Sergio Barni

Early acute necrosis, delayed apoptosis and cytoskeletal breakdown in cultured cerebellar granule neurons exposed to methylmercury

Cerebellar granule cells (CGCs) are a sensitive target for methylmercury (MeHg) neurotoxicity. In vitro exposure of primary cultures of rat CGCs to MeHg resulted in a time‐ and concentration‐dependent cell death. Within 1 hr exposure, MeHg at 5–10 μM caused impairment of mitochondrial activity, de‐energization of mitochondria and plasma membrane lysis, resulting in necrotic cell death. Lower MeHg concentrations (0.5–1 μM) did not compromise cell viability, mitochondrial membrane potential and function at early time points. Later, however, the cells progressively underwent apoptosis and 100% cell death was reached by 18 hr treatment. Neuronal network fragmentation and microtubule depolymerization were detected as early as within 1.5 hr of MeHg (1 μM) exposure, long before the occurrence of nuclear condensation (6–9 hr). Neurite damage worsened with longer exposure time and proceeded to the complete dissolution of microtubules and neuronal processes (18 hr). Microtubule stabilization by taxol did not prevent MeHg‐induced delayed apoptosis. Similarly ineffective were the caspase inhibitors z‐VAD‐fluoromethylketone and z‐DEVD‐chloromethylketone, the L‐type calcium channel inhibitor nifedipine, the calcium chelator EGTA and BAPTA, and the NMDA receptor antagonist MK‐801. On the other hand, insulin‐like growth factor‐I partially rescued CGCs from MeHg‐triggered apoptosis. Altogether these results provide evidence that the intensity of MeHg insult is decisive in the time of onset and the mode of neuronal death that follows, i.e., necrosis vs. apoptosis, and suggest that cytoskeletal breakdown and deprivation of neurotrophic support play a role in MeHg delayed toxicity. J. Neurosci. Res. 59:775–787, 2000

Clinical lessons from the first applications of BNCT on unresectable liver metastases.

After a long series of studies on the effects of neutron irradiation of 10 B loaded neoplastic cells both in culture and in animal experiments, we started the clinical application of BNCT on humans affected by liver metastases of a radically resected colon adenocarcinoma. The procedure we adopted includes a first surgical phase, with hepatectomy; a radiotherapeutic phase, in which the isolated liver, washed and chilled, is extracorporeally irradiated with thermal neutrons; and then a second surgical phase for the reconnection of the liver to the patient. Until now two patients have been subjected to the BNCT treatment. The first one survived 44 months with a good quality of life, and died because of diffuse recurrences of his intestinal tumour. The second patient had the same early perioperative course, but after 33 days a worsening of a dilatative cardiomyopaty, from which he was suffering, determined a cardiac failure and eventually death. This clinical experience, although limited, has shown that extracorporeal neutron irradiation of the liver is a feasible procedure, able to ensure the complete destruction of liver metastases and a possible long lasting survival. In our patients neutron irradiation caused massive cellular necrosis highly specific to tumour cells, whereas normal cells were mostly spared. Nevertheless, the impact of such a traumatic operation on the patients organism must be taken into account. Finally, we have to be aware that the fight against tumour rarely leads to a complete victory. We now have an innovative weapon which is both powerful and partly unsettled: it must be refined and above all used.

Increase in liver pigmentation during natural hibernation in some amphibians.

The amount/distribution of liver melanin in 3 amphibian species (Rana esculenta, Triturus a. apuanus, Triturus carnifex) was studied during 2 periods of the annual cycle (summer activity–winter hibernation) by light and electron microscopy, image analysis and microspectrofluorometry. The increase in liver pigmentation (melanin content) during winter appeared to be correlated with morphological and functional modifications in the hepatocytes, which at this period were characterised by a decrease in metabolic activity. These findings were interpreted according to the functional role (e.g. phagocytosis, cytotoxic substance inactivation) played by the pigment cell component in the general physiology of the heterothermic vertebrate liver and, in particular, in relation to a compensatory engagement of these cells against hepatocellular hypoactivity during the winter period.

Mechanisms of changes to the liver pigmentary component during the annual cycle (activity and hibernation) of Rana esculenta L.

The present study was performed to elucidate the mechanisms responsible for the changes of melanin content/distribution we had previously discovered in the liver parenchyma of Rana esculenta during natural hibernation. Melanomacrophagic component response was analysed using morphocytochemical methods. The results demonstrated that during the prehibernation period (October–November) the melanomacrophages reach the highest proliferative activity (BrdU, PCNA labelling) which is accompanied by an evident melanosynthesis (dopa‐oxidase activity). In contrast, after hibernation, the decrease of liver pigmentation was the consequence of a partial cell loss by apoptotic mechanisms (TUNEL labelling, pyknosis‐karyorhexis) accompanied by a decrease of melanosome content by autophagy and low melanosynthetic activity. On the basis of these findings, there is evidence that liver melanomacrophages represent a metabolically (melanin synthesis/degradation) and cytokinetically (proliferation/death) active cell population during the annual cycle of the frog. The results are also discussed in relation to the functional synergism between hepatocytes and pigment cells in the adaptation to environmental changes.

Static Cytofluorometry and Fluorescence Morphology of Mitochondria and DNA in Proliferating Fibroblasts

The shape, distribution, and content of mitochondria in individual cells were examined during the cell cycle phases (G(0)/G(1), S, G(2) mitosis) in living human fibroblasts by static cytofluorometry and fluorescence microscopy. The morphocytochemical evaluations were performed in cell cultures submitted to double supravital fluorochrome staining with Hoechst 33342 and DiOC(6) to label DNA and mitochondria, respectively. The staining modalities were based on the stability of mitochondrial labeling. The G(1) to early S phases were characterized by the presence of filamentous mitochondria, except during the early postmitotic period. During late S, G(2), and mitotic phases, mitochondrial mass reached its highest value and mitochondria became short and numerous. During the last stage of mitosis, mitochondria were distributed among daughter cells through a cytoplasmic bridge.

In situ assessment of oxidant and nitrogenic stress in bleomycin pulmonary fibrosis

Reactive oxygen species (ROS) and nitric oxide (NO) have a role in the development of pulmonary fibrosis after bleomycin administration. The ROS production induces an antioxidant response, involving superoxide dismutases (SODs), catalase, and glutathione peroxidases. We compared in situ oxidative burden and antioxidant enzyme activity in bleomycin-injured rat lungs and normal controls. ROS expression and catalase, glucose-6-phosphate-dehydrogenase (G6PHD), and NOS/NADPH-diaphorase activity were investigated by using histochemical reactions. Nitric oxide synthase (e-NOS and i-NOS) and SOD (MnSOD, Cu/ZnSOD, ECSOD) expression was investigated immunohistochemically. After treatment ROS production was enhanced in both phagocytes and in type II alveolar epithelial cells. Mn, Cu/Zn, and ECSOD were overexpressed in parenchymal cells, whereas interstitium expressed ECSOD. Catalase and G6PHD activity was moderately increased in parenchymal and inflammatory cells. NOS/NADPH-d activity and i-NOS expression increased in alveolar and bronchiolar epithelia and in inflammatory cells. It can be suggested that the concomitant activation of antioxidant enzymes is not adequate to scavenge the oxidant burden induced by bleomycin lung damage. Inflammatory cells and also epithelial cells are responsible of ROS and NO production. This oxidative and nitrosative stress may be a substantial trigger in TGF-β1 overexpression by activated type II pneumocytes, leading to fibrotic lesions.

Ethanol selectively interferes with the trophic action of NMDA and carbachol on cultured cerebellar granule neurons undergoing apoptosis

Exposure of mature rat cerebellar granule neurons to non-depolarizing conditions (5 mM K+) for 24 h resulted in the onset of apoptosis. NMDA, forskolin, carbachol and GABA attenuated low K+-induced toxicity, although to a different extent, with NMDA and GABA being the most effective agents. When cells were co-exposed for 24 h to ethanol, the survival promoting action of NMDA and carbachol, but not that of forskolin and GABA, was attenuated. By contrast, a 24 h cell pre-treatment with ethanol, followed by its removal prior to K+ deprivation, was ineffective towards the neurotrophic action of NMDA and carbachol. The concomitant presence of alcohol and neurotrophic factors was not required for the pro-apoptotic effect of ethanol to be manifest after a long-term alcohol exposure: inhibition of NMDA- and carbachol-mediated neurotrophism was still observed when cells were pre-exposed for 72 h to alcohol in depolarizing conditions, prior to the challenge with 5 mM K+-containing medium and the test compounds in the absence of ethanol. The present study shows that ethanol promotes apoptotic cell death of cultured cerebellar neurons by selectively inhibiting the neurotrophic effect of NMDA and carbachol, and suggests that alcohol may cause permanent changes in the control mechanisms of apoptosis: this finding may have significant implications for the in vivo toxicity of prenatal ethanol exposure on the developing cerebellum.

Correlation between the liver temperature employed during machine perfusion and reperfusion damage: Role of Ca2+

This study compares the effects of machine perfusion (MP) at different temperatures with simple cold storage. In addition, the role of Ca2+ levels in the MP medium was evaluated. For MP, rat livers were perfused for 6 hours with Krebs‐Henseleit (KH) solution (with 1.25 or 2.5 mM CaCl2) at 4°C, 10°C, 20°C, 25°C, 30°C, or 37°C. For cold storage, livers were perfused in situ and preserved with Celsior solution at 4°C for 6 hours. The reperfusion period (2 hours at 37°C) was performed under the same conditions used for MP‐preserved and cold storage–preserved livers. Hepatic enzyme release, bile production, adenosine triphosphate (ATP) levels, and morphology were evaluated during MP and reperfusion. MP at 37°C caused marked enzyme release; the same findings were obtained during reperfusion. By contrast, MP temperature lowering induced a significant decrease in liver damage. High levels of biliary gamma‐glutamyltransferase and lactate dehydrogenase were found with MP at 4°C and 10°C but not with MP at 20°C. When a KH–1.25 mM CaCl2 solution was used during MP at 20°C, very low enzyme release was observed and significantly lower hepatic damage was present at the end of the reperfusion period in comparison with cold storage. The same results were obtained when ruthenium red, a calcium uniporter blocker, was added to KH–2.5 mM CaCl2. ATP levels were higher and morphology was better in liver preserved with KH–1.25 mM CaCl2. MP at 20°C with KH–1.25 mM CaCl2 resulted in better quality liver preservation, improving hepatocyte and endothelial biliary cell survival, in comparison with cold storage. This raises the need to reconsider the temperature and calcium levels to be used during liver MP. Liver Transpl 14:494–503, 2008.

Pulmonary toxicity of instilled cadmium-doped silica nanoparticles during acute and subacute stages in rats.

Potential risk associated with new nanomaterial exposure needs to be assessed. This in vivo study investigated pulmonary effects of engineered cadmium-containing silica nanoparticles Cd/SiNPs (1 mg/rat), silica SiNPs (600 μg/rat) and CdCl₂ (400 μg/rat) 1, 7 and 30 days after intratracheal instillation. Comprehensive histopathological and immunocytochemical characterization of lung damage in terms of apoptosis, cell proliferation, inflammation, fibrosis and metabolism were obtained. After exposure to all treatments, lung parenchyma showed injury patterns characterized by collapsed alveoli, inflammation, granuloma formation, thickened alveolar septa and bronchiolar epithelium exfoliation. Type II pneumocytes, containing scarcely surfactant-lamellated bodies, were also observed. Apoptotic phenomena enhanced as following, Cd/SiNPs>CdCl₂> SiNPs. In parallel with these findings, a significant increase of PCNA-immunoreactive cells was detected together with high mitotic activity. Cellular localization and distribution of IL-6, IP-10 and TGF-β1 revealed an increased expression of these cytokines as evidence of an enhanced cellular inflammatory response. CYP450-immunoreactivity was also enhanced, at bronchiolar (e.g. Clara cells) and alveolar (e.g. macrophages) level after both Cd/SiNPs and CdCl₂. These overall effects were observed acutely and lasted until the 30th day, with Cd/SiNPs producing the most marked effects. Collagen-immunolabelling changed particularly 7 and 30 days after Cd/SiNPs, when a strong stromal fibrogenic reaction occurred. The present findings suggest that Cd/SiNPs produce significantly greater pulmonary alterations than either SiNPs or CdCl₂ under the present experimental conditions.

Frog hepatocyte modifications induced by seasonal variations: A morphological and cytochemical study

A correlated morphological and cytochemical approach was employed to study frog hepatocytes in different periods of their annual cycle, including the natural hibernating period. There were considerable changes in the distribution and organization of hepatic glycogen in different phases of the annual cycle, and distribution of organelles as well. The most striking findings were glycogen storage during the prehibernation and hibernation phases, followed by drastic glycogen depletion. Cytochemical staining of a number of enzymes (succinate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, paranitrophenyl phosphatase, acid phosphatase, and glucose-6-phosphatase) involved in a variety of metabolic pathways, showed various cytoplasmic localizations and differences in intensity of the reaction products as a function of seasonality. Morphological and cytochemical data were interpreted as evidencing different functional requirements during seasonal changes in the frog.