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Recent discoveries of at least two heart fields and dynamic nature of cardiac development as well as controversies regarding the participation of heart fields in development of different heart structures led us to investigate the dynamics of incorporation of the first and second heart fields and prospective fate of the straight heart tube by labeling chicken embryos in vivo with the fluorescent lipophilic dye DiI. The cephalic and caudal limits of the anterior and posterior segments of the straight heart tube were labeled in two groups of embryos. Labels were tracked along the "C," "S," and "U" loops up to the tetracavitary or mature heart (n = 30 embryos/group; torsion and looping stage). To determine whether the atria and atrioventricular canal are derived from the first heart field the straight heart tube was cultured in vitro and immunodetection of Sox-9 and troponin I was performed to identify the mesenchymal and myocardial lineages respectively. Proliferating cell nuclear antigen (PCNA) immunodetection was used to determine the involvement of cell proliferation in heart tube development during torsion and looping. Embryological constitution of the straight heart tube and heart looping (C, S, and U) were not consistent with current descriptions. In fact, right ventricle precursors were absent in the straight heart tube derived from the first heart field. During torsion and looping, the cephalic segment of the straight heart tube gradually shifted into the heart tube until it was located at the myocardial interventricular septum in the tetracavitary heart. In contrast, the caudal segment of the straight heart tube was elongated and remodeled to become the first heart field derived left ventricle and the proximal part of the ventricular inlets. The ventricular outflows, right ventricle, distal part of the ventricular inlets, and atria developed from the second heart field.

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Sebaceous tumors are common in dogs. These tumors include both benign and malignant lesions. Immunohistochemical evaluation of these tumors can aggregate information regarding the origin and degree of malignancy of the lesions. Focusing on this matter, sixty-one samples including normal skin and sebaceous tumors were selected from dogs of various breeds and ages, with no predilection for sex, from the archive of Veterinary Pathology Service of Federal Fluminense University, Niterói/RJ, Brazil. The samples underwent to histological processing, routine staining and immunohistochemistry with anti-PCNA (proliferating cell nuclear antigen). Descriptive statistical analysis was performed, the Wilcoxon-Mann-Whitney test was used to compare the distribution of anti-PCNA labelling in different groups of variables. In case there were more than two groups, the Analysis of Variance (ANOVA) test was performed. The mean age of the affected animals was 10.56 years. The most affected breeds were Caniches and Cocker Spaniels, as well as mixed breed animals. There was immunostaining of PCNA in both benign and malignant tumors, as well as in hyperplasic lesions with varying intensity. Most of the tumors were neoplasms which represented 67.27% of the total sample; within these, 75.00% were benign. The most frequent neoplasm was sebaceous adenoma (37.74%). Results indicated no statistical difference in the distribution of anti-PCNA labelling between the groups of sex, age, reproductive status, localization, size of tumor, and histopathological diagnosis. Although there are not many studies analyzing anti-PCNA labelling in sebaceous tumors, several of them pointed out to the predictive value in other neoplasms. With this matter in mind, we intended to evaluate the expression of anti-PCNA in canine sebaceous tumor and a possible association with the malignancy of the lesions.

Tumores sebáceos são comuns em cães. Tais tumores incluem lesões benignas e malignas. A avaliação imunohistoquímica desses tumores pode agregar informações sobre a origem e o grau de malignidade das lesões. Para este fim, sessenta e uma amostras, incluindo pele normal e tumores sebáceos foram selecionadas de cães de várias raças e idades, sem predileção por sexo, do arquivo do Serviço de Patologia Veterinária da Universidade Federal Fluminense, Niterói/RJ, Brasil. As amostras passaram por processamento histológico, coloração de rotina e imuno-histoquímica com anti-PCNA (proliferating cell nuclear antigen). Foram realizadas análises estatísticas descritivas além dos testes de Wilcoxon-Mann-Whitney para comparar a distribuição da marcação de anti-PCNA entre grupos de variáveis. Para variáveis com mais de dois grupos, aplicou-se a Análise de Variância (ANOVA). A idade média dos animais afetados foi de 10.56 anos. As raças mais afetadas foram Caniches e Cocker Spaniel, e ainda animais sem raça definida. Houve imunomarcação de PCNA em tumores benignos, malignos, e ainda em lesões hiperplásicas com intensidade variada. A maioria dos tumores eram neoplásicos representando 67.92% do total; destes, 75.00% eram benignos. O adenoma sebáceo foi a neoplasia mais frequente (37.74%). Não foram encontradas diferenças significativas nas distribuições de anti-PCNA entre os grupos das variáveis sexo, idade, status reprodutivo, localização e tamanho do tumor e diagnóstico histopatológico. Embora não haja estudos com anti-PCNA em tumores sebáceos caninos, numerosas publicações apontam seu valor preditivo em outras neoplasias. Com isso, a finalidade deste estudo foi avaliar a expressão de anti-PCNA em tumores sebáceos caninos e sua possível associação com a malignidade das lesões.

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OBJECTIVE: To observe the effects of different doses of propofol on the growth of transplanted liver tumor in BALB/C mice and check the expression of PCNA, CD34 and pAKT proteins to clarify the mechanism on molecule level. METHOD: Human primary liver cancer cells SMMC-7721 were subcutaneously cultured in BALB/C mice, and the transplanted tumor model of BALB/C mice was constructed. Forty mice successfully modeled were randomly divided into 5 groups (n = 8): the blank control group (group C), low-fat milk group (group I), low-dose (50 mg/kg) propofol group (P1), middle-dose (100 mg/kg) propofol group (P2) and high dose (150 mg/kg) propofol group (P3). Tumor volume changes were observed at 3, 6, 9, 12, 15 and 18 days (T1, T2, T3, T4, T5, T6 and T7) before and after administration of the drug, and tumor growth curves were plotted. After 19 days of administration, all mice were killed for tumor collection, tumor weight was measured, and the tumor inhibition rate of propofol was calculated. The protein expression of cluster of differentiation 34 (CD34) in transplanted tumor was detected by immunohistochemistry, and the protein expression of proliferating cell nuclear antigen (PCNA) and phospho-Akt (pAKT) was detected by immunofluorescence. RESULTS: Compared with group C, there was no significant difference in tumor volume in group I. At T2 ~ 7, the tumor volume of group P1, P2 and P3 decreased successively (P < 0.05). There was no significant difference in the inhibitory rate of tumor in group I, and the inhibitory rate of tumor in group P1, P2 and P3 increased successively (P < 0.05). There was no significant difference in PCNA, CD34, and pAKT protein expression in group I, while PCNA, CD34, and pAKT protein content in P1, P2, P3 groups were successively decreased (P < 0.05). CONCLUSION: Propofol had a dose-dependent effect on the growth of liver cancer xenografts in mice, inhibiting the expression of PCNA, CD34 and pAKT proteins, and the effect was most obvious in the 150 mg/kg propofol group.

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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.

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 50 mcg/dia; 3) T 50 mcg/dia; 4) BE 50 mcg + T 300 mcg/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.

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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.

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OBJECTIVE: To analyze the effect of thalidomide on the progression of endometriotic lesions experimentally induced in rats and to characterize the pattern of cell proliferation by immunohistochemical Proliferating Cell Nuclear Antigen (PCNA) labeling of eutopic and ectopic endometrium. METHODS: Fifteen female Wistar rats underwent laparotomy for endometriosis induction by resection of one uterine horn, isolation of the endometrium and fixation of a tissue segment to the pelvic peritoneum. Four weeks after, the animals were divided into 3 groups: control (I), 10mg/kg/day (II) and 1mg/kg/day (III) intraperitoneal thalidomide for 10 days. The lesion was excised together with the opposite uterine horn for endometrial gland and stroma analysis. Eutopic and ectopic endometrial tissue was submitted to immunohistochemistry for analysis of cell proliferation by PCNA labeling and the cell proliferation index (CPI) was calculated as the number of labeled cells per 1,000 cells. RESULTS: Group I showed a mean CPI of 0.248 ± 0.0513 in the gland and of 0.178 ± 0.046 in the stroma. In contrast, Groups II and III showed a significantly lower CPI, that is, 0.088 ± 0.009 and 0.080 ± 0.021 for the gland (p < 0.001) and 0.0945 ± 0.0066 and 0.075 ± 0.018 for the stroma (p < 0.001), respectively. Also, the mean lesion area of Group I was 69.2 mm2, a significantly higher value compared with Group II (49.4 mm2, p = 0.023) and Group III (48.6 mm2, p = 0.006). No significant difference was observed between Groups II and III. CONCLUSION: Thalidomide proved to be effective in reducing the lesion area and CPI of the experimental endometriosis implants both at the dose of 1 mg/kg/day and at the dose of 10 mg/kg/day.

OBJETIVO: Analisar o efeito da talidomida na progressão de lesões endometrióticas induzidas experimentalmente em ratas e caracterizar o padrão de proliferação celular pela marcação imunohistoquímica de Antígeno Nuclear de Célula Proliferativa (PCNA) no endométrio eutópico e ectópico. MéTODOS: Quinze ratas Wistar foram submetidas a laparotomia para indução de endometriose por ressecção de um corno uterino, isolamento do endométrio e fixação de um segmento do tecido ao peritônio pélvico. Após quatro semanas, os animais foram divididos em 3 grupos: controle (I), 10 mg/kg/dia (II) e 1 mg/kg/dia (III) de talidomida intraperitoneal por um período de 10 dias. As lesões foram resseccionadas juntamente com o corno uterino oposto para análise da glândula endometrial e do estroma. O tecido endometrial eutópico e ectópico foi submetido à imunohistoquímica para análise da proliferação celular por marcação com PCNA e o índice de proliferação celular (CPI) foi calculado como o número de células marcadas por 1.000 células. RESULTADOS: O grupo I apresentou média de CPI de 0,248 ± 0,0513 na glândula e de 0,178 ± 0,046 no estroma. Em contraste, os grupos II e III apresentaram CPI significativamente menor, isto é, 0,088 ± 0,009 e 0,080 ± 0,021 para a glândula (p < 0,001) e 0,0945 ± 0,0066 e 0,075 ± 0,018 para o estroma (p < 0,001), respectivamente. Além disso, a área de lesão média do Grupo I foi de 69,2 mm2, valor significativamente maior em relação ao Grupo II (49,4 mm2, p = 0,023) e Grupo III (48,6 mm2, p = 0,006). Não houve diferença estatisticamente significante entre os Grupos II e III. CONCLUSãO: A talidomida mostrou-se eficaz na redução da área da lesão e CPI dos implantes de endometriose experimental tanto na dose de 1 mg/kg/dia quanto na dose de 10 mg/kg/dia.

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Abstract Objective To analyze the effect of thalidomide on the progression of endometriotic lesions experimentally induced in rats and to characterize the pattern of cell proliferation by immunohistochemical Proliferating Cell Nuclear Antigen (PCNA) labeling of eutopic and ectopic endometrium. Methods Fifteen female Wistar rats underwent laparotomy for endometriosis induction by resection of one uterine horn, isolation of the endometrium and fixation of a tissue segment to the pelvic peritoneum. Four weeks after, the animals were divided into 3 groups: control (I), 10mg/kg/day (II) and 1mg/kg/day (III) intraperitoneal thalidomide for 10 days. The lesion was excised together with the opposite uterine horn for endometrial gland and stroma analysis. Eutopic and ectopic endometrial tissue was submitted to immunohistochemistry for analysis of cell proliferation by PCNA labeling and the cell proliferation index (CPI) was calculated as the number of labeled cells per 1,000 cells. Results Group I showed a mean CPI of 0.248 ± 0.0513 in the gland and of 0.178 ± 0.046 in the stroma. In contrast, Groups II and III showed a significantly lower CPI, that is, 0.088 ± 0.009 and 0.080 ± 0.021 for the gland (p < 0.001) and 0.0945 ± 0.0066 and 0.075 ± 0.018 for the stroma (p < 0.001), respectively. Also, the mean lesion area of Group I was 69.2mm2, a significantly higher value compared with Group II (49.4mm2, p = 0.023) and Group III (48.6mm2, p = 0.006). No significant difference was observed between Groups II and III. Conclusion Thalidomide proved to be effective in reducing the lesion area and CPI of the experimental endometriosis implants both at the dose of 1mg/kg/day and at the dose of 10 mg/kg/day.

Resumo Objetivo Analisar o efeito da talidomida na progressão de lesões endometrióticas induzidas experimentalmente em ratas e caracterizar o padrão de proliferação celular pela marcação imunohistoquímica de Antígeno Nuclear de Célula Proliferativa (PCNA) no endométrio eutópico e ectópico. Métodos Quinze ratas Wistar foram submetidas a laparotomia para indução de endometriose por ressecção de um corno uterino, isolamento do endométrio e fixação de um segmento do tecido ao peritônio pélvico. Após quatro semanas, os animais foram divididos em 3 grupos: controle (I), 10 mg/kg/dia (II) e 1 mg/kg/dia (III) de talidomida intraperitoneal por um período de 10 dias. As lesões foram resseccionadas juntamente com o corno uterino oposto para análise da glândula endometrial e do estroma. O tecido endometrial eutópico e ectópico foi submetido à imunohistoquímica para análise da proliferação celular por marcação com PCNA e o índice de proliferação celular (CPI) foi calculado como o número de células marcadas por 1.000 células. Resultados O grupo I apresentou média de CPI de 0,248 ± 0,0513 na glândula e de 0,178 ± 0,046 no estroma. Em contraste, os grupos II e III apresentaram CPI significativamentemenor, isto é, 0,088 ± 0,009 e 0,080 ± 0,021 para a glândula (p < 0,001) e 0,0945 ± 0,0066 e 0,075 ± 0,018 para o estroma (p < 0,001), respectivamente. Além disso, a área de lesãomédia do Grupo I foi de 69,2mm2, valor significativamentemaior em relação ao Grupo II (49,4mm2, p = 0,023) e Grupo III (48,6mm2, p = 0,006). Não houve diferença estatisticamente significante entre os Grupos II e III. Conclusão A talidomida mostrou-se eficaz na redução da área da lesão e CPI dos implantes de endometriose experimental tanto na dose de 1mg/kg/dia quanto na dose de 10 mg/kg/dia.

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Purpose: To investigate the effects of pine needle extract (PNE) on the expression of proliferating cell nuclear antigen (PCNA) and Ki-67 during liver regeneration induced by 70% partial hepatectomy (PH) in rat. Methods: Forty-eight male rats (SD, 7 weeks) had surgery (70% PH). They were randomly divided into two groups. PH + PNE group was only provided PNE diluted in water (10%) for drinking and PH group was provided water from 5 days before surgery to the time of sacrifice. PNE was made by pressing and filtering. Animals were sacrificed at 12h, 24h, 36h, 60h, 84h, 168h after PH, respectively. The expressions of PCNA and Ki-67 were determined as proliferation indices. Results: Immunohistochemistry turned out to increase the expression of PCNA and Ki-67. PCNA expression of PH+PNE group increased up to twice of that of PH group. Western blot also seemed to increase the PCNA expression. These results indicated the promotion of cell proliferation in liver tissue and hepatic regeneration. Conclusions: Pine needle extract stimulates the expression of some mitotic proteins during liver regeneration induced by 70% PH in rats. It suggests that administration of pine needle extract could accelerate the liver regeneration after partial hepatectomy.(AU)

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OBJECTIVE: Endometriosis is characterized by the growth of endometrial tissue outside the uterine cavity. The prevalence of endometriosis among women experiencing pain, infertility, or both is as high as 35% to 50%. The most common symptoms of endometriosis are dysmenorrhea, dyspareunia, chronic pelvic pain, and infertility. Evidence has suggested that endometriosis symptoms result from a local inflammatory peritoneal reaction caused by ectopic endometrial implants that undergo cyclic bleeding. On the other hand, regular physical exercise seems to have protective effects against diseases that involve inflammatory processes such as type 2 diabetes and colon and breast cancer. On this basis, it is possible that the practice of physical exercise may have beneficial effects on endometriosis. Therefore, the objective of this study was to evaluate the possible anti-inflammatory effect of physical exercise on endometriosis experimentally induced in rats. STUDY DESIGN: Seventy female Wistar rats were divided into 7groups of 10 animals each. Animals performed light exercise (swimming once a week), moderate exercise (swimming 3 times a week), and intense exercise (swimming 5 times a week) before or after endometriosis induction. RESULTS: At the end of the experimental protocol, a reduction in the size of endometriotic lesions was observed after physical exercise regardless of its frequency, with a greater reduction in the groups practicing moderate and intense activity; an increase in FAS levels and a decrease in matrix metalloproteinases 9 and proliferating cell nuclear antigen (PCNA)levels was also observed. The immunohistochemistry results did not lead to conclusive results. As expected, oxidative stress was reduced in all groups. These results show that the practice of physical exercise could be beneficial, at least in part, for the treatment of endometriosis.

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BACKGROUND: Allopregnanolone is a neurosteroid synthesized in the central nervous system independently of steroidogenic glands; it influences sexual behavior and anxiety. The aim of this work is to evaluate the indirect effect of a single pharmacological dose of allopregnanolone on important processes related to normal ovarian function, such as folliculogenesis, angiogenesis and luteolysis, and to study the corresponding changes in endocrine profile and enzymatic activity over 4 days of the rat estrous cycle. We test the hypothesis that allopregnanolone may trigger hypothalamus - hypophysis - ovarian axis dysregulation and cause ovarian failure which affects the next estrous cycle stages. METHODS: Allopregnanolone was injected during the proestrous morning and then, the animals were sacrificed at each stage of the estrous cycle. Ovarian sections were processed to determine the number and diameter of different ovarian structures. Cleaved caspase 3, proliferating cell nuclear antigen, α-actin and Von Willebrand factor expressions were evaluated by immunohistochemistry. Luteinizing hormone, prolactin, estrogen and progesterone serum levels were measured by radioimmunoassay. The enzymatic activities of 3ß-hydroxysteroid dehydrogenase, 3α-hydroxysteroid oxidoreductase and 20α-hydroxysteroid dehydrogenase were determined by spectrophotometric assays. Two-way ANOVA followed by Bonferroni was performed to determine statistical differences between control and treated groups along the four stages of the cycle. RESULTS: The results indicate that allopregnanolone allopregnanolone decreased the number of developing follicles, while atretic follicles and cysts increased with no effects on normal cyclicity. Some cysts in treated ovaries showed morphological characteristics similar to luteinized unruptured follicles. The apoptosis/proliferation balance increased in follicles from treated rats. The endocrine profile was altered at different stages of the estrous cycle of treated rats. The angiogenic markers expression increased in treated ovaries. As regards corpora lutea, the apoptosis/proliferation balance and 20α-hydroxysteroid dehydrogenase enzymatic activity decreased significantly. Progesterone levels and 3ß-hydroxysteroid dehydrogenase enzymatic activity increased in treated rats. These data suggest that allopregnanolone interferes with steroidogenesis and folliculogenesis at different stages of the cycle. CONCLUSION: Allopregnanolone interferes with corpora lutea regression, which might indicate that this neurosteroid exerts a protective role over the luteal cells and prevents them from luteolysis. Allopregnanolone plays an important role in the ovarian pathophysiology.

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