Abstract
Clinostats and Random Positioning Machines (RPMs) are valuable devices for microgravity simulations in order to study fundamental gravity-dependent mechanisms on ground and in preparation for space flights. Both devices have different modes of operation, which have to be carefully considered and comprehensively discussed with respect to their potential impact on the quality of (simulated) microgravity. Here, we used the well-studied oxidative burst reaction of the immune cell (macrophage) model system, in order to compare clinorotation with random positioning. The RPM was used in a clinorotation mode, rotating the sample with 60 rpm around the horizontal axis and in the “random speed mode” (2-10 rpm) with and without random direction, thus, two axes rotating either bi- or unidirectionally. The production of Reactive Oxygen Species (ROS) during oxidative burst of the macrophages was visualized by the luminescence luminol assay. During clinorotation the cells responded with a reduction of the ROS production which is fully in line with earlier studies (clinostat, parabolic flight). In contrast, the exposure to the two random positioning modes showed that the oxidative burst response during RPM-exposure differs significantly from that observed during clinorotation, i.e. a jittering, more variant form of ROS production. We can conclude from our work, that the RPM is not suitable in its real random mode (with and without random direction; 2-10 rpm) to simulate the conditions of microgravity for the chosen system. We recommend that investigators using microgravity simulators should carefully choose the device and mode of operation specifically for their cellular system of interest.
Highlights
To maintain human health during space flight, great effort has been made in the past years to study the effects of altered gravity on the immune system (Choukèr and Ullrich 2016)
In order to proof that the Random Positioning Machines (RPMs) is usable as a clinostat, we rotated the inner frame of the RPM with 60 rpm around its horizontal axis with the cell cuvette in the center of rotation, comparable to common 2D clinostats
We performed control experiments using phosphate buffered saline (PBS) as a negative control to see if vibrations etc. can induce a stress-related Reactive Oxygen Species (ROS) production: The control experiment showed clearly that no ROS production was occurring
Summary
To maintain human health during space flight, great effort has been made in the past years to study the effects of altered gravity on the immune system (Choukèr and Ullrich 2016). In preparation of experiments to be performed in microgravity and to increase the knowledge under normal gravity conditions, scientists design microgravity simulators (ground-based facilities) on which they expose their model system and study their question of interest. Space immunology studies have focused on the influence of altered gravity on macrophages due to their role in immune response. Reactive Oxygen Species (ROS) are produced during their oxidative burst reaction, as the immunological firstline of defense and this is an measurable parameter of the immune response, perfectly suited for a space experiment. Several investigators have used chemoluminescence assays to quantify the amount of produced ROS in different cell types, in order to ascertain the influence of gravity on this crucial host-defense mechanism. Several investigators have used chemoluminescence assays to quantify the amount of produced ROS in different cell types, in order to ascertain the influence of gravity on this crucial host-defense mechanism. Huber et al (2006) established the methodology of luminol measurement in rodent alveolar macrophages, whereas Unruh et al (2016) used the same assay to measure the ROS production in blue mussel hemocytes
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