Ultrahigh-Frequency Echocardiography of Autonomic Devoid Phox2B Homozygous Embryos Does Not Reveal a Significant Cardiac Phenotype before Embryo Death.

In vivo micro-imaging of mice is useful in studying the genetic basis of cardiac development in mutant embryos. We examined Phox2b-/- mutant mice, which lack autonomic innervation to the heart and die in utero, and investigated whether this lack of innervation causes cardiac dysfunction during embryogenesis. A VisualSonics Vevo 2100 ultrahigh-frequency linear array ultrasound machine with 30- and 40-MHz probes was used to analyze embryo size, gross characteristics, ventricular contractility and rhythm. Phox2b-/- mutant embryos underwent cessation of heartbeat and death at a greater rate than wild-type controls. We did not observe a hydrops phenotype or congenital heart defects in Phox2b-/- mutants. Analysis of heart rhythm revealed no significant correlation with genotype. Absent these signs of a progressive pathology, we suggest that Phox2b-/- mutant embryos likely die of sudden death secondary to acute arrhythmia. These data provide insight into the role of cardiac autonomic innervation during development.

Amphotericin B Penetrates into the Central Nervous System Through Focal Disruption of the Blood Brain Barrier in Experimental Hematogenous Candida Meningoencephalitis.

Hematogenous Candida meningoencephalitis (HCME) is a life-threatening complication of neonates and immunocompromised children. Amphotericin B (AmB) shows poor permeability and low cerebrospinal fluid (CSF) concentrations, but is effective in treatment of HCME. In order to better understand the mechanism of CNS penetration of AmB, we hypothesized that AmB may achieve focally higher concentrations in infected CNS lesions. An in vitro BBB model was serially infected with C. albicans. Liposomal AmB (LAMB) or deoxycholate AmB (DAMB) at 5 µg/ml were then provided, vascular and CNS compartments were sampled 4h later. For in vivo correlation, rabbits with experimental HCME received a single dose of DAMB 1 mg/kg or LAMB 5 mg/kg, and were euthanized after 1, 3, 6 and 24h. Evans blue solution (2%) 2 ml/kg administered IV one hour prior to euthanasia stained infected regions of tissue but not histologically normal areas. AmB concentrations in stained and unstained tissue regions were measured using UPLC. For selected rabbits, MRI scans performed on days 1-7 postinoculation were acquired before and after IV bolus Gd-DTPA at 15min intervals through 2h post-injection. The greatest degree of penetration of DAMB and LAMB through the in vitro BBB occurred after 24h of exposure (P=0.0022). In vivo the concentrations of LAMB and DAMB in brain abscesses were 4.35±0.59 and 3.14±0.89-times higher vs. normal tissue (P≤0.019). MRI scans demonstrated that Gd-DTPA accumulated in infected areas with disrupted BBB. Localized BBB disruption in HCME allows high concentrations of AmB within infected tissues, despite the presence of low CSF concentrations.

Comparing the Effects of Isoflurane and Alpha Chloralose upon Mouse Physiology.

Functional magnetic resonance imaging of mice requires that the physiology of the mouse (body temperature, respiration and heart rates, blood pH level) be maintained in order to prevent changes affecting the outcomes of functional scanning, namely blood oxygenation level dependent (BOLD) measures and cerebral blood flow (CBF). The anesthetic used to sedate mice for scanning can have major effects on physiology. While alpha chloralose has been commonly used for functional imaging of rats, its effects on physiology are not well characterized in the literature for any species. In this study, we anesthetized or sedated mice with isoflurane or alpha chloralose for up to two hours, and monitored physiological parameters and arterial blood gasses. We found that, when normal body temperature is maintained, breathing rates for both drugs decrease over the course of two hours. In addition, alpha chloralose causes a substantial drop in heart rate and blood pH with severe hypercapnia (elevated blood CO2) that is not seen in isoflurane-treated animals. We suggest that alpha chloralose does not maintain normal mouse physiology adequately for functional brain imaging outcome measures.

Postoperative hyperoxia (60%) worsens hepatic injury in mice.

BACKGROUND: Liver damage by ischemia and reperfusion injury is a risk factor for morbidity and mortality after liver surgery. Postoperative oxygen treatment is routinely applied in the postanesthesia and intensive care unit after liver surgery. The risks of aggravating the injury by increasing inspiratory oxygen from 21 to 60% in the postoperative period were investigated in mice. METHODS: Parameters of liver injury were compared after induction of hepatic ischemia-reperfusion injury, by clamping the left liver lobe for 45 min, and reperfusion for 24 h either under normoxic (21% oxygen) or hyperoxic (60% oxygen) conditions (n=22 per group). The extent of tissue injury and oxidative responses was analyzed in the presence or absence of polymorphonuclear leukocytes, functional Kupffer cells, and the p47phox unit of the nicotinamide adenine dinucleotide phosphate oxidase (n=6 to 11 per group). RESULTS: Compared with postoperative normoxic conditions, hyperoxia increased cell damage (glutamate-pyruvate transaminase: 1,870 [±968 SD] vs. 60% 2,981 [±1,038 SD], 21 vs. 60% oxygen, in U/l as mean±SD; P<0.01), liver weights (341±52 vs. 383±44, 21 vs. 60% oxygen, in mg as mean±SD; P=0.02), damage scores (1.9±0.8 vs. 3.1±1.0, 21 vs. 60% oxygen, score as mean±SD; P=0.02), and reactive oxygen species (15.0±12.0 vs. 30.4±19.2, 21 vs. 60% oxygen, in µmol/l as mean±SD; P<0.05). The aggravation of the tissue damaging effects as a result of hyperoxia was not seen in mice with depletions of polymorphonuclear leukocytes or Kupffer cells, or with nonfunctioning nicotinamide adenine dinucleotide phosphate oxidase. CONCLUSION: Liver injury after ischemia was significantly aggravated by hyperoxia as a consequence of immune cell-mediated oxidative burst. Further studies are needed to elucidate whether routine delivery of high inspirational oxygen concentrations postoperatively should be limited.

In vivo hypoxic preconditioning protects from warm liver ischemia-reperfusion injury through the adenosine A2B receptor.

BACKGROUND: Liver ischemia-reperfusion injury (IRI) is a known risk factor for the postoperative outcome of patients undergoing liver surgery/transplantation. Attempts to protect from organ damage require multidisciplinary strategies and are of emerging interest in view of patients with higher age and American Society of Anesthesiology status. Ischemic preconditioning has been successfully applied to prevent from IRI during liver resection/transplantation. Because even short periods of ischemia during preconditioning inevitably lead to hypoxia and formation of anti-inflammatory/cytoprotective acting adenosine, we reasoned that short nonischemic hypoxia also protects against hepatic IRI. METHODS: Mice underwent hypoxic preconditioning (HPC) by breathing 10% oxygen for 10 min followed by 10 min of 21% oxygen before left liver lobe ischemia (45 min) and reperfusion (4 hr). The interactions of hypoxia→adenosine→adenosine receptors were tested by pharmacologic antagonism at adenosine receptor (AR) sites in wild-type mice and in mice with genetic deletions at the A1, A2A, A2B, and A3 ARs. Hepatocellular damage, inflammation, and metabolic effects were quantified by enzyme activities, cytokines, liver myeloperoxidase, blood adenosine, and tissue AMP, respectively. RESULTS: Hepatoprotection by HPC was significant in wild-type and A1, A2A, and A3 AR knockout mice as quantified by lower alanine aminotransferase serum activities, cytokine levels, histologic damage scores, tissue myeloperoxidase concentrations, and preserved AMP concentrations. Protection by HPC was blunted in mice pretreated with the A2B AR antagonist MRS1754 or in A2B AR knockout mice. CONCLUSIONS: Because liver protective effects of HPC are negated when the A2B receptor is nonfunctional, the hypoxia→adenosine→A2B receptor pathway plays a critical role in the prevention of warm IRI in vivo. Hypoxic activation of this pathway warrants use of selective A2B AR agonists or even intermittent hypoxia (e.g., in deceased organ donors) to protect from liver IRI.

Rat model of metastatic breast cancer monitored by MRI at 3 tesla and bioluminescence imaging with histological correlation.

BACKGROUND: Establishing a large rodent model of brain metastasis that can be monitored using clinically relevant magnetic resonance imaging (MRI) techniques is challenging. Non-invasive imaging of brain metastasis in mice usually requires high field strength MR units and long imaging acquisition times. Using the brain seeking MDA-MB-231BR transfected with luciferase gene, a metastatic breast cancer brain tumor model was investigated in the nude rat. Serial MRI and bioluminescence imaging (BLI) was performed and findings were correlated with histology. Results demonstrated the utility of multimodality imaging in identifying unexpected sights of metastasis and monitoring the progression of disease in the nude rat. METHODS: Brain seeking breast cancer cells MDA-MB-231BR transfected with firefly luciferase (231BRL) were labeled with ferumoxides-protamine sulfate (FEPro) and 1-3 x 106 cells were intracardiac (IC) injected. MRI and BLI were performed up to 4 weeks to monitor the early breast cancer cell infiltration into the brain and formation of metastases. Rats were euthanized at different time points and the imaging findings were correlated with histological analysis to validate the presence of metastases in tissues. RESULTS: Early metastasis of the FEPro labeled 231BRL were demonstrated on T2*-weighted MRI and BLI within 1 week post IC injection of cells. Micro-metastatic tumors were detected in the brain on T2-weighted MRI as early as 2 weeks post-injection in greater than 85% of rats. Unexpected skeletal metastases from the 231BRL cells were demonstrated and validated by multimodal imaging. Brain metastases were clearly visible on T2 weighted MRI by 3-4 weeks post infusion of 231BRL cells, however BLI did not demonstrate photon flux activity originating from the brain in all animals due to scattering of the photons from tumors. CONCLUSION: A model of metastatic breast cancer in the nude rat was successfully developed and evaluated using multimodal imaging including MRI and BLI providing the ability to study the temporal and spatial distribution of metastases in the brain and skeleton.

Considerations for laboratory animal imaging center design and setup.

In vivo animal imaging is an outstanding noninvasive tool to study the pathophysiology of disease or response to therapy; additionally, serial imaging reduces the required number of experimental animals. Because of the tremendous capital investment, we recommend the imaging center be a shared resource to facilitate innovative and productive cross-disciplinary scientific collaborations. A shared center also enables a broader range of imaging, as equipment is often cost prohibitive for smaller facilities. A multitude of factors will determine the architectural design, facility efficiency, and functionality. Important considerations to determine during the planning stages include the types of animals to be imaged, types of imaging studies to be performed, types of imaging equipment and related services to be offered, and the location of the imaging center. Architects must work closely with manufacturers to accommodate equipment-related building specifications; facility planners and veterinarians can provide a practical logistical design that will ensure efficient functionality. Miscellaneous considerations include biosecurity levels, use of radioisotopes, and personnel safety in the imaging environment. The ideal imaging center will include space to house animals and perform necessary preimaging procedures, state-of-the-art in vivo imaging devices and the most up-to-date anesthesia, physiological support, and monitoring equipment. The center staff should include imaging specialists for technical development and data analysis. As it is difficult to provide a comprehensive manual for setting up an in vivo animal imaging center, we offer advice based on our experiences with the National Institutes of Health Mouse Imaging Facility. Because magnetic resonance imaging (MRI) is the most expensive imaging tool, requires specific building design considerations, and poses unique occupational health and safety risks, we focus on MRI as the foundation for an imaging facility design.

Comparison of Fenestra VC Contrast-enhanced computed tomography imaging with gadopentetate dimeglumine and ferucarbotran magnetic resonance imaging for the in vivo evaluation of murine liver damage after ischemia and reperfusion.

OBJECTIVES: Comparison of intravenous Fenestra VC-enhanced computed tomography (CT) with gadopentetate dimeglumine and Ferucarbotran contrast-enhanced magnetic resonance imaging (MRI) for the in vivo imaging of hepatic ischemia/reperfusion injury (IRI) in a murine model. MATERIAL AND METHODS: After induction of hepatic IRI by left liver lobe (LLL) ischemia (30, 45, and 75 minutes) and reperfusion (4 hours and 24 hours), a total of 130 mice were imaged either by Fenestra VC-enhanced 3-D CT or by dynamic, T1-weighed gadopentetate dimeglumine or static, T2*-weighed Ferucarbotran 2-D MRI (4.7 T). RESULTS: Detection of liver tissue damage as a consequence of IRI was not possible by CT or MRI without the use of contrast media. (1) Mice subjected to liver IRI (45 minutes of ischemia) and injected with Fenestra VC showed a distinct liver enhancement of the viable liver tissue or a nonenhancement of the necrotic tissue. The Fenestra VC CT-unenhanced liver volume increased as a function of time of ischemia and reperfusion. The unenhanced liver volume also correlated positively with serum liver enzyme activities and damage scores from liver histology. (2) The signal intensities (SI) between normal liver tissue and livers subjected to 30 minutes of ischemia were not different on dynamic gadopentetate dimeglumine-enhanced magnetic resonance images. More severe IRI as induced by 45 or 75 minutes of ischemia was characterized by (a) early hyperenhancement of regions in the LLL with rapid increase of SI higher than that observed in the undamaged liver within the first few minutes and (b) delayed hyperenhancement in the later course after gadopentetate dimeglumine injection, respectively. (3) Ferucarbotran MRI detected signs of IRI after only 30 minutes of liver ischemia and hence detected IRI earlier than Fenestra VC or gadopentetate dimeglumine. With longer duration of ischemia, Ferucarbotran SI increased in the LLL, but viable and necrotic tissues were not clearly distinguishable. CONCLUSIONS: MicroCT with Fenestra VC enhancement and MRI using either gadopentetate dimeglumine or Ferucarbotran enhancement of the liver revealed that all techniques allow in vivo determination of hepatic IRI as a function of the duration of ischemia and reperfusion of the liver. However, Fenestra VC-enhanced CT of the murine liver is superior to gadopentetate dimeglumine and Ferucarbotran for localization, quantification, and differentiation of viable from metabolically inactive/damaged liver tissue after hepatic ischemia/reperfusion but Fenestra VC is less sensitive than Ferucarbotran to detect the early onset of subtle consequences of hepatic IRI.

Anatomical and functional imaging of tumors in animal models: focus on pheochromocytoma.

This review focuses on anatomical (computed tomography, magnetic resonance imaging) and functional (positron emission tomography) imaging methods for tumor localization and identification of experimentally induced tumors in animal models, especially pheochromocytoma. Although anatomical imaging can precisely locate primary and metastatic tumors, functional imaging has high specificity for some tumors, especially those of endocrine origin. This is due to the fact that endocrine tumor cells take up hormone precursors, express hormone receptors and transporters, and synthesize, store, and release hormones. These characteristic properties of endocrine tumors enable investigators to create highly specific radiopharmaceuticals, particularly for positron emission tomography. For example, localization of pheochromocytoma involves [18F]-6F-dopamine. It is a highly specific radiopharmaceutical since it uses the norepinephrine transporter system expressed in most pheochromocytoma cells. Here we review both anatomical and functional imaging methods that are used conjointly in order to localize and identify specific characteristics of tumors.

MicroCT for high-resolution imaging of ectopic pheochromocytoma tumors in the liver of nude mice.

Successful outcomes for patients with cancer often depend on the early detection of tumor and the prompt initiation of active therapy. Despite major advances in the treatment of many cancers, early-stage lesions often go undetected due to the suboptimal resolution of current anatomical and functional imaging modalities. This limitation also applies to preclinical animal tumor models that are crucial for the evaluation and development of new therapeutic approaches to cancer. We report a new mouse model of metastatic pheochromocytoma, generated using tail vein injection of the mouse pheochromocytoma cell (MPC) line that reproducibly generated multiple liver tumors in the animals. Furthermore, we show that in vivo microCT imaging enhanced using a hepatobiliary-specific contrast agent, glyceryl-2-oleyl-1,3-di-7-(3-amino-2,4,6-triiodophenyl)-heptanoate (DHOG), detected tumors as small as 0.35 mm as early as 4 weeks after the injection of the tumor cells. This model may be useful for in vivo studies of tumor biology and for development of new strategies to treat metastatic pheochromocytoma.

Euthanasia of mouse fetuses and neonates.

We sought to determine whether any of the common methods of euthanasia for adult rodents would lead to an acceptable death for fetuses or neonates. We wanted to identify a method that was rapid, free of signs of pain or distress, reliable, and minimally distressful to the person performing the procedure and that minimized the amount of handling required to perform the procedure. We evaluated six methods of euthanasia, with and without anesthesia, in three age groups of mice: gravid mice (E14-20) and neonatal pups (P1-P7 and P8-P14). Euthanasia methods included: halothane inhalation, carbon dioxide inhalation, intraperitoneal sodium pentobarbital, intravenous potassium chloride, and cervical dislocation with and without anesthesia. Noninvasive echocardiography was used to assess heartbeat during euthanasia. With cardiac arrest as the definition of death, no method of euthanasia killed fetal mice. Halothane inhalation (5% by vaporizer) was not an acceptable method of euthanasia for mice of the age groups tested. Intraperitoneal administration of sodium pentobarbital for euthanasia required a higher dose than the previously established dose, and there is a risk of reduced efficacy in pregnant animals due to potential intrauterine injection. Carbon dioxide asphyxiation was the most efficient method of euthanasia for neonatal mouse pups P1-14. For pregnant adult mice, intravenous potassium chloride under anesthesia, carbon dioxide asphyxiation, and cervical dislocation alone or under anesthesia were excellent methods of euthanasia.

Considerations for setting up a small-animal imaging facility.

Imaging techniques allow for the conduct of noninvasive, in vivo longitudinal small-animal studies, but also require access to expensive and complex equipment, and personnel who are properly trained in their use. The authors describe their planning and staffing of the NIH Mouse Imaging Facility, and highlight important issues to consider when designing a similar facility.

In vivo bioluminescence imaging.

In vivo bioluminescent imaging (BLI) is a versatile and sensitive tool that is based on detection of light emission from cells or tissues. Bioluminescence, the biochemical generation of light by a living organism, is a naturally occurring phenomenon. Luciferase enzymes, such as that from the North American firefly (Photinus pyralis), catalyze the oxidation of a substrate (luciferin), and photons of light are a product of the reaction. Optical imaging by bioluminescence allows a low-cost, noninvasive, and real-time analysis of disease processes at the molecular level in living organisms. Bioluminescence has been used to track tumor cells, bacterial and viral infections, gene expression, and treatment response. Bioluminescence in vivo imaging allows longitudinal monitoring of a disease course in the same animal, a desirable alternative to analyzing a number of animals at many time points during the course of the disease. We provide a brief introduction to BLI technology, specific examples of in vivo BLI studies investigating bacterial/viral pathogenesis and tumor growth in animal models, and highlight some future perspectives of BLI as a molecular imaging tool.

Biomedical applications of fluorescence imaging in vivo.

Optical imaging can advance knowledge of cellular biology and disease at the molecular level in vitro and, more recently, in vivo. In vivo optical imaging has enabled real-time study to track cell movement, cell growth, and even some cell functions. Thus, it can be used in intact animals for disease detection, screening, diagnosis, drug development, and treatment evaluation. This review includes a brief introduction to fluorescence imaging, fluorescent probes, imaging devices, and in vivo applications in animal models. It also describes a quantitative fluorescence detection method with a reconstruction algorithm for determining the location of fluorophores in tissue and addresses future applications of in vivo fluorescence imaging.

Immune evasion by murine melanoma mediated through CC chemokine receptor-10.

Human melanoma cells frequently express CC chemokine receptor (CCR)10, a receptor whose ligand (CCL27) is constitutively produced by keratinocytes. Compared with B16 murine melanoma, cells rendered more immunogenic via overexpression of luciferase, B16 cells that overexpressed both luciferase and CCR10 resisted host immune responses and readily formed tumors. In vitro, exposure of tumor cells to CCL27 led to rapid activation of Akt, resistance to cell death induced by melanoma antigen-specific cytotoxic T cells, and phosphatidylinositol-3-kinase (PI3K)-dependent protection from apoptosis induced by Fas cross-linking. In vivo, cutaneous injection of neutralizing antibodies to endogenous CCL27 blocked growth of CCR10-expressing melanoma cells. We propose that CCR10 engagement by locally produced CCL27 allows melanoma cells to escape host immune antitumor killing mechanisms (possibly through activation of PI3K/Akt), thereby providing a means for tumor progression.