Postnatal Development of NPY and Somatostatin-28 Peptidergic Populations in the Human Angular Bundle

The angular bundle is a white matter fiber fascicle, which runs longitudinally along the parahippocampal gyrus. It is best known for carrying fibers from the entorhinal cortex (EC) to the hippocampus through the perforant and alvear pathways, as well as for carrying hippocampal output to the neocortex, and distributing fibers to polysensory cortex. The angular bundle is already present prenatally at the beginning of the fetal period. Connections between the EC and the hippocampus are established by the 20th gestational week (gw). In the postnatal period, it shows increasing myelination. The angular bundle, as well as other white matter portions of gyral surfaces in the brain, presents interstitial neurons, a remnant of subplate neurons. Those interstitial neurons show neurochemical phenotypes both prenatally and postnatally, among which, neuropeptide Y (NPY) and Somatostatin-28 (SOM-28) peptidergic populations are noticeable, and accompany the fiber connections in the maturation of the hippocampal formation. We sought to investigate the topography of the postnatal distribution and relative density of neurons immunoreactive for NPY or SOM in the angular bundle along the rostrocaudal axis of the hippocampus. The study was carried out in 15 cases, ranging from 35 gws, up to 14 year old. All cases showed positive neurons showing a polygonal or spindle shaped morphology for both peptides, scattered throughout the angular bundle. The highest number of positive neurons appeared around birth and the ensuing weeks. Up to one and a half years, the density of both peptidergic populations decreased slightly. However, cases older than 2 years of age showed a substantial decrease in density of immunolabeled neurons, density that did not showed a minor decrease in density of positive neurons in cases older than 2 years. In addition, a topography from caudal to rostral levels of the angular bundle was detected at all ages. The functional significance of interstitial cells is unknown, but the existence of SOM and NPY peptidergic neurons, presumably inhibitory, in the white matter of the angular bundle, could contribute to the basic wiring of the hippocampal formation, through which autobiographical and spatial memories can begin to be stored in the infant brain

Pressurized Extraction as an Opportunity to Recover Antioxidants from Orange Peels: Heat treatment and Nanoemulsion Design for Modulating Oxidative Stress

Orange peel by-products generated in the food industry are an important source of value added compounds that can be potentially reused. In the current research, the effect of oven-drying (50–70 ◦C) and freeze-drying on the bioactive compounds and antioxidant potential from Navelina, Salustriana, and Sanguina peel waste was investigated using pressurized extraction (ASE). Sixty volatile components were identified by ASE-GC-MS. The levels of terpene derivatives (sesquitenenes, alcohols, aldehydes, hydrocarbons, and esters) remained practically unaffected among fresh and freeze-dried orange peels, whereas drying at 70 ◦C caused significative decreases in Navelina, Salustri ana, and Sanguina peels. Hesperidin and narirutin were the main flavonoids quantified by HPLC-MS. Freeze-dried Sanguina peels showed the highest levels of total-polyphenols (113.3 mg GAE·g −1 ), total flavonoids (39.0 mg QE·g −1 ), outstanding values of hesperedin (187.6 µg·g −1 ), phenol acids (16.54 mg·g −1 DW), and the greatest antioxidant values (DPPH•, FRAP, and ABTS•+ assays) in comparison with oven-dried samples and the other varieties. Nanotechnology approaches allowed the formulation of antioxidant-loaded nanoemulsions, stabilized with lecithin, starting from orange peel extracts. Those provided 70–80% of protection against oxidative UV-radiation, also decreasing the ROS levels into the Caco-2 cells. Overall, pressurized extracts from freeze-drying orange peel can be considered a good source of natural antioxidants that could be exploited in food applications for the development of new products of commercial interest

Comparison of histological delineations of medial temporal lobe cortices by four independent neuroanatomy laboratories

The medial temporal lobe (MTL) cortex, located adjacent to the hippocampus, is crucial for memory and prone to the accumulation of certain neuropathologies such as Alzheimer's disease neurofibrillary tau tangles. The MTL cortex is composed of several subregions which differ in their functional and cytoarchitectonic features. As neuroanatomical schools rely on different cytoarchitectonic definitions of these subregions, it is unclear to what extent their delineations of MTL cortex subregions overlap. Here, we provide an overview of cytoarchitectonic definitions of the entorhinal and parahippocampal cortices as well as Brodmann areas (BA) 35 and 36, as provided by four neuroanatomists from different laboratories, aiming to identify the rationale for overlapping and diverging delineations. Nissl-stained series were acquired from the temporal lobes of three human specimens (two right and one left hemisphere). Slices (50 μm thick) were prepared perpendicular to the long axis of the hippocampus spanning the entire longitudinal extent of the MTL cortex. Four neuroanatomists annotated MTL cortex subregions on digitized slices spaced 5 mm apart (pixel size 0.4 μm at 20× magnification). Parcellations, terminology, and border placement were compared among neuroanatomists. Cytoarchitectonic features of each subregion are described in detail. Qualitative analysis of the annotations showed higher agreement in the definitions of the entorhinal cortex and BA35, while the definitions of BA36 and the parahippocampal cortex exhibited less overlap among neuroanatomists. The degree of overlap of cytoarchitectonic definitions was partially reflected in the neuroanatomists' agreement on the respective delineations. Lower agreement in annotations was observed in transitional zones between structures where seminal cytoarchitectonic features are expressed less saliently. The results highlight that definitions and parcellations of the MTL cortex differ among neuroanatomical schools and thereby increase understanding of why these differences may arise. This work sets a crucial foundation to further advance anatomically-informed neuroimaging research on the human MTL cortex

Neuropeptides in the developing human hippocampus under hypoxic–ischemic conditions

The perinatal period, sensitive for newborn survival, is also one of the most critical moments in human brain development. Perinatal hypoxia due to reduced blood supply to the brain (ischemia) is one of the main causes of neonatal mortality. Brain damage caused by perinatal hypoxia–ischemia (HI) can lead to neuro- and psychological disorders. However, its impact seems to be region-dependent, with the hippocampus being one of the most affected areas. Among the neuronal populations of the hippocampus, some interneuron groups – such as somatostatin- or neuropeptide Y-expressing neurons – seem to be particularly vulnerable. The limited information available about the effects of HI in the hippocampus comes mainly from animal models and adult human studies. This article presents an immunohistochemical analysis of somatostatin (SOM) and neuropeptide Y (NPY) expression in the developing human hippocampus after perinatal HI. Two rostrocaudal sections of the body of the hippocampus were analysed, and the number of immunostained cells in the polymorphic layer of the dentate gyrus (DG) and the pyramidal cell layer and stratum oriens of the CA3, CA2 and CA1 fields of the hippocampus proper were quantified. The results showed a lower density of both neuropeptides in hypoxic compared to control cases. In the HI group, the number of SOM-immunoreactive cell bodies was statistically significantly lower in the pyramidal cell layer and stratum oriens of CA1, while the number of NPY-expressing neurons was statistically lower in the pyramidal cell layer of CA2. Besides, the number of SOM-expressing neurons was significantly higher in the stratum oriens of CA1 compared to that in CA2. In sum, we observed a different vulnerability of SOM- and NPY-containing neurons in the developing human hippocampus following perinatal HI damage. Our results could contribute to a better understanding of the behaviour of these neuronal populations under stressful conditions during the perinatal period

Ex vivo, in situ perfusion protocol for human brain fixation compatible with microscopy, MRI techniques, and anatomical studies

We present a method for human brain fixation based on simultaneous perfusion of 4% paraformaldehyde through carotids after a flush with saline. The left carotid cannula is used to perfuse the body with 10% formalin, to allow further use of the body for anatomical research or teaching. The aim of our method is to develop a vascular fixation protocol for the human brain, by adapting protocols that are commonly used in experimental animal studies. We show that a variety of histological procedures can be carried out (cyto- and myeloarchitectonics, histochemistry, immunohistochemistry, intracellular cell injection, and electron microscopy). In addition, ex vivo, ex situ high-resolution MRI (9.4T) can be obtained in the same specimens. This procedure resulted in similar morphological features to those obtained by intravascular perfusion in experimental animals, provided that the postmortem interval was under 10 h for several of the techniques used and under 4 h in the case of intracellular injections and electron microscopy. The use of intravascular fixation of the brain inside the skull provides a fixed whole human brain, perfectly fitted to the skull, with negligible deformation compared to conventional techniques. Given this characteristic of ex vivo, in situ fixation, this procedure can probably be considered the most suitable one available for ex vivo MRI scans of the brain. We describe the compatibility of the method proposed for intravascular fixation of the human brain and fixation of the donor’s body for anatomical purposes. Thus, body donor programs can provide human brain tissue, while the remainder of the body can also be fixed for anatomical studies. Therefore, this method of human brain fixation through the carotid system optimizes the procurement of human brain tissue, allowing a greater understanding of human neurological diseases, while benefiting anatomy departments by making the remainder of the body available for teaching purposes.This work was supported in part by the National Institute of Health (Grant R01 AG056014-R01), a UCLM travel grant (to RI), a UCLM research grant to Human Neuroanatomy Laboratory (2020-GRIN-28837), and project EQC2019-006341-P (AEI/FEDER UE)

Unfolding the Medial Temporal Lobe Cortex to Characterize Neurodegeneration Due to Alzheimer’s Disease Pathology Using Ex vivo Imaging

Neurofibrillary tangle (NFT) pathology in the medial temporal lobe (MTL) is closely linked to neurodegeneration, and is the early pathological change associated with Alzheimer’s Disease (AD). In this work, we investigate the relationship between MTL morphometry features derived from high-resolution ex vivo imaging and histology-based measures of NFT pathology using a topological unfolding framework applied to a dataset of 18 human postmortem MTL specimens. The MTL has a complex 3D topography and exhibits a high degree of inter-subject variability in cortical folding patterns which poses a significant challenge for volumetric registration methods typically used during MRI template construction. By unfolding the MTL cortex, the proposed framework explicitly accounts for the sheet-like geometry of the MTL cortex and provides a two-dimensional reference coordinate space which can be used to implicitly register cortical folding patterns across specimens based on distance along the cortex despite large anatomical variability. Leveraging this framework in a subset of 15 specimens, we characterize the associations between NFTs and morphological features such as cortical thickness and surface curvature and identify regions in the MTL where patterns of atrophy are strongly correlated with NFT pathology

Integrating Color Deconvolution Thresholding and Weakly Supervised Learning for Automated Segmentation of Neurofibrillary Tangle and Neuropil Threads

Abnormally phosphorylated tau proteins are known to be a major indicator of Alzheimer's Disease (AD) with strong association with memory loss and cognitive decline. Automated generation of pixel-wise accurate neurofibrillary tangles (NFTs) and neuropil threads (NTs) segmentation is a challenging task, due to lack of ground truth segmentation data of these abnormal tau pathology. This problem is most prominent in the case of segmenting NTs, where the small threadlike morphology makes pixel-wise labeling a laborious task and unrealistic for large-scale studies. Lack of ground truth data poses a significant limitation for many learning-based methods to generate accurate segmentations of NFTs and NTs. This work presents an automated pipeline for pixel level segmentation of NFTs and NTs that does not rely on ground truth segmentation data. The pipeline is composed of four main steps: (1) color deconvolution is used to separate histopathology images into staining channels (DAB, Hematoxylin, and Eosin), (2) Otsu's thresholding is used on the DAB stain channel to generate pixel level segmentation of abnormal tau proteins staining, (3) a weakly-supervised learning paradigm (WildCat), using only global descriptors of images, is used to generate density maps of potential regions of NFTs and NTs, and (4) density maps and segmentations are then integrated using connected component analysis to localize NFTs and NTs in the detected tau segmentations. Our results show high global classification accuracy for NFTs (Acc:0.96) and NTs (Acc:0.91), and statistically significant distinctions when evaluating the percent area occupied of the detected NTs relative to expert ratings of NTs severity. Qualitative assessment of the NFTs and NTs results showed accurate pixel-level segmentations of the NFTs, while modest performance for NTs

Ex vivo MRI atlas of the human medial temporal lobe : characterizing neurodegeneration due to tau pathology

Tau neurofibrillary tangle (NFT) pathology in the medial temporal lobe (MTL) is closely linked to neurodegeneration, and is the early pathological change associated with Alzheimer’s disease (AD). To elucidate patterns of structural change in the MTL specifically associated with tau pathology, we compared high-resolution ex vivo MRI scans of human postmortem MTL specimens with histology-based pathological assessments of the MTL. MTL specimens were obtained from twenty-nine brain donors, including patients with AD, other dementias, and individuals with no known history of neurological disease. Ex vivo MRI scans were combined using a customized groupwise diffeomorphic registration approach to construct a 3D probabilistic atlas that captures the anatomical variability of the MTL. Using serial histology imaging in eleven specimens, we labelled the MTL subregions in the atlas based on cytoarchitecture. Leveraging the atlas and neuropathological ratings of tau and TAR DNA-binding protein 43 (TDP-43) pathology severity, morphometric analysis was performed to correlate regional MTL thickness with the severity of tau pathology, after correcting for age and TDP-43 pathology. We found significant correlations between tau pathology and thickness in the entorhinal cortex (ERC) and stratum radiatum lacunosum moleculare (SRLM). When focusing on cases with low levels of TDP-43 pathology, we found strong associations between tau pathology and thickness in the ERC, SRLM and the subiculum/cornu ammonis 1 (CA1) subfields of the hippocampus, consistent with early Braak stages

Comparison of histological delineations of medial temporal lobe cortices by four independent neuroanatomy laboratories

The medial temporal lobe (MTL) cortex, located adjacent to the hippocampus, is crucial for memory and prone to the accumulation of certain neuropathologies such as Alzheimer's disease neurofibrillary tau tangles. The MTL cortex is composed of several subregions which differ in their functional and cytoarchitectonic features. As neuroanatomical schools rely on different cytoarchitectonic definitions of these subregions, it is unclear to what extent their delineations of MTL cortex subregions overlap. Here, we provide an overview of cytoarchitectonic definitions of the cortices that make up the parahippocampal gyrus (entorhinal and parahippocampal cortices) and the adjacent Brodmann areas (BA) 35 and 36, as provided by four neuroanatomists from different laboratories, aiming to identify the rationale for overlapping and diverging delineations. Nissl-stained series were acquired from the temporal lobes of three human specimens (two right and one left hemisphere). Slices (50 µm thick) were prepared perpendicular to the long axis of the hippocampus spanning the entire longitudinal extent of the MTL cortex. Four neuroanatomists annotated MTL cortex subregions on digitized (20X resolution) slices with 5 mm spacing. Parcellations, terminology, and border placement were compared among neuroanatomists. Cytoarchitectonic features of each subregion are described in detail. Qualitative analysis of the annotations showed higher agreement in the definitions of the entorhinal cortex and BA35, while definitions of BA36 and the parahippocampal cortex exhibited less overlap among neuroanatomists. The degree of overlap of cytoarchitectonic definitions was partially reflected in the neuroanatomists' agreement on the respective delineations. Lower agreement in annotations was observed in transitional zones between structures where seminal cytoarchitectonic features are expressed more gradually. The results highlight that definitions and parcellations of the MTL cortex differ among neuroanatomical schools and thereby increase understanding of why these differences may arise. This work sets a crucial foundation to further advance anatomically-informed human neuroimaging research on the MTL cortex