Fatty liver oxidative events monitored by autofluorescence optical diagnosis: Comparison between subnormothermic machine perfusion and conventional cold storage preservation.

AIMS: Livers with moderate steatosis are currently recruited as marginal organs to face donor shortage in transplantation, even though lipid excess and oxidative stress increase preservation injury risk. Sensitive, real-time detection of liver metabolism engagement could help donor selection and preservation procedures, ameliorating the graft outcome. Hence, we investigated endogenous biomolecules with autofluorescence (AF) properties as biomarkers supporting the detection of liver oxidative events and the assessment of metabolic responses to external stimuli. METHODS: Livers from male Wistar rats fed a 12-day methionine/choline-deficient (MCD) diet were subjected to AF spectrofluorometric analysis (fiber-optic probe, 366-nm excitation) before and after organ isolation, and following preservation (cold storage or 20°C machine perfusion) and reperfusion. RESULTS: Innovative dynamic AF results on lipid oxidation to lipofuscin-like lipopigments, correlating with biochemical oxidative damage (thiobarbituric acid reactive substances) and antioxidant defense (glutathione) parameters, suggested lipid engagement in MCD livers counteracting reactive oxidizing species. The maintained MCD liver functionality was supported by limited changes in bilirubin AF spectral profile, reflecting bile composition balance, despite their intrinsic mitochondrial weakness, confirmed by adenosine 5'-triphosphate levels, and regardless of different preservation effects on energy metabolism revealed by conventional reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate and flavin AF data. CONCLUSION: Autofluorescence showed that, after a relatively short time on an MCD diet, livers are still able to face oxidizing events and maintain a functional balance. These results strengthen AF as a supportive diagnostic tool in experimental hepatology, to characterize marginal livers in real time, monitor their response to ischemia/reperfusion, and investigate protective therapeutic agents.

Autofluorescence discrimination of metabolic fingerprint in nutritional and genetic fatty liver models.

Liver tissue autofluorescence (AF) has been characterized in two models with a different potential to undergo disease progression to steatohepatitis: Wistar rats, administered with a methionine, choline deficient diet (MCD), and Zucker (fa/fa) rats, homozygous for a spontaneous mutation of leptin receptor. AF spectra were recorded from liver tissue cryostatic sections by microspectrofluorometry, under 366nm excitation. Curve fitting analysis was used to estimate the contribution of different endogenous fluorophores (EFs) to the overall AF emission: i) fluorescing fatty acids, a fraction of liver lipids up to now poorly considered and complicated to detect by conventional procedures; ii) lipofuscin-like lipopigments, biomarkers of oxidizing events; iii) NAD(P)H and flavins, biomarkers of energy metabolism and tissue redox state. AF data and biochemical correlates of hepatocellular injury resulted to depend more on rat strain than on intratissue bulk lipid or ROS levels, reflecting a different metabolic ability of the two models to counteract potentially harmful agents. AF analysis can thus be proposed for extensive applications ranging from experimental hepatology to the clinics. AF based diagnostic procedures are expected to help both the prediction of the risk of fatty liver disease progression and the prescreening of marginal organs to be recruited as donors for transplantation. A support is also foreseen in the advancement and personalization of strategies to ameliorate the donor organ preservation outcome and the follow up of therapeutic interventions.

[Pt(O,O'-acac)(γ-acac)(DMS)] versus cisplatin: apoptotic effects in B50 neuroblastoma cells.

Cisplatin is one of the most active chemotherapeutic agents used in the treatment of childhood and adult malignancies. Cisplatin induces cell death through different pathways. Despite its effectiveness, the continued clinical use of cisplatin is limited by onset of severe side effects (nephrotoxicity, ototoxicity and neurotoxicity) and drug resistance. Therefore, one of the main experimental oncology purpose is related to the search for new platinum-based drugs to create different types of adducts or more specific and effective subcellular targets. Thus, [Pt(O,O'-acac)(γ-acac)(DMS)], which reacts preferentially with protein thiols or thioether, was synthesized. In our research, different approaches were used to compare cisplatin and [Pt(O,O'-acac)(γ-acac)(DMS)] effects in B50 rat neuroblastoma cells. Our results, using immunocytochemical, cytometric and morphological techniques, showed that these compounds exert a cytostatic action and activate apoptosis with different pathways. Long-term effects demonstrated that [Pt(O,O'-acac)(γ-acac)(DMS)] exerts cytotoxic effects in neuronal B50 cell line not inducing drug resistance. Analysis was performed both to compare the ability of these platinum compounds to induce cell death and to investigate the intracellular mechanisms at the basis of their cytotoxicity.

Cerebellum neurotransmission during postnatal development: [Pt(O,O'-acac)(γ-acac)(DMS)] vs cisplatin and neurotoxicity.

Several chemotherapeutic drugs are known to cause neurotoxicity. Platinum-based agents in use or in clinical trials display neurotoxic potential accompanied by neurological complications; recent studies have identified a large number of behavioural issues in paediatric oncology patients. To understand the toxicity of platinum drugs at the molecular and cellular levels, this study compares the possible cytotoxic effects of an older platinum compound, cisplatin and a new platinum compound, [Pt(O,O'-acac)(γ-acac)(DMS)], on the CNS of postnatally developing rats, which is much more vulnerable to injury than the CNS of adult rats. Since several drugs interact with neurotransmitters during neuronal maturation, we performed immunostainings with antibodies raised against markers of glutamate and GABA, the major neurotransmitters in the cerebellum. After a single injection of cisplatin at postnatal day 10 (PD10), the labelling of Purkinje cells with the neurotransmitter markers evidenced alterations between PD11 and PD30, i.e. atrophy of the dendrite tree, changes in the distribution of synaptic contacts of parallel and climbing fibres, delay in the elimination of transient synapses on cell soma and severely impaired pinceau formation at the axon hillock. After treatment with [Pt(O,O'-acac)(γ-acac)(DMS)], the sole relevant change concerned the timing of climbing fibres elimination; the transient synapses disappearance on the Purkinje cell soma was delayed in some cells; instead, the growth of Purkinje cell dendrite tree was normal as was the formation of inhibitory synaptic contacts on these neurons. These findings add new evidence not only on the lower neurotoxicity of [Pt(O,O'-acac)(γ-acac)(DMS)] vs cisplatin but also on the involvement of neurotransmitters and relative synaptic connections in the maturation of central nerve tissue.

Integrated autofluorescence characterization of a modified-diet liver model with accumulation of lipids and oxidative stress.

Oxidative stress in fatty livers is mainly generated by impaired mitochondrial ß-oxidation, inducing tissue damages and disease progression. Under suitable excitation, light liver endogenous fluorophores can give rise to autofluorescence (AF) emission, the properties of which depend on the organ morphofunctional state. In this work, we characterized the AF properties of a rat liver model of lipid accumulation and oxidative stress, induced by a 1-9-week hypercaloric methionine-choline deficient (MCD) diet administration. The AF analysis (excitation at 366 nm) was performed in vivo, via fiber optic probe, or ex vivo. The contribution of endogenous fluorophores involved in redox reactions and in tissue organization was estimated through spectral curve fitting analysis, and AF results were validated by means of different histochemical and biochemical assays (lipids, collagen, vitamin A, ROS, peroxidised proteins, and lipid peroxidation -TBARS-, GSH, and ATP). In comparison with the control, AF spectra changes found already at 1 week of MCD diet reflect alterations both in tissue composition and organization (proteins, lipopigments, and vitamin A) and in oxidoreductive pathway engagement (NAD(P)H, flavins), with a subsequent attempt to recover redox homeostasis. These data confirm the AF analysis potential to provide a comprehensive diagnostic information on negative effects of oxidative metabolism alteration.

Platinum drugs and neurotoxicity: effects on intracellular calcium homeostasis.

[Pt(O,O'-acac)(γ-acac)(DMS)] (PtAcacDMS) is a new platinum compound showing low reactivity with nucleobases and specific reactivity with sulfur ligands intracellularly. It induces apoptosis in breast cancer cells, but appears to be less neurotoxic to the developing cerebellum than cisplatin (cisPt). The aim of this study was to assess the neurotoxicity of platinum compounds on calcium homeostasis in the dentate gyrus and Cornu Ammonis regions of the hippocampal formation during rat postnatal development. Two intracellular calcium homeostasis systems were taken for measurement, calbindin, a calcium buffer protein, and a plasma membrane calcium ATPase (PMCA1). The platinum compounds showed different effects on these markers in the two areas. One day after injection (PD11), cisPt decreased calbindin immunoreactivity and PMCA1 labeling in both regions; at PD17, the downregulation of PMCA1 persisted. Instead, PtAcacDMS produced varying effects on calbindin immunoreactivity in the two regions at PD11 and PD17; but in all cases, the changes incurred in calbindin immunoreactivity were counterbalanced by changes produced in PMCA1 expression. In conclusion, PtAcacDMS seems to affect calcium homeostasis in the central nervous system differently than cisPt. Both the platinum compounds act early to alter the calbindin buffering system. However, the most important difference between cisPt and PtAcacDMS is that, in vivo, the latter acts early to stimulate calcium efflux from nerve cells as reflected by its effect on PMCA1. The rapid onset of an activated calcium pump appears to be essential to cope with the excessive intracellular calcium concentration stemming from the downregulation of calbindin which could damage neuron function and morphology.

Developmental expression of cellular prion protein and apoptotic molecules in the rat cerebellum: effects of platinum compounds.

Programmed cell death is regulated by prototypes of a large family of Bcl-2-like proteins such as Bax and Bcl-2. A neuroprotective role for cellular prion protein (PrPc) on programmed cell death has been reported, although the cytosolic accumulation of PrPc correlates with toxicity and death of some neurons by apoptosis. In order to understand the signalling function of PrPc in promoting survival or suppressing cell death, we analyzed the expression and co-localization of PrPc, Bax and Bcl-2 proteins in the developing cerebellum of rats treated at PD10 (postnatal day 10) with the chemotherapeutic drug cisplatin (cisPt) or the new platinum (Pt) compound [Pt(O,O'-acac)(γ-acac)(DMS)] (PtAcacDMS). Differences in the expression of PrPc, Bax and Bcl-2 were found in proliferating cells and immature Purkinje neurons. One day after administration (PD11), cisPt markedly increased the apoptosis of the proliferating cells of the EGL (external granular layer); at the same time, several apoptotic bodies with strong Bax immunoreactivity were noticed. After PtAcacDMS, changes in PrPc and apoptotic proteins, with respect to the controls, were found but Bax immunopositive apoptotic bodies were not detectable, which could mean that apoptotic cell death of proliferating cells is preserved. Co-localization was clearly detected in the Purkinje cell population and may explain better the mechanisms by which PrPc and the apoptotic proteins function, and particularly the role of PrPc. Considering the reactivity of Purkinje neurons to these proteins at PD11 and Pd17, at least PrPc expression increased after cisPt and PtAcacDMS treatments or, if PrPc decreased, balanced itself with Bcl-2. The noteworthiness of this finding is that it emphasizes that most of the post-mitotic Purkinje cells need to be rescued, otherwise they undergo degeneration and are not replaced. Based on the effects of both Pt compounds on Purkinje cell differentiation, it should be emphasized that PrPc, together with the synergistic action of the co-localized anti-apoptotic protein, acts as a neuroprotective protein countering cytotoxicity in the postnatal critical phases of cerebellum development.

Postnatal development of the central nervous system: anomalies in the formation of cerebellum fissures.

A natural defect in rat cerebellum postnatal development has been found in the fissura prima, consisting in various complex configurations of the cerebellar layers. We investigated the genesis of fissure malformations through immunoreactions for PCNA, GFAP, GABAA alpha6, and calbindin to label proliferating cells of the external granular layer (egl), radial glial fibers, mature granule cells, and Purkinje cells, respectively. Results on critical stages of rat postnatal development provided interesting evidences on how the malformation develops in fissures prima and secunda. Early (postnatal day 10) at the site of malformation, the Bergmann radial glia was often retracted and showed distortions and irregular running. The interruption of GFAP-positive radial glial fibers could fit in with the presence of clusters of PCNA-unlabeled cells in the sites of fusion of the egl; the clusters of cells are granule cells since their soma is labeled by GABAA alpha6. Moreover, an altered migration of granule cell precursors to the internal granular layer was evident which, in turn, affected Purkinje cell differentiation and the growth of their dendrites. In summary, the changed relationship among glial fiber morphology, granule cell migration, and Purkinje cell differentiation suggests how the Bergmann glial fibers have a basic role in the foliation process, being the driving physical force in directing migration of the granule cells at the base of fissure.