Publications by authors named "Daniele Lana"

20 Publications

  • Page 1 of 1

Protective Effect of Adenosine A Receptor Agonist, BAY60-6583, Against Transient Focal Brain Ischemia in Rat.

Front Pharmacol 2020 11;11:588757. Epub 2021 Feb 11.

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Division of Pharmacology and Toxicology, University of Florence, Florence, Italy.

Cerebral ischemia is a multifactorial pathology characterized first by an acute injury, due to excitotoxicity, followed by a secondary brain injury that develops hours to days after ischemia. During ischemia, adenosine acts as an endogenous neuroprotectant. Few studies have investigated the role of A receptor in brain ischemia because of the low potency of adenosine for it and the few selective ligands developed so far. A receptors are scarcely but widely distributed in the brain on neurons, glial and endothelial cells and on hematopoietic cells, lymphocytes and neutrophils, where they exert mainly anti-inflammatory effects, inhibiting vascular adhesion and inflammatory cells migration. Aim of this work was to verify whether chronic administration of the A agonist, BAY60-6583 (0.1 mg/kg i.p., twice/day), starting 4 h after focal ischemia induced by transient (1 h) Middle Cerebral Artery occlusion (tMCAo) in the rat, was protective after the ischemic insult. BAY60-6583 improved the neurological deficit up to 7 days after tMCAo. Seven days after ischemia BAY60-6583 reduced significantly the ischemic brain damage in cortex and striatum, counteracted ischemia-induced neuronal death, reduced microglia activation and astrocytes alteration. Moreover, it decreased the expression of TNF-α and increased that of IL-10 in peripheral plasma. Two days after ischemia BAY60-6583 reduced blood cell infiltration in the ischemic cortex. The present study indicates that A receptors stimulation can attenuate the neuroinflammation that develops after ischemia, suggesting that A receptors may represent a new interesting pharmacological target to protect from degeneration after brain ischemia.
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http://dx.doi.org/10.3389/fphar.2020.588757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7905306PMC
February 2021

A Adenosine Receptors: When Outsiders May Become an Attractive Target to Treat Brain Ischemia or Demyelination.

Int J Mol Sci 2020 Dec 18;21(24). Epub 2020 Dec 18.

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Section of Pharmacology and Toxicology, University of Florence, 50139 Florence, Italy.

Adenosine is a signaling molecule, which, by activating its receptors, acts as an important player after cerebral ischemia. Here, we review data in the literature describing AR-mediated effects in models of cerebral ischemia obtained in vivo by the occlusion of the middle cerebral artery (MCAo) or in vitro by oxygen-glucose deprivation (OGD) in hippocampal slices. Adenosine plays an apparently contradictory role in this receptor subtype depending on whether it is activated on neuro-glial cells or peripheral blood vessels and/or inflammatory cells after ischemia. Indeed, ARs participate in the early glutamate-mediated excitotoxicity responsible for neuronal and synaptic loss in the CA1 hippocampus. On the contrary, later after ischemia, the same receptors have a protective role in tissue damage and functional impairments, reducing inflammatory cell infiltration and neuroinflammation by central and/or peripheral mechanisms. Of note, demyelination following brain ischemia, or autoimmune neuroinflammatory reactions, are also profoundly affected by ARs since they are expressed by oligodendroglia where their activation inhibits cell maturation and expression of myelin-related proteins. In conclusion, data in the literature indicate the ARs as putative therapeutic targets for the still unmet treatment of stroke or demyelinating diseases.
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http://dx.doi.org/10.3390/ijms21249697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766015PMC
December 2020

Space-Dependent Glia-Neuron Interplay in the Hippocampus of Transgenic Models of β-Amyloid Deposition.

Int J Mol Sci 2020 Dec 11;21(24). Epub 2020 Dec 11.

Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Viale Pieraccini 6, 50139 Firenze, Italy.

This review is focused on the description and discussion of the alterations of astrocytes and microglia interplay in models of Alzheimer's disease (AD). AD is an age-related neurodegenerative pathology with a slowly progressive and irreversible decline of cognitive functions. One of AD's histopathological hallmarks is the deposition of amyloid beta (Aβ) plaques in the brain. Long regarded as a non-specific, mere consequence of AD pathology, activation of microglia and astrocytes is now considered a key factor in both initiation and progression of the disease, and suppression of astrogliosis exacerbates neuropathology. Reactive astrocytes and microglia overexpress many cytokines, chemokines, and signaling molecules that activate or damage neighboring cells and their mutual interplay can result in virtuous/vicious cycles which differ in different brain regions. Heterogeneity of glia, either between or within a particular brain region, is likely to be relevant in healthy conditions and disease processes. Differential crosstalk between astrocytes and microglia in CA1 and CA3 areas of the hippocampus can be responsible for the differential sensitivity of the two areas to insults. Understanding the spatial differences and roles of glia will allow us to assess how these interactions can influence the state and progression of the disease, and will be critical for identifying therapeutic strategies.
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http://dx.doi.org/10.3390/ijms21249441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763751PMC
December 2020

An Overview on the Differential Interplay Among Neurons-Astrocytes-Microglia in CA1 and CA3 Hippocampus in Hypoxia/Ischemia.

Front Cell Neurosci 2020 11;14:585833. Epub 2020 Nov 11.

Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Florence, Italy.

Neurons have been long regarded as the basic functional cells of the brain, whereas astrocytes and microglia have been regarded only as elements of support. However, proper intercommunication among neurons-astrocytes-microglia is of fundamental importance for the functional organization of the brain. Perturbation in the regulation of brain energy metabolism not only in neurons but also in astrocytes and microglia may be one of the pathophysiological mechanisms of neurodegeneration, especially in hypoxia/ischemia. Glial activation has long been considered detrimental for survival of neurons, but recently it appears that glial responses to an insult are not equal but vary in different brain areas. In this review, we first take into consideration the modifications of the vascular unit of the glymphatic system and glial metabolism in hypoxic conditions. Using the method of triple-labeling fluorescent immunohistochemistry coupled with confocal microscopy (TIC), we recently studied the interplay among neurons, astrocytes, and microglia in chronic brain hypoperfusion. We evaluated the quantitative and morpho-functional alterations of the neuron-astrocyte-microglia triads comparing the hippocampal CA1 area, more vulnerable to ischemia, to the CA3 area, less vulnerable. In these contiguous and interconnected areas, in the same experimental hypoxic conditions, astrocytes and microglia show differential, finely regulated, region-specific reactivities. In both areas, astrocytes and microglia form triad clusters with apoptotic, degenerating neurons. In the neuron-astrocyte-microglia triads, the cell body of a damaged neuron is infiltrated and bisected by branches of astrocyte that create a microscar around it while a microglial cell phagocytoses the damaged neuron. These coordinated actions are consistent with the scavenging and protective activities of microglia. In hypoxia, the neuron-astrocyte-microglia triads are more numerous in CA3 than in CA1, further indicating their protective effects. These data, taken from contiguous and interconnected hippocampal areas, demonstrate that glial response to the same hypoxic insult is not equal but varies significantly. Understanding the differences of glial reactivity is of great interest to explain the differential susceptibility of hippocampal areas to hypoxia/ischemia. Further studies may evidence the differential reactivity of glia in different brain areas, explaining the higher or lower sensitivity of these areas to different insults and whether glia may represent a target for future therapeutic interventions.
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http://dx.doi.org/10.3389/fncel.2020.585833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686560PMC
November 2020

Ethanol neurotoxicity is mediated by changes in expression, surface localization and functional properties of glutamate AMPA receptors.

J Neurochem 2020 Oct 26. Epub 2020 Oct 26.

Department of Neuroscience, Psychology, Drug Research and Child Health (NeuroFarBa), Section of Pharmacology and Toxicology, University of Florence, Florence, Italy.

Modifications in the subunit composition of AMPA receptors (AMPARs) have been linked to the transition from physiological to pathological conditions in a number of contexts, including EtOH-induced neurotoxicity. Previous work from our laboratory showed that EtOH withdrawal causes CA1 pyramidal cell death in organotypic hippocampal slices and changes in the expression of AMPARs. Here, we investigated whether changes in expression and function of AMPARs may be causal for EtOH-induced neurotoxicity. To this aim, we examined the subunit composition, localization and function of AMPARs in hippocampal slices exposed to EtOH by using western blotting, surface expression assay, confocal microscopy and electrophysiology. We found that EtOH withdrawal specifically increases GluA1 protein signal in total homogenates, but not in the post-synaptic density-enriched fraction. This is suggestive of overall increase and redistribution of AMPARs to the extrasynaptic compartment. At functional level, AMPA-induced calcium influx was unexpectedly reduced, whereas AMPA-induced current was enhanced in CA1 pyramidal neurons following EtOH withdrawal, suggesting that increased AMPAR expression may lead to cell death because of elevated excitability, and not for a direct contribution on calcium influx. Finally, the neurotoxicity caused by EtOH withdrawal was attenuated by the non-selective AMPAR antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt as well as by the selective antagonist of GluA2-lacking AMPARs 1-naphthyl acetyl spermine. We conclude that EtOH neurotoxicity involves changes in expression, surface localization and functional properties of AMPARs, and propose GluA2-lacking AMPARs as amenable specific targets for the development of neuroprotective drugs in EtOH-withdrawal syndrome.
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http://dx.doi.org/10.1111/jnc.15223DOI Listing
October 2020

Neuroprotective effects of mGluR5 activation through the PI3K/Akt pathway and the molecular switch of AMPA receptors.

Neuropharmacology 2020 01 7;162:107810. Epub 2019 Oct 7.

Department of Health Sciences, Unit of Clinical Pharmacology and Oncology, University of Florence, Viale G. Pieraccini 6, 50139, Florence, Italy.

Previous studies have demonstrated that antagonists of mGluR1, but not mGluR5, are neuroprotective in models of cerebral ischemia. To investigate the individual roles of mGlu1 and mGlu5 receptors in in vitro model of cerebral ischemia we used low doses of the non-selective group I agonist DHPG and mGlu1 and mGlu5 selective positive allosteric modulators (PAMs). In hippocampal slices subjected to 30 min oxygen-glucose deprivation (OGD), DHPG (1 μM) and the mGluR5 PAM (VU0092273) significantly reduced OGD-induced CA1 injury monitored by propidium iodide staining of the slices and quantitative analysis of CA1 neurons. In contrast, the mGluR1 PAM (VU0483605) showed no neuroprotection. These protective effects of DHPG and VU0092273 were prevented by inhibition of PI3K/Akt pathway by LY294002. The mGluR5 PAM (VU0092273) also prevented GluA2 down-regulation triggered by ischemic injury, via PI3K/Akt pathway, revealing a further contribution to its neuroprotective effects by reducing the excitotoxic effects of increased Ca influx through GluA2-lacking AMPA receptors. Furthermore, immunohistochemical assays confirmed the neuroprotective effect of VU0092273 and revealed activation of glia, indicating the involvement reactive astrogliosis in the mechanisms of neuroprotection. Our data suggest that selective activation/potentiation of mGluR5 signalling represents a promising strategy for the development of new interventions to reduce or prevent ischemia-induced neuronal death.
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http://dx.doi.org/10.1016/j.neuropharm.2019.107810DOI Listing
January 2020

Neurotoxicity of Unconjugated Bilirubin in Mature and Immature Rat Organotypic Hippocampal Slice Cultures.

Neonatology 2019 15;115(3):217-225. Epub 2019 Jan 15.

Department of Neurosciences, Psychology, Drug Research and Child Health, University of Florence, Florence, Italy.

Background: The physiopathology of bilirubin-induced neurological disorders is not completely understood.

Objectives: The aim of our study was to assess the effect on bilirubin neurotoxicity of the maturity or immaturity of exposed cells, the influence of different unconjugated bilirubin (UCB) and human serum albumin (HSA) concentrations, and time of UCB exposure.

Methods: Organotypic hippocampal slices were exposed for 48 h to different UCB and HSA concentrations after 14 (mature) or 7 (immature) days of in vitro culture. Immature slices were also exposed to UCB and HSA for 72 h. The different effects of exposure time to UCB on neurons and astrocytes were evaluated.

Results: We found that 48 h of UCB exposure was neurotoxic for mature rat organotypic hippocampal slices while 72 h of exposure was neurotoxic for immature slices. Forty-eight-hour UCB exposure was toxic for astrocytes but not for neurons, while 72-h exposure was toxic for both astrocytes and neurons. HSA prevented UCB toxicity when the UCB:HSA molar ratio was ≤1 in both mature and immature slices.

Conclusions: We confirmed UCB neurotoxicity in mature and immature rat hippocampal slices, although immature ones were more resistant. HSA was effective in preventing UCB neurotoxicity in both mature and immature rat hippocampal slices.
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http://dx.doi.org/10.1159/000494101DOI Listing
December 2019

Microglial distribution, branching, and clearance activity in aged rat hippocampus are affected by astrocyte meshwork integrity: evidence of a novel cell-cell interglial interaction.

FASEB J 2019 03 29;33(3):4007-4020. Epub 2018 Nov 29.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Aging and neurodegenerative diseases share a condition of neuroinflammation entailing the production of endogenous cell debris in the CNS that must be removed by microglia ( i.e., resident macrophages), to restore tissue homeostasis. In this context, extension of microglial cell branches toward cell debris underlies the mechanisms of microglial migration and phagocytosis. Amoeboid morphology and the consequent loss of microglial branch functionality characterizes dysregulated microglia. Microglial migration is assisted by another glial population, the astroglia, which forms a dense meshwork of cytoplasmic projections. Amoeboid microglia and disrupted astrocyte meshwork are consistent traits in aged CNS. In this study, we assessed a possible correlation between microglia and astroglia morphology in rat models of chronic neuroinflammation and aging, by 3-dimensional confocal analysis implemented with particle analysis. Our findings suggest that a microglia-astroglia interaction occurs in rat hippocampus via cell-cell contacts, mediating microglial cell branching in the presence of inflammation. In aged rats, the impairment of such an interaction correlates with altered distribution, morphology, and inefficient clearance by microglia. These data support the idea that generally accepted functional boundaries between microglia and astrocytes should be re-evaluated to better understand how their functions overlap and interact.-Lana, D., Ugolini, F., Wenk, G. L., Giovannini, M. G., Zecchi-Orlandini, S., Nosi, D. Microglial distribution, branching, and clearance activity in aged rat hippocampus are affected by astrocyte meshwork integrity: evidence of a novel cell-cell interglial interaction.
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http://dx.doi.org/10.1096/fj.201801539RDOI Listing
March 2019

Different Patterns of Neurodegeneration and Glia Activation in CA1 and CA3 Hippocampal Regions of TgCRND8 Mice.

Front Aging Neurosci 2018 13;10:372. Epub 2018 Nov 13.

Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Florence, Italy.

We investigated the different patterns of neurodegeneration and glia activation in CA1 and CA3 hippocampal areas of TgCRND8 mice. The main feature of this transgenic model is the rapid development of the amyloid pathology, which starts already at 3 months of age. We performed immunohistochemical analyses to compare the different sensibility of the two hippocampal regions to neurodegeneration. We performed qualitative and quantitative evaluations by fluorescence immunohistochemistry with double or triple staining, followed by confocal microscopy and digital image analysis in stratum pyramidale (SP) and stratum radiatum (SR) of CA1 and CA3, separately. We evaluated time-dependent Aβ plaques deposition, expression of inflammatory markers, as well as quantitative and morphological alterations of neurons and glia in transgenic mice at 3 (Tg 3M) and 6 (Tg 6M) months of age, compared to WT mice. In CA1 SR of Tg 6M mice, we found significantly more Medium and Large plaques than in CA3. The pattern of neurodegeneration and astrocytes activation was different in the two areas, indicating higher sensitivity of CA1. In the CA1 SP of Tg 6M mice, we found signs of reactive astrogliosis, such as increase of astrocytes density in SP, increase of GFAP expression in SR, and elongation of astrocytes branches. We found also common patterns of glia activation and neurodegenerative processes in CA1 and CA3 of Tg 6M mice: significant increase of total and reactive microglia density in SP and SR, increased expression of TNFα, of iNOS, and IL1β in astrocytes and increased density of neurons-astrocytes-microglia triads. In CA1 SP, we found decrease of volume and number of pyramidal neurons, paralleled by increase of apoptosis, and, consequently, shrinkage of CA1 SP. These data demonstrate that in TgCRND8 mice, the responses of neurons and glia to neurodegenerative patterns induced by Aβ plaques deposition is not uniform in the two hippocampal areas, and in CA1 pyramidal neurons, the higher sensitivity may be related to the different plaque distribution in this area. All these modifications may be at the basis of memory loss, the peculiar symptom of AD, which was demonstrated in this transgenic mouse model of Aβ deposition, even at early stages.
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http://dx.doi.org/10.3389/fnagi.2018.00372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243135PMC
November 2018

Dexpramipexole enhances hippocampal synaptic plasticity and memory in the rat.

Neuropharmacology 2018 12 3;143:306-316. Epub 2018 Oct 3.

Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Italy.

Even though pharmacological approaches able to counteract age-dependent cognitive impairment have been highly investigated, drugs improving cognition and memory are still an unmet need. It has been hypothesized that sustaining energy dynamics within the aged hippocampus can boost memory storage by sustaining synaptic functioning and long term potentiation (LTP). Dexpramipexole (DEX) is the first-in-class compound able to sustain neuronal bioenergetics by interacting with mitochondrial F1Fo-ATP synthase. In the present study, for the first time we evaluated the effects of DEX on synaptic fatigue, LTP induction, learning and memory retention. We report that DEX improved LTP maintenance in CA1 neurons of acute hippocampal slices from aged but not young rats. However, we found no evidence that DEX counteracted two classic parameters of synaptic fatigue such as fEPSP reduction or the train area during the high frequency stimulation adopted to induce LTP. Interestingly, patch-clamp recordings in rat hippocampal neurons revealed that DEX dose-dependently inhibited (IC 814 nM) the I current, a rapidly-inactivating K current that negatively regulates neuronal excitability as well as cognition and memory processes. In keeping with this, DEX counteracted both scopolamine-induced spatial memory loss in rats challenged in Morris Water Maze test and memory retention in rats undergoing Novel Object Recognition. Overall, the present study discloses the ability of DEX to boost hippocampal synaptic plasticity, learning and memory. In light of the good safety profile of DEX in humans, our findings may have a realistic translational potential to treatment of cognitive disorders.
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http://dx.doi.org/10.1016/j.neuropharm.2018.10.003DOI Listing
December 2018

The Selective Antagonism of Adenosine A Receptors Reduces the Synaptic Failure and Neuronal Death Induced by Oxygen and Glucose Deprivation in Rat CA1 Hippocampus .

Front Pharmacol 2018 24;9:399. Epub 2018 Apr 24.

Department of Neuroscience, Psychology, Drug Research and Child Health, NEUROFARBA, Section of Pharmacology and Toxicology, University of Florence, Florence, Italy.

Ischemia is a multifactorial pathology characterized by different events evolving in time. Immediately after the ischemic insult, primary brain damage is due to the massive increase of extracellular glutamate. Adenosine in the brain increases dramatically during ischemia in concentrations able to stimulate all its receptors, A, A, A, and A. Although adenosine exerts clear neuroprotective effects through A receptors during ischemia, the use of selective A receptor agonists is hampered by their undesirable peripheral side effects. So far, no evidence is available on the involvement of adenosine A receptors in cerebral ischemia. This study explored the role of adenosine A receptors on synaptic and cellular responses during oxygen and glucose deprivation (OGD) in the CA1 region of rat hippocampus . We conducted extracellular recordings of CA1 field excitatory post-synaptic potentials (fEPSPs); the extent of damage on neurons and glia was assessed by immunohistochemistry. Seven min OGD induced anoxic depolarization (AD) in all hippocampal slices tested and completely abolished fEPSPs that did not recover after return to normoxic condition. Seven minutes OGD was applied in the presence of the selective adenosine A receptor antagonists MRS1754 (500 nM) or PSB603 (50 nM), separately administered 15 min before, during and 5 min after OGD. Both antagonists were able to prevent or delay the appearance of AD and to modify synaptic responses after OGD, allowing significant recovery of neurotransmission. Adenosine A receptor antagonism also counteracted the reduction of neuronal density in CA1 stratum pyramidale, decreased apoptosis at least up to 3 h after the end of OGD, and maintained activated mTOR levels similar to those of controls, thus sparing neurons from the degenerative effects caused by the simil-ischemic conditions. Astrocytes significantly proliferated in CA1 stratum radiatum already 3 h after the end of OGD, possibly due to increased glutamate release. Areceptor antagonism significantly prevented astrocyte modifications. Both A receptor antagonists did not protect CA1 neurons from the neurodegeneration induced by glutamate application, indicating that the antagonistic effect is upstream of glutamate release. The selective antagonists of the adenosine A receptor subtype may thus represent a new class of neuroprotective drugs in ischemia.
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http://dx.doi.org/10.3389/fphar.2018.00399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928446PMC
April 2018

Alterations in the Interplay between Neurons, Astrocytes and Microglia in the Rat Dentate Gyrus in Experimental Models of Neurodegeneration.

Front Aging Neurosci 2017 11;9:296. Epub 2017 Sep 11.

Department of Health Sciences, Section of Pharmacology and Clinical Oncology, University of FlorenceFlorence, Italy.

The hippocampus is negatively affected by aging and neurodegenerative diseases leading to impaired learning and memory abilities. A diverse series of progressive modifications in the intercellular communication among neurons, astrocytes and microglia occur in the hippocampus during aging or inflammation. A detailed understanding of the neurobiological modifications that contribute to hippocampal dysfunction may reveal new targets for therapeutic intervention. The current study focussed on the interplay between neurons and astroglia in the Granule Layer (GL) and the Polymorphic Layer (PL) of the Dentate Gyrus (DG) of adult, aged and LPS-treated rats. In GL and PL of aged and LPS-treated rats, astrocytes were less numerous than in adult rats. In GL of LPS-treated rats, astrocytes acquired morphological features of reactive astrocytes, such as longer branches than was observed in adult rats. Total and activated microglia increased in the aged and LPS-treated rats, as compared to adult rats. In the GL of aged and LPS-treated rats many neurons were apoptotic. Neurons decreased significantly in GL and PL of aged but not in rats treated with LPS. In PL of aged and LPS-treated rats many damaged neurons were embraced by microglia cells and were infiltrated by branches of astrocyte, which appeared to be bisecting the cell body, forming triads. Reactive microglia had a scavenging activity of dying neurons, as shown by the presence of neuronal debris within their cytoplasm. The levels of the chemokine fractalkine (CX3CL1) increased in hippocampal homogenates of aged rats and rats treated with LPS, and CX3CL1 immunoreactivity colocalized with activated microglia cells. Here we demonstrated that in the DG of aged and LPS-treated rats, astrocytes and microglia cooperate and participate in phagocytosis/phagoptosis of apoptotic granular neurons. The differential expression/activation of astroglia and the alteration of their intercommunication may be responsible for the different susceptibility of the DG in comparison to the CA1 and CA3 hippocampal areas to neurodegeneration during aging and inflammation.
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http://dx.doi.org/10.3389/fnagi.2017.00296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601988PMC
September 2017

The neuron-astrocyte-microglia triad in CA3 after chronic cerebral hypoperfusion in the rat: Protective effect of dipyridamole.

Exp Gerontol 2017 10 9;96:46-62. Epub 2017 Jun 9.

Department of Health Sciences, Section of Pharmacology and Clinical Oncology, University of Florence, Viale Pieraccini 6, 50139 Firenze, Italy. Electronic address:

We investigated the quantitative and morphofunctional alterations of neuron-astrocyte-microglia triads in CA3 hippocampus, in comparison to CA1, after 2 Vessel Occlusion (2VO) and the protective effect of dipyridamole. We evaluated 3 experimental groups: sham-operated rats (sham, n=15), 2VO-operated rats treated with vehicle (2VO-vehicle, n=15), and 2VO-operated rats treated with dipyridamole from day 0 to day 7 (2VO-dipyridamole, n=15), 90days after 2VO. We analyzed Stratum Pyramidalis (SP), Stratum Lucidum (SL) and Stratum Radiatum (SR) of CA3. 1) ectopic neurons increased in SL and SR of 2VO-vehicle, and 2VO-dipyridamole rats; 2) apoptotic neurons increased in SP of 2VO-vehicle rats and dipyridamole reverted this effect; 3) astrocytes increased in SP, SL and SR of 2VO-vehicle and 2VO-dipyridamole rats; 4) TNF-α expression increased in astrocytes, blocked by dipyridamole, and in dendrites in SR of 2VO-vehicle rats; 5) total microglia increased in SL and SR of 2VO-vehicle and 2VO-dipyridamole rats; 6) triads increased in SR of 2VO-vehicle rats and dipyridamole reverted this effect. Microglia cooperated with astrocytes to phagocytosis of apoptotic neurons and debris, and engulfed ectopic non-fragmented neurons in SL of 2VO-vehicle and 2VO-dipyridamole rats, through a new mechanism called phagoptosis. CA3 showed a better adaptive capacity than CA1 to the ischemic insult, possibly due to the different behaviour of astrocytes and microglial cells. Dipyridamole had neuroprotective effects.
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http://dx.doi.org/10.1016/j.exger.2017.06.006DOI Listing
October 2017

The neuron-astrocyte-microglia triad involvement in neuroinflammaging mechanisms in the CA3 hippocampus of memory-impaired aged rats.

Exp Gerontol 2016 10 25;83:71-88. Epub 2016 Jul 25.

Department of Health Sciences, Section of Pharmacology and Clinical Oncology, University of Florence, Viale Pieraccini 6, 50139 Firenze, Italy. Electronic address:

We examined the effects of inflammaging on memory encoding, and qualitative and quantitative modifications on proinflammatory proteins, apoptosis, neurodegeneration and morphological changes of neuron-astrocyte-microglia triads in CA3 Stratum Pyramidale (SP), Stratum Lucidum (SL) and Stratum Radiatum (SR) of young (3months) and aged rats (20months). Aged rats showed short-term memory impairments in the inhibitory avoidance task, increased expression of iNOS and activation of p38MAPK in SP, increase of apoptotic neurons in SP and of ectopic neurons in SL, and decrease of CA3 pyramidal neurons. The number of astrocytes and their branches length decreased in the three CA3 subregions of aged rats, with morphological signs of clasmatodendrosis. Total and activated microglia increased in the three CA3 subregions of aged rats. In aged rats CA3, astrocytes surrounded ectopic degenerating neurons forming "micro scars" around them. Astrocyte branches infiltrated the neuronal cell body, and, together with activated microglia formed "triads". In the triads, significantly more numerous in CA3 SL and SR of aged rats, astrocytes and microglia cooperated in fragmentation and phagocytosis of ectopic neurons. Inflammaging-induced modifications of astrocytes and microglia in CA3 of aged rats may help clearing neuronal debris derived from low-grade inflammation and apoptosis. These events might be common mechanisms underlying many neurodegenerative processes. The frequency to which they appear might depend upon, or might be the cause of, the burden and severity of neurodegeneration. Targeting the triads may represent a therapeutic strategy which may control inflammatory processes and spread of further cellular damage to neighboring cells.
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http://dx.doi.org/10.1016/j.exger.2016.07.011DOI Listing
October 2016

Clasmatodendrosis and β-amyloidosis in aging hippocampus.

FASEB J 2016 Apr 31;30(4):1480-91. Epub 2015 Dec 31.

*Department of Chemistry "Ugo Schiff," Department of Health Sciences, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," Department of Experimental and Clinical Medicine, and Department of Biology, University of Florence, Florence, Italy; National Institute of Optics, National Research Council (CNR), Florence, Italy; and Department of Psychology, The Ohio State University, Columbus, Ohio, USA

Alterations of the tightly interwoven neuron/astrocyte interactions are frequent traits of aging, but also favor neurodegenerative diseases, such as Alzheimer disease (AD). These alterations reflect impairments of the innate responses to inflammation-related processes, such as β-amyloid (Aβ) burdening. Multidisciplinary studies, spanning from the tissue to the molecular level, are needed to assess how neuron/astrocyte interactions are influenced by aging. Our study addressed this requirement by joining fluorescence-lifetime imaging microscopy/phasor multiphoton analysis with confocal microscopy, implemented with a novel method to separate spectrally overlapped immunofluorescence and Aβ autofluorescence. By comparing data from young control rats, chronically inflamed rats, and old rats, we identified age-specific alterations of neuron/astrocyte interactions in the hippocampus. We found a correlation between Aβ aggregation (+300 and +800% of aggregated Aβ peptide in chronically inflamed and oldvs.control rats, respectively) and fragmentation (clasmatodendrosis) of astrocyte projections (APJs) (+250 and +1300% of APJ fragments in chronically inflamed and oldvs.control rats, respectively). Clasmatodendrosis, in aged rats, associates with impairment of astrocyte-mediated Aβ clearance (-45% of Aβ deposits on APJs, and +33% of Aβ deposits on neurons in oldvs.chronically inflamed rats). Furthermore, APJ fragments colocalize with Aβ deposits and are involved in novel Aβ-mediated adhesions between neurons. These data define the effects of Aβ deposition on astrocyte/neuron interactions as a key topic in AD biology.-Mercatelli, R., Lana, D., Bucciantini, M., Giovannini, M. G., Cerbai, F., Quercioli, F., Zecchi-Orlandini, S., Delfino, G., Wenk, G. L., Nos, D. Clasmatodendrosis and β-amyloidosis in aging hippocampus.
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http://dx.doi.org/10.1096/fj.15-275503DOI Listing
April 2016

The integrated role of ACh, ERK and mTOR in the mechanisms of hippocampal inhibitory avoidance memory.

Neurobiol Learn Mem 2015 Mar 13;119:18-33. Epub 2015 Jan 13.

Department of Neuroscience, Psychology, Drug Research and Child Health, Division of Pharmacology and Toxicology, University of Florence, Viale Pieraccini 6, 50139 Firenze, Italy. Electronic address:

The purpose of this review is to summarize the present knowledge on the interplay among the cholinergic system, Extracellular signal-Regulated Kinase (ERK) and Mammalian Target of Rapamycin (mTOR) pathways in the development of short and long term memories during the acquisition and recall of the step-down inhibitory avoidance in the hippocampus. The step-down inhibitory avoidance is a form of associative learning that is acquired in a relatively simple one-trial test through several sensorial inputs. Inhibitory avoidance depends on the integrated activity of hippocampal CA1 and other brain areas. Recall can be performed at different times after acquisition, thus allowing for the study of both short and long term memory. Among the many neurotransmitter systems involved, the cholinergic neurons that originate in the basal forebrain and project to the hippocampus are of crucial importance in inhibitory avoidance processes. Acetylcholine released from cholinergic fibers during acquisition and/or recall of behavioural tasks activates muscarinic and nicotinic acetylcholine receptors and brings about a long-lasting potentiation of the postsynaptic membrane followed by downstream activation of intracellular pathway (ERK, among others) that create conditions favourable for neuronal plasticity. ERK appears to be salient not only in long term memory, but also in the molecular mechanisms underlying short term memory formation in the hippocampus. Since ERK can function as a biochemical coincidence detector in response to extracellular signals in neurons, the activation of ERK-dependent downstream effectors is determined, in part, by the duration of ERK phosphorylation itself. Long term memories require protein synthesis, that in the synapto-dendritic compartment represents a direct mechanism that can produce rapid changes in protein content in response to synaptic activity. mTOR in the brain regulates protein translation in response to neuronal activity, thereby modulating synaptic plasticity and long term memory formation. Some studies demonstrate a complex interplay among the cholinergic system, ERK and mTOR. It has been shown that co-activation of muscarinic acetylcholine receptors and β-adrenergic receptors facilitates the conversion of short term to long term synaptic plasticity through an ERK- and mTOR-dependent mechanism which requires translation initiation. It seems therefore that the complex interplay among the cholinergic system, ERK and mTOR is crucial in the development of new inhibitory avoidance memories in the hippocampus.
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http://dx.doi.org/10.1016/j.nlm.2014.12.014DOI Listing
March 2015

The selective antagonism of P2X7 and P2Y1 receptors prevents synaptic failure and affects cell proliferation induced by oxygen and glucose deprivation in rat dentate gyrus.

PLoS One 2014 19;9(12):e115273. Epub 2014 Dec 19.

Dept. of Neuroscience, Psychology, Drug Research and Child Health, NEUROFARBA, Division of Pharmacology and Toxicology, University of Florence, Florence, Italy.

Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X7 and P2Y1 receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y1 receptor) and BBG (selective antagonist of P2X7 receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2'-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU+ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU+ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y1 antagonist reduced the number of BrdU+ cells at this time. The data demonstrate that P2X7 and P2Y1 activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y1 receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115273PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272279PMC
December 2015

The neuron-astrocyte-microglia triad in a rat model of chronic cerebral hypoperfusion: protective effect of dipyridamole.

Front Aging Neurosci 2014 27;6:322. Epub 2014 Nov 27.

Section of Pharmacology and Clinical Oncology, Department of Health Sciences, University of Florence Florence, Italy.

Chronic cerebral hypoperfusion during aging may cause progressive neurodegeneration as ischemic conditions persist. Proper functioning of the interplay between neurons and glia is fundamental for the functional organization of the brain. The aim of our research was to study the pathophysiological mechanisms, and particularly the derangement of the interplay between neurons and astrocytes-microglia with the formation of "triads," in a model of chronic cerebral hypoperfusion induced by the two-vessel occlusion (2VO) in adult Wistar rats (n = 15). The protective effect of dipyridamole given during the early phases after 2VO (4 mg/kg/day i.v., the first 7 days after 2VO) was verified (n = 15). Sham-operated rats (n = 15) were used as controls. Immunofluorescent triple staining of neurons (NeuN), astrocytes (GFAP), and microglia (IBA1) was performed 90 days after 2VO. We found significantly higher amount of "ectopic" neurons, neuronal debris and apoptotic neurons in CA1 Str. Radiatum and Str. Pyramidale of 2VO rats. In CA1 Str. Radiatum of 2VO rats the amount of astrocytes (cells/mm(2)) did not increase. In some instances several astrocytes surrounded ectopic neurons and formed a "micro scar" around them. Astrocyte branches could infiltrate the cell body of ectopic neurons, and, together with activated microglia cells formed the "triads." In the triad, significantly more numerous in CA1 Str. Radiatum of 2VO than in sham rats, astrocytes and microglia cooperated in the phagocytosis of ectopic neurons. These events might be common mechanisms underlying many neurodegenerative processes. The frequency to which they appear might depend upon, or might be the cause of, the burden and severity of neurodegeneration. Dypiridamole significantly reverted all the above described events. The protective effect of chronic administration of dipyridamole might be a consequence of its vasodilatory, antioxidant and anti-inflammatory role during the early phases after 2VO.
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http://dx.doi.org/10.3389/fnagi.2014.00322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245920PMC
December 2014

Hippocampal long term memory: effect of the cholinergic system on local protein synthesis.

Neurobiol Learn Mem 2013 Nov 27;106:246-57. Epub 2013 Sep 27.

Department of Health Sciences, Section of Pharmacology and Clinical Oncology, University of Florence, Viale Pieraccini 6, 50139 Firenze, Italy.

The present study was aimed at establishing a link between the cholinergic system and the pathway of mTOR and its downstream effector p70S6K, likely actors in long term memory encoding. We performed in vivo behavioral experiments using the step down inhibitory avoidance test (IA) in adult Wistar rats to evaluate memory formation under different conditions, and immunohistochemistry on hippocampal slices to evaluate the level and the time-course of mTOR and p70S6K activation. We also examined the effect of RAPA, inhibitor of mTORC1 formation, and of the acetylcholine (ACh) muscarinic receptor antagonist scopolamine (SCOP) or ACh nicotinic receptor antagonist mecamylamine (MECA) on short and long term memory formation and on the functionality of the mTOR pathway. Acquisition test was performed 30 min after i.c.v. injection of RAPA, a time sufficient for the drug to diffuse to CA1 pyramidal neurons, as demonstrated by MALDI-TOF-TOF imaging. Recall test was performed 1 h, 4 h or 24 h after acquisition. To confirm our results we performed in vitro experiments on live hippocampal slices: we evaluated whether stimulation of the cholinergic system with the cholinergic receptor agonist carbachol (CCh) activated the mTOR pathway and whether the administration of the above-mentioned antagonists together with CCh could revert this activation. We found that (1) mTOR and p70S6K activation in the hippocampus were involved in long term memory formation; (2) RAPA administration caused inhibition of mTOR activation at 1 h and 4 h and of p70S6K activation at 4 h, and long term memory impairment at 24 h after acquisition; (3) scopolamine treatment caused short but not long term memory impairment with an early increase of mTOR/p70S6K activation at 1 h followed by stabilization at longer times; (4) mecamylamine plus scopolamine treatment caused short term memory impairment at 1 h and 4 h and reduced the scopolamine-induced increase of mTOR/p70S6K activation at 1 h and 4 h; (5) mecamylamine plus scopolamine treatment did not impair long term memory formation; (6) in vitro treatment with carbachol activated mTOR and p70S6K and this effect was blocked by scopolamine and mecamylamine. Taken together our data reinforce the idea that distinct molecular mechanisms are at the basis of the two different forms of memory and are in accordance with data presented by other groups that there exist molecular mechanisms that underlie short term memory, others that underlie long term memories, but some mechanisms are involved in both.
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http://dx.doi.org/10.1016/j.nlm.2013.09.013DOI Listing
November 2013

The neuron-astrocyte-microglia triad in normal brain ageing and in a model of neuroinflammation in the rat hippocampus.

PLoS One 2012 18;7(9):e45250. Epub 2012 Sep 18.

Department of Pharmacology, University of Florence, Florence, Italy.

Ageing is accompanied by a decline in cognitive functions; along with a variety of neurobiological changes. The association between inflammation and ageing is based on complex molecular and cellular changes that we are only just beginning to understand. The hippocampus is one of the structures more closely related to electrophysiological, structural and morphological changes during ageing. In the present study we examined the effect of normal ageing and LPS-induced inflammation on astroglia-neuron interaction in the rat hippocampus of adult, normal aged and LPS-treated adult rats. Astrocytes were smaller, with thicker and shorter branches and less numerous in CA1 Str. radiatum of aged rats in comparison to adult and LPS-treated rats. Astrocyte branches infiltrated apoptotic neurons of aged and LPS-treated rats. Cellular debris, which were more numerous in CA1 of aged and LPS-treated rats, could be found apposed to astrocytes processes and were phagocytated by reactive microglia. Reactive microglia were present in the CA1 Str. Radiatum, often in association with apoptotic cells. Significant differences were found in the fraction of reactive microglia which was 40% of total in adult, 33% in aged and 50% in LPS-treated rats. Fractalkine (CX3CL1) increased significantly in hippocampus homogenates of aged and LPS-treated rats. The number of CA1 neurons decreased in aged rats. In the hippocampus of aged and LPS-treated rats astrocytes and microglia may help clearing apoptotic cellular debris possibly through CX3CL1 signalling. Our results indicate that astrocytes and microglia in the hippocampus of aged and LPS-infused rats possibly participate in the clearance of cellular debris associated with programmed cell death. The actions of astrocytes may represent either protective mechanisms to control inflammatory processes and the spread of further cellular damage to neighboring tissue, or they may contribute to neuronal damage in pathological conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045250PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445467PMC
February 2013