Oxygen Therapy in Neonatal Resuscitation

Use of Oxygen in Perinatal Asphyxia and Resuscitation

Oxidative Stress: Pathophysiologic Background

Asphyxia is characterized by prolonged periods of ischemia and hypoxia, which lead to specific cellular changes affecting enzyme activities, mitochondrial function, cytoskeletal structure, membrane transport, and antioxidant defenses. All of these changes collectively predispose tissue to reoxygenation injury.5,13,23 During hypoxia, limited oxygen availability decreases oxidative phosphorylation, resulting in a failure to resynthesize energy-rich phosphates, including adenosine 5′-triphosphate (ATP) and phosphocreatine. As a consequence, there is a reduction in the activity of the membrane ATP-dependent Na⁺/K⁺ pump, thus favoring intracellular influx of Na⁺, Ca²⁺, and water, leading to cytotoxic edema. Moreover, Ca²⁺ elicits the activation of numerous metabolic pathways that render injury to structural components of the cell. Adenine nucleotide catabolism during hypoxia-ischemia results in accumulation of hypoxanthine, which is subsequently converted into toxic reactive oxygen species upon the reintroduction of molecular oxygen during resuscitation, provided that xanthine oxidase is available (Figure 34-1).²⁴

Figure 34-1 During hypoxia, ATP is exhausted, and complete rebuilding is not achieved. Purine derivatives (e.g., hypoxanthine) accumulate. During reoxygenation, specific proteases transform xanthine reductase (X_REDUCTASE) into xanthine oxidase (X_OXIDASE), which uses oxygen as a substrate, generating a burst of reactive oxygen species such as superoxide anion (O₂⋅⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (O⋅). In the presence of ferrous iron (Fe⁺⁺), Fenton chemistry generates great amounts of superoxide anion. Superoxide anion easily combines with abundant nitric oxide (NO), generating peroxynitrite (NOO⋅), an extremely aggressive nitrogen free radical. Free radicals are capable of damaging nearby molecules and organelles, but also act as signaling molecules causing changes in gene expression, promoting inflammation, altering immune response, and inducing apoptosis.

In the endothelium, ischemia promotes expression of certain proinflammatory gene products (e.g., leukocyte adhesion molecules, cytokines) and bioactive agents (e.g., endothelin, thromboxane A₂), while repressing other “protective” gene products (e.g., prostacyclin, nitric oxide). Ischemia induces a proinflammatory state that increases tissue vulnerability to further injury on reperfusion.¹³

Reperfusion and reoxygenation of ischemic tissues result in the formation of toxic reactive oxygen species, including superoxide anions (•O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radicals (•OH), and nitrogen reactive species, especially peroxynitrite (•ONOO-). Under physiologic conditions, hypoxanthine accumulated during the ischemia is further oxidized by xanthine dehydrogenase to uric acid in the cells containing this enzyme. However, during prolonged ischemia, xanthine dehydrogenase is converted to xanthine oxidase by specific proteases. Of note, xanthine oxidase uses oxygen as a substrate and during hypoxia-ischemia is unable to catalyze the conversion of hypoxanthine (resulting in the buildup of excess tissue levels of hypoxanthine). When oxygen is reintroduced during resuscitation, conversion of the excess hypoxanthine by xanthine oxidase results in the formation of reactive oxygen species, especially anion superoxide. Moreover, in the presence of nitric oxide, both superoxide and nitric oxide will combine in the formation of reactive nitrogen species, especially peroxynitrite. In tissues rich in ferrous iron such as the brain, Fenton chemistry will ensue, leading to the formation of the highly reactive hydroxyl radical. Interestingly, xanthine oxidase in humans is mainly restricted to the liver and intestine. It has been shown, however, that xanthine oxidase leaks out into the blood after hypoxia and hypotension, and the hypoxanthine-xanthine oxidase system may be detrimental in all parts of the body. Many other oxygen radical generating systems are presently well described.²⁴

Reactive oxygen species and reactive nitrogen species are potent oxidizing and reducing agents that directly damage cellular structures. They are able to peroxidize membranes, structural proteins and enzymes, and nucleic acids. In addition, they are known to be extremely important regulators of intracellular signaling pathways that modulate DNA and RNA synthesis, protein synthesis, and enzyme activation, and directly influence the cell.¹⁵ Increasingly, the concept of redox regulation has acquired more relevance. Thereby, oxidizable thiols are common elements for biologic processes. These control elements are functionally organized in redox circuits, which are controlled by central nodes constituted by sulphur/disulfide couples (e.g., reduced and oxidized glutathione). These circuits function independently and are highly responsive for redox conditions, thus signaling and regulating biologic processes.¹⁷

A vast array of enzymatic and nonenzymatic antioxidants has evolved in biologic systems to protect cellular structures against the deleterious action of free radicals. Antioxidant enzymes catalytically remove reactive oxygen species (ROS), thereby decreasing ROS reactivity, and protect proteins through the use of chaperones, transition metal-containing proteins (transferrin, ferritin, ceruloplasmin), and low molecular weight compounds that purposely function as oxidizing or reducing agents to maintain intracellular redox stability.⁴²

Clinically, antioxidant enzymes that have been most widely studied are the superoxide dismutases, catalases, and glutathione peroxidase. The most relevant nonenzymatic cytoplasmic antioxidant is reduced glutathione (GSH), a tripeptide (γ-glutamyl-cysteinyl-glycine). Hence, two molecules of GSH establishing a disulfide bond form oxidized glutathione and release one electron that is accepted by a free radical in order to stabilize the outer atomic shell. Thiol-disulfide strategy is extremely important in maintaining the reducing state in the cytoplasm and the cell redox status, essential for cell reproduction and maturation. Other systems to detoxify hydrogen peroxide in mitochondria and other organelles include glutaredoxin, thioredoxin, thioredoxin reductase, and the peroxiredoxins. Other enzymes with antioxidant and signaling functions are heme oxygenases (HO-1 and HO-2). HO-1 removes heme, a pro-oxidant, and generates biliverdin, an antioxidant, releasing iron and carbon monoxide. Finally, nonenzymatic antioxidants such as reduced glutathione, vitamin C, vitamin E, and β-carotene also function to protect cells from damaging effects of ROS.²

Oxidative stress in a biologic system is defined as the imbalance of pro-oxidants and antioxidants in favor of pro-oxidants.¹⁵ Different biomarkers of oxidative stress have been used in biology and medicine. An indirect way of measuring oxidative stress is the detection of increased activity of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, or glutathione redox cycle enzymes. Another measure is to analyze the oxidized form of a nonenzymatic molecule such as oxidized glutathione. An increased concentration of oxidized glutathione or a decreased ratio of reduced to oxidized glutathione may indirectly reflect a pro-oxidant status.⁴²

For clinical purposes, the most widely employed markers of oxidative stress are those derived from the oxidant alteration of biologic molecules that convey the following characteristics: being chemically stable, reproducible, and easily measurable in biologic fluids. Urinary markers of oxidative stress are extremely valuable in neonatology because urine sampling is readily available, allowing serial measurements without the need of supplementary blood sampling. In this regard, oxidation of circulating phenylalanine by hydroxyl radicals leads to the formation of ortho-tyrosine (O-tyr) and metatyrosine. These specific metabolites are not synthesized by any physiologic metabolic pathway; their concentration in urine specifically reflects the oxidation of phenylalanine by hydroxyl radicals. In addition, the action of hypochlorous acid and peroxynitrite upon phenylalanine produces byproducts such as chlorotyrosine and nitrotyrosine, which conspicuously reflect inflammatory processes and nitrosative stress.¹⁰ Other relevant urinary biomarkers are oxidized bases of DNA. Specifically, guanine is prone to oxidation by hydroxyl radicals, and its lesion product 8-hydroxyguanine (8-oxo-Gua) and its 2′-deoxynucleoside equivalent 7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) are highly mutagenic. Urinary elimination of 8-oxo-Gua and 8-oxo-dG perfectly reflect nuclear attack by hydroxyl radicals.⁶ In recent experiments performed in a piglet model of hypoxia and reoxygenation, urinary elimination of metabolites O-tyr and 8-oxo-dG correlated significantly with the amount of oxygen used on reoxygenation.³⁴ Finally, F2-isoprostanes and isofurans, which are non-cyclooxygenase oxidative derivatives of arachidonic acid, are considered at present the most reliable markers of lipid peroxidation. These compounds are chemically stable, formed in vivo, present in all organic fluids and tissues, and are not affected by dietary content of lipids. Isofurans reflect oxidation in a high oxygen atmosphere and isoprostanes in a normoxic environment. In addition, byproducts derived from the oxidation of docosahexanoic acid such as neuroprostanes and neurofurans, have also been considered very valuable biomarkers, especially related to oxidative damage of neuronal membranes.³⁵

Oxidative Stress: Differences between Resuscitation with 100% Oxygen and Room Air

In the absence of severe lung disease or cyanotic congenital heart disease, resuscitation with 100% oxygen has been shown to cause supraphysiological arterial partial pressures of oxygen in the newly born infant. By contrast, resuscitation with room air increases the Pao₂ to physiological levels only (i.e., approximately 70 to 80 mm Hg). Biomarkers of oxidative stress such as oxidized glutathione or antioxidant enzyme activity are significantly increased in patients receiving excessive oxygen. Thus, newborn infants resuscitated with pure oxygen exhibit higher oxidative stress after resuscitation than infants recovered with room air.³⁸ Conspicuously, oxidative stress derived from resuscitation with pure oxygen may cause a long-lasting pro-oxidant status. Hence, in newborn babies resuscitated with 100% oxygen, oxidative stress was detected 4 weeks after birth. These infants had a decreased ratio of total blood reduced glutathione to oxidized glutathione and oxidized DNA bases in urine at 1 month of life. No such effect has been observed in infants resuscitated with room air (Figure 34-2).³⁷

Figure 34-2 Ratio of oxidized glutathione to reduced glutathione in asphyxiated newborns resuscitated with room air (RAR) or 100% oxygen (OxR) determined at 0 (birth), 3, and 28 days of life. ** p < .01 versus control; ## p < .01 versus RAR.

Hyperoxemia has also been associated with a series of negative side effects, including increased oxygen consumption and metabolic rate, increased activation of leukocytes and endothelial cells, and increased formation of reactive oxygen species and reactive nitrogen species.¹⁴ Prolonged vasoconstriction of cerebral arteries has also been shown. In a study involving premature infants younger than 33 weeks’ gestational age at 24 hours, cerebral blood flow was reduced by 20% in infants given 80% oxygen compared with infants in whom room air was used. This finding is in line with studies in newborn rats showing that the use of 100% oxygen for resuscitation reduces cerebral blood flow compared with room air resuscitation.¹²

Room Air Versus Pure Oxygen for Resuscitation of the Newborn

Traditionally, postnatal resuscitation in the delivery room had been performed with 100% oxygen.¹ However, in 1998, the Resair 2 trial showed that it was feasible to resuscitate asphyctic newborn infants with room air.²⁸ Thereafter, a series of clinical studies summarized in a recent updated review and meta-analysis has shown not only the suitability of room air for the resuscitation of the asphyctic term neonate but also a series of advantages, among which the most relevant is a decrease in mortality.^30,31

Animal Studies

Animal studies have shown that after a period of severe hypoxia, physiologic functions such as blood pressure and blood flow to various organs, including the brain, are restored equally efficiently with 21% and 100% oxygen. Evoked potentials and biochemical indicators such as base deficit and hypoxanthine are restored efficiently using room air for resuscitation. Furthermore, experimental studies have also clearly shown that using 21% instead of 100% oxygen offers significant additional advantages. Hence, the use of high oxygen concentrations is not only toxic for the lungs, but also for several other organs such as the heart, liver, and brain. In a recent review the most relevant findings in animal models of hypoxia and reoxygenation with 21% versus 100% have been detailed.³² Thus, extracellular glycerol concentration from the striatum was significantly higher in hypoxic piglets resuscitated with pure oxygen as compared with those resuscitated with air. Moreover, in a mouse model, hyperoxia after hypoxic-ischemic brain injury increased secondary neuronal injury and interfered with myelination. Matrix metalloproteinases, which reflect tissue damage and repair, were measured in lung, liver, heart, and brain of hypoxic piglets recovered with 100% versus 21% oxygen. Remarkably, the use of elevated oxygen concentration caused a significant increase of metalloproteinases in all analyzed tissues.¹² The H₂O₂ concentration in leukocytes from the sagittal sinus increased significantly in newborn hypoxic piglets resuscitated with 100% oxygen, in contrast to piglets given 21% oxygen. The nitric oxide concentration in the brain also tended to become higher if pure oxygen was used compared with ambient air for resuscitation of piglets. The stage might be set for a higher production of reactive nitrogen species and peroxynitrite if 100% oxygen is used.^18,19,20

There are also clear indications that resuscitation with 100% oxygen augments inflammatory processes in the myocardium and the brain more than 21% oxygen. In a study using cord occlusion of fetal lambs, resuscitation with room air restored blood pressure as fast as when using 21% oxygen. In the cortex and subcortical area, significantly higher levels of proinflammatory cytokines and activities of Toll-like receptors 2 and 4 were found in animals given 100% oxygen.²⁵

Some animal studies have shown that cerebral brain microflow is reestablished faster with 100% oxygen. One study in newborn mice found that 100% versus 21% oxygen gave faster cerebral blood flow restoration and poorer short-term and improved long-term recovery.²⁷ In another study, the slower normalization of cerebral blood flow in animals resuscitated with room air versus animals resuscitated with 100% oxygen almost disappeared, however, if moderate hypercapnia was present. In birth asphyxia, hypercapnia always coincides with hypoxia. Hypercapnia might seem to be an important factor in reestablishing brain blood flow after asphyxia. As mentioned earlier, other studies have shown that 100% oxygen during normocapnia, in contrast to the use of 21% oxygen, reduces cerebral blood flow.¹²

Oxygen is a pulmonary vasodilator, but studies performed in animal models of hypoxia reoxygenation have shown that hyperoxia blunts vasodilator activity of oxygen probably mediated by the generation of reactive oxygen species especially H₂O₂ in the mitochondrial matrix. H₂O₂ diffuses to the cytoplasm, activating phosphodiesterase 5 (PDE5) which, acting upon the vascular smooth muscle cells, degrades cGMP thus inhibiting nitric oxide mediated vasorelaxation and favoring persistent vasoconstriction. Remarkably, resuscitation with 100% oxygen of term fetal lambs rendered asphyctic by cord occlusion initially induced a decrease in pulmonary vascular resistance (PVR) and subsequent increase in pulmonary blood flow. Nonetheless, subsequent values (between 2 and 30 min) for PVR did not differ between lambs resuscitated with air or 100% oxygen. Interestingly, the increased pulmonary artery contractility induced by 100% oxygen was reversed when superoxide anions were scavenged. Hence, the use of 100% oxygen increases partial pressure of oxygen in the pulmonary artery, yet it does not enhance oxygen uptake by lung tissue, does not decrease pulmonary vascular resistance, or increase systemic oxygen extraction ratios. Furthermore, 100% oxygen also induces oxidative stress and increases pulmonary artery contractility, thus favoring pulmonary hypertension.21,22 Animal studies have also shown that even in meconium aspiration, resuscitation with room air is as efficient as resuscitation with 100% oxygen, provided that a sufficient tidal volume is given.³⁶

The results from animal studies quite overwhelmingly indicate that room air resuscitation is, in most cases, beneficial when compared with the use of 100% oxygen. Still there might be clinical conditions such as prematurity with primary lung disease when limited initial supplemental oxygen should be added.

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