In this study, we found an early decrease (onset by week 3) in grip strength that coincided with the timing of increased Substance P and IL-1β immunostained cells, and IL-1β production in flexor digitorum tendons at the level of the carpal tunnel in rats exposed to a high demand voluntary repetitive reaching task, but not a low demand task (see also reference 20
). We observed in the HRHF tendons, a later increase (12 weeks) in infiltrating macrophages that coincided temporally with the peak in Substance P and tendon histopathology, including increased peritendon cellularity primarily due to increased peritendon CTGF and PLF immunostained fibroblasts, and increased endotendon collagen disorganization. There was also a trend toward neovascularization by week 12 in HRHF rat tendons.
Forelimb grip strength declined progressively in weeks 3–12, bilaterally, in HRHF animals compared to controls. This extends our previous observations showing decreased grip strength in week 12 in HRHF rats (only week 12 was examined in this earlier study; 22). The bilateral response was due to the use of each limb in performing the HRHF task, one as the preferred reach limb and the other as their postural support limb. Since decreased forelimb grip strength is an indicator of deep tissue hyperalgesia in animal models of muscle inflammation (25
), and the timing of IL-1β increases and grip strength declines are temporally matched, it is likely that tendon inflammation contributed to the grip strength losses. However, Substance P occurrence in tendons is also associated with nociceptive behaviors, such as hind paw thermal sensitivity after Achilles tendon injury (30
). It is likely then that Substance P also contributed to grip strength declines, especially since it showed a trend for an increase at week 3 matching the onset of grip strength declines.
We observed progressive increases in the pro-inflammatory cytokine, IL-1β, in HRHF tendons with continued task performance as well as increased inflammatory cells in the peritendon. We have previously found increased cytokines in forelimb flexor tendons of rats performing the HRNF tasks in week 8, but not earlier, and not with in tendons of LRNF rats (20
). These combined findings support our hypothesis of exposure dependent tissue and behavioral responses. Increased IL-1β has been shown to be an early response to tendon injury (21,31–33. Levels of mRNA for IL-1β increase only transiently on day 3 in tendon and sheath after a tensile loading injury (31
). Increased pro-inflammatory cytokines (IL-1β and TNFα), neutrophils and macrophages are observed early after Achilles tendon injury (21
), but not in tenosynovial sheaths collected from patients during carpal tunnel surgery which typically occurs long after the onset of pain (the most likely onset of injury and associated inflammation) in these patients (10
). However, in patients with lateral epicondylitis, IL-1 immunoreactive fibroblasts are observed in the extensor carpi radialis brevis muscle origin (33
). IL-1β is known to induce proliferation of fibroblasts (34
). Exposing tendon cells to IL-1β in vitro
results in an initiation of tendon matrix destructive pathways, such as matrix metalloproteinase pathways (35
). Therefore, our observed early increase in IL-1β is most likely contributing to the initiation of the subsequent proliferative and degenerative tendon changes.
Numerous papers have identified Substance P in tendon specimens from patients with painful chronic tendinopathies (7
) and in animal studies of tendon disorders (14
). For example, in tendons of patients with chronic medial and lateral epicondylalgia, Substance P immunoreactivity was present in tendon-associated nerve bundles and free nerve endings (7
). We also observed Substance P immunoreactivity in free nerve endings in tendon and blood vessel walls, as well as in tenocytes, mast cells and macrophages, cells identified by others to show Substance P immunoreactivity, especially with tendinosis (7
). Likewise, our observed Substance P increases were only in the HRHF task group, a group with histopathological signs of tendinosis. Substance P has several roles including facilitating histamine release from mast cells and thus enhancing vasodilation and extravasation of immune cells (40
). This hold true in our model as well, with both Substance P and extrinsic macrophages increases in week 12 peritendon regions. Since IL-1β facilitates Substance P release (42
), perhaps both contribute to inflammation as well as nociceptive behaviors with tendinopathies. Substance P has also been implicated in the induction of CTGF and proliferation of fibroblasts (43
). This would explain our temporal match in Substance P and peritendon fibrotic tissue changes.
Degenerative changes most commonly observed in overuse tendinopathies include pericellular thickening, fibrosis and hypercellularity (11
). Endotendon changes are also commonly observed, and include abnormal tenocyte morphology, increased CTGF immunoreactive cell densities, and collagen fibril disorganization (16
). Our findings are similar to findings by Nakama (17
), although our increase in CTGF immunoreactive cell densities was most prominent in the peritendon versus within the tendon matrix in Nakama’s study. This difference may be regional, as they examined tendon attachment sites to bone (the enthesis and just distal to it), and we examined unattached flexor tendon regions passing through wrist region (at least 4–5 mm proximal to the distal enthesis). An examination of the enthesis of flexor carpi radialis tendons and flexor carpal ulnaris tendons in our HRHF rats also shows an increase in CTGF in rounded tenocytes (unpublished data). However, we have chosen to focus on flexor digitorum tendons passing through the carpal tunnel since any thickening of these tendons would likely contribute to a presumed occupation of space by thickened (fibrotic) median nerve epineurium within the carpal tunnel in our model (22
), and therefore to our previously observed declines in conduction velocity in this section of the median nerve (22
). Like inflammation, the tendon histopathological changes in our repetitive strain injury model showed exposure dependence, being absent from LRNF rats, and in HRHF rats.
Our observed increase in CTGF immunostained fibroblasts in 12 week HRHF flexor digitorum peritendon is highly suggestive of proliferative fibrotic tissue changes in this tendon region. We also observed an increase in CTGF immunostained fibroblasts at the endotendon and thickening peritendon interface, changes that could be due to a fibroblast invasion of the endotendon. However, the presence of collagen type I around these CTGF immunostained (and therefore activated) fibroblasts is also suggestive of increased collagen deposition by the more internally located and perhaps more mature fibroblasts. This data matches our previous findings of increased CTGF and collagen type I in association with fibrosis-induced compression of the median nerve (22
). We also observed an increase in PLF immunostained fibroblasts and an increased presence PLF in the tendon matrix surrounding these fibroblasts. These findings combined with the CTGF and collagen results are suggestive of both peritendon fibroblast proliferative hyperplasia and matrix deposition with performance of a HRHF task, changes contributing to peritendon thickening and perhaps to some of the endotendon degenerative changes.
In conclusion, our findings support overuse as a factor in tendon injuries. We also found that the development of inflammatory, neuropeptide and histopathological changes were dependent both on level of task demands as well as on time spent engaged in an overuse activity. The timing of both tendon IL-1β and Substance P increases suggests that they contribute to grip strength declines. Finally, the early increase of tendon IL-1β, a cytokine with roles in initiating fibroblast proliferation and degenerative tendon changes, suggests that this early inflammatory cytokine response is one inducing factor of the later observed fibrotic tendon and degenerative changes.