Bu eser (HPL için) NIH hibe R01 GM087609, (MTL) (HPL için) Anthony Shu, NIH hibe U01 HL099776 onuruna Ingrid Lai ve Bill Shu bir Hediyesi, Hagey Laboratuvarı için bir hibe ile kısmen desteklenmiştir Pediatrik Rejeneratif Tıp ve (MTL ve HPL için) Meşe Vakfı. GGW Tıp Stanford School, Stanford Tıp Bilim Adamı Yetiştirme Programı tarafından desteklenen ve NIGMS eğitim hibe GM07365 oldu. MSH CIRM Klinik Fellow Eğitim Hibe TG2-01159 tarafından desteklenmiştir. Gxy Sarnoff Kardiyovasküler Vakfı fon tarafından desteklenmiştir.

NOT: Bu yazıda anlatılan tüm işlemler Laboratuar Hayvan Bakımı Stanford İdari Paneli (APLAC) tarafından oluşturulan kurallara göre yapılır. 1. Zamanlı Gebelikler - Süperovulasyon Tekniği (Şekil 1) NOT: Kesinlikle E16.5 E18.5 ve fetal cerrahi fare embriyoları gebelik yaşı zamanlama kritik önem taşımaktadır. Superovulasyon neden hamile kısrakların serum (PMS) ve insan koryonik gonadotropin (HCG) enjeksiyonları kullanarak fare gebeliklerin zamanlama bizim protokol bu bölümde detaylı olarak. <ol><li> 1:00 ve 1 gün için 3:00 arasında, 100 ul PBS hacminde PMS&#39;nin 3,0-5,0 uluslararası ünitedir (IU) ile dişi farelere (kafes başına &lt;5) intraperitonal olarak (İP) enjekte edilir. </li><li> 12:00 ve 3. gün (kırk beş PMS enjeksiyonundan sonra saat 47), 2:00 arasında, 100 ul PBS hacminde HCG 3,0-5,0 IU dişi fareler IP enjekte edilir. NOT: HCG enjeksiyonu endükleyicisi<html> yumurtlama yaklaşık 12 saat sonrası enjeksiyon. </li><li> Hemen HCG enjeksiyonları sonrasında 8-16 hafta yaş arası erkeklerde ile kadın dostum. NOT: Biz genellikle bireysel erkeklerin bir kafese iki kadın yerleştirin. </li><li> Fetal yaş E0.5 olarak - (10:00 6:00) ve kayıt günün 4 sabahı erkeklerden ayrı kadın. NOT: Vajinal fişler şu anda kontrol edilebilir; Ancak, bir vajinal fiş gözlem gebelik garanti etmez ve hiçbir fiş gözlenen zaman kadın hamile olabilir. Gebelik gün 4 sabahı vajinal fişleri kontrol, gebelik yaşları E16.5 E18.5 ve görsel muayene ve / veya palpasyonla genellikle gözlemlenebilir olduğu göz önüne alındığında kesinlikle gerekli değildir. Bizim deneyimimizde, ve zorlanma bağlı olarak, süper ovulasyon kadınların yaklaşık% 30-50 burada açıklanan tekniği kullanılarak gebe. </li></ol>pg &quot;/&gt; Şekil Süperovulasyon Tekniği 1. şematik. Farelerde superovulation zamanlı gebeliklerde şematik gösteren prosedürü. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> 2. Murin Fetal Cerrahi E16.5 E18.5 ve Embryolarının üzerinde (Dorsal Yaralama) (Şekil 2) <ol><li> İşlem öncesi,% 70 izopropil alkol ile ameliyathane ve ekipman tüm yüzeylerini temizleyin. Buna ek olarak, onları otoklav ile prosedüründe kullanılacak tüm cerrahi malzemeleri ve aletleri sterilize. Bazı kurumlar sıcak boncuk sterilizasyon sonraki kullanımına izin verebilir. Operasyon Başına, gazlı bez ve cerrahi aletler, steril paketleri kullanın. </li><li> Dakikada başına 1 L bakım anestezi ardından dk başına 2 L% 2.5 izofluran / oksijen karışımı altında gebe annelerin anestezi (fetal yaş E16.5 E18.5 veya) neden olur. </li><li> ConFirma uygun Anesthetization, fare derin pedal refleksleri bastırılmış sağlamak ve yüzüstü pozisyonda fare koyun. </li><li> Puralube prosedürü sırasında göz tahrişine veya kuruluğunu önlemek için böyle bir veteriner oftalmik merhem sürün. </li><li> Artık 30 sn (Şekil 2A) için tüy dökücü krem ışık uygulaması tatbik edilmesiyle karın hazırlayın. </li><li> Povidon-iyot ve alkol (Şekil 2B ve 2C) ile aseptik cerrahi karın hazırlayın. </li><li> Mikrocerrahi makas (Şekil 2B ve 2E) kullanarak mikroskop altında orta hat laparotomi gerçekleştirin. </li><li> Yavaşça cerrahi (Şekil 2F) için seçilen rahim ve fetus maruz. </li><li> Bir kör uçlu iğne kullanılarak sıcak (38 ° C), fosfat tamponlu tuz (PBS) ile cerrahi alanı yıkayınız NOT: Bir dikkatli bir şekilde büyük çaplı iğne ucu bükülmesiyle yapılabilir. (Şekil 2G ve 2H). </li><li> Izin şekilde fetus yerleştirinsırtında tam erişim s. </li><li> Amaçlanan dorsal yaralama (Şekil 2I) site örten rahim içinden 7-0 naylon sütür kullanarak bir çanta dize dikiş geçirin. Pozisyon kese sol veya omurilik sağındaki sırtı bir bölge üzerinde dizisi ve büyük kan damarlarının yoksun uterus duvarına bir bölgede. </li><li> Çanta dize (Şekil 2J) merkezinde rahim duvarı ve amniyon kesesi yoluyla 3 mm kesi yapmak. </li><li> Sıcak (38 ° C) PBS ile kesi sitesini yıkayınız. </li><li> Mikrocerrahi makas kullanarak, fetusun sırtında, tek bir tam kalınlıkta eksizyonel yara, uzunluğu yaklaşık 1 mm kesti. </li><li> Yavaşça leke kesi sitesi pamuk uçlu aplikatör ile kurulayın. </li><li> Yaranın konumunu (Şekil 2K) işaretlemek için yara içine 3 ul hacmi Hindistan mürekkep subkutan enjekte edilir. </li><li> Mürekkep yara site içinde muhafaza edilmiştir sağlamak için ılık (38 ° C) PBS ile sulayın. </li><li> Cerrahi asistanı i varnject çanta dizesi olarak amniyon kesesi içine künt uçlu 10 G şırınga aracılığıyla sıcak (38 ° C) PBS (Şekil 2L) kapalıdır. Çanta dize kapatma tamamlama (Şekil 2M) yaklaştığında olarak şırınga geri çekin. </li><li> Yavaşça karın boşluğuna (Şekil 2N) içine rahim döndürür. </li><li> Evert cilt ve periton. </li><li> Sıcak (38 ° C) PBS ile karın boşluğuna cerrahi asistanı sulamak var. </li><li> Cilt ve periton zımbalama hızla kapatmak karın (Şekil 2O) kapalı. standart kapama iki kat yapılmaktadır; periton ve ikinci tabaka içinde bir tabakanın, subkutan doku ve deride karın kası. Acil fedakarlık ve fetusun hasat için, bizim gösteri, bir katmanda kapatılmasını gösterir. </li><li> 30 dakika süre ile ya da hayvan sternum yatma muhafaza edilmesi için yeterli bilinci yerine kadar 37 ° C&#39;ye ayarlanmış sıcak bir kuluçka makinesi içinde gözlem altında hayvan yerleştirin. </li><li> Ret etmeyintamamen prosedür kurtarıldı kadar diğer hayvanların şirkete hayvan urn. </li><li> Anestezi ve sonraki 48 saat boyunca uyanırken, buprenorfin subkutan enjeksiyonu (0.05 mg / kg) ağrı değerlendirmesine göre gerektiğinde analjezi için her 12 saat yönetmek. Gerektiğinde ek bir post-operatif ağrı tedavisi için deri altı enjeksiyon yoluyla Karprofen (5 mg / kg) uygulayınız. </li><li> Kafes hayvanları dönün ve gıda ve su ad libitum ile bunları sağlamak. </li><li> Ağrı belirtileri yakından izleyin. </li><li> 48 saat ameliyat sonrası, izofluran ve hasat yaralı fetusun aşırı dozda hamile anne kurban. Bunu yapmak için,% 5 ya da daha yüksek bir izofluran konsantrasyonunu ayarlamak ve nefes kesildikten sonra 1 dakika için maruz kalma korumak. Servikal dislokasyon ile ötenazi onaylayın. Yaş uyumlu kontrol için bir yarasız embriyo Hasat. Geç embriyolar gibi IACUC tavsiyeleri, tutarlı ötenazi ayrı bir yöntem olmalıdır decizye, servikal dislokasyon veya kimyasal enjeksiyon. </li></ol><img alt="Şekil 2," src="/files/ftp_upload/52297/52297fig2highres.jpg" /> Şekil 2. şematik Kemirgen Fetal Cerrahi. E16.5 E18.5 ve fare embriyoları yaralama dorsal Genel adımlar. (A) fare karın Depilasyon. (B ve C) fare karın hazırlanması. (D) Mikroskop cerrahi için kullanılan rahim içinden çanta dize dikiş prosedürü. (E) orta hat laparotomi. (F) uterusun Pozlama. künt uçlu iğne (G) oluşturulması. ılık tuzlu su ile rahim (H) Sulama. (I) oluşturulması. (J) Kesi Duvar ve 1 mm kalınlıkta tam eksizyonel yara nesil. (K) Hindistan mürekkep cilt altı enjeksiyonu. (L ve M) kese dize dikiş kapatılması. (N ve O)Karın Kapatma. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

<ol>
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The authors have nothing to disclose.

Burada sunulan cerrahi protokol öncelikle laboratuvar 10 tarafından 2006 yılında yayınlanan fetal kemirgen skarsız şifa bir eksizyonel modelini açıklar. Eksizyonel yaralama 11 diğer kurulan modellere ek olarak, fetal kemirgen skarsız şifa insizyonel modelleri de 12,13 var. Maymun, koyun, tavşan, Chalk ve sıçan fetal skarsız yara iyileşmesinin araştırmalar 14-17 olarak bildirilmiştir. Ancak, fareler nedeniyle nispeten düşük başına diem kafes maliyeti ve...

Fetal cilt yaraları gebelik 1 sonlarına kadar hızlı ve scarlessly iyileşir. Fetal skarsız yara onarımı, normal doku yapısının ve fonksiyonunun rejenerasyonu ile karakterize edilir. yara izi fenotip bir Scarless geçiş insanlarda ve farelerde 2,3 embriyonik günde 18 (E18) etrafında gebeliğin üçüncü trimesterde ortaya çıkar. Yetişkin karşılaştırıldığında fetal yara onarım hızlı epitelizasyonu, bağ dokusu birikimi, ve fibroblast göç ile karakterize edilir. Birçok çalışma erken fetal gelişim sırasında skarsız yara iyileşmesinin olay için olası açıklamalar teklif var. Enflamasyon yetişkin yara tamir temel bileşenidir; Ancak fetal yaralar akut inflamasyon 4 olmaması ile karakterize edilir. Bu İster fetal aşamalarında bağışıklık sisteminin fonksiyonel olgunlaşmamış bir sonucudur belirsiz kalır olduğunu. Yeni yapılan bir çalışmada önerdi bolluk, mat farklılıklarurity ve E18, fetal deri genel E15 mast hücre fonksiyonu, en azından bir fare 3&#39;te skarsız fenotip geçiş sorumlu olabilir. Diğer çalışmalar özellikleri ve fetal ve erişkin yara makrofajlar bolluğu farklılıklar fetal yara onarımı 5 esnasında normal dışı matriks (ECM) reform sorumlu olduklarını varsaymak. Fetal ve erişkin gelişimi sırasında çevresel faktörlerin farklılıklar da yara onarım etkileyebilir. Longaker ve arkadaşları fetus, yara sıvısı 6 yara sıvısında yetişkin hiçbirine kıyasla hiyalüronik asit uyarıcı aktivitesi yüksek seviyelerde sahip olduğunu göstermiştir. Sonuç olarak fetal yara ortamında hiyalüronik asit, hücre motilitesi ve çoğalması için elverişli bir mikro-teşvik eden bir glikozaminoglikan, seviyelerinin erken cenin gelişimi sırasında görülen skarsız fenotipinden sorumlu olabilir. Feta gerçeği kanıtlar noktasının diğer hatlarıl yara ortamı nispeten hipoksemikti ve büyüme faktörleri 7 zengin steril amniyotik sıvı batmış olduğunu. Ancak, kesin bir cevap fibrotik onarım skarsız rejenerasyon geçişi tetikler embriyogenez sırasında kritik bir olay ya da faktörü sağlanmıştır. Fetusta skarsız şifa için mekanizmalar sorumlu anlama kesin ve tekrarlanabilir bir model gerektirir. Burada detay E16.5 (skarsız) ve E18.5 (skar) fare embriyoları sırtında fetal skarsız yara iyileşmesi bir tekrarlanabilir bir model. Buna ek olarak, bu modelin küçük varyasyonlar Fetal yaraların ve deri 8,9 gen ekspresyon analizi gibi ileri çalışmalar, bir dizi gerçekleştirmek için kullanılabilir. Tam zamanlı gebelikler bu fetal skarsız yara iyileşmesi modeli başarılı recapitulation için kritik olduğunu göz önüne alındığında, biz süperovulasyon zamanlanmış gebeliklerde de detay bizim protokol.

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">Name of Material/Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:155px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7-O MONOSOF Suture</td>
			<td>eSuture</td>
			<td>SN-1647G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Forceps</td>
			<td>Kent Scientific</td>
			<td>INS650916</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Autoclip 9mm</td>
			<td>Texas Scientific Instruments</td>
			<td>205060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-272-382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Black Pigment</td>
			<td>AIMS</td>
			<td>242</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD Safety-Lok 3ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309596</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPMI-MD Surgical Microscope</td>
			<td>Carl Zeiss Surgical Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pregnant Mares Serum (PMS)</td>
			<td>Millipore</td>
			<td>367222</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Chorionic Gonadotropin (HCG)</td>
			<td>Sigma-Aldrich</td>
			<td>CG10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nair (depilatory cream)</td>
			<td>Church and Dwight Co.</td>
			<td>22600267058</td>
			<td></td>
		</tr>
	</tbody>
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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.

Histolojik analiz için, E16.5 ve E18.5 fare embriyolarından sırt derisinde deri yaraları 48 saat% 4 PFA içinde sabit sonrası yaralanma, ve parafin gömülmüş hasat edilmesi. Floresan transgenik modellerde, Ekim ile dondurulması uygun olabilir. Hücresel ve bağ dokusu mimarisi görselleştirmek için kullanılan olabilir birkaç lekeler vardır. Hematoksilen ve eozin mavi çekirdekleri ve eozinofilik yapıları (yani., Sitoplazma ve hücre dışı kollajen) kırmızı, pembe, turuncu ve çeşitli tonla...

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.

Scarless Yara Repair Fare Fetal Cilt Modeli

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

a-mouse-fetal-skin-model-of-scarless-wound-repair

Research

JoVE Journal

Medicine

12.2K Görüntüleme.  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.

Video: A Mouse Fetal Skin Model of Scarless Wound Repair

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Tam Kan Stimülasyon Testi kullanarak Pro-inflamatuar sitokin IL-1β Doğru ve Basit Ölçme

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Tıp

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.

Sonsuz Horizon Darbeli kullanarak tek taraflı Servikal Spinal Kord Yaralanması bir Contusive Modeli

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

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.

Bir İskemik Yara Model üretmek için Basitleştirilmiş Tekniği

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.

Fetal Alkol Pozlama bir Zebra balığı Modelinde teratojenik değişiklikler değerlendirilmesi

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.

Bir Fetal Fare Modelinde Biyoaktif Ajan intratrakeal Yönetimi İçin Yeni Bir Cerrahi Yaklaşım

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.

Yara Şifa murin Modeli

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.

Rahim içi Büyüme Kısıtlama bir Tavşan Modelinde Fetal Ekokardiyografi ve Pulsed-wave Doppler Ultrason

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.

Endovasküler Aort Anevrizma Onarımı Yeni Bir murin Modeli

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.

Mouse Dorsal Skinfold Odası&#39;nda İskemik Doku Yaralanması: Bir Cilt Flap Model Akut Kalıcı iskemisi Araştırma

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.

Fare kaburga Rezeksiyonu için Cerrahi Prosedür: Büyük Ölçekli Uzun Kemik Onarım İçin Bir Model

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