Evaluation of region selective bilirubin-induced brain damage as a basis for a pharmacological treatment

Abstract

The neurologic manifestations of neonatal hyperbilirubinemia in the central nervous system (CNS) exhibit high variations in the severity and appearance of motor, auditory and cognitive symptoms, which is suggestive of a still unexplained selective topography of bilirubin-induced damage. By applying the organotypic brain culture (OBC: preserving in vitro the cellular complexity, connection and architecture of the in vivo brain) technique to study hyperbilirubinemia, we mapped the regional target of bilirubin-induced damage, demonstrated a multifactorial toxic action of bilirubin, and used this information to evaluate the efficacy of drugs applicable to newborns to protect the brain. OBCs from 8-day-old rat pups showed a 2–13 fold higher sensitivity to bilirubin damage than 2-day-old preparations. The hippocampus, inferior colliculus and cerebral cortex were the only brain regions affected, presenting a mixed inflammatory-oxidative mechanism. Glutamate excitotoxicity was appreciable in only the hippocampus and inferior colliculus. Single drug treatment (indomethacin, curcumin, MgCl2) significantly improved cell viability in all regions, while the combined (cocktail) administration of the three drugs almost completely prevented damage in the most affected area (hippocampus). Our data may supports an innovative (complementary to phototherapy) approach for directly protecting the newborn brain from bilirubin neurotoxicity.

Neurotoxicity of Unconjugated Bilirubin in Mature and Immature Rat Organotypic Hippocampal Slice Cultures

“…Therefore, our findings on the neurotoxic effect of UCB in mature slices when the UCB:HSA molar ratio is > 1 cannot be compared to previous studies [14-16], because these had a different design and, mainly, were carried out in immature slices [10]. However, in one study, UCB did not affect cell viability [14], while in others it induced neuronal necrosis [15, 16]. …”

“…Although the better potential of organotypic hippocampal slices compared to isolated cell cultures in investigating UCB-induced injuries has been well recognized [6], few studies were performed using this method [14-16], being mainly focused on UCB-induced modification of synaptic transmission. These studies are heterogeneous: donor rats were 6–8 days old [14], or 7–10 days old [15], or 2 and 8 days old [16]; rat hippocampal slices were maintained in vitro for 3 [14, 15] or 6 [16] days prior to use (to allow tissues to recover from experimental trauma caused by the isolation procedure) and, according to Bayer et al [10], represent models of immature tissues; slices were exposed to 1–10 µM UCB plus 2–20 µM HSA (molar ratio 0.5) for 24 or 48 h [14], or 50 µM UCB plus 100 µM HSA (molar ratio 0.5) for 24 h [15], or to 70, 140, or 300 nM of unbound bilirubin for 24 h [16].…”

“…These studies are heterogeneous: donor rats were 6–8 days old [14], or 7–10 days old [15], or 2 and 8 days old [16]; rat hippocampal slices were maintained in vitro for 3 [14, 15] or 6 [16] days prior to use (to allow tissues to recover from experimental trauma caused by the isolation procedure) and, according to Bayer et al [10], represent models of immature tissues; slices were exposed to 1–10 µM UCB plus 2–20 µM HSA (molar ratio 0.5) for 24 or 48 h [14], or 50 µM UCB plus 100 µM HSA (molar ratio 0.5) for 24 h [15], or to 70, 140, or 300 nM of unbound bilirubin for 24 h [16]. Therefore, our findings on the neurotoxic effect of UCB in mature slices when the UCB:HSA molar ratio is > 1 cannot be compared to previous studies [14-16], because these had a different design and, mainly, were carried out in immature slices [10]. However, in one study, UCB did not affect cell viability [14], while in others it induced neuronal necrosis [15, 16].…”

Human liver stem cells express UGT1A1 and improve phenotype of immunocompromised Crigler Najjar syndrome type I mice

“…6B, arrowheads and Fig. 6C), was reduced upon HLSC injection with respect to PBS-treated controls, showing that these cells efficiently prevented the pathological effects of unconjugated bilirubin when injected early in the newborns 19 . For serum bilirubin and brain and liver histology assessment, 21 days old HLSC-injected NSG/Ugt1 −/− mice could not be compared with 21 days old control NSG/Ugt1 −/− mice as the latter did not survive beyond 16 days despite PT.…”

“…Red neurons are representative of ischemic and hypoxic injury, which may be induced by unconjugated bilirubin toxicity in PBS-injected NSG/Ugt1 −/− mice, leading to death within 16 days after birth 27 . On the other hand, HLSC-injected NSG/Ugt1 −/− mice at 21 days after birth had no apparent motor deficits compared to their PBS-injected counterparts, showing that these cells efficiently prevented the pathological effects of unconjugated bilirubin when injected early in the newborns (Movie S2) 19,28,29 .…”