A multi drug resistant strain of Pseudomonas aeruginosa isolated from human burn wound was used for this study. Over night culture was diluted into fresh TSB and incubated for 2.5 h at 37°C. The subculture was centrifuged (10 min, 4°C, 880 g). The bacterial pellet was washed once and resuspended in a cold sodium phosphate buffer (J.T.Baker, Deventer, Holland, pH 7.4). CFU were calculated by OD600 nm measurement (UV-VIS-Spektrometer, Perkin Elmer 555).
Adult male Sprague-Dawley rats (250–300 g) were obtained from the Unit for Laboratory Animal Medicine (University of Michigan, Ann Arbor, MI) and maintained under standard laboratory conditions. In addition to a resting period at the institutional vivarium, animals were acclimated to the laboratory environment for at least 48 hours before treatment. After treatment, the rats were placed in individual cages in a temperature controlled room with food and water provided ad libitum and a 12 hour light and a 12 hour dark diurnal cycle. All experiments were performed in accordance with National Institute of Health guidelines and approval was obtained from the University of Michigan Animal Care and Use Committee.
Sprague-Dawley rats (n = 21) were randomized into three groups: 1) burn only; 2) burn and infection (108 CFU of multi-drug resistant Pseudomonas aeruginosa) or 3) sham burn. Animals in the first two groups received a 30% (TBSA) partial thickness burn. The animals were anesthetized prior to each intervention with intraperitoneal Ketamine hydrochloride (100 mg/kg; Fort Dodge Laboratories, Fort Dodge, Iowa) and Xylazine (13 mg/kg; Bayer Corporation, Shawnee Mission, Kansas) injection. The skin of the whole torso was clipped and treated with depilatory cream (Sally Hansen®Div. Del Laboratories, Inc., Farmingdale. NY). Twenty-four hours later, rats were placed in a mold, which exposed an area of 30% total body surface area (TBSA). Rats, except the sham burn group 3, sustained a superficial partial scald injury at the defined area on both flanks and back at 60°C for twenty seconds. This extend of injury in this animal model is time dependent and has been previously described . Additionally, the burn wound of the infected group was covered with a 2 cm × 2 cm gauze containing 108 CFU (colony forming units) of log-phase Pseudomonas aeruginosa. Injury was covered with Tegaderm™ HP (3 M Health Care, St. Paul, MN) and Flex-Wrap™ Self-Adherent Wrap (The Kendall Company, Mansfield, MA). After treatment, rats were resuscitated subcutaneously with 5 ml saline. For the remainder of the study, all animals received buprenorphine (0.3 mg/kg) intra peritoneal twice daily for pain control post-burn. In previous experiments we have not observed any abdominal injury with this scald burn model which could affect food intake or absorption [13–17]. Weight changes were monitored daily. Blood was collected after 12, 24, 48 and 72 h, then after 6 and 12 days. This was used for cytokine ELISA, LPS quantification, and peripheral blood analysis. After either 6 or 12 days the animals were sacrificed and treated areas of the infected and non infected wound tissues were harvested aseptically, weighed, homogenized, serially diluted and plated in triplicate on trypticase soy agar with 5% sheep blood and Pseudomonas isolation agar (both from Becton Dickinson). Bacterial plates were then incubated for 18 hours and the number of colony forming units were counted in blinded fashion. Results are expressed as CFU per gram infected skin tissue.
Peripheral blood analysis
At appropriate time points, 20 μl of EDTA anti-coagulated blood was collected from the rat tail vein. A Hemavet Mascot Multispecies Hematology System Counter 1500 R (CDC Technologies Inc., Oxford, CT) was used to measure the blood differential, WBC, RBC, hemoglobin and hematocrit. Furthermore, 200 μl of plasma was collected and stored at -20°C for cytokine and endotoxin screening.
For the purpose of determining the amount of endotoxin present in the plasma, a QCL-1000 Limulus Amebocyte Lysate Assay (Bio Whittaker Inc., Walkersville, MD) was performed. This assay uses a modified Limulus Amebocyte Lysate and a coloring substrate to detect gram-negative bacterial endotoxin chromogenically. Following the manufacturer's instruction, 50 μl of plasma was diluted 1:10 in order to quantify the endotoxin levels. Concentrations were photospectrometrically analyzed at 410 nm and calculated corresponding to the standard curve.
With the purpose of quantifying changes in cytokine levels, ELISA's were performed for IL-1β, IL-6, Il-10 and TNF-α using Cytoscreen™ Immunoassay Kits (BioSource International, Inc., Camarillo, CA). Following the manufacturer's instruction, 50 μl of plasma was used in a 1:1 dilution. ELISA plates were read on a plate reader (Bio-Tek Instruments, Windoski, VT) at dual wavelengths of 465 nm and 590 nm. Sample concentrations were determined by comparison to a standard curve.
Data with normal distribution was analyzed using ANOVA and Student's t-test (StatView®, Abacus Concepts/SAS® Institute, Cary, N.C.). Results were considered to be significant at p < 0.05 and expressed as the mean ± SEM.