Search

Bead-based immunoassays are multiparametric analysis allowing for the simultaneous quantification of a large number of biomarkers within a single sample. Mass cytometry is an emerging cytometric technique that offers a high multiplexing capacity in a high-throughput setting but has not yet been applied to bead-based assays. In this study, we developed a multiplex bead-based immunoassay of cytokines and CD163 designed for mass cytometry (MC). A set of 11 types of lanthanide-encoded microbeads were synthesized by two-stage dispersion polymerization as classifier candidates for the assay. These beads were then decorated with different Abs on the surface to capture the target cytokines in solution. Gold nanoparticles were employed as reporters to identify the binding of target cytokines on the classifier surface. As a proof-of-concept study, we first developed four-plex and nine-plex assays of mixtures of cytokines in standard solutions. The MC signal intensities of these immunoassays were responsive to the concentration differences in the standard solutions with high detection sensitivities at low analyte concentrations. Finally, we examined a sample of peripheral blood mononuclear cells (PBMCs) with the nine-plex assay, comparing an unstimulated sample with a sample stimulated to promote cytokine secretion., Competing Interests: The authors declare the following competing financial interest(s): Dr. Allo was an employee of Fluidigm Canada (now Standard BioTools Canada). He has since left the company., (© 2022 The Authors. Published by American Chemical Society.)

Lanthanide nanoparticles (LnNPs) have the potential to be used as high-sensitivity mass tag reporters in mass cytometry immunoassays. For this application, however, the LnNPs must be made colloidally stable in aqueous buffers, demonstrate minimal non-specific binding to cells, and have functional groups to attach antibodies or other targeting agents. One possible approach to address these requirements is by using lipid coating to modify the surface of the LnNPs. In this work, 39 nm diameter NaYF 4 :Yb, Er NPs (LnNPs) were coated with a lipid formulation consisting of egg sphingomyelin, 1,2-dioleoyl- sn -glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium propane, cholesterol-(polyethylene glycol-600), and 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[biotinyl(polyethylene glycol)-2000]. The resulting biotinylated lipid-coated LnNPs were characterized by dynamic light scattering to determine the hydrodynamic size and stability in phosphate buffered saline, and the composition of the lipid coatings was quantified by liquid chromatography-tandem mass spectrometry. The specific and non-specific binding of the biotinylated lipid-coated LnNPs to a model system of functionalized polystyrene microbeads were then tested by both suspension and imaging mass cytometry. We found that targeted binding with minimal non-specific binding can be achieved with the lipid-coated LnNPs and that the lipid composition of the coating has an impact on the performance of the LnNPs as mass cytometry reporters. These results additionally establish the importance of quantifying the composition of lipid-coated nanomaterials to optimize them more effectively for their desired application.

Background & Aims: Pancreatic ductal adenocarcinomas (PDACs) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). We investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule CDH11, which has been associated with other fibrotic disorders and is expressed by activated fibroblasts., Methods: We compared levels of CDH11 messenger RNA in human pancreatitis and pancreatic cancer tissues and cells with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11 +/+ and Cdh11 -/- mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11 +/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored., Results: Levels of CDH11 messenger RNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11 +/- and KPC/Cdh11 -/- mice survived significantly longer than KPC/Cdh11 +/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11 +/+ mice and incompletely in KPC/Cdh11 +/- and KPC/Cdh11 -/- mice, whose lesions also contained fewer FOXP3 + cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11 +/+ mice, tumors of KPC/Cdh11 +/- mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival of KPC/Cdh11 +/- and KPC/Cdh11 -/- mice only or reduced subcutaneous tumor growth in mT3 engrafted Cdh11 +/+ mice when given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice., Conclusions: Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer., (Copyright © 2021 AGA Institute. All rights reserved.)

Although most children survive B cell acute lymphoblastic leukemia (B-ALL), they frequently experience long-term, treatment-related health problems, including osteopenia and osteonecrosis. Because some children present with fractures at ALL diagnosis, we considered the possibility that leukemic B cells contribute directly to bone pathology. To identify potential mechanisms of B-ALL-driven bone destruction, we examined the p53 -/- ; Rag2 -/- ; Prkdc scid/scid double mutant (DM) mouse models of spontaneous B-ALL. In contrast to DM animals, leukemic TM mice displayed brittle bones, and the TM leukemic cells overexpressed p53 , encoding receptor activator of nuclear factor κB ligand. RANKL is a key regulator of osteoclast differentiation and bone loss. Transfer of TM leukemic cells into immunodeficient recipient mice caused trabecular bone loss. To determine whether human B-ALL can exert similar effects, we evaluated primary human B-ALL blasts isolated at diagnosis for RANKL expression and their impact on bone pathology after their transplantation into NOD. -/- /SzJ (NSG) recipient mice. Primary B-ALL cells conferred bone destruction evident in increased multinucleated osteoclasts, trabecular bone loss, destruction of the metaphyseal growth plate, and reduction in adipocyte mass in these patient-derived xenografts (PDXs). Treating PDX mice with the RANKL antagonist recombinant osteoprotegerin-Fc (rOPG-Fc) protected the bone from B-ALL-induced destruction even under conditions of heavy tumor burden. Our data demonstrate a critical role of the RANK-RANKL axis in causing B-ALL-mediated bone pathology and provide preclinical support for RANKL-targeted therapy trials to reduce acute and long-term bone destruction in these patients.Prkdc scid/scid double mutant (DM) mouse models of spontaneous B-ALL. In contrast to DM animals, leukemic TM mice displayed brittle bones, and the TM leukemic cells overexpressed Rankl , encoding receptor activator of nuclear factor κB ligand. RANKL is a key regulator of osteoclast differentiation and bone loss. Transfer of TM leukemic cells into immunodeficient recipient mice caused trabecular bone loss. To determine whether human B-ALL can exert similar effects, we evaluated primary human B-ALL blasts isolated at diagnosis for RANKL expression and their impact on bone pathology after their transplantation into NOD. Prkdc scid/scid Il2rg tm1Wjl /SzJ (NSG) recipient mice. Primary B-ALL cells conferred bone destruction evident in increased multinucleated osteoclasts, trabecular bone loss, destruction of the metaphyseal growth plate, and reduction in adipocyte mass in these patient-derived xenografts (PDXs). Treating PDX mice with the RANKL antagonist recombinant osteoprotegerin-Fc (rOPG-Fc) protected the bone from B-ALL-induced destruction even under conditions of heavy tumor burden. Our data demonstrate a critical role of the RANK-RANKL axis in causing B-ALL-mediated bone pathology and provide preclinical support for RANKL-targeted therapy trials to reduce acute and long-term bone destruction in these patients., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Mass cytometry is a highly multiplexed single-cell analysis platform that uses metal-tagged reagents to identify multiple cellular biomarkers. The current metal-tagged reagent preparation employs thiol-maleimide chemistry to covalently couple maleimide-functionalized metal-chelating polymers (MCPs) with antibodies (Abs), a process that requires partial reduction of the Ab to form reactive thiol groups. However, some classes of Abs (for example, IgM) as well as biomolecules lacking cysteine residues have been challenging to label using this method. This inherent limitation led us to develop a new conjugation strategy for labeling a wide range of biomolecules and affinity reagents. In this report, we present a metal tagging approach using a new class of azide- or transcyclooctene-terminated MCPs with copper(I)-free strain-promoted alkyne-azide cycloaddition or tetrazine-alkene click chemistry reactions, in which biomolecules with -NH 2 functional groups are selectively activated with a dibenzocyclooctyne or tetrazine moiety, respectively. This approach enabled us to generate highly sensitive and specific metal-tagged IgGs, IgMs, small peptides, and lectins for applications in immunophenotyping and glycobiology. We also created dual-tagged reagents for simultaneous detection of markers by immunofluorescence, mass cytometry, and imaging mass cytometry using a two-step conjugation process. The Helios mass cytometer was used to test the functionality of reagents on suspension human leukemia cell lines and primary cells. The dual-tagged Abs, metal-tagged lectins, and phalloidin staining reagent were used to visualize target proteins and glycans on adherent cell lines and frozen/FFPE tissue sections using the Hyperion Imaging System. In some instances, reagents produced by click conjugation showed superior sensitivity and specificity compared to those of reagents produced by thiol-maleimide chemistry. In general, the click chemistry-based conjugation with new MCPs could be instrumental in developing a wide range of highly sensitive metal-containing reagents for proteomics and glycomics applications.

Mass cytometry uniquely enables high-dimensional single-cell analysis of complex populations. This recently developed technology is based on inductively coupled time-of-flight mass spectrometry for multiplex proteomic analysis of more than 40 markers per cell. The ability to characterize the transcriptome is critical for the understanding of disease pathophysiology, medical diagnostics, and drug discovery. Current techniques allowing the in situ detection of transcripts in single cells are limited to a small number of simultaneous targets and are generally tedious and labor-intensive. In this report, we present the development of a multiplex method for targeted RNA detection by combining the mass cytometry and RNAscope® platforms. This novel assay, called Metal In Situ Hybridization (MISH), includes the hybridization of RNA-specific target probes followed by signal amplification achieved through a cascade of hybridization events, ending with the binding of amplifier-specific detector probes. The detector probes are tagged with isotopically pure metal atoms used for detection by mass cytometry. Proof-of-principle experiments show the simultaneous detection of three mRNA targets in Jurkat cells in suspension cell assay mode. The localization of transcripts was also investigated using the imaging mass cytometry platform in Jurkat and KG-1a cells. In addition, we optimized the antibody staining procedure to allow the co-detection of mRNA and cell surface markers. Our data demonstrate that MISH can be used to complement protein detection by mass cytometry as well as to investigate gene transcription and translation in single cells. © 2017 International Society for Advancement of Cytometry., (© 2017 International Society for Advancement of Cytometry.)

This study investigates the characteristics of porous calcium polyphosphate particulates (CPPp) formed using two different processing treatments as bone void fillers in non- or minimally load-bearing sites. The two calcium polyphosphate particulate variants (grades) were formed using different annealing conditions during particulate preparation to yield either more slowly degrading calcium polyphosphate particulates (SD-CPPp) or faster degrading particulates (FD-CPPp) as suggested by a previous degradation study conducted in vitro (Hu et al., Submitted for publication 2016). The two CPPp grades were compared as bone void fillers in vivo by implanting particulates in defects created in rabbit femoral condyle sites (critical size defects). The SD-CPPp and FD-CPPp were implanted for 4- and 16-week periods. The in vivo study indicated a significant difference in amount of new bone formed in the prepared sites with SD-CPPp resulting in more new bone formation compared with FD-CPPp. The lower bone formation characteristic of the FD-CPPp was attributed to its faster degradation rate and resulting higher local concentration of released polyphosphate degradation products. The study results indicate the importance of processing conditions on preparing calcium polyphosphate particulates for potential use as bone void fillers in nonload-bearing sites. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 874-884, 2017., (© 2016 Wiley Periodicals, Inc.)

Adynamic bone is the most frequent type of bone lesion in patients with chronic kidney disease; long-term use of antiresorptive therapy may also lead to the adynamic bone condition. The hallmark of adynamic bone is a loss of bone turnover, and a major clinical concern of adynamic bone is diminished bone quality and an increase in fracture risk. Our current study aims to investigate how bone quality changes with age in our previously established mouse model of adynamic bone. Young and old mice (4 months old and 16 months old, respectively) were used in this study. Col2.3Δtk (DTK) mice were treated with ganciclovir and pamidronate to create the adynamic bone condition. Bone quality was evaluated using established techniques including bone histomorphometry, microcomputed tomography, quantitative backscattered electron imaging, and biomechanical testing. Changes in mineral and matrix properties were examined by powder X-ray diffraction and Raman spectroscopy. Aging controls had a natural decline in bone formation and resorption with a corresponding deterioration in trabecular bone structure. Bone turnover was severely blunted at all ages in adynamic animals, which preserved trabecular bone loss normally associated with aging. However, the preservation of trabecular bone mass and structure in old adynamic mice did not rescue deterioration of bone mechanical properties. There was also a decrease in cortical bone toughness in old adynamic mice that was accompanied by a more mature collagen matrix and longer bone crystals. Little is known about the effects of metabolic bone disease on bone fracture resistance. We observed an age-related decrease in bone toughness that was worsened by the adynamic condition, and this decrease may be due to material level changes at the tissue level. Our mouse model may be useful in the investigation of the mechanisms involved in fractures occurring in elderly patients on antiresorptive therapy who have very low bone turnover., (© 2015 American Society for Bone and Mineral Research.)

Methylenedioxymethamphetamine (MDMA) causes a persistent loss of dopaminergic cell bodies in the substantia nigra of mice. Current evidence indicates that MDMA-induced neurotoxicity is mediated by oxidative stress probably due to the inhibition of mitochondrial complex I activity. In this study we investigated the contribution of dopamine (DA) to such effects. For this, we modulated the dopaminergic system of mice at the synthesis, uptake or metabolism levels. Striatal mitochondrial complex I activity was decreased 1 h after MDMA; an effect not observed in the striatum of DA depleted mice or in the hippocampus, a dopamine spare region. The DA precursor, L-dopa, caused a significant reduction of mitochondrial complex I activity by itself and exacerbated the dopaminergic deficits when combined with systemic MDMA. By contrast, no damage was observed when L-dopa was combined with intrastriatal injections of MDMA. On the other hand, dopamine uptake blockade using GBR 12909, inhibited both, the acute inhibition of complex I activity and the long-term dopaminergic toxicity caused by MDMA. Moreover, the inhibition of DA metabolism with the monoamine oxidase (MAO) inhibitor, pargyline, afforded a significant protection against MDMA-induced complex I inhibition and neurotoxicity. Taken together, these findings point to the formation of hydrogen peroxide subsequent to DA metabolism by MAO, rather than a direct DA-mediated mitochondrial complex I inhibition, and the contribution of a peripheral metabolite of MDMA, as the key steps in the chain of biochemical events leading to DA neurotoxicity caused by MDMA in mice., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Phosphodiesterase 5 (PDE5) inhibitors have recently been reported to exert beneficial effects against ischemia-reperfusion injury in several organs but their neuroprotective effects in brain stroke models are scarce. The present study was undertaken to assess the effects of sildenafil against cell death caused by intrastriatal injection of malonate, an inhibitor of succinate dehydrogenase; which produces both energy depletion and lesions similar to those seen in cerebral ischemia. Our data demonstrate that sildenafil (1.5mg/kg by mouth (p.o.)), given 30min before malonate (1.5μmol/2μL), significantly decreased the lesion volume caused by this toxin. This protective effect can be probably related to the inhibition of excitotoxic pathways. Thus, malonate induced the activation of the calcium-dependent protease, calpain and the cyclin-dependent kinase 5, cdk5; which resulted in the hyperphosphorylation of tau and the cleavage of the protective transcription factor, myocyte enhancer factor 2, MEF2. All these effects were also significantly reduced by sildenafil pre-treatment, suggesting that sildenafil protects against malonate-induced cell death through the regulation of the calpain/p25/cdk5 signaling pathway. Similar findings were obtained using inhibitors of calpain or cdk5, further supporting our contention. Sildenafil also increased MEF2 phosphorylation and Bcl-2/Bax and Bcl-xL/Bax ratios, effects that might as well contribute to prevent cell death. Finally, sildenafil neuroprotection was extended not only to rat hippocampal slices subjected to oxygen and glucose deprivation when added at the time of reoxygenation, but also, in vivo when administered after malonate injection. Thus, the therapeutic window for sildenafil against malonate-induced hypoxia was set at 3h., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background and Purpose: PD5 inhibitors have recently been reported to exert beneficial effects against ischaemia-reperfusion injury in several organs. However, there are few studies regarding their neuroprotective effects in brain ischaemia. The present study was designed to assess the effects of sildenafil against chemical hypoxia induced by malonate. Intrastriatal injection of malonate produces energy depletion and striatal lesions similar to that seen in cerebral ischaemia through mechanisms that involve generation of reactive oxygen species (ROS)., Experimental Approach: Volume lesion was analysed by cytochrome oxidase histochemistry. Generation of reactive species was determined by in situ visualization of superoxide production and nitrotyrosine measurement. Protein levels were determined by Western blot after subcellular fractionation., Key Results: Sildenafil, given 30 min before malonate, significantly decreased the lesion volume in the rat. This protective effect cannot be attributed to any effect on ROS production but to the inhibition of downstream pathways. Thus, malonate induced the activation of apoptosis signal-regulating kinase-1 (ASK1) and two MAPK kinases, MKK3/6 and MKK7, which lead to an increased phosphorylation of JNK and p38 MAPK, effects that were blocked by sildenafil. Selective inhibitors of p38 and JNK (SB203580 or SP600125, respectively) were used in combination with malonate in order to evaluate the plausible implication of these pathways in the protection afforded by sildenafil. While inhibition of p38 provided a significant protection against malonate-induced neurotoxicity, inhibition of JNK did not., Conclusions and Implications: Sildenafil protects against the chemical hypoxia induced by malonate through the regulation of the ASK1-MKK3/6-p38/MAPK signalling pathway., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

The aim of the present study was to investigate whether late pre-conditioning using 3-nitropropionic acid (3NP) prevents the 5-hydroxytryptamine (5-HT) deficits caused by the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA) in the rat. For this purpose we administered 3NP 24 h before MDMA (3 x 5 mg/kg i.p., every 2 h) and rats were killed 7 days later. Pre-treatment of 3NP afforded complete protection against MDMA-induced 5-HT deficits independent of any effect on MDMA-induced hyperthermia or 5-HT transporter activity. To identify the transductional mechanisms responsible for the neuroprotective effect of 3NP, we first examined the involvement of nitric oxide (NO) by using selective inhibitors of all three nitric oxide synthase isoforms. Inhibition of endothelial and neuronal nitric oxide synthase, but not inducible nitric oxide synthase, reversed 3NP-induced pre-conditioning. The NO donor S-Nitroso-N-acetylpenicilamine mimicked 3NP effects further suggesting the involvement of NO in mediating 3NP protection. To investigate the involvement of NOS/soluble guanylate cyclase (sGC)/protein kinase G/mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) signaling pathway we examined the effect of 5-hydroxydecanoate (5-HD), a selective mitoK(ATP) blocker, and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one, a potent inhibitor of sGC, on 3NP-induced tolerance. 5-hydroxydecanoate, but not 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one, suppressed 3NP-mediated protection suggesting that mitoK(ATP) opening, but not NO-mediated activation of sGC, participates in the mechanism underlying tolerance to MDMA. Our data also showed that the protective effect of 3NP was abolished by cycloheximide, supporting the involvement of de novo protein synthesis. In conclusion, 3NP-induced delayed tolerance against 5-HT deficits caused by MDMA occurs via NO production.

Background and Purpose: 3,4-methylenedioxymethamphetamine (MDMA) causes a persistent loss of dopaminergic cell bodies in the substantia nigra of mice. Current evidence indicates that such neurotoxicity is due to oxidative stress but the source of free radicals remains unknown. Inhibition of mitochondrial electron transport chain complexes by MDMA was assessed as a possible source., Experimental Approach: Activities of mitochondrial complexes after MDMA were evaluated spectrophotometrically. In situ visualization of superoxide production in the striatum was assessed by ethidium fluorescence and striatal dopamine levels were determined by HPLC as an index of dopaminergic toxicity., Key Results: 3,4-methylenedioxymethamphetamine decreased mitochondrial complex I activity in the striatum of mice, an effect accompanied by an increased production of superoxide radicals and the inhibition of endogenous aconitase. alpha-Lipoic acid prevented superoxide generation and long-term toxicity independent of any effect on complex I inhibition. These effects of alpha-lipoic acid were also associated with a significant increase of striatal glutathione levels. The relevance of glutathione was supported by reducing striatal glutathione content with L-buthionine-(S,R)-sulfoximine, which exacerbated MDMA-induced dopamine deficits, effects suppressed by alpha-lipoic acid. The nitric oxide synthase inhibitor, N(G)-nitro-L-arginine, partially prevented MDMA-induced dopamine depletions, an effect reversed by L-arginine but not D-arginine. Finally, a direct relationship between mitochondrial complex I inhibition and long-term dopamine depletions was found in animals treated with MDMA in combination with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine., Conclusions and Implications: Inhibition of mitochondrial complex I following MDMA could be the source of free radicals responsible for oxidative stress and the consequent neurotoxicity of this drug in mice.

Phosphodiesterase 5 (PDE5) inhibitors are often used in combination with club drugs such as 3,4-methylenedioxymethamphetamine (MDMA or ecstasy). We investigated the consequences of such combination in the serotonergic system of the rat. Oral administration of sildenafil citrate (1.5 or 8 mg/kg) increased brain cGMP levels and protected in a dose-dependent manner against 5-hydroxytryptamine depletions caused by MDMA (3 x 5 mg/kg, i.p., every 2 h) in the striatum, frontal cortex and hippocampus without altering the acute hyperthermic response to MDMA. Intrastriatal administration of the protein kinase G (PKG) inhibitor, KT5823 [(9S, 10R, 12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid, methyl ester)], suppressed sildenafil-mediated protection. By contrast, the cell permeable cGMP analogue, 8-bromoguanosine cyclic 3',5'-monophosphate, mimicked sildenafil effects further suggesting the involvement of the PKG pathway in mediating sildenafil protection. Because mitochondrial ATP-sensitive K(+) channels are a target for PKG, we next administered the specific mitochondrial ATP-sensitive K(+) channel blocker, 5-hydroxydecanoic acid, 30 min before sildenafil. 5-hydroxydecanoic acid completely reversed the protection afforded by sildenafil, thereby implicating the involvement of mitochondrial ATP-sensitive K(+) channels. Sildenafil also increased Akt phosphorylation, and so the possible involvement of the Akt/endothelial nitric oxide synthase (eNOS)/sGC signalling pathway was analysed. Neither the phosphatidylinositol 3-kinase inhibitor, wortmannin, nor the selective eNOS inhibitor, L-N5-(1-iminoethyl)-L-ornithine dihydrochloride, reversed the protection afforded by sildenafil, suggesting that Akt/eNOS/sGC cascade does not participate in the protective mechanisms. Our data also show that the protective effect of sildenafil can be extended to vardenafil, another PDE5 inhibitor. In conclusion, sildenafil protects against MDMA-induced long-term reduction of indoles by a mechanism involving increased production of cGMP and subsequent activation of PKG and mitochondrial ATP-sensitive K(+) channel opening.

Rationale: A close relationship appears to exist between 3,4-methylenedioxymethamphetamine (MDMA)-induced changes in core body temperature and long-term serotonin (5-HT) loss., Objective: We investigated whether changes in core body temperature affect MDMA metabolism., Materials and Methods: Male Wistar rats were treated with MDMA at ambient temperatures of 15, 21.5, or 30 degrees C to prevent or exacerbate MDMA-induced hyperthermia. Plasma concentrations of MDMA and its main metabolites were determined for 6 h. Seven days later, animals were killed and brain indole content was measured., Results: The administration of MDMA at 15 degrees C blocked the hyperthermic response and long-term 5-HT depletion found in rats treated at 21.5 degrees C. At 15 degrees C, plasma concentrations of MDMA were significantly increased, whereas those of three of its main metabolites were reduced when compared to rats treated at 21.5 degrees C. By contrast, hyperthermia and indole deficits were exacerbated in rats treated at 30 degrees C. Noteworthy, plasma concentrations of MDMA metabolites were greatly enhanced in these animals. Instrastriatal perfusion of MDMA (100 microM for 5 h at 21 degrees C) did not potentiate the long-term depletion of 5-HT after systemic MDMA. Furthermore, interfering in MDMA metabolism using the catechol-O-methyltransferase inhibitor entacapone potentiated the neurotoxicity of MDMA, indicating that metabolites that are substrates for this enzyme may contribute to neurotoxicity., Conclusions: This is the first report showing a direct relationship between core body temperature and MDMA metabolism. This finding has implications on both the temperature dependence of the mechanism of MDMA neurotoxicity and human use, as hyperthermia is often associated with MDMA use in humans.

The mechanisms underlying 3,4-methylenedioxymethamphetamine (MDMA)-induced serotonergic (5-HT) toxicity remain unclear. It has been suggested that MDMA depletes 5-HT by increasing brain tyrosine levels, which via non-enzymatic hydroxylation leads to DA-derived free radical formation. Because this hypothesis assumes the pre-existence of hydroxyl radicals, we hypothesized that MDMA metabolism into pro-oxidant compounds is the limiting step in this process. Acute hyperthermia, plasma tyrosine levels and concentrations of MDMA and its main metabolites were higher after a toxic (15 mg/kg i.p.) vs. a non-toxic dose of MDMA (7.5mg/kg i.p.). The administration of a non-toxic dose of MDMA in combination with l-tyrosine (0.2 mmol/kg i.p.) produced a similar increase in serum tyrosine levels to those found after a toxic dose of MDMA; however, brain 5-HT content remained unchanged. The non-toxic dose of MDMA combined with a high dose of tyrosine (0.5 mmol/kg i.p.), caused long-term 5-HT depletions in rats treated at 21.5 degrees C but not in those treated at 15 degrees C, conditions known to decrease MDMA metabolism. Furthermore, striatal perfusion of MDMA (100 microM for 5h) combined with tyrosine (0.5 mmol/kg i.p.) in hyperthermic rats did not cause 5-HT depletions. By contrast, rats treated with the non-toxic dose of MDMA under heating conditions or combined with entacapone or acivicin, which interfere with MDMA metabolism or increase brain MDMA metabolite availability respectively, showed significant reductions of brain 5-HT content. Altogether, these data indicate that although tyrosine may contribute to MDMA-induced toxicity, MDMA metabolism appears to be the limiting step.

Preconditioning has emerged as a valid strategy against different neurotoxic insults. Although the mechanisms underlying preconditioning are not fully understood, the activation of ATP-sensitive potassium (KATP) channels has been proposed to play a pivotal role in neuronal preconditioning. In the present work we examine whether minoxidil a KATP channel activator protects against the long-term toxicity caused by the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA) in rats. Our data show that intrastriatal administration of minoxidil prevents MDMA-induced long-term indole depletions in the rat striatum. This effect was not related to an effect on core temperature, as pre-treatment with minoxidil did not significantly alter MDMA-induced hyperthermia. Taking into account that minoxidil opens both sarcolemmal and mitochondrial KATP channels, we examined the role of each type of channels in the protective effects of minoxidil using specific inhibitors. The administration of HMR-1098, a blocker of the sarcolemmal KATP channels, along with minoxidil did not affect the protection afforded by the latter. On the contrary the selective mitochondrial KATP channel blocker 5-hydroxydecanoic acid completely reversed the protection afforded by minoxidil, thereby implicating the involvement of mitochondrial (but not sarcolemmal) KATP channels. Furthermore our data show the participation of Akt and extracellular signal-regulated kinases in minoxidil-afforded protection. Intrastriatal administration of wortmannin or PD98059 (inhibitors of phosphatidylinositol-3-kinase and mitogen-activated protein kinase/extracellular regulated protein kinase, respectively), along with minoxidil abolished the protective effect of minoxidil against the serotonergic toxicity caused by MDMA. These results demonstrate that minoxidil by opening mitochondrial KATP channels completely prevents MDMA toxicity and that Akt and MAP kinases are involved in minoxidil-afforded neuroprotection.

Amiloride and several of its congeners known to block the Na(+)/Ca(2+) and/or Na(+)/H(+) antiporters potentiate methamphetamine-induced neurotoxicity without altering methamphetamine-induced hyperthermia. We now examine whether amiloride also exacerbates 3,4-methylenedioxymethamphetamine (MDMA)-induced long-term serotonin (5-HT) loss in rats. Amiloride (2.5 mg/kg, every 2 h x 3, i.p.) given at ambient temperature 30 min before MDMA (5 mg/kg, every 2 h x 3, i.p.), markedly exacerbated long-term 5-HT loss. However, in contrast to methamphetamine, amiloride also potentiated MDMA-induced hyperthermia. Fluoxetine (10 mg/kg i.p.) completely protected against 5-HT depletion caused by the MDMA/amiloride combination without significantly altering the hyperthermic response. By contrast, the calcium channel antagonists flunarizine or diltiazem did not afford any protection. Findings with MDMA and amiloride were extended to the highly selective Na(+)/H(+) exchange inhibitor dimethylamiloride, suggesting that the potentiating effects of amiloride are probably mediated by the blockade of Na(+)/H(+) exchange. When the MDMA/amiloride combination was administered at 15 degrees C hyperthermia did not develop and brain 5-HT concentrations remained unchanged 7 days later. Intrastriatal perfusion of MDMA (100 microM for 8 h) in combination with systemic amiloride caused a small depletion of striatal 5-HT content in animals made hyperthermic but not in the striatum of normothermic rats. These data suggest that enhancement of MDMA-induced 5-HT loss caused by amiloride or dimethylamiloride depends on their ability to enhance MDMA-induced hyperthermia. We hypothesise that blockade of Na(+)/H(+) exchange could synergize with hyperthermia to render 5-HT terminals more vulnerable to the toxic effects of MDMA.

The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA) produces long-term toxicity to serotonin (5-HT) neurones in rats, which is exacerbated when combined with the mitochondrial inhibitor malonate. Moreover, MDMA, which does not produce dopamine depletion in the rat, potentiates malonate-induced striatal dopamine toxicity. Because the malonate/MDMA combination acutely causes a synergistic increase of 5-HT and dopamine release, in this study we sought to determine whether pharmacological blockade of MDMA- and/or malonate-induced dopamine release prevents neurotoxicity. Fluoxetine, given 30 min prior to the malonate/MDMA combination, afforded complete protection against 5-HT depletion and reversed MDMA-induced exacerbation of dopamine toxicity found in the malonate/MDMA treated rats. Protection afforded by fluoxetine was not related to changes in MDMA-induced hyperthermia. Similarly, potentiation of malonate-induced dopamine toxicity caused by MDMA was not observed in p-chlorophenylalanine-5-HT depleted rats. Finally, the dopamine transporter inhibitor GBR 12909 completely prevented dopamine neurotoxicity caused by the malonate/MDMA combination and reversed the exacerbating toxic effects of malonate on MDMA-induced 5-HT depletion without significantly altering the hyperthermic response. Overall, these results suggest that the synergic release of dopamine caused by the malonate/MDMA combination plays an important role in the long-term toxic effects. A possible mechanism of neurotoxicity and protection is proposed.

Akin to what has been reported for cocaine, systemic administration of the dopamine D1 receptor antagonist, SCH 23390 ((R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride), blocks the expression but not the induction of 3,4-methylenedioxymethamphetamine (MDMA)-induced behavioral sensitization. Since the medial prefrontal cortex (mPFC) appears to regulate the expression of sensitization to cocaine, this study examined whether microinjection of SCH 23390 into the mPFC would alter the expression of MDMA sensitization. Saline or MDMA was administered for 5 consecutive days. After 12 days of withdrawal, rats received a bilateral intra-mPFC microinjection of SCH 23390 or saline followed by an intraperitoneal (i.p.) challenge dose of MDMA. While SCH 23390 enhanced locomotion in MDMA-naïve rats, it completely suppressed the expression of sensitization in MDMA-pretreated animals. Since, SCH 23390 has a fairly good affinity for 5-HT(2C) receptors, we went further to study the role of mPFC D1 and 5-HT(2C) receptors in this, apparently, paradoxical effect shown by SCH 23390. Thus, the microinjection of both SKF 81297 (R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide) and MK 212 (6-chloro-2-(1-piperazinyl)pyrazine hydrochloride), a D1 and 5-HT(2C) receptor agonist, respectively, blocked MDMA sensitization. By contrast, the 5-HT(2C) receptor antagonist, RS 102221 (8-[5-(2,4-dimethoxy-5-(4-trifluoromethylphenylsulfonamido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4,5]decane-2,4-dione hydrochloride), had no effect in MDMA-naïve or MDMA-sensitized animals, but reversed the effects of SCH 23390 in MDMA-pretreated rats. These results demonstrate that suppression of MDMA-induced sensitization by SCH 23390 is mediated by 5-HT(2C) receptor stimulation in the mPFC and not by the blockade of mPFC D1 receptors. Furthermore, these data indicate that stimulation of 5-HT(2C) receptors by SCH 23390 is not a minor issue and should be considered when interpreting future data.

There is ample evidence that plastic changes in the nervous system require the excitatory amino acid transmission. This appears to be also the case for psychostimulant-induced behavioral sensitization. More specifically the glutamatergic input from the medial prefrontal cortex (mPFC) to the VTA and the NAc appears to be involved in behavioral sensitization processes. However, dissociations regarding the role of the mPFC with respect to the development and expression of sensitization, as well as with respect to the psychostimulant being studied (amphetamine versus cocaine) appear to exist. The present study examined the role of the dorsal mPFC in the development and expression of 3,4-methylenedioxymethamphetamine (MDMA)-induced sensitization. Bilateral ibotenic acid or sham lesions of the dorsal mPFC were performed 7 days prior to or 4 days after a context-dependent sensitization-inducing regimen of MDMA (15 mg/kg i.p.) or saline. Rats were then challenged with MDMA (5 mg/kg i.p.) after 12 days of withdrawal. Ibotenic acid lesions did not affect the activating effects of MDMA, but prevented the development and expression of MDMA sensitization. Thus, the distance traveled during the development phase of sensitization increased in sham-lesioned rats but not in ibotenic-lesioned animals. Similarly, sham-lesioned rats showed a sensitized response when challenged with MDMA after the withdrawal period, an effect not observed in ibotenic-lesioned animals. These data reinforce the view that the dorsal mPFC is involved in psychostimulant sensitization and more specifically they indicate that the dorsal mPFC plays a key role in the development and expression of MDMA-induced behavioral sensitization.

Minocycline has been shown to exert neuroprotection against a wide variety of toxic insults both in vitro and in vivo. However, contradictory results have recently been reported. We now report that minocycline affords no protection against the neurotoxicity caused by malonate or N-methyl-d-aspartate (NMDA). Rats were treated with minocycline (45 mg/kg i.p. x 7) every 12 h. Thirty minutes after the second dose of minocycline, an intrastriatal stereotaxic injection of malonate (1.5 mumol) or NMDA (0.1 mumol) was administered. Seven days later, the rats were killed, and lesion volumes were quantified using two different methods [triphenyltetrazolium chloride (TTC) staining or cytochrome oxidase histochemistry]. Our results show that minocycline does not prevent the lesions caused by either malonate or by NMDA. On the contrary, the putative NMDA receptor antagonist, MK-801, blocked the toxicity caused by both toxins indicating that, although by different mechanisms, excitotoxicity is mediating neuronal death. We conclude that minocycline, at least under our experimental conditions, is not neuroprotective against excitotoxicity caused by either malonate or NMDA.

Rationale: There is a large body of evidence indicating that the mesoaccumbens dopamine pathway is critically involved in the expression of behavioral sensitization to amphetamine and cocaine, but its role in the development of sensitization to psychostimulants is not that sound. Very few studies, however, have examined the role of dopamine transmission in 3,4-methylenedioxymethamphetamine (MDMA)-induced sensitization., Objectives: The effects of the D1 receptor antagonist SCH 23390 on the development and expression of MDMA-induced behavioral sensitization were investigated in rats., Methods: During the development phase of sensitization, SCH 23390 was administered 15 min before every administration of MDMA. After 12 days of withdrawal, a MDMA challenge dose was given and locomotor activity was measured. In separate experiments, 15 min before the challenge injection of MDMA, SCH 23390 was administered either systemically or directly into the core of the nucleus accumbens (NAc) of MDMA-pretreated rats., Results: SCH 23390 did not prevent the development of MDMA-induced behavioral sensitization but completely blocked the expression when given before the challenge dose of MDMA. The same results were obtained when SCH 23390 was locally applied into the core of the NAc., Conclusions: The present data suggest that D1 receptor stimulation is not critical for the development of long-term MDMA sensitization, in agreement with what has been reported for cocaine. By contrast, expression of sensitization depends on the activation of D1 receptors located in the NAc core.