Autori ringraziano la signora Risa Otsuka per l&#39;assistenza tecnica e la sperimentazione di aiuto. Questa ricerca è stata in parte sostenuta dal Ministero dell&#39;Istruzione, della Scienza, Sport e Cultura, Grant-in-Aid per giovani scienziati (B), 2010, 22700476 e Suzuken Memorial Foundation 2010 per KT E questo studio è stato supportato dalla Ricerca e Sviluppo di Next-Generation Simulazione integrata della materia vivente, una parte dello sviluppo ed uso della Next-Generation Supercomputer Progetto di MEXT, e in parte dal progetto JST ERATO Biologia Gas Suematsu.

1. Etichettatura Eritrociti con isotiocianato di fluorescina isomero I (FITC) Tutti i protocolli sperimentali che abbiamo usato sono stati approvati dal Comitato Animal Cura di Keio University School of Medicine. <ol><li> Anestetizzare una topi donatori, e dopo una incisione laparotomia prelevare sangue intero dalla vena cava inferiore con una siringa da 1 ml contenenti piccole quantità di eparina. Il topo è stato immediatamente eutanasia tramite iniezione sodio pentobarbital (100 mg / kg ip). </li><li> Lavare il sangue intero due volte con PBS (pH 7,4) mediante centrifugazione per 5 min a 400 g in una con rotori. </li><li> Sciogliere 10 mg di FITC in 5 ml di 100 mM Na 2 HPO 4 e filtrare con una membrana avente pori 0,22-micron. </li><li> In una provetta miscelare 1 ml della soluzione FITC con 0,15 ml di 3 mM di glucosio, 0,25 ml di 180 mM NaH 2 PO 4 ml, 1,5 e 100 mM di Na 2 HPO 4. Aggiungere 0,2 ml di RBC pellet, e toccare il tubo per la miscelazione. </li><li> Mantenere la provetta per 2 ore a 4 ° C e poi lavare gli RBC macchiati due volte in PBS. </li><li> Mettere una piccola quantità della sospensione RBC su un prato diapositiva e vedere se gli RBC aspetto sano (cioè, che la loro forma e dimensione sono normali) e che l&#39;intensità di fluorescenza è sufficiente. </li><li> Sospendere il 0,1 ml di globuli rossi in 0,9 ml di PBS a pH 7,4, e mantenere la sospensione a 4 ° C fino ad iniezione. </li></ol> 2. Preparazione di Ossigeno-sensitive Dye <ol><li> Sciogliere 500 mg di BSA in 10 ml di PBS a pH 7,4. </li><li> Aggiungere 30 mg di Pd (II)-meso-tetra (4-carbossifenil) porfina (Pd-TCPP) alla soluzione BSA e agitazione per una notte. </li><li> (Opzionale) Estratto BSA-bound Pd-TCPP utilizzando gravità-flow cromatografia o una colonna di selezione per separarlo dalla libera Pd-TCPP. </li><li> Centrifugare la soluzione per rimuovere indisciolto Pd-TCPP, e filtrare il surnatante con un filtro a membrana con pori 0,22-micron. &lt;/ Li&gt; <li> Conservare le aliquote da 1 ml in provette a -20 ° C. Evitare ripetuti congelamenti e scongelamenti. </li></ol> 3. Preparazione degli animali <ol><li> Per l&#39;osservazione microscopica del microcircolo preparare un piatto di plastica con un foro di 20 millimetri di diametro, e nastro di 30-mm-quadrato di vetro quadrato copertina sopra il foro. </li><li> Anestetizzare un mouse, togliere la pelliccia e preparazione della pelle. Inserire un catetere in coda vano per l&#39;iniezione del farmaco utilizzando un ago 30 G collegato ad un 10-cm polietilene (PE 10) catetere riempito con eparinizzata PBS. </li><li> Dopo l&#39;incisione mediana, estendere un lobo principale del fegato nel piatto di plastica, e posizionare il mouse in decubito sternale. </li><li> Preparare piccoli slittamenti cucina con l&#39;involucro (3 mm x 8 mm) e piastrelle intorno al bordo del lobo epatico per inibire spostamento del fegato con respirazione e tenerlo da essiccazione. </li><li> Osservare la microcircolazione epatica al microscopio (con luce trasmessa), e verificare che la bflusso lood non ha stasi nel campo di vista per almeno 15 min. </li><li> Iniettare lentamente 0,2 ml di globuli rossi fluorescente per l&#39;osservazione del flusso sanguigno o 0,2 ml di Pd-TCPP soluzione pO 2 misurazione. Questa quantità di globuli rossi marcata con FITC rappresenteranno per 1/50 di tutti i globuli rossi in circolazione nella regione visualizzato. </li></ol> 4. Visualizzazione del flusso sanguigno <ol><li> Eccitare il FITC mediante irradiazione con luce lampada al mercurio che è passata attraverso un filtro passa-banda (450-490 nm) 1. </li><li> Registrare l&#39;immagine fluorescente con una telecamera CCD. </li></ol> 5. pO 2 Misura Pd-TCPP fosforescenza è relativamente debole e deve essere rilevata con elevata sensibilità del rivelatore. Tutti gli esperimenti devono essere eseguite in una stanza buia. <ol><li> I picchi di assorbimento di Pd-TCPP sono a 410 nm e 532 nm, quindi la seconda armonica lunghezza d&#39;onda 532 nm è raccomandato per l&#39;eccitazione. Questa lunghezza d&#39;onda può essere generato da un Nd: YAG impulso laser 2. </li><li> Far passare il fascio dal laser Nd: YAG nella porta appropriata sul microscopio invertito, e regolare il punto di fascio ad una posizione centrale nel piano focale. La risoluzione spaziale dipende dalla dimensione del punto laser, che viene modificato facendo passare il fascio attraverso un forellino. </li><li> Per rilevare la fosforescenza, collegare un lungo filtro passa-(&gt; 620 nm) e un tubo fotomoltiplicatore (PMT) all&#39;altra porta del microscopio. La PMT dovrebbe essere sensibile alle lunghezze d&#39;onda rosse, in particolare intorno a 700 nm. </li><li> Campionare il segnale fosforescenza (500 punti campionati a 200 kHz) e calcolare la costante di tempo di decadimento inserendo una funzione esponenziale al decadimento. </li><li> Per la calibrazione preparare due serie di campioni di Pd-TCPP soluzione pO 2 150 mmHg e 0 mmHg. Aggiungere ditionito di sodio 1% in volume da campionare per produrre assenza di ossigeno. Mantenere il pH dei campioni a 7,4 e la temperatura a 37 ° C durante la calibrazione. </li><li> Fissare il Stern-Volmer costante k q e oxygen-free tempo di decadimento della luminescenza τ 0 nell&#39;equazione (1) 3. Nel nostro caso, con Pd-TCPP soluzioni a 37 ° C e un pH di 7,4, q k è 374 mmHg -1 s -1 e τ 0 è 0,74 ms. Questi parametri di misura dipendono dalla apparecchiatura (cioè laser, rivelatore, altri dispositivi ottici) e il metodo di elaborazione del segnale, quindi è necessaria la calibrazione in ciascun sistema di misurazione 4. τ 0 / τ = ​​1 + k q • τ 0 • pO 2 (1) </li><li> Impostare il mouse sul piatto sul microscopio scena, osservare la microcircolazione epatica, e regolare la posizione del bersaglio microvasi al punto del punto laser. </li></ol> 6. Epatite Modello <ol><li> Topi veloci con libero accesso all&#39;acqua durante la notte. </li><li> Preparare una20 mg per ml di soluzione di paracetamolo (APAP) in DMSO. </li><li> Iniettare la intraperitonealy a 1 ml/100 g di peso corporeo (cioè 200 mg / kg di peso corporeo). Dopo l&#39;iniezione, il mouse può essere dato libero accesso a cibo e acqua 5. </li><li> 24 ore dopo l&#39;iniezione, anestetizzare il mouse e esporre il fegato da una incisione mediana. Se si ottiene epatite, necrosi nella regione pericentral sarà facilmente visibili ad occhio nudo. </li></ol> 7. Risultati rappresentativi Esempio di immagini microcircolo epatico sono mostrati nella Figura 1, dove (a) è una luce trasmessa immagine e (b) è una immagine di fluorescenza. I singoli eritrociti FITC marcati si osservano nel film video, e venule portali, sinusoidi, e venule centrali sono riconosciuti dalla direzione del flusso sanguigno. Come mostrato in figura 2, il punto laser irradiare un venule centrale attraverso una lente di obiettivo x100 era approximtamente 10 um in diametro. Intravascolare pO 2 nel microcircolo epatica è stata misurata in maschio C57BL / 6 topi, e la figura 3 (a) mostra le relazioni tra pO 2 e diametro del vaso in venule portale e centrale. Il gradiente di pO 2 dal portale venule centrale indica che l&#39;ossigeno i globuli rossi comunicato in modo efficace durante il passaggio attraverso sinusoidi. Come mostrato nella Figura 3 (b), la media pO 2 era 59,8 mmHg in vasi portali e 48,2 mmHg in sinusoidi, e 38,9 mmHg in venule centrali. Quando abbiamo prodotto epatite acuta iniettando APAP intraperitonealy, intravascolare pO 2 è stato significativamente elevati in ogni parte della microcircolazione epatica: p &lt;0.05 nelle vene portali e p &lt;0.01 in sinusoidi e nelle vene centrali (Figura 3 (c)). <img src="/files/ftp_upload/3996/3996fig1.jpg" alt="Figura 1" /> Figura 1. Basso ingrandimento imaghi mostrando microcircolazione epatica con (a) luce trasmessa e (b) fluorescenza. <img src="/files/ftp_upload/3996/3996fig2.jpg" alt="Figura 2" /> Figura 2. Immagine di spot laser irradiare un venule mirato centrale per pO 2 misura. <img src="/files/ftp_upload/3996/3996fig3.jpg" alt="Figura 3" /> Figura 3. PO 2 gradiente nella microcircolazione epatica di topi sani di controllo e di topi con paracetamolo lesione epatica indotta. (A) mostra pO 2 rispetto al diametro del vaso per portale venule (PV) e venule centrale (CV), e (b) mostra il gradiente di ossigeno da PV a CV attraverso sinusoidi (S). La differenza di due pO tra PV e CV riflette il consumo di ossigeno di epatociti o di altre cellule parenchimali. (C) mostra che in condizioni di epatite indotta da APAP amministrazione, pO 2 è stato significativamente elevati nei PV, S, e il CV e la pO 2pendenza ridotta, indicando che il consumo di ossigeno epatica è stata compromessa.

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Non abbiamo nulla da rivelare.

Fornire ossigeno ai tessuti è un ruolo essenziale del microcircolo, e un sacco di articoli descrivono malattie legate all&#39;anatomia dei microvasi e la reologia 6. Il flusso ematico epatico è una combinazione di sangue arterioso e venoso. Circa il 30% del flusso sanguigno al fegato è ben sangue ossigenato fornito dalla arteria epatica e il restante 70%, fornito dalla vena portale, è poco ossigenato deflusso venoso sangue dalla milza e tratto gastrointestinale 7. Malattie del fegato come la st...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Pd (II) meso-Tetra (4-carbossifenil) porfina </td><td> Frontier Scientific, Inc. </td><td> PdT790 </td></tr><tr><td> I isotiocianato di fluorescina isomero </td><td> Sigma-Aldrich Co. </td><td> F7250 </td></tr><tr><td> Acetaminophen </td><td> Sigma-Aldrich Co. </td><td> A7085 </td></tr></tbody></table> <table border="1"><tbody><tr><td> Nome dell&#39;apparecchiatura </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td></td><td></td><td></td><td> Attrezzatura </td></tr><tr><td> tubo fotomoltiplicatore </td><td> Hamamatsu Photonics KK </td><td> H10722-20 </td></tr></tbody></table>

visualization and analysis of blood flow and oxygen consumption in hepatic microcirculation: application to an acute hepatitis model

C&#39;è una notevole discrepanza tra l&#39;offerta e la domanda di ossigeno nel fegato, perché il consumo di ossigeno epatico è relativamente alto, ma circa il 70% delle scorte di sangue epatico è poco vena porta sangue ossigenato derivato dal tratto gastrointestinale e la milza. L&#39;ossigeno viene trasportato al epatociti dal sangue che scorre da un ramo terminale della vena porta ad un venule centrale via sinusoidi, e questo rende un gradiente di ossigeno in lobuli epatici. Il gradiente di ossigeno è un importante parametro fisico che coinvolge l&#39;espressione di enzimi a monte ea valle in microcircolazione epatica, ma la mancanza di tecniche per la misurazione del consumo di ossigeno nel microcircolo epatico ha ritardato la delucidazione dei meccanismi relativi al metabolismo dell&#39;ossigeno nel fegato. Abbiamo quindi usato marcata con FITC eritrociti per visualizzare la microcircolazione epatica e usato laser assistita phosphorimetry per misurare la pressione parziale di ossigeno nel microvasi lì. Noncontact e continuo di misurazione ottica in grado di quantificare velocità di flusso, diametri dei vasi sanguigni e gradienti di ossigeno legati al consumo di ossigeno nel fegato. In un modello di epatite acuta che abbiamo fatto con la somministrazione di paracetamolo ai topi che abbiamo osservato un aumento della pressione di ossigeno in entrambe le venule portali e centrale, ma un gradiente di ossigeno è diminuita nei sinusoidi, indicando che la necrosi degli epatociti nella zona pericentral potrebbe spostare la pressione di ossigeno e influenzano l&#39;espressione degli enzimi nella zona periportale. In conclusione, i nostri metodi ottici per emodinamica epatica di misura e del consumo di ossigeno in grado di rivelare i meccanismi connessi con la malattia epatica.

Watch this Scientific Journal Video about Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model at JoVE.com

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Un sistema ottico è stato sviluppato per visualizzare microcircolazione epatica con eritrociti marcata con FITC e per misurare la pressione parziale di ossigeno nel microvasi con laser assistita phosphorimetry. Questo metodo può essere usato per studiare i meccanismi fisiologici e patologici analizzando struttura microvascolare, diametro, velocità del flusso ematico, e tensione di ossigeno.

Visualizzazione e analisi del flusso ematico e consumo di ossigeno nella microcircolazione epatica: Applicazione di un modello di epatite acuta

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

visualization-analysis-blood-flow-oxygen-consumption-hepatic

Research

JoVE Journal

Medicine

12.5K Views.  Keio University. Un sistema ottico è stato sviluppato per visualizzare microcircolazione epatica con eritrociti marcata con FITC e per misurare la pressione parziale di ossigeno nel microvasi con laser assistita phosphorimetry. Questo metodo può essere usato per studiare i meccanismi fisiologici e patologici analizzando struttura microvascolare, diametro, velocità del flusso ematico, e tensione di ossigeno.

Video: Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Bioluminescent modello murino epatica Tumori

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Ricerca

Medicina

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

Eterotopica ausiliario Ratto trapianto di fegato con flusso regolato Portal Vein arterialization in acuta insufficienza epatica

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Il Coniglio Blood-shunt modello per lo studio di acuta e sequele tardive di emorragia subaracnoidea: Aspetti tecnici

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

Ultrasuoni Imaging per la valutazione di Spinal Cord Blood Flow in Sperimentale midollo spinale Lesioni

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

L&#39;ossigeno-glucosio Deprivazione e riossigenazione come<em&gt; In Vitro</em&gt; Ischemia-riperfusione Injury modello per lo studio barriera sanguigna del cervello Erettile

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

Un modello acuta della retina per la valutazione sanguigni della retina barriera breccia e potenziali farmaci per il trattamento

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Analisi ottimizzata delle<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; Epatica steatosi

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Un modello di Ischemia cardiaca cronica nei suini utilizzando un Constrictor Ameroid

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Analizzando il tasso di consumo di ossigeno nei Cardiomyocytes neonatali coltivati primari del topo utilizzando un analizzatore di flusso extracellulare

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.