Sex-related correspondence between mechanical hypersensitivity and the discharge of medullary pain control neurons in neuropathic rats

Here we studied whether the sex-related difference in mechanical hypersensitivity induced by neuropathy is associated with the discharge rate of medullary pain control neurons. We performed experiments in male and female rats with spared nerve injury (SNI) model of peripheral neuropathy. Mechanical hypersensitivity was assessed behaviorally by monofilaments. Discharge rates of pain-control neurons were determined using in vivo single unit recordings under light anesthesia. Recording targets were two medullary nuclei involved in descending pain control: the rostral ventromedial medulla (RVM) and the medullary dorsal reticular nucleus (DRt). Based on the response to peripheral noxious stimulus, neurons were classified as pronociceptive RVM ON-like or DRt neurons, or antinociceptive RVM OFF-like neurons. Behavioral results indicated that the mechanical hypersensitivity induced by SNI was significantly stronger in females than males. The ongoing discharge rates of pronociceptive RVM ON-like neurons were higher and those of antinociceptive RVM OFF-like neurons lower in SNI females than SNI males. Ongoing discharge rates of pronociceptive DRt neurons were not significantly different between SNI females and males. The results suggest that a sex difference in the discharge rate of pain control neurons in the RVM but not DRt may contribute to the maintenance of stronger neuropathic hyper-sensitivity in females.


Introduction
Injuries of peripheral nerves often cause neuropathic pain that is typically associated with mechanical hypersensitivity [19]. Nerve injuries cause multiple changes in pain processing and modulating mechanisms that contribute to neuropathic pain [3]. Among these mechanisms are plastic changes in various brainstem nuclei involved in descending control of pain such as the rostral ventromedial medulla (RVM) [14], dorsal reticular nucleus of the medulla (DRt) [22], and a number of other nuclei involved in pain control [33]. Concerning the RVM, earlier findings indicate that it plays a role in neuropathic pain as indicated by the reduction of mechanical hypersensitivity following local anesthesia of the RVM in nerve-injured male animals [34]. Moreover, neurophysiological recordings in male rats have shown pronociceptive changes in the response characteristics of RVM neurons following peripheral nerve injury [15]. DRt is another medullary pain control nucleus that has predominantly a pronociceptive role [22], although it may also play a role in the conditioning noxious stimulationinduced remote inhibition of concurrent nociception [5]. Pronociceptive role of the DRt has been shown also in neuropathic male animals [24,26,37].
In the clinic, chronic neuropathic pain is more prevalent in females than males [6,30]. In line with this, there is experimental evidence indicating that peripheral nerve injuries in the rat induce stronger mechanical hypersensitivity in females than males [1,7,9,40]. Sexual dimorphism in descending controls provides one explanation for sex differences in pain. Namely, earlier studies have shown sex-related differences in descending control of pain under both non-neuropathic [21,23,27] and neuropathic conditions [40]. However, it is not yet known whether the discharge properties of neurons in medullary pain control nuclei that provide the final common pathway for descending pathways vary with sex and correspond to the sex difference of pain hypersensitivity under neuropathic conditions.
Here we propose the hypothesis that the stronger neuropathic pain Abbreviations: DRt, dorsal reticular nucleus of the medulla; RVM, rostral ventromedial medulla; SNI, spared nerve injury. behavior in females than males is associated with sex difference in the discharge rates of medullary neurons involved in descending pain control. We tested this hypothesis in male and female rats with spared nerve injury (SNI) of the sciatic nerve by assessing pain behaviors and neuronal discharge rates in two medullary pain control nuclei, the RVM and the DRt.

Animals
Experimental animals were adult, male and female Hannover-Wistar rats (weight: 180-230 g; Envigo, Horst, The Netherlands). The Ethical Committee on Animal Experiments of the regional government of Southern Finland approved the study protocol (permission # ESAVI-41116-2019). The experiments were performed according to the guidelines of European Communities Council Directive 2010/63/EU on the use of animals for scientific purposes. All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. The animals were housed in polycarbonate cages with a deep layer of sawdust. The temperature in the thermostatically controlled animal room was 24.0 ± 0.5 • C. The room was artificially illuminated from 8.30 a.m. to 8.30p.m. The home cages were environmentally enriched. The animals received commercial pelleted rat feed and tap water ad libitum.

Techniques for producing neuropathy
The spared nerve injury (SNI) model developed by Decosterd and Woolf [11] was used to induce peripheral neuropathy. Prior to surgery, the animal was anesthetized with sodium pentobarbital (60 mg/kg i.p. followed by 15-20 mg/kg more as needed; OrionPharma, Espoo, Finland). In the SNI operation, the common peroneal and tibial nerves were ligated with 4-0 silk, and sectioned distal to the ligation, after which the muscle and skin were sutured. For postoperative pain treatment, animals received buprenorphine for three days (0.01 mg/kg twice daily).

Assessment of mechanical sensitivity
The focus of the present behavioral assessments was on mechanical sensitivity, since in the clinic nerve injury most commonly produces tactile allodynia-like mechanical hypersensitivity condition [19]. Before behavioral testing, the rats were habituated to the experimental conditions 1-2 h daily during 2-3 days. Mechanical hypersensitivity was assessed with monofilaments while the rat was standing on a metal grid. The sural nerve area (lateral area of the foot) ipsilateral to the SNI was stimulated five times with an ascending series of calibrated monofilaments (1-10 g; North Coast Medical, Inc., Morgan Hill, CA, USA). The withdrawal threshold in grams was defined from psychometric function curves as the stimulus intensity evoking a 50% response rate to repetitive stimulation.

In vivo electrophysiological recording of pain-control neurons in the RVM and DRt
Single medullary neurons were recorded extracellularly using methods described in detail earlier [35]. Neural signals recorded with lacquer-coated tungsten microelectrodes (tip impedance 5-10 MΩ) were amplified using a custom-made amplifier, filtered, digitized (Micro 1401, Cambridge Electronic Design, Cambridge, UK), stored using a standard computer, and thereafter, analyzed offline (Spike2 software, Cambridge Electronic Design) that allowed evaluating separately discharge of more than one neuron in single recording session [35].
After induction of anesthesia (sodium pentobarbital 60 mg/kg i.p.) and placement of the animal in the stereotaxic frame, a hole was drilled in the skull for placement of the recording electrode in the RVM (AP − 2.3 mm; ML 0 mm; DV 10.0-10.5 mm from the dura mater) or the DRt (AP − 5.1 mm; ML 1.4 mm; DV 8.6 mm ventrally from the dura mater) [32]. Before starting recordings, the deep level of anesthesia needed for surgical procedures was allowed to wear off to the level where the animal did not have any spontaneous limb movements but noxious stimulation caused a brief flexion reflex without other behavioral responses. This level of anesthesia was kept by administering sodium pentobarbital 15-20 mg/kg/h. RVM neurons and DRt neurons responding to noxious stimulation typically have large receptive fields covering most of the body [14,39]. Neurons were classified based on the response to tail pinch at an approximate force of 350 g [15]. RVM neurons that increased discharge rate by ≥ 20 % when exposed to pinch were classified as pronociceptive RVM ON-like cells ( Fig. 1 A), whereas RVM neurons decreasing discharge rate by ≥ 20 % when exposed to pinch were classified as antinociceptive OFF-like cells (Fig. 1 B). We did not verify whether pinch-evoked responses of RVM neurons were associated with spinal reflex responses as in the original classification scheme [14]. Therefore, we use the terms RVM ON-like and OFF-like cell. Other types of RVM cells were not included in this study. When searching for DRt neurons, those responding only to noxious stimulus intensities at wide areas of the body [39] and increasing activity by > 20 % when exposed to tail pinch were considered (Fig. 1 C). After identifying an RVM ON-like or OFF-like cell or a DRt cell, its spontaneous activity was determined for 30 s.

Course of the study
Time course of the study is illustrated in Fig. 2. Briefly, after habituation the pre-SNI withdrawal threshold was determined. Next, SNI operation was performed under general anesthesia, after which the animals were allowed to recover for one week. Next, the withdrawal threshold was determined in unanesthetized animals at a time point (4th postoperative week), when mechanical hypersensitivity was expected to be maximal [1,7]. After this at the 4th week, medullary neurons were recorded under light anesthesia. RVM neurons and DRt neurons were studied in different animals. At the completion of the experiments, the animals were given a lethal dose of sodium pentobarbital (150 mg/kg), the recording site marked with electrolytic lesion, and the brains removed for verification of recording sites ( [32]; Fig. 1 D,E).

Statistical analyses
Preliminary statistical analyses indicated that both the behavioral and neurophysiological data failed to pass the Shapiro-Wilk normality test. Therefore, median and interquartile range were used for data reporting. Non-parametric Mann-Whitney U test was used for statistical comparisons. P < 0.05 (two-tailed) was considered to represent a significant difference.

Behavioral assessment of mechanical sensitivity
Before SNI operation, the monofilament-evoked limb withdrawal threshold was significantly lower in females (median 12.5 g, interquartile range 12.5 g -14.0 g) than in males (median 20.5 g, interquartile range: 15.5 g -21.0 g; Mann-Whitney U = 8.5, P = 0.013). By the fourth week after SNI operation, the withdrawal threshold was significantly decreased from the preoperative level both in females (Mann-Whitney U = 0, P = 0.002) and males (Mann-Whitney U = 0, P = 0.003). On the fourth postoperative week the withdrawal threshold was significantly lower in SNI females than SNI males (Mann-Whitney U = 2, P = 0.0029; Fig. 3 A).
The ongoing discharge rate of pronociceptive RVM ON-like cells was significantly higher in female than male SNI animals (Mann-Whitney U = 13, P = 0.049; Fig. 3 B). Conversely, the ongoing discharge rate of antinociceptive RVM OFF-like cells was significantly lower in female than male SNI animals (Mann-Whitney U = 16, P = 0.017; Fig. 3 C). Ongoing discharge rate of pronociceptive DRt neurons was not different between female and male SNI animals (Mann-Whitney U = 52.5, P = 0.93; Fig. 3 D).

Discussion
The present results in rats with SNI model of peripheral neuropathy show that neuropathic pain, as reflected by mechanical hypersensitivity, is stronger in females than males. This is in line with earlier findings reporting that peripheral nerve injuries produce stronger pain-related behavior in female rats [1,7,9,40]. A novel finding of the present study is that stronger mechanical hypersensitivity in female SNI rats was accompanied by a higher ongoing discharge rate in pronociceptive RVM ON-like neurons and a lower ongoing discharge rate in antinociceptive RVM OFF-like neurons. These associations between behavioral and neuronal findings are in line with the hypothesis that the discharges of RVM ON-and OFF-like neurons mediate a descending pronociceptive action that contributes to the maintenance of stronger neuropathic pain in SNI females. In another medullary pain control nucleus, the DRt, discharge rates, however, did not vary with the sex of the SNI animals  indicating that the properties of DRt neurons are not likely to contribute to the sex difference in neuropathic pain. There is abundant evidence that the RVM and its ON-and OFFneurons provide an important relay for descending control of pain [14]. Increased discharge rates of RVM ON-neurons and decreased discharge rates of RVM OFF-neurons have been associated with pronociception in various experimental conditions, whereas converse changes have been associated with antinociception [8,17]. However, decreased OFF cell activity is in some conditions associated with antinociception, through recruitment of descending noradrenergic pathways [12]. Concerning the role of the RVM in neuropathic pain, earlier results in SNI males showed that the development of neuropathic hypersensitivity was associated with increased ongoing discharge rate of RVM ON-neurons and decreased ongoing discharge rate of RVM OFF-neurons [15]. This earlier finding in SNI males is in line with the present results and the interpretation that RVM ON-and OFF-like neurons play a role in neuropathic hypersensitivity. The present results extend these earlier findings by showing a sex difference in the discharge of pro-and antinociceptive RVM neurons of SNI animals. The present results, however, do not reveal whether the discharge rate differences in the RVM are due to sex-dependent mechanisms within the RVM or whether they reflect sex-dependent differences elsewhere. For example, the midbrain periaqueductal gray that projects to the RVM [14,33] is involved in a sexually dimorphic descending control of pain [23]. Interestingly, a recent study in naïve control rats reported that there is no significant sex difference in pain behavior or responses of RVM ON-and OFF-cells evoked by mechanical stimulation [18].
In addition to the RVM, another medullary recording site in the present study was DRt that is also involved in descending regulation of pain [22]. The studied DRt neurons had large receptive fields and they gave excitatory responses to noxious stimulation, which is in line with earlier findings [10,39]. A number of studies have shown that activation of the DRt facilitates spinal nociception [2,10,13]. Of particular interest for the present study is that the DRt contributes to pain facilitation also in models of experimental neuropathy [24,26,37]. Against our expectations, however, there was no sex difference in the discharge rates of DRt neurons between the SNI males and females.
The present correlative results are in line with the earlier evidence indicating that differences in descending pain control mechanisms play a role in sexually dimorphic differences in pain. For example, the disappearance of the stronger spinal nociceptive response in females than males following spinal transection indicates involvement of descending pain control pathways in the sex difference of nociception [41]. Furthermore, previous studies have shown sex differences in various neurotransmitter mechanisms of descending pain modulatory pathways in non-neuropathic [21,23,27,36] and neuropathic conditions [40].
In contrast to the development of sexually dimorphic mechanical hypersensitivity following peripheral nerve injury in the present and some earlier rat studies [1,7,9,40], earlier mouse studies did not reveal sex-related differences in the mechanical hypersensitivity at the corresponding time point after nerve injury [29,31,38]. A plausible explanation for this difference is the floor effect that did not allow observing sex-differences in mice. In accordance with this explanation, the mechanical threshold of both male and female mice in the cited studies [29,31,38] had decreased to the level of the absolute mechanical activation threshold of mouse primary afferent nerve fibers innervating the tested sural nerve area [20], due to which it may not have been possible to observe sex-differences in thresholds of SNI mice. Alternatively, sexual dimorphism in the development of neuropathic hypersensitivity varies between mice and rats.
Among limitations of this study is that we performed the experiments only at one time point after nerve injury. Mechanisms underlying neuropathic pain may differ at various post-injury time points [4]. In addition, the present study focused on two specific pain control nuclei of the medulla, the RVM and the DRt. Thereby the present results leave open whether neurons in pain control nuclei that were not studied here show sexually dimorphic discharge rate differences in neuropathic pain conditions. Moreover, in addition to descending pain control mechanisms there are multiple other pain processing and modulating mechanisms at various levels of the neuraxis that we did not study here and that potentially play a role in the sex-difference of neuropathic as well as non-neuropathic pain [25,28,30]. It is noteworthy that the present study addressing behavioral and neurophysiological differences between males and females with an established SNI did not have sham-operated control groups. This was not considered critical for testing the current working hypothesis on sex differences, since numerous previous studies have demonstrated that SNI produces a dramatic decrease in the mechanical withdrawal threshold when compared with sham operation (e. g., [1,7,11,16,40]). In line with this, the median withdrawal threshold of both SNI males and females of the present study decreased from the pre-SNI threshold as much as by 76 %. Additionally, our earlier neurophysiological study comparing sham and SNI males showed that SNI induced pronociceptive changes in the ongoing discharge of RVM ONand OFF-cells [15].

Conclusions
The main finding of the present study is that discharge rates of medullary pain control neurons in the RVM but not in the DRt are associated in a sexually dimorphic fashion with mechanical hypersensitivity in neuropathic animals. A plausible explanation for this result is that a sex difference in the descending pain control originating in or relaying through the RVM is among mechanisms contributing to the maintenance of stronger neuropathic pain in females.