Imaging of adeno-associated viral capsids for purposes of gene editing using CEST NMR/MRI.

PURPOSE: Gene therapy using adeno-associated virus (AAV) vector-mediated gene delivery has undergone substantial growth in recent years with promising results in both preclinical and clinical studies, as well as emerging regulatory approval. However, the inability to quantify the efficacy of gene therapy from cellular delivery of gene-editing technology to specific functional outcomes is an obstacle for efficient development of gene therapy treatments. Building on prior works that used the CEST reporter gene lysine rich protein, we hypothesized that AAV viral capsids may generate endogenous CEST contrast from an abundance of surface lysine residues. METHODS: NMR experiments were performed on isolated solutions of AAV serotypes 1-9 on a Bruker 800-MHz vertical scanner. In vitro experiments were performed for testing of CEST-NMR contrast of AAV2 capsids under varying pH, density, biological transduction stage, and across multiple serotypes and mixed biological media. Reverse transcriptase-polymerase chain reaction was used to quantify virus concentration. Subsequent experiments at 7 T optimized CEST saturation schemes for AAV contrast detection and detected AAV2 particles encapsulated in a biocompatible hydrogel administered in the hind limb of mice. RESULTS: CEST-NMR experiments revealed CEST contrast up to 52% for AAV2 viral capsids between 0.6 and 0.8 ppm. CEST contrast generated by AAV2 demonstrated high levels of CEST contrast across a variety of chemical environments, concentrations, and saturation schemes. AAV2 CEST contrast displayed significant positive correlations with capsid density (R2 > 0.99, p < 0.001), pH (R2 = 0.97, p = 0.01), and viral titer per cell count (R2 = 0.92, p < 0.001). Transition to a preclinical field strength yielded up to 11.8% CEST contrast following optimization of saturation parameters. In vivo detection revealed statistically significant molecular contrast between viral and empty hydrogels using both mean values (4.67 ± 0.75% AAV2 vs. 3.47 ± 0.87% empty hydrogel, p = 0.02) and quantile analysis. CONCLUSION: AAV2 viral capsids exhibit strong capacity as an endogenous CEST contrast agent and can potentially be used for monitoring and evaluation of AAV vector-mediated gene therapy protocols.

Noninvasively differentiating acute and chronic nephropathies via multiparametric urea-CEST, nuclear Overhauser enhancement-CEST, and quantitative magnetization transfer MRI.

PURPOSE: Standardized blood tests often lack adequate sensitivity and specificity to capture the gradual progression of renal injuries. We suggest a multiparametric molecular MRI approach as a noninvasive tool for monitoring renal function loss and distinguishing different types of renal injuries. METHODS: CEST and quantitative magnetization transfer (qMT) imaging were performed on cisplatin (n = 16) and aristolochic acid (AA)-induced nephropathy (n = 22) mouse models at 7T with an infusion of either saline or urea. Seven-pool Lorentzian fitting was applied for the analysis of CEST Z-spectra, and the T1 -corrected CEST contrast apparent exchange-dependent relaxation (AREX) from urea (+1 ppm) and two nuclear Overhauser enhancement (NOE) pools (-1.6 and -3.5 ppm) were measured. Similarly, qMT spectra were fitted into two-pool Ramani equation and the relative semi-solid macromolecular pool-size ratio was measured. Histology of mouse kidneys was performed to validate the MR findings. RESULTS: AA model showed disrupted spatial gradients of urea in the kidney and significantly decreased NOE CEST and qMT contrast. The cisplatin model showed slightly decreased qMT contrast only. The orrelation of MR parameters to histological features showed that NOE CEST and qMT imaging are sensitive to both acute and chronic injuries, whereas urea CEST shows a significant correlation only to acute injuries. CONCLUSION: These results indicate that our multiparametric approach allows comprehensive and totally noninvasive monitoring of renal function and histological changes for distinguishing different nephropathies.

Individually tailored spatial-spectral pulsed CEST MRI for ratiometric mapping of myocardial energetic species at 3T.

PURPOSE: CEST MRI has been used to probe changes in cardiac metabolism via assessment of CEST contrast from Cr. However, B1 variation across the myocardium leads to spatially variable Cr CEST contrast in healthy myocardium. METHODS: We developed a spatial-spectral (SPSP) saturation pulsed CEST protocol to compensate for B1 variation. Flip angle maps were used to individually tailor SPSP pulses comprised of a train of one-dimensional spatially selective subpulses selective along the principal B1 gradient dimension. Complete Z-spectra in the hearts of (n = 10) healthy individuals were acquired using conventional Gaussian saturation and SPSP schemes and supported by phantom studies. RESULTS: In simulations, the use of SPSP pulses reduced the average SD of the effective saturation B1 values within the myocardium (n = 10) from 0.12 ± 0.02 µT to 0.05 ± 0.01 µT (p < 0.01) and reduced the average SD of Cr CEST contrast in vivo from 10.0 ± 4.3% to 6.1 ± 3.5% (p < 0.05). Results from the hearts of human subjects showed a significant reduction of CEST contrast distribution at 2 ppm, as well as amplitude, when using SPSP saturation. Corresponding phantom experiments revealed PCr-specific contrast generation at body temperature when SPSP saturation was used but combined PCr and Cr contrast generation when Gaussian saturation was used. CONCLUSION: The use of SPSP saturation pulsed CEST resulted in PCr-specific contrast generation and enabled ratiometric mapping of PCr to total Cr CEST contrast in the human heart at 3T.

T1ρ imaging as a non-invasive assessment of collagen remodelling and organization in human skeletal muscle after ligamentous injury.

Dysregulation and fibrosis of the extracellular matrix (ECM) in skeletal muscle is a consequence of injury. Current ECM assessment necessitates muscle biopsies to evaluate alterations to the muscle ECM, which is often not practical in humans. The goal of this study was to evaluate the potential of a magnetic resonance imaging sequence that quantifies T1ρ relaxation time to predict ECM collagen composition and organization. T1ρ imaging was performed and muscle biopsies obtained from the involved and non-involved vastus lateralis muscle on 27 subjects who had an anterior cruciate ligament (ACL) tear. T1ρ times were quantified via monoexponential decay curve fitted to a series of T1ρ-weighted images. Several ECM indices, including collagen content and organization, were obtained using immunohistochemistry and histochemistry in addition to hydroxyproline. Model selection with multiple linear regression was used to evaluate the relationships between T1ρ times and ECM composition. Additionally, the ACL-deficient and healthy limb were compared to determine sensitivity of T1ρ to detect early adaptations in the muscle ECM following injury. We show that T1ρ relaxation time was strongly associated with collagen unfolding (t = 4.093, P = 0.0007) in the ACL-deficient limb, and collagen 1 abundance in the healthy limb (t = 2.75, P = 0.014). In addition, we show that T1ρ relaxation time is significantly longer in the injured limb, coinciding with significant differences in several indices of collagen content and remodelling in the ACL-deficient limb. These results support the use of T1ρ to evaluate ECM composition in skeletal muscle in a non-invasive manner. KEY POINTS: Dysregulation and fibrotic transformation of the skeletal muscle extracellular matrix (ECM) is a common pathology associated with injury and ageing. Studies of the muscle ECM in humans have necessitated the use of biopsies, which are impractical in many settings. Non-invasive MRI T1ρ relaxation time was validated to predict ECM collagen composition and organization with aligned T1ρ imaging and biopsies of the vastus lateralis in the healthy limb and anterior cruciate ligament (ACL)-deficient limb of 27 subjects. T1ρ relaxation time was strongly associated with collagen abundance and unfolding in the ACL-deficient limb, and T1ρ relaxation time was strongly associated with total collagen abundance in the healthy limb. T1ρ relaxation time was significantly longer in the ACL-deficient limb, coinciding with significant increases in several indices of muscle collagen content and remodelling supporting the use of T1ρ to non-invasively evaluate ECM composition and pathology in skeletal muscle.

Delayed urea differential enhancement CEST (dudeCEST)-MRI with T1 correction for monitoring renal urea handling.

PURPOSE: We demonstrate a method of delayed urea differential enhancement CEST for probing urea recycling action of the kidney using expanded multi-pool Lorentzian fitting and apparent exchange-dependent relaxation compensation. METHODS: T1 correction of urea CEST contrast by apparent exchange-dependent relaxation was tested in phantoms. Nine mice were scanned at 7 Tesla following intraperitoneal injection of 2M 150 µL urea, and later saline. T1 maps and Z-spectra were acquired before and 20 and 40 min postinjection. Z-spectra were fit to a 7-pool Lorentzian model for CEST quantification and compared to urea assay of kidney homogenate. Renal injury was induced by aristolochic acid in 7 mice, and the same scan protocol was performed. RESULTS: Apparent exchange-dependent relaxation corrected for variable T1 times in phantoms. Urea CEST contrast at +1 ppm increased significantly at both time points following urea injection in the inner medulla and papilla. When normalizing the postinjection urea CEST contrast to the corresponding baseline value, both urea and saline injection resulted in identical fold changes in urea CEST contrast. Urea assay of kidney homogenate showed a significant correlation to both apparent exchange-dependent relaxation (R2 = 0.4687, P = .0017) and non-T1 -corrected Lorentzian amplitudes (R2 = 0.4964, P = .0011). Renal injury resulted in increased T1 time in the cortex and reduced CEST contrast change upon urea and saline infusion. CONCLUSION: Delayed urea enhancement following infusion can provide insight into renal urea handling. In addition, changes in CEST contrast at 1.0 ppm following saline infusion may provide insight into renal function.

Noninvasive imaging of renal urea handling by CEST-MRI.

PURPOSE: Renal function is characterized by concentration of urea for removal in urine. We tested urea as a CEST-MRI contrast agent for measurement of the concentrating capacity of distinct renal anatomical regions. METHODS: The CEST contrast of urea was examined using phantoms with different concentrations and pH levels. Ten C57BL/6J mice were scanned twice at 7 T, once following intraperitoneal injection of 2M 150 µL urea and separately following an identical volume of saline. Kidneys were segmented into regions encompassing the cortex, outer medulla, and inner medulla and papilla to monitor spatially varying urea concentration. Z-spectra were acquired before and 20 minutes after injection, with dynamic scanning of urea handling performed in between via serial acquisition of CEST images acquired following saturation at +1 ppm. RESULTS: Phantom experiments revealed concentration and pH-dependent CEST contrast of urea that was both acid- and base-catalyzed. Z-spectra acquired before injection showed significantly higher CEST contrast in the inner medulla and papilla (2.3% ± 1.9%) compared with the cortex (0.15% ± 0.75%, P = .011) and outer medulla (0.12% ± 0.58%, P = .008). Urea infusion increased CEST contrast in the inner medulla and papilla by 2.1% ± 1.9% (absolute), whereas saline infusion decreased CEST contrast by -0.5% ± 2.0% (absolute, P = .028 versus urea). Dynamic scanning revealed that thermal drift and diuretic status are confounding factors. CONCLUSION: Urea CEST has a potential of monitoring renal function by capturing the spatially varying urea concentrating ability of the kidneys.

Fractal Analysis of Cardiac Fibrosis Patterns Derived From Magnetization Transfer Contrast MRI in Patients With End-Stage Renal Disease.

OBJECTIVE. The purpose of this study is to use fractal analysis to characterize diffusely fibrotic, focally fibrotic, and healthy myocardium in patients with end-stage renal disease (ESRD) on the basis of previously published magnetization transfer (MT) contrast images. MATERIALS AND METHODS. The MT ratio values of patients with ESRD (n = 34) and healthy control subjects (n = 19) were used to construct anatomically faithful 3D left ventricular reconstructions. Established MT ratio threshold values were used to define healthy, diffusely enhanced, focally enhanced, and total enhanced tissue domains. The fractal dimension (FD) for reach domain was calculated using a 3D box-counting algorithm. RESULTS. Patients with ESRD showed a higher FD across all fibrotic domains compared with control subjects, in whom diffusely and focally enhanced myocardium showed largely planar distributions (mean [± SD] FD, 2.12 ± 0.02 and 1.92 ± 0.09, respectively), whereas the combined domain was fractal in 3D (mean FD, 2.41 ± 0.04). The FD and volume of fibrotic tissue were logarithmically correlated in the population with ESRD. Fractal analysis of MT-weighted cardiac MRI data revealed that the geometric characteristics of cardiac scar in patients with ESRD transition from fractal in 2D to planar in 2D to fractal in 3D as scar volume increases. CONCLUSION. Fatal arrhythmias in individuals with ESRD are increasingly attributed to cardiac fibrosis. Histologic analysis reveals that fibrosis progresses via a fractal expansion pattern. The method presented in this study can be applied to characterize the irregular space-filling morphometry of any pathologic substrate identified by contrast enhancement across noninvasive imaging modalities.

Cardiac Chemical Exchange Saturation Transfer MR Imaging Tracking of Cell Survival or Rejection in Mouse Models of Cell Therapy.

Purpose To examine whether cardiac chemical exchange saturation transfer (CEST) imaging can be serially and noninvasively used to probe cell survival or rejection after intramyocardial implantation in mice. Materials and Methods Experiments were compliant with the National Institutes of Health Guidelines on the Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. One million C2C12 cells labeled with either europium (Eu) 10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid (HP-DO3A) or saline via the hypotonic swelling technique were implanted into the anterior-lateral left ventricular wall in C57BL/6J (allogeneic model, n = 17) and C3H (syngeneic model, n = 13) mice. Imaging (frequency offsets of ±15 parts per million) was performed 1, 10, and 20 days after implantation, with the asymmetrical magnetization transfer ratio (MTRasym) calculated from image pairs. Histologic examination was performed at the conclusion of imaging. Changes in MTRasym over time and between mice were assessed by using two-way repeated-measures analysis of variance. Results MTRasym was significantly higher in C3H and C57BL/6J mice in grafts of Eu-HP-DO3A-labeled cells (40.2% ± 5.0 vs 37.8% ± 7.0, respectively) compared with surrounding tissue (-0.67% ± 1.7 vs -1.8% ± 5.3, respectively; P < .001) and saline-labeled grafts (-0.4% ± 6.0 vs -1.2% ± 3.6, respectively; P < .001) at day 1. In C3H mice, MTRasym remained increased (31.3% ± 9.2 on day 10, 28.7% ± 5.2 on day 20; P < .001 vs septum) in areas of in Eu-HP-DO3A-labeled cell grafts. In C57BL/6J mice, corresponding MTRasym values (11.3% ± 8.1 on day 10, 5.1% ± 9.4 on day 20; P < .001 vs day 1) were similar to surrounding myocardium by day 20 (P = .409). Histologic findings confirmed cell rejection in C57BL/6J mice. Estimation of graft area was similar with cardiac CEST imaging and histologic examination (R2 = 0.89). Conclusion Cardiac CEST imaging can be used to image cell survival and rejection in preclinical models of cell therapy. © RSNA, 2016 Online supplemental material is available for this article.

Noninvasive mapping of endothelial dysfunction in myocardial ischemia by magnetic resonance imaging using an albumin-based contrast agent.

Noninvasive preclinical methods for the characterization of myocardial vascular function are crucial to an understanding of the dynamics of ischemic cardiac disease. Ischemic heart disease is associated with myocardial endothelial dysfunction, resulting in leakage of plasma albumin into the extravascular space. These features can be harnessed in a novel noninvasive three-dimensional magnetic resonance imaging method to measure fractional blood volume (fBV) and vascular permeability (permeability-surface area product, PS) using labeled albumin as a blood pool contrast agent. C57BL/6 mice were imaged before and 3 days after myocardial infarction (MI). Following the quantification of endogenous myocardial R1 , the dynamics of intravenously injected albumin-based contrast agent, extravasating from permeable myocardial blood vessels, were tracked on short-axis magnetic resonance images of the entire heart. This study successfully discriminated between infarcted and remote regions at 3 days post-infarct, based on a reduced fBV and increased PS in the infarcted region. These findings were confirmed using ex vivo fluorescence imaging and histology. We have demonstrated a novel method to quantify blood volume and permeability in the infarcted myocardium, providing an imaging biomarker for the assessment of endothelial dysfunction. This method has the potential to three-dimensionally visualize subtle changes in myocardial permeability and to track endothelial function for longitudinal cardiac studies determining pathophysiological processes during infarct healing.

An interactive videogame designed to improve respiratory navigator efficiency in children undergoing cardiovascular magnetic resonance.

BACKGROUND: Advanced cardiovascular magnetic resonance (CMR) acquisitions often require long scan durations that necessitate respiratory navigator gating. The tradeoff of navigator gating is reduced scan efficiency, particularly when the patient's breathing patterns are inconsistent, as is commonly seen in children. We hypothesized that engaging pediatric participants with a navigator-controlled videogame to help control breathing patterns would improve navigator efficiency and maintain image quality. METHODS: We developed custom software that processed the Siemens respiratory navigator image in real-time during CMR and represented diaphragm position using a cartoon avatar, which was projected to the participant in the scanner as visual feedback. The game incentivized children to breathe such that the avatar was positioned within the navigator acceptance window (±3 mm) throughout image acquisition. Using a 3T Siemens Tim Trio, 50 children (Age: 14 ± 3 years, 48 % female) with no significant past medical history underwent a respiratory navigator-gated 2D spiral cine displacement encoding with stimulated echoes (DENSE) CMR acquisition first with no feedback (NF) and then with the feedback game (FG). Thirty of the 50 children were randomized to undergo extensive off-scanner training with the FG using a MRI simulator, or no off-scanner training. Navigator efficiency, signal-to-noise ratio (SNR), and global left-ventricular strains were determined for each participant and compared. RESULTS: Using the FG improved average navigator efficiency from 33 ± 15 to 58 ± 13 % (p < 0.001) and improved SNR by 5 % (p = 0.01) compared to acquisitions with NF. There was no difference in navigator efficiency (p = 0.90) or SNR (p = 0.77) between untrained and trained participants for FG acquisitions. Circumferential and radial strains derived from FG acquisitions were slightly reduced compared to NF acquisitions (-16 ± 2 % vs -17 ± 2 %, p < 0.001; 40 ± 10 % vs 44 ± 11 %, p = 0.005, respectively). There were no differences in longitudinal strain (p = 0.38). CONCLUSIONS: Use of a respiratory navigator feedback game during navigator-gated CMR improved navigator efficiency in children from 33 to 58 %. This improved efficiency was associated with a 5 % increase in SNR for spiral cine DENSE. Extensive off-scanner training was not required to achieve the improvement in navigator efficiency.

Gadolinium free cardiovascular magnetic resonance with 2-point Cine balanced steady state free precession.

BACKGROUND: Cardiovascular magnetic resonance (CMR) of ventricular structure and function is widely performed using cine balanced steady state free precession (bSSFP) MRI. The bSSFP signal of myocardium is weighted by magnetization transfer (MT) and T1/T2-relaxation times. In edematous and fibrotic tissues, increased T2 and reduced MT lead to increased signal intensity on images acquired with high excitation flip angles. We hypothesized that acquisition of two differentially MT-weighted bSSFP images (termed 2-point bSSFP) can identify tissue that would enhance with gadolinium similar to standard of care late gadolinium enhancement (LGE). METHODS: Cine bSSFP images (flip angles of 5° and 45°) and native-T1 and T2 maps were acquired in one mid-ventricular slice in 47 patients referred for CMR and 10 healthy controls. Afterwards, LGE images and post-contrast T1 maps were acquired and gadolinium partition coefficient (GPC) was calculated. Maps of ΔS/So were calculated as (S45-S5)/S5*100 (%), where Sflip_angle is the voxel signal intensity. RESULTS: Twenty three patients demonstrated areas of myocardial hyper-enhancement with LGE. In enhanced regions, ΔS/So, native-T1, T2, and GPC were heightened (p < 0.05 vs. non-enhanced tissues). ΔS/So, native-T1, and T2 all demonstrated association with GPC, however the association was strongest for ΔS/So. Bland-Altman analysis revealed a slight bias towards larger volume of enhancement with ΔS/So compared to LGE, and similar transmurality. Subjective analysis with 2-blinded expert readers revealed agreement between ΔS/So and LGE of 73.4 %, with false positive detection of 16.7 % and false negative detection of 15.2 %. CONCLUSIONS: Gadolinium free 2-point bSSFP identified tissue that enhances at LGE with strong association to GPC. Our results suggest that with further development, MT-weighted CMR could be used similar to LGE for diagnostic imaging.

2D cine DENSE with low encoding frequencies accurately quantifies cardiac mechanics with improved image characteristics.

BACKGROUND: Displacement Encoding with Stimulated Echoes (DENSE) encodes displacement into the phase of the magnetic resonance signal. The encoding frequency (ke) maps the measured phase to tissue displacement while the strength of the encoding gradients affects image quality. 2D cine DENSE studies have used a ke of 0.10 cycles/mm, which is high enough to remove an artifact-generating echo from k-space, provide high sensitivity to tissue displacements, and dephase the blood pool. However, through-plane dephasing can remove the unwanted echo and dephase the blood pool without relying on high ke. Additionally, the high sensitivity comes with the costs of increased phase wrapping and intra-voxel dephasing. We hypothesized that ke below 0.10 cycles/mm can be used to improve image characteristics and provide accurate measures of cardiac mechanics. METHODS: Spiral cine DENSE images were obtained for 10 healthy subjects and 10 patients with a history of heart disease on a 3 T Siemens Trio. A mid-ventricular short-axis image was acquired with different ke: 0.02, 0.04, 0.06, 0.08, and 0.10 cycles/mm. Peak twist, circumferential strain, and radial strain were compared between acquisitions employing different ke using Bland-Altman analyses and coefficients of variation. The percentage of wrapped pixels in the phase images at end-systole was calculated for each ke. The dephasing of the blood signal and signal to noise ratio (SNR) were also calculated and compared. RESULTS: Negligible differences were seen in strains and twist for all ke between 0.04 and 0.10 cycles/mm. These differences were of the same magnitude as inter-test differences. Specifically, the acquisitions with 0.04 cycles/mm accurately quantified cardiac mechanics and had zero phase wrapping. Compared to 0.10 cycles/mm, the acquisitions with 0.04 cycles/mm had 9 % greater SNR and negligible differences in blood pool dephasing. CONCLUSIONS: For 2D cine DENSE with through-plane dephasing, the encoding frequency can be lowered to 0.04 cycles/mm without compromising the quantification of twist or strain. The amount of wrapping can be reduced with this lower value to greatly simplify the input to unwrapping algorithms. The strain and twist results from studies using different encoding frequencies can be directly compared.

Ovarian carcinoma: quantitative biexponential MR imaging relaxometry reveals the dynamic recruitment of ferritin-expressing fibroblasts to the angiogenic rim of tumors.

PURPOSE: To quantitatively monitor the dynamic perivascular recruitment of ferritin heavy chain (FHC)-overexpressing fibroblasts to ovarian carcinoma xenografts by using R2 mapping and biexponential magnetic resonance (MR) relaxometry. MATERIALS AND METHODS: In vivo studies of female mice were approved by the institutional animal care and use committee. In vitro analysis included MR-based R2 relaxation measurements of monkey kidney cell line (CV1) fibroblasts that overexpress FHC, followed by inductively coupled plasma mass spectrometry to assess cellular iron content. For in vivo analysis, CV1-FHC fibroblasts were either mixed with fluorescent human ovarian carcinoma cells before subcutaneous implantation (coinjection) or injected intraperitoneally 4 days after the cancer cells were injected (remote recruitment). Dynamic changes in tumor R2 were used to derive CV1-FHC cell fraction in both models. In coinjection tumors, dynamic contrast material-enhanced MR imaging was used to measure tumor fractional blood volume. Whole-body fluorescence imaging and immunohistochemical staining were performed to validate MR results. One-way repeated measures analysis of variance was used to assess MR and fluorescence imaging results and tumor volume, and one-way analysis of variance was used to assess spectrometric results, fractional blood volume, and immunohistochemical evaluation. RESULTS: CV1-FHC fibroblasts (vs CV1 fibroblasts) showed enhanced iron uptake (1.8 mmol ± 0.5 × 10(-8) vs 0.9 mmol ± 0.5 × 10(-8); P < .05), retention (1.6 mmol ± 0.5 × 10(-8) vs 0.5 mmol ± 0.5 × 10(-8), P < .05), and cell density-dependent R2 contrast. R2 mapping in vivo revealed preferential recruitment of CV1-FHC cells to the tumor rim in both models. Measurement of fractional blood volume was similar in all tumors (2.6 AU ± 0.5 × 10(-3) for CV1, 2.3 AU ± 0.3 × 10(-3) for CV1-FHC, 2.9 ± 0.3 × 10(-3) for CV1-FHC-ferric citrate). Dynamic changes in CV1-FHC cell fraction determined at MR relaxometry in both models were confirmed at immunohistochemical analysis. CONCLUSION: FHC overexpression, when combined with R2 mapping and MR relaxometry, enabled in vivo detection of the dynamic recruitment of exogenously administered fibroblasts to the vasculature of solid tumors.

MRI reporter genes: applications for imaging of cell survival, proliferation, migration and differentiation.

Molecular imaging strives to detect molecular events at the level of the whole organism. In some cases, the molecule of interest can be detected either directly or with targeted contrast media. However many genes and proteins and particularly those located in intracellular compartments are not accessible for targeted agents. The transcriptional regulation of these genes can nevertheless be detected, although indirectly, using reporter gene encoding for readily detectable proteins. Such reporter proteins can be expressed in the tissue of interest by genetically introducing the reporter gene in the target cells. Imaging of reporter genes has become a powerful tool in modern biomedical research. Typically, expression of fluorescent and bioluminescent proteins and the reaction product of expressed enzymes and exogenous substrates were examined using in vitro histological methods and in vivo whole body imaging methods. Recent advances in MRI reporter gene methods raised the possibility that MRI could become a powerful tool for concomitant high-resolution anatomical and functional imaging and for imaging of reporter gene activity. An immediate application of MRI reporter gene methods was by monitoring gene expression patterns in gene therapy and in vivo imaging of the survival, proliferation, migration and differentiation of pluripotent and multipotent cells used in cell-based regenerative therapies for cancer, myocardial infarction and neural degeneration. In this review, we characterized a variety of MRI reporter gene methods based on their applicability to report cell survival/proliferation, migration and differentiation. In particular, we discussed which methods were best suited for translation to clinical use in regenerative therapies.

Displacement-encoded and manganese-enhanced cardiac MRI reveal that nNOS, not eNOS, plays a dominant role in modulating contraction and calcium influx in the mammalian heart.

Within cardiomyocytes, endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) are thought to modulate L-type calcium channel (LTCC) function and sarcoplasmic reticulum calcium cycling, respectively. However, divergent results from mostly invasive prior studies suggest more complex roles. To elucidate the roles of nNOS and eNOS in vivo, we applied noninvasive cardiac MRI to study wild-type (WT), eNOS(-/-), and nNOS(-/-) mice. An in vivo index of LTCC flux (LTCCI) was measured at baseline (Bsl), dobutamine (Dob), and dobutamine + carbacholamine (Dob + CCh) using manganese-enhanced MRI. Displacement-encoded MRI assessed contractile function by measuring circumferential strain (E(cc)) and systolic (dE(cc)/dt) and diastolic (dE(cc)/dt(diastolic)) strain rates at Bsl, Dob, and Dob + CCh. Bsl LTCCI was highest in nNOS(-/-) mice (P < 0.05 vs. WT and eNOS(-/-)) and increased only in WT and eNOS(-/-) mice with Dob (P < 0.05 vs. Bsl). LTCCI decreased significantly from Dob levels with Dob + CCh in all mice. Contractile function, as assessed by E(cc), was similar in all mice at Bsl. With Dob, E(cc) increased significantly in WT and eNOS(-/-) but not nNOS(-/-) mice (P < 0.05 vs. WT and eNOS(-/-)). With Dob + CCh, E(cc) returned to baseline levels in all mice. Systolic blood pressure, measured via tail plethysmography, was highest in eNOS(-/-) mice (P < 0.05 vs. WT and nNOS(-/-)). Mice deficient in nNOS demonstrate increased Bsl LTCC function and an attenuated contractile reserve to Dob, whereas eNOS(-/-) mice demonstrate normal LTCC and contractile function under all conditions. These results suggest that nNOS, not eNOS, plays the dominant role in modulating Ca(2+) cycling in the heart.

Monocyte and/or macrophage infiltration of heart after myocardial infarction: MR imaging by using T1-shortening liposomes.

PURPOSE: To test the hypothesis that magnetic resonance (MR) imaging R1 (R1 = 1/T1) mapping after selectively labeling monocytes with a T1-shortening contrast agent in vivo would enable the quantitative measurement of their spatiotemporal kinetics in the setting of infarct healing. MATERIALS AND METHODS: All procedures were performed in mice and were approved by the institutional committee on animal research. One hundred microliters of dual-labeled liposomes (DLLs) containing gadolinium (Gd)-diethylenetriaminepentaacetic acid (DTPA)-bis(stearylamide) and DiI dye were used to label monocytes 2 days before myocardial infarction (MI). MI was induced by occlusion of the left anterior descending coronary artery for 1 hour, followed by reperfusion. MR imaging R1 mapping of mouse hearts was performed at baseline on day -3, on day 0 before MI, and on days 1, 4, and 7 after MI. Mice without labeling were used as controls. ΔR1 was calculated as the difference in R1 between mice with labeling and those without labeling. CD68 immunohistochemistry and DiI fluorescence microscopy were used to confirm that labeled monocytes and/or macrophages infiltrated the postinfarct myocardium. Statistical analysis was performed by using two-way analysis of variance and the unpaired two-sample t test. RESULTS: Infarct zone ΔR1 was slightly but nonsignificantly increased on day 1, maximum on day 4 (P < .05 vs all other days), and started to decrease by day 7 (P < .05 vs days -3, 0, and 1) after MI, closely reflecting the time course of monocyte and/or macrophage infiltration of the infarcted myocardium shown by prior histologic studies. Histologic results confirmed the presence and location of DLL-labeled monocytes and/or macrophages in the infarct zone on day 4 after MI. CONCLUSION: R1 mapping after labeling monocytes with T1-shortening DLLs enables the measurement of post-MI monocyte and/or macrophage spatiotemporal kinetics.

Quantitative pancreatic ß cell MRI using manganese-enhanced Look-Locker imaging and two-site water exchange analysis.

Pancreatic ß-cell imaging would be useful in monitoring the progression of and therapies for diabetes. The purpose of this study was to develop and evaluate quantitative ß-cell MRI using manganese (Mn(2+)) labeling of ß cells, T1 mapping, and a two-site water exchange model. Normal, pharmacologically-treated, and severely diabetic mice underwent injection of MnCl(2). Pancreatic water proton T1 relaxation was measured using Look-Locker MRI, and two-site water exchange analysis was used to estimate model parameters including the intracellular water proton relaxation rate constant (R1(ic)) and the intracellular fraction as indicators of ß-cell function and mass, respectively. Logarithmic plots of T1 relaxation revealed two distinct proton pools relaxing with different T1s, and the two-site water exchange model fit the measured T1 relaxation data better than a monoexponential model. The intracellular R1(ic) time course revealed the kinetics of ß-cell Mn(2+) labeling. Pharmacological treatments with nifedipine, tolbutamide, and diazoxide altered R1(ic), indicating that beta cell function was a determinant of Mn(2+) uptake. Intracellular fraction was significantly higher in mice with normal ß cell mass than in diabetic mice (14.9% vs. 14.4%, P < 0.05). Two-site water exchange analysis of T1 relaxation of the Mn(2+)-enhanced pancreas is a promising method for quantifying ß cell volume fraction and function.

Cyclical depressurization degranulates platelets in an agonist-free mechanism of platelet activation.

Activation of circulating platelets by receptor binding and subsequent coagulation events are defined by a well characterized physiological response. However, the growing prevalence of chronic kidney disease (CKD) and implication of platelet-released factors in worsening cardiovascular outcomes with hemodialysis warrant further investigation into the mechanobiology of platelet degranulation. The significant drops in pressure caused by high friction across the hemodialysis flow circuit present an overlooked platelet stimulant not involving immobilization as a driver for cytoskeletal rearrangement. In this study, platelets from healthy and dialysis (pre- and post-treatment) donors were cyclically depressurized in static suspension to measure changes in physiology by integrin αIIbß3 activation and surface P-selectin expression. The progressive increase in CD62P with no changes in PAC1 over pressure-cycling duration regardless of uremia signifies that hydrostatic depressurization involves a novel agonist-free mechanism leading to platelet degranulation as a unique case in which CD62P and PAC1 do not interchangeably indicate platelet activation. Subsequent stimulation using ADP further suggests that sustained depressurization regimens desensitize integrin αIIbß3 activation. Variability in platelet response caused by uremia and CKD are observed by elevated baseline PAC1 in pre-dialysis samples, PAC1 retention after ADP exposure, and maximum CD62P with ADP independent of pressure. Theory for hydrostatic pressure-induced degranulation circumventing integrin-initiated signal transduction is here presented based on the Starling Equation.

Hemodialysis exacerbates proteolytic imbalance and pro-fibrotic platelet dysfunction.

Multi-organ fibrosis among end stage renal disease (ESRD) patients cannot be explained by uremia alone. Despite mitigation of thrombosis during hemodialysis (HD), subsequent platelet dysfunction and tissue dysregulation are less understood. We comprehensively profiled plasma and platelets from ESRD patients before and after HD to examine HD-modulation of platelets beyond thrombotic activation. Basal plasma levels of proteolytic regulators and fibrotic factors were elevated in ESRD patients compared to healthy controls, with isoform-specific changes during HD. Platelet lysate (PL) RNA transcripts for growth and coagulative factors were elevated post-HD, with upregulation correlated to HD vintage. Platelet secretome correlations to plasma factors reveal acutely induced pro-fibrotic platelet phenotypes in ESRD patients during HD characterized by preferentially enhanced proteolytic enzyme translation and secretion, platelet contribution to inflammatory response, and increasing platelet dysfunction with blood flow rate (BFR) and Vintage. Compensatory mechanisms of increased platelet growth factor synthesis with acute plasma matrix metalloproteinase (MMP) and tissue inhibitor of MMPs (TIMP) increases show short-term mode-switching between dialysis sessions leading to long-term pro-fibrotic bias. Chronic pro-fibrotic adaptation of platelet synthesis were observed through changes in differential secretory kinetics of heterogenous granule subtypes. We conclude that chronic and acute platelet responses to HD contribute to a pro-fibrotic milieu in ESRD.

Improved arterial spin labeling after myocardial infarction in mice using cardiac and respiratory gated look-locker imaging with fuzzy C-means clustering.

Experimental myocardial infarction (MI) in mice is an important disease model, in part due to the ability to study genetic manipulations. MRI has been used to assess cardiac structural and functional changes after MI in mice, but changes in myocardial perfusion after acute MI have not previously been examined. Arterial spin labeling noninvasively measures perfusion but is sensitive to respiratory motion and heart rate variability and is difficult to apply after acute MI in mice. To account for these factors, a cardiorespiratory-gated arterial spin labeling sequence using a fuzzy C-means algorithm to retrospectively reconstruct images was developed. Using this method, myocardial perfusion was measured in remote and infarcted regions at 1, 7, 14, and 28 days post-MI. Baseline perfusion was 4.9 +/- 0.5 mL/g min and 1 day post-MI decreased to 0.9 +/- 0.8 mL/g min in infarcted myocardium (P < 0.05 versus baseline) while remaining at 5.2 +/- 0.8 mL/g min in remote myocardium. During the subsequent 28 days, perfusion in the remote zone remained unchanged, while a partial recovery of perfusion in the infarct zone was seen. This technique, when applied to genetically engineered mice, will allow for the investigation of the roles of specific genes in myocardial perfusion during infarct healing.