Manganese‐enhanced MRI (MEMRI) via topical loading of Mn2+ significantly impairs mouse visual acuity: a comparison with intravitreal injection

Manganese‐enhanced MRI (MEMRI) with topical loading of MnCl₂ provides optic nerve enhancement comparable to that seen by intravitreal injection. However, the impact of this novel and non‐invasive Mn²⁺ loading method on visual function requires further assessments. The objective of this study is to determine the optimal topical Mn²⁺ loading dosage for MEMRI and to assess visual function after MnCl₂ loading. Intravitreal administration was performed to compare the two approaches of MnCl₂ loading. Twenty‐four hours after topical loading of 0, 0.5, 0.75, and 1 M MnCl₂, T₁‐weighted, T2‐weighted, diffusion tensor imaging and visual acuity (VA) assessments were performed to determine the best topical loading dosage for MEMRI measurements and to assess the integrity of retinas and optic nerves. Mice were perfusion fixed immediately after in vivo experiments for hematoxylin and eosin and immunohistochemistry staining. Topical loading of 1 M MnCl₂ damaged the retinal photoreceptor layer with no detectable damage to retina ganglion cell layers or prechiasmatic optic nerves. For the topical loading, 0.75 M MnCl₂ was required to see sufficient enhancement of the optic nerve. At this concentration the visual function was significantly affected, followed by a slow recovery. Intravitreal injection (0.25 μL of 0.2 M MnCl₂) slightly affected VA, with full recovery a day later. To conclude, intravitreal MnCl₂injection provides more reproducible results with less adverse side‐effects than topical loading. Copyright © 2014 John Wiley & Sons, Ltd.     

Assessing manganese efflux using SEA0400 and cardiac T1‐mapping manganese‐enhanced MRI in a murine model

The sodium–calcium exchanger (NCX) is one of the transporters contributing to the control of intracellular calcium (Ca²⁺) concentration by normally mediating net Ca²⁺ efflux. However, the reverse mode of the NCX can cause intracellular Ca²⁺ concentration overload, which exacerbates the myocardial tissue injury resulting from ischemia. Although the NCX inhibitor SEA0400 has been shown to therapeutically reduce myocardial injury, no in vivo technique exists to monitor intracellular Ca²⁺ fluctuations produced by this drug. Cardiac manganese‐enhanced MRI (MEMRI) may indirectly assess Ca²⁺ efflux by estimating changes in manganese (Mn²⁺) content in vivo, since Mn²⁺ has been suggested as a surrogate marker for Ca²⁺. This study used the MEMRI technique to examine the temporal features of cardiac Mn²⁺ efflux by implementing a T₁‐mapping method and inhibiting the NCX with SEA0400. The change in ¹H₂O longitudinal relaxation rate, ΔR₁, in the left ventricular free wall, was calculated at different time points following infusion of 190 nmol/g manganese chloride (MnCl₂) in healthy adult male mice. The results showed 50% MEMRI signal attenuation at 3.4 ± 0.6 h post‐MnCl₂ infusion without drug intervention. Furthermore, treatment with 50 ± 0.2 mg/kg of SEA0400 significantly reduced the rate of decrease in ΔR₁. At 4.9–5.9 h post‐MnCl₂ infusion, the average ΔR₁ values for the two groups treated with SEA0400 were 2.46 ± 0.29 and 1.72 ± 0.24 s^?1 for 50 and 20 mg/kg doses, respectively, as compared to the value of 1.27 ± 0.28 s^?1 for the control group. When this in vivo data were compared to ex vivo absolute manganese content data, the MEMRI T₁‐mapping technique was shown to effectively quantify Mn²⁺ efflux rates in the myocardium. Therefore, combining an NCX inhibitor with MEMRI may be a useful technique for assessing Mn²⁺ transport mechanisms and rates in vivo, which may reflect changes in Ca²⁺ transport. Copyright © 2009 John Wiley & Sons, Ltd.     

Fractionated manganese injections: effects on MRI contrast enhancement and physiological measures in C57BL/6 mice

Manganese‐enhanced MRI (MEMRI) is an increasingly used imaging method in animal research, which enables improved T₁‐weighted tissue contrast. Furthermore accumulation of manganese in activated neurons allows visualization of neuronal activity. However, at higher concentrations manganese (Mn²⁺) exhibits toxic side effects that interfere with the animals' behaviour and well‐being. Therefore, when optimizing MEMRI protocols, a compromise has to be found between minimizing side effects and intensifying image contrast. Recently, a low concentrated fractionated Mn²⁺ application scheme has been proposed as a promising alternative. In this study, we investigated effects of different fractionated Mn²⁺ dosing schemes on vegetative, behavioural and endocrine markers, and MEMRI signal contrast in C57BL/6N mice. Measurements of the animals' well‐being included telemetric monitoring of body temperature and locomotion, control of weight and observation of behavioural parameters during the time course of the injection protocols. Corticosterone levels after Mn²⁺ application served as endocrine marker of the stress response. We compared three MnCl₂ · 4H₂O application protocols: 3 times 60 mg/kg with an inter‐injection interval of 48 h, six times 30 mg/kg with an inter‐injection interval of 48 h, and 8 times 30 mg/kg with an inter‐injection interval of 24 h (referred to as 3 × 60/48, 6 × 30/48 and 8 × 30/24, respectively). Both the 6 × 30/48 and the 8 × 30/24 protocols showed attenuated effects on animals' well‐being as compared to the 3 × 60/48 scheme. Best MEMRI signal contrast was observed for the 8 × 30/24 protocol. Together, these results argue for a fractionated application scheme such as 30 mg/kg every 24 h for 8 days to provide sufficient MEMRI signal contrast while minimizing toxic side effects and distress. Copyright © 2010 John Wiley & Sons, Ltd.     

Regional specificity of manganese accumulation and clearance in the mouse brain: implications for manganese‐enhanced MRI

Manganese‐enhanced MRI has recently become a valuable tool for the assessment of in vivo functional cerebral activity in animal models. As a result of the toxicity of manganese at higher dosages, fractionated application schemes have been proposed to reduce the toxic side effects by using lower concentrations per injection. Here, we present data on regional‐specific manganese accumulation during a fractionated application scheme over 8 days of 30 mg/kg MnCl₂, as well as on the clearance of manganese chloride over the course of several weeks after the termination of the whole application protocol supplying an accumulative dose of 240 mg/kg MnCl₂. Our data show most rapid accumulation in the superior and inferior colliculi, amygdala, bed nucleus of the stria terminalis, cornu ammonis of the hippocampus and globus pallidus. The data suggest that no ceiling effects occur in any region using the proposed application protocol. Therefore, a comparison of basal neuronal activity differences in different animal groups based on locally specific manganese accumulation is possible using fractionated application. Half‐life times of manganese clearance varied between 5 and 7 days, and were longest in the periaqueductal gray, amygdala and entorhinal cortex. As the hippocampal formation shows one of the highest T₁‐weighted signal intensities after manganese application, and manganese‐induced memory impairment has been suggested, we assessed hippocampus‐dependent learning as well as possible manganese‐induced atrophy of the hippocampal volume. No interference of manganese application on learning was detected after 4 days of Mn²⁺ application or 2 weeks after the application protocol. In addition, no volumetric changes induced by manganese application were found for the hippocampus at any of the measured time points. For longitudinal measurements (i.e. repeated manganese applications), a minimum of at least 8 weeks should be considered using the proposed protocol to allow for sufficient clearance of the paramagnetic ion from cerebral tissue. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

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 MRI visualizes V1 in the non‐human primate visual cortex

MRI at 7 Tesla has been used to investigate the accumulation of manganese in the occipital cortex of common marmoset monkeys (Callithrix jacchus) after administering four fractionated injections of 30 mg/kg MnCl₂ · 4H₂O in the tail vein. We found a statistically significant decrease in T₁ in the primary (V1) and secondary (V2) areas of the visual cortex caused by an accumulation of manganese. The larger T₁ shortening in V1 (ΔT₁ = 640 ms) relative to V2 (ΔT₁ = 490 ms) allowed us to robustly detect the V1/V2 border in vivo using heavily T₁‐weighted MRI. Furthermore, the dorso‐medial (DM) and middle‐temporal (MT) areas of the visual pathway could be identified by their T₁‐weighted enhancement. We showed by comparison to histological sections stained for cytochrome oxidase (CO) activity that the extent of V1 is accurately identified throughout the visual cortex by manganese‐enhanced MRI (MEMRI). This provides a means of visualizing functional cortical regions in vivo and could be used in longitudinal studies of phenomena such as cortical plasticity, and for non‐destructive localization of cortical regions to guide in the implementation of functional techniques. Published in 2009 by John Wiley & Sons, Ltd.     

Manganese threonine chelate—a new enteric contrast agent for MRI: a pilot study on rats

Enteric contrast agents are important in gastrointestinal MRI. However, no currently available agent is well established as the standard of care. In this study, in vitro relaxivities of manganese threonine chelate (Mn‐Thr), a common nutritional food supplement, were measured at 1.5 T and 3 T with further investigation of its efficacy and safety in vivo as an enteric contrast agent. According to the calculated relaxivities, T₁W and T₂W TSE sequences of Mn‐Thr solutions at different concentrations were acquired, and the optimal concentration for dark lumen imaging on both T₁W and T₂W images was determined in vitro. To validate the optimal concentration in vivo, eight Sprague‐Dawley rats were randomly divided into two groups. Each group received rectal injection of either 2.00 g/L (about 3.80 mM) Mn‐Thr or saline as an enteric contrast agent and underwent MRI. After a time interval of one week, the same procedures were repeated with the alternative contrast agent. Animals were sacrificed after the second MRI. Tissue manganese quantification and histopathological examination were obtained. Qualitative MR image quality assessments were performed and compared between Mn‐Thr and saline. Measured T₁ and T₂ relaxivities of Mn‐Thr were significantly higher than those of MnCl₂ in vitro (p < 0.05). At the concentration of 2.00 g/L (about 3.80 mM), Mn‐Thr produced a dark lumen on T₁W and T₂W images both in vitro and in vivo. Compared with saline, Mn‐Thr showed significantly more homogenous luminal signal and increased bowel wall conspicuity in image quality assessments. Tissue manganese concentrations were not significantly different between two groups. Histopathological examinations were normal in both groups. Our data suggest that Mn‐Thr possesses favorable paramagnetic properties and can create a homogenous dark lumen on T₁W and T₂W images without obvious side effects in healthy rats. As a commercially available nutritional food supplement, Mn‐Thr appears to be a promising enteric contrast agent for MRI.     

Monitoring dynamic alterations in calcium homeostasis by T1‐mapping manganese‐enhanced MRI (MEMRI) in the early stage of small intestinal ischemia–reperfusion injury

Manganese‐enhanced MRI studies have proven to be useful in monitoring physiological activities associated with calcium ions (Ca²⁺) due to the paramagnetic property of the manganese ion (Mn²⁺), which makes it an excellent probe of Ca²⁺. In this study, we developed a method in which a Mn²⁺‐enhanced T₁‐map MRI could enable the monitoring of Ca²⁺ influx during the early stages of intestinal ischemia–reperfusion (I/R) injury. The Mn²⁺ infusion protocol was optimized by obtaining dose‐dependent and time‐course wash‐out curves using a Mn²⁺‐enhanced T₁‐map MRI of rabbit abdomens following an intravenous infusion of 50 mmol/l MnCl₂ (5–10 nmol/g body weight (BW)). In the rabbit model of intestinal I/R injury, T₁ values were derived from the T₁ maps in the intestinal wall region and revealed a relationship between the dose of the infused MnCl₂ and the intestinal wall relaxation time. Significant Mn²⁺ clearance was also observed over time in control animals after the infusion of Mn²⁺ at a dose of 10 nmol/g BW. This technique was also shown to be sensitive enough to monitor variations in calcium ion homeostasis in vivo after small intestinal I/R injury. The T₁ values of the intestinal I/R group were significantly lower (P < 0.05) than that of the control group at 5, 10, and 15 min after Mn²⁺ infusion. Our data suggest that MnCl₂ has the potential to be an MRI contrast agent that can be effectively used to monitor changes in intracellular Ca²⁺ homeostasis during the early stages of intestinal I/R injury. Copyright © 2015 John Wiley & Sons, Ltd.     

Morphological changes and manganese content in the brains of rat pups subjected to subchronic poisoning with manganese chloride

Morphological changes in neurons and the distributions of nerve and glial cells were studied, the glial index was calculated, and manganese (Mn) contents were determined in the caudate nucleus, the nucleus accumbens, the dorsal and ventral septal nuclei, and the frontoparietal areas of the cerebral cortex in the 40-day-old offspring of rats given different doses (10 and 20 mg/kg) of manganese chloride (MnCl₂·4H₂O) 15–20 days before pregnancy, during pregnancy, and for one month after parturitiion with the first portion of food. Mn poisoning increased Mn contents in the brains of rat pups, damaged a small proportion of neurons, and produced marked gliosis. These changes are believed to underlie previously described impairments to learning processes and emotional state in rat pups. Translated from Morfologiya, Vol. 133, No. 1, pp. 25–30, January–February, 2008.     

Three‐dimensional inversion recovery manganese‐enhanced MRI of mouse brain using super‐resolution reconstruction to visualize nuclei involved in higher brain function

The visualization of activity in mouse brain using inversion recovery spin echo (IR‐SE) manganese‐enhanced MRI (MEMRI) provides unique contrast, but suffers from poor resolution in the slice‐encoding direction. Super‐resolution reconstruction (SRR) is a resolution‐enhancing post‐processing technique in which multiple low‐resolution slice stacks are combined into a single volume of high isotropic resolution using computational methods. In this study, we investigated, first, whether SRR can improve the three‐dimensional resolution of IR‐SE MEMRI in the slice selection direction, whilst maintaining or improving the contrast‐to‐noise ratio of the two‐dimensional slice stacks. Second, the contrast‐to‐noise ratio of SRR IR‐SE MEMRI was compared with a conventional three‐dimensional gradient echo (GE) acquisition. Quantitative experiments were performed on a phantom containing compartments of various manganese concentrations. The results showed that, with comparable scan times, the signal‐to‐noise ratio of three‐dimensional GE acquisition is higher than that of SRR IR‐SE MEMRI. However, the contrast‐to‐noise ratio between different compartments can be superior with SRR IR‐SE MEMRI, depending on the chosen inversion time. In vivo experiments were performed in mice receiving manganese using an implanted osmotic pump. The results showed that SRR works well as a resolution‐enhancing technique in IR‐SE MEMRI experiments. In addition, the SRR image also shows a number of brain structures that are more clearly discernible from the surrounding tissues than in three‐dimensional GE acquisition, including a number of nuclei with specific higher brain functions, such as memory, stress, anxiety and reward behavior. Copyright © 2014 John Wiley & Sons, Ltd.     

Influence of peripheral nerve grafts on the expression of GAP-43 in regenerating retinal ganglion cells in adult hamsters

Summary We have examined the ability of axotomized retinal ganglion cells in adult hamsters, to regenerate axons into a peripheral nerve graft attached to the optic nerve and the expression of GAP-43 by these neurons. We also examined the effect on these events of transplanting a segment of peripheral nerve to the vitreous body. The left optic nerves in three groups of hamsters were replaced with a long segment of peripheral nerve attached to the proximal stump of the optic nerve 2 mm from the optic disc to induce regeneration of retinal ganglion cells into the peripheral nerve. An additional segment of peripheral nerve was transplanted into the vitreous of the left eye in the second group. The animals from the first and second groups were allowed to survive for 1–8 weeks and the number of regenerating retinal ganglion cells was determined by applying the retrograde tracer, Fluoro-Gold to the peripheral nerve graft and the expression of GAP-43 was studied by immunocytochemistry in the same retinas. As a control, a segment of optic nerve was transplanted into the vitreous body of the left eye in the third group of hamsters. These animals were allowed to survive for 4 weeks and the number of regenerating retinal ganglion cells was counted as in Groups 1 and 2. The percentages of the regenerating retinal ganglion cells which also expressed GAP-43 were very high at all time points in Group 1 (with no intravitreal peripheral nerve) and Group 2 (with intravitreal peripheral nerve) and at 4 weeks for the Group 3 (with intravitreal optic nerve) animals. In addition, the number of regenerating retinal ganglion cells, the number of retinal ganglion cells expressing GAP-43 and the number of regenerating retinal ganglion cells which also expressed GAP-43 were much higher in Group 2 than in Group 1 at all the time points and it was also much higher in Group 2 than in Group 3 at 4 weeks whereas there was no significant difference between the results from Groups 1 and 3 at 4 weeks. These data suggested that there was a close correlation between the number of the axotomized retinal ganglion cells regenerating axons into the peripheral nerve graft attached to the optic nerve and the expression of GAP-43. In addition, the intravitreal peripheral nerve, probably by releasing various neurotrophic factors and by acting synergistically, can enhance the expression of GAP-43 in some of the axotomized retinal ganglion cells and promote the regeneration of retinal ganglion cells into the peripheral nerve graft.     

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.     

Behavioral Impairments in Acute and Chronic Manganese Poisoning in White Rats

Single p.o. doses of manganese chloride (MnCl₂·4H₂O; 50 mg/kg) induced significant and reversible decreases in total activity in white rats, along with worsening of the acquisition of an avoidance reaction in response to unconditioned and conditioned stimuli, increases in the latent period of conditioned reflex activity, and a temporary worsening of the learning process. Chronic manganese poisoning (daily p.o. manganese chloride at 20 or 50 mg/kg for one month) led to significant impairment of learning processes in a multipath maze but had no significant effect on reproduction of previously acquired stereotypical behavior.     

Quantitative manganese tract tracing: dose-dependent and activity-independent terminal labelling in the mouse visual system

At concentrations sufficient for visualisation using MRI, manganese (Mn) is believed to behave as a calcium analogue. This study examines different concentrations of Mn for enhanced MR tract tracing. The premise of activity-dependent axonal transport was also examined by partial or complete blockade of retinal ganglion cell activity. Quantitative T(1) maps and semi-quantitative normalised signal intensities in the superior colliculi facilitated assessment of applied intraocular concentrations and activity dependence, respectively. Varying the concentration of applied Mn revealed a non-monotonic profile, with optimal, unfavourable and undesirable effects noted: 25 mM proved optimal, showing a maximal decrease in T(1), whereas 400 mM was associated with no terminal-field enhancement. The estimated vitreal concentration for optimal transport of Mn (2 mM) is substantially lower than that used in previous studies of the mouse. Both the partial blockade of inputs to 50% of retinal ganglion cells by a mGluR6 glutamate agonist and the complete blockade of all retinal ganglion cell activity with tetrodotoxin failed to decrease the relative enhancement in the superior colliculus. The failure to prevent axonal transport of Mn by blocking activity (and therefore theoretically the intracellular influx) appeared to be paradoxical. The optimal vitreal concentration of Mn has previously been shown to facilitate massive intracellular uptake of Mn, competitively blocking calcium, and 1 mM Mn blocks neurotransmission pre-synaptically. These results suggest that, at concentrations required for optimal Mn-enhanced MRI tract tracing in the visual system of the mouse, the uptake and transport of Mn may be dominated by passive mechanisms, which may also block neurotransmission. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Activity-induced manganese-dependent functional MRI of the rat visual cortex following intranasal manganese chloride administration

Manganese-enhanced MRI (MEMRI) of the brain requires delivery of manganese into the target brain regions. It was previously shown that, following intranasal application, ongoing olfactory stimulation facilitates manganese transport along the olfactory nerve into the olfactory bulb, so bypassing the blood–brain barrier (BBB). We report on experiments to evaluate whether visual stimulation can permit manganese transport onwards from the olfactory bulb to the visual cortex. Rats in intact olfactory bulb group were reserved intact olfactory bulb, while those in olfactory bulbectomy group received bilateral bulbectomy. After intranasal MnCl₂ administration, olfactory and visual stimulations were performed on all the animals for a consecutive 20 h. The visual cortex was then examined using MEMRI. Enhanced imaging on T1WI was noted in the visual cortex of the intact olfactory bulb group. Image subtraction revealed that the signal intensity in the visual cortex of the intact olfactory bulb group was significantly higher than that of olfactory bulbectomy group. Volume of interest (VOI) analysis also showed that normalized intensities in the visual cortex of the intact olfactory bulb group were significantly higher as compared with those of the olfactory bulbectomy group. Inductively coupled plasma mass spectrometry (ICP-MS) confirmed that the manganese content in the visual cortex of the intact olfactory bulb group was increased in comparison with that of the olfactory bulbectomy group. These findings indicate that activity-induced manganese-dependent functional MRI (AIM fMRI) of the rat visual cortex can be performed following intranasal administration of manganese and demonstrate that manganese can migrate from the olfactory bulb to the visual cortex.     

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. Copyright © 2015 John Wiley & Sons, Ltd.     

Inhibition of the sodium–calcium exchanger via SEA0400 altered manganese‐induced T1 changes in isolated perfused rat hearts

Manganese (Mn²⁺)‐enhanced MRI (MEMRI) provides the potential for the in vivo evaluation of calcium (Ca²⁺) uptake in the heart. Recent studies have also suggested the role of the sodium–calcium (Na⁺–Ca²⁺) exchanger (NCX) in Mn²⁺ retention, which may have an impact on MEMRI signals. In this study, we investigated whether MEMRI with fast T₁ mapping allowed the sensitive detection of changes in NCX activity. We quantified the dynamics of the Mn²⁺‐induced T₁ changes in isolated perfused rat hearts in response to SEA0400, an NCX inhibitor. The experimental protocol comprised 30 min of Mn²⁺ perfusion (wash‐in), followed by a 30‐min wash‐out period. There were three experimental groups: 1, NCX inhibition by 1 µ m SEA0400 during Mn²⁺ wash‐in only (SEAin, n = 6); 2, NCX inhibition by 1 µ m SEA0400 during Mn²⁺ wash‐out only (SEAout, n = 6); 3, no NCX inhibition during both wash‐in and wash‐out to serve as the control group (CNTL, n = 5). Rapid T₁ mapping at a temporal resolution of 3 min was performed throughout the perfusion protocol using a triggered saturation–recovery Look–Locker sequence. Our results showed that NCX inhibition during Mn²⁺ wash‐in caused a significant increase in relaxation rate (R₁) at the end of Mn²⁺ perfusion. During the wash‐out period, NCX inhibition led to less reduction in R₁. Further analysis of Mn²⁺ content in myocardium with flame atomic absorption spectroscopy was consistent with the MRI findings. These results suggest that Mn²⁺ accumulation and retention in rat hearts are, in part, dependent on NCX activity. Hence, MEMRI may provide an imaging method that is also sensitive to changes in NCX activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Fractionated manganese-enhanced MRI

We investigated the use of manganese-enhanced MRI (MEMRI) with fractionated doses as a way to retain the unique properties of manganese as a neuronal contrast agent while lessening its toxic effects in animals. First, we followed the signal enhancement on T1-weighted images of the brains of rats receiving 30 mg/kg fractions of MnCl2 . 4H2O every 48 h and found that the signal increased in regions with consecutive fractionated doses and ultimately saturated. Second, we used T1 mapping to test whether the amount of MRI-visible manganese that accumulated depended on the concentration of manganese in the fractions. For a fixed cumulative dose of 180 mg/kg MnCl2 . 4H2O, increasing fraction doses of 6 x 30 mg/kg, 3 x 60 mg/kg, 2 x 90 mg/kg and 1 x 180 mg/kg produced progressively shorter T1 values. The adverse systemic health effects, including complications at the injection site and poor animal well-being, also rose with the fraction dose. Thus, fractionated MEMRI can be used to balance the properties of manganese as a contrast agent in animals against its toxic effects.     

Urocortin 2 protects against retinal degeneration following bilateral common carotid artery occlusion in the rat

Urocortin 2 (Ucn 2) is corticotropin-releasing factor (CRF) paralog that preferentially activates CRF₂ receptors. Ucns exert CRF₂-mediated cytoprotective effects against ischemia-reperfusion injury in cardiomyocytes. However, little is known regarding potential retinoprotective effects of Ucns despite the known presence of CRF family peptides and their receptors (predominantly CRF_2α) in retina. Therefore, the present study investigated the effects of post-ischemic intravitreal Ucn 2 (2 nmol) administration on ischemia-induced retinal degeneration. Two-month-old rats were subjected to permanent bilateral common carotid artery occlusion, and their retinas were processed histologically after two weeks survival to determine the density of viable cells in the ganglion cell layer and the thickness of all retinal layers. In vehicle-treated subjects, carotid occlusion reduced retina thickness by approximately 60% as compared to sham-operated animals. In contrast, intraocular Ucn 2 treatment led to a marked amelioration of the retinal layers, and the thickness of all layers was significantly increased by 40% compared to ischemic vehicle-treated subjects. Ucn 2 treatment also increased the number of cells by 55% in the ganglion cell layer as compared to those from carotid-occluded retinas of vehicle-treated subjects. These findings suggest that intraocular Ucn 2 treatment may protect against ischemia-induced retinal degeneration, results with potential therapeutic implications for ophthalmic diseases.