Graziella Bernocchi

Cell proliferation, apoptosis and mitochondrial damage in rat B50 neuronal cells after cisplatin treatment

Abstract.  Objectives: Cisplatin (cisPt) is used as a chemotherapeutic agent for the treatment of a variety of human tumours; more recently, it has been demonstrated that tumour cell exposure to cisPt ultimately results in apoptosis, but the mechanism by which nuclear cisPt/DNA generates the cytoplasmic cascade of events involved has not been clarified. We have investigated the effects of cisPt on proliferation in the neuronal cell line B50, with particular attention being given to understand whether mitochondria are a target of cisPt and their involvement in the apoptotic process. Materials and methods: Rat neuronal B50 cells were used to investigate the mechanisms of cisPt‐induced cytotoxicity; this line has been used as a model system for neurotoxicity in vivo. Results: Changes in proliferation, induction of apoptosis, activation of caspase‐3 and DNA fragmentation were observed in the cells, as well as morphological and biochemical alterations of mithocondria. Activation of caspase‐9 confirmed that mitochondria are a target of cisPt. Conclusion: CisPt exerts cytotoxic effects in the neuronal B50 cell line via a caspase‐dependent pathway with mitochondria being central relay stations.

Bioactive peptides and serotonin immunocytochemistry in the cerebral ganglia of hibernating Helix aspersa

The role of some neuromodulators and neurotransmitters in the functioning of molluskan cerebral neurons and in their metabolic changes during hibernation has been considered. The cerebral ganglion of mollusks is a center for the integration of different inputs from the sensory areas of the head and for the generation of motor command impulses. During hibernation, animals are deprived of many external sensory stimuli and do not have locomotion and feeding. Immunocytochemistry for bioactive peptides (BAPs), such as SP (Substance P), CCK8 (Cholecystokinin 8/Gastrin), CGRP (Calcitonin-Gene-Related Peptide) and ET (Endothelin), and serotonin was performed on cerebral ganglia of active and hibernating Helix aspersa. The distribution of the immunopositivity was analyzed in different cell-containing areas (procerebrum, mesocerebrum, metacerebrum) and in the neuropiles. With all the antibodies raised against peptides, we observed that only a few neurons, mainly of small and medium size, had immunopositivity during the period of activity, the patterns of distribution being quite similar to those previously described in Helix or other gastropods. Fibers and varicosities with BAP immunopositivity were found in the procerebral and central neuropiles and sometimes around neurons. Serotonin-immunopositive neurons, including the giant neuron, were observed in the metacerebrum; numerous fibers and varicosities immunopositive for serotonin were present in the neuropile areas. In hibernating snails, the number of fibers with BAP and serotonin immunopositivity decreased in several areas of the neuropiles. Moreover, an increased number of neurons of the metacerebrum (two-to four-fold) and mesocerebrum (8- to 28-fold) had BAP-like immunopositivity, and the intensity of the immunoreaction for serotonin of the metacerebral neurons was also higher than in the active snails. These results are discussed, taking into account two hypotheses. The first hypothesis assumes that the increased immunocytochemical staining was really linked to accumulation of BAPs and serotonin. The second hypothesis considers that the antibodies for BAPs recognized a preprotein, the synthesis of BAPs being completed during the active period only. Both the hypotheses account for the co-occurrence and co-localization of two or ore peptides and serotonin and stress that the hibernation condition is of interest for studies on the actual function of single neurons in the cerebral ganglia. Finally, the data are consistent with the changes recently found in other markers of the morphological and functional activity of neurons, demonstrating that the neuromodulation and the neurotransmission are slowed during hibernation.

cisDDP treatment and development of the rat cerebellum.

CONTENTS Abbreviations 161

Reorganization of the rat cerebellar cortex during postnatal development following cisplatin treatment.

We examined the effects of the antitumor agent cisplatin on the development and plasticity of cerebellar cytoarchitecture. Since knowledge of the parallel and climbing fiber-Purkinje cell system is important in order to determine the architectural basis of cerebellar function, we used immunofluorescence for vesicular glutamate transporters (VGluT1 and VGluT2) to evaluate the trend of synaptogenesis of parallel and climbing fibers on Purkinje cells in the cerebellum vermis after a single injection of cisplatin to 10-day-old rats, i.e., during a crucial period of cerebellar development. The temporal and spatial patterns of VGluT1 and VGluT2 immunoreactivity after the early cisplatin injury provided evidence that remodeling of excitatory afferents and Purkinje cell dendrites occurs. After an early slow down of Purkinje cell dendrite growth, 7 days following the treatment, the extension of the molecular layer was reduced, as was parallel fiber innervation, but VGluT1 immunoreactive fibers contacted Purkinje cell dendrite branches extending within the external granular layer. VGluT2 immunopositive climbing fiber varicosities were still largely present on the soma and stem dendrites of Purkinje cells. Twenty days after the cisplatin injection, the thickness of the VGluT1 immunopositive molecular layer was reduced. VGluT2 climbing fiber varicosities were found on the remodeled Purkinje cell dendrites, as in controls, although at a lower density. Alterations in the immunoreactivity for polysialic acid neural cell adhesion molecule (PSA-NCAM) during the recovery phase suggest that this molecule plays a fundamental role not only during development, but also in the reorganization of neuroarchitecture. The changes were restricted to the neocerebellar vermis and were likely dependent on the different timing of lobule formation. The results of these investigations reveal the existence of vulnerability windows of the cerebellum to exposure to experimental or environmental cytotoxic agents during a critical period in development.

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.

Morphological Features of Organelles during Apoptosis: An Overview

An apoptotic program leading to controlled cell dismantling implies perturbations of nuclear dynamics, as well as changes affecting the organelle structure and distribution. In human cancer cells driven to apoptosis by different stimuli, we have recently investigated the morphological properties of several organelles, including mitochondria, lysosomes, endoplasmic reticulum and Golgi apparatus. In this review, we will discuss the body of evidence in the literature suggesting that organelles are generally relocated and/or degraded during apoptosis, irrespectively of the apoptogenic stimulus and cell type.

Developmental plasticity of rat cerebellar cortex after cisplatin injury: Inhibitory synapses and differentiating Purkinje neurons

A single injection of cisplatin, a cytostatic agent, (5 microg/g body weight) in 10-day old rats leads later to the reorganization of the cerebellar cortex in lobules VI-VIII of the vermis. Double immunofluorescence reaction for glutamate receptor (GluR)2/3, a ionotropic glutamate receptor that labels postsynaptically Purkinje neurons, and glutamic acid decarboxylase (GAD)65, an isoform of the GABA synthesis enzyme that labels presynaptically inhibitory terminals in the molecular layer, were employed. Less-differentiated Purkinje cells were present in rats treated on postnatal day (PD)11 at the top of lobule VI and in lobules VII-VIII, in comparison with the deep zones of the same lobules and lobule III. The changes were interpreted as due to loss of trophic factors of Purkinje cell growth, e.g. signaling molecules and granule cells. However, we have shown that a remodelling of Purkinje cell dendrites occurred on PD30 (20 days after cisplatin). In fact, despite of the GluR2/3 labeling of the entire Purkinje cell dendrites, the GAD65 immunofluorescent terminals were adjacent to the proximal parts of the dendrite, while they were scarce in the distal dendritic branchlets. The findings were discussed in relation to the changed cytoarchitecture of the cerebellar cortex, which from PD17 to PD30 includes regeneration of the external germinal layer, reorientation of the main dendritic branches and of the Purkinje cell branchlets, and the presence of ectopic cells.

Nitric oxide‐containing neurons in the nervous ganglia of Helix aspersa during rest and activity: Immunocytochemical and enzyme histochemical detection

Nitric oxide synthase (NOS) immunoreactivity and staining for nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐diaphorase) activity are two cytochemical markers for nitric oxide (NO)‐containing neurons. The authors examined the changes in the distribution of NOS immunolabeling and NADPH‐diaphorase reactivity in the cerebral and buccal ganglia of the terrestrial snail Helix aspersa during resting and active phases. During inactivity and after 1 day of activity, in the mesocerebrum and metacerebrum of the snails, there were several reactive neurons for both markers; after 7 days of activity, the number of reactive neurons was lower. Opposite results were obtained in the buccal ganglia, in which increased staining and numbers of reactive neurons were present in the active snails (after 1 day and 7 days of activity). Although the staining patterns for the two reactions were similar, colocalization was not always observed. The comparison between inactive and active animals provided a more precise survey of NOS‐containing neurons in the snail cerebral ganglia than previously described. Moreover, it suggested that not only is NO involved in distinct nervous circuits, but, as a ubiquitous molecule, it also plays a role in neuroprotection and neuropeptide release. J. Comp. Neurol. 409:274–284, 1999.

Mitochondrial fusion: A mechanism of cisplatin-induced resistance in neuroblastoma cells?

Cisplatin induces apoptosis through different pathways. The intrinsic apoptotic pathway is mediated by mitochondria, which, as a result of cisplatin treatment, undergo morphological alterations. The aim of this study was to investigate cisplatin-induced mitochondrial functional and morphological long-term effects in neuroblastoma B50 rat cells. To this purpose, we followed evaluated different several apoptotic markers by means of flow cytometry, confocal and electron microscopy and western blotting techniques. We applied different treatment protocols based on the incubation of the neuroblastoma B50 rat cells with 40 μM cisplatin: (i) for 48 h and harvesting of the cells at the end of the treatment; (ii) further recovery in drug-free medium for 7 days post-treatment; (iii) conditions as in (ii) followed by re-seeding in normal medium and growth for a further 4 days. We observed apoptosis induction after the first treatment and after the recovery from cell death after long-term culture in drug-free medium. Interestingly, the latter phenomenon was characterized by mitochondrial elongation and mitochondrial protein rearrangement. In recovered and re-seeded cells, mitochondrial equilibrium moved toward fusion, possibly protecting cells from apoptosis.

Morphohistochemical changes in hepatocytes during the life cycle of the European eel

The comparative analysis of morphological, histochemical and cytochemical patterns of eel (Anguilla anguilla L.) hepatocytes reveals clear differences between two stages of its life cycle, i.e. the trophic stage (yellow eel) and reproductive stage (silver eel). The storage of glycogen prevails in the yellow eel, whilst lipids appear to be remarkably increased in the silver eel, in which some hepatocytes also show glycogen-rich areas. Generally, in the silver eel dehydrogenase and acid phosphatase activities seem greater and different distribution of the reaction products is present; on the contrary, a lower G6PDH activity is observed. The electron microscopy characteristics and distribution of both cellular organelles and reserve materials reflect the modifications found at light microscopy. The ultrastructural patterns provide further evidence for the heterogeneity of liver parenchyma in silver eel. In particular, the coexistence of nuclei showing a different degree of chromatin compactness is also accounted for by the quantitative cytochemical data on the nuclear DNA after Feulgen reaction and intercalation with propidium iodide at low and high concentrations. With regard to the DNA content, the hepatocytes in the silver eel as well as in the yellow eel are mainly 2c. However, some 4c values are also found, which according to the literature can be ascribed to cells in G2 phase. The present data may express the onset of different functional requirements during the reproductive stage in comparison with the trophic one. Moreover, our results are consistent with modifications found by other authors as a consequence of interruption of nourishment and during gonad maturation, i.e. two phenomena characterizing the transition from yellow to silver eel.