S Galbiati

Retinoic acid-induced neuritogenesis of human neuroblastoma SH-SY5Y cells is ERK independent and PKC dependent

Retinoic acid (RA), an active metabolite of vitamin A, is a natural morphogen involved in development and differentiation of the nervous system. To elucidate signaling mechanisms involved in RA‐induced neuritogenesis, we used human neuroblastoma SH‐SY5Y cells, an established in vitro model for studying RA action, to examine the role of extracellular signal‐regulated kinase (ERK) 1 and 2 in RA‐induced neuritogenesis and cell survival. From immunoblotting experiments, we observed that RA induced delayed but persistent ERK1 and ERK2 phosphorylation (until 96 hr) that was reduced significantly by the specific mitogen‐activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126. For the subsequent studies we chose 24 hr as the reference time. Inhibition of ERK activation did not affect RA‐induced neuritogenesis (percentage of neurite‐bearing cells and neurite length) but significantly reduced cell survival. In addition, we analyzed the signaling pathway that mediates ERK activation. Our results suggest that RA‐induced ERK phosphorylation does not follow the classic Raf kinase‐dependent pathway. Protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI 3‐K) are possible alternative kinases involved in the ERK signaling pathway. In fact, in the presence of the specific PKC inhibitor GF 109203X, or the specific PI 3‐K inhibitor wortmannin, we observed a significant dose‐dependent reduction in ERK phosphorylation. RA‐induced neuritogenesis and cell survival were reduced by GF 109203X in a concentration‐dependent manner. These results suggest that rather than ERK1 and ERK2, it is PKC that plays an important role during early phases of RA‐induced neuritogenesis.

Neurotoxicity of platinum compounds: comparison of the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y

The main dose-limiting side effect of cancer treatment with platinum compounds is peripheral neurotoxicity. To investigate the intracellular mechanisms of platinum drugs neurotoxicity we have studied the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. Both platinum compounds are toxic causing cellular death by inducing apoptosis but oxaliplatin is less neurotoxic than cisplatin. The study of the proteins involved in the intracellular transduction pathways that may cause apoptotic death, revealed a very similar pattern of changes after exposure to cisplatin or oxaliplatin. In particular, as demonstrated by densitometric analysis, after exposure to both platinum compounds the total amount of the anti-apoptotic protein Bcl-2 was significantly reduced. Conversely, the amount of the pro-apoptotic protein p53 significantly increased. Caspases 3 and 7 were activated, but their activation was a late event, indicating a secondary role in the apoptotic process. Among the mitogen activated protein kinases, only the p38 protein was activated (phosphorylated) early enough to have a possible role in inducing apoptosis, possibly through p53 stabilization. The results of the present study and the data of the literature demonstrate that the ways in which cisplatin and oxaliplatin are neurotoxic are very similar and include not only DNA damage, but also the modulation of specific molecules involved in regulating the cellular equilibrium between apoptotic death and the cell cycle.

Pixantrone (BBR2778) reduces the severity of experimental allergic encephalomyelitis

Pixantrone is less cardiotoxic and is similarly effective to mitoxantrone (MTX) as an antineoplastic drug. In our study, pixantrone reduced the severity of acute and decreased the relapse rate of chronic relapsing experimental allergic encephalomyelitis (EAE) in rats. A marked and long-lasting decrease in CD3+, CD4+, CD8+ and CD45RA+ blood cells and reduced anti-MBP titers were observed with both pixantrone and MTX. In vitro mitogen- and antigen-induced T-cell proliferation tests of human and rodents cells evidenced that pixantrone was effective at concentrations which can be effectively obtained after i.v. administration in humans. Cardiotoxicity was present only in MTX-treated rats. The effectiveness and the favorable safety profile makes pixantrone a most promising immunosuppressant agent for clinical use in multiple sclerosis (MS).

Extracellular signal-regulated kinases 1 and 2 phosphorylated neurons in the tele- and diencephalon of rat after visceral pain stimulation: an immunocytochemical study

We aimed at verifying whether extracellular signal-regulated kinases (erks) 1 and 2 are activated, i.e. phosphorylated, in forebrain neurons after visceral pain stimulation (VPS). Ether and urethane anaesthetized rats received an intraperitoneal injection of acetic acid or were left untreated (ECT, UCT). After 2 h the animals were perfused. Paraffin embedded brain sections immunoreacted with an antibody selective for the phosphorylated erks. The light microscope analysis revealed only a few labelled neurons in ECT, while in UCT, positive cells were detected. In VPS rats (VPSR) positive cells were mainly distributed in regions, such as the hypothalamic anterior and thalamic paraventricular midline nuclei, amygdala, hippocampal and parahippocampal, insular and perirhinal cortex, involved in nociception and/or visceral activities. Our data suggest an association of erks activation with the emotional component of nociception; moreover, they show that erks activation is not suppressed by anaesthesia.

Immunocytochemical localization of extracellular signal-regulated kinases 1 and 2 phosphorylated neurons in the brainstem of rat following visceral noxious stimulation.

The aim of the present study was to determine the activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 in brainstem neurons following noxious visceral stimulation. Ether and urethane anaesthetized rats received an intraperitoneal injection of acetic acid (ENS, UNS) or were left untreated (ECT, UCT). Paraffin embedded brain sections immunoreacted with an antibody specific for phosphorylated ERKs. In noxious stimulated rats ERKs activated neuron profiles in the periaqueductal gray matter, parabrachial, dorsal raphe, solitary tract nucleus, area postrema and superior colliculus suggest that ERKs activation takes place mainly in brainstem nuclei in which nociception and visceral activities interact. The comparison between ENS and UNS rats shows that the long acting anaesthetic urethane attenuates the number of the ERKs activated neurons compared to the short acting ether.

Cationic liposomes target sites of acute neuroinflammation in experimental autoimmune encephalomyelitis.

The binding selectivity of charged liposomes to the spinal cord of rats affected by experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, was investigated. Positively and negatively charged liposomes were injected into the tail vein of rats, and blood/brain barrier (BBB) targeting was determined by confocal microscopy as a function of the temporal evolution of the inflammatory response. Accumulation in spinal cord endoneural vessels was observed for cationic, but not for anionic, liposomes, and only in EAE but not in healthy rats. The overall binding efficacy paralleled the severity of the clinical score, but targeting was observed already before clinical manifestation of inflammation. Preferential binding of positively charged liposomes in the course of acute EAE can be ascribed to subtle changes of BBB morphology and charge distribution in a similar way as for the binding of cationic particles to proliferating vasculature in chronic inflammation and angiogenesis. Our findings suggest that vascular changes related to increased binding affinity for cationic particles are very early events within the inflammatory reaction in acute EAE. Investigation of cationic vascular targeting can help to shed further light on these occurrences, and, potentially, new diagnostic and therapeutic options may become available. In neuroinflammatory diseases, cationic colloidal carrier particles may enable intervention at affected BBB by an approach which is independent from permeability increase.

Intraepidermal innervation and tail nerve conduction velocity in neurotoxicity models: results of a correlation study in normal and pathological conditions

Animal models of human diseases affecting the peripheral nervous system are widely used to assess the pathogenesis of neurotoxicity and to compare the effect of new agents. Several behavioural, pathological and neurophysiological methods have been used, and each has advantages and disadvantages. A major goal in the study of neurotoxicity would be to assess the damage in the same way in animal models and in humans. In this study we correlated the neurophysiological results obtained in normal rats and in rats treated with cisplatin 2 mg/kg q3d × 8 with the density of intraepidermal fibers (IEF) obtained in skin biopsy specimens. The aim was to investigate the possible role of a minimally invasive procedure such as skin biopsy as an alternative method to assess the peripheral neurotoxicity of antineoplastic drugs. The nerve conduction velocity (NCV) in the tail nerve was assessed in thirty‐six young adult female Wistar rats which were left untreated, or treated with erythropoietin (EPO), cisplatin (CDDP) or EPO + CDDP. CDDP and CDDP + EPO‐treated rats had a significantly reduced NCV vs. age‐matched untreated rats. At sacrifice, skin specimens were obtained. The density of IEF was calculated by 2 independent blinded examiners and the correlation existing between NCV and IEF was highly significant (r = 0.670, p < 0.001). This preliminary result suggests that IEF should be evaluated in other animal models and might represent a useful tool to study peripheral neurotoxicity also in humans.

RAT in vivo models of taxanes’ peripheral neurotoxicity following chronic intravenous administration

The “taxanes” family includes some widely used antineoplastic agents, such as paclitaxel and docetaxel. Treatment with these microtubule‐stabilizing drugs is often associated with neurotoxicity, a potentially severe side effect limiting the clinical utility of these agents. To study the pathogenesis of taxanes’ neurotoxicity and to compare it to the effect of new agents, the availability of reliable in vivo models is warranted. In this study we developed chronic iv models for the assessment of “taxanes” peripheral neurotoxicity. Forty‐eight adult Wistar rats were divided into six groups of 8 animals each and treated as follows: paclitaxel or docetaxel at doses of 5, 10, 12.5 mg/kg 1q7d × 4 via a chronic jugular vein implant. The evaluation was based on the assessment of body weight and survival as a measure of general tolerability, and on the measurement of tail nerve conduction velocity, a neurophysiological method previously used in animal models of toxic peripheral neuropathies. The results were compared with those obtained in untreated or vehicle‐treated control rats. A clear dose‐dependent effect was evident both on general toxicity, and on neurophysiological changes measured at the end of the treatment (untreated controls = 41,9 m/sec, vehicle = 40,3 m/sec; paclitaxel 5 mg/kg = 32,5 m/sec, 10 mg/kg = 28,5 m/sec, 12.5 m/kg = 27,4 m/sec; docetaxel 5 mg/kg = 33,6 m/sec, 10 mg/kg = 27,8 m/sec, 12.5 mg/kg = 27,0 m/sec), demonstrating the usefulness of this new model system to investigate peripheral neurotoxicity mediated by taxanes, and potentially other drugs, under chronic treatment schedules.