Fast and robust quantification of uncertainty in non-linear diffusion MRI models.

Diffusion MRI (dMRI) allows for non-invasive investigation of brain tissue microstructure. By fitting a model to the dMRI signal, various quantitative measures can be derived from the data, such as fractional anisotropy, neurite density and axonal radii maps. We investigate the Fisher Information Matrix (FIM) and uncertainty propagation as a generally applicable method for quantifying the parameter uncertainties in linear and non-linear diffusion MRI models. In direct comparison with Markov Chain Monte Carlo (MCMC) sampling, the FIM produces similar uncertainty estimates at much lower computational cost. Using acquired and simulated data, we then list several characteristics that influence the parameter variances, including data complexity and signal-to-noise ratio. For practical purposes we investigate a possible use of uncertainty estimates in decreasing intra-group variance in group statistics by uncertainty-weighted group estimates. This has potential use cases for detection and suppression of imaging artifacts.

MESMERISED: Super-accelerating T1 relaxometry and diffusion MRI with STEAM at 7 T for quantitative multi-contrast and diffusion imaging.

There is an increasing interest in quantitative imaging of T1, T2 and diffusion contrast in the brain due to greater robustness against bias fields and artifacts, as well as better biophysical interpretability in terms of microstructure. However, acquisition time constraints are a challenge, particularly when multiple quantitative contrasts are desired and when extensive sampling of diffusion directions, high b-values or long diffusion times are needed for multi-compartment microstructure modeling. Although ultra-high fields of 7 T and above have desirable properties for many MR modalities, the shortening T2 and the high specific absorption rate (SAR) of inversion and refocusing pulses bring great challenges to quantitative T1, T2 and diffusion imaging. Here, we present the MESMERISED sequence (Multiplexed Echo Shifted Multiband Excited and Recalled Imaging of STEAM Encoded Diffusion). MESMERISED removes the dead time in Stimulated Echo Acquisition Mode (STEAM) imaging by an echo-shifting mechanism. The echo-shift (ES) factor is independent of multiband (MB) acceleration and allows for very high multiplicative (ESxMB) acceleration factors, particularly under moderate and long mixing times. This results in super-acceleration and high time efficiency at 7 T for quantitative T1 and diffusion imaging, while also retaining the capacity to perform quantitative T2 and B1 mapping. We demonstrate the super-acceleration of MESMERISED for whole-brain T1 relaxometry with total acceleration factors up to 36 at 1.8 mm isotropic resolution, and up to 54 at 1.25 mm resolution qT1 imaging, corresponding to a 6x and 9x speedup, respectively, compared to MB-only accelerated acquisitions. We then demonstrate highly efficient diffusion MRI with high b-values and long diffusion times in two separate cases. First, we show that super-accelerated multi-shell diffusion acquisitions with 370 whole-brain diffusion volumes over 8 b-value shells up to b = 7000 s/mm2 can be generated at 2 mm isotropic in under 8 minutes, a data rate of almost a volume per second, or at 1.8 mm isotropic in under 11 minutes, achieving up to 3.4x speedup compared to MB-only. A comparison of b = 7000 s/mm2 MESMERISED against standard MB pulsed gradient spin echo (PGSE) diffusion imaging shows 70% higher SNR efficiency and greater effectiveness in supporting complex diffusion signal modeling. Second, we demonstrate time-efficient sampling of different diffusion times with 1.8 mm isotropic diffusion data acquired at four diffusion times up to 290 ms, which supports both Diffusion Tensor Imaging (DTI) and Diffusion Kurtosis Imaging (DKI) at each diffusion time. Finally, we demonstrate how adding quantitative T2 and B1+ mapping to super-accelerated qT1 and diffusion imaging enables efficient quantitative multi-contrast mapping with the same MESMERISED sequence and the same readout train. MESMERISED extends possibilities to efficiently probe T1, T2 and diffusion contrast for multi-component modeling of tissue microstructure.

Ultra-high resolution and multi-shell diffusion MRI of intact ex vivo human brains using kT-dSTEAM at 9.4T.

Diffusion MRI (dMRI) in ex vivo human brain specimens is an important research tool for neuroanatomical investigations and the validation of dMRI techniques. Many ex vivo dMRI applications have benefited from very high dMRI resolutions achievable on small-bore preclinical or animal MRI scanners for small tissue samples. However, the investigation of entire human brains post mortem provides the important context of entire white matter (WM) network systems and entire gray matter (GM) areas connected through these systems. The investigation of intact ex vivo human brains in large bore systems creates challenges due to the limited gradient performance and transmit radio-frequency (B1+) inhomogeneities, specially at ultra-high field (UHF, 7T and higher). To overcome these issues, it is necessary to tailor ex vivo diffusion-weighted sequences specifically for high resolution and high diffusion-weighting. Here, we present kT-dSTEAM, which achieves B1+ homogenization across whole human brain specimens using parallel transmit (pTx) on a 9.4T MR system. We use kT-dSTEAM to obtain multi-shell high b-value and high resolution diffusion-weighted data in ex vivo whole human brains. Isotropic whole brain data can be acquired at high b-value (6000-8000 s/mm2) at high resolution (1000 µm) and at moderate b-value (3000 s/mm2) at ultra-high isotropic resolution (400 µm). As an illustration of the advantages of the ultra-high resolution, tractography across the WM/GM border shows less of the unwanted gyral crown bias, and more high-curvature paths connecting the sulcal wall than at lower resolution. The kT-dSTEAM also allows for acquisition of T1 and T2 weighted images suitable for estimating quantitative T1 and T2 maps. Finally, multi-shell analysis of kT-dSTEAM data at variable mixing time (TM) is shown as an approach for ex vivo data analysis which is adapted to the strengths of STEAM diffusion-weighting. Here, we use this gain for multi-orientation modelling and crossing-fiber tractography. We show that multi-shell data allows superior multiple orientation tractography of known crossing fiber structures in the brain stem.

High resolution anatomical and quantitative MRI of the entire human occipital lobe ex vivo at 9.4T.

Several magnetic resonance imaging (MRI) contrasts are sensitive to myelin content in gray matter in vivo which has ignited ambitions of MRI-based in vivo cortical histology. Ultra-high field (UHF) MRI, at fields of 7T and beyond, is crucial to provide the resolution and contrast needed to sample contrasts over the depth of the cortex and get closer to layer resolved imaging. Ex vivo MRI of human post mortem samples is an important stepping stone to investigate MRI contrast in the cortex, validate it against histology techniques applied in situ to the same tissue, and investigate the resolutions needed to translate ex vivo findings to in vivo UHF MRI. Here, we investigate key technology to extend such UHF studies to large human brain samples while maintaining high resolution, which allows investigation of the layered architecture of several cortical areas over their entire 3D extent and their complete borders where architecture changes. A 16 channel cylindrical phased array radiofrequency (RF) receive coil was constructed to image a large post mortem occipital lobe sample (~80×80×80mm3) in a wide-bore 9.4T human scanner with the aim of achieving high-resolution anatomical and quantitative MR images. Compared with a human head coil at 9.4T, the maximum Signal-to-Noise ratio (SNR) was increased by a factor of about five in the peripheral cortex. Although the transmit profile with a circularly polarized transmit mode at 9.4T is relatively inhomogeneous over the large sample, this challenge was successfully resolved with parallel transmit using the kT-points method. Using this setup, we achieved 60µm anatomical images for the entire occipital lobe showing increased spatial definition of cortical details compared to lower resolutions. In addition, we were able to achieve sufficient control over SNR, B0 and B1 homogeneity and multi-contrast sampling to perform quantitative T2* mapping over the same volume at 200µm. Markov Chain Monte Carlo sampling provided maximum posterior estimates of quantitative T2* and their uncertainty, allowing delineation of the stria of Gennari over the entire length and width of the calcarine sulcus. We discuss how custom RF receive coil arrays built to specific large post mortem sample sizes can provide a platform for UHF cortical layer-specific quantitative MRI over large fields of view.

Robust and fast nonlinear optimization of diffusion MRI microstructure models.

Advances in biophysical multi-compartment modeling for diffusion MRI (dMRI) have gained popularity because of greater specificity than DTI in relating the dMRI signal to underlying cellular microstructure. A large range of these diffusion microstructure models have been developed and each of the popular models comes with its own, often different, optimization algorithm, noise model and initialization strategy to estimate its parameter maps. Since data fit, accuracy and precision is hard to verify, this creates additional challenges to comparability and generalization of results from diffusion microstructure models. In addition, non-linear optimization is computationally expensive leading to very long run times, which can be prohibitive in large group or population studies. In this technical note we investigate the performance of several optimization algorithms and initialization strategies over a few of the most popular diffusion microstructure models, including NODDI and CHARMED. We evaluate whether a single well performing optimization approach exists that could be applied to many models and would equate both run time and fit aspects. All models, algorithms and strategies were implemented on the Graphics Processing Unit (GPU) to remove run time constraints, with which we achieve whole brain dataset fits in seconds to minutes. We then evaluated fit, accuracy, precision and run time for different models of differing complexity against three common optimization algorithms and three parameter initialization strategies. Variability of the achieved quality of fit in actual data was evaluated on ten subjects of each of two population studies with a different acquisition protocol. We find that optimization algorithms and multi-step optimization approaches have a considerable influence on performance and stability over subjects and over acquisition protocols. The gradient-free Powell conjugate-direction algorithm was found to outperform other common algorithms in terms of run time, fit, accuracy and precision. Parameter initialization approaches were found to be relevant especially for more complex models, such as those involving several fiber orientations per voxel. For these, a fitting cascade initializing or fixing parameter values in a later optimization step from simpler models in an earlier optimization step further improved run time, fit, accuracy and precision compared to a single step fit. This establishes and makes available standards by which robust fit and accuracy can be achieved in shorter run times. This is especially relevant for the use of diffusion microstructure modeling in large group or population studies and in combining microstructure parameter maps with tractography results.

Lymphocyte subsets and their proliferation in a model for a delayed-type hypersensitivity reaction in the skin.

A delayed-type hypersensitivity (DTH) reaction was induced in the skin of young pigs, by local injection of phytohaemagglutinin, and evaluation was carried out on the resulting accumulation of lymphocyte subsets and lymphocyte production by incorporation of bromodeoxyuridine in the skin and the draining lymph node. There was a rapid increase in mononuclear cells, which were found in clusters around venules. These included very few B lymphocytes, and CD8+ lymphocytes far outnumbered CD4+ cells. Underlining the importance of determining absolute numbers, the relative and absolute numbers of lymphocyte subsets showed quite different patterns during the development of the skin reaction. Lymphocytes in the normal skin incorporated the DNA precursor bromodeoxyuridine at higher rates than have been found for peripheral lymphoid organs. After intradermal phytohaemagglutinin injections, all subsets showed high proliferation rates in the skin, with kinetics which differed from the reaction in the draining lymph node. The labelling indexes of cells labelled with bromodeoxyuridine in vitro and in vivo were comparable. The phytohaemagglutinin injections also caused a marked and rapid increase in the proliferation of the cells in the basal layer of the epidermis. This model DTH-like reaction in skin with major CD8+ T-cell accumulation and proliferation locally and in the lymph nodes provides a reliable model for study of such reactions and for investigation of the regulatory role of cytokines.

Proliferation of macrophage subpopulations in the adult rat: comparison of various lymphoid organs.

Adult male Lewis rats received a single intravenous injection of 5-bromo-2'-deoxyuridine (BRDU) to label all proliferating cells in the S-phase of the cell cycle. Various lymphoid organs were removed 1 and 24 hr after injection to assess local proliferation and migration of newly formed cells, respectively. In cell suspensions, surface staining was performed for macrophage subsets (ED1, ED2, ED3), and the DNA label BRDU was detected by a monoclonal antibody. Local proliferation of ED1+ macrophages occurred in all organs investigated with the exception of the blood. Bone marrow outweighed the other organs by far; in addition to the proliferating ED1+ promonocytes, the bone marrow also contained BRDU-labeled ED2+ macrophages. Newly formed ED1+ monocytes migrated into lymphoid organs such as the mesenteric lymph nodes and spleen where they comprised about 90% of newly formed macrophages. In the spleen, ED3+ macrophages seemed to be renewed by local proliferation, whereas in the mesenteric lymph nodes these cells were replaced by immigration. The heterogeneity of macrophages was further demonstrated by the different renewal of splenic macrophages. ED1+ and ED3+ cells were replaced in a matter of days, whereas it would probably take several months to renew ED2+ cells.

Proliferation of lymphocyte subsets in the adult rat: a comparison of different lymphoid organs.

Adult, male Lewis rats received a single injection of 5-bromo-2'-deoxyuridine (BrdUrd) i.v. to label proliferating cells in the S phase of the cell cycle. After 1 and 24 h the thymus, bone marrow, blood, spleen, peripheral, cervical and mesenteric lymph nodes as well as Peyer's patches were removed. In cell suspensions surface staining was performed for B, T, T helper (Th) and cytotoxic/suppressor (Tc/s) T lymphocytes by identifying kappa light chain, CD5+, CD4+ and CD8+ cells, respectively. On the same slide the DNA label BrdUrd was demonstrated by a monoclonal antibody. B, T, Th and Tc/s lymphocytes proliferate locally both in central lymphoid organs such as the thymus and the bone marrow, and in peripheral lymphoid organs such as the spleen, lymph nodes and Peyer's patches. Within an organ the amount of proliferation among the lymphocyte subsets is similar, differing not more than threefold. Although concerning only a small fraction of cells within the organ, an unexpected finding is the high percentage of BrdUrd-labeled cells among B lymphocytes in the thymus (3%) and among T lymphocytes in the bone marrow (3%). One day after injection of BrdUrd the thymus contains 25% BrdUrd+ T lymphocytes, while the other organs investigated do not show more than about 2% BrdUrd+ B and T lymphocytes. Many of the newly formed lymphocyte subsets leave their organ of birth within 24 h. Thus the amount of proliferation in the lymphocyte subsets investigated is very similar and the differences between central (thymus and bone marrow) and peripheral lymphoid organs are much smaller than expected.

The mucosa of the male genital tract; part of the common mucosal secretory immune system?

The selective migration of mucosal-derived lymphoid blasts to other mucosal organs is taken to be an essential part of the common secretory immune system. In rats, proliferating lymphoid cells from mesenteric lymph nodes (mLN) and peripheral lymph nodes (pLN) were labeled in vitro using two different techniques, in order to test the hypothesis that the mucosa of the male genital tract is a preferential site for mLN lymphoid blasts to home to. A low but significant migration to male genital organs was found, but with no difference between blasts from pLN and mLN. Thus there is no evidence to include the male genital tract in the common mucosal secretory immune system. Recirculating lymphocytes from the thoracic duct entered the male genital organs with a similar distribution to the pattern of lymphoid blasts. There is probably an exchange between these immigrating lymphocytes and the different subsets, which are localized in the epithelium (T suppressor) and interstitial tissue (T helper) in male genital organs. The lymphoid cells in the male genital tract might play an important role in the immune function of seminal fluid and in sexually transmissible diseases.

Migration pattern of lymphocyte subsets in the normal rat and the influence of splenic tissue.

Lymphocyte subsets leave the blood and appear in the thoracic duct of normal rats at different rates. The aim of the present study was to investigate their migration pattern through blood, spleen, bone marrow, mesenteric lymph nodes, and Peyer's patches in normal Lewis rats and to study the role of the spleen using splenectomized and spleen-transplanted animals. Fluorescein isothiocyanate (FITC)-labelled thoracic duct lymphocytes (TDL) were injected intravenously into rats and after 15 min, 1, 6, and 24 h the percentages of B, T, T helper (TH) and T-cytotoxic/suppressor (TC/S) lymphocytes in the FITC+ cells were determined in cell suspensions by means of monoclonal antibodies. B and T lymphocytes are preferentially localized in different organs, e.g. B cells in Peyer's patches and T cells in mesenteric lymph nodes. The migration of TH lymphocytes differed from that of TC/S lymphocytes in all the organs investigated. In the late phase after injection the migration of B and TH lymphocytes was influenced by the spleen, since after splenectomy the number of injected B lymphocytes increased and that of TH lymphocytes decreased in all organs investigated except the bone marrow. Splenic autotransplantation could not normalize the disturbed migration.

Numbers and heterogeneity of mast cells in the male genital tract of the rat.

Normal adult rats were used to quantitate and characterize mast cells in the male genital tract. The tissues were either fixed in a fixative containing formalin (Schaffer solution) or with basic lead acetate (BLA) to identify 'connective-tissue mast cells' and 'mucosal mast cells', respectively. In the epididymis and seminal vesicle small numbers of mast cells were identified without any obvious heterogeneity. In the prostate, however, a mean of 45.1 +/- 9.3 and 23.0 +/- 4.0 mast cells/mm2 was found after BLA and Schaffer fixation, respectively. This difference might be of functional and clinical significance.

Postnatal development and lymphocyte production of jejunal and ileal Peyer's patches in normal and gnotobiotic pigs.

The development of the number, size, structure and proliferative capacity of Peyer's patches (PP) in the jejunum and ileum has been studied during the early postnatal period of conventional and germ-free pigs. A mean of 15 discrete PP in the jejunum and upper ileum (jejPP) were counted at birth, and the number increased only gradually. A continuous PP is located in the terminal ileum (ileal PP). The length of both jejPP and ileal PP increased with age due to the increase in follicle size and in the number of follicles in the ileal PP. In older pigs, only the ileal PP regressed to small scattered follicles. In germ-free piglets at 39 and 59 days of age, longer PP were found than in normal new-born piglets, but they were significantly shorter than in age-matched controls. Lymphocyte production was studied by the metaphase-arrest technique using vincristine. Lymphocyte production in follicles increased dramatically with age, while in other compartments, such as the inter-follicular and dome area, a low age-independent production of lymphocytes was found. There were no differences in lymphocytopoiesis between jejPP and ileal PP. The present data show major differences in the development, structure and function of PP in pigs in comparison to other species. These species-specific aspects are important for future studies on the immunological function of PP.

Comparison of lymphocyte production in lymphoid organs and their compartments using the metaphase-arrest technique.

Lymphocyte proliferation was studied in normal young anesthetized pigs by the metaphase-arrest technique using vincristine (VCR). In each animal biopsies were taken simultaneously from the thymus, mesenteric lymph nodes, spleen, palatine tonsil and Peyer's patches from the ileum and jejunum. After taking the first samples, 0.25 mg VCR/kg body weight was injected i.v. and then four more biopsies were excised for up to 3.5 h after VCR. Imprints of the lymphoid organs were evaluated as an overall index for each organ, and histological sections were used to determine the mitotic index in typical B- and T-lymphocyte areas in these organs. In follicles of mesenteric lymph nodes, tonsils and the two types of Peyer's patches a comparable increase in the mitotic index was found, 3.62% per hour. In the corona the increase was also comparable but much lower, 0.43% per hour and in the interfollicular area similarly 0.38% per hour. In the spleen the mitotic rate was 0.69% for the white pulp and 0.42% per hour for the red pulp. In the thymic cortex the mitotic index increased by 0.49% and in the medulla by a surprisingly high value of 0.32% per hour. The metaphase-arrest technique in larger animals enables a comparison of lymphocyte production among organs and their different compartments, and demonstrates the important contribution of peripheral lymphoid organs to the renewal of the lymphocyte pools.