Magnesium sulphate reduces tertiary gliosis but does not improve EEG recovery or white or grey matter cell survival after asphyxia in preterm fetal sheep

Abstract Maternal magnesium sulphate (MgSO4) treatment is widely recommended before preterm birth for neuroprotection. However, this is controversial because there is limited evidence that MgSO4 provides long‐term neuroprotection. Preterm fetal sheep (104 days gestation; term is 147 days) were assigned randomly to receive sham occlusion with saline infusion (n = 6) or i.v. infusion with MgSO4 (n = 7) or vehicle (saline, n = 6) from 24 h before hypoxia–ischaemia induced by umbilical cord occlusion until 24 h after occlusion. Sheep were killed after 21 days of recovery, for fetal brain histology. Functionally, MgSO4 did not improve long‐term EEG recovery. Histologically, in the premotor cortex and striatum, MgSO4 infusion attenuated post‐occlusion astrocytosis (GFAP+) and microgliosis but did not affect numbers of amoeboid microglia or improve neuronal survival. In the periventricular and intragyral white matter, MgSO4 was associated with fewer total (Olig‐2+) oligodendrocytes compared with vehicle + occlusion. Numbers of mature (CC1+) oligodendrocytes were reduced to a similar extent in both occlusion groups compared with sham occlusion. In contrast, MgSO4 was associated with an intermediate improvement in myelin density in the intragyral and periventricular white matter tracts. In conclusion, a clinically comparable dose of MgSO4 was associated with moderate improvements in white and grey matter gliosis and myelin density but did not improve EEG maturation or neuronal or oligodendrocyte survival. Key points Magnesium sulphate is widely recommended before preterm birth for neuroprotection; however, there is limited evidence that magnesium sulphate provides long‐term neuroprotection. In preterm fetal sheep exposed to hypoxia–ischaemia (HI), MgSO4 was associated with attenuated astrocytosis and microgliosis in the premotor cortex and striatum but did not improve neuronal survival after recovery to term‐equivalent age, 21 days after HI. Magnesium sulphate was associated with loss of total oligodendrocytes in the periventricular and intragyral white matter tracts, whereas mature, myelinating oligodendrocytes were reduced to a similar extent in both occlusion groups. In the same regions, MgSO4 was associated with an intermediate improvement in myelin density. Functionally, MgSO4 did not improve long‐term recovery of EEG power, frequency or sleep stage cycling. A clinically comparable dose of MgSO4 was associated with moderate improvements in white and grey matter gliosis and myelin density but did not improve EEG maturation or neuronal or oligodendrocyte survival.

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EDITOR COMMENTS
Reviewing Editor: This study investigates the role of MgSO4 on brain development.Interestingly, although MGSO4 improves EEG outcomes there is still loss of neurons.But this is an important study in understanding the underlying mechanisms for the potential benefits of such treatment.
The key points section may be over the word limit.Comments to the Author: The reviewers have found merit in the study, but the manuscript will require revision based on their comments.Please state actual p values throughout including figures and include in figure legends the sample size(s) and statistical test(s) used in the figure .-----------------

REFEREE COMMENTS
Referee #1: General Comments This is a very well-done study in a complex model.It is meritorious that the investigators are able to maintain the fetal sheep after severe umbilical cord occlusion for 21 days.The model has the advantage that it more closely represents the human pregnancy and human brain than rodent models and physiological determinations can be quantified during the studies.
Magnesium sulfate is now widely used to reduce the development of cerebral palsy in women at risk for early preterm birth in countries worldwide.The authors have examined the ability of magnesium sulfate (MgSO4) to protect the immature fetal brain after exposure to severe umbilical cord occlusion.They have extended their previous work that examined short term outcomes after neuroprotection with MgSO4.In the current study they have examined the fetus over a prolonged recovery period of 21 days.They have used appropriate doses of MgSO4 which closely simulate those used to treat pregnant women before preterm birth.Physiological measures including EEG were performed over the 21 day period of study.Control animals were included.The post-mortem brains were obtained for analysis 21 days after umbilical cord occlusion.They found that MgSO4 did not improve long-term EEG recovery.However, MgSO4 infusion attenuated post-occlusion astrocytosis (GFAP+) and microgliosis in the premotor cortex and striatum but did not affect reactive (ameboid) microglia or improve neuronal survival.MgSO4 was associated with fewer total (Olig-2+) oligodendrocytes in the periventricular and intragyral white matter.The numbers of mature oligodendrocytes were also reduced in the occlusion groups compared to the sham occlusion.MgSO4 also improved myelin density in the intragyral and periventricular white matter tracts.Consequently, MgSO4 infusion had only modest effects on white matter in the immature fetal sheep after umbilical cord occlusion but did not contribute to neuronal sparing.
Since MgSO4 is widely used to treat pregnant women with threatened preterm labor, understanding the potential mechanisms underlying its effect is translationally relevant.The results of the study support the authors conclusions.The discussion is well written and addresses all of the important aspects of the data.There are only a few points that need to be considered.
3. Page 17, line 18.Where is the data to support the conclusion that MgSO4 reduced total seizures in this study.This reviewer searched for the data and was unable to locate it.Please provide the data to support this conclusion in the current study.

Did they not experience any fetal loss during the studies?
Referee #2: This work by Galinsky and colleagues examines whether antenatal administration of MgSO4 provides longer term neuroprotective benefit with the preterm brain, assessed using histological analyses combined with EEG outcomes.A comprehensive set of data are presented for grey and white matter brains regions, with results broadly showing that MGSO4 did not improve brain activity after severe asphyxic insult, and while MgSO4 modified the neuroinflammatory response in a region-specific manner, it was associated with cell loss of neurons and oligodendrocytes.
Overall this is an important and timely study, and the results are likely to be highly interpretable to the clinical situation.
There are however a few points that require revision: I found myself reading the Introduction a couple of times in an attempt for clarity on the rationale for the study.The way it is currently written, the study appears to be incremental given that you have previously demonstrated a lack of protection in preterm sheep with MgSO4 in brains collected at 3 days.However I think this current study has merit to fill a knowledge gap on the effects of preterm exposure to MgSO4 at term-equivalent brain age.Some questions that you might consider for clarity: In the Doyle 2009 meta-analysis, is there data on the cohort of infants who receive benefit from MgSO4 exposure?I cannot see the Koning et al 2018 (Int J Dev Neurosci) study in preterm rodents mentioned.How does this data relate to your results; ie preterm administration, and term-equivalent brain collection?
Have longer term preclinical studies to examine the effects of MgSO4 been undertaken?
The piglet studies are mentioned in the Discussion but not the Introduction, which support a lack of neuroprotection with MgSO4.Are the piglet studies representative of the preterm or term-equivalent brain?
The sample size for groups presented in the Results should be more clearly stated; Page 6 paragraph 3 states n=18 total fetuses; page paragraph 3 states HI+vehicle n=6, HI+MGSO4 n=7, or sham n=6.It would be useful to add a line into the results to redefine the groups and sample sizes, and whether all fetuses were used in all analysis.It appears in some graphs (eg GFAP area) that n=7 fetuses were included in the sham group.Please clarify.
Is the MgSO4 administered directly to the fetus?The Khatib et al 2022 (Placenta) study indicates that some benefits are mediated via a placental response.Is it possible that lack of positive results in the current study is due to route of administration?Please justify fetal administration and limitations.We have revised our manuscript in response to the editors' and reviewers' comments, provided a point-by-point response below and have uploaded all relevant files using the online submission system.

Reviewing Editor:
This study investigates the role of MgSO4 on brain development.Interestingly, although MGSO4 improves EEG outcomes there is still loss of neurons.But this is an important study in understanding the underlying mechanisms for the potential benefits of such treatment.
The key points section may be over the word limit.This section is within the word limit, word count: 140/150 words Page 4 -Should 'neuroprotective' be 'neuroprotection'?Corrected, thank you.
Page 4 -It would be helpful to mention glutamate in this paragraph.It comes into the discussion but associating it with NMDA here would improve clarity.It was confusing to read 'anti-excitatory'.Could rephrasing to something like '.. Mg inhibits the excitatory NT glutamate by inhibiting NMDA"?We have rephrased this section to read: The most likely mechanism for neuroprotection with magnesium is through its physiological role as an endogenous inhibitor of N-methyl-Daspartate (NMDA) receptors by excitatory amino acids such as glutamate.
Page 6 -Were there nineteen animals in the study (6, 7, 6)?Indeed, there were 19 animals in the study, thank you for alerting us to the typographical error.
Page 8 -It is stated that studies occurred between 103-125.Does this mean that day one could have been anywhere between 103-125 with tissue collection 21d later (124-146)?Or does this mean that every study started at d 103 and ended at d 125?If the former, this is a very wide range and brain development would be very different at tissue collection.If the later, please clarify.The latter is correct.We have clarified this section accordingly, thank you.Page 14 -JP does not have supplementary data.We have now incorporated the supplementary figure and tables into the MS file as Figure 1 and Tables 1 and 2  2. Page 7, line 12.Presumably, leads and catheters were exteriorized.That is correct, we stated this this in the Methods (page 7, paragraph 2) 3. Page 17, line 18.Where is the data to support the conclusion that MgSO4 reduced total seizures in this study.This reviewer searched for the data and was unable to locate it.Please provide the data to support this conclusion in the current study.We have clarified this section of the Discussion by stating we have previously shown that MgSO 4 reduces electrographic seizures using the same experimental paradigm and infusion protocol as the present study (page 18, paragraph 2).We apologise for any confusion this may have caused.

Did they not experience any fetal loss during the studies?
We have included a statement in the Methods to clarify that in this experimental protocol we experience a fetal loss rate of 20 % that did not vary between the experimental groups.In cases of fetal loss, the subject was excluded from study (page 9, paragraph 1).

Referee #2:
I found myself reading the Introduction a couple of times in an attempt for clarity on the rationale for the study.The way it is currently written, the study appears to be incremental given that you have previously demonstrated a lack of protection in preterm sheep with MgSO4 in brains collected at 3 days.However I think this current study has merit to fill a knowledge gap on the effects of preterm exposure to MgSO4 at term-equivalent brain age.We wholeheartedly agree with the reviewer and have revised the Introduction to clarify this important point (page 5, paragraph 2 and page 6, paragraph 1).Thank you.
In the Doyle 2009 meta-analysis, is there data on the cohort of infants who receive benefit from MgSO4 exposure?We have included the relative risk and confidence interval data that was reported in the metaanalysis for the cohort of infants who received benefit from MgSO 4 exposure in the revised Introduction (page 4, paragraph 1).I cannot see the Koning et al 2018 (Int J Dev Neurosci) study in preterm rodents mentioned.How does this data relate to your results; ie preterm administration, and term-equivalent brain collection?We thank the reviewer for pointing out the excellent paper by Koning et al. (Koning et al., 2018) which showed a reduction in MBP loss with MgSO 4 treatment 7 days after hypoxia ischaemia.Although the serum Mg 2+ concentration achieved in the study by .1 mmol/L) is much higher than the levels achieved in this study and in human cohort studies (1.89 mmol/L (page 13, paragraph 2)), the histological outcome showing an improvement in MBP+ staining with MgSO 4 is broadly consistent with our study.We have included this important study in the revised Discussion (page 23, paragraph 3).

Have longer term preclinical studies to examine the effects of MgSO4 been undertaken?
To the best of our knowledge, this is the first study in a large animal translational model of preterm hypoxia ischaemia to evaluate the impact of MgSO 4 on EEG maturation, tertiary gliosis, and white and grey matter cell survival after 21-days recovery to term equivalent age.Similarly, in neonatal rats exposed to hypoxia ischaemia on postnatal day 4 (Daher et al., 2018), after 40 days recovery postnatal MgSO 4 treatment was associated with reduced thalamic and hippocampal tissue loss in males only.However, the MgSO4-induced reduction in tissue loss was not associated with significant improvements in motor function or cognition (Daher et al., 2018).We have included this consideration in the revised Discussion (page 24, paragraph 2).
The piglet studies are mentioned in the Discussion but not the Introduction, which support a lack of neuroprotection with MgSO4.Are the piglet studies representative of the preterm or term-equivalent brain?
The piglet studies are representative of the term-equivalent brain, we have clarified this in the revised Discussion (page 19, paragraph 2).Thank you.
The sample size for groups presented in the Results should be more clearly stated; Page 6 paragraph 3 states n=18 total fetuses; page paragraph 3 states HI+vehicle n=6, HI+MGSO4 n=7, or sham n=6.It would be useful to add a line into the results to redefine the groups and sample sizes, and whether all fetuses were used in all analysis.It appears in some graphs (eg GFAP area) that n=7 fetuses were included in the sham group.Please clarify.We apologise and thank the reviewer for alerting us to the typographical error.We have revised the methods, results and figure legends to clearly state the study comprised 19 subjects, with n=6 sham control, n=6 vehicle+occlusion and n=7 MgSO 4 +occlusion.
Is the MgSO4 administered directly to the fetus?The Khatib et al 2022 (Placenta) study indicates that some benefits are mediated via a placental response.Is it possible that lack of positive results in the current study is due to route of administration?Please justify fetal administration and limitations.The reviewer raises an interesting and important point.We administered MgSO4 directly to the fetus since Mg 2+ transfer across the sheep placenta is limited (Akoury et al., 1997).It is reasonable to expect that direct infusion to the fetus also exposes the placenta to MgSO 4 , although this was not directly assessed in our study.We have incorporated this point into the revised Discussion (page 20, paragraph 2).Indeed, it is possible that MgSO4 promotes neuroprotection by modulating inflammation within the placenta, circulation or centrally.However, it is important to note that the study by Khatib et al. 2022 did not correlate placental inflammation with direct markers of brain inflammation or injury.Furthermore, two subgroup analyses focusing on infants exposed to clinical chorioamnionitis from a large randomised controlled trial (Rouse et al., 2008) showed MgSO4 was not associated with improvements in neurodevelopment at 2 years of age or reduced rates of intraventricular haemorrhage or periventricular leukomalacia (Kamyar et al., 2016;Edwards et al., 2018).Although, both data sets were relatively small, they suggest MgSO 4 did not modulate placental inflammation.Nevertheless, our data support the potential for MgSO4 to promote central anti-inflammatory effects which were linked to a modest improvement in myelin density.We have included consideration of this important point in the revised Discussion (page 21, paragraph 2) Are the variable results to date confounded by dosing?Where do your fetal plasma levels sit in terms of dose?Can you correlate plasma MgSO4 with specific effects in the brain?This is an interesting point, although it is something that out data set is unable to verify due to highly consistent fetal serum Mg2+ levels between subjects that were similar to cord blood levels in preterm infants after maternal MgSO 4 administration (page 23, paragraph 3).The dosing regime in this study produced fetal plasma levels of 1.89 ± 0.08 mmol/L in the MgSO 4 +occlusion group compared to 0.88 ± 0.07 mmol/L in the vehicle+occlusion group (page 13, paragraph 2).Nevertheless, this is an important point to consider with respect to human cohort studies where the variable duration of MgSO 4 infusion relative to the timing of delivery could result in varying circulating magnesium levels in the fetus/neonate.We have included this point in the revised Discussion (page 21, paragraph 2).

08-Mar-2023 1st Revision -Editorial Decision
Dear Dr Galinsky, Re: JP-RP-2023-284381R1 "MgSO4 reduces tertiary gliosis but does not improve EEG recovery, or white or grey matter cell survival after asphyxia in preterm fetal sheep" by Robert Galinsky, Simerdeep Kaur Dhillon, Sharmony B Kelly, Guido Wassink, Joanne Davidson, Christopher Arthur Lear, Lotte G. van den Heuij, Laura Bennet, and Alistair J. Gunn 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 revisions have been requested.
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Are the variable results to date confounded by dosing?Where do your fetal plasma levels sit in terms of dose?Can you correlate plasma MgSO4 with specific effects in the brain?Editors, J Physiol Re: MgSO 4 reduces tertiary gliosis but does not improve EEG recovery, or white or grey matter cell survival after asphyxia in preterm fetal sheep Authors: Robert Galinsky, Simerdeep K. Dhillon, Sharmony B. Kelly, Guido Wassink, Joanne O. Davidson, Christopher A. Lear, Lotte G. van den Heuij, Laura Bennet, Alistair J. Gunn Dear Dr Schultz, Thank you for the opportunity to respond to the editors' and reviewers' comments on our submission.
. Thank you Page 17 -Please remove 'strongly' from before infer.Removed, as requested.It would be helpful to use line numbers.Line numbers have been incorporated into the revised version of the MS.Please include the sample size in the figure legends.
Figure legends now include a sample size.Thank you.Senior Editor: Comments to the Author: Please state actual p values throughout including figures and include in figure legends the sample size(s) and statistical test(s) used in the figure.Specific P values are now reported in the results and figures.The sample sizes and statistical tests used are now reported in the revised figure legends.4, line 20: 'Neuroprotection' not 'neuroprotective'.Corrected.Thank you.
Comments for Authors to ensure the paper complies with the Statistics Policy: Please state actual p values throughout including figures.Please include sample size(s) and statistical test(s) used in figure legends.