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><tbody><tr><td>Prohance</td><td>Bracco</td><td></td><td>Gadoteridol radio contrast media</td></tr><tr><td>Bromophenol blue dye</td><td>Biorad</td><td>161-0404</td><td>Dye for infusate visualization</td></tr><tr><td>Agarose gel powder</td><td>Biorad</td><td>161-3101EDU</td><td>Agarose powder for creating gels</td></tr><tr><td>Medrad Veris MR Vital Signs Monitor</td><td>Medrad</td><td></td><td>MR safe infusion pressure monitor</td></tr><tr><td>16 G SmartFlow Catheter</td><td>SurgiVision</td><td></td><td>Infusion catheter</td></tr><tr><td>Medrad Continuum MR Infusion System</td><td>Medrad</td><td></td><td>MR safe infusion pump</td></tr><tr><td>SMART Frame MRI Guided trajectory frame</td><td>ClearPoint</td><td></td><td>Infusion catheter frame</td></tr><tr><td>Osirix imaging software and DICOM Viewer</td><td>Osirix Imaging Software</td><td></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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

11.4K 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

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

세미 두께 뇌 조각의 고해상도 형광 이미징에 대한 빠른 접근

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

연구

신경과학

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

두개 내 주입 및 자기 공명 이미징을 통해 뇌 전이를 모델링

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

암 연구학

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

This work describes the development of flexible interdigitated electrodes for implementation in 3D brain tumor models, namely, in vitro culture, in ovo model, and in vivo murine model. The proposed method can be used to evaluate the effects of pulsed electric fields on tumors at different levels of complexity.

교모세포종의 펄스 전기장 치료를 위한 유연한 유기 전자 장치

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed ...

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 ...

의학

A MRI-Based Toolbox for Neurosurgical Planning in Nonhuman Primates

In this paper, we outline a method for surgical preparation that allows for the practical planning of a variety of neurosurgeries in NHPs solely using data extracted from magnetic resonance imaging (MRI). This protocol allows for the generation of 3D printed anatomically accurate physical models of the brain and skull, as well as an agarose gel model of the brain modeling some of the mechanical properties of the brain. These models can be extracted from MRI using brain extraction software for the model of the brain, and custom code for the model of the skull. The preparation protocol takes advantage of state-of-the-art 3D printing technology to make interfacing brains, skulls, and molds for gel brain models. The skull and brain models can be used to visualize brain tissue inside the skull with the addition of a craniotomy in the custom code, allowing for better preparation for surgeries directly involving the brain. The applications of these methods are designed for surgeries involved in neurological stimulation and recording as well as injection, but the versatility of the system allows for future expansion of the protocol, extraction techniques, and models to a wider scope of surgeries.

The method outlined below aims to provide a comprehensive protocol for the preparation of nonhuman primate (NHP) neurosurgery using a novel combination of three-dimensional (3D) printing methods and MRI data extraction.

비인간 영장류의 신경 외과 계획을위한 MRI 기반 도구 상자

In this paper, we outline a method for surgical preparation that allows for the practical planning of a variety of neurosurgeries in NHPs solely using ...

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High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

혈액-뇌 장벽의 고해상도 Confocal 영상: 영상, 3 차원 재구성 및 Transcytosis의 정량화

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

전 Utero Electroporation 및 마이그레이션 GABAergic 수의 라이브 이미징 배아 마우스 두뇌의 Organotypic 조각 문화

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

종양 내 이질성, 온코스트림 및 침략의 공간 및 분자 특성화를 위한 신경교종 소구역의 레이저 캡처 미세 분해

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

시투에서 Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures 시각화

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

중시경에서 현미경 비늘에 이르기까지 신경 세포 이미징을위한 조직 제거 방법

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

뇌의 아가 로스 젤의 모델로 영상 유도 대류 강화 된 납품

요약

더 많은 비디오 탐색

이 비디오의 챕터

세미 두께 뇌 조각의 고해상도 형광 이미징에 대한 빠른 접근

두개 내 주입 및 자기 공명 이미징을 통해 뇌 전이를 모델링

교모세포종의 펄스 전기장 치료를 위한 유연한 유기 전자 장치

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

비인간 영장류의 신경 외과 계획을위한 MRI 기반 도구 상자

혈액-뇌 장벽의 고해상도 Confocal 영상: 영상, 3 차원 재구성 및 Transcytosis의 정량화

전 Utero Electroporation 및 마이그레이션 GABAergic 수의 라이브 이미징 배아 마우스 두뇌의 Organotypic 조각 문화

종양 내 이질성, 온코스트림 및 침략의 공간 및 분자 특성화를 위한 신경교종 소구역의 레이저 캡처 미세 분해

시투에서 Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures 시각화

중시경에서 현미경 비늘에 이르기까지 신경 세포 이미징을위한 조직 제거 방법

뇌의 아가 로스 젤의 모델로 영상 유도 대류 강화 된 납품

요약

더 많은 비디오 탐색

이 비디오의 챕터

세미 두께 뇌 조각의 고해상도 형광 이미징에 대한 빠른 접근

두개 내 주입 및 자기 공명 이미징을 통해 뇌 전이를 모델링

교모세포종의 펄스 전기장 치료를 위한 유연한 유기 전자 장치

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

비인간 영장류의 신경 외과 계획을위한 MRI 기반 도구 상자

혈액-뇌 장벽의 고해상도 Confocal 영상: 영상, 3 차원 재구성 및 Transcytosis의 정량화

전 Utero Electroporation 및 마이그레이션 GABAergic 수의 라이브 이미징 배아 마우스 두뇌의 Organotypic 조각 문화

종양 내 이질성, 온코스트림 및 침략의 공간 및 분자 특성화를 위한 신경교종 소구역의 레이저 캡처 미세 분해

시투에서 Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures 시각화

중시경에서 현미경 비늘에 이르기까지 신경 세포 이미징을위한 조직 제거 방법

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