INPP5A phosphatase is a synthetic lethal target in GNAQ and GNA11-mutant melanomas.

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.

Fiber alignment in 3D collagen networks as a biophysical marker for cell contractility.

Cells cultured in 3D fibrous biopolymer matrices exert traction forces on their environment that induce deformations and remodeling of the fiber network. By measuring these deformations, the traction forces can be reconstructed if the mechanical properties of the matrix and the force-free matrix configuration are known. These requirements limit the applicability of traction force reconstruction in practice. In this study, we test whether force-induced matrix remodeling can instead be used as a proxy for cellular traction forces. We measure the traction forces of hepatic stellate cells and different glioblastoma cell lines and quantify matrix remodeling by measuring the fiber orientation and fiber density around these cells. In agreement with simulated fiber networks, we demonstrate that changes in local fiber orientation and density are directly related to cell forces. By resolving Rho-kinase (ROCK) inhibitor-induced changes of traction forces, fiber alignment, and fiber density in hepatic stellate cells, we show that the method is suitable for drug screening assays. We conclude that differences in local fiber orientation and density, which are easily measurable, can be used as a qualitative proxy for changes in traction forces. The method is available as an open-source Python package with a graphical user interface.

Matrigel 3D bioprinting of contractile human skeletal muscle models recapitulating exercise and pharmacological responses.

A key to enhance the low translatability of preclinical drug discovery are in vitro human three-dimensional (3D) microphysiological systems (MPS). Here, we show a new method for automated engineering of 3D human skeletal muscle models in microplates and functional compound screening to address the lack of muscle wasting disease medication. To this end, we adapted our recently described 24-well plate 3D bioprinting platform with a printhead cooling system to allow microvalve-based drop-on-demand printing of cell-laden Matrigel containing primary human muscle precursor cells. Mini skeletal muscle models develop within a week exhibiting contractile, striated myofibers aligned between two attachment posts. As an in vitro exercise model, repeated high impact stimulation of contractions for 3 h by a custom-made electrical pulse stimulation (EPS) system for 24-well plates induced interleukin-6 myokine expression and Akt hypertrophy pathway activation. Furthermore, the known muscle stimulators caffeine and Tirasemtiv acutely increase EPS-induced contractile force of the models. This validated new human muscle MPS will benefit development of drugs against muscle wasting diseases. Moreover, our Matrigel 3D bioprinting platform will allow engineering of non-self-organizing complex human 3D MPS.

New insights into molecular changes in skeletal muscle aging and disease: Differential alternative splicing and senescence.

Progressive loss of muscle mass and function due to muscle fiber atrophy and loss in the elderly and chronically ill is now defined as sarcopenia. It is a major contributor to loss of independence, disability, need of long-term care as well as overall mortality. Sarcopenia is a heterogenous disease and underlying mechanisms are not completely understood. Here, we newly identified and used Tmem158, alongside Cdkn1a, as relevant senescence and denervation markers (SDMs), associated with muscle fiber atrophy. Subsequent application of laser capture microdissection (LCM) and RNA analyses revealed age- and disease-associated differences in gene expression and alternative splicing patterns in a rodent sarcopenia model. Of note, genes exhibiting such differential alternative splicing (DAS) are mainly involved in the contractile function of the muscle. Many of these splicing events are also found in a mouse model for myotonic dystrophy type 1 (DM1), underscoring the premature aging phenotype of this disease. We propose to add differential alternative splicing to the hallmarks of aging.

A Microphysiological Cell-Culturing System for Pharmacokinetic Drug Exposure and High-Resolution Imaging of Arrays of 3D Microtissues.

Understanding the pharmacokinetic/pharmacodynamic (PK/PD)-relationship of a drug candidate is key to determine effective, yet safe treatment regimens for patients. However, current testing strategies are inefficient in characterizing in vivo responses to fluctuating drug concentrations during multi-day treatment cycles. Methods based on animal models are resource-intensive and require time, while traditional in vitro cell-culturing methods usually do not provide temporally-resolved information on the effects of in vivo-like drug exposure scenarios. To address this issue, we developed a microfluidic system to 1) culture arrays of three-dimensional spheroids in vitro, to 2) apply specific dynamic drug exposure profiles, and to 3) in-situ analyze spheroid growth and the invoked drug effects in 3D by means of 2-photon microscopy at tissue and single-cell level. Spheroids of fluorescently-labeled T-47D breast cancer cells were monitored under perfusion-culture conditions at short time intervals over three days and exposed to either three 24 h-PK-cycles or a dose-matched constant concentration of the phosphatidylinositol 3-kinase inhibitor BYL719. While the overall efficacy of the two treatment regimens was similar, spheroids exposed to the PK profile displayed cycle-dependent oscillations between regression and regrowth. Spheroids treated with a constant BYL719 concentration regressed at a steady, albeit slower rate. At a single-cell level, the cell density in BYL719-treated spheroids oscillated in a concentration-dependent manner. Our system represents a versatile tool for in-depth preclinical characterization of PK/PD parameters, as it enables an evaluation of drug efficacy and/or toxicity under realistic exposure conditions.

Measurement of Skeletal Muscle Fiber Contractility with High-Speed Traction Microscopy.

We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels under auxotonic loading conditions. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Young's modulus of the matrix without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/mL Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 ± 0.15 µN and tetanic forces (100-Hz stimulation frequency) of 2.38 ± 0.71 µN, corresponding to a tension of 0.44 ± 0.25 kPa and 2.53 ± 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium stabilizer tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high-speed method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.

Peroxisome proliferator-activated receptor γ coactivator 1α regulates mitochondrial calcium homeostasis, sarcoplasmic reticulum stress, and cell death to mitigate skeletal muscle aging.

Age-related impairment of muscle function severely affects the health of an increasing elderly population. While causality and the underlying mechanisms remain poorly understood, exercise is an efficient intervention to blunt these aging effects. We thus investigated the role of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a potent regulator of mitochondrial function and exercise adaptation, in skeletal muscle during aging. We demonstrate that PGC-1α overexpression improves mitochondrial dynamics and calcium buffering in an estrogen-related receptor α-dependent manner. Moreover, we show that sarcoplasmic reticulum stress is attenuated by PGC-1α. As a result, PGC-1α prevents tubular aggregate formation and cell death pathway activation in old muscle. Similarly, the pro-apoptotic effects of ceramide and thapsigargin were blunted by PGC-1α in muscle cells. Accordingly, mice with muscle-specific gain-of-function and loss-of-function of PGC-1α exhibit a delayed and premature aging phenotype, respectively. Together, our data reveal a key protective effect of PGC-1α on muscle function and overall health span in aging.

A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues.

Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.

Glucocorticoid-loaded liposomes induce a pro-resolution phenotype in human primary macrophages to support chronic wound healing.

Glucocorticoids are well established anti-inflammatory agents, however, their use to treat chronic inflammatory diseases is limited due to a number of serious side effects. For example, long-term local treatment of chronic wounds with glucocorticoids is prohibited by dysregulation of keratinocyte and fibroblast function, leading to skin thinning. Here, we developed and tested liposome formulations for local delivery of dexamethasone to primary human macrophages, to drive an anti-inflammatory/pro-resolution phenotype appropriate for tissue repair. The liposomes were loaded with the pro-drug dexamethasone-phosphate and surface-modified with either polyethylene glycol or phosphatidylserine. The latter was used to mimic phosphatidylserine-harboring apoptotic cells, which are substrates for efferocytosis, an essential pro-resolution function. Both formulations induced a dexamethasone-like gene expression signature in macrophages, decreased IL6 and TNFα release, increased secretion of thrombospondin 1 and increased efferocytosis activity. Phosphatidylserine-modified liposomes exhibited a faster uptake, a higher potency and a more robust phenotype induction than polyethylene glycol-modified liposomes. Fibroblast and keratinocyte cell cultures as well as a 3D skin equivalent model showed that liposomes applied locally to wounds are preferentially phagocytosed by macrophages. These findings indicate that liposomes, in particular upon shell modification with phosphatidylserine, promote dexamethasone delivery to macrophages and induce a phenotype suitable to support chronic wound healing.

Stable Silenolates and Brook-Type Silenes with Exocyclic Structures.

The first silenolates with exocyclic structures [(Me3Si)2Si(Si2Me4)2SiC(R)O]-K+ (2a: R = 1-adamantyl; 2b: mesityl; 2c: o-tolyl) were synthesized by the reaction of the corresponding acylcyclohexasilanes 1a-c with KOtBu. NMR spectroscopy and single-crystal X-ray diffraction analysis suggest that the aryl-substituted silenolates 2b,c exhibit increased character of functionalized silenes as compared to the alkyl-substituted derivative 2a due to the different coordination of the K+ counterion to the SiC(R)O moiety. 2b,c, thus, reacted with ClSiiPr3 to give the exocyclic silenes (Me3Si)2Si(Si2Me4)2Si=C(OSiiPr3)R (3b: R = Mes; 3c: o-Tol), while 2a afforded the Si-silylated acylcyclohexasilane 1d. The thermally remarkably stable compound 3b, which is the first isolated silene with the sp2 silicon atom incorporated into a cyclopolysilane framework, could be fully characterized structurally and spectroscopically.

Age-dependent regulation of tendon crimp structure, cell length and gap width with strain.

The black-and-white patterning of tendon fascicles when visualized by light microscopy, also known as crimp, is a well-known feature of fiber-forming collagens. However, not much is known about its development, function and response to strain. The objective of this study is to investigate the interaction of tenocyte and crimp morphology as well as their changes with increasing age and acute strain. In contrast to previous studies, which used indirect measures, such as polarized light, to investigate the crimp structure, this study visualizes internal crimp structure in three dimensions without freezing, sectioning, staining or fixing the tissue, via two-photon imaging of green fluorescent protein expressing cells within mouse tail tendon fascicles. This technique further allows straining of the live tissue while visualizing changes in crimp morphology and cell shape with increasing specimen length. Combining this novel microscopy technique with computational image and data analysis revealed a complex relationship between tenocytes and the extracellular matrix that evolves with increasing age. While the reduction of crimping with strain was observed as expected, most of the crimps were gone at 0-1% strain already. Even relatively low strains of 3% led to pronounced changes in the crimp structure after relaxation, particularly in the young animals, which could not be seen with bright-field imaging. Cell length and gap width increased with strain. However, while the cells were able to return to their original length even after high strains of 6%, the gaps between the cells widened, which may imply modified cell-cell communication after overstretching.

Photoinduced Brook-Type Rearrangement of Acylcyclopolysilanes.

Previously unknown 1,1,4-tris(trimethylsilyl)-4-acyldodecamethylcyclohexasilanes (Me3Si)2Si6Me12(Me3Si)COR (16a, R = tert-butyl; 16b, R = 1-adamantyl) have been synthesized by the reaction of the potassium silanides (Me3Si)2Si6Me12(Me3Si)K with acid chlorides ClCOR, and their photochemical rearrangement reactions have been studied. The molecular structures of 16a,b as determined by single-crystal X-ray diffraction analysis exhibit an unusual twist-boat conformation of the cyclohexasilane ring. When 16a,b were photolyzed with λ >300 nm radiation, they underwent Brook type 1,3-Si → O migration reactions to generate the cyclohexasilanes 17a,b with exocyclic Si=C bonds along with smaller amounts of the ring-enlarged species 19a,b with endocyclic Si=C double bonds. While 17a,b were stable enough to allow characterization by NMR and UV absorption spectroscopy, the less stable products 19a,b could only be observed in the form of their methanol adducts.

Effect of Calstabin1 depletion on calcium transients and energy utilization in muscle fibers and treatment opportunities with RyR1 stabilizers.

Depletion of calstabin1 (FKBP12) from the RyR1 channel and consequential calcium leakage from the sarcoplasmic reticulum (SR) is found in certain disease conditions such as dystrophy, aging or muscle overuse. Here, we first assessed the effect of calstabin1 depletion on resting Ca(2+) levels and transients. We found that depletion of calstabin1 with the calstabin1-dissociation compound FK506 increased the release of calcium from the SR by 14 % during tetanic stimulation (50 Hz, 300 ms) and delayed cytosolic calcium removal. However, we did not find a significant increase in resting cytosolic Ca(2+) levels. Therefore, we tested if increased SERCA activity could counterbalance calcium leakage. By measuring the energy utilization of muscle fibers with and without FK506 treatment, we observed that FK506-treatment increased oxygen consumption by 125% compared to baseline levels. Finally, we found that pretreatment of muscle fibers with the RyR1 stabilizer JTV-519 led to an almost complete normalization of calcium flux dynamics and energy utilization. We conclude that cytosolic calcium levels are mostly preserved in conditions with leaky RyR1 channels due to increased SERCA activity. Therefore, we suggest that RyR1 leakiness might lead to chronic metabolic stress, followed by cellular damage, and RyR1 stabilizers could potentially protect diseased muscle tissue.

Establishment of a human skeletal muscle-derived cell line: biochemical, cellular and electrophysiological characterization.

Excitation-contraction coupling is the physiological mechanism occurring in muscle cells whereby an electrical signal sensed by the dihydropyridine receptor located on the transverse tubules is transformed into a chemical gradient (Ca2+ increase) by activation of the ryanodine receptor located on the sarcoplasmic reticulum membrane. In the present study, we characterized for the first time the excitation-contraction coupling machinery of an immortalized human skeletal muscle cell line. Intracellular Ca2+ measurements showed a normal response to pharmacological activation of the ryanodine receptor, whereas 3D-SIM (super-resolution structured illumination microscopy) revealed a low level of structural organization of ryanodine receptors and dihydropyridine receptors. Interestingly, the expression levels of several transcripts of proteins involved in Ca2+ homoeostasis and differentiation indicate that the cell line has a phenotype closer to that of slow-twitch than fast-twitch muscles. These results point to the potential application of such human muscle-derived cell lines to the study of neuromuscular disorders; in addition, they may serve as a platform for the development of therapeutic strategies aimed at correcting defects in Ca2+ homoeostasis due to mutations in genes involved in Ca2+ regulation.

Ascomycete derivative to MS therapeutic: S1P receptor modulator FTY720.

Fingolimod (FTY720) represents the first in a new class of immune-modulators whose target is sphingosine-1-phosphate (S1P) receptors. It was first identified by researchers at Kyoto University and Yoshitomi Pharmaceutical as a chemical derivative of the ascomycete metabolite ISP-1 (myriocin). Unlike its natural product parent, FTY720 does not interfere with sphingolipid biosynthesis. Instead, its best characterized mechanism of action upon in vivo phosphorylation, leading to the active principle FTY720-P, is the rapid and reversible inhibition of lymphocyte egress from peripheral lymph nodes. As a consequence of S1P1 receptor internalization, tissue-damaging T-cells can not recirculate and infiltrate sites of inflammation such as the central nervous system (CNS). Furthermore, FTY720-P modulation of S1P receptor signaling also enhances endothelial barrier function. Due to its mode of action, FTY720 effectively prevents transplant rejection and is active in various autoimmune disease models. The most striking efficacy is in the multiple sclerosis (MS) model of experimental autoimmune encephalomyelitis, which has now been confirmed in the clinic. FTY720 demonstrated promising results in Phase II trials and recently entered Phase III in patients with relapsing MS. Emerging evidence suggests that its efficacy in the CNS extends beyond immunomodulation to encompass other aspects of MS pathophysiology, including an influence on the blood-brain-barrier and glial repair mechanisms that could ultimately contribute to restoration of nerve function. FTY720 may represent a potent new therapeutic modality in MS, combined with the benefit of oral administration.

In vivo monitoring the fate of Cy5.5-Tat labeled T lymphocytes by quantitative near-infrared fluorescence imaging during acute brain inflammation in a rat model of experimental autoimmune encephalomyelitis.

T cells and macrophages directed against myelin proteins orchestrate the inflammation process in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). So far, assessment of macrophages infiltration or structural alterations has been achieved by in vivo imaging. In this work, we show the infiltration of Cy5.5-labeled T lymphocytes into the brains of EAE rats by reflectance near-infrared fluorescence imaging. T lymphocytes were labeled with Cy5.5-Tat and administered intravenously to naïve or EAE animals. The highest fluorescence signal was observed for EAE animals, which received myelin-activated T cells during the acute phase of the disease. The temporal profile of fluorescence in this group paralleled the pattern of neurological impairment during the acute phase, the remittance and first relapses of EAE. No disease specific fluorescence pattern was observed for EAE animals, which received naïve T cells. However, uptake of Cy5.5-Tat by scavenger cells (e.g. macrophages) following death of labeled T cells in vivo prevents prolonged longitudinal studies. Our work demonstrates that Cy5.5-Tat labeling of T cells is suitable for in vivo fluorescence imaging of inflammation initiation in the EAE model. This approach may particularly be useful for evaluation of novel anti-inflammatory therapies.

Feasibility and limits of magnetically labeling primary cultured rat T cells with ferumoxides coupled with commonly used transfection agents.

Visualization and quantification of inflammatory processes is of high importance for early diagnosis of a multitude of diseases. Magnetic resonance imaging (MRI) using iron oxide (FeO) nanoparticles as contrast agents allows the study of macrophage infiltration during inflammation in a variety of tissues. Macrophages are effectors of the immune response, their appearance being orchestrated by activated T lymphocytes. Therefore, tracking of labeled T lymphocytes, which initiate the immune process, should enable earlier detection of tissue inflammation. In this study, we investigate the feasibility of specifically labeling harvested T cells by using dextran-coated FeO nanoparticles and commonly available transfection agents (TAs). Physicochemical properties of the newly formed FeO/TA vesicles were determined as well as their cell toxicity and their T cell activation potential. The labeling efficiency of each FeO/TA combination was evaluated by measuring the transverse MRI relaxation rate R(2) by X-ray spectroscopy and magnetic selection. Toxicity and labeling efficacy differed significantly among TAs. The best results were achieved by using polyamine TAs and in particular by using poly-l-lysine at a concentration of 1.5 microg/mL administered in combination with 22.5 microg iron/mL. By using this protocol, up to 60% of harvested T cells could be labeled. Microscopic investigation revealed FeO/TA nanoparticles not only localized within the cytoplasma of the cells but also sticking to the outer membrane surface.

Quantitative dynamic contrast-enhanced MRI in tumor-bearing rats and mice with inversion recovery TrueFISP and two contrast agents at 4.7 T.

PURPOSE: To characterize tumor vascularization by dynamic-contrast enhanced (DCE) MRI using low and medium molecular weight paramagnetic contrast agents (CA) and inversion recovery (IR) true fast imaging with steady state precession (TrueFISP) in tumor-bearing rats and mice. MATERIALS AND METHODS: T(1) mapping was performed using IR True FISP in phantoms and in vivo at 4.7 T and validated with a segmented IR gradient-echo (IR GE) method. CA concentration in DCE-MRI studies in vivo was calculated from time-series T(1) maps using the CAs GdDOTA and P792 (low and medium molecular weight, respectively). Standard vascular input functions (VIFs) were measured in the jugular veins and were used for modeling of the CA kinetics with a two-compartment model. In rat breast tumors, vascular permeability (transfer constant K(trans)), fractional plasma volume v(p), and fractional leakage space v(e) were quantified and parametric maps were generated. RESULTS: The IR TrueFISP T(1) was slightly underestimated in phantoms and overestimated in vivo (10%) with respect to IR GE. VIFs showed only small interindividual variation. Mean K(trans) values were 0.062 +/- 0.017 min(-1) for GdDOTA and 0.015 +/- 0.005 min(-1) for P792 (N = 12). Mean v(e) and v(p) values were 0.15 +/- 0.04 (0.09 +/- 0.03) and 0.04 +/- 0.01 (0.03 +/- 0.01) for GdDOTA (P792). CONCLUSION: DCE-MRI with IR TrueFISP provided absolute values for K(trans), v(p), and v(e). Direct comparison between GdDOTA and P792 revealed significant differences in the VIF, model-fit-quality, permeability, leakage space, and plasma volume. The larger molecular weight CA P792 appears to be better for measuring tumor vascular parameters.

Therapy-induced plasticity of cognitive functions in MS patients: insights from fMRI.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system whose pathological mechanisms are still not completely understood. Physical as well as cognitive deterioration are consequences within the disease process that have an extensive impact on the patient's quality of life. Therefore, understanding the functional background of spontaneous as well as induced remission is of high relevance. Studies on visualization of therapeutic effects of pharmacological or cognitive treatment by functional magnetic resonance imaging (fMRI) are still rare. From fMRI studies on focal brain lesions hypotheses on mechanisms of brain reorganization can be derived. This contribution will first give an overview of the existing studies using fMRI in MS, on cognitive decline, on cognitive treatment studies and its therapeutic effects on behavioural readouts in MS, and on therapy-induced brain plasticity and its possible visualization by fMRI. Results of a study on correlating the effects of cognitive training with changes in brain organization in patients with mild to severe cognitive impairment will be reported.

Analysis of lesion development during acute inflammation and remission in a rat model of experimental autoimmune encephalomyelitis by visualization of macrophage infiltration, demyelination and blood-brain barrier damage.

In vivo tracking of macrophage migration is feasible by labeling cells with ultra-small particles of iron oxide (USPIO). It is demonstrated that it is possible to monitor distinct patterns of macrophage migration during the early states of inflammation in a rodent model of chronic relapsing experimental autoimmune encephalomyelitis (EAE). As previous MRI studies showed that EAE inflammation processes are clearly linked to macrophage infiltration in the brain, a longitudinal protocol for macrophage visualization was designed, where USPIOs were injected repeatedly during the acute phase of the disease, the remitting phase and the first relapse. In addition to USPIO-enhanced MRI, blood-brain barrier (BBB) damage, magnetization transfer ratios (MTRs) and neurological impairment were assessed as classical markers for central nervous system (CNS) inflammation and tissue damage. During the acute phase, animals showed severe paralysis of the hind paws, intense accumulation of macrophages in brain tissue and some diffuse patterns of BBB disruption. While USPIO-accumulation completely disappeared after the acute phase, residual damage of the BBB remained detectable in some lesions during the remitting phase. During the first relapse, the accumulation of USPIO-loaded cells was less pronounced but still detectable. The time course of MTR, which is used as a marker for myelin loss, was linked to the infiltration of macrophages during the acute phase.