The Performance of Flash Replenishment Contrast-Enhanced Ultrasound for the Qualitative Assessment of Kidney Lesions in Patients with Chronic Kidney Disease.

We investigated the accuracy of CEUS for characterizing cystic and solid kidney lesions in patients with chronic kidney disease (CKD). Cystic lesions are assessed using Bosniak criteria for computed tomography (CT) and magnetic resonance imaging (MRI); however, in patients with moderate to severe kidney disease, CT and MRI contrast agents may be contraindicated. Contrast-enhanced ultrasound (CEUS) is a safe alternative for characterizing these lesions, but data on its performance among CKD patients are limited. We performed flash replenishment CEUS in 60 CKD patients (73 lesions). Final analysis included 53 patients (63 lesions). Four readers, blinded to true diagnosis, interpreted each lesion. Reader evaluations were compared to true lesion classifications. Performance metrics were calculated to assess malignant and benign diagnoses. Reader agreement was evaluated using Bowker's symmetry test. Combined reader sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for diagnosing malignant lesions were 71%, 75%, 45%, and 90%, respectively. Sensitivity (81%) and specificity (83%) were highest in CKD IV/V patients when grouped by CKD stage. Combined reader sensitivity, specificity, PPV, and NPV for diagnosing benign lesions were 70%, 86%, 91%, and 61%, respectively. Again, in CKD IV/V patients, sensitivity (81%), specificity (95%), and PPV (98%) were highest. Inter-reader diagnostic agreement varied from 72% to 90%. In CKD patients, CEUS is a potential low-risk option for screening kidney lesions. CEUS may be particularly beneficial for CKD IV/V patients, where kidney preservation techniques are highly relevant.

Classifying Kidney Disease in a Vervet Model Using Spatially Encoded Contrast-Enhanced Ultrasound Perfusion Parameters.

Early stages of diabetic kidney disease (DKD) are difficult to diagnose in patients with type 2 diabetes. This work was aimed at identifying contrast-enhanced ultrasound (CEUS) perfusion parameters, a microcirculatory biomarker indicative of early DKD progression. CEUS kidney flash-replenishment data were acquired in control, insulin resistant and diabetic vervet monkeys (N = 16). By use of a mono-exponential model, time-intensity curve parameters related to blood volume (A), velocity (ß) and flow rate (perfusion index [PI]) were extracted from 10 concentric kidney layers to study spatial perfusion patterns that could serve as strong indicators of disease. Mean squared error (MSE) was used to assess model performance. Features calculated from the perfusion parameters were inputs for the linear regression models to determine which features could distinguish between cohorts. The mono-exponential model performed well, with average MSEs (±standard deviation) of 0.0254 (±0.0210), 0.0321 (±0.0242) and 0.0287 (±0.0130) for the control, insulin resistant and diabetic cohorts, respectively. Perfusion index features, with blood pressure, were the best classifiers between cohorts (p < 0.05). CEUS has the potential to detect early microvascular changes, providing insight into disease-related structural changes in the kidney. The sensitivity of this technique should be explored further by assessing various stages of DKD.

Association of Contrast-Enhanced Ultrasound-Derived Kidney Cortical Microvascular Perfusion with Kidney Function.

Background: Individuals with chronic kidney disease (CKD) have decreased kidney cortical microvascular perfusion, which may lead to worsening kidney function over time, but methods to quantify kidney cortical microvascular perfusion are not feasible to incorporate into clinical practice. Contrast-enhanced ultrasound (CEUS) may quantify kidney cortical microvascular perfusion, which requires further investigation in individuals across the spectrum of kidney function. Methods: We performed CEUS on a native kidney of 83 individuals across the spectrum of kidney function and calculated quantitative CEUS-derived kidney cortical microvascular perfusion biomarkers. Participants had a continuous infusion of the microbubble contrast agent (Definity) with a flash-replenishment sequence during their CEUS scan. Lower values of the microbubble velocity (ß) and perfusion index (ß×A) may represent lower kidney cortical microvascular perfusion. Multivariable linear regression models tested the associations of the microbubble velocity (ß) and perfusion index (ß×A) with estimated glomerular filtration rate (eGFR). Results: Thirty-eight individuals with CKD (mean age±SD 65.2±12.6 years, median [IQR] eGFR 31.5 [18.9-41.5] ml/min per 1.73 m2), 37 individuals with end stage kidney disease (ESKD; age 54.8±12.3 years), and eight healthy volunteers (age 44.1±15.0 years, eGFR 117 [106-120] ml/min per 1.73 m2) underwent CEUS without side effects. Individuals with ESKD had the lowest microbubble velocity (ß) and perfusion index (ß×A) compared with individuals with CKD and healthy volunteers. The microbubble velocity (ß) and perfusion index (ß×A) had moderate positive correlations with eGFR (ß: rs=0.44, P<0.001; ß×A: rs=0.50, P<0.001). After multivariable adjustment, microbubble velocity (ß) and perfusion index (ß×A) remained significantly associated with eGFR (change in natural log transformed eGFR per 1 unit increase in natural log transformed biomarker: ß, 0.38 [95%, CI 0.17 to 0.59]; ß×A, 0.79 [95% CI, 0.45 to 1.13]). Conclusions: CEUS-derived kidney cortical microvascular perfusion biomarkers are associated with eGFR. Future studies are needed to determine if CEUS-derived kidney cortical microvascular perfusion biomarkers have prognostic value.

Development of a Robotic Shear Wave Elastography System for Noninvasive Staging of Liver Disease in Murine Models.

Shear wave elastography (SWE) is an ultrasound-based stiffness quantification technology that is used for noninvasive liver fibrosis assessment. However, despite widescale clinical adoption, SWE is largely unused by preclinical researchers and drug developers for studies of liver disease progression in small animal models due to significant experimental, technical, and reproducibility challenges. Therefore, the aim of this work was to develop a tool designed specifically for assessing liver stiffness and echogenicity in small animals to better enable longitudinal preclinical studies. A high-frequency linear array transducer (12-24 MHz) was integrated into a robotic small animal ultrasound system (Vega; SonoVol, Inc., Durham, NC) to perform liver stiffness and echogenicity measurements in three dimensions. The instrument was validated with tissue-mimicking phantoms and a mouse model of nonalcoholic steatohepatitis. Female C57BL/6J mice (n = 40) were placed on choline-deficient, L-amino acid-defined, high-fat diet and imaged longitudinally for 15 weeks. A subset was sacrificed after each imaging timepoint (n = 5) for histological validation, and analyses of receiver operating characteristic (ROC) curves were performed. Results demonstrated that robotic measurements of echogenicity and stiffness were most strongly correlated with macrovesicular steatosis (R2  = 0.891) and fibrosis (R2  = 0.839), respectively. For diagnostic classification of fibrosis (Ishak score), areas under ROC (AUROCs) curves were 0.969 for ≥Ishak1, 0.984 for ≥Ishak2, 0.980 for ≥Ishak3, and 0.969 for ≥Ishak4. For classification of macrovesicular steatosis (S-score), AUROCs were 1.00 for ≥S2 and 0.997 for ≥S3. Average scanning and analysis time was <5 minutes/liver. Conclusion: Robotic SWE in small animals is feasible and sensitive to small changes in liver disease state, facilitating in vivo staging of rodent liver disease with minimal sonographic expertise.

Amphiphilic phospholipid-iodinated polymer conjugates for bioimaging.

Amphiphilic phospholipid-iodinated polymer conjugates were designed and synthesized as new macromolecular probes for a highly radiopaque and biocompatible imaging technology. Bioconjugation of PEG 2000-phospholipids and iodinated polyesters by click chemistry created amphiphilic moieties with hydrophobic polyesters and hydrophilic PEG units, which allowed their self-assemblies into vesicles or spiked vesicles. More importantly, the conjugates exhibited high radiopacity and biocompatibility in in vitro X-ray and cell viability measurements. This new type of bioimaging contrast agent with a Mn value of 11 289 g mol-1 was found to have a significant X-ray signal at 3.13 mg mL-1 of iodine equivalent than baseline and no cytotoxicity after 48 hours incubation of with HEK and 3T3 cells at 20 µM (20 picomoles) concentration of conjugates per well. The potential of adopting the described macromolecular probes for bioimaging was demonstrated, which could further promote the development of a field-friendly and highly sensitive bioimaging contrast agent for point-of-care diagnostic applications.

In Vivo Assessment of the Potential for Renal Bio-Effects from the Vaporization of Perfluorocarbon Phase-Change Contrast Agents.

Low-boiling-point perfluorocarbon phase-change contrast agents (PCCAs) provide an alternative to microbubble contrast agents. Although parameter ranges related to in vivo bio-effects of microbubbles are fairly well characterized, few studies have been done to evaluate the potential of bio-effects related to PCCAs. To bridge this gap, we present an assessment of biological effects (e.g., hemorrhage) related to acoustically excited PCCAs in the rodent kidney. The presence or absence of bio-effects was observed after sonication with various perfluorocarbon core PCCAs (decafluorobutane, octafluoropropane or a 1:1 mixture) and as a function of activation pulse mechanical index (MI; minimum activation threshold, which was a moderate MI of 0.81-1.35 vs. a clinical maximum of 1.9). Bio-effects on renal tissue were assessed through hematology and histology including measurement of blood creatinine levels and the quantity of red blood cell (RBC) casts present in hematoxylin and eosin-stained kidney tissue sections after sonication. Short-term (24 h) and long-term (2 and 4 wk) analyses were performed after treatment. Results indicated that bio-effects from PCCA vaporization were not observed at lower mechanical indices. At higher mechanical indices, bio-effects were observed at 24 h, although these were not observable 2 wk after treatment.

Experimental verification of theoretical equations for acoustic radiation force on compressible spherical particles in traveling waves.

Acoustophoresis uses acoustic radiation force to remotely manipulate particles suspended in a host fluid for many scientific, technological, and medical applications, such as acoustic levitation, acoustic coagulation, contrast ultrasound imaging, ultrasound-assisted drug delivery, etc. To estimate the magnitude of acoustic radiation forces, equations derived for an inviscid host fluid are commonly used. However, there are theoretical predictions that, in the case of a traveling wave, viscous effects can dramatically change the magnitude of acoustic radiation forces, which make the equations obtained for an inviscid host fluid invalid for proper estimation of acoustic radiation forces. To date, experimental verification of these predictions has not been published. Experimental measurements of viscous effects on acoustic radiation forces in a traveling wave were conducted using a confocal optical and acoustic system and values were compared with available theories. Our results show that, even in a low-viscosity fluid such as water, the magnitude of acoustic radiation forces is increased manyfold by viscous effects in comparison with what follows from the equations derived for an inviscid fluid.

Nucleation and growth synthesis of siloxane gels to form functional, monodisperse, and acoustically programmable particles.

Nucleation and growth methods offer scalable means of synthesizing colloidal particles with precisely specified size for applications in chemical research, industry, and medicine. These methods have been used to prepare a class of silicone gel particles that display a range of programmable properties and narrow size distributions. The acoustic contrast factor of these particles in water is estimated and can be tuned such that the particles undergo acoustophoresis to either the pressure nodes or antinodes of acoustic standing waves. These particles can be synthesized to display surface functional groups that can be covalently modified for a range of bioanalytical and acoustophoretic sorting applications.

A pilot study to assess markers of renal damage in the rodent kidney after exposure to 7 MHz ultrasound pulse sequences designed to cause microbubble translation and disruption.

Acoustic radiation force has been proposed as a mechanism to enhance microbubble concentration for therapeutic and molecular imaging applications. It is hypothesized that once microbubbles are localized, bursting them with acoustic pressure could result in local drug delivery. It is known that low-frequency, high-amplitude acoustic energy combined with cavitation nuclei can result in bioeffects. However, little is known about the bioeffects potential of acoustic parameters involved in radiation force and microbubble destruction pulse sequences applied at higher frequencies. In this pilot study, rat kidneys are exposed to high-duty cycle, low-amplitude pulse sequences known to cause substantial bubble translation due to radiation force, as well as high-amplitude short pulse sequences known to cause microbubble destruction. Both studies are performed at 7 MHz on a clinical ultrasound system, and implemented in three-dimensions (3-D) for entire kidney exposure. Analysis of biomarkers of renal injury and renal histopathology indicate that there was no significant renal damage due to these ultrasound parameters in conjunction with microbubbles within the study group.

Validation of dynamic contrast-enhanced ultrasound in rodent kidneys as an absolute quantitative method for measuring blood perfusion.

Contrast-enhanced ultrasound (CEUS) has demonstrated utility in the monitoring of blood flow in tissues, organs and tumors. However, current CEUS methods typically provide only relative image-derived measurements, rather than quantitative values of blood flow in milliliters/minute per gram of tissue. In this study, CEUS derived parameters of blood flow are compared with absolute measurements of blood flow in rodent kidneys. Additionally, the effects of contrast agent infusion rate and transducer orientation on image-derived perfusion measurements are assessed. Both wash-in curve and time-to-refill algorithms are examined. Data illustrate that for all conditions, image-derived flow measurements were well-correlated with transit-time flow probe measurements (R > 0.9). However, we report differences in the sensitivity to flow across different transducer orientations as well as the contrast analysis algorithm utilized. Results also indicate that there exists a range of contrast agent flow rates for which image-derived estimates are consistent.

Introduction to rodent cardiac imaging.

Imaging is a noninvasive complement to traditional methods (such as histology) in rodent cardiac studies. Assessments of structure and function are possible with ultrasound, microcomputed tomography (microCT), and magnetic resonance (MR) imaging. Cardiac imaging in the rodent poses a challenge because of the size of the animal and its rapid heart rate. Each aspect in the process of rodent cardiac imaging-animal preparation, choice of anesthetic, selection of gating method, image acquisition, and image interpretation and measurement-requires careful consideration to optimize image quality and to ensure accurate and reproducible data collection. Factors in animal preparation that can affect cardiac imaging are the choice of anesthesia regime (injected or inhaled), intubated or free-breathing animals, physiological monitoring (ECG, respiration, and temperature), and animal restraint. Each will vary depending on the method of imaging and the length of the study. Gating strategies, prospective or retrospective, reduce physiological motion artifacts and isolate specific time points in the cardiac cycle (i.e., end-diastole and end-systole) where measurements are taken. This article includes a simple explanation of the physics of ultrasound, microCT, and MR to describe how images are generated. Subsequent sections provide reviews of animal preparation, image acquisition, and measurement techniques in each modality specific to assessing cardiac functions such as ejection fraction, fractional shortening, stroke volume, cardiac output, and left ventricular mass. The discussion also includes the advantages and disadvantages of the different imaging modalities. With the use of ultrasound, microCT, and MR, it is possible to create 2-, 3-, and 4-dimensional views to characterize the structure and function of the rodent heart.

Hepatic transcript levels for genes coding for enzymes associated with xenobiotic metabolism are altered with age.

Metabolism studies are crucial for data interpretation from rodent toxicity and carcinogenicity studies. Metabolism studies are usually conducted in 6 to 8 week old rodents. Long-term studies often continue beyond 100 weeks of age. The potential for age-related changes in transcript levels of genes encoding for enzymes associated with metabolism was evaluated in the liver of male F344/N rats at 32, 58, and 84 weeks of age. Differential expression was found between the young and old rats for genes whose products are involved in both phase I and phase II metabolic pathways. Thirteen cytochrome P450 genes from CYP families 1-3 showed alterations in expression in the older rats. A marked age-related decrease in expression was found for 4 members of the Cyp3a family that are critical for drug metabolism in the rat. Immunohistochemical results confirmed a significant decrease in Cyp3a2 and Cyp2c11 protein levels with age. This indicates that the metabolic capacity of male rats changes throughout a long-term study. Conducting multiple hepatic microarray analyses during the conduct of a long-term study can provide a global view of potential metabolic changes that might occur. Alterations that are considered crucial to the interpretation of long-term study results could then be confirmed by subsequent metabolic studies.

Imaging techniques for small animal imaging models of pulmonary disease: micro-CT.

Microcomputed tomography (micro-CT) is ideal for quantifying pulmonary disease because of the inherent contrast between tissue and air that exists in the lungs. Both in vivo and in vitro studies can be performed using micro-CT. Live animal studies show function, while fixed specimen studies show structure. Through the use of image processing techniques, both acute and chronic lung diseases can be quantified. The information provided by micro-CT is complementary to histological evaluation, since CT is nondestructive. This paper discusses two examples, in vivo and in vitro, of how micro-CT can be used to assess pulmonary diseases in small animal models. With the use of micro-CT, we were able to quantify pulmonary fibrosis in the live rat and investigate the microstructure of the airway in fixed mouse lungs.

Application of magnetic resonance imaging in developmental neurotoxicity testing: a pilot study.

In a pilot developmental neurotoxicity study, a protocol was designed to utilize three-dimensional magnetic resonance (MR) images for linear and volumetric measurements of the developing rat brain. MR imaging, because of its non-destructive nature, provides a complement to traditional optical microscopy. Sprague-Dawley dams received 0, 1.25, 4.0 or 7.5mg/kg methylazoxymethanol acetate (MAM) by intraperitoneal injection during gestation days 13-15. At postnatal days (PND) 23 and 60, brains from representative male and female rats from two dams in each dose group were fixed with 10% neutral buffered formalin by transcardial perfusion for in situ MR imaging. A 7T small animal magnet system was used to obtain isotropic images at 100 microm resolution for PND 23 and 150 microm resolution for PND 60. Data from a rapid screening method based on midpoint MR slices of whole brain, cerebrum, cerebellum, and hippocampus showed a dose-related decreased volume of whole brain, cerebrum, and hippocampus in treated rats. Subsequent volumetric estimates using the Cavalieri method confirmed these findings. The brains were subsequently removed and processed for conventional histologic examination of hematoxylin and eosin-stained sections. It is concluded that MR imaging in rat developmental neurotoxicity studies offers the advantages of in situ volumetric measurements of brain structures while preserving the samples for conventional optical microscopy.

2-Butoxyethanol female-rat model of hemolysis and disseminated thrombosis: X-ray characterization of osteonecrosis and growth-plate suppression.

We recently proposed a chemically induced rat model for human hemolytic disorders associated with thrombosis. The objective of the present investigation was to apply a noninvasive, high-magnification X-ray analysis, the Faxitron radiography system, to characterize the protracted bone damage associated with this 2-butoxyethanol model and to validate it by histopathology. Groups of female Fischer 344 rats were given 0, 250, or 300 mg of 2-butoxyethanol/kg body weight daily for 4 consecutive days. Groups were then sacrificed 2 hours or 26 days after the final treatment. The treated animals displayed a darkened purple-red discoloration on the distal tail. Histopathological evaluation, including phosphotungstic acid-hematoxylin staining of animals sacrificed 2 hours after the final treatment, revealed disseminated thrombosis and infarction in multiple organs, including bones. The Faxitron MX-20 specimen radiography system was used to image selected bones of rats sacrificed 26 days posttreatment. Premature thinning of the growth plate occurred in the calcaneus, lumbar and coccygeal vertebrae, femur, and ilium of the treated animals. Areas of decreased radiographic densities were seen in the diaphysis of the femur of all treated animals. The bones were then examined histologically and showed a range of changes, including loss or damage to growth plates and necrosis of cortical bone. No thrombi were seen in the animals sacrificed at 30 days, but bone and growth plate changes consistent with prior ischemia were noted. The Faxitron proved to be an excellent noninvasive tool that can be used in future studies with this animal model to examine treatment modalities for the chronic effects of human thrombotic disorders.

New insights into functional aspects of liver morphology.

The liver is structurally and functionally complex and has been considered second only to brain in its complexity. Many mysteries still exist in this heterogeneous tissue whose functional unit of the lobule has continued to stump morphologists for over 300 years. The primary lobule, proposed by Matsumoto in 1979, has been gaining acceptance as the functional unit of the liver over other conceptual views because it's based on vessel architecture and includes the classic lobule as a secondary feature. Although hepatocytes comprise almost 80% of the liver, there are at least another dozen cell types, many of which provide "cross-talk" and play important functional roles in the normal and diseased liver. The distribution and functional roles of all cells in the liver must be carefully considered in both the analysis and interpretation of research data, particularly data in the area of genomics and "phenotypic anchoring" of gene expression results. Discoveries regarding the functional heterogeneity of the various liver cell types, including hepatocytes, hepatic stellate cells, sinusoidal endothelia, and Kupffer cells, are providing new insights into our understanding of the development, prevention and treatment of liver disease. For example, functional differences along zonal patterns (centrilobular or periportal) have been demonstrated for sinusoidal endothelium, Kupffer cells, and hepatocytes and can explain the gradients and manifestations of disease observed within lobules. Intralobular gradients of bile uptake, glycogen depletion, glutamine synthetase, and carboxylesterase by hepatocytes; widened fenestrations in centrilobular sinusoidal lining cells; and differences in the components of centrilobular extracellular matrix or function of Kupffer cells have been demonstrated. Awareness of the complexities and heterogeneity of the liver will add to a greater understanding of liver function and disease processes that lead to toxicity, cancer, and other diseases.