Amide proton signals as pH indicator for in vivo MRS and MRI of the brain-Responses to hypercapnia and hypothermia.

Using proton MRS and MRI of mouse brain at 9.4T, this work provides the first in vivo evidence of pH-dependent concurrent changes of three amide signals and related metabolic responses to hypercapnia and hypothermia. During hypercapnia, amide proton MRS signals of glutamine at 6.8-6.9ppm and 7.6ppm as well as of unspecific compounds at 8.1-8.3ppm increase by at least 50% both at 37°C and 22°C. These changes reflect a reduced proton exchange with water. They are strongly correlated with intracellular pH which ranges from 6.75±0.10 to 7.13±0.06 as determined from a shift in creatine phosphokinase equilibrium. In MRI, saturation transfer from aliphatic as well as aromatic and/or amide protons alters slightly during hypercapnia and significantly during hypothermia. The asymmetry in magnetization transfer ratios decreased slightly during hypercapnia and hypothermia. Regardless of pH or temperature, saturation transfer from aliphatic protons between -2 and -4ppm frequency offset to water protons is significantly greater than that from aromatic/amide protons at corresponding offsets between +2 and +4ppm. Irradiation of aliphatic compounds at -3.5ppm frequency offset from water predominantly saturates lipids and water associated with myelin. Taken together, the results indicate that, for the B1 power used in this study, dipolar coupling between aliphatic and water protons rather than proton exchange is the dominant factor in Z-spectra and magnetization transfer ratio asymmetry of the brain in vivo.

Axonal loss and neuroinflammation caused by peroxisome-deficient oligodendrocytes.

Oligodendrocytes myelinate axons for rapid impulse conduction and contribute to normal axonal functions in the central nervous system. In multiple sclerosis, demyelination is caused by autoimmune attacks, but the role of oligodendroglial cells in disease progression and axon degeneration is unclear. Here we show that oligodendrocytes harbor peroxisomes whose function is essential for maintaining white matter tracts throughout adult life. By selectively inactivating the import factor PEX5 in myelinating glia, we generated mutant mice that developed normally, but within several months showed ataxia, tremor and premature death. Absence of functional peroxisomes from oligodendrocytes caused widespread axonal degeneration and progressive subcortical demyelination, but did not interfere with glial survival. Moreover, it caused a strong proinflammatory milieu and, unexpectedly, the infiltration of B and activated CD8+ T cells into brain lesions. We conclude that peroxisomes provide oligodendrocytes with an essential neuroprotective function against axon degeneration and neuroinflammation, which is relevant for human demyelinating diseases.

Halogenated volatile anesthetics alter brain metabolism as revealed by proton magnetic resonance spectroscopy of mice in vivo.

Halogenated volatile anesthetics (HVA) are widely used in medicine and research but their effects on brain metabolism in intact organisms are still largely unknown. Here, localized proton magnetic resonance spectroscopy (MRS) of anesthetized mice was applied to evaluate HVA effects on cerebral metabolites in vivo. Experimental protocols combined different concentrations of isoflurane, halothane, sevoflurane, and desflurane with known modulators of adrenergic, GABAergic, and glutamatergic neurotransmission. As a most striking finding, brain lactate increased in individual mice from 1.0 ± 0.6 mM (awake state) to 6.2 ± 1.5 mM (1.75% isoflurane). In addition, relative to total creatine, there were significant isoflurane-induced increases of alanine by 111%, GABA by 20%, choline-containing compounds by 20%, and myo-inositol by 10% which were accompanied by significant decreases of glucose by 51% and phosphocreatine by 9%. The elevation of lactate was most pronounced in the striatum. The HVA effects correlated with the respective minimal alveolar concentrations and were mostly reversible within minutes. The observed alterations are best explained by an HVA-induced stimulation of adrenergic pathways in conjunction with an inhibition of the respiratory chain. Apart from casting new light on cerebral energy metabolism, the present results challenge brain studies of HVA-anesthetized animals.

Cell layers and neuropil: contrast-enhanced MRI of mouse brain in vivo.

Contrast-enhanced T1- and T2-weighted MRI at 9.4 T and in-plane resolutions of 25 and 30 µm has been demonstrated to differentiate between neural tissues in mouse brain in vivo, including granule cell layers, principal cell layers, general neuropil, specialized neuropil and white matter. In T1-weighted MRI of the olfactory bulb, hippocampus and cerebellum, contrast obtained by the intracranial administration of gadopentetate dimeglumine (Gd-DTPA) reflects the extra- and intracellular spaces of gray matter in agreement with histological data. General neuropil areas are highlighted, whereas other tissues present with lower signal intensities. The induced contrast is similar to that in plain T2-weighted MRI, but offers a 16-30-fold higher contrast-to-noise ratio. Systemic administration of manganese chloride increases the signal-to-noise ratio in T1-weighted MRI to a significantly greater extent in principal cell layers and specialized neuropil than in granule cell layers, whereas gadolinium-enhanced MRI indicates no larger intracellular spaces in these tissues. Granule cell layers are enhanced no more than general neuropil by manganese, whereas gadolinium-enhanced MRI indicates significantly larger intracellular spaces in the cell layers. These discrepancies suggest that the signal increase after manganese administration reflects cellular activity which is disproportionate to the intracellular space. As a result, principal cell layers and specialized neuropil become highlighted, whereas granule cell layers, general neuropil and white matter present with lower signal intensities.

FLASHlight MRI in real time-a step towards Star Trek medicine.

This work describes a dynamic magnetic resonance imaging (MRI) technique for local scanning of the human body with use of a handheld receive coil or coil array. Real-time MRI is based on highly undersampled radial gradient-echo sequences with joint reconstructions of serial images and coil sensitivity maps by regularized nonlinear inversion (NLINV). For this proof-of-concept study, a fixed slice position and field-of-view (FOV) were predefined from the operating console, while a local receive coil (array) is moved across the body-for the sake of simplicity by the subject itself. Experimental realizations with a conventional 3 T magnet comprise dynamic anatomic imaging of the head, thorax and abdomen of healthy volunteers. Typically, the image resolution was 0.75 to 1.5 mm with 3 to 6 mm section thickness and acquisition times of 33 to 100 ms per frame. However, spatiotemporal resolutions and contrasts are highly variable and may be adjusted to clinical needs. In summary, the proposed FLASHlight MRI method provides a robust acquisition and reconstruction basis for future diagnostic strategies that mimic the usage of ultrasound. Necessary extensions for this vision require remote control of all sequence parameters by a person at the scanner as well as the design of more flexible gradients and magnets.

Myelin mapping in the central nervous system of living mice using contrast-enhanced magnetization transfer MRI.

This work compares magnetization transfer (MT) MRI of living mice with contrast-enhanced MT MRI using intraventricular administration of gadopentetate dimeglumine (Gd-DTPA), systemic administration of MnCl2, and both. In MT MRI at 9.4 T, the contrast-to-noise ratio (CNR) between white matter (WM) and gray matter (GM) increased by 85% after Gd-DTPA injection into the lateral ventricle. When applied in conjunction with manganese-enhanced MT MRI (117 µm isotropic resolution, 6 min measuring time), Gd-DTPA boosted the CNR increase from +56% to +117%. Additional T1 measurements at 2.35 T revealed that intraventricular Gd-DTPA shortens the T1 of GM much more than that of WM, which corresponds to estimated extracellular spaces of 26% in GM and only 15% in WM. These results explain the additional MT contrast enhancement by Gd-DTPA and demonstrate that the T1 shortening by intracellular Mn2+ is well complemented by extracellular Gd-DTPA. The data suggest a high myelin and low water content to hinder access of hydrophilic paramagnetic agents, so that the resulting differential accumulation effectively reduces the MT saturation in water-rich tissues and thereby facilitates the mapping of myelin-rich tissues. Finally, a 156% CNR increase between GM and WM for contrast-enhanced MT MRI at 9.4T using both Gd-DTPA and manganese allowed for 60µm isotropic resolution (102 min measuring time), which delineated myelinated fibers and layers even within GM areas such as the thalamus and cerebellar cortex. Improved MT contrasts were also seen in the cervical spinal cord.

Anesthesia triggers drug delivery to experimental glioma in mice by hijacking caveolar transport.

BACKGROUND: Pharmaceutical intervention in the CNS is hampered by the shielding function of the blood-brain barrier (BBB). To induce clinical anesthesia, general anesthetics such as isoflurane readily penetrate the BBB. Here, we investigated whether isoflurane can be utilized for therapeutic drug delivery. METHODS: Barrier function in primary endothelial cells was evaluated by transepithelial/transendothelial electrical resistance, and nanoscale STED and SRRF microscopy. In mice, BBB permeability was quantified by extravasation of several fluorescent tracers. Mouse models including the GL261 glioma model were evaluated by MRI, immunohistochemistry, electron microscopy, western blot, and expression analysis. RESULTS: Isoflurane enhances BBB permeability in a time- and concentration-dependent manner. We demonstrate that, mechanistically, isoflurane disturbs the organization of membrane lipid nanodomains and triggers caveolar transport in brain endothelial cells. BBB tightness re-establishes directly after termination of anesthesia, providing a defined window for drug delivery. In a therapeutic glioblastoma trial in mice, simultaneous exposure to isoflurane and cytotoxic agent improves efficacy of chemotherapy. CONCLUSIONS: Combination therapy, involving isoflurane-mediated BBB permeation with drug administration has far-reaching therapeutic implications for CNS malignancies.

Myelin mapping in the living mouse brain using manganese-enhanced magnetization transfer MRI.

This work demonstrates manganese-enhanced magnetization transfer (MT) MRI to improve the contrast of myelinated structures in mouse brain in vivo. Systemic administration of manganese chloride led to a reduction of the MT ratio by 23% in white matter and 35% in gray matter. The effect increased their contrast-to-noise ratio by 48% and facilitated a mapping of myelin-rich white matter tissues. Relaxation time measurements revealed the manganese-induced shortening of T1 to be smaller in the corpus callosum (-42%) than in the cortex (-52%) or hippocampus (-60%). These findings are in line with the assumption that a high myelin and correspondingly low water content hinder the free diffusion and uptake of manganese ions. The resulting preferential accumulation of manganese in gray matter structures causes a stronger reduction of the MT saturation in gray matter than in white matter. Extending MRI assessments with conventional MT contrast, manganese-enhanced MT MRI at 76 x 80 x 160 microm(3) resolution and 2.35 T field strength allowed for a delineation of small myelinated structures such as the fornix, mammillothalamic tract, and fasciculus retroflexus in the living mouse brain.

Role of n-type voltage-dependent calcium channels in autoimmune optic neuritis.

OBJECTIVE: The aim of this study was to investigate the role of voltage-dependent calcium channels (VDCCs) in axon degeneration during autoimmune optic neuritis. METHODS: Calcium ion (Ca(2+)) influx into the optic nerve (ON) through VDCCs was investigated in a rat model of optic neuritis using manganese-enhanced magnetic resonance imaging and in vivo calcium imaging. After having identified the most relevant channel subtype (N-type VDCCs), we correlated immunohistochemistry of channel expression with ON histopathology. In the confirmatory part of this work, we performed a treatment study using omega-conotoxin GVIA, an N-type specific blocker. RESULTS: We observed that pathological Ca(2+) influx into ONs during optic neuritis is mediated via N-type VDCCs. By analyzing the expression of VDCCs in the inflamed ONs, we detected an upregulation of alpha(1B), the pore-forming subunit of N-type VDCCs, in demyelinated axons. However, high expression levels were also found on macrophages/activated microglia, and lower levels were detected on astrocytes. The relevance of N-type VDCCs for inflammation-induced axonal degeneration and the severity of optic neuritis was corroborated by treatment with omega-conotoxin GVIA. This blocker led to decreased axon and myelin degeneration in the ONs together with a reduced number of macrophages/activated microglia. These protective effects were confirmed by analyzing the spinal cords of the same animals. INTERPRETATION: We conclude that N-type VDCCs play an important role in inflammation-induced axon degeneration via two mechanisms: First, they directly mediate toxic Ca(2+) influx into the axons; and second, they contribute to macrophage/microglia function, thereby promoting secondary axonal damage. Ann Neurol 2009;66:81-93.

In vivo MRI of altered brain anatomy and fiber connectivity in adult pax6 deficient mice.

The impact of developmental ablation of Pax6 function on morphology and functional connectivity of the adult cerebrum was studied in cortex-specific Pax6 knockout mice (Pax6cKO) using structural magnetic resonance imaging (MRI), manganese-enhanced MRI, and diffusion tensor MRI in conjunction with fiber tractography. Mutants presented with decreased volumes of total brain and olfactory bulb, reduced cortical thickness, and altered layering of the piriform cortex. Tracking of major neuronal fiber bundles revealed a disorganization of callosal fibers with an almost complete lack of interhemispheric connectivity. In Pax6cKO mice intrahemispheric callosal fibers as well as intracortical fibers were predominantly directed along a rostrocaudal orientation instead of a left-right and dorsoventral orientation found in controls. Fiber disorganization also involved the septohippocampal connection targeting mostly the lateral septal nucleus. The hippocampus was rostrally extended and its volume was increased relative to that of the forebrain and midbrain. Manganese-induced MRI signal enhancement in the CA3 region suggested a normal function of hippocampal pyramidal cells. Noteworthy, several morphologic disturbances in gray and white matter of Pax6cKO mice were similar to observations in human aniridia patients. The present findings indicate an important role of Pax6 in the development of both the cortex and cerebral fiber connectivity.

MRI of cellular layers in mouse brain in vivo.

Noninvasive imaging of the brain of animal models demands the detection of increasingly smaller structures by in vivo MRI. The purpose of this work was to elucidate the spatial resolution and structural contrast that can be obtained for studying the brain of C57BL/6J mice by optimized T2-weighted fast spin-echo MRI at 9.4 T. As a prerequisite for high-resolution imaging in vivo, motion artifacts were abolished by combining volatile anesthetics and positive pressure ventilation with a specially designed animal bed for fixation. Multiple substructures in the cortex, olfactory bulb, hippocampus, and cerebellum were resolved at 30 to 40 microm in-plane resolution and 200 to 300 microm section thickness as well as for relatively long echo times of 65 to 82 ms. In particular, the approach resulted in the differentiation of up to five cortical layers. In the olfactory bulb the images unraveled the mitral cell layer which has a thickness of mostly single cells. In the hippocampus at least five substructures could be separated. The molecular layer, Purkinje layer, and granular layer of the cerebellum could be clearly differentiated from the white matter. In conclusion, even without the use of a contrast agent, suitable adjustments of a widely available T2-weighted MRI sequence at high field allow for structural MRI of living mice at near single-cell layer resolution.

Multi-slice real-time MRI of temporomandibular joint dynamics.

OBJECTIVES: The purpose of this work was to improve the clinical versatility of high-speed real-time MRI studies of temporomandibular joint (TMJ) dynamics by simultaneous recordings of multiple MRI movies in different sections. METHODS: Real-time MRI at 3 T was realized using highly undersampled radial FLASH acquisitions and image reconstruction by regularized nonlinear inversion (NLINV). Multi-slice real-time MRI of two, three or four slices at 0.75 mm resolution and 6 to 8 mm thickness was accomplished at 50.0 ms, 33.3 ms or 25.5 ms temporal resolution, respectively, yielding simultaneous movies at 2 × 10, 3 × 10 or 4 × 10 frames per second in a frame-interleaved acquisition mode. Real-time MRI movies were evaluated by three blinded raters for visibility of the anterior and posterior border of disc, shape of the disk body and condyle head as well as movement of the disc and condyle (1 = excellent, 5 = no visibility). RESULTS: Effective delineation of the disk atop the mandibular condyle was achieved by T1-weighted images with opposed-phase water-fat contrast. Compared to 8 mm sections, multi-slice recordings with 6 mm thickness provided sharper delineation of relevant structures as confirmed by inter-rater evaluation. Respective dual-slice and triple-slice recordings of a single TMJ as well as dual-slice recordings of both joints (one slice per TMJ) received the highest visibility ratings of ≤ 2 corresponding to high confidence in diagnostic content. CONCLUSIONS: The improved access to TMJ dynamics by multi-slice real-time MRI will contribute to more effective treatment of temporomandibular disorders.

SONU20176289, a compound combining partial dopamine D(2) receptor agonism with specific serotonin reuptake inhibitor activity, affects neuroplasticity in an animal model for depression.

We investigated the efficacy of SONU20176289, a member of a group of novel phenylpiperazine derivatives with a mixed dopamine D(2) receptor partial agonist and specific serotonin reuptake inhibitor (SSRI) activity, in a chronic stress model of depression in male tree shrews. Animals were subjected to a 7-day period of psychosocial stress before treatment for 28 days with SONU20176289 (6 mg/kg/day, p.o.), during which stress was maintained. Stress reduced the in vivo brain concentrations of N-acetyl-aspartate, total creatine, and choline-containing compounds, as measured by localized proton magnetic resonance spectroscopy. Post mortem analyses revealed a reduced adult dentate cell proliferation and a decreased GluR2 expression in the prefrontal cortex. All these alterations were prevented by concomitant administration of SONU20176289. The results provide further support to the concept that antidepressant treatments may act by normalizing disturbed neuroplasticity, and indicate that combining dopamine D(2) receptor agonism with SSRI activity may serve as an effective tool in the treatment of depressive/anxiety syndromes.

Early MRI changes in a mouse model of multiple sclerosis are predictive of severe inflammatory tissue damage.

MRI is routinely used for in vivo detection of multiple sclerosis (MS) lesions. Histopathological correlates of MRI signal alterations are still poorly defined. In the present study, we describe a mouse model of MS presenting with inflammatory brain lesions. During the acute disease phase, two independent lesion patterns were identified by T1- and T2-weighted high-resolution 3D MRI: lesions with reduced signal intensity on both T1- and T2-weighted images (type A) and lesions with slightly reduced signal intensity on T1-weighted images and increased signal intensity on T2-weighted images (type B). Type A lesions were characterized by significantly denser inflammatory cell infiltrates and more myelin loss than type B lesions. Lesion cellularity, myelin loss and immunoglobulin deposition correlated with MRI signal intensities in both lesion types. Gd-DTPA enhancement correlated with Ig deposition and spacially matched to areas with abundant activated microglia cells at the lesion border. Using serial MRI, type A lesions revealed a persistent hypointense pattern reflecting axon and myelin loss. Signal intensity increases on T2-weighted images of type B lesions decreased during lesion evolution, and no significant T1 signal alterations developed. Taken together, MRI of mouse EAE models with brain lesions provide new insights into lesion pathology and evolution and may prove useful for the in vivo assessment of new therapeutic strategies in MS.

High-field diffusion tensor imaging of mouse brain in vivo using single-shot STEAM MRI.

Information about the microstructural organization of cerebral white matter that is accessible by magnetic resonance diffusion tensor imaging (DTI) gains increasing importance for studies of animal brain. Particular challenges occur for in vivo conditions as well as at high magnetic fields. Here, we have employed a diffusion-weighted (DW) single-shot STEAM MRI sequence for DTI of mouse brain in vivo at 7 T. The approach exploits the increased longitudinal magnetization and prolonged T1 relaxation times of water protons at higher magnetic field strengths without suffering from susceptibility-induced artifacts. When compared to studies at 2.35 T, half Fourier DW STEAM MRI at 7 T yielded a substantial gain in signal-to-noise ratio (SNR) that could be invested either in a reduction of the measurement time or an increase of the spatial resolution. Thus, for a measurement time of 3h, DTI with a voxel size of 117 microm x 117 microm x 720 microm not only resulted in high-quality maps of the fractional anisotropy and main diffusion direction (MDD), but also allowed for fiber tracking of major mouse brain structures in vivo.

Global brain atrophy after unilateral parietal lesion and its prevention by erythropoietin.

In humans, neurotrauma is suspected to cause brain atrophy and accelerate slowly progressive neurodegenerative disorders, such as Alzheimer's disease or schizophrenia. However, a direct link between brain injury and subsequent delayed global neurodegeneration has remained elusive. Here we show that juvenile (4-week-old) mice that are given a discrete unilateral lesion of the parietal cortex, develop to adulthood without obvious clinical symptoms. However, when monitored 3 and 9 months after lesioning, using high-resolution three-dimensional MRI and behavioural testing, the same mice display global neurodegenerative changes. Surprisingly, erythropoietin, a haematopoietic growth factor with potent neuroprotective activity, prevents behavioural abnormalities, cognitive dysfunction and brain atrophy when given for 2 weeks after acute brain injury. This demonstrates that a localized brain lesion is a primary cause of delayed global neurodegeneration that can be efficiently counteracted by neuroprotection.

Towards MRI temperature mapping in real time-the proton resonance frequency method with undersampled radial MRI and nonlinear inverse reconstruction.

BACKGROUND: Optimal control of minimally invasive interventions by hyperthermia requires dynamic temperature mapping at high temporal resolution. METHODS: Based on the temperature-dependent shift of the proton resonance frequency (PRF), this work developed a method for real-time MRI thermometry which relies on highly undersampled radial FLASH MRI sequences with iterative image reconstruction by regularized nonlinear inversion (NLINV). As a first step, the method was validated with use of a temperature phantom and ex vivo organs (swine kidney) subjected to heating by warm water or a pulsed laser source. RESULTS: The temperature maps obtained by real-time PRF MRI demonstrate good accuracy as independently controlled by fiber-optic temperature sensors. Moreover, the dynamic results demonstrate both excellent sensitivity to single laser pulses (20 ms duration, 6 J energy output) and high temporal resolution, i.e., 200 ms acquisition times per temperature map corresponding to a rate of 5 frames per second. In addition, future extensions to in vivo applications were prepared by addressing the breathing-related motion problem by a pre-recorded library of reference images representative of all respiratory states. CONCLUSIONS: The proposed method for real-time MRI thermometry now warrants further developments towards in vivo MRI monitoring of thermal interventions in animals.

Manganese-enhanced 3D MRI of established and disrupted synaptic activity in the developing insect brain in vivo.

The antennal lobe of the sphinx moth Manduca sexta serves as a model for the development of the olfactory system. Here, the establishment of the glomerular synaptic network formed by the olfactory receptor axons and antennal lobe neurons at pupal stage P12 was followed by transection of the right antenna and - within 24 h - by injection of MnCl2 into the hemolymph. In vivo 3D MRI at 100 and 60 microm isotropic resolution was then performed at P13 to P17. Whereas the left antennal lobe revealed a pronounced increase of the signal-to-noise ratio (SNR) reflecting normal synaptic activity, the observation of only a small SNR increase within the right antennal lobe indicated the disruption of pertinent activity after antennal transection. The accumulation of manganese in the intact antennal system became observable within 3 h and lasted for at least 2 days after injection. Intra-individual comparisons between the right and left side yielded a statistically significant differential SNR increase in the left antennal lobe. Because such an effect was not observed in younger animals studied at pupal stages P10/P11, the MRI findings confirm the development of functional synapses in the antennal lobe of Manduca sexta by P13.

Mapping of the habenulo-interpeduncular pathway in living mice using manganese-enhanced 3D MRI.

This magnetic resonance imaging (MRI) study describes mapping of the habenulo-interpeduncular pathway in living mice based on manganese-induced contrast. Six hours after intracerebroventricular microinjection of MnCl2, T1-weighted 3D MRI (2.35 T) at 117 mum isotropic resolution revealed a continuous pattern of anterograde labeling from the habenula via the fasciculus retroflexus to the interpeduncular nucleus. Alternatively, the less invasive systemic administration of MnCl2 allowed for monitoring of the dynamic uptake pattern of respective neural components with even higher reproducibility across animals. Time courses covered the range from 42 min to 24 h after injection. In conclusion, manganese-enhanced MRI may open new ways for functional assessments of the habenulo-interpeduncular system in animal models with cognitive impairment.

In Vivo Brain MR Imaging at Subnanoliter Resolution: Contrast and Histology.

This article provides an overview of in vivo magnetic resonance (MR) imaging contrasts obtained for mammalian brain in relation to histological knowledge. Emphasis is paid to the (1) significance of high spatial resolution for the optimization of T1, T2, and magnetization transfer contrast, (2) use of exogenous extra- and intracellular contrast agents for validating endogenous contrast sources, and (3) histological structures and biochemical compounds underlying these contrasts and (4) their relevance to neuroradiology. Comparisons between MR imaging at subnanoliter resolution and histological data indicate that (a) myelin sheaths, (b) nerve cells, and (c) the neuropil are most responsible for observed MR imaging contrasts, while (a) diamagnetic macromolecules, (b) intracellular paramagnetic ions, and (c) extracellular free water, respectively, emerge as the dominant factors. Enhanced relaxation rates due to paramagnetic ions, such as iron and manganese, have been observed for oligodendrocytes, astrocytes, microglia, and blood cells in the brain as well as for nerve cells. Taken together, a plethora of observations suggests that the delineation of specific structures in high-resolution MR imaging of mammalian brain and the absence of corresponding contrasts in MR imaging of the human brain do not necessarily indicate differences between species but may be explained by partial volume effects. Second, paramagnetic ions are required in active cells in vivo which may reduce the magnetization transfer ratio in the brain through accelerated T1 recovery. Third, reductions of the magnetization transfer ratio may be more sensitive to a particular pathological condition, such as astrocytosis, microglial activation, inflammation, and demyelination, than changes in relaxation. This is because the simultaneous occurrence of increased paramagnetic ions (i.e., shorter relaxation times) and increased free water (i.e., longer relaxation times) may cancel T1 or T2 effects, whereas both processes reduce the magnetization transfer ratio.