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The cholinergic neurotransmitter system in the brain is crucial in processing information related to cognitive, behavioral, and motor functions. A cholinergic dysfunction has been correctly described as one of the primary causes of neurodegenerative diseases. Differences in levels of acetylcholine or expression and function of receptors in selected brain areas have been indicated as one of the causes of sexual dimorphism in neurotransmission. However, variability in results among studies based on different mice strains could affect conclusions on this topic. Visual evoked potentials (VEPs) of male and female DBA/2J and C57BL/6J mice, which are two of the most common strains backgrounds in use for developing transgenic mice models of neurological diseases, have been studied. Effects induced by a single low dose of physostigmine have also been performed to evaluate the cholinergic system involvement. VEPs responses to luminous stimuli in C57BL/6J mice have shown a consistently lower latency than in DBA/2J, confirming the previous observation of strain differences in cholinergic function. Interestingly, strains present an opposite-sex difference in VEP latency not apparently related to sensitivity to physostigmine. These findings point at paying extreme attention to the choice of the genetic background of the animal model, especially in those basic and pre-clinical experiments that involve visual functioning.

Alcohol dependence continues to be an important health concern and animal models are critical to furthering our understanding of this complex disease. A hallmark feature of alcoholism is a significant increase in alcohol drinking over time. While several different animal models of excessive alcohol (ethanol) drinking exist for mice and rats, a growing number of laboratories are using a model that combines chronic ethanol exposure procedures with voluntary ethanol drinking with mice as experimental subjects. In the last years several experimental evidences have shown an involvement of opioid system in alcoholism.

PURPOSE: It has been hypothesized that specific early-life stress (ES) procedures on CD-1 male mice produce diabetes-like alterations due to the failure of negative feedback of glucocorticoid hormone in the pituitary. The aim of this study is to investigate the possible mechanism that leads to this pathological model, framing it in a more specific clinical condition.METHODS: Metabolic and hypothalamic-pituitary-adrenal-related hormones of stressed mice (SM) have been analyzed immediately after stress procedures (21 postnatal days, PND) and after 70 days of a peaceful (unstressed) period (90 PND). These data have been compared to parameters from age-matched controls (CTR), and mice treated during ES procedures with oligonucleotide antisense for pro-opiomelanocortin (AS-POMC).RESULTS: At 21 PND, SM presented an increased secretion of hypothalamic CRH and pituitary POMC-derived peptides, as well as higher plasmatic levels of ACTH and corticosterone vs. CTR. At 90 PND, SM showed hyperglycemia, with suppression of hypothalamic CRH, while pituitary and plasmatic ACTH levels, as well as plasma corticosterone, were constantly higher than in CTR. These values are accompanied by a progressive acceleration in gaining total body weight, which became significant vs. CTR at 90 PND together with a higher pituitary weight. Treatment with AS-POMC prevented all hormonal and metabolic alterations observed in SM, both at 21 and 90 PND.CONCLUSIONS: These findings show that these specific ES procedures affect the negative glucocorticoid feedback in the pituitary, but not in the hypothalamus, suggesting a novel model of ACTH-dependent hypercortisolism that can be prevented by silencing the POMC gene.

von Frey hairs are important tools for the study of mechanisms of cutaneous stimulation-induced sensory input. Mechanical force is exerted via application of a particular hair to the cutaneous receptive field until buckling of the hair occurs. The most commonly used von Frey filaments are productive in evaluating behavioral responses of neuropathic pain in preclinical and clinical research. To reduce the potential experimenter bias, automated instruments are being developed for behavioral assessment.

Alcohol dependence continues to be an important health concern and animal models are critical to furthering our understanding of this complex disease. A hallmark feature of alcoholism is a significant increase in alcohol drinking over time. While several different animal models of excessive alcohol (ethanol) drinking exist for mice and rats, a growing number of laboratories are using a model that combines chronic ethanol exposure procedures with voluntary ethanol drinking with mice as experimental subjects. In the last years several experimental evidences have shown an involvement of opioid system in alcoholism.

von Frey hairs are important tools for the study of mechanisms of cutaneous stimulation-induced sensory input. Mechanical force is exerted via application of a particular hair to the cutaneous receptive field until buckling of the hair occurs. The most commonly used von Frey filaments are productive in evaluating behavioral responses of neuropathic pain in preclinical and clinical research. To reduce the potential experimenter bias, automated instruments are being developed for behavioral assessment.

Several chronic neuroinflammatory diseases, including Parkinson&rsquo, s disease (PD), have the so-called &lsquo, redox imbalance&rsquo, in common, a dynamic system modulated by various factors. Among them, alteration of the mitochondrial functionality can cause overproduction of reactive oxygen species (ROS) with the consequent induction of oxidative DNA damage and apoptosis. Considering the failure of clinical trials with drugs that eliminate ROS directly, research currently focuses on approaches that counteract redox imbalance, thus restoring normal physiology in a neuroinflammatory condition. Herein, we used SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA), a neurotoxin broadly employed to generate experimental models of PD. Cells were pre-treated with the Rho-modulating Escherichia coli cytotoxic necrotizing factor 1 (CNF1), before the addition of 6-OHDA. Then, cell viability, mitochondrial morphology and dynamics, redox profile as well as autophagic markers expression were assessed. We found that CNF1 preserves cell viability and counteracts oxidative stress induced by 6-OHDA. These effects are accompanied by modulation of the mitochondrial network and an increase in macroautophagic markers. Our results confirm the Rho GTPases as suitable pharmacological targets to counteract neuroinflammatory diseases and evidence the potentiality of CNF1, whose beneficial effects on pathological animal models have been already proven to act against oxidative stress through an autophagic strategy.

In hypertensive retinopathy, the retinal damage due to high blood pressure is accompanied by increased expression of Glial Fibrillary Acidic Protein (GFAP), which indicates a role of neuroinflammatory processes in such a retinopathy. Proteins belonging to the Rho GTPase family, particularly Rac1, are involved in the activation of Muller glia and in the progression of photoreceptor degeneration, and may thus represent a novel candidate for therapeutic intervention following central nervous system inflammation. In this paper, we have observed that topical administration as eye drops of Cytotoxic Necrotizing Factor 1 (CNF1), a Rho GTPase modulator, surprisingly improves electrophysiological and behavioral visual performances in aged spontaneously hypertensive rats. Furthermore, such functional improvement is accompanied by a reduction of Rac1 activity and retinal GFAP expression. Our results suggest that Rac1 inhibition through CNF1 topical administration may represent a new strategy to target retinal gliosis.

Adipose-derived stem cells (ADSCs) are multipotent mesenchymal stem cells that have the ability to differentiate into several cell types, including chondrocytes, osteoblasts, adipocytes, and neural cells. Given their easy accessibility and abundance, they became an attractive source of mesenchymal stem cells, as well as candidates for developing new treatments for reconstructive medicine and tissue engineering. Our study identifies a new signaling pathway that promotes ADSCs osteogenic differentiation and links the lipid signaling enzyme phospholipase C (PLC)-β1 to the expression of the cell cycle protein cyclin E. During osteogenic differentiation, PLC-β1 expression varies concomitantly with cyclin E expression and the two proteins interact. These findings contribute to clarify the pathways involved in osteogenic differentiation and provide evidence to develop therapeutic strategies for bone regeneration.

We describe a stress-derived type-2 diabetes model in male mice, and formulate new hypotheses on how the model was induced, how diabetes-like alterations were prevented through specific pharmacological treatments, and how its possible neuroendocrine pathogenesis could be hypothesized. Pregnant females arrived in our laboratory on their 14th day of conceptional age. After birth, control mice never showed any apparent behavioral-metabolic-endocrine alterations. However, application of postnatal stress (brief mother deprivation, plus sham injection, daily from birth to weaning), was followed in adult male mice by two series of diabetes-like alterations. Some alterations (e.g., body overweight, immune, neurophysiologic, neurobehavioral alterations) were selectively prevented by opioid antagonist naloxone daily administered during nursing period. The aforementioned alterations plus several others (e.g., hyperglycemia, neuroendocrine alterations) were prevented by administration of specific antisense oligodeoxinucleotide, which modulated synthesis-hyperfunction of proopiomelanocortin-derived corticotropin (ACTH)-corticosterone and endorphins in the pituitary. Surprisingly, together with metabolic alterations, enduring increment of neurophysiologic/neurobehavioral brain performances were observed, accompanied by energy compensative reactions, and brain mitochondria hyperfunction. Thus, increased glycemia/lipidemia appeared to furnish fuel necessary to cope with increased request of energy. Diabetes-like alterations were accompanied by enduring hyperfunction of opioid- and ACTH-corticosterone-endogenous structures in the brain, which were apparently due to failure of negative feedback hormone mechanisms in the pituitary, for the control of the hypothalamus-pituitary-adrenal axis. In conclusion, for the first time we can hypothesize that a diabetes-like syndrome is produced by enduring hyperfunction of two proopiomelanocortin-dependent endogenous systems (brain opioid- and ACTH-corticosterone systems), following failure of pituitary feedback hormonal control, after complex stress procedures.

In Alzheimer’s disease (AD) patients, apopoliprotein (APOE) polymorphism is the main genetic factor associated with more aggressive clinical course. However, the interaction between cerebrospinal fluid (CSF) tau protein levels and APOE genotype has been scarcely investigated. A possible key mechanism invokes the dysfunction of synaptic plasticity. We investigated how CSF tau interacts with APOE genotype in AD patients. We firstly explored whether CSF tau levels and APOE genotype influence disease progression and long-term potentiation (LTP)-like cortical plasticity as measured by transcranial magnetic stimulation (TMS) in AD patients. Then, we incubated normal human astrocytes (NHAs) with CSF collected from sub-groups of AD patients to determine whether APOE genotype and CSF biomarkers influence astrocytes survival. LTP-like cortical plasticity differed between AD patients with apolipoprotein E4 (APOE4) and apolipoprotein E3 (APOE3) genotype. Higher CSF tau levels were associated with more impaired LTP-like cortical plasticity and faster disease progression in AD patients with APOE4 but not APOE3 genotype. Apoptotic activity was higher when cells were incubated with CSF from AD patients with APOE4 and high tau levels. CSF tau is detrimental on cortical plasticity, disease progression and astrocyte survival only when associated with APOE4 genotype. This is relevant for new therapeutic approaches targeting tau.

Von Frey hairs are important tools for the study of mechanisms of cutaneous stimulation-induced sensory input. Mechanical force is exerted via application of a particular hair to the cutaneous receptive field until buckling of the hair occurs. The most commonly used Von Frey filaments are productive in evaluating behavioral responses of neuropathic pain in preclinical and clinical research. To reduce the potential experimenter bias, automated instruments are being developed for behavioral assessment.

Background To identify the correlation between high body mass index (BMI ≥ 25) and the risk of postoperative complications of thyroidectomy. Methods A comparative study between thyroidectomy performed in normal or overweigh-obese patients has been performed. Postoperative outcomes, including hypocalcemia, laryngeal nerve palsy, bleeding, operation time and hospital stay, were evaluated. Results A total of 266 patients underwent total thyroidectomy were included. Of them, 104 patients had a BMI below 25 and 162 patients had a BMI ≥ 25. There was no statistically significant difference in the occurrence of early or permanent hypoparathyroidism, recurrent laryngeal nerve palsy, bleeding complications, or postoperative duration of hospital stay. There was, however, a higher operative time in patients with a BMI ≥ 25. Conclusion Despite the longer operative time, thyroidectomy can be performed safely in patients with a BMI ≥ 25.

Little is known about the impact of weight loss interventions on pregnancy rate. A systematic review with meta-analysis of literature has been performed to evaluate the incidence of successful pregnancy after bariatric interventions in infertile women. By pooling together data from 589 infertile obese women, we have been able to provide an aggregate estimation of successful pregnancy after weight loss interventions. Our results showed an impressive high incidence (58 %) of infertile women who become spontaneously pregnant after surgery. Based on our results, bariatric interventions could be included in the treatment of obesity-related infertility. However, the results must be interpreted with caution owning to the quality of the data provided by included studies. Further ad hoc studies are needed to validate the effectiveness of bariatric interventions on reproductive function.

none 8 Epilepsy, one of the most common conditions affecting the brain, is characterized by neuroplasticity and brain cell energy defects. In this work, we demonstrate the ability of the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) to counteract epileptiform phenomena in inbred DBA/2J mice, an animal model displaying genetic background with an high susceptibility to induced- and spontaneous seizures. Via modulation of the Rho GTPases, CNF1 regulates actin dynamics with a consequent increase in spine density and length in pyramidal neurons of rat visual cortex, and influences the mitochondrial homeostasis with remarkable changes in the mitochondrial network architecture. In addition, CNF1 improves cognitive performances and increases ATP brain content in mouse models of Rett syndrome and Alzheimer's disease. The results herein reported show that a single dose of CNF1 induces a remarkable amelioration of the seizure phenotype, with a significant augmentation in neuroplasticity markers and in cortex mitochondrial ATP content. This latter effect is accompanied by a decrease in the expression of mitochondrial fission proteins, suggesting a role of mitochondrial dynamics in the CNF1-induced beneficial effects on this epileptiform phenotype. Our results strongly support the crucial role of brain energy homeostasis in the pathogenesis of certain neurological diseases, and suggest that CNF1 could represent a putative new therapeutic tool for epilepsy. Travaglione, Sara; Ballan, Giulia; Fortuna, Andrea; Ferri, Alberto; Guidotti, Marco; Campana, Gabriele; Fiorentini, Carla; Loizzo, Stefano Travaglione, Sara; Ballan, Giulia; Fortuna, Andrea; Ferri, Alberto; Guidotti, Marco; Campana, Gabriele; Fiorentini, Carla; Loizzo, Stefano

Overexpression of pro-inflammatory cytokines and cellular energy failure are associated with neuroinflammatory disorders, such as Alzheimer's disease. Transgenic mice homozygous for human ApoE4 gene, a well known AD and atherosclerosis animal model, show decreased levels of ATP, increased inflammatory cytokines level and accumulation of beta amyloid in the brain. All these findings are considered responsible for triggering cognitive decline. We have demonstrated that a single administration of the bacterial E. coli protein toxin CNF1 to aged apoE4 mice, beside inducing a strong amelioration of both spatial and emotional memory deficits, favored the cell energy restore through an increment of ATP content. This was accompanied by a modulation of cerebral Rho and Rac1 activity. Furthermore, CNF1 decreased the levels of beta amyloid accumulation and interleukin-1β expression in the hippocampus. Altogether, these data suggest that the pharmacological modulation of Rho GTPases by CNF1 can improve memory performances in an animal model of Alzheimer's disease via a control of neuroinflammation and a rescue of systemic energy homeostasis.

Mechanisms of vascular complications in type-2 diabetes patients and animal models are matter of debate. We previously demonstrated that a double-stress model applied to male mice during nursing period produces enduring hyperfunction of endogenous opioid and adrenocorticotropin (ACTH)-corticosteroid systems, accompanied by type-2 diabetes-like alterations in adult animals. Administration of the opioid receptor antagonist naloxone, or of an antisense oligodeoxynucleotide versus proopiomelanocortin mRNA, capable to block the pro-opiomelanocortin-derived peptides β-endorphin and ACTH, selectively prevent these alterations. Here, we investigated alterations produced by our stress model on aorta endothelium-dependent relaxation and contractile responses. Mice, stressed during nursing period, showed in the adulthood hormonal and metabolic type-2 diabetes-like alterations, including hyperglycemia, increased body weight and increased plasma ACTH and corticosterone levels. Ex vivo isolated aorta rings, gathered from stressed mice, were less sensitive to noradrenaline-induced contractions versus controls. This effect was blocked by nitric-oxide synthase-inhibitor l -NG-nitroarginine added to bath organ solution. Aorta rings relaxation caused by acetylcholine was enhanced in stressed mice versus controls, but following treatment with the nitric-oxide donor sodium nitroprusside, concentration-relaxation curves in aorta from stressed groups were similar to controls. Therefore, vascular response alterations to physiologic-pharmacologic stimuli were apparently due to nitric-oxide hyperfunction-dependent mechanisms. Aorta functional alterations, and plasma stress hormones enhancement, were prevented in mice stressed and treated with antisense oligodeoxinucleotide, addressed to reduce ACTH- and corticosteroid-mediated hyperfunction. This study demonstrates the key role of ACTH-corticosteroid axis hyperfunction for the triggering of vascular conditions in male adult rodents following postnatal stress in a type-2 diabetes model.

Neonatal manipulations (10 min of maternal separation plus s.c. sham injection, daily for the first 21 d of life) determine overweight in male adult mice. In this work, we investigated the mechanisms underlying mild obesity and the alteration of caloric balance. Neonatally manipulated mice become overweight after onset of maturity, showing increased fat tissue and hypertrophic epididymal adipocytes. Increase in body weight occurs in the presence of a small increase in daily food intake (significant only in the adult period) and the absence of a decrease in spontaneous locomotor activity, while the calculated caloric efficiency is higher in manipulated mice, especially in adulthood. Fasting adult animals show hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia, and hyperleptinemia. Soon after weaning and in the adulthood, plasma corticosterone and adrenocorticotropin (ACTH) are also significantly increased. Thus, neonatal manipulations in nongenetically susceptible male mice program mild obesity, with metabolic and hormonal alterations that are similar to those found in experimental models of diabetes mellitus, suggesting that this metabolic derangement may have at least part of its roots early on in life and, more interestingly, that psychological and nociceptive stimuli induce these features.

The double postnatal stress model (brief maternal separation plus sham injection daily applied from birth to weaning) induces metabolic alterations similar to type 2 diabetes in young-adult male mice. We verify whether 1) the stress also induces brain metabolic–functional alterations connected to diabetes and 2) different alterations are modulated selectively by two stress-damaged endogenous systems (opioid- and/or ACTH–corticosteroid-linked). Here, diabetes-like metabolic plus neurophysiologic–neurometabolic parameters are studied in adult mice following postnatal stress and drug treatment. Surprisingly, together with ‘classic’ diabetes-like alterations, the stress model induces in young-adult mice significantly enhanced brain neurometabolic–neurophysiologic performances, consisting of decreased latency to flash-visual evoked potentials (−∼8%); increased level (+∼40%) and reduced latency (−∼30%) of NAD(P)H autofluorescence postsynaptic signals following electric stimuli; enhanced passive avoidance learning (+∼135% latency); and enhanced brain-derived neurotrophic factor level (+∼70%). Postnatal treatment with the opioid receptor antagonist naloxone prevents some alterations, moreover the treatment with antisense (AS; AS vs proopiomelanocortin mRNA) draws all parameters to control levels, thus showing that some alterations are bound to endogenous opioid-system hyper-functioning, while others depend on ACTH–corticosterone system hyper-functioning. Our stress model induces diabetes-like metabolic alterations coupled to enhanced brain neurometabolic–neurophysiologic performances. Taken all together, these findings are compatible with an ‘enduring acute-stress’ reaction, which puts mice in favorable survival situations vs controls. However, prolonged hormonal–metabolic imbalances are expected to also produce diabetes-like complications at later ages in stressed mice.

Mild stress repeatedly applied to neonatal rodents induces alterations of central nervous system functions, persisting up to the adult age. Most alterations may be mediated through hormones and neuromediators active on the autonomic nervous system, therefore we tested the efficacy of selective opioid receptor agonists on the vas deferens of adult mice that, as neonates, had undergone daily mild stress until weaning (brief isolation and solvent injection). We found in the adult mouse (90 days old) decreased sensitivity of vas deferens to selective mu-, delta- and kappa-opioid receptor agonist drugs. The neonatal administration of an antisense oligodeoxynucleotide adrenocorticotropin-synthesis-inhibitor partly prevented these effects.

In a previous study we ascertained the presence of sigma1 and sigma2 recognition sites in the rabbit iris-ciliary body, an ocular structure involved in aqueous humor production and drainage. We characterized the sigma1 sites using the preferential ligand (+)-pentazocine, which caused a significant reduction of intraocular pressure (IOP). In the present study, flunarizine, a calcium channel blocker with a complex pharmacological profile, bound to sigma1 sites expressed in the iris-ciliary body with moderate affinity (K(i) = 68 nM). Unilateral topical flunarizine (0.01-0.1%) caused a dose-related reduction of IOP in ocular normotensive rabbits and in the alpha-chymotrypsin model of ocular hypertension, without altering the IOP of the contralateral eye. This activity was blocked by the sigma1 site antagonist NE-100 [N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine HCl] which, by itself, had no effect on IOP. Detection of flunarizine in rabbit iris-ciliary body homogenates, after topical instillation, showed that it adequately penetrates the rabbit eye. To investigate mechanisms that may contribute to ocular hypotension induced by sigma1 agonists, we carried out in vitro studies on the isolated rabbit iris-ciliary body. Flunarizine (IC50 = 5. 96 nM) and (+)-pentazocine (IC50 = 3. 81 nM) inhibited [3H]norepinephrine release. Moreover, flunarizine (IC50 = 6.34 nM) and (+)-pentazocine (IC50 = 27.26 nM) also antagonized isoproterenol-induced cAMP accumulation. The action of flunarizine and (+)-pentazocine was sensitive to NE-100 antagonism; however, this latter compound partially prevented their effect on [3H]norepinephrine and cAMP accumulation. These findings indicate that flunarizine and (+)-pentazocine interact with ocular sigma1 sites and may prove effective in the control of ocular hypertension.

Von Frey hairs are important tools for the study of mechanisms of cutaneous stimulation-induced sensory input. Mechanical force is exerted via application of a particular hair to the cutaneous receptive field until buckling of the hair occurs. The most commonly used Von Frey filaments are productive in evaluating behavioral responses of neuropathic pain in preclinical and clinical research. To reduce the potential experimenter bias, automated instruments are being developed for behavioral assessment.

Alcohol dependence continues to be an important health concern and animal models are critical to furthering our understanding of this complex disease. A hallmark feature of alcoholism is a significant increase in alcohol drinking over time. While several different animal models of excessive alcohol (ethanol) drinking exist for mice and rats, a growing number of laboratories are using a model that combines chronic ethanol exposure procedures with voluntary ethanol drinking with mice as experimental subjects. In the last years several experimental evidences have shown an involvement of opioid system in alcoholism.

Alcohol dependence continues to be an important health concern and animal models are critical to furthering our understanding of this complex disease. A hallmark feature of alcoholism is a significant increase in alcohol drinking over time. While several different animal models of excessive alcohol (ethanol) drinking exist for mice and rats, a growing number of laboratories are using a model that combines chronic ethanol exposure procedures with voluntary ethanol drinking with mice as experimental subjects. In the last years several experimental evidences have shown an involvement of opioid system in alcoholism.

Neurotrophins (NTs) play an essential role in modulating activity-dependent neuronal plasticity. In this context, the site and extent of NT secretion are of crucial importance. Here, we demonstrate that the activation of phospolipase C (PLC) and the subsequent mobilization of Ca(2+) from intracellular stores are essential for NT secretion initiated by both Trk and glutamate receptor activation. Mutational analysis of tyrosine residues, highly conserved in the cytoplasmic domain of all Trk receptors, revealed that the activation of PLC-gamma in cultured hippocampal neurons and nnr5 cells is necessary to mobilize Ca(2+) from intracellular stores, the key mechanism for regulated NT secretion. A similar signalling mechanism has been identified for glutamate-mediated NT secretion-which in part depends on the activation of PLC via metabotropic receptors-leading to the mobilization of Ca(2+) from internal stores by inositol trisphosphate. Thus, PLC-mediated signal transduction pathways are the common mechanisms for both Trk- and mGluRI-mediated NT secretion.

In previous experiments the activity-dependent secretion of nerve growth factor (NGF) from native hippocampal slices and from NGF-cDNA transfected hippocampal neurons showed unusual characteristics [Blochl and Thoenen (1995) Eur J Neurosci 7:1220-1228; (1996) Mol Cell Neurosci 7:173-190]. In both hippocampal slices and cultured hippocampal neurons the activity-dependent secretion proved to be independent of extracellular calcium, but dependent on the release of calcium from intracellular stores. Under different experimental conditions, Goodman et al. [(1996) Mol Cell Neurosci 7:222-238] reported that the high potassium-mediated secretion of brain-derived neurotrophic factor (BDNF) from hippocampal cultures was dependent on extracellular calcium. Mowla et al. [(1997) Proc 27th Annu Meet Soc Neurosci New Orleans 875.10] reported on even further-reaching differences between NGF and BDNF secretion, namely, that in hippocampal neurons and in pituitary cell lines NGF was secreted exclusively according to the constitutive pathway, whereas BDNF was exclusively sorted according to the activity-dependent regulated pathway. In view of the crucial importance of such potential differences between the processing, sorting, and secretory mechanisms of different neurotrophins for their modulatory roles in activity-dependent neuronal plasticity, a thorough analysis under comparable experimental conditions was mandatory. We demonstrate that in native hippocampal slices and adenoviral-transduced hippocampal neurons there are no differences between NGF and BDNF with respect to the subcellular distribution and mechanism of secretion; that the activity-dependent secretion of both NGF and BDNF is dependent on intact intracellular calcium stores; and that the differences between our own observations and those of Goodman et al. (ibid.) regarding the dependence on extracellular calcium do not reflect differences between NGF and BDNF sorting and secretion, but reflect the differing experimental conditions used.

Addition of Zn2+_ to cell medium inhibited the induction of ornithine decarboxylase (ODC) activity in ODC overproducing L1210-DFMOr cells. A significant effect was observed at a concentration as low as 0.01 mM, however a more marked inhibition was caused by the addition of 0.1 mM Zn2+. The inhibition of the induction of ODC activity was accompanied by a proportional decrease in the content of immunoreactive ODC protein, whereas the level of ODC mRNA, detemined by a solution hybridization RNase protection assay, was not affected signigicantly. Instead, some acceleration of ODC turnover was observed. the addition of 0.1 mM Co2+ or Mn2+, but not of other divalent metal ions, also inhibited ODC induction; differently from Zn2+ however, these metals affected cell viability and/or cell growth. Removal of endogenous Zn2+ by a chelator also provoked a strong decrease of ODC induction, which was reversed by Zn2+. However, addition of Zn2+ in excess of the chelator proved to be markedly inhibitory. These results indicate that both a restricted Zn2+ availability and an enhanced presence of the metal can inhibit the induction of ODC in L1210-DFMOr cells.

Dilution of quiescent L1210-DFMOr (difluoromethylornithine-resistant) cells in fresh medium containing serum led to the induction of ornithine decarboxylase (ODC) and to the expression of its mRNA, as determined by a sensitive solution-hybridization-RNase-protection assay. Addition of the chelating agent diethylenetriaminepentaacetic acid (DTPA) at seeding time caused an inhibition of the induction of ODC activity by up to 90%, and only Zn2+ of the bivalent metal ions tested was effective in reversing this effect. The inhibition of the induction of ODC activity was accompanied by a marked decrease, prevented by Zn2+ supplementation, of the accumulation of immunoreactive ODC protein and ODC mRNA. DTPA treatment also caused a slight acceleration of ODC turnover. These results indicate that a restricted Zn2+ availability in L1210-DFMOr cells impairs ODC induction remarkably, mainly by affecting the expression of the messenger.

Immunoreactive dynorphin A-like material (ir-dyn A) in human plasma was measured by a validated radioimmunoassay. In peripheral plasma extracts mean concentrations between 20 and 40 fmol/ml were determined in volunteers and in patients with pituitary adenomas. In this latter group superimposable levels were detected three days before and during transsphenoidal microsurgery. Interestingly, ir-dyn A levels evaluated in extracts of hypothalamic-hypophysial plasma obtained during surgery, just after tumor removal, were 4–5 times higher than in peripheral plasma. Reverse-phase high performance liquid chromatography (rp-HPLC) of extracts of peripheral plasma samples revealed two immunoreactive peaks. The major form had the same retention time of dyn A-(1–32); whereas a second, more lipophilic, peak eluted later and was not further characterized. In contrast, rp-HPLC analysis of extracts of plasma collected from the suprapituitary region displayed only one peak eluting in the position of synthetic dyn A-(1–17). The presence of dyn-related peptides in hypothalamic-hypophysial plasma supports the hypothesis that they may play a part in the regulation of hypothalamic and/or pituitary functions in humans.

Pediatric pain management underwent many changes since the undertreatment of pain in children was reported in the literature in 1980. Increasing data also suggest that long-term behavioural effects can be observed in children, following pain episodes as early as in the neonatal period. Therefore, the knowledge about safe and effective management of pain in children should be applied with greater effectiveness into clinical practice. Other advances in the field include the findings of long-term residual behavioural and metabolic effects induced by pain experienced during the critical periods of development in laboratory animals. Recent data in laboratory animals and clinical data in children suggest that early repeated and/or severe pain and other stressful procedures applied in the perinatal periods may produce not only behavioral, but also important hormonal, immune and metabolic long-term effects. In this paper we shall report data on some metabolic conditions described in adult humans following disruption of hormonal-metabolic programming produced in the peri-natal period. Quite similar signs can be found between animal models and human conditions, most of them being connected with hypothalamus-pituitary-adrenal hormones (HPA) dysfunction. In addition, some signs in animal models, such as overweight and abdominal overweight are prevented by treatment with the μ- and δ-opioid receptor antagonist naloxone during the lactating period. This indicates that some long-term consequences following stress received during the early phases of life in mammals may be bound to the HPA system dysregulation, whereas others are bound to different (e,g., opioid) endogenous brain receptors and/or neuromediators alteration.

Previously, we showed that our post-natal handling model induces pro-opiomelanocortin-derived (POMC) endogenous systems alterations in male mice at weaning. These alterations last up to adult age, and are at the basis of adult hormonal and metabolic conditions similar to mild metabolic syndrome/type-2 diabetes. Here, we evaluate how sex influences post-natal programming in these metabolic conditions. Subjects are adult control (non-handled) female (NHF) and male (NHM) CD-1 mice; adult post-natal handled female (HF) and male (HM) mice. Handling consists of daily maternal separation (10 min) plus sham injection, from birth to weaning (21 days). In adult handled males (90-days old) we find not only POMC-derived hormones alterations (enhanced basal plasma corticosterone (+91%) and ACTH (+109%)) but also overweight (+5.4%), fasting hyperglycemia (+40%), hypertriglyceridemia (+21%), enhanced brain mRNA expression of hydroxysteroid(11-beta)dehydrogenase type-1 (HSD11B1) (+49%), and decreased mRNA-HSD11B2 (-39%). Conversely, uric acid, creatinine, HDL(C), total cholesterol, glucose and insulin incremental area under-the-curve are not affected. In females, post-natal handling does not produce both hormonal and dysmetabolic diabetes-like changes; but handling enhances n3- and n6-poly-unsaturated, and decreases saturated fatty acids content in erythrocyte membrane composition in HF versus NHF. In conclusion, for the first time we show that female sex in mice exerts effective protection against the hypothalamus-pituitary-adrenal homeostasis disruption induced by our post-natal handling model on POMC cleavage products; endocrine disruption is in turn responsible for altered metabolic programming in male mice. The role of sex hormones is still to be elucidated.

Background Several studies have shown that opioids may suppress the immune system either by binding to mu-opioid receptors (MOR) expressed in immune cells or by activating these receptors within the central nervous system. Objective To assess the level of lymphocyte expression of MOR mRNA in patients with chronic non-cancer pain, who were treated with intrathecal morphine or with morphine plus bupivacaine via an intrathecal drug delivery system, and to investigate whether intrathecal morphine and the associated local anesthetic administration influences lymphocyte subpopulations. Methods In total, 29 people [10 controls (age range 59–85 years) and 19 patients (age range 47–89 years) with various chronic non-malignant pain conditions] were enrolled in the study. MOR mRNA levels were evaluated in peripheral lymphocytes, and lymphocyte subsets were determined by direct immunofluorescence using flow cytometry. Results After 12 months of treatment with intrathecal morphine (1.5–4 mg/day), there was an increase in MOR mRNA levels in lymphocytes of 65% compared with controls and 47% with pretreatment values. Even higher levels (increase of 142% compared with controls and 135% with pretreatment values) were observed in the patients treated with morphine plus bupivacaine (0.2–0.4 mg/day). Elevation of MOR mRNA levels was confirmed in patients after 24 months of treatment. At this time point, the percentage of natural killer cells was significantly decreased. Conclusion This preliminary study suggests that opioids must be used with care in patients who are already immunosuppressed by disease or by other, concurrently administered drugs.

In previous investigations we added a physical stress (mild pain) to the "classical" post-natal psychological stress in male mice, and we found that this combination produced a series of dysmetabolic signs very similar to mild human type-2 diabetes. Here, for the first time we demonstrate that within this diabetes model at least two groups of signs depend on the unbalance of two different endogenous systems. Newborn male mice were daily exposed to stressful procedures for 21 days (brief mother separation plus sham injection). Other groups underwent the same procedure, and also received naloxone (Na) to block μ-δ endogenous receptors, or a phosphorothioate antisense oligonucleotide (AS) directed against pro-opiomelanocortin (POMC)-mRNA [to block adrenocorticotropin (ACTH)- and POMC-derived opioid peptides]. Adult mice which received only post-natal stress increased body weight (+7.5%), abdominal overweight (+74%), fasting glycemia (+43%), plasma corticosterone (+110%), plasma (+169%) and pituitary (+153%) ACTH levels. Conversely, hypothalamic ACTH and corticotropin-releasing hormone (CRH) were reduced (-70% and -75%, respectively). Neonatal AS administration reverted all parameters to control values. Neonatal naloxone had little or no influence on glucose, corticosterone, ACTH, CRH levels, whereas it prevented body overweight and abdominal overweight. We conclude that, within this type-2 diabetes model in male mice at least two endocrino-neurohumoral systems are damaged, one concerning the opioid system, and the other concerning HPA hormones. The use of the two drugs was of primary importance to demonstrate this statement, and to demonstrate that these two groups of signs could be defined as "separate entities" following our complex post-natal stress model.

Early life events have been implicated in the programming of adult chronic diseases. Several investigations suggest that the role of early environment in influencing development mainly involves the hypothalamic-pituitary-adrenal axis. Therefore, we examined whether 1) daily neonatal handling, applied from birth to weaning induces HPA hormones alterations in mice lasting up to the adult age; and 2) if the administration of an antisense oligodeoxynucleotide versus pro-opiomelanocortin (As-POMC) prevents hormonal alterations observed in previously handled mice (Handled). In the adult phase (90 days), Handled are overweight and have higher basal plasma immuno-reactive (ir)-corticosterone and adrenocorticotropin (ir-ACTH), and higher pituitary ir-ACTH; while they have lower hypothalamic ir-ACTH and corticotropin-releasing hormone (ir-CRH) in comparison with the non-handled mice. As-POMC (0.05-0.1 nmol/g body weight per day) administered during the same period dose-dependently prevents the increase in body weight, in plasma ir-corticosterone, ir-ACTH, and pituitary ir-ACTH, also preventing the decrease in hypothalamic ir-CRH and ir-ACTH; while the mismatch oligonucleotide is nearly inactive. This data indicates that pharmacological treatment in neonatal life may have enduring effects, reducing the alterations in hormonal homeostatic programming mechanisms induced by early repeated handling.

Ursodeoxycholic acid (UDCA) is beneficial in cholestatic diseases but its molecular mechanisms of action remain to be clearly elucidated. Other bile acids, such as chenodeoxycholic (CDCA), are agonists for the nuclear farnesoid X receptor (FXR) and regulate the expression of genes relevant for bile acid and cholesterol homeostasis. In ileal cells CDCA, through the FXR, up-regulates the expression of the ileal bile acid-binding protein (IBABP), implicated in the enterohepatic circulation of bile acids. We report that UDCA (100 and 200 microM) induced a moderate increase of IBABP mRNA (approximately 10% of the effect elicited by 50 microM CDCA) in enterocyte-like Caco-2 cells and approximately halved the potent effect of CDCA (50 microM). On the contrary, UDCA reduced by 80-90% CDCA-induced IBABP transcription in hepatocarcinoma derived HepG2 cells. We confirmed that these effects on IBABP transcription required the FXR by employing a cell-based transactivation assay. Finally, in a receptor binding assay, we found that UDCA binds to FXR expressed in CHO-K1 cells (K(d)=37.7 microM). Thus, UDCA may regulate IBABP in Caco-2 cells, which express it constitutively, by acting as a partial agonist through a FXR mediated mechanism. The observation that in HepG2 cells, which do not express constitutively IBABP, UDCA was able to almost completely prevent CDCA-induced activation of IBABP promoter, suggests that tissue-specific factors, other than FXR, may be required for bile acid regulation of FXR target genes.

Bile acids may play a role in the pathogenesis of intestinal inflammation by activating the signalling pathways that control cell proliferation, among other cell systems. We investigated the action of different bile acids, particularly chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), on steady-state and transcriptional regulation of the protooncogene c-fos, involved in the regulation of cell proliferation and differentiation, in colon carcinoma Caco-2 cells. Specific bile acids had a stimulatory effect of on the expression of c-fos mRNA. This proved to be concentration- and time-dependent and may be partly due to an increase in the rate of transcription of the corresponding gene rather than to any change in the stability of mRNA. In Caco-2 cells exposed to 250 microM CDCA for 1 h a maximal increase of c-fos mRNA ( approximately 2.5-fold induction over the control) was observed; deoxycholic acid (DCA; 250 microM) and lithocholic acid (LCA; 250 microM) were less effective (approximately 2-fold induction over the control). UDCA and cholic acid (CA) did not modify c-fos gene expression in this cell line. Finally, we investigated the role of protein kinase C (PKC) in transcriptional regulation of the c-fos gene by bile acids. Although induction of c-fos by 12-O-tetradecanoyl 13-acetate (10 nM), a potent PKC activator, was completely antagonised by bis-indolyl-maleimide I (1 microM); only about 40% of the bile acid-mediated rise in c-fos mRNA was blocked. Thus it appears that PKC, as well as other signalling pathways, is involved in CDCA-, DCA- and LCA-induced c-fos gene expression.

In this study we characterized calcitonin (CT) receptors in human neuroblastoma IMR 32 cells. Saturation binding assays indicated that [125I]-human CT bound with high affinity to IMR 32 cell membranes (K(d) = 253.6 pM; Bmax = 3.84 fmol/ mg protein). In competition binding studies, human adrenomedullin displayed high affinity for these sites (IC50 = 30 nM) whereas human alpha calcitonin-gene related peptide (alphaCGRP; IC50 = 145 nM) and human amylin (IC50 = 415 nM) showed lower affinity. These peptides increased cAMP levels in viable cells; the relative potencies were: human CT > human adrenomedullin > human cCGRP > or = human amylin. The expression of mRNA coding for the published sequences of the human calcitonin receptor and of the human calcitonin receptor-like receptorwas evaluated by reverse transcriptase-polymerase chain reaction. Electrophoretic analysis did not confirm the occurrence of mRNA coding for the above mentioned receptors in these cells. This study suggests the presence of a novel, putative CT receptor in IMR 32 cells.

Neurotrophins, secreted in an activity-dependent manner, are thought to be involved in the activity-dependent refinement of synaptic connections. Here we demonstrate that in hippocampal neurons and the rat pheochromocytoma cell line PC12 application of exogenous neurotrophins induces secretion of neurotrophins, an effect that is mediated by the activation of tyrosine kinase neurotrophin receptors (Trks). Like activity-dependent secretion of neurotrophins, neurotrophin-induced neurotrophin secretion requires mobilization of calcium from intracellular stores. Because neurotrophins are likely to be released from both dendrites and axons, neurotrophin-induced neurotrophin release represents a potential positive feedback mechanism, contributing to the reinforcement and stabilization of synaptic connections.

Gastric tolerability, absorption and pharmacological activity of the non-steroidal anti-inflammatory drug 4-biphenylacetic acid (BPAA), as an inclusion complex with β-cyclodextrin (β-CyD) or chemically modified β-CyDs: 2,6-di-O-methyl-β-CyD (DM-β-CyD), 2,3,6-tri-O-methyl-β-CyD (TM-β-CyD) and 2-hydroxypropyl-β-CyD (HP-β-CyD), were investigated in the rat after oral administration. BPAA absorption, determined from area under the plasma concentration-time curve (AUC), was increased by complexation with all β-CyDs in the following order: DM-β-CyD > TM-β-CyD > HP-β-CyD > β-CyD. The carrageenan paw oedema test demonstrated a significant increase in anti-inflammatory activity of BPAA and the ED50 values, compared with BPAA alone, were reduced to about a third for the BPAA-DM-βCyD complex and halved for the others. BPAAA complexed with DM-β-CyD, HP-β-CyD or β-CyD showed better gastric tolerability compared with uncomplexed drug, whereas the BPAA-TM-β-CyD complex produced marked gastric lesions similar in extent to BPAA alone. TM-β-CyD (500mg kg−1) and DM-β-CyD (1000mg kg−1) caused gastric erosions 21 h after oral administration. The pharmacokinetic profiles of BPAA-β-CyD complexes have shown that DM-β-CyD is the most effective in enhancing the bioavailability of BPAA.

Gene expression in mammalian cells can be suppressed by oligonucleotides complementary to the target mRNA. This strategy was explored as a means of arresting translation of the prohormone precursor proopiomelanocortin (POMC), used as a model system of peptide messengers that are synthesized and released from endocrine and neuronal cells. The synthesis of the POMC-derived peptides adrenocorticotropin (ACTH) and beta-endorphin (beta-END) was markedly reduced by an oligodeoxynucleotide (ODN) complementary to a region of beta-END mRNA in AtT-20 cells, which retain many of the differentiated phenotypes of corticotrophs; this treatment did not affect the steady-state levels of POMC mRNA. Antisense ODN was stable in cell culture medium for 24 h, and cellular uptake was low (approximately 2.5% of the added ODN); however, the intracellular levels of the ODN were sufficient to form a ribonuclease-resistant duplex with complementary cellular mRNA. Addition of ODN to the cell culture did not affect the cellular levels of chromogranin A-(264-314)/pancreastatin or cell viability and proliferation, as evidenced by bromodeoxyuridine incorporation and ornithine decarboxylase activity. Microinfusion of the antisense ODN in the rat hypothalamic arcuate nucleus, where the majority of POMC-positive brain perikarya are located, significantly reduced ACTH- and beta-END-immunopositive neurons, and antisense ODN-treated rats showed substantially less of the grooming behavior usually observed in a novel environment.

We evaluated the pharmacodynamic and pharmacokinetic profile of Mipragoside, a monosialoganglioside isopropyl-ester (as 0.5% w/w ophthalmic gel), on allergic inflammation of the eye induced by reverse passive Arthus reaction, on a non-immune mast cell degranulation elicited by compound 48/80 and on ocular inflammation produced by horse serum. Conjunctiva was sensitized by injection of rabbit antisera to bovine proteins and the allergic conjunctivitis was triggered by intravenous administration of bovine gamma globulin. The permeability of the blood-conjunctival barrier was evaluated by a fluorometric method. Compound 48/80 was topically administered at concentration of 50mg/ml and histological analysis of conjunctiva was performed. Horse serum was administered by intravenous injection at different days. The pharmacokinetic profile of topical 3H-Mipragoside on 48/80 model was investigated and compared with untreated animals. Mipragoside treatment significantly reduced (p0.05 vs placebo) the conjunctival vasopermeability induced by reverse passive Arthus reaction as well as successfully reduced the eosinophil levels in the conjunctival epithelium (p0.01 vs placebo) elicited by compound 48/80. Further, Mipragoside successfully reduced the primary signs of ocular inflammation produced by horse serum administration. A radiotracer technique was used to evaluate the disposition of 3H-Mipragoside in the rabbit ocular tissues. Disposition of the drug was monitored at 30, 60, 120 and 240 min. 3H-Mipragoside levels in the inflamed conjunctiva were significantly higher (p0.01) than in the control eye.

We measured the release of immunoreactive (ir) dynorphin (dyn) A-(1-17) and dyn B from the rat hypothalamus by an in vitro superfusion technique. The system was validated on the basis of the recovery and stability of radiolabeled peptides added to the superfused hypothalami. These were detected as authentic peptides by reverse-phase high-performance liquid chromatography (rp-HPLC) only in the presence of a cocktail of peptidase inhibitors added to the superfusion medium. We observed spontaneous release of ir-dyn B, evaluated by a validated radioimmunoassay in the superfusates, that was increased by potassium and veratridine depolarization. It was calcium-dependent and tetrodotoxin-sensitive. We could not evaluate ir-dyn A-(1-17) directly in the superfusates, because the peptidase inhibitors added to the medium significantly altered the tracer-antibody reaction. To obviate this problem, pooled superfusate samples were purified on C18 cartridges and assayed by rp-HPLC. Rp-HPLC analysis of superfusates revealed two molecular forms with the same retention time as authentic dyn A-(1-17) and dyn B which were four times higher in K(+)-stimulated fractions. We could not detect dyn A-(1-32), comprising dyn A-(1-17) and dyn B, even though this peptide is recognized by the antibodies used in this study and is detected in acetic acid extracts of the rat hypothalamus. The spontaneous and K(+)-evoked release of ir-dyn A-(1-17) and ir-dyn B were significantly higher in 2-week ovariectomized rats, in parallel with the increase of their content in the anterior hypothalamus preoptic area.