Takdir Dr. T. Taki ve Dr. M. Funamoto ve teknik destek için genişletilir.

Deney öncesinde tüm rats bir hafta alışmana verilmesi gerekir. Hayvanlar üzerinde tüm cerrahi işlemler Kılavuzu uygun olarak bakım ve laboratuvar hayvanlarının kullanımı (www.nap.edu/catalog/5140.html) veya diğer uygun etik kurallar için yapılmalıdır. İletişim kuralları hayvan refahı Komitesi devam etmeden önce uygun Kurumu tarafından onaylanması. Tüm sonraki yordamlar aseptik koşullar altında gerçekleştirilmelidir.
1. CPB devre hazırlama
Not: kişisel koruyucu ekipman eldiven, gözlük ve temiz kat veya tek kullanımlık elbisesi giymek.
<ol><li>Set-up KPB devre<ol>
<li>Polivinil klorür tüpler KPB devre ve şekil 1' de gösterildiği gibi bir değiştirilmiş yenidoğan membran oxygenator için önceden tasarlanmış bir Venöz rezervuar bağlanmanız gerekir. Tüm bağlantıları sıkı ve su sızıntısı değil olun.</li>
		<li>CPB devre silindir pompa aygıt üreticinin iletişim kuralları göre ayarlayın.</li>
		<li>Silindir pompa yüksekliği ayarlanabilir bir tabloda tutmak ve deneysel masanın altına 10 cm için tablonun yüksekliğini ayarla.</li>
	</ol>
</li>
	<li>Astar KPB devre<ol>
<li>Hydroxyethyl nişasta çözüm mix 12 mL 0.1 mL heparin ve 0.5 mL KPB devre priming için % 7 karbonat çözeltisi ile.</li>
		<li>11 mL astar çözeltisi ile devre yavaşça dönen pompa bezi ile Başbakan. Bir 18'lik havalandırma iğne hava havalandırma için rezervuar içine koymak.</li>
		<li>Membran oxygenator deair, oxygenator devirme ile birkaç kez vurdu. Hava hava embolisi ve yetersiz oksijen önlemek için tamamen gizlenmiş olmalı. Devre astar sırasında tarafından havzanın ayarla bir elektrikli ısı lamba devre ısı.</li>
	</ol>
</li>
</ol>2. CPB önce yordamı
Not: Cerrahi alan ve aygıtları % 70 alkol veya dördüncül amonyum bileşik kullanmadan önce dezenfekte.
<ol><li>Anestezi ve hayvan ayarı<ol>
<li>Bir sıçan bir Buharlaştırıcı %3.0 isoflurane karışık hava soluma ile anestezi. 16 lik kanül nefes borusu entübe ve fare çalışma kürsüye ayarlayın. (Örn: analjezi dozaj ve sıklığı ile ilgili senin yerel hayvan bakımı yönergeleri izleyin buprenorfin 0.005 mg/kg SC) Not: Sıçan derin anestezi olmalı ve refleksleri kaybetmek. Solunum ritmik olmalı ama değil tutuklandı.</li>
		<li>Fareyi bir elektrikli Isıtma yastığı ile donatılmış bir işletim masa aktarın. Mekanik havalandırma 8 mL/kg tidal hacim, 70 devir/dk solunum ve oksijen sensörü tarafından izlenen ilham oksijen kesir % 30'u ile başlar.</li>
		<li>Anestezi ile 1.5-%2.0 isoflurane ve ketamin/xylazine KPB başlatma adlı ek bir yönetim ile koruyun.</li>
		<li>Rektal sıcaklık rektal sonda kullanarak izlemek. Normothermic vücut ısısını 37 ° c sıcaklık pedi sıcaklığını ayarlama ve ısı lamba devre yerleştirerek korumak.</li>
		<li>Fareyi sırtüstü pozisyonda ayarla ve dört bacaklarda iğne sabitleme ile germek. Kalp atışı ikili omuz ve sol karın ECG elektrot iğne ayarlayarak monitör. Nemli gazlı bez yerleştirin veya kuruluk önlemek için gözleri oftalmik merhem uygulamak.</li>
	</ol>
</li>
	<li>Cannulation<ol>
<li>Tüm vücut yüzeyinin % 70 etanol veya başka bir antiseptik çözüm püskürtme sprayingby tarafından dezenfekte sonra saç bilateral inguinal bölge ve şu servikal bölge üzerinde bir ustura ile tıraş ederim. Yerel Anestezi (örneğin lidokain) Cilt kesi yapmadan önce kullanılmalıdır. Gibi alternatif bir cerrahi bodur kesi sitenin tüm vücut yerine kullanılabilir Not: vücut ısısı bir düşüş önlemek için % 70 etanol sprey.</li>
		<li>Cilt (yaklaşık 5 mm) bilateral inguinal bölge ve şu servikal bölge makas tarafından deşmek ve açık açık sağ ana femoral arter ortaya çıkarmak için doku incelemek. Atardamar damar ve sinir yakın dikkatlice ayırın. Ortak femoral arter sonunda 4-0 ipek ve pozlama gerginliği ligate.</li>
		<li>Şu ortak femoral arter arter duvarına (yaklaşık 1 mm) arter için dikey yönde mikro makas kesilmiş ve dikkatli--dan belgili tanımlık kesme 24'lik intravenöz kateter sistemik arter izlemek için 1 cm derinliğe cannulate basınç ve arteriyel kan gazı kısmi basınç analiz.</li>
		<li>Heparin sodyum (500 IU/kg) kateter üzerinden yönetmek.</li>
		<li>2.2.2 ve 24'lik intravenöz kateter sol ortak femoral arter KPB devre için bir Arteryel infüzyon çizgisiyle cannulate 2.2.3 adımları izleyin.</li>
		<li>17'lik çoklu delik angiocatheter şu iç juguler ven yerleştirin ve sağ Kulakçık ve inferior vena kava (IVC) önceden. Kateter itmeyin kabaca gemi kolayca zarar verebilir. CPB devre Venöz drenaj için kateter takın.</li>
		<li>Her cannulated bölgesinin kirlenmesini önlemek için nemli bir gazlı bez ile kapak.</li>
	</ol>
</li>
</ol>3. işlem CPB sırasında
<ol><li>CPB sırasında 0, 8 L/dk oxygenator % 100 oksijen gazı sunmak ve 30 devir/dk solunum oranını düşürmek. Arteryel oksijen kısmi basıncı 200 ile 400 mmHg için gereklidir.</li>
	<li>CPB başında KPB başlatma sonra vücut ısısı hemen damla azaltmak için en fazla, 42 ° c ısı ped sıcaklık ayarını yükseltin. Vücut sıcaklığı 36 ° C'ye döndüğünde 37 ° c ayar sıcaklığı ayarlayın</li>
	<li>Dikkatle KPB akışını başlatmak ve havzanın kan kan hacmi göz kulak. Boş bir rezervuar hava embolisi neden olabilir. Havzanın kan kan hacmi azalır Eğer pompa akış tablo yüksekliğini ayarlayıp veya drenaj kateter konumunu değiştirerek düşürebilir. Kolayca delikli sağ atriyumu ve/veya kalp ritim bozukluğu neden olabilir Venöz drenaj kateter yeniden konumlandırmak değil.</li>
	<li>Artırmak ve 100 mL/kg pompa akışı tutmak/min, ise ortalama kan basıncı 70 mmHg korunur. Uygun kan basınç korunur, en az 1 ml rezervuar içinde küçük bir birim kabul edilebilir olur. Az 1 mL rezervuar kanında ise, organlarda bir hava embolisi neden olabilir.</li>
	<li>Kan basıncı sabit değil, 2-3 mL astar çözeltisi (KPB sonra anemi neden) devre ekleyin.</li>
</ol>4. işlem KPB sonrası
<ol><li>Venöz drenaj boru kelepçe ve devre kaldırın. Yavaş yavaş arter kan basıncı korumak için devre içinde kalan kan süzülür.</li>
	<li>70 devir/dk solunum hızı artırın.</li>
	<li>Venöz drenaj kateter ve sol atardamar kateter kaldırın ve sonra proksimal ve distal sitedeki gemi ligate.</li>
	<li>Arteryel satırını doğru femoral arterin 60 dk KPB bitiminden sonra kaldırın.</li>
	<li>Her yara serum fizyolojik ile temiz ve yaranın dikiş ile kapatın.</li>
	<li>Anestezi ve tüpü boğaza tüp hayvan spontan solunum kontrol ettikten sonra son bulur.</li>
	<li>Isıtılan steril izotonik sıvılar yönetmek ve ısı mat ve elektrik ısı lamba hayvanlar sıcak tutmak için kullanın. Koşulları hayvanın anestezi kurtarma kadar sık sık kontrol edin. Ne zaman gerekli solunum desteği sağlar. Not: hayvan hareket başladığında, ısı kaynağı sıcak veya soğuk tarafı seçmek hayvan izin vermek belgili tanımlık kafes bölümünden çıkarılmalıdır.</li>
	<li>Tam olarak solunum tekrar yerine geleceğinden hayvan şirketin diğer hayvanların dışında tutmak. Hayvan kadar tam kurtarma şirkete iade etmeyin.</li>
	<li>Anestezi kurtarma sonra yiyecek ve su alımını kontrol ve uygun beslenme desteği sağlar. Analjezikler yönetmek ve rahatsızlık veya ağrı belirtileri kontrol.</li>
</ol>

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instrumental+responses+in+rats+after+hypothermic+cardiopulmonary+by-pass.">Instrumental responses in rats after hypothermic cardiopulmonary by-pass.</a> P Soc Exp Biol Med. 126 (1), 225-228 (1967).</li><li>Fabre, O., 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+recovery+model+of+partial+cardiopulmonary+bypass+in+the+rat.">A recovery model of partial cardiopulmonary bypass in the rat.</a> Perfusion. 16 (3), 215-220 (2001).</li><li>Hirao, S., Masumoto, H., Minatoya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+cardiopulmonary+bypass+models+to+Investigate+multi-organ+injury.">Rat cardiopulmonary bypass models to Investigate multi-organ injury.</a> Clin Surg. 2, 1-6 (2017).</li><li>Jungwirth, B., 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=Neurologic+outcome+after+cardiopulmonary+bypass+with+deep+hypothermic+circulatory+arrest+in+rats:+description+of+a+new+model.">Neurologic outcome after cardiopulmonary bypass with deep hypothermic circulatory arrest in rats: description of a new model.</a> J Thorac Cardiov Sur. 131 (4), 805-812 (2006).</li><li>de Lange, F., Yoshitani, K., Podgoreanu, M. V., Grocott, H. P., Mackensen, G. 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An update.</a> Eur J Cardio-Thorac. 21 (2), 232-244 (2002).</li><li>Hirao, 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=Recombinant+human+soluble+thrombomodulin+prevents+acute+lung+injury+in+a+rat+cardiopulmonary+bypass+model.">Recombinant human soluble thrombomodulin prevents acute lung injury in a rat cardiopulmonary bypass model.</a> J Thorac Cardiov Sur. , (2017).</li><li>Yamazaki, S., Inamori, S., Nakatani, T., Suga, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+protein+C+attenuates+cardiopulmonary+bypass-induced+acute+lung+injury+through+the+regulation+of+neutrophil+activation.">Activated protein C attenuates cardiopulmonary bypass-induced acute lung injury through the regulation of neutrophil activation.</a> J Thorac Cardiov Sur. 141 (5), 1246-1252 (2011).</li><li>Wang, C. T., Zhang, L., Wu, H. W., Wei, L., Xu, B., Li, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxycycline+attenuates+acute+lung+injury+following+cardiopulmonary+bypass:+involvement+of+matrix+metalloproteinases.">Doxycycline attenuates acute lung injury following cardiopulmonary bypass: involvement of matrix metalloproteinases.</a> Int J Clin Exp Patho. 7 (11), 7460-7468 (2014).</li><li>Liu, K., 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=Curcumin+attenuates+cardiopulmonary+bypass-induced+lung+oxidative+damage+in+rats.">Curcumin attenuates cardiopulmonary bypass-induced lung oxidative damage in rats.</a> J Cardiovasc Pharm T. 17 (4), 395-402 (2012).</li><li>Taki, 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=Fetal+mesenchymal+stem+cells+ameliorate+acute+lung+injury+in+a+rat+cardiopulmonary+bypass+model.">Fetal mesenchymal stem cells ameliorate acute lung injury in a rat cardiopulmonary bypass model.</a> J Thorac Cardiov S. 153 (3), 726-734 (2017).</li><li>Zhu, X., 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=Establishment+of+a+novel+rat+model+without+blood+priming+during+normothermic+cardiopulmonary+bypass.">Establishment of a novel rat model without blood priming during normothermic cardiopulmonary bypass.</a> Perfusion. 29, 63-69 (2014).</li><li>Inoue, K., 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=Deep+anesthesia+worsens+outcome+of+rats+with+inflammatory+responses.">Deep anesthesia worsens outcome of rats with inflammatory responses.</a> Inflamm Res. 65 (7), 563-571 (2016).</li><li>Bradfield, J. 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Tüm yazarlar ile ilgili ticari destek ifşa gerek yok.

Bu sıçan KPB modelde, inflamatuar sitokinlerin ve HMGB-1, inflamatuar yanıt düzenleyen bir anahtar transkripsiyon faktörü serum ve akciğer ifade düzeyleri önemli ölçüde KPB sonra arttı. HMGB-1 düzeyine serum salgılanmasını kalp damar cerrahisi11geçiren hastalarda yükselmiş ve en yüksek serum HMGB-1 seviye CPB sırasında daha ağır sistemik inflamatuar yanıt sendromu ile ilişkili olduğu önceki klinik çalışmalar gösterdi ve akciğer oksijenasyonu bozulma KPB<sup class=...

1953 yılında Dr. John H. Gibbon Jr. başarıyla KPB1kullanarak ilk kalp cerrahisi yapılan ve daha sonra temel bir modalite kardiyovasküler cerrahi oldu. CPB için ilgili çoklu organ komplikasyonlar hala teknikleri ve cihazlar önemli ölçüde rafine edilmiş iken, kalp damar ameliyatları sonucu güvenliğini aşmasına ve ameliyat sonrası morbidite ve mortalite2etkileyebilir. KPB ile ilgili organ hasarı bağışıklık sistemi harekete geçirmek ve sentez proinflamatuar sitokinler, iltifat harekete geçirmek ve üretim oksijen serbest radikallerin2neden olur. Onun patofizyolojisi, ancak, tam aydınlatılmamıştır değil.
Hayvan modelleri KPB klinik kullanımı recapitulating ve KPB sonrası açıklama patofizyolojik süreçlerin etkinleştirmek; Bu stratejileri bu komplikasyonları önlemek için gelişmekte olan önceden klinik çalışmaları kolaylaştırabilir. Beri Popovic ve ark. ilk KPB rat sıçan KPB manken 19673' te rapor modelleri rafine edilmiş ve yavaş yavaş daha fazla maliyet-etkililik, uygun deneysel süreç ve yöntemleri test bir bolluk nedeniyle büyük hayvan modellerin genetik meydana gelmiş ve protein düzeyleri. Buna ek olarak, doğuştan farelerin genetik olarak özdeş, olası biyolojik önyargıları azaltmak olabilir.
Fabre ve ark. ilk kurulan birden fazla organ sekel KPB4uzun süreli çalışma izin bir kurtarma modeli. Bu basit hayatta kalma model avantajları esneklik (KPB akışı ve süre), hayati durumu ve tekrarlanabilirlik sistemik inflamasyon vardır. Sıçan KPB modelleri KPB5sırasında çoklu organ yaralanması önlemek amacı tedavi stratejileri incelenmesi için çok önemli hale gelmiştir ve son zamanlarda klinik durumlar CPB sırasında simülasyonu için çeşitli modeller geliştirilmiştir. De Lange ve ark. Miyokard hasarı7için ilgili enzimatik, genetik ve histolojik yanıt tanımlamak için kullanılan bir kardiyak arrest modeli geliştirdi. Engin ve ark. Miyokard İnfarktüsü ve kontrollü reperfüzyon kalp disfonksiyonu odak iskemi ve reperfüzyon hasarı8üzerinden analiz etmek için kırılan bir KPB modeli kullanılarak düzenlenmiştir. Jungwirth ve ark. ilk kurulan global iskemi ve reperfüzyon hasarı DHCA ve destekler potansiyel nöroprotektif stratejileri6tarafından aydınlatmak derin hipotermik dolaşım tutuklama (DHCA) modeli. DHCA kullanarak çalışmalar hipotermi, reperfüzyon ve/veya hemoliz tetikleyen sinyal olaylar9etkisini araştırmak. Derin hipotermi harekete geçirmek ve çeşitli enzim inactivation etkileyebilir ve yollar ve mekanizmaları bilinmeyen10kalır. Öte yandan, kardiyak arrest modelleri veya kalp iskemi modelleri iskemi ve reperfüzyon kalp yaralanması araştırmak için kullanılmalıdır. Son derece insan KPB özetlemek çeşitli bu sıçan KPB modeller patolojik süreçler için KPB ile ilgili açığa vurmak ve KPB ile ilgili komplikasyonlar etkisinin azaltılmasına yardımcı olur.
Bu iletişim kuralı kan nakli ya da bir fare inotropic ajanlar olmadan basit bir KPB modeli gösterir. Bu modeli birden fazla organ sekel KPB ile uzun süreli çalışma için verir.

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			<td height="21" style="height:21px;width:216px;">OxiQuant B</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CMA 450 Temperature Controller</td>
			<td style="width:140px;">CMA</td>
			<td align="right" style="width:145px;">8003759</td>
			<td>Temperature Controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CMA 450 Heating Pad</td>
			<td style="width:140px;">CMA</td>
			<td align="right" style="width:145px;">8003763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CMA 450 Rectal Probe</td>
			<td style="width:140px;">CMA</td>
			<td align="right" style="width:145px;">8003761</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DIN(8) to Disposable BP Transducer</td>
			<td style="width:140px;">ADInstruments</td>
			<td style="width:145px;">MLAC06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable BP Transducer</td>
			<td style="width:140px;">ADInstruments</td>
			<td style="width:145px;">MLT0670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IX-214 Data Recorder</td>
			<td style="width:140px;">iWorx Systems</td>
			<td style="width:145px;">IWX-214</td>
			<td>amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LabScribe software</td>
			<td style="width:140px;">iWorx Systems</td>
			<td style="width:145px;"></td>
			<td>software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Roller pump</td>
			<td style="width:140px;">Furue Science</td>
			<td style="width:145px;">Model RP-VT</td>
			<td>pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Happy Cath</td>
			<td style="width:140px;">Medikit</td>
			<td style="width:145px;">EB 19G 4HCLs PP</td>
			<td>17-gauge multiorifice angiocatheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SURFLO ETFE I.V. Catheter</td>
			<td style="width:140px;">Terumo</td>
			<td style="width:145px;">SR-OX2419CA</td>
			<td>24-gauge angiocatheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygenator</td>
			<td style="width:140px;">Mera</td>
			<td style="width:145px;">HPO-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CPB circuit</td>
			<td style="width:140px;">Mera</td>
			<td style="width:145px;">custom-made</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hespander fluid solution</td>
			<td style="width:140px;">Fresenius Kabi</td>
			<td style="width:145px;">3319547A4035</td>
			<td>Hydroxyethyl starch</td>
		</tr>
	</tbody>
</table>

a recovery cardiopulmonary bypass model without transfusion or inotropic agents in rats

Kardiyopulmoner bypass (KPB) kardiyovasküler cerrahide vazgeçilmezdir. CPB tekniği ve aygıtları, çoklu organ komplikasyonlar dramatik arıtma rağmen ilgili uzun süreli KPB hala kalp damar ameliyatları sonucu güvenliğini aşmasına ve ameliyat sonrası morbidite ve mortalite daha da kötüye gidebilir. Hayvan modelleri KPB klinik kullanımı recapitulating açıklama CPB sırasında meydana patofizyolojik süreçlerin etkinleştirin ve bu komplikasyonlar karşı koruma stratejileri geliştirmek için önceden klinik çalışmalar kolaylaştırmak. Sıçan KPB onların daha fazla maliyet-etkililik, uygun deneysel süreçler, genetik, bol test yöntemleri veya protein düzeyleri ve genetik tutarlılık nedeniyle avantajlı modellerdir. Bunlar bağışıklık sistemi harekete geçirmek ve sentez proinflamatuar sitokinler, iltifat harekete geçirmek ve üretim oksijen serbest radikallerin araştırılması için kullanılabilir. Fare modelleri rafine edilmiş ve yavaş yavaş büyük hayvan modelleri meydana gelmiş. Burada, kan nakli ve/veya bir sıçan inotropic ajanlar olmadan basit bir KPB modeli açıklar. Bu kurtarma modeli birden fazla organ sekel KPB ile uzun vadeli bir çalışma sağlar.

Tüm KPB devre şekil 1 gösterir. Bu modelde fizyolojik değişkenler Şekil 2' de gösterilen ve rektal ısı dahil, arteryel kan basıncı ve kalp hızı demek. Şekil 3 arteryel kan gazı analizleri de dahil olmak üzere kısmi basınç Arteryel oksijen, Arteryel karbon dioksit, Hematokrit, temel aşırı, serum potasyum ifadesidir ve potansiyel hidrojenin kısmi basınç CPB sırasında gösteri...

Watch this Scientific Journal Video about A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats at JoVE.com

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Kan nakli veya Sıçanlarda Inotropic ajanlar olmadan kurtarma Kardiyopulmoner Bypass modeli

Burada, kan nakli ya da bir fare inotropic ajanlar olmadan basit kurtarma Kardiyopulmoner bypass modeli tanımlamak için bir iletişim kuralı mevcut. Bu model birden fazla organ sekel Kardiyopulmoner baypas uzun vadeli bir çalışma sağlar.

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ ...

a-recovery-cardiopulmonary-bypass-model-without-transfusion-or

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.8K Görüntüleme.  Kyoto University. Burada, kan nakli ya da bir fare inotropic ajanlar olmadan basit kurtarma Kardiyopulmoner bypass modeli tanımlamak için bir iletişim kuralı mevcut. Bu model birden fazla organ sekel Kardiyopulmoner baypas uzun vadeli bir çalışma sağlar.

Video: A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Sıçanlarda Ortotopik Hind-Ekstremite Nakli

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

İmmünoloji ve Enfeksiyon

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Tanı Laboratuvarı Sıçanlar ve Fareler Ecto-Endoparasites

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

Bulaşıcı Mürin Norovirüs Ters Genetik Aracılı Kurtarma

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Immünkompetan Kemirgenler bir Sağlam Pnömoni Modeli karşı antibakteriyel etkinliğini değerlendirmektir<i&gt; S. pneumoniae</i&gt;<i&gt; H. influenza</i&gt;<i&gt; K. pneumoniae</i&gt;<em&gt; P. aeruginosa</em&gt; ya da<em&gt; A. baumannii</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

En iyi duruma getirilmiş Interferon-gamma ELISpot tahlil ölçü birimi T hücre yanıtları sonrasi hipererjik eskiden şiling şimdi domuz modelinde

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

Sekonder Bakteriyel Pnömoni Murine Modelleri için Hassas Patojen Dağıtım ve Kurtarma Sistemi

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

Zenginleştirilmiş İnsan Hafızası B Hücre Popülasyonlarından İstenilen Özgülliklerle Antikorların Seçimi ve Geri Kazanımı İçin Tek Hücreli Tarama Yöntemi

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Akış Sitometrisi Kullanılarak İnsan Lökositler tarafından Aspergillus fumigatus Conidia Fagositozunun ölçülmesi

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Visualization and Quantification of High-Dimensional Cytometry Data using Cytofast and the Upstream Clustering Methods FlowSOM and Cytosplore

The complexity of data generated by mass cytometry has necessitated new tools to rapidly visualize analytic outcomes. Clustering methods like Cytosplore or FlowSOM are used for the visualization and identification of cell clusters. For downstream analysis, a newly developed R package, Cytofast, can generate a rapid visualization of results from clustering methods. Cytofast takes into account the phenotypic characterization of cell clusters, calculates the cell cluster abundance, then quantitatively compares groups. This protocol explains the applications of Cytofast to the use of mass cytometry data based on modulation of the immune system in the tumor microenvironment (i.e., the natural killer [NK] cell response) upon tumor challenge followed by immunotherapy (PD-L1 blockade). Demonstration of the usefulness of Cytofast with FlowSOM and Cytosplore is shown. Cytofast rapidly generates visual representations of group-related immune cell clusters and correlations with immune system composition. Differences are observed in the clustering analysis, but separation between groups are visible with both clustering methods. Cytofast visually shows the patterns induced by PD-L1 treatment that include a higher abundance of activated NK cell subsets, expressing a higher intensity of activation markers (i.e., CD54 or CD11c).

Cytofast is a visualization tool used to analyze output from clustering. Cytofast can be used to compare two clustering methods: FlowSOM and Cytosplore. Cytofast can rapidly generate a quantitative and qualitative overview of mass cytometry data and highlight the main differences between different clustering algorithms.

Sitofast ve Upstream Kümeleme Yöntemleri FlowSOM ve Cytosplore kullanılarak Yüksek Boyutlu Sitometri Verilerinin Görselleştirilmesi ve Ölçülmesi

The complexity of data generated by mass cytometry has necessitated new tools to rapidly visualize analytic outcomes. Clustering methods like Cytosplore ...