application of pea reduces wdr rf size - final edit

Shaun Paul Croft Student I.D: 4065985 Project Study 2

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 causal factor 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 several studies[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 several ways 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 schematic representation 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 the spinal cord two sections rostrally from their entry point through the dorsal root ganglion (not shown). WDRs travel through lamina V in these spinal 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

rectal probe 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 metatarsal level 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. all three 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 response and 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-hoc tests. *** = significantly different from corresponding pre-carrageenan result (p<0.001).

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Table 1. Effects of carrageenan injection on ipsilateral/contralateral hindpaw circumference and volumeInjection of carrageenan into the ipsilateral hindpaw produced a significant increase in ipsilateral hindpaw circumference and volume (p<0.001) but had no effect on the contralateral 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 these carrageenan-induced increases.Paw circumference/paw volume data were analysed using Kruskal-Wallis and Dunn’s multiple comparison post-hoc tests.

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Figure 3. Effect of vehicle, PEA and PEA+GW6471 administration on WDR neurone RF sizes mapped with 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-hoc tests. * = 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 and in the presence of vehicle, PEA, or PEA+GW6471PEA treatment produced a significant reduction in RF size compared to vehicle (p<0.05). Blockade of PPAR-α with GW6471 reversed the effects 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-hoc tests.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 mapped with 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-hoc tests.# = 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 and in the presence of vehicle, PEA, or PEA+GW6471PEA treatment produced a significant reduction in RF size compared to vehicle. Concomitant administration of PEA and GW6471 reversed the effects 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-hoc tests. Data are 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 reduces inflammation-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/contralateral weight bearing in rat hindpawsSaline 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-hoc tests.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

several hours-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 central and 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

several days 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 basal PPAR-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 basal rates of

activation.

4.2. Mechanisms of 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

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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 central sensitisation 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 motor neurones in the spinal grey matter. Normal cell control of [ion]i through the use of Na+/K+ATPase and the Na+/K+/Cl- co-transporter is shown at the top of the figure; the effects of GABA release by the pre-synaptic inhibitory axon of a GABAergic spinal inter-neurone are also shown. Action potentials in the PAF result in the release of Glu and the subsequent activation of AMPA-Rs on the inter-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 potential shift towards the equilibrium potential of Na+, which is now present at the highest concentration of all three ions in the PAF. The voltmeter in the bottom left of the figure shows that the cell membrane potential has increased from c.-60mV to c.-35mV, enough to cross the firing threshold and elicit the propagation of an action potential. During PAD this depolarisation is maintained and prevents repeat firing of the PAF due to inhibition of the Ca2+ channels on the post-synaptic membrane. 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 central sensitisation [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

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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 several minutes[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-

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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 several limitations 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

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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, several issues 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

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AcknowledgmentsDue 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.

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GlossaryAβ-fibre – A large diameter, myelinated neurone that senses the innocuous feeling of “touch” (such as brushing the hairs on your skin) 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.

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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.

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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 of heat 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 of by some researchers as a causal mechanism for neuronal sensitisation.

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application of pea reduces wdr rf size - final edit

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