Molecular impact of launch related dynamic vibrations and static hypergravity in planarians

Abstract

Although many examples of simulated and real microgravity demonstrating their profound effect on biological systems are described in literature, few reports deal with hypergravity and vibration effects, the levels of which are severely increased during the launch preceding the desired microgravity period. Here, we used planarians, flatworms that can regenerate any body part in a few days. Planarians are an ideal model to study the impact of launch-related hypergravity and vibration during a regenerative process in a "whole animal" context. Therefore, planarians were subjected to 8.5 minutes of 4 g hypergravity (i.e. a human-rated launch level) in the Large Diameter Centrifuge (LDC) and/or to vibrations (20-2000 Hz, 11.3 G_rms) simulating the conditions of a standard rocket launch. The transcriptional levels of genes (erg-1, runt-1, fos, jnk, and yki) related with the early stress response were quantified through qPCR. The results show that early response genes are severely deregulated after static and dynamic loads but more so after a combined exposure of dynamic (vibration) and static (hypergravity) loads, more closely simulating real launch exposure profiles. Importantly, at least four days after the exposure, the transcriptional levels of those genes are still deregulated. Our results highlight the deep impact that short exposures to hypergravity and vibration have in organisms, and thus the implications that space flight launch could have. These phenomena should be taken into account when planning for well-controlled microgravity studies.

Keywords: Biophysics; Developmental biology.

Fig. 1. Experimental design.

a Animals were amputated the day before of the exposure to simulated launch loads (day −1). At day 0, animals were loaded into T25 flasks and the experiment was initiated. Immediately after the exposure to hypergravity, vibration or both, RNA was extracted from half of the animals (0 h). Four days after the exposure, the RNA of the rest of the animals was extracted (4d). The regenerated structures were imaged at 4 days after the exposure. b The four experimental groups of animals.

Fig. 2. Vibration system.

a Inside of the LDC gondola the vibration system consisting of the actuator, the amplifier and the data acquisition system was mounted in one gondola. The cooling system was placed in a second gondola (not visible in the image). b Detail of the top part of the actual actuator shown here with a T25 flask attached which contained five animals. The flasks were completely filled with planarian artificial medium, leaving no air bubbles. During simulated launch exposures the animals were at ambient conditions. During other periods the temperatures were 20–22 °C.

Fig. 3. In vivo phenotype of animals exposed to hypergravity and/or vibration 4 days after the exposure.

Animals in all groups were able to regenerate the head (the eyes are indicated with arrow heads). No alterations are observed between the animals from the four different conditions. n ≥ 10. Scale bar = 1 mm.

Fig. 4. qPCR analysis of planarians exposed to vibration or/and 4 g hypergravity compared to 1 g static controls directly after exposure or at 4 days post exposure.

The mRNA levels of the indicated genes are analyzed with respect to the levels of ura4. Values represent the means of at least two biological replicates each one with five animals. Error bars represent standard deviation. Data was analyzed by two-sided Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 5. The six red-colored swing-out and one central gondola from the 8-meter diameter Large Diameter Centrifuge (LDC).

a Both centrifuges are currently located at the technology center (ESTEC) from the European Space Agency ESA) in Noordwijk, the Netherlands. b The Acceleration Spectral Density (ASD) of the random vibration test specification profile from the 20 to 2000 Hz range as used for exposing planarians to a simulated launch load. This profile is based on the minimum workmanship levels for random vibration testing. The equipment set-up was divided over two gondolas where the actual actuator was placed in the outer gondola (see for further details Fig. 2).