Este trabalho foi financiado em parte por uma concessão do NIH concessão R01 GM087609 (para HPL), um presente de Ingrid Lai e Bill Shu em honra de Anthony Shu (para HPL), NIH concessão U01 HL099776 (para MTL), o Laboratório Hagey para Pediatric Regenerative Medicine e The Oak Foundation (para MTL e HPL). GGW foi apoiado pela Escola de Medicina de Stanford, o Programa de Formação de Stanford Medical Scientist, e uma bolsa de formação NIGMS GM07365. MSH foi apoiada por CIRM Clinical Fellow Training Grant TG2-01159. WXH foi apoiado pelo financiamento da Fundação Sarnoff Cardiovascular.

NOTA: Todos os procedimentos descritos neste trabalho são realizados de acordo com as diretrizes estabelecidas pelo Painel Administrativo Stanford em Laboratory Animal Care (APLAC). 1. Temporário Gestações - Técnica superovulação (Figura 1) NOTA: Precisamente cronometrar a idade gestacional de embriões de camundongos para a cirurgia fetal em E16.5 e E18.5 é de importância crítica. Nesta seção, detalhe nosso protocolo para cronometrar gestações rato usando grávida éguas soro (PMS) e gonadotrofina coriônica humana (HCG) injeções para induzir superovulação. <ol><li> Injectar camundongos fêmeas (&lt;5 por gaiola) por via intraperitoneal (IP) com 3,0-5,0 unidades internacionais (UI) de PMS em um volume de 100 l PBS 01:00 - 15:00 para o dia 1. </li><li> Entre as 12:00 e as 14:00 do dia 3 (quarenta e cinco a 47 horas após a injecção de PMS), injectar ratinhos fêmea IP com 3,0-5,0 UI de HCG em um volume de 100 ul de PBS. NOTA: Os induz injeção de HCG<html> ovulação, aproximadamente, 12 h pós-injecção. </li><li> Imediatamente após injeções de HCG, companheiro fêmeas com machos com idade entre 8-16 semanas. ATENÇÃO: Nós normalmente colocar duas fêmeas em uma gaiola de machos individuais. </li><li> Fêmeas separados de homens na manhã do dia 4 (06:00 - 10:00) e registro como E0.5 idade fetal. NOTA: plugs vaginais pode ser verificado neste momento; No entanto, a observação de um plug vaginal não garante a gravidez e as fêmeas podem engravidar quando nenhum plug é observado. Tendo em conta que a gravidez é normalmente observada por inspeção e / ou palpação visual em idades gestacionais E16.5 e E18.5, verificação de fichas vaginais na manhã do dia 4 não é estritamente necessário. Na nossa experiência, e, dependendo da estirpe, aproximadamente 30-50% das mulheres super-ovulado engravidar utilizando a técnica aqui descrita. </li></ol>pg &quot;/&gt; Figura 1. Esquema de Técnica superovulação. Procedimento exibição Esquema de superovulação cronometrado gestações em camundongos. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig1large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> 2. Cirurgia Fetal Murino (Dorsal o ferimento) em E16.5 e E18.5 embriões (Figura 2) <ol><li> Antes do procedimento, limpe todas as superfícies da sala de cirurgia e equipamentos com 70% de álcool isopropílico. Além disso, todos os fornecimentos esterilizar instrumentos cirúrgicos e que serão utilizados no processo de autoclavagem por eles. Algumas instituições podem permitir o uso posterior de esterilização quente talão. Por operação, use embalagens esterilizadas que incluem gaze e instrumentos cirúrgicos. </li><li> Induzir a anestesia em mães grávidas (E16.5 idade fetal ou E18.5) sob 2,5% de mistura de isoflurano / oxigênio a 2 L por min seguido de manutenção da anestesia em 1 L por min. </li><li> Para conempresa anesthetization adequado, garantir os reflexos profundos pedal do mouse são suprimidas e coloque o mouse na posição prona. </li><li> Aplique uma pomada oftálmica vet como Puralube para evitar irritação nos olhos ou secura durante o procedimento. </li><li> Prepare abdômen pela administração de uma leve aplicação de creme depilatório para não mais do que 30 segundos (Figura 2A). </li><li> Prepare abdômen para a cirurgia asséptica com iodopovidona e álcool (Figura 2B e 2C). </li><li> Execute linha média laparotomia sob o microscópio com tesoura microcirúrgica (Figura 2D e 2E). </li><li> Gentilmente expor útero e feto selecionados para a cirurgia (Figura 2F). </li><li> Lavar campo cirúrgico com água morna (38 ° C) solução salina tamponada com fosfato (PBS) utilizando uma agulha de ponta romba NOTA: Pode ser feita dobrando cuidadosamente a ponta de uma agulha de grande calibre. (Figura 2G e 2H). </li><li> Posicionar o feto de uma maneira que permitas acesso total ao dorso. </li><li> Passe um ponto cordão da bolsa utilizando sutura 7-0 nylon através do útero que cobre o local do ferimento dorsal pretendido (Figura 2I). Posição de fio de bolsa ao longo de uma região dorso para a esquerda ou direita da espinal medula, e em uma região da parede uterina desprovida de vasos sanguíneos grandes. </li><li> Faça uma incisão três milímetros através da parede uterina e saco amniótico, no centro do cordão da bolsa (Figura 2J). </li><li> Irrigar local da incisão com água morna (38 ° C) PBS. </li><li> Com uma tesoura de microcirurgia, cortar uma única ferida excisional de espessura total, aproximadamente 1 mm de comprimento, no dorso do feto. </li><li> Delicadamente, local da incisão secar com algodão-ponta do aplicador. </li><li> Injectar 3 ul volume de nanquim subcutaneamente no local da ferida para marcar o local da ferida (Figura 2K). </li><li> Lavar com água morna (38 ° C) para assegurar PBS tinta foi retido no interior do sítio da ferida. </li><li> Já cirúrgico assistente inject quente (38 ° C) PBS através de ponta romba 10 G seringa no saco amniótico como o cordão da bolsa é fechada (Figura 2L). Retrair seringa como cadeia de fecho da bolsa se ​​aproxima da conclusão (Figura 2 M). </li><li> Suavemente útero voltar para dentro da cavidade abdominal (Figura 2N). </li><li> Evert pele e peritônio. </li><li> Já assistente cirúrgico irrigar cavidade abdominal com água morna (38 ° C) PBS. </li><li> Fechar abdômen rapidamente por grampeamento pele e peritônio fechado (Figura 2O). O fecho de padrão é executada em duas camadas; peritoneu e da musculatura abdominal numa camada, tecido subcutâneo e pele na segunda camada. Para sacrifício imediato e colheita do feto, a nossa demonstração mostra o encerramento, em uma camada. </li><li> Colocar o animal sob observação numa incubadora quente regulado a 37 ° C durante 30 minutos ou até que o animal recupera a consciência suficiente para manter decúbito esternal. </li><li> Não return o animal para a companhia de outros animais até que ele está totalmente recuperado do procedimento. </li><li> Quando acordarmos da anestesia e durante a subsequente 48 horas, administrar a injecção subcutânea de buprenorfina (0,05 mg / kg) a cada 12 horas, para a analgesia conforme necessário, com base na avaliação da dor. Administrar carprofeno (5 mg / kg) via injecção subcutânea para o alívio da dor adicional de pós-operatório, conforme necessário. </li><li> Retorno animais na gaiola e dar-lhes comida e água ad libitum. </li><li> Acompanhar de perto para manifestações de dor. </li><li> 48 horas após a cirurgia, sacrificar mãe grávida com uma overdose de isoflurano e colheita feridos feto. A fim de fazer isso, ajustar a concentração de isoflurano a 5% ou superior e manter a exposição durante 1 min após a cessação da respiração. Confirme a eutanásia com luxação cervical. Colher um embrião sem ferimentos para o controle pareados por idade. Embriões tardias devem ter um método separado da eutanásia consistente com as recomendações IACUC, como decapitação, luxação cervical, ou injeção de produtos químicos. </li></ol><img alt="Figura 2" src="/files/ftp_upload/52297/52297fig2highres.jpg" /> Figura 2. Esquema de Cirurgia Murino Fetal. Etapas gerais para dorsal ferindo em E16.5 e E18.5 embriões de rato. (A) Depilação de rato abdômen. (B e C) Preparação de rato abdômen. (D) Microscópio usado para cirúrgico procedimento. (E) Midline laparotomia. (F) A exposição de útero. (G) Criação de agulha de ponta romba. (H) Irrigação do útero com soro fisiológico morno. (I) Criação de cordão da bolsa de sutura. (J) incisão através uterino de parede e 1 mm de geração ferida excisional espessura total. (K) A injecção subcutânea de nanquim. (L e M) Encerramento do cordão da bolsa de sutura. (N e O)Encerramento do abdômen. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig2large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a>

<ol>
	<li>Larson, B. J., Longaker, M. T., Lorenz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scarless+fetal+wound+healing:+a+basic+science+review.">Scarless fetal wound healing: a basic science review.</a> Plastic and reconstructive surgery. 126, 1172-1180 (2010).</li><li>Wilgus, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+healing+in+fetal+skin:+a+review+of+the+literature.">Regenerative healing in fetal skin: a review of the literature.</a> Ostomy/wound management. 53, 16-31 (2007).</li><li>Wulff, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells+contribute+to+scar+formation+during+fetal+wound+healing.">Mast cells contribute to scar formation during fetal wound healing.</a> The Journal of investigative dermatology. 132, 458-465 (2012).</li><li>Lorenz, H. P., Adzick, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scarless+skin+wound+repair+in+the+fetus.">Scarless skin wound repair in the fetus.</a> The Western journal of medicine. 159, 350-355 (1993).</li><li>Longaker, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+in+the+fetus.+Possible+role+for+inflammatory+macrophages+and+transforming+growth+factor-beta+isoforms.">Wound healing in the fetus. Possible role for inflammatory macrophages and transforming growth factor-beta isoforms.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 2, 104-112 (1994).</li><li>Longaker, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+in+fetal+wound+healing.+IV.+Hyaluronic+acid-stimulating+activity+distinguishes+fetal+wound+fluid+from+adult+wound+fluid.">Studies in fetal wound healing. IV. Hyaluronic acid-stimulating activity distinguishes fetal wound fluid from adult wound fluid.</a> Annals of surgery. 210, 667-672 (1989).</li><li>Colombo, J. A., Napp, M., Depaoli, J. R., Puissant, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+influences+of+human+and+rat+amniotic+fluid+on+neural+tube-derived+rat+fetal+cells.">Trophic influences of human and rat amniotic fluid on neural tube-derived rat fetal cells.</a> International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience. 11, 347-355 (1993).</li><li>Colwell, A. S., Longaker, M. T., Peter Lorenz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+differentially+regulated+genes+in+fetal+wounds+during+regenerative+repair.">Identification of differentially regulated genes in fetal wounds during regenerative repair.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 16, 450-459 (2008).</li><li>Hu, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+in+fetal+murine+keratinocytes+and+fibroblasts.">Gene expression in fetal murine keratinocytes and fibroblasts.</a> The Journal of surgical research. , (2014).</li><li>Colwell, A. S., Krummel, T. M., Longaker, M. T., Lorenz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+mouse+excisional+wound+model+of+scarless+healing.">An in vivo mouse excisional wound model of scarless healing.</a> Plastic and reconstructive surgery. 117, 2292-2296 (2006).</li><li>Wilgus, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+cyclooxygenase-2+mediated+inflammation+on+scarless+fetal+wound+healing.">The impact of cyclooxygenase-2 mediated inflammation on scarless fetal wound healing.</a> The American journal of pathology. 165, 753-761 (2004).</li><li>Iocono, J. A., Ehrlich, H. P., Keefer, K. A., Krummel, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan+induces+scarless+repair+in+mouse+limb+organ+culture.">Hyaluronan induces scarless repair in mouse limb organ culture.</a> Journal of pediatric surgery. 33, 564-567 (1998).</li><li>Chopra, V., Blewett, C. J., Krummel, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+from+fetal+to+adult+repair+occurring+in+mouse+forelimbs+maintained+in+organ+culture.">Transition from fetal to adult repair occurring in mouse forelimbs maintained in organ culture.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 5, 47-51 (1997).</li><li>Adzick, N. S., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+the+study+of+fetal+tissue+repair.">Animal models for the study of fetal tissue repair.</a> The Journal of surgical research. 5, 47-51 (1991).</li><li>Block, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+in+the+new-born+opossum+(Didelphis+virginianam).">Wound healing in the new-born opossum (Didelphis virginianam).</a> Nature. 187, 340-341 (1960).</li><li>Longaker, M. T., Dodson, T. B., Kaban, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rabbit+model+for+fetal+cleft+lip+repair.">A rabbit model for fetal cleft lip repair.</a> Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons. 48, 714-719 (1990).</li><li>Longaker, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+fetal+cleft+lip+repair+in+lambs.">A model for fetal cleft lip repair in lambs.</a> Plastic and reconstructive surgery. 90, 750-756 (1992).</li></ol>

The authors have nothing to disclose.

O protocolo cirúrgico aqui apresentado descreve um modelo excisional de fetal cura scarless murino publicado pela primeira vez em 2006 por nosso laboratório 10. Em adição a outros modelos estabelecidos da ferida excisional 11, modelos incisionais de cicatrização sem cicatrizes murino fetal existir como bem 12,13. As investigações sobre a cicatrização de feridas scarless fetal no macaco, cordeiro, coelho, gambá, e rato têm sido relatados 14-17. No entanto, os ratos ...

Feridas cutâneas fetais curar rapidamente e scarlessly até o final da gestação 1. Fetal reparação ferida scarless é caracterizada pela regeneração da arquitetura do tecido normal e função. A transição de uma scarless a cicatrizes fenótipo ocorre no terceiro trimestre da gravidez em seres humanos e por volta do dia embrionário 18 (E18) em ratos 2,3. Em comparação com a de adultos, a reparação de feridas fetal é caracterizada pela rápida epitelização, a deposição de tecido conjuntivo, e a migração de fibroblasto. Muitos estudos têm oferecido possíveis explicações para o fenômeno da cicatrização de feridas scarless durante o desenvolvimento fetal precoce. A inflamação é um componente fundamental de reparação de feridas de adultos; No entanto, as feridas do feto são caracterizadas por uma ausência de inflamação aguda 4. Se isto é uma conseqüência da imaturidade funcional do sistema imunológico durante os estágios fetais permanece obscuro. Um estudo recente sugere que as diferenças na abundância, maturity, e a função de mastócitos em E15 vs pele fetal E18 podem ser responsáveis ​​para a transição de um fenótipo sem cicatrizes, pelo menos no rato 3. Outros estudos sustentam que as diferenças nas propriedades e abundância de macrófagos feridas fetais e adultos são responsáveis ​​pela reforma da matriz extracelular normal (ECM) durante a reparação de feridas fetal 5. Diferenças em fatores ambientais durante o desenvolvimento fetal e adulto também pode afetar a reparação de feridas. Longaker e seus colegas mostraram que o líquido da ferida de o feto possui altos níveis de atividade estimulante ácido hialurônico comparado a nenhum adulto na ferida fluido 6. Consequentemente, os níveis mais elevados de ácido hialurónico, um glicosaminoglicano que promove um microambiente propício à motilidade e proliferação celular, no ambiente da ferida fetal pode ser responsável pelo fenótipo scarless visto durante o desenvolvimento fetal precoce. Outras linhas de evidência apontam para o fato de que o fetaambiente l ferida é relativamente hipoxêmica e submerso no líquido amniótico estéril rico em fatores de crescimento 7. No entanto, nenhuma resposta definitiva foi fornecido para um evento crítico ou fator durante a embriogênese que dispara a transição de regeneração scarless para reparação fibrótica. A compreensão dos mecanismos responsáveis ​​pela cura scarless no feto necessita de um modelo preciso e reprodutível. Aqui nós detalhe um modelo reprodutível de scarless cicatrização de feridas fetal no dorso de E16.5 (scarless) e embriões E18.5 (cicatrização) do mouse. Além disso, pequenas variações deste modelo pode ser utilizado para executar uma série de outros estudos, como a análise de feridas fetais e 8,9 pele expressão gênica. Dado que as gravidezes precisamente cronometradas são críticos para recapitulação sucesso deste modelo de cicatrização fetal scarless ferida, nós também detalhe nosso protocolo para gestações de superovulação cronometrado.

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			<td height="20" style="height:20px;">7-O MONOSOF Suture</td>
			<td>eSuture</td>
			<td>SN-1647G</td>
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			<td>Kent Scientific</td>
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			<td height="20" style="height:20px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
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			<td height="20" style="height:20px;">Autoclip 9mm</td>
			<td>Texas Scientific Instruments</td>
			<td>205060</td>
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			<td height="20" style="height:20px;">Insulin Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-272-382</td>
			<td></td>
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			<td height="20" style="height:20px;">Black Pigment</td>
			<td>AIMS</td>
			<td>242</td>
			<td></td>
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			<td height="20" style="height:20px;">BD Safety-Lok 3ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309596</td>
			<td></td>
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			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010-049</td>
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			<td height="20" style="height:20px;">OPMI-MD Surgical Microscope</td>
			<td>Carl Zeiss Surgical Inc</td>
			<td></td>
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			<td>Millipore</td>
			<td>367222</td>
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			<td height="20" style="height:20px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
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			<td height="20" style="height:20px;">Nair (depilatory cream)</td>
			<td>Church and Dwight Co.</td>
			<td>22600267058</td>
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a mouse fetal skin model of scarless wound repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Para a análise histológica, feridas cutâneas na pele dorsal de embriões de camundongos E16.5 e E18.5 devem ser colhidas 48 horas após o ferimento, fixadas em 4% PFA, e embebidos em parafina. Em modelos transgênicos fluorescentes, a criopreservação com outubro pode ser apropriado. Existem várias manchas que podem ser utilizados para visualizar arquitectura do tecido conjuntivo e celular. HE é uma mancha de duas cores que mancha núcleos azuis e estruturas eosinofílica (ie., Citoplasma e colágeno extr...

Watch this Scientific Journal Video about A Mouse Fetal Skin Model of Scarless Wound Repair at JoVE.com

A Mouse Fetal Skin Model of Scarless Wound Repair

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Um modelo de pele Rato Fetal de reparação de feridas Scarless

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

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Medicine

12.2K visualizações.  Stanford University School of Medicine. During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Vídeo: A Mouse Fetal Skin Model of Scarless Wound Repair

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Um Modelo contundente de lesão da medula espinhal cervical unilateral Usando o Impactor horizonte infinito

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Medicina

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

Uma técnica simplificada para a produção de um modelo de ferida isquêmica

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Avaliar as alterações teratogênicos em um modelo de Zebrafish de exposição fetal ao álcool

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Uma nova abordagem cirúrgica para administração intratraqueal de agentes bioativos em um modelo de camundongo Fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Modelo Murino de Cicatrização

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Ecocardiografia fetal e ultra-som Doppler pulsátil em um modelo de coelho de restrição do crescimento intra-uterino

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Um novo modelo murino de reparação endovascular de aneurisma da aorta

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Lesão tecido isquêmico na Skinfold Câmara do mouse Dorsal: Um Modelo retalho de pele para investigar aguda isquemia persistente

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Um procedimento cirúrgico para ressecção do mouse Rib: Um Modelo de Grande Escala Repair longo osso

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.