이 작품은 건강 연구의 캐나다 연구소 (CIHR), 타운센드 가족, 잭 브라운 가정 알츠하이머 연구 재단 (WAS)를 지원했습니다. WAS는 알츠하이머 병에있는 캐나다 연구 의자의 소유자입니다. PTTL는 자연 과학 및 공학 연구 상담 및 건강 연구 장학금에 대한 마이클 스미스 재단에서 지원됩니다.

1. 마우스 두뇌의 고정 및 cryosectioning <ol><li> 유전자 변형 생쥐는 잘린 및 추출 두뇌 뒤에 이산화탄소 가스를 사용하여 희생하고 있습니다. </li><li> 두뇌는 중앙에 길이 방향 해부하는 것입니다. 두뇌의 절반은 생화 학적 분석을위한 드라이 아이스에 냉동 플래시됩니다. 다른 4 적어도 48 시간 동안 4 % paraformaldehyde (PFA)에 빠져들 것이다 ° C. </li><li> 최소한 48 시간 4% PFA에 고정 후, 헤미의 두뇌는 30 % 자당 용액에 직접 전송하고 4 배치 ° C. 시점에서, 헤미의 두뇌는 자당 솔루션에 떠 있어야합니다. 헤미의 두뇌는 보통 48시간에 대한 요구, 아래로 침몰하면 cryosectioning 위해 OCT에 포함된 될 준비가 된 것입니다. </li><li> cryosectioning 전에 손잡이에서 OCT의 얇은 레이어를 탑재하고 -20 ° C.에서 프리즈 수 냉동 OCT의 레이어에 헤미의 두뇌와 장소의 나머지에서 소뇌를 제거합니다. 즉시 OCT와 헤미 두뇌를 커버 천천히 -20 ° C.에서 프리즈 수 두뇌가 불투명 켜져있는 OCT의 포함되면, 그것은 sectioned 될 준비가 된 것입니다. </li><li> 30 μm의 각 부분의 두께를 설정합니다. D&#39; Olomos 솔루션과 용기에 각각의 슬라이스를 수집합니다. </li></ol> 2. neuritic plaques에 대한 Immunohistochemistry 절차 (무료 부동 섹션) <ol><li> 15 분 동안 88% 개미 산성의 각 섹션을 처리합니다. 개미 산성 치료 후 섹션은 일시적으로 불투명 설정하고, 주름에 나타날 수 있습니다. </li><li> 부드러운 잡고 5 분 X 3 TX - PBS (0.3 % 트리톤 PBS에서 X - 100)와 접시와 세척에 각 섹션을 제거합니다. </li><li> 블록 내생 과산화수소는 0.5 % TX - PBS, RT에서 H 2 O 2 부드러운 잡고 30 분 차단. </li><li> 부드러운 잡고 10 분 X 3 TX - PBS로 섹션을 씻으십시오. </li><li> 부드러운 잡고 1 시간 동안 실온에서 TX - PBS에 용해 5% 아닌 지방 탈지 우유와 함께 블록 섹션을 참조하십시오. </li><li> TX - PBS는 4에서 5 %의 우유를 포함하는으로 희석 주 항체의 섹션 (4G8을 Biotinylated, 1:500-1:2000, 인장) ° C 부드러운 잡고 하룻밤. 품어 </li><li> 부드러운 잡고 10 분 X 3 TX - PBS로 섹션을 씻으십시오. </li><li> ABC 솔루션 다음과 같은 제조 업체의 프로토콜 (엘리트 ABC, 벡터 연구소를) 준비. 부드러운 잡고 30 분 ABC 혼합물의 섹션을 품어. </li><li> 부드러운 잡고 10 분 X 3 TX - PBS로 섹션을 씻으십시오. </li><li> 3,3 &#39;- diaminobenzidine의 tetrahydrochloride (DAB) (벡터 연구소) 다음과 같은 제조 업체의 프로토콜을 준비합니다. 50-10 분 DAB 혼합물의 섹션을 품어. 이 시점에서, 당신은 갈색 색상이 각 부분에 대한 개발 준수합니다. </li><li> 부드러운 잡고 10 분 X 3 TX - PBS로 섹션을 씻으십시오. </li><li> 자유 부동 뇌 섹션은 조직 부착을 도울 수 있도록 젤라틴 코팅 슬라이드에 장착됩니다. 섹션 공기가 크실렌의 일련 취소하여 다음과 Entellen에 탑재 건조 후 수 있습니다. </li><li> Plaques은 40X 배율의 빛을 현미경 하에서 시각 있으며, 각각의 마우스에 대한 슬라이스 당 의미 플라크 수를 평가하여 계량하고 있습니다. </li></ol> 3. Thioflavin S는 neuritic plaques의 검출을위한 절차를 염색법 <ol><li> 마우스 희생과 두뇌 추출 절차와 같은 섹션 1.1 1.5로 표시됩니다. </li><li> 산 4-5 헤미 뇌 유리 슬라이드에 섹션과 완전히 얼룩하기​​ 전에 건조 공기 수 있습니다. </li><li> 1 분 70 % 에탄올로 슬라이드를 씻으십시오. </li><li> 1 분 80 %를 에탄올로 슬라이드를 씻으십시오. </li><li> 15 분 동안 필터링 (0.2 μm의 필터) thioflavin S 솔루션 (에탄올 80 %에서 1 %)에 슬라이드를 품어. (Thioflavin S 솔루션은 각각의 사용하기 전에 필터링해야합니다.) 참고 : 빛과 thioflavin S를 보호하고 빛으로부터 스테인드 슬라이드를 보호합니다. </li><li> 1 분 80 %를 에탄올로 슬라이드를 씻으십시오. </li><li> 70 % 에탄올 일분과 슬라이드를 씻으십시오. </li><li> 증류수로 두 번 씻으십시오. </li><li> 수성 장착 미디어와 슬라이드 투명 매니큐어로 밀봉 coverslip 뒤에 최소한 2 시간 동안 짙은 어둠 속에서 건조 수 있도록 마운트 슬라이드. 스테인드 섹션 4의 어둠 속에 보관해야 ° C. </li><li> 녹색 형광 스테인드 plaques는 형광 microscropy과 시각 수 있습니다. 얼룩이 시간 함께 사라질 것이다 때문에 일주일 이내 슬라이드를 분석할 수 있습니다. </li></ol> 4. 대표 결과 안티 간질 약물과 neuritic 플라크 형성을 억제하는 기분 안정제 valproic 산성 (VPA)의 효능에 대한 최근 연구에서는, 우리는 위에서 설명한 immunostaining을 사용하고 thioflavin S는 광고 모델 마우스 (3)에서 neuritic plaques를 식별하는 절차를 염색법. 그림 1은 안티 Aβ 사용하여 biotinylated 4G8 물들일과 ABC 방식을 통해 감지 전형 9개월 된 APP23 형질 전환 마우스를 나타냅니다. Neuritic plaques는 명확하게 항체으로 표시되며 흰색 화살표로 표시됩니다. thioflavin S 얼룩을 사용하여, 우리는 2 달이 넘은 APP23xPS 트라 neuritic 플라크 증착을 감지nsgenic 마우스 (그림 2). Thioflavin S 바운드 Aβ 함유 neuritic의 plaques은 형광 현미경 (흰색 화살표로 표시)에서 녹색으로 나타납니다. 우리는 VPA 치료와 neuritic 플라크 형성이 상당히 (3) 감소는 것을 보여주었다. 또 다른 연구에서는, 우리는 분자 링크를 기본 hypoxia 및 광고 pathogenesis을 공부했다. 다시 두 4G8 방지 Aβ immunohistochemistry의 얼룩과 thioflavin S 염색법을 사용하여, 우리는 hypoxia가 neuritic plaques (4)이있는 Aβ의 형성을 촉진 발견했습니다. <img src="/files/ftp_upload/2831/2831fig1.jpg" alt="그림 1"/> 그림 1. AD 유전자 변형 생쥐에서 neuritic 플라크 형성 Immunohistochemical 분석 9. 개월 나이에 APP23 생쥐가 희생되었고 해부했다는 고정하고, sectioned. hippocampal에 Neuritic plaques는 안티 Aβ 항체 4G8를 사용하여 감지되었으며 ABC와 DAB 방법을 사용하여 개발되었습니다. plaques은 40X로 가벼운 현미경에 의해 시각되었습니다. 화이트 화살표가 Aβ의 neuritic plaques을 가리 킵니다. <img src="/files/ftp_upload/2831/2831fig2.jpg" alt="그림 2"/> 그림 2. AD 모델 생쥐에서 neuritic plaques의 Thioflavin S 얼룩. 올드 8주에서 APP23xPS45 두 유전자 변형 생쥐는 희생되었고 해부되었다, 고정하고, sectioned. hippocampal에 Neuritic 재앙은 thioflavin S 형광 염색법을 사용하여 확인 및 40X 배율에서 현미경에 의해 시각되었습니다 있습니다. 화이트 화살표 thioflavin S 스테인드 neuritic의 plaques를 나타냅니다.

<ol>
	<li>Glenner, G. G., Wong, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+initial+report+of+the+purification+and+characterization+of+a+novel+cerebrovascular+amyloid+protein.">Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein.</a> Biochem Biophys Res Commun. 120, 885-890 (1984).</li><li>Hardy, J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Qing, H., He, G., Ly, P. T., Fox, C. J., Staufenbiel, M., Cai, F., Zhang, Z., Wei, S., Sun, X., Chen, C. H., Zhou, W., Wang, K., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproic+acid+inhibits+Abeta+production,+neuritic+plaque+formation,+and+behavioral+deficits+in+Alzheimer's+disease+mouse+models.">Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models.</a> J Exp Med. 205, 2781-2789 (2008).</li><li>Sun, X., He, G., Qing, H., Zhou, W., Dobie, F., Cai, F., Staufenbiel, M., Huang, L. E., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+facilitates+Alzheimer's+disease+pathogenesis+by+up-regulating+BACE1+gene+expression.">Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression.</a> Proc Natl Acad Sci U S A. 103, 18727-18732 (2006).</li></ol>

모든 실험은 브리티시 컬럼비아 애니멀 케어 및 사용위원회 CIHR 지침의 대학에 따라 실시되었다.

biotinylated 표시 4G8 항체 얼룩에게 광고 neuritic plaques를 사용 Immunohistochemistry은 특이성과 쥐를 모델. 얼룩의 결과는 부량 및 다른 치료 그룹 사이의 플라크 부하의 비교를하실 수 있습니다. 결과에 영향을 미칠 수있는 몇 가지 중요한 단계가 있습니다. 헤미의 두뇌는 두개골에서 추출하기 전에 perfused 있지 않기 때문에,주의가 헤미 두뇌에 손상을 방지하기 위해 추출 과정에서 사용해야합니다. 헤미의 두뇌가 수동적으로 perfused이기 때문에 더욱이, 우리는 4 적어도 48 시간 4퍼센트 PFA에 잠복기 권장 ° C 이전에 30 % 자당 솔루션 잠수함이 잠수합니다. 또는 transcardial 재관류는 두뇌를 추출하기 전에 할 수 있습니다. 두뇌가 제대로 고정되면, 그것은 고무 질감이 있어야합니다. 두뇌가 제대로 고정되지 않은 경우에는 섹션 쉽게 D&#39; Olomos 솔루션이나 얼룩 절차 중에 눈물 것입니다. 4G8 단클론 항체는 아미노산 잔류물 Aβ의 17-24에 반응하고 핵심 시퀀스 (VFFAE)에 에피토프을 인식. 4G8가 마우스 수종에서 파생된 것이므로, 그것은 높은 배경 얼룩을주는 경향이있다. 따라서, 우리는 배경 얼룩을 최소화하면서 고려 진정한 신호를 달성하기 위해 함께 표시된 희석에서 섹션을 품어하기로 결정했습니다. 4G8 항체를 사용하여 neuritic plaques 감지 immunostaining 이외에도, thioflavin S 얼룩 방법도 plaques을 식별하는 데 사용됩니다. Thioflavin S는 동질적인 염료 혼합물이다 sulfonic의 산성과 dehydrothiotoluidine의 methylation 결과에서 해당. Thioflavin S가 아닌 선택과 같은 아밀로이드 oligomers 이들과 같은 단백질의 베타 시트 내용을 바인딩합니다. 바인딩시 thioflavin는 방출 스펙트럼의 특성 푸른 변화를 겪습. 반대 monomeric 양식 thioflavin S 바인딩은 파란 변화를 이끌어내는하지 않으며 형광 현미경으로 발견하지 못했습니다. 형광의 강도가 아밀로이드 예금 소량의 좋은 시각화를 허용 등 Thioflavin S 얼룩이 아밀로이드에 대한 화면으로 빠른 대안을 제공합니다. 그러나, thioflavin S 얼룩 얘기가 하나 단점은 특이성의 부족이다. 이러한 fibrinoids, 유리 모양의, 각질 등으로 포함하는 광범위한 베타 시트를 포함한 많은 다른 조직 구성이 염료에 대한 다소 친근감을 가지고 있습니다.

<table border="1"><tbody><tr><th> 시약 및 장비의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> Paraformaldehyde </td><td> 시그마 - 알드리치 </td><td> P6148 - 1KG </td></tr><tr><td> Polyvinylpyrrolidene </td><td> 시그마 - 알드리치 </td><td> PVP40 - 100G </td></tr><tr><td> 자당 </td><td> 피셔 사이 언티픽 </td><td> S5 - 3 </td></tr><tr><td> 에틸렌 글리콜 </td><td> 시그마 - 알드리치 </td><td> 324558 - 2L </td></tr><tr><td> 조직 테크 OCT 컴파 운드 </td><td> 사쿠라 </td><td> 4583 </td></tr><tr><td> 88% 개미 산성 </td><td> 피셔 사이 언티픽 </td><td> A118P - 500 </td></tr><tr><td> 트리톤 X - 100 </td><td> ICN의 Biomedicals </td><td> 807426 </td></tr><tr><td> H2O2 </td><td> 시그마 - 알드리치 </td><td> H - 1009 </td></tr><tr><td> 비오틴은 4G8 항체를 라벨 </td><td> 시더 레인 </td><td> SIG - 39240-500 </td></tr><tr><td> 엘리트 ABC 키트 </td><td> 벡터 </td><td> PK - 6100 </td></tr><tr><td> IMM 협정 DAB </td><td> 벡터 </td><td> SK - 4105 </td></tr><tr><td> 크실렌 </td><td> 피셔 사이 언티픽 </td><td> X4 - 4 </td></tr><tr><td> Entellan </td><td> EM 과학 </td><td> 65037-71 </td></tr><tr><td> Superfrost의 * 플러스 현미경 슬라이드 </td><td> 피셔 사이 언티픽 </td><td> 12-550-15 </td></tr><tr><td> 그라 이오 스탯 </td><td> Leica </td><td> CM - 3050 - S </td></tr><tr><td> MX35 프리미어 마이크로톰 블레이드 </td><td> 써모 과학 </td><td> 3,052,835 </td></tr><tr><td> Thioflavin S </td><td> 시그마 - 알드리치 </td><td> T1892 - 25G </td></tr></tbody></table>

detection of neuritic plaques in alzheimer's disease mouse model

알츠하이머 병 (AD)은 치매로 이어지는 가장 일반적인 neurodegenerative 무질서이다. Neuritic 플라크 형성이 알츠하이머 질환의 병리 학적 특징 중 하나입니다. neuritic plaques의 중심 구성 요소는 β - secretase와 γ - secretase에 의해 베타 아밀로이드 전구체 단백질 (APP)의 순차 proteolytic 절단에서 파생되는 작은 filamentous 단백질이라는 아밀로이드 β 단백질 (Aβ) 1입니다. 아밀로이드 가설 AD 병리 2 기본 독성 메커니즘으로 plaques을 Aγ 함유 것을 알아볼 수 있습니다. neuritic 플라크의 존재의 사후 분석 AD의 진단을 확인합니다. 광고 pathogenesis에 Aγ 신경 생물학 우리의 이해를 더하기 위해 인간의 APP의 유전자에 AD 관련 변이를 표현하는 다양한 마우스 종자가 생성되었습니다. 질병의 정도에 따라,이 마우스는 다른 연령대에 neuritic plaques을 개발합니다. 이 생쥐는 APP 처리 경로와 neuritic 플라크 형성에 영향을 미칠 수있는 pathogenesis 및 약물 개발 연구를위한 귀중한 도구 역할을합니다. 이 프로토콜에서는, 우리는 광고 모델 마우스에서 neuritic plaques의 구체적인 검출을위한 immunohistochemical 방법을 사용합니다. thiofalvin S 얼룩 방법을 사용하여 ABC​​와 DAB 방법과 형광 검출을 사용 immunohistochemical 감지 : 우리는 구체적으로 절반 뇌, paraformaldehyde 고정, cryosectioning 및 광고 형질 전환 마우스에서 신경질 plaques를 감지하는 두 가지 방법을 추출에서 준비에 대해 설명합니다.

Watch this Scientific Journal Video about Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model at JoVE.com

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

AD의 병리 학적 특징 중 하나는 아밀로이드 β 단백질의 긍정적인 neuritic plaques의 형성이다. immunohistochemical 감지 thioflavin S 얼룩 방법을 사용하여 ABC​​와 DAB 방법과 형광 검출을 사용하여이 프로토콜에서 우리는 유전자 변형 AD 모델 마우스에서 neuritic plaques를 감지하는 두 가지 방법을 설명합니다.

알츠하이머 질병 마우스 모델에서 Neuritic Plaques의 감지

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

detection-of-neuritic-plaques-in-alzheimer-s-disease-mouse-model

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JoVE Journal

Neuroscience

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

36.5K 조회수.  The University of British Columbia. AD의 병리 학적 특징 중 하나는 아밀로이드 β 단백질의 긍정적인 neuritic plaques의 형성이다. immunohistochemical 감지 thioflavin S 얼룩 방법을 사용하여 ABC​​와 DAB 방법과 형광 검출을 사용하여이 프로토콜에서 우리는 유전자 변형 AD 모델 마우스에서 neuritic plaques를 감지하는 두 가지 방법을 설명합니다.

비디오: Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

β - 아밀로이드 Oligomeric의 준비 1-42</subHippocampal 조각에 시냅틱 소성 장애의>와 유도

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

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Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &amp;#946;-Protein, the Causative Agent of Alzheimer&#039;s Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

아밀로이드 β 단백질, 알츠하이머 병의 원인이되는 에이전트에 대한 Aptamers의 선택

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

두 광자 현미경을 사용하여 마우스 척수의 생체내 이미징에

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

알츠하이머병 모델 마우스에서 학습 및 메모리 적자에 대한 모리스 워터 메이즈 테스트

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

내생 NADH 형광의 2 광자 이미징에 의한 마우스 대뇌 피질의 Microregional Hypoxia의 탐지

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

영구와 광범위한 신경 세포의 형질 도입에 대한 신생아 마우스 뇌의 뇌실 바이러스 감염

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

마우스 해마에 올리고머 아밀로이드 - 베타의 정위 주입

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of correlation of the BOLD signal in spatially independent regions of the brain defines the functional connectivity of those regions. During a cognitive fMRI task, a psychophysiological interaction (PPI) analysis can be used to examine changes in the functional connectivity during specific contexts defined by the cognitive task. An example of such a task is one that engages the memory system, asking participants to learn pairs of unrelated words (encoding) and recall the second word in a pair when presented with the first word (retrieval). In the present study, we used this type of associative memory task and a generalized PPI (gPPI) analysis to compare changes in hippocampal connectivity in older adults who are carriers of the Alzheimer's disease (AD) genetic risk factor apolipoprotein-E epsilon-4 (APOE&#949;4). Specifically, we show that the functional connectivity of subregions of the hippocampus changes during encoding and retrieval, the two active phases of the associative memory task. Context-dependent changes in functional connectivity of the hippocampus were significantly different in carriers of APOE&#949;4 compared to non-carriers. PPI analyses make it possible to examine changes in functional connectivity, distinct from univariate main effects, and to compare these changes across groups. Thus, a PPI analysis may reveal complex task effects in specific cohorts that traditional univariate methods do not capture. PPI analyses cannot, however, determine directionality or causality between functionally connected regions. Nevertheless, PPI analyses provide powerful means for generating specific hypotheses regarding functional relationships, which can be tested using causal models. As the brain is increasingly described in terms of connectivity and networks, PPI is an important method for analyzing fMRI task data that is in line with the current conception of the human brain.

Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer&#039;s Disease

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

메모리의 일반화 된 정신 상호 작용 (PPI) 분석 관련 Alzheimer의 질병을 위한 유전 모험에 개인에 연결

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

효소 기반의 바이오 센서를 사용하면 알츠하이머 병 마우스 모델에서 토닉과 Phasic이 품목을 측정하는

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

신생아 Hypoxic-허 혈 성 뇌 손상의 마우스 모델에서 Neurobehavioral 평가

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...