Addressing Spaceflight Biology through the Lens of a Histologist–Embryologist

Round 1

Reviewer 1 Report

Theotokis and colleagues are providing a well-presented and highly informative and extensively scrutinized with 156 references review from available experimental data on how the outer space conditions can allow or sustain file, with an emphasis on the early development stages.

It gives a fascinating and literally ahead of its time  topic of discussion.

Author Response

We thank Reviewer 1 for the diligent scrutiny of the submitted manuscript and appreciate the positive feedback.

Reviewer 2 Report

The manuscript by Theotokis et al is a well written and very interesting work. Literature review has been rigorously carried out. I believe addressing the following things will enrich the work:

1. Discuss about the aspect of stress and autophagy in this regard of histology development during  spaceflight biology

2. Also elaborate the limitation of this review in the discussion section and the further scope of this work from a translational viewpoint.

Author Response

We acknowledge reviewer’s assessment thus we were encouraged to include available information about the implication of stress and autophagy during spaceflight biology as a last paragraph in section four of the revised manuscript.

More specifically for 1, the following text has been added:

“This organ-based approach can be extended further and include tissues that are metabolically active since space environment can afflict vital biochemical reactions with the involvement of oxidative stress, autophagy and damage [1]. For instance, Blaber and colleagues showed that exposure to space conditions for 13.5 days resulted in elevated reactive oxygen species (ROS) and activated autophagy, including the proteasome system, in the mouse liver [2]. Ramifications of this exposure revealed hepatocyte senescence triggered by a buildup of oxidized proteins and profound mitochondrial dysfunction, a common denominator when the cell homeostasis is being perturbed [2, 3]. Interestingly, space radiation and microgravity can have opposite effects on specific molecular pathways; radiation, as shown with transcriptomic analysis, revealed to inhibit autophagy thereby promoting an aged-like phenotype whilst microgravity a proliferative phenotype [4]. Such nuances should be taken into account to create a tailored protection that safeguard the health of astronauts.”

[1] Goodwin, T.J. and M. Christofidou-Solomidou, Oxidative Stress and Space Biology: An Organ-Based Approach. Int J Mol Sci, 2018. 19(4).

[2] Blaber, E.A., M.J. Pecaut, and K.R. Jonscher, Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver. Int J Mol Sci, 2017. 18(10).

[3] Nguyen, H.P., et al., The effects of real and simulated microgravity on cellular mitochondrial function. NPJ Microgravity, 2021. 7(1): p. 44.

[4] Barravecchia, I., et al., Microgravity and space radiation inhibit autophagy in human capillary endothelial cells, through either opposite or synergistic effects on specific molecular pathways. Cell Mol Life Sci, 2021. 79(1): p. 28.

Furthermore, in accordance with the second point raised, although we referenced a couple of pros and cons of the review (such as “gaps in current gut microbiome knowledge” and “space medicine”) we modified the discussion by highlighting translational scopes and concomitant limitations, tailored to human aspects.

Particularly for 2, the following text has been added:

“From a translational viewpoint, along with the rise of “-omics” techniques, advanced research tools are highlighted and open up new horizons in quick, accurate and efficient tests with practical applications in monitoring real-time the genome, transcriptome or proteome of a recruited astronaut.

However, someone can argue that limitations accompany the narrative of this trajectory. Ethical reasons do not allow extensive manipulations, currently prohibited in humans, and even if this was permitted it would literally take years of monitoring to identify the experimental effect on any tissue or organ system.”

Again, we highly appreciate the reviewer’s valuable suggestions towards the enrichment of our work.

Reviewer 3 Report

Dear authors, 

This is a very interesting report describing different embryological outcomes on micro-and hyper gravity settings which is very well written and reads easily. 

I have the following minor comments:

Title: is there another word of choice for the last word? histologist-embryologist sounds a bit weird. Histo-embryologist? Is the word Histology necessary?

Line 53. Why is there specifically focused on proprioception and the vestibular system within this review. Is this by its role of gravity that governs organogenesis?

Line 68. Perhaps include the embryological layers (ecto-meso-endoderm)

Line 80. Begin the new sentence with “The utricle is responding….

Line 105-106. Why is it especially the saccule that was altered? Is this due to excessive vertical movements during flight?

Line 129-130; perhaps I should know this, but what are righting reactions?

line 131-132; Histological analysis of developing rat brain specimens of an 11-day spaceflight, suggested variations in the choroid plexus. Can you elaborate a bit more on this? Why and how does it change? If known.

Line 173-174; is it known if the microgravity alone does affect the down-regulated type I collagen and osteocalcin gene expression or could this also be due to radiation factors? A common comment, many studies are performed under micro-hypergravity settings, does the radiation levels also have effects? In other words, is this parameter simply left out in these experiments?

The same for line 185-188. Is it true that microgravity implies that much genetic variations? What is exactly meant by “profound differences”? Are these “differences” causing any morphological changes?  

Table. Quite strange for me, but I am a novel in space embryology, is that the early embryological processes (which are really important) seem to be unaffected by the experiments performed.

Line 196, please insert, beneath the neural tube

Line 200. Insert: The fetus

Line 264. Does it matter who wrote the manuscript?

 

Author Response

Title: is there another word of choice for the last word? histologist-embryologist sounds a bit weird. Histo-embryologist? Is the word Histology necessary?

All authors Profess the discipline of Histology and Embryology in relevant University departments namely Laboratory of Histology and Embryology in Aristotle University of Thessaloniki and Laboratory of Histology and Embryology in Democritus University of Thrace. Thus, we believe that the word Histology is essential since we address the aspects of space biology in the scope of such principle. Lastly, as far as we know there is no reference to Histo-embryologist in any online term-based search or applicable field apropos of Histology and Embryology literature.

Line 53. Why is there specifically focused on proprioception and the vestibular system within this review. Is this by its role of gravity that governs organogenesis?

The vestibular system is responsible for the body’s equilibrium, it maintains balance and provides awareness of the body’s spatial orientation. The sensory part is located in the inner and detect not the motion itself, but changes in the rate of motion, specifically acceleration or deceleration i.e. those produced by altered gravity. Then, vestibular information disseminates in diverse brain areas such as brainstem and cerebellum and other proprioceptive gravity-sensing neurons. Experimental data have shown that animals exposed to altered gravity conditions during critical developmental stages of organogenesis displayed miscellaneous perturbations in CNS and related structures, hence their dominant role in this review.

Line 68. Perhaps include the embryological layers (ecto-meso-endoderm)

The three primary germ layers have been named.

Line 80. Begin the new sentence with “The utricle is responding….

“The” has been added.

Line 105-106. Why is it especially the saccule that was altered? Is this due to excessive vertical movements during flight?

Saccule is primarily responding to vertical acceleration e.g., hair cells react to a gravitational force. It is therefore, part of the vestibular adaptation to either those long- or short-term exposure to extremely rapid changes in gravity, such as those that occur during shuttle launch and landing, affect the vestibulocerebellar system and more so the saccular nerve afferents which arise mainly from the inferior shank-like part of the macula of saccule.

Line 129-130; perhaps I should know this, but what are righting reactions?

The righting reaction or reflex, also known as the labyrinthine righting reflex, is a reflex that corrects the orientation of the body when it is taken out of its normal upright position. It is initiated by the vestibular system, which detects that the body is not erect and causes the head to move back into position as the rest of the body follows. The perception of head movement involves the body sensing linear acceleration or the force of gravity through the otoliths, and angular acceleration through the semicircular canals. The reflex uses a combination of visual system inputs, vestibular inputs, and somatosensory inputs to make postural adjustments when the body becomes displaced from its normal vertical position.

Line 131-132; Histological analysis of developing rat brain specimens of an 11-day spaceflight, suggested variations in the choroid plexus. Can you elaborate a bit more on this? Why and how does it change? If known.

Mani-Ponset and colleagues compared the choroid plexuses of developing normal rats with those of rat fetuses that had been growing in space for 11 days, from gestational day 9 to day 20. Immunocytochemistry was used to show that the distribution of ezrin (a cytoskeletal protein involved in apical cell differentiation in choroid plexus) and carbonic anhydrase II (which is partly involved in the cerebrospinal fluid production) were the two primary factors modified in fetuses grown in space. Results showed that ezrin was strongly found in the cytoplasm of ependymal cells that lining the choroid plexuses of fetuses growing in space and weakly expressed in the apical cytoplasmic membrane of the choroid plexuses from the fourth ventricle. These modifications showed that the rat fetal brain's choroid plexus exhibits delayed development in a microgravitational environment. As opposed to ground control fetuses, strong immunoreactions to anti-carbonic anhydrase II antibodies revealed that this enzyme is far more prevalent in rats grown in space.

More information about this experiment has been included in the relevant segment, as per suggestion of the Reviewer.

Line 173-174; is it known if the microgravity alone does affect the down-regulated type I collagen and osteocalcin gene expression or could this also be due to radiation factors? A common comment, many studies are performed under micro-hypergravity settings, does the radiation levels also have effects? In other words, is this parameter simply left out in these experiments?

Landis and and colleagues presented their findings of type I collagen and osteocalcin gene expression suggesting that osteoblasts subjected to flight followed a slower progression toward a differentiated function. In their own words, quoting “The summary of data indicates that spaceflight, including microgravity exposure, demonstrably affects bone cells by down-regulating type I collagen and osteocalcin gene expression and thereby inhibiting expression of the osteogenic phenotype notably by committed osteoblasts. The information is important for insight into the response of bone cells to changes of gravity and of force in general.” Simply put, they did not take into consideration space radiation, whatsoever.

The same for line 185-188. Is it true that microgravity implies that much genetic variations? What is exactly meant by “profound differences”? Are these “differences” causing any morphological changes? 

Wnorowski and colleagues published this excellent paper in Stem Cell Reports in 2019 in an effort to understand the cellular and physiological processes influenced by microgravity specifically in human heart cells. Employing a multi-disciplinary approach and access to the ISS, they utilized hiPSC-CMs (representing the first use of hiPSC technology in space) to provide insights into the effects of microgravity on human cardiac physiology, cell structure, and gene expression. In this work, they presented videos of altered hiPSC-CM contraction and calcium handling in vitro cultures (acknowledging morphological changes), altered hiPSC-CM RNA expression profiles by RNA sequencing, laying the groundwork for future studies to more accurately model cardiac and human physiology.

Table. Quite strange for me, but I am a novel in space embryology, is that the early embryological processes (which are really important) seem to be unaffected by the experiments performed.

Based on the data provided by the up-to-date research presented in this review, it seems indeed that very early developmental events can somehow counteract the perturbed, space-like conditions, be that either altered gravity or radiation. These undifferentiated, pluri- or multipotent cells (up to the gastrula) have the capacity to initiate amendatory mechanisms to offset potential damage whilst post-mitotic cells simply cannot. Additionally, as demonstrated in the fertility and reproduction repercussions section, species complexity is always a catalytic factor to potential preventive mechanisms. For example, its known that in humans, major developmental defects, also referred to as major congenital anomalies, arise during organogenesis (4-8^th week of gestation) and if they are severe like heart or neural tube defects they most probably end in intrauterine miscarriage (spontaneous abortion) or stillbirth. However, it is of vital importance to acknowledge that there is not a single experiment taking into account the chronicity of a potential damage simple because it could not be adequately monitored as most of these experiments are short-termed. Taken together, properly recorded experiments in bona fide species will eventually give us more answers as to what the true impact of space conditions are in a complex organism, including humans.

Line 196, please insert, beneath the neural tube

“the” has been added.

Line 200. Insert: The fetus

“The” has been added.

Line 264. Does it matter who wrote the manuscript?

It is indeed not a prerequisite so we excluded it from the conclusion section.

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All minor comments have been addressed and changes are now reflected in the revised manuscript.

All in all, we very much appreciate the encouraging, critical and constructive comments on this manuscript by Reviewer 3. The comments have been very thorough and useful in improving the manuscript.