Although it is not life-threatening, OA is incurable and ultimately results in chronic, debilitating symptoms. Complicating clinical OA treatment is the common finding that the severity of joint degeneration does not necessarily correlate to the symptomatic consequences of OA [19–21]. Clinically, debilitating symptoms can appear across a broad spectrum of joint degeneration, where patients may experience intense pain or joint dysfunction with little or no radiographic evidence of tissue damage or minimal symptoms with severe radiographic evidence of tissue degeneration [22, 23]. The lack of a unifying relationship between cartilage damage and symptoms in patients with OA may be attributed to psychosocial conditions and individual coping capabilities [19, 24–26]. Stress and environmental factors can clearly affect OA pain experiences; however, a potential exists that cartilage loss simply does not explain a significant portion of OA symptoms, even if environmental factors are well controlled. Our data in this study provide an example of this conundrum in a rodent OA model, where environmental factors were well controlled between the OA and non-OA groups.
Among the correlations identified, many are counterintuitive and likely arbitrary associations. First, many researchers would postulate that a thicker synovial lining would occur as a result of synovial inflammation, and therefore a negative relationship should occur between joint damage and the 50 % paw withdrawal threshold (heightened sensitivity). We identified a positive correlation, not a negative one. Because the synovial capsule was damaged with MCLT + MMT surgery but not with MCLT sham surgery, we speculate that the thickening of the synovial lining may not occur until a later time in MCLT sham animals. Thus, the correlation identified in the MCLT sham group may be an arbitrary relationship that results from the recovery of limb sensitivity to baseline levels following surgery and the delayed development of synovial damage following MCLT sham surgery. However, a skin incision sham (which was not included in this study) or age-matched naïve controls would be necessary to verify this hypothesis. Similarly, stance time imbalance was negatively correlated to the cartilage lesion width and depth in MCLT + MMT animals. Because a limb imbalance greater than 0 indicates that more time is spent on the left limb (contralateral) than on the right limb (injured), most OA researchers would postulate that this correlation would be positive; namely, as the lesion size increases, the gait sequence becomes more imbalanced. Thus, like medial joint capsule thickness, correlations between limb imbalance and joint histology could be driven by arbitrary correlations resulting from the recovery of imbalance parameters to baseline levels following the MCLT + MMT surgery.
Negative correlations were observed between multiple histological measures and the stride length residual and single-limb support residuals. Conceptually, these correlations follow the anticipated relationship: As the joint damage increases in severity, stride lengths decrease and periods of single-limb support reduce. However, these same gait compensations were identified with MCLT sham surgery, despite a lack of damage identified through the OARSI histological grading system (Fig. 10). When MCLT sham and MCLT + MMT data sets were assessed in conjunction, the correlations in the MCLT + MMT group also appeared to be coincidental, despite following the predicted pattern.
To be clear, this experiment was specifically designed to compare animals receiving an MCL sham surgery with animals receiving an MCLT + MMT surgery. Rats began the experiment as littermates and were cohoused, tested side by side on the same day, and fed the same diet throughout the experiment. Whereas the gait profiles in these animals did differ from the expected gait pattern of naïve controls, minimal differences were observed between the OA cohort (MCLT + MMT) and the non-OA cohort (MCLT alone). Moreover, whereas correlations could be identified between joint damage and changes in animal behavior in the OA cohort (MCLT + MMT group), the same behavioral changes, by and large, were found in the non-OA cohort (MCLT sham), despite the lack of significant joint damage. The exception may be tactile allodynia, which remained elevated in MCLT + MMT animals at 6 weeks but was not elevated in MCLT sham animals. However, the correlations between hind limb sensitivity and cartilage damage in the OA cohort were relatively weak (R = −0.4498, R
2 = 0.2023), and the range of sensitivities largely overlapped with the range in MCLT sham animals. Moreover, the tactile sensitivity difference between MMT + MCLT and MCLT sham animals did not appear until week 6, when cartilage damage was already very severe (>50 % of cartilage surface affected, cartilage lesions >75 % of the cartilage depth, and significant calcified cartilage damage). In combination, these data demonstrate the inherent limits of correlative relationships identified in our OA cohort. When taken in context with our MCLT sham results, correlations in the MCLT + MMT group appear to be largely coincidental or a relatively minor contributor to the behavioral phenotype in the OA group. Moreover, these data pose a fundamental question in the rat MCLT + MMT model of knee OA: Will therapeutics that prevent or reverse cartilage degeneration following a simulated meniscus injury have efficacy in treating OA-related symptoms and disability?
Although MCLT sham and MCLT + MMT animals were treated identically at each time point, data in a historical database of weight- and velocity-matched naïve animals were used as controls. Because of the strong correlations of most gait variables to animal weight and walking velocity (see Fig. 2), weight- and velocity-matched historical controls are advantageous relative to preoperative controls. Walking velocities can vary markedly between testing days and between trials, and most rats used in OA research gain 10–50 % body weight in the weeks after surgery. Failure to account for these covariates in the statistical analysis markedly reduces the sensitivity of the gait analysis [11]. The control database used in this study represents multiple experiments across a wide range of weights (307–425 g) and walking velocities (15.4–76.3 cm/s), allowing for the humane reduction of research animals by eliminating the need to replicate naïve data collected in prior work. Nonetheless, historical controls are not without limitations, as environmental factors can vary between the experimental animals and those in the control database. However, it is also worth noting that stride length and single-limb support time residuals consistently shifted down with postsurgical time, even though these data were collected from the cohorts in a random order (week 4, week 1, week 2, week 6). This indicates that the downward trend is unlikely to be due to a temporal change in the environment.
Unfortunately, the causes of gait abnormalities following MCLT sham and MCLT + MMT in the rat remain uncertain. Clearly, mechanical destabilization of the joint due to ligament injury of the MCL may cause a mechanical dysfunction that ultimately manifests in changes in the spatiotemporal gait pattern. However, if destabilization of the joint due to MCLT was the primary factor, gait compensations would be expected immediately after transection and for some dysfunction to occur consistently across the postsurgery time points. Instead, our data indicate a progressive development of bilateral gait compensations over time in both the MCLT sham and MCLT + MMT groups. This temporal shift seems to indicate that mechanisms other than mechanical loss of the MCL are involved in the development of the gait compensation found after these simulated joint injuries.
The act of cutting the skin, and not the injuries to the joint through either MCLT or MCLT + MMT, could cause the gait compensations and tactile sensitivity changes over the course of the 6-week experiment, and a skin incision sham would be needed to verify this hypothesis. Nonetheless, our primary conclusion is that, despite the development of full-thickness cartilage defects, calcified cartilage damage, and osteophyte formation in the MCLT + MMT group, there is no discernible gait pattern difference between the MCLT sham and MCLT + MMT groups, and differences in tactile sensitivity were limited to the 6-week time point. The lack of association between these histological parameters and rodent gait compensations would still hold even if the skin incision were the root cause of the gait abnormality, and this lack of association between cartilage damage and behavioral changes in a rodent model of OA highlights the lack of known unifying relationships between OA pathogenesis and the development of OA disease sequelae.
Another limit of this experiment includes the lack of preoperative data for von Frey testing; instead, data for naïve littermate controls were used. Although it would have been useful to understand how the tactile sensitivity of our animals changed over the course of the experiment, the primary intention of this experiment was to create a data set with behavioral profiles paired to a histological profile, such that correlations between behavior and histology could be constructed. In future studies, more sophisticated statistical correlation models may assist in identifying relationships between joint damage and behavioral changes, and assessment of changes in sensitivity, rather than raw sensitivity, may improve these correlations.
It is also worth noting that the von Frey test examines tactile sensitivity in the hind paw and that because the surgically simulated injury is at the knee, the von Frey test detects secondary (or referred) hypersensitivity. Knee bend or application of pressure to the knee could allow for more direct assessment of primary hypersensitivity in these models; however, it should be noted that these methods require animal restraint which may affect the behavioral measure.
For correlation analyses, the OARSI histopathology assessment scheme for the rat was advantageous because many of the parameters measured are real numbers rather than the ordinal ranks typical of many histological grading schemes. Nonetheless, the OARSI histopathology scheme still tends to be cartilage-centric and focuses largely on structural changes in the joint. This approach to grading joint damage may neglect the continuum of changes happening throughout the OA joint. First and foremost, proinflammatory and catabolic mediators are chronically upregulated in OA [6, 27, 28], and OA pain is often considered to be inflammatory. Inflammation also plays a critical role in MCL injury and repair after injury [29, 30], and upregulation of inflammatory mediators can affect muscle function. As such, behavioral changes associated with the MCL sham and MCLT + MMT surgery may be more closely linked to local inflammation at the site of each injury. Although joint inflammation was not directly assessed in this study, synovial capsule thickness (an indirect assessment of synovitis) did not appear to explain either gait or tactile sensitivity changes in the MCLT + MMT group. However, direct assessment of inflammatory cytokines and chemokines in the MCL, synovial fluid, synovial lining, or fat pad could be used in the future to more thoroughly evaluate the correlation between joint inflammation and behavioral changes.
In addition, strong evidence has emerged that OA pain has neuropathic pain components [31, 32], including evidence of damage to nociceptive fibers in the periphery of the joint [33, 34]. Joint innervation actively responds to the OA environment and joint injury [35, 36], and damaged sensory nerves can release neuropeptides that stimulate other nerve endings. Similarly, damage to the neuromuscular system can occur with MCL rupture [29, 37]. Because rats with MCLT sham surgery and MCLT + MMT surgery both experience chronic musculoskeletal injuries, behavioral changes observed in both models may be more indicative of nervous system remodeling, both in the periphery and centrally, rather than structural changes to the cartilage and bone within the joint. Again, direct assessment of changes in joint innervation and the associated dorsal root ganglia and dorsal horn of the spinal cord could be used in the future to more thoroughly evaluate the correlation between neuronal and behavioral changes in this model of OA.
Finally, movement-evoked pain is an early characteristic of OA, and, as a result, a person or animal may modify a gait pattern to protect an injured limb from loading and motion. If protective patterns are repeated over time, muscles surrounding the joint will adapt and a fear of specific movement patterns may develop. For chronic diseases such as knee OA, it is not yet clear if long-term use of a protective gait sequence promotes or prevents future joint degeneration; it is also not yet clear if the use of protective gaits or limb guarding will propagate OA-related disability. Thus, the gait abnormalities identified in this experiment are possibly learned behaviors that develop from the prior protection of an injured limb. In future experiments blocking joint afferents at the time of injury or after the onset of symptoms, researchers could begin to more thoroughly examine this hypothesis.