Effects of topiramate, bupropion and naltrexone isolated or combined on subcutaneous adipose tissue in obese rats

ABSTRACT Objective: To evaluate the effects of combining topiramate, bupropion and naltrexone in obesity-induced rats on their weight and subcutaneous adipose tissue. Methods: A total of 40 male Wistar rats were induced to obesity for 8 weeks and the animals were divided into 8 groups: Ctr - control, G0 - Sham, G1 - oral saline solution (1.0mL/day), G2 - topiramate (20.0mg/kg) and bupropion (5.0mg/kg), G3 - naltrexone (20.0mg/kg), G4 - topiramate (20.0mg/kg), G5 - bupropion (5.0mg/kg) and G6 - topiramate (20.0mg/kg), bupropion (5.0mg/kg) and naltrexone (20.0mg/kg). During the experiment, all animals were weighed weekly. After 30 days of treatment animals were euthanized and their skin fragments were processed and stained with hematoxylin and eosin for morphological, morphometric and biochemical analyzes. Results: The only group that presented a decrease in the volume of subcutaneous adipose tissue was G3, but this decrease was not significant when compared with the other groups. The G4, the G5 and the G6 presented increased adipose tissue volume. Data showed that until the eighth week all animals increased their weight by approximately 50%. After treatment animals of all groups, except G3, increased their weight from 4% to 9% approximately. The G3 was the only group that lost weight, but this decrease was not significant. Conclusion: The medicines studied were not efficient in reducing weight in obese rats. However, it should be considered that 30-day treatment period is not enough to observe the stronger effects of these drugs.

The effect of testosterone replacement on intramedullary, inguinal and visceral fat in ovariectomized rats / Efeito da reposição de testosterona na gordura intramedular, inguinal e visceral em ratas ovariectomizadas

Abstract Objective The present article aims to evaluate the impact of testosterone treatment on the expansion of visceral, subcutaneous and intramedullary adipose tissue of ovariectomized rats and the visceral and subcutaneous fat expression of peroxisome proliferator-activated receptors (PPARs) gamma. Methods In total 48 female Wistar rats were castrated and randomly divided into 6 treatment groups: group E2 was submitted to estradiol 5 μg/day; group T, to testosterone 5 μg/day; group E2+ T, to estradiol 5 μg/day + testosterone 5 μg/day; group TT, to testosterone 30 μg/day; group E2+ TT, to estradiol 5 μg/day+ testosterone 30 μg/day; and placebo was administered to group P. After 5 weeks, the rats were euthanized, the inguinal and visceral adipose tissues were harvested, weighted, and had their PPAR gamma expression evaluated by reverse transcription quantitative polymerase chain reaction (RTqPCR). The right femurs were harvested and histologically prepared to performthe number count of the intramedullary adipocytes. Results The expansion of visceral fat tissue was much higher in the TT group when compared with other treated groups (p < 0.001). The TT group also showed a higher expansion of inguinal fat (p < 0.01), and groups E2 +T and E2+ TT presented lower growth compared to the P group (p < 0.01). The number of femur intramedullary adipocytes only showed significant differences between groups TT and E2 + TT (p < 0.05). The expression of PPAR gamma showed no differences among the groups. Conclusion The use of testosterone in high doses leads to an important expansion in both visceral and inguinal adipose tissues. Association with estradiol exerts an expansion-repressive effect on the visceral and inguinal adipose tissues.

Resumo Objetivo Este artigo tem como objetivo avaliar o impacto do tratamento com testosterona na expansão dos tecidos adiposos visceral, subcutâneo e intramedular de ratas ovariectomizadas e a expressão de receptores ativados por proliferadores de peroxissoma (RAPPs) gama nas gorduras visceral e subcutânea. Métodos No total, 48 ratas Wistar foram castradas e divididas aleatoriamente em 6 grupos de tratamento: o grupo E2 recebeu estradiol 5 μg/dia; o grupo T, testosterona 5 μg/dia; o grupo E2 + T, estradiol 5 μg/dia + testosterona 5 μg/dia; o grupo TT, testosterona 30 μg/dia; o grupo E2 + TT, estradiol 5 μg/dia + testosterona 30 μg/dia; e o grupo P recebeu placebo. Após 5 semanas, as ratas foram submetidas a eutanásia, o tecido adiposo inguinal e visceral foi coletado, pesado, e se avaliou a expressão dos RAPP gama por reação em cadeia da polimerase via transcriptase reversa quantitativa (RCP-TRq). Os ossos do fêmur direito foram colhidos e processados histologicamente para contagem de números de adipócitos intramedulares. Resultados A expansão do tecido adiposo visceral foi muito maior no grupo TT quando comparado a outros grupos tratados (p < 0,001). O grupo TT também apresentou maior expansão da gordura inguinal (p < 0,01), e os grupos E2 +T e E2 + TT apresentaram menor crescimento em relação ao grupo P (p < 0,01). O número de adipócitos intramedulares no fêmur mostrou apenas diferenças significativas entre os grupos TT e E2 + TT (p < 0,05). A expressão de RAPP gama não mostrou diferenças entre os grupos. Conclusão O uso de testosterona emaltas doses leva a uma importante expansão nos tecidos adiposos visceral e inguinal. A associação com o estradiol exerce um efeito repressivo de expansão nos tecidos adiposos visceral e inguinal.

Effect of testosterone on proliferation markers and apoptosis in breasts of ovariectomized rats / Efeito da testosterona em marcadores de proliferação e apoptose em mamas de ratas ovariectomizadas

Abstract Objective To investigate the action of testosterone (T), isolated or associated with estradiol benzoate (EB), on the proliferation markers and apoptosis of breasts of ovariectomized rats. Methods A total of 48 castrated female Wistar rats were divided into 6 groups, and each of them were submitted to one of the following treatments for 5 weeks: 1) control; 2) EB 50 mcg/day + T 50 mcg/day; 3) T 50mcg/day; 4) EB 50 mcg +T 300 mcg/day; 5) T 300 mcg/day; and 6) EB 50 mcg/day. After the treatment, the mammary tissue was submitted to a histological analysis and immunoexpression evaluation of proliferation markers (proliferating cell nuclear antigen, PCNA) and apoptosis (caspase-3). Results There was a statistically significant difference among the groups regarding microcalcifications and secretory activity, with higher prevalence in the groups treated with EB. There was no difference among the groups regarding atrophy, but a higher prevalence of atrophy was found in the groups that received T versus those that received EB +T. There was a difference among the groups regarding the PCNA (p = 0.028), with higher expression in the group submitted to EB +T 300 mcg/day. Regarding caspase-3, there was no difference among the groups; however, in the group submitted to EB +T 300 mcg/day, the expression was higher than in the isolated T group. Conclusion Isolated T did not have a proliferative effect on the mammary tissue, contrary to EB. Testosterone in combination with EB may or may not decrease the proliferation, depending on the dose of T.

Resumo Objetivo Investigar a ação da testosterona (T) isolada ou associada ao benzoato de estradiol (EB) na proliferação e apoptose de mamas de ratas ovariectomizadas. Métodos Um total de 48 ratas Wistar castradas foram divididas em 6 grupos, e cada um foi submetido a um dos seguintes tratamentos durante 5 semanas: 1) controle; 2) BE 50 mcg/dia + T 50mcg/dia; 3) T 50 mcg/dia; 4) BE 50 mcg + T 300mcg/dia; e) T 300 mcg/dia; e f) BE 50 mcg/dia. Após o tratamento, o tecido mamário foi submetido a análise histológica e avaliação de imunoexpressão de marcadores de proliferação (antígeno nuclear de células proliferantes, PCNA) e apoptose (caspase-3). Resultados Houve diferença estatisticamente significante entre os grupos com relação às microcalcificações e à atividade secretora, com maior prevalência nos grupos tratados com BE. Não houve diferença entre os grupos quanto à atrofia, mas houve maior prevalência de atrofia nos grupos que receberam T versus os que receberam BE+ T. Houve diferença entre os grupos quanto ao ANCP (p= 0,028), com maior expressão no grupo BE+ T 300 mcg/dia. Com relação à caspase-3, não houve diferença entre os grupos, mas, no grupo BE+ T 300 mcg/dia, a expressão foi maior do que no grupo de T isolada. Conclusão A T isolada não apresentou efeito proliferativo do tecido mamário, contrariamente ao EB. A T em associação ao EB pode diminuir ou não a proliferação, a depender da dose de T.

Alteração do tecido conjuntivo orbitário após aplicação de bimatoprost: estudo experimental em ratos / Changes of orbital connective tissue after bimatoprost injection. Experimental study in rats

RESUMO O bimatoprost é utilizado comumente como a droga de primeira escolha no tratamento do glaucoma primário de ângulo aberto. Hiperemia conjuntival, crescimento dos cílios, enoftalmia, escurecimento cutâneo periocular, sulco palpebral profundo e prurido ocular têm sido relatados em pacientes que receberam bimatoprost em doses únicas diárias durante cerca de 3 meses. O mecanismo exato para estes efeitos adversos permanece desconhecido. Objetivo: Verificar em animais de experimentação, as alterações do tecido conjuntivo orbitário após injeção retrobulbar de bimatoprost 0,03%. Métodos Foram utilizados trinta e seis ratos machos (Rattus norvegicus albinus) submetidos a diferentes períodos de injeção retrobulbar de bimatoprost à direita. O material exenterado foi submetido à análise histológica, morfométrica (diâmetro, densidade numérica e densidade de volume dos adipócitos) e imunohistoquímica para marcação do VEGF. Os resultados destas análises foram submetidos à análise descritiva com o auxílio do software R. O nível de significância adotado foi 5%. Para as comparações foi proposto o modelo de regressão linear com efeitos mistos. Resultados: Na amostra estudada, as órbitas submetidas a injeções retrobulbares de bimatoprost apresentaram ao redor do nervo óptico tecido conjuntivo mais espesso, com inúmeros capilares e vasos de vários calibres e a redução da quantidade, diâmetro e volume das células adiposas estatisticamente significativo quando comparado à órbita contralateral e ao grupo controle. Conclusão: Neste estudo observaram-se as seguintes alterações potencialmente reversíveis do tecido conjuntivo orbitário nos ratos submetidos à injeção retrobulbar de bimatoprost: 1) redução da quantidade, do diâmetro e do volume das células adiposas orbitárias; 2) neovascularização local; 3) espessamento e remodelamento das fibras de colágeno na cavidade orbitária.

ABSTRACT Bimatoprost is commonly used as the drug of first choice in the treatment of primary open-angle glaucoma. Conjunctival hyperemia, eyelash growth, enophthalmos, periocular skin pigmentation, deep lid sulcus and itching eyes have been reported in patients that daily received single dosages during a three month period. The exact mechanism for these adverse effects remains unknown. Objective: to verify alterations, in test animals, of the orbital connective tissue after peribulbar injections of bimatoprost 0.03% using histological and immune-histochemical analysis. Methods: thirty six male test rats (Rattus norvegicus albinus) were subjected to various periods of periocular injections of bimatoprost 0.03% in the right eye.All extracted material was submitted to histological, morphometric (diameter, numeric density and density of adipocyte volume) and immune-histochemical analysis to mark the vascular endothelial growth factor (VEGF). These analysis results were then submitted to a descriptive analysis with the help of R software. The significance level used was 5%. For comparison, the model of linear regression with mixed effects was proposed. Results: In the sample studied, the eye sockets that were continuously submitted to bimatoprost had a denser conjunctival tissue around the optic nerve, with numerous capillaries and blood vessels of various sizes and a reduction of quantity, diameter and volume of adipose cells of statistic importance when compared to the contralateral eye socked and the control group. Conclusion: In this study, the following potentially reversible changes of orbital connective tissue were observed in test rats subjected to periocular injection of bimatoprost: 1) reduction of quantity, diameter and volume of orbital adipose cells; 2) local neovascularization; 3) thickening and remodeling of collagen fibers in the orbital cavity.

Study of Angiogenesis-Related Phosphodiesterase Inhibitors.

Angiogenesis is the process through which new capillaries form from pre-existing capillaries and venules. Its occurrence depends on the migration of vascular endothelial cells which is inhibited by high levels of cAMP. Such levels can be regulated by the degradation caused by the phosphodiesterases (PDEs). Therefore, by inhibiting the action of PDEs it is assumed that angiogenesis can be inhibited with the prevention of migration of these cells. The aim of this study was to evaluate the effect PDE inhibitors on angiogenesis in mice by using nonspecific inhibitors (aminophylline) and selective inhibitors of PDE4 (roflumilast) and PDE5 (sildenafil). BALB/c mice were used as a model; under anesthesia, the mice had a sponge of 0.5 x 0.5 cm introduced into their dorsal subcutaneous tissue; they were then divided into 4 groups and daily gavage treated: 1) control group (n=13) – treated with 0.3 mL of saline solution; 2) aminophylline group (n=16) – 50 mg/kg; 3) roflumilast group (n=14) – 5mg/kg; 3) sildenafil group (n=12) –100 mg/kg. After 7 days, with the animals anesthetized, a blood sample was drawn for hemoglobin (Hb) measurement, the sponge implant was removed, and its content was obtained in 2 mL of saline solution for hemoglobin measurement. Absorbance levels (A), the amount of Hb from the sponge (S) and the total blood Hb concentration levels from each mouse were evaluated. According to the results obtained, we concluded that aminophylline, roflumilast and sildenafil (phosphodiesterase inhibitors) did not cause any alteration in the angiogenesis evaluated by the sponge-implantation method.

Evaluation of palpebral fissure and orbital volume after bimatoprost 0.03% orbital injections. Experimental study in rats / Avaliação da fenda palpebral e do volume orbitário após aplicações orbitárias de bimatoprost 0.03%. Estudo experimental em ratos

Objective: To evaluate in experimental animals the changes of the palpebral fissure and the orbital volume after orbital injection of bimatoprost 0.03%. Methods: Two main groups of Wistar rats were analyzed, one after orbital injection of bimatoprost 0.03% and another, a control group, after orbital injection of saline solution. The calculation of the palpebral fissure was done on images by means of computer processing, using the program Image J. After taking photographs, the animals were submitted to bilateral orbital exenteration and the volume was calculated in all the animals by the water displacement method (Archimedes’ Principle). Results: While comparing the measurements of the palpebral fissure and the orbital volume among animals given an injection with bimatoprost 0.03% and the control group it was found that there were no statistically significant differences. Conclusions: In this study there were no statistically significant differences in the measurement of the vertical palpebral fissure and the orbital volume among animals given the orbital injection of bimatoprost 0.03% and the animals of the control group. .

Objetivo: Avaliar em modelos experimentais as alterações da fenda palpebral e do volume orbitário após aplicação orbitária de bimatoprost 0,03%. Métodos: Dois principais grupos compostos por ratos Wistar foram analisados, sendo comparados os animais submetidos à injeção orbitária de bimatoprost 0.03% com os submetidos à injeção orbitária de solução salina. O cálculo da fenda palpebral vertical foi obtido através de imagem computadorizada utilizando-se o programa Image J. Após serem fotografados os animais foram submetidos à exenteração bilateral e o volume orbitário foi calculado pelo método de deslocamento da coluna de água (Princípio de Archimedes). Resultados: Quando foram comparadas as medidas da fenda palpebral vertical e do volume orbitário entre os animais submetidos a injeção de bimatoprost 0.03% e o grupo controle não foi obsevada diferença estatisticamente significante. Conclusão: Neste estudo não houve diferença estatisticamente significante nas medidas da fenda palpebral vertical e no volume orbitário entre os animais submetidos à injeção orbitária de bimatoprost 0.03% e o grupo controle. .

Evaluation of Herbalife Nutritional Supplements in the Hepatic Function of Rats.

The intake of nutritional supplements among people who aim for weight loss has increased all over the world given the fact it is easily acquired without the need of a medical prescription.The goal of this study was to research the presence of toxicity due to the intake of Herbalife protein supplements and shakes in an animal model. Twenty-one male Wistar rats were divided in three groups: control, shake and protein. The shake and protein groups received 100 mg/kg/day of the supplement whereas the control group received 0.5 mg/kg/day of water. Supplementation regimen lasted two months and after this period laboratory exams and histological analysis were performed in order to evaluate hepatic lesions. The groups showed no differences in values of GGT, alkaline phosphatase, AST, ALT, cholesterol, HDL or triglycerides. All the toxicity reports related to Herbalife supplements are over longer periods of time of intake than the period established in this model. There are no reports on how the response of rats to this kind of supplementation works. The mechanism of hepatotoxicity in humans suggests an immune-mediated reaction. The presented model could not show the expected toxicity, it is reasonable to conclude that there is a direct relation between hepatic lesions in humans and the intake of Herbalife supplements.