저자는 셈스 - 머피 클리닉, 멤피스, 테네시뿐만 아니라 멤피스 테네시 건강 과학 센터의 대학, 테네시에서 신경 외과 부서에서 MRI 시설에서 직원에게 감사의 말씀을 전합니다.

1. 젤 팬텀의 준비 및 염료 <ol><li> 탈 이온수 1000 ml의 0.1 % 아가로 오스 분말 2g을 용해하여 0.2 % 아가 로스 겔을 제조. 적절한 혼합을 보장하기 위해 약 1 분간 저어; 즉시 간격 사이에 교반, 9 분 동안 3 분 간격으로하거나 해제 할 때까지 솔루션을 구비되어 있습니다. </li><li> 아가로 오스 겔은 액체이지만, 5cm X 5cm X 5cm 용기에 용액을 붓는다. 물을 추가하고 아가 로스 젤 냉각하고 정착 할 수 있도록 용기 상단에 공간을 허용합니다. </li><li> 아가 로스 젤 (약 1 ~ 2 시간)을 응고되면, 젤 및 냉장의 상단에 물을 1cm를 추가합니다. 그것은 혼합 24-48 시간 안에 젤을 사용하는 것이 최선이지만, 일주일에 최대 10 냉장을 위해 저장 될 수있다. </li><li> 0.017 % 브로 모 페놀 블루 염료 (BPB) 50 ㎖ 및 gadoteridol 무선 조영제의 2 ㎜로 구성된 60 ML의 주사기에서 무선 조영제를 준비합니다. <ol><li> 50m에 BPB 염료의 8.5 mg의 결합L은 0.017 % BPB 솔루션을 만들기 위해 물을 탈. </li><li> 2 밀리미터 gadoteridol 솔루션을 만들 수 50 ㎖ 0.017 % BPB 용액에 주식의 0.2 ml의 0.5 M gadoteridol를 추가합니다. </li></ol></li></ol> 주입 시스템 2. 준비 <ol><li> 주사기 펌프 주입 시스템 (기본 방법) : 주사기 펌프의 준비를 위해, 주입 라인의 죽은 볼륨을 줄이고, 압력 센서를 통해 주사기에 직접 주입 카테터를 연결합니다. 주사기 펌프의 퍼지 기능은 10 μL / 분의 속도로 카테터의 프라이밍 볼륨보​​다 큰 볼 루스를 사용하여 공기의 라인을 취소하기 위해 사용될 수있다. </li><li> 튜브 펌프 주입 시스템 (대체 방법) : 주입 펌프에 무선 조영제가 들어있는 주사기를 연결합니다. IV 모니터에 부착 된 트랜스 듀서와 펌프 출구에 압력 센서를 부착합니다. 압력 센서의 오픈 엔드에 16 G 주입 카테터를 연결합니다. 참고 : 16 G 주입 카테터의 끝은 내부 직경이0.2 mm 및 0.35 mm의 외경 측정기. 팁은 용융 실리카로 만든 팁 길이는 3mm입니다. 약 825 mm로 증가하고 15mm 계속, 카테터는 1.6 mm 또는 16 G.에 테이퍼 방식으로 최대 단계 </li><li> 임의의 기포를 제거하기 위해 16.667 μL / 분에서 약 15 분 동안 시스템을 퍼지하여 주입을 위해 준비한다. 기계가 높아 라인 압력으로 주입을 중단하므로, 16.667 μL / 분의 유속을 초과하지 않는다. 주입 펌프, 주입 펌프의 &quot;알약&quot;함수를 이용하여 공기의 퍼지 라인을 출사 선에 주입 카테터를 연결 다음. </li><li> 주입 카테터 마운트 장착하고 젤 팬텀 용기 (5cm X 5cm X 5cm)와 MRI의 장소에 궤도 프레임. </li></ol> 3. CED 젤 주입 및 MR 스캔 <ol><li> 주입을 시작하기 전에 IV 모니터에 의해 기록 된 압력 값 (mmHg로)를 제로. </li><li> 아가 로스 젤 (W)에 주입 카테터를 삽입i 번째 주입 펌프는이 경우 1.667 μL / 분에서, 가능한 최저 유량으로 실행. </li><li> 표 1에 열거 된 파라미터를 이용하여, MR 스캔을 시작하고, 1.667 μL / 분의 속도로 주입 계속. 주입 총 부피가 60 μL (약 38 분)에 도달 할 때까지 일정한 속도로 겔을 주입한다. </li><li> 3 분 50 초 간격으로 계속 젤을 스캔합니다. 압력 측정 값마다 60 초를 기록한다. 주입 부피가 60 μl를 도달하면, 주입 펌프를 끄; 완벽한 MR 스캔 압력 수치를 기록하는 계속하는 동안. </li></ol> 4. MR 데이터 분석 <ol><li> MR 이미지를 분석, ROI 분할 기능을 가진 적절한 DICOM 뷰어를 사용합니다. </li><li> 그림 1에서와 같이 카테터의 단면으로 표시 각각의 검사에서 올바른 프레임을 선택합니다. </li><li> &quot;ROI - 직사각형&quot;툴을 사용하여, 임의의 것을 포함하지 않는 겔의 가장 큰 부분을 선택주입 사이트의 부분입니다. 소프트웨어가 출력 표준 편차와 평균 픽셀 밀도. 평균에서 세 개의 표준 편차에 해당하는 값을 찾을 수 있습니다. 이 값은 콘트라스트가 99.7 %의 신뢰와 함께 존재할 때를 결정하기위한 임계치로서 사용된다. </li><li> &quot;투자 수익 (ROI) - 원&quot;을 사용하여 도구를 충분히 큰 원으로 주입 사이트를 둘러싸는이 고유 한 이름을 제공합니다. </li><li> 원을 선택하고 사용하는 &quot;투자 수익 (ROI) - 설정 픽셀 값에&quot;도구로 단계 4.3에서 발견 된 입력 임계 값 &quot;현재의 값보다 큰 경우 :&quot;상자 만이 줄에 체크 표시. 그런 다음 &quot;이 새로운 값&quot;상자에서 큰 값 (25,000)를 입력합니다. 이전에 정의 된 임계 값에 의해 둘러싸인 영역을 선택하는 픽셀 밀도를 리셋. </li><li> 다음으로, &quot;투자 수익 (ROI) - 지역 성장 (2D/3D 분할)&quot;을 사용하여 도구를 2D 성장 지역, = 2 초기​​ 반경 매개 변수를 신뢰 알고리즘 및 브러시 투자 수익 (ROI)을 선택합니다. 총 면적을 계산하기위한 소프트웨어를 주입 사이트의 내부를 클릭 OF이 지역. </li><li> 구형 주입 클라우드를 가정하기 식을 통해 확산 영역으로부터의 체적을 계산 : V = 4/3π (√ (면적 / π)) 3 </li></ol>

<ol>
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저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

<html> 주입의 성공을 보장하기위한 중요한 단계가있다 : 공기의 주입 라인 정화 아가 로스 겔을 혼합, MR 데이터를 분석, 작은 내부 도관의 직경을 사용하여 역류를 최소화하기 위해 카테터 디자인을 강화하고 느껴지는 압력을 최소화 약물이 주입되는 젤 또는 조직 처. 전술 한 바와 같이, 주입 성공 메인 손해는 주입 라인 공기이다. 정확하고 철저하게 공기의 주입 라인을 정화하는 공기가 주입을 ?...

대류 강화 된 배달 (CED)는 악성 뇌종양, 간질, 신진 대사 장애 (파킨슨 병 등) 신경 퇴행성 질환 1, 뇌졸중, 외상 2 등의 신경 질환의 광범위한 스펙트럼에 대한 치료 옵션으로 제안되었다. CED는 약물이나 다른 infusate의 분포에 대한 긍정적 인 압력 대량 흐름을 고용하고있다. CED는 임상 적으로 볼륨 3에서, 낮은에서 높은에 이르기까지, 분자량 화합물의 안전, 신뢰성, 균일 한 전달을 제공합니다. 뇌 조직에 기존의 약물 전달은 심각한 혈액 - 뇌 장벽 (4)에 의해 제한된다. 뇌 실질 들어가는 혈액 - 뇌 장벽 블록 극성 및 고 분자량 분자, 뇌의 모세 혈관을 이루는 내피 세포 간의 긴밀한 접합함으로써 형성. CED 통해 직접 뇌 실질 내출혈 주입 이전 치료 약물 전달 양식의 한계를 극복 할 수있다및 혈액 - 뇌 장벽을 통과하지 않을 치료제의 사용을 허용하고, 따라서 가능한 치료 옵션 5와 같은 이전에 사용할 수 있었다. 미국 국립 보건 연구소 (NIH)의 연구진은 혼자가 확산 6-8으로보다 큰 치료 약물 농도를 달성하는 수단으로 1990 년대 초에 CED을 설명했다. CED의 첫 번째 방법은, 뇌에 하나 이상의 카테터를 주입 카테터를 주입 펌프를 연결하고, 대상 지역에 직접 치료제 펌핑 참여. 증가 분배 분획 비교적 안정한 농도는 주입 펌프에 의해 생성 된 정압이 조직이 팽창 및 약물 (9)의 투과를 허용하는 원인으로 발생하는 것으로보고된다. CED위한 기본적인 기술은 주로이 제 한 바와 같이 동일하게 유지된다. 카테터 디자인 (10), 주입 기술의 진보 <suP&gt; 11 일, 뇌의 변화 (12, 13)을 보정 여러 선상에 주입 (14)을 최적화하며, infusate 손실 15을 모니터링하는 라인 압력 모니터링 (2) 및 실시간 MRI 모니터링 치료 (10)의 안전성과 효능을 증가하고있다. 추가의 중요성은 유량 등 카테터 디자인 및 주입 전략에 게재되어있다. 성공 CED는 제한 카테터 역류 및 조직 손상, 카테터 설계 및 주입 속도와 상관 관계가있다. 좁은 직경과 카테터 팁 (16)에서 카테터를 뇌 - 인터페이스를 따라 역류 제한 할뿐만 아니라 손상을 제한하기 위해 낮은 주입 속도를 가진 카테터의 사용. 또한 주입 역류 또는 탈선 배달 (17)의 보정을 허용하는 동시에 MR 영상은 시각적 주입 카테터 위치에 대한 정확한 위치를 확인하고, 따라서 약물 전달을 제공합니다. MR 영상은 VD (분포의 볼륨을 대략하고 추적하는 데 사용할 수 있습니다) 주입 된 약물. VD는 세그먼트 (18)에 대한 임계 값으로 둘러싼 비 주입 된 겔로부터 상기 세 평균 표준 편차보다 큰 MR 촬상 신호 강도 값을 이용하여 계산된다. 이 뇌에 분포 약물의 양을 나타 내기 때문에 VD는 CED 대한 유용한 측정이다. (VI) 주입 부피와 함께, 비 약물 주입에 의해 덮여 볼륨을 정량화 (VD / 바이올렛)를 생성 할 수있다. VD, 젤 카테터의 상호 작용, poroelastic 속성 및 주입 구름의 형태 10 : 아가 로스 젤 팬텀과 같은 CED를 이해하기위한 중요한 인간 두뇌의 여러 가지 중요한 기계적 특성을 모방. 0.2 % 아가 로스 젤의 혼합물로 인해 CED에 젤의 팽창에 의해 발생 지역의 기공 부분의 생체 내 변화를 모방하는 것으로 나타났다. 인간의 뇌와 유사한 기공 분율은 유사한 상호 작용과 VD (19)의 정확한 측정을 촉진한다. 의 또한 유사한 농도같은 0.6 %와 0.8 %로 garose 젤은 뇌 (20)와 유사한 주입 압력 프로파일을 보여 주었다. 또한, 반투명 아가 로스 겔 카테터 배치 및 주입 환류의 실시간 시각화의 이점을 제공한다. 아가로 오스 젤 팬텀은 생산 상대적으로 저렴합니다. 아가 로스 젤 팬텀의 비용은 신경 수술 전반에 걸쳐 미래의 광범위한 훈련에 열쇠가 될 수 있습니다. 이러한 속성으로 인해, 아가 로스 겔은 뇌 조직을 사용하지 않고 인간의 두뇌 주입의 주요 특성의 많은 복제, 유용한 서로 게이트를 제공한다. 전술 한 바와 같이, 아가 로스 젤 모델로 CED을 영상 유도 테스트, 연구 및 교육을위한 생체 외 방법에 도움을 제공합니다. 이 문서의 목적은, 아가 로스 젤 팬텀을 다시하는 방법을 설명하는 적절한 CED 테스트 및 분석 프로토콜 개요하고, 신경 질환의 치료를위한 CED 주입하는 동안 직면 일반적인 오류를 해결하는 것입니다.

<table border="0" cellpadding="0" cellspacing="0" width="590">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Prohance</td>
			<td style="width:71px;">Bracco</td>
			<td style="width:119px;"></td>
			<td style="width:195px;">Gadoteridol radio contrast media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Bromophenol Blue Dye</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">161-0404</td>
			<td>Dye for infusate visualization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Agarose Gel Powder</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">161-3101EDU</td>
			<td>Agarose powder for creating gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Medrad Veris MR Vital Signs Monitor</td>
			<td style="width:71px;">Medrad</td>
			<td style="width:119px;"></td>
			<td>MR safe infusion pressure monitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">16 Gauge SmartFlow Catheter</td>
			<td style="width:71px;">SurgiVision</td>
			<td style="width:119px;"></td>
			<td>Infusion catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Medrad Continuum MR Infusion System</td>
			<td style="width:71px;">Medrad</td>
			<td style="width:119px;"></td>
			<td>MR safe infusion pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">SMART Frame MRI Guided Trajectory Frame</td>
			<td style="width:71px;">ClearPoint</td>
			<td style="width:119px;"></td>
			<td>Infusion catheter frame</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:205px;">Osirix Imaging Software and DICOM Viewer</td>
			<td style="width:71px;">Osirix Imaging Software</td>
			<td style="width:119px;"></td>
			<td>OsiriX 32-bit DICOM Viewer</td>
		</tr>
	</tbody>
</table>

image-guided convection-enhanced delivery into agarose gel models of the brain

대류 향상된 우편 (CED)는 신경 질환의 광범위한 치료 옵션으로 제안되었다. Neuroinfusion 카테터 CED는 기존의 약물 전달 방법보다 두개 대상으로 치료제의 큰 수량을 제공하는 긍정적 인 압력 대량 흐름 수 있습니다. 실시간 MRI의 임상 적 유용성이 CED (rCED)를 유도 정확하게 표적 치료를 모니터링하고, 합병증을 식별하는 능력에있다. 교육, rCED는 효율적이고 합병증을 최소화 할 수있다. 뇌의 아가로 오스 겔 모델은 CED 테스트, 연구 및 교육에 대한 접근 도구를 제공합니다. 또한 주입의 시각적 인 피드백을 제공하면서 시뮬레이션 뇌 rCED는 모의 수술 연습을 할 수 있습니다. 주입의 분석은 인간 뇌 조직에 비해 훈련생이 모델의 유사도를 검증 할 수 있도록 분배 분획 (VD / VI)의 계산을 허용한다. 이 문서는 우리 아가 로스 젤 뇌 팬텀을 설명하고 중요한 날을 설명신경 질환의 치료를위한 CED 주입하는 동안 직면 한 일반적인 함정을 해결하면서 CED 주입 및 분석 프로토콜 동안 trics.

해석 및 CED 주입 분석은 분배 비율 및 infusate 역류와 같은 몇 가지 중요한 요소를 포함한다. 유통 부분의 계산은 VD의 계산에 크게 의존한다. 따라서 MR 영상의 정확한 해석은 매우 중요하다. 우리는 위에서 열거 된 신뢰성있는 측정을 재생하기위한 반 - 자동화 된 방법을 제안한다. 이러한 방법은 객관적으로 infusate 구름의 단면적과 대략 반경을 결정합니다. 변수는, 아가 로스 겔에 주입 클라우드는...

Watch this Scientific Journal Video about Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain at JoVE.com

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

대류 향상된 우편 (CED)는 신경 질환의 광범위한 치료 옵션으로 제안되었다. CED의 채택을위한 보건 의료 전문가를 준비하기 위해, 접근 교육 모델이 필요합니다. 우리는 테스트, 연구 및 교육에 대한 인간 두뇌의 같은 모델로 아가 로스 젤의 사용을 설명합니다.

뇌의 아가 로스 젤의 모델로 영상 유도 대류 강화 된 납품

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

image-guided-convection-enhanced-delivery-into-agarose-gel-models

Research

JoVE Journal

Medicine

11.3K 조회수.  University of Tennessee Health Science Center.  대류 향상된 우편 (CED)는 신경 질환의 광범위한 치료 옵션으로 제안되었다. CED의 채택을위한 보건 의료 전문가를 준비하기 위해, 접근 교육 모델이 필요합니다. 우리는 테스트, 연구 및 교육에 대한 인간 두뇌의 같은 모델로 아가 로스 젤의 사용을 설명합니다. 

비디오: Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

연구

의학

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

쥐의 뇌에 직접 약물 전달 이소성 점막 Engrafting 절차

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

마우스 신장 이식 : 동종 이식 거부의 모델

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

신생아 마우스의 뇌실 영역에 플라스미드 DNA의 트랜스포존 중재 통합 아교 ​​모세포종의 소설 모델을 생성합니다

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

생체 외에서 혈액-뇌 장벽 돼지 뇌 내 피 세포를 기반으로 하는 모델의 설립에 대 한 방법 개선

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

세포종과 치료 줄기 세포 시드 장비의 Intracranial 주입의 이미지 기반 절제

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

기계 학습을 사용 하 여 이미지 기반 요법 치료 반응 예측: 간세포 암 종의 트랜스 동맥 치료 예

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

쥐의 승모판 역류에서 제어된 볼륨 과부하의 이미지 유도 횡격모동 밸브 전단지 펑크 모델

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

쥐에서 건조 분말 제형의 장내 투여

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...