Long-lasting antinociceptive effects of green light in acute and chronic pain in rats

Abstract

Treatments for chronic pain are inadequate, and new options are needed. Nonpharmaceutical approaches are especially attractive with many potential advantages including safety. Light therapy has been suggested to be beneficial in certain medical conditions such as depression, but this approach remains to be explored for modulation of pain. We investigated the effects of light-emitting diodes (LEDs), in the visible spectrum, on acute sensory thresholds in naive rats as well as in experimental neuropathic pain. Rats receiving green LED light (wavelength 525 nm, 8 h/d) showed significantly increased paw withdrawal latency to a noxious thermal stimulus; this antinociceptive effect persisted for 4 days after termination of last exposure without development of tolerance. No apparent side effects were noted and motor performance was not impaired. Despite LED exposure, opaque contact lenses prevented antinociception. Rats fitted with green contact lenses exposed to room light exhibited antinociception arguing for a role of the visual system. Antinociception was not due to stress/anxiety but likely due to increased enkephalins expression in the spinal cord. Naloxone reversed the antinociception, suggesting involvement of central opioid circuits. Rostral ventromedial medulla inactivation prevented expression of light-induced antinociception suggesting engagement of descending inhibition. Green LED exposure also reversed thermal and mechanical hyperalgesia in rats with spinal nerve ligation. Pharmacological and proteomic profiling of dorsal root ganglion neurons from green LED-exposed rats identified changes in calcium channel activity, including a decrease in the N-type (CaV2.2) channel, a primary analgesic target. Thus, green LED therapy may represent a novel, nonpharmacological approach for managing pain.

Conflict of interest statement

– There is no conflict of interest for any of the authors.

Figures

Figure 1. Effect of light emitting diode (LED) exposure on thermal analgesia in naïve rats

Following measurement of baseline (BL) paw withdrawal latency (PWL, seconds), rats were randomly assigned (n=6 per group) to exposures of eight hours daily for five days to: dark; ambient room light; or white, green (λ=525 nm) or blue (λ=472 nm) LED. At the end of this exposure paradigm, PWLs were again measured. Blue and GLED exposure resulted in thermal analgesia. *p

Figure 2. Green light emitting diode (GLED)-induced thermal analgesia – time course and duration of effect

(A) Schematic representation of the experimental design, LED exposures, and Hargreaves testing. (B) Bar graph showing the paw withdrawal latency (seconds) of rats (n=6 per group) treated as shown in the schematic in A. BL indicates the baseline latency before GLED exposure. GLED exposure resulted in thermal analgesia starting at the second day (D2) of phototherapy and lasted 4 days after cessation (till D9) of LED exposure. *p<0.05 when comparing to BL (one-way ANOVA followed by Student-Newman-Keuls test).

Figure 3. Green light emitting diode (GLED)-induced thermal analgesia – effect of level of illuminance – without development of tolerance

(A) Bar graph of paw withdrawal latency (PWL, in seconds) of rats (n=6 per group) exposed to the indicated illuminance level of GLED for eight hours daily for five days. BL indicates the baseline latency before GLED exposure. LED exposure GLED exposure as low as 4 lux resulted in thermal analgesia. (B) Bar graph of PWL (seconds) of rats (n=6 per group) exposed to the indicated illuminance level of GLED for eight hours daily for five and twelve days. The PWLs were not different following 5 and 12 days of consecutive exposure to GLED. *p<0.05 when comparing to BL (one-way ANOVA followed by Student-Newman-Keuls test).

Figure 4. Involvement of the visual system in green light emitting diode (GLED)-induced thermal analgesia

(A) Bar graph of paw withdrawal latency (seconds) of rats (n=6 per group) prior to and after treatment with GLED as indicated. During the GLED exposure paradigm, the rats were fitted with clear or dark plastic lenses on their eyes. Blocking green LED absorbance to the eyes with dark contacts prevented the development of GLED-induced thermal analgesia. (B) Rats ‘wearing’ green plastic eye contacts and exposed to ambient room light for eight hours daily, developed thermal analgesia on days 3 and 4. *p<0.05 when comparing to baseline (BL) (one-way ANOVA followed by Student-Newman-Keuls test). (C) Absorbance spectra, in arbitrary units (a.u.), of clear, dark, or green contacts. Dark contacts absorbed light in all wavelengths while green contacts showed a peak absorbance in the 580–700 nm range.

Figure 5. Green light emitting diode (GLED)-induced thermal analgesia does not rely on skin pigmentation and occurs in both genders

Bar graph of paw withdrawal latency (seconds) of male and female Sprague-dawley rats (SD, white fur) and male long-evans (LE) rats (n=6 per group) prior to and after treatment with GLED as indicated. All rats developed thermal analgesia compared to their own baseline. *p

Figure 6. Green light emitting diode (GLED)-induced thermal analgesia involves activity of the descending pain pathways through endogenous opioid signaling

(A) Bar graph of paw withdrawal latency (seconds) of rats (n=6 per group) prior to and after treatment with GLED as indicated. BL indicates the baseline latency before GLED exposure. Inhibiting mu-opioid receptor (MOR) with naloxone (intraperitoneal (i.p.) or intrathecal (i.t.) administration) (see Table 1) reversed GLED-induced thermal analgesia. Un-treated (No Tx) rats or rats injected i.p. with saline (n=6 per group) developed GLED-induced thermal analgesia. (B) Inactivation of the descending pathway pain with an injection of a 2% solution of lidocaine into the rostral ventromedial medulla (RVM) of rats (n=6 per group) reversed GLED-induced thermal analgesia. *p<0.05 when comparing to BL (one-way ANOVA followed by Student-Newman-Keuls test).

Figure 7. Green light emitting diode (GLED)-induced thermal analgesia does not invoke a stress/anxiety response

(A) Bar graph of paw withdrawal latency (seconds) of rats (n=6 per group) prior to and after treatment with GLED as indicated. BL indicates the baseline latency before GLED exposure. Inhibiting the alpha- (with Phenoxybenzamine or Phentolamine) or beta- (with propranolol) adrenergic receptors (see Table 1) failed to reverse GLED-induced thermal analgesia. (B) Following verification that GLED exposure (8 hr × 5D) induces analgesia (B), rats were subjected to the elevated plus maze (EPM) test. (C) GLED exposure did not change the number of times the rats entered the open or closed arms in the EPM. (D) The anxiety index, an integrated measure of times and entries into the arms of the EPM, was not different between rats at baseline and after exposure to the GLED paradigm. (E) The GLED exposed rats spent significantly more time in the open arms, indicating that GLED exposure was anxiolytic. *p<0.05 when comparing to BL (one-way ANOVA followed by Student-Newman-Keuls test).

Figure 8. Motor function is not affected by green light emitting diode (LED) treatment

Bar graph of latency of rats (n=6 per group) to fall off the rotarod at incremental speed. Thermal analgesia induced by GLED exposure did not impair motoric performance as there were no differences between the fall off latencies between these and rats exposed to ambient room light. p>0.05 when comparing between the two conditions (paired Student’s t test).

Figure 9. Comparative proteomic analysis of dorsal root ganglia (DRG) and dorsal horn (DH) of the spinal cord from rats exposed ambient or green light emitting diode (GLED) using one-dimensional-liquid chromatography-electrospray ionization-tandem mass spectrometry

Venn diagram of the identified proteins in control and GLED exposed rat DRG (A) and dorsal horn of the spinal cord (E). Biological process (B, F), molecular function (C, G), and cellular component (D, H) were analyzed using the Proteome software Scaffold. The numbers indicate the percent of proteins detected in the proteomic study that are clustered in the annotated groups from naïve (black) or GLED exposed rats (green).

Figure 10. Exposure to green light emitting diode (GLED) reverses thermal hyperalgesia and mechanical allodynia induced in a model of neuropathic pain

(A) Seven days following a spinal nerve ligation (SNL) surgery on their left hind paw, rats (n=6 per group for all groups throughout this figure) displayed a significant decrease in their paw withdrawal latencies (PWLs, seconds), which was completely reversed by daily eight hours exposure to GLED exposure (4 lux). #p<0.05 when comparing to BL, *p<0.05 when comparing to BL or SNL (one-way ANOVA followed by Student-Newman-Keuls test). (B) Bar graph of paw withdrawal thresholds (PWTs, grams) of rats after receiving a SNL injury and after GLED exposure for 3 days (4 lux, 8 hours per day). BL indicates the baseline PWT before GLED exposure. PWTs were completely reversed 3 days of GLED exposure compared to post-surgery (post-Sx) levels. #p<0.05 compared to BL (one-way ANOVA followed by Student-Newman-Keuls test). Time course of return to baseline for PWLs (C) and PWTs (D). Some error bars are smaller than the bars. (E) Quantitative RT-PCR for the indicated genes from spinal cords of naïve rats as well rats with SNL + GLED or Sham + GLED. The mRNA levels for each gene were normalized to L27 mRNA (a ribosomal internal control gene). The L27-normalized values for each condition were divided by the L27-normalized values for naïve and are expressed as fold change over naïve levels. Data represent mean fold change ± S.E.M. (n=3 for each). A robust upregulation of PENK mRNA was observed in SNL + GLED or Sham + GLED condition (*, p<0.05, Student’s t-test vs. control).