Aging contributes to inflammation in upper extremity tendons and declines in forelimb agility in a rat model of upper extremity overuse

Abstract

We sought to determine if tendon inflammatory and histopathological responses increase in aged rats compared to young rats performing a voluntary upper extremity repetitive task, and if these changes are associated with motor declines. Ninety-six female Sprague-Dawley rats were used in the rat model of upper extremity overuse: 67 aged and 29 young adult rats. After a training period of 4 weeks, task rats performed a voluntary high repetition low force (HRLF) handle-pulling task for 2 hrs/day, 3 days/wk for up to 12 weeks. Upper extremity motor function was assessed, as were inflammatory and histomorphological changes in flexor digitorum and supraspinatus tendons. The percentage of successful reaches improved in young adult HRLF rats, but not in aged HRLF rats. Forelimb agility decreased transiently in young adult HRLF rats, but persistently in aged HRLF rats. HRLF task performance for 12 weeks lead to increased IL-1beta and IL-6 in flexor digitorum tendons of aged HRLF rats, compared to aged normal control (NC) as well as young adult HRLF rats. In contrast, TNF-alpha increased more in flexor digitorum tendons of young adult 12-week HRLF rats than in aged HRLF rats. Vascularity and collagen fibril organization were not affected by task performance in flexor digitorum tendons of either age group, although cellularity increased in both. By week 12 of HRLF task performance, vascularity and cellularity increased in the supraspinatus tendons of only aged rats. The increased cellularity was due to increased macrophages and connective tissue growth factor (CTGF)-immunoreactive fibroblasts in the peritendon. In conclusion, aged rat tendons were overall more affected by the HRLF task than young adult tendons, particularly supraspinatus tendons. Greater inflammatory changes in aged HRLF rat tendons were observed, increases associated temporally with decreased forelimb agility and lack of improvement in task success.

Figure 1. Motor performance changes in aged and young adult rats with high repetition low force (HRLF) task performance.

(A) Percentage of successful reaches. (B) A rat during the forehead sticker removal test. (C) Forehead sticker removal score (FHSR; 0 = no attempt at sticker removal to 5 = successful removal of sticker) with preferred reach or support forelimb. (D) FHSR in young adult HRLF and NC rats *: p<0.05, **: p<0.01, compared to aged-matched NC; &: p<0.05, compared to the same week in the other age group.

Figure 2. Pro-inflammatory cytokine levels in aged and young rat forelimb flexor digitorum tendons.

(A–D) IL1-β, TNF-α, IL-6 and IL-10 in aged rats’ preferred reach limb and support limb tendons. (E–H) IL1-β, TNF-α, IL-6 and IL-10 in young adult rat tendons. *: p<0.05 and **: p<0.01, compared to aged-matched NC rats; ##:p<0.01 compared to support limb of same-aged 12 week HRLF rats: &: p<0.05 and &&: p<0.01, compared to same week and limb of other age group.

Figure 3. Forelimb flexor digitorum tendons at wrist level of NC and HRLF rats.

(A,B) Cellularity scores in normal control (NC) and high repetition low force (HRLF) aged and young adult rats. *:p<0.05, compared to age-matched NC rats. (C) Photo of a flexor digitorum tendon (T) at wrist level from a young NC rat. Cap = capillary; CT = connective tissue. (D & E) Photos of flexor digitorum tendons (T) at wrist level from two different young 12-week HRLF rats. Arrows in D and E indicate sites of increased cellularity in the epitendon. Scale bar = 50 µm.

Figure 4. Supraspinatus tendons of NC and HRLF rats.

(A) A low power photo of the enthesis of a supraspinatus tendon attaching to greater tuberosity of humerus in an aged NC rat. Scale bar = 250 µm. (B) A higher power image of same section as shown in A. Inset is an enlargement of boxed area shown in A. (C&D). Photos of the enthesis of supraspinatus tendons in (C) an aged 12-week HRLF rat, and (D) a young adult 12-week HRLF rat. Arrows in C indicate increased site of cellularity, a region shown enlarged in inset. Scale bar in C is 50 µm; panels B and D are at same magnification. (E–H) Photos of distal cut ends of a supraspinatus tendon in: (E) an aged NC rat, (F) aged 12-week HRLF rat, (G) young adult NC rat, and (H) young adult 12-week HRLF rat. Arrows in F and H point out sites of increased cellularity and vascularity (cap = capillary bed). T = tendon; M = muscle fibers. Scale bar in F is 100 µm; panels E, G and H are at same magnification.

Figure 5. Histomorphometry and CTGF immunoreactivity in distal supraspinatus tendons.

(A) Cellularity scores of aged high repetition low force (HRLF) and aged normal control (NC) rat tendons. (B) Cellularity scores in young adult rat tendons. *p<0.05, compared to NC. (C) CTGF in distal supraspinatus tendon of aged NC, and (D) aged 12-week HRLF rat. (E) CTGF in distal supraspinatus epitendon of aged 12-week HRLF rat. Epi = epitendon, endo = endotendon; para = paratendon. Scale bar = 50 µm.

Figure 6. ED1-immunoreactive macrophages in flexor digitorum and supraspinatus peritendon regions of preferred reach limbs.

(A) ED1 cells in the flexor digitorum peritendon in aged rats, and (B) in young adult rats. (C) ED1 cells in supraspinatus endotendon of aged rats, and (D) in young adult rats. *p<0.05, compared to age-matched NC. (E) Photo of a flexor digitorum tendon of an aged-NC rat, and (F) in an aged 12-week HRLF rat. Arrow depicts ED1 cells in paratendon (ED1 is black; eosin counterstain), shown enlarged in the inset. (G) Photo of a distal cut end of a supraspinatus tendon of an aged 12-week HRLF rat. Arrows depict ED1 cells in paratendon, shown enlarged in inset. (H) Photo of the muscle mass of an aged 12-week HRLF rat showing ED1 cells. Arrow depicts ED1 cells (brown in color; eosin counterstain), shown enlarged in inset. Scale bar = 50 micrometers.