Structural Alterations of Skeletal Muscle Induced by Ischemia
and Reperfusion
Hans J. Appell(1, 2) , Sabine Glöser(2), José M.C. Soares (1, 3) and José A. Duarte (1, 3)
(1) The Muscle Atrophy Research Group (MARG), (2) Institute of Sport Orthopedics, German Sport University Cologne, Germany and (3) Department of Sport Biology, FCDEF, University of Porto, Portugal
Abstract
The purpose of the study was to undertake a series of experiments with varying time periods of ischemia followed by reperfusion to investigate the effects on muscle morphology.
Charles River mice received a tourniquet at their right limb for 60, 90, and 120 minutes, respectively, under general anesthesia which was followed by a reperfusion period of 60
minutes (n = 5 in each group). Their soleus muscles were removed, the left one serving as
contralateral control, and processed for light and electron microscopic examination. Muscle
fiber cross sectional areas and the thickness of the capillaries basement membranes were
evaluated morphometrically. Capillary endothelial ultrastructure showed considerable pathological alterations extending to intracellular edema, and the basement membrane was
thickened by about 50%. The muscle fibers were reduced in size, which depended on the
period of ischemia (15-23%). Skeletal muscle morphology showed mitochondrial disturbances, condensation of contractile material, presence of lysosomes, and focal fiber necrosis, which also seemed to be aggravated the longer the ischemic period had lasted. The interstitial space was widened speaking in favor of an intramuscular edema. It is concluded
that skeletal muscle morphology is severely affected under conditions of ischemia/reperfusion and that the capillary endothelium is mainly involved in the pathophysiological mechanisms.
Key words : capillary endothelium, ischemia, muscle damage, reperfusion, skeletal muscle.
Basic Appl. Myol. 9 (5): 263-268, 1999
When orthopedic surgeons administer a tourniquet
allowing for surgery under bloodless conditions, the respective muscles suffer from ischemia and following
reperfusion (I/R). This harmful situation has been studied predominantly with regard to functional [13, 17, 26]
and metabolic [1, 6, 7, 8, 19, 27] alterations in muscle or
focussing on the vascular bed [4, 9, 23, 28, 29, 32]. I/R
is associated with capillary dysfunction that leads to an
increased permeability during reperfusion thus allowing
proteins to escape from the vascular bed; consequently
the muscles become edematous [8, 9, 16]. These effects
most probably originate from oxidative stress during
reperfusion that is being triggered during the ischemic
period by conversion of the endothelial enzyme xanthine dehydrogenase to xanthine oxidase [12, 22, 30].
Compared to other tissues, like cardiac and cerebral
tissues, skeletal muscle has been described to be relatively resistant to ischemia because the maintenance of
its metabolic capacity is assumed to cease only after
five to seven hours [7, 15, 19, 27]. However, an ultrastructural study on biopsies obtained during surgery, i.e.
after various periods (15 min to 90 min) of ischemia,
but without reperfusion, clearly pointed out that already
under such conditions skeletal muscle ultrastructure
showed eventual pathological alterations, especially
extending to metabolically important organelles [2]. It
can be expected that these alterations will be aggravated
during reperfusion. However, more recent studies dealing with the effects of I/R on skeletal muscle morphology did not bring about very detailed informations [10,
11, 24, 26, 31].
Since the harmful effects of reperfusion appear to be
preconditioned during the ischemic phase, as outlined
above, more reperfusion damage may be expected with
prolonged periods of ischemia. The aim of the present
set of experiments was to study various periods of ischemia (60, 90, 120 min) each followed by a 60 min period of reperfusion and to morphologically describe
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Muscle morphology after ischemia/reperfusion
skeletal muscle reactions. Moreover, some morphometric data on muscle fiber diameters and capillary wall
architecture may shed some light on the role of edema
development during I/R resembling a kind of compart ment syndrome [9].
Results
Regarding the morphometric data, there were no differences between the overall controls and the contralateral controls of the respective experimental groups. The
quantitative data for muscle fiber cross sectional area
and capillary basement membrane thickness are shown
in Table 1.
The fiber cross sectional areas underwent a progressive reduction with prolonged periods of ischemia. The
small standard deviations speak in favor of a homogeneous fiber size as well as a homogeneous reaction of
the whole fiber population. This reduction amounted to
15 to 23% of the control fiber size. Basement membrane
thickness of the capillary wall increased by more than
half, with some tendency for more thickening with prolonged experimental periods, which, however, could not
be substantiated statistically.
The microscopic specimens of the overall controls and
all contralateral control muscles completely showed a
normal appearance which also holds true for the microvascular bed. However, the capillary ultrastructure was
conspicuously altered in all experimental groups. Beyond the thickening of the endothelial basement me mbrane (Tab. 1), the endothelial cells appeared swollen
and contained large vacuoles resembling confluent vesicles. In some cases, the endothelial cells contained large
edematous blebs (Fig. 1). They sometimes showed
pseudopodia-like flaps protruding into the lumen. The
capillary lumen frequently appeared narrowed or collapsed, and fenestrations of the (normally) continuous
endothelium occurred (Fig. 2). Even endothelial gaps or
breaks could be observed. These alterations were already present in the 60 I / 60 R group and did not seem
to increase in severity or frequency with prolonged periods of ischemia.
The pathological alterations of the muscle fibers appeared to become more severe the longer the muscles
had been ischemic, as far as could be judged by the frequency and conspicuousness of their occurrence. Some
muscle fibers showed a moderate intrafiber edema,
which, however, stood back against an edematous widening of the interstitial space (Figs. 3, 4, 5, 6). This in-
Materials and Methods
The experiments were performed on Charles-River
mice (aged 8 weeks with a body weight of 35-40 g) under standardized conditions with regard to housing environment and feeding and following the institutional
guidelines for the care and use of laboratory animals.
Three groups of mice (n = 5) received a tourniquet (rubber cuff) at the proximal part of their right hindlimb
taking several precautions into consideration [25] for
60, 90, or 120 minutes; respectively, and these periods
of ischemia were followed by a 60 min. reperfusion period after removal of the cuff. All experiments were
done under sodium-pentobarbital anesthesia (i.p. 50
mg/kg b.w.). One group of mice (n = 5) without any experimental intervention served as the overall control
group. The animals were finally sacrificed by cervical
dislocation after both soleus muscles had been completely removed, the right one serving as experimental
and the left one as intragroup control muscle.
Each muscle was divided into three pieces maintain ing constant muscle length and these were transferred to
2.5% glutaraldehyde for two hours. The specimen were
postfixed with 1% osmiumtetroxide, dehydrated in
graded alcohol, contrasted with 0.5% uranylacetate, and
embedded in Epon. Semithin sections for light microscopy (Leitz) were stained with toluidine blue, ultrathin
sections for electron microscopy (Zeiss EM 10A) were
contrasted with 0.2% lead citrate.
Morphometry was performed using the MOP Videoplan (Kontron). From every specimen, at least 300 fibers were measured for fiber area in cross section at a
light microscopic magnification of x250. To ensure the
evaluation of proper cross sections, two fiber diameters
were also measured (feret X, feret Y); the area measurements were only considered for further calculation, if
feret X and feret Y did not differ more than 10%. Electron micrographs from cross sectioned capillaries (20
capillaries per sample) were used for estimation of the
thickness of the vascular basement membrane at 4 randomly chosen capillary wall sites at primary magnifications of x10,000.
All quantitative data were expressed as means with
standard deviations after transformation into percentages from controls. For statistical analysis of percentage
differences from controls the Student t-test was applied.
For differences between the groups, the one-way
ANOVA for repeated measurements was used with post
hoc comparison using the Scheffe F-test. The level of
significance was set at an alpha of 5%.
Table 1. Means and standard deviations of the percentage
changes as compared to the contralateral controls
(= 100) for muscle fiber cross sectional areas and
capillary basement membrane thickness after
varying periods (min) of ischemia (I) and 60 min
reperfusion (R). Significant differences: § vs. contralateral control, # vs. 60 I / 60 R, + vs. 90 I / 60
R..
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Experimental
Group
60 I / 60 R
90 I / 60 R
Fiber
Area
85.1 ± 1.1 §
79.5 ± 2.3 § #
Basement
Membrane Thickness
152.2 ± 9.3 §
162.6 ± 9.6 §
Muscle morphology after ischemia/reperfusion
Figure 1. Electron miscrograph (90 I / 60 R) of a capillary with intraendothelial edematous blebs; bar
represents 2 µm.
Figure 3. Electron micrograph from a 60 I / 60 R muscle
with wide interstitual space and a lysosome in the
muscle fiber, the insert shows residual bodies
from a 120 I / 60 R muscle; bars represent 2 µm.
Figure 2. Electron micrograph (60 I / 60 R) of a narrowed capillary with fenestrated endothelium
(insert) surrounded by a thickened basement
membrane; bars represent 2 µm and 0.5 µm in
the insert.
terstitial edema seemed to become more pronounced
with longer periods of ischemia. The muscle fibers of
the 60 I / 60 R group showed a light vacuolization
mostly in their peripheral parts that originated from destructed mitochondria and contained some lysosomes.
Moreover, residual bodies were frequently found with
prolonged periods of ischemia (Fig. 3). Some fibers appeared necrotic with a complete disorganization of their
internal structures or showed peripherally condensed
contractile material while the fiber center was filled
with structureless precipitates (Fig. 4). In the 90 I / 60 R
group, such was encountered frequently with various
patterns of complete internal destruction of the fibers
(Fig. 5). With longer periods of ischemia (120 I / 60 R),
even more necrotic fibers were found (Fig. 6) contain-
Figure 4. Light micrograph (60 I / 60 R, above) with
two severly altered muscle fibers and insterstitial
edema, an electron micrograph of the marked
portion is shown below; bars represent 50 µm
(LM) and 5 µm (EM).
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Muscle morphology after ischemia/reperfusion
Figure 7. Electron micrograph (120 I / 60 R) of a necrotic muscle fiber with abundant lysosomes and
a phagocyte in its lower right corner; bar represents 10 µm.
Figure 5. Light micrograph (90 I / 60 R) of a necrotic
muscle fiber, its electron microscopic appearance is shown below; bars represent 30 µm (LM)
and 3 µm (EM).
Figure 6. Light micrograph (120 I / 60 R) showing three
necrotic muscle fibers and interstitial edema; bar
represents 40 µm.
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ing many lysosomes at the EM level, and phagocytes
were present in the vicinity or within those fibers (Fig.
7).
Discussion
In accordance with current metabolic concepts on ischemia/reperfusion [8, 12, 22, 30] the blood capillaries
are among the first structures to suffer from oxidative
stress during reperfusion which at least partly should be
derived from xanthine oxidase. Already during short
periods (15 min.) of ischemia (without reperfusion),
capillary obstruction and endothelial swelling has been
described [2]. This observation may be attributed to alterations in ionic exchanges across the endothelial
membrane [20], which is assumed to be a major cause
for microcirculatory failure during postischemic reperfusion [23]. Endothelial pathology is further manifested
by an impaired structural integrity of the microvascular
bed. Especially the occurrence of fenestrated or gap
capillaries is most uncommon in skeletal muscle [14].
Such results in an escape of proteins and other macro molecules from the bloodstream into the interstitial
space. The enlargement of the endothelial basement
membrane might be interpreted as a (yet obsolete) defense mechanism of the capillaries against increased
extravascular pressure [14], but should rather be looked
at as the morphological correlate of functionally puffed
constituents of the basement membrane due to extravasal water accumulation. Macromolecular leakage has
mainly been held responsible for the I/R tissue injury
Muscle morphology after ischemia/reperfusion
[16] and results in an increased osmotic pressure in the
interstitial space that is being aggravated during reperfusion with an edematous water accumulation. Especially for muscles contained in a tight fascial compart ment, such will result in a compartment syndrome [9].
Such, together with the “no reflow” phenomenon, may
lead to secondary ischemia during reperfusion [23].
These considerations receive support from earlier
studies using the same experimental protocol as present
[8] where the muscle wet weight increased by up to
90% and the protein content of muscle increased by up
to 40%. This is morphologically reflected by an edematous enlargement of the interstitial space and by the
morphometric muscle fiber data. It could have been expected that the muscle fibers, like endothelial cells, in creased in volume because of homeostatic disturbances
leading to intracellular edema. This mechanism cannot
be ruled out, but is most probably masked by the higher
osmotic pressure in the interstitial space forcing water to
escape from the muscle fibers. Consequently, the cross
sectional areas of the muscle fibers were reduced, and
this effect was increasing with the period of ischemia.
This finding correlates with earlier observations on the
intensity of oxidative stress and muscle protein content
as a temporal function of the ischemic period [8].
Since the formation of reactive oxygen species during
ischemia and reperfusion is assumed to play a major
role, several attempts have been made to reduce I/R
damage by administration of antioxidants or inhibitors
of xanthine oxidase (XO). The efficacy of allopurinol as
an XO inhibitor has been controversially reported in the
literature. While some experiments showed a protective
effect against oxidative stress and against muscle damage [1, 10], another study did not show major protective
effects [18]. Vitamin E as a radical scavenger, irrespective of the origin of reactive oxygen species, has been
successfully used in various I/R experiments [1, 11, 24].
In a series of experiments with the same experimental
protocol, administration of Vitamin E did not only prevent the muscles from oxidative stress, but especially
from endothelial damage, intramuscular edema. and
major muscle fiber damage [1].
Although the present pathological alterations finally
entering into fiber necrosis have not been quantified for
methodological reasons, it appears that the longer the
ischemic period had lasted the more the muscle fibers
were affected. Mitochondrial destruction, condensation
of the contractile material, and occurrence of lysosomes
probably represent a pathophysiological cascade. The
formation of reactive oxygen species, increasing with
time of ischemia [8] also affects the muscle fibers and
especially their sarcolemmal and mitochondrial me mbrane systems [24]. Consequently, an impaired sarcolemmal integrity facilitates an influx of calcium ions
into the muscle fibers leading to a loss of calcium ho -
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meostasis; this promotes the condensation of contractile
material and mitochondrial disturbances and also activates the lysosomal system entering into an autolytic
process [3, 21].
It seems, however, surprising that under the experimental conditions not all muscle fibers underwent the
described signs of pathological alterations and necrosis
to the same or a comparable extent. Similar phenomena
hold true for other types of muscle damage induced by
aggressive experimental protocols. In accordance with
recent concepts [5], the observed cellular damage may
have occurred only in an especially susceptible fiber
population. In this sense, the homeostatic disturbances
cannot sufficiently be tolerated by those fibers, while
other fibers obviously are more resistant. It remains unclear whether fiber necrosis represents a direct effect of
the above described mechanisms induced by ischemia
and reperfusion or whether some fibers enter into a programmed cell death (apoptosis) under such circumstances.
Address correspondence to:
Prof. Dr. Hans-Joachim Appell, Institute of Sport Orthopedics, German Sport University, D-50927 Cologne,
Germany, phone +49 221 4982543, fax +49 221 49
12001, Email [email protected].
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