Berit H. Munkeby

Resuscitation with 100% O2 Increases Cerebral Injury in Hypoxemic Piglets

Perinatal asphyxia is a major cause of immediate and postponed brain injury in the newborn. We hypothesized that resuscitation with 100% O2 compared with ambient air is detrimental to the cerebral tissue. We assessed cerebral injury in newborn piglets that underwent global hypoxia and subsequent resuscitation with 21 or 100% O2 by extracellular glycerol, matrix metalloproteinase (MMP) expression levels, and oxidative stress. Extracellular glycerol was sampled by cerebral microdialysis. MMP levels were analyzed in cerebral tissue by gelatin zymography, broad matrix degrading capacity, and real-time PCR. Total endogenous antioxidant capacity was measured by the oxygen radical absorbance capacity assay. Extracellular glycerol increased 50% after resuscitation with 100% O2 compared with 21% O2. Total MMP activity was doubled in resuscitated animals at endpoint compared with baseline (p = 0.018), and the MMP-2 activity was significantly increased in piglets that were resuscitated with 21% O2 (p = 0.003) and 100% O2 (p = 0.001) compared with baseline. MMP-2 mRNA level was 100% increased in piglets that were resuscitated with 100% O2 as compared with 21% O2 (p < 0.05). Oxygen radical absorbance capacity values in piglets that were resuscitated with 100% O2 were considerably reduced compared with both baseline (p = 0.001) and piglets that were resuscitated with 21% O2 (p = 0.001). In conclusion, our data show increased MMP-2 activity at both gene and protein levels, accompanied with cerebral leakage of glycerol, presumably triggered by augmented oxidative stress. Our findings suggest that resuscitation of asphyxiated piglets with 100% O2 is detrimental to the piglet brain compared with resuscitation with 21% O2.

Interleukin-10 reverses acute detrimental effects of endotoxin-induced inflammation on perinatal cerebral hypoxia-ischemia

UNLABELLED Perinatal brain injuries and the subsequent development of cerebral palsy are closely associated with intrauterine infections and inflammatory response. Antibiotics have proven futile in reducing perinatal brain injuries. We tested whether treatment with the anti-inflammatory cytokine IL-10 could have beneficial effects during a concomitant endotoxin and cerebral hypoxic-ischemic challenge. Thirty-three newborn piglets were randomized to pretreatment with: CONTROLS placebo, Endotoxin: 2 kU/kg bolus and infusion of 1 kU/kg per h of endotoxin, or Endotoxin+IL-10: endotoxin in addition to 50 microg/kg of porcine recombinant IL-10. We induced cerebral hypoxia-ischemia by bilateral clamping of the common carotid arteries and ventilation with 8% oxygen for 20 min followed by 3 h of reoxygenation/reperfusion. Extracellular lactate, pyruvate, glycerol and glutamate, microcirculation and tissue oxygenation were monitored in the striatum by microdialysis, laser Doppler flow and oxygen tension probe, respectively. During and/or after cerebral hypoxia-ischemia, Endotoxin caused marked deterioration of the cerebral metabolic situation with higher lactate/pyruvate ratio (P=0.003), compared to CONTROLS and Endotoxin+IL-10. This was caused mainly by very low levels of pyruvate (P=0.001). During the following reoxygenation, Endotoxin compromised cerebral microcirculation (P=0.038) and tissue oxygenation (P=0.012) compared to CONTROLS and Endotoxin+IL-10. After a period of remission, a secondary energy failure and a new rise in the lactate/pyruvate ratio was seen in Endotoxin (P=0.002), but not in CONTROLS or Endotoxin+IL-10. At the end of observation, only the Endotoxin+IL-10 group had regained their baseline values in all variables. Thus IL-10 counteracts acute effects of endotoxin on cerebral metabolism, microcirculation and oxygen tension during hypoxia-ischemia in the perinatal brain.

Comparison of Short- and Long-Duration Oxygen Treatment after Cerebral Asphyxia in Newborn Piglets

We tested whether reoxygenation with 100% O2 for 5 min after experimental asphyxia in newborn piglets was as efficient as 100% O2 for 20 min compared with room air. Forty-one anesthetized piglets, 1–3 d old, were randomized to cerebral hypoxemia-ischemia-hypercapnia (HIH) or control (n = 5). HIH was achieved by ventilation with 8% O2, temporary occlusion of the common carotid arteries, and adding of CO2. After 25 min, reoxygenation-reperfusion was started with 100% O2 for 20 min (group 1, n = 12), or 21% 12), 100% O2 for 5 min (group 2, n = 12). All piglets were observed for 2 h. During O2 (group 3, n = reoxygenation-reperfusion, significantly higher blood pressure and more complete restoration of microcirculation (laser Doppler flow) in the cerebral cortex was found in both groups reoxygenated with 100% O2 compared with 21% O2 (regional cerebral blood flow ≥ 100%versus 70% of baseline, p = 0.04). Reoxygenation with 100% O2 for 5 min was as efficient as 20 min. Oxygen delivery in cortex was significantly higher in groups 1 and 2 compared with group 3 (p = 0.03), but there were no significant differences in cerebral metabolic rate for oxygen. In the striatum, no significant differences in flow or extracellular glutamate, glycerol, and lactate/pyruvate ratio were found between the groups. In conclusion, after experimental asphyxia, newborn piglets can be reoxygenated as efficiently with 100% O2 for only 5 min as 100% O2 for 20 min compared with room air.

Morphological and hemodynamic magnetic resonance assessment of early neonatal brain injury in a piglet model

To investigate the utility of functional and morphological magnetic resonance imaging (MRI) to assess the extent of brain injury in a hypoxia‐ischemia (HI) piglet model and further to validate that the desired ischemic injury was successfully induced.

Resuscitation with 21 or 100% oxygen in hypoxic nicotine-pretreated newborn piglets: possible neuroprotective effects of nicotine.

Background: Perinatal asphyxia is a major concern in perinatal medicine. Resuscitation and ways to prevent and minimize adverse outcomes after perinatal asphyxia are subject to extensive research. Objectives: In this study we hypothesized that, prior to hypoxia, intravenously administered nicotine might have an effect on how newborn piglets tolerate hypoxia, with regard to the time and degree of damage inflicted, due to its suggested neuroprotective abilities, and further that resuscitation with 21 compared with 100% oxygen in nicotine-pretreated animals would cause less cerebral damage. Methods: Thirty anesthetized newborn piglets were randomized to either hypoxia or control groups, and pretreatment with either saline or nicotine. In addition, the nicotine/hypoxia group was randomized to resuscitation with either 21 or 100% oxygen for 15 min following hypoxia. Results: We found significantly more necrosis in the striatum and cortex combined (p = 0.036), and in the striatum alone (p = 0.026), in the animals pretreated with nicotine and resuscitated with 100% when compared to 21% oxygen. There was no significant difference in the cerebellum. We also found significantly increased tolerance to hypoxia as measured by the time interval that the animals endured hypoxia: 103.8 ± 28.2 min in the nicotine-pretreated animals vs. 66.5 ± 19.5 minin the saline-pretreated animals (p = 0.035). Conclusion: Nicotine enhances newborn piglets’ ability to endure hypoxia, and resuscitation with 21% oxygen inflicts less necrosis than 100% oxygen. The potential neuroprotective effects of nicotine in the newborn brain should be further investigated.

Effect of interleukin-10 on newborn piglet brain following hypoxia-ischemia and endotoxin-induced inflammation

Objective: Previous animal studies indicated that interleukin (IL)-10 attenuates the inflammatory response to a challenge by inflammation and hypoxia-ischemia, but the effect of IL-10 administration after onset of inflammation has not been studied. We wanted to assess (1) whether IL-10 had a beneficial effect on brain metabolism and microcirculation in newborn piglets after an inflammatory, hypoxic and ischemic challenge, and (2) whether IL-10 had any harmful effects per se. Methods: Anesthetized piglets were randomized to control (n = 8), IL-10 (n = 10), endotoxin (ETX) (n = 10), or ETX and IL-10 (ETX/IL-10) (n = 10) groups. IL-10 was administered after pretreatment with saline in the IL-10 group or ETX in the ETX/IL-10 group. Then, cerebral hypoxia and ischemia was induced by bilateral clamping of the common carotid arteries and ventilation with 8% O2 for 30 min, followed by 4 h of reoxygenation and reperfusion. Extracellular levels of lactate, pyruvate, and glycerol were measured with microdialysis in periventricular white matter and parasagittal subcortical tissue, and tissue oxygenation and microcirculation were measured with Doppler technique. We compared the areas under the concentration-time and flow-time curves and maximum concentrations between (1) the ETX/IL-10 and ETX groups, and (2) the control and IL-10 groups. Results: We found no differences between (1) the ETX/IL-10 and ETX groups, and also no differences between (2) the control and IL-10 groups. Conclusion: We could not show that the treatment with IL-10 after onset of inflammation had neuroprotective effects in the newborn piglet brain. IL-10 did not attenuate metabolism in the absence of ETX-induced inflammation.

Cerebral perfusion in perinatal hypoxia and resuscitation assessed by transcranial contrast-enhanced ultrasound and 3 T MRI in newborn pigs.

Objective:Cerebrovascular factors are crucially involved in the early injury after perinatal asphyxia. With magnetic resonance imaging (MRI) and ultrasonography (US), this study aimed to quantify microvascular perfusion changes due to hypoxia and resuscitation, by comparing contrast-enhanced ultrasound (CEUS) to dynamic susceptibility contrast-enhanced (DSC)-MRI and diffusion-weighted MRI. Material and Methods:Newborn piglets (n = 12/17) were reoxygenated with 21% (n = 6) or 100% O2 (n = 6) after global hypoxia. Five piglets served as controls. CEUS and MRI were performed before, during, and up to 7 hours after hypoxia. Following are the perfusion parameters for CEUS: peak intensity (PI), area under the curve (AUC), time to peak (TTP), and upslope a and perfusion/diffusion parameters for MRI: relative cerebral blood volume, relative cerebral blood flow, mean transit time, and apparent diffusion coefficient were compared between different regions in the brain across time points and also compared with histology at the end. Results:In CEUS, compared with the control group, perfusion changed significantly over time, in the hyperoxic group in all regions for PI, AUC in all regions of interests. The changes presented mainly as decreased perfusion during and shortly after resuscitation: for PI in the basal ganglia (BG), cortex, and the whole brain with 50 to 60% (P ≤ 0.001); for AUC in the BG and cortex with 90% (P ≤ 0.02) and in the whole brain with 70% (P = 0.004). In the injured brains (confirmed by histology), significant changes over time were seen in TTP and AUC with mainly increased perfusion during hypoxia: for TTP in the cortex, AUC in the BG and whole brain with 90 to 100% (P ≤ 0.04), and for TTP in the whole brain with 50% (P = 0.02). DSC-MRI showed the same trends in perfusion with regard to relative cerebral blood volume as CEUS. In all pigs exposed to hypoxia, perfusion returned toward baseline values at 7 hours after hypoxia in both methods. Apparent diffusion coefficient decreased significantly after 7 hours in the injured brains in the BG from 114.6 ± 1.2 × 10−5mm2/s to 90.3 ± 24 × 10−5 mm2/s (P = 0.03). Conclusions:CEUS and DSC-MRI can detect an early temporal evolution of cerebral perfusion in perinatal hypoxia and resuscitation, reversible after 7 hours. Hyperoxic resuscitation caused early decreased cerebral perfusion, not present in the normoxic group. The combined use of CEUS and DSC-MRI can provide important diagnostic information and give new insights into perinatal vascular hypoxia mechanisms.

Resuscitation of Hypoxic Newborn Piglets With Supplementary Oxygen Induces Dose-Dependent Increase in Matrix Metalloproteinase-Activity and Down-Regulates Vital Genes

The optimal oxygen concentration for newborn resuscitation is still discussed. Oxygen administration during reoxygenation may induce short- and long-term pathologic changes via oxidative stress and has been associated to later childhood cancer. The aim was to study changes in oxidative stress-associated markers in liver and lung tissue of newborn pigs after acute hypoxia followed by reoxygenation for 30 min with 21, 40, or 100% oxygen compared with room air or to ventilation with 100% oxygen without preceding hypoxia. Nine hours after resuscitation, we found a dose-dependent increase in the matrix metalloproteinase gelatinase activity in liver tissue related to percentage oxygen supply by resuscitation (100% versus 21%; p = 0.002) pointing at more extensive tissue damage. Receiving 100% oxygen for 30 min without preceding hypoxia decreased the expression of VEGFR2 and TGFBR3 mRNA in liver tissue, but not in lung tissue. MMP-, VEGF-, and TGFβ-superfamily are vital for the development, growth, and functional integrity of most tissues and our data rise concern about both short- and long-term consequences of even a brief hyperoxic exposure.

Early protective effect of hypothermia in newborn pigs after hyperoxic, but not after normoxic, reoxygenation

Abstract Mild hypothermia can attenuate the development of brain damage after asphyxia. Supplemental oxygen during resuscitation increases generation of reactive oxygen species, compared to room air. It is unknown if supplemental oxygen affects hypothermic neuroprotection. We studied the early effects of hyperoxic reoxygenation and subsequent hypothermia on tissue oxygenation, microcirculation, inflammation and brain damage after global hypoxia. Anesthetized newborn pigs were randomized to control (n=6), or severe global hypoxia (n=46). Three pigs died during hypoxia or reoxygenation. After 20-min reoxygenation with room air (n=22) or 100% oxygen (n=21), pigs were randomized to normothermia (deep rectal temperature 39°C, n=22) or total body cooling (35°C, n=21) for 6.5 h before the experiment was terminated. We demonstrated a differential effect of post-hypoxic hypothermia between animals reoxygenated with 100% oxygen and with room air, with reduced damage only in hypothermic animals reoxygenated with 100% oxygen (P=0.001). Hyperoxic reoxygenation resulted in a significant overshoot in striatal oxygen tension, without affecting microcirculation. Inflammatory response after the insult did not differ between groups. The results indicate an early protective effect of hypothermia which may vary with oxygen level used during reoxygenation.

Nicotine in a Small-to-Moderate Dose Does Not Cause a Significant Increase in Plasma Catecholamine Levels in Newborn Piglets

Background: Nicotine has a wide range of effects. Several studies are being undertaken investigating the positive effects on inflammation and apoptosis. Recently, nicotine has been investigated in a piglet model of perinatal asphyxia, where the question has been raised whether nicotine’s effect on the sympathetic nervous system can explain some of the positive effects. Objectives: We hypothesized that nicotine in small-to-moderate doses would not cause a significant increase in plasma catecholamine levels, whereas a higher dose would give a significant effect, confirming the believed dose-dependent matter in which nicotine exerts its effect on the sympathetic nervous system. Methods: Seventeen anesthetized newborn piglets were randomized to one of three doses of nicotine (130, 260 or 1,000 µg/kg/h) that was given intravenously for 1 h. Blood samples for catecholamine analyzes were drawn at baseline and at the end of the infusion. Catecholamines were determined using HPLC. Results: No significant increase in catecholamines was detected in the animals treated with the small or moderate nicotine doses, whereas the higher dose gave a significant increase in adrenaline (p = 0.019). Conclusion: Nicotine in small-to-moderate doses does not cause significant increase in plasma catecholamines, thus indicating that the positive effects of nicotine in studies using these doses most likely cannot be explained by the systemic release of catecholamines.