Oxidative Stress in the Amygdala Contributes to Neuropathic Pain

Accepted Manuscript Oxidative stress in the amygdala contributes to neuropathic pain B. Sagalajev, H. Wei, Z. Chen, I. Albayrak, A. Koivisto, A. Pertovaara PII: DOI: Reference:

S0306-4522(17)30881-3 https://doi.org/10.1016/j.neuroscience.2017.12.009 NSC 18179

To appear in:

Neuroscience

Received Date: Accepted Date:

8 May 2017 6 December 2017

Please cite this article as: B. Sagalajev, H. Wei, Z. Chen, I. Albayrak, A. Koivisto, A. Pertovaara, Oxidative stress in the amygdala contributes to neuropathic pain, Neuroscience (2017), doi: https://doi.org/10.1016/j.neuroscience. 2017.12.009

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1

OXIDATIVE STRESS IN THE AMYGDALA CONTRIBUTES TO NEUROPATHIC PAIN B. SAGALAJEVa,1, H. WEIa,1, Z. CHENa,b,1, I. ALBAYRAKa, A. KOIVISTOc, A. PERTOVAARAa,* a

Department of Physiology, Faculty of Medicine, Helsinki, Finland

b

Department of Neuroscience and Biomedical Engineering, Aalto University School

of Science and AMI Centre, Espoo, Finland c

OrionPharma Inc., Turku, Finland

1

These authors contributed equally to this work

*

Corresponding author: A. Pertovaara, Department of Physiology, Faculty of

Medicine, POB 63, University of Helsinki, 00014 Helsinki, Finland. E-mail: [email protected]

2 Abstract

Earlier studies indicate that the central nucleus of the amygdala (CeA) contributes to neuropathic pain. Here we studied whether amygdaloid administration of antioxidants or antagonists of TRPA1 that is among ion channels activated by oxidative stress attenuates nociceptive or affective pain in experimental neuropathy, and whether this effect involves amygdaloid astrocytes or descending serotonergic pathways acting on the spinal 5-HT1A receptor. The experiments were performed in rats with spared nerve injury (SNI). Drugs were administered through a chronic cannula in the CeA or internal capsule (control site), and an intrathecal catheter. Nociception was assessed using monofilaments and affective pain using conditioned place-aversion. Antioxidants or TRPA1 antagonists in the CeA attenuated both nociceptive and affective pain in SNI animals but not in sham controls or in a control injection site. Drugs influencing astroglia (a gap junction decoupler or a D-amino acid oxidase inhibitor) in the CeA had no effect on SNI rats, whereas local anesthesia of the CeA attenuated nociception. Spinally administered 5-HT1A receptor antagonist at a dose that had no effect alone prevented the antinociceptive effect of amygdaloid TRPA1 blockers. The results suggest that injury-induced amygdaloid oxidative stress that drives TRPA1 promotes neuropathic pain behavior. This pronociceptive effect involves suppression of medullospinal serotonergic feedback-inhibition acting on the spinal 5-HT1A receptor. While the CeA is involved in mediating the nerve injuryinduced pronociception, it may not be a critical relay for the recruitment of medullospinal feedback-inhibition.

3

Key words: amygdala; antioxidant; descending pain modulation; neuropathic pain; spinal 5-HT1A receptor; Transient receptor potential ankyrin 1; Chemical compounds studied in this article: A-967079 (PubChem CID: 60150207); AS-057278 (PubChem CID: 9822); Carbenoxolone (PubChem CID: 24892726); Chembridge-5861528 (PubChem CID: 2873523); Lidocaine (PubChem CID: 3676); Phenyl-N-tert-butylnitrone (PubChem CID: 638877); TEMPOL (PubChem CID: 137994); tert-butyl-hydroperoxide (PubChem CID: 6410); WAY-100635 (PubChem CID: 5684)

Abbreviations: CeA, central nucleus of the amygdala; Chem, Chembridge-5861528; DAAO, D-amino acid oxidase; LPBN, lateral parabrachial nucleus; PAG, periaqueductal gray; PBN, Phenyl-N-tert-butylnitrone; ROS, reactive oxygen species; RVM, rostroventromedial medulla; SNI, spared nerve injury; t-BOOH, tert-butylhydroperoxide; TRPA1, transient receptor potential ankyrin 1;

4 INTRODUCTION

Amygdala has an important role in the processing of primary emotions, such as fear (LeDoux, 2000). Moreover, a subdivision of the amygdala, the central nucleus (CeA) that has bidirectional connections with several cortical and subcortical structures is involved in processing and modulation of pain-related signals (Neugebauer, 2015). In animals with a peripheral nerve injury-induced neuropathy, enhanced nociception has been accompanied by amygdaloid changes in anatomical structure (Gonçalves et al., 2008), neuronal response properties (Gonçalves and Dickenson, 2012; Ikeda et al., 2007) and neurotransmitter receptor actions (Ansah et al., 2009, 2010; Bourbia et al., 2010; Ji et al., 2017; Pedersen et al., 2007; Wei et al., 2015). These amygdaloid changes possibly contribute to neuropathic pain and comorbid emotional disorders such as anxiety and depression. Studies on inflammatory pain have shown that reactive oxygen species (ROS) generated by oxidative stress facilitate nociceptive processing in the amygdala and contribute to enhancement of pain behavior (Ji et al., 2015; Li et al., 2011; Lu et al., 2016). It is well established that ROS are important endogenous agonists of transient receptor potential ankyrin 1 (TRPA1) (Andersson et al., 2008), a calcium-permeable non-selective cation channel expressed in the nervous system on a subpopulation of primary afferent nociceptors (story et al., 2003), astrocytes (Shigetomi et al., 2011), and oligodendrocytes (Hamilton et al., 2016). In the spinal dorsal horn of neuropathic animals, ROS has been shown to contribute to enhancement of pain behavior (Kim et al., 2004). This pronociceptive effect may involve multiple accompanying mechanisms, such as release of various pro-inflammatory cytokines (Sanna et al., 2017), but for many of which the spinal TRPA1 channel may be a final common pronociceptive target (Koivisto et al., 2014; Wei et al., 2011a). It is not yet clear whether peripheral neuropathy induces oxidative stress in the amygdala and whether amygdaloid ROS exert a role in neuropathic pain. While earlier studies suggested that neurons in the central nervous system of healthy animals do not express TRPA1 (Story et al., 2003), it is not yet known whether amygdaloid neurons or glial cells exposed to the nerve injury-induced plasticity express TRPA1, the ROS-induced drive of which might contribute to neuropathic symptoms. Here we studied whether amygdaloid ROS or ROS-driven TRPA1 might have a role in the maintenance of neuropathic pain. Therefore, we assessed sensory-

5 discriminative aspect of pain, mechanical nociception, and emotional-motivational aspect of pain, affective pain, following amygdaloid administration of antioxidants or TRPA1 antagonists in rats with an experimental model of peripheral neuropathy. To study whether astrocytes, a cell type expressing TRPA1 (Shigetomi et al., 2011) and having an important role in chronic pain at spinal level (Hansson, 2010), might be involved in the amygdala-driven facilitation of neuropathic pain, we determined the effect by amygdaloid administration of drugs acting on astrocytes in neuropathic animals. Moreover, there is evidence indicating that feedback-inhibition of pain by medullospinal serotonergic pathway acting on the spinal 5-HT1A is suppressed in neuropathic conditions (Sagalajev et al., 2017; Wei et al., 2006). Here, we raised the hypothesis that amygdaloid ROS or ROS-driven TRPA1 has a pronociceptive effect in neuropathy through inhibitory action on serotonergic feedback-inhibition of pain. To test this hypothesis, we determined whether blocking amygdaloid TRPA1 disinhibits serotonergic feedback-inhibition of pain and whether the antinociceptive effect resulting in the disinhibition can be prevented by administering spinally a 5HT1A receptor antagonist.

EXPERIMENTAL PROCEDURES

Experimental animals

The experiments were performed in adult, male Hannover-Wistar rats (weight: 180230 g; Envigo, Horst, The Netherlands). The experimental protocol was accepted by the Ethical Committee on Animal Experiments of the regional government of Southern Finland. 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 minimal number of animals to produce reliable scientific data. Food and water were available ad libitum and animals were maintained in a climate-controlled room, at the temperature of 22 ºC and humidity of 55%, and a 12 h light/dark cycle with lights on at 6:00 am. The experimental protocols were conducted during the light phase of the cycle. Animals that had an installed guide cannula in the brain were kept individually

6 in transparent boxes with soft bedding and with visual and auditory contact to animals in neighboring boxes.

Spared nerve injury model

Among peripheral nerve trauma-induced models of neuropathy (Honoré et al., 2011), the spared nerve injury (SNI) model (Decosterd and Woolf, 2000), was chosen, since it induces a robust and stable facilitation of withdrawal responses to mechanical stimulation. For the nerve operation, general anesthesia was induced with sodium pentobarbital (OrionPharma, Espoo, Finland) that was administered intraperitoneally at the dose of 60 mg/kg. Additional pentobarbitone doses (15-20 mg/kg) were given as needed to keep a deep level of anesthesia during which the animals did not react to noxious stimulation. Then, skin incision was made in the lateral surface of the left thigh, followed by a section through the biceps femoris muscle to expose the sciatic nerve trunk with its terminal branches: the sural, common peroneal and tibial nerves. The exposed common peroneal and tibial nerves were tightly ligated with 4-0 silk, and they were sectioned distal to the ligation. Finally, 3-4 mm of the distal nerve stump was removed. The sural nerve was left intact. For postoperative pain treatment animals were administered 0.01 mg/kg of buprenorphine twice daily up to the third postoperative day. After surgery, the animals were allowed to recover for at least a week prior staring the experiments. For the SNI group of this study, only animals with tactile allodynia-like behavior (threshold to monofilament stimulation in the nerveinjured side < 4 g, which is below the lower 95% confidence limit of the threshold in unoperated control animals). In the present sample of animals, SNI model produced mechanical hypersensitivity in all nerve-injured animals. In sham controls, operation was performed as in the SNI group, except that the common peroneal and tibial nerves were only exposed but not ligated.

Intracerebral microinjections

For amygdaloid drug administrations, a guide cannula was installed into the central nucleus of the right amygdala as described earlier (Sagalajev et al., 2015). The right amygdala was the target in this study, since previous rat studies indicate that the right amygdala has a more important role in pain processing than the left amygdala

7 (Carrasquillo and Gereau, 2008; Ji and Neugebauer, 2009; Kolber et al., 2010), although not in all conditions (Spuz et al., 2014). Moreover, it has been reported that the descending control of nociception by the amygdala is stronger on the contra- than ipsilateral side (Bourbia et al., 2010; Kolber et al., 2010). This earlier finding further supported the choice of the right amygdala for unilateral drug injections in the studied group of animals with a nerve injury in the left hind limb. For installation of the guide cannula (26 gauge; PlasticsOne, Roanoke, VA, USA), the skull was exposed for drilling a hole for the placement of the guide cannula under pentobarbital anesthesia (60 ml/kg i.p.). Guide cannula was installed in the same operation in which the peripheral nerve was ligated. The injection target in the right amygdala was the capsule lateral of the central nucleus of amygdala (CeA): 2.1 mm posterior from the bregma, 4.3 mm lateral from the midline, and 7.8 mm ventral from the dura mater (Paxinos and Watson, 1986). A separate group of animals had a guide cannula in a control injection site (the right internal capsule: 2.1 mm posterior from the bregma, 3.6 mm lateral from the midline, and 5.0 mm ventral from the dura mater; Paxinos and Watson, 1986). Control injection site was used to exclude the possibility that the studied compound induced its effects due to its spread to adjacent brain areas. The tip of the guide cannula was positioned 2 mm above the injection target. A dental screw and dental cement were used to fix the guide cannula on the skull. The minimum interval from the guide cannula installation to the actual experiments was one week. A dummy cannula was placed in the guide cannula between experiments. Studied drugs or vehicle were administered intracerebrally at a volume of 0.5 μl using 33 gauge injection needles (PlasticsOne) connected to a 10 µl Hamilton microsyringe (Hamilton Bonaduz AG, Bonaduz, Switzerland) by polyethylene (PE10) tubing.. The animals were gently restrained by the experimenter during the injection procedure. The duration of injection was 30 s. After the injection, the injection needle was retained within the cannula for an additional 20 s to prevent backflow of the studied drug into the cannula. In addition to a guide cannula in the right CeA, a group of animals were installed an intrathecal (i.t.) catheter for drug delivery to the lumbar spinal cord. The catheter (Intramedic PE-10, Becton Dickinson and Company, Sparks, MD, USA) was installed under pentobarbital anesthesia (60 mg/kg i.p.) as described elsewhere (Størkson et al., 1996) at the same time as nerve injury and installation of brain cannula were performed. After recovery from anesthesia, the correct placing of the

8 catheter was verified by injecting lidocaine (4%, 7–10 μl followed by a 10 μl of saline for flushing) with a 50 μl Hamilton syringe (Hamilton Bonaduz AG). Only rats that had no motor impairment prior to lidocaine injection but had a bilateral paralysis of hind limbs following i.t. lidocaine administration were included in the study. With i.t. drug administrations, the volume of drug injections was 5 μl followed by a flush with 15 μl of saline.

Assessment of pain behavior

Clinical observations indicate that nerve injury frequently produces tactile allodynia or hypersensitivity to mechanical skin stimulation (e.g., with brush) but only occasionally hyperalgesia to heat (Scadding and Koltzenburg, 2006). Since hypersensitivity to mechanical stimulation can be bothersome and prominent following traumatic nerve injuries (Jensen and Finnerup, 2014), the focus of this study was in the evaluation of tactile allodynia-like hypersensitivity that was done by determining withdrawal response to monofilament stimulation of the spared sural nerve area. Furthermore, central mechanisms that were studied in the present experiments have an important role in hypersensitivity to mechanical rather than heat stimulation (Treede et al., 1992). Before any testing, the experimental animals were habituated to the experimental conditions by allowing them to spend 1-2 h daily in the laboratory for 2-3 days.

Assessment of mechanically evoked withdrawal responses

Tactile allodynia-like hypersensitivity to mechanical stimulation was determined by assessing the hind limb withdrawal threshold to stimulation of the hind paw with monofilaments (von Frey-hairs). In this study, the term mechanical nociception is used to refer to mechanically evoked hind limb withdrawal response, independent whether the response is elicited by noxious or innocuous test stimulation. Testing of responses to mechanical stimulation was performed while the rat was standing on a metal grid. In each testing, the paw of the nerve-injured limb was stimulated five times using an ascending series of monofilaments (1-26 g; North Coast Medical, Inc., Morgan Hill, CA). At each test force, the frequency of the withdrawal response was assessed. An increased response rate was considered to represent mechanical

9 hypersensitivity. When compared with the traditional withdrawal threshold determination, the currently used method has the advantage that it allows assessing separately drug effects on responses to threshold and suprathreshold force levels. It is a limitation that the assessment of hypersensitivity was not blinded. However, it should be noted that our earlier study by the same experimenter indicated that the drug-induced effect using the same test stimulus procedure was of identical magnitude with and without formal blinding (Wei et al., 2012).

Evaluation of affective pain

Affective pain (emotional component of pain) induced by stimulation of the nerveinjured limb was assessed using place-avoidance paradigm (LaBuda and Fuchs, 2000). Prior to testing, the animals spent one to two hours daily for two days in the test box to get familiar with the experimental environment. In the testing, the rat was placed within a Plexiglas chamber (60 x 30 x 30 cm; half of which was painted black on the external surface) that was placed upon an elevated metal grid. At the start of the experiment, the animal was placed over the midline of the chamber and the plantar surface of the hind paw was stimulated with a 60 g monofilament (North Coast Medical, Inc.) at an interval of 15 s for 15 min. Rats that are not stimulated prefer to stay within the dark side of the chamber. When the animal was within the dark side of the chamber, its injured hind paw was stimulated. Conversely, the uninjured hind paw was stimulated when the animals was within the light side of the chamber. During testing period of 15 min, rats were allowed to move freely throughout the chamber. The percent time the animal spent in the light side of the chamber during the 15 min observation period was assessed in each condition for each animal. The more aversive the mechanical stimulation of the injured hind paw is, the more the animal is expected to spend time in the light side of the chamber; i.e., the place-avoidance test is considered to assess affective pain. A prerequisite for the currently used aversive place-avoidance paradigm is that one of the hind limbs is hypersensitive (LaBuda and Fuchs, 2000). Unlike SNI, sham operation did not influence nociceptive threshold. Therefore, the currently used aversive place-conditioning paradigm could not be used to assess affective pain in the sham group.

Evaluation of heat nociception

10

Heat nociception was assessed in a group of sham controls by determining withdrawal latency to noxious heat applied to the plantar skin using a radiant heat device (Plantar test model 7370, Ugo Basile, Varese, Italy). Heat nociception was not assessed in the injured limb of SNI animals, since SNI caused denervation of the optimal stimulation target, the plantar skin, and the currently used device does not allow optimal delivery of the heat beam to the spared sural nerve area in the lateral side of the plantar skin of the foot, near the border between the glabrous and hairy skin. The cut-off point was set at 15 s. During testing, rats were free to move inside a transparent box of thermal plantar test device. Each measurement was replicated once at one min interval, and the average of the two successive measurements was used in further calculations. Motor component of the limb withdrawal response evoked by heat stimulation is identical to that evoked by mechanical stimulation. Therefore, a differential drug effect on responses evoked by heat versus mechanical stimulation would help in revealing whether the drug effect could be explained by an action on the sensory rather than motor system.

Drugs

Antioxidants TEMPOL and Phenyl-N-tert-butylnitrone (PBN), ROS donor tert-butylhydroperoxide (t-BOOH), gap junction decoupler carbenoxolone, DAAO inhibitor AS-057278, and the 5-HT1A receptor antagonist WAY-100635 were purchased from Sigma-Aldrich (St. Louis, MO, USA), while the local anesthetic lidocaine was purchased from OrionPharma (Espoo, Finland). TRPA1 antagonist Chembridge5861528 (Chem; a TRPA1 channel antagonist and derivative of HC-030031) (Koivisto and Pertovaara, 2015) was synthetized by ChemBridge (San Diego, CA, USA). A-967079 (McGaraughty et al., 2010) a selective TRPA1 channel antagonist with a structure different from CHEM, was used for comparison. The doses of drug were chosen based on earlier studies showing that spinal administrations of TEMPOL at the dose of 100 or 200 µg, PBN at the dose of 110 µg (Wei et al., 2011b), and carbenoxolone (Wei et al., 2016), AS-057278 (Wei et al., 2013), Chem or A-967079 (Wei et al., 2011a) at the dose of 10 µg attenuated mechanical hypersensitivity, while intrathecally administered t-BOOH increased hypersensitivity of healthy controls at the dose of 1.1 µmol (Wei et al., 2011b). The

11 dose of WAY-100635 was 2.5 µg, since its spinal administration at this dose has prevented cortical stimulation-induced serotonergic anti-hypersensitivity effect in nerve-injured animals (Sagalajev et al., 2017).

Course of the study

Spinal nerve ligation (or sham operation) and installations of the brain cannula and in some animals also intrathecal catheter were done on the first day under general anesthesia. Habituation of the animals to the testing conditions was performed during the second postoperative week. Drug effects were tested two to three weeks after the nerve/sham injury and installations of the brain cannula and intrathecal catheter (Fig. 1). Nerve injury or sham operation was performed always on the left side, and the brain cannula was always in the right hemisphere. Test stimuli used for assessment of pain behavior were applied to the left (injured) limb, except in the aversive placeavoidance test also the right intact limb was stimulated when the animal was in the light compartment. In each drug testing condition, the assessment of pain behavior was performed before and at various time points up to one hour following drug administrations. Corresponding vehicle (with i.t. injections physiological saline and with intracerebral injections DMSO or saline, as appropriate) was used as control. The maximum effects of drugs alone or drug combinations were chosen for further analyses. In attempts to reverse the anti-hypersensitivity effect induced by amygdaloid administration of a TRPA1 antagonist, the spinal cord was pretreated with a 5-HT1A receptor antagonist or vehicle before amygdaloid drug/vehicle administration; the delay between the spinal and amygdaloid administrations was 5 min. These delays were chosen based on the pharmacokinetic properties of the studied compounds so that both compounds were expected to have their maximal effects at the same time. The experimental groups are described in the results section. While originally the same number of animals was chosen for the conditions to be compared with each other, due to accidental removal of the brain cannula or intrathecal catheter or anesthesia death, the size of groups that were compared with each other was unequal in some conditions. Each animal participated in two to four drug testing sessions at an interval of 2 days and in an order that varied among animals. Assessment of pre-drug responses in each session indicated that none of the drug treatments had long-term

12 effects. At the end of the experiments, the animals were given a lethal dose of pentobarbitone and the brains removed for verification of the injection sites according to the atlas of Paxinos and Watson (1986; Fig. 2).

Statistical analyses

Statistical evaluation of the behavioral data was performed using one-way ANOVA or two-way mixed-design ANOVA followed by t-test with a Bonferroni correction for multiple comparisons. When comparing behavioral results of only two groups, paired or unpaired t-test, as appropriate, was used. P < 0.05 (two-tailed) was considered to represent a significant difference.

RESULTS

Pain behavior in neuropathic animals following amygdaloid administration of antioxidants

SNI induced a significant facilitation of mechanical nociception when compared with sham-operated controls. This is indicated e.g. by the finding that at the stimulation force of 6 g, the lower 95% confidence interval of the hind limb response rate in SNI animals was above the upper 95% confidence interval of the corresponding response rate in sham controls. To test the hypothesis that SNI-induces oxidative stress in the amygdala that contributes to neuropathic pain behavior, we assessed whether amygdaloid administration of antioxidants attenuates pain behavior in SNI animals. CeA administration of TEMPOL, an antioxidant, produced attenuation of mechanical nociception in five minutes, after which the antinociceptive effect was gradually reduced and disappeared completely by the end of the observation period of 60 min (Fig. 3 A). The mechanical antinociceptive effect induced by TEMPOL was doserelated (100 µg and 200 µg: F2,17 = 4.0, P = 0.04; Fig. 3 B). CeA administration of TEMPOL (200 µg) also attenuated affective pain-like behavior in SNI animals (t6 = 4.9, P = 0.003; Fig. 3 C). The mechanical antinociceptive effect induced by TEMPOL

13 was replicated in SNI animals with CeA administration of another antioxidant PBN (110 µg: F1,11 = 5.7, P = 0.04; Fig. 3 D). To study whether amygdaloid administration of a ROS donor in sham controls recapitulates facilitation of nociception observed in neuropathic conditions, t-BOOH was microinjected into the CeA at a dose (1.1µmol) that has produced a strong facilitation of nociception when administered spinally in healthy controls (Wei et al., 2011b). However, in the CeA of control animals t-BOOH failed to influence mechanical nociception when compared with vehicle-treated controls (F1,8 = 0.02; Fig. 3 E).

Pain behavior in neuropathic animals following amygdaloid administration of TRPA1 antagonists

To test the hypothesis that amygdaloid TRPA1 is among downstream pronociceptive mechanisms driven by amygdaloid ROS, we assessed whether pharmacological blocking of amygdaloid TRPA1 attenuates pain behavior in SNI animals. Microinjection of Chembridge-5861528 (Chem; a TRPA1 antagonist) into the right CeA produced mechanical antinociceptive effect in the injured (left) hind limb of SNI animals. The antinociceptive effect was maximal 30 min after the injection and disappeared by 60 min after the injection (Fig. 4 A). The mechanical antinociceptive effect induced by Chem was dose-related (5 µg and 10 µg: F2,14 = 4.0, P = 0.04; Fig. 4 B). Post hoc testing indicated that the antinociceptive effect of Chem was significant at the dose of 10 µg but not yet at the dose of 5 µg (Fig. 4 B). Chem in the right CeA attenuated in a dose-related fashion also affective painlike behavior in SNI animals as indicated by the conditioned place-avoidance test (5 µg and 10 µg: F2,16 = 8.4, P = 0.003; Fig. 4 C). Post hoc testing indicated that Chem at a dose of 10 µg but not at the dose of 5 µg produced a significant suppression of affective pain-like behavior (Fig. 4 C). Chem at a dose of 10 µg failed to produce a significant attenuation of nociception when it was administered into a control injection site (the right internal capsule) in SNI animals (F1,6 = 0.03; Fig. 4 D). In non-neuropathic control animals, CeA administration of Chem at a dose of 10 µg failed to influence mechanically evoked responses in the contralateral (left) hind limb (F1,8 = 0.03; Fig. 4 E). In a group

14 of non-neuropathic controls, also heat nociception after CeA administration of Chem at the dose of 10 µg was assessed by determining latency to radiant heat stimulation of the hind paw. The mean heat-evoked withdrawal latency of control animals 15 min following Chem treatment was not different from that following vehicle treatment (6.1 + 0.1 s versus 6.1 + 0.1 s, respectively; + S.E.M., t4 = 0.8, not shown). To assess whether the mechanical antinociceptive effect induced by blocking amygdaloid TRPA1 with Chem can be replicated with a structurally different TRPA1 antagonist, A-967079 was administered into the right CeA of SNI animals. A-967079 produced a dose-related mechanical antinociceptive effect (5 µg and 10 µg: F2,15 = 8.0, P = 0.004; Fig. 4 F). Post hoc testing indicated that A-967079 attenuated mechanical nociception at the dose of 10 µg but not at the dose of 5 µg (Fig. 4 F).

Pain behavior in neuropathic animals following amygdaloid administration of compounds acting on astroglia

Since among cell types expressing TRPA1 is astrocyte (Shigetomi et al., 2011), we attempted to study potential role of amygdaloid astrocytes in mechanical nociception of SNI animals by administering drugs inhibiting activation or function of astrocytes. Carbenoxolone, a gap junction decoupler, at a dose (10 µg) that significantly attenuated mechanical nociception following spinal administration of neuropathic animals (Wei et al., 2016) failed to influence mechanical nociception when administered into the CeA of SNI animals (F1,10 = 0.4; Fig. 5 A). To further assess the potential role of amygdaloid astrocytes in mechanical hypersensitivity, AS-057278, an inhibitor of astroglial enzyme D-amino acid oxidase (DAAO), was administered into the CeA of SNI animals at a dose (10 µg) that has attenuated hypersensitivity following spinal administration (Wei et al., 2013). In the CeA, AS-057278 failed to influence mechanical hypersensitivity of SNI animals (F1,9 = 0.5; Fig. 5 B).

Medullospinal mechanism mediating the amygdala-driven hypersensitivity effect

To test the hypothesis that the antinociceptive effect induced by CeA administration of TRPA1 antagonists was due to disinhibition of descending pain-inhibitory

15 serotonergic pathways, we assessed whether blocking the spinal 5-HT1A receptors reverses the antinociceptive effect induced by Chem in the CeA of SNI animals. Main effect of drug treatments (treatment groups: vehicle in CeA; 10 μg of Chem alone in CeA; 2.5 μg of WAY-100635 i.t. + 10 μg of Chem in CeA; 2.5 μg of WAY-100635 i.t.) on mechanical nociception was significant (F3,19 = 10.0, P < 0.0004; Fig. 5 C). Post hoc testing indicated that spinal administration of WAY-100635, a 5-HT1A receptor antagonist, prevented the antinociceptive effect induced by CeA administration of Chem (10 µg; Fig. 5 C). At the currently used dose, WAY-100635 alone had no significant effect on nociception (Fig. 5 C).

Local anesthesia of the CeA

To test the hypothesis that under control conditions CeA or fibers passing through it do not have a general tonic effect on descending pain modulatory pathways, CeA as well as fibers passing though it were locally anesthetized with microinjection of lidocaine (4 %, 0.5 µl) in control animals. In line with the hypothesis, lidocaine failed to influence mechanically evoked limb withdrawal responses in control conditions (F1,8 = 0.02; Fig. 5 D). Next, by CeA administration of lidocaine in neuropathic animals we tested the hypothesis that in neuropathic conditions the net tonic effect of CeA or fibers passing through it is suppression of an inhibitory feedback loop driven by ascending injury discharge that recruits descending inhibition through a projection that does not involve a relay in or a pathway through the CeA. According to this hypothesis, lidocaine in the CeA should disinhibit the feedback loop attenuating pain, while leaving intact the ascending injury signal recruiting the feedback inhibition; e.g., through a direct projection from the lateral parabrachial nucleus to the rostroventromedial medulla (RVM; Roeder et al., 2016). In line with this hypothesis, lidocaine in the CeA of neuropathic animals had a significant mechanical antinociceptive effect (F1,9 = 5.6, P = 0.04; Fig. 5 E).

DISCUSSION

16 Main findings

Among main results of the present study was that blocking amygdaloid administration of antioxidants attenuated facilitation of mechanical nociception and affective pain in neuropathic animals. These findings suggest that amygdaloid ROS promotes maintenance of neuropathic pain in a tonic fashion. Downstream, amygdaloid TRPA1 contributed to the mediation of the pronociceptive effect of ROS, an established TRPA1 agonist (Andersson et al., 2008). This is indicated by the findings that pharmacological blocking of amygdaloid TRPA1 suppressed the neuropathyassociated enhancement of mechanical nociception and affective pain. The suppression of pain behavior induced by blocking amygdaloid TRPA1 was observed only in nerve-injured but not sham control animals. The antinociceptive effect of TRPA1 block in the brain was restricted to the amygdala, since TRPA1 antagonist in a control injection site, the internal capsule, failed to influence nociception. Astrocytes in the amygdala may not have been critical in the CeA-driven antinociceptive effect since amygdaloid administration of a gap junction decoupler or DAAO inhibitor failed to influence nociception in SNI animals. Spinal administration of a 5-HT1A receptor antagonist prevented the antinociceptive effect induced by blocking the amygdaloid TRPA1 suggesting that amygdaloid TRPA1 promoted in a tonic fashion nociception in neuropathy by suppressing nerve injury-induced serotonergic feedback-inhibition of nociception.

CeA in neuropathic pain

There are several reasons to assume that the amygdala might contribute to the development and maintenance of chronic neuropathic pain and its comorbidities. First, it is well established that the amygdala is important in processing of primary emotions, such as fear or anxiety (LeDoux, 2000). Second, amygdala is not only involved in processing of emotions but particularly its central nucleus (CeA), the main output nucleus of the amygdala, exerts a role in processing and controlling nociception (Neugebauer, 2015). The CeA receives ascending nociceptive inputs directly through the spinal dorsal horn-lateral parabrachial nucleus (LPBN)-pathway, and indirectly through the convergent thalamo-cortical pathway relaying in the basolateral nucleus of the amygdala (BLA) (Bernard and Besson, 1990; Neugebauer,

17 2015). Third, CeA has descending nociception-regulating projections predominantly through the midbrain periaqueductal gray (PAG) - RVM pathway to the spinal dorsal horn (Pertovaara and Almeida, 2006). Additionally, amygdala is connected to various cortical and subcortical structures, such as the medial prefrontal cortex and the anterior cingulate cortex that play a role in affect and cognition (Neugebauer, 2015; Yalcin et al., 2014). Fourth, peripheral nerve injury is associated with marked morphological, and functional changes of the BLA and CeA (see the Introduction). The present results are in line with this earlier evidence and support the concept that amygdala contributes to the facilitation of sensory-discriminative and emotionalmotivational components of pain in neuropathy. In line with this, amygdaloid administrations of antioxidants or TRPA1 antagonists suppressed pain-related behavior in tests of mechanical nociception and affective pain. Drug injections into the CeA were performed at a volume of 0.5 µl. At this volume, the injection spreads in cerebral tissue at least 1 mm (Myers, 1966). Therefore, it is probable that drug may have had direct effects also on the adjacent amygdaloid subnuclei, such as BLA. Still, since CeA is involved in pain processing and it is the main amygdaloid output nucleus to the brainstem, it may be proposed that changes in pain behavior following amygdaloid drug treatments influenced the function of CeA. It is noteworthy that drug injections into a brain control site, internal capsule, failed to influence pain behavior. This finding supports the proposal that the drug effects following CeA administrations were due to action on the amygdala rather than some other structure. However, the present results do not allow pinpointing the critical sites of action within amygdala. Nerve injury-induced plastic changes and/or sustained injury discharge in the amygdala were critical for the oxidative stress-driven and TRPA1-mediated pronociceptive effect, since amygdaloid administration of antioxidants or TRPA1 antagonists did not attenuate pain behavior in control animals. Nor did acute CeA administration of a ROS donor alone recapitulate neuropathic pain behavior in control animals.

Oxidative stress and TRPA1 in the amygdala of neuropathic animals

Earlier studies have demonstrated that inflammatory pain induces oxidative stress in the amygdala and amygdaloid oxidative stress facilitates nociceptive processing in the CeA that is accompanied by increased pain behavior (Ji et al., 2015; Li et al., 2011;

18 Lu et al., 2016). The present pharmacological results extend these earlier findings and suggest that also peripheral nerve injury induces oxidative stress in the amygdala that contributes to neuropathic pain, as indicated by the attenuation of nociception and affective pain following CeA administration of antioxidants. Since ROS is an endogenous agonist of TRPA1 (Andersson et al., 2008), it may be hypothesized that the facilitation of neuropathic pain induced by amygdaloid ROS might be mediated, at least partly, by amygdaloid TRPA1. In this study, attenuation of nociception and affective pain was shown in neuropathic animals using two selective TRPA1 antagonists that have different structures. This finding supports the proposal that TRPA1 in the amygdala is involved in the maintenance of neuropathic pain. Since neuronal TRPA1 has not been demonstrated in central neurons of healthy control animals (Story et al., 2003), it remains to be studied whether amygdaloid neurons express TRPA1 following peripheral nerve injuryinduced neural plasticity. Additionally or alternatively, amygdaloid TRPA1 that was involved in promoting neuropathic pain may have been expressed on glia. In line with this, in the central nervous system, astrocytes (Shigetomi et al., 2011) and oligodendroglia (Hamilton et al., 2016) have been shown to express TRPA1. Moreover, amygdaloid microglia has been shown to contribute to neuropathic pain and anxiety (Sawada et al., 2014) while TRPA1, at least in the spinal cord, has been reported to promote pain through activation of microglia (Meotti et al., 2017). These findings raise the question whether TRPA1 and microglia might have a pronociceptive interaction also in the amygdala. Oxidative stress has an interesting interaction with the glutamatergic system. On one hand, activation of ionotropic glutamate receptors, in particular that of the NMDA receptor, has been shown to induce ROS in the nervous system (Bondy and Lee, 1993; Reynolds and Hastings, 1995). On the other hand, ROS in the spinal dorsal horn has been shown to be involved in enhancement of NMDA receptor activation and thereby, in promotion of nociception (Gao et al., 2007). These findings raise the possibility that interaction of ROS with the amygdaloid glutamatergic system may have a role in promotion of neuropathic pain. In line with this, blocking amygdaloid NMDA receptors has been reported to attenuate pain behavior in neuropathic animals (e.g., Ansah et al., 2010), while non-NMDA receptors were shown to be involved in intra-amygdaloid synaptic plasticity following nerve injury (Ikeda et al., 2007). Group I metabotropic glutamatergic receptors (mGluRs) in the amygdala may also contribute

19 to nociception and affective pain in neuropathy as suggested by attenuation of pain behavior following block of amygdaloid group I mGluRs (e.g., Ansah et al., 2009 & 2010). The role of amygdaloid mGluRs has been studied more extensively in inflammatory pain conditions than in neuropathy (Neugebauer, 2015). For example, it has been shown that amygdaloid mGluR8 inhibits pain-related enhanced excitatory transmission in the CeA of arthritic animals. The amygdaloid mGluR8-induced inhibitory effect in arthritis is associated with suppression of nociceptive behavior and the corresponding discharge rate changes in medullary pain control neurons. In contrast, amygdaloid mGluR7 has inhibitory actions only in control but not arthritic animals (Palazzo et al., 2008 & 2011; Ren et al., 2011). Also amygdaloid mGluR5 has been shown to be involved in promotion of inflammatory pain (Kolber et al., 2010). In the BLA, mGluR1 has been shown to be involved in cross-talk between amygdala and the pre-infra-limbic cortex that contributes to inflammatory pain (Luongo et al., 2013). Among non-glutamatergic receptors of the BLA, 5-HT2C receptor was recently shown to increase synaptic excitation of CeA neurons of nerve-injured animals and thereby neuropathic pain behavior (Ji et al., 2017). The potential role of amygdaloid ROS in these findings still requires further studies. CeA administrations of carbenoxolone, a compound decoupling gap junctions between astrocytes or astrocytes and neurons (Alvarez-Maubecin et al., 2000), or AS057278, an inhibitor of astroglial DAAO, failed to influence nociception in SNI animals of the present study. In contrast, at the currently used doses both of these compounds attenuated nociception when administered spinally in neuropathic animals (Wei et al., 2011b, 2016). These findings do not support the hypothesis that TRPA1 expressed on amygdaloid astrocytes contributed to neuropathic pain. However, it should be noted that carbenoxolone and DAAO inhibitors, such as AS-057278, have also effects other than inhibition of astrocytes that provide a complicating factor to the interpretations. Among them are modulation of neuronal membrane properties (Chepkova et al., 2008) and synaptic transmission (Tovar et al., 2009) by carbenoxolone. Moreover, DAAO inhibition may cause accumulation of D-serine, an endogenous agonist on the glycine-binding B site of the predominantly pronociceptive N-methyl-D-aspartate receptor (Schell et al., 1995). Since the effects of carbenoxolone and AS-057278 were studied only in a test of nociception but not in the aversive place-conditioning test, the present results do not allow excluding the possibility that these compounds might have had a selective effect on affective pain.

20

Inhibition of descending serotonergic pathways by amygdala in neuropathy

Descending serotonergic pathways that originate predominantly in the RVM (Kwiat and Basbaum, 1992) are known to contribute to pain modulation (Millan, 2002; Pertovaara and Almeida, 2006). Descending serotonergic pathways may facilitate or inhibit nociception, depending on the subtype of the spinal 5-HT receptor. There is earlier evidence suggesting that the spinal 5-HT3 receptor is involved in descending pain facilitation (Sagalajev et al., 2015; Suzuki et al., 2002), whereas among subtypes of spinal 5-HT receptors inhibiting pain has been shown to be the 5-HT1A receptor (Gjerstad et al., 1996; You et al., 2005). Serotonergic feedback-inhibition acting on the spinal 5-HT1A receptor is suppressed in animals with neuropathic pain, which contributes to neuropathic pain. This is suggested by the findings that blocking the medullary 5-HT1A receptor that presumably auto-inhibits serotonergic cell bodies with descending projections (Wei and Pertovaara, 2006) or stimulation of the secondary somatosensory cortex (Sagalajev et al., 2017) disinhibited descending pathways inducing antinociceptive effect in neuropathic animals. Antinociception induced by disinhibition of descending pathways could be prevented by blocking the spinal 5HT1A receptor (Sagalajev et al., 2017; Wei and Pertovaara, 2006). It should be noted that also other subtypes of the 5-HT receptor, such as 5-HT5A or 5-HT1B/D that were not studied here, have been shown to attenuate neuropathic pain behavior in the spinal dorsal horn (Avila-Rojas et al., 2015). Moreover, it still remains to be studied whether descending non-serotonergic pathways, such as noradrenergic ones (Pertovaara, 2013), might have contributed to the attenuation of nociception following amygdaloid administration of antioxidants or TRPA1 agonists in SNI animals. The present study extends earlier findings on serotonergic pain regulation in neuropathy by showing that blocking the amygdaloid TRPA1 disinhibited the serotonergic feedback-inhibition. Disinhibition lead to a significant antinociceptive effect that could be prevented by blocking the spinal 5-HT1A receptor. CeA projects to the midbrain PAG that further projects to the RVM (Pertovaara and Almeida, 2006). Therefore, the pronociceptive pathway originating in the CeA and suppressing medullospinal serotonergic feedback-inhibition may have a relay in the midbrain.

21 The spinal dorsal horn – LPBN – CeA pathway is a major pathway for ascending nociceptive signals to the amygdala (Bernard and Besson, 1990). It is proposed that this pathway was likely to be involved in the injury discharge-induced generation of oxidative stress and a consequent ROS-induced drive of amygdaloid TRPA1 that led to the CeA-driven suppression of serotonergic feedback-inhibition. In line with this proposal, administration of antioxidants or TRPA1 antagonists in the CeA attenuated the CeA-induced pronociceptive effect and lead to disinhibition of medullospinal feedback-inhibition, as indicated by the reduction in nociception in SNI animals. On the other hand, the marked antinociceptive effect following local anesthesia of CeA suggests that the recruitment of medullospinal feedback-inhibition does not require a relay in or nerve fibers passing through the CeA but the ascending injury discharge may have activated medullospinal feedback-inhibition e.g. through recently shown direct LPBN projections to the RVM (Roeder et al., 2016; see graphical abstract).

CONCLUSIONS

The present pharmacological results suggest that ascending nerve injury discharge induces oxidative stress in the CeA. Reactive compounds generated by oxidative stress drive amygdaloid TRPA1, which leads to amygdala-induced suppression of medullospinal feedback-inhibition of pain mediated by serotonergic pathways and acting on the spinal 5-HT1A receptor. In converse, amygdaloid administration of antioxidants, TRPA1 antagonists or a local anesthetic disinhibits serotonergic feedback-inhibition leading to 5-HT1A receptor antagonist-reversible attenuation of enhanced nociception in neuropathy. CeA is involved in mediating the nerve injuryinduced suppression of serotonergic feedback-inhibition but not in its recruitment (see graphical abstract).

Acknowledgements - This work was financially supported by the Sigrid Jusélius Foundation, Helsinki, Finland.

22 Conflicts of Interest - One of the authors (AK) is an employee of a pharmaceutical company (Orion corporation, OrionPharma, Turku, Finland). Other authors declare no conflicts of interest concerning this work.

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31 LEGENDS FOR FIGURES

Fig. 1. Flow diagram showing time course of the study.

Fig. 2. Microinjections sites in the right amygdala (grey circles) and the right internal capsule (black diamonds). Each symbol in schematic graphs represents 1-3 overlapping injection sites. In the photographic example, the arrow indicates the injection site. CeA, central nucleus of the amygdala; BLA, basolateral nucleus of the amygdala; LaA, lateral amygdaloid nucleus.

Fig. 3. Effect of amygdaloid administration of TRPA1 antagonists on pain behavior in animals with a spared nerve injury (SNI). A)

Time course of the mechanical antihypersensitivity effect following

administration of Chembridge-5861528 (Chem; 10 µg), a TRPA1 channel antagonist. Test force 6 g. B) Dose-related antihypersensitivity effect of Chem at different stimulus forces. C) Dose-related attenuation of pain affect by Chem. D) Absence of antihypersensitivity effect by Chem administered in a control injection site (the internal capsule). E) Effect of Chem on mechanically evoked responses in nonneuropathic animals. F) Dose-related antihypersensitivity effect of A-967079, another TRPA1 channel antagonist. CeA, central nucleus of the amygdala. A decrease in the response rate in represents drug-induced attenuation of mechanical sensitivity, whereas in C a decrease in time spent in light represent attenuation of pain affect. Error bars represent S.E.M. (in A and B, nChem = 5 and nVeh = 6; in C, nVeh/Chem5 = 7 and nChem10 = 5; in D, n=4; in E, n=5, in F, n=6). *P < 0.05, **P < 0.01, ***P < 0.005 (t-test with Bonferroni correction; reference: the corresponding vehicle value).

Fig. 4. Effect by amygdaloid administration of antioxidants on pain behavior in animals with spared nerve injury.

32 A)

Time course of the antihypersensitivity effect following administration

TEMPOL, an antioxidant. Test force 6 g. B) Dose-related antihypersensitivity effect by TEMPOL. C) Attenuation of pain affect by TEMPOL. D) Antihypersensitivity effect by PBN, another antioxidant. E) An attempt to recapitulate mechanical hypersensitivity in healthy controls by amygdaloid administration of t-BOOH, a ROS donor. CeA, central nucleus of the amygdala. A decrease in the response rate in represents drug-induced attenuation of mechanical sensitivity, whereas in C a decrease in time spent in light represent attenuation of pain affect. Error bars represent S.E.M. (in A and B, nVeh = 6 and nTEMPOL = 7; in C, n=7; in D, nVeh = 6 and nPBN = 7; in E, n=5). *P < 0.05, ***P < 0.005 (t-test with Bonferroni correction; reference: the corresponding vehicle value).

Fig. 5. A) Attempt to attenuate mechanical hypersensitivity by amygdaloid administration of carbenoxolone (carb, a gap junction decoupler) in spared nerve injury (SNI) animals. B) Attempt to attenuate mechanical hypersensitivity by AS057278 (an inhibitor of D-amino acid oxidase) in SNI animals. C) Mechanical antihypersensitivity effect induced by amygdaloid administration of Chembridge5861528 (Chem; 10 µg) is prevented by blocking the spinal 5-HT1A receptor with WAY-100635 (WAY; 2.5 µg) in SNI animals. D) Effect by amygdaloid administration of lidocaine, a local anesthetic, on mechanical sensitivity in nonneuropathic control animals. E) Effect by lidocaine on mechanical hypersensitivity in SNI animals. CeA, central nucleus of the amygdala. Veh, vehicle. Lido, lidocaine. In A-E, error bars represent S.E.M. (in A, n=6; in B, nVeh = 6, nAS = 5; in C, n = 6, except that nChem = 5; in D, n = 5; in E, nVeh = 6 and nLido = 5). **P < 0.01, ***P < 0.005 (in C and D, Bonferroni corrected t-test; reference: unless specified, the corresponding vehicle value).

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Veh

8

Stimulus force [g]

10

Vehicle

60 40 20 0

4

6

8

10

15

Stimulus force [g]

26

10 mg Chem

A-967079 in CeA: hypersensitivity A-96 10 mg A-96 5 mg Vehicle

100

CHEM 10 mg

80

F 80 60 40

**

Chem in a control injection site

100

4

*

100

Stimulus force [g]

Time [min]

D

2

200

20 0

1

**

15

*

40

Chem in CeA: pain affect

300

**

5

*

60

Response rate [%]

20

80

C

***

40

Pre

Chem 10 mg Chem 5 mg Vehicle

***

60

0

Chem in CeA: hypersensitivity

100

Response rate [%]

Response rate [%]

80

B

***

Vehicle Chem 10 mg

Time spent in light [s]

Chem in CeA: time course

100

**

A

2

4

6

8

Stimulus force [g]

10

Response rate [%]

60 40 20 4

6

8

40 20 0

10

Stimulus force [g]

20

2

4

6

8

10

Veh

Stimulus force [g]

D

E

Lidocaine in CeA: Sham

100

Vehicle

60 40 20 4

6

8

10

15

Stimulus force [g]

26

Chem Way WAY+Chem

Lidocaine in CeA: SNI

100

Lido

80

0

40

0

1

**

60

Lido Vehicle

80

**

2

60

***

80

60

**

1

Response rate [%]

0

AS 10mg Vehicle

80

i.t. WAY-100635 + Chem in CeA

100

***

100

Carb 10mg Vehicle

80

C

AS-057278 in CeA

Response rate [%]

100 Response rate [%]

B

Carbenoxolone in CeA

Response rate [%]

A

40 20 0

1

2

4

6

8

Stimulus force [g]

10

CeA

TRPA1 antagonist/ Antioxidant/ Local anesthetic

PAG

CeA

?

? RVM

5-HT

Nerve injury

excitatory inhibitory

Injury discharge

Injury discharge

5-HT1A

LPBN

Spinal dorsal horn

Feedback-inhibition

Spinal dorsal horn

RVM

5-HT

Feedback-inhibition

LPBN

PAG

5-HT1A

Nerve injury

34

-

amygdaloid administration of antioxidants or TRPA1 antagonists attenuated nociception and affective pain in neuropathy

-

blocking spinal 5-HT1A receptors prevented the antinociception induced by the amygdaloid drug treatments

-

in neuropathy, amygdaloid oxidative stress driving TRPA1 suppressed serotonergic feedback-inhibition of nociception

-

antioxidants and TRPA1 antagonists in the amygdala disinhibited the serotonergic feedback-inhibition of nociception