Extracellular biomolecular free radical formation during injury

Abstract

Determine if oxidative damage increases in articular cartilage as a result of injury and matrix failure and whether modulation of the local redox environment influences this damage. Osteoarthritis is an age associated disease with no current disease modifying approaches available. Mechanisms of cartilage damage in vitro suggest tissue free radical production could be critical to early degeneration, but these mechanisms have not been described in intact tissue. To assess free radical production as a result of traumatic injury, we measured biomolecular free radical generation via immuno-spin trapping (IST) of protein/proteoglycan/lipid free radicals after a 2 J/cm² impact to swine articular cartilage explants. This technique allows visualization of free radical formation upon a wide variety of molecules using formalin-fixed, paraffin-embedded approaches. Scoring of extracellular staining by trained, blinded scorers demonstrated significant increases with impact injury, particularly at sites of cartilage cracking. Increases remain in the absence of live chondrocytes but are diminished; thus, they appear to be a cell-dependent and -independent feature of injury. We then modulated the extracellular environment with a pulse of heparin to demonstrate the responsiveness of the IST signal to changes in cartilage biology. Addition of heparin caused a distinct change in the distribution of protein/lipid free radicals at sites of failure alongside a variety of pertinent redox changes related to osteoarthritis. This study directly confirms the production of biomolecular free radicals from articular trauma, providing a rigorous characterization of their formation by injury.

Keywords: Cartilage; DMPO; Free radical; Injury; Osteoarthritis; Oxidation; Oxidative stress; Posttraumatic; Radical; Redox; SOD3; Trauma.

Figure 1:. Increased DMPO staining in ECM after injury suggests increased extracellular biomolecular free radical formation after injury.

a) Representative sections from no DMPO sample stained with anti-DMPO antibody, and sections from a DMPO sample with no primary antibody and with anti-DMPO antibody. The samples without DMPO or without primary antibody had minimal staining compared to the anti-DMPO stained sample. Scale 200 μm. b) Samples were co-incubated with DMPO, H₂O₂, and myoglobin alone or myoglobin and iron acetate. The open arrows denote chondrocytes. The pink chondrocytes are negative for DMPO in the no primary sample whereas the brown chondrocytes seen in the treated samples are positive for DMPO (arrows). The myoglobin and iron treated samples generated ECM free radicals without impact, a positive control of free radical damage to the ECM. Scale 50 μm. c) Assessment of stain reproducibility by using serial sections of a 2 J/cm² impacted femoral explant with 70 mM DMPO. Scale 100 μm. d) Representative images of extracellular DMPO staining demonstrate increased DMPO staining after the 2 J/cm² impact compared to non-impacted tissue. Scale 100 μm. e) Blinded scoring of IST images demonstrated increased extracellular DMPO staining between the impacted femur and tibia. IST stain scoring was higher in the femur compared to the tibia. *p < 0.05 Kolmogorov-Smirnov test, κ = 0.59, n = 4. f) DMPO staining is observed in ECM of collagen fibers from rat tails after injury. Collagen fibers which were cut with a scalpel had increased staining compared to the uninjured control. The samples which were manually torn had the most intense staining in all the groups. These results support biomolecular free radical formation as a result of ECM failure is not restricted to articular cartilage injury. Scale 200 μm.

Figure 2:. Extracellular biomolecular free radical production at the time of impact compared to the subsequent 24 hours.

a) Anti-DMPO western blot of culture media 24 h after impact injuries. The impacted DMPO treated samples had more staining than the unimpacted samples, supporting ECM radical formation increases with impact injury. b) Schematic of how exposures were arranged. By staggering the timing of DMPO incubation prior or post injury the timing for biomolecular free radical formation can be determined. c) DMPO incubation prior to injury has increased DMPO staining at sites of ECM failure (black arrows) compared to DMPO incubation after injury. Scale 200 μm. d) Semi-quantitative scoring demonstrated the pre-exposed samples did have more DMPO staining than the control and post exposure. Due to the low n value significance was not reached, p = 0.057. *p < 0.05 Kolmogorov-Smirnov test, κ = 0.15 n = 4.

Figure 3:. Extracellular DMPO staining is associated with viable chondrocytes.

a) Representative images illustrate increased extracellular staining (black arrows) in the viable chondrocyte sample compared to the ethanol-treated samples. This suggests that viable chondrocytes (open arrows) contribute to DMPO staining at sites of ECM failure. Scale 200 μm. b) Histological scoring of the images indicates viable chondrocytes contribute to increased DMPO staining at sites of failure. The scores also suggest that since removing the cells does not return the staining to baseline, the impact itself is also a contributor to the DMPO staining intensity. *p < 0.05 Kolmogorov-Smirnov test, κ = 0.33, n = 4.

Figure 4:. Heparin treatment delocalizes SOD3, produces extracellular 3-NT, and increases DMPO staining at site of failure.

a) Representative images demonstrate that SOD3 is present in the articular cartilage of our swine samples (back arrows) and that heparin treatment decreased SOD3 localization compared to untreated in the absence of injury. Scale 100 μm. b) Confirmation of extracellular 3-NT punctate staining (yellow arrows) observed in the ECM of heparin treated samples without injury. Red indicates safranin O counterstain and pink indicates anti-3NT staining. Scale 50 μm. c) The staining pattern surrounding ECM failure was different between the DMPO and heparin DMPO treated samples. The staining appeared sharper around the failure in the heparin DMPO group compared to the DMPO group. Scale 50 μm.

Figure 5:. Delocalization of SOD3 increases biomolecular free radical formation at the site of failure but decreases the biomolecular free radical formation radiating from the failure.

a) Using a custom MATLAB program, the cracks were traced. b) The image was inverted. The cells near the traced lines were masked (arrows) to avoid cellular contribution to calculations of staining intensity. c) Vectors perpendicular to the traced crack were generated along the length of the crack. Each vector was divided into distance ranges 0–5 (white), 5–10 (blue), 10–15 (red), 15–20 (pink) μm. Each vector had an average and maximum intensity reported. d) The difference in average intensity between the DMPO (blue) and Heparin DMPO (red) treated groups were significantly different at distances greater 5 μm from the crack. This suggests that the delocalization of SOD3 prevents biomolecular free radicals’ formation farther into the tissue. This is supported by the maximum intensity being highest within the first 5 μm and the maximum intensities decreasing farther from the crack. p < 0.05 scale 50 μm. n = 17 DMPO, n = 27 Heparin DMPO.