Rat blast model and tissue collection
Using a previously described hind limb rat protocol, Sprague–Dawley rats (n = 8) were first anesthetized with ketamine (80 mg/kg) and xylazine (7 mg/kg) delivered intraperitoneally, and pre-blast doses of enrofloxacin (5 mg/kg) and buprenorphine (0.05 mg/kg) were administered subcutaneously for antisepsis and analgesia, respectively, as previously described . Animals were then secured to an aluminum block with a 2.5 cm hole. The limb to be traumatically amputated was held in place over this hole . Immediately afterwards, these injured rats were transferred to sterile field for wound management and primary surgical closure. A detailed description of the blast setup, blast, surgical closure and post blast care has been previously reported . Four rats were sacrificed at 7 days post-injury and the tissue was collected for analysis. Under sterile conditions, muscle samples (~ 0.2 cm3) from the zone of the injury of the traumatized residual limb of the rats were surgically removed and analyzed as detailed below. Control uninjured muscle tissue RNA was harvested from uninjured lower extremities. In the acute post-operative period, rats were monitored for signs of distress, and given a 5-day course of buprenorphine (0.05 mg/kg administered subcutaneously twice a day) and enrofloxacin (5 mg/kg administered subcutaneously twice a day). All remaining animals (n = 4) were euthanized 42 days after the blast injury, to be used for radiographic evaluation of the injury site. Radiographs of the amputated limbs were performed with a digital Faxitron radiography machine (Faxitron X-Ray LLC, Lincolnshire, IL) as previously described . All animal procedures were performed under approved appropriate protocols by the Institutional Animal Care and Use Committee at the University of Maryland Medical Center.
Human tissue collection and radiographs
Discarded muscle tissues (~ 0.2 cm3 in volume) were obtained from the zone of injury of extremities from 5 wounded patients during surgical debridement procedures within 2-weeks of blast trauma (average 10-days post-injury). Tissue samples were divided into equal portions for histology and RNA extraction. This collection method was performed with patient consent according to a protocol approved by the Institutional Review Board at Walter Reed National Military Medical Center. Human control uninjured muscle tissue RNA was purchased from Amsbio (Cambridge, MA). Human radiographs of the injured extremity were performed following standard clinical protocols at the Walter Reed National Military Medical Center as part of the patient’s clinical care, including in preparation for the debridement procedure (average 10-days post-injury) and 42 days after injury, and evaluated by independent orthopaedic surgery residents under the direct supervision of at least one experienced orthopaedic surgeon.
Total RNA extraction from traumatized tissues, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and common cytokine PCR array
Total RNA from rat (n = 4, 7-days post-injury) and human (n = 5, average 10-days post-injury) blast injured tissues were prepared using the RNeasy mini Kit (Qiagen, Germantown, MD) following manufacturer’s instruction and as previously described . 500 ng of total RNA from each sample was converted into complementary DNA (cDNA) using the Superscript III First Strand Synthesis System for RT-PCR (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA). To investigate common genes related to osteogenesis and fibrosis between human and rat, real-time qRT-PCR was performed using commercially available TaqMan gene expression assays (Applied Biosystems/Thermo Fisher Scientific, Foster City, CA) for OPN, RUNX2 and COL1A1 (human samples) as well as Opn, Runx2 and Col1a1 (rat samples). Gene expression was normalized to HPRT1 (human samples) and Hprt1 (rat samples) levels, and relative gene expression was determined using the 2−ΔΔCt method . For the cytokine gene expression profiles, RNA integrity was assessed electrophoretically using a 6000 RNA Pico lab-on-a-chip in a Bioanalyzer (Agilent, Santa Clara, CA), and cDNA was synthesized using the RT2 First Strand Kit (SA Biosciences/Qiagen). PCR arrays were performed with a RT2 Profiler™ PCR Array Rat Common Cytokines (cat# PARN-021Z, SA Biosciences/Qiagen) and RT2 Profiler™ PCR Array Human Common Cytokines (cat# PAHS-021Z, SA Biosciences/Qiagen) following manufacturer’s instructions, and data analysis was performed using the RT2 Profiler PCR Array Data Analysis software (SA Biosciences/Qiagen). All PCR-based assays were performed using an ABI7900HT system (Applied Biosystems/Thermo Fisher Scientific).
Histology and picrosirius red (PSR) staining
Both sets of rat and human traumatized tissue and uninjured control tissue samples were separately fixed in phosphate-buffered 4% paraformaldehyde (FD Neuro Technologies Inc, Columbia, MD) followed by sequential ethanol dehydration infiltrated with xylenes and embedded in paraffin as previously described . Five-micron thick sections on glass slides from all tissues were used for hematoxylin and eosin (H&E) and Picrosirius Red (PSR) staining for histo-pathological evaluation and collagen immunohistochemistry, respectively. Sections were deparaffinized in xylenes, rehydrated using a graded series of ethanol and stained with H&E staining following standard laboratory procedures and PSR for 1 h as previously described [24, 26]. Stained H&E slides were analyzed in bright-field microscopy and stained PSR slides were analyzed by polarizing microscopy following standard procedures.
Scanning electron microscopy (SEM)
Approximately 5 mm3 pieces of both traumatized rat and human tissues were decellularized in 1% SDS solution for 30 min at 37 °C. Samples were fixed in 2.5% paraformaldehyde (PFA)/glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4; Electron Microscopy Sciences, Hatfield, PA) overnight at 4 °C. Fixed tissues were incubated with 1% osmium tetroxide (Electron Microscopy Sciences) for 20 min and dehydrated in a graded ethanol (25/50/75/85/95/100%) and hexamethyldisilazane (HMDS; Electron Microscopy Sciences) (25/50/75/100%). Samples were vacuum dried at room temperature. The desiccated samples were coated with gold using a sputter coater (Balzers; Schaumburg, IL) and surface topography was examined by scanning electron microscope (S-4800; Hitachi, Troy, MI, in the Biomedical Engineering and Physical Science Shared Resource, NIBIB, NIH) at 5 kV with various magnifications.
Paraformaldehyde fixed 5-μm thick tissue sections on glass slides underwent deparaffination and hydration and antigen retrieval. Primary antibodies used were Transforming Growth Factor-β1 (TGF-β1) and CD31 (Abcam, Cambridge, MA). After primary antibody incubation, the sections were washed 3 times with phosphate-buffered saline (PBS) for 5 min. The sections were than incubated with each respective secondary antibody (Alexa Fluor 488, Alexa Fluor 568-tagged secondary mouse or rabbit IgG antibodies, Invitrogen/Thermo Fisher Scientific, 1:250) for 30 min at room temperature. After secondary antibody incubation, Hoechst 33342 (1 μg/ml) was applied for 5 min onto section for nuclear staining. The sections were then washed 3 times in PBS for 5 min each and mounted in FluorSave reagent (Calbiochem, Billerica, MA). Slides with stained sections were viewed and analyzed using a Zeiss Axio Observer Z1 with Apotome optical sectioning device (Carl Zeiss, Thornwood, NY).
Western blot analysis
Rat (n = 4, 7-days post injury) and human (n = 5, average 10-days post-injury from HO patients) traumatized tissues were homogenized in RIPA Buffer (Sigma-Aldrich, St. Louis, MO) containing protease inhibitors (Roche, South San Francisco, CA). Total protein extracts were centrifuged at 13,000 rpm for 15 min at 4 °C, and the supernatants used for downstream analyses. Control uninjured human muscle tissue lysate was purchased from Abcam (Cambridge, MA) and control rat tissue was obtained from the lower uninjured extremities of Sprague–Dawley rats not exposed to blast injury. 10 µg of total protein extracts were separated by gel electrophoresis using a NuPAGE® 4–12% Bis–Tris Gel (Applied Biosystems/Life Technologies, Carlsbad, CA). Proteins were then transferred onto Immobilon-P membranes (Millipore, Burlington, MA) and stained with Ponceu S solution (Sigma-Aldrich) to assess transfer efficiency. Membranes were incubated with the indicated antibodies: Fibronectin (FN), SMAD family member 3 (SMAD3), TGF-β1, Plasminogen Activator Inhibitor 1 (PAI-1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) as loading control. Detection was performed by incubation with horseradish peroxidase-conjugated mouse or rabbit secondary antibodies (KPL, Gaithersburg, MD; 1:10,000) followed by Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore).
Replicates are expressed as mean ± SD values and significance was calculated by two-tailed Student’s t-test.