Ulrika Björklund

Exposure of cultured astroglial and microglial brain cells to 900 MHz microwave radiation

Abstract Thorlin, T., Rouquette, J.-M., Hamnerius, Y., Hansson, E., Persson, M., Björklund, U., Rosengren, L., Rönnbäck, L. and Persson, M. Exposure of Cultured Astroglial and Microglial Brain Cells to 900 MHz Microwave Radiation. Radiat. Res. 166, 409–421 (2006). The rapid rise in the use of mobile communications has raised concerns about health issues related to low-level microwave radiation. The head and brain are usually the most exposed targets in mobile phone users. In the brain, two types of glial cells, the astroglial and the microglial cells, are interesting in the context of biological effects from microwave exposure. These cells are widely distributed in the brain and are directly involved in the response to brain damage as well as in the development of brain cancer. The aim of the present study was to investigate whether 900 MHz radiation could affect these two different glial cell types in culture by studying markers for damage-related processes in the cells. Primary cultures enriched in astroglial cells were exposed to 900 MHz microwave radiation in a temperature-controlled exposure system at specific absorption rates (SARs) of 3 W/kg GSM modulated wave (mw) for 4, 8 and 24 h or 27 W/kg continuous wave (cw) for 24 h, and the release into the extracellular medium of the two pro-inflammatory cytokines interleukin 6 (Il6) and tumor necrosis factor-alpha (Tnfa) was analyzed. In addition, levels of the astroglial cell-specific reactive marker glial fibrillary acidic protein (Gfap), whose expression dynamics is different from that of cytokines, were measured in astroglial cultures and in astroglial cell-conditioned cell culture medium at SARs of 27 and 54 W/kg (cw) for 4 or 24 h. No significant differences could be detected for any of the parameters studied at any time and for any of the radiation characteristics. Total protein levels remained constant during the experiments. Microglial cell cultures were exposed to 900 MHz radiation at an SAR of 3 W/kg (mw) for 8 h, and Il6, Tnfa, total protein and the microglial reactivity marker ED-1 (a macrophage activation antigen) were measured. No significant differences were found. The morphology of the cultured astroglial cells and microglia was studied and appeared to be unaffected by microwave irradiation. Thus this study does not provide evidence for any effect of the microwave radiation used on damage-related factors in glial cells in culture.

μ-Opioid agonists inhibit the enhanced intracellular Ca2+ responses in inflammatory activated astrocytes co-cultured with brain endothelial cells

In order to imitate the in vivo situation with constituents from the blood-brain barrier, astrocytes from newborn rat cerebral cortex were co-cultured with adult rat brain microvascular endothelial cells. These astrocytes exhibited a morphologically differentiated appearance with long processes. 5-HT, synthetic mu-, delta- or kappa-opioid agonists, and the endogenous opioids endomorphin-1, beta-endorphin, and dynorphin induced higher Ca(2+) amplitudes and/or more Ca(2+) transients in these cells than in astrocytes in monoculture, as a sign of more developed signal transduction systems. Furthermore, stimulation of the co-cultured astrocytes with 5-HT generated a pronounced increase in intracellular Ca(2+) release in the presence of the inflammatory or pain mediating activators substance P, calcitonin gene-related peptide (CGRP), lipopolysaccharide (LPS), or leptin. These Ca(2+) responses were restored by opioids so that the delta- and kappa-opioid receptor agonists reduced the number of Ca(2+) transients elicited after incubation in substance P+CGRP or leptin, while the mu- and delta-opioid receptor agonists attenuated the Ca(2+) amplitudes elicited in the presence of LPS or leptin. In LPS treated co-cultured astrocytes the mu-opioid receptor antagonist naloxone attenuated not only the endomorphin-1, but also the 5-HT evoked Ca(2+) transients. These results suggest that opioids, especially mu-opioid agonists, play a role in the control of neuroinflammatory activity in astrocytes and that naloxone, in addition to its interaction with mu-opioid receptors, also may act through some binding site on astrocytes, other than the classical opioid receptor.

IN INFLAMMATORY REACTIVE ASTROCYTES CO-CULTURED WITH BRAIN ENDOTHELIAL CELLS NICOTINE-EVOKED Ca2+ TRANSIENTS ARE ATTENUATED DUE TO INTERLEUKIN-1β RELEASE AND REARRANGEMENT OF ACTIN FILAMENTS

The aim of this study was to investigate whether nicotine acetylcholine receptors (nAChRs) are expressed in a more pronounced way in astrocytes co-cultured with microvascular endothelial cells from adult rat brain, compared with monocultured astrocytes, as a sign of a more developed signal transduction system. Also investigated was whether nicotine plays a role in the control of neuroinflammatory reactivity in astrocytes. Ca(2+) imaging experiments were performed using cells loaded with the Ca(2+) indicator Fura-2/AM. Co-cultured astrocytes responded to lower concentrations of nicotine than did monocultured astrocytes, indicating that they are more sensitive to nicotine. Co-cultured astrocytes also expressed a higher selectivity for alpha7nAChR and alpha4/beta2 subunits and evoked higher Ca(2+) transients compared with monocultured astrocytes. The Ca(2+) transients referred to are activators of Ca(2+)-induced Ca(2+) release from intracellular stores, both IP(3) and ryanodine, triggered by influx through receptor channels. The nicotine-induced Ca(2+) transients were attenuated after incubation with the inflammatory mediator lipopolysaccharide (LPS), but were not attenuated after incubation with the pain-transmitting peptides substance P and calcitonin-gene-related peptide, nor with the infection and inflammation stress mediator, leptin. Furthermore, LPS-induced release of interleukin-1beta (IL-1beta) measured by enzyme-linked immunosorbent assay (ELISA) was more pronounced in co-cultured versus monocultured astrocytes. Incubation with both LPS and IL-1beta further attenuated nicotine-induced Ca(2+) response. We also found that LPS and IL-1beta induced rearrangement of the F-actin filaments, as measured with an Alexa488-conjugated phalloidin probe. The rearrangements consisted of increases in ring formations and a more dispersed appearance of the filaments. These results indicate that there is a connection between a dysfunction of nicotine Ca(2+) signaling in inflammatory reactive astrocytes and upregulation of IL-1beta and the rearrangements of actin filaments in the cells.

A new concept affecting restoration of inflammation-reactive astrocytes

Long-lasting pain may partly be a consequence of ongoing neuroinflammation, in which astrocytes play a significant role. Following noxious stimuli, increased inflammatory receptor activity, influences in Na(+)/K(+)-ATPase activity and actin filament organization occur within the central nervous system. In astrocytes, the Ca(2+) signaling system, Na(+) transporters, cytoskeleton, and release of pro-inflammatory cytokines change during inflammation. The aim of this study was to restore these cell parameters in inflammation-reactive astrocytes. We found that the combination of (1) endomorphin-1, an opioid agonist that stimulates the Gi/o protein of the μ-opioid receptor; (2) naloxone, an opioid antagonist that inhibits the Gs protein of the μ-opioid receptor at ultralow concentrations; and (3) levetiracetam, an anti-epileptic agent that counteracts the release of IL-1β, managed to activate the Gi/o protein and Na(+)/K(+)-ATPase activity, inhibit the Gs protein, and decrease the release of IL-1β. The cell functions of astrocytes in an inflammatory state were virtually restored to their normal non-inflammatory state and it could be of clinical significance and may be useful for the treatment of long-term pain.

Ifenprodil restores GDNF‐evoked Ca2+ signalling and Na+/K+‐ATPase expression in inflammation‐pretreated astrocytes

J. Neurochem. (2011) 119, 686–696.

Naloxone in ultralow concentration restores endomorphin-1–evoked Ca2+ signaling in lipopolysaccharide pretreated astrocytes

Long-term pain is a disabling condition that affects thousands of people. Pain may be sustained for a long time even after the physiological trigger has resolved. Possible mechanisms for this phenomenon include low-grade inflammation in the CNS. Astrocytes respond to inflammatory stimuli and may play an important role as modulators of the inflammatory response in the nervous system. This study aimed first to assess how astrocytes in a primary culture behave when exposed to the endogenous μ-opioid receptor agonist endomorphin-1 (EM-1), in a concentration-dependent manner, concerning intracellular Ca²⁺ responses. EM-1 stimulated the μ-opioid receptor from 10⁻¹⁵ M up to 10⁻⁴ M with increasing intensity, usually reflected as one peak at low concentrations and two peaks at higher concentrations. Naloxone, pertussis toxin (PTX), or the μ-opioid receptor antagonists CTOP did not totally block the EM-1-evoked Ca²⁺ responses. However, a combination of ultralow concentration naloxone (10⁻¹² M) and PTX (100 ng/ml) totally blocked the EM-1-evoked Ca²⁺ responses. This suggests that ultralow (picomolar) concentrations of naloxone should block the μ-opioid receptor coupled G(s) protein, and that PTX should block the μ-opioid receptor coupled G(i/o) protein. The second aim was to investigate exposure of astrocytes with the inflammatory agent lipopolysaccharide (LPS). After 4 h of LPS incubation, the EM-1-evoked Ca²⁺ transients were attenuated, and after 24 h of LPS incubation, the EM-1-evoked Ca²⁺ transients were oscillated. To restore the EM-1-evoked Ca²⁺ transients, naloxone was assessed as a proposed anti-inflammatory substance. In ultralow picomolar concentration, naloxone demonstrated the ability to restore the Ca²⁺ transients.

Potentiating effect of endothelial cells on astrocytic plasminogen activator inhibitor type-1 gene expression in an in vitro model of the blood–brain barrier

There is accumulating evidence of the importance of cellular communication between the cells that compose the blood-brain barrier (BBB). Astrocytes are known to affect the expression of tissue-type plasminogen activator (t-PA) and its inhibitor plasminogen activator inhibitor type-1 (PAI-1) in endothelial cells. We investigated the influence of endothelial cells on astrocytic gene expression of PAI-1, protease nexin-1 (PN-1) and t-PA using an in vitro model of the BBB. Primary rat astrocyte-enriched cultures were cocultured with primary adult rat brain microvascular endothelial cells on opposite sides of a transwell membrane. After coculturing for 9-11 days, the cultures were treated with lipopolysaccharide (LPS) for 8 h or 24 h. The levels of PAI-1, PN-1 and t-PA mRNA in untreated and treated monocultures and cocultures were analyzed by Real-Time RT-PCR. Cocultivation of astrocytes and endothelial cells increased astrocytic PAI-1 mRNA expression, and this response was further amplified by LPS treatment. The levels of PN-1 and t-PA mRNA expression in astrocytes were unaffected by cocultivation and/or LPS treatment. Analysis of endothelial PAI-1 and t-PA gene expression revealed increased PAI-1 mRNA levels in cocultured cells, whereas t-PA mRNA levels remained unchanged. These results demonstrate that the cocultivation of astrocytes and endothelial cells induces a pronounced increase in astrocytic PAI-1 gene expression, and that this effect is amplified by LPS treatment. These findings imply an important role for intercellular crosstalk in modulating PAI-1 gene expression within the BBB, under both physiologic and pathophysiologic conditions.

Ultralow concentrations of bupivacaine exert anti‐inflammatory effects on inflammation‐reactive astrocytes

Bupivacaine is a widely used, local anesthetic agent that blocks voltage‐gated Na+ channels when used for neuro‐axial blockades. Much lower concentrations of bupivacaine than in normal clinical use, < 10−8 m, evoked Ca2+ transients in astrocytes from rat cerebral cortex, that were inositol trisphosphate receptor‐dependent. We investigated whether bupivacaine exerts an influence on the Ca2+ signaling and interleukin‐1β (IL‐1β) secretion in inflammation‐reactive astrocytes when used at ultralow concentrations, < 10−8 m. Furthermore, we wanted to determine if bupivacaine interacts with the opioid‐, 5‐hydroxytryptamine‐ (5‐HT) and glutamate‐receptor systems. With respect to the μ‐opioid‐ and 5‐HT‐receptor systems, bupivacaine restored the inflammation‐reactive astrocytes to their normal non‐inflammatory levels. With respect to the glutamate‐receptor system, bupivacaine, in combination with an ultralow concentration of the μ‐opioid receptor antagonist naloxone and μ‐opioid receptor agonists, restored the inflammation‐reactive astrocytes to their normal non‐inflammatory levels. Ultralow concentrations of bupivacaine attenuated the inflammation‐induced upregulation of IL‐1β secretion. The results indicate that bupivacaine interacts with the opioid‐, 5‐HT‐ and glutamate‐receptor systems by affecting Ca2+ signaling and IL‐1β release in inflammation‐reactive astrocytes. These results suggest that bupivacaine may be used at ultralow concentrations as an anti‐inflammatory drug, either alone or in combination with opioid agonists and ultralow concentrations of an opioid antagonist.

Anti-inflammatory substances can influence some glial cell types but not others.

In rat microglial enriched cultures, expressing Toll-like receptor 4, we studied cytokine release after exposure with 1 ng/ml LPS for 0.5-24 h. Dexamethasone and corticosterone exposure served as controls. We focused on whether naloxone, ouabain, and bupivacaine, all agents with reported anti-inflammatory effects on astrocytes, could affect the release of TNF-α and IL-1β in microglia. Our results show that neither ultralow (10(-12) M) nor high (10(-6) M) concentrations of these agents had demonstrable effects on cytokine release in microglia. The results indicate that anti-inflammatory substances exert specific influences on different glial cell types. Astrocytes seem to be functional targets for anti-inflammatory substances while microglia respond directly to inflammatory stimuli and are thus more sensitive to anti-inflammatory substances like corticoids. The physiological relevance might be that astrocyte dysfunction influences neuronal signalling both due to direct disturbance of astrocyte functions and in the communication within the astrocyte networks. When the signalling between astrocytes is working, then microglia produce less pro-inflammatory cytokines.

PACAP attenuates 5-HT, histamine, and ATP-evoked Ca2+ transients in astrocytes

Pituitary adenylate cyclase-activating polypeptide (PACAP) has neuroprotective properties and plays an important role in neuroinflammation. PACAP38 interacts with its receptors, PAC1, and VPAC, on astrocytes at 10−8 M to induce biphasic Ca2+ transients, which were reduced to a single transient by the PAC1-blocking PACAP antagonist PACAP6-38. At 10−12 M even the single transient, corresponding to PAC1 was blocked. PACAP-induced Ca2+ transients were more pronounced in astrocytes cocultured with brain endothelial cells than in monocultured astrocytes, indicating that astrocytes that receive signals from microvessels develop more sensitive signal transduction systems for Ca2+. In this sensitive system, PACAP38 attenuated 5-HT, histamine, and ATP-evoked Ca2+ transients, showing the anti-inflammatory properties of PACAP.