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Brain interstitial pH (pH brain ) alterations play a crucial role in the development of hypoxic-ischemic (HI) encephalopathy (HIE) caused by asphyxia in neonates. The newborn pig is one of the most suitable large animal models for studying HIE, however, compared to rats, experimental data on pH brain alterations during HIE induction are limited. The major objective of the present study was thus to compare pH brain changes during HIE development induced by experimental normocapnic hypoxia (H) or asphyxia (A), elicited with ventilation of a gas mixture containing 6%O 2 or 6%O 2 /20%CO 2 , respectively for 20 min, under either normothermia (NT) or hypothermia (HT) (38.5 ± 0.5 °C or 33.5 ± 0.5 °C core temperature, respectively) in anesthetized piglets yielding four groups: H-NT, A-NT, H-HT, and A-HT. pH brain changes during HI stress and the 60 min reoxygenation period were measured using a pH-selective microelectrode inserted into the parietal cortex through an open cranial window. In all groups, the pH brain response to HI stress was acidosis, at the nadir pH brain values dropped from the baseline of 7.27 ± 0.02 to H-NT:5.93 ± 0.30, A-NT:5.90 ± 0.52, H-HT:6.81 ± 0.27, and A-HT:6.27 ± 0.24 indicating that (1) H and A elicited similar, severe brain acidosis under NT greatly exceeding pH changes in arterial blood (pH a dropped to 7.24 ± 0.07 and 6.78 ± 0.03 from 7.52 ± 0.06 and 7.50 ± 0.05, respectively), and (2) HT ameliorated more the brain acidosis induced by H than by A. In all four groups, pH brain was restored to baseline values without an alkalotic overshoot during the observed reoxygenation, Our findings suggest that under NT either H or A - both commonly employed HI stresses to elicit HIE in piglet models - would result in a similar acidotic pH brain response without an alkalotic component either during the HI stress or the early reoxygenation period., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Kynurenic acid (KYNA), an endogenous neuroprotectant with antiexcitotoxic, antioxidant, and anti-inflammatory effects, is synthesized through the tryptophan-kynurenine (KYN) pathway. We investigated whether brain KYN or KYNA levels were affected by asphyxia in a translational piglet model of hypoxic-ischemic encephalopathy (HIE). We also studied brain levels of the putative blood-brain barrier (BBB) permeable neuroprotective KYNA analogue SZR72, and whether SZR72 or therapeutic hypothermia (TH) modified KYN or KYNA levels. KYN, KYNA, and SZR72 levels were determined using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry in five brain regions 24 h after 20 min of asphyxia in vehicle-, SZR72- and TH-treated newborn piglets (n = 6-6-6) and naive controls (n = 4). Endogenous brain KYN levels (median range 311.2-965.6 pmol/g) exceeded KYNA concentrations (4.5-6.0 pmol/g) ~100-fold. Asphyxia significantly increased cerebral KYN and KYNA levels in all regions (1512.0-3273.9 and 16.9-21.2 pmol/g, respectively), increasing the KYN/Tryptophan-, but retaining the KYNA/KYN ratio. SZR72 treatment resulted in very high cerebral SZR72 levels (13.2-33.2 nmol/g); however, KYN and KYNA levels remained similar to those of the vehicle-treated animals. However, TH virtually ameliorated asphyxia-induced elevations in brain KYN and KYNA levels. The present study reports for the first time that the KYN pathway is altered during HIE development in the piglet. SZR72 readily crosses the BBB in piglets but fails to affect cerebral KYNA levels. Beneficial effects of TH may include restoration of the tryptophan metabolism to pre-asphyxia levels.

Hypoxic-ischemic encephalopathy (HIE) remains to be a major cause of long-term neurodevelopmental deficits in term neonates. Hypothermia offers partial neuroprotection warranting research for additional therapies. Kynurenic acid (KYNA), an endogenous product of tryptophan metabolism, was previously shown to be beneficial in rat HIE models. We sought to determine if the KYNA analog SZR72 would afford neuroprotection in piglets. After severe asphyxia (pHa = 6.83 ± 0.02, ΔBE = -17.6 ± 1.2 mmol/L, mean ± SEM), anesthetized piglets were assigned to vehicle-treated (VEH), SZR72-treated (SZR72), or hypothermia-treated (HT) groups ( n = 6, 6, 6; Tcore = 38.5, 38.5, 33.5 °C, respectively). Compared to VEH, serum KYNA levels were elevated, recovery of EEG was faster, and EEG power spectral density values were higher at 24 h in the SZR72 group. However, instantaneous entropy indicating EEG signal complexity, depression of the visual evoked potential (VEP), and the significant neuronal damage observed in the neocortex, the putamen, and the CA1 hippocampal field were similar in these groups. In the caudate nucleus and the CA3 hippocampal field, neuronal damage was even more severe in the SZR72 group. The HT group showed the best preservation of EEG complexity, VEP, and neuronal integrity in all examined brain regions. In summary, SZR72 appears to enhance neuronal activity after asphyxia but does not ameliorate early neuronal damage in this HIE model.

Hypoxic-ischemic encephalopathy (HIE) is still a major cause of neonatal death and disability as therapeutic hypothermia (TH) alone cannot afford sufficient neuroprotection. The present study investigated whether ventilation with molecular hydrogen (2.1% H 2 ) or graded restoration of normocapnia with CO 2 for 4 h after asphyxia would augment the neuroprotective effect of TH in a subacute (48 h) HIE piglet model. Piglets were randomized to untreated naïve, control-normothermia, asphyxia-normothermia (20-min 4%O 2 -20%CO 2 ventilation; T core = 38.5 °C), asphyxia-hypothermia (A-HT, T core = 33.5 °C, 2-36 h post-asphyxia), A-HT + H 2 , or A-HT + CO 2 treatment groups. Asphyxia elicited severe hypoxia (pO 2 = 19 ± 5 mmHg) and mixed acidosis (pH = 6.79 ± 0.10). HIE development was confirmed by altered cerebral electrical activity and neuropathology. TH was significantly neuroprotective in the caudate nucleus but demonstrated virtually no such effect in the hippocampus. The mRNA levels of apoptosis-inducing factor and caspase-3 showed a ~10-fold increase in the A-HT group compared to naïve animals in the hippocampus but not in the caudate nucleus coinciding with the region-specific neuroprotective effect of TH. H 2 or CO 2 did not augment TH-induced neuroprotection in any brain areas; rather, CO 2 even abolished the neuroprotective effect of TH in the caudate nucleus. In conclusion, the present findings do not support the use of these medical gases to supplement TH in HIE management.

Brain interstitial pH (pHbrain) alterations play an important role in the mechanisms of neuronal injury in neonatal hypoxic-ischemic encephalopathy (HIE) induced by perinatal asphyxia. The newborn pig is an established large animal model to study HIE, however, only limited information on pHbrain alterations is available in this species and it is restricted to experimental perinatal asphyxia (PA) and the immediate reventilation. Therefore, we sought to determine pHbrain over the first 24h of HIE development in piglets. Anaesthetized, ventilated newborn pigs (n = 16) were instrumented to control major physiological parameters. pHbrain was determined in the parietal cortex using a pH-selective microelectrode. PA was induced by ventilation with a gas mixture containing 6%O2-20%CO2 for 20 min, followed by reventilation with air for 24h, then the brains were processed for histopathology assessment. The core temperature was maintained unchanged during PA (38.4±0.1 vs 38.3±0.1°C, at baseline versus the end of PA, respectively; mean±SEM). In the arterial blood, PA resulted in severe hypoxia (PaO2: 65±4 vs 23±1*mmHg, *p<0.05) as well as acidosis (pHa: 7.53±0.03 vs 6.79±0.02*) that is consistent with the observed hypercapnia (PaCO2: 37±3 vs 160±6*mmHg) and lactacidemia (1.6±0.3 vs 10.3±0.7*mmol/L). Meanwhile, pHbrain decreased progressively from 7.21±0.03 to 5.94±0.11*. Reventilation restored pHa, blood gases and metabolites within 4 hours except for PaCO2 that remained slightly elevated. pHbrain returned to 7.0 in 29.4±5.5 min and then recovered to its baseline level without showing secondary alterations during the 24 h observation period. Neuropathological assessment also confirmed neuronal injury. In conclusion, in spite of the severe acidosis and alterations in blood gases during experimental PA, pHbrain recovered rapidly and notably, there was no post-asphyxia hypocapnia that is commonly observed in many HIE babies. Thus, the neuronal injury in our piglet model is not associated with abnormal pHbrain or low PaCO2 over the first 24 h after PA., Competing Interests: The authors have declared that no competing interests exist.

Cortical spreading depolarization (SD) involves activation of NMDA receptors and elicit neurovascular unit dysfunction. NMDA cannot trigger SD in newborns, thus its effect on neurovascular function is not confounded by other aspects of SD. The present study investigated if NMDA affected hypercapnia-induced microvascular and electrophysiological responses in the cerebral cortex of newborn pigs. Anesthetized piglets were fitted with cranial windows over the parietal cortex to study hemodynamic and electrophysiological responses to graded hypercapnia before/after topically applied NMDA assessed with laser-speckle contrast imaging and recording of local field potentials (LFP)/neuronal firing, respectively. NMDA increased cortical blood flow (CoBF), suppressed LFP power in most frequency bands but evoked a 2.5 Hz δ oscillation. The CoBF response to hypercapnia was abolished after NMDA and the hypercapnia-induced biphasic changes in δ and θ LFP power were also altered. MK-801 prevented NMDA-induced increases in CoBF and the attenuation of microvascular reactivity to hypercapnia. The neuronal nitric oxide synthase (nNOS) inhibitor (N-(4 S)-4-amino-5-[aminoethyl]aminopentyl-N'-nitroguanidin) also significantly preserved the CoBF response to hypercapnia after NMDA, although it didn't reduce NMDA-induced increases in CoBF. In conclusion, excess activation of NMDA receptors alone can elicit SD-like neurovascular unit dysfunction involving nNOS activity.

Cyclooxygenase-2 (COX-2) has an established role in the pathogenesis of hypoxic-ischemic encephalopathy (HIE). In this study we sought to determine whether COX-2 was induced by asphyxia in newborn pigs, and whether neuronal COX-2 levels were affected by H 2 treatment. Piglets were subjected to either 8 min of asphyxia or a more severe 20 min of asphyxia followed by H 2 treatment (inhaling room air containing 2.1% H 2 for 4 h). COX-2 immunohistochemistry was performed on brain samples from surviving piglets 24 h after asphyxia. The percentages of COX-2-immunopositive neurons were determined in cortical and subcortical areas. Only in piglets with more severe HIE, we observed significant, region-specific increases in neuronal COX-2 expression within the parietal and occipital cortices and in the CA3 hippocampal subfield. H 2 treatment essentially prevented the increases in COX-2-immunopositive neurons. In the parietal cortex, the attenuation of COX-2 induction was associated with reduced 8'-hydroxy-2'-deoxyguanozine immunoreactivity and retained microglial ramifcation index, which are markers of oxidative stress and neuroinfiammation, respectively. This study demonstrates for the first time that asphyxia elevates neuronal COX-2 expression in a piglet HIE model. Neuronal COX-2 induction may play region-specific roles in brain lesion progression during HIE development, and inhibition of this response may contribute to the antioxidant/anti-infiammatory neuroprotective effects of H 2 treatment.

Aims: Perinatal asphyxia (PA) often results in hypoxic-ischemic encephalopathy (HIE) in term neonates. Introduction of therapeutic hypothermia improved HIE outcome, but further neuroprotective therapies are still warranted. The present study sought to determine the feasibility of the activation of the cytoprotective PI-3-K/Akt and the MAPK/ERK signaling pathways in the subacute phase of HIE development in a translational newborn pig PA/HIE model., Main Methods: Phosphorylated and total levels of Akt and ERK were determined by Western blotting in brain samples obtained from untreated naive, time control, and PA/HIE animals at 24-48h survival (n=3-3-6,respectively). PA (20min) was induced in anesthetized piglets by ventilation with a hypoxic/hypercapnic (6%O 2 20%CO 2 ) gas mixture. Furthermore, we studied the effect of topically administered specific Akt1/2 and MAPK/ERK kinase inhibitors on Akt and ERK phosphorylation (n=4-4) in the cerebral cortex under normoxic conditions., Key Findings: PA resulted in significant neuronal injury shown by neuropathology assessment of haematoxylin/eosin stained sections. However, there were no significant differences among the groups in the high phosphorylation levels of both ERK and Akt in the cerebral cortex, hippocampus and subcortical structures. However, the Akt1/2 and MAPK/ERK kinase inhibitors significantly reduced cerebrocortical Akt and ERK phosphorylation within 30min., Significance: The major finding of the present study is that the PI-3-K/Akt and the MAPK/ERK signaling pathways appear to be constitutively active in the piglet brain, and this activation remains unaltered during HIE development. Thus, neuroprotective strategies aiming to activate these pathways to limit apoptotic neuronal death may offer limited efficacy in this translational model., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The neurovascular unit encompasses the functional interactions of cerebrovascular and brain parenchymal cells necessary for the metabolic homeostasis of neurons. Previous studies indicated marked but only transient (1-4 h) reactive oxygen species-dependent neurovascular dysfunction in newborn pigs after severe hypoxic/ischemic (H/I) stress contributing to the neuronal injury after birth asphyxia., Objectives: Our major purpose was to determine if neurovascular dysfunction would also occur later, at 24 h after a milder H/I stress. We also tested if the putative hydroxyl radical scavenger hydrogen (H2) exerted neurovascular protection., Methods: Anesthetized, ventilated piglets were assigned to three groups of 9 animals: time control, asphyxia/reventilation with air, and asphyxia/reventilation with air +2.1% H2 for 4 h. Asphyxia was induced by suspending ventilation for 8 min. Cerebrovascular reactivity (CR) of pial arterioles was determined using closed cranial window/intravital microscopy 24 h after asphyxia to the endothelium-dependent cerebrovascular stimulus hypercapnia, the neuronal function-dependent stimulus N-methyl-D-aspartate (NMDA), norepinephrine, and sodium nitroprusside. The brains were subjected to histopathology., Results: Hemodynamic parameters, blood gases, and core temperature did not differ significantly among the experimental groups. In the early reventilation period, the recovery of electroencephalographic activity was significantly better in H2-treated animals. Asphyxia/reventilation severely attenuated CR to hypercapnia and NMDA; however, reactivity to norepinephrine and sodium nitroprusside were unaltered. H2 fully or partially preserved CR to hypercapnia or NMDA, respectively. Histopathology revealed modest neuroprotection afforded by H2., Conclusions: Severe stimulus-selective delayed neurovascular dysfunction develops and persists even after mild H/I stress. H2 alleviates this delayed neurovascular dysfunction that can contribute to its neuroprotective effect., (Copyright © 2013 S. Karger AG, Basel.)

Cyclooxygenase (COX)-2 is the major constitutively expressed COX isoform in the newborn brain. COX-2 derived prostanoids and reactive oxygen species appear to play a major role in the mechanism of perinatal hypoxic-ischemic injury in the newborn piglet, an accepted animal model of the human term neonate. The study aimed to quantitatively determine COX-2 immunopositive neurons in different brain regions in piglets under normoxic conditions (n=15), and 4 hours after 10 min asphyxia (n=11). Asphyxia did not induce significant changes in neuronal COX-2 expression of any studied brain areas. In contrast, there was a marked regional difference in all experimental groups. Thus, significant difference was observed between fronto-parietal and temporo-occipital regions: 59±4% and 67±3% versus 41±2%* and 31±3%* respectively (mean±SEM, data are pooled from all subjects, n=26, *p<0.05, vs. fronto-parietal region). In the hippocampus, COX-2 immunopositivity was rare (highest expression in CA1 region: 14±2%). The studied subcortical areas showed negligible COX-2 staining. Our findings suggest that asphyxia does not significantly alter the pattern of neuronal COX-2 expression in the early reventilation period. Furthermore, based on the striking differences observed in cortical neuronal COX-2 distribution, the contribution of COX-2 mediated neuronal injury after asphyxia may also show region-specific differences.