Red LED photobiomodulation reduces pain hypersensitivity and improves sensorimotor function following mild T10 hemicontusion spinal cord injury

All animal work was approved by the ANU Animal Experimentation Ethics Committee. Hemicontusion spinal cord injuries were performed on 7-week-old Wistar rats under isoflurane (1.7–2.3 % v/v) anaesthesia. Following hair removal, a laminectomy of T10 vertebral body and removal of dura and arachnoid was performed, followed by a spinal cord hemicontusion using a customized impactor system [38] comprising of a cylindrical 10 g weight with a 1-mm diameter tip that was guided onto the right dorsal horn and dropped from 25 to 50 mm above the spinal cord.

Injured animals were divided into 670-nm-treated (SCI+670) and sham-treated (SCI) groups. SCI+670 rats received 30 min of 670 nm irradiation commencing 2 h after surgery and then every 24 h after locomotor assessment for the remainder of the recovery period. A commercially available 670 nm LED array (WARP 75A, Quantum Devices, Barneveld, WI; 75 mm² treatment area) was used for treatment. Spectral characteristics and power output (Fig. 1) of the LED were measured using a spectrometer (CCS175, Thorlabs) and custom made power meter that was calibrated against a commercially available power meter (PM100D, ThorLabs). Treatment was delivered through a transparent treatment box which was used to confine the animal within its home cage. This resulted in a 7-mm distance between the dorsal surface of the animal and the LED array and delivery of 35 mW/cm² (fluence = 63 J/cm²) of 670 nm at the contact surface of the animal’s dorsum. SCI rats (n = 29) were restrained in the identical way as the SCI+670 group (n = 29), but without the LED device switched on to control for 30 min restraint in the transparent treatment box. Three additional control groups were included: an intact uninjured group (control; n = 7) was untreated and did not receive any sham operations or sham treatment; a sham-injured group (shamSCI; n = 8) underwent the spinal surgery, but without the contusion, and was subjected to sham treatment; a sham-injured 670-nm-treated group (shamSCI+670; n = 10) underwent spinal surgery without the contusion and received daily 30 min treatments.

Uninjured, unshaven animals (n = 6) were euthanised with sodium pentobarbital solution (325 mg/ml; Virbac; dosage, 100 mg/kg). The overlaying heart, great vessels and muscles were detached from the anchoring connective tissues and retracted to the side to expose the underlying vertebral column. The T10 vertebral body was eroded with a dental drill to expose the spinal cord from the ventral surface. The cadaver was placed on its back in an inverted transparent treatment box so that the dorsum of the cadaver could be positioned over the 670 nm LED array and the ventral surface of the rat was accessible to enable placement of a custom-built light measuring device. This device comprised of a photodiode chip (surface area, 0.62 mm²; maximal response (>95 %) to 630–685 nm; ODD-660W, Opto Diode Corp.) that was fixed to the bottom of an aluminium cylinder (height, 7.0 mm; external diameter, 8.7 mm). The top of the cylinder was sealed with a glass coverslip, and the entire probe was painted with black paint but leaving a small circular window (4.0 mm diameter) centred over the chip sensor. This left a ~2.4-mm lip between the external edge of the glass window and the external circumference of the cylinder. When pressed onto the ventral surface of the spinal cord, no light could penetrate from the side because the chip was located 7.0 mm behind the 4.0-mm aperture; thus, only light rays between 71° and 90° are able to reach the surface of the sensor; angles deviating from 90° do not hit the entire surface of the photosensitive diode and therefore contribute less to the total power reading. The signal from the probe was amplified by a custom amplifier built for purpose. The key component was the logarithmic converter amplifier (AD8304, Analog Devices). The readings were then calibrated against a commercially available light power meter tuned at 670 nm (PM100D ThorLabs) by producing a calibration table for different radiant power (controlled by distance from the light source) and subsequently converted into intensity (power/unit area). The probe was used to determine light intensity from the 670 nm array through (i) the treatment box, (ii) the spinal cord and dorsal overlying structures and (iii) the equivalent space through the air to provide a measure of attenuation over the distance of the light path. Prior to activating the LED, ambient lights were switched off; however, we also confirmed that no photons were detected by the light meter with the ambient lights on. Three repeat readings were acquired for each measurement.

Example images were obtained with a D1X Nikon (5.3 megapixels) camera and 120-mm lens (Medical NIKKOR) with a ×2 adaptor and built in ring flash. Images were captured with both the ambient lights and LED array on and then repeated in the same position with the ambient lights off.

A temperature probe (ML309/MLT422, ADInstruments) connected to a data acquisition system (PowerLab 26T, LabChart v7.3.7, ADInstruments) was attached to the dorsum of the animals prior to, and 2 min after sham or light treatment on consecutive days from four sham- and four light-treated rats.

Sensitivity assessment was carried out on day 7 post-injury prior to locomotor and electrophysiological assessments. To assess hypersensitivity, a nylon filament (OD: 1.22 mm) was used to deliver innocuous tactile stimuli over six defined regions over the animals’ dorsum: Above-Level (dermatomes C6-T3), At-Level (dermatomes T9-T12) and Below-Level (dermatomes L2-L5) on ipsi- and contralateral sides relative to the injury. The boundary for each of the six regions was marked on the animals’ back, and 10 consecutive innocuous “pokes” were delivered in each boundary at an inter-poke-interval of approximately 1–2 s, or until the animal recovered from movement evoked from the previous poke if longer than 2 s. Prior to testing, the operator practiced the stimulus procedure. This ensured that each poke was as brief as possible, that the filament landed normal (90°) to the skin surface and that the final position of the filament handle was approximately half the distance to that of the distance at initial contact of the filament. This protocol ensured pokes of consistent duration and maximum force which was confirmed using a weighing balance (maximum bending force: 2.86 ± 0.09 g; n = 10 pokes). During sensitivity testing, animals were “semi-restrained” in a V-shaped plastic box. This restricted the animal’s ability to avoid the testing procedure and thereby facilitated the operator’s accuracy of each poke but enabled sufficient movement for the animal to display behavioural responses of interest. Testing was recorded using a webcam (Logitec HD Pro C920). Videos were assessed blind to the observer in slow motion play back by evaluating the response to each innocuous poke that was graded into one of four categories as (I) no response; (II) mild response characterised by acknowledgment of the stimulus, head turns, brief shuddering of the contacted skin, but no obvious pain avoidance behaviours; (III) medium response, characterised by moderate signs of pain perception, including moderate avoidance attempts by moving away from the stimulus and (IV) severe response, characterised by severe signs of pain perception, including attacking the stimulus and “desperate” avoidance attempts and escape behaviours including jumping, running, writhing or audible vocalization. The four categories, I–IV, were chosen because these behaviours are easily distinguishable. The frequency of each response category was multiplied by a weight; categories I–IV were multiplied by 0, 1, √2 and 2, respectively, to provide greater separation between ordinal pain behaviours between non-painful and painful [39], as well as to help minimise heteroscedasticity of the data. The sum of the 10 weighted responses provided a regional sensitivity score (RSS) for each region. This paradigm enables high-resolution measures of sensitivity to 10 innocuous pokes with each possible RSS ranging between 0 and 20. Scores from ipsi- and contralateral regions were pooled to determine level sensitivity scores (LSS) above, at and below the level of injury. An cumulative sensitivity score (CSS) was derived for each animal by summing the RSS from all six regions; the maximum CSS possible is therefore 120. The hypersensitivity threshold was defined by the mean + 2 standard deviations (confidence interval of 95.5 %) of CSSs calculated from uninjured intact rats (control group).

Animals were anaesthetised with urethane (12.5 % w/v; 1.4 g/kg; i.p.) and maintained at 37 °C on a heating mat. A tracheotomy was performed, and animals were placed in a stereotaxic frame. The gracile nuclei were exposed through the foramen magnum by head flection and removal of overlying muscles and meninges. Both left and right sciatic and sural nerves were exposed by the removal of the overlying skin followed by a splitting incision of the gluteus maximum and semimembranosus muscles, respectively. The exposed nerves were isolated from adjacent connective tissues and bathed in paraffin oil. Silver wire bipolar hook electrodes were used to stimulate sural nerves, and a single hook silver wire electrode was used to record from sciatic nerves to ensure complete recruitment of all sural nerve fibres upon electrical stimulation (square wave pulse, 0.5–1.1 mA, 0.05 ms). A platinum wire electrode was used to record from a single midline position on the brainstem at a location that was established to provide evoked potentials of equal magnitude and latency from left and right sural nerve stimulation. Thirty-three individual evoked potentials were recorded and averaged from the sciatic nerve and the brainstem in response to repeated sural nerve stimulations. Signals recorded from the brainstem were then processed offline (MATLAB, MathWorks). The averaged signal was band-pass filtered (500–3350 Hz) and response magnitudes calculated from the integral of rectified signals (integral limits: 5.00 ms before and 8.75 ms after the primary peak) after subtraction from baseline levels obtained prior to the stimulus. Latency was measured from the filtered signal where it first exceeded 3 standard deviations (confidence interval 99.7 %) of background levels.

Prior to surgery, animals were trained to run along an 80-cm custom build transparent walking-track with mirrors that reflected left and right sides and underneath of the animal. This enabled exquisite locomotor detail from all sides of interest to be video captured simultaneously from a single viewpoint. 2 h following surgery, initial recordings of animals running three consecutive times down the walking-track were acquired with a digital camera (Sony, NEX-VG20EH) at 50 frames per second, which provided adequate data for detailed gait analysis. Recordings were repeated every 24 h post-surgery for 7 consecutive days. Each video file was coded and assessed blind by one assessor. The BBB locomotor scale [40] for the left and right hind-limbs was used to generate locomotor scores from video files assessed in slow motion.

Animals from both groups (SCI, n = 15; SCI+670, n = 15) were divided into three recovery time points and sacrificed at 1, 3 and 7 days post-injury. At the end of designated recovery periods, animals were transcardially perfused with saline and 4 % buffered paraformaldehyde (w/v). Harvested spinal cords were cryoprotected in 30 % sucrose (w/v), cryosectioned at 20 μm in the longitudinal plane using a Leica CM1850 cryostat, and dorsal sections labelled with primary antibodies (1:200) against rat CD68 (ED-1 clone, MAB1435, Millipore), and Arginase-1 (AB60176, Abcam) or CD80 (AB53003, Abcam) to quantify microglia/macrophages (ED1⁺) and polarized subtypes M1 (CD80⁺ED1⁺) and M2 (Arginase1⁺ED1⁺), respectively. Tissue was subsequently incubated with the appropriate secondary antibodies (1:1000, Invitrogen, Alexa 594 conjugated chicken anti-goat #A21468, Alexa 488 conjugated goat anti-mouse #A31619, Alexa 594 conjugated goat anti-mouse #A31623, Alexa 488 conjugated donkey anti-rabbit #A21206). Slides were then incubated in Hoechst solution (2 μg/ml Sigma-Aldrich). Standard immunohistochemical controls were included.

To detect cells undergoing apoptosis/necrosis, a TUNEL assay was performed. Slides were incubated with 1:10 Terminal Deoxynucleotidyl Transferase (TdT) buffer (125 mM Tris-HCl, 1 M sodium cacodylate, 1.25 mg/ml BSA, pH 6.6) for 10 min and then 1-h incubation at 37 °C with reaction mixture [0.5 enzyme unit/μl TdT (Roche Applied Science) and 2.52 μM Biotin-16-dUTP (Roche Applied Science) diluted in 1:10 TdT buffer]. This was followed by 15 min incubation in 1:10 saline sodium citrate (SSC) buffer (175.3 mg/ml sodium chloride, 88.2 mg/ml sodium citrate, pH 7.0) and blocked with 10 % normal goat serum in 0.1 M PBS for 10 min before incubating with secondary antibody in 0.1 M PBS (1:1000 dilution, Invitrogen, Alexa 488 conjugated streptavidin S11223) at 37 °C for 30 min.

All image analysis was performed blind to the experimental group. 2D images were constructed from three colour channel (red, green and blue) images acquired from a LED fluorescent microscope (Carl Zeiss Colibri) with a ×20 objective and digital camera (AxioCam MRc 5) with all settings kept constant for each channel. Cells with co-labelling were quantified with ImageJ (v1.46r) using the Cell Counter plugin that enables the placement of different classes of markers onto an image. Cytoplasmic markers, a class for each channel, were used to tag positive label in a single focal plane for all green and red channels that were examined independently. To define ED1⁺ cells, the accompanying DAPI⁺ nucleus (blue channel) was tagged for cells where ED1 staining was clearly complementing the DAPI surface profile. Double-labelled cells (i.e., ED1⁺Arginase1⁺ or CD80⁺) were evaluated by scrutinising all tagged DAPI⁺ cells for co-labelling in red and green channels. These cells were tagged again with another marker class. All markers were automatically quantified for each class by the software. Cells out of focus were not included. Cell counts were obtained from dorsal horn regions with viable tissue and quantified as the mean of duplicate images, each covering a minimum area 0.05 mm². The areas of interest were defined and quantified prior to cell quantification and included the dorsal horn grey matter region as well as the white matter in the surrounding dorsal columns and lateral funiculus. Cell quantification is expressed as the number of cells per unit area (mm²).

We first set out to demonstrate that red light can pass through superficial and deep structures underlying the dorsal exterior surface and penetrate the entire spinal cord (Fig. 1). The penetrating light could be seen with the naked eye (example, Fig. 1a, b). The dorsal surface of uninjured rats (n = 6) was exposed to the LED array and 670 nm light intensity measured at the light source surface through the transparent treatment box which directly contacts the rat dorsum during treatment (Fig. 1c, intensity at dorsal surface; 35.4 ± 0.05 mW/cm²) and the ventral surface of the spinal cord, where light had to pass through an additional ~10 mm of the animals’ tissues from dorsal surface (Fig. 1c, intensity at ventral surface; 3.2 ± 0.6 mW/cm²). These data show that 91.1 ± 1.8 % of the light from the LED array was absorbed/dispersed by the tissues between the dorsal surface of the animal and the ventral surface of the spinal cord (Fig. 1c, black arrow). To indicate the approximate attenuation over the distance of light travelling from the light source through to the ventral spinal cord surface, we measured the intensity at the approximate distance (10 mm) through the air (33.0 ± 0.5 mW/cm²). This demonstrated that the expected attenuation (~7 %) of light is negligible over the distance required to travel to the ventral surface of the cord.

We measured the surface temperature of rats directly before and 2 min after treatment. Twenty-seven readings from sham-treated and 25 readings from light-treated animals were acquired from four animals in each group over consecutive days of treatment. While there was no significant difference in the surface temperature of sham-treated animals (before, 33.6 ± 0.23 °C; after, 33.6 ± 0.25 °C), there was a small but significant increase 2 min after light treatment (before, 32.8 ± 0.36 °C; after, 33.5 ± 0.22 °C; p = 0.038, paired t test).

To examine the effect of red light on the development of neuropathic pain, we assessed sensitivity on six regions over the rat dorsum using a T10 hemicontusion spinal cord injury model that results in clear development of hypersensitivity in most animals within 7 days. The T10 spinal hemicontusion resulted in 63 % of animals (n = 12) developing hypersensitivity in both sham-treated (SCI, n = 19) and light-treated (SCI+670, n = 19) groups at 7 days post-injury. The hypersensitive subpopulation of rats from the SCI group had a mean CSS (SCI, CSS: 25.3 ± 4.5) that was 3.7 × the hypersensitive threshold (Fig. 2a). The mean CSS was significantly reduced by 40 % (SCI+670, CSS: 14.5 ± 1.6; 2.1 × the hypersensitivity threshold) in the hypersensitive subpopulation of rats from the SCI+670 group. Light treatment significantly reduced At- (T9-T12 dermatomes) and Below- (L2-L5 dermatomes) LSSs, which arose from contralateral At-Level and both ipsi-and contralateral Below-Level regions (Fig. 2b). Compared to the uninjured control group (control, Fig. 2c), sham injury without light treatment (shamSCI, n = 8) had no significant effect on LSS or RSS despite two sham-injured animals developing At-Level hypersensitivity. Light treatment of sham-injured animals (shamSCI+670, n = 10) resulted in significant reductions of At- and Below-LSS compared to the shamSCI group (Fig. 2c). Thus, while the incidence of hypersensitivity was not altered by red light, the level of hypersensitivity was markedly reduced At- and Below-levels in T10 contused light-treated allodynic animals. Red light also caused a significant reduction in sensitivity in 670-treated sham-injured animals (shamSCI+670, CSS: 0.8 ± 0.5) compared to uninjured control animals (control, CSS: 2.8 ± 0.8) as well as normosensitive spinal cord injured animals (SCI, CSS: 3.5 ± 0.9), even though these animals were not hypersensitive.

Could red light cause an anaesthetic-like effect on somatosensation that resulted in reduced sensitivity scores? To rule out the possibility that red light causes a reduced responsiveness to innocuous stimuli by bringing about a generalized inhibitory effect on somatic neural pathway conduction, we quantified the functional integrity of the sensory dorsal column pathway, at 7 days post-injury. The dorsal column pathways were activated by electrical stimulation of the left and right sural nerves, and a recording electrode was placed on the midline of the gracile nuclei (Fig. 3a). Stimulation of left and right nerves from control animals (n = 7) evoke responses of equal magnitude (Fig. 3b; right side: 101 ± 8 % of left side) and latency (Fig. 3c; left-right side latency difference: 0.09 ± 0.03 ms) on both sides when recorded from the same midline-positioned recording electrode, while sham-treated T10 hemicontusion spinal cord injury (n = 7) resulted in a 37 % reduction in magnitude (right side: 63 ± 16 % of left side) and a 0.48 ± 0.09 ms delay of the injured (right) pathway, when comparing the intact (left) side. Red light treatment (n = 7) rescued both the magnitude (Fig. 3b; right side: 93 ± 17 % of left side) and latency (Fig. 3c; left-right side latency difference: −0.05 ± 0.35 ms) deficits otherwise observed in the SCI group, indicating that red light treatment restored sensory pathway conduction, rather than impeding it. Furthermore, the rescued magnitude and latency deficits in the SCI+670 group indicates that their reduced sensitivity scores (Fig. 2) were unlikely to have resulted from a generalized reduction of somatic neural conduction.

We performed a variety of control experiments to validate our interpretations. There was no observable difference of conduction magnitudes or latencies in any of the sham-injured animals (shamSCI, n = 4; shamSCI+670, n = 4). There was no significant difference between gracile nuclei potentials evoked from the left sural nerve in any of the groups (SCI, 15.9 ± 1.8 μV · ms; SCI+670, 11.9 ± 2.4 μV · ms; control, 16.2 ± 3.6 μV · ms; shamSCI, 10.8 ± 2.6 μV · ms; shamSCI+670, 15.0 ± 2.8 μV · ms; p = 0.70, one-way ANOVA). Similarly, there was no significant difference of response latencies when evoked on the left side for all groups (SCI, 33.7 ± 0.3 μV · ms; SCI+670, 34.0 ± 0.4 μV · ms; control, 34.0 ± 0.4 μV · ms; shamSCI, 34.2 ± 0.5 μV · ms; shamSCI+670, 34.7 ± 0.3 μV · ms; p = 0.51, one-way ANOVA). These control experiments indicated that dorsal column pathway response magnitudes and latencies were similar between the different groups and largely unaffected contralateral to the injury.

Could red light treatment cause motor deficits and thereby result in reduced sensitivity scores? To rule out the possibility that the red light impeded the animals’ ability to perform escaping locomotor behaviours, daily locomotor recovery was examined blind to the experimental group (Fig. 4). We found that rather than impeding locomotion, the SCI+670 group (n = 11) demonstrated improved locomotor recovery as early as 2 days post-injury on the ipsilateral side and 3 days post-injury on the contralateral side compared to the sham-treated group (n = 10). Although a group effect of red light improvement was evident on the ipsilateral side (p = 0.026, linear mixed effects model with repeated measures), this failed to reach significance on the contralateral side (p = 0.055). There was a highly significant effect of time for both sides (p < 2e-16). Locomotor improvements observed in the SCI+670 group indicate that reduced sensitivity scores in light-treated animals (Fig. 2) could not have resulted from locomotor deficits.

To examine the effect of red light on cell death following injury, the number of TUNEL⁺ cells was quantified at 1, 3 and 7 days post-injury in dorsal regions of the T10 spinal cord (Fig. 5, n = 5 for each time point). The SCI group resulted in an increased density of TUNEL⁺ cells in the dorsal spinal cord ipsilateral to the injury as early as day 1 (contralateral 1.5 ± 1.5 cells/mm²; ipsilateral 96.8 ± 41.1 cells/mm²), reaching maximum levels by day 3 (contralateral 13.1 ± 5.6 cells/mm²; ipsilateral 126.8 ± 41.5 cells/mm²). The contralateral side had much fewer cells where maximum levels were reached by day 7 (Fig. 5; contralateral 32.5 ± 32.5 cells/mm²; ipsilateral 74.2 ± 43.7 cells/mm²). Red light treatment resulted in a significant group reduction of TUNEL⁺ cells in the ipsilateral side, notably significant at the day 3 time point when TUNEL⁺ cells were maximal in the sham-treated group (1 dpi: 49.6 ± 25.2 cells/mm²; 3 dpi 18.2 ± 3.9 cells/mm²; 7 dpi 22.0 ± 6.1 cells/mm²). There was no significant difference in TUNEL labelling on the contralateral side between groups (1 dpi: 2 ± 2 cells/mm²; 3 dpi 6.2 ± 2.1 cells/mm²; 7 dpi 5.0 ± 3.9 cells/mm²).

Inflammation has long being implicated in the development of neuropathic pain [27]. We therefore quantified activated microglia/macrophages (ED1⁺ cells) at 1, 3 and 7 days post-injury in dorsal regions of T10 spinal cord (Fig. 6a–d, n = 5 for each time point). T10 spinal contusion resulted in an increase in ED1⁺ cell density as early as day 1 post-injury, reaching maximum levels by day 3 in the ipsilateral side. Maximum levels were also reached at day 3 on the contralateral side, but there were negligible ED1⁺ cells at days 1 and 7. Light treatment significantly reduced ED1 expression ipsilateral to the injury to approximately half that of the SCI group. Despite the low levels of ED1⁺ cells in the contralateral side, red light treatment also resulted in a significant reduction of ED1⁺ cells at the 3-day time point.

Microglia/macrophages can adopt pro- or anti-inflammatory states [30]. To determine the effect of red light treatment on the expression of pro-inflammatory (M1) cells, cells co-expressing CD80 and ED1 were quantified as a proportion of total ED1⁺ cells (Fig. 6e–h, n = 5 for each time point). The proportion of CD80⁺ED1⁺ cells ipsilateral to the injury was maximal at day 1 and remained greater than 40 % of the ED1 population at days 3 and 7 in more than half of animals. CD80⁺ED1⁺ cells were only found at day 3 on the contralateral side which coincided with the maximum number of ED1⁺ cells at that time point. Red light treatment did not have a significant impact on the proportion of M1 cells on either the ipsi- or contralateral sides. Note that no CD80⁺ED1⁺ cells were encountered at days 1 and 7 contralateral to the injury as ED1⁺ cells were also in small quantities at these time points (Fig. 6a).

To determine the effect of red light treatment on the expression of anti-inflammatory/wound-healing (M2) microglia/macrophages, ED1⁺ cells co-expressing Arginase-1 were quantified as a proportion of total ED1⁺ cells (Fig. 6i–l, n = 5 for each time point). In the SCI group, Arginase-1 expression increased with time ipsilateral to the injury (p = 0.0048, one-way ANOVA) but peaked at day 3 contralateral to the injury at the time when most ED1⁺ cells were present in that region. Ipsilateral to the injury, the SCI+670 group displayed significantly increased proportions of Arginase1⁺ED1⁺ cells from day 1, reaching approximately sevenfold that of the SCI group. This greater Arginase1⁺ED1⁺ proportion in light-treated animals was maintained at over one third of ED1⁺ cells for the entire duration investigated for the majority of animals. No Arginase1⁺ED1⁺ cells were detected contralateral to the injury in the SCI+670 group; however, there were very few ED1⁺ cells in this region (Fig. 6a). The group effect failed to reach significance contralateral to the injury (p = 0.0628) despite a significantly greater level of Arginase1⁺ED1⁺ cells in the SCI group.