Contribution of platelet P2Y12 receptors to chronic Complete Freund's adjuvant-induced inflammatory pain.

Essentials The role of platelet P2Y12 receptors in the regulation of chronic inflammatory pain is unknown. Complete Freund's Adjuvant (CFA)-induced chronic inflammatory pain model was used in mice. Gene deficiency and antagonists of P2Y12 receptors attenuate hyperalgesia and local inflammation. Platelet P2Y12 receptors contribute to these effects in the chronic phase of inflammation. SUMMARY: Background P2Y12 receptor antagonists are widely used in clinical practice to inhibit platelet aggregation. P2Y12 receptors are also known to regulate different forms of pain as well as local and systemic inflammation. However, it is not known whether platelet P2Y12 receptors contribute to these effects. Objectives To explore the contribution of platelet P2Y12 receptors to chronic inflammatory pain in mice. Methods Complete Freund's adjuvant (CFA)-induced chronic inflammatory pain was induced in wild-type and P2ry12 gene-deficient (P2ry12-/- ) mice, and the potent, direct-acting and reversible P2Y12 receptor antagonists PSB-0739 and cangrelor were used. Results CFA-induced mechanical hyperalgesia was significantly decreased in P2ry12-/- mice for up to 14 days, and increased neutrophil myeloperoxidase activity and tumor necrosis factor (TNF)-α and CXCL1 (KC) levels in the hind paws were also attenuated in the acute inflammation phase. At day 14, increased interleukin (IL)-1ß, IL-6, TNF-α and KC levels were attenuated in P2ry12-/- mice. PSB-0739 and cangrelor reversed hyperalgesia in wild-type mice but had no effect in P2ry12-/- mice, and PSB-0739 was also effective when applied locally. The effects of both local and systemic PSB-0739 were prevented by A-803467, a selective NaV1.8 channel antagonist, suggesting the involvement of NaV1.8 channels in the antihyperalgesic effect. Platelet depletion by anti-mouse CD41 antibody decreased hyperalgesia and attenuated the proinflammatory cytokine response in wild-type but not in P2ry12-/- mice on day 14. Conclusions In conclusion, P2Y12 receptors regulate CFA-induced hyperalgesia and the local inflammatory response, and platelet P2Y12 receptors contribute to these effects in the chronic inflammation phase.

Cell sorting in a Petri dish controlled by computer vision.

Fluorescence-activated cell sorting (FACS) applying flow cytometry to separate cells on a molecular basis is a widespread method. We demonstrate that both fluorescent and unlabeled live cells in a Petri dish observed with a microscope can be automatically recognized by computer vision and picked up by a computer-controlled micropipette. This method can be routinely applied as a FACS down to the single cell level with a very high selectivity. Sorting resolution, i.e., the minimum distance between two cells from which one could be selectively removed was 50-70 micrometers. Survival rate with a low number of 3T3 mouse fibroblasts and NE-4C neuroectodermal mouse stem cells was 66 ± 12% and 88 ± 16%, respectively. Purity of sorted cultures and rate of survival using NE-4C/NE-GFP-4C co-cultures were 95 ± 2% and 62 ± 7%, respectively. Hydrodynamic simulations confirmed the experimental sorting efficiency and a cell damage risk similar to that of normal FACS.

Astroglia-derived retinoic acid is a key factor in glia-induced neurogenesis.

Astroglial cells are essential components of the neurogenic niches within the central nervous system. Emerging evidence suggests that they are among the key regulators of postnatal neurogenesis. Although astrocytes have been demonstrated to possess the potential to instruct stem cells to adopt a neuronal fate, little is known about the nature of the glia-derived instructive signals. Here we propose that all-trans retinoic acid, one of the most powerful morphogenic molecules regulating neuronal cell fate commitment, may be one of the glia-derived factors directing astroglia-induced neurogenesis. According to data obtained from several complementary approaches, we show that cultured astrocytes express the key enzyme mRNAs of retinoic acid biosynthesis and actively produce all-trans retinoic acid. We show that blockage of retinoic acid signaling by the pan-RAR antagonist AGN193109 prevents glia-induced neuron formation by noncommitted stem cells. Therefore, we provide strong in vitro evidence for retinoic acid action in astroglia-induced neuronal differentiation.

Cultured astrocytes react to LPS with increased cyclooxygenase activity and phagocytosis.

Phagocytosis and prostaglandin E(2) production were investigated in purified cultures of perinatal rat forebrain astrocytes. Light and electron microscopic data indicated that astrocytes respond to bacterial endotoxin, lipopolysaccharide (LPS) by increased phagocytosis and by activating the cyclooxygenase enzyme-pathway. LPS-inducible phagocytosis of astrocytes was demonstrated by electron microscopic studies on colloidal gold uptake and by photometric determination of fluorescent bead ingestion. The internalisation of fragments of the plasma membrane was shown by histochemical detection of membrane-bound ecto-ATPase activity within intracellular vesicles. Activation of the cyclooxygenase pathway, a characteristic reaction of immune cells under inflammatory conditions, was also detected in astroglial cells upon treatment with LPS. The increased prostaglandin E(2) (PGE(2)) production by astrocytes in response to LPS was reduced by the non-steroid anti-inflammatory drug, indomethacin. Our data indicate that astrocytes display some tissue-protective reactions in response to inflammation inducing factors, even in the absence of peripheral immune cells or central microglia. The role of inducible astrocytic phagocytosis in a non-immune protection-pathway is discussed.

Proliferative and migratory responses of astrocytes to in vitro injury.

An in vitro "scratch-wound" model was used to evoke and investigate some astroglial responses to mechanical injury. The changes in the morphology, locomotion, and proliferation of injured astrocytes were analysed under culture conditions devoid of blood-derived cells responsible for activating the inflammatory cascade. The rate of proliferation was determined by immunocytochemical detection of BrdU-incorporating cells located next to or far from the wound. The motility of individual cells and the mass-advancement of cell-assemblies were monitored by computer controlled video-microscopy both in injured monolayers and in preparations of single cells or aggregates of astrocytes. The large sets of digitalized data allowed a reliable statistical evaluation of changes in cell positions providing a quantitative approach for studies on dynamics of cell locomotion. The results indicated that cultivated astrocytes respond to injury (1) with enhanced nestin immunoreactivity at the expanding processes, (2) with increased mitotic activity exceeding the rate caused by the liberation from contact inhibition, but (3) without specific, injury-induced activation of cell locomotion. Some advantages and drawbacks of "scratch-wound" models of astrocytic responses to mechanical injury are presented and discussed.

[Lead poisoning caused by lead-glazed ceramic household article, initially assumed to be of occupational origin]. / Kezdetben foglalkozási eredetünek imponáló familiaris alimentaris ólommérgezés ólommázas agyagedény háztartási használatát követóen.

The authors observed serious lead intoxication of a 18 year old female ceramic apprentice caused by tea with lemon stored in a glazed ceramic teapot made by the patient herself. The case history suggests: 1. the need of warning people to avoid the use of ceramic tablewares made by insufficient technology for alimentary purposes. 2. Patient with lead exposure can have also alimentary intoxication. 3. It seems advisable to perform screening tests of the ceramic apprentices who use lead glazes in order to detect the eventual increased absorption of lead in time.

Effects of retinoic acid on rat forebrain cells derived from embryonic and perinatal rats.

All-trans retinoic acid (RA), a potent inducer of neural development in non-committed neuroectodermal precursors and also, a teratogenic agent for early prosencephalic development is reported to promote the survival and differentiation of embryonic forebrain neurons, in vitro. In cultures of embryonic (E13, E15) rat forebrain cells, long-term (2-5 days) treatment with RA increased the number of neurons and the overall neurofilament immunoreactivity. Treatment with RA for periods longer than 1 h resulted in enhanced binding of the non-competitive NMDA-receptor antagonist, TCP, by embryonic and fetal (E17, E18) cells, but not by cells derived from perinatal (E19, P0) forebrains. As TCP binding-sites are localised within the channel-complex, treatment with RA was thought to result in an opening of the NMDA receptor channel. In direct binding assays, however, RA had no detectable effect, while conditioned media taken from RA-treated embryonic or fetal cells increased the TCP-binding, immediately. Analyses on conditioned media taken from control cultures of cells with various in vivo or in vitro ages revealed a stable extracellular glutamate level ([Glu]e) of 1-3 microM. This basal [Glu]e was restored within 24 h after addition of 100 microM exogenous glutamate. In the presence of RA, however, [Glu]e was stabilised at an approximately three-fold higher (4-10 microM) level by cells derived from embryonic and fetal brains. RA-treatment did not influence the [Glu]e in cultures of perinatal cells. The RA-induced rise in the neurofilament-immunoreactivity of embryonic brain cell cultures was prevented by simultaneous treatment with APV, a competitive antagonist of NMDA-receptors. The data suggest that a RA-induced shift in the set-point of extracellular glutamate-balance plays an important role in the promotion of survival and maturation of developing neurons, in culture.

Development of oxytocinergic neurons in monolayer cultures derived from embryonic, fetal and postnatal rat hypothalami.

Monolayer cultures from hypothalami of embryonic, fetal and postnatal rats were established to study the development of oxytocin-secreting neurons in vitro. Particular culture conditions, known to enhance the survival and growth of different types of neural cells in vitro, were used to investigate the conditions necessary for the appearance and survival of these peptide-producing cells in culture. They included increasing the concentration of potassium in the culture milieu and/or the addition of triiodothyronine (T(3) ). The use of immunocytochemical procedures with a monoclonal antibody that recognizes oxytocin-associated neurophysin (NP-OT) and polyclonal antibodies specific for oxytocin permitted us to identify the neurons. In cultures derived from embryonic (E16-E17) hypothalami, no NP-OT- or oxytocin-positive neurons were detected and their appearance could not be provoked by increasing extracellular potassium concentration or by administration of T(3) . On the other hand, in cultures obtained from fetal (E18-E19) rat hypothalami, a few neurons showed immunoreactivity for NP-OT (but not for oxytocin); the immunoreactivity was localized essentially in somata and proximal portions of neurites. When 10(8) M T(3) was included in the culture medium, there were cells showing immunoreactivity not only for NP-OT, but also for oxytocin, visible in somata and in dendritic- and axonal-like processes. In comparison, T(3) did not influence the total number of neurons developing in these cultures. Lastly, in cultures derived from young postnatal (PO-P2) rat hypothalami, NP-OT- and oxytocin-immunopositive neurons were found regularly; their appearance did not require any special pretreatment of the cultures. In all cultures, high extracellular potassium concentration (25 mM) resulted in a general improvement in the survival of neurons but did not induce the appearance of more oxytocin-immunoreactive cells. Our observations support in vivo results showing that although presumptive oxytocin-producing cells appear early in the development of the hypothalamus, their maturation, and in particular, their ability to produce oxytocin, occurs late at the time of birth. A factor that selectively enhances their differentiation, is the thyroid hormone, T(3) .