Vorremmo ringraziare Britta Kaltwasser per la sua eccellente assistenza tecnica e Mahesh Kumar Teli a organizzare la preparazione delle riprese video.

1. Animali <ol><li> Gli esperimenti sono stati effettuati secondo le linee guida NIH per la cura e l&#39;uso di animali da laboratorio e approvato dalle autorità locali. Per tutti gli esperimenti, C57Bl6 / J sorci di tipo selvatico, ApolipoproteinE - / - (ApoE KO) e SV129 topi (12-16 settimane, 26-30 g di peso corporeo, 5-6 animali per gruppo sperimentale) sono stati utilizzati. </li></ol> 2. La colorazione dei vasi cerebrali con lattice colorato <ol><li> Preparare una miscela di 25 pl di inchiostro nero di carbonio (Herlitz, Germania) con 0,5 ml della composizione di lattice (Pebeo, Francia) in un rapporto di 1:20 in una provetta EP e scaldare la miscela a 37 ° C in un bagno d&#39;acqua. Raccogliere il composto in una siringa 2 ml con un ago di 28-30G prima anestetizzante l&#39;animale. </li><li> Sciogliere 50 mg di polvere cloridrato papavarine in 1 ml di soluzione fisiologica sterile. Raccogliere la soluzione in una siringa da insulina. Rompere l&#39;ago da un&#39;altra siringa da insulina sterile. Posizionare questo ago al termine di un 20 cm PE10 tubo. Ora attaccate questo tubo PE10 sull&#39;ago della siringa contenente insulina soluzione papavarine cloridrato essere iniettato attraverso la vena femorale per garantire vasodilatazione e corretto riempimento dei vasi. </li><li> Preparare altri due siringhe da insulina ciascuna contenente 1 ml di soluzione fisiologica. Siringhe da insulina consentono una regolare erogazione del fluido iniettato in una posizione precisa. Tuttavia, a causa della elevata viscosità del lattice, alternativa 1 ml siringhe con aghi rimovibili ampia dovrebbero essere usati. </li><li> Prendere tutte le siringhe e strumenti sterilizzati dissezione vicino alla fase operativa. Stendere un foglio chirurgica mercante di stoffe intorno la fase di esercizio. La fase di esercizio deve essere sufficientemente illuminata e un microscopio operativo possono essere utilizzati, se necessario. </li><li> Ora misurare il peso corporeo di un C57Bl6 / J mouse e quindi indurre anestesia con isoflurano vaporizzato 5% in 70% N 2 O e 30% O 2. Continua l&#39;anestesia con 1% di isoflurano vaporizzato. Dopo POSIZIONAM correttag del mouse sul dorso e la fissazione degli arti, valutare la profondità dell&#39;anestesia. Poi tagliare la pelle sopra la vena femorale sinistra e localizzare la vena. Inserire la punta affilata dell&#39;ago posto sulla PE10 tubo e iniettare lentamente papavarine cloridrato soluzione ad una velocità di 100 pl per min (50 mg / kg di peso corporeo). </li><li> Dopo l&#39;iniezione, tagliare la cavità addominale e perforare la membrana. Tagliare le costole per esporre il cuore senza danneggiare o lividi esso. Agganciare l&#39;aorta toracica discendente con una pinza arterie. Fare un taglio netto sopra l&#39;atrio destro per consentire al sangue venoso defluire. Iniettare 2 ml di soluzione fisiologica nel ventricolo sinistro, seguita iniettando il lattice colorato manualmente con una leggera pressione sul 18-20 sec. Utilizzare le siringhe pre-riempite uno dopo l&#39;altro. Evitare di iniettare eventuali bolle. </li><li> Dopo iniezione di lattice, lasciare l&#39;animale per 5 min. Decapitare l&#39;animale e rimuovere con attenzione l&#39;intero cervello. Abbiate cura di non danneggiare l&#39;architettura navale e cortex Durante la rimozione dell&#39;osso del cranio e meningi. Mettere il cervello in un piatto cella 60 millimetri di coltura contenente 6-8 ml del 4% PFA per scattare una foto. Posizionare un righello di misura sotto il piatto trasparente. Scatta foto con un ingrandimento 25x della dorsale e la superficie ventrale del cervello con una macchina fotografica collegata al microscopio operatorio. Le foto devono essere prese a un angolo di 90 ° tra la piastra di Petri e l&#39;obiettivo microscopio al fine di quantificare la distanza originale. </li><li> Ripetere la procedura in altri animali 4-5. </li></ol> 3. La colorazione dei vasi cerebrali con miscela di inchiostri nero di carbonio <ol><li> Per confrontare il risultato di lattice a base colorazione vascolare cerebrale con il nostro protocollo, preparare una miscela di 100 pl di Herlitz Stempelfarbe inchiostro (CB1) con 900 microlitri di Pelican Scribtol Schwarz inchiostro (CB2) in una provetta da 1,5 ml EP. Preparare due tubi PE per fare un volume totale di 2 ml della miscela CB1 e CB2 in un rapporto 1:9. Raccogliere il composto in due siringhe da insulina separate (1ml ciascuna). Tappare le siringhe e metterli in un bagno d&#39;acqua per riscaldare fino a 37 ° C. Raccogliere 2 ml di soluzione fisiologica in altri due siringhe da insulina, e metterli in un bagno di acqua come bene. </li><li> Anestetizzare l&#39;animale e ripetere la stessa procedura descritta sopra, eccetto per iniezione di cloridrato papavarine. Dopo iniezione di 2 ml salaine nel ventricolo sinistro, iniettare una miscela di 2 ml di inchiostri nero di carbonio invece del lattice colorato. Eseguire le iniezioni a mano con una leggera pressione sul 18-20 sec per ml. Agganciare l&#39;aorta toracica discendente prima delle iniezioni. </li><li> Sacrificio l&#39;animale, rimuovere il cervello e prendere le foto. </li><li> Per conservazione a lungo termine del cervello, mettere il cervello in 4% PFA overnight seguita da incubazione con saccarosio con concentrazione aumenta gradualmente fino a quando il cervello galleggiante sommerge totalmente (5% seguito da 15% e 30%). Cervelli degli animali perfusi con il lattice colorato può essere conservato nello stesso modo. </li><li> Eseguire laprocedura in un altro 4-5 animali. Per confrontare la differenza di anatomia vascolare cerebrale a causa di differenti background genetico o ceppi, ripetere il protocollo in numero uguale di ApoE KO e SV129 topi, rispettivamente. </li><li> Per studiare l&#39;anatomia vascolare in condizioni ischemiche, inducono transitoria ischemia cerebrale focale per 45 min o 90 min secondo un protocollo standard di occlusione intraluminale del MCA sotto anestesia profonda (una descrizione dettagliata della chirurgia è oltre la portata di questo articolo, la si rimanda a Doeppner et al., 2010). Al termine del periodo di riperfusione programmato (1 giorno o 5 giorni), eseguire la colorazione vascolare con + CB1 CB2 solo gli inchiostri e macchiare l&#39;intero cervello con 2% di soluzione di TTC a 37 ° C per 5-10 minuti per identificare il volume dell&#39;infarto . TTC è ridotto a formazano rosso colorato dagli enzimi mitocondriali (succinato deidrogenasi in particolare). Dopo la colorazione TTC, le macchie dei tessuti metabolicamente attivi profondo rosso, mentre il tessuto infartuato rimane unstaiNed e appare bianca a causa di disfunzioni e denaturato enzima mitocondriale. Raccogliere immagini nello stesso modo come sopra descritto. </li></ol> 4. Studio dei territori vascolari cerebrali <ol><li> Per analizzare l&#39;anatomia dei vasi cerebrali, immagini della superficie dorsale del cervello sia macchiata di colore lattice o CB1 + CB2 inchiostri possono essere analizzati con il software ImageJ (un software basato su Java di dominio pubblico utilizzato per l&#39;elaborazione delle immagini e l&#39;analisi). I cervelli perfusi con il lattice di colore può mostrare gradi variabili di colorazione nave si trova sulla superficie dorsale, mentre CB1 + CB2 perfusione si macchia tutti gli arredi su entrambe le superfici ventrale e dorsale. </li><li> Aprire un file di immagine con il software ImageJ. Identificare tutti i punti anastomotiche tra l&#39;ACA e la MCA. Un punto anastomotico viene definito come il sito in cui il diametro del vaso è più stretta o la distanza tra il mezzo più vicini punti di ramificazione della ACA ei rami MCA, rispettivamente 2. <sup&gt; Disegnare una linea immaginaria (anastomosi linea) segnando i punti anastomotiche con l&#39;aiuto di strumento di disegno linea segmentata e il plugin &quot;Linea tratteggiata&quot;. </li><li> Impostare la scala in mm. Misurare la distanza tra la linea di anastomosi e la linea mediana a 4 mm caudalmente al polo frontale del cervello utilizzando retta strumento di disegno linee e strumento di &quot;misura&quot;. Fate lo stesso a 6 mm caudalmente dal polo frontale. Analizzare le immagini 3-4 per animale e calcolare il valore medio. Confrontare i valori tra i diversi gruppi di animali per identificare la variazione di anatomia cerebrovascolare. In C57Bl6 / J animali, la linea anastomotica tra ACA e l&#39;MCA può essere fatta più vicino alla linea mediana a 4 mm rispetto a 6 mm caudalmente dal polo frontale. ApoE topi KO presenterà alcuna differenza significativa in questo senso. Al contrario, in topi SV129 linea anastomotica giacerà millimetri ulteriormente e parallelamente alla linea mediana sia a 4 mm e 6 caudalmente dal polo frontale. </li><li> Per analizzare l&#39;anatomia vascolare in condizioni di ischemia, eseguire gli stessi calcoli di cui sopra. Identificare il confine infarto come il bordo rosso e bianco velina colorata che rappresenta un tessuto metabolicamente attivo e tessuto necrotico senza macchia, rispettivamente. Misurare la distanza del bordo infarto dalla linea mediana a 4 mm e di 6 mm caudale dal polo frontale del cervello. Raccogliere il valore medio a partire da immagini 3-4 per animale. Confrontare i risultati tra ischemia diverso e periodi di riperfusione. </li></ol>

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	<li>Meng, H., Peng, Y., 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=Nuclear+contrast+angiography:+A+simple+method+for+morphological+characterization+of+cerebral+arteries.">Nuclear contrast angiography: A simple method for morphological characterization of cerebral arteries.</a> Brain Research. 1261, 75-81 (2009).</li><li>Maeda, K., Hata, R., 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=Differences+in+the+cerebrovascular+anatomy+of+C57black/6+and+SV129+mice.">Differences in the cerebrovascular anatomy of C57black/6 and SV129 mice.</a> Neuroreport. 9 (7), 1317-1319 (1998).</li><li>Coyle, P., Jokelainen, P. 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R., Herz, 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=Visualization+of+macroscopic+cerebral+vessel+anatomy&#8212;A+new+and+reliable+technique+in+mice.">Visualization of macroscopic cerebral vessel anatomy&#8212;A new and reliable technique in mice.</a> Journal of Neuroscience Methods. 204, 249-253 (2012).</li><li>Doeppner, T. R., Nagel, F., 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=TAT-Hsp70-mediated+neuroprotection+and+increased+survival+of+neuronal+precursor+cells+after+focal+cerebral+ischemia+in+mice.">TAT-Hsp70-mediated neuroprotection and increased survival of neuronal precursor cells after focal cerebral ischemia in mice.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 29, 1187-1196 (2009).</li><li>Todo, K., Kitagawa, K., 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=Granulocyte-macrophage+colony-stimulating+factor+enhances+leptomeningeal+collateral+growth+induced+by+common+carotid+artery+occlusion.">Granulocyte-macrophage colony-stimulating factor enhances leptomeningeal collateral growth induced by common carotid artery occlusion.</a> Stroke. 39 (6), 1875-1882 (2008).</li><li>Sugiyama, Y., Yagita, Y., 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=Granulocyte+colony-stimulating+factor+enhances+arteriogenesis+and+ameliorates+cerebral+damage+in+a+mouse+model+of+ischemic+stroke.">Granulocyte colony-stimulating factor enhances arteriogenesis and ameliorates cerebral damage in a mouse model of ischemic stroke.</a> Stroke. 42 (3), 770-775 (2011).</li><li>Busch, H. J., Buschmann, I. R., 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=Arteriogenesis+in+hypoperfused+rat+brain.">Arteriogenesis in hypoperfused rat brain.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 23 (5), 621-628 (2003).</li><li>Maeda, K., Hata, R., 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=Regional+metabolic+disturbances+and+cerebrovascular+anatomy+after+permanent+middle+cerebral+artery+occlusion.">Regional metabolic disturbances and cerebrovascular anatomy after permanent middle cerebral artery occlusion.</a> in C57black/6 and SV129 mice. Neurobiology of Diseases. 6, 101-108 (1999).</li><li>Wang, Y., Kilic, 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=VEGF+overexpression+induces+post-ischaemic+neuroprotection,+but+facilitates+haemodynamic+steal+phenomena.">VEGF overexpression induces post-ischaemic neuroprotection, but facilitates haemodynamic steal phenomena.</a> Brain. 128 (1), 52-63 (2005).</li><li>ElAli, A., Doeppner, T. R., 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=Increased+blood-brain+barrier+permeability+and+brain+edema+after+focal+cerebral+ischemia+induced+by+hyperlipidemia:+Role+of+lipid+peroxidation+and+calpain+1/2,+matrix+metalloproteinase-2/9,+and+RhoA+overoxidation.">Increased blood-brain barrier permeability and brain edema after focal cerebral ischemia induced by hyperlipidemia: Role of lipid peroxidation and calpain 1/2, matrix metalloproteinase-2/9, and RhoA overoxidation.</a> Stroke. 42, (2011).</li><li>Coyle, P., Jokelainen, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+outcome+to+middle+cerebral+artery+occlusion+in+spontaneously+hypertensive+stroke-prone+rats+(SHRSP)+and+Wistar+Kyoto+(WKY)+rats.">Differential outcome to middle cerebral artery occlusion in spontaneously hypertensive stroke-prone rats (SHRSP) and Wistar Kyoto (WKY) rats.</a> Stroke. 14, 605-611 (1983).</li><li>Coyle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diameter+and+length+changes+in+cerebral+collaterals+after+middle+cerebral+artery+occlusion+in+the+young+rat.">Diameter and length changes in cerebral collaterals after middle cerebral artery occlusion in the young rat.</a> The Anatomical Record. 210 (2), 357-364 (1984).</li></ol>

Nessun conflitto di interessi dichiarati.

Perfusione di CB1 CB2 + per iniezione manuale può essere effettuata con successo da senza formazione intensiva in quanto non comporta alcun dispositivo specifico per implicare certa pressione 2,3. L&#39;eterogeneità dei risultati di perfusione nel nostro protocollo è anche trascurabile. Solo 1 animale su 16 non ischemiche animali e 3 su 20 animali ischemici hanno mostrato perfusione incompleta. In questi casi, l&#39;incorporazione di bolle durante la perfusione salina conseguente occlusione di vasi stata p...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Nome della ditta </td><td> Catalog No. </td></tr><tr><td> Scribtol Schwarz (CB2) </td><td> Pelican, Germania </td><td> 221 135 </td></tr><tr><td> Stempelfarbe (CB1) </td><td> Herlitz PBS AG, Germania </td><td> 10417202 </td></tr><tr><td> Gedeo lattice </td><td> Pebeo, Francia </td><td> 13042B </td></tr></tbody></table>

intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels

La struttura anatomica dei vasi cerebrali è un fattore determinante per l&#39;emodinamica del cervello così come la gravità delle lesioni in seguito insulti ischemici. Vascolarizzazione cerebrale risponde dinamicamente ai diversi stati patofisiologici e presenta notevoli differenze tra i ceppi e in condizioni di manipolazioni genetiche. Essenzialmente, una tecnica affidabile per la colorazione di vasi intracranici è essenziale per studiare la patogenesi di ictus ischemico. Fino a poco tempo, un insieme di tecniche diverse è stato impiegato per visualizzare la vascolarizzazione cerebrale compresa l&#39;iniezione di resina a bassa viscosità, Araldite F, gelatina miscelato con coloranti vari 1 (cioè rosso carminio, India inchiostro) o lattice con 2 o 3 senza nerofumo. Perfusione del bianco composizione di lattice attraverso l&#39;aorta ascendente è stata riportata da Coyle e Jokelainen 3. Maeda et al. 2 hanno modificato il protocollo con l&#39;aggiunta di carbon inchiostro nero per la composizione di lattice per la visualizzazione migliore contrasto delle navi dopo perfusione fisiologica del cervello. Tuttavia, perfusione inefficiente e inadeguato riempimento dei vasi sono spesso sperimentata a causa della elevata viscosità del composto lattice 4. Pertanto, abbiamo descritto una tecnica semplice ed efficace utilizzando una miscela di due inchiostri neri di carbonio disponibili in commercio (CB1 e CB2) per visualizzare la vascolarizzazione cerebrale in modo riproducibile 5. Abbiamo dimostrato che la perfusione con CB1 CB2 + in topi risultati in colorazione significativamente più piccoli vasi cerebrali ad una densità maggiore rispetto alla perfusione lattice 5. Qui, descriviamo il nostro protocollo di individuare i punti di anastomosi tra la parte anteriore (ACA) e medio arterie cerebrali (MCA) per studiare le variazioni delle navi nei topi con differenti background genetici. Infine, abbiamo dimostrato la fattibilità della nostra tecnica in un modello transitoria ischemia cerebrale focale in topi combinando CB1 +CB2-mediata colorazione vaso con colorazione TTC in vari gradi di lesioni ischemiche.

Il protocollo qui descritto supera le limitazioni tecniche di visualizzazione tradizionali a base di lattice di vascolarizzazione cerebrale dei roditori. Figura 1A mostra che la perfusione seguito del lattice colorato, solo i grandi vasi sulla superficie ventrale sono macchiati, lasciando l&#39;intera superficie dorsale senza macchia. Il risultato è anche molto variabile. Solo un animale su sei spettacoli colorazione parziale del ACA e la MCA visibile sulla superficie dorsale del cervello (dati non mos...

Watch this Scientific Journal Video about Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels at JoVE.com

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Analisi di anatomia roditore cerebrovascolari svolge un ruolo importante nella ricerca sperimentale ictus. In questo contesto, la perfusione intravascolare con lattice colorato è stato considerato come uno strumento standard per diversi anni. Tuttavia, questa tecnica comporta diverse limitazioni tecniche che mettono a repentaglio la sua riproducibilità. Qui, descriviamo un metodo semplice per visualizzare i vasi cerebrali in modo riproducibile. Iniezione di una miscela di due inchiostri carbon black commercialmente disponibili attraverso i risultati ventricolo sinistro del miocardio nel riempimento adeguato dei vasi cerebrali con visualizzazione ad alto contrasto. Abbiamo applicato con successo questa tecnica per identificare i punti di anastomosi tra territori vascolari cerebrali di topi con diversi background genetici. Abbiamo finalmente le prove che questo nuovo metodo e semplice per la colorazione del serbatoio può essere combinato con cloruro di trifeniltetrazolio (TTC) colorazione - uno strumento ampiamente utilizzato per osservare e analizzare i volumi di infarto nei topi.

Perfusione intravascolare di inchiostro nero di carbonio permette la visualizzazione affidabile dei vasi cerebrali

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

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Neuroscience

15.3K Views.  University of Duisburg-Essen Medical School. Analisi di anatomia roditore cerebrovascolari svolge un ruolo importante nella ricerca sperimentale ictus. In questo contesto, la perfusione intravascolare con lattice colorato è stato considerato come uno strumento standard per diversi anni. Tuttavia, questa tecnica comporta diverse limitazioni tecniche che mettono a repentaglio la sua riproducibilità. Qui, descriviamo un metodo semplice per visualizzare i vasi cerebrali in modo riproducibile. Iniezione di una miscela ...

Video: Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Modello di ischemia cerebrale focale da occlusione endovascolare Sutura della arteria cerebrale media nel ratto

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

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Neuroscienze

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Riproduzione automatica Interactive Video per gli Studi di comunicazione animale

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograda Etichettatura fluorescente Consente mirata extracellulare singola unità di registrazione da parte dei neuroni identificati<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualizzazione e manipolazione genetica di dendriti e spine nella corteccia cerebrale e nell&#39;ippocampo mouse utilizzando<em&gt; In utero</em&gt; Elettroporazione

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Due registrazioni elettrofisiologiche di sinapticamente-evocati Risposte astrogliale e neuronale in acute fettine di ippocampo

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Elettrofisiologia su isolati tronco cerebrale, midollo spinale Ammassi di Newborn Roditore Consente Neural Network respiratoria registrazione Uscita

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

Purificazione delle navi cervello di topo

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Isolamento e Incannulazione di cerebrale parenchimale Arteriole

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

Intracerebroventricular e intravascolare Iniezione di Viral particelle e fluorescenti microperle nel cervello neonatale

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Un modello del ratto dell'ipoperfusione lieve intrauterina con stenosi Microcoil

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...