Near-infrared spectroscopy measure of limb peripheral perfusion in neonatal arterial thromboembolic disease.

AIM: In critically ill neonates, peripheral perfusion and oxygenation assessment may provide indirect information on circulatory failure in limb arterial thromboembolic emergencies. Aims of our study were: 1) to evaluate the changes on tissue oxygenation index, oxyhemoglobin, deoxyhemoglobin and blood volume obtained by near-infrared spectroscopy (NIRS) on the infants legs; 2) to compare them with ultrasonographic data. METHODS: Tissue oxygenation index (TOI), oxyhemoglobin (O2Hb), deoxyhemoglobin (HHb) and blood volume (BV) differences were assessed by NIRS on the calf of 8 newborn infants (median weight 1995, range 585-3010 g; median gestational age 32.5, range 26-40 wks). An ultrasonographic scan of the arterial system was performed before the NIRS measurements, to validate the site of arterial occlusion. RESULTS: A t-test for independent samples showed lower values in the affected limb for all NIRS measurements. TOI measurements displayed lower values in the thromboembolic limb (mean 44.79±12%) versus unaffected (mean 47.95±17.08%) (P=0.0001). Mean (SD) peak systolic velocity in the patent artery below the occlusion decreased from 108±25 cm/s in the normal limb to 25.6±28 cm/s in the thrombus site (P=0.02). CONCLUSION: In neonatal intensive care units, measurement of limb peripheral perfusion and oxygenation seems to be clinically useful in arterial thromboembolic emergencies.

Fluoxetine may worsen hyperoxia-induced lung damage in neonatal rats.

Fluoxetine shows controversial lung effects as it prevents pulmonary hypertension in adult rats but exposure during gestation causes pulmonary hypertension in neonatal rats. In the present study, we tested the null hypothesis that the antidepressant drug fluoxetine does not modify the development of bronchopulmonary dysplasia (BPD) in neonatal rats. Experimental categories included I: room air (controls) with daily injection of saline; II: room air with daily injection of 10 mg/kg fluoxetine, i.p., during two weeks; III: 60% oxygen with daily injection of saline; and IV: 60% oxygen with daily injection of 10 mg/kg fluoxetine, i.p., during two weeks. Hyperoxia resulted in significant reduction in alveolar density and an increase in pulmonary endocrine cells, as well as increases in muscle layer areas of bronchi and arteries. Fluoxetine treatment generated a further increase in muscularisation and did not significantly modify the hyperoxia-induced reductions in alveolar density and increases in the endocrine cells. In hyperoxia, Real-Time PCR showed a lower pulmonary expression of vascular endothelial growth factor (VEGF) with no significant changes in the expression of matrix metalloproteinases (MMP) 2 and 12. Fluoxetine did not affect VEGF or MMP-2 expression but it significantly increased MMP-12 mRNA in both normoxic and hyperoxic groups. Zymographic analysis of MMP-2 activity in bronchoalveolar fluid showed a significantly reduced MMP-2 activity in hyperoxia, while fluoxetine treatment restored MMP-2 activity to levels comparable with the normoxic group. In conclusion, our data show that fluoxetine may worsen bronchial and arterial muscularisation during development of BPD and may up-regulate MMP expression or activity.

Experimental study on Satureja montana as a treatment for premature ejaculation.

AIM OF THE STUDY: Satureja montana (winter savory) is a medicinal plant traditionally used to treat different disorders including male sexual dysfunction. In this study we evaluated the effect of Satureja montana hydroalcoholic extract on copulatory behavior of sexually potent male rats. MATERIALS AND METHODS: The extract was orally administered acutely or repetitively for 8 consecutive days at the doses of 25 and 50 mg/kg. The main parameters of sexual behavior, mount (ML), intromission (IL), ejaculation (EL) latencies and post-ejaculatory interval (PEI), were evaluated in animals submitted to mating test and multiple ejaculations test. Testosterone serum levels were measured in rats acutely treated with Satureja montana extract dosed at 50 mg/kg. In addition the open field test was conducted to evaluate the locomotor behavior. RESULTS: When acutely administered at both dosages, the extract was able to significantly increase EL and decrease intromission frequency (IF) in comparison with controls. The significant increase in EL was found also when the extract was subacutely administered, daily for 8 consecutive days, at the dose of 25 mg/kg. In the multiple ejaculations test, EL values of treated rats were significantly increased during the 1st and 2nd sequence in comparison with controls; in addition only rats treated with the extract were able to reach the 4th ejaculation within 30 min. Testosterone serum level measured in rats acutely treated with Satureja montana at the dose of 50 mg/kg was significantly increased in rats in comparison with controls. Finally, the locomotor activity recorded in the open field test was not affected by the acute administration of the plant extract. CONCLUSIONS: These data suggest that Satureja montana could be considered as a natural remedy for the treatment of premature ejaculation delaying ejaculation latency without exerting any negative effect on the other parameters of sexual behavior and without exerting a sedative effect. In addition the increased serum level of testosterone confirms the positive influence of Satureja montana on male sexual function.

Evidence that isopropylthioxanthone (ITX) is devoid of anxiolytic and sedative effect.

Isopropylthioxanthone (ITX) is a well-known photo-initiator in ultraviolet light-cured inks frequently used in milk packaging materials, yoghurt, ready-to-feed infant formula, and other drinks. Traces of ITX have been found in milk and, as a consequence, there was considerable interest in studying the biological activity of this molecule and its potential hazard for the human health. Although the ITX genotoxic effects have been excluded by the European Food Safety Authority (EFSA), the US Environmental Protection Agency (USEPA) is still examining its possible toxic potential depending on a dose-effect ratio. Little is known about the ITX activity on the function of the central nervous system and cerebral neurotransmitters. Using behavioural, biochemical, and electrophysiological tests, the authors have found that: (1) ITX did not exert an in vivo anxiolytic or sedative effect when administered orally to rats; (2) ITX did not affect the binding characteristics of central and peripheral benzodiazepine receptors studied in vitro; and (3) ITX did not influence the ability of gamma-Aminobutyric acid (GABA) to increase the chloride channel permeability studied by patch clamp technique in a single neuron of cultured cerebellar granule cells.

Influence of Eurycoma longifolia on the copulatory activity of sexually sluggish and impotent male rats.

AIM OF THE STUDY: The root of Eurycoma longifolia Jack, native to South East Asia, has long been used as a male aphrodisiac remedy to treat sexual disorders. In the study we evaluated the influence of Eurycoma longifolia Jack on sexual behavior (including both motivation and copulatory performance) of sexually sluggish and impotent male rats. MATERIALS AND METHODS: The root powder of the plant was orally administered to adult Sprague-Dawley male rats, classified as sexually sluggish or impotent taking in account their behavior in pre-experimental tests. Groups of 8 animals each were submitted to three different types of treatment: (1) acute at 3 dose levels (250, 500 and 1000 mg/kg); (2) subacute (daily for 6 days) at the dose of 500 mg/kg and (3) subchronic (daily for 12 days) at the same dose (500 mg/kg). Mount, intromission and ejaculation latencies and post-ejaculatory interval were recorded during the mating test in order to evaluate sexual performance. In addition the partner preference test was used to assess sexual motivation. Testosterone serum levels were measured in subacutely treated rats and compared with the values of controls receiving vehicle. RESULTS: Concerning the copulatory activity of sexually sluggish rats, both acute (dosed at 500 and 1000 mg/kg) and subacute treatments with the root powder significantly reduced ejaculation latencies, increasing also the percentage of mounting and ejaculating animals; in addition the subacute administration reduced post-ejaculatory interval. In impotent rats both subacute and subchronic treatments increased the percentage of mounting and ejaculating rats. The motivational behavior of sluggish rats during the partner preference test was not affected by the treatments. Testosterone serum levels were increased in rats subacutely treated in comparison with controls. CONCLUSION: Eurycoma longifolia root improved sexual performance but not motivation in sluggish rats after acute or subacute administration. The effect could be mainly ascribed to increased testosterone levels.

Experimental evidence of the anaphrodisiac activity of Humulus lupulus L. in naïve male rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In the folk medicine Humulus lupulus L. (hops) is mainly recommended as a mild sedative with antispasmodic and digestive properties. It is also reputed to exert an anaphrodisiac effect but it is still lacking the experimental evidence of this activity. AIM OF THE STUDY: To evaluate the influence of Humulus lupulus extract on sexual behavior of both naïve and sexually potent male rats; thereafter to investigate the role of 8-prenylnarigenin (8-PN) in the effect displayed by the hop extract. MATERIALS AND METHODS: Sprague-Dawley male rats both naïve and sexually potent were acutely administered with the hop extract dosed at 5, 10, 25 and 50 mg/kg. In addition the extract was administered daily for 10 consecutive days at the dose of 0.25 mg/kg/day in sexually potent animals. The pure compound 8-PN was acutely administered in naïve rats at the dosages of 5, 12.5 and 25 microg/kg. All the animals were screened for their sexual behavior manifestation during the mating test. RESULTS: In naïve rats the acute administration of Humulus lupulus extract at the doses of 25 and 50 mg/kg significantly reduced the percentage of mounting and ejaculating animals, in comparison to vehicle controls. The other parameters recorded during the mating test were not affected by the hop extract. In sexually potent rats nor the acute neither the repeated administration of the extract modified their copulatory behavior. The pure compound 8-PN failed to influence male sexual behavior of naïve rats. CONCLUSION: Humulus lupulus extract exerted an anaphrodisiac effect only in naïve rats by inhibiting their mounting and ejaculating behavior. The presence of 8-PN in the extract could be only partially involved in the observed anaphrodisiac effect.

Natural endogenous ligands for benzodiazepine receptors in hepatic encephalopathy.

Benzodiazepines of natural origin (NBZDs) have been found in human blood and brains as well as in medicinal plants and foods. In plasma and brain tissue there are i.e. diazepam and nordiazepam equal to commercial drugs but there are also other benzodiazepine-like compounds termed "endozepines", which act as agonists at the benzodiazepine receptors of central type (CBR). A synthetic pathway for the production of NBZDs has not yet been found, but it has been suggested that micro-organisms may synthesize molecules with benzodiazepine-like structures. Hence NBZDs could be of both endogenous and exogenous source and be considered as natural anxyolitic and sedative. Interestingly there are also natural compounds, such as the polypeptide Diazepam Binding Inhibitor (DBI) acting as an "inversive agonist" implicated in fair and panic disorders. It has been suggested that NBZDs may play a role in the pathogenesis of hepatic encephalopathy (HE). Multidirectional studies evaluated NBZDs levels (1) in the blood of normal subjects, of cirrhotic with or without HE and in commercial benzodiazepine consumers; (2) in the blood of cirrhotic treated or not with a non-absorbable antibiotic; (3) in several constituents of our diet. In conclusion, NBZDs increase sometime in cirrhotics with or without HE but they reach concentrations not higher than those found in commercial benzodiazepines consumers. Hence NBZDs must be considered as occasional precipitating factor of HE and benzodiazepine antagonists only symptomatic drugs. The finding that NBZDs may be in part synthesized by intestinal bacterial flora and in part constituent of our diet underlines the importance to feed cirrhotic patients with selected food.

Evidence that the beta-acids fraction of hops reduces central GABAergic neurotransmission.

Humulus lupulus (hops) is traditionally used as a tranquilizing herbal remedy. Here, we investigated the in vivo and in vitro effect of hop beta-acids on central nervous system function. Oral administration of beta-acids (5-10mg/kg) in rats produced an increased exploratory activity in the open field, a reduction in the pentobarbital hypnotic activity and a worsening of picrotoxin-induced seizures. When dosed at 10mg/kg, beta-acids increased, in the elevated plus maze, open arm entries reducing in parallel those in closed arms. In the forced swimming test, we observed a reduction in the immobility time that could suggest an antidepressant-like activity. Electrophysiological studies performed on cerebellar granule cells in culture showed that the beta-acids fraction decreased GABA-evoked current in a dose-dependent way. The effect was not inhibited by the benzodiazepine antagonist Ro 15-1788. Benzodiazepine receptors involvement was also excluded by [(3)H]-Ro 15-1788 binding assay. In conclusion, the behavioral effects of beta-acids fraction could be explained by a reduction in the GABAergic activity although we cannot rule out the involvement of other neurotransmitter systems.

Evaluation of rifaximin, placebo and lactulose in reducing the levels of benzodiazepine-like compounds in patients with liver cirrhosis: a pilot study.

Benzodiazepine-like compounds (BZDs), either taken with the diet or synthesized by intestinal bacterial flora, may represent a precipitating factor for hepatic encephalopathy (HE) in cirrhotic patients. We evaluated whether a diet and/or treatment with rifaximin or lactulose can reduce serum concentrations of BZDs in 18 cirrhotic patients without HE. Patients were given a standard diet for 7 days to keep the dietary intake of BZDs constant and were then randomized to a 7-day treatment with rifaximin 1,200 mg/day, lactulose 10-20 g three times daily, or placebo. Blood samples were collected at enrollment, at the end of the diet and drug treatment periods, and 7 days after the drug was stopped (follow-up). Serum concentrations of BZDs were measured by a radioligand binding technique after high-performance liquid chromatography extraction and purification and were expressed as diazepam equivalents (DE). No change in serum BZD concentrations was observed during the diet, while a statistically significant decrease from 105.6 +/- 66.5 to 63.5 +/- 49.5 pmol DE/ml was achieved in rifaximin-treated patients (p < 0.05) but not in patients treated with lactulose or placebo. During the followup, serum BZD concentrations returned to 104.5 +/- 74.0 pmol DE/ml in rifaximin-treated patients (p < 0.05 vs. end-treatment values), while no significant change was observed in the lactulose- and placebo-treated patients. These data indicate that control of bacterial flora with cyclic administration of rifaximin plays a pivotal role in avoiding increased plasma concentrations of BZDs, which represent a precipitating factor for HE inpatients with severe liver disease.

New insight in the neuropharmacological activity of Humulus lupulus L.

The purpose of the present study was to investigate the effects of Humulus lupulus CO2 extract and its fraction containing alpha-acids on the central nervous system of rats. Both tested substances were able to prolong pentobarbital sleeping time, without affecting the latency to the loss of the righting reflex. This effect was dose-dependent, starting from a minimal dose of 10 mg/kg. Neither the extract nor its alpha-acid fraction affected the locomotor activity in the open field test or exerted an anxiolytic effect in rats submitted to the elevated plus-maze test. Interestingly both compounds reduced the immobility time during the behavioral despair test when administered three times (24, 5 and 1 h) before the test. In conclusion this report shows that Humulus lupulus CO2 extract exerts: (a) a pentobarbital sleep-enhancing property without influencing the motor behavior of rats; (b) an antidepressant activity. The same effects were elicited by the administration of the Humulus lupulus fraction containing alpha-acids, which can be considered as the major responsible for the enhanced pentobarbital effect and for the antidepressant property.

Activity of single components of Ferula hermonis on male rat sexual behavior.

The influence of the single components of Ferula hermonis extract on sexual behavior was studied in male rats. Sexually potent and sluggish/impotent animals were orally treated acutely (2.5 mg/kg) and subchronically (0.25 mg/kg/day for 10 days) with ferutinin, teferdin and teferin. Ferutinin alone acutely administered in potent rats was able to reduce mount and intromission latencies, while in sluggish/impotent animals, it induced the same effects and additionally shortened the ejaculation latency, as teferdin did. Both substances increased testosterone levels in rats. Unlike teferdin, ferutinin subchronically administered in potent rats negatively affected appetitive and consummatory sexual behavior, reducing also testosterone serum levels. In conclusion, if repetitively administered, ferutinin was able to stimulate sexual behavior after acute ingestion, but exerted a negative influence on the sexual capacity of potent male rats, whereas teferdin only improved copulatory performance of sluggish/impotent animals.

Nuclear location-dependent role of peripheral benzodiazepine receptor (PBR) in hepatic tumoral cell lines proliferation.

PBR is involved in numerous biological functions, including steroid biosynthesis, mitochondrial oxidative phosphorylation and cell proliferation. The presence of PBR at the perinuclear/nuclear subcellular level has been demonstrated in aggressive breast cancer cell lines and human glioma cells where it seems to be involved in cell proliferation. In our study we investigated the presence of perinuclear/nuclear PBR in different hepatic tumor cell lines with regard to binding to [3H] PK 11195 and protein analysis. The results obtained by saturation binding experiments and scatchard analysis of perinuclear/nuclear PBR density in parallel with the results on the growth curves of the cell lines tested, indicate that the perinuclear/nuclear PBR density correlates inversely with cell doubling time. Moreover, the cell line with high perinuclear/nuclear PBR proliferated in response to PBR ligand, whereas that with low perinuclear/nuclear PBR did not. Our results reinforce the idea that the subcellular localisation of PBR defines its function and that this receptor could be a possible target for new strategies against cancer.

Detection of levodopa, dopamine and its metabolites in rat striatum dialysates following peripheral administration of L-DOPA prodrugs by mean of HPLC-EC.

A high performance liquid chromatography (HPLC) method was developed to detected simultaneously L-dihydroxyphenylalanine (L-DOPA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum dilaysates following oral administration of L-DOPA or its prodrugs. The chromatographic system uses a reversed-phase C18 column with electrochemical detection at +0.30 V. Mobile phase consisted of 0.05 M citric acid, sodium EDTA 50 microM, sodium octylsulphonate 0.4 nM at pH of 2.9 and 8% methanol (v/v) at a flow rate of 1 ml/min. The calibration curves were linear over the concentration range of 10nm to 100 microM and the lower limits of detections were 125 fmol for L-DOPA, 50 fmol for DOPAC, 250 fmol for DA and 150 fmol for HVA at signal noise to ratio of 3. The repeatability (or intra-day precision), expressed by the relative standard deviation, were better than 4%. The construction of microdialysis probes has been described. The in vitro relative recoveries of each microdialysis probe were evaluated and the results show that they are similar and reproducible for all the analytes with CVs from 1 to 4%. The HPLC-EC method was applied to detect the extracellular levels of L-DOPA, DA, DOPAC and HVA in the striatum dialysates of freely moving rats after oral administration of six new potential L-DOPA prodrugs.

Opposite effect of acute and subchronic treatments with Ferula hermonis on copulatory behavior of male rats.

Sexually potent and sluggish/impotent male rats were orally treated with an extract of Ferula hermonis (30 and 60 mg/kg). The acute administration stimulated sexual motivation in potent rats and improved copulatory performance in sluggish/impotent rats. This last effect was elicited only by the higher dose, which, in parallel, increased serum testosterone levels in rats. On the contrary, when the extract was subchronically administered (10 days) a marked reduction in the percentage of rats achieving ejaculation was detected, together with a general impairment of the copulatory pattern. Furthermore, the repeated administration of the extract (6 mg/kg/day for 10 days) resulted in a significant reduction of testosterone levels in comparison with controls. The present results discourage a repeated assumption of F. hermonis, while suggesting its acute administration to improve the performance in sexual dysfunctions.

High-performance liquid chromatographic method for the quantification of anthranilic and 3-hydroxyanthranilic acid in rat brain dialysate.

Anthranilic acid (ANA) and 3-hydroxyanthranilic acid (3-HANA) have attracted considerable attention as two of the L-tryptophan kynurenine pathway metabolites in the central nervous system. In this study, a highly sensitive and accurate method for the quantification of ANA and 3-HANA has been developed using reversed-phase high performance liquid chromatography (HPLC) with fluorimetric detection. The HPLC assay was carried out using a C(18) column (5 microm, 250 x 4.6 mm i.d.). The mobile phase consisted of a mixture of 25 mM sodium/acetic acid buffer (pH 5.5) and methanol (90:10 v/v). Fluorimetric detection at lambda(ex)=316 nm and lambda(em)=420 nm was used. The assay was applied to the measurement of ANA and 3-HANA acid in rat brain dialysate following administration of L-tryptophan or L-kynurenine. 3-HANA and ANA levels were progressively increased during 90 min following administration of L-tryptophan, then decreased progressively to basal levels. 3-HANA levels were significantly higher than ANA levels after L-kynurenine administration. These findings suggest that the assay developed should provide an improved means for investigation of neurobiology of kynurenine pathway.

Pharmacological activity of hyperforin acetate in rats.

Hyperforin, the main antidepressant component of Hypericum extract, is not stable with regard to heat and light. Therefore, we investigated a newly synthetized derivative, hyperforin acetate. Herein we demonstrate its efficacy in animal models sensitive to antidepressant and anxiolytic drugs. In the forced swimming test, triple administration of hyperforin (5-20 mg/kg) significantly reduced the immobility time of rats, while in the learned helplessness test a daily treatment of 10 mg/kg for seven consecutive days was necessary to elicit an antidepressant effect. In the elevated plus-maze and in the light-dark test, the acute administration of hyperforin acetate (3-5 mg/kg) exerted an anxiolytic activity, which, however, was smaller than that of diazepam. The effect was inhibited by the pretreatment of rats with metergoline, a serotoninergic antagonist, but not with CGS-8216, a benzodiazepine receptor antagonist. Hyperforin acetate (3-10 mg/kg) was also able to reduce locomotion in rats without eliciting myorelaxant activity. As Hypericum extract was claimed to exert a potential influence on the liver drug metabolizing system, we showed that neither acute nor repeated oral doses of hyperforin acetate altered pentobarbital sleeping time in rats. Taken together, the present results show that hyperforin acetate is a pharmacologically active derivative of hyperforin and may be a starting point from which to develop new compounds for therapeutic purposes.

Antiproliferative effects of Ceratonia siliqua L. on mouse hepatocellular carcinoma cell line.

Extracts from pods and leaves of carob (Ceratonia siliqua L.) were tested for their ability to inhibit cell proliferation of mouse hepatocellular carcinoma cell line (T1). The two extracts showed a marked alteration of T1 cell proliferation in a dose-related fashion reaching the maximal effect at 1 mg/ml. Moreover, we demonstrated that leaf and pod extracts were able to induce apoptosis in T1 cell lines after 24-h treatment mediating a direct activation of the caspase 3 pathway. HPLC analysis revealed the presence of gallic acid, (-) epigallocatechin-3-gallate and (-) epicatechin-3-gallate in pod and leaf extracts, compounds well known to exert antiproliferative effects. Their concentration reached 6.28 mg/g in carob leaves and 1.36 mg/g in carob pods extract. The discovery that carob pod and leaf extracts contained antiproliferative agents could be of practical importance in the development of functional foods and/or chemopreventive drugs.

Extraction and purification from Ceratonia siliqua of compounds acting on central and peripheral benzodiazepine receptors.

The presence of molecules with high affinity for central and peripheral benzodiazepine receptors was determined in the pod and leaves of Ceratonia siliqua (carob). The amount of the substances able to selectively bind the central benzodiazepine receptor recovered from carob pods and leaves was respectively 12.17 and 18.7 ng diazepam equivalent/g. The amount of compounds active on peripheral benzodiazepine receptor in both pods and leaves was higher in comparison with the central one, being 49.83 and 40.00 PK 11195 equivalent/g, respectively. In particular the compounds acting on peripheral benzodiazepine receptors were found to be extremely concentrated in the young leaves (2572.57 ng PK 11195 equivalent/g). The presence of substances with central benzodiazepine activity in carob extracts seems of great importance in view of the possibility to use carob extract as potential natural products with anxiolytic-sedative effects. Moreover, the prevalence in leaves of substances acting on peripheral benzodiazepine receptor suggests the possible utilisation of leave extracts as chemopreventive agents.

Changes in kynurenic, anthranilic, and quinolinic acid concentrations in rat brain tissue during development.

Kynurenic, anthranilic, and quinolinic acid, brain tissue concentrations and indoleamine 2,3-dioxygenase [EC 1 13.11.17] activity were determined in rat brain, during pre- and postnatal development. Quinolinic acid brain tissue concentration was significantly increased at birth as compared with the prenatal level, then it declined rapidly in the postnatal period. By the contrary, kynurenic and anthranilic acids brain tissue concentrations in rat brain were significantly lower at birth as compared with those found prenatally; then kynurenic acid concentration decreased in the first postnatal week and increased thereafter, while anthranilic acid concentration increased in the first postnatal week and decreased thereafter. Indoleamine 2,3-dioxygenase [EC 1 13.11.17] activity were found unchanged in pre and post natal rat brain. The described opposite changes in quinolinic and kynurenic acids concentrations, occurring in pre- and postnatal period, despite the lack of knowledge on the precise role played by these compounds on the different neurotransmitter systems in the brain, could be involved in brain ontogenetic development.