Early predictors of outcome in infants treated with hypothermia for hypoxic ischaemic encephalopathy

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1 DEVELOPMENTAL MEDICINE & CHILD NEUROLOGY ORIGINAL ARTICLE Early predictors of outcome in infants treated with hypothermia for hypoxic ischaemic encephalopathy NAZAKAT MERCHANT 1,2 DENIS AZZOPARDI 1 1 Centre for the Developing Brain, Department of Perinatal Imaging, King s College London, St Thomas Hospital, London; 2 Department of Neonatology and Paediatrics, West Hertfordshire NHS Trust, London, UK. Correspondence to Denis Azzopardi at Department of Perinatal Imaging, Centre for the Developing Brain, King s College London, St Thomas Hospital, Westminster Bridge Road, London SE1 7EH, UK. denis.azzopardi@kcl.ac.uk PUBLICATION DATA Accepted for publication 12th September Published online ABBREVIATIONS aeeg Amplitude-integrated electroencephalography ADC Apparent diffusion coefficient AUC Area under the receiver operating characteristic curve CK-BB Creatine kinase brain band DWI Diffusion-weighted imaging GFAP Glial fibrillary acidic protein HIE Hypoxic ischaemic encephalopathy MRS Magnetic resonance spectroscopy NSE Neuron-specific enolase NICHD National Institute of Child Health and Development NPV Negative predictive value PPV Positive predictive value TNF Tumour necrosis factor UCHL1 Ubiquitin carboxyl-terminal hydrolase L1 Hypoxic ischaemic encephalopathy (HIE) is a leading cause of acquired neonatal brain injury. Assessment of the severity of cerebral injury and likely neurological outcome in infants with HIE is important for determining management and prognosis, for counselling parents, and for selection for neuroprotective trials. The condition of the infant at birth, the severity of HIE, neurophysiological tests, including amplitude-integrated electroencephalography (aeeg), biochemical markers, and neuroimaging have been used to assess prognosis and predict long-term outcome. The predictive accuracy of these indicators in the early postnatal period is modest. Neurophysiological assessment seems to be most helpful during the first 24 to 48 hours after birth whilst magnetic resonance imaging (MRI) seems most informative later. Several biochemical markers, including serum S100b and neuron-specific enolase (NSE), are also associated with HIE but their levels depend on the timing of sampling and their prognostic value is uncertain. Comprehensive neurophysiological assessment and neuroimaging may be limited to specialist centres. Therapeutic hypothermia is now standard care in infants with moderate to severe HIE so it is important to examine the influence of hypothermia on the assessment of prognosis in these infants. Hypoxic ischaemic encephalopathy (HIE) is a leading cause of acquired neonatal brain injury and may lead to long-term neurological sequelae or death. Assessment of the severity of cerebral injury, and the likely neurological outcomes, in infants with HIE is important for determining management and prognosis, for counselling parents, and for selection of infants for neuroprotective trials. As the clinical severity of HIE varies over time from birth, assessments are dependent on timing, and often have to be repeated. For assessing suitability for neuroprotective intervention, very early assessment, 3 to 6 hours after birth, is needed; however, the accuracy of assessments may be reduced the earlier they are performed. For some tests, for example the amplitude-integrated electroencephalography (aeeg), the false-positive rate is increased with very early assessment, whereas, if performed later, there may be an increase in the false-negative rate. The condition of the infant at birth, the severity of HIE, neurophysiological tests (including aeeg), biochemical markers, and neuroimaging have all been used for prognosis and to predict long-term outcomes. Predictive accuracy in the early postnatal period is modest. Of these, neurophysiological assessment seems most accurate during the first 24 to 48 hours after birth, whilst magnetic resonance imaging (MRI) seems most informative later. Several biochemical markers, including serum S100b and neuron-specific enolase (NSE), are also associated with HIE but depend on the timing of sampling, and their prognostic value is uncertain. Some of these tests, such as comprehensive neurophysiological assessment and neuroimaging, may be limited to specialist centres. 8 DOI: /dmcn The Authors. Journal compilation 2015 Mac Keith Press

2 Following positive results from randomized clinical trials, induced moderate hypothermia (core body temperature of 33.5 C), started within 6 hours of birth and continued for 72 hours, is now standard care for infants with moderate to severe HIE. 1 Because therapeutic hypothermia improves survival and reduces the rates of disability following HIE, it is possible that the prognostic values of predictors may be altered. Since the perceived prognosis greatly affects clinical management, it is important to examine the influence of hypothermia on the assessment of prognosis in infants with HIE. This paper describes predictors of outcome that may be useful during the first 7 days following perinatal asphyxia and HIE, and the influence of therapeutic hypothermia on these predictors. ASSESSING PROGNOSTIC INDICATORS Table I shows the measures used for assessing prognostic (and diagnostic) tests. Both sensitivity and specificity should be evaluated, since an ideal test would have a high sensitivity (true-positive rate) and a high specificity (true-negative rate). For continuous variables, the area under the receiver operating characteristic curve (AUC) is the best measure for determining the optimal level of the prognostic variable. Clinicians are particularly interested in the positive predictive value (PPV) and negative predictive value (NPV) of a test, as these indicate the rate at which the test correctly predicts outcome. A strong statistical association between a positive test result and an outcome of interest (such as survival or neurological outcomes) does not necessarily indicate a high prognostic accuracy of a test. For example, the severity of acidosis and the need for resuscitation at birth, although strongly associated with the occurrence of HIE and subsequent outcomes, are poor predictors in a population setting. The interpretation of reported data is hampered by the small size of most studies (the majority of which involve fewer than 50 cases), the lack of use of masked assessors, a high rate of loss to follow-up, and selection bias, particularly with retrospective analyses. PROGNOSTIC INDICATORS Response to resuscitation A number of clinical parameters reflecting the condition of a neonate at delivery are closely associated with subsequent outcomes but, individually, they have poor predictive accuracy. These include heart rate, Apgar score, the need for and duration of resuscitation, and the severity of acidaemia as determined by cord, early capillary, or arterial blood gas analysis. Overall, these variables have a low sensitivity and PPV but, when assessed soon after birth (e.g. 5min after birth for the Apgar score), they have a high specificity and NPV; the converse is true when they are assessed at a later time (e.g. 10min or more after birth for the Apgar score), 2 5 as shown in Table II. A combination of clinical variables is more predictive of subsequent outcome than individual analyses; for example, Apgar scores of 0 to 3, measured 5 minutes after birth, have greater prognostic value when combined with clinical presentations of peripartum complications, fetal acidaemia, and signs of neonatal encephalopathy. 4 In one study, logistic regression modelling of variables available up to 4 hours after birth identified the following three independent prognostic indicators: (1) administration of chest compressions 1 minute or more after birth; (2) onset of breathing more than 30 minutes after birth; and (3) a base deficit value of 16 or more in any blood gas measurement. The simple inclusion of these indicators improved the predictive power of variables with PPVs, increasing linearly with each additional prognostic indicator (Fig. 1). 6 Absence of all three of these prognostic indicators reduced the probability of severe adverse outcome by 20%. Information from randomized trials of cooling The entry criteria for randomized trials of cooling to reduce brain injury following asphyxia were chosen to identify infants with perinatal asphyxia and HIE with a high risk (>60%) of death or disabilities The criteria included a depressed Apgar score, prolonged resuscitation, and the occurrence of acidaemia, together with at least moderately severe HIE. The primary outcome measures of the trials were the rates of mortality and moderate or severe neurodevelopmental disabilities. Treatment with cooling resulted in a 15% reduction in the event rate, from 61% to 46%, so these can be considered PPVs of trial entry criteria (i.e. the combination of clinical signs of asphyxia at birth together with moderate/severe encephalopathy within 6h of birth) in infants treated with or without induced hypothermia. Table I: Important measures of a prognostic test Test outcome positive Test outcome negative Condition positive Condition negative True positive False positive Positive predictive value: TP/(TP+FP) False negative Sensitivity: TP/(TP+FN) True negative Specificity: TN/(FP+TN) Negative predictive value: TN/ (FN+TN) TP, true positive; FP, false positive; TN, true negative; FN, false negative. Table II: Odd ratios and prognostic measures for mortality in the first week after birth a Odds ratio (95% CI) Sensitivity (%) Heart rate <60 at 5min 16.5 ( ) Oxygen saturation 0.7 ( ) 0 98 <60 at 5min Apgar score 14.0 ( ) <4 at 5min HIE score ( ) a Adapted from Saugstad et al. 5 CI, confidence interval. Specificity (%) Early Predictors Following HIE Nazakat Merchant and Denis Azzopardi 9

3 scoring systems, based on the Sarnat score, are commonly used, such as a modified Sarnat encephalopathy score, 16 the Thompson score, 17 and others. 18 Problems with these scoring systems include difficulties in reliably measuring some clinical parameters soon after birth (e.g. assessing primitive reflexes, breathing, and seizures in sedated or paralysed ventilated infants) and the occurrence of intermediate signs that do not fit the defined classifications. The presence of moderate or severe encephalopathy, however graded or scored, has a strong association with an adverse clinical neurological outcome (e.g. odds ratios for death or disability of more than 20). 19 However, the level of HIE may change over the first few days after birth, and is affected by medication and biochemical abnormalities; therefore, the predictive accuracy of encephalopathy for subsequent neurological outcomes is variable. Figure 1: Stepwise prediction rule for severe adverse outcome. Error bars indicate 95% confidence intervals; horizontal line, observed adverse outcome rate of 68% for the entire cohort. Variables are administration of chest compressions >1 minute; onset of breathing >30 minutes; and base deficit value 16 in any blood gas analysis. 6 In a secondary analysis of data from the National Institute of Child Health and Development (NICHD) cooling trial, a scoring system derived from predictor variables, identified by logistic regression, correctly predicted death or disability in 78% of infants, and death in 80%, with no significant difference between the cooled and non-cooled groups. 12 The variables used in the scoring system were posture, activity, base deficit, and Apgar score at 5 minutes post partum. However, the performance of the scoring system has yet to be validated by a prospective study. In a subgroup analysis of cases from the same study, with Apgar scores recorded 10 minutes post partum, the rate of death and/or disability at 6 to 7 years of age was 75% in those with Apgar scores of 0 to 3 and 45% in children with scores of more than 3. The correlation between cooling and Apgar scores was not statistically significant, suggesting that the therapeutic effect of cooling is not dependent on the Apgar score recorded in the first few minutes after birth Clinical assessment of hypoxic ischaemic encephalopathy Although the definition of neonatal encephalopathy, including HIE, varies between studies, the key clinical features are abnormalities in posture, tone, alertness, and reflexes, and the occurrence of seizures. Several grading systems have been developed to allow systematic recording of the severity of encephalopathy that could be used for prognosis. The most widely quoted is the Sarnat score, which is based on changes in EEG characteristics in addition to standard clinical features. 15 Simpler grading or Influence of therapeutic hypothermia on the progression of hypoxic ischaemic encephalopathy and the subsequent neurological outcomes Studies carried out before the era of therapeutic cooling showed that the severity of HIE peaks around 24 hours after birth, and then, in surviving infants, resolves gradually over the subsequent few days. This pattern appears to be altered by therapeutic hypothermia. In the NICHD study, HIE improved steadily over the first 4 days in cooled infants, whilst improvements in HIE occurred later in non-cooled infants (Fig. 2). 13 A similar linear improvement in HIE score was reported by the UK TOBY cooling register of infants treated with cooling in the UK (Fig. 3). 20 These data suggest that hypothermia reduces or prevents worsening of encephalopathy in the 24 to 48 hours after birth, probably by reducing the development of secondary energy failure following resuscitation after asphyxia. In the NICHD study examining the predictive value of the stage of encephalopathy at different time points, the largest AUC for predicting death or disability was the grade of encephalopathy at 72 hours and at discharge (AUC 0.87). 13 In the Coolcap selective head cooling study, severe Figure 2: Evolution of encephalopathy in cooled and non-cooled infants HIE supplement 2015, 57 (Suppl. 3): 8 16

4 encephalopathy persisting 72 hours after birth was associated with death or severe disability, with these unfavourable outcomes being reported for 25 out of 28 infants (89%) in the cooled group, and 25 out of 25 infants (100%) in the non-cooled group. 21 In contrast, infants in the cooled group with moderate encephalopathy persisting 72 hours after birth had improved outcomes compared with infants in the non-cooled group, with favourable outcome rates of 69% (32/45) for infants in the cooled group, compared with 36% (12/36), for infants in the non-cooled group. 21 Amplitude-integrated EEG Cerebral function monitors are increasingly used to assess the occurrence and severity of neonatal encephalopathy. These devices display the time-compressed minimum and maximum peak-to-peak variability in the amplitude of the filtered EEG (aeeg), which appears as a band of activity moving slowly across the display screen. The lower edge of the band indicates the lowest peak-to-peak amplitude reached by the filtered EEG over a period of time, while the upper edge indicates the highest level. The width of the band indicates the variability in the EEG amplitude. In healthy neonates, the band fluctuates between 10 and 40lV, with the width changing regularly and correlating with phases of sleep and waking; the band becomes more variable during sleep, with alternating levels at the time of rapid eye movement and non-rapid eye movement. The appearance of the band of activity is related to the degree of continuity and activity of the EEG. With increasing discontinuity, as occurs in relation to prematurity, or in mild or moderate encephalopathy, the lower edge of the band falls below 5lV and the band appears wider (classified as moderately abnormal aeeg). With severe degrees of discontinuity, or suppression burst EEG, the band will be narrower and the upper edge will fall below 10lV (classified as severely abnormal aeeg). Bursts of higher voltage activity occur above the band of activity with a frequency and voltage that are dependent on the characteristics of the bursts in the EEG. Absent cerebral electrical activity results in an isoelectric EEG. The corresponding appearance of the aeeg is a straight line, or narrow band of activity, close to the baseline. Drift of the baseline may occur, especially in cases with isoelectric EEG. This is because the electrodes pick up cardiac activity. In clinical practice, these changes in aeeg following asphyxia are classified by (1) voltage criteria, namely normal, moderately abnormal, or severely abnormal, and (2) background patterns, namely continuous normal voltage, discontinuous normal voltage, burst suppression, continuous low voltage, or flat trace (Fig. 4). Many studies reporting the predictive value of the aeeg tend to group the criteria burst suppression, continuous low voltage, and flat trace as abnormal traces, and the normal continuous and discontinuous traces as normal or mildly abnormal traces. The two methods of Figure 3: Mean encephalopathy score during the first 4 days after birth compared with mean scores reported by Thompson et al. 17 Figure 4: Classification of amplitude-integrated EEG. classification seem to perform similarly for prognostication. Several studies carried out before the introduction of therapeutic hypothermia indicated that aeeg is an excellent early predictor of neurological outcomes following HIE. A recent systematic review reported that aeeg performed within 24 hours of birth has a sensitivity of 93% (95% confidence interval [CI] 85 97%) and specificity of 91% (95% CI 67 98%). Furthermore, individual studies report a positive predictive value of more than 80% for predicting death or disability in infants with HIE who were not cooled. 22,23 Combining the grade of clinically Early Predictors Following HIE Nazakat Merchant and Denis Azzopardi 11

5 assessed encephalopathy with the grade of aeeg abnormality further improves predictive accuracy. 16 Predictive value of aeeg in infants with HIE treated with therapeutic hypothermia Amplitude-integrated EEG recorded within 6 hours of birth. Some observational studies have shown that the predictive value of aeeg, recorded within 6 hours of birth, for adverse outcomes following HIE is approximately 25% lower in infants treated with cooling than that previously reported in the precooling era (Fig. 5). 24,25 In the TOBY randomized trial of cooling, the predictive value of aeeg within 6 hours of birth was lower in the cooled group than in the non-cooled group, but the PPV was lower in both groups than previously reported (PPV of 55% in the cooled group, compared with 63% in the non-cooled group). The best PPV was obtained when the low-voltage and flat trace patterns were combined to define the most severely abnormal patterns (PPV of 59% vs 71% for the cooled and non-cooled groups, respectively). 26 Evolution of the aeeg following asphyxia. The aeeg changes during the first few days following asphyxia depending on the duration and severity of the insult. The duration of aeeg abnormalities correlates with neurological outcome: in mild cases, there is a rapid recovery of aeeg within 6 to 12 hours, and the prognosis is excellent; in contrast, most infants with continuing severe suppression of aeeg, beyond 24 hours (low-voltage/flat trace patterns), ultimately have a very poor prognosis, with a high rate of death or severe neurodevelopmental abnormalities. Infants with prolonged intermediate abnormalities (moderately abnormal voltage or burst suppression patterns) have a more variable outcome. Treatment with hypothermia Figure 5: Positive predictive value of an abnormal amplitude integrated EEG (burst suppression, low voltage or flat trace) to predict poor outcome (death/disability) in hypothermia-treated or normothermia treated infants. 25 may alter the prognostic value of aeeg within 72 hours of birth: recovery occurred in approximately 50% of cooled infants with an abnormal aeeg (burst suppression or worse trace), persisting for 24 to 48 hours, whilst almost all infants with continuing abnormal aeeg at 48 hours had a poor outcome, despite cooling. 25,27 Electrographic and clinical seizures commonly occur in infants with moderate or severe HIE, and these probably contribute to neurological injury. 28,29 Hypothermia may lessen the frequency of seizures, but infants with continuous or very repetitive seizures usually have a severely suppressed interictal background EEG/aEEG and a poor prognosis, despite cooling. Neuroimaging: cranial ultrasound Cranial ultrasound scanning is routinely used in neonatal care. It has the advantage of safety and availability at the bedside, and can be done repeatedly; however, it is examiner dependent and there is poor interobserver agreement on its general use. 30 Normal cranial ultrasound findings can be reassuring, whereas abnormalities in the thalamus and basal ganglia, or evidence of cerebral oedema, are associated with abnormalities on MRI and abnormal neurodevelopmental outcomes. However, predictive accuracy is poor, with a high false-positive rate (Table III). 23 The combination of abnormal cranial ultrasound and neurological examination may improve prediction of neurological outcomes. 31 There are no data suggesting that hypothermia alters the interpretation of cranial ultrasonography. Doppler cerebral flow velocity indices are commonly used as markers of cerebral perfusion. The cerebral Pourcelot resistance index is calculated by dividing the difference between the peak systolic and diastolic flow velocities by the peak systolic flow velocity. In one study of 40 healthy term-born infants, the resistance index, averaged from measurements obtained from the main cerebral arteries, was 0.726, with a standard deviation of 0.057, within 24 hours of birth, 32 whilst a resistance index of 0.55 or less, between 24 and 72 hours after birth, had a high predictive value for abnormal outcome following perinatal asphyxia. 33 The PPV of the resistance index was only 60% (95% CI 45 74%) in infants treated for HIE with hypothermia, which is considerably less than the value previously reported for normothermic infants. 34 The NPV of the cerebral resistance index in the cooled infants was 78% (95% CI 67 86%), similar to that reported in previous studies of non-cooled infants with HIE. 34 Neuroimaging: magnetic resonance imaging Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) have been used in infants with perinatal asphyxia for more than 30 years. Initially primarily a research tool, MRI is now recognized as the optimal technique for imaging structural changes following cerebral insults, to support a diagnosis of hypoxic ischaemic injury, and for prognosis. Early MRS studies helped to characterize the cerebral biochemical and metabolic changes that 12 HIE supplement 2015, 57 (Suppl. 3): 8 16

6 Table III: Pooled sensitivities and specificities with confidence intervals for tests a Pooled sensitivity Pooled specificity Test No of studies No of patients Point estimate 95% CI Point estimate 95% CI aeeg within first 6h aeeg within first 24h aeeg within first 72h EEG within first 72h MRI DWI within first week ADC within first week T1/T2 MRI within first week T1/T2 MRI within first 2wk T1/T2 MRI within first 6wk MRS within first week MRS within first 2wk Cranial ultrasound VEP SEP first wk a Adapted from van Laerhoven et al. 23 CI, confidence interval; aeeg, amplitude-integrated EEG; MRI, magnetic resonance imaging; DWI, diffusion-weighted imaging; ADC, apparent diffusion coefficient; MRS, magnetic resonance spectroscopy; VEP, visual evoked potentials; SEP, somatosensory evoked potentials. occur following asphyxia, which led to the concept of delayed energy failure, often termed secondary energy failure, occurring several hours after reperfusion, offering a window of opportunity for neuroprotective interventions. Magnetic resonance imaging MRI has become the preferred neuroimaging method following HIE because of its sensitivity for detecting hypoxic ischaemic injury. Following moderate or severe HIE, abnormal signal intensity is most commonly detected in the basal ganglia and thalami, corticospinal tracts, the white matter, and cortex (Fig. 6). 35 These abnormalities closely correlate with pathology at autopsy and with the pattern of neurological abnormalities that develop in survivors, and have high predictive values for detecting adverse outcomes. Several studies have examined the prognostic accuracy of conventional T1- and T2-weighted MRI (T1/T2 MRI) following HIE. The timing of scanning is important since the characteristic abnormalities detected by conventional MRI occur progressively over several days, and the severity of injury may be underestimated during the first few days after birth. 36 Diffusion-weighted imaging (DWI) refers to MRI that is sensitive to the molecular diffusion of water. DWI can provide information about tissue microstructure. DWI will typically show changes following ischaemia earlier than conventional MRI, but may underestimate the extent of tissue injury during the first 24 hours following birth. Although DWI is complementary to conventional MRI, visual analysis of DWI does not seem to improve the prognostic accuracy of MRI in infants with HIE during the first week after birth: in a recent systematic review of the pooled sensitivity, prediction of outcome following HIE was 58% (95% CI 24 84%) for DWI and 84% (95% CI 27 99%) for T1/T2 MRI, whilst the specificity of the two techniques was similar (Table III). 23 A: Normal at term B: Loss of signal in posterior limb of internal capsule and abnormal signal in basal ganglia and thalamus (arrow) Figure 6: Magnetic resonance imaging changes following perinatal asphyxial encephalopathy. Quantitative MRI The degree of attenuation of the DWI signal is a measure of the amount of diffusion that has occurred. From this information, the diffusion coefficient can be calculated. However, since diffusion is not free but is hindered by many factors, in practice the apparent diffusion coefficient (ADC) is commonly used. In infants with HIE, ADC values may be reduced in white matter and, less consistently, in basal ganglia and thalami, but are influenced by the age of the infant at scanning. ADC values are typically reduced during the first week, but then return to normal and rise further after about 2 weeks; therefore, the postnatal age at scanning needs to be taken into account when assessing ADC values. In a meta-analysis, the sensitivity and specificity for prediction of neurological outcome by measurement of ADC during the first week were 79% (95% CI 50 93%) and 85% (95% CI 75 91%), respectively. 23 The directional diffusivity within a tissue is called anisotropy, and can be measured by diffusion tensor imaging. Fractional anisotropy may be a more reproducible quantitative measure of tissue microstructure than the ADC, but few Early Predictors Following HIE Nazakat Merchant and Denis Azzopardi 13

7 studies to assess this have been carried out in infants, and rarely during the first few days after birth. The development of tract-based spatial statistics has enabled observer-independent delineation of whole brain white matter, and group-wise analysis of fractional anisotropy. With this technique, fractional anisotropy following asphyxia has been observed to be lower than in with comparison infants, and to be lower in infants with HIE treated with hypothermia than in normothermic infants. 37 Changes in fractional anisotropy are significantly associated with subsequent neurodevelopmental outcomes, and there is a linear relationship between fractional anisotropy values and the mental development index of the Bayley Scales at 18 months. 38 Magnetic resonance spectroscopy MRS enables in vivo quantitative analysis of cerebral metabolites. For more than two decades, phosphorus-31 ( 31 P) and proton ( 1 H) MRS have been used to investigate cerebral metabolism following perinatal asphyxia. The initial pioneering studies using 31 P-MRS defined the biphasic pattern of impairment of cerebral energy metabolism in infants with HIE: impairment of cerebral metabolism occurred following a delay of several hours after resuscitation in moderately or severely encephalopathic infants. Subsequent clinical studies have used 1 H-MRS because the greater sensitivity of the 1 H nucleus allows more accurate regional measurements. Within the 1 H spectrum, peaks can be assigned, using appropriate scanning parameters, to N-acetyl aspartate, choline-containing compounds, creatine plus phosphocreatinine, lactate, myoinositol, alanine, glutamine, and glutamate. In agreement with findings of the cerebral metabolic disturbances that occur with asphyxia, characteristic changes in these spectral peaks are observed in infants with HIE, especially the elevation of lactate, which indicates tissue ischaemia and hypoxia, and a fall in N-acetyl aspartate, which is present primarily in neurons and reflects neuronal injury. Concentration or peak area ratios are commonly reported, but MRS is reported to have a low specificity (approximately 58%) during the first week after birth. The absolute concentration of N-acetyl aspartate was the best predictive MRS marker in one study. 39 Influence of hypothermia on MRI/MRS In two substudies of the randomized trials of therapeutic hypothermia in infants with HIE, the predictive accuracy of T1/T2 MRI in the cooled groups did not differ from that of the non-cooled groups, suggesting that MRI can be used to assess prognosis even following treatment with hypothermia; however, the median ages of infants at scanning in these substudies were 6 and 8 days. 39,40 The effect of hypothermia on MRS has not been reported. Biochemical biomarkers A number of biochemical markers have been evaluated for perinatal asphyxia. A good biomarker is one that can be tested for easily, rapidly, and early in the course of the disease, would be elevated in proportion to the level of injury, and is capable of predicting long-term outcomes early. However, a recent meta-analysis found that, unfortunately, most of the biochemical markers which have been evaluated are not specific for HIE, correlate poorly with neurological outcomes after brain injury, and have been evaluated only in single studies with few long-term follow-up studies. In addition, the optimal time for measuring biochemical markers is not known, making it difficult to recommend such biomarkers for routine clinical use. 41 Serum, urine, and cerebrospinal fluid have been used to measure biomarkers of brain injury. Some of the biomarkers which have been evaluated are urine lactate, urine S100, cord blood interleukin (IL)-6, serum non-protein-bound iron, serum CD14, NFjB activation, creatine kinase brain band (CK-BB), S100b, serum IL-8, serum ionized calcium, cerebrospinal fluid NSE, cerebrospinal fluid or serum IL-1b, and serum IL-6. Some of these biomarkers are discussed in the following sections. Creatine kinase brain band, neuron-specific enolase, and S100b CK-BB is an isoenzyme of creatine kinase and is found in neurons and astrocytes. Protein S100b is an isotype of S100, a dimeric calcium-binding protein with bb subunits found primarily in astrocytes and Schwann cells. NSE is a dimeric isoenzyme of the glyocytic enzyme enolase found in the cytoplasm of neurons and cells with neuroendocrine differentiation. Although these biomarkers are apparently specific for neural tissue, they have not been shown to be consistently elevated in HIE or correlated with outcome. Nagdyman et al. 42 found that CK-BB and S100b were elevated in term infants with HIE, while NSE levels were not. None of the biomarkers correlated with death or disability at 20 months of age. In contrast, Gazzolo et al. 43 found that urine S100b correlated with adverse outcomes at 12 months of age. Celtik et al. 44 also found that a rise in serum NSE did not correlate with neurological outcome. In contrast, Walsh et al. 45 found that cord CK-BB and serum CK-BB measured at 6 hours of age predicted seizures and neurological abnormalities at 7 months of age in 17 out of 22 infants (77%) with HIE. The evolution of serum NSE and S100b during the first 72 hours following birth has recently been reported in infants treated for HIE with hypothermia: NSE and S100b levels at baseline and at 72 hours strongly predicted death or neurological abnormalities, or MRI-detected brain injury, at 14 days of age. 46 Early measurements of NSE and S100b may be important biomarkers of brain injury following HIE, but information on long-term outcomes is lacking. Inflammatory cytokine markers Multiple organ dysfunction is commonly seen in HIE, and circulating pro-inflammatory cytokines are involved in the final common pathway of brain injury. Interleukins 1b, 6, 14 HIE supplement 2015, 57 (Suppl. 3): 8 16

8 and 8 and tumour necrosis factor (TNF) in cord, serum, and cerebrospinal fluid are associated with impaired cerebral oxidative metabolism and have been used to predict adverse outcomes following HIE. 47,48 Bartha et al. 49 showed that IL-1b, IL-6, IL-8, and TNF-a correlate with levels of lactate and choline in deep grey nuclei on MRS, but not with N-acetyl aspartate and choline. Infants with abnormal neurodevelopmental outcomes at 30 months of age had higher neonatal levels of IL-1b, IL-6, and IL-8, and lower levels of IL-12, but no change in IL-9, IL-13, or TNF. However, Savman et al. 48 did not find any correlation between IL-8 and adverse neurodevelopmental outcomes. Cytokine expression is reduced by hypothermia in experimental studies, but this has not been shown consistently in clinical studies. 50,51 Chalak et al. 50 found no difference in cord cytokine levels in the mild versus moderate to severe HIE groups, and also no difference in the cytokine levels during hypothermia and rewarming. However, in a subset of infants participating in a randomized controlled trial of hypothermia, Roka et al. 51 found that hypothermia was associated with a reduction in IL-6 and IL-4 levels. In the Chalak et al. 50 study, the cytokines IL-1, IL-6, and IL-8 were predictive of adverse neurodevelopmental outcomes following therapeutic hypothermia. Novel biomarkers of brain injury Novel biomarkers, possibly with better specificity for predicting brain injury, have recently been evaluated, along with the effects of therapeutic hypothermia. Glial fibrillary acidic protein (GFAP) is a cytoskeletal intermediary filament protein found in astrocytes. Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a neuron-specific cytoplasmic enzyme present in dendrites and neuronal cytoplasm. Phosphorylated axonal neurofilament heavy chain is a neurofilament protein found specifically in neurons as the major cytoskeletal element in nerve axons and dendrites. These three biomarkers are involved in the pathogenesis of traumatic brain injury, and have also been shown to reflect the severity of neuronal injury. 52,53 Cord GFAP and UCHL1 levels correlate with the severity of HIE; however, only GFAP levels remained significantly higher in moderate to severe HIE at later time intervals, in contrast to UCHL1, which did not show any difference in relation to severity by 6 to 24 hours. 50 In infants who were treated with induced hypothermia, there was no significant difference in GFAP and UCLH1 levels during hypothermia and rewarming. In the Chalak et al. 50 study of infants treated with hypothermia, GFAP values of more than 0.08pg/ml correctly identified all infants with abnormal outcomes at 20 months. Similarly, Massaro et al. 46 found higher UCHL1 levels initially and following 72 hours of cooling, and higher GFAP levels at 24 and 72 hours of age, in infants with adverse outcomes than in those with favourable outcomes. CONCLUSION Perinatal asphyxia, leading to moderate or severe HIE, causes several pathophysiological disturbances that are related to the severity of encephalopathy and subsequent neurodevelopmental outcomes. Some of these processes are attenuated by moderate hypothermia. The wide variety and variable evolution of these responses after asphyxia complicate their clinical utility as markers of disease and responses to therapy. Early prediction of outcome following HIE continues to depend primarily on repeated neurological evaluation, supported by neurophysiological tests and cerebral imaging. 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