Paola Lorenzini

Eicosapentaenoic acid stimulates the expression of myelin proteins in rat brain.

We have previously demonstrated that, in C6 glioma cells, eicosapentaenoic acid (EPA) stimulates the expression of proteolipid protein (PLP) via cAMP‐mediated pathways. In this study, we investigated whether n‐3 polyunsaturated fatty acids can affect myelinogenesis in vivo. A single dose of either EPA or docosahexaenoic acid (DHA) was injected intracerebroventricularly into 2‐day‐old rats, which were then killed after 3 days post‐injection (p.i.). Total RNA was isolated from the medulla, cerebellum, and cortex, and the expression of myelin‐specific mRNAs was analyzed by real‐time PCR. The levels of PLP, myelin basic protein, and myelin oligodendrocyte protein mRNAs increased in nearly all brain regions of DHA‐ and EPA‐treated animals, but the effect was more pronounced in EPA‐treated rats. The enhancement in PLP transcript levels was followed by an increase in PLP translation in EPA‐treated rats. A further indicator of accelerated myelination was the increase in 2′‐3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) protein levels. In EPA‐treated rats, the increased expression of myelin genes coincided with a decrease of cAMP‐response element‐binding protein (CREB)‐DNA binding in the cerebellum and cortex (1 hr p.i.). After 16 hr, this effect was still present in the same cerebral regions even though the decrease in EPA‐treated rats was less pronounced than in controls. The down‐regulation of CREB activity was due to a decrease in the levels of CREB phosphorylation. In conclusion, our data suggest that EPA stimulates the expression of specific myelin proteins through decreased CREB phosphorylation. These results corroborate the clinical studies of the n‐3 PUFA beneficial effects on several demyelinating diseases.

Influence of urethane and ketamine on rat hepatic cytochrome P450 in vivo

The aim of this study was to identify the effects of widely used laboratory anaesthetics on cytochrome (CYP) activity in male Sprague Dawley rats in vivo. The anaesthetics used were urethane and ketamine. 7-Ethoxyresorufin (EROD), 7-pentoxyresorufin (PROD), aniline and ethylmorphine were used as substrates for CYP 1A, CYP 2B, CYP 2E1 and CYP 3A, respectively. Urethane increased EROD (CYP 1A) activity by 40 % (p < 0.01), and hydroxylation of aniline (CYP 2E1) by 14 % in the early phase of anaesthesia and by 60 % (p < 0.01) in the later one. Urethane also reduced the demethylation of ethylmorphine by 37 % (p < 0.01), but did not affect CYP 2B activity significantly. Ketamine did not significantly affect CYP 1A, 2B or 2E1. However, it reduced the demethylation of ethylmorphine (i.e. CYP 3A) by 32 % (p < 0.01). From these data, we concluded that a single dose of urethane inhibits CYP 3A but increases CYP 2E1 and CYP 1A, and that a single dose of ketamine inhibits the activity of CYP 3A.

Synthesis and cholinesterase activity of phenylcarbamates related to Rivastigmine, a therapeutic agent for Alzheimer's disease.

In order to develop new cholinesterase agents effective against Alzheimers disease (AD) we synthesized some phenylcarbamates structurally related to Rivastigmine and evaluated their in vitro and in vivo biological activity. Among the compounds which displayed the most significant in vitro activity, 1-[1-(3-dimethylcarbamoyloxyphenyl)ethyl]piperidine (31b), in addition to a simple and cheaper synthesis, showed lower toxicity and very similar therapeutic index in comparison with Rivastigmine.

Ubiquitin-Mediated Stress Response in a Rat Model of Brain Transient Ischemia/Hypoxia

Ubiquitin (Ub) is a small 76-residue protein, involved in intracellular protein degradation through a specific ATP-dependent system, which uses Ub as a tag to label proteins committed to be hydrolyzed by a specific 26 S protease. PGP-9.5 is another important component of the Ub system, i.e. a neuron-specific carboxyl-terminal hydrolase, which recycles Ub from Ub-polypeptide complexes. We have investigated the expression of Ub and PGP-9.5 in rat hippocampal neurons in an early phase of reperfusion in a model of transient global brain ischemia/hypoxia (bilateral occlusion of common carotid arteries for 10 min accompanied by mild hypoxia—15% O2—for 20 min), by means of immunohistochemical methods using light and electron microscopy. The intensity of Ub and PGP-9.5 immunoreactivity was evaluated by image analysis. We have detected a marked increase of Ub immunoreactivity (UIR) in neurons of CA1, CA2, CA3, CA4, and dentate gyrus subfields 1 hr after ischemia/hypoxia (but not after hypoxia only), statistically significant as confirmed by image analysis. Such increase in immunoreactivity in ischemic/hypoxic rats was localized essentially in the nuclei of hippocampal neurons. There were no changes in PGP-9.5 immunoreactivity. The data suggest that in the present model of rat brain ischemia/hypoxia Ub is involved in the neuronal stress response.

Transient global brain hypoxia-ischemia in adult rats: neuronal damage, glial proliferation, and alterations in inositol phospholipid hydrolysis.

A model of ischemic-hypoxic brain injury which combines bilateral occlusion of common carotid arteries for 10 min and mild hypoxia (15% O2 for 10 min before and during occlusion) was developed. Global ischemia was assessed by a simplified EEG recording indicating isoelectric line, i.e. full arrest of cortical electrical activity. Histological examination of brain 7 days after ischemic insult showed from moderate to severe damage, mainly in the cerebral cortex (layers III, V and VI) and hippocampus (mainly CA1 subfield). The injury consisted of neuronal degeneration and necrosis with nuclear pyknosis and karyorrhexis. Immunohistochemical staining for gliofibrillar acidic protein showed a marked glial proliferation in the cerebral cortex and hippocampus. In the cortical slices, inositol phosphates accumulation stimulated by excitatory amino acid agonists (ACPD, ibotenate and quisqualate), as well as by norepinephrine and carbachol, was enhanced significantly (p < 0.01) with respect to sham-operated rats 7 days, but not 24 h, after the ischemic insult. The overall data show that the relatively simple transient brain hypoxia/ischemia rat model produces full arrest of cortical EEG, histopathological alterations and those relative to post-receptor neurochemical mechanisms characteristic of four-vessel occlusion model.

Transient global brain ischemia in young and aged rats: differences in severity and progression, but not localisation, of lesions evaluated by magnetic resonance imaging

A model of transient global brain ischemia consisting of bilateral occlusion of common carotid arteries for 10 min and mild hypoxia (15% O2−85% N2) for 20 min was studied by means of MRI in young and aged Fischer 344 rats (3–4 and 24–26 months, respectively). Ischemia was assessed by full suppression of spontaneous EEG activity, which reappeared and normalized similarly in the two age-groups. The survival of young with respect to aged rats was considerably higher both at 24 h (20/20, i.e. 100% vs 12/16, i.e. 75%) and at 48 h (16/20, i.e. 80% vs 6/16, i.e. 38%). The localisation of brain lesions, their severity and progression were evaluated by a diffusion-weighted MRI (DWI) sequence at 24 and 48 h post-ischemia. There were no DWI-detectable lesions in eight out of 20 young and two out of 12 aged rats. The localisation of DWI-detected lesions was rather similar in rats of the two age-groups. In fact, the cerebral cortex, mainly parietal, occipital and temporal lobes were damaged in 83% of young and 90% of aged rats. The respective percentages for the thalamus were 83 and 60%, for the striatum 58 and 50%, and for the hippocampus 25 and 30%. The lesions present in the cerebral cortex and the thalamus were considerably more severe in aged than in young rats. In conclusion, in spite of similar localisation of ischemic lesions in the two age-groups, their incidence was higher, appearance more rapid and severity more pronounced in aged with respect to young rats. This resulted in a considerably higher mortality of the former. The overall data indicate that the age issue is very important in experimental ischemia research.

Age-dependent MRI-detected lesions at early stages of transient global ischemia in rat brain

Although ischemic stroke has higher incidence and severity in aged than in young humans, the age factor is generally neglected in ischemia animal models. This study was aimed at comparing age-dependent effects at early stages of transient global cerebral ischemia (TGCI) in rats. TGCI was induced in two groups of rats (3–6 and 20–24 months old, respectively) by exposure to 15% oxygen and 15 min occlusion of the two common carotid arteries. Brains were analysed in vivo by MRI–apparent diffusion coefficient (ADC) and T2 maps–at 1–3 h post-TGCI and in vitro by histochemical examination of triphenyltetrazolium chloride (TTC)-stained slices. At 1–3 h post-TGCI, a higher incidence of lesions was found in aged than in young rats especially in the hippocampus and cortex (occipital plus parietal) but not in the thalamus. The lesioned regions showed lower ADC values in aged than in younger rats. The most substantial ADC decreases were associated with enhanced spin-spin relaxation and lower TTC staining. The different responses of the two age groups support the use of aged animals for investigations on different ischemia models. Our model of brain ischemia appears appropriate for further studies including drug effects.

Differential responsiveness of metabotropic glutamate receptors coupled to phosphoinositide hydrolysis to agonists in various brain areas of the adult rat

The effects of metabotropic glutamate receptor (mGluR) agonists on inositol phosphates (IP) accumulation were investigated in slices of the cerebral cortex, hippocampus, striatum and cerebellum of adult Sprague-Dawley rats. EC50 values for 1S, 3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) did not differ significantly between various brain areas (range 10−5 M), quisqualate was the most potent in all the brain areas (range 10−7−10−6 M), except the cerebellum (10−5 M), ibotenate was the most potent in the striatum (range 10−6 M) and the least potent in the cerebral cortex and hippocampus (range 10−4 M). The efficacy in the four brain areas showed the following trend of ranking order for ACPD and quisqualate: hippocampus > striatum > cerebral cortex > cerebellum, and for ibotenate: hippocampus > cerebral cortex > striatum > cerebellum, although the observed differences reached the level of statistical significance only in the case of ACPD (hippocampus and striatum vs cerebellum) and ibotenate (hippocampus vs cerebellum). Co-incubation of the agonists at maximally effective concentrations in any pairwise combination resulted in no substantial additivity of IP accumulation. D,L-1-amino-3-phosphonopropionic acid (AP3) and D,L-2-amino-4-phosphonobutyric acid (AP4) at 0.5 mM concentration antagonized ACPD-induced IP accumulation by about 70 and 45%, respectively, without differences between brain areas. On the other hand, the antagonistic effects ofl-serine-o-phosphate (SOP) at 1 mM concentration were the highest in the hippocampus (75%) and the lowest in the cerebellum (25%). The comparative data indicate considerable regional receptor heterogeneity, in terms of different ratios of response to the agonists (but not antagonists, except SOP). There is a robust responsiveness of mGluRs not only in the hippocampus and cerebral cortex, but also in the striatum which exhibits the highest affinity to both quisqualate and ibotenate.

Influences of hypothyroidism on lipid composition and inositol lipid-linked receptors responsiveness and protein kinase C (PKC) activity in the cerebral cortex of lewis rats

The influence of hypothyroidism (HO) induced by treatment with propylthiouracil on lipid composition, receptor responsiveness of MI-muscarinic receptors (M1AChRs) and metabotropic glutamate receptors (mGluRs) as well as on protein kinase C (PKC) activity was investigated in the cerebral cortex of Lewis rats. HO did not influence the lipid composition. There was a significant 2-fold increase of efficacy and 6-fold decrease of potency of carbachol-induced inositol phosphate (IP) accumulation in HO, with respect to control rats. The efficacy of trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD)-induced IP accumulation was also higher in HO (by 50%), without differences in EC50 values. The activities of soluble calcium-dependent and calcium-independent PKC were higher in HO than in control rats (both roughly 30%); membrane-associated PKCs were not modified. The data indicate that HO induces an increased responsiveness of M1AChRs and mGluRs and a rise in the soluble PKC activity that could be available and ready for translocation.