The Six-day-old Rat Air Pouch Model of Inflammation Characterization of the Inflammatory Response to Carrageenan

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 evalua- tion 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 Phar- maceutics, School of Pharmacy, University of Washington, Seattle, Washington 98195, USA.

Received November 1, 1993; revised and accepted May 1, 1994.

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 sam- piing, while also providing a sensitive measure for evalu- ating 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

Journal of Pharmacological and Toxicological Methods 32, 139-147 (1994) 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 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.

Methods

Animals

Male Sprague-Dawley rats (200-250 g) were used throughout.

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 hydro- chloride. The concentration of the methyl oxime deriva- tive 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.

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

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 subcu- taneously 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.

Results

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, dra- matic changes in the other inflammatory processes were observed in response to carrageenan administration.

Validation of the Model

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.

Cellular Accumulation in Pouch Fluid

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-

~eEv4

~3

o=2 o.

1

0 0

6OO

3

2

5OO

3oo

2oo o

lOO

p i i i i o - - - i

1 o 20 30 40 50 o 1 o 20 30 40 50

T ime (Hours) Time (Houm)

0 0

600

I [ I I I

10 20 30 40 50

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.

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.

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.

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

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

500

4OO

o~

u.I a_

200

100

I

5 10 15 20

T ime (Hours)

S. W. MARTIN ET AL. 141 THE SIX-DAY-OLD AIR POUCH MODEL OF INFLAMMATION

I

25

Figure 2. Time course of air pouch exudate concentration of PGE 2 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.

142 JPM Vol. 32, No, 3 November 1994:139-147

500

400

30O E

w o m 200

100

N.S.

N.S.

3

N.S.

l

,q

3 7 7 10

Time (Hours) 10

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 (0.1-

1 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 ap- parent, and by 21 hr the measured response in control and drug-treated animals was not significantly different (Fig- ure 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

Table 1. Leucocyte Accumulation in Air Pouch Exudate Following Injection of Carrageenan Together with Vehicle or rac-Ibuprofen

Time rac-Ibuprofen (hours) Control 0.1 0.5 (mg/kg) 1.0

1 0.4 ,+ 0.1 0.3 ,+ 0.04 (90%) 0.2 + 0.03 (70%) 0.3 + 0.02 (8I%) 3 0.7 _+ 0.1 0.6 _+ 0.2 (77%) 0.6 _+ 0.1 (77%) 0.3 _+ 0.05 (47%) 5 8.9 + 1.4 4.1 _+ 1.0 (45%) 4.1 + 1.2 (46%) 5.6 + 1.4 (63%) 7 22.1 ,+ 3.1 18.3 ,+ 4.5 (83%) I 1.8 ,+ 2.4 (53%) 19.5 ,+ 2.8 (88%) 24 94.2 ,+ 9.5 84.9 ,+ 11.7 (90%) 108 ,+ 25.5 (115%) 105 ,+ 19.2 (112%) n 14 7 5 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.


Tab le 2. Concentration of Protein in Air Pouch Exudate Following the Injection of Carrageenan Together with Vehicle or Ibuprofen

Exudate Protein Concentration (mg/mL)

Time rac-lbuprofen Hours Control 0.1 0.5 (mg/ml) 1.0

1 1.7 + 0.4 1.7 + 0.8 1.6 + 0.4 1.7 -+ 0.4 3 2.9 + 0.5 3.2 + 0.6 2.9 + 0.3 3.0 + 0.8 5 4.6 + 0.9 5.0 -+ 1.2 4.2 + 0.6 4.8 -+ 1.6 7 7.8 + 1.1 7.9 ___ 2.3 9.2 + 1.8 7.3 -+ 1.6 24 32.8 + 3.0 32.5 + 1.5 30.3 + 16.7 30.3 -+ 3.9 n 14 7 5 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-day- old 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 inflam- mogens 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-

501J

40C "7,

u~ 300

2OO

100

0

/

I P I

0 5 10 15 20 25

T ime (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.

100

8G

e-

g00

iii Q.

40

0

20

/(

I I

0.01 0.1 1

Dose 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 JPM Vol. 32, No. 3 November 1994:139 147

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 (Sedg- wick 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 continu- ing 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

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 eta 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 v ivo (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 pro- duces 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


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 Sedg- wick 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-day- old 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

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 nonaller- genic 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. How- ever, 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 dose- dependent 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-

146 JPM Vol. 32, No. 3 November 1994:139-147

erman et al., 1980; Hambleton and Miller, 1989). How- ever, 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 pharmaceuti- cal industry.

References Ackerman N, Tomolonis A, Miram L, Kheifets J, Martinez S, Carter A

(1980) Three day pleural inflammation: A new model to detect drug effects on macrophage accumulation. J Pharmacol Exp Ther 215:588- 595.

A1-Duaij AY, Sedgwick AD, Willoughby DA (1986) The effect of anti-inflammatory and rheumatoid disease modifying drugs on prolonged immune and non-immune inflammation in the six-day air pouch in rats. lnt J Tissue React 8:463-468.

Catanzaro PJ, Schwartz HJ, Graham RC (1971) Spectrum and possible mechanism of carrageenan cytotoxicity. Am J Pathol 64:387-404.

Chang W-C, Murota S-I, Tsurufuji S (1975) Contents of prostaglandin E in the exudate of rat carrageenin granuloma. Jap J Pharmacol 25:219- 221.

Edwards JCW, Sedgwick AD, Willoughby DA (1981) The formation of a structure with the features of synovial lining by subcutaneous injection of air: An in vivo tissue culture system. J Pathol 134:147-156.

Forrest MJ, Brooks PM, Tagaki T, Kowanko I (1988) The subcutaneous air-pouch model of inflammation. In Handbook of Animal Models for the Rheumatic Diseases, Vol 1. Eds., RA Greenwald and HS Diamond. London: CRC Press, pp. 125-134.

Fukuhara M, Tsurufuji S (1969) The effect of locally injected anti-inflammatory drugs on the carrageenin granuloma in rats. Biochem Pharma- col 18:475-484.

Gordon TP, Kowanko IC, James M, Roberts-Thomson PJ (1985) Monoso- dium urate crystal-induced prostaglandin synthesis in the rat subcutaneous air pouch. Clin Exp Rheumatol 3:291-296.

Hambleton P, Miller P (1988) Studies on immunological air pouch inflammation in the rat. lnt Arch Allergy Appl Immunol 87:70-75.

Hambleton P, Miller P (1989) Studies on carrageenin air pouch inflammation in the rat. Br J Exp Path 70:425-433.

Hirasawa N, Watanabe M, Mue S, Tsurufuji S, Ohuchi K (1991) Down- ward regulation of neutrophil infiltration by endogenous histamine without affecting vascular permeability responses in air-pouch-type carrageenin inflammation in rats. Inflammation 15:117-126.

Ishikawa H, Niinobe S, Tsurufuji S (1968) Studies on the mode of action of anti-inflammatory agents. 1 Quantitative analysis of anti-inflammatory effects by carboxymethyl cellulose pouch method. Yakugaku Zasshi 88:1472-1477.

Konno S, Tsurufuji S (1983) Induction of zymosan-air-pouch inflammation in rats and its characterization with reference to the effects of anticomplementary and anti-inflammatory agents. Br J Pharmacol 80:269-277.

Lowry OH, Rosenbrough N J, Farr AL, Randall RJ ( 1951 ) Protein determination with the Folin phenol reagent. J Biol Chem 193:265-275.

Mackay AR, Sedgwick AD, Dunn C J, Fleming WE, Willoughby DA (1985) The transition from acute to chronic inflammation. Br J Derma- tol 113(Suppl 28):34-48.

Martin SW, Stevens AJ, Houston JB (1993a) Use of the rat air pouch model of inflammation to evaluate regional drug delivery. Eur J Drug Metab Pharmacokinetics 18:236.

Martin SW, Stevens AJ, Reis ML, Houston JB (1993b) The rat pouch model of inflammation: Reduction of inflammation-enhanced plasma protein permeability by nonsteroidal antiinflammatory drugs. J Pharm Pharmacol 45(suppl 2):1116.

Mitjavila MT, Carbonell MT, S~iiz MP, Muntan6 J, Sfinchez J, Puig-Parel- lada P (1990) Role of iron in carrageenan-induced granuloma: action of desferrioxamine and indometacin. Pharmacology 40:236 240.

Nagase M, Baker DG, Schumacher Jr HR (1988) Prolonged inflammatory reactions induced by artificial ceramics in the rat air pouch model. J Rheumatol 15:1334 1338.

Ohuchi K, Levine L, Sato H, Tsurufuji S (1979) Prostaglandins E 2 F2~, 6-Keto-F~,~ and thromboxane B2 levels in carrageenin-induced inflammatory exudates in the rat air-pouch granuloma. Prostaglandins and Medicine 2:293-297.

Ohuchi K, Watanabe M, Levine L (1984) Arachidonate metabolites in acute and chronic allergic air pouch inflammation in rats and the anti-inflammatory effects of indomethacin and dexamethasone, lnt Arch Allergy Appl Immunol 75:157-163.

Ohuchi K, Yoshino S, Kanaoka K, Tsurufuji S, Levine L (1982) A possible role of arachidonate metabolism in allergic air pouch inflammation in rats. lnt Arch Allergy Appl lmmunol 68:326-331.

Ono N, Sunami A, Yamasaki Y, Yamamoto N, Miyake H (1987) Effects of proglumetacin maleate and its major metabolites on allergic air pouch inflammation in rats. EurJ Pharmacol 142:245 251.

Pugh-Humphreys RGP, Thomson AW (1979) An uhrastructural study of peritoneal mononuclear phagocytes from iota carrageenan injected mice. Cytobios 24:211-218.

Sato H, Hashimoto M, Sugio K, Ohuchi K, Tsurufuji S (1980) Compara- tive study between steroidal and non-steroidal anti-inflammatory drugs on the mode of their actions on vascular permeability in rat carrageenan air pouch inflammation. J Pharmacobiodyn 3:345-352.

Sedgwick AD, Lees P (1986a) Studies of eicosanoid production in the air pouch model of synovial inflammation. Agents Actions 18:429-438.

Sedgwick AD, Lees P (1986b) A comparison of air pouch sponge and pleurisy models of acute carrageenan inflammation in the rat. Agents Actions 18:439-446.

Sedgwick AD, Sin YM, Edwards JCW, Willoughby DA (1983) Increased inflammatory reactivity in newly formed lining tissue. J Pathol 141:483-495.

Sedgwick AD, Sin YM, Mackay AR, AI-Duaij A, Willoughby DA (1984) Studies into the model of action of non-steroidal anti-inflammatory drugs using a model of facsimile synovium. J Pharm Pharmacol 36:171-174.

Selye H (1953) Use of "'granuloma pouch" technic in the study of antiph- logistic corticoids. Proc Soc Exp Biol Med 82:328-333.

Stevens AJ, Martin SW, Houston JB (1993) Determination of a drug targeting index for two non-steroidal anti-inflammatory drugs when delivered regionally to the rat air pouch model of inflammation. Eur J Drug Metab Pharmacokinetics 18:235.

Thomson AW, Fowler EF, Pugh-Humphreys RGP (1979) Immunopharma- cology of the macrophage-toxic agent carrageenan, lnt J lmmunophar- macol 1:247-261.


Tsurufuji S, Sato H, Min KR, Ohuchi K (1978) Difference in the anti- inflammatory effect of indomethacin between acute and chronic stages of

carrageenin-induced inflammation. J Pharm Dyn 1:8-14.

Tsurufuji S, Yoshino S, Ohuchi K (1982) Induction of an allergic air-pouch inflammation in rats. lnt Arch Allergy Appl Immuno169:189-198.

Vinegar R, Truax JF, Selph JL, Voelker FA (1982) Pathway of onset, development and decay of carrageenan pleurisy in the rat. Fed Proc 41:2588-2595.

Whelan CJ (1974) Production of an inflammatory exudate containing prostaglandins. J Pharm Pharmaco126:355-356.

Willis AL (1970) Identification of prostaglandin E 2 in rat inflammatory exudate. Pharmacol Res Corn 2:297-304.

Yoshino S, Cromartie WJ, Schwab JH (1985) Inflammation induced by bacterial cell wall fragments in the rat air pouch: Comparison of rat strains and measurement of arachidonic acid metabolites. Am J Pathol 121:327-336.

Documents

The Six-day-old Rat Air Pouch Model of Inflammation Characterization of the Inflammatory Response to Carrageenan