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Alternatives to Laboratory Animals - ATLA

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Ischaemia–Reperfusion Injury in the Isolated Haemoperfused Bovine Uterus: An In Vitro Model of Acute Inflammation

Michael Braun and Manfred Kietzmann

Following on from previous studies on dermal inflammation in the isolated perfused bovine udder, a new in vitro model of the isolated haemoperfused bovine uterus was established for studies on acute inflammatory reactions (for example, eicosanoid synthesis and regulation of cyclooxygenase-1 [COX-1] and COX-2) caused by ischaemia–reperfusion (I–R) injury. The organs and blood used in this study were obtained from a slaughterhouse. Within 2 hours of slaughter, uterine perfusion was re-established, by using a mixture of homologous blood and Tyrode solution (4:1). After equilibration, several deposits of arachidonic acid (5mg and 0.1mg) and arachidonylethanolamide (0.1mg) were injected into the myometrial tissue. Tissue biopsies were taken from treated and untreated areas at 180 and 300 minutes after the onset of haemoperfusion, for measuring prostaglandin E2 (PGE2) levels. In addition, the regulation of COX-1 and COX-2 mRNA was investigated by using the reverse transcriptase-polymerase chain reaction. Eicosanoid levels were determined by using an enzyme immunoassay (ELISA). Because both an increase in PGE2 concentration and up-regulation of COX mRNA were observed, the inhibitory effects of dexamethasone, added to the perfusion medium, were studied. Dexamethasone caused a significant decrease in tissue PGE2 production, but did not induce down-regulation of COX-2 mRNA. In conclusion, the isolated haemoperfused bovine uterus was introduced as an in vitro model of acute inflammation, induced by I–R injury. The suitability of the model for investigating anti-inflammatory substances was demonstrated. Use of the isolated haemoperfused bovine uterus in pharmacological research and drug screening may contribute to reducing the number of animals used for testing.