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Corticotropin-releasing hormone potentiates neural injury induced by oxygen-glucose deprivation: a possible involvement of microglia
Neuroscience Letters 371 (2004) 133–137
Corticotropin-releasing hormone potentiates neural injury induced by oxygen-glucose deprivation: a possible involvement of microglia Wei Wang, Mark Solc, Ping Ji, Kimberly E. Dow∗ Department of Pediatrics, Apps Medical Research Centre, Kingston General Hospital, Queen’s University, Doran 3, Room 6-303, Kingston, Ont., Canada K7L 2V7 Received 10 June 2004; received in revised form 4 August 2004; accepted 24 August 2004
Abstract While corticotropin-releasing hormone (CRH) has been implicated in a variety of brain disorders such as ischemic injury, the molecular mechanism by which CRH elicits its activities is largely unclear. In the present study, we have determined the effect of CRH on oxygen-glucose deprivation (OGD) induced apoptosis in fetal hippocampal neurons. CRH alone at concentrations of 10–200 nM had no effect on neuronal apoptosis. However, when neurons were co-cultured with microglia, CRH alone at concentrations greater than 100 nM induced neuronal apoptosis and CRH potentiated significant neuronal apoptosis following exposure to OGD. The effect of CRH on neuronal apoptosis was inhibited in the presence of the CRH antagonist astressin. Real-time RT–PCR revealed an increase in mRNA levels of Fas ligand (Fas-L), a membrane protein related to the TNF family, in cultured microglia following OGD exposure. In the presence of CRH, OGD-induced Fas-L expression was significantly increased. The effect of CRH on Fas-L expression was inhibited by specific inhibitors of the extracellular signal-regulated protein kinase (PD98059) and p38 mitogen-activated protein kinase (SB203580). These results suggest that CRH potentiates neuronal apoptosis induced by OGD in the presence of microglia and that this effect may be mediated through the induction of proinflammatory mediators in microglia. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Microglia; Corticotropin-releasing hormone; Neurons; Oxygen-glucose deprivation; Apoptosis
CRH, a key mediator of the hypothalamic–pituitary– adrenocortical system response to stress, is up-regulated following multiple forms of brain injury [7,10,21,25,29]. Following brain insults such as excitotoxins, seizures, head injury, and forebrain ischemia, CRH antagonists have been shown to be neuroprotective [14–16,21,24]. A recent study using CRH-R1 receptor deficient mice has shown reduced cerebral injury following focal ischemia [23]. However, several studies have demonstrated a neuroprotective effect of CRH and one study has shown neither a neuroprotective nor a neurotoxic effect of CRH in rat cortical neuron cultures [5,12,19]. The mechanisms for these discrepancies may depend upon many factors such as CRH concentrations used, the specific CRH receptor expressed, and different ∗
Corresponding author. Tel.: +1 613 548 6046; fax: +1 613 548 1369. E-mail address: [email protected] (K.E. Dow).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.08.055
signal pathways activated in different experimental settings. In addition, CRH has been shown to play a role as a proinflammatory mediator in local inflammatory sites and in the central nervous system [1,3,11,13,28]. Thus, the effect of CRH on brain injury may result from its effect on the modulation of proinflammatory mediators released from brain astrocytes and microglia. Using an in vitro model of hypoxia/ischemia, we have determined the effect of CRH on neuronal apoptosis in cultured hippocampal neurons and on Fas ligand (Fas-L) expression in cultured microglia. Primary hippocampal neuron cultures were established from 19-day fetal Sprague–Dawley rat embryos as described previously, with modification [25]. Hippocampi were dissociated and the cells were plated in poly-d-lysine (0.1 mg/ml)coated dishes or plates in Dulbecco’s minimal essential medium containing 10% fetal calf serum. The medium was changed to neuron-defined, serum-free Neurobasal Medium
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(Gibco BRL) supplemented with B27, 25 M glutamate, 1 mM glutamine, 100 IU/ml penicillin and 100 g/ml streptomycin on the second day and maintained for 3 days at 37 ◦ C in a humidified 5% CO2 /95% air incubator. The medium was changed again and used for all experiments at days 8–10. Under this condition, purity of hippocampal neuron cultures is >98%. Cultures of microglia were established as described previously [26]. For co-cultures using transwell techniques, which permits cell-contact-independent communication via diffusible soluble factors only, freshly isolated microglia were plated in transwell inserts at 5 × 104 cells/insert. The inserts then were plated on top of neuron cultures in 24-well plates and subsequent experiments were performed. In vitro OGD experiments were performed as described previously with modifications [25]. Cultured hippocampal neurons in serum-free Neurobasal Medium were washed three times with a large quantity of phosphate buffered saline and replaced with deoxygenated glucose-free balanced salt solution. The cultures with or without microglia in the inserts were then placed into a chamber flushed with 0.5% CO2 /balance N2 until the O2 concentration fell below 0.1%. To reoxygenate neurons, the cultures were removed from the chamber after the designated exposure time and the culture medium was replaced with stored serumfree Neurobasal Medium and returned to normal incubating conditions. Neuronal apoptosis was assessed by quantification of DNA/histone mono- and oligonucleosomes using a sandwich enzyme-linked immunosorbent assay (ELISA) method (Cell Death Detection ELISAPLUS , Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions. Total RNA from cultured cells was isolated and onestep quantitative real-time RT–PCR was performed to determine mRNA expression levels using the QuantiTect SYBR® green RT–PCR kit (Qiagen, Inc., Mississauga, Ont.). Each reaction consisted of 0.25 g of total RNA, 12.5 l of 2× SYBR green PCR Master Mix, 1 l of sense and antisense primers, 0.25 l of QuantiTect® RT Mix. Quantitative RT–PCR was performed on a Cepheid Smart Cycler II (Cepheid, Sunnyvale, CA) by using 3-stage program parameters provided by the manufacturer as follows: 30 min at 50 ◦ C, 15 min at 95 ◦ C, and then 40 cycles of 15 s at 95 ◦ C, 30 s at 55 ◦ C, and 30 s at 72 ◦ C. Quantification of mRNA levels was performed by determining the thread hold cycle (CT ), which serves as a tool for calculation of the starting template amount in each sample. Results were analysed using the comparative method. The values were normalized to the housekeeping gene L32 by subtracting mean CT from mean target CT for each sample and converted into fold changes based on a doubling of PCR product in each PCR cycle according to the manufacturer’s guidelines. The sequence-specific primers for Fas-L: forward, 5 aaagaccacaaggtccaaca-3 ; reverse, 5 -cacagcagcccaaaacttta. For L32: forward, 5 -tgtcctctaagaaccgaaaagcc-3 ; reverse, 5 cgttgggattggtgactctga-3 . The oligonucleotide primers were synthesized from Qiagen, Inc.
Fig. 1. Effect of CRH on neuronal apoptosis in the absence (A) or presence (B) of microglia (MG). Hippocampal neurons were cultured with or without microglia in transwells and treated with CRH at different concentrations as indicated for 24 h. Neuronal apoptosis was determined by the apoptotic ELISA assay. Results are taken from three independent cultures. Data are mean ± S.D. ∗ p < 0.05 vs. neurons without CRH added.
Data are presented as means ± standard deviations. All experiments were repeated at least three times. Differences between groups were compared by student’s t test and analysis of variance or by Kruskal–Wallis one-way analysis of variance on ranks using SigmaStat software. Differences were considered to be statistically significant at a P value of <0.05. In the present study, the effect of CRH on neuronal apoptosis in cultured hippocampal neurons was first determined using a sandwich apoptotic ELISA assay. Addition of CRH at concentrations up to 200 nM had no effect on neuronal apoptosis. However, when neurons were co-cultured with microglia, CRH at concentrations of 100 to 200 nM induced significant neuronal apoptosis (approximately 20%, Fig. 1). The effect of CRH on apoptotic neuronal death was also observed by in situ terminal transferase-dUTP nick end labeling (TUNEL) assay. TUNEL staining and typical photomicrographs of TUNEL-positive cells are shown in Fig. 2. When hippocampal neurons were treated with 100 nM CRH, the number of TUNEL-positive cells was significantly increased in the presence of microglia (Fig. 2B) compared with in the absence of microglia (Fig. 2A). To determine the effect of CRH on OGD-induced neuronal apoptosis in the absence or presence of microglia, cultured neurons were first exposed to OGD and neuronal apoptosis was assessed by apoptotic ELISA assay. Exposure of cultured neurons to OGD for 2.5 h resulted in a 27% increase of fragmented chromatin compared with control neurons as determined by apoptotic ELISA assay (Fig. 3). Treatment of cultured neurons with 100 nM CRH during exposure to OGD did not influence OGD-induced neuronal apoptosis. However, in the presence
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Fig. 4. Effect of CRH on expression of Fas-L in cultured microglia. Cultured microglia were exposed to OGD for 6 h in the presence or absence of additives as indicated (100 nM CRH; 200 nM astressin, A; 50 M SB203580, SB; 50 M PD98059, PD). The mRNA levels of Fas-L were determined by real-time RT–PCR as described. Data are shown as Fas-L mRNA expression relative to L32 and the values of control have been normalized to 1. Results are given as the mean ± S.D. of values taken from three independent cultures. ∗ p < 0.05 vs. control; # p < 0.05 vs. OGD alone; and ˆ p < 0.05 vs. CRH with OGD.
Fig. 2. Effect of CRH on neuronal apoptosis in the absence (A) or presence (B) of microglia as determined by TUNEL staining. The image shown is representive of three separate experiments.
of microglia, 100 nM CRH significantly potentiated OGDinduced neuronal apoptosis (Fig. 3). The effect of CRH on neuronal apoptosis was inhibited in the presence of 200 nM of CRH antagonist astressin.
Fig. 3. Effect of CRH on OGD-induced neuronal apoptosis in the presence or absence of microglia (MG). Hippocampal neurons with or without microglia in transwells were exposed to OGD for 2.5 h in the presence or absence of 100 nM CRH or 200 nM astressin (A). Neuronal apoptosis was determined at 24 h following OGD exposure. Results are taken from three independent cultures. Data are mean ± S.D. ∗ p < 0.05 vs. control; # p < 0.05 vs. OGD with microglia; ˆ p < 0.05 vs. CRH with microglia.
The mechanisms by which CRH potentiates OGDinduced neuronal apoptosis in the presence of microglia are not clear. We have previously reported that exogenous administration of CRH promotes cell proliferation and induces TNF-␣ release in rat brain microglia that express the CRH receptor 1 and that these effects are possibly mediated via activation of the extracellular signal-regulated protein kinase (ERK1/2) and p38 mitogen-activated protein kinase (p38) [27]. In the present study, we further determined the effect of CRH on the expression of Fas-L, a membrane protein related to the TNF family, in cultured microglia using quantitative real-time RT–PCR techniques. CRH alone at concentrations up to 200 nM did not significantly effect Fas-L expression in cultured microglia but Fas-L expression was increased following exposure of cultured microglia to OGD for 6 h (Fig. 4). In the presence of CRH at 100 nM, mRNA levels of Fas-L were significantly increased following OGD exposure in microglial cultures and the effect of CRH on Fas-L expression was blocked by 200 nM astressin. To determine whether mitogen-activated protein kinase pathways are involved in the effect of CRH on Fas-L expression, microglial cells were treated with upstream inhibitors of ERK1/2 (PD98059) or p38 kinase (SB203580) at concentrations of 50 M for 1 h prior to the addition of CRH and OGD exposure. Both PD98059 and SB203580 attenuated CRH-mediated Fas-L expression (Fig. 4). It is well documented that inflammatory responses play an important role in mediating brain ischemic damage after an initial insult. These inflammatory responses involve activation of brain glial and microglial cells and production of proinflammatory cytokines. As demonstrated by our previous findings, microglia express numerous receptors for CNS signaling molecules including the CRH receptor [25,26]. Following brain injury, a number of proinflammatory mediators including TNF-␣ and Fas-L are released and neu-
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ronal death is accentuated by these mediators. Among them, the interaction of Fas and Fas-L is one of the major signal pathways mediating apoptotic cell death in a variety of cells and tissues. Studies have demonstrated that Fas and Fas-L play an important role in mediating delayed neuronal cell apoptosis after cerebral ischemia [18,22]. Fas-L is expressed in rat and human neurons, astrocytes and microglia [2,4]. Brain ischemic injury and oxidative stress increased Fas and Fas-L expression [8,9]. Recently, studies have also reported the effect of CRH on the regulation of Fas-L expression [6,17,20]. In undifferentiated PC12 cells that undergo apoptosis following serum deprivation, CRH induces FasL production and apoptosis via activation of p38 mitogenactivated protein kinase. The CRH-induced apoptotic effects are mediated through CRH-R1 [6]. Outside of the CNS, CRH has been shown to increase Fas-L expression in human extravillous trophoblast cells, probably mediated by the CRH-R1 receptor [17]. However, no data are available regarding the effect of CRH on Fas-L expression in central neural cells. The present study demonstrates that in addition to TNF-␣, CRH can upregulate the expression of Fas-L in cultured microglia in the presence of hypoxia/ischemia. Thus, by acting on the CRH-R1 receptor expressed in microglia, CRH may induce production of proinflammatory mediators such as TNF-␣ and Fas-L from microglia and subsequently promote neuronal apoptosis following brain ischemic injury. In conclusion, the results reported in this study suggest that CRH promotes Fas-L expression in microglia. The increased Fas-L may mediate CRH-potentiated neuronal apoptosis following OGD exposure. The study suggests that CRH increases neural death after brain injury via the induction of proinflammatory mediators in microglia. The interactions between CRH and proinflammatory mediators in brain may play a critical role in hypoxic/ischemic insult.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgements This work was supported by the Heart and Stroke Foundation of Ontario (NA5024 to K. Dow) and the Hospital for Sick Children (XG 02-064 to K. Dow).
[16]
[17]
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Corticotropin-releasing hormone potentiates neural injury induced by oxygen-glucose deprivation: a possible involvement of microglia Wei Wang, Mark Solc, Ping Ji, Kimberly E. Dow∗ Department of Pediatrics, Apps Medical Research Centre, Kingston General Hospital, Queen’s University, Doran 3, Room 6-303, Kingston, Ont., Canada K7L 2V7 Received 10 June 2004; received in revised form 4 August 2004; accepted 24 August 2004
Abstract While corticotropin-releasing hormone (CRH) has been implicated in a variety of brain disorders such as ischemic injury, the molecular mechanism by which CRH elicits its activities is largely unclear. In the present study, we have determined the effect of CRH on oxygen-glucose deprivation (OGD) induced apoptosis in fetal hippocampal neurons. CRH alone at concentrations of 10–200 nM had no effect on neuronal apoptosis. However, when neurons were co-cultured with microglia, CRH alone at concentrations greater than 100 nM induced neuronal apoptosis and CRH potentiated significant neuronal apoptosis following exposure to OGD. The effect of CRH on neuronal apoptosis was inhibited in the presence of the CRH antagonist astressin. Real-time RT–PCR revealed an increase in mRNA levels of Fas ligand (Fas-L), a membrane protein related to the TNF family, in cultured microglia following OGD exposure. In the presence of CRH, OGD-induced Fas-L expression was significantly increased. The effect of CRH on Fas-L expression was inhibited by specific inhibitors of the extracellular signal-regulated protein kinase (PD98059) and p38 mitogen-activated protein kinase (SB203580). These results suggest that CRH potentiates neuronal apoptosis induced by OGD in the presence of microglia and that this effect may be mediated through the induction of proinflammatory mediators in microglia. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Microglia; Corticotropin-releasing hormone; Neurons; Oxygen-glucose deprivation; Apoptosis
CRH, a key mediator of the hypothalamic–pituitary– adrenocortical system response to stress, is up-regulated following multiple forms of brain injury [7,10,21,25,29]. Following brain insults such as excitotoxins, seizures, head injury, and forebrain ischemia, CRH antagonists have been shown to be neuroprotective [14–16,21,24]. A recent study using CRH-R1 receptor deficient mice has shown reduced cerebral injury following focal ischemia [23]. However, several studies have demonstrated a neuroprotective effect of CRH and one study has shown neither a neuroprotective nor a neurotoxic effect of CRH in rat cortical neuron cultures [5,12,19]. The mechanisms for these discrepancies may depend upon many factors such as CRH concentrations used, the specific CRH receptor expressed, and different ∗
Corresponding author. Tel.: +1 613 548 6046; fax: +1 613 548 1369. E-mail address: [email protected] (K.E. Dow).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.08.055
signal pathways activated in different experimental settings. In addition, CRH has been shown to play a role as a proinflammatory mediator in local inflammatory sites and in the central nervous system [1,3,11,13,28]. Thus, the effect of CRH on brain injury may result from its effect on the modulation of proinflammatory mediators released from brain astrocytes and microglia. Using an in vitro model of hypoxia/ischemia, we have determined the effect of CRH on neuronal apoptosis in cultured hippocampal neurons and on Fas ligand (Fas-L) expression in cultured microglia. Primary hippocampal neuron cultures were established from 19-day fetal Sprague–Dawley rat embryos as described previously, with modification [25]. Hippocampi were dissociated and the cells were plated in poly-d-lysine (0.1 mg/ml)coated dishes or plates in Dulbecco’s minimal essential medium containing 10% fetal calf serum. The medium was changed to neuron-defined, serum-free Neurobasal Medium
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(Gibco BRL) supplemented with B27, 25 M glutamate, 1 mM glutamine, 100 IU/ml penicillin and 100 g/ml streptomycin on the second day and maintained for 3 days at 37 ◦ C in a humidified 5% CO2 /95% air incubator. The medium was changed again and used for all experiments at days 8–10. Under this condition, purity of hippocampal neuron cultures is >98%. Cultures of microglia were established as described previously [26]. For co-cultures using transwell techniques, which permits cell-contact-independent communication via diffusible soluble factors only, freshly isolated microglia were plated in transwell inserts at 5 × 104 cells/insert. The inserts then were plated on top of neuron cultures in 24-well plates and subsequent experiments were performed. In vitro OGD experiments were performed as described previously with modifications [25]. Cultured hippocampal neurons in serum-free Neurobasal Medium were washed three times with a large quantity of phosphate buffered saline and replaced with deoxygenated glucose-free balanced salt solution. The cultures with or without microglia in the inserts were then placed into a chamber flushed with 0.5% CO2 /balance N2 until the O2 concentration fell below 0.1%. To reoxygenate neurons, the cultures were removed from the chamber after the designated exposure time and the culture medium was replaced with stored serumfree Neurobasal Medium and returned to normal incubating conditions. Neuronal apoptosis was assessed by quantification of DNA/histone mono- and oligonucleosomes using a sandwich enzyme-linked immunosorbent assay (ELISA) method (Cell Death Detection ELISAPLUS , Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions. Total RNA from cultured cells was isolated and onestep quantitative real-time RT–PCR was performed to determine mRNA expression levels using the QuantiTect SYBR® green RT–PCR kit (Qiagen, Inc., Mississauga, Ont.). Each reaction consisted of 0.25 g of total RNA, 12.5 l of 2× SYBR green PCR Master Mix, 1 l of sense and antisense primers, 0.25 l of QuantiTect® RT Mix. Quantitative RT–PCR was performed on a Cepheid Smart Cycler II (Cepheid, Sunnyvale, CA) by using 3-stage program parameters provided by the manufacturer as follows: 30 min at 50 ◦ C, 15 min at 95 ◦ C, and then 40 cycles of 15 s at 95 ◦ C, 30 s at 55 ◦ C, and 30 s at 72 ◦ C. Quantification of mRNA levels was performed by determining the thread hold cycle (CT ), which serves as a tool for calculation of the starting template amount in each sample. Results were analysed using the comparative method. The values were normalized to the housekeeping gene L32 by subtracting mean CT from mean target CT for each sample and converted into fold changes based on a doubling of PCR product in each PCR cycle according to the manufacturer’s guidelines. The sequence-specific primers for Fas-L: forward, 5 aaagaccacaaggtccaaca-3 ; reverse, 5 -cacagcagcccaaaacttta. For L32: forward, 5 -tgtcctctaagaaccgaaaagcc-3 ; reverse, 5 cgttgggattggtgactctga-3 . The oligonucleotide primers were synthesized from Qiagen, Inc.
Fig. 1. Effect of CRH on neuronal apoptosis in the absence (A) or presence (B) of microglia (MG). Hippocampal neurons were cultured with or without microglia in transwells and treated with CRH at different concentrations as indicated for 24 h. Neuronal apoptosis was determined by the apoptotic ELISA assay. Results are taken from three independent cultures. Data are mean ± S.D. ∗ p < 0.05 vs. neurons without CRH added.
Data are presented as means ± standard deviations. All experiments were repeated at least three times. Differences between groups were compared by student’s t test and analysis of variance or by Kruskal–Wallis one-way analysis of variance on ranks using SigmaStat software. Differences were considered to be statistically significant at a P value of <0.05. In the present study, the effect of CRH on neuronal apoptosis in cultured hippocampal neurons was first determined using a sandwich apoptotic ELISA assay. Addition of CRH at concentrations up to 200 nM had no effect on neuronal apoptosis. However, when neurons were co-cultured with microglia, CRH at concentrations of 100 to 200 nM induced significant neuronal apoptosis (approximately 20%, Fig. 1). The effect of CRH on apoptotic neuronal death was also observed by in situ terminal transferase-dUTP nick end labeling (TUNEL) assay. TUNEL staining and typical photomicrographs of TUNEL-positive cells are shown in Fig. 2. When hippocampal neurons were treated with 100 nM CRH, the number of TUNEL-positive cells was significantly increased in the presence of microglia (Fig. 2B) compared with in the absence of microglia (Fig. 2A). To determine the effect of CRH on OGD-induced neuronal apoptosis in the absence or presence of microglia, cultured neurons were first exposed to OGD and neuronal apoptosis was assessed by apoptotic ELISA assay. Exposure of cultured neurons to OGD for 2.5 h resulted in a 27% increase of fragmented chromatin compared with control neurons as determined by apoptotic ELISA assay (Fig. 3). Treatment of cultured neurons with 100 nM CRH during exposure to OGD did not influence OGD-induced neuronal apoptosis. However, in the presence
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Fig. 4. Effect of CRH on expression of Fas-L in cultured microglia. Cultured microglia were exposed to OGD for 6 h in the presence or absence of additives as indicated (100 nM CRH; 200 nM astressin, A; 50 M SB203580, SB; 50 M PD98059, PD). The mRNA levels of Fas-L were determined by real-time RT–PCR as described. Data are shown as Fas-L mRNA expression relative to L32 and the values of control have been normalized to 1. Results are given as the mean ± S.D. of values taken from three independent cultures. ∗ p < 0.05 vs. control; # p < 0.05 vs. OGD alone; and ˆ p < 0.05 vs. CRH with OGD.
Fig. 2. Effect of CRH on neuronal apoptosis in the absence (A) or presence (B) of microglia as determined by TUNEL staining. The image shown is representive of three separate experiments.
of microglia, 100 nM CRH significantly potentiated OGDinduced neuronal apoptosis (Fig. 3). The effect of CRH on neuronal apoptosis was inhibited in the presence of 200 nM of CRH antagonist astressin.
Fig. 3. Effect of CRH on OGD-induced neuronal apoptosis in the presence or absence of microglia (MG). Hippocampal neurons with or without microglia in transwells were exposed to OGD for 2.5 h in the presence or absence of 100 nM CRH or 200 nM astressin (A). Neuronal apoptosis was determined at 24 h following OGD exposure. Results are taken from three independent cultures. Data are mean ± S.D. ∗ p < 0.05 vs. control; # p < 0.05 vs. OGD with microglia; ˆ p < 0.05 vs. CRH with microglia.
The mechanisms by which CRH potentiates OGDinduced neuronal apoptosis in the presence of microglia are not clear. We have previously reported that exogenous administration of CRH promotes cell proliferation and induces TNF-␣ release in rat brain microglia that express the CRH receptor 1 and that these effects are possibly mediated via activation of the extracellular signal-regulated protein kinase (ERK1/2) and p38 mitogen-activated protein kinase (p38) [27]. In the present study, we further determined the effect of CRH on the expression of Fas-L, a membrane protein related to the TNF family, in cultured microglia using quantitative real-time RT–PCR techniques. CRH alone at concentrations up to 200 nM did not significantly effect Fas-L expression in cultured microglia but Fas-L expression was increased following exposure of cultured microglia to OGD for 6 h (Fig. 4). In the presence of CRH at 100 nM, mRNA levels of Fas-L were significantly increased following OGD exposure in microglial cultures and the effect of CRH on Fas-L expression was blocked by 200 nM astressin. To determine whether mitogen-activated protein kinase pathways are involved in the effect of CRH on Fas-L expression, microglial cells were treated with upstream inhibitors of ERK1/2 (PD98059) or p38 kinase (SB203580) at concentrations of 50 M for 1 h prior to the addition of CRH and OGD exposure. Both PD98059 and SB203580 attenuated CRH-mediated Fas-L expression (Fig. 4). It is well documented that inflammatory responses play an important role in mediating brain ischemic damage after an initial insult. These inflammatory responses involve activation of brain glial and microglial cells and production of proinflammatory cytokines. As demonstrated by our previous findings, microglia express numerous receptors for CNS signaling molecules including the CRH receptor [25,26]. Following brain injury, a number of proinflammatory mediators including TNF-␣ and Fas-L are released and neu-
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ronal death is accentuated by these mediators. Among them, the interaction of Fas and Fas-L is one of the major signal pathways mediating apoptotic cell death in a variety of cells and tissues. Studies have demonstrated that Fas and Fas-L play an important role in mediating delayed neuronal cell apoptosis after cerebral ischemia [18,22]. Fas-L is expressed in rat and human neurons, astrocytes and microglia [2,4]. Brain ischemic injury and oxidative stress increased Fas and Fas-L expression [8,9]. Recently, studies have also reported the effect of CRH on the regulation of Fas-L expression [6,17,20]. In undifferentiated PC12 cells that undergo apoptosis following serum deprivation, CRH induces FasL production and apoptosis via activation of p38 mitogenactivated protein kinase. The CRH-induced apoptotic effects are mediated through CRH-R1 [6]. Outside of the CNS, CRH has been shown to increase Fas-L expression in human extravillous trophoblast cells, probably mediated by the CRH-R1 receptor [17]. However, no data are available regarding the effect of CRH on Fas-L expression in central neural cells. The present study demonstrates that in addition to TNF-␣, CRH can upregulate the expression of Fas-L in cultured microglia in the presence of hypoxia/ischemia. Thus, by acting on the CRH-R1 receptor expressed in microglia, CRH may induce production of proinflammatory mediators such as TNF-␣ and Fas-L from microglia and subsequently promote neuronal apoptosis following brain ischemic injury. In conclusion, the results reported in this study suggest that CRH promotes Fas-L expression in microglia. The increased Fas-L may mediate CRH-potentiated neuronal apoptosis following OGD exposure. The study suggests that CRH increases neural death after brain injury via the induction of proinflammatory mediators in microglia. The interactions between CRH and proinflammatory mediators in brain may play a critical role in hypoxic/ischemic insult.
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Acknowledgements This work was supported by the Heart and Stroke Foundation of Ontario (NA5024 to K. Dow) and the Hospital for Sick Children (XG 02-064 to K. Dow).
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