Hypergravity attenuates Reactivity in Primary Murine Astrocytes

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning, and memory as well as motion. Hence, disturbances of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we aim to understand the impact of gravitational loading induced by hypergravity to potentially modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the in vivo reactivity phenotype on custom-built centrifuges for cultivation as well as for live-cell imaging under hypergravity conditions in a physiological range (2g and 10g). We revealed spreading rates, migration velocities, and stellation to be diminished under 2g hypergravity. In contrast, proliferation and apoptosis rates were not affected. In particular, hypergravity attenuated reactivity induction. We observed cytoskeletal remodeling of actin filaments and microtubules under hypergravity. Hence, the reorganization of these key elements of cell structure demonstrates that fundamental mechanisms on shape and mobility of astrocytes are affected due to altered gravity conditions. In future experiments, potential target molecules for pharmacological interventions that attenuate astrocytic reactivity will be investigated. The ultimate goal is to enhance neuronal regeneration for novel therapeutic approache

Hypergravity attenuates Reactivity in Primary Murine Astrocytes

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning and memory as well as motion. Alterations or even disruptions of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we use increased gravitational (mechanical) loading induced by hypergravity to identify a potential method to modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the reactivity phenotype in vivo on custom-built centrifuges for cultivation as well as for livecell imaging under hypergravity conditions in a physiological range (2g and 10g). This resulted in significant changes to astrocyte morphology, behavior and reactivity phenotypes, with the ultimate goal being to enhance neuronal regeneration for novel therapeutic approaches

Effects of long-term immobilisation on endomysium of the soleus muscle in humans

Muscle fibres atrophy during conditions of disuse. Whilst animal data suggest an increase in endomysium content with disuse, that information is not available for humans. We hypothesised that endomysium content increases during immobilisation. To test this hypothesis, biopsy samples of the soleus muscle obtained from 21 volunteers who underwent 60 days of bed rest were analysed using immunofluorescence-labelled laminin γ-1 to delineate individual muscle fibres as well as the endomysium space. The endomysium-to-fibre-area ratio (EFAr, as a percentage) was assessed as a measure related to stiffness, and the endomysium-tofibre-number ratio (EFNr) was calculated to determine whether any increase in EFAr was absolute, or could be attributed to muscle fibre shrinkage. As expected, we found muscle fibre atrophy (P = 0.0031) that amounted to shrinkage by 16.6% (SD 28.2%) on day 55 of bed rest. ENAr increased on day 55 of bed rest (P < 0.001). However, when analysing EFNr, no effect of bed rest was found (P = 0.62). These results demonstrate that an increase in EFAr is likely to be a direct effect of muscle fibre atrophy. Based on the assumption that the total number of muscle fibres remains unchanged during 55 days of bed rest, this implies that the absolute amount of connective tissue in the soleus muscle remained unchanged. The increased relative endomysium content, however, could be functionally related to an increase in muscle stiffness

Microelectrode Array Electrophysiological Recording of Neuronal Network Activity during a Short-Term Microgravity Phase

During spaceflight, humans are subjected to a variety of environmental factors which deviate from Earth conditions. Especially the lack of gravity poses a big challenge to the human body and has been identified as a major trigger of many detrimental effects observed in returning astronauts but also in participants of spaceflight-analog studies. Structural alterations within the brain as well as declines in cognitive performance have been reported, which has brought the topic of brain health under microgravity into the focus of space research. However, the physiological mechanisms underlying these observations remain elusive. Every aspect of human cognition, behavior and psychomotor function is processed by the brain based on electro-chemical signals of billions of neurons, which relay information via neuronal networks throughout the body. Alterations in neuronal activity are the main cause of a variety of mental disorders and changed neuronal transmission may also lead to diminished human performance in space. Thus, understanding the functioning of these fundamental processes under the influence of altered gravity conditions on a cellular level is of high importance for any manned space mission. Previous electrophysiological experiments using patch clamp have shown that propagation velocity of action potentials (APs) is dependent on gravity. With this project, we aim to advance the electrophysiological approach from a single-cell level to a complex network level by employing Microelectrode array (MEA) technology. MEAs feature the advantage of real-time electrophysiological recording of a complex and mature neuronal network in vitro, without the need for invasive patch clamp insertion into cells. Using a custom-built pressure chamber, we were able to integrate and conduct our experiment on the ZARM Drop Tower platform, exposing the entire system to 4.7 s of high-quality microgravity (10-6 to 10-5 x g0). With this setup we were able to evaluate the functional activity patterns of iPSC-derived neuronal networks subjected to microgravity, while keeping them under controlled and stable temperature and pressure conditions. Activity data was acquired constantly - immediately before the drop, during the free-fall (microgravity) phase and during a subsequent post-drop recording phase. For neuronal activity analysis the action potential frequency in each experiment phase was calculated for the single electrodes. We found that during the 4.7 s lasting microgravity phase the mean action potential frequency across the neuronal networks was significantly elevated. Additionally, electrical activity readapted back to baseline level within 10 minutes of post-drop recordings. Our preliminary data shows that real-time, electrophysiological recording of neuronal network activity based on MEA technology is possible under altered gravity conditions and that differences in activity can be detected already in very short time frames in the second range. Furthermore, the observation that microgravity has an effect on the electrophysiological activity of neuronal networks is in line with previously published findings in single neurons and poses further questions with regards to astronaut brain health on manned space missions. The MEA payload system was approved for autonomous recording of redundant cellular electrophysiological data in the Drop Tower. It will be applied on other microgravity platforms such as sounding rockets and parabolic flights and thus increased experimental time. Apart from neurons, various other electrically active cellular systems such as myocytes or myotubes could be examined using this hardware

Effects of long‐term immobilisation on endomysium of the soleus muscle in humans

New findings: What is the central question of this study? While muscle fibre atrophy in response to immobilisation has been extensively examined, intramuscular connective tissue, particularly endomysium, has been largely neglected: does endomysium content of the soleus muscle increase during bed rest? What is the main finding and its importance? Absolute endomysium content did not change, and previous studies reporting an increase are explicable by muscle fibre atrophy. It must be expected that even a relative connective tissue accumulation will lead to an increase in muscle stiffness. Abstract: Muscle fibres atrophy during conditions of disuse. Whilst animal data suggest an increase in endomysium content with disuse, that information is not available for humans. We hypothesised that endomysium content increases during immobilisation. To test this hypothesis, biopsy samples of the soleus muscle obtained from 21 volunteers who underwent 60 days of bed rest were analysed using immunofluorescence-labelled laminin γ-1 to delineate individual muscle fibres as well as the endomysium space. The endomysium-to-fibre-area ratio (EFAr, as a percentage) was assessed as a measure related to stiffness, and the endomysium-to-fibre-number ratio (EFNr) was calculated to determine whether any increase in EFAr was absolute, or could be attributed to muscle fibre shrinkage. As expected, we found muscle fibre atrophy (P = 0.0031) that amounted to shrinkage by 16.6% (SD 28.2%) on day 55 of bed rest. ENAr increased on day 55 of bed rest (P < 0.001). However, when analysing EFNr, no effect of bed rest was found (P = 0.62). These results demonstrate that an increase in EFAr is likely to be a direct effect of muscle fibre atrophy. Based on the assumption that the total number of muscle fibres remains unchanged during 55 days of bed rest, this implies that the absolute amount of connective tissue in the soleus muscle remained unchanged. The increased relative endomysium content, however, could be functionally related to an increase in muscle stiffness

Neuronal regeneration of induced by exposure to hypergravity

Neuronal activity is the key modulator of nearly every aspect of behavior. Cognition, learning and memory as well as motion tasks are based on by neuronal transmission. Alterations or even disruptions of the transmission of synaptic signals are the main cause of many neurological disorders. A fundamental concern in the treatment of most neuropathologies is the re-integration of synaptic inputs to previously impaired neuronal networks that were affected e.g. by spinal cord injury, head trauma or other types of lesions to the nervous system. The regeneration of injured neuronal fibers is nearly completely inhibited by the scar tissue that forms upon damage to the surrounding tissue. Therefore regeneration of axonal projections through the lesion site is nearly impossible resulting in sustained damage of the tissue. To better understand the regeneration of nervous tissue, primary murine hippocampal neurons are used as a model closely related to human neural tissue. The influential role of increased gravity on neuronal development will be investigated by analyses of the different developmental stages, including neuritogenesis, neuronal polarization and further in maturation processes like synaptogenesis or synaptic integration in neural networks. Each of these developmental steps could play a role at a certain level in neuronal regeneration in vivo. Exposure of primary neurons to hypergravity conditions (2g) induced an increased number of neurites (about 30%) and longer projections (about 20%) compared to the control at 1g. At later stages of development mature synaptic contacts were formed under hypergravity conditions. In addition, the formation of glial scar tissues could be inhibited by exposure to hypergravity as well. Primary cultured astrocytes, the major cell type involved in glial scar formations, showed decreased lamellipodial protrusions and a deficit in cell spreading due to exposure to hypergravity conditions. Our results indicate hypergravity as an innovative measure to artificially stabilize cytoskeletal components of neuronal cells, enabling them to counteract the restricted process of neurite outgrowth and therefore enhance axon regeneration in previously traumatized networks

Induktion von neuronaler Regeneration durch Hypergravitation

Einleitung: Eine uneingeschränkte Funktion von neuronalen Zellen ist unerlässlich für jeden Aspekt des menschlichen Verhaltens. Wahrnehmung, Erinnerung, Lernen sowie jegliche Art von Bewegung sind grundsätzlich von der Aktivität unseres Nervensystems abhängig. Eine Ursache oder Konsequenz der meisten neurologischen Erkrankungen sind daher Veränderungen oder Störungen der synaptischen Aktivität. Um wirksame Behandlungen von neurologischen Störungen zu entwickeln, müssen synaptische Signale wieder in das zuvor verletzte neuronale Netzwerk integriert werden. Besonders nach schweren Läsionen, wie z.B. nach Traumata und Rückenmarksverletzungen, sowie durch Beeinflussung von Tumorgewebe oder epileptischen Anfällen wird vermehrt Narbengewebe gebildet, welches die Regeneration von neuronalen Ausläufern und damit synaptischen Signalen in das verletzte Gewebe nahezu vollständig inhibiert. Diese Art von Verletzung kann daher zu irreparablen Schäden der mentalen oder physischen Leistungsfähigkeit von Patienten führen. Fragestellung: Wir streben an, den sehr ineffizienten Vorgang der neuronalen Regeneration nach Verletzungen von Nervengewebe durch die Exposition in Hypergravitation zu induzieren. Unsere Hypothese ist, dass artifiziell erhöhte Schwerkraft eine potentiell sehr innovative und wirkungsvolle Methode darstellen könnte, um Komponenten des neuronalen Zytoskelettes zu stabilisieren, was weiterhin dazu führen sollte, dass die Projektionen der Nervenzellen der Inhibition des neuronalen Narbengewebes entgegenwirken und in gesteigertem Maße wachsen (regenerieren) können. Daher sollten die Ausläufer unter Einfluss von Hypergravitation viel ausgeprägter in der Lage sein auszuwachsen und sich anschließend neu in geschädigtes Gewebe zu integrieren. Weiterhin soll die neuronale Entwicklung und Aktivität bei Exposition mit ionisierender Strahlung untersucht werden, um Strahlung als Risikofaktor für neurodegenerative Erkrankungen z.B. in der bemannten Raumfahrt zu bewerten. Methodik: In der vorliegenden Studie werden primäre murine hippokampale Neuronen eingesetzt, welche ein nah-verwandtes Modell-System für humane Nervenzellen darstellen. Die neuronale Entwicklung unter dem Einfluss von Hypergravitation (2g) und Strahlenexposition wird bei allen Entwicklungsstadien im Vergleich zu Kontrollen (1g, unbestrahlt) untersucht, wie z.B. das Neuritenwachstum, die Polarisation, die Synaptogenese, sowie abschließend die Integration in ein maturiertes, funktionelles neuronales Netzwerk. Ergebnisse: Die Exposition von primären Neuronen an erhöhte Gravitation (2g) induzierte ein gesteigertes Auswachsen initialer Neuriten (ca. 30%), sowie ein erhöhtes Neuriten-Wachstum (Elongation) (ca. 20%) im Vergleich zu Kontrollen bei 1g. In späteren Entwicklungsstadien wurden trotz potentiellen Veränderungen des neuronalen Zytokslettes maturierte synaptische Kontakte ausgebildet. Weiterhin wurden primäre Astrozyten (Glia-Zellen, die neuronales Narbengewebe nach Verletzungen bilden) durch Hypergravitation in ihrem Wachstum und ihrer Ausbreitung (Narbenbildung) gehemmt. Diese Beobachtungen sind in großer Übereinstimmung mit einem stabilisierten Tubulin- und einem destabilisierten Aktin-Zytoskelett. Schlussfolgerungen: Unsere Ergebnisse belegen, dass neuronale Regeneration durch den Einfluss von Hypergravitation in primären Neuronen induziert und das Wachstum von primären Astrozyten (Glia-Zellen) durch die Kultivierung unter Hypergravitationsbedingungen gehemmt wird. Dieser Ansatz kann für weitere Studien angewandt werden, um die grundlegenden Mechanismen aufzuklären und die Effizienz neuronaler Regenerationsprozesse steigern zu können