Les auteurs tiennent à remercier Damien Gaillard pour inoculations et le suivi de l&#39;expérimentation animale. Ce travail a été soutenu par l&#39;Anses (Agence française pour l&#39;alimentation, l&#39;environnement et la santé et sécurité au travail) et par une subvention de la Fondation France Parkinson.

Toutes les procédures et protocoles impliquant des animaux ont été conformément à la directive CE 86/609 / CEE du Conseil et ratifiée par Cometh, le comité national français pour l&#39;examen de l&#39;éthique dans l&#39;expérimentation animale (protocole 11-0043). Les animaux ont été logés et soignés dans l&#39;Anses approuvé d&#39;installations expérimentales à Lyon (approbation B 69387 0801). 1. Préparation des souris <ol><li> Euthanasier souris par une injection intraperitoneale de dose létale de pentobarbital de sodium. </li><li> Récupérer l&#39;ensemble du cerveau de crâne de la souris et le placer dans une boîte de Petri en plastique de 35 mm sur la glace jusqu&#39;à l&#39;extraction. </li><li> Extraire la moelle épinière cervicale. REMARQUE: aS d&#39;extrait soit à partir de l&#39;une des moitiés du cerveau après sectionnement sagittal ou de cerveaux de souris disséquées, disponible après les expériences énumérées dans le tableau 1. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Experiment </td><td> Souris Inoculum (cerveau équivalent) </td><td> Période de survie (Dpi) </td><td> Median / survie maximale (vieux jours) </td><td> aS détection de d par ELISA / WB / IHC </td></tr><tr><td> 1 </td><td> Souris inoculées </td><td> 441 ± 166 </td><td> 419/736 </td><td> 8/8 </td></tr><tr><td> 2 </td><td> Souris inoculées (0,2 mg) </td><td> 150 ± 52 </td><td> 140/241 </td><td> 9/9 </td></tr></tbody></table> Tableau 1. Liste des expériences réalisées sur des souris M83. Les inoculations ont été réalisées à 6 semaines pour l&#39;expérience 2 dans la zone de striato-corticale avec 20 ul d&#39;un homogénat de cerveau de souris malade (1% poids / volume dans du glucose 5%), à la suite anesthésie de six semaines vieilles souris M83 homozygotes de 3% inhalation isoflurane. dpi: jours après inoculation. 2. aS Extraction de Brain moitiés <ol><li> Sagittalement coupé le cerveau pour obtenir deux moitiés. Peser chaque moitié dans un tube contenant ribolysis boulets de broyage. </li><li> Préparer High Salt (HS) un tampon contenant 50 mM Tris-HCl, pH 7,5, NaCl 750 mM, EDTA 5 mM, DTT 1 mM, 1% phosphatase et inhibiteurs de protéase cocktails. Ajouter tampon sel élevée aux moitiés du cerveau pour obtenir 20% (poids / volume) homogénats. </li><li> Préparer les échantillons provenant des moitiés du cerveau à l&#39;aide d&#39;un homogénéiseur mécanique à 6,0 m / s pendant 23 sec deux fois. Après la première homogénéisation de 23 sec, placer les tubes contenant les homogénats sur de la glace pendant 2 minutes avant le second cycle de 23 sec. </li><li> Centrifuger les échantillons à 1000 g pendant 5 min pour éliminer les fragments de cerveau non broyées. Récupérer les surnageants, diviser en 200 aliquotes et les conserver à -80 ° C pour analyse ultérieure ELISA. </li></ol> 3. aS Extraction de disséqués régions cérébrales <ol><li> Disséquer unensemble du cerveau dans une boîte de Petri en plastique de 35 mm sur la glace avec une loupe de faible puissance (8X grossissement) en utilisant deux forcipes dont les extrémités sont maintenues ensemble sauf lorsque disséquer l&#39;hippocampe. Ne pas dépasser 10 min pour préserver l&#39;intégrité du cerveau. Placez le côté droit du cerveau et récupérer les régions du cerveau suivants dans cet ordre: <ol><li> Séparé des deux bulbes olfactifs aide de pinces placées juste derrière l&#39;ampoule. Détacher du cerveau par un mouvement vers le bas. Répétez cette opération pour la deuxième ampoule. </li><li> Caler doucement la pince entre les deux cortex et le déplacer vers l&#39;avant pour faciliter la dissociation des deux cortex. Garder le cerveau en place avec une pince, utiliser un autre pour séparer le cortex de l&#39;hippocampe. </li><li> Placez la pince 2 mm en dessous du cortex. Maintenir une légère pression sur la pince jusqu&#39;à ce que le haut de l&#39;hippocampe est visible. Décoller la première partie du cortex, et répéter avec la deuxième partie. Utilisez la pince pour séparer les deux cortexà partir de l&#39;hippocampe et le déplacement vers l&#39;avant du cerveau. </li><li> Placez la pince ouverte autour de l&#39;un des hippocampes. Fermez la pince au bas de l&#39;hippocampe, puis retirez-le délicatement, récupérer autant que possible. Répétez la procédure pour la deuxième hippocampe. </li><li> Positionner les pinces ouvertes en dessous de la striata une douceur et le séparer du cerveau. Utilisez la pince pour enlever tout le cortex restant dans le striatum. Répétez cette procédure pour le deuxième striatum. </li><li> Utiliser la pince pour abaisser doucement par 2 mm le contour du cervelet pour faciliter la séparation du cervelet de cerveau. Placez la pince juste derrière le cervelet et le retirer en déplaçant la pince avant. </li><li> Utilisez la partie large de la pince à élever le mésencéphale afin de voir clairement où il rejoint le tronc cérébral. Faire une incision verticale à la jonction puis retirez le tronc cérébral. </li><li> Placez la pince derrière le mésencéphale, qui is composé de quatre structures arrondies. Inciser verticalement jusqu&#39;à ce que le mésencéphale a été complètement séparé du cerveau restant. </li></ol></li><li> Préparation des homogénats de variable% (poids / volume) dans du tampon HS, en fonction de la quantité de tissus disponibles, à savoir, 5% d&#39;homogénat pour un poids compris entre 10 et 30 mg, 10% pour un poids compris entre 30 et 80 mg, et 20% pour un poids de plus de 80 mg. <ol><li> Ajouter un volume suffisant de tampon HS aux régions du cerveau disséqués pour obtenir le% d&#39;homogénat attendu. </li><li> Vortex et vérifier que les tissus sont totalement immergés dans la mémoire tampon de HS. </li><li> Homogénéiser les échantillons préparés à partir des régions du cerveau disséqués ou la moelle épinière cervicale avec un broyeur de tissu composée d&#39;un tube en verre de borosilicate et deux pilons, A et B. </li><li> Pour chaque région du cerveau d&#39;être écrasé directement dans le tube. Un pilon insérer dans le tube et rétracter. Répétez ce mouvement une dizaine de fois pour dissocier le tissu. Ensuite, utilisez un pilon B à Continuer en broyant le tissu avec 20 autres mouvements. Transférer les homogénats dans un tube de 1,5 ml avec une pipette de transfert de 1 ml. </li></ol></li><li> Centrifuger les échantillons à 1000 g pendant 5 min à 4 ° C pour éliminer tous les fragments de cerveau non broyées. Récupérer les surnageants, les diviser dans 200 aliquotes et les conserver à -80 ° C pour analyse ultérieure ELISA. </li></ol> 4. Détection par ELISA de aS <ol><li> Diluer les anticorps de revêtement à 0,01 ng / ml. Utilisez soit anti-aS polyclonaux de lapin ou de l&#39;anticorps monoclonal clone 42 mM Na 2 CO 3 / tampon NaHCO3 50 (pH 9,6). </li><li> Enduire le microplaques de 96 puits avec 100 pi par puits de cette solution de revêtement, et laisser à 4 ° CO / N. Utilisation de l&#39;anticorps polyclonal de lapin anti-aS dans la solution de revêtement pour ELISA en utilisant des anticorps de détection syn514, clone 42, LB509, AS11, 4D6 ou 8A5. Utilisez la aS monoclonal anti-clone 42 d&#39;anticorps en tant que solution de revêtementen combinaison avec l&#39;anticorps de détection de l&#39;anti-pSer129. NOTE: Si nécessaire, les plaques peuvent être conservées à 4 ° C pendant une semaine avant le test ELISA est effectuée. </li><li> Utiliser un laveur de plaques pour laver les plaques cinq fois avec 300 ul de solution saline tamponnée au phosphate avec 0,05% de Tween 20 (PBST) par puits. De cette étape en avant, l&#39;incubation est à température ambiante. </li><li> Ajouter 200 pi de T20 tampon PBS par puits de blocage. Agiter pendant 1 heure à 150 tours par minute. Laver les plaques cinq fois avec du PBST. </li><li> Diluer les homogénats de cerveau (dilution de 1: 100 des 20% homogénats, 01:50 10% des broyats et 01h25 des broyats de 5% dans PBST BSA 1%), et ajouter 100 ul à chaque puits. Puis incuber pendant 2 heures tout en agitant à 150 tpm. Laver les plaques cinq fois avec du PBST. </li><li> Ajouter les différents anticorps de détection aS en PBS avec de la BSA 1% aux dilutions mentionnées dans la liste des matériaux. Incuber pendant 1 heure à 150 tours par minute. Laver les plaques cinq fois avec du PBST. </li><li> Ajouter soit anti--mouse ou anti-lapin conjugués IgG HRP dilué à 1: 8000 dans du PBST complété avec de la BSA 1% pendant 1 heure en agitant à 150 tpm. Laver les plaques cinq fois avec du PBST. </li><li> Ajouter 100 pi de 3,3 &#39;, 5,5&#39;-tétraméthylbenzidine (TMB) à chaque puits et incuber pendant 15 min dans l&#39;obscurité tout en agitant à 150 tpm. </li><li> Arrêter la réaction en ajoutant 100 pi de HCl 1 N par puits puis mesurer l&#39;absorbance à 450 nm avec le lecteur de microplaques. </li><li> Pour l&#39;analyse des données, il faut soustraire la valeur de DO obtenue dans un puits avec tous les réactifs à l&#39;exception des échantillons de cerveau de la souris (de puits vierge) à partir des valeurs de DO mesurées pour chacun des échantillons analysés. </li></ol> 5. Epitope Mapping <ol><li> Effectuer la cartographie d&#39;épitopes selon la méthode décrite par Osman 16. En bref, les peptides spot de la séquence α-synucléine humaine contenant 12 acides aminés sur nitrocellulose avec 10 acides aminés qui se chevauchent. </li><li> Bloc avec Tris 50 mM / NaCl 150 mM tampon à pH 10 contenant 0,05% de Tween 20 et 5% de lait en poudre. Incuber l&#39;anticorps dans une solution de blocage à une concentration de 2 ug d&#39;anticorps par ml à 2-10 ° CO / N. </li><li> Laver la membrane trois fois avec du Tris 50 mM / NaCl 150 mM tampon pH 10 contenant 0,05% de Tween 20. Incuber avec l&#39;anticorps de chèvre anti-IgG de souris conjugué à HRP. Laver la membrane cinq fois en utilisant le même tampon puis tache en utilisant un kit de coloration blot TMB occidentale. </li></ol> 6. Analyse statistique <ol><li> Utilisez le logiciel de R et nlme pour effectuer des régressions à effets mixtes pour modéliser OD. Pour chaque comparaison, effectuez un modèle d&#39;effet de régression mixte. Utilisez un effet fixe de distinguer symptomatique des groupes asymptomatiques. </li><li> Utilisez un effet aléatoire pour refléter la variabilité des répétitions pour une souris donnée. Vérifiez homoscédasticité en examinant les résidus et, si nécessaire, utiliser les fonctions de la variance pour modéliser la structure de variance des erreurs au sein des groupes en accord avec Pinheiro et Bates 17. Réglez 0,05 comme seuil de signification de P. </li></ol>

<ol>
	<li>Goedert, M., Spillantini, M. G., Del Tredici, K., Braak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=100+years+of+Lewy+pathology.">100 years of Lewy pathology.</a> Nat Rev Neurol. 9, 13-24 (2013).</li><li>Waxman, E. A., Giasson, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+and+regulation+of+casein+kinase-mediated+phosphorylation+of+alpha-synuclein.">Specificity and regulation of casein kinase-mediated phosphorylation of alpha-synuclein.</a> J Neuropathol Exp Neurol. 67, 402-416 (2008).</li><li>Anderson, J. P., 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=Phosphorylation+of+Ser-129+is+the+dominant+pathological+modification+of+alpha-synuclein+in+familial+and+sporadic+Lewy+body+disease.">Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease.</a> J Biol Chem. 281, 29739-29752 (2006).</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=Prion-like+acceleration+of+a+synucleinopathy+in+a+transgenic+mouse+model.">Prion-like acceleration of a synucleinopathy in a transgenic mouse model.</a> Neurobiol Aging. 33, 2225-2228 (2012).</li><li>Luk, K. C., 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=Intracerebral+inoculation+of+pathological+alpha-synuclein+initiates+a+rapidly+progressive+neurodegenerative+alpha-synucleinopathy+in+mice.">Intracerebral inoculation of pathological alpha-synuclein initiates a rapidly progressive neurodegenerative alpha-synucleinopathy in mice.</a> J Exp Med. 209, 975-986 (2012).</li><li>Giasson, B. I., 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=Neuronal+alpha-synucleinopathy+with+severe+movement+disorder+in+mice+expressing+A53T+human+alpha-synuclein.">Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein.</a> Neuron. 34, 521-533 (2002).</li><li>Luk, K. C., 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=Pathological+alpha-synuclein+transmission+initiates+Parkinson-like+neurodegeneration+in+nontransgenic+mice.">Pathological alpha-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice.</a> Science. 338, 949-953 (2012).</li><li>Masuda-Suzukake, M., 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=Prion-like+spreading+of+pathological+alpha-synuclein+in+brain.">Prion-like spreading of pathological alpha-synuclein in brain.</a> Brain : a journal of neurology. 136, 1128-1138 (2013).</li><li>Sacino, A. N., 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=Amyloidogenic+alpha-synuclein+seeds+do+not+invariably+induce+rapid,+widespread+pathology+in+mice.">Amyloidogenic alpha-synuclein seeds do not invariably induce rapid, widespread pathology in mice.</a> Acta neuropathologica. 127, 645-665 (2014).</li><li>Recasens, 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=Lewy+body+extracts+from+parkinson's+disease+brains+trigger+alpha-synuclein+pathology+and+neurodegeneration+in+mice+and+monkeys.">Lewy body extracts from parkinson's disease brains trigger alpha-synuclein pathology and neurodegeneration in mice and monkeys.</a> Ann Neurol. , (2013).</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=Phosphorylated+alpha-synuclein+can+be+detected+in+blood+plasma+and+is+potentially+a+useful+biomarker+for+Parkinson's+disease.">Phosphorylated alpha-synuclein can be detected in blood plasma and is potentially a useful biomarker for Parkinson's disease.</a> FASEB J. 25, 4127-4137 (2011).</li><li>El-Agnaf, O. M., 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+oligomeric+forms+of+alpha-synuclein+protein+in+human+plasma+as+a+potential+biomarker+for+Parkinson's+disease.">Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson's disease.</a> Faseb J. 20, 419-425 (2006).</li><li>Lee, H. 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.

L&#39;utilisation d&#39;un test ELISA a été démontrée pour détecter spécifiquement aS D directement à partir d&#39;homogénats de cerveau de souris au cours de la maladie dans le modèle de souris transgénique M83. En effet, cet ELISA pouvait facilement distinguer les souris malades de souris M83 M83 sains en utilisant des homogénats de cerveau ne entiers dans un tampon de sel élevée. Les étapes les plus critiques pour de bons résultats à l&#39;aide de cet ELISA sont...

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

En plus des méthodes établies comme Western blot, de nouvelles méthodes sont nécessaires pour quantifier rapidement et facilement associé à une maladie α-synucléine (aS D) dans des modèles expérimentaux de synucléopathies. Une lignée de souris transgéniques (M83) sur-exprimant les aS de A53T humaines et développer spontanément un phénotype clinique dramatique entre huit et 22 mois d&#39;âge, caractérisées par des symptômes tels que la perte de poids, une prostration, et sévère déficience motrice, a été utilisé dans cette étude. Pour les analyses moléculaires de aS D (aS associée à la maladie) chez ces souris, un test ELISA a été conçu pour quantifier spécifiquement aS D chez des souris malades. L&#39;analyse du système nerveux central dans ce modèle de souris a montré la présence de D aS principalement dans les régions du cerveau et caudale de la moelle épinière. Il n&#39;y avait pas de différences dans la distribution aS D entre différentes conditions expérimentales conduisant à la maladie clinique, à savoir, dans uninoculATED et le vieillissement normalement souris transgéniques et des souris inoculées avec des extraits de cerveau de souris malades. La détection spécifique de aS D immunoréactivité en utilisant un anticorps contre Ser129 phosphorylé aS par ELISA essentiellement en corrélation avec celle obtenue par transfert de Western et immunohistochimie. De façon inattendue, des résultats similaires ont été observés avec d&#39;autres anticorps dirigés contre la partie C-terminale de aS. La propagation de aS D, suggérant l&#39;implication d&#39;un mécanisme de «prion-like&quot;, peut donc être facilement contrôlée et quantifiée dans ce modèle de souris en utilisant une approche ELISA.

Dans cette étude, les tests ELISA utilisés spécifiquement identifiés aS associées à des maladies (aS D) dans des homogénats de cerveau préparés dans un tampon de sel élevée de souris M83 malades. En utilisant un anticorps reconnaissant spécifiquement pSer129 aS (p = 0,0074), l&#39;ELISA distingue facilement vieux, souris malades (&gt; 8 mois) à partir de jeunes (2-5 mois), les souris M83 sains (Figure 1). Plusieurs autres anticorps ont montré des signaux aussi élevés (&gt; 0,6...

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.

Détection de α-synucléine maladie associée par ELISA renforcée dans le cerveau de souris transgéniques surexprimant A53T humaine mutée α-synucléine

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

detection-disease-associated-synuclein-enhanced-elisa-brain

Research

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 vues.  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.

Vidéo: 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.

L&#39;utilisation de fibroblastes humains primaires pour la surveillance des phénotypes mitochondriales dans le domaine de la maladie de Parkinson

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 ...

Recherche

Médecine

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.

Induction du envahissantes Transitional cancer de la vessie cellulaire dans immunitaire humain MUC1 souris transgéniques intactes: un modèle pour le développement d&#39;immunothérapie

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.

Excitotoxiques stimulation de Microslices cerveau comme un<em&gt; In vitro</em&gt; Modèle de l&#39;AVC

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.

Guidée par l&#39;image convection-enhanced livraison en modèles Agarose Gel du cerveau

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.

Aiguë traumatisme cérébral chez la souris Suivi par longitudinale à deux photons imagerie

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).

Minimal intervention chirurgicale invasive d&#39;induire un infarctus du myocarde chez des souris

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.

Induction de l’athérosclérose accélérée chez les souris : le modèle « Wire-Injury »

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.

In Vitro et In Vivo la détection des Mitophagy dans les cellules humaines, C. Eleganset la souris

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.

Étude In Vivo du métabolisme du Glucose à haute teneur en graisses régime chez les souris nourries avec épreuve d’hyperglycémie provoquée par voie orale (HGPO) et Test de tolérance à l’insuline (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 ...

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.