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NGF Up-regulates TRPA1: Implications for Orofacial Pain

¹ Departments of Pharmacology and
² Endodontics, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA

View larger version (28K): [in this window] [in a new window]   Figure 1. NGF up-regulated the expression of TRPA1 mRNA in trigeminal ganglia. NGF concentration-dependently up-regulated the expression of TRPA1 mRNA (A) and CGRP mRNA (B) in the cultured trigeminal ganglia neurons. Trigeminal ganglia neurons were grown in culture in the presence of vehicle or NGF (1–100 ng/mL) for 24 hrs. Total RNA was isolated, and real-time RT-PCR was performed with primers specific for the TRPA1 or CGRP sequences. Data are presented as mean ± SEM (* = p < 0.05 and ** = p < 0.01 vs. vehicle group, one-way ANOVA with Bonferroni’s post hoc test; n = 3 independent cultures/group). Systemic administration of NGF significantly up-regulated the expression of TRPA1 mRNA (C) and CGRP mRNA (D) in trigeminal ganglia neurons. Male rats received a daily subcutaneous injection of vehicle or NGF (1 mg/kg) for 7 days. Total RNA was isolated from the trigeminal ganglia, and real-time RT-PCR was performed with primers specific for the TRPA1 or CGRP sequences. Data are presented as mean ± SEM (n = 3–4/group, ** = p < 0.01 and *** = p < 0.001 vs. vehicle group; two-tailed unpaired t test). Local administration of NGF significantly increased the expression of TRPA1 (E) and CGRP (F) mRNA in the trigeminal ganglia. Rats received intraganglionic (i.gl.) infusion of NGF into the left ganglia at a constant rate of 0.5 µg/hr/7 days or vehicle via a cannula attached to an osmotic mini-pump. Real-time RT-PCR was performed with specific primers against the TRPA1 or CGRP sequences, and the data were normalized to the mRNA levels found in the contralateral (right) ganglion for each rat/group. Data are presented as mean ± SEM (n = 4, * = p < 0.05 vs. vehicle group; two-tailed paired t test).

View larger version (51K): [in this window] [in a new window]   Figure 2. Systemic NGF increases the expression of TRPA1 in the TRPV1-positive subset of trigeminal ganglia neurons. In situ hybridization against the TRPA1 mRNA, combined with immunohistochemistry against the TRPV1 protein, was performed in trigeminal ganglia cryo-sections (20 µm in thickness) from rats injected with saline or NGF. TRPA1 mRNA (Panel A) was co-localized with TRPV1-containing neurons (B) in the trigeminal ganglia of vehicle (saline)-injected rats. The co-localization of TRPA1 mRNA (C) and TRPV1 (D) was significantly increased in the trigeminal ganglia of NGF-injected rats. Vertical white arrows show representative cells that contain detectable levels of TRPV1 but not of TRPA1 mRNA. Horizontal yellow arrows show cells that contain detectable levels of both TRPA1 and TRPV1. (E,F) Double-immunohistochemistry against TRPA1 and TRPV1 was performed in trigeminal ganglia neurons cultured in the presence of NGF or vehicle for 72 hrs. Immunoreactive TRPA1 protein was co-localized with TRPV1 in vehicle-treated trigeminal ganglia neurons (E). The co-localization of TRPA1 protein and TRPV1 was significantly increased in NGF-treated trigeminal ganglia neurons (F). Vertical white arrows show representative cells that contain detectable levels of TRPV1 but not of TRPA1. Horizontal yellow arrows show cells that contains detectable levels of both TRPA1 and TRPV1.

View larger version (13K): [in this window] [in a new window]   Figure 3. NGF treatment leads to an increase of TRPA1 activity measured as mustard-oil-evoked inward currents (IMO) in whole-cell patch clamp configuration. (A) The exposure of cultured trigeminal ganglia neurons to NGF (100 ng/mL) time-dependently increased the peak of IMO (black line) as compared with vehicle-treated neurons (grey line). Trigeminal ganglia neurons were grown in culture for a total of 72 hrs in the presence of NGF (100 ng/mL) or vehicle. IMO was recorded at 8, 24, 48, and 72 hrs of trigeminal ganglia culture. Data are presented as mean ± SEM (n = 8–16, *** = p < 0.001 for time 8 hrs vs. 24, 48, 72 hrs within each treatment group, and a = p < 0.01 NGF- vs. NGF+ for each time-point; two-way ANOVA with Bonferroni’s post hoc test). (B,C) Representative traces of IMO in trigeminal ganglia neurons grown in culture in the presence of vehicle (NGF–) or NGF+ for 8 hrs (top trace) and 48 hrs (bottom trace). Mustard oil (50 µM) was locally applied to trigeminal ganglia neurons for 60 sec.

Journal of Dental Research, Vol. 86, No. 6, 550-555 (2007)
DOI: 10.1177/154405910708600612


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