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Infection and Immunity, March 1999, p. 1079-1085, Vol. 67, No. 3
Clintec Technologies,
Received 29 June 1998/Returned for modification 2 November
1998/Accepted 1 December 1998
Most animal models of sepsis induced high mortality or early
recovery and do not mimic the long-lasting catabolic state observed in
patients. The purpose of this study is to develop a model of sepsis
which reproduces these disorders, especially the long-lasting muscle
wasting. This report summarizes our observations in a series of seven
experiments using this model with rats to study the route of live
Escherichia coli administration, dose of bacteria,
reproducibility of the model, bacterial count in tissues, comparison of
injection of live or dead bacteria, metabolic perturbations linked to
infection, and potential role of tumor necrosis factor alpha (TNF- Despite intensive care, sepsis is
still a major cause of death throughout the world. Sepsis is associated
with a very large range of disorders, such as profound hemodynamic and
nutritional derangements, organ failure and multiple metabolic
alterations with increased energy substrate turnover, altered hormonal
pattern, and intensive protein catabolism. Among these disturbances,
one of the most dramatic is the loss of lean body mass, especially muscle proteins. This problem is complicated by interactions with nutritional state (4). Moreover, these alterations can be
maintained for weeks in patients with trauma and sepsis. It is
therefore of primary importance to obtain a relevant model suitable for prolonged metabolic studies and sufficiently long-lasting to test the
efficiency of nutrition on outcome. Numerous models have been described, but most of them provide only short time observations because the majority of animals die within 24 to 48 h.
Most of the proposed models are very severe and present a high degree
of mortality. Cecal ligation with puncture (CLP) has been extensively
used, but animals die in 24 to 48 h (22, 36). Prolonged
survival has been described in a modification of the CLP model that
uses only one small puncture (35), but standardization of
this model is very difficult for metabolic studies due to a large
variability in the severeness of disease (13); furthermore, body weight loss is limited (35). In an effort to
standardize the bacterial strain used, peritonitis has been provoked
with known bacteria entrapped in a gelatin capsule or a fecal pellet made of sterile rat feces (12, 24), but high mortality was still observed. More recently, this latter model has been successfully used for studying the effect of septic abscess on protein synthesis rate measured in skeletal muscle 5 days after operation of animals (33). However, animals began to exhibit growth recovery 4 days after implantation of the fecal-agar pellet, and nutritional
status was different from clinical conditions since animals were not anorexic. Alexander et al. (2) induced peritonitis by means of continuous infusion of live bacteria with an osmotic pump, but body
weight loss of control rats was similar to that of septic animals. In
fact, all of these models involve an invasive surgical procedure which
contributes to the catabolic state, and differentiation of the effect
of sepsis from the effect of surgery is often difficult.
The administration of a high dose of endotoxin induced an overwhelming
aggression (22, 36). In contrast, moderate doses of
endotoxins are not lethal, and growth recovery of the animals occurred
after only 48 h. Prolonged or repeated administration leads to
endotoxin tolerance (3, 10, 20), which is also observed with
continuous infusion of endotoxin (11). Intravenous (i.v.)
administration of live bacteria has been criticized by numerous authors
because of the high mortality observed in few hours (24, 27, 28,
38). However, Perbellini et al. (26) and more recently
Shaw and Wolfe (30), clearly reproduced in dogs a situation
that hormonally, hemodynamically, and metabolically resembles human
sepsis. They provoked sepsis by injection of live Escherichia
coli organisms in a sublethal dose but gave no data on body weight
loss of the animals. With the same approach of a single i.v. injection
of live E. coli, we describe a highly reproducible sepsis
model which induces acute and prolonged body weight loss and
muscle atrophy and provides a new tool for studying the impact of
nutritional support in sepsis.
Animal care.
Male Sprague-Dawley rats (Iffa Credo, Saint
Germain sur l'Arbresle, France) weighing about 250 g were
individually housed in wire-bottom cages in a temperature-controlled
room (22 to 23°C) with a 12 h-12 h light-dark cycle. After 6 days of
acclimatization, animals were randomized into groups for injection of
live bacteria or saline. During the acclimatization period, all rats
had free access to water and to a semisynthetic diet containing 12%
protein described previously (25).
Preparation of bacteria.
An E. coli serotype
O153:K Experiments.
Experiment 1 was designed to compare the
effects of different routes of bacterial administration. A 0.5-ml
volume of the same preparation of bacteria (1.3 × 109
bacteria per ml) was injected either intraperitoneally (i.p.) or
i.v. into a lateral tail vein. Since rats in the i.v. group were
highly anorexic, a pair-fed (PF) control group was injected with
saline and fed with the same restricted food intake as the i.v. group.
Rectal temperature was measured at different times before and after
infection. Rats were studied for 2 days postinfection.
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Sustained Rat Model for Studying the
Long-Lasting Catabolic State of Sepsis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
)
in muscle wasting. After intravenous infection, animals were anorexic
and the catabolic state was long-lasting: body weight loss for 2 to 3 days followed by a chronic wasting state for several days. Liver, spleen, lung protein content, and plasma concentration of
2-macroglobulin were increased 2 and 6 days after
infection. At 6 days, muscle protein content was substantially (
40%)
reduced. The plasma TNF- level measured 1.5 h after infection
correlated with body weight loss observed 9 days later. The inhibition
of TNF- secretion by administration of pentoxifylline 1 h
before infection reduced muscle wasting and activation of proteolysis
at day 2 and abolished them at day 6. This septic model mimics in rats
the prolonged protein metabolism alterations and muscle atrophy
characteristics of infected patients and thus is useful for studying
the impact of nutritional support on outcome.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
:H strain isolated from calf septicemia was used. Bacteria
were grown in 10 ml of Minca broth (18) and incubated
overnight in a shaking incubator at 37°C. Chloramphenicol (Sigma,
L'Isle d'Abeau Chesnes, France) was added to culture medium to avoid
loss of the plasmid encoding antibiotic resistances (Kanr,
Strr, Cmpr, Tetr, Sulr)
and virulence properties (aerobactin [37] and surface
protein CS31A [14]). The morning after, the bacterial
suspension was used to inoculate fresh Minca broth. After a 2-h
incubation at 37°C, optical density of the bacterial suspension was
measured at 600 nm and the bacterial concentration was estimated,
assuming that an optical density of 1 represents 4 × 108 E. coli per ml. Bacteria were collected in
the logarithmic growth phase and centrifuged for 15 min at
6,000 × g, and the pellet was resuspended in saline.
Then, viable bacteria were counted by serial 10-fold dilutions plated
in duplicate on deoxycholate agarose (DCA; Difco, OSI, Paris, France).
Plates were incubated at 37°C for 15 h, and colonies were counted.
) in plasma. In a preliminary
kinetic study, we confirmed that the TNF- plasma concentration
peaked 90 min after infection. Rats were weighed every morning and
studied for up to 10 days after infection. The medium dose of bacteria
was used in subsequent experiments.
20°C until analysis.
Experiment 7 was designed to study the role of cytokines, and
particularly TNF-, in muscle wasting. At an initial body weight of
300 g, rats were divided into four groups: infected rats (INF group) and their PF controls, and infected rats treated with
pentoxifylline (PX; Torental; Hoechst, Paris, France) (PX-INF) and
their PF controls treated with PX (PX-PF). The INF group received
saline i.p. 1 h before injection of E. coli (7 × 108 bacteria per rat) into a lateral tail vein. PF animals
received an i.p. injection of saline 1 h before an i.v. saline
injection and were pair-fed the intake of infected rats. In the PX-INF
group, PX (100 mg/kg) was injected i.p. 1 h before administration
of bacteria. PX-PF animals received an i.p. injection of PX 1 h
before an i.v. injection of saline. Because PX treatment increases
voluntary food consumption in infected rats (5), the PX-PF
control group was pair-fed the intake of PX-INF animals. Blood samples
were taken 1.5 and 3 h after infection for cytokine measurements.
Between six and eight animals of each group were studied on days 2 and 6 after infection. After anesthesia with sodium pentobarbital (6.0 mg/100 g body weight), the epitrochlearis muscles were dissected intact
for incubation (see below). Gastrocnemius and soleus muscles were also
dissected and weighed.
Food intake. A controlled amount of food was distributed in six equal meals given at 03:00, 07:00, 11:00, 15:00, 19:00, and 23:00 by an automatic device. Infected rats were highly anorexic for 2 or 3 days and ate only 60% of the normal intake on day 7 after infection. In this context, pair-feedings were performed in experiments 1, 5, and 6. Since the food intake of PF rats was highly restricted, the time schedule distribution was used to maintain food intake throughout the day.
Food intake of ad libitum-fed rats was determined in a previous study, and the same daily amount of food was offered to infected animals. Food intake of each inoculated group was measured daily, and the same quantity of food was offered to PF rats with a time lag of 2 days after feeding of infected groups, except in experiment 6. To incubate simultaneously muscles of infected and control rats, the intake of controls was based on the intake of infected rats measured in previous experiments.Analytical procedures.
TNF- and interleukin-1
(IL-1) plasma concentrations were measured by using enzyme-linked
immunosorbent assay kits as instructed by the manufacturers (Genzyme
[Cambridge, Mass.] and Amersham [Buckinghamshire, England],
respectively). Biological activity of IL-6 was estimated in a bioassay
using the B-9 hybridoma cell line (1). Briefly, B-9 cells
(5,000/100 ml) were cultured in 96-well microtiter plates with serial
dilutions of test samples (15). The IL-6 standard was human
recombinant IL-6 (catalog no. 89/548; National Institute for Biological
Standards and Control, Hertfordshire, England), which was serially
diluted. After 48 h of incubation at 37°C with 5%
CO2, 20 ml of
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (2 mg/ml; Interchim, Montluçon, France), was added to
each well in the presence of phenazine methosulfate, and the mixture
was incubated for an additional 2 h to determine cell proliferation (7). The water-soluble formazan product was
quantitated at 490 nm in an MR 700 microplate reader (Dynatech
Laboratories, Inc., Guernsey, England).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Experiment 1 revealed the importance of the route of administration of bacteria (Fig. 1). The i.p. group showed a very limited body weight loss (2.2 ± 9.6 g) on the day of infection, and growth recovery began as early as day 2, reaching a weight gain of 3.4 ± 3.8 g. In contrast, for the 2 days following infection, the i.v. group showed a marked body weight loss (of 34.8 ± 2.8 g on day 2). The PF mice of the i.v. group also showed body weight loss during the 2 days postinfection, but only 67% of that for the i.v. group. Food intake was very different between the two infected groups: rats in the i.p. group ate 73 and 85% of preinfection food intake on days 1 and 2 postinfection, respectively, while corresponding values for rats in the i.v. group were only 17 and 13%.
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)
or i.v. (
). They had free access to alimentation. One control PF
group was injected i.v. with saline and ate the same quantity of food
as the i.v. group
(
). Body
weight changes were recorded for 2 days. Rats weighed 300 g at
infection. Data are means ± standard deviations. Intravenous
infection of rats resulted in the highest body weight loss: *,
P < 0.05 versus the PF group; 
, P < 0.05 versus the i.p. group.
Table 1 shows the effects of
infection on rectal temperature. In the i.p. group, infection produced
no change in rectal temperature. In contrast, the i.v. group showed a
marked hypothermia 1.5 h after infection, which had normalized
4 h after infection. At 24 and 48 h, these rats exhibited a
moderate fever (P < 0.05).
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Bacteria were given by the i.v. route in the following experiments, since i.p. administration failed to produce body weight loss. In these experiments, all infected rats showed symptoms of severe illness. After infection, they rapidly became lethargic and anorexic and exhibited strong piloerection. Most of the MDG animals and surviving HDG rats (experiment 2) exhibited chromodacryorrhea and diarrhea. There was an obvious mortality dose effect, since all LDG rats survived, only one MDG rat died (on day 1 postinfection), and all HDG rats died within 4 days (three on day 1, four on day 2, and one on day 4). Three days after inoculation, we observed an improvement of the clinical state of the rats: they became more active and no longer exhibited chromodacryorrhea and diarrhea.
Both LDG and MDG animals exhibited acute body weight loss (of about 40 g) during the first 2 days postinfection (Fig. 2). From days 2 to 5 postinfection, MDG rats lost 13 g of body weight whereas LDG rats gained 12 g. This dose effect was amplified from days 6 to 8 postinfection; growth recovery persisted in LDG rats, but body weights of MDG rats plateaued around 60 g below the initial body weight. MDG rats did not begin growth recovery until day 9 postinfection. They were 46 g below their day 0 body weight on day 10, while LDG rats had recovered their initial body weight. Body weight change of the MDG rats followed a triphasic course that can be summarized as follows: acute loss for 3 or 4 days (acute phase), stability from day 3 or 4 to 8 (chronic phase), and progressive recovery between days 8 and 10 postinfection (late phase).
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, low dose
([2.5 ± 0.3] × 108); 
, medium dose ([6.8 ± 0.9] × 108); 
, high dose ([2.0 ± 5] × 108). With the high dose, only one rat survived until day
4.
Plasma TNF- concentrations measured 1.5 h after infection
(experiment 2) were significantly higher (P < 0.05) in
rats that died (30.3 ± 4.6 ng/ml) than in rats that survived
(22.0 ± 8.5 ng/ml). Plasma TNF- concentrations were
significantly lower in LDG rats (14.8 ± 5.4 ng/ml) than in MDG
rats (29.0 ± 2.7 ng/ml), but MDG and HDG rats showed similar
TNF- concentrations (30.7 ± 4.9 ng/ml in HDG rats). In
surviving animals, there was a significant correlation between TNF-
concentration measured 1.5 h postinfection and body weight change
between day 0 (just before infection) and any day between days 6 and
10. The strongest relationship was found between TNF- concentration
and body weight change observed nine days after infection (Fig.
3).
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To test the reproducibility of the model in term of body weight changes (experiment 3), four groups of eight rats were infected with different bacterial preparations, using the medium dose of experiment 2. The amount of bacteria in the inoculum was between 88 and 103% of the theoretical dose. Two days after infection, body weight losses were 44 ± 5, 40 ± 3, 38 ± 4, and 34 ± 4 g for the four groups. The maximum weight losses were similar in the four groups, about 57 g. The days on which growth recovery began were also similar in the four groups (between days 7 and 9). Furthermore, repetition of administration of 15 successive preparations of bacterial inoculum allowed us to obtain a standard deviation of 20% around the mean value of CFU (data not shown).
In experiment 4, bacterial counts were determined in blood, liver, and spleen (Fig. 4). Bacteria disappeared quickly from blood, being found in all rats 2 days after infection but in no animals at 6 or 10 days. Bacteria were also detected in spleens and livers of all animals on day 2, the level observed in spleen being 100 times greater than that in liver. At 6 and 10 days after infection, we observed a great animal-to-animal variability in the number of bacteria in liver and spleen. For two animals, bacteria were still present in the spleen but not the liver on day 6; this could indicate better clearance of bacteria in the liver than in the spleen.
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The administration of dead bacteria failed to produce long-lasting body weight loss, since rats began growth recovery as soon as day 2 after injection (experiment 5 [Fig. 5]). Six days after administration of bacteria, rats injected with live bacteria had lost 50.5 ± 10.0 g of body weight, and rats injected with dead bacteria had gained 17.0 ± 6.4 g.
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Experiment 6 was designed to characterize organic and metabolic disturbances induced by the i.v. model of infection. Comparative variations in body weight change of control rats, infected rats, and PF rats of the infected group are given in Fig. 6. Body weight loss observed 6 days after infection (58.3 ± 16.6 g) was similar to that observed in rats of experiment 2 that received the same dose of bacteria (59.0 ± 16.7 g). During the same period, control rats exhibited a gain of 31.4 ± 4.8 g. PF animals showed the same loss as infected rats on the first day of pair-feeding. This was probably due to digestive tract emptying, since the food intake of these rats was greatly restricted (80% less than on the day before infection [Table 2]). As soon as day 2 postinfection, PF animals lost significantly less weight than infected rats.
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Sepsis induced an anabolic effect on liver, spleen, and lung since the
protein mass of these organs was significantly greater after infection
than in PF rats and often in controls (P < 0.05 [Table 3]). Kidney protein mass was
only slightly changed 6 days after infection.
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A pronounced catabolic effect was seen in muscle. At 2 and 6 days after infection, protein content of the gastrocnemius was 14 and 29% less in infected rats than in the corresponding PF animals. Six days after infection, this protein loss was 40% compared to controls. The atrophy observed in other muscles from different metabolic types (extensor digitorum longus, soleus, and tibialis anterior) was quantitatively and qualitatively similar to that observed in the gastrocnemius (data not shown).
Glycemia was greater in infected rats than in controls, but there was
no difference among other groups (Table 3). Two days after infection,
insulinemia was greater in infected rats than in controls and PF rats,
but this parameter was normalized 6 days after infection. Lactate was
not significantly modified in PF rats but was increased by infection.
Triglyceridemia was less in all treated groups than in controls, but 2 days after infection the levels of plasma triglycerides were higher in
infected rats than in their PF controls. Levels of plasma proteins were
the same in all groups, but an increase was observed 6 days
postinfection in infected rats. In contrast, the concentration of
2-macroglobulin, a protein characteristic of
inflammation in the rat, was dramatically elevated in infected animals
regardless of the time after infection; this concentration peaked
2 days after infection.
Experiment 7 was designed to further explore the role of
TNF- in our model in relation to the correlation presented in
Fig. 3. Administration of PX 1 h prior to infection suppressed the rise of the plasma TNF- level 1.5 h after infection (0.6 ± 0.6 versus 71.9 ± 19.0 ng/ml). IL-1 and IL-6 peaked 3 h after infection in our model (data not shown). PX treatment induced
84 and 61% reductions of plasma IL-1 and IL-6 concentrations,
respectively, 3 h after infection (0.22 ± 0.14 versus
1.38 ± 1.36 ng/ml for IL-1 and 1.98 ± 1.50 versus
5.09 ± 2.09 mg/ml for IL-6). Pretreatment of animals with PX
before infection reduced anorexia (Table
4). The body weight loss of rats in the
PX-INF group was always similar to that of their PF controls. Moreover,
PX-INF rats began to gain weight on day 3, although rats in the INF
group continued to lose weight. Thus, 6 days after infection, septic
rats had lost about 32 g of their initial body weight and infected
rats treated with PX had regained about 10 g.
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As shown in experiment 5, weights of the various muscles studied were significantly lower in infected animals than in control rats (Table 4). By contrast, PX treatment reduced atrophy of the gastrocnemius 14 to 16% versus respective PF rats (P < 0.05) and abolished atrophy of the soleus and epitrochlearis muscles at the end of the experiment. To determine the reason for the variations in muscle mass, we measured proteolysis rates in incubated epitrochlearis muscles. Muscle atrophy reflects protein loss, since protein concentration (milligrams per milligram of muscle) was not modified (data not shown). Protein degradation was significantly increased, 32% on day 2 and 21% on day 6, in infected rats compared with their controls (Table 4). After PX treatment, the increase of proteolysis in infected rats compared to their controls reached only 15% on day 2 and was completely abolished on day 6 (Table 4).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Previous animal models of sepsis induce body weight loss and muscle atrophy for a period of time too short to test the effect of nutritional support on body protein loss and recovery from infection. Our model succeeds in this objective since rats were in a catabolic state for up to 9 days.
Peritonitis models generally induced body weight loss for only 1 or 2 days (12, 24, 35, 36) and always exhibited high mortality (2, 12, 22, 24, 36). Endotoxin models induce a low mortality rate, but animals become rapidly resistant to endotoxin and exhibit a rapid growth recovery (8, 10, 20). Similarly, our data show that the injection of dead bacteria produced only a transient body weight loss. Numerous authors have criticized sepsis models produced by i.v. administration of bacteria on the grounds that hosts were suddenly overwhelmed with a bacterial challenge so massive that they were unable to respond with the full expression of their defense mechanisms (22, 24, 36). These models were highly lethal (27, 28, 38) but were generally designed to study death rate (27) or hypometabolic shock (28, 38). Furthermore, such models using very high doses of live organisms probably reflect an intoxication similar to those observed with endotoxin challenge rather than the response of the organism to a pathogen which colonizes and replicates significantly following challenge. The choice of pathogen is therefore an important consideration since a limited number of serogroups have the characteristics allowing replication and dissemination of bacteria (8). The bacterial strain used in this study probably has this capacity, since bacteria can be detected in tissues 10 days after infection, and we frequently observed abscesses in kidneys or testes.
Septic canine models using i.v. injection of live bacteria that reproduce a number of clinical disorders typically found in human sepsis, including hypermetabolism and hormonal perturbations, have been described (26, 30). However, the first model was designed to examine mortality and was therefore a model of shock (26). The period examined in the second study did not exceed 24 h after infection (30). Moreover, body weight loss or muscle atrophy was not reported in these studies. Our model focused on the latter problem for two main reasons: muscle atrophy acutely weaken septic patients, and protein recovery requires intensive nutritional support with long and high-cost convalescence.
The fall in rectal temperature observed 1.5 h after i.v. infection seems to indicate that rats exhibited shock at this time. However, this reaction was quite transient, and rectal temperature returned to baseline as early as 4 h after infection. On the other hand, rats exhibited moderate fever on days 1 and 2 postinfection, a reaction often noted in rats due to a relatively high surface area-to-mass ratio (11). Moreover, in contrast to observations in human sepsis, a hypothermic reaction was observed in several septic rat models (11, 17, 24). Increases in plasma glucose and insulin levels have often been found in humans and are difficult to reproduce in small-animal models (12, 20, 22, 24). As observed in humans, glucose, insulin, lactate, and temperature were increased at the same time (2 days after infection), indicating that our animals were probably in the hypermetabolic flow phase. In any case, they were not in the agonal phase of shock that has often been described (18, 19) or criticized as an undesirable effect of bolus injection of live bacteria or endotoxin (12, 22, 24). Indeed, profound hypoglycemia is always observed in the preterminal phase (27).
Infection induced acute anorexia for several days. This anorexia has been largely observed in human sepsis. Interactions between metabolic disorders and nutrition are apparently present in the chronic (days 3 to 7) and late (after day 7) phases of our model, since infected rats ate about 32 g of dry matter between days 6 and 10 postinfection and gained only 4 g in the same time. Data for other experiments in our laboratory indicate that PF rats should gain 14 to 16 g with the same food intake. This indicates an inefficiency of food intake, well known in stress situations in humans. Our model gives the opportunity to study such nutritional disorders.
It is well established that even a brief infection causes some
degree of malnutrition and that acute infection creates deficits in the
nutritional stores of the body (4). These deficits are thought to be induced by the acute-phase response, which increases nutritional requirements of organs like the liver (6, 19, 24,
35). In the absence of correct nutritional supply (due to
anorexia), the organism mobilizes muscle stores and induces a shift of
amino acids from the periphery to central organs (3, 6, 20,
24). A good septic model for studying this problem should
reproduce at the same time muscle atrophy, acute-phase response, and
anorexia over a long period of time. Such long-lasting perturbations
were observed in our model. Indeed, 6 days after infection, muscle
protein mass was 60% of that for controls and 70% of that for PF
rats, and liver protein mass was 129% of that for PF animals. The
protein content of other tissues such as the spleen and lung
increased after infection, suggesting an important role of these organs
for defense of the organism. Part of the anabolic response of liver is
thought to be associated with increase synthesis of acute-phase
proteins. In this study, plasma levels of
2-macroglobulin, a characteristic protein of the
acute-phase response in the rat, were increased 40- and 25-fold on days
2 and 6, respectively. Increased levels of acute-phase proteins are
probably due to increased synthesis rates. Since the amino acid
composition of acute-phase proteins is different from the mean amino
acid composition of whole-body proteins, the liver acute-phase response
could induce specific amino acid requirements (qualitatively and
quantitatively) (6, 29).
Plasma TNF- concentration measured 1.5 h after infection
was significantly higher in rats that died in the days after infection than in surviving animals. More interesting, in surviving animals, our
data show a strong correlation between plasma TNF- concentration measured 1.5 h after infection and body weight change observed 9 days later. This establishes a prognostic value of TNF-
concentration on outcome for rats. Clinical studies described
controversial results concerning the prognostic value of TNF-
concentration in human sepsis. This is probably due to the difficulty
in detecting sepsis enough early in patients. After infection, we found
an acute but transient TNF- peak, since plasma levels returned to baseline 4.5 h after infection. Such time-related variations have been reported for a murine endotoxemic model but not a peritonitis model obtained by CLP (9). It is possible that the CLP
surgical procedure resulted in elevation of endogenous glucocorticoids which inhibited secretion of cytokines, and particularly of TNF- (9). On the other hand, Martin et al. (23)
demonstrated recently that intravascular plastic catheters potentiated
TNF- release and exacerbated complications associated with sepsis.
Since cytokines are universally recognized as primary mediators of the
septic syndrome, it is advisable to use models that do not require a surgical procedure.
To further explore the involvement of TNF- in determining muscle
wasting, as suggested by the results shown in Fig. 3, we used PX, which
is known to inhibit the production of TNF- (5). PX
treatment minimized the difference in body weight between infected rats
and their PF controls over the entire course of the study and reduced
or abolished the muscle atrophy linked to infection. We showed
previously that muscle atrophy was mainly due to a persistent activation of proteolysis (34). The present study
demonstrates that PX treatment completely abolished the activation of
proteolysis observed at day 6. Breuillé et al. (5)
reported that PX treatment reduced the muscle protein synthesis
inhibition observed in the septic acute phase, as previously found
during chronic sepsis after administration of amrinone, another
inhibitor of TNF- secretion (21). Taken together,
these data suggest that the PX-induced improvement of muscle nitrogen
balance resulted from both reduced inhibition of protein synthesis and
depressed activation of proteolysis. Since an important effect of PX
treatment of septic rats is the suppression of the early appearance of
TNF- in plasma, our results lead us to conclude that TNF- is
an important determinant of muscle wasting and proteolysis
activation in sepsis. This observation is consistent with results
of Zamir et al. (39) showing in rats a decreased
proteolysis activation after anti-TNF- administration in acute
sepsis induced by CLP. However, the decrease of IL-1 production can
participate to the inhibition of muscle proteolysis in PX-treated
septic animals, since reduced activation of muscle proteolysis was
observed in septic animals treated with IL-1ra (40). On
the other hand, our study suggest that IL-6 could be a minor
determinant in sepsis-induced proteolysis. This contrasts to
recent data suggesting that IL-6 could directly activate muscle proteolysis and especially the lysosomal and
ATP-ubiquitin-dependent pathways (16, 32). The main
point underlined by our results is that TNF- secretion observed
in the first hours after infection has a pivotal role in supporting
activation of muscle proteolysis for days. However, the mode of action
of TNF-, direct or indirect through other cytokines, hormones,
or unknown compounds, as found in cancer patients
(31), remains to be determined.
In conclusion, we have developed a septic model in rats which reproduces sustained metabolic disturbances characteristics of sepsis. Our model is easy to use, since it requires no surgical procedure. Furthermore, we demonstrated that the response to a titrated dose of live E. coli was predictable and reasonably reproducible. Therefore, this model is suitable for studying possible nutritional interventions (prophylactic or therapeutic), especially the effect of specific nutritional support.
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ACKNOWLEDGMENTS |
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This work was supported by INRA and Clintec Technologies.
We thank Jean Pierre Girardeau for helpful scientific discussions and Caroline Buffière, Philippe Denis, Corinne Pouyet, and Fabienne Rambourdin for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire d'Etude du Métabolisme Azoté, INRA Theix, 63122 Ceyrat, France. Phone: (33) 04 73 62 42 10. Fax: (33) 04 73 62 47 55. E-mail: Breuille{at}Clermont.inra.fr.
Editor: P. J. Sansonetti
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, March 1999, p. 1079-1085, Vol. 67, No. 3
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