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1 Section of Cardiovascular Sciences and Cardiology, Department of Medicine, DeBakey Heart Center, Baylor College of Medicine and Methodist Hospital; and 2 Veterans Administration Medical Center, Winters Center for Heart Failure Research, Houston, Texas 77030
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Reperfusion of the ischemic myocardium
is associated with a cytokine cascade that reflects a cellular response
to injury. We studied this cascade in the mouse and found that acute
surgical trauma in sham-operated animals obscured early changes in
cytokine induction that occur during myocardial
ischemia-reperfusion (MI/R). Therefore, we utilized a new
implantable device that allows occlusion and reperfusion of the left
anterior descending coronary artery in a closed-chest mouse at any time
after instrumentation. Induction of interleukin (IL)-6 and tumor
necrosis factor (TNF)-
mRNA in the whole heart was examined by RNase
protection assay and quantitated by Phosphor- Imager. At 3 h after
instrumentation, levels of IL-6 mRNA in sham-operated animals increased
above those of control naive hearts, whereas this increase did not
occur until after 1 day for TNF- mRNA. The surgical trauma led to
exaggeration of I/R cytokine induction with greater variance in
response. At 3 days and 1 wk after instrumentation, levels of both IL-6
and TNF- mRNA in sham-operated animals were comparable to those of naive hearts and induction responses in I/R were much less variant. We
also found that 1 h of ischemia and 2 h of reperfusion at all time points of recovery (i.e., 3 h and 1, 3, and 7 days after instrumentation) led to a significant increase in IL-6 and TNF- mRNA
levels. In addition, 3 h of permanent occlusion, which did not induce
any mRNA increase after 1 wk postinstrumentation, caused marked
upregulation of IL-6 mRNA in an acutely prepared animal. This study of
early cytokine responses evoked by MI/R highlights the need for
dissipation of acute surgical trauma by using a chronic, closed-chest
mouse preparation.
murine; cytokine; interleukin; inflammation; surgery
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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IT IS WELL KNOWN that acute myocardial ischemia-reperfusion (MI/R) (6) is associated with an intense inflammatory reaction that plays a part in both acute extension of injury and repair of the myocardium. This inflammatory reaction is associated with a significant influx of leukocytes such as neutrophils (5, 10) and monocytes (1), which produce a vast array of mediators that orchestrate the sequelae of inflammation. These mediators, each of which has its own tightly regulated time course of synthesis, include oxygen-derived free radicals, proteases, chemokines, cytokines, and lipid-derived chemotactic agents. Thus many investigators are intensely studying the complex pathogenesis, time course, and factors involved in the inflammatory process to come to a clear understanding of this process and ultimately design useful and clinically relevant therapies.
A variety of different experimental paradigms were developed in both large (i.e., dog and pig) and small (i.e., rat and mouse) animals to examine the regulation of inflammatory mediators that results from reperfusing a previously ischemic vascular myocardial bed. Many of these experimental methods utilized anesthetized open-chest animals. In a previous experiment, Michael et al. (21) found that the acute surgical trauma associated with open-chest preparations often resulted in significant and highly variable background levels of inflammation in sham-operated animals. These background levels often were indistinguishable from those of ischemic-reperfused animals and, thus, compromised the ability to definitively assess inflammation strictly due to MI/R. This led to the development of a chronic model of I/R in dogs that allowed resolution of surgical trauma before I/R induction (21). This current paper describes our development of a closed-chest mouse model of MI/R that allows dissipation of the trauma and inflammation that occurs in the acute phase of surgical manipulations, permitting a more predictable and interpretable response.
Because of their prominent role in the orchestration of cardiac injury
response, the induction and molecular characteristics of the cytokines
tumor necrosis factor (TNF)- and interleukin (IL)-6 have been
extensively studied in animal models of I/R by Entman and co-workers
(7, 13, 14) as well as by other investigators (2, 15, 23). Therefore,
we utilized the molecular induction of these cytokines in the present
study to assess the affect of acute surgical trauma on extent and
variability of cytokine induction in MI/R.
The goals of the present study were to 1) develop a
closed-chest mouse model of MI/R with an implantable device that would allow ligation of the left anterior descending coronary artery (LAD)
after dissipation of acute surgical trauma, 2) analyze the time
course of TNF- and IL-6 mRNA upregulation in the myocardium that
results from acute surgical trauma in sham-operated animals, 3)
analyze the degree and variability of mRNA upregulation of TNF- and
IL-6 in the myocardium that results from MI/R and compare them with
those of time-matched sham-operated animals, and 4) determine
whether upregulation of these cytokines is reperfusion dependent.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Preparation and surgery of animals. Female C57BL/6 mice 8-12 wk of age (18.0-22.0 g body wt) were obtained from Harlan Sprague Dawley (Houston, TX). Mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (10 µl/g body wt of mouse). Each animal was placed in a supine position with paws taped to an electrocardiogram (ECG) board (lead II) to measure S-T segment elevations during ischemia and reperfusion. A 5-0 silk suture was placed around the upper front incisors and pulled taut to extend the neck, which facilitated access to the trachea for intubation. A midline incision was made in the skin from the submentum to the xyphoid process. The muscles overlying the trachea were gently separated to allow visualization of the endotracheal tube [polyethylene (PE)-90] placed in the trachea during intubation. The tip of the tube was placed 5-8 mm from the larnyx and taped in place. This tube was then inserted loosely into the PE-160 connection to the small animal ventilator (Harvard, S. Natick, MA). The animals were respirated with a volume of 2-4 ml and a tidal volume of 100-200 µl at a rate of ~110 strokes/min, with 100% O2 provided to the inflow of the ventilator. Normal chest expansion was noted as being comparable to that of a conscious mouse.
A microcoagulator (Codman, Randolph, MA) was used to coagulate blood vessels directly in the midline. Dissection was aided by a microscope (Zeiss, Jena, Germany), and the chest was opened with a lateral cut with tenotomy scissors along the left side of the sternum by cutting through the ribs to approximately midsternum. The chest walls were retracted by using 6-0 suture. The pericardium was then gently dissected to allow visualization of coronary artery anatomy. In pilot open-chest experiments in which background levels of TNF-
and IL-6 mRNA in sham-operated animals were highly variable, ligation
of the LAD was performed as previously described (20). Briefly,
ligation (1 h of ischemia followed by 2 h of reperfusion) proceeded with an 8-0 Surgipro monofilament polypropylene suture with a
tapered needle passed underneath the LAD ~1-3 mm from the tip of
the left auricle. This maneuver was easier with the needle modified to
form a U shape before its passage underneath the LAD. A 1-mm section of
PE-10 tubing was placed on top of the vessel, and a knot was tied on
top of the tubing to occlude the LAD. Absence of blood flow was
verified visually under the microscope, and the chest wall was
approximated and covered with a piece of moistened gauze to prevent
desiccation. Reperfusion (2 h) was induced by cutting the knot on top
of the PE-10 tubing with microscissors. This allowed release of the
occlusion and reperfusion of the formerly ischemic bed. The chest was
then closed with 6-0 Surgipro monofilament polypropylene suture with
one layer through the chest wall and muscle and a second layer through
the skin and subcutaneous material. In this study, the acute permanent
occlusion (PO) group (acute 3-h PO, n = 6 animals) was prepared
as described above (i.e., LAD occlusion 3 h after instrumentation). At
the end of 3 h of occlusion, the heart was immediately excised,
snap-frozen, and stored at 
80°C until mRNA analysis.
We developed a closed-chest method that is the subject of this report;
the surgical preparation of the animal was performed as follows. After
the thoracotomy was performed as described above, the pericardium was
dissected and an 8-0 Surgipro monofilament polypropylene suture with
the U-shaped tapered needle was passed under the LAD. The needle was
then cut from the suture, and the two ends of the 8-0 suture were then
threaded through a 0.5-mm piece of PE-10 tubing, forming a loose snare
around the LAD, as shown in Fig.
1A. The PE-10 tubing had been
previously soaked for 24 h in 100% ethanol. Each end of the suture was
then threaded through the end of a size 3 Kalt suture needle (Fine
Science Tools) and exteriorized through each side of the chest wall, as
shown in Fig. 1B. The chest was closed with four interrupted
stitches utilizing 6-0 suture, with care taken to avoid pneumothorax.
The ends of the exteriorized 8-0 suture were then tucked under the skin, which was then also closed with 6-0 suture. The animal was removed from the respirator, the endotracheal tube was withdrawn and
kept warm with a heat lamp, and the animal was allowed to breathe 100%
O2 via a nasal cone until full recovery of consciousness. At 3 h, 1 day, 3 days, or 1 wk after instrumentation, the animals were
reanesthetized with pentobarbital sodium. For animals randomized to the
MI/R groups (n = 6 at 3 h, n = 4 at 1 day, n = 6 at 3 days, n = 4 at 1 wk), the extremities were taped to a
lead II ECG board and the skin above the chest wall was reopened. The
8-0 suture, which had been previously exteriorized outside the chest
wall, was cleared of all debris from the skin and chest and carefully taped to heavy metal picks, as shown in Fig. 1C. Ligation of
the LAD for the specified time period, either 1 h of ischemia
followed by 2 h of reperfusion or 3 h of PO (n = 5), was
accomplished by gently pulling the heavy metal picks apart until an S-T
elevation appeared on the ECG, as shown in Fig.
2. The ECG was constantly monitored
throughout the entire ischemic interval to ensure persistent ischemia. At the end of ischemia, the 2 h of
reperfusion were accomplished by pushing the metal picks toward the
animal and cutting the suture close to the chest wall. Reperfusion was
confirmed by resolution of the S-T elevation, which usually occurred
very quickly, as shown in Fig 2. The skin was then closed with 6-0 suture, and the animal was allowed to recover in a warm cage. During
this procedure, the animal was neither reintubated nor administered
O2 but, rather, breathed room air under normal ventilation. At the end of the experiment (i.e., 2 h of reperfusion or 3 h of PO),
the chest was opened and the heart was immediately excised, snap-frozen, and stored at 80°C until mRNA isolation.
Sham-operated animals were prepared identically without undergoing the
I/R or PO protocol (n = 6 at 3 h, n = 3 at 1 day,
n = 8 at 3 days, n = 8 at 1 wk).
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and IL-6 mRNA.
These mice were anesthetized with pentobarbital sodium as was done for
all other mice. With no prior instrumentation, these hearts were
immediately excised, snap-frozen, and stored at 80°C until
mRNA isolation.
Measurement of S-T elevation in the MI protocol.
The S-T elevation was measured in millivolts from baseline to the top
of the T wave for each animal (Table 1).
Because there is no consistently distinguishable S-T segment in mice
because of their extremely high heart rates, the S-T elevation was
measured to the top of the T wave as a consistent approximation of the S-T segment. This was done at the end of the 1 h of ischemia
for the animals undergoing the I/R protocol. For the animals in the 3-h
PO protocol (both acute and 1 wk after instrumentation), the S-T
elevation was measured at both 1 and 3 h of ischemia. Because these two measurements were not different, the value listed in Table 1
is that taken at the end of the 3 h of ischemia.
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mRNA isolation. All solutions for RNA analysis were treated with 0.1% diethylpyrocarbonate and sterilized or prepared in diethylpyrocarbonate-treated water. Glassware was baked at 240°C for 5 h to remove trace RNases. Total RNA was isolated from whole heart according to the acid guanidinium thiocyanate-phenol-chloroform extraction developed by Chomczynski and Sacchi (4). Briefly, whole hearts were homogenized in RNA STAT-60 solution (Tel-Test, Friendswood, TX). For RNA extraction, 0.2 volumes of R-chloroform were then added per volume of homogenate. This mixture was incubated on ice for 15 min and then spun at 12,000 g for 15 min at 4°C. The supernatant was transferred to another tube, and an equal volume of isopropanol was added for RNA precipitation overnight at 4°C. The tubes were then spun at 12,000 g for 15 min at 4°C, and the supernatant was then decanted. The pellet was washed twice with 75% ethanol, briefly dried, and dissolved in 0.1% diethylpyrocarbonate-treated water. Quantification and purity of RNA were assessed by ultraviolet absorption (ratio of absorption at 260 nm to that at 280 nm), and RNA samples with ratios >1.9 were utilized for further analysis.
RNase protection assay and quantitation.
The expression levels of IL-6 and TNF- mRNA were determined using a
RNase protection assay (RPA). A commercially available kit (RiboQuant
kit; Pharmingen, San Diego, CA) and antisense RNA probe (mck-3b;
Pharmingen) according to the manufacturer's protocol. Briefly, for
synthesis of a radiolabeled antisense RNA probe that included loading
control L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the
final reaction mixture contained 10 µl
[-32P]UTP (740 MBq/ml, 20 mCi/ml; Amersham
Pharmacia Biotech, Piscataway, NJ), 1 µl of GTP, ATP, CTP, and UTP
(2.75 mmol each), 2 µl of dithiothreitol (100 mmol), 4 µl of
transcription buffer (1×), 1 µl of RNasin (40 units), 1 µl of
T7 polymerase (20 units), and an equimolar pool of linearized templates
(50 ng total). After 1 h at 37°C, the reaction mixture was treated
with 2 µl of RNase-free DNase (2 units) at 37°C for 30 min. The
probe was purified by extraction with phenol-chloroform (Acid
Phenol:Chloroform, pH 4.7; Ambion, Austin, TX), precipitated with
100% ethanol, and stored at 70°C for 30 min. After
centrifugation in a microfuge at 15,000 rpm (6,000 g) for 15 min at 4°C, the supernatant was discarded and the probe was washed
with 90% ethanol and dried at room temperature for 5 min. The pellet
was then dissolved in 50 µl of hybridization buffer (1×), 1 µl was quantitated in a scintillation counter, and the probe was
diluted to the appropriate concentration according to the
manufacture's protocol. Two microliters of the probe were added to the
tubes containing target RNA (20 µg) and dissolved in 8 µl of
hybridization buffer. The reaction mixtures were covered with mineral
oil, heated to 90°C, and then incubated at 56°C for 12-16
h. After overnight hybridization, unprotected RNA was digested with 100 µl of an RNase A + T1 mix (80 ng/µl A and 250 U/µl T1 in 60 µl
of 1× RNase buffer). After incubation for 60 min at 30°C, 18 µl of a proteinase K cocktail (1× proteinase K buffer, 10 mg/ml
proteinase K, and 2 mg/ml yeast tRNA) were added, and the reaction
mixtures were incubated for 15 min at 37°C. The protected RNA
fragments were isolated by extraction and precipitation, dissolved in 5 µl of 1× loading buffer, heated to 90°C, and resolved on a
denaturing 6% polyacrylamide sequencing gel (National Diagnostic,
Atlanta, GA). Dried gels were exposed overnight to radiographic film
(Kodak). Phosphoimaging of the gels was performed with a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Signals were
quantified using ImageQuant software and normalized to GAPDH.
Statistical analysis.
All values are given as means ± SE. Statistical significance between
sham groups versus naive control hearts for TNF- and IL-6 mRNA were
analyzed by two-tailed ANOVA with Bonferroni correction. Differences
between MI/R groups and time-matched sham-operated animals, as well as
between both PO groups, were analyzed by Student's t-test with
Bonferroni correction. To examine the homogeneity of variances for the
groups, the Fmax test was performed as described by
Sokal and Rohlf (24). Results were considered significant at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cardiac electrophysiological data.
Before ischemia, all electrophysiological variables were
similar among the five groups of mice. As shown in Table 1, a
significant and comparable peak elevation of the S-T segment occurred
in all MI groups, both with and without reperfusion, indicating that the ischemic insult was equivalent [P = not significant
(NS)] among the four ischemic groups. After reperfusion, the S-T
segment returned to near control values (i.e., 0) in all mice (except for the PO group), indicating a successful degree of reperfusion. Hence, IL-6 and TNF- mRNA levels cannot be explained by differences in ischemic damage as reflected by electrophysiological changes.
Analysis of IL-6 mRNA levels.
Figure 3A shows a representative
autoradiograph obtained by RPA of IL-6 mRNA levels isolated from the
whole heart of control as well as sham and MI/R groups at 3 h, 1 day, 3 days, and 1 wk after instrumentation. In addition, data are shown for
animals with permanent occlusion (3-h PO group) occurring both 3 h and 1 wk after instrumentation. The levels of IL-6 mRNA in the 3-h sham
group rose above those of the control hearts, gradually returning back
to control levels by 3 days after instrumentation and remaining at this
low level at 1 wk after instrumentation. At each time point, I/R led to
a pronounced increase in the levels of IL-6 mRNA above those of the
time-matched sham-operated animals. Three hours of PO after acute
preparation (i.e., LAD occlusion 3 h after instrumentation) led to a
significant IL-6 mRNA upregulation. In contrast, there was no increase
in IL-6 mRNA in the group of mice that underwent 3 h of PO 1 wk after
instrumentation.
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Analysis of TNF- mRNA levels.
Figure 4A shows a
representative autoradiograph obtained by RPA of TNF- mRNA levels
isolated from the whole heart of control as well as sham and MI/R
groups at 3 h, 1 day, 3 days, and 1 wk after instrumentation. Data from
3-h PO groups at 3 h and 1 wk after instrumentation are also shown.
There was no significant increase of TNF- mRNA in the sham group
until 1 day after instrumentation. This increase in TNF- mRNA in the
sham group returned to control values after 3 days and remained low
after 1 wk. At 3 h, 3 days, and 1 wk after instrumentation, MI/R
induced a significant increase of TNF- mRNA compared with
time-matched sham-operated animals. In contrast, TNF- mRNA was not
induced in PO mice.
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mRNA caused by MI/R after 1 day of instrumentation was
indistinguishable from that of the sham-operated animals at this time,
indicating that the inflammation from the surgery of the prior day was
still present. The data in Fig. 4B shows that waiting 3 days or
1 wk after instrumentation was enough time to allow dissipation of the
effects of acute surgical trauma and inflammation seen at 3 h and 1 day
after instrumentation. At 3 days and 1 wk, levels of TNF- mRNA in
sham-operated animals were comparable to those of naive control hearts
and allowed differentiation from the increase that resulted because of
MI/R (P < 0.05 vs. time-matched sham-operated animals). As
outlined above, animals with PO exhibited no rise in TNF- mRNA.
Thus, like that of IL-6, increased synthesis of TNF- mRNA was
dependent on reperfusion at this early time.
Comparison of time points.
Because of the unexpectedly large variations in TNF- and IL-6 mRNA
levels at 3 h, we tested to determine whether the data appeared to be
sampled from the same populations. Using the Fmax test, the variances for mRNA data at 3 h were clearly heterogeneous, implying that other factors were operant.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we describe for the first time the use of a closed-chest mouse model of MI/R that allows ligation and reperfusion of the LAD at any time after instrumentation. This method allows dissipation of the acute trauma and inflammation that occur during the initial surgical preparations of the animals, which may significantly influence experimental results when cytokine induction is examined during MI/R. The data suggest that the acute surgical trauma not only increases background of cytokine induction but also may accentuate or prime the response and cause significantly greater variability. The use of a chronic model obviates these difficulties.
Other investigators have previously developed closed-chest models of myocardial damage in the rat. Himori and Matsuura (8) utilized a preparation that allowed both occlusion and reperfusion of the myocardium after surgical recovery, but they did not examine the inflammatory response. Lepran et al. (17) developed a model in rats that was subsequently utilized by other groups (12, 19) and that allowed coronary artery ligation, but not reperfusion, of the myocardium. These investigators avoided the confounding effects of acute surgery and waited 7-10 days after instrumentation to start their experimental protocols, which were primarily aimed at avoiding the effects of anesthesia on electrophysiological states.
The most salient feature of the present report is that, starting at 3 days and continuing at 1 wk after instrumentation, the levels of both
TNF- and IL-6 mRNA found in sham-operated animals had decreased to
those expressed in naive control animals, as shown in Figs. 3B
and 4B.
This time course of downregulation of the inflammatory reaction (i.e., at least a 3-day wait) follows that found by Irwin et al. (9) in rats and Michael et al. (21) in a closed-chest dog model of MI/R. Michael et al. (21) assessed the time course of the release, after instrumentation, of creatine kinase (CK) and phosphorylase, both markers of cell injury and death, in the cardiac lymph effluent of dogs. They demonstrated that both CK and phosphorylase decreased to control levels at 3 days after initial surgery, thus allowing a careful and clear evaluation of the release of these enzymes in the cardiac lymph in control states as well as that resulting from MI/R. Another important feature of the mouse model presented in this study is that induction of MI/R at 3 days and 1 wk after instrumentation induced significant and consistent increases above levels of sham-operated animals in the mRNA levels of both cytokines tested. Therefore, the inflammation caused by MI/R could be clearly separated from that resulting from surgical manipulations of the animals.
The earlier times after instrumentation (i.e., 3 h and 1 day) were less
reliable for several reasons. First, the acute surgical trauma in
sham-operated mice at 3 h after instrumentation resulted in an increase
of IL-6 mRNA markedly above the level found in control hearts
(P < 0.05). In addition, inducing MI/R at this early time
caused a pronounced and highly variable exaggeration of the increase of
both TNF- and IL-6 mRNA (Figs. 3B and 4B). With 3 h
of PO in an acutely prepared animal (i.e., 3 h after instrumentation),
there was also an upregulation of IL-6 mRNA that was not observed in
the group that underwent 3 h of PO at 1 wk after instrumentation. The
phenomenon of inflammatory priming by a previous inflammatory response
is well described, but its mechanism is complex (11). In addition, the
nonhomogeneity of the relative mRNA levels supports the hypothesis that
the processes influencing cytokine regulation early (i.e., 3 h) were
not present at later time points. Finally, the large
variability of the data at 3 h has important practical implications,
forcing larger sample sizes to show effects of any intervention. At 1 day, a significant increase in TNF- mRNA occurred in sham-operated
animals, which was indistinguishable from the level seen in animals
subjected to MI/R. This would suggest that the inflammatory cascade
caused by the instrumentation surgery from the previous day was still continuing. The mechanism for the different pattern of expression of
IL-6 (18) and TNF- (25) mRNA early after surgery is not addressed by
this study, but the difference is highly consistent.
Another important finding in this study is that reperfusion augmented
both IL-6 and TNF- (Figs. 3B and 4B). In PO animals, no rise in cytokine mRNA was observed when surgical trauma was allowed
to dissipate (i.e., at 1 wk after instrumentation). In contrast,
1 h of ischemia and 2 h of reperfusion resulted in a marked upregulation of cytokine mRNA (P < 0.05). Reperfusion
dependence of IL-6 upregulation has been previously identified in
humans with myocardial infarction after coronary revascularization (22) and in both dog (7, 13) and, to some degree, rat models (3) of MI/R.
Similarly, reperfusion of ischemic myocardium in rats was associated
with increased plasma levels of TNF- (16). The present paper
confirms a similar phenomenon in mice once surgical trauma has dissipated.
An important report that corroborates the findings of the present study of the early surgery-induced upregulation of cytokines was published by Chandrasekar et al. (3). These investigators demonstrated in an acute rat model that IL-6 mRNA was detected in the myocardium as early as 15 min after surgery and lasted up to the end of the 6-h sampling period. Importantly, these levels were equivalent to those observed after 15 min of ischemia and 15 or 30 min of reperfusion. Only after 1 h of reperfusion did IL-6 mRNA significantly increase above the levels detected in the sham-operated animals (3). A similar trend of upregulation in sham-operated animals and early reperfusion was observed for IL-6 protein as assessed by Western blotting. As outlined above, these investigators also found reperfusion augmentation of IL-6 mRNA upregulation (3).
In conclusion, this investigation delineates a novel closed-chest mouse model of I/R with an implantable device that allows occlusion and reperfusion of the LAD at any time after instrumentation. The use of this model results in a more reproducible assessment of induction of inflammatory mediators in MI/R in mouse models. Also, this technique will be a potent tool in the study of responses to MI/R of the genetically altered mouse.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant P01-HL-42550 and a Medallion Foundation Grant. T. O. Nossuli is a postdoctoral fellow supported by NHLBI Grant T32-HL-07747-06.
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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: M. L. Entman, Dept. of Medicine, Section of Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza, M/S F-602, Houston, TX 77030-3498 (E-mail: mentman{at}bcm.tmc.edu).
Received 23 September 1999; accepted in final form 29 October 1999.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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