Brain interstitial pH (pH_brain) 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 pH_brain alterations is available in this species and it is restricted to experimental perinatal asphyxia (PA) and the immediate reventilation. Therefore, we sought to determine pH_brain over the first 24h of HIE development in piglets. Anaesthetized, ventilated newborn pigs (n = 16) were instrumented to control major physiological parameters. pH_brain 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 (P_aO₂: 65±4 vs 23±1*mmHg, *p<0.05) as well as acidosis (pH_a: 7.53±0.03 vs 6.79±0.02*) that is consistent with the observed hypercapnia (P_aCO₂: 37±3 vs 160±6*mmHg) and lactacidemia (1.6±0.3 vs 10.3±0.7*mmol/L). Meanwhile, pH_brain decreased progressively from 7.21±0.03 to 5.94±0.11*. Reventilation restored pH_a, blood gases and metabolites within 4 hours except for P_aCO₂ that remained slightly elevated. pH_brain 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, pH_brain 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 pH_brain or low P_aCO₂ over the first 24 h after PA.

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% H₂) or graded restoration of normocapnia with CO₂ 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%O₂–20%CO₂ ventilation; T_core = 38.5 °C), asphyxia-hypothermia (A-HT, T_core = 33.5 °C, 2–36 h post-asphyxia), A-HT + H₂, or A-HT + CO₂ treatment groups. Asphyxia elicited severe hypoxia (pO₂ = 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. H₂ or CO₂ did not augment TH-induced neuroprotection in any brain areas; rather, CO₂ 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.

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.

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.

The cerebral microcirculation undergoes dynamic changes regarding neurovascular responses through the perinatal period. CO ₂ is one of the most potent regulator of cerebral blood flow, however its effector mechanisms involve multiple possibly yet unknown pathways in the neonate. Acid-sensing ion channel-1A (ASIC1A) appears to play a decisive role in this cerebrovascular response in adult mice, however, cortical ASIC1A expression and its possible contribution to the response in the translational piglet model of term neonates is unknown. Therefore, we investigated neocortical ASIC1A expression and the effect of the specific ASIC1A-inhibitor psalmotoxin-1 on the neurovascular response to graded hypercapnia in piglets. Anesthetized, mechanically ventilated newborn pigs were equipped with a closed or open cranial window to assess the cortical blood flow (CoBF) with laser speckle contrast imaging or the neuronal activity with multichannel electrodes, respectively. Graded hypercapnia was elicited by ventilation of 5%–10% CO ₂ . ASIC1A was found to be ubiquitously expressed in neocortical neurons. Psalmotoxin-1 while unaffected the CoBF response to 5% CO ₂ , it significantly attenuated further increases in CoBF to 10% CO ₂ , accompanying the highly altered hypercapnia-induced changes in neuronal activity. We conclude that ASIC1A activation by hypercapnia-induced acidosis is partially responsible for the neurovascular response in the neonatal brain.