Silvia Lorrio

Nrf2 participates in depressive disorders through an anti-inflammatory mechanism

A causative relationship between inflammation and depression is gradually gaining consistency. Because Nrf2 participates in inflammation, we hypothesized that Nrf2 could play a role in depressive disorders. In this study, we have observed that Nrf2 deletion in mice results in: (i) a depressive-like behavior evaluated as an increase in the immobility time in the tail-suspension test and by a decrease in the grooming time in the splash test, (ii) reduced levels of dopamine and serotonin and increased levels of glutamate in the prefrontal cortex, (iii) altered levels of proteins associated to depression such as VEGF and synaptophysin and (iv) microgliosis. Furthermore, treatment of Nrf2 knockout mice with the anti-inflammatory drug rofecoxib reversed their depressive-like behavior, while induction of Nrf2 by sulforaphane, in an inflammatory model of depression elicited by LPS, afforded antidepressant-like effects. In conclusion, our results indicate that chronic inflammation due to a deletion of Nrf2 can lead to a depressive-like phenotype while induction of Nrf2 could become a new and interesting target to develop novel antidepressive drugs.

Galantamine Postischemia Provides Neuroprotection and Memory Recovery against Transient Global Cerebral Ischemia in Gerbils

Galantamine, currently used in Alzheimers patients, has shown neuroprotection in hippocampal slices subjected to oxygenglucose deprivation. Here, we present an in vivo study to evaluate the potential neuroprotective effects of galantamine in a transient global cerebral ischemia model in gerbils. Three treatment protocols were used. In the pretreatment protocol, gerbils were treated before ischemia and for 3 consecutive days thereafter. Eight groups of animals were included: sham operation plus placebo, 10 mg/kg mecamylamine and 10 mg/kg galantamine, respectively; and ischemia plus placebo, 10 mg/kg mecamylamine, 1 mg/kg galantamine, and 10 mg/kg galantamine and 10 mg/kg mecamylamine plus galantamine, respectively. Postischemia protocols included three groups of animals: sham operation, ischemia plus placebo, and ischemia plus 10 mg/kg galantamine; substances were administered 3 or 6 h after ischemia and for 2 consecutive days thereafter. Pyramidal neurons surviving in the cornus ammonis 1 region of the hippocampus were evaluated 72 h after reperfusion, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) histochemistry, caspase-3 and superoxide dismutase (SOD)-2 immunohistochemistries, and Western blottings were performed, and object placement tests were carried out. Galantamine significantly increased the number of living pyramidal neurons after ischemia-reperfusion injury. Galantamine significantly reduced TUNEL, active caspase-3, and SOD-2 immunoreactivity. The nicotinic antagonist mecamylamine blocked the protective effects of galantamine. The neuroprotective effects of galantamine were preserved even when first administered at 3 h postischemia. These results correlated with the performance in the object placement test. This study shows that galantamine provides in vivo neuroprotection and memory recovery against global cerebral ischemia, even when administration begins 3 h postischemia.

Nrf2-mediated haeme oxygenase-1 up-regulation induced by cobalt protoporphyrin has antinociceptive effects against inflammatory pain in the formalin test in mice.

&NA; This study investigated the effect of haeme oxygenase‐1 (HO‐1) in nociception induced by formalin injection in the mice hind paw. Intraperitoneal (i.p.) administration of cobalt protoporphyrin (CoPP, an HO‐1 inducer, 5 mg/kg) 24 h before the test, inhibited the nociceptive response during the second phase, but not during the first phase of the formalin test. The effect of CoPP was prevented by treatment with tin protoporphyrin (SnPP, an inhibitor of HO‐1 activity) administered either by i.p. (25 mg/kg, 30 min before the test) or intraplantar (400 nmol/paw, 5 min before the test) routes. Human embryonic kidney (HEK) 293T cells treated with 10 μM CoPP expressed 20‐fold higher HO‐1 levels when compared to controls; this effect was suppressed by transfection with the dominant negative for the nuclear factor‐erythroid 2‐related factor 2 (Nrf2). Western blot analysis also revealed that CoPP treatment induced a similar 20‐fold increase in HO‐1 expression in the paw; this effect was attenuated in knockout mice for Nrf2. CoPP treatment of wild‐type, but not in Nrf2 knockout mice, resulted in a striking increase of HO‐1 stained cells surrounding the muscular tissues of the hind limbs. HO‐1 positive cells were scarce in wild‐type and in Nrf2 knockout untreated mice. CoPP‐induced HO‐1 expression in Nrf2 knockout mice was lost and correlated with the loss of antinociceptive effects. In conclusion, Nrf2‐mediated HO‐1 expression induced an antinociceptive effect at peripheral sites. These results suggest that HO‐1 modulates the inflammatory pain pathways. Hence, the development of drugs that could raise peripheral HO‐1 could be relevant in inflammatory pain treatment.

Effects of memantine and galantamine given separately or in association, on memory and hippocampal neuronal loss after transient global cerebral ischemia in gerbils

Galantamine is an acetylcholinesterase inhibitor and memantine is a non competitive antagonist of NMDA receptors that are being used to treat Alzheimers disease (AD) patients. The fact that drugs with different mechanisms of action are available to treat AD introduces the prospect of prescribing drug combinations to amplify drug efficacy. This study was planed to evaluate the potential neuroprotective effects of galantamine combined with memantine in a transient global cerebral ischemia model in gerbils. Animal groups included in the study were: sham, ischemia, and ischemia plus galantamine (1 mg/kg and 10 mg/kg), memantine (10 mg/kg and 20 mg/kg), 1 mg/kg galantamine plus 10 mg/kg memantine, and 10 mg/kg galantamine plus 10 mg/kg memantine, respectively. Surviving pyramidal neurons in the CA1 subfield of the hippocampus, TUNEL, caspase-3 and SOD-2 immunohistochemistries, and the object placement test were evaluated 72 h after reperfusion. Memantine did not exert a clear neuroprotective effect, nor did it prevent spatial memory loss. In a previous study using the same experimental model, galantamine was neuroprotective and improved spatial memory. In this study, the association of 10 mg/kg memantine with 10 mg/kg galantamine increased the number of living pyramidal neurons, reduced TUNEL, active caspase-3 and SOD-2 immunoreactivity, and preserved spatial memory after ischemia-reperfusion injury; however, the effects of the combination were not statistically different from those observed in animals treated with galantamine alone. We believe these results are of interest from a clinical point of view because the association of both drugs is being used in clinical practice and in clinical trials to treat Alzheimers disease and vascular dementia.

Molecular MRI of Atherosclerosis

Despite advances in prevention, risk assessment and treatment, coronary artery disease (CAD) remains the leading cause of morbidity and mortality in Western countries. The lion’s share is due to acute coronary syndromes (ACS), which are predominantly triggered by plaque rupture or erosion and subsequent coronary thrombosis. As the majority of vulnerable plaques does not cause a significant stenosis, due to expansive remodeling, and are rather defined by their composition and biological activity, detection of vulnerable plaques with x-ray angiography has shown little success. Non-invasive vulnerable plaque detection by identifying biological features that have been associated with plaque progression, destabilization and rupture may therefore be more appropriate and may allow earlier detection, more aggressive treatment and monitoring of treatment response. MR molecular imaging with target specific molecular probes has shown great promise for the noninvasive in vivo visualization of biological processes at the molecular and cellular level in animals and humans. Compared to other imaging modalities; MRI can provide excellent spatial resolution; high soft tissue contrast and has the ability to simultaneously image anatomy; function as well as biological tissue composition and activity.

Chondroitin sulfate reduces cell death of rat hippocampal slices subjected to oxygen and glucose deprivation by inhibiting p38, NFκB and iNOS.

The glycosaminoglycan chondroitin sulfate (CS) is a major constituent of the extracellular matrix of the central nervous system where it can constitute part of the perineuronal nets. Constituents of the perineuronal nets are gaining interest because they have modulatory actions on their neighbouring neurons. In this study we have investigated if CS could afford protection in an acute in vitro ischemia/reoxygenation model by using isolated hippocampal slices subjected to 60min oxygen and glucose deprivation (OGD) followed by 120min reoxygenation (OGD/Reox). In this toxicity model, CS afforded protection of rat hippocampal slices measured as a reduction of lactate dehydrogenase (LDH) release; maximum protection (70% reduction of LDH) was obtained at the concentration of 3mM. To evaluate the intracellular signaling pathways implicated in the protective effect of CS, we first analysed the participation of the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2 by western blot. OGD/Reox induced the phosphorylation of p38 and dephosphorylation of ERK1/2; however, CS only inhibited p38 but had no effect on ERK1/2. Furthermore, OGD/Reox-induced translocation of p65 to the nucleus was prevented in CS treated hippocampal slices. Finally, CS inhibited iNOS induction caused by OGD/Reox and thereby nitric oxide (NO) production measured as a reduction in DAF-2 DA fluorescence. In conclusion, the protective effect of CS in hippocampal slices subjected to OGD/Reox can be related to a modulatory action of the local immune response by a mechanism that implies inhibition of p38, NFκB, iNOS and the production of NO.

Haeme oxygenase-1 overexpression via nAChRs and the transcription factor Nrf2 has antinociceptive effects in the formalin test

ABSTRACT Epibatidine has shown antinociceptive effects in various pain models, being 200‐fold more potent than morphine. Previous results from our laboratory demonstrated that HO‐1 overexpression has an antinociceptive effect in the formalin test. Furthermore, epibatidine was able to induce haeme oxygenase‐1 (HO‐1). So, the aim of this study was to investigate the effect of HO‐1 overexpression induced by epibatidine in nociception elicited by formalin injection in the mice hindpaw. Administration of epibatidine (4 μg/kg) 24 h before the test reduced the nociceptive response during the first phase and second phase of the formalin test. This effect was prevented by treatment with tin protoporphyrin (SnPP, an inhibitor of HO‐1 activity) administered via intraplantar 5 min before the test, suggesting a main role of HO‐1. Western blot analysis revealed that epibatidine treatment increased by 2‐fold HO‐1 expression in the paw; this effect was lost in knockout mice for nuclear factor‐erythroid 2‐related factor 2 (Nrf2) and was accompanied by the loss of its antinociceptive effect. Furthermore, the antinociceptive effect of epibatidine was related to the activation of alpha7 and/or alpha9 nAChRs since methyllycaconitine (MLA) and mecamylamine but not dihydro‐&bgr;‐erythroidine (DH&bgr;E) reverted this effect. Finally, we showed by flow cytometry and by immunofluorescence that white blood cells of the animals injected with epibatidine expressed more HO‐1 than control animals, and this expression was also reverted by MLA pre‐treatment. These findings demonstrate that HO‐1 induction by epibatidine has antinociceptive and anti‐inflammatory effects by the activation of MLA‐sensitive nAChRs.

Investigating the effect of tumor vascularization on magnetic targeting in vivo using retrospective design of experiment

Nanocarriers take advantages of the enhanced permeability and retention (EPR) to accumulate passively in solid tumors. Magnetic targeting has shown to further enhance tumor accumulation in response to a magnetic field gradient. It is widely known that passive accumulation of nanocarriers varies hugely in tumor tissues of different tumor vascularization. It is hypothesized that magnetic targeting is likely to be influenced by such factors. In this work, magnetic targeting is assessed in a range of subcutaneously implanted murine tumors, namely, colon (CT26), breast (4T1), lung (Lewis lung carcinoma) cancer and melanoma (B16F10). Passively- and magnetically-driven tumor accumulation of the radiolabeled polymeric magnetic nanocapsules are assessed with gamma counting. The influence of tumor vasculature, namely, the tumor microvessel density, permeability and diameter on passive and magnetic tumor targeting is assessed with the aid of the retrospective design of experiment (DoE) approach. It is clear that the three tumor vascular parameters contribute greatly to both passive and magnetically targeted tumor accumulation but play different roles when nanocarriers are targeted to the tumor with different strategies. It is concluded that tumor permeability is a rate-limiting factor in both targeting modes. Diameter and microvessel density influence passive and magnetic tumor targeting, respectively.

PP2A ligand ITH12246 protects against memory impairment and focal cerebral ischemia in mice.

ITH12246 (ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b][1,8]naphthyridine-3-carboxylate) is a 1,8-naphthyridine described to feature an interesting neuroprotective profile in in vitro models of Alzheimers disease. These effects were proposed to be due in part to a regulatory action on protein phosphatase 2A inhibition, as it prevented binding of its inhibitor okadaic acid. We decided to investigate the pharmacological properties of ITH12246, evaluating its ability to counteract the memory impairment evoked by scopolamine, a muscarinic antagonist described to promote memory loss, as well as to reduce the infarct volume in mice suffering phototrombosis. Prior to conducting these experiments, we confirmed its in vitro neuroprotective activity against both oxidative stress and Ca(2+) overload-derived excitotoxicity, using SH-SY5Y neuroblastoma cells and rat hippocampal slices. Using a predictive model of blood-brain barrier crossing, it seems that the passage of ITH12246 is not hindered. Its potential hepatotoxicity was observed only at very high concentrations, from 0.1 mM. ITH12246, at the concentration of 10 mg/kg i.p., was able to improve the memory index of mice treated with scopolamine, from 0.22 to 0.35, in a similar fashion to the well-known Alzheimers disease drug galantamine 2.5 mg/kg. On the other hand, ITH12246, at the concentration of 2.5 mg/kg, reduced the phototrombosis-triggered infarct volume by 67%. In the same experimental conditions, 15 mg/kg melatonin, used as control standard, reduced the infarct volume by 30%. All of these findings allow us to consider ITH12246 as a new potential drug for the treatment of neurodegenerative diseases, which would act as a multifactorial neuroprotectant.

Smaller quantal size and faster kinetics of single exocytotic events in chromaffin cells from the APP/PS1 mouse model of Alzheimer's disease.

The kinetics of single-amperometric exocytotic events has been measured in chromaffin cells of C57 mice and in an APP/PS1 mouse model of Alzheimers disease (AD). K(+) depolarisation causes a burst of spikes that indicate the quantal release of the single-vesicle content of catecholamine. The kinetic analysis of 278 spikes from 10 control cells and 520 spikes from 18 APP/PS1 cells shows the following features of the latter compared with the former: (i) 45% lower t(1/2); (ii) 60% smaller quantal size; (iii) 50% lower decay time. Spike feet also showed 60% smaller quantal size. Immunofluorescence and thioflavin staining showed no amyloid beta (Aβ) burden in adrenal medulla slices of APP/PS1 mice that however exhibited dense Aβ plaques in the cortex and hippocampus. Furthermore, acetylcholinesterase staining of adrenal medulla indicated no apparent differences in the innervation by splanchnic cholinergic nerve terminals of chromaffin cells from control and APP/PS1 mice. This is the first report identifying subtle differences in the last steps of exocytosis that could be an indication of synaptic dysfunction of the secretory machinery not linked to Aβ burden in AD.