Gli autori desiderano ringraziare Adriane Mosley e Libuse Jerabek (Stanford) per l&#39;assistenza con la tecnica chirurgica.

Tutti gli studi sugli animali sono stati eseguiti secondo le linee guida di cura degli animali e l&#39;uso del comitato di UCLA e il National Institutes of Health Guide per la cura e l&#39;uso di animali da laboratorio. La durata della procedura descritta di seguito è di circa 45-60 minuti dall&#39;inizio alla fine. 1. Preparazione del campo operatorio <ol><li> Eseguire la procedura in una sala operatoria degli animali pulita. </li><li> Equipaggiamento: isoflurano vaporizzatore, Gaymer Pompa T con il rilievo di riscaldamento. </li><li> Strumenti sterili: due pinze, forbici curve sottili, porta aghi. </li><li> Guanti sterili devono essere utilizzati durante l&#39;intera procedura. </li></ol> 2. Preparazione degli animali <ol><li> Inserire due topi maschi o femmine, di stesso background genetico, di peso e di dimensioni simili nella stessa gabbia e monitorare per almeno due settimane per garantire la convivenza armoniosa. Topi femmina sono preferiti a causa del loro comportamento meno aggressivo. </li><li> Anestetizzare gli animali dautilizzando un vaporizzatore isoflurano. Topi posto in una camera di induzione Posi-Seal collegati al vaporizzatore isoflurano (4-5% v / v). Una volta anestesia viene indotta, trasferire l&#39;animale alla zona rasatura pelo e mantenere l&#39;anestesia durante tutta la procedura attraverso un&#39;ogiva collegato a isoflurano (1,5-2% v / v). Applicare una pomata oftalmica con un Q-tip per prevenire gli occhi asciutti. </li><li> Posto l&#39;animale in posizione supina. Radere accuratamente il lato sinistro del mouse posizionata a sinistra e destra del mouse posizionata a destra a partire da circa 1 cm sopra il gomito a 1 cm al di sotto del ginocchio. </li><li> Asetticamente preparare le zone rasate dal fondo strofinando (2-3x) con salviettine Betadine intriso seguito da cotone imbevuto di alcol. Posizionare il mouse su un tappetino riscaldato coperto da un tampone sterile. </li><li> Per analgesia, amministrare Carprofene e buprenorfina per via intraperitoneale o sottocutanea alla dose di 10 mg / kg e 0,1 mg / kg, rispettivamente. </li><li> Posizionare gli animali dalla loro parte, schiena contro schiena, con undjacent aree rasate rivolti verso l&#39;alto. Per evitare qualsiasi contaminazione dell&#39;area chirurgica, coprire i topi con un telino sterile esponendo solo la zona di operazione. Creare una piccola apertura telo di rimanere sterile quando si esegue l&#39;intervento chirurgico. Abbiamo fatto la finestra drappo grande per avere una visione migliore durante la videoregistrazione. </li></ol> 3. Parabiosis <ol><li> Utilizzando una forbice tagliente, effettuare incisioni cutanee longitudinali ai lati rasato di ogni avviamento animale a 0.5 cm sopra il gomito fino a 0,5 cm al di sotto del ginocchio (Figura 1). Seguendo l&#39;incisione, staccare delicatamente la pelle dalla fascia sottocutanea tenendo la pelle con un paio di pinze curve e separare la fascia con una seconda coppia di creare 0,5 cm pelle libera. Eseguire questa separazione lungo tutta l&#39;incisione. </li><li> Iniziare la giunzione collegando all&#39;olecrano fianco di un animale a destra olecranon dell&#39;altro. Entrambi olecranons e le articolazioni del ginocchio sono chiaramente distinguibili seguente the incisione cutanea. Per facilitare l&#39;unione, piegare il gomito del primo mouse e passare l&#39;ago della non assorbibile 3-0 sutura sotto all&#39;olecrano. Analogamente, piegare il gomito del secondo mouse e passare la stessa sutura sotto di essa. Fissare i giunti saldamente da un doppio nodo chirurgico. </li><li> Collegare le articolazioni del ginocchio seguendo la stessa procedura. </li><li> Seguendo il fissaggio delle articolazioni, collegare la pelle dei due animali con un continuo 5-0 assorbibile Vicryl sutura ventrale partendo dal gomito verso il ginocchio. Per evitare la rottura della pelle e la separazione eseguire una sutura stretta della pelle nella zona intorno ai gomiti e le ginocchia. Una volta che l&#39;allegato pelle ventrale è stata completata, eseguire un doppio nodo chirurgico. Posizionare il mouse in posizione prona e proseguire la dorsale sutura termina con un doppio nodo chirurgico. Verificare la continuità della sutura e confermare la mancanza di aperture. </li><li> Somministrare 0,5 ml di NaCl allo 0,9% per via sottocutanea per ogni mouse per evitare deidrodration. </li></ol> 4. Recupero post-operatorio <ol><li> Tenere animali sul pad riscaldato fino al recupero. </li><li> Dopo il recupero, fornire analgesici carprofene e buprenorfina. Ripetere intraperitoneale o sottocutanea ogni hr 24 e 12, rispettivamente, per 48 ore alle stesse dosi descritte sopra (punto 2.5). Monitor animali per i segni di dolore e di angoscia, quali agitazione, letargia, masticazione di coda, inarcò la schiena, la mancanza di toelettatura, ecc giorno per due settimane. </li><li> Profilassi, trattare i topi con sulfametossazolo / trimetoprim sospensione orale nella loro bottiglia d&#39;acqua 2 mg sulfamidici / ml 0,4 mg / ml assetto per 10 giorni per prevenire le infezioni batteriche. </li><li> Casa ciascuna coppia parabiotic in una gabbia pulita con lettiera monolitico (ad es tovagliolo di carta o tampone sterile assorbente) per evitare l&#39;aspirazione di materiale di biancheria da letto. Ritorno agli animali di una gabbia piena di biancheria da letto quando sono in grado di mantenere decubito sternale con la loro testa. Per ridurre al minimo lo sforzodi raggiungere per il cibo durante la regolazione all&#39;esistenza parabiotic, posizionare le palline di cibo inumiditi sul pavimento della gabbia. Fornire materiale di nidificazione. In 1-2 settimane i topi parabiotic hanno la capacità di deambulare normalmente chirurgicamente coppie anteriori e posteriori degli arti. </li><li> Chimerismo sangue si verifica 10-14 giorni dopo l&#39;intervento. </li></ol> 5. Conferma e procedura inversa <ol><li> Dopo due settimane il sangue fluisce dalle vene coda di ciascun topo per analisi citofluorimetrica per confermare chimerismo sangue (Figura 3). </li><li> Trattamento del sangue per l&#39;analisi di citometria di flusso: diluire 2-3 gocce di sangue in 500 ml di EDTA 10 mM. Aggiungere 500 ml di 2% destrano (diluito in PBS), mescolare bene e incubare a 37 ° C per 30 min. Trasferire il surnatante in una nuova provetta, centrifugare a 1200 rpm per 5 min e risospendere le cellule pellet in PBS (tampone o altro) per analisi citofluorimetrica. </li><li> A seconda del disegno sperimentale, la coppia parabiotic può essere separato unt momenti successivi. Nella nostra esperienza, abbiamo mantenuto le coppie parabiosed fino a 9 mesi senza complicazioni. </li><li> Eseguire la procedura inversa su una superficie sterile chirurgica. Anestetizzare topi mettendoli in una camera di induzione collegato ad un vaporizzatore isoflurano (4-5%) e mantenere l&#39;anestesia con isoflurano 1,5-2%, come descritto sopra (passo 2.2). </li><li> Eliminare i peli dalla zona circostante la sutura iniziale e asettico preparare le zone rasate come descritto sopra (punti 2.3, 2.4). </li><li> Per analgesia, amministrare Carprofene e buprenorfina per via intraperitoneale o sottocutanea alla dose di 10 mg / kg e 0,1 mg / kg, rispettivamente. </li><li> Coprire area chirurgica con un telino sterile come sopra descritto (punto 2.6). </li><li> Usando forbici affilate, separare i topi attraverso un&#39;incisione longitudinale lungo la sutura laterale e staccare leggermente la fascia di nuova formazione tra i due topi con un paio di pinze curve. Per separare le articolazioni, tagliare i nodi di suturache li collega. Tagliare la pelle lungo l&#39;incisione per ottenere bordi lisci e riattaccare la pelle con una riassorbibile continuo 5-0 rivestita Vicryl sutura. </li><li> Per prevenire la disidratazione, somministrare 0,5 ml di NaCl allo 0,9% per via sottocutanea per ogni mouse. Tenere animali sul pad riscaldato fino al recupero. </li><li> Dopo il recupero, ripetere la somministrazione di analgesici, carprofene (10 mg / kg) ogni 24 ore e buprenorfina (0,1 mg / kg) ogni 12 ore per 48 ore. </li><li> Per prevenire le infezioni batteriche trattano topi con sospensione orale sulfametossazolo / trimetoprim (2 mg sulfamidici / ml 0,4 mg assetto / ml) nel loro bottiglia d&#39;acqua per 10 giorni. </li></ol>

<ol>
	<li>Bert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exp&#233;riences+et+consid&#233;rations+sur+la+greffe+animale.">Exp&#233;riences et consid&#233;rations sur la greffe animale.</a> Journal de l'Anatomie et de la Physiologie. 1, 69-87 .</li><li>Bunster, E., Meyer, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+of+parabiosis.">Improved methods of parabiosis.</a> Anat. Rec. 57, 339-380 (1933).</li><li>Waskow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+parabiotic+mice+for+the+study+of+DC+and+DC+precursor+circulation.">Generation of parabiotic mice for the study of DC and DC precursor circulation.</a> Methods Mol. Biol. 595, 413-428 (2010).</li><li>Finerty, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parabiosis+in+physiological+studies.">Parabiosis in physiological studies.</a> Physiol. Rev. 32, 277-302 (1952).</li><li>Aicher, A., Heeschen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonbone+marrow-derived+endothelial+progenitor+cells:+what+is+their+exact+location.">Nonbone marrow-derived endothelial progenitor cells: what is their exact location.</a> Circ. Res. 101, e102 (2007).</li><li>Abe, S., Boyer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+derived+from+the+circulation+contribute+to+the+repair+of+lung+injury.">Cells derived from the circulation contribute to the repair of lung injury.</a> Am. J. Respir. Crit. Care Med. 170, 1158-1163 (2004).</li><li>Donskoy, E., Goldschneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymocytopoiesis+is+maintained+by+blood-borne+precursors+throughout+postnatal+life.+A+study+in+parabiotic+mice.">Thymocytopoiesis is maintained by blood-borne precursors throughout postnatal life. A study in parabiotic mice.</a> J. Immunol. 148, 1604-1612 (1992).</li><li>Powell, A. E., Anderson, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+between+Intestinal+epithelial+cells+and+macrophages+in+a+cancer+context+results+in+nuclear+reprogramming.">Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.</a> Cancer Res. 71, 1497-1505 (2011).</li><li>Duyverman, A. M., Kohno, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transient+parabiosis+skin+transplantation+model+in+mice.">A transient parabiosis skin transplantation model in mice.</a> Nat. Protoc. 7, 763-770 (2012).</li><li>Ajami, B., Bennett, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life.">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nat. Neurosci. 10, 1538-1543 (2007).</li><li>Weissman, I. L., Jerabek, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+and+the+H-Y+antigen:+Requirement+for+male+T+cells,+but+not+B+cells,+to+induce+tolerance+in+neonatal+female+mice.">Tolerance and the H-Y antigen: Requirement for male T cells, but not B cells, to induce tolerance in neonatal female mice.</a> Transplantation. 37, 3-6 (1984).</li></ol>

Gli autori non hanno interessi finanziari concorrenti.

Il metodo parabiosis discusso qui presenta difficoltà tecniche minime e si traduce in tassi di mortalità bassi. Il fissaggio delle articolazioni del ginocchio e del gomito è un miglioramento significativo della Bunster e tecnica Meyer. Tuttavia, la procedura rimane così invasiva mantenimento di condizioni di sterilità durante l&#39;intervento chirurgico è imperativo. Per prevenire ulteriori infezioni del sito chirurgico, è importante che gli animali parabiosed ricevono una combinazione di antibiotici e monitorare...

Parabiosis, la chirurgica unione di due organismi, è stato descritto nel 1864 da Paul Bert come un modo per sviluppare un modello per studiare sistemi circolatorio condivisi e consisteva l&#39;unione della pelle e pareti muscolari dei due topi 1. Parabiosis promuove la formazione del microcircolo nel sito di infiammazione 3 e ha avuto diverse applicazioni in studi fisiologici, come la comunicazione ormonale tra l&#39;ipofisi e gonadi nonché il ruolo del rene nell&#39;ipertensione 4. È stato inoltre impiegato per investigare l&#39;assunzione e integrazione di cellule progenitrici in neovascolarizzazione 5, migrazione di cellule staminali ematopoietiche 6, e linfociti tratta 7, così come il ruolo e cinetica di cellule infiammatorie circolanti o staminali nel tumore metastasi 8,9, e le malattie neurodegenerative 10. Un vantaggio significativo di bugie parabiosisdal fatto che gli animali collaborato condividono antigeni circolanti comuni, consentendo la migrazione cellulare e la neovascolarizzazione senza innescare una reazione immunologica. Importante, Weissman et al. Hanno dimostrato che parabiosis tra topi maschio e femmina non porta alla formazione di anticorpi contro HY 11. Nel protocollo originale descritto da Paul Bert due animali sono stati uniti insieme mediante collegamento della pelle e pareti muscolari 1. Questo metodo però, causato sforzo significativo per gli animali e provocato elevata mortalità a causa di infezione della ferita. Da allora la tecnica parabiosis è stato rivisto da diversi gruppi con il più predominante è il protocollo proposto da Bunster e Meyer nel 1933 2. Il loro metodo incluso giunzione delle articolazioni scapola, cavità del corpo, e la pelle, consentendo un migliore supporto e meno dolore per gli animali. Allo stesso tempo, il nuovo metodo ha comportato minima cura post-operatoria e significativamente ridotto la mortalità. Il protocollo qui descritto è una modifica della tecnica Bunster e Meyer che è meno invasiva e permette più salda adesione. Vale a dire, i topi sono collegati attraverso le articolazioni del gomito e ginocchio, come pure la pelle. Questo impedisce l&#39;allungamento adesione della pelle e quindi provoca meno dolore e complicazioni. Qui si descrive l&#39;unione di una wild type (WT) topo adulto per un GFP costitutiva esprime mouse. Abbiamo dimostrato che due settimane dopo l&#39;intervento si possono raggiungere il 50% di chimerismo sangue dimostrare l&#39;efficacia di questa procedura chirurgica per creare un sistema circolatorio condiviso.

<table border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>Isoflurane-Phoenix</td>
			<td>Clipper</td>
			<td>NDC 57319-559-06</td>
		</tr>
		<tr>
			<td>Posi-Seal Induction Chamber</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0530-SM</td>
		</tr>
		<tr>
			<td>Portable Anesthesia System</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0007</td>
		</tr>
		<tr>
			<td>Gaymer T Pump</td>
			<td>Gaymar Industries, Inc</td>
			<td>TP650</td>
		</tr>
		<tr>
			<td>Warming Blanket (Heating pad)</td>
			<td>Kent Scientific Corp</td>
			<td>TP-22G</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Roboz</td>
			<td>RS-5101</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>FST 14063-09</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>FST</td>
			<td>FST 12501-13</td>
		</tr>
		<tr>
			<td>Electrical shaver</td>
			<td>Oster</td>
			<td>Golden A5</td>
		</tr>
	</tbody>
</table>

parabiosis in mice: a detailed protocol

Parabiosis è l&#39;unione chirurgica di due organismi che permettono la condivisione della circolazione sanguigna. Allegando la pelle di due animali promuove la formazione di microcircolo nel sito di infiammazione. Partner Parabiotic condividere i loro antigeni circolanti e quindi sono privi di reazione immunitaria avversa. In primo luogo descritto da Paul Bert nel 1864 1, l&#39;intervento parabiosis è stata affinata per Bunster e Meyer nel 1933 per migliorare la sopravvivenza degli animali 2. Nel protocollo corrente, due topi sono chirurgicamente uniti dopo una modifica della Bunster e tecnica Meyer. Gli animali sono collegati attraverso il gomito e del ginocchio seguita da attacco della pelle permettendo il supporto costante che impedisce sforzo sulla pelle suturata. Qui, descriviamo in dettaglio la parabiotic unione di un GFP onnipresente che esprime il mouse per una wild type (WT) del mouse. Due settimane dopo la procedura, la coppia viene separata e cellule positive GFP può essere rilevata mediante analisi citofluorimetrica nel cir sangueione del mouse WT. Il chimerismo sangue permette di esaminare il contributo delle cellule circolanti da un animale nell&#39;altro.

Il risultato atteso di parabiosis di due organismi è il pari contributo di sistema circolatorio di ogni animale ad una circolazione sanguigna comune (Figura 2). Si può facilmente verificare l&#39;equilibratura con successo il sangue del parabiosed WT e topi GFP positive con l&#39;analisi di citometria di flusso. Qui, il sangue venoso è stato ottenuto dalle code di entrambi parabionts a 2 settimane dopo l&#39;intervento chirurgico e stato frazionato per le cellule del sangue periferico (per escludere ...

Watch this Scientific Journal Video about Parabiosis in Mice: A Detailed Protocol at JoVE.com

Parabiosis in Mice: A Detailed Protocol

Parabiotic unione di due organismi porta allo sviluppo di un sistema circolatorio condiviso. In questo protocollo, si descrivono i passaggi chirurgici per formare una connessione parabiotic tra un topo wild-type e una costitutiva GFP che esprimono mouse.

Parabiosis in topi: un protocollo dettagliato

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Research

JoVE Journal

Medicine

73.7K Views.  University of California, Los Angeles.  Parabiotic unione di due organismi porta allo sviluppo di un sistema circolatorio condiviso. In questo protocollo, si descrivono i passaggi chirurgici per formare una connessione parabiotic tra un topo wild-type e una costitutiva GFP che esprimono mouse. 

Video: Parabiosis in Mice: A Detailed Protocol

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

The Social Trier Protocollo di prova dello stress per indurre stress psicologico

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

Ricerca

Medicina

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Protocollo per lungo Ipertermia Corpo intera durata in Mouse

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

Protocollo di raccolta per pancreas umano

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusi carotide di farmacocinetica e di farmacodinamica Analisi dei taxani nei topi

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Un semplice Critical dimensioni femorale Difetto Modello in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Eterotopico renale Autotrapianto in un modello di suino: un protocollo Step-by-Step

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Un passo per passo protocollo per la chirurgia sottoretinico in Conigli

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

Creazione di addominali aderenze nei topi

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Un protocollo dettagliato per la sudorazione monitoraggio Utilizzando un romanzo, Piccolo, dispositivo wireless

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

A Detailed Protocol for Physiological Parameters Acquisition and Analysis in Neurosurgical Critical Patients

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ICP waveform, amplitude of pulse (AMP), the correlation of ICP amplitude and ICP mean (RAP), pressure reactivity index (PRx), ICP and arterial blood pressure (ABP) wave amplitude correlation (IAAC), and so on, can reflect intracranial status, predict prognosis, and can also be used as guidance of proper treatment. However, most of the clinicians focus only on the mean ICP value while ignoring these parameters because of the limitations of the current devices. We have recently developed a multimodality monitoring system to address these drawbacks. This portable, user-friendly system will use a data collecting and storing device to continuously acquire patients&#39; physiological parameters first, i.e., ABP, ICP, and oxygen saturation, and then analyze these physiological parameters. We hope that the multimodality monitoring system will be accepted as a key measure to monitor physiological parameters, to analyze the current clinical status, and to predict the prognosis of the neurosurgical critical patients.

Recently, we have developed a portable multimodality monitoring system for the monitoring of various physiological parameters in neurosurgical critical patients. Detailed protocols on how to use this multimodality monitoring system are presented here.

Un protocollo dettagliato per i parametri fisiologici di acquisizione e analisi nei pazienti neurochirurgici critici

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ...