PEDIATRICS Vol. 106 No. 6 December 2000, pp. 1447-1451

Role of Carbon Monoxide and Nitric Oxide in Newborn Infants With Postasphyxial Hypoxic-Ischemic Encephalopathy

Yuan Shi, MD, Feng Pan, MSc, Huaqiang Li, MD, Jie Pan, BSc, Shiwen Qin, MD, and Chikao Shen, MD

From the Department of Pediatrics, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China.

	ABSTRACT

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Objective.  To investigate the role of carbon monoxide (CO) and nitric oxide (NO) in the pathogenesis of neonatal hypoxic-ischemic encephalopathy (HIE).

Methods.  Plasma CO and NO levels were studied in 33 asphyxiated term neonates, and 30 healthy neonates served as controls.

Results.  Among the 33 asphyxiated term neonates, plasma CO and NO levels in 28 neonates with HIE were significantly higher than those in the 5 infants without HIE and in the normal controls. The plasma CO and NO levels in the newborn infants with HIE stage 3 were found to be significantly higher than those in the neonates with HIE stage 1 and 2. Moreover, plasma CO and NO levels were significantly increased in neonates with brain damage and adverse outcome as compared with those in patients with normal neuroimaging and normal outcome.

Conclusion.  Plasma CO and NO levels after perinatal asphyxia are related to the severity of neonatal HIE, brain damage, and neurologic outcome. The present study suggests that CO and NO might play important roles in the pathogenesis of neonatal HIE.  Key words:  heme oxygenase, cerebral ischemia, cerebral anoxia.

Several lines of evidence indicate that a few endogenously produced mediators are involved in the pathogenesis of hypoxic-ischemic brain injury. Experimental studies suggest that a cytokine network orchestrates in the hypoxic-ischemic brain damage. There are a few reports about the significant changes of interleukin-1, interleukin-6, and tumor necrosis factor after brain hypoxia-ischemia.^1-3

Recently, biomedical interest in endogenously produced carbon monoxide (CO) and nitric oxide (NO) has grown rapidly. Both CO and NO are proposed to function as widespread transduction substances for the regulation of cell function and communication.⁴ Moreover, CO and NO are revealed to be neurotransmitters.⁵ The production of CO and NO is influenced by a variety of cytokines, including interleukin-1, interleukin-6, and tumor necrosis factor.^6-8 Although there is evidence that NO is involved in the pathogenesis of cerebral hypoxic-ischemic damage,⁹ few studies have examined the pathophysiology of CO in hypoxic-ischemic brain injury.¹⁰ Specifically, the roles of CO and NO in human newborn infants with hypoxic-ischemic encephalopathy (HIE) need additional study.

Despite the intense investigation that has been performed to understand the mechanisms of brain damage after perinatal asphyxia and to develop new therapeutic strategies, no data have been reported regarding CO and NO production and their clinical meanings in human neonates after perinatal asphyxia. Thus, we undertook this study 1) to investigate whether the plasma levels of CO and NO are affected by perinatal asphyxia and 2) to examine the relation of plasma CO and NO levels to the severity of HIE, brain damage, and neurologic outcome.

	MATERIALS AND METHODS

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Patients

The study population was comprised of asphyxiated term neonates admitted between 1996 and 1998 to the Neonatal Unit of Department of Pediatrics at the Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China. The neonates were identified to have experienced perinatal asphyxia when at least 3 of the following criteria were present: 1) fetal scalp blood pH <7.20, 2) umbilical artery blood pH<7.20 at birth, 3) Apgar scores <4 at 1 minute and/or <7 at 5 minutes after birth, 4) and requirement of >1 minute of positive pressure ventilation before sustained respiration occurred. The criteria for exclusion were: congenital malformations, metabolic disorders, congenital or acquired infections, maternal drug addiction, and absence of parental consent.¹¹

Complete obstetric histories were obtained and examinations were performed at the time of admission. The neonatal clinical course was followed prospectively and data were recorded on predetermined proform sheets. The informed consent was obtained from the parents.

Thirty randomly selected newborn infants who were born between 1996 and 1998 in the Department of Gynecology and Obstetrics, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China, served as the control group for plasma CO and NO measurements. The neurologic studies were not performed on the control group.

Clinical Assessment

A detailed and structured neurologic examination was performed at approximately 12, 36, and 72 hours of age, and then at 7 days of life.¹² The stage of encephalopathy was assessed according to a simplified Amiel-Tison and Ellison staging system.¹³ Stage 1 was diagnosed when hyperexcitability and/or hypotonia persisted for at least 72 hours after birth. Stage 2 was diagnosed in the presence of lethargy, hypotonia, and weak or partially absent primitive reflexes with or without seizures. Stage 3 was considered when there was coma or stupor in addition to severe tonus anomaly and frequent seizures.

Neurologic outcome was assessed in the patients at 3-month intervals by means of neurologic examinations according to the method of Amiel-Tison and Grenier and the Denver Developmental screen test.^14,15 Based on the outcome, the patients were classified as one of the following: 1) normal outcome; 2) mild motor impairment including slight abnormality in muscular tone or an abnormal pattern of motor development; or 3) adverse outcome when the patients developed cerebral palsy.

Neuroradiologic Examination

Cranial ultrasound examinations and computed tomography scans were performed in all the patients within 3 to 7 days of life. Unaware of the neonates' clinical status and CO and NO levels, the ultrasound scans were evaluated for size and configuration of ventricles, brain parenchymal echogenicity, and presence of cystic encephalomalacia. The results of the computed tomography scans were classified based on the extent of decreased attenuation in the brain parenchyma.¹⁶ According to the neuroimaging results, brain involvement was classified as one of the following: 1) normal; 2) mild changes revealed by isolated homogeneous echogenicity of brain parenchyma and virtual ventricles that normalized on serial imaging; or 3) brain damage indicated by severely abnormal echogenicity of brain parenchyma followed by parenchymal cysts or brain atrophy on serial imaging.

Sample Collection

Blood samples were obtained for analysis from the neonatal patients at the time of admittance to the neonatal unit. The samples were collected in heparinized tubes and centrifuged promptly at 3000 revolutions/minute for 15 minutes at 4°C to allow separation of the plasma. Then the samples were stored sealed on ice at 4°C for assay within 5 hours.

Assay for CO

CO concentration was measured using the simple, sensitive spectrophotometric method described by Chalmers.¹⁷ CO is trapped with hemoglobin (Hb) to form carboxyhemoglobin (COHb) and subsequently estimated by dithionite reduction. One mL of Hb solution (0.25 mL of fresh-packed erythrocytes in 50 mL of 0.24 mol/L ammonia solution) was mixed with 0.25 ~ 1.0 mL of a sample or an equivalent amount of water, which was used as a blank to measure the endogenous CO present in the Hb solution. Then 0.1 mL of 20% sodium dithionite solution was added to both the test sample and water-containing blank solution, vortex-mixed, and let stand 10 minutes. The absorbance at 541 and 555 nm against a reference curvette containing water was read and the ratio of the 541 to 555 readings was measured. Then the %COHb was calculated from a standard curve derived by mixing 100%HbO₂ and 100%COHb in different proportions: Then the CO concentration in x ml of the sample was given:

The detection limit of the assay for CO was 3.5 µmol/L. The concentration range of the standard curve was 3.5 to 500 µmol/L.

Assay for NO

Nitrite/nitrate (NO₂/NO₃) concentration has been confirmed to be a good indicator for NO production. The classic method described by Hegesh and Shiloah was used,¹⁸ and modified slightly in our laboratory.¹⁹ The measured samples were first deproteinated (30% ZnSO₄, 0.05 mL/mL of sample), then passed through a cadmium reduction column (100 × 8 mm) prewashed with HCl (0.1 mol/L) and ammonium hydroxide buffer (0.1 mol/L). After application of the sample, the column was eluted with ammonium chloride buffer (0.05 mol/L). A 5-mL fraction of the effluent was collected, and sulfanilamide solution (29.0 mmol/L) was added to it. The tube was mixed, and 3 minutes later, N-(l-naphthyl)-ethylenediamine dihydrochloride was added. Absorbance was measured after 20 minutes at 540 nm. Serial dilutions of sodium nitrite were used to prepare a standard curve. The detection limit of the assay for NO₂/NO₃ was 1.0 µmol/L. The concentration range of the standard curve was 1.0 to 500.0 µmol/L.

Statistical Analysis

The data are expressed as mean ± standard deviation, or median (range), for descriptive purpose. The Kruskall-Wallis test was used to analyze the differences among groups.

	RESULTS

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Thirty-three asphyxiated term neonates (19 boys, 14 girls) were studied. Five newborn infants had no HIE, 13 had HIE stage 1, 10 had stage 2, and 5 had stage 3. The main perinatal data of the sick neonates and the 30 healthy controls (17 boys, 13 girls) are shown in Table 1. No differences were found among groups regarding gestational age, birth weight, and type of delivery. Apgar scores at 1 and 5 minutes were significantly lower in the asphyxiated infants.

Plasma CO and NO Levels in HIE

As Table 2 shows, plasma CO and NO levels in the asphyxiated neonates with HIE were significantly increased as compared with those in the healthy infants and in the asphyxiated patients without HIE respectively, but there were no significant differences between the plasma CO and NO levels in the patients without HIE and in the normal controls.

Plasma CO and NO and Stage of Encephalopathy

Table 2 also shows that among the 28 neonatal patients with HIE, the infants with HIE stage 3 had significantly enhanced plasma CO levels as compared with those with HIE stage 1 and 2, although the plasma CO levels had no difference between stage 1 and 2. There were significant differences in the plasma NO levels among the 3 groups of patients with HIE stage 1, 2, and 3, which indicated that the more severe the condition of encephalopathy, the higher the plasma NO levels.

Plasma CO and NO Levels and Brain Damage

All 5 infants with no HIE and the 13 neonates with HIE stage 1 exhibited normal neuroradiologic examinations. Five of the 10 neonates with HIE stage 2 also showed normal brain conditions, but the other 5 neonates with HIE stage 2 revealed isolated homogeneous echogenicity of brain parenchyma and virtual ventricles. Brain damage was documented by neuroimaging techniques in all 5 neonates with HIE stage 3. As Table 3 indicates, significantly increased plasma CO and NO levels were found in the patients with brain damage.

Plasma CO and NO Levels and Outcome

Thirty neonates had a normal outcome, 2 had mild motor impairment, and 1 developed cerebral palsy. The 3 patients with mild motor impairment or with cerebral palsy all came from the group with HIE stage 3. The 1 patient with adverse outcome had the relatively highest CO and NO levels and showed atrophy of brain by computed tomography scans at 6 months of life. Table 4 shows the clinical information of the 5 patients with HIE stage 3. 

	DISCUSSION

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The results of this study provide the first evidence that the plasma CO and NO levels after perinatal asphyxia are related to the severity of neonatal HIE, brain damage, and neurologic outcome.

It is being increasingly appreciated that gas molecules such as NO function as new chemical messengers in the nervous system. Recent studies suggest that CO is another gas molecule that has similar biological actions as NO, which might be part of a complex cascade of mediators participating in the pathophysiology of the brain, although endogenous production of CO has been confirmed for over 30 years.²⁰ Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme to produce CO and bile pigments. Two forms of HO have been identified, including an inducible HO-1 and a constitutively expressed HO-2. The HO-1 isozyme is induced by a variety of agents such as stress reaction, heat shock, heme, and hydrogen peroxide.²¹ A portion of CO is present as COHb and a portion is dissolved in plasma which creates its physiologic and/or pathologic effect via the way of activation of guanylyl cyclase.^17,20 Both COHb values and CO levels in plasma have served as markers of CO production, but plasma CO levels might be more important and direct because CO, which freely exists in plasma, plays an important role. The endogenously produced CO has been suggested to play important roles in neuronal signaling and modulation of vascular tone. Additionally, CO is involved in some pathologic conditions such as endotoxic shock, hypoxia, and ischemia.^22-24

By in situ hybridization in brain slices, discrete neuronal localization of mRNA for HO-2 throughout the brain has been demonstrated. This localization is essentially the same as that for soluble guanylyl cyclase mRNA.²⁵ CO has also been implicated to take part in long-term potentiation, memory, and cognitive function.²⁶ Recently, a role in hypoxic-ischemic injury has also been demonstrated for CO. Evidence indicates that basal expression of HO-1 is elevated during hypoxia and endogenously generated CO is a physiologic modulator of the ventilatory response to hypoxia via its actions on carotid bodies and perhaps at brainstem neurons. In addition, CO might play a role in ventilatory adaptation to hypoxia.²⁷ In the brains of newborn rat pups exposed to 8% oxygen for 2.5 to 3 hours, and chronically hypoxic rat pups with congenital cardiac defects, HO immunostaining did not change after either acute or chronic hypoxia. Nevertheless, when 1-week old rats were subjected to right carotid coagulation and exposure to 8% oxygen/92% nitrogen for 2 hours, immunocytochemistry and Western blot analysis showed increased HO-1 staining in the ipsilateral cortex, hippocampus, and striatum at 12 to 24 hours up to 7 days after hypoxia-ischemia.²⁸ In a model of permanent middle cerebral artery occlusion in transgenic mice, expression of HO-1 has been suggested to be neuroprotective as determined by the stroke volumes and values of ischemic cerebral edema. The protective effects of pharmacologic stimulation of HO-1 activity may be partly through antioxidant action of HO-1.²⁹ Until now, there have been no studies about the changes of CO in hypoxic-ischemic injury in human beings. The present study showed that plasma CO levels were significantly increased in human newborn infants with HIE, and the highly increased plasma CO levels were related to the severity of neonatal HIE, brain damage, and neurologic outcome. What is the mechanism for CO involvement in neonatal HIE? First, HO-1 expressed by vascular smooth muscle cells and its product, CO, may regulate vascular tone under physiologic conditions, and when pathophysiologic hypoxia or/and ischemia conditions occur, HO-1 activity may be stimulated and CO production is markedly enhanced. The overproduced CO may change the cerebral vascular tone by regulating cGMP, which results in cerebral edema in the end. Second, HO-1 expressed by macrophages and its product, CO, as well as biliverdin, may have some protective effects by the way of antioxidant action, but overproduction of the substances may be toxic for the brain. Third, CO as a possible putative neural messenger may have some significant influence for the brain when its production is markedly changed after hypoxic-ischemic injury.

Until now, there have been some reports about the role of NO after cerebral hypoxic or/and ischemic injury, but a few problems still remain to be solved. Increase in NO in the brains of neonatal rats with hypoxic-ischemic damage has been found to have 2 peaks in the lesioned side of the cortex; one during hypoxia and the other during the reoxygenation.³⁰ NO contributed to irreversible membrane dysfunction caused by experimental ischemia in rat hippocampal CA1 neurons.³¹ NO has been revealed to regulate the cerebral blood flow response in hypoxia because inhibition of NO synthase increased cerebrovascular resistance and diminished the cerebral vasodilatory response during hypoxia or/and ischemia.³² The effect of posthypoxic-ischemic inhibition of NO synthesis on cerebral blood flow, metabolism, and electrocortical brain activity has also been studied in newborn lamb, which suggested that the immediate posthypoxic-ischemic blockade of NO might reduce brain damage.³³ Moreover, the posthypoxic-ischemic inhibition of NO synthase could diminish nonprotein-bound iron increment and preserve antioxidant capacity.³⁴ Nevertheless, NO production and activity were critical to the induction of ischemic tolerance in a neonatal rat model of hypoxic preconditioning,³⁵ and, if NO was inhibited after hypoxia-ischemia, pulmonary arterial pressure was elevated and oxygen need was increased.³⁶ Several lines of evidence have been indicated that the CO/HO system may function in concert with the NO/NO synthase system in the brain.³⁷ Expression of HO-1 in the human brain is induced by NO donors, whereas HO inhibitor suppresses NO production through a loss of L-arginine in rat cerebellar slices.³⁸ Our present study suggests that the elevations of NO in human newborn infants with HIE should be similar to the changes of CO, and that the increased NO also relates to the severity of the illness.

	CONCLUSION

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In summary, the present study indicates that CO and NO play some important roles in the pathophysiology of HIE. The assay of CO and NO levels may have some clinical values to estimate the severity and outcome of the illness. The regulation of HO and NO synthase in HIE needs additional investigation.

	FOOTNOTES

Received for publication Dec 9, 1999; accepted Apr 17, 2000.

Reprint requests to (Y.S.) Department of Pediatrics, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing 400042, China. E-mail: petshi{at}hotmail.com

	ABBREVIATIONS

CO, carbon monoxide; NO, nitric oxide; HIE, hypoxic-ischemic encephalopathy; COHb, carboxyhemoglobin; Hb, hemoglobin; NO₂/NO₃, nitrite/nitrate; HO, heme oxygenase.

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