MitoQ as an antenatal antioxidant treatment improves markers of lung maturation in healthy and hypoxic pregnancy

Abstract Chronic fetal hypoxaemia is a common pregnancy complication that increases the risk of infants experiencing respiratory complications at birth. In turn, chronic fetal hypoxaemia promotes oxidative stress, and maternal antioxidant therapy in animal models of hypoxic pregnancy has proven to be protective with regards to fetal growth and cardiovascular development. However, whether antenatal antioxidant therapy confers any benefit on lung development in complicated pregnancies has not yet been investigated. Here, we tested the hypothesis that maternal antenatal treatment with MitoQ will protect the developing lung in hypoxic pregnancy in sheep, a species with similar fetal lung developmental milestones as humans. Maternal treatment with MitoQ during late gestation promoted fetal pulmonary surfactant maturation and an increase in the expression of lung mitochondrial complexes III and V independent of oxygenation. Maternal treatment with MitoQ in hypoxic pregnancy also increased the expression of genes regulating liquid reabsorption in the fetal lung. These data support the hypothesis tested and suggest that MitoQ as an antenatal targeted antioxidant treatment may improve lung maturation in the late gestation fetus. Key points Chronic fetal hypoxaemia promotes oxidative stress, and maternal antioxidant therapy in hypoxic pregnancy has proven to be protective with regards to fetal growth and cardiovascular development. MitoQ is a targeted antioxidant that uses the cell and the mitochondrial membrane potential to accumulate within the mitochondria. Treatment of healthy or hypoxic pregnancy with MitoQ, increases the expression of key molecules involved in surfactant maturation, lung liquid reabsorption and in mitochondrial proteins driving ATP synthesis in the fetal sheep lung. There were no detrimental effects of MitoQ treatment alone on the molecular components measured in the present study, suggesting that maternal antioxidant treatment has no effect on other components of normal maturation of the surfactant system.

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EDITOR COMMENTS
Reviewing Editor: The clear strength of the present study is the model utilised to induce hypoxic pregnancy and the ability to assess the impact of MitoQ on multiple outcomes, as the Cambridge group have previously published.The main weakness however is that no data on pulmonary function or structure are provided to support the physiological impact of the changes in mRNA and protein expression.The manuscript would be substantially improved and more likely to proceed if the authors are able to expand their analysis and provide direct evidence of the effects of MitoQ on lung maturation or function.The reviewers have raised several additional concerns that need to be addressed.
Attention is needed to include exact p values in figures, text and tables.

Other points
Line 314 -please rephrase to acknowledge no effect on the components examined rather than suggesting all components.
As noted by the reviewers, blots are required to be included.

REFEREE COMMENTS
Referee #1: Thankyou for the opportunity to review this timely manuscripts.Mitochondrial based therapies and antioxidants are increasingly being recognized as the new frontier in neonatal medicine.As such this is a timely and well conducted study.
A generally well detailed methods section.Could the authors please provide additional detail on where lung samples were obtained from (i.e.left or right lobes) and if the sampling sight was standardized across all animals.I commend the authors on their extensive testing of the reference genes but note that the RT-PCR section does not currently meet MIQE guidelines (as stated by the authors).To meet the essential MIQE checks I request that the authors include the number of samples analyzed by RT-PCR in each group, and in a table the primer sequences and reaction conditions.For general readability the primary antibody details could be included as a supplementary Table .For non sheep researchers it would be useful for the authors to include reference to what the full gestation period for the species would be (i.e.~145d).
The authors are to be commended for their thoroughness regarding the number of proteins or genes that were measured.I do feel however that some tweaking may improve the impact of the results.For example, in the graphs it would be useful where both a western blot and PCR result is available for the same target to always have it in the order protein-PCR (there are swaps between figure 2 and 3 for example).I also found the use of the 'over' comparison in Figure 2A,B,C,E etc to be confusing.In terms of the post-hoc analysis which comparison groups do these represent?It would be useful if the same formatting used to compare the normaxia vs hypoxia for saline or MitoQ was used to show the significant comparisons between all blue or all red comparisons.Not essential but would have been useful to demonstrate the impact of mitoQ treatment on cell composition within the lung.Are the increases in SP-B due to more AT2 cells, or because the AT2 cells are induced by hypoxia and/or mitoQ to produce more protein?This could be included as a limitation within the discussion.
I am a little hesitant by the authors use of the the statement "Here we show maturational effects on the fetal lung of a mitochondria-targeted antioxidant therapy..." you were able to detect evidence of maturation such as SP-B, C, D production etc in all groups treated with saline, and without histological evidence I'm not sure there is a strong evidence that lung maturation was delayed by hypoxia.Rather much of the evidence is that mitoQ increases cellular function of some targets.The authors also alude to this in para 1, page 6.

Referee #2:
Lock M and colleagues investigated in their study MitoQ as a possible preventive strategy for chronic fetal hypoxemia.To this end, ewes were treated with MitoQ during late gestation and offspring's lungs were analysed with a focus on pulmonary surfactant maturation, mitochondrial respiration, ATP synthesis, and lung liquid reabsorption.The data demonstrate significant effects of hypoxia on lung maturation.MitoQ, however, showed only a mild preventive effect.
The research question is of great interest, the topic clinically very relevant, and the model appropriate.However, the data do not show marked effect of MitoQ on lung maturation after chronic fetal hypoxia.Overall, some additional aspects should be considered to strengthen the conclusion made by the authors: 1) The title implies a stronger effect of MitoQ than the data ultimately show.The authors might consider to rephrase.
2) The introduction is somewhat lengthy and could be more focused.There are some sections that belong in the discussion rather than in the introduction.This could certainly be improved to emphasize the focus of the study.
3) Animal protocol approval number should be included.Why did the only include male fetuses in this study?Did they expect sex-specific differences/response to hypoxia and/ MitoQ? 4) I am uncertain what the purpose of the surgical intervention was?The overall aim was to combine chronic hypoxia with MitoQ treatment.Why was the operation performed at day 100 of gestation?5) Was the pharmacological dose of MitoQ tested?Were markers of oxidative stress as a readout for sufficient MitoQ effect
6) The authors only show the protein quantification.The blots with bands for the respective protein and loading should be shown.

7) Was the wet lung/dry lung ratio measured as an indicator of impaired lung liquid absorption?
8) The authors show effects of hypoxia/MitoQ on the gene expression of surfactant proteins; was the protein expression assessed as well?9) Were hypoxia-induced changes in lung structure attenuated by MitoQ?10) Since the authors focus on surfactant proteins, it would be interesting to see if fraction of AT2 is changed by hypoxia+/-MitoQ.

"MitoQ as an antenatal antioxidant treatment improves lung development in healthy and hypoxic pregnancy"
Manuscript: JP-RP-2023-284786 Dear Prof Bennet, We thank the reviewing editor and reviewers for their critical appraisal of our manuscript.Please see below a detailed response to each comment.

EDITOR COMMENTS
Reviewing Editor:

1) The clear strength of the present study is the model utilised to induce hypoxic pregnancy and the ability to assess the impact of MitoQ on multiple outcomes, as the Cambridge group have previously published. The main weakness however is that no data on pulmonary function or structure are provided to support the physiological impact of the changes in mRNA and protein expression. The manuscript would be substantially improved and more likely to proceed if the authors are able to expand their analysis and provide direct evidence of the effects of MitoQ on lung maturation or function. The reviewers have raised several additional concerns that need to be addressed.
We thank the Reviewing Editor for their careful consideration and critique of the manuscript.Unfortunately, as the tissue used in this study was being generated as part of a programme of work designed with the primary objective of investigating cardiovascular physiology in the offspring (Brain et al., 2019;Botting et al., 2020) and on the placenta (Tong et al., 2022), no lung function studies were performed.Thus, only effects of hypoxic pregnancy with and without maternal treatment with MitoQ on fetal lung molecular studies could be determined.In addition, lung structure could not be assessed as the tissue was not appropriately instillation fixed to preserve airway structure.Despite the lack of functional data, these studies provide novel information of important physiological relevance in an ovine model of high translational relevance.The data in this paper would support the rationale for such functional analysis in future studies.In addition, we undertook these studies in line with the 3Rs to reduce the number of animals that undergo studies, and best make use of the valued experimental material.We have altered the title of the manuscript and expanded the Discussion section on technical limitations to address the Reviewing Editor's comment.
Line 401: "However, mitochondrial function and ROS production were not determined in this study due to the requirement of fresh tissue samples, and this may be an important avenue to explore in future studies.This limitation was due to tissues being generated as part of a programme of work designed with the primary objective of investigating cardiovascular physiology in the offspring (Brain et al., 2019;Botting et al., 2020) and on the placenta (Tong et al., 2022).Therefore, only effects of hypoxic pregnancy with and without maternal treatment with MitoQ on fetal lung molecular studies could be determined, without corroborating pulmonary function studies, or assessment of wet/dry lung ratio.Lung tissue was not instillation fixed to preserve airway structure, and therefore airspace ratio and quantification of type II alveolar epithelial cells was also not able to be assessed.The observed increase in surfactant protein expression may therefore be due to an increase in differentiation/larger population of type II alveolar epithelial cells, rather than an upregulation of surfactant protein production within the existing cells."

2) Attention is needed to include exact p values in figures, text and tables.
We thank the Reviewing Editor for this correction.P values have now been included for all values in text and in data tables.

3) Line 314 -please rephrase to acknowledge no effect on the components examined rather than suggesting all components.
We have adjusted phrasing to improve clarity.Line 324: "Importantly, there were no detrimental effects of MitoQ treatment alone on the molecular components measured in the present study, suggesting that maternal antioxidant does not impair the normal maturation of any of the examined pathways within the study."4) As noted by the reviewers, blots are required to be included.
We apologise for this omission.Images of the western blots have now been included within the figures.

5) The inclusion of only male fetuses is inappropriate.
To make the study viable ethically and economically, every singleton ovine pregnancy generated was used as in previous studies (McGillick et al., 2017;Botting et al., 2020;McGillick et al., 2021).Therefore, studies in the fetal period used the male offspring, while studies in the adult period used the female offspring, as ewe lambs are easier to group house and maintain compared to rams.Female fetuses were used for postnatal experiments (Brain et al., 2019).

REFEREE COMMENTS Referee #1: Thank you for the opportunity to review this timely manuscript. Mitochondrial based therapies and antioxidants are increasingly being recognized as the new frontier in neonatal medicine. As such this is a timely and well conducted study. 1) A generally well detailed methods section. Could the authors please provide additional detail on where lung samples were obtained from (i.e. left or right lobes) and if the sampling sight was standardized across all animals.
We thank the Reviewer for their praise and support of our study.All lung samples were collected from the lower right lobe and sampled from the same relative position from each animal.We have added this detail to the Methods section for clarity.
Line 133: "Lung tissue for molecular analysis was collected from the lower right lobe from the same position in each fetus and immediately frozen in liquid nitrogen for qRT-PCR and western blot analyses."

2) I commend the authors on their extensive testing of the reference genes but note that the RT-PCR section does not currently meet MIQE guidelines (as stated by the authors). To meet the essential MIQE checks I request that the authors include the number of samples analyzed by RT-PCR in each group, and in a table the primer sequences and reaction conditions.
We thank the Reviewer for indicating the requirements to meet the MIQE guidelines and apologise for this omission.The sample size for each individual primer has now been included in Table 1.The primer sequences have all previously been published within our group and are referenced within the Methods section.However, to address the Reviewer's comment, we have now included all sequences within Table 1 for clarity.

3) For general readability the primary antibody details could be included as a supplementary Table. For non sheep researchers it would be useful for the authors to include reference to what the full gestation period for the species would be (i.e. ~145d).
Thank you.The primary antibody details have now been included within Table 1.The length of gestation is stated at the beginning of the Methods section.
Line 83: "Pregnant ewes carrying a singleton pregnancy (determined by ultrasound scan at 80 days of gestation; term, 145 days) underwent surgery under general anaesthesia using aseptic conditions at 100 ± 1 days of gestation." 4) The authors are to be commended for their thoroughness regarding the number of proteins or genes that were measured.I do feel however that some tweaking may improve the impact of the results.For example, in the graphs it would be useful where both a western blot and PCR result is available for the same target to always have it in the order protein-PCR (there are swaps between figure 2

and 3 for example).
We thank the Reviewer for their suggestion and have changed the graphs to reflect protein expression before mRNA expression for the same target as a standard throughout the manuscript.We feel that this suggestion has markedly improved the presentation of the data.

5) I also found the use of the 'over' comparison in Figure 2A,B,C,E etc to be confusing. In terms of the post-hoc analysis which comparison groups do these represent? It would be useful if the same formatting used to compare the normaxia vs hypoxia for saline or MitoQ was used to show the significant comparisons between all blue or all red comparisons.
Thank you for the suggestion.We have changed the graphs to use the same symbols in Table 2 to more clearly display the significant changes per treatment.

6) Not essential but would have been useful to demonstrate the impact of mitoQ treatment on cell composition within the lung. Are the increases in SP-B due to more AT2 cells, or because the AT2 cells are induced by hypoxia and/or mitoQ to produce more protein? This could be included as a limitation within the discussion.
Thank you for the discussion point.Unfortunately, we were unable to assess the numerical density of type II AECs within this study.However, it is true that the observed increase in surfactant protein expression may be due to an increase in differentiation/larger population of type II AECs, rather than an upregulation of production within the existing cells.To address the Reviewer's point, this has been added to the Discussion section as a technical limitation of the study.
Line 408: "Lung tissue was not instillation fixed to preserve airway structure, and therefore airspace ratio and quantification of type II alveolar epithelial cells was also not able to be assessed.The observed increase in surfactant protein expression may therefore be due to an increase in differentiation/larger population of type II alveolar epithelial cells, rather than an upregulation of surfactant protein production within the existing cells."

7) I am a little hesitant by the authors use of the statement "Here we show maturational effects on the fetal lung of a mitochondria-targeted antioxidant therapy..." you were able to detect evidence of maturation such as SP-B, C, D production etc in all groups treated with saline, and without histological evidence I'm not sure there is a strong evidence that lung maturation was delayed by hypoxia. Rather much of the evidence is that mitoQ increases cellular function of some targets. The authors also allude to this in para 1, page 6.
Thank you.We agree.We have softened the relevant statements to more closely reflect the findings of the study.As mentioned in the Discussion section, the effect of fetal hypoxaemia on lung development is variable and depends on the timing, severity and duration of the insult.Using the current model of chronic hypoxia in the last third of pregnancy, our results are consistent with our previous work, showing increased pulmonary surfactant maturation (McGillick et al., 2017).This is in contrast to early-onset fetal growth restriction where surfactant maturation is impaired.It is therefore important to interrogate the effectiveness of MitoQ in a number of models of complicated pregnancy to determine if the same beneficial outcomes are observed.
Line 70: "Here, we tested the hypothesis that maternal treatment with MitoQ in late gestation will improve markers of maturation in the developing lung in both normal and hypoxic pregnancy in sheep, a species with similar fetal lung developmental milestones as humans (Lock et al., 2013;Morrison et al., 2018)."Line 305: "Here we show maturational effects on the fetal lung of a mitochondria-targeted antioxidant therapy at doses appropriate for human clinical translation."

Referee #2: Lock M and colleagues investigated in their study MitoQ as a possible preventive strategy for chronic fetal hypoxemia. To this end, ewes were treated with MitoQ during late gestation and offspring's lungs were analysed with a focus on pulmonary surfactant maturation, mitochondrial respiration, ATP synthesis, and lung liquid reabsorption. The data demonstrate significant effects of hypoxia on lung maturation. MitoQ, however, showed only a mild preventive effect. The research question is of great interest, the topic clinically very relevant, and the model appropriate. However, the data do not show marked effect of MitoQ on lung maturation after chronic fetal hypoxia. Overall, some additional aspects should be considered to strengthen the conclusion made by the authors: 1) The title implies a stronger effect of MitoQ than the data ultimately show. The authors might consider to rephrase.
We thank the Reviewer for their suggestion.We have modified the title to reflect the study more accurately.
"MitoQ as an antenatal antioxidant treatment improves markers of lung maturation in healthy and hypoxic pregnancy" 2) The introduction is somewhat lengthy and could be more focused.There are some sections that belong in the discussion rather than in the introduction.This could certainly be improved to emphasize the focus of the study.
We thank the Reviewer for their critique.To address the Reviewer's comment, we have reduced the length of the Introduction by removing an entire paragraph and focusing previous statements.

3) Animal protocol approval number should be included. Why did the only include male fetuses in this study? Did they expect sex-specific differences/response to hypoxia and/ MitoQ?
Thank you for these important comments.We have now added the appropriate animal ethics approval numbers to the manuscript.Line 74: "Experimental protocols for animal research were performed under the UK Animals (Scientific Procedures) Act 1986 and were approved by the Ethical Review Committee of the University of Cambridge under Home Office Project Licence PL70/7645 and PL80/2232." To make the study viable ethically and economically, every singleton ovine pregnancy generated was used.Therefore, studies in the fetal period used the male offspring, while studies in the adult period used the female offspring, as ewe lambs are easier to group house and maintain compared to rams.Female fetuses were used for other postnatal experiments (Brain et al., 2019).

4) I am uncertain what the purpose of the surgical intervention was? The overall aim was to combine chronic hypoxia with MitoQ treatment. Why was the operation performed at day 100 of gestation?
The surgical procedure was used to determine fetal sex to allow allocation to the present fetal component of the project if male (as presented within this study) or used for postnatal studies if female (as ewes are easier to group house than rams).Surgery was also performed to catheterise the maternal femoral artery and vein so that daily blood samples could be taken from the maternal artery.The degree of hypoxia within the chamber was titrated based on the PaO 2 of the mother, to achieve 50 mmHg.MitoQ treatment or saline were administered daily into the venous catheter.

5) Was the pharmacological dose of MitoQ tested? Were markers of oxidative stress as a readout for sufficient MitoQ effect investigated (e.g., 8-Oxo-dG, ROS,...)?
Measurement of MitoQ content in placentomes, fetal mid-brain, fetal biceps femoris, and fetal liver were performed using LC-MS/MS as previously described (Botting et al., 2020).To address the Reviewer's comment, the latter study has been referenced within the manuscript.
Line 229: "MitoQ treatment resulted in therapeutic concentrations (>25pmol g -1 ) in the placenta, fetal skeletal muscle, and fetal liver (Botting et al., 2020)." Maternal daily MitoQ treatment, from 105 to 138 days of gestation, resulted in greater therapeutic concentrations of MitoQ in the placenta, fetal skeletal muscle, and fetal liver than with single administration.Though oxidative stress was not assessed in these sheep, cardiac mitochondrial respiratory control ratio was assessed in hypoxic chicken embryos where it prevented the enhanced in vivo mitochondria-derived oxidative stress in the embryonic heart and restored the cardiac mitochondrial respiratory control ratio, left ventricular structure, and systolic function (Botting et al., 2020).

6) The authors only show the protein quantification. The blots with bands for the respective protein and loading should be shown.
We apologise.Western blot images have now been included within figures.

7) Was the wet lung/dry lung ratio measured as an indicator of impaired lung liquid absorption?
The tissue used in this study was being generated as part of a programme of work designed with the primary objective of investigating cardiovascular physiology in the offspring (Brain et al., 2019;Botting et al., 2020) or effects on the placenta (Tong et al., 2022).As collection of lung tissue was not the primary outcome, we were unable to assess wet lung/dry lung ratio within this study and were limited to only molecular studies.We have expanded on this within the Discussion section as a technical limitation to address the Reviewer's comment.
Line 406: Therefore, only effects of hypoxic pregnancy with and without maternal treatment with MitoQ on fetal lung molecular studies could be determined, without corroborating pulmonary function studies, or assessment of wet/dry lung ratio.

8) The authors show effects of hypoxia/MitoQ on the gene expression of surfactant proteins; was the protein expression assessed as well?
SP-B protein expression is displayed in Figure 2A alongside the corresponding mRNA expression.Unfortunately, due to limitations regarding antibody specificity for sheep, we were unable to assess other surfactant proteins within this study.In our previous studies, we have shown that changes in mRNA and protein expression have a consistent pattern of expression (Orgeig et al., 2010).

9) Were hypoxia-induced changes in lung structure attenuated by MitoQ?
Lung development was not the primary outcome of the project.Thus, only effects of hypoxic pregnancy with and without maternal treatment with MitoQ on fetal lung molecular studies could be determined within this study.Lung structure could not be assessed as the tissue was not appropriately instillation fixed to preserve airway structure.To address the Reviewer's comment, we have expanded on this within the Discussion section as a technical limitation.
Line 408: "Lung tissue was not instillation fixed to preserve airway structure, and therefore airspace ratio and quantification of type II alveolar epithelial cells was also not able to be assessed."

10) Since the authors focus on surfactant proteins, it would be interesting to see if fraction of AT2 is changed by hypoxia+/-MitoQ.
Thank you for the discussion point.Unfortunately, we were unable to assess the numerical density of type II AECs within this study.It is true however that the observed increase in surfactant protein expression my be due to an increase in differentiation/larger population of type II AECs, rather than an upregulation of production within the existing cells.To address the Reviewer's comment, this has been added to the Discussion section as a technical limitation of the study.
Line 408: "Lung tissue was not instillation fixed to preserve airway structure, and therefore airspace ratio and quantification of type II alveolar epithelial cells was also not able to be assessed.The observed increase in surfactant protein expression may therefore be due to an increase in differentiation/larger population of type II alveolar epithelial cells, rather than an upregulation of surfactant protein production within the existing cells."

11) Was lung cell proliferation or apoptosis assessed?
We were particularly interested in surfactant development within this study and without being able to assess the density of type II AECs as discussed above, we were also unable to assess proliferation or apoptosis of these cells within the fetal lungs due to a lack of instillation fixed tissue.To our knowledge there is no evidence of developmental hypoxia leading to apoptosis within the fetal lung.
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If you have any queries, please reply to this email and we will be pleased to advise.---------------EDITOR COMMENTS Reviewing Editor: Thank you for your revised manuscript, the reviewers have no further issues that need to be addressed.I however do have some minor issues to be addressed.

Regardless of the inclusion of p values in figure legends
, figures 1-6 can very easily be adapted to show the exact p values rather than summary symbols, as is the preferred style of the Journal.
The number of subjects varies in some results, eg Table 2. Please provide a brief explanation likely in methodology for why n has been reduced in some analyses.
Abstract: "maternal antioxidant therapy in hypoxic pregnancy has proven to be protective with regards to fetal growth and cardiovascular development" Please rewrite to be more cautious or clarify this evidence is from animal studies.
- --------------- Dear Prof Bennet, We thank the reviewing editor and reviewers for their approval of our manuscript.Please see below a detailed response to the remaining editor comments.

Reviewing Editor:
Regardless of the inclusion of p values in figure legends, figures 1-6 can very easily be adapted to show the exact p values rather than summary symbols, as is the preferred style of the Journal.
The P values have now been included on each graph as requested.
The number of subjects varies in some results, eg Table 2. Please provide a brief explanation likely in methodology for why n has been reduced in some analyses.
The missing samples were outliers that were removed from the analysis; this detail was previously included within the qRT-PCR method section but has now been moved to a more appropriate location within the statistical analysis methodology.
Line 217: "Outliers were determined using the ROUT method (Q=1%) and removed from the analysis." Abstract: "maternal antioxidant therapy in hypoxic pregnancy has proven to be protective with regards to fetal growth and cardiovascular development" Please rewrite to be more cautious or clarify this evidence is from animal studies.
Thank you for the suggestion, we have changed this sentence to clarify that the evidence is referring to animal studies.Line 2: "In turn, chronic fetal hypoxaemia promotes oxidative stress, and maternal antioxidant therapy in animal models of hypoxic pregnancy has proven to be protective with regards to fetal growth and cardiovascular development."

29-Jun-2023 2nd Revision -Editorial Decision
Dear Professor Morrison, Re: JP-RP-2023-284786R2 "MitoQ as an antenatal antioxidant treatment improves markers of lung maturation in healthy and hypoxic pregnancy" by Mitchell C Lock, Kimberley J Botting, Beth J Allison, Youguo Niu, Sage Ford, Michael P. Murphy, Sandra Orgeig, Dino A Giussani, and Janna L Morrison Thank you for submitting your revised Research Article to The Journal of Physiology.It has been assessed by the original Reviewing Editor and Referees and has been well received.Some final additions have been requested.Please see Required Items Section below.
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authors for their detailed response to the review.On extensive review it appears the authors have addressed and made changes in relations to the original reviewing comments appropriately.

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Please include an Abstract Figure legend text within the main article file.The abstract figure was labelled as Figure 7 in the manuscript.This has now been changed to "Abstract Figure" for clarity.An example of the Figure is below.

AbstractFigure:
Summary of molecular changes within the fetal lung as a result of MitoQ treatment.Changes in expression of pathways are indicated by direction of arrows; no change in expression is represented by purple sideways double-sided arrows.A main effect of MitoQ is represented by a green arrow.An interaction of hypoxia and MitoQ is indicated by yellow arrows.MitoQ treatment increased the expression of Mitochondrial Complex III and ATP Synthase (Complex V) and expression of Surfactant Proteins B & C.There was an interaction of MitoQ with hypoxia resulting in increased expression of sodium transporter SCNN1A and Surfactant Protein D within the fetal lung.