Giuseppina La Spada

Melatonin treatment mimics the antidepressant action in chronic corticosterone-treated mice.

Abstract:  Melatonin, involved in circadian cycle, provides protective effects on neuronal cells and acts as antidepressant by restoration of corticosterone levels. A mouse model of anxiety/depressive‐like behavior, induced by chronic corticosterone treatment, has been used to evaluate behavior and adult hippocampal neurogenesis in mice and their possible modulation under melatonin. With this aim, CD1 mice were subjected to 7 wk of corticosterone administration, and then behavioral tests as novelty‐suppressed feeding, open field and a forced swim test were performed. Cell proliferation in hippocampal dentate gyrus (DG) was investigated by 5‐bromo‐2′‐deoxyuridine and doublecortin immunohistochemistry techniques, and stereological procedure was used to quantify labeled cells. Golgi‐impregnated method was used to evaluate the changes of dendritic spines in DG neurons. A new therapeutic approach with antidepressant‐like substances (3 wk) such as melatonin (8 mg/kg) was employed to possibly modulate neural development in the adult hippocampus and the behavioral changes. The depressive‐like state caused by chronic corticosterone treatment was reversed by exogenous administration of melatonin; the proliferation of progenitor cells in mice hippocampus was significantly reduced under chronic corticosterone treatment (cort− 83.7 ± 20.3 versus cort+ 60.5 ± 18.2; P < 0.05), whereas long‐term treatment with melatonin prevented the corticosterone‐induced reduction in hippocampal cell proliferation (cort− 60.5 ± 18.2 versus mel 133.4 ± 26.9; P < 0.05). Corticosterone‐treated mice exhibited a reduced spine density, which was ameliorated by melatonin administration. These findings suggest a strong correspondence between behavior and neurogenesis, strengthening the hypothesis that neurogenesis contributes to the effects of melatonin as an antidepressant.

Oxidative stress induced by crude venom from the jellyfish Pelagia noctiluca in neuronal-like differentiated SH-SY5Y cells

Marine toxins are a suitable research model and their mechanism of action is intriguing and still under debate. Either a pore formation mechanism or oxidative stress phenomena may explain the damage induced by toxins. The effect of crude venom from isolated nematocysts of the jellyfish Pelagia noctiluca on neuronal-like cells derived from human neuroblastoma SH-SY5Y has been here studied. To prove the possible oxidative stress events, cell viability, assessed by MTT quantitative colorimetric assay, intracellular reactive oxygen species (ROS) quantified by the non-fluorescent probe H2DCF-DA and changes in mitochondrial transmembrane potential (ΔΨm) measured by the incorporation of a cationic fluorescent dye rhodamine-123 were verified on venom-treated cells (0.05-0.5μg/ml doses). A dose- and time-dependent reduction of all parameters was observed after venom treatment. NAC (N-acetyl-cysteine), antioxidant applied before crude venom application, significantly counteracted the decrease in cell viability and ROS production, while ΔΨm was only partially restored. The disruption of mitochondrial membrane by P. noctiluca crude venom may thus induce oxidative stress by inhibiting mitochondrial respiration and uncoupling oxidative phosphorylation, sensitizing mitochondria in SH-SY5H cells and facilitating membrane permeability. In sum, our findings suggest that P. noctiluca crude venom directly induces ΔΨm collapse with further generation of ROS and add novel information to the understanding of such toxins, still not completely clarified.

Protective effect of melatonin against the inflammatory response elicited by crude venom from isolated nematocysts of Pelagia noctiluca (Cnidaria, Scyphozoa).

Abstract:  Melatonin (N‐acetyl‐5‐methoxytryptamine) is an efficient free radical scavenger and antioxidant, both in vitro and in vivo. The role of melatonin as an immunomodulator is, in some cases, contradictory. In this study we have investigated the therapeutic efficacy of melatonin in rats subjected to Pelagia noctiluca crude venom (of the familia Pelaguiidae; and genus Pelagia) induced acute paw inflammation. In particular, injection of the venom into the paw of rats elicited an acute inflammatory response characterized by accumulation of fluid containing a large number of polymorphonuclear neutrophils in the paw and subsequent lipid peroxidation. Furthermore, the venom promoted an expression of iNOS, nitrotyrosine and the activation of the nuclear enzyme poly (ADP‐ribose) polymerase as determined by immunohistochemical analysis of paw tissues. Administration of melatonin 30 min, 1 and 6 hr after the challenge with the venom, caused a significant reduction in all the parameters of inflammation measured. Thus, based on these findings we propose that melatonin may be useful a treatment of local acute inflammation induced by P. noctiluca crude venom.

Melatonin’s stimulatory effect on adult hippocampal neurogenesis in mice persists after ovariectomy

Abstract:  In this study, we examined whether melatonin treatment would increase new cell formation in the hippocampus in ovariectomized (OVX) mice. Chronic exogenous melatonin administration increased bromodeoxyuridine (BrdU) (OVX‐sham 72 ± 3.2 versus OVX‐mel 122 ± 12.0; P < 0.05) and doublecortin (DCX) (OVX‐sham 88 ± 3.1 versus OVX‐mel 176 ± 9.9; P < 0.05) immunoreactive cells in the hippocampus of ovariectomized mice. This neuronal development was correlated with synaptic plasticity, identified using the Golgi impregnation method to quantify dendritic spines in mouse dentate gyrus (DG). Finally, the antidepressant‐like state of the animals was evaluated by the tail suspension test. The results indicate that melatonin acts on birth, survival, and differentiation of new neurons in the hippocampus, stimulates maturation of spines, and exerts an antidepressant‐like action under estrogen‐deprived conditions, in both a strain‐ and gender‐independent manner, suggesting that this indoleamine may be useful in improving brain functions.

Crude Venom from Nematocysts of the Jellyfish Pelagia noctiluca as a Tool to Study Cell Physiology

Marine animals represent a source of novel bioactive compounds considered as a good research model, whose mechanism of action is intriguing and still under debate. Among stinging animals, Cnidarians differentiated highly specialized cells, termed nematocytes, containing a capsule fluid with toxins and an inverted tubule, synergistically responsible for mechanisms of defence and predation. Such compounds include proteins and secondary metabolites with toxic action. With the aim of better elucidating the effects of Cnidarian venom upon cell targets, this short review reports on the current knowledge about the toxicological activity of venom extracted from nematocysts of the jellyfish Pelagia noctiluca, whose notable blooming is well known in the Strait of Messina (Italy). The effects on cultured cells, from both mammals and invertebrates, and erythrocytes are here being considered. What is known about the biological activity of Pelagia noctiluca crude venom accounts for a powerful biological activity at different levels, suggesting that cell damage may be due to a pore formation mechanism on cell membrane target leading to osmotic lysis, and /or to oxidative stress events. In this light, the study of venom activity may contribute to: i) validate suitable biological assays for venom testing; ii) elucidate cell function features; iii) understand the pathophysiology of envenoming.

Mechanisms of hyposmotic volume regulation in isolated nematocytes of the anthozoan Aiptasia diaphana.

The nature and role of potassium (K) and water transport mediating hyposmotically-induced regulatory volume decrease (RVD) were studied in nematocytes dissociated with 605 mM thiocyanate from acontia of the Anthozoan Aiptasia diaphana. Cell volume and hence RVD were calculated from the inverse ratios of the cross sectional areas of nematocytes (A/Ao) measured before (Ao) and after (A) challenge with 65% artificial sea water (ASW). To distinguish between K channels and K-Cl cotransport (KCC), external sodium (Na) and chloride (Cl) were replaced by K and nitrate (NO3), respectively. Inhibitors were added to identify K channels (barium, Ba), and putative kinase (N-ethylmaleimide, NEM) and phosphatase (okadaic acid, OA) regulation of KCC. In 65% NaCl ASW, nematocytes displayed a biphasic change in A/Ao, peaking within 4 min due to osmotic water entry and thereafter declining within 6 min due to RVD. Changing NaCl to KCl or NaNO3 ASW did not affect the osmotic phase but attenuated RVD, consistent with K channel and KCC mechanisms. Ba (3 mM) inhibited RVD. NEM and OA, applied separately, inhibited the osmotic phase and muted RVD suggesting primary action on water transport (aquaporins). NEM and OA together reduced the peak A/Ao ratio during the osmotic phase whereas RVD was inhibited when OA preceded NEM. Thus, both K channels and KCC partake in the nematocyte RVD, the extent of which is determined by functional thiols and dephosphorylation of putative aquaporins facilitating the preceding osmotic water shifts.

Evidence for Aquaporin-mediated Water Transport in Nematocytes of the Jellyfish Pelagia noctiluca

Nematocytes, the stinging cells of Cnidarians, have a cytoplasm confined to a thin rim. The main cell body is occupied by an organoid, the nematocyst, containing the stinging tubule and venom. Exposed to hypotonic shock, nematocytes initially swell during an osmotic phase (OP) and then undergo regulatory volume decrease (RVD) driven by K+, Cl- and obligatory water extrusion mechanisms. The purpose of this report is to characterize the OP. Nematocytes were isolated by the NaSCN/Ca2+ method from tentacles of the jellyfish Pelagia noctiluca, collected in the Strait of Messina, Italy. Isolated nematocytes were subjected to hyposmotic shock in 65% artificial seawater (ASW) for 15 min. The selective aquaporin water channel inhibitor HgCl2 (0.1-25 µM) applied prior to osmotic shock prevented the OP and thus RVD. These effects were attenuated in the presence of 1mM dithiothreitol (DTT), a mercaptide bond reducing agent. AgNO3 (1 µM) and TEA (tetraethylammonium, 100 µM), also reported to inhibit water transport, did not alter the OP but significantly diminished RVD, suggesting different modes of action for the inhibitors tested. Based on estimates of the nematocyte surface area and volume, and OP duration, a relative water permeability of ∼10-7 cm/sec was calculated and the number of putative aquaporin molecules mediating the OP was estimated. This water permeability is 3-4 orders of magnitude lower in comparison to higher order animals and may constitute an evolutionary advantage for Cnidarian survival.

Thiol-induced discharge of acontial nematocytes

The discharge of nematocytes, the stinging cells of Coelenterata, is a poorly understood phenomenon. In particular, little is known about the chemical stimuli that trigger the discharge. In this paper, we show that thiols are able to initiate the nematocyst discharge in isolated nematocytes. Among the thiols tested, reduced glutathione and cysteine were found to be the most effective. The effect of glutathione was likely two-fold: it formed mixed disulfides with membrane thiols, as shown by the ability of the mercapto-blocking reagent iodoacetamide to abolish its action; and it bound to the membrane through the glutamate moiety, as demonstrated by competitive experiments with free glutamate. Glutathione triggered the discharge at concentrations higher than those sufficient to activate the feeding response of Coelenterates. However, our results demonstrate for the first time that the modification of membrane thiols by selective agents may be a key event in the discharge of nematocytes.

Neuronal-like differentiated SH-SY5Y cells adaptation to a mild and transient H2O2-induced oxidative stress

Preconditioning (PC) is a cell adaptive response to oxidative stress and, with regard to neurons, can be considered as a neuroprotective strategy. The aim of the present study was to verify how neuronal‐like differentiated SH‐SY5Y cells adapt to a mild and transient H2O2‐induced oxidative stress and, hence, whether may be considered as more sensitive cell model to study PC pathways. A first screening allowed to define H2O2 concentrations for PC (10μM‐50μM), applied before damage(100μM H2O2). Cell viability measured 24 hours after 100μM H2O2–induced damage was ameliorated by 24‐hour pre‐exposure to low‐concentration H2O2 (10μM‐30μM) with cell size as well restored. Markers for apoptosis (Bcl‐2 and Bad), inflammation (iNOS), and redox system (MnSOD) were also determined, showing that, in cells pre‐exposed to 10μM H2O2 and then submitted to 100μM H2O2, Bcl‐2 levels were higher, Bad and iNOS levels were lower than those observed in damaged cells, and MnSOD levels were unchanged. Such findings show that (1) neuronal‐like differentiated SH‐SY5Y cells are a suitable model to investigate PC response and more sensitive to the effect of a mild and transient H2O2‐induced oxidative stress with respect to other neuronal cells; (2) 10μM H2O2–induced PC is mediated by apoptotic and inflammatory pathways, unlike antioxidant system; (3) such neuroprotective strategy and underlying signals proven in neuronal‐like differentiated SH‐SY5Y cells may contribute to understand in vivo PC mechanisms and to define a window for pharmacological intervention, namely, related to ischemic brain damage.