Este trabalho foi apoiado por bolsas do Instituto Nacional de Surdez e Outros Transtornos de Comunicação (R21DC010806 para CAM e JCS e R01DC011285 para CAM).

Use ratos machos Fischer 344-Brown Norway com 6 e 30 meses de idade para este protocolo. Os ratos foram obtidos da colônia de roedores do Instituto Nacional do Envelhecimento. Utilizamos ratos para este estudo porque a estrutura da laringe de ratos é semelhante à do ser humano, servindo funcionalmente para a proteção das vias aéreas e vocalizações específicas de espécies. Este estudo foi realizado de acordo com a Política PHS sobre Cuidados Humanos e Uso de Animais de Laboratório, O Guia NIH para o Cuidado e Uso de Animais de Laboratório, e a Lei de Bem-estar dos Animais (7 USC et seq.); O protocolo de uso de animais foi aprovado pelo Comitê Institucional de Cuidados e Uso Animal (IACUC) da Universidade do Kentucky. 1. Anestesia de ratos <ol><li> Prepare os anestésicos misturando cloridrato de cetamina (anestésico dissociativo) e cloridrato de xilazina (sedativo e analgésico) em solução salina tamponada. As concentrações de ketamina e xilazina na solução final são 100 mg / 8 mg por kgPeso corporal, respectivamente. </li><li> Injete os anestésicos no rato por administração intraperitoneal usando uma seringa com uma agulha de 25 G. </li><li> Determine que o rato está suficientemente anestesiado pressionando o pé ou o pé com fórceps. Se o rato não reagir à pitada, a cirurgia pode começar. Se o rato reage à prega do dedo do pé com contrações reflexas ou musculares, espere 1-2 minutos e repita o teste de pitada. Se o rato reage novamente, substitua o rato por um novo animal e repita o procedimento a partir do passo 1.2. </li><li> Aplique pomada oftálmica aos olhos do rato, depois que o rato estiver imóvel, para evitar que as córneas se sectem. </li></ol> 2. Implantação de bomba osmótica <ol><li> Coloque o ventral do rato na área cirúrgica asséptica. Administrar meloxicam como medicamento pré-anestésico. Administrar intraperitonealmente a uma dosagem de 1-4 mg / kg de peso corporal usando uma seringa com uma agulha de 25 G. </li><li> Use cortadores para remover uma aproximação1 &quot;x 1&quot; quadrado de pele da parte de trás do pescoço e aproximadamente 1 &quot;caudal de espaço entre os ombros. Rasgue o mais próximo possível da pele. </li><li> Molhe as costas eo pescoço com etanol desinfetante (70%). </li><li> Após o barbear, esfregue o aspecto dorsal do pescoço com 3 esfrega em sucessão de álcool iodo com o álcool. </li><li> Mantenha a temperatura corporal do rato, colocando-o sobre uma almofada de aquecimento ajustada para 34 ° C. </li><li> Preencha as bombas osmóticas preparadas assepticamente com 50 μL de NTF4 ou solução salina para tratamento NTF4 sistêmico ( Figura 1 ). <ol><li> Use um bisturi para fazer uma incisão horizontal de aproximadamente 2 cm de largura através da pele, apenas craniana para o espaço entre a escápula. Levante a extremidade posterior da incisão com fórceps com uma mão ao inserir a ponta dos hemostáticos e empurrando suavemente para posterior a incisão. </li><li> Depois que a ponta dos hemostatos é de aproximadamente 2 cm cRanial para a incisão, abra as alças nos hemostáticos, expandindo as pontas para formar um &quot;bolso&quot; oco subcutâneo para o local da incisão. Este será o site de colocação da bomba. </li></ol></li><li> Oriente o fim do portal de entrega da bomba primeiro após a inserção para minimizar qualquer interação do NTF4 e a cicatrização do site da incisão de bolso. </li><li> Entregue 50 μL de solução salina NTF4 durante 7 14 dias. O grupo de 7 dias recebeu 6,72 mg / dia de NTF4 para uma dose total de 47,04 mg. O grupo de 14 dias recebeu 6,72 mg / dia para uma dose total de 94,08 mg de NTF4 25 . </li><li> Use uma rosca de sutura de nylon 5-0, hemostáticos e fórceps para fechar a incisão feita para a colocação da bomba. </li><li> Observe os ratos durante um mínimo de 30 min à medida que se recuperam da anestesia. Os critérios para a conclusão do monitoramento incluem o animal se tornando ativo, movendo-se sobre a gaiola, bebendo água e iniciando outras atividades normais, como a preparação. </li><li> Monitore animais diariamenteDurante a primeira semana, observando a cicatrização do local cirúrgico, alimentação normal e consumo de água e passagem de urina / fezes e quaisquer sinais comportamentais anormais de estresse, dor ou outras complicações pós-operatórias. </li><li> Se o rato parece estar com dor ou angústia, forneça ao rato uma injeção subcutânea de 5 mg / kg de carprofeno uma vez a cada 24 h por até 5 dias para aliviar a dor. </li><li> Se parece haver uma infecção, consulte um veterinário para que a ferida se cure adequadamente. </li><li> Dependendo de qual grupo experimental o rato dentro, remova a sutura de nylon 5-0 7-10 dias após a cirurgia para evitar irritação da rosca. </li></ol> 3. Anestesia de ratos para injeção direta <ol><li> Retire o alimento dos ratos a noite anterior ao procedimento. Isso garante que não há alimentos para bloquear o endoscópio ou a agulha de injeção. </li><li> Pesar ratos e preparar acepromazina 1-2 mg / kg de peso corporal. Injetar por via intramuscular (a localização IM é aMúsculo tireo-arteronóide esquerdo). </li><li> Coloque o rato na caixa de indução. Induzir anestesia na caixa de indução com 5% de isoflurano e 1 LO 2 . </li><li> Mova o rato para um cone do nariz com 2% de isoflurano e 600 mL de O2. </li><li> Determine que o rato está suficientemente anestesiado pressionando o pé ou o pé com fórceps. Se o rato não reagir à pitada, o protocolo de injeção pode começar. Se o rato reage à prega do dedo do pé com contrações reflexas ou musculares, espere 1-2 minutos e repita o teste de pitada. Se o rato reage de novo, substitua o rato por um novo animal e repita o procedimento a partir do passo 3.4. </li></ol> 4. Injeção direta e visualização <ol><li> Coloque doses 50 μL preparadas assepticamente contendo NTF4 ou solução salina em um banho de H 2 O ajustado a 25 ° C durante 30 min antes da injeção. </li><li> Coloque o rato em posição supina e reclinada em uma plataforma de plexiglass ( Figura 2 ). SusColoque o rato na postura reclinada de seus incisivos superiores frontais através de um fio guia empurrado pela parte superior da plataforma. </li><li> Anexe uma seringa de 50 mm, 30 gauge, 100 μL a um endoscópio sinusal de 1,9 mm, 30 ° ( Figura 3 ). NOTA: A montagem da seringa está presa através de um gabarito que segura a cânula firmemente na parede externa do endoscópio. O endoscópio permite a visualização das pregas vocais e a orientação da seringa intra-oralmente. A posição da ponta da cânula é ajustada antes de cada animal para garantir que a ponta seja totalmente e claramente visível através da visão endoscópica ( Figura 4 ). </li><li> Use um par de fórmulas de borracha para estender a língua e movê-la lateralmente. Depois, insira um espéculo de plástico para manter a permeabilidade oral. Faça o espéculo de um tambor de seringa de plástico de 5 mL que é cortado para um comprimento de 1,5 a 2 cm, com as bordas cortadas deburadas e polidas lisas. </li><li> Desligue as luzes no rooM e coloque uma fonte de luz halógena no endoscópio. Ligue o gravador de vídeo para capturar o procedimento. </li><li> Mergulhe a extremidade distal do endoscópio em água morna por alguns segundos para minimizar o desenvolvimento de condensação na ponta de vidro quando inserido na boca do rato. </li><li> Usando feedback visual do monitor, guie cuidadosamente a agulha na área da prega vocal esquerda. </li><li> Tempo de injeção da solução com a fase inspiratória do ciclo de respiração dos animais para acessar completamente a prega vocal. Durante a fase inspiratória da respiração, a prega vocal é totalmente exposta. <ol><li> Uma vez que a prega vocal é totalmente visível, insira a agulha no tireoaritenóide esquerdo, encontrado lateral à borda mediana branca da prega vocal. Com a agulha no lugar, entregue o injeção através da depressão da seringa. </li></ol></li><li> Desligue a fonte de luz halógena no endoscópio e no reprodutor de vídeo e volte-se no roOm luzes. </li><li> Retorne o rato para a sua gaiola de casa e coloque em uma almofada de aquecimento. </li><li> Permita que o rato se recupere antes da remoção da almofada de aquecimento. Substitua alimentos e água na gaiola. </li><li> Monitorar ratos durante 7 dias após a injeção e, em seguida, eutanizar. Remova as laringes para crioesecção 24 . </li></ol> 5. Eutanização de ratos <ol><li> Anestesiar ratos com cloridrato de cetamina e cloridrato de xilazina (100 mg / 8 mg por kg de injeção intraperitoneal injetada no peso corporal). </li><li> Eutanizar por exsanguinação após uma toracotomia mediana. </li></ol>

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Os músculos laríngeos são vulneráveis ​​aos efeitos desfavoráveis ​​do envelhecimento. Estudos anteriores demonstraram alterações nos músculos da laringe envelhecidos que incluem mudanças no tamanho da fibra, número total de fibras, capacidade regenerativa, tamanho NMJ e alterações quantitativas, além de variações na função contrátil e mudanças da isoforma da miosina 4 , 11 , 27 , <sup clas...

Os músculos laríngeos se contraem de forma rápida e consistente e são vulneráveis ​​aos efeitos adversos do envelhecimento. Espera-se que essa atividade constante contribua para problemas de voz ou disfagia observados em pessoas com mais de 65 anos de idade 1 , 2 , 3 , 4 , 5 , 6 , 7 . Vários mecanismos moleculares e fisiopatológicos contribuem para esta disfunção relacionada à idade. Esses mecanismos podem incluir a remodelação da mucosa laríngea, a atrofia ou perda de fibras musculares, a falta de regeneração ou atrofia de fibras musculares que provoca curvatura das pregas vocais e incapacidade de fechamento gloteado 8 , 9 , 10 , 11 . Não há uma terapia médica comprovada neste momento que possa comPrever ou reabilitar essas mudanças relacionadas com a idade desses músculos. A modulação da eficácia da transmissão neuromuscular pode influenciar significativamente o desempenho neuromotor. A família das neurotrofinas inclui o fator de crescimento nervoso (NGF), o fator de crescimento do nervo derivado do cérebro (BDNF), neurotrofina 3 (NTF3) e NTF4 12 , 13 . Verificou-se que as neurotrofinas modulam a eficácia sináptica 1 , 4 . O fator de crescimento de hepatócitos, o fator de crescimento transformador beta e o fator de crescimento de fibroblastos foram recentemente utilizados em seres humanos para o tratamento de cicatrizes de pregas vocais de 15 a 17 . NTF4 também regula a eficácia NMJ; Ratos com falta de NTF4 mostram NMJs desmontados 11 , 18 , 19 . Esses estudos levam a efeitos promissores do tratamentoT do envelhecimento dos distúrbios do músculo laríngeo e da desnervação com fatores de crescimento. A terapêutica de injeção direta aos tecidos das pregas vocais não é sem precedentes em seres humanos. Por exemplo, as injeções locais de toxina botulínica são atualmente utilizadas como um tratamento eficaz para distúrbios do movimento neurológico que afetam os músculos da laringe, como a disfonia espasmódica e paralisia bilateral do nervo laríngeo 20 , 21 . O hidrogel de ácido hialurônico é outro injetável, que é usado para tratar a queima de dobras vocais e insuficiência glotal 22 , 23 . A laringoplastia por injeção pode ser usada para tratar uma variedade de distúrbios de comunicação 24 . Esses métodos de injeção direta são promissores para melhorar a função vocal e deglutição no envelhecimento populacional.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurotrophin 4</td>
			<td>Pepro Tech</td>
			<td>450-04</td>
			<td>200ng in 50&#956;l</td>
		</tr>
		<tr>
			<td>Alzet Osmotic Pump</td>
			<td>DURECT Corporation</td>
			<td>2001D</td>
			<td></td>
		</tr>
		<tr>
			<td>30&#176; endoscope</td>
			<td>Stoltz</td>
			<td>61029D</td>
			<td></td>
		</tr>
		<tr>
			<td>50mm 30 gauge 100-&#956;l syringe</td>
			<td>Hamilton</td>
			<td>84850 and 201812</td>
			<td></td>
		</tr>
		<tr>
			<td>saline (sodium chloride solution)</td>
			<td>Sigma-Aldrich</td>
			<td>S8776</td>
			<td></td>
		</tr>
		<tr>
			<td>ketamine hydrochloride</td>
			<td>Henry Schein</td>
			<td>56344</td>
			<td></td>
		</tr>
		<tr>
			<td>xylazine hydrochloride</td>
			<td>Henry Schein</td>
			<td>33198</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G 5/8 needle</td>
			<td>Becton-Dickinson</td>
			<td>305901</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Becton-Dickinson</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic ointment</td>
			<td>Henry Schein</td>
			<td>8897</td>
			<td></td>
		</tr>
		<tr>
			<td>clippers</td>
			<td>Oster</td>
			<td>44-018</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Decon</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>iodine (Betadine)</td>
			<td>Purdue Pharma L.P.</td>
			<td>606404</td>
			<td></td>
		</tr>
		<tr>
			<td>heating pad</td>
			<td>Sunbeam</td>
			<td>731-5</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 nylon suture thread</td>
			<td>AD Surgical</td>
			<td>PMN-518R6</td>
			<td></td>
		</tr>
		<tr>
			<td>crile hemostat</td>
			<td>Fine Science Tools</td>
			<td>13005-14</td>
			<td></td>
		</tr>
		<tr>
			<td>delicate suture tying forceps</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>meloxicam</td>
			<td>Henry Schein</td>
			<td>49756</td>
			<td></td>
		</tr>
		<tr>
			<td>carprofen</td>
			<td>Merritt Veterinary Supplies</td>
			<td>148700</td>
			<td></td>
		</tr>
		<tr>
			<td>antibiotic ointment</td>
			<td>Henry Schein</td>
			<td>57110</td>
			<td></td>
		</tr>
		<tr>
			<td>acepromizine Aceproject</td>
			<td>Henry Schein</td>
			<td>3845</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane Isothesia</td>
			<td>Henry Schein</td>
			<td>50033</td>
			<td></td>
		</tr>
		<tr>
			<td>induction box (anesthetizing box)</td>
			<td>Harvard Apparatus</td>
			<td>50-0116</td>
			<td></td>
		</tr>
		<tr>
			<td>oxygen compressed tank</td>
			<td>Scott Gross</td>
			<td>UN1072</td>
			<td></td>
		</tr>
		<tr>
			<td>plexiglas platform</td>
			<td>Small Parts Inc (Amazon)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rubber tipped forceps</td>
			<td>Fine science tools rubber</td>
			<td>11075-00</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid rubber for forceps above</td>
			<td>Lowe&#39;s</td>
			<td>42518</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic spectula (BD syringe cut to length)</td>
			<td>Becton-Dickinson</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>halogen light source rhino-laryngeal stroboscope</td>
			<td>Kay-Pentax</td>
			<td>RLS 9100 B</td>
			<td></td>
		</tr>
		<tr>
			<td>video recorder</td>
			<td>Kay-Pentax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>cryostat Mictotom HM525</td>
			<td>Thermo Scientific</td>
			<td>HM 525</td>
			<td></td>
		</tr>
		<tr>
			<td>Gill 1 hematoxylin</td>
			<td>VWR</td>
			<td>10143-142</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon eosin-Y alcoholic</td>
			<td>Thermo Fisher Scientific</td>
			<td>6766007</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-sodium channel Nav1.5 antibody produced in rabbit</td>
			<td>Sigma-Aldrich</td>
			<td>S0819</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas red-X phalloidin</td>
			<td>Sigma-Aldrich</td>
			<td>T7471</td>
			<td></td>
		</tr>
		<tr>
			<td>alpha- bungarotoxin alexa fluor 488 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>B-13422</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal anaesthesia machine</td>
			<td>Smiths Medical</td>
			<td>CDS 9000</td>
			<td></td>
		</tr>
	</tbody>
</table>

subcutaneous neurotrophin 4 infusion using osmotic pumps or direct muscular injection enhances aging rat laryngeal muscles

A disfunção laríngea em idosos é uma das principais causas de incapacidade, de distúrbios de voz à disfagia e perda de reflexos protetores das vias aéreas. Poucas, se houver, existem terapias que visam a disfunção do músculo laríngeo relacionado à idade. As neurotrofinas estão envolvidas na inervação muscular e na diferenciação das junções neuromusculares (NMJs). Pensa-se que as neurotrofinas melhoram a transmissão neuromuscular ao aumentar a liberação do neurotransmissor. As junções neuromusculares (NMJs) tornam-se menores e menos abundantes no envelhecimento dos músculos da laringe do rato, com evidência de desnervação funcional. Nós exploramos os efeitos do NTF4 para o uso clínico futuro como um procedimento terapêutico para melhorar a função no envelhecimento dos músculos da laringe humana. Aqui, fornecemos o protocolo detalhado para aplicação sistêmica e injeção direta de NTF4 para investigar a capacidade do músculo laríngeo do rato em envelhecimento de remodelar em resposta à aplicação NTF4. Neste método, os ratos receberam NTF4 sistematicamente via oBomba smotic ou por injeção direta através das pregas vocais. Os músculos laríngeos foram então dissecados e utilizados para exame histológico de morfologia e denervação relacionada à idade.

Os ratos foram eutanásicos após 2 semanas de infusão de bomba osmótica ou 1 semana após a injeção direta de NTF4. Larynges foram colhidos, colocados em crioprotectores (30% de sacarose e 70% de solução salina tamponada com fosfato) e depois seccionados em série em 10 μm de largura com um criostato. O envelhecimento dos músculos laríngeos é afetado pela administração de NTF4 25 . Além de ratos jovens e velhos, comparamos o lado injetado e não inje...

Watch this Scientific Journal Video about Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles at JoVE.com

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Aqui, apresentamos um protocolo para descrever o uso de neurotrofina 4 (NTF4) sistematicamente e diretamente para remodelar os músculos laríngeos do envelhecimento do rato.

A infusão subcutânea de neurotrofina 4 com bombas osmóticas ou injeção muscular direta melhora o envelhecimento dos músculos da laringe do rato

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if ...

subcutaneous-neurotrophin-4-infusion-using-osmotic-pumps-or-direct

Research

JoVE Journal

Developmental Biology

8.3K visualizações.  University of Kentucky.  Aqui, apresentamos um protocolo para descrever o uso de neurotrofina 4 (NTF4) sistematicamente e diretamente para remodelar os músculos laríngeos do envelhecimento do rato. 

Vídeo: Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Isolamento e caracterização de células-satélite de músculo de rato Cabeça branquiométrico

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Biologia do Desenvolvimento

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Geração de pluripotentes induzidas a partir de células-tronco Congelado Buffy Coats usando não-integração epissomais Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Isolamento e diferenciação de células tronco derivadas de tecido adiposo de Suínos Subcutânea tecidos adiposos

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

Análise de Desenvolvimento de rim Zebrafish com imagem Time-lapse Usando um microscópio de dissecação Equipado para Seccionamento Optical

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

Quantificando a densidade de ramificação em glândulas mamárias de ratos montadas inteiras usando o método de análise Sholl

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Isolamento de Pericitos Tipo I e Tipo II de Músculos Esqueléticos de Rato

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Indução e validação da senescência celular em células humanas primárias

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualizando o nó e placa Notochordal em Gastrulating embriões de rato usando microscopia eletrônica e imunofluorescência de montagem inteira

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Modelos de lesão adulto Zebrafish para estudar os efeitos de prednisolona na regeneração de tecido ósseo

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Direto de linhagem reprogramação de fibroblastos de rato adulto de progenitores eritroide

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...