universidade do minho - repositorium.sdum.uminho.pt · esta tese de mestrado representa um marco...

Patrícia Catarina Santos Rebelo

outubro de 2014

Comorbidity between experimental osteoarthritis and mood disorders in the rat: investigating the role of supraspinal galanin

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Universidade do Minho

Escola de Ciências da Saúde

Trabalho efetuado sob a orientação daProfessora Doutora Filipa Pinto-Ribeiro e co-orientação da Mestre Diana Amorim

Patrícia Catarina Santos Rebelo

outubro de 2014

Dissertação de MestradoMestrado em Ciências da Saúde

Comorbidity between experimental osteoarthritis and mood disorders in the rat: investigating the role of supraspinal galanin

Universidade do Minho

Escola de Ciências da Saúde

DECLARAÇÂO

Nome: Patrícia Catarina Santos Rebelo

Endereço eletrónico: [email protected]

Número do Bilhete de Identidade: 13904074

Título: Comorbidity between experimental osteoarthritis and mood disorders in the rat:

investigating the role of supraspinal galanin

Orientadores: Professora Doutora Filipa Pinto-Ribeiro e Mestre Diana Amorim

Ano de conclusão: 2014

Designação do Mestrado: Mestrado em Ciências da Saúde

É AUTORIZADA A REPRODUÇÃO INTEGRAL DESTA DISSERTAÇÃO APENAS PARA EFEITOS DE

INVESTIGAÇÃO, MEDIANTE DECLARAÇÃO ESCRITA DO INTERESSADO, QUE A TAL SE

COMPROMETE;

Universidade do Minho,

Assinatura: __________________________________________

mailto:[email protected]

III

AGRADECIMENTOS

Esta tese de Mestrado representa um marco importante na minha vida, um longo caminho

percorrido e sempre desejado. Cresci muito em termos de conhecimento e maturidade e, como

tal, não podia deixar de agradecer a todas as pessoas que contribuiram para que isso fosse

possível. Portanto, em homenagem a todos aqueles que sempre me apoiaram e que, direta ou

indiretamente, colaboraram na realização deste trabalho, deixo aqui os meus sinceros e

reconhecidos agradecimentos.

À minha orientadora, Doutora Filipa Pinto-Ribeiro, o meu maior agradecimento pela

oportunidade, apoio, orientação e paciência ao longo deste ano, por toda a liberdade de ação e

pelas sugestões e correções que permitiram o desenvolvimento de um trabalho melhor.

À minha co-orientadora, Mestre Diana Amorim, pela forma como me orientou no laboratório,

pela sua preciosa e disponível ajuda nunca negada, pela imensa paciência e amabilidade com

que sempre tentou responder às minhas questões e pelos experientes conselhos. Agradeço de

coração tudo aquilo que me ensinou e todo o tempo que dedicou a este trabalho.

À Vera Cardoso, um especial agradecimento, por todas as “aulas” de microscópio, pela sua boa

disposição e disponibilidade em colaborar sempre que solicitada a sua ajuda.

À Ana Pereira e Sónia Puga pela sua prontidão em ajudar.

A todos os docentes pelos conhecimentos transmitidos ao longo destes dois anos e por todos os

ensinamentos de vida que contribuiram para o meu crescimento académico e pessoal.

Aos meus pais, que sem eles nada seria possível, pelo amor incondicional, pela educação e por

toda a dedicação nesta minha caminhada. Serei eternamente grata por todo o esforço para que

nunca me faltasse nada e por todo o apoio. A eles, que renunciaram aos seus sonhos para que

eu pudesse realizar o meu, espero retribuir e compensar todo o conforto que sempre tive. Por

isso, a eles dedico todo este trabalho.

Ao meu irmão por toda a amizade e apoio, e aos meus tios, primas e avó pelo incentivo recebido

ao longo destes anos.

Ao meu namorado, pelo apoio e carinho diários, por toda a sua ajuda nas alturas mais difíceis,

pela força que sempre me transmitiu e pela paciência com que ouviu as minhas inquietações,

IV

dúvidas e desânimos. O meu agradecimento de coração pela forma como tornou especial este

ano e transformou momentos menos bons em momentos felizes.

Às meninas que levarei para sempre no meu coração:

- Cristiana, amiga e companheira de todas as horas de laboratório, pelos momentos felizes,

desânimos e angústias que ultrapassamos juntas. Não esquecerei todas as nossas horas de

microscópio juntas, assim como as nossas caminhadas noturnas.

- Maria João, que mesmo longe sempre deu o seu apoio e me lembrou que estava ali para tudo.

Exemplo de coragem e força, agradeço toda a sua amizade verdadeira, todos os momentos

divertidos e as longas conversas via skype.

- Joana, que por circunstâncias da vida nos últimos tempos nos vimos um pouco afastadas, mas

que existiu sempre entre nós uma carinhosa amizade.

A todos, um carinhoso muito obrigada!

“Quando não souberes para onde ir, olha para trás e sabe pelo menos de onde vens.” (Provérbio

africano)

V

Comorbidity between experimental osteoarthritis and mood disorders in the rat:

investigating the role of supraspinal galanin

ABSTRACT

Arthritis and depression are pathologies highly prevalent in society with great impact not only on

National Health Systems but also on the quality of life of patients. Several studies have shown

that anxiety and depression are common comorbidities of chronic pain patients while anxious

and/or depressed individuals also display altered perception of pain. This interplay has been

suggested to result from the fact that these pathologies share common modulatory pathways.

Indeed, acute changes in the neurochemistry of brain areas, such as the amygdala (AMY), have

been shown to transiently alter both mood and nociception. In chronic inflammatory pain, galanin

(GAL) and its receptors have been recently proposed as potential mediators of pain-depression

comorbidity as the expression of this neuropeptide is greatly increased not only in areas

mediating emotions (AMY), but also in areas mediating nociception, such as the dorsomedial

nucleus of the hypothalamus (DMH). Additionally, some studies using animal models of

depression, have also demonstrated that the differential availability/activation of galanin

receptors could induce a depressive profile in these animals. In the present work, we propose to

(i) evaluate how GAL in the DMH influences anxiety- and/or depressive-like behaviour; (ii) study

the role of the rostral ventromedial medulla (RVM) as a mediator of GAL effects and (iii)

investigate which supraspinal areas might be involved in relaying GAL effects through the

quantification of c-Fos expression. Wistar Han adult male rats were divided into two experimental

groups: animals with experimental osteoarthritis (ARTH) and control animals (SHAM). In a first

set of animals, emotional and nociceptive behaviours were assessed after GAL administration in

the DMH. In a second set, c-Fos expression in caudal brain areas that mediate nociception was

also quantified after GAL in the DMH and peripheral noxious stimulation of the right knee joint.

Our results showed ARTH animals display a depressive phenotype concomitant with alterations in

serotoninergic tone, a pathway mediated by GAL. Descending facilitation from the DMH after GAL

microinjection appears to be mediated both by the RVM and the dorsal reticular nucleus (DRt).

VII

Comorbidade entre a osteoartrite experimental e os distúrbios do humor: avaliação

do papel da galanina

RESUMO

A artrite e a depressão são patologias muito prevalentes na sociedade com um grande impacto

não só sobre os Sistemas Nacionais de Saúde mas também sobre a qualidade de vida dos

pacientes. Vários estudos demonstraram que a ansiedade e a depressão são comorbidades

comuns em pacientes que sofrem de dor crónica, enquanto indivíduos ansiosos e/ou deprimidos

também apresentam alterações na perceção da dor. Considera-se que esta interação entre

alterações emocionais e dor crónica resulta da partilha de vias moduladoras comuns entre as

duas patologias. De facto, já se demonstrou que alterações agudas na neuroquímica de áreas

cerebrais, como a amígdala (AMY), alteram tanto o humor como a perceção dolorosa. Na dor

inflamatória crónica, a galanina (GAL) e os seus receptores foram recentemente propostos como

potenciais mediadores da comorbidade dor-depressão, uma vez que a expressão deste

neuropeptídeo está aumentada não apenas em áreas que medeiam as emoções (AMY), mas

também em áreas que medeiam a nociceção, como o núcleo dorsomedial do hipotálamo (DMH).

Paralelamente, alguns estudos realizados em modelos animais de depressão, também

demonstraram que diferenças na disponibilidade/activação dos recetores de galanina

promoviam um fenótipo depressivo nestes animais. Neste trabalho, propusemo-nos a (i) avaliar o

efeito da GAL no DMH sobre o comportamento emocional e nocicetivo; (ii) estudar o papel da

medula rostral ventromedial (RVM) como mediadora do efeito facilitador da GAL, e (iii) estudar

quais as áreas supraespinhais que potencialmente medeiam a ação da GAL através da

quantificação da expressão de c-Fos. Os animais, ratos machos Wistar Han adultos, foram

divididos em dois grupos experimentais: animais osteoartríticos (ARTH) e animais controlo

(SHAM), nos quais se avaliou o comportamento emocional e nocicetivo após a administração de

GAL no DMH. Num segundo grupo avaliou-se a expressão de c-Fos em áreas cerebrais caudais

após a administração de GAL no DMH e a aplicação de estímulos nóxicos periféricos. Os nossos

resultados mostraram que a osteoartrite promove um fenótipo depressivo nos animais associado

a alterações nas vias serotoninérgicas, as quais se sabe serem mediadas pela GAL. Por fim,

verificou-se que a facilitação descendente após a ativação do DMH pela GAL é mediada pelo

RVM e pelo núcleo reticular dorsal (DRt).

IX

INDEX

CHAPTER 1: INTRODUCTION 1

1.1 Pain mechanisms 3

1.1.1 The role of the nociceptors 4

1.1.2 Ascending pain pathways 6

1.1.3 Supraspinal and descending pain modulation 7

1.1.3.1 Dorsomedial nucleus of the hypothalamus (DMH) 9

1.1.3.2 Periaqueductal gray matter (PAG) 9

1.1.3.3 Dorsal raphe nucleus (DRN) 10

1.1.3.4 Locus coeruleus (LC) 10

1.1.3.5 Rostral ventromedial medulla (RVM) 11

1.1.3.6 Dorsal reticular nucleus (DRt) 12

1.2 Chronic pain 12

1.2.1 Inflammatory pain 13

1.2.1.1 Osteoarthritis (OA) 13

1.2.1.1.1 Most common comorbidities in OA 15

1.2.2 Galanin (GAL) 16

1.2.2.1 Regulation of GAL in inflammation 17

CHAPTER 2: OBJECTIVES 19

CHAPTER 3: MATERIALS AND METHODS 23

3.1 Animals and ethical considerations 25

3.2 Anaesthesia and euthanasia 25

3.3 Induction of experimental osteoarthritis 26

3.4 Drugs 26

3.5 Evaluation of nociceptive behaviour 27

3.5.1 Vocalization 27

3.5.2 Pressure application measurement (PAM) 27

3.5.3 Paw-withdrawal (PW) test 28

3.6 Chronic implantation of intracerebral cannulae 28

X

3.7 Evaluation of mood-like behaviour 29

3.7.1 Open field (OF) test 29

3.7.2 Forced swimming test (FST) 29

3.8 Evaluation of c-Fos expression 30

3.8.1 Stimulation of c-Fos 30

3.8.2 c-Fos immunohistochemistry 30

3.8.3 c-Fos quantification 31

3.9 Experimental design 32

3.9.1 Impact of GAL in the DMH upon emotional, motor and nociceptive behaviour

(Experiment 1) 32

3.9.2 Evaluation of the expression of c-Fos after GAL in the DMH (Experiment 2) 33

3.10 Statistical analysis 34

CHAPTER 4: RESULTS 37

4.1 Histological confirmation of the injection sites 39

4.2 Nociceptive behaviour 40

4.2.1 Mechanical hyperalgesia 40

4.2.2 Heat hyperalgesia 40

4.2.3 Role of the RVM in behavioural hyperalgesia after GAL in the DMH 41

4.3 Locomotor activity 42

4.4 Mood-like behaviour 43

4.4.1 Anxiety-like behaviour 43

4.4.2 Depressive-like behaviour 44

4.5 c-Fos quantification 46

4.5.1 Ventrolateral periaqueductal gray matter (VLPAG) 46

4.5.2 Dorsal raphe nucleus (DRN) 47

4.5.3 Locus coeruleus (LC) 49

4.5.4 Rostral ventromedial medulla (RVM) 50

4.5.5 Dorsal Reticular Nucleus (DRt) 51

CHAPTER 5: DISCUSSION 53

5.1 Technical considerations 55

XI

5.1.1 The choice of animal strain 55

5.1.2 Anaesthesia 55

5.1.3 The choice of animal model 56

5.1.4 Evaluation of nociceptive behaviour 58

5.1.4.1 Mechanical hyperalgesia 59

5.1.4.2 Heat hyperalgesia 60

5.1.5 Pharmacological studies 60

5.1.6 Evaluation of mood-like behaviour 61

5.1.6.1 Anxiety-like behaviour 61

5.1.6.2 Depressive-like behaviour 62

5.1.7 Evaluation of c-Fos expression 63

5.1.7.1 c-Fos as neuronal marker 63

5.1.7.2 c-Fos stimulation protocol 64

5.2 The role of GAL in the DMH upon behaviour 65

5.2.1 Locomotor activity 65

5.2.2 Anxiety-like behaviour 66

5.2.3 Evaluation of depressive-like behaviour 66

5.3 The RVM as a relay of the GAL pronociceptive action in the DMH 68

5.4 c-Fos expression upon key areas of nociception 69

5.4.1 Ventrolateral periaqueductal gray matter (VLPAG) 69

5.4.2 Dorsal raphe nucleus (DRN) 70

5.4.3 Locus coeruleus (LC) 71

5.4.4 Rostral ventromedial medulla (RVM) 72

5.4.5 Dorsal Reticular Nucleus (DRt) 73

CHAPTER 6: CONCLUSION AND FUTURE PERSPECTIVES 75

CHAPTER 7: REFERENCES 79

XIII

ABREVIATIONS

5-HT – serotonin

ABC – avidin-biotin complex

AMY – amygdala

ANOVA2W – two-way analysis of variance

ARTH – osteoarthritic group

cAMP – cyclic adenosine monophosphate

CGRP – calcitonin gene-related peptide

CNS – central nervous system

DAB – diaminobenzine

DAG – diacylglycerol

DMH – dorsomedial nucleus of the hypothalamus

DNIC – diffuse noxious inhibitory controls

DRN – dorsal raphe nucleus

DRt – dorsal reticular nucleus

EPM – elevated plus maze

FBS – fetal bovine serum

FST – forced swimming test

GABA – gamma-aminobutyric acid

GAL – galanin

GALR – galanin receptors

GALR1 – galanin receptors type 1



GLU – glutamate

i.p – intraperitoneally

XIV

K/C – kaolin/carrageenan

LDB – light/dark box

LC – locus coeruleus

LIDO – lidocaine

LWT – limb-withdrawal threshold

MAPK – mitogen-activated protein kinase

MIA – monoiodoacetate

NA - noradrenaline

OA – osteoarthritis

OF – open field

PAG – periaqueductal gray matter

PAM – pressure measurement application

PBN – parabrachial nucleus

PBS – phosphate buffer saline

pERK – phosphorylated extracellular signal-regulated kinase

PFA – paraformaldehyde

PLC – phospholipase C

PVN – paraventriculat nucleus of the hypothalamus

PW – paw-withdrawal

PWL – paw-withdrawal latency

RVM – rostral ventromedial medulla

SAL – saline

SEM – standard error

SHAM – control group

SP – substance P

SPT – sucrose preference test

XV

STM – peripheral noxious stimulation

TF – tail-flick

VLPAG – ventrolateral periaqueductal gray matter

VPL – ventral posterolateral

VPM – ventral posteromedial

1

CHAPTER 1: INTRODUCTION

3

1. INTRODUCTION

The interest in the study of chronic pain and the search for therapies for its management has

increased in the last decades (Perez, 2006). Chronic pain is a major health issue all over the

world (Zhuo, 2008), affecting around 20% of the European population (McGuire and Kennedy,

2013; van Hecke et al., 2013). Unfortunately most of the available therapies have limited

success, highlighting the need to better understand the mechanisms underlying the development

and maintenance of chronic pain (Porreca et al., 2002).

The inability to satisfactorily treat pain has a profound economic impact on National Health

Systems and in the quality of life of patients with ramifications to their families and society

(Brennan et al., 2007). More importantly, prolonged longevity has also led to an increase in

age-related diseases, most of which are accompanied by chronic pain, e.g. osteoarthritis and

diabetes, again stressing the need for efficient therapies (Scholz and Woolf, 2002).

Pain is a multidimensional sensation that affects and is affected by emotional components such

as mood (Katz, 2002), and is considered the fifth vital sign (Lynch, 2001; Morone and Weiner,

2013). According to the definition adopted by the International Association for the Study of Pain

(IASP), pain is defined as “an unpleasant sensory and emotional experience associated with

actual or potential tissue damage, or described in terms of such damage” (Loeser and Treed,

2008). This definition clearly highlights that pain is a subjective experience and stresses the

notion that pain can occur without apparent reason or visible injury (Rainville, 2002).

1.1 Pain mechanisms

Pain can be divided into acute and chronic pain. Acute pain being considered an important

adaptive alarm system of short duration that usually disappears after healing, while chronic pain

lasts for at least 3 months, long after its original cause has been treated (Casey et al., 2008).

However, chronic pain differs from acute pain not only because of the duration of pain but, more

importantly, because of the inability to restore its function to normal homeostatic levels (Loeser

and Melzack, 1999).

Acute pain, defined as an adverse physiological response to a chemical, thermal or mechanical

stimulus often associated with surgery, trauma or certain diseases (Carr and Goudas, 1999),

4

comprises a motor and emotional response (Brennan et al. 2007) and varies according to the

intensity, type and duration of the stimulus (Voscopoulos and Lema, 2010).

Although the specific mechanisms underlying the transition from acute to chronic pain are mostly

unknown (Lavand’homme, 2011; Buchheit et al., 2012), peripheral inflammation and the

continuous activation of primary afferents play an important role in this process. Importantly, the

persistent activation of primary afferents winds-up neuronal activity in the spinal cord leading to

changes in the neurochemistry and signalling properties of neuronal networks not only within the

spinal cord but also in the brain, a process designated as secondary or central sensitization

(Julius and Basbaum, 2001).

Both, peripheral and central sensitizations are considered to play an important role in the

transition from acute to chronic pain conditions (Apkarian et al., 2009; Voscopoulos and Lema,

2010), as they contribute to the development of chronic pain syndromes such as hyperalgesia

(enhancement of the nociceptive response after a noxious stimulus), allodynia (a stimulus that

previous was innocuous is now perceived as painful) and spontaneous pain (sharp pain sensation

without any obvious source of stimulation) (Vadivelu et al., 2010).

Besides physical factors, life stressors and affective-cognitive factors have also been

demonstrated to play an important role towards the sustainability of chronic pain (Casey et al.,

2008; Kyranou and Puntillo, 2012). More recently Apkarian and colleagues (2011) proposed

pain-related long-term memories remained active long after healing and contributed to the

maintenance of chronic pain.

1.1.1 The role of the nociceptors

Pain is only perceived as such after the nociceptive signal is processed by the brain (Rainville,

2002), but nociceptors play an essential role in initiating it (Rainville, 2002). Nociceptors, or

primary afferents, are responsible for the detection, transduction and transmission of peripheral

stimuli to the spinal cord (Woolf and Ma, 2007).

Primary afferents are thus specialized receptors that detect noxious (painful) stimuli, such as

extreme temperatures (heat and cold) and pressure (Dubin and Patapoutian, 2010), and

5

represent the first line of defense against any potential threats to the organism (Woolf and Ma,

2007).

Cutaneous primary afferents involved in the transmission of sensory information include three

fibers types: Aβ-, A- and C-fibers (Julius and Basbaum, 2001), but only A- and C-fibers are

involved in acute nociceptive transmission (D’Mello and Dickenson, 2008; Basbaum et al.,

2009). A-fibers are small myelinated primary afferents responsible for fast nociceptive

transmission (Basbaum et al., 2009). These fibers project to the superficial lamina I and lamina

V of dorsal horn (Fig. 1B) and its activity is associated with the sensation of “first pain” (Basbaum

et al., 2009; Dubin and Patapoutian, 2010).

On the other hand, C-fibers, are unmyelinated fibers and are responsible for slow conduction,

whose activity is associated with “second pain” (Julius and Basbaum, 2001; Craig, 2003; Dubin

and Patapoutian, 2010). Interestingly, while A-fiber activity is usually evoked by one stimulatory

modality (only pressure or only temperatures), C-fibers respond simultaneously to mechanical,

thermal and chemical stimuli (Julius and Basbaum, 2001), being classified as polymodal (Dubin

and Patapoutian, 2010). In addition, C-fibers target mainly superficial laminae I and II (Fig. 1A)

(Basbaum et al., 2009; Dubin and Patapoutian, 2010).

Figure 1 – Schematic representation of the projections of A- (B) and C-fibers (A) to the spinal cord (Dubin and

Patapoutian, 2010).

B

A

6

1.1.2 Ascending pain pathways

Once primary afferents synapse in the superficial dorsal horn of spinal cord, the signal is

forwarded to the brain by projection neurons through multiple ascending pathways (Basbaum et

al., 2009) such as the spinothalamic, spinomesencephalic, spinoreticular, spinohypothalamic

and spinoreticulothalamic tracts (Lopes, 2007). These tracts relay nociceptive information to

several levels (Almeida et al., 2004) with the lateral column relaying the discriminative

component of pain (intensity, location, duration) while the medial column is associated with the

transmission of cognitive/affective components of pain (Almeida et al., 2004; Lopes, 2007).

Brain areas involved in the processing and modulation of pain are often referred to as the “pain

matrix” or “homeostatic afferent processing network” (Neugebauer et al., 2009).

The spinothalamic tract is a crucial pathway for pain transmission (Willis, 1985). It is composed

of a lateral component, that directly projects to the ventral posterolateral (VPL) and ventral

posteromedial (VPM) nuclei of the thalamus, relaying information about sensory-discriminative

aspects of pain to the somatosensory cortex (Almeida et al., 2006; Lopes, 2007). The medial

component projects to the medial thalamus that conveys information to the limbic system

concerning the emotional/cognitive aspects of pain (Almeida et al., 2004; Lopes, 2007).

The spinothalamic tract projects to multiple midbrain reticular areas that are involved in the

descending modulation of pain (Lopes, 2007) such as the periaqueductal gray matter (PAG)

(Millan, 1999), an area that plays a central role in the integration of supraspinal drives and

modulation of nociception (Lemke, 2004).

The spinomesencephalic tract projects specifically to mesencephalic nucleus also involved in

descending pain modulation (Lopes, 2007), namely the dorsal reticular nucleus (DRt) (Willis and

Westlund, 1997) and the parabrachial nucleus (PBN) (Millan, 1999). It is also known that the

stimulation of the regions innervated by the spinomesencephalic tract may evoke aversive

behaviours in the presence of noxious stimulation (Dougherty et al., 1999; Almeida et al., 2004).

The spinoreticular tract targets not only the lateral and medial reticular formation, involved in

motor control and nociception, respectively (Millan, 1999; Almeida et al., 2004), but also the

medial thalamic nucleus involved in the motivational-affective component of pain (Lopes, 2007;

Almeida et al., 2004). Its importance is related to the fact that it targets brainstem structures

responsible for the descending inhibition of pain (Almeida et al., 2004).

7

Finally, the spinohypothalamic tract projects to autonomic control centers in the hypothalamus,

thalamus and amygdala (Lemke, 2004; Lopes, 2007). This tract is responsible for contributing to

the activation of the neuroendocrine autonomic, motivational-affective responses to pain and for

activating the “fight or flight” response in life threatening situations (Almeida et al., 2004; Lemke,

2004).

As evidenced by the number of areas activated during and after noxious stimulation, the

transmission and modulation of nociception is not a linear process (Basbaum et al., 2009).

1.1.3 Supraspinal and descending pain modulation

The activation of areas processing nociception result in the activation of the descending pain

modulatory pathways (Fig. 2) that, through brainstem relays with spinal projections, will either

enhance or inhibit nociceptive transmission at the spinal cord level (Lima and Almeida, 2002).

Descending pain modulation is a dynamic and plastic process (Lima and Almeida, 2002). It

involves many brain regions that play an important role in the integration/processing of the

emotional, cognitive and autonomic components of pain (Lopes, 2007) towards the control of the

nociceptive transmission in the dorsal horn both in acute and chronic pain conditions (Millan,

1999; Heinricher et al., 2009). Literature shows that regions such as the medial and prefrontal

cortex has been associated with the cognitive aspects of the modulation (Oshiro et al. 2009), the

amygdala (AMY) with the emotional component processing (Neugebauer et al., 2009) and the

paraventricular nucleus of the hypothalamus (PVN) (Pinto-Ribeiro et al., 2008) and dorsomedial

nucleus of the hypothalamus (DMH) (Pinto-Ribeiro et al., 2013) with the autonomic and the

innate responses to pain (Borszcz, 2006).

Several studies have shown that most frontal and medial brain areas present few or no

projections to the spinal cord, these areas target relay nuclei such as the PAG (Fig. 2), the dorsal

raphe nucleus (DRN), the locus coeruleus (LC), the rostral ventromedial medulla (RVM) and the

DRt (Lemke, 2004; Tracey and Mantyh, 2007; Heinricher et al., 2009).

The PAG and the RVM, commonly known as the PAG-RVM system, (Lopes, 2007; Heinricher et

al., 2009) were considered, for a long time, as the sources of descending inhibitory control

(antinociception) (Heinricher et al., 2009). Since the PAG presents very few projections to the

8

spinal cord, its modulatory effects are relayed through spinal projecting RVM neurones (Hudson

and Lumb, 1996).

However, it is now evident that the descending pain modulatory system, depending on the

circumstances, can facilitate the spinal transmission of nociception (pronociception) (Ren and

Dubner, 2002; Tracey and Mantyh, 2007; Heinricher et al., 2009).

Figure 2 – Schematic representation of the ascending and descending pain modulatory circuits (Adapted from Ossipov et al., 2010).

9

1.1.3.1 Dorsomedial nucleus of the hypothalamus (DMH)

The DMH, located in the hypothalamic region (Stotz-Potter et al., 1996), is implicated in a wide

variety of functions including thermogenic responses to emotional stressors (DiMicco et al.,

2002; DiMicco et al., 2006), and cardiovascular, locomotor and stress/anti-anxiety responses

(Thompson et al., 1996). More importantly, the DMH is strongly involved in the “fight or flight”

response as it is responsible for the activation of areas mediating acute behaviour and autonomic

responses (DiMicco et al., 2002). Due to its role in acute stress and its projections to areas

mediating pain (ter Horst and Luiten, 1986; Wagner et al., 2013), the DMH was first considered

to inhibit nociception. Additionally, in 1996, anatomical studies by Thompson and colleagues,

showed that the DMH projects to areas such as PAG (Samuels et al., 2004; Martenson et al.,

2009), DRN and RVM.

However, more recently the DMH was proposed to mediate behavioural hyperalgesia (Martenson

et al., 2009; Pinto-Ribeiro et al., 2013). Studies by Martenson and colleagues (2009) and

Pinto-Ribeiro and colleagues (2013) showed the disinhibition of the DMH by bicucculine or its

activation by glutamate (GLU), respectively, decreased tail-flick (TF) latency which was

concomitant with changes in the activity of RVM pain modulatory cells towards the facilitation of

nociception. Interestingly, while the behavioural effect of the DMH disinhibition/activation

appears to be mediated the RVM in control animals, in an experimental model of monoarthritis it

was absent (Pinto-Ribeiro et al., 2013).

Interestingly, while DMH glutamatergic projections appear to be inhibited in monoarthritic

animals, galaninergic neurones remain active and the administration of galanin (GAL) in the DMH

decreased paw withdrawal (PW) latency - pronociceptive affect - in normal and arthritic animals

(Amorim et al., 2014).

1.1.3.2 Periaqueductal gray matter (PAG)

The PAG is a midbrain nucleus involved in several functions such as pain and analgesia, fear and

anxiety, autonomic regulation and reproductive behavior (Behbehani, 1995; Linnman et al.,

2012). However, it is for its role in pain modulation, both in acute and chronic conditions, that

the PAG is mostly known (Waters and Lumb, 2008).

10

In 1969, Reynolds demonstrated that the electrical stimulation of the PAG evoked behavioral

analgesia in rats (Loyd and Murphy, 2009), showing this area as an antinociceptive area able to

inhibit nociceptive transmission in the spinal cord (Waters and Lumb, 1997; Cui et al., 1999;

Loyd and Murphy, 2009). More recently, Waters and Lumb (2008) showed the neuronal

activation of the PAG may selectively “modulate” the activity of A and C-fibers by suppressing

the activity of latter while enhancing the activity of the former. These authors showed for the first

time, that the PAG could play both an antinociceptive and a pronociceptive role.

1.1.3.3 Dorsal raphe nucleus (DRN)

The DRN is another midbrain nucleus (Michelsen et al., 2008) where approximately half of the

brain’s serotonergic neurons can be found (Jacobs and Azmitia, 1992; McDevitt and Neumaier,

2011). The DRN has been strongly associated with a great variety of physiological and

behavioural functions, namely pain, sleep and mood disorders, such as depression (Peyron et al.,

1998; Michelsen et al., 2008).

This nucleus projects to sensory-motor areas in the rat (Kirifides et al., 2001; Lee et al., 2008)

and receives projections from many limbic areas, such as the prefrontal cortex, the

hypothalamus and the AMY (Nakamura, 2013). Its descending projections were shown to

modulate the behavioural responses evoked by noxious stimulations and may be involved in

analgesia (Wang and Nakai, 1994). It is also known that the inhibition of the activity of

serotonergic neurons in the DRN reduces anxiety in animal models while increasing it enhances

anxiety (Maier and Watkins, 2005).

1.1.3.4 Locus coeruleus (LC)

The LC, located in the dorsolateral pons (Liu et al., 2008) contains most of the noradrenergic

neurones in the brain and is involved in several biological functions including attention, vigilance,

brain plasticity, learning and memory (Berridge and Waterhouse, 2003; Liu et al., 2008). This

nucleus receives projections from several areas, including the PAG and the hypothalamus, and

targets the RVM and the spinal cord (Maeda et al., 2009; Ossipov et al., 2010).

11

Its role in pain modulation was first suggested by anatomical studies showing strong projections

to areas such as the PAG, the RVM and the spinal cord (Maeda et al., 2009). In addition,

electrophysiological studies showed an increase in the activity of LC neurones during peripheral

noxious stimulation, while its activation by morphine (Jones and Gebhart, 1988) inhibited

nociception (Liu et al., 2008; Maeda et al., 2009; Szabadi, 2012). Tsuruoka and Willis (1996)

and Maeda and colleagues (2009) showed that during chronic inflammation, the activation of the

LC decreased hyperalgesia, while LC bilateral lesion increased pain syndromes (hyperalgesia and

allodynia) and the expression of c-Fos protein, a marker of cell activation (Tsuruoka et al., 2003).

1.1.3.5 Rostral ventromedial medulla (RVM)

The RVM is considered as one of the brain effectors of pain descending modulation (Millan,

2002; Chai et al., 2012) as it projects to the dorsal horn of the spinal cord, where it is able to

directly and indirectly modulate the activity of primary afferents (Millan, 2002).

The RVM exerts a biphasic descending action either enhancing (facilitation) or inhibiting spinal

nociceptive transmission (Carlson et al., 2007; Lopes, 2007; Tillu et al., 2008; Aicher et al.,

2012), depending on the type and intensity of the initial stimulus (Urban and Gebhart, 1997; Bee

and Dickenson, 2007). Interesting, the inactivation of this nucleus by lidocaine (LIDO) attenuates

spinal hyperalgesia and mechanical allodynia (Géranton et al., 2010) in acute inflammation

(Ambriz-Tututi et al., 2011; Cleary and Heinricher, 2013) and neuropathic pain (Pertovaara et al.,

1996; Sanoja et al., 2008).

The facilitatory or inhibitory action of the RVM is associated with the activity of its heterogeneous

neurones that can be divided into: ON-, OFF- and NEUTRAL-cells (Carlson et al., 2007; Khasabov

et al., 2012). ON-cells are known to facilitate nociception since they increase their activity in

response to noxious stimuli just prior to the motor withdrawal reflex while OFF-cells inhibit

nociceptive transmission as their activity decreases immediately before the motor withdrawal

response (Fields et al., 1983; Martenson et al., 2009; Khasabov et al., 2012).

By contrast, NEUTRAL-cells do not respond to noxious or innocuous peripheral stimulation,

although a role in pain modulation could not be ruled out (Miki et al., 2002; Martenson, 2009).

In fact, Miki and colleagues (2002) observed that, during prolonged inflammation, NEUTRAL-cells

12

shifted their response profile from NEUTRAL- to ON- or OFF-like cells and thereby also

contributed to the descending modulation of pain in chronic disorders (Khasabov et al., 2012).

1.1.3.6 Dorsal reticular nucleus (DRt)

The DRt is located in the most caudal portion of the medullary dorsolateral reticular formation

(Leite-Almeida et al., 2006) and is one of the few nuclei that exclusively facilitate nociception

(Almeida et al., 1996; Tavares and Lima, 2007). Interestingly, this area is activated exclusively by

noxious stimuli independently of the body part stimulated (Almeida et al., 1996, 1999; Lima and

Almeida, 2002; Leite-Almeida et al., 2006). Anatomically, the DRt shares reciprocal projections

with the spinal cord and several brainstem areas such as the RVM, PAG and LC, and forebrain

areas (Lima and Almeida, 2002; Leite-Almeida et al., 2006). In addition to its activation during

acute stimulation, the DRt also displayed significant activation in chronic pain states

(inflammatory pain and neuropathic pain) (Pinto et al., 2006, 2008).

1.2 Chronic pain

The continuous activation of nociceptors increase synaptic excitability, decrease activation

thresholds and increase responsiveness of spinal neurons (Woolf and Ma, 2007; Woolf, 2011)

resulting in the plasticity of areas mediating nociception and leading to the development of pain

syndromes (hyperalgesia, allodynia and spontaneous pain) (Wall, 1979; Dubin and Patapoutian,

2010; Woolf, 2011).

During central sensitization, pain is no longer proportional to the intensity and duration of the

peripheral noxious stimuli (Woolf, 2011) due the occurrence of neuroplasticity in spinal and

supraspinal circuits mediating pain (Besson, 1999; Loeser and Melzack, 1999). Apkarian and

colleagues (2009) proposed plastic changes in the pain matrix led to “the inability to extinguish

the memory evoked by the initial injury” which imbalanced the facilitatory and inhibitory

descending pain drives towards the exacerbation of pain.

Chronic pain can be divided into (i) nociceptive or inflammatory, (ii) neuropathic and (iii)

neurogenic pain (Carr and Goudas, 1999; Scholz and Woolf, 2002). Chronic nociceptive pain is

usually associated with recurrent inflammatory processes due to tissue injury and/or the

13

activation of immune cells (Backonja, 2003), while neuropathic pain is associated with lesion of

the peripheral and central nervous system (Woolf, 2001). Neurogenic pain occurs when a cause

(physical) cannot be associated to pain (Bowsher, 1991).

1.2.1 Inflammatory pain

Inflammatory pain may be classified as acute or chronic (Lawrence and Gilroy, 2007) and

although chronic inflammatory pain shares many characteristics with it acute counterpart, it is

biologically distinct in what concerns exudation, cellular recruitment and the types of cells that

prevail in the chronic inflammatory response (Wakefield and Kumar, 2001).

Chronic inflammatory pain is characterized by long-term inflammation (or recurrent episodes of

prolonged inflammation) (Heap and van Heel, 2009), which induce the release of an

“inflammatory soup” from the affected tissues (Julius and Basbaum, 2001). This “inflammatory

soup” containing chemical mediators activates surrounding nerve endings and nociceptors

evoking pain (Scholz and Woolf, 2002; Kyranou and Puntillo, 2012). Besides external causes

(such as injuries) (Wakefield and Kumar, 2001), chronic inflammation might also be due to

aging, as tissue degeneration occurs (Backonja, 2003).

Age associated chronic inflammatory disorders include knee osteoarthritis, multiple sclerosis and

Chrohn’s disease (Wakefield and Kumar, 2001; Sommer and Kress, 2004; Heap and van Heel,

2009).

1.2.1.1 Osteoarthritis (OA)

OA is the leading cause of disability in the elderly population and affects a large proportion of

society (Dieppe and Lohmander, 2005; Arendt-Nielsen et al., 2010; Egloff et al., 2012;

Ferreira-Gomes et al., 2012). Epidemiological studies estimate that OA affects approximately 43

million people in the Unites States (Egloff et al., 2012) and 10% of the world’s population over 60

years (Adães et al., 2014). According to Monjardino and colleagues (2011), in Portugal 11,1% of

the population suffers from OA with 5,5% suffering from knee OA.

14

OA is a chronic multifactorial disease characterized by a progressive degradation of the articular

cartilage (narrowing joint space) associated with subchondral bone remodeling, bone sclerosis,

the formation of bone cysts, marginal osteophytes and secondary inflammation of synovial

membranes (Fig. 3) (van Laar et al., 2012; Hawamdeh and Al-Ajlouni, 2013). It is a degenerative

disease that involves nociceptive and non-nociceptive components due to peripheral

inflammation and central sensitization (Woolf, 2011; Arendt-Nielsen et al., 2010).

Figure 3 – Radiograph of a knee joint from a patient suffering from knee osteoarthritis. Note the development of osteophytes, the existence of bone sclerosis and the narrowing of the space between the adjacent joints (Adapted from Dieppe and Lohmander, 2005).

Amongst the possible affected joints, the knees present the higher prevalence of OA (Arden and

Nevitt, 2006; Jinks et al., 2007; Arendt-Nielsen et al., 2010). Knee OA leads to decreased knee

flexibility, constant pain and joint effusion, crepitus, bone deformities and loss of function

(Hawamdeh e Al-Ajlouni, 2013). OA patients display lower withdrawal thresholds during

mechanical and thermal stimulation when compared to healthy controls (van Laar et al., 2012).

They also display increased sensitivity to innocuous stimulation (Kosek e Ordeberg, 2000)

suggesting that in this disorder pain is centrally mediated (Lee et al., 2011).

Indeed, functional magnetic resonance imaging studies allowed the identification of several brain

regions involved in processing of pain in OA patients including the thalamus and AMY (Sofat et

al., 2011). Interestingly, the intensity of pain reported by patients is not proportional to the extent

15

of joint damage (van Laar et al., 2012) again suggesting the occurrence of central plasticity in

areas mediating pain.

The degeneration of knee structures induces a complex and dynamic cascade of biochemical and

cellular inflammatory events (Ji et al., 2011) affecting the normal activity of surrounding

nociceptors (Nagy et al., 2006) towards the enhancement on spinal nociceptive signaling through

the excessive release of substance P (SP) (Khasabov et al., 2012), calcitonin gene related-peptide

(CGRP) (Bird et al., 2006; Nagy et al., 2006; Orita et al., 2011) and GAL (Liu and Hökfelt, 2002).

1.2.1.1.1 Most common comorbidities in OA

In addition to the exacerbation of pain, the development of emotional impairments is common in

OA patients. Epidemiological studies show the incidence of chronic pain enhances the

development of mood disorders, such as anxiety and depression (Barton et al., 2007;

Sherbourne et al., 2009; Axford et al., 2010). Similarly, patients suffering from anxiety and/or

depression display changes in sensitivity and are more likely to develop chronic pain

(Neugebauer et al., 2009). In the case of OA patients, depression is the most common

comorbidity observed (Lin, 2008).

The comorbidity between chronic pain and emotional impairments is thought to result from the

existence of pathways that modulate both pain and emotions (Krishnan et al., 2008). This theory

is supported by the fact, that in elderly patients displaying both depression and arthritis, the

administration of antidepressants not only improved mood but also reduced the intensity of pain

contributing significantly to the improvement of the quality of life of these patients (Lin, 2008).

Although the mechanisms underlying chronic pain-depression comorbidities are still poorly

understood (Agarwal et al., 2013) GAL and its receptors are considered potential mediators of

this interplay, at least in rodents (Kuteeva et al., 2008). GAL exerts modulatory effects in

noradrenergic and serotonergic systems and Kuteeva and colleagues (2008) recently

demonstrated, in an animal model of depression, that the differential activation of GAL receptors

(GALR) could induce a more or less depressive-like profile. Interestingly, GAL receptors type-1

(GALR1) promoted “prodepressive”-like behaviours while GAL receptors type-2 (GALR2) promoted

an “antidepressant”-like phenotype.

16

1.2.2 Galanin (GAL)

GAL is a neuropeptide consisting of 29 or 30 amino acids (Xu et al., 2000; Elliot-Hunt et al.,

2004) involved in many diverse biological functions, including learning, feeding, memory,

cognition, neuroendocrine modulation and nociception (Jimenez-Andrade et al., 2004; Xu et al.,

2012a).

GAL is expressed widely throughout the central nervous system of various species, including the

rat (Lang et al., 2007), namely in the hypothalamus (preoptic, paraventricular, periventricular

and dorsomedial nuclei), DRN, LC, RVM, AMY (medial and lateral) and supraoptic nuclei (Fang et

al., 2012). This neuropeptide is also co-expressed with other neurotransmitters such as serotonin

(5-HT) in DR, norepinephrine (NA) in LC, SP and CGRP in dorsal root ganglia and

gamma-aminobutyric acid (GABA) in the dorsal horn (Landry et al., 2005; Lang et al., 2007). GAL

is released in the spinal cord predominantly by C-fiber (Liu and Hökfelt, 2002; Xu et al., 2012b)

during nerve injury and peripheral inflammatory processes (Liu and Hökfelt, 2002; Hulse et al.,

2012).

GAL is an important messenger for intercellular communication (Xiong et al., 2005) and its role

in pain modulation is complex (Holmes et al., 2003). It’s involved in the transmission and

modulation of nociception in the spinal cord (Fu et al., 2011) in a dose-dependent manner, with

high concentrations promoting antinociception and low concentrations pronociception (Hulse et

al., 2012).

GAL effect depends on the activation of three GAL G-protein-coupled receptor subtypes present in

primary afferent neurones: GALR1, GALR2 and GAL receptor type-3 (GalR3) (Fig. 4) with GALR1

and GALR3 being inhibitory and GalR2 excitatory (Xu et al., 2008, Hulse et al., 2012). The

signaling properties of GalR3 are still poorly defined (Lang et al., 2007) and its expression in

rodents is weak (Lu et al., 2005; Yu et al., 2013). By contrast GALR1 and GALR2 are highly

expressed in both humans and rodents (Hohmann et al., 2003; Yu et al., 2013) although their

expression between species varies significantly (Kuteeva et al., 2008).

In normal conditions, the activation of signaling pathways via GALR1 and GALR3 facilitate

hyperpolarization through Gi/G0 type G-proteins (Liu and Hökfelt, 2002) (Fig. 4) and,

consequently, contributes to the inhibition of circuits involved in nociception in spinal cord

(Landry et al., 2005). According to Landry and colleagues (2005), the activation of GALR1 and

17

GALR3 can lead to cell hyperpolarization through activation of Gi/Go proteins with a consequent

decrease of cyclic adenosine monophosphate (cAMP) intracellular levels, that is a second

messenger used in the ntracellular transduction, and opening of rectifying potassium (K+)

channels. GALR1 activating mitogen-activated protein kinase (MAPK) (Fig. 4), that is involved in

cellular responses to stimulation, for example factors proinflammatory. Contrary, GALR2 binds to

the alpha q/11 subunit and stimulate the phospholipase C (PLC), resulting in the increase of

inositol (1,4,5)-triphosphate (Ins(1,4,5)P3) and intracellular calcium (Ca2+), and in the activation

of protein kinase C via diacylglycerol (DAG) (Fig. 4). So, there is an enhancement of neural

excitation and the transmitter release is increased (Liu and Hökfelt, 2002; Landry et al., 2005).

Figure 4 – Transduction mechanisms of GAL receptors (K+-potassium; AC-adenylyl cyclase; ATP-adenosine triphosphate; cAMP- cyclic adenosine monophosphate; MAPK-mitogen-activated protein kinase; PLC-phospholipase C; Ptdlns(4,5)P2-phosphatidylinositol 4,5-bisphosphate; Ins(1,4,5)P3- inositol (1,4,5)-triphosphate; Ca2+ -calcium). (Liu and Hökfelt, 2002)

1.2.2.1 Regulation of GAL in inflammation

Several studies showed that GAL is involved in the regulation of inflammatory processes (Land

and Kofler, 2011) as this neuropeptide is strongly up-regulated (10x) during inflammatory events

(Liu and Hökfelt, 2002). This observation also suggested GAL could be modulating nociception at

the spinal cord (Sun et al., 2003; Lang and Kofler, 2011), a fact that was confirmed when the

intrathecal administration of GAL altered nociception (Lemons and Wiley, 2011). Furthermore,

during inflammatory processes, the pronociceptive effect of exogenous GAL in the spinal cord

18

was associated with the activation of GALR2 while activation of GALR1 was antinociceptive (Liu

and Hökfelt, 2002) (Fig. 5).

Figure 5 – Schematic representation of the effect of the activation of galanin receptors during inflammation. The pronociceptive role of GAL during inflammation is associated to a down-regulation of GALR1 and up-regulation of GALR2 in the spinal cord (Lundström et al. 2005).

Although, the action of GAL in nociceptive modulation has been intensively investigated in the

spinal cord using behavioural and electrophysiological techniques (Sun et al., 2003; Gu et al.,

2007), the role of GAL at the supraspinal level is still unclear (Yu et al., 2013).

In the brain, the intracerebroventricular administration of GAL activates areas that facilitate

nociception, such as the DMH and the AMY (Blackshear et al., 2007) as shown by increased

expression of c-Fos in these areas (Fang et al., 2012). Preliminary data from our group showed

that the activation of GALR in the DMH enhance nociception in rats (data not published). In

addition, Xiong and colleagues (2005) demonstrated that the intrathecal administration of GAL

increased hindpaw withdrawal latencies during the application of noxious thermal and

mechanical stimuli in rats with inflammation (Yu et al., 2013).

19

CHAPTER 2: OBJECTIVES

21

2. OBJECTIVES

OA is a chronic disease characterized by inflammation of the cartilage and adjacent structures as

a result of a gradual degeneration of articular structures. The release of proinflammatory factors

activates surrounding nerve endings causing pain. The prolonged activation of primary afferent

increases excitability at the spinal cord level promoting sensitization of spinal neurons and

supraspinal areas involved in pain modulation. These neuroplastic events lead to the

development of hyperalgesia, allodynia and spontaneous pain. In addition to the exacerbation of

pain, the occurrence of emotional disturbances is also common in chronic pain patients.

Although the mechanisms underlying this comorbidity are poorly understood, the galaninergic

pathways are one of the potential players in the correlation between these pathologies.

Taking the above mentioned into account and the fact that our group recently developed an

experimental model of OA in which animals display concomitant mood disorders, the objective of

this work was to evaluate the effect of the activation of DMH neurones expressing GAL receptors

upon the activity of caudal brain areas involved in the processing of nociception, through the use

of behavioural and molecular approaches. More specifically, our objectives were to:

1. Evaluate the effect of the activation of the DMH by GAL upon emotional, motor and

nociceptive behaviour in controls and animals with experimental OA;

2. Verify if the RVM mediates descending facilitation after GAL in the DMH;

3. Investigate which supraspinal areas are activated by the intracerebral administration of

GAL in the DMH through the quantification of c-Fos expression in the brain;

23

CHAPTER 3: MATERIALS AND METHODS

25

3. MATERIALS AND METHODS

3.1 Animals and ethical considerations

In this work we used Wistar Han adult male rats weighing between 235 – 285g (Charles River,

Barcelona, Spain) at the beginning of experiment. The animals were housed two per cage under

standard conditions at a constant ambient temperature of 20-24°C, relative humidity of 55+/-

10% with a 12h-12h light-dark cycle (light between 08.00h and 20.00h) and food and water

available ad libitum throughout the experiment. All procedures performed in this work were

approved by the European Community Council Directive 86/609/EEC and 2010/63/EU

concerning the use of animals for scientific purposes and by the ICVS Ethical Commission. The

experiments were designed to minimize animal suffering as well as the number of animals used.

Prior to performing any procedures, all animals were submitted to daily handling sessions

(10 min) and those animals used in behavioural assessments were habituated to the

experimental conditions by spending 1-2 hours every day of the week preceding the test in the

testing room.

3.2 Anaesthesia and euthanasia

For the induction of OA and intracerebral implantation of cannulae, the animals were

anaesthetized with a mixture of ketamine (1.5 mg/kg; Imalgene®, Merial, Lisbon, Portugal) and

medetomidine (1.0 mg/kg; Dorbene®, ESTEVE, Carnaxide, Portugal) administered

intraperitoneally (i.p.). After the surgery, the animals were recovered through the administration

of atipamezole (0.1 mL/kg i.p.; Antisedan®, Pfizer, Seixal, Portugal) and were monitored until

fully awake (feeding and grooming).

In order to perform the protocol to induce c-Fos expression, the animals were anaesthetized

using pentobarbitone (0.5 mL/kg; Eutasil®, CEVA, Algés, Portugal) delivered i.p. The level of

anaesthesia was monitored by pinching of the tail and dilatation of the pupils and was

maintained by infusing pentobarbitone diluted in saline solution (SAL; 0.25 mL/kg/h i.p.;

Unither, Amiens, France; pH=7.2).

26

At the end of the behavioural period and at the end of each experimental session of the c-Fos

stimulation protocol, the animals were injected with a lethal dose of pentobarbitone i.p.

(Eutasil®, CEVA) and transcardially perfused with 200 mL of 4% paraformaldehyde (PFA;

Panreac, Barcelona, Spain) in 0.1M phosphate buffer saline (PBS; pH=7.4) at room temperature

for easier diffusion. Afterwards, the brains were excised, post-fixed in the same fixative (PFA 4%)

for a week and then placed for 48 hours in a solution of 8% sucrose (Panreac). The contralateral

side of the brain was marked with a short cut and the brains were then sectioned in coronal

sections (50 µm of thickness) in a vibratome (Leica VT100, Freiburg, Germany). The sections

were mounted in microscope slides, counterstained, dehydrated, covered in mounting media

(Entellan New, Merck, Darmstadt, Germany) and cover slipped. This process was performed in

order to confirm the injection sites by comparing the coronal section with plates from the rat

brain atlas (Paxinos and Watson, 2007). In case of evaluation of c-Fos expression, the coronal

sections were not counterstained, they were collected in 12 wells culture plates previously filled

with PBS for c-Fos immunohistochemistry.

3.3 Induction of experimental osteoarthritis

The induction of osteoarthritis (OA) in rats was performed according to the protocol described

previously by Pinto-Ribeiro and colleagues. (2011). Briefly, a solution of 3% kaolin (Sigma-Aldrich,

St Louis, MO, USA) and 3% carrageenan (Sigma-Aldrich) dissolved in 0.9% sodium chloride (NaCl;

B. Braun, Barcarena, Portugal) was injected (0.1 mL) in the synovial capsule of the right knee of

animals in the osteoarthritic group (ARTH), while the control group (SHAM) animals were injected

with 0.1 mL SAL in the synovial capsule of the right knee. The injection of carrageenan induces

an inflammatory reaction that results in mechanical hyperalgesia, while kaolin is responsible for

the mechanical damage to the knee joint structures (Okamoto et al., 2013).

3.4 Drugs

Galanin (GAL; 1.0 nmol in 0.5 µL saline; Tocris, Ellisville, MO, USA) was prepared to perform

intracerebral injections in DMH. 2 % Lidocaine (LIDO; B. Braun) was used in the intracerebral

injections in the RVM. These drugs were administered in the DMH and RMV using a 33-gauge

injection cannula (Plastics One) connected to a syringe (5.0 µL Hamilton, Nevada, USA) with a

27

microinjection volume of 0.5 µL. The intracerebral injections lasted for 20s in order to prevent

activation of the DMH and RVM by pressure. After the injections, the guide cannula was left in

place for another 30s to minimize the return of the drug solution back to the injection cannula.

The expected spread of injecting 0.5 µL of the drugs in brain was around +/- 1 mm in diameter

(Myers, 1966). The drug doses were chosen in accordance to previous studies (Pinto-Ribeiro et

al., 2008) and control injections were performed with SAL as control values to eliminate any bias

that may result from injecting the solutions themselves.

All drug injections were performed taking into account that GAL and LIDO reached their peak

effect between 10 and 20 min after the injection (Gillis et al., 1973; Brock et al., 2001; Wang et

al., 2000; Sun and Yu, 2005; Xiong et al., 2005; Gu et al., 2007).

3.5 Evaluation of nociceptive behaviour

3.5.1 Vocalization

For each animal, the development of OA was confirmed by performing five consecutive

flexion-extension movements of the injected knee joint, 3 days after the induction. All ARTH

animals vocalized every time during each flexion-extension movement while SHAM animals did

not vocalized during the same procedure.

3.5.2 Pressure application measurement (PAM)

The pressure application measurement (PAM) is a novel behavioural test which allows measuring

mechanical hyperalgesia in rodent’s joints by the application of a force range of 0-1500g (Barton

et al., 2007). Briefly, the animals (n=19) were securely held and the force transducer was placed

on one side of the animal’s knee joint using the thumb and the forefinger on the other.

Afterwards, a gradually increasing force was applied across the joint until the animal showed

behavioural signs of discomfort (vocalization) or withdrew the limb. The value of the peak gram

force (gf) applied was recorded as the limb-withdrawal threshold (LWT). A total of two

measurements were made in the ipsilateral knee joint of both SHAM and ARTH animals and the

mean LWTs were calculated.

28

3.5.3 Paw-withdrawal (PW) test

The paw-withdrawal (PW) test is a tool that allows assessing thermal hyperalgesia by measuring

hind paw withdrawal latency (PWL) (Ossipov et al., 1999; Saegusa et al., 2000) as described by

Hargreaves and colleagues (1988). Firstly the animals (n=19) were habituated to the

experimental conditions, where they were placed on the test apparatus (Plantar Test Device

Model 37370, Ugo Basile, Comerio, Italy) for 30 min every day of the week preceding the test.

After, for assessing nociception in unanaesthetized animals, the PWL following radiant heat

stimulation onto the plantar aspect was determined before and 10, 20 and 30 min after the

intracerebral administration of the drugs, SAL and/or GAL and/or LIDO administration to the

DMH and/or RVM, according to the protocol for each experimental group (Fig. 7). In each

animal, the measurements were repeated twice and the mean of these values was used for

further calculations. A cut-off time of 14 s was used to prevent any tissue damage.

3.6 Chronic implantation of intracerebral cannulae

For drug administration, two stainless steel guide cannulae (26 gauge; Plastics One, Roanoke,

VA, USA) were implanted in the brain, one in the DMH and another in the RVM as described by

Pinto-Ribeiro and colleagues (2013). Briefly, the animals were anaesthetized as described in

section 3.2 and in order to avoid blindness, due to dehydratation of eyes, they were protected by

applying vaseline. The animals were, then, placed in a stereotaxic frame (KOPF Instruments,

Tujunga, California, USA), a longitudinal incision was made with a scalpel in the skin above the

skull and a sterilized stainless steel guide cannula was vertically positioned 1 mm above the

desired injection site in the DMH (rostrocaudal: -3,24 mm, lateromedial: -0,4 mm;

dorsoventral: -7,9 mm) and RVM (rostrocaudal: -10,92 mm; lateromedial: 0,0 mm; dorsoventral:

-9,4 mm) according to the coordinates of the atlas by Paxinos and Watson (2007). The guide

cannulae were firmly fixed into the skull with two anchoring screws and using dental acrylic

cement. Subsequently, the skin was sutured and a dummy cannula (Plastics One) was inserted

into each guide cannulae to prevent contamination and to maintain patency. At the end, the

animals were placed one per cage. Before any tests were performed, they were allowed to

recover for at least one week.

29

3.7 Evaluation of mood-like behaviour

3.7.1 Open field (OF) test

The open field (OF) test is used to assess anxiety-like behaviour and locomotor performance as

described by Leite-Almeida and colleagues (2009). In summary, 15 min after intracerebral

administration of SAL or GAL to the DMH, according to the protocol for each experimental group

(Fig. 7), each animal (n=18) was placed in the centre of a squared arena (43.2 x 43.2 cm; Med

Associates Inc., St. Albans, Vermont, USA) with light intensity of 240 lx in the central arena. The

animal’s behaviour was recorded for 5 min using a screen monitor connected to a video camera

and its exploratory activity was automatically registered by sensors. At the end of each session,

the arena (inner areas, floor and walls) was cleaned with a solution of 10% alcohol. The

anxiety-like behaviour was assessed by counting the number of faeces left in the arena at the end

of the OF test and measuring the time spent in the center of the arena (Mesquita et al., 2006;

Leite-Almeida et al., 2009; Amorim et al., 2014). The locomotor performance was assessed by

measuring the total distance travelled by the animals (Mesquita et al., 2006; Leite-Almeida et al.,

2009; Amorim et al., 2014).

3.7.2 Forced swimming test (FST)

The forced swimming test (FST) is a behavioural test that evaluates learned helplessness and

that is widely used for assessing antidepressant effect of drugs (Bessa et al., 2009; Slattery and

Cryan, 2012). This test was performed according to a report by Amorim and colleagues (2014).

On day 1, the rats (n=19) were submitted to a 5 min pre-test session, where they were

individually placed in transparent cylinders containing clean water, with a water depth such that

animals could not touch the base with their hind limbs or tails, (25°C; depth 30 cm). On day 2,

24h later, in each animal per experimental group (SHAM and ARTH), SAL or GAL was

administered to the DMH according to the protocol as shown in figure 7. Fifteen min after

intracerebral administration the rats were again placed in the cylinders under the same

conditions and the test session was recorded using a video camera. Posteriorly, the quantification

of the latency to immobility, the time spent swimming, climbing (escape behavior) and immobile

(floating) was performed for each rat using the Etholog® 2.2. Software (Ottoni, 2000). The

latency to immobility corresponds to the time elapsed between placing the animal in the water

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and observing a first immobile behaviour; swimming corresponds to active swimming motions

with large forepaw movements displacing around in the cylinder, more than necessary to merely

keep the head above water; climbing was defined as vigorous movements with front and hind

paws directed against the wall of cylinder in an attempt to climb out, and include diving;

immobile behaviour was observed when all the small movements are made only in the direction

of the animal to stay at the surface (Rénéric et al., 2002; Lino-de-Oliveira et al., 2005). Learned

helplessness behaviour was defined as a reduction in the latency to immobility and an increase

in time of immobility (Bessa et al., 2009; Amorim et al., 2014).

3.8 Evaluation of c-Fos expression

3.8.1 Stimulation of c-Fos

For stimulation of c-Fos, the animals (n=16) were placed in a stereotaxic frame, the skull was

exposed with a scalpel and a small hole was drilled using a manual drill above the DMH

(rostrocaudal: -3,24 mm, lateromedial: -0,4 mm; dorsoventral: -7,9 mm), according to the

coordinates of the atlas by Paxinos and Watson (2007), for the insertion of the guide cannula

(Plastics One) on the right hemisphere. The bregma was used as reference.

During 2 hours, the animals were submitted to a four stimulation protocols as shown in figure 8,

where it was injected SAL or GAL in the DMH at the beginning of the protocol and after 15 min.

These administrations were also accompanied by extension-flexion of the right knee 5 times every

2 minutes in two stimulation protocols.

3.8.2 c-Fos immunohistochemistry

The procedure followed to perform c-Fos immunohistochemistry was described previously by

Morgado and Tavares (2007) involving the avidin-biotin-peroxidase method. First, the sections

were washed twice in 0.1M PBS (pH=7.2) for 5 min each, incubated in hydrogen peroxidase

(H2O2, Panreac, Barcelona, Spain) (330 µL in 10 mL PBS 0.1M) for 30 min to inhibit endogenous

peroxidase activity, followed by 2 washes of 5 min in PBS 0.1M and PBS/T (3mL Triton X 100

(Sigma-Aldrich) in 1000 mL PBS 0.1M; pH=7.2). Sections were then incubated in a blocking

solution, 2.5% fetal bovine serum (FBS; Biochrom, Cambridge, United Kingdom) in PBS/T, for 2

h in order to avoid non-specific bindings followed by incubation in rabbit anti-c-Fos primary

31

antibody (Calbiochem, Merck, Algés, Portugal) (1:2000 in PBS/T and 2% FBS) overnight at room

temperature on an orbital shaker. The following day, sections were again washed in PBS/T

followed by incubation for 1 h in biotinylated swine anti-rabbit (Dako, Lisbon, Portugal) (1:200 in

PBS/T) at room temperature. After being washed 3 times for 10 min in PBS/T, the sections were

incubated in the avidin-biotin complex (ABC; Vectastain, Vector Laboratories, Peterborough, USA)

diluted in 1:200 in PBS/T for 30 min at room temperature. The sections were then washed 3

times for 5 minutes in PBS/T, PBS and Tris 0.05M (Trizma® base, Sigma-Aldrich; pH=7.6), and

stained with diaminobenzidine (DAB; Sigma-Aldrich; 20 mg DAB in solution of 40 mL Tris with 8

µL H2O2). Finally, the sections were washed twice in Tris and PBS to stop the staining reaction.

3.8.3 c-Fos quantification

c-Fos quantification was performed by counting the total number of c-Fos-immunoreactive

neurones occurring bilaterally in the brain with the aid of a Stereo Investigator 10 Software®

(MicroBrightField, Williston, VT, USA) coupled to a video camera attached to a Olympus Golgi

microscope. The brain sections were outline according to Paxinos and Watson (2007) stereotaxic

atlas and the quantification of c-Fos-positive neurones was performed blind. The cells in the

ipsilateral side of the coronal sections were marked in red and contralateral side were marked in

blue (Fig. 6).

Figure 6 – Coronal section of a SHAM animal showing c-Fos expression in the brain after the injection of GAL. The ipsilateral side was stained red and the contralateral side stained blue.

32

3.9 Experimental design

The present work was divided in two main experiments, experiment 1 and experiment 2 as

shown in figures 7 and 8, respectively. The animals we handled for a week previously to the

induction of OA. After this habituation period (as described in section 3.1), OA was induced (as

described in section 3.3) and its development was confirmed three days after.

3.9.1 Impact of GAL in the DMH upon emotional, motor and nociceptive behaviour (Experiment 1)

In experiment 1 (Fig. 7), three weeks after the induction of OA, the animals were reanaesthetized

(Section 3.2) to implant a cannula in the DMH and another in the RVM (Section 3.6) and were

allowed to recover for one week. To evaluate if the acute administration of GAL in the DMH

altered the performance emotional-like and nociceptive behaviours of SHAM and ARTH animals,

the rats were tested in (i) the OF test (Section 3.7.1) to assess anxiety/like and motor

behaviours, (ii) the FST (Section 3.7.2) to evaluate depressive-like behaviour and (iii) the PW test

(Section 3.5.3) to evaluate thermal hyperalgesia. During OF and FST the animals were

administered either with SAL (SHAM SAL and ARTH SAL) or GAL (SHAM GAL and ARTH GAL) in

the DMH 15 min before the beginning of the test. To evaluate nociceptive behaviour, basal PW

values were determined as the following protocols: (i) SAL+SAL administration in the RVM+DMH,

respectively; (ii) SAL+GAL administration in the RVM+DMH, respectively; (iii) LIDO+SAL

administration in the RVM+DMH, respectively; (iv) LIDO+GAL administration in the RVM+DMH,

respectively. The PW assessment was repeated at 10, 20 and 30 min after drugs administration.

At the end of the behavioural period the animals were sacrificed and the brains were removed for

further confirmation of injection sites.

33

Figure 7 – Schematic representation of the experimental design for experiment 1. One week preceding the induction of osteoarthritis (OA) using the kaolin/carrageenan (K/C) model, the animals were habituated to the experimenter and the testing apparatus. The development of OA was confirmed three days after induction. Three weeks after OA induction, the animals were reanaesthetized to implant an intracerebral cannula in the DMH and another in the RVM. After one week of recovery, the pressure application measurement (PAM) was performed (day 1). The Open field (OF) test was performed on day 2 and the forced swimming test (FST) on days 4 and 5, 15 min after the intracerebral microinjection of the drugs. To perform these tests, the animals were divided in four experimental groups: SHAM animals that injected with SAL (SHAM-SAL) or GAL (SHAM-GAL) and ARTH animals injected with SAL (ARTH-SAL) or GAL (ARTH-GAL). Finally, the rats performed the paw-withdrawal (PW) test in which they were injected with: (i) SAL+SAL in the RVM and DMH, respectively; (ii) SAL+GAL in the RVM and DMH, respectively; (iii) LIDO+SAL in the RVM and DMH, respectively; (iv) LIDO+GAL in the RVM and DMH, respectively. In this test, the PW Latency was assessed pre-injection (PI) of drugs and 10, 20 and 30 min following intracerebral drug injection. At the end, the animals were sacrificed and brains were removed to confirm the injection sites.

3.9.2 Evaluation of the expression of c-Fos after GAL in the DMH (Experiment 2)

In experiment 2 (Fig. 8), four weeks after the induction of OA the animals were reanaesthetized

(Section 3.2) to allow the evaluation of c-Fos expression induced by GAL administration in the

DMH (Section 3.8.2). To evaluate which the effect of the acute administration of the DMH, SHAM

and ARTH animals were placed in a stereotaxic frame (Section 3.8.1) and were submitted to one

of the following stimulation protocols for 2 h: (i) SAL administration in the DMH without

34

peripheral stimulation protocol; (ii) GAL administration in DMH without peripheral stimulation

protocol; (iii) SAL administration in DMH and extension-flexion of the right knee (5 times every 2

min); (iv) GAL administration in DMH and extension-flexion of the right knee (5 times every 2

min). SAL or GAL was administered twice during each protocol, with an interval of 15 min (time

point 0 and 15 min). Two weeks after the c-Fos stimulation protocol, the immunohistochemistry

against the c-Fos protein in coronal sections was performed (Section 3.8.2) followed by c-Fos

quantification (Section 3.8.3).

Figure 8 – Schematic representation of the experimental design for experiment 2. The animals were allowed to habituate to the experimenter and the testing apparatus for one week preceding the induction of OA through the kaolin/carrageenan (K/C) model. The development of OA was confirmed three days after induction. Four weeks after OA induction, the animals were reanaesthetized to perform the protocol to stimulate the expression of c-Fos. For this procedure the SHAM and ARTH animals were submitted to one of the four following protocols for 2h: (i) SAL administration in the DMH without peripheral stimulation; (ii) GAL administration in DMH without peripheral stimulation; (iii) SAL administration in DMH and extension-flexion of the right knee (5 times every 2 min); (iv) GAL administration in DMH and extension-flexion of the right knee (5 times every 2 min). SAL and GAL were administered twice during each protocol, with an interval of 15 min (time point 0 and 15 min).

3.10 Statistical analysis

The statistical analysis was performed using the GraphPad Prism® 6 Software (GraphPad

Software Inc, LaJolla, CA, USA). Differences in the behaviour between experimental groups in

experiment 1 was assessed using a two-way analysis of variance (ANOVA2W) followed by a t-test

with a Bonferroni correction, except in the PAM test were differences between SHAM and ARTH

animals were performed using a Student’s t-test for unpaired data. In experiment 2, differences

35

in c-Fos expression between experimental protocols and groups were assessed using a ANOVA2W

followed by a t-test with a Bonferroni correction for multiple comparisons. Statistical significant

was considered for P<0.05. Data in the results section are expressed as mean±standard error

(SEM).

37

CHAPTER 4: RESULTS

39

4. RESULTS

4.1 Histological confirmation of the injection sites

An example of injection sites in the DMH and in the RVM are shown in figure 9. In these figures it

is possible to confirm that the cannulae were correctly placed in DMH and RVM, and that the

injections were performed in the correct coordinates.

Figure 9 – Schematic representation of the DMH and the RVM superimposed on fotomicrographs of coronal sections of rat brain sections showing the injection site. (A) Example of a cannula track targeting the DMH (rostrocaudal:-3.24 mm; lateromedial:-0.4 mm; dorsoventral:-7.9 mm) and (B) the RVM (rostrocaudal:-10.92 mm; lateromedial: 0.0 mm; dorsoventral:-9.4 mm) according to the coordinates of the rat brain atlas by Paxinos and Watson (2007). (C-G) Schematic representation of other injection sites in the DMH (C: -3.00 mm; D: -3.12 mm; E: -3.24 mm; F: -3.36 mm; G: -3.48 mm). (H-L) Schematic representation of other injection sites in the RVM (H: -10.68 mm; I: -10.80 mm; J: -10.92 mm; K: -11.04 mm; L: -11.16 mm). (DMD - dorsomedial hypothalamic nucleus, compact; DMD - dorsomedial hypothalamic nucleus, dorsal; DMV - dorsomedial hypothalamic nucleus, ventral; GiA - gigantocellular reticular alpha; RMg - raphe magnus nucleus). Stain: cresyl violet.

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4.2 Nociceptive behaviour

4.2.1 Mechanical hyperalgesia

In order to confirm the development of mechanical hyperalgesia after the induction of

experimental OA, the limb withdrawal threshold (LWT) was evaluated. As shown in figure 10,

ARTH animals displayed a significance decrease in LWT when compared to SHAM (t(17)=3.314,

P=0.004) which confirms the development of mechanical hyperalgesia and thus correlates with

the establishment of experimental OA in these animals.

Figure 10 – Evaluation of the ipsilateral limb withdrawal latency (LWT) in SHAM and ARTH animals using the pressure application measurement (PAM) test. The LWT of the ARTH group was significantly decreased when compared to the SHAM group. Data presented as mean±SEM (**P<0.01) (SHAM, n=8; ARTH, n=11; SHAM - animals injected with SAL in the right knee joint; ARTH - animals injected with a mixture of kaolin and carrageenan in the right knee joint).

4.2.2 Heat hyperalgesia

To determine if experimental OA induced heat hyperalgesia, the paw withdrawal latency (PWL)

was evaluated. As shown in figure 11, no difference was found in the PWL between SHAM and

ARTH animals (F(1,20)=1.089, P=0.400) and between the ipsilateral and contralateral hindpaws

(F(1,20)=0.102, P=0.753).

41

Figure 11 – Evaluation of the ipsilateral and contralateral paw withdrawal latency (PWL) in SHAM and ARTH animals using the paw withdrawal test. No differences were found in PWL between ARTH and SHAM animals and between the ipsilateral and the contralateral hindpaws. Data presented as mean±SEM (n=6 per group). (SHAM - animals injected with saline in the right knee joint; ARTH - animals injected with a mixture of kaolin and carrageenan in the right knee joint).

As no differences were observed between the ipsilateral and the contralateral hindpaws in SHAM

and ARTH animals, from here onwards we will use the average of the two paws (right and left) in

further evaluations (Fig. 11).

4.2.3 Role of the RVM in behavioural hyperalgesia after GAL in the DMH

To verify if the RVM is mediating the pronociceptive effect of GAL in the DMH, a series of vehicle

and drug injections were performed in the RVM and the DMH, respectively, as shown in figure

12. Variation () of PWL was calculated by subtracting the value of basal PWL (pre-injection) from

the values of PWL at 20 min. PWL was not significantly different between experimental groups

(group effect: F(1,40)=0.072, P=0.790) but it varied with the drug combination (drug effect:

F(3,40)=20.57, P<0.001). Post hoc tests indicated that the injection of SAL+GAL in the RVM and

DMH, respectively, significantly increased PWL in SHAM and ARTH animals when compared

with the injection of SAL+SAL in these nuclei. In parallel, after the injection of LIDO+SAL in the

RVM and DMH, respectively, the PWL also increased in SHAM and ARTH animals when

compared with the injection of SAL+SAL (Fig. 12).

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Finally, the injection of LIDO+GAL in the RVM and DMH, respectively, significantly decreased the

PWL when compared with the injection of SAL+GAL in the RVM and DMH, but only in SHAM

animals (Fig. 12).

Figure 12 – Evaluation of paw withdrawal latencies (PWL) in SHAM and ARTH animals after a combination of drugs were simultaneously injected in the RVM and DMH. Data presented as mean±SEM (*P<0.05, ***P<0.001 (* corresponds to comparisons with SAL-injected animals); #P<0.05 (# corresponds to comparisons with GAL injected animals)). (n=6 per group; SHAM - animals injected with saline in the right knee joint; ARTH - animals injected with a mixture of kaolin and carrageenan in the right knee joint). (SAL - saline; GAL - galanin; LIDO - lidocaine). PWL was calculated by subtracting PWL pre injection values from PWL values at 20 min after

drug administration.

4.3 Locomotor activity

In order to evaluate if the induction of experimental OA and the administration of GAL affected

the locomotor activity of animals, the total distance travelled by the animals in the open field (OF)

arena was evaluated. As shown in figure 13, there were no significant differences in the total

distance travelled between ARTH and SHAM animals (group effect: F(1,15)=0.821, P=0.379).

Similary the injection of GAL in the DMH also didn’t alter the total distance travelled by the

animals in the OF arena (drug effect: F(1,15)=1.963, P=0.182).

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Figure 13 – Evaluation of the effect of the induction of experimental OA and of the injection of GAL in the DMH upon the motor activity of SHAM and ARTH animals in the OF arena. Data presented as mean±SEM (SHAM: SAL, n=4 and GAL, n=4; ARTH; SAL, n=5 and GAL n=6; SHAM - animals injected with saline in the right knee joint; ARTH - animals injected with a mixture of kaolin and carrageenan in the right knee joint; SAL - saline; GAL - galanin).

4.4 Mood-like behaviour

4.4.1 Anxiety-like behaviour

To determine if the induction of experimental OA promoted the anxiety-like behaviour in animals,

the ratio between the distance travelled in center versus the distance travelled in the periphery

and the number of fecal boli left in the arena at the end of behavioural session were evaluated in

the open field (OF) test. Identically, the potential effect of the injection of GAL in the DMH upon

anxiety-like behaviour was also assessed.

As shown in figure 14A the ratio between the distance travelled in the center/periphery was not

different between experimental groups (group effect: F(1,15)=1.650, P=0.219), but varied after

GAL microinjection in the DMH (drug effect: F(1,15)=1.996, P=0.007).

In parallel, no differences were found in the number of fecal boli left in the arena either between

experimental groups (group effect: F(1,15)=0.043, P=0.839) or after the administration of GAL in

the DMH (drug effect: F(1,15)=0.015, P=0.903) (Fig. 14B).

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Figure 14 – Evaluation of anxiety-like behaviour after the injection of SAL and GAL in the DMH in SHAM and ARTH animals. (A) Ratio of the total distance travelled by SHAM and ARTH animals in the OF test after the microinjection of SAL or GAL in the DMH. (B) Number of fecal boli left in the open field arena at the end of the experimental session by SHAM and ARTH animals microinjected with SAL and GAL in the DMH. Data presented as mean±SEM. (SHAM: SAL, n=4 and GAL, n=4; ARTH: SAL, n=5 and GAL, n=6; SHAM - animals injected with saline in the right knee joint; ARTH - animals injected with a mixture of kaolin and carrageenan in the right knee joint; SAL - saline; GAL - galanin).

4.4.2 Depressive-like behaviour

To determine if experimental OA induced a depressive-like behaviour, the latency to immobility,

the duration of the immobility time, the time spent swimming and climbing were evaluated in the

forced swimming test (FST) (Fig. 15). As before, the effect of injection of GAL in the DMH upon

the above mentioned parameters was also evaluated.

The latency to immobility was significantly different after the administration of GAL in the DMH

(interaction effect: F(1,14)=49.13, P<0.001) but this effect depended on the experimental group

(group effect: F(1,14)=45.60, P<0.001).

Post hoc tests showed that SAL-injected ARTH animals stop for the first time much earlier than

SAL-injected SHAM animals and that GAL in the DMH decrease the latency to immobility, but

only in SHAM animals (Fig. 15A).

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Figure 15 - Evaluation of depressive-like behaviour in SHAM and ARTH animals after the microinjection of SAL or GAL in the DMH. (A) Amount of time spent by the animals in active behaviours until they became immobile for the first time; (B) Time the animals spent immobile; (C) Time spent by the animals swimming ; (D) Time spent by the animals climbing. Data presented as mean±SEM *P<0.05, **P<0.01, ***P<0.001. (SHAM: SAL, n=4 and GAL, n=4; ARTH: SAL, n=4 and GAL, n=6; SHAM - animals injected with saline in the right knee joint; ARTH - animals injected with a mixture of kaolin and carrageenan in the right knee joint; SAL - saline; GAL - galanin).

The duration of the immobility time was significantly altered by the administration of GAL

(interaction effect: F(1,14)=13.95, P=0.002), an effect that varied with the experimental group

(group effect: F(1,14)=15.13, P=0.002). Post hoc tests showed that GAL in the DMH increased

the immobility time in SHAM animals and that the induction of arthritis had a similar effect on

rats (Fig. 15B).

The administration of GAL in the DMH altered the time spent swimming (interaction effect:

F(1,14)=25.44, P=0.002) but this effect was dependent on the experimental group (group effect:

F(1,14)=85.96, P=0.001). Post hoc tests showed that while ARTH animals spent significantly less

time swimming in comparison with SHAM animals, GAL in the DMH only decreased the time

spent swimming in the latter (Fig. 15C).

46

The time spent climbing was significantly different between experimental groups (group effect:

F(1,14) =26.55, P=0.001) and was altered by the administration of GAL in the DMH (drug effect:

F(1,14)=56.10, P<0.001). Post hoc tests showed that ARTH animals spent more time climbing

than SHAM animals and that GAL in the DMH decreased this parameter in both experimental

groups (Fig. 15D).

4.5 c-Fos quantification

c-Fos expression was quantified in order to determine which key caudal nuclei might be

mediating descending facilitation of nociception after the induction of experimental OA and the

administration of GAL in the DMH.

4.5.1 Ventrolateral periaqueductal gray matter (VLPAG)

In the ipsilateral side, the number of c-Fos expressing cells was significantly altered between

protocols (interaction effect: F(3,24)=18.87, P<0.001) but this effect was dependent on

experimental group (group effect: F(1,24)=7.064, P=0.014). Post hoc tests showed GAL in the

DMH and the simultaneous administration of GAL in the DMH while applying a peripheral noxious

stimulus increased c-Fos expression in VLPAG cells of ARTH animals when compared to SHAM

animals (Fig. 16A).

By contrast the application of a peripheral noxious stimulus decreased c-Fos expression in the

ARTH group when compared to the SHAM group. In addition in SHAM animals, peripheral

noxious stimulation increased c-Fos when compared to the administration of SAL while c-Fos

expression was decreased after GAL in the DMH and the simultaneous administration of GAL in

the DMH while applying a peripheral noxious stimulus when compared with peripheral noxious

stimulation (Fig. 16A).

47

Figure 16 – Evaluation of c-Fos expression in the VLPAG of SHAM and ARTH animals after the application of several stimulation protocols. (A) Ipsilateral side of the VLPAG and (B) contralateral side of the VLPAG. Data presented as mean±SEM. (*P<0.05, ***P<0.001 (* on top the bar corresponds to comparisons with the SAL-injected animals, while those on top of the horizontal line corresponds to comparisons between SHAM and ARTH in the same

protocol); ###P<0.001 # corresponds to comparisons with GAL-injected animals) P<0.01, P<0.001 (

corresponds to comparisons with the SAL+STM protocol)). (SHAM: SAL, n=4; GAL, n=4; SAL+STM, n=4 and GAL+STM,n=4; ARTH: SAL, n=4; GAL, n=4; SAL+STM, n=4 and GAL+STM, n=4; SHAM - animals injected with saline in the right knee joint; ARTH - animals injected with a mixture of kaolin and carrageenan in the right knee joint; SAL- saline; GAL - galanin; STM - peripheral noxious stimulation).

In the contralateral side of the VLPAG, the number of c-Fos expressing cells was significantly

altered between protocols (interaction effect: F(3,24)=7.464, P=0.001) but this effect was

dependent on experimental group (group effect: F(1,24)=17.85, P<0.001). Post hoc tests showed

the administration of GAL in the the DMH and the simultaneous administration of GAL in the

DMH while applying a peripheral noxious stimulus increased c-Fos expression in VLPAG cells of

ARTH animals when compared to SHAM animals (Fig. 16B). In addition, in SHAM animals,

peripheral noxious stimulation increased c-Fos expression when compared with the

administration of SAL in the DMH while the simultaneous administration of GAL in the DMH while

applying a peripheral noxious stimulus decreased c-Fos expression in the VLPAG when compared

with the application of a peripheral noxious stimulus (Fig. 16B).

4.5.2 Dorsal raphe nucleus (DRN)

The number of c-Fos expressing cells in ipsilateral side of DRN was not significantly different

between experimental groups (group effect: F(1,25)=2.997, P=0.096) however it was dependent

on the stimulation protocol (interaction effect: F(3,25)=10.020, P<0.001).

48

Post hoc tests showed that peripheral noxious stimulation and the simultaneous administration of

GAL in the DMH while applying a peripheral noxious stimulus increased c-Fos expression in DRN

cells of ARTH animals when compared to SHAM animals (Fig. 17A). In parallel, the application of

a peripheral noxious stimulation increased c-Fos expression in the DRN when compared with the

administration of SAL and GAL in the DMH (Fig 17A). In ARTH animals, the simultaneous

administration of GAL in the DMH while applying a peripheral noxious stimulus increased c-Fos

expression in the DRN when compared to the microinjection of GAL alone, to the application of a

peripheral noxious stimulation alone and when compared to SHAM animals in the same

conditions (Fig. 17A).

Figure 17 – Evaluation of c-Fos expression in the DRN of SHAM and ARTH animals after the application of several stimulation protocols. (A) Ipsilateral side of the DRN and (B) Contralateral side of the DRN. Data presented as mean±SEM. (*P<0.05, ***P<0.001 (* on top the bar corresponds to comparisons with the SAL-injected animals, while those on top of the horizontal line corresponds to comparisons between SHAM and ARTH in the same protocol); #P<0.05, ###P<0.001 (# corresponds to comparisons with GAL-injected animals); P<0.05 (

corresponds to comparisons with the SAL+STM protocol)). (SHAM: SAL, n=4; GAL, n=4; SAL+STM, n=4; GAL+STM, n=4; ARTH: SAL, n=4, GAL, n=4; SAL+STM, n=5; GAL+STM, n=4; SHAM - animals injected with saline in the right knee joint; ARTH - animals injected with a mixture of kaolin and carrageenan in the right knee joint; SAL - saline; GAL - galanin; STM - peripheral noxious stimulation).

In relation to the contralateral side of the DRN, no significant differences were found between

experimental groups (group effect: F(1,25)=0.000, P=0.002) although c-Fos expression was

significantly different depending on the stimulation protocol (interaction effect: F(3,25)=16.92,

P<0.001).

49

Post hoc tests showed the number of DRN cells expressing c-Fos is increased in ARTH animals

when compared to SHAM animals in basal conditions (after SAL in the DMH) and after the

application of a peripheral noxious stimulus (Fig. 17B). In SHAM animals, peripheral noxious

stimulation increased DRN c-Fos expression when compared to SAL and GAL in the DMH, while

the simultaneous administration of GAL in the DMH while applying a peripheral noxious stimulus

decreased c-Fos expression in the DRN when compared to peripheral noxious stimulation alone

(Fig. 17B).

4.5.3 Locus coeruleus (LC)

As shown in figure 18A, c-Fos expression on the ipsilateral LC was significantly different between

experimental group (group effect: F(1,24)=9.490, P=0.005) and its effect was dependent on the

stimulation protocol applied (interaction effect: F(3,24)=6.979, P=0.002). Post hoc tests showed

the induction of experimental OA significantly increased c-Fos expression in the LC (Fig. 18A). In

SHAM animals peripheral noxious stimulation increased LC c-Fos expression in ARTH animals

when compared with SAL in the DMH while, in ARTH animals, the simultaneous administration of

GAL in the DMH while applying a peripheral noxious stimulus decreased c-Fos expression in the

LC when compared to SAL in the DMH (Fig. 18A).

Figure 18 – Evaluation of c-Fos expression in the LC of SHAM and ARTH animals after the application of several stimulation protocols. (A) Ipsilateral side of the LC and (B) Contralateral side of the LC. Data presented as mean±SEM. (*P<0.05, **P<0.01, ***P<0.001 (* on top the bar corresponds to comparisons with the SAL-injected animals, while those on top of the horizontal line corresponds to comparisons between SHAM and ARTH in the same protocol); #P<0.05 (# corresponds to comparisons with GAL-injected animals)). (SHAM: SAL, n=4; GAL, n=4; SAL+STM, n=4; GAL+STM, n=4; ARTH: SAL, n=4, GAL, n=4; SAL+STM, n=4; GAL+STM, n=4; SHAM - animals injected with saline in the right knee joint; ARTH - animals injected with a mixture of kaolin and carrageenan in the right knee joint; SAL - saline; GAL - galanin; STM - peripheral noxious stimulation).

50

In relation to the contralateral side, c-Fos expression was significantly different between

experimental group (group effect: F(1,24)=5.047, P=0.034) but this effect was dependent on the

stimulation protocol applied (interaction effect: F(3,24)=7,438 P=0.001). Post hoc tests showed

the induction of experimental OA significantly increased c-Fos expression in the LC (Fig. 18B). In

SHAM animals peripheral noxious stimulation increased LC c-Fos expression in SHAM animals

when compared with SAL in the DMH while the simultaneous administration of GAL in the DMH

while applying a peripheral noxious stimulus decreased c-Fos expression in the LC when

compared to peripheral noxious stimulation alone (Fig. 18B). In ARTH animals, the simultaneous

administration of GAL in the DMH while applying a peripheral noxious stimulus decreased c-Fos

expression in the LC when compared to SAL in the DMH (Fig. 18B).

4.5.4 Rostral ventromedial medulla (RVM)

The number of c-Fos expressing cells on the ipsilateral side of the RVM was significantly different

between experimental groups (group effect: F(1,26)=13.77, P=0.001), an effect that depended on

the stimulation protocol applied (interaction effect: F(3,26)=64.66, P<0.001). Post hoc tests

indicated c-Fos expression was increased in ARTH animals when compared to SHAM animals

while GAL in the DMH decreased the number of c-Fos expressing cells in ARTH animals when

compared to SHAM animals (Fig. 19A).

In SHAM animals, GAL in the DMH and the simultaneous administration of GAL in the DMH while

applying a peripheral noxious stimulus decreased c-Fos expression in the LC when compared to

SAL in the DMH (Fig. 19A). In addition, the simultaneous administration of GAL in the DMH while

applying a peripheral noxious stimulus increased c-Fos expression in the LC when compared to

GAL in the DMH and peripheral noxious stimulation (Fig. 19A). In ARTH animals, GAL in the DMH

decreased c-Fos expression when compared to SAL in the DMH (Fig. 19A).

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Figure 19 – Evaluation of c-Fos expression in the RVM of SHAM and ARTH animals after the application of several stimulation protocols. (A) Ipsilateral side of the RVM and (B) Contralateral side of the RVM. Data presented as mean±SEM. (*P<0.05, **P<0.01, ***P<0.001 (* on top the bar corresponds to comparisons with the SAL-injected animals, while those on top of the horizontal line corresponds to comparisons between SHAM and ARTH in the same protocol); ###P<0.001 (# corresponds to comparisons with GAL-injected animals)). (SHAM: SAL, n=4; GAL, n=4; SAL+STM, n=5; GAL+STM, n=4; ARTH: SAL, n=4, GAL, n=4; SAL+STM, n=5; GAL+STM, n=4; SHAM - animals injected with saline in the right knee joint; ARTH - animals injected with a mixture of kaolin and carrageenan in the right knee joint; SAL - saline; GAl - galanin; STM - peripheral noxious stimulation).

In relation to the contralateral side of the RVM, the number of c-Fos expressing cells was

significantly different between experimental groups (group effect: F(1,26)=12,94, P=0.001), an

effect that depended on the stimulation protocol (interaction effect: F(3,26)=46.90, P<0.001).

Post hoc tests showed GAL in the DMH decreased RVM c-Fos expression in ARTH animals when

compared to SHAM animals. In SHAM animals, GAL in the DMH increased RVM c-Fos expression

when compared to SAL in the DMH. In addition, peripheral noxious stimulation and the

simultaneous administration of GAL in the DMH while applying a peripheral noxious stimulus

decreased c-Fos expression in the RVM when compared to GAL in the DMH (Fig. 19B).

4.5.5 Dorsal Reticular Nucleus (DRt)

In the ipsilateral side of the DRt, there were no significant differences in the number of c-Fos

expressing cells between experimental groups (group effect: F(1,25)=0.050, P=0.825) although its

expression depended on the stimulation protocol (interaction effect: F(3,25)=23.97, P<0.001) (Fig.

20A). Post hoc tests showed c-Fos is increased in ARTH animals when compared to SHAM

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animals (Fig. 20A). In addition, GAL in the DMH decreased DRt c-Fos expression in ARTH

animals when compared to SHAM animals (Fig. 20A).

In SHAM animals, GAL in the DMH increased DRt c-Fos expression when compared to SAL in the

DMH while peripheral noxious stimulation and the simultaneous administration of GAL in the

DMH while applying a peripheral noxious stimulus decreased c-Fos expression in the DRt when

compared to GAL in the DMH (Fig. 20A). In ARTH animals, peripheral noxious stimulation

decreased the number of c-Fos expressing cells in the DRt (Fig. 20A).

Figure 20 – Evaluation of c-Fos expression in the DRt of SHAM and ARTH animals after the application of several stimulation protocols. (A) Ipsilateral side of the DRt and (B) Contralateral side of the DRt. Data presented as mean±SEM. (*P<0.05, ***P<0.001 ((* on top the bar corresponds to comparisons with the SAL-injected animals, while those on top of the horizontal line corresponds to comparisons between SHAM and ARTH in the same protocol); ###P<0.001 (# corresponds to comparisons with GAL-injected animals)). (SHAM; SAL, n=4; GAL, n=4; SAL+STM, n=4; GAL+STM, n=5; ARTH: SAL, n=4, GAL, n=4; SAL+STM, n=4; GAL+STM, n=4). (SHAM - animals injected with saline in the right knee joint; ARTH - animals injected with a mixture of kaolin and carrageenan in the right knee joint; SAL - saline; GAL - galanin; STM – peripheral noxious stimulation).

On the contralateral side of the DRt, the number of c-Fos expressing cells was significantly

different between experimental groups (group effect: F(1,25)=7.687, P=0.010), an effect that

depended on the stimulation protocol (interaction effect: F(3,25)=41.35, P<0.001). Post hoc tests

showed c-Fos expression was decreased in ARTH animals when compared with SHAM animals

after GAL in the DMH (Fig. 20B). In SHAM animals, GAL in the DMH increased c-Fos expression

(Fig. 20B). In addition, peripheral noxious stimulation and the simultaneous administration of

GAL in the DMH while applying a peripheral noxious stimulus decreased c-Fos expression in the

DRt when compared to GAL in the DMH.

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CHAPTER 5: DISCUSSION

55

5. DISCUSSION

5.1 Technical considerations

5.1.1 The choice of animal strain

Research involving animals require good practices in order to guarantee the appropriate and

ethical use of animals in experimentation including the reduction of the number of animals used

per experiment and the refinement of procedures in order to minimize animal suffering and to

maximize the validity of the results (Little and Zaki, 2012; Longo et al., 2012).

In this study the experimental work was performed on Wistar Han adult male rats, a sociable

species, of easy management and handling and of low maintenance costs. This strain and the

Sprague Dawley strain are commonly used in Europe and the USA, respectively (Zmarowski et

al., 2012). However, the Wistar Han rats, in comparison to Sprague Dawley rats, display several

advantages such as (i) a smaller body size, which reduces the amount of space required; (ii)

longevity, which improve study integrity and (iii) nature, they are more resilient to insults (Son et

al., 2010; Zmarowski et al., 2012). These advantages lead this strain to be commonly used in

areas such as neuroscience (Amorim et al., 2014; Mateus-Pinheiro et al., 2014), toxicology

(Dunnick et al., 2012), aging (Pepeu, 2004) and oncology (Zmarowski et al., 2012).

In this work we opted for the use of male rats alone since studying female rats whose hormonal

changes can influence the results would require a longer study period and an increase in the

number of animals evaluated.

5.1.2 Anaesthesia

It is important to note the protocols for the stimulation of c-Fos were performed in anaesthetized

animals and that the anaesthesia may interfere with normal brain function and thus may, to

some extent, influence the expression of c-Fos. However, since all animals were anaesthetized

while the same protocol was performed we expect an identical effect of the anesthesia in all

experimental groups.

Also noteworthy is the fact that pentobarbitone anaesthesia has a depressive effect on neural

activity (Krukoff et al., 1992), namely in the function of cortical neurones (Wang et al., 2010) and

56

in the activation of GABA (Wan and Puil, 2002), a major inhibitory neurotransmitter in the brain.

To compensate for a potential confounding effect of using pentobarbitone we included a SHAM

and ARTH group of animals in which the basal expression of c-Fos was determined in animals

that were anaesthetized with pentobarbitone but only received an intracerebral injection of SAL in

the DMH.

In light of the above mentioned, one should also be careful while comparing data obtained using

awake or anaesthetized animals due to potential differences in the processing of nociception. As

shown by Wang and colleagues (2010), pentobarbitone anaesthesia significantly suppressed the

increase in neuronal activity of brain areas modulating nociception after noxious peripheral

stimulation by (i) decreasing the magnitude of the neuronal response, (ii) reducing the number of

responsive neurones and (iii) decreasing the response duration. Interestingly, pentobarbitone

anaesthesia increased the activity of neurones that were previously silent, in the

ventroposterolateral nucleus (Wang et al., 2010).

In what concerns the anaesthetic agent, we used pentobarbitone delivered intraperitoneally to

induce the initial anaesthesia and then administrated a reinforcement every hour until the end of

the experimental session. The hourly administration of reinforcements would have been

prevented if we had opted for intravenous continuous administration (Cleary et al., 2008),

however this techniques requires a surgery in the thoracic cavity which might bias our c-Fos

results since it is an invasive procedure that activates nociceptive pathways (Dubin and

Patapoutian, 2010). Another alternative would have been to use gas anaesthesia however

although it reduces the time necessary to anaesthetize the animal and there is less probability of

drug induced toxicity, this technique presents potential risks to the experimenter (Yasny and

White, 2012).

5.1.3 The choice of animal model

The interaction between the various joint tissues during the development and progression of OA

as well as its etiology and pathogenesis are not yet well known (Little and Smith, 2008; Longo et

al., 2012). As such, numerous animal models have been developed in an attempt to find new

strategies for the control and treatment of this disease (Gregory et al., 2012).

57

OA development is difficult to study in humans as it is a slowly progressive disease. In addition,

the extreme variability between individuals as a result of genetic variability, cultural and

environmental factors also greatly bias observations (Little and Smith, 2008). Hence, the use of

animal models of OA is advantageous because it allows (i) a greater control over its progression

and (ii) the study of specific aspects in the development of the pathology (Mastbergen and

Lafeber, 2009; Little and Zaki, 2012). More importantly, there is an analogy in what concerns the

overall organization of the nervous system in humans and rodents (Dubin and Patapoutian,

2010).

A perfect animal model has not been found yet (Longo et al., 2012) as no single model is able to

complete reproduce all the components of the human disorder (Gregory et al., 2012). Currently,

the available animal models of experimental OA include models of (i) spontaneous osteoarthritis

(Bendele, 2001), (ii) mechanically-induced OA (shear, compression and tension stresses (Poulet

et al., 2011; Wei et al., 2001)), (iii) chemically-induced models (intra-articular injection of

collagenase (Yeh et al., 2008; Adães et al., 2014), monoidoacetate (MIA) (Orita et al., 2011;

Ferreira-Gomes et al., 2012), complete Freund’s adjuvant (CFA) (Danziger et al., 1999; Hashmi

et al., 2010) and kaolin/carrageenan (K/C) (Kim et al., 2012; Amorim et al., 2014)) and (iv)

surgically-induced OA (anterior cruciate ligament transection combined with meniscetomy

(Kamekura et al., 2005; Hayami et al., 2006)). Moreover, to study the specific action of a

molecule, some genetically modified models have also been developed in mice (Helminen et al.,

2002).

The intra-articular injection of MIA is one of the most used models for the induction of

experimental OA (Pomonis et al., 2005; Schuelert and McDougall, 2009; Im et al., 2010; Orita et

al., 2011; Ferreira-Gomes et al., 2012; Kelly et al., 2013; Ogbonna et al., 2013) as it induces

histological changes and pain related behaviours identical to those of human OA patients

(Schualert and McDougall, 2009). On the downside, the extent of the degeneration of the joint

structures is mostly dependent on the concentration of MIA (Ogbonna et al., 2013) which often

causes sharp and large-scale cellular death and a disease severity higher than what is observed

in humans (Schuelert and McDougall, 2009).

In this work we used the model of K/C to induce experimental OA in rats through the injection of

a mixture of kaolin and carrageenan in the synovial capsule of the knee joint. This method was

chosen due to several advantages in relation to others models including (i) the pathology

58

develops rapidly within hours and persists for several weeks (Neugebauer et al., 2007), (ii)

sustained development of mechanical hyperalgesia, (iii) gradual degeneration of articular

structures and (iv) the development of comorbid mood-like disorders (Amorim et al., 2014).

The model of K/C was first established as model of acute monoarthritis that mimicked the acute

inflammatory phase of OA and was easily reproducible (Neugebauer et al., 2007). The

simultaneously administration of kaolin, responsible for mechanical damage to intra-joint

structures, and carrageenan, responsible for the inflammatory reaction, is also an advantage, in

relation to the majority of inflammatory animal models that use carrageenan alone since it

induces recurrent episodes of inflammation that are consistent with the human pathology

(Amorim et al., 2014). Finally, the injection of K/C into the synovial capsule causes mechanical

damage to the cartilage, inflammation of the synovia and synovial fluid exudate while

carrageenan alone only induces cartilage damage and to a lesser extent (Neugebauer et al.,

2007).

In the work herein we analyzed the animals four weeks after the induction of experimental OA.

The time frame between the induction and development of OA is important in this disorder as OA

is a slow progression disorder that needs time to fully develop (Amorim et al., 2014). In fact, until

recently and due to the use of smaller time frames only anxiety-like behaviour had been reported

in this model (Ji et al., 2007). Yet, Amorim and colleagues (2014) demonstrated that using the

K/C model animals would develop comorbid depressive-like behaviour four weeks of OA

induction post-induction. An observation that is in accordance with a recent report showing that

the development of mood disorders is a time-dependent process where anxiety-like behaviour

may precede the development of depressive-like behaviour (Yalcin et al., 2011).

5.1.4 Evaluation of nociceptive behaviour

For the evaluation of nociceptive behaviour it is important to use methods that are validated,

sensitive and specific for the assessment of nociception since pain cannot be directly quantified

in animals (Sandkühler, 2009). Since chronic pain is usually accompanied by the development of

abnormal sensory syndromes (hallmarks of pain) like spontaneous pain, allodynia and

hyperalgesia (Bridges et al., 2001; Dubin and Patapoutian, 2010), most evaluation methods have

been develop to assess changes in these parameters.

59

Spontaneous pain is a poorly understood aspect of chronic pain, and its detection and evaluation

are a major preclinical challenge (He et al., 2012). Recently, in mice models, He and colleagues

(2012) demonstrated that is possible to detect the presence of non-evoked ongoing pain in

animals with inflammation or peripheral nerve injury by evaluating negative reinforcement. This

“method” involves the placement of animals in a conditioned placement apparatus, where the

administration of different drugs was paired with a specific chamber in the apparatus.

Consequently, after free access to all chambers, the animals with inflammation or peripheral

nerve injury had a preference for the chamber where they were placed after the administration of

clonidine and LIDO (He et al., 2012). Spontaneous pain has a causal link with allodynia and

hyperalgesia (Djouhri et al., 2006), and in this work we evaluate hyperalgesia, a relevant form of

pain sensitization well defined in human chronic pain.

5.1.4.1 Mechanical hyperalgesia

Currently, there are two methods commonly used to evaluate the response to noxious

mechanical stimulation: the Randall-Sellito test and the pressure application measurement (PAM)

test. These tests are able to quantify the “sensitivity” of rodents by applying a known force on a

specific body part until the animals display a behavioural sign of discomfort. However, there are

several differences between the Randall-Sellito and the PAM tests.

The PAM test is a novel behavioural technique that was based in a classical approach proposed

by Randall-Sellito (1957) where a graded mechanical nociceptive stimulus is applied to the

inflamed paw (Keef et al., 1991; Auh and Ron, 2012; Santos-Nogueira et al., 2012). One of the

disadvantages of the PAM test is that it relies greatly on the expertize of the experimenter as the

force applied to the joint being tested depends on operator (Barton et al., 2007). However, the

PAM has the advantage of allowing the evaluation of most joints (Barton et al., 2007) while the

Randall-Sellito is restricted to evaluations in the paw (Lattanzi et al., 2012; Ferrari et al., 2013).

Moreover, in the PAM test we have the ability to change the size of the pressure application

surface and the force range of the transducer, making it a test easy to use and that allows rapid

and reproducible measurements (Barton et al., 2007).

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5.1.4.2 Heat hyperalgesia

Heat hyperalgesia, characterized by an increased in pain sensation in response to the application

of a noxious heat stimulus, can be measured using the paw-withdrawal (PW) test, the tail-flick

(TF) test and the hot-plate test where heat stimulation is applied to hindpaws, the tail and

simultaneously to forepaws and hindpaws, respectively.

In this work, to assess thermal hyperalgesia, the PW test was used and, the hind paw latency

was measured through the application of a radiant heat beam to the plantar aspect of the

hindpaw. Although this test is a nonspecific test that is applied to areas other than the affected

area (right knee), this test allowed us to evaluate an area which is also under descending

nociceptive control.

In the tail-flick test, the stimulus is applied to the tail, a body part that also is a thermoregulator

organ that can influence nociception itself. In fact, the withdrawal latency of the tail depends on

the temperature of the tail skin (Berg et al., 1988). Since to perform the pharmacological tests

we needed to repeat heat stimulation, we opted not to use this test.

Although the hot-plate test is a good method to assess supraspinal responses to noxious heat

and cold stimulation as it is performed in free moving animals (Gunn et al., 2011), this test

presents a steep learning curve and cannot be repeated more than twice. Another disadvantage

is the fact that the type of stimulation (forepaws and hindpaws are stimulated simultaneously)

evokes the activation of the “diffuse noxious inhibitory control” (DNIC) and can thus bias our

evaluation (van Wijk and Veldhuijzen, 2010; Staud et al., 2003).

5.1.5 Pharmacological studies

Pharmacological studies have been performed for several decades and set the basis for the

development of many pain management therapies. To study a specific neurotransmitter system

local or systemic administration of specific agonists and antagonists can be used. More

specifically, antagonists can be used to determine the tonic activity of a neurotransmitter by

blocking the activity of their receptors and thus impairing the activity of dependent circuits. On

the other hand, agonists are used to activate specific receptors and dependent pathways. In both

61

cases it is however important to keep in mind that the neurotransmitter and its receptors might

not be available in normal conditions.

5.1.6 Evaluation of mood-like behaviour

Anxiety- and depression- like behaviours can be assessed using a wide range of behavioural

testing paradigms. These behaviour paradigms are based on the assumption that rodents and

humans share, to some extent, innate behaviours when faced with new challenges (Bailey and

Crawley, 2009). However, it is necessary to take into account that each individual test assesses

only a fraction of the emotional profile of the animal (Ramos et al., 2008).

5.1.6.1 Anxiety-like behaviour

The open field (OF), the elevated plus maze (EPM) and the light/dark box (LDB) tests are the

most commonly used tests to measure anxiety-like behaviour in the rats (Ramos et al., 2008) as

they assess the conflict between curiosity, exploratory behaviour, and innate aversion to open

and brightly light areas (Keers et al., 2012).

The OF test besides evaluating anxiety-like behaviour (Walsh and Cummins, 1976; Leite-Almeida

et al., 2009) has the advantage of also allowing the simultaneous evaluation of locomotor

performance. While using this test, the experimenter should take into account several aspects

such as the physical characteristics of apparatus such as size, shape, color, floor texture, odor,

sound, nature and location of the starting area, as well as the intensity and position of the light

(Walsh and Cummins, 1976). In fact, the intensity of the lights used in the OF is of major

importance while evaluating anxiety as intense lights are considered a “stressor” and can impair

the animals’ vision and locomotor activity.

The EPM consist of an apparatus with two open and two closed arms, elevated approximately

1 m above the floor. In this test the time spent in the closed arms and open arms as well as in

the centre of the apparatus is quantified (Walf and Frye, 2007). The LDB, a precursor of the EPM

test (Bailey and Crawley, 2009), was composed of a small dark area and a large brightly light

area (Bourin and Hascoët, 2003). Here, the innate aversion of rodents to brightly light areas was

assessed. The advantages of both tests are their simplicity, the short duration of the test and the

62

fact that animals are free to move by themselves (Itoh et al., 1990). In addition, no training prior

training to test is required (Bourin and Hascoët, 2003). Again one should be careful while

evaluating the results as the animal’s performance might be affected by motor function or drug

administration. This problem can be resolved by performing a preliminary screening of locomotor

activity using, per example, the rotarod test (Bourin and Hascoët, 2003).

5.1.6.2 Depressive-like behaviour

One common symptom of depression is learned helplessness. Some learned helplessness

paradigms, expose animals to inescapable electroshocks while evaluating struggling/evasive

behaviour in aversive situations (Takamori et al., 2001; Vollmayr and Henn, 2001; Chourbaji et

al., 2005).

An alternative is the FST (Rénéric et al., 2002; Finn et al., 2003; Bessa et al., 2009;

Lino-de-Oliveira et al., 2005; Slattery and Cryan, 2012; Amorim et al., 2014) were animals are

placed in a cylinder filled with water and learn there is no possible escape - learned helplessness.

Two variables are currently evaluated during the 5 min testing session, the latency to immobility

and the time spent immobile.

One disadvantage of the FST is the fact that the results depend on the physical capability of the

animals. Thus, if the model interferes to some extent with the animals’ ability to swim, results

should be approached carefully. In addition, Takamori and colleagues (2001) evaluated the effect

of several antidepressants upon rats’ performance after inescapable shock and the FST and

concluded that the former displays greater sensitivity to a wider range of antidepressants when

compared to the latter.

We opted to use a modified version of the FST in our work (Amorim et al., 2014) as we also

quantified the time the animals spent climbing and swimming. The discrimination between

variables has been proposed to allow distinguishing between impairments in serotoninergic or

noradrenergic pathways, respectively (Rénéric et al., 2002; Lino-de-Oliveira et al., 2005).

Another component of depressive-like behaviour, anhedonic behaviour, is also commonly

evaluated using the sucrose preference test (SPT) (Anisman and Matheson, 2005; Overstreet,

2012). In this test, the preference for a sweet solution in relation to water is evaluated. However,

63

in the present work, as the peak effect of GAL is between 10-20 min, and the test session

requires at least 1 h to perform, the SPT test was not performed.

5.1.7 Evaluation of c-Fos expression

5.1.7.1 c-Fos as neuronal marker

c-Fos is a nuclear protein of the Fos protein family (Hoffman et al., 1993) and when its

proto-oncogene is rapidly and transiently activated in response to stimulation, the Fos protein is

expressed in cells (Harris, 1998; Coggeshall, 2005). c-Fos is involved in the signal transduction

cascade that links extracellular events to intracellular adaptations (Gao and Gi, 2009). Mapping

c-Fos expression is one of the most common methods to assess neuronal activation in pain

studies (Coggeshall, 2005).

The expression of c-Fos has been linked with nociception as most manipulations evoking

nociceptive responses alter the expression of c-Fos (Harris, 1998). The distribution of c-Fos has

been specifically associated with differences in the quality and intensity of noxious stimulation

(Cirelli and Tononi, 2000). Many stimuli can be used to induce c-Fos expression (Coggeshall,

2005) including noxious heat/cold or chemical, acoustic, thermal, visual, as well innocuous

stimuli (brushing of hairs and gentle manipulation of joints) (Cirelli and Tononi, 2000) and

depending on the experimental design this feature can either be an advantage or a disadvantage

of the technique. Interestingly, since under normal conditions the expression of c-Fos is low, this

parameter can also be used as a marker of activity in chronic pain conditions (Gao and Gi,

2009). Moreover, all neurotransmitters and neuromodulators, with the exception of GABA and

glycine, activate the expression of c-Fos (Cirelli and Tononi, 2000).

Currently, besides c-Fos, there are a considerable number of markers of neuronal activation that

may be used in pain research. The phosphorylated extracellular signal-regulated kinase (pERK) is

another commonly used marker of neuronal activity (Silva et al., 2010). ERK is a member of

MAPK family, it is activated via phosphorylation (Gao and Gi, 2009) and plays a major role in

regulating neuronal plasticity (Ji and Woolf, 2001). In 1999, Ji and colleagues verified that like

c-Fos, pERK expression is increased after peripheral noxious stimuli, however there are some

differences between c-Fos and pERK expression. First, while the c-Fos is induced by brief noxious

stimulation, stimuli lasting less than 10 seconds do not induce pERK (Gao and Gi, 2009).

64

Secondly, c-Fos expression increases between 30-60 min after noxious stimulation, peaking 1-2 h

and disappearing 8-24 h later. pERK however is detected within 1 min after stimulation, peaks at

3 min and returns to basal levels 2h later. Thirdly, while c-Fos is exclusively expressed in the

cell’s nuclei, pERK is also expressed in the cytoplasm, dendrites and axons (Gao and Gi, 2009).

The choice between these marker depends greatly on the type of stimulation used, short-term

intense stimuli would gain much from analyzing pERK while long-lasting less intense stimulation

period, like the ones used herein, gain from c-Fos analysis.

5.1.7.2 c-Fos stimulation protocol

Taking into account what was mentioned above, in our work, the expression of c-Fos protein was

used as a marker of neuronal activity in the central nervous system (CNS) either in basal

conditions or after mechanical stimulation of the right knee and/or GAL administration in the

DMH. The mechanical stimulus was chosen to mimic the impact of moving an osteoarthritic joint,

a stimulus known to alter the expression of c-Fos in the CNS (Bullitt, 1990).

Fos levels in the brain were determined by performing immunohistochemistry with a c-Fos

antibody. This technique is a powerful tool that allows analyzing dynamic short term changes in

neuronal activation (Hoffman et al., 1993) and it is easy to perform. Another advantage of this

technique is that it allows acquiring an integrated perspective of the impact of the procedures

that lead to changes in c-Fos expression in the CNS.

However, when using c-Fos expression for comparisons between different structures, it is

necessary to take into account that (i) c-Fos expression can be induced by a variety of peripheral

stimuli other than those at study, such as the stress induced by handling the animals, (ii)

neurones differ in their time to express c-Fos (Harris, 1998), thus failure to alter c-Fos expression

might not imply that there was no neuronal activation; (iii) the time lag between induction and

expression of c-Fos does not acutely allow to determine which event is responsible for c-Fos

expression (Harris, 1998), and finally (iv) c-Fos expression does not provide information

concerning the connectivity between activated areas/neurones (Kovács, 2008).

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5.2 The role of GAL in the DMH upon behaviour

5.2.1 Locomotor activity

OA causes physical disability limiting locomotion and movements (Steultjens et al., 2000) that

decrease the patient’ ability to perform basic daily life activities (Odding et al., 1998; Odding et

al., 2001) due to functional impairment of the affected joints (Axford et al., 2010).

Even though our ARTH animals displayed mechanical hyperalgesia in the affected knee, in

accordance with the hyperalgesia reported by OA patients, (Bajaj et al., 2001; Fernihough et al.,

2004; Wylde et al., 2012), these animals did not display locomotor impairments during the free

exploration task in the OF arena nor a clear behavioural phenotype (Teeple et al., 2013). It is

possible that the impact of knee OA in rodents is less pronounced than in humans, as rats are

quadruped and can, thus, more easily compensate weight bearing.

A detailed assessment of gait changes in this animal model of experimental OA should be

performed in order to better understand the extent of the impact of knee OA. It is important to

note that gait alterations are more pronounced in the early stages of experimental OA (acute

phase) (Boettger et al., 2011) and include a decreased flexion of the knee during strides (Landry

et al., 2007) and minimization of knee loading with compensational counter regulation of

locomotion in the contralateral knee joint (Kaufman et al., 2001). In addition the analysis of the

walking speed (Simjee et al., 2007; Vincelette et al., 2007), duration of stance and swing phases

(Orito et al., 2007), distances or angles between paw or footprint pressure (Ängeby-Möller et al.,

2008; Boettger et al., 2009) and, more importantly, the range of motion (Boettger et al., 2011)

could also provide more precise data concerning this model.

In relation to effect of the administration of GAL in the DMH upon locomotion, no differences

were found either between SHAM and ARTH animals or before and after GAL microinjection.

Although our results are in accordance with data showing the administration of GAL did not

induce motor impairments, sedation or toxicity (Xu et al., 2000), Kehr and colleagues (2002)

demonstrated that intracerebroventricularly administration of GAL reduced spontaneous

locomotor activity in rats. Our results suggest the activation of GAL-dependent descending

pathways in the DMH is not involved in the mediation of motor activity as GAL had no effect upon

this parameter.

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5.2.2 Anxiety-like behaviour

In humans, OA have been associated with the development of mood-like disorders, such as

anxiety and depression (Sharma et al., 2003; Axford et al., 2010) however in our work ARTH

animals only displayed depressive-like behaviour. This result contrast with reports from Axford

and colleagues (2010) and Song and colleagues (2014) where they demonstrated that chronic

pain enhances the development of anxiety in OA patients. In addition, Amorim and colleagues

(2014) also showed, using the OF test, that ARTH animals displayed an anxious-like phenotype.

One possibility that might explain the absence of anxiety is the fact that the arena might not be

large enough to cause an anxiogenic effect. Secondly, is it possible that our animals were no

longer anxious. In fact, Yalcin and colleagues (2011) demonstrated in a mouse model of

neuropathic pain that anxious-like behaviour was only observed in the early stages of the disease,

preceding the development of depressive-like behaviour. Although anxiety-like behaviour was not

tested during the first weeks in our experiment, an early onset of anxiety-like behaviour was

previously reported by Ji and colleagues (2007) using the same kaolin/carrageenan model in

adult male rats.

Again, the fact that the administration of GAL in the DMH did not alter anxiety-like behaviour in

our animals suggests that this behaviour might not be mediated by the pathways herein

activated. Nonetheless, as some studies show stress, and chronic pain is frequently considered a

stressful event, can change the expression of GAL (Sweerts et al., 2000; Christiansen et al.,

2011) and GAL can modulate anxiety- and depressive-like behaviours (Holmes et al., 2003; Zhao

et al., 2013), further studies should be performed to reevaluate anxiety-like behaviour using other

behavioural tests.

5.2.3 Evaluation of depressive-like behaviour

As previously mentioned, Yalcin and colleagues (2011) showed that the expression of

anxiety- and depressive-like behaviours is time-dependent. In line with this finding, although our

ARTH animals did not exhibit the former, they displayed a depressive-like phenotype when

compared with SHAM animals, through both a decrease in the latency to immobility and

increased immobility. Interestingly, while Yalcin and colleagues (2011) using an experimental

model of neuropathic pain observed the development of depressive-like behaviour only 6 to 8

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weeks post-induction, in our K/C model this phenotype was fully developed four weeks

post-induction hinting the inflammatory component of our model might contribute to challenge

the emotional status of the animals.

Increased immobility in the FST is interpreted as an indicator of learned helplessness (Amorim et

al., 2014), the animals’ equivalent of human “despair” (Castagné et al., 2011), as animals learn

that escape is impossible and adopt a passive behaviour (Lino-de-Oliveira et al., 2005).

Importantly, our results are in agreement with clinical data, showing that OA patients are more

vulnerable to developing symptoms of depression (Bair et al., 2003; Sheurborne et al., 2009;

Axford et al., 2010).

It might however be argued that the increased immobility of ARTH animals could be associated

with reduced motor competence, as shown by Wang and colleagues (2011) while testing

neuropathic animals in the FST. Yet, as previously discussed, our animals do not show any

locomotion impairment in the OF test four weeks after the induction of experimental OA.

Additional tests using the Rotarod apparatus at increasingly higher speeds, allowing the

endurance of the animals to be tested, should be performed to confirm the lack of motor

impairments in this animal model.

In the first experiment, the administration of GAL in the DMH decreased the latency to immobility

and increased the immobility time in SHAM, but not in ARTH, animals. Although these results are

in accordance with reports from Kuteeva and colleagues (2007) and Zhao and colleagues (2013)

where GAL plays a prodepressive role, the lack of effect in ARTH animals was unexpected. One

possibility is that in experimental OA, GAL-dependent circuits are already over-activated and thus

the administration of exogenous GAL provided no additional effect since in fact SAL-injected

ARTH animals already display a depressive-like phenotype.

Secondly, it is possible that DMH neurones expressing GAL receptors are tonically inhibited in

ARTH animals in an attempt to counteract the effects of experimental OA. Thirdly, it is also

possible that four week after the induction of the model, the availability of GAL receptors in the

DMH has decreased. In either case, although GAL clearly induces a prodepressive phenotype, as

shown in SHAM animals, its effect in this disorder depend on the plasticity of the circuits

mediating emotional behaviour in experimental OA.

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The dysfunction of serotonergic and noradrenergic system is strongly correlated with the

development of depression (Ressler and Nemeroff, 2000). In animal studies, the amount of time

spent swimming and climbing in the FST are two active behaviours that have been correlated

with changes in serotoninergic and noradrenergic tonus, respectively (Lino-de-Oliveira et al.,

2005; Rénéric et al., 2002).

The fact that our ARTH animals showed a significant decreased in the time spent swimming,

when compared to SHAM animals, suggests serotoninergic pathways are impaired in

experimental OA. In addition, as the administration of GAL in the DMH of SHAM animals also

reduced the time spent swimming, it is probable that DMH neurones expressing GAL receptors

are mediating serotonergic pathways involved in emotional processing (Li et al., 2013). This

hypothesizes is supported by a report showing GAL administered intracerebroventricularly

modulates the activity of serotoninergic neurones in the AMY (Blackshear et al., 2007).

Furthermore, another work showed GAL acts as an inhibitory modulator, inhibiting the release of

serotonin and norepinephrine in the AMY (Holmes et al., 2003).

In relation to the time spent climbing, our results were again unexpected as ARTH animals spent

more time climbing when compared to SHAM animals. While these results suggest that

noradrenergic pathways are not alter by experimental OA, taking into account that both serotonin

and noradrenalin receptors are targeted by antidepressants (Yokogawa et al., 2002) towards

increasing the concentration of these neurotransmitters in the synaptic cleft, the fact that the

function of noradrenergic pathways is preserved, may impact greatly on the therapies used for

the control of OA. As antidepressant usually inhibit the resorption and subsequent degradation of

a neurotransmitter, the excess of NA in the synaptic cleft could be deleterious that need to be

addressed in further studies.

5.3 The RVM as a relay of the GAL pronociceptive action in the DMH

Earlier results from Martenson et al. (2009) and Pinto-Ribeiro et al. (2013) demonstrated that

both the disinhibition and the activation of the DMH, respectively, enhanced nociception during

peripheral noxious stimulation, showing this nucleus plays a facilitatory role in the descending

modulation of nociception.

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Moreover, our behavioural data shows that GAL in the DMH has a pronociceptive action upon

thermociception in SHAM and ARTH animals, an effect that contrasts with previous works where

GAL microinjected in the arcuate nucleus (Sun et al., 2003; Sun et al., 2007), the PAG (Wang et

al., 1999; Wang et al., 2000), the nucleus accumbens (Xu et al., 2012a) and the

tuberomammillary nucleus (Sun et al., 2004) was antinociceptive.

As the DMH does not project directly to the spinal cord (Thompson et al., 1996), we opted to

evaluate the RVM as a potential relay area for this GAL-induced facilitatory effect since Martenson

et al. (2009) and Pinto-Ribeiro et al. (2013) showed DMH-induced behavioural hyperalgesia was

accompanied by the enhancement of the activity of pain-facilitating neurons in the RVM. This

hypothesis was further supported by the fact that in animal models of acute inflammation and

mononeuropathy (Urban and Gebhart, 1999; Vera-Portocarrero et al., 2006; Sanoja et al., 2008;

Saadé et al., 2012), behavioural hyperalgesia was temporarily decreased after inactivation of the

RVM with LIDO.

We were able to demonstrate that the RVM is involved in the descending facilition of nociception

after GAL in the DMH in both experimental groups since behavioural hyperalgesia was lost when

the RVM was inhibited with LIDO. Nonetheless, further studies are needed to investigate which

RVM cells might be mediating this facilitory effect.

5.4 c-Fos expression upon key areas of nociception

5.4.1 Ventrolateral periaqueductal gray matter (VLPAG)

The PAG is known to play an important role in inhibiting the perception of pain (Wu et al., 2014;

Loyd and Murphy, 2009). Many studies support its division into four different functional,

anatomical and neurochemical columns: dorsomedial, dorsolateral, lateral and ventrolateral

(Mitsui et al., 2003; Linman et al., 2012) which play different roles in chronic pain (Waters and

Lumb, 2008). The ventrolateral PAG (VLPAG) of the RVM is associated with pain relief and is

considered as part of the defence reaction used for animals (Green et al., 2006).

Interestingly the induction of experimental OA didn’t alter the activation of the VLPAG, a quite

surprising result taling into account that this area is part of the descending pain inhibitory

system.

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GAL in the DMH did not increase the activity of the VLPAG in both experimental groups when

compared to SAL-injected animals although c-Fos expression was significantly higher in

GAL-injected ARTH animals when compared GAL-injected SHAM, hinting DMH neurones

expressing GAL receptors do not mediate VLPAG activity.

As expected the activity of the VLPAG was enhanced after peripheral noxious stimulation, in line

with a report from Mitsui et al. (2003) that proposed a significant increase in the number of c-Fos

in the ventrolateral column of the PAG after noxious stimulation in structures such as the joint,

although this effect was restricted to SHAM animals. These results not only confirm the VLPAG as

a mediator of nociception but also suggest some remodeling of pain pathways might have

occurred in experimental OA.

The steep decrease in VLPAG activity in SHAM animals after the simultaneous administration of

GAL in the DMH and peripheral stimulation suggest DMH neurones expressing GAL receptors

exert a tonic descending effect over VLPAG neurones mediating nociception. The difference in

c-Fos expression between ARTH and SHAM animals in this protocol suggest GAL-dependent DMH

descending VLPAG inhibition is impaired in experimental OA.

5.4.2 Dorsal raphe nucleus (DRN)

The DRN is the largest serotoninergic nucleus (Kaehler et al., 1999; McDevitt and Neumaier et

al., 2011) recognized as an analgesic area (Segal, 1979; Inase et al., 1987; Biagioni et al.,

2013) as its descending projections modulate behavioural responses evoked by STM (Wang and

Nakai, 1994).

The increased in c-Fos expression in SAL-injected ARTH animals when compared to SAL-injected

SHAM animals is thus in accordance with the role of the DRN in the modulation of nociception. It

is probable that enhanced DRN activity induces the release of 5-HT as part of an attempt to

inhibit nociceptive transmission in the spinal cord although very little is known about the role of

the DRN and its serotonin projections in this disorder (Ito et al., 2013). The potential

envolvement of the DRN in the control of nociception is further supported by the significant

increase of DRN c-Fos expression after peripheral noxious stimulation. On the other hand the fact

that differences were observed only in SHAM animals suggests remodeling of serotonergic

pathways in experimental OA.

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Interestingly, the administration of exogenous GAL to the DMH didn’t alter the number of cells

expressing c-Fos in both experimental groups suggesting DMH neurones expressing GAL

receptors do not directly modulate the activity of the DRN while, by contrast, c-Fos expression in

the DRN increased significantly after the simultaneous administration of GAL in the DMH and the

application of peripheral noxious stimuli although only in ARTH animals. It would appear the in

SHAM animals the administration of GAL in the DMH had a tonic effect in decreasing DRN

activity while in ARTH it enhanced it. Since different GAL can either enhance or inhibit neuronal

activity depending on the GAL receptor being activated, further studies should evaluate whether

the expression of GAL receptors in the DMH is altered in experimental OA. Finally, a stronger

activation of the ipsilateral side when compared to the contralateral confirms descending

modulation from the DMH to the DRN is mostly ipsilateral (Biagioni et al., 2013).

5.4.3 Locus coeruleus (LC)

According to literature, the LC is the principal site for brain synthesis of norepinephrine (NA) (Liu

et al., 2008; Sara, 2009; Vazey and Aston-Jones, 2014) and an nucleus that strongly projects to

limbic areas (Samuels and Szabadi, 2008). Four weeks after the induction of experimental OA,

the increase in the c-Fos expression, when compared to SHAM animals, is in accordance with the

report from Tsuruoka and colleagues (2003) showing animals injected with carrageenan in the

hindpaw displayed a higher number of Fos-positive cells which suggests the LC is tonically

activated in experimental OA. Taking into account previous reports (Jasmin et al., 2002; Millan,

2002; Pertovaara, 2006) it is plausible that enhancing LC’s activity is a compensatory

mechanism to tonically and phasically inhibit spinal nociceptive transmission.

The administration of exogenous GAL to the DMH does not appear to influence LC activity by

itself as no significant differences in c-Fos expression were found between SAL- and GAL-injected

animals. Our results contrast with studies showing GAL and its receptors are co-expressed with

NA in the LC (Zachariou et al., 2000; Kuteeva et al., 2008) and the activation of GAL receptors

inhibit the spontaneous firing of LC neurones (Sevcik et al., 1993) and an effect in SHAM animals

would have been expected. Meanwhile, peripheral noxious stimulation enhances the activity of

the LC but ony in SHAM animals thus suggesting a role for this nucleus in the modulation of

nociception.

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Interestingly, in SHAM animals the simultaneous administration of GAL in the DMH while

applying peripheral noxious stimuli significantly decreased c-Fos expression in the LC when

compared to the application of the noxious stimuli alone suggesting DMH neurones expressing

GAL receptors are able to modulate the activity of LC neurones involved in the mediation of

noxious mechanical stimulation. As for ARTH animals, the decrease of c-Fos expression in the LC

after the simultaneous administration of GAL in the DMH while applying peripheral noxious

stimuli when compared to SAL-injected animals suggests the simultaneous activation of the

circuits mediating GAL/DMH-dependent and peripheral noxious stimuli-evoked effects inhibit the

activity of the LC leading to behavioural facilitation.

5.4.4 Rostral ventromedial medulla (RVM)

The RVM has since long been established as an area that contributes to the descending

modulation of nociception in chronic disorders (Khasabov et al., 2012). The greater expression of

c-Fos in SAL-injected ARTH animals when compared to SAL-injected SHAM suggests the RVM is

involved in the tonic mediation of nociception in experimental OA. Taking into account the

literature (Lang and Kofler, 2011), it is possible that RVM involvement is due to an attempt of the

pain modulatory system to counteract the effects of persistent activation of pain pathways in this

disorder (Pinto-Ribeiro et al., 2013).

Curiously, the administration of GAL in the DMH induced behavioural hyperalgesia in SHAM, but

not in ARTH, animals evidencing not only that DMH neurones expressing GAL receptors impact

on the activity of the RVM but that this effect is lost in experimental OA. As previously suggested

further studies should be performed in order to verify if the availability of GAL receptors in the

DMH is altered after experimental OA or if the DMH if the activity of DMH expressing GAL

receptors is being inhibited.

As expected in light of the role of the RVM in pain modulation (Cirelli and Tononi, 2000;

Coggeshall, 2005), c-Fos expression in SHAM animals after the application of mechanical

noxious stimulus on the knee was increased. In what concerns ARTH animals, it is not surprising

that c-Fos expression remained unchanged as this animals already displayed behavioural

hyperalgesia. On the other hand, it is also possible the RVM is unable to process additional

nociceptive inputs and/or that it has undergone neuroplasticity (Vanegas and Schaible, 2004).

73

Interestingly, in SHAM animals the number of c-Fos expressing cells in the DMH was significantly

decreased after the simultaneous application of a noxious stimuli and administration of

exogenous GAL to the DMH when compared to the microinjection of exogenous GAL in the DMH

alone. This result suggests that in SHAM animals GAL/DMH-induced behavioural hyperalgesia is

counteracted by the activation of pathways mediating peripheral noxious stimulation otherwise an

expression of c-Fos greater that what observed after the administration of exogenous GAL would

be expected.

5.4.5 Dorsal Reticular Nucleus (DRt)

The DRt is a pronociceptive center (Lima and Almeida, 2002) activated exclusively by noxious

stimulation (Almeida et al., 1996; Tavares and Lima, 2007). Thus, a greater activation of the DRt

would be expected in animals with experimental OA (Pinto et al., 2006, 2008). In fact, we verified

that the expression of c-Fos is significantly increased in the DRt of ARTH animals, when

compared to SHAM animals, suggesting this pronociceptive nucleus mediates descending

facilitation in experimental OA. A stronger activation of the ipsilateral DRt in comparison to the

contralateral side is also in accordance with the literature (Lima and Almeida, 2002) as

descending projections from the DRt are mainly ipsilateral.

The increase of c-Fos expression in the DRt of SHAM animals after the administration of GAL in

the DMH suggests DMH neurones expressing GAL receptors are involved in mediating

behavioural hyperalgesia after drug administration. Interestingly, noxious peripheral stimulation

by itself didn’t alter DRt activity, probably due to the activation of compensatory endogenous

descending inhibitory pathways (Ossipov et al., 2010).

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CHAPTER 6: CONCLUSION AND FUTURE PERSPECTIVES

77

6. CONCLUSION AND FUTURE PERSPECTIVES

Over the years several animal models have been developed to study OA and although there isn’t

a single model that mimics all the features of the human pathology, the K/C model of

experimental OA stands as an interesting pre-clinical tool to study the comorbidity between OA

and emotional impairments.

The results of this experimental work generally support the hypothesis that GAL plays a role in

the comorbidity between OA and mood disorders as the exogenous intracerebral microinjection of

this neuropeptide enhanced the expression of depressive-like behaviours while it also increased

the activity of pronociceptive nuclei. Furthermore, our results suggest that galaninergic pathways

target important serotoninergic, but not noradrenergic, areas again supporting a role for GAL in

the control of pain and emotions.

Future works should investigate the specific role of each GAL receptor in nociceptive facilitation

with a special emphasis on its impact upon serotoninergic areas. Another important aspect would

be to assess which pathways are mediating the GAL/DMH-induced descending facilitation in the

spinal cord.

79

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81

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