Central sensitization in the trigeminal nucleus caudalis produced by a conjugate of substance P and the A subunit of cholera toxin

Abstract

Individuals with chronic craniofacial pain experience symptoms that are consistent with central sensitization. In fact, central sensitization may constitute the major disease process in these conditions, particularly if the original injury has healed or the condition is idiopathic. To understand central sensitization we have developed a conjugate of substance P and cholera toxin (SP-CTA). SP-CTA is selectively taken up by cells that express neurokinin receptors. Twenty-four hours following intracisternal administration of SP-CTA, wild-type rats and mice demonstrated signs of persistent background nociception, but when tested for facial cold sensitivity, they did not differ from controls. However, treating the SP-CTA-injected animals with naloxone exposed cold hypersensitivity in the face. Mu-opioid receptor knockout mice treated with SP-CTA demonstrated hypersensitivity without naloxone treatment. These findings suggest that central sensitization leads to activation of an endogenous opioid system. The data also demonstrate that the intracisternal administration of SP-CTA in rodents is a useful model for studying central sensitization as a disease process without having to induce a peripheral injury.

Perspective: Central sensitization is a concern in many craniofacial pain conditions. In this project, we utilize a conjugate of substance P and the catalytic subunit of cholera toxin to induce central sensitization in the nucleus caudalis of rodents. The data indicate that the injected animals become hypersensitive in the face.

Figure 1

Localization of SP-CTA in rat brainstem following intracisternal injection. SP-CTA (10μg) was injected into the cisterna magna via percutaneous puncture (N = 2). One hour following the injections the brainstems were harvested and sectioned (20μ) on a cryostat and labeled for the NK-1 receptor and the A subunit of cholera toxin. The large image demonstrates immunohistochemical co-localization of NK-1 receptors (green) and SP-CTA (red)(bar = 50μ). Note that there is a significant amount of SP-CTA that still remained on the surface of the tissue at this time point. Labels: STV - Spinal Tract of V; TNC – Trigeminal Nucleus Caudalis. The insert illustrates individual TNC neurons labeling for both NK-1 receptors and SP-CTA (bar = 10μ).

Figure 2

Uptake of SP-CTA in NK1-KO mice. NK1-KO mice and Balb/c wild type mice were injected intracisternally with either SP-CTA (10μg, 1μl PBS) or with 1μl PBS (2 animals per treatment group). Two hours following the injections the brainstems were harvested, rinsed with PBS and run on western blots. The blots were probed with an anti-body to the A subunit of cholera toxin. The far left lane on the western blot was a 5 μg sample of the original SP-CTA stock solution.

Figure 3

Effect of SP-CTA and naloxone on nociception in rats at −4°C in the operant facial nociception assay. The rats were tested prior to injections (Baseline: vehicle treated, N = 10; SP-CTA, N = 10) and 24 hours following the intracisternal injection of either SP-CTA (10μg, 10μl) or Vehicle (10μl PBS). Thirty minutes prior to testing half of the animals received injections of naloxone (10mg/kg, sc.). N = 5 rats per treatment group. A. Number of licking events in the 30 min. trial. B. Number of contacts with the stimulus. Asterisks indicate p<0.05 ANOVA followed by Dunnett’s test when compared to baseline. Two way ANOVA p < 0.05 Vehicle versus SP-CTA, # indicates p < 0.05 Bonferroni’s post hoc test when comparing the naloxone treated animals that received SP-CTA versus those that did not receive SP-CTA.

Figure 4

SP-CTA dose response relationship in mice. Hairless SKH1-Hr^hr mice were tested in the operant facial nociception assay prior to treatment (Baseline, N = 36) and then 24 hours following intracisternal injections of SP-CTA (0.1, 1.0, or 10μg in 1μl PBS). Thirty minutes prior to testing half of the animal in each group received injections of naloxone (10mg/kg, sc.) and the other half received vehicle injections (PBS, 0.25mls, sc.) (N = 6 animals per treatment group). Asterisks indicate p < 0.05, ANOVA followed by Dunnett’s test when compared to the Baseline responses. Two way ANOVA p < 0.05 Vehicle versus Naloxone treatment and p < 0.05 for dose of SP-CTA.

Figure 5

Effect of intracisternal SP-CTA in mu opioid receptor knockout (MOR) mice. Baseline testing was completed at 10°C prior to intracisternal injections. Animals were treated with either 10μg SP-CTA or PBS via percutaneous intracisternal injection (1μl). The animals were tested 24 hrs later at 10°C using the operant facial nociception assay. A. MOR knockout mice (Baseline N = 23 mice; Vehicle N = 8 mice; SP-CTA N = 13 mice). Asterisks indicate p < 0.05, ANOVA followed by Dunnett’s test when compared to the Baseline responses. # indicates p < 0.05 Bonferroni’s test on SP-CTA versus vehicle treated animals. B. In wild type C57BL/6J mice some of the animals received subcutaneous injections of naloxone (10mg/kg) 30 minutes prior to testing. (Baseline N = 27 mice; Vehicle/Nal N = 7 mice; SP-CTA N = 7 mice; SP-CTA/Nal N = 7 mice). Asterisks indicate p < 0.05, ANOVA followed by Dunnett’s test when compared to the Baseline responses. # indicates p < 0.05 Bonferroni’s test on SP-CTA/Nal versus SP-CTA treated animals that did not receive naloxone.