Gunn Rats Research Articles

Impaired excitability of nucleus tractus solitarius neurons in a neonatal rat model of hyperbilirubinemia

Hyperbilirubinemia is associated with multiple morbidities including apnea of prematurity in preterm infants. Bilirubin, a byproduct of hemoglobin breakdown, is not excreted normally and results in high circulating levels giving rise to a state of hyperbilirubinemia. Free bilirubin (not bound to albumin) can cross the blood-brain barrier and be neurotoxic. Prior studies have shown that a systemic injection of bilirubin accumulates in the brainstem and is associated with an impaired ventilatory response to hypercapnia and an augmented hypoxic ventilatory depression. Here, using the Gunn rat model of endogenous hyperbilirubinemia, we investigated its effects on the excitability of neurons of the nucleus tractus solitarius (NTS). The homozygous Gunn rat (jj) model of hyperbilirubinemia lacks uridine diphosphate-glucuronosyltransferase-1A1, an enzyme which is needed to catabolize bilirubin. Heterozygous Nj Gunn rat pups were used for control. On postnatal (P) day 5, Nj and jj rat pups received either a 200mg/kg intraperitoneal injection of sulfadimethoxine (SDMX), which dissociates bilirubin from albumin resulting in increased levels of circulating free bilirubin, or an equivalent amount of saline as a vehicle control. Whole cell patch clamp was performed on NTS neurons two days later (P7) to assess characteristics of evoked excitatory postsynaptic currents (eEPSC).The amplitude of the evoked stimulus response to a 20Hz stimulus train was significantly increased in NTS neurons from jj+saline pups when compared to cells from Nj+saline and Nj+SDMX animals (p<0.05). This increased amplitude was no longer present after SDMX treatment of jj pups. Additionally, jj+saline NTS neurons did not exhibit any failure to solitary tract stimulation, whereas SDMX treatment significantly increased the failure rate during a 20Hz stimulus train (jj+saline =0.0%±0.0, jj+SDMX= 18.63%±8.82, p<0.05). Comparison of failure rate of jj+saline to Nj+saline (6.63%±3.12, p<0.05) was also statistically significant. The hyper-responsive NTS neurons of jj+saline rats may be an adaptive response to the deleterious effects of circulating (bound and unbound) bilirubin. However, the impaired response of neurons in jj+SDMX rats may reflect neurotoxic effects of increased free bilirubin leading to impaired neurotransmission. The latter is consistent with the high failure rate of NTS neurons in jj+SDMX rats. These data may have implications for impaired respiratory control mechanisms involved in mediating essential vagally mediated ventilatory defense responses to hypoxemia and may contribute to the apnea of prematurity associated with hyperbilirubinemia in preterm infants. William and Lois Briggs Endowment. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Severe motor dysfunction in Gunn rats, a model for neonatal bilirubin-induced damage, is not due to changes in brain bilirubin and total serum bilirubin

Severe motor dysfunction in Gunn rats, a model for neonatal bilirubin-induced damage, is not due to changes in brain bilirubin and total serum bilirubin

A change in risk assessment for treatment with statins in patients with familial hypercholesteremia and Gilbert's syndrome

Abstract Background Gilbert’s Syndrome (GS) is a heredity disease that mildly increases serum unconjugated bilirubin. GS is usually asymptomatic and considered to be benign. It is postulated to be present in 10-16% of the population but the exact incidence is unknown since it is usually an incidental finding that goes under diagnosed and under-reported. Conversely, heterozygous familial (FH) hypercholesteremia, estimated to be present in 1 in 250, is responsible for significant morbidity and mortality and never ignored. Patients with FH are especially at risk for atherosclerotic cardiovascular disease (ASCVD) due to the cumulative effect of high cholesterol starting at a young age. Recent studies report that mildly elevated serum bilirubin (especially the UGT1A1*28 allele), in patients with concurrent GS and FH, is protective against ASCVD. The deleterious effect of one liver mutation may be offset by the beneficial effect of the second mutation resulting in an inverse relationship between the two diseases. Due to that association, statins may be contraindicated in patients with concurrent FH and GH because they are more susceptible to toxicity and adverse events. Purpose Patients with coexisting FH and GS are postulated to have innate cardioprotection and therefore, treatment with statins may be unnecessary and should be avoided due to an increased risk for drug toxicity and intolerable adverse events. Methods This was a systematic review of 31 peer-reviewed publications identified by using databases: EBSCO Discovery, World Cat, PubMed, OVID, Google Scholar, Springer, Theime, Elsevier, Science Direct, and Cochrane Collaboration. Publications ranged from 2006-2021 and included subjects ages 18-80 and bench studies using Gunn rats. The quality of evidence was analyzed and evaluated. Results Recent retrospective and prospective studies indicate that mildly elevated serum bilirubin, in patients with concurrent GS and FH, is protective against ASCVD. Consequently, this population may not need statin therapy and may be more susceptible to statin toxicity and intolerable adverse events. Conclusion GS is postulated be innately cardioprotective for ASCVD. The identification of patients with concurrent FH and GS is not mainstream and is overdue. These are not uncommon diseases and their co-existence has been underestimated because GS is considered benign and underreported. The potential exists for patients with FH and silent Gilbert’s Syndrome to be overtreated or unnecessarily treated with statins. Medications that inhibit UDP-glucuronosyltranferase-enzyme activity and UG1A1 mediated glucuronidation may lead to toxicity and intolerable adverse events. Studies have shown an adverse relationship between statins and increased bilirubin. It is recommended that both FH and Gilbert’s Syndrome be routinely screened for in pediatric and adult populations, especially prior to statin prescribing.

Bilirubin-Induced Transcriptomic Imprinting in Neonatal Hyperbilirubinemia.

Recent findings indicated aberrant epigenetic control of the central nervous system (CNS) development in hyperbilirubinemic Gunn rats as an additional cause of cerebellar hypoplasia, the landmark of bilirubin neurotoxicity in rodents. Because the symptoms in severely hyperbilirubinemic human neonates suggest other regions as privileged targets of bilirubin neurotoxicity, we expanded the study of the potential impact of bilirubin on the control of postnatal brain development to regions correlating with human symptoms. Histology, transcriptomic, gene correlation, and behavioral studies were performed. The histology revealed widespread perturbation 9 days after birth, restoring in adulthood. At the genetic level, regional differences were noticed. Bilirubin affected synaptogenesis, repair, differentiation, energy, extracellular matrix development, etc., with transient alterations in the hippocampus (memory, learning, and cognition) and inferior colliculi (auditory functions) but permanent changes in the parietal cortex. Behavioral tests confirmed the presence of a permanent motor disability. The data correlate well both with the clinic description of neonatal bilirubin-induced neurotoxicity, as well as with the neurologic syndromes reported in adults that suffered neonatal hyperbilirubinemia. The results pave the way for better deciphering the neurotoxic features of bilirubin and evaluating deeply the efficacy of new therapeutic approaches against the acute and long-lasting sequels of bilirubin neurotoxicity.

Neonatal hyperbilirubinemia differentially alters the neurochemical profiles of the developing cerebellum and hippocampus in a preterm Gunn rat model.

Neonatal hyperbilirubinemia (NHB) can lead to brain injury in newborn infants by affecting specific regions including the cerebellum and hippocampus. Extremely preterm infants (EPT) are more vulnerable to bilirubin neurotoxicity, but the mechanism and extent of injury is not well understood. A preterm version of Gunn rat model was utilized to investigate severe preterm NHB. Homozygous/jaundiced Gunn rat pups were injected (i.p.) on postnatal day (P) 5 with sulfadimethoxine, which increases serum free bilirubin capable of crossing blood brain barrier and causing brain injury. The neurochemical profiles of the cerebellum and hippocampus were determined using in vivo 1 H MRS at 9.4T on P30 and compared with heterozygous/non-jaundiced control rats. Transcript expression of related genes were determined by real-time quantitative PCR. MRI revealed significant morphological changes in the cerebellum of jaundiced rats. The concentrations of myo-inositol (+54%), glucose (+51%), N-acetylaspartylglutamate (+21%), and the sum of glycerophosphocholine and phosphocholine (+17%) were significantly higher in cerebellum of the jaundiced group compared with control group. Despite the lack of morphologic changes in the hippocampus, the concentration of myo-inositol (+9%) was higher and the concentration of creatine (-8%) and of total creatine (-3%) were lower in the jaundiced group. In the hippocampus, expression of calcium/calmodulin dependent protein kinase II alpha (Camk2a), glucose transporter (Glut) 1, and Glut3 transcripts were downregulated in the jaundiced group. In the cerebellum, the expression of glial fibrillary acidic protein (Gfap), myelin basic protein (Mbp), and Glut1 transcript expression was upregulated in the jaundiced group. These results indicate osmotic imbalance, gliosis, changes in energy utilization and myelination and demonstrate that preterm NHB critically affects brain development in a region-specific manner, with the cerebellum more severely impacted than the hippocampus.

UDP Glucuronosyltransferase Gene Expression is Involved in the Stimulation of Ascorbic Acid Biosynthesis by Xenobiotics in Rats

Wistar-Shi (genotype +/+), heterozygous Gunn (j/+) and homozygous Gunn (j/j) rats was injected intraperitoneally with 3-methylcholanthrene (3MC) dissolved in corn oil. In rats of all genotypes the hepatic concentration of UDP glucuronosyltransferase (UDPGT) mRNA was increased at 48 and 96 h after the treatment with 3MC. Hepatic activity of 4-nitrophenol UDPGT was increased by 3MC in Wistar-Shi rats and heterozygous Gunn rats but not in homozygous Gunn rats. Urinary ascorbic acid excretion increased 72 and 96 h after the injection with 3MC in Wistar-Shi and heterozygous Gunn rats but not in homozygous Gunn rats. Ninety-six hours after the injection with 3MC, the hepatic concentration of ascorbic acid in Wistar-Shi rats was 90% higher than that in the corresponding control group, whereas in heterozygous and homozygous Gunn rats the increases were 70 and 30%, respectively. Wistar-Shi rats and homozygous Gunn rats were also injected daily for 3 d with sodium phenobarbital. In rats of both genotypes, the activity and hepatic concentration of chloramphenicol-UDPGT mRNA and liver and urine ascorbic acid concentration were increased by sodium phenobarbital. The data indicate that the stimulation of the expression of both the 4-nitrophenol and chloramphenicol UDPGT genes plays a key role in the ascorbic acid biosynthesis induced by 3MC and sodium phenobarbital.

Bilirubin-Induced Neurological Damage: Current and Emerging iPSC-Derived Brain Organoid Models.

Bilirubin-induced neurological damage (BIND) has been a subject of studies for decades, yet the molecular mechanisms at the core of this damage remain largely unknown. Throughout the years, many in vivo chronic bilirubin encephalopathy models, such as the Gunn rat and transgenic mice, have further elucidated the molecular basis of bilirubin neurotoxicity as well as the correlations between high levels of unconjugated bilirubin (UCB) and brain damage. Regardless of being invaluable, these models cannot accurately recapitulate the human brain and liver system; therefore, establishing a physiologically recapitulating in vitro model has become a prerequisite to unveil the breadth of complexities that accompany the detrimental effects of UCB on the liver and developing human brain. Stem-cell-derived 3D brain organoid models offer a promising platform as they bear more resemblance to the human brain system compared to existing models. This review provides an explicit picture of the current state of the art, advancements, and challenges faced by the various models as well as the possibilities of using stem-cell-derived 3D organoids as an efficient tool to be included in research, drug screening, and therapeutic strategies for future clinical applications.

Vascular network expansion, integrity of blood–brain interfaces, and cerebrospinal fluid cytokine concentration during postnatal development in the normal and jaundiced rat

BackgroundSevere neonatal jaundice resulting from elevated levels of unconjugated bilirubin in the blood induces dramatic neurological impairment. Central oxidative stress and an inflammatory response have been associated with the pathophysiological mechanism. Cells forming the blood–brain barrier and the choroidal blood–CSF barrier are the first CNS cells exposed to increased plasma levels of unconjugated bilirubin. These barriers are key regulators of brain homeostasis and require active oxidative metabolism to fulfill their protective functions. The choroid plexus-CSF system is involved in neuroinflammatory processes. In this paper, we address the impact of neonatal hyperbilirubinemia on some aspects of brain barriers. We describe physiological changes in the neurovascular network, blood–brain/CSF barriers integrities, and CSF cytokine levels during the postnatal period in normobilirubinemic animals, and analyze these parameters in parallel in Gunn rats that are deficient in bilirubin catabolism and develop postnatal hyperbilirubinemia.MethodsGunn rats bearing a mutation in UGT1a genes were used. The neurovascular network was analyzed by immunofluorescence stereomicroscopy. The integrity of the barriers was evaluated by [14C]-sucrose permeability measurement. CSF cytokine levels were measured by multiplex immunoassay. The choroid plexus-CSF system response to an inflammatory challenge was assessed by enumerating CSF leukocytes.ResultsIn normobilirubinemic animals, the neurovascular network expands postnatally and displays stage-specific regional variations in its complexity. Network expansion is not affected by hyperbilirubinemia. Permeability of the blood–brain and blood–CSF barriers to sucrose decreases between one- and 9-day-old animals, and does not differ between normobilirubinemic and hyperbilirubinemic rats. Cytokine profiles differ between CSF and plasma in all 1-, 9-, and 18-day-old animals. The CSF cytokine profile in 1-day-old animals is markedly different from that established in older animals. Hyperbilirubinemia perturbs these cytokine profiles only to a very limited extent, and reduces CSF immune cell infiltration triggered by systemic exposure to a bacterial lipopeptide.ConclusionThe data highlight developmental specificities of the blood–brain barrier organization and of CSF cytokine content. They also indicate that a direct effect of bilirubin on the vascular system organization, brain barriers morphological integrity, and inflammatory response of the choroid plexus-CSF system is not involved in the alteration of brain functions induced by severe neonatal jaundice.

Sexual dimorphism: increased sterol excretion leads to hypocholesterolaemia in female hyperbilirubinaemic Gunn rats.

Circulating bilirubin is associated with reduced serum cholesterol concentrations in humans and in hyperbilirubinaemic Gunn rats. However, mechanisms contributing to hypocholesterolaemia remain unknown. Therefore, this study aimed to investigate cholesterol synthesis, transport and excretion in mutant Gunn rats. Adult Gunn and control rats were assessed for daily faecal sterol excretion using metabolic cages, and water was supplemented with [1‐13C]‐acetate to determine cholesterol synthesis. Bile was collected to measure biliary lipid secretion. Serum and liver were collected for biochemical analysis and for gene/protein expression using RT‐qPCR and western blot, respectively. Additionally, serum was collected and analysed from juvenile rats. A significant interaction of sex, age and phenotype on circulating lipids was found with adult female Gunn rats reporting significantly lower cholesterol and phospholipids. Female Gunn rats also demonstrated elevated cholesterol synthesis, greater biliary lipid secretion and increased total faecal cholesterol and bile acid excretion. Furthermore, they possessed increased hepatic low‐density lipoprotein (LDL) receptor and SREBP2 expression. In contrast, there were no changes to sterol metabolism in adult male Gunn rats. This is the first study to demonstrate elevated faecal sterol excretion in female hyperbilirubinaemic Gunn rats. Increased sterol excretion creates a negative intestinal sterol balance that is compensated for by increased cholesterol synthesis and LDL receptor expression. Therefore, reduced circulating cholesterol is potentially caused by increased hepatic uptake via the LDL receptor. Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome).Key points Female adult hyperbilirubinaemic (Gunn) rats demonstrated lower circulating cholesterol, corroborating human studies that report a negative association between bilirubin and cholesterol concentrations.Furthermore, female Gunn rats had elevated sterol excretion creating a negative intestinal sterol balance that was compensated for by elevated cholesterol synthesis and increased hepatic low‐density lipoprotein (LDL) receptor expression.Therefore, elevated LDL receptor expression potentially leads to reduced circulating cholesterol levels in female Gunn rats providing an explanation for the hypocholesterolaemia observed in humans with elevated bilirubin levels.This study also reports a novel interaction of sex with the hyperbilirubinaemic phenotype on sterol metabolism because changes were only reported in females and not in male Gunn rats.Future studies are required to further evaluate the sexual dimorphism of this response and whether similar findings occur in females with benign unconjugated hyperbilirubinaemia (Gilbert's syndrome).

MGE-Like Neural Progenitor Cell Survival and Expression of Parvalbumin and Proenkephalin in a Jaundiced Rat Model of Kernicterus

Kernicterus is a permanent condition caused by brain damage from bilirubin toxicity. Dystonia is one of the most debilitating symptoms of kernicterus and results from damage to the globus pallidus (GP). One potential therapeutic strategy to treat dystonia in kernicterus is to replace lost GP neurons and restore basal ganglia circuits through stem cell transplantation. Toward this end, we differentiated human embryonic stem cells (hESCs) into medial ganglion eminence (MGE; the embryological origin of most of the GP neurons)-like neural precursor cells (NPCs). We determined neurochemical phenotype in cell culture and after transplanting into the GP of jaundiced Gunn rats. We also determined grafted cell survival as well as migration, distribution, and morphology after transplantation. As in the GP, most cultured MGE-like NPCs expressed γ-aminobutyric acid (GABA), with some co-expressing markers for parvalbumin (PV) and others expressing markers for pro-enkephalin (PENK). MGE-like NPCs survived in brains at least 7 weeks after transplantation, with most aggregating near the injection site. Grafted cells expressed GABA and PV or PENK as in the normal GP. Although survival was low and the maturity of grafted cells varied, many cells produced neurite outgrowth. While promising, our results suggest the need to further optimize the differentiation protocol for MGE-like NPC for potential use in treating dystonia in kernicterus.

Microfluidic photoreactor to treat neonatal jaundice

While in most cases, jaundice can be effectively treated using phototherapy, severe cases require exchange transfusion, a relatively risky procedure in which the neonate's bilirubin-rich blood is replaced with donor blood. Here, we examine extracorporeal blood treatment in a microfluidic photoreactor as an alternative to exchange transfusion. This new treatment approach relies on the same principle as phototherapy but leverages microfluidics to speed up bilirubin removal. Our results demonstrate that high-intensity light at 470 nm can be used to rapidly reduce bilirubin levels without causing appreciable damage to DNA in blood cells. Light at 470 nm was more effective than light at 505 nm. Studies in Gunn rats show that photoreactor treatment for 4 h significantly reduces bilirubin levels, similar to the bilirubin reduction observed for exchange transfusion and on a similar time scale. Predictions for human neonates demonstrate that this new treatment approach is expected to exceed the performance of exchange transfusion using a low blood flow rate and priming volume, which will facilitate vascular access and improve safety.

Potential of therapeutic bile acids in the treatment of neonatal Hyperbilirubinemia

Neonatal hyperbilirubinemia or jaundice is associated with kernicterus, resulting in permanent neurological damage or even death. Conventional phototherapy does not prevent hyperbilirubinemia or eliminate the need for exchange transfusion. Here we investigated the potential of therapeutic bile acids ursodeoxycholic acid (UDCA) and obeticholic acid (OCA, 6-α-ethyl-CDCA), a farnesoid-X-receptor (FXR) agonist, as preventive treatment options for neonatal hyperbilirubinemia using the hUGT1*1 humanized mice and Ugt1a-deficient Gunn rats. Treatment of hUGT1*1 mice with UDCA or OCA at postnatal days 10–14 effectively decreased bilirubin in plasma (by 82% and 62%) and brain (by 72% and 69%), respectively. Mechanistically, our findings indicate that these effects are mediated through induction of protein levels of hUGT1A1 in the intestine, but not in liver. We further demonstrate that in Ugt1a-deficient Gunn rats, UDCA but not OCA significantly decreases plasma bilirubin, indicating that at least some of the hypobilirubinemic effects of UDCA are independent of UGT1A1. Finally, using the synthetic, non-bile acid, FXR-agonist GW4064, we show that some of these effects are mediated through direct or indirect activation of FXR. Together, our study shows that therapeutic bile acids UDCA and OCA effectively reduce both plasma and brain bilirubin, highlighting their potential in the treatment of neonatal hyperbilirubinemia.

Normalizing hyperactivity of the Gunn rat with bilirubin-induced neurological disorders via ketanserin.

Severe neonatal hyperbilirubinemia has been known to cause the clinical syndrome of kernicterus and a milder one the syndrome of bilirubin-induced neurologic dysfunction (BIND). BIND clinically manifests itself after the neonatal period as developmental delay, cognitive impairment, and related behavioral and psychiatric disorders. The complete picture of BIND is not clear. The Gunn rat is a mutant strain of the Wistar rat with the BIND phenotype, and it demonstrates abnormal behavior. We investigated serotonergic dysfunction in Gunn rats by pharmacological analyses and ex vivo neurochemical analyses. Ketanserin, the 5-HT2AR antagonist, normalizes hyperlocomotion of Gunn rats. Both serotonin and its metabolites in the frontal cortex of Gunn rats were higher in concentrations than in control Wistar rats. The 5-HT2AR mRNA expression was downregulated without alteration of the protein abundance in the Gunn rat frontal cortex. The TPH2 protein level in the Gunn rat raphe region was significantly higher than that in the Wistar rat. It would be of value to be able to postulate that a therapeutic strategy for BIND disorders would be the restoration of brain regions affected by the serotonergic dysfunction to normal operation to prevent before or to normalize after onset of BIND manifestations. We demonstrated serotonergic dysregulation underlying hyperlocomotion in Gunn rats. This finding suggests that a therapeutic strategy for bilirubin-induced neurologic dysfunction (BIND) would be the restoration of brain regions affected by the serotonergic dysfunction to normal operation to prevent before or to normalize after the onset of the BIND manifestations. Ketanserin normalizes hyperlocomotion of Gunn rats. To our knowledge, this is the first study to demonstrate a hyperlocomotion link to serotonergic dysregulation in Gunn rats.

Life-Long Hyperbilirubinemia Exposure and Bilirubin Priming Prevent In Vitro Metabolic Damage

Background: Unconjugated bilirubin (UCB) is more than the final product of heme catabolism. Mildly elevated systemic bilirubin concentrations, such as in Gilbert syndrome (GS), protect against various oxidative stress-mediated and metabolic diseases, including cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, cancer, and age-related disease. The Gunn rat is an animal model of hereditary hyperbilirubinemia widely used in assessing the effect of high serum bilirubin concentration in various organs. The present work aims to understand if life-long hyperbilirubinemia and bilirubin-priming might contribute to protection against atherosclerosis and diabetic nephropathy (DN) at the cellular level. Methods: Primary aortic endothelial cells and podocytes obtained from hyperbilirubinemic homozygous jj and normobilirubinemic heterozygous Nj Gunn rats were exposed to Palmitic Acid (PA) and Angiotensin II (Ang II), respectively, and the effects on cell viability and the activation of damage-related metabolic pathways evaluated. Results were validated on immortalized H5V and HK2 cells exposed to damage after UCB pretreatment. Results: In both primary cell models, cells obtained from jj Gunn rats showed as significantly higher than Nj Gunn rats at any dose of the toxic agent. Reduction in CHOP expression and IL-6 release was observed in jj primary aortic endothelial cells exposed to PA compared to Nj cells. The same occurred on H5V pretreated with Unconjugated bilirubin. Upon Ang II treatment, primary podocytes from jj Gunn rats showed lower DNA fragmentation, cleaved caspase-3, and cleaved PARP induction than primary podocytes from Nj Gunn rats. In HK2 cells, the induction by Ang II of HIF-1α and LOXl2 was significantly reduced by UCB pretreatment. Conclusion: Our data suggest that in models of atherosclerosis and DN life–long hyperbilirubinemia exposure or bilirubin-priming significantly contribute to decrease the injury by enhancing thecellular defensive response,

Mitochondrial Function, Fatty Acid Metabolism, and Body Composition in the Hyperbilirubinemic Gunn Rat.

Background: Circulating bilirubin is associated with reduced adiposity in human and animal studies. A possible explanation is provided by in vitro data that demonstrates that bilirubin inhibits mitochondrial function and decreases efficient energy production. However, it remains unclear whether hyperbilirubinemic animals have similar perturbed mitochondrial function and whether this is important for regulation of energy homeostasis. Aim: To investigate the impact of unconjugated hyperbilirubinemia on body composition, and mitochondrial function in hepatic tissue and skeletal muscle. Materials and Methods: 1) Food intake and bodyweight gain of 14-week old hyperbilirubinemic Gunn (n = 19) and normobilirubinemic littermate (control; n = 19) rats were measured over a 17-day period. 2) Body composition was determined using dual-energy X-ray absorptiometry and by measuring organ and skeletal muscle masses. 3) Mitochondrial function was assessed using high-resolution respirometry of homogenized liver and intact permeabilized extensor digitorum longus and soleus fibers. 4) Liver tissue was flash frozen for later gene (qPCR), protein (Western Blot and citrate synthase activity) and lipid analysis. Results: Female hyperbilirubinemic rats had significantly reduced fat mass (Gunn: 9.94 ± 5.35 vs. Control: 16.6 ± 6.90 g, p < 0.05) and hepatic triglyceride concentration (Gunn: 2.39 ± 0.92 vs. Control: 4.65 ± 1.67 mg g−1, p < 0.01) compared to normobilirubinemic controls. Furthermore, hyperbilirubinemic rats consumed fewer calories daily (p < 0.01) and were less energetically efficient (Gunn: 8.09 ± 5.75 vs. Control: 14.9 ± 5.10 g bodyweight kcal−1, p < 0.05). Hepatic mitochondria of hyperbilirubinemic rats demonstrated increased flux control ratio (FCR) via complex I and II (CI+II) (Gunn: 0.78 ± 0.16 vs. Control: 0.62 ± 0.09, p < 0.05). Similarly, exogenous addition of 31.3 or 62.5 μM unconjugated bilirubin to control liver homogenates significantly increased CI+II FCR (p < 0.05). Hepatic PGC-1α gene expression was significantly increased in hyperbilirubinemic females while FGF21 and ACOX1 was significantly greater in male hyperbilirubinemic rats (p < 0.05). Finally, hepatic mitochondrial complex IV subunit 1 protein expression was significantly increased in female hyperbilirubinemic rats (p < 0.01). Conclusions: This is the first study to comprehensively assess body composition, fat metabolism, and mitochondrial function in hyperbilirubinemic rats. Our findings show that hyperbilirubinemia is associated with reduced fat mass, and increased hepatic mitochondrial biogenesis, specifically in female animals, suggesting a dual role of elevated bilirubin and reduced UGT1A1 function on adiposity and body composition.

Curcumin Prevents Cerebellar Hypoplasia and Restores the Behavior in Hyperbilirubinemic Gunn Rat by a Pleiotropic Effect on the Molecular Effectors of Brain Damage

Bilirubin toxicity to the central nervous system (CNS) is responsible for severe and permanent neurologic damage, resulting in hearing loss, cognitive, and movement impairment. Timely and effective management of severe neonatal hyperbilirubinemia by phototherapy or exchange transfusion is crucial for avoiding permanent neurological consequences, but these therapies are not always possible, particularly in low-income countries. To explore alternative options, we investigated a pharmaceutical approach focused on protecting the CNS from pigment toxicity, independently from serum bilirubin level. To this goal, we tested the ability of curcumin, a nutraceutical already used with relevant results in animal models as well as in clinics in other diseases, in the Gunn rat, the spontaneous model of neonatal hyperbilirubinemia. Curcumin treatment fully abolished the landmark cerebellar hypoplasia of Gunn rat, restoring the histological features, and reverting the behavioral abnormalities present in the hyperbilirubinemic rat. The protection was mediated by a multi-target action on the main bilirubin-induced pathological mechanism ongoing CNS damage (inflammation, redox imbalance, and glutamate neurotoxicity). If confirmed by independent studies, the result suggests the potential of curcumin as an alternative/complementary approach to bilirubin-induced brain damage in the clinical scenario.

Choline supplementation prevents the effects of bilirubin on cerebellar-mediated behavior in choline-restricted Gunn rat pups.

Background:Bilirubin is produced by the breakdown of hemoglobin, and is normally catabolized and excreted. Neurotoxic accumulation of serum bilirubin often occurs in premature infants. The homozygous Gunn rat lacks uridine diphosphate-glucuronosyltransferase-1A1 (UGT1A1), the enzyme needed to biotransform bilirubin. This rodent model of hyperbilirubinemia emulates many aspects of bilirubin toxicity observed in the human infant. We demonstrate that choline supplementation in early postnatal development is neuroprotective in the choline restricted Gunn rat, when hyperbilirubinemia is induced on postnatal day 5.Methods:We first compared behaviors and cerebellar weight of pups born to dams consuming regular rat chow to those of dams consuming choline-restricted diets. Secondly, we measured behaviors and cerebellar weights of pups born to choline-restricted dams, reared on a choline-restricted diet, supplemented with or without choline and treated with or without sulfadimethoxine (SDMX).Results:A choline-restricted diet did not change the behavioral outcomes, but cerebellar weight was reduced in the choline-restricted group regardless of genotype or SDMX administration. SDMX induced behavioral deficits in jj pups, and choline supplementation improved most behavioral effects and cerebellar weight in SDMX-treated jj rats.Conclusion:These results suggest that choline may be used as a safe and effective neuroprotective intervention against hyperbilirubinemia in the choline deficient premature infant.

Low Serum Levels of Fibroblast Growth Factor 2 in Gunn Rats: A Hyperbilirubinemia Animal Model of Schizophrenic Symptoms.

Fibroblast Growth Factor (FGF) 2 (also referred to as basic FGF) is a multifunctional growth factor that plays a pivotal role in the pro-survival, pro-migration and prodifferentiation of neurons. Because alterations in FGF2 levels are suggested to contribute to the pathogenesis of schizophrenia, we investigated serum levels of FGF2 in the Gunn rat, a hyperbilirubinemia animal model of schizophrenic symptoms. The enzyme-linked immunosorbent assay showed that the serum levels of FGF2 in Gunn rats were 5.09 ± 0.236 pg/mL, while those in the normal strain Wistar rats, serum levels were 11.90 ± 2.142 pg/mL. The serum FGF2 levels in Gunn rats were significantly lower than those in Wistar rats. We also measured serum levels of Unconjugated Bilirubin (UCB) and found a significant negative correlation between UCB and FGF2 in terms of serum levels in all the rats studied. Since it is known that FGF2 regulates dopaminergic neurons and have antineuroinflammatory effects, our finding suggests that low FGF2 levels may contribute to the pathogenesis of schizophrenia, in which imbalanced dopamin-ergic signaling and neuroinflammation are supposed to play certain roles.

Induction of fecal cholesterol excretion is not effective for the treatment of hyperbilirubinemia in Gunn rats.

Unconjugated hyperbilirubinemia, a feature of neonatal jaundice or Crigler-Najjar syndrome, can lead to neurotoxicity and even death. We previously demonstrated that unconjugated bilirubin (UCB) can be eliminated via transintestinal excretion in Gunn rats, a model of unconjugated hyperbilirubinemia, and that this is stimulated by enhancing fecal fatty acid excretion. Since transintestinal excretion also occurs for cholesterol (TICE), we hypothesized that increasing fecal cholesterol excretion and/or TICE could also enhance fecal UCB disposal and subsequently lower plasma UCB concentrations. To determine whether increasing fecal cholesterol excretion could ameliorate unconjugated hyperbilirubinemia, we treated hyperbilirubinemic Gunn rats with ezetimibe (EZE), an intestinal cholesterol absorption inhibitor, and/or a liver X receptor (LXR) and farnesoid X receptor (FXR) agonist (T0901317 (T09) and obeticholic acid (OCA), respectively), known to stimulate TICE. We found that EZE treatment alone or in combination with T09 or OCA increased fecal cholesterol disposal but did not lower plasma UCB levels. These findings do not support a link between the regulation of transintestinal excretion of cholesterol and bilirubin. Furthermore, induction of fecal cholesterol excretion is not a potential therapy for unconjugated hyperbilirubinemia. Increasing fecal cholesterol excretion is not effective to treat unconjugated hyperbilirubinemia. This is the first time a potential relation between transintestinal excretion of cholesterol and unconjugated bilirubin is investigated. Transintestinal excretion of cholesterol and unconjugated bilirubin do not seem to be quantitatively linked. Unlike intestinal fatty acids, cholesterol cannot "capture" unconjugated bilirubin to increase its excretion. These results add to our understanding of ways to improve and factors regulating unconjugated bilirubin disposal in hyperbilirubinemic conditions.

A Gunn rat model of preterm hyperbilirubinemia.

BackgroundThe impact of bilirubin in preterm infants is poorly understood. An animal model would assist in improving understanding. The Gunn rat lacks uridine diphosphate-glucuronylsyl transferase 1 and can be made acutely hyperbilirubinemic by injection of sulfodimethoxine (sulfa), a drug that displaces bilirubin from albumin and thus increases free bilirubin.MethodsOn postnatal day (P) 5, Gunn rats either heterozygous (Nj) or homozygous (jj) for glucuronosyltransferase activity were injected with either saline or sulfa. Behavior and cerebellar weight were measured.ResultsPups did not show any signs of acute bilirubin encephalopathy. Pup weight dropped significantly on P8 only in the jj-sulfa group. Behavior was affected only in the jj-sulfa group. Cerebellar weight was significantly less in the jj-sulfa group.DiscussionThe Gunn rat pup model may be a good model to study hyperbilirubinemia in preterm infants.