Sleep fragmentation exacerbates mechanical hypersensitivity and alters subsequent sleep-wake behavior in a mouse model of musculoskeletal sensitization

Abstract

Study objectives: Sleep deprivation, or sleep disruption, enhances pain in human subjects. Chronic musculoskeletal pain is prevalent in our society, and constitutes a tremendous public health burden. Although preclinical models of neuropathic and inflammatory pain demonstrate effects on sleep, few studies focus on musculoskeletal pain. We reported elsewhere in this issue of SLEEP that musculoskeletal sensitization alters sleep of mice. In this study we hypothesize that sleep fragmentation during the development of musculoskeletal sensitization will exacerbate subsequent pain responses and alter sleep-wake behavior of mice.

Design: This is a preclinical study using C57BL/6J mice to determine the effect on behavioral outcomes of sleep fragmentation combined with musculoskeletal sensitization.

Methods: Musculoskeletal sensitization, a model of chronic muscle pain, was induced using two unilateral injections of acidified saline (pH 4.0) into the gastrocnemius muscle, spaced 5 days apart. Musculoskeletal sensitization manifests as mechanical hypersensitivity determined by von Frey filament testing at the hindpaws. Sleep fragmentation took place during the consecutive 12-h light periods of the 5 days between intramuscular injections. Electroencephalogram (EEG) and body temperature were recorded from some mice at baseline and for 3 weeks after musculoskeletal sensitization. Mechanical hypersensitivity was determined at preinjection baseline and on days 1, 3, 7, 14, and 21 after sensitization. Two additional experiments were conducted to determine the independent effects of sleep fragmentation or musculoskeletal sensitization on mechanical hypersensitivity.

Results: Five days of sleep fragmentation alone did not induce mechanical hypersensitivity, whereas sleep fragmentation combined with musculoskeletal sensitization resulted in prolonged and exacerbated mechanical hypersensitivity. Sleep fragmentation combined with musculoskeletal sensitization had an effect on subsequent sleep of mice as demonstrated by increased numbers of sleep-wake state transitions during the light and dark periods; changes in nonrapid eye movement (NREM) sleep, rapid eye movement sleep, and wakefulness; and altered delta power during NREM sleep. These effects persisted for at least 3 weeks postsensitization.

Conclusions: Our data demonstrate that sleep fragmentation combined with musculoskeletal sensitization exacerbates the physiological and behavioral responses of mice to musculoskeletal sensitization, including mechanical hypersensitivity and sleep-wake behavior. These data contribute to increasing literature demonstrating bidirectional relationships between sleep and pain. The prevalence and incidence of insufficient sleep and pathologies characterized by chronic musculoskeletal pain are increasing in the United States. These demographic data underscore the need for research focused on insufficient sleep and chronic pain so that the quality of life for the millions of individuals with these conditions may be improved.

Keywords: Mice; pain; rodent; sleep restriction; von Frey.

Figure 1

Experimental protocol to determine the effect of sleep fragmentation (SF) in combination with musculoskeletal sensitization on mechanical hypersensitivity and subsequent sleep-wake behavior of mice. Mice in group 3 (n = 22) were used to determine the effect of sleep fragmentation combined with musculoskeletal sensitization on mechanical hypersensitivity. These mice were subjected to sleep fragmentation during the light period of the 5-day interval between the first and second intramuscular injections. A subset of mice (n = 16) was used to determine the effect of sleep fragmentation combined with musculoskeletal sensitization on subsequent sleep-wake behavior. These n = 16 mice were implanted with telemeters, and allowed 3 weeks of recovery. Baseline EEG and body temperature recordings were then obtained for 2 days. All mice (instrumented, uninstrumented) were habituated to the von Frey testing platform and underwent baseline testing, which was then followed by 1 day of habituation to the sleep disruption device. Mice then were randomized into an acidified or normal saline injection group (n = 11 per injection; n = 8 with telemeters per injection group). The first injection with acidified or normal saline was given at light onset, which was followed by sleep fragmentation for 5 consecutive light periods before the second injection was given. Testing with von Frey filaments took place on postsensitization days 1, 3, 7, 14, and 21.

Figure 2

Sleep fragmentation by itself does not induce mechanical hypersensitivity. Mice in group 1 (n = 12) that had sleep fragmented for 5 consecutive light periods (days) did not develop mechanical hypersensitivity. Response incidence to von Frey filament presentation did differ among baseline (BL), sleep fragmentation day 3 (SF3), or postfragmentation days 1, 3, and 7 in either the left or right hindpaw [F(1,22) = 0.001, P = 0.973]. Responses to von Frey filaments are plotted as mean ± standard error of the mean percent of total response incidence ([total responses / total filament presentations] × 100) per paw.

Figure 3

Musculoskeletal sensitization with or without sleep fragmentation induces bilateral mechanical hypersensitivity. (A) Mechanical hypersensitivity in mice was induced by two injections of acidified saline spaced 5 days apart. Mechanical hypersensitivity manifests as increased response incidence to von Frey filaments on postsensitization days 3 through 21 [F(1,20) = 22.633, P < 0.01]. Mice subsequently randomized to injection groups did not differ at preinjection baseline [F(1,20) = 1.099, P = 0.307] or on day 1 postinjection [F(1,20) = 0.129, P = 0.723] (n = 12 acidified saline, n = 10 normal saline). (B) Mice in which sleep was fragmented during the musculoskeletal sensitization period developed mechanical hypersensitivity that was apparent on day 1 postinjection and lasted for at least 21 days [F(1,17) = 129.998, P < 0.001] (n = 10 acidified saline, n = 9 normal saline). Responses to von Frey filaments are plotted as mean ± standard error of the mean percent of total response incidence ([total responses / total filament presentations] x 100) per paw for the ipsilateral paw. Asterisk, P ≤ 0.05 versus.normal saline. BL, baseline; Inj, injection; SF, sleep fragmentation. Closed circle denotes acidified saline injection; open circle denotes normal saline injection.

Figure 4

Sleep fragmentation combined with musculoskeletal sensitization exacerbates mechanical hypersensitivity. Comparisons were made between mice allowed undisturbed sleep during the musculoskeletal sensitization period (group 2) and mice in which sleep was fragmented during the sensitization period (group 3). Response incidence values did not differ among manipulation groups at baseline (BL). Mechanical hypersensitivity did not develop in mice injected with normal saline, irrespective of whether sleep was fragmented. Mice subjected to sleep fragmentation during the musculoskeletal sensitization period exhibited greater mechanical hypersensitivity on day 1 [F(1,20) = 4.427, P = 0.048] and day 21 [F(1,20) = 17.540, P < 0.001] postsensitization than did mice that were sensitized without sleep fragmentation. Responses to von Frey filaments are plotted as mean ± standard error of the mean percent of total response incidence ([total responses / total filament presentations] × 100) per paw for the leg ipsilateral to the injection site. Plus sign, P < 0.05 versus undisturbed sleep + acidified saline. Asterisk, P < 0.05 versus normal saline of the same sleep manipulation.

Figure 5

The combination of sleep fragmentation and musculoskeletal sensitization disrupts subsequent sleep for prolonged periods. (A) The average number of sleep-wake state transitions per hour across the 24-h light:dark period is plotted for preinjection baseline and postmanipulation days 2, 8, 15, and 22. All mice were subjected to sleep fragmentation (SF) with musculoskeletal sensitization (acidified saline). Sleep fragmentation combined with musculoskeletal sensitization significantly increased state transitions during the light [F(1,12) = 30.348, P < 0.001] and dark [F(1, 12) = 20.479, P = 0.001] periods. (B) The percent change from baseline (BL) for the 12-h light and the 12-h dark period is plotted for postsensitization days 2, 8, 15, and 22. On all postmanipulation days, mice with musculoskeletal sensitization (acidified saline) had significantly more sleep-wake state transitions than mice without musculoskeletal sensitization (normal saline), an effect apparent during the light period [F(1,12) = 20.782, P = 0.001] and during the dark [F(1,12) = 45.219, P < 0.001] periods. Values are the mean ± standard error of the mean for n = 7 mice per injection group. Number sign, P ≤ 0.05 versus preinjection baseline; asterisk, P ≤ 0.05 versus normal saline.

Figure 6

The combination of sleep fragmentation and musculoskeletal sensitization alters the duration and quality of subsequent sleep. The effect of sleep fragmentation (SF) during the musculoskeletal sensitization period on rapid eye movements (REM) sleep, nonrapid eye movement (NREM) sleep, wakefulness, and delta power during NREM sleep is presented as percent change from baseline (BL) values. Values are the mean ± standard error of the mean for n = 7 mice per injection group. Although there was a trend for increased REM sleep on the second post-sensitization day, no effect of treatment was revealed for REM sleep during the light period [F(1,12) = 0.525, P = 0.482] or dark period [F(1,12) = 0.254, P = 0.245]. NREM sleep of mice subjected to the combination of sleep fragmentation and musculoskeletal sensitization was decreased during the light period across all postmanipulation days [F(1,12) = 10.509, P = 0.007], and increased during the dark period [F(1,12) = 10.658, P = 0.007] on postmanipulation days 15 and 22. Wakefulness during the light period of mice in which sleep fragmentation was combined with musculoskeletal sensitization increased during the entire postsensitization period [F(1,12) = 16.903, P = 0.001]. Sensitized mice had significantly less wakefulness during the dark period [F(1,12) = 19.155, P = 0.001] on post-manipulation days 15 and 22. NREM delta power in mice subjected to sleep fragmentation and musculoskeletal sensitization significantly increased during light periods [F(1,12) = 13.555, P = 0.003] and decreased during dark periods [F(1,12) = 28.153, P < 0.001]. For all panels: number sign, P ≤ 0.05 versus premanipulation baseline; asterisk, P ≤ 0.05 versus normal pH saline injections. Inj, injection.