The developing brain is uniquely susceptible to oxidative stress, and endogenous antioxidant mechanisms are not sufficient to prevent injury from a hypoxic-ischemic challenge. Glutathione peroxidase (GPX1) activity reduces hypoxic-ischemic injury. Therapeutic hypothermia (HT) also reduces hypoxic-ischemic injury, in the rodent and the human brain, but the benefit is limited. Here, we combined GPX1 overexpression with HT in a P9 mouse model of hypoxia-ischemia (HI) to test the effectiveness of both treatments together. Histological analysis showed that wild-type (WT) mice with HT were less injured than WT with normothermia. In the GPX1-tg mice, however, despite a lower median score in the HT-treated mice, there was no significant difference between HT and normothermia. GPX1 protein expression was higher in the cortex of all transgenic groups at 30 min and 24 h, as well as in WT 30 min after HI, with and without HT. GPX1 was higher in the hippocampus of all transgenic groups and WT with HI and normothermia, at 24 h, but not at 30 min. Spectrin 150 was higher in all groups with HI, while spectrin 120 was higher in HI groups only at 24 h. There was reduced ERK1/2 activation in both WT and GPX1-tg HI at 30 min. Thus, with a relatively moderate insult, we see a benefit with cooling in the WT but not the GPX1-tg mouse brain. The fact that we see no benefit with increased GPx1 here in the P9 model (unlike in the P7 model) may indicate that oxidative stress in these older mice is elevated to an extent that increased GPx1 is insufficient for reducing injury. The lack of benefit of overexpressing GPX1 in conjunction with HT after HI indicates that pathways triggered by GPX1 overexpression may interfere with the neuroprotective mechanisms provided by HT.

The developing brain is uniquely susceptible to oxidative stress and subsequent injury [1‒3]. Hydrogen peroxide, in particular, accumulates in the neonatal, but not the adult, brain after hypoxia-ischemia (HI) [4]. Endogenous antioxidant mechanisms, such as catalase and glutathione peroxidase (GPX1), are not sufficient to prevent injury from a hypoxic-ischemic challenge. GPX1 activity has been shown to peak at day 1 (P1) of life in the mouse brain, decrease at P4, again at P7, and decline to adult levels by P14 [5]. Increased GPX1 at P7, however (via GPX1 overexpression in the mouse), reduces HI injury [6, 7]. Hypoxia preconditioning has been shown to protect the brain from subsequent insults, but we found that the GPX1-tg mice lost the protection afforded by GPX1 overexpression when given hypoxia preconditioning. WT littermates of these mice were protected by hypoxia preconditioning, however [7]. We also showed that ERK1/2 is transiently activated by hypoxia preconditioning in WT P7 mice, but this activation is blocked by GPX1 overexpression [8]. This indicates that redox balance depends on ERK1/2 activity, which subsequently protects against oxidative injury.

We previously determined GPX1 activity at P7 to be approximately 1.2-fold higher in the cortex of these GPX1-tg mice than in wild-type (WT) mice [6]. In addition, 24 h after HI, GPX1 activity rose 1.3-fold over naïve GPX1-tg cortex [6]. The P9-10 mouse brain is now considered by many to be more representative of the term-gestation human brain [9]. In the P9 GPX1-tg mouse, GPX1 protein expression was several times higher than WT in the cortex and hippocampus [10]. Since GPX1 targets hydrogen peroxide, its mechanism of action is presumably in the early, or acute, phase of HI injury, while hypothermia (HT), applied after injury onset, acts on the subsequent phase.

As a therapy, HT acts after injury is underway and has been shown to reduce HI injury, in the rodent [11] and the human [12] brain, but the benefit is limited and many questions remain regarding optimal treatment of newborns with HIE [13, 14]. The mechanisms of protection are not fully understood but are wide-ranging and include reduced metabolic rate, decreased production of free radicals, suppression of inflammation, and inhibition of excitotoxicity [14]. Consequently, additional treatments to HT may prove more effective, but they must be synergistic. Indeed, the challenge of inhibiting cell death without interfering with overall recovery indicates therapies should be timed to target the phases of injury and, ultimately, repair [15]. Here, we combined GPX1 overexpression with HT in a P9 mouse model of HI to test the effectiveness of enhanced antioxidant capacity with therapeutic cooling.

Mice

The GPX1-overexpressing mice used here (GPX1-tg) were developed with 1 additional copy of the transgene, resulting in expression in all brain regions analyzed in adult mice, including the cortex and striatum, as previously described [16]. The GPX1 activity ratio in these mice was approximately 1.5 for mesencephalon, and protein expression was 2.4-fold in cortex and 1.9-fold in striatum compared to WT [16]. GPX1-tg mice were bred and maintained at the UCSF Laboratory Animal Resource Center. Male mice heterozygous for GPX1 were bred with female WT (CD1) mice, and the genotype of resulting litters was determined by PCR, using standard methods as previously described [6, 16]. While we have used these mice in several previous studies [6, 7, 17] and have confirmed increased GPX activity and expression in the P7 mouse [6], we felt that the brain of the P9 mouse was sufficiently more mature to warrant confirmation of degree of increased GPX activity.

GPX Activity

To confirm overexpression in P9 GPX1-tg mice, selenium-dependent GPX1 enzymatic activity was measured in naïve cortex and hippocampus as previously described [6, 18]. Briefly, brains were removed from anesthetized GPX1-tg (n = 10) and WT mice (n = 13); cortices and hippocampi were quickly dissected on a cold surface and immediately frozen in methylbutane cooled by dry ice. Brain samples were stored at −80°C until assay, at which time they were homogenized in 50 mm potassium phosphate buffer with 1 mm EDTA (pH 7.0). GPX activity was measured in a coupled test system in which reduced glutathione and tert-butyl hydroperoxide were used as the substrates, and oxidized glutathione produced by GPX activity was measured by kinetic spectrophotometry (340 nm) of glutathione reductase-mediated NADPH oxidation. Units of GPX activity were determined by a standard curve of GPX expressed as units GPX/mg/min, where 1 unit (U) is defined as 1 nmol NADPH oxidized per minute. Protein was determined by Pierce BCA spectrophotometric assay (Pierce, Rockford, IL, USA).

HI/HT

Neonatal mice overexpressing GPX1 and their WT littermates (CD1 background) underwent HI on postnatal day 9 (P9) [11, 19, 20]. Briefly, under isoflurane anesthesia, the left common carotid artery was dissected and coagulated until severed. After recovery with the dam for 1 h, mice were exposed to hypoxia by exposure to 10% oxygen (balance nitrogen) for 40 min. After another 1 h period with the dam, mice were placed in chambers maintained at 36.5°C (normothermia [NT]) or 32°C (HT) for 3.5 h and gradually rewarmed over 30 min before returning to the dam. The temperature of each mouse was measured at the nape of the neck with a hand-held laser-guided remote thermometer which we have compared to a rectal probe and found this measurement to be 0.5°C less than the rectal temperature. Temperature was recorded every 30 min.

Histopathologic Analysis of Injury

For determination of degree of injury, 18 weeks after HI and HT or NT (a timepoint when injury is fully resolved), mice were anesthetized with Euthasol (Virbac AH, Fort Worth, TX, USA), perfused with 4% paraformaldehyde, brains were cut on a vibratome (50 μm), and alternate sections stained with cresyl violet and Perl’s iron stain. Brain injury in the Vannucci model is variable, from large areas of infarct to scattered areas of pyknosis. The Perl’s iron stain reveals these smaller areas of injury and thus provides a complement to the Nissl stain of the cresyl violet. Consequently, all sections are examined, and 11 regions are scored on a scale of 0–3: anterior, middle, and posterior cortex; anterior, middle, and posterior striatum; CA1, CA2, CA3, and dentate gyrus of the hippocampus; and thalamus, with 0 = no injury and 3 = severe cystic infarction, for a cumulative score of 0–33 [21]. In addition, injury volume was measured with the cresyl violet-stained sections using ImageJ software (NIH).

Protein Expression by Western Blot

At 30 min or 24 h after sham surgery, HI and NT, or HI and HT, brains were removed from anesthetized mice, and cortices and hippocampi were quickly dissected on a cold surface and immediately frozen in methylbutane cooled by dry ice. Brain samples were stored at −80°C. For 30 min: WT sham NT (n = 6), WT sham HT (n = 4), WT with HI and NT (n = 9), WT with HI and HT (n = 11), GPX1-tg sham NT (n = 6), GPX1-tg sham HT (n = 4), GPX1-tg with HI and NT (n = 10), GPX1-tg with HI and HT (n = 10). For 24 h: WT sham NT (n = 10), WT sham HT (n = 14), WT with HI and NT (n = 16), WT with HI and HT (n = 16), GPX1-tg sham NT (n = 10), GPX1-tg sham HT (n = 11), GPX1-tg with HI and NT (n = 18), GPX1-tg with HI and HT (n = 17). It should be noted that the timepoints used for Western blots here, 30 min and 24 h after NT or HT exposure, are 5 and 29 h after hypoxia, respectively. Thirty minutes is the point of recovery from HT and is the early timepoint for analysis.

Frozen brain tissue was homogenized in RIPA buffer (Sigma, St. Louis, MO, USA) with protease and phosphatase inhibitors (Thermo Fisher, Rockford, IL, USA) using dounce homogenizers. The homogenate was transferred to chilled Eppendorf tubes and left on ice for 30 min before centrifugation at 14,000 rpm for 15 min at 4°C. The supernatant was transferred to clean chilled tubes, and an aliquot was removed for determination of total protein by BCA assay (Thermo Fisher). 30 μg protein were separated by SDS-PAGE and transferred to PVDF membranes. After blocking for 1 h in 5% non-fat dry milk in TBS with 0.5% TWEEN, membranes were incubated in the following antibodies: goat-actin 1:2,000 (Abcam, Cambridge, MA, USA); rabbit-GPX1, 1:2,000 (Abcam, Cambridge, MA, USA); mouse-spectrin 1:4,000 (Millipore, Temecula, CA, USA); mouse-ERK1/2 1:4,000, Invitrogen; rabbit-phospho-ERK1/2 (Cell Signaling, Danvers, MA, USA); and corresponding secondary antibodies, all 1:2,000. The signal was visualized with enhanced chemiluminescence (Thermo Fisher) and blots exposed to film. Film was scanned, and mean optical densities were determined with ImageJ (NIH).

Statistical Analysis

GPX1 enzymatic assay results were analyzed by unpaired t-test and described as mean U GPX/mg protein ± SEM. Injury scores were evaluated by one-way ANOVA and Kruskal-Wallis with Dunn’s multiple comparison test and are shown as scatter plots with the median value of each group a horizontal line. Injury volumes were compared by t-test and are described as percent injured hemisphere compared to contralateral hemisphere ± S.D. Western blots were normalized to β-actin, evaluated with unpaired t-test, and presented as fold change relative to WT sham NT. Differences were considered significant at p < 0.05. Mortality was analyzed by contingency test and χ2. Graphpad Prism 7.0 (Carlsbad, CA) was used for all analysis except injury volumes which were by Excel.

Confirmation of GPX1 Overexpression in GPX1-tg Mice by Enzymatic Assay

GPX1 activity was higher at P9 in the GPX1-tg (n = 10) compared to WT (n = 13) littermates in both the cortex (GPX1-tg = 11.0 ± 2.02 U/mg vs. WT = 4.28 ± 0.59 U/mg; p < 0.002; Fig. 1a) and hippocampus (GPX1-tg = 11.1 ± 0.98 U/mg vs. WT = 7.92 ± 0.83 U/mg; p < 0.02; Fig. 1b).

Fig. 1.

GPX1 activity is higher in the GPX1-tg brain. a Cortex; **p < 0.002. b Hippocampus; *p < 0.02.

Fig. 1.

GPX1 activity is higher in the GPX1-tg brain. a Cortex; **p < 0.002. b Hippocampus; *p < 0.02.

Close modal

Brain Injury after HI

WT mice with HT were less injured than WT with NT (median scores 6 [n = 13] and 29 [n = 15], respectively; p < 0.03; Fig. 2a). In the GPX1-tg, however, despite a lower median score in the HT-treated mice, there was no significant difference between HT and NT (median scores 19 [n = 12] and 31 [n = 10], respectively; Fig. 2a). The pattern is similar in the brain regions analyzed separately: In the cortex, WT mice with HT were less injured than WT with NT (median scores 3 and 9, respectively; p < 0.03; Fig. 2b). In the GPX1-tg, there was no significant difference between HT and NT (median scores 5.5 and 9, respectively; Fig. 2b). In the hippocampus, WT mice with HT were less injured than WT with NT (median scores 3 and 10, respectively; p = 0.05; Fig. 2c). In the GPX1-tg, there was no significant difference between HT and NT (median scores 7 and 11, respectively; Fig. 2c). In the striatum, WT mice with HT were less injured than WT with NT (median scores 1 and 8, respectively; p < 0.02; Fig. 2d). In the GPX1-tg, there was no significant difference between HT and NT (median scores 5 and 8.5, respectively; Fig. 2d). In the thalamus, WT mice with HT were less injured than WT with NT (median scores 0 and 2, respectively; p < 0.006; Fig. 2e). In the GPX1-tg, the median scores were the same (2) for HT and NT (Fig. 2e). There were no differences in injury scores when comparing male and female of each treatment group (Fig. 2f). However, there were a small number of female mice in the WT with HT (n = 3) and male mice in the GPX1-tg with HT (n = 4) groups.

Fig. 2.

Histological injury scores. WT and GPX1-tg after HI with normothermia (NT) or hypothermia (HT) treatment. Solid horizontal line represents median. a Entire hemisphere (scale 0–33). WT with HT have lower median scores than WT with NT (*p < 0.03). b Cortex (scale 0–9). WT with HT have lower median scores than WT with NT (*p < 0.03). c Hippocampus (scale 0–12). WT with HT have lower median scores than WT with NT (*p = 0.05). d Striatum (scale 0–9). WT with HT have lower median scores than WT with NT (*p < 0.02). e Thalamus (scale 0–3). WT with HT have lower median scores than WT with NT (**p = 0.006). f Male versus female, entire hemisphere. There was a trend toward lower median scores for male GPX1-tg with HT compared to female GPX1-tg with HT (p < 0.09).

Fig. 2.

Histological injury scores. WT and GPX1-tg after HI with normothermia (NT) or hypothermia (HT) treatment. Solid horizontal line represents median. a Entire hemisphere (scale 0–33). WT with HT have lower median scores than WT with NT (*p < 0.03). b Cortex (scale 0–9). WT with HT have lower median scores than WT with NT (*p < 0.03). c Hippocampus (scale 0–12). WT with HT have lower median scores than WT with NT (*p = 0.05). d Striatum (scale 0–9). WT with HT have lower median scores than WT with NT (*p < 0.02). e Thalamus (scale 0–3). WT with HT have lower median scores than WT with NT (**p = 0.006). f Male versus female, entire hemisphere. There was a trend toward lower median scores for male GPX1-tg with HT compared to female GPX1-tg with HT (p < 0.09).

Close modal

Results of the volume measurements correspond with injury scores. HT reduced injury in the WT brain but not the GPX1-tg brain. Ipsilateral hemisphere/contralateral hemisphere (% ± S.D.): WT with HI and NT (63.9 ± 0.21) versus WT with HI and HT (86.3 ± 0.13) p < 0.04. GPX1-tg with HI and NT (69.0 ± 0.22) versus GPX1-tg with HI and HT (79.4 ± 0.17) p = 0.33.

Representative photomicrographs of a brain with median injury for each experimental group, cortex and hippocampus stained with cresyl violet and Perl’s iron stain, respectively, for WT with HI and NT (Fig. 3a), WT with HI and HT (Fig. 3b), GPX1-tg with HI and NT (Fig. 3c), and GPX1-tg with HI and HT (Fig. 3d).

Fig. 3.

Histological injury of WT and GPX1-tg cortex and hippocampus after HI and either NT or HT. Brains shown represent median injury score of each group. Alternate sections stained with cresyl violet (left) or Perl’s iron (right). a WT NT. b WT HT. c GPX-tg NT. d GPX-tg HT. Arrows depict focal areas of cell loss. Scale bar = 500 μm.

Fig. 3.

Histological injury of WT and GPX1-tg cortex and hippocampus after HI and either NT or HT. Brains shown represent median injury score of each group. Alternate sections stained with cresyl violet (left) or Perl’s iron (right). a WT NT. b WT HT. c GPX-tg NT. d GPX-tg HT. Arrows depict focal areas of cell loss. Scale bar = 500 μm.

Close modal

Mortality

There was no difference in mortality between the groups. Four mice died in WT NT, five in WT HT, four in GPX1-tg NT, and five in GPX1-tg HT. One was euthanized due to hydrocephalus (WT HT), three died before weaning (1 GPX1-tg NT, 2 GPX1-tg HT), and the remainder prior to scheduled perfusion.

GPX1 Protein Expression

GPX1 protein expression was increased not only in all GPX1-tg groups but also in WT with HI. Specifically, compared to WT sham NT, in the cortex 30 min after treatment, GPX1 was 3.1-fold higher in WT with HI and NT (p < 0.005), WT with HI and HT was 2.5-fold (p < 0.002), GPX1-tg sham with NT was 2.6-fold (p < 0.03), GPX1-tg sham with HT was 2.3-fold (p < 0.01), GPX1-tg with HI and NT was 2.8-fold (p < 0.01), and GPX1-tg with HI and HT was 3.3-fold (p < 0.02) (Fig. 4a). In the cortex 24 h after treatment, GPX1 was 5.0-fold higher in GPX1-tg sham with NT (p < 0.04), GPX1-tg sham with HT was 2.9-fold (p < 0.009), and GPX1-tg with HI and NT was 4.8-fold (p < 0.007), but not in GPX1-tg with HI and HT (p = 0.17) (Fig. 4b). In the hippocampus 30 min after treatment, there were no differences between groups (Fig. 4c). In the hippocampus 24 h after treatment, GPX1 was 1.70-fold higher in WT with HI and NT (p < 0.004), as well as all GPX1-tg groups: GPX1-tg sham and NT was 2.2-fold (p < 0.04), GPX1-tg sham and HT was 2.3-fold (p < 0.01), GPX1-tg with HI and NT was 2.6-fold (p < 0.007), and GPX1-tg with HI and HT was 1.7-fold (p < 0.02) (Fig. 4d).

Fig. 4.

GPX1 protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min. GPX1 is higher in WT HI NT (**p < 0.005), WT HI HT (**p < 0.002), GPX1-tg sham NT (*p < 0.03), GPX1-tg sham HT (*p < 0.01), GPX1-tg HI NT (*p = 0.01), and GPX1-tg HI HT (*p < 0.02). b Cortex 24 h. GPX1 is higher in GPX1-tg sham NT (* = p < 0.04), GPX1-tg sham HT (** = p < 0.009), and GPX1-tg HI NT (**p < 0.007) but not to GPX1-tg HI HT (p = 0.17). c Hippocampus 30 min. There were no differences between groups at 30 min. d Hippocampus 24 h. GPX1 is higher in WT HI NT (**p < 0.004), GPX1 sham NT (*p < 0.04), GPX1 sham HT (*p < 0.01), GPX1-tg HI NT (**p < 0.007), and GPX1-tg HI HT (*p < 0.02).

Fig. 4.

GPX1 protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min. GPX1 is higher in WT HI NT (**p < 0.005), WT HI HT (**p < 0.002), GPX1-tg sham NT (*p < 0.03), GPX1-tg sham HT (*p < 0.01), GPX1-tg HI NT (*p = 0.01), and GPX1-tg HI HT (*p < 0.02). b Cortex 24 h. GPX1 is higher in GPX1-tg sham NT (* = p < 0.04), GPX1-tg sham HT (** = p < 0.009), and GPX1-tg HI NT (**p < 0.007) but not to GPX1-tg HI HT (p = 0.17). c Hippocampus 30 min. There were no differences between groups at 30 min. d Hippocampus 24 h. GPX1 is higher in WT HI NT (**p < 0.004), GPX1 sham NT (*p < 0.04), GPX1 sham HT (*p < 0.01), GPX1-tg HI NT (**p < 0.007), and GPX1-tg HI HT (*p < 0.02).

Close modal

Spectrin Protein Expression

Spectrin expression was measured to determine the effect of HT and GPX1 overexpression on cell death mechanisms, as seen by the 145/150 kD fragments indicating the calpain-specific action of necrosis or the 120 kD fragments indicating the caspase-specific action of apoptosis, at an early (30 min) and late (24 h) stage of injury.

Spectrin 145/150 increased in all groups with HI, demonstrating the role of necrosis in neonatal HI injury, there was, however, no clear effect with either HT or GPX1 overexpression. In the cortex at 30 min, WT with HI and NT was 2.6-fold (p < 0.01), WT with HI and HT was 2.1-fold (p < 0.05), GPX1-tg with HI and NT was 3.4-fold (p < 0.04), and GPX1-tg with HI and HT was 3.9-fold (p < 0.006) (Fig. 5a). In the cortex at 24 h, WT with HI and NT was 6.9-fold (p < 0.003), WT with HI and HT was 4.9-fold (p < 0.03), GPX1-tg with HI and NT was 4.0-fold (p < 0.02) and GPX1-tg with HI and HT was 3.8-fold (p < 0.02) (Fig. 5b); in the hippocampus at 30 min, WT with HI and NT was 4.8-fold (p < 0.05), WT with HI and HT was 6.9-fold (p < 0.05), GPX1-tg with HI and NT was 7.0-fold (p < 0.03) and GPX1-tg with HI and HT was 3.4-fold (p < 0.04) (Fig. 5c); in the hippocampus at 24 h, WT with HI and NT was 3.6-fold (p < 0.03), WT with HI and HT was 4.8-fold (p < 0.002), GPX1-tg with HI and NT was 5.2-fold (p < 0.02) and GPX1-tg with HI and HT was 3.9-fold (p < 0.05) (Fig. 5d).

Fig. 5.

Spectrin 145/150 protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min. Spectrin 145/150 is elevated in WT HI NT (*p < 0.01), WT HI HT (*p < 0.05), GPX1-tg HI NT (*p < 0.04), and GPX1-tg HI HT (**p < 0.006). b Cortex 24 h. Spectrin 145/150 is elevated in WT HI NT (**p < 0.003), WT HI HT (*p < 0.03), GPX1-tg HI NT (*p < 0.02), and GPX1-tg HI HT (*p < 0.02). c Hippocampus 30 min. Spectrin 145/150 is elevated in WT HI NT (*p < 0.05), WT HI HT (*p < 0.05), GPX1-tg HI NT (*p < 0.03), and GPX1-tg HI HT (*p < 0.04). d Hippocampus 24 h. Spectrin 145/150 is elevated in WT HI NT (*p < 0.03), WT HI HT (**p < 0.002), GPX1-tg HI NT (*p < 0.02), and GPX1-tg HI HT (*p < 0.05).

Fig. 5.

Spectrin 145/150 protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min. Spectrin 145/150 is elevated in WT HI NT (*p < 0.01), WT HI HT (*p < 0.05), GPX1-tg HI NT (*p < 0.04), and GPX1-tg HI HT (**p < 0.006). b Cortex 24 h. Spectrin 145/150 is elevated in WT HI NT (**p < 0.003), WT HI HT (*p < 0.03), GPX1-tg HI NT (*p < 0.02), and GPX1-tg HI HT (*p < 0.02). c Hippocampus 30 min. Spectrin 145/150 is elevated in WT HI NT (*p < 0.05), WT HI HT (*p < 0.05), GPX1-tg HI NT (*p < 0.03), and GPX1-tg HI HT (*p < 0.04). d Hippocampus 24 h. Spectrin 145/150 is elevated in WT HI NT (*p < 0.03), WT HI HT (**p < 0.002), GPX1-tg HI NT (*p < 0.02), and GPX1-tg HI HT (*p < 0.05).

Close modal

Since spectrin 120 is a marker for apoptosis, which occurs in the later stages of HI injury, it is not surprising that there was no change in the cortex at 30 min (Fig. 6a). In the cortex at 24 h, however, all HI groups showed about a 20-fold increase over WT sham: WT with HI and NT was 27.1-fold (p < 0.02), WT with HI and HT was 24.6-fold (p = 0.05), GPX1-tg with HI and NT was 16.1-fold (p < 0.02), and GPX1-tg with HI and HT was 25.1-fold (p < 0.04) (Fig. 6b). Similar to the cortex, there was no change in the hippocampus at 30 min (Fig. 6c), but there was at 24 h: WT with HI and NT increased by 21.6-fold (p < 0.02), WT with HI and HT by 13.0-fold (p = 0.05), and GPX1-tg with HI and NT by 14.1-fold (p < 0.03) (Fig. 6d). In GPX1-tg with HI and HT, although the mean was more than 10 times higher than WT sham, it did not reach significance (11.5-fold, p = 0.07) (Fig. 6d).

Fig. 6.

Spectrin 120 protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min. There were no differences between groups. b Cortex 24 h. Spectrin 120 was elevated in WT HI NT (*p < 0.02), WT HI HT (*p = 0.05), GPX1-tg HI NT (*p < 0.02), and GPX1-tg HI HT (*p < 0.04). c Hippocampus 30 min. There were no differences between groups. d Hippocampus 24 h. Spectrin 120 was elevated in WT HI NT (*p < 0.02), WT HI HT (*p = 0.05), and GPX1-tg HI NT (*p < 0.03) but not GPX1-tg HI HT (p = 0.07).

Fig. 6.

Spectrin 120 protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min. There were no differences between groups. b Cortex 24 h. Spectrin 120 was elevated in WT HI NT (*p < 0.02), WT HI HT (*p = 0.05), GPX1-tg HI NT (*p < 0.02), and GPX1-tg HI HT (*p < 0.04). c Hippocampus 30 min. There were no differences between groups. d Hippocampus 24 h. Spectrin 120 was elevated in WT HI NT (*p < 0.02), WT HI HT (*p = 0.05), and GPX1-tg HI NT (*p < 0.03) but not GPX1-tg HI HT (p = 0.07).

Close modal

ERK1/2 Protein Expression

ERK1/2 is of interest primarily in comparison to its activated state. Thus, we do not expect to see significant differences between the groups. Indeed, there were no changes in ERK1/2 expression in the cortex at 30 min (Fig. 7a), the cortex at 24 h (Fig. 7b), the hippocampus at 30 min (Fig. 7c), or the hippocampus at 24 h (Fig. 7d).

Fig. 7.

ERK1/2 protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min. There were no differences in ERK1/2. b Cortex 24 h. There were no differences in ERK1/2. c Hippocampus 30 min. There were no differences in ERK1/2. d Hippocampus 24 h. There were no differences in ERK1/2.

Fig. 7.

ERK1/2 protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min. There were no differences in ERK1/2. b Cortex 24 h. There were no differences in ERK1/2. c Hippocampus 30 min. There were no differences in ERK1/2. d Hippocampus 24 h. There were no differences in ERK1/2.

Close modal

Phosphorylated ERK1/2 Protein Expression

In the cortex at 30 min, phosphorylation of ERK1/2 was lower in WT with HI and HT by −0.57-fold (p < 0.04), and while there was a trend toward lower phosphorylation of ERK1/2 in GPX1-tg with HI and HT, it did not reach significance (p = 0.054) (Fig. 8a). There were no differences in the levels of phosphorylation of ERK1/2 in the cortex at 24 h (Fig. 8b), the hippocampus at 30 min (Fig. 8c), or the hippocampus at 24 h (Fig. 8d).

Fig. 8.

Phospho-ERK1/2 (p-ERK) protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min p-ERK is lower in WT HI HT (*p < 0.04). There was a trend to lower p-ERK in GPX1-tg HI HT which was not significant (p = 0.054). b Cortex 24 h. There were no differences in p-ERK. c Hippocampus 30 min. There were no differences in p-ERK. d Hippocampus 24 h. There were no differences in p-ERK.

Fig. 8.

Phospho-ERK1/2 (p-ERK) protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min p-ERK is lower in WT HI HT (*p < 0.04). There was a trend to lower p-ERK in GPX1-tg HI HT which was not significant (p = 0.054). b Cortex 24 h. There were no differences in p-ERK. c Hippocampus 30 min. There were no differences in p-ERK. d Hippocampus 24 h. There were no differences in p-ERK.

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The Ratio of Phosphorylated ERK1/2/ERK1/2

The decreased phosphorylation of ERK1/2 is apparent in the ratio of phospho-ERK1/2/ERK1/2 early after TH. In the cortex at 30 min, WT with HI and NT was lower by −0.45-fold (p < 0.04), WT with HI and HT by −0.54-fold (p < 0.008), and GPX1-tg with HI and HT by −0.47-fold (p < 0.002). Also, GPX1-tg with HI and HT is lower than other GPX1-tg groups: compared to GPX1-tg sham with NT by −0.49-fold (p < 0.004), GPX1-tg sham with HT by −0.56-fold (p < 0.004), and GPX1-tg with HI and NT by −0.02-fold (p < 0.02) (Fig. 9a). In the cortex at 24 h, there were no changes in the ratio of phospho-ERK1/2/ERK1/2 (Fig. 9b). In the hippocampus at 30 min, phospho-ERK1/2/ERK1/2 was lower in WT with HI and NT by −0.29-fold (p < 0.05), in GPX1-tg with HI and NT by 0.36-fold (p < 0.03), and in GPX1-tg with HI and HT by −0.27-fold (p < 0.02). Also, phospho-ERK1/2/ERK1/2 is lower in GPX1-tg with HI and NT by 0.36-fold and in GPX1-tg with HI and HT by 0.27-fold, compared to GPX1-tg sham NT (p < 0.004 and p < 0.02, respectively) (Fig. 9c). In the hippocampus at 24 h, there were no changes in the ratio of phospho-ERK1/2/ERK1/2 (Fig. 9d).

Fig. 9.

Phospho-ERK/ERK. Protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min p-ERK/ERK is lower in WT HI NT (*p < 0.04), WT HI HT (**p < 0.008), and GPX1-tg HI HT (**p < 0.002). Also, GPX1-tg HI HT is lower than GPX1-tg sham NT (^^ p < 0.004), GPX1-tg sham HT (^^ p = 0.004), and GPX1-tg HI NT (^ p < 0.02). b Cortex 24 h. There were no differences in p-ERK/ERK. c Hippocampus 30 min p-ERK/ERK is lower in WT HI NT (*p = 0.05), GPX1-tg HI NT (*p < 0.03), and in GPX1-tg HI HT (*p < 0.02). Also, p-ERK/ERK is lower in GPX1-tg HI HT compared to GPX1-tg sham NT (^ p < 0.02). GPX1-tg HI NT is also lower than GPX1-tg sham NT (^^ p < 0.004). d Hippocampus 24 h. There were no differences in p-ERK/ERK.

Fig. 9.

Phospho-ERK/ERK. Protein expression in WT and GPX1-tg after HI and either NT or HT. Shown as fold change compared to WT sham NT. a Cortex 30 min p-ERK/ERK is lower in WT HI NT (*p < 0.04), WT HI HT (**p < 0.008), and GPX1-tg HI HT (**p < 0.002). Also, GPX1-tg HI HT is lower than GPX1-tg sham NT (^^ p < 0.004), GPX1-tg sham HT (^^ p = 0.004), and GPX1-tg HI NT (^ p < 0.02). b Cortex 24 h. There were no differences in p-ERK/ERK. c Hippocampus 30 min p-ERK/ERK is lower in WT HI NT (*p = 0.05), GPX1-tg HI NT (*p < 0.03), and in GPX1-tg HI HT (*p < 0.02). Also, p-ERK/ERK is lower in GPX1-tg HI HT compared to GPX1-tg sham NT (^ p < 0.02). GPX1-tg HI NT is also lower than GPX1-tg sham NT (^^ p < 0.004). d Hippocampus 24 h. There were no differences in p-ERK/ERK.

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This study expands upon our previous work on oxidative mechanisms in neonatal HI via GPX1 overexpression and with HT. The combination of two potentially protective interventions, GPX1 overexpression and HT, is a novel way of exploring the impact of HT on oxidative injury in the neonatal brain. This is the first study to show that there is no synergism of HT with GPX1 overexpression.

With a relatively moderate insult (40 min of 10% oxygen), we see a benefit with cooling in the WT but not in the GPX1-tg when the insult occurs at P9. The P9 mouse brain is now considered to more closely mimic the human brain at term birth, and the fact that we do not see a significant benefit with increased GPX1 here in the P9 model may be specific to this stage of development. It may indicate that oxidative stress in these slightly older mice is elevated to an extent that the degree of increase in the amount of GPX1 due to the transgene is insufficient for reducing injury, presumably due to an excess of hydrogen peroxide. It may also be a consequence of a surprisingly severe degree of injury overall, as demonstrated by the WT with NT group. We have shown the benefits of cooling in CD1 WT mice previously with a more severe insult (50 min 10% oxygen), in which a large number of NT-treated mice had severe injury [22]. Consequently, we reduced the duration of hypoxia to 40 min, aiming for a more moderate insult compared to 50 min of hypoxia. We have previously shown that GPX1 activity is higher in the naïve P7 GPX1-tg mouse cortex and hippocampus compared to WT and have now confirmed higher activity in the P9 GPX1-tg cortex and hippocampus. In the P7 model of HI, we previously showed decreased injury in GPX1-tg mice compared to WT littermates as a consequence of reduced hydrogen peroxide accumulation. The comparison to the P7 mouse may also suggest that increased antioxidant availability is more likely to reduce injury in the pre-term brain than the term brain.

While there are eight known forms of GPX [23], GPX1 (along with catalase) is considered to be the primary detoxifier of hydrogen peroxide in the brain [24]. Also, hydrogen peroxide accumulates in neonatal, but not adult, mouse brain after HI [4]. In recent years, however, there has been much interest in the phospholipid hydroperoxide GPX4. Also found in the brain, GPX4 reduces hydrogen peroxide at a much slower rate than GPX1 but, importantly, reduces hydroperoxides in cell membranes [25], thereby inhibiting ferroptosis [26]. The roles of GPX1 and GPX4 in HI, whether distinct or complementary, have yet to be fully determined.

Some previous studies using the Vannucci model of HI have found differences in injury severity associated with sex of the mice, with males being more injured than females [27, 28]. However, one study using P10 C57Bl/6 mice and 4 h of HT found no reduction in injury or seizure susceptibility overall with HT, but injury did correlate with seizure susceptibility in male mice only [29]. The fact that we did not find sex differences here could be attributable to low numbers in some groups, the relatively high degree of injury severity overall, strain of mouse used, or other factors yet to be understood. Regardless, sex differences in HI merit continued attention.

Whereas HT likely acts on a number of physiological mechanisms, delaying or diminishing secondary energy failure, edema, and inflammation during the hours after the initial injury, GPX1 is more specific in its action. It reduces the hydrogen peroxide that is produced by superoxide dismutase during the acute phase of HI injury as a consequence of excitotoxicity and oxidative stress, yet in GPX1-tg mice, the overexpression is ongoing. It is conceivable that HT applied during the later stages of the acute phase (beginning 1 h after mice resume breathing room air) negates the potential beneficial effects of ongoing overexpression of GPX1 in the brain. The increase in GPX1 protein seen in the WT cortex 30 min after HI and either NT or HT treatment (which is absent at 24 h) supports the idea that in this late acute phase, the brain is increasing its endogenous antioxidant defenses. HT may be synergistic to this increased GPX1 in the WT cortex but not the GPX1-tg. Regional differences in the timing of GPX1 expression, and perhaps antioxidant defense mechanisms overall, are suggested by the lack of increased GPX1 in the hippocampus in any group at 30 min, yet increased GPX1 is seen at 24 h.

Spectrin expression could provide a glimpse of the effects of HT and GPX1 overexpression on cell death mechanisms, whether necrotic or apoptotic. It is not surprising that spectrin 145/150, as a marker of necrosis, was present in all HI groups at 30 min and persists for 24 h, but it is notable that it was particularly high in the hippocampus. It is also not surprising that there is a lack of significant spectrin 120, a marker of apoptosis, at 30 min. The high levels of spectrin 120 at 24 h (approximately 10–25-fold) suggest ongoing apoptotic cell death which is not diminished by HT treatment or GPX overexpression.

We chose 30 min after HT or NT exposure as a timepoint for Western blot experiments in part due to our previous finding in the P7 mouse that hypoxia alone activates ERK1/2 in the WT cortex at 30 min, and GPX1 overexpression prevents this activation. In this study, HI transiently reduced ERK activation at 30 min in both the cortex and hippocampus in both WT and GPX1-tg, with and without HT. Since the ERK pathway is involved in many cellular processes, both pro-survival and pro-death, it is difficult to know what the consequence of this reduced activation is; however, this decrease mirrors the increase in spectrin 145/150 in the cortex at 30 min, suggesting that reduced ERK activation plays a role in necrotic cell death in HI.

In summary, this work contributes to our understanding of antioxidant mechanisms in neonatal HI, as well as the limitations and promise of HT in combination with genetic or pharmacological interventions.

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at UCSF under protocol AN187224 (D.M.F.) and carried out with standards of care in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

The authors have no conflicts of interest to disclose.

This study and all authors were supported by the grants NIH RO1 33997 and R35 NS097299 (to D.M.F.).

R. Ann Sheldon wrote the manuscript and acquired, analyzed, and interpreted data. Fuxin Lu performed animal experiments and procedures. Christine Windsor performed animal experiments and procedures and data acquisition and analysis. Nicholas Stewart did data acquisition and analysis. Xiangning Jiang provided oversight and assistance with experiments and helpful comments on the manuscript. Donna M. Ferriero conceived of and provided oversight of all aspects of the study and assisted with the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

1.
Gunn
AJ
,
Thoresen
M
.
Neonatal encephalopathy and hypoxic-ischemic encephalopathy
.
Handb Clin Neurol
.
2019
;
162
:
217
37
.
2.
Torres-Cuevas
I
,
Corral-Debrinski
M
,
Gressens
P
.
Brain oxidative damage in murine models of neonatal hypoxia/ischemia and reoxygenation
.
Free Radic Biol Med
.
2019 Oct
142
3
15
.
3.
Juul
SE
,
Ferriero
DM
.
Pharmacologic neuroprotective strategies in neonatal brain injury
.
Clin Perinatol
.
2014 Mar
41
1
119
31
.
4.
Lafemina
MJ
,
Sheldon
RA
,
Ferriero
DM
.
Acute hypoxia-ischemia results in hydrogen peroxide accumulation in neonatal but not adult mouse brain
.
Pediatr Res
.
2006 May
59
5
680
3
.
5.
Khan
JY
,
Black
SM
.
Developmental changes in murine brain antioxidant enzymes
.
Pediatr Res
.
2003 Jul
54
1
77
82
.
6.
Sheldon
RA
,
Jiang
X
,
Francisco
C
,
Christen
S
,
Vexler
ZS
,
Tauber
MG
et al
.
Manipulation of antioxidant pathways in neonatal murine brain
.
Pediatr Res
.
2004 Oct
56
4
656
62
.
7.
Sheldon
RA
,
Aminoff
A
,
Lee
CL
,
Christen
S
,
Ferriero
DM
.
Hypoxic preconditioning reverses protection after neonatal hypoxia-ischemia in glutathione peroxidase transgenic murine brain
.
Pediatr Res
.
2007 Jun
61
6
666
70
.
8.
Autheman
D
,
Sheldon
RA
,
Chaudhuri
N
,
von Arx
S
,
Siegenthaler
C
,
Ferriero
DM
et al
.
Glutathione peroxidase overexpression causes aberrant ERK activation in neonatal mouse cortex after hypoxic preconditioning
.
Pediatr Res
.
2012 Dec
72
6
568
75
.
9.
Northington
FJ
.
Brief update on animal models of hypoxic-ischemic encephalopathy and neonatal stroke
.
ILAR J
.
2006
;
47
(
1
):
32
8
.
10.
Sheldon
RA
,
Sadjadi
R
,
Lam
M
,
Fitzgerald
R
,
Ferriero
DM
.
Alteration in downstream hypoxia gene signaling in neonatal glutathione peroxidase overexpressing mouse brain after hypoxia-ischemia
.
Dev Neurosci
.
2015
37
4–5
398
406
.
11.
Carlsson
Y
,
Wang
X
,
Schwendimann
L
,
Rousset
CI
,
Jacotot
E
,
Gressens
P
et al
.
Combined effect of hypothermia and caspase-2 gene deficiency on neonatal hypoxic-ischemic brain injury
.
Pediatr Res
.
2012 May
71
5
566
72
.
12.
Jacobs
SE
,
Berg
M
,
Hunt
R
,
Tarnow-Mordi
WO
,
Inder
TE
,
Davis
PG
.
Cooling for newborns with hypoxic ischaemic encephalopathy
.
Cochrane Database Syst Rev
.
2013 Jan 31
1
CD003311
.
13.
Azzopardi
D
,
Strohm
B
,
Marlow
N
,
Brocklehurst
P
,
Deierl
A
,
Eddama
O
et al
.
Effects of hypothermia for perinatal asphyxia on childhood outcomes
.
N Engl J Med
.
2014 Jul 10
371
2
140
9
.
14.
Sabir
H
,
Bonifacio
SL
,
Gunn
AJ
,
Thoresen
M
,
Chalak
LF
et al
Newborn Brain Society Guidelines and Publications Committee
.
Unanswered questions regarding therapeutic hypothermia for neonates with neonatal encephalopathy
.
Semin Fetal Neonatal Med
.
2021 Jun 12
26
5
101257
.
15.
Wassink
G
,
Davidson
JO
,
Lear
CA
,
Juul
SE
,
Northington
F
,
Bennet
L
et al
.
A working model for hypothermic neuroprotection
.
J Physiol
.
2018 Dec
596
23
5641
54
.
16.
Mirault
ME
,
Tremblay
A
,
Furling
D
,
Trepanier
G
,
Dugre
F
,
Puymirat
J
et al
.
Transgenic glutathione peroxidase mouse models for neuroprotection studies
.
Ann N Y Acad Sci
.
1994 Nov 17
738
104
15
.
17.
Payton
KS
,
Sheldon
RA
,
Mack
DW
,
Zhu
C
,
Blomgren
K
,
Ferriero
DM
et al
.
Antioxidant status alters levels of Fas-associated death domain-like IL-1B-converting enzyme inhibitory protein following neonatal hypoxia-ischemia
.
Dev Neurosci
.
2007
29
4–5
403
11
.
18.
Flohe
L
,
Gunzler
WA
.
Assays of glutathione peroxidase
.
Methods Enzymol
.
1984
;
105
:
114
21
.
19.
Rice
JE
3rd
,
Vannucci
RC
,
Brierley
JB
.
The influence of immaturity on hypoxic-ischemic brain damage in the rat
.
Ann Neurol
.
1981 Feb
9
2
131
41
.
20.
Ditelberg
JS
,
Sheldon
RA
,
Epstein
CJ
,
Ferriero
DM
.
Brain injury after perinatal hypoxia-ischemia is exacerbated in copper/zinc superoxide dismutase transgenic mice
.
Pediatr Res
.
1996 Feb
39
2
204
8
.
21.
Sheldon
RA
,
Windsor
C
,
Ferriero
DM
.
Strain-related differences in mouse neonatal hypoxia-ischemia
.
Dev Neurosci
.
2018
40
5–6
490
6
.
22.
Koo
E
,
Sheldon
RA
,
Lee
BS
,
Vexler
ZS
,
Ferriero
DM
.
Effects of therapeutic hypothermia on white matter injury from murine neonatal hypoxia-ischemia
.
Pediatr Res
.
2017 Sep
82
3
518
26
.
23.
Brigelius-Flohe
R
,
Maiorino
M
.
Glutathione peroxidases
.
Biochim Biophys Acta
.
2013 May
1830
5
3289
303
.
24.
Liddell
JR
,
Dringen
R
,
Crack
PJ
,
Robinson
SR
.
Glutathione peroxidase 1 and a high cellular glutathione concentration are essential for effective organic hydroperoxide detoxification in astrocytes
.
Glia
.
2006 Dec
54
8
873
9
.
25.
Zhang
LP
,
Maiorino
M
,
Roveri
A
,
Ursini
F
.
Phospholipid hydroperoxide glutathione peroxidase: specific activity in tissues of rats of different age and comparison with other glutathione peroxidases
.
Biochim Biophys Acta
.
1989 Nov 6
1006
1
140
3
.
26.
Forcina
GC
,
Dixon
SJ
.
GPX4 at the crossroads of lipid homeostasis and ferroptosis
.
Proteomics
.
2019 Sep
19
18
e1800311
.
27.
Zhu
C
,
Xu
F
,
Wang
X
,
Shibata
M
,
Uchiyama
Y
,
Blomgren
K
et al
.
Different apoptotic mechanisms are activated in male and female brains after neonatal hypoxia-ischaemia
.
J Neurochem
.
2006 Feb
96
4
1016
27
.
28.
Lechner
CR
,
McNally
MA
,
St Pierre
M
,
Felling
RJ
,
Northington
FJ
,
Stafstrom
CE
et al
.
Sex specific correlation between GABAergic disruption in the dorsal hippocampus and flurothyl seizure susceptibility after neonatal hypoxic-ischemic brain injury
.
Neurobiol Dis
.
2021 Jan
148
105222
.
29.
McNally
MA
,
Chavez-Valdez
R
,
Felling
RJ
,
Flock
DL
,
Northington
FJ
,
Stafstrom
CE
.
Seizure susceptibility correlates with brain injury in male mice treated with hypothermia after neonatal hypoxia-ischemia
.
Dev Neurosci
.
2019 Feb 28
40
5–6
576
85
.