Angela Marino

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.

Administration of palmitoylethanolamide (PEA) protects the neurovascular unit and reduces secondary injury after traumatic brain injury in mice

Traumatic brain injury (TBI) is a major cause of preventable death and morbidity in young adults. This complex condition is characterized by significant blood brain barrier leakage that stems from cerebral ischemia, inflammation, and redox imbalances in the traumatic penumbra of the injured brain. Recovery of function after TBI is partly through neuronal plasticity. In order to test whether treatments that enhance plasticity might improve functional recovery, a controlled cortical impact (CCI) in adult mice, as a model of TBI, in which a controlled cortical impactor produced full thickness lesions of the forelimb region of the sensorimotor cortex, was performed. Once trauma has occurred, combating these exacerbations is the keystone of an effective TBI therapy. The endogenous fatty acid palmitoylethanolamide (PEA) is one of the members of N-acyl-ethanolamines family that maintain not only redox balance but also inhibit the mechanisms of secondary injury. Therefore, we tested whether PEA shows efficacy in a mice model of experimental TBI. PEA treatment is able to reduced edema and brain infractions as evidenced by decreased 2,3,5-triphenyltetrazolium chloride staining across brain sections. PEA-mediated improvements in tissues histology shown by reduction of lesion size and improvement in apoptosis level further support the efficacy of PEA therapy. The PEA treatment blocked infiltration of astrocytes and restored CCI-mediated reduced expression of PAR, nitrotyrosine, iNOS, chymase, tryptase, CD11b and GFAP. PEA inhibited the TBI-mediated decrease in the expression of pJNK and NF-κB. PEA-treated injured animals improved neurobehavioral functions as evaluated by behavioral tests.

Effect of various factors on Pelagia noctiluca (Cnidaria, Scyphozoa) crude venom-induced haemolysis

The haemolytic power of isolated nematocysts from the scyphozoan Pelagia noctiluca was studied with attention to the effect of osmotic protectants as carbohydrates at different MW, cations as Mg2+, Ca2+, Ba2+,Cu2+, K+; proteases as collagenase, trypsin, alpha-chymotrypsin, papain; and antioxidants. Crude venom was at first obtained by sonication of holotrichous-isorhiza nematocysts previously isolated from oral arms of P. noctiluca and then haemolytically tested upon human erythrocytes. Osmotic protectants were effective in inhibiting the haemolytic power depending on their molecular weight so that total inhibition of crude venom-induced haemolysis was observed after PEG treatment (polyethyleneglycol 6000Da). Amongst divalent cations only Ba2+ and Cu2+ significantly inhibited the haemolytic power of crude venom. Proteases seem not to alter the haemolytic activity while antioxidant compounds only slightly reduced the haemolytic power. Such findings may suggest a pore-forming mechanism for P. noctiluca crude venom rather than an oxidative damage to the cell membrane.

Reduction of ischemic brain injury by administration of palmitoylethanolamide after transient middle cerebral artery occlusion in rats

Stroke is the third leading cause of death and the leading cause of long-term disability in adults. Current therapeutic strategies for stroke, including thrombolytic drugs, such as tissue plasminogen activator offer great promise for the treatment, but complimentary neuroprotective treatments are likely to provide a better outcome. To counteract the ischemic brain injury in mice, a new therapeutic approach has been employed by using palmitoylethanolamide (PEA). PEA is one of the members of N-acyl-ethanolamine family maintain not only redox balance but also inhibit the mechanisms of secondary injury on ischemic brain injury. Treatment of the middle cerebral artery occlusion (MCAo)-induced animals with PEA reduced edema and brain infractions as evidenced by decreased 2,3,5-triphenyltetrazolium chloride (TTC) staining across brain sections. PEA-mediated improvements in tissues histology shown by reduction of lesion size and improvement in apoptosis level (assayed by Bax and Bcl-2) further support the efficacy of PEA therapy. We demonstrated that PEA treatment blocked infiltration of astrocytes and restored MCAo-mediated reduced expression of PAR, nitrotyrosine, iNOS, chymase, tryptase, growth factors (BDNF and GDNF) and GFAP. PEA also inhibited the MCAo-mediated increased expression of pJNK, NF-κB, and degradation of IκB-α. PEA-treated injured animals improved neurobehavioral functions as evaluated by motor deficits. Based on these findings we propose that PEA would be useful in lowering the risk of damage or improving function in ischemia-reperfusion brain injury-related disorders.

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.

Nematocyst discharge in Pelagia noctiluca (Cnidaria, Scyphozoa) oral arms can be affected by lidocaine, ethanol, ammonia and acetic acid

Nematocyst discharge and concomitant delivery of toxins is triggered to perform both defence and predation strategies in Cnidarians, and may lead to serious local and systemic reactions in humans. Pelagia noctiluca (Cnidaria, Scyphozoa) is a jellyfish particularly abundant in the Strait of Messina (Italy). After accidental contact with this jellyfish, not discharged nematocysts or even fragments of tentacles or oral arms may tightly adhere to the human skin and, following discharge, severely increase pain and the other adverse consequences of the sting. The aim of the present study is to verify if the local anesthetic lidocaine and other compounds, like alcohols, acetic acid and ammonia, known to provide pain relief after jellyfish stings, may also affect in situ discharge of nematocysts. Discharge was induced by a combined physico-chemical stimulation of oral arms by chemosensitizers (such as N-acetylated sugars, aminoacids, proteins and nucleotides), in the presence or absence of 1% lidocaine, 70% ethanol, 5% acetic acid or 20% ammonia, followed by mechanical stimulation by a non-vibrating test probe. The above mentioned compounds failed to induce discharge per se, and dramatically impaired the chemosensitizer-induced discharge response. We therefore suggest that prompt local treatment of the stung epidermis with lidocaine, acetic acid, ethanol and ammonia may provide substantial pain relief and help in reducing possible harmful local and systemic adverse reaction following accidental contact with P. noctiluca specimens.

Factors altering the haemolytic power of crude venom from Aiptasia mutabilis (Anthozoa) nematocysts

The effect of different agents upon the haemolytic power of Aiptasia mutabilis crude venom was studied inhuman erythrocytes to determine its toxicity and stability. Nematocysts were isolated from acontia of the Anthozoan A. mutabilis and submitted to sonication for extracting crude venom. Aliquots of venom were tested in 0.05% erythrocyte suspensions in the presence of various factors such as proteases (papain,collagenase, trypsin, alpha-chymotrypsin); cations (Ca2+, Mg2+, Ba2+, K+ and Cu2+), osmotic protectants as polyethylenglycole (PEG) of different MW and antioxidant compounds (GSH, cysteine and ascorbic acid).Results demonstrate the dose-response of the haemolytic effect of A. mutabilis. Haemolysis by the crude venom was prevented by Ca2+, Ba2+ and Cu2+ treatment, and to a minor extent by Mg2+ and K+. Papain and PEG with a molecular mass exceeding 1000 Da also prevented haemolysis. These findings are consistent with a pore-forming mechanism of crude venom in erythrocytes rather than an oxidative damage at the employed doses.

Morphological integrity and toxicological properties of Pelagia noctiluca (Scyphozoa) nematocysts

Isolated nematocysts of the Scyphozoan Pelagia noctiluca became diaphanous when incubated at low pH for 1 h or left at room temperature for a few hours. Diaphanous nematocysts were unable to undergo discharge when triggered by proper physico-chemical stimuli. The aim of the present study was to investigate the relationship between the morphological features of diaphanous nematocysts and the haemolytic power of their crude venom, obtained by sonication on ice. Nematocysts stored at−20 °C in neutral medium were used as a control. Our results show that the haemolytic power of crude venom from isolated nematocysts kept at low pH or at room temperature decreased significantly with respect to the control. From this study, it can be deduced that a neutral pH and low temperature conditions are recommended to store nematocysts properly in order to use them in toxicological investigations.