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Biomedical subjects

M Simonato

Publications and source records attributed to M Simonato.

At least 19 recordsLinked to original sources

Demographic history and population structure of the Antarctic silverfish Pleuragramma antarcticum.

The Antarctic silverfish Pleuragramma antarcticum (Nototheniidae) is the most abundant pelagic fish inhabiting Antarctic waters. In this study we investigated, through partial sequencing of the D-loop mitochondrial region, samples collected at four different locations in the Southern Ocean, three in the Atlantic and one in the Pacific sector. Sampling was replicated in two different years at two locations. Sequence analysis showed a remarkably high polymorphism, with 110 haplotypes over the 256 investigated specimens, and about 80% of haplotypes occurring only once. Neutrality tests indicated that all samples were not at mutation-drift equilibrium, and suggested a past population expansion. This result was supported by the presence of a star-like topology in the D-loop gene tree, and by results of mismatch distribution. The start of the expansion was dated, using a specifically calibrated clock, between 111 and 126 thousand years ago. This value corresponds to the start of the cooling period that led to the last glaciation peak, and is in close agreement with a recently suggested range expansion for pelagic Antarctic ecosystems. Analysis of molecular variation indicated a small, though highly significant, value of differentiation between samples. This result, together with the lack of association between clades and geographical locations, indicates a weak population structure for the species.

Animals↗

Induction of B1 bradykinin receptors in the kindled hippocampus increases extracellular glutamate levels: a microdialysis study.

A link between temporal lobe epilepsy (the most common epileptic syndrome in adults) and neuropeptides has been established. Among neuropeptides, the possible involvement of bradykinin has recently received attention. An autoradiographic analysis has shown that B1 receptors, which are physiologically absent, are expressed at high levels in the rat brain after completion of kindling, a model of temporal lobe epilepsy. Thus, the present work aimed at investigating the functional implications of this observation, by studying the effect of B1 receptor activation on extracellular glutamate levels in the kindled hippocampus. Microdialysis experiments have been performed in two groups of rats, control and kindled. Glutamate outflow has been measured under basal conditions and after chemical stimulation with high K+ (100 mM in the dialysis solution). Basal glutamate outflow in kindled animals was significantly higher than in controls. High K+-evoked glutamate outflow was also more pronounced in kindled animals, consistent with the latent hyperexcitability of the epileptic tissue. The B1 receptor agonist Lys-des-Arg9-BK induced an increase of basal and high K+-evoked glutamate outflow in kindled but not in control rats, and the selective B1 receptor antagonist R-715 prevented both these effects. Furthermore, R-715 significantly reduced high K+-evoked glutamate outflow when applied alone. These data suggest that the bradykinin system contributes to the modulation of epileptic neuronal excitability through B1 receptors.

Animals↗

Effects of defective herpes simplex vectors expressing neurotrophic factors on the proliferation and differentiation of nervous cells in vivo.

Neurotrophic factors (NTFs) are known to govern the processes involved in central nervous system cell proliferation and differentiation. Thus, they represent very attractive candidates for use in the study and therapy of neurological disorders. We constructed recombinant herpesvirus-based-vectors capable of expressing fibroblast growth factor-2 (FGF-2) and ciliary neurotrophic factor (CNTF) alone or in combinations. In vitro, vectors expressing FGF-2 and CNTF together, but not those expressing either NTF alone, caused proliferation of O-2A progenitors. Furthermore, based on double-labeling experiments performed using markers for neurons (MAP-2), oligodendrocytes (CNPase) and astrocytes (GFAP), most of the new cells were identified as astrocytes, but many expressed neuronal or oligodendrocytic markers. In vivo, vectors have been injected in the rat hippocampus. At 1 month after inoculation, a highly significant increase in BrdU-positive cells was observed in the dentate gyrus of animals injected with the vector expressing FGF-2 and CNTF together, but not in those injected with vectors expressing the single NTFs. Furthermore, double-labeling experiments confirmed in vitro data, that is, most of the new cells identified as astrocytes, some as neurons or oligodendrocytes. These data show the feasibility of the vector approach to induce proliferation and differentiation of neurons and/or oligodendrocytes in vivo.

Animals↗

Targeting kinin receptors for the treatment of neurological diseases.

Kinins (bradykinin, kallidin and their active metabolites) are peptide autacoids with established functions in cardiovascular homeostasis, contraction and relaxation of smooth muscles, inflammation and nociception. They are believed to play a role in disease states like asthma, allergies, rheumatoid arthritis, cancer, diabetes, endotoxic and pancreatic shock, and to contribute to the therapeutic effects of ACE inhibitors in cardiovascular diseases. Although kinins are also neuromediators in the central nervous system, their involvement in neurological diseases has not been intensively investigated thus far. This review analyzes the potential of central kinin receptors as therapeutic targets for neurological disorders. Initial data highlight potential roles for B(1) receptor antagonists as antiepileptic agents, and for B(2) receptor antagonists (and/or B(1) agonists) in the treatment of stroke. Functional B(1) receptors located on T-lymphocytes and on the blood brain-barrier are also putative targets for the management of multiple sclerosis. However, successful elucidation of the therapeutic value of these new pharmacological approaches will require refinement of our knowledge on the physiology and cellular localization of central kinin receptors.

Animals↗

Risk of hospitalization for upper gastrointestinal tract bleeding.

OBJECTIVE: This study evaluates the hospitalization risk for upper gastrointestinal bleeding (UGIB) with reference to the clinical characteristics of patients and drugs taken before admission. METHODS: This study is based on the GIFA (Italian Group for the Pharmacosurveillance in the Elderly) database. Cases with an ICD-9 code of esophagus, stomach or duodenum bleeding, or acute esophago-gastroduodenal disease associated with anemia have been classified as UGIB. Sex, age, year of observation, drugs taken at home, comorbidity, smoking, alcohol, and use of gastroprotectants have been also taken into account. Statistical analysis has been conducted using multivariate logistic regression models. RESULTS: 32,388 patients have been enrolled, 940 of which presented UGIB. Age, comorbidity, use of smoke and alcohol, hospitalization duration, and mortality during hospitalization were significantly higher in UGIB than nonUGIB patients. Increased UGIB risk has been found in patients taking NSAIDs (both when aspirin was included or excluded), acetaminophen, constipating agents, iron, ethacrynic acid, propranolol. Reduced UGIB risk has been found in patients taking nitrates. CONCLUSIONS: UGIB risk appears to correlate with clinical characteristics of the patient: it increases with age, comorbidity, and smoke and alcohol consumption. Among drugs, NSAIDs are associated with the highest UGIB risk, while nitrates with a reduction of risk.

Aged↗

Dendritic targeting of mRNAs for plasticity genes in experimental models of temporal lobe epilepsy.

PURPOSE: To analyze whether the subcellular localization of the messenger RNAs (mRNAs) coding for the neurotrophin brain-derived neurotrophic factor (BDNF), its receptor TrkB, and the alpha and beta subunits of calcium-calmodulin-dependent kinase II (CaMKII) are modified after pilocarpine and kindled seizures. METHODS: Epilepsy models: pilocarpine and kindling. Analysis of mRNA levels in the dendrites: high-resolution, nonradioactive in situ hybridization. RESULTS: Nonstimulated rats: BDNF, TrkB, and CaMKII-beta mRNAs localized in the soma and in the proximal dendrites of hippocampal pyramidal cells, and in the soma only of dentate gyrus (DG) granule cells; CaMKII-alpha mRNA localized throughout the dendritic length in neurons of all hippocampal subfields. Pilocarpine seizures: increased staining levels of CaMKII-alpha mRNA throughout the whole dendritic length in all hippocampal subfields; induction of CaMKII-beta, BDNF, and TrkB mRNAs dendritic targeting in CA1, CA3, and DG neurons. Class 2 kindled seizures: increase in dendritic staining intensity for CaMKII-alpha in CA1, CA3, and DG neurons; induction of dendritic localization of CaMKII-beta, BDNF, and TrkB mRNAs in CA3 neurons. Fully kindled seizures: no change in the subcellular distribution of BDNF, TrkB and CaMKII-beta mRNAs; reduction of CaMKII-alpha mRNA dendritic staining, as compared with unstimulated kindled animals. CONCLUSIONS: Data provide evidence that BDNF, TrkB, and CaMKII-alpha and -beta mRNAs are accumulated in the dendrites of specific hippocampal neurons during pilocarpine seizures and kindling development. The dendritic targeting of these genes may be causally involved in epileptogenesis and thus may represent a new therapeutic target for some forms of partial epilepsy.

Animals↗

Involvement of the neuropeptide orphanin FQ/nociceptin in kainate and kindling seizures and epileptogenesis.

PURPOSE: To investigate the role of orphanin FQ/nociceptin (OFQ/N) in epilepsy, we analyzed (a) proOFQ/N (the OFQ/N precursor) and ORL-1 (the OFQ/N receptor) messenger RNA (mRNA) levels in the kainate and in the kindling models of epilepsy in the rat; and (b) seizure expression in proOFQ/N knockout mice. METHODS: Epilepsy models: kainate and kindling. Northern blot analysis, radioactive in situ hybridization. RESULTS: Increased proOFQ/N mRNA levels were found in the thalamus (reticular nucleus) after kainate administration. In contrast, ORL-1 gene expression decreased dramatically in the amygdala, hippocampus, thalamus, and cortex after kainate administration. OFQ/N knockout mice displayed reduced susceptibility to kainate-induced seizures, in that (a) lethality was reduced, (b) latency to generalized seizure onset was significantly prolonged, and (c) behavioral seizure scores were significantly reduced. Furthermore, kindling progression was delayed in OFQ/N-/- mice. CONCLUSIONS: These data indicate that limbic seizures are associated with increased OFQ/N release in multiple brain areas, causing downregulation of ORL-1 receptors and activation of OFQ/N biosynthesis in selected areas, and support the notion that the OFQ/N-ORL-1 system may play a facilitatory role in ictogenesis and in epileptogenesis.

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Preclinical evaluation of CHF3381 as a novel antiepileptic agent.

CHF3381 [n-(2-indanyl)-glycinamide hydrochloride] has been selected on the basis of a screening program as the compound displaying the highest anticonvulsant activity in the maximal electroshock seizure (MES) test and the best therapeutic index with reference to the rotarod test in mice and rats. In this study, the antiepileptic activity and the behavioural toxicity of CHF3381 were characterised in multiple model systems. CHF3381 effectively prevented MES-induced convulsions when administered i.p. (ED50, 24 mg/kg and 7.5 mg/kg) or p.o. (ED50, 21 mg/kg and 21 mg/kg) in both mice and rats, respectively. The time course of oral anti-MES activity in the rat was related to the brain concentration profile of unchanged CHF3381. Interestingly, the brain drug levels were about 4-5 times higher than in plasma. CHF3381 was very effective in mice against picrotoxin-, and i.c.v. N-methyl-D-aspartate (NMDA)-induced hind limb tonic extension (ED50 Approximately/=10 mg/kg), but was a weaker antagonist of 4-amynopyridine- and bicuculline-induced tonic seizures (ED50 approximately/=100 mg/kg), and ineffective against pentylentetrazole- and picrotoxin-induced clonic seizures. CHF3381 antagonised the behavioural effects and lethality of i.p. administered NMDA (ED50 = 57 mg/kg p.o.), indicating that the compound may act as a functional NMDA antagonist. In keeping with this idea, CHF3381 weakly displaced [(3)H]-TCP from binding to NMDA receptor channels (Ki, 8.8 microM). In the rat amygdala kindling model, CHF3381 was more efficient against kindling development than against kindled seizures (minimally active dose = 80 vs. 120 mg/kg i.p). Furthermore, it significantly increased the seizure threshold in kindled rats at relatively low doses (40 mg/kg i.p.). In contrast with MK-801-induced hyperactivity, CHF3381 moderately reduced the spontaneous locomotor activity in mice at anticonvulsant doses. Toxic effects on motor performance (rotarod test) were found at high doses only (TD50 approximately/= 300 mg/kg p.o., congruent with 100 mg/kg i.p. in both mice and rats). Furthermore, CHF3381 did not impair passive avoidance and Morris water maze responding in the therapeutic range of doses. Finally, the development of tolerance after repeated doses was negligible. These data indicate that CHF3381 exerts anticonvulsant and antiepileptogenic effects in various seizure models and possesses good therapeutic window, with scarce propensity to cause neurological side-effects.

Animals↗

Circadian differences in the individual sensitivity to opiate overdose.

OBJECTIVE: The aim of the present study was to test whether circadian differences in the response to opiates exist in humans and, if so, whether they are synchronized with the well-known circadian variations in overdose frequency. DESIGN: Daily variations in opiate overdose frequency, total amount of naloxone necessary to treat the comatose state, and frequency of hospitalization were examined in pure, nonlethal, consecutive cases of opiate (presumably intravenous heroin) overdoses. Furthermore, daily variations in the frequency of lethal overdoses were examined in all cases observed during the same period. SETTING: An 8-yr prospective, observational study in the city and suburban area of Ferrara, Italy. PATIENTS: A total of 518 consecutive cases of nonlethal opiate overdoses in 327 different patients, plus 110 consecutive cases of lethal opiate overdoses with precise or presumptive time of death. RESULTS: Analysis of the circadian distribution of nonlethal overdoses showed a significant peak in the afternoon to early evening. Analysis of the distribution of the hourly average amount of naloxone used to rescue patients from coma showed an opposite circadian variation, with a significant peak in the early morning. The hospitalization risk was also significantly higher from 3:00 to 8:59 am. However, in a subset of representative cases, plasma morphine concentrations did not change significantly in different hours of the day. Analysis of circadian distribution of lethal overdoses showed a significant peak in the evening hours. The death risk (calculated as the percentage of lethal events in the total number of intoxications within a given time frame) was significantly higher from 3:00 to 8:59 am. CONCLUSION: The present data provide evidence for the existence of circadian variations in the individual sensitivity to opiate overdose.

Adult↗

Kindled seizure-evoked somatostatin release in the hippocampus: inhibition by MK-801.

The aim of this study was to evaluate the contribution of ionotropic glutamate receptors to kindled seizure-evoked somatostatin release in the hippocampus, using a microdialysis approach. Basal and amygdala stimulation-evoked somatostatin-like immunoreactivity (-LI) release was significantly greater in kindled compared to naive rats. In naive rats, neither hippocampal perfusion with the selective AMPA/kainate receptor antagonist GYKI 52466 nor with the selective NMDA receptor antagonist MK-801 affected behavior, EEG, or somatostatin-LI release. In kindled rats, GYKI 52466 was still devoid of any effect, while MK-801 significantly decreased stimulus-evoked (but not basal) somatostatin-LI efflux. MK-801 produced identical effects when injected i.p. This study provides the first direct evidence that kindled seizure-evoked somatostatin release in the hippocampus is partly NMDA receptor dependent.

Amygdala↗

Gene transfer into neurones for the molecular analysis of behaviour: focus on herpes simplex vectors.

The use of viral vectors to transfect genes into specific brain-cell populations is a novel approach that can be used to investigate the molecular and cellular basis of brain function. Ideal vectors should be targetable and capable of regulated transgene expression. From the viral vectors developed so far, this article focuses on herpes simplex virus 1 (HSV-1)-based vectors. HSV-1 vectors can be engineered for gene transfer to the brain, which makes them suitable for neuroscience research applications. In particular, genetic manipulations of the virus can almost eliminate toxicity and allow expression of multiple transgenes simultaneously. In some instances, transfection of selected neuronal populations is also possible. Specific alterations in behaviour and in disease models have been described after the viral-vector-mediated expression of specific genes within highly localized brain regions.

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Decreased levels of neuropeptide Y(5) receptor binding sites in two experimental models of epilepsy.

It has been suggested that the anticonvulsant effects of neuropeptide Y (NPY) could be mediated by the activation of Y(2) and/or Y(5) receptors. NPY Y(1) receptor levels are known to decrease and Y(2) to increase in rat models of epilepsy. By using an autoradiographic approach, we investigated whether epilepsy models (kainic acid and kindling) are also associated with changes in Y(5) receptors. Compared with naive controls, [125I][Leu(31), Pro(34)]PYY/BIBP3226-insensitive (Y(5)) binding sites in the hippocampus (strata oriens and radiatum of CA3 and CA1) and in the neocortex (superficial layers) were unchanged in sham-stimulated rats, but reduced by approximately 50% in kindled rats (seven days after the last stimulus evokes seizure), and further reduced (to approximately -90%) 1h after a kindled seizure. Additionally, Y(5) receptor binding sites in the hippocampus and in the neocortex were unchanged 6h after kainic acid injection, but were highly reduced at 12 and 24h. No changes in Y(5) binding levels were found in the dentate gyrus and the pyramidal cell layer of the hippocampus. The present data suggest that changes in Y(5) receptor levels occur in epilepsy models. These changes may play a role in seizure expression and/or in the maintenance of kindling hyperexcitability.

Animals↗

Somatostatin release in the hippocampus in the kindling model of epilepsy: a microdialysis study.

Somatostatin biosynthesis in the hippocampus is activated during and following kindling epileptogenesis. The aim of this study was to investigate whether this phenomenon is associated with enhanced somatostatin release in vivo. Experiments have been run in awake, freely moving rats, implanted with a bipolar electrode in the right amygdala (for kindling stimulation), and with a recording electrode and a microdialysis probe in the left hippocampus. Basal somatostatin-like immunoreactivity (-LI) release was significantly greater in kindled than naive rats. In naive rats, a 2-min perfusion with 100 mM K(+) did not affect behavior and EEG recordings and nonsignificantly increased somatostatin-LI release; a 10-min K(+) perfusion evoked numerous wet dog shakes, electrical seizures (class 0; latency congruent with 8 min, duration congruent with 8 min), and somatostatin-LI release ( congruent with 350% of basal); and a single kindling after-discharge (4 +/- 3-s duration in the hippocampus) also evoked somatostatin-LI release ( congruent with 200% of basal). In kindled rats, a 2-min 100 mM K(+) perfusion evoked hippocampal discharges in three of seven animals (latency congruent with 2 min, mean duration congruent with 1.5 min) and increased somatostatin-LI release ( congruent with 250% of basal); a 10-min K(+) perfusion evoked behavioral seizures (class 1 to 5, latency congruent with 4 min, mean duration congruent with 12 min) with numerous wet dog shakes and robust somatostatin-LI release ( congruent with 350% of basal); and a kindling stimulation evoked generalized seizures (class 4 or 5, 77 +/- 15-s duration in the hippocampus) with remarkable somatostatin-LI release ( congruent with 300% of basal). These data demonstrate that hippocampal somatostatin release is increased in the kindling model in vivo.

Amygdala↗

Different patterns of induction of fibroblast growth factor-2 and brain-derived neurotrophic factor messenger RNAs during kindling epileptogenesis, and development of a herpes simplex vector for fibroblast growth factor-2 gene transfer in vivo.

PURPOSE: To investigate the gene expression patterns of brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2) in the kindling model, and to construct a replication-defective herpes simplex virus vector to induce expression of FGF-2 in vivo. METHODS: RNase protection assay and herpes simplex virus vector (TH FGF-2) deleted in the immediate-early genes ICP4, ICP22, and ICP27, with FGF-2 inserted in tk under the control of the human cytomegalovirus immediate-early promoter. RESULTS: A single kindling stimulation did not modify BDNF gene expression, whereas it increased FGF-2 messenger RNA (mRNA) levels in the hippocampus, the cortex, and the hypothalamus. BDNF and FGF-2 gene expression were not altered in kindled animals left unstimulated for 1 week. In contrast, kindled seizures produced a great increase in hippocampal and cortical BDNF mRNA levels, but FGF-2 mRNA was increased only in the ipsilateral cortex. Infection of Vero cells with TH FGF-2 resulted in a long-lasting increase in FGF-2 levels. Protein extracts of infected cells induced neuronal differentiation of PC12 cells, indicating that the newly synthesized FGF-2 was biologically active. Robust transient transgene expression was observed in the rat hippocampus after inoculation with TH FGF-2 in the absence of significant toxicity. CONCLUSIONS: BDNF and FGF-2 are recruited at different stages of kindling and, accordingly, may play different roles in the adaptive changes taking place during epileptogenesis. TH FGF-2 is suitable for studies of FGF-2 involvement in kindling epileptogenesis.

Animals↗

Molecular analysis of behavior by gene transfer into neurons with herpes simplex vectors.

One goal of neuroscience is to define the molecular and cellular basis for behavior and neurological diseases. A novel approach to this problem is based on the use of viral vectors to transfect specific genes into specific brain cell populations. This review focuses on herpes simplex-based vectors. Major advances have recently been made to improve the characteristics of these vectors, in particular to reduce their toxicity, to modulate the greatness and the time-course of transgene expression, to precisely target specific cell populations, and to transfer multiple genes. Much remains to be done to obtain robust and prolonged transgene expression. However, specific alterations in the behavior and in disease models have already been described following the herpes simplex vector-mediated expression of specific genes within highly localized brain areas. Therefore, this research strategy is likely to provide new clues on the cellular and molecular basis of behavior and of neurological diseases.

Animals↗

Limbic seizures increase pronociceptin mRNA levels in the thalamic reticular nucleus.

We studied pronociceptin gene expression following limbic seizures. Northern blot analysis revealed increased pronociceptin mRNA levels in the thalamus (but not in the hippocampus) 3-24 h after kainate administration, with maximal effect (2-fold increase over basal levels) reached at 6 h. No variation in pronociceptin mRNA levels was observed 1-6 h after a stimulus-evoked kindled seizure. Carrageenan failed to affect pronociceptin gene expression in the thalamus, indicating that pain and/or acute stress do not account for kainate effects. In situ hybridization revealed that kainate evokes a dramatic (4-fold) increase in pronociceptin mRNA levels over the thalamic reticular nucleus. Kindled seizures evoked only a small, non-significant increase in pronociceptin gene expression over the dentate gyrus of the hippocampus.

Animals↗

Changes in hippocampal and cortical B1 bradykinin receptor biological activity in two experimental models of epilepsy.

An increased response to the activation of receptors mediating excitatory effects may be involved in some forms of epilepsy. In this study, it has been tested whether B1 bradykinin receptors (which mediate excitatory effects in the peripheral nervous system and have little constitutional expression in the central nervous system) may be proposed in this role. Two experimental models of epilepsy (kindling and kainate) have been employed, and glutamate outflow experiments have been performed in hippocampal and cortical slices taken from control, kindled and kainate-treated rats. The endogenous B1 receptor agonist Lys-des-Arg9-bradykinin (10(-7) M) did not affect electrically-evoked glutamate overflow in control animals, but concentration-dependently increased it in kindled rats (maximal effect +40 to + 50%) and, to a lesser extent (+20%), in kainate-treated rats. These effects were fully prevented by the selective B1 receptor antagonist R-715 (10(-6) M), but not by the selective B2 receptor antagonist Hoe 140 (10(-6) M). The observed changes in B1 bradykinin receptor biological activity may play a role in epileptic hyperexcitability.

Animals↗

Replication-defective herpes simplex virus vectors for neurotrophic factor gene transfer in vitro and in vivo.

We report here the construction and the use of two replication-defective herpes simplex virus vectors, SH FGF-2 and TH FGF-2, which efficiently transfer and express the cDNA for fibroblast-growth-factor-2 (FGF-2) in vitro and in vivo. One mutant was deleted in the immediate-early gene encoding ICP4; the other was deleted in ICP4, ICP22 and ICP27. FGF-2--or the control gene lacZ--were inserted in tk, under control of the human cytomegalovirus immediate-early promoter. Infection of Vero cells with SH FGF-2 induced a dramatic increase in FGF-2 protein levels in the first 2 days after infection, with a rapid return to baseline levels within day 4. In contrast, infection of Vero cells with TH FGF-2 displayed FGF-2 levels progressively increasing up to days 4-5, and slowly returning to baseline. Protein extracts of cells infected with TH FGF-2 induced neuronal differentiation of PC12 cells, indicating that the newly synthesized FGF-2 was biologically active. Robust transient transgene expression was also observed in the rat hippocampus after stereotaxical inoculation of TH FGF-2, but not of TH lacZ or of SH vectors. Enhanced gene expression both in vitro and in vivo by the triple immediate-early gene deletion mutant might be attributed to reduced vector cytotoxicity. The present data suggest that TH FGF-2 is suitable for studies of FGF-2 involvement in neurological disorders.

Animals↗