Male SV129 mice (Jackson Laboratory, Bar Harbor, Maine, local breeding colony, and Charles River laboratories) were utilized in the study.
Animal procedures were approved by the Johns Hopkins University Animal Care and Use Committee. During the experiments the animals were housed one per cage, maintained under controlled environmental conditions (12 hours light to dark cycle, temperature approximately 23°C), and provided with standard laboratory food and water ad libitum. Mice were anesthetized with ketamine hydrochloride (100 mg/kg) and xylazine (10 mg/kg), and the dorsum was shaved and then treated with a depilatory (Nair cream, Church & Dwight Co., Princeton, NJ). A burn wound protocol previously established in rats (15
) was adapted for use in mice. (14
) A custom-made 220-g aluminum rod was heated in a 100°C water bath for 5 minutes. One burn of 1.2-cm diameter was produced on the dorsum of each animal producing a 10% body surface area burn. Contact time of 4 seconds was measured with a standard metronome. Burn uniformity was assured, with only the weight of the aluminum template providing pressure to the shaved and depilated skin surface. No dressing was used after burn. The animals were resuscitated according to the Parkland formula (4 ml/kg × percent body area) by intraperitoneal injection of saline within one hour after burning. For example, to resuscitate a mouse weighing 25g with a burn covering 10% of body surface area, 2 ml Ringer’s lactate was administered. Skin tissues were collected at day 2 after burn for RNA extract. On days 1, 2, 3, 4, 7, 10, and 14 day after burn, tissues were collected and fixed in 10% formalin for 24 hours for IHC.
Mouse tissue harvest, RNA preparation
Using a template and scalpel, both dermal wound and normal skin tissues were collected at day 2 after burn. Total RNA was extracted from burned and unburned skin tissue of the same mouse with TRIzol (Invitrogen, Frederick, MD) and treated with DNase I (Ambion, Austin, TX) according to the manufacturer’s protocol.
Superarray RT2 Profiler PCR Array for Chemokines
One µg of each RNA sample was used for first-strand synthesis with the RT2 first strand kit (SABiosciences, Frederick, MD) according to the manufacturer’s protocol. Quantitative mRNA expression analysis of 84 chemokines and their receptors was performed with the mouse chemokine and receptor RT2 profiler PCR array PAMM-022 (SABiosciences, Frederick, MD) using the RT2 Real-Time SYBR Green PCR Master Mix (SABiosciences, Frederick, MD) and the iCycler Real-Time PCR Detection System (BioRad, Hercules, CA). The thermocycler parameters were 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The mRNA expression of each gene in burned tissue was normalized using the average expression of two housekeeping genes (HPRT1 and Hsp90ab1) and compared with the data obtained from the control group (unburned tissue) using the 2-DDCT method according to the manufacturer's protocol.
Quantitative reverse transcription PCR (qRT-PCR)
The results of RT2 Profiler PCR Arrays for HIF-1α and SDF-1 were confirmed by quantitative reverse transcription PCR (qRT-PCR) on the individual RNA samples. One µg of total RNA was used for first-strand synthesis with the iScript cDNA Synthesis system (BioRad, Hercules, CA). Real-time PCR was performed using iQ SYBR Green Supermix and the iCycler Real-Time PCR Detection System (BioRad, Hercules, CA). For each primer pair (sequences available upon request), annealing temperature was optimized by gradient PCR. The fold change in expression of each target mRNA relative to hypoxanthine phosphoribosyltransferase 1 (HPRT1) mRNA was calculated based on the threshold cycle (CT) for amplification as 2−(ΔCT), where ΔCT = CT,target − CT,HPRT1. The mRNA expression of each gene in burned tissue was compared with the data obtained from the unburned tissue of same mouse.
In vivo hypoxia detection
Hypoxyprobe™-1 was used to characterize hypoxia within the burn wound. Hypoxyprobe™-1 Omni Kit (Natural Pharmacial Intl. Inc., Burlington, MA) utilizes pimonidazole hydrochloride as a marker for hypoxia. This chemical distributes with the circulation and binds with thiol groups on amino acids at oxygen levels of pO2 < 10 mm Hg. The binding appears to be non-toxic to cells and permanent. The imaging is carried out using IHC with immunoperoxidase labeled antibodies against the pimonidazole-amino acid adducts. Hypoxyprobe™-1 (80 mg/kg body weight) is injected IP 90 minutes before sacrificing the mice for skin collection. The whole skin sample harvest process takes about 1 minute. The tissue is immediately put in formalin. This procedure does not generate significant background staining.
In vivo BrdU-labeling
For labeling S-phase mitotic cells, the thymidine analogue BrdU was used. Mice sustained a single 1.2 cm diameter burn wound on the dorsum. BrdU was injected IP at 120 mg/kg twice at a one-hour interval (240 mg/kg in total) at day 2 after burn, and mice were sacrificed at 1 hour, 24 hours, and 48 hours after BrdU injections. Wound skin tissues were fixed in formalin and processed for immunohistostaining for BrdU (Cat# KT-077, Kamiya Biomedical, Seattle, WA).
Burn wounds were harvested with 4mm of adjacent normal skin. Specimens from each site were bisected at the center and fixed in 10% buffered formalin solution overnight. Five-µm-thick paraffin-embedded sections were stained and analyzed by light microscopy. Heat-induced antigen retrieval was used for all antigens except K17, which does not need antigen retrieval. To prevent nonspecific binding, 100 µl of blocking solution containing 2% normal rabbit serum for Hypoxyprobe-1, HIF-1α, and VEGF or goat serum for SDF-1 was applied for 30 minutes. Then 100 µl of rabbit anti-Hypoxyprobe antibody (1:500, Natural Pharmacial Intl. Inc.), rabbit anti-HIF-1α(1:100, Abcam, Cambridge, MA Cat# ab65979), rabbit anti-VEGF (1:1000, Abcam, Cambridge, MA Cat# ab46154), goat anti-SDF-1 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA. Cat# sc-6193), rabbit polyclonal anti-ki67 (1:200, Abcam, Cambridge, MA Cat# ab15580), rat anti-F4/80 (macrophage marker) (1:100, Abcam, Cambridge, MA Cat#x00023; ab6640), rabbit anti-myeloperoxidase (neutrophil marker) (1:1, Abcam, Cambridge, MA Cat#x00023; ab15484), rabbit anti-pancytokeratin (1:100, Abcam, Cambridge, MA Cat#x00023; ab9377), rabbit polyclonal anti-E cadherin (1:100, Abcam, Cambridge, MA Cat#x00023;ab53033) and rabbit anti-keratin 17 (1:1000 dilution, a gift from P. Coulombe) were applied to the sections for 1 hour at room temperature. The sections were then incubated with biotinylated secondary antibody (1:500 dilution; Vector Laboratories, Burlingame, CA). Streptavidin-biotin-horseradish peroxidase was used for signal amplification and diaminobenzidine was used for staining (Vector Laboratories). Counterstaining was performed with hematoxylin and nuclear fast red for 30 seconds, respectively, and 3% H2O2 (Fisher Scientific, Fair Lawn, NJ) was used for blocking endogenous peroxidase activity.
Analysis of immunohistochemistry staining
There were at least three mice in each experimental group. The histological results were presented visually and quantitatively using Image-Pro 5.1 software (Media Cybernetics, Silver Spring, MD). Visually, it was clear that the leading zone of the healing area demonstrated high staining density for pimonidazole while the adjacent zone was less hypoxic. The healing area was defined as the area extending from the first hair follicle to the wound edge. Regions of interest (ROIs) were defined as the leading zone and the adjacent zone. () The leading zone is where hypoxia was found. The adjacent zone was within the healing area where it was not hypoxic, and later shown to contain proliferating cells by Ki67 staining. The zones were easily outlined as ROIs for quantification of staining using Image-Pro on pimonidazole staining. Staining intensity per area (staining density) was used to represent the degree of staining over the ROIs. Using the Image-Pro system, a segment of normal skin remote from wounding on each slide was used to determine the baseline staining. The same ROIs used to assess hypoxia on pimonidazole staining were then used to assess the expression of HIF-1α, VEGF, and SDF-1 on sequential sections of the same tissue block.
Figure 1 Photomicrograph showing the location of hypoxia in the burn wound at day 4. To the right is coagulation necrosis of the burn wound. To the left is normal skin with hair follicles outlined in yellow. The area between the burn wound and the first normal (more ...)
The results were reported as mean ± standard error. Statistical comparison between groups and within different zones was done using two-way ANOVA with Tukey post-tests. The results were considered statistically significant when p < 0.05.