Neurocognitive Changes in Patients with Post-COVID Depression.

Background: Depression and cognitive impairment are recognized complications of COVID-19. This study aimed to assess cognitive performance in clinically diagnosed post-COVID depression (PCD, n = 25) patients using neuropsychological testing. Methods: The study involved 71 post-COVID patients with matched control groups: recovered COVID-19 individuals without complications (n = 18) and individuals without prior COVID-19 history (n = 19). A post-COVID depression group (PCD, n = 25) was identified based on psychiatric diagnosis, and a comparison group (noPCD, n = 46) included participants with neurological COVID-19 complications, excluding clinical depression. Results: The PCD patients showed gender-dependent significant cognitive impairment in the MoCA, Word Memory Test (WMT), Stroop task (SCWT), and Trail Making Test (TMT) compared to the controls and noPCD patients. Men with PCD showed worse performances on the SCWT, in MoCA attention score, and on the WMT (immediate and delayed word recall), while women with PCD showed a decline in MoCA total score, an increased processing time with less errors on the TMT, and worse immediate recall. No differences between groups in Sniffin's stick test were found. Conclusions: COVID-related direct (post-COVID symptoms) and depression-mediated (depression itself, male sex, and severity of COVID-19) predictors of decline in memory and information processing speed were identified. Our findings may help to personalize the treatment of depression, taking a patient's gender and severity of previous COVID-19 disease into account.

Age-Related Decline in Brain Myelination: Quantitative Macromolecular Proton Fraction Mapping, T2-FLAIR Hyperintensity Volume, and Anti-Myelin Antibodies Seven Years Apart.

Age-related myelination decrease is considered one of the likely mechanisms of cognitive decline. The present preliminary study is based on the longitudinal assessment of global and regional myelination of the normal adult human brain using fast macromolecular fraction (MPF) mapping. Additional markers were age-related changes in white matter (WM) hyperintensities on FLAIR-MRI and the levels of anti-myelin autoantibodies in serum. Eleven healthy subjects (33-60 years in the first study) were scanned twice, seven years apart. An age-related decrease in MPF was found in global WM, grey matter (GM), and mixed WM-GM, as well as in 48 out of 82 examined WM and GM regions. The greatest decrease in MPF was observed for the frontal WM (2-5%), genu of the corpus callosum (CC) (4.0%), and caudate nucleus (5.9%). The age-related decrease in MPF significantly correlated with an increase in the level of antibodies against myelin basic protein (MBP) in serum (r = 0.69 and r = 0.63 for global WM and mixed WM-GM, correspondingly). The volume of FLAIR hyperintensities increased with age but did not correlate with MPF changes and the levels of anti-myelin antibodies. MPF mapping showed high sensitivity to age-related changes in brain myelination, providing the feasibility of this method in clinics.

Role of Demyelination in the Persistence of Neurological and Mental Impairments after COVID-19.

Long-term neurological and mental complications of COVID-19, the so-called post-COVID syndrome or long COVID, affect the quality of life. The most persistent manifestations of long COVID include fatigue, anosmia/hyposmia, insomnia, depression/anxiety, and memory/attention deficits. The physiological basis of neurological and psychiatric disorders is still poorly understood. This review summarizes the current knowledge of neurological sequelae in post-COVID patients and discusses brain demyelination as a possible mechanism of these complications with a focus on neuroimaging findings. Numerous reviews, experimental and theoretical studies consider brain demyelination as one of the mechanisms of the central neural system impairment. Several factors might cause demyelination, such as inflammation, direct effect of the virus on oligodendrocytes, and cerebrovascular disorders, inducing myelin damage. There is a contradiction between the solid fundamental basis underlying demyelination as the mechanism of the neurological injuries and relatively little published clinical evidence related to demyelination in COVID-19 patients. The reason for this probably lies in the fact that most clinical studies used conventional MRI techniques, which can detect only large, clearly visible demyelinating lesions. A very limited number of studies use specific methods for myelin quantification detected changes in the white matter tracts 3 and 10 months after the acute phase of COVID-19. Future research applying quantitative MRI assessment of myelin in combination with neurological and psychological studies will help in understanding the mechanisms of post-COVID complications associated with demyelination.

Challenges and Practical Solutions to MRI and Histology Matching and Measurements Using Available ImageJ Software Tools.

Traditionally histology is the gold standard for the validation of imaging experiments. Matching imaging slices and histological sections and the precise outlining of corresponding tissue structures are difficult. Challenges are based on differences in imaging and histological slice thickness as well as tissue shrinkage and alterations after processing. Here we describe step-by-step instructions that might be used as a universal pathway to overlay MRI and histological images and for a correlation of measurements between imaging modalities. The free available (Fiji is just) ImageJ software tools were used for regions of interest transformation (ROIT) and alignment using a rat brain MRI as an example. The developed ROIT procedure was compared to a manual delineation of rat brain structures. The ROIT plugin was developed for ImageJ to enable an automatization of the image processing and structural analysis of the rodent brain.

Global hypomyelination of the brain white and gray matter in schizophrenia: quantitative imaging using macromolecular proton fraction.

Myelin deficiency is commonly recognized as an important pathological feature of brain tissues in schizophrenia (SZ). In this pilot study, global myelin content abnormalities in white matter (WM) and gray matter (GM) of SZ patients were non-invasively investigated using a novel clinically-targeted quantitative myelin imaging technique, fast macromolecular proton fraction (MPF) mapping. MPF maps were obtained from 23 healthy subjects and 31 SZ patients using a clinical 1.5T magnetic resonance imaging (MRI) scanner. Mean MPF in WM and GM was compared between the healthy control subjects and SZ patients with positive and negative leading symptoms using the multivariate analysis of covariance. The SZ patients had significantly reduced MPF in GM (p < 0.001) and WM (p = 0.02) with the corresponding relative decrease of 5% and 3%, respectively. The effect sizes for the myelin content loss in SZ relative to the control group were 1.0 and 1.5 for WM and GM, respectively. The SZ patients with leading negative symptoms had significantly lower MPF in GM (p < 0.001) and WM (p = 0.003) as compared to the controls and showed a significant MPF decrease in WM (p = 0.03) relative to the patients with leading positive symptoms. MPF in WM significantly negatively correlated with the disease duration in SZ patients (Pearson's r = -0.51; p = 0.004). This study demonstrates that chronic SZ is characterized by global microscopic brain hypomyelination of both WM and GM, which is associated with the disease duration and negative symptoms. Myelin deficiency in SZ can be detected and quantified by the fast MPF mapping method.

Long-term monitoring of chronic demyelination and remyelination in a rat ischemic stroke model using macromolecular proton fraction mapping.

Remyelination is a key process enabling post-stroke brain tissue recovery and plasticity. This study aimed to explore the feasibility of demyelination and remyelination monitoring in experimental stroke from the acute to chronic stage using an emerging myelin imaging biomarker, macromolecular proton fraction (MPF). After stroke induction by transient middle cerebral artery occlusion, rats underwent repeated MRI examinations during 85 days after surgery with histological endpoints for the animal subgroups on the 7th, 21st, 56th, and 85th days. MPF maps revealed two sub-regions within the infarct characterized by distinct temporal profiles exhibiting either a persistent decrease by 30%-40% or a transient decrease followed by return to nearly normal values after one month of observation. Myelin histology confirmed that these sub-regions had nearly similar extent of demyelination in the sub-acute phase and then demonstrated either chronic demyelination or remyelination. The remyelination zones also exhibited active axonal regrowth, reconstitution of compact fiber bundles, and proliferation of neuronal and oligodendroglial precursors. The demyelination zones showed more extensive astrogliosis from the 21st day endpoint. Both sub-regions had substantially depleted neuronal population over all endpoints. These results histologically validate MPF mapping as a novel approach for quantitative assessment of myelin damage and repair in ischemic stroke.

Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain.

(1) Background: Neurogenesis is considered to be a potential brain repair mechanism and is enhanced in stroke. It is difficult to reconstruct the neurogenesis process only from the histological sections taken from different animals at different stages of brain damage and restoration. Study of neurogenesis would greatly benefit from development of tissue-specific visualization probes. (2) Purpose: The study aimed to explore if overexpression of ferritin, a nontoxic iron-binding protein, under a doublecortin promoter can be used for non-invasive visualization of neurogenesis using magnetic resonance imaging (MRI). (3) Methods: Ferritin heavy chain (FerrH) was expressed in the adeno-associated viral backbone (AAV) under the doublecortin promoter (pDCX), specific for young neurons, in the viral construct AAV-pDCX-FerrH. Expression of the enhanced green fluorescent protein (eGFP) was used as an expression control (AAV-pDCX-eGFP). The viral vectors or phosphate-buffered saline (PBS) were injected intracerebrally into 18 adult male Sprague-Dawley rats. Three days before injection, rats underwent transient middle-cerebral-artery occlusion or sham operation. Animals were subjected to In vivo MRI study before surgery and on days 7, 14, 21, and 28 days after injection using a Bruker BioSpec 11.7 T scanner. Brain sections obtained on day 28 after injection were immunostained for ferritin, young (DCX) and mature (NeuN) neurons, and activated microglia/macrophages (CD68). Additionally, RT-PCR was performed to confirm ferritin expression. (4) Results: T2* images in post-ischemic brains of animals injected with AAV-pDCX-FerrH showed two distinct zones of MRI signal hypointensity in the ipsilesioned hemisphere starting from 14 days after viral injection-in the ischemic lesion and near the lateral ventricle and subventricular zone (SVZ). In sham-operated animals, only one zone of hypointensity near the lateral ventricle and SVZ was revealed. Immunochemistry showed that ferritin-expressing cells in ischemic lesions were macrophages (88.1%), while ferritin-expressing cells near the lateral ventricle in animals both after ischemia and sham operation were mostly mature (55.7% and 61.8%, respectively) and young (30.6% and 7.1%, respectively) neurons. RT-PCR confirmed upregulated expression of ferritin in the caudoputamen and corpus callosum. Surprisingly, in animals injected with AAV-pDCX-eGFP we similarly observed two zones of hypointensity on T2* images. Cellular studies also showed the presence of mature (81.5%) and young neurons (6.1%) near the lateral ventricle in both postischemic and sham-operated animals, while macrophages in ischemic lesions were ferritin-positive (98.2%). (5) Conclusion: Ferritin overexpression induced by injection of AAV-pDCX-FerrH was detected by MRI using T2*-weighted images, which was confirmed by immunochemistry showing ferritin in young and mature neurons. Expression of eGFP also caused a comparable reduced MR signal intensity in T2*-weighted images. Additional studies are needed to investigate the potential and tissue-specific features of the use of eGFP and ferritin expression in MRI studies.

Neurodegeneration, Myelin Loss and Glial Response in the Three-Vessel Global Ischemia Model in Rat.

(1) Background: Although myelin disruption is an integral part of ischemic brain injury, it is rarely the subject of research, particularly in animal models. This study assessed for the first time, myelin and oligodendrocyte loss in a three-vessel model of global cerebral ischemia (GCI), which causes hippocampal damage. In addition, we investigated the relationships between demyelination and changes in microglia and astrocytes, as well as oligodendrogenesis in the hippocampus; (2) Methods: Adult male Wistar rats (n = 15) underwent complete interruption of cerebral blood flow for 7 min by ligation of the major arteries supplying the brain or sham-operation. At 10 and 30 days after the surgery, brain slices were stained for neurodegeneration with Fluoro-Jade C and immunohistochemically to assess myelin content (MBP+ percentage of total area), oligodendrocyte (CNP+ cells) and neuronal (NeuN+ cells) loss, neuroinflammation (Iba1+ cells), astrogliosis (GFAP+ cells) and oligodendrogenesis (NG2+ cells); (3) Results: 10 days after GCI significant myelin and oligodendrocyte loss was found only in the stratum oriens and stratum pyramidale. By the 30th day, demyelination in these hippocampal layers intensified and affected the substratum radiatum. In addition to myelin damage, activation and an increase in the number of microglia and astrocytes in the corresponding layers, a loss of the CA1 pyramidal neurons, and neurodegeneration in the neocortex and thalamus was observed. At a 10-day time point, we observed rod-shaped microglia in the substratum radiatum. Parallel with ongoing myelin loss on the 30th day after ischemia, we found significant oligodendrogenesis in demyelinated hippocampal layers; (4) Conclusions: Our study showed that GCI-simulating cardiac arrest in humans-causes not only the loss of pyramidal neurons in the CA1 field, but also the myelin loss of adjacent layers of the hippocampus.

Scan-Rescan Repeatability and Impact of B0 and B1 Field Nonuniformity Corrections in Single-Point Whole-Brain Macromolecular Proton Fraction Mapping.

BACKGROUND: Single-point macromolecular proton fraction (MPF) mapping is a recent quantitative MRI method for fast assessment of brain myelination. Information about reproducibility and sensitivity of MPF mapping to magnetic field nonuniformity is important for clinical applications. PURPOSE: To assess scan-rescan repeatability and a value of B0 and B1 field inhomogeneity corrections in single-point synthetic-reference MPF mapping. STUDY TYPE: Prospective. POPULATION: Eight healthy adult volunteers underwent two scans with 11.5 ± 2.3 months interval. FIELD STRENGTH/SEQUENCE: 3T; whole-brain 3D MPF mapping protocol included three spoiled gradient-echo sequences providing T1 , proton density, and magnetization transfer contrasts with 1.25 × 1.25 × 1.25 mm3 resolution and B0 and B1 mapping sequences. ASSESSMENT: MPF maps were reconstructed with B0 and B1 field nonuniformity correction, B0 - and B1 -only corrections, and without corrections. Mean MPF values were measured in automatically segmented white matter (WM) and gray matter (GM). STATISTICAL TESTS: Within-subject coefficient of variation (CV), intraclass correlation coefficient (ICC), Bland-Altman plots, and paired t-tests to assess scan-rescan repeatability. Repeated-measures analysis of variance (ANOVA) to compare field corrections. RESULTS: Maximal relative local MPF errors without correction in the areas of largest field nonuniformities were about 5% and 27% for B0 and B1 , respectively. The effect of B0 correction was insignificant for whole-brain WM (P > 0.25) and GM (P > 0.98) MPF. The absence of B1 correction caused a positive relative bias of 4-5% (P < 0.001) in both tissues. Scan-rescan agreement was similar for all field correction options with ICCs 0.80-0.81 for WM and 0.89-0.92 for GM. CVs were 1.6-1.7% for WM and 0.7-1.0% for GM. DATA CONCLUSION: The single-point method enables high repeatability of MPF maps obtained with the same equipment. Correction of B0 inhomogeneity may be disregarded to shorten the examination time. B1 nonuniformity correction improves accuracy of MPF measurements at 3T. Reliability of whole-brain MPF measurements in WM and GM is not affected by B0 and B1 field corrections. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2020;51:1789-1798.

Quantitative Imaging of White and Gray Matter Remyelination in the Cuprizone Demyelination Model Using the Macromolecular Proton Fraction.

Macromolecular proton fraction (MPF) has been established as a quantitative clinically-targeted MRI myelin biomarker based on recent demyelination studies. This study aimed to assess the capability of MPF to quantify remyelination using the murine cuprizone-induced reversible demyelination model. MPF was measured in vivo using the fast single-point method in three animal groups (control, cuprizone-induced demyelination, and remyelination after cuprizone withdrawal) and compared to quantitative immunohistochemistry for myelin basic protein (MBP), myelinating oligodendrocytes (CNP-positive cells), and oligodendrocyte precursor cells (OPC, NG2-positive cells) in the corpus callosum, caudate putamen, hippocampus, and cortex. In the demyelination group, MPF, MBP-stained area, and oligodendrocyte count were significantly reduced, while OPC count was significantly increased as compared to both control and remyelination groups in all anatomic structures (p < 0.05). All variables were similar in the control and remyelination groups. MPF and MBP-stained area strongly correlated in each anatomic structure (Pearson's correlation coefficients, r = 0.80-0.90, p < 0.001). MPF and MBP correlated positively with oligodendrocyte count (r = 0.70-0.84, p < 0.01 for MPF; r = 0.81-0.92, p < 0.001 for MBP) and negatively with OPC count (r = -0.69--0.77, p < 0.01 for MPF; r = -0.72--0.89, p < 0.01 for MBP). This study provides immunohistological validation of fast MPF mapping as a non-invasive tool for quantitative assessment of de- and remyelination in white and gray matter and indicates the feasibility of using MPF as a surrogate marker of reparative processes in demyelinating diseases.

New Neurons in the Post-ischemic and Injured Brain: Migrating or Resident?

The endogenous potential of adult neurogenesis is of particular interest for the development of new strategies for recovery after stroke and traumatic brain injury. These pathological conditions affect endogenous neurogenesis in two aspects. On the one hand, injury usually initiates the migration of neuronal precursors (NPCs) to the lesion area from the already existing, in physiological conditions, neurogenic niche - the ventricular-subventricular zone (V-SVZ) near the lateral ventricles. On the other hand, recent studies have convincingly demonstrated the local generation of new neurons near lesion areas in different brain locations. The striatum, cortex, and hippocampal CA1 region are considered to be locations of such new neurogenic zones in the damaged brain. This review focuses on the relative contribution of two types of NPCs of different origin, resident population in new neurogenic zones and cells migrating from the lateral ventricles, to post-stroke or post-traumatic enhancement of neurogenesis. The migratory pathways of NPCs have also been considered. In addition, the review highlights the advantages and limitations of different methodological approaches to the definition of NPC location and tracking of new neurons. In general, we suggest that despite the considerable number of studies, we still lack a comprehensive understanding of neurogenesis in the damaged brain. We believe that the advancement of methods for in vivo visualization and longitudinal observation of neurogenesis in the brain could fundamentally change the current situation in this field.

Plant polyprenols reduce demyelination and recover impaired oligodendrogenesis and neurogenesis in the cuprizone murine model of multiple sclerosis.

Recent studies showed hepatoprotective, neuroprotective, and immunomodulatory properties of polyprenols isolated from the green verdure of Picea abies (L.) Karst. This study aimed to investigate effects of polyprenols on oligodendrogenesis, neurogenesis, and myelin content in the cuprizone demyelination model. Demyelination was induced by 0.5% cuprizone in CD-1 mice during 10 weeks. Nine cuprizone-treated animals received daily injections of polyprenols intraperitoneally at a dose of 12-mg/kg body weight during Weeks 6-10. Nine control animals and other nine cuprizone-treated received sham oil injections. At Week 10, brain sections were stained for myelin basic protein, neuro-glial antigen-2, and doublecortin to evaluate demyelination, oligodendrogenesis, and neurogenesis. Cuprizone administration caused a decrease in myelin basic protein in the corpus callosum, cortex, hippocampus, and the caudate putamen compared with the controls. Oligodendrogenesis was increased, and neurogenesis in the subventricular zone and the dentate gyrus of the hippocampus was decreased in the cuprizone-treated group compared with the controls. Mice treated with cuprizone and polyprenols did not show significant demyelination and differences in oligodendrogenesis and neurogenesis as compared with the controls. Our results suggest that polyprenols can halt demyelination, restore impaired neurogenesis, and mitigate reactive overproduction of oligodendrocytes caused by cuprizone neurotoxicity.

Quantitative assessment of demyelination in ischemic stroke in vivo using macromolecular proton fraction mapping.

A recent MRI method, fast macromolecular proton fraction (MPF) mapping, was used to quantify demyelination in the transient middle cerebral artery occlusion (MCAO) rat stroke model. MPF and other quantitative MRI parameters (T1, T2, proton density, and apparent diffusion coefficient) were compared with histological and immunohistochemical markers of demyelination (Luxol Fast Blue stain, (LFB)), neuronal loss (NeuN immunofluorescence), axonal loss (Bielschowsky stain), and inflammation (Iba1 immunofluorescence) in three animal groups ( n = 5 per group) on the 1st, 3rd, and 10th day after MCAO. MPF and LFB optical density (OD) were significantly reduced in the ischemic lesion on all days after MCAO relative to the symmetrical regions of the contralateral hemisphere. Percentage changes in MPF and LFB OD in the ischemic lesion relative to the contralateral hemisphere significantly differed on the first day only. Percentage changes in LFB OD and MPF were strongly correlated (R = 0.81, P < 0.001) and did not correlate with other MRI parameters. MPF also did not correlate with other histological variables. Addition of T2 into multivariate regression further improved agreement between MPF and LFB OD (R = 0.89, P < 0.001) due to correction of the edema effect. This study provides histological validation of MPF as an imaging biomarker of demyelination in ischemic stroke.

Effects of Fluoxetine on Hippocampal Neurogenesis and Neuroprotection in the Model of Global Cerebral Ischemia in Rats.

A selective serotonin reuptake inhibitor, fluoxetine, has recently attracted a significant interest as a neuroprotective therapeutic agent. There is substantial evidence of improved neurogenesis under fluoxetine treatment of brain ischemia in animal stroke models. We studied long-term effects of fluoxetine treatment on hippocampal neurogenesis, neuronal loss, inflammation, and functional recovery in a new model of global cerebral ischemia (GCI). Brain ischemia was induced in adult Wistar male rats by transient occlusion of three main vessels originating from the aortic arch and providing brain blood supply. Fluoxetine was injected intraperitoneally in a dose of 20 mg/kg for 10 days after surgery. To evaluate hippocampal neurogenesis at time points 10 and 30 days, 5-Bromo-2'-deoxyuridine was injected at days 8-10 after GCI. According to our results, 10-day fluoxetine injections decreased neuronal loss and inflammation, improved survival and functional recovery of animals, enhanced neurogenesis, and prevented an early pathological increase in neural stem cell recruitment in the subgranular zone (SGZ) of the hippocampus without reducing the number of mature neurons at day 30 after GCI. In summary, this study suggests that fluoxetine may provide a promising therapy in cerebral ischemia due to its neuroprotective, anti-inflammatory, and neurorestorative effect.

Histological validation of fast macromolecular proton fraction mapping as a quantitative myelin imaging method in the cuprizone demyelination model.

Cuprizone-induced demyelination in mice is a frequently used model in preclinical multiple sclerosis research. A recent quantitative clinically-targeted MRI method, fast macromolecular proton fraction (MPF) mapping demonstrated a promise as a myelin biomarker in human and animal studies with a particular advantage of sensitivity to both white matter (WM) and gray matter (GM) demyelination. This study aimed to histologically validate the capability of MPF mapping to quantify myelin loss in brain tissues using the cuprizone demyelination model. Whole-brain MPF maps were obtained in vivo on an 11.7T animal MRI scanner from 7 cuprizone-treated and 7 control С57BL/6 mice using the fast single-point synthetic-reference method. Brain sections were histologically stained with Luxol Fast Blue (LFB) for myelin quantification. Significant (p < 0.05) demyelination in cuprizone-treated animals was found according to both LFB staining and MPF in all anatomical structures (corpus callosum, anterior commissure, internal capsule, thalamus, caudoputamen, and cortex). MPF strongly correlated with quantitative histology in all animals (r = 0.95, p < 0.001) as well as in treatment and control groups taken separately (r = 0.96, p = 0.002 and r = 0.93, p = 0.007, respectively). Close agreement between histological myelin staining and MPF suggests that fast MPF mapping enables robust and accurate quantitative assessment of demyelination in both WM and GM.

High-resolution three-dimensional quantitative map of the macromolecular proton fraction distribution in the normal rat brain.

The presented dataset provides a normative high-resolution three-dimensional (3D) macromolecular proton fraction (MPF) map of the healthy rat brain in vivo and source images used for its reconstruction. The images were acquired using the protocol described elsewhere (Naumova, et al. High-resolution three-dimensional macromolecular proton fraction mapping for quantitative neuroanatomical imaging of the rodent brain in ultra-high magnetic fields. Neuroimage (2016) doi: 10.1016/j.neuroimage.2016.09.036). The map was reconstructed from three source images with different contrast weightings (proton density, T1, and magnetization transfer) using the single-point algorithm with a synthetic reference image. Source images were acquired from a living animal on an 11.7 T small animal MRI scanner with isotropic spatial resolution of 170 µm3 and total acquisition time about 1.5 h. The 3D dataset can be used for multiple purposes including interactive viewing of rat brain anatomy, measurements of reference MPF values in various brain structures, and development of image processing techniques for the rodent brain segmentation. It also can serve as a gold standard image for implementation and optimization of rodent brain MRI protocols.

High-resolution three-dimensional macromolecular proton fraction mapping for quantitative neuroanatomical imaging of the rodent brain in ultra-high magnetic fields.

A well-known problem in ultra-high-field MRI is generation of high-resolution three-dimensional images for detailed characterization of white and gray matter anatomical structures. T1-weighted imaging traditionally used for this purpose suffers from the loss of contrast between white and gray matter with an increase of magnetic field strength. Macromolecular proton fraction (MPF) mapping is a new method potentially capable to mitigate this problem due to strong myelin-based contrast and independence of this parameter of field strength. MPF is a key parameter determining the magnetization transfer effect in tissues and defined within the two-pool model as a relative amount of macromolecular protons involved into magnetization exchange with water protons. The objectives of this study were to characterize the two-pool model parameters in brain tissues in ultra-high magnetic fields and introduce fast high-field 3D MPF mapping as both anatomical and quantitative neuroimaging modality for small animal applications. In vivo imaging data were obtained from four adult male rats using an 11.7T animal MRI scanner. Comprehensive comparison of brain tissue contrast was performed for standard R1 and T2 maps and reconstructed from Z-spectroscopic images two-pool model parameter maps including MPF, cross-relaxation rate constant, and T2 of pools. Additionally, high-resolution whole-brain 3D MPF maps were obtained with isotropic 170µm voxel size using the single-point synthetic-reference method. MPF maps showed 3-6-fold increase in contrast between white and gray matter compared to other parameters. MPF measurements by the single-point synthetic reference method were in excellent agreement with the Z-spectroscopic method. MPF values in rat brain structures at 11.7T were similar to those at lower field strengths, thus confirming field independence of MPF. 3D MPF mapping provides a useful tool for neuroimaging in ultra-high magnetic fields enabling both quantitative tissue characterization based on the myelin content and high-resolution neuroanatomical visualization with high contrast between white and gray matter.