Os autores gostariam de agradecer ao Dr. Hui Zheng (Baylor College of Medicine) para a APP cDNA. Este trabalho foi financiado pelo NIH R21AG031948 (DB, JMB), F30AG030878 (ESS), R01DK073394 (AFO), o John Sealy Memorial Fund Endowment for Biomedical Research (AFO), e Jean C. e D. William Willis Neuroscience Research Endowment (ESS). JMB é um estudioso do Programa de Pesquisa Translacional Scholar e membro da Universidade do Texas Medical Sucursal Claude E. Pimenta antiga americanos Independência Centro (apoiado pelo NIH UL1RR029876 e P30-AG-024832, respectivamente).

<html> 1. Expressão de domínio recombinante APP intracelular (AICD) <ol><li> E. Transformação coli BL21 com AICD humana (resíduos 649-695 de APP, isoforma neuronal de numeração) clonado no vector pGEX-4T-1 (GE Healthcare). Este vector expressará AICD como a porção C-terminal de uma proteína de fusão de glutationa-S-transferase (GST). Este vector também codifica uma sequência de clivagem pela trombina para facilitar a remoção da porção GST. Detalhes da clonagem AICD em pGEX-4T-1 pode ser encontrada na nossa publicação anterior 4. </li><li> A partir de uma colónia isolada, inocular 10 ml de caldo de LB com ampicilina (100 jig / ml) e incubar durante a noite a 37 ° C com agitação vigorosa. </li><li> Na manhã seguinte, inocular 400 ml de LB / ampicilina, num balão de um litro com 5 ml da cultura durante a noite. </li><li> Incubar a 37 ° C com agitação vigorosa até a densidade óptica a 600 nm ter atingido 0,4 e 0,6. Isto normalmente levar de 2 a 2,5 horas. </li></ol>&gt; content &quot;Neste ponto, pode ser desejável para remover uma pequena amostra (~ pi 10) para seguir a purificação por SDS-PAGE (ver Figura 1A, B). Cada passo de purificação subsequente deve ter também uma pequena alíquota removida para análise por SDS-PAGE. <ol start="5"><li> Induzem a expressão de GST-AICD pela adição de 0,4 mM de isopropil-beta-D-tiogalactopiranósido (IPTG). Incubar a 37 ° C com agitação vigorosa durante 5 horas. </li><li> Centrifugar as células a 6.000 xg durante 15 min a 4 ° C. O pelete de células pode ser armazenada a -80 ° C durante vários meses, neste ponto do procedimento. </li></ol> 2. Purificação de GST-AICD <ol><li> Preparar 50 ml de tampão de lise como se segue: 1 mM de ditiotreitol, 1 mM de ácido etilenodiaminotetracético (EDTA), 0,1% de Triton-X100, fluoreto de fenilmetilsulfonilo 1 mM (PMSF), cocktail inibidor de protease completo (Roche Applied Science), e solução salina tamponada com fosfato. Relaxar no gelo. </li><li> Descongelar pelete bacteriana (se previamente congelado) emgelo. </li></ol> Neste ponto, é importante que todos os passos são realizados a 4 ° C para limitar a proteólise. Todos os tubos, tampões, e outros reagentes devem ser pré-arrefecida. Se usar uma imprensa francesa ou emulsificante para lise, estes devem ser pré-refrigerados também. <ol start="3"><li> Completamente ressuspender o sedimento bacteriano em 20 ml de tampão de lise. Uma combinação de centrifugação e a trituração com 10 ml de pipeta em gelo assegurará completa ressuspensão do sedimento. </li><li> Passar as células ressuspensas bacterianas através de um emulsionante (por exemplo, Avestin EmulsiFlex-C3) ou célula de pressão Francesa, pré-refrigerada a 4 ° C a 30000 psi. Passar o lisado por o emulsionante uma segunda vez. Recolher o lisado para um tubo de ml pré-refrigerada 50. </li></ol> A principal vantagem destes dois instrumentos é que eles minimizar o aquecimento da amostra. A técnica normalmente usada de sonicação produz calor significativo na ponta da sonda de ultra-sons epode resultar em agregação de proteínas recombinantes. <ol start="5"><li> Centrifuga-se o lisado a 15.000 xg durante 30 min. Separa-se e manter-se o sobrenadante para um tubo de ml pré-refrigerada 50. </li><li> Adicionar 500 ul de uma suspensão a 50% de esférulas de glutationa-agarose (Sigma-Aldrich). Rodar durante 30 min a 4 ° C. Enquanto a amostra está a rodar, fazer de tampão de eluição fresco: 50 mM de Tris 7,8, 0,1% de Triton X100, 0,5 mM de ditiotreitol, 10 mM de glutationa reduzida. </li></ol> O único passo do protocolo, que é realizado à temperatura ambiente é o passo de eluição. Assim, o tampão de eluição é mantida à temperatura ambiente. Tampão de eluição deve ser preparada. <ol start="7"><li> Verter o lisado e lama numa coluna de cromatografia de 5 descartável &quot;poliestireno com grosseiras (90-130 mm) (filtros Evergreen Scientific). Lava-se a coluna 5 vezes com 3 ml de solução salina tamponada com fosfato. </li><li> Tapar a parte inferior da coluna e adicionar 500 ul de tampão de eluição de temperatura ambiente. Tapar o topo dacoluna e girar durante 5 min à temperatura ambiente. Recolher o eluato num refrigerados tubo de microcentrífuga de 1,5 ml. Repita a eluição mais duas vezes (1,5 ml eluato total). </li><li> Dialisar o eluato num molecular da membrana de diálise de corte de peso 10000 (por exemplo, Thermo Scientific Slide-A-Lyzer de diálise cassetes) em 4 litros de solução salina tamponada com fosfato durante a noite a 4 ° C. Dializar contra um adicional de 4 litros de tampão fosfato salino a 4 ° C durante 1 hora no dia seguinte. </li><li> Remover a proteína a partir da cassete de diálise e realizar um ensaio de quantificação de proteína para determinar o rendimento de GST-AICD. Uma purificação típica de 400 ml de cultura irá resultar em rendimentos de&gt; 20 mg de proteína purificada em concentrações de 10-20 mg / ml. Alíquota da proteína em alíquotas de 200 ul e congelar a -80 ° C. </li></ol> 3. A clivagem de trombina de GST-AICD e Purificação de AICD <ol><li> Ul descongelamento 200 das purificada GST-AICD e adicionar 20 ul de uma Thromb U / ulpol Incubar durante a noite a 37 ° C. </li></ol> A qualidade e pureza da trombina varia consideravelmente entre os fabricantes, e pode ser uma importante fonte de contaminação. Usamos trombina da GE Healthcare (ver tabela de reagentes). Após incubação durante a noite,&gt; 95% da proteína de fusão deve ser clivado (Figura 1B). <ol start="2"><li> Após a clivagem com trombina durante a noite, é provável que uma fracção da AICD libertada foi submetido a agregação e a solução pode parecer turva. Limpar o material agregado por centrifugação a 20.000 xg durante 30 min a 4 ° C. Remover o sobrenadante para um tubo de microcentrífuga de pré-arrefecida. </li><li> Adicionar 50 ul de 50% de esférulas de glutationa-agarose a suspensão aquosa e 50 ul de 50% de p-aminobenzamidina-agarose suspensão para remover GST e da trombina, respectivamente. Incubar com rotação durante 5 minutos à temperatura ambiente. </li><li> Centrifugar brevemente para sedimentar as esferas de agarose, e remover o sobrenadante para um novo tubo. Extrair tele GST mais quatro vezes com glutationa-agarose. A única extracção com p-aminobenzamidina-agarose é suficiente para remover o U 20 da trombina. </li><li> Executar um ensaio de quantificação de proteínas em 5 ul da AICD purificada. Os rendimentos típicos a partir de uma cultura de 400 ml são 50-100 ug, a uma concentração de 0,2 a 0,5 mg / ml. Rendimentos mais altos são possíveis por meio de clivagem de volumes maiores de GST-AICD. </li></ol> Purificada AICD deve ser preparado para a caracterização bioquímica / biofísica. Material não utilizado deve ser descartado. Também é possível concentrar a AICD por liofilização da amostra e ressuspensão em um pequeno volume de tampão 6. Se detectar AICD requer blotting (tal como após uma armadilha de filtro ou de ensaio dot blot), a recuperação do antigénio da membrana deve ser realizada como descrito por Pimplikar Suryanarayana e 12. 4. Agregação AICD de Microscopia de Força Atômica (AFM) <ol><li> Diluir preparado de fresco AICD em phosphate solução salina tamponada a uma concentração de 0,1 mg / ml num volume final de 15 ul. Neste ponto, vários compostos e / ou proteínas podem ser adicionados, para determinar se eles inibem a agregação AICD. Por exemplo, demonstramos que as concentrações equimolares de ubiquilin-1 prevenir a agregação de AICD neste ensaio 4. </li></ol> Também é possível acrescentar um maior volume de reacção para experiências bioquímicas, tais como ensaios de armadilha de filtragem e de espalhamento de luz 4. Tivemos sucesso realizando ensaios de armadilha de filtro em misturas reaccionais tão grande como 400 ul com AICD diluído para concentrações tão baixas como 1 uM. <ol start="2"><li> Induzir a agregação térmica por incubação das amostras a 43 ° C com agitação a 800 rpm num Thermomixer de Eppendorf durante 48 horas. Esta temperatura foi escolhida com base em ensaios que examinam a chaperones agregação térmica do modelo da citrato sintase chaperone substrato 4,13. </li></ol><p class="jove_content"&gt; Agregação térmica é um método vulgarmente utilizado para induzir a transição estrutural da proteína nativa a partir de uma estrutura para um não-nativo (que é geralmente folha beta rico), que resulta na formação de agregados intermoleculares. Agregação térmica também é utilizado para examinar a função de chaperonas moleculares, os quais protegem os segmentos hidrofóbicos de formar inadequado interacções intermoleculares (e, assim, retardar ou evitar a agregação térmica). <ol start="3"><li> Dilui-se a amostra 20 vezes em água desionizada e ul ponto 2 sobre mica clivada de fresco. Seca-se a amostra num exsicador durante a noite. Será necessário determinar empiricamente a diluição óptima para a imagiologia, e, portanto, várias diluições que variam de 10 - e 100 vezes seria prudente. </li><li> Visualize a AICD agregado utilizando a operação AFM em modo de tocar 14. As vigas são utilizados ouro revestidas com nitreto de micro alavancas afiados (MSNL, Bruker), com um raio da ponta nominal de 2 nm. Típico tocando durante amplitudesção de imagem são 10-20 nm, a frequência de ressonância das consolas (30-50 kHz). </li></ol> Nós descobrimos que a qualidade da imagem e do contraste de agregados de proteínas diferentes dependia da força aplicada e da duração da experiência. Para minimizar a perturbação da amostra pela ponta, é mantida a força aplicada relativamente baixo, mantendo uma relação de set-point acima de 95% e limitando o varrimento de cada uma das amostras de 5-10 min. Processamento de imagem foi realizada com WSxM software (Nanotec). Processamento de imagem padrão consiste em avião subtração e achatamento. Nossos laboratórios utilizam uma &quot;casa-construída&quot; AFM 15, interligados a um sistema de digitalização sonda comercial controle microscópio (Nanotec). 5. Resultados representativos A expressão e um enriquecimento relativo de GST-AICD é mostrado na Figura 1A. Após a lise, a maior parte da proteína de fusão está presente na fracção solúvel e, portanto, a extracção a partir de inclusiem órgãos não é necessária. O material eluído da coluna de agarose de glutationa é&gt; 95% puro. Na preparação mostrado na Figura 1, a concentração de proteína eluída da coluna foi de 14,5 mg / ml, e o rendimento foi de 22 mg (a partir de uma cultura inicial de 400 ml). A clivagem de 200 ul da preparação, durante a noite, com 20 U de trombina resultou em quase 100% de clivagem da proteína de fusão (Figura 1B). A quantidade de trombina utilizada é suficientemente baixo para não ser detectáveis ​​por coloração com azul de Coomassie (ver &quot;Clivadas&quot; pista Figura 1B). A remoção da trombina e GST resultou em alguma perda de material, no entanto, foi&gt; 90% puro com um rendimento nesta preparação de 70 ug de AICD, a uma concentração de 0,35 mg / ml. Figura 1C mostra o diagrama de fluxo para a agregação de AICD e subsequente análise. Agregados AICD fotografada por AFM são tipicamente esferoidais / amorfa, e variam em tamanho de 50 a 100 nm (Figura 1D, F). Agregadomaterial não é detectável, quando uma quantidade equimolar da chaperona ubiquilin-1 é adicionado à reacção de agregação (Figura 1E) 4. <img alt="Figura 1" src="/files/ftp_upload/4204/4204fig1.jpg" /> Figura 1. Purificação e agregação de AICD. A) Coloração com azul de Coomassie de amostras recolhidas em cada passo do processo de purificação. Para as amostras não induzidas e induzidas, 20 uL de bactérias totais foram corridas no gel. Para a fracção sobrenadante, 5 ul de lisado (a partir de um volume total de 20 ml), foram executados no gel. Para a fracção de sedimento, o sedimento foi ressuspenso em 20 ml de tampão de lise, e 5 ul deste material foram sonicadas num sonicador de banho de água durante 30 min a 4 ° C antes do carregamento para o gel. Para o fluxo contínuo, 5 pi foram corridos no gel. As quantidades de líquido eluído carregadas no gel são indicadas. B) de Coomassie azul do gel corado não clivada e clivada GST-AICD (2 ul) e 1 e 5 m &amp;u; l purificada AICD. C) Fluxograma de purificação AICD, agregação e análise. D, E) representativas de imagem AFM termicamente agregada AICD na ausência (D) e na presença (E) da chaperona molecular ubiquilin-1. F) Histograma da distribuição de tamanho de agregados AICD.

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Não há conflitos de interesse declarados.

Neste protocolo temos delineado um procedimento para obter AICD altamente puro para análises estruturais, biofísicos e bioquímicos. Este procedimento não requer equipamento sofisticado cromatografia e é, portanto, acessível para a maioria dos laboratórios. Outros grupos têm purificada AICD 5-7,16, incluindo GST-AICD 17-19, para as análises bioquímicas / estrutural. Desvantagens aos protocolos anteriores incluem fraca solubilidade do AICD 16, menos do que ideal pureza 17,</...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> pGEX-4T-1 </td><td> GE Healthcare </td><td> 28-9545-49 </td><td></td></tr><tr><td> Trombina </td><td> GE Healthcare </td><td> 27-0846-01 </td><td></td></tr><tr><td> Ampicilina </td><td> Fisher Scientific </td><td> BP1760 </td><td></td></tr><tr><td> Bradford reagente de ensaio de proteínas </td><td> Bio-Rad </td><td> 500-0002 </td><td></td></tr><tr><td> Coomassie azul </td><td> Bio-Rad </td><td> 161-0786 </td><td></td></tr><tr><td> IPTG (isopropil-beta-D tiogalactopiranósido) </td><td> Sigma-Aldrich </td><td> I6758 </td><td></td></tr><tr><td> Glutationa-agarose </td><td> Sigma-Aldrich </td><td> G4510 </td><td></td></tr><tr&gt; <td> p-aminobenzamidina-agarose </td><td> Sigma-Aldrich </td><td> A7155 </td><td></td></tr><tr><td> Cocktail inibidor de protease completo </td><td> Roche </td><td> 11836170001 </td><td></td></tr><tr><td> Slide-A-Lyzer cassetes de diálise </td><td> Thermo Scientific </td><td> 66380 </td><td></td></tr><tr><td> Colunas de cromatografia </td><td> Scientific Evergreen </td><td> 208-3367-050 </td><td></td></tr><tr><td> Emulsificante </td><td> Avestin, Inc </td><td> EmulsiFlex-C3 </td><td> Altamente recomendado </td></tr><tr><td> Eppendorf Thermomixer </td><td> Eppendorf </td><td> 022670107 </td><td></td></tr><tr><td> Discos de mica </td><td> Ted Pella </td><td> 50-12 </td><td></td></tr><tr><td> Cantilevers de AFM </td><td> Bruker </td><td> MSNL-10 </td><td></td></tr><tr><td> WSxM software </td><td> Nanotec </td><td> N / A </td><td> Free Download </td></tr></tbody></table>

purification and aggregation of the amyloid precursor protein intracellular domain

Proteína precursora amilóide (APP) é uma proteína transmembranar de tipo I associado à patogénese da doença de Alzheimer (AD). APP é caracterizada por um grande domínio extracelular e um domínio citosólico curto denominado o domínio intracelular de APP (AICD). Durante a maturação através da via secretora, APP pode ser clivada por proteases denominadas α, β e γ-secretases 1. A clivagem proteolítica da APP sequencial com β e γ-secretases conduz à produção de um péptido pequeno proteolítico, denominado de Ap, que é amiloidogénica e o constituinte principal das placas senis. A AICD também é libertada a partir da membrana, após o processamento secretase, e por meio de interacções com Fe65 e Tip60, pode translocar para o núcleo para participar na regulação da transcrição de genes alvo múltiplas 2,3. Interacções proteína-proteína envolvendo a AICD podem afectar o tráfico, o processamento e as funções celulares de holo-APP e seus C-terminfragmentos al. Mostrámos recentemente que AICD pode agregar in vitro, e este processo é inibido pelo ubiquilin-1-AD implicado chaperone molecular 4. Consistente com estes resultados, a AICD expôs domínios hidrofóbicos e está intrinsecamente desordenados in vitro 5,6, no entanto, obtém estrutura secundária estável quando ligado ao Fe65 7. Propusemos que ubiquilin-1 impede inadequado interacções inter-e intramolecular da AICD, prevenindo a agregação in vitro e em células intactas 4. Enquanto a maioria dos estudos focado o papel de APP na patogénese de AD, o papel da AICD neste processo não são claras. Expressão da AICD foi mostrado para induzir a apoptose 8, para modular as vias de sinalização 9, e para regular a sinalização de cálcio 10. A sobre-expressão da AICD e Fe65 num modelo de ratinho transgénico induz patologia de Alzheimer, como 11, e recentemente AICD foi detectado no braem lisados ​​por western blotting quando usando técnicas de recuperação de antigénios apropriados 12. Para facilitar os estudos estruturais, bioquímicas e biofísicas da AICD, desenvolvemos um procedimento para a produção de quantidades grandes de forma recombinante altamente pura proteína AICD. Iremos descrever um método para a indução in vitro da agregação térmica da AICD e análise por microscopia de força atómica. Os métodos descritos são úteis para a caracterização bioquímica, biofísica e estrutural da AICD e os efeitos das chaperones moleculares na agregação AICD.

Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Um método para a purificação em larga escala do domínio intracelular de APP (AICD) é descrita. Nós também descrevem metodologia de indução<em&gt; In vitro</em&gt; Agregação AICD e visualização por microscopia de força atômica. Os métodos descritos são úteis para a caracterização bioquímica / estrutural da AICD e os efeitos das chaperones moleculares sobre a sua agregação.

Purificação e Agregação do Domínio proteína precursora de amilóide intracelular

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

purification-aggregation-amyloid-precursor-protein-intracellular

Research

JoVE Journal

Medicine

11.7K visualizações.  University of Texas Medical Branch. Um método para a purificação em larga escala do domínio intracelular de APP (AICD) é descrita. Nós também descrevem metodologia de indução In vitro Agregação AICD e visualização por microscopia de força atômica. Os métodos descritos são úteis para a caracterização bioquímica / estrutural da AICD e os efeitos das chaperones moleculares sobre a sua agregação.

Vídeo: Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

Executar e Processamento de FNA Pad Fat Anterior para amilóide

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

Medicina

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Transplante subretinal de células precursoras do fotoreceptor purificado MACS na retina do rato adulto

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Use Synergetic de células neuronais precursoras e Peptídeos de auto-montagem em Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&amp;#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Avaliação de Alternância Espontânea, Reconhecimento de Objetos Novos e Clasping de Membros em Modelos de Rato Transgênico de Neuropatologia Amilóide-β e Tau

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetria em plaquetas humanas lavadas: O efeito do inibidor Pannexin1-Brilliant Blue FCF na agregação induzida por colagénio

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Avaliação da remoção de reserva e intracelular do cálcio diastólica cálcio sarcoplasmático em Cardiomyocytes Ventricular isolado

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Na Vivo Imagem de Cx3cr1gfp/gfp repórter ratos com a tomografia de coerência óptica espectral-domínio e digitalização Laser Oftalmoscopia

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Isolamento e purificação de Pericytes cardíacos murinos

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Isolamento baseado em ultracentrífuga de vesículas extracelulares de ADSCs humanas

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Autor em destaque: Padronizando e melhorando a extração e purificação de vesículas extracelulares de ADSCs humanas

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

Purificação e Caracterização de Vesículas Extracelulares de Células-Tronco Mesenquimais Derivadas de Tecido Adiposo Humano

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...