Manganese-enhanced MRI Signal Research Articles

Cell specificity of Manganese-enhanced MRI signal in the cerebellum

Magnetic Resonance Imaging (MRI) resolution continues to improve, making it important to understand the cellular basis for different MRI contrast mechanisms. Manganese-enhanced MRI (MEMRI) produces layer-specific contrast throughout the brain enabling in vivo visualization of cellular cytoarchitecture, particularly in the cerebellum. Due to the unique geometry of the cerebellum, especially near the midline, 2D MEMRI images can be acquired from a relatively thick slice by averaging through areas of uniform morphology and cytoarchitecture to produce very high-resolution visualization of sagittal planes. In such images, MEMRI hyperintensity is uniform in thickness throughout the anterior-posterior axis of sagittal sections and is centrally located in the cerebellar cortex. These signal features suggested that the Purkinje cell layer, which houses the cell bodies of the Purkinje cells and the Bergmann glia, is the source of hyperintensity. Despite this circumstantial evidence, the cellular source of MRI contrast has been difficult to define. In this study, we quantified the effects of selective ablation of Purkinje cells or Bergmann glia on cerebellar MEMRI signal to determine whether signal could be assigned to one cell type. We found that the Purkinje cells, not the Bergmann glia, are the primary of source of the enhancement in the Purkinje cell layer. This cell-ablation strategy should be useful for determining the cell specificity of other MRI contrast mechanisms.

Manganese-enhanced MRI reveals changes within brain anxiety and aversion circuitry in rats with chronic neuropathic pain- and anxiety-like behaviors

Chronic pain often predicts the onset of psychological distress. Symptoms including anxiety and depression after pain chronification reportedly are caused by brain remodeling/recruitment of the limbic and reward/aversion circuitries. Pain is the primary precipitating factor that has caused opioid overprescribing and continued overuse of opioids leading to the current opioid epidemic. Yet experimental pain therapies often fail in clinical trials. Better understanding of underlying pathologies contributing to pain chronification is needed to address these chronic pain related issues. In the present study, a chronic neuropathic pain model persisting 10 weeks was studied. The model develops both anxiety- and pain-related behavioral measures to mimic clinical pain. The manganese-enhanced magnetic resonance imaging (MEMRI) utilized improved MRI signal contrast in brain regions with higher neuronal activity in the rodent chronic constriction trigeminal nerve injury (CCI-ION) model. T1-weighted MEMRI signal intensity was increased compared to controls in supraspinal regions of the anxiety and aversion circuitry, including anterior cingulate gyrus (ACC), amygdala, habenula, caudate, ventrolateral and dorsomedial periaqueductal gray (PAG). Despite continuing mechanical hypersensitivity, MEMRI T1 signal intensity as the neuronal activity measure, was not significantly different in thalamus and decreased in somatosensory cortex (S1BF) of CCI-ION rats compared to naïve controls. This is consistent with decreased fMRI BOLD signal intensity in thalamus and cortex of patients with longstanding trigeminal neuropathic pain reportedly associated with gray matter volume decrease in these regions. Significant increase in MEMRI T2 signal intensity in thalamus of CCI-ION animals was indication of tissue water content, cell dysfunction and/or reactive astrogliosis. Decreased T2 signal intensity in S1BF cortex of rats with CCI-ION was similar to findings of reduced T2 signals in clinical patients with chronic orofacial pain indicating prolonged astrocyte activation. These findings support use of MEMRI and chronic rodent models for preclinical studies and therapeutic trials to reveal brain sites activated only after neuropathic pain has persisted in timeframes relevant to clinical pain and to observe treatment effects not possible in short-term models which do not have evidence of anxiety-like behaviors. Potential improvement is predicted in the success rate of preclinical drug trials in future studies with this model.

Longitudinal MEMRI analysis of brain phenotypes in a mouse model of Niemann-Pick Type C disease

Niemann-Pick Type C (NPC) is a rare genetic disorder characterized by progressive cell death in various tissues, particularly in the cerebellar Purkinje cells, with no known cure. Mouse models for human NPC have been generated and characterized histologically, behaviorally, and using longitudinal magnetic resonance imaging (MRI). Previous imaging studies revealed significant brain volume differences between mutant and wild-type animals, but stopped short of making volumetric comparisons of the cerebellar sub-regions. In this study, we present longitudinal manganese-enhanced MRI (MEMRI) data from cohorts of wild-type, heterozygote carrier, and homozygote mutant NPC mice, as well as deformation-based morphometry (DBM) driven brain volume comparisons across genotypes, including the cerebellar cortex, white matter, and nuclei. We also present the first comparisons of MEMRI signal intensities, reflecting brain and cerebellum sub-regional Mn2+-uptake over time and across genotypes.

Hippocampal changes in inflammasomes, apoptosis, and MEMRI after radiation-induced brain injury in juvenile rats

PurposeThe aim of this study was to characterize changes in hippocampal inflammasomes, pyroptosis and apoptosis in juvenile rats after brain irradiation and to assess whether manganese-enhanced magnetic resonance imaging (MEMRI) reflected those changes.Materials and methodsFour-week-old male Sprague-Dawley rats received a whole-brain radiation dose of 15 Gy or 25 Gy. Hippocampal inflammasomes and apoptosis were measured using Western blot analysis at 4 days and 8 weeks after irradiation. MEMRI and magnetic resonance spectroscopy (MRS) were performed at the same time points.ResultsNeither the 15 Gy nor 25 Gy group showed changes in the expression of inflammasome proteins absent in melanoma 2 (AIM2), gasdermin-D (GSDMD), nucleotide oligomerization domain-like receptor protein 1 (NLRP1) and NLRP3 at 4 days or 8 weeks after radiation injury (P > 0.05). Furthermore, the expression levels of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18 were not significantly different among the groups (P > 0.05). The expression levels of cleaved caspase-1 and -3, indicators of apoptosis, were higher in the irradiation groups than in the control group at 4 days post irradiation, especially for caspase-3 (P < 0.05), but this increase was slightly attenuated at 8 weeks after radiation injury. Four days post irradiation, the MEMRI signal intensity (SI) in the irradiation groups, especially the 25 Gy group, was significantly lower than that in the control group (P < 0.05). Eight weeks after radiation injury, the SI of the 15 Gy group and the 25 Gy group recovered by different degrees, but the SI of the 25 Gy group was still significantly lower than that of the control group (P < 0.05). On day 4 post irradiation, the metabolic ratio of N-acetylaspartate (NAA) to creatine (Cr) in the 15 Gy group and 25 Gy group was significantly lower than that in the control group (P < 0.05). The NAA/Cr ratio in the 15 Gy group recovered to control levels at 8 weeks (P > 0.05), but the NAA/Cr ratio in the 25 Gy group remained significantly lower than that in the control group (P < 0.05).ConclusionRadiation-induced brain injury is dose-dependently associated with apoptosis but not inflammasomes or pyroptosis, and the change in apoptosis can be detected by MEMRI.

Mapping accumulative whole-brain activities during environmental enrichment with manganese-enhanced magnetic resonance imaging

An enriched environment (EE) provides multi-dimensional stimuli to the brain. EE exposure for days to months induces functional and structural neuroplasticity. In this study, manganese-enhanced magnetic resonance imaging (MEMRI) was used to map the accumulative whole-brain activities associated with a 7-day EE exposure in freely-moving adult male mice, followed by c-Fos immunochemical assessments. Relative to the mice residing in a standard environment (SE), the mice subjected to EE treatment had significantly enhanced regional MEMRI signal intensities in the prefrontal cortex, somatosensory cortices, basal ganglia, amygdala, motor thalamus, lateral hypothalamus, ventral hippocampus and midbrain dopaminergic areas at the end of the 7-day exposure, likely attributing to enhanced Mn2+ uptake/transport associated with brain activities at both the regional and macroscale network levels. Some of, but not all, the brain regions in the EE-treated mice showing enhanced MEMRI signal intensity had accompanying increases in c-Fos expression. The EE-treated mice were also found to have significantly increased overall amount of food consumption, decreased body weight gain and upregulated tyrosine hydroxylase (TH) expression in the midbrain dopaminergic areas. Taken together, these results demonstrated that the 7-day EE exposure was associated with elevated cumulative activities in the nigrostriatal, mesolimbic and corticostriatal circuits underpinning reward, motivation, cognition, motor control and appetite regulation. Such accumulative activities might have served as the substrate of EE-related neuroplasticity and the beneficial effects of EE treatment on neurological/psychiatric conditions including drug addiction, Parkinson’s disease and eating disorder.

A New Tool for In Vivo Study of Astrocyte Connexin 43 in Brain

Astrocytes are glial cells organized in dynamic and structured networks in the brain. These plastic networks, involving key proteins such as connexin 43 (Cx43), are engaged in fine neuronal tuning and have recently been considered as emerging therapeutic targets in central nervous system disorders. We developed and validated a new application of the manganese-enhanced magnetic resonance imaging (MEMRI) technique allowing in vivo investigations of astrocyte-neuron interactions through quantification of brain Cx43 functional activity. The proof of concept has been achieved by quantification of MEMRI signals in brain after either local astrocyte-specific Cx43 knockdown with shRNA or systemic administration of Cx43 blockers. Unilateral hippocampal Cx43 genetical silencing was associated with an ipsilateral local increase of MEMRI signal. Furthermore, Cx43 blockers also enhanced MEMRI signal responses in hippocampus. Altogether, these data reveal the MEMRI technique as a tool for quantitative imaging of in vivo Cx43-dependent function in astrocytes under physiological and pathological conditions.

Primary Role of the Amygdala in Spontaneous Inflammatory Pain- Associated Activation of Pain Networks - A Chemogenetic Manganese-Enhanced MRI Approach.

Chronic pain is a major health problem, affecting 10–30% of the population in developed countries. While chronic pain is defined as “a persistent complaint of pain lasting for more than the usual period for recovery,” recently accumulated lines of evidence based on human brain imaging have revealed that chronic pain is not simply a sustained state of nociception, but rather an allostatic state established through gradually progressing plastic changes in the central nervous system. To visualize the brain activity associated with spontaneously occurring pain during the shift from acute to chronic pain under anesthetic-free conditions, we used manganese-enhanced magnetic resonance imaging (MEMRI) with a 9.4-T scanner to visualize neural activity-dependent accumulation of manganese in the brains of mice with hind paw inflammation. Time-differential analysis between 2- and 6-h after formalin injection to the left hind paw revealed a significantly increased MEMRI signal in various brain areas, including the right insular cortex, right nucleus accumbens, right globus pallidus, bilateral caudate putamen, right primary/secondary somatosensory cortex, bilateral thalamus, right amygdala, bilateral substantial nigra, and left ventral tegmental area. To analyze the role of the right amygdala in these post-formalin MEMRI signals, we repeatedly inhibited right amygdala neurons during this 2–6-h period using the “designer receptors exclusively activated by designer drugs” (DREADD) technique. Pharmacological activation of inhibitory DREADDs expressed in the right amygdala significantly attenuated MEMRI signals in the bilateral infralimbic cortex, bilateral nucleus accumbens, bilateral caudate putamen, right globus pallidus, bilateral ventral tegmental area, and bilateral substantia nigra, suggesting that the inflammatory pain-associated activation of these structures depends on the activity of the right amygdala and DREADD-expressing adjacent structures. In summary, the combined use of DREADD and MEMRI is a promising approach for revealing regions associated with spontaneous pain-associated brain activities and their causal relationships.

Iso-α-Acids, Bitter Components in Beer, Suppress Inflammatory Responses and Attenuate Neural Hyperactivation in the Hippocampus.

Due to the growth in aging populations worldwide, prevention and therapy for age-related cognitive decline and dementia are in great demand. We previously demonstrated that long-term intake of iso-α-acids, which are hop-derived bitter compounds found in beer, prevent Alzheimer’s pathology in a rodent model. On the other hand, the effects of iso-α-acids on neural activity in Alzheimer’s disease model mice have not been investigated. Here, we demonstrated that short-term intake of iso-α-acids suppresses inflammation in the hippocampus and improves memory impairment even after disease onset. Importantly, we demonstrated that short-term administration of iso-α-acids attenuated the neural hyperactivation in hippocampus. In 6-month-old 5 × FAD mice exhibiting hippocampus inflammation and memory impairment, oral administration of iso-α-acids for 7 days reduced inflammatory cytokines, including MIP-1α and soluble Aβ and improved object memory in the novel object recognition test. In 12-month-old J20 mice, intake of iso-α-acids for 7 days also suppressed inflammatory cytokines and soluble Aβ in the brain. Manganese-enhanced magnetic resonance imaging (MEMRI) of hippocampi of J20 mice showed increased manganese compared with wild type mice, but iso-α-acids canceled this increased MEMRI signal in J20 mice, particularly in the hippocampus CA1 and CA3 region. Taken together, these findings suggest that short-term intake of iso-α-acids can suppress hippocampus inflammation even after disease onset and improve hyper neural activity in Alzheimer’s disease model mice.

Prenatal Irradiation-Induced Hippocampal Abnormalities in Rats Evaluated Using Manganese-Enhanced MRI.

The aim of this study was to characterize hippocampal abnormalities in rats after prenatal x-ray irradiation using manganese-enhanced MRI (MEMRI). All radiation-exposed rat brains showed a reduced volume with prominent dilatation of lateral ventricles. Moreover, MEMRI-enhanced areas within the hippocampus were reduced in volumes by approximately 25% of controls, although the entire volume of hippocampus was decreased by approximately 50% of controls. MEMRI signals were enhanced strongly in the hilus and granular layer of the dentate gyrus (DG) and the pyramidal layer and infrapyramidal region of the CA3 region, and moderately along the CA1/2 pyramidal cell layer in the control rats. In radiation-exposed rats, MEMRI signals in the CA1/2 regions disappeared due to disrupting their laminar organization, although strong MEMRI signals were sustained in the DG and CA3 regions. Histopathological examinations in radiation-exposed rats revealed disorganizations of the DG granule cell layer and the CA3 pyramidal cell layer with reducing the cell density. The CA1/2 pyramidal cell layer was disrupted by invading ectopic cell mass. Neural cell adhesion molecule (NCAM)-positive fiber bundles were sustained in radiation-exposed rats, although they distributed aberrantly in the suprapyramidal CA3 region with a slight reduction of NCAM staining. Furthermore, glial components consisted largely by astrocytes and minor by microglia were densely distributed in the DG rather than in other hippocampal regions, and their density radiation-exposed rats. In conclusion, MEMRI signal enhancements could delineate different neuronal and/or glial components among hippocampal regions. We characterized microstructures of the deformed hippocampus as well as its macrostructures in a prenatally radiation-exposed rat model using in vivo MEMRI. The present findings provide advantageous information for detecting nondestructively hippocampal deformations in neurodevelopmental disorders.

Chemotherapy-Induced Cognitive Impairment Is Associated with Cytokine Dysregulation and Disruptions in Neuroplasticity.

Chemotherapy-induced cognitive impairment, often referred to as "chemobrain," is a common side effect. In this study, mice received three intraperitoneal injections of a combination of docetaxel, adriamycin, and cyclophosphamide (DAC) at 2-day intervals. A water maze test was used to examine cognitive performance, and manganese-enhanced magnetic resonance imaging (MEMRI) was used to examine hippocampal neuronal activity. The whole brain, prefrontal cortex, hippocampus, and blood samples were then collected for cytokine measurement. The DAC-treated mice displayed a significantly shorter duration spent in and fewer entries into the target quadrant of the water maze than the control mice and a pronounced decrease in MEMRI signal intensity in the hippocampal subregions. In a separate experiment using in vivo transcranial two-photon imaging, DAC markedly eliminated dendritic spines without changing the rate of spine formation, leading to a striking loss of spines in the medial prefrontal cortex. DAC treatment resulted in significant elevations in the levels of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) and in significant decreases in the levels of the anti-inflammatory cytokines IL-4 and IL-10 in most of the sera and brain tissues examined. The IL-6 and TNF-α levels of several sera and brain tissues showed strong inverse correlations with the duration and number of entries in the target quadrant of the water maze and with the hippocampal MEMRI signal intensity, but also showed striking positive correlations with spine elimination and loss. These results indicate that chemobrain is associated with cytokine dysregulation and disrupted neuroplasticity of the brain.

MEMRI detects neuronal activity and connectivity in hypothalamic neural circuit responding to leptin

Hypothalamus plays the central role in regulating energy homeostasis. To understand the hypothalamic neurocircuit in responding to leptin, Manganese-Enhanced MRI (MEMRI) was applied. Highly elevated signal could be mapped in major nuclei of the leptin signaling pathway, including the arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH) in fasted mice and the enhancement was reduced by leptin administration. However, whether changes in MEMRI signal reflect Ca2+ channel activity, neuronal activation or connectivity in the leptin signaling pathway are not clear. By blocking L-type Ca2+ channels, the signal enhancement in the ARC, PVN and DMH, but not VMH, was reduced. By disrupting microtubule with colchicine, signal enhancement of the secondary neural areas like DMH and PVN was delayed which is consistent with the known projection density from ARC into these regions. Finally, strong correlation between c-fos expression and MEMRI signal increase rate was observed in the ARC, VMH and DMH. Together, we provide experimental evidence that MEMRI signal could represent activity and connectivity in certain hypothalamic nuclei and hence may be used for mapping activated neuronal pathway in vivo. This understanding would facilitate the application of MEMRI for evaluation of hypothalamic dysfunction in metabolic diseases.

Spatial learning and memory impairments are associated with increased neuronal activity in 5XFAD mouse as measured by manganese-enhanced magnetic resonance imaging

Dysfunction of neuronal activity is a major and early contributor to cognitive impairment in Alzheimer's disease (AD). To investigate neuronal activity alterations at early stage of AD, we encompassed behavioral testing and in vivo manganese-enhanced magnetic resonance imaging (MEMRI) in 5XFAD mice at early ages (1-, 2-, 3- and 5-month). The 5XFAD model over-express human amyloid precursor protein (APP) and presenilin 1 (PS1) harboring five familial AD mutations, which have a high APP expression correlating with a high burden and an accelerated accumulation of the 42 amino acid species of amyloid-β. In the Morris water maze, 5XFAD mice showed longer escape latency and poorer memory retention. In the MEMRI, 5XFAD mice showed increased signal intensity in the brain regions involved in spatial cognition, including the entorhinal cortex, the hippocampus, the retrosplenial cortex and the caudate putamen. Of note, the observed alterations in spatial cognition were associated with increased MEMRI signal intensity. These findings indicate that aberrant increased basal neuronal activity may contribute to the spatial cognitive function impairment at early stage of AD, and may further suggest the potential use of MEMRI to predict cognitive impairments. Early intervention that targets aberrant neuronal activity may be crucial to prevent cognitive impairment.

Ability of Mn(2+) to Permeate the Eye and Availability of Manganese-enhanced Magnetic Resonance Imaging for Visual Pathway Imaging via Topical Administration.

Background:Manganese-enhanced magnetic resonance imaging (MEMRI) for visual pathway imaging via topical administration requires further research. This study investigated the permeability of the corneal epithelium and corneal toxicity after topical administration of Mn2+ to understand the applicability of MEMRI.Methods:Forty New Zealand rabbits were divided into 0.05 mol/L, 0.10 mol/L, and 0.20 mol/L groups as well as a control group (n = 10 in each group). Each group was further subdivided into epithelium-removed and epithelium-intact subgroups (n = 5 in each subgroup). Rabbits were given 8 drops of MnCl2 in 5 min intervals. The Mn2+ concentrations in the aqueous and vitreous humors were analyzed using inductively coupled plasma-mass spectrometry at different time points. MEMRI scanning was carried out to image the visual pathway after 24 h. The corneal toxicity of Mn2+ was evaluated with corneal imaging and pathology slices.Results:Between the aqueous and vitreous humors, there was a 10 h lag for the peak Mn2+ concentration times. The intraocular Mn2+ concentration increased with the concentration gradients of Mn2+ and was higher in the epithelium-removed subgroup than that in the epithelium-intact subgroup. The enhancement of the visual pathway was achieved in the 0.10 mol/L and 0.20 mol/L epithelium-removed subgroups. The corresponding peak concentrations of Mn2+ were 5087 ± 666 ng/ml, 22920 ± 1188 ng/ml in the aqueous humor and 884 ± 78 ng/ml, 2556 ± 492 ng/ml in the vitreous body, respectively. Corneal injury was evident in the epithelium-removed and 0.20 mol/L epithelium-intact subgroups.Conclusions:The corneal epithelium is a barrier to Mn2+, and the iris and lens septum might be another intraocular barrier to the permeation of Mn2+. An elevated Mn2+ concentration contributes to the increased permeation of Mn2+, higher MEMRI signal, and corneal toxicity. The enhancement of the visual pathway requires an effective Mn2+ concentration in the vitreous body.

Cocaine-induced locomotor sensitization in rats correlates with nucleus accumbens activity on manganese-enhanced MRI.

A long-standing goal of substance abuse research has been to link drug-induced behavioral outcomes with the activity of specific brain regions to understand the neurobiology of addiction behaviors and to search for drug-able targets. Here, we tested the hypothesis that cocaine produces locomotor (behavioral) sensitization that correlates with increased calcium channel-mediated neuroactivity in brain regions linked with drug addiction, such as the nucleus accumbens (NAC), anterior striatum (AST) and hippocampus, as measured using manganese-enhanced MRI (MEMRI). Rats were treated with cocaine for 5 days, followed by a 2-day drug-free period. The following day, locomotor sensitization was quantified as a metric of cocaine-induced neuroplasticity in the presence of manganese. Immediately following behavioral testing, rats were examined for changes in calcium channel-mediated neuronal activity in the NAC, AST, hippocampus and temporalis muscle, which was associated with behavioral sensitization using MEMRI. Cocaine significantly increased locomotor activity and produced behavioral sensitization compared with saline treatment of control rats. A significant increase in MEMRI signal intensity was determined in the NAC, but not AST or hippocampus, of cocaine-treated rats compared with saline-treated control rats. Cocaine did not increase signal intensity in the temporalis muscle. Notably, in support of our hypothesis, behavior was significantly and positively correlated with MEMRI signal intensity in the NAC. As neuronal uptake of manganese is regulated by calcium channels, these results indicate that MEMRI is a powerful research tool to study neuronal activity in freely behaving animals and to guide new calcium channel-based therapies for the treatment of cocaine abuse and dependence.

Manganese-Enhanced MRI for Preclinical Evaluation of Retinal Degeneration Treatments.

Apply manganese-enhanced magnetic resonance imaging (MEMRI) to assess ion channel activity and structure of retinas from mice subject to light-induced retinal degeneration treated with prophylactic agents. Abca4(-/-)Rdh8(-/-) double knockout mice with and without prophylactic retinylamine (Ret-NH2) treatment were illuminated with strong light. Manganese-enhanced MRI was used to image the retina 2 hours after intravitreous injection of MnCl2 into one eye. Contrast-enhanced MRIs of the retina and vitreous humor in each experimental group were assessed and correlated with the treatment. Findings were compared with standard structural and functional assessments of the retina by optical coherence tomography (OCT), histology, and electroretinography (ERG). Manganese-enhanced MRI contrast in the retina was high in nonilluminated and illuminated Ret-NH2-treated mice, whereas no enhancement was evident in the retina of the light-illuminated mice without Ret-NH2 treatment (P < 0.0005). A relatively high signal enhancement was also observed in the vitreous humor of mice treated with Ret-NH2. Strong MEMRI signal enhancement in the retinas of mice treated with retinylamine was correlated with their structural integrity and function evidenced by OCT, histology, and a strong ERG light response. Manganese-enhanced MRI has the potential to assess the response of the retina to prophylactic treatment based on the measurement of ion channel activity. This approach could be used as a complementary tool in preclinical development of new prophylactic therapies for retinopathies.

Manganese-Enhanced MRI Reflects Both Activity-Independent and Activity-Dependent Uptake within the Rat Habenulomesencephalic Pathway.

Manganese-enhanced magnetic resonance imaging (MEMRI) is a powerful technique for assessing the functional connectivity of neurons within the central nervous system. Despite the widely held proposition that MEMRI signal is dependent on neuronal activity, few studies have directly tested this implicit hypothesis. In the present series of experiments, MnCl2 was injected into the habenula of urethane-anesthetized rats alone or in combination with drugs known to alter neuronal activity by modulating specific voltage- and/or ligand-gated ion channels. Continuous quantitative T1 mapping was used to measure Mn2+ accumulation in the interpeduncular nucleus, a midline structure in which efferents from the medial habenula terminate. Microinjection of MnCl2 into the habenular complex using a protocol that maintained spontaneous neuronal activity resulted in a time-dependent increase in MEMRI signal intensity in the interpeduncular nucleus consistent with fast axonal transport of Mn2+ between these structures. Co-injection of the excitatory amino-acid agonist AMPA, increased the Mn2+-enhanced signal intensity within the interpeduncular nucleus. AMPA-induced increases in MEMRI signal were attenuated by co-injection of either the sodium channel blocker, TTX, or broad-spectrum Ca2+ channel blocker, Ni2+, and were occluded in the presence of both channel blockers. However, neither Ni2+ nor TTX, alone or in combination, attenuated the increase in signal intensity following injection of Mn2+ into the habenula. These results support the premise that changes in neuronal excitability are reflected by corresponding changes in MEMRI signal intensity. However, they also suggest that basal rates of Mn2+ uptake by neurons in the medial habenula may also occur via activity-independent mechanisms.

G.P.125: Manganese enhanced muscle MRI as a sensitive outcome measure of dystrophin restoration in the mdx mouse

A central component of dystrophic pathology in DMD patients as well as mdx mice is a persistent increase in steady-state calcium levels in muscle. Increased calcium load leads to loss of muscle strength, activation of calcium-dependent proteases and may increase muscle cell necrosis. As a key primary event in dystrophic pathology, measurement of calcium levels may offer a sensitive marker of the efficacy of therapeutic agents. Manganese is a divalent cation with very similar chemical properties to calcium and is thought to be taken up by cardiac and skeletal muscle by the same receptors as calcium ions. It is also a contrast agent in T1 weighted MRI, enabling calcium levels in muscle to be measured non-invasively. We have previously shown that manganese-enhanced MRI (MEMRI) contrast is elevated in cardiac pathology in mdx and can track the dynamics of calcium channel blockade in dystrophic models. Here, we have extended this analysis by infusing manganese intravenously and observing the uptake by skeletal muscle using MRI, showing that the mdx mouse has an increased steady-state level of MEMRI contrast in both chest wall and triceps. Furthermore, we treated a cohort ( n = 6) of 12-week old mdx mice with an increasing dosage (64 mg/kg or 133 mg/kg) of phosphorothioate Morpholino oligonucleotides (PMOs) to induce exon skipping. Animals were treated with a series of three i.v. injections spaced two weeks apart, scanned after a further two weeks, and muscle (heart, chest wall and triceps) harvested for dystrophin quantification by IF and Western blotting, as well as for histology. We show that inducing exon skipping, even at very low levels, can be detected by changes in the MEMRI signal in both cardiac and skeletal muscle, even in the absence of discernable changes in histology. These data suggest that muscle MEMRI may be a valuable tool for longitudinal studies on dystrophin restoration in mdx and may have increased sensitivity when compared to more established methods.

Reduced hippocampal manganese-enhanced MRI (MEMRI) signal during pilocarpine-induced status epilepticus: Edema or apoptosis?

Manganese-enhanced MRI (MEMRI) has been considered a surrogate marker of Ca(+2) influx into activated cells and tracer of neuronal active circuits. However, the induction of status epilepticus (SE) by kainic acid does not result in hippocampal MEMRI hypersignal, in spite of its high cell activity. Similarly, short durations of status (5 or 15min) induced by pilocarpine did not alter the hippocampal MEMRI, while 30 min of SE even reduced MEMRI signal Thus, this study was designed to investigate possible explanations for the absence or decrease of MEMRI signal after short periods of SE. We analyzed hippocampal caspase-3 activation (to evaluate apoptosis), T2 relaxometry (tissue water content) and aquaporin 4 expression (water-channel protein) of rats subjected to short periods of pilocarpine-induced SE. For the time periods studied here, apoptotic cell death did not contribute to the decrease of the hippocampal MEMRI signal. However, T2 relaxation was higher in the group of animals subjected to 30min of SE than in the other SE or control groups. This result is consistent with higher AQP-4 expression during the same time period. Based on apoptosis and tissue water content analysis, the low hippocampal MEMRI signal 30min after SE can potentially be attributed to local edema rather than to cell death.

Time series analysis of in vivo cardiac MRI-PET image fusion of the human amniotic mesenchymal stem cell (hAMSC) engraftment

Methods Porcine ischemia reperfusion (IR) injury model was employed to assess the successful stem cell engraftment signal by manganese enhanced MRI (MEMRI). To validate the MEMRI signal, hAMSCs were transduced by PET reporter gene (RG) utilizing the herpes simplex virus-thymidine kinase transgene construct, which is expressed only in engrafted cells to trap 18F-FHBG radiotracer. One week after IR injury, the RG transduced hAMSCs were delivered via transendocardial injection into periand intra-infarct regions. PET-CT and MR locator-MEMRI-delayed enhanced MRI (DEMRI) images were acquired post hAMSC injection on days 0, 7, and 45. All pairs of CT and MR locator images were registered by gradient descent to optimize mutual information between the images generating transformation matrixes. These transformation matrixes were used to align PET with MEMRI and DEMRI and longitudinal images into common frames of reference. Additionally image pairs were fused into a single display. An ROI around the engrafted stem cells on MEMRI was copied to the same location on PET-CT longitudinally and average intensity was sampled at each time point.

Sex-related long-term behavioral and hippocampal cellular alterations after nociceptive stimulation throughout postnatal development in rats

Early noxious stimuli may alter the neurogenesis rate in the dentate gyrus and the behavioral repertoire of adult rats. This study evaluated the long-term effects of noxious stimulation, imposed in different phases of development, on nociceptive and anxiety-like behaviors, hippocampal activation, cell proliferation, hippocampal BDNF and plasma corticosterone levels in 40 day-old male and female adolescents. Noxious stimulation was induced by intra-plantar injection of Complete Freund's adjuvant (CFA), on postnatal days (P) 1 (group P1), 8 (P8) or 21 (P21). Control animals were not stimulated in any way. On P21 a subset of animals from each group received BrdU and was perfused on P40 for identification of proliferating cells in the granule cell layer of the dentate gyrus. Another subset of rats was subjected to behavioral testing on P40 and one week later, to magnetic resonance imaging (MRI) acquisition. Noxious stimulation evoked hypoalgesia in adolescents, mainly in females (P < 0.02), reflected by greater latency to withdraw the paw and less paw lickings in the hot plate test than controls (P < 0.001). It also resulted in more time spent in the open arms, e.g., less anxiety-like behavior than controls (P < 0.01), especially in females (P < 0.01, compared with males). Proliferative cell rate in the dentate gyrus was the highest in P8 males and females (P < 0.001), with males exhibiting more proliferation than females on P1 and P8, which was directly related to the hippocampal levels of BDNF and inversely related to plasma corticosterone. Sex differences were also detected in manganese-enhanced MRI signal, which was more prominent in P1 females than males (P < 0.01). This study represents the first step of investigation on the cellular basis of the sex-dependent long-term consequences of nociceptive stimuli in newborns.