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Cataracts are a common consequence of aging; however, pathogenesis remains poorly understood. Here, we observed that after 3 months of age mice lacking the G protein-coupled leukocyte chemotactic receptor Fpr1 (N-formyl peptide receptor 1) began to develop bilateral posterior subcapsular cataracts that progressed to lens rupture and severe degeneration, without evidence of either systemic or local ocular infection or inflammation. Consistent with this, Fpr1 was detected in both mouse and human lens in primary lens epithelial cells (LECs), the only cell type present in the lens; however, expression was confined to subcapsular LECs located along the anterior hemispheric surface. To maximize translucency, LECs at the equator proliferate and migrate posteriorly, then differentiate into lens fiber cells by nonclassical apoptotic signaling, which results in loss of nuclei and other organelles, including mitochondria which are a rich source of endogenous N-formyl peptides. In this regard, denucleation and posterior migration of LECs were abnormal in lenses from Fpr1 -/- mice, and direct stimulation of LECs with the prototypic N-formyl peptide agonist fMLF promoted apoptosis. Thus, Fpr1 is repurposed beyond its immunoregulatory role in leukocytes to protect against cataract formation and lens degeneration during aging., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Pregnant women are at increased risk of thrombotic adverse events. Plasma derived immune globulin (IG) products, which are used in pregnancy for various indications, may contain procoagulant impurity activated coagulation factor XI (FXIa). Procoagulant IG products have been associated with increased thrombogenicity but their effect in pregnancy is unknown., Methods: Late pregnant (gestation days 17-20) or early lactation (days 1-3) and control female mice were treated with IGs supplemented with human FXIa then subjected to ferric chloride (FeCl 3 ) vessel injury. Occlusion of blood vessel was assessed by recording blood velocity in the femoral vein for 20 min using doppler ultrasound laser imaging. FXIa dose was selected by the ability to increase thrombin generation in mouse plasma in vitro., Results: FXIa produced robust thrombin generation in mouse plasma ex vivo. Following FeCl 3 injury, pregnant and non-pregnant mice receiving IG + FXIa exhibited faster reduction of blood velocity in femoral vein compared to IG alone or untreated controls. In vitro, thrombin generation in plasma samples collected after thrombosis in FXIa-treated animals was elevated and could be reduced by anti-FXI antibody., Conclusions: Our results suggest that intravenously-administered FXIa may contribute to thrombosis at the site of vascular injury in both pregnant and non-pregnant animals.

Background: Protein Phosphatase 2A (PP2A) function is controlled by regulatory subunits that modulate the activity of the catalytic subunit and direct the PP2A complex to specific intracellular locations. To study PP2A's role in signal transduction pathways that control growth and differentiation in vivo, a transgenic mouse lacking the B56γ regulatory subunit of PP2A was made., Results: Lack of PP2A activity specific to the PP2A-B56γ holoenzyme, resulted in the formation of an incomplete ventricular septum and a decrease in the number of ventricular cardiomyocytes. During cardiac development, B56γ is expressed in the nucleus of α-actinin-positive cardiomyocytes that contain Z-bands. The pattern of B56γ expression correlated with the cardiomyocyte apoptosis we observed in B56γ-deficient mice during mid to late gestation. In addition to the cardiac phenotypes, mice lacking B56γ have a decrease in locomotive coordination and gripping strength, indicating that B56γ has a role in controlling PP2A activity required for efficient neuromuscular function., Conclusions: PP2A-B56γ activity is required for efficient cardiomyocyte maturation and survival. The PP2A B56γ regulatory subunit controls PP2A substrate specificity in vivo in a manner that cannot be fully compensated for by other B56 subunits., (© 2014 Wiley Periodicals, Inc.)

The muscle-specific protein NRAP is concentrated at cardiac intercalated disks, plays a role in myofibril assembly, and is upregulated early in mouse models of dilated cardiomyopathy. Using a tet-off system, we developed novel transgenic lines exhibiting cardiac-specific NRAP overexpression ~2.5 times greater than normal. At 40-50 weeks, NRAP overexpression resulted in dilation and decreased ejection fraction in the right ventricle, with little effect on the left ventricle. Expression of transcripts encoding brain natriuretic peptide and skeletal α-actin was increased by cardiac-specific NRAP overexpression, indicative of a cardiomyopathic response. NRAP overexpression did not alter the levels or organization of N-cadherin and connexin-43. The results show that chronic NRAP overexpression in the mouse leads to right ventricular cardiomyopathy by 10 months, but that the early NRAP upregulation previously observed in some mouse models of dilated cardiomyopathy is unlikely to account for the remodeling of intercalated disks and left ventricular dysfunction observed in those cases., (Published by Elsevier Inc.)

Background: Similar to a subset of human patients who progress from monoclonal B lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL), New Zealand Black (NZB) mice have an age-associated progression to CLL. The murine disease is linked to a genetic abnormality in microRNA mir-15a/16-1 locus, resulting in decreased mature miR-15a/16., Methods: Spleens of aging NZB were analyzed for the presence of B-1 cells via flow cytometry and for the presence of a side population (SP) via the ability of cells to exclude Hoechst 33342 dye. The SP was assayed for the presence of hyperdiploid B-1 clones and for the ability to differentiate into B-1 cells in vitro and transfer disease in vivo. In addition, enhanced apoptosis of chemoresistant NZB B-1 cells was examined by restoring miR-16 levels in nutlin-treated cells., Results: Aging NZB mice develop a B-1 expansion and clonal development that evolves from MBL into CLL. An expansion in SP is also seen. Although the SP did contain increased cells with stem cell markers, they lacked malignant B-1 cells and did not transfer disease in vivo. Similar to B-1 cells, splenic NZB SP also has decreased miR-15a/16 when compared with C57Bl/6. Exogenous addition of miR-15a/16 to NZB B-1 cells resulted in increased sensitivity to nutlin., Conclusion: NZB serve as an excellent model for studying the development and progression of age-associated CLL. NZB SP cells do not seem to contain cancer stem cells, but rather the B-1 stem cell. NZB B-1 chemoresistance may be related to reduced miR-15a/16 expression., (© 2010 International Clinical Cytometry Society.)

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.

Nitric oxide (NO) locally regulates vascular resistance and blood pressure by modulating blood vessel tone. Thrombospondin-1 signaling via its receptor CD47 locally limits the ability of NO to relax vascular smooth muscle cells and increase regional blood flow in ischemic tissues. To determine whether thrombospondin-1 plays a broader role in central cardiovascular physiology, we examined vasoactive stress responses in mice lacking thrombospondin-1 or CD47. Mice lacking thrombospondin-1 exhibit activity-associated increases in heart rate, central diastolic and mean arterial blood pressure and a constant decrease in pulse pressure. CD47-deficient mice have normal central pulse pressure but elevated resting peripheral blood pressure. Both null mice show exaggerated decreases in peripheral blood pressure and increased cardiac output and ejection fraction in response to NO. Autonomic blockade also induces exaggerated hypotensive responses in awake thrombospondin-1 null and CD47 null mice. Both null mice exhibit a greater hypotensive response to isoflurane, and autonomic blockage under isoflurane anesthesia leads to premature death of thrombospondin-1 null mice. Conversely, the hypertensive response to epinephrine is attenuated in thrombospondin-1 null mice. Thus, the matricellular protein thrombospondin-1 and its receptor CD47 serve as acute physiological regulators of blood pressure and exert a vasopressor activity to maintain global hemodynamics under stress.

RGS5 is a potent GTPase-activating protein for G(ialpha) and G(qalpha) that is expressed strongly in pericytes and is present in vascular smooth muscle cells. To study the role of RGS5 in blood vessel physiology, we generated Rgs5-deficient mice. The Rgs5(-/-) mice developed normally, without obvious defects in cardiovascular development or function. Surprisingly, Rgs5(-/-) mice had persistently low blood pressure, lower in female mice than in male mice, without concomitant cardiac dysfunction, and a lean body habitus. The examination of the major blood vessels revealed that the aortas of Rgs5(-/-) mice were dilated compared to those of control mice, without altered wall thickness. Isolated aortic smooth muscle cells from the Rgs5(-/-) mice exhibited exaggerated levels of phosphorylation of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase in response to stimulation with either sodium nitroprusside or sphingosine 1-phosphate. The results of this study, along with those of previous studies demonstrating that RGS5 stability is under the control of nitric oxide via the N-end rule pathway, suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS (regulator of G protein signaling) family member known to inhibit G protein-coupled receptor-mediated vasoconstrictor signaling. Blocking the function or the expression of RGS5 may provide an alternative approach to treat hypertension.

Mutations in Notch receptors and their ligands have been identified as the cause of human congenital heart diseases, indicating the importance of the Notch signaling pathway during heart development. In our study, we use Cre-Lox technology to inactivate Notch2 in several cardiac cell lineages to determine the functional requirements for Notch2 during mammalian heart development. Inactivation of Notch2 in cardiac neural crest cells resulted in abnormally narrow aortas and pulmonary arteries due to a decrease in smooth muscle tissue. The reduction in smooth muscle tissue was not due to cell migration defects but instead was found to be caused by less proliferation in smooth muscle cells during mid to late gestation. Our findings demonstrate that Notch2 is required cell autonomously for proper formation of the heart outflow tract and provides insights into the role of Notch2 in vascular smooth muscle development and the cardiovascular defects associated with Alagille syndrome., ((c) 2008 Wiley-Liss, Inc.)

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.

We evaluated the relaxation enhancement and biodistribution of short- vs. long-circulating magnetoliposomes as a new contrast agent for magnetic resonance (MR) imaging of bone marrow. Magnetoliposomes with (ML-PEG) and without (ML) incorporation of polyethylene glycol (PEG, Mw 2000) were prepared, measuring 40 nm in diameter with 1-6 iron oxide crystals/vesicle. PEGylation selectively enhanced the T2 relaxivity of magnetoliposomes by 10% to 15%, with R1 and R2 values of 3 and 240 s(-1)/mM at 1.5 T and 37 degrees C. ML (n = 6) and ML-PEG (n = 6) preparations were administered IV into young (6-8 weeks old) and adult (>1 year old) Sprague-Dawley rats at 100 pmol Fe/kg. PEGylation increased blood half-life (P<0.05 for t > 30 minutes), following a biexponential clearance with a long half-life of 53.2+/-13.2 minutes. The clearance of ML was monoexponential, with a half-life 7.4+/-0.4 minutes. MR imaging revealed a pronounced uptake in bone marrow, including the iliac bone, femur, tibia, and upper and lower vertebrae. The bone marrow uptake of ML-PEG was comparable to that of ML, with both reaching a plateau within 30 minutes following injection. Fast spin-echo T2-weighted imaging was found to provide optimal contrast enhancement and allowed a clear depiction of red to yellow marrow conversion due to normal aging. While the use of magnetoliposomes can provide the added benefit of therapeutic drug or gene delivery, further investigation is warranted to assess their usefulness in differentiating normal vs. abnormal marrow conditions.

Gene therapy is a promising approach to the treatment of many forms of disease, including cancer. Of critical concern in its implementation is the ability to control the location, duration, and level of expression of the therapeutic gene. Here, we propose the use of local heat in combination with a heat-sensitive promoter to help accomplish this. Certain members of the family of heat shock protein (hsp) promoters display a regulation that depends strongly on temperature. We present a study of natural hsp70 induction in rat leg by MRI-guided focused ultrasound to investigate the hsp70 promoter as a possible candidate for use in control of gene expression with local heat. A temperature increase of 5-8 degrees C in the focal region for 45 minutes led to a differential expression of the hsp70 mRNA between the focal region and the surrounding tissue ranging from a factor of 3 to 67.

A gradient-echo echo planar imaging (EPI) sequence has been used to generate images of cat brain during respiratory challenges. Direct spectrophotometric measurements have been made simultaneously in order to correlate the changes in oxygen saturation as measured by spectrophotometry with the image intensity changes seen in the gradient-echo images. When blood volume remains approximately constant, as derived from the spectrophotometry data, good correlation is seen between calculated plots made of changes in the transverse relaxation rate, delta R2*, and the oxygen saturation as measured by spectrophotometry for much of the time course of the respiratory challenges of anoxia, apnea and hypercapnia. In some cases, the correlation is poorer during the recovery periods of the apnea and anoxia challenges. Those lower correlations can often be accounted for by changes in blood volume, which also affects the NMR relaxation rate. These results contribute to the understanding of the image intensity changes seen during functional brain imaging studies in humans.

Interpretation of NMR diffusion data is complicated in the presence of diffusion barriers. In this study, restricted diffusion in cylinders is evaluated. A method is presented to determine the diameter of the cylinder and the unrestricted diffusion coefficient from the dependence of the trace of the diffusion tensor with diffusion time. This method is valid even if the orientation of the cylinders is unknown and varies within the sample. An example is given for the diffusion of phosphocreatine in the cylindrically shaped fibers of rabbit skeletal muscle.

The occlusion of the middle cerebral artery was used as an experimental acute stroke model in 30 cats. The diffusion of water was followed by diffusion-sensitized MRI between 1 and 15 h after induction of stroke. It is demonstrated that images representing the trace of the diffusion tensor provide a much more accurate delineation of affected area than images representing the diffusion in one direction only. The reason is that the strong contrast caused by the anisotropy and orientation of myelin fibers is completely removed in the trace of the diffusion tensor. The trace images show a small contrast between white and gray matter. The diffusion coefficient of white matter is decreased in acute stroke to approximately the same extent as gray matter. It is further shown that the average lifetime of water in extra and intracellular space is shorter than 20 ms both for healthy and ischemic tissue indicating that myelin fibers are permeable to water. The anisotropy contrast did not change before or after induction of stroke, nor after sacrifice. Together, these observations are consistent with the view that the changes in water diffusion during acute stroke are directly related to cytotoxic oedema, i.e., to the change in relative volume of intra- and extracellular spaces. Changes in membrane permeability do not appear to contribute significantly to the changes in diffusion.

Purpose: To investigate the causal connections between ischemia and the hyperintensity in diffusion-weighted MR images that has been associated with it., Methods: Diffusion-weighted and T2-weighted MR imaging were used in a feline global cerebral ischemia/reperfusion model. Single 30-minute vascular occlusions followed by reperfusion were studied. Global occlusions were used to avoid interpretive complications associated with the temporally unstable hemodynamics of the penumbral zones around focal occlusions and the possible growth of the ischemic and penumbral regions with time., Results: Diffusion-weighted hyperintensity and the associated diffusional slowing were not attributable exclusively to the cessation of blood flow because: 1) it does not appear abruptly at the onset of ischemia; 2) it resolves slowly early in reperfusion; and 3) it reappears after prolonged reperfusion., Conclusion: The times during which diffusion-weighted hyperintensity is manifested during ischemia, and recovers with reperfusion, point to a role for energy metabolism failure.

Metabolism of [1-13C]-glucose was studied in situ in cat brain using gradient-enhanced proton-detected heteronuclear spectroscopy. Proton detection of [1-13C]-glucose, [3-13C]-lactate, 4-[13C]-glutamine, 4-[13C]-glutamate and the combined signals 2-[13C]-glutamate/glutamine and 3-[13C]-glutamate/glutamine was achieved, despite the fact that some of the associated proton resonances are close to the water signal. Two-dimensional [1H-13C]-spectra demonstrate the possibility of in situ spectral assignment with 1H sensitivity and 13C resolution. Spectroscopic images of glucose and its metabolic products were also acquired, showing the possibility to study spatial dependence of metabolism.

The present study describes results from an in vivo 19F NMR study on rats exposed to the xenobiotic compound 2-fluoroaniline. Qualitative pharmacokinetics and the biotransformation of 2-fluoroaniline were studied after exposure to 50 mg/kg body wt 2-fluoroaniline. Accumulation and elimination of the parent compound in and from the liver of exposed animals were readily observed. Metabolites formed in the liver were shown to be efficiently excreted from the liver, as the amount of metabolites in this organ was always less than 10% of the maximum amount of the parent compound observed. In the bladder, rapid accumulation of 2-fluoroaniline-derived metabolites was detected. The metabolite pattern was shown to change in time, with the parent compound being dominantly present during the first hours of exposure. N-acetylated products (4-acetamido-3-fluorophenyl sulphate and 4-acetamido-3-fluorophenyl glucuronide) were observed to accumulate more slowly in the bladder than the non-acetylated products (4-amino-3-fluorophenyl sulphate and 4-amino-3-fluorophenyl glucuronide). Urine metabolite patterns obtained from the bladder in vivo were compared to those obtained during metabolic cage experiments.

When deoxygenated, blood behaves as an effective susceptibility contrast agent. Changes in brain oxygenation can be monitored using gradient-echo echo-planar imaging. With this technique, difference images also demonstrate that blood oxygenation is increased during periods of recovery from respiratory challenge.

A genomic library of Sarcocystis cruzi sporozoite DNA was constructed in bacteriophage lambda gt10. Recombinant phages containing insert DNA were selected by growth on Escherichia coli strain C600 hflA150. Of 14 clones examined, 11 contained DNA inserts ranging in size from approximately 1.45 kilobase (kb) to 6.18 kb. Insert DNA from four of these clones specifically hybridized to 32P-labelled S. cruzi merozoite DNA. One of these insert DNA, clone SL41, was selected and labelled with 32P. This probe did not hybridize with the other ten DNA inserts nor with bovine cellular DNA, but it hybridized with sporozoite, merozoite and bradyzoite DNA preparations. The SL41 probe could detect merozoite DNA in as little as 17 ng total DNA. Genomic probes detecting developmental stages of Sarcocystis spp. could provide an improved means is diagnosis of acute bovine sarcocystosis.

The occlusion of the middle cerebral artery in cat brain was used as an experimental stroke model to investigate the physical basis of the recently reported lowered diffusion constant of water in acute infarcted brain tissue (Moseley et al., Magn. Reson. Med. 14, 330, 1990). The original findings were confirmed in this study of 12 animals investigated with the diffusion-sensitized stimulated echo sequence. The following additional results were obtained: First, the onset of significant lowering of the diffusion constant in the stroke area varied significantly (up to 2.5 h depending on the animal). Second, the affected area is much more clearly outlined in diffusion-weighted images than in T2-weighted images, even in the period between 3 to 12 h following occlusion. Third, for diffusion times between 50 and 2000 ms. the diffusion constant of water is independent of diffusion time in healthy tissue, as well as in the stroke area. Fourth, the diffusion anisotropy is similar in healthy and in stroke area and remains similar regardless of the diffusion time used.

Cat brain images sensitized to incoherent motion by additional gradient pulses were obtained on a 4.7 T magnetic resonance unit equipped with shielded gradient coils. The apparent diffusion coefficient of water in gray and white matter was accurately determined and imaged from the signal attenuation curve obtained as a function of gradient strength. Contrast in calculated diffusion images differed from typical T2-weighted contrast. Furthermore, in gray matter and in areas containing flowing CSF the attenuation curve was found to be biexponential. These results are interpreted in terms of a simple voxel model with microcirculation and diffusion contributions.

Apparent diffusion coefficients (Da) of individual metabolites can be studied in vivo by diffusion NMR spectroscopy using an echo sequence sensitized to molecular motion. The methods are based on the echo attenuation due to phase dispersion resulting from incoherent displacement during the diffusion time. As the displacement of metabolites by diffusion in vivo can be affected by compartment size, temperature, adsorption processes, etc., the presented methods are potentially useful in studying such phenomena in vivo. Here, the methods are applied to phosphocreatine in the rat quadriceps muscle. It is demonstrated that the displacement of phosphocreatine resembles free diffusion for short diffusion times but becomes limited as a result of boundaries due to compartmentation for longer diffusion times. The limit of the displacement indicates an apparent average size of 44 microns of the compartment in the direction of the diffusion gradient. As the gradient was applied approximately parallel (angle less than 25 degrees) to the muscle fiber, this result indicates that phosphocreatine moves freely in the cytosol but is limited by the boundaries of the muscle cells. Error analyses are performed with regard to motion artifacts and gradient performance. The methods were tested extensively for distilled water and free metabolites.