Paulo Roberto da Silva Lima

A diferença de média de hiperplasia mio-intimal

pós-angioplastia na artéria ilíaca externa de

coelhos com aterosclerose induzida e tratados

com Allium sativum comparados aos tratados

com Cilostazol

2013  

Paulo Roberto da Silva Lima

Maceió

1. Introdução

1.1. Contexto

A hiperplasia da íntima:

Migração e proliferação de células musculares

lisas vasculares

Remodelagem desse novo tecido (Davies,

2007)

É reversível apenas na primeira e segunda

semanas após o trauma vascular (Li, 1999;

Sterpetti, 1996)

Vascular. 6a ed. Rio de Janeiro: DiLivros; 2007;149-72.

Li JM, Brooks G. Cell cycle regulatory molecules (cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors) and the cardiovascular system; potential targets for therapy? Eur Heart J. 1999 Mar;20(6):406-20. Review. PubMed PMID: 10213344.

Sterpetti AV, Cucina A, Lepidi S, Randone B, Stipa F, Aromatario C, Travi D, D'Angelo LS, Cavallaro A, Stipa S. Progression and regression of myointimal hyperplasia in experimental vein grafts depends on platelet-derived growth factor and basic fibroblastic growth factor production. J Vasc

Surg. 1996 Apr;23(4):568-75. PubMed PMID: 8627890.

• 

A radiação ionizante é eficaz (Costa, 2002;

Ducasse, 2006)

•  O paclitaxel reduz a hiperplasia da íntima (Park,

2003; Shafiq, 2005)

Costa R, Fagundes D. Modelos experimentais de hiperplasia intimal: efeitos da radiação ionizante. Acta Cir Bras. 2002 May;17(3).

Ducasse E, Chevalier J, Cosset JM, Creusy C, Eschwege F, Speziale F, Sbarigia E, Midy D, Baste JC, Lartigau E. Ionizing radiation to prevent arterial intimal hyperplasia at the edges of the stent: induces necrosis and fibrosis. J

Surg Res. 2006 Oct;135(2):331-6. Epub 2006 May 22. PubMed PMID: 16716353.

Park SJ, Shim WH, Ho DS, Raizner AE, Park SW, Hong MK, Lee CW, Choi D, Jang Y, Lam R, Weissman NJ, Mintz GS. A paclitaxel-eluting stent for the prevention of coronary restenosis. N Engl J Med. 2003 Apr 17;348(16):1537-45.

PubMed PMID:12700373.

Shafiq N, Malhotra S, Pandhi P, Grover A, Uboweja A. A meta-analysis of clinical trials of paclitaxel- and sirolimus-eluting stents in patients with obstructive coronary artery disease. Br J Clin Pharmacol. 2005 PubMed PMID

15606446; PubMed Central PMCID: PMC1884956.

•  O cilostazol demonstrou ser eficaz na redução

da hiperplasia da íntima (Tsai, 2008; Robertson,

2012)

–  Tem ação antiplaquetária (Robertson, 2012;

Friedland, 2012)

–  Inibe o acúmulo de triglicerídeos e colesterol na

parede arterial (Ito, 2012)

Tsai CS, Lin FY, Chen YH, Yang TL, Wang HJ, Huang GS, Lin CY, Tsai YT, Lin SJ, Li CY. Cilostazol attenuates MCP-1 and MMP-9 expression in vivo in LPS-administrated balloon-injured rabbit aorta and in

vitro in LPS-treated monocytic THP-1 cells. J Cell Biochem. 2008 Jan 1;103(1):54-66. PubMed PMID: 17516547.

Robertson L, Ghouri MA, Kovacs F. Antiplatelet and anticoagulant drugs for prevention of restenosis/ reocclusion following peripheral endovascular treatment. Cochrane Database Syst Rev. 2012 Aug

15;8:CD002071. Review. PubMed PMID: 22895926.

Friedland SN, Eisenberg MJ, Shimony A. Meta-analysis of randomized controlled trials on effect of cilostazol on restenosis rates and outcomes after percutaneous coronary intervention. Am J Cardiol. 2012 May

15;109(10):1397-404. Epub 2012 Feb 28. Review. PubMed PMID: 22381162.

Ito H, Uehara K, Matsumoto Y, Hashimoto A, Nagano C, Niimi M, Miyakoda G, Nagano K. Cilostazol inhibits accumulation of triglyceride in aorta and platelet aggregation in cholesterol-fed rabbits. PLoS One

•  Efeitos do Allium sativum (Efendy,1997; Srilatha,

1997; Borek, 2001; Campbell, 2001):

– Anti-inflamatório

– Hipocolesterolêmico

– Anti-hipertensivo

– Antitumoral

Efendy JL, Simmons DL, Campbell GR, Campbell JH. The effect of the aged garlic extract, 'Kyolic', on the development of experimental atherosclerosis. Atherosclerosis. 1997 Jul 11;132(1):37-42. PubMed PMID: 9247357.

Srilatha B, Adaikan PG, Ng SC, Aulkumaran S. Effects of feeding egg yolk on the serum lipid levels in rabbits. Methods Find Exp Clin Pharmacol. 1997 Sep;19(7):489-92. PubMed PMID: 9413832.

Borek C. Antioxidant health effects of aged garlic extract. J Nutr. 2001 Mar;131(3s):1010S-5S. Review. PubMed PMID: 11238807.

Campbell JH, Efendy JL, Smith NJ, Campbell GR. Molecular basis by which garlic suppresses atherosclerosis. J Nutr. 2001 Mar;131(3s):1006S-9S. PubMed PMID: 11238806.

•  O Allium sativum é um produto seguro com DL

50 de 3034 mg/Kg (Mikail, 2010)

Mikail HG. Phytochemical screening, elemental analysis and acute toxicity of aqueous extract of Allium sativum L. bulbs in experimental rabbits. Journal of Medicinal Plants Research. 2010 Fev 18;4(4):322-6.

Pergunta de pesquisa

qual a diferença de média de hiperplasia mio-intimal

pós-angioplastia na artéria ilíaca externa de coelhos com

aterosclerose induzida tratados com Allium sativum

comparados aos tratados com Cilostazol ?

1.2. Hipótese

•  A hipótese é que o Allium sativum é mais eficaz na

redução da hiperplasia mio-intimal pós-angioplastia na

artéria ilíaca externa de coelhos com aterosclerose

induzida que os tratados com Cilostazol

1.3. Objetivo

•  Determinar a diferença de média da hiperplasia

mio-intimal pós-angioplastia na artéria ilíaca externa de

coelhos com aterosclerose induzida tratados com Allium

sativum comparados aos tratados com Cilostazol

2. Métodos

Comitê de Ética em Pesquisa

•  Aprovado no CEP/UNCISAL sob o nº 63-A em

19/11/2009

•  Aprovado no CEPA/LTF/UFPB sob o n˚ 0412/09 em

16/12/2009

2.1. Tipo de estudo

•  Ensaio aleatório em animais de experimentação

2.2. Local

•  Biotério do Laboratório de Tecnologia Farmacêutica

(LTF) do Campus I da Universidade Federal da Paraíba,

João Pessoa, PB

•  2.3. Amostra

•  2.3.1. Critérios de inclusão

•  Coelhos (Oryctolagus cuniculus) da raça Nova Zelândia com

aterosclerose induzida e submetidos à angioplastia da artéria ilíaca

externa direita

•  2.3.2. Critérios de exclusão

•  Morte antes da administração das drogas

•  Machos

•  Idade menor que 4 e maior que 6 meses

•  Peso corporal menor que 2 kg e maior que 5 kg de peso

•  Modificações na anatomia das estrutura a serem abordadas

•  Trombose

•  2.3.3. Amostragem

• 

Amostra probabilística por conveniência

•  2.3.4. Termo de Consentimento Livre e

Esclarecido

•  Não se aplica, cabe apenas o CEPA

2.4. Procedimentos

2.4.1. Grupos estudados

-  Grupo AS(Allium sativum) – tratados com 800 µg/kg de

Allium sativum por via oral por 35 dias (Efendy, 1997)

-  Grupo C (Cilostazol) – 50 mg/dia de cilostazol por via

oral por 35 dias (Tsai, 2008)

- Grupo SF (Soro Fisiológico) – tratados com solução

fisiológica com 10 mL/dia por via oral por 35 dias

Efendy JL, Simmons DL, Campbell GR, Campbell JH. The effect of the aged garlic extract, 'Kyolic', on the development of experimental atherosclerosis. Atherosclerosis. 1997 Jul 11;132(1):37-42. PubMed PMID: 9247357 Tsai CS, Lin FY, Chen YH, Yang TL, Wang HJ, Huang GS, Lin CY, Tsai YT, Lin SJ, Li CY. Cilostazol attenuates MCP-1

and MMP-9 expression in vivo in LPS-administrated balloon-injured rabbit aorta and in vitro in LPS-treated monocytic THP-1 cells. J Cell Biochem. 2008 Jan 1;103(1):54-66. PubMed PMID: 17516547. 11  

Q  

Quarentena40  

dias  

100  dias  

Angioplas>a  2  dias    

35  dias  

Dieta  hipercolesterolêmica  

20ml/dia  

A  

Tratamento  

Cil,  SF,  Al  

E  

T1  

T5  

T4  

T2   T3  

T0  

Eutanásia  

24h  

Microsphere Perfusion

The authors hypothesized that the

wound healing response would have

an immediate effect on the hindlimb

vessel perfusion. The authors

there-fore studied three animals in each

group on day 3 post-occlusion to

quantify these effects. Microsphere

analysis was performed as previously

described and validated (14). Briefly,

2.5 ! 10

_{stable radiolabeled 15}

_!

_m

microspheres (BioPhysics Assay

Labo-ratory, Worcester, MA) were injected

(1 mL) through a straight flush

cathe-ter in the left ventricle just prior to

sacrificing on day 3. Animals were

then sacrificed. The lateral head of the

gastrocnemius and the soleus were

harvested from both hindlimbs in each

animal. Three separate 1 g blocks from

each muscle were submitted for

mi-crosphere

analysis

and

average

counts/gram of tissue for each

hind-limb was calculated. A 1 g specimen

from the apex of the left ventricle

served as an internal control. Ratios of

left and right hindlimb to left ventricle

were calculated from the raw data.

Clinical Assessment

All animals were clinically

evalu-ated for muscle atrophy and distal

limb necrosis on days 0, 3, 7, 14, and

28. Muscle atrophy was evaluated by

measurement of calf circumference.

Atrophy was considered significant if

the size of the experimental calf was

reduced by more than 20% when

com-pared with the control. Distal limb

ne-crosis was evaluated by macroscopic

examination and considered to be

sig-nificant if skin necrosis was observed

at the level of the foot or ankle.

Immunohistochemistry/Histology

All animals were euthanized by an

intravenous injection of thiopental

(100 mg/kg). With a straight flush

catheter positioned in the distal

ab-dominal aorta, 500 mL of 10%

forma-lin were hand injected into both

hind-limbs. After disarticulating both hip

joints each hindlimb was removed and

placed in 10% formalin for 24 hours.

Two axial sections were obtained from

the entire leg, 4 – 4.5 cm and 4.5–5 cm

cephalad to the superior pole of the

patella and immediately fixed in 10%

phosphate-buffered saline formalin,

and then embedded in paraffin.

Five-

!

m thick sections were prepared

from paraffin-embedded samples at

the cephalad portion of each slide.

Immunohistochemical staining was

carried out with mouse monoclonal

antibody

against

human

CD31

(N1596; Dako Cytomation,

Carpinte-ria, CA) to detect vascular endothelial

cells, or with mouse monoclonal

anti-body against human alpha smooth

muscle actin (dilution 1:100

RDI-AC-TINabm-A4; Research Diagnostics,

Flanders,

NJ)

to

detect

vascular

smooth muscle cells. Microwave

irra-diation was performed to retrieve the

antigens. Sections were incubated in

0.3% hydrogen peroxide to block

en-dogenous peroxidase activity. Protein

blocking, incubation with secondary

biotinylated antibody and

avidin-bi-otin interaction were performed with a

Vectastain ABC kit (Vector

Laborato-ries, Burlingame, CA) according to the

manufacturer’s protocol. These

anti-bodies were visualized with

diamino-benzidine as a chromogen (DAB kit;

Vector Laboratories).

At !200 magnification, with a Carl

Zeiss AxioCam (Thornwood, NY)

dig-ital camera with the Zeiss Axioskop

(DIC and Neofluar optics) connected

to a personnel computer (Dell, Austin,

TX), 6 random fields were captured.

The images were imported into

J-Im-age (National Institutes of Health) to

calculate the average arteriolar

lumi-nal area per animal. The number of

capillaries and myocytes per field

were manually counted and expressed

as the capillary/muscle fiber ratio.

Hematoxylin and eosin staining

was done on the sections to identify

and

quantify

inflammatory

cells.

Stained sections were reviewed at !20

magnification, by an experienced

vet-erinary pathologist (K.L.G.), who was

blinded to sample group.

Inflamma-tion was scored subjectively on a scale

from 1 to 10 (1 " mild, 10 " severe)

based on both the area occupied by

inflammatory cells (mononuclear cells

including macrophages) and the

de-gree of hypercellularity.

Statistics

The data are presented as mean #

standard deviation. All P values were

Figure 2.

Lateral spot radiograph of the left proximal hindlimb demonstrating normal

vascular anatomy. EIA, external iliac artery; CFA, common femoral artery; SFA,

super-ficial femoral artery; PA, popliteal artery; SA, saphenous artery; HA, hypogastric artery;

PFA, profunda femoral artery.

994

•

Endovascular Model of Rabbit Hindlimb Ischemia

July 2005 JVIR

13  

Anatomia vascular normal: EIA, artéria ilíaca externa; CFA, artéria femoral

comum; SFA, artéria femoral superficial; PA, artéria poplítea; SA, artéria

safena; HA, artéria hipogástrica; PFA, artéria femoral profunda; LCA, artéria

circunflexa lateral

Liddell RP, Patel TH, Weiss CR, Lee DS, Matsuhashi T, Brown PR, Gabrielson KL, Rodriguez ER, Eng J, Kimura H, Hofmann LV. Endovascular model of rabbit hindlimb ischemia: a platform to evaluate therapeutic angiogenesis. J Vasc Interv Radiol. 2005 Jul;16(7):991-8. PubMed PMID: 16002507

Barone R, Pavaux C, Blin PC. Atlas D`Anatomie du Lapin. Paris: Masson et Cie; 1973. Pág 133

15  

2.4.2. Técnica de randomização

Por permutação simples (http://www.randomizer.org/

form.htm)

2.4.3.Técnica de mascaramento

Foi aplicada no momento da coleta tanto para o laboratório

como para histologia.

2.5.  Variáveis  

2.5.1.  Variável  primária  

- A diferença de média de hiperplasia mio-intimal

2.5.2  Variáveis  secundárias  

• A média de hiperplasia mio-intimal no grupo AS

• A média de hiperplasia mio-intimal no grupo C

• A média de hiperplasia mio-intimal no grupo SF

•  Foi mensurada por microscopia óptica

• Morfometria digital (Image J 1.46o, 64

bit, for Mac, Wayne Rasband,

National Institutes of Health, USA, the

public domain)

•  Cinco semanas após angioplastia

2.5.3. Dados complementares

•  A média de colesterol total

•  A média de HDL

•  A média de LDL

•  A média de VLDL

•  A média de triglicerídeo

•  Média de peso

•  Frequência de isquemia do membro

•  Frequência de perda do membro

•  Frequência de morte

•  Frequência de hematoma, equimose

•  Frequência de infecção

2.6. Método estatístico

2.6.1. Cálculo do tamanho da amostra

•  O tamanho da amostra foi arbitrado em 10 coelhos

por grupo (Cortelekoglu, 2006; Santos, 2005)

Cortelekoglu  T,  Bozkurt  AK,  Ustundag  N,  Koksal  C,  Sayin  AG.  The  effects  of  clopidogrel  and  calcium  dobesilate  on  in>mal  hyperplasia   following  vascular  injury.  Acta  Chir  Belg.  2006  Mar-­‐Apr;106(2):206-­‐10.  PubMed  PMID:  16761479.  

2.6.2. Análise estatística

•  Coleta de dados

–  Formulário padronizado

•  Armazenamento dos dados

–  Planilha de dados eletrônica (Microsoft Excel® for

Mac 2011, Versão 14.1.4 (111121)

•  Tabulação dos dados

–  Planilha de dados eletrônica

•  Estatística descritiva

– 

O  intervalo  de  confiança  de  95%  (Gardner,  1989)  

•  Aplicativo

– 

GraphPad  Prisma®  (Versão  6,  for  Mac  OS  X,  

GraphPad  Sojware  Inc.,  San  Diego,  CA),  3  de  

outubro  de  2012  

Gardner  MJ,  Altman  DJ.  Esta>s>c  with  confidence:  confidence  intervals  and  sta>s>cal  guidelines.  London:  BMJ  Publishing  Group;  1989  

•  Variável analisada

– 

A  diferença  de  média  de  hiperplasia  mio-intimal

•  Hipóteses Estatísticas

– 

H

_o

:  P

_AS

 =  P

_C

 =  P

_SF

– 

H

₁

:  P

_AS

 ≠  P

_C

 ≠  P

_SF

•  Testes Estatísticos

•  ANOVA (One-way e Two-way), Qui-quadrado

•  Valor de Alfa

– 

Menor  que  0,05  

3. Resultados

Morte&na&cirurgia&

n=2&

&

Animais&

selecionados&

n=&37&

Morte&na&

quarentena&

n=&5&

Animais&

randomizados&

n=32&

Allium&sativum&

n=&10&

Cilostazol&

n=&10&

Soro&fisiológico&

n=&10&

Desvio da pesquisa

Espessamento médio-intimal

pix

el x

10

5

Soro fisiológico

Cilostazol

Allium sativum

p < 0,0001

Hiperplasia da mio-intimal

Média do soro fisiológico:

35,74 x 10

_{pixels IC 95%, 31,76 a 39,71 x 10}

_pixels

Média do cilostazol:

16,21x 10

_{pixels IC 95%, 13,36 a 19,05 x 10}

_pixels

Média do Allium sativum:

Mediana de hiperplasia mio-intimal

Mediana do soro fisiológico:

36,43 x 10

_{pixels IC 95%, 29,23 a 45,46 x 10}

_pixels

Mediana do cilostazol:

15,46x 10

_{pixels IC 95%, 12,66 a 23,70 x 10}

_pixels

Mediana do Allium sativum:

19,79 x 10

_{pixels IC 95%, 12,07 a 31,40 x 10}

_pixels

Colest total

mg/dL

Cilostazol

Allium sativum

SF 0,9%

-200

0

200

400

600

Cilostazol

Allium sativum

SF 0,9%

P  =  0,0899    

Média do soro fisiológico:

62,30 IC 95%, 25,55 a 99,05

Média do cilostazol:

35,55 IC 95%, 31,72 a 39,38

Média do Allium sativum:

HDL

mg/dL

Cilostazol

Allium sativum

SF 0,9%

0

20

40

60

80

100

Cilostazol

Allium sativum

SF 0,9%

P = 0,5796

Média do soro fisiológico:

20,23 IC 95%, 12,65 a 27,80

Média do cilostazol:

20,80 IC 95%, 17,91 a 23,68

Média do Allium sativum:

LDL

mg/dL

Cilostazol

Allium sativum

SF 0,9%

-200

0

200

400

600

Cilostazol

Allium sativum

SF 0,9%

P = 0,0124

Média do soro fisiológico:

16,11 IC 95%, -8,75 a 40,97

Média do cilostazol:

2,19 IC 95%, -1,38 a 5,75

Média do Allium sativum:

VLDL

mg/dL

Cilostazol

Allium sativum

SF 0,9%

0

20

40

60

80

100

P = 0,8107

Média do soro fisiológico:

15,33 IC 95%, 10,50 a 20,08

Média do cilostazol:

12,60 IC 95%, 9,69 a 15,51

Média do Allium sativum:

Triglicerideo

mg/dL

Cilostazol

Allium sativum

SF 0,9%

0

100

200

300

400

500

Cilostazol

Allium sativum

SF 0,9%

P  =  0,2013    

Média do soro fisiológico:

101,3 IC 95%, 43,77 a 158,7

Média do cilostazol:

66,30 IC 95%, 47,61 a 84,99

Média do Allium sativum:

Peso

g

Cilostazol

Allium sativum

SF 0,9%

0

1000

2000

3000

4000

5000

P <0,0001

Média do soro fisiológico:

2986 g IC 95%, 2540 a 3432 g

Média do cilostazol:

3133 g IC 95%, 2702 a 3364 g

Média do Allium sativum:

37  

Infecção

N

d

e

c

o

e

lh

o

s

Sim

Não

0

2

4

6

8

10

SF 0,9%

Cilostazol

Allium sativum

P= 0,7866

38  

Frequência de isquemia do membro

-  4 em 30, sendo 3 no SF e 1 no Allium

Frequência de perda do membro

-  4 em 30, sendo 3 no SF e 1 no Allium

Frequência de morte

- Não houve

Frequência de hematoma, equimose

4. Conclusão

•  Não há diferença de média de hiperplasia  mio-­‐in>mal

 pós-angioplastia na artéria ilíaca externa de coelhos com

aterosclerose induzida tratados com Allium sativum

comparados aos tratados com Cilostazol