Questo lavoro è stato sostenuto da sovvenzioni da Peking Union Medical College Youth Fund e i fondi di ricerca fondamentali per le Università centrale (Grant No. 3332015200).

Tutti gli esperimenti sugli animali sono stati eseguiti nel rispetto di tutte le pertinenti linee guida, regolamenti e agenzie di regolamentazione. Il presente protocollo in questione è stato effettuato sotto la guida e l'approvazione dell'Istituto della microcircolazione animale etica Comitato (IMAEC) al Peking Union Medical College (PUMC).
1. gli animali
<ol><li>Prima dell'inizio dell'esperimento, è necessario tenere tre topi BALB/c per gabbia, con temperatura controllata (24 ± 1 ° C) e umidità (55 ± 5%), sotto un ciclo luce-buio di 12 h. I topi consentire il libero accesso al cibo normale e acqua.</li>
	<li>Dividere in modo casuale i topi in un gruppo di controllo non-diabetico ed un gruppo diabetico. Pesare ogni singolo mouse e calcolare il volume di iniezione utilizzando la massa corporea di ogni mouse preciso.</li>
	<li>Veloce i topi per 4 h prima iniezione di streptozotocin (STZ) e fornire acqua normale come di consueto il giorno sperimentale 1.</li>
	<li>Preparare il tampone di citrato di sodio 0,1 M a pH 4,3. Mettere 1 mL di soluzione in una provetta da microcentrifuga da 1,5 mL e avvolgere il tubo del microcentrifuge nella carta stagnola per evitare l'esposizione alla luce.</li>
	<li>Sciogliere il STZ in tampone citrato di sodio (pH 4.3) ad una concentrazione finale di lavoro di 5 mg/mL prima dell'uso.</li>
	<li>Dare i topi delle iniezioni intraperitoneali gruppo diabetico di STZ alla dose di 40 mg/kg, utilizzando una siringa da 1 mL e un ago 25-G. Iniettare i topi del controllo non-diabetico con lo stesso volume di tampone citrato di sodio (pH 4,3).</li>
	<li>Rimettere i topi nelle gabbie e fornire loro cibo normale e 10% di acqua di saccarosio.</li>
	<li>Ripetere i passaggi da 1.3-1.7 sperimentale giorni 2-5 (cioè, i prossimi 4 giorni consecutivi).</li>
	<li>Sostituire l'acqua di 10% di saccarosio con acqua normale dopo l'ultima iniezione di STZ.</li>
	<li>Velocemente i topi per 6 h, ma dare loro libero accesso all'acqua e misurare i livelli di glucosio del sangue nove giorni più tardi (14 giorno sperimentale). Raccogliere un campione di sangue dalla vena caudale per confermare l'iperglicemia utilizzando un sistema di monitoraggio del glucosio nel sangue. Nota: I topi con sangue glucosio livelli > 200 mg / dL sono considerati diabetici.</li>
</ol>2. preparazione dello strumento
<ol><li>Pulire le superfici ottiche della punta della sonda e il connettore della sonda dell'apparato Doppler laser con un panno morbido e non abrasivo per rimuovere polvere o particelle. Collegare il cavo alla porta dello strumento (Figura 1A).</li>
	<li>Montare il supporto di calibrazione permettendo il flusso standard per essere in equilibrio termico con un ambiente sperimentale (temperatura ambiente, solitamente per 30 min). Agitare il flusso standard delicatamente per 10 s e lasciate riposare per 2 min.</li>
	<li>Posizionare il contenitore standard di flusso al centro della base di calibrazione. Sistemare il morsetto per l'altezza massima e fissare la sonda nel morsetto tale che punti verso il basso al contenitore. Assicurarsi che lo standard di flusso sia correttamente posizionato sotto la sonda.</li>
	<li>Spostare lentamente la sonda verso il basso fino a quando la punta è sommerso correttamente nello standard flusso. Selezionare e premere "taratura" sul laser Doppler apparato e scegliere il canale di lavoro che la sonda è collegata al. Eseguire il programma di calibrazione fino a quando un avviso di "Calibrazione riuscita" viene visualizzato sullo schermo del laser Doppler apparato.</li>
	<li>Fissare la sonda utilizzando supporti di sonda. Proteggere manualmente la sonda per evitare movimenti.</li>
	<li>Mantenere la stanza sperimentale a temperatura costante (24 ± 1 ° C) e umidità (~ 50-60%).</li>
	<li>Disattivare qualsiasi luce esterna (come lampade fluorescenti e spot) prima di eseguire l'esperimento per evitare il cambiamento indotto dalla luce esterna.</li>
</ol>3. preparazione degli animali
<ol><li>Autoclave la chirurgico strumenti e lasciarli raffreddare a temperatura ambiente prima dell'uso.</li>
	<li>Dare i topi 10 min per acclimatarsi all'ambiente sperimentale prima rilevazione dell'isolotto pancreatico microvascolare vasomotion dal laser Doppler.</li>
	<li>Riempire una siringa da 1 mL con 1 mL di sodio pentobarbital 3%. Iniettare la soluzione di sodio pentobarbital (75 mg/kg i.p.) per anestetizzare i topi.</li>
	<li>Coprire gli occhi del mouse con garza medica pre-umidificata per prevenire la secchezza.</li>
	<li>Assicurarsi che il mouse perde completamente la coscienza e non risponde più alla coda o retropiede pizzica con il forcipe. Monitorare l'anestesia durante tutto l'evento di anestetico e intra-operatoria ogni 15 min. mantenere l'anestesia, completandola con il 10% del volume iniziale iniezione della soluzione pentobarbital quando necessario.</li>
	<li>Posizionare un rilievo di riscaldamento con uno strato semi-isolante sotto l'animale e metti l'animale in posizione supina e trasferimento per la stazione di lavoro del laser Doppler apparato. Difficoltà il mouse per la piattaforma di funzionamento con nastro chirurgico.</li>
	<li>Tampone pelle addominale del mouse con betadine e poi 75% etanolo viene utilizzato a tampone pulito la zona addominale.</li>
	<li>Iniettare per via sottocutanea 2% lidocaine/0.5% bupivacaine miscela (50/50).  Tagliare un cm ~ 3-foro di diametro al centro di una garza-spugna. Coprire la regione addominale con la spugna di garza.</li>
	<li>Sollevare la pelle addominale con il forcipe e praticare un'incisione verticale iniziale lungo la linea mediana dell'addome utilizzando un paio di forbici bisturi o pelle.</li>
	<li>Afferrare il muscolo sottostante con la pinzetta e incise per entrare nella cavità addominale. Non ferire qualsiasi altro organo. Piegare la pelle e il muscolo di fondo sopra la cassa per rivelare la cavità addominale. Delicatamente esporre il corpo del pancreas e la milza usando un paio di pinze dal naso smussato.</li>
</ol>4. dati acquisizione per l'analisi
<ol><li>Eseguire il software dell'apparato Doppler laser facendo clic su "File" → "Nuovo" per creare un nuovo file di misurazione. Per configurare il monitor connessi, nella scheda "Generale", impostare la durata del monitoraggio su "Free run". Utilizzare le impostazioni predefinite per la scheda "Monitor LDF" fare clic su "Avanti".</li>
	<li>Impostare la visualizzazione del grafico in the "display setup dialog box." Selezionare i canali di "Velocità di flusso, Conc," selezionando le rispettive caselle. Selezionare i seguenti parametri: "Origine dati per il canale" e "etichetta, unità e colore." Fare clic su "Avanti".</li>
	<li>Immettere le informazioni utente il soggetto e la misurazione (cioè, nome e numero di soggetto, operatore, monitoraggio tempo, commenti, ecc.) in "file informazioni nella finestra di dialogo" e fare clic su "Next" per terminare la configurazione di misura. Nota: Una finestra di misurazione viene creata automaticamente dal software (Figura 1B).</li>
	<li>Manualmente è possibile avanzare l'elettrodo al pancreas. Assicurarsi che la distanza tra la sonda e pancreas tessuti sia all'interno di 1mm. Una distanza inappropriata dà una lettura del flusso di sangue artificialmente aumentata o diminuita.</li>
	<li>Fare clic sull'icona della barra degli strumenti "Start" per iniziare a registrare i dati di unità (PU) aspersione sanguigno microvascolare. Raccogliere i dati di PU continuamente per 1 minuto ogni run. Fare clic su "Stop" per interrompere la misurazione. Selezionare "File" → "Salva come" per nome e salvare il file finito misura.</li>
	<li>Riposizionare manualmente la sonda dopo ogni corsa per evitare effetti additivi e l'esaurimento localizzata di contrattile e la capacità di rilassamento. Ripetere i passaggi 4.1-4.4 per raccogliere dati di PU microvascolari multi-punto (cioè, tre punti scelti a caso dal tessuto pancreatico) per ogni mouse. Misurare i dati dell'unità di elaborazione di un piatto antiriflesso come controllo della linea di base.</li>
	<li>Chiudere lo strato del muscolo addominale e lo strato della pelle con una sutura. Sistemare gli animali in gabbie pulite dopo gli esperimenti.</li>
	<li>Mantenere l'animale caldo inserendo la gabbia di recupero mezzo-il termoforo. Nota: Prestare attenzione al calore, igiene, fluido e l'ingestione di cibo e infezione. Amministrare i topi con 2 mg/kg Carprofen per 48 h come trattamento del dolore postoperatorio.  Eseguire l'eutanasia iniettando 150 mg/kg i.p. di sodio pentobarbital quando topi sono osservati per essere in uno stato di grave dolore o disagio che non può essere alleviato.</li>
</ol>5. calcolo dei parametri di Vasomotion microvascolare
<ol><li>Utilizzare il comando "Esporta" del laser Doppler software per esportare il tempo e dati grezzi PU come file *. xlsx e aprire il file in un foglio di calcolo.</li>
	<li>Calcolare l'unità di perfusione basale medio (PUb) (Vedi punto 4.6).</li>
	<li>Calcolare l'aspersione di sangue medio (PUun) per 1 min di una misura come segue: media aspersione del sangue (PUun) = PU - PUb (equazione 1).</li>
	<li>Calcolare la frequenza (cicli/min) per ogni 1 min di misurazione. Nota: La frequenza di vasomotion microvascolare è definita come il numero di picchi che si è verificato in un'onda di vasomotion microvascolare al minuto.</li>
	<li>Calcolare l'ampiezza (ΔPU) per ogni 1 min di misurazione.<ol>
<li>Calcolare l'ampiezza del vasomotion microvascolare come differenza tra il minimo (PUmin) e massimo (PUmax): ampiezza (ΔPU) = PUmax - PUmin (equazione 2).</li>
	</ol>
</li>
	<li>Calcolare la velocità relativa (PU) per ogni 1 min di misurazione.</li>
</ol>

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Nei casi che coinvolgono la disfunzione microvascolare (ad es., diabete, pancreatite acuta, malattie microvascolari periferiche, ecc.), alcune malattie portare alla riduzione del flusso sanguigno. Diverso da cambiamenti nel flusso sanguigno, non ci sono indicatori importanti, quali microvascolare vasomotion, che rispecchiano la condizione funzionale del microcircolo. L'indicatore specifico, vasomotion microvascolare, è generalmente definito come l'oscillazione del tono microvascolare in letti microvasc...

Come parametro della condizione funzionale della microcircolazione, vasomotion microvascolare si assume la responsabilità per la consegna e lo scambio di ossigeno, sostanze nutritive e ormoni ed è fondamentale per la rimozione dei prodotti metabolici, quali anidride carbonica e rifiuti cella 1. microvascolare vasomotion regola anche la distribuzione del flusso di sangue e la perfusione tissutale, interessando così la pressione sanguigna microcircolatoria locale e risposte ad infiammazione, che può indurre l'edema in molte malattie. Di conseguenza, vasomotion microvascolare è estremamente importanti per mantenere la funzione fisiologica di organi2,3,4, tessuti e cellule di componente. Il danno della vasomotion microvascolare potrebbe essere uno dei passaggi chiavi nello sviluppo delle malattie relative microcircolazione5.
Laser Doppler è stato inizialmente sviluppato per l'osservazione e la quantificazione nel campo della microcircolazione ricerca6. Questa tecnica, insieme ad altri approcci tecnici (ad es., laser speckle7, ossigeno transcutanea, ecc.), è stata considerata come lo standard d'oro per valutare il flusso di sangue nel microcircolo. Il razionale che l'aspersione del sangue di microcircolazione locale (cioè, capillari, arteriole, venule, ecc.) può essere determinata da apparato dotato di laser Doppler, si basa sul principio dell'effetto Doppler. La lunghezza d'onda e la frequenza della luce di emissione stimolata cambia quando particelle di luce incontro commovente globuli nei microvessels, o rimangono invariati. Pertanto, nella microcircolazione, il numero e la velocità delle cellule del sangue sono i fattori chiavi per la grandezza e la distribuzione di frequenza della luce Doppler-spostato, mentre la direzione del flusso sanguigno microvascolare è irrilevante. Utilizzando diversi metodi, una varietà di tessuti sono stati utilizzati per studi microcircolatori, compreso i mesenteries e dorsale dello skinfold alloggiamenti di topi, ratti, criceti e anche gli esseri umani8. Tuttavia, nel protocollo attuale, ci concentriamo sull'aspetto funzionale status di vasomotion microvascolare dell'isolotto pancreatico, che viene valutato utilizzando laser Doppler e un sistema di parametri di valutazione fatti in casa.
Microcircolazione dell'isolotto pancreatico è principalmente composto di isole pancreatiche microvasi e presenta caratteristiche peculiari. Una rete capillare di isole pancreatiche mostra una densità cinque volte più alto rispetto alla rete capillare della sua controparte esocrina9. Un condotto che prevede la consegna di ingresso del glucosio e di insulina divulgazione, le cellule endoteliali dell'isolotto forniscono ossigeno ai cellule metabolicamente attive nell'isolotto cellule β. Inoltre, la prova emergente dimostra inoltre che l'isolotto microvasi sono coinvolti non solo nella regolazione dell'espressione genica dell'insulina e sopravvivenza della β-cellula, ma anche nell'influenzare la funzione β-cellulare; promuovere la proliferazione delle cellule β; e producendo un numero di sostanze e fattori di crescita angiogenici vasoattivi,10. Pertanto, a questo proposito, si deduce che la condizione funzionale del vasomotion microvascolare dell'isolotto pancreatico può influenzare la funzione della β-cellula dell'isolotto ed essere coinvolta nella patogenesi di malattie come la pancreatite acuta/cronica, diabete e altri malattie del pancreas-relative.
Analizzare i cambiamenti patologici dello stato funzionale delle insule pancreatiche microvascolare vasomotion potrebbe essere una strategia fattibile per determinare i contributi della microcircolazione dell'isolotto pancreatico per le malattie di cui sopra. Fornire una descrizione dettagliata della procedura che descrive l'approccio per determinare isole pancreatiche vasomotion microvascolare in vivo . Misure tipiche vengono visualizzate nei Risultati di rappresentante. Infine, i vantaggi e le limitazioni del metodo sono evidenziate nella discussione, insieme a ulteriori applicazioni.

<table>
	<tbody>
		<tr>
			<td>MoorVMS-LDF2</td>
			<td>Moor Instruments</td>
			<td>GI80</td>
			<td>PeriFlux 5000 (Perimed Inc.) can be used as an alternative apparatus to harvest data</td>
		</tr>
		<tr>
			<td>MoorVMS-PC Software</td>
			<td>Moor Instruments</td>
			<td>GI80-1</td>
			<td>Software of MoorVMS-LDF2</td>
		</tr>
		<tr>
			<td>Calibration stand</td>
			<td>Moor Instruments</td>
			<td>GI-cal</td>
			<td>Calibration tool</td>
		</tr>
		<tr>
			<td>Calibration base</td>
			<td>Moor Instruments</td>
			<td>GI-cal</td>
			<td>Calibration tool</td>
		</tr>
		<tr>
			<td>Calibration flux standard</td>
			<td>Moor Instruments</td>
			<td>GI-cal</td>
			<td>Calibration tool</td>
		</tr>
		<tr>
			<td>One Touch UltraEasy glucometer</td>
			<td>Johnson and Johnson</td>
			<td>#1955685</td>
			<td>Confirm hyperglycemia</td>
		</tr>
		<tr>
			<td>One Touch UltraEasy strips</td>
			<td>Johnson and Johnson</td>
			<td>#1297006</td>
			<td>Confirm hyperglycemia</td>
		</tr>
		<tr>
			<td>Streptozotocin</td>
			<td>Sigma-Aldrich</td>
			<td>S0130</td>
			<td>Dissolve in sodium citrate buffer (pH 4.3)</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td>Working concentration 3 %</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm Inc.</td>
			<td>200121</td>
			<td>Working concentration 75 %</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Amresco</td>
			<td>335</td>
			<td>Working concentration 10 %</td>
		</tr>
		<tr>
			<td>Medical gauze</td>
			<td>China Health Materials Co.</td>
			<td>S-7112</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Blunt-nose forceps</td>
			<td>Shang Hai Surgical Instruments Inc.</td>
			<td>N-551</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Surgical tapes</td>
			<td>3M Company</td>
			<td>3664CU</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Gauze sponge</td>
			<td>Fu Kang Sen Medical Device CO.</td>
			<td>BB5447</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Yu Lin Surgical Instruments Inc.</td>
			<td>175C</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Skin scissor</td>
			<td>Carent</td>
			<td>255-17</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Ning Bo Surgical Instruments Inc.</td>
			<td>3325-77</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Syringe and 25-G needle</td>
			<td>MISAWA Inc.</td>
			<td>3731-2011</td>
			<td>Scale: 1 ml</td>
		</tr>
	</tbody>
</table>

laser doppler: a tool for measuring pancreatic islet microvascular vasomotion in vivo

Come una condizione funzionale della microcircolazione, vasomotion microvascolare è importante per la consegna di ossigeno e nutrienti e la rimozione di anidride carbonica e prodotti di scarto. Il danno della vasomotion microvascolare potrebbe essere un passo cruciale nello sviluppo delle malattie collegate alla microcircolazione. Inoltre, l'isolotto pancreatico altamente vascularized e adattato per supportare la funzione endocrina. A questo proposito, sembra possibile inferire che la condizione funzionale del vasomotion microvascolare dell'isolotto pancreatico potrebbe colpire la funzione delle isole pancreatiche. Analizzare i cambiamenti patologici della condizione funzionale di isole pancreatiche vasomotion microvascolare può essere una strategia fattibile per determinare i contributi che microcircolazione dell'isolotto pancreatico rende alle malattie correlate, come il diabete mellito, pancreatite, ecc. Di conseguenza, questo protocollo viene descritto utilizzando un monitor di flusso di anima di Doppler laser per determinare lo stato funzionale di isole pancreatiche microvascolare vasomotion e di stabilire parametri (tra cui la perfusione sanguigna media, ampiezza, frequenza e relativa velocità di isole pancreatiche microvascolare vasomotion) per la valutazione della condizione funzionale microcircolatoria. In un modello del mouse diabetici streptozotocin-indotti, abbiamo osservato un'alterata condizione funzionale della vasomotion microvascolare dell'isolotto pancreatico. In conclusione, questo approccio per la valutazione dell'isolotto pancreatico vasomotion microvascolare in vivo può rivelare meccanismi relativi alle malattie pancreatiche.

Una fotografia del laser di misurazione microvascolare vasomotion Doppler apparato dotato di un diodo laser a semiconduttore è mostrata in Figura 1A. Software di interfaccia utente è presentato in Figura 1B. Utilizzando il metodo di cui sopra, i parametri emodinamici di vasomotion microvascolare dell'isolotto pancreatico sono stati rilevati per controllo non-diabetico e topi diabetici. Una varietà di tecniche, tra cui flussome...

Watch this Scientific Journal Video about Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo at JoVE.com

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Laser Doppler: Uno strumento per la misurazione dell'isolotto pancreatico Vasomotion microvascolare In Vivo

Isole pancreatiche microvascolare vasomotion regola la distribuzione di sangue dell'isolotto e mantiene la funzione fisiologica delle cellule β dell'isolotto. Questo protocollo descrive l'utilizzo di un monitor Doppler laser per determinare lo stato funzionale di isole pancreatiche vasomotion microvascolare in vivo e a valutare il contributo della microcircolazione di isole pancreatiche per malattie del pancreas.

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

laser-doppler-tool-for-measuring-pancreatic-islet-microvascular

Research

JoVE Journal

Medicine

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

8.2K Views.  Chinese Academy of Medical Sciences & Peking Union Medical College. Isole pancreatiche microvascolare vasomotion regola la distribuzione di sangue dell'isolotto e mantiene la funzione fisiologica delle cellule β dell'isolotto. Questo protocollo descrive l'utilizzo di un monitor Doppler laser per determinare lo stato funzionale di isole pancreatiche vasomotion microvascolare in vivo e a valutare il contributo della microcircolazione di isole pancreatiche per malattie del pancreas.

Video: Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Intraluminali occlusione dell&#39;arteria cerebrale media (MCAO) Modello per l&#39;ictus ischemico con la guida flussimetria laser Doppler in topi

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Ricerca

Medicina

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

L&#39;endotelina-1 indotta cerebrale media occlusione dell&#39;arteria modello per l&#39;ictus ischemico con guida laser flussimetria Doppler in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocollo migliorato per microdissezione laser di isole pancreatiche umane da campioni chirurgici

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI per il tracciamento cellulare

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

Fundus Fotografia come un comodo strumento per studiare le risposte microvascolari a malattie cardiovascolari Fattori di rischio in studi epidemiologici

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

Misurare Crescente aortica Rigidità<em&gt; In Vivo</em&gt; In topi con ultrasuoni

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

A High-contenuti<em&gt; In Vitro</em&gt; La replica della piattaforma Discovery isole pancreatiche β-cellule

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Fosforo-31 spettroscopia di risonanza magnetica: uno strumento per misurare<em&gt; In Vivo</em&gt; Capacità fosforilazione ossidativa mitocondriale nel muscolo scheletrico umano

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Chirurgica dell'angiogenesi in porcina Allotransplantation Tibial: un nuovo grande osso animale vascolarizzato Allotransplantation composito modello

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...