RESUMEN
Poking palpebral conjunctiva evoked upper-eyelid retraction during ophthalmic surgery. Iatrogenic eyelid ptosis occurred if eyelid branch of lachrymal nerve was sectioned. Mesencephalic trigeminal nucleus (Vme) neurons were labeled when tracer injected into lachrymal nerve innervating eyelid Mueller's muscle. Masseter afferent Vme neurons projecting to oculomotor nucleus (III) was observed in toad and rat, which helps amphibians to stare prey when they open mouth widely to prey. We hypothesized single Vme neurons may have peripheral collaterals to both eyelid and masseter muscles. WGA-594 was injected into upper eyelid, and WGA-488 was simultaneously delivered into ipsilateral masseter muscle in the same rat. Then, double labeled Vme neurons were found under both conventional and confocal microscope. Meanwhile, contact of WGA-594 positive eyelid afferent Vme neurons with WGA-488 labeled masseter afferent ones were observed sometimes. Combined with our previous observation of oculomotor projection Vme neurons, we thought WGA-594/488 double labeled Vme cells, at least some of them, are oculomotor projecting ones. Contact between eyelid and masseter afferent Vme neurons are supposed to be electrotonically coupled, based on a line of previous studies. If exogenous or genetic factors make these Vme neurons misinterpret masseter input as eyelid afferent signals, these Vme neurons might feedforward massages to eyelid retractor motoneurons in the III. Besides, oculomotor projecting Vme neurons might be co-fired by adjacent masseter afferent Vme neurons through electrotonic coupling once the masseter muscle is activated. In these cases, Marcus Gunn Syndrome might occur. This finding leads to a new hypothesis for the Syndrome.
Asunto(s)
Blefaroptosis , Músculo Masetero , Ratas , Animales , Ratas Gunn , Neuronas Aferentes , Neuronas Motoras , Párpados , Tegmento Mesencefálico , Núcleos del Trigémino , Nervio Trigémino/fisiologíaRESUMEN
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.
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Células Madre Pluripotentes Inducidas , Traumatismos del Sistema Nervioso , Animales , Bilirrubina , Encéfalo , Humanos , Ratones , Organoides , Ratas , Ratas GunnRESUMEN
BACKGROUND: Severe 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. METHODS: Gunn 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. RESULTS: In 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. CONCLUSION: The 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.
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Barrera Hematoencefálica , Ictericia Neonatal , Animales , Bilirrubina/metabolismo , Barrera Hematoencefálica/metabolismo , Líquido Cefalorraquídeo/metabolismo , Plexo Coroideo/metabolismo , Citocinas/metabolismo , Humanos , Hiperbilirrubinemia/metabolismo , Recién Nacido , Ictericia Neonatal/metabolismo , Ratas , Ratas Gunn , SacarosaRESUMEN
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.
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Distonía , Ictericia , Kernicterus , Células-Madre Neurales , Animales , Encefalinas , Ictericia/terapia , Células-Madre Neurales/trasplante , Parvalbúminas/metabolismo , Precursores de Proteínas , Ratas , Ratas Gunn , Ácido gamma-Aminobutírico/metabolismoRESUMEN
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 [1-13 C]-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).
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Enfermedad de Gilbert , Hipercolesterolemia , Animales , Bilirrubina/metabolismo , Colesterol/metabolismo , Femenino , Enfermedad de Gilbert/metabolismo , Hiperbilirrubinemia/metabolismo , Hipercolesterolemia/metabolismo , Lipoproteínas LDL/metabolismo , Hígado/metabolismo , Masculino , Ratas , Ratas Gunn , Receptores de LDL/genética , Receptores de LDL/metabolismo , Caracteres Sexuales , Esteroles/metabolismoRESUMEN
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. IMPACT: 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.
Asunto(s)
Bilirrubina , Kernicterus , Animales , Humanos , Hiperbilirrubinemia/complicaciones , Kernicterus/prevención & control , Ketanserina/farmacología , Ratas , Ratas Gunn , Ratas WistarRESUMEN
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.
Asunto(s)
Ácido Quenodesoxicólico/análogos & derivados , Hiperbilirrubinemia Neonatal/tratamiento farmacológico , Ácido Ursodesoxicólico/uso terapéutico , Animales , Ácidos y Sales Biliares/uso terapéutico , Bilirrubina/sangre , Ácido Quenodesoxicólico/uso terapéutico , Hiperbilirrubinemia Neonatal/sangre , Íleon/efectos de los fármacos , Íleon/metabolismo , Isoxazoles/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratas Gunn , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismo , Resultado del TratamientoRESUMEN
BACKGROUND: 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. METHODS: 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. RESULTS: We found that EZE treatment alone or in combination with T09 or OCA increased fecal cholesterol disposal but did not lower plasma UCB levels. CONCLUSIONS: 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. IMPACT: 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.
Asunto(s)
Ácido Quenodesoxicólico/análogos & derivados , Colesterol/metabolismo , Síndrome de Crigler-Najjar/terapia , Ezetimiba/uso terapéutico , Heces/química , Hidrocarburos Fluorados/uso terapéutico , Hiperbilirrubinemia/terapia , Sulfonamidas/uso terapéutico , Animales , Bilis/química , Ácidos y Sales Biliares/metabolismo , Bilirrubina/química , Ácido Quenodesoxicólico/farmacología , Ácido Quenodesoxicólico/uso terapéutico , Síndrome de Crigler-Najjar/metabolismo , Grasas de la Dieta/farmacocinética , Ezetimiba/farmacología , Haptoglobinas/análisis , Hidrocarburos Fluorados/farmacología , Intestinos/efectos de los fármacos , Intestinos/metabolismo , Lípidos/sangre , Receptores X del Hígado/metabolismo , Masculino , PPAR delta/metabolismo , Distribución Aleatoria , Ratas , Ratas Gunn , Receptores Citoplasmáticos y Nucleares/metabolismo , Sulfonamidas/farmacologíaRESUMEN
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. Second, 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. CONCLUSIONS: These results suggest that choline may be used as a safe and effective neuroprotective intervention against hyperbilirubinemia in the choline-deficient premature infant. IMPACT: This article investigates the effect of neonatal jaundice/bilirubin neurotoxicity on cerebellar-mediated behaviors. This article explores the potential use of choline as an intervention capable of ameliorating the effect of bilirubin on the choline-restricted developing brain. This article opens the door for future studies on the action of choline in the presence of hyperbilirubinemia, especially in preterm neonates.
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Conducta Animal , Bilirrubina/metabolismo , Cerebelo/fisiología , Colina/administración & dosificación , Suplementos Dietéticos , Animales , Animales Recién Nacidos , Ictericia Neonatal/sangre , Ratas , Ratas GunnRESUMEN
Marcus Gunn Syndrome (MGS), mostly occurring in congenital ptosis patients, is characterized by jaw movement evoking ptotic eyelid retraction, followed by collapse. Inverted, bilateral and acquired MGS were also reported. Some cases manifest MGS only temporarily in life. These features suggest MGS may be due to multiple pathogeneses, which are still unclear. People also classify MGS as a kind of trigeminal oculomotor synkenesis (TOS), like Duane syndrome (DS), in which ocular adduction prompts eyelid moving or eyeball retraction. The most popular hypothesis for TOS is congenital miswiring, as evidence supporting this hypothesis is found in DS cases: hypoplasia abducens nerve fusing with a branch of oculomotor nerve is observed. Seven mutant genes have been identified associated with TOS and two of them are found among MGS cases. Accordingly, these mutant genes may dominate cranial nerve misconnection and generate TOS. However, unlike in DS case, evidence of miswiring is not encountered in most MGS cases. The fact is that two "MGS genes" are from congenital fibrosis of extraocular muscle (CFEOM) cases presenting with associated MGS. But most of MGS cases do not suffer CFEOM. Thus, mutant genes dominated congenital miswiring may not be the pathogenesis for the majority of MGS. As an alternate pathogenic pathway, a "release hypothesis" proposed that MGS is a primitive physiologic reflex that became suppressed during phylogenetic development but could be released under certain pathologic conditions. This hypothesis was and is overlooked because the hypothesized reflex arc has not been defined. Decades ago, a neural tract tracing study in Xenopus revealed a direct projection from masticator afferent mesencephalic trigeminal nucleus (Vme) neurons to oculomotor and trochlear nucleus (III/IV). In clinical studies, co-firing of pterygoid muscle and levator palpebrae was recorded by electromyography during onset of MGS, and stimulating pterygoid muscle nerve elicited eyelid retraction. Recently, retraction of the ipsilateral eyelid by stimulating the trigeminal motor root was even observed in cases without congenital ptosis and MGS, highlighting the existence of a latent pathway. In rats, recently we demonstrated projections from the Vme neurons to the III/IV, and to their premotor neurons in interstitial nucleus of Cajal by neural tract tracing and electrophysiologic studies. Fos expression in pre-oculomotor neurons was induced by repeated down stretching the lower jaw. Combining previous and our own studies, we assumed the Vme neurons is excited when jaw moves and in turn, some eyelid activity related III motoneurons are activated through projections of Vme to oculomotor system, like in Xenopus. Genetic factors may dominate to what extent this primitive reflex-arc is preserved, which consequently determines phenotype.
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Blefaroptosis , Anomalías Maxilomandibulares , Animales , Humanos , Músculos Oculomotores , Filogenia , Ratas , Ratas Gunn , Nervio TrigéminoRESUMEN
BACKGROUND: 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. METHOD: 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. RESULTS: 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. CONCLUSION: 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.
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Factor 2 de Crecimiento de Fibroblastos/sangre , Hiperbilirrubinemia/sangre , Esquizofrenia/sangre , Animales , Bilirrubina/sangre , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Gunn , Ratas WistarRESUMEN
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.
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Conducta Animal/efectos de los fármacos , Lesiones Encefálicas/prevención & control , Cerebelo/anomalías , Modelos Animales de Enfermedad , Hiperbilirrubinemia/fisiopatología , Malformaciones del Sistema Nervioso/prevención & control , Animales , Animales Recién Nacidos , Conducta Animal/fisiología , Bilirrubina/sangre , Lesiones Encefálicas/fisiopatología , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/patología , Sistema Nervioso Central/fisiopatología , Cerebelo/efectos de los fármacos , Cerebelo/patología , Cerebelo/fisiopatología , Discapacidades del Desarrollo/fisiopatología , Discapacidades del Desarrollo/prevención & control , Humanos , Inflamación/fisiopatología , Inflamación/prevención & control , Malformaciones del Sistema Nervioso/fisiopatología , Células de Purkinje/efectos de los fármacos , Células de Purkinje/patología , Ratas Gunn , Resultado del TratamientoRESUMEN
PURPOSE: The purpose of this study was to determine the importance of UGT1A1 activity on the metabolism and pharmacokinetics of a releasable PEG ~ SN-38 conjugate, PLX038A. Irinotecan (CPT-11) is converted to the topoisomerase 1 inhibitor SN-38 by first-pass hepatic metabolism and is converted to its glucuronide SN-38G by UGT1A1. With diminished UGT1A1 activity, the high liver exposure to SN-38 can cause increased toxicity of CPT-11. In contrast, releasable PEG ~ SN-38 conjugates-such as PLX038-release SN-38 in the vascular compartment, and only low levels of SN-38 are expected to enter the liver by transport through the OATP1B1 transporter. METHODS: We measured CPT-11 and PLX038A metabolites in plasma and bile, and determined pharmacokinetics of PLX038A in UGT1A-deficient and replete rats. RESULTS: Compared to CPT-11, treatment of rats with PLX038A results in very low levels of biliary SN-38 and SN-38G, a low flux through UGT1A, and a low SN-38G/SN-38 ratio in plasma. Further, the pharmacokinetics of plasma PLX038A and SN-38 in rats deficient in UGT1A is unchanged compared to normal rats. CONCLUSIONS: The disposition of PEGylated SN-38 is independent of UGT1A activity in rats, and PLX038 may find utility in full-dose treatment of patients who are UGT1A1*28 homozygotes or have metastatic disease with coincidental or incidental liver dysfunction.
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Camptotecina/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Glucuronatos/farmacología , Glucuronosiltransferasa/metabolismo , Irinotecán/farmacología , Polietilenglicoles/química , Profármacos/farmacología , Inhibidores de Topoisomerasa I/farmacología , Animales , Bilis/metabolismo , Camptotecina/farmacocinética , Camptotecina/farmacología , Glucuronatos/farmacocinética , Irinotecán/farmacocinética , Hígado/metabolismo , Profármacos/farmacocinética , Ratas , Ratas Gunn , Distribución Tisular , Inhibidores de Topoisomerasa I/farmacocinéticaRESUMEN
Pluripotent stem cells have been investigated as a renewable source of therapeutic hepatic cells, in order to overcome the lack of transplantable donor hepatocytes. Whereas different studies were able to correct hepatic defects in animal models, they focused on the most mature phenotype of hepatocyte-like cells (HLCs) derived from pluripotent stem cells and needed freshly prepared cells, which limits clinical applications of HLCs. Here, we report the production of hepatic stem cells (pHSCs) from human-induced pluripotent stem cells (hiPSCs) in xeno-free, feeder-free, and chemically defined conditions using as extracellular matrix a recombinant laminin instead of Matrigel, an undefined animal-derived matrix. Freshly prepared and frozen pHSCs were transplanted via splenic injection in Gunn rats, the animal model for Crigler-Najjar syndrome. Following cell transplantation and daily immunosuppression treatment, bilirubinemia was significantly decreased (around 30% decrease, P < .05) and remained stable throughout the 6-month study. The transplanted pHSCs underwent maturation in vivo to restore the deficient metabolic hepatic function (bilirubin glucuronidation by UGT1A1). In conclusion, we demonstrate for the first time the differentiation of hiPSCs into pHSCs that (a) are produced using a differentiation protocol compatible with Good Manufacturing Practices, (b) can be frozen, and (c) are sufficient to demonstrate in vivo therapeutic efficacy to significantly lower hyperbilirubinemia in a model of inherited liver disease, despite their immature phenotype. Thus, our approach provides major advances toward future clinical applications and would facilitate cell therapy manufacturing from human pluripotent stem cells.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Síndrome de Crigler-Najjar/terapia , Hepatocitos/fisiología , Hiperbilirrubinemia/terapia , Células Madre Pluripotentes Inducidas/fisiología , Hígado/fisiología , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular , Células Cultivadas , Criopreservación , Modelos Animales de Enfermedad , Humanos , Hígado/cirugía , Ratas , Ratas Gunn , Medicina Regenerativa/métodos , Trasplante HeterólogoRESUMEN
BACKGROUND: The 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. METHODS: On 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. RESULTS: Pups 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. CONCLUSION: The Gunn rat pup model may be a good model to study hyperbilirubinemia in preterm infants.
Asunto(s)
Bilirrubina/sangre , Hiperbilirrubinemia Neonatal/inducido químicamente , Sulfadimetoxina , Animales , Animales Recién Nacidos , Conducta Animal , Biomarcadores/sangre , Cerebelo/patología , Modelos Animales de Enfermedad , Femenino , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Hiperbilirrubinemia Neonatal/sangre , Hiperbilirrubinemia Neonatal/genética , Hiperbilirrubinemia Neonatal/patología , Masculino , Ratas Gunn , Factores de Tiempo , Pérdida de PesoRESUMEN
BACKGROUND: Although growing evidence indicates that ECT affects astrocytes, the exact mechanisms of the therapeutic effect of ECT are still unknown. Astrocytic endfeet express the water channel aquaporin (AQP) 4 abundantly and ensheath brain blood vessels to form gliovascular units. It has been shown that the coverage of blood vessels by AQP4-immunostained endfeet is decreased in the prefrontal cortex (PFC) of patients with major depression. This study was made to determine whether ECT restores the astrocytic coverage of blood vessels with amelioration of depressive symptoms. METHODS: After electroconvulsive shock (ECS) administration to rats, the forced swimming test (FST) and Y-maze test were performed. Subsequently, immunofluorescence analysis was conducted to measure the coverage of blood vessels by astrocytic endfeet in the PFC and hippocampus by using the endothelial cell marker lectin and anti-AQP4 antibody. We also performed Western blot to examine the effects of ECS on the hippocampal expression of AQP4 and the tight junction molecule claudin-5. RESULTS: Gunn rats showed learned helplessness and impaired spatial working memory, compared to normal control Wistar rats. ECS significantly improved the depressive-like behavior. Gunn rats showed a decrease in astrocytic coverage of blood vessels, that was significantly increased by ECS. ECS significantly increased expression of AQP4 and claudin-5 in Gunn rats. CONCLUSIONS: ECS increased the reduced coverage of blood vessels by astrocytic endfeet in the mPFC and hippocampus with amelioration of depressive-like behavior. Therefore, therapeutic mechanism of ECT may involve restoration of the impaired gliovascular units by increasing the astrocytic-endfoot coverage of blood vessels.
Asunto(s)
Astrocitos/metabolismo , Depresión/metabolismo , Electrochoque , Trastornos de la Memoria/metabolismo , Animales , Trastorno Depresivo Mayor/metabolismo , Hipocampo/metabolismo , Humanos , Masculino , Aprendizaje por Laberinto , Corteza Prefrontal/metabolismo , Ratas , Ratas Gunn , Ratas WistarRESUMEN
Decreased inflammatory status has been reported in subjects with mild unconjugated hyperbilirubinemia. However, mechanisms of the anti-inflammatory actions of bilirubin (BR) are not fully understood. The aim of this study is to assess the role of BR in systemic inflammation using hyperbilirubinemic Gunn rats as well as their normobilirubinemic littermates and further in primary hepatocytes. The rats were treated with lipopolysaccharide (LPS, 6 mg/kg intraperitoneally) for 12 h, their blood and liver were collected for analyses of inflammatory and hepatic injury markers. Primary hepatocytes were treated with BR and TNF-α. LPS-treated Gunn rats had a significantly decreased inflammatory response, as evidenced by the anti-inflammatory profile of white blood cell subsets, and lower hepatic and systemic expressions of IL-6, TNF-α, IL-1ß, and IL-10. Hepatic mRNA expression of LPS-binding protein was upregulated in Gunn rats before and after LPS treatment. In addition, liver injury markers were lower in Gunn rats as compared to in LPS-treated controls. The exposure of primary hepatocytes to TNF-α with BR led to a milder decrease in phosphorylation of the NF-κB p65 subunit compared to in cells without BR. In conclusion, hyperbilirubinemia in Gunn rats is associated with an attenuated systemic inflammatory response and decreased liver damage upon exposure to LPS.
Asunto(s)
Hiperbilirrubinemia/complicaciones , Inflamación/complicaciones , Animales , Apoptosis/efectos de los fármacos , Bilirrubina/farmacología , Biomarcadores/sangre , Células Cultivadas , Citocinas/sangre , Citocinas/genética , Citocinas/metabolismo , Citoprotección/efectos de los fármacos , Femenino , Hepatocitos/metabolismo , Hiperbilirrubinemia/sangre , Leucocitos/metabolismo , Lipopolisacáridos , Hígado/metabolismo , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Gunn , Transducción de SeñalRESUMEN
BACKGROUND: Advances in prenatal screening and early diagnosis of genetic disease will potentially allow for preemptive treatment of anticipated postnatal disease by in utero cell transplantation (IUCT). This strategy carries potential benefits over postnatal treatment, which might allow for improved engraftment and function of the transplanted cells. Congenital metabolic disorders may be an ideal target for this type of therapy, as in most cases, they require replacement of a single deficient hepatic enzyme, and multiple small-animal models exist for preclinical testing. METHODS: The Gunn rat, a Crigler-Najjar syndrome model animal lacking UDP-glucuronosyltransferase (UGT1A1), was used as recipient. Human amniotic epithelial cells (hAECs), which possess hepatic differentiation potential, were transplanted into the midgestation fetal Gunn rat liver via ultrasound-guided IUCT. The impact of IUCT on live birth and postnatal survival was evaluated. Human cell engraftment was immunohistochemically analyzed on postnatal day 21. RESULTS: Ultrasound-guided IUCT was conducted in rat fetuses on embryonic day 16. Following IUCT, the antihuman mitochondria-positive cells were detected in the liver of recipient rats at postnatal day 21. CONCLUSIONS: Here, we have introduced ultrasound-guided IUCT of hAEC using a small-animal model of a congenital metabolic disorder without immunosuppression. The immunological advantage of IUCT was demonstrated with xenogeneic IUCT. This procedure is suitable to conduct preclinical studies for exploring the feasibility and efficacy of ultrasound-guided transuterine cell injection using rodent disease models.
Asunto(s)
Síndrome de Crigler-Najjar/cirugía , Terapias Fetales , Hígado/cirugía , Placenta/citología , Trasplante de Células Madre , Ultrasonografía Intervencional , Animales , Supervivencia Celular , Síndrome de Crigler-Najjar/diagnóstico por imagen , Síndrome de Crigler-Najjar/embriología , Síndrome de Crigler-Najjar/metabolismo , Modelos Animales de Enfermedad , Femenino , Terapias Fetales/efectos adversos , Edad Gestacional , Supervivencia de Injerto , Humanos , Hígado/diagnóstico por imagen , Hígado/embriología , Hígado/metabolismo , Embarazo , Ratas Gunn , Trasplante de Células Madre/efectos adversos , Factores de Tiempo , Trasplante HeterólogoRESUMEN
BACKGROUND: Phototherapy (PT) is the standard treatment of neonatal unconjugated hyperbilirubinemia. Fluorescent tube (FT)-emitted PT light is known to induce oxidative DNA damage in neonates. Nowadays, however, FTs have largely been replaced by light-emitting diodes (LEDs) for delivering PT. Until now, it is unknown whether LED-PT causes oxidative DNA damage. We aim to determine whether LED-PT induces oxidative DNA damage in hyperbilirubinemic rats. METHODS: Adult Gunn rats, with genetically unconjugated hyperbilirubinemia, received LED-PT in the clinically relevant doses of 10 or 30 µW/cm2/nm. Urine was collected at 0, 24, and 48 h of PT. A group of young Gunn rats received intensive LED-PT of 100 µW/cm2/nm for 24 h. Urine was collected every 8 h and analyzed for the levels of oxidative DNA damage marker 8-hydroxy-2'deoxyguanosine (8-OHdG) and creatinine. DNA damage was evaluated by immunohistochemistry (γH2AX) of skin and spleen samples. RESULTS: LED-PT of 10 and 30 µW/cm2/nm did not affect urinary concentrations of 8-OHdG and creatinine or the 8-OHdG/creatinine ratio. Likewise, intensive LED-PT did not affect the 8-OHdG/creatinine ratio or the number of γH2AX-positive cells in the skin or spleen. CONCLUSIONS: Our results show that LED-PT does not induce oxidative DNA damage in hyperbilirubinemic Gunn rats either at clinically relevant or intensive dosages.
Asunto(s)
Daño del ADN , Estrés Oxidativo , Fototerapia/métodos , Animales , Hiperbilirrubinemia Neonatal , Ratas , Ratas GunnRESUMEN
Gunn rats bear a mutation within the uridine diphosphate glucuronosyltransferase-1a1 (Ugt1a1) gene resulting in high serum bilirubin levels as seen in Crigler-Najjar syndrome. In this study, the Gunn rat was used as an animal model for heritable liver dysfunction. Induced mesenchymal stem cells (iMSCs) derived from embryonic stem cells (H1) and induced pluripotent stem cells were transplanted into Gunn rats after partial hepatectomy. The iMSCs engrafted and survived in the liver for up to 2 months. The transplanted iMSCs differentiated into functional hepatocytes as evidenced by partially suppressed hyperbilirubinemia and expression of multiple human-specific hepatocyte markers such as albumin, hepatocyte nuclear factor 4α, UGT1A1, cytokeratin 18, bile salt export pump, multidrug resistance protein 2, Na/taurocholate-cotransporting polypeptide, and α-fetoprotein. These findings imply that transplanted human iMSCs can contribute to liver regeneration in vivo and thus represent a promising tool for the treatment of inherited liver diseases.