Prenatal Hypoxic-Ischemic Insult Changes the Distribution and Number of NADPH-Diaphorase Cells in the Cerebellum

Astrogliosis, oligodendroglial death and motor deficits have been observed in the offspring of female rats that had their uterine arteries clamped at the 18th gestational day. Since nitric oxide has important roles in several inflammatory and developmental events, here we evaluated NADPH-diaphorase (NADPH-d) distribution in the cerebellum of rats submitted to this hypoxia-ischemia (HI) model. At postnatal (P) day 9, Purkinje cells of SHAM and non-manipulated (NM) animals showed NADPH-d+ labeling both in the cell body and dendritic arborization in folia 1 to 8, while HI animals presented a weaker labeling in both cellular structures. NADPH-d+ labeling in the molecular (ML), and in both the external and internal granular layer, was unaffected by HI at this age. At P23, labeling in Purkinje cells was absent in all three groups. Ectopic NADPH-d+ cells in the ML of folia 1 to 4 and folium 10 were present exclusively in HI animals. This labeling pattern was maintained up to P90 in folium 10. In the cerebellar white matter (WM), at P9 and P23, microglial (ED1+) NADPH-d+ cells, were observed in all groups. At P23, only HI animals presented NADPH-d labeling in the cell body and processes of reactive astrocytes (GFAP+). At P9 and P23, the number of NADPH-d+ cells in the WM was higher in HI animals than in SHAM and NM ones. At P45 and at P90 no NADPH-d+ cells were observed in the WM of the three groups. Our results indicate that HI insults lead to long-lasting alterations in nitric oxide synthase expression in the cerebellum. Such alterations in cerebellar differentiation might explain, at least in part, the motor deficits that are commonly observed in this model

Inhibition of glutamate NMDA receptor in a prenatal hipoxia-ischemia model: morphofunctional analises of cerebellum

Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorLesões sistêmicas peri e pré-natais alteram o desenvolvimento do SNC, levando a problemas cognitivos e motores em crianças que podem perdurar por toda a vida. Um tipo particular de lesão é a hipóxia-isquemia (HI), caracterizada pela interrupção momentânea ou permanente do fluxo sanguíneo. Um dos mecanismos propostos para as lesões decorrentes da HI é a excitotoxicidade glutamatérgica. O uso de inibidores da neurotransmissão glutamatérgica tem sido estudados em diversos modelos de HI. Neste trabalho, avaliamos os efeitos morfofuncionais da administração de um antagonista não-competitivo do receptor de glutamato NMDA sobre o desenvolvimento do cerebelo. Ratas no 18 dia de gestação foram anestesiadas, os cornos uterinos expostos e as 4 artérias uterinas obstruídas por 45 minutos (Grupo H). Animais controle tiveram os úteros expostos, sem a obstrução (Grupo S). Após a cirurgia a gestação prosseguiu. Somente animais nascidos a termo foram utilizados. Um dia após o nascimento, metade de cada ninhada foi designada para receber MK801, 0,3mg/kg/dia, (grupos SM e HM) e a outra metade recebeu solução salina (grupos SS e HS), por 5 dias. Após anestesia e perfusão-fixação com paraformaldeído 4% aos 9, 23, 30 e 60 dias pós-natais, cortes parassagitais do cerebelo foram obtidos em criótomo e submetidos à imunohistoquímica para calbindina, GFAP, GLAST, PDGFRα e MBP. A partir de 45 dias de vida, os animais foram testados em vários de testes comportamentais: labirinto em cruz elevado (LCE), campo vazado (CV), ROTAROD, teste de caminhada sobre barras (ladder test) e teste do comprimento da passada (stride length). Aos 9 dias, a espessura da árvore dendrítica era menor nos animais SM, HS/HM, demonstrando efeitos deletérios tanto do MK801 quanto da HI. Menor número de células PDGFRα+ foi observado nos animais HS/HM, sem efeitos da administração de MK801. Aos 23 dias, maior número de células PDGFRα+ foi observado nos animais HM comparado aos outros 3 grupos, indicando efeito neuroprotetor do MK801. Nessa idade, menor número de fibras mielinizadas (MBP+) foi observada nos animais HS, e a administração de MK801 parece reverter estes efeitos. Aos 9 dias a distribuição de GLAST estava alterada nos animais HS, com os efeitos da HI parcialmente revertidos pelo MK801. Não foram observados efeitos da HI ou do MK801 sobre comportamentos relacionados a ansiedade pelo LCE, assim como na latência de queda no ROTAROD. HI piora a performance motora no ladder test. No teste do CV, não observamos efeitos da HI sobre a busca por novidade assim como sobre a atividade locomotora espontânea. No entanto, MK801 diminui comportamentos de autolimpeza e a atividade locomotora espontânea. Menor variação das passadas foi observada em decorrência da administração de MK801 no stride length, com nenhum efeito da HI. Nossos resultados demonstram que a inibição do receptor NMDA tem um efeito neuroprotetor sobre os progenitores de oligodendrócitos e mielinização, provavelmente pela manutenção da capacidade proliferativa por um período maior. A atividade do receptor NMDA exerce importante papel na diferenciação das células de Purkinje, assim como na distribuição do transportador GLAST, corroborando a importância deste receptor na gênese das lesões causadas pela HI.Peri and prenatal systemic lesions alter CNS development leading to motor and cognitive problems in children that might persist throughout life. A particular kind of injury, the hypoxic ischemic (HI), is characterized by a permanent or temporary blockage of blood flow. One of the proposed mechanisms downstream from a HI event is called glutamatergic excitotoxicity. The administration of glutamate inhibitors has been studied in HI models for several years. In this work, we evaluated the effects of administration of a non-competitive antagonist of glutamate receptor, NMDA, on cerebellar development and behavioral tests of HI animals. Pregnant rats in the 18th gestational day were anesthetized, the uterine horns were exposed and the four uterine arteries were clamped for 45 minutes (group H). Sham controls had the uterine horns exposed, but no arteries were clamped (group S). Gestation proceeded after surgery. Only full term animals were used. One day after birth half the animals was assigned to receive either SALINE (groups SS and HS) or MK801 (groups SM and HM). Animals were anesthetized and perfused with 4% paraformaldehyde at 9, 23, 30 and 60 days of age. Parasagittal cerebellar sections were submitted to Calbindin, GFAP, GLAST, PDGFRα and MBP immunohistochemistry. Beginning at P45 animals were subjected to a battery of behavioral tests: elevated plus maze (EPM), hole board (HB), ROTAROD, ladder test and stride length. At P9 the dendritic tree of Purkinje cells were thinner in SM, HS/HM animals, indicating that both HI and MK801 are deleterious regarding this Purkinje cell differentiation. A lower number of PDGFRα+ cells was observed in HS/HM animals, with no effects of MK801 administration. At P23 a greater number of PDGFRα+ cells was found in HM animals when compared to the other 3 groups, demonstrating a neuroprotector effect of MK801. A lower number of myelinated fibers (MBP+) was observed in HS animals at P9, and MK801 administration reverse this effect. At P9, GLAST distribution was altered in HS animals, and MK801 partially reverse this altered distribution. No effects of HI and MK801 were observed in the EPM and ROTAROD tests. HI decreased motor performance of hind limbs in the ladder test, though no effect of MK801 was noted. In the HB test, we do not observe HI effects regarding the novelty seeking behavior and locomotor activity, otherwise the administration of MK801 decreased the number of grooming and locomotor activity. In the stride length test, we do not observed effects of HI although MK801 augmented the length variation of the fore limbs. Our results show that inhibition of NMDA receptors exerts a neuroprotector effect on oligodendrocyte progenitor cells and myelination, probably by temporarily inhibiting differentiation of those, providing more time to proliferate. NMDA activity exerts a crucial role in Purkinje cell differentiation as well as in GLAST distribution. Taken together our results lead us to conclude that NMDA receptor activity has an important role in the genesis of lesions caused by HI events

Environmental and food chain contamination sunscreens: a potential risk to human health

Submitted by Alexandre Sousa ([email protected]) on 2015-09-29T14:19:44Z No. of bitstreams: 1 Analytica_2015_45-54.pdf: 943897 bytes, checksum: d0a60d9cb63bba728b5d2ec0c0721108 (MD5)Approved for entry into archive by Alexandre Sousa ([email protected]) on 2015-09-29T14:19:53Z (GMT) No. of bitstreams: 1 Analytica_2015_45-54.pdf: 943897 bytes, checksum: d0a60d9cb63bba728b5d2ec0c0721108 (MD5)Approved for entry into archive by Alexandre Sousa ([email protected]) on 2015-09-29T14:34:42Z (GMT) No. of bitstreams: 1 Analytica_2015_45-54.pdf: 943897 bytes, checksum: d0a60d9cb63bba728b5d2ec0c0721108 (MD5)Made available in DSpace on 2015-09-29T14:34:42Z (GMT). No. of bitstreams: 1 Analytica_2015_45-54.pdf: 943897 bytes, checksum: d0a60d9cb63bba728b5d2ec0c0721108 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Farmacologia e Toxicologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Farmacologia e Toxicologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Farmacologia e Toxicologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Farmacologia e Toxicologia. Rio de Janeiro, RJ, Brasil.Evidências cientí­ficas têm apontado fortemente para a presença de uma vasta gama de­ filtros solares orgânicos numa variedade de alimentos, água e leite materno. Vários trabalhos demonstram que a exposição oral de roedores a estes compostos é capaz de promover alterações reprodutivas e desregulação do eixo tireoideano levando principalmente ao hipotireoidismo. Essa condição em humanos gera uma série de comprometimentos como défi­cit cognitivo, hiperatividade e alterações motoras. As crianças, neste contexto, são mais vulneráveis a essa exposição, pois alterações nos níveis dos hormônios tireoideanos durante janelas críticas do desenvolvimento geram retardo mental caso não haja a reposição hormonal adequada. A recomendação do uso de filtros solares para prevenção do câncer de pele e o crescente uso por parte da população, principalmente de regiões costeiras e ribeirinhas acaba por contaminar a água, peixes e outros alimentos através da irrigação. A julgar pelos potenciais danos causados pelo consumo destas substâncias, é necessário o estabelecimento de políticas de vigilância voltadas para o monitoramento da contaminação de alimentos com fi­ltros solares afim de garantira saúde e segurança da população.Accumulating evidence indicates the presence of a large number of organic sunscreens in food, water and breast-milk. Several works has shown that oral exposure of rodents to sunscreens induce reproductive disorders and thyroid disruption leading to hypothyroidism. This condition in humans has been shown to cause several damages like cognitive disabilities, hyperactivity and motor changes. The children, in this context, are more vulnerable to this exposure, because the reduction of thyroid hormones during critical periods of development induce mental retardation if no hormonal replacement has been given. The sunscreens prescription for prevention of skin cancer and the growing use of these products by population, mainly in costal and river side regions leading to water and ¬shes contamination as well as other foods through of irrigation. Whereas the potencial damages induced for consumption of this substances, it is needed the establishment of surveillance policies for monitoring food contamination with sunscreens to ensure the health and safety of population

Potency evaluation of unfractionated heparins commercialized in Brazil through anti-factor Xa and anti-factor IIa chromogenic tests and coagulation assay: Vigil Sanit Debate, Rio de Janeiro, 2023, v.11: e01923 | Publicado em: 11/05/2023

Introduction: Heparin is a drug that has anticoagulant activity, binding to antithrombin and accelerating the rate of inhibition of several proteases involved in the coagulation process. In the 2000s, the world market faced a troubled period regarding heparins after reports of allergic reactions and deaths caused by its use, requiring more rigorous quality control. Objective: The main goal of this work was to perform quality control of unfractionated sodium heparins of porcine origin commercialized in Brazil and heparin raw material on a dry basis, of both porcine and bovine origin, through potency assays. Method: Sixty-four samples of the final product (commercialized) were analyzed: 39 of brand A and 25 of brand B, and six samples of raw materials. Samples were assayed through anti-factor Xa and anti-factor IIa, according to United States  Pharmacopeia (USP), and coagulation assay, described in the 5th edition of Brazilian Pharmacopeia (BP). Results: In the present study, 40 heparin samples were approved in all potency assays, while 24 samples were non-approved, 23 of brand A and one of brand B. All samples of porcine-origin raw materials were considered approved, while the three of bovine origin showed lower potency. Conclusions: Almost all non-approved samples presented potency above 110%, which may represent a bleeding risk for patients. Thus, it is necessary to monitor the quality control of heparins and assess the clinical condition of patients undergoing their use to identify and reduce risks and safeguard public health.TÍTULO PT: Avaliação da potência das heparinas não fracionadas comercializadas no Brasil por meio dos ensaios cromogênicos antifator Xa e antifator IIa e teste de coagulação Introdução: A heparina é um fármaco que apresenta atividade anticoagulante, ligando-se à antitrombina e acelerando a taxa de inibição de diversas proteases envolvidas no processo de coagulação. Na década de 2000, o mercado mundial enfrentou um período conturbado em relação às heparinas após relatos de reações alérgicas e de mortes causadas pelo seu uso, o que exigiu um controle de qualidade mais rigoroso. Objetivo: Realizar o controle de qualidade das heparinas sódicas não fracionadas de origem suína comercializadas no Brasil e da matéria-prima  heparina em base seca, tanto de origem suína quanto bovina, por meio de ensaios de potência. Método: Foram analisadas 64 amostras do produto final (comercializado), sendo 39 da marca A e 25 da marca B, e seis amostras de matérias-primas. As amostras foram testadas por antifator Xa e antifator Iia, de acordo com a Farmacopeia dos Estados Unidos (USP), e por teste de coagulação, descrito na 5ª edição da Farmacopeia Brasileira (BP). Resultados: Quarenta amostras de heparina foram aprovadas em todos os ensaios de potência e 24 amostras não foram aprovadas, sendo 23 da marca A e uma da marca B. Todas as amostras de matérias-primas de origem suína foram consideradas aprovadas, enquanto as três de origem bovina apresentaram menor potência. Conclusões: Quase todas as amostras não aprovadas apresentaram potência acima de 110%, o que pode representar risco de sangramento para os pacientes. Assim, é necessário monitorar o controle de qualidade das heparinas e avaliar a condição clínica dos pacientes em uso para identificar e reduzir os riscos e salvaguardar a saúde pública

Double labeling with NADPH-d histochemistry (dark-blue) and microglia or astroglia immunoidentification (brown) in the vermis region of the cerebellar WM (0.5 mm mediolateral distance), during development.

<p>A–D - P9; E-H - P23. A, C, E and G (SHAM); B, D, F and H (HI); A–B and E–F – double labeled with anti-ED1 antibody; C–D and G–H – double labeled with anti-GFAP antibody. In both groups at P9, we can observe small, rounded NADPH-d+/ED1+ cells (A and B) or NADPH-d+/GFAP+ cells (C and D), as indicated by arrows. In D, observe a blood vessel, transversally cut, which presents NADPH-d staining, surrounded by GFAP+ astrocytic endfeet (asterisk). At P23, observe small rounded NADPH-d+/ED1+ cells in both groups, as indicated by arrows. HI animals display NADPH-d+/GFAP+ cells with typical reactive astrocyte morphology (arrows). SHAM animals do not present NADPH-d+/GFAP+ cells resembling reactive astrocytes. Arrowheads point to typical GFAP+ astrocytes, with no NADPH-d labeling. Notice the presence of NADPH-d+ blood vessels (asterisks in G and H) that are surrounded by GFAP+ astrocytic processes in HI animals (H) but not in SHAM animals (G). Calibration bar: 50 µm.</p

NADPH-d labeling in the cerebellar white matter (at the vermis region - 0.5 mm mediolateral distance) during development.

<p>A, B and C - P9; D, E and F - P23. A e D (NM); B and E (SHAM); C and F (HI). Observe that NADPH-d staining shows a higher number of cells (shown here in black - arrows) in HI animals, when compared to NM and SHAM ones. Observe in F that the morphology of the positive cells (arrowheads) in P23 HI animal is different from the NADPH-d stained cells in NM/SHAM animals at the same age and from all groups at P9. Calibration bar: 50 µm.</p

Distribution of NADPH-d stained cells, shown here in black after gray-scale conversion of the original color image, in the molecular layer (ML) of different folia in the vermis region (0.5 mm mediolateral distance) of the cerebellum at P23 and P90.

<p>A, C, E and G (SHAM); B, D, F and H (HI). A–D: P23 - A and B, folium 6; C and D, folium 10; E–H: P90 - E and F, folium 6; G and H, folium 10. At P23 and P90, SHAM animals presented a diffuse NADPH-d staining in the ML and a great number of NADPH-d+ cell bodies in folium 6 (A and E - arrows). This same pattern is observed in HI animals (B and F - arrows). In folium 10, no labeled cells were seen in the ML of SHAM animals (C and G), whereas HI animals presented a weaker staining in the ML in the same folium and ectopic NADPH-d+ cells near the PC, but also in the superficial ML (arrows in D and H). Granular cell layer showed a maintained NADPH-d staining in both groups. ML = molecular layer; PC = PC. Calibration bar: 50 µm.</p

Pattern of NADPH-d labeling in Purkinje cells at P9 shown here after gray-scale conversion of the original color image.

<p>The scheme in the uppermost part of the figure indicates in bold and italic the folia shown in the figures A to H, at the vermis region (0.5 mm mediolateral distance). Med - medial (fastigial) cerebellar nucleus; Pcuf - preculminate fissure; Prf - primary fissure; Psf - posterior superior fissure; Ppf - prepyramidal fissure; Sf - secondary fissure; Uf - uvular fissure Plf - posterolateral fissure; ML - molecular layer. A, B: At folium 3 in SHAM animals (A), Purkinje cell bodies have a strong labeling and the dendritic trees are well defined by NADPH-d staining (arrows), while in HI animals (B), cell bodies have a weaker staining with a less defined staining pattern in the dendritic arbors (arrows). C, D: At folium 1 in SHAM animals (C), both cell bodies and processes have a strong labeling (arrowheads), while in HI ones (D), the cell bodies do not present NADPH-d activity (asterisks in the presumptive PC). Observe in D that the ML presents a strong and diffuse staining that corresponds to the location of the dendritic trees of the Purkinje cells. E, F: At folium 10, none of the groups presented NADPH-d staining in Purkinje cell bodies or in the dendritic arbors (asterisks in the presumptive PC). The presence of Purkinje cells was verified by hemaetoxilin staining in an adjacent section (G – SHAM; H – HI, arrows). WM = white matter; IGL = internal granular layer; EGL = external granular layer. Calibration bar: A–D, G and H = 50 µm, E and F - 100 µm.</p

Average cell numbers per 100 µm² in the cerebellar white matter of HI, SHAM and NM animals at P9, P23 and P45.

<p>5 animals in each age from at least 3 different litters per group were used (maximum of 2 pups per dam). Note that, at P23, the number of cells in HI group is significantly higher than in the SHAM and NM and that no difference was observed between these last two groups. At P45 no NADPH-d+ cells were observed in all groups. *** P<0.001.</p