R.V. lavoro è stato supportato da una borsa di studio post-dottorato Roche (2014-2017).

<ol>
	<li>Blanchette, M., Daneman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+maintenance+of+the+BBB.">Formation and maintenance of the BBB.</a> Mech Dev. 138, 8-16 (2015).</li><li>Chow, B. W., Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Molecular+Constituents+of+the+Blood-Brain+Barrier.">The Molecular Constituents of the Blood-Brain Barrier.</a> Trends Neurosci. 38 (10), 598-608 (2015).</li><li>Niewoehner, 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=Increased+Brain+Penetration+and+Potency+of+a+Therapeutic+Antibody+Using+a+Monovalent+Molecular+Shuttle.">Increased Brain Penetration and Potency of a Therapeutic Antibody Using a Monovalent Molecular Shuttle.</a> Neuron. 81 (1), 49-60 (2014).</li><li>Yu, Y. 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=Therapeutic+bispecific+antibodies+cross+the+blood-brain+barrier+in+nonhuman+primates.">Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates.</a> Sci Transl Med. 6 (261), 154 (2014).</li><li>Preston, J. E., Joan Abbott, N., Begley, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcytosis+of+macromolecules+at+the+blood-brain+barrier.">Transcytosis of macromolecules at the blood-brain barrier.</a> Adv Pharmacol. 71, 147-163 (2014).</li><li>De Bock, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Into+rather+unexplored+terrain-transcellular+transport+across+the+blood-brain+barrier.">Into rather unexplored terrain-transcellular transport across the blood-brain barrier.</a> Glia. 64 (7), 1097-1123 (2016).</li><li>Bien-Ly, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferrin+receptor+(TfR)+trafficking+determines+brain+uptake+of+TfR+antibody+affinity+variants.">Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants.</a> J Exp Med. 211 (2), 233-244 (2014).</li><li>Sade, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+blood-brain+barrier+transcytosis+assay+reveals+antibody+transcytosis+influenced+by+pH-dependent+receptor+binding.">A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding.</a> PLoS One. 9 (4), 96340 (2014).</li><li>Tian, X., 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=LRP-1-mediated+intracellular+antibody+delivery+to+the+Central+Nervous+System.">LRP-1-mediated intracellular antibody delivery to the Central Nervous System.</a> Sci Rep. 5, 11990 (2015).</li><li>Hsu, J., Rappaport, J., Muro, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+binding,+uptake,+and+transport+of+ICAM-1-targeted+nanocarriers+across+endothelial+and+subendothelial+cell+components+of+the+blood-brain+barrier.">Specific binding, uptake, and transport of ICAM-1-targeted nanocarriers across endothelial and subendothelial cell components of the blood-brain barrier.</a> Pharm Res. 31 (7), 1855-1866 (2014).</li><li>Siupka, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+apical-basal+traffic+of+the+cation-independent+mannose-6-phosphate+receptor+in+brain+endothelial+cells.">Bidirectional apical-basal traffic of the cation-independent mannose-6-phosphate receptor in brain endothelial cells.</a> J Cereb Blood Flow Metab. , (2017).</li><li>Zlokovic, B. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+saturable+mechanism+for+transport+of+immunoglobulin+G+across+the+blood-brain+barrier+of+the+guinea+pig.">A saturable mechanism for transport of immunoglobulin G across the blood-brain barrier of the guinea pig.</a> Exp Neurol. 107 (3), 263-270 (1990).</li><li>Deane, 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=IgG-assisted+age-dependent+clearance+of+Alzheimer's+amyloid+beta+peptide+by+the+blood-brain+barrier+neonatal+Fc+receptor.">IgG-assisted age-dependent clearance of Alzheimer's amyloid beta peptide by the blood-brain barrier neonatal Fc receptor.</a> J Neurosci. 25 (50), 11495-11503 (2005).</li><li>Cabezon, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+applied+to+study+the+trafficking+of+8D3-coated+gold+nanoparticles+at+the+blood-brain+barrier.">Serial block-face scanning electron microscopy applied to study the trafficking of 8D3-coated gold nanoparticles at the blood-brain barrier.</a> Histochem Cell Biol. , (2017).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. (65), (2012).</li><li>Collin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+uptake+of+tau/pS422+antibody+and+reduced+progression+of+tau+pathology+in+a+mouse+model+of+Alzheimer's+disease.">Neuronal uptake of tau/pS422 antibody and reduced progression of tau pathology in a mouse model of Alzheimer's disease.</a> Brain. 137 (10), 2834-2846 (2014).</li><li>Villasenor, 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=Trafficking+of+Endogenous+Immunoglobulins+by+Endothelial+Cells+at+the+Blood-Brain+Barrier.">Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier.</a> Sci Rep. 6, 25658 (2016).</li><li>Villasenor, 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=Region-specific+permeability+of+the+blood-brain+barrier+upon+pericyte+loss.">Region-specific permeability of the blood-brain barrier upon pericyte loss.</a> J Cereb Blood Flow Metab. , (2017).</li><li>Armulik, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+regulate+the+blood-brain+barrier.">Pericytes regulate the blood-brain barrier.</a> Nature. 468 (7323), 557-561 (2010).</li><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+Tissue+Clearing.">Clarifying Tissue Clearing.</a> Cell. 162 (2), 246-257 (2015).</li><li>Sibarita, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Deconvolution Microscopy. 95, 201-243 (2005).</li><li>Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comparison of Widefield/Deconvolution and Confocal Microscopy for Three-Dimensional Imaging. , 453-467 (2006).</li><li>Chenouard, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective+comparison+of+particle+tracking+methods.">Objective comparison of particle tracking methods.</a> Nat Methods. 11 (3), 281-289 (2014).</li><li>Foret, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+theoretical+framework+to+infer+endosomal+network+dynamics+from+quantitative+image+analysis.">A general theoretical framework to infer endosomal network dynamics from quantitative image analysis.</a> Curr Biol. 22 (15), 1381-1390 (2012).</li><li>Daneman, R., Zhou, L., Kebede, A. A., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+are+required+for+blood-brain+barrier+integrity+during+embryogenesis.">Pericytes are required for blood-brain barrier integrity during embryogenesis.</a> Nature. 468 (7323), 562-566 (2010).</li><li>Bell, R. 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=Pericytes+control+key+neurovascular+functions+and+neuronal+phenotype+in+the+adult+brain+and+during+brain+aging.">Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging.</a> Neuron. 68 (3), 409-427 (2010).</li><li>Ben-Zvi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mfsd2a+is+critical+for+the+formation+and+function+of+the+blood-brain+barrier.">Mfsd2a is critical for the formation and function of the blood-brain barrier.</a> Nature. 509 (7501), 507-511 (2014).</li><li>Stewart, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+vesicles+in+the+blood-brain+barrier:+are+they+related+to+permeability.">Endothelial vesicles in the blood-brain barrier: are they related to permeability.</a> Cell Mol Neurobiol. 20 (2), 149-163 (2000).</li><li>Follain, G., Mercier, L., Osmani, N., Harlepp, S., Goetz, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing+-+multi-scale+spatio-temporal+imaging+towards+in+vivo+cell+biology.">Seeing is believing - multi-scale spatio-temporal imaging towards in vivo cell biology.</a> J Cell Sci. 130 (1), 23-38 (2017).</li></ol>

Gli autori sono sotto lavoro retribuito da Roche.

Il protocollo di cui sopra descrive la preparazione di sezioni digalleggiante del cervello, macchiatura immunofluorescente, acquisizione di immagini e parametri di analisi per microscopia ad alta risoluzione della BBB. Questo metodo è stato recentemente utilizzato per studiare la localizzazione di anticorpo consegna piattaforme3, il trasporto di IgG endogeno attraverso la BBB17e l'eterogeneità della BBB su Pericita perdita18. Diverse fasi del protocollo possono essere modificati per adattarsi all'obiettivo specifico dell'esperimento. In primo luogo, l'uso di sezioni spesse (100 µm) facilita la loro manipolazione durante le procedure di montaggio e di immunostaining. Permette anche per la ricostruzione 3D della rete capillare, l'unità neurovascolare e per la generazione di sezioni trasversali NVU e capillare. Tuttavia, penetrazione di anticorpi all'interno delle sezioni del tessuto può variare e alcuni macchiatura dell'anticorpo può essere limitata allo strato superficiale del tessuto vicino il vetrino coprioggetti. Il protocollo può essere modificato aumentando la concentrazione di detersivo durante il passaggio di permeabilizzazione e/o la lunghezza del passo permeabilizzazione per migliorare la penetrazione dell'anticorpo nel tessuto. In secondo luogo, la qualità dell'immagine può essere compromessa quando si tenta di acquisire immagini più profonde all'interno del tessuto (di solito 20-30 µm sotto la superficie) a causa di dispersione della luce così come aberrazioni ottiche dalla mancata corrispondenza di indice di rifrazione. Per ovviare a questo problema, nuovi metodi per schiarimento del tessuto e dell'anticorpo attivo penetrazione20 possono essere combinati con questo protocollo per grandi volumi di immagini del tessuto. In terzo luogo, deconvoluzione viene eseguita dopo l'acquisizione di immagini per migliorare la risoluzione assiale dell'immagine. La scelta dell'algoritmo di deconvoluzione cieco utilizzato in questo protocollo si basava su (i) la sua facilità d'uso, come richiesto, nessun pre-calcolo della funzione di diffusione del punto (ii) la sua robustezza per il miglioramento della qualità di immagine21e (iii) la mancanza di manufatti su mIgG strutture intracellulari dopo l'implementazione. A seconda delle strutture intracellulari visualizzate nell'esempio, altri algoritmi di deconvoluzione potrebbero causare immagini di qualità superiore. I seguenti riferimenti21,22 forniscono un ampio dibattito sui vantaggi e limitazioni degli ulteriori algoritmi di deconvoluzione di immagine. Infine, l'uso di un pacchetto di software di analisi di immagine ha permesso la segmentazione dei capillari e strutture intracellulari in tre dimensioni. Chiaramente l'analisi dell'immagine non è limitato al software descritto nel protocollo e pacchetti alternativi, ad esempio quelli discussi in riferimento23, può essere utilizzato per immagini del segmento. L'idoneità dei programmi software diversi per l'analisi delle strutture intracellulari attraverso la Bee deve essere verificata empiricamente valutando la precisione di segmentazione di immagini.
Poiché questo metodo si basa su campioni fissati, non fornisce informazioni dirette sulle dinamiche di transcitosi attraverso la Bee. Tuttavia, si può abbinare con esperimenti di tempo-corso24, ad esempio per via endovenosa iniettando la molecola di interesse e misurando il suo accumulo all'interno di BECs in diversi momenti dopo l'iniezione, per ricostruire la cinetica di trasporto intracellulare. Il vantaggio di questo approccio è che permette l'analisi delle regioni profonde del cervello, come mostrato in18, che sono attualmente inaccessibile al videomicroscopia approcci di imaging dal vivo. Un passaggio fondamentale durante il protocollo è l'attento monitoraggio della fissazione del tessuto. Fissaggio con 4% PFA riduce drasticamente l'immunogenicità degli organelli intracellulari e di immunoglobuline endogena o marginalmente amministrato17 (Figura 1B). Una limitazione di questo protocollo è relativo requisito degli anticorpi di alta qualità (cioè, bassa colorazione aspecifica, bassa reattività crociata) adatti a immunofluorescenza. Purché tali reagenti sono disponibili, il metodo può essere applicato per studiare la localizzazione intracellulare di tutta la proteina di interesse. Ad esempio, il protocollo è stato utilizzato per identificare i lisosomi nel cervello cellule endoteliali17. Si deve inoltre osservare che, poiché questo protocollo si basa su microscopia confocal, la risoluzione laterale è limitata dalla diffrazione e non è possibile risolvere strutture più piccoli di circa 175 a 250 nm (Figura 2).
Gli studi precedenti hanno effettuato analisi dettagliata della composizione cellulare dell'unità neurovascolare usando microscopia confocale25,26. Tuttavia, indagando il trasporto intracellulare a BBB si basa principalmente sull'uso di microscopia elettronica di trasmissione19,27,28. Mentre questo metodo offre la massima risoluzione laterale di strutture intracellulari, microscopia elettronica rimane una tecnica impegnativa con bassa velocità effettiva. Inoltre, il numero di bersagli molecolari differenti che possono essere visualizzate da EM è molto limitato. Questo protocollo offre un'alternativa accessibile per studiare il trasporto intracellulare a BBB. La procedura completa, dall'accumulazione del cervello all'analisi di immagine, può essere eseguita in 5 o 6 giorni. Se gli anticorpi adatti sono disponibili, immunofluorescenza consente la rilevazione simultanea di più tipi/molecole delle cellule all'interno del campione stesso. Inoltre questo protocollo potrebbe essere combinato con tecniche di microscopia di Super-risoluzione per superare i limiti nella risoluzione spaziale29. Nel complesso, il protocollo sopra descritto consente la quantificazione dei cambiamenti nella localizzazione intracellulare di proteine di interesse all'interno dell'unità neurovascolare. Sua applicazione per diverse perturbazioni genetiche o farmacologiche permetterà di indagare le funzioni struttura e trasporto intracellulare di BBB in vivo.

Emato - encefalica (BBB) è una barriera cellulare continua formata da neuroni, astrociti, cellule endoteliali e periciti che separa il sistema nervoso centrale (SNC) dal sangue circolazione1. Il regolamento di trasporto attraverso la Bee svolge un ruolo cruciale nel mantenimento dell'omeostasi del cervello ed è mediato dalla proprietà specializzate delle cellule endoteliali cerebrali (BECs). La presenza di giunzioni intercellulari strette tra BECs e un basso tasso basale di transcitosi limitare il trasporto paracellulare e transcellulare di molecole ematica, rispettivamente2. Recentemente, la via di transcitosi in BECs è stata sfruttata per migliorare la fornitura di grandi molecole terapeutiche al cervello3,4. Tuttavia, i meccanismi di transcitosi attraverso la Bee non sono ancora stati completamente caratterizzato5,6.
Vasto lavoro è stato fatto in vitro per decifrare i meccanismi cellulari e molecolari che regolano il trasporto intracellulare attraverso BECs7,8,9,10, 11, ma tali sistemi non riescono a ricapitolare l'architettura complessa e la fisiologia dell'unità neurovascolare (NVU). D'altra parte, gli studi in vivo12,13 fornire dettagliate informazioni quantitative sui tassi di trasporto attraverso la Bee ma non forniscono le comprensioni nei meccanismi intracellulari di trasporto. Di conseguenza, indagando i componenti cellulari e intracellulari il NVU in vivo ed ex vivo rimane molto impegnativo14. Solo un numero limitato di tecniche è suscettibile di analizzare strutture subcellulari all'interno delle cellule il nvu. Maggior parte degli studi utilizza la microscopia elettronica, ma questa tecnica è limitata dai protocolli complessi necessari per la corretta preparazione e trattamento dei campioni. Quindi, abbiamo stabilito una metodologia basata sulla microscopia confocale ad alta risoluzione che faciliterebbe l'elaborazione dei campioni del cervello, l'analisi e la quantificazione dei compartimenti subcellulari all'interno delle cellule il nvu.
Qui, descriviamo un protocollo che utilizza sezioni digalleggiante di cervello di topo per eseguire imaging quantitativo del BBB e NVU a livello cellulare e subcellulare. Abbiamo testato e approvato un certo numero di anticorpi anti-immagine e ricostruire il NVU in tre dimensioni. Inoltre, questo protocollo permette la formazione immagine la risoluzione ottica massima di diffrazione limitata degli organelli all'interno dei capillari del cervello. Insieme con analisi dell'immagine, questo protocollo consente di indagare il trasporto intracellulare di macromolecole attraverso la Bee in diverse condizioni sperimentali, ad esempio in modelli murini di malattia di neurodegenerazione.

<table style="border-collapse:collapse;width:665pt" width="886">
	<colgroup>
		<col style="width:223pt" width="297" />
		<col style="width:98pt" width="130" />
		<col style="width:93pt" width="124" />
		<col style="width:251pt" width="335" />
	</colgroup>
	<tbody>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rat monoclonal antibody clone ER-MP12 against CD31/PECAM</td>
			<td class="xl18" style="width:98pt" width="130">Novus Biologicals</td>
			<td class="xl18" style="width:93pt" width="124">MCA2388</td>
			<td class="xl18" style="width:251pt" width="335">Labels Brain Endothelial Cells 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rat monoclonal antibody against Podocalyxin</td>
			<td class="xl18" style="width:98pt" width="130">R&#38;D Systems</td>
			<td class="xl18" style="width:93pt" width="124">MAB1556</td>
			<td class="xl18" style="width:251pt" width="335">Labels Lumen of capillaries 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rabbit polyclonal antibody against CollagenIV</td>
			<td class="xl18" style="width:98pt" width="130">Biotrend</td>
			<td class="xl18" style="width:93pt" width="124">BT21-5014-70</td>
			<td class="xl18" style="width:251pt" width="335">Labels Basement membrane of capillaries 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rabbit IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Goat polyclonal antibody against CD13</td>
			<td class="xl18" style="width:98pt" width="130">R&#38;D Systems</td>
			<td class="xl18" style="width:93pt" width="124">AF2335</td>
			<td class="xl18" style="width:251pt" width="335">Labels pericytes 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-goat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl22" height="42" style="width:223pt;height:31.5pt" width="297">Mouse monoclonal antibody clone G-A-5 against GFAP coupled to Cy3</td>
			<td class="xl18" style="width:98pt" width="130">Abcam</td>
			<td class="xl18" style="width:93pt" width="124">ab49874</td>
			<td class="xl18" style="width:251pt" width="335">Labels Astrocytes 
			Use at dilution 1:100</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rabbit polyclonal antibody against Iba-1</td>
			<td class="xl18" style="width:98pt" width="130">Wako</td>
			<td class="xl18" style="width:93pt" width="124">019-19741</td>
			<td class="xl18" style="width:251pt" width="335">Labels Microglia 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rat monoclonal antibody ABL93 gainst LAMP2</td>
			<td class="xl18" style="width:98pt" width="130">Fitzgerald</td>
			<td class="xl18" style="width:93pt" width="124">10R-CD107BBMSP</td>
			<td class="xl18" style="width:251pt" width="335">Labels Lysosomes 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl23" height="42" style="width:223pt;height:31.5pt" width="297">donkey polyclonal antibody against mouse IgG (H+L) AlexaFluor594</td>
			<td class="xl18" style="width:98pt" width="130">LifeTechnologies</td>
			<td class="xl18" style="width:93pt" width="124">A21203</td>
			<td class="xl21">Use at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">donkey polyclonal antibody against mouse IgG (H+L) AlexaFluor488</td>
			<td class="xl18" style="width:98pt" width="130">LifeTechnologies</td>
			<td class="xl18" style="width:93pt" width="124">A21202</td>
			<td class="xl21">Use at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">goat polyclonal antibody against mouse IgG (H+L) AlexaFluor555</td>
			<td class="xl18" style="width:98pt" width="130">LifeTechnologies</td>
			<td class="xl18" style="width:93pt" width="124">A21422</td>
			<td class="xl21">Use at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Filter paper</td>
			<td class="xl18" style="width:98pt" width="130">Sigma-Aldrich</td>
			<td class="xl18" style="width:93pt" width="124">WHA10334347</td>
			<td class="xl18" style="width:251pt" width="335">Whatman prepleated qualitative filter paper 
			Grade 0858 1/2, grained</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="21" style="width:223pt;height:15.75pt" width="297">Cyanoacrylate glue</td>
			<td class="xl18" style="width:98pt" width="130">Roth Carl</td>
			<td align="right" class="xl18" style="width:93pt" width="124">258.1</td>
			<td class="xl18" style="width:251pt" width="335">Roti Coll 1</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="21" style="width:223pt;height:15.75pt" width="297">Fluorescent Mounting medium</td>
			<td class="xl18" style="width:98pt" width="130">Dako</td>
			<td class="xl18" style="width:93pt" width="124">S3023</td>
			<td class="xl18" style="width:251pt" width="335">Dako fluorescent mounting medium</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Adult mice</td>
			<td class="xl18" style="width:98pt" width="130">The Jackson Laboratory</td>
			<td class="xl18" style="width:93pt" width="124">000664</td>
			<td class="xl18" style="width:251pt" width="335">C57BL/6J male or female mice 
			between 9 and 19 months of age</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="21" style="width:223pt;height:15.75pt" width="297">Vibratome</td>
			<td class="xl18" style="width:98pt" width="130">Leica Biosystems</td>
			<td align="right" class="xl18" style="width:93pt" width="124">14047235612</td>
			<td class="xl21">Leica VT100S vibrating blade microtome</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Laser Scanning Confocal microscope</td>
			<td class="xl18" style="width:98pt" width="130">Leica Microsystems</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl18" style="width:251pt" width="335">Leica TCS SP8 X with HyD detectors 
			and White light laser</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Image processing software</td>
			<td class="xl18" style="width:98pt" width="130">Leica Microsystems</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl21">Leica Application Suite AF version 3.1.0 build 8587</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Image analysis software</td>
			<td class="xl18" style="width:98pt" width="130">Bitplane scientific software</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl21">Imaris version 7.6.5 build 31770 for x64</td>
		</tr>
		<tr height="99" style="height:74.25pt">
			<td class="xl18" height="99" style="width:223pt;height:74.25pt" width="297">Fluorescence SpectraViewer</td>
			<td class="xl18" style="width:98pt" width="130">ThermoFisher Scientific</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl25" style="width:251pt" width="335">https://www.thermofisher.com/ch/en/home/life-science 
			/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html</td>
		</tr>
	</tbody>
</table>

high-resolution confocal imaging of the blood-brain barrier: imaging, 3d reconstruction, and quantification of transcytosis

Emato - encefalica (BBB) è un'interfaccia dinamica multicellulare che regola il trasporto delle molecole tra la circolazione e il cervello. Transcitosi attraverso la Bee regola la consegna di ormoni, metaboliti e gli anticorpi terapeutici al parenchima del cervello. Qui, presentiamo un protocollo che unisce immunofluorescenza delle sezioni digalleggiante con scansione laser confocale e analisi delle immagini per visualizzare gli organelli sottocellulari all'interno delle cellule endoteliali a BBB. Combinando questo set di dati con software di analisi di immagine 3D consente la segmentazione semi-automatizzata e quantificazione del volume capillare e superficie, nonché il numero e intensità degli organelli intracellulari a BBB. La rilevazione dell'immunoglobulina endogeno del mouse (IgG) all'interno di vescicole intracellulari e loro quantificazione a BBB viene utilizzata per illustrare il metodo. Questo protocollo può essere applicato potenzialmente all'indagine dei meccanismi di regolazione BBB transcitosi di molecole differenti in vivo.

Come esempi rappresentativi delle immagini ottenute dal protocollo descritto qui, sezioni di cervello di topo sono stati macchiati con anticorpi che riconoscono diverse componenti di NVU compreso della membrana dello scantinato, astrociti, periciti e cellule endoteliali (Vedi Tabella materiali per anticorpi specifici utilizzati) (Figura 1A, D ed E). A questa risoluzione è possibile distinguere i singoli processi astrocitari e fine-piedi che sono a diretto contatto con i vasi capillari.
Per evidenziare l'idoneità di questo protocollo per la rilevazione di strutture intracellulari, sezioni di cervello da animali iniettati perifericamente con l' anticorpo umano anti-Tau mAb8616 sono state macchiate con un anticorpo anti-umano fluorescente etichettato ( Figura 1B). mAb86 è noto in particolare i neuroni bersaglio che esprimono una forma patologica di Tau16. Mediante il protocollo descritto nel presente documento, mAb86 è stato rilevato con diffrazione limitata risoluzione all'interno di singole strutture vescicolari all'interno dei neuroni (Figura 1B-C). Inoltre, IgG di topo endogeno è stato rilevato in strutture intracellulari all'interno delle cellule endoteliali ma non nei periciti (D-E diFigura 1e Figura 2).
L'acquisizione di z-stack confocale ad alta risoluzione del vasculature cerebrale permette per la segmentazione tridimensionale dei vasi capillari e vescicole intracellulari a BBB. La figura 2 Mostra un esempio del processo di rendering e segmentare un capillare etichettato con CollagenIV e mouse IgG positivi intracellulare vescicole. Quantificando un set di dati completo di immagini segmentate, ad esempio misurando il numero di vescicole per capillare di volume, è possibile studiare i cambiamenti nei processi di trasporto intracellulare in condizioni diverse. Figura 3 B-C Mostra le differenze d'intensità di fluorescenza e numero vescicola di mIgG corrispondente mIgG al parenchima del cervello, rispettivamente, su Pericita svuotamento nel modello del topo di pdgf-bret/ret come precedentemente segnalato 17. lo stesso approccio è stato anche recentemente utilizzato per analizzare i cambiamenti nel trasporto intracellulare al BBB tra differenti del cervello regioni18.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56407/56407fig1.jpg"> 
Figura 1: Etichettatura di più tipi di cellule e strutture subcellulari del neurovascolare Unit Immagini rappresentative dell'unità neurovascolare (A e D) e vescicole intracellulari contenute all'interno dei neuroni (B) o cellule endoteliali (E) ottenute con questo protocollo. L'immagine di proiezione massima intensità in A (in alto) Mostra la distribuzione degli astrociti GFAP-positivo (rossi) che circondano capillari etichettati da CollagenIV (verdi). Frecce puntano ai singoli processi astrocytic. Barra della scala = 20 µm. A questa risoluzione, i singoli Astrocita processi e puntali sono chiaramente visibili, come mostrato nell'immagine ingrandita della regione "in box" (in basso). Punte di freccia indicano puntali astrocytic. Barra della scala = 10 µm. L'immagine in B Mostra l'accumulo di un anticorpo iniettato perifericamente, mAb86 (verde), all'interno di un neurone hippocampal. Le punte delle frecce scegliere singole mAb86-positivi vescicole. Barra della scala = 10 µm. Il grafico c Mostra l'intensità del profilo di linea di una singola vescicola. La dimensione della vescicola è stata stimata da tutta la larghezza a metà altezza di una misura gaussiana (linea nera continua) della curva di intensità (linea verde e cerchi). Le immagini d mostrano una ricostruzione tridimensionale di una cellula endoteliale (verde) circondata da un Pericita (rosso) all'interno della lamina basale (CollagenIV, grigio). I pannelli inferiori mostrano i canali individuali di fluorescenza. Barra della scala = 10 µm. Le immagini in E mostrano la localizzazione di mIgG (rosso) in vescicole intracellulari all'interno delle cellule endoteliali (pannello, CD31 in verde a sinistra) ma non in periciti (pannello di destra, CD13 in verde). In tutte le immagini, DAPI macchiato i nuclei sono mostrati in blu. Barra della scala = 5 µm. pannelli D ed E sono stati modificati dal riferimento17. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56407/56407fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56407/56407fig2.jpg"> 
Figura 2 : Rendering tridimensionale dei vasi capillari e vescicole intracellulari presso la barriera sangue - cervello. Con il protocollo descritto, immagini ad alta risoluzione confocal di capillari CollagenIV-positivo (verdi) e mouse IgG vescicole intracellulari (rosso) sono state acquisiti (A). Il pannello di sinistra mostra una singola sezione ottica con sezioni trasversali. Le frecce puntano ai singole mIgG-positivi vescicole all'interno delle cellule endoteliali del cervello. Il pannello sulla destra mostra il 3D ricostruzione del z-stack completo utilizzando software elaborazione immagini (Vedi Tabella materiali). Il volume capillare (B) e vescicole individuali (C) sono stati resi in tre dimensioni e quantificati tramite software di elaborazione immagine (Vedi Tabella materiali). In tutte le immagini, DAPI macchiato i nuclei sono mostrati in blu. Scala bar = 5 µm. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56407/56407fig2large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56407/56407fig3.jpg"> 
Figura 3 : Quantificazione di mIgG localizzazione intracellulare al BBB. Immagini rappresentative, mostrando (A) ricostruzioni tridimensionali dei capillari (etichettati con collagenIV, verde) e la distribuzione di mIgG (rosso) in topi C57BL/6 e pdgf-bret/ret Pericita impoverito topi precedentemente descritto in19. Barre di scala = 10 µm. Le immagini in B mostrano la segmentazione delle vescicole intracellulari all'interno della maschera di CollagenIV. THe grafico in B , la quantificazione e il confronto del numero delle vescicole mIgG per volume di capillare. Ogni punto rappresenta misure da singoli segmenti capillari. La linea continua indica la media e le barre di errore rappresentano la deviazione standard dei dati. Le immagini in C Visualizza la segnale di fluorescenza mIgG fuori la maschera di CollagenIV. Allo stesso modo, il grafico c Mostra la quantificazione e il confronto di intensità di fluorescenza mIgG nel parenchima del cervello fra i topi C57BL/6 e pdgf-bret/ret Pericita impoverito topi. Unità di intensità di fluorescenza sono stati normalizzati dall'intensità del parenchima mIgG medio misurato in tutti i topi C57BL/6. Questa figura è stata modificata dal riferimento17. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56407/56407fig3large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis at JoVE.com

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

Imaging ad alta risoluzione confocale della barriera sangue - cervello: Imaging, ricostruzione 3D e quantificazione di transcitosi

Qui presentiamo un protocollo basato su microscopia per imaging ad alta risoluzione e una ricostruzione tridimensionale del mouse unità neurovascolare e barriera emato - encefalica utilizzando sezioni digalleggiante del cervello. Questo metodo consente la visualizzazione, analisi e quantificazione degli organelli intracellulari a BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

high-resolution-confocal-imaging-blood-brain-barrier-imaging-3d

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.6K Views.  Roche Innovation Center Basel. Qui presentiamo un protocollo basato su microscopia per imaging ad alta risoluzione e una ricostruzione tridimensionale del mouse unità neurovascolare e barriera emato - encefalica utilizzando sezioni digalleggiante del cervello. Questo metodo consente la visualizzazione, analisi e quantificazione degli organelli intracellulari a BBB.

Video: High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Un saggio Slice organotipica per High-Resolution Time-Lapse Imaging della migrazione neuronale nel cervello postnatale

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Ricerca

Neuroscienze

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

Un metodo rapido per High-Resolution Imaging di fluorescenza in semi-spessa fettine di cervello

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Casting cerebrovascolare del topo adulto per l&#39;imaging 3D e analisi morfologica

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Ad alta risoluzione magnetica funzionale per immagini Metodi risonanza per mesencefalo umano

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Imaging ad alta risoluzione dal vivo del comportamento delle cellule in via di sviluppo neuroepitelio

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Alta risoluzione<em&gt; In Vivo</em&gt; Manuale Protocollo Segmentazione for Human ippocampale Sottocampi Utilizzando 3T Risonanza Magnetica

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex Vivo Imaging di postnatale cerebellare granuli migrazione cellulare Utilizzando confocale macroscopia

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

Ad alta risoluzione strutturale Risonanza Magnetica del subcortex Umana<I&gt; In Vivo</I&gt; E Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Risoluzione alta analisi quantitativa Immunogold di Recettori di membrana a retina nastro Sinapsi

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

Imaging ad alta risoluzione confocale della barriera sangue - cervello: Imaging, ricostruzione 3D e quantificazione di transcitosi

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Alta risoluzione

Ex Vivo Imaging di postnatale cerebellare granuli migrazione cellulare Utilizzando confocale macroscopia

Ad alta risoluzione strutturale Risonanza Magnetica del subcortex Umana

Risoluzione alta analisi quantitativa Immunogold di Recettori di membrana a retina nastro Sinapsi

Utilizzando un dispositivo di microfluidica per stimolazione meccanica e alta risoluzione Imaging di c. elegans