Effectiveness of conservative interventions for sickness and pain behaviors induced by a high repetition high force upper extremity task

Abstract

Background: Systemic inflammation is known to induce sickness behaviors, including decreased social interaction and pain. We have reported increased serum inflammatory cytokines in a rat model of repetitive strain injury (rats perform an upper extremity reaching task for prolonged periods). Here, we sought to determine if sickness behaviors are induced in this model and the effectiveness of conservative treatments.

Methods: Experimental rats underwent initial training to learn a high force reaching task (10 min/day, 5 days/week for 6 weeks), with or without ibuprofen treatment (TRHF vs. TRHF + IBU rats). Subsets of trained animals went on to perform a high repetition high force (HRHF) task for 6 or 12 weeks (2 h/day, 3 days/week) without treatment, or received two secondary interventions: ibuprofen (HRHF + IBU) or a move to a lower demand low repetition low force task (HRHF-to-LRLF), beginning in task week 5. Mixed-effects models with repeated measures assays were used to assay duration of social interaction, aggression, forepaw withdrawal thresholds and reach performance abilities. One-way and two-way ANOVAs were used to assay tissue responses. Corrections for multiple comparisons were made.

Results: TRHF + IBU rats did not develop behavioral declines or systemic increases in IL-1beta and IL-6, observed in untreated TRHF rats. Untreated HRHF rats showed social interaction declines, difficulties performing the operant task and forepaw mechanical allodynia. Untreated HRHF rats also had increased serum levels of several inflammatory cytokines and chemokines, neuroinflammatory responses (e.g., increased TNFalpha) in the brain, median nerve and spinal cord, and Substance P and neurokinin 1 immunoexpression in the spinal cord. HRHF + IBU and HRHF-to-LRLF rats showed improved social interaction and reduced inflammatory serum, nerve and brain changes. However, neither secondary treatment rescued HRHF-task induced forepaw allodynia, or completely attenuated task performance declines or spinal cord responses.

Conclusions: These results suggest that inflammatory mechanisms induced by prolonged performance of high physical demand tasks mediate the development of social interaction declines and aggression. However, persistent spinal cord sensitization was associated with persistent behavioral indices of discomfort, despite use of conservative secondary interventions indicating the need for prevention or more effective interventions.

Keywords: Aggression; Cytokines; Inflammation; Mechanical hypersensitivity; Repetitive loading; Social interaction; Upper extremity; Von Frey; Work-related musculoskeletal disorders.

Fig. 1

Duration of social interaction, aggression, forepaw withdrawal behavioral changes and reach performance abilities. a Duration of positive social interaction (in seconds) of adult rats with a novel juvenile rat during a 300 s observation period. b Incidence of aggression of the adult rat with a novel juvenile rat during social interaction period (which ended the test, thus lowering the duration of positive social interaction in a). c Forepaw withdrawal response to light probing of glabrous skin at midpoint of forepaw. Y-axis shows size of von Frey filament (in grams) required to elicit a withdrawal response (mean of 5 probings shown). d Reach impulse (Newton’s of reach force multiplied by grasp time in seconds). HRHF and LRLF targets are indicated by dashed lines. e Example of a successful reach. The upper orange line indicates the maximum force threshold, the blue line depicts the lower force threshold; the green line depicts the target force threshold; and the pink line above the time marker shows the force deflection. The reward line shows that a food reward was delivered to a trough for rats to lick up. f Example of three unsuccessful reaches in one 500 ms cueing cycle, in which all reaches were below the target threshold (green line); thus, no food reward was dispensed. Results of two-way ANOVAs for each behavioral parameter are shown. *p < 0.05 in a–c, compared to age-matched FRC rats; **p < 0.01 in d, compared to week 1 results of same group; ^†,††p < 0.05 and p < 0.01, respectively, compared to age-matched untreated HRHF rats

Fig. 2

Serum levels of several cytokines and chemokines, assayed using multiplex ELISA. Results of two-way ANOVAs for each cytokine are shown in each panel, using the factors treatment group and week of experiment. Serum levels of a IL-1beta, b IL-6, c CSCL1 (Gro/KC), d TNFalpha, e CCL20 (MIP3a), and f IFNgamma are shown for each group, across weeks of training and task performance, as indicated. *p < 0.05, compared to food restricted control (FRC) rats euthanized after 1 week of food restriction; ^†p < 0.05, compared to age-matched untreated TRHF or untreated HRHF rats

Fig. 3

Inflammatory cytokine immunoreactivity in brain ependymal and endothelial cells. A–C There was an absence of IL-1beta and TNFalpha in these cell types in FRC rat brains. D–F IL-1beta and TNFalpha immunoreactivity increased in these cell types in 12-week HRHF rats. In D and E, arrows indicate positively stained ependymal cells surrounding the ventricle as well as small stained glial-like cells within the brain parenchyma. In F, inset shows IL-1beta stained endothelial cell. G, H Percent area with IL-1beta and TNFalpha immunoreactivity, respectively, in ependymal cells. Results for one-way ANOVAs are shown. **p < 0.01, compared to FRC rats; ^†p < 0.05, compared to untreated HRHF rats

Fig. 4

Inflammatory cytokines in the median nerve. A, B TNFalpha immunoreactivity in the median nerve at the level of the wrist of TRHF and TRHF + IBU rats, respectively. Images were taken with a 40× objective. C, D IL-1alpha and TNFalpha levels in the median nerve, respectively, assayed using ELISA. Results for one-way ANOVAs are shown. **p < 0.01, compared to age-matched untreated FRC rats; ^†p < 0.05, compared to age-matched untreated HRHF rats

Fig. 5

Substance P and NK-1R immunostaining in superficial lamina of dorsal horns of spinal cords at cervical 7 segmental level. A–D Representative images of dorsal horns showing punctuate substance P immunofluorescence (green) staining distributed across entire zone, medial to lateral (right to left side, respectively, in each panel). 12-week HRHF and HRHF + IBU rats show increased immunoexpression laterally. Insets in D shows location of substance P immunostaining at low power. Scale bars 50 μm, and applies to all panels except insets. E–H Dorsal horns showing neurokinin-1 receptor (NK-1R) immunostaining (red). Inset in E shows localization of NK-1R immunostaining at low power. I, J Quantification of percent area with immunofluorescence staining for substance P and NK-1R, respectively, in C7 dorsal horn superficial lamina. Results for two-way ANOVAs are shown. *p < 0.05, compared to age-matched untreated FRC rats

Fig. 6

TNFalpha and GFAP immunostaining in the superficial lamina (lamina I and II) of dorsal horns of the spinal cord at cervical 7 segmental level. A–C Representative immunoreactivity in FRC rat for TNFalpha, GFAP, or both together. Arrow indicates same cells in each panel, showing a lack of overlap between TNFalpha and GFAP. Scale bars 50 μm, and applies to all other panels except insets. D–F Representative immunoreactivity in 12-week HRHF rat for TNFalpha, GFAP, or both together. Arrows and lettering a–c indicate cells shown enlarged in insets of each panel and depicts cells with TNFalpha and GFAP co-localization. G, H Quantification of percent area with TNFalpha and GFAP immunostaining. **p < 0.01, compared to food restricted control (FRC) rats

Fig. 7

TNFalpha immunostaining (green) in dorsal horn deep lamina (DH) and ventral horns (VH) of the spinal cord at cervical 7 segmental level. NeuN, a nuclear stain, shown in red. A, E Representative immunoreactivity in FRC rat. B, F Representative immunoreactivity in untreated 12-week HRHF rats. Arrow in B indicates same cells as in upper inset, showing TNFalpha in NeuN stained neurons. Lower insets shows areas of assay for dorsal horn deep lamina and ventral horns, respectively. C, G Representative immunoreactivity in 12-week HRHF + IBU rat. Inset in C shows TNFalpha in NeuN stained neurons. D, H Representative immunoreactivity in 12-week HRHF-to-LRLF rat. Insets show TNFalpha staining in NeuN stained neurons. I, J Quantification of number of TNFalpha immunostained cells per mm². **,*p < 0.05 and p < 0.01, compared to food restricted control (FRC) rats; ^††p < 0.01, compared to age-matched untreated HRHF rats