Brian B. Roman

Functional MR microimaging of pancreatic β-cell activation

The increasing incidence of diabetes and the need to further understand its cellular basis has resulted in the development of new diagnostic and therapeutic techniques. Nonetheless, the quest to noninvasively ascertain β-cell mass and function has not been achieved. Manganese (Mn)-enhanced MRI is presented here as a tool to image β-cell functionality in cell culture and isolated islets. Similar to calcium, extracellular Mn was taken up by glucose-activated β-cells resulting in 200% increase in MRI contrast enhancement, versus nonactivated cells. Similarly, glucose-activated islets showed an increase in MRI contrast up to 45%. Although glucose-stimulated Ca influx was depressed in the presence of 100 μM Mn, no significant effect was seen at lower Mn concentrations. Moreover, islets exposed to Mn showed normal glucose sensitivity and insulin secretion. These results demonstrate a link between image contrast enhancement and β-cell activation in vitro, and provide the basis for future noninvasive in vivo imaging of islet functionality and β-cell mass.

In vivo biodistribution and clearance of peptide amphiphile micelles

UNLABELLED Peptide amphiphiles (PAs) are promising biomaterials for medical applications. To translate the use of PAs successfully from laboratories to clinics, in vivo studies regarding the safety of these nanomaterials are required. To examine the toxicity and clearance of PA biomaterials, we intravenously administered cy7-labeled, spherical PA micelles, control micelles without a peptide sequence, or PBS in a murine model and investigated biocompatibility, biodistribution, and clearance. Both peptide and non-peptide labeled micelles were approximately 8 nm in diameter, but of opposite surface charge. Neither micelle type caused aggregation or hemolysis of red blood cells. All micelles primarily accumulated in the bladder and were present in urine samples confirming elimination through renal clearance. Ex vivo imaging showed that micelles were also found in the liver suggesting some involvement of the reticuloendothelial system. However, no evidence of toxicity was found within the liver, spleen, kidney, bladder, intestines, lung, and heart. FROM THE CLINICAL EDITOR Safety studies related to peptide amphiphile biomaterials are discussed in this paper, demonstrating that organotoxicity is unlikely with these materials, however, RES activation in the liver may be of consideration in further studies and needed for potential applications.

Functional MRI characterization of isolated human islet activation.

The noninvasive assessment of pancreatic islets would be an invaluable tool in advancing the treatment of type I diabetes and in understanding its pathophysiology. As shown previously in rodents, manganese‐enhanced MRI (MEMRI) can be successfully used to quantify β‐cell function. In this study, we successfully applied this technique to isolated human pancreatic islets in both a static and, more significantly, MRI‐compatible perfusion set‐up. Unlike rodent islets, which produced a significant increase in the signal‐to‐noise ratio (SNR) when treated with 25 µM MnCl2 or less, human islets demonstrated significant manganese uptake when exposed to an extracellular concentration of 50 µM MnCl2. Nonspecific passive manganese uptake was present and quantified in a 15% SNR increase over the control group. However, glucose‐induced manganese uptake caused an SNR increase equal to 45% over nonactivated islets. This corresponds to a statistically significant decrease in the T1 relaxation time from 1501 ms for untreated islets to 1362 ms following passive uptake, and to 861 ms following glucose stimulation. As expected, no manganese cytotoxicity was measured, as shown by normal insulin secretion profiles. These data confirm the viability of MEMRI to assess isolated human islet functionality in vitro, and this technique shows promise for the monitoring of their performance in vivo following transplantation. Copyright

Comparison and evaluation of mouse cardiac MRI acquired with open birdcage, single loop surface and volume birdcage coils

Although the quality and speed of MR images have vastly improved with the development of novel RF coil technologies, the engineering expertise required to implement them is often not available in many animal in vivo MR laboratories. We present here an open birdcage coil design which is easily constructed with basic RF coil expertise and produces high quality images. The quality and advantages of mouse cardiac MR images acquired with open birdcage coils were evaluated and compared to images acquired with a bent single loop surface, and standard birdcage coils acquired at 4.7 Tesla. Two low pass open birdcage coils, two single loop surface coils, and a low pass volume birdcage coil were constructed and their B(1) distributions were evaluated and compared. The calculated average signal-to-noise ratio for the left ventricular wall was 10, 23 and 32 for the volume birdcage coil, single loop surface coil and open birdcage coil, respectively. The results demonstrate that the open birdcage coil provides greater sensitivity than the volume coil and a higher signal/contrast-to-noise ratio and B(1) homogeneity than the single loop surface coil. The open birdcage coil offers easy access and better quality mouse cardiac imaging than both the single loop surface coil and volume birdcage coil and does not require extensive RF engineering expertise to construct.

Genetic background influences adaptation to cardiac hypertrophy and Ca2+ handling gene expression

Genetic variability has a profound effect on the development of cardiac hypertrophy in response to stress. Consequently, using a variety of inbred mouse strains with known genetic profiles may be powerful models for studying the response to cardiovascular stress. To explore this approach we looked at male C57BL/6J and 129/SvJ mice. Hemodynamic analyses of left ventricular pressures (LVPs) indicated significant differences in 129/SvJ and C57BL/6J mice that implied altered Ca2+ handling. Specifically, 129/SvJ mice demonstrated reduced rates of relaxation and insensitivity to dobutamine (Db). We hypothesized that altered expression of genes controlling the influx and efflux of Ca2+ from the sarcoplasmic reticulum (SR) was responsible and investigated the expression of several genes involved in maintaining the intracellular and sarcoluminal Ca2+ concentration using quantitative real-time PCR analyses (qRT-PCR). We observed significant differences in baseline gene expression as well as different responses in expression to isoproterenol (ISO) challenge. In untreated control animals, 129/SvJ mice expressed 1.68× more ryanodine receptor 2(Ryr2) mRNA than C57BL/6J mice but only 0.37× as much calsequestrin 2 (Casq2). After treatment with ISO, sarco(endo)plasmic reticulum Ca2+-ATPase(Serca2) expression was reduced nearly two-fold in 129/SvJ while expression in C57BL/6J was stable. Interestingly, β (1) adrenergic receptor(Adrb1) expression was lower in 129/SvJ compared to C57BL/6J at baseline and lower in both strains after treatment. Metabolically, the brain isoform of creatine kinase (Ckb) was up-regulated in response to ISO in C57BL/6J but not in 129/SvJ. These data suggest that the two strains of mice regulate Ca2+ homeostasis via different mechanisms and may be useful in developing personalized therapies in human patients.

Curcumin aggravates CNS pathology in experimental systemic lupus erythematosus.

Complement activation and inflammation are key disease features of systemic lupus erythematosus. Curcumin is an anti-inflammatory agent that inhibits the complement cascade. Therefore, we hypothesized that curcumin will be protective in CNS lupus. To assess the effect of curcumin on CNS-lupus, MRL/lpr mice were used. Brain MRI showed that curcumin (30mg/kg body wt. i.p. from 12-20 weeks) worsened regional brain atrophy. The volumes of the lateral and third ventricles are significantly increased (150%-213% and 107%-140%, without and with treatment respectively compared to MRL+/+ controls). The hippocampus was reduced further (83%-81%) by curcumin treatment. In line with increased brain atrophy, there were edematous cells (41% increase in cell size in MRL/lpr compared to MRL+/+ mice. The cell size was further increased by 28% when treated with curcumin; p<0.02) in the cortex. In line with increased atrophy and edema, there was a significant increase (p<0.02) in the mRNA and protein expression of the water channel protein, aquaporin 4 in these mice. The increase in the matrix proteins, glial fibrillary acidic protein and vimentin in lupus mice in the hippocampus was prevented by curcumin. Curcumin increased IgG deposits and decreased C3 deposits in brain with a corresponding increase in immune complexes and decrease in C3 concentration (by 60% in MRL/lpr mice Vs. MRL+/+ mice and a further 26% decrease when treated with curcumin) in circulation. Decrease in C3 could alter the transport of immune complexes leading to an increase in IgG deposits which could induce inflammatory pathways thereby leading to worsening of the disease. The neurological outcome as measured by maze performance indicates that the curcumin treated mice performed poorly compared to the untreated counterparts. Our results for the first time provide evidence that at the dose used in this study, curcumin aggravates some CNS disease manifestations in experimental lupus brain. Therefore, until a safe dose range is established by additional studies, and the validity of the findings is determined in human patients, caution may be warranted in the use of curcumin, even as adjuvant therapy for CNS lupus.

β-Cell subcellular localization of glucose-stimulated Mn uptake by X-ray fluorescence microscopy: implications for pancreatic MRI.

Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β-cells and showed that, in the presence of MnCl(2), glucose-activated pancreatic islets yield significant signal enhancement in T(1)-weigheted MR images. In this study, we exploited for the first time the unique capabilities of X-ray fluorescence microscopy (XFM) to both visualize and quantify the metal in pancreatic β-cells at cellular and subcellular levels. MIN-6 insulinoma cells grown in standard tissue culture conditions had only a trace amount of Mn, 1.14 ± 0.03 × 10(-11)µg/µm(2), homogenously distributed across the cell. Exposure to 2 mM glucose and 50 µM MnCl(2) for 20 min resulted in nonglucose-dependent Mn uptake and the overall cell concentration increased to 8.99 ± 2.69 × 10(-11) µg/µm(2). When cells were activated by incubation in 16 mM glucose in the presence of 50 µM MnCl(2), a significant increase in cytoplasmic Mn was measured, reaching 2.57 ± 1.34 × 10(-10) µg/µm(2). A further rise in intracellular concentration was measured following KCl-induced depolarization, with concentrations totaling 1.25 ± 0.33 × 10(-9) and 4.02 ± 0.71 × 10(-10) µg/µm(2) in the cytoplasm and nuclei, respectively. In both activated conditions Mn was prevalent in the cytoplasm and localized primarily in a perinuclear region, possibly corresponding to the Golgi apparatus and involving the secretory pathway. These data are consistent with our previous MRI findings, confirming that Mn can be used as a functional imaging reporter of pancreatic β-cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast.

If the skull fits: magnetic resonance imaging and microcomputed tomography for combined analysis of brain and skull phenotypes in the mouse

The mammalian brain and skull develop concurrently in a coordinated manner, consistently producing a brain and skull that fit tightly together. It is common that abnormalities in one are associated with related abnormalities in the other. However, this is not always the case. A complete characterization of the relationship between brain and skull phenotypes is necessary to understand the mechanisms that cause them to be coordinated or divergent and to provide perspective on the potential diagnostic or prognostic significance of brain and skull phenotypes. We demonstrate the combined use of magnetic resonance imaging and microcomputed tomography for analysis of brain and skull phenotypes in the mouse. Co-registration of brain and skull images allows comparison of the relationship between phenotypes in the brain and those in the skull. We observe a close fit between the brain and skull of two genetic mouse models that both show abnormal brain and skull phenotypes. Application of these three-dimensional image analyses in a broader range of mouse mutants will provide a map of the relationships between brain and skull phenotypes generally and allow characterization of patterns of similarities and differences.

Manganese-enhanced MRI detection of impaired calcium regulation in a mouse model of cardiac hypertrophy.

The aim of this study was to use manganese (Mn)‐enhanced MRI (MEMRI) to detect changes in calcium handling associated with cardiac hypertrophy in a mouse model, and to determine whether the impact of creatine kinase ablation is detectable using this method. Male C57BL/6 (C57, n = 11) and male creatine kinase double‐knockout (CK‐M/Mito–/–, DBKO, n = 12) mice were imaged using the saturation recovery Look–Locker T1 mapping sequence before and after the development of cardiac hypertrophy. Hypertrophy was induced via subcutaneous continuous 3‐day infusion of isoproterenol, and sham mice not subjected to cardiac hypertrophy were also imaged. During each scan, the contrast agent Mn was administered and the resulting change in R1 (=1/T1) was calculated. Two anatomical regions of interest (ROIs) were considered, the left‐ventricular free wall (LVFW) and the septum, and one ROI in an Mn‐containing standard placed next to the mouse. We found statistically significant (p < 0.05) decreases in the uptake of Mn in both the LVFW and septum following the induction of cardiac hypertrophy. No statistically significant decreases were detected in the standard, and no statistically significant differences were found among the sham mice. Using a murine model, we successfully demonstrated that changes in Mn uptake as a result of cardiac hypertrophy are detectable using the functional contrast agent and calcium mimetic Mn. Our measurements showed a decrease in the relaxivity (R1) of the myocardium following cardiac hypertrophy compared with normal control mice. Copyright

Empirical mathematical model for dynamic manganese-enhanced MRI of the murine pancreas for assessment of β-cell function

Autoimmune ablation of pancreatic β-cells and alteration of its microvasculature may be a predictor of Type I diabetes development. A dynamic manganese-enhanced MRI (MEMRI) approach and an empirical mathematical model were developed to monitor whole pancreatic β-cell function and vasculature modifications in mice. Normal and streptozotocin-induced diabetic FVB/N mice were imaged on a 9.4T MRI system using a 3D magnetization prepared rapid acquisition gradient echo pulse sequence to characterize low dose manganese kinetics in the pancreas head, body and tail. Average signal enhancement in the pancreas (head, body, and tail) as a function of time was fit by a novel empirical mathematical model characterizing contrast uptake/washout rates and yielding parameters describing peak signal, initial slope, and initial area under the curve. Signal enhancement from glucose-induced manganese uptake was fit by a linear function. The results demonstrated that the diabetic pancreatic tail had a significantly lower contrast uptake rate, smaller initial slope/initial area under the curve, and a smaller rate of Mn uptake following glucose activation (p<0.05) compared to the normal pancreatic tail. These observations parallel known patterns of β-cell loss and alteration in supportive vasculature associated with diabetes. Dynamic MEMRI is a promising technique for assessing β-cell functionality and vascular perfusion with potential applications for monitoring diabetes progression and/or therapy.