Subarachnoid Hemorrhage Depletes Calcitonin Gene-Related Peptide Levels of Trigeminal Neurons in Rat Dura Mater.

Subarachnoid hemorrhage (SAH) remains a major cause of cerebrovascular morbidity, eliciting severe headaches and vasospasms that have been shown to inversely correlate with vasodilator calcitonin gene-related peptide (CGRP) levels. Although dura mater trigeminal afferents are an important source of intracranial CGRP, little is known about the effects of SAH on these neurons in preclinical models. The present study evaluated changes in CGRP levels and expression in trigeminal primary afferents innervating the dura mater 72 h after experimentally induced SAH in adult rats. SAH, eliciting marked damage revealed by neurological examination, significantly reduced the density of CGRP-immunoreactive nerve fibers both in the dura mater and the trigeminal caudal nucleus in the medulla but did not affect the total dural nerve fiber density. SAH attenuated ex vivo dural CGRP release by ~40% and in the trigeminal ganglion, reduced both CGRP mRNA levels and the number of highly CGRP-immunoreactive cell bodies. In summary, we provide novel complementary evidence that SAH negatively affects the integrity of the CGRP-expressing rat trigeminal neurons. Reduced CGRP levels suggest likely impaired meningeal neurovascular functions contributing to SAH complications. Further studies are to be performed to reveal the importance of impaired CGRP synthesis and its consequences in central sensory processing.

Inflammatory Orofacial Pain Activates Peptidergic Neurons and Upregulates the Oxytocin Receptor Expression in Trigeminal Ganglion.

The majority of orofacial pain is caused by musculoskeletal and neuropathological diseases related to inflammatory processes that lead even to transcriptional alterations in the trigeminal ganglion (TG) neurons. The hypothalamic nonapeptide oxytocin has been reported to modulate nociception via binding and activating its receptor in primary sensory neurons. The purpose of this study was to analyze the gene expression of the oxytocin receptor (OTR), c-Fos, an indicator of neuronal activity, and α-calcitonin gene-related peptide (αCGRP), a characteristic neurotransmitter of the peptidergic trigeminal primary afferents in an animal model of inflammation-induced orofacial pain. Carrageenan was unilaterally injected into the vibrissal pads of male and female adult Wistar rats. RT-qPCR was performed to analyze the levels of mRNA expression in TGs 24 h after injection. The gene expression analysis revealed higher fold changes regarding the c-Fos (mean ± S.E: ♀: 3.9 ± 0.19; ♂: 3.55 ± 0.18) and αCGRP (♀: 2.84 ± 0.13; ♂: 3.39 ± 0.47) expression levels of mRNA, and a moderate rise in the expression of the OTR mRNA (♀: 1.52 ± 0.07; ♂: 1.49 ± 0.07) was observed in comparison to both vehicle(saline)-treated and untreated controls. Our results furnish evidence for inflammation-induced activation of peptidergic neurons, and it is suggested that oxytocin modulates inflammation-induced nociception by enhancing their signaling capacity due to its elevated expression in the sensory ganglion cells, thus providing new therapies for orofacial pain relief that target the OTRs.

Peripheral Branch Injury Induces Oxytocin Receptor Expression at the Central Axon Terminals of Primary Sensory Neurons.

Considerable evidence suggests that oxytocin, as a regulatory nonapeptide, participates in modulatory mechanisms of nociception. Nonetheless, the role of this hypothalamic hormone and its receptor in the sensory pathway has yet to be fully explored. The present study performed immunohistochemistry, enzyme-linked immunosorbent assay, and RT-qPCR analysis to assess changes in the expression of the neuronal oxytocin receptor in female rats following tight ligation of the sciatic nerve after 1, 3, and 7 days of survival. Oxytocin receptor immunoreactivity was present in both dorsal root ganglia and lumbar spinal cord segments, but not accumulated at the site of the ligation of the peripheral nerve branch. We found a time-dependent change in the expression of oxytocin receptor mRNA in L5 dorsal root ganglion neurons, as well as an increase in the level of the receptor protein in the lumbar segment of the spinal cord. A peak in the expression was observed on day 3, which downturned slightly by day 7 after the nerve ligation. These results show that OTR expression is up-regulated in response to peripheral nerve lesions. We assume that the importance of OTR is to modify spinal presynaptic inputs of the sensory neurons upon injury-induced activation, thus to be targets of the descending oxytocinergic neurons from supraspinal levels. The findings of this study support the concept that oxytocin plays a role in somatosensory transmission.

Role of Gangliosides in Peripheral Pain Mechanisms.

Gangliosides are abundantly occurring sialylated glycosphingolipids serving diverse functions in the nervous system. Membrane-localized gangliosides are important components of lipid microdomains (rafts) which determine the distribution of and the interaction among specific membrane proteins. Different classes of gangliosides are expressed in nociceptive primary sensory neurons involved in the transmission of nerve impulses evoked by noxious mechanical, thermal, and chemical stimuli. Gangliosides, in particular GM1, have been shown to participate in the regulation of the function of ion channels, such as transient receptor potential vanilloid type 1 (TRPV1), a molecular integrator of noxious stimuli of distinct nature. Gangliosides may influence nociceptive functions through their association with lipid rafts participating in the organization of functional assemblies of specific nociceptive ion channels with neurotrophins, membrane receptors, and intracellular signaling pathways. Genetic and experimentally induced alterations in the expression and/or metabolism of distinct ganglioside species are involved in pathologies associated with nerve injuries, neuropathic, and inflammatory pain in both men and animals. Genetic and/or pharmacological manipulation of neuronal ganglioside expression, metabolism, and action may offer a novel approach to understanding and management of pain.

Distinct changes in chronic pain sensitivity and oxytocin receptor expression in a new rat model (Wisket) of schizophrenia.

Clinical studies have shown that schizophrenia is accompanied by hypoalgesia. Accordingly, we have previously reported that a chronic schizophrenia-related rat substrain (Wisket) showed decreased acute heat pain sensitivity. The aim of the present study was to determine the mechanical pain sensitivity and the effects of opioid ligands in a chronic osteoarthritic pain model generated using Wisket rats. Our previous molecular biological studies indicated that the impairment in opioid and cannabinoid receptor functions observed in these animals did not explain their altered pain sensitivity. Therefore, we aimed to investigate another endogenous antinociceptive system, i.e., the oxytocinergic system (which is also implicated in schizophrenia) via the determination the brain-region specific oxytocin receptor mRNA expression in Wisket rats. Osteoarthritis was induced in male adult control Wistar rats without any interventions and in Wisket rats after juvenile social isolation and ketamine treatment. The degree of allodynia and the effects of systemic morphine or intrathecal endomorphin-1 administration were determined. Furthermore, the expression of the oxytocin receptor mRNA was assessed in different brain structures (prefrontal cortex, striatum, diencephalon, brainstem, and olfactory bulb). A lower degree of allodynia was observed in the Wisket group compared with control animals 1 and 2 weeks after the induction of osteoarthritis, which was accompanied by a comparable degree of edema. Systemically or intrathecally applied opioids caused similar time-response curves in both groups, with apparently shorter effects in Wisket animals. The expression of the oxytocin receptor mRNA was lower in most of the brain regions (with the exception of the diencephalon) investigated in Wisket rats vs. the control animals. In summary, both acute and chronic hypoalgesia (as nonspecific symptoms in patients with schizophrenia) can be simulated in Wisket animals as endophenotypes despite the impairment of the endogenous antinociceptive systems evaluated. Thus, this model might be an appropriate tool for further investigation of the molecular basis of altered pain perception in schizophrenia.

Chronic adriamycin treatment impairs CGRP-mediated functions of meningeal sensory nerves.

Adriamycin is a potent anthracycline-type antitumor agent, but it also exerts potentially serious side effects due to its cardiotoxic and neurotoxic propensity. Multiple impairments in sensory nerve functions have been recently reported in various rat models. The present experiments were initiated in an attempt to reveal adriamycin-induced changes in sensory effector functions of chemosensitive meningeal afferents. Meningeal blood flow was measured with laser Doppler flowmetry in the parietal dura mater of adult male Wistar rats. The dura mater was repeatedly stimulated by topical applications of capsaicin, a transient receptor potential vanilloid 1 (TRPV1) receptor agonist, or acrolein, a transient receptor potential ankyrin 1 (TRPA1) receptor agonist, which induce the release of calcitonin gene-related peptide (CGRP) from meningeal afferents. The blood flow increasing effects of CGRP, histamine, acetylcholine and forskolin were also measured. Capsaicin- and acrolein-induced CGRP release was measured with enzyme-linked immunoassay in an ex vivo dura mater preparation. TRPV1 content of trigeminal ganglia and TRPV1-, CGRP- and CGRP receptor component-immunoreactive structures were examined in dura mater samples obtained from control and adriamycin-treated rats. The vasodilator effects of capsaicin, acrolein and CGRP were significantly reduced in adriamycin-treated animals while histamine-, acetylcholine- and forskolin-induced vasodilatation were unaffected. Measurements of CGRP release in an ex vivo dura mater preparation revealed an altered dynamic upon repeated stimulations of TRPV1 and TRPA1 receptors. In whole-mount dura mater preparations immunohistochemistry revealed altered CGRP receptor component protein (RCP)-immunoreactivity in adriamycin-treated animals, while CGRP receptor activity modifying protein (RAMP1)-, TRPV1- and CGRP-immunostaining were left apparently unaltered. Adriamycin-treatment slightly reduced TRPV1 protein content of trigeminal ganglia. The present findings demonstrate that adriamycin-treatment alters the function of the trigeminovascular system leading to reduced meningeal sensory neurogenic vasodilatation that may affect the local regulatory and protective mechanisms of chemosensitive afferents leading to alterations in tissue integrity.

The osmotically and histamine-induced enhancement of the plasma vasopressin level is diminished by intracerebroventricularly administered orexin in rats.

The effects of the centrally administered neuropeptides orexin-A on water intake and vasopressin (VP) secretion were studied in male Wistar rats (180-250 g). Different doses (10, 30, and 90 µg/10 µl) of the orexins and the specific orexin receptor-1 (OX(1)) antagonist SB 408124 (30 µg/10 µl) were administered intracerebroventricularly (i.c.v.) under anaesthesia, and the water consumption was measured during 6 h. A plasma VP level elevation was induced by histamine (10 mg/kg) or 2.5% NaCl (10 ml/kg) administered intraperitoneally (i.p.). The plasma VP levels were measured by radioimmunoassay. Increased water consumption was observed after the administration of 30 µg/10 µl orexin-A. There were no changes in basal VP secretion after the administration of different doses of the orexins. A significant increase in plasma VP concentration was detected following histamine administration. After 2.5% NaCl administration, there was a moderate VP level enhancement. Intracerebroventricularly administered orexin-A (30 µg/10 µl) blocked the VP level increase induced by either histamine or 2.5% NaCl administration. The inhibitory effects were prevented by the specific OX(1) receptor antagonist. In conclusion, the orexins increased water consumption. After 30 µg/10 µl orexin-A administration, the polydipsia was more pronounced. The OX(1) receptor antagonist significantly decreased the polydipsia. Histamine or hyperosmotic VP release enhancement was blocked by previously administered orexin. This inhibition was not observed following OX(1) receptor antagonist administration. Our results suggest that the effects of the orexins on water consumption or blockade of the histamine and osmosis-induced VP level increase are mediated by the OX(1) receptor.

Effects of orexin-monoaminergic interactions on oxytocin secretion in rat neurohypophyseal cell cultures.

The effects of orexin-monoaminergic compound interactions on oxytocin release were studied in 14-day rat neurohypophyseal cell cultures prepared by an enzymatic dissociation technique. The oxytocin contents of the supernatants were determined by radioimmunoassay. Following the administration of orexin-A or orexin-B in increasing doses, significant changes were not observed in the oxytocin content of the supernatant media. The oxytocin level increased substantially in response to adrenaline, noradrenaline, serotonin, histamine, dopamine or K(+) treatment. Preincubation with orexin-A or orexin-B reduced the adrenaline-, histamine- or serotonin-induced oxytocin level increases, but the oxytocin concentrations of the supernatant media remained above the control level. There was no significant difference in decreasing effect between orexin-A and orexin-B. Neither orexin-A nor orexin-B induced changes in oxytocin release following monoaminergic compound treatment. The results indicate that the changes in oxytocin secretion induced by the monoaminergic system can be directly influenced by the orexin system. The effects of orexin on oxytocin release can be antagonized by an orexin-1 receptor-specific antagonist. It may be presumed that the orexins can play a role in the pathogenetic process of metabolic diseases (e.g. obesity) by reducing the effects of increased oxytocin release caused by monoaminergic compounds. The interactions between the monoaminergic and orexin systems regarding oxytocin secretion occur at both the hypothalamic and the neurohypophyseal levels.

The effects of orexins on monoaminerg-induced changes in vasopressin level in rat neurohypophyseal cell cultures.

The effects of orexin-monoaminergic compound interactions on vasopressin release were studied in 14-day neurohypophyseal cell cultures from adult rats, prepared by an enzymatic dissociation technique. The vasopressin contents of the supernatants were determined by radioimmunoassay. Following administration of either orexin-A or orexin-B in increasing doses, significant changes were not observed in the vasopressin levels of the supernatant media. The vasopressin level substantially increased after epinephrine, norepinephrine, serotonin, histamine, dopamine or K(+) treatment. Preincubation with either orexin-A or orexin-B reduced the epinephrine-, histamine- or serotonin-induced increases in vasopressin level, but the vasopressin concentrations of the supernatant media remained above the control level. There was no significant difference in decreasing effect between orexin-A and orexin-B. Neither orexin-A nor orexin-B induced changes in vasopressin release following monoaminergic compound treatment. The results indicate that the changes in vasopressin secretion induced by the monoaminergic system can be directly influenced by orexin system. It may be presumed that the orexins can play a physiological role in the regulation of the water metabolism by reducing the effect of increased vasopressin release caused by monoaminergic compounds. The interactions between the monoaminergic and orexin systems regarding vasopressin secretion occur at both the hypothalamic and the neurohypophyseal level.

Optimization of PCR amplification for B- and T-cell clonality analysis on formalin-fixed and paraffin-embedded samples.

In many cases, particularly in retrospective studies, only formalin-fixed and paraffin-embedded (FFPE) tissue samples are available for molecular studies. DNA recovered from FFPE tissues generally consists of fragmented small target sequences with chemical alterations. Clonality analysis is not easy on FFPE samples, in fact, it requires even more experience than that of performed on fresh samples or is more complicated than most genomic PCR amplifications for somatic genes. In our study, we have performed a multi-parameter PCR evaluation investigating immunoglobulin heavy chain gene (IgH) and T-cell receptor gamma gene (TCRgamma) rearrangements on non-purified crude lysates of FFPE samples, in order to establish the significance of different variables on performance of PCR amplification. The results showed that a slight decrease in the concentration of primers in combination with a slight increase in MgCl2 and Taq polymerase concentrations, as well as the use diluted crude template and a standard amount of dNTPs can be the modifications of choice while adjusting IgH and TCRgamma clonality tests on poor quality DNA FFPE samples. Using our improved protocol, 74% (17/23) of the tested B-cell lymphomas and 68% (31/46) of the tested T-cell lymphomas demonstrated monoclonal PCR product, proving the applicability of our optimized method. Our experience may be of help during the optimization process in technically difficult cases as well as to determine which parameters and how should be changed to minimize false-negative and false-positive results.

[A comparison of chemical composition and nutritional value of organically and conventionally grown plant derived foods]. / Az ökológiai (bio) és konvencionális termesztésu növényi élelmiszerek beltartalmának, táplálkozási értékének összehasonlítása.

The consumption of organic food has been increasing all over the world. Due to this fact, there are growing numbers of scientific studies examining the nutritional value of organic food. The aim of this review is to provide an overall picture of the beneficial and harmful nutritional content of organically and conventionally produced crops based on existing international comparative surveys. Furthermore, the authors attempt to define the relationship between organic and conventional food production systems and the nutritional value of food products as well as the consumption of organic and conventional diets which have important human health implications. Organic crops contain a significantly higher amount of certain antioxidants (vitamin C, polyphenols and flavonoids) and minerals, as well as have higher dry matter content than conventional ones. Moreover, there is a lower level of pesticide residues, nitrate and some heavy metal contaminations in organic crops compared to conventional ones. There is a relationship between the different fertilisation and plant protection methods of these two plant production systems and the nutritional composition of crops. Consequently, it can be concluded that organically produced plant derived food products have a higher nutritional value, including antioxidants than conventional ones. Furthermore, due to the fact that there is a lower level of contamination in organic crops, the risk of diseases caused by contaminated food is significantly reduced.