Animals

Pathogen-free adult male Sprague-Dawley rats (Rattus norvegicus; 400-550 g; 91 animals) were purchased from Taconic Farms (Germantown, New York, USA). All rats were paired and housed in clean standard plastic cages and kept on a 12-h light/dark cycle with unlimited access to food (standard rodent chow), fresh water, and appropriate enrichment. Rats were allowed to acclimate to the vivarium conditions for at least three days before any handling or experimental procedures. After injury, rats were returned to clean home cages with soft bedding. All experimentation involving rats was performed in accordance with institutional standard guidelines and approved (SUR-20-997) by the Uniformed Services University Institutional Animal Care and Use Committee (IACUC) in compliance with all applicable Federal regulations governing the protection of animals in research.

Rats were divided into one of four experimental groups (n = 21/group) (Fig. 1) consisting of (1) blast overpressure exposure only (B); (2) complex orthopaedic injury consisting of right femur fracture and soft tissue crush injury (COI) with 3 h of tourniquet-induced hind limb ischemia and reperfusion injury with tourniquet release (IRI) followed by limb amputation (HLA) through the zone of injury (ZOI); (3) B and COI followed by a 1 h delayed hind limb amputation (dHLA); (4) B and COI with IRI followed by dHLA. Additionally, samples from normal, healthy naïve rats (n = 7) were included in the study for comparison.

Fig. 1

Diagram of experimental groups and sequence of trauma injury patterns. Rats were randomly assigned to undergo blast (B), complex orthopaedic injury and ischemia reperfusion injury followed by hind limb amputation through the zone of injury (ZOI) (COI + IRI + HLA), B + COI + 1 h delayed (d) HLA, or B + COI + IRI + dHLA

Trauma injury patternsAnesthesia

Rats were initially anesthetized with 4% isoflurane and administered a ketamine-xylazine (75 mg/kg ketamine (Henry Schein Animal Health, Dublin, Ohio, USA), 10 mg/kg xylazine (Akorn, Inc., Lake Forest, Illinois, USA)) mixture, with appropriate re-dosing as necessary for the duration of the procedure.

Blast overpressure exposure (B)

To induce a whole-body blast injury, rats were secured and placed in an Advanced Blast Simulator overpressure shock tube (ORA Inc., Fredericksburg, Virginia, USA), and exposed to a blast overpressure of 125 ± 10 kPa as previously described [37].

Complex orthopaedic injury (COI)

Animals undergoing extremity injuries first had the right hind limb shaved. A Lateral Long Bone Ballistic System (University of Alabama-Birmingham, Birmingham, Alabama, USA) was used to create a comminuted femur fracture in a similar fashion previously described [38]. The right hind limb of the rat was internally rotated and placed between the two anvils on the support stage, then a weight was dropped from a height of 88 cm to reliably produce a mid-shaft comminuted femur fracture. Immediately following the fracture, the anvils and support stage were adjusted to apply a soft tissue crush injury to the fracture site. A pressure of 20 psi was applied for one minute, as determined using a Chatillon DF series force gauge (AMETEK Inc., Berwyn, Pennsylvania, USA).

Tourniquet-induced ischemia/reperfusion (IRI)

For pneumatic tourniquet application, a pneumatic cuff (Hokanson, Bellevue, Washington, USA) inflated to 300 mmHg was applied as proximally as possible on the right hind limb to induce prolonged ischemia for 3 h, then released to cause reperfusion injury [39].

Hind limb amputation (HLA)

Amputation of the right hind limb was performed through the fracture site with appropriate hemostasis and debridement of devitalized tissue and bone fragments, followed by hamstring and quadriceps myoplasty over the exposed residual femur in a manner similar to that previously described [37, 40].

Post-operative monitoring

All rats received sustained released buprenorphine (1.2 mg/kg; Zoopharm, Windsor, Colorado, USA) for postoperative pain management, with repeat dosing three days post-injury. Rats were assessed twice daily for three days post-injury using IACUC approved pain charts.

Serum collection

Whole blood was collected at various timepoints post-injury (n = 5-7 rats/injury group). Lateral tail vein sampling occurred at 1 h, 3 h, and 3-day post injury. At 6 h, 24 h, and 7-day post-injury, blood was collected though exsanguination via cardiac puncture. Serum was separated by centrifugation (2000x g, 10 min, room temperature), then aliquoted and stored at − 80 °C.

Clinical chemistry

Whole serum chemistries were measured using an Element DCSX Veterinary Chemistry Analyzer (Heska, Loveland, Colorado, USA). Kidney function was assessed by measuring blood urea nitrogen (BUN), creatinine (Cr), and the BUN:Cr ratio. Liver function was assessed by measuring alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin levels.

Isolation and characterization of exosomes

Serum exosomes were isolated from 250 μL of freshly isolated whole serum according to manufacturer recommendations using the ExoQuick ULTRA EV Isolation Kit for Serum and Plasma (System Biosciences, LLC, Palo Alto, California, USA). Exosomes were pelleted, and the supernatant was aspirated and saved as the exosome-depleted serum (EDS). The exosome pellet was then re-suspended and subject to column purification for depletion of highly abundant proteins such as albumin and immunoglobulin. EDS and exosome samples were aliquoted and stored at − 80 °C.

Size of isolated exosomes was characterized by dynamic light scattering size analysis using a DynaPro NanoStar and Dynamic 7.9 software (Wyatt Technology Corporation, Santa Barbara, California, USA). The acquisition time of each exosome preparation (4 μL) was five seconds with 10 acquisitions performed in triplicate, and a specific refractive index increment (dn/dc) of 0.185.

Imaging of exosomes was performed by transmission electron microscopy (TEM). Re-suspended exosomes were applied to formvar-coated carbon-stabilized 3 mm copper grids (Electron Microscopy Sciences, Hatfield, Pennsylvania, USA) for one minute and then the excess was wicked away with filter paper. Grids were rinsed gently and briefly in nanopure water to remove buffer salts and 2% aqueous uranyl acetate was then applied for 1 min before the excess was wicked away. Grids were allowed to air dry and then were examined in a JEOL JEM-1011 transmission electron microscope (JEOL USA, Inc., Peabody, Massachusetts, USA). Images were recorded on an AMT XR50 digital camera (Advanced Microscope Techniques, Woburn, Massachusetts, USA).

Protein analysis of whole serum and serum exosomes was performed using SDS-PAGE under reducing and denaturing conditions with protease inhibitors (Promega, Madison, Wisconsin, USA) according to manufacturer instructions (NuPAGE 4-12% Bis-Tris gradient gels; Invitrogen, Carlsbad, California, USA). Sample protein concentrations were determined using a BCA Protein Assay (Pierce BCA Protein Assay kit; Thermo Scientific, Rockford, Illinois, USA).

Exosomal markers were assessed by Western blot analysis of tetraspanins CD9 and CD81, tumor susceptibility gene 101 (TSG101), and programmed cell death 6-interacting protein (ALIX) in total protein preparations of matched serum (10 μg) and serum exosomes (5 μg). Proteins were transferred to a nitrocellulose membrane 30 min using NuPAGE Transfer Buffer (Invitrogen, Carlsbad, California, USA) with a Bio-Rad TransBlot Turbo machine (Bio-Rad, Hercules, California, USA). Nitrocellulose membranes were blocked for one hour at room temperature in blocking buffer (5% BSA (Sigma-Aldrich Co., St. Louis, Missouri, USA) in 0.05% Tween 20 in TBS (Invitrogen, Carlsbad, California, USA)). The membranes were quickly washed with washing buffer (0.05% Tween 20 in TBS), followed by two consecutive washes, then were incubated with primary antibodies in the blocking buffer. Rabbit anti-rat monoclonal antibodies against CD9 (ab109201; Abcam Inc., Cambridge, Massachusetts, USA), CD81 (ab92726; Abcam Inc., Cambridge, Massachusetts, USA), TSG101 (ab133586; Abcam Inc., Cambridge, Massachusetts, USA), and ALIX (ab86429; Abcam Inc., Cambridge, Massachusetts, USA) expression were used at 1:1000 dilution. After overnight incubation at 4 °C and washing, the membranes were incubated with secondary antibody (goat anti-rabbit IgG/HRP conjugate, ab6721; Abcam Inc., Cambridge, Massachusetts, USA) at 1:10,000 dilution at room temperature for one hour, followed by washing. The protein bands were developed using chemiluminescence kit reagents (Immobilon Western Chemiluminescent, Millipore Sigma, St. Louis, Missouri, USA) and visualized on BioRad ChemiDoc (Hercules, California, USA). The intensity of specific bands was quantified by densitometry and Image Lab software (version 6.0.1; BioRad, Hercules, California, USA).

Total protein visualization was performed using a silver nitrate statin of equal total protein preparations of matched serum and serum exosomes. The gel was rinsed in de-ionized water, then placed in a fixative (10% acetic acid, 40% methanol). After fixing, the gel was washed in de-ionized water and sensitized in 12.5% glutaraldehyde. The gel was then washed again in de-ionized water followed by a wash in 20% ethanol. Next, the gel was stained using a silver stain composed of 0.4% silver nitrate, 0.25% ammonium hydroxide, 0.2% sodium hydroxide, and 18.6% ethanol. Following staining, the gel was washed in 20% ethanol, then protein bands were developed using a solution of 20% ethanol, 0.04% formaldehyde, and 0.0006 M citric acid.

Proinflammatory mediator analysis

At various timepoints post-injury, proinflammatory mediator levels (IFN-γ, IL-1β, IL-10, IL-4, IL-5, IL-6, KC/GRO (CXCL1), IL-13, and TNF-α) in whole serum, isolated serum exosomes, and EDS samples were simultaneously measured using a commercial 9-plex multiplex protein array (V-PLEX Proinflammatory Panel 2 Rat Kits; catalog K15059D; Meso Scale Diagnostics, Rockville, Maryland, USA). Prior to assaying, exosome samples were lysed using a non-ionic lysis buffer (50 mM Tris-HCl pH 7.4, 1% IGEPAL-CA630, 1 mM EDTA, 150 mM NaCl). Samples were analyzed in duplicate, and assays were performed per manufacturer kit instructions. Data acquisition was performed using a Meso Sector S600 (Meso Scale Diagnostics, Rockville, Maryland, USA) and quantitative results were generated using Methodical Mind software (version MMPR 1.0.27; Meso Scale Diagnostics, Rockville, Maryland, USA). Exosome and EDS samples were corrected to account for dilution effects in the isolation process. Quality control and data visualization was accomplished using the Workbench Software (version LSR_4_0_13; Meso Scale Diagnostics, Rockville, Maryland, USA). Data sets from some non-related unpublished studies performed in our laboratory demonstrate that the Meso Scale Diagnostics immunoassay platform used in this study offers a broader linear dynamic range, higher sensitivity, minimal background signals, and significantly less preparatory and processing time with greater throughput and reproducibility than standard ELISA and multi-analyte Luminex (bead-based) immunoassay platforms.

In determining the compartmentalization of proinflammatory mediators, the following assumptions were made regarding the detection of mediators in each of the serum fractions:

1.

Previous studies have demonstrated that EV-encapsulated mediators are not detected without disruption of the membrane [36]. Therefore, the whole serum measurement illustrated in Fig. 2A represents a standard, conventional immunoassay detection of mediators without treatment with a lysis buffer and the encapsulated mediators are not expected to be detected. The measured whole serum proinflammatory mediators include freely circulating (FC) and exosome membrane-associated (MA) proinflammatory mediators: Whole Serum = FC + MA

2.

After precipitation of serum exosomes, measured proinflammatory mediators in the EDS are, by definition, FC. EDS = FC

3.

The measured serum exosome proinflammatory mediators include both encapsulated (EC) and membrane-associated (MA) proinflammatory mediators: Serum Exosomes = EC + MA

4.

MA and EC fraction cytokine levels must then be calculated from the Whole Serum, Serum Exosome, and EDS measured values (Fig. 2B).

Fig. 2

Calculation of concentrations of proinflammatory mediators within each serum compartment. A Diagram of serum fraction and associated proinflammatory mediators detected. The gray circles represent exosomes and are not to scale. B Equations for calculating proinflammatory mediators in each compartment

Statistical analysis

Graphing and statistical analysis were performed using GraphPad Prism (version 9.0.2, GraphPad software, San Diego, California, USA). Outliers were determined using the GraphPad Prism ROUT analysis (Q = 1%). Welch’s t-test was used when comparing two groups. Statistical significance was defined as p-value < 0.05.