A new animal model in the study of UCB metabolism and neurotoxicity

2007/2008Hyperbilirubinemia is the most common clinical situation during neonatal life and it is observed in 60% of full−term and 80% of pre−term infants. A combination of factors still not well defined such as: prematurity, infections, genetic disorders, brest-feed under-nourishing, may cause hazardous, toxic levels of UnConjugated Bilirubin (UCB) during neonatal period (neonatal jaundice) that pose a direct threat of brain bamage (kernicterus). The deposition of UCB in the Central Nervous System (CNS) causes Bilirubin Encephalopathy (BE) with lifelong motor, auditory and mental impairment. The in vivo knowledge on kernicterus derives almost totally from the investigation on Gunn rat that is a natural model for BE. In this animal model the genetic lesion are closely parallel those present in the Crigler-Najjar syndrome type I and the neuropathological lesions are also similar to those found in humans. The Gunn rat is a mutant strain of Wistar rats that lack the uridin di phospho glucoronosyl transferase (UDPGT) activity toward bilirubin. Although the Gunn rat the classical laboratory model for bilirubin encephalopathy its use for the study of molecular mechanisms involved and the determination of other genes modulating the disease is limited by the existence of different strains and by the impossibility to generate targeted mutations in rats, preventing the in vivo study of the role of other genes in BE (i.e. Mrp1). The aim of my PhD project was to generate a mouse model of hyperbilirubinemia due to a one base deletion in the UGT1a1 gene, identical to the one present in the Gunn rat. To reach this goal, we took advantage to the “Gene Targeting” technique. This genetic technique uses the homologous recombination to modify an endogenous gene. First we constructed the targeting vector specific for the gene of interest. To target genes in mice, the targeting vector was inserted into mouse embryonic stem cells (ES) in culture. At the same time we set up two screening strategies to verify the presence of the targeted mutation in electroporated ES cells (Southern blot and Multiplex PCR). Two positive clones were identified (A9 and G7). Then the positive ES clones were amplified and injected into the blastocysts. Blastocysts were implanted in to a foster mother to obtain the so-called “chimera.” Chimeric mice have two different populations of genetically distinct type of cells originated from different mouse strains and can be selected by the fur colour. We obtained two chimeras, one deriving from A9 clone and one from G7. These chimeras were mated with wild type mice to check for germ line transmission. If the modified ES cells made up the reproductive organ, the offspring will inherit the mutated allele (heterozygous). At the present time we are screening the offspring of the chimeras, to check for germ line transmission. Mating the heterozygous mice, the offspring will have the entire body based on the previously mutated embryonic stem cell (homozygous). Obtaining this new animal model for bilirubin neurotoxicity (Gunn mouse) will be crucial to understand the mechanisms regulating the disease, together with an improvement of the diagnosis, prediction of the prognosis, and development of new therapeutic strategies.Più del 60% dei neonati a termine e l’80% dei neonati prematuri sviluppa un ittero fisiologico nella prima settimana di vita a causa dell’immaturità dei processi fisiologici correlati al metabolismo della bilirubina durante il periodo neonatale. Tuttavia, una combinazione di fattori non ancora ben definiti quali: prematurità, disidratazione, sepsi, disordini di tipo genetico e/o malnutrimento durante l’allattamento, possono causare livelli di bilirubina non coniugata (UCB) eccezionalmente alti durante il periodo neonatale (iperbilirubinemia neontale). Questa condizione fa si che l’UCB possa attraversare in maniera massiva la barriera ematoncefalica (BBB) e depositarsi in specifiche aree cerebrali, ponendo il neonato a rischio di sviluppare encefalopatia da bilirubina (kernittero). Questa grave patologia (tipica della sindrome di Crigler-Najjar-I) si caratterizza per: sordità e disfunzioni uditive, gravi disordini motori (atetosi, spasticità muscolare e ipotonia), disfunzioni visive e displasia dentale. Il modello animale classico per il kernittero è rappresentato dal ratto Gunn. In questo ceppo di ratti è presente una mutazione nel gene dell’UGT che determina la completa inattività dell’enzima epatico bilirubin-glucuronil transferasi (UGT1A1) responsabile della coniugazione dell’UCB a due acidi glucuronici e la successiva eliminazione della bilirubina coniugata attraverso la bile. Gli effetti fisiologici della mutazione a carico dell’UGT1A1 sono molto simili a quelli riscontrati nei pazienti affetti da sindrome di Crigler-Najjar I. L’esistenza di questo modello animale ha consentito un approccio sperimentale al problema della neurotossicità da bilirubina. Tuttavia il modello del ratto Gunn possiede innumerevoli limitazioni, come: l’esistenza di diversi ceppi e, ancora più importante, l’impossibilità di ottenere ceppi mutanti per altri geni coinvolti nel metabolismo della bilirubina (i.e. Mrp1). Pertanto l’obiettivo del mio progetto di dottorato è stata la generazione di un modello murino di iperbilirubinemia dovuto ad una delezione di una base nel gene UGT1, identica a quella presente nel ratto Gunn. La tecnica utilizzata per raggiungere tale scopo è denominata “Gene Targeting”; tale tecnica biotecnologica si serve della ricombinazione omologa per modificare uno specifico gene d’interesse. Il primo passo è stata la costruzione di un vettore specifico per il gene di interesse (UGT1a1) contenete la delezione di una base nell’Esone 4. Parallelamente alla costruzione del vettore di targeting abbiamo messo a punto due tecniche che permettano lo screening dei cloni resistenti alla doppia selezione (positiva e negativa): Soutern blot e Multiplex PCR. Il vettore è stato poi inserito all’intero del genoma murino di cellule staminali embrionali (ES). Dopo lo screening dei cloni resistenti abbiamo identificato due cloni positivi, ovvero che hanno subito ricombinazione omologa del vettore di targeting: A9 e G7. Questi cloni sono stati ulteriormente vagliati per escludere la presenza di eventi di ricombinazione non omologa. Successivamente i positivi cloni sono stati amplificati ed iniettati all’interno di blastocisti. Le blastocisti sono state impiantate in una madre adottiva (forster mother), da esse si originano topi chimera. Abbiamo ottenuto due chimere, una dal clone A9 ed una dal clone G7. Queste chimere sono state incrociate a loro volta con topi wilde type per verificare la trasmissione della mutazione per via germinale (in eterozigosi). Al momento stiamo analizzando la progenie delle due chimere ottenute per trovare degli eterozigoti. I topi eterozigoti verranno poi incrociati tra lo per ottenere l’omozigote. Questo nuovo modello animale permetterà di studiare i meccanismi coinvolti nella neuro-tossicità da bilirubina, analizzando in vivo gli eventi biologici che si sviluppano in caso di mutazione a carico del gene UGT1 ed in futuro potrà anche essere usato per testare nuovi approcci diagnostici e terapeutici per il trattamento di neonati affetti da questa malattia.XXI Ciclo198

Pengembangan Perangkat Pembelajaran Flipped Classroom Pada Materi Getaran Harmonis

The aims of this research are to determine the validity of the product by experts and practitioners as well as to describe the attractiveness, the easiness and the expediency of the product. Development models used in this research is the ADDIE model which consists of five stages: analysis, design, development, implementation, and evaluation. The stage at this research explains only at the stage of development. The results obtained from expert validation test with an average score of 3.60 with the qualifying products is very valid, practitioners test results obtained an average score of 3.28 with very valid qualifying, and one by one test results of the product showed that the product was very attractive, very easy to use, and helpful. Penelitian ini bertujuan untuk mengetahui validitas produk oleh ahli dan praktisi serta untuk mendeskripsikan kemenarikan, kemudahan, dan kemanfaatan dari produk pengembangan. Model pengembangan yang digunakan dalam penelitian ini adalah model ADDIE yang terdiri dari lima tahapan, yaitu analisis, desain, pengembangan, implementasi, dan evaluasi. Pada penelitian ini, tahapan yang dilakukan hanya sampai pada tahap pengembangan. Hasil penelitian yang diperoleh dari uji validasi ahli yaitu rata-rata skor produk 3,60 dengan kualifikasi sangat valid, hasil uji praktisi diperoleh rata-rata skor 3,28 dengan kualifikasi sangat valid, dan hasil uji 1-1 menunjukkan bahwa produk memiliki kualitas sangat menarik, sangat mudah digunakan, dan bermanfaat

Albumin administration prevents neurological damage and death in a mouse model of severe neonatal hyperbilirubinemia

Therapies to prevent severe neonatal unconjugated hyperbilirubinemia and kernicterus are phototherapy and, in unresponsive cases, exchange transfusion, which has significant morbidity and mortality risks. Neurotoxicity is caused by the fraction of unconjugated bilirubin not bound to albumin (free bilirubin, Bf). Human serum albumin (HSA) administration was suggested to increase plasma bilirubin-binding capacity. However, its clinical use is infrequent due to difficulties to address its potential preventive and curative benefits, and to the absence of reliable markers to monitor bilirubin neurotoxicity risk. We used a genetic mouse model of unconjugated hyperbilirubinemia showing severe neurological impairment and neonatal lethality. We treated mutant pups with repeated HSA administration since birth, without phototherapy application. Daily intraperitoneal HSA administration completely rescued neurological damage and lethality, depending on dosage and administration frequency. Albumin infusion increased plasma bilirubin-binding capacity, mobilizing bilirubin from tissues to plasma. This resulted in reduced plasma Bf, forebrain and cerebellum bilirubin levels. We showed that, in our experimental model, Bf is the best marker to determine the risk of developing neurological damage. These results support the potential use of albumin administration in severe acute hyperbilirubinemia conditions to prevent or treat bilirubin neurotoxicity in situations in which exchange transfusion may be required.</p

Impairment of enzymatic antioxidant defenses is associated with bilirubin-induced neuronal cell death in the cerebellum of Ugt1 KO mice

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration. \ua9 2015 Macmillan Publishers Limited All rights reserved

Fludarabine increases nuclease-free AAV- and CRISPR/Cas9-mediated homologous recombination in mice

: Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold. Short-term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease- and CRISPR/Cas9-mediated AAV-HR two- to sevenfold in the murine liver, without causing overt toxicity. Fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Notably, the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice, suggesting that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing AAV-HR efficiency in mice. These results suggest that use of a clinically approved RNR inhibitor can potentiate AAV-HR-based genome-editing therapeutics

Serum Bilirubin Levels and Promoter Variations in HMOX1

The aim of our study was to assess the possible relationships among heme oxygenase (HMOX), bilirubin UDP-glucuronosyl transferase (UGT1A1) promoter gene variations, serum bilirubin levels, and Fabry disease (FD). The study included 56 patients with FD (M : F ratio = 0.65) and 185 healthy individuals. Complete standard laboratory and clinical work-up was performed on all subjects, together with the determination of total peroxyl radical-scavenging capacity. The (GT)n and (TA)n dinucleotide variations in the HMOX1 and UGT1A1 gene promoters, respectively, were determined by DNA fragment analysis. Compared to controls, patients with FD had substantially lower serum bilirubin levels (12.0 versus 8.85 μmol/L, p=0.003) and also total antioxidant capacity (p<0.05), which showed a close positive relationship with serum bilirubin levels (p=0.067) and the use of enzyme replacement therapy (p=0.036). There was no association between HMOX1 gene promoter polymorphism and manifestation of FD. However, the presence of the TA7 allele UGT1A1 gene promoter, responsible for higher systemic bilirubin levels, was associated with a twofold lower risk of manifestation of FD (OR = 0.51, 95% CI = 0.27–0.97, p=0.038). Markedly lower serum bilirubin levels in FD patients seem to be due to bilirubin consumption during increased oxidative stress, although UGT1A1 promoter gene polymorphism may modify the manifestation of FD as well

Meta-Review of Metanalytic Studies with Repetitive Transcranial Magnetic Stimulation (rTMS) for the Treatment of Major Depression

BACKGROUND: Major Depression (MD) and treatment-resistant depression (TRD) are worldwide leading causes of disability and therapeutic strategies for these impairing and prevalent conditions include pharmacological augmentation strategies and brain stimulation techniques. In this perspective, repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique with a favorable profile of tolerability which, despite being recently approved by the Food and Drug Administration (FDA) for the treatment of patients with medication-refractory unipolar depression, still raises some doubts about most effective parameters of stimulation.METHODS: A literature search was performed using PubMed for the years 2001 through February 2011 in order to review meta-analytic studies assessing efficacy and safety issues for rTMS in depressive disorders. Fifteen meta-analyses were identified and critically discussed in order to provide an updated and comprehensive overview of the topic with specific emphasis on potentially optimal parameters of stimulation.RESULTS: First meta-analyses on the efficacy of rTMS for the treatment of MD and TRD have shown mixed results. On the other hand, more recent meta-analytic studies seem to support the antidepressant efficacy of the technique to a greater extent, also in light of longer periods of stimulation (e.g. > 2 weeks).CONCLUSION: rTMS seems to be an effective and safe brain stimulation technique for the treatment of medication refractory depression. Nevertheless, further studies are needed to better define specific stimulation-related issues, such as duration of treatment as well as durability of effects and predictors of response

Age-dependent pattern of cerebellar susceptibility to bilirubin neurotoxicity in vivo in mice

Neonatal jaundice is caused by high levels of unconjugated bilirubin. It is usually a temporary condition caused by delayed induction of UGT1A1, which conjugates bilirubin in the liver. To reduce bilirubin levels, affected babies are exposed to phototherapy (PT), which converts toxic bilirubin into water-soluble photoisomers that are readily excreted out. However, in some cases uncontrolled hyperbilirubinemia leads to neurotoxicity. To study the mechanisms of bilirubin-induced neurological damage (BIND) in vivo, we generated a mouse model lacking the Ugt1a1 protein and, consequently, mutant mice developed jaundice as early as 36 hours after birth. The mutation was transferred into two genetic backgrounds (C57BL/6 and FVB/NJ). We exposed mutant mice to PT for different periods and analyzed the resulting phenotypes from the molecular, histological and behavioral points of view. Severity of BIND was associated with genetic background, with 50% survival of C57BL/6‑Ugt1−/− mutant mice at postnatal day 5 (P5), and of FVB/NJ-Ugt1−/− mice at P11. Life-long exposure to PT prevented cerebellar architecture alterations and rescued neuronal damage in FVB/NJ-Ugt1−/− but not in C57BL/6-Ugt1−/− mice. Survival of FVB/NJ-Ugt1−/− mice was directly related to the extent of PT treatment. PT treatment of FVB/NJ-Ugt1−/− mice from P0 to P8 did not prevent bilirubin-induced reduction in dendritic arborization and spine density of Purkinje cells. Moreover, PT treatment from P8 to P20 did not rescue BIND accumulated up to P8. However, PT treatment administered in the time-window P0–P15 was sufficient to obtain full rescue of cerebellar damage and motor impairment in FVB/NJ-Ugt1−/− mice. The possibility to modulate the severity of the phenotype by PT makes FVB/NJ-Ugt1−/− mice an excellent and versatile model to study bilirubin neurotoxicity, the role of modifier genes, alternative therapies and cerebellar development during high bilirubin conditions

Bilirubin-Induced Oxidative Stress Leads to DNA Damage in the Cerebellum of Hyperbilirubinemic Neonatal Mice and Activates DNA Double-Strand Break Repair Pathways in Human Cells

Unconjugated bilirubin is considered a potent antioxidant when present at moderate levels. However, at high concentrations, it produces severe neurological damage and death associated with kernicterus due to oxidative stress and other mechanisms. While it is widely recognized that oxidative stress by different toxic insults results in severe damage to cellular macromolecules, especially to DNA, no data are available either on DNA damage in the brain triggered by hyperbilirubinemia during the neonatal period or on the activation of DNA repair mechanisms. Here, using a mouse model of neonatal hyperbilirubinemia, we demonstrated that DNA damage occurs in vivo in the cerebellum, the brain region most affected by bilirubin toxicity. We studied the mechanisms associated with potential toxic action of bilirubin on DNA in in vitro models, which showed significant increases in DNA damage when neuronal and nonneuronal cells were treated with 140 nM of free bilirubin (Bf), as determined by γH2AX Western blot and immunofluorescence analyses. Cotreatment of cells with N-acetyl-cysteine, a potent oxidative-stress inhibitor, prevented DNA damage by bilirubin, supporting the concept that DNA damage was caused by bilirubin-induced oxidative stress. Bilirubin treatment also activated the main DNA repair pathways through homologous recombination (HR) and nonhomologous end joining (NHEJ), which may be adaptive responses to repair bilirubin-induced DNA damage. Since DNA damage may be another important factor contributing to neuronal death and bilirubin encephalopathy, these results contribute to the understanding of the mechanisms associated with bilirubin toxicity and may be of relevance in neonates affected with severe hyperbilirubinemia