Neonatal jaundice is the most common clinical manifestation in the neonate period, which is mainly caused by the increase of serum bilirubin in neonates. In the prenatal period, due to the hypoxic environment, the number of red cells is increased and the hemoglobin content in the fetal is high. After birth, due to the hyperoxic environment, the survival time of red blood cells becomes short. The fetal hemoglobin is gradually converted to adult hemoglobin, producing a large amount of bilirubin, which exceeds the ability of the liver to treat bilirubin. When the bilirubin level is above 139 μmol/L, skin or organ xanthochromia may appear, which is called neonate jaundice. It has been previously proposed that if the concentration of bilirubin in the blood is above 342 μmol/L, serious damage to the nervous system of the neonates may occur, thus triggering the acute-stage manifestation of acute bilirubin encephalopathy (ABE) or chronic kernicterus and BIND [9,10]. Brain damage caused by neonatal hyperbilirubinemia (NHB) is not always reversible, but may lead to cerebral palsy and hearing loss. The most common types of nerve injuries are the sequelae of severe neurological dyskinesia and hearing impairment [2,11-13]. Acute bilirubin encephalopathy or kernicterus and BIND mainly affects subcortical regions, such as globus pallidus, hypothalamus, substantia nigra, cerebellar dentate nucleus, hippocampus, brainstem, etc [14,15]. However, the underlying mechanisms are still unknown [16,17]. Most of the ABE neonatal patients still have a chance to be recovered by timely treatment of reducing serum bilirubin levels, while only a few suffer from sequelae like kernicterus.
The feature of MRI manifestation to bilirubin encephalopathy is the high signal of globus pallidus in the acute stage of T1-weighted imaging (T1WI). With the development of the disease, it changes from the high signal in the acute stage of T1WI to the high symmetric signal of bilateral globus pallidus and subthalamic nucleus in the chronic stage and FLAIR sequence signal[18-20]. In the neonatal period, the basal ganglia nerve cells have strong physiological and biochemical metabolism and oxygen consumption is increasing. Especially, the middle and later parts of the globus pallidus are the most sensitive . This leads to the selective deposition of serum bilirubin in the globus pallidus, and the middle and later parts of the globus pallidus become more sensitive[21-23]. Besides, it will cause damage to neurons and glial cells, the apoptosis of neurons and the change of glial cells’ mitochondria function[24,25]. Basal ganglia pallidus injury caused by hyperbilirubinemia can be efficiently detected by MRI. The high and symmetric signal of bilateral T1WI pallidal is an important imaging feature of neonatal ABE, while the change from a high signal of T1WI to a high signal of T2-weighted imaging (T2W1) is the imaging feature of nuclear jaundice on MRI, indicating neuronal cell necrosis and poor prognosis. Presently, the mechanism of MRI signal change remains unclear. This may be related to the reduction of the T1 value, which is caused by the deposition of bilirubin in glial cells and the destruction of bilirubin to the nerve cell membrane .
Most of the MRI results mentioned above were obtained from brain MRI findings of the severe or profound NHB patients with bilirubin encephalopathy or non-bilirubin encephalopathy. So far, there is no report about whether the MRI results of mild and moderate NHB patients without bilirubin encephalopathy manifestation are abnormal. Therefore, this study retrospectively analyzes the bilirubin level and MRI results of 103 patients with jaundice to understand whether there is abnormal brain MRI at different bilirubin levels, as well as whether there is a statistical difference of abnormal brain MRI results in bilirubin levels, gestational age, and birth weight.
It is reported in the literature that some mild levels of bilirubin may cause temporary or permanent neurological sequelae under the condition that a certain level of bilirubin is considered safe by people. Based on total serum bilirubin concentration (TSB), the 103 patients in our research were divided into three groups, including 16 cases in the mild group (TSB: 0.0—229.0μmol/L), 49 cases in the moderate group (TSB: 229.0—342.0μmol/L) and 38 cases in the severe group (TSB ≥342.0μmol/L). There were 21 cases with abnormal MRI results, including 5 (31.25%) cases in the mild group, 8 (16.33%) cases in the moderate group and 8 (21.05%) cases in the severe group(Table 3). The comparison of TSB among the three groups indicated that the difference was statistically significant (P＜0.01) (Table 1), whereas the comparison of abnormal brain MRI results among the three groups indicated that the difference was not statistically significant (P＞0.05). This implies that bilirubin brain nerve damage may occur in patients without the manifestation of ABE even at a low level of bilirubin, and the rate of MRI abnormality does not grow with the rising bilirubin level. The study findings are not consistent with the reports of EI Houchi, S.Z. et al. that the higher the total bilirubin level is, the higher the proportion of abnormal MRI is. We think that the low number of cases in the mild group may also be an important factor. Under Taoka 140 et al.’s follow-up of the observed subjects for 2 years, it was found that the infants had been developing normally whether the high symmetric T1WI signal was demonstrated in globus pallidus (GP) and substantia niga (STN) regions or not. In accordance with reports in the literature, it may be related to the development of gray matter mass in GP and STN after the birth of neonates [30-33]. Besides, substantial research has reported that this manifestation is one of the brain MRI findings of nuclear jaundice [34-36]. Along with the extensive clinical application of MRI, the manifestation is becoming more and more common in neonates including normal neonates. Thus, some scholars recently have raised different opinions[29-31]. For example, Harris et al.  reported that a high symmetric signal in the globus pallidus region of four 5-21 day neonates with acute kernicterus disappeared during the follow-up. This manifestation was thought transient, showing no correlation with the prognosis of the patients. This manifestation also appeared in the cases of neonatal hypoxic-ischemic encephalopathy, hypoglycemia and other cases [38,39].
In this study, the neonate patients, with a high signal of globus pallidus in T1WI or T2W1 caused by the diseases of neonatal hypoxic-ischemic encephalopathy, hypoglycemia, hepatolenticular degeneration, or other diseases, were excluded. Of our 21 patients with MRI, 12 were followed up for one month, and 9 cases were followed up for 3 months. Moreover, 6 cases of patients had normal brain MRI results in the reexamination of one month later. And 3 cases of patients were not reexamined of the MRI, one with neurological abnormalities 2 months later, who was delivered with a gestational age of 40 weeks, a birth weight of 3.31kg, clear amniotic fluid, an Apgar score of 8 at 1 min and 9 in 5 min, a maximum bilirubin Value of 327.3μmol/L, and an uneven signal of the brain MRI in bilateral basal ganglia region. However, one case of this study showed no brain MRI abnormalities with a maximum bilirubin value of 502.3 μmol/L, a birth weight of 3.05 kg, a gestational age of 36+3 weeks, clear amniotic fluid and an Apgar score of 9 at 1 min and 9 in 5 min. In addition, another case in the study may have ABE with a gestational age of 40+4 weeks, a birth weight of 3.5 kg, meconium-stained amniotic fluid during birth, an Apgar score of 9 at 1 min and 9 in 5 min, the maximum bilirubin value of 130.4μmol/L and a high symmetric T1WI signal of bilateral basal ganglia indicated by his brain MRI. The high symmetric signal of bilateral globus pallidus is not unique to the neonatal ABE, but can also be seen in some neonatal patients with hypoxic-ischemic encephalopathy[40,41], or even in normal neonates. However, the MRI manifestations of hypoxic-ischemic encephalopathy (HIE) involved a more extensive scope, which was characterized by internal capsule, the putamen and the thalami, and accompanied by cortical and subcortical, deep leukoplakia plaque abnormal signals, diffuse brain edema, intracranial hemorrhage, and so on. These accompanied manifestations were rarely reported in bilirubin encephalopathy .
Blood-brain barrier injury might be caused by anoxia or other factors in antepartum and intrapartum, increasing the permeability of the blood-brain barrier and causing more free bilirubin to enter into brain tissue through the injured blood-brain barrier and to deposit in the basal nerve nucleus, cerebral ganglia, subthalamic nucleus, parietal nucleus, ventricular nucleus, caudate nucleus, cerebellum, oblongata, cerebral cortex, spinal cord and brainstem, etc. Accordingly, the utilization of oxygen in brain tissue was inhibited, 176 leading to brain damage. It is also believed that bilirubin deposition causes the influx of neuron cells Ca2+, stimulates the increase of proteolytic enzyme activity, oxidative damage, immune stimulation, immunotoxicity, increased cytoglutamate and excitotoxicity, inflammatory damage and other pathways, leading to neuronal necrosis and apoptosis. It has been reported in many studies that the occurrence of neonatal BE is related to such factors as gestational age, birth weight, bilirubin binding state and bilirubin level, etc. In this study, 103 patients were divided into the pre-term group (26 cases) and the full-term group (77 cases), for the comparison of their bilirubin values (289.70±85.38vs 310.36±72.32, P =0.232) and MRI abnormal result (P =0.16), showing no statistically significant difference (P＞0.05). It indicated that there may be no difference in the toxicity of bilirubin to the central nervous system between the pre-term group and the full-term group without BE clinical manifestation. Moreover, premature infants with younger gestational age appeared not to be more susceptible to bilirubin toxicity. The explanations of this result may be as follows: (1) Premature delivery patients, who are usually admitted to the hospital after birth, are treated for jaundice in time during hospitalization; while full-term infants are admitted to the hospital only when their bilirubin value reaches a higher level. On the one hand, a high level of bilirubin can easily cause nerve damage by passing through the blood-brain barrier; on the other hand, the longer the high level of bilirubin remains in the body, the more neurotoxicity the bilirubin is. (2) The statistical analysis in this study may be biased, on account of the fewer cases in the pre-term delivery group, especially the lack of pre-term patients less than 31 weeks of gestational age due to obstetric factors. Meanwhile, we also observed whether there was a statistical difference in birth weight and brain MRI abnormality among jaundiced patients. Based on birth weight, 103 patients were divided into two groups, one group with a birth weight of < 2500g (16 cases, including 6 abnormal cases) and the other group with a birth weight of ≥2500g (87 cases, including 15 abnormal cases) (Table 2). The comparison of brain MRI abnormality between the two groups was P=0.09, P >0.05, indicating that the difference was not statistically significant.
In addition, the comparison of the bilirubin value between the abnormal MRI group and the normal MRI group was 303.56±83.04 vs 305.55±74.54 (P=0.92, P>0.05) (Table 1). The difference was not statistically significant, which indicated that there was no obvious difference in the bilirubin level between the abnormal MRI group and the normal MRI group of jaundiced patients without ABE.
Moreover, we measured the T1WI signal values of the patients with abnormal brain MRI and the signal values of the patients with normal brain MRI in the three groups, and performed statistical comparison. The results showed that the T1WI signal values of the patients with abnormal MRI were all higher than those of the patients with normal MRI (P<0.05), and the T1WI signal values of the patients with MRI abnormalities in the three groups also had statistically difference (P<0.05) (Table 4). With the increase of serum bilirubin level, the T1WI signal value of the patients with MRI abnormalities also increased, which was consistent with the report of Yan Ruifang et al  that there was a linear correlation between the mean signal value of globus pallidus and serum total bilirubin levels in the lesion group. This indicated that with the increase in the bilirubin level, the more bilirubin deposited on neuron such as globus pallidus, the more serious damage would be made to nerve tissue such as neuron.
NHB can cause not only acute bilirubin encephalopathy and kernicterus but also bilirubin induced neurological dysfunction (BIND) [55,56], which includes mild neurological abnormalities, cognitive disorder, auditory neuropathy spectrum disorder (ANSD) and so on. Auditory complications, a disabling neurological finding in kernicterus, are typically characterized by varying degrees of auditory neuropathy/dys-synchrony (AN/AD) ranging from central auditory processing difficulties with normal hearing to severe AN/AD with absent auditory brainstem responses, and possibly accompanying severe hearing loss and deafness. In fact, the brainstem cochlear nuclei are said to be one of the first structures affected by elevated total bilirubin, followed by the auditory nerve[58,59]. Thus, we performed hearing tests for the 103 patients with brainstem auditory evoked potential (BAEP) devices. From Table 5, it was found that 15 cases of 82 patients with normal MRI results were abnormal, accounting for 18.29%. In the mild group, 2 cases of 5 patients with abnormal MRI were abnormal, accounting for 40.00%. In the moderate group, 4 cases of 8 patients with abnormal MRI were abnormal, accounting for 50.00%. In the severe group, 6 cases of 8 patients with abnormal MRI were abnormal, accounting for 75.00%. The change of BAEP is closely related to bilirubin concentration and duration, but BAEP may be abnormal in children with a moderate bilirubin level. In other words, BAEP may be abnormal in children with related safe bilirubin abnormalities that are improved after clinical treatment, even if there is no clinical manifestation [61,62]. BAEP was improved with the decrease of serum bilirubin or after the process intervention such as phototherapy and blood exchange transfusion[63-65]. Vitoria Akinpelu, et al. reported in 2013 that the abnormal BAEP rate fluctuated between 9% and 83.3% before the clinical intervention, and about over half of the BAEP abnormalities were improved after clinical treatment. After admission, all 103 patients were treated with medication and/or phototherapy, and their jaundice decreased significantly. After a 1-3 month followed-up, the reexamination was carried out, showing that the patients with hearing abnormalities in the MRI normal group and the mild group all returned to the normal level, while 1 case in the moderate group and 2 cases in the severe group remained abnormal. Sharma, et al. and Agrawal, et al. showed that BAEP improved in 77.2% and 76.5% of the children surveyed. This result indicated that on the one hand, the BAEP abnormal rate of the patients with abnormal MRI was higher than that of the patients with normal MRI, and the BAEP abnormal rate of the patients with abnormal MRI increased with the rising bilirubin level. It also showed that with the increase of bilirubin, the hearing damage became more obvious. On the other hand, it proved the application value of MRI in neonatal jaundice patients, which could timely detect the abnormalities of the central nervous system of the patients with jaundice and provide imaging evidence for early diagnosis and early intervention.
Based on this study, we found abnormalities in the neonatal brain’s MRI and BAEP at different bilirubin concentration levels. It indicates that in some conditions, even a low level of bilirubin concentration might lead to neurological damage in the neonatal period. Therefore, strengthening the monitoring of neonatal jaundice and using the MRI and BAEP to test the bilirubin induced neurological damage for neonates are recommended.
Limitations of the Present Study
Although the findings of this study indicate that in babies with jaundice, the brain MRI may show abnormal changes at mild and moderate levels of bilirubin, and there is no statistically significant difference between brain MRI abnormalities in the severe group, our results have been affected by some issues. First, we failed to measure the free bilirubin levels, internal environmental conditions such as serum albumin levels, pH values, etc. of the jaundice newborns participating in the study at that time. Therefore, it cannot be said with certainty that the baby's brain was exposed to bilirubin crossing the blood-brain barrier Prime type. Secondly, the sample size in this study is relatively small, especially in the mild group and the group with a birth weight of less than 2500g. Finally, the craniocerebral pathology test of the newborn animal Jaundice model and long-term follow-up was not performed to validate our findings. This issue should be addressed in future research.
In conclusion, in the presence of certain factors, such as premature, hypoproteinemia, potential intrauterine hypoxia, etc., central nervous system damage may also occur at a low level of bilirubin and result in an abnormality on MRI and BAEP. Meanwhile, MRI and BAEP can also be used to provide early abnormal information for the judgment of central nervous system damage to NHB neonatal patients without clinical manifestations of ABE, and offer clues for early treatment and early intervention to prevent the occurrence of severe brain tissue damage.