Sodium chloride solution

Adult (clitellate) earthworms (0.55–0.75 g body weight) of Eienia andrei (Sav.) were collected from the stockbreeding maintained in the Institute of Zoology of the Jagiellonian University, kept in controlled laboratory conditions (21 ± 1 °C; 12:12 LD) in a commercial soil (PPUH Biovita, Poland). On the experimental day, each earthworm was washed with water, and then injected with 20 µl of stimulant solution into the coelomic cavity (1 cm behind the clitellum). The stimulants were prepared in sodium chloride solution 0.9 % (Baxter Terpol, Poland) and the following compounds were used: phorbol 12-myristate 13-acetate-PMA (PMA, 0.1 µg/ml), lipopolysaccharide from E. coli 0111:B4 (LPS, 1 mg/ml), zymosan A from Saccharomyces cerevisiae (Z, 1 mg/ml) or Micrococcus luteus (Ml, 1 mg/ml). All stimulants were purchased from Sigma (St. Louis, Mo., USA). LPS was used, rather than live E. coli, as it is the strongest compound/stimulating agent of all Gram⁻ bacteria. After injection, earthworms were placed individually in 15 ml vials filled with filter paper that was soaked with water (Homa et al. 2013 (link), modified). Subsequently, coelomocytes were collected 24 or 72 h later for analyses. Control (CTR) animals were injected by sodium chloride solution (0.9 % NaCl). In addition, some control animals were kept in a commercial soil.

LPS derived from Pseudomonas aeruginosa (Sigma) was solubilized in 0.9% sodium chloride solution (Baxter) to a final concentration of 1 mg/ml. A total of 7 doses (1 mg/ml, 15 ml each) were administered to ferrets via a nebulizer, each dose delivered for 1 h daily for 7 days (Figure 1b). LPS was delivered to conscious ferrets using Allied Schuco® S5000 Nebulizer (Allied) in a tightly sealed chamber supplied with O_2, (4 L/min) (Figure 1c). Daily animal monitoring for adverse events included observations of food/water intake, bowel movement, animal activity, and signs of pain.
A total of 15 ml of saline was administered to ferrets via a nebulizer in control groups. Lung function measurements were performed at baseline and day 7 post LPS or saline delivery. BAL samples were collected on day 7 post LPS or saline administration. Baseline PFTs were performed 1 day before the first exposure to LPS.
Ferrets were closely monitored pre‐, intra‐, and post‐nebulization for any signs of erythema/swelling of the sclera and conjunctiva. We also watched for tearing and blepharospasm (blinking/winking). None of these signs were observed in any animal. We used a damp towel to wipe down the pelage of each animal post‐nebulization with the intent to minimize skin exposure. This also mitigated the risk of the animal getting LPS from its coat onto its mucous membranes as well.