Waldemar Ladno

In Vivo Quantitation of Glucose Metabolism in Mice Using Small-Animal PET and a Microfluidic Device

The challenge of sampling blood from small animals has hampered the realization of quantitative small-animal PET. Difficulties associated with the conventional blood-sampling procedure need to be overcome to facilitate the full use of this technique in mice. Methods: We developed an automated blood-sampling device on an integrated microfluidic platform to withdraw small blood samples from mice. We demonstrate the feasibility of performing quantitative small-animal PET studies using 18F-FDG and input functions derived from the blood samples taken by the new device. 18F-FDG kinetics in the mouse brain and myocardial tissues were analyzed. Results: The studies showed that small (∼220 nL) blood samples can be taken accurately in volume and precisely in time from the mouse without direct user intervention. The total blood loss in the animal was <0.5% of the body weight, and radiation exposure to the investigators was minimized. Good model fittings to the brain and the myocardial tissue time–activity curves were obtained when the input functions were derived from the 18 serial blood samples. The R2 values of the curve fittings are >0.90 using a 18F-FDG 3-compartment model and >0.99 for Patlak analysis. The 18F-FDG rate constants \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{1}^{{\ast}}\) \end{document}, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{2}^{{\ast}}\) \end{document}, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{3}^{{\ast}}\) \end{document}, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{4}^{{\ast}}\) \end{document}, obtained for the 4 mouse brains, were comparable. The cerebral glucose metabolic rates obtained from 4 normoglycemic mice were 21.5 ± 4.3 μmol/min/100 g (mean ± SD) under the influence of 1.5% isoflurane. By generating the whole-body parametric images of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{FDG}^{{\ast}}\) \end{document} (mL/min/g), the uptake constant of 18F-FDG, we obtained similar pixel values as those obtained from the conventional regional analysis using tissue time–activity curves. Conclusion: With an automated microfluidic blood-sampling device, our studies showed that quantitative small-animal PET can be performed in mice routinely, reliably, and safely in a small-animal PET facility.

An integrated microfluidic blood sampler for determination of blood input function in quantitative mouse microPET studies

In this study, we developed and constructed a blood sampling system on an integrated microfluidic platform to withdraw sub-microliter blood samples from mice up to a rate of one to two samples per second. On one microfluidic chip, eighteen blood samples can be collected on designated wells and can be retrieved later for further analysis. The chip is flushed with Heparin lock flush between sample collections. The device is remotely controlled by a user-friendly interface and can be programmed to take samples at specific time interval. Preliminary results show that both saline solution and FDG blood samples (/spl sim/220 nano-liter/sample) can be taken with a consistent volume (variation < 2.2% s.d.) at a rate of one sample per second. Although the volumes are small, the FDG activities in blood samples are detectable with low variability (<1.2%). Our study demonstrates the feasibility of deriving input function from mice using a microfluidic blood sampling device. The blood loss (<100 /spl mu/l) and the impact on physiological change is expected to be minimized.

A semi-automated vascular access system for preclinical models

Murine models are used extensively in biological and translational research. For many of these studies it is necessary to access the vasculature for the injection of biologically active agents. Among the possible methods for accessing the mouse vasculature, tail vein injections are a routine but critical step for many experimental protocols. To perform successful tail vein injections, a high skill set and experience is required, leaving most scientists ill-suited to perform this task. This can lead to a high variability between injections, which can impact experimental results. To allow more scientists to perform tail vein injections and to decrease the variability between injections, a vascular access system (VAS) that semi-automatically inserts a needle into the tail vein of a mouse was developed. The VAS uses near infrared light, image processing techniques, computer controlled motors, and a pressure feedback system to insert the needle and to validate its proper placement within the vein. The VAS was tested by injecting a commonly used radiolabeled probe (FDG) into the tail veins of five mice. These mice were then imaged using micro-positron emission tomography to measure the percentage of the injected probe remaining in the tail. These studies showed that, on average, the VAS leaves 3.4% of the injected probe in the tail. With these preliminary results, the VAS system demonstrates the potential for improving the accuracy of tail vein injections in mice.

First-pass angiography in mice using FDG-PET: a simple method of deriving the cardiovascular transit time without the need of region-of-interest drawing

In this study, we developed a simple and robust semi-automatic method to measure the right ventricle to left ventricle (RV-to-LV) transit time (TT) in mice using 2-[/sup 18/F]fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET). The accuracy of the method was first evaluated using a 4-D digital dynamic mouse phantom. The RV-to-LV TTs of twenty-nine mouse studies were measured using the new method and compared to those obtained from the conventional ROI-drawing method. The results showed that the new method correctly separated different structures (e.g., RV, lung, and LV) in the PET images and generated corresponding time activity curve (TAC) of each structure. The RV-to-LV TTs obtained from the new method and ROI method were not statistically different (p=0.20; r=0.76). We expect that this fast and robust method is applicable to the pathophysiology of cardiovascular diseases using small animal models such as rats and mice.

Cardiovascular transit times in mice by high temporal resolution microPET

In this study, we evaluated the temporal resolution of a new microPET system in mice. We then applied the high resolution capability for determining the cardiovascular transit time which is an important physiological parameter in the study of animal models of cardiovascular disease. Two groups of mice, group 1 (n=12) with high body weight and group 2 (n=10) with low body weight, were studied. Each mouse was injected with a 18F-deoxyglucose (FDG; 40-50 /spl mu/L; 8-48 MBq) bolus and a list mode PET acquisition was started. MicroCT was performed for attenuation correction. We histogrammed the first 9 seconds of the list mode data into frames of 0.2 s-0.5 s duration. After the images were reconstructed and attenuation corrected, the ROIs were assigned to the following structures: vena cava, right and left ventricle blood pool (RV&LV), left lung and aorta. A time activity curve (VAC) of each ROI was generated. To correct for the recirculation, the end of each TAC was approximated by an exponential decay curve. The transit time (TT) was then calculated as the difference of the mean arrival times of the bolus in RV and LV. Short time frames of 0.3 s proved to be the best in terms of reducing noise and at the same being able to follow the rapid physiological processes. The peaks of the TACs showed good separation in various structures if the time spread of the bolus in the vena cava measured as full width of half maximum (FWHM) was shorter than 2 s. Group 1 had significantly (p<0.05) longer TT as expected than those of group 2.

A new method of deriving the mice cardiovascular transit time without the need of region-of-interest drawing

In this study, we developed a simple and robust semiautomatic method to measure the right ventricle to left ventricle (RV-to-LV) transit time (TT) in mice using 2-[18F]fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET). The accuracy of the new method was first evaluated using a 4-D digital dynamic mouse phantom. The RV-to-LV TTs of thirty-two mice studies were measured using the new method and compared to those obtained from the conventional ROI-drawing method. The results showed that the new method accurately separated different structures (e.g. RV, lung and LV) in the PET images and generated corresponding time activity curve (TAC) of each structure. The RV-to-LV TTs obtained from the new method and ROI method were not statistically different (p=0.31; r=0.77). We expect that this fast and robust method is applicable to study the pathophysiology of cardiovascular diseases using small animal models such as rats and mice.