Metoda Lipnik-Štangelj

*University of Ljubljana, Faculty of Medicine, Department of Pharmacology and Experimental Toxicology Slovenia* 

#### **1. Introduction**

606 Pharmacology

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Oancea, M., Mani, A., Hussein, MA., Almasan, A. (2004) Apoptosis of multiple myeloma*.* 

Otsuki, T., Yamada, O., Yata, K., Sakaguchi, H., Kurebayashi, J., et al. (2000) Expression and

Otsuki, T., Yata, K., Sakaguchi, H., Uno, M., Fujii, T., et al. (2002) IL-10 in myeloma cells.

Papadaki, H., Kyriakou, D., Foudoulakis, A., Markidoum F., Alexandrakis, M. (1997) Serum

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Shinwari, Z., Manogaran, PS., Alrokayan, SA., Al-Hussein, KA., Aboussekhra, A. (2007)

Stokke, T., Holte, H., Smedshammer, L., Smeland, EB., Kaalhus, O., et al. (1998) Proliferation

Tsujimoto, Y. & Shimizu, S. (2007) Role of mitochondrial membrane permeability transition

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85, 2521–27.

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a preliminary report. *Pancreas* 36, 15-23.

42.

123-132.

proliferation factor but not a differentiation factor for human myeloma cells*. Blood* 

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levels of interleukin-6 receptor in multiple myeloma as indicator of disease activity.

pancreatic cell line MIA PaCa2 proliferation byHA-But, a hyaluronic butyric ester:

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and apoptosis in malignant and normal cells in B-cell non-Hodgkin's lymphomas.

Ethanol consumption has for a long time been associated with brain damage. Experimental studies and necropsy examinations of chronic alcoholics have shown a variety of structural and functional alterations in the neurons as well as in the glial cells. Such alterations are seen also in children with the alcoholic foetal syndrome. Ethanol is known to be a teratogen. Its abusage can result as dysfunction of the central nerve system (CNS), growth deficiency and facial malformation in the fetus, and behavioural, learning, sensory and motor disabilities (Barret et al., 1996; González & Salido, 2009; Šarc & Lipnik-Štangelj, 2009a). Chronic ethanol consumption in the adult is also intimately associated with brain atrophy. Accumulating evidence indicates that ethanol-induced neurobehavioral dysfunctions may be related to disruptions in the patterns of neuronal and glial developments such as depression of neurogenesis, aberrant migration of neurons and alterations in late gliogenesis and neurogenesis. These changes can further reduce the populations of cortical neurons and glial cells, trigger the biochemical alterations in glial cells and deleterious consequences for neuronal-glial interactions, and eventually lead to damage or apoptosis of these cells (González & Salido, 2009; Šarc & Lipnik-Štangelj, 2009b; Sofroniew & Vinters, 2010).

As the most abundant type of glial cells in the brain, astrocytes provide metabolic and trophic support to neurons, modulate synaptic activities and have a strong capacity to scavenge oxidants and suppress cellular apoptosis. However, when the capacity of cells to eliminate the oxidants is overwhelmed, overproduction of reactive oxygen species (ROS) can cause morphological and functional alterations in the cells, including cellular Ca2+ homeostasis and some active molecules tightly associated with neuronal activity (Allansson et al., 2001; Halassa et al., 2007; Sofroniew & Vinters, 2010).

Although astrocytes are more resistant than neurons to the oxidative and neurotoxic stresses and to the chemical and toxic damages in the surrounding environment, any impairment of astrocytes can dramatically affect neuronal functions. The ethanol-induced detrimental alterations of astrocytes would lead to perturbances in neuron–astroglia interactions and developmental defects of the brain (González & Salido, 2009; Šarc & Lipnik-Štangelj, 2009b). Given this important role of astroglial cells in neuronal functioning, they have become a significant object of toxicological evaluation.

Ethanol Toxicity in the Brain: Alteration of Astroglial Cell Function 609

operate within astrocytes (Araque et al., 2001; Fellin et al., 2004; Gonzales et al., 2006a;

Released messengers, in turn, activate Ca2+ entry or Ca2+ release from intracellular stores by acting on ionotropic and metabotropic receptors, respectively. By this way, ATP and glutamate are the major active neurotransitters involved in the cell-to-cell communication of Ca2+ signals in astrocytes and other cell types in the CNS (Bowser & Khakh, 2007; Percea & Araque, 2007). Another putative intercellular signalling molecule for cel-to-cell communication is nitric oxide which is synthesized by enzymatic oxidation of L-arginine by nitric oxide synthase (Willmott et al., 2000). Nitric oxide (NO) activates guanylilcyclase and increases cytoplasmic cyclic

Communication between astrocytes thus seems to rely on any communication systems and signalling molecules, which act in parallel or display regional and cellular specialisation. From this point of view, there is a bidirectional signal communication system within the CNS, which might be mainly carried out by extracellular messengers, released from any type of cells. Because of their close apposition to neurons, signalling molecules released by astrocytes can modulate synaptic transmission and neuronal excitability, as well as neuronal plasticity and survival. Even it could be possible that astrocytes could play roles in higher cognitive functions like learning and memory. It is not therefore estrange that an alteration in Ca2+ signalling, and hence in the function of astrocytes, could affect synaptic activity and

Close physical relationship between astrocytes and neurones provides an opportunity for many functional interactions. There is a bidirectional signalling pathway between astrocytes and neurons on one side, and astrocytes and blood vessels on the other, which opens the possibility to an exchange of a huge amount of information in the CNS. There are several mechanisms that have been suggested to underline the release of signalling molecules from astrocytes: reverse operation of glutamate transporters, volume-regulated anion channels, gap-junctional hemichannels, diffusional release through purinergic receptors and Ca2+ dependent exocytosis (Araque et al., 2001; Haydon & Carmignoto, 2006; Montana et al., 2006; Parpura et al., 2004). Among the different molecules released, two major signalling messengers, released by astrocytes, are ATP and glutamate (Gonzalez & Salido, 2009).

The mechanisms, by which astrocytes release ATP, appear to be diverse, employing vesicular release, connexion hemi-channels, cystic fibrosis transmembrane regulator, or the P-glycoprotein (Braet et al., 2004). On the other hand, astrocytic glutamate release can be carried out through connexion hemi-channel, excitatory amino acid transporters (EAAT), anion transporter, via P2X7 receptor channels or exocytosis. Depending on the mechanism employed, ATP and/or glutamate release by astrocytes can be Ca2+-dependent or

Besides the mechanisms for ATP and/or glutamate release from astrocytes, exocytosis constitutes the mechanism that has recently received special attention, since it was initially considered to occur only in neurons (Bowser & Khakh, 2007; Fellin et al., 2006; Gonzalez et

guanosine monophosphate (cGMP) signalling cascades (Galione et al, 1993).

plasticity and bran homeostasis (Gonzalez & Salido, 2009).

independent (Bowser & Khakh, 2007; Braet et al., 2004).

al., 2006a; Perea & Araque, 2007).

**2.2 Release of intercellular messengers** 

Malarkey & Parpura, 2008; Montana et al. 2006).

## **2. Astrocytes in the central nerve system**

Central nerve system is a complex network, constitutes from several types of cells. Besides neuronal cells, where the information is received, integrated and sent as an output signal, there are several other cell types in the CNS. Oligodendrocytes are specialized for the myelin formation, astrocytes have multiple support functions to neurons, and microglial cells play an important role in defence and inflammation, and act as scavengers when tissue is destroyed. Some other types of cells in the CNS are ependimal cells, which are epithelial cells that line brain ventricles and central canal of spinal and assist in secretion and circulation of cerebral spinal fluid, and endothelial cells which create a blood-brain barrier (González & Salido, 2009).

Glial cells were discovered by the pathologist Rudolf Virchow in 1856. They represent the majority cell population in the CNS. There is a number between 12 and 15 billion neurons in cerebral cortex and about a billion neurons in spinal cord, whereas there are 10 to 50 times more glial cells than neurons in the CNS. When they were discovered, glial cells have been recognised as brain glue. They surround neurons and hold them in place. Later it has been realized that glial cells play a number of other functions in the brain. Astrocytes are the most abundant type of glial cells, and present numerous projections that anchor neurons to their blood supply (Braet et al., 2001; González & Salido, 2009; Grafstein et al., 2000; Haydon, 2001).

#### **2.1 Molecular aspects of astrocyte function**

Astrocytes signal each other using Ca2+ ions (Verkhratsky et al., 1998). This type of cell-to cell communication has been termed "calcium excitability" that occurs as transient or prolonged elevations in intracellular concentration of Ca2+ ions. It can be spontaneous or triggered in response to specific neurotransmitters (Araque et al., 2001; Cornell-Bell et al., 1990). The membrane potential of glia is relatively stable, and although they can express voltage-gated channels (Verkhratsky et al., 1998), they exhibit little or no fluctuation in membrane potential.

Astrocytes respond to a variety of extracellular stimuli by raising intracellular concentration of Ca2+ ions that modulates different intracellular processes like differentiation, cytoskeleton reorganisation, and secretion of neuroactive molecules (Araque et al., 1998; Sofroniew & Vinters, 2010; Verkhratsky & Kettenmann, 1996). A rise in intracellular concentration of Ca2+ ions, localize to one part of an astrocyte can propagate through-out the entire cell, and Ca2+ resposes may be transmitted from one astrocate to others, leading to regenerative Ca2+ signal that spread within astrocyte networks (Cornell-Bell et al., 1990; Fam et al., 2000). This cell-to-cell communication could effectively signal to neurons, endothelial or other cell type in the CNS. Obviously, Ca2+ signalling in astrocytes is complementary to and interacts with signalling in vascular brain cells (Leybaert et al., 2004) and electrical signalling in neurons (Araque et al., 1999; Parpura et al., 1994). Besides calcium excitability, there are also other mechanisms for transmitting signals between astrocytes, such as releasing of diffusible extracellular messengers. Extracellular release of neurotransmitters like glutamate or adenosine triphosphate (ATP), and consequent activation of specific receptors on neighbouring astrocytes, may also mediate Ca2+ wave propagation (Bowser & Khakh, 2007). In addition, astrocytes are able to release other signalling molecules like D-serine and eicosanoids, and more than one of describing mechanisms for neurotransmitter release does

Central nerve system is a complex network, constitutes from several types of cells. Besides neuronal cells, where the information is received, integrated and sent as an output signal, there are several other cell types in the CNS. Oligodendrocytes are specialized for the myelin formation, astrocytes have multiple support functions to neurons, and microglial cells play an important role in defence and inflammation, and act as scavengers when tissue is destroyed. Some other types of cells in the CNS are ependimal cells, which are epithelial cells that line brain ventricles and central canal of spinal and assist in secretion and circulation of cerebral spinal fluid, and endothelial cells which create a blood-brain barrier

Glial cells were discovered by the pathologist Rudolf Virchow in 1856. They represent the majority cell population in the CNS. There is a number between 12 and 15 billion neurons in cerebral cortex and about a billion neurons in spinal cord, whereas there are 10 to 50 times more glial cells than neurons in the CNS. When they were discovered, glial cells have been recognised as brain glue. They surround neurons and hold them in place. Later it has been realized that glial cells play a number of other functions in the brain. Astrocytes are the most abundant type of glial cells, and present numerous projections that anchor neurons to their blood supply (Braet et al., 2001; González & Salido, 2009; Grafstein et al., 2000; Haydon, 2001).

Astrocytes signal each other using Ca2+ ions (Verkhratsky et al., 1998). This type of cell-to cell communication has been termed "calcium excitability" that occurs as transient or prolonged elevations in intracellular concentration of Ca2+ ions. It can be spontaneous or triggered in response to specific neurotransmitters (Araque et al., 2001; Cornell-Bell et al., 1990). The membrane potential of glia is relatively stable, and although they can express voltage-gated channels (Verkhratsky et al., 1998), they exhibit little or no fluctuation in

Astrocytes respond to a variety of extracellular stimuli by raising intracellular concentration of Ca2+ ions that modulates different intracellular processes like differentiation, cytoskeleton reorganisation, and secretion of neuroactive molecules (Araque et al., 1998; Sofroniew & Vinters, 2010; Verkhratsky & Kettenmann, 1996). A rise in intracellular concentration of Ca2+ ions, localize to one part of an astrocyte can propagate through-out the entire cell, and Ca2+ resposes may be transmitted from one astrocate to others, leading to regenerative Ca2+ signal that spread within astrocyte networks (Cornell-Bell et al., 1990; Fam et al., 2000). This cell-to-cell communication could effectively signal to neurons, endothelial or other cell type in the CNS. Obviously, Ca2+ signalling in astrocytes is complementary to and interacts with signalling in vascular brain cells (Leybaert et al., 2004) and electrical signalling in neurons (Araque et al., 1999; Parpura et al., 1994). Besides calcium excitability, there are also other mechanisms for transmitting signals between astrocytes, such as releasing of diffusible extracellular messengers. Extracellular release of neurotransmitters like glutamate or adenosine triphosphate (ATP), and consequent activation of specific receptors on neighbouring astrocytes, may also mediate Ca2+ wave propagation (Bowser & Khakh, 2007). In addition, astrocytes are able to release other signalling molecules like D-serine and eicosanoids, and more than one of describing mechanisms for neurotransmitter release does

**2. Astrocytes in the central nerve system** 

**2.1 Molecular aspects of astrocyte function** 

(González & Salido, 2009).

membrane potential.

operate within astrocytes (Araque et al., 2001; Fellin et al., 2004; Gonzales et al., 2006a; Malarkey & Parpura, 2008; Montana et al. 2006).

Released messengers, in turn, activate Ca2+ entry or Ca2+ release from intracellular stores by acting on ionotropic and metabotropic receptors, respectively. By this way, ATP and glutamate are the major active neurotransitters involved in the cell-to-cell communication of Ca2+ signals in astrocytes and other cell types in the CNS (Bowser & Khakh, 2007; Percea & Araque, 2007). Another putative intercellular signalling molecule for cel-to-cell communication is nitric oxide which is synthesized by enzymatic oxidation of L-arginine by nitric oxide synthase (Willmott et al., 2000). Nitric oxide (NO) activates guanylilcyclase and increases cytoplasmic cyclic guanosine monophosphate (cGMP) signalling cascades (Galione et al, 1993).

Communication between astrocytes thus seems to rely on any communication systems and signalling molecules, which act in parallel or display regional and cellular specialisation. From this point of view, there is a bidirectional signal communication system within the CNS, which might be mainly carried out by extracellular messengers, released from any type of cells. Because of their close apposition to neurons, signalling molecules released by astrocytes can modulate synaptic transmission and neuronal excitability, as well as neuronal plasticity and survival. Even it could be possible that astrocytes could play roles in higher cognitive functions like learning and memory. It is not therefore estrange that an alteration in Ca2+ signalling, and hence in the function of astrocytes, could affect synaptic activity and plasticity and bran homeostasis (Gonzalez & Salido, 2009).

#### **2.2 Release of intercellular messengers**

Close physical relationship between astrocytes and neurones provides an opportunity for many functional interactions. There is a bidirectional signalling pathway between astrocytes and neurons on one side, and astrocytes and blood vessels on the other, which opens the possibility to an exchange of a huge amount of information in the CNS. There are several mechanisms that have been suggested to underline the release of signalling molecules from astrocytes: reverse operation of glutamate transporters, volume-regulated anion channels, gap-junctional hemichannels, diffusional release through purinergic receptors and Ca2+ dependent exocytosis (Araque et al., 2001; Haydon & Carmignoto, 2006; Montana et al., 2006; Parpura et al., 2004). Among the different molecules released, two major signalling messengers, released by astrocytes, are ATP and glutamate (Gonzalez & Salido, 2009).

The mechanisms, by which astrocytes release ATP, appear to be diverse, employing vesicular release, connexion hemi-channels, cystic fibrosis transmembrane regulator, or the P-glycoprotein (Braet et al., 2004). On the other hand, astrocytic glutamate release can be carried out through connexion hemi-channel, excitatory amino acid transporters (EAAT), anion transporter, via P2X7 receptor channels or exocytosis. Depending on the mechanism employed, ATP and/or glutamate release by astrocytes can be Ca2+-dependent or independent (Bowser & Khakh, 2007; Braet et al., 2004).

Besides the mechanisms for ATP and/or glutamate release from astrocytes, exocytosis constitutes the mechanism that has recently received special attention, since it was initially considered to occur only in neurons (Bowser & Khakh, 2007; Fellin et al., 2006; Gonzalez et al., 2006a; Perea & Araque, 2007).

Ethanol Toxicity in the Brain: Alteration of Astroglial Cell Function 611

(ATP and adenosine), gamma-aminobutyric acid (GABA), and D-serine. The release of such gliotransmitters occurs in response to changes in neuronal synaptic activity, involves astrocyte excitability as reflected by increases in intracellular concentration of Ca2+ ions, and can alter neuronal excitability (Halassa et al., 2007; Perea et al., 2009). Such evidence has given rise to the 'tripartite synapse', which posits that astrocytes play direct and interactive roles with neurons during synaptic activity in a manner that is essential for information processing by neural circuits (Araque et al., 1999; Halassa et al., 2007; Perea et al., 2009).

Astrocytes importantly contribute to creation of immune response in the brain. They are an important source of several cytokines and neurotrophic factors in the CNS that have a crucial immunoregulatory role and also promote neuronal survival and neurite growth (Lipnik-Štangelj, 2006). Moreover, cytokines have an impact on neurotoxicity, synaptic transmission and synaptic plasticity in the brain (Allan & Rothwell, 2001). Activation of astrocytes leads to up-regulation of pro-inflammatory cytokines like interleukin-1 beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX2) (Gonzalez & Salido, 2009; Sofroniew &

Research into the actions of IL-1beta in the brain initially focused on its role in host defence responses to systemic disease. IL-1beta can also elicit an array of responses which could

TNF-alpha has an important function in neurotoxicity, synaptic transmission and synaptic plasticity. It influences homeostatic synaptic scaling by inducing the insertion of AMPA receptors at post-synaptic membranes (Stellwagen & Malenka, 2006). In addition, TNF-alpha may have a pivotal role in augmenting intracerebral immune responses and inflammatory demyelination due to its diverse functional effects on glial cells, such as oligodendrocytes

Unlike TNF-alpha, which is a prototypical pro-inflammatory cytokine, IL-6 affects inflammation and neuronal regeneration via a number of mechanisms. In this sense, besides its immunoregulatory role, IL-6 can also promote neuronal survival and neurite growth.IL-6 can be induced by a variety of molecules including IL-1beta, TNF-alpha, transforming growth factor-beta and prostaglandins, and many other mediators such as beta-amyloid, interferon-g and IL-4 can potentiate these primary inducers, highlighting the complex

After brain injury, such as a stroke or trauma, astrocytes become reactive, and can undergo to profound proliferation, forming gliosis near or at the site of damage. Astrocyte activity is marked by hypertrophy, resulting in an expression of protein such as glial fibrillary acidic protein (GFAP), adhesion molecules and antigen presenting capabilities, including major histocompatibility antigens. Reactive astrocytes represent an obstacle preventing establishment of normal neural contact and circuitry. On the other hand, reactive astrocytes produce a myriad of neurotoxic substances in various brain pathologies (Mori et al., 2006).

nature of IL-6 modulation (Šarc et al., 2011; Gonzalez & Salido, 2009).

inhibit, exacerbate or induce neuronal damage and death (Gonzalez & Salido, 2009).

**2.3.5 Immune response** 

Vinters, 2010).

and astrocytes themselves (Šarc et al., 2011).

**2.4 Reactive gliosis and glial scar formation** 

#### **2.3 The role of astroytes in the central nerve system**

#### **2.3.1 Astrocytes and development of central nerve system**

The developmental generation of astrocytes tends to occur after the initial production of neurons in many CNS regions (Sofroniew & Vinters, 2010). During development of the brain, astrocytes (radial glia) take part in guiding the migration of developing axons and certain neuroblasts (Powel & Geller, 1999). In addition, substantive evidence is accumulating that astrocytes are essential for the formation and function of developing synapses by releasing molecular signals such as thrombospondin (Barres, 2008; Christopherson et al., 2005). Astrocytes appear also to influence developmental synaptic pruning by releasing signals that induce expression of complement C1q in synapses and thereby tag them for elimination by microglia (Barres, 2008).

#### **2.3.2 Blood-brain barrier and regulation of blood flow**

Together with brain microvascular endothelial cells astrocytes create the blood-brain barrier that protects the brain from toxic substances in the blood, supplies the brain tissues with nutrients, and filters harmful substances from the brain back to the bloodstream, enabling the proper environment in the CNS. Astrocytes may regulate endothelial cell metabolism, and vasoconstriction and vasodilatation by producing substances with angiogenic properties, such as endothelial growth factor (Proia et al., 2008), ATP (Leybaert et al., 2004), and arachidonic acid, prostagladins and nitric oxide, (Gabryel et al., 2007; Sofroniew & Vinters, 2010), that can increase or decrease CNS blood vessel diameter and blood flow in a coordinated manner. Moreover, astrocytes may be primary mediators of changes in local CNS blood flow in response to changes in neuronal activity (Koehler et al., 2009). Thus, astrocytes play important functions at the level of arterioles where blood flow is controlled, at the level of capillaries where blood-brain barrier is located and at the level of blood immune cells (Leybaert et al., 2004).

#### **2.3.3 Energy, metabolism and homeostasis**

Astrocytes play a number of other functions which are crucial for the maintenance of homeostasis and neuronal function. They provide energy supply to neurons and coordinate metabolic reactions. Astrocytes are the principal storage sites of glycogen granules in CNS. The greatest accumulation of astrocytic glycogen occurs in areas of high synaptic density, and its utilisation can sustain neuronal activity during hypoglicemia and during periods of high neuronal activity (Sofroniew & Vinters, 2010).

Astrocytes regulate the external chemical environment by removing excess ions notably potassium, regulate brain cell volume, and participate in recycling neurotransmitters released during synaptic transmission by expressing high levels of transporters for neurotransmitters such as glutamate, GABA, histamine and glycine, that serve to clear the neurotransmitters from the synaptic space. Astrocytes also represent the major site for the detoxification or bioactivation of neurotoxins (Perdan et al., 2009).

#### **2.3.4 Synapse function**

There is accumulating evidence that astrocytes play direct roles in synaptic transmission through the regulated release of synaptically active molecules including glutamate, purines (ATP and adenosine), gamma-aminobutyric acid (GABA), and D-serine. The release of such gliotransmitters occurs in response to changes in neuronal synaptic activity, involves astrocyte excitability as reflected by increases in intracellular concentration of Ca2+ ions, and can alter neuronal excitability (Halassa et al., 2007; Perea et al., 2009). Such evidence has given rise to the 'tripartite synapse', which posits that astrocytes play direct and interactive roles with neurons during synaptic activity in a manner that is essential for information processing by neural circuits (Araque et al., 1999; Halassa et al., 2007; Perea et al., 2009).
