Characterization of an animal model of Bladder painful ... · Characterization of an animal model...

Characterization of an animal model of Bladder Painful Syndrome/ Interstitial Cystitis

Rita Daniela Santos Matos

Mestrado em Biologia Celular e Molecular Departamento de Biologia Faculdade de Ciências da Universidade do Porto 2014

Orientador Ana Cristina Estrela de Oliveira Charrua Cordeiro, PhD Faculdade de Medicina da Universidade do Porto

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Characterization of an animal model of Bladder painful syndrome/ Interstitial cystitis 2

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Todas as correções determinadas pelo júri, e só essas, foram efetuadas. O Presidente do Júri,

Porto, ______/______/_________

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Dissertação para a candidatura ao grau de

Mestre em Biologia Celular e Molecular

submetida à Faculdade de Ciências da

Universidade do Porto.

A presente dissertação foi orientada pela

Doutora Ana Cristina Estrela de Oliveira Charrua

Cordeiro e foi realizada no Centro de

Investigação Médica da Faculdade de Medicina

da Universidade do Porto.

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Acknowledgments

First, I would like to thank to Dr. Ana Charrua not only for supervising this

thesis, but also for all comprehension, sympathy, care, support, interest, knowledge

and time dispensed with me. Thank you so much.

I also would like to thank Prof. Francisco Cruz to accept me in this great group

and allow me the contact with this amazing research area.

I would like to thank Prof. Célia Cruz and Prof. António Avelino for their

availability since the first time.

I must not forget to thanks to “lunch friends” Dr. Bárbara Frias, Dr. Ana Coelho,

Raquel Oliveira, Isabel Regadas, Lígia Almeida, Diana Nascimento, Gisela Borges,

José Pedro Castro and César Monteiro, for their constant good mood, sympathy and

for the excellent way that welcomed me.

I also would like to thank to Anabela Silvestre and Elisa Nova for their technical

support.

At last, but not the least, I would like to thank to my family. To my parents and

my brother that while away, always supported me. To my father, thank you for your

words of advice, care and support. To my mother and my brother, thank you for your

happiness and to make me laugh every day, even when I want to cry.

To my grandmother, for her love and care.

I would like to thank to Daniel for his care, love, friendship, support and for all

the hours that we spent in front of computer working. Thank you to being present in my

life whenever I need.

Thank you!

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Resumo

O Síndrome Doloroso Vesical/Cistite Intersticial (BPS/IC, do inglês bladder pain

syndrome/ interstitial cystitis) é um síndrome crónico e doloroso, cuja etiologia

permanece desconhecida. Doentes com esta patologia apresentam uma

hiperatividade do sistema nervoso simpático, caracterizada pelo aumento da

expressão de tirosina hidroxilase na bexiga, pelo aumento dos níveis de noradrenalina

na urina e pelo aumento da pressão sanguínea e ritmo cardíaco durante a distensão

da bexiga. Estes doentes também apresentam um aumento da expressão do TRPV1

na bexiga. A hiperatividade simpática pode também ser observada em várias

patologias dolorosas em humanos e em modelos animals de dor.

Estudos anteriores demonstraram que a estimulação crónica adrenérgica com

fenilefrina em ratos induz um comportamento de dor, aumenta a frequência urinária e

disfunção do urotélio, o que simula o BPS/IC. A dor e o aumento da frequência urinária

são dependentes dos aferentes primários sensíveis à capsaicina, um agonista do

TRPV1. A desensibilização destes aferentes primários por doses elevadas de

capsaicina reverte os efeitos induzidos pela fenilefrina, sugerindo uma possível relação

entre a atividade adrenérgica e a atividade nociceptiva (como o próprio TRPV1 ou com

outros recetores como os recetores canabinóides, por exemplo).

Este trabalho teve três objetivos principais. O primeiro objetivo foi demonstrar o

envolvimento dos adrenocetores no aparecimento dos sintomas de BPS/IC,

caracterizando qual o subtipo de adrenorecetor que está envolvido no aparecimento

da dor e nas alterações histológicas observadas em animais submetidos a estimulação

crónica adrenérgica com fenilefrina. O segundo objetivo foi avaliar a relação entre os

adrenocetores e o TRPV1 durante a estimulação crónica adrenérgica com fenilefrina.

O terceiro objetivo foi verificar como é que os endocanabinóides modulam a dor

vesical e a hiperatividade durante a cistite.

Para avaliar qual o subtipo de adrenocetor que está envolvido no aparecimento

da dor, aos ratos Wistar fêmeas injetados diariamente com 2.5 mg/Kg de fenilefrina

(subcutânea durante 14 dias) foi-lhes administrado oralmente os seguintes

antagonistas dos α1 – adrenocetores: Silodosina, Naftopidil ou Prazosina. Ao dia 15,

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os animais foram anestesiados com uretano (1.2 g/Kg) e as cistometrias foram

realizadas durante 2h. Depois deste período, os animais foram perfundidos com

paraformaldeído e a bexiga e o segmento L6 da medula espinhal foram colhidos. A

bexiga dos animais foi cortada e corada com Hematoxilina-Eosina para analisar a

morfologia do urotélio e com Azul de Toluidina para estudar os mastócitos. Os cortes

de bexigas foram também sujeitos a uma reação de imunofluorescência contra a

caspase 3, para analisar a apoptose do urotélio. Os segmentos da região L6 da

medula espinal foram sujeitos a reação de imunohistoquímica contra a proteína Fos.

Para estudar a relação entre os adrenocetores e as vias nociceptivas, um

grupo de ratinhos mutantes para o TRPV1 foram injetados diariamente com fenilefrina,

durante 14 dias. O comportamento doloroso foi avaliado nestes animais. Mais ainda,

um grupo de ratos Wistar fêmeas e respetivos controlos foram injetados diariamente

com fenilefrina durante 14 dias. No fim do tratamento os animais foram submetidos a

cistometria com capsaicina.

Para avaliar se os endocanabinóides modulam a dor vesical e a hiperatividade

durante a cistite, um grupo de ratos Wistar fêmeas foram instilados com

lipopolisacarídeo (LPS) e com URB (inibidor da FAAH, do inglês fatty acid amide

hydrolase), e foi avaliado o comportamento doloroso, bem como o funcionamento da

bexiga através da cistometria. Estas experiências foram repetidas em animais tratados

com LPS, URB e antagonistas do recetor canabinóide 1 (CB1), recetor canabinóide 2

(CB2) e TRPV1 (MJ15, SR144528 e SB366791, respetivamente). Os segmentos L6 da

medula espinal foram sujeitos a uma reação de imunohistoquímica contra a proteína

Fos.

Este trabalho confirmou que a estimulação crónica adrenérgica com fenilefrina

induz um comportamento de dor, infiltração de mastócitos, apoptose de células

uroteliais e ativação das vias nociceptivas. A Silodosina (antagonista dos α1A-

adrenorecetores) reduz os efeitos induzidos pela fenilefrina. Para além disso, o TRPV1

e o CB1 parecem estar envolvidos na hiperatividade da bexiga.

Assim sendo, podemos afirmar que BPS/IC resulta da interação entre várias

vias, nomeadamente dos α1A-adrenoreceptores, do TRPV1 e do CB1.

Palavras-chave: Síndrome doloroso vesical/ cistite intersticial; alfa-adrenorecetores;

TRPV1; CB1; vias nociceptivas

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Abstract

Bladder painful syndrome/ interstitial cystitis (BPS/IC) is a chronic debilitating

condition with unknown etiology. Patients with BPS/IC present an increased

sympathetic activity, characterized by an increase in tyrosine hydroxylase expression in

the bladder, increased noradrenaline levels in urine and elevated mean blood pressure

and heart rate during bladder hydrodistention. These patients also present an increase

in expression of TRPV1 in the bladder. Sympathetic overactivity also can be observed

in several human painful diseases and in rat pain models.

Previous studies showed that chronic adrenergic stimulation with phenylephrine

(PHE) in rats induced pain behavior, increased voiding frequency and urothelial

dysfunction, which mimic BPS/IC. Pain and increased frequency where dependent of

capsaicin sensitive primary afferents activation since desensitization of these fibers

with high levels of capsaicin reverse the phenylephrine mediated effect, demonstrating

a possible relation between adrenergic activity and nociceptive activity (like TRPV1 or

other receptors like cannabinoid receptors, for example).

Hence, this work had three main goals. The first goal was to demonstrate the

involvement of adrenoceptors in the appearance of BPS/IC symptoms, characterizing

which subtypes are involved in the appearance of pain and histological changes

observed in animals submitted to chronic adrenergic stimulation with phenylephrine.

The second aim was to evaluate the cross-talk between adrenoceptors and TRPV1

during chronic adrenergic stimulation with phenylephrine. The third aim was to verify

how endocannabinoids modulate the bladder pain and hyperactivity during cystitis.

To assess which adrenoceptor subtypes are involved in the appearance of pain,

to female Wistar rats injected with 2.5 mg/Kg/day of phenylephrine (PHE;

subcutaneously for 14 days) was administrated orally the follow antagonists of

adrenoceptors: Silodosin, Naftopidil or Prazosin. The visceral pain behavior and

voiding pattern were analyzed before and 14 days after PHE treatment. At day 15,

animals were anaesthetized with urethane (1.2 g/Kg) and cystometries were performed

for 2h. Animals were then perfused with paraformaldehyde and the bladder and L6

spinal cord segments were harvested. Bladder sections from these animals were

stained with Hematoxylin-eosin to analyze urothelium morphology, with Toluidine Blue

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to study mast cells, and immunoreacted (IR) against caspase 3, to investigate

urothelial apoptosis. L6 spinal cord segments were IR against Fos.

Furthermore, to study the cross-talk between adrenoceptors and nociceptive

pathways, a group of TRPV1 knockout mice were injected with PHE daily for 14 days.

Visceral pain behavior and cystometry were performed in these animals. Further, a

group of female Wistar rats was daily injected with phenylephrine for 14 days. At the

end of treatment, animals were submitted to cystometry with capsaicin.

To assess if endocannabinoids modulate bladder pain and hyperactivity during

cystitis, a group of female Wistar rats were instilled with lipopolysaccharide (LPS) and

URB (inhibitor of FAAH, fatty acid amide hydrolase). Visceral pain behavior and

cystometry were performed. These experiments were repeated in rats co-treated with

LPS, URB and antagonists of CB1, CB2 and TRPV1 (MJ15, SR144528 and

SB366791, respectively). L6 spinal cord segments were IR against Fos.

The results confirm that chronic adrenergic stimulation with PHE induce visceral

pain, mast cells infiltration, apoptosis of urothelial cells and activation of nociceptive

pathways. Silodosin (antagonist of α1A-adrenoceptor subtype) seem to be more

effective than naftopidil or prazosin (α1D and α1-adrenoceptor antagonists, respectively)

to reduce the effects induced by PHE. This work also shows that TRPV1 and CB1 are

involved in bladder hyperactivity.

Thus, it can be affirmed that BPS/IC result the interaction between several

pathways, namely those involving α1A-adrenoceptors, TRPV1 and CB1.

Keywords: Bladder painful syndrome/ interstitial cystitis; alpha-adrenoceptors; TRPV1;

CB1; nociceptive pathways

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Table of Contents

Table list ......................................................................................................................... 15

Figure list ........................................................................................................................ 16

Abbreviations ................................................................................................................. 18

I. Introduction ............................................................................................................. 21

1. Bladder painful syndrome/ Interstitial cystitis (BPS/IC) ..................................... 22

1.1 Definition of BPS/IC .................................................................................... 22

1.2 Etiology of BPS/IC ...................................................................................... 24

1.3 Diagnosis of BPS/IC .................................................................................... 24

1.4 Epidemiology of BPS/IC .............................................................................. 25

1.5 Treatment of BPS/IC .................................................................................... 25

2. Sympathetic nervous system dysfunction in BPS/IC .......................................... 26

2.1. Nociceptive pathway modulation by adrenoceptors ........................................ 27

II. Aims ........................................................................................................................ 32

III. Material and Methods .......................................................................................... 34

1. Experimental Animals ......................................................................................... 36

2. Chronic adrenergic stimulation ........................................................................... 36

2.1. Blockade of alpha1-adrenoceptors ............................................................... 36

2.1.1. Pain behavior test ..................................................................................... 37

2.1.2. Organ motility test .................................................................................... 38

2.1.3. Cystometry ............................................................................................... 38

2.1.4. Tissue harvesting and processing ............................................................. 39

2.1.5. Immunohistochemistry ............................................................................. 39

2.1.6. Bladder Histological Evaluation ............................................................... 40

2.2 Cross-talk between adrenoceptors and TRPV1 ............................................ 40

3. Cannabinoid system and TRPV1 cross-talk during cystitis ................................ 40

3.1 LPS-induced cystitis ..................................................................................... 40

3.2 Pain behavior test ......................................................................................... 41

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3.3 Cystometries ................................................................................................. 41

4. Data presentation ................................................................................................. 41

IV. Results ................................................................................................................. 43

1. Blockade of alpha1-adrenoceptors ...................................................................... 45

1.1 Pain behavior test .............................................................................................. 45

1.2 Organ motility test ........................................................................................ 46

1.3 Cystometry ........................................................................................................ 46

1.4 Immunohistochemistry ................................................................................. 47

1.5 Bladder histological evaluation ......................................................................... 49

2. Cross-talk between adrenoceptors and TRPV1 ................................................... 50

2.1 Pain behavior test .............................................................................................. 50

2.2 Cystometry ................................................................................................... 51

3. Cannabinoid system and TRPV1 cross-talk during cystitis ................................ 52

3.1 Cystometries ................................................................................................. 52

3.2 Pain behavior test ......................................................................................... 55

3.3 Immunohistochemistry ................................................................................. 56

V. Discussion ............................................................................................................... 59

VI. Conclusion ........................................................................................................... 67

VII. Future work ......................................................................................................... 71

VIII. References ........................................................................................................... 76

IX. Attachments ......................................................................................................... 86

1. Phenylephrine and antagonist solutions .............................................................. 88

2. Cystometry, tissue harvesting and processing solutions ..................................... 88

3. Immunoreaction against Fos in spinal cord segments ......................................... 89

4. Immunoreaction against caspase 3 in bladder sections ....................................... 90

5. Preparation of solutions for immunoreaction ...................................................... 91

6. Hematoxylin-Eosin staining in bladder sections ................................................. 92

7. Toluidine Blue staining in bladder sections ........................................................ 92

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Table list

Table 1. Experimental group of Wistar rats submitted to chronic adrenergic stimulation

and alpha-adrenoceptors antagonists……………………………………………..…….38

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Figure list

Figure 1. Hunner’s lesion in bladder from patients with BPS/IC, adapted from

ESSIC……………………………………………………………………………….….24

Figure 2. Animals placed in individual chambers to perform visceral pain behavior test

(figure 2a), in lower abdominal region using a series of Von Frey monofilaments (figure

2b)…………………………………………………………………………………..…..38

Figure 3. Animals placed into boxes with filter paper to analyze alterations on bladder

motility……………………………………………………………………………...…..39

Figure 4. Animals under cystometry, over a heating pad and with a 20-gauge needle

inserted in the bladder dome to infuse saline solution……………………………...…..40

Figure 5. Graph bar showing lower abdominal mechanical pain threshold using Von

Frey filaments in control animals, PHE group, PHE +Silodosin group, PHE +Naftopidil

group and PHE +Prazosin group………………………….…………………...……….46

Figure 6. Graph bar showing the voiding frequency (urinary spots/ h) of control

animals, animals submitted to chronic adrenergic stimulation with PHE and animals

submitted to chronic adrenergic stimulation with PHE and α-adrenoceptors

antagonists……………………………………………………………………………...47

Figure 7. Graph bar showing the bladder reflex activity (bladder contractions/ minute)

of control animals and animals submitted to chronic adrenergic stimulation with PHE

and α-adrenoceptors antagonists………………………………………………………..48

Figure 8. Graph bar showing the spinal Fos expression (number of Fos immunopositive

cells/ section) in control animals and animals submitted to chronic adrenergic

stimulation with PHE and α-adrenoceptors

antagonists.......................................................................................................................49

Figure 9. Bladder sections immunoreacted against caspase 3 in control animals and

animals submitted to chronic adrenergic stimulation with PHE and α-adrenoceptors

antagonists……………………………………………………………………………...49

Figure 10. Graph bar showing urothelium integrity (percentage of low urothelium) of

bladder sections stained with Hematoxylin-Eosin………...............................................50

Figure 11. Graph bar showing the mast cells in bladder sections stained with Toluidine

Blue……………………………………………………………………………………..51

Figure 12 Graph bar showing lower abdominal mechanical pain threshold using Von

Frey filaments in WT saline animals and WT and KO TRPV1 animals under chronic

adrenergic stimulation……………………………………………...…………………..52

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Figure 13. Graph bar of bladder reflex activity evaluated during cystometry with saline

and capsaicin in control Wistar rats and in Wistar rats submitted to chronic adrenergic

stimulation with PHE…………………………………………………………………...52

Figure 14. Line graph of bladder reflex activity of control animals and LPS-treated

animals submitted to different concentrations of URB937..…………………………...53

Figure 15. Graph bar of bladder reflex activity of control animals and LPS-treated

animals submitted to URB937 and SR144528…………….…………………………...54


animals submitted to URB937 and MJ15………………….…………………………...55


animals submitted to URB937 and SB366791……………..……..……………………56

Figure 18. Graph bar showing lower abdominal mechanical pain threshold using Von

Frey filaments in control animals, LPS-inflamed animals and animals administrated

with URB937………………………………...…………………………………………57

Figure 19. Graph bar of expression of Fos protein in control animals, LPS-inflamed and

in LPS and URB937..…………………………………………………………………..58

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Abbreviations

ACh – Acetylcholine

APF – Antiproliferative factor

ATP – Adenosine triphosphate

BDNF – Brain derived neurotrophic factor

AEA - Anandamide

BPS - Bladder painful syndrome

BPS/IC – Bladder painful syndrome/ Interstitial cystitis

CAP - Capsaicin

CaMKII – Calmodulin-dependent Kinase II

CB1 – Cannabinoid receptor 1

CB2 – Cannabinoid receptor 2

EEC – European Economic Community

ESSIC – European Society for the Study of Interstitial Cystitis

FAAH – Fatty acid amide hydrolase

GAG-layer – Glycosaminoglycan-layer

IC – Interstitial Cystitis

ICS – International Continence Society

KO – Knockout

LPS – Lipopolysaccharide

L6 – Lumbar 6

NGF – Nerve growth factor

P2X3 – P2X purine receptor

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PBS – Painful bladder syndrome

PHE – Phenylephrine

PIP2 – Phosphothylinositol (4,5)-biphosphate

PKA – Protein kinase A

PKC – Protein kinase C

PLA2 – Phopholipase A2

PLC – Phospholipase C

PLD – Phospholipase D

TH – Tyrosine hydroxylase

TRPV1 – Transient receptor potential vanilloid 1

SP – Substance P

WT – Wild type

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I. Introduction

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I. Introduction

1. Bladder painful syndrome/ Interstitial cystitis (BPS/IC)

1.1 Definition of BPS/IC

Bladder Painful Syndrome/Interstitial cystitis (BPS/IC) is a chronic pathology

characterized by supra-pubic pain related to bladder filling, frequently accompanied by

other lower urinary tract symptoms such as increased daytime and night-time

frequency, in the absence of proven urinary infection or other obvious pathology [1]. It

is a chronic debilitating and multifactorial syndrome with no long-term effective therapy

[1, 2]. The principal symptom of this syndrome is suprapubic pain [3], which might be

felt in other regions rather than the bladder [1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15].

BPS/IC is more predominant in women [1]. BPS/IC has a great impact in the

psychological, social, economic, professional and personal life of patients with this

disease, so much that sometimes small and easy actions like travelling, recreation

activities with family and sleeping become very hard [1, 2].

This syndrome can be divided into two different subtypes: the classic subtype

and the nonclassic/ nonulcerative subtype [7, 11, 12, 16]. They differ in various

aspects, like histopathological characteristics, age distribution (patients with non-

ulcerative form being younger than patients with classic form), complications pattern

and response to treatment [12, 13]. Patients with classic BPS/IC exhibit pelvic pain, an

increase in urinary frequency and urgency and blood in urine [14]. Five to fifty patients

develop Hunner´s lesion (figure 1), which is a “circumscript, reddened mucosal area

with small vessels radiating towards a central scar, with a fibrin deposit or coagulum

attached to this area. With an increase in bladder distension this site can rupture and

lead to an edema” [14, 17]. These lesions can be confirmed by cystoscopy with

hydrodistension under general, epidural or local anesthesia by experienced urologists

and in some cases it may be necessary to make a biopsy to confirm Hunner´s lesion

and exclude a carcinoma [14]. Hunner’s lesion may also be confoundable with

glomerulations. However, the latter are areas of bleeding in the bladder wall and do not

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lead automatically to a diagnosis of BPS/IC [14]. Presently it is still unclear if the non-

ulcer and classic subtypes represent different stages of a single disease or if they are

different diseases entities [14].

Figure 1. Hunner´s lesion in bladder from patients with BPS/IC, adapted from ESSIC (European Society for the study of BPS/IC) in

http://www.essic.eu/video_028images.html.

The designation of this syndrome has changed over the years. Initially, the

syndrome was called Interstitial Cystitis (IC) due to the presence of “typical cystoscopic

and histological features” on the bladder wall, which include inflammation in the deeper

layers of the bladder wall [1, 14, 18]. However, not all patients presented bladder

inflammation signs. Hence, the term IC was progressively changed to Painful Bladder

Syndrome (PBS) evidencing bladder pain instead of inflammation. The International

Continence Society (ICS) define PBS as “the complaint of suprapubic pain related to

bladder filling, accompanied by other symptoms such as increased daytime and night-

time frequency, in the absence of proven urinary infection or other obvious pathology”

[19]. However, this definition excluded 34% of the patients having bladder pain and

being classified by experts to have IC. So, the ESSIC group propose to give it the

name of bladder painful syndrome (BPS). ESSIC defines that “Bladder painful

syndrome would be diagnosed on the basis of chronic pelvic pain, pressure or

discomfort perceived to be related to the urinary bladder accompanied by at least one

other urinary symptom like persistent urge to void or urinary frequency, of more than

six weeks duration, in the absence of infection or other identifiable causes. Confusable

diseases as the cause of symptoms must be excluded. Further documentation and

classification of BPS might be performed according to findings at cystoscopy with

hydrosdistention and morphological findings in bladder biopsies” [14, 20]. However,

http://www.essic.eu/video_028images.html

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omitting the name “Interstitial cystitis” might cause serious problems in different health

systems, so it was decided that the name of Bladder Painful Syndrome/Interstitial

Cystitis could be used during the transition time [14].

Therefore, in this thesis, the term BPS/IC will be used now on, even when

referring to research where the anterior nomenclature was used.

1.2 Etiology of BPS/IC

The etiology of BPS/IC remains unknown, although several hypotheses have

been proposed, mostly linked to a dysfunction in the urothelium: Infection [1, 10, 11,

21, 22], Inflammation [1, 10, 13, 21, 22], Mast cell activation [1, 2, 11, 12, 21, 22, 23,

24], Urothelial dysfunction/ glycosaminoglycan (GAG)-layer defect [1, 2, 10],

Autoimmune mechanisms [1, 10, 13, 21, 22], Toxic agents [1, 22], among others.

1.3 Diagnosis of BPS/IC

The absence of a consensus definition for BPS/IC and the fact that the

symptoms are not specific (symptoms are very similar to other conditions, called

confoundable diseases) difficult the diagnosis of these patients. So, the diagnosis of

BPS/IC is a diagnosis of exclusion and the patients can remain undiagnosed for years

[24]. The diagnosis of BPS/IC, due to the lack of specific markers, is based on

symptoms described by the patients, such as pain, discomfort or pressure, and

exclusion of any identifiable infection [2, 7, 10, 22]. BPS/IC diagnosis also includes

cystoscopy with or without hydrodistention and may include biopsy to analyze mast cell

infiltration [7]. However, a negative result in all of these parameters does not exclude

BPS/IC. Occasionally, some patients do not exhibit abnormalities in the bladder, but

they have other BPS/IC symptoms. So, it is important to exclude all other confoundable

diseases, that could be the causes of the symptoms [5, 7, 22].

Furthermore, not all initial symptoms appear simultaneously but rather gradually

[24]. In the early stage of the disease, symptoms may be mild and intermittent, but they

tend to become constant and severe while the disease progresses [24]. Nocturia, for

example, tends to appear later in the progress of the disease and, commonly, women

report exacerbation of the symptoms during the premenstrual week [24]. Although the

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median age of diagnosis of patients is 42-46 years old [24], the non-ulcer subtype of

BPS/IC is usually associated with younger age at diagnosis [22].

1.4 Epidemiology of BPS/IC

The prevalence of BPS/IC is difficult to estimate because of diagnosis

demands. Hence, the available information about BPS/IC prevalence is based on

different criteria. It is widely accepted that BPS/IC is more frequent in women than in

men, with a female/male ratio that ranges from 5:1 to 10:1 [12, 25]. Also, some studies

show that BPS/IC seems to be more frequent in Caucasians [7, 8, 10, 12, 24, 26].

However, one should not exclude that the cultural difference may reflect the different

frequency observed in those studies. Ito et al. say that BPS/IC is rare in Asian

countries with an incidence of 2 per 100000 urological patients [7, 27]. On the other

hand, the prevalence of BPS/IC in United States is 3-fold greater than the prevalence

in Europe [28]. Looking at the overall population, Moutzouris et al. estimate a

prevalence of 52 to 197 per 100000 women and 40 to 70 per 100000 men, diagnosed

by a physician [24].

1.5 Treatment of BPS/IC

Treatment of BPS/IC is not effective; it aims to reduce the symptoms and

improve the patient´s quality of life, focusing in pain and urological symptoms

(frequency and urgency). Presently, the most used treatments can be divided into four

groups: conservative therapy, oral medication, intravesical therapy and interventional

treatment and surgery [1, 2, 7, 11, 13, 18, 22, 26]. The conservative therapy includes

alterations in the quotidian of patients diagnosed with BPS/IC to improve their life´s

quality [1, 2, 7, 10, 22]. The oral medication includes a large variety of oral therapies,

aiming at reducing symptoms. However, this type of treatments present reduced

efficacy. Intravesical therapy consists on the application of a local anesthetic, alone or

with other drugs, directly into the bladder. Interventional treatment for patients with

BPS/IC include bladder distension, electromotive drug administration, transurethral

resection, botulinum toxin A and hyperbaric oxygen. Surgery is only used as a last

option [1, 2, 7, 10, 12, 14, 22].

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2. Sympathetic nervous system dysfunction in BPS/IC

The relation between BPS/IC and the sympathetic nervous system was never

evaluated. However, it is known that patients with BPS/IC present high levels of urinary

catecholamines [29]. In 1999, Stein et al., showed that patients with BPS/IC have high

levels of urinary catecholamines when compared to urine from normal volunteers [29],

which may diffuse from sprouted sympathetic nerves in the mucosa and the detrusor,

and may contribute to alterations in the permeability of the GAG-layer [23, 29, 30].

Peeker and co-workers explored the neurogenic nature of BPS/IC,

determining by immunohistochemistry the presence and density of nerve fibers

containing tyrosine hydroxylase (TH, a sympathetic fibers marker) [12]. These authors

observed that the number of TH immunoreactive fibers was significantly increased in

BPS/IC patients when compared to control groups, suggesting an increase in the

activity of the peripheral sympathetic nervous system, and therefore indicating a

primary etiology [11, 12].

BPS/IC patients often exhibit an exaggerated response to stress, as observed

in patients with anxiety disorders [30]. This can be related with dysfunction of the

sympathetic nervous system [30, 31]. Furthermore, Charrua and co-workers

demonstrated through the TILT test (tilt table test) that BPS/IC patients presented an

increased sympathetic activity and confirmed an increase in noradrenaline levels in the

urine of BPS/IC patients [9, 29, 30]. These observations presume overstimulation of

adrenergic receptors present in the urinary bladder. The urinary bladder expresses

different subtypes of α-adrenoceptors and β-adrenoceptors. The most abundantly

expressed are the α1A, α1D and β3 subtypes. The α1A subtype is expressed in the

bladder urothelium and body [32, 33]. Curiously, it is thought that the α1A adrenoceptor

is activated only when an excess of norepinephrine exists in the urine or in the blood

[32]. The α1D subtype is expressed in the detrusor muscle, in the urothelium, and has

almost exclusively intracellular expression [32, 34, 35]. Both α- and β-adrenoceptors

are involved in mediating the responses to the endogenous noradrenaline [36]. β3

subtype is mainly expressed in the urothelium and when stimulated by mirabegron, a

β3 –adrenoceptor agonist, it decreases detrusor contractility and the urinary frequency.

So, the β3 subtype seems to not be involved in bladder pain or any change in bladder

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morphology [36]. Therefore, the present study will focus on α-adrenoceptors, namely

on α1A and α1D.

2.1. Nociceptive pathway modulation by adrenoceptors

The overstimulation of adrenoceptors, in an animal model of rats chronically

treated with phenylephrine, leads to pain, vesical hyperactivity, alterations in urothelium

and mastocytosis, all signs and symptoms observed in patients with BPS/IC [30]. Pain

and increased void frequency were dependent on capsaicin-sensitive primary afferents

activation since desensitization of these fibers with high doses of capsaicin reverses

the phenylephrine-mediated effects [37, 38], demonstrating a possible relation between

the adrenergic and nociceptive activity. Hence, one of the aims of the present study is

to demonstrate the involvement of adrenoceptors in the appearance of BPS/IC

symptoms and that these receptors cross-talk with nociceptive receptors. In this study

we will focus on TRPV1, a receptor expressed in afferent bladder nerves, forming a

close contact with the bladder epithelium, in urothelial cells and in myofibroblasts [37,

38, 39, 40]. It has been suggested that TRPV1 is overexpressed in the bladder of

patients with BPS/IC [39, 41, 42]. Furthermore, it has been shown that the TRPV1

receptor is essential for pain arousal and for an increase in bladder frequency during

cystitis [43, 44].

One possible mechanism through which the adrenoceptor could act over TRPV1

is by an intracellular mechanism that will lead to its later sensitization. TRPV1 can be

sensitized through phosphorylation by different kinases, such as protein kinase A

(PKA), protein kinase C (PKC) and calmodulin-dependent kinase II (CaMKII), or by

membrane lipids such as phosphothylinositol (4,5)-biphosphate (PIP2), which is

cleaved by phospholipase (PLC) [45]. All these enzymes can be activated upon

adrenoceptors stimulation.

Another possible mechanism is that adrenoceptor activity can indirectly modulate

TRPV1 activity by acting on other receptors. Cannabinoid receptors are one example

[46]. The expression of cannabinoid receptor 1 (CB1), which co-localizes with TRPV1,

increases in patients with chronic bladder pain syndromes [47]. Furthermore, systemic

administration of cannabinoid agonists reduced pain induced by bladder inflammation

and increased the voiding interval [47], a mechanism that involves inhibition of bladder

afferent nerves [48]. TRPV1 and CB1 have common agonists, such as anandamide

[46, 49]. Anandamide is a lipid, which is an endocannabinoid, and can have analgesic

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effects in animal models of inflammatory pain [49, 50]. Anandamide can be degraded

by FAAH (fatty acid amide hydrolase), an enzyme present in the human and rat

urothelium [50, 51, 52]. It is thought that when administrated at low doses, anandamide

acts on CB1, but at high doses, anandamide acts on TRPV1 [46, 47]. Although the

inhibition of FAAH by URB597, an inhibitor of FAAH, has been referred to suppress

peripheral hyperalgesia induced by bladder inflammation [50], no studies have been

conducted in BPS/IC.

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II. Aims

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II. Aims

This work has three main goals:

The first goal is to demonstrate the involvement of adrenoceptors in the

appearance of BPS/IC symptoms, by characterizing the adrenoceptors subtypes

involved in the appearance of pain and organ motility changes and by characterizing

which adrenoceptor subtypes are involved in the bladder histological changes

observed in rats submitted to chronic adrenergic stimulation.

The second aim is to evaluate the cross-talk between adrenoceptors and

TRPV1 during chronic adrenergic stimulation.

The third aim is to understand how endocannabinoids modulate bladder pain

and hyperactivity during cystitis.

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III. Material and Methods

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III. Material and Methods

1. Experimental Animals

Adult female Wistar rats (175-200g) and adult C57BL/6J mice (20-25g) were

bought from Charles River Laboratories (Barcelona, Spain). TRPV1 Knockout (KO)

mice (20-25g) were bought from Jackson Laboratories (Bar Harbor, Maine, USA). All

animals were maintained at 22 ᵒC, with 60% humidity, under inverted light-dark cycle

(12h/12h), and provided with water and food ad libitum. All experiments were

performed according to the European Communities Council Directive of 24 November

1986 (86/609/EEC), after approval from Ethical Committee of the Faculty of Medicine

of University of Porto and from Direcção Geral de Alimentação e Veterinária.

2. Chronic adrenergic stimulation

Chronic adrenergic stimulation was induced in mice and rats, by performing

daily subcutaneous injections, for 14 days, with 2.5 mg phenylephrine (PHE)/Kg body

weight (Sigma-Aldrich).

2.1. Blockade of alpha1-adrenoceptors

For α1-adrenoceptor subtype blockade, rats orally received specific α1-

adrenoceptor subtypes antagonists, concomitantly with the daily chronic adrenergic

stimulation (n=6 for each group) (see table 1). Rats receiving vehicle were used as

negative controls (group A). Rats receiving phenylephrine were used as positive control

(group B) (see attachment 1).

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Table 1. Experimental groups of Wistar rats submitted to chronic adrenergic stimulation and alpha-adrenoceptors antagonists.

Group Treatment Dose

C α1A-AR antagonista (Silodosin (Urorec®, Recordati) 0.2 mg/ Kg

D α1D-AR antagonist (Naftopidi, *) 0.9375 mg/ Kg

E α1-AR antagonist (Prazosin, Sigma-Aldrich) 0.05 mg/ Kg

*Naftopidil was a kind gift from Prof. Igawa from Department of Continence Medicine, University of Tokyo, Tokyo, Japan

2.1.1. Pain behavior test

Visceral pain behavior tests were performed before and 14 days after beginning

the treatment, in all animals from all groups and treatments used.

Visceral pain behavior was evaluated using a mechanical hyperalgesia test,

adapted from Laird et al., 2001 [53]. Animals were placed in individual chambers (230x

170x 140 mm) with a wire mesh floor and allowed to acclimate for 20 min or until cage

exploration stopped (Figure 2a). This test was performed in the lower abdominal

region, and consists in touching this region with one of a series of Von Frey

monofilaments (rated at 4, 6, 8, 10, 15, 26 and 60g for rats, or rated 0.008, 0.02, 0.04,

0.07 and 0.16g for mice). Filaments were applied 5 seconds perpendicularly with

enough strength to cause the monofilament to slightly bend (see figure 2b). Each

monofilament was tested 5 times with an interval of 30 seconds between

monofilaments. It was considered a positive response when the animal reacted to the

filament (abdomen retraction or jump) in at least three of the five filament applications.

In case of no response to the filament, the next-stronger monofilament was applied.

Figure 2. Animals placed in individual chambers to perform visceral pain behavior test (figure 2a), in the lower abdominal region

using a series of Von Frey monofilaments (figure 2b), according to Laird et al., 2001 [53].

A

B

B

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2.1.2. Organ motility test

Organ motility tests were also assessed before and 14 days after beginning the

treatment, in all animals from all groups and treatments used.

Urinary spots were used to analyze voiding frequency (urinary spots/ h). For

this, the animals were placed in boxes with filter paper during one hour (figure 3).

Figure 3. Animals placed into boxes with filter paper to analyze alterations on bladder motility.

2.1.3. Cystometry

In animals submitted to chronic adrenergic stimulation with PHE, cystometries

were performed at day 15 after beginning the treatment with PHE alone or with each

antagonist of α1-adrenoceptor subtypes, under anesthesia with urethane (1.2 g/ Kg

body weight, subcutaneously), with the body temperature maintained at 37ᵒC with a

heating pad. The bladder dome was exposed through a small, low abdominal incision.

A 20 gauge needle was inserted in the bladder dome and saline solution was infused

(6 mL/ h) (figure 4). After a period of 30 minutes of stabilization, the infusion of saline

solution was recorded for 2 hours, to analyze bladder reflex activity (number of bladder

contractions/ minute).

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Figure 4. Animals under cystometry, over a heating pad and with a 20 gauge needle inserted in the bladder dome to infuse a saline solution.

2.1.4. Tissue harvesting and processing

Two hours after the beginning of cystometry, animals were transcardially

perfused with Tyrode´s solution followed by paraformaldehyde 4% (see attachment 2).

L6 spinal cord segments were harvested and sectioned at 40 µm. The urinary bladders

were harvested, fixed by immersion in 10% buffered formalin, for 24 hours, embedded

in paraffin, sectioned at 10-12 µm and mounted on poly-L-lysine-coated slip.

2.1.5. Immunohistochemistry

L6 spinal cord segments were sectioned and immunoreacted against Fos

protein, a pain and neuronal activity marker (see attachment 3 and 5 for a more

detailed protocol). The number of Fos immunopositive cells were determined under the

light microscopy Zeiss Axioskop 40.

Bladder sections were immunoreacted against caspase 3, a marker of cellular

apoptosis, to analyze urothelium integrity (see attachment 4 and 5 for a more detailed

protocol). Slides were observed under fluorescence microscope Zeiss Apotome and

images were acquired.

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2.1.6. Bladder Histological Evaluation

To assess urothelium integrity, bladder sections were stained for Hematoxylin-

Eosin (see attachment 6 for a more detailed protocol) and the thickness of the

urothelium and the extension of lesion was determined using AxioVision 4.0.

To analyze mast cell infiltration, bladder sections were stained with Toluidine

Blue (see attachment 7 for a more detailed protocol). Images were acquired and the

number of mast cells was counted. Bladder mucosa areas were determined using

ImageJ 1.47v software.

2.2 Cross-talk between adrenoceptors and TRPV1

To evaluate the cross-talk between α-adrenoceptor and TRPV1, TRPV1

knockout mice (n=6) were submitted to chronic adrenergic stimulation with

phenylephrine for 14 days. Furthermore, a control group with wild type mice injected

with saline solution and a group of wild type mice injected with phenylephrine were

used (n=6 for each group). Pain behavioral tests (as described in 2.1.1) were

performed.

To verify if chronic adrenergic stimulation enhances TRPV1 response, adult

female Wistar rats (n=6) were submitted to chronic adrenergic stimulation with

phenylephrine daily for 14 days. At the end of treatment, animals were submitted to a

cystometry (as described in 2.1.3). After saline infusion, 50µM capsaicin (TRPV1

agonist, Sigma-Aldrich) was infused. This procedure was repeated in another group of

four animals injected with saline, used as control group (n=4).

3. Cannabinoid system and TRPV1 cross-talk during cystitis

3.1 LPS-induced cystitis

Cystitis was induced by intravesical instillation of 0.5 mL of lipopolysaccharide

(LPS, Sigma-Aldrich) 5 mg/ mL, for 1h, in female Wistar rats (n=6 for each drug

application). Control animals were instilled with saline solution (n=6).

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3.2 Pain behavior test

Visceral pain behavior tests (as described in 2.1.1) were performed before

inflammation, 24h after LPS instillation and after 1 µM URB937 injection.

3.3 Cystometries

Twenty-four hours after LPS or saline instillation (n=6 for each drug application),

cystometry was performed as described in 2.1.3. After a period of 30 minutes of

stabilization, URB397 (Cayman Chemical Company) was given intravenously in doses

of 0.01, 0.1, 1, 5 and 10 µM (cumulative doses) to LPS- and saline-treated animals.

Since the dose most effective to reduce bladder reflex activity induced by

cystitis was 1µM of URB, it was used 1µM of URB937 + 10 µM MJ15 (CB1 antagonist,

Tocris Bioscience), 1µM URB937 + 0.3 mg/ Kg SR144528 (CB2 antagonist, Cayman

Chemical Company) to determine the involvement of each cannabinoid receptor in

FAAH induced blockade.

Since 7 mg/kg induced bladder hyperactivity, 1µM URB937 + 1.4 µg/ Kg

SB366791 (TRPV1 antagonist, Tocris Bioscience) were used to analyze the

involvement of TRPV1.

At the end of the cystometry, bladders were harvested and the cystitis was

confirmed histologically.

4. Data presentation

Data are presented as mean ± standard deviation and comparison between the

paired data were performed by using the Student´s t test. For multiple comparisons, the

statistical analysis was performed using the Dunn’s Multiple Comparisons Test

(ANOVA – One-way analysis of variance non-parametric). P values inferior to 0.05

were considered statistically significant.

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IV. Results

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IV. Results

1. Blockade of alpha1-adrenoceptors


Control animals presented a score of 41.2 ± 21. It was observed that animals

treated with PHE had a decreased mechanical pain threshold compared with the

control, with a score of 6.7 ± 2.4 (P<0.001, figure 5). The blockade with Silodosin or

Prazosin reversed the effect of PHE to 20.8 ± 19.3 and 14.2 ±7.0, respectively.

Naftopidil was unable to reverse PHE induced referred pain, 6.0 ± 2.5 (P<0.001, figure

5).

Figure 5. Graph bar showing lower abdominal mechanical pain threshold using Von Frey filaments. After chronic adrenergic

stimulation with 2.5 mg of PHE/kg body weight, animals presented a decrease in the mechanical pain threshold (*** P < 0.001 comparing to control). Co-treatment with silodosin and prazosin abolished abdominal mechanical pain threshold. Naftopidil was

unable to reverse PHE effect (*** P<0.001).The statistical analysis were performed using the Dunn’s Multiple Comparisons Test

(ANOVA – One-way analysis of variance non-parametric).

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

Control PHE PHE +Silodosin

PHE +Naftopidil

PHE +Prazosin

Fila

me

nt

(g)

Visceral pain test

***

***

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1.2 Organ motility test

Control animals had 2.8 ± 1.0 urinary spots/ h. PHE treatment induced an

increase in the number of urinary spots/ h to 7.8 ± 3.3, when compared to control group

(P<0.05, figure 6). In animals submitted to treatment with PHE concomitantly with

Silodosin or Prazosin the number of urinary spots/ h is similar to control animals, 4.8 ±

3.3 and 4.3 ± 4.6 urinary spots/h, respectively (figure 6). Animals treated with Naftopidil

had an increase in the number of urinary spots/h to 9.2 ± 2.6 (P<0.01) (figure 6).

Figure 6. Graph bar showing the voiding frequency (urinary spots/ h) of control animals, animals submitted to chronic adrenergic stimulation with PHE (PHE group) and animals submitted to chronic adrenergic stimulation with PHE and α-adrenoceptors

antagonist (group PHE+ Silodosin, PHE+ Naftopidil and PHE+ Prazosin). The statistical analysis were performed using the

Dunn’s Multiple Comparisons Test (ANOVA – One-way analysis of variance non-parametric).

1.3 Cystometry

Control animals had 0.4 ± 0.1 bladder contractions/ min. PHE treated animals

had an increased bladder reflex activity, reading values of 1.9 ± 0.9 bladder

contractions/min (P<0.001, figure 7). Adrenoceptors antagonists Silodosin or Prazosin

were able to reverse PHE-induced bladder reflex activity to values of 0.7 ± 0.2 and 0.7

± 0.2 bladder contractions/ min, respectively (figure 7). Co-treatment with Naftopidil

also increased bladder reflex activity to 1.9 ± 0.4 bladder contractions/ min (P<0.001)

(figure 7).

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0


PHE +Naftopidil

PHE +Prazosin

Uri

nar

y sp

ots

/ h

Urinary spots

*

**

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Figure 7. Graph bar showing the bladder reflex activity (bladder contractions/ minute) of control animals, animals submitted to

chronic adrenergic stimulation with PHE (PHE group) and animals submitted to chronic adrenergic stimulation with PHE and α-adrenoceptors antagonist (group PHE+ Silodosin, PHE+ Naftopidil and PHE+ Prazosin). The statistical analysis were performed

using the Dunn’s Multiple Comparisons Test (ANOVA – One-way analysis of variance non-parametric).

1.4 Immunohistochemistry

The L6 spinal cord segment of control animals had 5 ± 3 immunopositive cells.

PHE treated animals had an increase in the number of Fos immunopositive cells/

section to 33 ± 6 (P<0.01, figure 8). The blockade with Silodosin or Prazosin reverses

the effect induced by PHE to 8 ± 3 and 4 ± 0.5, respectively. Blockade with Naftopidil

also increased the number of Fos immunoreactive cells for 21 ± 7 (P<0.05) (figure 8).

0,00

0,50

1,00

1,50

2,00

2,50

3,00


PHE +Naftopidil

PHE + Prazosin

Bla

dd

er

con

trac

tio

ns/

min

Bladder reflex activity

***

***

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Figure 8. Graph bar showing the spinal Fos expression (number of Fos immunopositive cells/ section). Chronic adrenergic

stimulation with 2.5 mg PHE/ kg body weight, increase the number of Fos immunopositive cells/ section (** P<0.01).Co-treatment with Silodosin or Prazosin reverse the effect induced by PHE. Treatment with Naftopidil also increase spinal Fos expression. The

statistical analysis were performed using the Dunn’s Multiple Comparisons Test (ANOVA – One-way analysis of variance non-

parametric).

.

Bladder sections from control animals showed no staining for caspase 3 (figure

9A). The bladder of animals that received chronic adrenergic stimulation presented

caspase 3 staining in the urothelium (figure 9B). Naftopidil induced cellular apoptosis,

as observed by caspase 3 staining (figure 9D). Bladder of animals that received

Silodosin or Prazosin, were not stained for caspase 3 (figure 9C and 9E, respectively).

Figure 9. Bladder sections immunoreacted against caspase 3. A) control group. B) PHE group. C) PHE and Silodosin. D) PHE and

Naftopidil. E) PHE and Prazosin.

0

5

10

15

20

25

30

35

40

45

Control PHE PHE+Silodosin

PHE+Naftopidil

PHE+Prazosin

Nº

of

Fos

IR+

cells

/se

ctio

n

Spinal Fos expression

A B

C

D E

**

*

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1.5 Bladder histological evaluation

Bladder sections from controls stained for Hematoxylin-Eosin exhibited an

urothelium without disruption. Treatment with PHE and antagonists (Naftopidil or

Prazosin) increased the percentage of low urothelium to 53% ± 15, 82% ± 14 and

46.9% ± 17,7, respectively (P<0.001, figure 10). Adrenoceptor antagonist Silodosin

reversed the effect of PHE for 12% ± 13 (figure 10).

Figure 10. Graph bar showing the urothelium integrity (percentage of low urothelium) of bladder sections stained with

Hematoxylin-Eosin. PHE, PHE +Naftopidil and PHE +Prazosin increase the percentage of low urothelium (*** P<0.001). Administration of Silodosin or Silodosin + Naftopidil reverses the effect induced by PHE. The statistical analysis were performed


Bladder sections from control animals stained for Toluidine Blues presented 8 ±

4 mast cells/ mm2 (figure 11). Treatment with PHE increased the number of mast cells

in mucosa layer to 17 ± 6 (P<0.05, figure 11). Co-administration of PHE and Naftopidil

also increased the number of mast cells/ mm2 to 20 ± 5 (P<0.01, figure 11).

Adrenoceptors antagonists Silodosin or Prazosin reversed the effect of PHE to values

of 14 ± 4 and 16 ± 8, respectively (figure 11).

0,0

20,0

40,0

60,0

80,0

100,0


PHE +Naftopidil

PHE +Prazosin

Pe

rce

nta

ge o

f lo

w u

roth

eliu

m

Urothelium Integrity

***

***

***

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Figure 11. Graph bar showing the mast cells (mast cells/ mm2) in bladder sections stained with Toluidine Blue. PHE increase the

number of mast cells (* P<0.05).Co-treatment with Naftopidil also increase the number of mast cells (** P< 0.01). Administration

of Silodosin or Prazosin reverses the effect induced by PHE. The statistical analysis were performed using the Dunn’s Multiple Comparisons Test (ANOVA – One-way analysis of variance non-parametric).

2. Cross-talk between adrenoceptors and TRPV1


Control animals (WT saline) had a score of 0.09 ± 0.06 (figure 12). Chronic

adrenergic stimulation in wild type animals decreased mechanical pain threshold

compared to control to 0.008 ± 0.0 (P<0.05, figure 12). Administration of PHE to

knockout (KO) TRPV1 animals increased the pain threshold to values similar to those

of the control group (figure 12).

0

5

10

15

20

25

30

control PHE PHE +Silodosin PHE +Naftopidil PHE +Prazosin

Mas

t ce

lls/

mm

2

Mast cells

*

**

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Figure 12. Graph bar showing lower abdominal mechanical pain threshold using Von Frey filaments. PHE administration to WT animals decrease pain threshold (*P<0.05). The statistical analysis were performed using the Dunn’s Multiple Comparisons Test

(ANOVA – One-way analysis of variance non-parametric).

2.2 Cystometry

Control animals under cystometry presented a value of 0.58 ± 0.21 bladder

contractions/ minute. Administration of 50 µM of capsaicin to these animals increased

the number of bladder contractions to 0.90 ± 0.18, presenting a variation of 0.33 ± 0.15

(figure 13). Animals treated with PHE had 1.73 ± 1.02 bladder contractions/ minute

(figure 13). Capsaicin administration increased the number of bladder contractions to

2.68 ± 1.28, when compared to animals treated with PHE (figure 13), presenting a

variation of 0.95 ±0.37 (figure 13).

Figure 13. Graph bar of bladder reflex activity (bladder contractions/ minute) evaluated during cystometry with saline and capsaicin

(CAP) in control Wistar rats and in female Wistar rats submitted to chronic adrenergic stimulation with PHE.

0,00

0,05

0,10

0,15

0,20

WT saline WT PHE KO TRPV1 PHE

Fila

me

nt

(g)

Visceral pain test

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

Control PHE

DFr

eq

ue

ncy


*

*

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3. Cannabinoid system and TRPV1 cross-talk during cystitis

3.1 Cystometries

Control animals presented 0.5 ± 0.1 bladder contractions/ minute. Animals with

cystitis presented an increase in the number of bladder contractions/ minute to 2.1 ±

0.6 (figure 14). 1µM of URB937 reversed the bladder hyperactivity (0.7 ± 0.2 bladder

contractions/ minute in LPS-treated animals, figure 14). 5 µM and 10 µM of URB937

increased bladder hyperactivity (1.3 ± 0.4 and 2.4 ± 0.8 bladder contractions/ minute,

respectively, in LPS-treated animals, figure 14).

Figure 14. Line graph of bladder reflex activity of control animals and LPS-treated animals submitted to different concentrations of URB937.

Control animals instilled with vehicle had 0.5 ± 0.1 bladder contractions/ minute.

Administration of URB937 1µM alone or concomitantly with CB2 antagonist SR144528

to control animals did not change the bladder reflex activity (0.5 ± 0.2 and 0.4 ± 0.1,

respectively, figure 15).

Animals with cystitis (n=6) had 0.9 ± 0.1 bladder contractions/ minute.

Administration of URB937 alone or co-administrated with CB2 antagonist SR144528 in

these animals did not change significantly the number of bladder contractions (0.7 ±

0.2 and 0.55 ± 0.07, respectively) (figure 15).

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

saline 0.01 µM 0.1 µM 1 µM 5 µM 10 µM

Bla

dd

er

con

trac

tio

ns/

min

URB (mM)


LPS

control

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Figure 15. Graph bar of bladder reflex activity of control animals and LPS-treated animals submitted to URB937 and SR144528 (CB2 antagonist). Administration of URB937 alone or with CB2 antagonist decrease the number of bladder contractions/ minute.

The statistical analysis were performed using the Dunn’s Multiple Comparisons Test (ANOVA – One-way analysis of variance non-

parametric).

Control animals instilled with vehicle had 0.5 ± 0.1 bladder contractions/ minute

(figure 16). Administration of URB937 1µM alone or concomitantly with CB1 antagonist

MJ15 to these animals did not change the bladder reflex activity (0.5 ± 0.2 and 0.6 ±

0.2, respectively, figure 16).

Animals with cystitis (n=6) had 1.42 ± 0.23 bladder contractions/ minute

(P<0.001 compared to control). Administration of URB937 in these animals decreased

the number of bladder contractions to 0.7 ± 0.2 (figure 16). Animals administrated with

URB and the CB1 antagonist (MJ15) had an increase in bladder reflex activity to 1.9 ±

0.8 (P<0.01) compared with LPS-inflamed animals instilled with vehicle (figure 16).

0,0

0,2

0,4

0,6

0,8

1,0

Vehicle URB 1uM URB 1uM +SR144528(CB2 antagonist)

Bla

dd

er

con

trac

tio

ns/

min


Naive animals

LPS-inflamedanimals

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Figure 16. Graph bar of bladder reflex activity of control animals and LPS-inflamed animals submitted to URB937 and MJ15 (CB1

antagonist). LPS-inflamed animals have an increase in the number of bladder contractions, when compared to control (*** P<0.001). Administration of URB937 in LPS-inflamed animals reduces the number of bladder contractions/ minute. Co-

administration of URB937 and CB1 antagonist increase bladder reflex activity (** P<0.01). The statistical analysis were performed


Control animals (n=3) instilled with vehicle had 0.4 ± 0.2 bladder contractions/

minute (figure 17). Administration of URB937 1 µM alone or concomitantly with TRPV1

antagonist SB366791 did not change the bladder reflex activity (0.4 ± 0.2 and 0.4 ± 0.2,

respectively, figure 17).

Animals instilled with LPS (n=3) and vehicle had 1.2 ± 0.2 bladder contractions/

minute. Administration of URB937 on animals with cystitis increased the number of

bladder contractions to 2.4 ± 0.8 (figure 17). Co-administration of URB937 and TRPV1

antagonist SB366791 in LPS-inflamed animals decreased the number of bladder

contractions/ minute to 1.3 ± 0.6 (figure 17).

0,0

0,5

1,0

1,5

2,0

2,5

3,0

Vehicle URB 1uMURB 1 uM +MJ15 (CB1 antagonist)

Bla

dd

er

con

trac

tio

ns/

min


Naive animals

LPS-inflamedanimals

***

**

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Figure 17. Line graph of bladder reflex activity of control animals and LPS-inflamed animals submitted to URB937 and SB366791

(TRPV1 antagonist). Administration of URB937 in LPS-inflamed animals increases the number of bladder contractions/ minute. Co-administration of URB937 and TRPV1 antagonist in LPS-inflamed animals decrease the number of bladder contractions/

minute. The statistical analysis were performed using the Dunn’s Multiple Comparisons Test (ANOVA – One-way analysis of

variance non-parametric).


Control animals presented a score of 18 ± 7. Animals with cystitis had a

decreased mechanical pain threshold compared with the control, with values of 1 ± 1

(P<0.001, figure 18). Administration of 1µM of URB937 increased the pain threshold to

7 ± 3 (figure 18), when compared to control group.

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

Vehicle URB 10 uM URB 10uM+SB366791 (TRPV1

antagonist)

Bla

dd

er

con

trac

tio

ns/

min


naive animals

LPS- treated animals

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Figure 18. Graph bar showing lower abdominal mechanical pain threshold using Von Frey filaments. Instillation of LPS decreases

the pain score (*** P<0.001). Administration of URB937 increased pain threshold. The statistical analysis were performed using the

Dunn’s Multiple Comparisons Test (ANOVA – One-way analysis of variance non-parametric).

3.3 Immunohistochemistry

Control animals had 5 ± 3 Fos immunopositive cells/ section (figure 19).

Instillation of LPS increased the number of Fos immunopositive cells/ section to 31 ±

12 (P<0.01, figure 19).

Animals instilled with LPS and administrated with URB937 had a decrease in

the number of Fos immunopositive cells/ section to 15 ± 8 (P<0.05, figure 19).

0

5

10

15

20

25

30

Control LPS URB

Fila

me

nt

(g)

Visceral pain test ***

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Figure 19. Graph bar of expression of Fos protein in control animals, LPS-inflamed animals and in LPS+1µM URB937. Instillation

of LPS increase the number of Fos immunopositive cells, when compared to control (** P<0.01). Animals instilled with LPS and administrated with URB937 had a decrease in the number of Fos immunopositive cells, when compared to LPS-inflamed animals (*

P<0.05). The statistical analysis were performed using the Dunn’s Multiple Comparisons Test (ANOVA – One-way analysis of

variance non-parametric).

0

5

10

15

20

25

30

35

40

45

Control LPS LPS+ 1 uM URB

Nu

mb

er

of

Fos-

exp

ress

ing

cells

Expression of Fos protein

** *

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V. Discussion

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V. Discussion

The major outcomes of the present work were:

1. Alpha1A-adrenoceptors mediate pain induced by chronic adrenergic

stimulation;

2. Chronic adrenergic stimulation-mediated effects occur through a TRPV1-

dependent mechanism, which involves the receptor sensitization;

3. Endocannabinoids modulate visceral pain and bladder activity, through CB1

and TRPV1 receptor, during cystitis.

1. Alpha1A-adrenoceptors mediate pain induced by chronic adrenergic

stimulation

The present experiments confirm that chronic adrenergic stimulation with PHE

induces visceral pain behavior in animals and bladder hyperactivity, which are a

characteristic exhibited by BPS/IC patients [30]. Administration of PHE and Silodosin

(α1A-adrenoceptor antagonist) reversed visceral pain behavior and reversed the

bladder hyperactivity induced by PHE. It is known that urinary bladder express alpha-

adrenoceptors [54, 55, 56, 57, 58, 59]. The α1-adrenoceptors can be found throughout

the urinary bladder [34, 35, 60]. In the bladder of healthy individuals, there is no

evidence of an outstanding alpha-adrenergic stimulation. However, under pathologic

conditions, it is known that there is an increase in the density of α-adrenoceptors,

insomuch, that the noradrenaline-induced response in the bladder is converted from

relaxation to contraction [54], which can contribute to the bladder hyperactivity

observed in some disorders like BPS/IC.

In the present work, the animals under chronic adrenergic stimulation also

presented an increase in spinal Fos protein expression, indicating that PHE induces

activation of nociceptive pathways coming from the bladder. Patients with BPS/IC

present an increase both in nociceptive fibers containing substance P (SP) and in

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urinary substance P [2, 61, 62]. This increase has a positive correlation with the

severity of pain felt by those patients [2]. The reduction of Fos expression by the

blockade of α1A-adrenoceptor suggest that this receptor alters the activity of primary

afferents. Its blockade may lead to a decrease the transmission of nociceptive

information to central nervous system, which could result in a decrease in pain and

vesical activity.

Chronic adrenergic stimulation with PHE induced mast cell infiltration, a sign

similar to the one observed in patients with BPS/IC [1, 2, 6, 9, 26]. This could be a

consequence of nociceptive fibers activation since the release of SP is known to cause

degranulation and activation of mast cells present nearby nerve terminals [2]. The

blockade of α1A-adrenoceptor inhibited mast cell migration induced by mast cell

activation, showing that this receptor is somehow involved in the chemotaxis of these

inflammatory cells. Hence, α1A-adrenoceptor blockers present themselves as good

targets to overcome pain and inflammation associated with BPS/IC.

Chronic adrenergic stimulation also induced urothelium disruption as observed

by an increase in the percentage of degraded urothelium and the immunoreactivity for

the pro-apoptotic molecule caspase 3. Patients with BPS/IC exhibit disruption in the

urothelium similar to the ones observed in the present work [1, 2, 6, 9, 23]. In fact, it

was observed that the urothelium from BPS/IC patients was immunopositive for

caspase 3 [63, 64]. Charrua et al. has observed that the urothelium from animals

submitted to chronic adrenergic stimulation with PHE was immunopositive for caspase

3 [30]. The blockade of α1A-adrenoceptor reversed the urothelium damage induced by

PHE, possible by blocking apoptosis, showing that this effect resulted from adrenergic

overactivity. The urothelium has a barrier function, to prevent the diffusion of urine

constituents for the underlying tissues [14, 65]. Urothelial damage allows the diffusion

of urinary solutes which may activate sensory nerve endings, leading to pain,

inflammation and urinary frequency. Hence, the reversion of urothelium damage will

possible contributes to an improvement of all other symptoms observed in BPS/IC

patients. In fact, patients that receive pentosan polysulfate sodium, known to decrease

the permeability of urothelium, reported an improvement of pain, urgency and

frequency [1, 7, 13, 18, 22].

Chronic adrenergic stimulation with phenylephrine increased bladder reflex

activity, as previous demonstrated by Charrua et al. [30]. The increase in bladder reflex

activity can be translated as an increase in the number of bladder contraction per

minute, which means that the urinary frequency increases. This increase in urinary

frequency is also reported in patients with BPS/IC [1, 2, 4, 22]. The blockade of α1A-

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adrenoceptor reverses the increase in bladder reflex activity, which means that, under

pathologic conditions, this receptor is involved in the increase of urinary frequency.

2. Chronic adrenergic stimulation-mediated effects occur through a TRPV1-

dependent mechanism, which involves the receptor sensitization

Pain and increased frequency were dependent of capsaicin sensitive primary

afferents activation since desensitization of these fibers with capsaicin reverses the

phenylephrine mediated effect [40, 41]. These results indicate that there is relation

between adrenergic activity and nociceptive activity. TRPV1 has been shown to be

essential for the development of bladder pain and hyperactivity during cystitis [38, 43,

46] being the urinary bladder nociceptive receptor per excellence [37, 38, 39, 63]. In

fact, TRPV1 is overexpressed in the urinary bladder of patients with BPS/IC [39, 41,

42].

Hence, in this work it was hypothesized that adrenoceptors interact with TRPV1

inducing the PHE-mediated effects. In fact, TRPV1 knockout mice chronically treated

with PHE did not feel pain nor presented any of the bladder changes observed in their

wild type littermates. These results show that TRPV1 activity is downstream to α-

adrenoceptors activation.

It was also hypothesized that adrenoceptor sensitized TRPV1 by an intracellular

mechanism. In the present work it was observed that PHE-treated animals had higher

bladder frequency upon capsaicin stimulation than control animals. This seems to point

to a sensitization of TRPV1. In fact, α-adrenoceptors activation is known to trigger the

Gq/11 signaling pathway, therefore promoting the activation of phospholipase C (PLC),

phospholipase A2 (PLA2) and phospholipase D (PLD) [37, 38]. In turn, these enzymes

lead to mobilization of Ca2+ from intracellular stores and activation of mitogen-activated

kinase and phosphathylinositol (4,5)-biphosphate (PIP2) kinase pathways, which

causes vasoconstriction. The influx of calcium and the consequential increase of

intracellular calcium concentration are necessary for the desensitization of TRPV1.

Many signaling pathways, like calmodulin (CaM) and the decrease of PIP2, are

implicated in desensitization of TRPV1 [37, 66]. The process of sensitization of TRPV1

is made by phosphorylation, by different kinases, like protein kinase A (PKA), protein

kinase C (PKC) and calmodulin-dependent kinase II (CaMKII), or by membrane lipids

such as PIP2, which is cleaved by phospholipase (PLC). It is known that TRPV1 needs

to be phosphorylated by CaMKII to be activated by vanilloids [67]. All these enzymes

are known to sensitize TRPV1 [68].

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3. Endocannabinoids modulate visceral pain and bladder activity, through CB1

and TRPV1 receptor, during cystitis

The administration of URB937 did not produce any effect on the bladder

frequency nor pain behavior of control animals. Furthermore, administration of URB937

concomitantly with exogenous cannabinoids, such as SR144528, MJ15 or SB366791,

also did not produce any effect on bladder frequency.

The administration of 1 mM URB937 to animals with cystitis reversed bladder

hyperactivity. However, at doses of 5 and 10 mM URB937 induced an increase of

bladder frequency. FAAH blockade increases endocannabinoids levels [49].

Anandamide (AEA) is one of those molecules. AEA is known to act both in CB1 and

TRPV1 receptors, which are co-expressed in most nociceptive primary sensory

neurons in bladder [46, 47].

In fact, previous work has demonstrated that anandamide could contribute to

the development of hyperalgesia and hyperreflexia by activating TRPV1 [46]. Hence, it

was hypothesized that when low doses of URB are used, endocannabinoids levels may

reach a plateau that lead to CB1 activation. Increasing endocannabinoids levels by

further blocking FAAH, they will act on TRPV1. In fact, the reversion of 1 mM URB

effect by blocking CB1 receptor demonstrated the involvement of this receptor.

It is known that administration of cannabinoid agonists reduce pain induced by

bladder inflammation [47]. In this work, FAAH inhibition reversed bladder pain induced

by inflammation. This result is corroborated by a decrease in Fos expression at L6

spinal cord level after URB937 application. Altogether these results suggest that FAAH

inhibition and, subsequently, endocannabinoids are involved in the activation of

nociceptive pathways.

In summary, it is possible that during bladder painful syndrome/ interstitial

cystitis there is an interaction between several pathways, namely α1A-adrenoceptors,

TRPV1 and CB1, as shown in the following schematic drawing:

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VI. Conclusion

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VI. Conclusion

The present work confirmed that chronic adrenergic stimulation with

phenylephrine induces visceral pain, mast cell infiltration, apoptosis of urothelial cells

and activation of nociceptive pathways. Silodosin (antagonist of α1A-adrenoceptor

subtype) seems to be more effective than naftopidil or prazosin (α1D and α1-

adrenoceptor antagonist) to reduce the effects induced by chronic adrenergic

stimulation with phenylephrine.

According to previous studies, this work showed that TRPV1 are involved in

bladder hyperactivity, as well as CB1. However, it is necessary more studies to better

understand the role of these receptors in bladder hyperactivity, a characteristic of

BPS/IC.

Thus, we can conclude that BPS/IC results from the activation and the cross-

talk between different pathways, possibly involving α-adrenoceptors, TRP channels

(TRPV1 and possibly TRPV4) and cannabinoid receptors (CB1).

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VII. Future work

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VII. Future work

This work allows characterizing the adrenoceptors subtypes involved in pain,

organ motility and bladder histological changes in an animal model of BPS/IC. These

results suggest that silodosin (antagonist of α1A-adrenoceptor subtype) seems to be

more effective to reduce the effects induced by chronic adrenergic stimulation with

phenylephrine. Furthermore, BPS/IC seems to be a result of the cross-talk between

different pathways, involving α-adrenoceptors, TRPV1 and cannabinoid receptors

(CB1). However the role of these receptors in bladder hyperactivity is not completely

understood.

Besides TRPV1 and CB1, there are more nociceptive receptors such as P2X3,

which could be also involved in bladder hyperactivity [69]. Activation of P2X3 and TRP

channels by bladder distension or chemical stimuli can release some mediators like

ATP, Ach (acetylcholine) and NGF (nerve growth factor) [70]. It is also known that

patients with BPS/IC present altered levels of APF (anti-proliferative factor), NGF,

BDNF (brain derived nerve factor), and interleukins, among others [71].

Some authors, observed a decrease in the microvascular density in the

suburothelium in patients with BPS/IC, suggesting that hypoxia can be a cause of the

disease [1, 25]. To clarify this idea, would be interesting to study the effect of chronic

bladder ischemia in bladder function and morphology and in sympathetic activity, using

a rat model of atherosclerosis-induced chronic bladder ischemia.

Thus, there are many questions that can be studied in a future work. Which are

the connections between the adrenoceptor pathways and the nociceptive pathways,

namely at the expression and function of TRP (TRPV1 and TRPV4) receptors? What

are the roles of CB1 and P2X3 in this process? Will NGF, BDNF and interleukins be

involved in BPS/IC? How ischemia influences BPS/IC characteristics? Can ischemia

stimulus induce sympathetic overactivity and how these can induce the changes

observed in patients with BPS/IC?

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The role of TRPV4 and P2X3 can be assessed using PHE-treated animals

administrated with agonists and antagonists of these receptors and using co-cultures of

DRG and urothelial cells treated with PHE. The activity of these receptors can be

determined by calcium imaging technique.

To assess the role of neurothrophins and interleukins, control animals and

ovariectomized animals can be submitted to chronic adrenergic stimulation with PHE

for 14 days. Urine samples can be collected before, at day 8 and day 14 after

beginning the treatment, to determine neurothrophins and interleukins levels by ELISA.

The levels of neurothrophins and interleukins can also be determined in urine from

BPS/IC patients and aged matched normal volunteers.

To verify if an ischemic stimulus induces chronic sympathetic overactivity and

verify whether this sympathetic overactivity induces bladder changes observed in

BPS/IC patients, that an animal model of atherosclerosis-induced chronic bladder

ischemia, like described by Nomiya et al., 2012 [72], can be used.

After ischemic insult and at different time points, the levels of plasmatic and

urine noradrenaline can be measured by ELISA and HPLC. Organ expression of

tyrosine hydroxylase and dopamine-β-hydroxylase, by immunohistochemistry and

western blot, respectively, can be also assessed. Furthermore, visceral pain behavior

and organ motility and integrity can be evaluated at different point times, as well as

spinal Fos expression and inflammatory signs.

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VIII. References

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IX. Attachments

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IX. Attachments

1. Phenylephrine and antagonist solutions

The phenylephrine (Sigma-Aldrich P6126-5G) solution used for chronic

adrenergic stimulation both in rat and mice was daily injected at 2.5 mg/ Kg body

weight. This solution vehicle was saline solution.

Silodosin (antagonist of alpha1A- adrenoceptor) were used in a concentration of

0.2 mg/ Kg. Naftopidil (antagonist of alpha1D- adrenoceptor) were used in a

concentration of 0.9375 mg/ Kg. The solution vehicle was saline solution.

Prazosin (non-specific antagonist of alpha1-adrenoceptor) were used in a

concentration of 0.05 mg/ Kg. The solution vehicle was saline solution.

2. Cystometry, tissue harvesting and processing solutions

During cystometry, saline solution was infusecd (1.8 mL/ h) into the bladder

dome of anaesthetized animals (urethane 1.2 mg/ Kg).

During perfusion, the fixative and Tyrode´s solution were prepared as follows:

- Paraformaldehyde 4% (for 1L): 250 mL paraformaldehyde 16%, 250 mL

Phosphate buffer 0,4M pH 7.2 and 500 mL bidestilled water);

- Tyrode’s solution (for 1L): 6.80g NaCl; 0.40g KCl; 0.32g MgCl2.6H2O; 0.10g

MgSO4.7H2O; 0.17g NaH2PO4.H20; 1.00g glucose and 2.20g NaHCO3).

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L6-S1 spinal cord segments were sectioned at 40 µm and the cuts were stored

at -20ᵒC in crioprotectant. To prepare this solution, 500 mL of phosphate buffer 0.1M

pH 7.2, 300g of sucrose and 300 mL of ethyleneglycol were used

3. Immunoreaction against Fos in spinal cord segments

L6-S1 spinal cord segments were sectioned and immunostained for Fos protein,

like described below:

The segments were washed 3 times with PBS (5 minutes each), following a

wash with ethanol 50% in elix for 10 minutes, to permeabilize the membranes and

allow better entry of the substrate.

The slides were incubated in ethanol 50% and 330 µL of H2O2 for 30 minutes, to

inhibit the endogenous peroxidase. After this, the sections were washed with ethanol

50% for 10 minutes and with PBS for 5 minutes.

Next, the slides were incubated with PBST/ NSS 10% for 1 hour.

Afterwards, there was an incubation in the primary antibody α- c Fos

(Calbiochem) diluted 1: 10000 in PBST/ NSS 2% for 2 days at 4ᵒC.

The slides were washed 3 times (10 minutes each) with PBST/ NSS 2% and

incubated with secondary antibody biotinylated swine α-rabbit (Palex Medical) diluted

1:200 in PBST/NSS 2% for 1 hour.

Then, the slides were washed twice (10 minutes each) with PBST/ NSS 2%,

with PBST for 10 minutes and incubated with ABC, during at least one hour, to amplify

the final signal (avidin binds to the biotin of the secondary antibody).

Next, the slides were washed 3 times with PBST (for 10 minutes each time),

with PBS for 10 minutes and with Tris-HCl 0,1M for 10 minutes, to remove the excess

of secondary antibody- ABC complex, or just ABC.

The slides were incubated with diaminobenzidine (DAB), which is a

chromogenic substrate of peroxidase. In the presence of H2O2, the enzyme forms a

brown and insoluble precipitate where there is enzyme, and therefore the antigen

under study.

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Finally, the sections were washed with Tris-HCl 0,1 M for 5 minutes and with

PBS for 5 minutes. Finally, the slides were mounted with EuKit (National Diagnostics)

and observed on light microscopy (Zeiss Axioskop 40).

4. Immunoreaction against caspase 3 in bladder sections

Bladder sections were immunoreacted against caspase 3, a marker of cellular

apoptosis, to analyze urothelium integrity.

Briefly, the slides were washed 3 times with xylene (5 minutes each time),

washed with ethanol 100% 3 times (5 minutes each), 3 times with ethanol 90% (for 5

minutes each), 3 times with ethanol 75% (for 5 minutes each) and 3 times with distilled

water (for 5 minutes each).

Then, slides were incubated with citrate buffer 2 g/L pH 6, in the microwave for

15 minutes, and left to cool, for 30 minutes.

After this, the slides were washed with PBS 2 times (for 10 minutes each), with

1% H2O2 in methanol for 10 minutes and with PBS for 10 minutes.

Next, the slides were incubated with PBST/NSS 10% for 1h and, after this, with

primary antibody rabbit anti-caspase 3 (Abcam) diluted 1:500 in PBST/NSS 2% for 2

days at 4ᵒC.

The slides were washed with PBST/NSS 2% 3 times for 10 minutes each time,

and incubated with secondary antibody 488 goat anti-rabbit 1:3000 (Invitrogen) in

PBST/NSS 2% for 1h.

After this, the slides were washed with PBST/NSS 2% 2 times (10 minutes

each), with PBST for 10 minutes and with PBS for 10 minutes.

Finally, the slides were mounted with glycerol and observed on fluorescence

microscope (Zeiss Apotome).

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5. Preparation of solutions for immunoreaction

Phosphate buffered saline (PBS) is a buffered solution used in biological

research, and containing sodium chloride, sodium phosphate and, in some cases,

potassium chloride and potassium phosphate. This solution helps to maintain a

constant pH. PBST results from addition of Tween 20 or Triton X-100 to PBS. The

Triton X-100 permeabilizes the cells, facilitating the entry of the antibody into the cells.

PBS 0.1M (for 1L): 100 mL of stock solution of PBS 10x and 900 mL of distilled

water.

PBST (for 1L): 888 mL of distilled water, 100 mL of PBS 10x and 12 mL of

Triton X-100 at 25%.

Triton X-100 25% (for 12 ml): 3 mL of Triton X-100 and 9 mL of distilled water.

PBST/ NSS 10% contains PBST and NSS (normal swine serum).

PBST/NSS 10% (for 8 mL): 800 µL of NSS and 7.2 mL of PBST.

PBST/NSS 2% (for 8 mL): 160 µL of NSS and 7.84 mL of PBST.

For incubation with ABC (Vector Laboratories), use 1 µL of A and 1 µL of B per

200 µL of PBST. For example, to prepare 3.2 mL of PBST is necessary to add 16 µL of

A and 16 µL of B. This solution needs to be prepared, at least, 30 minutes prior to use

and placed in the shaker.

To prepare DAB (diaminobenzidine), first add 5 µL of H2O2 at 30% in 20 mL of

Tris-HCl (0,05 M pH=7.6), and then add 10 mg of DAB.

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6. Hematoxylin-Eosin staining in bladder sections

Bladder sections were washed with xylene for 10 minutes, to remove paraffin.

After this, the slides were washed with ethanol 100% (2 minutes), ethanol 90%

(2 minutes) and ethanol 70% (2 minutes).

The slides were washed with water for 10 minutes, and then the cuts were

stained with Hematoxylin for 3 minutes.

After staining, the slides were placed under current water for 15 minutes.

Then, the slides were stained with eosin for 30 seconds, and washed with

ethanol 70%, ethanol 90% and ethanol 100% (only for some seconds in each one).

The slides were washed with xylene for 10 minutes, mounted with EuKit and

observed under the light microscope.

7. Toluidine Blue staining in bladder sections

Bladder sections were washed with xylene for 10 minutes to remove paraffin.

After this, the slides were washed with ethanol 100% (2 minutes), ethanol 90%

(2 minutes) and ethanol 70% (2 minutes).

The slides were washed with water for 10 minutes, and then the cuts were

stained with Toluidine Blue for 4 minutes.

After staining, the slides were placed under current water for 15 minutes.

Then, the slides were washed with ethanol 70%, ethanol 90% and ethanol

100% (only for some seconds in each one).

The slides were washed with xylene for 10 minutes, mounted with EuKit and

observed under the light microscope.

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