The exploration of a new environment leads to the modulation of gene expression for prolonged times in the rat.

In the present study we performed a transcriptional analysis in order to evaluate changes in gene expression induced by exploration in prolonged times. The analysis was carried out 3, 10 and 20 days after exploration. We analyzed the modulation of the expression levels of Pfn2, Casp3, Pdrg1, Pea15, Ywhaz genes which previously were found not modulated 2 days after exploration. Our data show that the expression of Pfn2, Casp3, Pdrg1, Pea15, Ywhaz genes was modulated at 10 or 20 days. The transcript, whose expression had been evaluated with the qRT-PCR, code for proteins which belong to the following functional categories: synaptic modulation, apoptosis, signal transduction. It is interesting to note that the modulation of the expression of these genes was evident some days after environmental exploration, and not previously at 2 days after conditioning as occurred after contextual fear conditioning (CFC). Hence it is possible to hypothesize that the spatial memory processes require a longer period of elaboration than the emotional ones, fundamental for the survival of the species.

Contextual fear conditioning modulates the gene expression over time.

Contextual fear conditioning (CFC) is a quick cognitive test based on the association context-aversive stimulus in which a single training leads to a long-term memory. Previously, we showed that 2 days after conditioning the expression of the genes Napa, Pnf2, Casp3, Pdrg1, Ywhaz, Stmn1, Bpgm, were positively modulated in CFC rats respect to naïve rats, explor rats which had freely explored the experimental apparatus and SO rats to which the same number of aversive shocks used in CFC paradigm had been administered in the same CFC apparatus in less time to prevent the association between painful stimuli and apparatus, whereas the genes Actr3, Pea15 and Tiprl were more expressed in SO rats and Cplx1, Trim32 and Ran genes were more expressed in explor rats. At 2 days, Tomm20 gene expression resulted positively modulated in both CFC and explor rats. Herein, we have tested the expression of these genes for a period longer than 2 days, by monitoring the modulation of transcripts within 20 days after conditioning. The expression of the transcripts was assessed by qRT-PCR.We found that three days after CFC only the genes Tiprl and Trim32 were positively modulated in CFC rats whereas the gene Tomm20 was negatively modulated in CFC rats as well as in SO and explor rats. Ten days after CFC, the expression of Trim32 was still positively modulated whereas the genes Tiprl and Tomm20 returned to the constitutive level, and the gene Ran was significantly more expressed in CFC rats than in naïve, SO and explor rats. Interestingly, 20 days after CFC, the genes Stmn1 and Tiprl again became significantly more expressed in CFC rats compared with naïve, SO and explor rats.

Physical Exercise Improves Total Antioxidant Capacity and Gene Expression in Rat Hippocampal Tissue.

Exercise may exert beneficial effects on cognitive functions and play an important role in the prevention of neurodegenerative diseases. Such effects seem to be mediated by changes in anti-oxidative status, but limited information is available on the nature of molecular pathways supporting the antioxidant effects of exercise in the brain. In this study 3-5-month-old male Wistar albino rats were subjected to three times/week moderate intensity exercise on a rodent treadmill for a period of 6 weeks. The tissue antioxidant activity towards various reactive oxygen species (ROS) was determined in the hippocampus. In addition, to identify the molecular pathways that may be involved in ROS metabolism, the expression of nerve growth factor (NGF) and sirtuins (SIRT1 and SIRT3) were measured. Our results showed a higher antioxidant activity in the hippocampus of physically trained rats compared to sedentary controls. Furthermore, exercise induced an up-regulation of NGF, possibly related to an improved redox balance in the hippocampus. These results suggest that physical exercise might prevent age-induced oxidative damage in the hippocampus.

Further evidence of a prolonged hypotensive and a bradycardic effect after mandibular extension in normal volunteers.

We previously reported that in normotensive humans submaximal mouth opening (mandibular extension) obtained by an ad hoc dilator (spring device), associated with partial masticatory movements and prolonged for 10 minutes is followed by a long-lasting reduction of blood pressure (BP) and heart rate (HR). Similar results were obtained by us in anesthetized rats. A recent independent study failed to confirm the results in the normotensive human. We reassessed, in 25 normotensive volunteers, the effects on BP and HR of mandibular extension obtained by the spring device associated with partial masticatory movements compared to a control procedure, consisting in keeping a tongue depressor between the incisor teeth. Both procedures were applied for 10 minutes and systolic BP (SBP), diastolic BP (DBP) and HR were measured every 10 minutes by an automatic recorder, for 30 minutes before and 120 minutes after the procedures in seated subjects watching nature documentary films on laptop screen.Baseline levels (mean of the last 3 measurements before procedure) did not significantly differ between the experimental and control sessions. Two way repeated measures ANOVA on absolute (recorded) values did not reveal a significant main effect of treatment for SBP, DBP and HR, but a significant main effect of time (P<0.001) for BP and HR. In addition, a significant interaction of time and treatment was found for SBP (P<0.001) and DBP (P=0.005), but not for HR. In addition, two way repeated measures ANOVA was done on changes from baseline obtaining a significant main effect of treatment (P<0.001) and time (P<0.001) and a significant interaction of time and treatment for SBP (P<0.001) and DBP (P<0.01). Post-hoc comparisons revealed significantly lower values for SBP and DBP in experimental compared to control values at almost all times and this decrement was by about 5 mmHg. Furthermore, for both absolute values and changes from baseline, the interaction effect was, for BP, of a qualitative type as indicated by an opposite effect in the time-course between control and experimental sessions. This study thus provides confirmatory evidence that submaximal mouth opening for a relatively brief time is followed by prolonged albeit small reductions of BP in normotensive human volunteers.

Persistent effects after trigeminal nerve proprioceptive stimulation by mandibular extension on rat blood pressure, heart rate and pial microcirculation.

The trigemino-cardiac reflex is a brainstem reflex known to lead to a decrement in heart rate and blood pressure, whereas few data have been collected about its effects on the cerebral hemodynamic. In this study we assess the in vivo effects of trigeminal nerve peripheral stimulation by mandibular extension on pial microcirculation and systemic arterial blood pressure in rats. Experiments were performed in male Wistar rats subjected to mandibular extension obtained inserting an ad hoc developed retractor between the dental arches. Mean arterial blood pressure and heart rate were recorded and the pial arterioles were visualized by fluorescence microscopy to measure the vessel diameters before (15 minutes) during (5-15 minutes) and after (80 minutes) mandibular extension. While in control rats (sham-operated rats) and in rats subjected to the dissection of the trigeminal peripheral branches mean arterial blood pressure, heart rate and pial microcirculation did not change during the whole observation period (110 minutes), in rats submitted to mandibular extension, mean arterial blood pressure, heart rate and arteriolar diameter significantly decreased during stimulation. Afterward mean arterial blood pressure remained reduced as well as heart rate, while arteriolar diameter significantly increased evidencing a vasodilatation persisting for the whole remaining observation time. Therefore, trigeminal nerve proprioceptive stimulation appears to trigger specific mechanisms regulating systemic arterial blood pressure and pial microcirculation.

Acetyl-L-carnitine affects nonassociative learning processes in the leech Hirudo medicinalis.

Acetyl-L-carnitine is a natural molecule widely distributed in vertebrate and invertebrate nervous system. It is known to have significant effects on neuronal activity playing a role as neuroprotective and anti-nociceptive agent, as well as neuromodulatory factor. About its capability of affecting learning processes the available data are controversial. In the present study, we utilized the simplified model system of the leech Hirudo medicinalis to analyze the effects of acetyl-L-carnitine, assessing whether and how it might affect elementary forms of nonassociative learning processes. In leeches with the head ganglion disconnected from the first segmental ganglion, repetitive application of weak electrical shocks onto the caudal portion of the body wall induces habituation of swim induction whereas brush strokes on the dorsal skin produces sensitization or dishabituation when the nociceptive stimulus is delivered on previously habituated animals. Herein, the effects of different concentrations of acetyl-L-carnitine (2 mM - 0.05 mM) have been tested at different times on both sensitization and dishabituation. The results show that a single treatment of acetyl-L-carnitine blocked the onset of sensitization in a dose- and time-dependent manner. In fact, the most effective concentration able to block this process was 2 mM, which induced its major effects 11 days after the treatment, whereas 0.05 mM was unable to affect the sensitization process at all considered time points. On the contrary, acetyl-L-carnitine did not completely abolish dishabituation at the tested concentrations and at every time point. Finally, acetyl-L-carnitine also impaired the habituation of swim induction, but only 11 days after treatment.

Reduction of myocardial infarct size in rat by IRFI-048, a selective analogue of vitamin E.

The effects of IRFI-048 (2,3-dihydro-5-methoxy-4,6,7-trimethyl-2-benzofuranyl acetic acid), a selective analogue of Vitamin E, on myocardial tissue injury were examined in anaesthetized rats subjected to 60-min occlusion of the left coronary artery followed by 60-min reperfusion. Infarct size (Evan's blue and tetrazolium stain), serum creatinphosphokinase (CPK), plasma malonaldehyde (MAL), cardiac myeloperoxidase (MPO) activity, and ST-segment of electrocardiogram (ECG) and survival rate were evaluated. Postischaemic reperfusion produced severe cardiac necrosis, caused neutrophil (PMNs) infiltration (evaluated by MPO activity) in the jeopardized tissue, increased serum CPK and plasma MAL, raised ST-segment of ECG, and decreased survival rate. IRFI-048, (200 and 400 mg/kg o.s.) given to the rats 6 h before occlusion, caused a reduction of necrotic area expressed as a percentage of either the area at risk or the total left ventricle, decreased MPO activity both in the area at risk (from 3.2 +/- 0.3 U x 10(-3)/g tissue to 1.1 +/- 0.4 U x 10(-3)/g tissue; p < .005) and in the necrotic area (from 5.7 +/- 0.9 U x 10(-3)/g tissue to 1.8 +/- 0.5 U x 10(-3)/g tissue; p < .001), attenuated the rise of ST-segment of ECG (from 0.51 +/- 0.14 mV in the vehicle group to 0.28 +/- 0.11 mV in the treated group; p < .005), reduced the increase of plasma MAL and serum CPK during reperfusion (from 42 +/- 5.3 nmol/ml to 15 +/- 3.1 nmol/ml and 139 +/- 13 IU/100 ml to 58 +/- 7.5 IU/100 ml, respectively; p < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Octopamine and Leydig cell stimulation depress the afterhyperpolarization in touch sensory neurons of the leech.

In touch sensory neurons of the leech, a train of spikes evoked by intracellular electrical stimulation leads to an afterhyperpolarization, mainly due to the activation of the Na+/K+ electrogenic pump and partly to a Ca(2+)-activated K+ conductance. It has been found that serotonin is able to reduce the afterhyperpolarization through the inhibition of the Na+/K+ electrogenic pump. We have investigated the possible modulation of the afterhyperpolarization by other endogenous neurotransmitters and we have found that octopamine is also able to reduce its amplitude. The electrical stimulation of the octopaminergic Leydig neurons mimics this effect. We have compared the actions of the two amines and found that the effect of serotonin is blocked by methysergide but not by high [Mg2+] or by phentolamine, and it is still present in touch cells isolated in culture. On the contrary, the octopamine modulation of the afterhyperpolarization does not occur in single touch cells in culture and it is blocked by all these treatments. These data suggest that while serotonin should act monosynaptically, octopamine should act through a serotonergic pathway.

Caulerpenyne, a toxin from the seaweed Caulerpa taxifolia, depresses afterhyperpolarization in invertebrate neurons.

The massive invasion of the Mediterranean Sea by the tropical seaweed Caulerpa taxifolia (Vahl) C. Agardh has stimulated several investigations in order to test the environmental risk from an ecotoxicological point of view. The studies carried out on various experimental models have shown that caulerpenyne, the major metabolite synthesized by the seaweed, affects several cellular and molecular targets. In addition, neurological disorders have been reported in patients who accidentally ate C. taxifolia, but no evidence about the potential effects of the seaweed and of its metabolites on nerve cells were up to now available. Herein we describe that caulerpenyne modifies the electrical properties of touch mechanosensory cells of the leech Hirudo medicinalis. The physiological firing of these cells causes an afterhyperpolarization that is mainly due to the activity of the Na+/K+-ATPase and to a lesser extent to a calcium-dependent potassium current. Caulerpenyne depressed this afterhyperpolarization; the effect was dose-dependent and partially reversible. Experiments have been carried out in order to understand the mechanism through which caulerpenyne reduced the afterhyperpolarization. The action of the biotoxin has been tested in the presence of pharmacological blockers of calcium-dependent potassium channels such as cadmium and apamin. In these experimental conditions, caulerpenyne still reduced the residual afterhyperpolarization, suggesting a direct effect of the toxin on the Na+/K+-ATPase. In order to test this hypothesis, we have performed experiments where the Na+/K+-ATPase was activated by the intracellular injection of sodium and where also its basal activity was modified as well. From the data collected we suggest that caulerpenyne inhibits both the basal and the sodium-induced activity of the Na+/K+-ATPase in leech touch neurons.

Acetyl-L-carnitine induces a sustained potentiation of the afterhyperpolarization.

Acetyl-L-carnitine is known to improve many aspects of the neural activity even if its exact role in neurotransmission is still unknown. This study investigates the effects of acetyl-L-carnitine in T segmental sensory neurons of the leech Hirudo medicinalis. These neurons are involved in some forms of neural plasticity associated with learning processes. Their physiological firing is accompanied by a large afterhyperpolarization that is mainly due to the Na+/K+ ATPase activity and partially to a Ca2+ -dependent K+ current. A clear-cut hyperpolarization and a significant increase of the afterhyperpolarization have been recorded in T neurons of leeches injected with 2 mM acetyl-L-carnitine some days before. Acute treatments of 50 microM acetyl-L-carnitine induced similar effects in T cells of naive animals. In the presence of apamin, a pharmacological blocker of Ca2+ -dependent K+ channel, acetyl-L-carnitine still enhanced the residual afterhyperpolarization, suggesting an effect of the drug on the Na+/K+ATPase. Acetyl-L-carnitine also increased the hyperpolarization induced by intracellular injection of Na+ ions. Therefore, acetyl-L-carnitine seems to be able to exert a positive sustained effect on the Na+/K+ ATPase activity in leech T sensory neurons. Moreover, in these cells, widely arborized, the afterhyperpolarization seems to play an important role in determining the action potential transmission at neuritic bifurcations. A computational model of a T cell has been previously developed considering detailed data for geometry and the modulation of the pump current. Herein, we showed that to a larger afterhyperpolarization, due to the acetyl-L-carnitine-induced effects, corresponds a decrement in the number of action potentials reaching synaptic terminals.

Neurotoxic effects of caulerpenyne.

1. In this paper the authors tested the effect of caulerpenyne (CYN), a sesquiterpene synthesized by the green alga Caulerpa taxifolia onto the central nervous system of the leech Hirudo medicinalis. Investigations have been performed with three different approaches: neuroethological, electrophysiological and neurochemical techniques. 2. CYN application mimics the effect of a nociceptive stimulation (brushing), eliciting a clear-cut potentiation of the animal swim response to the test stimulus (non associative learning process such as sensitization). This effect is similar to that one induced by the endogenous neurotransmitter serotonin (5HT). 3. CYN strongly reduces the after-hyperpolarization (AHP) recorded from T sensory neurons. This effect overlaps that one produced by 5HT, but it is not affected by the serotonergic antagonist methysergide. 4. The decrease of AHP amplitude due to CYN application is observed also in presence of apamin, a blocking agent of Ca++-dependent K+ channels, suggesting that CYN is acting through the inhibition of the Na+/K+ electrogenic pump. 5. The depression of the AHP driven by CYN is not prevented by application of MDL 12330A, an adenylate cyclase inhibitor. On the other hand MDL 12330A counteracts the reduction of AHP due to 5HT application. 6. Incubation of the leech central nervous system with CYN induces the phosphorylation of proteins of 29, 50, 66 and 100 kDa. This pattern of phosphorylation is similar to that one elicited by 5HT treatment. 7. The data demonstrate that CYN exerts remarkable effects on leech neurons by acting onto specific molecular targets such as the Na+/K+ ATPase. This effect may influence important neural integrative functions and may explain the sensitizing action produced by the toxin on swim induction. Finally, caulerpenyne does not act through the pathways involved in the 5HT action, and its effect is not mediated by the second messenger cyclic AMP. The mechanism of action of CYN are still under investigations.

Effects of fructose 1,6-diphosphate on splanchnic artery occlusion shock in the rat.

Splanchnic artery occlusion (SAO) shock, produced by clamping splanchnic arteries for 45 min followed by the release of occlusion, was induced in male rats, treated 15 min before surgery, with fructose 1,6-diphosphate (FDP) or with equivalent doses of fructose or inorganic phosphate. Survival rate, peritoneal macrophage phagocytosis and plasma levels of myocardial depressant factor (MDF) were measured. Shocked animals pretreated with vehicle exhibited 24.6 +/- 0.9% phagocytic activity, 110 +/- 3.9 units/ml MDF plasma levels and 0% survival. Sham animals showed the following values: survival 100%; phagocytosis, 49.5 +/- 1.3%; MDF, 22 +/- 2.9 units/ml. Pretreatment with FDP (25 mg/kg/i.v.) significantly improved survival rate (50%) and macrophage phagocytosis (37.9 +/- 0.4%) and reduced plasma MDF levels (77 +/- 3 units/ml). Equivalent doses of fructose and inorganic phosphate did not improve survival, as well as lower doses of FDP. These results suggest a beneficial effect of FDP in SAO shock.

Hexaprazol antagonism to gastric electrical potential fall by acetylsalicylic acid in rat.

Hexaprazol was tested against the gastric barrier breaking effect of aspirin (ASA), using the electric potential difference (PD) test in rats without pyloric ligation. The activity of hexaprazol was evident at a dose lower than the antisecretory one and also at the same range of antiulcer doses. The drug's activity in reducing the PD fall induced by ASA was compared with that of carbenoxolone, pirenzepine, cimetidine and ranitidine administered by the same route. Their relative activity, measured by AUB (area under baseline) index, followed this order: carbenoxolone greater than hexaprazol greater than pirenzepine greater than cimetidine greater than ranitidine. Measured by RI (irritancy index) inhibition, the order of activity was similar: carbenoxolone greater than hexaprazol greater than or equal to pirenzepine greater than cimetidine greater than ranitidine. It was demonstrated that hexaprazol had an anti-barrier breaker activity of marked intensity and duration; this efficacy was always dose-dependent.

2-(2,3-Dihydro-5-acetoxy-4,6,7-tribenzofuranyl)acetic acid (IRFI 016): a new antioxidant mucoactive drug.

IRFI 016 has demonstrated significant antioxidant activity, inhibiting hepatic lipid peroxidation (rat intoxicated by CCl4) and the formation of gastric lesions by ethanol (rat). This activity proved equal to or better than that exhibited by the most investigated antioxidant/radical scavenger agents (such as BHA, BHT, Vitamin E). The drug markedly increased mucus production (rabbit, mouse) by all the administration routes used (os, i.v. and inhalatory) and proved more active than, or overlapping, the most noted mucoregulatory/mucolytic drugs (sobrerol, bromexine, thiopronine, ambroxol, N-acetylcysteine) which were chosen for comparison. The tracheo-bronchial mucus viscosity was also significantly reduced (bronchitic animals) as was the fucose and total protein content. In the pigeon, IRFI 016 improved mucociliary clearance. Moreover IRFI 016 evidenced anti-inflammatory activity nearly equal to that exhibited by ASA and phenylbutazone (carrageenin oedema, abcesses and inflammatory pain).

Effect of erdosteine and its metabolites on tracheobronchial mucus production and transport.

The effect of erdosteine and its metabolites on tracheobronchial mucus production and transport was studied. Erdosteine showed important muco-regulating action (increase of mucus production), and also influenced muciliary clearance. Erdosteine, after intravenous administration, was more active than the muco-regulating drugs used for comparative purposes (about 4 times as active as N-acetyl-cysteine; about 1.8 times as active as sobrerol and bromexine; and about 1.4 times as active as ambroxol). After oral administration erdosteine showed significant action on mucus production, causing an effect quantitatively the same as that produced by bromexine. Erdosteine and its metabolites also increased TMV in pigeons after intravenous administration. Moreover, erdosteine and its metabolites were significantly active and their acute toxicity was extremely low.

Nesosteine: a new mucoregulatory agent.

A new, original molecule, nesosteine, modified both the rheology and the production of tracheobronchial mucus in rabbits. The drug highly significantly reduced the viscosity of tracheobronchial mucus in animals made bronchitic by H2SO4 aerosol and markedly increased mucoproduction in healthy animals. Nesosteine was more active than the best known mucolytic/mucoregulatory drugs, such as sobrerol, N-acetylcysteine, bromexine, ambroxol, S-carboxymethylcysteine and mercaptopropionylglycine. The fluidifying activity of the drug was also demonstrated in vitro (pig's gastric mucin), although this proved to be less marked than in vivo. Nesosteine reduced the amount of total proteins of the tracheobronchial mucus, acting on albumin, alpha 1, alpha 2, beta and gamma mucoproteins.

Compared kinetics of 2-(2,3-dihydro-5-acetoxy-4,6,7-trimethylbenzofuranyl) acetic acid (IRFI 016) and its active metabolite 2-(2,3-dihydro-5-hydroxy-4,6,7-trimethylbenzofuranyl) acetic acid (IRFI 005) in plasma and bronchial alveolar liquid in mice.

The concentration of 2-(2,3-dihydro-5-acetoxy-4,6,7-trimethylbenzofuranyl) acetic acid (IRFI 016) and its active metabolite 2-(2,3-dihydro-5-hydroxy-4,6,7-trimethylbenzofuranyl) acetic acid (IRFI 005) in bronchial alveolar liquid (BAL) and plasma of mice were studied. IRFI 016 and its active metabolite IRFI 005 are both present in BAL and plasma after oral administration of IRFI 016. In BAL no delay times were noted, in comparison with plasma, regarding Cmax time nor significant variation of t1/2 and the elimination constant (Kel). IRFI 016, orally administered, is very rapidly absorbed and, both in unaltered form and as its active metabolite, reaches the anatomic site where it carries out its principal pharmacological activity, according to the same kinetic course observed in plasma.

A new antioxidant drug limits brain damage induced by transient cerebral ischaemia.

Restoration of blood flow after an ischaemic event generates the formation of oxygen radicals which could augment brain damage. The authors studied the effects of different doses (50, 100, 200 mg/kg/i.p.) of a new antioxidant, IRFI-016, [2(2,3-dihydro-5-acetoxy-4,6,7-trimethylbenzofuranyl) acetic acid] on brain damage in the Mongolian gerbil induced by 5 min of bilateral carotid occlusion (BCO) followed by reperfusion. Post-ischaemic brain malondialdehyde (MDA) levels and locomotor activity at different times and delayed neuronal death of hippocampal CA1 area on the fourth day after occlusion were evaluated. During reperfusion, after BCO, enhancement of brain MDA occurs (37.5%, 62.5% and 100% at 15, 30 and 60 min of reperfusion, respectively). Brain MDA postischaemic increases were reduced at 15 min of reperfusion to 15.4% and 44.4% by IRFI-016, 100 and 200 mg/kg, respectively. After 30 min of reperfusion brain MDA was reduced to 31.25% and 53.13% by IRFI-016 100 and 200 mg/kg, respectively. Hyperactivity and delayed neuronal death of CA1 were significantly reduced in postischaemic gerbils treated with the highest doses of IRFI-016. Results indicate that pretreatment with the antioxidant IRFI-016 improves in a dose-dependent manner brain damage induced by ischaemia and reperfusion in the gerbil.

Review on sobrerol as a muco-modifying drug: experimental data and clinical findings in hypersecretory bronchopulmonary diseases.

Sobrerol is an agent with a terpenic structure used to modify mucus characteristics in patients with hypersecretory bronchopulmonary diseases and this review is aimed at listing and discussing all the scientific evidence on this drug. All the toxicological findings testify its excellent tolerability and very low toxicity. In the pharmacological section of the review the effects of sobrerol on mucus production and its characteristics are discussed together with its effect on experimental models. A number of open clinical trials suggesting a therapeutic potential activity of the drug is examined together with a series of randomized double-blind clinical trials confirming its efficacy in relieving obstructive symptoms in chronic bronchitic patients.

Synthesis and A1-adenosine receptors affinities of purino[7,8-c]quinazoline-8,10(9H,11H)-diones as rigid analogues of 8-arylxanthines.

Title compounds were prepared by a cyclocondensation reaction between 8-(2-aminophenyl)xanthines and trialkyl orthoesters. Some of them showed activities as A1-adenosine receptor antagonists with binding values in the micromolar range. Results are discussed with reference to 1,3-dialkyl-8-arylxanthines. Considerations on the role played by both electronic and conformational factors are also reported.