Interleukin-1ß promotes the neurogenesis of carotid bodies by stimulating the activation of ERK1/2.

The carotid body (CB) is a complex sensory organ that functions to sense homeostatic O2 in the blood. Previous studies have shown that CBs express interleukin (IL)-1 receptor type I and that the chemosensitivity of CBs is increased following stimulation with pro-inflammatory cytokines. However, the effects of pro-inflammatory cytokines, such as IL-1ß, on the neurogenesis of CB are unclear. Thus, in this study, we aimed to assess the effects of IL-1ß and intermittent hypobaric hypoxia (IHH) plus IL-1ß on the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, tyrosine hydroxylase (TH) and the expression of nestin, a well-established stem cell marker in the nervous system. The results showed that TH, nestin expression and ERK1/2 phosphorylation were increased in the rat CB following intraperitoneal injection of IL-1ß. Moreover, IL-1ß had additive effects on IHH. These results suggested that the plasticity of CB was increased following treatment with IL-1ß and that ERK1/2 may be involved in neurogenic signaling in CBs.

Sertraline promotes hippocampus-derived neural stem cells differentiating into neurons but not glia and attenuates LPS-induced cellular damage.

Sertraline is one of the most commonly used antidepressants in clinic. Although it is well accepted that sertraline exerts its action through inhibition of the reuptake of serotonin at presynaptic site in the brain, its effect on the neural stem cells (NSCs) has not been well elucidated. In this study, we utilized NSCs separated from the hippocampus of fetal rat to investigate the effect of sertraline on the proliferation and differentiation of NSCs. The study demonstrated that sertraline had no effect on NSCs proliferation but it significantly promoted NSCs to differentiate into serotoninergic neurons other than glia cells. Furthermore, we found that sertraline protected NSCs against the lipopolysaccharide-induced cellular damage. These data indicate that sertraline can promote neurogenesis and protect the viability of neural stem cells.

A therapeutic strategy for choroidal neovascularization based on recruitment of mesenchymal stem cells to the sites of lesions.

Choroidal neovascularization (CNV) is a common cause of severe and irreversible visual loss; however, the treatment of CNV has been hindered by its complex and poorly understood pathogenesis. It has been postulated that bone marrow (BM)-derived cells (BMCs) contribute to CNV, but little is known about the role of mesenchymal stem cells (MSCs) in CNV and their therapeutic potential for CNV treatment. We found that BM-derived MSCs transplanted by intravenous injection into laser-induced CNV mouse models were specifically recruited into CNV lesions, where they differentiated into multiple cell types and participated in the development of neovascularization, without stagnation in other organs. By taking advantage of this recruitment potential, engineered MSCs were used to produce the antiangiogenic pigment epithelial-derived factor (PEDF) at the CNV sites, thereby inhibiting the growth of CNVs and stimulating regressive features. Further studies indicated that the effect may be mediated, at least partly, by retinal pigment epithelial (RPE) cells, which function as important regulators for CNV development. These results suggest that MSCs contribute to CNV and could serve as delivery vehicles of antiangiogenic agents for the treatment of a range of CNV-associated diseases.

Interleukin-6 increases intracellular Ca2+ concentration and induces catecholamine secretion in rat carotid body glomus cells.

Although abundant evidence indicates mutual regulation between the immune and the central nervous systems, how the immune signals are transmitted to the brain is still an unresolved question. In a previous study we found strong expression of proinflammatory cytokine receptors, including interleukin (IL)-1 receptor I and IL-6 receptor alpha in the rat carotid body (CB), a well-known arterial chemoreceptor that senses a variety of chemostimuli in the arterial blood. We demonstrated that IL-1 stimulation increases intracellular calcium ([Ca(2+)](i)) in CB glomus cells, releases ATP, and increases the discharge rate in carotid sinus nerve. To explore the effect of IL-6 on CB, here we examine the effect of IL-6 on [Ca(2+)](i) and catecholamine (CA) secretion in rat CB glomus cells. Calcium imaging showed that extracellular application of IL-6 induced a rise in [Ca(2+)](i) in cultured glomus cells. Amperometry showed that local application of IL-6 evoked CA release from glomus cells. Furthermore, the CA secretory response to IL-6 was blocked by 200 microM Cd(2+), a well-known Ca(2+) channel blocker. Our experiments provide further evidence for the responsiveness of the CB to proinflammatory cytokines and indicate that the CB might play a role in inflammation sensing and transmission of such information to the brain.

[Toll-like receptor 4 expression and cytokine secretion in microglial cells induced by IgG stimulation].

AIM: To investigate the effects of immunoglobulin G (IgG) on the expression of toll-like receptor 4 (TLR4) and secretion of cytokines in microglial cells in vitro. METHODS: Cultured primary rat microglial cells were stimulated with different concentrations of rat IgG (2 mg/L, 20 mg/L, 200 mg/L) and lipopolysaccharide (LPS) 10 mg/L for 24 h, respectively. The TLR4 expression in the microglial cells was examined by immunofluorescence staining and tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) levels in the culture medium were assayed by ELISA. RESULTS: IgG stimulation induced a significant TLR4 expression and TNF-alpha secretion in cultured microglial cells in a dose-dependent manner, while IFN-gamma was not detected in the same medium samples. As a positive control, LPS caused increases of TLR4 expression and both IFN-gamma and TNF-alpha production in the microglial cells. CONCLUSION: TLR4 expression could be induced in microglia in vitro by non-pathogenic protein, IgG from the same species. Therefore, congeneric IgG stimulation might lead to proinflammmatory cytokine production, probably via MyD88-dependent pathway. This finding suggests that TLR4 may play more roles than pathogen recognition of innate immune reactivity, at least in the central nervous system.

Action potential modulates Ca2+-dependent and Ca2+-independent secretion in a sensory neuron.

Neurotransmitter release normally requires calcium triggering. However, the somata of dorsal root ganglion (DRG) neurons possess a calcium-independent but voltage-dependent secretion (CIVDS) in addition to the classic calcium-dependent secretion (CDS). Here, we investigated the physiological role of CIVDS and the contributions of CIVDS and CDS induced by action potentials (APs) in DRG soma. Using membrane capacitance measurements, caged calcium photolysis, and membrane capacitance kinetics analysis, we demonstrated that AP-induced secretion had both CIVDS and CDS components. Following physiological stimuli, the dominant component of AP-induced secretion was either CIVDS for spontaneous firing or CDS for high-intensity stimuli. AP frequency modulates CDS-coupled exocytosis and CIVDS-coupled endocytosis but not CIVDS-coupled exocytosis and CDS-coupled endocytosis. Finally, CIVDS did not contribute to excitatory postsynaptic currents induced by APs in DRG presynaptic terminals in the spinal cord. Thus, CIVDS is probably an essential physiological component of AP-induced secretion in the soma. These findings bring novel insights into primary sensory processes in DRG neurons.

[Neuroprotective effects of combined application of JAK-STAT signal pathway inhibitor and free radical scavenger on focal cerebral ischemia/reperfusion injury in rats].

OBJECTIVE: To investigate the neuroprotective effects and dose-response relation by combining JAK-STAT signal pathway inhibitor (AG490) with free radical scavenger dimethylthiourea (DMTU) in rats subjected to focal cerebral ischemia/reperfusion (I/R) injury. METHODS: In all rats, the middle cerebral artery occlusion (MCAO) was produced by occlusion of right internal carotid artery with a nylon monofilament. One hundred male Sprague-Dawley (SD) rats were divided into ten groups according to random digits table, 10 rats were in each group. The first experiment involved I/R model control, dimethyl sulfoxide (DMSO) control, normal saline (NS) control, AG490, DMTU and combination of AG490 and DMTU (A+D) groups. The second experiment involved model group and three experimental groups in which various doses of DMTU and AG490 were administered. The neurological behavior scores (NBS) were assessed at 24, 48 and 72 hours after reperfusion respectively in both experiments, and all the animals were then decapitated to determine the brain infarct volume after 72 hours. RESULTS: The values of NBS in A+D group, AG490 group and DMTU group were higher than those in model group at 24, 48 and 72 hours after I/R, and their brain infarct volumes were obviously smaller than model group as well (all P<0.05). The brain infarct volume in A+D group was obviously smaller compared with AG490 and DMTU alone (all P<0.05). The values of NBS were higher and the brain infarct volumes were smaller in both high dose and medium dose combination groups than those in low dose combination and model groups respectively (all P<0.05). In addition, brain infarct volumes in high dose group were smaller than medium dose group (P<0.05), but there was no statistically significant difference between low dose and model groups. CONCLUSION: The combined application of AG490 and DMTU produces a dose-dependent synergistic neuroprotective effect.

Alterations of Fc gamma receptor I and Toll-like receptor 4 mediate the antiinflammatory actions of microglia and astrocytes after adrenaline-induced blood-brain barrier opening in rats.

Blood-brain barrier (BBB) opening occurs under many physiological and pathological conditions. BBB opening will lead to the leakage of large circulating molecules into the brain parenchyma. These invasive molecules will induce immune responses. Microglia and astrocytes are the two major cell types responsible for immune responses in the brain, and Fc gamma receptor I (FcgammaRI) and Toll-like receptor 4 (TLR4) are the two important receptors mediating these processes. Data suggest that activation of the FcgammaRI pathway mediates antiinflammatory processes, whereas activation of TLR4 pathway leads to proinflammatory activities. In the present study, we tested the hypothesis that BBB opening could lead to alterations in FcgammaRI and TLR4 pathways in microglia and astrocytes, thus limiting excessive inflammation in the brain. The transient BBB opening was induced by adrenaline injection through a caudal vein in Sprague-Dawley rats. We found that the FcgammaRI pathway was significantly activated in both microglia and astrocytes, as exhibited by the up-regulation of FcgammaRI and its key downstream molecule Syk, as well as the increased production of the effector cytokines, interleukin (IL)-10 and IL-4. Interestingly, after transient BBB opening, TLR4 expression was also increased. However, the expression of MyD88, the central adapter of the TLR4 pathway, was significantly inhibited, with decreased production of the effector cytokines IL-12a and IL-1beta. These results indicate that, after transient BBB opening, FcgammaRI-mediated antiinflammatory processes were activated, whereas TLR4-mediated proinflammatory activities were inhibited in microglia and astrocytes. This may represent an important neuroprotective mechanism of microglia and astrocytes that limits excessive inflammation after BBB opening.

Nicotine promotes contribution of bone marrow-derived cells to experimental choroidal neovascularization in mice.

Nicotine can increase size and severity of experimental choroidal neovascularization (CNV); however, the mechanism is uncertain. Recent studies demonstrated that the development of CNV involves the contribution of bone marrow-derived cells (BMCs). This study aims to investigate the effects and the potential mechanism of nicotine on BMCs' contribution to CNV. Green fluorescent protein (GFP) chimeric mice were developed by transplanting bone marrow cells from GFP transgenic mice to C57BL/6J mice. CNV was induced by lasering. Nicotine was administered orally in drinking water. Histopathologic study and choroidal flatmount were performed to measure the CNV severity and BMCs recruitment. BMCs expressing different cell markers in CNV and local expressions of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and vascular cell adhesion molecule-1 (VCAM-1) were detected by immunofluorescence. Nicotine administration resulted in larger diameter and surface area of CNV (P<0.05). Nicotine-exposed mice demonstrated increased area and density of GFP+ cells, increased GFP+ vascular cells area, and decreased ratio of BMCs expressing F4/80 in CNV (P<0.05). Furthermore, the expression of VEGF and bFGF within CNV and VCAM-1 in choroid beneath CNV was up-regulated in nicotine-exposed mice. Our results suggest that nicotine promotes recruitment and incorporation of BMCs into CNV and affects differentiation of BMCs in CNV. These effects may be partly due to indirect actions of nicotine on BMCs via other factors (e.g. VEGF or VCAM-1). It is helpful to understand the mechanism of the effect of nicotine in CNV development.

Lipopolysaccharide up-regulates IL-6R alpha expression in cultured leptomeningeal cells via activation of ERK1/2 pathway.

To clarify the response of leptomeningeal cells to immune stimulation, the effect of lipopolysaccharide (LPS) on expression of IL-6 receptors in the cultured leptomeningeal cells was investigated. The results showed that the expression of IL-6R alpha was invisible in the purified leptomeningeal cells while it was seen in the cells when they were co-cultured with astrocytes. On the other hand, GP130 was moderately expressed in both conditions. Following incubation with different doses of LPS, IL-6R alpha expression in purified leptomeningeal cells was increased in a time- and dose-dependent manner, while GP130 level remained unchanged. Concomitantly, phosphorylated ERK1/2 level was increased following LPS stimulation and its inhibition by PD98059 attenuated the LPS-induced increase of IL-6R alpha expression. These data indicate that leptomeningeal cells can respond to immunogenic stimuli as manifested by expression of cytokine receptors. Moreover, ERK1/2 pathway seems to be involved in the process of LPS-induced IL-6R alpha up-regulation in leptomeningeal cells.

Neuroprotective effects of (-)-epigallocatechin-3-gallate (EGCG) on paraquat-induced apoptosis in PC12 cells.

Green tea, owing to its beneficial effect on health, is becoming more and more popular worldwide. (-)-Epigallocatechin-3-gallate (EGCG), the main ingredient of green tea polyphenols, is a known protective effect on injured neurons in neurodegenerative disease, such as Alzheimer's disease and Parkinson's disease. Paraquat (PQ) is a widely used herbicide that possesses a similar structure to MPP(+) and is toxic to mesencephalic dopaminergic neurons. In the present study, PQ-injured PC12 cells were chosen as an in vitro cell model of Parkinson's disease and the neuroprotective effects of EGCG were investigated. The results showed that EGCG attenuated apoptosis of PC12 cells induced by PQ. The possible mechanism may be associated with maintaining mitochondrial membrane potential, inhibiting caspase-3 activity and downregulating the expression of pro-apoptotic protein Smac in cytosol. The present study supports the notion that EGCG could be used as a neuroprotective agent for treatment of neurodegenerative diseases.

Up-regulation of IL-1 receptor type I and tyrosine hydroxylase in the rat carotid body following intraperitoneal injection of IL-1beta.

It is well established that reciprocal modulation exists between the central nervous system and immune system. Interleukin (IL)-1beta, a proinflammatory cytokine secreted at early stage of immune challenge, has been recognized as one of the informational molecules in immune-to-brain communication. However, how this large molecule is transmitted to the brain is still unknown. In recent years it has been reported that the cranial nerves, especially the vagus, may play a pivotal role in this regard. It is proposed that IL-1beta may bind to its corresponding receptors located in the glomus cells of the vagal paraganglia and then elicit action potentials in the nerve. The existence of IL-1 receptor type I (IL-1RI) in the vagal paraganglia has been shown. The carotid body, which is the largest peripheral chemoreceptive organ, is also a paraganglion. We hypothesize that the carotid body might play a role similar to the vagal paraganglia because they are architectonically similar. Recently we verified the presence of IL-1RI in the rat carotid body and observed increase firing in the carotid sinus nerve following IL-1beta stimulation. The aim of this study was to observe the changes in expression of IL-1RI and tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, in the glomus cells of the rat carotid body following intraperitoneal injection of IL-1beta. The radioimmunoassay result showed that the blood IL-1beta level was increased after the intraperitoneal injection of rmIL-1beta (750 ng/kg) from 0.48+/-0.08 to 0.78+/-0.07 ng/ml (P<0.05). Immunofluorescence and Western blot analysis showed that the expression of IL-1RI and TH in the rat carotid body was increased significantly following peritoneal IL-1beta stimulation. In addition, double immunofluorescence labeling for TH and PGP9.5, a marker for glomus cells, or TH immunofluoresence with hematoxylin-eosin (HE) counterstaining revealed that a considerable number of glomus cells did not display TH immunoreactivity. These data provide morphological evidence for the response of the carotid body to proinflammatory cytokine stimulation. The results also indicate that not all of the glomus cells express detectable TH levels either in normal or in some abnormal conditions.

Coordinated expression of Ets-1, pERK1/2, and VEGF in retina of streptozotocin-induced diabetic rats.

PURPOSE: To investigate the role played by E26 transformation-specific-1 (Ets-1), a transcription factor, and extracellular signal-regulated kinase 1/2 (ERK1/2) in the expression of vascular endothelial growth factor (VEGF), and the interaction of Ets-1 and ERK1/2 in the retina of diabetic rats. METHODS: Diabetes was induced in rats by an intraperitoneal injection of streptozotocin (STZ). To follow the time course in the expression of Ets-1, phosphorylated ERK1/2 (pERK1/2), and VEGF, rats were killed at 1, 2, 4, and 8 weeks after the injection of STZ, and total proteins were extracted from the isolated retinas. An adenovirus vector encoding dominant-negative Ets-1 and an inhibitor of PD98059 was injected intravitreally to investigate the effects of Ets-1 blockade and ERK1/2 inhibition on the expression of VEGF. Four weeks after the first intravitreal injection, total proteins and total RNA were extracted from the retinas for Western blot and Northern blot analyses. RESULTS: The expression of Ets-1, pERK1/2, and VEGF in the retina increased in a time-dependent manner after STZ injection. The phosphorylation of ERK1/2 and protein level of VEGF were significantly reduced following intravitreal Ets-1. Inhibition of ERK1/2 phosphorylation resulted in a significant reduction in the expression of Ets-1 and the level of VEGF protein. CONCLUSIONS: These results indicate that in the retina of STZ-induced diabetic rats: (1) the alterations of Ets-1, pERK1/2, and VEGF are approximately synchronized; (2) the phosphorylation of ERK1/2 is regulated by the expression of Ets-1; (3) the production of Ets-1 protein is dependent on the ERK1/2 pathway, and (4) the protein level of VEGF is regulated by both Ets-1 expression and ERK1/2 phosphorylation. We propose that VEGF, Ets-1, and ERK1 act synergistically in the development of diabetic retinopathy.

IL-1beta inhibits IK and increases [Ca2+]i in the carotid body glomus cells and increases carotid sinus nerve firings in the rat.

Increasing evidence indicates that there exists a reciprocal communication between the immune system and the brain. Interleukin 1beta (IL-1beta), a proinflammatory cytokine produced during immune challenge, is believed to be one of the mediators of immune-to-brain communication, but how it gets into the brain is unknown because of its large molecular weight and difficulty in crossing the blood-brain barrier. Our previous work has demonstrated that IL-1 receptor type I is strongly expressed in the glomus cells of rat carotid body (CB), a well characterized polymodal chemoreceptive organ which serves not only for the detection of hypoxia, hypercapnia and acidity, but also for low temperature and blood glucose. The present study was designed to test whether IL-1beta could stimulate the CB glomus cells and alter the discharge properties in the carotid sinus nerve, the afferent nerve innervating the organ. The results from whole-cell patch-clamp recordings and calcium imaging showed that extracellular application of IL-1beta significantly decreased the outward potassium current and triggered a transient rise in [Ca(2+)](i) in the cultured glomus cells of rat CB. Furthermore, by using extracellular recordings and pharmacological intervention, it was found that IL-1beta stimulation of the CB in the anaesthetized rat in vivo significantly increased the discharge rate in the carotid sinus nerve, most probably mediated by ATP release. This experiment provides evidence that the CB responds to cytokine stimulation and proposes the possibility that the CB might play a role in immune-to-brain communication.

Role of extracellular signal-regulated kinase in glutamate-stimulated apoptosis of rat retinal ganglion cells.

PURPOSE: To investigate the involvement of the extracellular signal-regulated kinase (ERK) signaling pathway after intravitrevous injection of glutamate in rat retina. METHODS: Three groups of five Sprague-Dawley rats each were studied. Group I was a normal control group, intravitreal saline injections. In Group II, one eye received an intravitreal glutamate injection (375 nmol, dissolved in saline) while the contralateral eye served as control. In Group III, intravitreal PD98059 (100 micro mol, an inhibitor of ERK) injections were administered 1 hr before glutamate injections. Seven days after injections, phosphorylated (activated) ERK in retina was localized by immunohistochemistry and fluorescent double labeling of retinal cryosections. Specific ERK blockade was documented to assess the functional significance of activated ERK. TUNEL staining was performed to assess apoptotic cell death. RESULTS: Expression of phosphorylated ERK in rat retina was observed in the inner nuclear layer, the outer nuclear layer, and the nerve fiber layer after 3 days intravitreous injection of glutamate, increasing significantly after 7 days. Double immunofluorescence labling demonstrated that the increased retinal immunostaining for phospho-ERK was predominantly localized to the retinal Müller cells after 7 days intravitreous injection of glutamate. Moreover, blocking activation of ERK significantly improved the number of TUNEL-positive cells in the eyes receiving intravitreal PD98059 injections compared with the eyes receiving glutamate injections. CONCLUSIONS: The ERK pathway is involved in signal transduction in the retina after excessive stimulation by glutamate, which may contribute to the antiapoptotic role in retinal ganglion cell death induced by glutamate.

PC12 cells express IL-1 receptor type I and response to IL-1beta stimulation.

PC12 cell line has been widely used in a diverse array of neurophysiological studies including in exploration of oxygen-sensing mechanism. In present study, we first identified with immunocytochemistry and Western blot methods that interleukin-1 receptor type I was expressed in the PC12 cells. We then demonstrated with patch clamping technique that extracellular application of IL-1beta dose-dependently inhibited the outward voltage-dependent and TEA-sensitive potassium currents (I(K)) in the PC12 cells, and pre-incubation with the interleukin-1 receptor antagonist almost completely abolished this inhibitory effect. In addition, application of IL-1beta shifted steady-state inactivation of I(K) in hyperpolarizing direction, but did not alter its steady-state activation. Furthermore, IL-1beta-induced inhibition of I(K) led to a membrane depolarization and a transient increase of [Ca(2+)](i) in PC12 cells. Taking together, the present study elucidates that PC12 cells bear interleukin-1 receptor and response to IL-1beta stimulation.

ERK1/2 and p38 mitogen-activated protein kinase mediate iNOS-induced spinal neuron degeneration after acute traumatic spinal cord injury.

The enhanced production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of neuronal apoptosis after acute traumatic spinal cord injury (SCI). In the present study, to further characterize the pathways mediating the synthesis and release of NO, we examined activation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK) in microglia/macrophages in the injured area of adult rats subjected to a complete transection at the T10 vertebrae level and assessed their role in NO production and survival of neurons by using immunohistochemistry, Western blot, RT-PCR and pharmacological interventions. Results showed activation of microglia/macrophages featured by morphological changes, as visualized immunohistochemically with the marker OX-42, in the areas adjacent to the lesion epicenter 1 h after surgery. Concomitantly, iNOS mRNA and its protein in the activated microglia/macrophages were also significantly upregulated at early hours after surgery. Their levels were maximal at 6 h, persisted for at least 24 h, and returned to basal level 72 h after SCI. Furthermore, phosphorylated ERK1/2 and p38 MAPK were activated as well in microglia/macrophages in injured area with a similar time course as iNOS. With administration of L-NAME, a NOS inhibitor, the number of apoptotic neurons was clearly decreased, as assessed with TUNEL method at 24 h after SCI. In parallel, loss of neurons induced by SCI, assessed with NeuN immunohistochemistry, was also diminished. Moreover, the effect of inhibition of phosphorylation ERK1/2 and p38 MAPK by corresponding inhibitors PD98059 and SB203580 administered before and after SCI was also investigated. Inhibition of p38 effectively reduced iNOS mRNA expression and rescued neurons from apoptosis and death in the area adjacent to the lesion epicenter; whereas the inhibition of ERK1/2 had a smaller effect on decrease of iNOS mRNA and no long-term protective effect on cell loss. These results indicate the ERK1/2 and p38 MAPK signaling pathway, especially the latter, play an important role in NO-mediated degeneration of neuron in the spinal cord following SCI. Strategies directed to blocking the initiation of this cascade prove to be beneficial for the treatment of acute SCI.

[Effects of electroacupuncture at "Zusanli" (ST 36) on ERK1/2 phosphorylation in the dorsal horn of spinal cord of the rat].

OBJECTIVE: To observe the effect of electroacupuncture at Zusanli (ST 36) on phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in the dorsal horn of spinal cord induced by plantar inflammation in the rat. METHODS: All the rats were randomly divided into 5 groups: normal control group, simple electroacupuncture group, formalin group, formalin plus ipsilateral electroacupuncture group and formalin plus contralateral electroacupuncture group. The acute inflammation animal model was made by injection of 100 microL of 4% formalin into the right posterior foot pad. Electroacupuncture was given at "Zusanli" (ST 36) for 30 min, with sparse-dense waves, frequency 2-15 Hz, and intensity 2-3 mA. One and a half hours latter, the rats were killed under anesthesia, and pERK1/2 expression in the lumbar dorsal horn were investigated with immunohistochemical method. RESULTS: The positive cells were rarely seen (6.45 +/- 1.05) in the superficial spinal cord in the control group; a few cells (14.07 +/- 3.19) in ipsilateral superficial spinal cord were found in the electroacupuncture group. The number of pERK1/2-positive neurons (26.57 +/- 4.93) in lamina I - II0 of the ipsilateral dorsal horn in the formalin group increased significantly. After electroacupuncture at ipsilateral Zusanli (ST 36), the number of positive cells (20.79 +/- 5.21) had a tendency to decrease, but with no statistically significant difference. However, after electroacupuncture at contralateral Zusanli (ST 36), the number of positive cells (14.75 +/- 3.03) significantly decreased as compared with the non-acupuncture group (P < 0.05). CONCLUSION: The inhibition of ERK1/2 phosphorylation in the spinal cord dorsal horn by electroacupuncture is possibly involved in acupuncture analgesic effect.

Morphological evidence for existence of IL-6 receptor alpha in the glomus cells of rat carotid body.

The carotid body (CB) senses changes in arterial blood PO2 and modulates respiratory movement. It is generally accepted that the dopaminergic type I cells in the CB are chemoreceptors. However, it has not been clarified whether the carotid body has the ability to perceive the stimulation of proinflammatory cytokines. Interleukin 6 (IL-6) as a multifunctional cytokine plays a pivotal role in host defense mechanism. In the present study, we observed the expression of IL-6Ralpha mRNA and protein in the carotid body using immunohistochemistry, Western blots, and in situ hybridization. The results confirmed the presence of IL-6Ralpha proteins and mRNAs in the glomus cells of rat carotid body. These results suggest that the function of the carotid body may be influenced by the proinflammatrory cytokines through their receptors.

Different signaling molecules responsible for IL-1beta-induced oxytocinergic and vasopressinergic neuron activation in the hypothalamic paraventricular nucleus of the rat.

It has been well known that oxytocin (OT)-ergic and arginine vasopressin (AVP)-ergic neurons located in the hypothalamic paraventricular nucleus (PVN) and super optic nucleus (SON) are two kinds of neuroendocrine cells with diverse functions. It has also been demonstrated that immune stimuli can activate these neurons to secret OT and AVP. However, the intracellular signal transduction molecules responsible for the activation of these OT-ergic and AVP-ergic neurons in PVN by immune stimuli are still unclear. In this experiment, the roles of Fos, a protein product of immediate early gene c-fos, and extracellular signal-regulated protein kinase (ERK) 1/2, a signal transduction molecule of mitogen-activated protein kinase (MAPK) family, in these processes were studied in the PVN of the rat following IL-1beta stimulation. The Sprague-Dawley rats were received either 750 ng/kg IL-1beta or equal volume normal saline (NS) injection intravenously (i.v.), and perfused transcardially by 4% paraformaldehyde 3h later. Fos and phosphorylated ERK1/2 (pERK1/2)-immunoreactivity (-ir) was observed in PVN by ABC immunohistochemical staining. Meanwhile, the double staining for OT/Fos, AVP/Fos, OT/pERK1/2 and AVP/pERK1/2 were also processed. The ABC immunohistochemical staining results showed that after an i.v. injection of IL-1beta, the expressions of Fos and pERK1/2 increased evidently in the PVN. Double-staining results showed that a large number of OT-ir cells contained strong Fos-ir products in their nuclei, while only a few of OT cells were double labeled with pERK1/2. As to AVP neurons, great quantities of AVP cells were strongly double labeled with pERK1/2 while there were nearly no Fos-ir nuclei in AVP-ir cells. We conclude from these results that the intracellular IL-1beta-induced events in OT and AVP neurons in PVN are quite different. The OT neurons are mainly activated via Fos without involvement of ERK1/2 pathway, while the latter, but not Fos, involves the intracellular event in AVP neurons activated by IL-1beta.