Agents Actions 36 (1992)
0065-4299/92/020093-06 $1.50+0.20/0 9 1992 Birkh/iuser Verlag, Basel
Changes in corticosterone levels under different degrees of acute inflammation in mice S. L. Chio and Y. M. Sin 1 Department of Zoology,National Universityof Singapore, Singapore0511
Abstract
Carrageenan of different concentrations was injected into the 6-day-old air pouch in mice. It was found that 12 mg carrageenan caused a significant increase of plasma and exudate corticosterone levels at 24 h, while 1 and 3 mg carrageenan could only induce a significant increase of exudate corticosterone at 4 h. Elevation of corticosterone in both plasma and inflammatory exudate appeared to be correlated, suggesting that the exudate corticosterone was derived from the blood circulation. Injection of exogenous histamine and PGE 2 into the air pouch induced a significant increase in exudate levels of corticosterone. However, plasma corticosterone increased significantly only after histamine administration, although a slight increase was observed in those injected with PGE 2. These findings thus suggest that endogenous histamine and PGE z which are released during carrageenan-induced acute inflammation, as shown in our previous work, might be responsible for the increase of corticosterone in both plasma and inflammatory exudate.
Introduction
Corticosterone has long been known as an antiinflammatory steroid. Pretreatment with adrenal glucocorticoids prevents the rapid appearance of the polymorphonuclear leucocytes in the inflamed tissues of the mouse [-1, 2], rat [3], guinea pig [-4] and man [5]. Similarly, corticosterone is also able to inhibit interleukin-1 production [6], arachidonic acid production from injured cells [7] and the release of histamine from mast cells and basophils [8, 9]. However, little work has been done to clarify the cause of corticosterone release during inflammation. In our previous studies [10], we have shown that corticosterone levels in the inflammatory exudate and plasma increased significantly
after injecting carrageenan into the 6-day-old air pouch. It was found that the increased exudate corticosterone was closely related to the exudate cell accumulation. Thus, it would be interesting at this point to find out, firstly, if corticosterone levels in the pouch exudate and plasma are dependent on the amount of carrageenan used and, secondly, whether pharmacological mediators, namely, histamine and PGE2, which are released during tissue injury will play any part in modulating the corticosterone levels in the plasma and inflammatory exudate. Materials and methods Animals
1Author for correspondence.
Female Swiss albino mice weighing 20-25 g were used. They were supplied with mouse pellets and
Agents Actions36 (1992)
94 tap water was given ad libitum. For all experiments, 6 animals were used for each group.
Experimental procedures Simple air pouches were created according to the method previously described [11]. Mice were anaesthetized and their backs were swabbed with 70% ethanol. Five millilitres of air were then injected subcutaneously along the midline of the back. Three days later, 2.5 ml of air was re-injected into the pouch to ensure that the cavity remained open. Six days after initial injection, various concentrations (1, 3, 12 mg/ml) of sterile carrageenan (kindly provided by Marine Colloid, Springfield, USA) were injected into the test animals as previously discussed [12]. In control mice, the pouch was injected with 1 ml of sterile physiological saline. Animals were killed at various time intervals after injection of carrageenan or saline. The total number of exudate leucocytes, exudate volume and corticosterone levels in the plasma and exudate were determined. Acute inflammation was also induced by injecting free base histamine (Sigma) and PGE 2 (ICN Biochemicals). Different concentrations of exogenous histamine (3, 30, 300, 3000 ~tg/ml) or PGE 2 (0.1, 1, 5/ag/ml) were prepared in physiological saline and the resultant solutions were sterilized by passing through an autoclaved Millipore filter chamber. One millilitre of each prepared solution was injected into the air pouch. On the other hand, control animals were injected with 1 ml of sterile physiological saline. In our preliminary experiments, it was found that exogenous histamine and PGE2 caused significant changes in the plasma and exudate corticosterone at 1 and 0.5 h, respectively. Thus, animals injected with histamine and PGEz were all killed at 1 and 0.5 h, respectively, after initial injection. Corticosterone levels of both plasma and exudate were analysed in all the animals.
Exudate leucocyte count Mice of both control and test animals were anaesthetized with ether and bled to death through the jugular vein. The blood was collected in EDTA. One millilitre of cold physiological saline was injected into the pouch. The exudate was collected and cooled in an ice bath. The exudate volume was recorded and then the total leucocytes in the ex-
udate fluid were determined in a Coulter counter. Mice with untreated air pouches were also killed immediately at time zero for leucocyte and corticosterone analysis. This serves as a background control.
Determination of corticosterone Blood and pouch exudate were obtained for the analysis of corticosterone and the procedure is similar to that described in our previous work [10].
Statistical analysis Results were expressed as means_+ SE. The significance of the results was determined by Student's t-test and 1-way analysis of variance followed by Student-Newlman-Keuls test. A value of P < 0.05 was considered to be significant. Results
The effect of different concentrations of carrageenan at inducing leucocyte infiltration and fluid exudation into the 6-day-old air pouch was examined. Figure 1 showed the number of leucocytes present in the inflammatory exudate after injecting saline or different concentrations of carrageenan into the air pouch. It was found that the number of leucocytes accumulated in the pouch treated with 12 mg of carrageenan was significantly higher (P
0065-4299/92/020093-06 $1.50+0.20/0 9 1992 Birkh/iuser Verlag, Basel
Changes in corticosterone levels under different degrees of acute inflammation in mice S. L. Chio and Y. M. Sin 1 Department of Zoology,National Universityof Singapore, Singapore0511
Abstract
Carrageenan of different concentrations was injected into the 6-day-old air pouch in mice. It was found that 12 mg carrageenan caused a significant increase of plasma and exudate corticosterone levels at 24 h, while 1 and 3 mg carrageenan could only induce a significant increase of exudate corticosterone at 4 h. Elevation of corticosterone in both plasma and inflammatory exudate appeared to be correlated, suggesting that the exudate corticosterone was derived from the blood circulation. Injection of exogenous histamine and PGE 2 into the air pouch induced a significant increase in exudate levels of corticosterone. However, plasma corticosterone increased significantly only after histamine administration, although a slight increase was observed in those injected with PGE 2. These findings thus suggest that endogenous histamine and PGE z which are released during carrageenan-induced acute inflammation, as shown in our previous work, might be responsible for the increase of corticosterone in both plasma and inflammatory exudate.
Introduction
Corticosterone has long been known as an antiinflammatory steroid. Pretreatment with adrenal glucocorticoids prevents the rapid appearance of the polymorphonuclear leucocytes in the inflamed tissues of the mouse [-1, 2], rat [3], guinea pig [-4] and man [5]. Similarly, corticosterone is also able to inhibit interleukin-1 production [6], arachidonic acid production from injured cells [7] and the release of histamine from mast cells and basophils [8, 9]. However, little work has been done to clarify the cause of corticosterone release during inflammation. In our previous studies [10], we have shown that corticosterone levels in the inflammatory exudate and plasma increased significantly
after injecting carrageenan into the 6-day-old air pouch. It was found that the increased exudate corticosterone was closely related to the exudate cell accumulation. Thus, it would be interesting at this point to find out, firstly, if corticosterone levels in the pouch exudate and plasma are dependent on the amount of carrageenan used and, secondly, whether pharmacological mediators, namely, histamine and PGE2, which are released during tissue injury will play any part in modulating the corticosterone levels in the plasma and inflammatory exudate. Materials and methods Animals
1Author for correspondence.
Female Swiss albino mice weighing 20-25 g were used. They were supplied with mouse pellets and
Agents Actions36 (1992)
94 tap water was given ad libitum. For all experiments, 6 animals were used for each group.
Experimental procedures Simple air pouches were created according to the method previously described [11]. Mice were anaesthetized and their backs were swabbed with 70% ethanol. Five millilitres of air were then injected subcutaneously along the midline of the back. Three days later, 2.5 ml of air was re-injected into the pouch to ensure that the cavity remained open. Six days after initial injection, various concentrations (1, 3, 12 mg/ml) of sterile carrageenan (kindly provided by Marine Colloid, Springfield, USA) were injected into the test animals as previously discussed [12]. In control mice, the pouch was injected with 1 ml of sterile physiological saline. Animals were killed at various time intervals after injection of carrageenan or saline. The total number of exudate leucocytes, exudate volume and corticosterone levels in the plasma and exudate were determined. Acute inflammation was also induced by injecting free base histamine (Sigma) and PGE 2 (ICN Biochemicals). Different concentrations of exogenous histamine (3, 30, 300, 3000 ~tg/ml) or PGE 2 (0.1, 1, 5/ag/ml) were prepared in physiological saline and the resultant solutions were sterilized by passing through an autoclaved Millipore filter chamber. One millilitre of each prepared solution was injected into the air pouch. On the other hand, control animals were injected with 1 ml of sterile physiological saline. In our preliminary experiments, it was found that exogenous histamine and PGE2 caused significant changes in the plasma and exudate corticosterone at 1 and 0.5 h, respectively. Thus, animals injected with histamine and PGEz were all killed at 1 and 0.5 h, respectively, after initial injection. Corticosterone levels of both plasma and exudate were analysed in all the animals.
Exudate leucocyte count Mice of both control and test animals were anaesthetized with ether and bled to death through the jugular vein. The blood was collected in EDTA. One millilitre of cold physiological saline was injected into the pouch. The exudate was collected and cooled in an ice bath. The exudate volume was recorded and then the total leucocytes in the ex-
udate fluid were determined in a Coulter counter. Mice with untreated air pouches were also killed immediately at time zero for leucocyte and corticosterone analysis. This serves as a background control.
Determination of corticosterone Blood and pouch exudate were obtained for the analysis of corticosterone and the procedure is similar to that described in our previous work [10].
Statistical analysis Results were expressed as means_+ SE. The significance of the results was determined by Student's t-test and 1-way analysis of variance followed by Student-Newlman-Keuls test. A value of P < 0.05 was considered to be significant. Results
The effect of different concentrations of carrageenan at inducing leucocyte infiltration and fluid exudation into the 6-day-old air pouch was examined. Figure 1 showed the number of leucocytes present in the inflammatory exudate after injecting saline or different concentrations of carrageenan into the air pouch. It was found that the number of leucocytes accumulated in the pouch treated with 12 mg of carrageenan was significantly higher (P
