Roberto Avola

The hippocampus in spontaneously hypertensive rats: an animal model of vascular dementia?

Hypertension is a main risk factor for cerebrovascular disease, including vascular dementia. The present study was designed to evaluate if hypertension-dependent changes of the hippocampus of spontaneously hypertensive rats (SHR) of different ages were related with those occurring in vascular dementia. The hippocampus was chosen as the brain area involved in learning and memory. Systolic pressure was slightly increased in 2-month-old SHR in comparison with age-matched normotensive Wistar-Kyoto (WKY) rats and augmented progressively with age in SHR. No microanatomical changes were observed in the hippocampus of SHR of 2 months in comparison with age-matched WKY rats. A limited decrease of white matter volume was observed in 4-month-old SHR. In SHR of 6 months, a reduction of grey matter volume both in the CA1 subfield and in the dentate gyrus occurred. Evaluation of phosphorylated 200-kDa neurofilament immunoreactivity revealed a decreased immune reaction area in the CA1 subfield of 6-month-old SHR compared to age-matched WKY rats and no changes in the expression and localization of the dendritic marker microtubule associated protein (MAP)-2. In 6-month-old SHR, an increase of glial fibrillary acidic protein (GFAP)-expression was found by Western blot analysis. Immunohistochemistry revealed an increase in number (hyperplasia), but not in size of astrocytes. These findings indicate the occurrence of cytoskeletal breakdown and astroglial changes primarily in the CA1 subfield of the hippocampus of SHR of 6 months. The occurrence in the hippocampus of SHR of regressive changes and astroglial reaction similar to those occurring in neurodegenerative disorders with cognitive impairment suggests that they represent an animal model of vascular dementia.

Propofol attenuates peroxynitrite-mediated DNA damage and apoptosis in cultured astrocytes: an alternative protective mechanism.

Background:The concentration of peroxynitrite in the brain increases after central nervous system injuries. The authors hypothesized that propofol, because of its particular chemical structure, mitigates the effects of peroxynitrite-mediated oxidative stress and apoptosis by the induction of heme oxygenase (HO)-1 in primary cultured astroglial cells. Methods:Primary cultured astroglial cells were incubated for 18 h with a known peroxynitrite donor (3 mm SIN-1) in the presence or absence of propofol (40 &mgr;m, 80 &mgr;m, 160 &mgr;m, and 1 mm). The protective effects of propofol were evaluated by 3(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide cytotoxicity assay, lactic dehydrogenase release, DNA ladderization by Comet assay, and caspase-3 activation by Western blot analysis. Results:Appropriate propofol concentrations (ranging from 40 &mgr;m to 1 mm) significantly increased HO-1 expression and attenuated SIN-1–mediated DNA ladderization and caspase-3 activation. The protective effects of propofol were mitigated by the addition of tin mesoporphyrin, a potent inhibitor of HO activity. The addition of a specific synthetic inhibitor of nuclear factor &kgr;B abolished propofol-mediated HO-1 induction, suggesting a possible role of this nuclear transcriptional factor in our experimental conditions. Conclusions:The antioxidant properties of propofol can be partially attributed to its scavenging effect on peroxynitrite as well as to its ability to increase HO-1 expression at higher concentrations, a property that might be relevant to neuroprotection during anesthesia.

Neuroendocrine-immune (NEI) circuitry from neuron-glial interactions to function: Focus on gender and HPA-HPG interactions on early programming of the NEI system.

Bidirectional communication between the neuroendocrine and immune systems during ontogeny plays a pivotal role in programming the development of neuroendocrine and immune responses in adult life. Signals generated by the hypothalamic–pituitary–gonadal axis (i.e. luteinizing hormone‐releasing hormone, LHRH, and sex steroids), and by the hypothalamic–pituitary–adrenocortical axis (glucocorticoids (GC)), are major players coordinating the development of immune system function. Conversely, products generated by immune system activation exert a powerful and long‐lasting regulation on neuroendocrine axes activity. The neuroendocrine–immune system is very sensitive to preperinatal experiences, including hormonal manipulations and immune challenges, which may influence the future predisposition to several disease entities. We review our work on the ongoing mutual regulation of neuroendocrine and immune cell activities, both at a cellular and molecular level. In the central nervous system, one chief compartment is represented by the astroglial cell and its mediators. Hence, neuron–glial signalling cascades dictate major changes in response to hormonal manipulations and pro‐inflammatory triggers. The interplay between LHRH, sex steroids, GC and pro‐inflammatory mediators in some physiological and pathological states, together with the potential clinical implications of these findings, are summarized. The overall study highlights the plasticity of this intersystem cross‐talk for pharmacological targeting with drugs acting at the neuroendocrine–immune interface.

Increased Expression of Glial Fibrillary Acidic Protein in the Brain of Spontaneously Hypertensive Rats

Astrogliosis, consisting in astroglial proliferation and increased expression of the specific cytoskeletal protein glial fibrillary acid protein (GFAP) is common in several situations of brain damage. Arterial hypertension, which induces cerebrovascular changes, can cause also brain damage, neurodegeneration and dementia (vascular dementia). This study was designed to assess astroglial reaction in different brain areas (frontal cortex, occipital cortex, hippocampus and striatum) of spontaneously hypertensive rats (SHR) in the pre‐hypertensive phase (2 months of age), in the developing phase of hypertension (4 months of age) and in established hypertension (6 months of age). SHR were compared to age‐matched normotensive Wistar‐Kyoto (WKY) rats. Analysis included reverse transcription‐polymerase chain reaction (RT‐PCR) of GFAP mRNA, GFAP immunochemistry (Western blot analysis) and immunohistochemistry. A significant increase of GFAP mRNA and an increase of GFAP immunoreactivity were noticeable in different brain areas of SHR compared to normotensive WKY rats at 6, but not at 2 or 4 months of age. Immunohistochemistry revealed a numerical augmentation (hyperplasia) and an increase in size (hypertrophy) of GFAP‐immunoreactive astrocytes in frontal cortex, occipital cortex and striatum of SHR. In the hippocampus of SHR only a numerical increase of GFAP‐immunoreactive astrocytes was found. These finding demonstrating the occurrence of astrogliosis in the brain of SHR with established hypertension suggest that hypertension induces a condition of brain suffering enough to increase biosynthesis and expression of GFAP similarly as reported in several neurodegenerative disorders and in brain ischemia.

Basic fibroblast growth factor (bFGF) acts on both neurons and glia to mediate the neurotrophic effects of astrocytes on LHRH neurons in culture

Luteinizing hormone‐releasing hormone (LHRH) neurons play a pivotal role in the neuroendocrine control of mammalian reproduction. Astrocytes were shown to be involved in the regulation of LHRH neuronal function, but little is known about the contribution of astroglial‐derived factors in the regulation of LHRH neuron development. In order to gain insight into the mechanisms regulating the development of these cells, at morphological and biochemical levels we characterized the neurotrophic effects exerted by young astrocytes (maintained in culture for 8 days in vitro) and old astrocytes (maintained 26 days) on the differentiation, proliferation, and phenotypic expression of immortalized hypothalamic LHRH (GT1‐1) neurons in vitro. Culturing GT1‐1 cells in the presence of young glia for different time intervals caused a marked acceleration in the acquisition of their neuronal phenotype. At all times examined, GT1‐1 cells cocultured with young glia exhibited a significantly greater extension of processes/cell, larger number of processes/cell and greater surface area of growth cones than GT1‐1 cells grown over nonglial adhesive substrates (polylysine). By contrast, when GT1‐1 neurons were cocultured with old glia, the length of neuronal processes and the growth cone surface area were significantly lower than in control GT1‐1 neurons cultured in the absence of glia. At 3 days in vitro (DIV), GT1‐1 neurons cocultured with young glia exhibited a 50% lower incorporation of [3H]thymidine than GT1‐1 neurons cultured without glia. By contrast, in the presence of old glia [3H]thymidine incorporation was significantly higher in cells cocultured with glia than in GT1‐1 neurons cultured alone. Localization of the proliferating cells by dual immunohistochemical staining revealed that the incorporation of bromodeoxiuridine (BrdU) was restricted to nuclei of GT1‐1 neurons when these were cocultured with young glia, but associated with both neurons and astrocytes in the presence of old glia. At the functional level, coculture of GT1‐1 neurons with young glia increased the spontaneous release of LHRH as compared to GT1‐1 neurons grown in the absence of glia. By contrast, in the presence of old glia LHRH release in the medium was significantly lower than in controls. Conditioned medium of young glia (ACM‐Y) induced significant neurotrophic and functional effects on GT1‐1 cells, but these effects were 50% less potent than the coculture itself. Heat denaturation of ACM‐Y totally abolished its neurotrophic and functional properties, indicating that they involved a peptide factor. Suppression of bFGF activity in ACM‐Y reduced its neurotrophic activity by approximately 40%, but did not affect its LHRH release‐promoting effects. By contrast, neutralization of endogenous bFGF activity in GT1‐1 neurons cocultured with young glia counteracted both neurotrophic and functional effects of young glia. Treatment of old glia with bFGF rescued its neurotrophic and functional effects on GT1‐1 cells. Moreover, the ACM of aged bFGF‐treated old glia was the most powerful neurotrophic stimulus for GT1‐1 neurons. These results suggest that: 1) soluble peptidic factors, including bFGF, and mechanism(s) requiring coculture are responsible for the highly potent neurotrophic and functional effects of young glia; 2) the inhibitory effects of old glia on neurite outgrowth and LHRH release are mediated in part by soluble inhibitory molecules and in part by factors requiring coculture with old glia; 3) old glia may revert to a growth‐supporting state when treated with bFGF and this functional shift involves a diffusible molecule with potent neurotrophic and functional effects on immortalized LHRH neurons. Synapse 36:233–253, 2000.

The dopaminergic system in hypertension.

Dopamine exerts cardiovascular and renal actions mediated through interaction with specific dopamine receptors. Dopamine receptors are cell surface receptors coupled to G-proteins and classified into two main super families based on biochemical, pharmacological and molecular characteristics. The dopamine D1-like receptor super family includes D1 and D5 receptors, known also in rodents as D1A and D1B sites. These receptors are linked to stimulation of adenylate cyclase. The dopamine D2-like receptor super family includes D2, D3 and D4 receptors. These receptors are linked to inhibition of adenylate cylase or not related with this enzyme activity. They also interfere with opening of Ca+2 channels and are linked to stimulation of K+ receptors. Dopamine receptor subtypes are expressed in brain as well as in extracerebral structures such as the heart, blood vessels, carotid body, kidney, adrenal gland, parathyroid gland and gastrointestinal tract. In the kidney, which represents the peripheral organ where dopamine receptors were more extensively investigated, dopamine receptors are involved in regulation of hemodynamic, electrolyte and water transport, as well as renin secretion. Hypertension-related dopamine receptor changes were also investigated primarily in the kidney. Defective renal dopamine production and/or dopamine receptor function have been reported in human primary hypertension as well as in genetic models of animal hypertension. There may be a primary defect in D1-like receptors and an altered signalling system in the proximal tubules that lead to reduced dopamine-mediated effects on renal sodium excretion in hypertension. Studies on the influence of hypertension on dopamine D2-like receptors are sparse Disruption of either D1A or D3 receptors at the gene level causes hypertension in mice. Using peripheral blood lymphocytes as possible markers of the status of dopamine receptors in essential hypertension, no changes of dopamine D1-like receptors were noticeable, whereas an increase of dopamine D2-like receptors likely representing an up-regulation mechanism was reported. Available information collectively indicates an involvement of peripheral dopaminergic system in hypertension consisting either in impaired receptor transduction mechanisms and/or in receptor loss. A better knowledge of molecular bases of these changes may contribute to the development of specific therapeutic approaches in the future.

Glial fibrillary acidic protein and vimentin expression is regulated by glucocorticoids and neurotrophic factors in primary rat astroglial cultures.

The neurotrophic factors epidermal growth factor (EGF), basic fibroblast growth factor, (bFGF), insulin‐like growth factor I (IGF‐I) and insulin (INS) regulate neural and astroglial cell functions. Glucocorticoids may influence the metabolism of astroglial compartment and are key hormones in neurodegenerative events. This study was designed to assess the interactions between growth factors and dexamethasone (DEX) on cytoskeletal proteins (GFAP and vimentin) expression in 25 days in vitro (DIV) astrocyte cultures. An increase in GFAP and vimentin expression was observed after 12 h pretreatment with bFGF and subsequent treatment for 60 h with DEX. GFAP immunoreactivity was decreased after 24 h progression growth factors (EGF, IGF‐I and INS) addition, when compared to control 36 h DEX and bFGF‐pretreated cultures for the last 12 h. Vimentin immunoreactivity was decreased after 12 h bFGF pretreatment and subsequent 60 h DEX addition in astrocyte cultures compared to 12 h bFGF‐pretreated ones. Pretreatment for 36 h with DEX plus bFGF in the last 12 h and subsequent treatment for 24 h with DMEM (Dulbeccos modified Eagle medium; DMEM) + BSA (bovine serum albumine) (harvesting), or with progression growth factors (EGF, IGF‐I or INS) alone or two of them together, stimulated GFAP expression, compared to untreated controls. Immunochemical analysis of the mitogen‐activated protein kinase ERK2 suggests an involvement of this enzyme in the control of GFAP expression. The above findings support the view of an interactive and complex dialogue between growth factors and glucocorticoids during astroglial cell proliferation and maturation in culture. This may have implications in therapeutic approach of neurologic disorders associated with astrogliosis, including cerebrovascular disease.

Nuclear factor‐κb activation is associated with glutamate‐evoked tissue transglutaminase up‐regulation in primary astrocyte cultures

We have previously demonstrated that alterations of cell redox state, evoked by glutamate, are associated with tissue transglutaminase increases in primary astrocyte cultures. Furthermore, glutamate exposure activated the nuclear factor (NF)‐κB pathway, and its effects were significantly reduced by antioxidants. Here, we investigated the possible involvement of activated NF‐κB pathway in glutamate‐evoked tissue transglutaminase up‐regulation in primary astrocytes. The presence of DNA binding activity by NF‐κB in nuclear extracts of astrocytes, treated for 24 hr with glutamate (500 μM) or untreated, was assessed by EMSA, using an oligonucleotide probe containing the NF‐κB consensus sequence present in the tissue transglutaminase promoter. Supershifting with monoclonal antibodies revealed that activated NF‐κB dimer complexes were composed of p50 and p65 subunits. Interestingly, the specific NF‐κB inhibitor SN50 (but not its inactive analogue SN50M), when added to cell cultures 30 min prior to glutamate treatment, was able gradually to reduce glutamate‐induced NF‐κB activation. Western blot analysis confirmed the reduction of the p50 amount in nuclear extracts. Notably, the preincubation with SN50 also diminished glutamate‐increased tissue transglutaminase expression, as showed by both RT‐PCR and Western blotting. Competition experiments, carried out with an excess of a probe containing the NF‐κB consensus sequence present in the κ‐light‐chain promoter, demonstrated a preferential binding of the tissue transglutaminase specific NF‐κB probe in the nuclear extracts of glutamate‐treated astrocytes compared with untreated astrocytes. These preliminary data suggest that NF‐κB activation, which has been demonstrated to be involved in astrocyte response to glutamate, could also be associated with the molecular pathway leading to glutamate‐evoked tissue transglutaminase up‐regulation.

Effect of growth factors on nuclear and mitochondrial ADP-ribosylation processes during astroglial cell development and aging in culture

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and insulin (INS) are powerful mitogens and may regulate gene expression in cultured astrocytes by ADP-ribosylation process. Nuclear poly-ADP ribose polymerase (PARP) and mitochondrial monoADP-ribosyltransferase (ADPRT) are the key enzymes involved in poly-ADP-ribosylation and mono ADP-ribosylation, respectively. In this investigation the effect of EGF, bFGF, IGF-I or INS on nuclear PARP and mitochondrial ADPRT activities were assessed in nuclei and mitochondria purified from developing (30 DIV) or aging (90 and 190 DIV) primary rat astrocyte cultures. A marked increase of PARP activity in bFGF or IGF-I treated astroglial cell cultures at 30 DIV was found. Nuclear PARP and mitochondrial ADPRT activities were greatly stimulated by treatment with EGF or INS alone or together in astrocyte cultures at 30 DIV. Nuclear PARP and mitochondrial ADPRT activities showed a more remarkable increase in control untreated astrocyte cultures at 190 DIV than at 90 DIV. These findings suggest that ADP-ribosylation process is involved in DNA damage and repair during cell differentiation and aging in culture. Twelve hours treatment with EGF, INS or bFGF significantly stimulated nuclear PARP and mitochondrial ADPRT activities in 190 DIV aging astrocyte cultures. The above results indicate that EGF, INS and bFGF may play a crucial role in the post-translational modification of chromosomal proteins including ADP-ribosylation process in in vitro models. This suggests that growth factors regulate genomic stability in glial cells during development and maturation, stimulating nuclear and mitochondrial ADP-ribosylation processes in developing or aging astrocyte cultures.

Cross-talk between luteinizing hormone-releasing hormone (LHRH) neurons and astroglial cells: developing glia release factors that accelerate neuronal differentiation and stimulate LHRH release from GT1-1 neuronal cell line and LHRH neurons induce astroglia proliferation

Recent evidences indicate that the bidirectional flow of informations governing neuron-astrocyte interactions plays a crucial role during the development and in the adult brain. In the present study, we have used the immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neuronal cell line (GT1-1, subclone) to investigate LHRH-astroglial cell interactions and addressed the following questions: (a) does the astroglial cell compartment influence GT1-1 neuron morphology, LHRH secretion and/or proliferation?; (b) does the bidirectional flow of informational molecules released during neuron-astroglia interactions influence one or both cell compartments?; (c) are receptor-mediated cell-cell interactions between neurons and astroglia involved in such crosstalk? In this experimental design, GT1-1 neuronal cells were grown either: (1) in Dulbeccos modified eagles medium (DMEM); (2) in the presence of conditioned medium from astroglial cell (ACM) cultures at different stages of glia differentiation and maturationin vitro; 93) in the presence of astroglial cells, in co-cultures or mixed-cultures; and (4) in the absence or the presence of antibodies (Abs) for neural cell adhesion molecule, (N-CAM) receptor. This work shows that during its maturation and differentiationin vitro (8–40 days, DIV), astroglial cells in primary culture release factors able to markedly influence GT1-1 cell morphology and accelerate LHRH cell secretory potential, with a potency depending on both the ‘age’ of astroglia and the degree of GT1-1 neuron differentiationin vitro. Regional differences in glial-derived factors that promote LHRH neuronal differentiation and secretion were observed, with hypothalamic astroglia being the most potent neurotrophic stimulus. Such effects were specific for astroglia conditioned medium (CM), since oligodendrocyte CM was without effect. Boiling of the ACM for 10 min completely abolished stimulatory activity on neuronal cells. When immature astroglial cells (12 DIV) were co-cultured with GT1-1 neurons, LHRH release increased by about 2- to 3-fold over basal levels and GT1-1 neuron proliferation was doubled. Astroglial cells responded to GT1-1 neuronal signals with an almost doubling of the [3H]-thymidine incorporation and DNA synthesis. Extensive neurite outgrowth and establishment of cell-cell contacts between the two cell compartments were observed in the mixed culture preparation, accompanied by a marked stimulatory effect on both cell proliferation and LHRH secretion. Addition of N-CAM-Ab in the GT1-1-astroglial cell mixed cultures resulted in a dramatic disruption of GT1-1-astroglia morphology and a 95% suppression of the stimulatory effect on both cell proliferation and LHRH release suggesting the local adhesive mechanisms are importantly involved in the crosstalk between GT1-1 neurons and astroglial cellsin vitro. This work shows for the first time the presence of a bidirectional interaction between the LHRH neurons and astroglial cells and suggest a potential interplay between the two compartments in the regulation of LHRH neuronal physiology.