Ex vivo relaxation rates and magnetic susceptibility changes of corpus callosum in aging rats

Department of Biomedical EngineeringMyelin, the main component of white matter (WM), is a lipid-protein membrane structure that surrounds axon compactly in the nervous systems of vertebrates. Myelin exists in the form of a multi-lamellar sheath consisting of repeating units of the myelin bilayers and most of myelinated axons are distributed in WM. The main role of myelin is the electrical insulator for neurons, which increases the speed and efficiency of signal conduction. Since speed of action potential transmission is necessary to promote various neuronal functions, the measurements of myelin content are important for studies of normal development and neurodegenerative diseases. In the evaluation of myelin, magnetic resonance imaging (MRI) is widely used as a noninvasive imaging technique that provides detailed anatomical images with various contrast mechanisms. Due to the limitation in MRI resolution and the size of myelinated axons (~ 1 ??m), myelin cannot be directly resolved by MRI. Also, the T2 value of non-aqueous protons of myelin (50 ??s < T2 < 1 ms) is too short to measure the signal in conventional MRI, making direct imaging difficult. Therefore, most MRI techniques currently used for myelin imaging are mainly based on indirect estimation of myelin. Currently, various MRI techniques for indirectly examining the myelin content are being studied with their respective strengths and weaknesses. However, there is still no method that is considered gold standard in the field of myelin MRI. Studies comparing and analyzing the effects of volumetric parameters on myelin through various validation methods are still lacking. Therefore, the purpose of this study is to quantify volumetric changes in myelin, such as myelin volume fraction (MVF), in the corpus callosum (CC) of post-mortem aging rat brains through MRI-based measurements and histological/theoretical validations. In the first section, the relationship with MVF was established through the MRI-derived values: longitudinal relaxation rate R1 and the magnetic susceptibility values obtained through quantitative susceptibility mapping (QSM). The absolute MVF values were determined by transmission electron microscopy (TEM) as a gold standard measure for comparison with the values obtained by the aforementioned MRI techniques. Also, QSM simulations were performed based on the TEM-derived structures to theoretically evaluate and understand the MR signal properties. Correlations of MVF versus MRI-derived values (R1 and magnetic susceptibility) showed a strong linear relationship. In addition, QSM simulation results established a linearly proportional relationship between simulated magnetic susceptibility and MVF. Statistically significant linear correlations between MRI-derived values and MVF demonstrated that variable myelin content in WM (i.e., CC) could be quantified across different stages of aging. These results further support that both MRI techniques (R1 and QSM) provide an efficient means to study the brain aging process with accurate volumetric quantification of myelin content in the WM. In the second section, multiple spin echo sequence-based MRI-R2 values were measured to confirm that myelin volume information could be detected even when the short-T2 component (myelin water signal) was not detected due to the fixation effect. TEM-based quantification of MVF and corresponding Monte-Carlo simulation to estimate relaxation rates (R2,IE) due to diffusion in the presence of inhomogeneous magnetic field perturbation in intra- and extra-cellular (IE) spaces were respectively performed. A significant correlation between mean MRI-R2 and MVF values was observed, and the estimated R2,IE values of Monte-Carlo simulations in IE water signals were also positively correlated with MVF values. However, the magnitude of R2,IE values were much smaller than that those observed for MRI-R2 values, indicating that R2-related changes in MVF are likely dominated by the myelin water content. Such comparisons between independent parameters from MRI, TEM, and simulations support the suggestion that myelin water signals were indistinguishably mixed to exhibit mono-exponential R2, and still reflect the volumetric information of myelin. In conclusion, it was confirmed that the proposed MRI-based measurements (R1, R2 and QSM) can be usefully used for the quantification of myelin volume in the post-mortem rat CC regions based on histological/theoretical validations (TEM and simulation).clos

Empirical relationship between TEM-derived myelin volume fraction and MRI-R2 values in aging ex vivo rat corpus callosum

Ex vivo ratiometric measurements of short- and long-T2 components using the multiple spin echo sequence of MRI are often employed to evaluate alterations in myelin content in the white matter (WM) of the brain. However, the relationship between absolute MRI-T2 values (long-T2 component) and myelin volumetric information in aged ex vivo rodent WM appears to be influenced by factors such as animal species, field strength, and fixation durations/washing. Here, multiple spin echo sequence-based MRI-R2 (the reciprocal of T2) values were measured in the corpus callosum (CC) region in the post-mortem rat brains (n = 9) of different age groups with common fixation techniques without washing at 7 T. Transmission electron microscopy (TEM)-based quantification of myelin volume fraction (MVF) and corresponding Monte-Carlo simulation to estimate relaxation rates (R2,IE) due to diffusion in the presence of inhomogeneous magnetic field perturbation in intra- and extra-cellular (IE) spaces were respectively performed. To determine whether the short-T2 components originating from myelin water were mixed with long-T2 components from IE water or were undetectable, the MVF values obtained from TEM results were respectively compared with MRI-R2 and R2,IE values. A significant correlation (Pearson&apos;s correlation coefficient r = 0.8763; p &lt; 0.01) of average MRI-R2 and MVF values was observed. Estimated R2,IE values from Monte-Carlo simulations in IE water signals were also positively correlated (r = 0.8281; p &lt; 0.01) with MVF values. However, the magnitudes of R2,IE values were much smaller than those observed for MRI-R2 values, indicating that changes in R2 related MVF are likely dominated by myelin water components. Such comparisons between independent parameters from MRI, TEM, and simulations support the suggestion that myelin water signals were indistinguishably mixed to exhibit mono-exponential T2 relaxation, and multiple spin echo sequence-based MRI-R2 values in aging ex vivo rat CC without prolonged washing still reflect the volumetric information of myelin, likely due to enhanced water exchange across the myelin

Quantitative susceptibility mapping and R1 measurement: Determination of the myelin volume fraction in the aging ex vivo rat corpus callosum

In studies of the white matter (WM) in aging brains, both quantitative susceptibility mapping (QSM) and direct R-1 measurement offer potentially useful ex vivo MRI tools that allow volumetric characterization of myelin content changes. Despite the technical importance of such MRI methods in numerous age-related diseases, the supposed linear relationship between the estimates of either the QSM or R-1 method and age-affected myelin contents has not been validated. In this study, the absolute myelin volume fraction (MVF) was determined by transmission electron microscopy (TEM) as a gold standard measure for comparison with the values obtained by the aforementioned MR methods. To theoretically evaluate and understand the MR signal characteristics, QSM simulations were performed using the finite perturber method (FPM). Specifically, the simulation geometry modeling was based on TEM-derived structures aligned orthogonally to the main magnetic field, the construct of which was used to estimate the magnetic field shift (Delta B) changes arising from the conjectured myelin structures. Experimentally, ex vivo corpus callosum (CC) samples from rat brains obtained at 6 weeks (n = 3), 4 months (n = 3), and 20 months (n = 3) after birth were used to establish the relationship between changes quantified by either QSM or R-1 with the absolute MVF by TEM. From the ex vivo brain samples, the scatterplot of mean MVF versus R-1 was fitted to a linear equation, where R-1mean = 0.7948 x MVFmean + 0.8118 (Pearson&apos;s correlation coefficient r = 0.9138; p &lt; 0.01), while the scatterplot of mean MVF versus MRI-derived magnetic susceptibility (chi) was also fitted to a line where chi(measured,mean) = -0.1218 x MVFmean - 0.006345 (r = -0.8435; p &lt; 0.01). As a result of the FPM-based QSM simulations, a linearly proportional relationship between the simulated magnetic susceptibility, chi(simulated,mean), and MVF (r = -0.9648; p &lt; 0.01) was established. Such a statistically significant linear correlation between MRI-derived values by the QSM (or R-1) method and MVF demonstrated that variable myelin contents in the WM (i.e., CC) can be quantified across multiple stages of aging. These findings further support that both techniques based on QSM and R-1 provide an efficient means of studying the brain-aging process with accurate volumetric quantification of the myelin content in WM

The robustness of T2 value as a trabecular structural index at multiple spatial resolutions of 7 Tesla MRI

Purpose: To evaluate the robustness of MR transverse relaxation times of trabecular bone from spin-echo and gradient-echo acquisitions at multiple spatial resolutions of 7 T. Methods: The effects of MRI resolutions to T-2 and T-2* trabecular bone were numerically evaluated by Monte Carlo simulations. T-2, T-2* and trabecular structural indices from multislice multi-echo and UTE acquisitions were measured in defatted human distal femoral condyles on a 7 T scanner. Reference structural indices were extracted from high-resolution microcomputed tomography images. For bovine knee trabecular samples with intact bone marrow, T-2 and T-2* were measured by degrading spatial resolutions on a 7 T system. Results: In the defatted trabecular experiment, both T-2 and T-2* values showed strong (vertical bar r vertical bar&gt; 0.80) correlations with trabecular spacing and number, at a high spatial resolution of 125 mu m(3). The correlations for MR image-segmentation-olutions of 250 and 500 mu m(3). The correlations for T-2* rapidly dropped (vertical bar r vertical bar&lt; 0.50) at a spatial resolution of 500 mu m(3), whereas those for T-2* remained consistently high (vertical bar r vertical bar&gt; 0.85). In the bovine trabecular experiments with intact marrow, low- resolution (approximately 1 mm(3), 2 minutes) T-2 values did not shorten (vertical bar r vertical bar&gt; 0.95 with respect to approximately 0.4mm(3), 11 minutes) and maintained consistent correlations (vertical bar r vertical bar &gt; 0.70) with respect to trabecular spacing (turbo spin echo, 22.5 minutes). Conclusion: T-2 measurements of trabeculae at 7 T are robust with degrading spatial resolution and may be preferable in assessing trabecular spacing index with reduced scan time, when high-resolution 3D micro-MRI is difficult to obtain

Automated Volumetric Determination of High R2* Regions in Substantia Nigra : A Feasibility Study of Quantifying SN Atrophy in Progressive Supranulcear Palsy

The establishment of an unbiased protocol for the automated volumetric measurement of iron-rich regions in the substantia nigra (SN) is clinically important for diagnosing neurodegenerative diseases exhibiting midbrain atrophy, such as progressive supranuclear palsy (PSP). This study aimed to automatically quantify the volume and surface properties of the iron-rich 3D regions in the SN using the quantitative MRI-R2* map. Three hundred and sixty-seven slices of R2* map and susceptibility-weighted imaging (SWI) at 3-T MRI from healthy control (HC) individuals and Parkinson&apos;s disease (PD) patients were used to train customized U-net++ convolutional neural network based on expert-segmented masks. Age- and sex-matched participants were selected from HC, PD, and PSP groups to automate the volumetric determination of iron-rich areas in the SN. Dice similarity coefficient values between expert-segmented and detected masks from the proposed network were for R2* maps and for SWI. Reductions in iron-rich SN volume from the R2* map (SWI) were observed in PSP with area under the receiver operating characteristic curve values of 0.96 (0.89) and 0.98 (0.92) compared with HC and PD, respectively. The mean curvature of the PSP showed SN deformation along the side closer to the red nucleus. We demonstrated the automated volumetric measurement of iron-rich regions in the SN using deep learning can quantify the SN atrophy in PSP compared with PD and HC

Adaptive cellular response of the substantia nigra dopaminergic neurons upon age-dependent iron accumulation

Progressive iron accumulation in the substantia nigra in the aged human brain is a major risk factor for Parkinson&amp;#039;s disease and other neurodegenerative diseases. Heavy metals, such as iron, produce reactive oxygen species and consequently oxidative stress in cells. It is unclear, however, how neurons in the substantia nigra are protected against the age-related, excessive accumulation of iron. In this study, we examined the cellular response of the substantia nigra against age-related iron accumulation in rats of different ages. Magnetic resonance imaging confirmed the presence of iron in 6-month-old rats; in 15-month-old rats, iron accumulation significantly increased, particularly in the midbrain. Transcriptome analysis of the region, in which iron deposition was observed, revealed an increase in stress response genes in older animals. To identify the genes related to the cellular response to iron, independent of neurodevelopment, we exposed the neuroblastoma cell line SH-SY5Y to a similar quantity of iron and then analyzed their transcriptomic responses. Among various stress response pathways altered by iron overloading in the rat brain and SH-SY5Y cells, the genes associated with topologically incorrect protein responses were significantly upregulated. Knockdown of HERPUD1 and CLU in this pathway increased susceptibility to iron-induced cellular stress, thus demonstrating their roles in preventing iron overload-induced toxicity. The current study details the neuronal response to excessive iron accumulation, which is associated with age-related neurodegenerative diseases

Analysis of Extracellular Vesicles Using Coffee Ring

Extracellular vesicles are categorized in subsets according to their biogenesis processes. To facilitate the investigation of subsets, an effective method is needed for isolating subpopulations. The efficacy of existing density and size-based isolation methods is limited, and as a result, the correlation of properties within separated subpopulations is modest. Here, we introduced size separation with ∼48 nm resolution that exploits Marangoni flow and the coffee-ring effect in microdroplets in which extracellular vesicles are spatially deposited at different location according to size of extracellular vesicle. Interestingly, the analysis of tetraspanin proteins of the extracellular vesicles facilitated by this method reveals that the size of extracellular vesicles is correlated with expression of tetraspanin proteins (CD9, CD63, CD81) that are associated with the size of extracellular vesicles. The findings show that CD9 and CD81 are uniformly expressed regardless of size, CD63 is highly expressed only in larger extracellular vesicles. This evidence indicates that extracellular vesicles can be classified based on size and expression of CD63