Development and initial application of a fully integrated photoacoustic micro-ultrasound system.

Photoacoustic (PA) imaging for biomedical applications has been under development for many years. Based on the many advances over the past decade, a new photoacoustic imaging system has been integrated into a micro-ultrasound platform for co-registered PA-ultrasound (US) imaging. The design and implementation of the new scanner is described and its performance quantified. Beamforming techniques and signal processing are described, in conjunction with in vivo PA images of normal subcutaneous mouse tissue and selected tumor models. In particular, the use of the system to estimate the spatial distribution of oxygen saturation (sO2) in blood and co-registered with B-mode images of the surrounding anatomy are investigated. The system was validated in vivo against a complementary technique for measuring partial pressure of oxygen in blood (pO2). The pO2 estimates were converted to sO2 values based on a standard dissociation curve found in the literature. Preliminary studies of oxygenation effects were performed in a mouse model of breast cancer (MDA-MB-231) in which control mice were compared with mice treated with a targeted antiangiogenic agent over a 3 d period. Treated mice exhibited a >90% decrease in blood volume, an 85% reduction in blood wash-in rate, and a 60% decrease in relative tissue oxygenation.

New approaches in small animal echocardiography: imaging the sounds of silence.

Systolic and diastolic dysfunction of the left ventricle (LV) is a hallmark of most cardiac diseases. In vivo assessment of heart function in animal models, particularly mice, is essential to refining our understanding of cardiovascular disease processes. Ultrasound echocardiography has emerged as a powerful, noninvasive tool to serially monitor cardiac performance and map the progression of heart dysfunction in murine injury models. This review covers current applications of small animal echocardiography, as well as emerging technologies that improve evaluation of LV function. In particular, we describe speckle-tracking imaging-based regional LV analysis, a recent advancement in murine echocardiography with proven clinical utility. This sensitive measure enables an early detection of subtle myocardial defects before global dysfunction in genetically engineered and rodent surgical injury models. Novel visualization technologies that allow in-depth phenotypic assessment of small animal models, including perfusion imaging and fetal echocardiography, are also discussed. As imaging capabilities continue to improve, murine echocardiography will remain a critical component of the investigator's armamentarium in translating animal data to enhanced clinical treatment of cardiovascular diseases.

Echocardiographic speckle-tracking based strain imaging for rapid cardiovascular phenotyping in mice.

RATIONALE: High-sensitivity in vivo phenotyping of cardiac function is essential for evaluating genes of interest and novel therapies in small animal models of cardiovascular disease. Transthoracic echocardiography is the principal method currently used for assessing cardiac structure and function; however, standard echocardiographic techniques are relatively insensitive to early or subtle changes in cardiac performance, particularly in mice. OBJECTIVE: To develop and validate an echocardiographic strain imaging methodology for sensitive and rapid cardiac phenotyping in small animal models. METHODS AND RESULTS: Herein, we describe a modified echocardiographic technique that uses speckle-tracking based strain analysis for the noninvasive evaluation of cardiac performance in adult mice. This method is found to be rapid, reproducible, and highly sensitive in assessing both regional and global left ventricular (LV) function. Compared with conventional echocardiographic measures of LV structure and function, peak longitudinal strain and strain rate were able to detect changes in adult mouse hearts at an earlier time point following myocardial infarction and predicted the later development of adverse LV remodeling. Moreover, speckle-tracking based strain analysis was able to clearly identify subtle improvement in LV function that occurred early in response to standard post-myocardial infarction cardiac therapy. CONCLUSIONS: Our results highlight the utility of speckle-tracking based strain imaging for detecting discrete functional alterations in mouse models of cardiovascular disease in an efficient and comprehensive manner. Echocardiography speckle-tracking based strain analysis represents a method for relatively high-throughput and sensitive cardiac phenotyping, particularly in evaluating emerging cardiac agents and therapies in mice.

Enhancing repair of the mammalian heart.

The injured mammalian heart is particularly susceptible to tissue deterioration, scarring, and loss of contractile function in response to trauma or sustained disease. We tested the ability of a locally acting insulin-like growth factor-1 isoform (mIGF-1) to recover heart functionality, expressing the transgene in the mouse myocardium to exclude endocrine effects on other tissues. supplemental mIGF-1 expression did not perturb normal cardiac growth and physiology. Restoration of cardiac function in post-infarct mIGF-1 transgenic mice was facilitated by modulation of the inflammatory response and increased antiapoptotic signaling. mIGF-1 ventricular tissue exhibited increased proliferative activity several weeks after injury. The canonical signaling pathway involving Akt, mTOR, and p70S6 kinase was not induced in mIGF-1 hearts, which instead activated alternate PDK1 and SGK1 signaling intermediates. The robust response achieved with the mIGF-1 isoform provides a mechanistic basis for clinically feasible therapeutic strategies for improving the outcome of heart disease.

Non-invasive real-time imaging of atherosclerosis in mice using ultrasound biomicroscopy.

There are increasing needs to develop imaging techniques to study in vivo vascular morphology and function in various mouse models of atherosclerosis. Using ultrasound biomicroscopy (UBM), we developed and validated a new imaging protocol to follow lesion progression in atherosclerotic mice. ApoE and LDL receptor double knockout mice (DKO) with various degree of atherosclerosis and normal control mice were imaged at the level of the ascending aorta using UBM. Average plaque thickness, as well as plaque area were delineated in the short-axis images, and were subsequently compared with histological measurements. We showed that plaque area at this vascular site was closely correlated to total plaque burden from en face measurement (p<0.0001). UBM-measured plaque thickness and area correlated with indices for histology measures from the same vascular region (p<0.0001 respective p<0.0001). Furthermore, in 16 DKO mice aged from 32 to 35 weeks, UBM showed significantly weekly increases of IMT in the ascending aorta from 0.106+/-0.108 mm at 32 weeks of age to 0.256+/-0.345 mm at 35 weeks of age (p=0.0002). In conclusion, this novel imaging protocol provides us with a non-invasive, accurate and inexpensive way to follow lesion progression in mice in vivo.

Calcitriol regulates the expression of the genes encoding the three key vitamin D3 hydroxylases and the drug-metabolizing enzyme CYP3A4 in the human fetal intestine.

BACKGROUND AND AIMS: The human fetal jejunum has been shown to harbour the vitamin D3 (D3) nuclear receptor (VDRn) and to be responsive to calcitriol/1,25-dihydroxyvitamin D3[1,25(OH)2D3] through modulation of proliferation and differentiation processes. The aim of the study was to evaluate the presence as well as the effect of 1,25(OH)2D3 exposure on the expression levels of the three key D3-hydroxylase gene transcripts (25-hydroxylase, CYP27A; 24-hydroxylase, CYP24; 1alpha-hydroxylase, CYP27B1) as well as that of the 1,25(OH)2D3-responsive endobiotic/xenobiotic metabolizing enzyme CYP3A4 (which is also considered a major detoxifiying enzyme) in the human proximal and distal intestine.methods Specimens from normal fetuses ranging from 15 to 20 weeks of gestation were obtained following elective termination of normal pregnancies. Intestinal explants were cultured for a period of 24 h or 48 h with 10-7 m 1,25(OH)2D3. All data were compared to paired-control cultures without 1,25(OH)2D3. Total RNA was extracted and cDNA synthesized by RT-PCR. The cDNA obtained was amplified by radioactive PCR, the signal intensity evaluated by densitometric analyses and expressed in relation to the levels of GAPDH. RESULTS: Data indicate that VDRn, the three D3-hydroxylases as well as CYP3A4 are expressed in all segments of the human fetal small intestine and in the colon. Basal expression levels of VDRn, CYP27A, CYP24 and CYP3A4 were found to be similar in the proximal, median and distal jejunum as well an in the proximal and distal colon. In contrast, basal 1alpha-hydroxylase CYP27B1 expression levels were found to be 65% higher in the colon than in the small intestine (P < 0.02). The 1alpha-hydroxylase was also found to be sensitive to 1,25(OH)2D3 with a 31% decrease in its expression levels within 24 h of 1,25(OH)2D3 exposure to reach a 55% decrease after 48 h of incubation in the presence of the hormone (P < 0.05). Furthermore, the levels of the 25-hydroxylase gene transcript were also decreased by 10% within the first 24 h and by 29% after 48 h of incubation in the presence of 1,25(OH)2D3 (P < 0.003). VDRn expression levels were also found to be reduced following incubation in the presence of 1,25(OH)2D3. In contrast, exposure to 1,25(OH)2D3 contributed to a 4.8 fold increase in the expression of the 24-hydroxylase gene transcript within the first 24 h of exposure (P < 0.03), and to a highly significant induction (24, 22 and 1.5 fold over basal values) of the CYP3A4 gene transcript in 3 of the 4 specimens studies. CONCLUSIONS: Collectively, the data illustrate that at mid-gestation 1,25(OH)2D3 is fully active in the modulation of all D3-hydroxylases in the human developing intestine. They also show that the detoxifying enzyme CYP3A4 is not only present along the intestinal tract but is also sensitive to 1,25(OH)2D3, indicating that the hormone may be a key element in intestinal development and in the maintenance of the intestinal mucosa integrity in the basal state and in response to damage-inducing agents.

High sensitivity of rat hepatic vitamin D3-25 hydroxylase CYP27A to 1,25-dihydroxyvitamin D3 administration.

CYP27A is considered the main vitamin D(3) (D(3))-25 hydroxylase in humans. Our purpose was to evaluate the effect of the D(3) nutritional and hormonal status on hepatic CYP27A mRNA, cellular distribution, transcription rate, and enzyme activity. Studies were carried out in normal and in D-depleted rats supplemented with D(3), 25OHD(3), or 1,25(OH)(2)D(3). CYP27A exhibited a significant gender difference and was observed throughout the hepatic acinus not only in hepatocytes but also in sinusoidal endothelial, stellate, and Kupffer cells. Neither D(3) nor 25OHD(3) influenced CYP27A mRNA levels. However, 1,25(OH)(2)D(3) repletion led to a 60% decrease in CYP27A mRNA, which was accompanied by a 46% decrease in mitochondrial D(3)-25 hydroxylase activity. The effect of 1,25(OH)(2)D(3) was mediated by a significant decrease in CYP27A transcription, whereas its mRNA half-life remained unchanged. Our data indicate that CYP27A is present in hepatic parenchymal and sinusoidal cells and that the gene transcript is not influenced by the D(3) nutritional status but is transcriptionally regulated by 1,25(OH)(2)D(3) exposure.