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'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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure change. With the increase of AD patients worldwide, the pathology and treatment of the disease has become a focus in the International Pharmaceutical Industry. Thus, the establishment of the animal model to mimic AD in the laboratory is of great importance.
Here, we describe a detailed protocol for establishing a mimic of AD in a rat animal model though intracerebroventricular injection of amyloid beta protein 25-35 (A&#946;25-35) combined with aluminum trichloride (AlCl3) and anterodorsal thalamic nucleus injection of recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) to rats. The related markers of AD were measured, including: spatial memory, neuronal structure and substructure, neuronal A&#946;, and neurofibrillary tangles (NFT) production. This rat model demonstrates spatial memory impairment, neuronal structure and substructure pathological changes, neuron intracellular A&#946; burden, and NFT aggregation, and provides a close mimic of the neuronal structure and function disorder to that of clinical AD patients. Thus, the presented AD rat modelprovides a valuable in vivo tool for exploring neuronal function, neuronal pathology, and drug screening of AD.

This is a protocol to mimic Alzheimer&#39;s Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (A&#946;) burden, and neurofibrillary tangles aggregation, induced by the injection of A&#946;25-35 combined with aluminum trichloride and recombinant human transforming growth factor-&#946;1.

합성 아 밀 로이드 베타 단백질의 Intracerebroventricular 주입에 의해 쥐 동물 모델에서 Alzheimer의 질병의 귀중 한 모방의 설립

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

연구

행동분석학

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Cisterna 마그나 마우스에서 뇌척수의 직렬 그룹에 대한 기술

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

생물학

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 ...

신경과학

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

인간 A53T를 과발현 형질 전환 마우스의 뇌에 강화 된 ELISA에 의한 질병 관련 α-synuclein의 검출은 α-synuclein의 돌연변이

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

의학

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

일반 마우스의 아밀로이드-β의 펩티드의 뇌 실내 주사는 급성 치매와 같은인지 적자를 유도합니다

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

상호 빛과 전자 현미경은 β - 아밀로이드 플라크와 소교의 상호 작용을 연구하는

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

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.

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 ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

양적 3D<em&gt; 인 실리코</em알츠하이머 병의 쥐 모델에서 뇌 아밀로이드 - 베타 탐식&gt; 모델링 (q3DISM)

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

아밀로이드 -β 및 타우 신경 병증의 유전자 변형 마우스 모델에서 자발적 교대, 새로운 대상 인식 및 사지 걸림의 평가

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

알츠하이머 질병 마우스 모델에서 Neuritic Plaques의 감지

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