Rotenone prevents impact-induced chondrocyte death

Abstract

Mechanical insult to articular cartilage kills chondrocytes, an event that may increase the risk of posttraumatic osteoarthritis. Recent reports indicate that antioxidants decrease impact-induced chondrocyte death, but the source(s) of oxidants, the time course of oxidant release, and the identity of the oxidative species generated in response to injury are unknown. A better understanding of these processes could lead to new treatments of acute joint injuries. To that end, we studied the kinetics and distribution of oxidant production in osteochondral explants subjected to a single, blunt-impact injury. We followed superoxide production by measuring the time-dependent accumulation of chondrocyte nuclei stained with the superoxide-sensitive probe dihydroethidium. The percentage of chondrocytes that were dihydroethidium-positive was 35% above baseline 10 min after impact, and 65% above baseline 60 min after impact. Most positive cells were found within and near areas contacted directly by the impact platen. Rotenone, an electron transport chain inhibitor, was used to test the hypothesis that mitochondria contribute to superoxide release. Rotenone treatment significantly reduced dihydroethidium staining, which remained steady at 15% above baseline for up to 60 min postimpact. Moreover, rotenone reduced chondrocyte death in impact sites by more than 40%, even when administered 2 h after injury (p < 0.001). These data show that much of the acute chondrocyte mortality caused by in vitro impact injuries results from superoxide release from mitochondria, and suggest that brief exposure to free radical scavengers could significantly improve chondrocyte viability following joint injury.

Figure 1

Experimental Design. All explants (n= 44) were harvested and cultured for 2 days prior to starting the experiments. (A) The time course of oxidant production: DHE staining was measured in impact and control sites at various times after impact injury (1, 3, 6, 24, 48 hours). Four different explants were used for each time point (n =20). (B) The effects of rotenone on oxidant production: DHE staining was repeatedly measured in the same explants during the first hour after impact. The 4 groups included 2 rotenone treatments, one in which treatment was initiated before impact and carried on throughout the 60 minute imaging session (Group 1) and another in which treatment was delayed until 30 minutes post-impact (Group 2). Controls for the study included a set of explants that were impacted but not treated with rotenone (Group 3) and a set that was not impacted and not treated (Group 4). Each group consisted of 3 explants (n = 12) (C) Effects of rotenone on viability: Impact sites and control sites on the same explants were imaged in explants stained for viability (Calcein AM/ethidium homodimer) 24 hours after impact. A control group (no rotenone) and 3 rotenone treatment groups were included. The treatment groups differed with respect to the timing of rotenone dosing relative to impaction. 3 explants were used for each group.

Figure 2

Structural Damage Caused by Impact. (A) A safranin-O and fast green stained sagittal section shows disruption of the cartilage surface near the border of an impact site. (B) Closer view of the surface of the section in A shows vertical clefts (indicated by arrows) extending through the superficial zone and into the transitional zone. The bar in A is 0.6 mm in length.

Figure 3

Injury and Rotenone effects on Superoxide Production. (A) The diagram represents a typical osteochondral explant (2.5 cm × 2.5 cm square) under laser illumination on the scanning confocal microscope. A 5 mm diameter impact site (70 minutes post-impact) is shown on the surface of the explant (white circle). A z-axis projection (0-200 μm-deep) of a low magnification scan (4× objective) covering the border of an impact site is shown on the right. The field subtends ~ 1/4 of the impact site area. Low magnification z-projected images show DHE (red) and calcein AM (green) staining in an impact site on an untreated explant (B) and on an explant treated with rotenone (C). The bar in B represents 1 mm. Panels D and E show higher magnification images (20× objective) from within the sites shown in B and C. The bar in E is 150 μm long.

Figure 4

Time Lapse Imaging of Dihydroethidium Staining at an Impact Site. Panels A-F show a single 1.2 × 1.2 mm field imaged at 5, 10, 20, 30, 45 and 60 minutes after injury. The insets in the bottom right corner are enlargements from each image showing the same small group cells, all of which remained positive until the end of the experiment.

Figure 5

Time Course of Impact-Induced Superoxide Production. (A)The percentage of dihydroethidium-stained chondrocytes (% DHE Positive) measured repeatedly in the first 60 minutes post-impact in the same explants. Closed squares show results for impact sites in untreated explants. Closed triangles represent impact sites in explants treated with rotenone starting 1 hour before impact and during the imaging session. Open circles show data for impact sites in explants that were treated with rotenone starting at 30 minutes post-impact. Open triangles show results for untreated, non-impacted controls. Error bars indicate standard deviations based on 3 explants per group. (B) DHE positive staining at impact and control sites at the indicated times post-impact (1, 3, 6, 24, 48 hours). Asterisks indicate significant differences between impact sites and control sites. Error bars show standard deviations based on 4 explants per time point. Chondrocyte death rates over this time interval are shown in the line/symbol plot overlying the column plot.

Figure 6

Rotenone Effects on Impact Site Viability. Chondrocyte viabilities are shown for explants that were treated with rotenone for varying times before and after impact. Black columns show results for impact sites and white columns for non-impact sites. Treatment times (indicated below the X axis) were 2 hours before and after impact (2/2), 1 hour before and after impact (1/1), or 2 hours after impact (0/2). Results for untreated controls (0/0) are included for reference. Columns and error bars show means and standard deviations based on 3 explants. Asterisks indicate significant differences between impact sites that were rotenone-treated and those that were not treated. (p < 0.001).