Subarachnoid Hemorrhage Depletes Calcitonin Gene-Related Peptide Levels of Trigeminal Neurons in Rat Dura Mater.

Subarachnoid hemorrhage (SAH) remains a major cause of cerebrovascular morbidity, eliciting severe headaches and vasospasms that have been shown to inversely correlate with vasodilator calcitonin gene-related peptide (CGRP) levels. Although dura mater trigeminal afferents are an important source of intracranial CGRP, little is known about the effects of SAH on these neurons in preclinical models. The present study evaluated changes in CGRP levels and expression in trigeminal primary afferents innervating the dura mater 72 h after experimentally induced SAH in adult rats. SAH, eliciting marked damage revealed by neurological examination, significantly reduced the density of CGRP-immunoreactive nerve fibers both in the dura mater and the trigeminal caudal nucleus in the medulla but did not affect the total dural nerve fiber density. SAH attenuated ex vivo dural CGRP release by ~40% and in the trigeminal ganglion, reduced both CGRP mRNA levels and the number of highly CGRP-immunoreactive cell bodies. In summary, we provide novel complementary evidence that SAH negatively affects the integrity of the CGRP-expressing rat trigeminal neurons. Reduced CGRP levels suggest likely impaired meningeal neurovascular functions contributing to SAH complications. Further studies are to be performed to reveal the importance of impaired CGRP synthesis and its consequences in central sensory processing.

The effects of CO2 levels and body temperature on brain interstitial pH alterations during the induction of hypoxic-ischemic encephalopathy in newborn pigs.

Brain interstitial pH (pHbrain) 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 pHbrain alterations during HIE induction are limited. The major objective of the present study was thus to compare pHbrain changes during HIE development induced by experimental normocapnic hypoxia (H) or asphyxia (A), elicited with ventilation of a gas mixture containing 6%O2 or 6%O2/20%CO2, 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. pHbrain 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 pHbrain response to HI stress was acidosis, at the nadir pHbrain 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 (pHa 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, pHbrain 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 pHbrain response without an alkalotic component either during the HI stress or the early reoxygenation period.

Differential Effects of Hypothermia and SZR72 on Cerebral Kynurenine and Kynurenic Acid in a Piglet Model of Hypoxic-Ischemic Encephalopathy.

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.

Quantitative elemental mapping of biological tissues by laser-induced breakdown spectroscopy using matrix recognition.

The present study demonstrates the importance of converting signal intensity maps of organic tissues collected by laser-induced breakdown spectroscopy (LIBS) to elemental concentration maps and also proposes a methodology based on machine learning for its execution. The proposed methodology employs matrix-matched external calibration supported by a pixel-by-pixel automatic matrix (tissue type) recognition performed by linear discriminant analysis of the spatially resolved LIBS hyperspectral data set. On a swine (porcine) brain sample, we successfully performed this matrix recognition with an accuracy of 98% for the grey and white matter and we converted a LIBS intensity map of a tissue sample to a correct concentration map for the elements Na, K and Mg. Found concentrations in the grey and white matter agreed the element concentrations published in the literature and our reference measurements. Our results revealed that the actual concentration distribution in tissues can be quite different from what is suggested by the LIBS signal intensity map, therefore this conversion is always suggested to be performed if an accurate concentration distribution is to be assessed.

The Kynurenic Acid Analog SZR72 Enhances Neuronal Activity after Asphyxia but Is Not Neuroprotective in a Translational Model of Neonatal Hypoxic Ischemic Encephalopathy.

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.

Hydrogen-induced Neuroprotection in Neonatal Hypoxic-ischemic Encephalopathy.

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.

Correction: Brain interstitial pH changes in the subacute phase of hypoxic-ischemic encephalopathy in newborn pigs.

[This corrects the article DOI: 10.1371/journal.pone.0233851.].

Inhaled H2 or CO2 Do Not Augment the Neuroprotective Effect of Therapeutic Hypothermia in a Severe Neonatal Hypoxic-Ischemic Encephalopathy Piglet 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; Tcore = 38.5 °C), asphyxia-hypothermia (A-HT, Tcore = 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.

Brain interstitial pH changes in the subacute phase of hypoxic-ischemic encephalopathy in newborn pigs.

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.

NMDA attenuates the neurovascular response to hypercapnia in the neonatal cerebral cortex.

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.

Molecular hydrogen alleviates asphyxia-induced neuronal cyclooxygenase-2 expression in newborn pigs.

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 H2 treatment. Piglets were subjected to either 8 min of asphyxia or a more severe 20 min of asphyxia followed by H2 treatment (inhaling room air containing 2.1% H2 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. H2 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 H2 treatment.

Active forms of Akt and ERK are dominant in the cerebral cortex of newborn pigs that are unaffected by asphyxia.

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%O220%CO2) 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.

Delayed neurovascular dysfunction is alleviated by hydrogen in asphyxiated newborn pigs.

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.

Depolarization of mitochondria in endothelial cells promotes cerebral artery vasodilation by activation of nitric oxide synthase.

OBJECTIVE: Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. APPROACH AND RESULTS: Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca(2+)]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca(2+)]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. CONCLUSIONS: Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca(2+)]i as well as by phosphoinositide-3 kinase-protein kinase B-induced eNOS phosphorylation. Both mitochondrial reactive oxygen species-dependent and -independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.

Cerebral microcirculatory responses of insulin-resistant rats are preserved to physiological and pharmacological stimuli.

OBJECTIVE: Previously, we have shown that IR impairs the vascular reactivity of the major cerebral arteries of ZO rats prior to the occurrence of Type-II diabetes mellitus. However, the functional state of the microcirculation in the cerebral cortex is still being explored. METHODS: We tested the local CoBF responses of 11-13-week-old ZO (n = 31) and control ZL (n = 32) rats to several stimuli measured by LDF using a closed cranial window setup. RESULTS: The topical application of 1-100 µm bradykinin elicited the same degree of CoBF elevation in both ZL and ZO groups. There was no significant difference in the incidence, latency, and amplitude of the NMDA-induced CSD-related hyperemia between the ZO and ZL groups. Hypercapnic CoBF response to 5% carbon-dioxide ventilation did not significantly change in the ZO compared with the ZL. Topical bicuculline-induced cortical seizure was accompanied by the same increase of CoBF in both the ZO and ZL at all bicuculline doses. CONCLUSIONS: CoBF responses of the microcirculation are preserved in the early period of the metabolic syndrome, which creates an opportunity for intervention to prevent and restore the function of the major cerebral vascular beds.

Regional Differences in the Neuronal Expression of Cyclooxygenase-2 (COX-2) in the Newborn Pig Brain.

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.

Evaluation of laser-speckle contrast image analysis techniques in the cortical microcirculation of piglets.

A new laser speckle-contrast analysis (LASCA) technique based on multi-exposure imaging was employed to simultaneously study pial arteriolar responses with cerebrocortical perfusion changes to various vasodilator (5-10% CO(2) ventilation, bradykinin (1-10 µM), N-methyl-D-aspartate (100 µM)) vasoconstrictor (10-100 µM noradrenaline, 1M KCl), or neutral (2.1% H(2) ventilation) stimuli as well as to asphyxia in the newborn piglet. Anesthetized, ventilated animals (n=20) were fitted with closed cranial windows. Multiple exposure laser-speckle image series (1-100 ms) were obtained using a near infrared diode laser (λ=808 nm). The autocorrelation decay time (τ) of speckle fluctuations was determined over pial arterioles and parenchymal areas to express 1/τ being proportional to blood flow velocity by two different LASCA techniques: our novel multi-exposure or a single exposure (2 and 20 ms) approach. 1/τ values yielded by different LASCA techniques were not significantly different at most points. LASCA easily detected both increases and decreases in cortical blood flow (CoBF). Cortical 1/τ changes to hypercapnia closely matched quantitative CoBF data determined previously, and were also in accordance with increases of pial arteriolar blood flow, calculated from arteriolar flow velocity and cross sectional area changes. In summary, LASCA emerges as an appealing method to simultaneously study microvascular reactivity and cortical perfusion changes in the piglet.

Hydrogen supplemented air inhalation reduces changes of prooxidant enzyme and gap junction protein levels after transient global cerebral ischemia in the rat hippocampus.

Transient global cerebral ischemia (TGCI) occurs during acute severe hypotension depriving the brain of oxygen and glucose for a short period of time. During reperfusion, several mechanisms can induce secondary neuronal damage, including the increased production of reactive oxygen species (ROS). Hydrogen gas-enriched air inhalation is a neuroprotective approach with proven antioxidant potential, which has not yet been examined in TGCI. Accordingly, we set out to describe the effect of inhalation of 2.1% hydrogen supplemented room air (H(2)-RA) in comparison with a well studied neuroprotective agent, rosiglitazone (RSG) in a TGCI rat model. Male Wistar rats were exposed to TGCI (n=26) or sham operation (n=26), while a third group served as intact control (naive, n=5). The operated groups were further divided into non-treated, H(2)-RA, RSG (6 mg/kg i.v.) and vehicle treated animals. Tissue samples from the hippocampus and frontal cortex were taken 3 days following surgery. Western blot analysis was applied to determine the expressions of cyclooxygenase-2 (COX-2), neuronal and endothelial nitric oxide synthase (nNOS and eNOS, respectively), manganese superoxide dismutase (MnSOD) and glial connexin proteins: connexin 30 and connexin 43. The expressions of COX-2, and connexin proteins were upregulated, while nNOS was downregulated 3 days after TGCI. Both RSG and H(2)-RA prevented the changes of enzyme and connexin levels. Considering the lack of harmful side effects, inhalation of H(2)-RA can be a promising approach to reduce neuronal damage after TGCI.

Impaired vascular responses of insulin-resistant rats after mild subarachnoid hemorrhage.

Insulin resistance (IR) impairs cerebrovascular responses to several stimuli in Zucker obese (ZO) rats. However, cerebral artery responses after subarachnoid hemorrhage (SAH) have not been described in IR. We hypothesized that IR worsens vascular reactions after a mild SAH. Hemolyzed blood (300 µl) or saline was infused (10 µl/min) into the cisterna magna of 11-13-wk-old ZO (n = 25) and Zucker lean (ZL) rats (n = 25). One day later, dilator responses of the basilar artery (BA) and its side branch (BA-Br) to acetylcholine (ACh, 10(-6) M), cromakalim (10(-7) M, 10(-6) M), and sodium nitroprusside (10(-7) M) were recorded with intravital videomicroscopy. The baseline diameter of the BA was increased both in the ZO and ZL rats 24 h after the hemolysate injection. Saline-injected ZO animals showed reduced dilation to ACh (BA = 9 ± 3 vs. 22 ± 4%; and BA-Br = 23 ± 5 vs. 37 ± 7%) compared with ZL rats. Hemolysate injection blunted the response to ACh in both the ZO (BA = 4 ± 2%; and BA-Br = 12 ± 3%) and ZL (BA = 7 ± 2%; and BA-Br = 11 ± 3%) rats. Cromakalim (10(-6) M)-induced dilation was significantly reduced in the hemolysate-injected ZO animals compared with the saline control (BA = 13 ± 3 vs. 26 ± 5%; and BA-Br = 28 ± 8 vs. 44 ± 9%) and in the hemolysate-injected ZL rats compared with their saline control (BA = 24 ± 4 vs. 32 ± 4%; but not BA-Br = 39 ± 6 vs. 59 ± 9%). No significant difference in sodium nitroprusside reactivity was observed. Western blot analysis of the BA showed a lower baseline level of neuronal nitric oxide synthase expression and an enhanced cyclooxygenase-2 level in the hemolysate-injected ZO animals. In summary, cerebrovascular reactivity to both endothelium-dependent and -independent stimuli is severely compromised by SAH in IR animals.

Hydrogen is neuroprotective and preserves cerebrovascular reactivity in asphyxiated newborn pigs.

Hydrogen (H2) has been reported to neutralize toxic reactive oxygen species. Oxidative stress is an important mechanism of neuronal damage after perinatal asphyxia. We examined whether 2.1% H2-supplemented room air (H2-RA) ventilation would preserve cerebrovascular reactivity (CR) and brain morphology after asphyxia/reventilation (A/R) in newborn pigs. Anesthetized, ventilated piglets were assigned to one of the following groups: A/R with RA or H2-RA ventilation (A/R-RA and A/R-H2-RA; n = 8 and 7, respectively) and respective time control groups (n = 9 and 7). Asphyxia was induced by suspending ventilation for 10 min, followed by reventilation with the respective gases for 4 h. After euthanasia, the brains were processed for neuropathological examination. Pial arteriolar diameter changes to graded hypercapnia (5-10% CO2 inhalation), and NMDA (10(-4) M) were determined using the closed cranial window/intravital microscopy before and 1 h after asphyxia. Neuropathology revealed that H2-RA ventilation significantly reduced neuronal injury induced by A/R in virtually all examined brain regions including the cerebral cortex, the hippocampus, basal ganglia, cerebellum, and the brainstem. Furthermore, H2-RA ventilation significantly increased CR to hypercapnia after A/R (% vasodilation was 23 ± 4% versus 41 ± 9%, p < 0.05). H2-RA ventilation did not affect reactive oxygen species-dependent CR to NMDA. In summary, H2-RA could be a promising approach to reduce the neurologic deficits after perinatal asphyxia.