Biz onu mükemmel teknik yardım için Krista den Ouden teşekkür ederim. Bu teknik, mali, Hollanda Böbrek vakıf, hibe C06.2174 tarafından desteklenmiştir. MCV Bilimsel Araştırma Hollanda örgütü (NWO) Vidi-hibe 016.096.359 tarafından desteklenmektedir.

Tüm deneyler Utrecht deneysel hayvan komitenin hayvan deneysel etik kılavuz çizgileri uygun olarak yürütülür. Protokol yazarın kurumun hayvan bakımı ve kullanımı komitesi rehberlik ve onay altında yapılır. CKD 8 hafta yaşında erkek doğuştan Lewis sıçan (Charles River, Sulzfeld, Almanya) indüklenir. Sıçanlar bir ışık, sıcaklık ve nem kontrollü bir ortamda, standart koşullar altında muhafaza edilmektedir. 1. Cerrahi Hazırlık <ol><li> Cerrahi aletler sterilize: <ul><li> 1 öğrenci doku 1-2 diş 12 cm forseps </li><li> 1 öğrenci standart desen forseps </li><li> 2 Semken forseps </li><li> 1 Mayo makas </li><li> 1 öğrenci iris makası </li><li> Makasla 1 Olsen-Hegar iğne tutucu </li><li> Battaniye </li></ul></li><li> Envanter listesini kontrol edin: <ul><li> Isınma pad ve lamba ile masa Çalışma </li><li> O <sub&gt; 2, izofluran </li><li> Dokular </li><li> Tıraş makinesi </li><li> Terazi </li><li> Steril çalışma seti </li><li> Steril gazlı bez 5x5 ve 10x10 </li><li> % 70 alkol </li><li> % 0.9 &#39;luk NaCl çözeltisi </li><li> 1 ml şırınga </li><li> Iğneler (25G) </li><li> Jel köpük pedler: spongostan </li><li> Vicryl 4.0 ve 5.0 </li><li> Buprenorfin, 0.03 mg / kg (1:10 fizyolojik tuz içinde seyreltilmiş) </li><li> Ameliyat sonrası sıçanlar için temiz bir kafes </li></ul></li></ol> 2. Sağ Taraf Uninephrectomy <ol><li> % 70 alkol ile masaya dezenfekte edin. </li><li> Yeri indüksiyon kutusunda fare ve% 4 izofluran ve O 2 1 litre akışı ile anestezi neden. Not: Kullanılan sıçan suşu bağlı olarak, ön ve perioperatif analjezi reçete edilir, yerel veteriner hekim ile görüşülmelidir bu ihtiyacı. </li><li> Koni içine masa ve yer burun sıçan aktarın. Sıçan ve burun konisi sıçan vücut ısısını korumak için bir ısıtma pedi üzerine yerleştirilir. Sıçan is O 2 izofluran ve 1 litre% 2 karışımı ile havalandırılan. </li><li> Sıçan kanadını Tıraş ve alkol ile dezenfekte edin. Yıkayın ve dezenfekte el ve steril cerrahi eldiven giyin. Kafa kapağı ve bir maske. 1 olun - 1.5 cm kesi paralel anatomik forseps ve künt makas kullanılarak kaburga. Arka kasların künt diseksiyon ile böbrek Açığa. </li><li> Böbreğin alt kutbu görünür, dikkatle perirenal yağ dokusu kavrama küçük künt forseps kullanabilir. Hafifçe forseps ile perirenal yağ çekerek böbrek dışa. Bu işlem sırasında böbreküstü bezi rahatsız dikkat edin. Böbrek üstü bezi kolayca yağ dokusunda medial sitesinde bir forseps koyarak çıkarıldı ve hafifçe böbreküstü bezi ve Şekil 2&#39;de gösterildiği gibi, böbrek arasında yukarı doğru hareket ettirilebilir. </li><li> Gazlı bez ve yağ ve bağ dokusu çevreleyen açık hafifçe böbrek yerleştirin. Renal arter ve ven belirlenmesi ve bir bağ (5.0 yervikril) gemilerin etrafında tek bir düğüm ile, ancak düğüm kapalı kravat yok. </li><li> Böbrek ve düğüm arasında boşluk oluşturmak için, yaklaşık 0.5 cm aorta doğru damarları boyunca dikkatle gevşek düğüm taşıyın. Bu kesim sonra kaymasını bağ önlemek için yapılır. </li><li> Iki çift düğüm ile düğüm. Perfüzyon durduruldu ise böbreğin renk hemen kahverengiye kadar değişecektir. Bağ ucunda kesmeyin. </li><li> Böbrek yakın böbrek damarları kesin ve böbrek çıkarın. Yavaşça böbrek damarlarının kanama kontrol etmek için dikilen uzun ucunda çekin. Bağ gemide yerde kalmak gerekir. Bağ uzun biter kesin. Not, kalan böbrek damarlarının hemen karın boşluğuna geri çekilecektir. </li><li> Leke eksize böbrek kuru ve tartın. </li><li> Vicryl 5.0 ile sütürlerinin ile iskelet kası kesi kapatın. Arka bacak, 0.10 mL (0.03 mg / kg) enjekte buprenorfin im. Yakınintrakutanöz dikişler ile Vicryl 4.0 ile cilt. Bu yara açmasını sıçan engeller. Not: Lewis sıçanlarda solunum depresyonu nedeniyle, sadece sadece izofluran kapatmadan önce post-operatif analjezi verir. </li><li> Yiyecek ve su kolay erişim ile temiz bir tek kafeste sıçan ev. Isınma pad O / N kafes yarım yerleştirin ve dikkatli bir şekilde sıçan ertesi gün kontrol edin. Yara kapatıldı ve sıçan ameliyat sonrası 6 ile 12 saat arasında normal olan, aktif olduğunda, sıçan grubu gövde içine tekrar yerleştirilebilir. </li></ol> 3. Sol Taraf Ara toplam nefrektomi <ol><li> Yedi gün sonra, aynı preparatlar olarak da 2.1 &#39;den 2.5&#39; e tarif sağ tarafı için de sol tarafta yapılır. Ameliyattan önce, sağ böbrek ağırlığının yaklaşık 2/3rds karşılık kaldırılması gerekiyor böbrek doku miktarı hesaplanır. Ameliyat öncesi jel köpük küçük parçalar (Spongostan, Johnson &amp; Johnson, New Jersey) hazırlayın. </li&gt; <li> Sol böbrek üst kutup etrafında keskin bir makas yerleştirin ve bir inme (makas konumu için, bkz. Şekil 3) en üst kutup rezeke. Jel köpük bir parça ile hemen kapak ve steril gazlı bez ile hafif baskı. </li><li> 3.3 &#39;de olduğu gibi böbrek alt kutup ile tekrarlayın. </li><li> Cilt üzerinde pıhtılaşma önlemek için böbrek kaldırın. Kanama durana kadar steril gazlı bez ile jel köpük pedler hem de hafif basınç koruyun. Kanama devam zaman, başka bir köpük yastık ekleyin. </li><li> Karın içinde yapışık jel köpük pedler ile kalan böbrek yerleştirin. Kas ve deri kapatın ve 2,9-2,12 izleyin. </li></ol> 4. Sham Cerrahi <ol><li> Sham çalışan kontrolleri böbrekler ortaya koymak için aynı prosedürü tabi. Bunun yerine böbrek kökünü kazıma ya da bir hafta sonra direkleri kesme, böbrekler her iki böbreküstü bezleri rahatsız etmemek için özen, bir hafta arayla decapsulated vardır. Yara kapatılması ve ameliyat sonrası bakım kimliği olan2.11 ve 2.12 için entical. </li></ol>

<ol>
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Yazarlar ifşa hiçbir şey yok.

Yüksek tuz diyeti ve geçici NOS inhibisyonu ile birlikte Lewis sıçanlarda renal kitle, bir 5/6th cerrahi olarak çıkarılması insan CKD benzer CKD bir modeli yol açar ve CKD medikal tedavinin nedensel mekanizmaları ve etkinliğinin çalışma sağlar. 5/6th nefrektomi modeli CKD, iyi bilinen ve geniş çapta tarif modelidir. Ancak, sadece böbrek kitle 5/6th kaldırarak tüm sıçan suşları acil böbrek yetmezliğine yol açmaz. Biz GFP + Lewis sıçan 21 durumu gibi CKD...

Ilerici doğa, takip eden son dönem böbrek hastalığı ve ilişkili kardiyovasküler morbidite ve mortalite nedeniyle, CKD giderek büyüyen bir halk sağlığı sorunu 1&#39;dir. CKD ilerleme Yavaşlama bu nedenle önemli bir sağlık önceliktir. CKD dengesinin karmaşık bozukluklar ile karakterize olduğundan, bütünleştirici hayvan modellerinde CKD gelişimi ve ilerlemesi çalışma için gereklidir. Böbrek birbirleri ile etkileşim farklı hücre tipleri arasında, geniş bir aralık oluşur. Bu karmaşıklık in vitro taklit edilemez. CKD yeni tedavi girişimleri incelemek, insan CKD 2,3 çeşitli yönleri benzeyen 5/6th nefrektomi ablasyon modeli, ilerleyen böbrek hastalığının iyi bilinen bir deneysel model, kullanın. Renal kitle brüt azalma ilerici glomerüler ve tübülo-interstisyel yaralanma, kalan nefron kaybı ve sistemik ve glomerüler hipertansiyon gelişimine neden olur. Bu Progr ile ilişkiliessive böbrek içi kılcal kaybı 4, inflamasyon ve glomeruloskleroz. Için risk faktörleri her zaman endotel fonksiyonu 5 üzerindeki etkisi CKD. Biz CKD gelişimi nispeten dirençli olan bir fare suşu (Lewis) kullanılan ve bu nedenle nitrik oksit (NO) azalması 6, 7, 8 ve yüksek tuz diyeti 9, renal kitle 5/6th çıkarılması birleştirilir. Varış ve iklimlendirme sonra, hayvanlar iki gün sonra L-NNA devamı ile sağ taraflı uninephrectomy (UNX) takip 4 haftalık bir süre için içme suyu (20 mg / L) için ilave bir NO sentaz inhibitörü (L-NNA), almak . Bir hafta sonra, renal kitle 2/3rds subtotal nefrektomi (SNX) yani kaldırma sol tarafında yapılır. SNX sonra, hayvanlar, 4 haftalık bir süre için içme suyu içinde 20 mg / L, LNNA yine ardından 2 gün süre ile iyileşmeye bırakıldı edilir. Zemin chow (zaman çizgisi bkz. Şekil 1) de ilave bir yüksek tuz diyeti (% 6), deney boyunca devam edilir. TO yandaki UNX gerçekleştirmek için bir neden ve sol taraftaki SNX böbrek damarlarının daha kolay böbrek vücut dışında maruz bırakıldığında çok damarları germeden böbrek erişimi kolaylaştırır sol taraftaki uzun olmasıdır . Literatürde, model bir hafta sonra 10,11,12 sağ böbrek UNX ardından, sol böbrek kutup önce kaldırılır olduğu tarif edilmektedir. Bizim ellerde bu model böbrek yetmezliği çok daha hızlı bir gelişme gösterdi, ama aynı zamanda böbrek fonksiyon kaybı çok daha büyük bir değişim. Böbrek yetmezliğinin ilerlemesi plazma üre, sistolik kan basıncı ve proteinüri ölçerek zaman içinde takip eder. SNX sonra altı hafta, böbrek yetmezliği, geliştirmiştir glomerüler filtrasyon hızında belirgin bir azalma (% 69) ve etkin renal plazma akışı (% 62) 13 hipertansiyon (sistolik kan basıncı&gt; 150 mmHg), proteinüri (ile karakterize&gt; 50 mg/24 saat) ve hafif üremi (&gt; 10 mM). Histolojik özellikleri tubul içeriro-interstisyel zarar inflamasyon, tübüler atrofi ve fibrozis ve kalan nüfus (&lt;% 10) içinde sağlıklı glomerül büyük azalmaya yol açan fokal glomerüloskleroz yansıyan. Takip 12 hafta sonrasına kadar SNX terapötik girişimlerin değerlendirilmesi için bir fırsat penceresi sağlayan, CKD daha fazla ilerleme gösterir.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>L-NNA</td>
			<td>Sigma-aldrich</td>
			<td>N5501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spongostan dental: gel foam pads 1x1x1 cm</td>
			<td>Johnson&amp;Johnson</td>
			<td>Ms0005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethicon Vicryl FS-2S naald 4/0 V392H p/36</td>
			<td>Ethicon</td>
			<td>V303H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethicon Vicryl RB-1+ naald 5/0 V303H p/36</td>
			<td>Ethicon</td>
			<td>V392H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buprenorphine (0.3 mg/ml)</td>
			<td>Via local pharmacist ordered by Reckitt Benckiser pharmaceuticals</td>
			<td>unknown</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Student Tissue Forceps - 1x2 Teeth 12 cm</td>
			<td>Fine Science Tools (FST)</td>
			<td>91121-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Student Standard Pattern Forceps</td>
			<td>FST</td>
			<td>91100-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>FST</td>
			<td>14010-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2X Semken Forceps</td>
			<td>FST</td>
			<td>11008-13</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Student Iris Scissors</td>
			<td>FST</td>
			<td>91460-11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder</td>
			<td>FST</td>
			<td>12002-14</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

5/6th nephrectomy in combination with high salt diet and nitric oxide synthase inhibition to induce chronic kidney disease in the lewis rat

Kronik böbrek hastalığı (KBH) küresel bir sorundur. CKD ilerleme Yavaşlama önemli bir sağlık önceliktir. CKD dengesinin karmaşık bozukluklar ile karakterize olduğundan, bütünleştirici hayvan modellerinde CKD gelişimi ve ilerlemesi çalışma için gereklidir. CKD CKD ve yeni terapötik girişimlerin gelişimini incelemek için, insan CKD çeşitli yönleri benzeyen, 5/6th nefrektomi ablasyon modeli, ilerleyen böbrek hastalığının bilinen bir deneysel model kullanın. Renal kitle brüt azalma ilerici glomerüler ve tübülo-interstisyel yaralanma, kalan nefron kaybı ve sistemik ve glomerüler hipertansiyon gelişimine neden olur. Ayrıca, ilerleyen böbrek içi kılcal kaybı, enflamasyon ve glomerüloskleroz ile ilişkilidir. Için risk faktörleri her zaman endotel fonksiyonu üzerindeki etkisi CKD. Bu taklit için, nitrik oksit (NO) azalması ve yüksek tuz diyeti ile renal kitle 5/6th çıkarılması birleştirir. Varış ve iklimlendirme sonra, hayvanlar yenidenSağ taraflı uninephrectomy takiben 4 haftalık bir süre için içme suyu (20 mg / L) ile desteklenmiş bir NO sintaz inhibitörü (NG-nitro-L-arginin) (L-NNA), alma işlemine. Bir hafta sonra, bir alt toplam nefrektomi (SNX) sol tarafta gerçekleştirilir. SNX sonra, hayvanlar, 4 haftalık bir süre için daha içme suyu (20 mg / L) LNNA takiben iki gün süre ile iyileşmeye bırakıldı edilir. Zemin chow (zaman çizgisi bkz. Şekil 1) de ilave bir yüksek tuz diyeti (% 6), deney boyunca devam edilir. Böbrek yetmezliğinin ilerlemesi plazma üre, sistolik kan basıncı ve proteinüri ölçerek zaman içinde takip eder. SNX sonra altı hafta, böbrek yetmezliği geliştirdi. Böbrek fonksiyonu &#39;altın standart&#39; inulin ve para-amino hippurik asit (PAH) temizlik teknolojisi kullanılarak ölçülür. CKD Bu model bir glomerüler filtrasyon hızı (GFH) azalma ve etkin renal plazma akışı (ERPF), hipertansiyon (sistolik kan basıncı&gt; 150 mmHg), proteinüri (&gt; 50 mg/24 saat ile karakterizedir) Ve hafif üremi (&gt; 10 mM). Histolojik özellikleri inflamasyon, tübüler atrofi ve fibrozis ve kalan nüfus (&lt;% 10) içinde sağlıklı glomerül büyük azalmaya yol açan fokal glomerüloskleroz yansıyan tübülo-interstisyel zarar içerir. Takip SNX CKD daha fazla ilerleme gösteren 12 hafta sonrasına kadar.

Subtotal nefrektomi sonra, toplam böbrek kütlesinin yaklaşık 1/6th bırakılır. Şekil 4 önceki iki deneylerde ortalama ve standart sapma ile doğru böbrek Çıkarılan parçanın ağırlığını gösterir. Bir UNX sonra hafta, sol böbrek hipertrofisi oluşur tutmalı; belirten 5/6th kaldırma daha az doğru böbrek her zaman sonuçlarının ağırlığına göre hesaplanan kaldırılması gerekiyor ağırlık. Bu ameliyat sırasında sol böbrek ağırlığını belirlemek mümkün olmadığı...

Watch this Scientific Journal Video about 5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat at JoVE.com

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Cerrahi renal kitle 5/6th kaldırarak Lewis sıçan kronik böbrek hastalığı (KBH) kurmak için bir iki aşamalı yöntemi açıklanmıştır. Cerrahi işlem kombinasyonu, NOS-inhibisyonu ve yüksek tuz diyeti nedensel mekanizmaları ve yeni tedavi yaklaşımları gelişimi çalışma izin, insan CKD benzeyen bir model yol açar.

Yüksek Tuz Diyet ve Lewis Rat Kronik Böbrek Hastalığı tetikleyebilecek Nitrik Oksit Sentaz inhibisyonu ile bağlantılı 5/6th nefrektomi

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

56th-nephrectomy-combination-with-high-salt-diet-nitric-oxide

Research

JoVE Journal

Medicine

23.4K Görüntüleme.  University Medical Center Utrecht. Cerrahi renal kitle 5/6th kaldırarak Lewis sıçan kronik böbrek hastalığı (KBH) kurmak için bir iki aşamalı yöntemi açıklanmıştır. Cerrahi işlem kombinasyonu, NOS-inhibisyonu ve yüksek tuz diyeti nedensel mekanizmaları ve yeni tedavi yaklaşımları gelişimi çalışma izin, insan CKD benzeyen bir model yol açar.

Video: 5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

Nitrik Oksit Biyoaktivite içlin Analitik Teknikler

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

Tıp

Catheter Ablation in Combination With Left Atrial Appendage Closure for Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and disordered electrical activity in the atria gives rise to palpitations, fatigue, dyspnea, chest pain and dizziness with or without syncope 4, 5. Patients with AF have a five-fold higher risk of stroke 6.
Oral anticoagulation (OAC) with warfarin is commonly used for stroke prevention in patients with AF and has been shown to reduce the risk of stroke by 64% 7. Warfarin therapy has several major disadvantages, however, including bleeding, non-tolerance, interactions with other medications and foods, non-compliance and a narrow therapeutic range 8-11. These issues, together with poor appreciation of the risk-benefit ratio, unawareness of guidelines, or absence of an OAC monitoring outpatient clinic may explain why only 30-60% of patients with AF are prescribed this drug 8.
The problems associated with warfarin, combined with the limited efficacy and/or serious side effects associated with other medications used for AF 12,13, highlight the need for effective non-pharmacological approaches to treatment. One such approach is catheter ablation (CA), a procedure in which a radiofrequency electrical current is applied to regions of the heart to create small ablation lesions that electrically isolate potential AF triggers 4. CA is a well-established treatment for AF symptoms 14, 15, that may also decrease the risk of stroke. Recent data showed a significant decrease in the relative risk of stroke and transient ischemic attack events among patients who underwent ablation compared with those undergoing antiarrhythmic drug therapy 16.
Since the left atrial appendage (LAA) is the source of thrombi in more than 90% of patients with non-valvular atrial fibrillation 17, another approach to stroke prevention is to physically block clots from exiting the LAA. One method for occluding the LAA is via percutaneous placement of the WATCHMAN LAA closure device. The WATCHMAN device resembles a small parachute. It consists of a nitinol frame covered by fabric polyethyl terephthalate that prevents emboli, but not blood, from exiting during the healing process. Fixation anchors around the perimeter secure the device in the LAA (Figure 1). To date, the WATCHMAN is the only implanted percutaneous device for which a randomized clinical trial has been reported. In this study, implantation of the WATCHMAN was found to be at least as effective as warfarin in preventing stroke (all-causes) and death (all-causes) 18. This device received the Conformit&eacute; Europ&eacute;enne (CE) mark for use in the European Union for warfarin eligible patients and in those who have a contraindication to anticoagulation therapy 19.
Given the proven effectiveness of CA to alleviate AF symptoms and the promising data with regard to reduction of thromboembolic events with both CA and WATCHMAN implantation, combining the two procedures is hoped to further reduce the incidence of stroke in high-risk patients while simultaneously relieving symptoms. The combined procedure may eventually enable patients to undergo implantation of the WATCHMAN device without subsequent warfarin treatment, since the CA procedure itself reduces thromboembolic events. This would present an avenue of treatment previously unavailable to patients ineligible for warfarin treatment because of recurrent bleeding 20 or other warfarin-associated problems.
The combined procedure is performed under general anesthesia with biplane fluoroscopy and TEE guidance. Catheter ablation is followed by implantation of the WATCHMAN LAA closure device. Data from a non-randomized trial with 10 patients demonstrates that this procedure can be safely performed in patients with a CHADS2 score of greater than 1 21. Further studies to examine the effectiveness of the combined procedure in reducing symptoms from AF and associated stroke are therefore warranted.

Catheter ablation is combined with placement of the WATCHMAN Left Atrial Appendage Closure Device to prevent ischemic stroke in a patient with non-valvular atrial fibrillation.

Atriyal fibrilasyon için Apendikse Kapatma ile kombinasyonu Kateter Ablasyon

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Polikistik Böbrek Hastalığı Olan İnsanlar ve Fareler kromozom anomalileri Eğitim Spektral Karyotipik

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Sıçan Kronik Besin İnfüzyon Bir Model

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Kronik Böbrek Hastalarında Vasküler Fonksiyon Değerlendirilmesi

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Rat Retina için Elvax 40W ile yavaş salınımlı İlaç Dağıtım: Kronik Koşullar Tedavisinde Etkileri

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

Farelerde Kararlı Yüksek seviyeli Kemik İliği Kimerizm Yaratma için miyelosupresif Ajan olarak Busulfan

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

Sıçan Böbrek için Biologies intravasküler teslim

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Fare modelleri Glomerular hastalığı böbrek fonksiyonlarının değerlendirilmesi

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Nitrik oksit VASP fosforilasyon ölçme tarafından kan trombosit tabanlı algılama

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...