Histology and histochemistry of the accessory gland of the female reproductive tract of Rhodnius neglectus Lent, 1954 (Hemiptera: Reduviidae) / Histologia e histoquímica das glândulas acessórias do sistema reprodutor feminino de Rhodnius neglectus Lent, 1954 (Hemiptera: Reduviidae)

Rhodnius neglectus is a wild triatomine, vector of the protozoan Trypanosoma cruzi, which causes Chagas' disease, and feeds on the blood of small mammals, being essential for its growth and reproduction. Accessory glands of the female reproductive tract are important in insect reproduction, but their anatomy and histology in R. neglectus are poorly studied. The aim of this work was to describe the histology and histochemistry of the accessory gland of the female reproductive tract of R. neglectus. The reproductive tract of five females of R. neglectus was dissected and the accessory glands transferred to Zamboni's fixative solution, dehydrated in a crescent series of ethanol, embedded in historesin, sectioned at 2 µm thick, stained with toluidine blue for histological analysis or mercury bromophenol blue for detection of total proteins. The accessory gland R. neglectus is tubular, without branches, opening in the dorsal region of the vagina and differing along its length in proximal and distal regions. In the proximal region, the gland is lined by the cuticle with a layer of columnar cells associated with muscle fibers. In the distal region of the gland, the epithelium has spherical secretory cells with terminal apparatus and conducting canaliculi opening in the lumen through pores in the cuticle. Proteins were identified in the gland lumen, terminal apparatus, nucleus and cytoplasm of secretory cells. The histology of the R. neglectus gland is similar to that found in other species of this genus, but with variations in the shape and size of its distal region.

Rhodnius neglectus é um triatomíneo silvestre, vetor do protozoário Trypanosoma cruzi, causador da doença de Chagas. Este inseto se alimenta do sangue de pequenos mamíferos, que é essencial para o seu crescimento e reprodução. As glândulas acessórias do sistema reprodutor feminino são importantes na reprodução de insetos, mas sua anatomia e histologia em R. neglectus são pouco conhecidas. O objetivo deste trabalho foi descrever a histologia e histoquímica da glândula acessória do aparelho reprodutor feminino de R. neglectus. O sistema reprodutor de cinco fêmeas de R. neglectus foi dissecado e as glândulas acessórias transferidas para solução fixadora de Zamboni, desidratadas em série crescente de etanol, embebidas em historesina, seccionadas com 2 µm de espessura, coradas com azul de toluidina para análise histológica ou submetidas ao teste de mercúrio-bromofenol para detecção de proteínas totais. O sistema reprodutor de R. neglectus tem uma glândula acessória tubular, sem ramificações, abrindo-se na região dorsal da vagina, sendo diferenciada em regiões proximal e distal. Na região proximal, a glândula é revestida internamente pela cutícula com uma camada de células colunares associadas a fibras musculares. Na região distal ocorrem células secretoras esféricas com aparato terminal e canalículos condutores que se abrem no lúmen da glândula através de poros na cutícula. O teste histoquímico revelou a presença de proteínas no lúmen da glândula e no aparato terminal, núcleo e citoplasma das células secretoras. A histologia da glândula de R. neglectus é semelhante à das espécies desse gênero, mas com variações na forma e no tamanho de sua região distal.

Study of the integument that covering back and stinger of the freshwater stingray Potamotrygon rex (Chondricthyes, Potamotrygonidae) / Estudo do tegumento que reveste o dorso e ferrão da arraia de água doce Potamotrygon rex (Chondricthyes, Potamotrygonidae)

The objective of this study was to describe the histology and histochemistry of the integument covering the back and stinger of the freshwater stingray Potamotrygon rex, endemic to the Middle Upper Tocantins River. The species has a dark back and yellowish circular spots that extend to the tail, which has one to two stings located in the median portion of the tail. Through histological study it was observed that the epithelia of the back and stinger are composed of non-keratinized stratified pavement epithelial tissue, and are organized in three layers: basal, intermediate and superficial. The differences between the tissues are related to the cell types that compose them. The back is organized with epithelial cells, mucus cells, granulocyte cells and chromatophores. The mucus cells are distributed in different layers along the animal's back, influencing the thickness of the tissue. The tissue that covers the stinger is composed of epithelial cells, chromatophores and specialized cells in protein synthesis. In the histochemistry, the stinger epithelial cells were stained with Bromophenol Blue, especially those of the intermediate layer, which were called specialized cells. In the back the epithelial cells were stained with Bromophenol Blue, Alcian Blue and PAS, and the mucous cells with PAS. In both tissues the presence of protein reserves was detected, areas so called because they are stained strongly with Bromophenol Blue. The results show that the stinger presents activity directed to the production of proteins, and that the back is organized to produce different components, which constitute the cuticle that covers the animal's body.

O objetivo deste estudo foi descrever a histologia e histoquímica do tegumento que reveste o dorso e o ferrão da arraia de água doce Potamotrygon rex, endêmica do Médio Alto Tocantins. A espécie possui o dorso escuro e manchas circulares amareladas que se estendem até a cauda, que possui de um a dois ferrões localizados na porção mediana da cauda. Através do estudo histológico observou-se que os epitélios do dorso e do ferrão são compostos por tecido epitelial pavimentoso estratificado não queratinizado, e estão organizados em três camadas: basal, intermediária e superficial. As diferenças entre os tecidos estão relacionadas aos tipos de células que os compõem. O dorso é organizado com células epiteliais, células mucosas, células de granulócitos e cromatóforos. As células mucosas estão distribuídas em diferentes camadas ao longo do dorso do animal, influenciando na espessura do tecido. O tecido que reveste o ferrão é composto por células epiteliais, cromatóforos e células proteicas. Na histoquímica, as células epiteliais do ferrão foram coradas com Azul de Bromofenol, principalmente as da camada intermediária, que foram denominadas células proteicas. No dorso as células epiteliais foram coradas com Azul de Bromofenol, Azul Alcian e PAS, e as células mucosas com PAS. Em ambos os tecidos também foi detectada a presença de reservatórios de proteínas, áreas assim denominadas por estarem fortemente coradas com Azul de Bromofenol. Os resultados mostram que o ferrão apresenta atividade direcionada à produção de proteínas, e que o dorso se organiza para produzir diferentes componentes que constituem a cutícula que reveste o corpo do animal.

Lysosomal storage disease induced by Sida planicaulis (Sin. Sida carpinifolia) (Malvaceae) in sheep in the state of Rio de Janeiro / Doença do armazenamento lisossomal induzida por Sida planicaulis (Sin. Sida carpinifolia) (Malvaceae) em ovinos no estado do Rio de Janeiro

Ingestion of S. planicaulis (Sin. S. carpinifolia) causes lysosomal storage disease in sheep. The main toxic compound of this plant, swainsonine, inhibits the enzymatic activity of α-mannosidase I and II, resulting in lysosomal storage of glycoproteins. We describe a case of spontaneous poisoning by S. planucaulis in Rio de Janeiro state, Brazil. Physical examination of affected animals revealed proprioceptive deficit, motor incoordination, staggering movement, and head tremors. Histopathological evaluation showed severe swelling/cytoplasmic vacuolization in Purkinje neurons, with a foamy appearance and occasional karyolysis or karyopyknosis, and intense vacuolization of acinar cells of the pancreas and, less markedly, thyroid follicular cells. The positive lectin-histochemistry labeling for Con A, WGA and sWGA lectins characterized the disease as a glycoproteinosis. The ultrastructural evaluation revealed numerous vacuoles up to 2.5µm in diameter bounded by membranes up to 20nm thick in pancreatic acinar cells. The diagnosis of S. planicaulis toxicity was established based on epidemiological data, clinicopathological, lectino-histochemical, and ultrastructural findings. This is the second report of spontaneous poisoning of sheep by S. planicaulis in Brazil, but the first in the Southeastern Brazil.

A ingestão de S. planicaulis (Sin. S. carpinifolia) tem sido responsabilizada por doença do armazenamento lisossomal em ovinos. O principal composto tóxico dessa planta, a swainsonina, inibe atividade enzimática da α-manosidase I e II, que redunda no armazenamento de glicoproteínas no interior de lisossomos. Descreveu-se um caso de intoxicação espontânea por S. planicaulis no estado do Rio de Janeiro, Brasil. O exame físico dos animais afetados revelou déficit proprioceptivo, incoordenação motora, cambaleio à movimentação e tremores de intenção e na cabeça. A avaliação histopatológica evidenciou severa tumefação/vacuolização citoplasmática em neurônios de Purkinje, com aspecto espumoso e eventual cariólise ou cariopicnose e intensa vacuolização de células acinares do pâncreas e, menos marcadamente, foliculares da tireoide. O exame lectino-histoquímico positivo para as lectinas Con A, WGA e sWGA foi capaz de caracterizar a enfermidade como uma glicoproteinose. A avaliação ultraestrutural revelou numerosos vacúolos de até 2,5µm de diâmetro, delimitados por membranas de até 20nm de espessura em células acinares do pâncreas. O diagnóstico da intoxicação por S. planicaulis foi estabelecido com base nos dados epidemiológicos, nos achados clínico-patológicos e confirmado pelas avaliações lectino-histoquímica e ultraestrutural. Esta é segunda descrição da intoxicação natural por S. planicaulis em ovinos no Brasil e a primeira na região Sudeste do país.

Distribution of extracellular matrix related proteins in normal and cryptorchid ziwuling black goat testes

The Ziwuling black goat is an indigenously in China, their offspring are frequently affected by congenital cryptorchidism. The extracellular matrix (ECM) contains cytokines and growth factors that regulate the development of the testis, and component changes often result in pathological changes. Cryptorchidism is closely related to structural changes in ECM. In this study, the histochemical staining, immunohistochemical, immunofluorescence and Western blot combined with semi-quantitative analysis was used to describe the distribution of the important ECM components Collagen type IV (Col IV), laminin (LN)and heparan sulfate proteoglycans (HSPG) in the normal and cryptorchid testes of Ziwuling black goats. Results showed that: The histochemical staining showed that the dysplasia of seminiferous tubules and decreased number of Sertoli cells in cryptorchidism, as well as sparse collagen fiber. Meanwhile, the distribution of reticular fibers is relatively rich. Furthermore, the PAS and AB staining in the interstitial vessels and lamina propria of seminiferous tubules is weak. The immunohistochemical and immunofluorescence revealed that Col IV, LN was strongly expressed in Leydig, Sertoli cells of normal testes and moderately positive in the spermatogonia and spermatids, but HSPG was not expressed in the spermatogonia. However, cryptorchidism, the expression of Col IV, LN and HPSG in Leydig, Sertoli cells significantly decreased, as well as the expression of Col IV and LN in capillary endothelial cells, but HSPG was moderately expressed in spermatogonia. Based on these data, the underdevelopment of spermatogenic epithelium, decreased synthesis function of collagen fibers and Leydig cells develop usually in the cryptorchidism were shown to be closely related to the abnormal metabolism of Col IV and LN. The positive expressed of HSPG in the spermatogonia of cryptorchid testes is related to the compensatory development of spermatogonia.(AU)

Leishmania infantum in the reproductive organs of dogs / Leishmania infantum em órgãos reprodutivos de cães

ABSTRACT: Leishmania infantum causes canine leishmaniasis. Using parasitological and molecular analyses, we identified L. infantum in the reproductive organs of male and female dogs. Using histochemistry, immunohistochemistry, and PCR, we examined tissue samples from the reproductive organs of 8 male dogs and 16 female dogs diagnosed with leishmaniasis. Despite the absence of macroscopic or microscopic lesions in these organs, we observed L. infantum amastigotes in tissue samples from the testis and the uterus. PCR and sequencing of these tissues revealed sequences that matched 100% with L. infantum DNA available at GenBank. The presence of L. infantum amastigotes and DNA in testicular and uterine tissue samples suggested that these organs can harbor the parasite without associated macroscopic or microscopic lesions, and this can be especially important in the vertical and venereal transmission of leishmaniasis in dogs.

RESUMO: Leishmania infantum é agente etiológico da leishmaniose canina. Por meio de análises parasitológicas e moleculares, a presença do parasita foi investigada em órgãos reprodutivos de cães machos e fêmeas. Amostras de tecidos dos órgãos reprodutivos de 8 cães machos e 16 fêmeas diagnosticados com leishmaniose foram avaliadas por histoquímica, imunohistoquímica e PCR. Apesar de não terem sido observadas lesões macroscópicas ou microscópicas nos órgãos reprodutivos desses cães, formas amastigotas de L. infantum foram observadas em amostras teciduais do testículo e útero. A PCR e o sequenciamento do DNA extraído desses tecidos revelaram sequências 100% idênticas a L. infantum depositadas no GenBank. Nossos resultados sugerem que os testículos e o útero podem abrigar o parasita, sem associação com lesões macroscópicas ou microscópicas, o que pode ter uma grande importância na transmissão venérea e vertical da leishmaniose entre cães.

Gibberellicacid promotes dormancy-breakingof riceseeds and the formation of abnormal seedlings / O ácido giberélico promove a superação da dormência de sementes de arroz e a formação de plântulas anormais

Rice is one species that present dormancy after harvest and can be prolonged during seed storage. This work aimed to determine whether gibberellic acid (GA3) is an efficient promoter of dormancy-breaking in rice seeds and evaluate changes in biological structures via histochemistry. The cultivar used was SCS122 Miura submitted to 0 mg L-1, 500 mg L-1, and 1000 mg L-1 of GA3. Germination, viability, root, shoot and seedling length, starch optical microscopy, and quantification of total soluble sugars were performed. The use of 500 mg L-1 and 1000 mgL-1 of GA3 was efficient in dormancy-breaking rice seeds, reducing the percentage of dormant seeds to 4% and 1%, respectively. Despite lowering the dormancy percentage, the presence of GA3 causes an increase in the percentage of abnormal seedlings. Therefore, it cannot be recommended as a method of dormancy-breaking rice seeds at the concentrations used. Optical microscopy is efficient to verify that with the dormancy-breaking, the degradation of starch granules occurs, increasing the availability of totalsoluble sugars for the growth and development of seedlings.

O arroz é uma das espécies que apresenta dormência após a colheita, podendo esta ser prolongada durante o armazenamento das sementes. Este trabalho teve por objetivo determinar se o ácido giberélico (GA3) é um promotor eficiente da superação de dormência em sementes de arroz e avaliar mudanças nas estruturas biológicas via histoquímica.A cultivar utilizada foi aSCS122 Miura submetida a 0 mg L-1, 500 mg L-1e 1000 mg L-1de GA3. Foram realizadas análises de germinação, viabilidade, comprimento de raiz, parte aérea e plântula, microscopia óptica do amido e quantificação dos açúcares solúveis totais. A utilização de 500 mg L-1 e 1000 mg L-1de GA3foi eficiente para a superação da dormência de sementes de arroz, reduzindo o percentual de sementes dormentes para 4% e 1% respectivamente. Apesar de reduzir o percentual dormência, a presença de GA3provoca aumento do percentual de plântulas anormais, e por isso, nas concentrações utilizadas, não pode ser recomendado como método de superação em sementes de arroz. A microscopia óptica é eficiente para verificar que com a superação de dormência, ocorre a degradação dos grânulos de amido, aumentando a disponibilidade de açúcares solúveis totais para o crescimento e desenvolvimento de plântulas.

Gibberellicacid promotes dormancy-breakingof riceseeds and the formation of abnormal seedlings / O ácido giberélico promove a superação da dormência de sementes de arroz e a formação de plântulas anormais

Rice is one species that present dormancy after harvest and can be prolonged during seed storage. This work aimed to determine whether gibberellic acid (GA3) is an efficient promoter of dormancy-breaking in rice seeds and evaluate changes in biological structures via histochemistry. The cultivar used was SCS122 Miura submitted to 0 mg L-1, 500 mg L-1, and 1000 mg L-1 of GA3. Germination, viability, root, shoot and seedling length, starch optical microscopy, and quantification of total soluble sugars were performed. The use of 500 mg L-1 and 1000 mgL-1 of GA3 was efficient in dormancy-breaking rice seeds, reducing the percentage of dormant seeds to 4% and 1%, respectively. Despite lowering the dormancy percentage, the presence of GA3 causes an increase in the percentage of abnormal seedlings. Therefore, it cannot be recommended as a method of dormancy-breaking rice seeds at the concentrations used. Optical microscopy is efficient to verify that with the dormancy-breaking, the degradation of starch granules occurs, increasing the availability of totalsoluble sugars for the growth and development of seedlings.(AU)

O arroz é uma das espécies que apresenta dormência após a colheita, podendo esta ser prolongada durante o armazenamento das sementes. Este trabalho teve por objetivo determinar se o ácido giberélico (GA3) é um promotor eficiente da superação de dormência em sementes de arroz e avaliar mudanças nas estruturas biológicas via histoquímica.A cultivar utilizada foi aSCS122 Miura submetida a 0 mg L-1, 500 mg L-1e 1000 mg L-1de GA3. Foram realizadas análises de germinação, viabilidade, comprimento de raiz, parte aérea e plântula, microscopia óptica do amido e quantificação dos açúcares solúveis totais. A utilização de 500 mg L-1 e 1000 mg L-1de GA3foi eficiente para a superação da dormência de sementes de arroz, reduzindo o percentual de sementes dormentes para 4% e 1% respectivamente. Apesar de reduzir o percentual dormência, a presença de GA3provoca aumento do percentual de plântulas anormais, e por isso, nas concentrações utilizadas, não pode ser recomendado como método de superação em sementes de arroz. A microscopia óptica é eficiente para verificar que com a superação de dormência, ocorre a degradação dos grânulos de amido, aumentando a disponibilidade de açúcares solúveis totais para o crescimento e desenvolvimento de plântulas.(AU)

Leishmania infantum in the reproductive organs of dogs / Leishmania infantum em órgãos reprodutivos de cães

Leishmania infantum causes canine leishmaniasis. Using parasitological and molecular analyses, we identified L. infantum in the reproductive organs of male and female dogs. Using histochemistry, immunohistochemistry, and PCR, we examined tissue samples from the reproductive organs of 8 male dogs and 16 female dogs diagnosed with leishmaniasis. Despite the absence of macroscopic or microscopic lesions in these organs, we observed L. infantum amastigotes in tissue samples from the testis and the uterus. PCR and sequencing of these tissues revealed sequences that matched 100% with L. infantum DNA available at GenBank. The presence of L. infantum amastigotes and DNA in testicular and uterine tissue samples suggested that these organs can harbor the parasite without associated macroscopic or microscopic lesions, and this can be especially important in the vertical and venereal transmission of leishmaniasis in dogs.

Leishmania infantum é agente etiológico da leishmaniose canina. Por meio de análises parasitológicas e moleculares, a presença do parasita foi investigada em órgãos reprodutivos de cães machos e fêmeas. Amostras de tecidos dos órgãos reprodutivos de 8 cães machos e 16 fêmeas diagnosticados com leishmaniose foram avaliadas por histoquímica, imunohistoquímica e PCR. Apesar de não terem sido observadas lesões macroscópicas ou microscópicas nos órgãos reprodutivos desses cães, formas amastigotas de L. infantum foram observadas em amostras teciduais do testículo e útero. A PCR e o sequenciamento do DNA extraído desses tecidos revelaram sequências 100% idênticas a L. infantum depositadas no GenBank. Nossos resultados sugerem que os testículos e o útero podem abrigar o parasita, sem associação com lesões macroscópicas ou microscópicas, o que pode ter uma grande importância na transmissão venérea e vertical da leishmaniose entre cães.

Leishmania infantum in the reproductive organs of dogs / Leishmania infantum em órgãos reprodutivos de cães

Leishmania infantum causes canine leishmaniasis. Using parasitological and molecular analyses, we identified L. infantum in the reproductive organs of male and female dogs. Using histochemistry, immunohistochemistry, and PCR, we examined tissue samples from the reproductive organs of 8 male dogs and 16 female dogs diagnosed with leishmaniasis. Despite the absence of macroscopic or microscopic lesions in these organs, we observed L. infantum amastigotes in tissue samples from the testis and the uterus. PCR and sequencing of these tissues revealed sequences that matched 100% with L. infantum DNA available at GenBank. The presence of L. infantum amastigotes and DNA in testicular and uterine tissue samples suggested that these organs can harbor the parasite without associated macroscopic or microscopic lesions, and this can be especially important in the vertical and venereal transmission of leishmaniasis in dogs.(AU)

Leishmania infantum é agente etiológico da leishmaniose canina. Por meio de análises parasitológicas e moleculares, a presença do parasita foi investigada em órgãos reprodutivos de cães machos e fêmeas. Amostras de tecidos dos órgãos reprodutivos de 8 cães machos e 16 fêmeas diagnosticados com leishmaniose foram avaliadas por histoquímica, imunohistoquímica e PCR. Apesar de não terem sido observadas lesões macroscópicas ou microscópicas nos órgãos reprodutivos desses cães, formas amastigotas de L. infantum foram observadas em amostras teciduais do testículo e útero. A PCR e o sequenciamento do DNA extraído desses tecidos revelaram sequências 100% idênticas a L. infantum depositadas no GenBank. Nossos resultados sugerem que os testículos e o útero podem abrigar o parasita, sem associação com lesões macroscópicas ou microscópicas, o que pode ter uma grande importância na transmissão venérea e vertical da leishmaniose entre cães.(AU)

Diminuição do índice de necrose em enxertos cutâneos em coelhos mediante a aplicação de células tronco mesenquimais xenógenas / Reducing of the necrosis index in skin grafts in rabbits through application of xenogenous mesenchymal stem cells

O presente estudo teve como objetivo avaliar a inflamação em auto-enxertos cutâneos obtidos no terceiro, sétimo e décimo quarto dia de pós-operatório, além disso, buscou-se determinar diferenças no processo de cicatrização no grupo tratado com células tronco mesenquimais xenógenas em relação ao grupo controle utilizando a avaliação microscópica e imuno-histoquímico. A avaliação microscópica foi realizada utilizando cortes histológicos corados pela técnica de histoquímica com hematoxilina-eosina (HE), e a imuno-histoquímica, com cortes submetidos a anticorpos específicos. As variáveis analisadas foram quantidade de vasos, células inflamatórias, COX-2, Macrófagos e presença de necrose. Os dados foram analisados estatisticamente pelo software R. A quantidade de vasos foi maior (p<0,0001) no grupo tratamento (GT) durante o dia 3, enquanto no grupo controle (GC) foi maior no dia 7. No dia 3 houve menor porcentagem de necrose no grupo tratamento (GT) (p = 0,038). Nos demais dias avaliados não houve diferença entre a porcentagem de necrose observada nos dois tratamentos (p = 0,98), sendo de 53% para o grupo controle (GC) e 47% para o grupo tratamento (GT). Em relação ao número de macrófagos não houve diferença entre os grupos (p = 0,5637). Entretanto, entre os dias houve diferença significativa (p = 0,0223), sendo menor número de macrófagos no terceiro dia. A imunomarcação de COX-2 foi similar entre os grupos (p = 0,5637) e entre os dias (p = 0,9843). Portanto, o emprego das células tronco mesenquimais xenógenas em enxertos cutâneos promoveu menor ocorrência de necrose, favorecendo sua cicatrização, e não induziu o processo inflamatório, sendo assim factível seu uso em cirurgias reconstrutivas.

The present study aimed to assess inflammation in skin autografts obtained on the third, seventh and fourteenth postoperative day, in addition, it sought to determine differences in the healing process in the group treated with xenogenous mesenchymal stem cells in relation to to the control group using microscopic and immunohistochemical evaluation. Microscopic evaluation was performed using histological sections, stained by the hematoxylin-eosin (HE) histochemistry technique, and immunohistochemistry with sections were subjected to specific antibodies. The variables analyzed were the number of vessels, inflammatory cells (COX-2 and Macrophages) and the presence of necrosis. The data were analyzed statistically by software R. The number of vessels was higher (p< 0.0001) ) in the treatment group (GT) during day 3, while in the control group (CG) it was higher on day 7. On day 3 there was a lower percentage of necrosis in the treatment group (GT) (p = 0.038). On the other evaluated days, there was no difference between the percentage of necrosis observed in the two treatments (p = 0.98), being 53% for the control group (CG) and 47% for the treatment group (GT). Regarding the number of macrophages, there was no difference between groups (p = 0.5637). However, between days there was a significant difference (p = 0.0223), with a lower number of macrophages on the third day. The immunostaining of COX-2 was similar between groups (p = 0.5637) and between days (p = 0.9843). Therefore, the use of xenogenous mesenchymal stem cells in skin grafts promoted a lower occurrence of necrosis, favoring its healing, and did not induce the inflammatory process, thus making its use in reconstructive surgery feasible.

Diminuição do índice de necrose em enxertos cutâneos em coelhos mediante a aplicação de células tronco mesenquimais xenógenas / Reducing of the necrosis index in skin grafts in rabbits through application of xenogenous mesenchymal stem cells

O presente estudo teve como objetivo avaliar a inflamação em auto-enxertos cutâneos obtidos no terceiro, sétimo e décimo quarto dia de pós-operatório, além disso, buscou-se determinar diferenças no processo de cicatrização no grupo tratado com células tronco mesenquimais xenógenas em relação ao grupo controle utilizando a avaliação microscópica e imuno-histoquímico. A avaliação microscópica foi realizada utilizando cortes histológicos corados pela técnica de histoquímica com hematoxilina-eosina (HE), e a imuno-histoquímica, com cortes submetidos a anticorpos específicos. As variáveis analisadas foram quantidade de vasos, células inflamatórias, COX-2, Macrófagos e presença de necrose. Os dados foram analisados estatisticamente pelo software R. A quantidade de vasos foi maior (p<0,0001) no grupo tratamento (GT) durante o dia 3, enquanto no grupo controle (GC) foi maior no dia 7. No dia 3 houve menor porcentagem de necrose no grupo tratamento (GT) (p = 0,038). Nos demais dias avaliados não houve diferença entre a porcentagem de necrose observada nos dois tratamentos (p = 0,98), sendo de 53% para o grupo controle (GC) e 47% para o grupo tratamento (GT). Em relação ao número de macrófagos não houve diferença entre os grupos (p = 0,5637). Entretanto, entre os dias houve diferença significativa (p = 0,0223), sendo menor número de macrófagos no terceiro dia. A imunomarcação de COX-2 foi similar entre os grupos (p = 0,5637) e entre os dias (p = 0,9843). Portanto, o emprego das células tronco mesenquimais xenógenas em enxertos cutâneos promoveu menor ocorrência de necrose, favorecendo sua cicatrização, e não induziu o processo inflamatório, sendo assim factível seu uso em cirurgias reconstrutivas.(AU)

The present study aimed to assess inflammation in skin autografts obtained on the third, seventh and fourteenth postoperative day, in addition, it sought to determine differences in the healing process in the group treated with xenogenous mesenchymal stem cells in relation to to the control group using microscopic and immunohistochemical evaluation. Microscopic evaluation was performed using histological sections, stained by the hematoxylin-eosin (HE) histochemistry technique, and immunohistochemistry with sections were subjected to specific antibodies. The variables analyzed were the number of vessels, inflammatory cells (COX-2 and Macrophages) and the presence of necrosis. The data were analyzed statistically by software R. The number of vessels was higher (p< 0.0001) ) in the treatment group (GT) during day 3, while in the control group (CG) it was higher on day 7. On day 3 there was a lower percentage of necrosis in the treatment group (GT) (p = 0.038). On the other evaluated days, there was no difference between the percentage of necrosis observed in the two treatments (p = 0.98), being 53% for the control group (CG) and 47% for the treatment group (GT). Regarding the number of macrophages, there was no difference between groups (p = 0.5637). However, between days there was a significant difference (p = 0.0223), with a lower number of macrophages on the third day. The immunostaining of COX-2 was similar between groups (p = 0.5637) and between days (p = 0.9843). Therefore, the use of xenogenous mesenchymal stem cells in skin grafts promoted a lower occurrence of necrosis, favoring its healing, and did not induce the inflammatory process, thus making its use in reconstructive surgery feasible.(AU)

Avaliação da eficácia do sistema rigeneracon no tratamento de lesões de calvária em ratos / [Evaluation of the efficacy of the rigeneracon system in the treatment of calvary injuries in rats]

Defeitos ósseos constituem um problema de saúde global. O sistema Rigenera permite a extração de microenxertos ricos em células-tronco mesenquimais (CTMs). Objetivou-se avaliar o processo de regeneração óssea por enxertos obtidos pelo sistema Rigenera em defeitos críticos na calvária de ratos. Foram utilizados 18 ratos Wistar, machos, pesando 285±29g, distribuídos em três grupos (n=6), sendo cada animal controle de si mesmo, denominados G15-Controle e G15-Tratado (15 dias); G30-Controle e G30-Tratado (30 dias) e G60-Controle e G60-Tratado (60 dias). Foram realizadas duas lesões de 5mm de diâmetro em cada antímero da calvária. Nos grupos tratados, foram utilizados microenxertos autólogos de cartilagem xifoide, obtidos pelo sistema Rigenera. O defeito contralateral serviu como controle em todos os animais. Os animais foram eutanasiados aos 15, 30 e 60 dias após a cirurgia, e as amostras foram processadas para a histoquímica. Nos grupos controle, não foram observados sinais de regeneração óssea, enquanto nos grupos tratamento foram verificadas áreas de formação óssea e tecido mesenquimal ativado. O sistema Rigenera foi eficiente na obtenção de microenxertos autólogos, para terapia celular em defeito crítico de calvária de ratos. Com o aprimoramento do protocolo, o sistema Rigenera poderá ser amplamente utilizado no tratamento de lesões ósseas.(AU)

Bone defects are a global health problem. The Rigenera system allows the extraction of micro grafts rich in mesenchymal stem cells (MSCs). The objective of this study was to evaluate the bone regeneration process by grafts obtained by the Rigenera system in defects in the rats calvarian. Eighteen male Wistar rats were used, weighing 285 ± 29g, distributed in three groups (n = 6), where each animal was treatment and control, called G15-Control and G15-Treated (15 days); G30-Control and G30-Treated (30 days) and G60-Control and G60-Treated (60 days). Two 5mm diameter lesions were performed on each calvaria side. In the treated groups, autologous micrograft from xiphoid cartilage, obtained by the Rigenera system, were used. The other defect served as a control in all animals. The animals were euthanized at 15, 30 and 60 days after the surgery and the samples were processed for histochemistry. In the control groups, no signs of bone regeneration were observed, while in the treatment groups, areas of bone formation and activated mesenchymal tissue were verified. The Rigenera system was efficient in obtaining autologous micrograft for cell therapy in a critical calvaria defect in rats. Rigenera system can be widely used in the treatment of bone injuries.(AU)

Bovine tuberculosis: diagnosis in dairy cattle through the association of analyzes

ABSTRACT: Tuberculosis is a chronic anthropozoonosis of worldwide occurrence, caused by the bacterium Mycobacterium tuberculosis and its variants. In Brazil, the National Program for the Control and Eradication of Brucellosis and Tuberculosis in cattle, is responsible for diagnosing and the correctly allocate positive animals, but there is still a lack of definitive diagnosis of the disease. This study described the use of five diagnostic tools that can be used, preferably together, for the confirmation of suspected cases. These tools included the clinical examination comparative cervical tuberculin test, macroscopic findings during the slaughtering and histopathology of the damaged tissues followed by histochemistry. We evaluated a total of 211 dairy cattle, where 15.1% (32/211) had classic clinical signs of bovine tuberculosis, 74 (35%) showed reactivity in the comparative cervical tuberculin test. Of the total number of animals, 141 (66.8%) were referred for sanitary slaughter due to legal and control issues in the outbreaks of the disease. In the follow-up of slaughtering and inspection of viscera and carcasses, 74 (52.5%) had macroscopic lesions compatible with bovine tuberculosis, while 67 (47.5%) showed no visible changes. During the inspection, fragments of lymph nodes and liver and lung parenchyma were collected from five cattle with macroscopic lesions and five with no lesions. The histopathological analysis showed numerous areas of caseous necrosis with or without central calcification and granulomatous inflammatory infiltrate. In the special staining of Ziehl-Neelsen, numerous acid-fast bacilli were evidenced in all cases.

RESUMO: A tuberculose é uma antropozoonose crônica de ocorrência mundial, causada pela bactéria Mycobacterium tuberculosis e suas variantes. No Brasil existe o Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose em bovinos que viabiliza o diagnóstico e a destinação correta dos animais positivos, porém ainda há carência quanto ao diagnóstico da doença. Assim, este trabalho descreve a utilização de cinco ferramentas diagnósticas para a confirmação de casos suspeitos de tuberculose. As ferramentas utilizadas compreenderam o exame clínico, teste tuberculínico cervical comparativo, os achados macroscópicos durante o abate sanitário e a histopatologia dos tecidos lesados seguido de histoquímica. O estudo avaliou um total de 211 bovinos leiteiros, dos quais 15,1% (32/211) apresentaram sinais clínicos clássicos de tuberculose bovina, 35,1% (74/211) apresentaram reatividade no teste tuberculínico cervical comparativo, e 143 animais (67,8%) foram encaminhados para abate sanitário devido a questões legais e de controle nos focos da doença. No acompanhamento do abate e inspeção sanitária de vísceras e carcaças verificou-se que 51,8% (74/143) dos bovinos abatidos apresentavam lesões macroscópicas compatíveis com tuberculose bovina, enquanto 48,2% (69/143) não apresentavam alterações visíveis. Durante a inspeção foram coletados fragmentos de linfonodos e parênquima de fígado e pulmão de cinco bovinos com lesões macroscópicas e de cinco sem lesões, que na análise histopatológica apresentaram numerosas áreas de necrose caseosa com ou sem calcificação central e infiltrado inflamatório granulomatoso. Na coloração de Ziehl-Neelsen foram evidenciados numerosos bacilos álcool-ácido resistentes em todos os casos. Assim, diante dos resultados obtidos verifica-se que as análises empregadas no presente estudo foram de extrema importância para o diagnóstico acurado de tuberculose em bovinos.

Morphology, histology and histochemistry of the digestive tract of the Banded tilapia, Tilapia sparrmanii (Perciformes: Cichlidae)

This study described anatomical, histological and histochemical features of the mucosal layer of the digestive tract of Tilapia sparrmanii Smith, 1840, an omnivorous freshwater fish endemic to Southern Africa. This species exhibited a short thick oesophagus with long deep longitudinal folds (466.68 ± 16.91 µm), and a thick (173.50 ± 10.92 µm) muscular layer that allow the passage of large food items. The mucosa was lined with stratified secretory epithelium rich in goblet cells that secreted neutral and acid mucins. The stomach was a sac-like structure with simple tubular glands surrounded by connective tissue. The mucosa was lined with simple columnar epithelium and the lamina propria exhibited a well-developed layer of gastric glands that occupied the entire length of the cardio-fundic region. The stomach mucosa consisted of epithelial cells with intense neutral mucin secretion which protects against gastric juice. Neck cells of gastric glands synthesized neutral and acid mucins. The intestine was highly coiled and presented a complex pattern of transversal folds internally (villi). Villi length decreased progressively from the anterior to the posterior intestine (p 0.0001). Tunica muscularis of the mid-intestine had the thinnest thickness among all parts of the intestine (p 0.0001). Goblet cells whose numbers increased towards the rectum secreted both acid and neutral mucins. The results indicate structural similarities of T. sparrmanii GIT with other tilapia species and will be useful for understanding the physiology of the digestive systems as well as functional components of the GIT.

Morphology, histology and histochemistry of the digestive tract of the Banded tilapia, Tilapia sparrmanii (Perciformes: Cichlidae)

This study described anatomical, histological and histochemical features of the mucosal layer of the digestive tract of Tilapia sparrmanii Smith, 1840, an omnivorous freshwater fish endemic to Southern Africa. This species exhibited a short thick oesophagus with long deep longitudinal folds (466.68 ± 16.91 µm), and a thick (173.50 ± 10.92 µm) muscular layer that allow the passage of large food items. The mucosa was lined with stratified secretory epithelium rich in goblet cells that secreted neutral and acid mucins. The stomach was a sac-like structure with simple tubular glands surrounded by connective tissue. The mucosa was lined with simple columnar epithelium and the lamina propria exhibited a well-developed layer of gastric glands that occupied the entire length of the cardio-fundic region. The stomach mucosa consisted of epithelial cells with intense neutral mucin secretion which protects against gastric juice. Neck cells of gastric glands synthesized neutral and acid mucins. The intestine was highly coiled and presented a complex pattern of transversal folds internally (villi). Villi length decreased progressively from the anterior to the posterior intestine (p 0.0001). Tunica muscularis of the mid-intestine had the thinnest thickness among all parts of the intestine (p 0.0001). Goblet cells whose numbers increased towards the rectum secreted both acid and neutral mucins. The results indicate structural similarities of T. sparrmanii GIT with other tilapia species and will be useful for understanding the physiology of the digestive systems as well as functional components of the GIT.(AU)

Influence of environment on the leaf morpho-anatomy and histochemical of the ayahuasca leaf: Populations cultivated in extra-Amazonian regions

Psychotria viridis Ruiz & Pav (Rubiaceae) occurs naturally throughout the Amazon and it is traditionally used by indigenous communities, being incorporated into religious use in urban contexts over the last few decades. It is known and cultivated in many regions of South America for possessing valuable bioactive alkaloids. In this paper, we described P. viridis leaf morphology, anatomy and histochemistry from three populations cultivated in the southeastern Brazil, in order to identify possible adaptations to local environment and management. All plants presented terminal stipules and basic morpho-anatomical patterns of leaves, consistent with most species of the genus, as heterogeneous dorsiventral mesophyll, uniseriate epidermis, presents large cells with prominent vacuoles and druses. Unicellular non-glandular trichomes and multicellular starry trichomes were present in the primary and secondary veins. Amphi-hypostomatic leaf pattern, not yet described for the species, was common in all studied plants. Variation in the presence of domatia in the same population indicates that this structure cannot be used for taxonomic determination of P. viridis, as already described for other species of the genus. Presence of secretory ducts and reduction in stomata density and leaf area represent the main morpho-anatomic adaptations of plants from drier and warmer climates. Histochemical tests were positive for alkaloids, polysaccharides, proteins and phenolic compounds, being negative for starch only in plants subjected to water stress. We concluded that the morpho-anatomical and histochemical alterations found in the plants of this study resulted from seasonal water deficit adaptations and to maintain or attract mutualistic organisms

Influence of environment on the leaf morpho-anatomy and histochemical of the ayahuasca leaf: Populations cultivated in extra-Amazonian regions

Psychotria viridis Ruiz & Pav (Rubiaceae) occurs naturally throughout the Amazon and it is traditionally used by indigenous communities, being incorporated into religious use in urban contexts over the last few decades. It is known and cultivated in many regions of South America for possessing valuable bioactive alkaloids. In this paper, we described P. viridis leaf morphology, anatomy and histochemistry from three populations cultivated in the southeastern Brazil, in order to identify possible adaptations to local environment and management. All plants presented terminal stipules and basic morpho-anatomical patterns of leaves, consistent with most species of the genus, as heterogeneous dorsiventral mesophyll, uniseriate epidermis, presents large cells with prominent vacuoles and druses. Unicellular non-glandular trichomes and multicellular starry trichomes were present in the primary and secondary veins. Amphi-hypostomatic leaf pattern, not yet described for the species, was common in all studied plants. Variation in the presence of domatia in the same population indicates that this structure cannot be used for taxonomic determination of P. viridis, as already described for other species of the genus. Presence of secretory ducts and reduction in stomata density and leaf area represent the main morpho-anatomic adaptations of plants from drier and warmer climates. Histochemical tests were positive for alkaloids, polysaccharides, proteins and phenolic compounds, being negative for starch only in plants subjected to water stress. We concluded that the morpho-anatomical and histochemical alterations found in the plants of this study resulted from seasonal water deficit adaptations and to maintain or attract mutualistic organisms(AU)

The epiphyseal plate closure phenomena for male and female Japanese quail ( Coturnix coturnix japonica ): histological and biochemical alterations

This study was conducted to determine the effect of sex on ossification processes in quail by determining the exact closure timing of the epiphyseal plate in the proximal region of the femur. This was done by investigating the histological and biochemical parameters affecting the osteogenesis process that takes place following quail hatching to observe if any variation existed between males and females in this regard. For this purpose, blood samples were collected from six male and female specimens via IV catheters every week for the first 42 days that followed hatching. The samples were transferred into serum tubes, and PTH (PTH), triiodothyronine (T3), thyroxine (T4), inorganic phosphorus, calcium, and vitamin D values in the samples, which are known to have an impact on ossification, were analyzed. The specimens from which the blood samples were collected were then euthanized, and histological cut-sections that covered the epiphyseal growth plate were collected, along with the bone sections of the proximal regions of the right femur. Considering decalcification, these histological sections were kept in an ethylene diamine tetra acetic acid (EDTA) solution. Routine histological examinations were then conducted on these sections, after which they were embedded in paraffin. Crossman's modified triple staining method was used to prepare them, and Wilcoxon Signed Ranks Test was used to statistically evaluate whether the inspected biochemical parameters played a role in the ossification process of quail and whether a statistical difference existed between sexes in this regard. The findings of our study revealed that poultry animals also have five zones in the epiphyseal plate as do mammals, and they have calcified cartilage areas. The findings also indicate that, while ossification starts to occur both in male and female quail specimens, calcification occurs more frequently in females. It was determined that the proximal epiphyseal plate regions of the femur close at the end of the sixth week in both sexes of quail, although the calcification and ossification are more advanced in some females compared with males.(AU)

Understanding the trade-off between the environment and fertility in cows and ewes

The environment contributes to production diseases that in turn badly affect cow performance, fertility and culling. Oestrus intensity is lower in lame cows, and in all cows 26% potential oestrus events are not expressed (to avoid getting pregnant). To understand these trade-offs, we need to know how animals react to their environment and how the environment influences hypothalamus-pituitary-adrenal axis (HPA) interactions with the hypothalamus-pituitary-ovarian axis (HPO). Neurotransmitters control secretion of GnRH into hypophyseal portal blood. GnRH/LH pulse amplitude and frequency drive oestradiol production, culminating in oestrus behaviour and a precisely-timed GnRH/LH surge, all of which are disrupted by poor environments. Responses to peripheral neuronal agents give clues about mechanisms, but do these drugs alter perception of stimuli, or suppress consequent responses? In vitro studies confirm some neuronal interactions between the HPA and HPO; and immuno-histochemistry clarifies the location and sequence of inter-neurone activity within the brain. In both species, exogenous corticoids, ACTH and/or CRH act at the pituitary (reduce LH release by GnRH), and hypothalamus (lower GnRH pulse frequency and delay surge release). This requires inter-neurones as GnRH cells do not have receptors for HPA compounds. There are two (simultaneous, therefore fail-safe?) pathways for CRH suppression of GnRH release via CRH-Receptors: one being the regulation of kisspeptin/dynorphin and other cell types in the hypothalamus, and the other being the direct contact between CRH and GnRH cell terminals in the median eminence. When we domesticate animals, we must provide the best possible environment otherwise animals trade-off with lower production, less intense oestrus behaviour, and impaired fertility. Avoiding life-time peri-parturient problems by managing persistent lactations in cows may be a worthy trade-off on both welfare and economic terms – better than the camouflage use of drugs/hormones/feed additives/intricate technologies? In the long term, getting animals and environment in a more harmonious balance is the ultimate strategy.

Morphological and immunohistochemical comparison of the pituitary gland between a tropical Paracheirodon axelrodi and a subtropical Aphyocharax anisitsi characids (Characiformes: Characidae)

Cardinal tetra Paracheirodon axelrodi and bloodfin tetra Aphyocharax anisitsi are two species of characids with high trade value as ornamental fish in South America. Although both species inhabit middle water layers, cardinal neon exhibits a tropical distribution and bloodfin tetra a subtropical one. Generally, these species are difficult to grow, so it becomes essential to know some key components of the neuroendocrine system to achieve their reproduction in captivity. Considering the importance of deepening the knowledge of the reproductive physiology through functional morphology, for the first time in this work we performed an anatomical, morphological and immunohistochemical analysis of the pituitary gland of these two species. In both species, a leptobasic type pituitary is found in the ventral zone of the hypothalamus and it is characterized by a neurohypophysis which has a well-developed pituitary stalk and a globular adenohypophysis. The pituitary components, characterized by histochemistry and immunohistochemistry, shows a distribution pattern of cells types similar to other teleost species, with only slight differences in the distribution of βFSH and βLH for P. axelrodi.(AU)

El cardenal tetra Paracheirodon axelrodi y el tetra Aphyocharax anisitsi son dos especies de carácidos con alto valor comercial como peces ornamentales en América del Sur. Aunque ambas especies habitan en las capas medias de agua, el neón cardenal exhibe una distribución tropical, mientras que el tetra cola roja una distribución subtropical. En general estas especies son difíciles de cultivar, por lo que es esencial conocer algunos componentes clave de los sistemas neuroendocrinos para lograr su reproducción en cautiverio. Considerando la importancia de profundizar en el conocimiento de la fisiología reproductiva a través de la morfología funcional, en este trabajo realizamos, por primera vez, un análisis anatómico, morfológico e inmunohistoquímico de la glándula pituitaria de estas dos especies. En ambas especies, la hipófisis, del tipo leptobásica, se encontró en la zona ventral del hipotálamo y se caracteriza por una neurohipófisis con un tallo hipofisario bien desarrollado y una adenohipófisis globular. Los componentes hipofisarios, caracterizados por la histoquímica y la inmunohistoquímica, mostraron un patrón de distribución de tipos de células similares a otras especies de teleósteos, con solo pequeñas diferencias en la distribución de βFSH y βLH para P. axelrodi.(AU)