Fluorocitrate Treatment Research Articles

Involvement of iNOS-induced reactive enteric glia cells in gastrointestinal motility disorders of postoperative Ileus mice

Postoperative ileus (POI) is the cessation or reduction of gastrointestinal (GI) motility after surgery. Reactive enteric glial cells (EGCs) are critical for maintaining bowel function. However, the triggering mechanisms and downstream effects of reactive EGCs in POI were poorly understood. The goal of this current study was to investigate whether the inducible nitric oxide synthase (iNOS)-driven reactive EGCs participated in GI motility disorders and mechanisms underlying altered GI motility in POI. Intestinal manipulation (IM)-induced POI mice and iNOS−/− mice were used in the study. Longitudinal muscle and myenteric plexuses (LMMPs) from the distal small intestine were stained by immunofluorescence. Our results found that the GI motility disorders occurred in the IM-induced POI mice, and reactive EGCs were observed in LMMPs. Glial metabolic inhibitor gliotoxin fluorocitrate (FC) treatment or iNOS gene knockout attenuated GI motility dysfunction. In addition, we also found that FC treatment or iNOS gene knockout significantly inhibited the fluorescence intensity macrophage colony-stimulating factor (M-CSF), which reduced M2 phenotype macrophages activation in LMMPs of IM-induced POI mice. Our findings demonstrated that iNOS-driven reactive EGCs played a key role and were tightly linked to the MMs homeostasis in the POI mice. EGCs are emerging as a new frontier in neurogastroenterology and a potential therapeutic target.

A1 astrocytes contribute to murine depression-like behavior and cognitive dysfunction, which can be alleviated by IL-10 or fluorocitrate treatment

BackgroundAstrocytes are crucial regulators in the central nervous system. Abnormal activation of astrocytes contributes to some behavior deficits. However, mechanisms underlying the effects remain unclear. Here, we studied the activation of A1 astrocytes and their contribution to murine behavior deficits.MethodsA1 astrocytes were induced by treatment with lipopolysaccharide (LPS) in vitro. The functional phenotype of astrocytes was determined by quantitative RT-PCR, ELISA, and immunohistochemistry. To assess the role of A1 astrocytes in vivo, mice were injected intraperitoneally with LPS. Then, murine behaviors were tested, and the hippocampus and cortex were analyzed by quantitative RT-PCR, ELISA, and immunohistochemistry. The function of IL-10 and fluorocitrate on A1 astrocyte activation was also examined.ResultsOur results show that astrocytes isolated from B6.129S6-Il10tm1Flv/J homozygotes (IL-10tm1/tm1) were prone to characteristics of A1 reactive astrocytes. Compared with their wild-type counterparts, IL-10tm1/tm1 astrocytes exhibited higher expression of glial fibrillary acidic protein (GFAP). Whether or not they were stimulated with LPS, IL-10tm1/tm1 astrocytes exhibited enhanced expression of A1-specific transcripts and proinflammatory factors IL-1β, IL-6, and TNFα. In addition, IL-10tm1/tm1 astrocytes demonstrated hyperphosphorylation of STAT3. Moreover, astrocytes from IL-10tm1/tm1 mice showed attenuated phagocytic ability and were neurotoxic. IL-10tm1/tm1 mice demonstrated increased immobility time in the forced swim test and defective learning and memory behavior in the Morris water maze test. Moreover, enhanced neuroinflammation was found in the hippocampus and cortex of IL-10tm1/tm1 mice, accompanying with more GFAP-positive astrocytes and severe neuron loss in the hippocampus. Pretreatment IL-10tm1/tm1 mice with IL-10 or fluorocitrate decreased the expression of proinflammatory factors and A1-specific transcripts in the hippocampus and cortex, and then alleviated LPS-induced depressive-like behavior.ConclusionThese results demonstrate that astrocytes isolated from B6.129S6-Il10tm1Flv/J homozygotes are prone to A1 phenotype and contribute to the depression-like behavior and memory deficits. Inhibiting A1 astrocyte activation may be an attractive therapeutic strategy in some neurodegenerative diseases.

Anti-nociceptive roles of the glia-specific metabolic inhibitor fluorocitrate in paclitaxel-evoked neuropathic pain.

Paclitaxel (Taxol) is a powerful chemotherapy drug used in breast cancers, but it often causes neuropathic pain, leading to the early cessation of therapy and poor treatment outcomes. Approaches for the management of paclitaxel-induced neuropathic pain are urgently needed. The involvement of spinal astrocytes in the pathogenesis of paclitaxel-induced neuropathy has been reported, but little is known about the role of fluorocitrate (FC), a selective inhibitor of astrocyte activation, during neuropathic pain related to paclitaxel treatment. In this study, we investigated the effects of FC on paclitaxel-induced neuropathic pain. Glial fibrillary acidic protein (GFAP) expression was determined to assess astrocyte activation. To explore the mechanisms involved, the expression of glial glutamate transporter 1 (GLT-1) and the activation of mitogen-activated protein kinases in the spinal dorsal horn were analyzed. The results showed that paclitaxel decreased the mechanical nociceptive thresholds and increased GFAP expression, leading to spinal astrocyte activation. After paclitaxel treatment, GLT-1 was significantly down-regulated, and the phosphorylation of ERK1/2 and JNK were obviously up-regulated. However, paclitaxel treatment did not increase p38 phosphorylation. Additional studies showed that paclitaxel-evoked mechanical hypersensitivity was reduced by FC treatment. Moreover, FC treatment inhibited the activation of astrocytes and reversed the changes in GLT-1 expression and MAPK phosphorylation. Further study indicated that FC did not influence the antitumor effect of paclitaxel, suggesting that FC blocked paclitaxel-induced neuropathic pain without antagonizing its antitumor effect. Together, these results suggested that paclitaxel induced astrocyte-specific activation, which may contribute to mechanical allodynia and hyperalgesia, and that FC could be a potential therapeutic agent for paclitaxel-induced neuropathic pain.

Fluorocitrate induced the alterations of memory-related proteins and tau hyperphosphorylation in SD rats

Astrocytes provide structural, metabolic and trophic supports for neurons. However, there are no direct evidences whether astrocytes involve in the regulation of synaptic proteins expression and tau phosphorylation until now. Here, we injected 1nmol fluorocitrate (FC), which preferentially taken up by astrocytes and results in reversible inhibition of the astrocytic tricarboxylic acid cycle, into the left lateral ventricle of the brain in the SD rats for 1h, and found that FC treatment decreased several memory-related proteins levels, such as AMPA receptor GluR1/2, postsynaptic density protein 93/95, Arc and phosphorylated cAMP response element binding proteins, while increased synaptophysin and synapsin I levels in the hippocampus. FC treatment also increased the levels of phosphorylated tau at multiple Alzheimer-related phosphorylation sites, as well as activation of glycogen synthase kinase-3β and inactivation of protein phosphatase-2A. Similar effects were also observed in the primary hippocampal neurons, which were cultured with the conditioned media from FC-treatment primary astrocytes. Our data suggest that astrocytes regulate neuronal tau phosphorylation and several synaptic proteins expression.

Characterization of 14C-acetate uptake in cultured rat astrocytes

To investigate the regulation mechanism of 14C-acetate uptake in astrocytes, uptake experiments with cultured rat astrocytes were performed. In this study, we used a relatively low concentration (0.0185 mM) of 14C-acetate. The uptake was dependent on pH and acetate concentration, and saturated by 10 mM acetate. Furthermore, the selective inhibition by p-(chloromercuri) benzenesulfonic acid (pCMBS) but not by α-cyano-3-hydroxycinnamate (CHC) showed that the monocarboxylate transporter-1 (MCT-1)-mediated transport system is essentially important in cultured rat astrocytes. A significant reduction (more than 30%) in 14C-acetate uptake was observed with 0.5 mM fluorocitrate treatment, which indicated 14C-acetate uptake in this study might reflect not only the transport process, but also the metabolic process. Glutamate significantly increased 14C-acetate uptake. An increase in extracellular potassium concentration had no effect on 14C-acetate uptake. The Ca 2+ ionophore A23187 caused a 40% reduction, and ouabain (inhibitor of Na +-K +-ATPase) and monensin (Na + ionophore) significantly decreased 14C-acetate uptake by astrocytes. The combined use of ouabain and monensin caused significantly greater decreases than the addition of either chemical alone. These results suggest that glutamate stimulation and changes in Ca 2+ and Na + concentrations might have important roles in regulation of 14C-acetate uptake in cultured rat astrocytes.

Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function

The role of enteric glia in gastrointestinal physiology remains largely unexplored. We examined the actions of the gliotoxin fluorocitrate (FC) on intestinal motility, secretion, and inflammation after assessing its efficacy and specificity in vitro. FC (100 microM) caused a significant decrease in the phosphorylation of the glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diaz-4-yl)amino]-2-deoxyglucose in enteric glial cultures and a reduction in glial uptake of the fluorescent dipeptide Ala-Lys-7-amino-4-methylcoumarin-3-acetic acid in both the ileum and colon. Dipeptide uptake by resident murine macrophages or guinea pig myenteric neurons was unaffected by FC. Incubation of isolated guinea pig ileal segments with FC caused a specific and significant increase in glial expression of the phosphorylated form of ERK-1/2. Disruption of enteric glial function with FC in mice reduced small intestinal motility in vitro, including a significant decrease in basal tone and the amplitude of contractility in response to electrical field stimulation. Mice treated with 10 or 20 micromol/kg FC twice daily for 7 days demonstrated a concentration-dependent decrease in small intestinal transit. In contrast, no changes in colonic transit or ion transport in vitro were observed. There were no changes in glial or neuronal morphology, any signs of inflammation in the FC-treated mice, or any change in the number of myenteric nitric oxide synthase-expressing neurons. We conclude that FC treatment causes enteric glial dysfunction, without causing intestinal inflammation. Our data suggest that enteric glia are involved in the modulation of enteric neural circuits underlying the regulation of intestinal motility.

Effects of the gliotoxin fluorocitrate on spreading depression and glial membrane potential in rat brain in situ.

DC extracellular potential shifts (deltaVo) associated with spreading depression (SD) reflect massive cell depolarization, but their cellular generators remain obscure. We have recently reported that the glial specific metabolic poison fluorocitrate (FC) delivered by microdialysis in situ caused a rapid impairment of glial function followed some hours later by loss of neuronal electrogenic activity and neuron death. We have used the time windows for selective decay of cell types so created to study the relative participation of glia and neurons in SD, and we report a detailed analysis of the effects of FC on evoked SD waves and glial membrane potential (Vm). Extracellular potential (Vo), interstitial potassium concentration ([K+]o), evoked potentials, and transmembrane glial potentials were monitored in the CA1 area before, during, and after administration of FC with or without elevated K+ concentration in the dialysate. SD waves propagated faster and lasted longer during FC treatment. DeltaVo in stratum pyramidale, which normally are much shorter and of smaller amplitude than those in stratum radiatum, expanded during FC treatment to match those in stratum radiatum. The coalescing SD waves that develop late during prolonged high-K+ dialysis and are typically limited to stratum radiatum, also expanded into stratum pyramidale under the influence of FC. SD provoked in neocortex normally does not spread to the CA1, but during FC treatment it readily reached CA1 via entorhinal cortex. Once neuronal function began to deteriorate, SD waves became smaller and slower, and eventually failed to enter the region around the FC source. Slow, moderately negative deltaVo that mirrored [K+]o increments could still be recorded well after neuronal function and SD-associated Vo had disappeared. Glial cell Vm gradually depolarized during FC administration, beginning much before depression of neuronal antidromic action potentials. Calculations based on the results predict a large decrease in glial potassium content during FC treatment. The results are compatible with neurons being the major generator of the deltaVo associated with SD. We conclude that energy shortage in glial cells makes brain tissue more susceptible to SD and therefore it may increase the risk of neuron damage.

Changes in extracellular nitrite and nitrate levels after inhibition of glial metabolism with fluorocitrate

The role of glial cells in nitric oxide production in the cerebellum of conscious rats was investigated with a glial selective metabolic inhibitor, fluorocitrate. The levels of nitric oxide metabolites (nitrite plus nitrate) in the dialysate following in vivo microdialysis progressively increased to more than 2-fold the basal levels during a 2-h infusion of fluorocitrate (1 mM), and the increase persisted for more than 2 h after the treatment. Pretreatment with N G-nitro- l-arginine methyl ester attenuated the fluorocitrate-induced increase in nitric oxide metabolite levels. None of the glutamate receptor antagonists, including d(−)-2-amino-5-phosphonopentanoic acid, 6,7-dinitroquinoxaline-2,3-dione, and (±)- α-methyl-4-carboxyphenylglycine, inhibited the fluorocitrate-induced increase. The l-arginine-induced increase was significantly reduced by fluorocitrate treatment, while N-methyl- d-aspartate, (+)- α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and trans-(±)-1-amino-(1S,3R)-cyclopentane-dicarboxylic acid increased nitric oxide metabolites levels in the fluorocitrate-treated rats, as much as in control animals. These results suggest that glial cells play an important role in modulating nitric oxide production in the cerebellum by regulating l-arginine availability.

Effects of energy deprivation induced by fluorocitrate in immortalised rat brain microvessel endothelial cells

The effects of the mitochondrial aconitase inhibitor, fluorocitrate on the immortalised rat brain endothelial cell line (RBE4) were investigated. Treatment with different concentrations of fluorocitrate (0–1 mM) for 24 h induced a significant, concentration-dependent decrease in the MTT reduction (an index of mitochondrial function), intracellular ATP content, glucose consumption and lactate production by RBE4 cell monolayers but did not alter the glucose to lactate ratio at concentrations lower than 0.5 mM. At all concentrations, fluorocitrate induced a significant decrease in the protein content per well. Fluorocitrate treatment of confluent RBE4 cells induced a marked redistribution of the F-actin cytoskeleton from a characteristic marginal band to a more diffuse cytosolic pattern. This redistribution of the cytoskeleton coincided with a reduction in the total cellular F-actin content of the RBE4 cells at fluorocitrate concentrations greater than 0.5 mM. Treatment of confluent RBE4 cells with fluorocitrate had no significant effect on RBE4 cell monolayer permeability measured by FITC-dextran or [ 14C]sucrose. These results show that whilst energy deprivation following fluorocitrate treatment induces significant changes in the RBE4 cell F-actin cytoskeleton and cellular metabolism, it does not have any significant effect on endothelial cell monolayer permeability. These results demonstrate that profound toxic effects on endothelial cell structure and metabolism are not necessarily accompanied by changes in endothelial cell monolayer permeability.

The effect of depressing glial function in rat brain in situ on ion homeostasis, synaptic transmission, and neuron survival

The supporting role of glial cells in maintaining neurons and in ion homeostasis has been studied in situ by perfusing the gliotoxin fluorocitrate (FC) through a microdialysis fiber in the CA1 area of urethane-anesthetized rats. Extracellular direct current potential, extracellular potassium concentration ([K+]o) and amino acid levels, extracellular pH (pHo), and evoked field activity were studied. Histology verified the swelling of glial cells after 4 hr of FC treatment. Massive neuron damage was evident after 8 hr. FC dialysis caused the rapid decrease of glutamine, pHo became progressively more acid, and [K+]o moderately elevated. Orthodromic transmission was variably blocked within 30 min to 4 hr. After 4 hr, spreading depression (SD) waves that originated from the neocortex invaded hippocampal CA1, [K+]o increased to higher levels, pHo became very acid, and there were steep increases in taurine, glutamate, and GABA levels. Simultaneously, the antidromic population spike (a-PS) became depressed and eventually disappeared. When a shorter dialysis probe that spared cortex was used to sample CA1, no SD was seen, a-PS was not abolished, and ion homeostasis was altered less markedly. Repeated SD provoked in hippocampus in the absence of FC caused only mild depression of a-PS. Dialysis of high-K+ solution in healthy neocortex or hippocampus caused only slight elevation of [K+]o at distances of 200-400 microns from the dialysis membrane. After treatment with FC, similar high-K+ dialysis raised [K+]o much more. We conclude the following: (1) recurrent SD waves injure neurons if and only if glial function has failed; (2) neurons can regulate [K+]o, albeit imperfectly; (3) glia is required for the normal fine tuning of [K+]o and particularly for the recovery of pathologically elevated [K+]o; and (4) glia are required for the regulation of pHo. The similarities between glial poisoning by FC and the reported changes in the penumbra of ischemic infarcts suggest that the extension of neuron loss into the penumbral region might depend on failure of glial protection.

Selective inhibition of glial cell metabolism in vivo by fluorocitrate

The effect of fluorocitrate on glial and neuronal amino acid metabolism was studied. One nmol of fluorocitrate administered intrastriatally in the rat caused a 95% reduction of glutamine formation from [ 14C]acetate, a substrate which enters the glial cells selectively. The metabolism of [ 14C]glucose which enters neurons, was unaffected by fluorocitrate treatment except for the glutamine formation. This is evidence that fluorocitrate is a selective inhibitor of the glial Krebs' cycle. [ 14C]Citrate and 2-oxoglutarate labelled amino acids in a manner similar to [ 14C]acetate, which shows that these substrates are taken up and metabolized by glial cells. Differences in the labelling of γ-aminobutyric acid (GABA) from [ 14C]acetate and citrate suggest that astrocytes associated with GABAergic and glutamatergic nerve terminals may differ in their preference for amino acid precursors.

The effect of fluorocitrate on transmitter amino acid release from rat striatal slices.

In order to study the role of glutamine from glial cells for the synthesis of transmitter amino acids, the effect of the gliotoxic-substance fluorocitrate on amino acid release from slices was investigated. In vivo treatment with 1 nmol fluorocitrate reduced the Ca2+ dependent K+ evoked release of endogenous glutamate and GABA from the slices, whereas the glutamine efflux decreased and alanine efflux increased. The K+ evoked release of [3H]D-aspartate increased during fluorocitrate treatment. The latter is consistent with an inhibited uptake of D-aspartate into glial cells. Incubation of striatal slices with fluorocitrate (0.1 mM) decreased the glutamine efflux and increased the alanine efflux. Similar to the in vivo condition, fluorocitrate increased the K+ evoked [3H]D-aspartate release, but the K+ evoked release of endogenous glutamate and GABA increased rather than decreased. The ratio between the K+ evoked release of exogenous D-aspartate to endogenous glutamate increased in both cases. The results suggest an important role of glial cells in the synthesis and inactivation of transmitter amino acids.