Lumen expansion is initially driven by apical actin polymerization followed by osmotic pressure in a human epiblast model.

Post-implantation, the pluripotent epiblast in a human embryo forms a central lumen, paving the way for gastrulation. Osmotic pressure gradients are considered the drivers of lumen expansion across development, but their role in human epiblasts is unknown. Here, we study lumenogenesis in a pluripotent-stem-cell-based epiblast model using engineered hydrogels. We find that leaky junctions prevent osmotic pressure gradients in early epiblasts and, instead, forces from apical actin polymerization drive lumen expansion. Once the lumen reaches a radius of ∼12 µm, tight junctions mature, and osmotic pressure gradients develop to drive further growth. Computational modeling indicates that apical actin polymerization into a stiff network mediates initial lumen expansion and predicts a transition to pressure-driven growth in larger epiblasts to avoid buckling. Human epiblasts show transcriptional signatures consistent with these mechanisms. Thus, actin polymerization drives lumen expansion in the human epiblast and may serve as a general mechanism of early lumenogenesis.

Clots reveal anomalous elastic behavior of fiber networks.

The adaptive mechanical properties of soft and fibrous biological materials are relevant to their functionality. The emergence of the macroscopic response of these materials to external stress and intrinsic cell traction from local deformations of their structural components is not well understood. Here, we investigate the nonlinear elastic behavior of blood clots by combining microscopy, rheology, and an elastic network model that incorporates the stretching, bending, and buckling of constituent fibrin fibers. By inhibiting fibrin cross-linking in blood clots, we observe an anomalous softening regime in the macroscopic shear response as well as a reduction in platelet-induced clot contractility. Our model explains these observations from two independent macroscopic measurements in a unified manner, through a single mechanical parameter, the bending stiffness of individual fibers. Supported by experimental evidence, our mechanics-based model provides a framework for predicting and comprehending the nonlinear elastic behavior of blood clots and other active biopolymer networks in general.

Comparative Study of Terminal Cortical Potentials Using Iridium and Ag/AgCl Electrodes.

Brain ischemia induces slow voltage shifts in the cerebral cortex, including waves of spreading depolarization (SD) and negative ultraslow potentials (NUPs), which are considered as brain injury markers. However, different electrode materials and locations yield variable SD and NUP features. Here, we compared terminal cortical events during isoflurane or sevoflurane euthanasia using intracortical linear iridium electrode arrays and Ag/AgCl-based electrodes in the rat somatosensory cortex. Inhalation of anesthetics caused respiratory arrest, associated with hyperpolarization and followed by SD and NUP on both Ir and Ag electrodes. Ag-NUPs were bell shaped and waned within half an hour after death. Ir-NUPs were biphasic, with the early fast phase corresponding to Ag-NUP, and the late absent on Ag electrodes, phase of a progressive depolarizing voltage shift reaching -100 mV by two hours after death. In addition, late Ir-NUPs were more ample in the deep layers than at the cortical surface. Thus, intracortical Ag and Ir electrodes reliably assess early manifestations of terminal brain injury including hyperpolarization, SD and the early phase of NUP, while the late, giant amplitude phase of NUP, which is present only on Ir electrodes, is probably related to the sensitivity of Ir electrodes to a yet unidentified factor related to brain death.

Molecular Characterisation and Phylogeny of Tula Virus in Kazakhstan.

Orthohantaviruses are zoonotic pathogens that play a significant role in public health. These viruses can cause haemorrhagic fever with renal syndrome in Eurasia. In the Republic of Kazakhstan, the first human cases were registered in the year 2000 in the West Kazakhstan region. Small mammals can be reservoirs of orthohantaviruses. Previous studies showed orthohantavirus antigens in wild-living small mammals in four districts of West Kazakhstan. Clinical studies suggested that there might be further regions with human orthohantavirus infections in Kazakhstan, but genetic data of orthohantaviruses in natural foci are limited. The aim of this study was to investigate small mammals for the presence of orthohantaviruses by molecular biological methods and to provide a phylogenetic characterization of the circulating strains in Kazakhstan. Small mammals were trapped at 19 sites in West Kazakhstan, four in Almaty region and at seven sites around Almaty city during all seasons of 2018 and 2019. Lung tissues of small mammals were homogenized and RNA was extracted. Orthohantavirus RT-PCR assays were applied for detection of partial S and L segment sequences. Results were compared to published fragments. In total, 621 small mammals from 11 species were analysed. Among the collected small mammals, 2.4% tested positive for orthohantavirus RNA, one sample from West Kazakhstan and 14 samples from Almaty region. None of the rodents caught in Almaty city were infected. Sequencing parts of the small (S) and large (L) segments specified Tula virus (TULV) in these two regions. Our data show that geographical distribution of TULV is more extended as previously thought. The detected sequences were found to be split in two distinct genetic clusters of TULV in West Kazakhstan and Almaty region. TULV was detected in the common vole (Microtus arvalis) and for the first time in two individuals of the forest dormouse (Dryomys nitedula), interpreted as a spill-over infection in Kazakhstan.

Shape multistability in flexible tubular crystals through interactions of mobile dislocations.

We study avenues to shape multistability and shape morphing in flexible crystalline membranes of cylindrical topology, enabled by glide mobility of dislocations. Using computational modeling, we obtain states of mechanical equilibrium presenting a wide variety of tubular crystal deformation geometries, due to an interplay of effective defect interactions with out-of-tangent-plane deformations that reorient the tube axis. Importantly, this interplay often stabilizes defect configurations quite distinct from those predicted for a two-dimensional crystal confined to the surface of a rigid cylinder. We find that relative and absolute stability of competing states depend strongly on control parameters such as bending rigidity, applied stress, and spontaneous curvature. Using stable dislocation pair arrangements as building blocks, we demonstrate that targeted macroscopic three-dimensional conformations of thin crystalline tubes can be programmed by imposing certain sparse patterns of defects. Our findings reveal a broad design space for controllable and reconfigurable colloidal tube geometries, with potential relevance also to architected carbon nanotubes and microtubules.

Modeling mechanochemical pattern formation in elastic sheets of biological matter.

Inspired by active shape morphing in developing tissues and biomaterials, we investigate two generic mechanochemical models where the deformations of a thin elastic sheet are driven by, and in turn affect, the concentration gradients of a chemical signal. We develop numerical methods to study the coupled elastic deformations and chemical concentration kinetics, and illustrate with computations the formation of different patterns depending on shell thickness, strength of mechanochemical coupling and diffusivity. In the first model, the sheet curvature governs the production of a contractility inhibitor and depending on the threshold in the coupling, qualitatively different patterns occur. The second model is based on the stress-dependent activity of myosin motors and demonstrates how the concentration distribution patterns of molecular motors are affected by the long-range deformations generated by them. Since the propagation of mechanical deformations is typically faster than chemical kinetics (of molecular motors or signaling agents that affect motors), we describe in detail and implement a numerical method based on separation of timescales to effectively simulate such systems. We show that mechanochemical coupling leads to long-range propagation of patterns in disparate systems through elastic instabilities even without the diffusive or advective transport of the chemicals.

Mechanochemical induction of wrinkling morphogenesis on elastic shells.

Morphogenetic dynamics of tissue sheets require coordinated cell shape changes regulated by global patterning of mechanical forces. Inspired by such biological phenomena, we propose a minimal mechanochemical model based on the notion that cell shape changes are induced by diffusible biomolecules that influence tissue contractility in a concentration-dependent manner - and whose concentration is in turn affected by the macroscopic tissue shape. We perform computational simulations of thin shell elastic dynamics to reveal propagating chemical and three-dimensional deformation patterns arising due to a sequence of buckling instabilities. Depending on the concentration threshold that actuates cell shape change, we find qualitatively different patterns. The mechanochemically coupled patterning dynamics are distinct from those driven by purely mechanical or purely chemical factors, and emerge even without diffusion. Using numerical simulations and theoretical arguments, we analyze the elastic instabilities that result from our model and provide simple scaling laws to identify wrinkling morphologies.

Shape-morphing architectures actuated by Janus fibers.

We describe a combined experimental and theoretical investigation of shape-morphing structures assembled by actuating composite (Janus) fibers, taking into account multiple relevant factors affecting shape transformations, such as strain rate, composition, and geometry of the structures. Starting with simple bending experiments, we demonstrate the ways to attain multiple out-of-plane shapes of closed rings and square frames. Through combining theory and simulation, we examine how the mechanical properties of Janus fibers affect shape transitions. This allows us to control shape changes and to attain target 3D shapes by precise tuning of the material properties and geometry of the fibers. Our results open new perspectives of design of advanced mechanical metamaterials capable to create elaborate structures through sophisticated actuation modes.

Inhibition of Cortical Activity and Apoptosis Caused by Ethanol in Neonatal Rats In Vivo.

Alcohol consumption during pregnancy causes fetal alcohol spectrum disorder, which includes neuroapoptosis and neurobehavioral deficits. The neuroapoptotic effects of alcohol have been hypothesized to involve suppression of brain activity. However, in vitro studies suggest that ethanol acts as a potent stimulant of cortical activity. We explored the effects of alcohol (1-6 g/kg) on electrical activity in the rat somatosensory cortex in vivo at postnatal days P1-23 and compared them with its apoptotic actions. At P4-7, when the peak of alcohol-induced apoptosis was observed, alcohol strongly suppressed spontaneous gamma and spindle-bursts and almost completely silenced neurons in a dose-dependent manner. The dose-dependence of suppression of neuronal activity strongly correlated with the alcohol-induced neuroapoptosis. Alcohol also profoundly inhibited sensory-evoked bursts and suppressed motor activity, a physiological trigger of cortical activity bursts in newborns. The suppressive effects of ethanol on neuronal activity waned during the second and third postnatal weeks, when instead of silencing the cortex, alcohol evoked delta-wave electrographic activity. Thus, the effects of alcohol on brain activity are strongly age-dependent, and during the first postnatal week alcohol profoundly inhibits brain activity. Our findings suggest that the adverse effects of alcohol in the developing brain involve suppression of neuronal activity.

The Nature of the Sensory Input to the Neonatal Rat Barrel Cortex.

UNLABELLED: Sensory input plays critical roles in the development of the somatosensory cortex during the neonatal period. This early sensory input may involve: (1) stimulation arising from passive interactions with the mother and littermates and (2) sensory feedback arising from spontaneous infant movements. The relative contributions of these mechanisms under natural conditions remain largely unknown, however. Here, we show that, in the whisker-related barrel cortex of neonatal rats, spontaneous whisker movements and passive stimulation by the littermates cooperate, with comparable efficiency, in driving cortical activity. Both tactile signals arising from the littermate's movements under conditions simulating the littermates' position in the litter, and spontaneous whisker movements efficiently triggered bursts of activity in barrel cortex. Yet, whisker movements with touch were more efficient than free movements. Comparison of the various experimental conditions mimicking the natural environment showed that tactile signals arising from the whisker movements with touch and stimulation by the littermates, support: (1) a twofold higher level of cortical activity than in the isolated animal, and (2) a threefold higher level of activity than in the deafferented animal after the infraorbital nerve cut. Together, these results indicate that endogenous (self-generated movements) and exogenous (stimulation by the littermates) mechanisms cooperate in driving cortical activity in newborn rats and point to the importance of the environment in shaping cortical activity during the neonatal period. SIGNIFICANCE STATEMENT: Sensory input plays critical roles in the development of the somatosensory cortex during the neonatal period. However, the origins of sensory input to the neonatal somatosensory cortex in the natural environment remain largely unknown. Here, we show that in the whisker-related barrel cortex of neonatal rats, spontaneous whisker movements and passive stimulation by the littermates cooperate, with comparable efficiency, in driving cortical activity during the critical developmental period.

The serotonin reuptake inhibitor citalopram suppresses activity in the neonatal rat barrel cortex in vivo.

Inhibition of serotonin uptake, which causes an increase in extracellular serotonin levels, disrupts the development of thalamocortical barrel maps in neonatal rodents. Previous in vitro studies have suggested that the disruptive effect of excessive serotonin on barrel map formation involves a depression at thalamocortical synapses. However, the effects of serotonin uptake inhibitors on the early thalamocortical activity patterns in the developing barrel cortex in vivo remain largely unknown. Here, using extracellular recordings of the local field potentials and multiple unit activity (MUA) we explored the effects of the selective serotonin reuptake inhibitor (SSRI) citalopram (10-20mg/kg, intraperitoneally) on sensory evoked activity in the barrel cortex of neonatal (postnatal days P2-5) rats in vivo. We show that administration of citalopram suppresses the amplitude and prolongs the delay of the sensory evoked potentials, reduces the power and frequency of the early gamma oscillations, and suppresses sensory evoked and spontaneous neuronal firing. In the adolescent P21-29 animals, citalopram affected neither sensory evoked nor spontaneous activity in barrel cortex. We suggest that suppression of the early thalamocortical activity patterns contributes to the disruption of the barrel map development caused by SSRIs and other conditions elevating extracellular serotonin levels.

Isoflurane suppresses early cortical activity.

OBJECTIVE: Isoflurane and other volatile anesthetics are widely used in children to induce deep and reversible coma, but they may also exert neurotoxic actions. The effects of volatile anesthetics on the immature brain activity remain elusive, however. METHODS: The effects of isoflurane on spontaneous and sensory-evoked activity were explored using intracortical extracellular field potential and multiple unit recordings in the rat barrel cortex from birth to adulthood. RESULTS: During the first postnatal week, isoflurane suppressed cortical activity in a concentration-dependent manner. At surgical anesthesia levels (1.5-2%), isoflurane completely suppressed the electroencephalogram and silenced cortical neurons. Although sensory potentials evoked by the principal whisker deflection persisted, sensory-evoked early gamma and spindle-burst oscillations were completely suppressed by isoflurane. Isoflurane-induced burst-suppression pattern emerged during the second postnatal week and matured through the first postnatal month. Bursts in adolescent and adult rats were characterized by activation of entire cortical columns with a leading firing of infragranular neurons, and were triggered by principal and adjacent whiskers stimulation, and by auditory and visual stimuli, indicating an involvement of horizontal connections in their generation and horizontal spread. INTERPRETATION: The effects of isoflurane on cortical activity shift from total suppression of activity to burst-suppression pattern at the end of the first postnatal week. Developmental emergence of bursts likely involves a development of the intracortical short-and long-range connections. We hypothesize that complete suppression of cortical activity under isoflurane anesthesia during the first postnatal week may explain neuronal apoptosis stimulated by volatile anesthetics in the neonatal rats.

Quantification of cerebrospinal fluid transport across the cribriform plate into lymphatics in rats.

A major pathway by which cerebrospinal fluid (CSF) is removed from the cranium is transport through the cribriform plate in association with the olfactory nerves. CSF is then absorbed into lymphatics located in the submucosa of the olfactory epithelium (olfactory turbinates). In an attempt to provide a quantitative measure of this transport, 125I-human serum albumin (HSA) was injected into the lateral ventricles of adult Fisher 344 rats. The animals were killed at 10, 20, 30, 40, and 60 min after injection, and tissue samples, including blood (from heart puncture), skeletal muscle, spleen, liver, kidney, and tail were excised for radioactive assessment. The remains were frozen. To sample the olfactory turbinates, angled coronal tissue sections anterior to the cribriform plate were prepared from the frozen heads. The average concentration of 125I-HSA was higher in the middle olfactory turbinates than in any other tissue with peak concentrations achieved 30 min after injection. At this point, the recoveries of injected tracer (percent injected dose/g tissue) were 9.4% middle turbinates, 1.6% blood, 0.04% skeletal muscle, 0.2% spleen, 0.3% liver, 0.3% kidney, and 0.09% tail. The current belief that arachnoid projections are responsible for CSF drainage fails to explain some important issues related to the pathogenesis of CSF disorders. The rapid movement of the CSF tracer into the olfactory turbinates further supports a role for lymphatics in CSF absorption and provides the basis of a method to investigate the novel concept that diseases associated with the CSF system may involve impaired lymphatic CSF transport.

Development of cerebrospinal fluid absorption sites in the pig and rat: connections between the subarachnoid space and lymphatic vessels in the olfactory turbinates.

The textbook view that cerebrospinal fluid (CSF) absorption occurs mainly through the arachnoid granulations and villi is being challenged by quantitative and qualitative studies that support a major role for the lymphatic circulation in CSF transport. There are many potential sites at which lymphatics may gain access to CSF but the primary pathway involves the movement of CSF through the cribriform plate foramina in association with the olfactory nerves. Lymphatics encircle the nerve trunks on the extracranial surface of the cribriform plate and absorb CSF. However, the time during development in which the CSF compartment and extracranial lymphatic vessels connect anatomically is unclear. In this report, CSF-lymphatic connections were investigated using the silastic material Microfil and a soluble Evan's blue-protein complex in two species; one in which significant CSF synthesis by the choroid plexus begins before birth (pigs) and one in which CSF secretion is markedly up regulated within the first weeks after birth (rats). We examined a total of 46 pig fetuses at embryonic (E) day E80-81, E92, E101, E110 (birth at 114 days). In rats, we investigated a total of 115 animals at E21 (birth at 21 days), postnatal (P) day P1-P9, P12, P13, P15, P22, and adults. In pigs, CSF-lymphatic connections were observed in the prenatal period as early as E92. Before this time (E80-81 fetuses) CSF-lymphatic connections did not appear to exist. In rats, these associations were not obvious until about a week after birth. These data suggest that the ability of extracranial lymphatic vessels to absorb CSF develops around the time that significant volumes of CSF are being produced by the choroid plexus and further support an important role for lymphatic vessels in CSF transport.

Integration of the subarachnoid space and lymphatics: is it time to embrace a new concept of cerebrospinal fluid absorption?

In most tissues and organs, the lymphatic circulation is responsible for the removal of interstitial protein and fluid but the parenchyma of the brain and spinal cord is devoid of lymphatic vessels. On the other hand, the literature is filled with qualitative and quantitative evidence supporting a lymphatic function in cerebrospinal fluid (CSF) absorption. The experimental data seems to warrant a re-examination of CSF dynamics and consideration of a new conceptual foundation on which to base our understanding of disorders of the CSF system. The objective of this paper is to review the key studies pertaining to the role of the lymphatic system in CSF absorption.

Lymphatic vessels gain access to cerebrospinal fluid through unique association with olfactory nerves.

BACKGROUND: Physiological studies suggest that a major portion of cerebrospinal fluid (CSF) drainage is associated with transport along cranial and spinal nerves with absorption taking place into lymphatic vessels external to the central nervous system. Especially important is CSF transport through the cribriform plate in association with the olfactory nerves. This study examined the anatomical connections that link the CSF and extracranial lymphatics at the base of the brain. METHODS AND RESULTS: The contrast agent, Yellow Microfil, was infused into the cranial sub-arachnoid compartment of 2- to 7-day-old lambs postmortem. In some animals, blue Microfil was perfused into the carotid arteries. Yellow Microfil was observed in extensive lymphatic networks in the submucosa associated with the olfactory and respiratory epithelium. Since little of the contrast agent was present within the interstitium of the olfactory submucosa, there appeared to be direct continuity between the subarachnoid space, the perineurial spaces of the olfactory nerve fibers that penetrated the cribriform plate, and the lumens of the lymphatic vessels within the olfactory submucosa. Lymphatics encircled the olfactory nerves at the level of the emerging nerve rootlets (in many cases providing the outer limit of the perineurial space) and then dispersed freely in the submucosa at greater distance from the crib-riform plate. These vessels converged into larger collecting ducts that emptied into various lymph nodes in the head and neck. CONCLUSIONS: Lymphatic vessels gain access to the brain extracellular fluid (CSF) in an unusual anatomical association with the olfactory nerves external to the cranial vault. This study highlights the important role played by lymphatic vessels in CSF absorption.

Evidence of connections between cerebrospinal fluid and nasal lymphatic vessels in humans, non-human primates and other mammalian species.

BACKGROUND: The parenchyma of the brain does not contain lymphatics. Consequently, it has been assumed that arachnoid projections into the cranial venous system are responsible for cerebrospinal fluid (CSF) absorption. However, recent quantitative and qualitative evidence in sheep suggest that nasal lymphatics have the major role in CSF transport. Nonetheless, the applicability of this concept to other species, especially to humans has never been clarified. The purpose of this study was to compare the CSF and nasal lymph associations in human and non-human primates with those observed in other mammalian species. METHODS: Studies were performed in sheep, pigs, rabbits, rats, mice, monkeys and humans. Immediately after sacrifice (or up to 7 hours after death in humans), yellow Microfil was injected into the CSF compartment. The heads were cut in a sagittal plane. RESULTS: In the seven species examined, Microfil was observed primarily in the subarachnoid space around the olfactory bulbs and cribriform plate. The contrast agent followed the olfactory nerves and entered extensive lymphatic networks in the submucosa associated with the olfactory and respiratory epithelium. This is the first direct evidence of the association between the CSF and nasal lymph compartments in humans. CONCLUSIONS: The fact that the pattern of Microfil distribution was similar in all species tested, suggested that CSF absorption into nasal lymphatics is a characteristic feature of all mammals including humans. It is tempting to speculate that some disorders of the CSF system (hydrocephalus and idiopathic intracranial hypertension for example) may relate either directly or indirectly to a lymphatic CSF absorption deficit.