The Study of the Caudal Vertebrae of Thick-Toed Geckos after a Prolonged Space Flight by X-ray Phase-Contrast Micro-CT.

The proximal caudal vertebrae and notochord in thick-toed geckos (TG) (Chondrodactylus turneri, Gray, 1864) were investigated after a 30-day space flight onboard the biosatellite Bion-M1. This region has not been explored in previous studies. Our research focused on finding sites most affected by demineralization caused by microgravity (G0). We used X-ray phase-contrast tomography to study TG samples without invasive prior preparation to clarify our previous findings on the resistance of TG's bones to demineralization in G0. The results of the present study confirmed that geckos are capable of preserving bone mass after flight, as neither cortical nor trabecular bone volume fraction showed statistically significant changes after flight. On the other hand, we observed a clear decrease in the mineralization of the notochordal septum and a substantial rise in intercentrum volume following the flight. To monitor TG's mineral metabolism in G0, we propose to measure the volume of mineralized tissue in the notochordal septum. This technique holds promise as a sensitive approach to track the demineralization process in G0, given that the volume of calcification within the septum is limited, making it easy to detect even slight changes in mineral content.

Neuromorphological Atlas of Human Prenatal Brain Development: White Paper.

Recent morphological data on human brain development are quite fragmentary. However, they are highly requested for a number of medical practices, educational programs, and fundamental research in the fields of embryology, cytology and histology, neurology, physiology, path anatomy, neonatology, and others. This paper provides the initial information on the new online Human Prenatal Brain Development Atlas (HBDA). The Atlas will start with forebrain annotated hemisphere maps, based on human fetal brain serial sections at the different stages of prenatal ontogenesis. Spatiotemporal changes in the regional-specific immunophenotype profiles will also be demonstrated on virtual serial sections. The HBDA can serve as a reference database for the neurological research, which provides opportunity to compare the data obtained by noninvasive techniques, such as neurosonography, X-ray computed tomography and magnetic resonance imaging, functional magnetic resonance imaging, 3D high-resolution phase-contrast computed tomography visualization techniques, as well as spatial transcriptomics data. It could also become a database for the qualitative and quantitative analysis of individual variability in the human brain. Systemized data on the mechanisms and pathways of prenatal human glio- and neurogenesis could also contribute to the search for new therapy methods for a large spectrum of neurological pathologies, including neurodegenerative and cancer diseases. The preliminary data are now accessible on the special HBDA website.

Micromorphology of pineal gland calcification in age-related neurodegenerative diseases.

BACKGROUND: The formation of concrements in human pineal gland (PG) is a physiological process and, according to many researchers, is associated with the involution of PG structures. The majority of scientific publications concern progressive calcification of PG, leaving out studies on the destruction of already formed calcified concrements. Our study fills the gap in knowledge about calcified zones destruction in PG in normal aging and neuropathological conditions, which has not been addressed until now. PURPOSE: Our objective is to gain insight into human PG tissue impairment in both normal aging and neurodegenerative conditions. X-ray phase-contrast tomography (XPCT) allowed us to study PG tissue degeneration at high spatial resolution and, for the first time, to examine the damaged PG concrements in detail. Our research finding could potentially enhance the understanding of the PG involvement in the process of aging as well as in Alzheimer's disease (AD) and vascular dementia (VD). METHODS: The research was carried out on human PG autopsy material in normal aging, VD, and AD conditions. Laboratory-based micro-computed tomography (micro-CT) was used to collect and evaluate samples of native, uncut, and unstained PG with different degrees of pineal calcification. The detailed high-resolution 3D images of the selected PGs were produced using synchrotron-based XPCT. Histology and immunohistochemistry of soft PG tissue confirmed XPCT results. RESULTS: We performed via micro-CT the evaluation of the morphometric parameters of PG such as total sample volume, calcified concrements volume, and percentage of concrements in the total volume of the sample. XPCT imaging revealed high-resolution details of age-related PG alteration. In particular, we noted signs of moderate degradation of concrements in some PGs from elderly donors. In addition, our analysis revealed noticeable degenerative change in both concrements and soft tissue of PGs with neuropathology. In particular, we observed a hollow core and separated layers as well as deep ragged cracks in PG concrements of AD and VD samples. In parenchyma of some samples, we detected wide pinealocyte-free fluid-filled areas adjacent to the calcified zones. CONCLUSION: The present work provides the basis for future scientific research focused on the dynamic nature of PG calcium deposits and PG soft tissue in normal aging and neurodegenerative diseases.

Deep Learning-Based Segmentation of Post-Mortem Human's Olfactory Bulb Structures in X-ray Phase-Contrast Tomography.

The human olfactory bulb (OB) has a laminar structure. The segregation of cell populations in the OB image poses a significant challenge because of indistinct boundaries of the layers. Standard 3D visualization tools usually have a low resolution and cannot provide the high accuracy required for morphometric analysis. X-ray phase contrast tomography (XPCT) offers sufficient resolution and contrast to identify single cells in large volumes of the brain. The numerous microanatomical structures detectable in XPCT image of the OB, however, greatly complicate the manual delineation of OB neuronal cell layers. To address the challenging problem of fully automated segmentation of XPCT images of human OB morphological layers, we propose a new pipeline for tomographic data processing. Convolutional neural networks (CNN) were used to segment XPCT image of native unstained human OB. Virtual segmentation of the whole OB and an accurate delineation of each layer in a healthy non-demented OB is mandatory as the first step for assessing OB morphological changes in smell impairment research. In this framework, we proposed an effective tool that could help to shed light on OB layer-specific degeneration in patients with olfactory disorder.

Cytoskeleton Markers in the Spinal Cord and Mechanoreceptors of Thick-Toed Geckos after Prolonged Space Flights.

Spaceflight may cause hypogravitational motor syndrome (HMS). However, the role of the nervous system in the formation of HMS remains poorly understood. The aim of this study was to estimate the effects of space flights on the cytoskeleton of the neuronal and glial cells in the spinal cord and mechanoreceptors in the toes of thick-toed geckos (Chondrodactylus turneri GRAY, 1864). Thick-toed geckos are able to maintain attachment and natural locomotion in weightlessness. Different types of mechanoreceptors have been described in the toes of geckos. After flight, neurofilament 200 immunoreactivity in mechanoreceptors was lower than in control. In some motor neurons of flight geckos, nonspecific pathomorphological changes were observed, but they were also detected in the control. No signs of gliosis were detected after spaceflight. Cytoskeleton markers adequately reflect changes in the cells of the nervous system. We suggest that geckos' adhesion is controlled by the nervous system. Our study revealed no significant disturbances in the morphology of the spinal cord after the prolonged space flight, supporting the hypothesis that geckos compensate the alterations, characteristic for other mammals in weightlessness, by tactile stimulation.

Prenatal development of sympathetic innervation of the human pancreas.

BACKGROUND: The sympathetic nervous system plays an important role in the regulation of pancreatic exocrine and endocrine secretion. The results of experimental studies also demonstrate the involvement of the sympathetic nervous system in the regulation of endocrine cell differentiation and islet formation during the development of the pancreas. However, the prenatal development of sympathetic innervation of the human pancreas has not yet been studied. MATERIAL AND METHODS: Pancreatic autopsy samples from 24 human fetuses were examined using immunohistochemistry with antibodies to tyrosine hydroxylase (TH). The density, concentration, and size (width, length, perimeter and area) of the TH-positive sympathetic nerves were compared in four developmental periods: pre-fetal (8-11 weeks post conception (w.p.c.), n = 6), early fetal (13-20 gestational weeks (g.w.), n = 7), middle fetal (21-28 g.w., n = 6) and late fetal (29-40 g.w., n = 5) using morphometric methods and statistical analysis (Multiple Comparisons p values). Double immunofluorescence with antibodies to TH and either insulin or glucagon and confocal microscopy were applied to analyze the interaction between the sympathetic nerves and endocrine cells, and the co-localization of TH with hormones. RESULTS: TH-positive sympathetic nerves were detected in the fetal pancreas starting from the early stages (8 w.p.c.). The developmental dynamics of sympathetic nerves was follows: from the pre-fetal period, the amount of TH-positive nerves gradually increased and their branching occurred reaching the highest density and concentration in the middle fetal period, followed by a decrease in these parameters in the late fetal period. From the 14th g.w. onwards, thin TH-positive nerve fibers were mainly distributed in the vicinity of blood vessels and around the neurons of intrapancreatic ganglia, which is similar in adults. There were only rare TH-positive nerve fibers adjacent to acini or located at the periphery of some islets. The close interactions between the TH-positive nerve fibers and endocrine cells were observed in the neuro-insular complexes. Additionally, non-neuronal TH-containing cells were found in the pancreas of fetuses from the pre-fetal and early fetal periods. Some of these cells simultaneously contained glucagon. CONCLUSIONS: The results demonstrate that sympathetic innervation of the human pancreas, including the formation of perivascular and intraganglionic nerve plexuses, extensively develops during prenatal period, while some processes, such as the formation of sympathetic innervation of islet capillaries, may occur postnatally. Non-neuronal TH-containing cells, as well as the interactions between the sympathetic terminals and endocrine cells observed in the fetal pancreas may be necessary for endocrine pancreas development in humans.

Micro-CT Study of Mongolian Gerbil Humeral Bone After Prolonged Spaceflight Based on a New Algorithm for Delimitation of Long-Bone Regions.

The Mongolian gerbil displays unique physiological and anatomical features that make this species an attractive object for biological experiments in space. However, until recently, the Mongolian gerbil has remained a novel, mostly unstudied animal model in investigating bone loss in weightlessness (G0). After 12 days of orbital Foton-M3 mission, the humerus of Mongolian gerbils has been studied here via micro-computed tomography (micro-CT) to quantify bone morphometric parameters. The samples from the flight group, delayed synchronous ground-control group, and basal control group were investigated, and main morphometric parameters were reported in the article. The accurate selection of a region of interest is an essential step for a correct assessment of bone parameters. We proposed a new, easy and efficient method for delimiting the bone's basic regions in the humerus. It is based on quantitative estimation of X-ray attenuation in the cortical bone as a function of humerus bone length. The micro-CT analysis of the basic bone regions revealed a difference in bone morphometric parameters between the flight and control gerbils. The most significant bone loss was observed in the cortical part of the proximal humeral zone in the flight group. No statistically significant changes of volume fraction in the cancellous tissue of proximal and distal epiphyses and metaphyses were observed. A statistically significant increase in both cancellous bone volume and bone X-ray attenuation in the flight group was detected in the proximal part of the diaphyses. We assume that enhanced calcium deposition in the diaphyseal cancellous tissue occurred due to a bone response to G0 conditions.

Immunohistochemical Characteristics of the Human Carotid Body in the Antenatal and Postnatal Periods of Development.

The evolutionary and ontogenetic development of the carotid body is still understudied. Research aimed at studying the comparative morphology of the organ at different periods in the individual development of various animal species should play a crucial role in understanding the physiology of the carotid body. However, despite more than two centuries of study, the human carotid body remains poorly understood. There are many knowledge gaps in particular related to the antenatal development of this structure. The aim of our work is to study the morphological and immunohistochemical characteristics of the human carotid body in the antenatal and postnatal periods of development. We investigated the human carotid bodies from 1 embryo, 20 fetuses and 13 adults of different ages using samples obtained at autopsy. Immunohistochemistry revealed expression of ßIII-tubulin and tyrosine hydroxylase in the type I cells and nerve fibers at all periods of ontogenesis; synaptophysin and PGP9.5 in the type I cells in some of the antenatal cases and all of the postnatal cases; 200 kDa neurofilaments in nerve fibers in some of the antenatal cases and all of the postnatal cases; and GFAP and S100 in the type II cells and Schwann cells in some of the antenatal cases and all of the postnatal cases. A high level of tyrosine hydroxylase in the type I cells was a distinctive feature of the antenatal carotid bodies. On the contrary, in the type I cells of adults, the expression of tyrosine hydroxylase was significantly lower. Our data suggest that the human carotid body may perform an endocrine function in the antenatal period, while in the postnatal period of development, it loses this function and becomes a chemosensory organ.

Cerebellar morphology and behavioural correlations of the vestibular function alterations in weightlessness.

In humans and other vertebrates, the range of disturbances and behavioural changes induced by spaceflight conditions are well known. Sensory organs and the central nervous system (CNS) are forced to adapt to new environmental conditions of weightlessness. In comparison with peripheral vestibular organs and behavioural disturbances in weightlessness conditions, the CNS vestibular centres of vertebrates, including the cerebellum, have been poorly examined in orbital experiments, as well as in experimental micro- and hypergravity. However, the cerebellum serves as a critical control centre for learning and sensory system integration during space-flight. Thus, it is referred to as a principal brain structure for adaptation to gravity and the entire sensorimotor adaptation and learning during weightlessness. This paper is focused on the prolonged spaceflight effects on the vestibular cerebellum evidenced from animal models used in the Bion-M1 project. The changes in the peripheral vestibular apparatus and brainstem primary vestibular centres with appropriate behavioural disorders after altered gravity exposure are briefly reviewed. The cerebellum studies in space missions and altered gravity are discussed.

Reproduction and the Early Development of Vertebrates in Space: Problems, Results, Opportunities.

Humans and animals adapt to space flight conditions. However, the adaptive changes of fully formed organisms differ radically from the responses of vertebrate embryos, foetuses, and larvae to space flight. Development is associated with active cell proliferation and the formation of organs and systems. The instability of these processes is well known. Over 20 years has passed since the last systematic experiments on vertebrate reproduction and development in space flight. At the same time, programs are being prepared for the exploration of Mars and the Moon, which justifies further investigations into space flight's impact on vertebrate development. This review focuses on various aspects of reproduction and early development of vertebrates in space flights. The results of various experiments on fishes, amphibians, reptiles, birds and mammals are described. The experiments in which our team took part and ontogeny of the vertebrate nervous and special sensory systems are considered in more detail. Possible causes of morphological changes are also discussed. Research on evolutionarily and taxonomically different models can advance the understanding of reproduction in microgravity. Reptiles, in particular, geckos, due to their special features, can be a promising object of space developmental biology.

Investigation of the human pineal gland 3D organization by X-ray phase contrast tomography.

Pineal gland (PG) is a part of the human brain epithalamus that plays an important role in sleep, circadian rhythm, immunity, and reproduction. The calcium deposits and lesions in PG interfere with normal function of the organ and can be associated with different health disorders including serious neurological diseases. At the moment, the detailed mechanisms of PG calcifications and PG lesions formation as well as their involvement in pathological processes are not fully understood. The deep and comprehensive study of the structure of the uncut human PG with histological details, poses a stiff challenge to most imaging techniques, due to low spatial resolution, low visibility or to exceedingly aggressive sample preparation. Here, we investigate the whole uncut and unstained human post-mortem PGs by X-ray phase contrast tomography (XPCT). XPCT is an advanced 3D imaging technique, that permits to study of both soft and calcified tissue of a sample at different scales: from the whole organ to cell structure. In our research we simultaneously resolved 3D structure of parenchyma, vascular network and calcifications. Moreover, we distinguished structural details of intact and degenerated PG tissue. We discriminated calcifications with different structure, pinealocytes nuclei and the glial cells processes. All results were validated by histology. Our research clear demonstrated that XPCT is a potential tool for the high resolution 3D imaging of PG morphological features. This technique opens a new perspective to investigate PG dysfunction and understand the mechanisms of onset and progression of diseases involving the pineal gland.

Pancreatic endocrine cell arrangement during human ontogeny.

In the human pancreas, various forms of endocrine cell arrangement are found: single endocrine cells, endocrine cell clusters, and mantel, bipolar and mosaic cell (mixed) islets. Our aim was to analyse the distribution and dynamics of insulin-, glucagon- and somatostatin-containing cells within the various forms of endocrine pancreas arrangement during human prenatal development and in adults and to suggest a mechanism of change in the endocrine cell ratio in adult islets. Pancreatic autopsies derived from human foetuses from the 10th to the 40th weeks of development and from adults were examined using histological, immunohistochemical and morphometric methods. During development, the human endocrine pancreas undergoes not only de novo differentiation of endocrine cells and islet formation, but morphogenetic restructuring, which is revealed as a change of the α-, ß- and δ-cell ratio in the islets. In particular, increased proportion of glucagon- and somatostatin-containing cells and decreased proportion of ß-cells were shown in the largest mosaic islets in adults. Our results indicate that the distribution and proportion of α-, ß- and δ-cells depend on the islets size and vascularisation. Studying of the mechanism of such restructuring may contribute to the development of new approaches in the treatment of diabetes mellitus.

Reptiles in Space Missions: Results and Perspectives.

Reptiles are a rare model object for space research. However, some reptile species demonstrate effective adaptation to spaceflight conditions. The main scope of this review is a comparative analysis of reptile experimental exposure in weightlessness, demonstrating the advantages and shortcomings of this model. The description of the known reptile experiments using turtles and geckos in the space and parabolic flight experiments is provided. Behavior, skeletal bones (morphology, histology, and X-ray microtomography), internal organs, and the nervous system (morphology, histology, and immunohistochemistry) are studied in the spaceflight experiments to date, while molecular and physiological results are restricted. Therefore, the results are discussed in the scope of molecular data collected from mammalian (mainly rodents) specimens and cell cultures in the parabolic and orbital flights and simulated microgravity. The published data are compared with the results of the gecko model studies after the 12-44.5-day spaceflights with special reference to the unique peculiarities of the gecko model for the orbital experiments. The complex study of thick-toed geckos after three spaceflights, in which all geckos survived and demonstrated effective adaptation to spaceflight conditions, was performed. However, future investigations are needed to study molecular mechanisms of gecko adaptation in space.

Immunohistochemical detection of vimentin in pancreatic islet ß- and α-cells of macrosomic infants of diabetic and nondiabetic mothers.

BACKGROUND: Expression of the intermediate filament protein vimentin has been recently observed in the pancreatic islet ß- and α-cells of humans with type 2 diabetes mellitus. It was suggested that the presence of vimentin in endocrine cells may indicate islet tissue renewal, or potentially represent the dedifferentiation of endocrine cells, which could contribute to the onset of type 2 diabetes or islet cell dysfunction. AIM: To analyze the expression of vimentin in pancreatic ß- and α-cells of macrosomic infants of diabetic and nondiabetic mothers. SUBJECTS: Pancreatic samples of five macrosomic infants (gestational age 34-40weeks) from three diabetic and two nondiabetic mothers were compared to six control infants (32-40weeks, weight appropriate for gestational age) from normoglycemic mothers. METHODS: Pancreatic autopsy samples were examined by double immunofluorescent labeling with antibodies against vimentin and either insulin or glucagon. Alterations in the endocrine pancreas were measured using morphometric methods, then data were statistically analyzed. RESULTS: In the pancreatic islets of macrosomic infants from diabetic and nondiabetic mothers, we observed vimentin-positive cells, some of which simultaneously contained insulin or glucagon. We also quantitatively showed that the presence of such cells was associated with hypertrophy and hyperplasia of the islets, and with an increase in ß- and α-cell density. CONCLUSIONS: We speculate that the appearance of vimentin-positive islet cells may reflect induction of differentiation in response to the increased insulin demand, and vimentin may serve as an early marker of endocrine pancreas disorders.

Structure of neuro-endocrine and neuro-epithelial interactions in human foetal pancreas.

In the pancreas of many mammals including humans, endocrine islet cells can be integrated with the nervous system components into neuro-insular complexes. The mechanism of the formation of such complexes is not clearly understood. The present study evaluated the interactions between the nervous system components, epithelial cells and endocrine cells in the human pancreas. Foetal pancreas, gestational age 19-23 weeks (13 cases) and 30-34 weeks (7 cases), were studied using double immunohistochemical labeling with neural markers (S100 protein and beta III tubulin), epithelial marker (cytokeratin 19 (CK19)) and antibodies to insulin and glucagon. We first analyse the structure of neuro-insular complexes using confocal microscopy and provide immunohistochemical evidences of the presence of endocrine cells within the ganglia or inside the nerve bundles. We showed that the nervous system components contact with the epithelial cells located in ducts or in clusters outside the ductal epithelium and form complexes with separate epithelial cells. We observed CK19-positive cells inside the ganglia and nerve bundles which were located separately or were integrated with the islets. Therefore, we conclude that neuro-insular complexes may forms as a result of integration between epithelial cells and nervous system components at the initial stages of islets formation.

Monochromatic computed microtomography using laboratory and synchrotron sources and X-ray fluorescence analysis for comprehensive analysis of structural changes in bones.

A combination of X-ray tomography at different wavelengths and X-ray fluorescence analysis was applied in the study of two types of bone tissue changes: prolonged presence in microgravity conditions and age-related bone growth. The proximal tail vertebrae of geckos were selected for investigation because they do not bear the supporting load in locomotion, which allows them to be considered as an independent indicator of gravitational influence. For the vertebrae of geckos no significant differences were revealed in the elemental composition of the flight samples and the synchronous control samples. In addition, the gecko bone tissue samples from the jaw apparatus, spine and shoulder girdle were measured. The dynamics of structural changes in the bone tissue growth was studied using samples of a human fetal hand. The hands of human fetuses of 11-15 weeks were studied. Autonomous zones of calcium accumulation were found not only in individual fingers but in each of the investigated phalanges. The results obtained are discussed.

Ontogeny of neuro-insular complexes and islets innervation in the human pancreas.

The ontogeny of the neuro-insular complexes (NIC) and the islets innervation in human pancreas has not been studied in detail. Our aim was to describe the developmental dynamics and distribution of the nervous system structures in the endocrine part of human pancreas. We used double-staining with antibodies specific to pan-neural markers [neuron-specific enolase (NSE) and S100 protein] and to hormones of pancreatic endocrine cells. NSE and S100-positive nerves and ganglia were identified in the human fetal pancreas from gestation week (gw) 10 onward. Later the density of S100 and NSE-positive fibers increased. In adults, this network was sparse. The islets innervation started to form from gw 14. NSE-containing endocrine cells were identified from gw 12 onward. Additionally, S100-positive cells were detected both in the periphery and within some of the islets starting at gw 14. The analysis of islets innervation has shown that the fetal pancreas contained NIC and the number of these complexes was reduced in adults. The highest density of NIC is detected during middle and late fetal periods, when the mosaic islets, typical for adults, form. The close integration between the developing pancreatic islets and the nervous system structures may play an important role not only in the hormone secretion, but also in the islets morphogenesis.

Computed microtomography and X-ray fluorescence analysis for comprehensive analysis of structural changes in bone.

This paper presents the results of a comprehensive analysis of structural changes in the caudal vertebrae of Turner's thick-toed geckos by computer microtomography and X-ray fluorescence analysis. We present algorithms used for the reconstruction of tomographic images which allow to work with high noise level projections that represent typical conditions dictated by the nature of the samples. Reptiles, due to their ruggedness, small size, belonging to the amniote and a number of other valuable features, are an attractive model object for long-orbital experiments on unmanned spacecraft. Issues of possible changes in their bone tissue under the influence of spaceflight are the subject of discussions between biologists from different laboratories around the world.

Dopamine D1 receptor-dependent regulation of extracellular citrulline level in the rat nucleus accumbens during conditioned fear response.

Nucleus accumbens (N.Acc) contains a subclass of nitric oxide (NO)-generating interneurons that are presumably regulated by the dopamine input. Receptor mechanisms underlying dopamine-NO interaction in the N.Acc are poorly understood. In the current study, we used in vivo microdialysis combined with high-performance liquid chromatography to examine participation of dopamine D1 receptors in regulation of extracellular levels of citrulline (an NO co-product) in the medial N.Acc of Sprague-Dawley rats during both pharmacological challenge and a conditioned fear response. The intraaccumbal infusion of the D1 receptor agonist SKF-38393 (100-500 microM) increased dose-dependently the local dialysate citrulline levels. The SKF-38393-induced increase in extracellular citrulline was prevented by intraaccumbal infusions of 500 microM 7-nitroindazole, a neuronal NO synthase inhibitor. In behavioral microdialysis experiment, the accumbal levels of extracellular citrulline markedly increased in rats given a mild footshock paired with tone. The presentation of the tone previously paired with footshock (the conditioned fear response) produced a "conditioned" rise of extracellular citrulline levels in the N.Acc which was attenuated by intraaccumbal infusion of 100 microM SCH-23390, a dopamine D1 receptor antagonist, and prevented by intraaccumbal infusion of 500 microM 7-nitroindazole. The results suggest that in the N.Acc, the dopamine D1 receptors might regulate the neuronal NO synthase activity; this dopamine-dependent mechanism seems to participate in activation of the neuronal NO synthase and probably NO formation in this brain area during the conditioned fear response.