Wound trauma mediated inflammatory signaling attenuates a tissue regenerative response in MRL/MpJ mice

Mice were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg), xylazine (15 mg/kg), and acepromazine (2.5 mg/kg). After shaving the dorsum, the exposed skin was washed gently with room temperature sterile water and prepped with Betadine (a 10% povidone-iodine solution for skin disinfection). The Betadine solution from the prep area was wiped off using 3 series of sponge gauzes containing 70% isopropyl alcohol. A single full thickness circular burn (15 mm diameter; ~15% total body) was introduced with electrocautery Bovie (370-400°C for 1.5 sec; Bovie Aaron Medical, St. Petersburg, FL) on the mid-dorsum of each mouse. This protocol produces a histologically proven well demarcated, full thickness, anesthetic injury that is non-lethal (< 0.5% mortality). Wounds were treated with bacitracin (Pharmaderm, Melville, NY) (applied topically) immediately after wounding, left uncovered, and allowed to desiccate. Once mice recovered from anesthesia, they were placed alone in separate cages and maintained under standard conditions in the animal facility (as described above). Buprenorphine (Reckitt Benckiser Pharmaceuticals, Richmond, VA) 0.1 mg/kg SC BID was given on post-operative days 1 through 3 for pain management. No additional therapeutic intervention was provided, as administration of anti-inflammatory or analgesic drugs may introduce complications into the assessment of inflammatory responses. Wounds became covered with inflammatory eschar, and no infection was evident macroscopically. At various time points post injury, cohorts of mice were killed by carbon dioxide inhalation and subsequent cervical dislocation. At the time of killing, cardiac blood was collected and serum was obtained and aliquoted and stored at -80°C until use. Lungs and wound edge/margin tissue from the ear and skin were collected and stored in RNALater (Ambion, Austin, TX). All samples were kept at 4°C until use.

A 2-mm through-and-through circular hole was made in the center of the cartilaginous part of each ear using a standard surgical biopsy punch (Acuderm, Inc, Ft. Lauderdale, FL) immediately following thermal injury. Ear-hole diameter size was determined using a 7X Bausch & Lomb ocular magnifier (Fisher Scientific) immediately following the wounding procedure (day 0) and at frequent intervals over a 31-day evaluation period. Mice with ear-holes that were not cleanly cut or altered in configuration by the mice through scratching within a few days of injury were excluded from the study. Typically, in non-injured mice, the circular wounds healed in a circular fashion whereby the mean hole diameter was calculated by taking the average of the longest and the corresponding perpendicular measurement. Wound size was calculated based on the formula A = (d/2)² (π), where A is the area of the wound in square millimeters and d is the mean ear-hole diameter.

Quantification of murine IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IP-10, Eotaxin, IFN-γ, GM-CSF, MCP-1, MIP-1α, RANTES, TNFα and MIP-2α in mouse serum was determined in duplicative measurements using Luminex-100 technology (Austin, TX) with Fluorokine MAP Multiplex Mouse Cytokine Panels (R&D Systems, Minneapolis, MN) and Mouse Proinflammatory (7-plex) Arrays (Mesoscale Discovery, Gaithersburg, MD) in accordance to manufacturer's instructions. Sample concentrations were interpolated from standard curves, and results were expressed in pg/ml.

Total RNA was extracted from skin, lung and ear tissue and stored in RNALater (Ambion, Austin, TX). Briefly, tissue was homogenized using Trizol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated using Qiagen RNeasy Lipid Tissue Mini Kit (QIAGEN Inc. Valencia, CA) according to manufacturer's instructions. Sample quantity, and quality was assessed by determining the A_260/280, A_260/230 ratio on a Nanodrop 2000 Spectrophotometer (NanoDrop Technologies Inc. Wilmington, DE) and by measuring 28S/18S ribosomal RNA ratios and RNA Integrity Number (RIN) using an Agilent 2100 BioAnalyzer (Agilent Technologies Inc., Santa Clara, CA). Agilent RNA integrity values for all sampled wound specimens in this study were ≥ 8.5. Reverse transcription was performed with Roche 1^st Strand Synthesis kit (Roche Diagnostics Corporation, Indianapolis, IN). Briefly, 1.0 μg sample RNA was added to a master mix containing 1x reaction buffer, 5 mM MgCl₂, 1 mM deoxynucleotide mix, 6.4 μg random primers, 100 units RNase inhibitor, and 40 units AMV reverse transcriptase. 10 mM Tris buffer, pH 7.5 was used to reach 40 μl final reaction volume. Then, final reaction mixture was subjected to a single reverse-transcription cycle of 25°C for 10 min, 42°C for 60 min, 99°C for 5 min, and 4°C for at least 10 min.

Quantitative real-time polymerase chain reaction (RT-PCR) was performed using the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA). Custom designed 'Wound Repair' TaqMan^® Low Density Array (TLDA) cards (Applied Biosystems, Foster City, CA) and catalogued "Primer Assays" (SABiosciences, Rockville, MD) were used to assess gene expression of a small cohort of pro-inflammatory and/or anti-inflammatory cytokines: (IL-1α, IL-1β, IL-6, TNFα, MCP-1, MIP-2α, IL-10, PGE-2, MIP-1α, MIP-1β, PF-4, ENA-78, IP-10, I-TAC, and iNOS). Amplification parameters were as follows: one cycle of 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 30 sec and 60°C for 1 min.

RT-PCR data were analyzed using the Sequence Detection System version 2.3 included with the ABI Prism 7900 HT RT-PCR system or using Microsoft Excel. The threshold cycle (C_t) for each sample was manually set to 0.2 and the baseline was set between 3 and 15 cycles. 18 S ribosomal RNA was used as an endogenous housekeeping control for normalization and the comparative C_t method was used to calculate the relative fold expression by . Assays with C_t values greater than 35 cycles were excluded from analysis [27, 28].

Full-thickness ear-hole biopsies were made in the pinnae of both ears within 1 hr after burn wounds (~177 mm²) were created on the mid-dorsum of each mouse. As shown in Figure 1A, ear-hole closure was significantly impaired in thermally injured mice. The average wound closure response was delayed and reduced to 65% of that of sham-treated mice. By 1-4 days post wounding, the wound edge and adjacent tissue surrounding the ear-holes became transiently inflamed (observed macroscopic reddening) in the majority of the mice (Figure 1B). Moreover, in 50-60% of the thermally injured mice the inflammatory response gradually became more progressive resulting in an augmented inflammatory cell infiltration (data not shown), which lead to extensive necrosis and fibrosis development (Figure 1B-C). Within 10-21 days wounding a large percentage (38%) had to be euthanized early during the study due to extensive necrosis, inflammation and ear deformation. The wound closure measurements for these mice were not included in the results depicted in Figure 1A. In contrast, no such ear lesions were observed in mice that were sham-injured or burned only (no ear wounds).

mRNA transcript expression levels were determined by RT-PCR and normalized to gene transcript levels in normal uninjured naïve skin (Figure 2). The expression of mRNA transcripts for IL-1α, IL-1β, TNFα, PGE₂, MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), MIP-2α (CXCL2), PF4 (CXCL4), and ENA-78 (CXCL5) was significantly increased at days 1 and 3 post thermal injury. By contrast, IL-6, IP-10 (CXCL10) and I-TAC (CXCL11) expression peaked at day 1 post injury and then gradually declined over the subsequent 6 days. Compared with burn wounds on Balb/c mice (data not shown), MRL/MpJ burn wounds had increased and prolonged transcription of IL-6, IL-1β and decreased transcription of IL-10 and chemokines MIP-2α, ENA-78, IP-10 and I-TAC in burn wound margin tissue [26].

At 2, 6 and 24 hours post burn injury, the levels of various inflammatory mediators were quantified in the serum. Four cytokines/chemokines (IL-6, TNFα, KC (CXCL1), and MIP-2α (CXCL2)) were significantly elevated within the first 24 hr post wounding compared with unburned (sham-treated) mice (Figure 3). A similar pattern was observed in dually injured (ear punch and burn) mice. IL-1β, and IL-10 were not detectable in thermally injured mice at any of the studied time points, (data not shown) and measurement of MIP-2α levels was not performed in the dually-injured group.

Significantly upregulated expression of mRNA transcripts for proinflammatory cytokine/chemokines IL-6, IL-1β, TNFα, MIF-1α, MIP-2α and MCP-1 as well as the anti-inflammatory cytokine IL-10 and inducible nitric oxide synthase (i NOS) were measured in the wounded lung and ear at 6 and 24 hrs post thermal injury (Figures 4 and 5). Marked inflammation, as evidenced by the gross and histological appearance of the wound (Figures 1B-C) and increased global expression of potent inflammatory mediators (Figure 5), was seen in the early ear wound healing in the thermally injured mouse.