Nicola Costa

Systemic Administration of Nω-Nitro-l-Arginine Methyl Ester and Indomethacin Reduces the Elevation of Brain PGE2Content and Prevents Seizures and Hippocampal Damage Evoked by LiCl and Tacrine in Rat☆

Administration of tacrine (5 mg/kg i.p.), an anticholinesterase agent, in rats pretreated (24 h beforehand) with lithium chloride (LiCl; 12 mEq/kg i.p.) enhances the expression of neuronal nitric oxide (NO) synthase (NOS), increases NO, and causes seizures and hippocampal damage. Here we report immunohistochemistry evidence showing that in rat LiCl and tacrine enhance the expression of cyclooxygenase type 2 (COX-2) enzyme protein in the dorsal hippocampus and elevate brain PGE2 content during the preconvulsive period. The latter effect, but not enhanced COX-2 expression, is inhibited by previous (30 min before tacrine) administration of N omega-nitro-L-arginine-methyl ester (L-NAME; 10 mg/kg i.p.), an inhibitor of NO synthesis, thus implicating NO in the mechanism of stimulation of COX activity leading to elevation of brain PGE2 content. Indomethacin (10 mg/kg given i.p. 30 min before tacrine), an inhibitor of COX activity, prevented brain PGE2 elevation and abolished the expression of seizures and hippocampal damage thus supporting a role for this metabolite of the arachidonic acid cascade in the mechanisms of LiCl and tacrine-evoked neurotoxicity in rat.

Effects of compounds acting on GABAB receptors in the pentylenetetrazole kindling model of epilepsy in mice

The involvement of GABA(B) receptors in the behavioural and epileptic electrocortical discharges occurring in chemical kindling induced by repeated treatments with a subconvulsant dose of pentylenetetrazole (25 mg/kg i.p.) has been investigated in CD1 mice. Behavioural and electrocorticographic epileptic seizures following kindling induced by pentylenetetrazole (25 mg/kg i.p.) were attenuated or completely antagonized in a dose-dependent manner by the GABA(B) receptor agonist R-baclofen (2 and 6 mg/kg) whilst the GABA(B) receptor antagonist 3-amino-propyl-diethoxy-methyl-phosphinic acid (CGP 35348, 25, 50 or 100 mg/kg) and 3-[1-(S)-(3, 4-dichloro-phenyl-ethyl]amino-2-(S)-hydroxy-propyl-benzyl-phosphinic acid (CGP 55845A, 10 or 20 mg/kg) produced a more rapid development of kindling and an increase in behavioural and electrocorticographic epileptic changes. In addition, all GABA(B) receptor antagonists were able to induce an increase in Fos and Jun protein expression in pentylenetetrazole (25 mg/kg i.p.) treated mice whilst the GABA(B) receptor agonist R-baclofen (2 or 6 mg/kg) attenuated the expression of Fos and Jun protein, at cortical and limbic structures. In order to study the persistence of changes induced by pentylenetetrazole kindling, different groups of mice were rechallenged with a kindling stimulus 15 or 30 days after withdrawal from the last injection of vehicle+pentylenetetrazole, R-baclofen+pentylenetetrazole or GABA(B) receptor antagonists+pentylenetetrazole. The groups receiving GABA(B) receptor antagonists+pentylenetetrazole showed a higher incidence of seizures following the kindling stimulus than mice receiving vehicle+pentylenetetrazole whilst animals treated with R-baclofen were protected from the kindling stimulus. The different effects observed following repeated treatment with the GABA(B) receptor agonist and antagonist used revealed that GABA(B) receptors are able to affect the development of the epileptic kindling state induced by pentylenetetrazole.

N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction

BackgroundHIV envelope gp 120 glycoprotein is released during active HIV infection of brain macrophages thereby generating inflammation and oxidative stress which contribute to the development of the AIDS-Dementia Complex (ADC). Gp120 has also been found capable to generate excitotoxic effect on brain tissue via enhancement of glutamatergic neurotransmission, leading to neuronal and astroglial damage, though the mechanism is still to be better understood.Here we investigated on the effect of N-acetylcysteine (NAC), on gp120-induced damage in human cultured astroglial cells and the possible contribution of gp120-related reacting oxygen species (ROS) in the imbalanced activity of glutamine synthase (GS), the enzyme that metabolizes glutamate into glutamine within astroglial cells playing a neuroprotective role in brain disorders.ResultsIncubation of Lipari human cultured astroglial cells with gp 120 (0.1–10 nM) produced a significant reduction of astroglial cell viability and apoptosis as evaluated by TUNEL reaction and flow cytometric analysis (FACS). This effect was accompanied by lipid peroxidation as detected by means of malondialdehyde assay (MDA). In addition, gp 120 reduced both glutamine concentration in astroglial cell supernatants and GS expression as detected by immunocytochemistry and western blotting analysis. Pre-treatment of cells with NAC (0.5–5 mM), dose-dependently antagonised astroglial apoptotic cell death induced by gp 120, an effect accompanied by significant attenuation of MDA accumulation. Furthermore, both effects were closely associated with a significant recovery of glutamine levels in cell supernatants and by GS expression, thus suggesting that overproduction of free radicals might contribute in gp 120-related dysfunction of GS in astroglial cells.ConclusionIn conclusion, the present experiments demonstrate that gp 120 is toxic to astroglial cells, an effect accompanied by lipid peroxidation and by altered glutamine release. All the effects of gp120 on astroglial cells were counteracted by NAC thus suggesting a novel and potentially useful approach in the treatment of glutammatergic disorders found in HAD patients.

The effect of inflammatory stimuli on NMDA-related activation of glutamine synthase in human cultured astroglial cells

Removal of glutamate from the synaptic cleft by astroglial glutamine synthase (GS) is a crucial step in the regulation of glutamate turnover and metabolism, thus participating in endogenous neuroprotective processes occurring within brain tissues. Here we investigated on the effect of inflammatory cytokines on GS activity in astroglial cells undergoing NMDA receptors stimulation. Incubation of human cultured astroglial cells with NMDA (100 microM) enhanced GS expression, an effect driven by the generation of nitric oxide (NO) since l-NAME (500 microM), an inhibitor of NO synthase, reversed this effect. NMDA-related increase of GS activity and glutamine concentration was antagonised by previous incubation of astroglial cells with a mixture of LPS plus gammaIFN, an effect counteracted by dexamethasone, the latter effect being accompanied by inhibition of inducible NO synthase. These results show that LPS plus gammaIFN inhibit elevation of GS activity subsequent to NMDA receptor stimulation in astroglial cells via enhancement of inducible NO synthase, and this may represent the site of interaction between pro-inflammatory and excitotoxic stimuli in the brain.

Bergamot polyphenol fraction prevents nonalcoholic fatty liver disease via stimulation of lipophagy in cafeteria diet-induced rat model of metabolic syndrome

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries. Defective autophagy of lipid droplets (LDs) in hepatocytes, also known as lipophagy, has recently been identified as a possible pathophysiological mechanism of NAFLD. Experimental and epidemiological evidence suggests that dietary polyphenols may prevent NAFLD. To address this hypothesis and analyze the underlying mechanisms, we supplemented bergamot polyphenol fraction (BPF) to cafeteria (CAF) diet-fed rats, a good model for pediatric metabolic syndrome and NAFLD. BPF treatment (50 mg/kg/day supplemented with drinking water, 3 months) potently counteracted the pathogenic increase of serum triglycerides and had moderate effects on blood glucose and obesity in this animal model. Importantly, BPF strongly reduced hepatic steatosis as documented by a significant decrease in total lipid content (-41.3% ± 12% S.E.M.), ultrasound examination and histological analysis of liver sections. The morphometric analysis of oil-red stained sections confirmed a dramatic reduction in LDs parameters such as total LD area (48.5% ± 15% S.E.M.) in hepatocytes from CAF+BPF rats. BPF-treated livers showed increased levels of LC3 and Beclin 1 and reduction of SQSTM1/p62, suggesting autophagy stimulation. Consistent with BPF stimulation of lipophagy, higher levels of LC3II were found in the LD subcellular fractions of BPF-expose livers. This study demonstrates that the liver and its lipid metabolism are the main targets of bergamot flavonoids, supporting the concept that supplementation of BPF is an effective strategy to prevent NAFLD.

In vivo targeting and growth inhibition of the A20 murine B-cell lymphoma by an idiotype-specific peptide binder.

B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-beta cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 treatment was associated with an increased number of tumor-infiltrating, activated CD8+ T cells that exerted a tumor-specific cytolytic activity. These findings show that a short peptide that binds specifically to the complementarity-determining regions of the A20 BCR allows in vivo detection of neoplastic cells together with significant inhibition of tumor growth in vivo.

Amino acid levels in some brain areas of inducible nitric oxide synthase knock out mouse (iNOS −/− ) before and after pentylenetetrazole kindling

Inducible nitric oxide synthase knock-out (iNOS(-/-)) mice are valid models of investigation for the role of iNOS in patho-physiological conditions. There are no available data concerning neuroactive amino acid levels of iNOS(-/-) mice and their behaviour in response to pentylenetetrazole (PTZ). We found no significant differences in the convulsive dose 50 (CD(50)) between iNOS(-/-) and control (iNOS(+/+)) mice, however, iNOS(-/-) mice reach the kindled status more slowly than control, suggesting that in basal condition the GABA-benzodiazepine inhibitory inputs are unaltered by iNOS mutation. Clear differences between iNOS(+/+) and iNOS(-/-) mice amino acid concentrations were evident both in basal conditions and after kindling. Our results show that aspartate was significantly lower in all brain areas studied except the brain stem whereas glutamate and glutamine were significantly higher in the cortex, hippocampus and brain stem. GABA was slightly and not significantly higher in the cortex, hippocampus and brain stem, whereas taurine was significantly higher in all areas except diencephalon and glycine was significantly lower in the diencephalon and cerebellum. In this context, the inability of iNOS(-/-) mice to increase the NO levels following PTZ administrations indicate that NO might play a pro-epileptogenic role in the genesis and development of some types of epilepsy. Since there is no correlation between neurotransmitter levels and the development of kindling, it is possible to exclude that the difference between the two strains is due to an imbalance between the considered neurotransmitters, and it is then possible that this difference is due to the presence of iNOS, which might be involved in long term plasticity of the brain.

CCK-8 prevents the development of kindling and regulates the GABA and NPY expression in the hippocampus of pentylenetetrazole (PTZ)-treated adult rats.

Neuronal loss and irreversible brain damage often cause the worsening of symptoms and the decreased efficacy of pharmacological treatment occurring in epileptic patients and animal models of kindling. Recently we reported that the neurotransmitter/neuromodulatory peptide Cholecystokinin-8 (CCK-8) is able to induce the structural and functional neuronal recovery of chemical- and surgical-induced lesions when i.p. injected in rodents. The present study therefore, was aimed at verifying the hypothesis that treatment with a CCK-8 dose having a neuroprotective action might affect brain alterations and the development of kindling in adult rats receiving the convulsant agent pentylenetetrazole (PTZ). Compared to rats receiving Saline prior to PTZ, which manifested clonic-tonic seizures (Class 5 behavioural change scale) after three weeks of treatment, rats pre-treated with CCK-8 showed an improvement of behavioural score exhibiting myoclonus and occasionally tonic seizures (Class 3/4). This decreased susceptibility to develop convulsions was associated with the recovery of PTZ-induced reduction of ChAT levels in forebrain and GABA/GAD expression in the hippocampus. Furthermore, NPY immunoreactivity distribution and NPY mRNA levels were also increased in the hippocampus of rats receiving CCK-8 injection before each PTZ treatment. These data indicate that CCK-8 possesses the ability to prevent and/or suppress the convulsant effects of PTZ by stimulating the synthesis of neurotransmitters/peptides involved in the inhibition of hippocampal hyper-excitability. Our findings suggest that CCK-8 may have anticonvulsant and neuroprotective properties that merit further investigation.

The human immunodeficiency virus type 1 (HIV-1) glycoprotein gp120 reduces the expression of neuronal nitric oxide synthase in the hippocampus but not in the cerebral cortex and medial septal nucleus of rat

The expression of neuronal type of nitric oxide synthase (nNOS) has been studied by immunocytochemistry in the brain of rats receiving a single daily (for up to 14 consecutive days) intracerebroventricular (i.c.v.) injection of the HIV-1 recombinant protein gp 120 (100 ng/day). This treatment failed to affect nNOS expression in the cerebral cortex and medial septal nucleus whereas it reduced nNOS immunopositive neurones in the CA1 hippocampal cell layer of rats treated with gp120 for 14 days; the latter effect was accompanied by a parallel decrease in Ca(2+)-dependent NOS enzyme activity in hippocampal brain tissue homogenates. In conclusion, the present data demonstrate that in rats gp120 reduces the expression of nNOS in the hippocampus, an area of the brain involved in memory formation.

CCK-8 induces NGF and BDNF synthesis and modulates TrkA and TrkB expression in the rat hippocampus and septum: Effects on kindling development.

In our previous studies, we demonstrated that intraperitoneal (i.p.) injections with the neurotransmitter/neuromodulatory peptide Cholecystokinin-8 (CCK-8) stimulate the synthesis of the neurotrophin nerve growth factor (NGF) resulting in the structural and functional recovery of neuronal damage. This neurotrophin-mediated neuroprotective action of CCK-8 has opened a new perspective for a better understanding of the CCK neurobiological and pharmacological properties. To explore the possible beneficial effects of the CCK-induced increase of neurotrophin availability in brain, we compared the effects of i.p. CCK-8 in healthy rats and in a chemical kindling model using a subconvulsive dose of pentylenetetrazol (PTZ). Behavioural changes were monitored during treatment and classified according to a six-point scale. After 3 weeks of treatment (12 trials), the PTZ group of rats manifested generalized clonic-tonic seizures (Class 5 behaviour). For this reason, this time point was chosen to compare the effects of CCK-8 treatment on the expression of NGF, the brain derived neurotrophin factor (BDNF) and their receptors in the septum and hippocampus. We found that repeated i.p. injections with CCK-8 in adult rats result in: (1) an increase of NGF and BDNF protein and mRNA levels in the septum and hippocampus; (2) a down-regulation of TrkA and p75NTR and an up-regulation of TrkB; (3) reduced susceptibility to develop chemical kindling; (4) recovery of the PTZ-induced changes in the expression of neurotrophin receptors in the septal and hippocampal tissues. This data clearly indicates that CCK-induced variation of neurotrophin synthesis in brain is able to influence the susceptibility to develop seizures in adult rats most probably by counteracting the progressive neuronal dysfunction and/or damage.