Expression of TRPV1 channels after nerve injury provides an essential delivery tool for neuropathic pain attenuation

Abstract

Increased expression of the transient receptor potential vanilloid 1 (TRPV1) channels, following nerve injury, may facilitate the entry of QX-314 into nociceptive neurons in order to achieve effective and selective pain relief. In this study we hypothesized that the level of QX-314/capsaicin (QX-CAP)--induced blockade of nocifensive behavior could be used as an indirect in-vivo measurement of functional expression of TRPV1 channels. We used the QX-CAP combination to monitor the functional expression of TRPV1 in regenerated neurons after inferior alveolar nerve (IAN) transection in rats. We evaluated the effect of this combination on pain threshold at different time points after IAN transection by analyzing the escape thresholds to mechanical stimulation of lateral mental skin. At 2 weeks after IAN transection, there was no QX-CAP mediated block of mechanical hyperalgesia, implying that there was no functional expression of TRPV1 channels. These results were confirmed immunohistochemically by staining of regenerated trigeminal ganglion (TG) neurons. This suggests that TRPV1 channel expression is an essential necessity for the QX-CAP mediated blockade. Furthermore, we show that 3 and 4 weeks after IAN transection, application of QX-CAP produced a gradual increase in escape threshold, which paralleled the increased levels of TRPV1 channels that were detected in regenerated TG neurons. Immunohistochemical analysis also revealed that non-myelinated neurons regenerated slowly compared to myelinated neurons following IAN transection. We also show that TRPV1 expression shifted towards myelinated neurons. Our findings suggest that nerve injury modulates the TRPV1 expression pattern in regenerated neurons and that the effectiveness of QX-CAP induced blockade depends on the availability of functional TRPV1 receptors in regenerated neurons. The results of this study also suggest that the QX-CAP based approach can be used as a new behavioral tool to detect dynamic changes in TRPV1 expression, in various pathological conditions.

Figure 1. The effect of QX-CAP application on the escape threshold of NP and non-NP group at different time points after IAN transection.

The changes in escape threshold following subcutaneous application of QX-CAP in sham-operated group (A); Only CAP injected sham-operated group (B); 2-weeks NP group (C); 2-weeks non-NP group (D); 3-weeks NP group (E); 3-weeks non-NP groups (F); 4-weeks NP group (G); 4-weeks non-NP group (H). The measurement were performed before the transection, 3 days after transection, 2, 3, and 4 weeks after transection/sham operation (depending on groups) and at various time points after injection of QX-CAP or CAP (n = 15 for each group, ANOVA followed by Dunnett’s test, *p<0.05). QX: QX-314; CAP: Capsaicin; Preop.: Preoperation; Preinj.: Preinjection; Pretrans.: Pretransection.

Figure 2. Photomicrographs of immunohistochemistry of TG cells labeled for TRPV1, NF200 and FG in sham-operated group and in 2-; 3-and 4-week NP groups and in 2-; 3- and 4 weeks non-NP groups.

Expanded view of TG in the sham-operated group (D1–D4). Arrow points on an example of TRPV1⁺+FG⁺+NF^- cell. Arrowhead points on an example of TRPV1⁺+FG⁺+NF⁺ cell. Note that TRPV1-positive cells increased with time after transection. Scale bar: 50 µm.

Figure 3. IAN transection both in NP and non-NP groups changes the expression profile of TRPV1 to myelinated neurons of a larger diameter.

The total number of TG cells labeled for the fluoro-gold (FG⁺) (A: NP group, B: Non-NP group); TG cells that labeled for TRPV1 and FG (TRPV1⁺+FG⁺) in 2-; 3-and 4-week NP groups and in sham-operated group (C: NP group, D: Non-NP group). The ratio of TRPV1⁺+FG⁺ to all FG+ positive cells (E: NP group, F: Non-NP group). n = 5 for each group, (ANOVA followed by the Student–Newman–Keuls test, *p<0.05). The number of cells positive for TRPV1, FG and NF200 (TRPV1⁺+FG⁺+NF⁺) (G: NP group, H: Non-NP group); positive for TRPV1 and FG but not for NF200 (TRPV1⁺+FG⁺+NF^-) (I: NP group, J: Non-NP group); positive for NF200 and FG (NF⁺+FG⁺) (K: NP group, L: Non-NP group) in 2-; 3-and 4-week NP groups and in sham-operated group revealed by immunohistochemistry. ANOVA followed by the Student–Newman–Keuls test. # indicates non-significant difference. TRPV1⁺+FG⁺+NF^- and TRPV1⁺+FG⁺+NF⁺ positive cells between the same groups are compared by paired t-test and the statistical significances are shown in the figure (G and H). 1*−4* indicate significant difference. 1: Sham TRPV1⁺+FG⁺+NF⁺ Vs TRPV1⁺+FG⁺+NF^-, 2: 2-wk non-NP TRPV1⁺+FG⁺+NF⁺ Vs TRPV1⁺+FG⁺+NF^-, 3: 3-wk non-NP TRPV1⁺+FG⁺+NF⁺ Vs TRPV1⁺+FG⁺+NF^-, 4: 4-wk non-NP TRPV1⁺+FG⁺+NF⁺ Vs TRPV1⁺+FG⁺+NF^-. p<0.05. n = 5 for each group.

Figure 4. The pattern of distribution of TRPV1 was altered in non-NP groups.

The distribution area of TRPV1⁺+FG⁺+NF⁺ positive cells for all experimental groups. A cell area >1000 µm² was considered large, while that <1000 µm² was considered medium. Note that most of the cells were in the medium range, and the peak distribution shifted to the right in the transected groups. n = 5 for each group.