muscl e
fi bres
dem onstr ated remarkable di sorgani zation and
fragm entation,
i ncrem ent
of
nucl ear
peripheralizati on and pyknosis together wit h
sarcoplasmic
vacuol ati on.
Also,
marked
congestion and dilati on of the m yocardi al
blood vessels were displ ayed (Fig. 4a). The
alt erati ons becam e more prominent in the
cardiac m uscle fibres of rats stressed for 30
days (Fig. 5a, b).
Treatm ent with diazepam at a dose of
0.1mg/kg b.w. for 30 days of the rats stressed
for 15 &30 days exhi bit ed an obvious
recovery and restorati on of the norm al
architecture of the m uscl e fi bres (Fi gs. 4b &
6).
Fig. 4. Longitudinal section of the cardiac muscle of :
a) a rat stress ed f or 15 days showing a mar ked
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dis oriented muscle fibres (* ), s arc oplasmic
vac uolation (thin arr ows), degenerated muscle
fibres (thic k arr ows), megalic nuclei, dilated and
c ongested blood vess els ( BV), b) a r at stress ed
for 15 days and treated with diazep am f or 30
days s howing the improvement of architecture of
c ardiac myof ibres and normal nuc lei (N ). H&E,
Bar = 6.25 µm.
Fig. 5. Longitudinal s ect ion of the cardiac muscle of
rat
stress ed f or
30
days
showing:
a)
dis arr angem ent of muscle fibres, vacuolated
s arc oplas m (arr ow) and pyknot ic nuc lei(N ), b)
more dis arr angement of c ardiomyoc ytes with
marked s arc oplasmic vacuolation and pyknotic
nuclei (thick arr ows), dense fibres (thin arr ows)
and congestion of blood vess els (B V).H&E, Bar =
6.25 µm & 12 µm, respectively
Fig. 6. Longitudinal s ect ion of the cardiac muscle of
a rat stress ed f or 30 days and treated with
diazepam for 30 days showing appr oximat el y
normal hist ological appear anc e of c ardiac
myoc yte (arr ows). Some pyknotic nuc lei (N) are
still s een. H &E, Bar = 6. 25 µm.
Azan stain:
Sections of cardiac muscle fi bres of
control rat s stained wit h azan revealed
norm al distributi on of bl ue coloured collagen
fi bres in the endom ysium between the m uscle
fi bres and around the bl ood vessels (Fig. 7a).
Unstr essed rats treated with di azepam only
for 30 days exhibit ed norm al di stri buti on of
coll agen fibres as i n control ones (Fig. 7b).
In rat groups stressed for 5, 15, and 30
days, t here were an excess d epositi on of
coll agen fibres in the endom ysi um of
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El-Desouki et al., The Cardiac Muscle of the Albino Rat under Immobilization Stress and the Curative Role of Diazepam
cardiomyofibres and around the blood vessels
(Figs 8a, 9a & 10a). The i ncrem ent of
collagen fi bres was tim e-dependent. Aft er
tr eatm ent of rats wit h diazepam, a marked
decrease in the di stri buti on of coll agen fi bres
was observed and appeared approximately
similar to the contr ol ones, indicating the
curative role of di azepam (Figs 8b, 9b, &10b).
Fig. 7. Longitudinal s ection of the c ardiac muscle
showing normal dens e distribut ion of collagen
fibres in endomysium (arrows): a) a control r at,
b) a stressed-r at treated with diazep am f or 30
days. Azan, Bar = 6.25µm.
Fig. 8. Longitudinal section of the cardiac muscle of :
a) a rat stressed f or 5 days s howing increas e of
collagen f ibres in endomys ium (arr ows), b) a
stress ed-r at f or 5 days treat ed wit h diazep am
showing normal appearanc e of c ollag en fibres
(arr ows) approxim ately s imilar to c ontrol. Azan,
Bar = 6.25 µm.
277
c ompact dense of c ollag en fibres around blood
vess els and in endomys ium of cardiomyocyte, b)
a rat stress ed f or 15 days and treated with
diazepam
f or
30
days
showing
normal
distribution of collagen fibres (arr ows). Azan,
Bar = 6.25 µm.
Fig. 10. Longitudinal section of t he c ardiac muscle of
r at stress ed f or 30 days showing: a) th e
increment of c ollagen c ompact fibres in
endomys ium and ar ound blood vess el (arr ow),
b) stress ed-r ats f or 30 days treat ed with
diazepam
f or
30
days
showing
normal
distribution of c ollag en fibres between c ardiac
myocytes (arrows). Azan, Bar = 6.25 µm.
Histochemical observations:
Glycogen:
The glycogen distribution in the cardiac
muscl e of contr ol rats is stained strong pink
colour wit h PAS technique (Fig. 11a) as seen
in unstr essed rats tr eated wit h diazepam for
30 days (Fig. 11b).The rats stressed for 5, 15,
and 30 days showed gradual marked
reduction in the distribution of glycogen
materials in the cardiac muscle fibres
(Figs12a,13a, &14a). Aft er tr eatm ent wit h
di azepam, partial recovery of the glycogen
distr ibution in t he m uscle fibres was
dem onstrated ( Figs12b, 13b, &14b).
Fig. 11. Longit udinal s ect ion of the cardiac muscle
showing nor mal strong distribution of glyc ogen in
the c ardiac m uscle fibr es: a) in a c ontrol rat, b)
in unstressed rat treat ed with diazepam f or 30
days.
PAS, Bar = 6. 25µm.
Fig. 9. Longitudinal section of the cardiac muscle of :
a) a rat stress ed f or 15
days s howing high
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Egypt. J. Exp. Biol. (Zool.), 8(2): 273 – 285 (2012)
Total protein:
In contr ol and unstr essed rat groups
tr eated wit h diazepam, the total protein
contents appeared as deeply st ained granules
wit h bromophenol blue in the sarcoplasm and
nuclei of all the m uscl e fi bres (Fig.15a&b).
The rats stressed for different durati ons (5,
15, and 30 days) showed gradual decrease in
the protei n contents of m uscl e fi bres in the
sarcoplasm and nuclei (Figs16a, 17a, &18a).
Aft er tr eatment wit h diazepam, the m uscle
fi bres rest ored their proteinic contents and
appeared rat her similar t o the contr ol ones
(Figs16b, 17b, & 18b).
Fig. 12. Longitudinal s ect ion of the cardiac muscle of:
a) a rat stress ed for 5 days showing marked
decreas e of glyc ogen, b) treatment of stressed rat
(f or 5 days) with diazep am for 30 days s howing
partial rec overy of glyc ogen distribution. PAS, Bar
= 6.25 µm.
Fig. 15. Longit udinal s ection of the c ar diac muscle
showing normal strong distribution of total
proteins content in: a) a c ontrol rat, b)
unstressed rat treated with diazep am for 30
days. BPB, Bar = 6.25 µm.
Fig. 13. Longitudinal s ect ion of the cardiac muscle of:
a) a rat str ess ed f or 15 days showing an obvious
reduct ion of glyc ogen mater ials, b) stressed- rat
(f or 15 days) treated with diaz epam f or 30 days
showing partial restoration of the glycog en
distribut ion. PAS, Bar = 6.25 µm.
Fig. 14. Longitudinal s ect ion of the cardiac muscle of:
a) a rat stress ed f or 30 days s howing t he
depletion of glyc ogen, b) a rat stress ed for 30
days treated with diazepam for 30 days s howing
partial restoration of the glycog en distribution.
PAS, Bar = 6.25 µm.
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Fig. 16. Longit udinal s ection of the c ar diac muscle
of: a) a rat stressed for 5 days s howing a
marked reduct ion in the distribut ion of protein
c ontents in the cardiac musc le fibr es, b) a r at
stress ed f or 5 days and treated with diazepam
f or 30 days showing restoration of normal strong
protein distribution in the c ardiac myof ibres.
BPB, Bar = 6.25 µm.
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El-Desouki et al., The Cardiac Muscle of the Albino Rat under Immobilization Stress and the Curative Role of Diazepam
Fig. 17. Longitudinal s ection of the c ardiac muscle
of : a) a rat stress ed for 15 days s howing a slight
reduction in the distribution of total protein
contents in the c ardiac muscle f ibres, b) a r at
stress ed f or 15 days and treat ed with diaz epam
for 30 days s howing a restoration of total protein
with high distr ibution in the c ardiac muscle
fibres. BPB, Bar = 6.25 µm.
Fig. 18. Longitudinal s ection of the c ardiac muscle
of : a) a rat stress ed f or 30 days showing
moderate decr eas e of protein c ontents in the
cardiac musc le fibres, b) a rat stress ed f or 30
days and treated with diaz epam for 30 days
showing normal strong protein c ont ents. BPB,
Bar = 6.25 µm.
Immunohistochemical observations:
Immunostaining for desmin showed
evident brown colour i n the i ntercalated discs
and Z li nes of cardiac muscle fi bres in the
control and unstr essed rats tr eated wit h
diazepam for 30 days (Fig.19a & b). The rats
str essed for 5, 15, and 30 days showed an
obvious reducti on in immunoreactivity of
desmin of cardiom yocytes
and expressed
faint brown reacti on i n i ntercalat ed discs and
Z lines (Figs 20a, 21a, and 22a). Aft er
tr eatm ent of the str essed rats wit h diazepam
for 30 days,
a marked increase of
immunoreacti on intensity f or desmin w as
expressed, indicati ng the recovery of desmin
to a l evel approximatel y similar to the contr ol
(Figs 20b, 21b, and 22b).
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279
Fig. 19. Longit udinal s ection of the c ar diac muscle
showing positive intens e desmin expr ess ion in
the int ercalated discs (arr ows) and Z lines (short
thick arrows): a) a control r at, b) unstr ess ed r at
treated with diaz epam f or 30 days. Immunostain,
Bar = 6.25 µm.
Fig. 20. Longit udinal s ection of the c ar diac muscle
of: a) a rat stressed for 5 days s howing a
marked decreas e of immunoreactivity to desmin
and seen wit h faint brown reaction in
int ercalated discs (arrows) and Z lines (short
thick arr ows), b) a r at stress ed for 5 days and
treated with diaz epam f or 30 days s howing an
obvious rec overy and increas e im munopos it ive
reaction to desmin in interc alated discs (arr ows)
and Z lines (arr ow heads). Immunost ain, Bar =
6.25 µm.
Fig. 21. Longit udinal s ection of the c ar diac muscle
of: a) a rat str ess ed for 15 days showing a
noticeable decreas e of immunopositive reaction
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280
Egypt. J. Exp. Biol. (Zool.), 8(2): 273 – 285 (2012)
to des min and express ed with f aint brown
reaction in interc alated discs (arr ows) and Z
lines (short thick arr ows), b) a rat stressed f or
15 days and tr eated wit h diazepam for 30 days
showing a marked increas e of immunop os itive
reaction to desmin and it appears normal in
interc al at ed discs (arrows) and Z lines (short
thick arrows Imm unost ain, Bar = 6. 25 µm.
Fig. 22. Longitudinal section of the cardiac muscle of
a): a rat stressed f or 30 days revealing an
obvious r eduction of immunopos itive reaction to
des min in interc alat ed discs (arr ows) and Z lines
(arr owheads), b): a rat stress ed for 30 days and
treated with diaz epam f or 30 days s howing an
obvious rec overy of im munopos it ive r eaction to
des min in interc alat ed discs (arr ows) and Z lines
(short thick arrows). Immunostain, Bar = 6.25
µm.
DISC USSION:
It has been reported that str ess involved
in the pathogenesi s of a vari ety of diseases
like anxi ety and depression (Metz et al. ,
2005; Sevgi et al., 2006; Kulkarni and
Juvekar, 2008). Exposure t o str ess usually
aff ects motor activity and causes pain
percepti on in skeletal muscl e (Torres et al. ,
2001; Appell et al., 2004) and evokes
histopathological changes in the gastric
mucosa (G abry et al. , 2011)
A familiar stress response i n vertebrat es
is the activati on of adrenocortical activity.
Acut e or chronic stress causes an el evati on
in corticosterone l evel s in mammals, hence
increased
adrenocorti cal
acti vit y
is
consi dered as an index of str ess response in
vertebrates (Nirupama et al., 2010). Cortisol
(a natural steroid hormone) i s made by the
adrenal gland in response to str ess such as
fasting f or prol onged tim e (B eszczynska,
2005), noise stress (Swami et al., 2007), hi gh
ambient temperature (Mazroa and Asker,
2010), and immobili zation str ess (El- Desouki
et al., 2011).
In the present st udy, the level of the
cortisol horm one i ncreased aft er exposure t o
different peri ods (5, 15 and 30 days) of
immobilizati on
stress
and
was
tim edep endent. In accordance, Gesi et al. (2001)
reported a m arked i ncrease of the cortisol
levels in response to noise stressful stimulus
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that activated the hypothalamic-pituit aryadrenal axis l eading to a rel ease of adrenocorticotr opic hormone (ACTH). Sim ilarl y,
immobili zati on
str ess
response-r elated
disorders resulted in the hypersecreti on of
both
ACTH
and
corti cost erone
to
a
subsequent str essor (Armari o et al., 2008).
Mazroa and Asker (2010) recorded the
increm ent of corti sol levels i n rats aft er
exposure to high ambi ent temperature. The
disturbances m ay vary by type, int ensity, and
duration of a stressor, and the strain / sex
diff erenti ation of the exp erimental subjects
(Ki oukia-Fuougi a et al., 2002). The length of
stress peri od may aff ect
neurol ogical,
behavioral, and biochemi cal param eters,
possibly in different ways (Rai et al., 2003).
The present w ork showed that dail y 2 hr
immobili zed-str ess of rats for 5,10 and 30
days provoked structural changes in the
cardiac muscle such as degeneration and
disorgani zati on
of
the
muscle
fi bres,
vacuolation of the sarcoplasm, pyknosis of
the nuclei and congesti on of t he blood
vessels. The alt eration was ti me-dependent.
Thes e results are in agreem ent with Gesi et
al. (1999) who observed t hat aft er 6 hr of
noise exposure; only the atrial myocardi al
tissue underwent ultrastr uctural changes,
whereas aft er 12 hr the m yocardial damage
involved both atria and ventri cles. Kušl eikait e
et al. ( 2004) dem onstr ated that immobil ization
stress decreased the relaxati on of the smooth
muscl es
and
damaged
cardiomyocyte
ultr astr ucture
as
m anif ested
by
karyopyknosis, karyorrhexis, desintegrati on of
myofibrils, vacuoli zation of mitochondri a and
endopl asmic reti culum.
Moreover,
Antona
et
al.
(2003)
dem onstrated that restr ai nt stress produced
num erous painful pathologies, such as
fi bromyal gia,
characteri zed
by
diff use
muscul ar
pain
(hyperalgesi a)
and/or
tenderness
(all odyni a).
With
foll owing
immobili zati on, t he decrease i n m uscle
power, w hich is shown as muscle weakness,
might depend not only on a quantitative
m echani sm (loss of mass), but also on
qualit ative mechanisms, i. e. loss of the
intrinsic capacity of m uscl e fi bres to develop
force and, in aged muscl e, sl owing of
shortening vel ocity.
Additionally, acute restr aint stress f or 6
hr caused severe anxi ety like behaviour and
im pair ed locomotor acti vity as compared wit h
unstressed animals (Kumar et al., 2010).
Also, the hist opathol ogical changes were
induced in stomach, intestine, testi s and
adrenal gland under immobilizati on stress in
male rat s (Gabry et al., 2002; El-Refai y,
2010; El-Desouki et al., 2011).
Exposure to stress can stimulate
num erous pat hways leadi ng to i ncreased
production of fr ee radicals that contribut e to
the occurr ence of pathol ogical condit ions
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El-Desouki et al., The Cardiac Muscle of the Albino Rat under Immobilization Stress and the Curative Role of Diazepam
(Bagchi et al., 1999; Rai et al., 2003).
Restrai nt stress may also impair the
anti oxidant defence system, leading to
oxidati ve damage, by changing the bal ance
between oxi dant and anti oxidant fact ors
(G anesan et al., 2011). Malonaldhyde level (a
biomarker of lipi d peroxidation) was al so
significantly i ncreased whil e glut athione level
(a bi omarker of protecti ve oxi dati ve i njury)
was significantl y decreased in all ti ssues aft er
exposure t o str ess (Erosy et al., 2008).
In the curr ent study, collagen fi bres
increased in endomysium and around blood
vessels aft er exposure to immobilizati on
str ess for diff erent times. The enhancem ent
of fi bres was tim e-dependant. I n agreem ent
with the present results, Rai et al. (2003 &
2004) and El-R efaiy (2010) found that
fi broblasts and coll agen fibres in str essed rats were increased and this leads to the
thickening of testi s basement m embrane.
Moreover, El-Dri eny and Mousa (2006)
report ed that the colonic mucosa of rats
exposed to str ess showed features indicati ng
fi brosis as a result of increased coll agen
synthesis by fi broblasts. El-Desouki et al.
(2011) - in their ultrastructural st udy report ed t hat the immobil ized str essed- rats
showed i ncrem ent of collagen fibres i n the
adrenal cort ex.
The findings of the present study
il lustr ated that the admini strati on of diazepam
could improve the hist opathological changes
and di sturbance of collagen fi bres result ed
under the effect of immobil izati on str ess in
the cardi ac myocytes. The present result s
were in agreement with the previous studi es
reporting that benzodiazepines can reduce or
suppress the eff ects induced by various
stress stimuli. In fact, diazepam treatm ent
reduced the ultr astr uctural alt erations in the
atrial tissue (Pellegrini et al., 1996), and al so
reduced disturbances in the brain, kidney,
adrenals and heart that were induced by
chronic mild stress in rats (Nirmal et al. ,
2008; A bdel Baky and Ali (2009). Additionally,
ultrastr uctural
changes
induced
by
immobil izati on str ess in rat adrenal gl ands
(El-Desouki et al., 2011) and i n gastric
ultrastr ucture alteration (Nagi., 2012) were
modulated by di azepam.
The prev enti ve effect of diazepam is
suggested to be attributed to its abil ity to
inhibit the str ess-i nduced acti vation of HPAaxis
and
sympathetic
stimulation
and
functi onal alt erati on of cell m embranes due to
steroids (G ehlot et al., 1997). B oth central
and peri pheral BDZ li gands are able to
prevent the m yocardial damage induced by
noise exposure, the extent of this protecti on
dep ends on the sp ecific drug used and the
duration of str ess exposure (Gesi et al. ,
1999). El-Desouki et al. (2011) report ed t hat
diazepam
admini str ation
im proved
the
histological and ultrastr uctural alt erations of
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281
the adr enal cort ex of imm obil iztion stressedrats. Moreover, Gabry et al. (2011) reported
that diazepam treatm ent of str essed-r ats
show ed an obvious im provem ent in the
st omach alt erations and reducti on of collagen
fibres
after
immobilization
stress.
Anti depressant
drugs
have
also
been
report ed to elevate antioxidant enzym e
defence
system
particularly
superoxide
en zym e
and
cat alase
activity.
These
antioxi dant enzym es raised the level of
oxidati ve defence against str ess (Kolla et al.,
2005; Kumar et al., 2010).
The hi stochemical observations in the
present work dem onstr ated that the st ressedrats for different durati ons showed a
significant decrease in gl ycogen i n cardiac
myocyt es and this was ti me-dependant.
Similarl y, Leivo et al. (1998) found t hat the
immobili zati on str ess l eads to abnorm al
mitochondri a and sarcoplasmic changes in
rabbit skelet al muscles. Rosochacki et al.
(2000) recorded that immobili zation str ess
caused a decrease of glycogen content in
longissimus dorsi muscle by 27% in duroc
pi gs and by 44% in pi etr ain pi gs. Nader
and E sser (2001) decl ared that aft er an acute
bout of exercise, signif icant reducti on in
muscl e glycogen was observed in the ti bi alis
anterior and soleus muscle. Da Sil va et al.
(2006) and B osi et al. (2008) found that
immobili zati on
si gnificantly
reduces
the
glycogen content in all skeletal muscl e.
Such results in eventual str uctural
disturbances and degeneration are probably
triggered by a loss of calcium hom eostasis
(Soares et al., 1993).
Brown et al. (1995)
explai ned the reducti on of glycogen in muscle
may due to t he role of reactive oxyg en
species
that
cause
damage
to
the
m echani sms of carbohydrates synthesis in
cell s and might be due to inhibition of
mitochondri al energy m etabolizati on and the
inability of cells to store glycogen and convert
lactate and pyruvate to glycogen. Amoroso et
al. (2000) illustr at ed that oxidati ve str ess
induces m any damaging processes i n str ess
disorders such as disrupti on of energy
pathways, mitochondrial dysfuncti on and
dysregulation of calcium hom eostasis.
The pr esent work dem onstrated that the
immobili zati on str essed-rats for dif ferent
durations showed si gnificant depletion in the
protein contents in cardiac myocytes and that
was tim e-dependant. In agreem ent with the
present findings, Michelsson et al. (1990) and
Graves et al. (1997) demonstrated that
several intrinsic tools of fi bre wasti ng could
be responsible for muscle atr ophy induced by
immobili zation, contributing to an i ncreased
degradation of proteins and a marked
reduction in protei n synthesis.
The reducti on in total protein w as also
report ed by Li u et al. (1996) who showed that
the immobil izati on str ess for 8 hr caused
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282
Egypt. J. Exp. Biol. (Zool.), 8(2): 273 – 285 (2012)
oxidati ve damage to protein in rat. Also,
significant
reducti on
of
prot eins
in
sperm atogenic cell s of rat test es exposed to
oxidati ve str ess was recorded by Sakr and ElAbd (2009)
and in rat
testes by
immobilizati on stress for different durations
(El -Refai y, 2010). Additionall y, the reducti on
in total protein was dem onstr ated i n rat
adrenal cortical cell s under immobil izati on
str ess ultrastructurally (El-Desouki et al. ,
2011) and in gastric mucosa (Nagi, 2012).
Aft er tr eatment of str essed-r ats wit h
diazepam in t he present work, the glycogen
and
proteinic
contents
appeared
approxim at ely norm al as i n control ones.
Similar result s were report ed in testes,
adrenal gland and gastric mucosa aft er
treatm ent with di azepam to restraint stressedrats (El-Refaiy, 2010; El-Desouki et al., 2011;
Gabry et al., 2011; Nagi, 2012).
The interm ediate filam ents (IFs) are
one of the three abundant cytoskeletal
proteins. The interm ediate filam ents i nclude
cytokeratin, vimenti n, desmin, gli al fi brill ary
acidic
proteins,
neurofilam ent
prot eins,
nuclear lam ins and nestin (Alberts et al. ,
1989). They pl ay an import ant role in the
str uctural int egrity and in the tr ansport i nto
and out of the cel ls. The interm ediate
fi lam ents
provide
fl exible
intr acell ular
scaff olding whose functi on is to str ucture
cytoplasm and to resist str esses externall y
appli ed to the cell (Djabali, 1999; Omary et
a l., 2004). Cytoskelet al IFs are typi cally used
in animals as an indir ect marker of tissue
injur y (Ananth et al. , 2003). Desmin is a 53
kDa of IFs that exhibits a high degree of
ti ssue specificity, it s expression bei ng
predominantl y confined to all types of muscl e
cells (cardiac, skeletal and smooth muscle).
Desmin is l ocalized i n the Z-disk region and
at the i ntercalated disk i n skeletal and cardi ac
muscl e cells and acts to stabilize sarcom eres
in sti mulat ed muscle (S chaart et al., 1989).
Paulin and Li (2004) show ed that desmin is a
muscle-specifi c protein and a key subunit of
the interm ediate fil ament i n cardiac, skeletal
and smooth muscles. Desmin fi lam ents are
mainl y located at the peri phery of Z-disk of
striated m uscles and at the dens e bodies of
smooth muscl e cells and they have been
postulated to play a criti cal rol e in the
maint enance of str uctural and m echanical
integrity of the contr actil e apparatus in
muscul ar tissues.
In
the
present
work,
the
immunohistochemical result s showed an
obvious l oss of desmin imm unostaini ng in
intercalated di scs and periphery of Z l ines in
cardiomyocytes of rats str essed for dif ferent
durations (5,15, and 30 days). In accordance,
Barash et al. (2002) found loss of desmin
immunostaini ng aft er a bout of eccentri c
exercise in the rat ti bialis anterior muscl es.
Such a l arge-scale change could be the result
of
depolym eri zation
due
to
desmin
phosphorylation (I nada et al., 1999) or the
result of covalent modificati on such as ADPri bosyl ati on (Graves et al., 1997), both of
whi ch have been observed in ot her muscle
system s.
In t he present study, the tr eatm ent
of str essed-r ats with diazepam for 30 days
revealed a rem arkable improvement and
recovery of desmin alm ost up to the contr ol
form in int ercalat ed and Z discs. In
agreem ent wit h the present fi ndings, El- Baely
(2011) found that tr eatm ent of rats wit h
di azepam at a therapeuti c dose (0.1 mg/kg
b.w.) f or 30 days improved the adrenal
cytoskeletal IFs (cytokeratin and vim entin) of
rats stressed for 30 days, and restored them
to the norm al expression in the adrenal
gl ands (cort ex and m edull a) and i n t he gastri c
mucosa of albi no rats (Nagi, 2012).
In conclusi on, the immobili zati on str ess
may be responsible for the histol ogical,
histochemical
and
imm unohistochemical
alterations induced in the cardiac muscl e of
al bino rats, and diazepam could improve
these alt erati ons.
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