Antioxidants in Hypoxic Ischemic Encephalopathy

Introduction: Perinatal asphyxia and related injuries are leading cause of morbidity and mortality in India. These babies are at risk to develop hypoxic ischemic encephalopathy (HIE) due to multiple organ including brain damage. The role of malondialdehyde (MDA) as a marker of free radical injury has been well established. Vitamin C and alpha Tocopherols have neutralizing effects on the free radicals. Considering these facts, it was decided to estimate serum MDA level in cases of HIE and the neutralizing effects of Vitamin C and Vitamin E. Material and Methods: The study was conducted in a tertiary care teaching hospital. Neonates with an Apgar score of <6 at 5 minutes and birth weight >2500g were included in the study. They were randomized into two groups. One group was given Vit E and vitamin C and the other was not given any of the anti oxidants. Serum MDA level were measured in both groups. Result: Serum MDA level was found to be increasing in both groups, but the increase was significantly higher in group II where antioxidants were not given. It was also found that serum MDA level was significantly low in antioxidants groups with HIE than those without antioxidants. Conclusion: Antioxidants supplementation in cases of HIE is associated with lesser production of free radicals and their neutralization is affected by antioxidants resulting into lesser damage to brain.


Introduction
P erinatal asphyxia is a signifi cant cause of neonatal mortality and morbidity.It is the brain which suff ers the most because it u lizes more oxygen and energy for its func on.The brain, therefore, is highly suscep ble to asphyxia and hypoxic ischemic changes, which is brought on due to defect in oxida ve phosphoryla on and ATP produc on.There is also abnormal accumula on of electrolytes in cell leading to forma on of reac ve oxygen free radicals.This free radical oxygen reacts with biological molecules causing neuronal apoptosis and necrosis 1 .
Hypoxic Ischemic Encephalopathy (HIE) resul ng due to perinatal asphyxia is characterized by necrosis of neurons of central nervous systems.Oxida ve stress plays a signifi cant role in the mechanism of hypoxic ischemic damage to neuronal ssues.Serum Malondialdehyde (MDA) which is the end product of lipid peroxida on is a marker of oxida ve stress 2,3 .An oxidants play a defensive role in protec ng ssues damage.Low level of an oxidants during oxida ve stress leads to forma on of free radicals 4,5 .
The aim of this study was to study eff ect of an oxidants in hypoxic ischemic encephalopathy(HIE) and the objec ves were to es mate serum MDA level (as marker of free radicals) in HIE and to study neutralisa on eff ects of an oxidants in cases of HIE compared with cases of HIE not exposed to an oxidants

Materials and Methods
The study was carried out to assess the role of an oxidants in cases of hypoxic ischemic encephalopathy.The most common and primary representa ve of dietary an oxidants are Vit C, Tocopherols, and Carotenoid.Vit C and Vit E are nutrients which have free radical neutralizing proper es 4 .This prospec ve case control study was conducted in paediatrics Department of a ter ary care rural teaching Hospital from January 2013 to December 2013.Term neonates with Apgar score of <6 at 1 minute and 5 minute a er birth and weighing ≥2500g were included in the study.Babies having any congenital defects, preterm, post term, small for gesta onal age (SGA) and Large for gesta onal age and evidence of early onset sepsis or any other non asphyxiated illnesses likely to aff ect the newborn, were excluded from the study.
A er ini al stabiliza on, babies were shi ed to NICU and were managed for hypoxic ischemic encephalopathy which also included oxygen inhala on.Three milliliters of peripheral venous blood was collected in a sterile plain vial within two hours of stabiliza on, at 24 hours of birth and on 5 th post natal day (at about 120 hours).Serum was separated from blood sample immediately by centrifuga on at 1300 rpm for 10 min and stored in separate deionised vials at -20 degree C. The serum was analyzed for MDA level by Thio-barbituric acid reac ve substances assay (T3RSA) method 6 .
The result were analysed by means of student's t test by epi info 7.

Results
Fi y two neonates were included in the study.They were randomly divided into two groups.There were 22 neonates in group I and 30 neonates in group II.Neonates in group I were given Vit E drops100 IU (Evion Drops, 50 IU /ml, MERCK, Made in Usagaon Ponta Goa, India) and Vit C 200 mg (Syrup Limcee 100 mg/ml, Piramal Health Care Pvt Ltd, made in Solan, HP, India) a er 2 hours of birth and Vit C 200 mg once daily orally for next 5 days.Babies in group II were managed for HIE without having been given any an oxidants.Serum MDA level was es mated at 2 hours of birth, at 24 hours and on 5 th postnatal day.
The mean MDA level within two hours of birth was 1.7347±0.38μmol/L in group I and 1.925± 0.53 μmol /L in group II.A rise in mean serum MDA was observed at 24 hours sample and again on 5 th post natal day in both groups (Table 1)

Discussion
Perinatal asphyxia is an important cause of fresh s llbirth and early neonatal death 7 .According to Na onal Neonatal Perinatal database of India, 23% of all neonates in our country suff er from asphyxia 7 .Mul organ involvement and neuronal necrosis is seen in HIE 8 .During early phase of brain injury due to HIE, a large cascade of events follows such as excitatory amino acid receptor over ac va on, impaired uptake of glutamate, increase in intracellular calcium concentra on, ac va on of NO synthetase (NOS), ac va on of protein and DNA damage 9,10 .
During the reperfusion period, free radical produc on increases.Free radical damage is further aggravated in neonates due to immature an oxidant defenses 11,12 .Free radical can lead to lipid peroxida on as well as DNA and protein damage and can trigger  13,14 .An oxidant level has been found to be low in HIE 15 .Frequent use of oxygen and ven la on has been proved to be life savings yet it is also proved that excess oxygen and defi ciency of an oxidant may lead to genera on of free radical and neuronal brain damage 4,16 .
Of the 22 neonates, fi ve neonates had an Apgar score of three, seven had four and ten neonates had Apgar score of 5 at 5 min.We compared serum MDA level as oxida ve stress marker at birth (a er 2 hours), at 24 hours and on 5 th postnatal day in both groups.There was no signifi cant diff erence in serum MDA levels at two hours in both groups Table-1.Nivedita studied the serum level of oxida ve stress markers and reported that serum MDA in cord blood was signifi cantly higher among cases than controls.MDA level was signifi cantly higher at 48 hours of life in cases than in control 3 .
There was progressive increase in level of serum MDA level in both groups at 24 hours age.Diff erence in increase was signifi cant (p <0.001).Serum MDA level on 5 th postnatal day was also found to be signifi cantly lower in group I when compared with group II (table II).The concentra on of MDA declined in interven onal group where an oxidants were used and this decline was signifi cant (p < 0.005).
Serum MDA level was also compared with stages of HIE in both groups.Three babies in group I and nine babies in group II died.The level was found to be progressively increasing as the severity of HIE increased though it was lower in group I babies than in group II.However, the diff erences were signifi cant.Manoj et al also found increasing serum MDA level from stage I to stage III of HIE in their study 10 .Kumar et al studied 43 neonates with perinatal asphyxia who subsequently developed HIE.They found mean plasma MDA level in control group as 0.59 ± 0.24 μmol/L whereas in HIE I it was 3.38 ±1.80 μml/L, 2.98 + 1.92 in stage II and 3.60 + 2.23 in HIE stage III.Plasma MDA level in infants with HIE was higher compared with control subjects.Although there was a progressive increment in plasma level of MDA with increasing severity of HIE the diff erences were not sta s cally signifi cant 17 .Saroj also found rising trend of serum MDA level with increasing severity of HIE 18 .Krimi et al studied 69 babies of HIE for serum MDA level and found signifi cantly higher level of serum MDA in Sarnat grade II and III than in Sarnat grade I.He also found signifi cantly higher level of serum MDA level in babies who died than those who survived 19 .However, Kumar did not fi nd any correla on of serum MDA level with severity of HIE 18 .
It is concluded that an oxidant ac vity is low among cases of HIE.An oxidant supplementa on in newborn with birth asphyxia results in decrease in serum MDA level reducing neuronal death of newborn.Use of an oxidant for e.g.Vit C and Vit E appears to be in order to reduce neonatal deaths and morbidity.Serum MDA level and mortality and morbidity in HIE cases can be safely predicted based on higher serum MDA level.A long term study is required to predict cut off level of serum MDA level in various stages of HIE.The limita on of our study is that it is a short term study and babies have not been followed up for long term mortality, morbidity and developmental delay, it will be unwise to comment on protec ve aspect of these an oxidants in case of birth asphyxia.

Conclusion
We concluded that the an oxidant supplementa on in the newborn with birth asphyxia results in decrease in the serum MDA levels which can be the bases for proposing early an oxidant supplementa on in the management of birth asphyxia.Therefore con nued use of an oxidants appears to be in order ll we have a short and long term clinical study to substan ate these facts or prove it otherwise.
Limita ons of the study are small sample size and short dura on of follow-up.As we do not followed the pa ents for long, we are not in posi on to comment on the fact wheather decrease in the serum MDA levels of the cases as result of an oxidant supplementa on does results in the changes in the morbidity and mortality associated with birth asphyxia.Further studies with similar objec ve, with longer follow-up dura on and larger sample size are proposed.

Table 1 :
Mean serum MDA level

Table 2 :
Mean serum MDA level in HIE on 5th post natal day