Hydrogen (H 2

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.

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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 treatment. Piglets were subjected to either 8 min of asphyxia or a more severe 20 min of asphyxia followed by H treatment (inhaling room air containing 2.1% H 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 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 treatment.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are neuroprotective in numerous models. Impairment of cerebrovascular reactivity (CR) contributes to ischemia/reperfusion (I/R)-induced neuronal damage. We tested whether PACAP and/or VIP preserve CR to I/R-sensitive dilator responses dependent on endothelial and/or neuronal function. Accordingly, changes in pial arteriolar diameters in response to hypercapnia (5–10% CO2 ventilation) or topical N-methyl-d-aspartate (NMDA, 10–4 M) were determined before and after I/R via intravital microscopy in anesthetized/ventilated piglets. Local pretreatment with non-vasoactive doses of PACAP (10–8 M) and VIP (10–9 M) prevented the attenuation of postischemic CR to hypercapnia; to 10% CO2, the CR values were 27±8% vs 92±5%* vs 88±13%* (vehicle vs PACAP38 vs VIP, CR expressed as a percentage of the response before I/R, mean±SEM, n=8–8, *p<0.05). PACAP, but not VIP, preserved CR to NMDA after I/R, with CR values of 31±10% vs 87±8%* vs 35±12% (vehicle vs PACAP38 vs VIP, n=6–6). Unlike PACAP, VIP-induced vasodilation has not yet been investigated in the piglet. We tested whether VIP-induced arteriolar dilation was sensitive to inhibitors of cyclooxygenase (COX)-1 (SC-560, 1 mg/kg), COX-2 (NS-398, 1 mg/kg), indomethacin (5 mg/kg), and nitric oxide synthase (L-NAME, 15 mg/kg). VIP (10–8–10–7–10–6 M, n=8) induced reproducible, dose-dependent vasodilation of 16±3%, 33±6%*, and 70±8%*. The response was unaffected by all drugs, except that the vasodilation to 10–8 M VIP was abolished by SC-560 and indomethacin. In conclusion, PACAP and VIP differentially preserve postischemic CR; independent of their vasodilatory effect.