Questo lavoro è stato sostenuto dal National Institutes of Health sovvenzioni K08 NS061039-01A2, il Programma per Neurology Research &amp; Discovery, e The A. Alfred Taubman Medical Research Institute presso l&#39;Università del Michigan. Questo lavoro utilizza la Morfologia e Image Analysis Nucleo del Michigan Diabetes Research e centro di formazione, finanziato dal National Institutes of Health di Grant 5P90 DK-20572 presso l&#39;Istituto Nazionale del Diabete e Malattie Digestive e Renali. Gli autori desiderano ringraziare Robinson Singleton e Gordon Smith (University of Utah) per la generosa donazione di campioni di pelle umana per sostenere lo sviluppo iniziale della tecnica immunoistochimica biomarcatore nocicettivo.

Parte A: Immunoistochimica Preparazione della piastra a 96 pozzetti e la prevenzione di colorazione di fondo Punch biopsie cutanee sono raccolti da soggetti umani e incubato per 12-24 ore in una soluzione di fissativo (paraformaldeide al 2% con 0,75 M soluzione di L-lisina (pH 7.4) e 0,05 mm di sodio periodate) a 4 ° C, come descritto in precedenza 8. I campioni vengono poi crioprotetti in tampone fosfato (PBS) con il 20% di glicerolo a 4 ° C per un massimo di 1 settimana, incorporato nel supporto di montaggio della temperatura ottimale di taglio (OCT), poi sezionato in 50 micron sezioni spesse su un criostato. Il protocollo descritto di seguito è progettato per 8 sezioni di pelle, il numero massimo di sezioni cutanee possibile sottoporsi immunoistochimica libero di fluttuare in una piastra a 96 pozzetti. 1 ° GIORNO: 1. Prevenzione della immunoreattività non specifica nello strato corneo <ol><li> Etichetta piastra da 96 pozzetti, come mostrato in Figura 1. </li&gt; <li> Aggiungere 150 ml di Immagine-IT FX Signal (Immagine-IT), efficace per bloccare la colorazione di fondo 11,12, in tutti i pozzetti in riga 1 della piastra a 96 pozzetti. </li><li> Aggiungere 150 microlitri di PBS 1X in ciascun pozzetto della fila 2 e 3 della piastra a 96 pozzetti. </li><li> Acquisire due 50 sezioni micron per paziente: una sezione della biopsia prese a DL, una sezione della biopsia prese a PT. Di un&#39;ansa da inoculo (LeLoop) è utilizzato per trasferire sezioni da un risciacquo all&#39;altro per evitare danni morfologici. Delicatamente trasferire ogni sezione 50 micron, con un ansa, in un individuo ben di Riga 1, contenente immagini-IT. Incubare le sezioni in Image-IT per 30 min a RT su un rocker piatto. </li><li> Sciacquare le sezioni due volte con PBS 1X (righe 2 e 3) per 10 min a RT. Collocare piastra bene su una sedia a dondolo piatta tra risciacqui. </li></ol> 2. Preparazione del 5% BSA Blocking Solution e 1% soluzione di risciacquo <ol><li> Mentre le sezioni di biopsia sono incubando in Image-IT, preparare 5% soluzione bloccantezione (5% di BSA, 0,3% TX-100, 0,1 M soluzione di PBS-Vortex fino BSA si scioglie completamente). </li><li> Aggiungere 150 ml di soluzione di blocco BSA 5% in ogni pozzetto della fila 4. </li><li> L&#39;utilizzo di un ansa, trasferire le sezioni in singoli pozzetti della soluzione bloccante 5% di BSA. Incubare le sezioni in soluzione di blocco BSA 5% per 1-2 ore a temperatura ambiente su un rocker piatto. </li></ol> 3. Preparazione di 1% BSA risciacquo soluzione e diluizione degli anticorpi primari <ol><li> Mentre le sezioni sono incubando in una soluzione bloccante 5% BSA, preparare la soluzione di risciacquo 1% (1% BSA, TX-100, 0,1 M soluzione di PBS-Vortex 0,3% BSA fino scioglie completamente). </li><li> Mentre le sezioni sono incubando in una soluzione bloccante 5% BSA, diluire anticorpi primari in 1% soluzione di risciacquo. <ol><li> Per otto sezioni (8 x 150 ml = 1,200 microlitri) è necessario un totale di 1.200 microlitri. Gli anticorpi primari sono costituiti i 1.500 microlitri (circa il 20% del volume extra). </li><li> Le diluizioni degli anticorpi primari: PGP, 1:500; Trk A,1:500 in 1% BSA soluzione di risciacquo. </li></ol></li></ol> 4. L&#39;incubazione delle biopsie sezionati in anticorpo primario <ol><li> Aggiungere 150 ml di anticorpi primari diluiti nei pozzetti designate della piastra a 96 pozzetti (riga 5). </li><li> Trasferire sezioni da bloccare soluzione al 5% (riga 4) nei pozzetti di anticorpi primari designati (riga 5). </li><li> Sigillare la piastra a 96 pozzetti con parafilm e foglio di alluminio per evitare l&#39;essiccazione e l&#39;esposizione alla luce. </li><li> Incubare la piastra a 96 pozzetti O / N a 4 ° C su un rocker piatto. </li></ol> GIORNO 2: 5. Sciacquare biopsie in 1% BSA soluzione di risciacquo <ol><li> Aggiungere 150 microlitri di soluzione di risciacquo BSA 1% in ogni pozzetto con riga 6, 7, e 8. </li><li> Risciacquare sezioni tre volte con soluzione di risciacquo BSA 1% (righe 6, 7, e 8) per 1 ora ogni volta a RT. Coprire la piastra bene con un foglio di alluminio e posto su bilanciere piatta tra risciacqui. </li></ol> 6. Diluizione degli anticorpi secondari &lt;ol&gt; <li> Mentre le sezioni sono incubate nell&#39;ultimo risciacquo della soluzione di risciacquo BSA 1% (riga 8), diluire anticorpi secondari nella soluzione di risciacquo BSA 1%: <ol><li> Per otto sezioni (8 x 150 ml = 1,200 microlitri) è necessario un totale di 1.200 microlitri. Gli anticorpi secondari sono costituiti i 1.500 microlitri (circa il 20% del volume extra). </li><li> Le diluizioni degli anticorpi secondari: per PGP (Alexa Fluor 488 asino anti-coniglio, 1:250), per Trk A (Alexa Fluor 647 asino anti-capra, 1:250) in 1% BSA soluzione di risciacquo. </li></ol></li></ol> 7. L&#39;incubazione delle biopsie sezionati in anticorpo secondario <ol><li> Aggiungere 150 ml di anticorpi secondari diluiti nei rispettivi pozzetti designate della piastra a 96 pozzetti (riga 9). </li><li> Trasferire le sezioni da 1% soluzione di BSA blocco (riga 8) nel anticorpo secondario bene (riga 9). </li><li> Sigillare la piastra a 96 pozzetti con parafilm e foglio di alluminio per evitare l&#39;essiccazione e l&#39;esposizione alla luce. </li><li> Incubare sezioni del designato anticorpi secondari O/ N a 4 ° C su un rocker piatto. </li></ol> 8. Sciacquare biopsie in 1% BSA soluzione di risciacquo <ol><li> Aggiungere 150 microlitri di soluzione di BSA 1% risciacquo in ciascun pozzetto della fila 10, 11, e 12. </li><li> Risciacquare sezioni tre volte con soluzione di risciacquo BSA 1% (righe 10, 11 e 12) per 1 ora ogni volta a RT. Coprire la piastra bene con un foglio di alluminio e posto su un bilanciere piatta tra risciacqui. </li></ol> 9. Preparazione di preparati microscopici e montaggio Biopsie sezionate <ol><li> Mentre le sezioni di biopsia sono incubando nell&#39;ultimo risciacquo della soluzione di risciacquo BSA 1% (riga 12), preparare vetrini da microscopio. </li><li> Porre 50 ml di soluzione di risciacquo 1% di BSA in una diapositiva alla volta. </li><li> Rimuovere le sezioni da 1% soluzione di risciacquo BSA (riga 12) e posizionarlo nella 50 microlitri goccia di soluzione di risciacquo BSA 1% sul vetrino da microscopio designato. Dopo ottimizzando la posizione della sezione, rimuovere l&#39;eccesso di soluzione di risciacquo BSA 1% con una pipetta di vetro bulbed, prendendo precauzioniper evitare di toccare il campione. </li></ol> NOTA: Assicurarsi che la sezione non è ripiegato su, il campione deve essere piatto contro la superficie del vetrino da microscopio. <ol start="4"><li> Mettere 1 goccia di reagente Prolungare Oro montaggio con DAPI pressi della biopsia sul vetrino da microscopio antifade. Prendete un 22x22 mm microscopio vetrino di vetro e posizionare delicatamente sopra la biopsia e una goccia di reagente Prolungare antifade Oro con DAPI goccia. Ripetere l&#39;operazione per ogni sezione. </li><li> Lasciate vetrini da microscopio secco O / N a temperatura ambiente al buio. </li></ol> NOTA: Rimuovere eventuali bolle d&#39;aria utilizzando un puntale. Asciugare eccesso Prolungare reagente antifade Oro. Parte B: confocale Imaging 10. Confocale Imaging <ol><li> Segnali di fluorescenza immagine utilizzando un Olimpo FluoView 500 microscopio confocale a scansione laser con una immersione in olio (1,3 NA) obiettivo zoom e due volte con la versione del software FluoView 5.0 40X. </li&gt; <li> Utilizzare Alexa Fluor 488 e Alexa Fluor 647 per eccitare il 543-nm HeNe laser verde e il 633-nm HeNe laser rosso, rispettivamente. Il segnale Alexa Fluor 488 è rappresentato da uno sguardo verde up table (LUT) e la Alexa Fluor 647 da una LUT rosso. </li><li> Impostare le aperture confocale per ogni rivelatore a 400 micron per migliorare i segnali. Scansioni sequenziali dovrebbero essere prese ad una risoluzione di 1.024 x 1.024 per massimizzare la separazione del segnale. </li><li> Cattura tridimensionale Z-serie con intervalli di 1,2 micron z-step (in base all&#39;unità di scansione ottimale calcolato dal software FluoView) con Kalman media (due fotogrammi). </li></ol> Parte C: visualizzazione tridimensionale e animazione 11. Tridimensionale (3D) di visualizzazione e animazione <ol><li> Aprire i file FluoView in software Imaris x64 (versione 7.3, Bitplane AG) di visualizzare i set di immagini 3D. </li><li> Regolare la visualizzazione come necessario per migliorare il contrasto del segnale specifico nervo. </li><li> Creare un surfaccia per visualizzare il confine dermo-epidermica. Utilizzare lo strumento Contour in modalità di disegno per disegnare il contorno. </li><li> Assegnare una superficie semitrasparente al contorno per vedere i segnali fluorescenti sottostanti. </li><li> Cattura immagini fisse dei segnali fluorescenti 3D per creare immagini istantanee del film in 3D. </li><li> Utilizzare la funzione di animazione per creare un film in 3D. Le immagini possono essere ruotate di 360 gradi più volte per mostrare separatamente e unito ogni segnale (figure 2, 3 e 4). Impostare l&#39;animazione per creare filmati composti da 200 fotogrammi animati a 15 fotogrammi al secondo. Salvare ogni filmato come file AVI raw. </li><li> Comprimere il filmato finale AVI con VirtualDub software (versione 1.9.4). </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+nerve+fiber+density:+normative+reference+range+and+diagnostic+efficiency.">Epidermal nerve fiber density: normative reference range and diagnostic efficiency.</a> Arch. Neurol. 55 (12), 1513-1520 (1998).</li><li>Griffin, J. W., McArthur, J. C., Polydefkis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cutaneous+innervation+by+skin+biopsies.">Assessment of cutaneous innervation by skin biopsies.</a> Curr. Opin. Neurol. 14 (5), 655-659 (2001).</li><li>Cheng, H. T., Dauch, J. R., Hayes, J. M., Yanik, B. M., Feldman, E. 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Cytochem. 58 (2), 207-218 (2010).</li><li>Polydefkis, M., Hauer, P., Griffin, J. W., McArthur, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+biopsy+as+a+tool+to+assess+distal+small+fiber+innervation+in+diabetic+neuropathy.">Skin biopsy as a tool to assess distal small fiber innervation in diabetic neuropathy.</a> Diabetes Technol. Ther. 3 (1), 23-28 (2001).</li><li>Lauria, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+fibre+neuropathies.">Small fibre neuropathies.</a> Curr. Opin. Neurol. 18 (5), 591-597 (2005).</li><li>Sorensen, L., Molyneaux, L., Yue, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+among+pain,+sensory+loss,+and+small+nerve+fibers+in+diabetes.">The relationship among pain, sensory loss, and small nerve fibers in diabetes.</a> Diabetes Care. 29 (4), 883-887 (2006).</li><li>Lauria, G., Morbin, 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=Axonal+swellings+predict+the+degeneration+of+epidermal+nerve+fibers+in+painful+neuropathies.">Axonal swellings predict the degeneration of epidermal nerve fibers in painful neuropathies.</a> Neurology. 61 (5), 631-636 (2003).</li><li>Herrmann, D. N., McDermott, M. 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=Epidermal+nerve+fiber+density,+axonal+swellings+and+QST+as+predictors+of+HIV+distal+sensory+neuropathy.">Epidermal nerve fiber density, axonal swellings and QST as predictors of HIV distal sensory neuropathy.</a> Muscle Nerve. 29 (3), 420-427 (2004).</li><li>Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+27:+Neural+plasticity+after+nerve+injury+and+regeneration.">Chapter 27: Neural plasticity after nerve injury and regeneration.</a> Int. Rev. Neurobiol. 87, 483-505 (2009).</li></ol>

Nessun conflitto di interesse a dichiarare.

Misurazione di IENFD è stato ampiamente utilizzato per determinare il grado di neuropatie periferiche 13,14. Attualmente, il protocollo più comunemente usato misura solamente le densità di fibre nervose che penetrano la membrana basale dell&#39;epidermide, ma non prende in considerazione ramificazione assonale e / o modifiche morfologiche dei nervi. Inoltre, l&#39;analisi IENFD corrente non ha mostrato di correlare IENFD con la presenza di dolore in PN 15. Abbiamo pr...

Allo stato attuale, è pratica comune per i medici di quantificare intraepidermiche densità delle fibre nervose, (IENFD) da biopsie cutanee, che possono essere utilizzati per diagnosticare piccole neuropatie fibra 3, 6-8. Le biopsie sono presi dalla gamba distale (DL), 10 cm sopra il malleolo laterale, e la coscia prossimale (PT), 20 cm al di sotto della spina iliaca anteriore 9. Tutto IENF vengono etichettati utilizzando prodotto gene della proteina 9.5 (PGP), un marker neuronale padella 10-12. Al momento, si tratta di una pratica standard per la diagnosi di piccole neuropatie in fibra con IENFD determinata colorando PGP con campo chiaro microscopia 6. Inoltre, diversi gruppi di ricerca hanno utilizzato protocolli di immunofluorescenza per PGP immunoistochimica 7-9. Neuropatia Piccolo fibra è comunemente associato con dolore neuropatico. Per comprendere ulteriormente il ruolo di IENF essenziale per l&#39;elaborazione del dolore, abbiamo sviluppato una tecnica di co-label IENF totale con fibre che generano dolore. Nociceptiva IENF, specificamente Aδ e fibre C, possono essere studiati attraverso la co-etichettatura dei IENF con PGP e il marcatore nocicettivo, tropomiosina-recettore-chinasi A (Trk A) 5. Trk A è il recettore ad alta affinità per il fattore di crescita nervoso che è essenziale per lo sviluppo della nocicezione. Le fibre nervose nocicettive A-positivi Trk sono fibre peptidergici che esprimono la sostanza P (SP) e la calcitonina peptide correlato al gene (CGRP). In precedenza, Lauria e colleghi hanno applicato la tecnica della doppia etichettatura per studiare PN, co-etichettatura IENF PGP-positivo con un pennarello nocicettivo 10. Nel nostro precedente studio, abbiamo dimostrato che Trk IENF A-positivo, ma non Trk IENF A-negativo, sono stati sovraregolati in un modello animale di neuropatia diabetica dolorosa 5. Questa tecnica di co-etichettatura offre la possibilità di confrontare la quantificazione di IENFD nocicettivo al totale IENFD e la capacità di studiare i cambiamenti morfologici associati a PN. La capacità di visualizzare nocicettivo IENF e compare quantificazione di IENFD totale a IENFD nocicettivo potrebbe fornire evidenza oggettiva per la presenza di dolore, e possibilmente comprensione della gravità del dolore associato a PN. Questa tecnica è applicabile alla pelle anche di modelli animali. Rispetto agli studi precedenti, l&#39;attuale protocollo descrive i metodi per l&#39;analisi delle immagini 3D, creando l&#39;opportunità di evitare errori che potrebbero verificarsi in analisi di immagini 2D.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Fisher Scientific</td>
			<td>BP399-4</td>
			<td>To make up 1X PBS</td>
		</tr>
		<tr>
			<td>Image-IT FX Signal</td>
			<td>Invitrogen</td>
			<td>I36933</td>
			<td>Image-IT</td>
		</tr>
		<tr>
			<td>Protein Gene Product 9.5 (Polyclonal rabbit)</td>
			<td>AbD Serotec</td>
			<td>7863-0504</td>
			<td>PGP</td>
		</tr>
		<tr>
			<td>Tropomyosin Related-Kinase A (Polyclonal goat)</td>
			<td>R&amp;D Systems</td>
			<td>AF1056</td>
			<td>Trk A</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey &alpha;-rabbit</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>AF488 donkey &alpha;-goat</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 donkey &alpha;-goat</td>
			<td>Invitrogen</td>
			<td>A21447</td>
			<td>AF647 donkey &alpha;-goat</td>
		</tr>
		<tr>
			<td>Albumin, from Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>Triton X- 100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>TX-100</td>
		</tr>
		<tr>
			<td>Non-calibrated Loop</td>
			<td>LeLoop</td>
			<td>MP 199025</td>
			<td>inoculating Loop</td>
		</tr>
		<tr>
			<td>96-well assay plate</td>
			<td>Corning Incorporated</td>
			<td>3603</td>
			<td>Well plate</td>
		</tr>
		<tr>
			<td>Prolong Gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass 22x22 mm</td>
			<td>Fisher Scientific</td>
			<td>12-541-B</td>
			<td>Coverslips</td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>Microscope Slides</td>
		</tr>
		<tr>
			<td>Olympus Fluoview Laser Scanning Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV500</td>
			<td>Confocal Microscope</td>
		</tr>
		<tr>
			<td>Optimum Cutting Temperature</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>OCT</td>
		</tr>
		<tr>
			<td>Leica cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>Cryostat</td>
		</tr>
	</tbody>
</table>

three-dimensional imaging of nociceptive intraepidermal nerve fibers in human skin biopsies

Una biopsia della pelle è comunemente utilizzato per quantificare intraepidermiche densità delle fibre nervose (IENFD) per la diagnosi di polineuropatia 1,2 periferico. Attualmente, è pratica comune per raccogliere 3 millimetri biopsie dalla gamba distale (DL) e la coscia prossimale (PT) per la valutazione di polyneuropathies lunghezza-dipendenti 3. Tuttavia, a causa della natura di IENFs multidirezionale, è impegnativo per esaminare sovrapposizione strutture nervose attraverso l&#39;analisi di formazione immagine bidimensionale (2D). Alternativamente, l&#39;imaging tridimensionale (3D) potrebbe fornire una soluzione migliore per questo dilemma. Nella presente relazione, presentiamo i metodi per applicare l&#39;imaging 3D per lo studio neuropatia dolorosa (PN). Per identificare IENFs, campioni di pelle vengono elaborati per l&#39;analisi di immunofluorescenza di proteine ​​prodotto genico 9.5 (PGP), un marcatore neuronale padella. Al momento, si tratta di una pratica standard per la diagnosi di piccole neuropatie in fibra con IENFD scoraggiareminato da PGP immunoistochimica utilizzando campo chiaro microscopia 4. In questo studio, abbiamo applicato all&#39;analisi doppia immunofluorescenza per identificare IENFD totale, utilizzando PGP, e IENF nocicettiva, attraverso l&#39;uso di anticorpi che riconoscono tropomiosina-recettore-chinasi A (Trk A), il recettore ad alta affinità per il fattore di crescita nervosa 5. I vantaggi di co-colorazione IENF con anticorpi A PGP e Trk avvantaggia lo studio del PN dalla colorazione chiaramente PGP-positivi, le fibre nocicettive. Questi segnali fluorescenti possono essere quantificati per determinare nocicettivi cambiamenti IENFD e morfologiche IENF associate a PN. Le immagini fluorescenti sono acquisite da microscopia confocale ed elaborati per l&#39;analisi 3D. 3D Imaging fornisce capacità di rotazione per analizzare ulteriormente i cambiamenti morfologici associati a PN. Nel loro insieme, fluorescente co-colorazione, confocale e analisi 3D chiaramente beneficiare studio della PN.

Abbiamo applicato l&#39;attuale protocollo per studiare la morfologia di IENF in PT e DL biopsie cutanee di pazienti con PN. La pelle, da tre soggetti, è stato raccolto presso l&#39;Università dello Utah per dimostrare la Pathomorphology associata a PN. I soggetti sono: Caso 1: un 51-anno-vecchio maschio con una storia di PN di diabete di tipo 2 (durata: 14 mesi; punteggio del dolore: 51); Caso 2: un 56-anno-vecchio maschio con una storia di PN di diabete di tipo 2 (durata: 108 mesi; punteggio del dolore: 47), e causa...

Watch this Scientific Journal Video about Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies at JoVE.com

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

Per studiare i cambiamenti di fibre nervose nocicettive intraepidermiche (IENFs) nelle neuropatie dolorose (PN), abbiamo sviluppato protocolli che potrebbe esaminare direttamente le variazioni morfologiche tridimensionali osservati in IENFs nocicettivi. Analisi tridimensionale IENFs ha il potenziale di valutare le variazioni morfologiche del IENF in PN.

Imaging tridimensionale di fibre nervose nocicettive intraepidermiche in biopsie cutanee umane

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

three-dimensional-imaging-nociceptive-intraepidermal-nerve-fibers

Research

JoVE Journal

Medicine

13.0K Views.  University of Michigan. Per studiare i cambiamenti di fibre nervose nocicettive intraepidermiche (IENFs) nelle neuropatie dolorose (PN), abbiamo sviluppato protocolli che potrebbe esaminare direttamente le variazioni morfologiche tridimensionali osservati in IENFs nocicettivi. Analisi tridimensionale IENFs ha il potenziale di valutare le variazioni morfologiche del IENF in PN.

Video: Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrini umani cella linee tumorali come un modello tridimensionale per lo studio dei diritti dell&#39;uomo e terapia dei tumori neuroendocrini

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Ricerca

Medicina

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Three-dimensional Tissue Culture modello per studiare Primario Osso Osseo e le sue Malignità

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensionale (3D) Tumore Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensionale alginato-tallone coltura di cellule umane adenoma ipofisario

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Visualizzazione quantitativa di leucociti infiltrati in un modello di murina della miocardite fulminante mediante microscopia a foglio chiaro

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and long-term consequences of unresolved pain or excessive analgosedation, adequate nociceptive monitoring remains challenging in non-communicative, critically ill adults. In the intensive care unit (ICU), routine nociceptive evaluation is carried out by the attending nurse using the Behavior Pain Scale (BPS) in mechanically ventilated patients. This assessment is limited by medication use (e.g., neuromuscular blocking agents) and the inherent subjective character of nociceptive evaluation by third parties.
Here, we describe the use of two nociceptive reflex testing devices as tools for objective pain evaluation: the pupillary dilation reflex (PDR) and nociception flexion reflex (NFR). These measurement tools are non-invasive and well tolerated, providing clinicians and researchers with objective information regarding two different nociceptive processing pathways: (1)&#160;the pain-related autonomic reactivity&#160;and (2) the ascending component of the somatosensory system. The use of PDR and NFR measurements are currently limited to specialized pain clinics and research institutions because of impressions that these are technically demanding or time-consuming procedures, or even because of a lack of knowledge regarding their existence.
By focusing on the two abovementioned nociceptive reflex assessments, this study evaluated their feasibility as a physiological pain measurement method in daily practice. Pursuing novel technologies for evaluating the analgesia level in unconscious patients may further improve individual pharmacological treatment and patient related outcome measures. Therefore, future research must include large well-designed clinical trials in a real-life environment.

Pain assessment in anesthetized patients who cannot communicate with the outside world in any way remains challenging despite the development of innovative objective pain evaluation tools. In this project, the pupillary dilation reflex and the nociception flexion reflex are assessed in critically ill, mechanically ventilated adult patients.

Valutazione obiettiva nocicettivo nei pazienti di ICU ventilato: uno studio di fattibilità utilizzando pupillometria e il riflesso di flessione nocicettivo

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

Stampa tridimensionale di un'anomalia aortica complessa

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy.

Raffinata compensazione dei tessuti basata su CLARITY per l'organizzazione tridimensionale dei fibroblasti nei cuori di topo sani e feriti

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Imaging tridimensionale ad alta risoluzione della vascolarizzazione del footpad in un modello di cancrena posteriore murina

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

Guided Endodontics: Three-Dimensional Planning and Template-Aided Preparation of Endodontic Access Cavities

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, pulpitis or apical periodontitis may develop in the long term. Root canal treatment of teeth with severe PCO and pulpal or periapical pathosis is challenging for general practitioners and even for well-equipped endodontic specialists. To ensure detection of the calcified root canal and avoid excessive loss of tooth structure or root perforation, static navigation using templates ("Guided Endodontics") was introduced a few years ago. The general workflow includes three-dimensional imaging using cone-beam computed tomography (CBCT), a digital surface scan, and superimposition of both in a planning software. This is followed by virtual planning of the access cavity and the design of a template that will guide the drill to the desired target point. To do this, a true-to-scale virtual image of the drill must be placed in a way that the tip of the drill reaches the orifice of the calcified root canal. Once the template has been fabricated using computer-aided design and computer-aided manufacturing (CAD/CAM) or a 3D printer, guided preparation of the access cavity can be performed clinically. For research purposes, a postoperative CBCT image can be used to quantify the accuracy of the access cavity performed. This work aims to present the technique of static guided endodontics from imaging to clinical implementation.

Guided endodontics describes a template-aided approach for access cavity preparation. The procedure requires cone-beam computed tomography and a surface scan to produce a template. An incorporated sleeve guides the drill to the target point. This allows the preparation of minimally invasive endodontic access cavities in calcified teeth.

Endodonzia guidata: pianificazione tridimensionale e preparazione assistita da template di cavità di accesso endodontico

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, ...