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Effects of central nervous system free fatty acids, prostaglandins and lysophospholipids on allodynia in a mouse model of orofacial pain

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b Phase 2 Inflammatory Pain If a noxious stimulus is intense or prolonged, leading to tissue damage and inflammation, there is increased afferent inflow to the CNS from the injured area

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INTRODUCTION

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Phospholipases A2 (PLA2, EC 3.1.1.4) are a diverse group of enzymes

that catalyze hydrolysis of acyl ester bonds at the sn-2 position of the glycerol

moiety of membrane phospholipids, to produce free fatty acids and

lysophospholipids These enzymes are subdivided into several groups

depending upon their structure, enzymatic properties, subcellular localization and cellular function Cytosolic PLA2 (cPLA2) catalyzes the hydrolysis of

arachidonic acid (AA) from neural membrane phospholipids Secretory PLA2(sPLA2) catalyzes the hydrolysis of neural membrane phospholipids with no strict fatty acid selectivity Brain cytosolic fraction also contains an 80 kDa calcium-independent phospholipase A2 (iPLA2) activity which preferentially hydrolyzes linoleoyl acyl chain than palmitoyl and arachidonyl acyl chains from

membrane phospholipids (Yang et al., 1999)

AA is a major unsaturated fatty acid in neural membranes It is released from membrane phospholipids by a number of enzymatic mechanisms involving the receptor-mediated stimulation of PLA2 and phospholipase C / diacylglycerol

lipase pathways (Farooqui et al., 1989) AA can be reincorporated into neural membranes or metabolized to prostaglandins or thromboxanes (Farooqui et al.,

2000; Farooqui and Horrocks, 2006) The metabolites of AA play important

roles in sensitization of dorsal horn circuitry in pain states (Samad et al., 2001;

Svensson and Yaksh, 2002)

Lysophospholipids are important signaling molecules (Sasaki et al.,

1993; Farooqui and Horrocks, 2006), and some have their own receptors

(Bazan and Doucet, 1993; Moolenaar, 1994; Steiner et al., 2002) They can be

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hydrolyzed to fatty acids and glycerophosphocholine or

glycerophosphoethanolamine by lysophospholipases (Farooqui et al., 1985) or

reacylated to the native phospholipids by CoA-dependent or CoA-independent

acyltransferases (Farooqui et al., 2000) These reactions not only prevent an

increase in lysophospholipid levels in brain tissue but also help maintain normal

phospholipid composition (Ross and Kish, 1994; Farooqui et al., 2000) High

concentrations of lysophospholipids may act as detergents to disrupt

membrane structures (Weltzien, 1979) and contribute to neural cell injury

(Farooqui et al., 2000; Farooqui and Horrocks, 2006) A major

lysophospholipid in mammalian brain, lysophosphatidylcholine (LPC) is

metabolized to 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (commonly

known as platelet-activating factor, PAF) The latter is not only involved in inflammatory responses and pathophysiology of many neurodegenerative diseases (Farooqui and Horrocks, 2004) but also plays an important role in pain

sensitivity (Bonnet et al., 1981) Subplantar injections of PAF into the rat

hindpaw increase pain sensitivity (Dallob et al., 1987), whilst systemic

administration of PAF antagonists decreases inflammatory nociceptive

responses in rats (Teather et al., 2002)

Allodynia is defined as innocuous somatosensory stimulation that

evokes abnormally intense, prolonged pain sensations (Kugelberg and

Lindblom, 1959; Lindblom and Verillo, 1979), or pain due to a stimulus that is not normally painful (Walters, 1994) Recent studies have shown that inhibitors

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that received facial carrageenan injections (Yeo et al., 2004) The latter is used

as a model of orofacial pain (Ng and Ong, 2001; Vahidy et al., 2006 under

revision) The PLA2 inhibitors could act by modulating free fatty acids, and their metabolites: prostaglandins, or lysosphospholipid levels, therefore the present study was carried out to determine which of these compounds might have a pro- or perhaps anti-allodynic effect after facial carrageenan injections

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LITERATURE REVIEW

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Pain

Pain is an unpleasant sensory and emotional experience which is primarily associated with tissue damage or describe in terms of tissue damage, or both (International Association for the Study of Pain 2004)

Recent advances in the field of pain research have revealed that pain is not

a single sensory experience; different forms of pain are mediated by different neurological mechanisms Moreover it has been argued that the

neurophysiological mechanisms for all the various pain states are not the same and that normal (nociceptive) and abnormal (neuropathic) pain represent the endpoints of a sequence of possible changes that can occur in the nervous system Normally, a steady state is maintained in which there is a close

correlation between injury and pain But changes induced by nociceptive input

or by changes in the environment can result in variations in the quality and quantity of the pain sensation produced by a particular noxious stimulus

These changes are temporary as the system would always tend to restore the normal balance However, long lasting or very intense nociceptive input would distort the nociceptive system to such an extent that the close correlations between injury and pain would be lost

There are three major stages or phases of pain, each with a different

neurophysiological mechanism These are (1) the processing of a brief noxious stimulus; (2) the consequences of prolonged noxious stimulation, leading to tissue damage and peripheral inflammation; and (3) the consequences of

neurological damage, including neuropathies and central pain states

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1 Phases of Pain

There are three phases of pain known:

(a) Phase 1 (Acute Nociceptive Pain)

The mechanism involved can be viewed as a simple and direct route of transmission centrally toward the thalamus and cortex and thus the conscious perception of pain, however there is possibility of modulation occurring at

synaptic relays along the way It has been suggested that Phase 1 pain can best be explained by models based on the specificity interpretation of pain mechanisms, that is, the existence within the peripheral and central nervous systems (CNS) of a series of neuronal elements concerned solely with the processing of these simple noxious elements

(b) Phase 2 (Inflammatory Pain)

If a noxious stimulus is intense or prolonged, leading to tissue damage and inflammation, there is increased afferent inflow to the CNS from the injured area due to the increased activity and responsiveness of sensitized

nociceptors In this phase, the subject experiences spontaneous pain, a

change in the sensations evoked by stimulation of the injured area, and also of the undamaged areas surrounding the injury This change in evoked sensation

is known as hyperalgesia, defined as an increased response to a stimulus which is normally painful (IASP 2004) or a leftward shift of the stimulus-

response function that relates magnitude of pain to stimulus intensity or an increased response to a stimulus that is normally painful

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Many cases of hyperalgesia have features of allodynia The term allodynia pertains when there is not an increased response to a stimulus that normally provokes pain However, when there is also a response of increased pain to a stimulus that normally is painful, hyperalgesia is the appropriate word With allodynia the stimulus and the response are in different modes, whereas with hyperalgesia they are in the same mode (IASP 2004) Hyperalgesia in the area

of injury is known as primary hyperalgesia, and in the area of normal tissue surrounding the injury site, as secondary hyperalgesia

(c) Phase 3 (Neuropathic Pain)

These are abnormal pain states and are defined as pain initiated or caused

by a primary lesion or dysfunction in the nervous system In clinical terms, Phase 1 and 2 pains are symptoms of peripheral injury, whereas Phase 3 pain

is a symptom of neurological diseases that include lesions of peripheral nerves

or damage to any portion of the somatosensory system within the CNS These pains are spontaneous, triggered by innocuous stimuli, or are exaggerated responses to noxious minor stimuli

2 Role of Peripheral Mechanism of Hyperalgesia

An injury to the skin or to an internal organ evokes the initial discharge in the nociceptive afferents that innervate the damaged area and, as a

consequence of the ensuing inflammatory process, sensitizes these nociceptive

endings (Treede et al., 1992) During the initial injury and for the duration of the

repair process there will be increased nociceptive activity from the injured

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region It is well known that sensitized nociceptors respond to peripheral stimuli with a lower threshold and an increased excitability, hence the possibility that the afferent discharges during the inflammatory process will be greater in

magnitude and duration than the initial injury-related storm

These afferent storms cause, in turn, central changes in excitability mediated by positive feedback loops between spinal and supra spinal neurons and by the enhanced synaptic actions of certain neurotransmitters, possibly

involving N-methyl-D-aspartate (NMDA) receptor mechanism (Woolf and

Thompson, 1991; Dubner and Ruda, 1992; Cervero, 1995) The central

changes are maintained by the incoming discharges in sensitized nociceptors

so that, in the absence of such discharges, the central alterations decline and the system returns to normal sensory processing

There is an increase in the afferent inflow to the CNS from damaged or inflamed areas due to the increased activity and responsiveness of sensitized nociceptors Moreover, the nociceptive neurons in the spinal cord modify their responsiveness and increase their excitability (Woolf and King, 1989; Cervero

et al , 1992; Dubner and Ruda, 1992; Woolf et al., 1994) All of these changes

indicate that due to the noxious input generated by the tissue injury and

inflammation, the CNS has moved to an excitable state

Primary Hyperalgesia and Sensitization

Within the area of primary hyperalgesia, low intensity mechanical or thermal stimuli evoke pain It is known that an injury induces a process of

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but lowered thresholds (Burgess and Perl, 1967; Bessou and Perl, 1969; Meyer and Campbell, 1981) Sensitization is defined as a leftward shift of the

stimulus-response function that relates magnitude of the neural response to stimulus intensity. The sensitization of the peripheral nociceptors is

characterized by two main changes in their response properties: appearance of spontaneous activity that provides a continuous afferent barrage that is

believed to contribute to spontaneous pain and decrease in threshold to an extent that non-noxious stimuli will activate the sensitized receptor The drop in

threshold is generally agreed to underlie primary hyperalgesia (Treede et al.,

1992)

Mechanical Hyperalgesia

Hyperalgesia to mechanical stimuli are of two different types One form

is evident when the skin is gently stroked with a cotton swab and may be called stroking hyperalgesia, dynamic hyperalgesia, or allodynia The second form is evident when punctuate stimuli, such as von Frey probes, are applied and thus has been turned punctuate hyperalgesia

Secondary Hyperalgesia

Primary hyperalgesia is characterized by the presence of enhanced pain

to heat and mechanical stimuli, whereas secondary hyperalgesia is

characterized by enhanced pain to only mechanical stimuli The changes responsible for secondary hyperalgesia have two different components: (1) a change in the modality of the sensation evoked by low-threshold

mechanoreceptors, from touch to pain, and (2) an increase in the magnitude of

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the pain sensations evoked mechanical sensitive nociceptors (LaMotte et al., 1991; Cervero et al., 1994) The first change results in touch-evoked pain

(allodynia); that is the perception of painful sensations following the activation

of low-threshold mechanoreceptors The second alteration produces

hyperalgesia These changes are mediated by alterations in the central

processing of the sensory input and are induced by the arrival of the initial

injury-related afferent storm, in the CNS (LaMotte et al., 1991; Torebjork et al.,

1992) Importantly, there is no thermal allodynia in areas of secondary

hyperalgesia (Raja et al., 1984; LaMotte et al., 1991)

Activation of the nociceptors leads to a flare response which extends well outside the area of initial injury Activation of one nociceptor leads to the release of excitatory neuropeptide, substance P Tachykinin is the adopted generic name for the family of peptides to which substance P belongs There are three main mammalian tachykinin peptides: substance P itself, neurokinin

A, and neurokinin B, although in the animal kingdom there are many other peptides that are structurally and functionally similar (Iversen, 1994) Elevated levels of substance P are found in the periphery following nerve injury

(Donnerer et al., 1993; Carlton et al., 1996) It is released only after intense, constant peripheral stimuli (Morton et al., 1990) It has been seen that by using

substance P itself, or close analogues, intrathecal injections can be shown to produce hyperalgesia to a variety of noxious stimuli (Cridland and Henry,

1986) In the dorsal horn, both nociceptive and low-threshold neurons are

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3 Role of Central Mechanism of Hyperalgesia

Capsaicin is a naturally occurring vanilloid, that selectively activates and

at higher doses selectively deactivates, and ultimately damages, several types

of fine sensory C and Aδ-fibers Intense experimental pain produced by a burn injury, or by chemical stimulation of nociceptors with capsaicin changes the responsiveness of an area of undamaged skin surrounding the injury site such that normally innocuous stimuli, such as brushing and touch, are painful

(allodynia), and normally mild painful stimuli, such are pinprick, are more painful

(hyperalgesia) (LaMotte et al., 1991) This phenomenon of “secondary

hyperalgesia” is now known to be due to a central change, induced and

maintained by input from nociceptors, such that activity in large myelinated afferents connected to low-threshold mechanoreceptors provokes painful

sensations as well as tactile sensations (Torebjork et al., 1992), and input from nociceptors evokes enhanced pain sensations (Cervero et al., 1994)

Animal studies show that the expression of both spontaneous pain and hyperalgesia are dependant on ongoing afferent activity from the injury site

(LaMotte et al., 1991; Torebjork et al., 1992)

Central Sensitization

Experiments in laboratory animals have provided evidence for the

concept that noxious input to the CNS sets off a central process of

enhancement of responsiveness that continues independently of peripheral afferent drive Short periods of electrical stimulation at noxious intensity

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produces increased excitability The time course of the increased excitability is similar to that of short-term potentiation in single dorsal horn neurons (Carvero

et al , 1984; Cook et al., 1987) Increases in excitability of dorsal horn

C-fiber-evoked potentials, with a time course similar to that of long term potentiation, have also been described (Liu and Sandkuhler, 1995) These increases in central excitability are also referred to as “central sensitization” (Woolf, 1983; Woolf and Thompson, 1991) Central sensitization refers to the increased synaptic efficacy established in somatosensory neurons in the dorsal horn of the spinal cord following intense peripheral noxious stimuli, tissue injury or nerve damage This heightened synaptic transmission leads to a reduction in pain threshold, an amplification of pain responses and a spread of pain

sensitivity to non-injured areas (Ji et al., 2003)

4 Trigeminal System

(a) Trigeminal Nerves and Ganglion

Peripheral Nerves

Trigeminal nerve of fifth cranial nerve is a general sensory nerve

carrying touch, temperature nociception, and proprioception from the superficial and deep structures of the face It contains both sensory (general somatic afferent [GSA]) and motor (special visceral efferent [SVE]) fibers The sensory innervations are provided to the face and oral cavity and the motor innervations

to the muscles of mastication The three main trigeminal nerve divisions:

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cranial nerves (reviewed Shankland, 2000) The mandibular division is the largest with some 78,000 myelinated fibers compared with 50,000 fibers in the

maxillary and only 26,000 fibers in the ophthalmic divisions (Pennisi et al.,

1991) Each division supplies a distinct dermatome on the head and face and the adjacent mucosal and meningeal tissues (Brodal, 1965; reviewed Usunoff

et al., 1997) The ophthalmic innervates the forehead, dorsum of the nose, upper eyelid, orbit (cornea and conjunctiva), mucous membranes of the nasal vestibule and frontal sinus, and the cranial dura The maxillary nerve

innervates the upper lip and cheek, lower eyelid, anterior portion of the temple, paranasal sinuses, oral mucosa of the upper mouth, nose, pharynx, gums, maxillary teeth, hard palate, soft palate, and cranial dura The mandibular nerve has a sensory and a motor component The motor component

innervates the muscles of mastication; the temporalis, masseter, lateral and medial pterygoids, mylohyoid, the anterior belly of the digastric muscle, the tensors tympani and veli palatine The sensory component innervates the lower lip and chin, posterior portion of the temple, external auditory meatus and tympanic membrane, external ear teeth of the lower jaw, oral mucosa of the cheeks and the floor of the mouth, anterior two thirds of the tongue,

temporomandibular joint and cranial dura

Fibers from the autonomic nervous system join trigeminal nerve to reach peripheral tissues Sympathetic fibers from the superior cervical ganglion join the peripheral trigeminal nerves to reach the sweat and mucosal glands in the facial skin and oral and nasal cavities Each trigeminal branch also receives

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parasympathetic fibers: the ophthalmic nerve from the ciliary ganglion, the maxillary from the pterygopalatine ganglion, the mandibular nerve from the submandibular and otic ganglia

Trigeminal Ganglion

The trigeminal (Gasserian) ganglion is crescent shaped and lies in the Meckel’s cave adjacent to the petrosal bone Ganglion cells are

pseudounipolar and invested by satellite cells, with some showing complex

interdigitations with the neuronal membrane (Beaver et al., 1965) In the dorsal

root ganglia, trigeminal ganglion cells can be classed as large, light (type A) cells; smaller, dark (type B) cells (Lieberman, 1976) and small type C cells (Kai-Kai, 1989) A variety of peptides are known to be present in the ganglion, particularly in the smaller cells For the humans, these include calcitonin gene related peptide (CGRP), substance P, somatostatin, galanin and enkephalins (Del Fiacco and Quartu, 1994; Quartu and Del Fiacco, 1994) In the human ganglion, nearly half of the cells contain CGRP, with about 15% containing substance P, and some showing colocalization (Helme and Fletcher, 1983;

Quartu et al., 1992) Such cells are thought to be associated with nociceptive

transmission Besides the peptides, another transmitter for the trigeminal

ganglion, like dorsal root ganglion, is likely to be glutamate (Wanaka et al.,

1987)

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(b) Trigeminal Sensory Nuclei

The brainstem trigeminal sensory nuclei consist of the trigeminal sensory nuclear complex (the principal and spinal trigeminal nuclei), the mesencephalic nucleus (Me5), and a number of smaller collections of cells (paratrigeminal and peritrigeminal nuclei), which are thought to be sensory in function The motor trigeminal and intertrigeminal nuclei are associated with the motor functions of the jaw muscles

Trigeminal Sensory Nuclear Complex

The trigeminal nuclear complex is in the dorsolateral brainstem and extends from the rostral pons to the C2 level The complex is subdivided into for subnuclei; the principal or main sensory trigeminal nucleus (Pr5) and the three spinal trigeminal nuclei- oralis (Sp5O), interpolaris (Sp5I) and caudalis (Sp5C) (Capra and Dessem, 1992) A major transmitter throughout the

sensory complex is glutamate, with NMDA and non-NMDA receptors at all

levels (Petralia et al., 1994; Tallaksen-Greene et al., 1992) In addition

peptides such as substance P and CGRP are important transmitters (Sessle, 2000)

Principal Sensory Trigeminal Nucleus

Pr5 is located in the lateral pontine tegmentum It is narrow and

elongated dorsoventrally with mostly small neurons; large neurons are seen along the lateral border Animal studies have shown that cells in Pr5 are

mechanoreceptive with low thresholds and small receptive fields (Jacquin et al.,

1988) It is therefore thought to be analogous to the dorsal column nuclei in

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providing for discriminative tactile sensations for the face Pr5 contains many projection neurons, with its predominant projection being to the ventroposterior medial nucleus (VPM) of the thalamus

Spinal Trigeminal Nucleus Oralis

Sp5O has been shown to receive extensive intraoral projections

(Arvidsson and Gobel, 1981; Takemura et al., 1991) and this is consistent with loss of oral sensation after vascular lesions in humans (Graham et al., 1988)

Responses generally have very widespread receptive fields and can show modality convergence, for example from both cutaneous and tooth receptors (Jacquin and Rhoades, 1990) In the rat Sp5O has extensive projections to the facial motor nuclei and spinal cord with less dense projections to the trigeminal

motor nucleus, thalamus and cerebellum (Ruggiero et al., 1981; Jacquin and

Rhoades, 1990)

Spinal Trigeminal Nucleus Interpolaris

Located in the medulla, Sp5I has extensive inputs from the intraoral

structures, including tooth pulp (Takemura et al., 1991) Cells responsive to

both low-threshold mechanoreceptors and nociceptors in the skin and the

periodontium have been described in rats (Jacquin et al., 1989) Sp5I projects

to the thalamus, cerebellum, superior colliculus and spinal cord (Phelan and Falls, 1991)

Spinal Trigeminal Nucleus Caudalis

The Sp5C extends from the obex to the C2 level where it becomes

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threshold nociceptive specific responses, thermosensitive specific responses, HPC (heat, pinch, cold) cells, and wide dynamic range neurons are all present

in Sp5C (Hu, 1990; Sessle, 2000)

Mesencephalic Trigeminal Nucleus

The Me5 comprises a band of scattered cells that extends from the level

of rostral Pr5 through the pons and midbrain Me5 cells project to the motor trigeminal nucleus forming a monosynaptic reflex arc There are also

projections to Pr5, which may provide a relay to the thalamus for proprioceptive

sensation (Lou et al., 1991, 1995) Projections to various brainstem nuclei as

well as cerebellum and spinal cord have also been studied in animals

(Raappana and Arvidsson, 1993; Shigenaga et al., 1990)

(c) Ascending Trigeminothalamic Tracts

These are of two types, ventral and dorsal, which convey the GSA information from the face, oral cavity and the dura mater to the thalamus

The ventral trigeminothalamic tract is a pathway for pain sensation, temperature and light touch sensations from the face and oral cavity It also contains GSA fibers from the cranial nerves VII, IX and X it receives input from the free nerve endings and Merkel tactile disks The ventral tract ascends to the contralateral sensory cortex via three the first, second and third-order neurons The second-order neurons are located in the trigeminal nucleus and mediate painful stimuli and are found in the caudal third of the spinal trigeminal nucleus They give rise to decussating axons that terminate in the contra

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lateral ventral posterio-medial (VPM) nucleus of the thalamus The third-order neurons are located in the VPM nucleus of the thalamus and project via the posterior limb of the internal capsule to the face area of the post-central gyrus

The dorsal trigeminothalamic tract is an uncrossed tract which sub serves the discriminative tactile and pressure sensation from the face and oral cavity via the GSA fibers of cranial nerve V It receives input from Meissner and Pacini corpuscles It ascends to the sensory cortex via the first, second and third-order neurons The second-order neurons are located in the principal sensory nucleus of cranial nerve V and project to the ipsilateral VPM nucleus of the thalamus The third-order neurons are located in the VPM nucleus and project via the posterior limb of the internal capsule to the face area of the post-central gyrus

5 Orofacial Pain

Diagnosis of orofacial pain is complicated because of the region’s

density of anatomical structures, rich innervations and high vascularity Most prevalent pain in this area originates from the teeth and their surrounding

structures Pain can arise due to local injury that can result from trauma,

infection and neoplasms Neuralgic pain is generally expressed in the facial area as idiopathic trigeminal neuralgia

The ill-defined category of atypical oral and facial pain includes a variety

of pain descriptions like phantom tooth pain (Turp, 2005), atypical odontalgia

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(Marbach et al., 1982; Aguggia, 2005), and the burning mouth syndrome

(Grushka et al., 2003) The pains in these cases usually have a burning quality

that occasionally intensifies to produce throbbing sensation The pain is not triggered by remote stimuli, but may be intensified by stimulation of the affected area itself

6 Tissue Injury Models of Persistent Pain

There are different models of tissue injury and inflammation that produce responses that mimic human clinical pain conditions in which the pain lasts for longer periods of time for example formalin injected beneath the footpad of a rat

or a cat (Dubuisson and Dennis, 1977) Models of inflammation that produce more persistent pain include the injection of carrageenan or complete Freud’s

adjuvant into the footpad (Iadarola et al., 1988) or into the joint of the limb (Schaible et al., 1987) or facial carrageenan injections (Ng and Ong, 2001; Yeo

et al., 2004) These models result in rapid, short-lasting, acute pain responses similar in duration to the formalin method In the inflammation model elicited by injection of carrageenan into the footpad, the cutaneous inflammation appears within 2 hours and peaks within 6-8 hours Hyperalgesia and edema are

present for approximately 1 week to 10 days The physiological and

biochemical effects are limited to the effected limb (Iadarola et al., 1988) and

there are no signs of systemic disease

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7 Brainstem Mechanism in Trigeminal Nociception

Nuclear Localization of Nociceptive Responses

Clinical findings are consistent with the findings in experimental animals indicating that Sp5C is the most important component of the trigeminal nuclear complex for perception of noxious stimuli applied to craniofacial region (Sessle, 2000) Cells in lamina 1 respond to noxious mechanical, thermal and chemical stimulation of structures such as the cornea, cerebral vasculature, oral and nasal mucosa, teeth and temporomandibular joint

Besides the involvement of Sp5C in trigeminal nociceptive activity,

recordings from Sp5O and Sp5I indicate nociceptive responses are present in more rostral levels of the spinal trigeminal complex Lesions or injections of analgesic chemicals into these levels can also interfere with nociceptive

behavior, particularly if the stimulus is applied to intra- or perioral regions (Dallel

et al , 1998; Takemura et al., 1993) It has also been suggested that Sp5O is

particularly important for processing information about sort duration nociceptive stimuli, whereas Sp5C is more important for processing tonic nociceptive

information (Raboisson et al., 1995)

Nociceptive Transmitters

The afferents that terminate in Sp5C contain neuropeptides and amino acids that have been implicated as excitatory neurotransmitters or

neuromodulators (e.g substance P, glutamate, nitric oxide) in central

nociceptive transmission (Sessle, 2000) Antagonism of the substance P

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chemical stimulation of dural afferents (Shepheard et al., 1995) Studies have

also indicated central involvement of excitatory amino acids in trigeminal

nociception (Parada et al., 1997)

8 Methods of Assessing Pain in Animals

Pain research and therapy during the past century evolved from

Descartes’ concept of pain as a direct transmission system from ‘pain

receptors’ in the body tissues to a ‘pain center’ in the brain It is assumed that injury or any other pathology will lead to pain Due to this the early history of pain measurement was focused on the psychophysical relationship between the extent of the injury and perceived pain Nearly all studies of pain, till the publication of the gate control theory of pain (Melzack and Wall, 1965),

concentrated on the measurement of the pain intensity However the gate control theory of pain led to the recognition that pain rarely has a one-to-one relationship to a stimulus Acute pain can be proportional to the extent of the injury but along with the contribution of psychological factors, complex

relationships are revealed that are influenced by fear, anxiety, cultural

background and the meaning of the situation to the person (Melzack and Wall, 1988)

Pain, acute or chronic, has a distinctly unpleasant affective quality It becomes overwhelming and disrupts ongoing behavior and thought The measurement of pain is therefore essential to determine pain intensity, quality and duration, to aid in diagnosis and to help decide on the choice of therapy

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Recent advances in the technology of neuro-imaging have reinforced the concept that the realization of pain in humans is a comprehensive process that involves the parallel integration of sensory, emotional and perceptual noxious information by multiple brain structures (Rainville, 2002) Similar brain

structures are involved in the process of nociception and related expression of nociceptive behaviors in injured animals (Chang and Shyu, 2001) Thus,

spontaneous and/or evoked nociceptive behaviors in animals are described frequently as either ‘pain’ or ‘pain-like’ behaviors A recognized problem

associated with testing the analgesic properties of pre-existing drugs and new chemical entities in animal nociceptive models, is that the experimenter is usually required to evoke a reflex threshold-pain response using a range of noxious stimuli, principally mechanical, but also thermal and chemical

Standard evaluation methods in use are the hot-plate and tail-flick tests (Le

Bars et al., 2001) and the use of von Frey monofilaments to assess mechanical

allodynia These methods test for the presence of pain-like behaviors at a particular moment in time The initial diagnosis of neuropathic pain in humans utilizes the gentle brushing of soft brushes and cotton buds across the skin to assess the presence and spread of positive signs of sensory disturbance This dynamic, ‘brush-evoked’ allodynia is mediated by low-threshold-fiber inputs; unlike punctuate stimuli such as von Frey monofilaments, which assess the presence of static, mechanical allodynia that appears to be mediated by high-

threshold-fiber inputs (Field et al., 1999; Dworkin et al., 2003)

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9 Phospholipase A 2

Phospholipase A2 (PLA2) belongs to a family of enzymes that catalyzes

the hydrolysis of the sn-2 position of membrane glycerophospholipids to

liberate arachidonic acid (AA), a precursor of eicosanoids and leukotrienes The same reaction also produces lysophosholipids, which represent another class of lipid mediators (Murakami and Kudo, 2002) These enzymes are divided into 14 groups that comprise four major types: the cytosolic, calcium-dependant PLA2s (cPLA2), the cytosolic, calcium-independent PLA2s (iPLA2), the secreted, calcium-dependant PLA2s (sPLA2) and plasmalogen-selective phospholipase A2 (PlsEtn-PLA2), (Six and Dennis, 2000; Balsinde et al., 2002;

Murakami and Kudo, 2002) It has been found that inhibitors to secretory phospholipase A2 (sPLA2): 12-epi-scalaradial, cytosolic phospholipase A2

(cPLA2): AACOCF3, or calcium-independent phospholipase A2 (iPLA2): BEL,

significantly decrease allodynia in a mouse model of orofacial pain (Yeo et al.,

2004) However it was not determined whether the effect of PLA2 inhibition was due to inhibition of formation of free fatty acids or lysophospholipids

10 Free Fatty Acids

Arachidonic acid (AA) is a major unsaturated fatty acid in neural

membranes It is released from membrane phospholipids by a number of enzymatic mechanisms involving the receptor-mediated stimulation of PLA2and phospholipase C / diacylglycerol lipase (PLC/ DAG lipase) pathways

(Farooqui et al., 1989) Linoleic acid, an 18-carbon, n-6 essential fatty acid, is

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the major precursor of AA, which is a 20-carbon, n-6 fatty acid with four double bonds AA is converted to prostaglandins, leukotrienes and thromboxanes, collectively called eicosanoids; which differ from other intracellular messengers

in one important way: they can cross the cell membrane and leave the cell in which they are generated to act on neighboring cells because of their

amphiphilic nature (Wolfe and Horrocks, 1994) AA is involved in normal

receptor function and signal transduction High concentrations of AA are

known to produce a variety of detrimental effects on neural membrane

structures and activities of membrane bound enzymes (Chan et al., 1983; Yu et

al., 1986; Farooqui et al., 1997) However central effects of AA on behavioral

patterns in animals were unknown till the present study Incubation of AA with cortical brain slices produced brain edema (Chan and Fisherman, 1978) and it inhibits the re-uptake of glutamate in primary cultures of neurons and

astrocytes and induces astrocytic swelling in vitro (Yu et al., 1986) Moreover it

has been found that AA can also be reincorporated into neural membranes

(Farooqui et al., 2000, Farooqui and Horrocks, 2006) It has been reported that intracerebroventricular (i.c.v.) injection of AA per se had no effect on pain

perception in rats (Murillo-Rodriguez et al., 1998) Injection of the vaccine

preservative, thimerosal, also resulted in an anti-nociceptive effect, presumably

by raising arachidonic levels (Ates et al., 2003)

Oleic acid (OA) is released by astrocytes and is used by neurons for the synthesis of phospholipids and is specifically incorporated into growth cones

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(Tabernero et al., 2001) OA promotes axonal growth, neuronal clustering, and induces neuronal differentiation (Tabernero et al., 2001)

DHA and EPA are released from plasmalogens (vinylether containing phospholipids) by the action of plasmalogen-selective-PLA2 (Farooqui and Horrocks, 2004) EPA protects hippocampus from age-related and irradiation-induced changes that lead to impairment in synaptic function; the evidence suggests that this is due to its anti-inflammatory effects, specifically preventing changes induced by the pro-inflammatory cytokine, interleukin-1beta (IL-1beta)

(Lonergan et al., 2004) DHA has an important role in neuronal development; it

promotes neuronal differentiation in rat embryonic hippocampal primary

cultures (Calderon and Kim, 2004)

11 Eicosanoids/ Prostaglandins

AA is a major precursor of eicosanoids, which refers collectively to group

of oxygenated 20-carbon compounds, including prostaglandins, thromboxanes

and leukotrienes (Smith et al., 1991) The metabolites of AA, including PGs,

play important roles in sensitization of dorsal horn circuitry in pain states

(Samad et al., 2001; Svensson and Yaksh, 2002) Primary PGs play a direct

role in neural activity by modulating the release of neurohormones and

neurotransmitters, whereas others are involved in regulating circulatory

functions (Wainwright, 1997) Intraspinal administration of cyclooxygenase-2 (Cox-2) inhibitor decreases inflammation-induced central Prostaglandin E2(PGE2) levels and mechanical hyperalgesia (Samad et al., 2001) PGE2 and

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the receptor for PGE2 (EP receptor) are key factors contributing to the

generation of hyperalgesia caused by inflammation, it has been found that activation of spinal EP1 receptors are crucial in a carrageenan-induced

mechanical hyperalgesia model and that some of the mechanisms underlying inflammation-induced plastic changes are mediated by time-dependent

increase in PGE2 concentration, activation of EP1 receptors, and increase in [Ca2+]i in the spinal dorsal horn (Nakayama et al., 2002) More studies have

found that spinal PGE2 activates the EP1 receptors existing on the central terminals of primary afferents, subsequently increasing [Ca2+]i in the spinal dorsal horn, which are involved in the mechanisms of spinal PGE2-induced

nociceptive transmission (Nakayama et al., 2004)

12 Lysophospholipids

Lysophospholipids are generated along with fatty acids during the

hydrolysis of membrane phospholipids by phospholipases A1 (EC 3.1.1.32) and

A2 (PLA2) (EC 3.1.1.4) These enzymes hydrolyze acyl groups from the sn-1 and sn-2 positions of the glycerol moiety of phospholipids respectively The

lysophospholipids can then be hydrolyzed to fatty acid and

glycerophospho-base by lysophospholipases (Farooqui et al., 1985) or reacylated to the native

phospholipids by CoA-dependent or CoA-independent acyltransferases

(Farooqui et al., 2000) These reactions help to prevent an increase in

lysophospholipid levels in brain tissue (Ross and Kish, 1994; Farooqui et al.,

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calcium but also increase neural membrane fluidity and permeability At higher concentrations, lysophospholipids disrupt membrane structures by acting as detergents (Weltzien, 1979) and may be responsible for neural cell injury and

neurodegeneration (Farooqui et al., 2000, Farooqui and Horrocks, 2006)

Lysophosphatidylcholine (LPC) is a major lysophospholipid in

mammalian brain and is the major component of oxidized low-density

lipoprotein (Kugiyama et al., 1990; Mangin et al., 1993) LPC is well known for

injuring myelin sheaths by rehydration at the intraperiodic line (Blakemore, 1977; Hall, 1972) However little is known about its central effect on allodynia

tissue - a remodeling pathway, de novo synthesis, and an oxidative

fragmentation pathway (Farooqui and Horrocks, 2004) PAF is involved in inflammatory responses and pathophysiology of many neurodegenerative diseases (Farooqui and Horrocks, 2004) Exogenous PAF-acethar also plays

an important role in pain sensitivity and has ability to generate signs of

inflammation and hyperalgesia by standard subplanter injections in the rat paw

(Bonnet et al., 1981) and increases pain sensitivity (Dallob et al., 1987) while

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systemic administration of PAF antagonists decreases inflammatory

nociceptive responses in rats and might be useful for treating patients with

acute or chronic pain (Teather et al., 2002)

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OBJECTIVES

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Recent studies of NUS laboratories have shown that inhibitors to

secretory phospholipase A2 (sPLA2): 12-epi-scalaradial, cytosolic

phospholipase A2 (cPLA2): arachidonoyl trifluoromethane, and

calcium-independent phospholipase A2 (iPLA2): bromoenol lactone, exerted pronounced anti-nociceptive effects in mice that received facial carrageenan injections (Yeo

et al., 2004) The latter was used as a model of orofacial pain (Ng and Ong, 2001) The PLA2 inhibitors could act by modulating free fatty acids, their

metabolites or lysosphospholipid levels, therefore the present study was carried out to determine which of these compounds might have a pro- or perhaps anti-allodynic effect after facial carrageenan injections

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HYPOTHESES

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Null Hypothesis

There is no difference in behavioral responses of mice injected with facial carrageenan and intracerebroventricular free fatty acids, prostaglandins and lysophospholipids compared to control mice

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MATERIALS

AND METHODS

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1 Ethical concerns

Animals in this study were cared for and treated according to the ethical standards and guidelines for investigations of experimental pain in animals prescribed by the Committee for Research and Ethical Issues of the

International Association for the Study of Pain (1983) Keeping in consideration the discomforting disorder, the number of animals studied was restricted to n =

4 per group All procedures involving the mice were reviewed and approved by the Institutional Animal Care and Use Committee

2 Chemicals

The compounds tested in this study include four free fatty acids, four lysophospholipids and one PAF antagonist

Oleic acid {OA, CH3(CH2)7CH=CH(CH2)7COOH}, arachidonic acid (AA,

C20H32O2), eicosapentaenoic acid (EPA, C20H30O2), docosahexaenoic acid (DHA, C22H32O2); lysophosphatidylethanolamine (LPE), lysophosphatidic acid (LPA, C21H39Na2O7P), and lysophosphatidylcholine (LPC), were purchased from Sigma (St Louis, USA) Platelet-activating factor (PAF, C26H55N2O7P), Prostaglandin E1 (PGE1, C20H34O5), Prostaglandin E2 (PGE2, C20H32O5) and Prostaglandin E3 (PGE3, C20H30O5) were purchased from Cayman Chemicals (Ann Arbor, USA) Ginkgolide B (a PAF receptor antagonist, C20H24O10) was purchased from Biomol International (Plymouth Meeting, USA)

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Dimethyl sulfoxide (DMSO, Sigma) was used as a vehicle for free fatty acids, LPE, LPA, PAF and gingkolide B Normal saline was used as a vehicle for LPC, since the latter is insoluble in DMSO

Lambda carrageenan was also purchased from Sigma (St Louis, USA)

3 Subjects

Sixty adult male Balb/c mice (7-8 weeks of age; 25-30g at arrival) were purchased from the Laboratory Animal Centre, Singapore The mice were housed in stainless steel cages (6 mice per cage) under a 12:12 hr dark/light cycle in an animal house with an ambient temperature of 25 ± 2°C Water and

food were available ad libitum

They were randomly assigned to one of fifteen groups Each group consists of four mice All mice were labeled with a coding system to allow repeated readings of the behavioral responses of an individual mouse to be followed before injection, and at different time intervals after injection The entire study was carried out in 10 batches The investigator was blinded to the type of compound injected in each mouse

4 Surgery

The surgery consists of two parts: the intracerebroventricular injection and the facial carrageenan injection

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(a) Intracerebroventricular injection

These were carried out to test possible effects of free fatty acids,

prostaglandins, lysophophospholipids and a PAF receptor antagonist on

carrageenan induced allodynia (explained below) These compounds

presumably diffuse via the cerebrospinal fluid to act on the trigeminal nuclei in the medulla oblongata Allodynia was quantified by measuring the response of mice to facial mechanical stimulation by Von Frey hair filaments

Mice were deeply anesthetized with intraperitoneal injection of 0.2-0.3 ml

of 3.5% chloral hydrate This was followed by asepsis and a midline scalp incision to expose the cranial vault A small bur hole was made in the skull, and 5 µl of a 2 mM solution (i.e 10 nmol) of free fatty acids, prostaglandins, lysophospholipids or vehicle controls were injected into the right lateral ventricle over 1 minute, using a microliter syringe mounted on a stereotaxic apparatus (coordinates: 0.7 mm caudal to bregma, 1.0 mm lateral to midline, 3.0 mm from the surface of the cerebral cortex) The needle was withdrawn 10 min later, and the scalp sutured The injection of this amount of solution is estimated to result in final extracellular concentration of 5 µM of compounds, assuming diffusion in the cerebrospinal fluid and distribution in 1.5 ml of central nervous

tissue This concentration is similar to that previously used in vivo, where

approximately 5 µM of lysophospholipids was shown to have biological effects

(Nishikawa et al., 1989; Lee et al., 2004)

Verification of injection site: These coordinates had earlier been verified by

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(b) Facial carrageenan injection

All mice received a facial injection of carrageenan to induce pain

sensitization after the i.c.v injections This was carried out by injecting 50 µl of

a 2 mg / 50 µl saline solution of lambda carrageenan into the subcutaneous tissue over the right maxilla (Ng and Ong, 2001) while the mice were still under anesthesia The injection of carrageenan produced a constant swelling of approximately 4mm in diameter, and is presumed to cause allodynia via

activation of the trigeminothalamic and thalamocortical pathways

5 Behavioral testing by applying mechanical stimulation

Mice were tested 24hr before and 8, 24 and 72 hr after the surgery Testing was conducted from 0830 to 1400 hr The testing procedure consisted

of assessment of the animal’s response to mechanical stimulation of the face

(a) Stimulus

For mechanical stimulation a von Frey hair (Touch-Test Sensory

Evaluator, North Coast Medical, Morgan Hill, USA) was used The von Frey

hair consisted of plastic monofilament of length 4cm for which the force

required to bend was approximately 1.4 g (or converted to log units, 4.17 log units) The stimulus was applied over the subcutaneous tissue of the right maxillary region (the location of injected carrageenan)

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(b) Testing procedures

To observe the response to mechanical stimulation, mice were tested individually in a deep rectangular stainless steel tank (60 X 40 X 25cm) Before the actual stimulation session began, the mice were habituated to the tank for

at least 10 minutes The observer reached into the cage with the von Frey Hair and touched the walls every 30 seconds These reaching movements were carried out slowly Once the animal was habituated, the stimulations were applied when the mouse was in a no locomotion state, with four paws placed

on the ground, neither moving nor freezing, but exhibiting sniffing behavior A new stimulus was applied only when the mouse resumed this position and at least 30 seconds after the preceding stimulation The experimenter was

blinded to the treatments allocated to the mice

During the testing session of each mouse the carrageenan-injected area

of the face was probed 20 times with the von Frey hair filament The scoring

procedure used in this study has been modified from Vos et al., 1994

(c) Categories of Response

The four elements of response have been previously described (Vos et

al., 1994) as follows: 1) detection: mouse turns head toward stimulating object, and the stimulus object is then explored (sniffing, licking), 2) withdrawal

reaction: mouse turns head slowly away or pulls it briskly backward when stimulation is applied, 3) escape/ attack: mouse avoids further contact with the stimulus object, either passively, by moving its body away from the stimulating

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the head buried under the body, or actively by attacking the stimulus object making biting or grabbing movements, 4) asymmetric face grooming: mouse displays an uninterrupted series of face-wash strokes directed to the stimulated facial area

Response calculation by Vos et al., (1994)

In the study by Vos et al., (1994), it was observed that the rats

performed either one or more of the above mentioned elements of behavioral response in the exact same order In addition, a withdrawal reaction was assumed to include a ‘detection’ element preceding the head withdrawal

reaction, therefore consisting of two response elements Similarly a ‘face grooming’ reaction included the first three elements of behavioral response, hence consisting of four response elements Therefore they formulated rank-ordered descriptive response categories: no response, mild aversive response, strong aversive response and prolonged aversive response; assigning each response category a grade from 0-4 respectively This way the ‘response’ was classified in an ordinal scale

Response calculation in this study

The responses observed after each stimulus in this study were scored based on the same criteria for increasing nociceptive responses, described by

Vos et al., (1994) However, it was observed that with each probe the

behavioral categories mentioned above were performed either in isolation or in

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