우리는 그녀의 우수한 기술 지원을 크리스타 서재 Ouden 감사합니다. 이 기술은 재정적 네덜란드의 신장 재단 보조금 C06.2174에 의해 지원되었다. MCV는 과학 연구에 대한 네덜란드기구 (NWO) 비디 - 부여 016.096.359에 의해 지원됩니다.

모든 실험은 위트레흐트 실험 동물위원회의 동물 실험 윤리 가이드 라인에 따라 실행됩니다. 프로토콜은 저자의 기관의 동물 관리 및 사용위원회의지도 및 승인하에 수행됩니다. CKD ~ 8 주 세의 나이에 남성 근친 루이스 쥐 (찰스 리버, Sulzfeld, 독일)에서 유도된다. 쥐 빛, 온도 및 습도 제어 환경의 표준 조건 하에서 보관되어 있습니다. 1. 수술 준비 <ol><li> 수술기구를 소독 : <ul><li> 1 학생 조직은 1-2 치아 12cm를 포셉 </li><li> 1 학생의 표준 패턴 집게 </li><li> 2 Semken 포셉 </li><li> 1 마요 가위 </li><li> 1 명 홍채 가위 </li><li> 가위로 1 올슨-Hegar 바늘 홀더 </li><li> 담요 </li></ul></li><li> 재고 목록을 확인 : <ul><li> 온난화 패드와 램프 테이블 운영 </li><li> O <suB&gt; 2, isoflurane을 </li><li> 조직 </li><li> 면도기 </li><li> 저울 </li><li> 무균 조작 세트 </li><li> 멸균 거즈 5X5 및 10 × 10 </li><li> 70 % 알코올 </li><li> 0.9 % 염화나트륨 용액 </li><li> 1 ML의 주사기 </li><li> 바늘 (25G) </li><li> 젤 폼 패드 : spongostan </li><li> Vicryl 4.0 및 5.0 </li><li> 부 프레 노르 핀 0.03 ㎎ / ㎏ (1:10 생리 소금에 희석) </li><li> 수술 후 쥐 깨끗한 케이지 </li></ul></li></ol> 2. 오른쪽 Uninephrectomy <ol><li> 70 % 알코올로 테이블을 소독. </li><li> 장소는 유도 상자에 쥐 4 % isoflurane을하고 O 2 1 리터의 흐름으로 마취를 유도. 주 : 사용 된 쥐의 변형에 따라 사전 및 수술 진통제를 처방, 지역 수의사와상의이 필요. </li><li> 원뿔 테이블과 장소 코 쥐를 전송합니다. 쥐와 코 콘은 쥐의 체온을 유지하기 위해 가열 패드에 배치됩니다. 쥐 I들 O 2 isoflurane을하고 1리터 2 %의 혼합물로 환기. </li><li> 쥐의 측면을 면도하고 알코올로 소독. 세척 및 소독 손을 멸균 수술 장갑을 착용하십시오. 머리 모자와 마스크를 착용하십시오. 만들기 1 - 1.5 cm의 절개 병렬 해부 집게와 무딘 가위를 사용하여 갈비뼈합니다. 등 근육의 무딘 절개하여 신장을 노출합니다. </li><li> 신장의 하부 극 볼 수 있습니다,주의 신 주위 지방 조직을 그립에있는 작은 무딘 집게를 사용합니다. 부드럽게 집게로 신 주위 지방을 당겨 신장을 구체화. 이 절차를 수행하는 동안 부신을 방해하지 않도록주의하십시오. 부신 쉽게 지방 조직에서 중간 사이트에 집게를 배치하여 제거하고 부드럽게 부신과 그림 2로 신장 사이의 위쪽으로 이동할 수 있습니다. </li><li> 가제 지방과 결합 조직을 둘러싼 명확한 부드럽게 신장을 놓습니다. 신장 동맥과 정맥을 확인하고 합자 (5.0 배치vicryl) 혈관 주위에 하나의 매듭하지만, 매듭을 묶을하지 않습니다. </li><li> 신장과 매듭 사이의 공간을 만들기 위해, 약 0.5 cm가 대동맥쪽으로 혈관을 따라 조심스럽게 느슨한 매듭을 이동합니다. 이 절단 후에 떨어져 미끄러지는 합자을 방지하기 위해 수행됩니다. </li><li> 두 배 매듭으로 매듭을 묶어. 혈류가 중단되는 경우, 신장의 색은 바로 갈색으로 변경됩니다. 합자의 끝을 절단하지 마십시오. </li><li> 신장에 가까운 신장 혈관을 차단하고 신장을 제거합니다. 부드럽게 신장 혈관의 출혈을 확인하는 합자의 긴 끝냅니다. 끈은 선박에 그 자리에 체재한다. 합자의 긴 끝을 잘라. 참고 남은 신장 혈관 즉시 복강에 철회 할 것이다. </li><li> 얼룩 절제 신장 건조하고 무게. </li><li> Vicryl 5.0에 봉합을 실행하는 골격 근육 절개를 닫습니다. 뒷다리에 0.10 ML (0.03 ㎎ / ㎏) 부 프레 노르 핀 메신저를 주입. 를 닫습니다피내 봉합 Vicryl 4.0 피부. 이 상처를 열지 쥐를 방지 할 수 있습니다. 참고 : 루이스 쥐에서 호흡 저하로 인해, 우리는 단지 isoflurane을을 전환하기 전에 수술 후 진통을 제공합니다. </li><li> 음식과 물에 쉽게 접근 할 수있는 깨끗한 독방 케이지의 쥐를 집입니다. 온난화 패드 O / N에 케이지 절반을 놓고 조심스럽게 쥐 다음날 확인합니다. 상처가 닫혀 있고 쥐가 수술 후 6 ~ 12 시간 사이에 일반적입니다, 활성화 될 때, 쥐가 그룹 하우징에 다시 배치 할 수 있습니다. </li></ol> 3. 왼쪽 소계 절제 <ol><li> 7 일 후에 같은 준비가로 2.1에서 2.5에 설명 된 오른쪽 측면으로 왼쪽에 만들어집니다. 수술 전, 오른쪽 신장의 무게의 약 2/3rds에 해당 제거해야 신장 조직의 양이 계산됩니다. 수술 전 젤 거품의 작은 조각 (Spongostan, 존슨 앤 존슨, 뉴저지) 준비합니다. </lI&gt; <li> 왼쪽 신장의 상단 기둥 주위에 날카로운 가위를 배치 한 뇌졸중 (가위 위치를 그림 3 참조) 상부 기둥을 잘라 내다. 젤 거품의 조각으로 즉시 뚜껑을 덮고 멸균 거즈와 가벼운 압력을 발휘. </li><li> 3.3에서와 같이 신장의 하부 극 반복합니다. </li><li> 피부에 혈액 응고 방지하기 위해 신장을 올립니다. 출혈이 멈출 때까지 멸균 거즈로 젤 거품 패드 모두에서 가벼운 압력을 유지합니다. 출혈이 지속되면 다른 폼 패드를 추가합니다. </li><li> 복부 안쪽에 부착 젤 거품 패드로 남은 신장을 놓습니다. 근육과 피부를 닫고 2.9-2.12를 따릅니다. </li></ol> 4. 가짜 수술 <ol><li> 가짜 작동 컨트롤은 신장을 노출하기 위해 동일한 절차를 겪는다. 대신 신장 extirpating 또는 일주일 후 채를 절단, 신장 모두 부신 분비를 방해하지 돌보는, 일주일 간격으로 캡슐화가 있습니다. 상처 봉합 수술 후 관리는 ID이다2.11와 2.12에 entical. </li></ol>

<ol>
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저자는 공개 아무것도 없어.

높은 소금 다이어트 및 임시 NOS 억제와 결합 루이스 쥐에서 신장 질량의 5/6th의 외과 제거는 인간의 CKD 유사한 CKD의 모델에 이르게 및 CKD의 치료 개입의 인과 관계 메커니즘과 효능을 연구 할 수 있습니다. 5/6th의 절제 모델은 CKD에 대한 잘 알려진 광범위하게 설명하는 모델입니다. 그러나 단순히 신장 질량의 5/6th을 제거하면 모든 쥐의 변종 즉시 신부전으로 이어질하지 않습?...

그 진보적 성격, 계속되는 말기 신장 질환과 관련된 심혈 관계 이환율과 사망률로 인해 CKD는 성장 공중 보건 문제 1. CKD의 진행을 둔화 따라서 중요한 건강 우선 순위입니다. CKD는 항상성의 복잡한 derangements 특징 때문에, 통합 동물 모델은 CKD의 개발 및 진행을 연구 할 필요가있다. 신장은 서로 상호 작용하는 다른 세포 유형의 다양한으로 구성되어 있습니다. 이러한 복잡성은 체외에서 모방 할 수 없습니다. CKD에 새로운 치료 개입을 연구하기 위해, 우리는 인간의 CKD 2,3의 여러 측면을 닮은 5/6th 절제 절제 모델, 진보적 인 신장 질환의 잘 알려진 실험 모델을 사용합니다. 신장 질량 총 감소는 진보적 인 사구체와 tubulo-간질 부상, 남은 네프론의 손실과 조직 및 사구체 고혈압의 개발을 발생합니다. 이 progr을과 연관되어essive intrarenal 모세관 손실 4, 염증과 사구체. 위험 요인은 결국 내피 세포 기능 5에 미치는 영향을 CKD. 우리는 CKD의 개발에 상대적으로 저항하는 쥐의 변형 (루이스)를 사용하기 때문에 우리는 산화 질소 (NO) 고갈 6, 7, 8, 높은 소금 다이어트 9에서 신장 질량의 5/6th의 제거를 결합했다. 도착 적응 한 후, 동물 이틀 후 L-NNA의 지속과 오른쪽면 uninephrectomy (UNX) 다음에 사주의 기간 동안 물 (20 ㎎ / L)을 첨가 NO 합성 효소 억제제 (L-NNA), 수신하지 . 일주일 후, 신장 질량의 2/3rds의 소계 절제 (SNX) 즉, 제거는 왼쪽에 수행됩니다. SNX 후, 동물을 4 주 동안 마시는 물에 20 ㎎ / L LNNA에 이어 다시 이틀 복구 할 수 있습니다. 지상 차우 (시간 선 그림 1 참조) 보충 높은 소금 다이어트 (6 %)을, 실험을하는 동안 계속됩니다. 티그는 오른쪽에 UNX을 수행 할 수있는 이성과 왼쪽에 SNX은 신장 혈관이 쉽게 신장이 신체 외부에 노출되었을 때 너무 많은 혈관을 스트레칭하지 않고 신장에 액세스 할 수 있습니다 왼쪽에 더 이상 것입니다 . 문학, 모델은 일주일 후 10,11,12 오른쪽 신장 UNX 다음, 왼쪽 신장의 극을 먼저 제거되는 설명되어 있습니다. 우리의 손이 모델은 신장 장애의 더 빠른 발전을 보였다, 또한 신장 기능의 손실에 훨씬 더 큰 변화. 신부전의 진행은 혈중 요소, 수축기 혈압과 단백뇨를 측정하여 시간이 지남에 따라한다. SNX 후 6 주까지, 신부전, 개발 사구체 여과 속도의 현저한 감소 (69 %)하고 효과적인 신장 플라즈마 흐름 (62 %) 13 고혈압 (수축기 혈압&gt; 150 mmHg로), 단백뇨 (특징&gt; 50 mg/24 HR) 및 가벼운 요독증 (&gt; 10 MM). 조직 학적 특징은 tubul 포함오 - 간질 손상, 염증, 관 위축 및 섬유화와 나머지 인구 (&lt;10 %)에서 건강 사구체의 대규모 감소로 이어지는 초점 사구체 경화증으로 반영됩니다. 후속 최대 12 주 후까지 SNX는 치료 개입의 평가를위한 기회의 창을 제공 CKD의 추가 진행을 보여줍니다.

<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

만성 신장 질환 (CKD)는 세계적인 문제입니다. CKD의 진행을 느리게하는 주요 건강 우선 순위입니다. CKD는 항상성의 복잡한 derangements 특징 때문에, 통합 동물 모델은 CKD의 개발 및 진행을 연구 할 필요가있다. CKD의 CKD 및 새로운 치료 개입의 개발을 연구하기 위해, 우리는 인간의 CKD의 여러 측면을 닮은 5/6th 절제 절제 모델, 진보적 인 신장 질환의 잘 알려진 실험 모델을 사용합니다. 신장 질량 총 감소는 진보적 인 사구체와 tubulo-간질 부상, 남은 네프론의 손실과 조직 및 사구체 고혈압의 개발을 발생합니다. 또한 진보적 인 intrarenal 모세 혈관 감소, 염증과 사구체 경화증과 연관되어 있습니다. 위험 요인은 결국 내피 세포 기능에 미치는 영향을 CKD. 이 모방하기 위해, 우리는 산화 질소 (NO) 고갈과 높은 소금 다이어트와 신장 질량의 5/6th의 제거를 결합합니다. 도착 적응 한 동물은 다시오른쪽면 uninephrectomy 다음에 사주의 기간 동안 물 (20 ㎎ / L)을 첨가 NO 합성 효소 억제제 (NG-니트로-L-아르기닌) (L-NNA)를 가압하지 않습니다. 일주일 후, 소계 절제 (SNX)이 왼쪽에서 수행됩니다. SNX 후, 동물을 4 주 더 동안 물 (20 ㎎ / L)에 LNNA 다음 이틀 동안 복구 할 수 있습니다. 지상 차우 (시간 선 그림 1 참조) 보충 높은 소금 다이어트 (6 %)을, 실험을하는 동안 계속됩니다. 신부전의 진행은 혈중 요소, 수축기 혈압과 단백뇨를 측정하여 시간이 지남에 따라한다. SNX 후 6 주까지, 신부전을 개발하고있다. 신장 기능이 &#39;황금 표준&#39;이눌린 및 파라 - 아미노 히 푸르 산 (PAH) 정리 기술을 사용하여 측정됩니다. CKD의이 모델은 사구체 여과율 (GFR)의 감소와 효과적인 신장 플라즈마 흐름 (ERPF), 고혈압 (수축기 혈압&gt; 150 mmHg로), 단백뇨 (&gt; 50 mg/24 시간이 특징입니다)와 가벼운 요독증 (&gt; 10 MM). 조직 학적 특징은 염증, 관 위축 및 섬유화와 나머지 인구 (&lt;10 %)에서 건강 사구체의 대규모 감소로 이어지는 초점 사구체에 반영 tubulo-간질 손상이 있습니다. 후속 SNX는 CKD의 추가 진행을 보여줍니다 12주 후까지.

부분합을 절제 한 후, 총 신장 질량의 약 1/6th이 남아 있습니다. 그림 4는 이전의 두 실험에서 평균과 ​​표준 편차의 오른쪽 신장 제거 된 부품의 무게를 보여줍니다. 하나는 UNX 후 주에, 왼쪽 신장의 비대가 발생하는 것을 염두에 두어야한다 나타내는 것을 5/6th 제거 미만 오른쪽 신장 항상 결과의 무게에 근거하여 산출 제거해야합니다 무게. 이 수술 중 좌측 신장의 무게를 결정하는...

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

수술 신장 질량의 5/6th를 제거하여 루이스 쥐에서 만성 신장 질환 (CKD)을 설정하는 두 단계 방법을 설명합니다. 수술의 조합, NOS-억제와 높은 소금 다이어트 인과 관계 메커니즘과 새로운 치료 개입의 개발 연구를 허용, 인간의 CKD를 닮은 모델에 연결됩니다.

높은 소금 다이어트와 루이스 쥐에서 만성 신장 질환을 유발하는 산화 질소 합성 효소 억제와 함께 5/6th의 절제

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 조회수.  University Medical Center Utrecht. 수술 신장 질량의 5/6th를 제거하여 루이스 쥐에서 만성 신장 질환 (CKD)을 설정하는 두 단계 방법을 설명합니다. 수술의 조합, NOS-억제와 높은 소금 다이어트 인과 관계 메커니즘과 새로운 치료 개입의 개발 연구를 허용, 인간의 CKD를 닮은 모델에 연결됩니다.

비디오: 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.

질소 산화물 Bioactivity을 시금을위한 분석 기법

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 ...

연구

의학

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.

심방 세동에 대한 왼쪽 심방 부위 요 폐쇄와 함께 카테 테르 박리

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.

Polycystic 신장 질환과 함께 인간과 마우스의 염색체 이상을 공부하는 스펙트럼 Karyotyping

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.

쥐의 만성 영양 주입의 모델

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.

만성 신장 질환 환자에서 혈관 기능의 평가

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.

쥐의 망막에 Elvax 40W를 통해 서방 약물 전달 : 만성 질환의 치료를위한 시사점

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.

마우스에서의 높은 수준의 안정적인 골수 키메라를 생성하기위한 에이전트로서 골수 억제 술판

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.

쥐의 신장에 생물의 혈관 배달

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.

신장 기능 사 질병 마우스 모델에서의 평가

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.

VASP 인 산화를 측정 하 여 혈액에 산화 질소의 혈소판 기반 탐지

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