1. Introduction
Neuropathic pain arises because of the damage to either peripheral or central nerves; however, the mechanisms responsible for the resulting pain are unclear. As many as 10% of the general population suffer from neuropathic pain, with the majority being females.9,15 Emerging clinical evidence suggests that a subset of osteoarthritis (OA) and rheumatoid arthritis patients present with neuropathic-like pain as scored on the PainDetect scale,17,35 and these symptoms are due to joint nerve damage.14 Although there are many variables that contribute to the processing and perception of pain, an abundance of clinical and preclinical evidence indicates that sex is a vital factor.12,42 Females are more likely to suffer from a chronic pain condition such as arthritis37 as well as present with neuropathic pain symptoms.10 To date, the exact mechanisms underlying the sex differences seen in pain processing have yet to be elucidated. Moreover, it has been demonstrated that males and females respond differently to certain classes of analgesic medications,11 thus highlighting a need for sex-specific therapies.
The voltage-gated sodium channel, Nav1.8, has emerged as a promising target for analgesia because this ion channel is exclusively expressed on nociceptors and is critical for the neurotransmission of nociceptive information.10 Preclinically, an upregulation of Nav1.8 has been associated with nociceptor hyperexcitability and ectopic firing in a variety of chronic pain conditions including arthritis.10,19,32,39 Furthermore, in a model of end-stage OA, selective blockade of joint Nav1.8 ion channels using A-803467 reversed afferent hypersensitivity, improved hindlimb weight-bearing deficits, and hindpaw secondary allodynia.32
Recently, a link has been described between Nav1.8 and the lipid mediator lysophosphatidic acid (LPA),26 which has been identified as a crucial promoter of nerve demyelination and neuropathic pain.40 Peripheral administration of LPA can cause an increase in the expression of Nav1.8 and enhanced Nav1.8 currents in isolated dorsal root ganglia (DRG).26 With respect to arthritis, the concentration of LPA in the synovial fluid of OA patients was found to be correlated with disease severity.23 Lysophosphatidic acid has also been linked to inflammatory joint disease where synovial tissues harvested from rheumatoid arthritis patients exhibited a higher expression of LPA and LPA receptors.24 Intra-articular injection of LPA in rat knees is known to cause saphenous nerve demyelination, joint afferent sensitisation, weight-bearing deficits, and mechanical allodynia consistent with peripheral and central sensitisation.23 Blocking the activity of articular LPA1 and LPA3 receptors with the antagonist Ki16425 prevented LPA-induced demyelination and neuropathic-like pain.23
The aim of this study was to determine whether LPA induces sex-specific neuropathy and associated pain in rat knee joints. The role of Nav1.8 in mediating these responses will also be compared between the sexes.
2. Methods
2.1. Animals
Eighty-nine male and female Wistar rats (222-284 g and 179-231 g, respectively; Charles River, Quebec, Canada) were housed in ventilated racks at 22 ± 2°C on a 12-hour light:dark cycle. After arrival at the animal care facility, all rats were permitted at least 1 week to acclimate to their environment before entry into the study. Standard laboratory chow and water were provided ad libitum. All experimental protocols were approved by the Dalhousie University Committee on the Use of Laboratory Animals, which acts in accordance with ARRIVE and the standards put forth by the Canadian Council for Animal Care.
2.2. Lysophosphatidic acid–induced joint neuropathy
Animals were deeply anaesthetised (2%-4% isoflurane; 100% oxygen at 1 L/minute) until cessation of all sensory reflexes. The right (ipsilateral) knee joint was shaved, swabbed with 100% ethanol and 50 μL of LPA (50 μg in 5% ethanol/saline) was injected into the knee joint capsule through the patellar ligament (intra-articular; i.artic.). The knee was then manually extended and flexed for 30 seconds to disperse the solution throughout the joint.
2.3. Behavioural pain measurements
2.3.1. Von Frey hair algesiometry
Von Frey hair algesiometry was used as a measure of secondary allodynia. Alert, unanaesthetised animals were placed in a Plexiglas chamber with a metal mesh flooring, which allowed access to the plantar surface of the hindpaws. After allowing the animal to acclimate, until exploratory behaviour ceased (approximately 10 minutes), ipsilateral hindpaw mechanosensitivity was assessed using a modification of the Dixon up-down method.5 A von Frey hair was applied perpendicular to the plantar surface of the ipsilateral hindpaw (avoiding the toe pads) until the hair flexed; the filament was then held in place for 3 seconds. If there was a positive response (ie, withdrawal, shake, or lick of the hindpaw), the next lower strength hair was applied; if there was a lack of response, the next higher strength hair was applied up to a cutoff of 15-g bending force.
2.3.2. Exploratory activity
Activity was used to measure nonevoked or spontaneous pain. Alert, unanaesthetised animals were placed singly in a 30 × 30-cm transparent chamber with overhead camera (ImagingSource DFK22AUC03 camera; ImagingSource, Charlotte, NC) and allowed to move freely. The floor of the chamber was divided into quadrants. Exploratory activity of each animal was recorded over 3 minutes and analysed offline. Outcome measures recorded were (1) the time the animal spent rearing on their hindlimbs, and (2) the number of quadrant crosses completed in the 3-minute recording.
2.3.3. Experimental timeline
Forty animals underwent baseline von Frey hair algesiometry testing and exploratory activity analysis. Fourteen male and 14 female rats were then induced with LPA and the remaining animals (n = 6 male; n = 6 female) were used as naive controls. These cohorts were tested on days 7, 14, and 21 throughout the development of the LPA model.
Separate cohorts of male and female rats were treated on day 21 after LPA with a subcutaneous injection of either vehicle (100 μL; dimethyl sulfoxide[DMSO]:cremophor:saline; 1:1:18) or A-803467 (5 mg/100 μL) over the knee joint. Von Frey hair algesiometry assessments were conducted at 30, 60, 120 minutes after drug administration. Activity was not assessed during the 120-minute time course as animals become habituated to the testing chamber and do not move freely.
2.4. Electrophysiological recording of joint afferents
Single-unit joint afferent recordings were performed as previously described.30 Naïve (n = 21) and LPA-treated (n = 19) animals (21-24 days after LPA) were deeply anaesthetised using urethane (25% solution; 2 g/kg intraperitoneally). Core body temperature was measured through a rectally inserted thermometer and maintained at 37 ± 1°C by a thermostatically controlled heating blanket (CWE Inc., Ardmore, PA). After loss of the pedal withdrawal reflex, the trachea was cannulated to allow for artificial ventilation with a Harvard rodent respiratory pump (Harvard Apparatus, Holliston, MA) with 100% O2 (stroke volume: 2.5 mL; breath frequency: 52 breaths/minute). The jugular vein was cannulated for administration of the muscle relaxant gallamine triethiodide (50 mg/kg), which eliminated hindlimb neuromuscular activity, and in the LPA-treated animals, the distal saphenous artery was cannulated for close intra-arterial (i.a.) administration of A-803467 or vehicle to the knee joint. The right hind paw was placed in a shoe-like holder that was connected to a force transducer and torque meter (Data Track 244-1-R; Intertechnology, Toronto, Ontario, Canada) to standardise the amount of rotational force being applied to the knee joint. A longitudinal skin incision was made along the medial aspect of the hindlimb, and the reflected skin was sutured to a metal “O” ring to create a pool, which was filled with warm mineral oil to prevent tissue desiccation. The medial articular branch of the saphenous nerve was isolated and transected in the inguinal region to eliminate spinal reflexes. The epineurium was removed, and the nerve teased to isolate fine neurofilaments, which were then placed on a platinum recording electrode to measure single-unit activity. To identify a joint afferent fibre and its receptive field, the knee joint was gently probed with a blunt glass rod. The mechanosensitivity of the joint fibre was assessed by applying noxious outward rotations to the knee and counting the number of action potentials elicited during the rotation. Noxious rotation refers to torque occurring outside the normal working range of the knee, but not severe enough to cause soft-tissue injury. Three sets of noxious rotations, each lasting 5 seconds, were applied 5 minutes apart as a baseline measurement of afferent activity. The LPA-treated animals were given close i.a. infusion of A-803467 (500 µg) or vehicle (DMSO:cremophor:saline; 1:1:18), and joint mechanosensitivity was assessed for an additional 15 minutes. The percentage change in afferent activity before and after administration of A-803467 or vehicle was calculated off-line using Spike2 software (Cambridge Electronic Design, Cambridge, United Kingdom). The effect of A-803467 was not tested on afferent fibres from naive controls because no sensitisation was observed in these fibres.
2.5. Histology
2.5.1. G-ratio analysis of the saphenous nerve
A segment of the saphenous nerve was isolated proximal to the ipsilateral knee joint from naive, day 7 LPA, and day 21 LPA animals. It was then processed as described previously for electron microscopy.28 The saphenous nerve sections were inserted into a JEOL JEM 1230 transmission electron microscope (JEOL Corp, Ltd, Tokyo, Japan). One nerve cross-section was visually partitioned into 9 quadrants, and 3 images were captured (from quadrants 1, 5, and 9) at magnification of 2500×. All fibres were assessed using the G-ratio plugin in ImageJ processing software. Fibres were separated based on internal axonal diameter, where those with an internal diameter of <3 μm were considered small diameter, and those with an internal diameter of >3 μm were classified as large diameter fibres.2 The G-ratio was calculated using the equation ;)
where a is the internal axonal area, and A is the total axonal area of the fibre. The higher the G-ratio, the higher the degree of demyelination.
2.6. Immunohistochemistry
Knee joint afferents were labelled using the retrograde fluorescent dye, Fluoro-Gold. Male and female rats were deeply anaesthetised (2%-4% isoflurane; 100% oxygen at 1 L/minute) 5 days before histological endpoints for naive, day 7 LPA, and day 21 LPA cohorts (n = 3-4 per group). The ipsilateral knee was shaved and swabbed with 100% ethanol. Using a 30G needle attached to a Hamilton syringe, 10 µL of Fluoro-Gold (2% solution in saline; Fluorochrome, Denver, CO) was injected through the patellar ligament into the knee joint cavity. Ten microliter of a 0.2-M phosphate-buffered saline solution was subsequently injected, and the knee was flexed and extended for 30 seconds. Five days after Fluoro-Gold administration, anaesthetised animals were transcardially perfused with cold 1% heparinised saline followed by 4% paraformaldehyde. Dorsal root ganglia (DRGs) from L3 were removed and fixed in 4% paraformaldehyde overnight. After fixation, DRGs were placed in 20% sucrose followed by 40% sucrose for 48 hours each to dehydrate and cryopreserve the tissue. Dorsal root ganglia were then embedded in OCT (Sakura Finetek, Torrance, CA) and frozen at −20°C until sectioning. The DRGs were sectioned at 12 µm and mounted on Superfrost Plus slides (Fischer Scientific, Nepean, Ottawa, Canada) with each slide containing 5 consecutive sections from the DRG.
The expression of ATF3 and Nav1.8 channels by Fluoro-Gold–labelled neurones innervating the knee joint was assessed using commercially available antibodies. Dorsal root ganglion sections were treated with 10% normal goat serum for 1 hour to block nonspecific binding sites on the tissue. Slides were then incubated with rabbit-anti-ATF3 (1:500; Santa Cruz Biotechnology, Dallas, TX) or rabbit-anti-Nav1.8 (1:250; Abcam, Cambridge, MA) overnight at 4°C. Sections were washed 4 times with PBS after which they were incubated with Alexa fluor 488 goat-anti-rabbit (1:500 and 1:750, respectively; Invitrogen, Carlsbad, CA) for 1 hour in the dark. Slides were mounted with Fluoromount-G (Invitrogen) and viewed under a Ziess Axio Imager 2 (Zeiss, Oberkochen, Germany) at 80× magnification. Photomicrographs were taken using an AxioCam HRm camera (Zeiss) and analysed offline using Image J software. The total number of Fluoro-Gold–positive neurones was counted, and the percentage that expressed ATF3 or Nav1.8 was calculated.
2.7. Drugs
1-Oleoyl-LPA sodium salt (Abcam) was dissolved in a 5% ethanol/saline solution to a final concentration of 50 µg/50 µL. A-803467 (Tocris Bioscience, Abingdon, United Kingdom) was prepared in vehicle (1:1:18; DMSO:cremophor:saline) at a concentration of 5 mg/100 µL for behavioural experiments and 500 µg/100 µL for electrophysiological experiments. A-803467 solutions were used immediately after preparation. Urethane (Sigma Aldrich, St. Louis, MO) was dissolved in saline to produce a 25% solution.
2.8. Statistical analysis
All data were expressed as mean ± SEM. Data were tested for Gaussian distribution by the Kolmogorov–Smirnov test. Data from behavioural pain, electrophysiological, immunohistochemistical, and G-ratio experiments were normally distributed and were therefore analysed using parametric statistics (2-way analysis of variance [ANOVA], 1-way ANOVA, unpaired 2-tailed Student t test, χ2 test). A P value less than 0.05 was considered statistically significant.
3. Results
3.1. Sex differences in lysophosphatidic acid–induced peripheral nerve damage
Saphenous nerves were harvested from naive, day 7 LPA, and day 21 LPA animals for qualitative histological analysis (Fig. 1). Assessment of saphenous nerve electron micrographs, using G-ratio analysis, showed that the myelin thickness around both the small diameter (Fig. 1B) and large diameter (Fig. 1C) nerve fibres of LPA-treated female animals at day 7 and day 21 after induction were decreased when compared with naive controls (P < 0.0001; n = 365-385 small diameter fibres from 4 to 7 animals per group; 1-way ANOVA; P < 0.0001; n = 197-373 large diameter fibres from 4 to 7 animals per group; 1-way ANOVA). In male animals, myelin thickness around small diameter fibres was significantly decreased 7 days (P < 0.0001; n = 366-394 fibres from 4 to 7 animals per group) and 21 days after LPA administration (P < 0.0001; n = 394-450 fibres from 4 to 7 animals per group) when compared with naive controls. Myelin thickness around large diameter fibres was only found to be significantly decreased 21 days after LPA (P < 0.0001; n = 292-329 fibres from 4 to 7 animals per group) when compared with naive male controls. Furthermore, LPA-induced demyelination of small and large diameter fibres was found to be significantly greater in female animals on day 7 (P < 0.0001; n = 366-374 small diameter fibres from 4 to 7 animals per group; P < 0.0001; n = 138-197 large diameter fibres from 4 to 7 animals per group) and day 21 (P < 0.0001; n = 365-450 small diameter fibres from 4 to 7 animals per group; 1-way ANOVA; P < 0.0001; n = 322-329 large diameter fibres from 4 to 7 animals per group) when compared with male animals. Finally, there was no significant difference in the myelination states of small and large diameter fibres between naive male and naive female rats (P > 0.05; n = 385-394 small diameter fibres from 4 to 7 animals per group; P > 0.05; n = 292-373 large diameter fibres from 4 to 7 animals per group).
Figure 1.: Sex differences in LPA-induced demyelination of the saphenous nerve. Representative electron photomicrographs of saphenous nerve sections from naive and LPA-treated animals (day 7 and day 21) (A). G-ratios were calculated as a measure of myelin thickness and separated into small (B) and large (C) diameters based on internal axonal size. On day 7 and day 21, LPA caused demyelination of small diameter fibres in both sexes compared with naive controls (male; ****P < 0.0001; 1-way ANOVA; n = 366-450 fibres from 4 to 7 animals per group; female; ****P < 0.0001; 1-way ANOVA; n = 365-385 fibres from 4 to 7 animals per group). For large diameter fibres, LPA caused demyelination in females on day 7 (****P < 0.0001; 1-way ANOVA; n = 197-373 fibres from 4 to 7 animals per group) and day 21 (****P < 0.0001; 1-way ANOVA; n = 322-373 fibres from 4 to 7 animals per group) when compared with naive controls; however, LPA only caused significant demyelination in males on day 21 when compared with naive counterparts (****P < 0.0001; 1-way ANOVA; n = 292-329 fibres from 4 to 7 animals per group). When treated with LPA, female animals presented with a greater magnitude of demyelination in both small and large diameter fibres compared with males on day 7 (****P < 0.0001; 1-way ANOVA; n = 138-374 fibres from 4 to 7 animals per group) and on day 21 (****P < 0.0001; 1-way ANOVA; n = 322-450 fibres from 4 to 7 animals per group) of the model. Naïve G-ratio values did not significantly differ between sexes for small (P > 0.05; 1-way ANOVA; n = 385-394 fibres from 7 animals per group) and large (P > 0.05; 1-way ANOVA; n = 292-373 fibres from 7 animals per group) diameter fibres. Data are mean ± SEM. Thick arrows indicate large diameter fibres and thin arrows indicate small diameter fibres. ANOVA, analysis of variance; LPA, lysophosphatidic acid.
3.2. Characterisation of sex differences in lysophosphatidic acid–induced joint mechanosensitivity
All electrophysiological experiments were completed on days 21 to 24 after LPA induction and in weight-matched naive animals. All recorded fibres were mechanosensitive nociceptive Aδ- and C-fibres (characteristics summarised in Table 1). The magnitude of evoked firing, mechanical thresholds, and percentage of fibres that exhibited spontaneous firing were compared between naive and LPA animals. During baseline rotations of the knee joint, fibres from male and female naive animals fired on average 12 ± 2 and 13 ± 3 action potentials, respectively, over the 5-second movement. Lysophosphatidic acid–treated female animals fired on average 31 ± 4 action potentials, whereas fibres from LPA-treated male animals fired 27 ± 4 action potentials. Fibres from LPA-treated male and female animals fired significantly more than their naive counterparts (P < 0.001; n = 9-11 fibres per group; 2-way ANOVA; Table 1). Mechanical thresholds were significantly reduced in female LPA animals (P < 0.05; n = 9 fibres per group; 2-way ANOVA; Table 1) but not male LPA animals (P > 0.05; n = 10 fibres per group; 2-way ANOVA; Table 1). Spontaneous firing was observed in 50% of male LPA and 33% of female LPA animals, whereas none was observed in naive controls (P < 0.05; n = 9-11 fibres per group; chi-squared test; Table 1). There was no sex difference in any of the electrophysiological parameters measured in either naive or LPA-treated animals.
Table 1: Mechanosensitive characteristics of fibres recorded by single-unit electrophysiology.
3.3. Characterisation of sex differences in lysophosphatidic acid–induced neuropathic pain
Intra-articular injection of LPA produced secondary allodynia in the ipsilateral hindpaw throughout the 21-day development of the model in both males and females. Significant decreases in withdrawal threshold were seen in female LPA (P < 0.0001; n = 6-14; 2-way ANOVA; Fig. 1A) and male LPA (P < 0.01; n = 6-14; 2-way ANOVA; Fig. 1A) when compared with naive controls. When LPA-treated females and males were compared, the females had a more pronounced decrease in hindpaw withdrawal threshold over the 21 days (P < 0.001; n = 14; 2-way ANOVA; Fig. 2A). There were no significant differences in withdrawal threshold between naive male and female animals (P > 0.05; n = 6; 2-way ANOVA; Fig. 1A).
Figure 2.: Pain behaviour in the LPA model of joint neuropathy is sex-specific. A progressive decrease in hindpaw withdrawal threshold is exhibited in both sexes over 21 days after LPA administration compared with naive controls (male; P < 0.01; 2-way ANOVA; n = 6-14; female; P < 0.0001; 2-way ANOVA; n = 6-14) (A). The decrease in withdrawal threshold was more pronounced in females compared with males (P < 0.0001; 2-way ANOVA; n = 14). A significant decrease in time rearing in female animals (B) is observed over 21 days after LPA when compared with naive females (P < 0.0001; 2-way ANOVA; n = 6-14). There was no significant difference in time rearing throughout 21 days between male LPA-treated and male naive animals (C) (P > 0.05; 2-way ANOVA; n = 6-14). Both sexes exhibited decreases in quadrant crosses (D and E) when compared with naive control animals (male; P < 0.05; 2-way ANOVA; n = 6-14; female; P < 0.05; 2-way ANOVA; n = 6-14). Data are mean ± SEM. Pound symbols (#) indicate post hoc statistics between female naive and female LPA-treated animals (#P < 0.05). Asterisks (*) indicate post hoc statistics between male LPA- and female LPA-treated animals *P < 0.05, **P < 0.01). ANOVA, analysis of variance; LPA, lysophosphatidic acid.
Injection of LPA significantly decreased time rearing (P < 0.0001; n = 6-14; 2-way ANOVA; Fig. 2B) and quadrant crosses (P < 0.05; n = 6-14; 2-way ANOVA; Fig. 2D) in females when compared with their naive counterparts. Conversely, in male animals, LPA only had a significant effect on quadrant crosses when compared with naive animals (P < 0.05; n = 6-14; 2-way ANOVA; Fig. 2E). Male and female comparisons were not made for activity analyses because of their inherent differences in baseline activity state.
3.4. Expression of Nav1.8 and ATF3 in males and females
Immunohistochemical analysis of the expression of ATF3 in Fluoro-Gold–positive DRG neurones from naive, day 7 LPA-, and day 21 LPA-treated animals demonstrated that, after administration of LPA into the knee joint, ATF3 expression significantly increases in both sexes (Figs. 3A and B). In naive male and female DRGs, 28 ± 5% and 25 ± 5% of neurones from the joint express ATF3, respectively. An increase to 51 ± 7% 7 days after LPA treatment (P < 0.05; n = 3-4 animals per group, 2-way ANOVA; Fig. 3B) was observed in both sexes, and expression levels remained elevated (49 ± 5%-59 ± 1%) on day 21 after treatment (P < 0.05-0.01; n = 3-4 animals per group, 2-way ANOVA; Fig. 3B). No sex difference was observed in naive or LPA animals.
Figure 3.: Comparative expression of ATF3 and Nav1.8 in knee joint DRGs from males and females. Representative photomicrographs showing ATF3 (A) and Nav1.8 (C) expression in FG-positive cells. Neurones that innervate the joint expressed increased levels of nerve damage marker ATF3 at 7 and 21 days after LPA administration compared with naive animals (P < 0.05; 2-way ANOVA; n = 60-141 neurones from 3 to 4 animals per group). ATF3 levels were not significantly different between male and female animals (B) (P > 0.05; 2-way ANOVA; n = 60-141 neurones from 3 to 4 animals per group). The expression of Nav1.8 in these population of neurones also did not differ between experimental groups or between males and females (D) (P > 0.05; 2-way ANOVA; n = 44-140 neurones from 3 to 4 animals per group). White arrows indicate positive expression of ATF3 or Nav1.8 and yellow arrows indicate a lack of their expression. ANOVA, analysis of variance; ATF3, activating transcription factor-3; FG, Fluoro-Gold; LPA, lysophosphatidic acid.
The expression of Nav1.8 in Fluoro-Gold–positive DRG neurones was also assessed in naive, day 7 LPA-, and day 21 LPA-treated animals (Figs. 3C and D). The percentage of joint neurones that expressed Nav1.8 was between 60 ± 6% and 68 ± 5%. No changes in expression were observed between naive and LPA-treated animals or between sexes (P > 0.05; n = 3-4 animals per group, 2-way ANOVA; Fig. 3D). The number of Fluoro-Gold–positive neurones was similar between sexes.
3.5. Effect of local administration of A-803467 on lysophosphatidic acid–induced joint mechanosensitivity
Close i.a. administration of A-803467 significantly attenuated rotation-induced firing in both male (P < 0.0001; n = 9-10; 2-way ANOVA) and female (P < 0.0001; n = 6-8; 2-way ANOVA) joint afferents compared with vehicle (Fig. 4). In males, A-803467 decreased firing by 14% relative to baseline firing over the 15-minute period recorded (Fig. 5). A-803467 was more efficacious in females than males (P < 0.0001; n = 9-10; 1-way ANOVA), decreasing firing by 37% relative to baseline firing over the 15-minute period recorded (Fig. 5).
Figure 4.: A-803467 attenuates joint afferent firing in the LPA model of joint neuropathy in male and female animals. Example of single-unit recordings in vehicle (top panel)– and A-803467 (bottom panel)–treated knees in males (A) and females (C). A-803467 significantly decreased afferent firing relative to vehicle over the 15-minute period recorded in males (B) (P < 0.0001; 2-way ANOVA; n = 10 fibres in vehicle group and n = 9 fibres in A-803467 group) and females (D) (P < 0.0001; 2-way ANOVA; n = 6 fibres in vehicle group and n = 8 fibres in A-803467 group). *P < 0.05 post hoc analysis. Data are mean ± SEM. ANOVA, analysis of variance; LPA, lysophosphatidic acid.
Figure 5.: A-803467 administration attenuates joint afferent firing more profoundly in female than male animals. When the mean percent change in firing rate over 15 minutes was compared between groups, A-803467 significantly decreased firing in afferent fibres recorded from male (****P < 0.0001; 1-way ANOVA; n = 10 fibres in vehicle group and n = 9 fibres in A-803467 group) and female (****P < 0.0001; 1-way ANOVA; n = 6 fibres in vehicle group and n = 8 fibres in A-803467 group) animals compared with vehicle-treated knees. A-803467 caused a larger decrease in mechanonociception from female compared with male animals (****P < 0.0001; 1-way ANOVA; n = 6-10 fibres per group). Data are mean ± SEM. ANOVA, analysis of variance.
3.6. Effect of local administration of A-803467 on lysophosphatidic acid–induced neuropathic pain
On day 21 after LPA induction, baseline withdrawal thresholds differed between male and female rats, 12.66 ± 0.77 g and 10.58 ± 0.82 g, respectively. When the raw data for the male and female time courses were analysed independently, it showed that the effect of A-803467 was significant for both sexes (male: P < 0.05; n = 7; female: P < 0.01; n = 7; 1-way ANOVA; data not shown). However, because of the different baselines, to compare the potential sex difference in response to A-803467, the data were represented as percent change from day 21 LPA baseline. When using the percent change from day 21 LPA baseline data, the response to A-803467 was greater in females compared with males over the 120-minute time course tested (P < 0.05; n = 7; 2-way ANOVA; Fig. 6). The females reached a maximum of 42% increase after A-803467 treatment, with a maximum improvement in withdrawal threshold from 10.58 ± 0.82 g to 14.93 ± 0.07 g. The males reached a maximum of 8% increase, with an improvement in withdrawal threshold from 12.66 ± 0.77 g to 15.00 ± 0.00 g after drug administration. Although both sexes arrived at similar withdrawal thresholds after A-803467 treatment, a weakness of the study is that von Frey hair cutoff was set at 15 g, which limits the ability to test the maximum analgesic capacity of the drug. Vehicle control for A-803467 did not alter hindpaw withdrawal threshold in either sex throughout the time course (P > 0.05; n = 7; 1-way ANOVA; data not shown). The effect of A-803467 was not assessed in naive animals because we did not observe any indication that they were in pain and therefore would not benefit from treatment.
Figure 6.: Local administration of A-803467 attenuated hindpaw secondary allodynia more effectively in female animals. When animals were treated with a subcutaneous injection (over the knee joint) of A-803467 (5 mg) on day 21 of the LPA model, female animals had a larger increase in hindpaw withdrawal threshold than males (*P < 0.05; 2-way ANOVA; n = 7) over the 120-minute time course. Data are mean ± SEM. ANOVA, analysis of variance; LPA, lysophosphatidic acid.
4. Discussion
The epidemiology of painful arthropathies favours women over men with the prevalence of arthritis increasing with each 10 years of age.21,31 Similarly, the incidence of neuropathic pain in females is significantly greater than males3,12 confirming that there are sex differences in the occurrence and neurobiological processing of chronic pain conditions. Emerging evidence indicates that arthritis has a neuropathic component in about 30% of patients, and therapies used to treat neuropathic pain have been found to be effective in some arthritis patients.1,25,27 Taken together, this study aimed to assess sex differences in joint neuropathy and associated pain in LPA-treated rats.
On days 7 and 21 after administration, LPA caused a significant decrease in joint nerve myelin thickness in both male and female rats compared with naive controls, confirming the previously reported demyelinating properties of LPA.13,23 Both small and large diameter axons of the saphenous nerve were responsive to LPA-induced myelin loss suggesting that there may be an impairment in joint proprioception and neuropathic pain in this model. Demyelinated large diameter afferents may also change their phenotype and now act as nociceptors signalling pain in the joint.6 Ueda et al. has previously shown that LPA causes demyelination of sensory nerve fibres by causing downregulation of myelin proteins such as myelin basic protein, myelin-associated glycoprotein, and myelin 0 protein, which are all critical for maintaining myelin integrity.13 Activation of neuronal LPA1 receptors also leads to the stimulation of calpain, which is responsible for myelin protein degradation.43 Although it has previously been shown that there are sex differences in nerve regeneration in healthy rats,38 the data presented here show for the first time that LPA-induced neuropathy is sex-specific. Intra-articular injection of LPA in female rats resulted in a greater level of saphenous nerve demyelination than measured in male animals on day 7 and day 21. Sex hormones are a possible explanation for this disparity in the demyelinating effect of LPA. Testosterone and dihydrotestosterone have been shown to be necessary for maintaining peripheral nerve integrity.29 Metabolites of testosterone, including dihydrotestosterone, were neuroprotective in a model of diabetic neuropathy29 leading to an amelioration of mechanical allodynia and thermal hyperalgesia.4 It should be noted, however, that female sex hormones can also impart neuroprotection and prevent the development of experimental neuropathic pain.8,33,41 The influence of different sex hormones on peripheral neuropathy and neuropathic pain could be disease-specific and may involve other metabolic or inflammatory factors, which require further elucidation.
Immunohistochemistry corroborated a previous study, which reported that intra-articular injection of LPA produced nerve damage as evinced by expression of the neuronal damage marker ATF3.23 The current study extended this finding by showing that LPA-induced neuropathy was indeed occurring in the knee as ATF3 was coexpressed with Fluoro-Gold–labelled joint afferent neurones. Comparison of the sexes revealed no significant difference in ATF3 expression suggesting that, although demyelination was greater in females, the number of damaged fibres was not disparate between the sexes.
This study found that LPA produces secondary tactile allodynia as demonstrated by a reduction in von Frey hair withdrawal threshold. Prolonged or enhanced nociceptor activity causes central sensitisation, which is the neurophysiological origin of the secondary allodynia described here. In addition, this study shows that LPA causes a reduction in exploratory behaviours when compared with naive control animals. In a previous study, local injection of LPA into the knee joint has been shown to cause joint pain as demonstrated by a shift in hindlimb weight bearing away from the affected side.21 Together these findings suggest that LPA confers a peripheral spontaneous pain phenotype in these animals. This change in stance and locomotor activity is likely due to pain directly at the joint. This observation was confirmed here by electrophysiological recordings of joint afferents from LPA animals, which were sensitised compared with naive controls. In male animals, LPA caused a less severe pain response compared with females. Although ATF3 analysis revealed a similar level of expression between the sexes, the G-ratio data indicate enhanced demyelination in female animals. Although a similar number of joint neurones seem to be damaged due to LPA, the degree of demyelination is playing a larger role in driving the pain responses and heightened mechanosensitivity. The electrophysiology data presented here confirmed that joint afferent nerves were spontaneously active in LPA-treated animals, a common characteristic of neuropathy, but this phenomenon was not observed in naive animals. The existence of demyelination shown in this study was corroborated by previously published electrophysiological experiments, which revealed reduced joint afferent conduction velocity consistent with demyelination and neuropathic pain in this model.23 Lysophosphatidic acid–induced demyelination of peripheral nerves likely results in an enlargement of the nodes of Ranvier leading to greater sodium channel availability and consequently ectopic neuronal firing, as seen in other models of peripheral neuropathy.16,20
As discussed previously, sex hormones have been shown to contribute to changes in nerve integrity and pain processing. Thus, the numerous actions of hormones may explain the sex-specific pain response seen in our study. Most studies have focused on the role estradiol plays in pain modulation, leaving research on the effects of testosterone and progesterone relatively sparse in comparison. Estradiol can exert both antinociceptive and pronociceptive effects, whereas testosterone seems to be more antinociceptive and protective in painful conditions.7,34 These mixed results as well as the toxicity associated with prolonged exogenous hormone administration limits these studies and further complicates the true role of sex hormones in pain. In addition, recent findings have supported the notion that differing cell signaling pathways are responsible for neuropathic pain processing between males and females. Using the spared-nerve injury model in mice, it was found that while central sensitisation occurred in both sexes, the mediators of this response were different. In female mice, central plastic changes were T-cell dependent, whereas in male mice, microglia were responsible.22,36 These studies, taken together with the data shown here, merit further exploration into sex-specific mechanisms of demyelination and pain as well as the cellular and molecular signatures responsible for these differences.
Voltage-gated sodium channels present on nociceptors has emerged as an attractive target for the treatment of neuropathic pain. Systemic administration of nonselective sodium channel blockers such as lidocaine produces a potent long-lasting analgesia in neuropathic pain patients.44 The limitations of this approach, however, include complete loss of sensation, cardiotoxicity, sedation, and cognitive impairment.18 The development of specific NaV channel blockers, which only act on nociceptive nerves could circumvent these unwanted side effects and allow for a safer more targeted analgesia. The Nav1.8 channel blocker A-803467 has been shown to reduce joint mechanosensitivity and pain in a rat model of OA.32 In corroboration, this study demonstrated that local administration of A-803467 successfully reduced joint afferent hypersensitivity and inhibited tactile secondary allodynia in the LPA model of joint neuropathy. Interestingly, the antinociceptive effect of A-803467 was greater in female rats despite there not being any difference in Nav1.8 expression levels between the sexes. Together these data suggest that the gating properties of joint Nav1.8 ion channels are different in females compared with males, although future experiments are required to test this hypothesis. Pan et al. showed that hindpaw injection of LPA in female rats caused an upregulation of Nav1.8 ion channels in ipsilateral L4 to L6 DRG neurones in conjunction with the development of mechanical allodynia. Furthermore, they showed that LPAr1 and Nav1.8 are coexpressed, and that LPA potentiated Nav1.8 currents measured in DRG neurones.26 Thus, LPA could be a promoter of Nav1.8 channel opening and sensitivity in a sex-specific manner. An alternative explanation could be related to the greater extent of demyelination in female joint nerves exposing a larger number of Nav1.8 ion channels at nodes of Ranvier along the length of the saphenous nerve. This in conjunction with a possible higher overall affinity to A-803467 could account for the greater antinociceptive effect of A-803467 observed in female knee joints.
Treating pain in a multifactorial disease such as arthritis is complicated by the heterogeneity of the patient population. The mixed phenotype of arthritis pain consisting of nociceptive, neuropathic, and inflammatory components requires the use of multiple analgesics that target various aspects of an individual's pain signature. Sex differences are known to exist in pain processing, yet analgesics are prescribed to males and females without this consideration in mind. The data presented here demonstrate that joint neuropathy and neuropathic pain in response to LPA treatment was greater in females. The percent reduction in joint mechanonociception and secondary allodynia with A-803467 was more effective in females indicating a possible sex-specific sensitivity to Nav1.8 ion channel blockade. In conclusion, targeting the Nav1.8 ion channel could be beneficial for the treatment of arthritic neuropathic pain, especially in women.
Conflict of interest statement
The authors have no conflict of interest to declare.
All experimental protocols were approved by the Dalhousie University Committee on Laboratory Animals, which acts in accordance with the standards put forth by the Canadian Council for Animal Care.
The data sets used and/or analysed during the current study available from the corresponding author on reasonable request.
Acknowledgements
The technical assistance of Mary-Ann Trevors and Stephen Whitefield in the CORES facility at Dalhousie University is gratefully acknowledged.
This work was funded by an Arthritis Society of Canada strategic operating Grant (SOG-17-0108) and a Dalhousie University Department of Anaesthesia Research Grant. M.S. O'Brien is the recipient of an Arthritis Society of Canada studentship.
Author contributions: All authors conceived the study and participated in its design. H.T.A. Philpott conducted the pain behaviour experiments, performed the G-ratio measurements, analysed data, and helped draft the manuscript. M.S. O'Brien conducted electrophysiology experiments, performed the immunohistochemical experiments, analysed data, and helped draft the manuscript. J.J. McDougall coordinated the experiments, helped analyse data, and helped draft the manuscript. All authors read and approved the final manuscript.
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