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.

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.

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.

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.

Longitudinal manganese-enhanced magnetic resonance imaging of delayed brain damage after hypoxic-ischemic injury in the neonatal rat.

BACKGROUND: Hypoxia-ischemia (HI) in the neonatal brain results in a prolonged injury process. Longitudinal studies using noninvasive methods can help elucidate the mechanisms behind this process. We have recently demonstrated that manganese-enhanced magnetic resonance imaging (MRI) can depict areas with activated microglia and astrogliosis 7 days after hypoxic-ischemic brain injury. OBJECTIVE: The current study aimed to follow brain injury after HI in rats longitudinally and compare manganese enhancement of brain areas to the development of injury and presence of reactive astrocytes and microglia. METHODS: The Vannucci model for hypoxic-ischemic injury in the neonatal rat was used. Pups were injected with either MnCl(2) or saline after 6 h and again on day 41 after HI. Longitudinal MRI (T(1) weighted) was performed 1, 3, 7 and 42 days after HI. The brains were prepared for immunohistochemistry after the final MRI. RESULTS: There was severe loss of cerebral tissue from day 7 to day 42 after HI. Most manganese-enhanced areas in the hippocampus, thalamus and basal ganglia at day 7 were liquefied after 42 days. Manganese-enhancement on day 42 corresponded to areas of activated microglia and reactive astrocytes in the remaining cortex, hippocampus and amygdala. However, the main area of enhancement was in the remaining thalamus in a calcified area surrounded by activated microglia and reactive astrocytes. CONCLUSION: Manganese-enhanced MRI can be a useful tool for in vivo identification of cerebral tissue undergoing delayed cell death and liquefaction after HI. Manganese enhancement at a late stage seems to be related to the accumulation of manganese in calcifications and gliotic tissue.

A comparison of retrospectively self-gated magnetic resonance imaging and high-frequency echocardiography for characterization of left ventricular function in mice.

Non-invasive imaging methods like echocardiography and magnetic resonance imaging (MRI) are very valuable in longitudinal follow-up studies of cardiac function in small animals. To be able to compare results from studies using different methods, and explain possible differences, it is important to know the agreement between these methods. As both self-gated high-field MRI and high-frequency echocardiography (hf-echo) M-mode are potential methods for evaluation of left ventricular (LV) function in healthy mice, our aim was to assess the agreement between these two methods. Fifteen healthy female C57BL/6J mice underwent both self-gated MRI and hf-echo during the same session of light isoflurane anaesthesia. LV dimensions were estimated offline, and agreement between the methods and reproducibility for the two methods assessed using Bland-Altman methods. In summary, hf-echo M-mode had better inter-observer repeatability than self-gated MRI for all measured parameters. Compared with hf-echo, systolic posterior wall thicknesses were significantly higher when measured by MRI, while diastolic anterior wall thicknesses were found to be significantly smaller. MRI measurements of diastolic LV diameter were also higher using MRI, resulting in larger fractional shortening values compared with the values obtained by hf-echo. In conclusion, hf-echo M-mode is easy to apply, has high temporal and spatial resolution, and good reproducibility. Self-gated MRI might be advantageous in cases of abnormal LV geometry and heterogeneous regional myocardial function, especially with improvements in spatial resolution. The moderate agreement between the methods must be taken into account when comparing studies using the two modalities.

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.

Manganese-enhanced magnetic resonance imaging of hypoxic-ischemic brain injury in the neonatal rat.

Hypoxic-ischemic injury (HI) to the neonatal brain results in delayed neuronal death with accompanying inflammation for days after the initial insult. The aim of this study was to depict delayed neuronal death after HI using Manganese-enhanced MRI (MEMRI) and to evaluate the specificity of MEMRI in detection of cells related to injury by comparison with histology and immunohistochemistry. 7-day-old Wistar rat pups were subjected to HI (occlusion of right carotid artery and 8% O(2) for 75 min). 16 HI (HI+Mn) and 6 sham operated (Sham+Mn) pups were injected with MnCl(2) (100 mM, 40 mg/kg) and 10 HI-pups (HI+Vehicle) received NaCl i.p. 6 h after HI. 3D T(1)-weighted images (FLASH) and 2D T(2)-maps (MSME) were acquired at 7 T 1, 3 and 7 days after HI. Pups were sacrificed after MR-scanning and brain slices were cut and stained for CD68, GFAP, MAP-2, Caspase-3 and Fluorojade B. No increased manganese-enhancement (ME) was detectable in the injured hemisphere on day 1 or 3 when immunohistochemistry showed massive ongoing neuronal death. 7 days after HI, increased ME was seen on T(1)-w images in parts of the injured cortex, hippocampus and thalamus among HI+Mn pups, but not among HI+Vehicle or Sham+Mn pups. Comparison with immunohistochemistry showed delayed neuronal death and inflammation in these areas with late ME. Areas with increased ME corresponded best with areas with high concentrations of activated microglia. Thus, late manganese-enhancement seems to be related to accumulation of manganese in activated microglia in areas of neuronal death rather than depicting neuronal death per se.

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.

In vivo mapping of temporospatial changes in manganese enhancement in rat brain during epileptogenesis.

Mesial temporal lobe epilepsy is associated with structural and functional abnormalities, such as hippocampal sclerosis and axonal reorganization. The temporal evolution of these changes remains to be determined, and there is a need for in vivo imaging techniques that can uncover the epileptogenic processes at an early stage. Manganese-enhanced magnetic resonance imaging may be useful in this regard. The aim of this study was to analyze the temporospatial changes in manganese enhancement in rat brain during the development of epilepsy subsequent to systemic kainate application (10 mg/kg i.p.). MnCl(2) was given systemically on day 2 (early), day 15 (latent), and 11 weeks (chronic phase) after the initial status epilepticus. Twenty-four hours after MnCl(2) injection T1-weighted 3D MRI was performed followed by analysis of manganese enhancement. In the medial temporal lobes, there was a pronounced decrease in manganese enhancement in CA1, CA3, dentate gyrus, entorhinal cortex and lateral amygdala in the early phase. In the latent and chronic phases, recovery of the manganese enhancement was observed in all these structures except CA1. A significant increase in manganese enhancement was detected in the entorhinal cortex and the amygdala in the chronic phase. In the latter phase, the structurally intact cerebellum showed significantly decreased manganese enhancement. The highly differentiated changes in manganese enhancement are likely to represent the net outcome of a number of pathological and pathophysiological events, including cell loss and changes in neuronal activity. Our findings are not consistent with the idea that manganese enhancement primarily reflects changes in glial cells.

Antitumor efficacy improved by local delivery of species-specific endostatin.

OBJECT: Conflicting results have been reported concerning the antitumor efficacy of the angiogenesis inhibitor endostatin. This may be due to differences in the biological distribution of endostatin between studies or to the varying biological efficacies of the different protein forms that were examined. To address this issue, the authors used a local delivery approach in which each tumor cell secreted endostatin, providing uniform endostatin levels throughout the tumors. This allowed a direct assessment of the biological efficacy of soluble endostatin in vivo. METHODS: The authors genetically engineered BT4C gliosarcoma cells so that they would stably express and secrete either the human or murine form of endostatin. Endostatin-producing cells or mock-infected cells were implanted intracerebrally in syngeneic BD-IX rats. The antitumor efficacy of endostatin was evaluated on the basis of survival data and tumor volume comparisons. In addition, microvascular parameters were assessed. The authors confirmed the continuous release of endostatin by the BT4C cells. A magnetic resonance imaging-assisted comparison of tumor volumes revealed that local production of murine endostatin significantly inhibited tumor growth. Notably, 40% of the animals in this treatment group experienced long-term survival without histologically verifiable tumors 7 months after cell implantation. After local treatment with murine endostatin, tumor blood plasma volumes were reduced by 71%, microvessel density counts by 84%, and vascular area fractions by 75%. In contrast, human endostatin did not inhibit tumor growth significantly in this model. Centrally located regions of necrosis were present in tumors secreting both the human and the murine species-specific form of endostatin. CONCLUSIONS: The results suggest that endostatin inhibits tumor angiogenesis in vivo in a species-specific manner.

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.

Collagenase increases the transcapillary pressure gradient and improves the uptake and distribution of monoclonal antibodies in human osteosarcoma xenografts.

Cancer therapy based on tumor-selective macromolecules may fail due to the elevated interstitial fluid pressure (IFP) that reduces the transvascular and interstitial convection in solid tumors. Modulation of the tumor extracellular matrix (ECM) may reduce IFP and enhance transvascular filtration and interstitial transport of macromolecules. We therefore measured the effect of the ECM-degrading enzyme collagenase on IFP and microvascular pressure (MVP) in human osteosarcoma xenografts using the wick-in-needle and micropipette methods, respectively. The tumor uptake and distribution of a systemically administered osteosarcoma-associated monoclonal antibody (TP-3) after i.v. injection of collagenase were analyzed using confocal laser scanning microscopy. Collagenase (0.1%) reduced both IFP (45%) and MVP (60%), but the kinetics of the recoveries differed, because MVP had recovered by the time IFP reached its minimum level. Thus, collagenase increased the transcapillary pressure gradient, inducing a 2-fold increase in the tumor uptake and improving the distribution of the monoclonal antibody, which was localized further into the tumor. To study the mechanism of the reduction in MVP, mean arterial blood pressure was measured and found not to be affected by the collagenase treatment. The reduction in MVP was rather due to reduced vascular resistance because microvascular-associated collagen was totally or partially disintegrated. Although collagenase may favor metastasis and thus not be clinically relevant, this study shows proof of principle that degradation of the ECM leads to a favorable change in the transvascular pressure gradient, thereby increasing antibody penetration and binding to tumor cells.

Radiation improves the distribution and uptake of liposomal doxorubicin (caelyx) in human osteosarcoma xenografts.

Liposomal drug delivery appears to improve the antitumor effect and reduce toxicity compared with the free drug. The therapeutic index may be improved further by combining cytotoxic drugs and radiotherapy. Successful therapy requires that the cytotoxic agents reach the tumor cells. Therefore, we studied tumor growth and the microdistribution of liposomal doxorubicin (Caelyx) with and without additional ionizing radiation in human osteosarcoma xenografts in athymic mice. Caelyx was injected i.v. 1 day before single or fractionated radiotherapy. Both chemoirradiation regimens induced significant tumor growth delays and worked synergistically. Confocal laser scanning microscopy showed that intact liposomes were located in close proximity to endothelial cells, and the distribution of released doxorubicin was heterogeneous. Before radiotherapy, hardly any doxorubicin was localized in the central parts of the tumor. Radiotherapy increased the tumor uptake of doxorubicin by a factor of two to four, with drug being redistributed farther from the vessels in the tumor periphery and located around vessels in the central parts of the tumor. Colocalization of doxorubicin and hypoxic cells showed no distribution of drug into hypoxic areas. Dynamic contrast-enhanced magnetic resonance imaging (MRI) 1 day before the injection of Caelyx and 2 days after treatment start showed that the combined treatment reduced the vascular volume and the vascular transfer rate of the MRI tracer. The results show that chemoirradiation with Caelyx induces synergistic treatment effects. Improved intratumoral drug uptake and distribution are responsible to some extent for the enhanced antitumor effect.