https://orcid.org/0000-0002-1898-1973
Abstract
This study aimed to investigate the effect of cannabidiol (CBD) on type 4 Toll-like receptors (TLR4), glial cells and pro-inflammatory cytokines during the neuropathic pain induced by the chemotherapy agent paclitaxel (PTX), as well as the involvement of the endocannabinoid system in this process.
Male C57BL6 mice were subjected to PTX-induced neuropathic pain. To evaluate the involvement of the TLR4, glial cells and cannabinoid CB2 receptor, specific inhibitors or antagonists were intrathecally administered. The western blotting and immunofluorescence assay was performed to evaluate the spinal expression of TLR4, microglia, astrocytes and cannabinoid CB2 receptor. The levels of spinal pro-inflammatory cytokines and endocannabinoids were determined by enzyme-linked immunosorbent assay and liquid chromatography-mass spectrometry analysis, respectively.
CBD prevented PTX-induced neuropathic pain, and the cannabinoid CB2 receptor antagonist AM630 reversed this effect. In addition, CBD treatment inhibited the spinal expression of TLR4 and Iba1 in mice with neuropathic pain. CBD also increased spinal levels of endocannabinoids anandamide and 2-arachidonoylglycerol, and reduced levels of cytokines in mice with neuropathic pain.
CBD was efficient in preventing PTX-induced neuropathic pain, and this effect may involve inhibition of the TLR4 on microglia spinal with activation of the endocannabinoid system.
Introduction
Worldwide, approximately 18–34% of people suffer from some form of chronic pain, of which 7–8% is neuropathic in origin.[1] Neuropathic pain is defined by the International Association for the Study of Pain as ‘pain caused by a lesion or disease of the somatosensory nervous system’.[2] Neuropathic pain can also be caused by several other factors, such as direct traumatic injuries, surgical interventions, neurodegenerative diseases or infections and chemotherapy.[2] In the case of chemotherapy-induced neuropathic pain (CINP), it has been shown that 30–60% of patients undergoing chemotherapy develop peripheral neuropathy and, consequently, pain.[1, 3]
Several classes of chemotherapeutic agents have been found to induce neuropathic pain, and their severity depends mainly on the cumulative dose administered over the course of treatment.[4] CINP – which may appear from the beginning of treatment and persist for years – is one of the main factors responsible for the interruption of chemotherapy.[5] This symptom as well as some sequelae are mainly due to chemotherapeutic agents acting through intracellular mechanisms, which interfere with nucleic acid synthesis or its function and subsequently affect the division of cells and their growth.[4] Among these chemotherapeutic agents, paclitaxel (PTX) – a compound derived from the bark of the Pacific yew tree (Taxus brevifolia) – is an antineoplastic agent that is routinely included in chemotherapy treatments, especially for patients with breast, ovarian, head, neck and lung cancer.[6] Although its effectiveness as an antineoplastic agent has been demonstrated, the development of neuropathic pain is one of the main side effects resulting from its use.[6]
Despite PTX-induced neuropathic pain being widely known, few mechanisms involved in this process have been thoroughly examined. One of them is the involvement of type 4 Toll-like receptors (TLR4).[7] Studies have shown the involvement of TLR4 in spinal glial cells in relation to the onset and continuation of neuropathic pain.[8, 9]
Using pharmacological agents to treat neuropathic pain remains ineffective for various individuals. This pain type is resistant to conventional analgesic regimens such as non-steroidal anti-inflammatory drugs.[10] Opioids have limited efficacy, and co-analgesics (antidepressant and anticonvulsant medications) are often the first-line treatment, but they lead to several side effects.[10]
Thus, new treatment strategies for neuropathic pain, including cannabidiol (CBD), have been investigated. CBD is the major non-psychotropic component extracted from Cannabis sativa, and studies have shown its efficiency in controlling neuropathic pain.[11, 12] Although CBD does not have a high affinity for cannabinoid receptors, some studies have shown that this cannabinoid may increase the availability of endocannabinoids, which have proven antinociceptive efficacy,[13] through inhibition of the metabolizing enzyme fatty acid amide hydrolase (FAAH).[14]
Thus, this study aimed to evaluate in an unprecedented way the effect that CBD on PTX-induced neuropathic pain as well as the spinal participation of TLR4 and of the endocannabinoid system in this process.
Methods
Animals
Male Swiss mice (8–10 weeks old at the beginning of the experiments) from our breeding colony at the Federal University of Alfenas (UNIFAL) in, Alfenas, Brazil, were used. Mice were housed individually in standard hanging cages, at 22 ± 1°C and 50 ± 5% relative humidity, using a reversed 12 h light/dark cycle, with lights on at 7:30 p.m. The animals were provided with standard rodent food and tap water ad libitum. All experiments were conducted between 9:00 a.m. and 5:00 p.m. This study was conducted with the approval of the UNIFAL’s Animal Use Ethics Committee at UNIFAL, Minas Gerais, Brazil (protocol number 676/2015, approved on 22 December 2015). Furthermore, all experiments were done in accordance with the International Association for the Study of Pain’s guidelines on the use of laboratory animals.[15]
In-vivo procedures were performed following the ARRIVE guidelines[16] attempting to minimize the number of animals employed and their discomfort.
Drugs
The following drugs were used: minocycline (a selective microglia inhibitor), fluorocitrate (FC) (an inhibitor of astrocytes), morphine sulfate, an opioid analgesic and paclitaxel (PTX), which were both purchased from Sigma-Aldrich (St Louis, MO, USA); Lipopolysaccharide Rhodobacter sphaeroides (LPS-RS), a TLR4 antagonist, purchased from Invivogen (Toulouse, France); 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl] (4-methoxyphenyl) methanone (AM630), which is a cannabinoid CB2 receptor antagonist, was purchased from Invivogen Tocris (Minneapolis, MN, USA) and cannabidiol was purchased from THC-Pharm-GmbH (Frankfurt, Germany). Minocycline, FC, AM630, LPS-RS were diluted in sterile saline. CBD was solubilized in ethanol, cremophor and sterile saline at a ratio of 1:1:18.
Injections
Intrathecal injection
Before intrathecal injections, the animals were lightly anaesthetized with isoflurane (2%) and O2 (2 l/min). Thereafter, the lumbar region was shaved and cleaned. The mice were positioned in ventral decubitus to facilitate palpation of the L5–L6 intervertebral spaces. Intrathecal injection of the mice was done in this intervertebral space using a 10 µl Hamilton syringe connected to a 28-gauge needle (Hamilton, USA). A flick of the tail was indicative of successful intrathecal administration. Lidocaine (2%, 0.5 μl) was administered to a group of animals using temporary paralysis of the hind limbs as an endpoint to confirm the effectiveness of the injection technique.[17]
Intraperitoneal injection
For intraperitoneal injections, the mice were restrained with their heads tilted lower than the body to avoid injury to internal organs or major blood vessels. After swabbing the lower right quadrant with alcohol, a 25-gauge needle was introduced slowly through the skin, subcutaneous tissue and abdominal wall. CBD or PTX was administered in a volume of 10 ml/kg of body mass.[18]
Model for chemotherapy-induced neuropathic pain
To induce neuropathic pain, mice were treated with PTX (Sigma-Aldrich, MO, USA) – solubilized with sterile saline – from the original stock concentration of 6 mg/ml (at a 1:1 ratio of cremophor EL to ethanol) to 1 mg/ml, which was administered intraperitoneally, at a dose of 2 mg/kg, every other day for a total of six injections – method adapted from Masocha.[19]
In the first experiments in which the effect of PTX on the nociceptive threshold was evaluated, as well as the effect of CBD on mechanical allodynia induced by PTX, the PTX was i.p. administered for 6 alternate days (days 2, 4, 6, 8, 10 and 12) to CBD (days 1, 3, 5, 7, 9 and 11). After verifying significant mechanical allodynia after the third day of treatment with PTX, for the next experiments that investigated the participation of cannabinoid CB2 receptors, TLR4, microglia and astrocytes, treatment with PTX was only on 3 alternate days.
Mechanical nociceptive threshold assessment
The von Frey filament test (Stoeling, Wood Dale, IL, USA) was used to measure the mechanical nociceptive threshold. For this test, the mice were placed on an elevated wire mesh platform in individual glass compartments, and they were allowed to acclimate for at least 30 min. A mechanical stimulus was applied to the middle of the plantar surface, using a series of von Frey filaments with increasing bending forces (0.07, 0.16, 0.4, 0.6, 1.0, 1.4 and 2.0 g), and the pressure value was recorded upon paw withdrawal.[20] The results reported for the test represent the mean values of three consecutive tests performed at intervals of 3 min.
Experimental protocol
To assess the effect of CBD on PTX-induced neuropathic pain, an experiment was performed, which involved firstly measuring baseline latency, and then administering PTX intraperitoneally on 6 alternate days (days 2, 4, 6, 8, 10 and 12) to CBD (days 1, 3, 5, 7, 9 and 11). Other measurements of the nociceptive threshold were performed 4, 7, 14, 21 and 28 days after baseline latency. The nociceptive threshold was measured on the fourth and seventh days before the administration of PTX and CBD, respectively (Figure 1). For this experiment, the following groups (n = 6) were used: CBD (5 mg/kg) + PTX group, consisting of animals that received alternate injections of CBD (5 mg/kg dose) and PTX; CBD (10 mg/kg) + PTX group, consisting of animals that received alternate injections of CBD (10 mg/kg dose) and PTX; CBD (10 mg/kg) + Veh group, consisting of animals that received alternate injections of CBD (10 mg/kg dose) and PTX vehicle; Veh + PTX group, consisting of animals that received alternate injections of CBD vehicle and PTX; Veh + Veh group, consisting of animals that received alternate injections of CBD vehicle and PTX vehicle and the MORP + PTX group, consisting of animals that received alternate injections of morphine at the 4 mg/kg dose (positive control) and PTX.
Experimental protocol for evaluation of the antinociceptive effect of cannabidiol (CBD), and investigation of the involvement of spinal microglia, astrocytes, TLR4 and cannabinoid CB2 receptors. The baseline latency (BL) of the nociceptive threshold of each mouse was firstly measured using the von Frey filament test, and paclitaxel (PTX) and/or CBD were subsequently injected on alternate days. During and after treatment with these substances, other nociceptive threshold measurements were performed on the 4th, 7th, 14th, 21st and 28th days (dotted lines). To evaluate the involvement of the spinal microglia, astrocytes, TLR4 and cannabinoid CB2 receptors; minocycline, fluorocitrate, LPS-RS and AM630 were administered intrathecally on the 7th day of neuropathic pain, and the nociceptive threshold was then measured 20, 60, 180, 300, 420 and 1.440 min after administration. LPS-RS, lipopolysaccharide Rhodobacter sphaeroides.
Due to identifying mechanical allodynia after the sixth alternate day of PTX administration (i.e. three injections), the neuropathic pain induction protocol was changed to only 3 days of administration of PTX alternating with CBD treatment, and the seventh day was chosen and used for the subsequent experiments (Figure 1).
For the experiments that investigated the involvement of cannabinoid CB2 receptors, microglia, astrocytes and the TLR4, the baseline latency of the nociceptive threshold was measured, and after the last PTX and/or CBD (10 mg/kg) treatment, the AM630, minocycline, FC and LPS-RS, respectively, were intrathecally administered, and new measurements of nociceptive threshold were taken at 20 min, and 1, 3, 5, 12 and 24 h after injection. The following groups were used (n = 6): AM630 (2 µg/5 µl) + CBD + PTX group, consisting of animals with neuropathic pain pretreated with AM630 followed by a 2 µg/5 µl dose of AM630 before the last CBD injection; AM630 (4 µg/5 µl) + CBD + PTX group, consisting of animals with neuropathic pain pretreated with AM630 followed by a 4 µg/5 µl dose of AM630 before the last CBD injection; AM630 (4 µg/5 µl) + PTX group, consisting of animals with neuropathic pain pretreated with AM630 followed by a 4 µg/5 µl dose of AM630 before the last PTX injection; LPS-RS (500 ng/5 µl) + PTX group, consisting of animals that received LPS-RS at a 500 ng dose before the last PTX injection; LPS-RS (1000 ng/5 µl) + PTX group, consisting of animals that received LPS-RS at a 1000 ng dose before the last PTX injection; Mino (500 ng/5 µl) + PTX group, consisting of animals that received minocycline at a 500 ng dose before the last PTX injection; Mino (1000 ng/5 µl) + PTX group, consisting of animals that received minocycline at a 1000 ng dose before the last PTX injection; FC (500 pmol/5 µl) + PTX group, consisting of animals that received FC at a 500 pmol dose before the last PTX injection and the FC (1000 pmol/5 µl) + PTX group, consisting of animals that received FC at a 1000 pmol dose before the last PTX injection. The groups were used to evaluate the vehicle of all these substances regarding the nociceptive threshold of mice with neuropathic pain. Groups similar to the groups described above were also used for molecular assays (n = 4–5 per group).
To perform the molecular experiments (western blot, liquid chromatography-mass spectrometry, immunofluorescence and enzyme-linked immunosorbent assay [ELISA]), the spinal cord tissues were collected on the seventh day of PTX and/or CBD treatments.
Western blotting
Western blot analysis was performed to evaluate the cannabinoid CB2 receptor, TRL4, Iba-1 and expression of the glial fibrillary acidic protein’s (GFAP’s) protein levels. Thus, after the seventh day of PTX-induced neuropathic pain, the animals from groups similar to those described above were killed by decapitation. Thereafter, the spinal cord samples (segments L4–L6) were carefully harvested, homogenized in radioimmunoprecipitation assay buffer with a cocktail of protease inhibitors (Sigma-Aldrich, MO, USA), and immediately snap-frozen in a bath of liquid nitrogen and stored at −80°C for further analysis. The protein concentration in the lysate was measured using the Bradford reagent. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%) and transferred onto polyvinylidene difluoride nylon membranes using a semi-dry electrophoretic system (Bio-Rad, CA, USA). The membranes were blocked with 5% bovine serum albumin in phosphate-buffered saline (PBS), for 2 h at room temperature, and then incubated overnight at 4°C in primary anti-cannabinoid CB2 receptor (1:500, Cayman, MI, USA), anti-Iba1 (1:500, Wako, Osaka, Japan), anti-GFAP (1:400, Boster Bio, CA, USA) and anti-TRL4 (1:400, Abcam, MA, USA) antibodies. The membranes were then incubated for a further 2 h at room temperature, with specific secondary antibodies, and after this incubation period, a detection kit (enhanced chemiluminescence detection kit, Bio-Rad, CA, USA) was applied for 3 min. Immunoblot analysis images were captured on a chemiluminescence imaging analyzer (Chemidoc, Bio-Rad, CA, USA), and band intensities were quantified using specific software (Image Lab, Bio-Rad, CA, USA). The intensity of each band was expressed relative to that of β-actin. Data are expressed as fold changes in band intensity normalized to the control.
Immunofluorescence
To the co-localize cannabinoid CB2 receptor and TLR4 expression in the dorsal horn of the spinal cord, staining immunofluorescence was performed in sections of the spinal cord (L4–L6 segments). After 7 days of neuropathic pain, the mice were anaesthetized with tribromoethanol and then perfused, intracardially, with saline followed by 4% paraformaldehyde in 0.1 M PBS. After the perfusion, spinal cord segments (L5–L6) were harvested and post-fixed for 2 h in 4% paraformaldehyde and then stored for 48 h in 30% sucrose solution in PBS for cryoprotection. Subsequently, using a cryostat, coronal sections (30 μm) of each sample were obtained, which were then washed and incubated in 0.1 M of glycine, and then washed again and incubated for 1 h in 5% bovine serum albumin (Sigma-Aldrich, MO, USA) and 0.1 M of PBS (pH 7.4) containing 0.4% Triton X-100. The coronal sections were then incubated in primary anti-cannabinoid CB2 receptor (1:100, Santa Cruz Biotechnology, CA, USA) and anti-TRL4 (1:400, Abcam, MA, USA) antibodies, after which the tissue sections were washed in PBS and then incubated with Alexa 488 goat anti-rabbit IgG (1:200; Santa Cruz Biotechnology, CA, USA) and Alexa 647 donkey anti-mouse IgG (1:200; Abcam, Cambridge, MA, USA) for 1 h. After these procedures, the sections were mounted using fluoromount (Sigma-Aldrich, MO, USA). The sections – which were analysed via epifluorescence, using a confocal microscope (Nikon, Tokyo, Japan) – were visually chosen using a 20× objective.
Liquid chromatography-mass spectrometry analysis of endocannabinoids
Spinal cords (segments L4–L6) from groups similar to those previously described were collected on the seventh day of neuropathic pain, and immediately snap-frozen in a bath of liquid nitrogen and stored at −80°C for further analysis. For liquid chromatography-mass spectrometry (LC-MS/MS) analysis, samples were spiked with 10 pmol of internal standard for anandamide (AEA)-d8 and 2-arachidonylglycerol (2-AGd8), followed by homogenization in H2O/MeOH, and then purification.[21] Following the extraction, the samples were dried, suspended in 100 μl of MeOH, and injected into the LC-MS/MS. Mobile phases were water and acetonitrile, both containing 0.1% formic acid. The gradient condition was: 0–1 min, 5% B; 5 min, 70% B and 9.5–11 min, 98% B. A flow rate of 0.5 ml/min and Ascentis Express C8 (150 × 2.1 mm; 2.7 µm) column were used. Mass spectrometry was operated in positive mode for high-resolution multiple reaction monitoring analysis. Results are expressed as pmol per mg of tissue.
Quantification of IL-1β and TNF-α levels in the spinal cord
Spinal cord samples (L4–L6 segments) of the mice were collected on the seventh day of neuropathic pain, to evaluate the IL-1β and necrosis factor-alpha (TNF-α) levels via enzyme linked-immunosorbent assay (ELISA). Right gastrocnemius muscles were also collected from the mice. The concentrations of IL-1β and TNF-α in the spinal cord, expressed in pg/mg, were determined using a commercially available IL-1β and TNF-α ELISA kit (R&D Systems, MN, USA). The samples were transferred to 1.5 ml tubes, placed on dry ice, resuspended in 1 ml of PBS per 200 mg of tissue, and then homogenized using a Polytron tissue homogenizer (Kinematica Inc., Switzerland) for 20 s. Thereafter, spinal cord homogenates were centrifuged for 10 min at 400 g and 4°C. Supernatants were transferred to 1.5 ml microtubes (Eppendorf, NY, USA), centrifuged at 15,000 rpm for 5 min, and then collected for cytokine analysis using the specific kits described above. The detection limit of the assays was 15.6 pg/µl for IL-1β and TNF-α.
Statistical analysis
The data are presented as the mean ± SEM of the evaluated parameter and were analysed for statistical significance as follows: by two-way analysis of variance (ANOVA) for repeated measurements, followed by Bonferroni’s post hoc test for multiple comparisons of nociceptive threshold measurements; and by one-way ANOVA followed by Bonferroni’s post hoc test for analysis of ELISA, western blot and liquid chromatography-mass spectrometry results. The minimum level of significance considered was P < 0.05. Statistical analyses and preparation of figures were done using version 5 of the GraphPad Prism software (GraphPad Software, La Jolla, CA).
Results
Antinociceptive effect of CBD on PTX-induced neuropathic pain
PTX-induced neuropathic pain has been an important model for pain induced by chemotherapeutic agents and enables investigation of both treatment strategies and action mechanisms involved in this symptom.[6] Our results indicated mechanical allodynia induced by PTX both during (fourth and seventh days, P < 0.001, F5.26 = 32.56) and after induction periods (14th, 21st and 28th days, P < 0.001, F5.26 = 32.56) (Figure 2a). This allodynia was significantly reduced (P < 0.001, F5.26 = 32.56) after CBD treatment with the 10 mg/kg dose (Figure 2a). Figure 2b shows that CBD-induced antinociception was longer than morphine-induced (positive control) antinociception (28 vs 7 days, P < 0.001, F5.26 = 32.56), in mice with neuropathic pain. Notably, CBD at the 5 mg/kg dose did not alter allodynia induced by PTX. Additionally, CBD at the 10 mg/kg dose did not alter the nociceptive threshold in mice without neuropathic pain.
Effect of cannabidiol (a) and morphine (b) on the paclitaxel (PTX)-induced neuropathic pain. Data are expressed as the mean ± SEM of six animals per group. ***P < 0.001 indicates statistical difference between PTX group and control group (Co); ##P < 0.01 and ###P < 0.001 indicate statistical difference between PTX group and group pretreated with cannabidiol (CBD, 10 mg/kg) plus PTX; ππP < 0.01 and πππP < 0.001 indicate statistical difference between the group pretreated with CBD (10 mg/kg) plus PTX and the group pretreated with morphine (MORP, 4 mg/kg) plus PTX. Two-way ANOVA followed by the Bonferroni test (factors: time and treatment). BL, baseline latency.
These datasets show that CBD treatments were efficient in preventing and reducing mechanical allodynia induced by PTX.
CBD promotes an antinociception effect, through the cannabinoid CB2 receptor mechanism, on PTX-induced neuropathic pain
After demonstrating the antinociceptive effect of CBD, we evaluated the spinal involvement of the cannabinoid CB2 receptor in this process. The cannabinoid CB2 receptor antagonist AM630, administered intrathecally at the dose of 4 µg, significantly reversed the CBD-induced antinociception on the seventh day of neuropathic pain induction, and this effect lasted for 5 h (P < 0.001, F5.41 = 24.18 for 20, 60 and 180 min; and P < 0.01, F5.41 = 24.18 for 300 min – see Figure 3a). The administration of vehicles does not change the allodynia. AM630 did not alter the nociceptive threshold of animals without neuropathic pain. Additionally, after seven alternate days of CBD and PTX treatment, there was a significant increase (P < 0.05, F4.15 = 5.876) in the expression of the cannabinoid CB2 receptor’s protein levels in the spinal cord, which was reversed (P < 0.05, F4.15 = 5.876) by AM630 (Figure 3b). These findings suggest spinal involvement of the cannabinoid CB2 receptors in the CBD-induced antinociception.
Evaluation of spinal involvement of the cannabinoid CB2 receptors and TLR4 in the antinociceptive effect of the cannabidiol (CBD) on the paclitaxel (PTX)-induced neuropathic pain. Data are expressed as the mean ± SEM of 4 (b and d) – 6 (a and c) animals per group. In (a and c), ***P < 0.001 indicates statistical difference between PTX group and control group (Co); ##P < 0.01 and ###P < 0.001 indicate statistical difference between PTX group and group pretreated with CBD (10 mg/kg) plus PTX; ππP < 0.01 and πππP < 0.001 indicate statistical difference between the group pretreated with PTX plus CBD (10 mg/kg) and the group pretreated with the cannabinoid CB2 receptor antagonist AM630 plus PTX and CBD (10 mg/kg). Two-way ANOVA followed by the Bonferroni test (factors: time and treatment). BL, baseline latency. In (b), *P < 0.05 indicates statistical difference between PTX plus CBD group and the control group (Co); #P < 0.001 indicates statistical difference between PTX plus CBD group and the group pretreated with AM630 and CBD (10 mg/kg) plus PTX. In D, ***P < 0.001 indicates statistical difference between PTX group and control group (Co); ###P < .001 indicates statistical difference between PTX plus CBD group and PTX group; or PTX plus LPS-RS and PTX group. One-way ANOVA followed by the Bonferroni test. LPS-RS, lipopolysaccharide Rhodobacter sphaeroides.
CBD modulates spinal TRL4 activation in PTX-induced neuropathic pain
Previous studies have shown the involvement of TLR4 in the genesis of neuropathic pain.[9] Thus, the next step of the study was to evaluate the effect of CBD on these receptors during the induction of neuropathy by PTX.
The initial pharmacological results of this study showed that a 1000 ng dose of the TLR4 antagonist LPS-RS reversed (P < 0.001, F5.25 = 35.16 for 20, 60, 180 and 300 min; and P < 0.01, F5.25 = 35.16 for 12 h) the PTX-induced neuropathic pain (Figure 3c). A similar effect was found when using a 500 ng dose of LPS-RS, which lasted 5 h (P < 0.001, F5.25 = 35.16). These results suggest the participation of spinal TLR4 in this pain model. LPS-RS did not alter the nociceptive threshold of animals without neuropathic pain.
Additionally, CBD treatment (P < 0.001, F4.15 = 18.44) prevented an increase in the expression of the TLR4’s protein levels in the spinal cords of mice with neuropathic pain (Figure 3d). Furthermore, a reduction (P < 0.001, F4.15 = 18.44) in the expression of the TLR4’s protein levels was verified by LPS-RS.
These results suggest a reduction in spinal TLR4 activation by CBD during the induction of neuropathic pain by PTX.
Spinal glial cells involved in PTX-induced neuropathic pain
Spinal TLR4 is expressed in glial cells, and once activated, it stimulates these cells to release cytokines and neurotransmitters, thus potentiating the nociceptive process.[8] Figure 4a and c show that intrathecal administration of the microglia activation inhibitor, minocycline (1000 ng dose), and the astrocyte inhibitor, FC (1000 pmol dose), significantly reduced (P < 0.001, F5.25 = 37.54) the mechanical allodynia produced by the PTX treatment, and this effect lasted for 12 h.
Evaluation of spinal involvement of the microglia and astrocytes in the paclitaxel (PTX)-induced neuropathic pain. Data are expressed as the mean ± SEM of 4 (b and d) – 6 (a and c) animals per group. In (a and c), ***P < 0.001 indicates statistical difference between PTX group and control group (Co); #P < 0.05 and ###P < 0.001 indicate statistical difference between PTX group and group pretreated with PTX plus minocycline (Mino, 1000 ng/5 µl), or PTX plus fluorocitrate (FC, 1000 pmol/5 µl) group. Two-way ANOVA followed by the Bonferroni test (factors: time and treatment). In (b and d), ***P < 0.001 indicates statistical difference between PTX group and control group (Co); #P < 0.05, ##P < 0.01 and ###P < 0.001 indicate statistical difference between PTX group and PTX group plus LPS-RS. One-way ANOVA followed by the Bonferroni test. BL, baseline latency.
Additionally, during the seventh day of the PTX-induced neuropathic pain, there was an increase (P < 0.001, F4.15 = 10.55) in the protein level expression of the microglia activation marker, Iba1, in the spinal cord, which was inhibited by a 1000 ng dose of minocycline and a 10 mg/kg dose of CBD (Figure 4b). The protein level expression of the astrocyte marker, GFAP, was not altered by PTX; however, the basal expression was reduced (P < 0.05, F4.15 = 3.862) by a 1000 pmol dose of FC (Figure 4b). CBD did not alter the expression of the GFAP’s protein levels in mice with and without neuropathic pain (P > 0.05).
Although spinal microglia and astrocytes may be involved in PTX-induced neuropathic pain, CBD did not influence this process.
CBD modulates spinal endocannabinoids and pro-inflammatory cytokines levels during PTX-induced neuropathic pain
As cannabinoid CB2 receptors have previously been shown to participate in the antinociceptive effect of CBD, we evaluated whether possible ligands for them, such as AEA and 2-AG, would be involved in this process.
On the seventh day of the neuropathic pain induction protocol involving the alternating of PTX with CBD, a significant increase (P < 0.001, F4.15 = 3.862) in spinal levels of AEA and 2-AG was observed (Figure 5a and b). Furthermore, the AEA increase induced by CBD mice with neuropathic pain was reversed by AM630 treatment (P < 0.001, F4.15 = 3.862). CBD also increased these endocannabinoid levels in mice without neuropathic pain (treated with saline). Minocycline, FC or LPS-RS did not alter AEA or 2-AG levels (Figure 5a and b).
Effect of cannabidiol (CBD) on the spinal endocannabinoids and pro-inflammatory cytokine levels during paclitaxel (PTX)-induced neuropathic pain. Data are expressed as a mean ± SEM of the spinal anandamide (AEA) (a), 2-AG (b), TNF-α (c) and IL1-β (d) levels on the seventh day of neuropathic pain. In (a and b), ***P < 0.001 indicates a statistical difference between control group (Co) and CBD group, or CBD plus PTX group; &&&P < 0.001 indicates a statistical difference between CBD plus PTX group and group pretreated with AM630 and CBD plus PTX group. In (c and d), ***P < 0.001 indicates a statistical difference between control group (Co) and PTX group; #P < 0.05 ##P < 0.01 and ###P < 0.001 indicate a statistical difference between PTX group and the following groups: CBD plus PTX, AM630 and CBD plus PTX, minocycline plus PTX, fluorocitrate plus PTX and LPS-RS plus PTX; &P < 0.05 indicates a statistical difference between the CBD plus PTX group and AM630 and CBD plus PTX group. One-way ANOVA followed by the Bonferroni test. Mino, minocycline; FC, fluorocitrate; AM, cannabinoid CB2 receptor antagonist AM630, LPS-RS, lipopolysaccharide Rhodobacter sphaeroides. n = 5 mice per group.
As mentioned earlier, spinal pro-inflammatory cytokines play an important role in potentiating the nociceptive impulse, mainly through the sensitization of second-order neurons.[8] In this context, we evaluated spinal levels of the pro-inflammatory cytokines TNF-α and IL-1β during PTX-induced neuropathic pain, as well as the effect of CBD on these levels. On the seventh day of neuropathic pain, there was a significant increase (P < 0.001, F4.32 = 7.360) in TNF-α and IL-1β levels (Figure 5c and d), which was prevented by pretreatment with CBD (P < 0.001, F4.32 = 7.360). Interestingly, the AM630 blocked (P < 0.05, F4.32 = 7.360) the inhibitory effect of CBD on TNF-α levels in the spinal cord the mice with neuropathic pain (Figure 5c). Minocycline (P < 0.001, F4.32 = 7.360), FC (P < 0.001, F4.32 = 7.360) and LPS-RS (P < 0.05, F4.32 = 7.360) also inhibited the increase of TNF-α and IL-1β levels in the spinal cords of mice with neuropathic pain (Figure 5c and d).
These results suggest that, through the involvement of cannabinoid CB2 receptors, CBD can modulate the release of endocannabinoid and pro-inflammatory cytokines during the induction of neuropathic pain by chemotherapy.
CBD reduces TRL4 expression in the spinal cord during neuropathic pain
As the previous results showed that TLR4 is involved in nociception induced by PTX and that cannabinoid CB2 receptors are involved in CBD-induced antinociception, we performed an immunofluorescence assay to co-localize and evaluate the expression of these receptors in the dorsal horn of the spinal cord. Figure 6 shows an increase in the expression of both cannabinoid CB2 receptors and TLR4 in the spinal cords of mice with neuropathic pain. In addition, this increased TLR4 expression was reduced by pretreatment with CBD.
Effect of cannabidiol (CBD) on the cannabinoid CB2 receptor and TLR4 expression in the spinal cord’s dorsal horn. Representative immunofluorescence and confocal micrographs of cannabinoid CB2 receptor and TLR4 in the spinal cord’s dorsal horn after 7 days of paclitaxel (PTX)-induced neuropathic pain. The sections were double immune-labelled against cannabinoid CB2 receptor (green) and TLR4 (red). Images at 20× magnification, scale bar: 100 μm. n = 4 mice per group. Co = control group.
Taken together, these results show that, during PTX-induced neuropathic pain, there is an increase in TLR4 expression, which can be modulated by CBD via cannabinoid CB2 receptors in the spinal cord.
Discussion
This study shows that treatment with CBD prevented the development of mechanical allodynia induced by PTX. This cannabinoid also seems to inhibit spinal TLR4 and microglia activation in mice with PTX-induced neuropathic pain. Additionally, the involvement of the endocannabinoid system in this process was verified.
Our pharmacological approaches also demonstrated that spinal microglia and astrocytes are involved in PTX-induced neuropathic pain. However, only an increase in microglia expression was found after PTX injections. Spinal microglia activation occurs during the early phase of neuropathic pain and precedes astrogliosis[22, 23]; thus, microglia and astrocytes are important for the beginning and maintenance, respectively, of neuropathic pain. As there is crosstalk between these cells, the basal maintenance of astrocyte action by microglia may respond to the lack of increased GFAP expression in the animals in this study that were pretreated with PTX.
Besides glial cell activation, some studies have shown the important role performed by TLR4 during PTX-induced neuropathic pain.[7, 24] A study conducted by Li et al.[7] indicated a significant increase in the expression of TLR4’s protein levels in rats, at 1, 3, 7 and 21 days after 4 alternate days of 2 mg/kg of PTX treatment. However, the authors did not find differences between the days of expression. Furthermore, a single dose of PTX (1 mg/kg) also resulted in increased spinal expression of TLR4.[23] Although these studies show the involvement of TLR4 in PTX-induced neuropathic pain, this study also evaluated the involvement of these receptors, but in a short neuropathic pain induction protocol, which was modified to three alternate injections of the chemotherapeutic agent, and was performed with mice. In addition, our pharmacological experiments using the TLR4 antagonist LPS-RS and molecular assays confirmed the involvement of this receptor.
In response to TLR4 activation, an intracellular transduction cascade is initiated, which culminates in the activation of nuclear factor-kappa B transcription factor and mitogen-activated protein kinases, resulting in the production and release of pro-inflammatory cytokines, including TNF-α, IL-1β and interleukin-6 (IL-6).[25] In this study, the spinal levels of TNF-α and IL-1β increased due to the PTX treatment, which was reversed by the TLR4 antagonist LPS-RS. This inhibition was also detected after minocycline and FC injection, thus showing the involvement of microglia, astrocytes and TLR4 in the release of pro-inflammatory cytokines. Once released, cytokines sensitize second-order neurons in the dorsal horn of the spinal cord, which potentiates the nociceptive impulse.[26]
In addition to confirming the involvement of glial cells and the TLR4 receptor in the proposed model of PTX-induced neuropathic pain, we also verified an inhibition of this mechanism by CBD, which led to antinociception. However, the mechanisms involved in CBD-induced antinociception, especially in neuropathic pain, are still poorly understood. A study conducted by Ward et al.[27] indicated the systemic involvement of 5TH1A receptors in the prevention of neuropathic pain produced by PTX after CBD treatment of female mice. Another study found a blocking of the antinociceptive effect of CBD in glycine receptor α3 subunit knockout mice with neuropathic pain induced by spinal nerve ligation, which is an important target for cannabinoids.[28] Adding to the little evidence in the literature, our study is the first to suggest the inhibition of spinal TLR4 by CBD in the pain model evaluated.
Several studies have shown inhibition of TLR4-induced signalling by CBD, but all of them have evaluated this effect in vitro, using different cell types. Fitzpatrick et al.[29] found a reduction in TLR4 activation and, consequently, of levels of the cytokine TNF-α in human Tamm–Horsfall protein-1 (THP-1)-derived macrophages. Similarly, IL-1β production by TLR4 activation in BV-2 microglial cells was blocked by CBD (5 and 10 µM dose).[30] In another study, CBD (3 µM dose) reduced TLR4-induced oxidative stress in mesenchymal stromal cells.[31] Although these studies did not directly assess TLR4, the CBD inhibited the action of lipopolysaccharide (LPS), which is a selective agonist for these receptors.
A target receptor that can be activated by CBD and inhibit TLR4 is the cannabinoid CB2 receptor. Our results showed that the cannabinoid CB2 receptor antagonist AM630 reduced antinociception and the expression of the cannabinoid CB2 receptors’ protein levels induced by CBD, in mice that received PTX. Moreover, AM630 prevented the increase in spinal AEA levels and blocked the reduction of the spinal pro-inflammatory cytokine TNF-α after CBD treatment of animals with neuropathic pain. Although CBD has a low affinity for cannabinoid CB2 receptors,[32] it has structural properties that may interact with the FAAH enzyme, which is the one most involved in inhibiting AEA metabolism. Additionally, a study determined an IC50 value of 1520 nM in the FAAH inhibition promoted by CBD in rats.[30]
As a result of this inhibition, there is greater availability of AEA and, consequently, greater activation of cannabinoid CB2 receptors. Despite AEA having a low binding affinity for CB2 cannabinoid receptors, its metabolite, 5,6-epoxide of anandamide (5,6-EET-EA), which is oxygenated by the cytochrome P450 enzyme, is a potent cannabinoid CB2 receptor agonist,[33] which may justify the activation of this receptor by the AEA.
Cannabinoid CB2 receptors are present in microglial cells,[34] and their activation can inhibit them during neuropathic pain.[35] A study using a specific cannabinoid CB2 receptor agonist (JWH-133) showed that this mechanism occurs via inhibition of spinal TLR4.[36] An earlier study had already shown the prevention of LPS-upregulated TLR4 expression in the dendritic cells of mouse bone marrow occurred through JWH-133 treatment.[37] JWH-133 also protected against mortality and decreased pro-inflammatory cytokine levels in mice with acute liver failure induced by LPS.[38] In-vivo and in-vitro studies have also shown that LPS upregulates cannabinoid CB2 receptor expression.[39, 40] Increased endocannabinoid levels were also found after LPS administration.[41] Our results with immunofluorescence also indicated an increase in cannabinoid CB2 receptor and TLR4 in the spinal cord’s dorsal horn, in animals with neuropathic pain. This evidence suggests that the endocannabinoid system may be activated against inflammatory and infectious processes to suppress the immune response.
Taken together, the antinociceptive effect of CBD on PTX-induced neuropathic pain may involve an indirect endocannabinoid system activation that will modulate the spinal TLR4 on microglia cells and, consequently, release pro-inflammatory cytokines.
Additionally, our results indicated that CBD-induced antinociception was superior and longer lasting than that of morphine, which is an opioid analgesic widely used to control cancer pain.[42] Morphine treatment for 6 alternate days probably promoted tolerance, which was not found with CBD, thus strengthening the hypothesis that CBD can be a treatment strategy during chemotherapy.
Conclusions
In conclusion, this study showed that CBD prevented PTX-induced neuropathic pain; therefore, it could be an important treatment strategy when chemotherapy is being used against cancer. Furthermore, we suggest that the CBD-induced antinociception may involve the inhibition of spinal TLR4 by endocannabinoid system activation; however, future studies will be necessary to better unravel this mechanism.
Authors Contribution
R.S. and G.G. conceived the study. F.V., L.E., G.N., C.S. and L.F. performed the experiments and analysed the data. R.S., F.V. and L.E. wrote the manuscript with support from G.G. that critically revised the work. All authors discussed the results and contributed to the final manuscript.
Funding
This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas (FAPEMIG); and by National Council for the Improvement of Higher Education [grant 001].
Conflict of Interest
The authors declare that there are no conflicts of interest.
Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.
