Long-Term Depression Induced by Optogenetically Driven Nociceptive Inputs to Trigeminal Nucleus Caudalis or Headache Triggers.

The peripheral trigeminovascular pathway mediates orofacial and craniofacial pain and projects centrally to the brainstem trigeminal nucleus caudalis (TNc). Sensitization of this pathway is involved in many pain conditions, but little is known about synaptic plasticity at its first central synapse. We have taken advantage of optogenetics to investigate plasticity selectively evoked at synapses of nociceptive primary afferents onto TNc neurons. Based on immunolabeling in the trigeminal ganglia, TRPV1-lineage neurons comprise primarily peptidergic and nonpeptidergic nociceptors. Optical stimulation of channelrhodopsin-expressing axons in the TRPV1/ChR2 mouse in TNc slices thus allowed us to activate a nociceptor-enriched subset of primary afferents. We recorded from lamina I/II neurons in acutely prepared transverse TNc slices, and alternately stimulated two independent afferent pathways, one with light-activated nociceptive afferents and the other with electrically-activated inputs. Low-frequency optical stimulation induced robust long-term depression (LTD) of optically-evoked EPSCs, but not of electrically-evoked EPSCs in the same neurons. Blocking NMDA receptors or nitric oxide synthase strongly attenuated LTD, whereas a cannabinoid receptor 1 antagonist had no effect. The neuropeptide PACAP-38 or the nitric oxide donors nitroglycerin or sodium nitroprusside are pharmacologic triggers of human headache. Bath application of any of these three compounds also persistently depressed optically-evoked EPSCs. Together, our data show that LTD of nociceptive afferent synapses on trigeminal nucleus neurons is elicited when the afferents are activated at frequencies consistent with the development of central sensitization of the trigeminovascular pathway.SIGNIFICANCE STATEMENT Animal models suggest that sensitization of trigeminovascular afferents plays a major role in craniofacial pain syndromes including primary headaches and trigeminal neuralgia, yet little is known about synaptic transmission and plasticity in the brainstem trigeminal nucleus caudalis (TNc). Here we used optogenetics to selectively drive a nociceptor-enriched population of trigeminal afferents while recording from superficial laminae neurons in the TNc. Low-frequency optical stimulation evoked robust long-term depression at TRPV1/ChR2 synapses. Moreover, application of three different headache trigger drugs also depressed TRPV1/ChR2 synapses. Synaptic depression at these primary afferent synapses may represent a newly identified mechanism contributing to central sensitization during headache.

Dynamic changes in CGRP, PACAP, and PACAP receptors in the trigeminovascular system of a novel repetitive electrical stimulation rat model: Relevant to migraine.

Migraine is the seventh most disabling disorder globally, with prevalence of 11.7% worldwide. One of the prevailing mechanisms is the activation of the trigeminovascular system, and calcitonin gene-related peptide (CGRP) is an important therapeutic target for migraine in this system. Recent studies suggested an emerging role of pituitary adenylate cyclase-activating peptide (PACAP) in migraine. However, the relation between CGRP and PACAP and the role of PACAP in migraine remain undefined. In this study, we established a novel repetitive (one, three, and seven days) electrical stimulation model by stimulating dura mater in conscious rats. Then, we determined expression patterns in the trigeminal ganglion and the trigeminal nucleus caudalis of the trigeminovascular system. Electrical stimulation decreased facial mechanical thresholds, and the order of sensitivity was as follows: vibrissal pad >inner canthus >outer canthus (P < 0.001). The electrical stimulation group exhibited head-turning and head-flicks (P < 0.05) nociceptive behaviors. Importantly, electrical stimulation increased the expressions of CGRP, PACAP, and the PACAP-preferring type 1 (PAC1) receptor in both trigeminal ganglion and trigeminal nucleus caudalis (P < 0.05). The expressions of two vasoactive intestinal peptide (VIP)-shared type 2 (VPAC1 and VPAC2) receptors were increased in the trigeminal ganglion, whereas in the trigeminal nucleus caudalis, their increases were peaked on Day 3 and then decreased by Day 7. PACAP was colocalized with NEUronal Nuclei (NeuN), PAC1, and CGRP in both trigeminal ganglion and the trigeminal nucleus caudalis. Our results demonstrate that the repetitive electrical stimulation model can simulate the allodynia during the migraine chronification, and PACAP plays a role in the pathogenesis of migraine potentially via PAC1 receptor.

The thalamic reticular nucleus is activated by cortical spreading depression in freely moving rats: prevention by acute valproate administration.

This study investigated the effect of repetitive cortical spreading depression (CSD) on behaviour and the anatomical and physiological patterns of cellular activation of cortical and subcortical areas in awake, moving rats. Rat behaviours in response to repetitive CSD events evoked by the application of KCl were quantified with electrophysiological recording. Immunohistochemistry was used to quantify anatomical regions of cellular activation. The effects of acute valproic acid administration on the behavioural parameters and cellular activation were evaluated. CSD significantly decreased locomotor activity and induced freezing in awake, moving rats, and stimulated c-Fos expression in the cortex, trigeminal nucleus caudalis (TNC), and amygdala. CSD also resulted in a prominent increase in c-Fos expression in the ipsilateral thalamic reticular nucleus (TRN) visual sector. Electrophysiological recordings revealed propagation of CSD into the TRN. Valproic acid pretreatment decreased the duration of CSD-induced freezing episodes and reversed the CSD-induced reduction in locomotor activity. Acute valproic acid administration also significantly blocked CSD-induced c-Fos expression in the TNC and TRN. These findings show that CSD events cause consistent behavioural responses and activate specific brain regions in awake, freely moving rats. Selective activation of TRN by CSD and the suppression of this activation by valproic acid suggest that this brain region may play an important role in migraine pathogenesis and may represent a novel target for migraine therapy.

Pain. Part 2a: Trigeminal Anatomy Related to Pain.

In order to understand the underlying principles of orofacial pain it is important to understand the corresponding anatomy and mechanisms. Paper 1 of this series explains the central nervous and peripheral nervous systems relating to pain. The trigeminal nerve is the 'great protector' of the most important region of our body. It is the largest sensory nerve of the body and over half of the sensory cortex is responsive to any stimulation within this system. This nerve is the main sensory system of the branchial arches and underpins the protection of the brain, sight, smell, airway, hearing and taste, underpinning our very existence. The brain reaction to pain within the trigeminal system has a significant and larger reaction to the threat of, and actual, pain compared with other sensory nerves. We are physiologically wired to run when threatened with pain in the trigeminal region and it is a 'miracle' that patients volunteer to sit in a dental chair and undergo dental treatment. Clinical Relevance: This paper aims to provide the dental and medical teams with a review of the trigeminal anatomy of pain and the principles of pain assessment.

Referred pain.

Comprehending orofacial referred pain requires an understanding of the neuroanatomy of the trigeminal nerve and associated cranial nerves. It also requires knowledge of the concept of neuronal convergence as well as the recognition that the caudalis is laminated and is therefore responsible for sensory receptive fields-that one interneuron may receive multiple sensory inputs and that structures within a lamina have sensory neurons which project into the caudalis and may share the same interneuron.

Effects of a liquid diet on the response properties of temporomandibular joint nociceptive neurons in the trigeminal subnucleus caudalis of growing rats.

OBJECTIVE: To investigate whether low mechanical loading on the temporomandibular joint (TMJ) when ingesting a liquid diet affects the response properties of neurons in the trigeminal spinal tract subnucleus caudalis (Sp5C) in growing rats. MATERIALS AND METHODS: Shortly after weaning, 2-week-old male rats were fed chow pellets (control) or a liquid diet (experimental). Firing activities of single sensory units were recorded from the Sp5C at 4, 5, 7, and 9 weeks. Neurons were functionally classified by their responsiveness to TMJ stimuli. The responses of Class II and III neurons to TMJ stimuli were investigated. RESULTS: In both neuron classes, the firing threshold in the experimental group was significantly lower than in the control group at all time points, but remained static in the control group throughout the experimental period, whereas it peaked in the experimental group at 4 weeks, decreased at 5 weeks, and remained stable thereafter until 9 weeks. Similarly, the initial firing frequency was significantly higher in the experimental group than in the control group, but remained static in the control group throughout the experimental period, whereas in the experimental group, it was at its lowest at 4 weeks, increased at 5 weeks, and stayed stable thereafter until 9 weeks. CONCLUSION: Differences in TMJ loading arising from variable diet consistency during growth may affect the functional characteristics of Sp5C neurons.

Participation of calbindin-D28K in nociception: results from calbindin-D28K knockout mice.

Since calbindin-D(28K) (CB-D(28K))-positive neurons have been related to nociceptive sensory processing, we have hypothesized that altered CB-D(28K) expression could alter nociceptive transmission. We have used +/+ and -/- knockout (KO) mice for CB-D(28k) in different behavioral models of pain and sensory responses at the caudalis subdivision of the trigeminal spinal nucleus in order to understand how this protein may participate in nociception. Behavioral responses to formalin injection in the hind paw or at the whisker pad or in the hind paw glutamate or i.p. acetic acid tests showed an increase of the pain threshold in CB-D(28k) -/- mice. KO mice showed a diminution of the inhibitory activity at Sp5C nucleus and a marked reduction of GABA content. Sp5C neurons from CB-D(28k) -/- mice did not change their spontaneous activity or tactile response after formalin injection in the whisker pad. In contrast, Sp5C neurons increased their spontaneous firing rate and tactile response after formalin injection in their receptive field in CB-D(28k) +/+ mice. The results of this study demonstrate the active role played by CB-D(28k) in nociceptive sensory transmission. The lack of this calcium binding protein, associated to deficient GABAergic neurotransmission, translates into dysfunction of sensory processing of nociceptive stimuli.

Trigeminal interpolaris/caudalis transition neurons mediate reflex lacrimation evoked by bright light in the rat.

Abnormal sensitivity to bright light can cause discomfort or pain and evoke protective reflexes such as lacrimation. Although the trigeminal nerve is probably involved, the mechanism linking luminance to somatic sensory nerve activity remains uncertain. This study determined the effect of bright light on second-order ocular neurons at the ventral trigeminal interpolaris/caudalis transition (Vi/Vc) region, a major termination zone for trigeminal sensory fibers that innervate the eye. Most Vi/Vc neurons (80.9%) identified by responses to mechanical stimulation of the ocular surface also encoded bright light intensity. Light-evoked neural activity displayed a long latency to activation (> 10 s) and required transmission through the trigeminal root ganglion. Light-evoked neural activity was inhibited by intravitreal injection of phenylephrine or l-N(G) -nitro-arginine methyl ester (L-NAME), suggesting a mechanism coupled to vascular events within the eye. Laser Doppler flowmetry revealed rapid light-evoked increases in ocular blood flow that occurred prior to the increase in Vi/Vc neural activity. Synaptic blockade of the Vi/Vc region by cobalt chloride prevented light-evoked increases in tear volume, whereas blockade at the more caudal spinomedullary junction (Vc/C1) had no effect. In summary, Vi/Vc neurons encoded bright light intensity and were inhibited by drugs that alter blood flow to the eye. These results support the hypothesis that light-responsive neurons at the Vi/Vc transition region are critical for ocular-specific functions such as reflex lacrimation, whereas neurons at the caudal Vc/C1 junction region probably serve other aspects of ocular nociception.

A naturalistic glyceryl trinitrate infusion migraine model in the rat.

BACKGROUND AND AIM: Glyceryl trinitrate (GTN) infusion is a reliable method to provoke migraine-like headaches in humans. Previous studies have simulated this human model in anaesthetized or in awake rodents using GTN doses 10,000 times higher than used in humans. The relevance of such toxicological doses to migraine is not certain. Anaesthesia and low blood pressure caused by high GTN doses both can affect the expression of nociceptive marker c-fos. Therefore, our aim was to simulate the human GTN migraine model in awake rats using a clinically relevant dose. METHODS: Awake rats were infused with GTN (4 µg/kg/min, for 20 min, i.v.), a dose just 8 times higher than in humans. mRNA and protein expression for c-fos were analysed in the trigeminal vascular system at various time points using RT-PCR and immunohistochemistry, respectively. RESULTS: A significant upregulation of c-fos mRNA was observed in the trigeminal nucleus caudalis at 30 min and 2 h that was followed by an upregulation of Fos protein in the trigeminal nucleus caudalis at 2 h and 4 h after GTN infusion. Pre-treatment with sumatriptan attenuated the activation of Fos at 4 h, demonstrating the specificity of this model for migraine. CONCLUSION: We present a validated naturalistic rat model suitable for screening of acute anti-migraine drugs.

K(ATP) channel openers in the trigeminovascular system.

BACKGROUND: The ATP-sensitive K(+) (K(ATP)) channel openers levcromakalim and pinacidil are vasodilators that induce headache in healthy people. The neuropeptide calcitonin gene-related peptide (CGRP) induces headache in healthy people and migraine in migraineurs, potentially through a mechanism that involves opening of vascular or neuronal K(ATP) channels and mast cell degranulation. Using rat as a model, we studied the molecular presence of K(ATP) channels in the trigeminovascular system. Furthermore, we examined whether K(ATP) channel openers stimulate the in vitro release of CGRP and whether they degranulate dural mast cells. METHODS: mRNA and protein expression of K(ATP) channel subunits were studied in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) by qPCR and western blotting. In vitro CGRP release was studied after application of levcromakalim (1 µM) and diazoxide (10 µM) to freshly isolated rat dura mater, TG and TNC. Rat dural mast cells were challenged in situ with levcromakalim (10(-5) M) to study its potential degranulation effect. RESULTS: mRNA and protein of K(ATP) channel subunits Kir6.1, Kir6.2, SUR1 and SUR2B were identified in the TG and TNC. K(ATP) channel openers did not release or inhibit capsaicin-induced CGRP release from dura mater, TG or TNC. They did also not induce dural mast cell degranulation. CONCLUSIONS: K(ATP) channel openers do not interact with CGRP release or mast cell degranulation. Activation of these channels in the CNS is antinociceptive and therefore cannot explain the headache induced by K(ATP) channel openers. Thus, they are likely to induce headache by interaction with extracerebral K(ATP) channels, probably the SUR2B isoforms.

Facial injections of pruritogens or algogens elicit distinct behavior responses in rats and excite overlapping populations of primary sensory and trigeminal subnucleus caudalis neurons.

In the present study, we investigated whether intradermal cheek injection of pruritogens or algogens differentially elicits hindlimb scratches or forelimb wipes in Sprague-Dawley rats, as recently reported in mice. We also investigated responses of primary sensory trigeminal ganglion (TG) and dorsal root ganglion (DRG) cells, as well as second-order neurons in trigeminal subnucleus caudalis (Vc), to pruritic and algesic stimuli. 5-HT was the most effective chemical to elicit dose-dependent bouts of hindlimb scratches directed to the cheek, with significantly less forelimb wiping, consistent with itch. Chloroquine also elicited significant scratching but not wiping. Allyl isothiocyanate (AITC; mustard oil) elicited dose-dependent wiping with no significant scratching. Capsaicin elicited equivalent numbers of scratch bouts and wipes, suggesting a mixed itch and pain sensation. By calcium imaging, ∼ 6% of cultured TG and DRG cells responded to 5-HT. The majority of 5-HT-sensitive cells also responded to chloroquine, AITC, and/or capsaicin, and one-third responded to histamine. Using a chemical search strategy, we identified single units in Vc that responded to intradermal cheek injection of 5-HT. Most were wide dynamic range (WDR) or nociceptive specific (NS), and a few were mechanically insensitive. The large majority additionally responded to AITC and/or capsaicin and thus were not pruritogen selective. These results suggest that primary and second-order neurons responsive to pruritogens and algogens may utilize a population coding mechanism to distinguish between itch and pain, sensations that are behaviorally manifested by distinct hindlimb scratching and forelimb wiping responses.

Evaluation of cutaneous allodynia following induction of cortical spreading depression in freely moving rats.

BACKGROUND: Cortical spreading depression (CSD) is a wave of depolarization followed by depression of bioelectrical activity that slowly propagates through the cortex. CSD is believed to be the underlying mechanism of aura in migraine; however, whether CSD can elicit pain associated with migraine headache is unclear. METHODS: Awake, freely moving rats were monitored for both CSD events and behavioral responses resulting from dural-cortical pinprick and/or KCl injection to the occipital cortex. RESULTS: We observed tactile allodynia of the face and hindpaws, as well as enhanced Fos expression within the trigeminal nucleus caudalis (TNC) following CSD induced by KCl injection into the cortex, but not by pinprick. Application of KCl onto the dura elicited cutaneous allodynia and increased Fos staining in the TNC but did not elicit CSD events. CONCLUSIONS: These data suggest that sustained activation of trigeminal afferents that may be required to establish cutaneous allodynia may not occur following CSD events in normal animals.

[Effect of migrepin on activity of trigeminal nucleus caudalis neurons].

Neurophysiological experiments on anesthetized rats were used to study the effects of various doses (12.5, 25, 37.5 mg/kg, i.v.) of drug composition migrepin (representing a combination of potassium-2,4-dichlorobenzoate, carbamazepine, and caffeine) on background firing of the trigeminal nucleus caudalis neurons and their responses to electrical stimulation of the dura mater. It was found that migrepin produces direct, dose-dependent inhibitory action on functional activity of TNC neurons. The results confirmed anti-migraine properties of the drug but did not exclude the necessity to study its action in clinical trials.

Astroglia in medullary dorsal horn (trigeminal spinal subnucleus caudalis) are involved in trigeminal neuropathic pain mechanisms.

The aim of this study was to investigate whether astroglia in the medullary dorsal horn (trigeminal spinal subnucleus caudalis; Vc) may be involved in orofacial neuropathic pain following trigeminal nerve injury. The effects of intrathecal administration of the astroglial aconitase inhibitor sodium fluoroacetate (FA) were tested on Vc astroglial hyperactivity [as revealed by glial fibrillary acid protein (GFAP) labeling], nocifensive behavior, Vc extracellular signal-regulated kinase phosphorylation (pERK), and Vc neuronal activity in inferior alveolar nerve-transected (IANX) rats. Compared with sham-control rats, a significant increase occurred in GFAP-positive cells in ipsilateral Vc at postoperative day 7 in IANX rats, which was prevented following FA administration. FA significantly increased the reduced head withdrawal latency to high-intensity heat stimulation of the maxillary whisker pad skin in IANX rats, although it did not significantly affect the reduced escape threshold to low-intensity mechanical stimulation of the whisker skin in IANX rats. FA also significantly reduced the increased number of pERK-like immunoreactive cells in Vc and the enhanced Vc nociceptive neuronal responses following high-intensity skin stimulation that were documented in IANX rats, and glutamine administration restored the enhanced responses. These various findings provide the first documentation that astroglia is involved in the enhanced nociceptive responses of functionally identified Vc nociceptive neurons and in the associated orofacial hyperalgesia following trigeminal nerve injury.

Large conductance calcium-activated potassium channels (BKCa) modulate trigeminovascular nociceptive transmission.

Migraine is a common, disabling, neurological problem whose acute management would benefit from the development of purely neurally acting therapies. The trigeminocervical complex is pivotal in nociceptive signaling in migraine, and is an accepted target for putative antimigraine agents. Whole-cell patch-clamp or extracellular recordings were made of trigeminal neurons identified in rat brainstem slices. Bath application of the large conductance calcium-activated potassium (BKCa) channel opener NS1619 caused a dramatic decrease of cell firing that could be reversed by the co-application of iberiotoxin. NS1619 hyperpolarized the resting membrane potential and reduced the frequency of spontaneous action potentials in these neurons. These data suggest the presence of BKCa channels in the trigeminocervical complex. In vivo in cat L-glutamate-evoked firing was facilitated in nociceptive neurons, also responding to stimulation of the superior sagittal sinus, in the trigeminal nucleus caudalis by the BKCa peptide antagonists, iberiotoxin and slotoxin. Of units tested, 70% responded to microiontophoretic application of the blockers, identifying a subpopulation of trigeminal neurons expressing toxin-sensitive BKCa channels. NS1619 inhibited 74% of cells tested, and this was reversed by slotoxin, suggesting that the action of NS1619 in these cells was mediated through BKCa channels. These data are consistent with the presence of BKCa channels in the trigeminal nucleus caudalis that are potential targets for the development of antimigraine treatments, and may also offer insights into receptor mechanisms involved in sensitization and thus allodynia, in migraine.

Central sensitization induced in trigeminal and upper cervical dorsal horn neurons by noxious stimulation of deep cervical paraspinal tissues in rats with minimal surgical trauma.

OBJECTIVE: This study investigated if central sensitization is induced in the trigeminal subnucleus caudalis (also termed the medullary dorsal horn) and C1 and C2 dorsal horns by noxious stimulation of deep upper cervical paraspinal tissues in a preparation relatively free of surgical trauma. METHODS: Adult male Sprague-Dawley rats (275-450 g) were anesthetized intraperitoneally. Animals were then placed in a stereotaxic frame; a small cutaneous incision was made 3 to 4 mm near the bregma in the midline, and an opening into the skull was prepared by a 1/32-inch drill, 1 mm to the left from the midline. An epoxylite-coated tungsten microelectrode was introduced at an 18 degrees angle to enter this small opening on the skull and was then carefully advanced about 16 mm through cortex, cerebellum, and brainstem to reach subsequently histologically confirmed sites in the Vc and upper cervical (C1 and C2) dorsal horn region. Thirty-three, 27, and 15 neurons recorded in medullary, C1, and C2 dorsal horns, respectively, of chloralose/urethane-anesthetized rats were activated by noxious stimulation of mechanoreceptive fields involving V1, V2, and/or V3 trigeminal nerve territories. The inflammatory irritant mustard oil was injected into the deep paraspinal tissues at the level of the left C1-C2 joint. Pre and postinjection receptive field (RF) sizes were mapped by nonnoxious mechanical stimuli and noxious mechanical and heat stimuli. RESULTS: A 30- to 50-minute increase (mean, 165% +/- 38.1%) in RF size postinjection for 62% of neurons tested was demonstrated, suggesting central sensitization; for most (>70%) neurons, the RF expanded caudally into cervically innervated tissues. CONCLUSIONS: These findings provide the first documentation that deep cervical nociceptive inputs can induce central sensitization in medullary and C1/C2 dorsal horns and suggest that these effects may reflect mechanisms contributing to deep cervical pain and its referral.

Cross-desensitization of responses of rat trigeminal subnucleus caudalis neurons to cinnamaldehyde and menthol.

Most cold-sensitive subnucleus caudalis (Vc) neurons are also excited by the TRPM8 agonist menthol and the TRPA1 agonist cinnamaldehyde (CA). We investigated how interactions among menthol, CA and noxious cooling and heating of the tongue affected responses of superficial Vc units recorded in thiopental-anesthetized rats. Units responded to 1% CA which enhanced cold- and heat-evoked responses 5 min later. They responded more strongly to 10% CA which initially depressed cold responses, followed by enhancement at 5 min without affecting responses to heat. Following 10% CA, the mean response to 1% menthol was significantly lower than when menthol was tested first. After menthol, the subsequent response to CA was significantly weaker compared to the mean CA-evoked response when it was tested first. These results demonstrate mutual cross-desensitization between CA and menthol. The response to CA was enhanced following prior application of 10% ethanol (menthol vehicle). Prior application of menthol did not prevent the biphasic effect of 10% CA on cold-evoked responses, nor did prior application of CA prevent menthol enhancement of cold-evoked responses. Responses to noxious heat were unaffected by 10% CA and menthol regardless of the order of chemical presentation. These data indicate that superficial Vc neurons receive convergent input from primary afferents expressing TRPM8 and TRPA1. The mutual cross-desensitization between CA and menthol, and differential modulation of cold- vs. heat-evoked responses, suggests a direct inhibition of TRPM8 and TRPA1 expressed in peripheral nerve endings by CA and menthol, respectively, rather than a central site of interaction.

[Disturbed microvascular permeability in the eye caused by capsaicin microinjections into the trigeminal caudalis nucleus].

Capsaicin microinjection into the trigeminal caudalis nucleus (the central projection area of trigeminal capsaicin-sensitive nerve) increase extravasation of proteins in rat eye. The effect was inhibited by ruthenium red introduction (a capsaicin receptor antagonist) and by blocking the effector functions of capsaicin-sensitive nerve endings. It is suggested that capsaicin stimulation of central terminations of trigeminal capsaicin-sensitive afferents induce an increase in the microvascular permeability of the eye, which is mediated through the effector function of capsaicin-sensitive nerves.

Translating science into the art of acute pain management.

Pain is a multistep process that originates in the peripheral nervous system at the site of injury, is transmitted and processed within the central nervous system, and is perceived at the level of the cerebral cortex. Each of these steps in pain transmission is subject to intervention, with the possibility of reducing or blocking the nociceptive information to result in decreased pain. Based on knowledge of pain processes, dentists can use analgesic strategies to prevent or reduce pain.