James C. Lacefield

Three-dimensional High-Frequency Ultrasound Imaging for Longitudinal Evaluation of Liver Metastases in Preclinical Models

Liver metastasis is a clinically significant contributor to the mortality associated with melanoma, colon, and breast cancer. Preclinical mouse models are essential to the study of liver metastasis, yet their utility has been limited by the inability to study this dynamic process in a noninvasive and longitudinal manner. This study shows that three-dimensional high-frequency ultrasound can be used to noninvasively track the growth of liver metastases and evaluate potential chemotherapeutics in experimental liver metastasis models. Liver metastases produced by mesenteric vein injection of B16F1 (murine melanoma), PAP2 (murine H-ras-transformed fibroblast), HT-29 (human colon carcinoma), and MDA-MB-435/HAL (human breast carcinoma) cells were identified and tracked longitudinally. Tumor size and location were verified by histologic evaluation. Tumor volumes were calculated from the three-dimensional volumetric data, with individual liver metastases showing exponential growth. The importance of volumetric imaging to reduce uncertainty in tumor volume measurement was shown by comparing three-dimensional segmented volumes with volumes estimated from diameter measurements and the assumption of an ellipsoid shape. The utility of high-frequency ultrasound imaging in the evaluation of therapeutic interventions was established with a doxorubicin treatment trial. These results show that three-dimensional high-frequency ultrasound imaging may be particularly well suited for the quantitative assessment of metastatic progression and the evaluation of chemotherapeutics in preclinical liver metastasis models.

A New Three-Dimensional Ultrasound Microimaging Technology for Preclinical Studies Using a Transgenic Prostate Cancer Mouse Model

Prostate cancer is the most common cancer in adult men in North America. Preclinical studies of prostate cancer employ genetically engineered mouse models, because prostate cancer does not occur naturally in rodents. Widespread application of these models has been limited because autopsy was the only reliable method to evaluate treatment efficacy in longitudinal studies. This article reports the first use of three-dimensional ultrasound microimaging for measuring tumor progression in a genetically engineered mouse model, the 94-amino acid prostate secretory protein gene-directed transgenic prostate cancer model. Qualitative comparisons of three-dimensional ultrasound images with serial histology sections of prostate tumors show the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. Ultrasound imaging identified tumors ranging from 2.4 to 14 mm maximum diameter. The correlation coefficient of tumor diameter measurements done in vivo with three-dimensional ultrasound and at autopsy was 0.998. Prospective tumor detection sensitivity and specificity were both >90% when diagnoses were based on repeated ultrasound examinations done on separate days. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated volume doubling times of 5 and 13 days for two prostate tumors. Compared with other microimaging and molecular imaging modalities, the application of three-dimensional ultrasound imaging to prostate cancer in mice showed advantages, such as high spatial resolution and contrast in soft tissue, fast and uncomplicated protocols, and portable and economical equipment that will likely enable ultrasound to become a new microimaging modality for mouse preclinical trial studies.

Novel Small Interfering RNA–Containing Solution Protecting Donor Organs in Heart Transplantation

Background— Ischemia/reperfusion injury is a major factor in graft quality and subsequent function in the transplantation setting. We hypothesize that the process of RNA interference may be used to “engineer” a graft to suppress expression of genes associated with inflammation, apoptosis, and complement, which are believed to cause ischemia/reperfusion injury. Such manipulation of pathological gene expression may be performed by treatment of the graft ex vivo with small interfering RNA (siRNA) as part of the preservation procedure. Methods and Results— Heart grafts from BALB/c mice were preserved in UW solution (control) or UW solution containing siRNAs targeting tumor necrosis factor-&agr;, C3, and Fas genes (siRNA solution) at 4°C for 48 hours and subsequently transplanted into syngeneic recipients. Tumor necrosis factor-&agr;, C3, and Fas genes were elevated by ischemia/reperfusion injury after 48 hours of preservation in UW solution. Preservation in siRNA solution knocked down gene expression at the level of messenger RNA and protein in the grafts after transplantation. All grafts preserved in siRNA solution showed strong contraction, whereas grafts preserved in control solution demonstrated no detectable contraction by high-frequency ultrasound scanning. siRNA solution–treated organs exhibited improved histology and diminished neutrophil and lymphocyte infiltration compared with control solution–treated organs. Furthermore, the treated heart grafts retained strong beating up to the end of the observation period (>100 days), whereas all control grafts lost function within 8 days. Conclusion— Incorporation of siRNA into organ storage solution is a feasible and effective method of attenuating ischemia/reperfusion injury, protecting cardiac function, and prolonging graft survival.

Therapeutic inhibition of mitochondrial reactive oxygen species with mito-TEMPO reduces diabetic cardiomyopathy.

AIMS The mitochondria are important sources of reactive oxygen species (ROS) in the heart. Mitochondrial ROS production has been implicated in the pathogenesis of diabetic cardiomyopathy, suggesting that therapeutic strategies specifically targeting mitochondrial ROS may have benefit in this disease. We investigated the therapeutic effects of mitochondria-targeted antioxidant mito-TEMPO on diabetic cardiomyopathy. METHODS The mitochondria-targeted antioxidant mito-TEMPO was administrated after diabetes onset in a mouse model of streptozotocin-induced type-1 diabetes and type-2 diabetic db/db mice. Cardiac adverse changes were analyzed and myocardial function assessed. Cultured adult cardiomyocytes were stimulated with high glucose, and mitochondrial superoxide generation and cell death were measured. RESULTS Incubation with high glucose increased mitochondria superoxide generation in cultured cardiomyocytes, which was prevented by mito-TEMPO. Co-incubation with mito-TEMPO abrogated high glucose-induced cell death. Mitochondrial ROS generation, and intracellular oxidative stress levels were induced in both type-1 and type-2 diabetic mouse hearts. Daily injection of mito-TEMPO for 30 days inhibited mitochondrial ROS generation, prevented intracellular oxidative stress levels, decreased apoptosis and reduced myocardial hypertrophy in diabetic hearts, leading to improvement of myocardial function in both type-1 and type-2 diabetic mice. Incubation with mito-TEMPO or inhibition of Nox2-containing NADPH oxidase prevented oxidative stress levels and cell death in high glucose-stimulated cardiomyocytes. Mechanistic study revealed that the protective effects of mito-TEMPO were associated with down-regulation of ERK1/2 phosphorylation. CONCLUSIONS Therapeutic inhibition of mitochondrial ROS by mito-TEMPO reduced adverse cardiac changes and mitigated myocardial dysfunction in diabetic mice. Thus, mitochondria-targeted antioxidants may be an effective therapy for diabetic cardiac complications.

Design, calibration and evaluation of a robotic needle-positioning system for small animal imaging applications

A needle-positioning robot has been developed for image-guided interventions in small animal research models. The device is designed to position a needle with an error < or =100 microm. The robot has two rotational axes (pitch and roll) to control needle orientation, and one linear axis to perform needle insertion. The three axes intersect at a single point to create a remote centre of motion (RCM) that acts as a fulcrum for the orientation of the needle. The RCM corresponds to the skin-entry point of the needle into the animal. The robot was calibrated to ensure that the three axes intersected at a single point defining an RCM and that the needle tip was positioned at the RCM. Needle-positioning accuracy and precision were quantified in Cartesian coordinates at ten target locations in the plane of each rotational axis. The measured needle-positioning accuracy in free space was 54 +/- 12 microm for the pitch axis plane and 91 +/- 21 microm for the roll axis plane. The measured needle-positioning precision was 15 and 17 microm for the pitch and roll axes planes, respectively. The robots ability to insert a needle into a tumour in a euthanized mouse was demonstrated.

Deficiency of Capn4 Gene Inhibits Nuclear Factor-κB (NF-κB) Protein Signaling/Inflammation and Reduces Remodeling after Myocardial Infarction

Background: The causal role of calpain in myocardial remodeling after infarction has not been addressed. Results: Deficiency of Capn4 inhibited NF-κB signaling and inflammation and reduced myocardial remodeling, dysfunction, and mortality after infarction. Conclusion: Calpain contributes to myocardial inflammation and remodeling after infarction. Significance: This study provides a significant insight into the function of calpain in post-myocardial infarction remodeling. Calpain has been implicated in acute myocardial injury after myocardial infarction (MI). However, the causal relationship between calpain and post-MI myocardial remodeling has not been fully understood. This study examined whether deletion of Capn4, essential for calpain-1 and calpain-2 activities, reduces myocardial remodeling and dysfunction following MI, and if yes, whether these effects of Capn4 deletion are associated with NF-κB signaling and inflammatory responses in the MI heart. A novel mouse model with cardiomyocyte-specific deletion of Capn4 (Capn4-ko) was employed. MI was induced by left coronary artery ligation. Deficiency of Capn4 dramatically reduced the protein levels and activities of calpain-1 and calpain-2 in the Capn4-ko heart. In vivo cardiac function was relatively improved in Capn4-ko mice at 7 and 30 days after MI when compared with their wild-type littermates. Deletion of Capn4 reduced apoptosis, limited infarct expansion, prevented left ventricle dilation, and reduced mortality in Capn4-ko mice. Furthermore, cardiomyocyte cross-sectional areas and myocardial collagen deposition were significantly attenuated in Capn4-ko mice, which were accompanied by down-regulation of hypertrophic genes and profibrotic genes. These effects of Capn4 knock-out correlated with restoration of IκB protein and inhibition of NF-κB activation, leading to suppression of proinflammatory cytokine expression and inflammatory cell infiltration in the Capn4-ko heart after MI. In conclusion, deficiency of Capn4 reduces adverse myocardial remodeling and myocardial dysfunction after MI. These effects of Capn4 deletion may be mediated through prevention of IκB degradation and NF-κB activation, resulting in inhibition of inflammatory responses.

Detectability of Small Blood Vessels with High-Frequency Power Doppler and Selection of Wall Filter Cut-Off Velocity for Microvascular Imaging

Power Doppler imaging of physiologic and pathologic angiogenesis is widely used in preclinical studies to track normal development, disease progression and treatment efficacy but can be challenging given the presence of small blood vessels and slow flow velocities. Power Doppler images can be plagued with false-positive color pixels or undetected vessels, thereby complicating the interpretation of vascularity metrics such as color pixel density (CPD). As an initial step toward improved microvascular quantification, flow-phantom experiments were performed to establish relationships between vessel detection and various combinations of vessel size (160, 200, 250, 300 and 360 microm), flow velocity (4, 3, 2, 1 and 0.5 mm/s) and transducer frequency (30 and 40 MHz) while varying the wall filter cut-off velocity. Receiver operating characteristic (ROC) curves and areas under ROC curves indicate that good vessel detection performance can be achieved with a 40-MHz transducer for flow velocities > or =2 mm/s and with a 30-MHz transducer for flow velocities > or =1 mm/s. In the second part of the analysis, CPD was plotted as a function of wall filter cut-off velocity for each flow-phantom data set. Three distinct regions were observed: overestimation of CPD at low cut-offs, underestimation of CPD at high cut-offs and a plateau at intermediate cut-offs. The CPD at the plateau closely matched the phantoms vascular volume fraction and the length of the plateau corresponded with the flow-detection performance of the Doppler system assessed using ROC analysis. Color pixel density vs. wall filter cut-off curves from analogous in vivo experiments exhibited the same shape, including a distinct CPD plateau. The similar shape of the flow-phantom and in vivo curves suggests that the presence of a plateau in vivo can be used to identify the best-estimate CPD value that can be treated as a quantitative vascularity metric. The ability to identify the best CPD estimate is expected to improve quantification of angiogenesis and anti-vascular treatment responses with power Doppler.

Establishment of a Serum Tumor Marker for Preclinical Trials of Mouse Prostate Cancer Models

Current prostate cancer research in both basic and preclinical trial studies employ genetically engineered mouse models. However, unlike in human prostate cancer patients, rodents have no counterpart of prostatic-specific antigen (PSA) for monitoring prostate cancer initiation and progression. In this study, we established a mouse serum tumor marker from a mouse homologue of human prostate secretory protein of 94 amino acids (PSP94). Immunohistochemistry studies on different histologic grades from both transgenic and knock-in mouse prostate cancer models showed the down-regulation of tissue PSP94 expression (P < 0.001), the same as for PSA and PSP94 in humans. The presence of mouse serum PSP94 was shown by affinity column and immunoprecipitation purification using a polyclonal mouse PSP94 antibody. A competitive ELISA protocol was established to quantify serum PSP94 levels with a sensitivity of 1 ng/mL. Quantified serum levels of mouse PSP94 ranged from 49.84 ng/mL in wild-type mice to 113.86, 400.45, and 930.90 ng/mL in mouse prostatic intraepithelial neoplasia with microinvasion, well differentiated, moderately differentiated, and poorly differentiated prostate cancer genetically engineered prostate cancer mice, respectively (P < 0.01, n = 68). This increase in serum PSP94 is also well correlated with age and tumor weight. Through longitudinal monitoring of serum PSP94 levels of castrated mice (androgen ablation therapy), we found a correlation between responsiveness/refractory prostate tissues and serum PSP94 levels. The utility of mouse serum PSP94 as a marker in hormone therapy was further confirmed by three-dimensional ultrasound imaging. The establishment of the first rodent prostate cancer serum biomarker will greatly facilitate both basic and preclinical research on human prostate cancer.

Volume measurement variability in three-dimensional high-frequency ultrasound images of murine liver metastases

The identification and quantification of tumour volume measurement variability is imperative for proper study design of longitudinal non-invasive imaging of pre-clinical mouse models of cancer. Measurement variability will dictate the minimum detectable volume change, which in turn influences the scheduling of imaging sessions and the interpretation of observed changes in tumour volume. In this paper, variability is quantified for tumour volume measurements from 3D high-frequency ultrasound images of murine liver metastases. Experimental B16F1 liver metastases were analysed in different size ranges including less than 1 mm3, 1-4 mm3, 4-8 mm3 and 8-70 mm3. The intra- and inter-observer repeatability was high over a large range of tumour volumes, but the coefficients of variation (COV) varied over the volume ranges. The minimum and maximum intra-observer COV were 4% and 14% for the 1-4 mm3 and <1 mm3 tumours, respectively. For tumour volumes measured by segmenting parallel planes, the maximum inter-slice distance that maintained acceptable measurement variability increased from 100 to 600 microm as tumour volume increased. Comparison of free breathing versus ventilated animals demonstrated that respiratory motion did not significantly change the measured volume. These results enable design of more efficient imaging studies by using the measured variability to estimate the time required to observe a significant change in tumour volume.

Exercise training enhances insulin-stimulated nerve arterial vasodilation in rats with insulin-treated experimental diabetes

Insulin stimulates nerve arterial vasodilation through a nitric oxide (NO) synthase (NOS) mechanism. Experimental diabetes reduces vasa nervorum NO reactivity. Studies investigating hyperglycemia and nerve arterial vasodilation typically omit insulin treatment and use sedentary rats resulting in severe hyperglycemia. We tested the hypotheses that 1) insulin-treated experimental diabetes and inactivity (DS rats) will attenuate insulin-mediated nerve arterial vasodilation, and 2) deficits in vasodilation in DS rats will be overcome by concurrent exercise training (DX rats; 75-85% VO2 max, 1 h/day, 5 days/wk, for 10 wk). The baseline index of vascular conductance values (VCi = nerve blood flow velocity/mean arterial blood pressure) were similar (P ≥ 0.68), but peak VCi and the area under the curve (AUCi) for the VCi during a euglycemic hyperinsulinemic clamp (EHC; 10 mU·kg(-1)·min(-1)) were lower in DS rats versus control sedentary (CS) rats and DX rats (P ≤ 0.01). Motor nerve conduction velocity (MNCV) was lower in DS rats versus CS rats and DX rats (P ≤ 0.01). When compared with DS rats, DX rats expressed greater nerve endothelial NOS (eNOS) protein content (P = 0.04). In a separate analysis, we examined the impact of diabetes in exercise-trained rats alone. When compared with exercise-trained control rats (CX), DX rats had a lower AUCi during the EHC, lower MNCV values, and lower sciatic nerve eNOS protein content (P ≤ 0.03). Therefore, vasa nervorum and motor nerve function are impaired in DS rats. Such deficits in rats with diabetes can be overcome by concurrent exercise training. However, in exercise-trained rats (CX and DX groups), moderate hyperglycemia lowers vasa nervorum and nerve function.