<p class="jove_content"&gt; 작가의 작품 연구에 연구를 부여 G.0136.11 및 G.0130.11 의한 앤트워프 대학의 부여 ID-BOF 2006 (PPO와 AVdL 부여), (AVdL, ZB와 PPO 부여) 및 1.5.021.09에 의해 지원되었다. 과학 기술에 대한 플랑드르 연구소 BRAINSTIM (ZB와 AVDL 부여)보기 : 학술 연구 - 플랑드르 SBO 연구 기금 IWT-60838에 의한 (FWO-Vlaanderen, 벨기에)에 대한 기금의 N.00 (PPO에 부여) EC-FP6-가지고와 DiMI (LSHB-CT-2005-512146)에 의해 부분 플레미쉬 정부에서 Methusalem 연구 기금 (ZB 부여), EC-FP6-가지고와 에밀 (LSHC-CT-2004-503569)에 의한 부분 그리고 남북 대학 매력 폴란드에 의한 IUAP-NIMI-P6/38 (AVDL 부여). 나탈리 드 Vocht는 FWO-Vlaanderen에서 박사 학위 - 학생이기을 보유하고 있습니다. 피터 Ponsaerts는 FWO-Vlaanderen의 포스트 박사 동료입니다.</p

1. 셀 준비 <ol><li> 실험은 유전자 루시페라제 및 eGFP 기자 단백질을 표현하는 설계 전직 생체내 교양 줄기 세포 인구를 사용하여 시작되어야합니다. 여기는 이전 Bergwerf 외. 2, 5에 기술된 / 루시페라제 eGFP - 표현 murine 골수 파생 stromal 세포 (BMSC-Luc/eGFP)을 사용합니다. </li><li> 1 μg / ML의 Puromycine로 보충 15 ML 전체 확장 매체 (CEM)에 T75 문화 플라스크 당 8 × 10 5 세포의 밀도의 전지 라벨, 플레이트 BMSC-Luc/eGFP 세포 이틀전 일. </li><li> 세포가 37에서 48 시간 동안 성장할 수 있도록 허용 ° C에서 5 % CO 2. </li><li> 5 추가 X 10 6 빙하 블루 마이크론 크기의 철 산화물 입자 (GB MPIO) 성장 세포 배양에 당 ML 배지 (총 : 75 × 10 T75 문화 플라스크에서 15 ML 문화 매체 당 6 입자). </li><li> endocytosis를 통해 입자 이해 가능하도록 16 시간 동안 알을 품다. </li><li> 남은 입자를 멀리 씻으그리고 세포에 GB MPIO의 세포 confluency 및 균질 분포를 얻기 위해 추가로 24 시간 동안 성장할 수 있습니다. </li><li> eGFP + 세포의 총 금액 eGFP + 및 GB MPIO + 세포의 비율을 계산하여 형광 현미경을 사용하여 시각적으로 입자 이해를 확인합니다. </li><li> 워시 기가바이트 MPIO-라벨 BMSC-Luc/eGFP 전지 두번 트립신-EDTA (에틸렌 다이아 민 테트라 초산) 치료 (5 분 T75 문화 플라스크 당 5 ML)에 따라 10 ML PBS 및 수확 세포와. </li><li> 133 X 10 6 세포 / PBS에서 ML의 최종 농도에서 수확한 세포 resuspend 씻으십시오. </li><li> 이식까지 얼음 세포를 유지. </li></ol> 2. 마우스의 CNS의 세포 주입 <ol><li> 300ml의 intraperitoneal 주사 (80 밀리그램 / ㎏) + xylazine (16 밀리그램 / ㎏) 혼합하여 쥐를 마취시키다. </li><li> 쥐가 잠들 수 있도록 5 분 동안 기다리십시오. </li><li> 멸균 조작을 할 수 있도록 마우스 머리를 면도. </li><li> stereotactic 프레임에 마우스를 놓습니다. </li><li> 를 Desinfect피부와 생쥐의 눈이 탈수를 방지하기 젖었어. </li><li> 두개골을 노출하는 중간선 두피 절개를 만들고, 주어진 좌표에 치과 드릴 버를 사용하여 두개골에 구멍을 뚫을 : 후부 2mm와 Bregma 옆으로 2mm합니다. </li><li> 간단히 와동 세포 현탁액을하고 주사기의 세포 현탁액을 기음. </li><li> 경질 아래에 2.5 mm의 깊이에서 주사기 바늘을 넣고 1 분 압력 평형을 허용합니다. </li><li> 자동화된 마이크로 주입 펌프를 사용하여 경질하에 2mm의 깊이에서와 0.70 μl / 분 속도로 3 μl 세포 현탁액 (4 × 10 5 세포)를 주사. </li><li> 주입된 세포 현탁액의 역류를 방지하기 위해 추가로 5 분 기다리십시오. </li><li> 천천히 바늘을 철회하고 피부를 suturing 전에 피부 테두리를 소독. </li><li> 탈수를 방지하기 위해 subcutaneously 300 μl 0.9 % NaCl 용액을 주사하고, 마취로부터 회복하기 위해 히팅 램프 아래에 마우스를 놓습니다. P를 관리제도적 기준에 따라 OST-요원은 진통. </li></ol> 3. 생체내 Bioluminescence에서 <ol><li> isoflurane과 산소 3%의 혼합물에 의해 생쥐를 마취시키다. </li><li> 광자 영상기에서 쥐를 넣은 isoflurane과 산소 1.5 %로 마취 수준을 줄일 수 있습니다. </li><li> 정맥 kg의 체중 당 150 MG D-luciferin을 주사. </li><li> 사진 비전 소프트웨어를 사용하여 5 분 영상을 취득. </li><li> M3vision 소프트웨어를 사용하여 이미지 처리를 수행합니다. 관심 고정 영역을 사용하여 관찰된 신호를 계량. </li></ol> 4. 생체내 자기 공명 영상에서 <ol><li> N 2 O (3시 7분) : O 2 혼합물의 isoflurane 3퍼센트로 쥐를 마취시키다. </li><li> 수평 9.4T MR 시스템의 restrainer에 생쥐를 삽입하고 마취 수준 O 2 혼합물의 isoflurane 1 % 감소 : N 2 O (3시 7분을). </li><li> dehydra을 방지하기 위해 생쥐의 눈을 물에 적셔기는 마우스 배꼽 아래에 센서를 배치하여 체온을 모니터하고 호흡 속도를 모니터링하는 직장​​ 프로브를 연결합니다. </li><li> 110 ± 분당 10 심호흡으로 호흡 율을 유지하고 ± 0.5가 ° C. 37 좁은 범위 내에서 체온이 일정하게 유지 </li><li> 마우스 머리와 자석의 중간 위치에 마우스를 위에 표면의 RF 코​​일을 배치합니다. </li><li> 70 μm의 2 인 - 비행기 해상도로 줄기 세포 이주를 공부하기 위해 구체적인 해부 학적 정보 및 T2 *-가중 기울기 에코 (GE) 이미지를 취득하는 10 코로나 T2-가중 스핀 에코 (SE) 이미지 세트를 취득. 반복 시간 (TR)를 : 다음과 같이 순서대로 매개 변수를 설정 500 MS, 에코 시간 (TE) : 8 MS (GE 순서) 및 반복 시간 (TR) : 4,200 MS, 에코 시간 (TE) : 12.16 MS (SE 순서); 18x18 mm 2, 매트릭스 : 256x256, 1 밀리미터 슬라이스 두께 1mm 슬라이스 분리 (Paravision 5.1 소프트웨어) 뷰 (FOV)의 필드. </li><li> 데이터 처리를 수행Amira 4.0 소프트웨어를 사용합니다. </li></ol> 5. 해부 조직학 <ol><li> isofluorane (4 %), 산소 (0.5 L / 분)과 질소 (1 L / 분) 2 분 동안 혼합물을 흡입하여 매우 깊이 쥐를 마취시키다. 경추 탈구로 쥐를 희생. </li><li> 두개골에서 마우스 두뇌를 제거하고 2 시간 PBS에서 4 % paraformaldehyde에서 뇌 조직을 흥분시키는. </li><li> PBS의 20 % 자당의 하룻밤 PBS에서 10 %의 자당에 PBS, 2 시간의 5 % 자당에 2 시간 : 자당의 다양한 그라디언트 년 이후 두뇌를 배치하여 뇌 조직을 탈수. </li><li> 액체 질소를 사용하여 뇌 조직을 고정시키고 sectioning 때까지 -80 ° C에서 조직을 저장합니다. </li><li> 그라 이오 스탯을 사용하여 10 μm의 두께는 섹션의 섹션 뇌 조직. </li><li> 화면에 GB MPIO 입자와 형광 현미경을 사용하여 eGFP 표현 세포에서 녹색 형광의 청색 형광을 위해 cryosections를 흠없는. </li><li> 화면에 대한 cryosections를 흠없는염증성 세포에서 적색 / 녹색 배경 형광. </li><li> 세포 이식은 상호 작용 내생 세포 인구를 (예 : microglia, astrocytes, T 세포, ...)를 식별하는 데 더욱 immunohistochemical 및 immunofluorescent stainings를 수행합니다. </li></ol> 6. 대표 결과 우리는 여기서 시각 생쥐의 CNS에서 루시페라제 / eGFP-표현 (줄기) 세포 집단의 성공 multimodal 이미징을위한 이벤트를 최적화된 순서를 표현했습니다. 처음에는 쉽게 확인할 수 있습니다 BMSC-Luc/eGFP 세포의 효율 라벨에 설명된 기가바이트 MPIO의 라벨링 절차 결과는 형광 현미경 (그림 2A)를 사용. 다음으로, 기가바이트 엠피오의 접목에 따라 생쥐의 CNS에서 BMSC-Luc/eGFP이라는, 세포 이식의 생존은 루시페라제 활동 (그림 2B)을 기반으로 생체내 BLI에 의해 모니터할 수 있습니다. 또한, 이식할 세포의 정확한 지방화는 생체내에 의해 모니터할 수 있습니다 </em&gt;를 기가바이트 MPIO (그림 2C)의 철분 함유량을 기준으로 MRI. 마지막으로, histological 분석 BLI와 MRI로 확인한 결과의 검증을 허용합니다. 안정적인 생존은 시간에 안정 eGFP 표현 (그림 2D)에 의해 확인되었다. 본 내용의 colocalization eGFP은 - 표현 청색 형광 엠피오와 세포 것은 현지화 및 이식할 세포의 생존의 정확한 결정을 허용합니다. 또한, 줄기 세포의 이중 형광 라벨은 염증 세포가 5로 만든 배경 형광을 기반으로 거짓 긍정적인 결과를 관찰할 수있는 가능성을 줄여줍니다. <img alt="1 그림." src="/files/ftp_upload/3906/3906fig1.jpg" /> 그림 1. 처리 순서의 개요 <ol class="lroman"><li> 휴대폰 라벨 : BMSC-Luc/eGFP은 ML 문화 매체 당 5 × 10 6기가바이트 MPIO 입자로 분류된다. 16 부화의 시간과 24 시간의 다음과 같은 휴식 기간이 지난 후에 세포를 위해 수확하고했습니다만 주입. </li><li> 세포 이식 : bregma -2 음, 경질 미만의 중간선과 2mm에서 2 밀리미터 오른쪽 : 기가바이트 엠피오는 BMSC-Luc/eGFP는 다음과 같은 좌표에서 마우스의 뇌에 이식할 있습니다 분류. </li><li> 생체내 BLI 년 : 체중을 D-luciferin/kg 150 밀리그램의 아래에 정맥 주사 관리, 생성된 빛이 광자 영상기를 사용 오분 동안 획득한 것입니다. </li><li> 생체내 MRI 년 : 70 μm의 2 인 - 비행기 해상도를 갖춘 10 개 코로나 T2-가중 스핀 에코 (SE) 영상과 T2 *-가중 기울기 에코 (GE)의 이미지 집합 인수되었다. 반복 시간 (TR)를 : 다음과 같이 시퀀스 매개 변수가 있었 500 MS, 에코 시간 (TE) : 8 MS (GE 순서) 및 반복 시간 (TR) : 4,200 MS, 에코 시간 (TE) : 12.16 MS (SE 순서); 18x18 mm 2, 매트릭스 : 256x256, 1 밀리미터 슬라이스 두께 1mm 슬라이스 분리 뷰 (FOV)의 필드. </li><li> 사후 조직학 : 10μm 두께 cryosection는 표현 eGFP과 청색 fluorescently 분류 BMSC U 동안 상영되었다형광 현미경을 노래. </li></ol><img alt="그림 2." src="/files/ftp_upload/3906/3906fig2.jpg" /> 줄기 세포 이식의 그림 2. Multimodal 이미징 <ol class="ualpha"><li> 빙하 파란 (GB) 엠피오의 직접 immunofluorescence 현미경은 BMSC-Luc/eGFP을 분류. </li><li> 4x10 5 주입된 생쥐의 생체내 BLI에서는 기가바이트 엠피오는 주 1 주입 후 주 2 시에 BMSC-Luc/eGFP 세포를 분류. 주 1, 주 2 사후 이식에서 발견 BLI 신호의 통계적 분석. 데이터가 5 분 기간 동안의 cm2 당 steradian 당 초 당 광자의 평균 개수 (산도 / S / SR / cm 2)로 표현됩니다 + / - 맑은 BLI 신호를 보여주는 생쥐의 관심을 고정 영역에서 계산된 표준 오차 사출 사이트. </li><li> 그리고 레이블이 지정되지 않은 BMSC-L : 기가바이트 MPIO의 코로나 2 차원 MRI는 BMSC-Luc/eGFP를 ((I) T2-가중 스핀 에코 이미지 그리고 (ii) T2 *-가중 기울기 에코 영상 왼쪽 패널)로 분류UC / eGFP (오른쪽 패널 : (3) T2-가중 스핀 에코 영상과 (VI) T2 *-가중 기울기 에코 이미지) 마우스 뇌에 이식. </li><li> 기가바이트 MPIO의 Histological 분석은 주 2 사후 주입에 BMSC-Luc/eGFP 이식이라는. eGFP - 표현 및 GB MPIO 분류 BMSC-Luc/eGFP 이식술의 지방화를위한은 (i) 직접 immunofluorescence 분석. 의 (2) 높은 배율 세부 사항은 (i). 기가바이트 MPIO의 주변에서 astrocytic 반흔 조직의 발전을 나타내는 GFAP 항원에 대한 (3) Immunofluorescent 염색법은 BMSC-Luc/eGFP 라벨. Iba-1 항원에 대한 (VI) Immunofluorescent 염색법, 침략을 나타내는 및 GB MPIO의 주변은 microglia 의해 BMSC-Luc/eGFP이라는. </li></ol>

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저자는 이익 갈등을 선언 없습니다.

이 보고서에서는 면역 관할 생쥐의 CNS의 세포 이식의 세부 특성화 세 보완 이미징 modalities의 조합 (BLI, MRI 및 조직학)에 최적화된 프로토콜을 설명합니다. 리포터 유전자 반딧불 루시페라제 및 eGFP, 및 GB 엠피오와 직접적인 셀 라벨과 유전자 조작을 바탕으로 세포의 리포터 유전자 라벨,의 조합이 생체내에서 줄기 세포 이식의 정확한 평가로 안내합니다. BMSC의 입자 라?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> IMDM </td><td> Lonza </td><td> BE12-722F </td><td> 세포 성장 매체 CEM의 구성 요소 </td></tr><tr><td> 태아 소 혈청 </td><td> Gibco </td><td> 10270-106 </td><td> 세포 성장 매체 CEM의 구성 요소 </td></tr><tr><td> 말 혈청 </td><td> Gibco </td><td> 1605-122 </td><td> 세포 성장 매체 CEM의 구성 요소 </td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> Gibco </td><td> 15,140 </td><td> 세포 성장 매체 CEM의 구성 요소 </td></tr><tr><td> Fungizone </td><td> Gibco </td><td> 15290-018 </td><td> 세포 성장 매체 CEM의 구성 요소 </td></tr><tr><td> PBS </td><td> Gibco </td><td> 14,190 </td><td></td></tr><tr><td> Puromycine </td><td> Invivogen </td><td> 개미-PR-1 </td><td></td></tr><tr><td> 트립신 </tD&gt; <td> Gibco </td><td> 25,300 </td><td></td></tr><tr><td> 기가바이트 MPIO </td><td> 앞머리 연구소 </td><td> ME04F/7833 </td><td></td></tr><tr><td> D-luciferin </td><td> Promega </td><td> E1601 </td><td></td></tr><tr><td> 케타민을 (Ketalar) </td><td> 화이자 </td><td></td><td></td></tr><tr><td> Xylazine (Rompun) </td><td> 바이엘 헬스케어 </td><td></td><td></td></tr><tr><td> Isoflurane </td><td> Isoflo </td><td> 05260-05 </td><td></td></tr><tr><td> 0.9 % NaCl 용액 </td><td> 벡스터 </td><td></td><td></td></tr><tr><td> paraformaldehyde </td><td> 머크 </td><td> 1.04005.1000 </td><td></td></tr><tr><td> 자당 </td><td> Applichem </td><td> A1125 </td><td></td></tr><tr><td> 미세 분사 펌프 </td><td> KD 과학 </td><td> KDS100 </td><td></td></tr><tr><td> 광자 영상기 </td><td> Biospace 연구소 </td><td></td><td></td></tr><tr&gt; <td> 9.4T MR 스캐너 </td><td> Bruker Biospin </td><td> Biospec 20분의 94 USR </td><td></td></tr><tr><td> BX51 현미경 </td><td> 올림포스 산 </td><td> BX51 </td><td></td></tr><tr><td> Mycrom HM 그라 이오 스탯 </td><td> Prosan </td><td> HM525 </td><td></td></tr><tr><td> 주사기 </td><td> 해밀턴 </td><td> 7635-01 </td><td></td></tr><tr><td> 30 게이지 바늘 </td><td> 해밀턴 </td><td> 7762-03 </td><td></td></tr><tr><td> 포토 비전 소프트웨어 </td><td> Biospace 연구소 </td><td></td><td></td></tr><tr><td> M3vision 소프트웨어 </td><td> Biospace 연구소 </td><td></td><td></td></tr><tr><td> Paravision 5.1 소프트웨어 </td><td> Bruker Biospin </td><td></td><td></td></tr><tr><td> Amira 4.0 소프트웨어 </td><td> 얼굴 이미징 </td><td></td><td></td></tr></tbody></table>

multimodal imaging of stem cell implantation in the central nervous system of mice

지난 10 년 동안 줄기 세포 이식은 잠복기 및 임상 연구에서 두 질환의 다양한 주 또는 보조 치료 양상 등의 증가하고 관심을 받고있다. 그러나, 기능적 결과 및 / 또는 조직 재생 다음과 줄기 세포 이식에 관한 결과가 매우 다양 날짜입니다. 일반적으로 임상 혜택은 기본 메커니즘 (들) 1의 깊은 이해없이도 관찰된다. 따라서 여러 노력은 정확하게 생존, 운명 및 이식할 줄기 세포 및 / 또는 그들의 마이크로 환경의 생리를 평가하는 데 궁극적인 목표로 이식 줄기 세포를 모니터링하는 다른 분자 이미징 modalities의 발전에 주도했습니다. 분자 이미징에 의해 결정 하나 이상의 매개 변수에서 관찰 변경 관찰된 임상 효과에 관한 수 있습니다. 이러한 맥락에서, 우리의 연구는 bioluminescence 이미징의 통합 이용 (BLI), 자기 공명 영상 (MRI) 및 histological analysi에 초점S는 이식 줄기 세포를 평가합니다. BLI는 일반적으로 비 invasively 세포 루시페라제-리포터 유전자를 표현하는 생화 학적 반응을 바탕으로 이식 2-7, 다음 셀 추적을 수행하고 시간에 세포 생존을 모니터링하는 데 사용되는 것은 그것의 기판에 빛을 다음과 같은 상호 작용을 방출 수 있습니다 (예 : D- luciferin) 8, 9. 반면 MRI는 민감도가 높은 MRI 대비 에이전트와 함께 세포 라벨 이후에 생성되는 반면에 달려 있지만, 10 임상 적용하고 정확하게 매우 높은 해상도를 11-15로 세포 이식을 찾습니다하는 데 사용할 수있는 비침습 기술이다 . 마지막으로, 사후 histological 분석은 높은 해상도와 감도있는 비침습 방법으로 얻은 연구 결과를 확인하기 위해 선택한 방법입니다. 또한 엔드 포인트 histological 분석은 우리가 이식할 세포 및 / 또는 주변 조직, 바의 상세한 phenotypic 분석을 수행할 수 있습니다형광 기자 단백질 및 / 또는 특정 항체와 직접 셀 라벨링의 사용에 SED. 요약하면, 우리는 여기에 시각적으로 서로 다른 줄기 세포 및 / 또는 환경 관련 생쥐의 CNS에서 이식 줄기 세포에 따라 특성을 해명하는 BLI, MRI 및 조직학의 complementarities를 보여줍니다. 예를 들어, 뼈가 골수는-파생된 유전자 향상된 녹색 형광 단백질 (eGFP)와 반딧불 루시페라제 (fLuc) 및 청색 형광 마이크론 크기의 철 산화물 입자 (MPIOs)로 레이블을 표현할 수 있도록 설계 stromal 세포를,에 이식할 예정 면역 - 관할 마우스 및 결과의 CNS는 BLI, MRI 및 조직학 (그림 1)에 의해 모니터링됩니다.

Watch this Scientific Journal Video about Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice at JoVE.com

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

생체내 bioluminescence과 자기 공명 영상에서 (I), 그리고 (ii) 해부 histological 분석 :이 문서를 사용하여 쥐 두뇌에있는 세포 이식의 multimodal 이미징을위한 이벤트를 최적화된 순서를 설명합니다. 하나의 동물에서 이러한 이미징 modalities를 결합하면 높은 해상도, 민감도와 특이성과 세포 이식 평가를하실 수 있습니다.

마우스의 중추 신경계에서 줄기 세포 주입의 Multimodal 이미징

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

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11.0K 조회수.  University of Antwerp. 생체내 bioluminescence과 자기 공명 영상에서 (I), 그리고 (ii) 해부 histological 분석 :이 문서를 사용하여 쥐 두뇌에있는 세포 이식의 multimodal 이미징을위한 이벤트를 최적화된 순서를 설명합니다. 하나의 동물에서 이러한 이미징 modalities를 결합하면 높은 해상도, 민감도와 특이성과 세포 이식 평가를하실 수 있습니다.

비디오: Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

시신경의 절개 : 중추 신경계에서 신경 세포의 Apoptosis 성인의 모델

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

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신경과학

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

중추 신경계에서 Transducing 전지 Lentiviral 벡터 제작

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

의 중추 신경계에 단일 세포의 라벨<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Polyribosome 분별에 의한 중추 신경계 Translatome의 생체 심문

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

설치류 중추 신경계의 생체 Optogenetic 자극

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

줄기의 네트워크 문화 생물 신경 독소의 기능 평가 중추 신경계 신경 세포가 유래

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

장용 신경계의 제자리 칼슘 이미징에서

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

수동 CLARITY를 사용하여 마우스 중추 신경계의 광학 지우기

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

제 3-탈피 초파리 Melanogaster 의 중앙 신 경계 활동의 electrophysiological 녹음

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...