This work was supported by a National Imaging Facility Subsidised Access Grant to BJC, a National Health and Medical Research Council of Australia program grant (482800) to KAR and PJB. The authors would like to thank Andrew Arthur, Hasar Hazme and Marie-Claude Gregoire for support in developing this method.

Alle in dieser Studie beschriebenen Verfahren wurden von dem Sydney Local Health District und University of Sydney Tierethikkomitees und folgten den NIH Leitfaden für die Pflege und Verwendung von Labortieren, Achte Auflage (2011) genehmigt. 1. Vorbereitung der Tiere HINWEIS: In diesem Protokoll männlichen db / db - Mäuse (BKS.Cg- Dock7 m + / + Lepr db / J) wurden in Gruppenhaltung mit ad libitum Zugang zu Futter und Wasser bis zum Alter von 6 Wochen gehalten. Zum Zeitpunkt der Bildgebung, Mäuse gewogen ~ 30 g. Alle Mäuse in diesem Protokoll verwendet hatten Nüchternblutzuckerspiegel zwischen 10 und 14 mmol / L. <ol><li> Bei Bedarf schnell die Mäuse. In dem vorliegenden Beispiel schnell die Mäuse für 5 Stunden vor dem experimentellen Verfahren. </li><li> Behandlung von Mäusen mit dem gewünschten Wirkstoffe (zB Medikament, Protein, Peptid) vor dem Beginn der Bilderzeugung. In diesem Beispiel verabreicht eine subkutane Injektion von Insulin (3U / kg Humaninsulin) oder äquivalente Volumen PBS 30 min vor dem Beginn der Bildgebung. </li></ol> 2. Richten Sie Workflows Hinweis: Dieses Protokoll auf einem PET / CT-Scanner durchgeführt wurde. Erwerben PET-Daten zunächst durch Akquisition von CT-Daten gefolgt. <ol><li> PET - Einstellungen: <ol><li> Select - Isotop wie 18 F, eingestellte Scandauer bis 3.600 s, und die obere und untere Ebene Energiediskriminierung bis 350 keV - 650 keV (default) mit einem Koinzidenzzeitfenster von 3,432 ns (Standard). Histogram list-Modus-Daten in 16 Rahmen (6 x 10 s, 4 x 60 s, 1 x 300 s, 5 x 600 s) für den Zeitraum 0-60 min nach Tracerinjektion. Rekonstruieren Emission Sinogramme mit Hilfe von 2D-FBP mit einem Zoom-1.5. HINWEIS: Die rekonstruierten Bilder bestanden aus 16 dynamischen Rahmen, die jeweils mit 128 × 128 × 159 Voxel und eine Voxel - Größe von 0,52 × 0,52 × 0,796 mm 3. </li></ol></li><li> CT - Einstellungen: <ol><li> Zumeine Ganzkörper-CT-Scan, stellte den Strom bei 500 A, Spannung bei 50 kV, Belichtungszeit von 500 ms und 200 Projektionen über eine Umdrehung 360. Stellen Sie das Detektorsichtfeld (FOV) zu 30.722.048, Anzahl der Bettpositionen bis 3 (zur Deckung volle PET FoV Bereichs), Überlappung zwischen Bettpositionen = 30,234713% und Detektor Binning bis 4. HINWEIS: CT-Rekonstruktion wurde mit Kegelstrahl-Tomographie-Bildrekonstruktionssoftware mit HU-Kalibrierung, bilinearer Interpolation und Shepp-Logan-Filter durchgeführt. </li></ol></li><li> 18 F-FDG: <ol><li> Um ein ausreichenden 18 F-FDG (zum Beispiel 450 MBq in 0,5 ml) von einem lokalen Provider ankommen ~ 30 min vor der ersten Injektion. Aliquot und verdünnt den 18 F-FDG so dass Tiere erhalten ~ 10 MBq von 18 F-FDG in einem Endvolumen von 0,1 ml. </li></ol></li></ol> 3. Imaging Protocol <ol><li> Abwischen Induktionskammer und Aufnahmebett mit 80% (v / v) Ethanol aseptische Bedingungen aufrechtzuerhalten. PlaCE, um die Maus in einer Ansaugkammer und anästhesieren mit 5% Isofluran in Sauerstoff. </li><li> Platzieren Sie die Maus auf ein Aufnahmebett mit einem elektrischen Heizkissen angebracht auf Körpertemperatur und einen Präzisions Verdampfer Bugkonus zu halten liefern Isofluran (Wartung, 1,5 bis 2%) bei einer Strömungsgeschwindigkeit von 1 l / min. Bewerben Augensalbe auf die Augen Trockenheit während der Narkose zu verhindern. </li><li> Bewegen Sie die Maus in Bauchlage auf einem Sensorfeld, um die Atmung zu überwachen und sicherzustellen, dass eine angemessene Ebene der Narkose gehalten wird. </li><li> Erwärmen Sie den Schwanz unter Verwendung einer Wärmepackung für 1 bis 2 min, um die laterale Schwanzvene aufzuweiten. Katheterisieren die laterale Schwanzvene eine 30-Gauge-Nadel in die laterale Schwanzvene durch Einfügen. Sichern die Nadel an Ort und Stelle mit chirurgischem Klebstoff und den Katheter sichern. </li><li> Last Bildgebung in die Scanner-Bett und das Bett durch die Maschine bewegen, so dass der Katheter von der Rückseite der Maschine zugegriffen werden kann. </li><li> Befestigen des Katheters an der 18 F-FDG Spritze in einer SYRinge Fahrer. Berechnen die genaue 18 F-FDG - Dosis (10 MBq) , basierend auf der Aktivität in der Spritze vor der Injektion und das Volumen verabreicht werden (&lt;100 ul, injizierte über 10 s). </li><li> Um die Auswirkungen der Narkose auf der Variabilität der Glukoseaufnahme zu minimieren, um eine konstante Zeit zwischen der Einleitung der Narkose und der Injektion von 18 F-FDG (zB 30 min) sicherzustellen. </li><li> Commence die PET - Scan unmittelbar vor der Injektion von 18 F-FDG. Nach Abschluss des PET-Scan (3,600 s), einen CT-Scan (~ 10 min) durchführen für die Co-Registrierung von Radiotracer-Aufnahme mit Geweben zu ermöglichen. </li><li> Bewegen des Aufnahmebetts in die Ausgangsposition, entfernen Sie das Tier aus dem Bett. </li><li> An diesem Punkt das Tier einschläfern oder lassen Sie es sich erholen: <ol><li> Für Euthanasie, Genickbruch, während noch unter Narkose durchführt und Organe von Interesse für eine spätere Analyse sammeln. </li><li> Wenn so dass die Maus zu erholen, legen Sie die Maus in einzelnen Gehäusen auf einem Heizkissen odervor einer Heizlampe. Überwachen Sie die Maus, bis es genügend Bewusstsein wiedererlangt Brustlage zu halten. Lassen Sie die Maus für 1 h erholen, bevor auf Gruppenhaltung zurück. </li></ol></li></ol> 4. PET Bildverarbeitung HINWEIS: Die Bildrekonstruktion wurde mit Erwerb Arbeitsplatzsoftware v1.5.0.28 und Analyse in der Forschung am Arbeitsplatz Software v4.2 durchgeführt. <ol><li> Co-Register Bilder CT und PET und stellen Sie sicher, dass die Ausrichtung in allen drei Dimensionen korrekt ist. <ol><li> Im Menü ‚Datei‘, wählen Sie ‚Ordnersuche / Import‘ und wählen Sie den Ordner mit den Daten enthalten. Wählen Sie die gewünschten PET und CT-Daten ein und klicken Sie auf den ‚General Analysis‘ Tab. </li><li> Sortieren Sie die Daten, so dass die CR bezeichnet ist ‚Quelle‘ und PET bezeichnet ‚Ziel‘. Im Menü ‚Arbeitsablauf‘, wählen Sie ‚Registrierung‘. Wenn die Bilder angepasst werden müssen richtig zusammen registriert, verwenden Sie die Werkzeuge in th seine &#39;Registration&#39; Menü. </li></ol></li><li> Im Menü ‚Arbeitsablauf‘, wählen Sie ‚ROI Quantifizierung‘. <ol><li> Verwenden Sie die ‚Pan‘ und ‚Zoom‘ Funktionen in der Registerkarte ‚Bild‘ die gewünschte Region zu suchen. Im Menü ‚Extras‘, wählen Sie die ‚Erstellen‘ Registerkarte und klicken Sie auf das Pinsel-Symbol. Zeichnen Sie den ROI auf das Bild </li></ol></li><li> Extrahieren Zeitaktivitätskurven von ‚Save ROI Quantifizierung‘ von den ‚Speichern‘ Menü auswählen. Speichern Sie die Daten als CSV-Datei. </li><li> Quantifizieren Radioaktivität Aufnahme als Bq pro cm 3 des Gewebes. Konvertieren Werte in Prozent der injizierten Dosis pro cm 3 (% ID / cm 3) durch die CSV in eine Tabellenkalkulationsdatei lädt. </li></ol> 5. Eingangsfunktion <ol><li> Um für das System Punktverwaschungsfunktion zu korrigieren, entfalten die geschätzte PSF System für 5 Iterationen die reblurred Van Cittert Entfaltungsverfahren unter Verwendung von wie zuvor 9 beschrieben. HINWEIS: Dies aufgrund der geringen Größe der Hohlvene in einer Maus erforderlich ist. </li><li> Verwenden Post Dekonvolution Bilder eine Bluteingangsfunktion zeitAktivitätsKurve zu erzeugen, wie oben beschrieben. </li></ol> 6. Kinetic Modeling HINWEIS: Das FDG Zwei Gewebekompartiment Modell (1) erfordert die Plasmaeingangsfunktion. <ol><li> Konvertieren der Bluteingangsfunktion in der CSV - Datei an die Plasmaeingangsfunktion unter Verwendung der folgenden Gleichung 10: Input_plasma = Input_blood × (0,386 e - 0.191t + 1,165). </li><li> Bei dem kinetischen Modellierungstool klicken Sie auf die ‚Kinetic‘ klicken. Importieren Sie das Gewebe und Plasma-Gesamtaktivität zählen CSV-Dateien in das kinetische Modellierungswerkzeug von ‚Load Time Activity Curve‘ aus dem ‚Menü‘ auswählen. </li><li> Im ‚Model‘ Menü 2 Gewebekompartimenten wählen. Stellen Sie sicher, dass das Kästchen neben k4 nicht ausgewählt istund gibt einen Wert von 0. Für Erstmontage, deaktivieren die Box für vB (Blutvolumenanteil), und einen Wert von 2% ein. </li><li> Klicken Sie auf die &quot;Fit aktuelle Region. Korrigieren der extrahierten interessierenden Bereich für die Dispersion 11, 12. Sie erreichen dies durch den Chi-Quadrat-Wert für das FDG-Modell für verschiedene Dispersionszeiten zu minimieren. </li><li> Durchführen einer zweiten schwimmenden Sitz vB Wert mit (Kontrollkästchen neben dem Feld für vb) und den optimierten Dispersionswert der regionalen Geschwindigkeitskonstanten (k 1 -k 3) zu berechnen. Berechne den regionalen Zufluß konstant wie K i = (k 1 x k 3) / (k 2 + k 3). </li></ol>

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The authors have nothing to disclose.

Das hier beschriebene Protokoll stellt eine robuste, nicht-invasive Methode, die Kinetik der Glukoseaufnahme aus dem Blut in dem Gewebe und anschließenden Metabolismus bei Mäusen zu bestimmen. Die db / db - Maus ist ein ist ein etabliertes Tiermodell von Diabetes Typ 2 , die 14 extensiv Insulinresistenz und entsprechende Eingriffe verwendet worden ist , zu untersuchen. Allerdings haben frühere Studien nur Endpunkt Aufnahme im Herzen quantifizierten <sup class="xref...

Der Zweck dieser Studie war es, eine Positronen - Emissions - Tomographie / Computertomographie (PET / CT) basierend Methodik zu entwickeln , die in - vivo - Echtzeit - Aufnahme von Glucose aus dem Blutstrom in spezifische Gewebe in Mäusen zu quantifizieren. Dies wurde unter Verwendung erreicht 18 F-markierten fluorodeoxyglucose (FDG) Glukoseaufnahme und kinetische Modellierung zur Messung der Raten von 18 F-FDG - Aufnahme aus dem Plasma zu dem intrazellulären Raum zu schätzen, die Geschwindigkeit des Transports von intrazellulären Raum zu Plasma und die Rate der 18 F-FDG - Phosphorylierung. Bei Nagetieren 18F-FDG ist in der präklinischen Bewertung zahlreicher Krebstherapien 1, Studien der Tumorprogression 2 und Tumor - Stoffwechsel 3 sowie Abbildung von braunen Fettdepots 4, neuroinflamation 5 und dem Stoffwechsel im Gehirn 6 verwendet </sup&gt;. Traditionelle Methoden verwendet , um die Gewebe - spezifische Aufnahme von Glucose in Mäusen zu untersuchen (und Ratten) In der Regel beinhaltet die Behandlung mit 2-Desoxyglucose radiomarkiert mit entweder 3 H oder 14 C , gefolgt von Euthanasie, Gewebeentnahme und Messung der Radioaktivität in jedem Gewebe 7. Die Verwendung von PET / CT ermöglicht eine nicht-invasive Bestimmung der Glukoseaufnahme und Stoffwechsel in mehreren Organen und Regionen gleichzeitig in lebenden Tieren. Zusätzlich ist, wie Euthanasie nicht erforderlich ist, ist diese Technik für den Einsatz in Langzeitstudien geeignet. Typ 2 Diabetes mellitus (T2DM) durch gestörte Glukosestoffwechsel und Hyperglykämie sekundär zu reduzierte Gewebe Ansprechbarkeit auf Insulin (Insulinresistenz) und die Unfähigkeit von pankreatischen -Cells gekennzeichnet ausreichenden Mengen von 8 Insulin zu produzieren. Die kinetische Analyse der Glukoseaufnahme und Stoffwechsel kann wichtige Einblicke inder Mechanismus der Wirkung und Wirksamkeit von therapeutischen Interventionen sowie für erweiterte Überwachung des Krankheitsverlaufs ermöglichen.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PET/CT Scanner</td>
			<td style="width:152px;">Siemens</td>
			<td style="width:119px;">Inveon&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">18F-FDG</td>
			<td style="width:152px;">PETNET Solutions</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td style="width:152px;">Pharmachem</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30 guage needle</td>
			<td style="width:152px;">BD</td>
			<td align="right" style="width:119px;">305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PMOD modelling software</td>
			<td style="width:152px;">PMOD Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BKS.Cg-Dock7m&#160;+/+&#160;Leprdb/J&#160; mice</td>
			<td style="width:152px;">Jackson Laboratory</td>
			<td style="width:119px;">000642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Human insulin</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

determining glucose metabolism kinetics using 18f-fdg micro-pet/ct

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Wir haben zuvor mithilfe der db / db - Maus - Modell die Auswirkungen der Erhöhung der Plasma - Apo-A-I - Spiegel auf die Kinetik der Glukoseaufnahme und Stoffwechsel 13 zu untersuchen. In dieser Studie verwenden wir db / db - Mäuse , die mit Insulin behandelt , um die Nützlichkeit von PET / CT - Bildgebung zu zeigen , die Aufnahme von 18 F-FDG aus dem Plasma in den Gastrocnemius - Muskel in Echtzeit zu überwachen. <p class="jove_content" fo:kee...

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Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

Die Bestimmung Glucose Metabolism Kinetics Verwendung<sup&gt; 18</sup&gt; F-FDG Micro-PET / CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

determining-glucose-metabolism-kinetics-using-18f-fdg-micro-petct

Research

JoVE Journal

Medicine

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

13.8K Aufrufe.  UNSW Australia. This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

Serie: Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

Nicht-invasive Bildgebung des akuten Abstoßung nach Nierentransplantation mit Rat<sup&gt; 18</sup&gt; F-FDG PET

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

Forschung

Medizin

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Non-invasive optische Messung des zerebralen Metabolismus und Hämodynamik bei Säuglingen

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

Nichtinvasive Intratracheale Intubation, die Pathologie und Physiologie der Maus Lung Study

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Nicht-invasive Bildgebung und Analyse der zerebralen Ischämie in lebenden Ratten mit Hilfe der Positronen-Emissions-Tomographie mit<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical profiles, inclusive of blood glucose levels in both the fed and fasted state, HbA1c, insulin levels, clinical history of diet, body composition, and body weight status, an assessment of normal and abnormal glycemic control can be made. Interpretation of an IVGTT is done through measurement of changes in glucose and insulin levels over time in relation to the dextrose challenge. Critical components to be considered are: peak glucose and insulin levels reached in relation to T0 (end of glucose infusion), the glucose clearance rate K derived from the slope of rapid glucose clearance in the first 20 min (T1 to T20), the time to return to glucose baseline, and the area under the curve (AUC). These IVGTT measures will show characteristic changes as glucose homeostasis moves from a healthy to a diseased metabolic state5. Herein we will describe the characterization of nonhuman primates (Rhesus and Cynomolgus macaques), which are the most relevant animal model of Type II diabetes (T2D) in humans and the IVGTT and clinical profiles of these animals from a lean healthy, to obese dysmetabolic, and T2D state 8, 10, 11.

The goal of this protocol is to present a standard method to perform intravenous glucose tolerance tests (IVGTTs) to assess glycemic control in nonhuman primates and assess their metabolic status from healthy to dysmetabolic.

Charakterisierung von metabolischen Status in nicht-menschlichen Primaten mit der intravenösen Glukosetoleranztest

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Ein organ High Throughput-System zur Charakterisierung von Arzneimittelempfindlichkeit von Primary Multiple Myelomzellen

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

Studie von In Vivo Metabolismus von Glukose in fettreichen Diät gefüttert Mäuse mit oralen Glukosetoleranztest (OGTT) und Insulin-Toleranz-Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

Ganzen Körper und regionale Quantifizierung der aktiven Menschen braune Fettgewebe mit 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of disorders of consciousness, disrupted higher brain function, and verbal disturbances. As a consequence of an insufficient ability to communicate, objective evaluations are needed from family members, medical staff, and caregivers. One such evaluation is the assessment of functioning brain areas. Recently, multimodal brain imaging has been used to explore the function of damaged brain areas. [18F]-fluorodeoxyglucose positron emission tomography-computed tomography ([18F]FDG-PET/CT) is a successful tool for examining brain function. However, the assessment of brain glucose metabolism based on [18F]FDG-PET/CT is not standardized and depends on several varying parameters, as well as the patient&#39;s condition. Here, we describe a series of semiquantitative assessment protocols for a region-of-interest (ROI) image analysis using self-produced [18F]FDG tracers in patients with sTBI. The protocol focuses on screening the participants, preparing the [18F]FDG tracer in the hot lab, scheduling the acquisition of [18F]FDG-PET/CT brain images, and measuring glucose metabolism using the ROI analysis from a targeted brain area.

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

[18F]-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography is useful for studying glucose metabolism related to brain function. Here, we present a protocol for an [18F]FDG tracer set-up and semiquantitative assessment of the region-of-interest analysis for targeted brain areas associated with clinical manifestations in patients with severe traumatic brain injury.

Semi-quantitative Bewertung mit [18F] FDG Tracer bei Patienten mit schweren Hirnverletzungen

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Visualisierung von endogenen Mitophagie-Komplexen in Situ in menschlichen Pankreas-Betazellen unter Verwendung von Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

Die Bestimmung Glucose Metabolism Kinetics Verwendung

Zusammenfassung

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Ein organ High Throughput-System zur Charakterisierung von Arzneimittelempfindlichkeit von Primary Multiple Myelomzellen

Studie von In Vivo Metabolismus von Glukose in fettreichen Diät gefüttert Mäuse mit oralen Glukosetoleranztest (OGTT) und Insulin-Toleranz-Test (ITT)

Ganzen Körper und regionale Quantifizierung der aktiven Menschen braune Fettgewebe mit ¹⁸F-FDG PET/CT

Semi-quantitative Bewertung mit [¹⁸F] FDG Tracer bei Patienten mit schweren Hirnverletzungen

Visualisierung von endogenen Mitophagie-Komplexen in Situ in menschlichen Pankreas-Betazellen unter Verwendung von Proximity Ligation Assay