Impact of intrauterine fetal resuscitation with oxygen on oxidative stress in the developing rat brain

Abstract

Use of maternal oxygen for intrauterine resuscitation is contentious because of the lack of evidence for its efficacy and the possibility of fetal harm through oxidative stress. Because the developing brain is rich in lipids and low in antioxidants, it remains vulnerable to oxidative stress. Here, we tested this hypothesis in a term pregnant rat model with oxytocin-induced fetal distress followed by treatment with either room air or 100% oxygen for 6 h. Fetal brains from both sexes were subjected to assays for biomarkers of oxidative stress (4-hydroxynonenal, protein carbonyl, or 8-hydroxy-2ʹ-deoxyguanosine), expression of genes mediating oxidative stress, and mitochondrial oxidative phosphorylation. Contrary to our hypothesis, maternal hyperoxia was not associated with increased biomarkers of oxidative stress in the fetal brain. However, there was significant upregulation of the expression of select genes mediating oxidative stress, of which some were male-specific. These observations, however, were not accompanied by changes in the expression of proteins from the mitochondrial electron transport chain. In summary, maternal hyperoxia in the setting of acute uteroplacental ischemia-hypoxia does not appear to cause oxidative damage to the developing brain.

Full text

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
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Impact of intrauterine fetal
resuscitation with oxygen
on oxidative stress
in the developing rat brain
Jia Jiang1,4, Tusar Giri1, Nandini Raghuraman2, Alison G. Cahill3 & Arvind Palanisamy1,2*
Use of maternal oxygen for intrauterine resuscitation is contentious because of the lack of evidence
for its ecacy and the possibility of fetal harm through oxidative stress. Because the developing
brain is rich in lipids and low in antioxidants, it remains vulnerable to oxidative stress. Here, we
tested this hypothesis in a term pregnant rat model with oxytocin-induced fetal distress followed by
treatment with either room air or 100% oxygen for 6 h. Fetal brains from both sexes were subjected
to assays for biomarkers of oxidative stress (4-hydroxynonenal, protein carbonyl, or 8-hydroxy-
2ʹ-deoxyguanosine), expression of genes mediating oxidative stress, and mitochondrial oxidative
phosphorylation. Contrary to our hypothesis, maternal hyperoxia was not associated with increased
biomarkers of oxidative stress in the fetal brain. However, there was signicant upregulation of
the expression of select genes mediating oxidative stress, of which some were male-specic. These
observations, however, were not accompanied by changes in the expression of proteins from the
mitochondrial electron transport chain. In summary, maternal hyperoxia in the setting of acute
uteroplacental ischemia-hypoxia does not appear to cause oxidative damage to the developing brain.
Maternal oxygen administration is one of the most widely practiced interventions for intrauterine resuscitation
of a distressed fetus1–5. However, whether such an intervention improves fetal or neonatal outcomes is question-
able. Recent meta-analyses suggest that maternal oxygen does not improve either fetal oxygenation or acid–base
status3,5. In addition, recent evidence from a randomized controlled trial suggested that room air resuscitation
was non-inferior to oxygen therapy in the management of labor with Category II fetal heart rate tracing6. Taken
together with the added concern that maternal oxygen therapy and the relative hyperoxic environment could
increase plasma biomarkers of oxidative stress during delivery4,7,8, it is plausible that oxygen might be harmful
rather than helpful. An unanswered question in this regard is whether the oxidative stress aects the develop-
ing fetal brain. is is important because the fetal brain is rich in lipids and low in antioxidants which could
make it vulnerable to the eects of oxidative stress9,10. A meaningful inquiry in human subjects, however, is not
possible because of ethical limitations. To address this knowledge gap, we utilized our pragmatic term pregnant
rat model designed to induce fetal distress by stimulating aberrant uterine contractility with oxytocin (OXT)
11. Using this model, we investigated the eect of fetal resuscitation with either room air or 100% oxygen with
the hypothesis that resuscitation with oxygen, compared to room air, would increase oxidative stress in the fetal
brain. Furthermore, considering the sex-dierences in the response to oxidative stress12–16, we speculated that
the male brain would be especially vulnerable.
Results
Dams from both room air and oxygen groups tolerated the experiment and all pups were noted to be alive at
the time of cesarean delivery. e number of dams per treatment condition and their litter data are shown in
Table1. We rst assessed whether maternal hyperoxia was associated with increased fetal oxygenation. Maternal
exposure to 100% oxygen was associated with a signicant increase in the PaO2 and oxygen content of fetal le
OPEN
           
USA. 
USA. Department of Women’s Health, Dell Medical School, The University of Texas at Austin, Austin, TX,
USA. Present address: Department of Anesthesiology, Beijing Chaoyang Hospital, Capital Medical University,
Beijing, China. *
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ventricular blood (Table2 and Fig.1). However, there were no signicant dierences in the pH, paCO2, HCO3,
base decit, or lactate levels between the two groups.
We then assessed whether fetal resuscitation with maternal oxygen induces oxidative damage in the devel-
oping brain using validated biomarkers of oxidative stress (Fig.2). We did not observe either treatment- or
sex-specic dierences in the concentration of 4-HNE, protein carbonyl, and 8-OHdG in the fetal cortex
(Fig.2A–C). Similarly, the magnitude of the antioxidant response, assessed with GSSG/GSH ratio was also no
dierent between the treatment conditions of either fetal sex (Fig.2D).
We next examined the impact of fetal resuscitation with oxygen on the dierential expression of genes
associated with oxidative stress and mitochondrial oxidative phosphorylation in the developing brain of both
sexes (Fig.3). Unlike the oxidative stress biomarkers, we observed a signicant upregulation in the expression
of genes mediating oxidative stress (Ucp3, Nox1), antioxidant response (Sod3, Cat, Prdx3, Txnrd2) and oxidative
Table 1. Details of animal use. One male and female pup/unique dam/treatment condition was used for
experiments unless stated otherwise, with the dam as the experimental unit. M: male ospring; F: female
ospring.
Control
(RA) Oxygen
(100% O2) P value
E 20 dams 8 8
Litter size (mean ± S.D) 10.6 ± 2.4 11.4 ± 1.8 0.48
Sex of the pups (mean ± S.D.)
M4.5 ± 2.3 5.5 ± 1.8 0.35
F6.1 ± 1.8 5.9 ± 1.6 0.77
Survival 100% 100%
Table 2. Blood gas analysis of the pups aer in utero exposure to maternal hyperoxia. Le ventricular blood
from three pups/unique dam/treatment condition was pooled prior to blood gas analysis, with the dam
considered as the experimental unit. *p ≤ 0.05; **p ≤ 0.01
RA (n = 3) 100% O2 (n = 3) p-value
pH 7.1 ± 0.04 7.2 ± 0.07 0.28
pO2 (mmHg) 33 ± 2.6 86 ± 12 0.02*
pCO2 (mmHg) 71 ± 7.3 55 ± 6.8 0.14
HCO3 (mmol/L) 22 ± 0.18 21 ± 1.7 0.71
Lactate (mmol/L) 12 ± 0.7 14 ± 1.1 0.21
Base excess (mmol/L) −8.2 ± 0.72 −9.7 ± 1.4 0.41
O2 content (mL/dL) 6.3 ± 1.3 13 ± 0.73 0.006**
Figure1. Maternal hyperoxia is associated with increased fetal oxygenation. Scatter plots showing the extent
of increase in fetal PaO2 (A) and oxygen content (B) aer maternal hyperoxia with 100% oxygen for 6h. Data
are presented as mean ± S.E.M and analyzed with Welch’s t-test; *p ≤ 0.05 and **p ≤ 0.01 (n = 3 per treatment
condition).
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phosphorylation (Mt-cyb) in the developing brain aer oxygen exposure (Fig.3A). Among these genes, Nox1,
Sod3, Cat, Prdx3, and Txnrd2 were dierentially upregulated in the male vs. female brain aer resuscitation with
oxygen (Fig.3B).
Considering our previous work showing changes in mitochondrial biogenesis aer acute intrapartum
hypoxemia11, we were interested in understanding the additive impact of maternal administration of oxygen
on mitochondrial oxidative phosphorylation. ere were neither treatment nor sex-related dierences in the
expression of proteins related to any of the mitochondrial electron transport chain complexes in the maternal
hyperoxia group (Fig.4).
Discussion
Our investigations in a term pregnant rat model show that maternal hyperoxia in the setting of induced fetal
distress did not appear to be associated with oxidative damage to the developing brain. ough we observed male
sex-specic upregulation of select genes mediating oxidative stress and antioxidant defense, this was not accom-
panied by any changes in the mitochondrial proteins involved in the oxidative phosphorylation pathway. Taken
together with our previous data showing a signicant eect of acute uteroplacental ischemia on oxidative stress
and behavioral outcomes even with room air treatment11, our results suggest that the magnitude of the initial
hypoxemic insult is more likely to impact outcomes rather than the choice of oxygen or room air resuscitation.
Our targeted preclinical research provides important novel data on oxidative stress in the fetal brain during
intrauterine resuscitation with maternal oxygen aer placental ischemia-hypoxemia. ough maternal hyperoxia
is known to increase biomarkers of oxidative stress in the umbilical cord blood7,17, it is unclear whether the puta-
tive oxidative stress aects the fetal brain, arguably the most important organ of interest. By adopting a pragmatic
model of acute, reversible uteroplacental ischemia caused by oxytocin-induced aberrant uterine contractility, our
studies were designed to reect a relatively common and clinically relevant scenario during labor. e absence
Figure2. Oxidative stress biomarkers in the fetal brain aer maternal hyperoxia. Scatter plots showing the
extent of oxidative damage to lipids, proteins, and DNA as quantied by 4-hydroxynonenal (A), protein
carbonyl (B), and 8-OHdG (C), respectively. Maternal hyperoxia was not associated with an increase in any
of the biomarkers in either sex. e GSSG/GSH ratio (D), indicative of the collective glutathione antioxidant
response, was no dierent between the groups. Data are presented as mean ± S.E.M and analyzed with 2-way
ANOVA with Sidak’s multiple comparisons test to assess for sex dierences in the treated group (n = 8 male and
female pups from 8 unique dams/treatment condition).
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Figure3. Dierentially expressed oxidative stress genes in the fetal brain aer maternal hyperoxia. (A) Scatter
plots showing dierential expression of genes involved in the oxidative stress response and prevention aer
maternal hyperoxia. ere was a signicant eect of treatment for Ucp3 ([F (1, 24) = 5.7]) and Mt-cyb ([F (1,
20) = 5.06]), and a signicant treatment vs. sex interaction for Nox1 ([F (1, 22) = 4.32]), Sod3 ([F (1, 21) = 5.5]),
Cat ([F (1, 23) = 7.79]), Prdx3 ([F (1, 21) = 4.46]), and Txnrd2 ([F (1, 23) = 4.76]). (B) Bar graphs highlighting
the sex-dependent dierences in gene expression involving oxidative stress (le) and antioxidant defense (right)
aer maternal hyperoxia with 100% oxygen (ETC: electron transport chain). Data are presented as mean ± S.E.M
and analyzed with 2-way ANOVA with Sidak’s multiple comparisons test to assess for sex dierences in the
treated group; *p ≤ 0.05 and **p ≤ 0.01 (n = 7 male and female pups from 7 unique dams/treatment condition).
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of oxidative damage in the fetal brain aer maternal hyperoxia was contrary to our hypothesis. is unexpected
result could partly be explained by dynamic changes in fetal cerebral blood ow and oxygenation during acute
ischemia-hypoxia. For example, acute hypoxemia followed by reperfusion and a relatively hyperoxic environment
is likely to result in enhanced oxidative stress. So, what could protect the fetal brain from maternal hyperoxia?
Preserved ‘brain-sparing’ blood ow in a previously uncompromised fetus could reduce the fetal brain impact of
acute uteroplacental ischemia and minimize oxidative stress by reducing the magnitude of initial hypoxemia18.
Conversely, it is equally plausible that oxygen delivery to the brain is not signicantly enhanced aer maternal
hyperoxia, thereby limiting the generation of oxygen free radicals. Support for this possibility comes from elegant
BOLD MRI imaging studies in pregnant subjects showing that maternal hyperoxia does not increase the signal
in the fetal brain despite an increase in signal in the extra-cranial fetal organs19. Furthermore, there is evidence
Figure4. Changes in mitochondrial oxidative phosphorylation in the fetal cortex aer maternal hyperoxia.
(A) Representative immunoblots showing all 5 electron transport chain complexes in the cerebral cortical
homogenates of both male and female ospring aer maternal exposure to either room air or hyperoxia, with rat
heart mitochondria as positive control. Exposure time had to be increased from 1 to 3min to visualize Complex
I. (B) Scatter plots highlighting the lack of signicant changes in mitochondrial OXPHOS proteins in the fetal
brain aer exposure to 6h of maternal hyperoxia with 100% oxygen. Data are presented as mean ± S.E.M and
analyzed with 2-way ANOVA with Sidak’s multiple comparisons test to assess for sex dierences in the treated
group (n = 3 each for all experiments).
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that maternal hyperoxia might directly increase fetal cerebral vascular resistance and redistribute blood ow
away from the brain20,21. However, unlike the compromised pups in utero in our study, these imaging studies
were performed in normoxic fetuses at baseline, which might inuence the magnitude and direction of the
‘brain-sparing’ eect. Finally, humoral responses typically associated with altered vasoreactivity aer hyperoxia,
such as decreased production of nitric oxide and inhibition of endothelial prostaglandin synthesis observed
in other settings, could be at play22. Taken together, we are convinced that, unlike the well-characterized fetal
consequences of maternal hypoxia, the fetal eects of maternal hyperoxia are nuanced and need more targeted
mechanistic investigations. Nevertheless, we are reassured with the observation that maternal hyperoxia in the
setting of fetal compromise does not increase oxidative stress biomarkers in the developing brain.
e substantial upregulation of genes related to both the oxidative stress and the antioxidant pathway aer
maternal hyperoxia is a novel nding. We could not ascertain whether the antioxidant response was commensu-
rate with the degree of oxidative stress. However, considering the relatively benign impact of maternal hyperoxia
on oxidative stress biomarkers, we are inclined to favor the possibility of a proportionate antioxidant response.
More interesting to us was the sex dierence in the oxidative stress response, with male fetuses showing a marked
upregulation of Nox1 gene, accompanied by a substantial increase in antioxidant enzymes suggestive for a com-
pensatory response. is is perhaps not surprising considering the sex dierences in oxidative stress response,
mitochondrial biology, and gonadal steroid milieu in the developing brain23–27. For example, females appear to
have increased intracellular glutathione28, increased level of the antioxidant paraoxonase 229, and more robust
mitochondrial biogenesis in males possibly predisposing them to oxidative stress30. A majority of these changes,
however, are known to be mediated by estradiol in the post-pubertal brain, so it remains unclear if the hormonally
neutral intrauterine environment results in such changes in the redox environment of the developing brain. e
male-specic upregulation of Nox1 (NADPH oxidase 1) warrants further studies because of its integral role as a
dedicated mammalian enzyme system involved in the generation of superoxide anions31, a major form of reactive
oxygen species. Upregulation of Nox1 is widely observed during recovery from ischemic stroke32–34, suggesting
that there could be mechanistic parallels between the impact of acute intrapartum ischemia-hypoxemia on the
fetal brain and ischemic stroke. Future studies are required to determine whether these redox responses are
persistent and enduring, and whether they lead to functionally variant outcomes in the ospring.
We had previously shown that mitochondrial oxidative phosphorylation was permanently upregulated in the
cingulate cortex of adolescent male, but not female, rat ospring aer uteroplacental ischemia induced by aber-
rant uterine contractility even without maternal hyperoxia11. Considering the changes in oxidative stress gene
expression with maternal hyperoxia, we sought to determine if this was accompanied by changes in mitochon-
drial oxidative phosphorylation. ough we limited our investigations to the immediate post-hyperoxia period,
we did not observe related changes in the expression of proteins from the mitochondrial electron transport chain.
Whether these sex-dependent changes in oxidative stress gene expression are consequential, enduring, and cause
altered neurobehavioral outcomes need to be determined.
e biggest strength of the study is that it provides the basic scientic foundation regarding the impact of
maternal hyperoxia on oxidative stress in the fetal brain. By replicating an acute in utero clinical scenario, our
study is distinct from other studies in the eld that investigate the eect of hypoxia in newborn pups. e only
comparable study is by Boksa etal. which reported a nuanced eect of oxytocin on redox biology in the fetal
brain35, oxytocin increased the lactate levels but reduced the concentration of brain malondialdehyde, an oxida-
tive stress marker. ough reduced oxidative stress may appear to be a counter-intuitive observation, oxytocin
was administered in this study as a continuous, low-dose infusion which presumably had negligible eects
on uteroplacental perfusion, and therefore fetal oxidative stress, compared to the acute placental ischemia-
hypoxemia noted with our model. Furthermore, it is unclear whether such changes were caused by oxytocin per
se or the 6–15min of induced anoxia at birth that was part of the experimental paradigm. In addition, a direct
eect of oxytocin could not be ruled out. In contrast, our previously established paradigm clearly demonstrated
signicant uteroplacental ischemia by inducing uterine hypercontractility with a dose of oxytocin that is not
known to cross the placenta11,36, resulting in increased condence in our results.
Our study needs to be interpreted in the context of a few limitations. First, the lack of assessment of blood
gases in the dam, and therefore, conrmation of maternal hyperoxia, could be cited as a limitation. However,
previous data from our lab had shown that maternal PaO2 was approximately 350mmHg aer 4h of 100%
oxygen in spontaneously breathing dams37, providing reassurance that maternal hyperoxia was achieved. e
more relevant unanswered question was whether maternal hyperoxia was associated with fetal hyperoxia which
we addressed comprehensively with blood gas studies showing increased partial pressure of oxygen and oxygen
content in the fetal circulation. Second, our assessment time point of 6h may have been either too early or too
late resulting in a failure to capture the magnitude of oxidative stress. However, corroborative evidence suggests
that biomarkers of oxidative damage such as 4-HNE and protein carbonyl can be detected as early as 1–3h and
can be elevated up to 12 h38–41, rendering that possibility unlikely. If 6h of maternal hyperoxia does not cause
oxidative damage, it is probably safe to assume that short-term administration of oxygen in the clinical setting is
unlikely to have a major impact. ird, we did not perform histological analysis of the fetal brain to determine if
there was neuronal cell death. ough we did not observe neuronal apoptosis aer placental ischemia-hypoxemia
in our previous study11, direct exposure to 100% oxygen can be neurotoxic to the developing brain especially
during prolonged or repetitive exposure42,43, and therefore, needs to be ascertained in future experiments. ird,
our study does not exclude the possibility of oxidative stress in fetal organs other than the brain. Similarly, a
brain-region specic change in oxidative stress could not be investigated because of the diculty in ensuring
accurate anatomic brain dissection at this developmental age. Finally, the lack of functional neurobehavioral
outcomes might be considered a limitation, but our study was primarily designed to be a molecular biological
examination of the developing brain aer in utero exposure to maternal hyperoxia.
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In summary, we report that there is no major oxidative damage to the fetal brain aer maternal oxygen therapy
in the setting of intrapartum fetal compromise. Although we noted signicant dierences in the expression
of genes in the oxidative stress-mitochondrial oxidative phosphorylation pathway, of which some were sex-
dependent, there were no changes in the expression of emblematic proteins from the mitochondrial oxidative
phosphorylation pathway. Collectively, our data provide foundational knowledge to better understand the impact
of the choice of resuscitative agent for fetal distress and raises the biological possibility that sex-specic dier-
ences in neonatal neurological outcomes could potentially be amplied by the choice of maternal resuscitative
agent. erefore, follow up studies are required to determine if these gene expression changes are persistent and
whether they lead to worsening of functional neurobehavioral outcomes.
Materials and methods
All experimental procedures were approved by the Institutional Animal Care and Use Committee at Washington
University in St. Louis (#20170010) and comply with the Animal Research: Reporting of InVivo Experiments
(ARRIVE) guidelines. All methods proposed here were performed in accordance with relevant institutional
guidelines and regulations.
Animals and treatment. Timed pregnant Sprague–Dawley (SD) rats (SAS 400, Charles River Laborato-
ries, Wilmington, MA) were acquired on embryonic day (E) 13 and housed under standard housing conditions
until experimentation. On E20, randomized dams were administered 100 mcg/kg oxytocin (OXT) (1mg/mL in
sterile normal saline, Selleck Chemicals, Houston, TX) through a 25G tail vein catheter under brief isourane
anesthesia (with compressed 21% oxygen as the carrier gas) to induce a 10min tetanic uterine contraction and
acute placental ischemia-hypoxemia as described by us previously11. In this paradigm, fetal distress was con-
rmed with elevated fetal brain lactate. We chose this dose of OXT because of minimal transplacental transfer36,
thereby excluding the possibility of a direct eect of OXT on the developing brain. Following treatment, dams
were immediately randomly assigned to two groups during the ischemia–reperfusion period: room air (RA) and
100% oxygen (O). In group RA, treated dams (n = 8) were exposed to 21% oxygen administered at a ow rate of
3L/min into a standard rat container (Kent Scientic Corporation, Torrington, CT) for 6h. Similarly, in Group
O, dams (n = 8) were exposed to 100% oxygen at the same ow rate for 6h. All containers (3 L total volume) were
pre-lled with the respective gases at a rate of 5L/min for at least 3min to reach the satised oxygen concentra-
tion. A small outlet in the container allowed for monitoring of gases and prevented buildup of carbon dioxide.
All dams tolerated the treatment with OXT and the subsequent exposure to dierent gases. 6h aer either RA
or O exposure, fetuses were removed via cesarean delivery under brief isourane anesthesia (in 21% oxygen for
group RA and 100% oxygen for group O, respectively, to maintain treatment group-specic oxygen concentra-
tion). Pups were quickly sexed based on dierences in anogenital distance between males and females, and both
male and female brains were extracted and snap-frozen in liquid nitrogen for storage at −80°C. One pup of
either sex was used per treatment condition.
Collection of fetal left ventricular blood for blood gas analysis. To assess whether maternal hyper-
oxia was associated with an increase in fetal oxygenation, we performed a separate set of experiments. Briey, 6
E20 timed-pregnant Sprague Dawley dams were administered 100 mcg/kg OXT through a 25G tail vein catheter
followed immediately by random assignment to either room air or 100% oxygen treatment (n = 3 each) for 6h as
described above. Subsequently, at least 3 fetuses were collected per dam via cesarean delivery and immediately
dissected to access the thoracic cavity. Because le ventricular puncture was technically challenging due to the
rapid heart rate and the extremely low residence time of blood, we transected the le ventricle, allowed the blood
to pool in the thoracic cavity, and aspirated it immediately into a heparin-coated 1mL syringe. We were able
to collect approximately 100 µL per fetus, and the entire 300 µL from all three fetuses per dam was thoroughly
admixed in the same syringe before blood gas analysis with the Stat Prole Prime® Analyzer (Nova Biomedical,
Waltham, MA).
Assays for biomarkers of oxidative stress. To assess whether exposure to maternal hyperoxia causes
oxidative damage to the fetal brain, we rst assayed for biomarkers of oxidative stress. Specically, we investi-
gated oxidative damage to lipids (4-hydroxynonenal, a lipid peroxidative product), proteins (protein carbonyl),
and DNA (8-hydroxy-2′-deoxyguanosine [8-OHdG]) in the brains of both sexes. In addition, we assayed for the
glutathione (GSSG/GSH) ratio as a marker of the antioxidant response. Approximately 25–50mg of brain tissue
from the le cerebral cortex was used for all experiments. Protein concentration was determined using Pierce™
BCA Protein Assay Kit (ermoFisher Scientic). e extent of oxidative damage to lipids and proteins was
assessed with OxiSelect™ HNE Adduct Competitive ELISA and OxiSelect™ Protein Carbonyl ELISA kits (Cell
Biolabs), respectively, according to manufacturer’s instructions but with slight modications (addition of PEI to
a nal concentration of 0.5% for the protein carbonyl assay). 8-OHdG was quantied in specially prepared corti-
cal tissue lysates. Briey, approximately 25mg of cortical tissue was homogenized to powder with liquid nitrogen
using a pestle. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, USA) and the concentration
was measured using a NanoDrop 2000 spectrophotometer (ermo Scientic™, USA). Oxidative DNA damage
marker 8-oxoguanine (8-OHdG) was quantied using the OxiSelect™ Oxidative DNA Damage ELISA kit (Cell
Biolabs) according to manufacturer’s instructions. Absorbance for all assays was measured at 450nm. For the
total glutathione (GSSG/GSH) assay, lysates were prepared with approximately 50mg of cortical tissue treated
with 5% metaphosphoric acid followed by quantication with OxiSelect™ Total Glutathione (GSSG/GSH) Assay
kit (Cell Biolabs) according to manufacturer’s instructions. Absorbance was immediately read at 405nm at
1min intervals for 10min to determine the slopes for nal calculations.
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Taqman RT-qPCR for dierential expression of oxidative stress genes. Total RNA was iso-
lated from the right fetal cerebral cortex (approx. 30mg) using RNeasy Mini Kit (Qiagen, Germantown, MD).
Genomic DNA was eliminated using QIAshredder (Qiagen, Germantown, MD) and an on-column treatment
with RNase-Free DNase Set (Qiagen, Germantown, MD). 2.5µg of puried RNA (260/280 ratio ≥ 2.0) was
reverse transcribed to cDNA using SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme (ermoFisher
Scientic, Waltham, MA) in a nal volume of 20μl at 37°C for 30min, 50 °C for 10min, 85°C for 5min,
and 4°C for 30min. Subsequently, we performed planned comparisons of treatment-related dierences in the
expression of select target genes related to oxidative stress (Nox1, Nox3, Nos2, Ucp3), antioxidant defense (Cat,
Gpx1, Gpx4, Gsr, Prdx1, Prdx2, Prdx3, Prdx6, Sod1, Sod2, Sod3, Srxn1, Cygb, Ngb, Txnip, Txnrd2), and the mito-
chondrial electron transport chain (ETC) (Mt-cyb, Mt-nd1, Mt-nd2, Mt-nd4, Mt-nd5, Mt-atp8, Mt-co1, Mt-co3).
We included mitochondrial ETC genes because of the integral role of mitochondria in the generation of reactive
oxygen species during oxidative stress. A customized TaqMan Array 96-well FAST plate (ermoFisher Scien-
tic, Waltham, MA) containing these 28 prevalidated probes and 4 endogenous genes (18S rRNA, Actb, Gapdh,
Pgk1) was used for qPCR. Each 10μl reaction contained 5μl of TaqMan Master Mix (ermoFisher Scientic,
Waltham, MA), 3μL of ultrapure water, and 2μl of cDNA. qPCR was performed in an Applied Biosystems
7500 Real-Time PCR System (ermoFisher Scientic, Waltham, MA) with the following cycling conditions:
preincubation at 50°C for 2min and then at 95°C for 10min, followed by 40 amplication cycles (95°C, 15s
and 60°C, 1min) and cooling (40°C, 10s). All reactions were performed in triplicate. Of the 4 endogenous
reference genes, only Actb and Gapdh were stably expressed across experimental conditions (geNorm algorithm;
qbase + version 3.2). Relative mRNA expression, normalized to the geometric mean of Actb and Gapdh, was
calculated using the 2−ΔΔCT method with sex-matched control samples as reference.
Western blot for mitochondrial oxidative phosphorylation. Next, we performed immunoblots of
the developing cortex to quantify the eect of maternal oxygen resuscitation on the mitochondrial oxidative
phosphorylation system (OXPHOS) in the developing brain. Briey, we isolated mitochondria from approxi-
mately 100mg of the fetal cortex using the Mitochondria Isolation Kit (ermoFisher Scientic) followed by
lysis and homogenization with RIPA buer containing protease inhibitor and phosphatase inhibitor in 1X PBS.
Protein concentrations were determined using a BCA protein assay kit (ermoFisher Scientic, USA). 15µg
of each sample was treated with reduced LDS buer and heated at 50°C for 5min, then loaded onto a 10% Bolt
gel (ermoFisher Scientic). Rat heart mitochondria was used as positive control. Aer separation in MES
SDS running buer (ermoFisher Scientic), proteins were transferred to nitrocellulose membrane and sub-
sequently blocked for 2h at room temperature in 5% non-fat dry milk and 1X Tween 20 in Tris-buered saline
(TBS-T). Membrane was incubated with primary mouse anti-OXPHOS antibody (OriGene; 1:3000 diluted in
5% BSA and 1X TBS-T) overnight at 4°C, followed by HRP-conjugated anti-mouse secondary antibody (1:5000
diluted in 5% non-fat dry milk and 1X TBS-T) for 1h at room temperature. A chemiluminescent detection
reagent (ECL Prime, GE Healthcare) was used to visualize the proteins. Aer stripping for 45min followed by
blocking with 5% non-fat dry milk and 1X TBS-T for 30min, the membrane was reprobed with rabbit anti-
VDAC1 antibody (Cell Signaling, US. 1:5000 diluted in 5% non-fat dry milk and 1X TBS-T) for 1h at room
temperature, followed by HRP-conjugated anti-rabbit secondary antibody (1:5000 diluted in 5% non-fat dry
milk and 1X TBS-T) for 1h at room temperature. Images were subsequently processed with Image Studio ver
5.2 (LI-COR) for densitometric quantication. Full-length western blot images are presented as Supplementary
Information.
Statistical analysis. Data outliers were detected and eliminated using ROUT (robust regression and out-
lier analysis) with Q set to 10%. Normality of residuals was checked with D’Agostino-Pearson omnibus test
followed by Welch’s t-test for blood gas data and the 2-way ANOVA and Sidak’s multiple comparisons test for
all other data where sex of the ospring was a variable. RT-qPCR data with non-normal residuals (Nox1, Nos2,
Prdx3, Sod3, Srxn1, Mt-atp8, Mt-co3) were Box-Cox transformed prior to statistical testing. Data are presented
as mean ± SEM and analyzed with Prism 8 for Mac OS X (Graphpad Soware, Inc, La Jolla, CA). A two-tailed P
value ≤ 0.05 was accorded statistical signicance.
Conference presentation. is abstract was presented at the 40th Annual Pregnancy Meeting, Society for
Maternal Fetal Medicine, Grapevine, TX, 76501, February 3–8, 2020.
Received: 26 January 2021; Accepted: 23 April 2021
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Author contributions
J.J. was responsible for conceptualization, investigation, data curation, and writing the initial dra; T.G. assisted
J.J. in the experiments and provided supervision and project administration; N.R. and A.G.C. helped with criti-
cal review and editing, and provided the clinical context; A.P. planned the experiments with J.J., provided the
technical and intellectual resources, performed formal statistical analysis, and critically reviewed and revised
the manuscript.
Funding
Departmental startup funds to AP.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 021- 89299-w.
Correspondence and requests for materials should be addressed to A.P.
Reprints and permissions information is available at www.nature.com/reprints.
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