Inhibition of Firefly Luciferase by General Anesthetics: Effect on In Vitro and In Vivo Bioluminescence Imaging

<div><h3></h3><p>Bioluminescence imaging is routinely performed in anesthetized mice. Often isoflurane anesthesia is used because of its ease of use and fast induction/recovery. However, general anesthetics have been described as important inhibitors of the luciferase enzyme reaction.</p> <h3>Aim</h3><p>To investigate frequently used mouse anesthetics for their direct effect on the luciferase reaction, both in vitro and in vivo.</p> <h3>Materials and Methods</h3><p>isoflurane, sevoflurane, desflurane, ketamine, xylazine, medetomidine, pentobarbital and avertin were tested in vitro on luciferase-expressing intact cells, and for non-volatile anesthetics on intact cells and cell lysates. In vivo, isoflurane was compared to unanesthetized animals and different anesthetics. Differences in maximal photon emission and time-to-peak photon emission were analyzed.</p> <h3>Results</h3><p>All volatile anesthetics showed a clear inhibitory effect on the luciferase activity of 50% at physiological concentrations. Avertin had a stronger inhibitory effect of 80%. For ketamine and xylazine, increased photon emission was observed in intact cells, but this was not present in cell lysate assays, and was most likely due to cell toxicity and increased cell membrane permeability. In vivo, the highest signal intensities were measured in unanesthetized mice and pentobarbital anesthetized mice, followed by avertin. Isoflurane and ketamine/medetomidine anesthetized mice showed the lowest photon emission (40% of unanesthetized), with significantly longer time-to-peak than unanesthetized, pentobarbital or avertin-anesthetized mice. We conclude that, although strong inhibitory effects of anesthetics are present in vitro, their effect on in vivo BLI quantification is mainly due to their hemodynamic effects on mice and only to a lesser extent due to the direct inhibitory effect.</p> </div

Quantification of Calcium Amount in a New Experimental Model: A Comparison between Ultrasound and Computed Tomography.

PURPOSE:Calcification is an important prognostic factor in aortic valve stenosis. However, there is no ultrasound (US) method available to accurately quantify calcification in this setting to date. We aimed to validate a new US method for measuring the amount of calcium in an in vitro model, and compare it to computed tomography (CT), the current imaging gold standard. MATERIALS AND METHODS:An agar phantom (2% agar) was made, containing 9 different amounts of calcium-hydroxyapatite Ca5(PO4)3OH (2 to 50 mg). The phantoms were imaged with micro-CT and US (10 MHz probe). The calcium area (areacalcium) and its maximum pixel value (PVmax) were obtained. These values were summed to calculate CT and US calcium scores (∑(areacalcium × PVmax)) and volumes (∑areacalcium). Both US- and CT-calcium scores were compared with the calcium amounts, and with each other. RESULTS:Both calcium scores correlated significantly with the calcium amount (R2 = 0.9788, p<0.0001 and R2 = 0.8154, p<0.0001 for CT and US respectively). Furthermore, there was a significant correlation between US and CT for calcium volumes (R2 = 0.7392, p<0.0001) and scores (R2 = 0.7391, p<0.0001). CONCLUSION:We developed a new US method that accurately quantifies the amount of calcium in an in vitro model. Moreover it is strongly correlated with CT

Correlation of calcium amount with US calcium volume and CT calcium volume.

<p>(A) The correlation between the calcium amount and the US calcium volume. There is a significant correlation between the amount of calcium (2-50mg) and the US calcium volume, with R² = 0.8161, p<0.0001. (B) The correlation between the calcium amount and the CT calcium. There is a significant correlation between the amount of calcium (2-50mg) and the CT calcium volume, with R² = 0.9788, p<0.0001.</p

Schematic overview of the position of phantom and ultrasound probe during ultrasound image acquisition.

<p>Schematic overview of the position of phantom and ultrasound probe during ultrasound image acquisition. Left. Position to obtain long axis images. The long axis of the probe (LA probe) is parallel to the long axis of the calcium (LA calcium). The phantom moves in direction along the short axis of the probe (SA probe). A = agar phantom with 5 increasing amounts of calcium, B = ultrasound probe mounted on support stand. Right. Position to obtain short axis images. The long axis of the probe (LA probe) is perpendicular to the long axis of the calcium (LA calcium). The phantom moves in direction along the short axis of the probe (SA probe). A = agar phantom with 5 increasing amounts of calcium, B = ultrasound probe mounted on support stand.</p

The correlation between the US calcium volume and CT calcium volume.

<p>The correlation between the calcium volume measured with CT and ultrasound, for different amounts of calcium (2-50mg). There is a significant correlation between the two techniques with R² = 0.7392, p<0.0001.</p

The correlation between cIB calcium score and US calcium score.

<p>The correlation between calcium scores measured with cIB (cIB calcium score) and ultrasound (US calcium score) for different amounts of calcium (2-50mg). There is a significant correlation between the two techniques with R² = 0.9439, p <0.0001.</p

Echocardiographic integrated backscatter for detecting progression and regression of aortic valve calcifications in rats.

ABSTRACT: BACKGROUND: Calcification is an independent predictor of mortality in calcific aortic valve disease (CAVD). The aim of this study was to evaluate the use of non-invasive, non-ionizing echocardiographic calibrated integrated backscatter (cIB) for monitoring progression and subsequent regression of aortic valvular calcifications in a rat model of reversible renal failure with CAVD, compared to histology. METHODS: 28 male Wistar rats were prospectively followed during 21 weeks. Group 1 (N=14) was fed with a 0.5% adenine diet for 9 weeks to induce renal failure and CAVD. Group 2 (N=14) received a standard diet. At week 9, six animals of each group were killed. The remaining animals of group 1 (N=8) and group 2 (N=8) were kept on a standard diet for an additional 12 weeks. cIB of the aortic valve was calculated at baseline, 9 and 21 weeks, followed by measurement of the calcified area (Ca Area) on histology. RESULTS: At week 9, cIB values and Ca Area of the aortic valve were significantly increased in the adenine-fed rats compared to baseline and controls. After 12 weeks of adenine diet cessation, cIB values and Ca Area of group 1 decreased compared to week 9, while there was no longer a significant difference compared to age-matched controls of group 2. CONCLUSIONS: cIB is a non-invasive tool allowing quantitative monitoring of CAVD progression and regression in a rat model of reversible renal failure, as validated by comparison with histology. This technique might become useful for assessing CAVD during targeted therapy

In vivo comparison of anesthetics.

<p>(<b>A</b>) represents the maximum BLI intensity obtained from the same mice anesthetized with isoflurane or the test anesthetic mentioned below the curve. Values were normalized to isoflurane signal intensities. (<b>B</b>) shows a scatter plot of the time to the peak signal intensity for the different anesthetics. Each data point represents a unique bioluminescent acquisition from the in vivo comparison of anesthetics study. Lines indicate the mean. There was a significantly later peak for ketamine/medetomidine and isoflurane as compared to avertin, pentobarbital and unanesthetized animals. <i>** p<0.01 between indicated conditions</i>; <i>‡ p<0.05 compared to both isoflurane and ketamine/medetomidine</i>.</p

Reversibility of photon emission inhibition by high substrate concentration.

<p>Photon emission and its inhibition by isoflurane (<b>A</b>) and avertin (<b>B</b>) was examined at normal concentration of D-luciferin (D-luc 150 mg/L) and a 10× higher concentration (D-luc 1500 mg/L) to assess the reversibility of the inhibition. * p<0.05, ** p<0.01, *** p<0.0001.</p

Effect of ketamine and xylazine on BLI signal of intact cells and cell lysates.

<p>The effect on photon emission (PE) of the anesthetics ketamine (<b>A</b>) and xylazine (<b>B</b>) are shown using intact cells (full line) and cell lysates (dotted line). All data are represented as mean % of control (absence of anesthetic) ± SD. Grey intervals in the curves indicate the concentrations used <i>in vivo</i>, as reported in literature for mice.</p