Non-invasive diode laser activation of transient receptor potential proteins in nociceptors

Abstract

We investigated diode laser (980 nm) evoked activation of transient receptor potential proteins (TRPV1 and TRPV2). C and A-delta (Aδ) nociceptor families are primarily responsible for pain mediation in the peripheral nervous system. TRPV1 proteins have been associated with heat evoked pain in C fibers while Aδ fibers have been associated with TRPV2. Diode laser stimulation allows a margin of safety between non-invasive activation and damage (19, 22, 34). Laser pulses (20-50 ms, 0.1-10 W, 980 nm) were used to stimulate: A) in vitro: excised patches from HEK293 cells expressing TRPV1; B) in vitro: rat DRG nociceptors expressing either TRPV1 or TRPV2; and C) in vivo: C-fibers of the rat saphenous nerve (SN) trunk. Cell currents were recorded using standard patch clamp methods. The SN was also stimulated electrically with bipolar electrodes. Stimulation (20-50 ms) of HEK and DRG cells expressing TRPV1 was highly reproducible. Activation and peak currents were achieved at estimated peak temperatures of 55°C and 70°C. Threshold activation was also observed in DRG neurons expressing TRPV2. The conduction velocity for laser-activated saphenous nerve afferents was in the C fiber range (0.5-1 m/s). Electrically stimulated nerve contained stimulation artifacts and complex neural components with conduction velocities ranging from 0.3-30 m/s. Diode laser activation of TRPV1 protein is a reproducible and effective means to probe TRP activity in both in vivo and in vitro preparations.

Fig. 1

Currents evoked by repetitive laser stimulation of excised membrane patches HEK293 cells expressing TRPV1.

Fig. 2

The kinetics of laser induced currents type 2 (TRPV1) and type 4 (TRPV2) expressing nociceptors. laser stimulus: 20 ms, 2.41 W (1.200 mA of laser pumping current)

Fig. 3

Laser induced current of TRPV1 positive (type 2) cells evoked by laser pulses: 20 ms, 2.41 W (laser current 1200 mA) before and after TRPV1 antagonist capsazepine (10 uM). Two minutes between repeated tests. Bath temperature was maintained at 32 °C between tests.

Fig. 4

TRPV1 positive (type 2) cells n=8 were activated by laser pulses: 20 ms, 1–6 W

Fig. 5

A, B Compound action potential (CNAP) evoked by laser and electrical stimulation of the trunk of the saphenous nerve. (A) electrical stimulation (0.25 ms); (B) laser stimulation (20 ms). Note the reduction in artifact and neural components with laser stimulation. Thick arrows indicate the probable C fiber components. Thin arrows indicate stimulus onset.