델라웨어 밸리의 라이온스 안구 은행의 지원은이 작품을 위해 귀중한했습니다. ICP8에 대한 기본 마우스 단클론 항체 박사 데이비드 Knipe (하버드 의과 대학)에서 일종의 선물이었다. 우리는 또한 도움이 토론과 전문 지식에 대한 박사 스티븐 제닝스 (의학 Drexel 대학 대학) 박사 피터 Laibson (유언장 안구 연구소)을 감사드립니다.

모든 시약 및 장비 무균 구입 또는 이전 절차 살균되었다. 이 문서는 미리 enucleated 눈으로 시작 전 생체 모델을 설정하는 단계를 설명합니다. 우리의 실험에서, 우리는 젊은 (8-12 wks) 흰둥이 토끼 (펠 - Freez 체액, 로저스, AR)에서 신선한 전체 눈알을 사용합니다. 또한, 우리는 지역 안구 은행에서 얻은 인간의 각막을 사용합니다. 핵의 제거를 직접 수행하는 경우, 절차를 수행하는 동안 각막 또는 limbus 손상을 방지하는데주의를 기울여야. 토끼 핵의 제거 절차가 다른 곳이 찾을 수 있습니다. 1 부 : Corneoscleral 버튼의 절단 <ol><li> PBS와 거즈의 좁은 스트립을 습식 및 절차를 수행하는 동안 잡고 용이하게하기 위해 안구 두르고. </li><li> 약 0.5 cm 떨어진 limbus에서 공막에 접선 절개를하기 위해 날카로운 메스를 사용하십시오. </li><li> 절개를 확장하는 날카로운 가위를 사용하여 C 완전히 소비세orneoscleral 버튼을 클릭합니다. </li><li> corneoscleral 버튼이 분리되면 집게와 scleral 림을 누른 상태, 아이리스 거리 애타게. 내피를 손상하거나 각막에 과도한 스트레스를 발휘하지 마십시오. </li></ol> 2 부 : Organotypic 문화에 각막 소개 <ol><li> Explanted 각막은 비 필수 아미노산 (1X), L-글루타민 (2 ㎜), 페니실린 (200 U / ML) 및 스트렙토 마이신 (200 μg / ML)로 보충 최소 필수 매체 (가상)에 배양되어 있습니다. 필요한 경우에는 amphotericin 같은 항 곰팡이 요원이도 추가 할 수 있습니다. </li><li> 55 ° C.에서 문화 매체의 1 % 아가로 오스 솔루션을 준비하고 준비를 해 놓도록 </li><li> 철저히 멸균 PBS 함유 페니실린 (200 U / ML) 및 스트렙토 마이신 (200 μg / ML)에서 각막을 씻어. </li><li> 멸균 현장 판의 잘으로 각막 상피​​ 편을 배치합니다. </li><li> 에 충분히 각막의 내피 오목에 아가로 오스 함유 미디어를 추가완전히 채우십시오. 아가로 오스를위한 ~ 1 분 더욱 강화 할 수 있습니다. </li><li> 상피 표면을 커버 할 수있는 35mm 조직 배양 접시에 젤 비계 상피 쪽을 지원하고 추가 할 문화 매체로 각막를 놓습니다. </li><li> 각막은 중간 변경 사항을 모든 48 시간으로 일주일 넘게이 방법으로 배양 할 수 있습니다. </li></ol> HSV-1 감염 및 실험 약물로 치료 : 제 3 부 <ol><li> 35mm 조직 배양 접시에 바이러스의 원하는 금액을 포함하는 매체 1 ML을 추가합니다. 우리는 보통 부담 KOS 각막 당 HSV-1 1x10 4 PFU로 감염. 우리 주식 titers는 CV-1 monolayers에 플라크의 assays를 수행하여 결정됩니다. </li><li> 바이러스가 포함 된 매체로 다운 아가로 오스 발판 상피 측과 함께 각막를 놓습니다. </li><li> 간헐적 흔들 때마다 10-15 분으로 1 시간에 감염 조직 문화 인큐베이터로 각막를 놓습니다. </li><li> 1 시간 후, 바이러스 contai를 대기음매체 닝 및 잔류 바이러스 입자를 제거하는 PBS로 2 ~ 3 배를 헹굴. </li><li> 다시 이전 위치로 각막을 놓고 상피 표면을 충당하기 위해 새로운 문화 매체를 추가 할 수 있습니다. </li><li> 실험 치료 실험의 특성에 따라, 감염 전이나 후에 시작 할 수 있습니다. </li><li> 문화 매체마다 48 시간으로 변경됩니다. </li></ol> 제 4 부 : 샘플 수집 <ol class="lalpha"><li> 상패 분석 분석을위한 매체가. 잘 매체를 섞어서 -80에서 상패 분석 또는 상점에 대한 즉시 사용 ° C 다른 시간 지점에서 샘플을 수집하면 나중에 사용하기 위해. </li><li> DNA, RNA, 단백질, 또는 각막 상피 세포 <ol><li> 아가로 오스 비계를 제거하고 PBS로 잘 각막을 씻어. </li><li> 만 각막 상피​​ 자료를 확보 할 수있는 각막에서 scleral 조직의 소비세 반지는 수집됩니다. </li><li> 유메스를 노래 격리 할 재료의 종류에 따라 적절한 버퍼로 각막 상피​​ 세포를 다 쳤어요. DNA와 RNA의 분리를 들어, 우리는 DNeasy 혈액 및 조직 Kit 및 RNeasy 미니 키트, 각각 (QIAGEN, Hilden, 독일)을 사용합니다. 단백질 lysates를 수집, 우리는 Laemmli 버퍼를 사용합니다. 각막 상피​​ 세포의 유동 세포 계측법 분석을 위해, 우리는 세포를 느슨하게하는 트립신의 작은 금액을 각막을 사전에 배양 한 후 메스로 트립신으로를 제거. 조직의 동등한 양을 얻을하지 않습니다 각막 상피​​를 스크랩 때문에 샘플을 분석 할 때 적절한 컨트롤을 포함하는 것이 중요합니다. 우리는 서양 blots을위한로드 컨트롤로 참조 qPCRs의 유전자, 그리고 nucleolin 등의 qRT-PCRs, GAPDH에서 참조 녹취록 형식으로 18S rRNA의를 사용합니다. </li></ol></li><li> immunohistochemistry 또는 immunofluorescence을위한 조직 샘플 <ol><li> 아가로 오스 비계를 제거하고 옥수수를 헹굼PBS과 잘 EA. </li><li> 동영상에서와 같이 실험 목표에 따라, 파라핀 조직 블록 또는 냉동 조직 블록을 만들기 위해 각막을 사용합니다. 각막 조직 섹션을 준비 할 때 우리는 표준 절차를 따르십시오. </li></ol></li></ol> 대표 결과 조직 문화의 세포를 연구에 사용할 수있는 방법의 대부분은 쉽게 HSV-1 예를 생체에 감염된 각막에 사용하기 위해 적용 할 수 있습니다. 여기에 표시는 HSV-1 감염을 공부에 적용되는 가장 일반적인 기술의 몇 가지에 대한 대표적인 결과입니다 - qPCR (그림 1D), qRT-PCR (그림 1B), 서양 얼룩 (그림 1 층), 상패 분석 (그림 1C) 및 조직 immunofluorescence (그림 1E). <img src="/files/ftp_upload/3631/3631fig1.jpg" alt="그림 1" /> 1 그림. HSV-1 예 V에 감염된 각막 분석이보. 급성 각막 상피 HSV-1 감염의 전 생체 모델의 (A) 도식 표현. (BF)이 비디오 문서에서 설명하는 방법을 사용하여 감염된 각막의 분석. Explanted 토끼 각막이 변형 KOS HSV-1 1x10 4 PFU / 각막에 감염의 존재 (PAA) 또는 phosphonoacetic 산 (400 μg / ML), HSV 복제의 알려진 억제제의 부재 (모의)에서 배양 하였다. (B) 총 RNA는 지정된 시간 지점에서 수집되었고, 바이러스 전사는 HSV-1 효소에 대한 프리 머와 qRT-PCR에 의해 평가되었다. 18S rRNA에 대한 프리 머이는 참조로 사용되었습니다. N = 3. 오류 바 ± SEM을 나타냅니다. (C) 문화 매체 48 hpi에서 수집되었고, 전염성 입자 생산 CV-1 monolayers에서 상패 분석에 의해 평가되었다. (D) 총 DNA 48 hpi에서 수집되었고, 바이러스 게놈 복제에 평가 된 HSV-1 게놈에 대한 프리 머와 qPCR 분석. PRGAPDH에 대한 imers은 참조로 사용되었습니다. N = 3. 오류 바 ± SEM을 나타냅니다. (E) 모의 - 처리 각막이 sectioned 48 hpi,에 - 플래시 고정하고 감염된 상피 내에 바이러스 초기 단백질 (ICP8)의 존재에 대한 간접 immunofluorescence에 의해 분석 하였다. 핵은 Höchst 33,258으로 counterstained 있습니다. (F) 단백질 lysates 48 hpi에서 수집되었고, 바이러스 단백질 (ICP0)의 표현은 서양 얼룩에 의해 평가되었다. BCA 분석은 샘플의 동등한 로딩을 보장하기 위해 사용되었다. hpi는 = 시간 후 감염.

<ol>
	<li>Kaye, S., Choudhary, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+keratitis.">Herpes simplex keratitis.</a> Prog. Retin. Eye Res. 25, 355-380 (2006).</li><li>Holmberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+of+exotic+pets.">Enucleation of exotic pets.</a> Journal of Exotic Pet Medicine. 16, 88-94 (2007).</li><li>Remeijer, L., Osterhaus, A., Verjans, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+herpes+simplex+virus+keratitis:+the+pathogenesis+revisited.">Human herpes simplex virus keratitis: the pathogenesis revisited.</a> Ocul. Immunol. Inflamm. 12, 255-285 (2004).</li><li>Foreman, D. M., Pancholi, S., Jarvis-Evans, J., McLeod, D., Boulton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+organ+culture+model+for+assessing+the+effects+of+growth+factors+on+corneal+re-epithelialization.">A simple organ culture model for assessing the effects of growth factors on corneal re-epithelialization.</a> Exp. Eye Res. 62, 555-564 (1996).</li><li>Xu, K. P., Li, X. F., Yu, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+organ+culture+model+for+assessing+epithelial+responses+to+surfactants.">Corneal organ culture model for assessing epithelial responses to surfactants.</a> Toxicol. Sci. 58, 306-314 (2000).</li><li>Klausner, E. A., Peer, D., Chapman, R. L., Multack, R. F., Andurkar, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+gene+therapy.">Corneal gene therapy.</a> J. Control Release. 124, 107-133 (2007).</li><li>Halford, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICP0+antagonizes+Stat+1-dependent+repression+of+herpes+simplex+virus:+implications+for+the+regulation+of+viral+latency.">ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency.</a> Virol. J. 3, 44 (2006).</li><li>Bhattacharjee, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+treatment+of+ocular+HSK+with+a+human+apolipoprotein+E+mimetic+peptide+in+a+mouse+eye+model.">Effective treatment of ocular HSK with a human apolipoprotein E mimetic peptide in a mouse eye model.</a> Invest Ophthalmol Vis. Sci. 49, 4263-4268 (2008).</li><li>Novitskaya, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difficulties+imaging+herpes+simplex+keratitis+with+fluorescein+isothiocynate-labeled+anti-HSV-1+antibodies+in+an+ex+vivo+model.">Difficulties imaging herpes simplex keratitis with fluorescein isothiocynate-labeled anti-HSV-1 antibodies in an ex vivo model.</a> Cornea. 28, 421-425 (2009).</li><li>Sharma, A., Shimeld, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+immunofluorescence+to+diagnose+herpes+simplex+virus+keratitis+in+mice.">In vivo immunofluorescence to diagnose herpes simplex virus keratitis in mice.</a> Br. J. Ophthalmol. 81, 785-788 (1997).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 동영상 글에서 우리는 전직 생체 모델에서 급성 각막 상피 HSV-1 감염을 공부하는 방법을 설명합니다. 이 세포 배양 결과의 생리 학적 정확한 검증 가능하며,이를 통해, 수행해야합니다 동물 실험의 양을 제한하기 때문에이 모델은, 생체 모델의 체외에서와 사이에 유용한 스테핑 돌입니다. 또한, 전 생체 모델은 그대로 인간의 각막 상피에 헤르페스 감염을 연구 할 수?...

<table border="1"><tbody><tr><td> 시약 / 용품 </td><td> 회사 </td><td> 카탈로그 # </td></tr><tr><td> 최소 필수 매체 (가상) </td><td> Mediatech </td><td> 10-010 - CV </td></tr><tr><td> 비 필수 아미노산 (100x 솔루션) </td><td> Mediatech </td><td> 25-025 - CI </td></tr><tr><td> L-글루타민 </td><td> Mediatech </td><td> 25-005 - CI </td></tr><tr><td> 페니실린 G 나트륨 소금 </td><td> 시그마 </td><td> P3032 </td></tr><tr><td> 스트렙토 마이신의 황산 소금 </td><td> 시그마 </td><td> S9137 </td></tr><tr><td> 아가로 오스 </td><td> Invitrogen </td><td> 15510-027 </td></tr><tr><td> 인산염은 식염 (PBS)를 버퍼 </td><td> 실험실에서 준비 </td><td> N / A </td></tr><tr><td> 트립신 EDTA (에틸렌 다이아 민 테트라 초산) (1X) &lt;/ TD&gt; <td> Mediatech </td><td> 25-053 - CI </td></tr><tr><td> DNeasy 혈액 및 조직 키트 </td><td> QIAGEN </td><td> 69,504 </td></tr><tr><td> RNeasy 미니 키트 </td><td> QIAGEN </td><td> 74,104 </td></tr><tr><td> Laemmli 버퍼 </td><td> 실험실에서 준비 </td><td> N / A </td></tr><tr><td> 최적의 절삭 온도 (OCT) 컴파운드 </td><td> 조직 - 테크 </td><td> 4583 </td></tr><tr><td> 전체 토끼 눈 (영) </td><td> 펠 - Freez </td><td> 41211-2 </td></tr><tr><td> 인간 각막 및 전체 눈 </td><td> 지역 안구 은행 </td><td> N / A </td></tr><tr><td> 12 자 자기 스포트 플레이트 </td><td> 아보가드로의 실험실 공급 </td><td> 1877 </td></tr><tr><td> 35mm 조직 문화 요리 </td><td> 매 </td><td> 353001 </td></tr><tr><td> 음 6세포 배양 플레이트 </td><td> Greiner 바이오 하나 </td><td> 657160 </td></tr><tr><td> Cryomold 중급 </td><td> 조직 - 테크 </td><td> 4566 </td></tr></tbody></table>

ex vivo organotypic corneal model of acute epithelial herpes simplex virus type i infection

헤르페스 각막염은 헤르페스 심플 바이러스 타입 1 (HSV-1)과 관련된 가장 심각한 pathologies 중 하나입니다. 헤르페스 각막염은 현재 선진국에서 모두 각막 - 파생 및 감염 관련 실명의 가장 큰 원인입니다. 헤르페스 각막염의 전형적인 프리젠 테이션, 흉터 등 영구적 인 각막 pathologies로 이어지는 시닝, 그리고 불투명도 1, 각막 상피의 감염, 때로는 깊은 각막 기질과 내피가 포함되어 있습니다. 각막 HSV-1 감염은 전통적으로 실험 모델의 두 가지 유형에서 공부하고 있습니다. 각막 상피 세포의 배양 monolayers는 배양 접시에 감염되는 체외 모델에이 간단, replicability의 높은 수준, 빠른 실험, 그리고 상대적으로 낮은 비용을 제공합니다. 한편, 토끼 나 쥐와 같은 동물이 각막에 직접 주사되는 생체 모델에는 매우 복잡한 생리를 제공합니다시스템,하지만 높은 비용, 더 이상 실험 필요한 동물 보호, 그리고 변화의 큰 학위를 취득했습니다. 이 비디오 문서에서는, 우리는 생체 모델의 체외과 모두의 장점을 결합하여 각막 상피 HSV-1 감염의 새로운 전직의 생체 모델에 대한 자세한 데모를 제공합니다. 예 생체 모델은 organotypically 문화에 유지 관리 및 HSV-1에 감염된 그대로 각막을 활용합니다. 예 생체 모델의 사용은 높은 생리 학적 기반의 결론은 가능하지만 그건 오히려 저렴하고 체외 모델에서의 비교 시간 약속을해야합니다.

Watch this Scientific Journal Video about Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection at JoVE.com

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

이 동영상 글에서 우리는 새로운의 사용을 설명<em&gt; 예 생체</em급성 헤르페스의&gt; 모델은 바이러스 유형 I 각막 상피​​ 감염을 단순.

급성 상피 헤르페스의 예 VIVO Organotypic 각막 모델은 바이러스 종류 나 감염을 단순

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

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JoVE Journal

Neuroscience

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

12.6K 조회수.  Drexel University College of Medicine. 이 동영상 글에서 우리는 새로운의 사용을 설명 예 생체 모델은 바이러스 유형 I 각막 상피​​ 감염을 단순.

비디오: Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

전체의 전의 생체내의 Culturing, 개발 Drosophila의 뇌는

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

연구

신경과학

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

본래 Vomeronasal 기관 및 액세서리 후각 망울의 생체 준비

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

<em&gt; 전의 VIVO</em&gt; 레이저에 의한 척수 손상 모델은 실시간으로 축삭 변성의 메커니즘을 평가하기 위해

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

맥락총 상피 세포의 섬모 운동의 관측<em&gt; 전의 VIVO</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

뇌 조각의 공포 회로의 예 생체 Optogenetic 해부

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

용매 클리어 뇌 조직에서 광견병 바이러스 감염의 고해상도 3D 이미징

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

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

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

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

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

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

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

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

인간 줄기 세포 이식 및 검증을 위한 생체 외 모델로서의 성인 인간 피질의 기관형 배양

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

AMD 연구를 위한 인간 기증자 안구의 OCT 기반 멀티모달 이미징

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

저자 스포트라이트: 연령 관련 황반변성 연구를 위한 인간 기증자 눈의 Ex Vivo OCT 기반 다중 모드 이미징  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

Ex Vivo (생체 외 ) 연령 관련 황반변성 연구를 위한 인간 기증자 눈의 OCT 기반 다중 모드 이미징

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...