Fundação 데 Amparo 지원 Pesquisa 에스타는 리우데 자네이루 (FAPERJ)와 Conselho 나셔널 드 Desenvolvimento Científico 전자 Tecnológico (CNPq)을해야합니까. 우리는 누구의 의견이 우리가 원고와 비디오를 동시에 개선하는 데 도움 익명 주심에 감사합니다.

등 6 OHDA과 apomorphine으로 고체 형태로 저장된 모든 화학 물질은, 무균 주입 물에 희석하여 오염을 피하기 위해, 층류 후드 내부 필터링되었습니다. 또한, 신경 및 neuroactive 물질은 저장 준비 처리 및 국제 지침 6 지역 Biosafety위원회가 정한 규정에 따라 처리되었습니다. 1 - 마취과와 수술 성인 남성 스위스 생쥐 (25~30그램)은 vagal 톤을 줄이기 위해 아트로 핀 (0.2 MG / kg, SC)와 premedicated와 케타민을 (90-120 밀리그램 / 킬로그램)과 Xylazine (10 MG / kg)의 intraperitoneal 주사와 함께 다음 anesthetized 아르 . 동물은 체온이 37 ° C 유지를 진정 작용의 유도가 몇 분 정도 걸립으로 보장하기 위해 전자 제어 손난로 (통찰력)에 배치됩니다. 마취 수준은 탈퇴 반사의 부재를 테스트하여 프로 시저를 통해 확인합니다. 마취 주사 량은 각 변형 / 식민지에 대한 ajusted해야합니다. 수술의 동물과 기간에 따라, 300ml의 추가 복용이 필요할 수 있습니다. 그 후, 생리 (NaCl 0,9 %)이 밖으로 건조에서 각막을 보호하고 첫번째 순간에있는 the blink 반사의 부재를 테스트하는 두 눈에 적용됩니다. 전체 머리 가죽 unsterile 영역 최소 접촉을 보장하기 위해 트리머로 면도있다. 국소 마취제 (Xylocaine)는 불편을 방지하기 위해 귀 바에 적용됩니다. 철수와 깜박 반사가 사라졌다는 한 번만, 마취의 수준을 나타내는 것은 깊은만큼 수술 (단계 3)이고, 동물은 특별히이 종 (인사이트)을위한 입 피스와 귀 막대를 사용하여 stereotaxic 프레임에 배치됩니다. 일단 positionated, ophthalmologic 연고 (마르카)는 모든 수술에 걸쳐 동물 눈을 보호하기 위해 적용됩니다. 눈을 제대로 보호하고 있는지 확인 될 경우에만, 절차 후 불임 코튼스 면봉을 사용하여 70 % 에탄올 다음 povidone 요오드 세 응용 프로그램을 반복하여 수술 영역의 피부의 소독과 함께 계속 귀를 바의 적절한 배치가 머리의 측면 움직임에 대한 테스트에 의해 보장 (머리가 ​​바가 안전하게 고정되는 것을 보장하기 위해 이동하지합니다.) 두개골의 랜드마크가 발표 때 머리 적절한 dorsoventral 경사는 나중에 확인됩니다. 화살 절개 (1.5 cm)는 멸균 메스 (# 15 블레이드)로 이루어집니다. 관심 분야를 통해 골막은 부드럽게 새로운 블레이드와 거리 스크랩한이며, 뼈가 두개골 명소를 발굴하기 위해 무균 시선과 목화 면봉로 닦아입니다 bregma는 화살과 코로나 봉합의 교차 지점으로 정의되어있는 동안 람다 화살 봉합사의 교차로와 lambdoid 봉합의 왼쪽과 오른쪽 부분을 통과 가장 맞는 라인의 요점입니다. 세로 정렬 기준 바늘의 팁을 먼저 수술 현미경을 통해 시험에서 bregma에서 제로와 람다 지점을 터치로 다음 이동합니다. 머리가 올바르게 위치하는 경우, 람다는 bregma 같은 dorso - 복부 수준에 있어야하고, 두 랜드마크 rostro - 꼬리 축을 따라 4.2 mm에서 멀리 떨어져 있어야합니다. 그렇지 않다면, 귀 바, 코받침이요은 느슨하게해야하며 머리가 있으니까 stereotaxical 조치에 대해 &quot;평면 두개골&quot;참조를 정의 원하는 위치를 달성하기 위해, interaural 축을 중심으로 기울어져. 참조 바늘 끝은 다음 원하는 좌표에 위치하고 있습니다 앞쪽에 - 후부 (AP 0.5 mm)과 (L -2.0 mm, 왼쪽) 측면. 위치는 두개골에 표시된이며 소형 (1.2 mm 직경) 구멍 열 영역을 방지하기 위해 간헐 동작을 사용하여 소독 드릴 비트와 함께 열립니다. 뼈 조각은 신중하게 치과 퀴렛로 제거하고 멸균 따뜻하고 0.9 % NaCl로 세탁합니다. 멸균 5 μl 해밀턴 주사기 (26s 게이지, 0.47 mm 외경)은 6 - OHDA 솔루션 (또는 제어 동물 차량)와로드 및 수직 stereotaxic 장치에 정렬됩니다. 바늘의 팁 (2 형 또는 12 ° 베벨)은 다음 dorso - 복부 좌표의 기준점를 따지기, 터치 pial 표면까지 열린 구멍에 삽입됩니다. 일단이 좌표를 따지기, 바늘이 천천히 (:; : -2.0 및 DV 아래의 -3.0 mm, pial 표면 7 L 0.5 AP = 연합 뉴스) striatum의 좌표에 도달할 저하됩니다. 준비 및 층류 후드 내부 필터링, 그리고 절차를 통해 알루미늄 호일에 의해 빛으로부터 보호되어야합니다되었다 6 - OHDA 솔루션 (0.02 %의 아스코르비 산 10 μg 6 - OHDA 0.9 % NaCl)을, 다음에 주입 0.1μL/min의 유량. 컨트롤 동물, 차량의 동일한 볼륨 (0.02 %의 아스코르비 산 및 멸균 물의 NaCl 0.9 %)가 주입됩니다. 전체 2.0 μL를 주입되면 매우 느리게 두뇌에서 철회되기 전에, 주사기는 5 분 이상 이하에서 5 분 장소에 보관됩니다. 두개골은 청소되고 절개는 # 6-0 나일론 봉합사 (Shalon)를 사용하여 닫힙니다. 추가을 위해감염에 대한 치료는 이러한 네오 마이신과 같은 항생제 연고 지역은, 봉합 피부에 적용될 수 있습니다. 동물은 탈수를 방지하기 위해 호수 (sc.)의 0.5 ML 주어진 프레임에서 제거하고, 따뜻하고 완전 복구까지 가까운 전망대 아래에 보관됩니다. 그들은 그 음식과 물을 광고 libitum와 함께 지내게됩니다. 진통제는 수술 후 처음 두 일 (5.5 ML의 일일 평균 물 소비량 부여에 대한 식수 병 및 바디 무게 / 하루 0.03 MG의 이부 프로펜 / g의 권장 복용량에 추가됩니다; 20 MG의 0,82 ML을 추가 / ML 이부 프로펜 - 애보트 서스펜션 100 ML의 식수로). 마우스는 일반적으로 방황하고 쉽게 물 식품 알약에 대한 도달, 절차에서 신속하게 복구합니다. 명백한 불편, 또는 감염의 서명의 경우에는 동물 지방 지침에 따라 euthanized해야합니다. 2 - 행동 검사 모터 장애가 주 후 관찰 될 수 있지만, 1 개월 후 병변 간격은 회전 테스트를 신뢰할 수있는 부량 전에 허용됩니다. 동물 DA의 작용제의 apomorphine (0.5 밀리그램 / 킬로그램)의 피하 주사를 수신하고, 30cm 직경의 불투명 실린더에 배치됩니다, 녹음 카메라 아래에 45cm를 배치. 각 테스트하기 전에 이전에 시험 동물에서 어떤 잔류물과 냄새를 제거하는 것이 중요합니다. 5 분 요법 이니 기간 후 운동은 5 분 기간 동안 기록됩니다. 모두 콘트라 및 ipsilateral 전신 순환은 측정 및 제어 그룹 (차량 주입)과 비교됩니다. 절차가 올바르게 수행 경우 작용제는 주로 lesioned 측면에 과민한 denervated striatum을 활성화 이후 6 OHDA lesioned 동물, contralateral 측면으로 회전하기 시작합니다 반면, 제어 동물은 어떤 회전을 전시하지 않습니다. 7 RPM (분당 최소 7 전신 contralateral 회전을 표시)을 통해 점수 동물이 성공적으로 lesioned로 간주됩니다. 동물이 안전하게 매주 반복 ​​수있는 시험 후 자신의 주택 30 분에 제공될 수 있습니다. 우리 손에, lesioned 생쥐 미만 5 % suboptimal 회전 속도를 표시합니다.

<ol>
	<li>Ungerstedt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6-hydroxy-dopamine+induced+degeneration+of+central+monoamine+neurons.">6-hydroxy-dopamine induced degeneration of central monoamine neurons.</a> Eur. J. Pharmacol. 5, 107-110 (1968).</li><li>Akerud, P., Canals, J. M., Snyder, E. Y., Arenas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+through+delivery+of+glial+cell+line-derived+neurotrophic+factor+by+neural+stem+cells+in+a+mouse+model+of+Parkinson's+Disease.">Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's Disease.</a> J. Neurosci. 21, 8108-8118 (2001).</li><li>Ghosh, A., Roy, A., Liu, X., Kordower, J. H., Mufson, E. J., Hartley, D. M., Ghosh, S., Mosley, R. L., Gendelman, H. E., Pahan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+inhibition+of+NF-kappaB+activation+prevents+dopaminergic+neuronal+loss+in+a+mouse+model+of+Parkinson's+Disease.">Selective inhibition of NF-kappaB activation prevents dopaminergic neuronal loss in a mouse model of Parkinson's Disease.</a> Proc. Natl. Acad. Sci. USA. 104, 18754-18759 (2007).</li><li>Alvarez-Fischer, D., Henze, C., Strenzke, C., Westrich, J., Ferger, B., H&#246;glinger, G. U., Oertel, W. H., Hartmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+striatal+6-OHDA+model+of+Parkinson's+disease+in+wild+type+and+a-synuclein-deleted+mice.">Characterization of the striatal 6-OHDA model of Parkinson's disease in wild type and a-synuclein-deleted mice.</a> Exp. Neurol. 210, 182-193 (2008).</li><li>Ungerstedt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6-Hydroxydopamine-induced+degeneration+of+the+nigrostriatal+dopamine+pathway:+The+turning+syndrome.">6-Hydroxydopamine-induced degeneration of the nigrostriatal dopamine pathway: The turning syndrome.</a> Pharmacology and Therapeutics Part B: General and Systematic Pharmacology. 2, 37-40 (1976).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidelines for the Care and Use of Mammals in Neuroscience and Behaviour Research. , 209 (2003).</li><li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li><li>Hudson, J. L., Horne, C. G. v. a. n., Str&#246;mberg, I., Brock, S., Clayton, J., Masserano, J., Hoffer, B. J., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+apomorphine-+and+amphetamine-induced+turning+with+nigrostriatal+dopamine+content+in+unilateral+6-hydroxydopamine+lesioned+rats.+Brain+Res.">Correlation of apomorphine- and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats. Brain Res.</a> , 626-6167 (1993).</li><li>Truong, L., Allbutt, H., Kassiou, M., Henderson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+a+preclinical+model+of+Parkinson's+disease:+A+study+of+behaviour+in+rats+with+graded+6-OHDA+lesions.">Developing a preclinical model of Parkinson's disease: A study of behaviour in rats with graded 6-OHDA lesions.</a> Behav. Brain Res. 169, 1-9 (2006).</li><li>Harvey, B. K., Wang, Y., Hoffer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+rodent+models+of+Parkinson's+disease.">Transgenic rodent models of Parkinson's disease.</a> Acta Neurochir. (Wien.). 101, 89-92 (2008).</li><li>Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ole+Isacson:+Development+of+New+Therapies+for+Parkinson's+Disease.">Ole Isacson: Development of New Therapies for Parkinson's Disease.</a> JoVE. 3, (2007).</li></ol>

실험 국제 신경 과학에있는 포유류의 관리 및 사용에 대한 지침과 행동 연구에 따라 수행되었습니다<sup&gt; 6</sup&gt; 기관 애니멀 케어 및 사용위원회의 승인 후 (Comissão 데 Avaliação 데 우소 데 동물 그들 Pesquisa : UFRJ / CAUAP 프로토콜 # DAHEICB 027). 적절한 조치는 실험 동물의 고통과 불편을 최소화하기 위해 이동했다.

nigro - striatal 경로를 광범위하게 일방적으로 병변이 striatum에 6 - OHDA의 단일 stereotaxic 주사로 쥐에서 안정적으로 얻을 수 있습니다. 병변의 범위는 같은 동영상에 그림, (DA의 합성의 단계를 제한 속도, dihydroxyphenylalanine에 L - 티로신의 즉, 변환을 catalyses)을 티로신 Hydroxylase에 대한 immunohistochemistry하여 사후 확인하실 수 있습니다. 생체내에서 apomorphine에 의해 유발 contralateral 회?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ketamine hydrochloride</td>
<td>&nbsp;</td>
<td>K&ouml;nig</td>
<td>&nbsp;</td>
<td>90-120 mg/Kg</td>
</tr>
<tr>
<td>Xylazine hydrochloride</td>
<td>&nbsp;</td>
<td>K&ouml;nig</td>
<td>&nbsp;</td>
<td>10 mg/Kg</td>
</tr>
<tr>
<td>Atropine Sulfate</td>
<td>&nbsp;</td>
<td>Isofarma</td>
<td>&nbsp;</td>
<td>Isofarma</td>
</tr>
<tr>
<td>6 &#x2013; Hydroxydopamine Hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>5 &mu;g/&mu;L</td>
</tr>
<tr>
<td>L - Ascorbic Acid</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>&nbsp;</td>
<td>0.2%</td>
</tr>
<tr>
<td>Xylocaine (Lidocaine Chloridrate)</td>
<td>&nbsp;</td>
<td>ASTRA Chemistry</td>
<td>&nbsp;</td>
<td>5% 50 mg</td>
</tr>
<tr>
<td>Nebacetin (Neomycin Sulfate + Bacitracin)</td>
<td>&nbsp;</td>
<td>Altana Pharma</td>
<td>&nbsp;</td>
<td>5 mg/g + 250 Ul/g</td>
</tr>
<tr>
<td>R-(-)-Apomorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>0.5 mg/Kg</td>
</tr>
<tr>
<td>GenTeal lOphthalmic ointment Hypromellose</td>
<td>&nbsp;</td>
<td>NOVARTIS</td>
<td>&nbsp;</td>
<td> 0.33%</td>
</tr>
<tr>
<td>Ibuprofen</td>
<td>&nbsp;</td>
<td>Abbott</td>
<td>&nbsp;</td>
<td>4 mg/ 100 ml drinking water</td>
</tr>
<tr>
<td>Povidine</td>
<td>&nbsp;</td>
<td>JohnsonDiversey</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>R-(-)-Apomorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>0.5 mg/Kg</td>
</tr>		</tbody>
		</table>
		</div>

murine model for parkinson's disease: from 6-oh dopamine lesion to behavioral test

파킨슨병 (PD)는 성별에 관계없이 전세계적으로 최소한 6백50만명, 사회적, 민족, 경제, 또는 지리적 경계에 영향을 미칩니다. 이러한 떨림, 강성 및 bradikinesia 같은 주요 증상, dopaminergic 세포 3 / 약 4는 substantia 니그라에서 길을 잃었 때 개발하고, striatal 모터 회로의 부드러운 조정 규정을 제공하기 위해 실패합니다. 우울증 및 환각이 일반적이며, 치매는 결국 환자의 20 %에서 발생합니다. 이때 PD의 진행을 지연하거나 중지시킬 치료가 없습니다. 이러한 증상의 경감을 향해 오히려 약물은 현재 사용 가능한 목표 더. 새로운 외과 전략 reversibly 깊은 뇌 구조의 전기 자극을 통해 기능적으로 손상된 회로에 스위치를 수 있지만 깊은 두뇌 자극이 주요 발전이지만, 그것은 모든 환자에 대해 적합하지 않습니다. 그것은 따라서 잠복기 모델에 새로운 세포 치료 방법을 테스트하는 데 필요한 남아 있습니다. dopaminergic 경로의 선택 neurotoxic 장애는 마약과 산화 - 유해한 화학 물질을 파괴하는 것이 있습니다 반면 (1 - 메틸 -4 - 페닐 - 1 ,2,3,6 - tertahydropyridine) 6 - hydroxydopamine (6 - OHDA) 또는 MPTP의 주입에 의해 복제 수 있습니다 또한 설치류에 PD의 특정 기능을 재생산한다. MPTP와는 달리, 6 - OHDA의 병변은 거대한 돌이킬 수의 연결 손실을 발생하고, 단일 또는 양자 수 있습니다. 6 - OHDA 병변의 모델은 신뢰할 수있다, 강력한 모터 적자로 연결하고, 가장 널리 rats1 연구의 40 년 후 사용됩니다. 이식할 세포와 호스트 사이의 상호 작용 지금 아니라 쥐보다 마우스에 더 철저하게 공부를 할 수 있듯이, 모델은 그것이 최근 4 특징되었습니다 마우스 2,3로 바뀜되었습니다. 이 비디오에서는, 우리는 어떻게 천천히 stereotaxically 삽입된 마이크로 주사기 바늘을 통해 6 - OHDA 2.0 μL를 제공함으로써 병변 anesthetized 마우스의 왼쪽 nigro - striatal 통로를 보여줍니다. dopaminergic 입력 손실 일 이내에 발생하고, 기능 장애가 dopaminergic 요원 5 유도 등급 동물 회전에 의해 게시물 수술 주 개월 동안 모니터링할 수 있습니다. 여기, 우리는 apomorphine의 단일 피하 관리 후 10 분 발생하는 전신 contralateral 회전은 병변 후 한 달 측정 보여줍니다. 결과과 단점은 아래에 설명되어 있습니다.

Watch this Scientific Journal Video about Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test at JoVE.com

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

파킨슨 질환은 실험적으로 6 - OH - 도파민에 의해 유도된 수있는 striatum에 dopaminergic innervation의 손실로 인해 발생합니다. 우리는 stereotaxic 병변을 수행하고 생쥐에서 apomorphine 유발 회전 동작을 모니터링하는 방법에 대해 설명합니다. 이 모델은 유용하고 파킨슨 질환에 대한 새로운 치료법을 테스트하기 위해 신뢰할 수 있습니다.

파킨슨병에 대한 Murine 모델 : 6 - OH 도파민의 병변에서 행동 테스트

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

murine-model-for-parkinson-s-disease-from-6-oh-dopamine-lesion-to

Research

JoVE Journal

Biology

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

31.4K 조회수.  Universidade Federal do Rio de Janeiro, Brasil. 파킨슨 질환은 실험적으로 6 - OH - 도파민에 의해 유도된 수있는 striatum에 dopaminergic innervation의 손실로 인해 발생합니다. 우리는 stereotaxic 병변을 수행하고 생쥐에서 apomorphine 유발 회전 동작을 모니터링하는 방법에 대해 설명합니다. 이 모델은 유용하고 파킨슨 질환에 대한 새로운 치료법을 테스트하기 위해 신뢰할 수 있습니다.

비디오: Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson: Development of New Therapies for Parkinson&#039;s Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

올 Isacson : 파킨슨병을위한 새로운 요법 개발

연구

생물학

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

쥐 Neonates에서 기본 Dissociated Midbrain의 도파민 세포 문화

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

잠재적인 파킨슨병의 유전자 검증을위한 C. 엘레간스 도파민 신경 세포의 변성 분석의 응용

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus의 번역 손상을 식별 할 수있는 모델로 laevis의

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

마그네틱 활성화 전략을 분리 하 고 정화 활 액 액체 파생 된 중간 엽 줄기 세포는 토끼 모델에서 정렬 셀

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

궁의 분리 마우스 모델에서 병 변 크기의 비 침 투 적인 모니터링

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). The most common pattern observed in the clinic is the peak-dose dyskinesia which consists of clinical manifestations of choreiform, dystonic, and ballistic movements. The 6-hydroxydopamine (6-OHDA) rat model of PD mimics several characteristics of LIDs. After repeated L-DOPA administration, 6-OHDA-lesioned rats exhibit dyskinetic-like movements (e.g., abnormal involuntary movements, AIMs). This protocol demonstrates how to induce and analyze AIMs in 6-OHDA-lesioned rats with 90%-95% dopaminergic depletion in the nigrostriatal pathway. Repeated administration (3 weeks) of L-DOPA (5 mg/kg, combined with 12.5 mg/kg of benserazide) can induce the development of AIMs. The time course analysis reveals a significant increase in AIMs at 30-90 min (peak-dose dyskinesia). Rodent models of LIDs are an important preclinical tool to identify effective antidyskinetic interventions.

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson&#039;s Disease

Rodent models of L-DOPA-induced dyskinesias are invaluable tools to identify therapeutic interventions to attenuate the development or alleviate the manifestations that emerge due to the repeated administration of L-DOPA. This protocol demonstrates how to induce and analyze dyskinetic-like movements in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease.

파킨슨 병의 일방적 인 6-OHDA 병변 쥐 모델에서 L-DOPA 유발 운동 이상증 평가

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). ...

Murine Intestinal Crypts 및 Single Stem Cell의 오가노이드 3D 배양

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

저자 스포트라이트: The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research (오가노이드 연구를 위한 쥐 장낭화낭수에서 오가노이드의 3D 배양 및 단일 줄기 세포)  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

쥐 장 선와에서 오가노이드와 오가노이드 연구를 위한 단일 줄기 세포의 3D 배양

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...