Green Light Antinociceptive and Reversal of Thermal and Mechanical Hypersensitivity Effects Rely on Endogenous Opioid System Stimulation

Abstract

Benefits of phototherapy were characterized in multiple diseases including depression, circadian rhythm disruptions, and neurodegeneration. Studies on migraine and fibromyalgia patients revealed that green light-emitting diodes (GLED) exposure provides a pragmatic and safe therapy to manage chronic pain. In rodents, GLED reversed hypersensitivity related to neuropathic pain. However, little is known about the underlying mechanisms of GLED efficacy. Here, we sought to understand how green light modulates the endogenous opioid system. We first characterized how exposure to GLED stimulates release of β-endorphin and proenkephalin in the central nervous system of male rats. Moreover, by individually editing each of the receptors, we found that µ- and δ-opioid receptors are required for green light's antinociceptive effect in naïve rats and a model of HIV-induced peripheral neuropathy. We investigated how GLED could increase pain thresholds, and explored its potential in reversing hypersensitivity in a model of HIV-related neuropathy. Through behavioral and gene editing approaches, we identified that green light provides antinociception via modulation of the endogenous opioid system in the spinal cord. This work identifies a previously unknown mechanism by which GLED can improve pain management. Clinical translation of these results will advance the development of an innovative therapy devoid of adverse effects. PERSPECTIVE: Development of new pain management therapies, especially for HIV patients, is crucial as long-term opioid prescription is not recommended due to adverse side effects. Green light addresses this necessity. Characterizing the underlying mechanisms of this potentially groundbreaking and safe antinociceptive therapy will advance its clinical translation.

Keywords: GP120; Phototherapy; endogenous opioids; green light; neuropathic pain.

Figure 1:. Green LED light (GLED) stimulates secretion of β-endorphin, proenkephalin, but not dynorphin in CSF of naïve rats.

Endogenous opioid levels in rats after exposure to white (WLED) or GLED. Rats were exposed to GLED or WLED for 6 days; 8h/day, 100 Lux. Blood and CSF were collected at the end of exposure. Levels of β-endorphin (A), Proenkephalin (B) and Dynorphin (C) were analyzed through ELISA in serum, as well as in CSF (D, E, F, respectively) (n=7–9, Mann-Whitney nonparametric test, **p<0.01, ***p<0.001). GLED exposure resulted in increased levels of β-endorphin and Proenkephalin in CSF, but not in serum. No changes were observed in Dynorphin levels. Results are expressed in mean±SEM.

Figure 2:. Validation of genomic editing of opioid receptors in the spinal cord.

Rats were intrathecally injected with plasmids encoding Cas9 and gRNAs targeting μ- (MOR), δ- (DOR), or κ-opioid receptors (KOR). Two weeks following injection, dorsal horns of the spinal cord were collected, and levels or each opioid receptor were assessed by western blot. (A) Sequences of gRNA specifically targeting MOR, DOR, or KOR. Representative western blots illustrating levels of MOR (B), DOR (C), or KOR (D) in rats injected with CRISPR/Cas9 constructs, and quantification of their respective levels of expression. Targeting opioid receptors resulted in a robust decrease of receptor expression in the dorsal horn of the spinal cord. Results are expressed in mean±SEM, n=4, Mann-Whitney nonparametric test, *p<0.05.

Figure 3:. μ- and δ-opioid receptors are required for GLED-mediated antinociception.

(A) Experimental design. After baseline testing (BL) for thermal sensitivity (Hargreaves test), rats were injected intrathecally with plasmids carrying control, μ- (MOR), δ- (DOR) or κ-opioid receptor (KOR) gRNA, and the CRISPR-associated endonuclease 9 (Cas9). Thermal sensitivity was measured before light exposure at days 13 and 26, and after exposure to white LED (WLED) or GLED at days 28 and 32. Rats were exposed to 100 Lux light over 6 days after D26 for 8h/day. (B) Thermal withdrawal latencies in rats injected with control or KOR gRNA, before and after exposure to WLED or GLED. KOR editing did not alter GLED antinociceptive effect (n=8, parametric two-way ANOVA followed by Tukey’s posthoc test, *p<0.05). (C) Thermal withdrawal latencies in rats injected with control or DOR gRNA, before and after exposure to WLED or GLED. DOR editing reduced GLED antinociceptive effect (n=8, parametric two-way ANOVA followed by Tukey’s posthoc test, *p<0.05). (D) Thermal withdrawal latencies in rats injected with control or MOR gRNA, before and after exposure to WLED or GLED. MOR editing blocked GLED antinociceptive effect (n=6–8, parametric two-way ANOVA followed by Tukey’s posthoc test, **p<0.01).

Figure 4:. GLED induces long-lasting antinociceptive effect in the GP120 model of chronic neuropathic pain.

Both mechanical (Von Frey test) and thermal (Hargreaves test) hypersensitivity were assessed in rats to examine GLED’s antinociceptive effect. Neuropathic pain was induced with three intrathecal injections of GP120. Seven days after the last injection of GP120 or saline solution, rat sensitivities to thermal and mechanical stimuli were measured. Following measurement of baseline (BL), rats were exposed to white LED (WLED) or GLED, 100 Lux 8h/day, for 6 days (black bars). (A) Mechanical hypersensitivity was assessed every day for 15 days. Light exposure started after BL measurements. 6 days of GLED exposure produced significant antinociception over 11 days compared to WLED exposed animals (n_SHAM=4, n_WLED=6, n_GLED=7, mean±SEM, parametric two-way ANOVA, followed by Tukey’s posthoc, *p<0.05). (B) Thermal hypersensitivity was measured over 15 days. 6 days of GLED exposure produced significant antinociception over 8 days compared to WLED exposed animals (n_SHAM=4, n_WLED=6, n_GLED=7, mean±SEM, parametric two-way ANOVA, followed by Tukey’s posthoc, *p<0.05).

Figure 5:. GLED antinociception in GP120 neuropathic pain model requires μ- and δ-opioid receptors.

Rats underwent intrathecal (IT) catheter surgery (Sx), and baseline mechanical sensitivity thresholds (BL) were measured two weeks after surgery using Von Frey Filaments. After baseline testing, rats were injected intrathecally with plasmids encoding control, μ- (MOR), δ- (DOR) or κ-opioid receptor (KOR) gRNAs, and the CRISPR-associated endonuclease 9 (Cas9). Mechanical sensitivity was measured two weeks after gene editing, and rats received three IT injections of GP120 (3×300 ng) over a week. One week after the last injection (at D26), hypersensitivity was evaluated to confirm establishment of neuropathic pain. Then rats were then exposed to WLED or GLED for 6 days (8h/day, 100 Lux), and mechanical thresholds were measured at days 28 and 32. (A) Mechanical withdrawal thresholds in rats injected with control or KOR gRNA, before and after exposure to WLED or GLED. KOR editing did not alter GLED antinociceptive effect (n=6, parametric two-way ANOVA followed by Tukey’s posthoc test, *p<0.05, ***p<0.001). (B) Mechanical withdrawal thresholds in rats injected with control or DOR gRNA, before and after exposure to WLED or GLED. DOR editing significantly reduced GLED antinociceptive effect in GP120 rats but did not completely block antinociception compared to WLED rats (n=7–8, parametric two-way ANOVA followed by Tukey’s posthoc test, *p<0.05, **p<0.01, ***p<0.001). (C) Mechanical withdrawal thresholds in rats injected with control or MOR gRNA, before and after exposure to WLED or GLED. MOR editing blocked GLED antinociceptive effect (n=6, parametric two-way ANOVA followed by Tukey’s posthoc test, **p<0.01, ***p<0.001).