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

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
to cPLA2, sPLA2 and iPLA2 exerted pronounced anti-nociceptive effects in mice

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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 stimulusresponse 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

8

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
nociceptor sensitization with an increase in the excitability of the nociceptors

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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
readily excited by substance P (Salt et al., 1983; Dougherty and Willis, 1991).

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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-fiberevoked 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:
ophthalmic, maxillary and mandibular, together constitute the largest of the

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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 (KaiKai, 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
continuous with the dorsal horn. Low-threshold mechanical responses, high

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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 postcentral 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
(Grushka et al., 2003; Baad-Hansen et al., 2005), atypical facial neuralgia

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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
receptor mechanisms (NK1) clocks c-fos expression induced by noxious

21


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

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9. Phospholipase A2
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, calciumdependant 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 PLA2
and 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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