Stretching of the back improves gait, mechanical sensitivity and connective tissue inflammation in a rodent model

Abstract

The role played by nonspecialized connective tissues in chronic non-specific low back pain is not well understood. In a recent ultrasound study, human subjects with chronic low back pain had altered connective tissue structure compared to human subjects without low back pain, suggesting the presence of inflammation and/or fibrosis in the low back pain subjects. Mechanical input in the form of static tissue stretch has been shown in vitro and in vivo to have anti-inflammatory and anti-fibrotic effects. To better understand the pathophysiology of lumbar nonspecialized connective tissue as well as potential mechanisms underlying therapeutic effects of tissue stretch, we developed a carrageenan-induced inflammation model in the low back of a rodent. Induction of inflammation in the lumbar connective tissues resulted in altered gait, increased mechanical sensitivity of the tissues of the low back, and local macrophage infiltration. Mechanical input was then applied to this model as in vivo tissue stretch for 10 minutes twice a day for 12 days. In vivo tissue stretch mitigated the inflammation-induced changes leading to restored stride length and intrastep distance, decreased mechanical sensitivity of the back and reduced macrophage expression in the nonspecialized connective tissues of the low back. This study highlights the need for further investigation into the contribution of connective tissue to low back pain and the need for a better understanding of how interventions involving mechanical stretch could provide maximal therapeutic benefit. This tissue stretch research is relevant to body-based treatments such as yoga or massage, and to some stretch techniques used with physical therapy.

Figure 1. Methods for injection, stretch technique, sham technique, gait testing and ultrasound measurements.

A: Cartoon illustrating the tissue layers in the low back of the rodent from the dermis to the deep back muscles. The syringe indicates the location 1 cm lateral of L3 where carrageenan or vehicle was injected. B: In vivo stretch technique showing animals held by the tail and lifted. C: Sham technique (to account for the additional handling associated with stretch) showing animal held by the tail but not lifted. D: Method used for gait analysis. The distance, in centimeters, between ipsilateral walking tracks represents the stride length (black arrows). The diagonal distance between consecutive right and left walking tracks produces the intrastep distance (yellow arrows). E: Ultrasound imaging tissue thickness measurements (indicated by yellow stars).

Figure 2. Gait analysis measurements.

Stride length (A) and intrastep distance (B) across experimental groups. Open and closed circles respectively denote the average measurements for vehicle and carrageenan groups. Standard errors (S.E.) are indicated with error bars. N = 4 carrageenan/sham, N = 8 all other groups. (***p<.001).

Figure 3. Von Frey filament testing for mechanical sensitivity in the low back.

Bar graphs illustrate the mean percentage response out of ten trials for each experimental group at two weeks with the 1 g filament. Open and closed bars respectively represent means ±S.E for vehicle and carrageenan groups. For each group, the average response at two weeks was adjusted for baseline Von Frey measurements. N = 4 carrageenan/sham, N = 8 all other groups. (**p<.01, ***p<.001).

Figure 4. Ultrasound imaging and measurements.

A–D: Examples of ultrasound images from vehicle/no treatment (A), vehicle/stretch (B), carrageenan/no treatment (C) and carrageenan/stretch (D) groups. White arrows indicate the location where ultrasound measurements of tissue thickness were obtained. E: Open and closed bars respectively represent the mean ± S.E tissue thickness (E) for vehicle and carrageenan groups. N = 4 Carrageenan/sham, N = 8 all other groups. (**p<.01, ***p<.001).

Figure 5. Histology and macrophage expression in the connective tissues of the low back.

A–D: Representative images of Hematoxylin/Eosin staining of soft tissues from the lumbar region in a rodent in vehicle (A), vehicle/stretch (B), carrageenan (C) and carrageenan/stretch (D). Tissue structures from top to bottom (superficial to deep) include the dermis, pannicular muscle, nonspecialized connective tissues and the deep back muscles. E–H: Representative images of macrophage immunolabeling within the nonspecialized connective tissues from vehicle/no treatment (E), vehicle/stretch (F), carrageenan/no treatment (G), and carrageenan/stretch (H) groups. I: Macrophage expression quantified by calculating the percent CD 68 immunostaining area within each image. Bar graph represents means for each group ± S.E. Scalebars: black = 1 mm, white = 25 µm. Results are means = 4 carrageenan/sham, N = 8 all other groups. (**p<.01, ***p<.001).