Manganese-Loaded Liposomes: An In Vitro Study for Possible Diagnostic Application.

The present study investigates the possible use of manganese (Mn)-based liposomal formulations for diagnostic applications in imaging techniques such as magnetic resonance imaging (MRI), with the aim of overcoming the toxicity limitations associated with the use of free Mn2+. Specifically, anionic liposomes carrying two model Mn(II)-based compounds, MnCl2 (MC) and Mn(HMTA) (MH), were prepared and characterised in terms of morphology, size, loading capacity, and in vitro activity. Homogeneous dispersions characterised mainly by unilamellar vesicles were obtained; furthermore, no differences in size and morphology were detected between unloaded and Mn-loaded vesicles. The encapsulation efficiency of MC and MH was evaluated on extruded liposomes by means of ICP-OES analysis. The obtained results showed that both MC and MH are almost completely retained by the lipid portion of liposomes (LPs), with encapsulation efficiencies of 99.7% for MC and 98.8% for MH. The magnetic imaging properties of the produced liposomal formulations were investigated for application in a potential preclinical scenario by collecting magnetic resonance images of a phantom designed to compare the paramagnetic contrast properties of free MC and MH compounds and the corresponding manganese-containing liposome dispersions. It was found that both LP-MC and LP-MH at low concentrations (0.5 mM) show better contrast (contrast-to-noise ratios of 194 and 209, respectively) than solutions containing free Mn at the same concentrations (117 and 134, respectively) and are safe to use on human cells at the selected dose. Taken together, the results of this comparative analysis suggest that these liposome-containing Mn compounds might be suitable for diagnostic purposes.

Brain Network Allostasis after Chronic Alcohol Drinking Is Characterized by Functional Dedifferentiation and Narrowing.

Alcohol use disorder (AUD) causes complex alterations in the brain that are poorly understood. The heterogeneity of drinking patterns and the high incidence of comorbid factors compromise mechanistic investigations in AUD patients. Here we used male Marchigian Sardinian alcohol-preferring (msP) rats, a well established animal model of chronic alcohol drinking, and a combination of longitudinal resting-state fMRI and manganese-enhanced MRI to provide objective measurements of brain connectivity and activity, respectively. We found that 1 month of chronic alcohol drinking changed the correlation between resting-state networks. The change was not homogeneous, resulting in the reorganization of pairwise interactions and a shift in the equilibrium of functional connections. We identified two fundamentally different forms of network reorganization. First is functional dedifferentiation, which is defined as a regional increase in neuronal activity and overall correlation, with a concomitant decrease in preferential connectivity between specific networks. Through this mechanism, occipital cortical areas lost their specific interaction with sensory-insular cortex, striatal, and sensorimotor networks. Second is functional narrowing, which is defined as an increase in neuronal activity and preferential connectivity between specific brain networks. Functional narrowing strengthened the interaction between striatal and prefrontocortical networks, involving the anterior insular, cingulate, orbitofrontal, prelimbic, and infralimbic cortices. Importantly, these two types of alterations persisted after alcohol discontinuation, suggesting that dedifferentiation and functional narrowing rendered persistent network states. Our results support the idea that chronic alcohol drinking, albeit at moderate intoxicating levels, induces an allostatic change in the brain functional connectivity that propagates into early abstinence.SIGNIFICANCE STATEMENT Excessive consumption of alcohol is positioned among the top five risk factors for disease and disability. Despite this priority, the transformations that the nervous system undergoes from an alcohol-naive state to a pathologic alcohol drinking are not well understood. In our study, we use an animal model with proven translational validity to study this transformation longitudinally. The results show that shortly after chronic alcohol consumption there is an increase in redundant activity shared by brain structures, and the specific communication shrinks to a set of pathways. This functional dedifferentiation and narrowing are not reversed immediately after alcohol withdrawal but persist during early abstinence. We causally link chronic alcohol drinking with an early and abstinence-persistent retuning of the functional equilibrium of the brain.

LA-ICP-MS bioimaging demonstrated disturbance of metal ions in the brain of Parkinson's disease model mouse undergoing manganese-enhanced MRI.

Manganese-enhanced MRI (MEMRI) is a powerful tool to study neuronal activity and microarchitecture in vivo. Yet the influence of exogenous manganese on the brain of the Parkinson's disease (PD) model mouse is poorly understood. Laser ablation connected to inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging for tissue section is an ideal tool to simultaneously analyze the metabolism of endogenous metal ions. In this study, DJ-1 knockout PD model mice were subjected to an MnCl2 saline treatment and the distribution of Mn and several other endogenous metal ions in brain regions was assessed by MEMRI and LA-ICP-MS imaging. The results demonstrated that Mn mainly deposited in subcortical regions, such as ventricles, hippocampus (HC), medial preoptic nucleus (MPO), lateral septal nucleus (LS), and ventromedial hypothalamic nucleus (VMH). The enhanced signal-to-noise ratio (S/N) determined by MEMRI for Mn is closely related to the signal in LA-ICP-MS imaging. Significantly, the treatment of MnCl2 disturbs the homeostasis of iron, zinc, copper, and calcium in the DJ-1 mouse, which could result in more severe symptoms of PD. Therefore, the application of MEMRI in the study of neurological disease must be made with caution.

Adult brain activation in response to pain is changed by neonatal painful stimulation according to sex: A manganese-enhanced MRI study.

Although it is known that nociceptive stimulation in the first postnatal week in rats is useful to model preterm pain, resulting in activation of specific brain areas, as assessed in vivo using manganese-enhanced magnetic resonance imaging (MEMRI), little is known about its long-term effects and sex specificity. Here we aimed to investigate whether inflammatory pain induced in male and female adult rats modify the pattern of brain activation between animals subjected or not to neonatal pain. For this, Complete Freund's adjuvant (CFA) was injected into the left hind paw of rat pups on postnatal day 1 (P1) or P8 to induce inflammatory response. During adulthood, CFA-treated and control animals were injected with CFA 1 hr prior MRI. MEMRI has the ability to enhance the contrast of selective brain structures in response to a specific stimulus, as the pain. MEMRI responses were consistent with activation of nociceptive pathways and these responses were reduced in animals treated with CFA on P1, but increased in animals treated on P8, mainly in the female group. In agreement, P8 female group showed exacerbated responses in the thermal nociceptive test. Using MEMRI, we conclude that the natural ability of adult rats to recognize and react to pain exposition is modified by neonatal painful exposition, mainly among females.

A hierarchy of manganese competition and entry in organotypic hippocampal slice cultures.

Contrast agents improve clinical and basic research MRI. The manganese ion (Mn2+ ) is an essential, endogenous metal found in cells and it enhances MRI contrast because of its paramagnetic properties. Manganese-enhanced MRI (MEMRI) has been widely used to image healthy and diseased states of the body and the brain in a variety of animal models. There has also been some work in translating the useful properties of MEMRI to humans. Mn2+ accumulates in brain regions with high neural activity and enters cells via voltage-dependent channels that flux calcium (Ca2+ ). In addition, metal transporters for zinc (Zn2+ ) and iron (Fe2+ ) can also transport Mn2+ . There is also transfer through channels specific for Mn2+ . Although Mn2+ accumulates in many tissues including brain, the mechanisms and preferences of its mode of entry into cells are not well characterized. The current study used MRI on living organotypic hippocampal slice cultures to detect which transport mechanisms are preferentially used by Mn2+ to enter cells. The use of slice culture overcomes the presence of the blood brain barrier, which limits inferences made with studies of the intact brain in vivo. A range of Mn2+ concentrations were used and their effects on neural activity were assessed to avoid using interfering doses of Mn2+ . Zn2+ and Fe2+ were the most efficient competitors for Mn2+ uptake into the cultured slices, while the presence of Ca2+ or Ca2+ channel antagonists had a more moderate effect. Reducing slice activity via excitatory receptor antagonists was also effective at lowering Mn2+ uptake. In conclusion, a hierarchy of those agents which influence Mn2+ uptake was established to enhance understanding of how Mn2+ enters cells in a cultured slice preparation.

Broad Kinase Inhibition Mitigates Early Neuronal Dysfunction in Tauopathy.

Tauopathies are a group of more than twenty known disorders that involve progressive neurodegeneration, cognitive decline and pathological tau accumulation. Current therapeutic strategies provide only limited, late-stage symptomatic treatment. This is partly due to lack of understanding of the molecular mechanisms linking tau and cellular dysfunction, especially during the early stages of disease progression. In this study, we treated early stage tau transgenic mice with a multi-target kinase inhibitor to identify novel substrates that contribute to cognitive impairment and exhibit therapeutic potential. Drug treatment significantly ameliorated brain atrophy and cognitive function as determined by behavioral testing and a sensitive imaging technique called manganese-enhanced magnetic resonance imaging (MEMRI) with quantitative R1 mapping. Surprisingly, these benefits occurred despite unchanged hyperphosphorylated tau levels. To elucidate the mechanism behind these improved cognitive outcomes, we performed quantitative proteomics to determine the altered protein network during this early stage in tauopathy and compare this model with the human Alzheimer's disease (AD) proteome. We identified a cluster of preserved pathways shared with human tauopathy with striking potential for broad multi-target kinase intervention. We further report high confidence candidate proteins as novel therapeutically relevant targets for the treatment of tauopathy. Proteomics data are available via ProteomeXchange with identifier PXD023562.

Maternal immune activation during pregnancy impacts on brain structure and function in the adult offspring.

Gestational infection constitutes a risk factor for the occurrence of psychiatric disorders in the offspring. Activation of the maternal immune system (MIA) with subsequent impact on the development of the fetal brain is considered to form the neurobiological basis for aberrant neural wiring and the psychiatric manifestations later in offspring life. The examination of validated animal models constitutes a premier resource for the investigation of the neural underpinnings. Here we used a mouse model of MIA based upon systemic treatment of pregnant mice with Poly(I:C) (polyriboinosinic-polyribocytidilic acid), for the unbiased and comprehensive analysis of the impact of MIA on adult offspring brain activity, morphometry, connectivity and function by a magnetic resonance imaging (MRI) approach. Overall lower neural activity, smaller brain regions and less effective fiber structure were observed for Poly(I:C) offspring compared to the control group. The corpus callosum was significantly smaller and presented with a disruption in myelin/ fiber structure in the MIA progeny. Subsequent resting-state functional MRI experiments demonstrated a paralleling dysfunctional interhemispheric connectivity. Additionally, while the overall flow of information was intact, cortico-limbic connectivity was hampered and limbic circuits revealed hyperconnectivity in Poly(I:C) offspring. Our study sheds new light on the impact of maternal infection during pregnancy on the offspring brain and identifies aberrant resting-state functional connectivity patterns as possible correlates of the behavioral phenotype with relevance for psychiatric disorders.

Effect of early experience on neuronal and behavioral responses to con- and heterospecific odors in closely related Mus taxa: epigenetic contribution in formation of precopulatory isolation.

BACKGROUND: The most effective learning occurs during sensitive periods. Olfactory plasticity to main social olfactory cues is limited to a critical period to a large degree. The objective was to evaluate the influence of early olfactory experience on the behavioral and neuronal responses of males to con- and heterospecific odors of receptive females in two species, M. musculus (subspecies musculus, wagneri) and M. spicilegus, and thus to determine the potential role of epigenetic contribution in the formation of precopulatory isolation. RESULTS: Males were reciprocally cross-fostered shortly after the birth and were tested for response to con- and heterospecific urine odors of estrus females using two-choice tests at 70-85 days of age. Neuronal activity of non- and cross-fostered males was evaluated at 90-110 days of age in the MOB and AOB to con- and heterospecific female odor using fMRI (MEMRI). Non-cross-fostered males of three taxa demonstrated a strong preference for odor of conspecific females compared to odor of heterospecific ones. Spicilegus-nursed musculus preferred odor of heterospecific females. Wagneri-nursed spicilegus and spicilegus-nursed wagneri did not demonstrate significant choice of con - or heterospecific female odor. The level of MRI signal obtained from the evaluation of manganese accumulation in AOB neurons was significantly higher when the odor of conspecific estrus females was exposed, compared to urine exposure of heterospecific females. The response pattern changed to the opposite in males raised by heterospecific females. Response patterns of neuronal activity in the MOB to con- and heterospecific female odors were different in cross-fostered and control males. CONCLUSION: The maternal environment, including odor, had a greater effect on the level of MRI signal in the AOB than the genetic relationships of the recipient and the donor of the odor stimulus. Behavioral and neuronal responses to con- and heterospecific odors changed in closely related Mus taxa as a result of early experience. We demonstrated the importance of early learning in mate choice in adulthood in mice and the possibility of epigenetic contribution in the formation of precopulatory reproductive isolation.

Cerebral hunger maps in rodents and humans by diffusion weighted MRI.

We design, implement and validate a novel image processing strategy to obtain in vivo maps of hunger stimulation in the brain of mice, rats and humans, combining Diffusion Weighted Magnetic Resonance Imaging (DWI) datasets from fed and fasted subjects. Hunger maps were obtained from axial/coronal (rodents/humans) brain sections containing the hypothalamus and coplanar cortico-limbic structures using Fisher's Discriminant Analysis of the combined voxel ensembles from both feeding situations. These maps were validated against those provided by the classical mono-exponential diffusion model as applied over the same subjects and conditions. Mono-exponential fittings revealed significant Apparent Diffusion Coefficient (ADC) decreases through the brain regions stimulated by hunger, but rigorous parameter estimations imposed the rejection of considerable number of pixels. The proposed approach avoided pixel rejections and provided a representation of the combined DWI dataset as a pixel map of the "Hunger Index" (HI), a parameter revealing the hunger score of every pixel. The new methodology proved to be robust both, by yielding consistent results with classical ADC maps and, by reproducing very similar HI maps when applied to newly acquired rodent datasets. ADC and HI maps demonstrated similar patterns of activation by hunger in hypothalamic and cortico-limbic structures of the brain of rodents and humans, albeit with different relative intensities, rodents showing more intense activations by hunger than humans, for similar fasting periods. The proposed methodology may be easily extended to other feeding paradigms or even to alternative imaging methods.

Continuous manganese delivery via osmotic pumps for manganese-enhanced mouse MRI does not impair spatial learning but leads to skin ulceration.

Manganese-enhanced magnetic resonance imaging (MEMRI) is a widely used technique in rodent neuroimaging studies. Traditionally, Mn2+ is delivered to animals via a systemic injection; however, this can lead to toxic effects at high doses. Recent studies have shown that subcutaneously implanted mini-osmotic pumps can be used to continuously deliver manganese chloride (MnCl2), and that they produce satisfactory contrast while circumventing many of the toxic side effects. However, neither the time-course of signal enhancement nor the effect of continuous Mn2+ delivery on behaviour, particularly learning and memory, have been well-characterized. Here, we investigated the effect of MnCl2 dose and route of administration on a) spatial learning in the Morris Water Maze and b) tissue signal enhancement in the mouse brain. Even as early as 3 days after pump implantation, infusion of 25-50 mg/kg/day MnCl2 via osmotic pump produced signal enhancement as good as or better than that achieved 24 h after a single 50 mg/kg intraperitoneal injection. Neither route of delivery nor MnCl2 dose adversely affected spatial learning and memory on the water maze. However, especially at higher doses, mice receiving MnCl2 via osmotic pumps developed skin ulceration which limited the imaging window. With these findings, we provide recommendations for route and dose of MnCl2 to use for different study designs.

Transcranial manganese delivery for neuronal tract tracing using MEMRI.

There has been a growing interest in the use of manganese-enhanced MRI (MEMRI) for neuronal tract tracing in mammals, especially in rodents. For this MEMRI application, manganese solutions are usually directly injected into specific brain regions. Recently it was reported that manganese ions can diffuse through intact rat skull. Here the local manganese concentrations in the brain tissue after transcranial manganese application were quantified and the effectiveness of tracing from the area under the skull where delivery occurred was determined. It was established that transcranially applied manganese yields brain tissue enhancement dependent on the location of application on the skull and that manganese that enters the brain transcranially can trace to deeper brain areas.

Continuous infusion of manganese improves contrast and reduces side effects in manganese-enhanced magnetic resonance imaging studies.

The ability to administer systemically high doses of manganese as contrast agent while circumventing its toxicity is of particular interest for exploratory MRI studies of the brain. Administering low doses either repeatedly or continuously over time has been shown to enable the acquisition of satisfactory MRI images of the mouse brain without apparent side effects. Here we have systematically compared the obtained MRI contrast and recorded potential systemic side effects such as stress response and muscle strength impairment in relation to the achieved contrast. We show in mice that administering MnCl2 via osmotic infusion pumps allows for a side-effect free delivery of a high cumulative dose of manganese chloride (480mg/kg bodyweight in 8 days). High contrast in MRI was achieved while we did not observe the weight loss or distress seen in other studies where mice received manganese via fractionated intraperitoneal injections of lower doses of manganese. As the normal daily conduct of the mice was not affected, this new manganese delivery method might be of particular use to study brain activity over several days. This may facilitate the phenotyping of new transgenic mouse models, the study of chronic disease models and the monitoring of changes in brain activity in long-term behavioral studies.

Hippocampal to basal forebrain transport of Mn2+ is impaired by deletion of KLC1, a subunit of the conventional kinesin microtubule-based motor.

Microtubule-based motors carry cargo back and forth between the synaptic region and the cell body. Defects in axonal transport result in peripheral neuropathies, some of which are caused by mutations in KIF5A, a gene encoding one of the heavy chain isoforms of conventional kinesin-1. Some mutations in KIF5A also cause severe central nervous system defects in humans. While transport dynamics in the peripheral nervous system have been well characterized experimentally, transport in the central nervous system is less experimentally accessible and until now not well described. Here we apply manganese-enhanced magnetic resonance (MEMRI) to study transport dynamics within the central nervous system, focusing on the hippocampal-forebrain circuit, and comparing kinesin-1 light chain 1 knock-out (KLC-KO) mice with age-matched wild-type littermates. We injected Mn2+ into CA3 of the posterior hippocampus and imaged axonal transport in vivo by capturing whole-brain 3D magnetic resonance images (MRI) in living mice at discrete time-points after injection. Precise placement of the injection site was monitored in both MR images and in histologic sections. Mn2+-induced intensity progressed along fiber tracts (fimbria and fornix) in both genotypes to the medial septal nuclei (MSN), correlating in location with the traditional histologic tract tracer, rhodamine dextran. Pairwise statistical parametric mapping (SPM) comparing intensities at successive time-points within genotype revealed Mn2+-enhanced MR signal as it proceeded from the injection site into the forebrain, the expected projection from CA3. By region of interest (ROI) analysis of the MSN, wide variation between individuals in each genotype was found. Despite this statistically significant intensity increases in the MSN at 6h post-injection was found in both genotypes, albeit less so in the KLC-KO. While the average accumulation at 6h was less in the KLC-KO, the difference between genotypes did not reach significance. Projections of SPM T-maps for each genotype onto the same grayscale image revealed differences in the anatomical location of significant voxels. Although KLC-KO mice had smaller brains than wild-type, the gross anatomy was normal with no apparent loss of septal cholinergic neurons. Hence anatomy alone does not explain the differences in SPM maps. We conclude that kinesin-1 defects may have only a minor effect on the rate and distribution of transported Mn2+ within the living brain. This impairment is less than expected for this abundant microtubule-based motor, yet such defects could still be functionally significant, resulting in cognitive/emotional dysfunction due to decreased replenishments of synaptic vesicles or mitochondria during synaptic activity. This study demonstrates the power of MEMRI to observe and measure vesicular transport dynamics in the central nervous system that may result from or lead to brain pathology.

Orotracheal manganese-enhanced MRI (MEMRI): An effective approach for lung tumor detection.

Lung cancer is a primary cause of cancer deaths worldwide. Timely detection of this pathology is necessary to delay or interrupt lung cancer progression, ultimately resulting in a possible better prognosis for the patient. In this context, magnetic resonance imaging (MRI) is especially promising. Ultra-short echo time (UTE) MRI sequences, in combination with gadolinium-based contrast agents, have indeed shown to be especially adapted to the detection of lung neoplastic lesions at submillimeter precision. Manganese-enhanced MRI (MEMRI) increasingly appears to be a possible effective alternative to gadolinium-enhanced MRI. In this work, we investigated whether low-dose MEMRI can effectively target non-small-cell lung cancer in rodents, whilst minimizing the potential toxic effect of manganese. Both systemic and orotracheal administration modalities allowed the identification of tumors of submillimeter size, as confirmed by bioluminescence imaging and histology. Equivalent tumor signal enhancements and contrast-to-noise ratios were observed with orotracheal administration using 20 times lower doses compared with the more conventional systemic route. This finding is of crucial importance as it supports the observation that higher performances of contrast agents can be obtained using an orotracheal administration route when targeting lung diseases. As a consequence, lower concentrations of contrast media can be employed, reducing the dose and potential safety issues. The non-detectable accumulation of ionic manganese in the brain and liver following orotracheal administration observed in vivo is extremely encouraging with regard to the safety of the orotracheal protocol with low-dose Mn2+ administration. To our knowledge, this is the first time that a study has clearly allowed the high-precision detection of lung tumor and its contours via the synergic employment of a strongly T1 -weighted MRI UTE sequence and ionic manganese, an inexpensive contrast agent. Overall, these results support the growing interest in drug and contrast agent delivery via the airways to target and diagnose several diseases of the lungs.

Manganese-enhanced MRI (ME MRI) in evaluation of the auditory pathway in an experimental rat model.

This study aimed to explore the optimal dose and manner of administration for visualization of the auditory pathway on manganese-enhanced MRI (ME MRI). Twenty-four healthy male Sprague-Dawley rats were randomly divided into three experimental groups (n = 8 for Groups A, B and C). The rats in Groups A, B and C were subjected to MnCl2 injection through the tympanum, inner ear endolymph and perilymph, respectively (0.2 M for four rats and 0.4 M for the others in each group) and observed at 1, 2, 3, 4, 7 and 10 days after the operation with 3.0 T MRI. The signal intensity (SI) and dynamic changes of the auditory pathways at various times, and at two doses through three injection routes, were compared by statistical analysis. Administration of MnCl2 through the perilymph best showed the complete auditory pathway (P < 0.01), whereas administration though the tympanum only demonstrated part of the pathway. The SI was highest at 24 h after administration of the tracer and began to decline at 48 h. The SI of the auditory cortex was higher after the injection of 0.4 M MnCl2 than that of 0.2 M MnCl2 . ME MRI best demonstrated the whole auditory pathway at 24 h after the injection of 0.4 M MnCl2 through the perilymph in the rat, which provided an optimal method for the study of ME MRI of the auditory pathway in the animal model.

Manganese-enhanced magnetic resonance imaging reveals increased DOI-induced brain activity in a mouse model of schizophrenia.

Maternal infection during pregnancy increases the risk for schizophrenia in offspring. In rodent models, maternal immune activation (MIA) yields offspring with schizophrenia-like behaviors. None of these behaviors are, however, specific to schizophrenia. The presence of hallucinations is a key diagnostic symptom of schizophrenia. In mice, this symptom can be defined as brain activation in the absence of external stimuli, which can be mimicked by administration of hallucinogens. We find that, compared with controls, adult MIA offspring display an increased stereotypical behavioral response to the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI), an agonist for serotonin receptor 2A (5-HT2AR). This may be explained by increased levels of 5-HT2AR and downstream signaling molecules in unstimulated MIA prefrontal cortex (PFC). Using manganese-enhanced magnetic resonance imaging to identify neuronal activation elicited by DOI administration, we find that, compared with controls, MIA offspring exhibit a greater manganese (Mn(2+)) accumulation in several brain areas, including the PFC, thalamus, and striatum. The parafascicular thalamic nucleus, which plays the role in the pathogenesis of hallucinations, is activated by DOI in MIA offspring only. Additionally, compared with controls, MIA offspring demonstrate higher DOI-induced expression of early growth response protein 1, cyclooxygenase-2, and brain-derived neurotrophic factor in the PFC. Chronic treatment with the 5-HT2AR antagonist ketanserin reduces DOI-induced head twitching in MIA offspring. Thus, the MIA mouse model can be successfully used to investigate activity induced by DOI in awake, behaving mice. Moreover, manganese-enhanced magnetic resonance imaging is a useful, noninvasive method for accurately measuring this type of activity.

Eliminating Nox2 reactive oxygen species production protects dystrophic skeletal muscle from pathological calcium influx assessed in vivo by manganese-enhanced magnetic resonance imaging.

KEY POINTS: Inhibiting Nox2 reactive oxygen species (ROS) production reduced in vivo calcium influx in dystrophic muscle. The lack of Nox2 ROS production protected against decreased in vivo muscle function in dystrophic mice. Manganese-enhanced magnetic resonance imaging (MEMRI) was able to detect alterations in basal calcium levels in skeletal muscle and differentiate disease status. Administration of Mn2+ did not affect muscle function or the health of the animal, and Mn2+ was cleared from skeletal muscle rapidly. We conclude that MEMRI may be a viable, non-invasive technique to monitor molecular alterations in disease progression and evaluate the effectiveness of potential therapies for Duchenne muscular dystrophy. ABSTRACT: Duchenne muscular dystrophy (DMD) is an X-linked progressive degenerative disease resulting from a mutation in the gene that encodes dystrophin, leading to decreased muscle mechanical stability and force production. Increased Nox2 reactive oxygen species (ROS) production and sarcolemmal Ca2+ influx are early indicators of disease pathology, and eliminating Nox2 ROS production reduces aberrant Ca2+ influx in young mdx mice, a model of DMD. Various imaging modalities have been used to study dystrophic muscle in vivo; however, they are based upon alterations in muscle morphology or inflammation. Manganese has been used for indirect monitoring of calcium influx across the sarcolemma and may allow detection of molecular alterations in disease progression in vivo using manganese-enhanced magnetic resonance imaging (MEMRI). Therefore, we hypothesized that eliminating Nox2 ROS production would decrease calcium influx in adult mdx mice and that MEMRI would be able to monitor and differentiate disease status in dystrophic muscle. Both in vitro and in vivo data demonstrate that eliminating Nox2 ROS protected against aberrant Ca2+ influx and improved muscle function in dystrophic muscle. MEMRI was able to differentiate between different pathological states in vivo, with no long-term effects on animal health or muscle function. We conclude that MEMRI is a viable, non-invasive technique to differentiate disease status and might provide a means to monitor and evaluate the effectiveness of potential therapies in dystrophic muscle.

Combining systemic and stereotactic MEMRI to detect the correlation between gliosis and neuronal connective pathway at the chronic stage after stroke.

BACKGROUND: The early dysfunction and subsequent recovery after stroke, characterized by the destruction and remodeling of connective pathways between cortex and subcortical regions, is associated with neuroinflammation. As major components of the inflammatory process, reactive astrocytes have double-edged effects on pathological progression. The temporal patterns of astrocyte and neuronal pathway activity can be revealed by systemic and stereotactic manganese-enhanced magnetic resonance imaging (MEMRI), respectively. In the present study, we aimed to detect an association between astrocyte activity and recovery of neuronal connective pathways by combining systemic with stereotactic MEMRI. METHODS: Fifty adult rats, divided into two groups, underwent a 60-min occlusion of the middle cerebral artery. The groups were given either a systemic administration or stereotactic injection of MnCl2 at 1, 3, 7, and 14 days after stroke and underwent MRI 4 and 2 days later, respectively. Immunofluorescence (IF) of group 1 was conducted to corroborate the results. Repetitive behavioral testing was also performed with all rats at 1, 3, 7, and 14 days to obtain a functional score. RESULTS: Ring- or crescent-shaped enhancements formed in the striatal peri-infarct regions (STR) at 11 and 18 days. This was concurrent with the activity of glial fibrillary acidic protein (GFAP)-positive astrocytes, which mainly localized at the peri-infarct region and significantly increased in number at 11 and 18 days after stroke. Microglia/macrophages, detected by IF, mainly localized in the lesion core, rather than in the region of enhancement. The ipsilateral substantia nigra (SN) revealed Mn-related signal enhancement reduction and subsequent signs of the recovery process at 3 to 5 days and 9 to 16 days, respectively. Behavioral testing showed that sensorimotor functions were initially disturbed, but subsequently recovered at 7 and 14 days. CONCLUSIONS: We found a positive temporal correlation between astrogliosis and the recovery of neuronal connective pathways at the chronic stage by using the in vivo method of MEMRI. Our results highlighted the potential contribution of astrocytes to the neuronal recovery of these connective pathways.

Investigation of the functional difference between the pathological itching and neuropathic pain-induced rat brain using manganese-enhanced MRI.

BACKGROUND: There is a remarkable similarity in the central sensitization of itch and pain. However, the interactions between itch and pain are only partially understood. PURPOSE: To investigate the functional activity of cerebral regions to provide clear information on the neuronal pathways related to both pathological itching (PI) and neuropathic pain (NP). MATERIAL AND METHODS: Sprague-Dawley rats were used in this study. PI was induced via neonatal capsaicin treatment, and scratching behavior was counted. NP was induced via lumbar spinal nerve 5 (L5) ligation, and mechanical allodynia was measured. The activated cerebral regions in the control, PI, and NP rats were measured using a 4.7 T magnetic resonance imaging (MRI) system and manganese-enhanced MRI (MEMRI). Subsequently, the cerebral activation regions were identified, and the signal intensity was compared. RESULTS: Cerebral activities of the PI-induced rats were found in three regions -7.10 and -4.20 mm, and two regions -2.45 mm from the bregma. In the NP-induced rats, cerebral activities were found in two regions 7.10 and -2.45 mm, and one region -4.20 mm from the bregma. Comparing the PI and NP rats, the cerebral activities were different in one region -7.10 mm and -2.45 mm, and two regions -4.20 mm from the bregma. The different regions were the midbrain area, the geniculate complex, the hypothalamic area, and the amygdala area. CONCLUSION: Our MEMRI investigation indicates functionally different activity of cerebral regions due to the effect of PI or NP. These findings provide clear information of the signal transduction in the brain regarding PI or NP that share a similar neuronal pathway.

Engineering an effective Mn-binding MRI reporter protein by subcellular targeting.

PURPOSE: Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely used for Mn-enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as a genetic reporter for MEMRI. METHODS: The bacterial Mn-binding protein, MntR was identified as a candidate reporter protein. MntR was engineered for expression in mammalian cells, and targeted to different subcellular organelles, including the Golgi Apparatus where cellular Mn is enriched. Transfected HEK293 cells and B16 melanoma cells were tested in vitro and in vivo, using immunocytochemistry, MR imaging and relaxometry. RESULTS: Subcellular targeting of MntR to the cytosol, endoplasmic reticulum and Golgi apparatus was verified with immunocytochemistry. After targeting to the Golgi, MntR expression produced robust R1 changes and T1 contrast in cells, in vitro and in vivo. Co-expression with the divalent metal transporter DMT1, a previously described Mn-based reporter, further enhanced contrast in B16 cells in culture, but in the in vivo B16 tumor model tested was not significantly better than MntR alone. CONCLUSION: This second-generation reporter system both expands the capabilities of genetically encoded reporters for imaging with MEMRI and provides important insights into the mechanisms of Mn biology which create endogenous MEMRI contrast.