Published online August 25, 2008
PEDIATRICS Vol. 122 No. 3 September 2008, pp. e722-e727 (doi:10.1542/peds.2008-0269)
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ARTICLE

Free Radical Injury and Blood-Brain Barrier Permeability in Hypoxic-Ischemic Encephalopathy

Ashok Kumar, MDa, Roopali Mittal, MDb, Hari Dev Khanna, MDc and Sriparna Basu, MDa

a Division of Neonatology
b Department of Pediatrics
c Department of Biophysics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
OBJECTIVES. The purpose of this work was to evaluate the extent of free radical injury in newborns with hypoxic ischemic encephalopathy by measuring plasma levels of malondialdehyde and nitric oxide and to assess the blood-brain barrier permeability by measuring the cerebrospinal fluid albumin/plasma albumin ratio.

METHODS. This prospective observational study was conducted over a period of 2 years at Sir Sundarlal Hospital, Banaras Hindu University. The study population consisted of 43 term neonates with perinatal asphyxia who subsequently developed hypoxic ischemic encephalopathy. Twenty normal gestational age- and gender-matched healthy infants without any perinatal asphyxia served as control subjects. Peripheral venous blood samples were analyzed for malondialdehyde, total plasma nitrates/nitrites, and albumin levels between 12 and 24 hours of life. To assess the blood-brain barrier permeability, the cerebrospinal fluid albumin/plasma albumin ratio was measured. Correlation among the levels of malondialdehyde, nitrates/nitrites, and blood-brain barrier permeability was calculated. Data were analyzed by using SPSS 10 software.

RESULTS. Plasma malondialdehyde and nitrate/nitrite levels were significantly higher in infants with hypoxic ischemic encephalopathy compared with control subjects. Although there was a progressive increment in plasma levels of malondialdehyde with increasing severity of hypoxic ischemic encephalopathy, the differences were not statistically significant. Plasma nitrate/nitrite levels were almost similar in all stages of hypoxic ischemic encephalopathy. Plasma albumin levels were comparable in infants with hypoxic ischemic encephalopathy and control subjects, whereas cerebrospinal fluid albumin levels and blood-brain barrier permeability were significantly higher in infants with hypoxic ischemic encephalopathy. Significant correlation was observed between plasma malondialdehyde and nitrate/nitrite levels with blood-brain barrier permeability.

CONCLUSIONS. Increased plasma levels of malondialdehyde and nitrates/nitrites are found to be associated with hypoxic ischemic encephalopathy, indicating the possible role of free radical injury in its causation. Increased blood-brain barrier permeability may be another contributory factor to the progression of the disease.

Key Words: blood-brain barrier permeability • hypoxic ischemic encephalopathy • lipid peroxidation • malondialdehyde • newborn • nitric oxide • perinatal asphyxia

Abbreviations: HIE—hypoxic ischemic encephalopathy • NO—nitric oxide • BBB—blood-brain barrier • CSF—cerebrospinal fluid • SNK—Student-Newman-Keuls • NOS—nitric-oxide synthase • nNOS—neuronal nitric-oxide synthase • eNOS—endothelial nitric-oxide synthase • iNOS—inducible nitric-oxide synthase • VEGF—vascular endothelial growth factor

Perinatal asphyxia and the resultant hypoxic ischemic encephalopathy (HIE) remain a leading cause of morbidity and mortality in the perinatal period in Third-World countries. Biochemical events, such as energy failure, membrane depolarization, brain edema, production of oxygen-free radicals, and lipid peroxidation, may lead to brain dysfunction and neuronal death.1 Hypoxia followed by reoxygenation of tissue leads to production of various free radicals. These free radicals set up a chain of reactions that injure membranes by lipid peroxidation, inactivation of enzymes damage of DNA, and degradation of structural proteins.2 The polyunsaturated fatty acid of the biological cell membrane is extremely vulnerable to free radical–induced peroxidation.3 Malondialdehyde, a stable product formed out of lipid peroxidation, can be measured in various tissue fluids to assess the extent of free radical–induced damage.

Nitric oxide (NO) has been found to mediate a variety of functions at the cellular level. NO plays a complex role in free radical–mediated injury during cerebral reperfusion.4 Most of the animal research has pointed to the role of NO to determine the extent of HIE.5 Several human studies also found the role of NO in HIE.6, 7

Blood-brain barrier (BBB) protects the integrity and function of the brain by selectively regulating the entry and exit of biologically important substances. Disruption of the BBB results in increased vascular permeability, brain edema, and secondary brain damage. There have been conflicting reports regarding the damage to the integrity of the BBB in perinatal asphyxia. BBB breakdown or alterations in transport systems play an important role in the pathogenesis of hypoxic injuries.8 In stressed neonates, hypoxia has been found to damage the BBB integrity and increase BBB permeability.9

The present study was undertaken to evaluate free radical injury in HIE by measuring the plasma levels of malondialdehyde and NO in newborns with perinatal asphyxia who subsequently developed HIE and to assess the permeability of BBB by measuring the cerebrospinal fluid (CSF) albumin/plasma albumin ratio.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Participants
The study was conducted in Sir Sundarlal Hospital, Banaras Hindu University, over a period of 2 years. Study subjects were composed of 43 consecutively born term infants (gestational age ≥ 37 weeks) with perinatal asphyxia who subsequently developed HIE. All of the infants were resuscitated as per the guidelines of the Newborn Resuscitation Program of the American Academy of Pediatrics and American Heart Association.10 Perinatal asphyxia was defined as a requirement of positive pressure ventilation for >1 minute during postnatal resuscitation and an Apgar score ≤6 at 5 minutes. Because all of the infants were term, only 100% oxygen was used during resuscitation. Twenty normal gestational age- and gender-matched healthy infants without any perinatal asphyxia (who cried immediately after birth and did not require positive pressure ventilation for resuscitation) served as control subjects. Exclusion criteria of the study were a history of maternal drug addiction and maternal anesthesia/analgesia, which can cause neonatal depression, and the presence of congenital or acquired infections, metabolic disorders, and major congenital malformations in the infant. The study was approved by the hospital ethics committee, and informed consent was taken from the parents. The gestational age was assessed from the first day of the last menstrual period and confirmed by the modified Ballard Score.11 All of the newborns were followed up closely during the hospital stay for progression to HIE, which was categorized into different stages according to Fenichel classification (modified by Ellis and Costello).12 Fenichel classification provides a syndromic diagnosis of HIE. It uses 7 clinical criteria (consciousness level, tone, suck, primitive reflexes, seizures, brainstem reflexes, and respiration) to classify HIE in 3 different stages with increasing severity (stages I, II, and III).

All of the infants with perinatal asphyxia were admitted and managed according to our unit protocol. A detailed account of each infant, including history, clinical examination, and daily progress, was recorded serially in a predesigned proforma.

Collection of Samples
Peripheral venous blood of all of the infants (case and control subjects) was collected by disposable syringes in sterile, heparinized, deionized polyethylene vials between 12 and 24 hours of life. Plasma was separated from the blood samples immediately by centrifugation at 2000 rpm for 5 minutes and was stored in separate deionized vials at –20°C. Samples were analyzed for malondialdehyde, NO, and albumin levels.

CSF was collected by lumbar puncture using a sterile technique in sterile plain vials between 12 and 24 hours of life. CSF was examined, and those samples suggestive of meningitis were excluded from the study. CSF samples were stored at –20°C until they were analyzed for albumin.

Laboratory Analysis
Malondialdehyde levels were measured by the thiobarbituric acid assay.13 NO in the plasma was estimated indirectly by measuring the amount of nitrates and nitrites formed in the body from NO. NO is a highly unstable compound, which, within few seconds of its formation, gets converted to nitrates. The quantitative estimation of nitrates thus corresponds with the level of NO in plasma. Nitrates in the plasma are reduced to nitrites. The nitrites give a pink color with a Griess reagent, the absorbance of which was measured by an enzyme-linked immunosorbent assay reader at 520 nm.14 Plasma albumin levels were measured by Autospan commercial kit (Span Diagnostics Ltd, Surat, Gujarat, India), and CSF albumin levels were measured by CHEMA diagnostic kit (Chema Diagnostica, Jesi, Marche, Italy).

Statistical Analysis
The data were analyzed by using SPSS 10 (SPSS Inc, Chicago, IL) statistical software. The statistical analysis was performed by Student's t test and 1-way analysis of variance (F) to find out the significant difference between the groups. Student-Newman-Keuls (SNK) test was applied to test the significance of difference among the means of different groups. Correlation coefficients between different variables were also calculated.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Maternal details and anthropometric parameters were comparable between the study and control groups of infants. For statistical calculations, patients were divided in 4 groups. Group A consisted of the control subjects (n = 20); group B consisted of the patients of the study group who progressed to stage I of HIE (n = 14); group C consisted of the patients who progressed to stage II (n = 16); and group D consisted of the patients who progressed to stage III (n = 13). Table 1 compares the plasma levels of malondialdehyde (µmol/mL) and nitrates/nitrites (µmol/mL) in the study population with control subjects. Plasma malondialdehyde and nitrates/nitrites were significantly higher in infants with HIE compared with control group (P < .001). Although there was progressive increment of plasma levels of malondialdehyde with increasing severity of HIE, the intragroup differences were not statistically significant (P > .05) in the SNK test. Plasma nitrate/nitrite levels were almost similar in all of the stages of HIE. A significant correlation was observed between plasma malondialdehyde and plasma nitrate/nitrite levels (r = 0.307; P < .05).


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  		TABLE 1 Levels of Plasma Malondialdehyde and Nitrates/Nitrites

 
Plasma albumin levels (g/dL) were comparable in infants with HIE and control subjects, whereas CSF albumin (mg/dL) and the BBB permeability were significantly higher in infants with HIE (P < .001; Table 2). There was significant increase in CSF albumin levels and the BBB permeability as the stage of HIE increased. Significant intragroup differences were observed in the SNK test (group B versus C, P < .001; group B versus D, P < .05; and group C versus D, P < .001). Significant correlation was observed between the BBB permeability and plasma malondialdehyde (r = 0.480; P < .001) and between the BBB permeability and plasma nitrate/nitrite levels (r = 0.547; P < .001).


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  		TABLE 2 CSF Albumin, Plasma Albumin, and CSF Albumin/Plasma Albumin Ratio (BBB Permeability x 10–3)

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Hypoxia and ischemia during perinatal asphyxia are the major causes of brain injury in newborn infants.15 The reperfusion or reoxygenation in the immediate post-hypoxia-ischemia period is one of the initial factors responsible for brain injury in asphyxiated infants by increased production of NO.16, 17 In addition to NO, posthypoxia-ischemia reperfusion and reoxygenation can also lead to an increase in the production of superoxide and hydrogen peroxide in neonatal brain tissue and in the cerebral microcirculation.18 Although these substances are relatively poorly reactive free radical species themselves, superoxide and NO are able to form peroxynitrite, which can decompose to form the powerful and cytotoxic oxidants hydroxyl and nitrogen dioxide. These oxidants are highly diffusible and can easily cross the BBB to exert their destructive action on brain tissue.18, 19 Peroxynitrite itself is able to initiate lipid peroxidation and react directly with sulfhydryl groups at physiologic pH values, causing the breakdown of polyunsaturated fatty acid and inflicting brain injury.20

In the present study, significantly higher concentrations of plasma malondialdehyde were found to be associated with HIE, indicating increased lipid peroxidation in these infants. Progressively higher levels of malondialdehyde were noted with the progression of the disease. Increased lipid peroxidation after hypoxia and/or ischemia has been confirmed by other authors both in clinical observation20–22 and in experimental investigations.15, 23

In our study we found increased plasma nitrate/nitrite levels in newborns suffering from HIE between 12 and 24 hours of age compared with control subjects. Infants who developed HIE had significantly higher nitrate/nitrite levels compared with those who did not develop HIE. This finding highlights the importance of NO in the causation of tissue injury in HIE. Higher CSF and serum nitrate/nitrite levels in asphyxiated newborns have been documented in other studies as well.6, 7, 20, 23

A significant linear correlation between plasma malondialdehyde and nitrate/nitrite levels was noticed in the present study. This finding can be explained by the fact that a likely progressive increase in NO has enhanced the degree of lipid peroxidation, which has been reflected by the increased levels of malondialdehyde.

The role of NO in hypoxic ischemic injury is actually far more complex than conceived. In recent years, it has become increasingly clear that NO has a critical but complex role in the pathophysiology of cerebral ischemia. NO is increased during cerebral ischemia and reperfusion, but it has been observed that this can have differing effects in different models.24 Numerous studies have been performed to clarify the functional role of NO in cerebral ischemic injury. The results of these studies are complex and contradictory in some cases, and it has been observed that NO released in response to hypoxia-ischemia in the newborn brain may mediate both protective and pathologic responses.25–27 In acute hypoxia, excess NO may be harmful, because it causes vasodilatation and reperfusion injury, but in chronic hypoxia, augmentation of NO-dependent increases in cerebral blood flow may serve to improve neurologic outcome. In experimental rat models, NO is found responsible in periventricular white matter damage in response to hypoxia.5 On the other hand, Gardner et al28 have shown that fetal pre-exposure to a reversible period of adverse intrauterine conditions upregulates NO-dependent vasodilator mechanisms and markedly reduces the peripheral vasoconstrictor response to a subsequent episode of acute hypoxia, even when induced up to a week after the end of the period of adversity. NO can also act as a superoxide radical scavenger and may inhibit platelet aggregation and neutrophil adhesion.29

In the brain, there are 2 major isoforms of NO synthase (NOS) expressed constitutively, the neuronal isoform (nNOS) present in neurons and the endothelial isoform (eNOS) present primarily within the vascular endothelium. NO derived from nNOS in the ischemic neonatal brain is cytotoxic, contributing to neurodegeneration after hypoxic-ischemic injury because of its reaction with the superoxide radical with the formation of the potent oxidant peroxynitrite, but NO generated from eNOS within the vascular endothelium may be protective through induction of vasodilation, which, in turn, decreases the severity of ischemia.24 A third isoform, inducible NOS (iNOS), is synthesized after induction by stimuli such as endotoxin or proinflammatory cytokines.30 It has been suggested that this variable effect of NO in cerebral ischemia may be because of the amount of NO, the time course of NO production, the isoform or cellular location of NOS, or the redox state of NO or NO-derived species.24 Studies using NO donors demonstrated that, in the early stages after focal cerebral ischemia, the vascular actions of NO are beneficial in promoting collateral circulation and microvascular flow.31 It was also observed that glutamate-induced Ca2+ overload in ischemic neurons leads to persistent activation of nNOS, resulting in continuous NO production.32

The BBB is a physical and metabolic barrier that separates the peripheral circulation from the central nervous system and serves to regulate and protect the microenvironment of the brain.33 Structurally, the BBB is formed by tight junctions between a monolayer of microvessel endothelial cells. Astrocytes, pericytes, and perivascular microglia surround the endothelial cells contributing to proper functioning of the BBB. Disruption of BBB results in increased vascular permeability, brain edema, and secondary brain damage. The disruption of BBB in hypoxic conditions is multifactorial and may involve factors such as oxidative stress, enhanced production of vascular endothelial growth factor (VEGF), NO, and inflammatory cytokines. The BBB is highly susceptible to oxidative stress, and hydrogen peroxide is an important mediator of oxidative cell injury. Hypoxia/reoxygenation cause opening of the BBB and endothelial release of hydrogen peroxide, in turn, increases lipid peroxidation and accumulation of malondialdehyde. An increased malondialdehyde level causes destruction the tight junctions of the endothelial monolayer of BBB and increases its permeability.34 VEGF is an angiogenic mitogen characterized as an inducer of vascular leakage promoting the leakage of plasma proteins from blood vessels. VEGF has been shown to induce increased vascular permeability via synthesis or release of NO predominantly derived from eNOS and iNOS.35, 36

In this study we assessed the permeability of BBB by measuring the CSF albumin/plasma albumin ratio. Many of the previous studies have used similar methods to assess BBB integrity.37–41 We have found a significant increase in BBB permeability with progression of HIE, implying more severe disruption of BBB in severe asphyxia causing exacerbated neuronal damage. This finding was in accordance with the findings of Anagnostakis et al9 and van der Flier et al,41 who found increased BBB permeability in infants suffering from meningitis and asphyxia. The presence of a higher level of albumin in the CSF may also be indicative of altered CSF secretion by the choroid plexus. Sivakumar et al42 have shown increased expression of VEGF, nNOS, eNOS, and iNOS in the epithelial cells of the choroid plexus and altered CSF secretion in animal models of hypoxic injury. They demonstrated ultrastructural changes and increased vascular permeability in the choroids plexus correlated with the increase in NO production and VEGF concentration after a hypoxic exposure in the neonatal period.

There are a few limitations to the present study. First, we measured malondialdehyde as an indicator of lipid peroxidation. Estimation of F2-isoprostanes, compounds derived from the nonenzymatic oxidation of arachidonic acid, could have provided an accurate assessment of lipid peroxidation.43 This facility was not available with us. Second, NO in the serum was estimated only indirectly by measuring the amount of nitrates and nitrites. Moreover, we did not measure malondialdehyde and nitrate/nitrite levels in the CSF, which could have helped us to find the correlation between plasma and CSF levels. Lastly, we could not use any exogenous marker or immunocytochemical method for the assessment of BBB permeability.


   CONCLUSIONS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
In our study, increased plasma levels of malondialdehyde and nitrates/nitrites are found to be associated with HIE, indicating the possible role of free radical injury in its causation. Increased BBB permeability may be another contributory factor to the progression of the disease. Additional studies are necessary to evaluate the role of antioxidants to reduce free radical injury and to consider administration of an NO inhibitor to reduce neurologic injury after a hypoxic-ischemic insult.


   FOOTNOTES
 
Accepted Apr 29, 2008.

Address correspondence to Sriparna Basu, MD, Banaras Hindu University, Division of Neonatology, Department of Pediatrics, Institute of Medical Sciences, Varanasi 221005, India. E-mail: drsriparnabasu{at}rediffmail.com

The authors have indicated they have no financial relationships relevant to this article to disclose.


What's Known on This Subject

Free radical–mediated injury to brain and other tissues may account for the development of HIE and progression of the disease.

 

What This Study Adds

Increased BBB permeability is directly correlated with plasma malondialdehyde and nitrate/nitrite levels in HIE, indicating the possible role of free radical injury in its causation and progression of the disease.

 


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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