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Hypothermia attenuates the severity of oxidative stress development in asphyxiated newborns
Journal of Critical Care (2012) 27, 469–473
Hypothermia attenuates the severity of oxidative stress development in asphyxiated newborns☆ Hiroki KakitaMD, PhD a , Mohamed Hamed HusseinMD, PhD a,b,c,⁎, Shin KatoMD, PhD a , Yasumasa Yamada MD, PhD d , Yoshiaki Nagaya MD a , Hayato Asai MD a , Tatenobu Goto MD, PhD a , Koichi Ito MD, PhD a , Tokio Sugiura MD, PhD a , Ghada Abdel-Hamid Daoud MD, PhD e , Tetsuya Ito MD, PhD a , Ineko Kato MD, PhD a , Hajime Togari MD, PhD a a
Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan Neonatal Intensive Care Unit, Pediatric Hospital, Cairo University, Cairo, Egypt c Medical Research Department, EgyBlood, VACSERA, Giza, Egypt d Department of Neonatology, Aichi Human Service Center Central Hospital, Japan e Obstetrics and Gynecology Department, EgyBlood, VACSERA, Giza, Egypt b
Keywords: Oxidative stress; Total hydroperoxide; Biological antioxidant potential; Asphyxia; Brain hypothermia
Abstract Purpose: This retrospective case-control study aimed to examine the development of oxidative stress in asphyxiated infants delivered at more than 37 weeks of gestation. Material and Methods: Thirty-seven neonates were stratified into 3 groups: the first group experienced hypothermia (n = 6); the second received hypothermia cooling cup treatment for 3 days, normothermia (n = 16); and the third was the control group (n = 15). Serum total hydroperoxide (TH), biological antioxidant potential, and oxidative stress index (OSI) (calculated as TH/biological antioxidant potential) were measured within 3 hours after birth. Results: Serum TH and OSI levels gradually increased after birth in hypothermia and normothermia cases. At all time points, serum TH and OSI levels were higher in hypothermia and normothermia cases than in control cases. Serum TH and OSI levels were higher in normothermia cases than in hypothermia cases at days 3, 5, and 7. Conclusion: This study demonstrated that hypothermia attenuated the development of systemic oxidative stress in asphyxiated newborns. © 2012 Elsevier Inc. All rights reserved.
☆ This study had no financial support from extramural sources. ⁎ Corresponding author. Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Tel.: +81 52 853 8246; fax: +81 52 842 3449. E-mail addresses: [email protected], [email protected] (M. Hamed Hussein).
0883-9441/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcrc.2011.12.013
1. Introduction Perinatal asphyxia encephalopathy is associated with high morbidity and mortality rates worldwide and is a major burden for patients, their families, and society. There is an urgent need to improve outcomes in affected infants. Perinatal asphyxia is
470 an insult caused by a lack of oxygen or lack of perfusion in various organs. Almost every organ of the body is affected by asphyxia, which leads to multiorgan failure, but the predominant insult occurs in the central nervous system [1]. The mechanism of cellular injury after hypoxia or ischemia is poorly understood, but it is probably mediated by an excess concentration of neurotransmitters, oxygen free radicals, and lipid peroxidation, which, in turn, leads to a cascade of damaging events [2]. We have previously reported that cerebrospinal fluid total hydroperoxide (TH) and oxidative stress index (OSI) are higher in severely asphyxiated newborns than in others [3]. Oxidative injury has been implicated as a causal factor in several complications of newborns, including bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis, intraventricular hemorrhage, periventricular leukomalacia, and perinatal asphyxia encephalopathy [4,5]. Neonates are at high risk of oxidative stress and are extremely susceptible to oxidative damage from reactive oxygen species (ROS) [5]. There is now increasing evidence that brain hypothermia reduces the brain injury caused by asphyxia [6,7]. Hypothermia attenuates blood-brain barrier damage, the release of excitatory neurotransmitters is reduced, free radical production is lessened, and anti-inflammatory cytokine levels are increased [2,8]. The mechanism of protection remains unclear. The present study was undertaken to evaluate the level of systemic TH and OSI in neonates with asphyxia and to ascertain whether or not hypothermia treatment has an effect on oxidative stress.
2. Materials and methods This retrospective case-control study examined asphyxiated infants delivered at more than 37 weeks of gestation who had been admitted to the neonatal intensive care unit at Aichi Human Service Center Central Hospital between January 2008 and September 2009. The inclusion criterion was a diagnosis of asphyxia. Perinatal asphyxia was defined as the need for positive pressure ventilation for more than 1 minutes during postnatal resuscitation and an Apgar score of 6 or less at 5 minutes. During this period, infants without perinatal asphyxia who cried immediately after birth and did not require positive pressure ventilation for resuscitation served as the control subjects. The control group included 15 cases, of which, 4 had initial vomiting and/or poor feeding, 2 had hypoglycemia, and 9 had mild transient tachypnea, and for which a brain magnetic resonance imaging (MRI) was obtained around the age of 12 months after informed parental consent to estimate the brain development and to be enrolled in the study as part of a control group. Clinical inclusion criteria of hypothermia were an Apgar score of 5 or less at 10 minutes after birth; a continued need for resuscitation, including endotracheal or mask ventilation at 10 minutes after birth; or severe acidosis and/or severe lactic acidosis, defined as pH less than 7.0 within 60 minutes
H. Kakita et al. of birth, and lactic acid more than 8 mmol/L after 60 minutes of birth, in an arterial or venous blood sample. Exclusion criteria were infants older than 6 hours after birth at the time of hypothermia commencement, major congenital abnormalities, refractory hypotension, persistent pulmonary hypertension, and disseminated intravascular coagulation.
2.1. Hypothermia treatment We fitted a cooling cap (Medi Cool MC-2100; Mac8, Tokyo, Japan) around the head for 72 hours in eligible infants. The system consisted of a small thermostatically controlled cooling unit and pump that circulated water through the cap. All infants were nursed under a radiant overhead heater, which was servocontrolled to the infant's abdominal skin temperature and adjusted to the nasopharynx temperature at 34°C. At the start of hypothermia, the overhead heater was turned off for a few hours to accelerate cooling; it was subsequently turned back on once the nasopharynx temperature had fallen to around 34°C. At the end of the 72-hour cooling period, the infants were slowly rewarmed at no more than 0.5°C/h until their temperature was within the normal temperature range. Arterial blood samples were obtained from the radial artery or umbilical cord blood sample (≤6 hours after birth). Arterial blood gases were analyzed using a Rapid Lab 348 analyzer (Chiron, Emeryville, Canada). Serial arterial or venous blood samples were obtained until 7 days after birth. Total hydroperoxide and biological antioxidant potential (BAP) were measured, without knowing the diagnosis, using a d-ROMs kit and a commercial assay kit, respectively (Diacron SRL, Parma, Italy), as previously described [9,10]. Oxidative stress index was determined as the ratio of TH to BAP because the shift in the oxidative/antioxidative balance toward the oxidative side is considered to be indicative of oxidative stress [9]. Magnetic resonance imaging was performed for all patients with perinatal asphyxia when they were around a month of age. Infants were assessed during their neonatal intensive care unit stay by a pediatric neurologist until they were discharged. Abnormal findings were defined as a loss of the posterior limb of the internal capsule, abnormal signal intensity in the basal ganglia and thalami, brain stem lesions, loss of gray/white matter differentiation, and cortical highlighting at T1 or T2 weighting. Infants were assessed again at approximately 12 months of age. Abnormal outcomes included death, severe disability, hearing loss, and cerebral palsy. Written informed consent was obtained for each infant from their parents. All study protocols were approved by the ethics committee of Aichi Human Service Center Central Hospital.
2.2. Statistical analysis Statistical analyses were performed using SPSS for Windows version 13.0 software (SPSS, Chicago, Ill).
Hypothermia and oxidative stress development Table 1
471
Baseline characteristics of infants
Characteristics
Control (n = 15)
Normothermia (n = 16)
Hypothermia (n = 6)
Birth weight (g) Gestational age (wk) Apgar score (1 min) Apgar score (5 min) Male pH at birth Lactate at birth (mmol/L)
3150 (2868-3490) 39 (37.1-39.5) 9 (8-10) 9 (9-10) 10 (66%) – –
3057 (2572-4588) 40.2 (38-41.3) 3 ⁎ (1-3) 5 ⁎ (3-6) 14 (87%) 7.005 (6.75-7.28) 10.1 (3.1-16.2)
3371 (2520-4000) 39.3 (38-41.3) 1.5 ⁎ (1-3) 4 ⁎ (2-6) 6 (100%) 6.93 (6.75-7.29) 14.9 (9-18)
Data are expressed as medians with ranges in parentheses. Analysis of variance was used to compare continuous data, followed by Bonferroni post hoc testing, and the χ2 test or Fisher exact test to compare dichotomous outcomes. ⁎ P b .05 compared with control.
Analysis of variance was used to compare continuous data, followed by Bonferroni post hoc testing, with dichotomous outcomes compared using the χ2 test or Fisher exact test, in different groups. A comparison of the means of the observations in the same group at different times was achieved using analysis of variance for repeated measures, followed by Tukey-Kramer post hoc testing. Data are reported as mean ± standard error of the mean, unless mentioned otherwise. P values less than .05 were considered statistically significant.
3. Results During the study period, 22 of the 37 enrolled infants were diagnosed as having perinatal asphyxia; 15 infants developed normally and were considered as controls. In 22 perinatal asphyxia cases, 6 infants received hypothermia therapy, and the remaining 16 infants did not. Baseline characteristics of the infants are presented in Table 1. Birth weight, gestational age, Apgar score at 1 and 5 minutes, pH, and lactate at birth did not differ significantly among the 3 groups. Hypothermia and normothermia cases were significantly lower than the control cases, but there were no significant differences between hypothermia and normothermia cases in terms of Apgar score at 1 and 5 minutes, pH, and lactate at birth. Although Table 2
all patients with hypothermia were male, there were no significant differences in the ratio of male patients among all 3 groups. Postnatal characteristics of the infants are presented in Table 2. The duration of ventilation and oxygen therapy in hypothermia and normothermia cases was significantly longer than in the control and was significantly longer in hypothermia than normothermia cases. The ratio of abnormal MRI findings and poor neurologic outcome was significantly higher in hypothermia and normothermia than in control cases. However, there were no significant differences between hypothermia and normothermia cases in the ratio of abnormal MRI findings and poor neurologic outcome. Serum TH and OSI levels are depicted in Figs. 1 and 2, respectively. Serum TH and OSI levels gradually increased significantly after birth in hypothermia and normothermia cases but not in control cases (hypothermia: P b .05, normothermia: P b .05, control: not significant). Serum TH and OSI levels at days 3, 5, and 7 were significantly higher than at day 0 in normothermia and hypothermia cases but not in the controls (hypothermia: P b .05, normothermia: P b .05, control: not significant). At all time points, serum TH and OSI levels were significantly higher in hypothermia and normothermia cases than in control cases. Serum TH and OSI levels were significantly higher in normothermia cases than in hypothermia cases at days 3, 5, and 7 (TH: normothermia 158 ± 14 Carr units and hypothermia 130 ±
Postnatal characteristics of infants
Characteristics
Control (n = 15)
Normothermia (n = 16)
Hypothermia (n = 6)
Duration of ventilation (d) Duration of oxygen treatment (d) Abnormal brain MRI findings Age at time of brain MRI
0 1 (0-3) 0 11 M + 25 D (10 M + 21 D − 13 M + 10 D) 0 12 M + 3 D (11 M + 0 D − 12 M + 17 D)
1 (0-7) ⁎ 3 (1-11) ⁎ 5 (31%) ⁎ 12 M + 18 D (11 M + 5 D − 13 M + 10 D) 6 (37.5%) ⁎ 12 M + 5 D (11 M + 15 D − 14 M + 1 D)
8.5 (4-100) ⁎,⁎⁎ 9 (5-100) ⁎,⁎⁎ 2 (33%) ⁎ 12 M + 9 D (11 M + 5 D − 14 M + 0 D) 2 (33%) ⁎ 13 M + 0 D (11 M + 11 D − 13 M + 5 D)
Poor Neurologic outcome Age at time of neurologic evaluation
Data are expressed as medians with ranges in parentheses. Analysis of variance was used to compare continuous data, followed by Bonferroni post hoc testing, and the χ2 test or Fisher exact test to compare dichotomous outcomes. M indicates months; D, days. ⁎ P b .05 compared with control. ⁎⁎ P b .05 compared with normothermia.
472
Fig. 1 Serum TH in control (n = 15), hypothermia (n = 16), and normothermia (n = 6) cases. Values shown are means ± SEM. Analysis of variance was used to compare continuous data, followed by Bonferroni post hoc test. Closed circles indicate control cases; open circles, hypothermia cases; and closed triangles, normothermia cases. ‡P b .05, compared with control; ⫧ P b .05, compared with normothermia.
H. Kakita et al. exhibit an initial transient recovery of cerebral oxidative metabolism, followed by a secondary deterioration with cerebral energy failure 6 to 15 hours. This delay offers a therapeutic window for neuroprotective strategy [12]. Mild hypothermia for 72 hours is currently introduced as the standard therapeutic intervention for infants with perinatal asphyxia encephalopathy. The results of several clinical trials support the safety and efficacy of hypothermia to decrease death and disability in infants with perinatal asphyxia [7,8,13,14]. Our present data did not reveal any significant differences in MRI findings and neurologic outcomes between hypothermia and normothermia cases because of a small sample size. Primary antioxidants prevent oxygen radical formation either by removing free radical precursors or by inhibiting catalysis, such as glutathione peroxidase, superoxide dismutase, and catalase. Secondary antioxidants such as vitamins C and E react with ROS that have already formed to achieve either removal or inhibition. Endogenous antioxidants exist intracellularly on the cell membrane and extracellularly [15,16]. Although many antioxidants can be present in the human body, circulating antioxidants cannot be viewed as simple chemicals, and measurement of individual components is unlikely to yield a complete picture of the in vivo situation. In pathological conditions with an oxidant/antioxidant imbalance, the oxidative stress concept suggests that high oxidant levels and low antioxidant levels are involved in poor outcomes [17,18]. The present
24 Carr units at day 3, normothermia 161 ± 17 Carr units and hypothermia 131 ± 13 Carr units at day 5, normothermia 173 ± 14 Carr units and hypothermia 143 ± 20 Carr units at day 7; OSI: normothermia 0.052 ± 0.006 and hypothermia 0.047 ± 0.0069 at day 3, normothermia 0.058 ± 0.0065 and hypothermia 0.046 ± 0.0065 at day 5, normothermia 0.059 ± 0.0067 and hypothermia 0.050 ± 0.0060 at day 7). There were no significant differences in serum BAP levels among control, hypothermia, and normothermia cases (data not shown).
4. Discussion The present study demonstrated that OSI and TH levels in hypothermia and normothermia cases gradually increased after birth, but those in hypothermia cases were significantly lower than in normothermia and control cases. These data suggested that hypothermia might ameliorate oxidative stress in asphyxiated infants. Hypoxia and ischemia during perinatal asphyxia are the major causes of brain injury in newborn infants. The reperfusion or reoxygenation in the immediate postischemia period is one of the initial factors responsible for brain injury in asphyxiated infants because of the increased production of ROS [11]. Immediately after birth, infants with asphyxia
Fig. 2 Serum OSI in control (n = 15), hypothermia (n = 16), and normothermia (n = 6) cases. Values shown are means ± SEM. Analysis of variance was used to compare continuous data, followed by Bonferroni post hoc test. Closed circles indicate control cases; open circles, hypothermia cases; closed triangles, normothermia cases. ‡P b .05, compared with control; ⫧ P b .05, compared with normothermia.
Hypothermia and oxidative stress development study found that serum BAP levels did not have any significant differences among the 3 groups. To evaluate oxidative stress status, we, therefore, measured the ratio of TH to BAP as an indicator of the degree of oxidative stress. Hasegawa et al [19] demonstrated that hypothermia effectively attenuated the release of apoptotic proteins, delayed the activation of caspase-3, and inhibited oxidative stress in PC cells. Katz et al [20] demonstrated that regulated hypothermia reduced oxidative stress in the hippocampus of rats that had experienced asphyxial cardiac arrest. Although several reports have demonstrated that hypothermia reduces the production of ROS and protects cells against damage [8,19-21], the effects of hypothermia on oxidative stress are still unclear, particularly in human neonates. Perrone et al [21] demonstrated that serum TH levels in a hypothermic group have a slower and smaller elevation until 72 hours after birth. Our data supported their findings. In addition, the present study revealed that in hypothermia cases, both TH and OSI have a slower and smaller elevation compared to normothermia cases after 72 hours (the end of the cooling period). This suggests that hypothermia may ameliorate the development of systemic oxidative stress in asphyxiated newborns; this effect lasted until at least day 7 after cooling. Although our study sample was small and retrospective, not prospective, TH and OSI levels were significantly lower in the hypothermia-treated cases, which could have led to the better neurologic outcome observed.
5. Conclusion This study documented that hypothermia may inhibit an increase in systemic oxidative stress in asphyxiated newborns. Although these findings require confirmation and amplification with a larger number of patients, the data provide insight into potential pathogenic mechanisms and may lead to protective therapies against perinatal asphyxia encephalopathy.
6. Limitations • The inclusion criteria and the lack of patient samples and data limited the number of enrolled newborns. • Approximately half of the enrolled infants moved to distant areas with their families, and a later neurologic assessment at 18 months of age was unable to be obtained.
References [1] Bracci R, Perrone S, Buonocore G. The timing of neonatal brain damage. Biol Neonate 2006;90(3):145-55.
473 [2] Alvarez-Diaz A, Hilario E, de Cerio FG, Valls-i-Soler A, Alvarez-Diaz FJ. Hypoxic-ischemic injury in the immature brain—key vascular and cellular players. Neonatology 2007;92(4):227-35. [3] Hussein MH, Daoud GA, Kakita H, Kato S, Goto T, Kamei M, et al. High cerebrospinal fluid antioxidants and interleukin 8 are protective of hypoxic brain damage in newborns. Free Radic Res 2010;44(4): 422-9. [4] Saugstad OD. Oxidative stress in the newborn—a 30-year perspective. Biol Neonate 2005;88(3):228-36. [5] Saugstad OD. Mechanisms of tissue injury by oxygen radicals: implications for neonatal disease. Acta Paediatr 1996;85(1):1-4. [6] Gluckman PD, Wyatt JS, Azzopardi D, Ballard R, Edwards AD, Ferriero DM, et al. Selective head cooling with mild systemic hypothermia after neonatal encephalopathy: multicentre randomised trial. Lancet 2005;365(9460):663-70. [7] Shankaran S, Pappas A, Laptook AR, McDonald SA, Ehrenkranz RA, Tyson JE, et al. Outcomes of safety and effectiveness in a multicenter randomized, controlled trial of whole-body hypothermia for neonatal hypoxic-ischemic encephalopathy. Pediatrics 2008;122(4):e791-8. [8] Edwards AD, Azzopardi DV. Therapeutic hypothermia following perinatal asphyxia. Arch Dis Child Fetal Neonatal Ed 2006;91(2): F127-31. [9] Kakita H, Hussein MH, Yamada Y, Henmi H, Kato S, Kobayashi S, et al. High postnatal oxidative stress in neonatal cystic periventricular leukomalacia. Brain Dev 2009;31(9):641-8. [10] Kakita H, Hussein MH, Daoud GA, Kato T, Murai H, Sugiura T, et al. Total hydroperoxide and biological antioxidant potentials in a neonatal sepsis model. Pediatr Res 2006;60(6):675-9. [11] Kumar A, Ramakrishna SV, Basu S, Rao GR. Oxidative stress in perinatal asphyxia. Pediatr Neurol 2008;38(3):181-5. [12] Buonocore G, Groenendaal F. Anti-oxidant strategies. Semin Fetal Neonatal Med 2007;12(4):287-95. [13] Azzopardi DV, Strohm B, Edwards AD, Dyet L, Halliday HL, Juszczak E, et al. Moderate hypothermia to treat perinatal asphyxial encephalopathy. N Engl J Med 2009;361(14):1349-58. [14] Azzopardi D, Strohm B, Edwards AD, Halliday H, Juszczak E, Levene M, et al. Treatment of asphyxiated newborns with moderate hypothermia in routine clinical practice: how cooling is managed in the UK outside a clinical trial. Arch Dis Child Fetal Neonatal Ed 2009;94(4):F260-4. [15] Victor VM, Rocha M, De la Fuente M. Immune cells: free radicals and antioxidants in sepsis. Int Immunopharmacol 2004;4(3):327-47. [16] Yu BP. Cellular defenses against damage from reactive oxygen species. Physiol Rev 1994;74(1):139-62. [17] Wolkow PP. Involvement and dual effects of nitric oxide in septic shock. Inflamm Res 1998;47(4):152-66. [18] Goode HF, Cowley HC, Walker BE, Howdle PD, Webster NR. Decreased antioxidant status and increased lipid peroxidation in patients with septic shock and secondary organ dysfunction. Crit Care Med 1995;23(4):646-51. [19] Hasegawa M, Ogihara T, Tamai H, Hiroi M. Hypothermic inhibition of apoptotic pathways for combined neurotoxicity of iron and ascorbic acid in differentiated PC12 cells: reduction of oxidative stress and maintenance of the glutathione redox state. Brain Res 2009;1283:1-13. [20] Katz LM, Young AS, Frank JE, Wang Y, Park K. Regulated hypothermia reduces brain oxidative stress after hypoxic-ischemia. Brain Res 2004;1017(1-2):85-91. [21] Perrone S, Szabo M, Bellieni CV, Longini M, Bango M, Kelen D, et al. Whole body hypothermia and oxidative stress in babies with hypoxicischemic brain injury. Pediatr Neurol 2010;43(4):236-40.
Hypothermia attenuates the severity of oxidative stress development in asphyxiated newborns☆ Hiroki KakitaMD, PhD a , Mohamed Hamed HusseinMD, PhD a,b,c,⁎, Shin KatoMD, PhD a , Yasumasa Yamada MD, PhD d , Yoshiaki Nagaya MD a , Hayato Asai MD a , Tatenobu Goto MD, PhD a , Koichi Ito MD, PhD a , Tokio Sugiura MD, PhD a , Ghada Abdel-Hamid Daoud MD, PhD e , Tetsuya Ito MD, PhD a , Ineko Kato MD, PhD a , Hajime Togari MD, PhD a a
Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan Neonatal Intensive Care Unit, Pediatric Hospital, Cairo University, Cairo, Egypt c Medical Research Department, EgyBlood, VACSERA, Giza, Egypt d Department of Neonatology, Aichi Human Service Center Central Hospital, Japan e Obstetrics and Gynecology Department, EgyBlood, VACSERA, Giza, Egypt b
Keywords: Oxidative stress; Total hydroperoxide; Biological antioxidant potential; Asphyxia; Brain hypothermia
Abstract Purpose: This retrospective case-control study aimed to examine the development of oxidative stress in asphyxiated infants delivered at more than 37 weeks of gestation. Material and Methods: Thirty-seven neonates were stratified into 3 groups: the first group experienced hypothermia (n = 6); the second received hypothermia cooling cup treatment for 3 days, normothermia (n = 16); and the third was the control group (n = 15). Serum total hydroperoxide (TH), biological antioxidant potential, and oxidative stress index (OSI) (calculated as TH/biological antioxidant potential) were measured within 3 hours after birth. Results: Serum TH and OSI levels gradually increased after birth in hypothermia and normothermia cases. At all time points, serum TH and OSI levels were higher in hypothermia and normothermia cases than in control cases. Serum TH and OSI levels were higher in normothermia cases than in hypothermia cases at days 3, 5, and 7. Conclusion: This study demonstrated that hypothermia attenuated the development of systemic oxidative stress in asphyxiated newborns. © 2012 Elsevier Inc. All rights reserved.
☆ This study had no financial support from extramural sources. ⁎ Corresponding author. Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Tel.: +81 52 853 8246; fax: +81 52 842 3449. E-mail addresses: [email protected], [email protected] (M. Hamed Hussein).
0883-9441/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcrc.2011.12.013
1. Introduction Perinatal asphyxia encephalopathy is associated with high morbidity and mortality rates worldwide and is a major burden for patients, their families, and society. There is an urgent need to improve outcomes in affected infants. Perinatal asphyxia is
470 an insult caused by a lack of oxygen or lack of perfusion in various organs. Almost every organ of the body is affected by asphyxia, which leads to multiorgan failure, but the predominant insult occurs in the central nervous system [1]. The mechanism of cellular injury after hypoxia or ischemia is poorly understood, but it is probably mediated by an excess concentration of neurotransmitters, oxygen free radicals, and lipid peroxidation, which, in turn, leads to a cascade of damaging events [2]. We have previously reported that cerebrospinal fluid total hydroperoxide (TH) and oxidative stress index (OSI) are higher in severely asphyxiated newborns than in others [3]. Oxidative injury has been implicated as a causal factor in several complications of newborns, including bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis, intraventricular hemorrhage, periventricular leukomalacia, and perinatal asphyxia encephalopathy [4,5]. Neonates are at high risk of oxidative stress and are extremely susceptible to oxidative damage from reactive oxygen species (ROS) [5]. There is now increasing evidence that brain hypothermia reduces the brain injury caused by asphyxia [6,7]. Hypothermia attenuates blood-brain barrier damage, the release of excitatory neurotransmitters is reduced, free radical production is lessened, and anti-inflammatory cytokine levels are increased [2,8]. The mechanism of protection remains unclear. The present study was undertaken to evaluate the level of systemic TH and OSI in neonates with asphyxia and to ascertain whether or not hypothermia treatment has an effect on oxidative stress.
2. Materials and methods This retrospective case-control study examined asphyxiated infants delivered at more than 37 weeks of gestation who had been admitted to the neonatal intensive care unit at Aichi Human Service Center Central Hospital between January 2008 and September 2009. The inclusion criterion was a diagnosis of asphyxia. Perinatal asphyxia was defined as the need for positive pressure ventilation for more than 1 minutes during postnatal resuscitation and an Apgar score of 6 or less at 5 minutes. During this period, infants without perinatal asphyxia who cried immediately after birth and did not require positive pressure ventilation for resuscitation served as the control subjects. The control group included 15 cases, of which, 4 had initial vomiting and/or poor feeding, 2 had hypoglycemia, and 9 had mild transient tachypnea, and for which a brain magnetic resonance imaging (MRI) was obtained around the age of 12 months after informed parental consent to estimate the brain development and to be enrolled in the study as part of a control group. Clinical inclusion criteria of hypothermia were an Apgar score of 5 or less at 10 minutes after birth; a continued need for resuscitation, including endotracheal or mask ventilation at 10 minutes after birth; or severe acidosis and/or severe lactic acidosis, defined as pH less than 7.0 within 60 minutes
H. Kakita et al. of birth, and lactic acid more than 8 mmol/L after 60 minutes of birth, in an arterial or venous blood sample. Exclusion criteria were infants older than 6 hours after birth at the time of hypothermia commencement, major congenital abnormalities, refractory hypotension, persistent pulmonary hypertension, and disseminated intravascular coagulation.
2.1. Hypothermia treatment We fitted a cooling cap (Medi Cool MC-2100; Mac8, Tokyo, Japan) around the head for 72 hours in eligible infants. The system consisted of a small thermostatically controlled cooling unit and pump that circulated water through the cap. All infants were nursed under a radiant overhead heater, which was servocontrolled to the infant's abdominal skin temperature and adjusted to the nasopharynx temperature at 34°C. At the start of hypothermia, the overhead heater was turned off for a few hours to accelerate cooling; it was subsequently turned back on once the nasopharynx temperature had fallen to around 34°C. At the end of the 72-hour cooling period, the infants were slowly rewarmed at no more than 0.5°C/h until their temperature was within the normal temperature range. Arterial blood samples were obtained from the radial artery or umbilical cord blood sample (≤6 hours after birth). Arterial blood gases were analyzed using a Rapid Lab 348 analyzer (Chiron, Emeryville, Canada). Serial arterial or venous blood samples were obtained until 7 days after birth. Total hydroperoxide and biological antioxidant potential (BAP) were measured, without knowing the diagnosis, using a d-ROMs kit and a commercial assay kit, respectively (Diacron SRL, Parma, Italy), as previously described [9,10]. Oxidative stress index was determined as the ratio of TH to BAP because the shift in the oxidative/antioxidative balance toward the oxidative side is considered to be indicative of oxidative stress [9]. Magnetic resonance imaging was performed for all patients with perinatal asphyxia when they were around a month of age. Infants were assessed during their neonatal intensive care unit stay by a pediatric neurologist until they were discharged. Abnormal findings were defined as a loss of the posterior limb of the internal capsule, abnormal signal intensity in the basal ganglia and thalami, brain stem lesions, loss of gray/white matter differentiation, and cortical highlighting at T1 or T2 weighting. Infants were assessed again at approximately 12 months of age. Abnormal outcomes included death, severe disability, hearing loss, and cerebral palsy. Written informed consent was obtained for each infant from their parents. All study protocols were approved by the ethics committee of Aichi Human Service Center Central Hospital.
2.2. Statistical analysis Statistical analyses were performed using SPSS for Windows version 13.0 software (SPSS, Chicago, Ill).
Hypothermia and oxidative stress development Table 1
471
Baseline characteristics of infants
Characteristics
Control (n = 15)
Normothermia (n = 16)
Hypothermia (n = 6)
Birth weight (g) Gestational age (wk) Apgar score (1 min) Apgar score (5 min) Male pH at birth Lactate at birth (mmol/L)
3150 (2868-3490) 39 (37.1-39.5) 9 (8-10) 9 (9-10) 10 (66%) – –
3057 (2572-4588) 40.2 (38-41.3) 3 ⁎ (1-3) 5 ⁎ (3-6) 14 (87%) 7.005 (6.75-7.28) 10.1 (3.1-16.2)
3371 (2520-4000) 39.3 (38-41.3) 1.5 ⁎ (1-3) 4 ⁎ (2-6) 6 (100%) 6.93 (6.75-7.29) 14.9 (9-18)
Data are expressed as medians with ranges in parentheses. Analysis of variance was used to compare continuous data, followed by Bonferroni post hoc testing, and the χ2 test or Fisher exact test to compare dichotomous outcomes. ⁎ P b .05 compared with control.
Analysis of variance was used to compare continuous data, followed by Bonferroni post hoc testing, with dichotomous outcomes compared using the χ2 test or Fisher exact test, in different groups. A comparison of the means of the observations in the same group at different times was achieved using analysis of variance for repeated measures, followed by Tukey-Kramer post hoc testing. Data are reported as mean ± standard error of the mean, unless mentioned otherwise. P values less than .05 were considered statistically significant.
3. Results During the study period, 22 of the 37 enrolled infants were diagnosed as having perinatal asphyxia; 15 infants developed normally and were considered as controls. In 22 perinatal asphyxia cases, 6 infants received hypothermia therapy, and the remaining 16 infants did not. Baseline characteristics of the infants are presented in Table 1. Birth weight, gestational age, Apgar score at 1 and 5 minutes, pH, and lactate at birth did not differ significantly among the 3 groups. Hypothermia and normothermia cases were significantly lower than the control cases, but there were no significant differences between hypothermia and normothermia cases in terms of Apgar score at 1 and 5 minutes, pH, and lactate at birth. Although Table 2
all patients with hypothermia were male, there were no significant differences in the ratio of male patients among all 3 groups. Postnatal characteristics of the infants are presented in Table 2. The duration of ventilation and oxygen therapy in hypothermia and normothermia cases was significantly longer than in the control and was significantly longer in hypothermia than normothermia cases. The ratio of abnormal MRI findings and poor neurologic outcome was significantly higher in hypothermia and normothermia than in control cases. However, there were no significant differences between hypothermia and normothermia cases in the ratio of abnormal MRI findings and poor neurologic outcome. Serum TH and OSI levels are depicted in Figs. 1 and 2, respectively. Serum TH and OSI levels gradually increased significantly after birth in hypothermia and normothermia cases but not in control cases (hypothermia: P b .05, normothermia: P b .05, control: not significant). Serum TH and OSI levels at days 3, 5, and 7 were significantly higher than at day 0 in normothermia and hypothermia cases but not in the controls (hypothermia: P b .05, normothermia: P b .05, control: not significant). At all time points, serum TH and OSI levels were significantly higher in hypothermia and normothermia cases than in control cases. Serum TH and OSI levels were significantly higher in normothermia cases than in hypothermia cases at days 3, 5, and 7 (TH: normothermia 158 ± 14 Carr units and hypothermia 130 ±
Postnatal characteristics of infants
Characteristics
Control (n = 15)
Normothermia (n = 16)
Hypothermia (n = 6)
Duration of ventilation (d) Duration of oxygen treatment (d) Abnormal brain MRI findings Age at time of brain MRI
0 1 (0-3) 0 11 M + 25 D (10 M + 21 D − 13 M + 10 D) 0 12 M + 3 D (11 M + 0 D − 12 M + 17 D)
1 (0-7) ⁎ 3 (1-11) ⁎ 5 (31%) ⁎ 12 M + 18 D (11 M + 5 D − 13 M + 10 D) 6 (37.5%) ⁎ 12 M + 5 D (11 M + 15 D − 14 M + 1 D)
8.5 (4-100) ⁎,⁎⁎ 9 (5-100) ⁎,⁎⁎ 2 (33%) ⁎ 12 M + 9 D (11 M + 5 D − 14 M + 0 D) 2 (33%) ⁎ 13 M + 0 D (11 M + 11 D − 13 M + 5 D)
Poor Neurologic outcome Age at time of neurologic evaluation
Data are expressed as medians with ranges in parentheses. Analysis of variance was used to compare continuous data, followed by Bonferroni post hoc testing, and the χ2 test or Fisher exact test to compare dichotomous outcomes. M indicates months; D, days. ⁎ P b .05 compared with control. ⁎⁎ P b .05 compared with normothermia.
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Fig. 1 Serum TH in control (n = 15), hypothermia (n = 16), and normothermia (n = 6) cases. Values shown are means ± SEM. Analysis of variance was used to compare continuous data, followed by Bonferroni post hoc test. Closed circles indicate control cases; open circles, hypothermia cases; and closed triangles, normothermia cases. ‡P b .05, compared with control; ⫧ P b .05, compared with normothermia.
H. Kakita et al. exhibit an initial transient recovery of cerebral oxidative metabolism, followed by a secondary deterioration with cerebral energy failure 6 to 15 hours. This delay offers a therapeutic window for neuroprotective strategy [12]. Mild hypothermia for 72 hours is currently introduced as the standard therapeutic intervention for infants with perinatal asphyxia encephalopathy. The results of several clinical trials support the safety and efficacy of hypothermia to decrease death and disability in infants with perinatal asphyxia [7,8,13,14]. Our present data did not reveal any significant differences in MRI findings and neurologic outcomes between hypothermia and normothermia cases because of a small sample size. Primary antioxidants prevent oxygen radical formation either by removing free radical precursors or by inhibiting catalysis, such as glutathione peroxidase, superoxide dismutase, and catalase. Secondary antioxidants such as vitamins C and E react with ROS that have already formed to achieve either removal or inhibition. Endogenous antioxidants exist intracellularly on the cell membrane and extracellularly [15,16]. Although many antioxidants can be present in the human body, circulating antioxidants cannot be viewed as simple chemicals, and measurement of individual components is unlikely to yield a complete picture of the in vivo situation. In pathological conditions with an oxidant/antioxidant imbalance, the oxidative stress concept suggests that high oxidant levels and low antioxidant levels are involved in poor outcomes [17,18]. The present
24 Carr units at day 3, normothermia 161 ± 17 Carr units and hypothermia 131 ± 13 Carr units at day 5, normothermia 173 ± 14 Carr units and hypothermia 143 ± 20 Carr units at day 7; OSI: normothermia 0.052 ± 0.006 and hypothermia 0.047 ± 0.0069 at day 3, normothermia 0.058 ± 0.0065 and hypothermia 0.046 ± 0.0065 at day 5, normothermia 0.059 ± 0.0067 and hypothermia 0.050 ± 0.0060 at day 7). There were no significant differences in serum BAP levels among control, hypothermia, and normothermia cases (data not shown).
4. Discussion The present study demonstrated that OSI and TH levels in hypothermia and normothermia cases gradually increased after birth, but those in hypothermia cases were significantly lower than in normothermia and control cases. These data suggested that hypothermia might ameliorate oxidative stress in asphyxiated infants. Hypoxia and ischemia during perinatal asphyxia are the major causes of brain injury in newborn infants. The reperfusion or reoxygenation in the immediate postischemia period is one of the initial factors responsible for brain injury in asphyxiated infants because of the increased production of ROS [11]. Immediately after birth, infants with asphyxia
Fig. 2 Serum OSI in control (n = 15), hypothermia (n = 16), and normothermia (n = 6) cases. Values shown are means ± SEM. Analysis of variance was used to compare continuous data, followed by Bonferroni post hoc test. Closed circles indicate control cases; open circles, hypothermia cases; closed triangles, normothermia cases. ‡P b .05, compared with control; ⫧ P b .05, compared with normothermia.
Hypothermia and oxidative stress development study found that serum BAP levels did not have any significant differences among the 3 groups. To evaluate oxidative stress status, we, therefore, measured the ratio of TH to BAP as an indicator of the degree of oxidative stress. Hasegawa et al [19] demonstrated that hypothermia effectively attenuated the release of apoptotic proteins, delayed the activation of caspase-3, and inhibited oxidative stress in PC cells. Katz et al [20] demonstrated that regulated hypothermia reduced oxidative stress in the hippocampus of rats that had experienced asphyxial cardiac arrest. Although several reports have demonstrated that hypothermia reduces the production of ROS and protects cells against damage [8,19-21], the effects of hypothermia on oxidative stress are still unclear, particularly in human neonates. Perrone et al [21] demonstrated that serum TH levels in a hypothermic group have a slower and smaller elevation until 72 hours after birth. Our data supported their findings. In addition, the present study revealed that in hypothermia cases, both TH and OSI have a slower and smaller elevation compared to normothermia cases after 72 hours (the end of the cooling period). This suggests that hypothermia may ameliorate the development of systemic oxidative stress in asphyxiated newborns; this effect lasted until at least day 7 after cooling. Although our study sample was small and retrospective, not prospective, TH and OSI levels were significantly lower in the hypothermia-treated cases, which could have led to the better neurologic outcome observed.
5. Conclusion This study documented that hypothermia may inhibit an increase in systemic oxidative stress in asphyxiated newborns. Although these findings require confirmation and amplification with a larger number of patients, the data provide insight into potential pathogenic mechanisms and may lead to protective therapies against perinatal asphyxia encephalopathy.
6. Limitations • The inclusion criteria and the lack of patient samples and data limited the number of enrolled newborns. • Approximately half of the enrolled infants moved to distant areas with their families, and a later neurologic assessment at 18 months of age was unable to be obtained.
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