Inhibition of p38 MAPK improves intestinal disturbances and oxidative stress induced in a rabbit endotoxemia model

Background Lipopolysaccharide (LPS) decreases intestinal contractility and induces the release of reactive oxygen species, which play an important role in the pathogenesis of sepsis. p38 mitogen‐activated protein kinase (MAPK) can be activated by a variety of stimuli such as LPS. The aims of this study were: (i) to investigate the role of p38 MAPK in the effect of LPS on (a) the acetylcholine, prostaglandin E₂ and KCl‐induced contractions of rabbit duodenum and (b) the oxidative stress status; (ii) to localize the active form of p38 in the intestine. Methods Rabbits were injected with (i) saline, (ii) LPS, (iii) SB203580, a specific p38 MAPK inhibitor or (iv) SB203580 + LPS. Duodenal contractility was studied in an organ bath. SB203580 was also tested in vitro. The protein expression of p‐p38 and total p38 was measured by Western blot and p‐p38 was localized by inmunohistochemistry. The formation of products of oxidative damage to proteins (carbonyls) and lipids (MDA+4‐HDA) was quantified in intestine and plasma. Key Results ACh, PGE₂ and KCl‐induced contractions decreased with LPS. LPS increased phospho‐p38 expression and the levels of carbonyls and MDA+4‐HDA. SB203580 blocked the effect of LPS on the ACh, PGE₂ and KCl‐induced contractions in vivo and in vitro and the levels of carbonyls and MDA+4‐HDA. P‐p38 was detected in neurons of the myenteric plexus and smooth muscle cells of duodenum. Conclusions & Inferences Lipopolysaccharide decreases the duodenal contractility in rabbits and increases the production of free radicals. p38 MAPK is a mediator of these effects.     

Activation of EP2 receptor suppresses poly(I: C) and LPS‐mediated inflammation in primary microglia and organotypic hippocampal slice cultures: Contributing role for MAPKs

Brain inflammation is a critical factor involved in neurodegeneration. Recently, the prostaglandin E₂ (PGE₂) downstream members were suggested to modulate neuroinflammatory responses accompanying neurodegenerative diseases. In this study, we investigated the protective effects of prostaglandin E₂ receptor 2 (EP₂) during TLR3 and TLR4‐driven inflammatory response using in vitro primary microglia and ex vivo organotypic hippocampal slice cultures (OHSCs). Depletion of microglia from OHSCs differentially affected TLR3 and TLR4 receptor expression. Poly(I:C) induced the production of prostaglandin E₂ in OHSCs by increasing cyclooxygenase (COX‐2) and microsomal prostaglandin E synthase (mPGES)‐1. Besides, stimulation of OHSCs and microglia with Poly(I:C) upregulated EP₂ receptor expression. Co‐stimulation of OHSCs and microglia with the EP₂ agonist butaprost reduced inflammatory mediators induced by LPS and Poly(I:C). In Poly(I:C) challenged OHSCs, butaprost almost restored microglia ramified morphology and reduced Iba1 immunoreactivity. Importantly, microglia depletion prevented the induction of inflammatory mediators following Poly(I:C) or LPS challenge in OHSCs. Activation of EP₂ receptor reversed the Poly(I:C)/LPS‐induced phosphorylation of the mitogen activated protein kinases (MAPKs) ERK, p38 MAPK and c‐Jun N‐terminal kinase (JNK) in microglia. Collectively, these data identify an anti‐inflammatory function for EP₂ signaling in diverse innate immune responses, through a mechanism that involves the mitogen‐activated protein kinases pathway.     

Lipopolysaccharides enhance the action of bradykinin in enteric neurons via secretion of interleukin‐1β from enteric glial cells

Functional changes of the enteric nervous system have been observed under inflammatory states of inflammatory bowel disease increasing the endotoxin level. The aim of the present study was to determine the effect of lipopolysaccharides (LPS) on myenteric neuron–glia interaction in vitro. We examined the increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the release of interleukin‐1β (IL‐1β) or prostaglandin E₂ (PGE₂) and COX‐2 expression in myenteric plexus cells from the rat intestine induced by LPS. LPS potentiated BK‐induced [Ca²⁺]_i increases in both myenteric neurons and enteric glial cells, which were suppressed by a B1R antagonist. Only in enteric glial cells, a B1R agonist increased [Ca²⁺]_i. The effects of LPS were blocked by pretreatment with an interleukin‐1 receptor antagonist or by reducing the density of enteric glial cells in culture. LPS prompted the release of IL‐1β from enteric glial cells. The augmenting effects of IL‐1β on the BK‐induced neural [Ca²⁺]_i increase and PGE₂ release from enteric glial cells were abolished by a phospholipase A₂ (PLA₂) inhibitor and a COX inhibitor, and partly suppressed by a COX‐2 inhibitor. IL‐1β up‐regulated the COX‐2 expression in enteric glial cells. LPS promotes IL‐1β secretion from enteric glial cells, resulting in augmentation of the neural response to BK through PGE₂ release via glial PLA₂ and COX‐2. The alteration of the regulatory effect of glial cells may be the cause of the changes in neural function in the enteric nervous system in inflammatory bowel disease. © 2009 Wiley‐Liss, Inc.     

Female Sex Hormones Influence the Febrile Response Induced by Lipopolysaccharide,Cytokines and Prostaglandins but not by Interleukin‐1β in Rats

There are differences in the immune response, and particularly fever, between males and females. In the present study, we investigated how the febrile responses induced by lipopolysaccharide (LPS) and different endogenous pyrogens were affected by female gonadal hormones. The febrile response to i.p. injection of LPS (50 μg/kg) was 40% lower in female rats compared to male or ovariectomised (OVX) female rats. Accordingly, oestrogen replacement in OVX animals reduced LPS‐induced fever. Treatment with the prostaglandin synthesis inhibitor indomethacin (2 mg/kg, i.p. 30 min before) reduced the febrile response induced by LPS in both OVX (88%) and sham‐operated (71%) rats. In line with the enhanced fever in OVX rats, there was increased expression of cyclooxygenase‐2 (COX‐2) in the hypothalamus and elevated levels of prostaglandin E₂ (PGE₂). In addition, OVX rats were hyper‐responsive to PGE₂ injected i.c.v. By contrast to the enhanced fever in response to LPS and PGE₂, the febrile response induced by i.c.v. injection of interleukin (IL)‐1β was unaffected by ovariectomy, whereas the responses induced by tumour necrosis factor (TNF)‐α and macrophage inflammatory protein (MIP)‐1α were completely abrogated. These results suggest that the mediators involved in the febrile response in females are similar to males, although the reduction of female hormones may decrease the responsiveness of some mediators such as TNF‐α and MIP‐1α. Compensatory mechanisms may be activated in females after ovariectomy such as an augmented synthesis of COX‐2 and PGE₂.     

Sustained activation of ERK signaling in astrocytes is critical for neuronal injury‐induced monocyte chemoattractant protein‐1 production in rat corticostriatal slice cultures

We previously demonstrated that N‐methyl‐d ‐aspartate (NMDA) treatment (50 μm , 3 h) induced astrocytic production of monocyte chemoattractant protein‐1 (MCP‐1, CCL2), a CC chemokine implicated in ischemic and excitotoxic brain injury, in rat corticostriatal slice cultures. In this study, we investigated the signaling mechanisms for NMDA‐induced MCP‐1 production in slice cultures. The results showed a close correlation between NMDA‐induced neuronal injury and MCP‐1 production, and an abrogation of NMDA‐induced MCP‐1 production in NMDA‐pretreated slices where neuronal cells had been eliminated. These results collectively indicate that NMDA‐induced neuronal injury led to astrocytic MCP‐1 production. NMDA‐induced MCP‐1 production was significantly inhibited by U0126, an inhibitor of extracellular signal‐regulated kinase (ERK). Immunostaining for phosphorylated ERK revealed that transient neuronal ERK activation was initially induced and subsided within 30 min, followed by sustained ERK activation in astrocytes. Treatment with U0126 during only the early phase (U0126 was washed out at 15 or 30 min after NMDA administration) suppressed early activation of ERK in neuronal cells, but not later activation of ERK in astrocytes. In this case, MCP‐1 production was not suppressed, suggesting that activation of neuronal ERK is not necessary for MCP‐1 production. In contrast, delayed application of U0126 at 3 h after the beginning of NMDA treatment inhibited MCP‐1 production to the same degree as that observed when U0126 was applied from 3 h before NMDA administration. These findings suggest that sustained activation of the ERK signaling pathway in astrocytes plays a key role in neuronal injury‐induced MCP‐1 production.     

Microsomal prostaglandin E synthase‐1 is induced in alzheimer's disease and its deletion mitigates alzheimer's disease‐like pathology in a mouse model

Epidemiological studies have suggested that long‐term use of nonsteroidal anti‐inflammatory drugs that inhibit cyclooxygenase (COX) activity can moderate the onset or progression of Alzheimer's disease (AD). Thus it has been suggested that prostaglandin E₂ (PGE₂), a major end‐product of COX, may play a pathogenic role in AD, but the involvement of PGE synthase (PGES), a terminal enzyme downstream from COX, has not been fully elucidated. Here we found that, among three PGES enzymes, only microsomal PGES‐1 (mPGES‐1) is induced, and its expression is associated with β‐amyloid (Aβ) plaques in the cerebral cortex in human AD patients and in Tg2576 mice, a transgenic AD mouse model. Furthermore, to investigate whether mPGES‐1 contributes to AD‐like pathology, we bred mPGES‐1‐deficient mice with Tg2576 mice. We found that mPGES‐1 deletion reduced the accumulation of microglia around senile plaques and attenuated learning impairments in Tg2576 mice. These results indicated that mPGES‐1 is induced in the AD brain and thus plays a role in AD pathology. Blockage of mPGES‐1 could form the basis for a novel therapeutic strategy for patients with AD. Inc. © 2013 Wiley Periodicals, Inc.     

Tripchlorolide protects neuronal cells from microglia‐mediated β‐amyloid neurotoxicity through inhibiting NF‐κB and JNK signaling

Recent research has focused on soluble oligomeric assemblies of β‐amyloid peptides (Aβ) as the proximate cause of neuroinflammation, synaptic loss, and the eventual dementia associated with Alzheimer's disease (AD). In this study, tripchlorolide (T₄), an extract of Tripterygium wilfordii Hook. F (TWHF), was studied as a novel agent to suppress neuroinflammatory process in microglial cells and to protect neuronal cells against microglia‐mediated oligomeric Aβ toxicity. T₄ significantly attenuated oligomeric Aβ(1‐42)‐induced release of inflammatory productions such as tumor necrosis factor‐α, interleukin‐1β, nitric oxide (NO), and prostaglandin E₂. It also downregulated the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in microglial cells. Further molecular mechanism study demonstrated that T₄ inhibited the nuclear translocation of nuclear factor‐κB (NF‐κB) without affecting I‐κBα phosphorylation. It repressed Aβ‐induced JNK phosphorylation but not ERK or p38 MAPK. The inhibition of NF‐κB and JNK by T₄ is correlated with the suppression of inflammatory mediators in Aβ‐stimulated microglial cells. These results suggest that T₄ protects neuronal cells by blocking inflammatory responses of microglial cells to oligomeric Aβ(1‐42) and that T₄ acts on the signaling of NF‐κB and JNK, which are involved in the modulation of inflammatory response. Therefore, T₄ may be an effective agent in modulating neuroinflammatory process in AD. © 2009 Wiley‐Liss, Inc.     

17β‐Oestradiol Anti‐Inflammatory Effects in Primary Astrocytes Require Oestrogen Receptor β‐Mediated Neuroglobin Up‐Regulation

Neuroglobin (Ngb), so named after its initial discovery in brain neurones, has received great attention as a result of its neuroprotective effects both in vitro and in vivo. Recently, we demonstrated that, in neurones, Ngb is a 17β‐oestradiol (E₂) inducible protein that is pivotal for hormone‐induced anti‐apoptotic effects against H₂O₂ toxicity. The involvement of Ngb in other brain cell populations, as well as in other neuroprotective effects of E₂, is completely unknown at present. We demonstrate Ngb immunoreactivity in reactive astrocytes located in the proximity of a penetrating cortical injury in vivo and the involvement of Ngb in the E₂‐mediated anti‐inflammatory effect in primary cortical astrocytes. Upon binding to oestrogen receptor (ER)β, E₂ enhances Ngb levels in a dose‐dependent manner. Although with a lesser degree than E₂, the pro‐inflammatory stimulation with lipopolysaccharide (LPS) also induces the increase of Ngb protein levels via nuclear factor‐(NF)κB signal(s). Moreover, a negative cross‐talk between ER subtypes and NFκB signal(s) has been demonstrated. In particular, ERα‐activated signals prevent the NFκB‐mediated Ngb increase, whereas LPS impairs the ERβ‐induced up‐regulation of Ngb. Therefore, the co‐expression of both ERα and ERβ is pivotal for mediating E₂‐induced Ngb expression in the presence of NFκB‐activated signals. Interestingly, Ngb silencing prevents the effect of E₂ on the expression of inflammatory markers (i.e. interleukin 6 and interferon γ‐inducible protein 10). Ngb can be regarded as a key mediator of the different protective effects of E₂ in the brain, including protection against oxidative stress and the control of inflammation, both of which are at the root of several neurodegenerative diseases.     

IL‐12 p40 homodimer,the so‐called biologically inactive molecule,induces nitric oxide synthase in microglia via IL‐12Rβ1

Earlier we have demonstrated that IL‐12 p40 homodimer (p40₂) induces the expression of inducible nitric oxide synthase (iNOS) in microglia. This study was undertaken to investigate underlying mechanisms required for IL‐12 p40₂‐ and IL‐12 p70‐induced expression of iNOS in microglia. IL‐12 p40₂ alone induced the activation of both extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK). Interestingly, the ERK pathway coupled p40₂ to iNOS expression via C/EBPβ, but not NF‐κB, whereas the p38 pathway relayed the signal from p40₂ to iNOS expression via both NF‐κB and C/EBPβ. Furthermore, by using microglia from IL‐12Rβ1 (?/?) and IL‐12Rβ2 (?/?) mice or siRNA against IL‐12Rβ1 and IL‐12Rβ2, we demonstrate that p40₂ induced the expression of iNOS in microglia via IL‐12Rβ1–(ERK+p38)–(NF‐κB +C/EBPβ) pathway. In contrast, both IL‐12Rβ1 and IL‐12Rβ2 were involved for IL‐12 p70‐induced microglial expression of iNOS. Although IL‐12Rβ1 coupled p70 to NF‐κB and C/EBPβ, IL‐12Rβ2 was responsible for p70‐mediated activation of GAS. This study delineates a new role of IL‐12Rβ1 and IL‐12Rβ2 for the expression of iNOS and production of NO in microglia that may participate in the pathogenesis of neuroinflammatory diseases. © 2009 Wiley‐Liss, Inc.     

Involvement of cannabinoid‐1 and cannabinoid‐2 receptors in septic ileus

Background Cannabinoid (CB) receptors are involved in the regulation of gastrointestinal (GI) motility under physiological and pathophysiological conditions. We aimed to characterize the possible influence of CB₁ and CB₂ receptors on motility impairment in a model of septic ileus. Methods Lipopolysaccharide (LPS) injections were used to mimic pathophysiological features of septic ileus. Spontaneous jejunal myoelectrical activity was measured in rats in vivo, and upper GI transit was measured in vivo by gavaging of a charcoal marker into the stomach of mice, in absence or presence of LPS, and CB₁ and CB₂ receptor agonists and antagonists. Tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 levels were measured using enzyme‐linked immunosorbent assay. Histology was performed with haematoxylin–eosin staining. Key Results Lipopolysaccharide treatment significantly reduced amplitude and frequency of myoelectric spiking activity and GI transit in vivo in a dose‐dependent manner. TNF‐α and IL‐6 were increased in LPS‐treated animals and histology showed oedema and cell infiltration. Both, the CB₁ agonist HU210 and the CB₂ agonist JWH133 reduced myoelectrical activity whereas the CB₁ antagonist AM251 caused an increase of myoelectrical activity. Pretreatment with AM251 or AM630 prevented against LPS‐induced reduction of myoelectrical activity, and also against the delay of GI transit during septic ileus in vivo. Conclusions & Inferences The LPS model of septic ileus impairs jejunal myoelectrical activity and delays GI transit in vivo. Antagonists at the CB₁ receptor or the CB₂ receptor prevent the delay of GI transit and thus may be powerful tools in the future treatment of septic ileus.     

The terminal prostaglandin synthases mPGES‐1, mPGES‐2, and cPGES are all overexpressed in human gliomas

Prostaglandin E2 has been connected to processes promoting tumor growth in several human malignancies including gliomas. The terminal prostaglandin synthases mPGES‐1, mPGES‐2, and cPGES convert PGH2 into prostaglandin E2. The inhibition of their function could significantly reduce PGE2 levels in tumors while avoiding some side effects related to the inhibition of the upstream enzymes COX‐1 and COX‐2. In this study, the immunohistochemical staining of mPGES‐1 and, for the first time, the staining of mPGES‐2 and cPGES are characterized and compared with COX‐1 and COX‐2 staining in the same tumor samples of 94 human gliomas. The main results demonstrate over‐expression of all three proteins, including cPGES and mPGES‐2 that are commonly considered non‐inducible, in both low‐ and high‐grade tumors. For all three proteins, average expression in tumor cells was higher in grade III tumors than grade II tumors. The analysis showed no correlation between tumor grade and staining of tumor cells or vascular endothelium with any of the antibodies except in oligodendrogliomas where moderate correlation (linear correlation coefficient 0.6; P < 0.01) could be found between tumor grade and tumor cell staining with mPGES‐1 and cPGES. In grade II tumors which recurred and were reoperated upon during the data gathering period, average expression of COX‐2, mPGES‐1, and cPGES was higher than in tumors that were operated on only once. Our results demonstrate the significance of all three terminal prostaglandin synthases, mPGES‐1, mPGES‐2, and cPGES, as a possible future target of inhibition in glioma therapy.     

Endothelial microsomal prostaglandin E synthase‐1 exacerbates neuronal loss induced by kainate

Prostaglandin E₂ (PGE₂) is increased in the brain after kainic acid (KA) treatment. We previously demonstrated that KA also induces PG synthase cyclooxygenase‐2 (COX‐2) expression rapidly in neurons of the brain and slowly in astrocytes and endothelia. Prevention of KA‐induced neuronal damage by nonneuronal COX‐2 inhibition suggests a novel modulatory mechanism for neuronal injury by nonneuronal PGs. It remains unclear, however, which PG synthase is responsible for this modulation following COX‐2 synthesis after neuronal insult. In addition, the PG receptor subtype that is involved in neuronal loss remains controversial. Here we demonstrate that microinjection of KA induces microsomal prostaglandin E synthase‐1 (mPGES‐1) in venous endothelial cells but not in neurons or astrocytes. We found that mPGES‐1 plays a central role in delayed production of PGE₂ and that mPGES‐1‐deficient mice exhibit significantly less neuronal loss induced by KA. Furthermore, KA injection caused an increase in the immunoreactivity for the EP3 receptor in the astrocytic endfeet that surround vascular endothelia. Neurons form intimate interactions with astrocytes via glutamate, and astrocytes contact vascular endothelia through endfeet. These findings suggest that endothelial cells may control neuronal excitotoxicity, most likely by regulating astrocytes via inducible PGE₂. © 2009 Wiley‐Liss, Inc.     

The selective oestrogen receptor modulator,bazedoxifene, mimics the neuroprotective effect of 17β‐oestradiol in diabetic ischaemic stroke by modulating oestrogen receptor expression and the MAPK/ERK1/2 signalling pathway

Because neuroprotection in stroke should be revisited in the era of recanalisation, the present study analysed the potential neuroprotective effect of the selective oestrogen receptor modulator, bazedoxifene acetate (BZA), in an animal model of diabetic ischaemic stroke that mimics thrombectomy combined with adjuvant administration of a putative neuroprotectant. Four weeks after induction of diabetes (40 mg kg^‐1 streptozotocin, i.p.), male Wistar rats were subjected to transient middle cerebral artery occlusion (intraluminal thread technique, 60 minutes) and assigned to one of three groups treated with either: vehicle, BZA (3 mg kg^‐1 day^‐1, i.p.) or 17β‐oestradiol (E₂) (100 μg kg^‐1 day^‐1, i.p.). At 24 hours post‐ischaemia‐reperfusion, brain damage (neurofunctional score, infarct size and apoptosis), expression of oestrogen receptors (ER)α, ERβ and G protein‐coupled oestrogen receptor), and activity of the mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK)1/2 and phosphoinositide 3‐kinase/Akt pathways were analysed. At 24 hours after the ischaemic insult, both BZA‐ and E₂‐treated animals showed lower brain damage in terms of improved neurofunctional condition, decreased infarct size and decreased apoptotic cell death. Ischaemia‐reperfusion induced a significant decrease in ERα and ERβ expression without affecting that of G protein‐coupled oestrogen receptor, whereas BZA and E₂ reversed such a decrease. The ischaemic insult up‐regulated the activity of both the MAPK/ERK1/2 and phosphoinositide 3‐kinase/Akt pathways; BZA and E₂ attenuated the increased activity of the ERK1/2 pathway, without affecting that of the Akt pathway. The results of the present study lend further support to the consideration of BZA as an effective and safer alternative overcoming the drawbacks of E₂ with respect to improving diabetic ischaemic stroke outcome after successful reperfusion.     

Reorientation of prostanoid production accompanies “activation” of adult microglial cells in culture

Using morphological, immunocytochemical, and functional parameters we have previously shown that highly purified adult rat microglial cells undergo a process of “activation” when cultured in a serum-containing medium in the absence of added proinflammatory substances or other factors (Slepko and Levi: Glia 16:241–246, 1996). Here we studied the lipopolysaccharide (LPS)-evoked production of two prostanoids, thromboxane A₂ (measured as thromboxane B₂) (TXB₂) and prostaglandin E₂ (PGE₂), as a function of microglial “activation.” LPS induced a greater time- and dose-dependent release of TXB₂, compared to PGE₂, in the less “activated” cells. Further “activation” led to amplified synthesis of PGE₂ and not of TXB₂, so that the TXB₂/PGE₂ ratio changed from 2.2 to 0.25 between the 2nd and 4th day in culture. Western blot experiments showed that the LPS-evoked expression of the inducible form of cyclooxygenase (COX) was markedly higher in cells exhibiting a more “activated” phenotype. The expression of the constitutive isoform of COX was low in all conditions, was slightly greater in more “activated” cells, and was not affected by LPS. Neither progression in microglial “activation” nor LPS treatment enhanced thromboxane synthase activity. We hypothesize that reorientation of prostanoid synthesis toward a major production of PGE₂ in the more “activated” cells can be largely attributed to an increased inducibility of cellular COX expression, combined with the inability of thromboxane synthase to cope with the increased availability of the COX product prostaglandin H₂ (PGH₂), the common precursor of TXA₂ and PGE₂. In view of the different, and at times opposite, functional activity of TXB₂ and PGE₂, the described change in prostanoid production pattern may contribute to the role of “activated” microglia in inflammation and host defense. J. Neurosci. Res. 49:292–300, 1997. © 1997 Wiley-Liss, Inc.     

Modulation of extracellular signal‐related kinase,cyclin D1, glial fibrillary acidic protein,and vimentin expression in estradiol‐pretreated astrocyte cultures treated with competence and progression growth factors

The present study seeks to elucidate the interactions between the “competence” growth factor basic fibroblast growth factor (bFGF) and/or estrogen 17β‐estradiol and the “progression” growth factors epidermal growth factor (EGF), insulin‐like growth factor‐I (IGF‐I), and insulin (INS) on DNA labeling and also cyclin D1, extracellular signal‐related kinase 1/2 (ERK1/2), glial fibrillary acidic protein (GFAP), and vimentin expression in astroglial cultures under different experimental conditions. Pretreatment for 24 hr with bFGF and subsequent exposure for 36 hr to estradiol (E₂) and EGF, IGF‐I, or INS stimulated DNA labeling in the last 12 hr, especially when the cultures were treated with progression growth factors. bFGF pretreatment and subsequent treatment with E₂ for 36 hr stimulated DNA labeling. The 36‐hr E₂ treatment alone did not significantly decrease DNA labeling, but contemporary addition of E₂ with two or three growth factors stimulated DNA labeling remarkably. When E₂ was coadded with growth factors, a significantly increased DNA labeling was observed, demonstrating an astroglial synergistic mitogenic effect evoked by contemporary treatment with growth factors in the presence of estrogens. Cyclin D1 expression was markedly increased when astrocyte cultures were pretreated for 36 hr with E₂ and subsequently treated with two or three competence and progression growth factors. A highly significant increase of ERK1/2 expression was observed after all the treatments (EGF, bFGF, INS, IGF‐I alone or in combination with two or three growth factors). GFAP and vimentin expression was markedly increased when the cultures were treated with two or three growth factors. In conclusion, our data demonstrate estradiol–growth factor cross‐talk during astroglial cell proliferation and differentiation in culture. © 2015 Wiley Periodicals, Inc.     

Differential Kv1.3, KCa3.1, and Kir2.1 expression in “classically” and “alternatively” activated microglia

Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) promote differentiation into classically activated M1‐like microglia, which produce high levels of pro‐inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL‐4 in contrast induces a phenotype associated with anti‐inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K⁺ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL‐4) microglia and studying their K⁺ channel expression by whole‐cell patch‐clamp, quantitative PCR and immunohistochemistry. We identified three major types of K⁺ channels based on their biophysical and pharmacological fingerprints: a use‐dependent, outwardly rectifying current sensitive to the K_V1.3 blockers PAP‐1 and ShK‐186, an inwardly rectifying Ba²⁺‐sensitive K_ir2.1 current, and a Ca²⁺‐activated, TRAM‐34‐sensitive K_Ca3.1 current. Both K_V1.3 and K_Ca3.1 blockers inhibited pro‐inflammatory cytokine production and iNOS and COX2 expression demonstrating that K_V1.3 and K_Ca3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN‐γ microglia exhibited high K_V1.3 current densities (～50 pA/pF at 40 mV) and virtually no K_Ca3.1 and K_ir currents, while microglia differentiated with IL‐4 exhibited large K_ir2.1 currents (～ 10 pA/pF at ?120 mV). K_Ca3.1 currents were generally low but moderately increased following stimulation with IFN‐γ or ATP (～10 pS/pF). This differential K⁺ channel expression pattern suggests that K_V1.3 and K_Ca3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106–121