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JPM Vol. 32, No.

3
November 1994:139-147

ORIGINAL ARTICLES

The Six-Day-Old Rat Air Pouch Model of Inflammation:


Characterization of the Inflammatory Response to Carrageenan
S. W. Martin, A. J. Stevens, B. S. Brennan, D. Davies, M. Rowland, and J. B. Houston
Department of Pharmacy, University of Manchester, Manchester, England, United Kingdom

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.

Journal of Pharmacological and ToxicologicalMethods 32, 139-147 (1994)


1994 Elsevier Science Inc.
655 Avenue of the Americas,New York, NY 10010

cation of cellular response, volume, and composition


changes (Forrest et al., 1988). However, the air pouch
model has the added advantage of not involving internal
organs which can be damaged or perforated during sampiing, while also providing a sensitive measure for evaluating agents which inhibit the arachidonic acid cascade
(Sedgwick and Lees, 1986b).
The 1-day-old air pouch model, first described by
Selye in 1953, has been widely used to investigate the
inflammatory reaction to a variety of agents such as
zymosan (Konno and Tsurufuji, 1983), carboxymethyl
cellulose (Ishikawa et al., 1968), monosodium urate
crystals (Gordon et al., 1985), ceramics used in artificial
organs (Nagase et al., 1988), carrageenan (Fukuhara and
Tsurufuji, 1969), Freunds adjuvant (Ohuchi et al., 1982),
and bacterial infection (Yoshino et al., 1985). Studies

1056-8719/94/$7.00

140

JPM Vol. 32, No. 3


November 1994:139-147

have indicated that the inflammatory reactivity of the air


pouch lining increases markedly with time as the lining
develops a more organized surface layer of mononuclear
phagocytes and fibroblastic cells, closely resembling a
synovial cavity on day 6 (Edwards et al., 1981; Sedgwick
and Lees, 1986a; Sedgwick et al., 1983). Therefore, the
6-day-old air pouch closely simulates many aspects of
joint inflammation and would seem to be a better model
than would the 1-day-old pouch for studying the antiinflammatory effects of steroidal (SAID) and nonsteroidal
drugs (NSAID). This antiinflammatory activity has been
poorly characterized in the 6-day-old pouch, although it
has been reported in the 1-day-old pouch (Ohuchi et al.,
1984; Tsurufuji et al., 1978). The aim of this study was to
fully characterize the acute inflammatory reaction to
carrageenan in the 6-day-old air pouch by using frequent
serial sampling and monitoring changes in exudate; and
volume, protein content, cell influx, and inflammatory
mediators in the presence and absence of antiinflammatory drugs.

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.

Production of Air Pouch

Results

Air pouches were produced on the dorsal surface of


rats using a modification of the method by Edwards et al,
(1981) in which they used a 20-mE air pouch. Rats were
lightly anesthetized with halothane on day 0 and 10 mL
of air, sterilized by passing through a 0.2-txM filter (sterile acrodisc, Gelman Sciences) and was injected subcutaneously on the dorsal surface. On day 3, air pouches
were reinflated with 4-6 mL of sterile air. On day 6, the
inflammogen carrageenan (5 mL; 4 mg/mL in phosphate-buffered saline, pH 7.4) was injected into the
pouch.

Proliferative Inflammatory Responses to Carrageenan


The volume of fluid recovered from the pouch did not
change significantly during the 48 hr of the study [Figure
l(a)]. The volume of pouch fluid recovered at 15 min
(4.0 + 0.1 mL) was not significantly different from that
recovered from animals at 48 hr (4.1 _+ 0.4 mE) (p >
0.82). Although exudate volume remained constant, dramatic changes in the other inflammatory processes were
observed in response to carrageenan administration.

Validation of the Model

Cellular Accumulation in Pouch Fluid

Rats (n = 6) were killed at intervals of up to 48 hr and


all pouch fluid was collected in order to determine exudate volume; the analysis of cell-free protein was carried
out according to the method first described by Lowry et
al. (1951). Total leucocyte numbers were counted using a
Neaubauer counting chamber after staining with Giemsa.
Pouches were dissected out and the wet weight determined, and, following desiccation to constant mass (60
C), their dry weights were recorded.

The time course of leucocyte influx into the air pouch


after carrageenan administration is summarised in Figure
l(b). After an initial lag period of 3 hr, the influx of
leucocytes was time related with a rapid influx of cells
between 3 hr (8.5 x 106 mL) and 8 h (183 106 mE). Cell
numbers continued to increase progressively with time
reaching 418 106 mE at 48 hr. Differential cell counts
indicated that the cell response during the first 24 hr was
predominantly polymorphonuclear (87%), with mononu-

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.

clear cell numbers increasing gradually to 150 106 mL


representing 25% of the total cell count at 48 hr.

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~

Protein Levels in Cell-Free Pouch Exudate


Immediately following the administration of carrageenan, the protein concentration of the pouch exudate
rapidly increased from 2.1 _+ 0.2 mg/mL at 1 hr to 13.7
+ 1.0 mg/mL at 8 hr; thereafter, the rate of increase was
much slower reaching levels of 26.7 + 1.9 mg/mL at 24
hr and 32.6 _+ 1.5 mg/mL at 48 hr.

u.I
a_
200

100

Pouch Exudate Levels of Prostaglandin E2


The concentration-time course of PGE 2 in air pouch
exudate following carrageenan administration is shown
in Figure 2. After an initial lag period of 3 hr, a rapid

10
Time

15

20

25

(Hours)

Figure 2. Time course of air pouch exudate concentration of


PGE2 after injection of carrageenan. Serial samples of air pouch
exudate were taken at times indicated and each point represents the
mean SEM for six animals.

JPM Vol. 32, No, 3


November 1994:139-147

142

500

N.S.
400

30O
E
N.S.

o
m 200

100

,q
N.S.

Time

10

10

(Hours)

Figure 3. PGE2 response time courses to carrageenan in control


animals spanning a 3-year interval. Clear and hatched bars indicate
responses obtained in 1990 and 1993, respectively. Each bar represents the mean _+ SEM for groups of between six and nine animals.

response to carrageenan has been consistent with no


significant difference between the 3-, 7-, and 10-hr responses to carrageenan in experiments performed in 1990
and 1993 (Figure 3).

Antiinflammatory Effects of lbuprofen


Bolus air pouch administration of rac-ibuprofen (0.11 mg/kg) at the same time as carrageenan reduced the
influx of leucocytes into the air pouch by 5 hr. Drug-induced reductions in cell numbers then gradually diminished, and, by 24 hr, no difference in cell influx was
observed between control and treatment groups (Table 1).
Bolus air pouch administration of rac-ibuprofen in the
range from 0.1 to 1 mg/kg did not significantly alter (p >

0.113) the amount of pouch cell-free protein during the


24 hr following carrageenan administration (Table 2).
Protein concentrations in rac-ibuprofen (l mg/kg) treated
animals reached 30.3 3.9 mg/mL at 24 hr and was not
significantly different from that in control animals (32.8
3.0 mg/mL; p < 0.113).
Rac-ibuprofen at 1 mg/kg significantly (p < 0.003)
reduced the volume of air pouch exudate recovered 24 hr
after carrageenan administration from 3.9 _+ 0.2 to 2.5
0.3 mL. Similarly, lower doses of 0.5 and 0.1 mg/kg
rac-ibuprofen reduced the exudate volume at 24 hr to 3.4
_+ 0.5 and 2.9 0.5 mL, respectively, but were not
significantly different from the control (p < 0.29; p <
0.06).
Of all the inflammatory processes studied, the prostaglandin response was found to be the most sensitive
marker to ibuprofen, with doses as low as 0.05 mg/kg
significantly reducing air pouch levels of PGE2. Bolus air
pouch administration of [S+]-ibuprofen (0.05-1 mg/kg)
at the same time as carrageenan-reduced PGE2 levels
dose dependently during the first 15 hr after which,
drug-induced reductions in PGE 2 levels became less apparent, and by 21 hr the measured response in control and
drug-treated animals was not significantly different (Figure 4). [S+]-Ibuprofen was found to reduce pouch levels
of PGE2 without altering the temporal profile of the
prostaglandin response to carrageenan.
The area under the PGE 2 response-time curve between 3 and 10 hr was found to be a more reproducible
means of quantifying drug effects. For example, the
PGE 2 response measured by area from 3 and 10 hr had a
lower coefficient of variation (CV = 30%; n = 20) compared to the 10-hr time point (CV = 53%; n -- 20). Areas
are expressed relative to the control to give percentage
inhibition (Figure 5).

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

Exudate Protein Concentration (mg/mL)


Time
Hours

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.,

1983). As different research groups have each modified


the air pouch model, strict comparisons cannot be made.
Tsurufuji and colleagues routinely performed experiments in the 1-day-old air pouch using various inflammogens and were the first to develop an allergic air
pouch inflammatory model (Tsurufuji et al., 1982).
These investigators have observed a slow development of
edema in response to carrageenan administration, with
less than 1 mL of exudate accumulating over 5 days
(Tsurufuji et al., 1978). In addition, other groups have
shown that a 1-day-old pouch is unable to retain fluid,
probably due to the poor cellularity of the air pouch
lining (Isaji et al., 1989; Sedgwick et al., 1983). How-

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

Figure 5. Shows the dose effect of intrapouch [S+]-ibuprofen


(0.05-1 mg/kg) upon the concentration of PGE2 in air pouch
exudate. Each point represents the percentage decrease of AUC
(3-10 hr) for PGE2 (ng/mL x min) mean _ SEM in drug-treated
animals relative to control synthesis for groups of between 6 and 9
animals.

144

ever, 3- and 6-day-old air pouch models are able to retain


fluid and they produce a much greater edematous response to carrageenan. The increased inflammatory response with pouch age reflects pouch development
changing from an incomplete poorly cellular and collagenous layer on day 1 to one with well organized and
densely arranged laminated collagenous fibers, with increasing numbers of fibroblasts on days 3-6 (Edwards et
al., 1981). The inner pouch lining similarly develops a
more organized surface layer of mononuclear phagocytes
and fibroblastic cells (Edwards et al., 1981 ).
Using the 6-day-old pouch, we found that the recovery
of air pouch exudate remained relatively constant during
the 48-hr study period (80%). This observation is in
agreement with previous studies, as marked increases in
exudate volume only begin to occur 2-3 days following
carrageenan administration and is believed to be associated with a transition from acute to chronic inflammation (Hambleton and Miller, 1989; Sedgwick et al.,
1984). An explanation for incomplete fluid recovery is
indicated by histological studies of edema formation in
the pouch wall and surrounding tissue (Isaji et al., 1989;
Konno and Tsurufuji, 1983). Although exudate volume
remained constant in control animals, we recovered a
significantly lower amount of exudate from rac-ibuprofen (1 mg/kg) treated animals at 24 hr, suggesting that an
edematous reaction to carrageenan administration may
have been occurring in control animals. Other NSAIDs,
such as indomethacin, piroxicam, aspirin, and benoxaprofen, have also been found to prevent edema formation
in carrageenan-induced air pouch inflammation (Sedgwick et al., 1984). In the chronic carrageenan-induced
inflammatory air pouch model, the effect of NSAIDs is
uncertain. Some investigators report that only steroids
are capable of reducing edema (Hambleton and Miller,
1989; Sato et al., 1980), whereas others found that multiple dosing with 1 mg/kg indomethacin significantly reduced the edema formation, 3, 7, and 14 days after
carrageenan administration (AI-Duaij et al., 1986).
Although the volume of air pouch exudate remained
relatively constant during the acute phase of our studies,
its composition changed dramatically over the same time
period. Cellular response to carrageenan depends upon
air pouch age, probably associated with better retention
of inflammatory fluid by older pouches and the continuing development of the pouch lining (Edwards et al.,
1981; Sedgwick et al., 1983). In the l-day-old pouch, a
very minor cellular response to 10 mg carrageenan was
observed at 6 hr (< 5 x l06 cells mL), in contrast to the
high leucocyte response at 6 hr in 3-day-old pouches (75
x 106 cells mL) and in 6-day-old pouches ( 110 x 106 cells
ml) (Sedgwick et al., 1983). Cellular response in the
1-day-old pouch can be increased markedly by raising
the dose of carrageenan to 80 mg, thus amplifying the
chemotactic response from approximately 5 x 106 cells

JPM Vol. 32, No. 3


November 1994:139 147

mL to 200 x 106 mL cells at 8 hr (Hirasawa et al., 1991;


Sedgwick et al., 1983).
In the 6-day-old air pouch we have characterized the
initial phase of the temporal profile for cellular accumulation. Earlier studies indicated that the initial influx of
leucocytes into the air pouch occurs between 3 and 5 hr
(Hambleton and Miller, 1989; Sedgwick et al., 1984), a
profile similar to that observed in the pleurisy model
(Ackerman et al., 1980; Vinegar et al., 1982). Cellular
response to carrageenan has been shown to be dose dependent but independent of volume of irritant administered; our results of 150 + 50 x 106 mE leucocytes at 7 hr
compares well with 110 to 180 x 106 mL at 6 hr reported
by Sedgwick e t a l . (1983, 1984) and 100 x 106 mL at 6 hr
reported by Hambleton and Miller (1989). The leucocyte
response to carrageenan was predominantly polymorphonuclear cells (87%), whereas the mononuclear cells
response was not observed until 9 hr and then gradually
increased to 25% of the cell total at 24 hr, observations
that equate well with those of Sedgwick et al. (1983,
1984). Hambleton and Miller (1989) reported a transition
on day 3 from a predominance of polymorphonuclear
cells to monocytes; this transition to monocytes is believed to be involved in the mechanism underlying the
change from acute to chronic inflammation (Mackay et
al., 1985).
One problem associated with the use of carrageenan is
its cytotoxicity to macrophages both in vitro and in vivo
(Catanzaro et al., 1971 ; Pugh-Humphreys and Thomson,
1979; Thomson et al., 1979). Evidence for the cytotoxicity of carrageenan in the air pouch is the rapid increase
in exudate levels of lactate dehydrogenase (Sedgwick
and Lees, 1986b). The cellular damage carrageenan produces probably accounts for the high concentration of
cell-free protein we measured in the air pouch exudate
which was, as expected, unaltered by ibuprofen (1
mg/kg). Proteins such as albumin have been shown to
enter the air pouch from the vasculature following carrageenan administration (Hambleton and Miller, 1989;
Isaji et al., 1989; Martin et al., 1993b). However, the
influx of plasma albumin reported by Martin et al.
(1993b) only contributes a minor fraction of the total
protein observed in this study. Therefore, the majority of
exudate protein measured is probably released from
damaged leucocytes within the exudate and cells lining
the air pouch.
Numerous investigators have reported a variety of
eicosanoids formed in response to carrageenan administration such as PGFe~, thromboxane B2, prostacyclin,
leukotriene B4, and PGE 2 (Ohuchi et al., 1979; Sedgwick
et al., 1986a). We have chosen to study PGE 2 levels in
pouch exudate as this has been the most widely studied
eicosanoid. PGE 2 production is influenced by the stage of
development of the air pouch lining with 6-day-old
pouches producing 20 times more PGE 2 than l-day-old

S. W. MARTIN ET AL.
THE SIX-DAY-OLD AIR POUCH MODEL OF INFLAMMATION

air pouches (Sedgwick and Lees, 1986a). This may be


accounted for by the greater influx of polymorphonuclear
leucocytes into the pouch exudate and the larger numbers
of phagocytic cells present in the pouch lining in the
6-day-old pouch (Edwards et al., 1981; Sedgwick et al.,
1983). Large variations in the PGE2 response in the
1-day-old pouch have been reported with exudate levels
ranging between 4 and 100 ng/mL (Chang et al., 1975;
Ohuchi et al., 1979; Tsurufuji et al., 1978; Whelan, 1974;
Willis, 1970). Similarly, in the 6-day-old air pouch large
differences in the PGE2 response to carrageenan have
been reported by different research groups. PGE2 levels
reported in this study are much higher than that in most
other reports. For example, Sedgwick and Lees (1986a)
reported a 6-hr PGE 2 response to carrageenan of 11
ng/mL, whereas we obtained levels of 150 ng/mL at 7 hr.
These seem to be genuine differences in PGE2 response
between groups, as both have found their model to be
very consistent over many years. There seems no obvious
explanation for these differences as similar experimental
methods and dose of Carrageenan Viscarin (20 mg) were
used in both studies. The large interanimal variability in
PGE2 response often observed with this model can probably be accounted for by variability in both the onset and
magnitude of the inflammatory response. The only reported PGE2 response time course data in the 6-day-old
pouch that is comparable with our own is that of Sedgwick and Lees (1986a). However, we have better characterized the first 24 hr of the acute phase due to more
frequent serial sampling. This may account for the difference in the time to reach maximal response. Whereas we
observed a maximal response of 450 ng/mL at 15 hr,
Sedgwick and Lees (1986a) reported a maximal PGE2
response of 11 ng/mL at 6 hr; however, they did not
sample between 6 and 24 hr, and so may have missed the
true time to maximum response.
Granuloma formation during the fist 24 hr of the acute
phase has not previously been quantified in this 6-dayold model, although it has been reported in the 1-day-old
air pouch (Fukuhara and Tsurufuji, 1969). Interestingly,
the temporal profile of granuloma formation is similar to
that of the influx of leucocytes with an initial lag period
of 3 hr followed by a continual weight increase up to 48
hr; it has been reported to reach a maximum 3 or 4 days
following administration of carrageenan or Freunds adjuvant (Hambleton and Miller, 1988, 1989). Histological
studies have also indicated an association between granuloma development and white cell influx. Histological
studies have shown that during the first 24 hr, edema
develops in the subcutaneous tissue surrounding the
pouch, with aggregation of polymorphonuclear leucocytes and monocytes at the inner surface of the pouch
wall (Konno and Tsurufuji, 1983).
Despite being widely investigated, the mechanism by
which NSAIDs exert their effects still remains unclear. In

145

this study we have concentrated on the dynamic response


to a NSAID. In particular, we have investigated the
effects of rac-ibuprofen on a number of proliferative
inflammatory processes in order to characterize the air
pouch model, and due to its restricted supply, we have
limited the use of (S+)-ibuprofen when studying the
concentration-effect relationship for PGE2. Although
local administration of NSAIDs and SAIDs has been
widely shown to reduce PGE 2 levels following nonallergenic and allergenic-induced inflammation (Forrest et
al., 1988; Ohuchi et al., 1982; Ono et al., 1987), this
reduction has not been fully quantified, and the duration
of NSAID-effect has not been well documented. However, we have been able to better characterize both the
acute PGE 2 response to inflammation and the duration of
inhibition by a NSAID in an individual animal. We have
previously used this model (Stevens et al., 1993) to
determine the pharmacokinetics of NSAIDs in both
plasma and pouch exudate. Therefore, we are now able to
study the relationship between the pharmacokinetics and
pharmacodynamics of a NSAID in the same animal; this
reduces the interindividual variability as well as the number of animals used.
(S+)-Ibuprofen reduced the intensity of the PGE2 response to carrageenan dose dependently in the range
from 0.1 to 0.5 mg/kg without changing the temporal
profile of the response. After demonstrating these dosedependent effects over 24 hr, we restricted subsequent
studies with (S+)-ibuprofen to the first 10 hr to minimize
the additional use of animals. The area under the PGE 2
response-time curve gave a more reproducible relationship to drug effect than did specific time-point estimates,
and such comparisons indicated the PGE 2 response to be
the most sensitive inflammatory marker to ibuprofen.
Preliminary studies suggest that the dose-dependent reduction in the temporal profile of PGE2 to be a direct
reflection of the concentration of drug in the air pouch, as
we have previously demonstrated that, irrespective of
mode (bolus or infusion) or route (intravenous or air
pouch) of (S+)-ibuprofen administration, the resulting
antiinflammatory response was related to the average
concentration of drug in the pouch during the inflammatory reaction (3-10 hr) (Martin et al., 1993a).
Ibuprofen-induced inhibition of leucocyte migration
occurred during the initial 3-5 hr following administration, after which the drug effects gradually diminished.
This profile of NSAID activity has also been observed
for indomethacin, piroxicam, aspirin, and benoxaprofen
in the 6-day-old air pouch (Sedgwick et al., 1984). In
multiple dosing studies, the effects of NSAIDs and
SAIDs seem uncertain. Some reports suggest that indomethacin and prednisolone potentiate the leucocyte response to carrageenan on day 3 by prolonging the polymorphonuclear response; this has similarly been
observed in the 3-day carrageenan pleurisy model (Ack-

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November 1994:139-147

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.

This study was supported by the LINK Programme in Selective Drug


Delivery and Targeting, funded by SERC/DTI/MRC and the pharmaceutical industry.

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