Funding for the production of this manuscript was provided by Nottingham Trent University. The authors wish to thank the University yard manager, Anna Gregory for the use of her horses and provision of fecal samples for use in the protocol. Thanks also to Chester Zoo Wildlife Endocrinology Laboratory for use of their facilities.

Etica dichiarazione: procedure che coinvolgono soggetti campo di campionamento e di animali sono stati approvati dalla Scuola di animali, rurale e Scienze Ambientali (ARES) alla Nottingham Trent University. 1. Raccolta di campioni di feci NOTA: I guanti devono essere indossati durante la manipolazione dei campioni fecali e metanolo. Se c&#39;è un forte sospetto che un animale possa essere affetto da una zoonosi, indumenti protettivi, come un camice da laboratorio dovrebbe essere indossati. <ol><li> Raccogliere i campioni fecali appena possibile (entro min a qualche ora) dopo la defecazione e metterli in un sacchetto di campionamento. Prendere tre a cinque sotto-campioni di qualsiasi campione defecato con un peso totale di circa 10 g. Etichettare il sacchetto con l&#39;ora e la data di raccolta, ID individuale, e congelare il campione a -20 ° C fino al momento dell&#39;analisi. </li><li> Per ridurre al minimo errore, congelare tutti i campioni a -20 ° C fino a quando lo studio è completo quindi di raccogliere i campioni insieme per l&#39;analisi in un unico lotto. &lt;/ Li&gt; </ol> 2. fecale Hormone Estrazione <ol><li> Lasciare i campioni di feci per scongelare a temperatura ambiente. </li><li> Assicurarsi che i campioni sono omogeneizzati miscelando manualmente ogni campione nel proprio sacchetto di campionamento. </li><li> Per ogni campione pesa 0.50 (± 0,003 g) di materiale fecale su una microbilancia in una barca pesare poi trasferire le feci in una fiala di vetro 8 ml. Utilizzare una nuova barca pesare per ogni campione e pulire le pinzette con il 30% di metanolo tra ogni campione. </li><li> Unire la 0,5 g di materiale fecale con 5 ml di metanolo al 90%: acqua e agitare O / N in un agitatore orbitale a RT. </li><li> La mattina seguente, Vortex il campione e quindi si centrifuga per 20 minuti a 600 g. </li><li> Decantare la frazione di metanolo in un tubo di vetro 16 x 125 mm 2 e posto in un rack in un bagnomaria regolato ad una temperatura di 37 ° C. Portare a secco con aria sotto cappa. Sciacquare la fiala di estrazione e posizionare il pellet fecale rimanente in un sacchetto di rifiuti clinici per lo smaltimento. </li><li> Risospendere gli estratti fecali in 100%, 1 ml di metanolo e trasferire in una provetta di polipropilene da 12 x 75 mm 2. Immediatamente tappo e conservare a -20 ° C fino al momento dell&#39;analisi. </li></ol> 3. Analisi Hormone <ol><li> Effettuare l&#39;analisi con un collegato-immuno-enzimatico (EIA) utilizzando antisiero policlonale corticosterone CJM006. NOTA:. Un secondo EIA è stato testato (cortisolo R4866 antisiero, ma questo non è riuscito a raggiungere la fase di validazione biochimica (vedere paragrafo 4.1) L&#39;antisiero policlonale corticosterone CJM006 e cortisolo R4866 antisiero e la rispettiva HRP del sono forniti da CJ Munro, University of California, Davis, CA. </li><li> Diluire l&#39;antisiero a 1: 15.000 in tampone di rivestimento (0,05 M NaHCO3, pH 9.6) con una pipetta ripetitiva e un puntale 50 microlitri, caricare una micropiastra a 96 pozzetti con 50 microlitri / bene. Coprire la piastra con un sigillante ed incubare O / N a 4 ° C. Lascia due pozzi non rivestiti per rappresentare i pozzi legame non specifico (NSB). </li><li> Immediatamente prima dell&#39;uso lavare i piatti microtitolazione cinque volte utilizzando un lavatore micropiastra automatizzato (soluzione di lavaggio; 0,15 M di NaCl, 0,05% di Tween 20). </li><li> Caricare le piastre con 50 microlitri per bene per la NSB di e pozzi [tampone EIA (0,1 M NaPO 4, 0,149 M di NaCl, 0,1% di albumina sierica bovina, pH 7,0) soltanto] &#39;zero&#39;, standard (corticosterone, 3.9 - 1000 pg / bene) o campioni fecali estratto (opportunamente diluiti a 1:20 tampone EIA, vedere paragrafo 4.1). Norme e zeri devono essere eseguiti in triplice copia e quadruplice, rispettivamente. Diluito campioni estratto fecale e NSB di dovrebbero essere eseguiti in duplicato. </li><li> Seguire questa immediatamente con l&#39;aggiunta di 50 ml / pozzetto dell&#39;etichetta perossidasi coniugato ᵻ diluito in tampone EIA a 1: 70.000. </li><li> Incubare le piastre microtitolazione al buio per 2 ore a temperatura ambiente. </li><li> Lavare le piastre microtitolazione con soluzione di lavaggio 5 volte e incubare le piastre a RT al buio con 100 l / well di RT substrato [0,4 mM 2,2&#39;-Azino-di- (3-ethylbenzthiazoline solfonico) sale diammonico, 1.6 mM H 2 O 2, 0,05 M citrato, pH 4,0]. Preparare il substrato immediatamente prima dell&#39;uso. </li><li> Utilizzare un Spettrofotometro alla lunghezza d&#39;onda di 405 nm per leggere le piastre di microtitolazione. L&#39;incubazione delle piastre è considerata completa quando la densità ottica dei pozzetti di zero raggiunge da 0,8 o 1,0. </li><li> Per assicurare la ripetibilità, utilizzare i controlli [standard EIA o un campione biologico diluiti in tampone EIA di consolidare a circa il 30% (standard EIA), il 50% (un campione per esempio biologica, pool di cavallo domestico estratto fecale) e il 70% (standard EIA) ] a dimostrare un intra e inter-assay coefficienti di variazione di &lt;10% e &lt;15%, rispettivamente. </li></ol> 4. Enzyme Linked-immunologico: convalida per la specie di studio e sesso <ol><li> Biochimica Validation (parallelismo) <ol><li> Utilizzando tampone EIA, eseguire una diluizione seriale del pool fecale in piùct, idealmente preso da presunti campioni di alta e bassa concentrazione di ormoni da diversi individui (range: pulito, 1: 2 a 1: 8192) ed eseguire sulla VIA, come descritto nella sezione 3. NOTA: Un parallelismo si considera raggiunto quando le diluizioni seriali di estratti fecali producono una curva spostamento parallelo ai risultati della curva e di regressione lineare standard di dimostrare R2&gt; 0,90, una pendenza&gt; 0,50 e p &lt;0.05. L&#39;estratto fecale diluito seriale che lega circa il 50% sulla curva standard dovrebbe essere considerata la diluizione ideale per eseguire tutti gli estratti fecali (ad esempio, 1:20). Non vi è alcuna interferenza considerato sul test quando i risultati di regressione lineare dimostrano R2&gt; 0,90 e p &lt;0.05. </li></ol></li><li> Biochimica Validation (Interference) <ol><li> Spike 200 ml di standard diluite in serie (zero, 3.9 a 1000 ng / pozzetto corticosterone) con 200 ml di estratto fecale opportunamente diluito in pool [diluito al 50% legame determinati da Parallelism (1:20)] ed eseguire sulla VIA, come al punto 3. A seguito di trama analisi EIA la concentrazione osservata meno il fondo (concentrazione del campione zero) rispetto alla concentrazione prevista. NOTA: se l&#39;aggiunta di estratto fecale pool diluito per gli standard non altera in modo significativo la quantità prevista e lineari risultati della regressione produce R 2&gt; 0,90, un pendio vicino al 1.0 e p &lt;0.05. Nessuna interferenza con l&#39;EIA è stato raggiunto. </li></ol></li><li> Validazione biologica: NOTA: Una dimostrazione che la VIA scelta ha la capacità di misurare le variazioni biologicamente rilevanti di ormone di interesse [ad esempio, l&#39;aumento delle concentrazioni di glucocorticoidi a seguito di un farmacologica (ACTH è spesso una procedura di licenza) o una sfida biologica (ad esempio, psicologica e / o ambientale; più probabile che sia un evento non regolamentata opportunistica)]. <ol><li> Selezionare, estrarre e analizzare i campioni di feci per le concentrazioni del metabolita glucocorticoidiutilizzando il processo descritto nelle sezioni 1 - 3 prima e dopo un evento impegnativo. NOTA: Un evento impegnativo sarà determinate specie. Un metodo esempio è riportato qui sotto cavallo domestico. Esempi di eventi impegnativi per i cavalli sono stati pubblicati in precedenza compreso l&#39;uso di gestione 9 e allevamento procedure 16. Un aumento significativo della concentrazione di metaboliti glucocorticoidi dopo l&#39;evento impegnativo deve essere osservato e confermato con l&#39;analisi statistica. Questo deve incorporare, se del caso, l&#39;effetto delle misure ripetute, data campione e singoli. validazione biologica è quindi considerato raggiunto. </li></ol></li></ol>

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The authors have nothing to disclose.

Analisi corticosterone fecale fornisce un mezzo per valutare i modelli a lungo termine di attività surrenalica nei cavalli. La natura non invasiva del metodo supera gli effetti confondenti di altri metodi di campionamento utilizzati per valutare l&#39;attività del surrene tra cui saliva e nel plasma analisi 9. Inoltre la tecnica ha un vantaggio non invasivo chiaro se lo studio cavalli liberi che vanno. Ci sono diversi punti chiave da discutere riguardo a questo metodo e il suo u...

Gli obiettivi metodo descritto per analizzare le concentrazioni di corticosterone nelle feci equine per fornire una valutazione non invasiva dell&#39;attività surrenale. Misurazione dell&#39;attività (HPA) dell&#39;asse ipotalamo-ipofisi-surrene è un approccio accettato di studiare la risposta a situazioni potenzialmente avversi in entrambe le specie in cattività e nazionali. La tecnica di riferimento e il metodo più diffuso è l&#39;uso di plasma sanguigno 1 Tuttavia, metodi alternativi come l&#39;analisi fecale sono state sviluppate per superare lo stress indotto mediante campionamento sangue stesso e permettere la possibilità di monitorare le specie che vanno. Durante una situazione di avversione, l&#39;omeostasi fisiologica è interrotta. L&#39;ipotalamo nel cervello rilascia ormone di rilascio della corticotropina (CRH), che agisce sulla ghiandola pituitaria anteriore e stimola il rilascio di ormone adrenocorticotropo (ACTH). ACTH entra nel flusso sanguigno e stimola la corteccia surrenale a secernere species glucocorticoidi specifici (GC). I glucocorticoidi sono strettamente legate ad eventi stressanti piuttosto che essere prodotta in modo coerente in tutti gli stati di energia accresciuta quindi sono spesso misurati in preferenza rispetto ad altri di stress legato ormoni 2. I glucocorticoidi sono responsabile di diversi effetti di adattamento nei cavalli. Energia viene rapidamente mobilizzato da siti di stoccaggio nel corpo sotto forma di acidi grassi e glucosio, assunzione di ossigeno viene aumentata, funzione sensoriale è migliorata 3 e il flusso di sangue è diminuita ad aree non necessarie per il movimento 4. Così come in qualità di un meccanismo di coping, l&#39;aumento indotto stress nella glucocorticoidi può anche aiutare a preparare l&#39;animale per il prossimo 5 fattore di stress. Valutare livelli dell&#39;ormone nel plasma e nella saliva coinvolge misurare l&#39;ormone effettivo circolante Tuttavia, misurando i metaboliti nelle misure feci il prodotto finale del metabolismo dell&#39;ormone. steroidi circolanti sono catabolizzati nella liver prima di escrezione della bile dove subiscono ulteriori modifiche facilitati dalle attività enzimatiche della flora batterica intestinale all&#39;interno della traccia 6. Pertanto, saggi immunologici diretti verso glucocorticoidi nel sangue può non essere adatto per l&#39;analisi dei metaboliti fecali glucocorticoidi 7. Come raccolta fecale può essere effettuata con nessun disturbo al cavallo, analisi delle feci per corticosterone, è stato ampiamente utilizzato per monitorare l&#39;attività HPA in una serie di circostanze. Corticosterone elevata nelle feci dei cavalli è stata riportata in risposta a situazioni potenzialmente avversi anche durante post-operatorio trattamento veterinario 8 e nelle abitazioni restrittiva 9. Campionamento fecale riflette un livello di glucocorticoide pool nel corso del tempo, piuttosto che il punto di campionamento in tempo offerto dal plasma e la saliva che lo rende adatto per il monitoraggio a lungo termine, cronica o modelli stagionali 10. A causa della non-invasivanatura del metodo, i campioni possono essere ritirati ripetutamente per un individuo senza la necessità di cattura o di ritenuta 11. Tuttavia, specie specifica il tempo di transito intestinale deve essere preso in considerazione quando si pianifica un protocollo di campionamento. Nei cavalli, il tempo di transito intestinale è di circa 18 ore 12 Pertanto, la risposta surrenale e successive metaboliti corticosterone possono essere rilevati nelle feci un giorno dopo attivazione iniziale dell&#39;asse HPA. Quando utilizzando tecniche non invasive immunodosaggio una validazione accurata per le specie oggetto di indagine è essenziale 13. Inoltre, le differenze di sesso ormone metaboliti sono stati segnalati probabilmente a causa di differenze nel tasso metabolico e il tipo di metabolita corticosterone escreto nelle varie specie, tra cui topi, 14 e polli 15. Era quindi importante nell&#39;ambito di questo metodo che il test è stato convalidato per l&#39;uso in maschi e femmine cavalli domestici come dettagliato in the protocollo. Questa differenza nel metabolismo ormonale tra i generi ha conseguenze per la qualità dei dati eppure raramente si rivolge e incluso come parte di convalida del test. Questo metodo non invasivo consente la valutazione a lungo termine di attività surrenalica nei cavalli domestici. I dettagli del protocollo sia la validazione del test e la tecnica di analisi in sé.

<table>
	<tbody>
		<tr>
			<td>Corticosterone antibody &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</td>
			<td>No longer available through UC Davis - please see Arbor Assays</td>
		</tr>
		<tr>
			<td>Cortisol antibody &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</td>
			<td>No longer available through UC Davis - please see Arbor Assays</td>
		</tr>
		<tr>
			<td>Corticosterone synthetic standard hormone</td>
			<td>Sigma Aldrich</td>
			<td>50-23-7</td>
			<td>Harnful if ingested or with skin contact. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Cortisol synthetic standard hormone</td>
			<td>Sigma Aldrich</td>
			<td>15087-01-1</td>
			<td>Harnful if ingested or with skin contact. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>67-56-1</td>
			<td>Irritant. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>144-55-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Carbonate Anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>497-19-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma Aldrich</td>
			<td>7558-79-4</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma Aldrich</td>
			<td>10049-21-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>9048-46-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>9005-64-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma Aldrich</td>
			<td>77-92-9</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>ABTS</td>
			<td>Sigma Aldrich</td>
			<td>30931-67-0</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide 30%</td>
			<td>Sigma Aldrich</td>
			<td>7722-84-1</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>7647-14-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 4</td>
			<td>VWR</td>
			<td>332732B</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 7</td>
			<td>VWR</td>
			<td>332742D</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 10</td>
			<td>VWR</td>
			<td>332762H</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma Aldrich</td>
			<td>435570</td>
			<td>Irritant. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Fisher Scientific</td>
			<td>BFS-525-010A</td>
			<td></td>
		</tr>
		<tr>
			<td>Air compressor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer +printer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fridge-freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drying apparatus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>+tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flammable liquid storagecabinet</td>
			<td>VWR</td>
			<td>649-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fume cupboard</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hot-plate stirrer</td>
			<td>VWR</td>
			<td>640-282</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate washer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Research&#174; pipettes - multipack option 2</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 1000ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 200ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 20ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater pipette</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette filler</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Progen Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Hilsonic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water purification system</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fecal glucocorticoid analysis: non-invasive adrenal monitoring in equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

cavalli domestici (n = 16, 8 cavalle, 8 castroni), con un&#39;età media di 15 anni (± 3) sono stati raggruppati in base al sesso e sottoposti a quattro disegni abitative con crescenti livelli di isolamento sociale (n = 4 cavallo / trattamento). Housing 1 coinvolti cavalli che vivono in un ambiente mandria, simulando attentamente il loro habitat naturale. Housing 2 coinvolti cavalli che vivono in coppie in una stalla coperta. Housing 3 cavalli coinvolti alloggiati solo nelle stalle, ma ...

Watch this Scientific Journal Video about Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids at JoVE.com

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecale Analisi glucocorticoidi: non invasivo di monitoraggio surrenale in equidi

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

fecal-glucocorticoid-analysis-non-invasive-adrenal-monitoring

Research

JoVE Journal

Biology

9.6K Views.  Nottingham Trent University. Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Video: Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

Topo surrenale cromaffini cella di isolamento

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

Ricerca

Biologia

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Etichettatura hESC e hMSCs con nanoparticelle di ossido di ferro per i non-invasiva in vivo inseguimento con la RM

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Etichettatura Cellule staminali con le tinture fluorescenti per la rilevazione non invasiva con Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Non invasive di visualizzazione 3D con sub-micron Risoluzione Utilizzando sincrotrone a raggi-X, la tomografia

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Un reversibile, metodo non invasivo per le misure di resistenza delle vie aeree e liquido di lavaggio broncoalveolare di campionamento nei topi

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A lungo termine del test di tossicità letale con il crostaceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Rapida analisi genetica di epitelio-mesenchimale segnalazione durante la rigenerazione dei capelli

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Una procedura di screening chimico per glucocorticoidi segnalazione con un reporter del sistema Zebrafish Larva Luciferase

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.

This protocol provides a convenient set of methods, which enables extremely fast, easy, non-invasive, reliable and low-cost, molecular sex determination of birds and their non-invasive, quick, safe and easily recognizable marking shortly after hatching. Only limited handling of chicks is required. This convenient toolbox of methods complies entirely with the RRR-guidelines.

Chi è Chi? Metodi non invasivi per Sex autonomamente e Mark altricial Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

Analisi di assorbimento di colesterolo LDL utilizzando Live Cell Imaging analisi con cella di monitoraggio dello stato

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...