Stretch-induced Ca2+ independent ATP release in hippocampal astrocytes.

KEY POINTS: Similar to neurons, astrocytes actively participate in synaptic transmission via releasing gliotransmitters. The Ca2+ -dependent release of gliotransmitters includes glutamate and ATP. Following an 'on-cell-like' mechanical stimulus to a single astrocyte, Ca2+ independent single, large, non-quantal, ATP release occurs. Astrocytic ATP release is inhibited by either selective antagonist treatment or genetic knockdown of P2X7 receptor channels. Our work suggests that ATP can be released from astrocytes via two independent pathways in hippocampal astrocytes; in addition to the known Ca2+ -dependent vesicular release, larger non-quantal ATP release depends on P2X7 channels following mechanical stretch. ABSTRACT: Astrocytic ATP release is essential for brain functions such as synaptic long-term potentiation for learning and memory. However, whether and how ATP is released via exocytosis remains hotly debated. All previous studies of non-vesicular ATP release have used indirect assays. By contrast, two recent studies report vesicular ATP release using more direct assays. In the present study, using patch clamped 'ATP-sniffer cells', we re-investigated astrocytic ATP release at single-vesicle resolution in hippocampal astrocytes. Following an 'on-cell-like' mechanical stimulus of a single astrocyte, a Ca2+ independent single large non-quantal ATP release occurred, in contrast to the Ca2+ -dependent multiple small quantal ATP release in a chromaffin cell. The mechanical stimulation-induced ATP release from an astrocyte was inhibited by either exposure to a selective antagonist or genetic knockdown of P2X7 receptor channels. Functional P2X7 channels were expressed in astrocytes in hippocampal brain slices. Thus, in addition to small quantal ATP release, larger non-quantal ATP release depends on P2X7 channels in astrocytes.

The proform of glia cell line-derived neurotrophic factor: a potentially biologically active protein.

Growing evidences have revealed that the proforms of several neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3), by binding to p75 neurotrophin receptor and sortilin, could induce neuronal apoptosis and are implicated in the pathogenesis of various neurodegenerative diseases. The glial cell line-derived neurotrophic factor (GDNF), one of the most potent useful neurotrophic factors for the treatment of Parkinson's disease (PD), is firstly synthesized as the proform (proGDNF) like other neurotrophin NGF, BDNF, and NT3. However, little is known about proGDNF expression and secretion under physiological as well as pathological states in vivo or in vitro. In this study, we investigated the expression profile and dynamic changes of proGDNF in brains of aging and PD animal models, with the interesting finding that proGDNF was a predominant form of GDNF with molecular weight of about 36 kDa by reducing and nonreducing immunoblots in adult brains and was unregulated in the aging, lipopolysaccharide (LPS), and 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) insult. We further provided direct evidence that accompanied activation of primary astrocytes as well as C6 cell line induced by LPS stimulation, proGDNF was increasingly synthesized and released as the uncleaved form in cell culture. Taken together, our results strongly suggest that proGDNF may be a biologically active protein and has specific effects on the cells close to its secreting site, and a potentially important role of proGDNF signaling in the brains, in the glia-neuronal interaction or in the pathogenesis of PD, should merit further investigation.

LPS-induced proNGF synthesis and release in the N9 and BV2 microglial cells: a new pathway underling microglial toxicity in neuroinflammation.

PURPOSE: While aberrant activation of microglial cells was evidently involved in neuroinflammation and neurotoxicity in the neurodegenerative diseases such as Alzheimer's and Parkinson's disease, objective of study was to address if activated microglias deliver their effect by releasing pro-neurotrophins. MATERIALS AND METHODS: By in vitro culture of N9 and BV2 cell lines and lipopolysaccharide (LPS) stimulation model, generation and release of proNGF, proBDNF and MMP-9 was studied in the activated microglial cells by immunocytochemistry, western blotting and bioassay methods. RESULTS: Activation of microglial cells was observed with obvious increasing iba1-immunoreactivity following LPS stimulation in cell culture. Synthesis and up-regulation of proNGF protein significantly occurred in N9 and BV2 cells 12h-48h after LPS exposure, whereas no significant changes of proBDNF and MMP9 were observed in these microglial cell lines with LPS insult. More interestingly, extracellular release or secretion of proNGF molecule was also detected in culture medium of N9 cells after LPS stimulation. Finally, bioassay using MTT, Hoechst/PI and TUNEL staining in SH-SY5Y cells further confirmed that proNGF treatment could result in apoptotic cell death but it did not significantly influence cell viability of SH-SY5Y cells. CONCLUSIONS: This in vitro study revealed LPS-stimulated proNGF synthesis and release in activated N9/BV2 microglial cell lines, also suggesting that proNGF may appeal a new pathway or possible mechanism underlying microglial toxicity in the neuroinflammation and a potential target for therapeutic manipulation of the neurodegenerative diseases.

Ginsenoside Rd protects neurons against glutamate-induced excitotoxicity by inhibiting ca(2+) influx.

Our previous studies have demonstrated that ginsenoside Rd (GSRd), one of the principal ingredients of Pana notoginseng, has neuroprotective effects against ischemic stroke. However, the possible mechanism(s) underlying the neuroprotection of GSRd is/are still largely unknown. In this study, we treated glutamate-injured cultured rat hippocampal neurons with different concentrations of GSRd, and then examined the changes in neuronal apoptosis and intracellular free Ca(2+) concentration. Our MTT assay showed that GSRd significantly increased the survival of neurons injured by glutamate in a dose-dependent manner. Consistently, TUNEL and Caspase-3 staining showed that GSRd attenuated glutamate-induced cell death. Furthermore, calcium imaging assay revealed that GSRd significantly attenuated the glutamate-induced increase of intracellular free Ca(2+) and also inhibited NMDA-triggered Ca(2+) influx. Thus, the present study demonstrates that GSRd protects the cultured hippocampal neurons against glutamate-induced excitotoxicity, and that this neuroprotective effect may result from the inhibitory effects of GSRd on Ca(2+) influx.

Glutamate release through connexin 43 by cultured astrocytes in a stimulated hypertonicity model.

We investigated the role of connexin 43 (Cx43) hemichannels in the release of glutamate by astrocytes after hypertonic stimulus. Mechanical, osmotic and oxidative stress, and changes in the extracellular or intracellular Ca(2+) levels induce connexin hemichannels located in the plasma membrane to open and release small ions and molecules with signaling potential such as glutamate, ATP, etc. In our past studies, we primarily found that acute hypertonic stimulus induced the release of glutamate. Since glutamate release was involved with several routes, we studied its release routes by astrocytes incubated in a hypertonic media for various periods. The glutamate release was increased after hypertonic stimulus. Glutamate release in hypertonic stimulus was inhibited by gap junction or Cx43 hemichannel blockers, but not by antagonists of purinergic receptor (P2XnR), glutamate transport inhibitors, intracellular Ca(2+) blockers, and pannexin 1(Panx1) hemichannel. The results suggest that glutamate release by the Cx43 hemichannels is likely to feature in the response of cultured astrocytes to hypertonic stimulus.

Effects of vagotomy, splanchnic nerve lesion, and fluorocitrate on the transmission of acute hyperosmotic stress signals to the supraoptic nucleus.

The response to hyperosmotic stresses in the abdominal cavity is regulated, in part, by vasopressin (VP)-secreting neurons in the supraoptic nucleus (SON). How osmotic stress signals are transmitted to the brain is incompletely understood, and whether the transmission routes for osmotic stress signals differ between acute and chronic stresses is unknown. Here we investigated the role of the vagus, splanchnic nerves, and astrocytes in the SON in transducing acute hyperosmotic-stress signals from the abdominal cavity. We found that acute administration of hyperosmotic saline triggered the activation of neurons as well as astrocytes in the SON and the adjoining ventral glia limitans (SON-VGL). Severing the subdiaphragmatic vagal nerve (SDV) prevented the normal response of cells in the SON to HS treatment and attenuated the release of VP into the bloodstream. Lesioning the splanchnic nerves (SNL) diminished HS-induced release of VP, but to a much lesser extent than SDV. Furthermore, SNL did not significantly affect the up-regulation of Fos in SON neurons or the up-regulation of Fos and GFAP in SON and SON-VGL astrocytes that normally occurred in response to HS and did not affect HS-induced expansion of the SON-VGL. Inhibiting astrocytes with fluorocitrate (FCA) prevented the response of the SON to HS and attenuated the release of VP, similarly to SDV surgery. These results suggest that the vagus is the principle route for the transmission of hyperosmotic signals to the brain and that astrocytes in the SON region are necessary for the activation of SON neurons and the release of VP into the bloodstream.

Effects of infrasound on cell proliferation in the dentate gyrus of adult rats.

Adult rats were used to identify the effects of infrasound on neurogenesis in the hippocampal dentate gyrus. After 7 consecutive days' exposure to infrasound of 16 Hz at 130 dB, immunostaining of 5-bromo-2'-deoxyuridine (BrdU) and doublecortin (DCX) was preformed. Compared with those in normal groups, the numbers of BrdU+ and DCX+/BrdU+ cells in the subgranular zone in infrasound groups were significantly decreased at 3, 6, 10 and 14 days and returned to normal at 18 days. The percentage of BrdU+ cells that were co-labeled with DCX showed no significant differences between the infrasound and normal groups. These data suggest that infrasound inhibits the cell proliferation in adult rat dentate gyrus but has no effects on early migration and differentiation of these newborn cells.

Acute hyperosmotic stimulus-induced Fos expression in neurons depends on activation of astrocytes in the supraoptic nucleus of rats.

Acute hyperosmolarity induced a time-dependent expression of Fos protein in both neurons and astrocytes of the rat supraoptic nucleus, with peak Fos expression occurring at 45 min in astrocytes and at 90 min in neurons after hypertonic stimulation in vivo. To determine whether the two cell types were activated separately or in an integrated manner, animals were pretreated with fluorocitrate, a glial metabolic blocker or carbenoxolone, a gap junction blocker followed by an acute hypertonic stimulation similar to that of the controls. Antibodies against glial fibrillary acidic protein, connexin 43, vasopressin, and oxytocin were used in serial sections to identify the cellular elements of the supraoptic nucleus. It was found that interruption of astrocyte metabolism with fluorocitrate significantly reduced Fos protein expression in both astrocytes and neurons, whereas blockage of gap junctions with carbenoxolone clearly reduced Fos protein expression in neurons, but not in astrocytes. These results indicate that both neurons and astrocytes in the rat supraoptic nucleus are involved in regulating osmolarity. Astrocytes are activated first, whereas connexin 43 functional hemichannels in SON astrocytes are required for the subsequent activation of the neurons.

A NMDA receptor antagonist, MK-801 impairs consolidating extinction of auditory conditioned fear responses in a Pavlovian model.

BACKGROUND: In auditory fear conditioning, repeated presentation of the tone in the absence of shock leads to extinction of the acquired fear responses. The glutamate N-methyl-D-aspartate receptor (NMDAR) is thought to be involved in the extinction of the conditioned fear responses, but its detailed role in initiating and consolidating or maintaining the fear extinction memory is unclear. Here we investigated this issue by using a NMDAR antagonist, MK-801. METHODS/MAIN FINDINGS: The effects of immediate (beginning at 10 min after the conditioning) and delayed (beginning at 24 h after conditioning) extinctions were first compared with the finding that delayed extinction caused a better and long-lasting (still significant on the 20(th) day after extinction) depression on the conditioned fear responses. In a second experiment, MK-801 was intraperitoneally (i.p.) injected at 40 min before, 4 h or 12 h after the delayed extinction, corresponding to critical time points for initiating, consolidating or maintaining the fear extinction memory. i.p. injection of MK-801 at either 40 min before or 4 h after delayed extinction resulted in an impairment of initiating and consolidating fear extinction memory, which caused a long lasting increased freezing score that was still significant on the 7th day after extinction, compared with extinction group. However, MK-801 administered at 12 h after the delayed extinction, when robust consolidation has been occurred and stabilized, did not affect the established extinction memory. Furthermore, the changed freezing behaviors was not due to an alteration in general anxiety levels, since MK-801 treatment had no effect on the percentage of open-arm time or open-arm entries in an Elevated Plus Maze (EPM) task. CONCLUSIONS/SIGNIFICANCE: Our data suggested that the activation of NMDARs plays important role in initiation and consolidation but not maintenance of fear extinction memory. Together with the fact that NMDA receptor is very important for memory, our data added experimental evidence to the concept that the extinction of conditioned fear responses is a procedure of initiating and consolidating new memory other than simply "erasing" the fear memory.

Involvement of medullary dorsal horn glial cell activation in mediation of masseter mechanical allodynia induced by experimental tooth movement.

OBJECTIVE: To investigate the involvement of microglial and astrocytic activation in the medullary dorsal horn (MDH) during the mediation of masseter area allodynia induced by experimental tooth movement (ETM). DESIGN: Five groups of adult Sprague-Dawley rats (n=60) were divided into control (CON), minocycline (MIN), ETM, and 10mg/kg or 30mg/kg MIN plus ETM (METM) groups. The upper-first-molar was moved mesially for rats in ETM and METM groups. Rats were pre-injected with minocycline in the MIN (30mg/kg) and METM (10mg/kg or 30mg/kg) groups. Pressure pain threshold (PPT) in masseter area was tested from day 0 to 14 for all 5 groups. Immunohistochemistry against OX42 (microglial marker) or GFAP (astrocytic maker) in the MDH was examined at days 1, 3, 7 and 14 for CON, MIN and 30mg/kg METM groups. RESULTS: Baseline PPT was expectedly seen in either CON or MIN groups, masseter mechanical allodynia was detected in the ETM group from day 4 to 13 (P<0.05). OX42 expression level at days 1, 3 and 7, and GFAP expression level at days 3, 7 and 14 were higher in ETM (P<0.05), but not in 30mg/kg METM, than in CON group. Minocycline reduced activation of microglia and astrocytes, and significantly attenuated the development of masseter mechanical allodynia in this model. CONCLUSIONS: These results indicate that mechanical allodynia in the masseter area induced by ETM can be attenuated by minocycline. Activation of microglia, possibly together with subsequent activation of astrocytes, seems to contribute to masseter mechanical allodynia.

Neuroprotective effects of ginsenoside Rd against oxygen-glucose deprivation in cultured hippocampal neurons.

We previously found that ginsenoside Rd (GSRd), one of the main active ingredients in Panax Ginseng, attenuates H(2)O(2)-induced oxidative injury in PC12 cells. Mounting evidence suggests that the oxidative stress is crucially involved in the pathophysiologic process of ischemia. In the present study, we examined the protective role of GSRd to attenuate ischemic neuronal injury in vitro. Cultured hippocampal neurons were exposed to oxygen-glucose deprivation (OGD) for 2h followed by a 24-h reoxygenation. GSRd exhibited remarkable neuroprotection when presented during OGD and reoxygenation, which may be ascribed to its antioxidative properties by reducing the intracellular reactive oxygen species and malondialdehyde production; increasing glutathione content; and enhancing the antioxidant enzymatic activities of catalase, superoxide dismutase and glutathione peroxidase. Additionally, GSRd could stabilize the mitochondrial membrane potential and attenuate apoptotic death of hippocampal neurons after OGD exposure. These findings suggested that GSRd may be a potential neuroprotective agent for cerebral ischemic injury and should encourage further in vivo studies on stroke to explore the potential neuroprotective efficacy of GSRd.

Involvement of activated astrocyte and microglia of locus coeruleus in cardiac pain processing after acute cardiac injury.

OBJECTIVE: It is still not known whether the glial cell activation of locus coeruleus (LC) is involved in the neurophysiologic mechanism of the acute phase of heart disease. The aim of this study was to investigate whether the glial cell activation of LC responds to acute cardiac injury (ACI). METHODS: In this study, ACI was established by intramyocardial injection of formalin. Afterward, we analysed c-Fos, OX42, GFAP and P2X(4)R expression levels in the LC of the rats by immunofluorescence staining or Western blot analysis. RESULTS: There was no significant difference in the levels of these markers in the LC between the normal control and the sham-operated groups. Following ACI, up-regulation of GFAP, OX42 and P2X(4)R expression levels were observed in locus coeruleus of the rats. The peak expression time was at hour 24. P2X(4)R was colocalized with OX42 in activated microglias, but not with GFAP in activated astrocytes. Compared with the control group, the ACI group showed a high expression level of c-Fos at hour 1 with a peak expression level at hour 2. CONCLUSION: The results showed that LC glia cells, like neurons, could sensitively respond to cardiovascular nociception induced by ACI at different time points. Results of this study may provide insights into the role of glial activation in response to ACI and may represent a potential strategy for investigation of neurophysiologic mechanism of cardiac pain.

Reciprocal pathway between medullary visceral zone and hypothalamic supraoptic nucleus or paraventricular nucleus involved in hyperosmotic regulation.

In this study we try to simultaneously investigate the response of neurons and astrocytes of rats following hyperosmotic stimulation and test the possibility that the reciprocal pathways between medullary visceral zone (MVZ) and hypothalamic paraventricular nucleus (PVN) or supraoptic nucleus (SON). Hyperosmotic pressure animal model was established by administering 3% sodium chloride as drinking water to rats. The distribution and expression of the HRP retrogradely labeled neurons, Fos, tyrosine hydroxylase (TH) or vasopressin (VP) positive neuron and glial fibrillary acidic protein (GFAP) positive astrocytes in the MVZ, SON and PVN were observed by quadruplicate-labeling methods of WGA-HRP retrograde tracing combined with anti-Fos, TH (or VP) and GFAP immunohistochemical technique. Fos positive neurons within the MVZ, PVN and SON increased markedly. There were also a large number of GFAP positive structures in the brain and their distribution pattern was fundamentally similar or analogous to Fos positive neurons in the above-mentioned areas. The augmented GFAP reactivities took on hypertrophic cell bodies, thicker and longer processes. Quadruplicate immunohistochemical staining showed that a neuron could be closely surrounded by many astrocytes and they formed neuron-astrocytic complex (N-ASC). Fos+/TH+/HRP+/GFAP+ and Fos+/VP+/HRP+/GFAP+ quadruplicate labeled N-ASC could be found in the MVZ, PVN and SON, respectively. The present results indicated that the neurons and astrocytes might be very active following hyperosmotic pressure and N-ASC as a functional unit might serve to modulate osmotic pressure. There were reciprocal osmoregulation pathways between the MVZ and SON or PVN in the brain.

Hypertonic stimulation induces synthesis and release of glutamate in cultured rat hypothalamic astrocytes and C6 cells.

OBJECTIVE: To investigate whether hypertonic saline (HS) can induce the synthesis and release of glutamate in cultured hypothalamic astrocytes or C6 cell line. METHODS: Astrocytes were isolated, cultured, purified and identified from the hypothalamus of newborn rat (1 day). The astrocytes were randomly divided into five groups: isotonic (IS) and HS groups, astrocytes were incubated by IS and HS (320 mosM NaCl) medium, respectively, for 1, 3, 5, 10 or 15 min; carbenoxolone (CBX)+IS and CBX+HS groups, astrocytes were pre-treated with CBX (100 mmol/L) for 1 h at 37 degrees C in a 5% CO(2) / 95% atmosphere, then removed to IS and HS medium, respectively, for 1, 3, 5, 10 or 15 min; Ca(2+)+HS group, astrocytes were pre-incubated with Ca Ca(2+) (1,000 micromol/L) for 1 h at 37 degrees C in a 5% CO(2) / 95% atmosphere, followed by a wash with isotonic FBS/DMEM, and then removed to hypertonic saline for 1, 3, 5, 10 or 15 min. The media of five groups were collected to analyze the medium glutamate concentration with high performance liquid chromatography. The astrocytes were fixed and double immunofluorescent stained with anti-glial fibrillary acidic protein (GFAP) and anti-glutamate. The C6 cells were divided into four groups: IS, HS, CBX+IS and CBX+HS groups, and used for quantitative measurement of glutamate in cells by flow cytometry (FCM). RESULTS: (1) Anti-GFAP immunofluorescent signal revealed no significant difference among various time points in each group, or among the five groups. (2) The anti-glutamate immunofluorescent signal was increased in HS group and peaked at 5 min, and decreased and returned to the level of IS group at 15 min (P < 0.01 vs the 5 min of HS group). In CBX+HS group, the glutamate intensity was higher than that in CBX+IS and HS groups. (3) The medium glutamate concentration had no change after treatment with HS for 1 and 3 min, while increased markedly after treatment for 5 min to 15 min (P< 0.01 vs 1 min and 3 min). On the contrary, the medium glutamate concentrations in the CBX+HS or Ca(2+)+HS group were significant lower than that in the HS group (P < 0.01). (4) FCM showed HS and CBX+HS induced glutamate increase in C6 cells. CONCLUSION: HS induced cultured rat hypothalamic astrocytes or C6 cells to synthesize and release glutamate; CBX could block glutamate release, but could not disrupt glutamate synthesis.

Protective effects of ginsenoside Rd on PC12 cells against hydrogen peroxide.

Oxidative stress-induced cell damage has been implicated in a variety of neurodegenerative disorders. In the current study, we investigated the protective role of ginsenoside Rd against the cytotoxicity induced by exposure to hydrogen peroxide (H(2)O(2)) and the underlying mechanism in the PC12 cell line. The protective effects of ginsenoside Rd (1, 10 microM) on H(2)O(2)-induced cytotoxicity may be ascribed to its antioxidative properties by reducing the intracellular reactive oxygen species level; decreasing malondialdehyde production, a common index of lipid peroxidation; and enhancing the antioxidant enzymatic activities of superoxide dismutase and glutathione peroxidase. Additionally, ginsenoside Rd could stabilize the mitochondrial membrane potential after H(2)O(2) exposure. These findings suggested that ginsenoside Rd may be considered a potential antioxidant agent and should encourage further research in neurodegenerative diseases to explore the potential neuroprotective effects of ginsenoside Rd.

[Changes in plasticity of rat hypothalamic neurons and astrocytes in humid and hot environment].

OBJECTIVE: To study the changes in the plasticity of the neurons and astrocytes in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus of rats exposed to a humid and hot environment. METHODS: The rats were subjected to stimulation with a humid and hot environment for 120 min in a climate chamber (dry bulb temperature of 40.0-/+0.5 degrees C with relative humidity of 60-/+5%). During the exposure, the behavioral responses of the rats were observed, and the changes in the expressions of Fos and GFAP in the PVN and SON in response to the exposure evaluated using immunohistochemical ABC methods. RESULTS: Exposure to a humid and hot environment caused restlessness and agitation in the rats, which showed increased respiratory frequency and scratching of the face with the forelimbs. Two rats died after the 120-min exposure. Significantly increased expressions of Fos and GFAP were detected in the PVN and SON following the exposure as compared with the control group. CONCLUSION: The neurons and astrocytes in the PVN and SON both participate in the regulation of responses to exposure to a humid and hot environment.

Electro-acupuncture of Tsusanli and Shangchuhsu regulates gastric activity possibly through mediation of the vagus-solotary complex.

BACKGROUND/AIMS: Acupuncture has been reportedly used to treat gastrointestinal diseases, however, its precise mechanism remains unknown. METHODOLOGY: In our study, the effects and mechanism of electro-acupuncture (EA) at Tsusanli (ST 36), Shangchuhsu (ST 37) on regulation of gastric activity were observed. RESULTS: EA at Tsusanli showed that gastric electric change was the most obvious, with significantly higher frequency and wave amplitude compared with that of the Shangchuhsu group and other groups. EA at Shangchuhsu demonstrated that the change of gastric electric level was much higher than that of the non-acupoint group and control group. After bilateral vagotomy, Tsusanlis was electro-acupunctured, the changes of electro-gastric graph (EGG) weren't significant with the control group. The frequency of electro-physiological activity in nucleus of solitary tract (NTS) and dorsal motor nucleus of the vagus nerve (DMV) in the Tsusanli group was markedly increased compared with that in the other group. Fos and GFAP expression in NTS and DMV in the Tsusanli group was significantly higher than that in other groups and control group. The results have indicated that EA at Tsusanli and Shangchuhsu cannot only regulate gastric activity, but also activate neurons and astrocytes in NTS and DMV. The effects on regulation and activation of EA at Tsusanli were very obvious. CONCLUSIONS: Our study suggests that this electroacupuncture regulation of gastric activity may partially depend upon integrated nerve pathway and related central neurons and astrocytes in the vagus-solitary complex.

[Effect of carbenoxolone on expression of Fos, NMDAR2 and GFAP in the hippocampus of pentylenetetrazo-kindled epileptic rats].

OBJECTIVE: Gap junctions, the clusters of intercellular channels, play an important role in synchronizing electrical activity. This study investigated the effect of gap junction blocker carbenoxolone (CBX) on epileptic activity in pentylenetetrazo (PTZ)-kindled rats. METHODS: Thirty adult male SD rats were randomly divided into three groups: control, PTZ-kindled and CBX-treated groups (n=10 each). The rats from the PTZ-kindled and the CBX-treated groups were intraperitoneally injected with PTZ (35 mg/kg x d) to induce epilepsy. After epilepsy kindling, they were intraperitoneally injected for 3 days with CBX (10 mg/kg) (CBX-treated group) or with normal saline (PTZ-kindled group). The control group received intraperitoneal injections of normal saline. Anti-GFAP, anti-Fos, and anti-NMDARZ immunohistochemical ABC methods were used to detect the expression of GFAP-Li, Fos-Li and NMDAR2-Li in the hippocampus respectively. RESULTS: Spontaneous seizures occurred in PTZ-kindled epileptic rats. CBX administration reduced spontaneous seizures. The NMDAR2-Li and Fos-Li neurons as well as GFAP-Li astrocytes in hippocampi increased in PTZ-kindled epileptic rats compared with controls. The numbers of Fos-Li (93.75 +/-7.94 vs 165.25 +/-15.87, P < 0.05) and NMDAR2-Li neurons (61.47 +/-3.62 vs 148.72 +/-14.53, P < 0.01) in the CBX-treated group were significantly less than in the PTZ-kindled group. There were no significant differences in the GFAP-Li expression between the CBX-treated and the PTZ-kindled groups. CONCLUSIONS: CBX may inhibit spontaneous seizures and decrease the numbers of Fos-Li and NMDARZ-Li neurons, thus providing anti-epileptic effects.

[Influence of intrathecal injection of fluorocitrate on the protective effect of electroacupuncture on gastric mucosa in high humid heat stress rats].

OBJECTIVE: To study if the spinal glial cells involve in the protective effect of acupuncture on gastric mucosa in high humid heat stress rats. METHODS: Thirty-six male SD rats were randomized into 6 groups: control, stress model, electroacupuncture (EA), Fluorocitrate(FCA, intrathecal injection of FCA. 1 microL, 60 min before humid heat stress), EA+ normal saline (NS, intrathecal injection of NS) and EA+ FCA groups. Stress model was established by putting the rats in a container with higher temperature and higher humidity [(40.0 +/- 0.5) degrees C, relative humidity (60 +/- 5)%] for 60 min. EA (50 Hz, intermittent waves, 2-5 V) was applied to bilateral "Zusanli" (ST 36) for 60 min. Using immunofluorescent methods, we observed glial fibrillary acidic protein (GFAP) and microglia OX42 immunoreactivity (OD value) in the tissue of the lumbar enlargement segment of the spinal cord. We evaluated and recorded the damage index (DI) of gastric mucosa of rats according to Guth's method. RESULTS: There were clear dot-line-like hemorrhage foci with formation of ulcer in the gastric mucosa 60 minutes after high humid heat stimulation. Compared with model group, DI of gastric mucosa in EA and EA+ NS groups decreased significantly (P < 0.05, suggesting a protective effect of EA on the gastric mucosa under stress), OD values of EA and EA + NS groups increased considerably (P < 0.05). Comparison between EA and EA+ FCA groups showed that DI of EA + FCA group was higher than that of EA group, while the OD values of GFAP and OX42 in EA+ FCA group was markedly lower than those in EA group (P < 0.05), suggesting an inhibitory effect of FCA on the effects of EA in improving stress-induced damage of gastric mucosa and upregulation of GFAP and OX42 expression. CONCLUSION: EA at "Zusanli" (ST 36) can prevent the gastric mucosa from injury caused by high humid heat stimulation. The lumbar spinal glial cells may play a role in EA's protective function.

Electro-acupuncture of Foot YangMing regulates gastric activity possibly through mediation of the dorsal vagal complex.

Acupuncture at some specific acupoints of Foot Yangming can regulate gastric activity. However, its precise mechanism remains unknown. In our study, the effects and mechanism of electro-acupuncture (EA) at Tsusanli (ST 36), Shangchuhsu (ST 37) on the regulation of gastric activity were observed. EA at Tsusanli showed that gastric electric change had a significantly higher frequency and wave amplitude as compared to that of the Shangchuhsu group and other groups. EA at Shangchuhsu demonstrated the change of gastric electric was greater than that of the non-acupoint group and the control group. After bilateral vagotomy, the change of electro gastric graph (EGG) of EA at Tsusanlis was not significant compared to the control group. In the mean time, we have observed the electric discharge of the neurons in NTS and DMV. The frequency of electro-physiological activity in nucleus of solitary tract (NTS) and dorsal motor nucleus of the vagus nerve (DMV) in Tsusanli group and Shangchuhsu group were markedly increased compared with that in other groups. The results have indicated that EA at Tsusanli and Shangchuhsu not only regulate gastric activity, but also activate neurons in NTS and DMV significantly. Our study suggests that the effect of EA at Tsusanli and Shangchuhsu on the gastric activity may partially depend upon integrated nerve pathway and related central neurons in dorsal vagal complex.