Efficacy of different treatment times of mild cerebral hypothermia on oxidative factors and neuroprotective effects in neonatal patients with moderate/severe hypoxic-ischemic encephalopathy.

OBJECTIVE: To investigate the efficacy of different treatment times of mild cerebral hypothermia for treating moderate/severe hypoxic-ischemic encephalopathy (HIE) in neonatal patients and its effects on oxidative factors.
METHODS: This prospective, randomized, controlled study included 92 neonatal patients with moderate/severe HIE and 30 controls. The patients with HIE received routine treatment, 48 hours of hypothermia, or 72 hours of hypothermia.
RESULTS: Superoxide dismutase (SOD) values were significantly lower and malondialdehyde (MDA) and neuron-specific enolase (NSE) values were higher in patients with HIE than in controls before the study. After 24, 48, and 72 hours of treatment, SOD values in all patients with HIE gradually increased and MDA and NSE values gradually decreased. At 3, 7, and 10 days, the Neonatal Behavioral Neurological Assessment scores were highest in the mild hypothermia for 72 hours group than in the other groups. The Mental and Psychomotor Development Indices scores of the Bayley Scales were significantly higher in the mild hypothermia for 72 hours group than in the other groups.
CONCLUSION: Hypothermia treatment of 72 hours is better than 48 hours for improving oxidative conditions, reducing NSE values, and improving neurological behavior and development for neonates with moderate/severe HIE.

RCT Entities:   Population Interventions Outcomes

Introduction

Hypoxic–ischemic encephalopathy (HIE) is a type of craniocerebral injury induced by perinatal hypoxia, including pathogens of fetal distress in the uterus, pregnancy-induced hypertension syndrome, eclampsia, and severe anemia.[1],[2] Studies have shown that the incidence of perinatal asphyxia can be as high as 3% to 5%,[3] and the incidence of HIE is estimated as 0.15% in developed countries and 0.23% to 2.65% in developing countries.[4],[5] Moreover, HIE-induced neural system injury accounts for 25% to 28% of infants’ neurological impairment.[6]

The mechanisms of HIE are still unclear. Generally, the mechanisms of HIE mainly include reperfusion injury, calcium overload, release of inflammatory factors, and dysfunction of the blood–brain barrier.[7-9] Traditional treatment of HIE includes life-sustaining treatments, medication treatment, such as monosialotetrahexosylganglioside (GM1) and cerebrolysin, and mesenchymal stromal cell therapy.[10],[11] In recent decades, application of mild hypothermia in treatment of HIE has been reported.[12] Mild hypothermia can improve the neonates’ neural function and improve the patients’ prognosis.[13],[14] However, clinical evidence for mild hypothermia is still inadequate and how it protects neonates against neural injury remains unclear.

The present randomized, controlled study aimed to investigate the efficacy of different treatment times of mild cerebral hypothermia in treatment of moderate/severe HIE in neonatal patients and its effects on oxidative factors. Our findings will add to the clinical evidence and provide new insight into mild hypothermia in treatment for HIE.

Methods and materials

Patients

The present prospective, randomized controlled study included neonatal patients who were diagnosed with moderate/severe HIE during January 2017 to April 2019. The diagnosis of moderate/severe HIE was according to the diagnostic criteria for HIE of the Society of Pediatrics, Chinese Medical Association in 2005. The inclusion criteria were as follows: 1) neonates who were within 6 hours of birth; 2) gestational age ≥37 weeks and weight ≥2500 g; 3) a 1-minute Apgar score ≤3 and 5-minute Apgar score ≤5, an umbilical artery pH <7.0 or base excess ≤−16 mmol/L, or resuscitation or mechanical ventilation was continued for 5 minutes after birth; 4) symptoms of HIE were observed within 6 hours after birth, such as convulsions, coma, abnormal muscle tone and irregular breathing, and obvious abnormality of an electroencephalogram. Exclusion criteria included the following: 1) patients who were diagnosed with convulsions caused by electrolyte disorder, intracranial hemorrhage and birth injury, as well as brain injury caused by intrauterine infection, genetic and metabolic diseases, and other congenital diseases; 2) neonates with congenital malformation or congenital metabolic abnormality; and 3) neonates with intrauterine infection and high suspicion of prenatal and intrapartum infection.

The following formula was used for calculating sample size: . We used the neuron-specific enolase (NSE) level 24 hours after treatment as the main outcome. Treatment was considered effective when there was a decrease in NSE levels >3. The mean NSE level is 30.0 ± 5.0 after 24 hours of treatment according to clinical experience. Therefore, δ = 3, s = 5 α = 0.05, and β = 0.10. The minimal sample size was 30. Additionally, 30 healthy neonates who were born in our hospital during the same period were enrolled as controls. Written informed consent was obtained from all parents of the study. The present study was approved by The First People’s Hospital of Lianyungang Affiliated to Xuzhou Medical University in November 2016.

Treatment

All neonates with HIE were randomly divided into three groups using an SPSS software (version 20; IBM Corp., Armonk, NY, USA) generated number list as follows: 1) support treatment (n = 30); 2) hypothermia for 48 hours (hypothermia 48-hour group) (n = 29); and 3) hypothermia for 72 hours (hypothermia 72-hour group) (n = 33). All neonates received normal routine treatment, including maintaining normal blood pressure and arterial blood gases, maintaining acid–base balance and limiting fluid intake, controlling convulsions, reducing intracranial pressure and nutritional support, and respiratory and nutritional support if necessary. For producing mild hypothermia, a ZJL-2000 II hypothermia instrument (Changchun Antai Electronic Products Co., Ltd., Changchun, China) was used. A cooling cap was used to wrap around the neonate’s head and the temperature of the head skin was maintained at 28°C to 30°C, with the body surface skin temperature at 34.5°C ± 0.5°C and anal temperature at 35.5°C ± 0.5°C. The treatment lasted for 48 hours or 72 hours for the hypothermia 48-hour group and hypothermia 72-hour group, respectively. After treatment, the temperature recovered using far-infrared radiation with 0.5°C/hour. The temperature recovered to normal within 6 hours. The support treatment group only received routine treatment as described above.

Measurement of serum factors

Blood samples of all patients with HIE were collected before treatment, as well as at 24 hours, 48 hours, 72 hours, and 7 days after treatment. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured using MDA and SOD kits (Nanjing Jiancheng Bio-Technology Co., Ltd., Nanjing, China), respectively. NSE levels were determined using an ELISA kit (Abcam, Cambridge, MA, USA). For controls, blood samples were collected at the same time points as the other groups.

Data collection

Basic clinical characteristics of all neonates were recorded, including sex, body weight, gestational age, and delivery mode. Neonatal Behavioral Neurological Assessment (NBNA) scores were used for measuring the neonates’ neurological behavior at 3, 7, and 10 days after treatment. The Mental (MDI) and Psychomotor Development Indices (PDI) scores of the Bayley Scales were recorded at 8 months after birth. Complications within 7 days after treatment were also recorded.

Statistical analysis

Continuous data are expressed by mean ± standard deviation. The chi-square test was used to compare count data and rates. Comparisons among three or more groups were conducted using ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered as statistically different. All calculations were performed using SPSS version 20.0 (IBM Corp.).

Results

Basic characteristics of all neonates

The present study enrolled 92 neonates with mild/moderate HIE. Among the patients, the mean gestational age was 38.98 ± 2.69 weeks, the male: female ratio was 49: 43, mean body weight was 3.45 ± 0.62 kg, and mean age of beginning treatment of neonates was 3.36 ± 1.65 hours. No significant differences in basic characteristics were found among the different groups of neonates (Table 1). During the study period, no patient was removed from the study or was lost to follow-up.

Table 1.

Basic characteristics of all neonates.

Variables	Support treatment, n = 30	Mild hypothermia for 48 hours, n = 29	Mild hypothermia for 72 hours, n = 33	Controls, n = 30	P value*
Gestational age, weeks	39.10 ± 2.56	39.06 ± 2.95	38.21 ± 2.53	39.63 ± 2.61	0.209
Age, hours	2.75 ± 1.59	3.70 ± 1.60	3.63 ± 1.78	3.30 ± 1.51	0.106
Sex, male : female	17 : 13	15 : 14	17 : 16	15 : 15
Body weight, g	3.41 ± 0.60	3.42 ± 0.64	3.31 ± 0.61	3.66 ± 0.59	0.148
Delivery mode, n (%)					0.918
Vaginal delivery	18 (60.00)	16 (55.17)	19 (57.57)	17 (56.67)
Cesarean section	12 (40.00)	13 (44.83)	14 (42.42)	13 (43.33)

Data are mean  ±  standard deviation, n, or n (%). *The chi-square test was used to compare count data and rates. Comparisons among three or more groups were conducted using ANOVA followed by Tukey’s post-hoc test.

Serum levels of SOD, MDA, and NSE in neonates with HIE treated by mild hypothermia for different times

To evaluate differences in different treatment times of mild hypothermia, serum levels of SOD, MDA, and NSE in the different groups were measured. There were no significant differences in serum SOD, MDA, and NSE levels among the HIE groups before the study (Figure 1). However, SOD values were significantly lower and MDA and NSE values were higher in patients with HIE than in controls before the study (all P < 0.05). After 24, 48, and 72 hours of treatment, SOD values in the HIE groups gradually increased and MDA and NSE values gradually decreased. At 24, 48, and 72 hours after treatment, the mild hypothermia 72-hour group showed the highest SOD value, as well as the lowest MDA and NSE values, compared with the mild hypothermia 48-hour group and the support treatment group (all P < 0.05). This trend was the opposite in the support treatment group. After 7 days of treatment, there were no significant differences in serum SOD, MDA, and NSE levels among the groups. These findings suggest that mild hypothermia treatment improves the oxidative condition and neural injury in patients with HIE, and the efficacy of treatment for 72 hours is better than treatment of 48 hours.

Figure 1.

Dynamic changes in serum levels of SOD, MDA, and NSE in neonates with hypoxic–ischemic encephalopathy who were treated with mild hypothermia for different times. NES: neuron-specific enolase; MDA: malondialdehyde; SOD: superoxide dismutase. *P<0.05 vs. support treatment (normal); #P<0.05 vs. mild hypothermia for 48 hours; &P<0.05 vs. mild hypothermia for 72 hours.

NBNA and Bayley scores in neonates with HIE who were treated by mild hypothermia for different treatment times

At 3, 7, and 10 days after treatment, NBNA scores were significantly higher in the mild hypothermia 72-hour group than in the other groups (all P < 0.05). Additionally, the support treatment group had significantly lower NBNA scores than those in the hypothermia groups and the control group (all P < 0.05) (Table 2). The MDI and PDI Bayley scores were significantly higher in the mild hypothermia 72-hour group than in the other groups (all P < 0.05). These scores were significantly lower in the support treatment group than in the hypothermia groups and the control group (P < 0.05). These results indicate that hypothermia treatment improves neurological behavior and is beneficial for neonates’ development, and that treatment of 72 hours of hypothermia has the best efficacy.

Table 2.

NBNA and Bayley scores in neonates with hypoxic–ischemic encephalopathy.

Variables	Support treatment, n = 30	Mild hypothermia for 48 hours, n = 29	Mild hypothermia for 72 hours, n = 33	Controls, n  =  30	P value*
NBNA score at 3 days	28.59 ± 1.45bcd	29.97 ± 1.52acd	31.55 ± 1.06abd	37.33 ± 1.02abc	<0.001
NBNA score at 7 days	28.93 ± 1.33bcd	30.07 ± 1.14acd	32.76 ± 1.00abd	37.17 ± 1.05abc	<0.001
NBNA score at 10 days	30.59 ± 1.11bcd	32.47 ± 1.16acd	33.94 ± 0.82abd	37.60 ± 1.22abc	<0.001
MDI score	82.00 ± 4.56bcd	86.80 ± 4.39acd	94.70 ± 3.60abd	103.93 ± 4.21abc	<0.001
PDI score	81.48 ± 3.72bcd	88.40 ± 5.40acd	96.45 ± 4.80abd	101.83 ± 4.68abc	<0.001

Data are mean  ±  standard deviation. *Comparisons among three or more groups were conducted using ANOVA followed by Tukey’s post-hoc test. aP<0.05 vs the support treatment group, bP<0.05 vs the mild hypothermia 48-hour group, cP<0.05 vs the mild hypothermia 72-hour group, dP<0.05 vs the control group. NBNA: Neonatal Behavioral Neurological Assessment; MDI: Mental Development Index; PDI: Psychomotor Development Index.

Complications and mortality of neonates with HIE treated by different treatment times of mild hypothermia

We compared complications and the mortality rate among patients with HIE. No significant differences were found in complications among the different groups (Table 3). During treatment, one patient died in the mild hypothermia 72-hour group, one died in the 48-hour group, and two died in the support treatment group.

Table 3.

Complications of neonates with hypoxic–ischemic encephalopathy.

Complications, n (%)	Support treatment, n = 30	Mild hypothermia for 48 hours, n = 29	Mild hypothermia for 72 hours, n = 33	P value*
Infection	2 (6.67)	2 (6.90)	1 (3.03)	0.406
Renal dysfunction	1 (3.33)	1 (3.45)	2 (6.06)	0.559
Electrolyte disorder	2 (6.67)	1 (3.45)	2 (6.06)	0.564
Thrombocytopenia	1 (3.33)	2 (6.90)	1 (3.03)	0.334
Impaired glucose	4 (13.33)	3 (10.34)	3 (8.33)	0.515

*The chi-square test was used for analysis.

Discussion

Moderate/severe HIE usually causes neurological sequelae and is one of the main causes of neonatal disability. Therefore, early diagnosis and treatment is important for improving infants’ neural function and protecting against neural injury.[15],[16] In recent years, mild hypothermia for treatment of neonates with HIE has been gradually widely used in the clinic. However, the mechanisms for mild hypothermia in neural protection are still unclear and clinical evidence remains inadequate. In the present study, we conducted two treatment strategies of different treatment times of mild hypothermia. We found that 72 hours of hypothermia was better than 48 hours of hypothermia for improving the oxidative condition and neural injury, as well as improving neurological behavior and development in neonates with moderate/severe HIE.

Application of mild hypothermia in treatment of patients with HIE has been reported in several studies. A recent large-scale study that included 1089 neonates with HIE showed that patients with HIE who received hypothermia treatment were more easily outborn and had lower odds of brain injury on magnetic resonance imaging compared with those who received standard care.[17] Another study showed that early mild hypothermia reduced interleukin-10 and interleukin-18 levels, as well as improved NBNA scores, in neonates with HIE.[18] However, a 2-year clinical study showed that the cognitive composite scores of children with mild HIE treated by therapeutic hypothermia were lower than those of a contemporaneous control group.[19] However, these scores might not have been different from those of survivors of moderate HIE treated with therapeutic hypothermia. In the present study, we found that mild hypothermia reduced NSE levels and enhanced the patients’ neurological behavior, as well as mid-term development. We also showed that 72 hours of treatment had better efficacy than 48 hours of treatment.

Changes in oxidative stress are considered as an important part of hypoxic–ischemic brain injury. A previous study showed a three to four-fold increase in oxidative protein carbonylation in the cortex, perirhinal cortex, and hippocampus of injured male rats with HIE, and increased glutathione peroxidase was found in female rats with HIE.[20] In another study, Kim et al.[21] reported that cerebral hypoxia–ischemia deactivated complex I. Curcumin can increase SOD levels and decrease MDA levels in hypoxic–ischemic brain injury in neonatal rats.[22] NSE levels are also an important factor in neurological function. A previous study showed that S100B and NSE levels were significantly higher in asphyxiated infants and treatment of hypothermia might reduce these levels.[23] However, few studies have focused on the effects of mild hypothermia on oxidative stress in patients with HIE. In this study, we showed that mild hypothermia decreased MDA and NSE levels and increased SOD levels in patients with HIE. This might be one of the molecular mechanisms for mild hypothermia treatment.

The present study has some limitations. First, this was a single-center study with a limited number of cases. Second, the long-term efficacy of the neonates’ neural function and development was not investigated. Further studies are still required to provide deeper insight into neonatal treatment with hypothermia for HIE.

Conclusion

We conducted a randomized, controlled study to investigate the efficacy of mild cerebral hypothermia on oxidative factors and neuroprotective effects in neonatal patients with moderate/severe HIE. Our study shows that mild hypothermia reduces oxidative stress and NSE levels, and improves neurological behavior and development in neonates with moderate/severe HIE. These results provide more clinical evidence and new insight for applications of mild hypothermia in treatment of HIE.