Application of PEA reduces WDR RF size - Final edit

Shaun Paul Croft Student I.D: 4065985 Project Study 2
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Administration of N-palmitoylethanolamine reduces and reverses carrageenan-induced wide
dynamic range neurone receptive field expansion and attenuates hyperalgesia in rats
Shaun Paul Croft
Institute of Neuroscience,School of Biomedical Sciences,University of Nottingham, Medical School,
Queen’s Medical Centre, Nottingham, UK
Abstract
The fatty acid amide (FAA) N-palmitoylethanolamine (PEA) has been shown to reduce inflammation
via activation of the peroxisome proliferator-activated receptor-α (PPAR-α),inhibiting the
transcription of pro-inflammatory cytokines and stimulating the transcription of anti-inflammatory
cytokines. Inflammation has been shown to be a causalfactor of neuropathic/neurogenic pain, and
aggravates many diseases such as asthma and stroke. Inflammation has also been shown to cause an
expansion of wide dynamic range neurone (WDR) receptive fields (RFs).
PEA and PPAR-α were investigated for a possible role in reducing inflammation-induced RF
expansion and hyperalgesia.
Adult male Sprague Dawley rats were anaesthetised with 2-3% isoflurane (1.5% during
electrophysiology), and L4-L5 segments of the spinal cord exposed. Microelectrodes were lowered
into the spinal cord to find single WDRs. Rats received intraplantar injection (i.pl) of either PEA
(50µg/50µl, n=6), PEA+GW6471 (PEA-50µg/50µl, GW6471-30µg/50µl, n=5) or vehicle (50µl, n=6),
followed 30mins later by λ-carrageenan (100µL, 2% in saline).
RFs were mapped using 8/26g von Frey hairs applied to the hindpaw at 0mins, 30mins, and every
20mins thereafter for 180mins.
For weight bearing, adult male Sprague Dawley rats were injected (i.pl) with the same treatments,
(n=4 for PEA and vehicle, n=5 for PEA+GW6471) plus one extra group given solely GW6471
(30µg/50µl, n=5) and each hindpaw was placed on a separate sensor of an Incapacitance tester.
PEA significantly reduced the carrageenan-induced RF expansion (p<0.001) and attenuated the
decrease in ipsilateral weight bearing at 150mins (p<0.05 vs. vehicle, p<0.01 vs. PEA+GW6471 and

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GW6471), indicating a reduction in ipsilateral hyperalgesia. GW6471, a PPAR-α selective
antagonist, blocked the effects of PEA and increased hyperalgesia, though not significantly vs.
vehicle. These results suggest that PEA alleviates the inflammation-induced hyperalgesia and
expansion of WDR RFs via PPAR-α activation. Furthermore they give evidence to support the use of
a PPAR-α agonist such as fenofibrate as a clinically effective anti-inflammatory agent with possible
use against arthritis, stroke, and other inflammatory diseases.
Key words: Carrageenan, inflammation, PEA,PPAR-α, receptive field, WDR, hyperalgesia
For an explanation of terminology used in this dissertation please refer to the glossary section.
1. Introduction
1.1. Roles of PEA and the PPAR-α in inflammation
PEA is an endogenous FAA and PPAR-α agonist[1-2]
. PEA has been proven to have an anti-
inflammatory action in severalstudies[2-6]
, and its activation of PPAR-α has been proposed as a
mechanism for this effect[1-2]
.
PPAR-α mediated anti-inflammatory effects are due to the change in gene transcription that results
from its activation[7]
. There are severalways in which gene transcription is altered following PPAR-α
activation; firstly there is ligand-dependent transcription, the transcription of genes following the
binding of activated PPAR-α, in a heterodimeric complex with a retinoid X receptor (RXR), to the
promoter region (PPAR-response elements - PPRE) of its target genes[7]
. This results in the
subsequent recruitment of co-activators and hence gene transcription[7]
.
This mechanism increases production of various anti-inflammatory cytokines such as interleukin-4
(IL-4), IL-5 and IL-10.
Another mechanism is via ligand-dependent transrepression, the prevention of gene transcription
following the binding of activated PPAR-α to other transcription factors (such as nuclear factor κβ –
NF-κβ), inhibiting this transcription factor and preventing it from recruiting co-activators to the
promoter regions of its own target genes[7-8]
. By inhibiting NF-κβ, PPAR-α can reduce the production
of cytokines and other pro-inflammatory mediators (PIMs) including IL-1β, IL-6, tumour necrosis
factor-α (TNF-α) and the enzymes cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase

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(iNOS)[8]
. Several of the proteins transcribed by PPAR-α then modulate the transcription of various
other genes, such as IL-4 inhibiting the production of IL-1β and increasing the production of the
interleukin-1 receptor antagonist (IL-ra) by preventing or inducing transcription of the responsible
genes respectively[9]
.
1.2. Wide dynamic range neurones (WDRs) and receptive fields (RFs)
WDRs are somatosensory neurones located mainly in lamina 5 of the spinal cord[10]
that respond to
both low and high threshold inputs, due to converging inputs from Aβ-fibres (low threshold), Aδ-
fibres (intermediate threshold) and C-fibres (high threshold)[10]
. Because of this anatomical
arrangement WDRs can respond to many stimuli and can produce either innocuously or noxiously
perceived outputs depending on the firing frequency of their action potentials[10]
. Figure 1 shows the
anatomical regions of the spinal cord and the laminae divisions of the spinal cord grey matter.
RFs are the areas of tissue innervated by a particular neurone, i.e. the area of tissue that, once
stimulated, results in the firing of that neurone. Their size is mediated by a mixture of glutamatergic,
GABAergic and serotinergic pathways[11-12]
. RF size control will be explained in more detail later.
It has also been found that direct application of PIMs to exposed neuronal axons produces central
sensitisation and an increase in RF size[13-14]
.
1.3. Hypotheses of investigation
During inflammation the RF size of neurones innervating the inflamed tissue increases markedly[14]
.
This experiment will investigate whether PEA injection into inflamed tissue can reduce the
inflammation-induced RF expansion of WDR neurones innervating the tissue. Secondly, it will also
investigate whether this effect is linked to PEAs anti-inflammatory effects via the activation of the
PPAR-α receptors in this tissue, resulting in the subsequent activation of the ligand-dependent
transcription and ligand-dependant transrepression mechanisms.

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Figure 1[15]
. Spinal cord gross anatomy and schematicrepresentation of spinal cord laminae
Electrophysiological recordings of WDR neurones were taken from lamina V (shown on right) of lumbar section 4/5 (shown
on left, the two sections above the sacrum). The spinothalamic and spinoreticular tracts are mirrored on both sides of the
spinal cord and somatosensory fibres cross over to the ipsilateral side of thespinal cord two sections rostrally from their
entry point through thedorsal root ganglion (not shown). WDRs travel through lamina V in thesespinal tracts.
2. Methods
2.1. Animals
All animal procedures were in accordance with the UK Home Office Animals (Scientific Procedures)
Act 1986 and International Association for the Study of Pain (IASP) guidelines.
2.1.1. Electrophysiology
Adult male Sprague Dawley rats weighing 180-200g, (Charles River, UK, n=17) were group housed
in a light controlled room with 12hr light/dark cycles and ad libitum access to food and water.
At the end of all procedures rats were sacrificed humanely using 5% isoflurane.
2.1.2. Behavioural tests
Behavioural testing used adult male Sprague Dawley rats weighing 210-280g (Charles River, UK,
n=17). Rats were group housed for 1week prior to behavioural tests and individually housed during
testing. In both cases rats had 12hr light/dark cycles and ad libitum access to food and water.
Following behavioural tests,rats were sacrificed using a transcardial perfusion of sodium
pentobarbital (0.9%) in saline.

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2.2. In vivo electrophysiology
Anaesthesia was induced by 2-3% isoflurane in 66% N2O/33% O2 followed by insertion of a cannula
into the trachea. Rats were then placed in a stereotaxic frame and the spinal cord exposed at lumbar
segments L4-L5 via a laminectomy. The exposed spinal section was held still by the use of clamps
located rostrally and caudally to the opening. Following surgery isoflurane was reduced to 1.5% to
maintain rats in a constant state of conscious areflexia. Core body temperature was monitored using a
rectalprobe and maintained at 37±1°C throughout surgery and recordings using a heating pad placed
underneath the rat.
Extracellular single-unit recordings of wide dynamic range (WDR) deep dorsal horn neurones (500-
1000µm, laminae IV-V) were made using glass-coated tungsten microelectrodes, produced in-house,
lowered through the spinal cord in 10µm steps using a SCAT-01 microdrive (Digitimer, Welwyn
Garden City, UK).
Action potentials were digitised and analysed using a CED micro1401 interface and Spike 2 data
acquisition software (Cambridge Electronic Design, Cambridge, UK).
All selected neurones had multiple inputs consisting of a short latency Aβ-fibre-evoked response (0-
20ms post-stimulus), an Aδ-fibre-evoked response (20-90ms post-stimulus) and a long latency C-
fibre-evoked response (90-300ms post-stimulus).
2.3. Receptive field mapping
WDR RFs were identified using mechanical brush and pinch stimuli induced by application of von
Frey hairs, and RFs usually extended over one or two toes of the hindpaw.
Individual von Frey hairs (Semmes-Weinstein Monofilaments; North Coast Medical Inc., USA, via
Linton Instrumentation, Norfolk, UK) of 8g and 26g bending forces were applied to the toes at
0mins (drug injection time point), 30mins (directly before carrageenan injection) and at 20min
intervals for the following 180mins to map the size of the RF after carrageenan-induced inflammation.
Total time that RF sizes were measured was 210mins.
8g von Frey hairs represented a non-noxious stimulus whereas 26g represented a noxious stimulus,
based on the noxious withdrawl threshold in conscious rats being 15g[16]
.

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2.4. Behavioural testing
Rats were anaesthetised with isoflurane (3% in 66% N2O -33% O2) before application of drug
treatments. The effects of vehicle, PEA,GW6471 and PEA+GW6471 on ipsilateral (left) and
contralateral (right) hindpaw weight bearing in rats with carrageenan-induced inflammation was
tested.
Weight (in grams) applied through the ipsilateral/contralateral hindpaw was measured using an
Incapacitance tester (Linton Instrumentation, U.K.) with each hindpaw placed on a different sensor.
Measurements were averaged over a period of 3secs. Data points represent the mean of three 3sec
readings.
2.5. Carrageenan inflammation
Following identification of a single suitable WDR neurone, or on commencing of the behavioural
testing, λ-carrageenan (100 µL, 2% in saline; Sigma, Poole, UK) was injected into the plantar surface
of the hindpaw 30mins after drug application (30mins).
Inflammation was measured via calculation of the hindpaw circumference using a thread suture,
looped around the paw at metatarsallevel and gently tightened until it contacted the entire outside
area of the paw. This suture was then opened out and measured to the nearest mm.
Paw circumference was measured prior to carrageenan injection (30mins) and at the end of recording
(210mins). 180mins was selected as the time frame for the pharmacological/carrageenan studies on
the basis of previous findings indicating 180mins post-carrageenan as the time at which maximum
hyperalgesia is observed[17]
.
Paw volume was also measured during the behavioural experiments. This was measured by dipping
the ipsilateral and contralateral hindpaws into a measuring cylinder of water. The water displacement
in cm3
was measured and recorded for each hindpaw.
2.6. Drug treatments
All compounds were administered via intraplantar injection into the hindpaw innervated by the WDR
which was being recorded. Administration took place 30mins prior to carrageenan injection
(at 0mins).

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In electrophysiological experiments there were two drugs that were tested. PEA is an endogenous
FAA and PPAR-α ligand, whereas GW6471 is a PPAR-α specific antagonist. In a separate control,
vehicle (3% Tween 80 in 0.9% saline) was injected into the hindpaw of rats instead of drugs.
Effects of PEA (50µg/50µl), PEA+GW6471 (PEA-50µg/50µl, GW6471-30µg/50µl) or vehicle (50µl)
administration on λ-carrageenan-induced RF expansion were measured every 20mins for 180mins
(50-210mins) and standardised to %control RF size. RF size was also measured at 0mins and again at
30mins to determine whether drug/vehicle alone had any effect on RF size.
During behavioural testing the same treatments were used at the same concentrations. However,a
separate group received injections of GW6471 alone.
2.7. Statistical analysis
Statistical analysis comparing carrageenan-induced expansion of WDR neurone RFs following
drug treatment was performed using an area under the curve analysis and one-way ANOVA. A
Bonferroni multiple comparison post-hoc test was applied to the one-way ANOVA analysis. These
analyses were applied to both 8g and 26g mechanically evoked RF mapping data.
Paw circumference data obtained from the electrophysiology experiment was analysed using one-way
ANOVA and Bonferroni multiple comparison post-hoc tests to determine significant differences
between all 6 data sets (pre-carrageenan vs. allthree drug treatments after 180mins). During
behavioural testing paw circumferences and paw volumes were analysed using Kruskal-Wallis and
Dunn’s multiple comparison post-hoc tests to determine differences between pre and post-carrageenan
ipsilateral/contralateral paw circumferences and volumes.
Behavioural data were evaluated using a two-way ANOVA analysing the effects of time and
treatment group on carrageenan-induced hindpaw weight bearing. Bonferroni multiple comparison
post-hoc tests were used to determine significant differences between results based on these two
variables.
Significance was set at p<0.05.

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2.8. Materials
Isoflurane was obtained from Abbott Laboratories Ltd (Maidenhead, UK). GW6471 and PEA were
purchased from Tocris Bioscience (Bristol, UK),and were stocked in ethanol on arrival.
3. Results
3.1. Intraplantar carrageenan injection increases rat hindpaw circumference and volume
Injection of λ-carrageenan produced a significant increase in paw circumference in all separate study
groups (p<0.001, Figure 2, Table 1). The significant increases in paw circumference in all three
electrophysiology study groups and increase in paw circumference and volume across all four
behavioural study groups, shows that λ-carrageenan injection produced an inflammatory response in
the rat paw that was accompanied by tissue oedema.
Drug treatments had negligible effects on paw circumference,and although PEA reduced the oedema
slightly more than PEA+GW6471, this difference was not significant (Figure 2, Table 1).
3.2. PEA attenuates carrageenan-induced RF expansion in WDRs
3.2.1. Pre-carrageenan RF sizes of spinal cord neurones
RFs of WDR neurones were mapped directly after intraplantar injection of vehicle, PEA or
PEA+GW6471 and 30mins prior to injection of λ-carrageenan (0mins). RFs were mapped directly
before λ-carrageenan injection (30mins) to establish whether these compounds elicited an increase in
RF size in a carrageenan-independent fashion. It was found that RF size fluctuated between 0mins
and 30mins after addition of all three compounds, but the change was not significant (Figures 3 and
5). Each compound also induced a different level and/or direction of change in RF size for 26g than it
did for 8g, so the fluctuations were discounted as drug/vehicle independent physiological processes.
3.2.2. RF size of spinal cord neurones following carrageenan injection: 8g mechanical stimuli
In vehicle and PEA+GW6471 treated rats,injection with λ-carrageenan caused a significant
expansion of neuronal RFs on the ipsilateral hindpaw (p<0.05, Figures 3 and 4). Treatment with PEA
completely attenuated the carrageenan-induced RF expansion vs. vehicle (p<0.05, Figures 3 and 4)
but concomitant injection with GW6471 led to carrageenan-induced expansion of the WDR RFs

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(p<0.001 vs. PEA alone, Figures 3 and 4).
3.2.3. RF size of spinal cord neurones following carrageenan injection: 26g mechanical stimuli
In similar fashion to 8g mechanical stimuli, λ-carrageenan caused a significant expansion of the 26g
evoked RFs on the hindpaw of both vehicle and PEA+GW6471 treated rats (p<0.05, Figures 5 and 6).
Once again, treatment with PEA completely attenuated the carrageenan-induced RF expansion
(p<0.001 vs. vehicle and PEA+GW6471, Figures 5 and 6). Unlike the 8g evoked responses PEA
treatment did not decrease the size of the RF past the pre-treatment baseline at any point.
Neither the total nor peak increases seen in PEA treated rats throughout recording was significant
compared to the baseline reading at 0mins.
Figure 2. Effects of intraplantar carrageenan injection on paw circumference
Carrageenan injection caused a robust increase in mean paw circumference compared with pre-carrageenan circumferences
(p<0.05). All three treatments (vehicle, PEA and PEA+GW6471) failed to prevent the carrageenan-induced inflammatory
responseand subsequent oedema. Mean increases in paw circumference were 6.17mm, 4.83mm and 6.20mm for vehicle,
PEA and PEA+GW6471 respectively. Paw circumference data were analysed using one-way ANOVA and Bonferroni
multiple comparison post-hoctests. *** = significantly different from corresponding pre-carrageenan result (p<0.001).

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Table 1. Effects of carrageenan injection on ipsilateral/contralateralhindpawcircumference and volume
Injection of carrageenan into the ipsilateral hindpaw produced a significant increase in ipsilateral hindpaw circumference and
volume (p<0.001) but had no effect on thecontralateral hindpaw.
The mean increase in circumference of the ipsilateral hindpaw between all rats and treatment groups was 9.3mm (p<0.001).
The circumference of the contralateral hindpaw decreased by a mean of 0.83mm, which was not significant.
The mean increase in ipsilateral hindpaw volume between all rats and treatment groups was 0.91cm3
(p<0.001). The
contralateral hindpaw showed a mean decrease in volume of 0.07cm3
. This was not significant.
Pre-treatment with vehicle, PEA, GW6471 or PEA+GW6471 failed to attenuate thesecarrageenan-induced increases.
Paw circumference/paw volume data were analysed using Kruskal-Wallis and Dunn’s multiple comparison post-hoctests.

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Figure 3. Effect of vehicle, PEA and PEA+GW6471 administration on WDR neurone RF sizes mappedwith an 8g
von Frey hair
Vehicle+carrageenan treatment lead to a significant increase in RF size (p<0.05). Injection of PEA alone prevented the
carrageenan-induced increase in RF size whereas PEA+GW6471 injection reversed the attenuation of carrageenan-induced
neuronal RF expansion caused by PEA treatment alone.
All results are expressed as means with SEM. Statistical analysis of data were performed using one-way ANOVA and
Bonferroni multiple comparison post-hoctests. * = first time point at which vehicle significantly different from PEA
(p<0.05). *** = first time point at which PEA+GW6471 significantly different from PEA (p<0.001).
Figure 4. Area under the curve analysis of the effects of carrageenan inflammation on the RF size of WDR neurones
following application of 8g stimuli andin the presence of vehicle, PEA, or PEA+GW6471
PEA treatment produced a significant reduction in RF size compared to vehicle (p<0.05).
Blockade of PPAR-α with GW6471 reversed theeffects of PEA on carrageenan-induced RF expansion, resulting in an RF
size significantly greater than that for PEA treatment alone (p<0.001). PEA+GW6471 RFs were also greater, though not
significantly, than pre-treatment with vehicle.
Data were statistically analysed using one-way ANOVA and Bonferroni multiple comparison post-hoctests.
All results are expressed as means with SEM. * = PEA significantly different from vehicle (p<0.05). *** = PEA
significantly different from PEA+GW6471 (p<0.001).

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Figure 5. Effect of vehicle, PEA and PEA+GW6471 administration on WDR neurone RF sizes mappedwith a 26g von
Frey hair
Vehicle+carrageenan treatment lead to a significant increase in RF size (p<0.001). Injection of PEA alone prevented the
carrageenan-induced increase in RF size. Like vehicle, PEA+GW6471 injection also resulted in a significant carrageenan-
induced increase in RF size (p<0.001).
Data are expressed as means with SEM. Data were statistically analysed using one-way ANOVA and Bonferroni multiple
comparison post-hoctests.
# = first time point at which vehicle significantly different from PEA (p<0.001). *** = first time point at which
PEA+GW6471 significantly different from PEA (p<0.001).
Figure 6. Area under curve analysis of the effects of carrageenan inflammation on the RF size of WDR neurones
following application of 26g stimuli andin the presence of vehicle, PEA, or PEA+GW6471
PEA treatment produced a significant reduction in RF size compared to vehicle. Concomitant administration of PEA and
GW6471 reversed theeffects of PEA, leading to a significant carrageenan-induced increase in RF size compared to PEA
treatment alone (p<0.001).
Data were analysed using one-way ANOVA and Bonferroni multiple comparison post-hoctests. Dataare expressed as
means with SEM. *** = significantly different from all other results (p<0.001).

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3.3. Carrageenan injection leads to an imbalance of ipsilateral/contralateral weight bearing and
ipsilateral hyperalgesia in rats
The second series of experiments measured the behavioural response following hindpaw injection of
carrageenan. Previous studies have shown that carrageenan produces an increase in contralateral
weight bearing and a decrease in ipsilateral weight bearing, indicative of hyperalgesia on the
ipsilateral hindpaw. Saline injection did not produce hyperalgesia (as shown in previous studies),
suggesting that hyperalgesia was carrageenan-induced,and may have been linked to the carrageenan-
induced initiation of the inflammatory response.
In all treatment groups there was a significant effect of time on carrageenan-induced hyperalgesia
(p<0.001, Figure 7), with a strong correlation between time and change in weight bearing/increasing
hyperalgesia (F=24.41).
There was a significant effect of PEA treatment (p<0.05 vs. vehicle, p<0.01 vs. GW6471 and
PEA+GW6471, Figure 7) at 120mins post-carrageenan (150mins), where it was observed that
ipsilateral hyperalgesia was reduced to pre-carrageenan levels. However,by 210mins the effect of
PEA treatment on carrageenan-induced hyperalgesia was no longer apparent and there was no
difference between the effects of vehicle and PEA on carrageenan-induced hyperalgesia.
4. Discussion
4.1. PPAR-α activation via PEA reducesinflammation-induced expansion of RFs and ipsilateral
hindpaw hyperalgesia
In this study, PEA injection prevented the carrageenan-induced expansion of RFs (Figures 3 and 5)
and reduced carrageenan-induced hindpaw hyperalgesia at 120mins post-carrageenan (Figure 7).
Moreover, PEAs effect was blocked with concomitant application of GW6471, a selective PPAR-α
antagonist. In previous studies, PPAR-α agonists have been shown to reduce the RF size of primary
afferent fibres (PAFs) in animal models[18]
. Data collected in this study also confirmed this, and
indicate that PPAR-α is the receptor by which PEA prevents this expansion.
The anti-inflammatory effects of PEA are hypothesised to be due to PPAR-α activation and gene

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Figure 7. Effects of λ-carrageenan on ipsilateral/contralateralweight bearing in rat hindpaws
Saline injection resulted in no increase in contralateral weight bearing and therefore no hyperalgesia (personal
communication with Dr James Burston).
Vehicle and GW6471 treatment lead to an increase in ipsilateral hyperalgesia vs. baseline levels from 60mins post-
carrageenan (90mins), whereas PEA+GW6471 showed an increase in ipsilateral hyperalgesia from 0mins vs. baseline levels.
Time had a significant effect on ipsilateral hyperalgesia in all treatment groups (p<0.001).
PEA treatment lead to a significant reduction in hyperalgesia at 150mins vs. all other treatment groups, with a reduction in
contralateral weight bearing of ~39g vs. vehicle (p<0.05), but had negligible effects at all other time points.
Data were analysed using two-way ANOVA and Bonferroni multiple comparison post-hoctests.
All results are expressed as means with SEM. * = PEA significantly different from vehicle (p<0.05) ** = PEA significantly
different from GW6471 and PEA+GW6471 treatment groups (p<0.01).
transcription modulation, namely via decreased PIM production and increased synthesis of anti-
inflammatory cytokines[7]
. The data gathered in this experiment support this hypothesis.
As stated earlier, PPAR-α activation has anti-inflammatory effects by two separate mechanisms,
ligand-dependent transcription and ligand-dependent transrepression[7]
. Of these two mechanisms,
ligand-dependent transrepression can occur much sooner, due to the fact that gene transcription takes
severalhours-several days to be completed whereas inhibition of transcription can theoretically occur
as soon as PEA activated PPAR-α is in proximity to the target transcription factor.
Previous studies suggest that PIMs produced under inflammatory conditions can initiate neuronal
sensitisation[13]
. This means that centraland peripheral sensitisation, the driving forces behind
induction of dorsal root reflexes (DRRs) and hence the expansion of neuronal RFs[7]
, is prevented by
the inhibition of transcription of these PIMs. However,because ligand-dependent transcription takes
severaldays to conclude, anti-inflammatory cytokines are not produced during the 3hour timeframe of
these experiments. This suggests why the circumferences and volumes of the ipsilateral hindpaws

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remained greater than normal in the presence of PEA,as local inflammation and oedema may have
been halted, but not reversed.
Figure 7 shows that GW6471 treatment alone leads to the greatest weight bearing on the contralateral
paw. This suggests that rats injected with GW6471 may have experienced more profound ipsilateral
hindpaw hyperalgesia than rats in other treatment groups; this indicates a role for basalPPAR-α
activity in the regulation of weight bearing and pain sensitivity. In contrast to this, PEA reduced the
overall contralateral weight bearing, although this was only significant 120mins after carrageenan
injection; 180mins post-carrageenan there was no significant difference between PEA and GW6471
treatment groups. As with the prevention of RF expansion, this could be due to PEA preventing the
transcription of PIMs and hence preventing neuronal sensitisation and mechanical hyperalgesia.
Another possibility is that PEA is reducing hyperalgesia indirectly via its entourage effect[19]
. This
effect is due to PEA increasing both cannabinoid type-1 receptor (CB1-R) and transient receptor
potential vanilloid type-1 receptor (TRPV1-R) affinity for the endocannabinoid anandamide (AEA),
indirectly increasing CB1-R activation and desensitising the TRPV1-Rs at lower [AEA],which has
been shown to be anti-nociceptive[19]
. PEA can also increase [AEA] by acting as a competitive
substrate at the AEA degradation enzyme fatty acid amide hydrolase (FAAH)[20]
. This increased
[AEA] and CB1-R/TRPV1-R affinity for AEA, coupled with the fact that CB1-R activation has been
shown to have anti-nociceptive effects in several studies[21-22]
, could point to a different mechanism by
which PEA reduces the pain and contralateral weight bearing seen in this investigation.
However,the ability of GW6471 to abolish the anti-nociceptive effects of PEA in this study suggests
that PPAR-α activation is necessary for these anti-nociceptive effects,although CB1-R activation
could play a minor role. There is however one more consideration to make. AEA has also been
shown to be a PPAR-α ligand[23]
, so it is possible that PEA’s anti-nociceptive effects are mediated
first via the entourage effect,leading to a decrease in AEA degradation by FAAH and subsequent
increase in [AEA]i,followed by PPAR-α activation by AEA.
The large standard error bars seen during RF mapping with the PEA+GW6471 treatment group
(Figures 3 and 5) and during weight bearing with the GW6471 and PEA+GW6471 treatment groups
(Figure 7) may be indicative of a facilitation of the inflammatory response, perhaps due to GW6471

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blocking the PPAR-α receptors in the rat hindpaws and preventing endogenous PEA or AEA from
activating them. GW6471 treatment also causes the largest standard error bars in other studies
investigating the anti-nociceptive effects of PPAR-α activation[18]
.
In investigating only the effects of PPAR-α agonists and antagonists on hyperalgesia and RF
expansion, this study provides strong evidence for PPAR-α involvement in anti-nociception, RF
control during inflammation and the regulation of hindpaw weight bearing via basalrates of
activation.
4.2. Mechanismsof RF expansion: Why does PEA prevent carrageenan-induced RF expansion?
RF expansion is controlled mainly by γ-amino-butyricacid (GABA) via GABAergic spinal inter-
neurones, and application of GABA antagonists lead to RF expansion[11]
. Spinal inter-neurones of
the dorsal horn control nociceptive transmission in primary afferent fibres (PAFs) by causing PAF-
synaptic inhibition[12]
.
GABAA-Rs are ionotropic receptors,and activation by GABA opens a Cl-
selective ion channel.
GABAB-Rs are metabotropic G-protein coupled receptors (GPCRs),and are linked to K+
channels via
a Gi G-protein subtype[24]
. Activation of GABAB-Rs causes a signal cascade that decreases[cAMP]i
and opens the K+
channel. Together the prolonged efflux of Cl-
and K+
lead to sustained
depolarisation of the PAF which in turn inhibits Ca2+
influx via Ca2+
channels, in turn preventing
further action potential propagation[11][25]
.
It has been stipulated that GABA release from inter-neurones is stimulated by Glu release from the
PAF. This Glu activates AMPA-Rs on the inter-neurone, leading to depolarisation via an influx of
Na+
and Ca2+
into the inter-neurone through the AMPA-R non-selective cation channel[26]
. Thus,
increased PAF activation leads to increased GABA release and increased PAF depolarisation[27]
.
In PAFs,this inhibitory mechanism is known as primary afferent-depolarisation (PAD),the
mechanisms of which are highlighted on Figure 8.
When tissue damage occurs the PIMs and cytokines released can cause sensitisation of the
nociceptors in the damaged tissue[13]
. However,the increased firing of these neurones due to the
damaging stimuli itself can lead to multi GABAergic innervations. This can produce an even stronger

Page 17 of 28
depolarisation of the PAF terminal than normal PAD.
When the membrane potential becomes too positive (i.e. when depolarisation becomes too strong), the
PAFs transmit spontaneous, high frequency, intermittent anti-dromic action potentials, known as
DRRs. These DRRs have been shown to increase inflammation and oedema in animal models of
rheumatoid arthritis, and also increase hyperalgesic states in tissue innervated by the PAFs
transmitting these DRRs,due mainly to the induction of centralsensitisation through the high
frequency action potentials. Furthermore, they cause an expansion of the RF of the affected PAFs,as
bicuculline, a GABAA-R antagonist, caused a cessation of DRRs and a concomitant reduction in RF
size[25]
.
Figure 8[11]
. GABAergic mechanisms of PAD on PAFs stimulating motorneurones in the spinal grey matter.
Normal cell control of [ion]i through the use of Na+
/K+
ATPaseand the Na+
/K+
/Cl-
co-transporter is shown at the top of the
figure; the effects of GABA release by the pre-synapticinhibitory axon of a GABAergic spinal inter-neurone are also shown.
Action potentials in thePAF result in the release of Glu and thesubsequent activation of AMPA-Rs on theinter-neurone.
The release of GABA from the inter-neurone activates GABAA/GABAB-Rs on the PAF, leading to an efflux of Cl-
and K+
ions, depolarising the neurone as a result of a membrane potentialshift towards the equilibrium potentialof Na+
, which is
now present at the highest concentration of all three ions in the PAF. Thevoltmeter in thebottomleft of the figure shows
that the cell membrane potentialhas increased from c.-60mV to c.-35mV, enough to cross thefiring threshold and elicit the
propagation of an action potential. During PAD this depolarisation is maintained and prevents repeat firing of thePAF due
to inhibition of the Ca2+
channels on thepost-synapticmembrane. Although this figure shows motor neurone innervation the
same process can occur between two spinal somatosensory neurones.
Since DRRs initiate peripheral sensitisation which in turn can lead to centralsensitisation[13]
and RF
expansion, and the fact that PEA was injected directly into the hindpaw, it seems likely that PEA acts
peripherally to prevent DRRs from occurring. As stated in section 4.1, by activating PPAR-α in the

Page 18 of 28
inflamed hindpaw, PEA is able to prevent the transcription and production of PIMs,decreasing PAF
excitability and preventing burst firing. This in turn prevents the onset of peripheral and central
sensitisation[13]
and attenuates the inflammation-induced RF expansion.
However, WDRs,the neurones used to map RFs in this study, are also susceptible to wind up. When
normal neurones fire action potentials their ionic gradients are reset by Na+
/K+
ATPases[28]
,restoring
the membrane potential back to that of a resting neurone. In WDRs,high frequency action potentials
from C-fibre inputs[29]
can depolarise the neurone before the resting potential is reached,resulting in a
slight depolarisation that increases with subsequent inputs[30]
. When this depolarisation reaches too
high a potential, prolonged burst firing is evoked in the WDR[30]
. This prolonged burst firing is
known as wind up. Wind up leads to many characteristics of central sensitisation, including the
expansion of RFs and hyperalgesia seen in this experiment[29]
, meaning wind up cannot be discounted
as the mechanism by which RF expansion and hyperalgesia were induced in this investigation.
It is possible that carrageenan-induced inflammation produced a DRR in the PAFs that triggered wind
up in the WDRs used for electrophysiological recording. In this case,pre-treatment with PEA would
prevent DRRs in the PAFs innervating the ipsilateral hindpaw and thus prevent the induction of WDR
wind up. Despite the mechanism being slightly different, the overall result of local PEA injection
would still be an attenuation of RF expansion and a reduction in hyperalgesia.
Although wind up cannot be wholly disproved as the mechanism by which RF expansion and
hyperalgesia occurred,it is much more plausible that neuronal sensitisation caused these effects,due
to the fact that the experiments were carried out over a 3hr period. Wind up is usually short lived,
lasting for a period of only severalminutes[31]
.
4.3. Accuracy and reproducibility of results
In all rats carrageenan produced a profound inflammatory response, leading to tissue oedema,
hyperalgesia and RF expansion. Pre-treatment with PEA both alleviated hyperalgesia and prevented
RF expansion. Although hyperalgesia and RF expansion did occur with PEA treatment,it was much
less profound than pre-treatment with vehicle, GW6471 alone, or concomitant injection of
PEA+GW6471. As a documented anti-inflammatory[1, 3-5]
PEA was expected to reduce inflammation-

Page 19 of 28
induced hyperalgesia and RF expansion, and since it did so almost without exception the accuracy of
data collected regarding the effects of PEA on carrageenan-induced inflammation can be assumed to
be accurate. It is also well established that PPAR-α activation attenuates inflammation[2, 6-7]
,and that
PEA is an endogenous ligand at this receptor[1]
,so the fact that PPAR-α blockade via GW6471 led to
the highest levels of hyperalgesia and RF expansion, again almost without exception, can also be
considered accurate.
In terms of reproducibility, many different studies have used the same basic surgical and WDR
electrophysiological recording techniques as this experiment, albeit with minor differences depending
on the type of recordings being taken[32-35]
and the use of Incapacitance testers in the measurement of
hindpaw weight bearing is also widespread[32-33]
. The use of carrageenan to induce inflammation and
of PEA to reduce it has also been performed many times and against many different disease states,
e.g. inflammatory bowel disorder, multiple sclerosis, etc[34-38]
,including several assays where the anti-
nociceptive effects of PEA via the entourage effect were investigated[39-40]
. These studies achieved
similar PEA and carrageenan-mediated results to those seen in this investigation.
There were however severallimitations encountered during these experiments. The first is that, due
to time restrictions, it was not possible to have a GW6471 only treatment group in the
electrophysiology tests. This is offset by the fact that the PEA+GW6471 group results proved that a)
GW6471 inhibits the PEA-induced prevention of RF expansion and b), given that GW6471 is a
PPAR-α antagonist, provides the mechanism, (PPAR-α activation), for how PEA produces this effect.
As stated earlier, the entourage effect of PEA may also play a part in the attenuation of carrageenan-
induced hyperalgesia and RF expansion. Further studies could investigate PEA’s ability to prevent
carrageenan-induced RF expansion and hyperalgesia in the presence of a CB1-R antagonist such as
AM251[41]
, in order to determine the proportion of analgesia, if any, that is mediated by CB1-R
activation.
5. Conclusion
The findings of this experiment indicate a role for PEA in the modulation of the inflammatory
response. Inflammation is a major cause or complication of many diseases,including, but not limited

Page 20 of 28
to, osteoarthritis[36]
, spinal cord injury[37]
, stroke[38]
, contact allergic dermatitis[39]
, inflammatory bowel
diseases[40, 42]
,asthma[43]
and multiple sclerosis[44]
. Inflammation is also a known cause of neurogenic
and neuropathic pain[13, 21, 22, 45-47]
, hyperalgesia[17]
and tactile and thermal allodynia[16]
. Given the vast
range of conditions for which attenuation of inflammation would be of benefit to the patient it would
seem likely that a compound such as PEA,proved to have clinical efficacy as an anti-inflammatory,
would be considered as a possible drug candidate.
There are,however, severalissues to take into consideration. One issue is that there is likely to be a
maximum therapeutic dose for PEA that cannot be exceeded,based on two factors; the first is the total
amount of PPAR-α present in the tissue due to gene-transcription taking a long time to complete.
There will be a saturation point during which all PPAR-α in the tissue is bound to the PPAR-response
elements (PPRE) located on the promoter regions of its target genes[7]
, leaving none spare for PEA to
bind with. The second factor is that PEA has been shown to be cytotoxic in concentrations larger than
30µM[48]
. Another problem is that PEA is rapidly metabolised to form its constituents, palmitic acid
and ethanolamine[49]
by the enzymes FAAH and N-acylethanolamide-hydrolysing acid amidase
(NAAA)[49]
,making long term treatment with PEA virtually impossible.
Perhaps a more promising treatment method for inflammatory conditions is a synthetic PPAR-α
agonist. Synthetic agonists are often developed to withstand rapid metabolism, and allow long term
treatment or slow release formulations to be developed. One such drug, fenofibrate, is often used by
patients suffering from hyperlipidaemia, hypercholesterolemia and mixed dyslipidaemia, especially
those at risk of cardiovascular diseases or metabolic diseases such as type-2 diabetes mellitus[50]
.
However,a recent study has found that fenofibrate reduces inflammation and skeletal muscle atrophy
in the chronic inflammatory condition rheumatoid arthritis[51]
. Although rheumatoid arthritis
represents just one inflammatory condition of many, it paves the way for future clinical trials of
fenofibrate in other inflammatory diseases, particularly those previously mentioned for which PEA
showed profound efficacy.
Word count: 4353

Page 21 of 28
Acknowledgments
Due to the need for an animal experimental licence, data used in this dissertation was collected by
PhD students Okine B and Burston J using the methods described. Electrophysiological recordings of
WDR neurones were demonstrated by PhD student Woodhams S.

Page 22 of 28
Glossary
Aβ-fibre – A large diameter, myelinated neurone that senses the innocuous feeling of “touch” (such as brushing
the hairs on yourskin) that is usually separate from, but can become inducted into, the pain pathway. These are
the fibres responsible for mechanical allodynia. Transmits information at high velocities.
Aδ-fibre – A medium diameter, myelinated neurone that transmit thermal and mechanical sensations at high
velocities. Responsible for the transmission of some nociceptive signals.
Ad libitum – Free access to food and water whenever the animals wanted.
Allodynia (mechanical/tactile) - Pain due to an innocuous stimulus which does not normally provoke pain.
Anaesthesia – Loss of bodily sensation with or without loss of consciousness.
Antagonist – A molecule that binds to a receptor but does not produce a response.
Anti-dromic firing – Action potentials that travel down an axon in the opposite direction to that expected.
Areflexia – A state of anaesthesia where neurological motor reflexes such as the knee jerk reaction are
abolished.
C-fibre – A small diameter, unmyelinated neurone responsible for the transmission of noxious stimuli. Action
potential transmission is slow through these unmyelinated fibres, leading to the post-stimulus inputs seen in
wide dynamic range neurone electrophysiological recordings.
Central sensitisation – See neuronal sensitisation.
Co-activators – Molecules which bind to transcription factor/promoter region complexes and allow the
formation of the transcription enzyme.
Contralateral – A part of the body on the opposite side to the body part being discussed.E.g. if discussing the
left side of the spinal cord then the contralateral paw is the paw on the right.
Cytokines – Pro/anti-inflammatory proteins released from various cells in response to inflammation.
Dorsal horn – The grey matter portion of the spinal cord at each level of the vertebrae.
Dorsal root reflex (DRR) – Anti/Orthodromic burst firing of primary afferent fibres induced when primary
afferent depolarisation crosses the depolarisation threshold and leads to neuronal excitation.
Electrophysiology – The intra or extracellular recording of neurones using microelectrodes, usually by
measuring action potentials evoked by a mechanical or electrical response.Different types of
electrophysiological recordings can be made, including single channel, membrane patch or whole cell
recordings.
Extracellular fluid (ECF) – The fluid found outside and between cells.
Gene transcription – The process of producing mRNA copies from DNA, which are then turned into proteins
such as receptors, cytokines, etc.
Hyperalgesia - An increased response to a noxious stimulus. Also includes primary and secondary
hyperalgesia induced by inflammation.
Inflammation – The physiological change in tissue after damage, whereby pro-inflammatory mediators,
cytokines and chemokines are released, hyperalgesia is induced, and tissue volume increases due to an influx
of tissue fluid (extracellular fluid) in the damaged area.
Innocuous stimulus – A stimulus that is not damaging to tissue,such as brushing the skin.

Page 23 of 28
Inter-neurone – An inhibitory neurone (usually GABAergic) which inhibits neuronal firing in the excitatory
neurone adjacent to it. Can be found in the brain or in the dorsal horn of the spinal cord.
Intraplantar injection – An injection into the sole of the paw.
Ipsilateral – A part of the body on the same side as the body part being discussed.E.g. if discussing the left side
of the spinal cord then the ipsilateral paw is the paw on the left.
Lamina – A division of the grey matter of the spinal cord.
Laminectomy – A surgical procedure where the spinal cord is exposed via removal of the surrounding tissue on
the back of the animal.
Ligand – A molecule which binds to a protein (usually a receptor or enzyme) in order to induce a chemical or
biological response.
Mechanical stimulus – Stimuli such as touch,pinch, brush, etc.
Microelectrode – A small diameter glass tube that creates a tight, high resistance seal around either a single ion
channel or a patch of ion channels.It is used to investigate the conductance and opening/closing kinetics of ion
channels in a range of conditions and under pharmacological manipulation by recording ion channel/cell
membrane potentials.Used in electrophysiology.
Neuronal sensitisation – Sensitisation is when a neurone becomes hyper-excitable, eliciting a larger than
normal response due to increased neurotransmitter release or increased receptor expression. Can occur in CNS
neurones (central sensitisation), or peripheral neurones (peripheral sensitisation).
Nociception – The transmission of noxious stimuli perceived as pain.
Noxious stimulus - A noxious stimulus is one which is damaging to normal tissues,e.g. a burn. They are
perceived by the brain as pain.
Oedema – Part of the inflammatory process,the increase in extracellular fluid volume around
damaged/inflamed tissue due to changes in capillary permeability which allows blood plasma to escape the
capillary and enter the extracellular fluid.
Orthodromic firing – Action potentials that travel down axons in the expected (normal) direction.
Peripheral sensitisation – See neuronal sensitisation.
Primary afferent depolarisation (PAD) – GABAergic inhibition of neuronal excitation in primary afferent
fibres.
Primary afferent fibres (PAFs) – neurones involved in nociceptive transmission that are part of the pain
pathway.
Primary hyperalgesia – The increased response seen in nociceptive neurones whose receptive field lies within
an area of tissue damage.
Pro-inflammatory mediators (PIMs) – Molecules that initiate and prolong the inflammatory response by
binding to specific receptors.Released mainly by microglia, macrophages and mast cells upon neuronal/tissue
damage. Their application onto neurones can cause the increase of receptive field size of the neurone in
question and also lead to neuronal sensitisation.
Promoter region – The area of the gene that promotes transcription of the gene when bound to transcription
factors.
Receptive field (RF) – The area of tissue innervated by a particular neurone, i.e. the area of tissue that,once
stimulated, results in the firing of that neurone.

Page 24 of 28
Secondary hyperalgesia – The increased response seen in neurones whose receptive fields lie adjacent to the
area of tissue damage. Can affect nociceptive neurones or Aβ-fibres, which convey their innocuous “touch”
sensation as a noxious stimulus after induction into the pain pathway. Induction into the pain pathway can be
down to pro-inflammatory mediator, chemokine/cytokine exposure, or as a response to hyperactivity and
neuronal sensitisation in surrounding neurones.
Thermal sensation – sensation ofheat stimuli perceived as cold, cool, warm or hot.
Transcription – See gene transcription.
Transcription enzyme – A multi-sub unit enzyme that is needed for gene transcription. Makes mRNA copies
from the DNA template. E.g. mRNA synthase.
Transcription factor – A molecule necessary for the transcription of genes.Binds to the promoter region of
the gene and allows the formation of the transcription enzyme.
Transactivation - Of a transcription factor or nuclear receptor, the ability to initiate gene transcription by
binding to the promoter region of its target gene,inducing the recruitment of co-activators and allowing the
formation of transcription enzymes.
Transrepression – Of a transcription factor or nuclear receptor, the ability to prevent the transcription of
other genes by binding to that gene’s transcription factor, preventing the binding of co-activators and the
subsequent formation of transcription enzymes.
Von Frey hairs – Thin metal rods of varying bending force used to induce mechanically evoked firing of a
neurone via application to the receptive field of the neurone being recorded.
Wide dynamic range neurone (WDR) – A neurone that responds to both innocuous and noxious responses,
and can be either high or low threshold.Receives input from Aβ, Aδ, and C-fibres, and so can respond to light
and heavy mechanical deformation (innocuous light touch to noxious pinch), a wide range of thermal
sensations (cold, cool, warm, hot), and many chemical insults. Their response is graded – higher frequency
action potentials mean a more painfully perceived stimulus. They are susceptible to wind up.
Wind up – Of a WDR, repeated, high frequency, long lasting spontaneous discharges caused by high frequency
inputs that do not allow baseline ionic gradients to be established in the resting phase of the action potential
cycle. Is thought ofby some researchers as a causalmechanism for neuronal sensitisation.

Page 25 of 28
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