Uncovering microstructural architecture from histology.

Microstructural tissue organization underlies the complex connectivity of the brain and controls properties of connective, muscle, and epithelial tissue. However, discerning microstructural architecture with high resolution for large fields of view remains prohibitive. We address this challenge with computational scattered light imaging (ComSLI), which exploits the anisotropic light scattering of aligned structures. Using a rotating lightsource and a high-resolution camera, ComSLI determines fiber architecture with micrometer resolution from histological sections across preparation and staining protocols. We show complex fiber architecture in brain and non-brain sections, including histological paraffin-embedded sections with various stains, and demonstrate its applicability on animal and human tissue, including disease cases with altered microstructure. ComSLI opens new avenues for investigating fiber architecture in new and archived sections across organisms, tissues, and diseases.

Longitudinal Alterations of Cerebral Blood Flow in High-Contact Sports.

OBJECTIVE: Repetitive head trauma is common in high-contact sports. Cerebral blood flow (CBF) can measure changes in brain perfusion that could indicate injury. Longitudinal studies with a control group are necessary to account for interindividual and developmental effects. We investigated whether exposure to head impacts causes longitudinal CBF changes. METHODS: We prospectively studied 63 American football (high-contact cohort) and 34 volleyball (low-contact controls) male collegiate athletes, tracking CBF using 3D pseudocontinuous arterial spin labeling magnetic resonance imaging for up to 4 years. Regional relative CBF (rCBF, normalized to cerebellar CBF) was computed after co-registering to T1-weighted images. A linear mixed effects model assessed the relationship of rCBF to sport, time, and their interaction. Within football players, we modeled rCBF against position-based head impact risk and baseline Standardized Concussion Assessment Tool score. Additionally, we evaluated early (1-5 days) and delayed (3-6 months) post-concussion rCBF changes (in-study concussion). RESULTS: Supratentorial gray matter rCBF declined in football compared with volleyball (sport-time interaction p = 0.012), with a strong effect in the parietal lobe (p = 0.002). Football players with higher position-based impact-risk had lower occipital rCBF over time (interaction p = 0.005), whereas players with lower baseline Standardized Concussion Assessment Tool score (worse performance) had relatively decreased rCBF in the cingulate-insula over time (interaction effect p = 0.007). Both cohorts showed a left-right rCBF asymmetry that decreased over time. Football players with an in-study concussion showed an early increase in occipital lobe rCBF (p = 0.0166). INTERPRETATION: These results suggest head impacts may result in an early increase in rCBF, but cumulatively a long-term decrease in rCBF. ANN NEUROL 2023;94:457-469.

Imaging crossing fibers in mouse, pig, monkey, and human brain using small-angle X-ray scattering.

Myelinated axons (nerve fibers) efficiently transmit signals throughout the brain via action potentials. Multiple methods that are sensitive to axon orientations, from microscopy to magnetic resonance imaging, aim to reconstruct the brain's structural connectome. As billions of nerve fibers traverse the brain with various possible geometries at each point, resolving fiber crossings is necessary to generate accurate structural connectivity maps. However, doing so with specificity is a challenging task because signals originating from oriented fibers can be influenced by brain (micro)structures unrelated to myelinated axons. X-ray scattering can specifically probe myelinated axons due to the periodicity of the myelin sheath, which yields distinct peaks in the scattering pattern. Here, we show that small-angle X-ray scattering (SAXS) can be used to detect myelinated, axon-specific fiber crossings. We first demonstrate the capability using strips of human corpus callosum to create artificial double- and triple-crossing fiber geometries, and we then apply the method in mouse, pig, vervet monkey, and human brains. We compare results to polarized light imaging (3D-PLI), tracer experiments, and to outputs from diffusion MRI that sometimes fails to detect crossings. Given its specificity, capability of 3-dimensional sampling and high resolution, SAXS could serve as a ground truth for validating fiber orientations derived using diffusion MRI as well as microscopy-based methods. STATEMENT OF SIGNIFICANCE: To study how the nerve fibers in our brain are interconnected, scientists need to visualize their trajectories, which often cross one another. Here, we show the unique capacity of small-angle X-ray scattering (SAXS) to study these fiber crossings without use of labeling, taking advantage of SAXS's specificity to myelin - the insulating sheath that is wrapped around nerve fibers. We use SAXS to detect double and triple crossing fibers and unveil intricate crossings in mouse, pig, vervet monkey, and human brains. This non-destructive method can uncover complex fiber trajectories and validate other less specific imaging methods (e.g., MRI or microscopy), towards accurate mapping of neuronal connectivity in the animal and human brain.

High-resolution hippocampal diffusion tensor imaging of mesial temporal sclerosis in refractory epilepsy.

OBJECTIVE: We explore the possibility of using diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI) to discern microstructural abnormalities in the hippocampus indicative of mesial temporal sclerosis (MTS) at the subfield level. METHODS: We analyzed data from 57 patients with refractory epilepsy who previously underwent 3.0-T magnetic resonance imaging (MRI) including DTI as a standard part of presurgical workup. We collected information about each subject's seizure semiology, conventional electroencephalography (EEG), high-density EEG, positron emission tomography reports, surgical outcome, and available histopathological findings to assign a final diagnostic category. We also reviewed the radiology MRI report to determine the radiographic category. DTI- and NODDI-based metrics were obtained in the hippocampal subfields. RESULTS: By examining diffusion characteristics among subfields in the final diagnostic categories, we found lower orientation dispersion indices and elevated axial diffusivity in the dentate gyrus in MTS compared to no MTS. By similarly examining among subfields in the different radiographic categories, we found all diffusion metrics were abnormal in the dentate gyrus and CA1. We finally examined whether diffusion imaging would better inform a radiographic diagnosis with respect to the final diagnosis, and found that dentate diffusivity suggested subtle changes that may help confirm a positive radiologic diagnosis. SIGNIFICANCE: The results suggest that diffusion metric analysis at the subfield level, especially in dentate gyrus and CA1, maybe useful for clinical confirmation of MTS.

Conditioned medium from amniotic fluid mesenchymal stem cells could modulate Alzheimer's disease-like changes in human neuroblastoma cell line SY-SY5Y in a paracrine manner.

BACKGROUND: Alzheimer's disease is usually diagnosed by significant extracellular deposition of beta-amyloid and intracellular neurofibrillary tangle formation. Here, we investigated the paracrine effect of amniotic fluid-derived mesenchymal stem cells on AD changes in human SH-SY5Y cells. METHODS: SH-SY5Y cells were divided into five groups: Control, 0.1 µg/ml LPS, 10 µg/ml LPS, 0.1 µg/ml LPS + conditioned medium, and 10 µg/ml LPS + conditioned medium. Cells were incubated with 0.1% and 10 µg/ml LPS for 48 h, followed by incubation with the conditioned medium of amniotic fluid-derived mesenchymal stem cells for the next 24 h. Beta-amyloid plaques were monitored by Congo-red staining. Survival and apoptosis were assessed by the MTT assay and flow cytometric analysis of Annexin-V. ELISA was used to measure the levels of neprilysin, angiotensin-converting enzyme, and Matrix Metalloproteinase-9. A PCR array was used to measure the expression of genes involved in neurogenesis. RESULTS: Bright-field imaging showed beta-amyloid plaques in the group treated with 10 µg/ml LPS. We found minimal effects in groups receiving 0.1 µg/ml LPS. The data showed that the reduction in the levels of neprilysin, angiotensin-converting enzyme, and Matrix Metalloproteinase-9 in the LPS-treated cells was attenuated after incubation with the stem cell secretome (p < 0.05). Amniotic fluid stem cell secretome increased the viability of LPS-treated SH-SY5Y cells (p 0.05) and was associated with a decrease in apoptotic changes (p < 0.05). We found the modulation of several genes involved in neurogenesis in the 10 µg/ml LPS + conditioned medium group compared to cells treated with 10 µg/ml LPS alone. CONCLUSION: Amniotic fluid stem cell secretion reduces AD-like pathologies in the human neuronal lineage.

Effect of Mineral Pitch on the Proliferation of Human Adipose Derived Stem Cells on Acellular Scaffold.

Purpose: Acellular scaffold extracted from extracellular matrix (ECM) have been used for constructive and regenerative medicine. Adipose derived stem cells (ADSCs) can enhance the vascularization capacity of scaffolds. High mobility group box 1 (HMGB1) and stromal derived factor1 (SDF1) are considered as two important factors in vascularization and immunologic system. In this study, the effect of mineral pitch on the proliferation of human ADSCs was evaluated. In addition to HMGB1 and SDF1, factors expression in acellular scaffold was also assessed. Methods: To determine acellular scaffold morphology and the degree of decellularization, hematoxylin & eosin (H&E), 6-diamidino-2-phenylindole (DAPI), and Masson's trichrome staining were applied. The scaffolds were treated with mineral pitch. Also, ADSCs were seeded on the scaffolds, and adhesion of the cells to the scaffolds were assessed using field emission scanning electron microscopy (FE-SEM). In addition, the efficiency of mineral pitch to induce the proliferation of ADSCs on the scaffolds was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. To measure HMGB1 and SDF1 mRNA expression, real-time polymerase chain reactions (RT-PCR) was used. Results: FE-SEM showed that decellularized matrix possesses similar matrix morphology with a randomly oriented fibrillar structure and interconnecting pores. No toxicity was observed in all treatments, and cell proliferation were supported in scaffolds. The important point is that, the proliferation capacity of ADSCs on Mineral pitch loaded scaffolds significantly increased after 48 h incubation time compared to the unloaded scaffold (P<0.001). Conclusion: The results of this study suggest that mineral pitch has potentials to accelerate proliferation of ADSCs on the acellular scaffolds.