Acidic saline-induced primary and secondary mechanical hyperalgesia in mice

Abstract

Most of our knowledge about chronic musculoskeletal pain is based on cutaneous pain models. To test the hypothesis that animals develop chronic muscular hyperalgesia following intramuscular acidic saline injections, primary hyperalgesia within the gastrocnemius muscle was analyzed compared to secondary cutaneous hyperalgesia in the hind paw that develops following intramuscular acid saline injection. Two acidic saline (pH 4) injections were administrated into the gastrocnemius of female CF-1 mice. The results indicate that mice developed a robust hypersensitivity bilaterally in primary (gastrocnemius muscle) secondary (cutaneous hind paw) sites that lasted up to 2 weeks. In addition, primary hyperalgesia correlated well with levels of Fos expression. Fos expression patterns in the spinal cord were different for primary secondary site stimulation. Hind-paw palpation stimulated ipsilateral Fos expression in the superficial spinal laminae at L4/L5 levels, bilaterally in deep laminae at L2-L5 spinal levels. In contrast, gastrocnemius compression stimulated widespread Fos expression in all regions of the ipsilateral dorsal horn within L2-L6 spinal segments. These findings indicate that acidic saline injection induces primary hyperalgesia in muscle that the patterns of Fos expression in response to primary vs secondary stimulation are strikingly different.

Perspective: This study assesses primary site muscular pain, which is the main complaint of people with musculoskeletal conditions, and identifies spinal patterns activated by noxious mechanical stimuli to the gastrocnemius. This study demonstrates approaches to test nociception arising from muscle aids in our understanding of spinal processing of primary secondary site hyperalgesia.

Figure 1

Photograph of the forceps device used to measure muscle compression threshold. Arrows indicate the pressure sensor (which is also the contact area with the tissue) and the site where force was applied by the examiner.

Figure 2. Quantification of Hind Paw Withdrawal to Von Frey Stimulation of the Paw and Compression of the Gastrocnemius Muscle

Mice displayed significant increase in cutaneous withdrawal responses from pre-injection to post-injection on the ipsilateral (A) and on the contralateral (B) sides following pH 4.0 saline injection. Likewise, mice displayed significant reductions in muscle withdrawal thresholds on both ipsilateral (C) and contralateral (D) sides following pH 4.0 saline injection. Arrows illustrate the time point at which pH 4.0 saline was injected into the right gastrocnemius. Data represented as mean ± S.E.M. Asterisks denote significant differences between neutral saline-and acidic saline-injected mice (P <0.05). Neutral saline-injection (pH 7.4) represents control group and acidic saline-injection (pH 4.0) represents experiment group.

Figure 3. Acidic Saline-Induced Fos Expression in Ipsilateral Lumbar Dorsal Horn Following Paw Palpation or Gastrocnemius Compression

Expression of Fos positive cells in ipsilateral L2/3, L4/5 and L6 dorsal horn of neutral and acidic saline-injected animals following paw palpation (left side) and gastrocnemius compression (right side). Images are displayed with scale bar equals 100 μms.

Figure 4. Schematic Representation of Fos Expression in the Lumbar Spinal Cord

A representation of the segmental distribution of the entire lumbar spinal cord following mechanical stimulation to hind paw (A) and gastrocnemius (B) anatomical sites. Following paw palpation, the largest number of Fos-positive cells was concentrated in superficial dorsal horn of segment L4/5 on ipsilateral side and deeper layers from L2-L5. Following gastrocnemius compression, widespread Fos activity was observed throughout the rostrocaudal extent of the lumbar spinal cord, L2-L6.

Figure 5. Correlation of Behavioral Responses and Fos Expression

Correlations between von Frey-induced withdrawal responses and paw palpation-induced Fos expression (secondary hyperalgesia; A, B, C) and between gastrocnemius compression peak forces and gastrocnemius compression-induced Fos expression (primary hyperalgesia; D, E, F) in the ipsilateral superficial dorsal horn. Correlations were significant only with primary hyperalgesia at L4/5 and L6 segments.