이 작품은 국립 신경 장애 뇌졸중 연구소 (NS - 19108), 편두통 연구 재단, 그리고 리처드 P. Kraig로 화이트 재단 교부금에 의해 투자되었다. 미스 마샤 P. Kraig는 문화 매체와 슬라이스 문화의 유지 보수의 준비에 도움. 우리는이 문서의 최종 버전에 대한 그녀의 의견과 수정에 대한 옐레나 Grinberg 감사합니다.

<ol>
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관심 없음 충돌 선언하지 않습니다.

냉간 preconditioning neuroprotection에 참여 시토킨 신호 시스템의 묘사에 중요한 두 가지 근본적으로 중요한 개념은 그림 6과 그림 7에 있습니다. 첫째, 크린 시토킨 정상적인 두뇌의 분자 신호를 매우 낮은 농도입니다. 그럼에도 불구하고, 생리학 시토킨 농도 변화 때문에 유전자 발현을 변경하는 능력 (그림 6)의 뇌 구조와 기능 (즉, 표현형)을 변경하는 엄청난 잠재력이 있습니다. 또한, 크린 시토킨 비​​슷한 효과와 단일 시토킨 변수 효과 (그림 7)을 가질 수있을 수 높은 중복하고 여러 크린 시토킨에 pleiotropic 있습니다. 따라서, 정확하게 차가운 - preconditioning, (또는 다른 생리 preconditioning 자극)와 관련된 신호 변수의 복합 분석 것은 결정되어야합니다에서 neuroprotection에 대한 본래 시토킨 - 기지를 설립. 이것은 멀티 플렉스 전략 분석을 통해 수행됩니다. 이것은 차가운 - preconditioning neuroprotection의 시토킨 &quot;서명&quot;을 수립합니다. <img src="/files/ftp_upload/2192/2192fig6.jpg" alt="그림 6" /> 그림 6. 두뇌 시토킨 신호의 파워. 삽화 다른 잘 인식 &#39;매쉬&#39;의 농도에 비해 뇌 크린 시토킨의 생리적 농도의 거대한 신호 전력을 전달. 농도는 기준점으로 하나의 고양이 수염으로 시작, 거리의 역수로 표현된다. 나트륨 (10 -1 M은 고추의 입자에 의해 그림)와 칼륨이 (토마토로 그림 10 -3 M) 각각 150 주변 3 MM의 수준에서 중간 뇌 공간에 존재, 및 잘 인식 역할을 신경 세포 electrophysiological 기능을 수행합니다. 마찬가지로, PH (즉, ~ 10 -7 M 수소 이온의 수준과 시카고의 레이크 프런트를 따라 780m로 그림)과 칼슘 (즉, 7.8 km는 맥코믹에서 시카고의 레이크 프런트 따라 위성 Google 이미지에서 본 그림과 같이 ~ 10 -8 M 수준 ) 시카고 대학 근처에 하이드 파크 (Hyde Park) 프로 몬 토리 포인트에 놓습니다. 또한,이 농도 이상의 중간 공간에 출시 신경 전달 물질은 현지 뇌 영역의 활동에 영향을 미칩니다. 반대로, 크린 시토킨는 (지구에서 화성의 거리로 표시) 이상의 천만 배 적은 농도에서 뇌 기능을 변경할 수 있습니다. <img src="/files/ftp_upload/2192/2192fig7.jpg" alt="그림 7" /> 그림 7. 타고난 시토킨 경로의 인터랙티브 신호. 크린 시토킨 상당히 중복하고 여러 크린 시토킨에 pleiotropic 아르 비슷한 효과를 가질 수 있으며 하나의 시토킨 변수 영향을 미칠 수 있습니다. 이러한 다양성은 리간드, 수용체, 그리고 phosphoproteins의 수준에서 발생하는 복잡한 상호 작용 신호 때문이다. 또한 복잡한 두뇌 세포 특정 타고난 시토킨, 수용체, 관련 phosphoprotein 변경 각각의 능력과 다양한 세포 유형으로 구성되어있다는 사실에서 유래. 여기에 설명을 위해 경로를 (면역 세포 연구에서 파생된) 신호에만 타고난 시토킨가 표시됩니다. 단순 들어, 두뇌는 본래 크린 시토킨에 대한 잠재적인 상호 작용을 보여주는 하나의 셀 (흰색 라인) (IL - 1α와 IL - 1β () IL - 1α / β, TNF - α, IL - 6로 여기에 언급로 그려진 것입니다 IFN - γ와 IL - 10), 수용체 (IL - 1R1, TNFR1, IL-6/gp130, IFNγR 및 IL - 10R) 및 phosphoproteins (즉, kinases ERK1 / 2, P38 (P38 - MAPK)와 JNK) 및 전사 요인 (ATF - 2, NFκB와 STAT3). ATF - 2를 통해 유전자 발현을 실행 JNK, p38 - MAPK 및 ERK1 / 2에 TNFR1 통해 예를 들어, TNF - α는 신호. TNF - α는 NFκB 활성화를 통해 직접 유전자 발현을 바꿀지도 모르겠어. 함께, 이러한 전사 인자의 활성화로 표시 크린 시토킨 및 수용체의 표현 (뭉툭한 끝) 증가 (화살표) 및 감소 보여주고 있습니다. 중요한 것은, 이러한 경로 하나 시토킨에 변화 (예를 들어, TNF - α) 다른 (예를 들어, IL - 1β) 크린 시토킨의 영향 생산을 보여줍니다. 따라서, 정확하게 차가운 - preconditioning, 관련 신호 변수의 복합 분석을 결정해야합니다에서 neuroprotection에 대한 시토킨 - 기지를 설립. 이것은 감기 - preconditioning의 본래 시토킨 &quot;서명&quot;을 수립합니다. (이미지 참조 # 25 데이터에서 컴파일되었습니다.)

슬라이스 문화의 준비 <table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 / 모델 번호 </td><td> 댓글 </td></tr><tr><td> Millicell - CM 조직 문화를 삽입 (30 ㎜) </td><td> 써모 피셔 </td><td> PICM0 - 3050 </td><td></td></tr><tr><td> 기초 중간 이글 </td><td> Invitrogen </td><td> 21,010 </td><td></td></tr><tr><td> 얼의 밸런스드 소금 솔루션 </td><td> 시그마 </td><td> E2888 </td><td></td></tr><tr><td> 호스 혈청 </td><td> Invitrogen </td><td> 26050-088 </td><td></td></tr><tr><td> Glutamax </td><td> Invitrogen </td><td> 35,050 </td><td></td></tr><tr><td> Gentamicin </td><td> Invitrogen </td><td> 15710-064 </td><td></td></tr><tr><td> Fungizone </td><td> Invitrogen </td><td> 15,295 </td><td></td></tr><tr><td> D - 글루코오스 </td><td> 시그마 </td><td> G8769 </td><td></td></tr><tr><td> BSL - 1 퓸 후드 </td><td> 빵 굽는 사람 </td><td></td><td></td></tr><tr><td> 맥일웨인 조직 헬기 </td><td> Brinkmann </td><td></td><td></td></tr><tr><td> 테플론 디스크 </td><td> Brinkmann </td><td> 023-49-310-1 </td><td></td></tr><tr><td> 주걱 </td><td> 써모 피셔 </td><td> 14-373 - 25A </td><td></td></tr><tr><td> 곡선 홍채 가위 </td><td> 파인 과학 도구 </td><td> 14091-09 </td><td></td></tr><tr><td> 롱 스트레이트 가위 </td><td> 파인 과학 도구 </td><td> 14002-14 </td><td></td></tr><tr><td> 롱 포셉 </td><td> 써모 피셔 </td><td> 13-812-40 </td><td></td></tr><tr><td> 직각 포셉 </td><td> 파인 과학 도구 </td><td> 11808-02 </td><td></td></tr><tr><td> 짧은 포셉 </td><td> 써모 피셔 </td><td> 13-812-38 </td><td></td></tr><tr><td> # 5 Inox 포셉 </td><td> 파인 과학 도구 </td><td> 11254-20 </td><td></td></tr><tr><td> 이 이리스 spatulae </td><td> 파인 과학 도구 </td><td> 10094-13 </td><td></td></tr><tr><td> 150 X 15mm 페트리 요리 </td><td> 써모 피셔 </td><td> 08-757-148 </td><td></td></tr><tr><td> 60 X 15mm 페트리 요리 </td><td> 써모 피셔 </td><td> 08-757 - 100B </td><td></td></tr><tr><td> 와일드 M8 stereomicroscope </td><td> 와일드 Heerbrugg </td><td></td><td></td></tr><tr><td> Gey의 균형 소금 솔루션 </td><td> 시그마 </td><td> G9779 </td><td></td></tr><tr><td> Blak - 레이 단파 자외선 측정 미터 </td><td> UVP </td><td> J - 225 </td><td></td></tr><tr><td> 6 - 잘 팰컨 문화 요리 </td><td> 써모 피셔 </td><td> 08-772 - 1B </td><td></td></tr><tr><td> Neurobasal </td><td> Invitrogen </td><td> 21,103</td><td></td></tr><tr><td> B27 보충 </td><td> Invitrogen </td><td> 17,504 </td><td></td></tr><tr><td> Gem21 Neuroplex </td><td> 제미 Bioproducts </td><td> 400-160-010 </td><td></td></tr><tr><td> 아스코르비 산 </td><td> 시그마 </td><td> A4544 </td><td></td></tr><tr><td> CO 2 분석기 </td><td> 바카라 </td><td> # 2820 </td><td></td></tr></tbody></table> 슬라이스 문화의 활력을위한 사전 검사 <table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 / 모델 번호 </td><td> 댓글 </td></tr><tr><td> Sytox 그린 </td><td> Invitrogen </td><td> S7020 </td><td></td></tr><tr><td> DMIBRE 거꾸로 현미경 </td><td> Leica </td><td></td><td></td></tr></tbody></table> 콜드 - Preconditioning <table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 / 모델 번호 </td><td> 댓글 </td></tr><tr><td> BSL - 2 퓸 후드 </td><td> NuAire </td><td></td><td></td></tr></tbody></table> Excitotoxic 상해 <table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 / 모델 번호 </td><td> 댓글 </td></tr><tr><td> CoolSnap FX CCD 카메라 </td><td> Photometrics </td><td></td><td></td></tr><tr><td> Fluoroscein </td><td> Invitrogen </td><td> F36915 </td><td></td></tr><tr><td> MetaMorph 소프트웨어 </td><td> 분자 장치 </td><td> V. 7.5.04 </td><td></td></tr><tr><td> 100 μm의 깊이 hemacytometer </td><td> Hausser 과학 </td><td></td><td></td></tr><tr><td> N - 메틸 - D - aspartate </td><td> Calbiochem </td><td> 454575 </td><td></td></tr><tr><td> SigmaStat 소프트웨어 </td><td> Systat 소프트웨어 주식 회사 </td><td> V. 3.5 </td><td></td></tr></tbody></table> 콜드 Preconditioning 적용된 공동 트리 트먼트 <table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 / 모델 번호 </td><td> 댓글 </td></tr><tr><td> sTNFR1 </td><td> R &amp; D 시스템 </td><td> 425 - R1 - 050 </td><td></td></tr><tr><td> 소 혈청 알부민 </td><td> 시그마 </td><td> A - 4503 </td><td></td></tr></tbody></table> RNA 격리 <table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 / 모델 번호 </td><td> 댓글 </td></tr><tr><td> RNAl ater </td><td> 앰비온 </td><td> AM7021 </td><td></td></tr><tr><td> 마이크로 RNEasy 키트 </td><td> Qiagen </td><td> 74,004 </td><td></td></tr><tr><td> β - 메르 캅 토 에탄올 </td><td> 어부 </td><td> 03446-100 </td><td></td></tr><tr><td> Diposable 1.5 RNAse - 무료 페슬 </td><td> 사우스 제르 정밀 도구 및 몰드 주식 회사 </td><td> 749521-1590 </td><td></td></tr><tr><td> RNasin </td><td> Promega </td><td> N261A </td><td></td></tr><tr><td> 트리스 [히드록시 메틸] aminomethane </td><td> 시그마 </td><td> T3069 </td><td></td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) (ethylenediaminetetraacetic 산성 나트륨 소금 하이드 레이트 </td><td> 시그마 </td><td> # E7889 </td><td></td></tr></tbody></table> RNA의 양을 정함 <table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 / 모델 번호 </td><td> 댓글 </td></tr><tr><td> RiboGreen 어세이 </td><td> Invitrogen </td><td> R11491 </td><td></td></tr><tr><td> 효모 tRNA </td><td> Invitrogen </td><td> 15401-011 </td><td></td></tr><tr><td> 1.5 ML 원심 분리기 튜브 </td><td> 미국 과학 </td><td> 16155500 </td><td></td></tr><tr><td> 96 - 웰 플레이트 형광 분석 </td><td> Nunc </td><td> DK - 4000 </td><td></td></tr><tr><td> 96 - 웰 플레이트 형광 판독기 </td><td> Tecan </td><td> Safire II </td><td></td></tr></tbody></table> 정량 PCR <table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 / 모델 번호 </td><td> 댓글 </td></tr><tr><td> iScript 역방향 전송 키트 </td><td> 바이오 래드 </td><td> 170-8891 </td><td></td></tr><tr><td> SYBR 그린 </td><td> Invitrogen </td><td> S7563 </td><td></td></tr><tr><td> 플래티넘 DNA 형성 촉매 효소 </td><td> Invitrogen </td><td> RNA Ultrasense 11732-927 </td><td></td></tr><tr><td> iCycler thermocycler </td><td> 바이오 래드 </td><td> iCycler 광학 모듈 </td><td></td></tr><tr><td> iCycler 시스템 소프트웨어 </td><td> 바이오 래드 </td><td> V. 3.1 </td><td></td></tr></tbody></table> Microarrays에 대한 정량 PCR <table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 / 모델 번호 </td><td> 댓글 </td></tr><tr><td> 어웨이 RNase </td><td> 분자 BioProducts </td><td> # 7000 </td><td></td></tr><tr><td> 알티 2 퍼스트 스트랜드 키트 </td><td> SABiosciences </td><td> C - 03 </td><td></td></tr><tr><td> SYBR 그린 기반의 PCR 믹스 </td><td> SABiosciences </td><td> PA - 011 </td><td> iCycler에 대한 특정 </td></tr><tr><td> RT PCR이 프로파일의 배열 </td><td> SABiosciences </td><td> PARN - 011A </td><td></td></tr></tbody></table> Proteomic 어세이 <table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그/ 모델 번호 </td><td> 댓글 </td></tr><tr><td> 세포 용해 키트 </td><td> 바이오 래드 </td><td> 171-304011 </td><td></td></tr><tr><td> Microcentrifuge </td><td> 피셔 사이 언티픽 </td><td> 마이크로 7 </td><td></td></tr><tr><td> BSA 단백질 재고 </td><td> 바이오 래드 </td><td> 500-0007 </td><td></td></tr><tr><td> BCA 단백질 분석 키트 </td><td> 써모 과학 </td><td> 23,225 </td><td></td></tr><tr><td> 단백질 96 MicroWell 플레이트 </td><td> Nunc </td><td> 449824 </td><td></td></tr><tr><td> 실링 테이프 </td><td> 바이오 래드 </td><td> 2,239,444 </td><td></td></tr><tr><td> 96 - 웰 플레이트 리더 </td><td> 퍼어킨 엘머 </td><td> 1420 - mulilabel 카운터 </td><td></td></tr><tr><td> Bioplex 랫 TNF - α의 시토킨 분석 </td><td> 바이오 래드 </td><td> X0000001T </td><td></td></tr></tbody></table> Immunostaining <table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 / 모델 번호 </td><td> 댓글 </td></tr><tr><td> 16 % Paraformaldehyde </td><td> 알파 Aesar </td><td> 43,368 </td><td></td></tr><tr><td> 라이신 </td><td> 시그마 </td><td> L - 6001 </td><td></td></tr><tr><td> 인산 나트륨 </td><td> 피셔 사이 언티픽 </td><td> S374 - 1 </td><td></td></tr><tr><td> 나트륨 periodate </td><td> 시그마 </td><td> S - 1878 </td><td></td></tr><tr><td> 나트륨 azide </td><td> 시그마 </td><td> S - 2002 </td><td></td></tr><tr><td> 과산화수소 (30 %) </td><td> 시그마 </td><td> H1009 </td><td></td></tr><tr><td> 염소 혈청 </td><td> 콜로라도 세럼 회사 </td><td> CS 0920 </td><td></td></tr><tr><td> 트리톤 X - 100 </td><td> 시그마 </td><td> T - 8787 </td><td></td></tr><tr><td> 얼룩 요리 </td><td> 테드 펠라 (주) </td><td> 14,511 </td><td></td></tr><tr><td> 플레이트 쉐이크 </td><td> 랩 - 라인 인 스트 루먼트 (주) </td><td> 3520 </td><td></td></tr><tr><td> # 1 필터 보고서 (185mm) </td><td> 워트 먼지 </td><td> 1001-185 </td><td></td></tr><tr><td> 3,3 - diaminobenzidine </td><td> 시그마 </td><td> D8001 </td><td></td></tr><tr><td> 디메틸 sulfoxide </td><td> 시그마 </td><td> D8418 </td><td></td></tr><tr><td> 에탄올 </td><td> Pharmco - AAPER </td><td> 111000200 </td><td></td></tr><tr><td> 크실렌 </td><td> 피셔 사이 언티픽 </td><td> X3S - 4 </td><td></td></tr><tr><td> Permount 설치 미디어 </td><td> 피셔 사이 언티픽 </td><td> SP15 - 100 </td><td></td></tr><tr><td> 그라 이오 스탯 </td><td> Leica </td><td> cm 3 050 S </td><td></td></tr><tr><td> 조직 테크 </td><td> 테드 펠라 (주) </td><td> 27,050 </td><td></td></tr><tr><td> 미세 팁 페인트 브러쉬 </td><td> 테드 펠라 </td><td> 11,806 </td><td></td></tr><tr><td> PAP 차단 펜 </td><td> 테드 펠라 (주) </td><td> 22,309 </td><td></td></tr><tr><td> SFX 신호 향상제 </td><td> Invitrogen </td><td> A31631 </td><td></td></tr><tr><td> Antifade을 연장 </td><td> Invitrogen </td><td> P7481 </td><td></td></tr></tbody></table>

strategies for study of neuroprotection from cold-preconditioning

신경 부상 관리가 용이​​하고 안전 preconditioning 치료 효과의 개발에 의해 완화 수있는 전신 마취와 관련 수술 절차의 병적 상태와 사망률의 자주 원인입니다. 우리는 부상 발병하기 전에 두뇌를 보호하는 치료제 개발을위한 새로운 목표를 식별하는 수단으로 추위 preconditioning에 대한 책임 신경 면역 신호를 정의하기 위해 추구합니다. 시간이 지남에 따라 변화를 신호 낮은 수준의 프로 염증 중재자 감기 - preconditioning neuroprotection을 위해 필수적입니다. 이 신호는 자극성의 자극이 분명 될 보호를위한 자극에 적응을위한 충분한 시간 임계값 크기에 도달할 것을 요구 생리 조절 hormesis의 기본 신조와 일치합니다. 따라서, 냉간 preconditioning neuroprotection에 관련된 면역 신호의 묘사는 생물 학적 시스템과 실험 조작 플러스 기술 역량이 높은 재현성과 구분이 필요합니다. 우리의 접근 방식은 밀접하게 자신의 성숙하고 정지 macroglia / microglia의 영향을 다중 시냅스 신경 네트워크와 생체내 매쉬의를 반영 체외 모델로 hippocampal 슬라이스 문화를 사용하는 것입니다. 그들이 femtomolar 범위에서 작동중인 크린 시토킨의 주요 소스되므로이 glial 상태 microglia에 특히 중요합니다. 또한, 슬라이스 문화가 몇 주 동안 체외에서 유지 될 수있는가 활성화 자극을 보여주고과 적응 반응을 평가할 수있는 충분한 시간입니다. 마지막으로, 환경 조건이 정확하게 그 시토킨가의 신호 때문에 슬라이스 문화를 사용하여 제어할 수 있습니다 추​​위 preconditioning 것은, 측정 했었 및 중요한 노드 측면을 해부하다하기 위해 변조된 수 있습니다. 시토킨 신호 시스템 분석은 민감하고 재현성 멀티 플렉스 기술의 사용을 필요로합니다. 우리는 조직 전체 시토킨 변화를 평가하는 정량 실시간 qPCR 배열 검사 다음 microglial 정품 인증 화면으로 TNF - α에 대한 양적 PCR을 사용합니다. 후자는 동시에 변화를 신호 여러 시토킨 시스템을 측정하는 가장 민감하고 재현성을 의미합니다. 중요한 변경 사항은 타겟 qPCR 그리고 단백질 감지와 함께 확인하고 있습니다. 우리는 Luminex 기술을 사용하여 다중 microsphere 흐름 cytometric assays를 사용하여 조직 기반 시토킨 단백질의 변화에​​ 대한 조사. 셀 - 특정 시토킨 생산은 이중 레이블 immunohistochemistry로 결정됩니다. 함께 촬영, 제안 조사 전략을 결합이 뇌 조직 준비 및 사용의 스타일, 차가운 - preconditioning의 장점을 모방 수있는 새로운 치료제의 개발을위한 잠재적인 목표를 식별을위한 최적의 접근법 수 있습니다.

Watch this Scientific Journal Video about Strategies for Study of Neuroprotection from Cold-preconditioning at JoVE.com

Strategies for Study of Neuroprotection from Cold-preconditioning

우리는 부상 발병하기 전에 두뇌를 보호하는 치료제 개발을위한 새로운 목표를 식별하는 수단으로 추위 preconditioning에 대한 책임 신경 면역 신호를 정의하기 위해 추구합니다. 우리는 생물 학적 시스템, 실험 조작 플러스 높은 재현성 및 민감 기술 용량을 필요로 이러한 작업을위한 전략을 제시한다.

콜드 preconditioning에서 Neuroprotection 연구를위한 전략

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

strategies-for-study-of-neuroprotection-from-cold-preconditioning

Research

JoVE Journal

Neuroscience

14.8K 조회수.  The University of Chicago Medical Center. 우리는 부상 발병하기 전에 두뇌를 보호하는 치료제 개발을위한 새로운 목표를 식별하는 수단으로 추위 preconditioning에 대한 책임 신경 면역 신호를 정의하기 위해 추구합니다. 우리는 생물 학적 시스템, 실험 조작 플러스 높은 재현성 및 민감 기술 용량을 필요로 이러한 작업을위한 전략을 제시한다.

비디오: Strategies for Study of Neuroprotection from Cold-preconditioning

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="http://www.jove.com/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

인광의 산소에 의존 담금질을 바탕으로 대뇌 혈액 산소 측정

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

연구

신경과학

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

의 레이저 캡처 Microdissection Drosophila 주변 뉴런

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

노화와 아밀로이드 병리 동안 시냅틱 얼터 레이션의 연구에 대한 고양이와 트렌스 제닉 마우스의 급성 Hippocampal 조각의 준비

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

쥐 뇌 조각에서 뉴런의 Vibrodissociation는 시냅틱 전송 및 이미지 Presynaptic 터미널 공부

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

연속 신경 근육 분기점에서 싱글 Schwann 세포를 윤곽을 그리다하는 사진 표백

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

막 성인 쥐에서 Ventromedial 시상 하부 뉴런의 잠재적 인 염료 이미징은 포도당 감지를 연​​구하는

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

해마 조각에서 CA1 핵 농축 분획의 분리는 Synapto 핵 메신저 단백질의 활동에 의존하는 핵 가져 오기를 공부합니다

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

신경 세포주에 신경 독성과 신경에 관여하는 분자 진학의 윤곽을하는 도구로 실시간 임피던스 기반의 세포 분석기

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic Hippocampal 슬라이스 문화 연구 Neuroprotection 및 종양 세포의 침입을 모델로

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

미세 전극 배열에서 신경 세포 배양의 자극에 대한 네트워크 반응의 시간 의존적 증가

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...