Hypoxic-ischemic encephalopathy (HIE) remains to be a major cause of morbidity, mortality and severe neurodevelopmental disability in term neonates. Moderate whole body hypothermia is an established, effective neuroprotective therapy to reduce mortality and long-term disability associated with HIE, however, research for adjunct therapies is still warranted to complement the effect of hypothermia. In the last decade, molecular hydrogen emerged as a simple, available, inexpensive substance with advantageous pharmacokinetics to ameliorate hypoxic-ischemic cellular damage. The present review examines the preclinical studies employing hydrogen to combat the deleterious consequences of hypoxic-ischemic insults in rodent and piglet HIE models. Hydrogen exerted unequivocal neuroprotective actions shown by preserved neurovascular function, neuronal viability, and neurocognitive functions in virtually all model species and hypoxic-ischemic insult types tested. Administration of hydrogen started in most studies after the hypoxic-ischemic insult enhancing the translational value of the findings. Among the explored mechanisms of hydrogen-induced neuroprotection, antioxidant, anti- apoptotic and anti-inflammatory effects appeared to be dominant. Unfortunately, the additive neuroprotective effect of hydrogen and therapeutic hypothermia has not yet been demonstrated, thus such studies are warranted to promote the clinical testing of molecular hydrogen as an adjunct neuroprotective treatment of HIE.

Reduced availability of reactive oxygen species is a key component of neuroprotection against various toxic stimuli. Recently we showed that the hydrogen peroxide scavenger catalase plays a central role in delayed preconditioning induced by the mitochondrial ATP-sensitive potassium channel opener BMS-191095. The purpose of the experiments discussed here was to investigate the neuroprotective effect of catalase in vitro using a recombinant adenoviral catalase gene transfer protocol. To induce catalase overexpression, cultured rat cortical neurons were infected with the adenoviral vector Ad5CMVcatalase and control cells were incubated with Ad5CMVntLacZ for 24 h. Gene transfer effectively increased catalase protein levels and activity, but did not influence other antioxidants tested. Ad5CMVcatalase, with up to 10 plaque forming units (pfu) per neuron, did not affect cell viability under control conditions and did not protect against glutamate excitotoxicity or oxygen-glucose deprivation. In contrast, catalase overexpression conferred a dose-dependent protection against exposure to hydrogen peroxide (viability: control, 33.02+/-1.09%; LacZ 10 pfu/cell, 32.85+/-1.51%; catalase 1 pfu/cell, 62.09+/-4.17%*; catalase 2 pfu/cell, 98.71+/-3.35%*; catalase 10 pfu/cell, 99.68+/-1.99%*; *p<0.05 vs. control; mean+/-SEM). Finally, the protection could be antagonized using the catalase inhibitor 3-aminotriazole. Our results support the view that enhancing cellular antioxidant capacity may play a crucial role in neuroprotective strategies.

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.

Background and Purpose —Cerebral arteriolar dilation to N -methyl- d -aspartate (NMDA) is a neuronally mediated process that is sensitive to cerebral ischemia. We tested the hypothesis that pretreatment with transcription or translation inhibitors preserves the vascular response to NMDA after global cerebral ischemia. Methods —Pial arteriolar diameters were measured in anesthetized piglets by use of a closed cranial window and intravital microscopy. Arteriolar responses to NMDA (10 ⁻⁵ and 10 ⁻⁴ mol/L) were measured before and 1, 2, and 4 hours after 10 minutes of ischemia. Ischemia was induced by increasing intracranial pressure. Subgroups were pretreated with vehicle, topical actinomycin D (Act-D) 10 ⁻⁵ or 10 ⁻⁶ mol/L, or intravenous cycloheximide (CHX) 1.0 or 0.3 mg/kg 15 minutes before ischemia. The effects of Act-D and CHX on vascular responses to NMDA without preceding ischemia were also examined. Results —In the vehicle group, arteriolar responses to NMDA were clearly attenuated 1 hour after ischemia but returned to baseline at 2 to 4 hours. Preischemic compared with 1 hour postischemic arteriolar dilation to NMDA was 10±2% versus 1±0% at 10 ⁻⁵ mol/L and 40±4% versus 20±4% at 10 ⁻⁴ mol/L NMDA (mean±SEM; both P <0.05, n=7). In contrast, pretreatment with Act-D resulted in preservation of the arteriolar responses to NMDA 1 hour after ischemia. For 10 ⁻⁶ mol/L (n=5) of Act-D, dilations were 6±2% versus 6±2% at 10 ⁻⁵ mol/L and 51±9% versus 39±10% at 10 ⁻⁴ mol/L of NMDA. For 10 ⁻⁵ mol/L (n=5) of Act-D, arterioles dilated by 7±2% versus 7±2% at 10 ⁻⁵ mol/L and 38±4% versus 35±4% at 10 ⁻⁴ mol/L NMDA. Similarly, CHX preserved NMDA-induced vasodilation. For 0.3 mg/kg of CHX (n=5), dilations were 8±2% versus 8±1% at 10 ⁻⁵ mol/L and 39±4% versus 28±6% at 10 ⁻⁴ mol/L NMDA. For 1.0 mg/kg of CHX (n=5), arterioles dilated by 10±2% versus 6±2% at 10 ⁻⁵ mol/L and 37±7% versus 35±6% at 10 ⁻⁴ mol/L NMDA. In experiments without ischemia, NMDA-induced vasodilation before and 85 minutes after administration of Act-D or CHX was not significantly different. Conclusions —Vascular responses of cerebral arterioles to NMDA after ischemia are preserved by pretreatment with either Act-D or CHX. Without preceding ischemia, Act-D and CHX do not potentiate neuronal-vascular responses to NMDA. Our results suggest that continued or augmented protein synthesis is involved in the transient attenuation of NMDA-induced dilation during the early reperfusion phase and that inhibitors of protein synthesis may protect neurons against ischemic stress.