4.4.6. Zymosan Induced Arthritis of the Knee

All experimental procedures were approved by the Ethical Review Process Committee and the UK Home Office, in accordance with the 1986 Animals (Scientific Procedures) Act (permission # 30/3441). Male C57BL/6J mice aged 8–10 weeks were used. Mice were housed in ventilated cages, maintained at 21 °C ± 2 °C and a 12-h light/12-h dark cycle, with food and water available ad libitum. Mice were anesthetized with 2% isoflurane and both knees were shaved. ZIA was induced by intra-articular injection of 180 µg of Zymosan A (Sigma) suspended in PBS as previously described [9]. Left knee joints received a vehicle control injection. For IVIS imaging, mice received an intravenous injection of 4 nmol Neutrophil Elastase 680 FAST imaging probe (Perkin Elmer, Waltham, MA, USA) and were imaged 4 h post intravenous injection using the IVIS Spectrum (Perkin Elmer) [54]. Images were analyzed using Living Image 4.7 software (Perkin Elmer) to obtain the average fluorescence intensities of a circular region of interest encompassing the knee joint. Mice were humanely culled 8 h post zymosan administration and knee joints were snap-frozen for gene expression analysis. RNA was extracted using the TRIzol method, as previously described [54]. Reverse transcription of 1 ug of total RNA was conducted using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) with random primers and following the manufacturer’s protocol. Real-time PCR was carried out using the power SYBR Green Master Mix in a real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Data are expressed as relative units calculated by 2−ΔΔCt by normalization relative to RPL32 and to fold change over vehicle-treated control samples.