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Pediatrics International (2014) ••, ••–••
doi: 10.1111/ped.12520
Original Article
Altered endothelial nitric oxide synthesis in preterm and small for gestational age infants Saadat Arif Huseynova,1 Nushaba Farkhad Panakhova,1 Pusta Ali Orujova,3 Safikhan Shamil Hasanov,4 Mehman Rustam Guliyev2 and Vafa Ilyas Yagubova2 Departments of 1Neonatology and 2Biochemistry, Azerbaijan Medical University, 3Sh. Alasgarova Maternity Hospital, and 4 K. Farajova Pediatrics Institute, Baku, Azerbaijan Abstract
Background: Preterm infants are often exposed to neuronal and endothelial damage. The aim of the present study was to investigate the correlation between endothelial dysfunction and neuronal injury in preterm infants. Methods: We compared serum nitric oxide (NO), endothelial nitric oxide synthase (eNOS) and neuron-specific enolase (NSE) concentrations in 33 moderate preterm (MP) and 47 late preterm (LP) infants using standard ELISA. Each group was classified as appropriate for gestational age (AGA) or small for gestational age (SGA). Results: Compared to the AGA infants, the SGA infants had higher NO on day 1 (MP: mean, 72.3 ng/mL, range, 50.9–99.5 ng/mL vs 52.2 ng/mL, range, 28.1–68.2 ng/mL, P < 0.05; LP: mean, 58.4 ng/mL, range, 25.7–66.4 ng/mL vs 43.7 ng/mL, range, 21.2–60.6 ng/mL, P < 0.05), lower eNOS concentration on day 3 in the MP group (mean, 5.8 IU/mL, range, 1.2–7.9 IU/mL vs 8.9 IU/mL, range, 4.2–14.6 IU/mL, P < 0.05), and on day 1 in the LP group (mean, 5.5 IU/mL, range, 1.5–8.1 IU/mL vs 7.7 IU/mL, range, 4.4–13.8 IU/mL, P < 0.05). The NO/eNOS ratio was higher in SGA infants compared with the AGA subgroups (MP: mean, 13.8, range, 9.9–20.2 vs mean, 9.9, range, 4.7–13.1, P < 0.05; LP: mean, 12.2, range, 9.2–19.9 vs mean, 9.9, range, 5.4–14.4, P < 0.05). AGA infants had lower NSE concentration compared with the SGA infants on day 1 in the LP group (mean, 27.4 ng/mL, range, 20–43 ng/mL vs mean, 40.89 ng/mL, range, 34–51 ng/mL, P < 0.05). A positive correlation was found between NO/eNOS ratio and NSE concentration (r = 0.75, P < 0.05 and r = 0.64, P < 0.05 on days 1 and 3, respectively). Conclusion: High NO concentration in the context of low eNOS activity suggests a possible role of NO in the development of neuronal injury in SGA infants.
Key words endothelial function, growth restriction, neuronal injury, nitric oxide, preterm.
Endothelial function plays an important role in the pathogenesis of diseases in the newborn and in vascular homeostasis. Endothelial activity is regulated through the actions of locally produced agents and reflects the balance of constricting and dilating factors. Among the vasodilators, nitric oxide (NO) appears to be the most important contributor toward the acute regulation of vascular tone. NO, which is generated by endothelial cells, is an important messenger molecule, with a wide range of actions controlling cerebral blood flow and metabolism.1,2 The altered production of NO by the vascular endothelium plays an important role in the pathogenesis of infant diseases and may influence organism growth.3,4 NO is formed via endothelial (eNOS) and neuronal NO synthase (nNOS); under physiological conditions, the dominant NOS isoform in the vasculature is eNOS.5 eNOS is the major source of NO, acts in endothelial dysfunction, leads to vascular and metabolic
Correspondence: Saadat Arif Huseynova, PhD, AZ 1022, Bakikhanov 23, Baku, Azerbaijan. Email: [email protected] Received 9 April 2013; revised 14 September 2014; accepted 17 September 2014.
© 2014 Japan Pediatric Society
disorders,6 and participates in hypoxic–ischemic brain injury.7 There is much to be investigated regarding how NO synthesis is regulated under physiological and pathological conditions. The impact of chronic hypoxia on the NOS isoenzymes in specific brain structures is unknown. Many specific biochemical markers of neuronal injury are being investigated to assess brain damage after perinatal asphyxia in neonates. Markers such as serum protein S-100, brain-specific creatine kinase, neuron-specific enolase (NSE), and interleukin-6 appear promising in identifying patients at risk of perinatal encephalopathy,8 but other studies determined these markers as of limited value in predicting severe brain damage after birth asphyxia.9,10 Previous studies have stated that biochemical markers of brain damage appear in altered fetomaternal blood flow to vital organs, including the brain, and that they are found in higher concentrations in infants with intrauterine growth restriction.11 Intrauterine growth restriction results in more severe and longer-lasting neuronal injuries,12 which are most likely a consequence of a disorder of the cerebral vascular blood circulation. Research shows that growth-retarded fetuses are characterized by decreased vascular growth and endothelial dysfunction in vitro.13
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Furthermore, intrauterine growth restriction has been associated with increased NO synthesis in placental tissue and fetal blood.14 In the context of broad-spectrum investigations confirming a significant effect of NO on the physiology and pathology of vascular homeostasis, the role of NO in the pathogenesis of perinatal brain injury has not been investigated completely. A study clarifying the role of NO in the impaired dilation of cerebral vasculature in small for gestational age (SGA) infants is reasonable. The results of previous studies have shown that different levels of inflammatory reactions in asphyxiated newborn infants depend both on growth restriction and on gestational age. This difference appears in the expression of endothelial inflammatory mediators in hypoxic conditions, which causes different structural and functional pathological changes in the infants with different morphological and physiological maturity levels.15,16 The aim of the present study was to determine the role of endothelial NO activity in neuronal injury and investigate the parameters, depending on the gestational age and growth restriction of newborns, that may contribute to the pathophysiology of endothelial dysfunction. We comparatively analyzed the changes in eNOS and NO as the markers of endothelial activity, and we examined changes in NSE concentration to evaluate neuronal injury in preterm infants during the first week of extrauterine adaptation.
Methods Patients and study design
This prospective study was conducted at the Baku Sh. Aleskerova Maternity Hospital. The Problem Commission on Pediatric Research at Azerbaijan Medical University and the Azerbaijan National Committee on Bioethics and Ethics of Science and Technology approved this study. Eighty infants born between November 2011 and January 2012 at a gestational age of 31–36 weeks and with a high risk of hypoxic–ischemic encephalopathy (HIE) were recruited for this study. Obstetric data were collected from the hospital records. Intrapartum and neonatal data were collected prospectively. The data on maternal pre-eclampsia, gender, type of delivery, resuscitation measures in the delivery room, and anthropometric measurements (e.g. weight, body length, head and chest circumference) were included on an individual research card for each infant. The diagnosis of asphyxia was determined according to Apgar score (≤5 at 5 min of life), initial capillary or arterial pH 7.00 mmol/L, according to the American Academy of Pediatrics guidelines.17,18 Blood gases were detected within 30 min after delivery. Gestational age was based on the date of the mother’s most recent menstrual period and ultrasonogram, and was confirmed using the Ballard et al. scale.19 Growth restriction was defined as estimated fetal anthropometric parameters, confirmed at birth, below the 10th percentile for gestational age and gender.20 The severity of neonatal encephalopathy was estimated based on the clinical behavior of a neonate at 24 h rated on the Sarnat score.21 Cranial ultrasound was done on day 3 of life using the 5 and 7.5 MHz sector transducers. Intraventricular hemorrhage (IVH) was clas© 2014 Japan Pediatric Society
sified into four grades according to the Papile description: grade I, confined to the germinal matrix; grade II, mild or no dilatation of the lateral ventricle; grade III, patent ventricular dilatation; and grade IV, extension into the adjacent brain parenchyma.22 The study exclusion criteria included death of a newborn within the first 3 days of life, transfer to other units, clinical or laboratory evidence of congenital infection, neonatal sepsis, or congenital malformation. Two patient groups were identified: moderate preterm (MP), birth at 31–33 weeks of gestation (n = 34); and late preterm (LP), birth at 34–36 weeks of gestation (n = 49). Because of a technical problem with the blood samples being insufficient for biomarker analysis, we lost the data for one infant from the MP group and for two infants from the LP group. The MP and LP infants were further classified into two subgroups: appropriate for gestational age (AGA; MP, n = 20; LP, n = 28) and SGA (MP, n = 13; LP, n = 19). Blood collection
Venous blood was collected on days 1 and 3 of life. No venous punctures were performed for the sole purpose of study-related analysis. The blood samples were collected in EDTA tubes and centrifuged for 15–20 min. The plasma samples were frozen at −70°C. Grossly hemolyzed samples were not included in the analysis. NO concentration in peripheral blood
The NO concentration was quantified using the Griess reaction in a commercial kit (Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA). This test is based on the conversion of nitrate to nitrite via the action of the nitrate reductase enzyme. The NO level in a given sample was measured by determining the nitrate and nitrite concentrations in the sample. The samples were ultra-filtered through a 10 000 molecular weight cut-off filter and directly assayed. Nitrite concentration was determined using the nitrite standard curve. Nitrate concentration was calculated by subtracting the initial nitrite concentration of the sample from the measured nitrite concentration following the enzymatic conversion of nitrate. eNOS and NSE in the peripheral blood on ELISA
The eNOS (antibodies-online, Aachen, Germany) and NSE (Life Science, Wuhan, China) plasma concentrations were measured using the aforementioned commercial kits, based on a standard enzyme immunoassay procedure.23 The specimens were diluted according to the manufacturer’s instructions for the ELISA kits to obtain the optimal density. NSE is expressed in ng/mL; eNOS activity is expressed in IU/mL. Statistical analysis
The data in the subgroups of both groups were tested for a normal distribution and found to be non-parametric. Significant differences between the AGA and SGA preterm groups were determined using the Mann–Whitney U-test to assess the differences in NO, eNOS, and NSE production. The same test was used to assess the difference in the quantitative variables between the
NO in neuronal injury of SGA infants
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Table 1 Maternal cha-racteristics MP, mean (range) or n (%) Age (years) Gravidy Premature rupture of membranes Chronic hypertension Oligohydramnios Pre-eclampsia Cesarean section
AGA (n = 20) 24.2 (19–33) 2.3 (1–6) 6 (30) 1 (5) 2 (10) 4 (20) 6 (30)
SGA (n = 13) 26.5 (19–32) 2.1 (1–5) 2 (15.4) 3 (23.1) 5 (38.5) 5 (38.5) 7 (53.8)
LP, mean (range) or n (%) AGA (n = 28) 26.9 (20–33) 2.3 (1–7) 6 (21.4) 1 (3.6) 2 (7.1) 1 (3.6) 6 (21.4)
SGA (n = 19) 27.3 (18–34) 3.1 (1–8) 2 (10.5) 4 (21.1) 6 (31.6) 8 (42.1)* 9 (47.4)
*P < 0.05 between subgroups of the same group. AGA, appropriate for gestational age; LP, late preterm; MP, moderate preterm; SGA, small for gestational age.
subgroups of the same study groups and between appropriate subgroups of MP and LP infants. Qualitative variables such as gender, maternal pre-eclampsia, cesarean section, resuscitation measures in the delivery room, and the degree of HIE and IVH were compared using Fisher’s exact test. Spearman rank-order correlation coefficient was used to determine the associations between the appropriate variables. In all instances, significance was established at P < 0.05.
Results Maternal characteristics are listed in Table 1. Mothers of the MP SGA and LP SGA infants, although of similar age and gravidy as the respective two subgroups, were more likely to have medical illnesses and pregnancy complications known to be associated with intrauterine growth restriction. Table 2 lists neonatal characteristics and clinical data. Perinatal asphyxia was frequently observed in both subgroups of MP infants. The majority of the SGA infants in each group required bag and mask ventilation or intubation in the delivery room. The majority of the SGA infants(69.2%, n = 9) in MP group were classified with severe HIE and IVH grade II–III, which was significantly higher compared with the AGA infants of the same group and respective subgroup of LP infants.
Changes in the concentrations of endothelial dysfunction and neuronal injury markers during the early neonatal period were different in the respective subgroups of MP and LP infants. As shown in Figure 1, NO was significantly elevated in the SGA MP and LP infants. The highest NO concentration was found in the MP SGA infants compared with the MP AGA infants and within both subgroups of LP infants. We did not observe a statistically significant difference between the NO level of the MP AGA and LP AGA subgroups. The eNOS activity changes were not consistent with the NO concentration in the study subgroups. Unlike in the case of NO, higher eNOS was observed in the AGA infants in the MP and LP groups than in the respective SGA subgroups. eNOS activity was significantly elevated in both subgroups of MP infants compared with the respective subgroups of LP newborns. NSE activity was not differentiated in the study subgroups. Only the AGA infants in the LP group had lower concentration of this neuronal injury marker compared with the SGA infants in the same group (P < 0.05) and the appropriate subgroup of MP newborns (P < 0.05). On Spearman’s rank-order correlation, NO and NSE results differed in the study subgroups. We found a significant positive correlation only on day 1 in the MP SGA subgroup and a negative correlation in the LP AGA infants on day 1 (Fig. 2).
Table 2 Infant characteristics and clinical parameters MP, mean (range) or n (%) Birthweight (g) Gender M/F Perinatal asphyxia Mean arterial blood pressure (mmHg) Free flow oxygen† Bag and mask ventilation† Intubation† Degree of encephalopathy Mild Moderate Severe IVH Grade I Grade II–III
AGA (n = 20) 1643.2b (1100–2005) 11/9 12 (60) 31.8 (26.4–35.20) 6 (30) 5 (25) 1 (5)
LP, mean (range) or n (%)
SGA (n = 13) 1110.4a (980–1310) 7/6 8 (61.5) 28.8 (24–31.3) 1 (7.7) 6 (46.2) 3 (23.1)
AGA (n = 28) 2394.3 (2000–2580) 14/14 10 (35.7) 34.5 (31.2–39.2) 11 (39.3) 6 (21.4) 0 (0)
SGA (n = 19) 1625.5a (1250–1800) 10/9 9 (47.4) 29.9 (26.1–35.80) 2 (10.5) 6 (31.6) 1 (5.3)
4 (20) 6 (30) 3 (15)a
2 (15.4) 2 (15.4) 9 (69.2)b
9 (32.1) 3 (10.7) 0 (0)
5 (26.3) 5 (26.3) 5 (26.3)
2 (10) 3 (15)a
2 (15.4) 9 (69.2)b
2 (7.1) 0 (0)
2 (10.5) 3 (15.8)
a P < 0.05 between subgroups of the same group; bP < 0.05 between AGA MP and LP subgroups. †Resuscitation required in the delivery room. AGA, appropriate for gestational age; IVH, intraventricular hemorrhage; LP, late preterm; MP, moderate preterm; SGA, small for gestational age.
© 2014 Japan Pediatric Society
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NO (µmol/L)
100
(a) *#
80 60
#
*
#
40 20
eNOS (IU/mL)
0 12 10 8 6 4 2 0
NSE (ng/mL)
50
(b)
# *# *
(c) *
40 30 20 10 0
MP AGA
MP SGA
LP AGA
LP SGA
Fig. 1 Mean (a) total nitric oxide (NO), (b) endothelial NO synthase (eNOS) and (c) neuron-specific enolase (NSE) concentration in moderate preterm (MP) and late preterm (LP) appropriate for gestational age (AGA) and small for gestational age (SGA) infants. Bars, SEM. ■, Day 1 results; □, day 3 results. *P < 0.05 AGA and SGA infants in the same group; #P < 0.05 vs the appropriate subgroup of LP infants.
With regard to mean NO/eNOS ratio in the newborns, a substantial difference was noted between the SGA and AGA infants from both groups (Fig. 3). NO/eNOS ratio did not change with regard to gestational age, but this ratio was higher in the MP SGA infants (P < 0.05) and in the LP SGA (P < 0.05) newborns compared with the respective subgroups in each group. To determine the correlation between the NO/eNOS ratio and neuronal injury, we analyzed the day 1 and day 3 ratios of NO/eNOS in both study subgroups (a total of 80 cases) and found a significant positive correlation between NSE and the NO/eNOS ratio (Fig. 4).
Discussion Intrauterine growth restriction is a common health problem that can lead to obesity, arterial hypertension, cardiovascular diseases, impaired glucose tolerance and other serious metabolic consequences later in life.24–26 NO is important in the physiology and pathology of fetomaternal and neonatal blood circulation.27–29 The role of NO in the pathogenesis of intrauterine growth restriction and brain injury is unclear. The use of biochemical markers to assess brain injury in newborns with NO changes is controversial. Previous studies show that elevated NO expression was found to be related to significantly lower antioxidant activity, increased lipid peroxidation and impaired neuronal function in growth-restricted infants.30 NO supplementation was useful to © 2014 Japan Pediatric Society
increase uteroplacental circulation31 and decrease the levels of neurospecific injury markers in the cord blood of SGA infants.32 We determined the concentrations of endothelial activation and neuronal injury markers at 5–6 h after birth because a high probability of low systemic perfusion in preterm infants occurs in day 1 of extrauterine life,33 and on day 3 of life to assess the stabilization of autoregulatory mechanisms.34 In the present study, the SGA and AGA newborns had different and conflicting correlations between NO and NSE. With increased NO concentration, we found high NSE activity in the MP SGA infants and a low risk of neuronal injury in the LP AGA infants (Fig. 2). The interpretation of these results was possible through the parallel determination of NO and eNOS levels in the AGA and SGA subgroups. We investigated the balance of NO/eNOS and its effect on neuronal injury in preterm infants. An important finding is the significant difference between the NO/eNOS balance in the SGA and AGA infants, which reflects the severity of neuronal injury and provides a basis for understanding different NO and NSE connections in the study subgroups. We found that a significantly higher NO/eNOS ratio is the result of higher NO concentration and lower eNOS activity in SGA infants compared to AGA infants. It is possible that the activation of neuronal and inducible NOS sources in these newborns occurs as the result of long-term and severe intrauterine and birth distress. This is suggested by the high occurrence of severe birth asphyxia and severe HIE (Table 1) in the SGA newborns, which is consistent with an increased NSE concentration in these infants (Fig. 1). In contrast, in AGA infants (particularly in the LP group), the lowest risk for severe HIE (Table 1) was observed in conjunction with decreased NSE activation (Fig. 1). In the context of a high degree of eNOS activation in LP AGA infants, increased NO might be a compensative or defensive strategy for the preterm brain, in which a low NO/eNOS ratio is associated with a negative correlation between NO and NSE. In contrast, a high NO concentration without adequate eNOS activation causes a more serious neuronal injury: there is a positive correlation between NO and NSE in the MP SGA infants. The statistically significant positive correlation between NSE and NO/eNOS ratio (Fig. 4) corresponds to the aforementioned result. Despite the results of previous studies confirming the dependence of neuronal injury on brain maturity,15,16 we found no significant difference in NSE level in the MP and LP infants. Therefore, neuronal injury did not depend on the gestational age but on the activation of endothelial and non-endothelial sources of NO synthesis; additionally, the growth-restricted infants in both groups undergo more significant endothelial dysfunction and neuronal injury. The intrauterine growth of the SGA infants was characterized by more serious pathologic conditions leading to endothelial damage. We detected larger changes of endothelial nitric oxide synthesis and a higher probability of neuronal injury in the growth-restricted infants than in the newborns with normal growth. The present study had limitations. We were unable to measure the concentration of all types of NOS, which would help confirm the hypothesis; an experimental study determined that chronic hypoxia decreases eNOS in the hippocampus and increases
NO in neuronal injury of SGA infants
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Fig. 2 Correlation between nitric oxide (NO) and neuron-specific enolase (NSE) in (a,b) moderate preterm (MP) appropriate for gestational age (AGA), (c,d) MP small for gestational age (SGA), (e,f) late preterm (LP) AGA and (g,h) LP SGA infants. (a,c,e,g) Day 1 results; (b,d,f,h) day 3 results. (a) r = 0.29; (b) r = 0.38; (c) r = 0.70*; (d) r = 0.37; (e) r = –0.67*; (f) r = –0.38; (g) r = 0.46; (h) r = 0.07. *P < 0.05.
nNOS in the neuronal and glial cells of the thalamus.35 We found significantly lower eNOS activity in the context of increased NO concentration in the SGA infants, compared with the AGA subgroups, and hypothesize that the long-term and deeper hypoxic conditions result in the suppression of eNOS and activation of other sources of NOS activation; therefore, eNOS is suppressed when NO is high. To our knowledge, no study has been done on eNOS suppression in the context of high NO concentration. Wei et al., however, determined that the endothelial NO production by eNOS can decrease ischemic injury by inducing vasodilation, while increasing the NO production by neurons can cause neuronal injury.36 Consistent with this observation, Liu et al. have suggested a neuroprotective effect of nNOS inhibitors.37 The present results are consistent with these studies and confirm in vivo that intrauterine growth restriction results in the rise of NO in the context of non-endothelial sources of activation.
Fig. 3 Mean nitric oxide/endothelial nitric oxide synthase (NO/ eNOS) ratio in the moderate preterm (MP) and late preterm (LP) appropriate for gestational age (AGA) and small for gestational age (SGA) infants. *P < 0.05 vs AGA infants. © 2014 Japan Pediatric Society
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Fig. 4 Spearman’s rank-order correlation between nitric oxide/ endothelial nitric oxide synthase (NO/eNOS) ratio and neuronspecific enolase (NSE) concentration in the moderate and late preterm infants (n = 80) on (a) day 1 and (b) day 3. (a) r = 0.75*; (b) r = 0.64* (*P < 0.05).
Preterm birth and intrauterine growth restriction are characterized by long-term neurodevelopmental problems later in life.38–40There is currently no perfect method for predicting the risk of behavior, speech, cognitive and other insufficiencies resulting from preterm birth and growth retardation. The lack of sensitive perinatal markers for the early and late prediction of neurologic outcome and a wide spectrum of clinical and experimental studies justify the need for further investigation in this area. We propose that an impaired NO/eNOS ratio may be involved in the pathogenesis of brain damage and neonatal disadaptation in SGA infants. The present results form the basis of a hypothesis that the NO/eNOS balance can serve as a predictive marker for preterm infants and may be accepted as a sensitive marker for neuronal injury in newborns. The early assessment of the severity of an acute cerebral lesion in preterm infants may provide a useful tool in therapeutic decision making and may prevent neurologic complications in these infants.
Acknowledgments The authors sincerely thank Science Development Foundation under the President of the Azerbaijan Republic for providing nitric oxide, endothelial nitric oxide synthase and neuron-specific enolase reagent kits. We also thank the staff of the Clinical Biochemistry Laboratory of Azerbaijan Medical University for assistance with biomarker analysis. © 2014 Japan Pediatric Society
1 Prado R, Watson BD, Kuluz J, Dietrich WD. Endothelium-derived nitric oxide synthase inhibition: Effects on cerebral blood flow, pial diameter artery, and vascular morphology in rat. Stroke 1992; 23: 1118–23. 2 Toda N, Ayajiki K, Okamura T. Cerebral blood flow regulation by nitric oxide. Recent advances. Pharmacol. Rev. 2009; 61: 62–97. 3 Vannuci SJ, Hagberg H. Hypoxia–ischemia in the immature brain. J. Exp. Biol. 2004; 207: 3149–54. 4 Lai M-C, Yang S-N. Perinatal Hypoxic ischemic encephalopathy. J. Biomed. Biotechnol. 2011; 2011: 609813. 5 Rafikov R, Fonseka FV, Kumar S et al. eNOS activation and NO function: Structural motifs responsible for the posttranslational control of endothelial nitric oxide synthase activity. J. Endocrinol. 2011; 210: 271–84. 6 Dumont I, Hou X, Hardy P et al. l. Developmental regulation of endothelial nitric oxide synthase in cerebral vessels of newborn pig by prostaglandin E2. J. Pharmacol. Exp. Ther. 1999; 291: 2627– 33. 7 Fabian RH, Perez-Polo JR, Kent AT. Perivascular nitric oxide and superoxide in neonatal cerebral hypoxia-ischemia. Am. J. Physiol. Heart Circ. Physiol. 2008; 295: 1809–14. 8 Naithani M, Simalti AK. Biochemical markers in perinatal asphyxia. J. Nepal Pediatr. Soc. 2011; 31: 151–6. 9 Nagdyman N, Grimmer I, Scholz T, Muller C, Obladen M. Predictive value of brain-specific proteins in serum for neurodevelopmental outcome after birth asphyxia. Pediatr. Res. 2003; 54: 270–75. 10 Wijnberger LDE, Nikkels PGJ, van Dongen AJCM et al. Expression in the placenta of neuronal markers for perinatal brain damage. Pediatr. Res. 2002; 4: 492–6. 11 Gazzolo D, Vinesi P, Marinoni E et al. S100B protein concentrations in cord blood: Correlations with gestational age in term and preterm deliveries. Clin. Chem. 2000; 46: 998–1000. 12 Figueras F, Cruz-Martinez R, Sanz-Cortes M et al. Neurobehavioral outcomes in preterm, growth-restricted infants with and without prenatal advanced signs of brain-sparing. Ultrasound Obstet. Gynecol. 2011; 38: 288–94. 13 Rozance PJ, Seedorf GJ, Brown A et al. Intrauterine growth restriction decreases pulmonary alveolar and vessel growth and causes pulmonary artery endothelial cell dysfunction in vitro in fetal sheep. AJP Lung Physiol. 2011; 6: 860–71. 14 Tikvica A, Jukic MK, Pintaric I et al. Nitric oxide synthesis in placenta is increased in intrauterine growth restriction and fetal hypoxia. Coll. Antropol. 2008; 2: 565–70. 15 Giuseppe D, Sergio C, Pasqua B et al. Preterm neonates leads to serum changes in protein S-100 and neuron specific enolase. Curr. Neurovasc. Res. 2009; 6: 110–16. 16 Mahmoud A, Gehad S, Mohamed E. Transcranial US of preterm neonates: High risk gestational age and birth weight for perinatal asphyxia. Egypt. J. Radiol. Nucl. Med. 2012; 43: 265–74. 17 Chen ZL, He RZ, Peng Q, Guo KY, Zhang YQ, Yuan HH. Clinical study on improving the diagnostic criteria for neonatal asphyxia. Zhonghua Er Ke Za Zhi 2006; 44: 167–72. 18 Yildizdas D, Yapicoglu H, Yilmaz HL, Sertdemir Y. Correlation of simultaneously obtained capillary, venous and arterial blood gases of patients in a paediatric intensive care unit. Arch. Dis. Child. 2004; 89: 176–80. 19 Ballard JL, Khoury JC, Wedig K. New Ballard Score, expanded to include extremely premature infants. J. Pediatr. 1991; 119: 417– 23. 20 Mandruzzato G, Meir YJ, Natale R, Maso G. Antepartal assessment of IUGR fetuses. Perinat. Med. 2001; 3: 222–29. 21 Sarnat HB, Sarnat MS. Neonatal encephalopathy following fetal distress: A clinical and electroencephalographic study. Arc. Neurol. 1976; 33: 695–706.
NO in neuronal injury of SGA infants 22 Papile LA, Burstein J, Burstein R. Incidence and evolution of the subependymal intraventricular hemorrhage: A study of infants with weights less than 1500 grams. J. Pediatr. 1978; 92: 529–34. 23 Lequin R. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin. Chem. 2005; 12: 2415–18. 24 Chakraborty S, Joseph DV, Bankart MJG, Petersen SA, Wailoo MP. Fetal growth restriction: Relation to growth and obesity at the age of 9 years. Arch. Dis. Child. Fetal Neonatal Ed. 2007; 92: 479–83. 25 Szostak-We˛gierek D, Szamotulska K. Fetal development and risk of cardiovascular diseases and diabetes type 2 in adult life. Med. Wieku Rozwoj. 2011; 15: 203–15. 26 Varvarigou AA. Intrauterine growth restriction as a potential risk factor for disease onset in adulthood. J. Pediatr. Endocrinol. Metab. 2010; 23: 215–24. 27 Lyalla F, Greera IA, Younga A, Myattb L. Nitric oxide concentrations are increased in the feto-placental circulation in intrauterine growth restriction. Placenta 1996; 17: 165–8. 28 Lopez-Jaramillo P, Arenas WD, Garcia RG, Rincon MY, Lopez M. The role of the l-arginine-nitric oxide pathway in preeclampsia. Ther. Adv. Cardiovasc. Dis. 2008; 2: 261–75. 29 Pearce W. Hypoxic regulation of the fetal cerebral circulation. J. Appl. Physiol. 2006; 100: 731–8. 30 Hracsko Z, Hermesz E, Ferencz A et al. Endothelial nitric oxide synthase is up-regulated in the umbilical cord in pregnancies complicated with intrauterine growth retardation. In vivo 2009; 23: 727–32. 31 Lyall F, Greer IA, Young A, Myatt L. Nitric oxide concentrations are increased in the feto-placental circulation in intrauterine growth restriction. Placenta 1996; 17: 165–8.
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32 Gazzolo D, Bruschettini M, Di Lorio R et al. l. Maternal nitric oxide supplementation decreases cord blood S100B in intrauterine growth-retarded fetuses. Clin. Chem. 2002; 4: 647–50. 33 McLean CW, Gayabyab RG, Noori S, Seri I. Cerebral circulation and hypotension in the premature infant: Diagnosis and treatment. In: Perlman JM (ed). Neurology Questions and Controversies. Elsevier Saunders, Philadelphia, 2008; 3–26. 34 Greisen G. Autoregulation of cerebral blood flow in newborn babies. Early Hum. Dev. 2005; 81: 423–8. 35 Dong Y, Yu Z, Sun Y et al. Chronic fetal hypoxia produces selective brain injury associated with altered nitric oxide synthases. Am. J. Obstet. Gynecol. 2011; 3: 16–28. 36 Wei G, Dawson VL, Zweier JL. Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia. Biochim. Biophys. Acta 1999; 20: 23–34. 37 Liu PK, Robertson CS, Valadka A. The association between neuronal nitric oxide synthase and neuronal sensitivity in the brain after brain injury. Ann. N. Y. Acad. Sci. 2002; 962: 226–41. 38 Klaric´ AŠ, Kolundžic´ Z, Galic´ S, Bošnjak VM. Language development in preschool children born after asymmetrical intrauterine growth retardation. Eur. J. Paediatr. Neurol. 2012; 16: 132–7. 39 Sung IK, Vohr B, Oh W. Growth and neurodevelopmental outcome of very low birth weight infants with intrauterine growth retardation: Comparison with control subjects matched by birth weight and gestational age. J. Pediatr. 1993; 123: 618–24. 40 Gratacos E. The problem of predicting neurological outcome in early-onset intrauterine growth restriction. Ultrasound Obstet. Gynecol. 2009; 33: 5–7.
© 2014 Japan Pediatric Society
Pediatrics International (2014) ••, ••–••
doi: 10.1111/ped.12520
Original Article
Altered endothelial nitric oxide synthesis in preterm and small for gestational age infants Saadat Arif Huseynova,1 Nushaba Farkhad Panakhova,1 Pusta Ali Orujova,3 Safikhan Shamil Hasanov,4 Mehman Rustam Guliyev2 and Vafa Ilyas Yagubova2 Departments of 1Neonatology and 2Biochemistry, Azerbaijan Medical University, 3Sh. Alasgarova Maternity Hospital, and 4 K. Farajova Pediatrics Institute, Baku, Azerbaijan Abstract
Background: Preterm infants are often exposed to neuronal and endothelial damage. The aim of the present study was to investigate the correlation between endothelial dysfunction and neuronal injury in preterm infants. Methods: We compared serum nitric oxide (NO), endothelial nitric oxide synthase (eNOS) and neuron-specific enolase (NSE) concentrations in 33 moderate preterm (MP) and 47 late preterm (LP) infants using standard ELISA. Each group was classified as appropriate for gestational age (AGA) or small for gestational age (SGA). Results: Compared to the AGA infants, the SGA infants had higher NO on day 1 (MP: mean, 72.3 ng/mL, range, 50.9–99.5 ng/mL vs 52.2 ng/mL, range, 28.1–68.2 ng/mL, P < 0.05; LP: mean, 58.4 ng/mL, range, 25.7–66.4 ng/mL vs 43.7 ng/mL, range, 21.2–60.6 ng/mL, P < 0.05), lower eNOS concentration on day 3 in the MP group (mean, 5.8 IU/mL, range, 1.2–7.9 IU/mL vs 8.9 IU/mL, range, 4.2–14.6 IU/mL, P < 0.05), and on day 1 in the LP group (mean, 5.5 IU/mL, range, 1.5–8.1 IU/mL vs 7.7 IU/mL, range, 4.4–13.8 IU/mL, P < 0.05). The NO/eNOS ratio was higher in SGA infants compared with the AGA subgroups (MP: mean, 13.8, range, 9.9–20.2 vs mean, 9.9, range, 4.7–13.1, P < 0.05; LP: mean, 12.2, range, 9.2–19.9 vs mean, 9.9, range, 5.4–14.4, P < 0.05). AGA infants had lower NSE concentration compared with the SGA infants on day 1 in the LP group (mean, 27.4 ng/mL, range, 20–43 ng/mL vs mean, 40.89 ng/mL, range, 34–51 ng/mL, P < 0.05). A positive correlation was found between NO/eNOS ratio and NSE concentration (r = 0.75, P < 0.05 and r = 0.64, P < 0.05 on days 1 and 3, respectively). Conclusion: High NO concentration in the context of low eNOS activity suggests a possible role of NO in the development of neuronal injury in SGA infants.
Key words endothelial function, growth restriction, neuronal injury, nitric oxide, preterm.
Endothelial function plays an important role in the pathogenesis of diseases in the newborn and in vascular homeostasis. Endothelial activity is regulated through the actions of locally produced agents and reflects the balance of constricting and dilating factors. Among the vasodilators, nitric oxide (NO) appears to be the most important contributor toward the acute regulation of vascular tone. NO, which is generated by endothelial cells, is an important messenger molecule, with a wide range of actions controlling cerebral blood flow and metabolism.1,2 The altered production of NO by the vascular endothelium plays an important role in the pathogenesis of infant diseases and may influence organism growth.3,4 NO is formed via endothelial (eNOS) and neuronal NO synthase (nNOS); under physiological conditions, the dominant NOS isoform in the vasculature is eNOS.5 eNOS is the major source of NO, acts in endothelial dysfunction, leads to vascular and metabolic
Correspondence: Saadat Arif Huseynova, PhD, AZ 1022, Bakikhanov 23, Baku, Azerbaijan. Email: [email protected] Received 9 April 2013; revised 14 September 2014; accepted 17 September 2014.
© 2014 Japan Pediatric Society
disorders,6 and participates in hypoxic–ischemic brain injury.7 There is much to be investigated regarding how NO synthesis is regulated under physiological and pathological conditions. The impact of chronic hypoxia on the NOS isoenzymes in specific brain structures is unknown. Many specific biochemical markers of neuronal injury are being investigated to assess brain damage after perinatal asphyxia in neonates. Markers such as serum protein S-100, brain-specific creatine kinase, neuron-specific enolase (NSE), and interleukin-6 appear promising in identifying patients at risk of perinatal encephalopathy,8 but other studies determined these markers as of limited value in predicting severe brain damage after birth asphyxia.9,10 Previous studies have stated that biochemical markers of brain damage appear in altered fetomaternal blood flow to vital organs, including the brain, and that they are found in higher concentrations in infants with intrauterine growth restriction.11 Intrauterine growth restriction results in more severe and longer-lasting neuronal injuries,12 which are most likely a consequence of a disorder of the cerebral vascular blood circulation. Research shows that growth-retarded fetuses are characterized by decreased vascular growth and endothelial dysfunction in vitro.13
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Furthermore, intrauterine growth restriction has been associated with increased NO synthesis in placental tissue and fetal blood.14 In the context of broad-spectrum investigations confirming a significant effect of NO on the physiology and pathology of vascular homeostasis, the role of NO in the pathogenesis of perinatal brain injury has not been investigated completely. A study clarifying the role of NO in the impaired dilation of cerebral vasculature in small for gestational age (SGA) infants is reasonable. The results of previous studies have shown that different levels of inflammatory reactions in asphyxiated newborn infants depend both on growth restriction and on gestational age. This difference appears in the expression of endothelial inflammatory mediators in hypoxic conditions, which causes different structural and functional pathological changes in the infants with different morphological and physiological maturity levels.15,16 The aim of the present study was to determine the role of endothelial NO activity in neuronal injury and investigate the parameters, depending on the gestational age and growth restriction of newborns, that may contribute to the pathophysiology of endothelial dysfunction. We comparatively analyzed the changes in eNOS and NO as the markers of endothelial activity, and we examined changes in NSE concentration to evaluate neuronal injury in preterm infants during the first week of extrauterine adaptation.
Methods Patients and study design
This prospective study was conducted at the Baku Sh. Aleskerova Maternity Hospital. The Problem Commission on Pediatric Research at Azerbaijan Medical University and the Azerbaijan National Committee on Bioethics and Ethics of Science and Technology approved this study. Eighty infants born between November 2011 and January 2012 at a gestational age of 31–36 weeks and with a high risk of hypoxic–ischemic encephalopathy (HIE) were recruited for this study. Obstetric data were collected from the hospital records. Intrapartum and neonatal data were collected prospectively. The data on maternal pre-eclampsia, gender, type of delivery, resuscitation measures in the delivery room, and anthropometric measurements (e.g. weight, body length, head and chest circumference) were included on an individual research card for each infant. The diagnosis of asphyxia was determined according to Apgar score (≤5 at 5 min of life), initial capillary or arterial pH 7.00 mmol/L, according to the American Academy of Pediatrics guidelines.17,18 Blood gases were detected within 30 min after delivery. Gestational age was based on the date of the mother’s most recent menstrual period and ultrasonogram, and was confirmed using the Ballard et al. scale.19 Growth restriction was defined as estimated fetal anthropometric parameters, confirmed at birth, below the 10th percentile for gestational age and gender.20 The severity of neonatal encephalopathy was estimated based on the clinical behavior of a neonate at 24 h rated on the Sarnat score.21 Cranial ultrasound was done on day 3 of life using the 5 and 7.5 MHz sector transducers. Intraventricular hemorrhage (IVH) was clas© 2014 Japan Pediatric Society
sified into four grades according to the Papile description: grade I, confined to the germinal matrix; grade II, mild or no dilatation of the lateral ventricle; grade III, patent ventricular dilatation; and grade IV, extension into the adjacent brain parenchyma.22 The study exclusion criteria included death of a newborn within the first 3 days of life, transfer to other units, clinical or laboratory evidence of congenital infection, neonatal sepsis, or congenital malformation. Two patient groups were identified: moderate preterm (MP), birth at 31–33 weeks of gestation (n = 34); and late preterm (LP), birth at 34–36 weeks of gestation (n = 49). Because of a technical problem with the blood samples being insufficient for biomarker analysis, we lost the data for one infant from the MP group and for two infants from the LP group. The MP and LP infants were further classified into two subgroups: appropriate for gestational age (AGA; MP, n = 20; LP, n = 28) and SGA (MP, n = 13; LP, n = 19). Blood collection
Venous blood was collected on days 1 and 3 of life. No venous punctures were performed for the sole purpose of study-related analysis. The blood samples were collected in EDTA tubes and centrifuged for 15–20 min. The plasma samples were frozen at −70°C. Grossly hemolyzed samples were not included in the analysis. NO concentration in peripheral blood
The NO concentration was quantified using the Griess reaction in a commercial kit (Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA). This test is based on the conversion of nitrate to nitrite via the action of the nitrate reductase enzyme. The NO level in a given sample was measured by determining the nitrate and nitrite concentrations in the sample. The samples were ultra-filtered through a 10 000 molecular weight cut-off filter and directly assayed. Nitrite concentration was determined using the nitrite standard curve. Nitrate concentration was calculated by subtracting the initial nitrite concentration of the sample from the measured nitrite concentration following the enzymatic conversion of nitrate. eNOS and NSE in the peripheral blood on ELISA
The eNOS (antibodies-online, Aachen, Germany) and NSE (Life Science, Wuhan, China) plasma concentrations were measured using the aforementioned commercial kits, based on a standard enzyme immunoassay procedure.23 The specimens were diluted according to the manufacturer’s instructions for the ELISA kits to obtain the optimal density. NSE is expressed in ng/mL; eNOS activity is expressed in IU/mL. Statistical analysis
The data in the subgroups of both groups were tested for a normal distribution and found to be non-parametric. Significant differences between the AGA and SGA preterm groups were determined using the Mann–Whitney U-test to assess the differences in NO, eNOS, and NSE production. The same test was used to assess the difference in the quantitative variables between the
NO in neuronal injury of SGA infants
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Table 1 Maternal cha-racteristics MP, mean (range) or n (%) Age (years) Gravidy Premature rupture of membranes Chronic hypertension Oligohydramnios Pre-eclampsia Cesarean section
AGA (n = 20) 24.2 (19–33) 2.3 (1–6) 6 (30) 1 (5) 2 (10) 4 (20) 6 (30)
SGA (n = 13) 26.5 (19–32) 2.1 (1–5) 2 (15.4) 3 (23.1) 5 (38.5) 5 (38.5) 7 (53.8)
LP, mean (range) or n (%) AGA (n = 28) 26.9 (20–33) 2.3 (1–7) 6 (21.4) 1 (3.6) 2 (7.1) 1 (3.6) 6 (21.4)
SGA (n = 19) 27.3 (18–34) 3.1 (1–8) 2 (10.5) 4 (21.1) 6 (31.6) 8 (42.1)* 9 (47.4)
*P < 0.05 between subgroups of the same group. AGA, appropriate for gestational age; LP, late preterm; MP, moderate preterm; SGA, small for gestational age.
subgroups of the same study groups and between appropriate subgroups of MP and LP infants. Qualitative variables such as gender, maternal pre-eclampsia, cesarean section, resuscitation measures in the delivery room, and the degree of HIE and IVH were compared using Fisher’s exact test. Spearman rank-order correlation coefficient was used to determine the associations between the appropriate variables. In all instances, significance was established at P < 0.05.
Results Maternal characteristics are listed in Table 1. Mothers of the MP SGA and LP SGA infants, although of similar age and gravidy as the respective two subgroups, were more likely to have medical illnesses and pregnancy complications known to be associated with intrauterine growth restriction. Table 2 lists neonatal characteristics and clinical data. Perinatal asphyxia was frequently observed in both subgroups of MP infants. The majority of the SGA infants in each group required bag and mask ventilation or intubation in the delivery room. The majority of the SGA infants(69.2%, n = 9) in MP group were classified with severe HIE and IVH grade II–III, which was significantly higher compared with the AGA infants of the same group and respective subgroup of LP infants.
Changes in the concentrations of endothelial dysfunction and neuronal injury markers during the early neonatal period were different in the respective subgroups of MP and LP infants. As shown in Figure 1, NO was significantly elevated in the SGA MP and LP infants. The highest NO concentration was found in the MP SGA infants compared with the MP AGA infants and within both subgroups of LP infants. We did not observe a statistically significant difference between the NO level of the MP AGA and LP AGA subgroups. The eNOS activity changes were not consistent with the NO concentration in the study subgroups. Unlike in the case of NO, higher eNOS was observed in the AGA infants in the MP and LP groups than in the respective SGA subgroups. eNOS activity was significantly elevated in both subgroups of MP infants compared with the respective subgroups of LP newborns. NSE activity was not differentiated in the study subgroups. Only the AGA infants in the LP group had lower concentration of this neuronal injury marker compared with the SGA infants in the same group (P < 0.05) and the appropriate subgroup of MP newborns (P < 0.05). On Spearman’s rank-order correlation, NO and NSE results differed in the study subgroups. We found a significant positive correlation only on day 1 in the MP SGA subgroup and a negative correlation in the LP AGA infants on day 1 (Fig. 2).
Table 2 Infant characteristics and clinical parameters MP, mean (range) or n (%) Birthweight (g) Gender M/F Perinatal asphyxia Mean arterial blood pressure (mmHg) Free flow oxygen† Bag and mask ventilation† Intubation† Degree of encephalopathy Mild Moderate Severe IVH Grade I Grade II–III
AGA (n = 20) 1643.2b (1100–2005) 11/9 12 (60) 31.8 (26.4–35.20) 6 (30) 5 (25) 1 (5)
LP, mean (range) or n (%)
SGA (n = 13) 1110.4a (980–1310) 7/6 8 (61.5) 28.8 (24–31.3) 1 (7.7) 6 (46.2) 3 (23.1)
AGA (n = 28) 2394.3 (2000–2580) 14/14 10 (35.7) 34.5 (31.2–39.2) 11 (39.3) 6 (21.4) 0 (0)
SGA (n = 19) 1625.5a (1250–1800) 10/9 9 (47.4) 29.9 (26.1–35.80) 2 (10.5) 6 (31.6) 1 (5.3)
4 (20) 6 (30) 3 (15)a
2 (15.4) 2 (15.4) 9 (69.2)b
9 (32.1) 3 (10.7) 0 (0)
5 (26.3) 5 (26.3) 5 (26.3)
2 (10) 3 (15)a
2 (15.4) 9 (69.2)b
2 (7.1) 0 (0)
2 (10.5) 3 (15.8)
a P < 0.05 between subgroups of the same group; bP < 0.05 between AGA MP and LP subgroups. †Resuscitation required in the delivery room. AGA, appropriate for gestational age; IVH, intraventricular hemorrhage; LP, late preterm; MP, moderate preterm; SGA, small for gestational age.
© 2014 Japan Pediatric Society
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NO (µmol/L)
100
(a) *#
80 60
#
*
#
40 20
eNOS (IU/mL)
0 12 10 8 6 4 2 0
NSE (ng/mL)
50
(b)
# *# *
(c) *
40 30 20 10 0
MP AGA
MP SGA
LP AGA
LP SGA
Fig. 1 Mean (a) total nitric oxide (NO), (b) endothelial NO synthase (eNOS) and (c) neuron-specific enolase (NSE) concentration in moderate preterm (MP) and late preterm (LP) appropriate for gestational age (AGA) and small for gestational age (SGA) infants. Bars, SEM. ■, Day 1 results; □, day 3 results. *P < 0.05 AGA and SGA infants in the same group; #P < 0.05 vs the appropriate subgroup of LP infants.
With regard to mean NO/eNOS ratio in the newborns, a substantial difference was noted between the SGA and AGA infants from both groups (Fig. 3). NO/eNOS ratio did not change with regard to gestational age, but this ratio was higher in the MP SGA infants (P < 0.05) and in the LP SGA (P < 0.05) newborns compared with the respective subgroups in each group. To determine the correlation between the NO/eNOS ratio and neuronal injury, we analyzed the day 1 and day 3 ratios of NO/eNOS in both study subgroups (a total of 80 cases) and found a significant positive correlation between NSE and the NO/eNOS ratio (Fig. 4).
Discussion Intrauterine growth restriction is a common health problem that can lead to obesity, arterial hypertension, cardiovascular diseases, impaired glucose tolerance and other serious metabolic consequences later in life.24–26 NO is important in the physiology and pathology of fetomaternal and neonatal blood circulation.27–29 The role of NO in the pathogenesis of intrauterine growth restriction and brain injury is unclear. The use of biochemical markers to assess brain injury in newborns with NO changes is controversial. Previous studies show that elevated NO expression was found to be related to significantly lower antioxidant activity, increased lipid peroxidation and impaired neuronal function in growth-restricted infants.30 NO supplementation was useful to © 2014 Japan Pediatric Society
increase uteroplacental circulation31 and decrease the levels of neurospecific injury markers in the cord blood of SGA infants.32 We determined the concentrations of endothelial activation and neuronal injury markers at 5–6 h after birth because a high probability of low systemic perfusion in preterm infants occurs in day 1 of extrauterine life,33 and on day 3 of life to assess the stabilization of autoregulatory mechanisms.34 In the present study, the SGA and AGA newborns had different and conflicting correlations between NO and NSE. With increased NO concentration, we found high NSE activity in the MP SGA infants and a low risk of neuronal injury in the LP AGA infants (Fig. 2). The interpretation of these results was possible through the parallel determination of NO and eNOS levels in the AGA and SGA subgroups. We investigated the balance of NO/eNOS and its effect on neuronal injury in preterm infants. An important finding is the significant difference between the NO/eNOS balance in the SGA and AGA infants, which reflects the severity of neuronal injury and provides a basis for understanding different NO and NSE connections in the study subgroups. We found that a significantly higher NO/eNOS ratio is the result of higher NO concentration and lower eNOS activity in SGA infants compared to AGA infants. It is possible that the activation of neuronal and inducible NOS sources in these newborns occurs as the result of long-term and severe intrauterine and birth distress. This is suggested by the high occurrence of severe birth asphyxia and severe HIE (Table 1) in the SGA newborns, which is consistent with an increased NSE concentration in these infants (Fig. 1). In contrast, in AGA infants (particularly in the LP group), the lowest risk for severe HIE (Table 1) was observed in conjunction with decreased NSE activation (Fig. 1). In the context of a high degree of eNOS activation in LP AGA infants, increased NO might be a compensative or defensive strategy for the preterm brain, in which a low NO/eNOS ratio is associated with a negative correlation between NO and NSE. In contrast, a high NO concentration without adequate eNOS activation causes a more serious neuronal injury: there is a positive correlation between NO and NSE in the MP SGA infants. The statistically significant positive correlation between NSE and NO/eNOS ratio (Fig. 4) corresponds to the aforementioned result. Despite the results of previous studies confirming the dependence of neuronal injury on brain maturity,15,16 we found no significant difference in NSE level in the MP and LP infants. Therefore, neuronal injury did not depend on the gestational age but on the activation of endothelial and non-endothelial sources of NO synthesis; additionally, the growth-restricted infants in both groups undergo more significant endothelial dysfunction and neuronal injury. The intrauterine growth of the SGA infants was characterized by more serious pathologic conditions leading to endothelial damage. We detected larger changes of endothelial nitric oxide synthesis and a higher probability of neuronal injury in the growth-restricted infants than in the newborns with normal growth. The present study had limitations. We were unable to measure the concentration of all types of NOS, which would help confirm the hypothesis; an experimental study determined that chronic hypoxia decreases eNOS in the hippocampus and increases
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Fig. 2 Correlation between nitric oxide (NO) and neuron-specific enolase (NSE) in (a,b) moderate preterm (MP) appropriate for gestational age (AGA), (c,d) MP small for gestational age (SGA), (e,f) late preterm (LP) AGA and (g,h) LP SGA infants. (a,c,e,g) Day 1 results; (b,d,f,h) day 3 results. (a) r = 0.29; (b) r = 0.38; (c) r = 0.70*; (d) r = 0.37; (e) r = –0.67*; (f) r = –0.38; (g) r = 0.46; (h) r = 0.07. *P < 0.05.
nNOS in the neuronal and glial cells of the thalamus.35 We found significantly lower eNOS activity in the context of increased NO concentration in the SGA infants, compared with the AGA subgroups, and hypothesize that the long-term and deeper hypoxic conditions result in the suppression of eNOS and activation of other sources of NOS activation; therefore, eNOS is suppressed when NO is high. To our knowledge, no study has been done on eNOS suppression in the context of high NO concentration. Wei et al., however, determined that the endothelial NO production by eNOS can decrease ischemic injury by inducing vasodilation, while increasing the NO production by neurons can cause neuronal injury.36 Consistent with this observation, Liu et al. have suggested a neuroprotective effect of nNOS inhibitors.37 The present results are consistent with these studies and confirm in vivo that intrauterine growth restriction results in the rise of NO in the context of non-endothelial sources of activation.
Fig. 3 Mean nitric oxide/endothelial nitric oxide synthase (NO/ eNOS) ratio in the moderate preterm (MP) and late preterm (LP) appropriate for gestational age (AGA) and small for gestational age (SGA) infants. *P < 0.05 vs AGA infants. © 2014 Japan Pediatric Society
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Fig. 4 Spearman’s rank-order correlation between nitric oxide/ endothelial nitric oxide synthase (NO/eNOS) ratio and neuronspecific enolase (NSE) concentration in the moderate and late preterm infants (n = 80) on (a) day 1 and (b) day 3. (a) r = 0.75*; (b) r = 0.64* (*P < 0.05).
Preterm birth and intrauterine growth restriction are characterized by long-term neurodevelopmental problems later in life.38–40There is currently no perfect method for predicting the risk of behavior, speech, cognitive and other insufficiencies resulting from preterm birth and growth retardation. The lack of sensitive perinatal markers for the early and late prediction of neurologic outcome and a wide spectrum of clinical and experimental studies justify the need for further investigation in this area. We propose that an impaired NO/eNOS ratio may be involved in the pathogenesis of brain damage and neonatal disadaptation in SGA infants. The present results form the basis of a hypothesis that the NO/eNOS balance can serve as a predictive marker for preterm infants and may be accepted as a sensitive marker for neuronal injury in newborns. The early assessment of the severity of an acute cerebral lesion in preterm infants may provide a useful tool in therapeutic decision making and may prevent neurologic complications in these infants.
Acknowledgments The authors sincerely thank Science Development Foundation under the President of the Azerbaijan Republic for providing nitric oxide, endothelial nitric oxide synthase and neuron-specific enolase reagent kits. We also thank the staff of the Clinical Biochemistry Laboratory of Azerbaijan Medical University for assistance with biomarker analysis. © 2014 Japan Pediatric Society
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32 Gazzolo D, Bruschettini M, Di Lorio R et al. l. Maternal nitric oxide supplementation decreases cord blood S100B in intrauterine growth-retarded fetuses. Clin. Chem. 2002; 4: 647–50. 33 McLean CW, Gayabyab RG, Noori S, Seri I. Cerebral circulation and hypotension in the premature infant: Diagnosis and treatment. In: Perlman JM (ed). Neurology Questions and Controversies. Elsevier Saunders, Philadelphia, 2008; 3–26. 34 Greisen G. Autoregulation of cerebral blood flow in newborn babies. Early Hum. Dev. 2005; 81: 423–8. 35 Dong Y, Yu Z, Sun Y et al. Chronic fetal hypoxia produces selective brain injury associated with altered nitric oxide synthases. Am. J. Obstet. Gynecol. 2011; 3: 16–28. 36 Wei G, Dawson VL, Zweier JL. Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia. Biochim. Biophys. Acta 1999; 20: 23–34. 37 Liu PK, Robertson CS, Valadka A. The association between neuronal nitric oxide synthase and neuronal sensitivity in the brain after brain injury. Ann. N. Y. Acad. Sci. 2002; 962: 226–41. 38 Klaric´ AŠ, Kolundžic´ Z, Galic´ S, Bošnjak VM. Language development in preschool children born after asymmetrical intrauterine growth retardation. Eur. J. Paediatr. Neurol. 2012; 16: 132–7. 39 Sung IK, Vohr B, Oh W. Growth and neurodevelopmental outcome of very low birth weight infants with intrauterine growth retardation: Comparison with control subjects matched by birth weight and gestational age. J. Pediatr. 1993; 123: 618–24. 40 Gratacos E. The problem of predicting neurological outcome in early-onset intrauterine growth restriction. Ultrasound Obstet. Gynecol. 2009; 33: 5–7.
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