Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO).
Male specific pathogen-free Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing approximately 250–300 g were used in all experiments. All experiments were performed in accordance with National Institutes of Health guidelines for care and use of animals. Approval for the experimental protocol was obtained from the University of Michigan Animal Care and Use Committee.
The procedure was performed according to a previously established method to produce partial thickness burn injury.12,13
Briefly, animals were anesthetized with a 40 mg/kg intraperitoneal (ip) injection of sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL). Dorsal hair was closely clipped and removed using Nair depilatory cream (Church & Dwight Inc., Princeton, NJ). Each rat was placed in an insulated, custom-made mold, which exposes the dorsal region over 20% of the total body surface area. Partial thickness scald burn injury was achieved by placing the exposed skin of the rat in a 60°C water bath for 25 seconds. Sham burn animals received the same treatment except they were immersed in room temperature water (21–24°C). The burn wound was scrub-débrided with dry sterile gauze and rinsed with 0.9% sterile NaCl. Each animal was resuscitated with 4 mL Ringer’s lactate / % total body surface area burn / kg body weight. One half of this fluid volume was given intraperitoneally and half subcutaneously immediately following the burn injury. After drying, an occlusive dressing of sterile Telfa (Kendall Co., Tyco Healthcare Group LP, Mansfield, MA) and Tegaderm HP (3M Health Care, St Paul, MN) was applied to prevent wound contamination. During the experiment each rat was singly housed and received 0.01 mg/kg buprenorphine subcutaneously at the time of burn and at 16 hours for postburn pain control.
Local wound treatment
Stock nanoemulsion compound NB-201 was obtained from NanoBio Corporation (Ann Arbor, MI). This nanoemulsion was manufactured by emulsification of super-refined soybean oil and water with surfactants and alcohol. The resultant droplets had a mean particle diameter of 350 nm. The experimental solution was made by diluting 1 mL of the 60% stock formulation with 4.88 mL sterile saline and adding 120 µL of 1 M ethylenediaminetetraacetic acid (EDTA) giving a final concentration of 10% NB-201 and 20 mM EDTA. A placebo nanoemulsion compound was manufactured in the same manner as the NB-201, but one of the active ingredients was deleted from the formulation (benzalkonium chloride). 5% Sulfamylon (UDL Laboratories, Inc., Rockford, IL) solution was formulated by mixing 50 g of mafenide acetate powder in 1 L of 0.9% sterile saline. The control reagent used was 0.9% sterile saline. Experimental groups consisted of sham, burn, burn + NB-201, burn + bacteria + saline, burn + bacteria + placebo, burn + bacteria + NB-201, and burn + bacteria + Sulfamylon. Sixteen hours following burn injury animals were anesthetized with inhaled isoflurane. The occlusive dressing and Telfa was removed. Nanoemulsion (NB-201), placebo, Sulfamylon or sterile saline was applied in a uniform fashion to the burn wound surface using a spray bottle. Animals in the sham or burn group received no topical treatment, but did undergo dressing change under anesthesia. The burn wound was then redressed with Telfa and a Tegaderm occlusive dressing. This treatment and dressing change was repeated at 24 hours following burn injury.
Bacterial culture and inoculation
Pseudomonas aeruginosa isolated from a human burn patient was provided by the Department of Pathology at the University of Michigan. This bacterial isolate is sensitive to the topical agent Silvadene and Sulfamylon. A bacterial inoculum was prepared by thawing an aliquot (0.5 mL, stored in 50% skim milk at −80°C) in 40 mL of Trypticase soy broth (Becton Dickinson, Franklin Lakes, NJ) and grown overnight at 37°C with constant shaking at 275 rpm. A sample of the resulting stationary-phase culture was transferred to 35 mL of fresh Trypticase soy broth and incubated for 2.5 hours to reach the log-phase. This subculture was transferred to a 50 mL conical polystyrene tube and centrifuged for 10 minutes at 4°C and 880 g. The bacterial pellet was washed with 0.9% sterile saline, and resuspended in 10 mL of ice-cold saline. The optical density of the suspension was measured at 620 nm and bacterial concentration (colony forming units [CFU] /mL) calculated using the formula OD620×2.5×108. The bacterial suspension was diluted with 0.9% sterile saline to a final concentration of 1×106 CFU per 100 µL. Eight hours following burn injury animals were anesthetized with inhaled isoflurane. The rats then underwent topical application of 1×106 CFUs of log-phase Pseudomonas aeruginosa in 100 µL of sterile saline pipetted onto a piece of Telfa in a uniform fashion followed by coverage with a Tegaderm occlusive dressing.
Thirty-two hours after thermal injury the animals were sacrificed and skin tissue samples were harvested using sterile technique. Skin samples were used immediately or frozen in liquid nitrogen.
Quantitation of bacterial wound infection
A 100 mg piece of excised skin tissue was mechanically homogenized in 1 mL of 0.9 NaCl. This homogenate was then further diluted with 9 mL of sterile saline. Serial dilutions were performed and skin homogenates plated in triplicate on blood agar plates (Becton Dickinson, Franklin Lakes, NJ). Culture plates were incubated for 24 hours at 37°C and CFUs counted.
Dermal cytokine analysis (ELISA)
A 100 mg sample of dorsal skin was homogenized in 1 mL of ice-cold lysis buffer consisting of 50 mL of PBS and protease inhibitor (Complete X, Roche, Indianapolis, IN) and 50 µL of Triton X (Roche). Homogenates were centrifuged at 3000g for 5 minutes and the supernatants collected and stored frozen at −80°C until use. Rat IL1-β, IL-6, TNF-α, CINC-1, CINC-3, IL-10 and TGF-β were measured by sandwich enzyme-linked immunosorbent assay (ELISA) using antibodies and reagents from R&D Systems, Inc. (Minneapolis, MN). Results were adjusted for previous dilution and expressed as pg/mL.
Detection of neutrophil sequestration (Myeloperoxidase assay)
100 mg of skin tissue was mechanically homogenized in 1 mL ice cold potassium phosphate buffer consisting of 115 mM monobasic potassium phosphate (Sigma Aldrich, Milwaukee, WI). Homogenates were centrifuged at 3000g for 10 min at 4°C, the supernatants were removed and the pellets were re-suspended in 1 mL C-TAB buffer consisting of dibasic potassium phosphate, cetyltrimethylammonium bromide, and acetic acid (Sigma Aldrich, Milwaukee, WI). The suspensions were sonicated (Branson Sonifier 250, Danbury, CT) on ice for 40 seconds. Homogenates were centrifuged at 3000g for 10 min at 4°C and the supernatant collected. Supernatants were incubated in 60°C water bath for 2 hours (Shaker Bath, 2568; Forma Scientific, Marietta, OH). Samples were stored at −80°C until needed or assayed immediately.
20 µL standards (Calbiochem, Gibbstown, NJ) or samples were added to a 96- well immunosorbent micro-plates (NUNC, Rochester, NY), followed by the addition of 155 µL of 20mM TMB/DMF consisting of 3,3`,5,5`-tetramethylbenzidine/N,N-dimethylformamide in 115 mM potassium phosphate buffer (Fischer Scientific, Pittsburgh, PA) to each well. The samples were mixed well, after which 20 µL of 3 mM H2O2 was rapidly added to each well. The reaction was stopped immediately by adding 50 µL/well of 0.061 mg/mL Catalase (Roche, Indianapolis, IN). The plates were read using a microplate reader at 620 nm. Myeloperoxidase (MPO) concentrations were calculated using a linear standard curve and adjusted for previous dilution. The final concentrations were expressed as µg/mL.
Determination of dermal capillary leak and tissue edema (Evans blue)
Burn wounds are associated with significant levels of capillary leak. This can lead to depletion of the intravascular volume and a need for large amounts of intravenous crystalloid fluid administration. To assess whether our therapy reduced capillary leak in conjunction with reducing inflammation we utilized the Evans blue assay, which is a measure of vascular permeability.13
Animals were anesthetized 90 minutes before tissue harvest. 50 mg/kg body weight of 10% Evans blue (Merck KgaA, Darmstadt, Germany) was injected ip into the burned animal at time 30.5 hours following thermal injury. At the tissue harvest time point animals were exsanguinated by incision of the inferior vena cava. Systemic Evans blue was flushed out with a total of four times the blood volume (7.46 mL/100 g body weight) of 0.9 NaCl with 100 units/mL heparin. Dorsal skin samples were harvested and a 100 mg sample was placed in 4 mL 99.5% formamide in polyethylene tubes. Tubes were placed on a shaker at room temperature for 48 hours for Evans blue extraction. Supernatants were collected and the absorbance read on a microplate reader at 620 nm. Concentrations were calculated from an Evans blue in formamide standard curve. Results are expressed as micrograms of Evans blue per mg of skin tissue.
Skin samples were fixed in 10% buffered formalin and embedded in paraffin. Eight µm thick sections were affixed to slides, deparaffinized, and stained with hematoxylin and eosin to assess morphologic changes.
Detection of hair follicle cell apoptosis (TUNEL assay)
Animals were anesthetized and underwent creation of a 20% partial thickness scald burn wound or sham injury. Treatment groups consisted of sham, burn + saline, burn + placebo, and burn + NB-201. Treatment and dressing changes were performed at 0 and 8 hours post-burn. No bacterial infection was created in this experiment. Full-thickness skin samples were taken from three locations across the entire burn wound at 12, and 24 hours post thermal injury for determination of hair follicle cell apoptosis. There were four animals per treatment group per time sample.
As described previously, apoptosis was detected in situ with fluorescein based labeling of DNA strand breaks using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (ApopTag, CHEMICON International, Inc, Temecula, CA).13
The three fresh skin samples for each animal were placed in disposable vinyl cryomolds filled with optimal cutting temperature compound (Sakura Finetek, U.S.A., Inc., Torrance, CA), and frozen at −80°C until ready for use. Frozen embedded skin specimens were cut into 4-µm-thick serial sections in a cryostat and collected on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PN). Sections were fixed and stained according to the manufacturer’s instructions.
The TUNEL assay slides were blinded to groups, and under the microscope appropriate hair follicle cells in a randomly chosen high-power field were identified. Appropriate hair follicles for analysis were those sectioned in the mid-sagittal or midcoronal plane. A total of 3–6 hair follicles were selected from among the three skin samples present on a slide. Fluorescent-labeled TUNEL slides were captured digitally at identical time post-labeling to control fading of fluorescence using an Olympus BX-51 fluorescence microscope at fixed image capture settings and 40× magnification. Each hair follicle was selected and first digitally captured by visualizing counterstained nuclei present using the DAPI excitation/emission channel. Then for each hair follicle analyzed, the excitation/filter channel was changed to visualize the fluorescein-labeled TUNEL-positive cells, and images again digitally captured. Within the captured images a region of interest (ROI) was digitally defined, set to include only hair follicle cells and exclude bright fluorescing hair shafts and surrounding cells (NIH Image J software, NIH, Bethesda, Md). Fluorescence of TUNEL-positive cells was quantified, normalized to ROI size, and expressed as pixels/area fraction, controlling for differences in ROI size.
All statistical analysis and graphs were performed using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA). Results are presented as mean values ± the SEM unless otherwise noted. Continuous variables were analyzed using an unpaired two-tailed Student’s t-test and/or One-way ANOVA followed by Tukeys posttest comparisons. The Kruskal-Wallis test with Dunn’s multiple comparisons was used to evaluate differences in medians for data with a non-parametric distribution. Discrete variables were compared using Fisher’s exact test. Statistical significance was defined as a p-value < 0.05.