저자는 예방 접종에 대한 데미안 가일 라드 감사 및 후속 동물 실험을하고 싶습니다. 이 작품은 ANSES (식품, 환경 및 산업 보건 및 안전을위한 프랑스 기관)에 의해 재단 프랑스 파킨슨에서 교부금에 의해 지원되었다.

모든 절차와 동물을 포함하는 프로토콜은 EC 지침 609분의 86 / EEC 및 오는, 동물 실험 (프로토콜 11-0043)에서 윤리의 고려에 대한 프랑스의 국가위원회의 비준을 따라했다. 동물 보관 및 ANSES의가 (0801 69387 승인 B) 리옹에 실험 시설을 승인에 마음에 든다고했다. 마우스 1. 준비 <ol><li> 펜토 바르 비탈 나트륨의 치사량의 복강 내 주사하여 마우스를 안락사. </li><li> 마우스 두개골에서 뇌 전체를 검색 및 추출까지 얼음에 35mm 플라스틱 페트리 접시에 넣습니다. </li><li> 자궁 경부 척수의 압축을 풉니 다. 참고 : 추출 αS 중 하나를 표 1에 나와있는 실험을 사용할 수 없습니다 시상 단면 후 또는 해부 마우스의 뇌에서 뇌 반쪽의 하나에서. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Experimen티 </td><td> 마우스 접종 (뇌에 해당) </td><td> 생존 기간 수 (dpi) </td><td> 중간 / 최대 생존 (일 이전) </td><td> 엘리사에 의해 D 감지 αS / WB / IHC </td></tr><tr><td> 1 </td><td> Uninoculated 마우스 </td><td> 441 ± 166 </td><td> 736분의 419 </td><td> 8/8 </td></tr><tr><td> (2) </td><td> 접종 한 마우스 (0.2 mg)을 </td><td> 150 ± 52 </td><td> 241분의 140 </td><td> 9/9 </td></tr></tbody></table> M83 마우스에서 수행 실험 표 1. 목록은. 예방 접종은 후, (포도당 1 % 중량 / 권 5 %) 아픈 마우스의 뇌 균질의 20 μL와 striato - 대뇌 피질의 영역에서 실험이 6 주에 실시 하였다 3 % 이소 플루 란 흡입 6 주 오래 된 동형 접합 M83 마우스의 마취. dpi로는 : 일 inocul 게시ATION. 뇌 반쪽 2. αS 추출 <ol><li> Sagittally 두 개의 반쪽을 얻기 위해 뇌를 잘라. 그라인딩 볼을 넣은 ribolysis 튜브에 각각 절반을 단다. </li><li> 에 50 mM Tris-HCl, pH 7.5, 750 mM의 염화나트륨, 5mM의 EDTA, 1mM의 DTT, 1 % 인산 및 프로테아제 저해제 칵테일을 함유 고염 (HS) 버퍼를 준비한다. 20 % (중량 / 부피) 균질를 얻기 위해 뇌의 절반에 높은 소금 버퍼를 추가합니다. </li><li> 6.0 M에서 기계 균질화를 사용하여 뇌의 반쪽에서 샘플을 준비 / S 23 초 동안 두 번. 첫 번째 23 초 균질화 한 후, 두 번째 23 초주기 전에 2 분 동안 얼음에 균질을 포함하는 튜브를 배치합니다. </li><li> 5 분 unground 뇌 조각을 제거하기 위해 1,000 XG에서 샘플을 원심 분리기. 200 μL 씩에 분할 이후 ELISA 분석을 위해 -80 ° C에서 그들을 유지, 상층 액을 복구 할 수 있습니다. </li></ol> 해부 두뇌 지역에서 3 αS 추출 <ol><li> 해부끝 해마를 해부 할 때를 제외하고 함께 보관이 forcipes를 사용하여 낮은 전력 돋보기 (8 배 확대)와 얼음에 35mm 플라스틱 페트리 접시에 전체 뇌. 뇌의 무결성을 유지하기 위해 10 분을 초과하지 마십시오. 뇌를 놓고 바로 옆이 순서대로 다음과 같은 뇌 영역을 검색 : <ol><li> 단지 전구 뒤에 배치 집게를 사용하여 두 개의 후각 전구 별도의 하나. 아래쪽으로 이동에 의해 뇌에서 분리. 두 번째 전구에 대해이 작업을 반복합니다. </li><li> 조심스럽게 두 대뇌 피질 사이에 집게를 쐐기 두 대뇌 피질의 분리를 용이하게하기 위해 앞으로 이동합니다. 한 집게와 장소에 두뇌를 유지, 해마에서 대뇌 피질을 분리하는 또 다른를 사용합니다. </li><li> 집게에게 피질 아래 2mm를 놓습니다. 해마의 상단이 보일 때까지 집게에 부드러운 압력을 유지한다. 피질의 첫 번째 부분을 벗겨, 두 번째 부분 반복합니다. 두 개의 대뇌 피질을 분리 집게를 사용하여해마에서 시작하여 뇌의 앞쪽으로 이동. </li><li> 해마 중 하나 주위에 오픈 집게를 놓습니다. 부드럽게 최대한 복구, 제거 해마의 하단에있는 집게를 닫습니다. 두 번째 해마에 대해이 절차를 반복합니다. </li><li> 선조체의 하나 아래 열린 집게를 놓고 부드럽게 뇌에서 분리. 선조체에서 남아있는 피질을 제거하기 위해 집게를 사용합니다. 두 번째 선조체에 대해이 절차를 반복합니다. </li><li> 부드럽게 뇌에서 소뇌의 분리를 용이하게하기 위해 2mm 소뇌의 윤곽에 의해 우울하기 위해 집게를 사용합니다. 다만 소뇌 뒤에 집게를 놓고 앞으로 집게를 이동하여 제거합니다. </li><li> 이 뇌간에 가입되는 시점을 명확하게보기 위해 중뇌 인상 집게의 넓은 부분을 사용합니다. 교차점에서 수직 절개 후 뇌간을 제거합니다. </li><li> , 중뇌 뒤 집게를 배치하는 나는네 개의 둥근 구조로 구성 s의. 중뇌가 완전히 남아있는 뇌에서 분리 될 때까지 수직으로 절개. </li></ol></li><li> 가변 % HS 버퍼 (중량 / 부피), 가능한 조직의 양에 따라, 즉, 10 및 30 mg의 간 중량 5 % 파쇄, 30 및 80 mg의 간 중량 10 %, 및 20 %의 균질 준비 80 mg의 위의 무게. <ol><li> 균질 예상 %를 얻기 위해 해부 뇌 영역에 HS 버퍼의 적절한 볼륨. </li><li> 소용돌이와 조직을 완전히 고속 버퍼에 포장되어 있는지 확인합니다. </li><li> 붕규산 유리 튜브 및 두 방앗, 및 B. 이루어지는 조직 분쇄기로 해부 뇌 영역 또는 자궁 척수로부터 제조 된 샘플 균질화 </li><li> 튜브에 직접 분쇄 할 각 뇌 영역을 따르십시오. 튜브에 유봉을 삽입하고 철회. 조직을 떼어 놓다하기 위해이 운동을 약 10 회 반복한다. 그리고 C에 유봉 (B)를 사용상기 (20)의 움직임과 함께 조직 연마 ontinue. 1 ml의 전송 피펫 1.5 ML 튜브에 균질을 전송합니다. </li></ol></li><li> 어떤 unground 뇌 조각을 제거하기 위해 4 ℃에서 5 분 동안 1,000 XG에서 샘플을 원심 분리기. , 상층 액을 검색 200 μL 씩에 그들을 분할 이후 ELISA 분석을 위해 -80 ℃에서 보관하십시오. </li></ol> ELISA에 의해 αS 4. 검출 <ol><li> 0.01 ng를 / ㎖으로 코팅 된 항체를 희석. 50 mM의 나 2 CO 3 / NaHCO3를 완충액 (pH 9.6)에 반 αS 토끼 폴리 클로 날 또는 단일 클론 클론 (42) 항체를 사용합니다. </li><li> 코트는 96 웰 마이크로이 코팅 용액을 잘 당 100 μL, 그리고 4 ° CO / N에 둡니다. 검출 항체를 사용하여 ELISA를 syn514 용 코팅 용액에 안티 αS 토끼 폴리 클로 날 항체를 사용하여 42 LB509, AS11, 4D6 또는 8A5 클론. 코팅 용액으로 안티 αS 모노클로 날 항체 클론 (42)을 사용하여항 pSer129 αS 검출 항체와 조합. 주 : 플레이트 ELISA 일주일 전에 4 ℃에서 유지 될 수있다 필요한 경우 수행된다. </li><li> 물론 당 0.05 % 트윈 20 (PBST)와 플레이트를 인산 완충 생리 식염수 300 μL로 5 회 씻어 접시 세척기를 사용합니다. 이후이 단계에서 배양 실온에 있습니다. </li><li> T20 PBS를 잘 당 버퍼를 차단의 200 μl를 추가합니다. 150 rpm에서 1 시간 동안 흔들어. PBST로 판 다섯 번 씻으십시오. </li><li> (: 20 % 균질 100을, 10 %의 균질 1:50 PBST BSA 1 %에서 5 %의 균질 1시 25분 희석 1)를 각 웰에 100 μl를 추가 뇌 균질 액을 희석한다. 150 rpm으로 진탕하면서 다음 2 시간 동안 품어. PBST로 판 다섯 번 씻으십시오. </li><li> 자료 목록에 언급 된 희석에서 BSA와 PBST 1 %를 다른 αS 검출 항체를 추가합니다. 150 rpm에서 1 시간 동안 품어. PBST로 판 다섯 번 씻으십시오. </li><li> 안티를 추가150 rpm에서 진탕하면서 1 시간 동안 1 % BSA로 보충 PBST로 8000 : - 마우스 또는 항 - 토끼의 IgG HRP 접합체 (1) 희석 하였다. PBST로 판 다섯 번 씻으십시오. </li><li> 각 웰에, 5,5&#39;- 테트라 메틸 벤지딘 (TMB) 솔루션 &#39;3,3의 100 μl를 추가하고 150 rpm으로 흔들어 동안 어둠 속에서 15 분 동안 품어. </li><li> 그럼 마이크로 플레이트 리더와 450 nm에서 흡광도 측정에 1 N의 HCl 100 μl를 첨가하여 반응을 정지. </li><li> 데이터 분석을 위해, 분석 시료의 각각에 대해 측정 된 OD 값으로부터 임의의 마우스 뇌 샘플 (블랭크 웰)을 제외하고 모든 시약 웰에서 얻어진 OD 값을 뺀다. </li></ol> 5. 에피토프 매핑 <ol><li> 오스만 (16)에 의해 기술 된 방법에있어서, 에피토프 맵핑을 수행한다. 간략하게, 10 중첩 아미노산 니트로 셀룰로오스 12 개의 아미노산을 함유하는 인간 α-synuclein의 시퀀스의 스폿 펩타이드. </li><li> 50mM 트리스 / 150 mM의 염화나트륨 완충액 pH 1 차단0, 0.05 % 트윈 20, 5 % 우유 분말을 함유. 2-10 ° CO / ml의 N에서 당 2 μg의 항체 농도에서 차단 용액에 항체 인큐베이션. </li><li> 세포막을 50mM 트리스 / 150 염소 항 - 마우스 IgG HRP 컨쥬 게이트 0.05 % 트윈 20을 함유하는 인큐베이션 mM의 염화나트륨 완충액 pH 10을 사용하여 세 번 씻는다. 다음 같은 버퍼를 사용하여 다른 5 배 웨스턴 블롯 TMB 염색 키트를 사용하여 얼룩 막 씻으십시오. </li></ol> 6. 통계 분석 <ol><li> 외경을 모델로 혼합 효과 회귀 분석을 수행하기 위해 R 소프트웨어와 NLME 패키지를 사용합니다. 각각의 비교를 위해, 혼합 효과 회귀 모형을 수행합니다. 무증상 그룹에서 증상을 구분하는 고정 효과를 사용합니다. </li><li> 주어진 마우스 반복의 변화를 반영하기 위해 임의의 효과를 사용합니다. 잔류 물을 검사하여 homoscedasticity 확인하고, 필요한 경우 및 핀 헤이 베이츠 1에 아울러 집단 내 에러의 분산 구조를 모델링 분산 함수를 사용7. (P)의 중요성 임계 값 0.05을 설정 </li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-linked+immunosorbent+assays+for+alpha-synuclein+with+species+and+multimeric+state+specificities.">Enzyme-linked immunosorbent assays for alpha-synuclein with species and multimeric state specificities.</a> J Neruosci Meth. 199, 249-257 (2011).</li><li>Unterberger, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+disease-associated+alpha-synuclein+in+the+cerebrospinal+fluid:+a+feasibility+study.">Detection of disease-associated alpha-synuclein in the cerebrospinal fluid: a feasibility study.</a> Clin Neuropathol. 33, 329-334 (2014).</li><li>Betemps, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+spreading+in+M83+mice+brain+revealed+by+detection+of+pathological+alpha-synuclein+by+enhanced+ELISA.">Alpha-synuclein spreading in M83 mice brain revealed by detection of pathological alpha-synuclein by enhanced ELISA.</a> Acta Neuropathol. (Berl). 2, 29 (2014).</li><li>Osman, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+monoclonal+antibody+that+recognizes+a+potential+coeliac-toxic+repetitive+pentapeptide+epitope+in+gliadins).">A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins).</a> Eur J Gastroenterol Hepatol. 13, 1189-1193 (2001).</li><li>Pinheiro, J. C., Bates, D. M., Chambers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+5.">Ch. 5.</a> Mixed-Effects Models in S and S-PLUS. 5, 206-225 (2000).</li><li>Mougenot, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+monoclonal+antibody,+against+human+alpha-synuclein,+in+a+subpopulation+of+C57BL/6J+mice,+presenting+a+deletion+of+the+alpha-synuclein+locus.">Production of a monoclonal antibody, against human alpha-synuclein, in a subpopulation of C57BL/6J mice, presenting a deletion of the alpha-synuclein locus.</a> J Neruosci Meth. 192, 268-276 (2010).</li><li>Specht, C. G., Schoepfer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+alpha-synuclein+locus+in+a+subpopulation+of+C57BL/6J+inbred+mice.">Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice.</a> BMC Neurosci. 2, 11 (2001).</li><li>Lee, B. R., Matsuo, Y., Cashikar, A. G., Kamitani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Ser129+phosphorylation+of+alpha-synuclein+in+melanoma+cells.">Role of Ser129 phosphorylation of alpha-synuclein in melanoma cells.</a> J Cell Sci. 126, 696-704 (2013).</li><li>Perrin, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+mapping+and+specificity+of+the+anti-alpha-synuclein+monoclonal+antibody+Syn-1+in+mouse+brain+and+cultured+cell+lines.">Epitope mapping and specificity of the anti-alpha-synuclein monoclonal antibody Syn-1 in mouse brain and cultured cell lines.</a> Neurosci Lett. 349, 133-135 (2003).</li><li>Emmanouilidou, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+alpha-synuclein+secretion+in+mouse+and+human+brain+parenchyma.">Assessment of alpha-synuclein secretion in mouse and human brain parenchyma.</a> PLoS One. 6, e22225 (2011).</li><li>Mougenot, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+prion+strains+in+a+transgenic+mouse+models+overexpressing+human+A53T+mutated+alpha-synuclein.">Transmission of prion strains in a transgenic mouse models overexpressing human A53T mutated alpha-synuclein.</a> J Neuropathol Exp Neurol. 70, 377-385 (2011).</li><li>Foulds, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+longitudinal+study+on+alpha-synuclein+in+blood+plasma+as+a+biomarker+for+Parkinson's+disease.">A longitudinal study on alpha-synuclein in blood plasma as a biomarker for Parkinson's disease.</a> Sci Rep. 3, 2540 (2013).</li></ol>

The authors have no competing interests to disclose.

ELISA의 사용은 구체적 M83 트랜스 제닉 마우스 모델에서 질병 중에 마우스 뇌 균질 물로부터 직접 αS D를 검출하는 것으로 입증되었다. 사실,이 ELISA 쉽게 높은 소금 버퍼 만 전체 뇌 균질를 사용하여 건강한 M83 마우스에서 아픈 M83 마우스를 구별 할 수있다. 이 ELISA를 사용하여 성공적인 결과를 위해 가장 중요한 단계는 다음과 같습니다 제대로 해부하는 동안 손상을 방...

Most current methods for detecting disease-associated &#945;-synuclein (&#945;SD) in experimental models of Parkinson's disease (PD), such as immunohistochemistry or Western blot, are time-consuming and not quantitative. This neurodegenerative disease is characterized by alpha-synuclein aggregation mainly in the form of inclusions containing an aggregated form of the normally soluble presynaptic protein &#945;S1,2 (Lewy bodies and Lewy neurites). Normally only marginally phosphorylated, &#945;S is hyperphosphorylated at its serine 129 residue in these inclusions3 and can be monitored by antibodies specifically directed against Ser129 phosphorylated &#945;S, thus providing a reliable marker of the pathology. 
Recent research suggests that a &#8220;prion-like&#8221; mechanism could be involved in the propagation of &#945;S aggregation within the nervous system of an affected patient4,5. These studies reported the acceleration of a synucleinopathy by inoculating brain extracts containing &#945;SD into a transgenic mouse model (M83) expressing an A53T mutated human &#945;S protein associated with a severe motor impairment occurring as the mice age6. In the same manner, intra-cerebral inoculation of aggregated recombinant &#945;S in the same M83 mouse model confirmed the acceleration of aggregation5. The induction of deposits of phosphorylated &#945;S has also been reported after inoculation of C57Bl/6 wild-type mice with either fibrillar recombinant &#945;S or brain extracts from human DLB patients7,8. Sacino et al.9 recently pointed out that after injection of fibrillar human &#945;S, a widespread and progressive cerebral &#945;S inclusion formation could be induced in M83 mice, but not in E46K transgenic mice or non-transgenic mice in which induced &#945;S inclusions were transient, and mainly restricted to the site of injection. Recent studies on monkeys confirmed propagation of &#945;S aggregates after inoculation of PD-derived extracts in species closer to humans10.
The link between &#945;S alterations and Parkinson&#8217;s disease suggest that &#945;SD is a potential biomarker for Parkinson&#8217;s disease11. A recent study showed the detection of oligomeric soluble aggregates of &#945;-synuclein in human cerebro-spinal fluid (CSF) and plasma as a potential biomarker for Parkinson&#8217;s disease based on a conventional sandwich system ELISA using the same antibody to capture and detect &#945;S12. Based on the same method, multimeric proteins were recognized in biological samples, including the brain, because there are multiple copies of epitopes present in the assembled forms13. Very recently, pathological &#945;S in the CSF of patients with a proven Lewy body pathology was detected using both an ELISA kit with a highly specific antibody against &#945;SD (5G4) and an immunoprecipitation assay14. These methods could differentiate patients with PD/DLB from other types of dementia.
The &#8220;prion-like&#8221; propagation of &#945;S aggregation was further studied in transgenic mouse model M83 using an ELISA approach that was designed to specifically identify &#945;SD15. In this study, we report the detailed ELISA protocol used to quantitatively detect &#945;SD in sick mice (whether or not inoculated with &#945;SD from sick M83 mice) and more especially in the brain regions specifically targeted by the pathological process in this M83 transgenic mouse model4.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col span="2" />
	</colgroup>
	<tbody>
		<tr>
			<td>LB509&#160;</td>
			<td></td>
			<td>Abcam</td>
			<td>&#160;ab27766</td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>AS11</td>
			<td></td>
			<td>Produced at Anses</td>
			<td></td>
			<td>Detection antibody 1/1000</td>
		</tr>
		<tr>
			<td>4D6&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>ab1903</td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>PSer129&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>&#160;ab59264</td>
			<td>Detection antibody 1/3000</td>
		</tr>
		<tr>
			<td>PSer129&#160;EP1536Y</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>ab51253</td>
			<td>Detection antibody 1/1000</td>
		</tr>
		<tr>
			<td>syn514&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>&#160;ab24717</td>
			<td>Detection antibody 1/500</td>
		</tr>
		<tr>
			<td>clone 42</td>
			<td></td>
			<td>BD Biosciences&#160;</td>
			<td>610787</td>
			<td>Coating and detection antibody (1/2000)</td>
		</tr>
		<tr>
			<td>8A5&#160;</td>
			<td></td>
			<td>Provided by Dr. Anderson</td>
			<td></td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>polyclonal anti-&#945;syn antibody</td>
			<td></td>
			<td>Millipore</td>
			<td>&#160;AB5038P</td>
			<td>Coating&#160; antibody</td>
		</tr>
		<tr>
			<td>Anti-mouse&#160; IgG HRP conjugate</td>
			<td></td>
			<td>Southern Biotech</td>
			<td>1010-05</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Anti-rabbit IgG HRP conjugate</td>
			<td></td>
			<td>Southern Biotech</td>
			<td>4010-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse&#160; IgG HRP conjugate</td>
			<td></td>
			<td>Dianova</td>
			<td>115-035-164</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="4">HS buffer</td>
			<td>Tris-HCl 50 mM&#160;</td>
			<td>Euromedex</td>
			<td>26-128-3094-B</td>
			<td rowspan="4">Adjust at pH 7.5 and keep at 4&#176;C</td>
		</tr>
		<tr>
			<td>NaCl 750 mM&#160;</td>
			<td>Euromedex</td>
			<td>1112-A</td>
		</tr>
		<tr>
			<td>EDTA 5 mM&#160;</td>
			<td>Euromedex</td>
			<td>EU0007-B</td>
		</tr>
		<tr>
			<td>DTT 1 mM&#160;</td>
			<td>Sigma</td>
			<td>43815</td>
		</tr>
		<tr>
			<td rowspan="4">PBS</td>
			<td>Na2HPO4 1 mM</td>
			<td>Euromedex</td>
			<td>1309</td>
			<td rowspan="4">Adjust at pH 7.5</td>
		</tr>
		<tr>
			<td>KH2PO4 1,5 mM</td>
			<td>Euromedex</td>
			<td>2018</td>
		</tr>
		<tr>
			<td>NaCl&#160; 137 mM</td>
			<td>Euromedex</td>
			<td>1112-A</td>
		</tr>
		<tr>
			<td>KCl 2,7 mM</td>
			<td>Euromedex</td>
			<td>P017</td>
		</tr>
		<tr>
			<td>Tween 20&#160;</td>
			<td></td>
			<td>Euromedex</td>
			<td>2001-C</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td></td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT 1 mM</td>
			<td></td>
			<td>Sigma</td>
			<td>43815</td>
			<td>stock solution 100 mM, toxic</td>
		</tr>
		<tr>
			<td>1% phosphatase cocktail</td>
			<td></td>
			<td>Pierce</td>
			<td>78428</td>
			<td></td>
		</tr>
		<tr>
			<td>1% protease inhibitor cocktail</td>
			<td></td>
			<td>Roche</td>
			<td>04&#160;693&#160;132&#160;001</td>
			<td>50 X concentrated</td>
		</tr>
		<tr>
			<td>Microplate MaxiSorpTM</td>
			<td></td>
			<td>Thermo Scientific&#160;</td>
			<td>442404</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Tampon carbonate 50 mM pH 9.6&#160;</td>
			<td>&#160;Na2CO3, 10H2O&#160;</td>
			<td>Sigma</td>
			<td>71360</td>
			<td>2.86 g/L</td>
		</tr>
		<tr>
			<td>&#160;NaHCO3&#160;</td>
			<td>Merk</td>
			<td>6329</td>
			<td>3.36 g/L, pH9.6</td>
		</tr>
		<tr>
			<td>Superblock T20 PBS blocking buffer&#160;</td>
			<td></td>
			<td>Pierce</td>
			<td>E6423H</td>
			<td>10 X concentrated</td>
		</tr>
		<tr>
			<td>TMB&#160;</td>
			<td></td>
			<td>Sigma</td>
			<td>T0440</td>
			<td>Used for ELISA</td>
		</tr>
		<tr>
			<td>TMB&#160;</td>
			<td></td>
			<td>Analytik Jena AG</td>
			<td>847-0104200302</td>
			<td>Used for epitope mapping</td>
		</tr>
		<tr>
			<td>HCl 1N&#160;</td>
			<td></td>
			<td>Chimie plus</td>
			<td>40030</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribolyser</td>
			<td></td>
			<td>Thermo</td>
			<td>Fast prep FP120</td>
			<td>keep on ice at this step</td>
		</tr>
		<tr>
			<td>Grinding tubes</td>
			<td></td>
			<td>Biorad</td>
			<td>355-1197</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate washer</td>
			<td></td>
			<td>Tecan</td>
			<td>Columbus Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td></td>
			<td>Biorad</td>
			<td>Model 680</td>
			<td></td>
		</tr>
		<tr>
			<td>Low power magnifier&#160;</td>
			<td></td>
			<td>VWR</td>
			<td>630-1062</td>
			<td>X8 magnification</td>
		</tr>
		<tr>
			<td>Forceps Dumont#7</td>
			<td></td>
			<td>WPI</td>
			<td>14097</td>
			<td>For dissection steps</td>
		</tr>
		<tr>
			<td>Transfer pipette 1ml Samso</td>
			<td></td>
			<td>Samso</td>
			<td>043231</td>
			<td></td>
		</tr>
		<tr>
			<td>1,5 ml tubes</td>
			<td></td>
			<td>Dutscher</td>
			<td>033290</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of disease-associated &#945;-synuclein by enhanced elisa in the brain of transgenic mice overexpressing human a53t mutated &#945;-synuclein

웨스턴 블롯 등의 설립 방법과 더불어, 새로운 방법을 쉽고 빠르게 synucleopathies의 실험 모델에서 질병 관련 α-synuclein의 (αS D)를 정량화하기 위해 필요하다. 과발현 인간 A53T의 αS를 자발적으로 체중 감소, 쇠약, 및 심각한 운동 장애 등의 증상을 특징으로 나이 여덟 22 개월 사이 극적인 임상 표현형을 개발하는 트랜스 제닉 마우스 선 (M83)는이 연구에 사용 하였다. 이 마우스 αS D (질병 관련 αS)의 분자 분석을 위해, ELISA 특별히 아픈 쥐에서 αS D를 정량화하기 위해 설계되었습니다. 이 마우스 모델에서 중추 신경계의 분석은 주로 꼬리 뇌 영역과 척수 αS D의 존재를 보여 주었다. 즉, 임상 질환으로 이어지는 다른 실험 조건 사이의 αS D 분포의 차이는 uninocul에 없었다ated 정상적으로 형질 전환 마우스를 노화와 병 마우스의 뇌 추출물 접종 생쥐에서. Ser129에 대한 항체를 사용하여 αS D 면역 반응의 구체적인 검출은 본질적으로 웨스턴 블롯 및 면역 조직 화학에 의해 얻어진 그 상관 관계를 ELISA로 αS를 인산화. 예기치 않게, 유사한 결과 αS의 C 말단 부위에 대해 여러 가지 다른 항체를 관찰 하였다. &quot;프리온 같은&quot;기구의 관련을 암시 αS D의 전파는, 쉽게 감시되고 따라서, ELISA 접근법을 사용하여이 마우스 모델에서 정량화 할 수있다.

이 연구에서 사용 된 ELISA를 특별히 아픈 M83 마우스에서 높은 소금 버퍼에 준비 뇌 균질 질병 관련 αS (αS D)를 확인했다. 특히 pSer129 αS (P = 0.0074)를 인식하는 항체를 사용하여 ELISA 쉽게 젊은 (구 2-5개월), 건강 M83 마우스 (그림 1)에서 이전, 아픈 마우스 (&gt; 8 개월)를 구별한다. 몇 가지 다른 항체는 유사하게 높은 신호 만 아픈 마우스의 뇌 균질 (&gt; 0.6 OD)를 보여 주었다. 이 4D6?...

Watch this Scientific Journal Video about Detection of Disease-associated Î±-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated Î±-synuclein at JoVE.com

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

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) ...

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JoVE Journal

Medicine

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

10.6K 조회수.  ANSES - French Agency for Food, Environmental and Occupational Health & Safety. An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated α-synuclein (αSD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated αS form or the C-terminal part of the protein.

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

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

파킨슨 병 분야에서 Mitochondrial Phenotypes을 모니터링하기위한 기본 인간 섬유 아세포의 사용

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

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Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

면역 본래대로 사람 MUC1 유전자 변형 마우스의 침략 과도 셀 방광 암의 유도 : 면역 요법 개발을위한 모델

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

같은 뇌 Microslices의 흥분 독성 자극<em&gt; 체외</em&gt; 스트로크의 모델

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

뇌의 아가 로스 젤의 모델로 영상 유도 대류 강화 된 납품

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

세로 두 광자 이미징 다음 마우스의 급성 뇌 외상

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

쥐 심근 경색을 유도 최소 침습 수술

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

마우스에서 가속 죽상 경화증의 유도: "와이어 부상" 모델

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

생체 외에서 그리고 Vivo에서 인간 세포, C. 선 충, 쥐에서 Mitophagy의 검출

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

높은 지방 다이어트를 먹이 쥐 경구 포도 당 내성 검사 (OGTT)과 인슐린 내성 검사 (ITT)를 사용 하 여 포도 당 대사 Vivo에서 의 연구

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.