이 작품은 한국국립연구재단(NRF)과 한림대학교 연구기금(NRF)의 연구보조금(NRF-2018-R1D1A1B07050175, HURF-2017-66)에 의해 지원되었다.

ICR 마우스와 관련된 모든 연구 프로토콜은 원주의 의과대학의 연세대학교 기관동물관리및이용위원회(IACUC)의 승인을 받았습니다. 실험은 세계 의학 협회의 윤리 강령에 따라 수행되었다. 이 프로토콜에서 임신 한 ICR 마우스는 음식과 물에 무료로 액세스 할 수있는 12/12 h 빛 / 어두운 주기에 보관되었습니다.
1. 달팽이관 해부
<ol><li>30분 동안 자외선을 켜서 라미나르 유동 조직 배양 후드를 살균하고, 사용하기 전에 70% 에탄올로 모든 표면을 분무한다. 표면이 건조하도록 합니다.</li>
	<li>해부 악기를 70% 에탄올에 10분 동안 놓고 사용하기 전에 건조하십시오.</li>
	<li>수술 블레이드를 사용하여 산후 3-4 일 된 마우스(그림 2A)를참수하십시오.</li>
	<li>라미나르 플로우 후드의 스테레오현미경 아래에 두개골을 놓고 70% 에탄올에 티슈를 담그세요.</li>
	<li>조직을 빠르게 흡수하여 조직 해부 용액(1x 행크의 균형 잡힌 소금 용액, 1mM HEPES)을 사용하여 에탄올을 제거합니다.</li>
	<li>수술 블레이드(그림 2B,C)로두개골의 중심선을 잘라.</li>
	<li>피부를 앞쪽으로 당기고 귀의 외부 청각 운하를 절단하여 두개골을 노출시다(도2D).</li>
	<li>전방에서 눈선을 가로질러 두개골의 후방 부분으로 잘라냅니다(그림2E).</li>
	<li>두개골을 열고 무딘 집게로 전뇌, 소뇌 및 뇌간을 제거합니다(그림 2F,G).</li>
	<li>마이크로 포셉을 사용하여 달팽이를 측두골(도2H)에서분리한다.</li>
	<li>달팽이관을 조직 해부 용액을 함유한 페트리 접시에 옮기다.</li>
	<li>모든 달팽이관 오틱 캡슐을 신중하게 해부하여 내부 달팽이관 연조직(도2I,J)만남깁니다.</li>
	<li>다른 한 쌍의 집게로 달팽이관과 달팽이관의 골수를 잡고 두 조직(도 2K)을천천히 분리합니다.</li>
	<li>그들을 부드럽게 벗겨내어 황반 혈관 및 지각 막을 제거하십시오(그림2L,M).</li>
	<li>살균 플라스틱 커버슬립을 새로운 조직 해부 용액에 넣고 코르티 의 장기를 직경 9mm의 커버슬립에 놓고 바실라 멤브레인이 아래쪽으로 향하게 합니다(그림2N-P).</li>
	<li>리스너의 막과 나머지 모골 조직을 집게로 커버슬립에 눌러 조직을 고정시킵니다(도2N-P).</li>
	<li>임베디드 조직으로 커버슬립을 직경 35mm의 공초점 접시의 중심으로 옮기.</li>
	<li>유리 클론 실린더를 접시중앙에 달팽이관이 배치하고, 100μL의 축축배 배지(DMEM/F12, 10% 태아소 세럼(FBS), 1% N2 보충제, 암피실린(10 μg/mL))을 접시에 놓는다.</li>
	<li>플레이트 5 × 103 개의 마우스 골수 유래 녹색 형광 단백질 (GFP)-태그 된 MSC 배양 매체 2 mL (45% DMEM + 45% DMEM/F12, 10% FBS, 1% N2 보충제, 10 μg/mL 의 암피실린) 외부 유리 실린더(그림2R).<ol>
<li>MSC가 80-90 % 컨캔트 일 때 트립신 에틸렌디아민 테트라아세트산으로 분리하여 통과시하십시오.</li>
	</ol>
</li>
	<li>공초점 접시를 가습된 인큐베이터로 조심스럽게 옮기고 5%CO2 분위기에서 37°C에서 하룻밤 동안 배양한다.</li>
	<li>실린더 내부와 바깥쪽에 있는 모든 매체를 흡인한 다음 공초점 접시에서 유리 실린더를 제거합니다.</li>
	<li>공초점 접시에 신선한 배양 배지 2mL를 추가하고 분석 준비가 될 때까지 가습 된 인큐베이터에 조직 문화 접시를 배양하십시오.</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61947/61947fig2v2.jpg"> 그림 2. 코르티와 MSC의 기관의 마우스 달팽이관 및 공동 문화의 해부. (A)마우스의 참수,(B)및(C)머리의 중간선 처질 해부,(D)및(E)뇌의 관상 해부,(F)및(G)뇌및 측두골의 제거,(H)달팽이벽의 제거,(J)달팽이관벽의 제거, (J) 달팽이관의 분리,(J)달팽이,(J)달팽이의 분리(L)코르티의 오르간으로부터 의 트리아 혈관(SV) 및 나선형 인대(SL)의 분리,(M)지각막 제거,(N-P)플라스틱 커버 슬립에 달팽이관 고정,(Q)공초점 요리에서 커버슬립 및 유리 실린더의 위치, (R) 공초점 요리에서,(R)현미경의 백색 배율 주황색(F, G, P)및 노란색 스케일 바(H,I)= 1mm; 녹색 스케일 바(J-O) = 0.5mm. <a href="https://www.jove.com/files/ftp_upload/61947/61947fig2v2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
2. 타임랩스 이미징
<ol><li>여기에 제시 된 실험의 경우 스테이지 탑 인큐베이터 시스템을 갖춘 공초점 현미경 시스템을 사용하십시오.</li>
	<li>공초점 현미경, 형광등 및 컴퓨터를 켭니다.</li>
	<li>공초점 현미경의 단계에 배치 된 스테이지 탑 인큐베이터의 조건을 37 °C 및 5 % CO2 대기로 설정합니다.</li>
	<li>접시 고정 용기에 샘플 접시를 놓고 접시 고정 뚜껑으로 덮고 상단 히터 뚜껑으로 챔버를 닫습니다.</li>
	<li>줌 및 포커스를 조정하여 시야에서 Corti 및 MSC의 장기를 현지화합니다.</li>
	<li>이미지 처리 소프트웨어를 엽니다. 찾기 옵션에서 20x 플랜-아포크로마트 목표(숫자 조리개 0.8)와 0.5배 자르기 영역을선택합니다.</li>
	<li>인수에서 스마트 설정을 클릭하고 EGFP를 선택합니다.</li>
	<li>획득하에 채널 탭을 열고 레이저 전원을 0.2%로 설정하고 핀홀을 44 μm로설정하고 마스터 게인을 750V로설정하고 디지털 게인을 1.0으로설정합니다.</li>
	<li>이미징 설정에서 ESID를 클릭하고 ESID 게인을 4로 설정하고 디지털 게인을 7.5로설정합니다.</li>
	<li>타일과 스테이크를 클릭하여 210개의 타일을 생성합니다.</li>
	<li>포커스 전략을 열고 포커스 모드를 선택합니다.</li>
	<li>기간 중, 지속 시간을 24시간으로 설정하고 간격을 10분으로 설정합니다.</li>
	<li>획득하에프레임 크기를 512 x 512픽셀로 설정하고, 속도를 8로, 양방향 방향, 평균을 4배로, 픽셀당 비트는 16으로설정한다.</li>
	<li>실험을 시작하려면 실험을 시작합니다.</li>
</ol>3. 이미지 파일 수정
<ol><li>처리 중, 스티치를클릭하고, 5 %로 최소 오버레이를 설정하고 최대 이동을 10 %로 설정합니다.</li>
	<li>영화 내보내기를클릭하고 압축되지 않은설정하고 속도를 7.5로설정합니다.</li>
</ol>4. 면역 염색
<ol><li>배지를 주의 깊게 흡입하고 인산완충식염(PBS)으로 샘플을 5분 간 세척합니다.</li>
	<li>PBS에서 4% 포르말린으로 샘플을 15분 동안 수정하고 PBS로 샘플을 3회 5분 동안 세척합니다.</li>
	<li>PBS에서 0.1% 트리톤 X-100으로 샘플을 10분 동안 세척하고 PBS로 3회 5분 간 세척합니다.</li>
	<li>250 μL의 phalloidin-iFluor 647 시약 (PBS에서 1:1000 희석)을 추가하고 셰이커의 실온에서 1 시간 동안 샘플을 배양합니다.</li>
	<li>PBS로 샘플을 3회 3회 세척하여 5분 동안 세척합니다.</li>
	<li>커버슬립을 유리 슬라이드에 옮기고 2방울의 장착 용액을 추가합니다.</li>
	<li>슬라이드에 커버슬립을 부드럽게 놓습니다.</li>
	<li>명확한 매니큐어로 커버 슬립을 밀봉하고 세포가 관찰 될 때까지 어둠 속에서 4 °C에 저장합니다.</li>
	<li>내분/방출(Ex/Em)=650/665 nm에서 적절한 필터를 사용하여 공초점 현미경을 사용하고, EGFP의 경우 Ex/Em=488/507 nm에서 슬라이드를 이미지합니다.</li>
</ol>

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저자는 공개 할 것이 없습니다.

손상된 세포의 재생을 촉진하기 위해 손상된 부위로 MSC를 이식하는 것은 광범위하게 연구되었으며 치료 효과가 분명합니다. MSC의 이식 및 후속 분화는 3-니트로프로피온산(13)에 의해 유도된난청을 가진 쥐의 청력을 회복하는 것으로 보고되었다. Lee et al.은 인간에게 MSC를 적용했지만,14번 청각에서 상당한 개선을 이루지 못했습니다. 최근까지, MSC 이식에 의한 ?...

청력 손실은 선천적으로 발생할 수 있으며 노화, 약물 및 소음을 포함한 여러 가지 요인에 의해 점진적으로 발생할 수 있습니다. 청력을 담당하는 모발 세포가 손상되면 손상된 기능을 복원하기가 매우 어렵기 때문에 청력 손실은 종종 치료하기가어렵습니다. 세계보건기구(WHO)에 따르면 전 세계 4억 6,100만 명이 청력 손실을 입은 것으로 추정되며, 이는 전 세계 인구의 6.1%를 차지합니다. 청력 상실을 가진 사람들 중 93%는 성인이고 7%는 어린이입니다.
청력 손실을 치료하기 위해 여러 가지 접근 법이 시도되었습니다. 특히, MSC를 이용한 재생 접근법은 유망한 치료법으로 부상했습니다. 조직이 손상되면, MSC는 자연적으로 순환 계통으로 방출되고 다양한 분자를 분비하는 상해 부위로 이동하여 재생을 촉진하는 미세 환경을 형성한다2. 따라서, 손상된 세포 기능3, 4, 5의 복원을 향상시키기 위해 강력한 면역 조절, 혈관신생 및 항 세포멸을 유발하는 분자의 장기 및 후속 분비를 표적으로 하는 외부 이식된 MSC의 이동을 통해 손상된 조직을 치료하는 방법을 개발하는 것이 중요하다3,4,5.
MSC가 손상된 조직으로 마이그레이션하는 호밍 프로세스가 극복해야 할 가장 중요한 장애물이 될 수 있습니다. MSC는 테더링 /롤링, 활성화, 체포, 트랜스 마이그레이션 / diapedesis 및 마이그레이션6,7,8의순차적 단계를 갖는 전신 호밍메커니즘을가지고 있습니다. 현재 이러한 단계를 개선할 방법을 파악하기 위한 노력이 진행 중입니다. 유전자 변형, 세포 표면 공학, 체외 프라이밍 및 자기 지도를 포함한 다양한 전략이6,7로테스트되었습니다. 또한 손상된 달팽이관 부위에 MSC를 호밍하여 청각 모발 세포의 보호 및 재생을 촉진하기 위한 여러 시도가 있었습니다. 그러나 생체 내에서 MSC를 추적하는 것은 시간이 많이 걸리고 노동 집약적이며 고도로 전문화된 기술9이필요합니다.
이러한 문제점을 해결하기 위해, 몇 시간 동안 세포의 이주를 촬영한 시간 경과 공초점 현미경검사를 통해 달팽이관내의 MSC의 호밍을 관찰하는 방법이 개발되었다(도1). 그것은 20세기 초에 개발 되었으며 최근 특정 세포의 마이그레이션을 공부 하기 위한 강력한 도구가 되고있다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;"> 도 1: 그래픽 추상화. (A)코르티의 해부 기관이 집게를 사용하여 플라스틱 커버슬립에 부착된 후, 커버슬립은 35mm 유리 바닥의 공초점 미세한 접시에 배치되고(B)유리 실린더가 배치된다. (C)유리 실린더 내부를 중간으로 채운 후,(D)GFP 라벨이 부착된 MSC를 매체로 기르기 바깥에 신중하게 첨가한다. (E)하룻밤 동안 인큐베이션 후유리실린더가 제거되고, 이미지는 공초점 현미경으로 촬영된다. 약어: GFP = 녹색 형광 단백질; MSC = 중간 엽 줄기 세포. <a href="https://www.jove.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<table><tbody><tr><td>10X PBS Buffer</td><td>GenDEPOT</td><td>P2100-104</td><td></td></tr><tr><td>4% Formalin</td><td>T&amp;amp;I</td><td>BPP-9004</td><td></td></tr><tr><td>Ampicillin</td><td>sigma&amp;nbsp;</td><td>A5354-10ml</td><td></td></tr><tr><td>BSA</td><td>sigma&amp;nbsp;</td><td>A4503-100G</td><td></td></tr><tr><td>confocal dish</td><td>SPL</td><td>200350</td><td></td></tr><tr><td>confocal microscope&amp;nbsp;</td><td>ZEISS</td><td>LSM800</td><td></td></tr><tr><td>coverslip</td><td>SPL</td><td>20009</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>10565-018</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher scientific</td><td>16140071</td><td></td></tr><tr><td>Fluorsheild with DAPI</td><td>sigma&amp;nbsp;</td><td>F6057</td><td></td></tr><tr><td>Forcep</td><td>Dumont</td><td>0508-L5-P0</td><td></td></tr><tr><td>HBSS</td><td>Thermo Fisher scientific</td><td>14065056</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher scientific</td><td>15630080</td><td></td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Phalloidin-iFluor 647 Reagent</td><td>abcam</td><td>ab176759</td><td></td></tr><tr><td>Stage Top Incubator</td><td>TOKAI HIT</td><td>WELSX</td><td></td></tr><tr><td>Strain C57BL/6 mouse messenchymal stem cells with GFP</td><td>cyagen</td><td>MUBMX-01101</td><td></td></tr><tr><td>Triton X-100</td><td>sigma&amp;nbsp;</td><td>T8787</td><td></td></tr></tbody></table>

in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti

세포 재생 및 치료에 대한 중간엽 줄기 세포 (MSC)의 효과를 연구하기 위해이 방법은 달팽이관 상피와 공동 배양 후 MSC 마이그레이션 및 형태 학적 변화를 추적합니다. 코르티의 장기는 해부 중에 생성된 Reissner 막의 일부를 눌러 플라스틱 커버슬립에 고정되었습니다. 실린더를 제거할 때 유리 실린더에 의해 수감된 MSC가 달팽이관 상피로 이동했습니다. 그들의 지배적인 국소화는 코르티의 기관의 modiolus에서 관찰되었다, 신경 섬유의 것과 유사한 방향으로 정렬. 그러나 일부 MsMC는 림푸스 지역에 국한되어 수평으로 길쭉한 모양을 보였습니다. 또한, 모발 세포 영역으로의 이동이 증가되었고, MSC의 형태는 가나마이신 치료 후 다양한 형태로 변경되었다. 결론적으로, 본 연구의 결과는 달팽이관 상피와 MSC의 공존이 세포 이식을 통해 치료의 발달과 다양한 조건과 요인을 검사 할 수있는 세포 재생연구에 유용 할 것임을 나타냅니다.

3차원 모드에서 MSC의 체외 이동은 트랜스웰 시스템 또는 전통적인 상처 치유 방법에 의해 2차원(2D)모드(11)에서마이그레이션을 관찰하도록 평가되었다. 코르티의 기관은 보처 세포, 클라우디우스 세포, 디터 세포, 기둥 세포, 헨센의 세포, 외부 헤어 셀, 내부 모발 세포, 신경 섬유, 바실라 막 및 망상 라미나12와같은 다양한 세포로 구성된...

Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

본 연구에서는, 우리는 코르티의 기관을 포함하는 달팽이관 상피와 전 생체 배양에 의해 손상된 조직을 향해 이동하는 세포를 관찰하는 공초점 현미경을 사용하여 실시간 이미징 방법을 제시한다.

코르티 의 장기를 향해 세포 마이그레이션을 탐구하는 체외 시간 경과 라이브 세포 이미징

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

in-vitro-time-lapse-live-cell-imaging-to-explore-cell-migration

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JoVE Journal

Medicine

3.7K 조회수.  Yonsei University Wonju College of Medicine. 본 연구에서는, 우리는 코르티의 기관을 포함하는 달팽이관 상피와 전 생체 배양에 의해 손상된 조직을 향해 이동하는 세포를 관찰하는 공초점 현미경을 사용하여 실시간 이미징 방법을 제시한다.

비디오: In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

3 차원 매트릭스에서 찬란 태그가 추가된 단백질을 표현 마이 그 레이션 전지의 라이브 세포 이미징

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

연구

생체공학

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

출생 후의 두뇌에의 연결을 마이 그 레이션의 고해상도 시간 경과 이미징을위한 Organotypic 슬라이스 분석

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

신경과학

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

분석<em&gt; 생체</em&gt; 셀 마이그레이션 Zebrafish의 배아에서 세포 이식 및 시간 경과 이미징을 사용하여

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

발생학

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

성인 마우스 Forebrain의 급성 슬라이스에 Neuroblast 마이그레이션의 시간 경과 영상

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

개발 병아리 광섬유 Tectum에 접선 셀 이동의 시각화

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

마우스 급성 뇌 조각의 Neuroblast 마이그레이션의 생체 내 출생 일렉트로 및 시간 경과 영상

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

마우스 인공 와우의 Explants의 장기 시간 경과 영상

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

공 초점 Macroscopy을 사용하여 출생 후의 소뇌 과립 세포 이동의 생체 이미징

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

배아 마우스 두뇌의 organotypic 슬라이스 문화에서 마이 그 레이션 뉴런의 저속 공 촛점 이미징<em&gt; Utero에서</em&gt; 일렉트로 포 레이션

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

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 ...