Os autores agradecem a Sra Dalia Sela eo Sr. Emi Sharon por seu trabalho técnico profissional e excelente no laboratório.

Os animais foram tratados de acordo com a resolução da ARVO sobre o uso de animais em pesquisa. O protocolo do estudo foi aprovado pelo Comitê institucional para o Animal Research Laboratory (aprovação sem 022-4598-2;. 021.211). 1. Preparação para a cirurgia # 1 <ol><li> Pesar e anestesiar um coelho branco da Nova Zelândia 13 dias de idade, com uma injecção intramuscular de cloridrato de cetamina 100 mg / ml (100 mg / kg) e cloridrato de xilazina 25 mg / ml (12,5 mg / kg). Essas doses são utilizados devido à duração do processo cirúrgico. Assegurar nível adequado de anestesia com a falta de um reflexo dor. </li><li> Usando uma mecha de algodão estéril, aplicar uma pequena quantidade de pomada oftálmica para o olho não operado para impedir que a córnea de secar. Aplicar 0,9% de solução salina normal de gotas sobre a córnea do olho operado durante a operação para evitar que as córneas de secar. </li><li> Realizar três medições de comprimento axial em cada olho, após anestesia tópica (oxybuprocaine cloridrato de 0,4%) usando um ultra-som Um scanner e, em seguida, calcular a média das medições. Aplicar a sonda perpendicularmente à córnea central. </li></ol> 2. Cirurgia # 1 - Pré Cruz Passo Linking <ol><li> Utilizando uma pinça oftálmicos cirúrgicos e tesouras sob um microscópio cirúrgico oftálmico, fazer uma peritomia 360 ° conjuntival com uma pinça oftálmicos cirúrgicos e tesouras. </li><li> Usando uma pinça em ângulo ou um gancho muscular identificar os quatro músculos retos extra-oculares e isolá-los com um 2-0 trançado de seda suturas não agulhados. NOTA: As suturas de seda vai ajudar com mover o mundo em direção desejada. O globo ocular é dividido em quadrantes entre os quatro músculos retos (quatro quadrantes). <ol><li> Mark pele com um marcador de duas zonas, em cada quadrante, um na esclera equatorial e um na esclera posterior. Estas são as zonas de irradiação. </li></ol></li><li> Prepare de 3 ml a seringa de 5 ml contendo 0,1% de riboflavina-5-Phosphat livre de dextranoe. Ligar a seringa a uma cânula lacrimal 26 G ou 25 G hydrodelineator cônico. </li><li> Prepara-se o dispositivo de irradiação que inclui uma fonte de luz UV A (370 nm) ligado a um chanfrada para baixo feito por medida de fibra óptica. Após a medição da potência de energia e de calibração (ver nota abaixo), configurar o dispositivo para 57 mW / cm². NOTA: A calibração foi realizada comercialmente pelo fabricante. Este método fornece uma dose total de luz UVA de 57 J / cm². (57 mW / cm 2 em 0,2 cm 2 é de 11,4 mW / 0,2 cm 2 utilizando 11,4 mW / 0,2 cm 2 para 200 segundos é uma carga acumulada de 2,2 J por 0,2 cm 2 spot) </li></ol> 3. Cirurgia # 1 - Cross Linking <ol><li> Mover o globo ocular, puxando as suturas de seda no sentido oposto do quadrante escolhido para ser tratada (por exemplo, Pull-se e quando o tratamento nasal quadrante temporal inferior). Realize o movimento do globo ocular usando uma sutura de seda 2-0 trançado não-needled que isolates cada músculo reto. </li><li> Aplicar a solução fotossensibilizador contendo 0,1% de riboflavina-5-fosfato livre de dextrano para a zona de irradiação de 20 segundos antes de iniciar a irradiação. </li><li> Irradiar o zona de irradiação usando um chanfrado para baixo feita sob encomenda da fibra óptica por um período de irradiação de 200 seg. Medir a área de cada uma das zonas de 0,2 cm com um raio de 4 mm. </li><li> Aplicar a solução fotossensibilizador contendo 0,1% de riboflavina-5-fosfato livre de dextrano para a zona de irradiação cada 20 seg durante o período de irradiação de 200 seg. NOTA: Executar a irradiação e o lançamento de riboflavina simultaneamente por dois cirurgiões. </li><li> Repita os passos de 3,1-3,4 para cada um dos quatro quadrantes do globo ocular. </li></ol> 4. Cirurgia # 1 - Post Cruz Passo Linking <ol><li> Raspar a pele ao redor dos olhos. Aparar as margens da pálpebra com uma tesoura oftálmicos cirúrgicos e em seguida, delicadamente suturar a parte superior e as pálpebras inferiores usando 4-0 trançado cor marfim de seda - 3/8 círculo recomprimento 13 milímetros verso cutting- C-3 de agulha (tarsorrafia). Aplicar uma pequena quantidade de pomada oftálmica na margem da pálpebra, no final do procedimento (cloranfenicol 5%). </li></ol> 5. Cirurgia # 1 - Cuidados pós-operativo <ol><li> Mantenha os animais sob uma lâmpada de aquecimento durante a recuperação. Não deixe um animal sem supervisão até que tenha recuperado a consciência suficiente para manter decúbito esternal. </li><li> Colocar o animal em uma gaiola limpa com novas roupas até que esteja totalmente recuperado. Após a recuperação, o animal será devolvido para a sala de animal. Devolver o animal para sua mãe apenas quando acordado. </li><li> Durante as primeiras 48 horas, examinar os animais relativas ao estado geral e sinais de doença ou infecções. Administrar analgesia conforme a secção 1. analgesia Administrar conforme necessário, se uma mudança no comportamento do coelho é observado. (Metamisole cai 50 mg / kg a cada 6 horas). </li></ol> 6. Cirurgia # 2 <ol><li> Cinquenta e cinco dias após surgery # 1, pesar e anestesiar o coelho com uma injecção intramuscular de cloridrato de cetamina 100 mg / ml (100 mg / kg) e cloridrato de xilazina 25 mg / ml (12,5 mg / kg). </li><li> Com uma tesoura oftalmológicas cirúrgicas sob um microscópio cirúrgico oftálmico, remover a tarsorrafia. Realizar três medições de comprimento axial em cada olho após anestesia tópica (cloridrato oxibuprocaína 0,4%) usando um ultra-som A-scanner e, em seguida, calcular a média das medições. Aplicar a sonda perpendicularmente à córnea central. </li><li> Eutanásia dos coelhos com fenobarbital de sódio intraperitoneal (200 mg / 1,5 kg de peso corporal). </li></ol>

<ol>
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Assoc. 85, 1032-1038 (1986).</li><li>Funata, M., Tokoro, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scleral+change+in+experimentally+myopic+monkeys.">Scleral change in experimentally myopic monkeys.</a> Graefes Arch. Clin. Exp. Ophthalmol. 228, 174-179 (1990).</li><li>McBrien, N. A., Norton, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+collagen+cross-linking+increases+form+deprivation+myopia+in+tree+shrew.">Prevention of collagen cross-linking increases form deprivation myopia in tree shrew.</a> Exp. Eye Res. 59, 475-486 (1994).</li><li>Iomdina, E. N., Daragan, V. A., Ilyina, E. 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Os autores declaram que não têm interesses financeiros concorrentes.

Apresenta-se o primeiro estudo in vivo da prevenção da miopia axial num modelo de coelho utilizando a tecnologia de ligação cruzada com riboflavina e irradiação UVA. Embora diferentes animais de laboratório pode ser usado neste tipo de estudo, escolhemos coelhos principalmente devido ao tamanho dos olhos e a necessidade de realizar a reticulação na superfície escleral. Descobrimos que a exposição da esclera de coelhos e suturando as pálpebras superior e inferior para ser processos de interpelação. Recomendamos aparar as margens da pálpebra e usar as suturas mencionados no nosso protocolo para garantir que as pálpebras são bem fechada para o período determinado, sem necessidade de correções. Um ponto importante a considerar é a necessidade de calibrar o dispositivo de irradiação e a fibra óptica para a energia de alimentação necessária. H &amp; E lâminas histológicas não revelaram alterações tóxicas para a retina em olhos submetidos ao processo de reticulação. Novas investigações devem ser realizadas para tratar a toxicidade potencial de riboflavina e irradiação UVA na retina, coróide e esclera, com foco na posição, a quantidade de energia e tempo de exposição. Várias limitações deste estudo consideração mandado. Apenas pré-operatório e pós-operatório comprimentos axiais foram medidos. O comprimento axial do globo ocular foi medida por A-scan de ultra-sons. Nós não avaliar as propriedades biométricos, análise de refração ou propriedades biomecânicas da esclera. Tomados em conjunto, pensamos que o efeito da reticulação escleral com riboflavina e UVA no alongamento axial do olho de coelho terá um impacto positivo na pesquisa de miopia.

Miopia é a mais comum das desordens de refracção. A prevalência de miopia nos EUA e na Europa é relatado para ser em torno de 30%, e em países asiáticos que afeta até 60% da população em geral 1,2. Progressão da miopia ocorre em até 50% dos míopes, geralmente a uma taxa de cerca de -0,5 dioptrias ao longo de um intervalo de dois anos 3. Os custos de saúde impostas por miopia são consideráveis, incluindo as despesas para óculos, lentes de contato e cirurgia refrativa e custos relacionados com o aumento dos riscos para a saúde de glaucoma, catarata, descolamento de retina e deficiência visual 4-6. Em estudos com animais de miopia, a redução da visão é induzida por sutura pálpebra 7-10, a colocação de um oclusor a uma curta distância a partir do olho e tatuagem da córnea 11. No entanto, para a miopia artificial para ocorrer nestes estudos, o processo de oclusão tem de ser realizado em animais muito jovens, como não há experiências de privação de vista carrIED para fora em espécimes adultos têm tido êxito. Uma das características importantes da miopia grave é uma alteração patológica da esclerótica com adelgaçamento progressivo da esclera, provavelmente devido a um mecanismo de feedback perturbado de emetropizaï¿½o após privação visuais 12 ou devido a alguma perturbação metabólica da esclerótica, tal como em Ehlers Danlos 13. Em última análise, ambos os mecanismos levam ao estiramento e afinamento da esclera, retina e coróide devido a anomalias estruturais da esclera míope como a diminuição do colágeno diâmetro da fibra 14,15 e os distúrbios fibrilogênese 16. Vários estudos têm mostrado que prejudicada reticulação do colagénio é um factor importante para o processo de enfraquecimento da esclera míope 17-18. Wollensak et ai. 19-21 induzida colagénio de ligação cruzada através da aplicação do fotossensibilizador riboflavina e irradiação ultravioleta-A (UVA) (370 nm)e observou um aumento significativo de 157%, na rigidez de porcino e humano in vitro esclera 19, e um aumento de 465% em rigidez escleral coelho in vivo (módulo de Young) 20. A reticulação também teve um efeito a longo prazo sobre esclera coelho in vivo: rigidez aumentada em 320,4% após 3 dias, 277,6% depois de 4 meses, e 502% após 8 meses (módulo de Young) 22. Tentativas terapêuticas para deter a progressão da miopia foram publicados 23-26 mas o sucesso destes métodos é controversa. Não há meios eficazes de prevenir a miopia progressiva foi encontrado até o momento. A etiologia da miopia ainda é controversa, e seu tratamento representa um desafio. Com base nestes resultados, é a hipótese de que a reticulação da esclerótica pode servir como um meio para o tratamento à base de esclerótica de progressão da miopia. O objetivo deste estudo é examinar a crosslinki colágeno escleralng efeito sobre o desenvolvimento da miopia axial induzida por oclusão visual.

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			<td height="21" style="height:21px;width:244px;">Skin marker</td>
			<td style="width:137px;">Devon</td>
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scleral cross-linking using riboflavin and ultraviolet-a radiation for prevention of axial myopia in a rabbit model

indivíduos míopes, especialmente aqueles com miopia severa, apresentam risco mais elevado do que o normal de catarata, glaucoma, descolamento de retina e alterações coriorretinianas. Além disso, miopia patológica é uma causa irreversível comum de deficiência visual e cegueira 1-3. O nosso estudo demonstra o efeito de reticulação escleral utilizando riboflavina e ultravioleta A radiação sobre o desenvolvimento da miopia axial num modelo de coelho. O comprimento axial do globo ocular foi medida por A-scan de ultra-sons em coelhos brancos New Zealand com idade de 13 dias (macho e fêmea). O olho, em seguida, foram submetidos a 360 ° peritomia conjuntival com reticulação escleral, seguido por tarsorrafia. alongamento axial foi induzida em 13 dias de idade coelhos Nova Zelândia por sutura as pálpebras do olho direito (tarsorrafia). Os olhos foram divididos em quadrantes e cada quadrante tinha duas zonas de irradiação esclerais, cada um com uma área de 0,2 cm² e um raio de 4 mm. A ligação cruzada foi realizada pela queda de 0,1%riboflavina-5-fosfato para a zonas de irradiação de 20 segundos antes de ultravioleta-A irradiação e a cada 20 segundos durante o tempo de irradiação de 200 seg-dextrano livre. A radiação UVA (370 nm) foi aplicado perpendicularmente à esclerótica a 57 mW / cm² (dose total de luz UVA, 57 J / cm). Tarsorrhaphies foram removidos no dia 55, seguida de medições repetidas comprimento axial. Este estudo demonstra que a reticulação escleral com ultravioleta e riboflavina-A radiação impede eficazmente induzida por oclusão alongamento axial em um modelo de coelho.

As Figuras 1 e 2 mostram graficamente as medições de comprimento axial de dois grupos. Grupo 1 coelhos foram submetidos a ligação cruzada de esclerótica e tarsorrafia no olho direito, enquanto o olho esquerdo não foi operado (Figura 1). Grupo 2 coelhos foram submetidos somente peritomia e tarsorrafia no olho direito, enquanto o olho esquerdo não foi operado (Figura 2). No grupo 1, que sofreu reticulação escleral e tarsorrafia, o comprimento axial significa no olho direito medido 10,68 ± 0,74 mm, antes da sutura da pálpebra e 14,29 ± 0,3 milímetro 55 dias mais tarde, por uma diferença média de 3,61 ± 0,76 mm. Valores correspondentes no olho não operado / suturar esquerda foram 10,70 ± 0,79 mm e 15,14 ± 0,32 mm, uma diferença média de 4,44 ± 0,81 mm (Figura 1). A comparação dos comprimentos axiais da suturadae olhos por suturar no final da fase de oclusão revelaram um aumento líquido menor nos olhos suturados. No grupo 2, que foram submetidos somente peritomia e tarsorrhaphies, significa comprimento axial do olho direito medido 10,50 ± 0,67 mm, antes da sutura da pálpebra e 15,69 ± 0,39 milímetros 55 dias mais tarde, por uma diferença média de 5,19 ± 0,85 mm. Valores correspondentes no olho não operado / suturar esquerda foram 10,54 ± 0,71 mm e 14,74 ± 0,38 mm, uma diferença média de 4,20 ± 0,67 mm (Figura 2). A comparação dos comprimentos axiais dos olhos suturados e suturar no final da fase de oclusão revelaram um aumento líquido maior nos olhos suturados. A comparação da alteração média do comprimento axial do olho direito entre o grupo 2 (5,19 ± 0,85 mm) e o grupo 1 (3,61 ± 0,76 milímetros), produziu um valor significativamente mais baixo no final da oclusão pHase (55 dias) nos olhos que se submeteram ao procedimento de reticulação (p &lt;0,001, teste não paramétrico de Mann-Whitney). A diferença entre os grupos no comprimento axial significativo nos olhos do lado esquerdo (4,20 ± 4,44 vs 0,67 milímetros ± 0,81 milímetros) não foi estatisticamente significativa (p = 0,39, teste de Mann-Whitney não-paramétrico). <img alt="figura 1" src="/files/ftp_upload/53201/53201fig1.jpg" /> Figura 1. Olho medições axiais certas antes e depois de reticulação escleral e tarsorrafia vs. medições axiais olho esquerdo. A média do comprimento axial do olho direito antes de reticulação escleral e tarsorrafia (início RE) e após a remoção do tarsorrafia 55 dias mais tarde (fim RE). O comprimento axial média do olho esquerdo na linha de base (LE iniciar) e 55 dias depois (no final LE). O olho esquerdo não foi operado e foi deixada em aberto. As barras de erro indicam o erro padrão da média. (Re-impresso com Permission da referência 27). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53201/53201fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53201/53201fig2.jpg" /> Figura 2. Olho medições axiais certas antes e depois tarsorrafia vs. medições axiais olho esquerdo. A média do comprimento axial do olho direito antes da tarsorrafia (início RE) e após a remoção do tarsorrafia 55 dias mais tarde (fim RE). O comprimento axial do olho esquerdo na linha de base média (LE começar) e 55 dias mais tarde (fim LE) .A olho esquerdo não foi operado e foi deixada em aberto. As barras de erro indicam o erro padrão da média. (Re-impresso com a permissão da referência 27). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53201/53201fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura</a>.

Watch this Scientific Journal Video about Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model at JoVE.com

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Nós demonstramos o efeito de reticulação escleral com riboflavina e UVA em um olho alongamento coelho axial. alongamento axial foi induzida em coelhos 13 dias de idade Nova Zelândia (masculino e feminino) suturando as pálpebras do olho direito (tarsorrafia).

Escleral reticulação Usando ultravioleta e riboflavina-A radiação de Prevenção de Axial Miopia em um modelo de coelho

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

scleral-cross-linking-using-riboflavin-ultraviolet-radiation-for

Nanyang Technological University

Research

JoVE Journal

Medicine

8.8K visualizações.  Rabin Medical Center, Beilinson Campus.  Nós demonstramos o efeito de reticulação escleral com riboflavina e UVA em um olho alongamento coelho axial. alongamento axial foi induzida em coelhos 13 dias de idade Nova Zelândia (masculino e feminino) suturando as pálpebras do olho direito (tarsorrafia). 

Vídeo: Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Na imagem de bioluminescência vivo Tumor de mama Dynamics Hipóxia metástase do cancro cerebral em um modelo de rato

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Medicina

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Implantação estereotáxica intracraniana e<em&gt; Em vivo</em&gt; Imaging Bioluminescent de xenoenxertos de tumores em um Sistema Modelo de Rato Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

Matrix-assistida Transplante autólogo de condrócitos para remodelação e reparação de lesões condrais em um modelo de coelho

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Triagem para Modificadores Melanoma usando um modelo de tumor Zebrafish autóctone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

O<em&gt; Em ovo</em&gt; CAM-ensaio como um Xenograftmodel para o Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

O coelho Modelo Blood-shunt para o Estudo da aguda e tardia Sequelas de hemorragia subaracnóidea: Aspectos Técnicos

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Difusa espectroscopia óptica para a avaliação quantitativa da Aguda de Radiação Ionizante toxicidade cutânea induzida pelo uso de um modelo de rato

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Segunda geração harmônica sinais na esclera coelho como uma ferramenta para avaliação do tecido terapêutico do cross-linking (TXL) para miopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

Tratamento orientado e seletivo de Teratomas células-tronco derivadas de pluripotentes usando radiação de feixe externo em um modelo animal de pequeno

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...