The role of TRPV1 in different subtypes of dorsal root ganglion neurons in rat chronic inflammatory nociception induced by complete Freund's adjuvant

Abstract

Background: The present study aims to investigate the role of transient receptor potential vanilloid 1 (TRPV1) in dorsal root ganglion (DRG) neurons in chronic pain including thermal hyperalgesia and mechanical allodynia. Chronic inflammatory nociception of rats was produced by intraplantar injection of complete Freund's adjuvant (CFA) and data was collected until day 28 following injection.

Results: Thermal hyperalgesia was evident from day 1 to day 28 with peak at day 7, while mechanical allodynia persisted from day 1 to day 14 and was greatest at day 7. Intrathecal administration of AMG 9810 at day 7, a selective TRPV1 antagonist, significantly reduced thermal hyperalgesia and mechanical allodynia. TRPV1 expression in DRG detected by Western blotting was increased relative to baseline throughout the observation period. Double labeling of TRPV1 with neuronal marker neurofilament 200 (NF200), calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4) was used to distinguish different subtypes of DRG neurons. TRPV1 expression was increased in the medium-sized myelinated A fiber (NF200 positive) neurons and in small non-peptidergic (IB4 positive) neurons from day 1 to day 14 and was increased in small peptidergic (CGRP positive) neurons from day 1 to day 28.

Conclusion: TRPV1 expression increases in all three types of DRG neurons after CFA injection and plays a role in CFA-induced chronic inflammatory pain including thermal hyperalgesia and mechanical allodynia.

Figure 1

Antagonizing effect of AMG 9810, a specific TRPV1 antagonist, on thermal hyperalgesia and mechanical allodynia on day 7 after CFA injection. A, C, and E in left panel for thermal hyperalgesia presented by hot plate latency (HPL); B, D and F in right panel for mechanical allodynia presented by paw withdrawal latency (PWT). A, Hot plate latency (HPL); C, Reversal of HPL (%); E, Area under the curve (AUC) of percentage of reversal of HPL. B, Paw withdrawal threshold; D, % Reversal of PWT; F, Area under the curve (AUC) of percentage of reversal of PWT. After AMG 9810 application, HPL and % reversal of HPL significantly increased at doses of 5 to 45 μg (p < 0.05, 0.01 or 0.001) from 1 h to 4 h; PWT and the reversal of PWT (%) displayed similar results, the reversal of HPL (%) significantly increased at doses of 5 to 45 μg (p < 0.05 or 0.01) at 4 h. *, $ and # represent comparison of AMG 9810 at 5, 15 or 45 μg with vehicle, respectively. n = 5–13.

Figure 2

Western blotting detection of TRPV1 protein in L4~L6 DRG. A, Western blotting bands. Upper row: TRPV1; Lower row: β-actin. B, Optical band density analysis. TRPV1 protein increased significantly after CFA injection from day 1 to day 28 (* p < 0.05). n = 3.

Figure 3

Double immunofluorescent staining of TRPV1 in DRG with neuronal markers of three subtypes at day 3 as an example. A, with NF200. In control animals, rare double staining of TRPV1 with NF200 was seen; after CFA injection, double staining of TRPV1 with NF200 significantly increased. B, with CGRP. Double staining of TRPV1 with CGRP significantly increased in CFA rats. C, with IB4. In control animals, double staining of TRPV1 with IB4 was ~50%; after CFA injection, double staining with IB4 significantly increased. Arrows indicate the doubly-labeled staining. Scale bar: 50 μm.

Figure 4

Quantification analysis of DRG neurons doubly stained with TRPV1 and neuronal markers. A, B and C show the percentage of TRPV1 positive neurons to NF200-, CGRP-, IB4-positive neurons, respectively. A, In control animals, rare double staining with NF200 was seen; after CFA injection, double staining of TRPV1 with NF200 significantly increased at days 1, 3, 7 and 14. B, Double staining of TRPV1 with CGRP significantly increased in CFA rats from day 1 to day 28. C, In control animals, double staining of TRPV1 with IB4 was ~50%, significantly increased from day 1 to day 14 after CFA injection. D, Area frequency distribution of TRPV1 and NF200 double stained neurons at day 3. After CFA injection, double staining neurons significantly increased in the medium-sized neurons with cell area of 600–1200 μm². * p < 0.05, ** p < 0.01, *** p < 0.001. n = 3.