Phosphorylation of CREB and mechanical hyperalgesia is reversed by blockade of the cAMP pathway in a time-dependent manner after repeated intramuscular acid injections

Abstract

Spinal activation of the cAMP pathway produces mechanical hyperalgesia, sensitizes nociceptive spinal neurons, and phosphorylates the transcription factor cAMP-responsive element binding protein (CREB), which initiates gene transcription. This study examined the role of the cAMP pathway in a model of chronic muscle pain by assessing associated behavioral changes and phosphorylation of CREB. Bilateral mechanical hyperalgesia of the paw was induced by administering two injections of acidic saline, 5 d apart, into the gastrocnemius muscle of male Sprague Dawley rats. Interestingly, the increases in immunoreactivity for CREB and phosphorylated CREB (p-CREB) in the spinal dorsal horn occur 24 hr, but not 1 week, after the second injection of acidic saline compared with pH 7.2 intramuscular injections. Spinal blockade of adenylate cyclase prevents the expected increase in p-CREB that occurs after intramuscular acid injection. The reversal of mechanical hyperalgesia by adenylate cyclase or protein kinase A inhibitors spinally follows a similar pattern with reversal at 24 hr, but not 1 week, compared with the vehicle controls. The p-CREB immunoreactivity in the superficial dorsal horn correlates with the mechanical withdrawal threshold such that increases in p-CREB are associated with decreases in threshold. Therefore, activation of the cAMP pathway in the spinal cord phosphorylates CREB and produces mechanical hyperalgesia associated with intramuscular acid injections. The mechanical hyperalgesia and phosphorylation of CREB depend on early activation of the cAMP pathway during the first 24 hr but are independent of the cAMP pathway by 1 week after intramuscular injection of acid.

Figure 3.

Immunohistochemistry of CREB and p-CREB 24 hr after the second intramuscular injection of pH 4.0, pH 7.2 control, or the preabsorption control. Both CREB and p-CREB densities increase 24 hr after the second intramuscular injection of pH 4.0 (A, D) compared with the animals that received pH 7.2 saline intramuscular injections (B, E). No staining was observed in tissue sections incubated with the immunizing peptide (C, F).

Figure 1.

Line graphs showing the effects of the inhibitors of the cAMP pathway on the median mechanical withdrawal threshold 24 hr after the second intramuscular acid injection. A, B, Intrathecal administration of SQ22536 (0.7 μmol) increases mechanical withdrawal threshold bilaterally compared with the 16% DMSO control 15 min after drug administration and remains increased for the next 2 hr. C, D, Intrathecal administration of PKI (60 nmol) increases mechanical withdrawal threshold bilaterally compared with the intrathecal saline control. E, F, A dose-dependent increase in mechanical withdrawal threshold is observed 45 min after spinal blockade of adenylate cyclase (SQ22536) or protein kinase A (PKI). INJ1, Before first intramuscular injection of pH 4.0 saline; INJ2, before second intramuscular injection of pH 4.0 saline; 24 hr INJ2, 24 hr after second intramuscular injection of pH 4.0 saline. Data are presented as the median with the 25th and 75th percentiles. *Contralaterally and ^#ipsilaterally significantly different from vehicle control; p ≤ 0.05.

Figure 2.

Line graphs showing the effects of the inhibitors of the cAMP pathway on the median mechanical withdrawal threshold 1 week after the second intramuscular acid injection. A, B, Intrathecal administration of SQ22536 (0.7 μmol) has no effect on mechanical withdrawal threshold compared with the 16% DMSO control. C, D, Intrathecal administration of PKI (60 nmol) has no effect on mechanical withdrawal threshold compared with the intrathecal saline control. INJ1, Before first intramuscular injection of pH 4.0 saline; INJ2, before second intramuscular injection of pH 4.0 saline; 1wk INJ2, 1 week after second intramuscular injection of pH 4.0 saline. Data are presented as the median with the 25th and 75th percentiles.

Figure 4.

Density of CREB and p-CREB immunoreactivity 24 hr and 1 week after the second intramuscular injection of pH 4.0 or 7.2 control. A, B, CREB and p-CREB increase in the spinal cord dorsal horn bilaterally 24 hr after the second intramuscular injection of pH 4.0 saline compared with pH 7.2 saline injections. C, D, CREB and p-CREB do not significantly differ 1 week after the second intramuscular injection of pH 4.0 compared with pH 7.2 saline injections. I–II, Laminas I and II; III–VI, laminas III–VI; 24 h, 24 hr; 1 wk, 1 week. Data are presented as the average with the SEM. *p, significantly different from the pH 7.2 control group; p < 0.05.

Figure 5.

Density of CREB and p-CREB immunoreactivity after intrathecal treatment with the adenylate cyclase inhibitor, SQ 22536, or saline. A, CREB remains unchanged after spinal inhibition of adenylate cyclase with SQ22536. B, The increase in p-CREB is prevented after spinal inhibition of adenylate cyclase with SQ22536 compared with the intrathecal saline control. I–II, Laminas I and II; III–VI, laminas III–VI. Data are presented as the average with the SEM. *Significantly different from saline control; p ≤ 0.05.

Figure 6.

Scatter plots showing the correlation of the mechanical withdrawal threshold ipsilaterally (A) and contralaterally (B) to the density of p-CREB immunoreactivity of the superficial dorsal horn at 24 hr. All animals at 24 hr were included in the analysis, and each point represents an individual animal.