Figure 6

Effects of collagenase exposure on the collagen network and neuronal dysregulation in an in vitro co-culture model mimicking the capsular ligament. (a) Type I collagen labeling (green) after a 20 min collagenase (col) incubation (n = 4 gels/group; n = 4 images/gel) is significantly reduced from levels with vehicle (veh) exposure (Wilcoxon test; *p = 0.008); in contrast, fiber orientation is unchanged with collagenase exposure (two-tailed t-test; p = 0.380). (b) Time-lapse captures of fluorescent calcium imaging show DRGs with segmented individual neurons labeled as active (blue; ≥ 1 calcium events) or inactive (yellow; no calcium events) in images from both before the addition of either collagenase (7 DRGs) or vehicle (4 DRGs) solution and after 20 min of exposure. Neuronal firing profiles captured across 28–33 neurons/DRG are the same in both groups at baseline (Wilcoxon test; p = 0.562), shown by frequency distributions of the percentage of neurons versus the firing magnitude per minute. Summary data in the bar plot also show no difference in number of calcium events at baseline. However, firing frequency patterns differ with treatment after 20 min, with vehicle-exposed neurons quieting as shown by a left-shift of the frequency curve peak toward zero and the summary data detailed in the bar plot (Wilcoxon test; *vs. vehicle p = 0.002). (c) Collagenase exposure increases neuronal MMP-1 expression (collagenase n = 4 gels; vehicle n = 2 gels; Wilcoxon test; *p < 0.001) quantified by the co-localization of the neuronal structural protein βIII-tubulin (green) and MMP-1 (red); a DAPI label for nuclei is also shown (blue). Images show the maximum projection image of acquired confocal stacks (n = 5/gel) in regions with DRG axons and/or soma. Box-and-whisker plots in (a) and (c) show horizontal lines representing the first (lower) quartile, median, and third (upper) quartile of the data; whiskers are the minimum and maximum.