Astrocytes are the neural cells mainly responsible for the maintenance of brain homeostasis. They form highly organized anatomical domains which are integrated by extensive networks of gap junctions and interconnected in a glial information-transfer system. One signalling pathway in this system propagates Ca2+ waves (Blomstrand et al. 1999; Scemes and Giaume 2006). Regulation of Ca2+ waves may be a mechanism by which the astrocyte networks detect changes in the CNS microenvironment and regulate brain activities under various physiological and pathophysiological conditions. This ability, along with the expression of a wide array of receptors, transporters, and ion channels, means that astrocyte networks are ideally positioned to sense and modulate neuronal activity. The glial cell line-derived neurotrophic factor (GDNF) produced and secreted by astrocytes (Nicole et al. 2001; Kuno et al. 2006) has neuroprotective functions that enhance the survival of dopaminergic, motor, and primary sensory neurons (Lin et al. 1993; Airaksinen and Saarma 2002). GDNF regulates the permeability of the blood–brain barrier (BBB) and activates the barrier function of capillary endothelial cells (Igarashi et al. 2000; Abbott et al. 2006). GDNF has been discussed to play an important role in the modulation of nociceptive signals and dysfunction of this system may contribute to the development and/or maintenance of neuropathic pain states (Nagano et al. 2003; Wang et al. 2003; Dong et al. 2005). This is especially relevant because long-term neuropathic pain is considered to result from a low-grade neuroinflammation in the CNS (Saadé and Jabbur 2008). Inflammatory mediators may penetrate the BBB in central glial response to injury, and inflammatory cytokines, such as interleukin 1-β (IL-1β) released from glia, may modulate neuronal activity and facilitate pain transmission. High levels of IL-1β produced under conditions of injury, stress, or disease evoke increased NMDA receptor phosphorylation and sensitivity in inflammatory pain models (Viviani et al. 2003; Guo et al. 2007). GDNF has protecting capabilities for cortical neurons by reducing the NMDA-induced Ca2+ influx (Nicole et al. 2001; Wang et al. 2002). Different NMDA antagonists were shown to enhance the production of GDNF in astrocytes (Toyomoto et al. 2005; Wu et al. 2009).
Ifenprodil acts as an NMDA receptor antagonist at the NR2B subunit (Iwata et al. 2007). It is used clinically as a neuroprotective agent in head ischemia, Parkinson’s disease, and stroke (Kato et al. 2006). Ifenprodil also enhances the production of GDNF in cultured astrocytes (Toyomoto et al. 2005).
Parameters associated with neuroinflammation are down-regulation of Na+ transporters, changing of Ca2+ signalling in the astrocyte networks, and release of cytokines (Morita et al. 2003; Schmidt et al. 2007; Hansson et al. 2008; Delbro et al. 2009; Vallejo et al. 2010). With lipopolysaccharide (LPS), NMDA, and IL-1β as inflammatory inducers, we wanted to evaluate how astrocytes behave concerning GDNF-evoked Ca2+ signalling, after inflammatory stimulation. We hypothesized that when astrocyte function is influenced by one or several of the inflammation inducers; it is possible to reverse some of the cell’s inflammatory dysfunction with an anti-inflammatory substance. During inflammation, NMDA receptor phosphorylation is increased and in addition IL-1β release from astrocytes occurs. As ifenprodil was shown to reduce astrocytic swelling and polyamine levels we wanted to evaluate if ifenprodil has any capabilities to restore some parameters, which are affected in inflammatory activated astrocytes.
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- Materials and methods
- Authors’ contributions
Regulation of Ca2+ dynamics by transmitters and soluble factors is a possible mechanism by which the astrocyte network detects changes in the CNS microenvironment, such as inflammatory processes, and regulates brain activities. The complexity of Ca2+ signalling has made it difficult to determine the physiological role of these phenomena although Ca2+ signalling is an important function of astrocytes. Intra- and intercellular Ca2+ signalling have been proposed to play important roles in information processing. It is a well-known element of signalling pathways implicated in activity dependent neuronal survival. Neurotrophic factors as GDNF induce a small and rapid increase in intracellular Ca2+, which plays an important role for neuronal survival (Pérez-García et al. 2004). Ca2+ signalling over long distances is analogous to, but much slower than, the propagation of action potentials in neurons (Cornell-Bell et al. 1990).
In the present study, GDNF was shown to induce Ca2+ transients, and the responses were better at low concentrations. When these Ca2+ transients were influenced by LPS, few astrocytes responded to GDNF. LPS is a potent inflammatory activator (Nakamura 2002), commonly used in experimental neuroinflammation (Zielasek and Hartung 1996). Attenuation in Ca2+ signalling when astrocytes were exposed to LPS has been shown earlier (Morita et al. 2003; Hansson et al. 2008; Delbro et al. 2009). When astrocytes are exposed to inflammatory stimuli the TLR4 is activated, which is expressed by astrocytes and LPS has been shown to be a TLR4 agonist (Kielian 2006; Forshammar et al. 2011). It has been proposed that activation of TLR4 drives inflammation that gives rise to symptoms and promotes BBB disruption through injury to endothelial cells (Krasowska-Zoladek et al. 2007). From observations in different cellular systems, a down-regulation of Na+ transporters is observed when cells were exposed to LPS, which implies dysfunction of the Na+/K+-ATPase activity. Astrocytes have strong resistance to Na+ influx only when Na+/K+-ATPase activity is maintained. Protein expression of Na+/K+-ATPase was reduced in astrocytes in primary culture after 8 h, as an indication that the Na+ transporters were down-regulated (Forshammar et al. 2011).
Astrocytic swelling is part of a cytotoxic response that characterizes brain damage and a number of mediators have been identified that initiate this process. Inflammatory stimuli induce elevation of extracellular K+, increased release of neurotransmitters such as glutamate and elevation in polyamine levels, which all induces swelling (Kempski et al. 1991; Hansson 1994; Kimelberg et al. 1995; Trout et al. 1995). The intracellular Ca2+ release in astrocytes is then decreased which also has an influence on the polymerization of the actin cytoskeleton (Hansson 1994; Allansson et al. 2001; Forshammar et al. 2011). Ifenprodil is a non-competitive antagonist and an inhibitor of the polyamine binding site on the NR2B subunit of the NMDA receptor (Chizh et al. 2001; Iwata et al. 2007), and it reduces both astrocytic swelling and polyamine levels (Trout et al. 1995). Addition of ifenprodil to astrocytes incubated with LPS, resulted in restoration of the attenuated GDNF-evoked Ca2+ transients as well as more cells responding in a concentration dependent manner. Changes of the NR2B subunit at the NMDA receptor have been observed in rats treated with LPS (Harréet al. 2008). It is possible that receptors like the NR2B subunit of the NMDA receptor are activated by inflammatory stimuli, which may obstruct the GDNF-induced Ca2+ signalling.
NMDA induces intracellular Ca2+ release in our model astrocytes. NMDA receptors are involved in numerous physiological and pathological processes, including synaptic plasticity, chronic pain, psychosis, ischemic insults, and several degenerative disorders. Agents that target and alter NMDA receptor function may thus have therapeutic benefit. Allosteric sites, which differ from agonist-binding and channel-permeation sites, can be modulated either positively or negatively. Astrocytes express both the NR1 and NR2 subunits of the NMDA receptor (Conti et al. 1996; Schipke et al. 2001; Verkhratsky and Kirchhoff 2007) in a relationship between polyamine synthesis and binding to the NMDA receptor (Trout et al. 1995). NR2B has been shown to be over-expressed during pain in mice, and blocking this subunit has been a focus in research on pain targets (Chizh et al. 2001). During pain states, enhanced extracellular concentrations of polyamines have been observed in the CNS (Chizh et al. 2001).
Interactions between GDNF and NMDA receptors have been demonstrated on neurons, showing that GDNF, through activation of the extracellular signal regulated kinases pathway, modulates the activity of the NMDA receptor by reducing the Ca2+ influx (Nicole et al. 2001). In the present study, the GDNF-evoked Ca2+ signalling was attenuated by NMDA. Ifenprodil blocked the NR2B subunit, and the GDNF-evoked Ca2+ transients were restored, and also a larger number of astrocytes responded.
The ability of IL-1β to influence astrocyte function not only depends on the expression of the appropriate receptors, but also on the activation of specific intracellular signalling pathways. Thus, the proinflammatory cytokine IL-1β mediates its effects on immunity and inflammation by interacting with the type 1 IL-1R receptor expressed on astrocytes particularly after injury, suggesting a specific association with inflammatory responses (Friedman 2001). IL-1β has been shown to modulate the voltage-dependent Na+ currents in an IL-1 receptor dependent manner in neurons (Liu et al. 2006). We show in the present study interactions between GDNF and IL-1β, and the GDNF-evoked Ca2+ transients were attenuated by IL-1β. We show that even in the interactions between GDNF and IL-1β, ifenprodil could restore the GDNF-evoked Ca2+ transients.
Ifenprodil has not only antagonistic effects on the NMDA receptor, but has also been shown to inactivate tetrodotoxin-resistant Na+ channels in rat dorsal root ganglion neurons (Tanahashi et al. 2007). This effect can be another mechanism by which ifenprodil demonstrated positive effects on the attenuated Ca2+ transients evoked by GDNF, and in combination with LPS, NMDA or IL-1β. The effects of ifenprodil seem rather specific for GDNF-evoked Ca2+ transients because they were not observed in response to endomorphin-1 or nicotine stimulation.
For in vitro cortical duct cells, proinflammatory cytokines as TNF-α and IL-1β have been shown to decrease the expression of Na+/K+-ATPase during severe experimental sepsis (Schmidt et al. 2007). Stimulation of inflammatory reactive receptors, such as TLR4, NMDA or IL-1R, seems to change the GDNF-evoked Ca2+ signalling and thereby disturb the Ca2+ transients in the astrocytic networks. One explanation can be that the Na+ transporters are down-regulated by LPS.
What, if something, can help us to identify new methods for treating neuroinflammation and regulating cellular balance? Because astrocytes are intimate co-players with neurons in the CNS, more knowledge of astrocyte responses to inflammatory activators may give new insight to our understanding of mechanisms underlying neuroinflammation and help us learn how to attenuate neuroinflammation and restore glial cell function. One strategy would be to block inflammatory active receptors or receptor sites.
In conclusion, using the co-cultured model of astrocytes, we have shown that ifenprodil restored the GDNF-evoked Ca2+ transients and increased the Na+/K+-ATPase expression. We conclude that this is a step in understanding astrocyte response and neuroinflammatory mechanisms. More study is needed to find means to attenuate neuroinflammation and restore glial cell function.