Marcelo Tadahiro Wayama
Efeito do raloxifeno na lesão periapical em ratas
ovariectomizadas.
ARAÇATUBA-SP
Marcelo Tadahiro Wayama
Efeito do raloxifeno na lesão periapical em ratas
ovariectomizadas.
Dissertação apresentada à Faculdade de Odontologia da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Araçatuba, para obtenção do título de MESTRE em Ciência Odontológica – Área de Concentração: Endodontia.
Orientador: Prof. Adj.João Eduardo Gomes Filho
Co-orientadora: Profª Ass. Rita Cássia Menegati Dornelles
ARAÇATUBA-SP
Catalogação na Publicação (CIP)
Serviço Técnico de Biblioteca e Documentação – FOA / UNESP
Wayama, Marcelo Tadahiro.
W357e Efeito do raloxifeno na lesão periapical de ratasovariecto- mizadas / Marcelo Tadahiro Wayama. - Araçatuba, 2014
114f. : il. + 1 CD-ROM
Dissertação (Mestrado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba
Orientador: Prof. João Eduardo Gomes Filho
Coorientadora: Profa. Rita Cássia Menegati Dornelles
1. Raloxifeno 2. Estrogênios 3. Periodontite periapical 4. Ovariectomia 5. Remodelação Óssea I. Título
Black D24
Dados Curriculares
MARCELO TADAHI RO WAYAMA
Nascim ent o: 26 de j aneiro de 1989 - São Paulo/ SP
Filiação: Michiko Yam ane; Hirot o Wayam a
2008
–
2011: Curso de Graduação em Odont ologia
–
Faculdade de Odont ologia de Araçat uba
–
Universidade
Est adual
Paulista “Júlio de Mesquita Filho”
- UNESP
2012
–
2014: Curso de Pós- Graduação em Endodontia,
nível de Mest rado
–
Faculdade de Odont ologia de Araçat uba
DEDICATÓRIA
Dedico este trabalho,
À m inha m ãe Michiko, あさん い も わいく あ う
い もにこ わ い あさんだいす す い しょに んばろ！
Ao m eu pai Hiroto, que sem pre m e deu apoio e força durante
essa traj et ória e pelos conselhos e ensinam entos da vida.
Ao m eu irm ão Junya, pelo seu com panheirism o com o irm ão e
am igo que sem pre esteve ao m eu lado nos m om entos tristes e
alegres da vida.
A m inha vó Dona Toshie, apesar de nos encontrar raram ente,
m as que a alegria esplandece quando nos encontram os. Aos m eus
avôs Dona Tokio, Sr. Rikio e Sr. I sam i, que nos deixou há m uito
tem po, m as que nos deixaram m arcas até hoj e.
Aos m eus tios Dona Sueli e Sr. Geraldo, por sem pre m e aj udar
nos m om entos difíceis da vida e pelo carinho im enso que tem por
AGRADECIMENTOS
A Deus
Por estar sem pre m e guiando durant e todo percurso da m inha
vida e fazer acontecer as coisas para o m elhor de m im .
Ao m eu orientador Prof. João Eduardo Gom es Filho, pela m inha
orientação, paciência e conselhos profissionais. Adm iro sua
sim plicidade e o carinho que tem por m im . Guardarei suas palavras.
A m inha co- orientadora Profª Rita Cássia Menegat i Dornelles,
por estar trabalhando j unto com igo desde a I C. Obrigado pela
paciência e colaboração nos trabalhos. Adm iro sua sabedoria e
dedicação à pesquisa com o docente.
Ao Professor Edilson Ervolino, por m e ensinar e colaborar na
part e histológica do trabalho. Continue sendo este professor
organizado e perfeccionist a pelos trabalhos que realiza.
Ao Professor Gilbert o Aparecido Coclete, em ceder a
oport unidade de realizar a m onitoria na radiologia e tam bém em
part icipar do m eu trabalho enriquecendo ainda m ais o proj eto.
Aos Professores Robert o Holland, Mauro Juvenal Nery, José
Arlindo Ot oboni Filho, Eloi Dezan Júnior e Luciano Tavares Angelo
Cintra, que m e t ransm it iram m uito dos seus conhecim entos e
Aos Professores Luciano Tavares Angelo Cintra e Carla Renata
Sipert por aceitarem o convite com o banca exam inadora da m inha
dissertação e por acrescentar ao m eu trabalho.
A Nelci, Cláudia, Elaine, Peterson e Grazi por m e aj udar nos
nossos experim entos, a paciência que tiveram e pela convivência
durante esta traj etória.
Aos am igos de pós- graduação, Gustavo, Sim one, Paulo,
Luciana, Ludm illa, Aguinaldo, Diego, Renata, Í ndia, Annelise,
Mariane, Loiane, Carlos, Gabrielly, Karina e Franscine pelo
com panheirism o e colaboração.
Aos am igos do Laborat ório das Ciências Básicas que sem pre m e
incent ivaram , aj udaram e deram o m aior apoio.
A todos os m eus am igos de Araçatuba e São Paulo, por m e
acom panharem durante todo esse cam inho. Obrigado pela
sinceridade e pelo am or de cada um .
À Fundação de Am paro a Pesquisa do Estado de São Paulo
( FAPESP) , pelo auxílio concedido para a realização desse trabalho.
A todos aqueles que, direta ou indiret am ente, contribuíram
Epígrafe
“Quanto maiores somos em humildade, tanto m ais próxim os
estamos da grandeza.”
Rabindranat h Tagore
“Saber não basta, devemos aplicar.
Desejar não basta, devemos fazer.”
Sumário
Página
Resum o
Abstract
Lista de figuras
Lista de gráficos
Listade tabelas
Lista de siglas e abreviaturas
I ntrodução... 20
Proposição... ... 24
Art igo 1... 25
Art igo 2... 49
Conclusão e referência... 76
Wayam a, MT. Efeito do raloxifeno na lesão periapical de rat as
ovariectom izadas. Araçatuba, 2014. 114p. Dissertação ( Mestrado em
Endodont ia) – Faculdade de Odontologia, Cam pus de Araçatuba,
Universidade Estadual Paulista “Júlio de Mesquita Filho”.
Resumo
O obj etivo deste estudo foi avaliar o efeito do raloxifeno RLX em
lesões periapicais em rat as ovariectom izadas ( OVX) . Ratas Wistar ( 6
m eses) foram distribuídas nos grupos: SHAM/ VEI , OVX/ VEI e
OVX/ RLX e receberam , por 60 dias, veículo ( VEI ) ou RLX, por
gavagem . Durante o trat am ento, a polpa do prim eiro m olar inferior
direito e esquerdo foram expostas ao am biente oral perm itindo a
análise da lesão aos 7 e 30 dias. Colet a sanguínea foi realizada para
análise bioquím ica. Logo após os anim ais foram eutanasiados e as
m andíbulas separadas, radiografadas, processadas para análises
histopatológica, histom étrica e im unoistoquím ica. Os dados foram
subm etidos ao teste de Tukey ou Dunn ( p< 0,05) . A concentração
plasm ática de estradiol evidenciou o hipoestrogenism o em rat as OVX.
A análise radiográfica m ostrou diferença significativa apenas entre os
períodos, as lesões com 7 dias apresentaram m enor extensão que
aquelas com 30 dias. Aos 7 dias, a at ividade da fosfatase alcalina e o
cálcio plasm ático foram superiores em OVX/ RLX em relação ao grupo
SHAM/ VEI . O fósforo plasm át ico foi m aior no grupo OVX/ RLX em
am bos os períodos. O grupo OVX/ VEI apresentou inflam ação m ais
F1-alfa e células TRAP- positivas que outros grupos em am bos os
períodos. Enquanto o trat am ento com RLX reverteu este quadro com
padrão sem elante à SHAM/ VEI . Não houve diferença estatística na
im unorreatividade de OPG e BALP entre os grupos em am bos sos
períodos. Os resultados m ostram que o hipoestrogenism o potencializa
os efeitos da lesão periapical a qual é am enizada pela ação do RLX
devido aos seus efeitos com binados em dim inuir a atividade
osteoclástica e am enizar o turnover ósseo.
Palavras- chave: Raloxifeno, estrogênios, periodont ite apical,
Wayam a, MT. Effect of raloxifene on periapical lesion in
ovariectom ized rat s. Araçatuba, 2014. 114p . Dissertation ( Master in
Endodont ics) – Dental School of Araçatuba, São Paulo State
University “Júlio de Mesquita Filho”.
Abstract
The aim of this study was to evaluate the effect of raloxifene ( RLX)
on periapical lesions in ovarietom ized rat s ( OVX) . Wistar rat s ( 6
m onths) were dist ributed into groups: SHAM/ veh, OVX/ veh and
OVX/ RLX that received for 60 days vehicle or RLX by gavage. During
treatm ent, t he pulp of lower first m olar was exposed to the oral
environm ent to enable lesion analysis on 7 and 30 days.Blood
collection was perform ed for biochem ical analysis. Soon after the
anim als were killed, the j aws separated, radiographed and processed
for histological, hist om etric and im m unohistochem ical analyzes. Data
were subm itted to Tukey or Dunn test ( p < 0.05) . The plasm a
concentrat ion of estradiol showed hypoestrogenism in OVX
rat s.Radiographically, the groups were sim ilar but lesions on day 7
were sm aller than lesions on day 30. On day 7, t he alkaline
phosphat ase activit y and plasm a calcium were higher in OVX- RLX
than the SHAM- veh. After 30 days of lesion, significant decrease was
observed in calcium level in the raloxifene group. The alkaline
phosphat ase activit y was higher in groups wit hout RLX aft er 30 days
group, in both tim e points. The OVX/ veh group presented m ore
intense inflam m at ion, larger area of periapical lesion and
im m unostaining of RANKL, HI F- 1alpha and TRAP- positive cells than
other groups in bot h tim e points. While treatm ent wit h RLX reversed
this condit ion and was sim ilar to SHAM/ veh. There was no statistical
difference in im m unoreactivity of OPG and BALP between groups in
both tim e points. The results showed that hypoestrogenism
potentiates the effects of periapical lesions, and t his potentiation is
dim inished by RLX due its effects in decreasing osteoclast activity and
soften bone turnover.
Keywords: Raloxifene, estrogens, apical periodontitis, ovariectom y,
Lista de Figuras
Página
Figura 1 –Aspectos radiográficos e histológicos da lesão periapical aos
7 e 30 dias após exposição pulpar ao am biente oral...44
Figura 2 –Área da lesão periapical ( m m ² ) , núm ero de células
m ult inucleares TRAP- positivas por m m , m icrofot ografias m ostrando
células m ultinucleadas TRAP- positivas...46
Figura 3 – Microfot ografias m ostrando as im unom arcações de RANKL,
Lista de gráficos
Página
Gráfico 1 – Concentração plasm ática de cálcio, fósforo e fosfatase
alcalina em diferent es grupos...72
Gráfico 2 – Padrão de im unoreatividade para RANKL, OPG, HI F- 1α
Lista de tabelas
Página
Tabela 1 – Art igo 1–Concentação plasm ática de estrógeno, pesagem
do útero, densidade radiográfica, áreas da lesões periapicais e células
TRAP- posit ivas de acordo com os grupos...48
Tabela 2 – Art igo 2–Concentração plasm ática de estrógeno e
Lista de siglas e abreviaturas
Abs = Absorbância
ANOVA = Análise de Variância
Al = Alum ínio
Cat.= catálogo
d = dias
C = cem ento
DR = Densitom etria radiográfica
EDTA = Ácido et ilenodiam inotetracético
I L = I nterleucina
EPM = Erro Padrão de Média
ALP = Alkaline phosphatase
HE = hem atoxicilina e eosina
i.m .= intram uscular
i.p. intraperitoneal
Kg = Kilogram a
Kv = Kilovolt
m A = Miliam pere
m m Al = Milím etro de alum ínio
Mo = Mouse
MSRE = Moduladores seletivos do receptor de estrógeno
N0 = Núm ero
NaCl = Cloreto de Sódio
AO = osso alveolar
OPG = Ost eoprotegerina
OVX = Ovariectom izada
P = Fósforo
p.c.= peso corporal
pH = Potencial hidrogeniônico
RANK = Receptor do fator ativador nuclear kappa- b
RANKL = Receptor ativador do fator nuclear kappa- b ligant e
Rb = Rabbit
RLX = raloxifeno
RPM = Rotação por Minuto
TRAP = Fosfatase ácida resistente ao tart rato
Sh = Sham
U/ L = Unidade por Litro
Vei = Veículo
20 Introdução
O aum ento da expectativa de vida está fazendo com que a
proporção de idosos cresça m ais rapidam ente do que qualquer outra
faixa etária no m undo ( Fabrício et al., 2008) . Com o passar da idade,
há m aior vulnerabilidade e incidência de processos patológicos, com o
a osteoporose. Estas condições interferem na qualidade de vida e no
aum ento do índice de m ortalidade, representando im port ante
problem a para a saúde das pessoas nesta faixa etária ( Ferreira et al.,
2012; Pinheiro et al., 2010) . Neste contexto, há grande preocupação
com a saúde dos idosos e com a prevenção de patologias decorrentes
do processo de envelhecim ento ( Sousa et al., 2007) , form ulando
novos estudos que possam m elhorar a qualidade de vida destes
indivíduos.
No indivíduo adult o, o esqueleto é m antido devido à sua
regeneração contínua que ocorre pelo processo cham ado
rem odelação óssea, isso ocorre através da rem oção e posterior
subst ituição do osso existente por tecido ósseo neoform ado
( Manolagas, 2000) . A rem odelação óssea é regulada por diferent es
m ecanism os, tais com o os m em bros da superfam ília do fator de
necrose tum oral, o receptor ativador do fator nuclear kappa B/ ligante
( RANK/ RANKL) e osteoprotegerina ( OPG) , que são essenciais para a
osteoclastogênese ( Honm a et al., 2014) . RANKL está expressa em
21 essencial na form ação de osteoclastos ( Lerner, 2006) . O RANKL se
liga a RANK que está presente nos pré- osteoclastos e estim ula a
diferenciação em osteoclastos, enquant o que a ligação do RANKL com
OPG inibe a sua diferenciação (Kotake et al., 2001) .
O idoso pode apresentar diversas alterações neste processo que
predispõem condições patológicas t ípicas do envelhecim ento ( Yazbek
et al., 2008) . A ost eoporose é um processo patológico decorrent e ao
declínio na concentração plasm ática de estrógeno, que ocorre em
m ulheres na m enopausa ( Bedell et al., 2012) . Estas alterações
resultam do aum ento de cavidades de reabsorção, que por sua vez
não são com pletam ente preenchidas pela sua form ação, resultando
na dim inuição da densidade óssea e consequente risco de fratura
( Arm as et al., 2012; Anbinder et al., 2006) .
O estrógeno é um dos principais horm ônios que part icipa do
processo fisiológico do indivíduo, com o crescim ento e
desenvolvim ento celular, regulação do sistem a reprodutor, neuronal,
im une, cardiovascular e esquelético ( Pettersson et al., 2001; Couse
et al., 1999) . Sobre o tecido esquelético o estrógeno prom ove a
dim inuição da reabsorção óssea, atuando diretam ente ou
indiretam ente sobre células do m et abolism o ósseo. Além disso, o
estrógeno desem penha ação im port ante nas doenças inflam atórias,
podendo interferir na produção de citocinas ( Millán et al., 2013;
22 A deficiência do est rógeno pode influenciar regiões específicas
com o, por exem plo, os sítios locais de doenças periodontais e
periapicopat ias, fazendo com que o nível de reabsorção aum ente.
( Xiong et al., 2007) . Nestes processos patológicos da região
periodontal são envolvidas diversas células com o osteoblastos e
osteoclastos cuj as funções determ inam o desenvolvim ento da perda
da m assa óssea ( Zhang et al., 2007) . Nas periapicopat ias, além das
células ósseas, citocinas com o I L- 1 e TNF- alfa tam bém part icipam
deste processo, os quais tam bém são observados na osteoporose.
Assim , os m ecanism os celulares e m oleculares que levam a perda
óssea, são sem elhantes entre o processo inflam atório e a osteoporose
( Palom o et al., 2007) . Estes dois quadros envolvem reabsorção
óssea, entretanto a osteoporose não é a causa prim ordial da
periodontite apical, m as que pode contribuir para o progresso da
lesão ( Xiong et al., 2007) .
Diversos fárm acos têm sido estudados com o possíveis agentes
terapêuticos para suprir a deficiência do estrógeno. Dentre estes,
destacam - se os m oduladores seletivos do receptor de estrógeno
( MSRE) que são da classe de m oléculas não horm onais e que
dependendo do receptor de estrógeno que se liga, podem
desencadear efeitos agonistas ou ant agonistas sobre o receptor de
estrógeno presente no tecido- alvo ( Rossi et al., 2010) . O cloridrat o
de raloxifeno ( RLX) , MSRE de segunda geração, m im etiza os efeitos
23 endom étrio ( Lewis et al., 2005) . Est udos m ostram que a t erapia com
raloxifeno resulta no aum ento da densidade m ineral óssea,
dim inuindo significativam ente a incidência de frat uras ( Siris et al.,
2005; Delm as et al., 2002; Ettinger et al., 1999) .
O m ecanism o de ação m olecular deste MSRE envolve alta
afinidade de ligação com o receptor de estrógeno, provocando
alteração conform acional na estrutura do receptor, sua dim erização e
associação com elem entos resposta do DNA específicos para o RLX
( Dutert re et al., 2000; ) . Devido a expressão dos receptores em
órgãos distintos, há seleção de tecidos para sua at uação. Port anto
dependendo de qual for receptor que se liga o efeito pode ser
antagonista ( com o em m am a e útero) não estim ulando as vias
estrogênicas, assim não causando tum ores nestes tecidos, ou pode
ter o efeito agonist a exercendo sua função anti- reabsortiva ( Perez,
2006; Sliwinski et al., 2005) .
Diante do exposto e considerando a im port ância da terapêutica
com MSRE é im portante à realização de estudo experim ental para
análise da ação do raloxifeno sobre a lesão periapical em organism os
24 Proposição
Obj etivo geral:
Analisar a atuação do raloxifeno na lesão periapical em rat as
ovariectom izadas;
Obj etivos específicos:
Avaliar e com parar a lesão periapical de 7 e 30 dias em rat as
adultas sham e ovariectom izadas ( OVX) pela coloração de
hem atoxilina e eosina;
Analisar e com parar a ação de raloxifeno na região periapical de
rat as sham e OVX, utilizando a expressão das proteínas
RANKL,TRAP, OPG, HI F-1α e BALP com o indicadores celulares
de predisposição à reabsorção ou form ação de tecido ósseo;
Analisar e com parar as concentrações dos m arcadores de
atividade celular do m etabolism o ósseo ( cálcio, fósforo e
25 Artigo 1
Effect of raloxifene on periapical lesions in ovariectomized rats
Abstract
I ntroduct ion: The aim of this st udy was to evaluate the effect of
raloxifene ( RLX) on periapical lesions in ovariectom ized ( OVX) rat s.
Methods: Fem ale Wistar rat s were OVX or subj ected to sham surgery
and received vehicle or RLX by gavage for 60 days. The treatm ent
groups were as follows: sham / vehicle, ( SHAM- veh) , OVX- veh and
OVX- RLX. During treatm ent, t he pulp of lower first m olar was
exposed to the oral environm ent allowing the lesions analysis on day
7 and 30. Blood sam ples were taken, the rat s were killed, and the
m andibles rem oved and prepared for radiographic, histopathological,
histom etric, and im m unohistochem ical analysis. Result s: Estradiol
plasm a concentration showed hypoestrogenism in OVX rat s. The
OVX- veh group showed larger periapical lesions with m ore resorption
lacunae and cells positive for tart rate- resistant acid phosphatase
( TRAP) . This condit ion was revert ed in OVX/ RLX group being sim ilar
to sham . Radiographically, t he groups were sim ilar but lesions on day
7 were sm aller than lesions on day 30. Conclusion: The results
showed that hypoestrogenism potentiates the progression of
periapical lesions, and this potentiation is dim inished by RLX due its
26 Keywords: Raloxifene hydrochloride, periapical lesion, bone
m etabolism , ovariectom ized.
Introduction
The growing elderly population has led to increasing interest in
the treatm ent and prevention of diseases associated with the aging
process ( 1) . This interest has encouraged a large num ber of
investigators to conduct new studies on anim al m odels of t he disease
processes related to aging. I n the current study, the effects of
raloxifene ( RLX) on periapical lesions in ovariectom ized rat s ( OVX)
were exam ined as a m odel of post - m enopausal osteoporotic bone
loss.
Periapical lesion st art s with inflam m ation and necrosis of the
tooth pulp. Bacterial growt h reaches the channel system recruiting
inflam m atory cells, inducing osteoclasts and prom ot ing bone
resorption ( 2) . Bone m etabolism is also regulated by estrogen that
interacts with cells involved in bone rem odeling: osteocytes,
osteoblasts, and osteoclasts ( 3) . Therefore, these cells can be
influenced by bot h system ic ( e.g., estrogen) and local factors ( e.g.,
inflam m at ion and necrosis) (4) .
Low estrogen concentrat ion in plasm a produces changes in
27 are extrem ely susceptible to changes in estrogen levels ( 5) . Estrogen
has been shown to influence bone resorption and its deficiency
aggravates osteopenia ( 6) and apical periodontitis ( 7) . Thus, it is
im port ant to underst and the m echanism s involved in t he interaction
between system ic and local factors.
Many t herapies have been studied t o treat and prevent t he
condit ions resulting from m enopause. However, som e of these
treatm ents have been associated with side effects like bloating and
breast tenderness ( 8) , or even breast and uterine tum ors ( 9) . RLX, a
benzothiophene analogue, has been approved for the treatm ent and
prevention of osteoporosis in postm enopausal wom en ( 10) . RLX has
been shown to increase bone m ineral density and reduce the
incidence of bone fractures ( 10- 11) ; it has not been associated with
breast and endom etrial t um ors due to its specific receptor activity; it
does not activate estrogen receptors in breast or uterine tissues ( 12) .
Therefore, RLX is indicated in pat ients with a history of breast and/ or
endom etrial neoplasm s, or other factors that contraindicate horm one
replacem ent therapy ( 13) .
Alt hough knowing the indication, there are no studies
evaluating t he effects of RLX on periapical lesions in anim als with
hypoestrogenism . Therefore, the aim of the present st udy was to
28 Material and methods
Anim als
Fort y- eight fem ale Wistar rat s ( six m onths of age) were used.
The experim ental procedures were approved by the instit utional
ethics com m ittee ( Ethics Com m it tee on Anim al Use – Univ Estadual
Paulista - 00799- 2012) . The anim als were distributed into 6 groups:
sham surgery plus vehicle treatm ent with 7 days ( SHAM- veh 7d) or
30 days of pulp exposure ( SHAM- veh 30d) ; OVX plus vehicle
treatm ent with 7 days ( OVX- veh 7d) or 30 days of pulp exposure
( OVX- veh 30d) ; and OVX plus RLX with 7 days ( OVX- RLX 7d) or 30
days of pulp exposure (OVX- RLX 30d) .
Estrous cycle
Det erm ination of t he estrous cycle status of t he rat s was
perform ed by swab vaginal collection ( 9 am ) . The swabs were
exam ined using an optical m icroscope, according the technique of
Long and Evans ( 14) . Fem ales with regular cycle ( 12 days) were
random ly distributed into the experim ental groups. Fem ales that did
not have a regular estrous cycle were excluded from t he study.
Ovariectom y
Rats were OVX or subj ected to sham surgery. The anim als were
29 Health Ltd, São Paulo, Brazil) and xylazine ( 25 m g/ kg; Coopazine,
Coopers Ltd. Brazil, São Paulo, Brazil) by intraperitoneal inj ection. An
abdom inal incision was m ade to expose the distal port ions of the
fallopian t ubes. Then the ovaries were rem oved in the OVX groups. I n
the sham groups, after the incision t he ovaries were exposed but not
rem oved. All anim als had their incisions closed with sutures and
received an intram uscular dose of ant ibiot ics ( 1 m L/ kg, Pentabiot ic
Veterinary, Fort Dodge Anim al Health Ltd, São Paulo, Brazil) .
Raloxifene or vehicle treatm ent
Ten days after OVX or sham surgery, vehicle ( dist illed water 0.3
m L) or raloxifene (Sigm a- Aldrich, Munich, Germ any) ( 1 m g/ kg in 0.3
m L dist illed water) was adm inistered daily by gavage, for 60 days
( 15- 17) .
Periapical lesion induction
During the treatm ent it was perform ed the periapical lesion
induction under general anesthesia. The right and left m andibular
first m olars had their pulp exposed to t he oral environm ent with the
aid of carbon drill bur ( Drill Long Neck Ln, Maillefer, Dentsply,
30 Sam ple Collection
Under general anesthesia, blood was collected from the j ugular
vein ( 18) , centrifuged ( 3,000 rpm for 20 m in at 2° C) and t he plasm a
stored in a freezer at - 20° C for m easurem ent of estradiol. The
anim als were killed by anesthet ic overdose and the m andibles
rem oved for radiographic, histopathological, histom etric, and
im m unohistochem ical analysis. The uteruses were also rem oved and
weighed.
Plasm a estradiol level m easurem ent and uterus weight
Plasm a estradiol concentrat ion was m easured in duplicate using
a Biom edicals estradiol kit by radioim m unoassay ( Cost a Mesa, CA,
USA) . The m inim um detectable dose of estradiol was 5.0 pg/ m L and
the intra- assay value was 3.9% . Uterus weights were determ ined on
a precision balance ( Mett ler Toledo, Barueri, SP – Brazil) .
Radiographic analysis
The m andibles were fixed in 4% form aldehyde for 24 hours.
The radiographs were obtained using a digital X- ray m achine ( Dabi
Atlante Spectro 70/ 10 ® , Ribeirão Preto, São Paulo, Brazil) with
calibration at 70 kV and 10 m A, 12 pulses and 40 cm of focal length.
The radiation incidence was focused perpendicular to the film - obj ect
31 Orion Corporat ion, Helsinki, Finland) and an alum inum penetrom eter
( 6063 alloy) were used to capture im ages ( 24 bits in TI FF form at
( tagged im age file form at ) . The m ost central port ion of the lesion was
selected for analysis and ten repeated m easurem ents were perform ed
to determ ine the average bone densitom etry ( pixel) ( 19) .
Histopathological and im m unohistochem ical analysis
Mandibles were decalcified in 10% ethylenediam inetetraacet ic
acid ( EDTA) for 60 days, subj ected to conventional histological
processing, em bedded in paraffin, and cut into sem i- serial sect ions.
Sections were either stained wit h hem atoxylin and eosin or subm it ted
to im m unohistochem istry using an indirect im m unoperoxidase
technique for tart rate- resistant acid phosphat ase ( TRAP) ( prim ary
antibody goat anti- TRAP SC 30832, Santa Cruz Biotechnology, Santa
Cruz, CA) following previously described protocol ( 20) .
Histological analysis was conducted by a certified histologist
( EE) using the following param et ers: nature and ext ension of
inflam m at ion, presence and extension of necrosis, vasculature state,
and pattern of cellularity of dental and periodontal t issues.
For the histom etric analysis of the periapical lesion, the distal
root of the m andibular first m olar was exam ined using Leica
32 periapical lesion were external cem entum surface, periodontal
ligam ent, and the external alveolar bone surface. The area of the
periapical lesion in m m ² was calculat ed at five equidistant sect ions
and t he widest area was selected.
Only m at ure osteoclasts were quantified as TRAP- positive
m ult inucleated cells that were quant ified in the perim eter of lesion.
The results were expressed as m ultinucleated TRAP- positive cells per
m m2.
Stat ist ical Analysis
Data were tabulated and statistically analyzed using analysis of
variance (ANOVA) for multiple comparisons. Tukey’s test was used for
pairwise com parisons using Sigm aplot software ( San Jose, CA – USA)
33 Results
Serum estradiol levels and uterus weight
At 7th and 30th days, OVX rat s showed levels significant ly lower
than rat s in t he sham groups ( p< 0.001) . Moreover, the uteruses of
the OVX rat s weighed less than the ut eruses of sham rat ( p< 0.001) .
The veh 30d group showed higher serum estradiol than
SHAM-veh 7d group ( p< 0.0001) . Estradiol level or uteruses weigh were not
affected by RLX treatm ent in OVX rats ( p> 0.05) (Tab. 1) .
Radiographic analysis
The radiographic density of the periapical lesion at 30 days
( Fig.1 D- F) after exposure to the oral environm ent was significantly
lower than after 7 days ( Fig.1 A- C) . However, there were no
significant differences between groups at the sam e tim e point
( Tab.1) .
Histological analysis
Histology im ages of periapical lesions in t he different
experim ental groups are shown in Figure 1 ( G- R) .
On day 7, SHAM- veh 7d and OVX- RLX 7d showed the root canal
34 rem nants ( Fig.1 G- H, O- P) . The periapical region showed an
inflam m atory infiltrat e com posed predom inantly by
polym orphonuclear neutrophils. The surface of the alveolar bone
dem onstrat es large num bers of active osteoclasts. The root canal was
occupied by necrotic debris and t he periapical region had an intense
polym orphonuclear inflam m atory infiltrate that reached t he alveolar
bone. The alveolar bone surface showed large num ber of active
osteoclasts and resorption lacunae in alm ost of t he whole extent of
the lesion.
On day 30, the root canal was com posed by necrotic t issue in
m ost specim ens. The inflam m atory infiltrate was m ore intense with
m any lym phocytes and som e neutrophils. On the surface of the
alveolar bone, a large num ber of active osteoclasts were observed.
The OVX- veh 30d group had necrotic pulp rem nants in the root canal
and a large periapical lesion ( Fig.1 M- N) . The inflam m atory infiltrat e
was com posed of lym phocytes and neutrophils and was larger area
than the infiltrat es in the other treatm ent groups. The surface of the
alveolar bone was irregular due to the large num ber of resorption
lacunae with active osteoclasts.
Histom etric analysis
The periapical lesion areas were larger in OVX- veh groups when
35 periapical lesions in OVX- RLX groups was sim ilar to that in SHAM- veh
( p> 0.05) ( Tab 1) . There was an increase in the size of periapical
lesions in all experim ental groups when com paring day 30 with day 7
of pulp exposure to the oral environm ent (Fig. 2A) ( Tab.1) .
TRAP im m unohistochem istry
I m m unohistochem ical technique for TRAP was highly specific to
osteoclasts. Labeling was confined to the cytosolic com part m ent of
predom inant ly m ultinucleated cells. I m m unostaining of TRAP is
shown in Fig 2B- E.
The num ber of TRAP- positive m ultinucleated cells per m illim eter
on the periapical lesion perim eter in OVX- veh was higher than that
observed in SHAM- veh and OVX- RLX ( p< 0.001) ( Tab.1) . The num ber
of TRAP- positive cells in OVX- RLX groups was sim ilar to that in
SHAM- veh ( p> 0.05) ( Tab.1) . I n OVX- veh, t here was an increase in
the num ber of TRAP- positive m ult inucleated cells on day 30
com pared with day 7. This difference between the tim es points did
not occur in the ot her groups.
Discussion
OVX rat s are an experim ental m odel used to sim ulate the bone
36 the efficacy of OVX in producing hypoest rogenism in t his st udy can be
seen in plasm a levels of estradiol as OVX rat s showed levels
significantly lower than rat s in t he sham groups. Moreover, the
uteruses of the OVX rat s weighed less t han the uteruses of sham rat ,
showing t he decrease in serum estradiol concentrat ion resulted in
uterine atrophy.
Serum estradiol concentrat ion of SHAM- veh 30d rat s was higher
than SHAM- veh 7d rat s. This was probably due to the phase of the
estrous cycle ( 22) . The SHAM- veh 30d rat s were in the proestrus
phase and SHAM- veh 7d rat s in diestrus. These results characterize
the anim als at different stages of the estrous cycle with serum
estradiol very oscillating during the est rous cycle (23) .
I n the present study, the uteruses of rat s in t he OVX- RLX 7d
and OVX- RLX 30d groups were not significantly different in weight
when com pared to the OVX- veh at both tim e points. This finding
indicates t hat RLX had no effect on t he uterus, which support s its
report ed estrogen antagonism act ion on the receptor present in this
organ ( 24) . This lack of effect on the uterus is an im port ant aspect to
be considered when selecting treatm ent with RLX for patients wit h a
history of uterine cancer.
Radiographic analysis showed differences between groups at
day 7 and day 30. However, there were no significant differences
37 radiographic analysis techniques that are not sufficient ly sensitive to
detect differences. More sensitive m ethods ( e.g., m icro- or cone beam
com puterized tom ography ( 25- 26) ) m ay be useful in future studies.
I n this study, the data indicated an im balance in bone
rem odeling after OVX, with exacerbat ion of the resorptive process
due to a large num ber of active osteoclastic cells and resorption
lacunae and the larger size of the periapical lesion when com pared to
sham rat s. Som e studies indicate that OVX m ay trigger changes in
bone m etabolism , increasing bone turnover as well as cytokines and
osteoclast num ber ( 27- 28) . Studies with postm enopausal wom en
have dem onstrat ed that they have significantly lower bone m ineral
density when com pared to prem enopausal wom en ( 29) which could
also indicate an exacerbation of bone resorption processes.
The reduction in estrogen plasm a concentrat ion during
m enopause, results in higher bone m etabolic activity ( 30) , which can
influence the survival of osteoclast s ( 31) . I t was shown that the
treatm ent with estrogen dim inished bone resorption by prom oting a
reduction in the num ber of osteoclasts. Rats t hat did not receive
estrogen therapy showed m ore TRAP- positive cells ( 32) . This was in
agreem ent with t he results of the present study, in which
im m unostaining for TRAP showed fewer TRAP- posit ive cells in the
38 Raloxifene in post m enopausal wom en increased bone density
by its agonist effect, dim inishing the action of osteoclast s ( 33- 34) .
Ost eoclast activity and lesion size was dim inished wit h RLX treatm ent
being sim ilar to t he SHAM group, dem onstrat ing the role of RLX in
decreasing resorptive activity and dim inishing bone loss. These were
sim ilar effects to those seen in a previous study ( 35) .
More studies are needed to better underst and the m echanism s
involved in the m oderat ion of periapical lesions in anim als wit h
hypoestrogenism treated with RLX. Nevertheless, the results of the
present study showed that RLX was effective in m im icking the
protective effect of estrogen, preventing the deleterious effects of
estrogen deficiency and decreasing the osteoclasts activit y and bone
turnover.
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44 Figure 1
Radiographic and histologic aspects of periapical lesions at 7 and 30
days after pulp exposure to the oral environm ent. A – F: Periapicial
radiographs of the m andibular first m olar in groups SHAM- veh7d ( A) ,
45 OVX- RLX30d ( F) . Periapical lesions of greater severity were observed
in OVX- veh 7d rat s ( B) and OVX- veh 30d rat s ( E) than in SHAM or
RLX treated. G – R: Photom icrographs showing t he histological
appearance and m agnitude of inflam m ation in periapical lesion at 7
and 30 days after pulp exposure to t he oral environm ent in groups
SHAM- veh7d ( G – H) , SHAM- veh30d ( I – J) , OVX- veh7d ( K – L) ,
OVX- veh30d ( M – N) , OVX- RLX7d ( O – P) , OVX- RLX30d ( Q – R) . A
greater m agnit ude of inflam m atory response was seen in OVX- veh 7d
( K – L) and OVX- veh 30d ( M – N) com pared to other treatm ent
groups. Periapical inflam m atory responses were sim ilar in SHAM- veh
( G – J) and OVX- RLX ( O – R) groups at day 7 and day 30 days after
pulp exposure to the oral environm ent. Abbreviations and sym bols: * ,
inflam m atory infiltrate; ab, alveolar bone; ce, cem entum ; n, necrotic
pulp rem nants. G – H: H&E staining; scale bars: G, I , K, M, O and Q,
300 μm; H, J, L, N, P, and R, 100 μm. Original magnification: G, I, K,
46 Figure 2
A: Periapical lesion area ( m m ² ) in dist al root of the m andibular first
m olar in different treatm ent groups. B: The num ber of TRAP- positive
m ult inucleated cells per m m in t he perim eter of the periapical lesion
of the distal root of the m andibular first m olar in different treatm ent
groups. C – E: Photom icrographs showing TRAP- positive
47 to the oral environm ent in groups SHAM- veh7d ( C) , OVX- veh7d ( D)
and OVX- RLX7d (E). Abbreviat ion: ab, alveolar bone; bv, blood
vessel. Hem atoxylin counterst aining; scale bars: C – E, 20 μm.
Original m agnificat ion: C – E, x1000. Sym bols: #p<
0.05vsSHAM-veh7d and OVX- RLX7d; §p< 0.05 vs SHAM- veh30d and OVX- RLX30d
48 Table 1
Experimental groups
Serum estradiol levels (pg/mL)
Uterine weight (g)
Radiographic density (pixel)
Periapical lesion (mm2)
TRAP (cells/mm2) SHAM-veh 7d 331.2 ± 146.9b 0.54 ± 0.12b 82.84 ± 4.49a 0.37 ± 0.04a 4.48 ± 1.60a
OVX-veh 7d 146.9 ± 39.8a 0.25 ± 0.15a 82.58 ± 5.03a 0.57 ± 0.06b 7.68 ± 1.34b
OVX-RLX 7d 136.0 ± 45.1a 0.21 ± 0.06a 83.24 ± 5.23a 0.31 ± 0.07a 3.34 ± 1,46a
SHAM-veh 30d 818.0 ± 159.9c 0.59 ± 0.16b 77.04 ± 5.24b 1.26 ± 0.24c 5.55 ± 2.07a
OVX-veh 30d 125.8 ± 31.1a 0.19 ± 0.14a 77.05 ± 5.13b 2.55 ± 0.45d 9.63 ± 5.15c
OVX-RLX 30d 119.9 ± 34.4a 0.19 ± 0.06a 78.82 ± 5.12b 1.39 ± 0.26c 4.45 ± 1.03a
Serum estradiol levels, uterus weight, radiographic density, periapical
lesion areas and TRAP- positice cells according to the groups. I n a
49 Artigo 2
Raloxifene decreases osteoclast activity in apical lesion
Abstract
I n the present study, it was evaluat ed the raloxifene ( RLX) action
during the periapical lesions developm ent in ovariectom ized ( OVX)
rat s. Wistar rat s ( 6 m onths) were distributed into groups: SHAM- veh,
OVX- VEI e OVX- RLX that received for 60 days vehicle or RLX by
gavage. During treatm ent it was perform ed the exposition of the pulp
of lower first m olar to the oral environm ent on the scheduled day
allowing lesion analysis after 7 and 30 days. Ninety days after the
treatm ent, blood was collected for m easurem ent of calcium ,
phosphorus, alkaline phosphatase and estradiol. The anim als were
killed and m andibles were subm it ted to im m unohistochem ical
analysis. Estradiol plasm a concentrat ion showed hypoestrogenism in
OVX rat s. On day 7, the alkaline phosphatase activity and plasm a
calcium were higher in OVX- RLX than SHAM- veh group ( p< 0.0002) .
The plasm a concent rat ion of phosphorus was higher in RLX group, in
both tim e points ( p< 0.0001) . On day 7, OVX- veh group showed m ore
im m unostaining j ust for receptor activator of nuclear fact or kappa- B
ligand ( RANKL) and hypoxia inducible factor - 1 alpha ( HI F1- alpha)
than other groups ( p< 0.05) . On 30 days, OVX- veh showed higher
im m unostaining j ust for RANKL than OVX- RLX ( p< 0.05) . The RANKL
50 sim ilar to SHAM- veh in both tim e points. There was no stat ist ical
difference in im m unoreactivity for osteoprogeterin ( OPG) and
bone-specific alkaline phosphatase ( BALP) between all groups in both tim e
points ( p> 0.05) . The results showed that RLX therapy m ay be
beneficial due decreasing osteoclast act ivity and bone turnover
preventing the delet erious effects of estrogen deficiency.
Keywords: RLX hydrochloride, periapical lesion, bone
m etabolism , OVX.
Introduction
The num ber of elderly is increasing faster than any other age
group in the world ( Kinsella & He, 2008) . Elderly presents several
physiological changes that predispose pathological condit ions of
aging, such as osteoporosis ( Christenson et al. 2012) . Thus, studies
are needed to im prove the life quality of these individuals.
The skeleton is m aintained by continuous regenerat ion that
occurs by the bone rem odeling process ( Manolagas 2000) . Bone
rem odeling is regulated by different m echanism s, such as m em bers
of the tum or necrosis factor superfam ily, the receptor activator of
nuclear factor kappa- B ( RANK) , RANKL and OPG, which are essential
51 RANKL is expressed on osteoblasts, fibroblasts and B and T cells
being a key cytokine in osteoclastogenesis ( Teitelbaum 2000, Lerner
2006) . The RANKL binds to RANK that is present in the
pre-osteoclasts, and st im ulates osteoclast differentiat ion, whereas the
binding of RANKL to OPG inhibits osteoclastogenic act ivity ( Yasuda et
al 1998, Takayanagi et al. 2000, Kotake et al. 2001) . Several
system ic and local factors, such as horm one presence, influence the
RANK/ RANKL/ OPG system ( Horowitz et al. 2001, Lerner 2004) . The
estrogen decrease bone resorption and increase bone form ation by
stim ulat ing OPG secretion and RANKL inhibition ( Kohli & Kohli 2011) .
Ot her m ediators part icipate in the regulation of bone
m etabolism such as HI F- 1 alpha, tart rate- resistant acid phosphatase
( TRAP) and alkaline phosphatase besides the TNF superfam ily
m em bers ( Wan et al. 2008, Bezerra et al. 2011, Press et al. 2014) .
The bone m etabolism is m ainly regulat ed by estrogen, which is
essential for the bone tissue m aintenance because it int eracts with
cells involved in bone rem odeling, as osteocytes, osteoblasts and
osteoclasts ( Khosla et al. 2012) , which in turn are influenced by
system ic and local factors ( Raisz & Rodan 1998) .
Ost eoporosis is a pathological process resulting from the
estrogen plasm a concentrat ion decrease, which occurs in m enopausal
wom en ( Bedell et al. 2012) . These changes increase resorption,
52 decreased bone density and subsequent fracture risk ( Anbinder et al.
2006, Arm as & Recker 2012) . Ost eoporosis interacts with local
factors, such as periapical lesion, aggravating t he bone loss ( Pallos et
al. 2006) .
Estrogen deficiency affects specific regions such as apical
periodontit is increasing t he absorption sites ( Xiong et al. 2007).
During the periapical lesion form at ion, the bone cells and m ediators
such as RANK/ RANKL/ OPG part icipate in this process, which are also
observed in osteoporosis ( Zupan et al. 2012, Wan et al. 2014) . Thus,
the cellular and m olecular m echanism s that lead to bone loss, are
sim ilar between inflam m at ion process and osteoporosis ( Palom o et al.
2007) .
The RLX m im ics t he beneficial effects of estrogens without
stim ulat ing t issues such as breast and endom etrium ( Lewis & Jordan
2005) once it binds to different receptors ( Rey et al., 2009) . St udies
dem onstrat e that RLX treatm ent results in increased bone m ineral
density and significantly reduce the fractures incidence ( Carneiro et
al. 2012, Mirkin et al. 2014, Pinkerton et al. 2014) . Raloxifene
influences bone rem odeling cells as osteoblasts and ost eoclast and
inflam m atory m ediators as RANK/ RANKL/ OPG ( Mencej -Bedrač et al.
2014) .
I t was report ed that the progression of periapical lesion was
53 bisphosphonates ( Xiong et al. 2007) . However, there is no study
evaluating t he effect of RLX on periapical lesion progression. This
way, the aim of the present study was to evaluate the RLX action on
periapical lesions in hypoestrogenic organism s.
Material and methods
Anim als
For this study, 48 fem ale Wistar rat s ( six m onths of age) from
the Faculty of Dentistry of Araçat uba/ UNESP were used. The
experim ental procedures proposed in this study were approved by
inst itutional ethics com m it tee ( Ethics Com m ittee on Anim al Use –
Univ Estadual Paulista - 00799- 2012) . The anim als were distributed
into 6 groups according to the pulp exposure tim e point , ovariectom y
and treatm ent : sham surgery plus vehicle treatm ent with 7 days
( SHAM- veh 7d) or 30 days of pulp exposure ( SHAM- veh 30d) ; OVX
plus vehicle treatm ent with 7 days ( OVX- veh 7d) or 30 days of pulp
exposure ( OVX- veh 30d) ; and OVX plus RLX with 7 days ( OVX- RLX
7d) or 30 days of pulp exposure (OVX- RLX 30d) .
Estrous cycle
The Long and Evans technique ( 1922) was perform ed for the
54 collection was perform ed ( 9: 00 am ) and exam ined using an opt ical
m icroscope. Rats presenting regular est rous cycle ( twelve days) were
selected and random ly divided into the experim ental groups. Fem ales
showing irregular estrous cycle were excluded from the study.
Ovariectom y
The surgical procedure was perform ed under anest hesia with
ket am ine ( 75 m g/ kg – by intraperit oneal; Vetaset - Fort Dodge
Anim al Health Lt d - São Paulo - Brazil) and xylazine ( 25 m g/ kg – by
intraperitoneal; Coopazine - Coopers Ltda Brazil - São Paulo - Brazil) .
I n the ovariectom ized group the distal port ion of the fallopian t ubes
were exposed and the ovaries were rem oved. I n t he sham groups,
the ovaries were exposed but not rem oved. After the surgery, all
anim als had their incisions closed with sut ure and received an
intram uscular dose of antibiot ics ( 1m L/ kg, Pentabiot ic Veterinary -
Fort Dodge Anim al Health Ltd - Sao Paulo - Brazil) .
Raloxifene ou vehicle trat m ent
Ten days after ovariectom y or sham surgery, the rat s received
daily vehicle ( dist illed water - 0.3 m L) or RLX ( Sigm a- Aldrich, Muhich,
Germ any) ( 1 m g/ kg in 0.3 m L dist illed water) by gavage for 60 days
55 Periapical lesion induction
During the treat m ent t he pulp exposition to t he oral
environm ent was perform ed for lesion analysis on 7 and 30 days. All
pulpal exposures of m andibular first m olars were standardized with
0.1 m m diam eter, with the aid of carbon drill bur ( Drill Long Neck Ln
- Maillefer, Dentsply - Catanduva - Brazil) , under general anesthesia.
Sam ple Collection
Ninety days after the beginning of adm inistrat ion of RLX or
vehicle, anim als were anesthetized for blood collection from the
j ugular vein ( Harm s & Oj eda, 1974) . The blood was centrifuged
( 3.000 rpm - 20 m in – 20 C) and plasm a stored in a freezer at - 20 º C
for m easurem ent of calcium , phosphorus, alkaline phosphatase and
estradiol. The anim als were killed by anesthetic overdose and the
m andibles rem oved for im m unohistochem ical analysis. The uteruses
were also rem oved used and weighed.
Plasm a estradiol level and uterus weight
The estradiol m easurem ent was perform ed according to
Biom edicals estradiol kit protocol by radioim m unoassay ( Costa Mesa,
CA - USA) . All sam ples were assayed in duplicate and in the sam e
56 estradiol was 5.0 pg/ m L and the intra- assay value was 3.9% . The
uterus weights were determ ined on a precision balance ( Mettler
Toledo, Barueri, SP – Brazil) .
Biochem ical Analysis
Biochem ical analysis of calcium , phosphorus and alkaline
phosphat ase were perform ed to analyze cellular activity of bone
m etabolism . Measurem ents were perform ed using com m ercial kit s
( Calcium : Labtest catalog 90 – Lagoa Santa/ MG – Brazil; Phosphorus:
Labtest Catalog 40 – Lagoa Santa/ MG – Brazil; Alkaline phosphatase:
Labtest Catalog 40 – Lagoa Santa/ MG – Brazil) .
I m m unohistochem ical processing
Mandibles were decalcified in 10% ethylenediam inetetraacet ic
acid ( EDTA) for 60 days, subj ected to conventional histological
processing, em bedded in paraffin, and sliced into sem i- serial
sect ions. Sections were either stained with hem atoxylin and eosin or
subm itted to im m unohistochem istry using an indirect
im m unoperoxidase technique for OPG ( Rabbit anti- opg – SC11383 –
Santa Cruz Biotechnology, Santa Cruz, CA), RANKL ( Goat anti- rankl –
SC7627 - Santa Cruz Biotechnology, Santa Cruz, CA), HI F- 1α ( Rabbit
57
alkaline phosphatase ( Goat – SC15065 - Santa Cruz Biot echnology,
Santa Cruz, CA) following previously described protocol ( Garcia et al.
2013) .
RANKL, OPG, HI F- 1α and BALP were analyzed in the proxim ity
of the periapical lesion with a 400x m agnification ( Leica
Microsystem s, Wetzlar, Germ any). A sem i- quant itative
im m unolabeling analysis was perform ed. Three histologic sect ions
from each anim al was used and the criteria for establishing the
im m unoreactivity pattern was m odified ( Kim et al. 2007) : score 0
( zero im m unoreactivity pat tern) : total absence of cells
im m unoreactive ( I R) ; score 1 ( low im m unoreactivity patt ern) : about
1/ 4 I R cells per area; score 2 ( m oderat e im m unoreactivity pat tern) :
around 1/ 2 I R cells per area; score3 ( high sim m unoreactivity
pattern) : about 3/ 4 I R cells per area.
Stat ist ical Analysis
Data were tabulated and analyzed statist ically using Sigm aplot
software ( San Jose, CA – USA) . Multiple com parisons were perform ed
using Kruskal- wallis followed by Dunn test for nonparam etric data.
And analysis of variance ( ANOVA) followed by Tukey test w as
58 Results
Serum estradiol levels and uterus weight
I n both tim e points, the sham rat s showed levels significantly
higher t han in the OVX rat s groups ( p< 0.001) . I n addit ion, the
uteruses of the sham rat s weighed m ore than the uteruses of OVX
rat s ( p< 0.001) . The SHAM- veh 7d group showed lower serum
estradiol than SHAM- veh 30d group ( p< 0.0001) . Uteruses weigh and
estradiol level were not affected by RLX treatm ent in OVX rat s
( p> 0.05) (Tab. 1) .
Biochem ical analysis
The results obtained of plasm a concentrat ions of calcium ,
phosphorus and alkaline phosphat ase of the experim ental anim als are
shown in Figure 1.
The plasm a calcium concentrat ion of OVX- RLX- 7 d was higher
than SHAM- veh 7d ( p= 0.0002) . I n the SHAM- veh 30d, t he plasm a
concentrat ion of calcium was higher t han SHAM- veh 7 ( p= 0.0002) .
After 30 days, the plasm a calcium concentrat ion was lower in the
OVX- RLX com pared with SHAM- veh 30 d ( p= 0.0002) and OVX- RLX 7
59 I n OVX- RLX group in both tim e points, the plasm a phosphorus
concentrat ion was higher than veh 7 d ( p< 0.0001) ,
SHAM-veh- 30 d (p< 0.0001) and OVX- veh 30 d (p< 0.0001) .
The alkaline phosphatase act ivity was higher in OVX- RLX 7 d
than SHAM- veh 7 d ( p< 0.0001) . The enzym e activit y was higher in
SHAM- veh 30d and OVX- Veh 30 d when com pared with t he
corresponding groups, with 7 days (p< 0.0001) .
I m m unohistochem istry
I m m unohistochem ical technique for RANKL, OPG, BALP and
HI F- 1α was highly specific in the detect ion of t hese proteins. Labeling
was confined to t he cytosolic com partm ent of the im m unoreactive
cells and the extracellular m atrix.
Labeling for RANKL, OPG and BALP was confined predom inantly
in osteoblasts, while HI F- 1α was expressed in fibroblasts, endothelial
cells and osteoblast s.
The photom icrographs showing t he histological appearance of
im m unolabeling and the im m unoreact ivit y pattern for RANKL, OPG,
60 SHAM- veh
On day 7 and 30, the im m unostaining for OPG and BALP
prevailed low. On day 7, the im m unolabeling for RANKL prevailed low
and for HI F- 1α prevailed m oderat e . On day 30, the im m unoreactivity
pattern for RANKL prevailed m oderat e and HI F- 1α was low to
m oderat e.
OVX- veh
The im m unostaining pat tern for OPG and BALP prevailed low on
day 7 and 30. I t was noted im m unost aining pattern high for HI F- 1α
and RANKL on day 7. On day 30, the im m unoreactivity pattern for
RANKL was m oderat e to high and for HI F- 1α prevailed m oderat e.
OVX- RLX
The im m unostaining for OPG and BALP prevailed low on day 7
and 30. On day 7, the im m unolabeling pattern for RANKL prevailed
low and for HI F- 1α m oderat e. On day 30, the im m unoreactivity
61 Discussion
Conventionally, st udies involving ovariectom y perform the
determ ination of plasm a estrogen and weighing of uterus for
confirm atory procedure ( García- Pérez et al. 2006) . I n t he present
study, the estrogen level in the OVX group was lower com pared with
sham rat s, indicating that ovariectom y result ed in decrease in plasm a
estrogen. I n addition, it was noted that the OVX rat s uterus were also
lighter than sham rat s uterus, confirm ing again the efficacy of
ovariectom y, since t he absence of serum concentrat ion of estrogen
results in atrophy of this organ ( Lopez- Belm onte et al. 2012) . The
uterus of rat s treated with RLX did not suffer any m odification
com pared to the sham rat s, showing t heir antagonist ic effect on the
receptor present in this organ. Based on such observation, RLX has
been indicated for patient s with a hist ory of uterine cancer due their
antagonist ic effect on this organ ( Jolly et al. 2003) .
The plasm a concentrat ion of estrogen in sham rat s, after 30
days of pulpar exposition, was higher and corresponds to proestrus
phase and t he anim als of the 7 days in diestrus. These results
characterize the anim als at different st ages of the estrous cycle with
plasm a level of est rogen is very oscillating during the estrous cycle
( Marcondes et al. 2002) .
I t could be observed that serum calcium level in OVX- RLX was
