Funziona questo è stato sostenuto attraverso sovvenzioni dal scienze naturali e ingegneria Research Council del Canada (RGPIN 238382-2011) e Canadian Institutes of Health Research (MOP115076). Heather Paul è stato sostenuto da una borsa di studio post-laurea scienze naturali e ingegneria Research Council del Canada e una borsa di studio Alberta innova Health Solutions. Megan Hallam è stato sostenuto da un scienze naturali e ingegneria Research Council Postgraduate borse di studio, una borsa di studio Frederick Banting e Charles Best Canada laureato e premio un bambini Alberta Hospital Research Training Institute in Genetica, Child Development, e la salute.

Questo protocollo è stato approvato dalla University of Calgary Comitato cura degli animali e conforme alla Guida per la cura e l&#39;uso di animali da laboratorio. 1. Diga separato da Offspring <ol><li> Separare la diga dalla sua prole per un minimo di 5 minuti prima mungitura 12. NOTA: La diga può essere munta fino a 5-6 ore dopo la separazione 1,6,13, ma periodi di separazione più lunghi di 4 ore possono alterare la composizione del latte 14. Mentre tempo di separazione non sembra impattare latte volume di raccolta 12, è consigliabile che un tempo di separazione coerente essere mantenuta per tutto lo studio. Composizione del latte può cambiare tutta la lattazione 15, quindi i tentativi dovrebbe essere fatto per mantenere il giorno di raccolta del latte coerente. La produzione massima di latte si consiglia di verificare in lattazione giorno 14 12. </li><li> Usando una camera di riscaldamento, verificare i cuccioli sono in grado di mantenere la corretta tempera corpori senza la presenza della madre per la durata della procedura di mungitura. NOTA: Nello studio presentato di seguito, la mungitura è stata eseguita allo svezzamento, quando le dighe erano circa 22 settimane di età e le prole 21 giorni di età, quindi nessuna camera di riscaldamento è stato utilizzato. </li></ol> 2. Set-up e preparazione <ol><li> Raccogliere tutti i materiali necessari per la procedura di mungitura. NOTA: Tutti i materiali possono essere trovati nella tabella materiali ed apparecchiature. </li><li> Posizionare una piastra elettrica sulla panca dove la mungitura avrà luogo e coprire il pad con un banco assorbenti sotto-pad. </li><li> Impostare il sistema anestetico. Assicurarsi che il sistema ha ossigeno e isoflurano prima dell&#39;inizio sufficiente. Fissare la maschera anestetico che verrà utilizzato per l&#39;induzione di anestesia iniziale alla macchina. Posizionare la maschera che verrà utilizzata per l&#39;anestesia di mantenimento vicino se diversa dalla maschera anestetico iniziale. </li><li> Collegare un ago da 25 G per una siringa da 1 ml per ossitocinainiezione con tecnica asettica. </li><li> Accendere il rilievo di riscaldamento in modo che la temperatura del corpo della madre è mantenuta durante la procedura di mungitura. NOTA: Monitorare la temperatura della piastra elettrica per assicurare il pad non diventi troppo calda e causare ustioni. In alternativa, utilizzare una fonte di calore, ad esempio un tavolo operatorio riscaldato, che può essere impostato ad una temperatura specifica. </li></ol> 3. Anestetizzare la diga di Utilizzo Isoflurane <ol><li> Aprire il serbatoio di ossigeno e ruotare il flusso a 1 L (1.000 cc) per min. Accendere il flusso di isoflurano e impostare al 5%. ATTENZIONE: Evitare l&#39;inalazione diretta di anestetico e prevenire l&#39;accumulo di vapori anestetici. </li><li> Anestetizzare la diga. </li><li> Passare alla maschera di manutenzione, se necessario, ponendo la supina diga sul tampone assorbente panca. Confermare anestesia dalla mancanza di pedale reflex. </li><li> Ridurre il flusso di isoflurano al 2-3% per il mantenimento dell&#39;anestesia. Monitorare continuamente diga durante tutta la procedura per garantire la depressione di respirazione non si verifica. NOTA: Una volta sotto anestesia, gli occhi della diga devono essere protetti con un lubrificante occhio sterile per evitare che gli occhi si secchi o di diventare graffiato. </li></ol> 4. L&#39;ossitocina Iniezione <ol><li> Assicurarsi che il ossitocina (20 USP Unità / ml) non ha superato la data di scadenza. Disinfettare la fiala di ossitocina con un alcool sterile wipe / alcol detergente pad. </li><li> Utilizzando una tecnica asettica, elaborare 2 UI (0,1 ml) di ossitocina nella siringa. Usare un nuovo ago e la siringa per ogni diga che verranno munte. NOTA: L&#39;ossitocina dosi variano generalmente da singole iniezioni di 1-5 UI 1,6,12,16. In alternativa, una dose di 4 UI / kg di peso corporeo può essere utilizzato 12. Una singola dose di 2 UI può essere ripetuta una volta se si incontra difficoltà di mungitura. </li><li> Iniettare l&#39;ossitocina per via intraperitoneale. Inserire l&#39;ago nel quadrante in basso a destra dell&#39;addome con l&#39;ago rivolto verso la testa, con un angolodi 15-30 °, circa 0,5 cm di profondità. </li><li> Tirare indietro lo stantuffo per garantire pressione negativa prima dell&#39;iniezione. Se il liquido (sangue, urine, contenuto intestinale, ecc) è aspirato nella siringa, estragga l&#39;ago e tentare l&#39;iniezione con un nuovo ago e la siringa. Se nessun fluido viene aspirato iniettare l&#39;ossitocina e scartare il ago e la siringa immediatamente in un contenitore a rischio biologico. </li><li> Attendere circa 5-15 min per il ossitocina per stimolare il latte delusione. </li></ol> 5. Preparazione di siti mungitura <ol><li> Scegli i siti / mammelle da cui verrà raccolto il latte. Il latte può essere raccolto da ogni capezzolo 12. </li><li> Rimuovere delicatamente il pelo intorno ai capezzoli da mungere con i trimmer, come la pelliccia può causare difficoltà nella raccolta dei campioni a causa dell&#39;umidità del latte. Essere gentile - la pelle intorno i capezzoli è estremamente sensibile e può essere secco e come tale è soggetta a graffi e rotture. </li><li> Sterilizzazione della tettarella iNon s necessario, ma opzionalmente, pulire il capezzolo con acqua tiepida dopo il pelo viene rimosso. Preparare almeno due siti come più di un sito può essere richiesta per la raccolta del latte. NOTA: Se l&#39;analisi del latte comprende profiling microbica, la zona del capezzolo può richiedere la sterilizzazione con iodio 5. </li></ol> 6. Latte Collection <ol><li> Premere delicatamente la base del capezzolo, espellendo manualmente il latte per la raccolta. NOTA: Se l&#39;analisi del latte comprende profiling microbica, le prime gocce di latte devono essere eliminate. </li><li> Raccogliere le gocce di latte in un tubo capillare, riempiendo il tubo capillare. Tubi capillari che possono ospitare volumi maggiori (ad esempio, 50 microlitri) facilitare il processo. NOTA: Se si riscontra difficoltà nella raccolta del latte, una seconda dose di ossitocina può essere somministrata. Si raccomanda di non superare la dose di 4 UI totale. </li><li> Erogare il latte dal tubo capillare in una provetta steriletoccare l&#39;estremità del tubo che è stato utilizzato per disegnare il latte dal capezzolo al lato della provetta - osservare il latte viene estratto dal tubo capillare tramite azione capillare. NOTA: &#39;Soffiare&#39; il latte che non aspirata nel tubo con un ago 18 G attaccato ad una siringa da 1 ml. </li><li> Monitorare continuamente la diga per segni di dolore o di depressione respiratoria e regolare il flusso di isoflurano conseguenza. </li><li> Continuare a raccogliere il latte, come descritto in questa sezione fino a quando il latte sufficiente sono stati raccolti per l&#39;analisi del latte prescelto. Per la creamatocrit e proteine ​​determinazione della concentrazione descritto di seguito, raccogliere 0,25 ml di latte. NOTA: utilizzare un sito di mungitura diverso se il latte delusione rallenta o il sito scelto non espelle il latte a sufficienza. Un massimo di circa 2,5 ml di latte per animale può essere raccolto 17, o fino a 0,5 ml per tettarella. Gli autori raccomandano che il tempo totale in anestesia essere limitato a circa 45-60 minuti, or 45 min di mungitura. </li><li> Raccogliere latte in una provetta per la misura creamatocrit microematocrito da campioni di latte fresco, e sigillare l&#39;estremità del tubo con argilla sigillante. Etichettare la provetta microematocrito con l&#39;ID diga e conservare il campione di latte in posizione verticale. </li><li> Quando la mungitura è completata, spegnere il flusso di isoflurano e ossigeno. Rimuovere la maschera dalla diga e continuare a monitorare diga fino sveglio. Si raccomanda che se non pienamente cosciente, la diga deve essere collocato su un tampone assorbente panchina durante il periodo di recupero, anziché direttamente sulla biancheria gabbia, per evitare che la biancheria venga aspirato o graffiare gli occhi della diga durante il recupero. NOTA: Non lasciare incustodita la diga fino a che non ha ripreso conoscenza sufficiente a mantenere recumbancy sternale. </li><li> Se non sono necessarie ulteriori analisi, congelare il latte a -80 ° C. Altri hanno suggerito che il latte può essere conservato fino a 3 ore a 4 ° C o 5 mesi a -20 ° C 18. </li></ol> 7. Creamatocrit misura <ol><li> Stimare il contenuto di grasso nel latte calcolando la creamatocrit latte (la percentuale di crema nel campione di latte) 19. NOTA: Le misurazioni con latte umano hanno dimostrato che il latte fresco o congelato può essere utilizzato per una misurazione creamatocrit, tuttavia latte fresco è più strettamente correlata (r = 0,92 rispetto r = 0,90) con concentrazione di lipidi 11. L&#39;uso di latte fresco o congelato per misurazioni creamatocrit deve essere mantenuto costante attraverso lo studio, come il latte scongelato è associato ad una piccola diminuzione creamatocrit valori 11. </li><li> Per il latte fresco, raccogliere un campione di latte dal capezzolo in un tubo microematocrito; riempire almeno ¾ (circa 15-20 ml). In alternativa, disegnare il latte fresco dal campione raccolto nel tubo capillare dopo mescolando bene. Sigillare la fine con l&#39;argilla sigillante. </li><li> Posizionare il tubo capillare nella filatore ematocrito, con ilestremità sigillata orientate verso l&#39;esterno, assicurando la centrifuga è equilibrata. </li><li> Iniziare lo spin ematocrito (120 sec a 13.700 xg). NOTA: il tempo di rotazione o la velocità possono cambiare a seconda del modello di centrifuga utilizzata. </li><li> Rimuovere il tubo dalla centrifuga dopo lo spin è completa ed eseguire le misurazioni per calcolare il creamatocrit. Osservare separazione del campione in uno strato di crema e uno strato trasparente. </li><li> Misurare e registrare la lunghezza totale del fluido nel tubo e la lunghezza del grasso (crema) strato utilizzando pinze o un righello. NOTA: Il creamatocrit è espresso come percentuale dello strato di crema all&#39;interno del campione del latte 19, calcolato come (lunghezza dello strato di crema / lunghezza totale della colonna latte) x 100 (Figura 1A). Calcolare valori di concentrazione e di energia grasso da valutazione creamatocrit come segue: concentrazione di grasso (g / L) = (creamatocrit (%) - 0,59) /0.146 (Figura 1B) 19; Valore energetico (kcal / L) = 290 + (66,8 * creamatocrit (%) (Figura 1C) 19. </li></ol> 8. Proteine ​​Concentrazione Determinazione <ol><li> Utilizzando Bovine Serum Albumin come standard proteine, determinare la concentrazione totale di proteine ​​del latte usando un test standard di proteine, come una proteina test Lowry 6. NOTA: La diluizione del latte può essere necessario per le misurazioni della proteina del latte a cadere all&#39;interno della curva standard del dosaggio. </li></ol>

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Gli autori non hanno conflitti di interesse da dichiarare. Tutti gli esperimenti sugli animali sono stati condotti secondo protocolli approvati CCAC.

Investigations into maternal milk components have increased as interest in early life development research rises. As the sole source of nutrition during the neonatal period, the bioactive compounds in milk are essential for ideal growth and development, especially in the context of intestinal and immune health3. The method presented here is a simple, non-invasive method of collecting milk from the lactating rat in amounts sufficient for downstream analysis, such as oligosaccharide profiling6. The me...

Il latte è l&#39;unica fonte di nutrimento per i mammiferi neonati, fornendo energia e nutrienti per la crescita infantile e lo sviluppo 1,2. Mentre il latte è composto principalmente da cellule, lipidi e proteine ​​1, contiene anche una pletora di composti bioattivi che modulano lo sviluppo primi anni di vita della prole compresi gli enzimi, carboidrati, ormoni, anticorpi, fattori di crescita, citochine, esosomi, microvescicole, e piccoli RNA tali come microRNA 1,2. Il ruolo fondamentale del latte materno nello stabilimento della prole immunitario e la salute intestinale 3, insieme con la prova che allattati al seno i neonati sono meno suscettibili alle malattie 2, sottolinea l&#39;importanza di identificare i componenti del latte associati ai processi di malattia nella prima infanzia ed i meccanismi molecolari coinvolti nelle loro azioni. Il ratto di sviluppo è un modello popolare per studiare l&#39;effetto di vari nutrizionale, fisiologico, e interventi chimici sulle precocesviluppo -life 4. L&#39;analisi del latte di ratto può quindi fornire romanzo spaccato la salute materna e prole. Progressi scientifici attuali forniscono ora aumentare le opportunità per le approfondite indagini sugli effetti di specifici componenti del latte sulla salute e la malattia. Ad esempio, il sequenziamento di latte profili batterici ha chiarito il loro ruolo nella prima colonizzazione intestinale dell&#39;intestino infantile 5, analisi di spettrometria di massa di oligosaccaridi del latte hanno fornito spaccato l&#39;alterazione dei profili latte oligosaccaridi via dieta materna 6, e sequenziamento profondo di microRNA secreto in i globuli di grasso del latte materno evidenzia possibili ruoli nella trascrizione genica, il metabolismo, e la funzione immunitaria 7. Modelli di ratto rappresentano uno dei più popolari organismi modello usati in studi materni 8,9. Un vantaggio è la loro gestazione e allattamento periodi brevi, che durano solo approximately 21 giorni ciascuno; il tempo totale dall&#39;inizio della gravidanza per allattamento rappresenta un breve periodo di tempo in cui i dati importanti possono essere generati. Le maggiori dimensioni dei ratti rispetto ai topi, nel contesto di raccolta del latte, può fornire un vantaggio significativo rispetto al volume di latte e facilità di raccolta del latte; produzione di latte nel topo, per esempio, sembra essere dipende dal peso corporeo totale con topi pesanti producono più latte 10. Qui, è fornita una descrizione generale per la raccolta manuale del latte di ratti che allattano. Questo protocollo richiede attrezzatura minima, non è invasiva, poco costoso, e può essere utilizzato per raccogliere adeguati volumi di latte per ulteriori analisi a valle. In breve, la diga è anestetizzato con isoflurano, latte letdown è stimolata da ossitocina, e latte viene raccolto in provette capillari tramite spremitura manuale del latte. Infine, come due componenti principali di latte sono grassi e proteine, una breve description di stimare latte tenore di materia grassa utilizzando misurazioni creamatocrit 11 e la quantificazione della concentrazione proteica totale usando un test standard di proteine ​​è presentato.

<table border="0" cellpadding="0" cellspacing="0" width="829">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Milking</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringes</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ul Microdispenser Capillary Tubes</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">21-169D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (20 USP Units/ml)</td>
			<td style="width:191px;">Bimeda-MTC</td>
			<td style="width:119px;">1OXY015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PPC Vet Isoflurane Inhalation Anesthetic, 250 ml</td>
			<td style="width:191px;">Fresenius Kabi</td>
			<td style="width:119px;">M60302</td>
			<td>Used on the order of a veterinarian</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pad</td>
			<td style="width:191px;">Dukal</td>
			<td style="width:119px;">853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absorbent Bench Underpad</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">82020-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Maxi-Therm Hyper/Hypothermia Blanket</td>
			<td style="width:191px;">Cincinnati Sub-Zero</td>
			<td style="width:119px;">274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Rodent Anesthesia Machine with Vaporizer</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;"></td>
			<td>Subject to individual laboratory needs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">Animal Masks</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;">50100/50102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge Tubes</td>
			<td style="width:191px;">Axygen</td>
			<td style="width:119px;">MCT-060-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChroMini Professional Trimmer</td>
			<td style="width:191px;">Wahl</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Creamatocrit</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin SafeCrit Plastic Microhematocrit Tubes (Untreated)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">22-274-914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Critoseal Capillary Tube Sealant Tray</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">470161-478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin CritSpin Microhematocrit Centrifuge</td>
			<td style="width:191px;">Beckman Coulter, Inc</td>
			<td style="width:119px;">X00-004999-001</td>
			<td></td>
		</tr>
	</tbody>
</table>

milk collection in the rat using capillary tubes and estimation of milk fat content by creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

Il latte è stato raccolto come descritto allo svezzamento da dighe Wistar (circa 22 settimane di età, peso 350 a 400 g) che consuma un controllo (AIN-93G, n = 5), ad alto contenuto proteico (40% di caseina peso / peso, n = 5) , o di fibre prebiotiche (21,6% peso / peso, rapporto 1: 1 di oligofruttosio e l&#39;inulina, n = 4) dieta durante la gravidanza e l&#39;allattamento. La dose di ossitocina era 2 UI. Il latte è stato raccolto in provette capillari, e un tubo è stato filato usando un filatore ematocrito per dete...

Watch this Scientific Journal Video about Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit at JoVE.com

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Milk Collection nella Rat Utilizzando tubi capillari e stima di tenore di materia grassa per Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

milk-collection-rat-using-capillary-tubes-estimation-milk-fat-content

Research

JoVE Journal

Biology

12.2K Views.  University of Calgary. Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Video: Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Ricerca

Biologia

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Isolamento dei fibroblasti Portal ratto<em&gt; In situ</em&gt; Fegato perfusione

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imaging funzionale del grasso bruno nei topi con FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk Collection Methods for Mice and Reeves&#039; Muntjac Deer

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Metodi Latte di raccolta per topi e Reeves &#39;Muntjac

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

Raccolta e analisi dei<em&gt; Arabidopsis</em&gt; Floema Essudati Utilizzo del metodo EDTA-agevolata

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Profilatura cellulare Redox usando la microscopia ad alto contenuto

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Determinazione degli acidi sialici nei campioni di fegato e latte di tipo selvatico e<em&gt; CMAH</em&gt; Mouse Knock-out.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

Analisi dell'autofagia indotta dalla fame nel corpo grasso larvale di Drosophila melanogaster

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

Quantificazione dell'infiltrazione adiposa del muscolo scheletrico mediante decellularizzazione

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Autore in primo piano: Quantificazione basata sulla decellularizzazione dell'infiltrazione adiposa del muscolo scheletrico  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.