Influence of chronic alcoholism and estrogen deficiency on the immunohistochemical expression of regulatory proteins of the bone resorption process in the periodontium of Wistar rats.

OBJECTIVE: The aim of this study was to investigate possible changes in immunohistochemical expression of proteins regulating the bone resorption process in the periodontium of rats subjected to alcoholism and/or estrogen deficiency. The investigated proteins were receptor activator of nuclear factor-kappa ß ligand (RANKL), a protein that stimulates bone resorption, and osteoprotegerin (OPG), a protein that inhibits bone resorption. At the molecular level, decreased OPG expression and/or increased RANKL expression are consistent with a greater predisposition to bone resorption. DESIGN: Wistar female rats were divided into ovariectomized (ovx) and non-ovariectomized (sham) groups, and subdivided into ad libitum diet (free diet), alcoholic diet (20% solution), and isocaloric diet (diet with a similar amount of calories as compared with groups ingesting an alcoholic diet). The alveolar bone crest and adjacent tissues were evaluated by immunohistochemical analyses for detection of OPG and RANKL. RESULTS: A significant decrease in OPG expression and a significant increase in RANKL expression were observed in ovariectomized animals which received alcohol as compared with non-ovariectomized animals which received isocaloric diet (experimental control). When estrogen deficiency was evaluated independently of the diet type, a significant decrease in OPG expression and a significant increase in RANKL expression were observed in ovariectomized animals as compared with non-ovariectomized animals. CONCLUSIONS: Estrogen deficiency associated with alcoholic diet, as well as estrogen deficiency (analyzed independently of diet type), decreased the immunostaining for OPG and increased the immunostaining for RANKL in the periodontium of rats.

Remodelação óssea do fêmur de ratas submetidas ao consumo de álcool e/ou à deficiência de estrógeno: análise imunoistoquímica e histomorfométrica / Bone remodeling in the femur of rats submitted to alcohol and/or estrogen deficiency: Immunohistochemical and histomorphometric analysis

O consumo excessivo de álcool constitui importante problema de saúde pública e possui ainda relação direta com a perda óssea mediante desequilíbrio da remodelação óssea, diminuição das taxas de reabsorção e também da osteogênese. A deficiência estrogênica também está diretamente associada à osteoporose, pois leva ao aumento da formação de osteoclastos e diminuição da síntese de osteoblastos, gerando um desequilíbrio no processo de remodelação óssea. A combinação de osteoporose e consumo de álcool pode ter efeito sinérgico e deletério sobre o tecido ósseo e tem sido objeto de estudos. O objetivo deste trabalho foi verificar possíveis alterações no metabolismo ósseo em fêmures de ratas submetidas ao alcoolismo crônico e deficiência estrogênica induzida por ovariectomia, por meio de análise histomorfométrica e imunoistoquímica. Foram utilizadas 90 ratas (Rattus norvegicus, variação albinus, Wistar) com 3 meses de idade, divididas em 6 diferentes grupos de igual número (15), conforme o tipo de dieta e quanto à presença ou ausência hormonal: Grupo 1: ovariectomia simulada (Sham), água e dieta livre; Grupo 2: Sham tratado com doses diárias de solução alcoólica a 20%; Grupo 3: Sham e alimentação isocalórica aos grupos associados ao álcool, fornecida por meio de solução aquosa de sacarose e dieta sólida; Grupo 4: ovariectomia, água e dieta sólida livre; Grupo 5: ovariectomia tratado com doses diárias de solução alcoólica a 20%; Grupo 6: ovariectomia e alimentação isocalórica semelhante ao grupo 3. Após 8 semanas do início da dieta, fez-se a eutanásia de todos os animais, e os fêmures foram removidos. A análise da dieta mostrou que o grupo Ovz dieta livre foi o que mais ganhou peso e o que mais ingeriu ração, apresentando diferenças significativas com relação aos demais grupos. Os animais dos grupos álcool consumiram em média 16 gramas de álcool por dia, sendo que o Sham álcool consumiu mais álcool, quando comparado ao Ovz álcool. Através da histomorfometria foi observado que os animais ovariectomizados apresentaram menor quantidade de osso trabecular em porcentagem, do que os sham operados, porém, sem diferença significativa. Os marcadores da remodelação óssea, RANKL, Osteoprotegerina e Osteocalcina, utilizados nas reações imunoistoquímicas, não mostraram alterações significativas no processo de remodelação óssea. Concluiu-se que ratas adultas jovens, esqueletalmente imaturas, submetidas ao alcoolismo crônico moderado, à remoção dos ovários ou à associação de ambos, mantêm as características de remodelação óssea cortical e trabecular do fêmur preservadas(AU).

Excessive alcohol consumption is an important public health problem and also has direct relation with bone loss by imbalancing bone turnover. Estrogen deficiency is directly associated with osteoporosis, because it leads to an unequal bone resorption and formation by increased osteoclast formation and decreased synthesis of osteoblasts. The combination of osteoporosis and consumption of alcohol can have deleterious and synergistic effect on bone tissue and has been the subject of several studies. The objective of this study will be to investigate possible changes in bone metabolism in femurs of rats submitted to chronic alcoholism and estrogen deficiency induced by ovariectomy. We used 90 rats (Rattus norvegicus, Albinus variation, Wistar) with 3 months old. The animals were divided into six different groups of equal number, according to the type of diet and the presence or absence of hormones: Group 1: sham ovariectomized (sham), water and free diet, Group 2: Sham treated daily with alcoholic solution of 20% and feed ad libitum; Group 3: Sham isocaloric nutritional control group ­ treated with liquid diet containing sucrose with the same average calories ingested on the eve by the alcohol group and solid diet; Group 4: ovariectomy, water and solid diet free; Group 5: ovariectomy treated daily with alcoholic solution of 20% and feed ad libitum; Group 6: ovariectomy isocaloric nutritional control group like group 3. After 8 weeks from the beginning of the diet, all animals were sacrificed and femurs were removed. Diet analysis showed that the Ovx ad libitum was the group that ingested more feed and gained more weight, showing significant differences with the other groups. The animals of alcohol groups, consuming on average 16 grams of alcohol per day, Sham alcohol group consumed more alcohol, as compared to OVZ alcohol. Femurs were evaluated by histomorphometric analysis and immunohistochemical analysis, using the markers, RANKL, Osteoprotegerin and Osteocalcin. The histomorphometric analysis evidenced that ovariectomized animals showed, in percentage, a lower amount of trabecular, than the sham operated, but without significant differences. Markers of bone turnover in immunohistochemical reactions showed no significant changes in bone remodeling process. It was concluded that the combination of estrogen deficiency and moderate chronic alcohol consumption did not cause deleterious effects on bone remodeling of the femur of young adult rats(AU)

Effects of estrogen deficiency combined with chronic alcohol consumption on rat mandibular condyle

Estrogen deficiency and chronic alcohol consumption may have a synergistic and deleterious effect on bone tissue. AIM: To investigate the effects of estrogen deficiency associated with chronic alcohol consumption on the mandibular condyle in rats. METHODS: Fifty-four female rats were first divided equally into two groups: ovariectomized Ovx and simulated ovariectomy Sham. One month after the surgeries, these groups were equally sub-divided according to their dietary treatment: G1: Sham/ad-libitum diet; G2: Sham/alcohol; G3: Sham/isocaloric; G4: Ovx/ad-libitum diet; G5: Ovx/alcohol, G6: Ovx/isocaloric. Eight weeks after starting the diets, all animals were anesthetized and sacrificed. The condyles were analyzed histologically, histomorphometrically, and immunohistochemically using the antibodies for bone sialoprotein BSP, osteocalcin OCC and receptor activator of nuclear factor kappa-B ligand RANKL. RESULTS: Histological analysis of the mandibular condyles showed that Ovx and Sham groups presented almost the same characteristics. The histomorphometric analysis showed that there was a statistically significant difference only between Ovx/isocaloric and Ovx/ad-libitum groups p=0.049. No difference was observed in the intensity of BSP, OCC, and RANKL antibody staining between the Ovx/alcohol and the other groups. CONCLUSIONS: It may be concluded that there was no histomorphometric, histological, or RANKL, BSP, and OCC staining differences between the Ovx/alcohol group and other experimental groups...

Alcoholic and isocaloric diet, but not ovariectomy, influence the apoptosis of bone cells within the alveolar bone crest of rats.

OBJECTIVE: Studies suggest that chronic alcoholism as well as oestrogen deficiencies may affect bones in general, including alveolar bone and, by doing so, increase individuals' susceptibility to develop progressive periodontal disease. This paper aims to verify the influence of chronic alcoholism and/or oestrogen deficiencies in the apoptosis of bone cells of the alveolar bone crest region in rats. DESIGN: Initially, 54 rats were divided into ovariectomized (Ovx) and Sham operated (Sham) groups. Thirty days after surgery, these two groups were equally sub-divided, and received, for 56 days, the following dietary intervention: alcoholic diet (with 20% alcohol solution,), isocaloric diet and ad libitum diet (free diet). Analysis was undertaken by immunohistochemistry, using an antibody to detect apoptosis (anti PARP p-85). RESULTS: When comparing the six experimental groups, no significant differences were observed in the apoptosis of bone cells. Also, there was no significant difference in the quantity of cells undergoing apoptosis when the animals from Ovx groups were compared with those from Sham groups. However, when comparing only different dietary groups, differences were observed between the groups ad libitum and isocaloric, to osteoblasts (p=0.045); and ad libitum and alcohol, to osteocytes (p=0.007). CONCLUSION: It is concluded that ovariectomy was not able to influence the rate of apoptosis of bone cells of the alveolar bone crest region in rats and that a possible influence of diet on apoptosis of osteoblasts and osteocytes cannot be ruled out.

Efeito da deficiência estrogênica associada ao consumo de álcool no côndilo mandibular de ratas / Effect of estrogen deficiency associated with alcohol intake in the mandibular condyle of rats

Tanto a deficiência estrogênica quanto o consumo excessivo de álcool apresentam efeito negativo no tecido ósseo. Considerando a ausência de trabalhos que investiguem a influência da osteoporose associada ao alcoolismo na região do côndilo mandibular, o objetivo desse estudo foi testar a hipótese de que a associação da deficiência estrogênica e consumo crônico de álcool apresentaram um efeito deletério na estrutura óssea do côndilo mandibular. Cinqüenta e quatro ratas (Ratthus norvegicus, variação albinus, Wistar), com 3 meses de idade, foram inicialmente divididas em 2 grupos: ovariectomizadas, ou Sham operadas. Um mês após as cirurgias os dois primeiros grupos foram subdivididos e receberam por 8 semanas diferentes dietas (Sham+dieta livre; Sham+álcool 20%; Sham+isocalórico; OVZ+dieta livre; OVZ+álcool 20%; OVZ+isocalórico). Foi possível avaliar que em todos os grupos ocorreu ganho no peso médio dos animais, sendo que o OVZ dieta livre foi o que apresentou maior ganho e o Sham isocalórico o menor. Os grupos Sham e OVZ álcool, consumiram em média o equivalente a 8,43 g de álcool absoluto por kg de peso/dia. Após o término do período de tratamento os animais foram sacrificados e as mandíbulas removidas. Após realizada a descalcificação, os côndilos foram incluídos em parafina. Realizaram-se secções de 3μm e 4μm para procedimentos imunoistoquímicos e análise histológica, respectivamente. Através da análise histológica, observou-se que os animais dos grupos Sham apresentaram mais osteoclastos em relação aos animais dos grupos Ovz. O grupo Ovz isocalórico foi o que apresentou maior porcentagem de área de osso trabeculado (65,10%) enquanto o Ovz dieta livre apresentou a menor (55,42%). A imunoistoquímica realizada com os anticorpos RANKL, BSP e OCC, apresentou positividade em osteoblastos, lacunas de osteócitos e condrócitos. Os osteoclastos foram marcados apenas pelo RANKL e BSP. Nas condições deste experimento, a ...

Both estrogen deficiency as excessive consumption of alcohol have a negative effect on bone. Given the absence of studies that investigate the influence of osteoporosis associated with alcoholism in the region of the mandibular condyle, the objective of this study was to test the hypothesis that the association of estrogen deficiency and chronic consumption of alcohol had a deleterious effect on bone structure of the mandibular condyle . Fifty-four rats (Ratthus norvegicus, Albinus variation, Wistar), aged 3 months, were initially divided into 2 groups: ovariectomized and Sham operated. A month after surgery the first two groups were subdivided and received different diets for 8 weeks (Sham + ad libitum diet; Sham + 20% alcohol; + Sham isocaloric; OVZ + ad libitum diet; OVZ + 20% alcohol; OVZ + isocaloric). The evaluation that occurred in all groups in mean weight gain of the animals, the OVZ ad libitum diet showed the highest gain and the lowest was Sham isocaloric. The groups Sham alcohol and Ovz alcohol consumed an average to 8.43 g of absolute alcohol per kg/day. After the treatment period the animals were sacrificed and the jaws removed. After de decalcification, the condyles were embedded in paraffin, to perform sections with 3μm and 4μm thick, for histological analysis and immunohistochemical procedures, respectively. Over histological analysis, it was observed that animals from the Sham groups had more osteoclasts than the animals from the Ovz groups. The Ovz isocaloric group showed the highest percentage of trabecular bone area (65.10%) while the Ovz ad libitum diet had the lowest. Immunohistochemistry performed with antibodies RANKL, BSP and OCC, was positive in osteoblasts, chondrocytes and osteocytes gaps. Osteoclasts were marked only by the BSP and RANKL. In this experiment conditions, the association of alcohol and estrogen deficiency caused no deleterious effects on the mandibular condyle of rats