Refinement as ethics principle in animal research: Is it necessary to standardize the Environmental enrichment in laboratory animals?

The Environmental enrichment technique, although scientifically recognized for raising the level of animal welfare, has led to the questioning of its influence on the results of experimental research. Thus, the goal is to promote reflection about the need for standardization of these procedures. For that, documents and experimental analysis were done, in order to quantify and characterize the types of environmental enrichment used and to evaluate the effect of that in the social behavior of Rattus norvegicus. Data from the document review confirmed the hypothesis that the researchers have used a variety of methods, not demonstrating a concern for standardization and prior assessment of its effects on the search results. Demand was corroborated in the experimental study in which, although there was available a simple object acting as refuge promotes behavioral improvements, the presence of the co-specific, as well as characteristics of the micro and macro environment can compromise the homogeneity of the sample. The data from this study endorse the need for validation procedures of environmental enrichment for specific proposals, to investigative data comparison, are possible and contribute to the refinement of the search to reduce the number of animals targeted for this purpose.

Morphometric and Molecular Muscle Remodeling after Passive Stretching in Elderly Female Rats.

OBJECTIVES: To determine the effects of three sessions of a passive stretching exercise protocol on the muscles of elderly female rats. METHODS: The effects of the stretching exercises on the soleus muscle were analyzed using immunohistochemistry [tissue inhibitors of matrix metalloproteinases (TIMP), the tumor necrosis factor-alpha (TNF-α), and the gene expression levels using real-time PCR of the transforming growth factor-beta 1 (TGF-ß1), collagen type 1 (COL1), and collagen type 3 (COL3)]. Fifteen 26-month-old female Wistar rats were randomly divided into two groups, namely, Stretching (SG, n=8) and Control (CG, n=7). The passive mechanical stretching protocol consisted of a set of 4 1-minute repetitions, with 30 seconds between each repetition (total treatment of 4 minutes), three times a week for 1 week. RESULTS: Immunohistochemical analysis revealed an increase of 71.4% in the TNF-α (p=0.04) gene expression levels for the SG and a 58% decrease in the TGF-ß1 gene expression levels (p=0.005) in the SG compared to that in the CG. No significant differences were observed between the groups for the immunostaining of TIMP-1 or the gene expression levels of COL1 and COL3. CONCLUSION: Three sessions of static stretching reduced the gene expression level of TGF-ß1, which, owing to its anti-fibrotic role, might contribute to the remodeling of the intramuscular connective tissue of the aging muscle. In addition, immunostaining revealed that TNF-α levels increased in the aging muscle tissue in response to stretching, indicating its effect on stimulating extracellular matrix degradation. These outcomes have important clinical implications in reinforcing the use of stretching exercises in the elderly, considering that the aging muscle presents an infiltration of connective tissue.

Morphometric and molecular muscle remodeling after passive stretching in elderly female rats

OBJECTIVES: To determine the effects of three sessions of a passive stretching exercise protocol on the muscles of elderly female rats. METHODS: The effects of the stretching exercises on the soleus muscle were analyzed using immunohistochemistry [tissue inhibitors of matrix metalloproteinases (TIMP), the tumor necrosis factor-alpha (TNF-α), and the gene expression levels using real-time PCR of the transforming growth factor-beta 1 (TGF-β1), collagen type 1 (COL1), and collagen type 3 (COL3)]. Fifteen 26-month-old female Wistar rats were randomly divided into two groups, namely, Stretching (SG, n=8) and Control (CG, n=7). The passive mechanical stretching protocol consisted of a set of 4 1-minute repetitions, with 30 seconds between each repetition (total treatment of 4 minutes), three times a week for 1 week. RESULTS: Immunohistochemical analysis revealed an increase of 71.4% in the TNF-α (p=0.04) gene expression levels for the SG and a 58% decrease in the TGF-β1 gene expression levels (p=0.005) in the SG compared to that in the CG. No significant differences were observed between the groups for the immunostaining of TIMP-1 or the gene expression levels of COL1 and COL3. CONCLUSION: Three sessions of static stretching reduced the gene expression level of TGF-β1, which, owing to its anti-fibrotic role, might contribute to the remodeling of the intramuscular connective tissue of the aging muscle. In addition, immunostaining revealed that TNF-α levels increased in the aging muscle tissue in response to stretching, indicating its effect on stimulating extracellular matrix degradation. These outcomes have important clinical implications in reinforcing the use of stretching exercises in the elderly, considering that the aging muscle presents an infiltration of connective tissue.

The development of a mechanical device to stretch skeletal muscle of young and old rats.

OBJECTIVE: How much force is needed to stretch skeletal muscle is still unknown. The aim of this study was to develop a device that mechanically stretches rat muscle to compare the force (N) required to stretch the soleus muscle of young and aged rats and the tibio-tarsal angle joint at neutral and stretched positions. METHODS: Twelve female Wistar rats were divided into two groups: a young group (YG, n=6, 311±11 g) of rats 3 months old and an aged group (AG, n=6, 351±43 g) of rats 15 months old. The left soleus muscle was mechanically held in full dorsal flexion and submitted to mechanical passive stretching: 1 bout of 10 repetitions, each repetition lasted 60 seconds with an interval of 45 seconds between repetitions, performed once a day, twice a week, for 1 week. The force required during stretching was measured by a load cell, and the tibio-tarsal angle joint was measured by photometry. RESULTS: The load cell calibration showed excellent reliability, as confirmed by the intraclass correlation coefficient value of 0.93. A decrease in delta force was found in the comparison between YG and AG (0.11±0.03 N vs 0.08±0.02 N, p<0.05, repeated measures ANOVA). There was no difference between the YG and the AG in the tibio-tarsal angle at resting position (87.1±3.8° vs 87.1±3.5°, p=0.35, Kruskal Wallis) and at the end of the stretching protocol (43.9±4.4° vs 42.6±3.4°, p=0.57, Kruskal Wallis). CONCLUSION: The device presented in this study is able to monitor the force necessary to stretch hindlimb rat muscles. Aged rats required less force than young rats to stretch the soleus muscle, and there was no difference regarding the tibio-tarsal angle between the two groups.

The development of a mechanical device to stretch skeletal muscle of young and old rats

OBJECTIVE: How much force is needed to stretch skeletal muscle is still unknown. The aim of this study was to develop a device that mechanically stretches rat muscle to compare the force (N) required to stretch the soleus muscle of young and aged rats and the tibio-tarsal angle joint at neutral and stretched positions. METHODS: Twelve female Wistar rats were divided into two groups: a young group (YG, n=6, 311±11 g) of rats 3 months old and an aged group (AG, n=6, 351±43 g) of rats 15 months old. The left soleus muscle was mechanically held in full dorsal flexion and submitted to mechanical passive stretching: 1 bout of 10 repetitions, each repetition lasted 60 seconds with an interval of 45 seconds between repetitions, performed once a day, twice a week, for 1 week. The force required during stretching was measured by a load cell, and the tibio-tarsal angle joint was measured by photometry. RESULTS: The load cell calibration showed excellent reliability, as confirmed by the intraclass correlation coefficient value of 0.93. A decrease in delta force was found in the comparison between YG and AG (0.11±0.03 N vs 0.08±0.02 N, p<0.05, repeated measures ANOVA). There was no difference between the YG and the AG in the tibio-tarsal angle at resting position (87.1±3.8° vs 87.1±3.5°, p=0.35, Kruskal Wallis) and at the end of the stretching protocol (43.9±4.4° vs 42.6±3.4°, p=0.57, Kruskal Wallis). CONCLUSION: The device presented in this study is able to monitor the force necessary to stretch hindlimb rat muscles. Aged rats required less force than young rats to stretch the soleus muscle, and there was no difference regarding the tibio-tarsal angle between the two groups.

Inhalation therapy with M1 inhibits experimental melanoma development and metastases in mice.

BACKGROUND: M1 is a homeopathic medicine with immunostimulatory properties used mainly by cancer patients to complement current therapies. Metastatic melanoma is a skin-originated form of cancer without a single therapy able to produce high rate and sustained responses, which attracts the use of complementary therapies such as M1. However, M1's anti-melanoma effects remain to be pre-clinically demonstrated. Therefore in the present work, we utilized a pulmonary metastatic melanoma model and a subcutaneous melanoma growth model to investigate the potential benefits of treatment with M1. METHODS: C57BL/6 mice were injected intravenously or subcutaneously with B16F10 mouse melanoma cells. After 24 h, mice were treated with either M1 or vehicle (water) for 14 days, euthanized and harvested for multi-parameter pulmonary and tumor analyses. RESULTS: Mice treated with M1 had significantly lower tumor burden in the lungs and subcutaneous tissue than control mice. Furthermore, tumors were impaired in proliferation and tumor related angiogenesis by the inhibition of myeloid derived suppressor cells (MDSC) positive for angiotensin II type 1 receptor (AT1R). CONCLUSION: Altogether these data suggest M1 is an efficient candidate for melanoma therapy to be considered for future clinic studies as this study is the first supporting the idea that melanoma patients may benefit with the treatment. The treatment with M1 provides advantages considering the highly-diluted properties and a cost effective alternative to costly chemotherapeutic approaches with, if any, lower toxicity.

Acute effects of stretching exercise on the soleus muscle of female aged rats.

UNLABELLED: It has been shown that stretching exercises can improve the flexibility and independence of the elderly. However, although these exercises commonly constitute training programs, the morphological adaptations induced by stretching exercises in aged skeletal muscle are still unclear. OBJECTIVE: To assess the acute effects of passive mechanical static stretching on the morphology, sarcomerogenesis and modulation of important components of the extracellular matrix of the soleus muscle of aged female rats. METHODS: Fifteen old female rats with 26 months were divided into two groups: stretching (n=8, SG) and control (n=7, CG): The stretching protocol consisted of 4 repetitions each of 1 min with 30s interval between sets. Stretching was performed on the left soleus muscle, 3 times a week for 1 week. After three sessions, the rats were anesthetized to remove the left soleus muscle, and then euthanized. The following analyses were carried out: muscle fiber cross-sectional area and serial sarcomere number; immunohistochemistry for the quantification of collagen I, III and TGFß-1. RESULTS: a decrease in muscle fiber cross-sectional area of the SG was observed when compared to the CG (p=0.0001, Kruskal-Wallis); the percentage of type I collagen was significantly lower in the SG when compared to the CG (p=0.01, Kruskal-Wallis), as well as the percentage of TGFß-1 (p=0.04, Kruskal-Wallis); collagen III was significantly higher in the SG than in the CG (7.06±6.88% vs 4.92±5.30%, p=0.01, Kruskal-Wallis). CONCLUSION: Although the acute stretching induced muscle hypotrophy, an antifibrotic action was detected.

[Increased VEGFR-1 immunoreactivity in the choroid-scleral complex in hypercholesterolemia experimental model]. / Aumento da imunorreatividade ao VEGFR-1 no complexo coroido-escleral em modelo experimental de hipercolesterolemia.

PURPOSE: The aim of this study is to investigate the expression of vascular endothelial growth factor (VEGF) in the choroid and sclera using hypercholesterolemia experimental model. METHODS: New Zealand rabbits were divided into two groups: 8 rabbits (8 eyes), in the normal diet group (NG), were fed by a standard diet for 4 weeks; and 13 rabbits (13 eyes), in the hypercholesterolemic group (HG), were fed by a 1% cholesterol-enriched diet for 8 weeks. Total serum cholesterol, triglyceride, HDL cholesterol and fasting blood glucose exams were performed at the initiation of the experiment and at the euthanasia time. After hypercholesterolemic group 8th week and NG 4th week, animals were euthanized and their eyes underwent immunohistochemical analysis with the RAM-11 and VEGFR-1). RESULTS: The diet has induced a significant increase in total cholesterol and triglyceride levels in HG when compared with NG (p<0.001). There was a significant increase in the RAM-11 and VEGFR-1 expressions in hypercholesterolemic group choroid and sclera in relation to NG (p<0,001). CONCLUSION: This study has revealed that the hypercholesterolemic diet in rabbits induces an increase in the macrophage concentration and immunoreactivity to VEGFR-1 in the choroid and sclera, resembling human age-related macular degeneration (ARMD).

Aumento da imunorreatividade ao VEGFR-1 no complexo coroido-escleral em modelo experimental de hipercolesterolemia / Increased VEGFR-1 immunoreactivity in the choroid-scleral complex in hypercholesterolemia experimental model

OBJETIVO: O objetivo deste trabalho é investigar a expressão do fator de crescimento vascular endotelial (VEGF) na coroide e esclera, utilizando um modelo experimental de hipercolesterolemia. MÉTODO: Coelhos New Zealand foram organizados em dois grupos: O grupo dieta normal (GN), composto por 8 coelhos (8 olhos), recebeu ração padrão para coelhos, durante 4 semanas; e o grupo hipercolesterolêmico (GH), composto por 13 coelhos (13 olhos), recebeu dieta rica em colesterol a 1% por 8 semanas. Foi realizada a dosagem sérica de colesterol total, triglicerídeos, HDL colesterol, glicemia de jejum no início do experimento e no momento da eutanásia. Ao final da 8ª semana para o GH e 4ª semana para o GN foi realizada a eutanásia dos animais e os olhos foram submetidos à análise imuno-histoquímica com os anticorpos RAM-11 e VEGFR-1. RESULTADOS: Observou-se significativo aumento do colesterol total e triglicerídeos do GH em relação ao GN (p<0,001). Houve significativo aumento da expressão da RAM-11 e VEGFR-1 na coroide e esclera dos animais do GH em relação ao GN (p<0,001). CONCLUSÃO: Este estudo demonstra que a dieta hipercolesterolêmica em coelhos induz ao aumento da concentração de macrófagos e da imunorreatividade ao VEGFR-1 na coroide e esclera, expressando similaridade com a degeneração macular relacionada à idade (DMRI) humana.

PURPOSE: The aim of this study is to investigate the expression of vascular endothelial growth factor (VEGF) in the choroid and sclera using hypercholesterolemia experimental model. METHODS: New Zealand rabbits were divided into two groups: 8 rabbits (8 eyes), in the normal diet group (NG), were fed by a standard diet for 4 weeks; and 13 rabbits (13 eyes), in the hypercholesterolemic group (HG), were fed by a 1% cholesterol-enriched diet for 8 weeks. Total serum cholesterol, triglyceride, HDL cholesterol and fasting blood glucose exams were performed at the initiation of the experiment and at the euthanasia time. After hypercholesterolemic group 8th week and NG 4th week, animals were euthanized and their eyes underwent immunohistochemical analysis with the RAM-11 and VEGFR-1). RESULTS: The diet has induced a significant increase in total cholesterol and triglyceride levels in HG when compared with NG (p<0.001). There was a significant increase in the RAM-11 and VEGFR-1 expressions in hypercholesterolemic group choroid and sclera in relation to NG (p<0,001). CONCLUSION: This study has revealed that the hypercholesterolemic diet in rabbits induces an increase in the macrophage concentration and immunoreactivity to VEGFR-1 in the choroid and sclera, resembling human age-related macular degeneration (ARMD).

Tratamento in vivo com M8, uma solução complexa altamente diluída, reduziu a malignidade de um modelo de melanoma de camundongo / In vivo treatment with M8, a highly diluted tinctures complex, reduced the malignancy of a mouse melanoma model

Background: Cancer is a class of disease responsible for 13% of death cause worldwide. Among all types of cancers, one of the most aggressive and with the highest death rate is melanoma. It is highly metastatic and current treatments with chemotherapeutic drugs do not yield satisfactory results. Therefore, the interest on new therapeutics for cancer treatment has been increasing on research. Highly diluted tinctures (HDT) are intended to enhance immune system responses resulting in reduced frequency of various diseases, and often present no risk of serious side-effects due to its low toxicity. Previous results have demonstrated in vitro inhibition of invasion ability and in vivo anti-metastatic potential of B16F10 lung metastasis model after mice treatment with M8 inhalation.Conclusion: Even though further investigation are necessary to elucidate the mechanisms of action of M8 treatment there is an indication that these highly diluted tinctures could be a promising therapy to treat metastatic melanoma.(AU)

Introdução: O Câncer é uma classe de doenças responsáveis por 13% das causas de mortes no mundo todo. Entre todos os tipos de cânceres, um dos mais agressivos e com maior índice de mortalidade é o melanoma. Ele é altamente metastático, e os tratamentos atuais com drogas quimioterapeuticas não geram resultados satisfatórios. Portanto, o interesse em novos agentes terapeuticos para o tratamento do câncer tem aumentado na pesquisa. Soluções altamente diluídas (CAD) são destinadas à aumentar a resposta do sistema imunológico resultando em menores frequencias de várias doenças, e também não apresentam riscos de graves efeitos colaterais, devido à sua toxicidade reduzida. Resultados anteriores demostraram a inibição in vitro da habilidade de invasão e potencial antimetastático in vivo do modelo de metástase pulmonar da B16F10, após o tratamento dos camundongos pela inalação do M8.Conclusão: Mais pesquisas são necessárias para esclarecer os mecanismos de ação do tratamento com M8. Entretanto, há um indicativo de que soluções altamente diluídas podem ser terapias coadjuvantes para o tratamento do melanoma metastático.(AU)