Ce travail est financé en sciences naturelles et en génie du Canada (subvention no 249669, 482986 et #) et la Fondation des maladies du Canada (subvention no 485508). Les auteurs tiennent à remercier Youssef El-Hayek et le Dr Qi Wan pour leur aide dans l&#39;élaboration des protocoles expérimentaux.

Toutes les procédures impliquant la manipulation des animaux ont été approuvés par l&#39;Université de Toronto Comité de protection des animaux, conformément aux directives institutionnelles (protocole n °. 20009387). Les méthodes suivantes doivent être effectuées à l&#39;aide des outils et des techniques stériles, dans une hotte à flux laminaire, le cas échéant. Dans le texte du protocole ci-dessous, l&#39;expression «EFH-SFM» se réfère à un milieu sans sérum supplémenté avec facteur de croissance épidermique, le facteur de croissance des fibroblastes et l&#39;héparine. EFH-SFM est utilisé lorsqu&#39;il examine la galvanotaxie de PNJ indifférenciées parce que ces mitogènes maintenir PNJ dans leur 4 état ​​indifférencié. Lorsque l&#39;on étudie la galvanotaxie de PNJ induites à se différencier en types de cellules matures, &quot;FBS-SFM» se réfère à un milieu sans sérum supplémenté avec 1% de sérum de veau foetal. FBS favorise la différenciation des PNJ dans les phénotypes neuraux matures 5. 1. Isolement et culture des précurseurs neuronaux (Nonmontré dans la vidéo) <ol><li> Anesthésier la souris CD1 (6-8 semaines) avec isofluorane et le sacrifice par dislocation cervicale. </li><li> Arrosez la tête dans l&#39;éthanol à 70% et décapiter l&#39;animal avec des ciseaux pointus dissection. </li><li> Tout en maintenant la tête avec une pince chirurgicale, enlever la peau sur la surface dorsale pour exposer le crâne. </li><li> L&#39;aide d&#39;un scalpel et non. 11 lames, marquer le crâne au niveau du sinus frontal dans l&#39;axe médio-latéral, et aussi le long de la suture sagittale dans le sens rostro-caudal. </li><li> Peler les os pariétaux loin de la tête sans. 7 pinces courbes, en prenant soin de ne pas percer le tissu cérébral. </li><li> Insérez une spatule mince en dessous du cerveau à partir de sous le cervelet et avancer vers les bulbes olfactifs. Tout en tenant le crâne en place avec une pince, tirez doucement sur le cerveau du crâne et placez-la immédiatement dans de la glace-froid liquide céphalo-rachidien artificiel (voir les recettes ci-dessous). </li><li> Sous un microscope de dissection, en utilisant stériledes ciseaux et des pinces à dissection couper le cerveau en deux le long de la ligne médiane. Tournez chaque hémisphère de sorte que la médiane (couper) la surface orientée vers le haut. </li><li> Sélectionnez un hémisphère, et avec la surface interne tournée vers le haut, localisez le bourrelet du corps calleux (région postérieure du corps calleux). </li><li> Faire une incision de la surface du cortex pour le bourrelet du corps calleux long de l&#39;axe dorso-ventral. </li><li> Peler le cortex incisée vers le bulbe olfactif pour exposer les parois médiale et latérale du ventricule latéral. </li><li> Tournez l&#39;hémisphère sorte que la surface dorsale orientée vers le haut, et l&#39;utilisation microciseaux courbes à découper et à recueillir les murs exposés médial et latéral, qui contiennent la région périventriculaire où résident les PNJ 6. </li><li> Répétez les étapes 1.8 à 1.11 pour l&#39;autre hémisphère. </li><li> Pipeter le tissu isolé dans 7 ml de solution de trypsine (voir recette ci-dessous) dans un tube de 15 ml, et placer le tube sur une bascule dans un 37 ° Cincubateur pendant 25 min. </li><li> Centrifuger le tube à 1500 tours par minute pendant 5 minutes, aspirer le surnageant et remettre en suspension le tissu dans 2 ml de solution d&#39;inhibiteur de trypsine (voir recette ci-dessous). </li><li> Doucement triturer le tissu avec un petit trou de forage pipette Pasteur 30-50 fois avec soin pour éviter les bulles d&#39;air. </li><li> Centrifuger le tube à 1500 tours par minute pendant 5 minutes, aspirer le surnageant et remettre en suspension dans 1-2 ml de GDF (voir recette ci-dessous) en triturant le culot 3-5 fois. </li><li> Centrifuger le tube à 1500 tours par minute pendant 3 minutes, aspirer le surnageant et remettre en suspension dans 1 ml de GDF + EFH. </li><li> Comptez vivre densité cellulaire avec un hémocytomètre et la plaque des cellules dans un flacon T25 culture à une densité de 10 cellules par ml dans GDF + EFH. </li><li> Permettre à la culture de se développer pendant 7 jours non perturbées pour produire flottants neurosphères primaires composées de PNJ. </li></ol> 2. Galvanotaxie Chambre Préparation <ol><li> Placez verre 3 carrés non. 1 lamelles (22 x 22 x 0,17 mm) ina bouteille d&#39;acide chlorhydrique 6N pendant la nuit. </li><li> Le lendemain, utilisez un diamant à pointe de verre cutter pour couper 6 bandes rectangulaires (22 x 5 x 0,17 mm) de verre à partir de carré. 1 lamelles. </li><li> Transférez les bordereaux lavés à l&#39;acide carrés et rectangulaires glisse dans une hotte à flux laminaire. Laver les barrettes rectangulaires et carrées d&#39;abord avec l&#39;éthanol à 70%, puis avec la culture de tissus de qualité de l&#39;eau en autoclave, et laisser sécher sur une Kim Wipe (pour la stérilité ajouté, le verre peut être autorisé à air sec). </li><li> Appliquer de la graisse à vide à la périphérie d&#39;une surface des lames de verre carrés, et les sceller à la base de boîtes de 60 mm de Pétri en plastique. </li><li> Appliquer de la graisse à vide le long de l&#39;axe long d&#39;une surface de la bande de verre rectangulaires, et les sceller aux bords opposés de la lame de verre carrés (de telle sorte qu&#39;ils soient parallèles les unes aux autres), afin de créer un creux central. </li><li> UV-stériliser les chambres pendant au moins 15 min dans la hotte à flux laminaire. </li><li> Pipette 250-300 ul de poly-L-Lysine sur la cuvette centrale des chambres et incuber à température ambiante pendant 2 heures. </li><li> Environ 15 min avant la fin de la période d&#39;incubation, préparer la solution de Matrigel (voir recette ci-dessous). </li><li> Aspirer le poly-L-lysine, se laver les bacs centrales avec 1 ml d&#39;eau autoclave, et la pipette 250-300 ul de solution de Matrigel sur les creux central. </li><li> Incuber les chambres à 37 ° C pendant 1 heure. </li><li> Aspirer la solution de Matrigel et laver délicatement les creux central avec 1-2 ml de GDF. </li><li> Pipette 100 ul d&#39;EFH-GDF ou FBS-GDF sur les creux central et transférer les chambres galvanotaxie sur la scène d&#39;un comptage au microscope. </li><li> Pipette 3-4 ml de la culture contenant de neurosphères dans une boîte de Petri de 60 mm et transférer la boîte de Pétri à l&#39;étape de comptage au microscope. </li><li> Un objectif de vision de 5x, utiliser une pipette P10 pour transférer 5-8 neurosphères entiers (jusqu&#39;à quatre à la fois) sur la cuvette centrale de chaque galvanotaxiechambre sans les dissocier, et soigneusement répartis les neurosphères autour de la cuvette centrale, sans perturber le substrat Matrigel. </li><li> Ajouter un supplément de 150 à 200 pi d&#39;EFH-GDF ou FBS-GDF sur les creux central. </li><li> Transférez les chambres galvanotaxie dans un 37 ° C, 5% de CO 2, 100% incubateur humidifié pour 17-20 heures (si l&#39;analyse PNJ indifférenciées) pour permettre à l&#39;neurosphères à adhérer au substrat Matrigel et se dissocier en cellules individuelles comme le montre la figure 1 . Si l&#39;analyse de PNJ différenciés, la période d&#39;incubation devrait être étendu aux 69-72 h pour permettre la différenciation des cellules. </li></ol> 3. Vivez cellulaire imagerie time-lapse <ol><li> Permettre au système d&#39;imagerie de cellules vivantes s&#39;équilibrer à 37 ° C, 5% de CO 2 pendant au moins 30 minutes avant le début de l&#39;enregistrement time-lapse. </li><li> Coupez deux 12 pièces de 1 cm de diamètre mm Argent fil, bobine eux d&#39;un bout, et les placer dans Clorox blixiviation pendant 20 min pour former électrodes Ag / AgCl. </li><li> Transférez les chambres galvanotaxie sur la scène d&#39;un comptage au microscope et sélectionnez cette chambre sera utilisé pour des cellules vivantes analyse de la migration d&#39;imagerie basée sur les critères suivants: i) les neurosphères devrait être presque complètement dissocié dans des cellules individuelles et ii) les cellules doivent posséder morphologies rondes avec petits-à-pas de processus qui s&#39;étend des corps cellulaires. </li><li> Transférer la chambre galvanotaxie sélectionné dans une hotte à flux laminaire, avec un pas de carré séparé. 1 lamelle de verre et de graisse à vide. </li><li> Laver le capot coulissant première à l&#39;éthanol 70%, puis avec de l&#39;eau en autoclave, et à appliquer une bande de graisse à vide sur deux bords parallèles de la lamelle. </li><li> Aspirer les milieux de culture à partir de la goulotte centrale de la chambre, puis de placer rapidement la lamelle (graisse du côté orienté vers le bas) sur la chambre de telle sorte que le reste des bandes de graisse sur les deux bandes parallèles de verre rectangulaires, créant ainsi un toità la chambre. </li><li> Pipeter 100 pi de frais EFH-GDF ou FBS-SFM dans le creux central par capillarité. </li><li> Utiliser la graisse à vide pour créer des bordures pour les piscines de milieux de culture sur chaque extrémité de la goulotte centrale, comme représenté sur la figure 2. </li><li> Coupez deux 15 pièces cm de tuyau en PVC, et d&#39;utiliser une seringue de 10 cc avec une aiguille de calibre 18 pour injecter la solution d&#39;agarose soigneusement dans le tube, veiller à ce qu&#39;aucune des bulles se forment dans les tubes, et laisser solidifier le gel pendant 5 min. </li><li> Transférer la chambre galvanotaxie pour le système d&#39;imagerie de cellules vivantes, ainsi que le gel d&#39;agarose à tubes, Ag / AgCl, et une paire de boîtes de Petri vides 60 mm qui sera utilisé comme milieu de culture et des réservoirs contient de l&#39;Ag / AgCl. Permettre à la chambre galvanotaxie à reposer à l&#39;intérieur de la 37 ° C, 5% CO 2 environnement de 20-30 min. </li><li> Pendant ce temps, préparez les couvercles des boîtes de Pétri vides 2 et le couvercle de la boîte de Petri de la chambre galvanotaxie par des trous de forageen y dermiques ou un outil similaire à celui représenté sur la figure 3. </li><li> Pipette 1-1,5 ml d&#39;EFH-GDF ou FBS-SFM sur chaque côté de la cuvette centrale, et 7-8 ml de la GDF dans chaque boîte de Pétri vide. Placez une boîte de Pétri de chaque côté de la cuvette centrale de la chambre galvanotaxie et placez un Ag / AgCl dans chaque boîte. Combler l&#39;écart entre la chambre galvanotaxie et les boîtes de Pétri à établir une continuité électrique avec les ponts gel d&#39;agarose, comme le montre la figure 4. </li><li> Connecter les électrodes Ag / AgCl à une alimentation externe, avec un ampèremètre en série pour mesurer le courant électrique, et allumez l&#39;alimentation. Utiliser un voltmètre pour mesurer la force du champ électrique directement à travers la cuvette centrale, et ajuster le débit de l&#39;alimentation en énergie jusqu&#39;à ce que l&#39;intensité du champ électrique souhaité est atteint (les dosages effectués dans ce laboratoire utiliser une résistance DCEF de 250 mV / mm courant électrique entre 1 et 1,5 mA). </li><li> Initiativete du module time-lapse sur le système d&#39;imagerie de cellules vivantes, et de permettre l&#39;expérience de courir pour la durée souhaitée. Après l&#39;achèvement de l&#39;essai, fixer les cellules dans du paraformaldéhyde à 4% pour une analyse immunohistochimique standard. </li></ol>

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Aucun conflit d&#39;intérêt déclaré.

Ce protocole a été adapté à partir des méthodes bien établies d&#39;études antérieures 7-9. Galvanotactic chambres peuvent être construits en utilisant une variété de techniques différentes, y compris la construction d&#39;un verre séparé bien pour le confinement de l&#39;ensemencement des cellules, ou par ablation laser CO 2 pour la microfabrication de la cuvette centrale 10,11. Certaines techniques peuvent être plus laborieux ou coûteux que d&#39;autres. Nous avons déc...

<table border="1"><tbody><tr><td height="44"> Nom de l&#39;objet </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td></td><td></td><td></td><td> Neural Isolement de cellules précurseurs </td></tr><tr><td> 2M NaCl </td><td> Sigma </td><td> S5886 </td><td> 11,688 g dans 100 ml dH O 2 </td></tr><tr><td> 1M KCl </td><td> Sigma </td><td> P5405 </td><td> 7,456 g dans 100 ml dH O 2 </td></tr><tr><td> 1M MgCl 2 </td><td> Sigma </td><td> M2393 </td><td> 20,33 g dans 100 ml dH O 2 </td></tr><tr><td> 155 mM NaHCO 3 </td><td> Sigma </td><td> S5761 </td><td> 1,302 g dans 100 ml dH O 2 </td></tr><tr><td> Glucose 0,5 M </td><td> Sigma </td><td> G6152 </td><td> 9,01 g dissous dans 100ml dH 2 O </td></tr><tr><td> 108 mM de CaCl 2 </td><td> Sigma </td><td> C7902 </td><td> 1,59 g dans 100 ml dH O 2 </td></tr><tr><td> Pénicilline-streptomycine </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Trypsine pancréatique bovine </td><td> Sigma </td><td> T1005 </td><td></td></tr><tr><td> Sheep testicules hyaluronidase </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> L&#39;acide Kynurenic </td><td> Sigma </td><td> K3375 </td><td></td></tr><tr><td> Ovomucoïde inhibiteur de trypsine </td><td> Worthington </td><td> LS003086 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12100046 </td><td></td></tr><tr><td> F12 </td><td> Invitrogen </td><td> 21700075 </td><td></td></tr><tr><td> Glucose à 30% </td><td> Sigma </td><td> G6152 </td><td></td></tr><tr><td> 7,5% de NaHCO 3 </td><td> Sigma </td><td> S5761 </td><td></td></tr><tr><td> HEPES 1M </td><td> Sigma </td><td> H3375 </td><td> 23,83 g dans 100 ml dH O 2 </td></tr><tr><td> L-glutamine </td><td> Gibco </td><td> 25030 </td><td></td></tr><tr><td> FEM </td><td> Invitrogen </td><td> PMG8041 </td><td> Reconstituer dans 1 ml de mélange d&#39;hormones et aliquote en 20 unités ul. </td></tr><tr><td> FGF </td><td> Invitrogen </td><td> PHG0226 </td><td> Reconstituer dans 0,5 ml de mélange d&#39;hormones et aliquote en 20 unités ul. </td></tr><tr><td> L&#39;héparine </td><td> Sigma </td><td> H3149 </td><td></td></tr><tr><td> Apo-transferrine </td><td> R &amp; D Systems </td><td> 3188-AT </td><td> 0,1 g dissous dans 4 ml dH 2 0 </td></tr><tr><td> Putrescine </td><td> Sigma </td><td> P7505 </td><td> Dissoudre 9,61 mg dans Apo-transferrine afinlution </td></tr><tr><td> Insuline </td><td> Sigma </td><td> I5500 </td><td> Dissolvez 25 mg dans 0,5 ml de HCl 0,1 N et ajouter à 3,5 ml de dH 2 0 </td></tr><tr><td> Sélénium </td><td> Sigma </td><td> S9133 </td><td></td></tr><tr><td> La progestérone </td><td> Sigma </td><td> P6149 </td><td></td></tr><tr><td> Outils de dissection standard </td><td> Outils Fine Science </td><td></td><td></td></tr><tr><td> Microscope à dissection </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td></td><td></td><td></td><td> Galvanotaxie Chambre Préparation </td></tr><tr><td> Carrés diapositives couvercle en verre </td><td> VWR </td><td> 16004 </td><td></td></tr><tr><td> Acide chlorhydrique 6N </td><td> VWR </td><td> BDH3204-1 </td><td></td></tr><tr><td> Graisse à vide élevé </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> 60 mm boîtes de Pétri </td><td> Fisher Scientific </td><td> 0875713A </td><td></td></tr><tr><td> La poly-L-lysine </td><td> Sigma </td><td> P4707 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td> Décongeler et aliquote de 150 unités en pl </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10082139 </td><td> N&#39;utiliser que si induction de la différenciation PNJ, sinon utilisez GDF + milieux de culture EFH comme indiqué ci-dessus </td></tr><tr><td> Compter microscope </td><td> Olympe </td><td> CKX41 </td><td></td></tr><tr><td></td><td></td><td></td><td> Vivez cellulaire imagerie time-lapse </td></tr><tr><td> Fil d&#39;argent </td><td> Alfa Aesar </td><td> 11434 </td><td></td></tr><tr><td> UltraPure Agarose </td><td> Invitrogen </td><td> 15510-027 </td><td></td></tr><tr><td> La chaleur inactivitéFBS ATED </td><td> Sigma </td><td> 16140071 </td><td></td></tr><tr><td> Tubes en PVC </td><td> Fisher Scientific </td><td> 80000006 </td><td> 3/32 &quot;ID x 5/32&quot; OD </td></tr><tr><td> Javellisant </td><td> Clorox </td><td></td><td></td></tr><tr><td> Seringue de 10 cc </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> Aiguille 18 de calibre </td><td> BD </td><td> 305195 </td><td></td></tr><tr><td> Perceuse Dremel </td><td> Dremel </td><td> Modèle 750 </td><td></td></tr><tr><td> Microscope inversé équipé humidifié, chambre incubé </td><td> Zeiss </td><td> Axiovert-200M </td><td></td></tr><tr><td></td><td></td><td></td><td> Recettes <table border="1"><tbody><tr><td> Article </td><td> Volume </td></tr><tr><td> 2M NaCl </td><td> 6,2 ml </td></tr><tr><td>1M KCl </td><td> 0,5 ml </td></tr><tr><td> 1M MgCl 2 </td><td> 0,32 ml </td></tr><tr><td> 155mm NaHCO 3 </td><td> 16,9 ml </td></tr><tr><td> Le glucose 1M </td><td> 1 ml </td></tr><tr><td> 108 mM de CaCl 2 </td><td> 0,09256 ml </td></tr><tr><td> Pénicilline-streptomycine </td><td> 1 ml </td></tr><tr><td> L&#39;eau autoclavée </td><td> 74 ml </td></tr></tbody></table> Liquide céphalo-rachidien artificiel <table border="1"><tbody><tr><td> Article </td><td> Volume ou de masse </td></tr><tr><td> Liquide céphalo-rachidien artificiel </td><td> 30 ml </td></tr><tr><td> Trypsine pancréatique bovine </td><td> 40 mg </td></tr><tr><td> Sheep testicules hyaluronidase </td><td> 22,8 mg </td></tr><tr><td> L&#39;acide Kynurenic </td><td> 5 mg </td></tr></tbody></table> &lt;strong&gt; Solution de trypsine <table border="1"><tbody><tr><td> Article </td><td> Volume ou de masse </td></tr><tr><td> GDF </td><td> 15 ml </td></tr><tr><td> Ovomucoïde inhibiteur de trypsine </td><td> 10 mg </td></tr></tbody></table> Solution inhibiteur de la trypsine <table border="1"><tbody><tr><td> Article </td><td> Volume </td></tr><tr><td> L&#39;eau autoclavée </td><td> 37 ml </td></tr><tr><td> 10X DMEM/F12 </td><td> 10 ml </td></tr><tr><td> Glucose à 30% </td><td> 2 ml </td></tr><tr><td> 7,5% de NaHCO 3 </td><td> 1,5 ml </td></tr><tr><td> HEPES 1M </td><td> 0,5 ml </td></tr><tr><td> Transferrine, putrescine solution </td><td> 4 ml </td></tr><tr><td> Une solution à 25 mg d&#39;insuline </td><td> 4 ml </td></tr><tr><td> Sélénium </td><td> 100 &amp; mu; L </td></tr><tr><td> La progestérone </td><td> 100 pi </td></tr></tbody></table> Mix Hormone (100 ml au total, conserver à -20 ° C) <table border="1"><tbody><tr><td> Article </td><td> Volume </td></tr><tr><td> L&#39;eau autoclavée </td><td> 37,5 ml </td></tr><tr><td> DMEM/F12 10X (3:1) </td><td> 5 ml </td></tr><tr><td> Glucose à 30% </td><td> 1 ml </td></tr><tr><td> 7,5% de NaHCO 3 </td><td> 0,75 ml </td></tr><tr><td> HEPES 1M </td><td> 0,25 ml </td></tr><tr><td> Mélange hormone </td><td> 5 ml </td></tr><tr><td> L-glutamine </td><td> 0,5 ml </td></tr><tr><td> Pénicilline-streptomycine </td><td> 0,5 ml </td></tr></tbody></table> Sérum Free Media EFH-GDF: ajouter 10 ul d&#39;EGF, 10 pi de FGF, et 3,66 ul d&#39;héparine FBS-GDF: ajouter 0,5 ml de FBS <tafrontière ble = &quot;1&quot;&gt; <tbody><tr><td> Article </td><td> Volume </td></tr><tr><td> Matrigel </td><td> 150 pi </td></tr><tr><td> GDF </td><td> 3,6 ml </td></tr></tbody></table> Matrigel Solution Matrigel aliquote doit être placé dans une boîte de glace et de laisser se décongeler lentement au fil 4-5 heures pour former un liquide visqueux avant mélange avec l&#39;AFD. Cela permettra d&#39;assurer la formation d&#39;une fine couche de substrat Matrigel. Si ce n&#39;est pas décongelé lentement, le substrat résultant contiendra des touffes de Matrigel, éventuellement entraver la migration des cellules. <table border="1"><tbody><tr><td> Article </td><td> Volume ou de masse </td></tr><tr><td> UltraPure Agarose </td><td> 300 mg dans 10 ml ddH 2 0 </td></tr><tr><td> GDF FBS inactivé chaleur </td><td> 8 ml 2 ml </td></tr></tbody></table> MatrigelSolution Mix 8 ml de GDF avec 2 ml de FBS inactivé de la chaleur dans un tube de 15 cc faucon. Mix d&#39;agarose avec 10 ml ddH 2 0 dans un erlenmeyer, et de la chaleur au micro-ondes pendant 30 secondes à 10 sec intervalles, en veillant à éliminer la solution du micro-ondes après chaque intervalle de 10 sec et bien mélanger. Suite à la dernière période de 10 secondes à micro-ondes, mélanger la solution d&#39;agarose avec la SFM / solution FBS et le ranger dans un bain de 57 ° C de l&#39;eau. </td></tr></tbody></table>

a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field

La découverte des cellules souches et des cellules progénitrices neurales (collectivement appelées cellules précurseurs neurales) (PNJ) dans le cerveau des mammifères adultes a conduit à un ensemble de recherches visant à utiliser les propriétés multipotentes et de prolifération de ces cellules pour le développement de stratégies neuroregenerative. Une étape cruciale pour le succès de ces stratégies est la mobilisation des PNJ vers un site de lésion consécutive à une transplantation ou exogène pour améliorer la réponse des précurseurs endogènes que l&#39;on trouve dans la région périventriculaire du système nerveux central. Par conséquent, il est essentiel de comprendre les mécanismes qui favorisent, d&#39;orienter et d&#39;améliorer la migration PNJ. Notre travail se concentre sur l&#39;utilisation du courant continu champs électriques (dcEFs) de promouvoir et de migration directe PNJ - un phénomène connu sous le nom galvanotaxie. Endogènes physiologiques des champs électriques fonctionnent comme des repères essentiels pour la migration cellulaire au cours du développement normal et la cicatrisation des plaies. La perturbation pharmacologique dutension du tube neural chez trans-embryons axolotl provoque de graves malformations du développement 1. Dans le contexte de la cicatrisation, le taux de réparation de la cornée blessés est directement corrélée à l&#39;ampleur du potentiel de blessure épithéliale qui se pose après une blessure, comme le montre l&#39;amélioration pharmacologique ou l&#39;interruption de ce dCEF 2-3. Nous avons démontré que les adultes subissent PNJ sous-épendymaires rapide et dirigé la migration cathodique in vitro lorsqu&#39;elles sont exposées à un dCEF appliqué de l&#39;extérieur. Dans ce protocole, nous allons décrire les techniques de notre laboratoire pour la création d&#39;un test galvanotaxie simple et efficace à haute résolution, observation à long terme de la translocation de cellule du corps dirigé (migration) sur un seul niveau de cellules. Ce test serait approprié pour étudier les mécanismes qui régulent la transduction dCEF dans la motilité cellulaire grâce à l&#39;utilisation de souris transgéniques ou knock-out, courts ARN interférents ou des agonistes / antagonistes des récepteurs spécifiques.

L&#39;analyse cinématique révèle que, en présence d&#39;une mesure de 250 mV / mm dCEF, les PNJ indifférenciées présentent galvanotaxie très rapide et dirigés vers la cathode (figure 5A, Film 1). En l&#39;absence d&#39;un dCEF, le mouvement aléatoire des cellules est observée (figure 5B, Film 2). A ce champ,&gt; 98% des PNJ indifférenciées migrer pour la de 6-8 heures pour lesquelles ils sont imagés, et depuis les cellules mortes ne migrent pas ce qui suggère qu&#39;ils r...

Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Dans ce protocole, nous allons montrer comment construire des chambres personnalisées qui permettent l&#39;application d&#39;un champ électrique à courant continu pour permettre imagerie time-lapse de cerveau adulte dérivé de neurones translocation cellule précurseur pendant galvanotaxie.

Un test galvanotaxie pour l&#39;analyse de la cinétique précurseurs neuronaux migration des cellules dans un champ appliqué de l&#39;extérieur électrique à courant continu

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

a-galvanotaxis-assay-for-analysis-neural-precursor-cell-migration

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Neuroscience

12.4K vues.  University of Toronto. Dans ce protocole, nous allons montrer comment construire des chambres personnalisées qui permettent l'application d'un champ électrique à courant continu pour permettre imagerie time-lapse de cerveau adulte dérivé de neurones translocation cellule précurseur pendant galvanotaxie.

Vidéo: A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural-Formant Colonie dosage cellulaire: Un test pour distinguer Bona Fide Cellules souches neuronales à partir de cellules progénitrices neurales

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

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Neurosciences

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Analyse de la migration cellulaire Tronc Crête neurale en utilisant une modification Zigmond Chambre Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Reproductible écrasement du nerf sciatique de souris et l&#39;évaluation subséquente de la régénération par l&#39;analyse du Mont muscle entier

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Analyse de la migration des crêtes neurales et la différenciation par la Croix-espèces transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Le test neuroblaste: Un test pour la génération et l&#39;enrichissement des cellules souches neuronales à partir de différenciation des cellules souches neurales Progeny Par cytométrie en flux

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolement et culture de cellules de crêtes neurales de tube neural embryonnaire murin

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Un test migration cellulaire quantitative pour murins de neurones entériques progéniteurs

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Enregistrements simultanés des potentiels corticaux champ Local, électrocardiogramme, électromyogramme et respiration rythmique d’un Rat librement mobile

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Enregistrement optique à grand champ à un seul photon dans des tranches de cerveau à l'aide de teinture sensible à la tension

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

Analyse de la différenciation et de la maturation de criblage à haute teneur de cultures de cellules souches neurales fœtales et adultes dérivées de cellules souches oligodendrocytes

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...