Does MgSO4 protect the preterm brain? Dissecting its role in the pathophysiology of hypoxic ischemic encephalopathy

Robert Galinsky, Laura Bennet, Alistair J.Gunn

Mitigating preterm encephalopathy continues to be one of the greatest challenges in perinatal medicine.Preterm encephalopathy is associated with high mortality, serious morbidity, and significant socio-economic impacts on the individuals, their families, and public health sectors and welfare systems that last a lifetime.The cost of disability associated with preterm brain injury continues to rise.Prevention of this injury, and disability, would significantly reduce this socioeconomic burden.

Magnesium sulfate (MgSO4) has become widely recommended for neuroprotection in preterm infants.This recommendation is based on a meta-analysis of randomized controlled trials of antenatal administration of magnesium sulfate to women at risk of preterm birth that found this intervention was associated with a small but significant reduction in the risk of cerebral palsy [relative risk 0.68; 95% confidence interval 0.53–0.87, 3988 babies from 4 trials] (Crowther et al., 2017).However, there was no significant effect on death or disability (relative risk 0.95;95% confidence interval 0.80–1.13, 4448 babies from 4 trials) (Crowther et al., 2017), raising the possibility that exposure to MgSO4might have been associated with a partial shift between outcomes rather than overall improvement.

The causes of preterm brain injury are multifactorial.However, hypoxia ischemia (HI) as shown by metabolic acidosis and the need for resuscitation at birth, remains common among preterm babies and is associated with increased risk of death, subcortical brain injury, and disability.Furthermore, excessive glutaminergic excitation and central nervous system inflammation during the evolution of hypoxic ischemic encephalopathy(HIE) are implicated in preterm white and grey matter injury.

The most likely mechanism for neuroprotection with magnesium sulfate is through its physiological role as an endogenous inhibitor of N-methyl-D-aspartate (NMDA) receptor activation by excitatory amino acids, such as glutamate.This is supported by evidence in preterm fetal sheep and neonatal piglets for suppression of baseline electroencephalogram (EEG) activity and reduced seizures with intravenous magnesium infusions after experimentally induced HIE (Galinsky et al., 2017; Lingam et al., 2019).However, there is evidence for other potential neuroprotective mechanisms linked to magnesium sulfate,including anti-inflammatory, anti-oxidative stress,and improved cardiovascular stability (Sugimoto et al., 2012; Galinsky et al., 2015).Despite these potential beneficial effects, preclinical animal studies and human trials have found that the effect of MgSO4treatment on HI or infection/inflammation-induced brain injury and neurodevelopmental outcomes are inconsistent between studies (Galinsky et al., 2020).The inconsistent effects in small animal studies most likely reflect confounding with iatrogenic hypothermia mediated by MgSO4-induced peripheral vasodilation.Furthermore, studies of HI in large animals at term equivalent age showed MgSO4had a modest-to-no benefit on histological brain injury (Galinsky et al., 2020).Collectively, the lack of evidence for MgSO4-induced protection in the preterm-equivalent brain highlighted the need to improve our understanding of whether MgSO4could mitigate cerebral injury and improve functional recovery during the evolution of preterm HIE.

Using an established translational large animal model of preterm HIE in fetal sheep, where neural development is comparable to human brain development at 28–30 weeks of gestation, our team has systematically evaluated the effect of a clinically comparable increase in fetal plasma magnesium concentration for 24 hours before and after hypoxia ischemia on functional recovery and histological brain injury throughout the secondary(Galinsky et al., 2017) and tertiary phases (Galinsky et al., 2023) of preterm HIE (as summarized in Figure 1).

Excitotoxicity and maturation of the electroencephalogram: From approximately 10 hours after HI, in the vehicle-treated group, we observed large amplitude stereotypical seizures,similar to previous studies (Galinsky et al., 2017).These stereotypical seizures are preceded by secondary loss of mitochondrial function and clinically are associated with adverse neurological outcomes.Magnesium is a potent inhibitor of neural glutamatergic activity.We showed that MgSO4was associated with a marked reduction in the number of seizures and seizure burden,strongly supporting a significant central effect on the NMDA receptor (Galinsky et al., 2017).These data confirm that the MgSO4infusion protocol,whilst achieving comparable fetal circulating levels of Mg2+to those reported in human studies, was sufficient to provide a central anti-excitatory effect.Despite reducing electrographic seizures, MgSO4was not associated with improved maturation of EEG power or frequency for 21 days after asphyxia(Galinsky et al., 2023).Indeed, we observed significantly lower EEG power and higher spectral edge frequency during the latter stages of recovery(days 17–21) in both MgSO4and vehicle groups exposed to HI compared to sham control (Galinsky et al., 2023).The higher spectral edge frequency at this age is consistent with impaired maturation of sleep state cycling observed after HI (Galinsky et al., 2023), and is most likely attributable to loss of inhibitory GABAergic neurons in the cortical and deep grey matter.Collectively, these data suggest that the anti-excitatory effects of MgSO4do not improve the functional maturation of EEG activity.

Histological outcomes:MgSO4 does not improve neuronal survival during and after secondary energy failure:The model of HIE in preterm fetal sheep used in these studies is consistent with the common clinical pattern of impaired oligodendrocyte maturation and diffuse white matter loss, with evolving loss of cortical and subcortical grey matter.Despite the significant anti-excitatory effects of MgSO4, it was not associated with improved survival of cortical or basal ganglia neurons at 3 or 21 days after HI.These data are consistent with previous reports that showed a lack of effect of antenatal MgSO4treatment on neurological damage assessed during secondary energy failure (2–3 days after HI) after HI in newborn piglets (Lingam et al., 2019).These findings support the concept that electrographic seizures during secondary energy failure are,most likely, a marker of neural injury rather than a substantial cause of secondary neuronal loss.

MgSO4 mitigates tertiary gliosis after hypoxiaischemia:Despite a lack of effect on neuronal survival during the secondary and tertiary phases of HIE, MgSO4was associated with reduced astrogliosis in the motor cortex and basal ganglia and reduced numbers of microglia in the caudate nucleus when compared to the vehicle group(Galinsky et al., 2023).Similarly, in the intragyral and periventricular white matter tracts, MgSO4reduced gliosis (Galinsky et al., 2023).Interestingly,the MgSO4-induced reduction in gliosis was only observed during the tertiary phase of HIE (21 days after HI); histological analyses at the time of secondary energy failure (3 days after HI)showed no effect of MgSO4on gliosis in the motor cortex, basal ganglia, hippocampus, thalamus or white matter (Galinsky et al., 2017).The exact mechanisms underpinning the MgSO4-induced reduction in gliosis are unclear, but MgSO4has been associated with anti-inflammatory effects linked to direct modulation of nuclear factor-κB signaling and inhibition of L-type calcium channels(Lin et al., 2010; Sugimoto et al., 2012).It is well established that inflammation is an important extrinsic mechanism involved in the activation of pro-apoptotic pathways during the evolution of HIE.Encouragingly, these observations suggest that MgSO4is moderately effective at modulating persistent grey and white matter inflammation after HI.

Impact of MgSO4 on white matter injury and myelination:Multiple studies have demonstrated that recovery from HI in small and large animal models, and human infants is associated with initial loss of immature oligodendrocytes followed by proliferation of oligodendrocyte progenitors that results in restoration of total cell numbers,but impaired lineage maturation and reduced myelination.We demonstrated that MgSO4was associated with a greater reduction in total(oligodendrocyte transcription factor-2; Olig-2+) oligodendrocytes during secondary energy failure (3 days after HI) in preterm fetal sheep,but increased cell proliferation compared to the vehicle group (Galinsky et al., 2017).There was no difference in the proportion of immature and mature oligodendrocytes between groups after 3 days of recovery, suggesting that the greater reduction in oligodendrocyte numbers in MgSO4-treated fetuses was not unique to a specific stage of oligodendrocyte development.However, the white matter tracts of the preterm fetal sheep brain at 0.7 of gestation predominantly include immature pre-myelinating oligodendrocytes,like the preterm human brain at 28–30 weeks of gestation.Consistent with this, we subsequently showed that at 21 days after preterm HI, at atime where myelination in the fetal sheep brain is comparable to the term human brain, there was a greater loss of total (Olig-2+) oligodendrocytes in MgSO4-treated fetuses exposed to HI compared to vehicle-treated fetuses.However, the number of mature myelinating adenomatous polyposis coli (CC1+) oligodendrocytes in the periventricular white matter was similarly reduced in the MgSO4and vehicle groups that were exposed to HI compared to sham control (Galinsky et al., 2023).The specific mechanism response for the loss of total (Olig-2+) oligodendrocytes in the MgSO4group is not known.However, NMDA receptors are present on oligodendrocytes and are activated during HI.Both immature and mature oligodendrocytes exhibit glutamate evoked currents, which can be inhibited by magnesium(Káradóttir et al., 2005).Indeed, neurons send synaptic inputs to oligodendrocytes residing within grey and white matter structures.This form of neuronal oligodendrocyte signaling may contribute to oligodendrocyte survival, differentiation, and stimulation of axonal myelination.The lack of longterm benefit to total oligodendrocyte survival after MgSO4treatment suggests that prolonged NMDA glutamate receptor blockade by magnesium cannot salvage acute loss of oligodendrocytes after HI.

Despite the greater reduction in total (Olig-2+) oligodendrocytes and a similar reduction in mature (CC1+) oligodendrocytes after MgSO4compared with vehicle, we showed an intermediate improvement in MBP+myelin density in MgSO4-treated fetuses 21 days after HI.The exact mechanisms underpinning this MgSO4-induced improvement in myelin density are unclear.However, it is reasonable to speculate that the anti-excitotoxic and anti-inflammatory effects of MgSO4demonstrated by us and others(Daher et al., 2018; Lingam et al., 2019) might have improved the milieu for myelin deposition by surviving oligodendrocytes.Alternatively, it is possible that surviving oligodendrocytes had improved myelination capacity, as shown by a study in adult mice that reported improved myelination during motor learning by surviving oligodendrocytes after a demyelinating injury(Bacmeister et al., 2020).

Does sex affect functional and histological outcomes? Although randomized controlled trials of antenatal MgSO4have not reported sexrelated differences in neonatal outcomes (Doyle et al., 2014), recent preclinical studies in small and large animals have found sexual dimorphisms relating to functional and histological outcomes with MgSO4treatment.In preterm fetal sheep,MgSO4-treated males showed greater suppression of neuronal excitation compared to females,as shown by a greater loss of high-frequency neural activity during normoxia and increased suppression of seizures during secondary energy failure after HI (Bennet et al., 2018).Our study into the effects of MgSO4on tertiary brain injury after HI was not designed to test the effect of sex and was not large enough to determine whether there were sex-specific effects of MgSO4in histological or functional outcomes.However,in neonatal rats exposed to hypoxia-ischemia,MgSO4treatment was associated with reduced thalamic and hippocampal tissue loss in males but no significant improvements in motor function or cognition (Daher et al., 2018).Collectively, these data raise the possibility that sex-specific effects of MgSO4may arise in preterm infants and should be considered in future preclinical and clinical studies.

Conclusion: We have shown that administration of MgSO4to preterm fetal sheep before and after HI reduced excitotoxicity during secondary energy failure but was not associated with improved neuronal survival during the secondary (after 3 days of recovery) and tertiary phases (after 21 days of recovery) of HIE.Of concern, MgSO4was associated with a persistent reduction in total numbers of oligodendrocytes in the white matter tracts, with no improvement in survival of mature,myelinating oligodendrocytes.Despite this, MgSO4reduced white and grey matter gliosis and was associated with a localized partial improvement in myelin density, suggesting a small potential benefit (Figure 1).We speculate this could be mediated through reduced maturational inhibition by reduced gliosis, or improved function of the remaining mature oligodendrocytes.Further investigation to understand the mechanisms behind the intermediate improvement in myelination with MgSO4is now essential.Given that acute neuroprotection tends to reduce mortality as seen with therapeutic hypothermia, if confirmed, we speculate that this lack of improvement in cell survival combined with improved myelination could explain the reduced rates of cerebral palsy despite the lack of improvement in survival in clinical randomized controlled trials of MgSO4.

This work was supported by Health Research Council of New Zealand (grants 17/601 and 22/559), the Auckland Medical Research Foundation, the Lottery Health Grants Board of New Zealand, the C.J.Martin Postdoctoral Fellowship and project grant from the National Health and Medical Research Council of Australia(APP1090890 and APP1164954) and the Victorian Government’s Operational Infrastructure Support Program (to RG).

Robert Galinsky*, Laura Bennet,Alistair J.Gunn

The Ritchie Centre, Hudson Institute of Medical Research, Melbourne, VIC, Australia (Galinsky R)Department of Obstetrics and Gynaecology,Monash University, Melbourne, VIC, Australia(Galinsky R)

Department of Physiology, The University of Auckland, Auckland, New Zealand (Bennet L,Gunn AJ)

*Correspondence to: Robert Galinsky, PhD,Robert.Galinsky@hudson.org.au.

https://orcid.org/0000-0002-6374-9372(Robert Galinsky)Date of submission: August 11, 2023

Date of decision: October 18, 2023

Date of acceptance: November 10, 2023

Date of web publication: December 15, 2023

https://doi.org/10.4103/1673-5374.390977

How to cite this article:Galinsky R, Bennet L,Gunn AJ (2024) Does MgSO4 protect the preterm brain?Dissecting its role in the pathophysiology of hypoxic ischemic encephalopathy.Neural Regen Res 19(9):1861-1862.

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