Efeitos da estimulação elétrica de baixa intensidade sobre o metabolismo ósseo de ratas ovarectomizadas
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Data
2008
Autores
Lirani-Galvão, Ana Paula Rebucci [UNIFESP]
Orientadores
Lazaretti-Castro, Marise [UNIFESP]
Tipo
Tese de doutorado
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Resumo
A estimulação elétrica de baixa intensidade (EE) tem sido utilizada para o reparo
ósseo, mas pouco se sabe sobre seus efeitos no tecido ósseo após a
menopausa. Os osteócitos provavelmente desempenham um importante papel
mediando este estímulo físico, e poderiam agir como transdutores através da
liberação de sinais bioquímicos como o óxido nítrico (NO). O objetivo deste
trabalho foi investigar os efeitos da EE no metabolismo ósseo de ratas
ovarectomizadas, e se o NO poderia ser um mediador destes efeitos. Sessenta
ratas (200-220g) foram divididas em 6 grupos: SHAM; SHAM tratado com 6mg/d
de L-NAME, um inibidor da NO sintase (SHAM-L-NAME); ovarectomizado (OVX);
OVX tratado com L-NAME (OVX-L-NAME) ou submetido a uma estimulação
elétrica no corpo todo do animal (OVX-EE) ou ambos (OVX-EE-L-NAME) por 12
semanas. A densidade mineral óssea (DMO) global, de coluna e membro
posterior foram medidas através de densitometria óssea antes e ao final do
protocolo. Após o sacrifício, as tíbias foram retiradas para análises
histomorfométricas e imunohistoquímicas. A apoptose de osteócitos (técnicas de
caspase-3 e TUNEL) e expressões da NO sintase endotelial (eNOS) e indutível
(iNOS) foram analisadas por imunohistoquímica. As ratas OVX demonstraram
significante (p<0.05 vs SHAM) diminuição da DMO final global, de coluna e de
membro posterior, medidas por densitometria, redução de volume ósseo (10% vs
25%) e número de trabéculas (1.7 vs 3.9), e aumento das superfícies de
reabsorção (4.7% vs 3.2%) e mineralização (15.9% vs 7.7%), medidas por
histomorfometria. No entanto, após a EE, todos estes parâmetros foram
semelhantes aos valores do grupo SHAM e significantemente diferentes de OVX.
A EE não foi capaz de prevenir a redução de volume ósseo e número de trabéculas causadas pela OVX na presença de L-NAME (OVX-L-NAME vs OVXEE-L-NAME),
como o fez na ausência deste inibidor da NOS. Porém, o L-NAME
não bloqueou os efeitos da EE na reabsorção óssea (superfícies de reabsorção e
de osteoclastos) em ratas OVX. A eNOS e iNOS foram: expressas de forma
semelhante no córtex das tíbias de SHAM, não expressas em OVX, e
similarmente expressas em OVX-EE quando comparadas ao SHAM. As
expressões de eNOS e iNOS não foram detectadas no tecido ósseo de nenhum
grupo tratado com L-NAME. Nas ratas OVX, a porcentagem de osteócitos
apoptóticos (24%) foi significantemente maior do que em SHAM (11%) e OVX-EE
(8%). Todos os grupos tratados com L-NAME tiveram uma diminuição na
porcentagem de osteócitos apoptóticos. Assim, nosso estudo evidenciou que a
EE previne alguns efeitos causados pela OVX no tecido ósseo preservando a
DMO, estrutura e microarquitetura ósseas, expressão de eNOS e iNOS e
viabilidade de osteócitos. Além disso, foi demonstrado que o L-NAME bloqueia
parcialmente os efeitos da EE na estrutura óssea (mas não na reabsorção) e na
expressão de eNOS e iNOS em ratas OVX, sugerindo que o NO possa ser um
mediador dos efeitos positivos da EE no tecido ósseo. No entanto, não foi
possível identificar se os efeitos positivos da EE na viabilidade de osteócitos
foram mediados pelo NO, pois os efeitos do L-NAME nestas células foram
semelhantes àqueles causados pela EE.
Low Intensity Electrical Stimulation (LIES) has been used for bone repair but little is known about its effects on bone after menopause. Osteocytes probably play a role in mediating this physical stimulus and they could act as transducers through the release of biochemical signals, such as nitric oxide (NO). The aim of the present study was to investigate the effects of LIES on bone metabolism in ovariectomized rats, and if NO could be a mediator of these effects. Sixty rats (200-220g) were divided into 6 groups: SHAM; SHAM treated with 6mg/d of LNAME, an inhibitor of NO synthase (SHAM-L-NAME); ovariectomized (OVX); OVX treated with L-NAME (OVX-L-NAME) or subjected to a whole body electrical stimulation (OVX-LIES) or both (OVX-LIES-L-NAME) for 12 weeks. Global, spine and posterior limb bone mineral density (BMD) were measured by bone densitometry before and at the end of protocol. After sacrifice, tibias were collected for histomorphometric and immunohistochemistry analysis. Osteocyte apoptosis (caspase-3 and TUNEL techniques) and expressions of endothelial NO synthase (eNOS) and inducible NOS (iNOS) were assessed by immunostaining. OVX rats showed significant (p<0.05 vs SHAM) decreased final global, spine and limb BMDs, measured by densitometry, decreased bone volume (10% vs 25%) and trabecular number (1.7 vs 3.9), and increased eroded surfaces (4.7% vs 3.2%) and mineralization surfaces (15.9% vs 7.7%), measured by histomorphometry. In contrast, after LIES, all these parameters tended to be similar to SHAM and significantly different from OVX. LIES was not able to prevent the reduction of bone volume and trabecular number caused by OVX in the presence of L-NAME (OVX-L-NAME vs OVX-LIES-L-NAME), as it did in the absence of this NOS inhibitor. However, L-NAME did not block the effects of LIES on bone resorption (eroded surface and osteoclasts surface) in OVX rats. eNOS and iNOS were: similarly expressed in tibiae cortices of SHAM, not expressed in OVX and correspondingly expressed in OVX+LIES when compared to SHAM. eNOS and iNOS expressions were not detected on bone of none of L-NAME treated groups. In OVX, the percentage of apoptotic osteocytes (24%) was significantly increased when compared to SHAM (11%) and OVX-LIES (8%). All LNAME treated groups showed a diminished percentage of apoptotic osteocytes. In conclusion, our study showed that LIES counteracts some effects of OVX on bone tissue preserving BMD, bone structure and microarchitecture, iNOS and eNOS expression and osteocyte viability. Moreover, it was demonstrated that L-NAME partially blocks the effects of LIES on bone structure (but not on bone resorption) and on iNOS and eNOS expressions in OVX rats, suggesting that NO may be a mediator of the positive effects of LIES on bone. However, it was not possible to identify if the known positive effects of LIES on osteocyte viability were mediated by NO, since the effects of L-NAME on these cells were similar to those caused by LIES.
Low Intensity Electrical Stimulation (LIES) has been used for bone repair but little is known about its effects on bone after menopause. Osteocytes probably play a role in mediating this physical stimulus and they could act as transducers through the release of biochemical signals, such as nitric oxide (NO). The aim of the present study was to investigate the effects of LIES on bone metabolism in ovariectomized rats, and if NO could be a mediator of these effects. Sixty rats (200-220g) were divided into 6 groups: SHAM; SHAM treated with 6mg/d of LNAME, an inhibitor of NO synthase (SHAM-L-NAME); ovariectomized (OVX); OVX treated with L-NAME (OVX-L-NAME) or subjected to a whole body electrical stimulation (OVX-LIES) or both (OVX-LIES-L-NAME) for 12 weeks. Global, spine and posterior limb bone mineral density (BMD) were measured by bone densitometry before and at the end of protocol. After sacrifice, tibias were collected for histomorphometric and immunohistochemistry analysis. Osteocyte apoptosis (caspase-3 and TUNEL techniques) and expressions of endothelial NO synthase (eNOS) and inducible NOS (iNOS) were assessed by immunostaining. OVX rats showed significant (p<0.05 vs SHAM) decreased final global, spine and limb BMDs, measured by densitometry, decreased bone volume (10% vs 25%) and trabecular number (1.7 vs 3.9), and increased eroded surfaces (4.7% vs 3.2%) and mineralization surfaces (15.9% vs 7.7%), measured by histomorphometry. In contrast, after LIES, all these parameters tended to be similar to SHAM and significantly different from OVX. LIES was not able to prevent the reduction of bone volume and trabecular number caused by OVX in the presence of L-NAME (OVX-L-NAME vs OVX-LIES-L-NAME), as it did in the absence of this NOS inhibitor. However, L-NAME did not block the effects of LIES on bone resorption (eroded surface and osteoclasts surface) in OVX rats. eNOS and iNOS were: similarly expressed in tibiae cortices of SHAM, not expressed in OVX and correspondingly expressed in OVX+LIES when compared to SHAM. eNOS and iNOS expressions were not detected on bone of none of L-NAME treated groups. In OVX, the percentage of apoptotic osteocytes (24%) was significantly increased when compared to SHAM (11%) and OVX-LIES (8%). All LNAME treated groups showed a diminished percentage of apoptotic osteocytes. In conclusion, our study showed that LIES counteracts some effects of OVX on bone tissue preserving BMD, bone structure and microarchitecture, iNOS and eNOS expression and osteocyte viability. Moreover, it was demonstrated that L-NAME partially blocks the effects of LIES on bone structure (but not on bone resorption) and on iNOS and eNOS expressions in OVX rats, suggesting that NO may be a mediator of the positive effects of LIES on bone. However, it was not possible to identify if the known positive effects of LIES on osteocyte viability were mediated by NO, since the effects of L-NAME on these cells were similar to those caused by LIES.
Descrição
Citação
LIRANI-GALVÃO, Ana Paula Rebucci. Efeitos da estimulação elétrica de baixa intensidade sobre o metabolismo ósseo de ratas ovarectomizadas. 2008. 190 f. Tese (Doutorado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2008.