modello per invio relazione di metà e fine periodo

MODELLO PER INVIO RELAZIONE DI METÀ E FINE PERIODO
NOME E COGNOME: Esi Domi
UNIVERSITÀ: Hospital University of Linkoping (Sweden)
DIPARTIMENTO (in caso di borsa per soggiorno all’estero specificare l’ente presso cui si è svolta la
ricerca): Clinical and Experimental Medicine (IKE)
TUTOR (in caso di borsa per soggiorno all’estero specificare il tutor dell’ente presso cui si è svolta la
ricerca): MD PhD Markus Heilig, Director of the Center for Social and Affective Neuroscience
Dept. Of Clinical and Experimental Medicine, Linkoping University.
TIPOLOGIA DI BORSA RICEVUTA: Borsa di ricerca SIF per soggiorno all´estero
TIPOLOGIA DI RELAZIONE (es.: metà periodo o finale): Finale
TITOLO DELLA RELAZIONE: “Exploring new mechanisms responsible for the progression to
alcohol addiction: Searching for novel medications”
Da inviare a: Società Italiana di Farmacologia – e-mail: [email protected]; [email protected]
Alla segreteria organizzativa della Societa´ Italiana di Farmacologia (SIF)
Relazione di meta´ periodo sull´ attivita´ svolta per la realizzazione del Progetto di Ricerca:
“Exploring new mechanisms responsible for the progression to alcohol
addiction: Searching for novel medications”
Borsista
Responsabile del progetto
PhD Domi Esi
MD PhD Markus Heilig,
Professor of Psychiatry
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RELAZIONE:
Introduction
Alcohol addiction is a chronic relapsing disorder frequently associated with increased sensitivity to
stress. Available treatments have limited efficacy and new more effective pharmacotherapies are
needed. Growing evidences correlate neuroinflammatory states to the neurobiological processes
involved in alcoholism. Alcohol exposure leads to neuroinflammatory response characterized by an
enhanced production of proinflammatory molecules such as cytokines and chemokines. For example,
in mice, administration of proinflammatory agents such as lipopolissaccaride (LPS) increases
voluntary alcohol intake (Blednov et al., 2011). Moreover, it has been demonstrated that Interleukin1β (IL-1β), tumor necrosis factor-α (TNFα) and interleukin 6 (IL-6), critical factors of the innate
immune response, increase after alcohol intake (Zou & Crews, 2010) and that serum levels of
cytokine and inflammatory endotoxins show positive correlations with alcohol craving. Activation of
innate immune signalling cascade can enhance alcohol consumption. In our laboratory it has been
recently shown that pioglitazone, a Peroxisome Proliferator Activated Receptor Gamma (PPARγ)
agonist, characterized by strong anti-inflammatory properties, reduces alcohol consumption in
genetically selected alcohol preferring msP rats and prevents neuronal cell death following binge-like
exposure to alcohol. Additionally, altered expression of genes involved in the neuroinflammatory
pathways may play an important role in alcohol abuse. Moreover, expression of different immunerelated genes is altered in alcoholic brains and in rodent lines that show high preference for alcohol.
In addition polymorphisms of genes encoding for IL1 B and IL1 and IL 10 are associated with an
increased susceptibility to alcoholism (Mulligan et al., 2006).
All these data support the evidence that the neuroimmune system plays an important role in alcohol
abuse. A picture emerge in which in a normal functioning immune system is less prone to precipitate
into alcohol dependence, whereas an over-activated immune system may predispose an individual to
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abuse excessive amounts of alcohol. Exploring the role of the neuroimmune system in shaping the
vulnerability to alcohol addiction can help in the development of more efficacious pharmacotherapies
for the treatment of this psychiatric condition.
The overarching hypothesis of the present project is that alcohol abuse is linked to a dysregulation,
innate or acquired, of the neuroimmune system. Corollary to this hypothesis, is that pharmacological
treatments able to normalize the neuroinflammatory function are able to treat aspects of alcohol
addiction.
In here we used a binge drinking model to induce alcohol dependence in male Wistar rats and to
explore the main inflammatory markers activated after the alcohol injury. The binge model of
drinking, which mimics a single cycle of binge intoxication in human alcoholics (Majchrowicz,
1975), is further validated by the fact that during alcohol intoxication animals reach sustained, high
blood alcohol levels (BALs), commonly observed among alcoholics. Furthermore, neuronal deficits
in animals treated with this binge alcohol model are associated with significant cognitive
dysfunctions, such as learning and memory impairment as well as behavioral deficits including
maladaptive perseverant behaviour (Obernier et al., 2002; Cippitelli et al., 2010a; Cippitelli et al.,
2010b). Binge alcohol intoxication analyzed by Fluorojade-B immunostaining (FJ-B positive cells)
induced a substantial neurodegeneration in the dentate gyrus (DG) of the Hippocampus and
Entorhinal Cortex. Chronic PPARγ activation by the selective agonist, pioglitazone reduced dosedependetly the neuronal damage induced by alcohol intoxication (Fig 3, unpublished data). It has
been proposed that alcohol-induced brain damage may result by imbalance in expression and
activation of transcription factors that regulate pro-survival versus pro-inflammatory states (Crews &
Nixon, 2009). Indeed, both in vitro and in vivo evidence have shown that alcohol shifts this balance
toward inflammation by decreasing function of the pro-survival cAMP responsive element-binding
protein (CREB)-mediated signalling (Bison & Crews, 2003; Zou & Crews, 2006), while increasing
the nuclear factor κB (NF-κB) transcriptional factor-dependent signaling that leads to induction of
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pro-inflammatory cytokines and enzymes that promote a pro-inflammatory cascade by further
activating NF-κB transcription (Davis & Syapin, 2004; Crews et al., 2006; Zou & Crews, 2010).
Based on the literature data and data from our laboratory that show a marked neurodegeneration
induced by binge alcohol intoxication in the hippocampus (HIPP) and entorhinal cortex (EC) and
considering that the circuitry from the EC to the hippocampal formation is critical for memory
formation and spatial learning (Aggleton et al., 2000) we analysed alcohol-induced alterations in the
levels of neuroimmune factors in these two regions. Moreover we examined PPARγ activation on the
neuroinflammatory markers elicited by the binge drinking model.
By Real-Time quantitative RT-PCR we examined mRNA expression of interleukin 1β, (IL-1β), the
chemokine (C-C motif) ligand 2, (CCL2), the tumor necrosis factor alfa, (TNF-α), interleukine 6,
(IL-6), transforming growth factor (TGF-β1 and 2) since these agents are known to play a pivotal role
in the neuroinflammation initiated by excessive alcohol use (Crews & Nixon, 2009; Alfonso-Loeches
et al., 2010).
Methods
RNA Isolation, cDNA Synthesis and Real Time Polymerase Chain Reaction. Adult male rats (Charles
River) were decapitated three hours after the last alcohol gavage, the same time point used to harvest
samples for histochemical analysis of FJ-B. Brains were quickly removed, areas of interest were
dissected and snap frozen in -40°C isopentane, and stored at -80°C until use. RNA isolation and
cDNA synthesis was executed as previously described (Drew et al, 2015). Briefly, tissue was
homogenized using a BBX24B Bullet Blender Blue homogenizer with 0.5mm RNase-free beads for
approximately 6 minutes at speed 8 (Next Advance, Averill Park, NY). RNA was isolated from tissue
homogenate using the RNeasy Lipid Tissue Mini Kit and optional on-column DNA digestion using
the supplementary RNase-free DNase set according to the manufacturer’s instructions (Qiagen,
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Valencia, CA). RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer
with its associated RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA). The iScript™
cDNA synthesis kit was used to prepare cDNA as described by the manufacturer (Bio-Rad, Hercules,
CA). Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to measure mRNA
expression using a CFX96 qRT-PCR detection system (Bio-Rad, Hercules, CA). TaqMan® Assays
were synthesized by Life Technologies (Grand Island, NY), and these FAM labeled primers were
used to perform PCR in duplicate 20μl reactions containing SsoAdvanced™ Universal Probes
Supermix (Bio-Rad, Hercules, CA). Data were calculated as the mean ∆Ct relative to the
housekeeping gene β-actin. The ∆∆Ct method was employed to generate fold expression variance of
ethanol and drug treated groups compared to control.
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Results
Fig.1 Changes in body weight and blood alcohol levels (BALs) during binge alcohol drinking.
***p<0.001 vs control.
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Somatic withdrawal signs
***
Fig. 2 Somatic withdrawal signs after alcohol cessation. Data are expressed as (Mean ± SEM)
***p<0.001 vs control.
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Fig. 3 Binge alcohol-induced a neurotoxicity in the dentate gyrus (DG) and the entorhinal cortex (EC)
and its prevention by pioglitazone (0, 30, 60 mg/kg os). Sections were stained by Fluoro Jade B (FJB) to visualize neurodegeneration. Panels (A), (B), (C) and (D) show representative sections (-6.1mm
from Bregma, 20 X magnification) visualizing labelled DG neurons in animals non-alcohol exposed
treated with vehicle (distilled water-PIO 0-CON), alcohol exposed treated with vehicle (PIO 0-ALC)
and pioglitazone 30 and 60 mg/kg respectively. In panels (F), (G), (H), (I) is represented the EC FJB
labeled neurons in PIO 0-CON, PIO 0-ALC, PIO 30-ALC and PIO 60-ALC treated rats. Panels (E)
and (J) show quantification of the histological data demonstrating alcohol-induced neurodegeneration
and its prevention by pioglitazone in DG and EC respectively. Data are the mean number of FJ-B
positive cells/mm2 ± SEM (N= 4-6 per group). #p<0.05 vs. PIO 0-CON group; *p<0.05 vs. PIO 0ALC group.
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Fig. 4 Effect of alcohol on expression of interleukin 1β, (IL-1β), TNF α; CCL2; (IL-6), TGFβ1 and
TGFβ1genes in the hippocampus after alcohol binge drinking. Data are expressed as (Mean ±
SEM); *p<0.05; ** p<0.01; p<0.001 vs control.
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Fig. 5 Effect of alcohol on expression of interleukin 1β, (IL-1β), TNF α; CCL2; (IL-6), TGFβ1 and
TGFβ1 and TGFβ2 genes in the entorhinal cortex after alcohol binge drinking. Data are expressed as
(Mean ± SEM); *p<0.05; ** p<0.01; p<0.001 vs control.
Conclusions
Our data support the evidence that a short period of binge alcohol intoxication induces alcohol
dependence as observed by the high blood alcohol levels during the treatment and the comparison of
the somatic withdrawal signs after alcohol cessation. We show that neuroinflammation importantly
contributes to alcohol-induced neurotoxicity as dynamic changes in the expression of proinflammatory markers were detected following alcohol treatment.
A picture that emerges from this study is that excessive alcohol induces a clear up-regulation of IL-6
expression in both HIPP and EC. While Il-1 expression was significant increased only in the
entorhinal cortex. No changes in TNFα mRNA levels were observed and ethanol exposure induced
a decrease of CCL2 only in the EC. The observed changes of the pro-inflammatory signal and in
specific the upregulation of IL-1 and Il-6 could be a hypothetical mechanism of action of alcohol
induced neurodegeneration after a binge drinking exposure observed in our previous experiments.
PPARgamma activation by the specific agonist, pioglitazone, was able to decrease the
neuroinflammation induced by excessive drinking.
Drugs with anti-inflammatory and antioxidant properties will be evaluated in order to contrast the
neuroinflammation induced by excessive alcohol consumption which is known to be key factor in the
development of alcohol dependence, neurodegeneration and a severe cognitive impairment in both
rodents and humans. Here, for the first time we provided evidence that PPAR-γ activation is also
neuroprotective against binge alcohol drinking, possibly with mechanisms involving inhibition of
neuroimmune response (Kane et al., 2011; Drew et al., 2015). To determine whether
neuroinflammation was responsible for the observed alcohol-induced neurotoxicity we hypothesized
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that activation of PPAR-γ could prevent expression of genes encoding neuroinflammatory molecules.
In order to confirm this hypothesis we examined mRNA expression of IL-1β and IL-6, cytokines
known to play a pivotal role in neuroinflammation initiated by excessive alcohol use (AlfonsoLoeches et al., 2010; Crews et al., 2009; Crews et al., 2011). We found that both the DG and the EC
are sensitive to neuroinflammatory processes as dynamic changes were observed in the expression of
these inflammatory cytokines in response to alcohol treatment. However, these alcohol-induced
changes were more prominent in the EC. There was robust alcohol-induced upregulation of IL-6 and
IL-1β expression in the EC that was completely abolished by concurrent administration of
pioglitazone. This suggests that, on the one hand these cytokines may contribute to alcohol-induced
neurodegeneration and on the other that the effect of pioglitazone may be related to its ability to blunt
alcohol-induced activation of these immune response meditors. These findings, together with our
previous evidence describing the ability of PPARγ agonists to reduce excessive alcohol intake and
vulnerability to relapse into alcohol seeking (Stopponi et al., 2011), provide a strong rationale for
consideration of PPARγ activation or its congeners as effective treatments for alcohol use disorders.
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