Balance of Antioxidants vs. Oxidants in Perinatal Asphyxia

Abstract

Perinatal asphyxia refers to an acute event of cerebral ischemia and hypoxia during the perinatal period, leading to various degrees of brain injury. The mechanisms involved in perinatal asphyxia include the production of reactive oxygen species (ROS), accumulation of intracellular calcium, lipid peroxidation, excitatory amino acid receptor overactivation, energy failure, and caspase-mediated cell death. Both primary and secondary neuronal damage are caused by the overproduction of ROS following a hypoxic/ischemic event. ROS can react with nearly any type of molecule, including lipids, proteins, polysaccharides, and DNA. Neonates who suffer from perinatal asphyxia are prone to oxidative stress, which is characterized by a disruption in the oxidant/antioxidant balance, favoring oxidants over the intracellular and extracellular antioxidant scavenging mechanisms. Current research has focused on developing treatment strategies that potentially improve the endogenous antioxidant neuroprotective mechanisms or minimize injury resulting from hypoxia/ischemia. In this narrative review, we aim to present evidence regarding the contribution of oxidant/antioxidant balance to the pathogenesis and progression of perinatal asphyxia. Also, we aim to explore the role of potential antioxidant therapies as promising treatment strategies for perinatal asphyxia, especially as an adjunct to therapeutic hypothermia in infants with perinatal asphyxia. The current literature on antioxidant treatments in newborns is limited; however, allopurinol, melatonin, and erythropoietin have shown some positive effects in clinical trials. Inhibitors of nitric oxide synthase, N-acetylcysteine, and docosahexaenoic acid have shown promising neuroprotective effects in preclinical studies. Finally, nanotherapeutics could potentially modulate oxidative stress in hypoxemic/ischemic brain injury by targeted medication delivery. Future research on neuroprotectants and their processes is warranted to develop innovative treatments for hypoxia/ischemia in clinical practice.

1. Introduction

Neonatal encephalopathy (NE) predominantly occurs during the perinatal period as a result of a sentient event that affects cerebral blood flow and oxygen supply and leads to impaired brain function. The prevalence of NE varies significantly; in developed countries, the prevalence is estimated to 1 to 8 per 1000 live births, while in underdeveloped regions it is much higher, at 26 per 1000 live births [1]. Although therapeutic hypothermia has been established as a standard therapy, NE continues to be a significant contributor to neonatal mortality and developmental disorders among term infants globally [2].

The transition from fetal to neonatal life introduces physiological oxidative stress, which stimulates the body’s antioxidant defense mechanisms. Birth represents a significant challenge to newborns, marked by a rapid adjustment from the relatively low-oxygen intrauterine environment to the more oxygen-rich external environment. This transition exposes newborns to reactive free radicals (RFRs), produced more intensely in response to the newborn’s less efficient natural antioxidant systems [3]. Evidence suggests that an imbalance between antioxidant and oxidant systems can result in oxidative damage, further highlighting the complexity of oxidative metabolism. Newborns, especially those born prematurely, have an immature antioxidant defense system (ADS) and thus, they are particularly vulnerable to oxidative stress and oxidative damage from RFRs [4]. Perinatal hypoxic/ischemic damage initiates a series of detrimental biochemical processes, including elevated neurotransmitter levels, overproduction of oxygen free radicals, heightened intracellular calcium concentrations, and activation of inflammatory mediators and signaling pathways, all of which contribute to tissue damage [5].

Previous reviews of the antioxidant/oxidant balance in asphyxiated neonates have focused on the biochemical basis of oxidative metabolism and the pathogenesis of NE [6,7,8], while several biomarkers have been identified with a potential role in the diagnosis and prognosis of injury in NE [8,9,10]. Moreover, the currently available treatments and adjuvant neuroprotective approaches based on antioxidant therapies have not been extensively discussed [6,7,8,9,11]. As research in NE is still evolving, clinical evidence and preclinical experimental data emerge, offering potential novel adjuvant therapeutic options that may improve the outcomes of hypothermia. Furthermore, limited data have been presented about novel techniques that could potentially modulate the oxidative stress in hypoxemic/ischemic brain injury by targeted delivery of medication, such as nanotherapeutics.

Hence, this narrative review seeks to offer a comprehensive elucidation of the pathophysiology of antioxidant/oxidant balance and the significance of free radicals and oxidative biomarkers in the context of NE, as well as to explore potential novel adjuvant therapies that could mitigate RFR production and enhance the outcomes of therapeutic hypothermia.

2. Materials and Methods

The Literature Search Strategy

A literature search was conducted in August 2024 in PubMed. Only English-language articles with full text available were considered. The terms ‘oxidate stress’, ‘oxidants’, ‘antioxidants’, ‘free radicals’ and ‘perinatal asphyxia’, ‘hypoxic-ischemic encephalopathy’, ‘neonatal encephalopathy’ were utilized. The search strategy is presented in

Table 1. The search strategy of the study.

Item	Description
Search date	31 August 2024
Time frame	From initiation to 31 August 2024
Search terms	‘Oxidate stress’, ‘oxidants’, ‘antioxidants’, ‘free radicals’ and ‘perinatal asphyxia’, ‘hypoxic-ischemic encephalopathy’, ‘neonatal encephalopathy’
Database	Pubmed
Inclusion and exclusion criteria	English, full-text articles

.

Our review is organized as follows: (1) the basics of the pathophysiology of oxidative stress in NE, (2) evidence from animal models of hypoxia/ischemia, (3) evidence of free radical biomarkers in NE, (4) a clinical assessment of neonates with encephalopathy, and (5) a summary of the latest research in the treatment options in NE (Figure 1).

3. Pathophysiology

3.1. Aerobic Metabolism and Oxidative Phosphorylation

Human beings utilize aerobic metabolism and oxidative phosphorylation to generate energy. These processes are executed within the mitochondria and require oxygen. This metabolic pathway releases electrons, which are transferred to the electron transport chain via nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The electron transport chain consists of the enzyme complexes I, III, and IV, which create membrane potential by transferring protons (H+) from the inner to the outer membrane. Consequently, protons flow back into the matrix due to the membrane potential, thus releasing energy that is stored in the form of adenosine triphosphate (ATP). Oxygen is crucial in this pathway for accepting these electrons, thereby protecting mitochondria from damage [12,13].

The central nervous system is known as the “oxygen regulator” because it requires substantial energy to sustain membrane potentials. Consequently, the central nervous system relies on aerobic metabolism which, compared to anaerobic metabolism, provides more ATP molecules [14]. Within the central nervous system, ion pumps that facilitate action potentials are ATP-dependent and thus require a significant amount of energy. Nevertheless, the brain is unable to store energy in forms like glycogen or phosphocreatine that could be quickly released. Therefore, in circumstances of low oxygen, glucose energy reserves are depleted rapidly and cells die within minutes [15,16].

3.2. Biological Antioxidant Systems

Oxidative stress represents a metabolic imbalance caused by an excess of free radical production or a reduced capacity of the antioxidant defense system (ADS) [3]. While free radicals are necessary for cell communication and the proper operation of biochemical pathways, an extreme imbalance of the ADS and free radicals can damage tissues and lead to acute or chronic illnesses [17]. The ADS is developed later in pregnancy to prepare the fetus for oxygenation after birth and encompasses both enzymatic and non-enzymatic components. Antioxidant enzymes neutralize reactive species by undergoing chemical reactions. Non-enzymatic antioxidant systems consist of proteins that sequester metals (including ferritin, transferrin, and ceruloplasmin), vitamins that prevent the oxidation of lipids (like A, C, D, and E), and small molecules that diminish reactive species.

Sufficient levels of vitamin A in the tissues could provide a defense against infections and the harmful effects of oxygen free radicals [18]. A previous study found significantly lower levels of vitamin A in neonates with reduced antioxidant defenses, as indicated by their increased need for delivery room resuscitation compared to controls [18]. In another study, low vitamin A levels during the first month of birth were found to greatly increase the likelihood of developing long-term respiratory impairment [19]. The antioxidant characteristics of vitamin C include the production of ascorbyl radicals, a terminal reactive oxygen species (ROS) with a relatively minimal harmful effect on biological systems. Although vitamin C is mainly considered an antioxidant, under specific circumstances, vitamin C may show prooxidant qualities in the presence of free (unbound) redox metals, such as iron or copper [20]. Numerous antioxidant processes are linked to vitamin D; when the condition of vitamin D and oxidation was assessed in neonates with NE, vitamin D levels were found to be lower than normal [21]. Additionally, the administration of vitamin D significantly reduced the malondialdehyde (MDA) content generated by hypoxemia/ischemia and elevated glutathione (GSH) and SOD (superoxide dismutase) activity, both of which were suppressed by hypoxemia/ischemia. Among numerous mechanisms of cellular injury that have been linked to the creation of ROS [22], it has been shown that calcitriol, an active form of vitamin D, suppresses ROS formation by interfering with the hydrogen peroxide pathway [23]. Vitamin E protects cellular membranes from ROS-caused lipid peroxidation, with the hydroxyl group on its six-membered ring situated outside of the membrane and scavenging ROS in the aqueous environment.

Furthermore, there has been great interest in the content of trace elements and their potential role as key regulators of antioxidant defense and oxidative stress. Copper is a cofactor in numerous detoxification enzymes and proteins, including SODs, ceruloplasmin, and copper–thioneine [24]. In preterm newborns, a decrease in SOD activity was linked to low copper levels [25]. Regarding ferrum, studies have shown that children with ferrum-deficiency anemia exhibit a decrease in antioxidant activities, such as that of catalase [26]. Zinc is essential for cell differentiation, metabolism, and growth, while promoting antioxidant enzyme activities like Zinc–SOD and Zinc–thioneine, hormone structures, and brain development [24]. Moreover, the importance of selenium for the best possible operation of the body’s natural antioxidant defense mechanisms is becoming more widely recognized [27]. The “selenoenzymes” glutathione peroxidases (GPx), thioredoxin reductases, and selenoprotein P are produced only when selenium is present [28,29]. Through selenoproteins, selenium is linked to the regulation of ROS levels and redox balance in nearly every tissue. Due to its significant antioxidant effect, low selenium levels have been linked to impaired oxidative system defenses [30]. Evidence suggests that supplementing with selenium may lessen pregnancy-related oxidative stress [31]. Overall, adequate selenium levels potentially strengthen endogenous antioxidant defenses to minimize the consequences of oxidant stress, even if a lack of selenium is unlikely to entirely account for free radical injury in neonates [32]. Mercury exists in three different forms, with a unique toxicological profile of each: elemental mercury can damage the nervous system and kidneys; organic mercury compounds, such as methylmercury, can cross the blood–brain barrier; and inorganic mercury salts can damage the kidneys and nervous system [33].

Moreover, GSH can combine with another GSH molecule to form oxidized glutathione (GSSG), which then donates two electrons to free radicals, aiding in their neutralization. The enzyme GSH reductase facilitates the conversion of GSSG back to GSH. SODs are important antioxidant enzymes that convert superoxides to hydrogen peroxide. Catalases and GPx catalyze the conversion of hydrogen peroxide to water and oxygen. The haem complex is broken down and converted to biliverdin by haem oxygenases, glutaredoxins, thioredoxins, peroxiredoxins, and other components of the ADS in red blood cells [13,17].

3.3. Hypoxia/Ischemia Reperfusion Injury

In conditions of ischemia and decreased oxygen supply followed by reperfusion, free radicals containing oxygen (ROS) or nitrogen (reactive nitrogen species, RNS) can be generated. Free radicals are extremely reactive and unstable due to an unpaired electron that they contain in their external membrane [34]. When the number of RFRs surpasses the capacity of the body’s endogenous ADS to neutralize them, it can lead to oxidative damage. This damage may affect DNA, polysaccharides, proteins, and lipids, compromising cellular function and potentially causing cell death through either apoptosis or necrosis [35]. The developing brain is characterized by a high oxygen requirement, a low antioxidant defense of polyunsaturated fatty acids which are the main component of phospholipids, a high concentration of metals that catalyze ROS production, and a large number of immature cells; thus, it is particularly vulnerable to ROS/RNS-induced injury [36].

The progression of NE can be segmented into three stages, though in actuality, these stages overlap, with events often occurring simultaneously (Figure 2). The initial stage lasts 6 h and is marked by a decrease in blood flow to the fetus, resulting in systemic and cerebral hypoperfusion. Cerebral hypoperfusion leads to brain ischemia, hypoxia, acidosis, and cellular damage due to energy depletion. During hypoxia/ischemia, the brain areas with the highest metabolic activity and numerous excitatory glutamatergic synapses are primarily affected [37]. A key factor in hypoxic/ischemic-related brain damage is the overstimulation of postsynaptic receptors due to the excitotoxicity of glutamate neurotransmission, leading to cell death [38]. During prolonged hypoxia/ischemia, neurons may face a depletion of high-energy metabolites, an increase in cytotoxic edema, and an extracellular accumulation of excitatory amino acids [39]. Some neurons may die immediately or shortly after the injury, while others might initially recover at different stages, eventually leading to cell death [40]. Cerebral hypoxia triggers anaerobic metabolism, which results in an intracellular increase in lactic acid, sodium, calcium, and water; a decrease in ATP levels; and the reuptake of neurotransmitters, leading to secondary excitotoxicity. Intracellular calcium triggers the release of lipases, nitric oxide synthase (NOS), and substances that promote cell death; enhances the production of free radicals; and causes direct damage to the mitochondria.

The second stage, which can last from 6 to 48 h, is characterized by ongoing excitotoxicity, a decrease in mitochondrial function and oxidative stress due to alterations in the membrane potential, a decrease in ATP production, and an alkaline environment despite sufficient oxygen supply. Immediately after a severe injury, there is a recovery of blood flow to the brain, which is vital for survival but paradoxically leads to reperfusion injury due to an overproduction of radicals [41]. This causes not just an initial burst of oxidative stress but also a gradual breakdown of the mitochondrial electron transport chain, a depletion in ATP levels, and further damage to brain cells [8]. During reperfusion, reactive molecules are mainly generated by the mitochondrial respiratory chain complex and the xanthine oxidase (XO) system. After reperfusion/reoxygenation, hypoxanthine is gradually increased, leading to the conversion of xanthine dehydrogenase (XD) to XO; in turn, XO is the main source of ROS by producing superoxide radicals using oxygen [8]. Additionally, the NADPH oxidase system and NOS uncoupling processes are also engaged, though their contributions are comparatively minor [12]. NOS and mitochondrial electron leak are identified as key factors in the production of ROS and RNS [42,43]. Moreover, N-methyl-D-aspartate (NMDA) and glutamate receptors trigger the production of nitric oxide (NO) through the activation of NOS. Peroxynitrite (ONOO−) and hydroxyl radicals (OH−) can be created when NO combines with superoxide. This leads to further cell damage through protein oxidation, lipid peroxidation, DNA damage, and mitochondrial inactivation [6]. Additionally, damage from overactive calcium channels can also result in the production of oxygen through NADPH oxidase system activity [44]. Additionally, non-protein-bound iron from hemoglobin increases the formation of hydroxyl radicals through the Fenton reaction, which significantly increases ROS levels [45]. These radicals and RNS exacerbate the damage to the mitochondria that started with the initial decrease in ATP levels and the overstimulation of glutamate receptors, leading to a further breakdown of oxidative phosphorylation, which results in a worsening of the energy crisis and the death of neurons [46]. Several studies have indicated that an increase in ROS can promote the release of cytochrome C from the mitochondria, which catalyzes the oxidation of cardiolipin [47,48]. Subsequently, pro-apoptotic proteins increase in the cytoplasm and then bind to Apaf-1, forming the Apaf-1/caspase-9/Sitka complex, which in turn inactivates caspase-3, resulting in apoptosis and the death of neurons [49].

The third stage, which can continue for several days or even weeks, is marked by inflammation and altered gene expression that result in defects in the formation of new neurons, the development of synapses, and the growth of axons [50,51]. Oxidative stress interacts with the immune system, creating a “vulnerability period” for NE. Initially, oxidative stress is triggered by immune cells at the site of residence, such as microglia. These microglia are highly responsive to hypoxia/ischemia and are activated by an increase in NO, glutamate, and ROS, leading to the production of a large number of inflammatory molecules [52]. Astrocytes are also activated, producing interleukins (IL-1α, IL-1β, IL-6), tumor necrosis factor-alpha, and interferon-gamma, which hinder the growth of new neurons, directly damage immune cells, and cause neuronal cell death [53].

4. Animal Models of Neonatal Hypoxia/Ischemia

The most commonly employed model for neonatal hypoxic/ischemic injury in animals is the model established by Rice-Vannucci [54]. Although initially developed for rats [54], this model has also been applied to other rodents [55,56,57]. In this model, after blocking one carotid artery, the piglets are exposed to a period of hypoxia, reproducing the injury of stroke of the middle cerebral artery, which is rare in preterm neonates [58]. Other methods for inducing brain injury from hypoxia/ischemia in piglets include the combination of hypoxia and hypotension-induced ischemia [59], cardiac arrest followed by cardiopulmonary resuscitation [60], and bilateral common carotid artery occlusion in conjunction with hypoxia [61].

In previous studies, it was shown that six hours after the asphyxic event, post-asphyxic hypothermia significantly reduced the formation of protein carbonyls in the parietal cortex and the striatum of newborn piglets [62,63]. On the other hand, when comparing hypothermic versus normothermic piglets 48 h after the hypoxic insult, no significant changes in cerebral protein carbonyl levels were found [64]. This evidence indicated that the role of oxidative stress in brain damage was crucial during the initial hours after the hypoxic event, which then normalized over time. Nonetheless, newborn piglets exposed to hypoxemia followed by 29 h of hypothermia had higher levels of carbonylated proteins compared to those that remained normothermic. Similar findings were also recorded in the cerebral white matter of newborn piglets subjected to cardiac arrest followed by hypothermia of limited duration [65]. Additionally, significantly lower levels of antioxidant enzymes like SOD, GPx, or catalase activities were found in the cerebral tissue of newborn rats exposed to asphyxia [66]. Of note, there was a reduction in antioxidant enzyme activities in newborn rats that were asphyxiated, both in normoxic and hypoxic conditions; however, the activities of these enzymes were lower in asphyxiated rats compared to controls [66].

Following an ischemic event, mild hypothermia delayed the utilization of SOD, GPx, and GSH in the brain tissue [67]. In newborn rats exposed to hypoxemia/ischemia followed by hypothermia, a notable increase in antioxidant protection was noted, characterized by higher levels of SOD and GPx expression [68]. Hypothermia resulted in an increase in the expression of SOD-1 in the neocortex and the caudate putamen, although the expression of GPx-1 increased only in the caudate putamen but not in the neocortex [69].

Finally, the expression of DNA-repair genes (DNA glycosylases OGG1, NEIL1, and NEIL3) was reduced in hypothermic pigs compared to controls or resuscitated pigs [70]. Decreased expression of these genes was primarily detected in the hippocampus but also included the cortex, cerebellum, and liver [70].

5. Free Radical Biomarkers in NE

The effectiveness of a biomarker in NE is affected by the timing of the brain insult; however, the period following birth does not necessarily equate to the period following the insult [71]. Regrettably, the timing of the insult, whether it occurs before or during birth, is often not known. Even the time between fetal death and delivery can vary significantly, with cell death occurring at different times after the insult [72]. This lack of precise timing complicates the categorization of cerebral injuries as primary (acute energy failure), secondary (following a second energy failure and subsequent cell death), or tertiary (increased sensitivity to subsequent cell death) [73]. Currently, with more advanced techniques for detecting ROS and RNS, it may be possible to enhance free radical biomarkers and more accurately detect the timing of the insult (

Table 2. Free radical biomarkers in neonatal encephalopathy.

Biomarker	Mechanism of Action	Fluid to Measure	Phase of Hypoxemia/Ischemia
Xanthine oxidase	Converts hypoxanthine and xanthine to an oxygen radical-producing form of xanthine dehydrogenase	Blood	Hypoxia/ischemia
Superoxide dismutase	Increases acutely to defend against ROS	Blood, CSF	Hypoxia/ischemia
Glutathione peroxidase	Increases acutely to defend against ROS	Blood, CSF	Hypoxia/ischemia
Catalase	Increases acutely to defend against ROS	Blood, CSF	Hypoxia/ischemia
Nitric oxide	Increases due to the upregulation of NOS during ischemia	Blood, CSF	Hypoxia/ischemia
Perfusion-assisted breathing	Assay to evaluate oxidative stress	Blood	Hypoxia/ischemia
Malondialdehyde	Biomarker of damage to n-3 and n-6 fatty acids	Blood, CSF	Hypoxia/ischemia followed by reperfusion/reoxygenation
4-hydroxynonenal	Byproduct of damage to n-6 fatty acids	Blood	Hypoxia/ischemia followed by reperfusion/reoxygenation
Prostaglandin-like compounds (isoprostanes, neuroprostanes, neurofurans)	Formed from the free radical-induced breakdown of arachidonic acid and docosahexaenoic acid	Blood	Hypoxia/ischemia followed by reperfusion/reoxygenation
Protein oxidation markers (protein carbonyls)	Formed through protein carbonylation, nitration, crosslinking, or the loss of thiol groups by free radicals	Blood	Hypoxia/ischemia followed by reperfusion/reoxygenation
Uric acid	Increased lately during the development of oxidative damage	Urine	Reperfusion/reoxygenation
Iron not-bound by proteins	Iron interacts with O2− and H2O2 forming highly reactive OH	Blood	Reperfusion/reoxygenation

CSF, cerebrospinal fluid.

).

5.1. Enzymes Against Free Radicals

XO is an enzyme containing molybdenum that produces free radicals in the presence of purines hypoxanthine and xanthine. XO increases with hypoxia/ischemia, leading to the breakdown of adenine monophosphate into these purines [10]. The main source of XO is likely from endothelial cells rather than the brain tissue itself, and the term XO refers to two forms, xanthine oxidoreductase, which is present in healthy cells, and xanthine dehydrogenase, which is converted to an oxygen radical-producing form during ischemia. Although XO is barely detectable in the adult human brain [74], studies have shown that newborns have low levels of both total xanthine dehydrogenase and XO [75].

SOD, GPx, and catalase are the initial line of defense against oxidative stress. After experiencing hypoxia/ischemia, their activity sharply increases to counteract the rise in ROS and their detrimental effects. Research has shown that asphyxiated newborns have significantly higher levels of SOD, catalase, and GPx in their cord blood compared to non-asphyxiated newborns [76]. However, by 24 h, only catalase and SOD levels remain elevated in the blood of infants who have experienced NE compared to those who have not [21]. Studies have also found a correlation between the levels of catalase and SOD in the cord blood and the severity of neonatal asphyxia [76]. Therefore, the measurement of these antioxidant enzymes in the cord blood could provide valuable insights into the timing and severity of neonatal asphyxia [76]. Of note, these findings are based on small groups of participants, so further research is needed to confirm these results in larger studies. Regarding the levels of these antioxidant enzymes in other body fluids, a previous study has shown that the activity of SOD was significantly higher in the cerebrospinal fluid up to 72 h after birth in term newborns who have experienced neonatal asphyxia, whereas the activities of GPx and catalase increased only in the most severely affected cases [77]. Most of the research on these topics was conducted before the standard use of therapeutic hypothermia, which raises questions about how hypothermia might affect the levels of these antioxidant enzymes.

5.2. Nitric Oxide

The upregulation of NOS during ischemia leads to increased production of NO and RNS. Studies have shown that newborns with neonatal asphyxia have higher levels of NO in their blood and cerebrospinal fluid, in addition to a higher ratio of nitrate to nitrite in their blood, which is a measure of NO levels [78,79]. Evidence suggested that higher NO levels were found in infants with early signs of cerebral injury compared to those without brain damage; thus, the increase in NO was related to the severity of neonatal asphyxia [78].

5.3. Perfusion-Assisted Breathing

The perfusion-assisted breathing (PAB) assay has been utilized in adults to evaluate the oxidative status in various conditions associated with oxidative stress, including diabetes mellitus, acute coronary syndrome, and stroke [80,81,82]. In neonates, it was shown that PAB, when used together with the severity of hypoxic/ischemic encephalopathy, could more accurately predict the outcome and the risk of developing neurological issues in term newborns who have suffered from asphyxia [83]. The study indicated that PAB, the fifth-minute Apgar score, and the pH level in the first hour after birth were crucial factors in determining the outcome for neonates who had experienced asphyxia. Additionally, the measurement of PAB in the blood could be a useful tool in assessing the prognosis of asphyxia during birth [83].

5.4. Markers of Lipid Damage (Malondialdehyde and 4-Hydroxynonenal)

The amount of damage to lipids during periods of hypoxemia/ischemia can exceed the body’s ability to self-repair, leading to negative effects on the structure and function of cell membranes, loss of function, and even cell death. Once this damage begins, several products of lipid damage are released, many of which can be detected in the brain due to its high fat content.

Malondialdehyde (MDA), which is a sign of damage to n-3 and n-6 fatty acids, has been extensively studied because it is easy to measure using common spectrophotometry. Evidence is consistent that serum MDA levels are higher in severely asphyxiated compared to non-asphyxiated newborns [21,79,81,83,84]. As for predicting outcomes, higher MDA levels in the blood at 24 h are linked to more severe NE [21], with the highest levels found in infants who died or had long-term neurological problems [85,86]. A potential issue with using MDA in the blood is that it might not be specific to the brain, as it could also reflect damage to other tissues due to widespread organ damage. Using MDA in the cerebrospinal fluid might provide a better idea of the damage to the brain in asphyxiated infants. Studies have shown that MDA levels in the cerebrospinal fluid are higher in asphyxiated compared to non-asphyxiated term infants, with a stronger link to the severity of NE [76]. This is also seen in infants who have died or have long-term neurological issues compared to those who have recovered normally [86]. However, more research is needed to confirm that MDA in the cerebrospinal fluid is a reliable indicator of brain damage in asphyxiated infants. Regarding 4-hydroxynonenal (4-HNE), a byproduct of damage to n-6 fatty acids, it was found that 4-HNE levels in the blood increased following a period of hypoxia [87]. However, evidence in this biomarker is limited because 4-HNE is difficult to measure and requires advanced liquid chromatography techniques as it is unstable [88].

5.5. Peroxidation-like Compounds

Prostaglandin-like compounds, including isoprostanes, neuroprostanes, and neurofurans, are formed from the free radical-induced breakdown of arachidonic acid and docosahexaenoic acid (DHA), which are types of polyunsaturated fatty acids. F2-isoprostanes appear to be the most reliable indicators of the extent of oxidative stress following hypoxia–reperfusion events [89], and they can be measured accurately using ultra-high-performance liquid chromatography–mass spectrometry techniques, even in newborns. Elevated levels of 8-iso-15(R)-PGF2α and total isoprostanes were found in infants with acidosis and depression compared to those without these conditions; 8-iso-15(R)-PGF2α levels were correlated with the severity of asphyxia [90]. However, a study that longitudinally measured serum F2-isoprostanes within the first 5 days after birth in neonates who experienced severe, moderate, or mild asphyxia found no differences between the groups and no correlation with brain damage detected through neuroimaging [91]. This limited and conflicting evidence suggests that more research is needed to determine if prostaglandin-like compounds can serve as biomarkers following perinatal asphyxia.

5.6. Protein Oxidation Markers

Protein carbonyls and products of advanced protein oxidation are formed through protein carbonylation, nitration, crosslinking, or the loss of thiol groups by free radicals. Elevated levels of protein carbonyls were found in asphyxiated term newborns at birth and 48 h after birth [85]. Higher levels of protein carbonyls were seen in neonates who developed seizures; however, there was no difference in the Sarnat stage or neurodevelopmental outcome at 9 months of age [85]. Moreover, higher levels of advanced-oxidation protein products were found in asphyxiated compared to non-asphyxiated preterm infants, with a positive correlation between this marker and plasma hydroperoxides [92]. It has been reported that higher plasma advanced-oxidation protein product levels at 4–6 days after birth have been seen in term infants with severe NE, who required therapeutic hypothermia, compared to those with milder or moderate NE [91]. However, subsequent measurements in hypothermic asphyxiated infants and controls showed no difference in advanced-oxidation protein product levels between the two groups [21]. Given this lack of difference in advanced-oxidation protein product levels during and after hypothermia, a possible role for hypothermia in regulating protein oxidation, which warrants further investigation, is suggested. Additionally, a significant independent association between magnetic resonance imaging (MRI) scores indicating brain injury and blood levels of advanced-oxidation protein products over the first 5 days of life in neonates with NE has also been reported, with a stronger correlation seen in male infants, indicating a potential role for advanced-oxidation protein products as a biomarker for cerebral oxidative injury [91].

5.7. Uric Acid

Uric acid is crucial in the development of oxidative damage in the brain during reperfusion/reoxygenation. Therefore, the amount of uric acid excreted through urine has been suggested as a simple and non-invasive way to measure the production of ROS related to oxygen deprivation in newborns. Several studies have consistently found that the ratio of uric acid to creatinine in the urine increases within 48–72 h of birth in both term and preterm newborns who have experienced neonatal asphyxia [84,93]. Additionally, there is a positive correlation between this ratio and the severity of neonatal asphyxia, as indicated by the Sarnat stage [84].

5.8. Iron Not Bound by Proteins

After experiencing hypoxemia followed by reoxygenation, the iron that was previously bound to hemoglobin interacts with O₂− and H₂O₂, forming highly reactive OH. Prior research showed that as the severity of asphyxia increased, so did the levels of iron not bound by proteins in the blood [94]. However, it was also noted that even infants who were moderately or severely asphyxiated had low or undetectable levels of this iron. Nonetheless, the presence of low or undetectable levels of this iron was not linked to any neurological problems at one year of age, even though the study only included a small number of infants [94].

6. Clinical Assessment of Neonates with Encephalopathy

Several scoring methods can be used to evaluate NE and predict the outcomes with clinical examination. There is a clear correlation between neuro-developmental outcomes and the Sarnat staging of encephalopathy at less than six hours of life [95]. However, a multicenter prospective analysis of newborns with hypothermia and NE revealed that a clinical neurological exam performed before six hours of life and showing moderate to severe encephalopathy was insufficient to predict unfavorable outcomes [96]. Studies on hypoxemic/ischemic newborns treated with hypothermia have also demonstrated that the Thompson score is a useful prognostic tool for predicting outcomes at 4 to 5 years of age [97]. To appropriately predict outcomes in infants with NE, clinical indicators alone are not sufficient unless clinical examination is combined with multiorgan dysfunction and Apgar score.

Regarding laboratory findings, it has been previously demonstrated that poor outcomes were linked to low initial newborn arterial pH (umbilical or arterial, within the first hour after birth). According to a meta-analysis, poor newborn outcomes were significantly correlated with low initial arterial pH in encephalopathic neonates prior to the hypothermia era [98]. Also, a lower initial pH was linked to more severe brain injury on MRI in a cohort of hypoxic/ischemic infants treated with therapeutic hypothermia [99]. Moreover, in a follow-up to the NICHD randomized trial of therapeutic hypothermia, early hypocarbia was linked to death or worse outcomes at 18 to 22 months of age [100]. Also, it was shown that worse 2-year outcomes were related to substantial variability in PCO₂ during hypothermia [101]. Additionally, a longer time for serum lactate to normalize was correlated with the severity of encephalopathy and the manifestation of seizures in neonates with NE [102]. Finally, hypo- and hyperglycemia in the early postnatal period have also been correlated with neurologic adverse outcomes [103].

Neuroimaging remains the cornerstone in the evaluation of asphyxiated newborns. In at-risk groups, electroencephalography (EEG) and amplitude-integrated EEG (aEEG) enable real-time evaluation of brain development and function in addition to seizure detection. The predictive accuracy of aEEG is time-dependent, such that a burst-suppression pattern persisting beyond 36 to 48 h suggests a significant risk of an aberrant neurodevelopmental outcome [104]. Also, suppressed aEEG tracings longer than 48 h increase the likelihood of severe impairments in survivors, albeit they are not a valid early single predictor in hypothermic neonates [105]. When combined with an aberrant early MRI, a persistently abnormal 48 h EEG background pattern was highly indicative of an unfavorable prognosis [106]. Evoked potentials, including visual, brain-stem auditory, and somatosensory, are electrical reactions to sensory input. In term newborns with NE, delayed or absent responses on the somatosensory-evoked potentials during the first few weeks of life were associated with poorer neurological outcomes at 18 to 24 months of age [107]. Although evoked potentials are effective supplemental prognostic tools in severe cases, the lack of rigorous studies and consensus regarding their prognostic efficacy has limited their utilization in clinical practice. Near-infrared spectroscopy can be utilized for the noninvasive assessment and continuous bedside monitoring of cerebral hemodynamics and oxygenation. Previous evidence has shown that at six and twenty-four hours postpartum, cerebral blood volume and cerebral oxygenation levels were higher in neonates with aberrant MRI findings [108]. Also, persistent higher levels of cerebral oxygenation in hypothermic neonates with NE were correlated with poor neurological outcomes [109]. Finally, brain MRI is a standard procedure used for the diagnosis and prognosis of newborns with NE. MRI is also becoming a common procedure for assessing the type and extent of brain damage in newborns with NE. According to a previous meta-analysis, brain MRI during the newborn era has a high sensitivity but moderate specificity for predicting unfavorable neurodevelopmental outcomes at one year [110], whereas a normal brain MRI is correlated with normal neurological results and little chance of significant neurodevelopmental impairment [111].

7. Therapeutic Strategies

7.1. Hypothermia

The effectiveness of hypothermia in reducing brain damage [112] and lessening severe neurological effects [113] in newborns with moderate to severe NE has been well-documented and is now the standard treatment for term and early preterm neonates with NE [50]. The main advantages of hypothermia arise from decreasing brain metabolism, which lessens the damage from secondary brain energy failure [114]. Furthermore, hypothermia prevents brain cell death, reduces harmful effects from overstimulation, controls the activity of glial cells [115], and stimulates certain RNA molecules [116]. Additionally, hypothermia has been shown to directly decrease the levels of oxygen-based free radicals after hypoxia/ischemia and reperfusion, as seen in both in vivo [42] and in vitro studies [117] (

Table 3. Treatment options for neonatal encephalopathy.

Option	Mechanism of Action	Phase of Hypoxemia/Ischemia
Hypothermia	Decreases ROS Decreases brain metabolism Controls activity of glial cells Prevents cell death	Within six hours of hypoxia/ischemia Reperfusion phase
Mitochondrial therapy:
1. Metformin.	Suppresses mitochondrial complex I in the respiratory chain Affects inflammatory T cells	Hypoxia/ischemia phase Reperfusion phase
2. Coenzyme Q10.	Decreases the production of ROS	Hypoxia/ischemia phase Reperfusion phase
3. Mitoquinone.	Reduces superoxide production, lipid peroxidation, and ROS generation	Hypoxia/ischemia phase Reperfusion phase
Antioxidants
1. Free radical initiators and scavengers:
I. Allopurinol.	Inhibits xanthine oxidase Chelator for non-protein-bound iron	Reperfusion phase
II. Erythropoietin.	Reduces inflammation Decreases ROS Decreases caspase activation Prevents cell death	Hypoxia/ischemia phase Reperfusion phase
III. Melatonin.	Antioxidant, anti-inflammatory, and anti-apoptotic action	Hypoxia/ischemia phase Reperfusion phase
IV. N-acetylcysteine.	Antioxidant (neutralizes ROS) Reduces the severity of hypoxic–ischemic damage Reduces the extent of demyelination in the corpus callosum Enhances myelin production	Hypoxia/ischemia phase Reperfusion phase
V. Docosahexaenoic acid.	Antioxidant (neutralizes ROS) Reduces glutamate excitotoxicity Decreases nitric oxide levels Enhances antioxidant enzyme activities	Hypoxia/ischemia phase Reperfusion phase
VI. Edaravone.	Antioxidant (neutralizes ROS)	Reperfusion phase
VII. Phenobarbital.	Reduces the levels of lipid peroxides and antioxidant enzymes	Within two hours of hypoxia/ischemia Reperfusion phase
VIII. Molecular hydrogen.	Antioxidant Maintains the reactivity of blood vessels	Hypoxia/ischemia phase Reperfusion phase
IX. Deferoxamine.	Removes iron excess Decrease excitatory amino acids Increases VEGF and erythropoietin	Hypoxia/ischemia phase Reperfusion phase
X. Superoxide dismutase.	Antioxidant (neutralizes ROS)	Reperfusion phase
2. Nitric oxide inhibitors.	Maintains cerebral blood flow	Hypoxia/ischemia phase
3. Magnesium.	Blocks NMDA receptors Reduces excessive calcium or glutamate Reduces inflammation Reduces oxidative stress	Hypoxia/ischemia phase Reperfusion phase
Stem cells	Reduce inflammation	Hypoxia/ischemia phase Reperfusion phase
Nanomaterials	Targeted therapeutic material	Hypoxia/ischemia phase Reperfusion phase

ROS, reactive oxygen species; VEGF, vascular endothelial growth factor; NMDA, N-methyl-D-aspartate.

).

7.2. Mitochondrial Therapy

There has been a growing interest in the application of small molecule inhibitors and activators aimed at mitochondria [118], such as tetra- and triphenylphosphonium, as well as the combination of various methods for delivering mitochondria-penetrating peptides [119]. Bioactive therapeutics and antioxidants, as an alternative to targeting mitochondria, are slowly becoming a viable approach for developing supplementary therapies. Metformin has been shown to limit mitochondrial respiration by directly suppressing mitochondrial complex I in the respiratory chain [120]. Metformin can also affect inflammatory T cells by blocking the utilization of 2-deoxyglucose in glycolysis, as well as reducing the production of IL-1β [121]. In animal studies with metabolic syndrome, treatment of the mother with metformin was effective in preventing fetal inflammation, which is a risk factor for neonatal hypoxic/ischemic encephalopathy [122]. Coenzyme Q10 (CoQ10) can decrease the production of ROS through the mitochondrial complex and research has shown that CoQ10 acts as a neuroprotective agent in various animal models, including rodent models of Alzheimer’s disease [123], middle cerebral artery occlusion [124], and traumatic brain injury [125]. However, the potential role of CoQ10 in neonatal hypoxia/ischemia has not yet been fully explored. Finally, mitoquinone, a ubiquinone derivative conjugated to triphenylphosphonium cation, is efficient in reducing superoxide production, lipid peroxidation, and subsequent ROS generation, and thus, mitoquinone could be used to treat structural mitochondrial damage [126].

7.3. Antioxidant Methods for Protecting the Brain from Injury Due to Low Oxygen Levels

The involvement of free radicals in the onset of hypoxia/ischemia brain damage, followed by reperfusion, has led to the investigation of antioxidant treatment methods for NE, with the goal of either enhancing the body’s natural ADS or reducing the production of ROS and RNS at various stages. Although the use of atmospheric air for the resuscitation of newborns has become a standard procedure in neonatal care, the application of other antioxidant substances is primarily confined to research environments, with only limited evidence from preclinical and/or clinical studies. Antioxidant treatments are possible approaches to treating perinatal asphyxia, in combination with therapeutic hypothermia for infants with moderate to severe NE [127].

7.4. Reduction in Free Radical Production Initiators and Scavengers

Allopurinol is a specific inhibitor of XO, competing with this enzyme to reduce the production of ROS during reperfusion [128,129]. Allopurinol, by blocking the activity of XO, also reduces the production of NO from nitrites, while acting as a chelator for non-protein-bound iron and a direct scavenger for free radicals [128,129]. These characteristics position it as a potential candidate in the treatment of hypoxic/ischemic encephalopathy. Maternal administration of allopurinol to fetuses experiencing hypoxia resulted in a decrease in the levels of the S-100B protein, an indicator of brain injury, in the infant’s cord blood [130]. Previous evidence has shown that allopurinol reduced oxidative stress and improved blood flow and brain activity in newborns without causing toxicity [131]. However, a later study found no benefits in newborns given allopurinol within the first four hours after birth compared to those given a placebo, likely due to the delay in its administration [132].

Erythropoietin, essential for brain development, is a promising agent in treating NE by increasing the expression of genes that prevent cell death, reducing inflammation, lowering levels of oxygen free radicals, decreasing the activation of caspases, lessening the effects of glutamate excitotoxicity, and enhancing signaling pathways [133,134]. Under conditions of hypoxia, the expression of erythropoietin receptors and the secretion of erythropoietin increase, making it a particularly effective option for treatment [135].

Melatonin, a powerful natural compound, has multiple beneficial effects including antioxidant, anti-inflammatory, and anti-apoptotic properties. It can easily cross the blood–brain barrier, making it an attractive option for treating hypoxic/ischemic encephalopathy [136,137].

N-acetylcysteine (NAC), a precursor of cysteine, possesses antioxidant effects, including the neutralization of ROS and the replenishment of glutathione in cells [138]. These attributes, along with its ability to dissolve in fat and its low toxicity [139], have positioned NAC as a promising candidate for neuroprotection in NE. NAC reduces the severity of hypoxic/ischemic damage in newborn rats [140] and can be used in conjunction with hypothermia to lessen brain damage [141,142]. Administering NAC to rats alongside hypothermia has been shown to enhance myelin production [141], improve brain function [141], and lead to better long-term motor outcomes [142].

DHA is known for its ability to neutralize free radicals and its role in reducing glutamate excitotoxicity and decreasing NO levels. Evidence of enhanced antioxidant enzyme activities in cultures of neurons enriched with DHA has suggested a potential neuroprotective function for DHA [143].

Edaravone acts as a neutralizer of free radicals and has been investigated for its multiple antioxidant effects across various experimental models, including those involving animal models of NE [144].

Phenobarbital administered within the first two hours of life in term newborns with perinatal asphyxia has been associated with lower levels of lipid peroxides and antioxidant enzymes in the cerebrospinal fluid [145].

Molecular hydrogen, a compound recently recognized for its antioxidant properties, has shown neuroprotective effects and maintains the reactivity of blood vessels in newborn pigs exposed to asphyxia [146].

Deferoxamine is an iron-binding antioxidant that crosses the blood–brain barrier and helps remove excess iron due to its capacity to bind free iron in the brain. Deferoxamine has been shown to lessen brain edema and injury in animal models of hypoxia/ischemia, lower the levels of excitatory amino acids, and improve the histological condition of the hippocampus in newborn rats following hypoxia/ischemia [127,147]. Following birth asphyxia, iron is released from ferritin, hemosiderin, or transferrin [148,149], leading to the production of free radicals [150]. Ferrous ions can also trigger the formation of MDA, which is a marker of lipid oxidation and oxidative damage to tissues [151]. By binding to iron, deferoxamine stops the creation of the harmful peroxynitrite anion and hydroxyl radicals and thus reduces the risk of iron-dependent free radical-induced damage, especially to polyunsaturated lipids [11].

SOD, an enzyme that neutralizes free radicals, could be beneficial in reducing brain damage; however, its use is restricted as SOD has limited ability to cross the blood–brain barrier. Encapsulation of SOD with biodegradable poly nanoparticles has been shown to effectively lower the volume of brain infarcts, edema, and the formation of ROS in an animal model of cerebral ischemia/reperfusion injury [152].

7.5. Nitric Oxide Inhibitors

NO can be both protective and harmful for the brain during periods of hypoxia/ischemia. The epithelial cells of blood vessels release NO that helps maintain the blood flow to the brain and protects the brain from ischemic injury. However, increased levels of NO can lead to overstimulation, cell edema, and cell death, in addition to injury in the blood vessel epithelial cells themselves [153]. The production of NO is controlled by neuronal, endothelial, and inducible NOS. These types of NOS are more active under conditions of hypoxia, leading to the suggestion that blocking NO production could be a way to protect the brain from injury [154].

7.6. Magnesium

Magnesium is a promising therapeutic agent for brain injury due to its ability to block NMDA receptors, which helps prevent harmful effects from excessive calcium or glutamate. Magnesium also has a significant role in helping reduce inflammation and damage from oxidative stress [46].

7.7. Stem Cells

Recent research indicates that bone marrow and umbilical cord blood stem cells might be beneficial in treating brain injury. Specifically, stem cells from umbilical cord blood offer protection against inflammation, cell death, and oxidative damage and control immune regulation. Certain types of cells, like endothelial progenitor cells and mesenchymal stem cells found in umbilical cord blood and tissue, are effective in reducing inflammation caused by brain damage. Mesenchymal stem cells have a strong ability to change the immune response, protecting against harmful immune reactions triggered by brain injury [155,156]. Administration of stem cells from umbilical cord blood can also lessen the damage to brain white matter by combining with other treatments for brain injury [49].

7.8. Nanomaterials

The application of nanotechnology or techniques based on nanocarriers is rapidly growing as a promising tool for the detection and treatment of cerebral illnesses [157,158]. Nanoparticles are carriers or delivery tools that enhance the bioavailability of therapeutics because of their enhanced solubility, reduced degradation by enzymes, decreased protein binding, and extended and targeted delivery at the injury site [159,160]. Therapeutic medicines can be more effectively created to counteract excitotoxicity by utilizing the advantages of nanoparticle drug delivery and comprehending the biomolecular mechanisms behind neurological disease processes. Polymeric nanoparticles, liposomes, hydrogels, and dendrimers are just a few of the many nanoparticle platforms that are utilized to deliver medications to the brain [161]. Polymeric nanoparticles have demonstrated the ability to traverse both an intact and compromised blood–brain barrier, and they can offer controlled drug release, targeting capabilities, and sustained drug action by protecting against proteases [162]. To avoid steric clearance and non-specific binding, nanoparticles can be modified in terms of size, shape, flexibility, and surface charge to change pharmacokinetics and enhance brain accumulation [163,164]. Densely polyethylene glycol-coated nanoparticles can move through the brain parenchyma by diffusion [165]. Among the drug-incorporating techniques are covalent conjugation to surface end groups and loading into lipophilic bilayers, cores, or nanoparticle matrices [166]. Nanoparticles can also be adorned with surface ligands or surfactants to enhance pharmacokinetics or cellular absorption [162].

According to current evidence, the endogenous antioxidant enzyme SOD, which breaks down SOX into H₂O₂ and water, is one of the most promising enzyme therapy possibilities. Reducing SOX levels at the expense of increasing H₂O₂ levels has a clear therapeutic effect by lowering mitochondrial oxidative stress and fragmentation, even if SOD transforms SOX into H₂O₂ [167]. Several of the following processes leading to neuronal death are mitigated by antioxidant intervention through the scavenging of ROS and injury-associated SOX. Numerous investigations have already demonstrated the therapeutic potential of different antioxidants as well as SOD-loaded nanoparticles or SOD-mimetic platforms. Though the suppression of excitotoxic damage appears promising, more research is necessary to determine the optimal therapeutic intervention point [157,161].

8. Discussion

The neonatal brain, due to its unique properties, is more vulnerable to oxidative stress injury. Furthermore, as the pathophysiology of neonatal brain injury is exceedingly complex, oxidative stress is becoming a major concern in neonatal brain damage, especially since a precise and efficient treatment is lacking. Only modest progress has been made in clinical practice despite a growing understanding of the pathophysiology of hypoxic/ischemic encephalopathy. One of the biggest obstacles is identifying this crucial situation as soon as possible and initiating treatment promptly. Before being implemented in clinical settings, more research is necessary to explore the mechanisms underlying the neuroprotective impact of treatment alternatives and how they can enhance antioxidant defense under hypoxic/ischemic situations.

The optimization of protocols in the utilization of antioxidant treatment is needed to achieve optimal and long-term protection, including timing and proper dose, taking into consideration the amount of injury, age, gender of the newborn, and comorbidities. Additionally, it is feasible that a combination of pharmaceutical treatment and therapeutic hypothermia would be better for hypoxic/ischemic injury than a single regimen. Melatonin and erythropoietin have shown some positive effects in clinical trials but need to be confirmed in larger studies before they can be used routinely. The effectiveness of allopurinol in treating neonatal encephalopathy is still uncertain. Promising neuroprotective effects of inhibitors of NOS, NAC, and DHA have been observed in preclinical studies, suggesting they should be tested in clinical trials. However, the current literature on antioxidant treatments in newborns is limited by small and varied sample sizes, often from samples taken before cooling, and by differences in the dosages used and the outcomes measured. Research in this field could advance through integrated analyses that combine insights from animal and human studies with those from in vitro investigations. Future research on neuroprotectants and their processes would lead to the development of innovative treatments for illnesses caused by hypoxia/ischemia, as a result of new technical advancements.

9. Conclusions

Free radicals are a key factor in brain damage following a period of hypoxia, ischemia, and reperfusion in newborns with asphyxia. Over the last few decades, various markers of oxidative and nitrosative damage from different body fluids have been suggested to measure the level of free radical damage after birth asphyxia. Among these markers, the antioxidant enzymes, the rate at which uric acid is excreted, the levels of NO, MDA, and the neuroprotective biomarkers have been more thoroughly studied both in vitro and in vivo, showing potential for predicting the severity and outcome of NE. Meanwhile, other markers have recently gained attention and need further evaluation. Various approaches to using antioxidants as protective measures for neonatal encephalopathy have been explored. Although it is standard practice to resuscitate newborns with asphyxia using room air and therapeutic hypothermia is now the preferred treatment, there is ongoing research into other molecules with antioxidant properties.

Based on the current research, it is evident that the body’s protective mechanisms play a role in determining the severity of brain damage caused by hypoxia/ischemia. From a treatment standpoint, it is important to develop strategies that either enhance the brain’s protective mechanisms or reduce the damage caused by hypoxia/ischemia. Antioxidants appear to be a promising target for both experimental and clinical therapy. Since free radicals are involved in many processes leading to cell death, by altering the level of oxidative stress, it is possible to influence the survival of cells. Further research is therefore warranted, focusing on how oxidative stress could have beneficial effects on how cells adapt to stress and their survival.