Free oxygen radicals and proinflammatory cytokines are important causes for brain injury in neonates with hypoxic ischemic encephalopathy (HIE). Our objectives were to test the hypothesis that a combination of antioxidants (ascorbic acid) and anti-inflammatory agents (ibuprofen) can ameliorate the brain injury in HIE and improve neurodevelopmental outcomes when given to term infants immediately after birth.
In a prospective, randomized, double-blinded controlled trial, 60 asphyxiated term infants were assigned to one of two groups, intervention and control. The intervention group (n=30) received intravenous ascorbic acid and oral ibuprofen for 3 days; and the control group (n=30) received similar volumes of a placebo. We measured a panel of cytokines at enrollment and administered the treatment drugs within 2 h after birth. Neurological evaluations and developmental screenings were performed for all survivors at 6 months of age.
The Intervention and Control groups did not differ in the severity of HIE at enrollment, the concentrations of IL-1β and IL-6, the incidence of mortality (37 vs 33%), the incidence of neurological abnormalities at hospital discharge (47 vs 55%) and the incidence of developmental delay at 6 months of age (32 vs 40%), respectively. None of the observed complications were related to intervention. Serum interleukin (IL)-1β and IL-6 concentrations correlated positively with the severity of HIE at birth (P<0.01), whereas only serum IL-6 correlated with neurodevelopmental outcome at 6 months (P<0.001).
Early administration of ascorbic acid and ibuprofen did not affect outcomes in infants with perinatal asphyxia. This study does not explain whether our intervention was not effective in blocking free radicals and inflammatory cytokines, if the dosing and route of administration were inadequate, or if other mediators existed that could have a more powerful role in brain injury during hypoxia–ischemia.
Perinatal hypoxia–ischemia (HI) is considered the single most important cause of brain injury in the newborn worldwide.1 Insult from HI causes immediate neuronal cell injury and exhaustion of cellular energy stores. Several hours after the initial insult, a secondary phase of neuronal injury emerges as a result of the production of oxygen-free radicals, inflammatory mediators and apoptosis. This late phase theoretically allows a window of opportunity for interventions to attenuate brain damage.
The free radicals and reactive oxygen species that are released during reperfusion and reoxygenation of the brain can induce significant cell necrosis and apoptosis.2, 3 Ascorbic acid (vitamin C) at very low concentrations scavenges and neutralizes these reactive oxygen species4 and regenerates antioxidants, such as α-tocopheroxyl, urate and β-carotene.5, 6 In animal studies, systemic injection with low doses results in high concentrations of ascorbic acid in brain tissue and a significant neuroprotection against hypoxic ischemic encephalopathy (HIE).7, 8
Interleukin-1β (IL-1β), IL-6 and other cytokines are released in HIE,9 and the treatment with recombinant IL-1 receptor antagonist (IL-1ra) attenuates neuronal damage in animal models.10 Ibuprofen can inhibit the production of proinflammatory cytokines and oxyradicals,11 and poses a long-lasting protective effect to the brain against global and focal ischemia in animals.12, 13
In spite of the experimental evidence for a potentially neuroprotective role of ascorbic acid and ibuprofen, the efficacy of these two drugs has not been studied in infants with HIE. As the cascade of events following HI injury is a complex one, it is unlikely that a single agent will be successful in ameliorating the effect of HI injury. In this prospective, double-blinded, randomized controlled trial, we hypothesized that when given immediately after birth, a combination of antioxidants (ascorbic acid) and anti-inflammatory agents (ibuprofen) can ameliorate the severity of encephalopathy and improve neurodevelopmental outcomes (NDOs) of infants with asphyxia.
Term infants (gestational age >37 weeks) diagnosed with perinatal asphyxia were eligible for the study if they met all of the following criteria: (1) Apgar score at 5 min <6; (2) profound metabolic or mixed acidosis with pH⩽7 in the initial blood gas obtained on admission to the NICU <2 h of age; and (3) any evidence of encephalopathy, such as coma, seizures or hypotonia. Infants were excluded from the study if they had major congenital anomalies, early sepsis, gastrointestinal bleeding, renal failure or thrombocytopenia. We planned to stop the intervention and withdraw subjects from the study if any of the exclusion criteria were met during treatment. This prospective, double-blinded, randomized controlled trial was conducted at Bab El-Shariya Hospital and was approved by the Review Board at Al-Azhar University, Cairo. Parental consents were obtained before enrollment. All subjects were born at the obstetric ward with no transports from outside.
Enrolled subjects were randomly assigned to one of two groups within 2 h after delivery. The Intervention group received a combination of intravenous ascorbic acid (vitamin C=100 mg kg−1 day−1 for 3 days) and oral ibuprofen (day 1: 10 mg kg−1, days 2 and 3: 5 mg kg−1). The Control group received similar volumes of placebo.
Primary outcomes and clinical assessments
The primary outcome of this study was the presence of abnormal NDO as detected by neurological examination or by Denver Developmental Screening Test (DDST II) at 6 months of age. Complete obstetrical history and physical examinations were obtained on admission. In addition to the routine daily assessment, detailed neurological examinations were performed in the first 24 h of life. During this initial evaluation, HIE was classified into mild, moderate and severe grades using Sarnat and Sarnat staging.14 Infants were reexamined at discharge and were then followed every 2 months until the age of 6 months. All clinical assessments for the subjects were performed by a single investigator (AE), who was unaware of the infant's group assignment.
Denver developmental screening test (DDST II)
The test includes multiple items to examine four major categories: gross motor, language, fine motor-adaptive and personal-social. Infants are scored for each test item as advanced, normal, caution or delayed. The overall developmental assessment of an infant was categorized as: Failed, if there are two or more ‘delays’; Questionable, if there is one delay and/or two or more ‘cautions’; and Normal, if no delays and maximum of one ‘caution’.15
Blood tests and cytokines measurement
On admission to the neonatal unit, blood was drawn for culture, C-reactive protein, complete blood count, a renal profile, IL-1β and IL -6. The renal profile and complete blood count were repeated at 3 days of age. Blood samples for cytokines were collected in sterile plastic tubes, left to clot and centrifuged for 5 min at room temperature. Sera were aspirated and passed through sterile 0.2 μm filters, and stored in aliquots at −70 °C until analyzed. Concentrations of IL-1β and IL-6 in serum were measured by enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (Dia-Med Eurogen Research ELISA kits, Turnhout, Belgium).16 Cytokines were also measured from a group of control normal infants (n=30) for comparison. Laboratory investigators were unaware of the infant's neurological status when analyses were performed.
Statistical analysis and sample size
To detect a difference between the two treatment groups in NDO that equals one standard deviation, a sample size of 17 subjects in each group was required (Power=80%, β=20% and α=0.05). On the basis of a previous study of similar population, 45% of HIE infants died before the age of 6 months, and 70% of survivors had abnormal NDO. Therefore, using a sample of 30 infants in each group would be sufficient to detect differences in NDO abnormalities between groups at 6 months of age. The standard two-tailed t-test and Mann–Whitney U-test were used to compare parametric and nonparametric continuous variables in the two groups. χ2 test was used to compare categorical variables between groups. Spearman's rank correlation coefficient and one-way ANOVA were performed to assess the correlation of cytokine levels with clinical outcomes. Receiver operating curve was used to determine a cutoff point for cytokines in the prediction of abnormal NDO and death. Sensitivity and specificity analyses were then calculated.
Sixty infants with asphyxia were included in the trial, with 30 infants assigned to each group. The two groups did not differ in demographic characteristics, complications at delivery, or severity of encephalopathy. The clinical and biochemical characteristics of the two groups are presented in Table 1.
The intervention and the Control groups did not differ in the use of mechanical ventilation (n=17 vs n=15, P=0.6), or the administration of continuous inotropic medications (n=16 vs n=14, P=0.61), respectively. We did not report any gastrointestinal bleeding/perforations, or other complications related to intervention. Following the treatment, there were no significant changes between the groups in serum creatinine, hemoglobin concentration and platelet count (Figure 1). Intervention and Control groups did not differ in the incidences of mortality (37 vs 33%) and abnormal examination at hospital discharge (30 vs 33%), respectively (Table 2). Neurological abnormalities at the age of 6 months were similar in both groups (Table 3).
Serum IL-1β and IL-6 concentrations were increased in infants with HIE (n=60) (10.5±3.2 and 114.6±75.7 pg per 100 ml) when compared with controls (6.1±2.3 and 29.3±12.5 pg per 100 ml, respectively) (P< 0.001); and they both correlated with the severity of HIE grades (P<0.01). Increased serum IL-6, but not IL-1β, correlated with poor NDO at 6 months (P<0.001) (Figure 2). IL-6 was a better predictor for poor NDO and death at 6 months (Figure 3), with a sensitivity of 91% and specificity of 88% (at cutoff level=49.5 pg per 100 ml).
This study showed that a regimen of combined ibuprofen and ascorbic acid did not improve neurological outcomes of infants with HIE. To our knowledge, this is the first trial to investigate such an approach.
Ascorbic acid has been shown to ameliorate the course of HIE in animals.8 Our study did not show a beneficial role for its use in term infants with HIE when combined with ibuprofen. It is uncertain if the absence of efficacy was related to inadequacy of the dose administered. Although a dose of 30 mg kg−1 was neuroprotective in rats,7 we used a dose of 100 mg kg−1, as this dose has been shown to be safe in neonates without any pro-oxidant or hemolytic effects in preterm infants.17 Our study population did not encounter any hemolytic side effects; however, using a bigger dose cannot be recommended until safety data at a bigger scale becomes available.
Ibuprofen has been shown to be protective to the adult brain in models for focal and global ischemia in animals.12, 13 Intravenous injection of 10 mg kg−1 in children is associated with good penetration of cerebrospinal fluid, with a peak concentration attained after 30–40 min.18 The use of the oral form of ibuprofen was advocated in our study because of its affordable pricing that would make it widely available, especially in the regions with the highest incidence of HIE. The bioavailability of ibuprofen when administered orally has been assured by different studies that proved the efficacy of oral ibuprofen in the treatment of ductus arteriosus in preterm infants. These same studies provided data to support the safety of oral ibuprofen in neonates.19 However, there is no data on bioavailability and adequacy of intestinal absorption in HIE infants who might have compromised gut perfusion and inadequate mesenteric blood flow. It could have been helpful if we measured serum concentrations of ibuprofen in these infants, but it was not feasible to perform these assays at our institution. Another explanation for the absence of efficacy in our intervention is the possible presence of mediators that are important for brain injury, but cannot be controlled by ascorbic acid or ibuprofen. Activation of complement system could be an example in such case.20 Finally, the population of this study included severely asphyxiated infants who were resistant to efforts of neuroprotection in previous trials.21 It might be helpful if future trials study our regimen only in mildly and moderately asphyxiated infants.
Infants in the intervention group did not have any significant changes in their renal functions or platelet counts, and the study did not report any adverse events related to either of the two medications. We did not report any side effect in subjects exposed to the intervention. Potential complications of ibuprofen include renal impairment, isolated intestinal perforation, necrotizing enterocolitis, pulmonary hypertension and bleeding disorders. Ascorbic acid has a theoretical pro-oxidant effect that has not been proven under physiological conditions.
We used DDST II for developmental evaluation of infants at 6 months of age. This test is often used by health professionals to assess asymptomatic populations. It is a noncomplex test that has been shown to accurately predict severe adverse outcomes in HIE infants.22 Other studies used more sophisticated tests, such as Bayley Scales for Infant Development, but those tests require standardized certification for examiner and has not been formulated for a non-English-speaking population.
Serum concentrations of IL-6 correlated with NDO. Although this association has been described earlier,9 the role of IL-6 in HIE as a neurotrophic vs a neuroprotective mediator needs further exploration. Serum concentrations of IL-1β did not correlate with adverse NDO. Previous studies showed a strong correlation of IL-1β in the cerebrospinal fluid but not in the serum with NDO, indicating a major role of its local CNS production.9
Although a randomized, double-masked controlled trail can provide the most accurate evidence-based information, our study did have some limitations due to limited resources. We did not remeasure cytokine concentrations after completing the treatment regimen. That step could have provided better explanations for why the treatment regimen was not efficacious. We did not measure ibuprofen concentrations following enteral administration, although such measurement would have been crucial in determining the bioavailability and absorbability of this drug in asphyxiated infants. Finally, we could have achieved more meaningful results if subjects were followed for a longer period than 6 months. Despite these limitations, we believe our pilot study still provides helpful information that can be used when designing future trials.
We conclude that although oxidative stress and inflammatory cytokines are involved in the process of HIE, the early administration of ascorbic acid and ibuprofen do not reduce mortality or improve NDO. It is not clear whether our treatment regimen was inadequate in blocking free radicals and inflammatory cytokines or whether other mechanisms responsible for HIE injury have the upper hand in the pathogenesis of brain damage in these infants. The combination of the two drugs was not associated with immediate side effects; that may encourage further studies using them at different dosages and route of administration.
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We thank Inderjeet Sandhu for her efforts in editing this manuscript.
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Aly, H., Abd-Rabboh, L., El-Dib, M. et al. Ascorbic acid combined with ibuprofen in hypoxic ischemic encephalopathy: a randomized controlled trial. J Perinatol 29, 438–443 (2009). https://doi.org/10.1038/jp.2009.1
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