Abstract
Objective
Jaundice (icterus) is the visible manifestation of the accumulation of bilirubin in the tissue and is indicative of potential toxicity to the brain. Since its very first description more than 2000 years ago, many efforts have been undertaken to understand the molecular determinants of bilirubin toxicity to neuronal cells to reduce the risk of neurological sequelae through the use of available chemicals and in vitro, ex vivo, in vivo, and clinical models. Although several studies have been performed, important questions remain unanswered, such as the reasons for regional sensitivity and the interplay with brain development. The number of new molecular effects identified has increased further, which has added even more complexity to the understanding of the condition. As new research challenges emerged, so does the need to establish solid models of prematurity.
Methods
This review critically summarizes the key mechanisms of severe neonatal hyperbilirubinemia and the use of the available models and technologies for translational research.
Impact
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We critically review the conceptual dogmas and models used for studying bilirubin-induced neurotoxicity.
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We point out the pitfalls and translational gaps, and suggest new clinical research challenges.
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We hope to inform researchers on the pro and cons of the models used, and to help direct their experimental focus in a most translational research.
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Free bilirubin: the only moiety of serum bilirubin that can enter the brain
In the clinical setting, the decision of treating a neonate with hyperbilirubinemia is based on the level of total serum bilirubin (TSB). TSB is the sum of circulating conjugated bilirubin (CB, or direct bilirubin), unconjugated bilirubin (UCB, or indirect bilirubin) bound to serum albumin, and the tiny fraction of UCB not bound to albumin, the so-called free bilirubin (Bf). Bf is lipophilic and can interact with lipids and cross cellular membranes entering into the cells mainly by passive diffusion,1,2,3 while a carrier-mediated mechanism is probably at work at low Bf concentrations.4 Several studies based on animal models investigated the ability of the different “bilirubins” (CB, UCB, Bf = UCB not bound to albumin) to enter the brain. In 1966 Diamond performed an in vivo study that addressed several questions.5 Young and adult animals were injected with radiolabeled (C14) CB, Bf (UCB not bound to albumin), or UCB firmly bound to human albumin (UCB + h-Alb). While C14-CB and C14-UCB + h-Alb were not detectable in the brain of injected animals, C14-Bf rapidly accumulated in the CNS. Moreover, addition of chemicals able to displace C14-UCB from albumin (thus rising Bf), drastically increased bilirubin concentrations in brain parenchyma. In contrast, in the animals injected with albumin, the level of bilirubin in the brain was reduced.5 This study demonstrated that: (1) only the fraction of UCB not bound to albumin (Bf) rapidly diffuses into the brain; and (2) that albumin is protective toward bilirubin accumulation in the brain. Thus, low serum albumin, which is common in preterm infants, increases the risk of developing neurological sequelae even at TSB levels under established risk thresholds;6 while the infusion of albumin in animal models of hyperbilirubinemia decreases bilirubin levels in the brain.7,8,9,10 (3) Bilirubin-albumin displacers, including some common treatments used in neonatal care, can increase Bf and are important additional variables to consider in assessing the risk of developing neurological sequelae.11,12,13 Despite it is recognized that Bf is responsible for damage (Fig. 1a),14,15,16,17,18 Bf is routinely quantified only in hyperbilirubinemic neonates in Japan.19,20,21 Elsewhere, quantification of Bf in the clinic is an unmet need, and risk management continues to be based solely on total bilirubin levels (TSB). A most user-friendly method than the oxidase-peroxidase technique is necessary to make a routine quantification of Bf. In this respect, the development of a fluorescent sensor is promising.12,18,22,23
a–h The major molecular mechanisms and effectors of bilirubin toxicity to brain. a Free bilirubin data; b demonstrated molecular mechanisms of bilirubin neurotoxicity, c bilirubin effects on neurotransmission, d brain development, e cellular membranes, f glucose utilization, g UCB transporters, and h ABCb1, ABCc1, & blood–brain interfaces. In vitro: chemical studies. Cells: cell lines and primary cell cultures. OBCs: organotypic brain cultures. In vivo: animal models and humans. Green check: to draw attention to results confirmed among the models; red check: to draw attention to disagreement among the models. Red target: to draw attention to a clinical goal to reach. ER endoplasmic reticulum, BAX BCL2-associated X, BBB blood–brain barrier, BCSFB blood-cerebrospinal fluid barrier, ABCb1 ATP-binding cassette subfamily B member 1, ABCc1 ATP-binding cassette subfamily C member 1, P450 cytochrome P450 mono-oxygenase, UCB unconjugated bilirubin, DIV days in vitro.
While a risk threshold for TSB has been proposed,24,25 the minimal amount of bilirubin that can cause brain damage is still unknown. The recent improvement of sensitivity and reproducibility of high-pressure liquid chromatography technology for quantifying bilirubin content in tissues26,27 can help to explore the relationship between blood (TSB, but possibly also Bf) and brain bilirubin content, and the effects of therapeutic approaches (phototherapy, albumin infusion, bilirubin chelators, etc.).7,8,9,27,28,29
Differential brain sensitivity to UCB toxicity: a paradigm shift
Selective bilirubin accumulation in specific regions of the brain is not required for developing damage
Displacers have frequently been used in studies on BIN. When used in spontaneously hyperbilirubinemic (jj) Gunn rats, displacers increasing the fraction of UCB not bound to albumin in the blood (Bf), can rapidly shift bilirubin from the vascular space to tissues,27,30 inducing the accumulation of bilirubin in specific regions of the brain and leading to the development of symptoms of BIN.27,31,32,33,34 Accumulation of bilirubin usually occurs in the cerebellum, hippocampus, inferior colliculi, and basal ganglia (see Fig. 2, yellow regions),27,31,32,33 a phenomenon known as jaundice of the ganglia (or “kern-icterus”; where kern = kernel, and icterus = yellow), a term coined by Christian Schmorl in 1903 (reviewed in ref. 35) In vitro models using immortalized cells, primary cultures, co-cultures, and organotypic brain cultures (OBCs) have demonstrated that the intracellular amount of bilirubin is proportional to the damage.36,37,38,39,40,41,42 MRI studies on severely hyperbilirubinemic infants have revealed a damage to brain structures similar to that shown in animal models injected with bilirubin displacers.24,43,44,45,46,47,48,49,50,51 Thus, the conclusion that accumulation of bilirubin in specific regions of the brain causes damage was believed to be gospel.
Upper: a sagittal view of a human brain; lower: a sagittal view of a rodent brain, shown to appreciate and compare the localization of the main brain areas reported to be affected by bilirubin. In yellow, the areas of the brain corresponding to the concept of kern-icterus (the distribution of bilirubin accumulation). In red: the regions reported to be affected by bilirubin neurotoxicity as observed in humans and models (in addition to the yellow ones).
However, in the last 20 years, this concept has been questioned. In jj Gunn rat and Ugt1a-/- mice not challenged with displacers, the amount of bilirubin is identical in all regions of the brain, even though BIN is present up to lethality.5,27,52,53,54,55 Most importantly, in the autopsies of infants who died with severe hyperbilirubinemia, only 5% of the cases showed a bilirubin accumulation in restricted areas of the brain.35,48,56 The regions of the brain sensitive to bilirubin toxicity appear to be even numerous (see Fig. 2, red regions). Regions such as the cerebral cortex (humans43 and animal models,36,57,58,59 plus the large majority of the studies using primary cultures), hippocampus (humans24,51,60 and animal models36,61,62) pons and medulla (humans24,51 and animal models53), and cerebellum (humans24,50,60,63 and animal models32,33,64,65,66,67) have been reported to be sensitive to bilirubin toxicity. This evidence supports the concept that bilirubin accumulation in specific regions of the brain is not required for developing damage.
The two apparently contradictory interpretations have been combined and explained by Christian Schmorl. As early as 1903, He suggested that “damaged brain tissue has a greater propensity to bind or retain bile pigment”, indicative of the fact that “selective areas of the brain are a priori more vulnerable to bilirubin and, once damaged, retain the pigment more avidly”.35,48,56
The neurological manifestations of BIN appear to be progressive as shown by the early and transient auditory dysfunctions, possibly leading to severe and permanent sequelae later on.45,46,68 In in vitro models, differential responses were dependent on the duration of the challenge, and varied from an early, possibly protective reaction, to irreversible damage.37,40,59,69,70,71,72,73 Similarly, in OBCs and in vivo (Gunn rat) models of BIN, damage to the cerebellum required a more prolonged exposure to bilirubin.27,36,64,65 This agrees with the development of cerebellar hypoplasia in rodents (in vivo models),33,64,65,66,74 and supports the concept that the duration of the toxic challenge may contribute to regional sensitivity. This evidence suggests that the damage observed in models may be progressive as observed clinically, and indicate a regional variability in response to bilirubin, despite being exposed to the same bilirubin concentration.75
Regional sensitivity
Several studies support the conclusion that bilirubin toxicity varies according to the regional sensitivity of the brain. Solid evidence of the biomolecular events of BIN has been established in cell lines and primary cultures (see section “Cellular and BBB defenses to bilirubin accumulation and toxicity” and Fig. 1). In vitro models are performed in tumoral or immortalized cell lines. Primary neurons can be isolated only from the whole brains of embryos, while astrocytes and oligodendrocytes are usually isolated from the cerebral cortex of perinatal animals. Therefore, the use of in vitro models cannot reproduce the significant heterogeneity of the brain, where each region possesses not only a different percentage of neurons and glia,76 but specialized populations of neural cells (e.g., granular, pyramidal, Purkinje neurons; radial, fibrous, protoplasmic, Bergmann glia), possibly presenting different levels of sensitivity against bilirubin toxicity. Despite these limitations, these models can still be used. It has been found that neuroblastoma and oligoblastoma cell lines are more sensitive to BIN as compared with hepatic and fibroblast cell lines,77 suggesting a significant sensitivity of the brain to bilirubin. Moreover, neurons and oligodendrocytes are much more sensitive to bilirubin toxicity than astrocytes and microglia.78 However, the neuronal composition (percentage of astrocytes, neurons, microglia, oligodendrocytes) of each brain region cannot explain the different sensitivities.76 Primary neuronal cells obtained from differential brain regions showed a different sensitivity and ability to oxidize bilirubin and a different pro-oxidant status.59,79 This different topographical sensitivity was confirmed in models more close to the whole brain as OBCs, where the hippocampus is more prone to bilirubin injury than the cerebral cortex and inferior colliculi, which in turn, are more sensitive than cerebellum to bilirubin injury.36 Localized difference in the dynamics of UCB clearance, alterations in the production of brain metabolites, and use of glucose (mainly in the regions belonging to the auditory system, Fig. 1f) has been reported in animal models of BIN.27,57,75,80
Developmental sensitivity
Apart from the observed regional sensitivity, in vitro, ex vivo, and in vivo models of BIN strongly suggest that bilirubin can target critical steps in brain development such as cell division, differentiation, migration, myelination, etc. Mature in vitro grown (DIV: days in vitro) neurons and microglia are more resistant to bilirubin compared with young (DIV) cells,78,81,82,83 and mitochondria isolated from young (DIV) neurons have a greater membrane permeability and cytochrome C release than mitochondria isolated from older (DIV) neurons.78 The opposite is observed in mitochondria isolated from animals at different ages78 (Fig. 1c).
OBCs obtained from P8 (P: post-natal age in days) animals were more prone to damage than slices from P2 rats, independently from the region of the brain analyzed.36 This finding agrees with an in vivo study using both Gunn rats and Ugt1A-/- mice33 where a window of maximal susceptibility to bilirubin toxicity is observed between P6 and P10.84,85 Additional studies supporting a developmental sensitivity to BIN demonstrate an age-dependent utilization of glucose (Fig. 1f),75,80 and the differential deposition of bilirubin after exposure to bilirubin displacers.32 Recent evidence obtained in vivo show that bilirubin could also affect the epigenetic control of brain development. The analysis of the acetylated chromatin immuno-precipitated from a cerebellum of the jaundiced Gunn rat revealed that almost 50% of the identified genes were involved in brain maturation, synaptogenesis, neurogenesis, myelination, etc., suggesting a possible mechanism for the development of the characteristic cerebellar hypoplasia.64
Cellular and BBB defenses to bilirubin accumulation and toxicity
Although most questions remain unanswered, toxicity of bilirubin to the brain is well known.
As confirmed in almost all models, bilirubin induces redox stress, inflammation, and cell death, and modulates multiple signaling pathways (Fig. 1b).27,36,39,61,63,67,79,84,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102 Bilirubin affects also neurotransmission (Fig. 1c),62,103,104,105 brain development (Fig. 1d),53,61,64,97,106,107,108 and glucose utilization (Fig. 1f).27,57,75,80 Membranes appear to be the main target of bilirubin (Fig. 1c).2,62,93,109,110,111
Still debated is the effect of bilirubin on the blood–brain interfaces, namely BBB and BCSFB. After the in vitro (transfected cell lines, vesicles) identification of ABCB1 (MDR1, Pgp) and ABCC1 (Mrp1) as UCB transporters (Fig. 1g, h),112 their potential contribution to protection against bilirubin toxicity has been a matter of debate for more than one decade. ABCC1 has been demonstrated to play a major role in cellular defense against UCB toxicity in vitro (silencing),113 protecting neurons and astrocytes (cell lines and primary cultures).114,115,116 However, ABCC1 is only marginally expressed in brain parenchyma in vivo.117 On the contrary, the transporter is strongly expressed at the BCSFB, but it is downregulated by bilirubin.117 Notably, the BCSFB is quite overlooked in the study of BIN, despite the ventricles being suggested as a way of entry of bilirubin into the brain.118
ABCB1 expression can be detected in vitro on neurons, astrocytes, and models of the BBB,113,114,119 where the transporter is downregulated by bilirubin (Fig. 1h). In vivo, ABCB1 is specific to BBB, and is up-regulated in hyperbilirubinemia from the post-natal age to adult life.117 Contrary to what was observed in vitro, ABCB1 looks to be the major contributor to reducing bilirubin brain entry in vivo29 (Fig. 1h). Contradictory results are certainly associated with the use of in vitro models that do not encapsulate the brain environment. Nevertheless, despite reducing the content of bilirubin in the brain compared with ABCC1/B1 knockout or chemical inhibition studies,120,121 the activity of the two bilirubin transporters in wild-type animals is insufficient to avoid the entry of bilirubin and development of BIN.120,121
The role of damage or a leaky BBB is still debated. While in vitro studies support the opening (leaked or even disrupted) of BBB,122,123 in vivo data do not support this concept55,75,124 (Fig. 1h), but suggest alteration of ABC transporters, cytokine, chemokine, and/or neutrophil infiltration.117,124
Therapeutical approaches
Despite a large number of still unanswered questions, knowledge obtained by using different in vivo models62,65,66,74,105,120,125,126,127,128 has been used as pre-clinical platforms for evaluating new therapeutic approaches in BIN (Fig. 3).
The paradigm of research models used to study the clinical problems of the hyperbilirubinemic newborn.
Reduction of TSB is the main treatment approach for BIN and has been achieved through phototherapy,84,85 phototherapy combined with the infusion of albumin,7,9,10 gene therapy,65,129 increasing UCB clearance,28 or inhibiting its production.130 In addition, clinical trials to repair neuronal damage using stem cell therapy are ongoing.131
An alternative strategy is to directly protect the brain from bilirubin toxicity with drugs. Minocycline has been demonstrated to fully protect Gunn rats from developing cerebellar hypoplasia, significantly improve neuronal loss, behavior (acute brainstem auditory evoked potentials abnormalities, and motor coordination tested by rotarod and open field), and reduce lethality in Ugt1a-/- mice,125,132,133,134 but cannot be used clinically due to its side effects. Anti-inflammatory and anti-oxidant drugs have also been shown to decrease the extent of damage from bilirubin toxicity.134,135 Recently, natural products such as choline, which can protect the membranes,136 and curcumin,67 which has a pleiotropic effect on the main in vivo molecular effectors of BIN (inflammation, redox, and glutamate neurotoxicity), were shown to be effective in the Gunn rat. The role of such agents to protect and reduce bilirubin toxicity is promising and must be further investigated for possible clinical use.
Summary and future challenges
In addition to the incomplete understanding of the observed regional sensitivity of the brain to bilirubin and the variabilities in the symptoms of BIN, there are still several clinical challenges that need to be addressed.
Because of improvements in neonatal care, preterm infants are an emerging population with an extreme vulnerability to bilirubin neurotoxicity.48 Unfortunately, ideal models of prematurity are not yet available and are not easy to develop. A part of the known different timescale in a human vs. rodent brain development (years vs. few months), the major problem is that the brain is extremely heterogeneous. Each region of the brain develop at a different time, thus cell division, differentiation, migration, physiological apoptosis, synaptogenesis, myelination, etc.; all suggested target of bilirubin, are largely time and region-dependent.137,138,139,140 The best solution may be to focus on targeted approaches, by selecting a specific brain region that represents the stage and the molecular process of interest. Alternatively, premature non-human primates may be the closest model to humans, but these models require specific technical expertise and appropriate equipment to properly perform the experiments, and may be hampered by complex ethical rules.
Moreover, we have also still to understand why some infants are vulnerable to “safe” TSB and normal serum albumin levels, while other infants do not develop neurological sequelae despite having extremely high TSB levels, suggesting the existence of individual genetic variability in brain sensitivity,141 that has to be explored.
Far from being a single organ, the brain is a conglomerate of different areas, each one with specific functions. These regions are intimately inter-connected allowing the complex functioning of the brain, the so-called connectome. Thus, we need a multidisciplinary interactive approach to study BIN including the field of imaging, genetics, biofunction, “omics” and the use of artificial intelligence.
Data availability
All data generated or analyzed during this study are included in this published article.
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Acknowledgements
We sincerely thank Prof. Jon F. Watchko for his inspiring presentation at the “Yellow seminars” of December 6, 2021 (http://bit.ly/YW_Watchko), and Dr Lorraine Kay Cabral for critical reading of this review.
Funding
S.J., C.T., and S.G. were financed in part by an internal grant of the Fondazione Italiana Fegato – Onlus. S.J. was financed by a fellowship from the Lembaga Pengelola Dana Pendidikan (Indonesia Endowment Fund for Education), in part by an internal grant of the Fondazione Italiana Fegato – Onlus.
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S.G. made substantial contribution to conception and design, acquisition, and revision of the literature, and wrote the article. S.J. contributed to acquisition and revision of the literature and wrote the article. C.T. discussed the results, revised the article for intellectual content, and contributed to the final revision of the English. All the authors read and approved the final version of the article.
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Gazzin, S., Jayanti, S. & Tiribelli, C. Models of bilirubin neurological damage: lessons learned and new challenges. Pediatr Res 93, 1838–1845 (2023). https://doi.org/10.1038/s41390-022-02351-x
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DOI: https://doi.org/10.1038/s41390-022-02351-x
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