Decreased physical function and increased pain sensitivity in mice deficient for type IX collagen

Abstract

Objective: In mice with Col9a1 gene inactivation (Col9a1(-/-)), osteoarthritis (OA) and intervertebral disc degeneration develop prematurely. The aim of this study was to investigate Col9a1(-/-) mice for functional and symptomatic changes that may be associated with these pathologies.

Methods: Col9a1(-/-) and wild-type mice were investigated for reflexes, functional impairment (beam walking, pole climbing, wire hang, grip strength), sensorimotor skills (rotarod), mechanical sensitivity (von Frey hair), and thermal sensitivity (hot plate/tail flick). Gait was also analyzed to determine velocity, stride frequency, symmetry, percentage stance time, stride length, and step width. Postmortem, sera obtained from the mice were analyzed for hyaluronan, and their knees and spines were graded histologically for degeneration.

Results: Col9a1(-/-) mice had compensatory gait changes, increased mechanical sensitivity, and impaired physical ability. Col9a1(-/-) mice ambulated with gaits characterized by increased percentage stance times and shorter stride lengths. These mice also had heightened mechanical sensitivity and were deficient in contact righting, wire hang, rotarod, and pole climbing tasks. Male Col9a1(-/-) mice had the highest mean serum hyaluronan levels and strong histologic evidence of cartilage erosion. Intervertebral disc degeneration was also detected, with Col9a1(-/-) mice having an increased incidence of disc tears.

Conclusion: These data describe a Col9a1(-/-) behavioral phenotype characterized by altered gait, increased mechanical sensitivity, and impaired function. These gait and functional differences suggest that Col9a1(-/-) mice select locomotive behaviors that limit joint loads. The nature and magnitude of behavioral changes were largest in male mice, which also had the greatest evidence of knee degeneration. These findings suggest that Col9a1(-/-) mice present behavioral changes consistent with anatomic signs of OA and intervertebral disc degeneration.

Figure 1. Col9a1^-/- mice have deficient sensorimotor skills (rotarod)

In rotarod tests, Col9a1^-/- mice under-performed wildtype (WT) controls. In accelerating trials, Col9a1^-/- mice fell from the rotarod sooner than their sex-matched WT controls (male-Col9a1^-/- vs. male-WT: p<0.001; female-Col9a1^-/- vs. female-WT: p<0.03). Although latencies to fall from the accelerating rotarod for female-Col9a1^-/- mice were lower than for female-WTs, differences in constant-speed trials were only significant in males (male-Col9a1^-/- vs. male-WT: p<0.01). Data are presented as mean plus standard deviation (n=5 for each sex-genotype); the female-WT constant-speed trial 4 does not have a standard deviation as all female-WTs remained on the rod for the 5 min limit.

Figure 2. Col9a1^-/- mice use an altered gait during unprompted trials

In gait trials where mice were freely exploring an open arena (unprompted trial), Col9a1^-/- mice had statistically significant differences in velocity, hind-limb percentage stance time, stride length, and step widths. Both male and female-Col9a1^-/- mice locomoted at slower speeds (* p<0.001) and used higher hind-limb percentage stance times (* p<0.001) than WT controls. Col9a1^-/- mice also had differences in stride geometries, using shorter stride lengths (* p<0.001), wider hind-limb step widths (* p<0.001), and narrower fore-limb step widths (^# p<0.01). Data are presented as quartile box blots of 14 male-WT, 13 male-Col9a1^-/-, 15 female-WT, and 18 female-Col9a1^-/- trials measured from 5 mice in each sex-genotype.

Figure 3. Gait differences for Col9a1^-/- mice exist in prompted trials, but are less extensive than in unprompted trials

In prompted gait trials, mice were startled into movement by brushing their hind quarters with a swab. When prompting this movement, most gait differences between Col9a1^-/- mice and wildtype (WT) controls diminished relative to those observed in unprompted trials (See Figure 2). For velocity, male-Col9a1^-/- mice tended to locomote at slower velocities than male-WTs; however, these differences were not statistically significant. Col9a1^-/- mice did use higher hind-limb percentage stance times (* p<0.02), and mutant males used narrower fore-limb step widths than WT controls (^# p<0.001). When comparing these data with the unprompted results, differences in prompted trials were smaller in magnitude and less significant. Data are presented as quartile box blots of 11 male-WT, 9 male-Col9a1^-/-, 9 female-WT, and 10 female-Col9a1^-/- trials measured from 5 mice in each sex-genotype.

Figure 4. Representative Histology Images for the Knee Structural Scores Observed

Representative images for the range of structural changes observed for the knee are shown for Safranin O-Fast Green stained sections. Levels of degeneration increase from left to right. Higher levels of structural changes associated with degeneration were more common in Col9a1^-/- mice relative the WTs (See Table 2).