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3
November 1994:139-147
ORIGINAL ARTICLES
Inflammation was induced in the 6-day-old rat air pouch by injection of carrageenan. The model
was characterized in terms of exudate volume, leucocyte influx, cell free protein, prostaglandin E2
levels, and granuloma formation. The time course of all these inflammatory markers, except
prostaglandin E> showed a 3-hr lag followed by a rapid increase to 8 hr. Thereafter, the rate of
increase was much slower to 48 hr. Differential cell counts indicated a predominantly polymorphonuclear cell response (75%) during the first 48 hr. Prostaglandin E2 levels increased rapidly
after a 3-hr lag, to a maximum of 440 +_ 140 ng/mL at 15 hr and thereafter quickly declined to 140
-+ 60 ng/mL at 21 hr. Prostaglandin E2 levels were the most sensitive inflammatory marker to
(S+)-ibuprofen and were reduced dose dependently in the range 0.05 to 1 mg/kg. We have
demonstrated the time course for duration of NSAID-induced reduction of prostaglandin E2 levels
during inflammation in an individual animal. Rac-ibuprofen (0.1-1 mg/kg) reduced leucocyte
influx at 3 and 5 hr, after which drug effects gradually diminished by 24 hr. Rac-ibuprofen at 1
mg/kg significantly reduced the volume of air pouch exudate recovered at 24 hr but had no effect
on protein levels.
Key words: Air-pouch inflammation; Leucocyte; PGE2; Granuloma; Protein; Ibuprofen
Introduction
Many animal models of inflammation, such as the
sponge implant, paw edema, peritoneal, pleurisy, and air
pouch have been developed for the discovery and evaluation of novel therapeutic agents. Each model has advantages and disadvantages for studying the effects of drugs
on different facets of the inflammatory process. For example, the peritoneal, pleurisy, and air pouch cavity
models of inflammation have the beneft of a readily
harvested inflammatory exudate which allows quantifi-
Address reprint requests to Dr. S. W. Martin, Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle,
Washington 98195, USA.
Received November 1, 1993; revised and accepted May 1, 1994.
1056-8719/94/$7.00
140
Anti-inflammatory Studies
Rac-ibuprofen in the range from 0.05-1 mg/kg (n =
5-14) was administered into the air pouch at the same
time as carrageenan. Serial samples (n = 5; 250 ~xL) of
pouch exudate were taken at intervals up to 24 hr for
determination of leucocyte numbers, protein concentration, and final exudate volume.
[S+]-Ibuprofen in the range from 0.05-1 mg/kg (n =
6-9) was administered into the air pouch at the same time
as carrageenan. Serial samples (n = 5; 250 jxL) of pouch
exudate were taken at intervals up to 21 hr for determination of PGE 2 concentration.
The PGE2 concentration was determined by adding a
50-~xL aliquot of exudate to 25 ~M indomethacin to halt
any further eicosanoid synthesis. Preformed eicosanoids
were then converted to their methyl oximate derivatives
by the further addition of 50 IxL methoxyamine hydrochloride. The concentration of the methyl oxime derivative of PGE 2 in pouch exudate was determined by RIA
(Amersham RPA530). Lowest detectable amount of
PGE2 = 8 pg/mL; intraassay sensitivity at midrange 29 +_
0.7 pg/tube CV = 2.3% n = 5.
Methods
Animals
Male Sprague-Dawley rats (200-250 g) were used
throughout.
Statistical Analysis
Results were expressed as the mean _+ SEM throughout and compared using one-way analysis of variance
and Student's t test.
Results
S. W. MARTIN ET AL.
THE SIX-DAY-OLD AIR POUCH MODEL OF INFLAMMATION
141
6OO
5OO
~eEv4
3
~3
3oo
o=2
o.
2oo
2
o
0
0
lOO
o---
1o
20
30
40
50
Time
1o
(Hours)
20
30
40
50
0
0
10
20
30
40
50
Time (Houm)
Time (Hours)
Figure 1. Time course of various air pouch parameters after injection of 40 mg carrageenan in 5 mL phosphate-buffered saline. Rats were
killed at times indicated, and the pouch contents were collected. Each point represents the mean _+SEM for six animals. (a) Shows the time
course of air pouch exudate volume. (b) Shows the time course of air pouch leucocyte accumulation. Polymorphonuclear ( ) mononuclear
cells (A), and totals (0) were expressed as total cell number per pouch. (c) Shows air pouch dry ( ) and wet (A) weight.
increase in PGE 2 levels was observed reaching a maximum concentration of 440 _+ 140 ng/mL at 15 hr; this
then quickly declined to a concentration of 190 + 60
ng/mL at 21 hr. In our 3 years experience, the PGE 2
Granulation Tissue
Distinct granulation of the pouch wall lining developed following the administration of carrageenan [Figure
l(c)]. The time course of granuloma formation was similar to that of leucocyte infiltration. After an initial lag
period of 3 hr, there was a rapid increase in pouch dry
weight between 3 (0.2 _+ 0.1 g) and 8 hr (0.5 _+ 0.1 g);
the weight continued to increase reaching 0.6 _+ 0.1 g at
48 hr.
600
500
4OO
o~
u.I
a_
200
100
10
Time
15
20
25
(Hours)
142
500
N.S.
400
30O
E
N.S.
o
m 200
100
,q
N.S.
Time
10
10
(Hours)
Discussion
We believe our data to be the first to characterize the
time course for duration of NSAID-induced reduction of
PGE 2 during inflammation in an individual animal, in
addition, we have fully described the initial phase of the
T a b l e 1. Leucocyte Accumulation in Air Pouch Exudate Following Injection of Carrageenan Together with Vehicle or rac-Ibuprofen
Time
(hours)
1
3
5
7
24
n
Control
0.4
0.7
8.9
22.1
94.2
,+ 0.1
_+ 0.1
+ 1.4
,+ 3.1
,+ 9.5
14
0.1
0.3
0.6
4.1
18.3
84.9
,+ 0.04 (90%)
_+ 0.2 (77%)
_+ 1.0 (45%)
,+ 4.5 (83%)
,+ 11.7 (90%)
7
rac-Ibuprofen
0.5 (mg/kg)
0.2
0.6
4.1
I 1.8
108
+ 0.03 (70%)
_+ 0.1 (77%)
+ 1.2 (46%)
,+ 2.4 (53%)
,+ 25.5 (115%)
5
1.0
0.3
0.3
5.6
19.5
105
+ 0.02 (8I%)
_+ 0.05 (47%)
+ 1.4 (63%)
,+ 2.8 (88%)
,+ 19.2 (112%)
7
Note: Serial samples of air pouch exudate were taken at times indicated and leucoycytes counted. Each value represents the mean + SEM with percent
of control in parenthesis for groups of between 5 and 14 animals. Significant difference between drug-treated and control group was only observed at 5
h (p < 0.017) as determined by one-way ANOVA.
S. W. MARTIN ET AL.
THE SIX-DAY-OLD AIR POUCH MODEL OF INFLAMMATION
143
T a b l e 2. Concentration of Protein in Air Pouch Exudate Following the Injection of Carrageenan Together with Vehicle or Ibuprofen
Control
1.7 + 0.4
3
5
7
24
n
2.9
4.6
7.8
32.8
+
+
+
+
14
rac-lbuprofen
0.5 (mg/ml)
0.1
1.7 + 0.8
0.5
0.9
1.1
3.0
3.2
5.0
7.9
32.5
1.0
1.7 -+ 0.4
1.6 + 0.4
+ 0.6
-+ 1.2
___2.3
+ 1.5
7
2.9
4.2
9.2
30.3
+
+
+
+
5
0.3
0.6
1.8
16.7
3.0
4.8
7.3
30.3
+ 0.8
-+ 1.6
-+ 1.6
-+ 3.9
7
Note: Serial samples of air pouch exudate were taken at times indicated and protein concentration determined. Each value represents the mean +__SEM
for groups of between 5 and 14 animals. There is no statistical significant difference between the protein concentration among groups at each time
point (one-way ANOVA).
acute inflammatory reaction to carrageenan in the 6-dayold air pouch, unlike previous studies. We have investigated the antiinflammatory effects of ibuprofen upon a
variety of inflammatory processes, such as volume of
pouch fluid, number, and type of inflammatory cells, the
protein and PGE 2 concentration of inflammatory fluid,
and granuloma formation. Of the inflammatory processes
studied, the PGE2 response was found to be the most
sensitive to ibuprofen-induced inhibition.
The inflammatory response of the air pouch has been
shown to change dramatically due to both a variety of
stimuli and the age of the air pouch, as assessed by
exudate volume and composition (Sedgwick et al.,
100
/(
501J
8G
40C
"7,
u~ 300
e-
g00
iii
Q.
40
2OO
20
100
0
0
10
15
20
I
25
T i m e (Hours)
Figure 4. Shows the effect of intrapouch administration of [S+]ibuprofen (A) 0.05 mg/kg; (V) 0.1 mg/kg; (0) 0.5 mg/kg; (11) 1
mg/kg; (0) control, upon the concentration of PGE2 in air pouch
exudate between 3 and 21 hr. Serial samples of air pouch exudate
were taken at times indicated and each point represents the mean
SEM for groups of between 6 and 9 animals.
0.01
0.1
D o s e mg kg "1
144
S. W. MARTIN ET AL.
THE SIX-DAY-OLD AIR POUCH MODEL OF INFLAMMATION
145
146
erman et al., 1980; Hambleton and Miller, 1989). However, others have reported that daily injection of 1 mg/kg
indomethacin significantly inhibits the influx of leucocytes 7 and 14 days after carrageenan administration
(A1-Duaij et al., 1986).
In summary, the 6-day-old air pouch seems to have a
number of advantages over other models of inflammation
as it provides a readily harvested inflammatory exudate
which allows the investigation of drug effects upon many
aspects of the inflammatory process. We have measured
changes in cell influx, volume, protein content, and PGE2
levels with time, in the presence and absence of antiinflammatory drugs. Using a serial sampling procedure, we
have been able to investigate the time course for duration
of antiinflammatory effects of NSAIDs. This approach
also allows the determination of drug levels in the inflammatory exudate and hence the investigation of drug
concentration-effect relationships.
References
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immune and non-immune inflammation in the six-day air pouch in rats.
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Catanzaro PJ, Schwartz HJ, Graham RC (1971) Spectrum and possible
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S. W. MARTIN ET AL.
THE SIX-DAY-OLD AIR POUCH MODEL OF INFLAMMATION
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147