Stefano Fumagalli è un collega della Fondazione Monzino.

1. Immunofluorescenza Il seguente protocollo è eseguita su criosezioni cerebrali coronali ottenuti da topi transcardially perfusi (20 ml di PBS, 0,1 mol / l, pH 7,4, seguita da 50 ml di paraformaldeide refrigerate 4% in PBS). Dopo perfusione, i cervelli sono accuratamente rimossi e trasferiti al 30% di saccarosio in PBS a 4 ° C per una notte per crioprotezione. I cervelli sono poi rapidamente congelati per immersione in isopentano a - 45 ° C per 3 minuti prima di essere sigillato in fiale e conservato a -70 ° C fino all&#39;utilizzo. Criosezioni cerebrali coronali (20 micron) sono tagliati in serie e sottoposti a protocollo immunofluorescenza 11, 6. Portare tutti i reagenti ed i campioni a temperatura ambiente prima dell&#39;uso. Si prega di notare che le diluizioni di lavoro presentati di anticorpi e siero risultato di studi effettuati al fine di ottenere le migliori prestazioni. Quando vengono utilizzati diversi anticorpi e siero, il protocollo deve essere convalidato. <pclass = &quot;jove_content&quot;&gt; Nota che, poiché gli anticorpi primari sia anti-CD11b e anti-CD68 sono realizzati nel ratto, per evitare segnale cross dalla destra anti-ratto Alexa 546, abbiamo usato una elevata diluizione di anti-CD11b seguita da amplificazione del segnale fluorescente con kit TSA (Cy5 Tyramide). Abbiamo istituito la diluizione di lavoro ottimale per anti-CD11b eseguendo il protocollo descritto tranne passo 1.17 e utilizzando almeno 7 diverse diluizioni di anti-CD11b come riportato in tabella 1. Come risultato, 1:30.000 è stata scelta essendo l&#39;unico diluizione conseguire: 1) segnale visibile con Cy5 alla lunghezza d&#39;onda di eccitazione 646 nm, 2) nessun segnale con Alexa 546 a lunghezza d&#39;onda di eccitazione 532 nm. In questo modo, il segnale fluorescente Alexa 546 è selettivamente associata con l&#39;espressione di CD68. Le diluizioni ottimali per anti-CD11b e anticorpi anti-CD68 possono variare a seconda del tipo di tessuto e devono essere definiti prima di iniziare il protocollo co-etichettatura. <ol><li> Lavare criosezioni cervello due volte con PBS. </li><li> Incriosezioni cubate per 5 min in PBS contenente 1% H 2 O 2 (passo richiesto, poiché l&#39;amplificazione fluorescente bisogno incubazione con perossidasi di rafano, streptavidina-HRP, vedere il passo 1.12). </li><li> Criosezioni Lavare 2x con PBS. </li><li> Incubare criosezioni per 60 min in PBS contenente il 10% NGS e 0,3% Triton. </li><li> Incubare criosezioni a 4 ° C per una notte in PBS contenente primario Ab Rat anti-CD11b [1:30.000], il 10% NGS e 0,3% Triton. </li><li> Lavare criosezioni 2x (5 min) con PBS. </li><li> Incubare criosezioni per 60 min in PBS contenente biotinilato secondario anti-Rat Ab [1:200] e 1% NGS. </li><li> Criosezioni Lavare 2x con PBS. </li><li> Lavare criosezioni con TNT (Tris-HCl, NaCl, Tween: 0,1 M Tris-HCl, pH 7,4, 0,15 M NaCl, 0,05% Tween 20). </li><li> Incubare criosezioni per 1,5 ore a TNB (tampone Tris-HCl-NaCl-Blocking: 0.1 M TRIS-HCl, pH 7.4, 0.15 M NaCl, 0,5% Blocco reagente da apposito kit (vedi tabella dei reagenti)). </li><li> Wash criosezioni 3x volte con TNT. </li><li> Incubare criosezioni in TNB contenente streptavidina-HRP [1:100]. </li><li> Criosezioni Lavare 3x con TNT. </li><li> Incubare criosezioni per 8 minuti in Amplification diluente contenente Cyanine 5 Tyramide [1:300]. </li><li> Criosezioni Lavare 3x con PBS. </li><li> Incubare criosezioni per 60 min in PBS contenente il 10% NGS e 0,1% Triton. </li><li> Incubare criosezioni a 4 ° C per una notte in PBS contenente primario Ab Rat anti-CD68 [1:200], 3% NGS e 0,3% Triton. </li><li> Criosezioni Lavare 3x con PBS. </li><li> Incubare criosezioni per 60 min in PBS contenente fluorconjugated secondario Ab Alexa 546 anti-Rat [1:500] e 1% NGS. </li><li> Criosezioni Lavare 3x con PBS. </li><li> Incubare criosezioni per 10 min in PBS contenente Hoechst 1 ug / ml. </li><li> Criosezioni Lavare 3x con PBS. </li><li> Montare criosezioni in Prolungare oro. </li></ol> 2. Acquisizione di immagini tridimensionali per Microscopia confocale <p class = &quot;jove_content&quot;&gt; Il microscopio utilizzato qui è stato un microscopio IX81 dotato di FV500 confocale unità di scansione con 3 linee laser: Ar-KR (488nm), He-Ne rosso (646nm), e He-Ne verde (532nm) e un diodo UV. <ol><li> Selezionare i laser di eccitazione a seconda delle lunghezze d&#39;onda dei coloranti fluorescenti per essere entusiasti (He-Ne rosso per Cy5, CD11b, He-Ne verde per Alexa546, CD68 e il diodo UV per la Hoechst, nuclei). </li><li> Selezionare la migliore combinazione specchio dicroico per la raccolta segnale luminoso. </li><li> Impostare la risoluzione delle immagini ad un minimo di 800 x 600 pixel. </li><li> Individuare una zona di interesse utilizzando epi-fluorescenza e progressivamente aumentare l&#39;ingrandimento con l&#39;obiettivo 40X. </li><li> Passare a Laser Scanning Microscopy (LSM) modalità. </li><li> Eseguire scansioni ripetitive per regolare il fotomoltiplicatore (PMT) e il guadagno per ogni canale. I laser possono essere attivati ​​individualmente per facilitare il set up. Mantenere guadagno più basso possibile per evitare segnali non specifici indesiderati. </li><li> Con repetitivo scansione in esecuzione, spostare il controllo del fuoco per definire gli estremi inferiore e superiore della z (lunghezza totale asse z = 10 micron). </li><li> Smettere di scansione ripetitiva. </li><li> Definire passo. Dovrebbe essere il più vicino possibile alla dimensione dei pixel (0,225 micron) per ottenere un rapporto 1:1 formato standard sopra l&#39;asse z. </li><li> Attivare filtro di Kalman almeno 2 volte. </li><li> Attivare la modalità di scansione sequenziale per evitare effetti sanguinare-through. </li><li> Vai a metà strada lungo l&#39;asse z ed eseguire una scansione sequenziale xy. Controllare il set-up di PMT e di guadagno (buon rapporto segnale / rumore). Se non è soddisfacente, ripetere il punto 2.6. </li><li> Eseguire acquisizione xyz. </li><li> Esportare dati come file multitiff. Ogni file multitiff genere contiene tre canali di colore (blu, verde e rosso) e 44 piani focali. </li></ol> 3. Tridimensionale Vista Acquisizioni confocale e rendering tridimensionale Caricare file multitiff al software Imaris ed elaborarli come segue: <ol><li>Software Open. </li><li> Selezionare la vista sorpassare. </li><li> Caricare il file multitiff. </li><li> Selezionare il colore desiderato per ogni canale. </li><li> Rimuovere il rumore di fondo da aumentando il valore minimo sul pannello di regolazione del display. Regolare singolarmente ciascun canale. </li><li> Vai alla vista di sezione. Muovetevi lungo l&#39;asse z in cerca di co-localizzazione (pixel giallo). </li><li> Istruzioni su un&#39;area gialla per vedere se la colocalizzazione è presente lungo l&#39;asse z (cioè appartiene ad un oggetto solido). Proiezioni asse Z sono visibili nella parte destra e inferiore della figura. </li><li> Prendete uno snapshot. </li><li> Torna a superare vista. </li><li> Raccolto 3D per isolare una cella o un gruppo di celle. </li><li> Selezionare un canale sul pannello di regolazione del display. </li><li> Selezionare la sorpassare / superfici algoritmo costruttore. Passi algoritmo: <ol><li> Selezionare il canale. </li><li> Definire soglia (utilizzare lo stesso valore minimo in tegli visualizzare pannello di regolazione). </li><li> Definire ridimensionamento. </li><li> Definire smoothing (migliore = 0,200). </li><li> Definire l&#39;aspetto del colore. </li><li> Fine algoritmo. </li></ol></li><li> Ripetere passo 3.12 per ogni canale. </li><li> Deselezionare i canali di fluorescenza sul pannello di regolazione del display e selezionare tutte le tre superfici. </li><li> Spostare il volume di trovare la migliore vista. </li><li> Prendete uno snapshot. </li></ol>

<ol>
	<li>Davalos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+mediates+rapid+microglial+response+to+local+brain+injury+in+vivo.">ATP mediates rapid microglial response to local brain injury in vivo.</a> Nat. Neurosci. 8, 752-758 (2005).</li><li>Yenari, M. A., Kauppinen, T. M., Swanson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+in+stroke:+therapeutic+targets.">Microglial activation in stroke: therapeutic targets.</a> Neurotherapeutics. 7, 378-391 (2010).</li><li>Iadecola, C., Anrather, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunology+of+stroke:+from+mechanisms+to+translation.">The immunology of stroke: from mechanisms to translation.</a> Nat. Med. 17, 796-808 (2011).</li><li>Jin, R., Yang, G., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mechanisms+in+ischemic+stroke:+role+of+inflammatory+cells.">Inflammatory mechanisms in ischemic stroke: role of inflammatory cells.</a> J. Leukoc. Biol. 87, 779-789 (2010).</li><li>Schilling, M., Besselmann, M., Muller, M., Strecker, J. K., Ringelstein, E. B., Kiefer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+phagocytic+activity+of+resident+microglia+over+hematogenous+macrophages+following+transient+focal+cerebral+ischemia:+an+investigation+using+green+fluorescent+protein+transgenic+bone+marrow+chimeric+mice.">Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice.</a> Exp. Neurol. 196, 290-297 (2005).</li><li>Perego, C., Fumagalli, S., De Simoni, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+pattern+of+expression+and+colocalization+of+microglia/macrophage+phenotype+markers+following+brain+ischemic+injury+in+mice.">Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice.</a> Journal of Neuroinflammation. 8, 174-193 (2011).</li><li>Zanier, E. 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=Human+umbilical+cord+blood+mesenchymal+stem+cells+protect+mice+brain+after+trauma.">Human umbilical cord blood mesenchymal stem cells protect mice brain after trauma.</a> Crit. CareMed. 39 (11), 2501-2510 (2011).</li><li>Capone, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere+derived+cells+exert+a+neuroprotective+action+by+changing+the+ischemic+microenvironment.">Neurosphere derived cells exert a neuroprotective action by changing the ischemic microenvironment.</a> PLoS ONE. 2, e373 (2007).</li><li>Bhatia, S., 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=Rapid+host+defense+against+Aspergillus+fumigatus+involves+alveolar+macrophages+with+a+predominance+ofalternatively+activated+phenotype.">Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance ofalternatively activated phenotype.</a> PLoS One. 6, e15943 (2011).</li><li>Raes, G., Noel, W., Beschin, A., Brys, L., de Baetselier, P., Hassanzadeh, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FIZZ1+and+Ym+as+tools+to+discriminate+between+differentially+activated+macrophages.">FIZZ1 and Ym as tools to discriminate between differentially activated macrophages.</a> Dev. Immunol. 9, 151-159 (2002).</li><li>Gesuete, 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=Recombinant+C1+inhibitor+in+brain+ischemic+injury.">Recombinant C1 inhibitor in brain ischemic injury.</a> Ann. Neurol. 66, 332-342 (2009).</li><li>Sica, A., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+plasticity+and+polarization:+in+vivo+veritas.">Macrophages plasticity and polarization: in vivo veritas.</a> J. Clin. Invest. 122 (3), 787-795 (2012).</li></ol>

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Vi presentiamo qui protocolli basati immunofluorescenza, seguita da analisi confocale tridimensionale come un approccio efficace per indagare la localizzazione e la co-espressione di M / M marcatori fenotipici nella zona ischemica (per un&#39;analisi più dettagliata vedi rif 6). Questo metodo combina colorazione specifica pertinente marker di attivazione M / M con confocale tridimensionale. La messa a punto di anticorpi, siero e fluorconjugated diluizioni di lavoro permette di segnale ottimale rumore dei mar...

Dopo il danno cerebrale acuto, microglia stanno rapidamente attivata e sottoposti drammatica morfologica e fenotipica cambia 1-3. Questa risposta intrinseca è associata al reclutamento di macrofagi del sangue nato che migrano nel infortunato 4,5 parenchima cerebrale. Il ruolo della microglia e macrofagi che non sono antigenicamente distinguibili (d&#39;ora in poi denominato M / M) nel trauma cranico è ancora dibattuta. Un numero crescente di studi indicano che, analogamente a quanto descritto per macrofagi periferici, microglia e macrofagi reclutati cervello possono assumere diversi fenotipi i cui estremi conforme a classico pro-infiammatoria tossico (M1) o anti-infiammatoria protettiva (M2) fenotipo. I diversi stati di attivazione, compreso secrezione di fattori pro o anti-infiammatorie, rilascio di molecole neurotrofici e dell&#39;attività lisosomiale sono caratterizzate da un modello specifico di marcatori fenotipici, la cui espressione dipende dal temporaleevoluzione dell&#39;ambiente circostante. La caratterizzazione di questi fenotipi M / M nel cervello danneggiato è ancora scarsa. Abbiamo utilizzato un modello murino consolidata di pMCAo per analizzare l&#39;espressione M / M e l&#39;evoluzione dopo l&#39;ictus. Protocolli di immunofluorescenza basato presentate qui mirano ad ottenere comprensione nella comparsa di specifici marcatori fenotipici M / M, la loro localizzazione e cellulari co-espressione nella zona ischemica. Abbiamo studiato alcune molecole associate diverso stato di attivazione o fenotipo, cioè CD11b, un marker di superficie espressa da leucociti e un indicatore ampiamente usato di M / M di attivazione / reclutamento 6-8, CD68 un marcatore dei lisosomi 6,7 e YM1 un secretoria proteina espressa alternativamente attivati ​​(M2) e macrofagi associati al recupero e funzione di ripristino 9-10. Quando due marcatori sono espresse dalla stessa cellula, ma in differenti compartimenti subcellulari, solo colocalizzazione non può essere molto informative. In questo caso, l&#39;analisi di coexpression può essere eseguita utilizzando un&#39;unica vista in pianta e rendering tridimensionali. Siamo qui descriviamo un protocollo per ottenere un&#39;approfondita analisi tridimensionale del marcatore coexpression.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD11b</td>
			<td>Kindly provided by Dr. A. Doni, Istituto Clinico Humanitas, Milan, Italy</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD68 </td>
			<td>AbD Serotec</td>
			<td>MCA 1957</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rabbit Anti-mouse Ym1 </td>
			<td>Stem Cell Technologies</td>
			<td>1404</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hoechst 33342</td>
			<td>Life technologies</td>
			<td>H21492</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse Anti-Neural Nuclei (NeuN)</td>
			<td>CHEMICON</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biotinilated Goat Anti-Rat antibody</td>
			<td>Jackson Immuno Research</td>
			<td>112-065-143</td>
			<td></td>
		 </tr>
		 <tr>
			<td>TSA Cyanine 5 System </td>
			<td>Perkin Elmer</td>
			<td>NEL705A001KT</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Prolong Gold </td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rat alexa 546</td>
			<td>Invitrogen</td>
			<td>A-11081</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse Alexa 488</td>
			<td>Invitrogen</td>
			<td>A-21121</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rabbit Alexa 594</td>
			<td>Invitrogen</td>
			<td>A-11037</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Normal Goat Serum</td>
			<td>Vectors Laboratories</td>
			<td>S-1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tritin X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma</td>
			<td>P4417-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Cryostat CM1850</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Olympus IX81 confocal microscope </td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>AnalySIS software</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaris software 5.0</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Photoshop cs2</td>
			<td>Adobe Systems</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software packages GraphPad Prism version 4.0</td>
			<td>GraphPad Software Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

three-dimensional confocal analysis of microglia/macrophage markers of polarization in experimental brain injury

Dopo l&#39;ictus cerebrale microglia / macrofagi (M / M) sottoposti attivazione rapida con i cambiamenti morfologici e fenotipici drammatici che includono l&#39;espressione di antigeni di superficie innovativi e la produzione di mediatori che si accumulano e mantengono la risposta infiammatoria. Prove emergenti indica che M / M sono cellule altamente plastica che possono assumere classico antinfiammatorio (M2) attivazione pro-infiammatoria (M1) o alternativo dopo danno cerebrale acuto. Tuttavia una caratterizzazione completa di M / M espressione marcatore fenotipo, la loro colocalizzazione e l&#39;evoluzione temporale nel cervello danneggiato è ancora mancante. Protocolli di immunofluorescenza specificamente colorazione pertinenti marcatori di attivazione M / M possono essere eseguite nel cervello ischemico. Presentiamo qui protocolli basati immunofluorescenza-seguita da analisi confocale tridimensionale come un approccio efficace per indagare il pattern di localizzazione e co-espressione di M / M marcatori fenotipici qualiCD11b, CD68, YM1, nel modello murino di ischemia focale indotta da occlusione permanente dell&#39;arteria cerebrale media (pMCAO). Analisi bidimensionale della zona macchiata rivela che ciascun marcatore è associato un definito M / M morfologia e ha una data localizzazione nella lesione ischemica. Modelli di M / M marcatore fenotipo co-espressione può essere valutata da tridimensionale confocale nella zona ischemica. Le immagini possono essere acquisite nel corso di un volume definito (10 micron asse z e una dimensione 0,23 um passo, corrispondente ad un volume di 180 x 135 x 10 micron) con una modalità di scansione sequenziale per minimizzare gli effetti di spurgo-through della lunghezza d&#39;onda ed evitare sovrapposizioni. Le immagini vengono poi elaborati per ottenere il rendering tridimensionale per mezzo di software Imaris. Vista solida di tre rendering tridimensionale consente la definizione di espressione marcatore in ammassi di cellule. Abbiamo dimostrato che le M / M hanno la capacità di differenziare verso una moltitudine di fenotipi, a seconda della posizione del sito di lesione e time dopo l&#39;infortunio.

Un esempio dei risultati ottenuti quando vengono effettuate etichettatura protocolli e acquisizioni confocali fuori nella regione ischemica è illustrato nelle figure 1A e 1B. Una visione bidimensionale di immagini acquisite mostra che a ventiquattro anni ore dopo ischemia (A), il marcatore CD68 lisosomiale (verde) viene espresso in cellule ipertrofiche ameboide CD11b (rosso) presenti nel nucleo ischemico. Nella zona di confine cellule positive (B) CD11...

Watch this Scientific Journal Video about Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury at JoVE.com

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

Un modo per acquisire nuove conoscenze nella complessità della risposta infiammatoria del cervello è presentato. Descriviamo protocolli basati immunofluorescenza-seguita da analisi confocale tridimensionale per indagare il pattern di co-espressione di marcatori fenotipici microglia / macrofagi in un modello murino di ischemia focale.

Tridimensionale confocale Analisi dei marcatori microglia / macrofagi di polarizzazione in Experimental Brain Injury

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

three-dimensional-confocal-analysis-microgliamacrophage-markers

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Neuroscience

11.5K Views.  IRCCS - Istituto di Ricerche Farmacologiche Mario Negri.  Un modo per acquisire nuove conoscenze nella complessità della risposta infiammatoria del cervello è presentato. Descriviamo protocolli basati immunofluorescenza-seguita da analisi confocale tridimensionale per indagare il pattern di co-espressione di marcatori fenotipici microglia / macrofagi in un modello murino di ischemia focale. 

Video: Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

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 ...

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Neuroscienze

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

Protocollo per tridimensionale confocale morfometrica Analisi di astrociti

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

Imaging tridimensionale e l'analisi dei mitocondri all'interno di fibre nervose umane Intraepidermal

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Le reti neurali ricalcolate anatomicamente tridimensionali dei micro-tessuti per la ricostruzione, la modulazione e la modellazione del sistema nervoso

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

Analisi automatizzata di andatura in topi con lesione da costrizione cronica

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

Formazione immagine tridimensionale su larga scala di organizzazione cellulare nella neocorteccia Mouse

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Impianto cronico di tutto-corticale Electrocorticographic Array nel Marmoset comune

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

Modellazione tridimensionale delle forme e analisi delle strutture cerebrali

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

Analisi dei sistemi della risposta neuroinfiammatoria ed emodinamica alla lesione cerebrale traumatica

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

Generazione organoide nervosa motoria tridimensionale

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...