J Appl Physiol
91: 1828–1835, 2001.
Effects of ovariectomy and estrogen on
ischemia-reperfusion injury in hindlimbs of female rats
NICOLE STUPKA AND PETER M. TIIDUS
Department of Kinesiology and Physical Education, Wilfrid Laurier University, and
Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3C5
Received 12 June 2001; accepted in final form 9 July 2001
inflammatory cells; myeloperoxidase; calpain; creatine kinase; skeletal muscle
ISCHEMIA-REPERFUSION (I/R) injury to skeletal muscle is
characterized by increased oxidative stress, altered
metabolite homeostasis, efflux of cellular proteins into
the circulation, disruptions in intracellular calcium
Original submission in response to a special call for papers on
“Genome and Hormones: Gender Differences in Physiology.”
Address for reprint requests and other correspondence: P. M.
Tiidus, Dept. of Kinesiology & Physical Education, Wilfrid Laurier
Univ., Waterloo, ON, Canada N2L 3C5 (E-mail: [email protected]).
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handling, increased proteolysis, leukocyte accumulation, and an inflammatory response (30, 32). Estrogen
administration has been shown to decrease I/R injury
in experimental models of stroke (31, 43) and myocardial infarction (23, 34). Whether estrogen can protect
skeletal muscle following I/R is not known. However,
gender-based differences in exercise-induced muscle
damage have been observed in humans and animal
models after an acute bout of aerobic or eccentric exercise (7). Attenuated plasma creatine kinase (CK)
activity (1), inflammatory response and leukocyte infiltration (35), and ultrastructural disruptions (18)
have been reported in female compared with male
animals. These differences have been attributed to the
female sex steroid hormone 17␤-estradiol, which may
act as an antioxidant or membrane stabilizer (39).
Estrogen administration has also been previously reported to attenuate postexercise skeletal muscle neutrophil infiltration and myeloperoxidase (MPO) activity in male and ovariectomized female rats (40, 41).
Estrogen has been reported to attenuate I/R-induced
damage and leukocyte infiltration in cardiac muscle
(22, 27, 34). However, no previous studies have examined the effects of estrogen on I/R-induced injury or
leukocyte infiltration in skeletal muscle. Thus this
study was undertaken to investigate the potential for
17␤-estradiol to attenuate indexes of skeletal muscle
damage and neutrophil infiltration after 2 h of complete hindlimb ischemia and 2 h of reperfusion in rats.
Female Sprague-Dawley rats were divided into three
experimental groups, regular with ovaries intact (R),
ovariectomized with placebo pellet implant (OP), and
ovariectomized with a 17␤-estradiol pellet implant
(OE), to asses the protective effects estrogen may have
on markers of muscle damage following I/R. It was
hypothesized that indexes and consequences of skeletal muscle disruption, including serum CK activity,
neutrophil infiltration into skeletal muscle, calpainlike activity, and lipid peroxidation, would be greater
in the OP animals compared with the R and OE rats.
The hindlimb I/R model was chosen because it has
relevance to skeletal muscle injury induced by reconstructive plastic surgery or thrombosis.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
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Stupka, Nicole, and Peter M. Tiidus. Effects of ovariectomy and estrogen on ischemia-reperfusion injury in hindlimbs of female rats. J Appl Physiol 91: 1828–1835,
2001.—The effects of estrogen and ovariectomy on indexes of
muscle damage after 2 h of complete hindlimb ischemia and
2 h of reperfusion were investigated in female SpragueDawley rats. The rats were assigned to one of three experimental groups: ovariectomized with a 17␤-estradiol pellet
implant (OE), ovariectomized with a placebo pellet implant
(OP), or control with intact ovaries (R). It was hypothesized
that following ischemia-reperfusion (I/R), muscle damage
indexes [serum creatine kinase (CK) activity, calpain-like
activity, inflammatory cell infiltration, and markers of lipid
peroxidation (thiobarbituric-reactive substances)] would be
lower in the OE and R rats compared with the OP rats due to
the protective effects of estrogen. Serum CK activity following I/R was greater (P ⬍ 0.01) in the R rats vs. OP rats and
similar in the OP and OE rats. Calpain-like activity was
greatest in the R rats (P ⬍ 0.01) and similar in the OP and
OE rats. Neutrophil infiltration was assessed using the myeloperoxidase (MPO) assay and immunohistochemical staining for CD43-positive (CD43⫹) cells. MPO activity was lower
(P ⬍ 0.05) in the OE rats compared with any other group and
similar in the OP and R rats. The number of CD43⫹ cells was
greater (P ⬍ 0.01) in the OP rats compared with the OE and
R rats and similar in the OE and R rats. The OE rats had
lower (P ⬍ 0.05) thiobarbituric-reactive substance content
following I/R compared with the R and OP rats. Indexes of
muscle damage were consistently attenuated in the OE rats
but not in the R rats. A 10-fold difference in serum estrogen
content may mediate this. Surprisingly, serum CK activity
and muscle calpain-like activity were lower (P ⬍ 0.05) in the
OP rats compared with the R rats. Increases in serum insulin-like growth factor-1 content (P ⬍ 0.05) due to ovariectomy
were hypothesized to account for this finding. Thus both
ovariectomy and estrogen supplementation have differential
effects on indexes of I/R muscle damage.
EFFECTS OF OVARIECTOMY AND ESTROGEN ON MUSCLE DAMAGE
METHODS
Animals
This study was approved by the University of Waterloo
Committee on Animal Ethics and Care and was performed in
accordance with the guiding principles of the Canadian
Council on Animal Care. Regular (with ovaries) and ovariectomized female Sprague-Dawley rats (Harlan) were housed
in cages of five to six animals in an environmentally controlled room with reversed light-dark cycles. The ovariectomy
was performed by the animal supplier at 8 wk of age. Animals were allowed free access to food and water and fed an
AIN-93-purified rodent diet. All animals were weighed before
death.
Experimental Design
J Appl Physiol • VOL
ical analysis. Sol is composed primarily of slow-twitch muscle
fiber types and thus differs from the gastrocnemius, TA, and
PLT, which are of mixed-fiber type. It is currently not known
whether muscle fiber type can influence post-I/R neutrophil
infiltration. After removal, the Sol was frozen in isopentane
cooled with liquid nitrogen after being placed on a block with
embedding medium (OCT). The tourniquet remained in place
and inflated while the hindlimb muscles were excised from
the I-only animals to avoid reperfusion, after which blood
was drawn from the descending aorta. All muscle samples
and serum were stored at ⫺70°C until the time of analysis.
Analysis
Serum. Serum was assayed for 17␤-estradiol and insulinlike growth factor-1 (IGF-1) content using a commercially
available radioimmunoassay kit (TKE21, Diagnostic Products, Los Angeles, CA, and 022-IGF-R20, Alpco Diagnostics,
Windham, NH, respectively). Serum was also assayed spectrometrically (wavelength ⫽ 340 nm) for CK activity using a
commercially available kit (DG147-UV, Sigma Diagnostics,
Columbus, OH).
Calpain-like activity. Calpain-like, calcium-dependent proteolytic activity in the PLT was determined by a microplate
reader (SpectraMax Plus 384, Molecular Devices) assay using casein as the substrate (29). A reaction mixture containing PLT extract, 2 mg/ml casein, 50 mM Tris (pH 7.5), 20 mM
dithiothreitol, and either 5 mM free calcium or 5 mM EDTA
was incubated at 30°C. After 30 min, a 40-␮l aliquot from
each sample was assessed for calcium-dependent proteolysis
using 20 ␮l of a concentrated Bio-Rad protein dye reagent
(500-0006, Bio-Rad Laboratories, Hercules, CA) diluted in
120 ␮l of double-distilled H2O. Calpain-like activity was
determined by calculating the difference between the absorbance values (at 595 nm) from the reaction mixture containing calcium and that containing EGTA and expressed as
caseinolysis per gram tissue wet weight over 30 min (2).
MPO. MPO activity was determined at a concentration of
6 mM H2O2 by continuously monitoring the oxidation of
o-dianisidine didrochloride at 480 nm at 37°C using a recording spectrophotometer (3, 40). One unit of MPO activity was
defined as a change in 1.0 unit of absorption at 480 nm/min
expressed per gram tissue wet weight.
CD43⫹ cells. CD43 is expressed by polymorphs and has
been used to detect the presence of neutrophils in skeletal
muscle following muscle following bupivacaine-induced myonecrosis (26) and hindlimb unloading (37). The frozen, OCTembedded Sol was serially cross sectioned to 5-␮m thickness
using a cryostat (Microm HM 500 OM, Microm International,
Walldorf, Germany). Negative control sections were included
in all analyses. The slides were dried overnight and stored at
⫺80°C until analysis. Slides were fixed in cold acetone for 15
min. Endogenous peroxidase activity was blocked using a
liquid diaminobenzidine substrate kit (00-2014, Zymed Laboratories, San Francisco, CA). The slides were blocked with
1% goat serum (D3002S, Dako Diagnostics Canada, Mississauga, ON) for 15 min to control for nonspecific binding. The
primary mouse anti-rat CD43 antibody (MCA54R, Serotech)
was diluted 1:50 in 1% goat serum, and positive slides were
incubated for 60 min. Slides were then incubated with a
secondary goat anti-mouse antibody (65-6440, Zymed Laboratories) for 45 min and with peroxidase (95-6543-B, Zymed
Laboratories) for an additional 15 min. A kit (00-2007, Zymed
Laboratories) was used for color development. All slides were
counterstained with hematoxylin solution (MHS-16, Sigma
Chemical).
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After 1 wk of acclimatization, the ovariectomized animals
were implanted with either a 0.25-mg, 21-day-release 17␤estradiol pellet (n ⫽ 16; E-121, Innovative Research of America) or a corresponding, 21-day-release placebo pellet (n ⫽ 16;
C-111, Innovative Research of America). The pellets were
implanted subcutaneously (neck skin fold) under halothane
anesthesia using a 10-gauge trochar needle (MP182, Innovative Research of America) (40, 41). Two weeks later, at ⬃12
wk of age, all of the ovariectomized animals underwent the
2 h of complete hindlimb ischemia and 2 h of reperfusion
protocol (I/R). After the 2 h ischemia period, reperfusion was
initiated by immediate reduction of cuff pressure to zero.
Similar I/R protocols have been previously reported to influence skeletal muscle leukocyte accumulation and oxidative
stress (e.g., Ref. 32).
R animals, also ⬃12 wk old, were assigned to one of three
experimental groups: no intervention (R-sham; n ⫽ 10), 2 h
of complete ischemia only (R-I only, n ⫽ 10), and 2 h of
complete ischemia and 2 h of reperfusion (R-I/R; n ⫽ 16). The
purpose of the R-sham group was to provide baseline values
for CK, MPO, thiobarbituric-reactive substances (TBARS),
calpain activity, and muscle neutrophil content. Because we
have previously seen no effect of ovariectomy and estrogen
replacement on these parameters before intervention (39,
41), the measures in R-sham group could be used as a
baseline for the OE and OP groups as well. Preliminary
power calculation estimates had suggested that the number
of animals used in this study would be adequate for statistical power.
One hindlimb from the I/R or I-only animals was selected
to undergo the experimental procedure. The animals were
anesthetized via intraperitoneal injection of pentobarbital
sodium (100 ␮l/100 g body wt) (Somnotol, MTC Pharmaceuticals, Cambridge, ON). Anesthesia was confirmed with the
loss of withdrawal reflex from toe web pinch and maintained
by pentobarbital sodium injections as required. The animals
were kept warm with a heating pad, and vital signs were
monitored throughout the procedure. Total ischemia was
induced by placing a finger tourniquet (Digikit, Kinetikos
Medical, San Diego, CA) over the upper hindlimb and inflating the cuff to a pressure of 350 mmHg (13) for 2 h of ischemia
and 2 h reperfusion or 2 h of ischemia only. Previous studies
have demonstrated significant skeletal muscle neutrophil
infiltration within 1–2 h after exercise or I/R-induced damage (3, 32, 41). After I/R intervention, blood was drawn from
the descending aorta of the rats and allowed to clot in a glass
test tube. The gastrocnemius, tibialis anterior (TA), and
plantaris (PLT) were excised from the experimental hindlimb, rinsed in saline, blotted, and snap frozen in liquid
nitrogen. The soleus (Sol) was allotted for immunohistochem-
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EFFECTS OF OVARIECTOMY AND ESTROGEN ON MUSCLE DAMAGE
Table 1. Animal characteristics
R-sham
R-I only
R-I/R
OP-I/R
OE-I/R
Weight, g
Days With Pellet
237.9 ⫾ 3.4
236.2 ⫾ 4.0
241.1 ⫾ 2.9
274.2 ⫾ 3.2*
213.8 ⫾ 2.1†
15.8 ⫾ 0.5
15.5 ⫾ 0.7
lighter compared with the OP animals (P ⬍ 0.05) (see
Table 1). The average number of days with pellet
implant was similar among the OE and OP rats (see
Table 1).
Estrogen
Values are means ⫾ SE. R-Sham, normal ovaries and no ischemia/
reperfusion (I/R); R-I only, normal ovaries with 2 h ischemia and no
reperfusion; R-I/R, normal ovaries with 2 h of ischemia and 2 h of
reperfusion; R, normal ovaries; OP, ovariectomized with placebo
pellet implant and 2 h of ischemia and 2 h of reperfusion; OE,
ovariectomized with estrogen pellet implant and 2 h of ischemia and
2 h of reperfusion. The weights of the rats in the R-sham, R-I only,
and R-I/R groups at the time of death were similar. The average
number of days with the pellet implant was similar for the OE and
OP groups. The animals in the * OP-I/R group weighed more and the
rats in the † OE group weighed less compared with the other groups
(P ⬍ 0.05).
IGF-1
The OP rats had a significantly greater serum IGF-1
content compared with the R and OE rats (P ⬍ 0.05).
The OE group had the lowest serum IGF-1 content
(P ⬍ 0.05).
Serum CK Activity
Serum CK activity was elevated after I/R in all groups
compared with R-sham and R-I only (P ⬍ 0.01). The CK
elevation following I/R was greater in the R group (P ⬍
0.05) than in the OP or OE groups (see Fig. 1).
Calpain
Calpain-like activity in the PLT was significantly
greater in the R-I only rats and R-I/R rats compared with
R-sham rats (P ⬍ 0.05). Calpain-like activity was lower in
the OE and the OP rats following I/R compared with the
R-I only rats (P ⬍ 0.05) and the R-I/R rats (P ⬍ 0.05).
After I/R, calpain-like activity was similar among the OE
and OP rats (P ⫽ 0.57) (see Fig. 2).
MPO
Statistics
MPO activity in the PLT was similar among the
R-sham, R-I only , and R-I/R rats. There was also no
Data were analyzed using one-way ANOVA with five factors (R-sham, R-I only, R-I/R, OP-I/R, and OE-I/R). A Neuman-Keuls post hoc analysis was performed if a significant
main effect was obtained to identify which groups differed
(Statistica v5.0, Statsoft, Tulsa, OK). The level of significance
was P ⬍ 0.05. All data are presented as means ⫾ SE.
Table 2. Circulating 17␤-estradiol and IGF-1 content
RESULTS
R
OP
OE
Animal Characteristics
There was no significant difference in weight at the
time of death among the three groups of female rats
with intact ovaries (R-sham, R-I only, and R-I/R). However, these animals were significantly heavier compared with the OE animals (P ⬍ 0.05) and significantly
J Appl Physiol • VOL
17␤-Estradiol, pg/ml
IGF-1, pg/ml
20.5 ⫾ 1.15
15.3 ⫾ 1.67
209.1 ⫾ 50.3*
759.8 ⫾ 26.7
941.3 ⫾ 18.7†
599.6 ⫾ 15.3‡
Values are means ⫾ SE. IGF-1, insulin-like growth factor-1. * The
OE rats had significantly greater 17␤-estradiol content compared
with the OP and R rats (P ⬍ 0.05). However, there was no significant
difference in 17␤-estradiol content between the OP and R rats. † OP
rats had greater serum IGF-1 content compared with the R and OE
rats (P ⬍ 0.05). ‡ R rats had greater serum IGF-1 content compared
with the OE rats (P ⬍ 0.05).
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The number of CD43⫹ cells in muscle cross section were
counted manually and expressed as number of positive cells
per square millimeter of muscle. Only those CD43⫹ cells that
were surrounded by muscle fibers were counted, and those
localized to the lumen of blood vessels or connective tissue
were excluded from analysis. Entire muscle cross sections
were captured with a digital camera (Spot, v2.2, Diagnostic
Instruments, Sterling Heights, MI), and area was quantified
using a computerized image analysis system (ImagePro Plus,
v4.0, Media Cybernetics, Silver Spring, MD). The countrecount variability was ⬍10%.
TBARS. To estimate levels of lipid peroxidation in the TA,
the TBARS assay developed by Ohkawa et al. (25) was used.
Fresh TA homogenates (20:1 volume of 0.154 M KCl buffer/g
wet wt) were added to a reaction mixture containing 0.2 ml of
0.28 M SDS, 1.5 ml of 3.5 M acetic acid (pH 3.5), and 55.5 mM
thiobarbituric acid solution and heated at 95°C for 1 h. The
resultant TBARS were extracted using a n-butanol-pyridine
(15:1 vol/vol) mixture, and the absorbance was determined
spectrophotometrically at 532 nm. The level of TBARS in
tissue was calculated using 0.5- to 5.0-nmol samples of
1,1,3,3-tetramethoxypropane as standards, and tissue lipid
peroxidation levels were expressed as nanomoles TBARS per
gram tissue wet weight.
Lactate and glycogen. Analysis of lactate and total glycogen were done on freeze-dried, perchloric acid-extracted TA
muscle using standard fluorometric techniques (15).
Serum estrogen content was significantly greater in
the OE animals compared with the OP and R animals
(P ⬍ 0.05). However, serum estrogen content was similar in OP animals and the R animals (See Table 2).
This lack of difference in estrogen content could not be
accounted for by the cessation of estrus in R rats.
Estrus may cease if female rats are housed together
and if there are no mature, breeding male rats in the
neighboring cages. To confirm the presence of estrus,
four R-sham rats were randomly chosen to undergo
vaginal cell smears for 4 consecutive days before death
(the average length of a rat estrus cycle) (10). These
cells were examined under a light microscope. Changes
in cell type and number were observed in all four of the
animals during the 4 days, suggesting that the rats
were indeed cycling (10).
EFFECTS OF OVARIECTOMY AND ESTROGEN ON MUSCLE DAMAGE
significant difference in MPO activity in the OP rats
and the R rats. MPO activity in the PLT of the OE
animals was significantly lower compared with any
other experimental group following I/R (P ⬍ 0.05) (see
Fig. 3).
CD43⫹ Cells
The number of CD43⫹ cells/mm2 tissue was greater
in the OP group following I/R compared with any other
experimental group. The number of CD43⫹ cells were
significantly elevated in the OP group compared with
R-sham animals (P ⬍ 0.05), and there was a trend
toward an increase in the OE (not significant, P ⫽ 0.08)
and R (not significant, P ⫽ 0.10) groups following I/R.
Also, the number of CD43⫹ cells was greater in the R,
OP, and OE groups after I/R compared with the R-I
only group. Thus neutrophil infiltration occurred dur-
Fig. 2. Muscle calpain-like activity. ww, Wet wt. Calpain-like activity was significantly greater in the R-2I only and R-I/R rats compared
with R-sham rats. Calpain-like activity was significantly lower in the
*OP-I/R and ⫹OE-I/R rats compared with the R-I/R and R-2I only
rats. Significance was set at P ⬍ 0.05. Results are from plantaris
muscle.
J Appl Physiol • VOL
Fig. 3. Muscle myeloperoxidase (MPO) activity. *MPO activity was
significantly lower in the OE-IR rats after I/R compared with any
other group (P ⬍ 0.05). Results are from plantaris muscle.
ing reperfusion only and not during ischemia (see
Fig. 4).
TBARS
TBARS content in the TA did not increase in response to ischemia only or I/R as it was similar among
the R-sham, R-I only, and R-I/R rats. TBARS content
was significantly greater in the OP-I/R group compared
with any other experimental group (P ⬍ 0.05). TBARS
content was ⬃1.5 times greater in the R rats compared
with the OE rats (not significant, P ⫽ 0.12) (see Fig. 5).
Muscle Lactate and Glycogen
Lactate and glycogen were run as confirmational
markers for I/R. As expected, in the R rats, muscle
lactate increased significantly after 2 h of ischemia and
returned to baseline values after 2 h of reperfusion
(P ⬍ 0.05). After I/R, muscle lactate content was similar in the R, OP, and OE rats. Ischemia decreased
Fig. 4. Muscle CD43-positive cells. *Number of CD43-positive cells
per mm2 of muscle was significantly greater in the OP-I/R rats
following I/R compared with any other experimental group (P ⬍
0.05). **Number of CD43-positive cells per mm2 of muscle was
greater in the OP-I/R and OE-I/R rats compared with the R-2I only
rats (P ⬍ 0.05). Results are from soleus muscle.
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Fig. 1. Serum creatine kinase (CK) activity. R-sham, normal ovaries
with no ischemia-reperfusion (I/R); R-2I only, normal ovaries with
2 h of ischemia only; R-I/R, normal ovaries with 2 h of ischemia and
2 h of reperfusion; OP-I/R, ovariectomized with placebo pellet and
I/R, OE-I/R; ovariectomized with estrogen pellet and I/R. *Serum CK
activity was significantly elevated compared with R-sham and R-2I
only. ⫹Serum CK activity was significantly greater in the R-I/R rats
compared with the OP-I/R and OE-I/R rats following I/R. Significance was set at P ⬍ 0.05.
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EFFECTS OF OVARIECTOMY AND ESTROGEN ON MUSCLE DAMAGE
Fig. 5. Thiobartituric acid-reactive substance (TBARS) content in
muscle. *Muscle TBARS content was significantly greater in the
OP-I/R rats following I/R compared with any other group (P ⬍ 0.05).
Results are from tibialis anterior muscle.
DISCUSSION
Estrogen administration to OE rats attenuated skeletal muscle MPO activity, CD43⫹ content, TBARS
content, and serum CK activity after I/R compared
with OP rats. This confirmed our initial hypothesis of a
protective effect of estrogen on skeletal muscle following I/R. However, after 2 h of ischemia and 2 h of
reperfusion, serum CK activity and skeletal muscle
MPO and calpain-like activity in the R rats were
greater than or to equal to that of the OP rats. These
findings were contrary to our initial hypothesis and
forced us to reevaluate the possible confounding effect
that ovariectomy itself may have on indexes of muscle
damage. Although similarities between R and OP rats
in circulating estrogen level may be related to these
findings, other factors may also play a role. Among
other effects, ovariectomy can stimulate an increase in
growth hormone release and IGF-1 production (11).
IGF-1 has been shown to attenuate injury after I/R in
the heart (6, 14) and brain (16) and in skeletal muscle
exposed to oxidants in vivo (21). Hence, serum was
analyzed not only for 17␤-estradiol content but also for
IGF-1 content; results were further interpreted by taking into account the potentially protective effects of
both estrogen and IGF-1. Although IGF-1 is only one of
many factors that may be altered due to ovariectomy,
for reasons cited above, we felt it to be a most likely
candidate to potentially influence some of the variables
measured in this experiment.
Typical to estrogen effects on body weight, the OP
animals were heavier than the R animals, which in
turn were heavier compared with the OE animals (P ⬍
0.05) (see Table 1). Ovariectomy will increase growth
hormone release, IGF-1 synthesis, and food intake but
J Appl Physiol • VOL
Table 3. Muscle metabolites
R-sham
R-I only
R-I/R
OP-I/R
OE-I/R
Lactate, mmol/kg dry wt
Glycogen, mmol/kg dry wt
24.12 ⫾ 2.07
104.6 ⫾ 21.9*
26.19 ⫾ 4.54
17.84 ⫾ 1.86
31.97 ⫾ 13.1
120.6 ⫾ 5.3‡
62.83 ⫾ 14.55†
164.1 ⫾ 11.64
187.4 ⫾ 8.86
196.5 ⫾ 8.04
Values are means ⫾ SE; results are from gastrocnemius muscle.
After 2 h of ischemia in the R-I only rats, there was a significant
increase in * muscle lactate and a significant decrease in † glycogen
compared with the R-sham, R-I/R, OP-I/R, and OE-I/R rats
(P ⬍ 0.05). ‡ There was a supercompensation of glycogen resynthesis
after reperfusion and glycogen content was significantly greater in
the R-I/R, OP-I/R, and OE-I/R rats (P ⬍ 0.05).
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total glycogen content in the R rats; after 2 h of reperfusion, muscle recovered to significantly greater than
baseline values (P ⬍ 0.05). Total glycogen values were
also greater in the OP and OE rats following I/R compared with the R-sham rats (P ⬍ 0.05) (see Table 3).
These changes in muscle lactate and glycogen are indicative and characteristic of the occurrence of I/R.
cause a decrease in spontaneous cage activity, hence
the greater body weight in OP animals (11, 12). Furthermore, estrogen can downregulate the growth hormone-IGF-1 axis (11, 12). There was a positive relationship between serum IGF-1 content and body
weight. Serum IGF-1 content was greatest in the OP
rats and lowest the OE rats, which incidentally also
had the greatest serum estrogen content. The serum
IGF-1 values obtained by us are similar to those reported by others in normal and ovariectomized rats
(12).
Although serum estrogen content was ⬃10-fold
greater in the OE animals compared with the OP and
R animals (P ⬍ 0.05), there was no significant difference among the OP animals and the R animals. This
lack of difference could not be attributed to the cessation of estrus in the R rats. Circulating estrogen content in OP rats has been reported to be similar to the
low end of physiological estrogen fluctuations in R rats
(27, 43). The estrogen values seen in our OP rats are
similar to those in other studies (27, 41). However, a
wide range of circulating estrogen values for normal,
female rats has been reported in the literature. Similar
to our findings, Persky et al. (27) found normal female
rats (equivalent to our R rats) to have circulating
estrogen values of 15–20 pg/ml. Zhai et al. (46) measured mean estrogen values of ⬃60 pg/ml, whereas
others have reported values as high as 285 pg/ml (23).
These discrepancies have, to our knowledge, not been
fully addressed and may influence markers of muscle
damage following ischemia and reperfusion.
Serum CK activity was elevated in the R-I/R rats but
not in the R-I only rats. The latter is likely due to
circulatory occlusion limiting CK efflux from muscle.
After I/R, serum CK activity was greater in the R rats
compared with the OE and OP rats. The protective
effects of both estrogen and IGF-1 may account for this
finding. IGF-1 has been shown to attenuate CK release
from cardiac muscle following I/R (14) and from muscle
that has been oxidatively stressed (with 2,4-dinitrophenyl) in vitro.
In the R rats, calpain-like activity was increased
after both ischemia only and I/R relative to R-sham.
Prolonged ischemia and I/R have been shown to disrupt calcium handling and increase intracellular calcium content in cardiac and skeletal muscle (46). In-
EFFECTS OF OVARIECTOMY AND ESTROGEN ON MUSCLE DAMAGE
J Appl Physiol • VOL
reperfusion in female rats (17) and after 4 h of ischemia
and 4 h of reperfusion in male rats (9).
The number of CD43⫹ cells/mm2 tissue in the R, OP,
and OE rats was significantly elevated after I/R compared with that shown in the R-I only rats. Similar to
our MPO results, the number of CD43⫹ cells was lower
in the OE rats compared with the OP rats. Thus estrogen appears to attenuate leukocyte infiltration into
skeletal muscle following I/R. However, other factors
may confound the relationship between estrogen,
ovariectomy, and inflammatory cell infiltration. Circulating estrogen values were similar in the OP rats and
the R rats; however, after I/R, the number of CD43⫹
cells was almost twofold greater in the OP rats compared with the OE rats. Furthermore, the number of
CD43⫹ cells was similar in the OE rats and the R rats,
despite an ⬃10-fold difference in circulating estrogen
content. Although circulating IGF-1 levels were elevated in the OP rats, the effect of this on post-I/R
skeletal muscle leukocyte infiltration cannot be determined from this study.
Our MPO data (as an index of muscle neutrophil
infiltration) did not always correlate exactly with our
CD43⫹ immunohistochemically determined neutrophil counts in muscle tissue. Although MPO data have
often been used as indirect markers of muscle neutrophil infiltration (3, 33, 40, 41), these data may not be as
accurate as direct histochemical determination of
neutrophil presence in muscle (32). We have also previously seen larger changes in postexercise muscle
neutrophil infiltration via direct count using histochemistry than inferred by changes to muscle MPO
activity (41). It is also possible that differences in
muscle fiber type may influence post-I/R neutrophil
infiltration and account for some of the discrepancy in
our MPO data [determined in PLT (mixed muscle)] and
CD43⫹ data [determined in Sol (slow muscle)].
A compelling reason for measuring both calpain-like
activity as well as neutrophil infiltration is the hypothesis put forward by Belcastro et al. (4), which suggests
that calpain may stimulate neutrophil infiltration following skeletal muscle damage. A positive correlation
between elevations in calpain-like and MPO activities
in cardiac and skeletal muscle has been observed in
rats after 1 h of treadmill running (24). Furthermore,
cleaved peptide fragments of calpain have been shown
to have neutrophil chemotactic potential (19, 20). We
have previously reported a correspondence between
estrogen administration to ovariectomized rats and
attenuation of both calpain activity and neutrophil
infiltration 1 h after running exercise in female rats
(41). OE rats did have attenuated muscle calpain activity compared with OP rats following I/R. However,
with overall results using the rodent hindlimb I/R
model, no correlation between calpain-like and MPO
activities or CD43⫹ cell counts was observed. It appears that there are important differences between
exercise-induced muscle damage and that caused by
I/R as it relates to induction of calpain-like activity.
Lipid peroxidation was assessed using the TBARS
assay. In the R rats, TBARS content in muscle did not
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creases in intracellular calcium can activate calpain (2,
38). Elevated ␮-calpain and m-calpain activities following I/R have been measured in cardiac muscle (42, 45).
Skeletal muscle calpain activity has also been reported
to be elevated after acute exercise (3, 41). This is the
first report, to our knowledge, that demonstrates increased calpain activity in skeletal muscle in vivo following hindlimb I/R.
Both ovariectomy and the estrogen pellet implant
appear to attenuate calpain-like activity following I/R.
Calpain-like activity was lower in the OE and the OP
rats following I/R compared with the R-I only rats (P ⬍
0.05) and the R-I/R rats (P ⬍ 0.05). Following I/R,
calpain-like activity was similar in the OE and OP
group (P ⫽ 0.57). Decreased calpain-like activity in the
OP rats compared with the R rats may be due to
increased IGF-1 expression following ovariectomy. In a
murine model of muscular dystrophy, IGF-1 administration (in conjunction with a high-protein diet) resulted in an attenuation in skeletal muscle calpain
activity (44). The mechanism by which estrogen could
attenuate calpain activity is unknown. Estrogen may
act directly on muscle membranes to preserve ultrastructural stability and intracellular calcium homeostasis (39, 41). We have previously reported that
estrogen replacement in ovariectomized rats attenuates postexercise calpain activity in skeletal muscle
(41).
Inflammatory cell infiltration into skeletal muscle
was assessed using the MPO assay and immunohistochemical staining for CD43. MPO activity was similar
among the R-sham, R-I only, R-I/R, and OP rats, but it
was significantly lower in the OE rats (P ⬍ 0.05).
Estrogen has been shown to attenuate leukocyte adhesion in cerebral blood vessels both under resting conditions and after I/R (31). Furthermore, after exercise,
estrogen administration has been shown to attenuate
MPO activity and inflammatory cell infiltration in
male and ovariectomized female rats (40, 41). However, others have suggested that estrogen does not
attenuate inflammatory cell infiltration; rather, it
merely delays it (35). Estrogen decreases adhesion
molecule expression and increases endothelial and
neuronal NO synthase content (5). Nitric oxide produced by endothelial and neuronal NO synthase has
potent anti-adhesive properties (5). Infiltrating neutrophils as well as leukocyte adhesion may contribute to
total MPO activity. Thus the attenuation in MPO activity observed in the OE group may also reflect decreased leukocyte adhesion to blood vessels due to
estrogen administration.
MPO activity did not increase in the R rats following
ischemia only or I/R. There are conflicting reports in
the literature about whether MPO activity does increase in skeletal muscle after I/R. Increases in MPO
activity have been measured after 4 h of hindlimb
ischemia and 1 h of reperfusion in mongrel dogs (33)
and after 2 h of ischemia and 2 h of reperfusion in male
rats (28). Others, however, did not detect elevations in
MPO activity after 30 min of ischemia and 1 h of
1833
1834
EFFECTS OF OVARIECTOMY AND ESTROGEN ON MUSCLE DAMAGE
We thank M. Burnett, D. McCutcheon, and Dr. R. Tupling for
advice and technical assistance with this study. We also thank Dr. H.
Green for use of the rat hindlimb I/R equipment and Dr. M. Tarnopolsky for laboratory facilities used for histochemistry.
This study was supported by a research grant from the Natural
Sciences and Engineering Research Council of Canada to P. M. Tiidus.
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