Aerobic interval training reduces inducible ventricular arrhythmias in diabetic mice after myocardial infarction.

Diabetes mellitus (DM) increases the risk of heart failure after myocardial infarction (MI), and aggravates ventricular arrhythmias in heart failure patients. Although exercise training improves cardiac function in heart failure, it is still unclear how it benefits the diabetic heart after MI. To study the effects of aerobic interval training on cardiac function, susceptibility to inducible ventricular arrhythmias and cardiomyocyte calcium handling in DM mice after MI (DM-MI). Male type 2 DM mice (C57BLKS/J Lepr (db) /Lepr (db) ) underwent MI or sham surgery. One group of DM-MI mice was submitted to aerobic interval training running sessions during 6 weeks. Cardiac function and structure were assessed by echocardiography and magnetic resonance imaging, respectively. Ventricular arrhythmias were induced by high-frequency cardiac pacing in vivo. Protein expression was measured by Western blot. DM-MI mice displayed increased susceptibility for inducible ventricular arrhythmias and impaired diastolic function when compared to wild type-MI, which was associated with disruption of cardiomyocyte calcium handling and increased calcium leak from the sarcoplasmic reticulum. High-intensity exercise recovered cardiomyocyte function in vitro, reduced sarcoplasmic reticulum diastolic calcium leak and significantly reduced the incidence of inducible ventricular arrhythmias in vivo in DM-MI mice. Exercise training also normalized the expression profile of key proteins involved in cardiomyocyte calcium handling, suggesting a potential molecular mechanism for the benefits of exercise in DM-MI mice. High-intensity aerobic exercise training recovers cardiomyocyte function and reduces inducible ventricular arrhythmias in infarcted diabetic mice.

Dynamic contrast enhanced MRI detects early response to adoptive NK cellular immunotherapy targeting the NG2 proteoglycan in a rat model of glioblastoma.

There are currently no established radiological parameters that predict response to immunotherapy. We hypothesised that multiparametric, longitudinal magnetic resonance imaging (MRI) of physiological parameters and pharmacokinetic models might detect early biological responses to immunotherapy for glioblastoma targeting NG2/CSPG4 with mAb9.2.27 combined with natural killer (NK) cells. Contrast enhanced conventional T1-weighted MRI at 7±1 and 17±2 days post-treatment failed to detect differences in tumour size between the treatment groups, whereas, follow-up scans at 3 months demonstrated diminished signal intensity and tumour volume in the surviving NK+mAb9.2.27 treated animals. Notably, interstitial volume fraction (ve), was significantly increased in the NK+mAb9.2.27 combination therapy group compared mAb9.2.27 and NK cell monotherapy groups (p = 0.002 and p = 0.017 respectively) in cohort 1 animals treated with 1 million NK cells. ve was reproducibly increased in the combination NK+mAb9.2.27 compared to NK cell monotherapy in cohort 2 treated with increased dose of 2 million NK cells (p<0.0001), indicating greater cell death induced by NK+mAb9.2.27 treatment. The interstitial volume fraction in the NK monotherapy group was significantly reduced compared to mAb9.2.27 monotherapy (p<0.0001) and untreated controls (p = 0.014) in the cohort 2 animals. NK cells in monotherapy were unable to kill the U87MG cells that highly expressed class I human leucocyte antigens, and diminished stress ligands for activating receptors. A significant association between apparent diffusion coefficient (ADC) of water and ve in combination NK+mAb9.2.27 and NK monotherapy treated tumours was evident, where increased ADC corresponded to reduced ve in both cases. Collectively, these data support histological measures at end-stage demonstrating diminished tumour cell proliferation and pronounced apoptosis in the NK+mAb9.2.27 treated tumours compared to the other groups. In conclusion, ve was the most reliable radiological parameter for detecting response to intralesional NK cellular therapy.

Late gadolinium enhancement in the assessment of the infarcted mouse heart: a longitudinal comparison with manganese-enhanced MRI.

PURPOSE: To evaluate late gadolinium-enhanced (LGE) assessment of infarct size, a comparison with manganese-enhanced magnetic resonance imaging (MEMRI), and histology was performed in a permanent infarction model in the mouse at the acute and chronic stage. MATERIALS AND METHODS: In a paired fashion at the acute and chronic stage after infarction (3-4 days and 21 days, respectively), LGE and MEMRI was performed using a self-gated fast low flip angle shot (FLASH). Infarct size was evaluated as the enhanced area relative to the complete myocardial wall area in a mid-ventricular slice. Paired comparisons were made between contrast agents and between timepoints, as well as to histology. RESULTS: At the acute stage, LGE delineated a larger infarct size as compared to both MEMRI and histology. Infarct size from LGE decreased from the acute to chronic stage, a temporal development not seen with MEMRI. At the chronic stage, no significant differences in infarct size were found between the methods. CONCLUSION: This study indicates an overenhancement of infarct size when using LGE, supported by an initial overestimation at the acute stage and a temporal decrease in infarct size from the acute to chronic stage, as compared to infarct size from MEMRI.

Simulated dive in rats lead to acute changes in cerebral blood flow on MRI, but no cerebral injuries to grey or white matter.

In this study, the effect of a simulated dive on rat brain was investigated using several magnetic resonance imaging (MRI)-methods and immunohistochemistry. Rats were randomly assigned to a dive- or a control group. The dive group was exposed to a simulated air dive to 600 kPa for 45 min. Pulmonary artery was monitored for vascular gas bubbles by ultrasound. MRI was performed 1 h after decompression and at one and 2 weeks after the dive with a different combination of MRI sequences at each time point. Two weeks after decompression, rats were sacrificed and brains were prepared for histology. Dived rats had a different time-curve for the dynamic contrast-enhanced MRI signal than controls with higher relative signal intensity, a tendency towards longer time to peak and a larger area under the curve for the whole brain on the acute MRI scan. On MRI, 1 and 2 weeks after dive, T2-maps showed no signal abnormalities or morphological changes. However, region of interest based measurements of T2 showed higher T2 in the brain stem among decompressed animals than controls after one and 2 weeks. Microscopical examination including immunohistochemistry did not reveal apparent structural or cellular injuries in any part of the rat brains. These observations indicate that severe decompression does not seem to cause any structural or cellular injury to the brain tissue of the rat, but may cause circulatory changes in the brain perfusion in the acute phase.

Labelling of olfactory ensheathing cells with micron-sized particles of iron oxide and detection by MRI.

A crucial issue in transplant-mediated repair of the damaged central nervous system (CNS) is serial non-invasive imaging of the transplanted cells, which has led to interest in the application of magnetic resonance imaging (MRI) combined with designated intracellular magnetic labels for cell tracking. Micron-sized particles of iron oxide (MPIO) have been successfully used to track cells by MRI, yet there is relatively little known about either their suitability for efficient labelling of specific cell types, or their effects on cell viability. The purpose of this study was to develop a suitable MPIO labelling protocol for olfactory ensheathing cells (OECs), a type of glia used to promote the regeneration of CNS axons after transplantation into the injured CNS. Here, we demonstrate an OEC labelling efficiency of >90% with an MPIO incubation time as short as 6 h, enabling intracellular particle uptake for single-cell detection by MRI without affecting cell proliferation, migration and viability. Moreover, MPIO are resolvable in OECs transplanted into the vitreous body of adult rat eyes, providing the first detailed protocol for efficient and safe MPIO labelling of OECs for non-invasive MRI tracking of transplanted OECs in real time for use in studies of CNS repair and axon regeneration.

Evaluation of intracellular labeling with micron-sized particles of iron oxide (MPIOs) as a general tool for in vitro and in vivo tracking of human stem and progenitor cells.

Magnetic resonance imaging (MRI)-based tracking is increasingly attracting attention as a means of better understanding stem cell dynamics in vivo. Intracellular labeling with micrometer-sized particles of iron oxide (MPIOs) provides a practical MRI-based approach due to superior detectability relative to smaller iron oxide particles. However, insufficient information is available about the general utility across cell types and the effects on cell vitality of MPIO labeling of human stem cells. We labeled six human cell types from different sources: mesenchymal stem cells derived from bone marrow (MSCs), mesenchymal stem cells derived from adipose tissue (ASCs), presumptive adult neural stem cells (ad-NSCs), fetal neural progenitor cells (f-NPCs), a glioma cell line (U87), and glioblastoma tumor stem cells (GSCs), with two different sizes of MPIOs (0.9 and 2.84 µm). Labeling and uptake efficiencies were highly variable among cell types. Several parameters of general cell function were tested in vitro. Only minor differences were found between labeled and unlabeled cells with respect to proliferation rate, mitotic duration, random motility, and capacity for differentiation to specific phenotypes. In vivo behavior was tested in chicken embryos and severe combined immunodeficient (SCID) mice. Postmortem histology showed that labeled cells survived and could integrate into various tissues. MRI-based tracking over several weeks in the SCID mice showed that labeled GSCs and f-NPCs injected into the brain exhibited translocations similar to those seen for unlabeled cells and as expected from migratory behavior described in previous studies. The results support MPIO-based cell tracking as a generally useful tool for studies of human stem cell dynamics in vivo.

Mn-alginate gels as a novel system for controlled release of Mn2+ in manganese-enhanced MRI.

The aim of the present study was to test alginate gels of different compositions as a system for controlled release of manganese ions (Mn(2+)) for application in manganese-enhanced MRI (MEMRI), in order to circumvent the challenge of achieving optimal MRI resolution without resorting to high, potentially cytotoxic doses of Mn(2+). Elemental analysis and stability studies of Mn-alginate revealed marked differences in ion binding capacity, rendering Mn/Ba-alginate gels with high guluronic acid content most stable. The findings were corroborated by corresponding differences in the release rate of Mn(2+) from alginate beads in vitro using T(1)-weighted MRI. Furthermore, intravitreal (ivit) injection of Mn-alginate beads yielded significant enhancement of the rat retina and retinal ganglion cell (RGC) axons 24 h post-injection. Subsequent compartmental modelling and simulation of ivit Mn(2+) transport and concentration revealed that application of slow release contrast agents can achieve a significant reduction of ivit Mn(2+) concentration compared with bolus injection. This is followed by a concomitant increase in the availability of ivit Mn(2+) for uptake by RGC, corresponding to significantly increased time constants. Our results provide proof-of-concept for the applicability of Mn-alginate gels as a system for controlled release of Mn(2+) for optimized MEMRI application.

In vivo MRI of olfactory ensheathing cell grafts and regenerating axons in transplant mediated repair of the adult rat optic nerve.

The purpose of the present study was to use magnetic resonance imaging (MRI) as a tool for monitoring transplant-mediated repair of the adult rat visual pathway. We labelled rat olfactory ensheathing cells (OECs) using micron-sized particles of iron oxide (MPIO) and transplanted them by: i) intravitreal injection (ivit) and ii) intra-optic nerve (ON) injection (iON) in adult rats with ON crush (ONC) injury. We applied T(2)-weighted MRI and manganese-enhanced MRI (MEMRI) to visualise transplanted cells and ON axons at specific times after injury and cell engraftment. Our findings demonstrate that ivit MPIO-labelled OECs are unequivocally detected by T(2)-weighted MRI in vivo and that the T(1)-weighted 3D FLASH sequence applied for MEMRI facilitates simultaneous visualisation of Mn(2+-) enhanced regenerating retinal ganglion cell (RGC) axons and MPIO-labelled OEC grafts. Furthermore, analysis of MRI data and ultrastructural findings supports the hypothesis that iON OEC transplants mediate regeneration and remyelination of RGC axons post injury.

Axonal tracing of the normal and regenerating visual pathway of mouse, rat, frog, and fish using manganese-enhanced MRI (MEMRI).

PURPOSE: To assess optic nerve (ON) regeneration after injury by applying manganese-enhanced MRI (MEMRI) in a study of comparative physiology between nonregenerating rat and mouse species and regenerating frog and fish species. MATERIALS AND METHODS: The normal visual projections of rats, mice, frogs, and fish was visualized by intravitreal MnCl(2) injection followed by MRI. Rats and mice with ON crush (ONC) were divided into nonregenerating (ONC only), and regenerating animals with peripheral nerve graft (ONC+PNG; rats) or lens injury (ONC+LI; mice) and monitored by MEMRI at 1 and 20 days post-lesion (dpl). Frog and fish with ON transection (ONT) were monitored by MEMRI up to 6 months postlesion (mpl). RESULTS: Signal intensity profiles of the Mn(2+)-enhanced ON were consistent with ON regeneration in the ONC+PNG and ONC+LI rat and mice groups, respectively, compared with the nonregenerating ONC groups. Furthermore, signal intensity profiles of the Mn(2+)-enhanced ON obtained between 1 mpl and 6 mpl in the fish and frog groups, respectively, were consistent with spontaneous, complete ON regeneration. CONCLUSION: Taken together, these results demonstrate that MEMRI is a viable method for serial, in vivo monitoring of normal, induced, and spontaneously regenerating optic nerve axons in different species.

Manganese transport in the rat optic nerve evaluated with spatial- and time-resolved magnetic resonance imaging.

PURPOSE: 1) To evaluate a novel theoretical model for in vivo axonal Mn(2+) transport with MRI data from the rat optic nerve (ON); and 2) to compare predictions from the new model with previously reported experimental data. MATERIALS AND METHODS: Time-resolved in vivo T(1)-weighted magnetic resonance imaging (MRI) of adult female Sprague-Dawley rat (n = 9) ON was obtained at different timepoints after intravitreal MnCl(2) injection. A concentration-dependent and a rate-dependent function for the Mn(2+) retinal ganglion cell (RGC) axon entrance was convolved with three different transport functions and each model system was optimized to fit the ON data. RESULTS: The rate-limited input function gave a better fit to the data than the concentration-limited input. Simulations showed that the rate-limited input leads to a semilogarithmic relationship between injected dose and Mn(2+) concentration in the ON, which is in agreement with previously reported in vivo experiments. A random walk transport model and an anterograde predominant slow model gave a similar fit to the data, both better than an anterograde predominant fast model. CONCLUSION: The results indicate that Mn(2+) input into RGC axons is limited by a maximum entrance rate into the axons. Also, a wide range of apparent Mn(2+) transport rates seems to be involved, different from synaptic vesicle transport rates, meaning that manganese does not depict synaptic vesicle transport rates directly.

Characterization of tumor microvascular structure and permeability: comparison between magnetic resonance imaging and intravital confocal imaging.

Solid tumors are characterized by abnormal blood vessel organization, structure, and function. These abnormalities give rise to enhanced vascular permeability and may predict therapeutic responses. The permeability and architecture of the microvasculature in human osteosarcoma tumors growing in dorsal window chambers in athymic mice were measured by confocal laser scanning microscopy (CLSM) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). Dextran (40 kDa) and Gadomer were used as molecular tracers for CLSM and DCE-MRI, respectively. A significant correlation was found between permeability indicators. The extravasation rate K(i) as measured by CLSM correlated positively with DCE-MRI parameters, such as the volume transfer constant K(trans) and the initial slope of the contrast agent concentration-time curve. This demonstrates that these two techniques give complementary information. Extravasation was further related to microvascular structure and was found to correlate with the fractal dimension and vascular density. The structural parameter values that were obtained from CLSM images were higher for abnormal tumor vasculature than for normal vessels.

Combination of Mn(2+)-enhanced and diffusion tensor MR imaging gives complementary information about injury and regeneration in the adult rat optic nerve.

PURPOSE: To evaluate manganese (Mn(2+))-enhanced MRI (MEMRI) and diffusion tensor imaging (DTI) as tools for detection of axonal injury and regeneration after intravitreal peripheral nerve graft (PNG) implantation in the rat optic nerve (ON). MATERIALS AND METHODS: In adult Fischer rats, retinal ganglion cell (RGC) survival was evaluated in Flurogold (FG) back-filled retinal whole mounts after ON crush (ONC), intravitreal PNG, and intravitreal MnCl(2) injection (150 nmol) at 0 and 20 days post lesion (dpl). MEMRI and echo-planar DTI (DTI-EPI) was obtained of noninjured ON one day after intravitreal MnCl(2) injection, and at 1 and 21 dpl after ONC, intravitreal PNG, and intravitreal MnCl(2) injections given at 0 and 20 dpl. GAP-43 immunohistochemistry was performed after the last MRI. RESULTS: ONC reduced RGC density in retina by 94% at 21 dpl compared to noninjured ON without MnCl(2) injections. Both intravitreal PNG and intravitreal MnCl(2) injections improved RGC survival in retina, which was reduced by 90% (ONC+MnCl(2)), 82% (ONC+PNG), and 74% (ONC+PNG+MnCl(2)) compared to noninjured ON. DTI-derived parameters (fractional anisotropy [FA], mean diffusivity, axial diffusivity lambda( parallel), and radial diffusivity lambda( perpendicular)) were unaffected by the presence of Mn(2+) in the ON. At 1 dpl, CNR(MEMRI) and lambda( parallel) were reduced at the injury site, while at 21 dpl they were increased at the injury site compared to values measured at 1 dpl. GAP-43 immunoreactive axons were present in the ON distal to the ONC injury site. CONCLUSION: MEMRI and DTI enabled detection of functional and structural degradation after rat ON injury, and there was correlation between the MRI-derived and immunohistochemical measures of axon regeneration.

Manganese-enhanced MRI of the rat visual pathway: acute neural toxicity, contrast enhancement, axon resolution, axonal transport, and clearance of Mn(2+).

PURPOSE: To provide dose-response data for the safe and effective use of MnCl(2) for manganese (Mn(2+)) -enhanced MRI (MEMRI) of the visual pathway. MATERIALS AND METHODS: Retinal ganglion cell (RGC) toxicity, CNR in MEMRI, axon density resolution for MEMRI, mode of axonal transport and clearance of Mn(2+) from the vitreous after ivit were investigated. After 0, 30, 150, 300, 1500, and 3000 nmol ivit MnCl(2), neural toxicity was measured by counting surviving RGC back-filled with FluroGold (FG), CNR of the vitreous body and visual pathway by three-dimensional (3D) MEMRI, resolution of ON axon density by correlating CNR with axon density, and axonal transport of Mn(2+) by studying CNR in 3D MEMRI of the ON after ion of 200 nmol MnCl(2). RESULTS: There were no changes in RGC density after ivit MnCl(2) 0 were recorded distally from the ion site, but there was no signal in the retina. At ivit doses >1500 nmol, clearance from the vitreous body was impaired. CONCLUSION: The optimal dose for MEMRI of the rat visual pathway was found to be 150-300 nmol ivit MnCl(2). Higher doses are toxic, causing RGC death, impair active clearance from the vitreous, and loss of Mn(2+) enhancement throughout the visual pathway. Mn(2+) traffic within RGC axons is mediated mainly by anterograde transport.

Axon tracing in the adult rat optic nerve and tract after intravitreal injection of MnDPDP using a semiautomatic segmentation technique.

PURPOSE: To develop and validate an objective technique for 3D segmentation of manganese-enhanced MR images of the optic nerve/tract (ON) in adult rats to improve contrast-to-noise (CNR) calculations and use the technique to ascertain if manganese dipyridoxyl diphosphate (MnDPDP) gives sufficient Mn(2+) enhancement compared to MnCl(2) when used for functional imaging of the visual pathway. MATERIALS AND METHODS: Intravitreous injection of the manganese-releasing MR contrast agent MnDPDP (30 nmol Mn(2+)) was performed to trace the ON in adult rats (n = 4). A positive control group of rats (n = 5) received a standard preparation of MnCl(2) (200 nmol Mn(2+)), while gadodiamide (1500 nmol Gd(3+)) was administered in negative control rats (n = 2). An objective technique for 3D segmentation of the enhanced ON was developed. CNR profiles along the ON were calculated by resampling the 3D image-volume in 2D planes perpendicular to the Mn(2+) enhanced ON in 0.2 mm steps, 4 mm along the segmented ON measured from the lamina cribrosa. RESULTS: The ON was successfully segmented and CNR calculated accurately within 2 minutes in a representative 3D MR image volume. Intravitreal MnDPDP injection resulted in significant MRI contrast enhancement of the retina and ON after 12-24 hours similar to that of MnCl(2) injection. CONCLUSION: 3D semiautomated image segmentation and the use of MnDPDP can improve in vivo axon tracing based on MRI. Mn(2+) was found to be released from MnDPDP after intravitreal injection in sufficient amounts to obtain functional tracing of the adult rat primary visual pathway.

Fluctuations in pO2 in irradiated human melanoma xenografts.

Several studies have demonstrated that untreated tumors may show significant fluctuations in tissue oxygen tension (pO(2)). Radiation treatment may induce changes in the tumor microenvironment that alter the pO(2) fluctuation pattern. The purpose of the present study was to investigate whether pO(2) fluctuations may also occur in irradiated tumors. A-07 human melanoma xenografts were irradiated with single doses of 0, 5 or 10 Gy. Fluctuations in pO(2) were recorded with OxyLite probes prior to irradiation and 24 and 72 h after the radiation exposure. Radiation-induced changes in the tumor microenvironment (i.e. blood perfusion and extracellular volume fraction) were assessed by dynamic contrast-enhanced magnetic resonance imaging. Seventy-two hours after 10 Gy, tumor blood perfusion had decreased to approximately 40% of that prior to irradiation, whereas the extracellular volume fraction had increased by approximately 25%. Fluctuations in pO(2) were seen in most tumors, irrespective of radiation dose and time after irradiation. The mean pO(2), the number of fluctuations around the mean pO(2), the number of fluctuations around threshold pO(2) values of 1, 2, 3, 5, 7 and 10 mmHg, and the amplitude of the fluctuations were determined for each pO(2) trace. No significant differences were detected between irradiated and unirradiated tumors. The results showed that pO(2) fluctuations may occur in irradiated tumors and that the pO(2) fluctuation pattern in A-07 tumors exposed to 5 or 10 Gy is similar to that in untreated tumors. Consequently, these doses did not induce changes in the tumor microenvironment that were sufficient to cause detectable alterations in the pO(2) fluctuation pattern.

Manganese-enhanced MRI of the optic visual pathway and optic nerve injury in adult rats.

PURPOSE: To evaluate manganese (Mn2+)-enhanced MRI in a longitudinal study of normal and injured rat visual projections. MATERIALS AND METHODS: MRI was performed 24 hours after unilateral intravitreal injection of MnCl2 (150 nmol) into adult Fischer rats that were divided into four groups: 1) controls (N = 5), 2) dose-response (N = 10, 0.2-200 nmol), 3) time-response with repeated MRI during 24-168 hours post injection (N = 4), and 4) optic nerve crush (ONC) immediately preceding the MnCl2 injection (N = 7). Control and ONC animals were reinjected with MnCl2 20 days after the first injection, and MRI was performed 24 hours later. RESULTS: In the control group, the optic projection was visualized from the retina to the superior colliculus, with indications of transsynaptic transport to the cortex. There was a semilogarithmic relationship between the Mn2+ dose and Mn2+ enhancement from 4 to 200 nmol, and the enhancement decayed gradually to 0 by 168 hours. No Mn2+-enhanced signal was detected distal to the ON crush site. In the control group, similar enhancement was obtained after the first and second MnCl2 injections, while in the ONC group the enhancement proximal to the crush site was reduced 20 days post lesion (20 dpl). CONCLUSION: Mn2+-enhanced MRI is a viable method for temporospatial visualization of normal and injured ON in the adult rat. The observed reduction in the Mn2+ signal proximal to the ONC is probably a result of retrograde damage to the retinal ganglion cells, and not of Mn2+ toxicity.