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1 Metrohealth Medical Center, Free radicals are
known to play an important role in modulating the development of
respiratory muscle dysfunction during sepsis. Moreover,
neutrophil numbers increase in the diaphragm after endotoxin administration. Whether or not superoxide derived from infiltrating white blood cells contributes to muscle dysfunction during sepsis is,
however, unknown. The purpose of the present study was to examine the
effect of apocynin, an inhibitor of the superoxide-generating neutrophil NADPH complex, on endotoxin-induced diaphragmatic
dysfunction. We studied groups of rats given saline, endotoxin,
apocynin, or both endotoxin and apocynin. Animals were killed 18 h
after injection, a portion of the diaphragm was used to assess force
generation, and the remaining diaphragm was used for determination of
4-hydroxynonenal (a marker of lipid peroxidation) and nitrotyrosine
levels (a marker of free radical-mediated protein modification). We
found that endotoxin reduced diaphragm force generation and that
apocynin partially prevented this decrease [e.g., force in
response to 20 Hz was 23 ± 1 (SE), 12 ± 2, 23 ± 1, and 19 ± 1 N/cm2, respectively, for
saline, endotoxin, apocynin, and endotoxin/apocynin groups;
P < 0.001]. Apocynin also
prevented endotoxin-mediated increases in diaphragm 4-hydroxynonenal
and nitrotyrosine levels (P < 0.01).
These data suggest that neutrophil-derived free radicals contribute to
diaphragmatic dysfunction during sepsis.
free radicals; skeletal muscle; diaphragm; respiratory muscles
A NUMBER OF RECENT STUDIES have indicated that free
radicals play an important role in modulating the induction of
respiratory muscle dysfunction during endotoxin-induced sepsis (10, 13, 14, 17). In the first of these studies, Shindoh et al. (10) demonstrated that endotoxin administration to hamsters was associated with both the development of contractile dysfunction and an increase in
lipid peroxidation within the diaphragm. In addition, these investigators found that administration of polyethylene
glycol-superoxide dismutase (a free radical scavenger) attenuated both
the diaphragmatic contractile dysfunction and lipid peroxidation
induced by endotoxin (10). In subsequent reports, it has been
demonstrated that administration of a number of other free radical
scavengers (e.g., N-acetylcysteine, catalase, dimethylsulfoxide) also protects the diaphragm from the
development of endotoxin-mediated respiratory muscle dysfunction and
lipid peroxidation (13, 14, 17).
The source of the oxygen-derived free radicals responsible for altering
respiratory muscle dysfunction after endotoxin administration has not,
however, been determined. One possibility is that diaphragmatic constituents (i.e., myocytes, endothelial cells) are altered in such a
way after endotoxin administration that these cells generate free
radicals, and the diaphragm, in effect, is damaged by its own metabolic
by-products. Another possibility is that activated white blood cells in
the diaphragm vasculature or interstitium provide an exogenous source
of damaging free radicals. In support of this latter possibility, we
have recently shown that both intravascular and interstitial neutrophil
numbers are markedly increased in the diaphragm after endotoxin
administration (15).
If white blood cells play a role in mediating free radical-related
diaphragmatic dysfunction after endotoxin administration, then agents
that inhibit white cell free radical formation should, in theory, also
reduce endotoxin-mediated diaphragmatic dysfunction. The purpose of the
present study was to test this hypothesis by examining the effect of
apocynin, an inhibitor of the neutrophil cell surface NADPH oxidase
complex, on endotoxin-induced diaphragmatic dysfunction (6, 11). We
studied groups of rats given saline, endotoxin, apocynin, or both
endotoxin and apocynin. Animals were killed 18 h after injection of
these substances, and a portion of the diaphragm was used to assess
muscle physiology. The remaining diaphragm was examined for white cell
infiltration (by using histochemical techniques), 4-hydroxynonenal
(4-HNE) formation (a marker of lipid peroxidation), and nitrotyrosine
formation (a marker of free radical-mediated protein modification).
Protocol.
Studies were performed on muscles taken from 25 adult (~6-mo-old)
male Sprague-Dawley rats weighing 400-500 g, which were cared for
in the Case Western Reserve University Animal Resource Center in
accordance with Association for Assessment and Accreditation of
Laboratory Animal Care guidelines. Diaphragms were studied from animals assigned to one of four groups:
1) a control group of animals that
received a 1-ml intraperitoneal injection of saline 18 h before death,
2) a group that received 1 ml of
saline containing 8 mg/kg endotoxin intraperitoneally 18 h before
death, 3) a group that received an
intraperitoneal injection of 4 mg/kg apocynin in 1 ml of saline 18 h
before death, and 4) a group that
received a mixture of both apocynin and endotoxin as an intraperitoneal injection 18 h before death. All groups also received a subcutaneous fluid bolus of 4 ml/kg 18 h before death (to minimize dehydration).
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70°C for subsequent immunoblot analysis. The remaining
portion of the right hemidiaphragm was fixed overnight with 4%
paraformaldehyde in 0.1 mM cacodylic buffer (pH 7.4). The
paraformaldehyde-fixed samples were embedded in OCT resin, frozen in
liquid nitrogen-cooled isopentane, and stored for later histological examination.
Diaphragmatic force-generating capacity. Small fiber bundle strips were dissected from the left hemidiaphragm for the purpose of assessing diaphragm force-generating capacity (15). When this dissection was performed, care was taken both to include the central tendinous portion of the diaphragm and also to include that portion of the ribs into which the diaphragm strip inserted. Muscle strips were then mounted vertically in a water-jacketed organ bath (Radnotti Glass, Monrovia, CA) filled with oxygenated Krebs-Henseleit solution containing 50 mM curare (pH.7.4 at 27°C). The rib end of each strip was attached to a hook at the bottom of the bath, whereas the central tendon end of each strip was tied to a metal rod connected to a vertically adjustable force transducer (Grass FT 10) fixed above the organ bath. A platinum mesh field electrode was placed on either side of each muscle strip, and these electrodes were connected to a constant-current amplifier (Biomedical Technology, Cleveland, OH) attached to a Grass S-48 stimulator.
Muscle strip length was then adjusted to optimum length (i.e., that length resulting in maximal force output), and current was adjusted to a supramaximal level (i.e., to 120% of the current required to attain maximal twitch force generation). Muscle strips were then allowed to equilibrate for a period of 10 min before the assessment of diaphragm force-generating capacity. After this equilibration period, twitch kinetics were assessed, and diaphragms were then sequentially stimulated with trains (800-ms duration) of 1-, 10-, 20-, 50-, and 100-Hz stimuli with a 5-s rest between adjacent trains. After an additional 30-s rest, each muscle strip underwent a 5-min repetitive stimulation trial (a test to assess muscle fatigability), consisting of contraction in response to trains of 20-Hz stimuli, with a train duration of 500 ms and a train rate of 15/min. Diaphragm strip length and weight were recorded after each experiment and used to normalize muscle force to cross-sectional area (4).Detection of white blood cells.
To assess neutrophil infiltration into the diaphragm, ultrathin muscle
sections (4 µm) were made from OCT-embedded diaphragm samples by
using a cryostat cooled to 20°C. These cross sections were
then stained for white blood cells by using Leukostat staining solutions (Fisher Scientific) (18). Cross sections were then viewed
under oil immersion at ×100 magnification, and white blood cells
were counted in 30 random fields per section. The location (intravascular or extravascular) of each white blood cell was also recorded.
Assessment of lipid peroxidation and protein nitrosylation. Lipid peroxidation and protein nitrosylation within the diaphragm were assessed by using immunoblot techniques (18-20). To assess lipid peroxidation, we quantified tissue levels of 4-HNE using rabbit polyclonal anti-4-HNE antibodies (supplied by Dr. L. Szweda, Case Western Reserve Univ.). To assess formation of nitrotyrosine, a reaction product of peroxynitrite with tyrosine residues in proteins, diaphragm muscle homogenates were incubated with rabbit polyclonal anti-nitrotyrosine antibodies (Upstate Biochemistry, Lake Placid, NY). The following is a description of this technique.
First, diaphragm muscle samples were vigorously homogenized in ice-cold 62.5 mM Tris buffer (pH 6.8) at 1:1 (wt/vol). The protein content of each homogenate was measured (Bio-Rad protein assay), and homogenate volume was adjusted so as to achieve a protein concentration of 0.5 mg/1 ml. After this step, all procedures were performed in duplicate. We next applied aliquots of tissue homogenates containing 50 µg of protein to freshly washed nitrocellulose membranes and allowed these samples to dry, leaving proteins bound to membranes. Blotted membranes were then washed with 5% milk in Tris-buffered saline (TBS) for 1 h with constant agitation to block nonspecific protein binding. After blocking, membranes were washed an additional two times in TBS. Membranes were incubated overnight with agitation and at 4°C in TBS and milk containing either anti-4-HNE polyclonal IgG (1:500) or anti-nitrotyrosine polyclonal IgG (at 2 µg/ml). Membranes were then washed twice in TBS and then incubated for 1.5 h with secondary antibodies (goat anti-rabbit) conjugated to horseradish peroxidase. After the membranes were repeatedly washed in TBS, 4-HNE and nitrotyrosine antibody staining was visualized by developing the membranes in a horseradish peroxidase detection solution (Calbiochem, La Jolla, CA) for 15 min. Immunoblotted membranes were allowed to dry, membrane images were then digitized by using a flatbed scanner, and scanned images were stored in a computer for later quantitative analysis. The average intensity of each individual blot was measured by using densitometry software (Sigma Scan Image, Jandel, San Rafael, CA); nonspecific background shading was subtracted when this image analysis was performed. The average densitometric intensity of each sample was expressed as a ratio to sample protein content, and this value was used as an index of 4-HNE or nitrotyrosine formation (depending on which antibody was used in a given assessment) in tissue samples.Statistical analysis. A one-way ANOVA was used to compare single variables across animal groups, with post hoc testing used to determine statistical differences between individual groups.
A repeated-measures ANOVA was used for comparisons in which repeated measurements of a given variable were made under different conditions (e.g., force-frequency curves from different groups). Data are presented as means ± 1 SE. A P value < 0.05 was taken to indicate statistical significance.| |
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Force production by in vitro diaphragm strips.
Diaphragm twitch contraction time was not affected by either endotoxin
or apocynin administration, as shown in Table
1. Similarly, twitch relaxation time was
also similar across the four experimental groups (Table 1).
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Quantification of diaphragm white cell numbers.
There were virtually no white blood cells found in extravascular
regions of diaphragms taken from saline-treated control animals and
only a small number of intravascular white blood cells in tissue
sections from this group. In contrast, white blood cells were readily
found in the perivascular regions of muscles taken from
endotoxin-treated animals (see Figs. 2 and
3), and somewhat higher numbers of
intravascular white blood cells were also found in diaphragms of
endotoxin-treated animals compared with saline-treated controls. On
average, total white cell numbers (i.e., the sum of intra- and
extravascular white blood cells) were fivefold greater for endotoxin-
compared with saline-treated animals
(P < 0.001), and the extravascular
white cell number was 27-fold greater for the endotoxin group compared
with the saline group (P < 0.002).
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Lipid peroxidation and protein nitrosylation in the diaphragm.
Both 4-HNE and nitrotyrosine concentrations were increased for
diaphragms from endotoxin-treated animals compared with saline-treated controls (see Figs. 4 and 5
for 4-HNE and Figs. 6 and 7 for
nitrotyrosine representative example and group mean data,
respectively). On average, 4-HNE, an index of lipid
peroxidation, was 99% higher in diaphragms of the endotoxin group
compared with saline-treated controls, whereas nitrotyrosine, an index
of peroxynitrite-mediated protein modification, was 88% higher in the
endotoxin group compared with saline controls
(P < 0.01). Apocynin administration
blunted the effects of endotoxin, reducing nitrotyrosine and 4-HNE to the levels seen in diaphragms from saline-treated control animals (P < 0.001 for comparison of
nitrotyrosine levels for apocynin/endotoxin-treated animals and
endotoxin-treated animals; P < 0.001 for comparison of 4-HNE levels in these two groups).
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Overview of major findings. This study found that 1) endotoxin administration to rats elicited a reduction in the force-generating capacity of excised diaphragmatic muscle strips; 2) this reduction in force was accompanied by biochemical alterations indicating increased lipid peroxidation in the septic diaphragm and formation of nitrotyrosine side groups, an index of peroxynitrite-mediated protein modification; 3) muscles from endotoxin-treated animals contained increased numbers of white blood cells; 4) administration of apocynin reduced endotoxin-related alterations in force and muscle biochemistry; and 5) white blood cells were also present in the diaphragm of apocynin-treated animals, albeit at lower concentrations than for animals given endotoxin alone.
Comparison with previous studies. A number of previous experiments have examined the relationship between infection and respiratory muscle function. One of the first of these was a study by Hussain et al. (7) that found that endotoxin administration produced a profound reduction in diaphragmatic force-generating capacity. In fact, this study noted that nonventilated dogs given endotoxin developed respiratory failure and subsequently died because of respiratory muscle dysfunction; i.e., diaphragm force-generating capacity became depressed in these animals before significant alterations in lung mechanics developed.
Further investigation by Boczkowski et al. (2) and Shindoh et al. (10) confirmed the fact that endotoxin administration depressed diaphragm contractile function and demonstrated, in addition, that much of this contractile dysfunction could be prevented by giving animals free radical scavengers at the time of endotoxin administration. These latter two studies found that the development of contractile dysfunction was associated with evidence of increased lipid peroxidation in the respiratory muscles and that these lipid biochemical modifications induced by endotoxin administration were also prevented by the administration of free radical scavengers. Additional work has revealed the fact that scavengers of several free radical species (superoxide dismutase, catalase, dimethylsulfoxide, N-acetylcysteine) and inhibitors of nitric oxide synthesis can each inhibit sepsis-induced diaphragmatic dysfunction, suggesting that a number of free radical species (i.e., superoxide anions, hydrogen peroxide, hydroxyl radicals, peroxynitrite anions) may interact to induce endotoxin-related respiratory muscle dysfunction (3, 13, 14). The present study investigated the possibility that free radical-mediated muscle dysfunction may occur as a consequence of white blood cell infiltration into the diaphragm. Apocynin is a potent inhibitor of the free radical-generating NADPH enzyme complex on the neutrophil surface and has been used previously as a probe to identify the contribution of neutrophil-mediated free radical formation to different forms of tissue injury (6, 11, 20). This agent is thought to ablate white cell free radical generation without inhibiting white cell phagocytosis, exocytosis, or intracellular killing (20) and is thought to act only on activated phagocytes (6, 11). In one recent study, this agent was shown to inhibit white blood cell-mediated lung damage during sepsis (as assessed by examining lung permeability to albumin) at doses similar to those employed in the present study, arguing that in vivo free radical generation by white blood cells is effectively inhibited by this agent at this particular dosage. It should also be noted that we have previously shown that apocynin administration does not alter contraction-related formation of free radicals in the diaphragm, suggesting that this agent does not affect myocyte-specific pathways of free radical formation (8). In the present work, apocynin administration both produced a degree of protection against the effects of endotoxin on diaphragmatic dysfunction and reduced the degree of endotoxin-related lipid peroxidation (one marker of free radical-mediated alteration of lipid cellular components) and endotoxin-related nitrotyrosine formation in the diaphragm (one marker of free radical-mediated alteration of protein cellular components). Although it is likely that one mechanism by which apocynin produced this effect was by directly preventing neutrophil generation of free radicals in muscle, this agent also markedly reduced migration of white blood cells into the diaphragm. This finding is consistent with previous reports examining white blood cell infiltration into the lung in sepsis and in other tissues during ischemia-reperfusion (5, 20). Specifically, Wang et al. (20) found that apocynin administration inhibited movement of white blood cells into the guinea pig lung during endotoxin-induced sepsis. The mechanism by which white blood cells move into tissue during sepsis appears to be due to three processes: 1) cytokine-mediated effects on endothelium to increase expression of adhesion molecules, 2) cytokine effects to activate white blood cells and potentiate white cell adhesion and diapedesis, and 3) an effect of free radicals to amplify the first two processes (20). The third factor acts, essentially, as a "positive feedback" loop, with an initial white cell free radical release leading to additional white cell recruitment and additional free radical formation (20). Apocynin is thought to inhibit white blood cell recruitment by preventing this third process, i.e., reducing neutrophil-mediated alterations in endothelium, subsequently reducing white blood cell attachment and infiltration (20).Comparison with limb muscle studies. The present report is the first to identify a potential role for white blood cells in mediating respiratory muscle dysfunction in sepsis. Whereas no previous study has examined the effects of apocynin in limb muscle, several reports have provided evidence that white blood cells may contribute to the induction of limb muscle functional derangements in sepsis and other pathophysiological states (5, 12). A few reports have presented evidence of neutrophil accumulation in limb muscles during systemic bacterial infections (9). Neutrophil infiltration also appears to be a contributor to the development of limb muscle injury after periods of ischemia-reperfusion (12). In this latter condition, tissue myeloperoxidase concentrations (a marker of white cell degranulation) were found to be increased after ischemia-reperfusion, with a correlation between tissue myeloperoxidase levels and the degree of tissue injury. The "late" phase of muscle damage seen after bouts of eccentric exercise also appears to be modulated, at least in part, by neutrophil infiltration (1).
Interaction between free radical-generating pathways in sepsis. The present findings suggest that an appreciable component of endotoxin-induced respiratory muscle dysfunction is due to free radicals generated by an apocynin-sensitive NADPH complex. We believe that our findings cannot be explained by an effect of apocynin to block myocyte-derived free radical formation because 1) we have previously shown that apocynin administration does not alter contraction-related formation of free radicals in the diaphragm (8), and 2) in recent work (unpublished observations), we have found that there is heightened free radical formation by the mitochondria of both diaphragm myocytes and sepsis that cannot be reduced by administration of agents that inhibit NADPH oxidase. We, therefore, believe that the present findings are most consistent with an effect of apocynin to inhibit white blood cell-mediated free radical formation.
We should emphasize, however, that the present findings do not preclude the participation of multiple pathways of free radical formation to respiratory muscle dysfunction in sepsis. In fact, a number of other sources of radical species have been reported. For example, it is known that endotoxin administration causes cytokine-mediated increases in nitric oxide synthase (NOS) levels in the respiratory muscles (2), and it is possible that heightened nitric oxide formation leads to peroxynitrite formation and tissue damage, in part, by this latter mechanism. In addition, we have also shown that diaphragmatic muscles taken from septic muscles contracting in vitro generate greater levels of reactive oxygen species (i.e., superoxide anions, hydrogen peroxide) than do control muscles, indicating that contraction-related free radical generation is upregulated in septic muscles (8). The superoxide anions produced by muscle contraction could, in theory, cause muscle damage either directly or by providing substrate for reactions producing hydroxyl and peroxynitrite anions. As a result, it appears that free radical production in the diaphragm in sepsis occurs by at least three different mechanisms (an apocynin-sensitive component, free radical generation by contracting myocytes, enhanced nitric oxide formation because of NOS upregulation). It is difficult, at this point, to elucidate the relative contributions of these various pathways. The degree of inhibition of diaphragm dysfunction in the present study was striking (i.e., force at 20 Hz was increased by 90% in animals given apocynin and endotoxin compared with the endotoxin-alone group), but previous reports have indicated that equally striking protection from endotoxin-induced muscle dysfunction can be achieved by administration of either NG-nitro-L-arginine methyl ester (this agent was found to increase 20 Hz force by 90%) or a host of free radical scavengers (i.e., 80% increases have been reported in response to treatment with catalase) (3, 10, 13, 14). Moreover, neither this nor previous studies have examined dose-response relationships for any of these agents; therefore, the maximum potential "benefit" that could accrue by inhibiting various pathways is not known. It is possible, however, that a synergism exists in terms of cellular damage by free radical species generated by these different mechanisms. It seems possible that free radical species generated by white blood cells may interact with species formed by other mechanisms (e.g., with nitric oxide formed as a consequence of NOS upregulation and free radical species formed during contraction by myocytes) to alter lipid and protein constituents within the diaphragm. For example, in otherwise normal muscle tissue, the superoxide anion levels generated by small numbers of neutrophils may have no appreciable effect on cell function. In the presence of heightened nitric oxide formation due to NOS upregulation, however, superoxide anions generated by the same number of white blood cells could greatly increase peroxynitrite formation and thereby inflict much greater levels of tissue damage. The presence of such synergistic interactions could account for the fact that the effects of endotoxin administration on respiratory muscle function can be blunted by apocynin, NOS inhibitors, or free radical scavengers.| |
ACKNOWLEDGEMENTS |
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This study was supported by National Institutes of Health Grants 54825 and 38926.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. Supinski, Metrohealth Medical Center, 2500 Metrohealth Dr., Cleveland, OH 44109.
Received 29 May 1998; accepted in final form 22 April 1999.
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 G. Supinski, D. Nethery, T. M. Nosek, L. A. Callahan, D. Stofan, and A. DiMarco Endotoxin administration alters the force vs. pCa relationship of skeletal muscle fibers Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2000; 278(4): R891 - R896. [Abstract] [Full Text] [PDF]
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), endotoxin-treated animals (
),
apocynin-treated animals (
), and animals given both endotoxin and
apocynin (
). Error bars indicate 1 SE.
