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

Ibrahim, Mohab M.; Patwardhan, Amol; Gilbraith, Kerry B.; Moutal, Aubin; Yang, Xiaofang; Chew, Lindsey A.; Largent-Milnes, Tally; Malan, T. Philip; Vanderah, Todd W.; Porreca, Frank; Khanna, Rajesh

doi: 10.1097/j.pain.0000000000000767
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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.

Supplemental Digital Content is Available in the Text.Repeated exposure to 525 nm wavelength green light is antinociceptive in naive rats and antihyperalgesic in rats with experimental neuropathic pain.

Departments of aAnesthesiology and

bPharmacology, College of Medicine, University of Arizona, Tucson, AZ, USA

Corresponding author. Address: Department of Anesthesiology, College of Medicine, University of Arizona, 1501 North Campbell Dr, PO Box 245050, Tucson, AZ 85724, USA. Tel.: (520) 626-4281; fax: (520) 626-2204. E-mail address: mibrahim@anesth.arizona.edu (M. M. Ibrahim).

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.painjournalonline.com).

Received May 27, 2016

Received in revised form October 24, 2016

Accepted October 26, 2016

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1. Introduction

In the United States, over 100 million patients suffer from chronic pain.36 On average, 261 to 300 billion dollars are spent annually to manage pain, and 297 to 336 billion dollars are lost to work non-productivity.36 Additionally, the emotional tax from chronic pain has resulted in life-altering events ranging from shattering family unity71 to suicide attempts.26,29,32 Options for treatment of acute, postoperative pain, as well as for chronic pain, are limited, and many drugs are marginally effective22 and often accompanied by severe side effects.19 Nonpharmacological options for the treatment of pain would be highly desirable and advantageous in safety, including lack of tolerance, somnolence, addiction, gastrointestinal disturbances and, perhaps, other factors such as convenience and cost.

Light therapy has been reported to be useful for multiple medical conditions. For example, light therapy, has been used to control depression,20,25 to increase daytime circadian stimulation, to improve sleep quality, and mitigate depression in Alzheimer disease.21 Additionally, bright light improved the mood of adolescents taking antidepressants compared with those without light therapy.62 Exposing patients to bright light of more than 6000 lux significantly improved seasonal affective disorder, a serious condition with increased risk of suicidality.20,25 Pain has sensory, cognitive, and affective dimensions.9,59 The emotional state of patients significantly influences pain.24 For example, chronic psychological stress and depression increased pain perception in humans when painful and nonpainful heat stimuli were applied to their forearms.76 A similar response is also reported in rodents. In a model of social defeat, rats demonstrated enhanced nociceptive response to subcutaneous formalin administration.2 By contrast, elevated emotional status may decrease the perception of pain47; general happiness and laughter therapy significantly decreased chronic pain scores in elderly patients.79 Similar findings were observed in primary care patients who were enrolled in positive psychological intervention programs, which decreased their depression and improved overall functionality, as well as decreased pain perception.42 It is possible that light therapy may have psychological effects, possibly arising from physiological changes such as increased endogenous opioid release, as shown in this study, which affect pain response or its perception.

Few studies have investigated possible effects of light therapy on pain. A randomized clinical trial investigated the effect of bright light exposure in managing nonspecific back pain where patients received light exposure once a week for 3 weeks reduced pain as assessed by the Brief Pain Inventory suggesting an active role for light in controlling pain.43 Preclinical studies on the possible modulation of pain by light therapy have not been reported. Here, we investigated the effect of light in modulation of acute nociception as well as in a model of chronic neuropathic pain. We found that green light produced antinociceptive and antihyperalgesic effects that involved descending, opioid-sensitive inhibition.

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2. Methods

2.1. Animals

Pathogen-free, adult, male and female Sprague Dawley (SD) as well as male Long Evans (LE) rats (weight at testing 250-350 g; Harlan–Sprague–Dawley, Indianapolis, IN) were housed in a climate-controlled room on a 12-hour light/dark cycle and were allowed to have food and water ad libitum. All procedures were approved by the University of Arizona Animal Care and Use Committee and conformed to the guidelines of the National Institutes of Health (publication no. 80–23, 1966) for the use of laboratory animals. All behavioral experiments were conducted by experimenters blinded to the treatment conditions. The experiments were replicated a minimum of 2 times with independent cohorts of animals.

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2.2. Chemicals

All experimental compounds, doses, sources, and catalog numbers are described in Table 1.

Table 1

Table 1

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2.3. Light-emitting diodes

Visible-spectrum light-emitting diode (LED) flex strips were purchased from ledsupply.com (Randolph, VT). The specifications of the LEDs were (1) #LS-AC60-6-BL, 472 nm wavelength (ie, blue), 8 W, 120 V, 120° beam angle; (2) #LS-AC60-6-GR, 525 nm wavelength (ie, green), 8 W, 120 V, 120° beam angle; and (3) #LS-AC60-66-WH, white, 9.6 W, 120 V, 120° beam angle. Light-emitting diode strips were affixed to the outside of clear plastic cages that housed the rats so as to prevent the strips from being chewed. Rats were exposed to the various LEDs in these cages with full access to food and water in a dark room devoid of any other source of light. After behavioral assessment, the rats were returned to their cages for additional LED exposure. At the end of daily testing, the rats were returned to their regular animal room where they were exposed to room light (regular florescent bulbs). A lux meter (Tondaj LX1010B) was used to determine the illuminance and luminous emittance of the LED strips.

Because there are no previously published data reporting the effects of green LEDs on antinociception in rats, as a first step, we performed “pilot” experiments using a fixed amount (330 lux—which represents 1 full “strip”) of green LEDs over time, noting that antinociception developed only after 3 to 5 hours of exposure for at least 3 days. This antinociception continued to gradually increase over the next 2 days of exposure, reaching its zenith between days 4 and 5 (data not shown). The bulk of the studies were thus performed with 8 hours daily exposure for 5 days. Next, we performed a light-intensity vs antinociception analysis, which revealed that lower lux intensity was more effective than higher lux exposure for 8 hours daily for 5 days. These pilot experiments formed the basis of the exposure paradigm used in these studies—lux level, number of hours of exposure per day, and number of days of exposure.

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2.4. Thermal sensory thresholds

Paw withdrawal latencies (PWLs) used were determined as described by Hargreaves et al.28 Rats were acclimated within Plexiglas enclosures on a clear glass plate maintained at 30°C. A radiant heat source (high-intensity projector lamp) was focused onto the plantar surface of the hind paw. When the paw was withdrawn, a motion detector halted the stimulus and a timer. A maximal cut-off of 33.5 seconds was used to prevent tissue damage.

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2.5. Tactile thresholds

The assessment of tactile sensory thresholds was determined by measuring the withdrawal response to probing the hind paw with a series of calibrated fine (von Frey) filaments. Each filament was applied perpendicular to the plantar surface of the paw of rats held in suspended wire mesh cages. Withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength (the “up and down” method), and data were analyzed with the nonparametric method of Dixon, as described by Chaplan et al.,12 and expressed as the mean withdrawal threshold.

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2.6. Elevated plus maze

The elevated plus maze (EPM) consists of 4 elevated (50 cm) arms (50 cm long and 10 cm wide) with 2 opposing arms containing 30 cm high opaque walls. Elevated plus maze testing occurred in a quiet testing room with ambient lighting at ∼500 lux. On the day of testing, the rats were allowed to acclimate to the testing room for 20 minutes. Each rat was placed in a closed arm, facing the entry platform and cage mates started in the same closed arm. Each rat was allowed 5 minutes to explore the EPM and then returned to its home cage. Between animals, the EPM was cleaned thoroughly with Versa-Clean (Fisher Scientific, Waltham, MA). Elevated plus maze performance was recorded using an overhead video camera (MHD Sport 2.0 WiFi Action Camera; Walmart.com) for later quantification. Open- and closed-arm entries were defined as the front 2 paws entering the arm, and open-arm time began the moment the front paws entered the open arm and ended on exit. An anxiety index was also calculated; the index combines EPM parameters into one unified ratio with values ranging from 0 to 1, with a higher value indicating increased anxiety.34 The following equation was used for calculation of the anxiety index:

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2.7. Implantation of intrathecal catheters

For intrathecal (i.t.) drug administration, rats were implanted with catheters as described by Yaksh and Rudy.83 Rats were anesthetized with isoflurane and placed in a stereotactic device. The occipital muscles were separated from their occipital insertions and retracted caudally to expose the cisternal membrane at the base of the skull. Polyethylene tubing was passed caudally from the cisterna magna to the level of the lumbar enlargement. Animals were allowed to recover and were examined for evidence of neurologic injury. Animals with evidence of neuromuscular deficits were excluded.

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2.8. Fabricating rat contact lenses and imaging of eyes postmortem

All plastic materials were purchased from Evergreen Scale Models (Des Plaines, IL). We used the method developed by Levinson et al.,44 with the following modifications. In brief, 0.25 mm sheets were cut into 2 cm2 pieces held by forceps over a 6 mm ball bearing, shaped when malleable with a copper pipe of 9 mm internal diameter and then trimmed into a truncated hemisphere with iris scissors and sanded with a fine grit and emery cloth to a depth of 3.5 ± 0.2 mm and a base diameter of 7.0 ± 0.2 mm. We used a Weller 1095-1000 W dual temperature heat gun, instead of the Bunsen burner used by Levinson et al., as a heat source for the fabrication process. The rats where anesthetized using isoflurane just long enough to place the contact lens in their eyes and were allowed to recover from anesthesia. The rats were anesthetized again with isoflurane to remove the contact lens at the end of each 8-hour exposure.

To examine if the contact lens induced any pathological damage, at the end of the experiment, the corneas were excised with the small rim of the sclera and then fixed for 30 minutes with 4% paraformaldehyde in phosphate-buffered saline as described previously.64 Next, the corneas were transferred to a 30% sucrose solution until staining with Evans blue solution for 1 minute. The Evans blue dye solution was prepared by mixing 1 mL of a commercially prepared 0.5% Evans blue sterile aqueous solution with 9 mL of normal saline. This yielded a final solution of 0.05% Evans blue, with a pH of 6.75. After staining, the excess dye was removed by gently passing the tissue through 2 baths of normal saline solution. Light microscope images of the stained corneas were obtained on an Olympus BX51 microscope with a Hamamatsu C8484 digital camera using a 4X UplanFL N, 0.13 numerical aperture, or a 20X UplanSApo 0.75 numerical aperture objective. The freeware image analysis program ImageJ (http://rsb.info.nih.gov/ij/) was used to generate merged images.

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2.8.1. Spinal nerve ligation

Nerve ligation injury produces tactile allodynia and thermal hypersensitivity.40 All nerve operations occurred 5 days after i.t. catheter implantation. Rats were anesthetized with 2% isoflurane in O2 anesthesia delivered at 2 L/min (total time under anesthesia was <60 minutes). The skin over the caudal lumbar region was incised and the muscles retracted. The L5 and L6 spinal nerves were exposed, carefully isolated, and tightly ligated with 4-0 silk distal to the dorsal root ganglion (DRG) without limiting the use of the left hind paw of the animal. All animals were allowed 7 days to recover before any behavioral testing. Any animals exhibiting signs of motor deficiency were euthanized. Sham control rats underwent the same operation and handling as the spinal nerve ligation (SNL) animals, but without the nerve ligation.

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2.8.2. Rostral ventromedial medulla cannulation

Rats received a bilateral guide cannula (26GA, #C235-1.2 mm; Plastics One Inc, Roanoke, VA) directed to the rostral ventromedial medulla (RVM). The cannula was placed at: −11.0 from bregma, −7.5 mm from the dura, and 0.6 mm on either side of the midline. Injections were made by expelling 0.5 μL through an injection cannula protruding 1 mm beyond the tip of the guide. Cannula placement was confirmed with 0.5 μL Evans blue injected into both sides of the cannula, and microscopic examination of medullary sections and data from rats with misplaced cannula were eliminated from the experiment. Acute single injections into the RVM were performed by inserting an injector (#C235I-SPC; Plastics One Inc) attached to a 2-μL Hamilton syringe and expelling 0.5 μL at the same coordinates.

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2.9. Rotarod

Rats were trained to walk on a rotating rod (10 rev/min; Rotamex 4/8 device, Columbus, OH) with a maximal cutoff time of 180 seconds. Training was initiated by placing the rats on a rotating rod and allowing them to walk until they either fell off or 180 seconds was reached. This process was repeated 6 times and the rats were allowed to recover for 24 hours before beginning green LED exposure. Before treatment, the rats were run once on a moving rod to establish a baseline value. Assessment consisted of placing the rats on the moving rod and timing them until either they fell off or reached a maximum of 180 seconds.

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2.10. Sample preparation for proteomics

The DRG and dorsal horn of the spinal cord from rats exposed to ambient light or GLEDs (4 lux, 5 days, 8 hours/day) were isolated and lysates were prepared by sonication in RIPA buffer (50mM Tris-HCl, pH 7.4, 50mM NaCl, 2mM MgCl2, 1% [vol/vol] nonidet P40, 0.5% [mass/vol] sodium deoxycholate, and 0.1% [mass/vol] sodium dodecyl sulfate and protease (Cat#B14002; Biotool) and phosphatase inhibitors (Cat#B15002, Biotool), and BitNuclease (Cat#B16002, Biotool)). Protein concentrations were determined using the BCA protein assay (Cat#PI23225, Thermo Fisher Scientific, San Jose, CA). Proteins were precipitated using 100% ice-cold acetone and centrifuged at 15,000g at 4°C for 10 minutes. The pellets, containing solubilized proteins, were resuspended in 100 mM Tris pH = 7.4, 8 M urea (to eliminate carrying forward any salt contamination) and their protein content analyzed by mass spectrometry at the Arizona Proteomics Consortium after trypsin digestion.

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2.10.1. Database searching

Tandem mass spectra were extracted. Charge state deconvolution and deisotoping were not performed. All tandem mass spectrometry (MS/MS) samples were analyzed using Sequest (version 1.3.0.339; Thermo Fisher Scientific, San Jose, CA). Sequest was set up to search RattusNorvegicus_UniprotKB_2016_0406_cont.fasta assuming the digestion enzyme trypsin. Sequest was searched with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 10.0 PPM. Oxidation of methionine and carbamidomethyl of cysteine were specified in Sequest as variable modifications.

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2.10.2 Criteria for protein identification

Scaffold (version 4.5.1; Proteome Software Inc, Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 20.0% probability to achieve a false discovery rate less than 0.1% by the Scaffold Local false discovery rate algorithm. Protein identifications were accepted if they could be established at greater than 100.0% probability and contained at least 5 unique peptides. Protein probabilities were assigned by the Protein Prophet algorithm.60 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Proteins were annotated with gene ontology terms from gene_association.goa_uniprot.3

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2.11. Semiquantitative real-time polymerase chain reaction

Semiquantitative real-time polymerase chain reaction (RT-PCR) was performed as described previously.8,57 After GLED treatment or control, rat spinal cords were dissected and the lumbar region isolated before flash freezing in liquid N2. The cords were kept at −80°C until analysis. RNA was extracted from tissues using Tri reagent (Cat# TR118; MRC, Cincinnati, OH) according to the manufacturer's protocol. Briefly, Tri reagent was added to the cords before homogenization. The homogenates were centrifuged at 5000g for 5 minutes at 4°C to eliminate insoluble materials, then chloroform was added to the supernatants at 20% (vol/vol). After centrifugation at 12,000g for 15 minutes at 4°C, the upper, RNA-containing, aqueous fraction was harvested in a new tube and total RNA was subsequently precipitated by adding isopropanol (50%, vol/vol). The precipitates were subjected to a final centrifugation at 20,000g for 10 minutes at 4°C, and the RNA pellets were washed twice with 75% ethanol. Finally, purified RNA was resuspended in ultrapure water and stored at −20°C until analysis. To generate cDNA, 2 μg of purified RNA were retrotranscribed using Maxima Reverse Transcriptase (Cat# EP0743; Thermo Fisher San Jose, CA) according to the manufacturer's instructions. Quantitative RT-PCR analysis was performed using 5X HOT FIREPol Evagreen (Cat# 08-24-00020; Solis Biodyne, Tartu, Estonia) on a CFX connect (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Specific primer sequences used are given in Table 2. L27 expression was measured as a housekeeping gene to normalize target mRNA expression between samples. The relative mRNA level for each gene (x) relative to L27 mRNA (internal control) was calculated using the ΔΔCt method.49

Table 2

Table 2

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2.12. Data analysis

The statistical significance of differences between mean was determined by parametric analysis of variance followed by post hoc comparisons (Student–Newman–Keuls test) using GraphPad Software. Animal numbers required to achieve statistical power for each in vivo experiment were determined by G.Power 3.1, using post hoc variance estimates from pilot experiments. Pharmacological profiling of sensory neuron data was analyzed using SigmaPlot 12.5 and compared by z-test. Differences were considered to be significant if P ≤ 0.05. All data were plotted in GraphPad Prism 6.

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3. Results

3.1. Antinociceptive effects of green light-emitting diodes

We first determined if various kinds of light conditions affected responses to a noxious thermal stimulus in naive rats. Rats were exposed, for 8 hours, to ambient room light that consisted of white fluorescent lights and ambient sunlight through glass windows (600 lux), white LEDs (575 lux), or darkness (0 lux), and PWL was measured immediately after termination of illumination. As shown in Figure 1, the PWLs were unchanged across all conditions. By contrast, naive rats exposed to GLEDs (525 nm wavelength; 110 lux) for 8 hours daily for 5 days demonstrated a time-dependent increase in PWL that was significantly higher than rats exposed to ambient room light. Testing was performed immediately after termination of GLEDs (Fig. 1). Paw withdrawal latencies were also higher in rats exposed to blue LEDs (472 nm wavelength; 110 lux) but less than in those rats exposed to GLEDs (Fig. 1). The increase in PWLs, elicited by GLED exposure (Fig. 2A), plateaued by the second day and was unchanged thereafter until the fifth day (Fig. 2B). The GLED exposure was terminated on day 5 and the PWLs were measured on subsequent days to determine whether the increase in PWLs was transient or long lasting. The increase in PWLs induced by GLED exposure was maintained for 4 days before returning to baseline (Fig. 2B).

Figure 1

Figure 1

Figure 2

Figure 2

The preceding experiments were conducted with 110 lux GLEDs. However, whether this lux level was sufficient to elicit the antinociceptive behavior is not known. Consequently, we exposed the rats to GLEDs spanning several lux intensities. Exposing rats for 8 hours to 4 lux GLED levels was sufficient for increasing PWLs compared with nonexposed rats (Fig. 3). Similar levels of PWLs were observed with rats exposed to 12, 36, or 110 lux GLED levels (Fig. 3). Exposure of rats to GLED 330 lux level significantly increased PWLs compared with baseline ambient light-exposed rats but resulted in only a half-maximal response when compared with the other lux conditions (Fig. 3). As the 4-lux GLED exposure for 8 hours was sufficient for achieving maximal antinociception, we used this lux level for all subsequent experiments.

Figure 3

Figure 3

We also tested if prolonged exposure to GLEDs could elicit tolerance to the antinociceptive effect. Rats exposed to GLEDs for an additional 7 days past the initial 5 days exposure (4 lux, 8 h/d)—total of 12 days of GLED exposure—did not develop tolerance (Fig. 3B). This is in contrast with published data with morphine where analgesic tolerance can be observed within 3 days67 and causes rats to be hyperalgesic within 5 days.82

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3.2. Characterization of the role of the visual system in the antinociceptive effects of green light-emitting diodes

We determined whether the mechanism of GLED-induced antinociception requires direct activation of the visual pathway by fashioning dark, opaque, plastic contact lenses that permitted no light penetration (confirmed by measuring light intensity). These were fitted onto the rats' eyes under anesthesia. As a control, transparent clear lenses were also installed onto control rats' eyes. The application of contact lenses did not alter the baseline PWL. Both groups of rats were then exposed to GLEDs for 8 hours daily for 5 days and their PWLs were monitored. Following this exposure paradigm, rats fitted with the dark, opaque, contact lenses failed to develop antinociception, whereas rats fitted with clear, transparent, contact lenses developed antinociception similar to rats with no contacts (Fig. 4A). Consistent with the importance of the visual system in the development of GLED-induced antinociception, rats fitted for 8 hours with “green” contacts that permit light transmission in the green part of the visual spectrum (Fig. 4B), developed antinociception when exposed to room light (Fig. 4C). Importantly, histological analysis of the eyes of the rats at the end of the experiments revealed no damage caused by either contact lens (data not shown). These results support a role for the visual system in mediating the GLED-mediated antinociception.

Figure 4

Figure 4

To test if pigmented skin is involved in the antinociceptive effects of GLEDs, we selected the pigmented LE rats, which are different from the albino SD rats that lack pigmentation. A similar level of antinociception was observed in SD or LE rats exposed to GLEDs for 8 hours daily for 5 days; the PWLs were significantly higher than the respective strains exposed to ambient light (Fig. 5). The antinociception was not restricted to male rats as female SD rats exposed to the same GLED paradigm also exhibited increased PWLs (Fig. 5). Collectively, these results suggest that pigmentation is not important for developing antinociception.

Figure 5

Figure 5

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3.3. Pharmacological characterization of the antinociceptive effects of green light-emitting diodes

The mechanisms that might underlie the antinociceptive effects of GLEDs were investigated by determining possible contributions of endogenous pain transmitters and circuits as well as mediators of stress. Naive rats exposed to GLEDs for 8 hours daily for 5 days received antagonists immediately after light termination, and then PWLs were measured 20 to 30 minutes later. The GLED-induced antinociception was reversed, to baseline levels, after administration of the mu-opioid receptor (MOR) antagonist naloxone (20 mg/kg intraperitoneally [i.p.]). Twenty microgram, i.t., of naloxone also reversed the antinociception (Fig. 6A). These results suggest that GLED exposure likely elicits antinociception through release of endogenous opioids.

Figure 6

Figure 6

Neurons within the RVM are known to project to the spinal or medullary dorsal horns to directly or indirectly enhance or diminish nociceptive traffic.85 We examined the possible contribution of the RVM in the GLED-induced antinociceptive response by microinjection of 2% lidocaine. Rostral ventromedial medulla lidocaine reversed the development of antinociception when the rats were exposed to GLEDs (Fig. 6B).

Although our data, thus far, suggest a role for the endogenous opioid system, it is possible that the exposure conditions may result in stress-induced analgesia that could involve the sympathetic nervous system with engagement of alpha- and beta-adrenergic receptors by norepinephrine or epinephrine.5,10,45,51,74,78 Thus, to elucidate the role of the adrenergic system, several adrenergic receptors antagonists were tested including phenoxybenzamine (1 mg/kg i.p.), a nonselective irreversible alpha-blocker; phentolamine (3 mg/kg i.p.), a nonselective reversible alpha-blocker; and propranolol (10 mg/kg s.c.), a nonselective beta-blocker. All these drugs given immediately after light termination and tested 15 to 20 minutes afterwards failed to prevent or reverse the antinociceptive effect of GLEDs (Fig. 7A). Additionally, rats exposed to GLEDs or ambient light were observed to have normal ambulation and huddling, as well as maintained regular grooming behaviors (data not shown), which is in contrast with diminished grooming observed in stressed rats.38

Figure 7

Figure 7

To address the possibility of the antinociception being the result of anxiety due to overstimulation of the visual system, we performed EPM experiments. A cohort of rats exposed to GLEDs for 8 hours daily for 5 days developed antinociception as before (Fig. 7B), showed no signs of stress/anxiety as there were no differences in closed- or open-arm entries, or in the anxiety index (Fig. 7C, D); the index combines EPM parameters into one unified ratio with values ranging from 0 to 1, with a higher value indicating increased anxiety.34 In fact, rats exposed to GLEDs spent a significantly higher amount of time in the open arms than rats exposed to ambient room light (Fig. 7E), suggesting that GLED exposure was anxiolytic. Collectively, these results show that GLED-induced antinociception is not due to an anxiety-related response in these rats.

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3.4. Exposure to green light-emitting diodes does not impair motor performance

If, after exposure to GLEDs, the rats had a reduced motor activity, then this could contribute to the antinociception. To test this possibility, we investigated if the GLED exposure affected motor performance using the rotarod assay. After verification of antinociceptive behaviors induced by exposure to GLEDs for 8 hours daily for 5 days exhibited antinociception, we observed no change in the ability of the rats to stay on a rotating rod (Fig. 8). Thus, repeated GLED exposure does not affect motor performance or sedation.

Figure 8

Figure 8

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3.5. Proteomics reveals green light-emitting diode effects in the dorsal root ganglion and spinal dorsal horn

We used an unbiased proteomics approach to identify possible protein alterations, using liquid chromatography–MS/MS, in tissues from ambient vs GLED-exposed rats. Among 559 nonredundant proteins identified in our DRG samples (Lumbar levels 4, 5, and 6 were pooled), 65 proteins were detected only in DRG from GLED-treated rats (Fig. 9A). Gene ontology terms corresponding to the biological processes (Fig. 9B), molecular functions (Fig. 9C), and cellular components (Fig. 9D) of these proteins identified in DRG were extracted and compared between ambient and GLED-exposed rats. We noted a higher number of proteins associated with the “response to stimulus” category and a fewer proteins associated with “growth” category in DRG from GLED-exposed rats compared with DRG from ambient room light-exposed rats (Fig. 9B). The molecular functions associated with these proteins suggest a decreased “structural molecule activity” and an increased “antioxidant activity” in DRG from rats with GLED-induced thermal analgesia compared with controls (Fig. 9C). Finally, fewer proteins from GLED DRG appear to be localized in “organelle part” and “membrane” (Fig. 9D).

Figure 9

Figure 9

A total of 430 nonredundant proteins were identified in dorsal horn tissues, of which 43 were found only in the samples from rats with GLED-induced thermal analgesia (Fig. 9E). We observed a higher number of proteins associated with “metabolic process” and “cellular process” categories in the dorsal horn from GLED-exposed rats compared with the dorsal horn from ambient room light-exposed rats (Fig. 9F). The molecular functions associated with these proteins suggest a decreased “transporter activity” and an increased “binding” functions in the dorsal horn from rats with GLED-induced thermal analgesia compared with controls (Fig. 9G). Finally, more proteins were observed to be in “cytoskeleton” and “intracellular organelles,” whereas fewer proteins noted in “membrane,” “plasma membrane,” and “organelle membrane” in the dorsal horn from GLED-exposed rats compared with the dorsal horn from ambient room light-exposed rats (Fig. 9H). Thus, fewer proteins are localized in membranes in the dorsal horn, which is correlated, with fewer proteins implicated in localization processes in DRG.

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3.6. Analgesic effects of green light-emitting diode exposure in neuropathic pain

Having determined that GLEDs are antinociceptive in naive animals, we next asked if this nonpharmacological paradigm could be effective in reversing allodynia and hyperalgesia associated with the SNL model of experimental neuropathic pain. Probing the plantar surface of the hind paw ipsilateral to the side of nerve injury in SNL rats, 7 days postinjury, revealed thermal hyperalgesia (Fig. 10A) and tactile allodynia (Fig. 10B). Exposing SNL-injured rats for 8 hours daily to 4 lux GLED levels resulted in complete reversal of thermal hyperalgesia; the latencies were significantly higher than baseline so as to be antinociceptive (Fig. 10A). Paw withdrawal thresholds were also maximally reversed by a 3-day exposure to GLEDs (Fig. 10B). To address the duration of the GLED effect in SNL rats, we terminated GLED exposure and continued to measure antihyperalgesic and antiallodynic behaviors. After termination of GLEDs, it took 10 days for the thermal latencies (Fig. 10C) and 4 days for mechanical thresholds to return to postsurgical values (Fig. 10D).

Figure 10

Figure 10

Using quantitative RT-PCR, we determined expression levels of mRNAs for various endogenous opioids in spinal cords of naive rats as well as rats with SNL or sham in the presence of GLED exposure. Expression of the following genes was measured: (1) prepronociceptin (PNOC) coding for nocistatin, nociceptin, and orphanin17; (2) pro-opiomelanocortin (POMC) coding for N-terminal peptide of proopiomelanocortin (NPP), melanotropin gamma, corticotropin, melanotropin alpha, lipotropin beta, lipotropin gamma, melanotropin beta, beta-endorphin, and met-enkephalin11; (3) proenkephalin-A (PENK) coding for synenkephalin, met-enkephalin (4 copies), met-enkephalin-arg-gly-leu, met-enkephalin-arg-phe, and leu-enkephalin18; (4) proenkephalin-B (PDYN) coding for alpha-neoendorphin, beta-neoendorphin, and leu-enkephalin (3 copies), big dynorphin, dynorphin A (1-17), dynorphin A (1-13), dynorphin A (1-8), leumorphin, and rimorphin17; and (5) mu-type opioid receptor (OPRM1) coding for prepronociceptin and pro-opiomelanocortin.61 These analyses demonstrate that expression of PENK gene, but not others, is significantly increased after GLED exposure (Fig. 10E), suggesting that the increased enkephalin levels in the spinal cord of GLED-exposed rats could be one contributing factor for their analgesia.

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4. Discussion

We characterized the antinociceptive and antihyperalgesic effects of GLED phototherapy. In naive rats, the antinociceptive effect of GLEDs involves (1) visual system, (2) mu-opioid receptor pathways and descending pain inhibitory pathways from the RVM, (3) increased spinal cord expression of enkephalins, and (4) alterations in spinal cord and nociceptor proteomes. We were unable to link GLED-mediated antinociception to stress/anxiety. We demonstrate GLED phototherapy's ability to reverse reduced sensory thresholds in a model of neuropathic pain, supporting its use as a possible novel, nonpharmacological approach in managing chronic pain.

A key issue in interpreting photically induced antinociception is delineating the route of entry of light and its possible relationship with pain modulatory circuits. Here, whole-body illumination with GLEDs for 8 hours resulted in antinociception. By contrast, previous studies reported reversal of pain in mice after 30 to 150 seconds of infrared LED exposure directly touching the skin.15,16 The differences in exposure times (seconds vs hours) and “routes of administration” with either direct skin exposure or whole-body illumination could result in engagement of different mechanisms. Additionally, lower-intensity GLEDs was more antinociceptive. These results are consistent with a recent study,63 which reported that low-intensity green light reduced the intensity of migraines by ∼15% compared with lights of other colors and higher intensities.

Blocking GLED access to the eyes prevented antinociception, arguing for an important role by the visual system in the development of antinociception. Our data point to a role for the visual system in the antinociceptive and antiallodynic effects of GLEDs. However, exactly how this occurs is unknown. Electrical stimulation of the optic nerves in rats has been reported to increase blood pressure and heart rate, and to decrease baroreceptor-mediated vagal bradycardia. This effect was largely attenuated by inactivation of the periaqueductal grey (PAG), an anatomical region known to modulate pain,33,54 suggesting mechanistic neural links between the optic nerve and the PAG.13 Light-sensitive melanopsin-containing ganglion cells in the eyes69 project directly to the circadian rhythm controlling suprachiasmatic nucleus31,80 and PAG30; these structures are connected.41 Importantly, the suprachiasmatic nucleus has been linked with pain as inferred from disruption of the circadian rhythm of thermoregulation in models of chronic inflammatory pain in rats75 and increased c-fos levels in rats with cystitis pain.6 Finally, a retrograde labeling of the PAG demonstrated glutamate-like immunoreactivity in several regions of the brain including the occipital cortices,4 further supporting a link between the visual system and pain response. Martenson et al.52 have recently identified a possible circuitry for photic stimulation that included pain-modulating “ON-cells” and “OFF-cells” in the RVM which project to the dorsal horn of the spinal cord where they are postulated to modulate somatosensory processing. An imbalance of these may lead to enhanced or diminished pain with ON-cells facilitating nociception and OFF-cells inhibiting nociception. Our studies support a role for the RVM as its chemical inactivation prevented GLED-induced antinociception.

We demonstrated that GLED-induced antinociception involves opioid receptors. However, whether this occurs through a peripheral or central mechanism of action is at present unknown. Suppression of GLED-induced antinociception with the opioid receptor antagonist naloxone, administered systemically, may support a role of peripheral opioid receptors, which is consistent with previous reports demonstrating that administration of naloxone prevents infrared spectrum therapy–induced antinociception in models of postoperative16 or inflammatory pain.53 However, the ability of naloxone given i.t. to reverse the antinociception effect argues in favor of a central site of action for the GLEDs since this dose and volume would not be expected to have a significant effect if the site of action was in the periphery. It is conceivable that GLEDs may have both central and peripheral effects. Future studies will explore these possibilities.

Although overstimulation of the visual system could cause stress-induced antinociception, our data suggest that GLED-induced antinociception is not linked to stress/anxiety for several reasons. First, pharmacological antagonism of receptors implicated in stress did not affect antinociception. Second, behavioral studies did not link GLED-induced antinociception to anxiety in the EPM experiments. Finally, grooming behaviors of rats with GLED exposures were unaffected, thus implying no link to stress. Future studies will assess neurochemical correlates of stress (eg, glucocorticoids) in GLED antinociception.

Sensory neuronal adaptations, due to possible changes in activities of voltage-gated calcium and/or sodium channels, may help explain the molecular mechanisms underlying GLED-induced antinociception. N-type voltage-gated calcium channels (CaV2.2) are key for antinociception.73 The overall functional competence of neurons for calcium influx, as assessed by their ability to respond to agonists of various receptors using a high-throughput calcium imaging platform,56 was not different between sensory neurons from rats exposed to ambient light or GLEDs (Supplementary Fig. 1, available online at http://links.lww.com/PAIN/A358). However, we observed a decrease in depolarization-induced Ca2+ influx through CaV2.2 in sensory neurons from GLED-exposed rats (Supplementary Fig. 1E, http://links.lww.com/PAIN/A358). No changes were observed in sodium channels (Supplementary Fig. 2, available online at http://links.lww.com/PAIN/A358) ruling out their involvement in neuronal adaptations and GLED-induced analgesia. It is possible that the prolonged antinociceptive effect of GLEDs may, in part, be attributed to long-term changes in CaV2.2 or other calcium channel subtypes. Further studies will investigate the role of calcium and other channels in mediating the long-lasting effect of GLEDs.

Unbiased proteomics identified a protein signature in the dorsal root ganglia and dorsal horn of GLED-exposed antinociceptive rats. Stratification of proteins based on gene ontology terms corresponding to biological processes, molecular functions, and cellular components revealed an enrichment of proteins related to antioxidant activity in ganglia of GLED-exposed rats, a finding consistent with reported reduction in antioxidant activities (eg, catalase and superoxide dismutase) in rats with inflammatory pain.53 Additionally, low-level light therapy reduces levels of inflammatory mediators (eg, interleukins 6 and 8) associated with intervertebral disc degeneration, a key cause of low back pain.35 We noted a reduction in the number of membrane-localized proteins in the dorsal horn of GLED-exposed rats, which likely correlates with decreased signaling capabilities of neurons. Our findings uncovered proteins enriched in tissues from GLED-exposed rats that are reportedly linked to antinociception (Table 3). For example, purine nucleoside phosphorylase breaks down adenosine into inosine, which has antinociceptive properties through actions on the adenosine A1 receptor58 and through pertussis toxin–sensitive G protein–coupled receptors and the subsequent inactivation of calcium channels.50 Aspartyl aminopeptidase, which converts angiotensin II to angiotensin III, is reported to activate inhibitory pain-descending pathways from the PAG.66 Our findings of chemical inactivation of the RVM further support a role of inhibitory pain-descending pathways in GLED-mediated antinociception. Another example is lactoylglutathione lyase (ie, glyoxalase-1), which catalyzes the detoxification of methylglyoxal, a cytotoxic ketoaldehyde, which is directly linked to activating nociceptors.1 Moreover, expression of glyoxalase-1 is reduced in diabetic neuropathy.37 These lines of evidence, along with those listed in Table 3, build a picture consistent with global changes induced by GLEDs to produce antinociception.

Table 3

Table 3

We used a model of neuropathic pain to illustrate the possible use of GLEDs as a nonpharmacological treatment. Spinal nerve ligation is a commonly used model of neuropathic pain in rats.40 Common neuropathic pain medications reverse hypersensitivity associated with neuropathic conditions, but do not usually produce antinociception. For example, gabapentin blocks the N-type calcium channel48 to reverse tactile allodynia and thermal hyperalgesia in diabetic neuropathy; however, it failed to produce antinociception.70 Carbamazepine, an antiepileptic medication used for neuropathic pain, blocks sodium channels to reverse thermal hyperalgesia in tail-flick test in a model of cisplatin-induced hyperalgesia, but fails to produce antinociception.55 By contrast, GLEDs reversed tactile and thermal hypersensitivity associated with SNL and produced long-lasting antinociception. We observed that GLEDs decreased calcium influx through the N-type calcium channel (supplementary Fig. 1F, http://links.lww.com/PAIN/A358), which may account for both the antiallodynic and antihyperalgesic effects; however, the exact mechanisms contributing to the long-lasting nature of the effect is presently unknown. We also observed that GLEDs activate opioid receptors, which may account for antiallodynia, antihyperalgesia, and antinociception. The complete reversal of the effects of GLEDs using naloxone also argues for a key role for opioid receptors in producing antiallodynia, antihyperalgesia, and antinociception. It is conceivable that multiple mechanisms of actions may act synergistically to produce the effects seen with GLEDs. In contrast with the side effects observed with opioids, an advantage of the GLED approach is lack of side effects, such as development of tolerance, sedation, respiratory depression, and motor coordination. Equally important was the lack of hyperalgesia after prolonged GLED exposure. The lack of these side effects suggests that using GLEDs may be a safe treatment for neuropathic and other pain states, and may also complement the use of pain medications.

Our findings identify the cellular and molecular basis of GLED-mediated antinociception. From a translational perspective, the discovery that GLED exposure is antinociceptive and antiallodynic, opens up routes for the development of a noninvasive therapeutic approach to pain. Consequently, modulating the duration and intensity of GLEDs may prove useful, clinically, for reducing opioid in pain management. Additionally, the long-lasting and nontolerance-inducing effects of GLEDs may improve patient compliance.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

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Acknowledgements

The authors thank Dr Chris Atcherley for help with determining the absorbance properties of the fabricated lenses and Molly M. Ryan for assistance with analysis of proteomics data.

M. M. Ibrahim and A. Patwardhan are co-first authors. M. M. Ibrahim and R. Khanna contributed equally. F. Porreca and R. Khanna are co-senior authors.

This work was supported, in part, by a Career Development Award from the University of Arizona Health Sciences (to A. Patwardhan), and a Children's Tumor Foundation NF1 Synodos grant (to R. Khanna), and start-up seed funds (to M. M. Ibrahim and R. Khanna). A. Moutal was partially supported by a Young Investigator Award from the Children's Tumor Foundation.

Supplemental Digital Content associated with this article can be found online at http://links.lww.com/PAIN/A358.

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Supplemental Digital Content Supplemental media

Video content associated with this article can be found online at http://links.lww.com/PAIN/A385

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      Keywords:

      Green-light phototherapy; Constellation pharmacology; Calcium imaging; Proteomics; Neuropathic pain; Thermal antinociception; Mechanical allodynia

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