Cellular and molecular basis of TNFa, IL-1ß and LPS mediated signaling in rat dorsal root ganglion

The proinflammatory cytokines TNFa and IL-1ß as well as bacterial lipopolysaccharide (LPS) are known to affect primary afferent functions related to pain and neurogenic inflammation. However, it is not completely understood how these molecules signal to primary sensory neurons of the dorsal root ganglion (DRG). In order to clarify this question RT-PCR, Northern blot, Western blot, RT-PCR in combination with laser capture microdissection (LCM) and in situ hybridization (ISH) with radioactive-labeled probes as well as double ISH were employed. These methods were used to determine the cell-specific expression pattern of TNF, IL-1 and their functional receptors as well as of LPS-related receptors in neuronal and non-neuronal cells of rat DRG as well as in the sensory cell line F11. The following essential new findings and conclusions have been obtained. (1) For the first time, the rat TNFR2 gene was characterized with 10 exons and 9 introns, which are located in chromosome 5q36. Three cDNAs for the rat TNFR2 gene were identified. Their full coding region was found to be identical. Three transcripts of the rat TNFR2 gene were observed in neural tissues (i.e. DRG, spinal cord and brain) and in peripheral tissues (i.e. spleen, lung and kidney). The regulation of TNFR2 transcripts by LPS seemed to occur in a tissue- and cell-specific manner as demonstrated for the spleen and DRG. (2) TNFR1 mRNA was found to be constitutively expressed in all DRG neurons including presumed nociceptive neurons coding for neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP) or vanilloid receptor 1 (VR1) and to be increased after LPS. In contrast to the literature, TNFR2 mRNA was found to be totally absent from DRG neurons of control rats and of rats after LPS challenge. TNFR1 mRNA and TNFR2 mRNA were found to be constitutively expressed in DRG non-neuronal cells and to be increased after systemic LPS. The data provided by this study suggest that TNF may influence DRG sensory functions by directly acting on TNFR1 in neurons or by indirectly acting on both TNFR1 and TNFR2 in non-neuronal cells. (3) Like DRG neurons, the sensory cell line F-11 was found to express TNFR1 but not TNFR2. Therefore, the F11 cell line is uniquely suited to study TNFR1-mediated intracellular signaling and cellular functions independent from that of TNFR2 effects. (4) There was no evidence for but strong evidence against constitutive or LPS-induced expression of TNF and IL-1 mRNAs in DRG neurons. LPS-induced expression of TNF and IL-1 mRNAs in DRG occurred exclusively in DRG non-neuronal cells. Thus, the previously reported concept that TNF and IL-1 are synthesized by DRG neurons should be dismissed. To the contrary, the present data indicate that endogenous TNF and IL-1 in DRG are exclusively synthesized by non-neuronal cells implicating that they may act on DRG neurons in a paracrine manner. (5) In contrast to a previous report indicating that IL-1R1 is expressed in all DRG cells, the present study demonstrated that IL-1R1 mRNA is expressed only in a subpopulation of DRG neurons and in some DRG non-neuronal cells as well. IL-1R1 exhibited substantial coincidence with presumed nociceptive neurons expressing VR1, SP or CGRP. The results of the present study suggest that endogenous and exogenous IL-1 may directly activate DRG neurons via IL-1R1 to preferentially modulate nociceptive functions. In addition, IL-1 may act on DRG non-neuronal cells to cause further release of IL-1. (6) For the first time, the functional LPS receptor-TLR4 was demonstrated to be expressed in DRG neuronal and non-neuronal cells at the mRNA level. The neuronal expression of TLR4 was limited to a subset of DRG neurons where it exhibited substantial coincidence with presumed nociceptive neurons expressing VR1, SP or CGRP. The mRNA coding for the LPS receptor accessory protein CD14 was totally absent from DRG neurons of control rats and of rats after systemic LPS. LPS-induced expression of CD14 occurred in DRG non-neuronal cells. The present data indicate that LPS may directly act on primary sensory neurons via TLR4 or indirectly act on primary sensory neurons via TLR4 and CD14. This implies that primary sensory neurons of DRG may detect an infectious state by directly sensing LPS via TLR4. Taken together, this study provides new insights into the cellular and molecular basis of TNF, IL-1 and LPS mediated primary sensory neurotransmission related to pain and neurogenic inflammation. In addition, the present study provides new evidence that the primary sensory neurons of DRG may have an important role as immunosensors to detect and control microbial infection and inflammation

Cellular and molecular basis of TNFa, IL-1ß and LPS mediated signaling in rat dorsal root ganglion

The proinflammatory cytokines TNFa and IL-1ß as well as bacterial lipopolysaccharide (LPS) are known to affect primary afferent functions related to pain and neurogenic inflammation. However, it is not completely understood how these molecules signal to primary sensory neurons of the dorsal root ganglion (DRG). In order to clarify this question RT-PCR, Northern blot, Western blot, RT-PCR in combination with laser capture microdissection (LCM) and in situ hybridization (ISH) with radioactive-labeled probes as well as double ISH were employed. These methods were used to determine the cell-specific expression pattern of TNF, IL-1 and their functional receptors as well as of LPS-related receptors in neuronal and non-neuronal cells of rat DRG as well as in the sensory cell line F11. The following essential new findings and conclusions have been obtained. (1) For the first time, the rat TNFR2 gene was characterized with 10 exons and 9 introns, which are located in chromosome 5q36. Three cDNAs for the rat TNFR2 gene were identified. Their full coding region was found to be identical. Three transcripts of the rat TNFR2 gene were observed in neural tissues (i.e. DRG, spinal cord and brain) and in peripheral tissues (i.e. spleen, lung and kidney). The regulation of TNFR2 transcripts by LPS seemed to occur in a tissue- and cell-specific manner as demonstrated for the spleen and DRG. (2) TNFR1 mRNA was found to be constitutively expressed in all DRG neurons including presumed nociceptive neurons coding for neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP) or vanilloid receptor 1 (VR1) and to be increased after LPS. In contrast to the literature, TNFR2 mRNA was found to be totally absent from DRG neurons of control rats and of rats after LPS challenge. TNFR1 mRNA and TNFR2 mRNA were found to be constitutively expressed in DRG non-neuronal cells and to be increased after systemic LPS. The data provided by this study suggest that TNF may influence DRG sensory functions by directly acting on TNFR1 in neurons or by indirectly acting on both TNFR1 and TNFR2 in non-neuronal cells. (3) Like DRG neurons, the sensory cell line F-11 was found to express TNFR1 but not TNFR2. Therefore, the F11 cell line is uniquely suited to study TNFR1-mediated intracellular signaling and cellular functions independent from that of TNFR2 effects. (4) There was no evidence for but strong evidence against constitutive or LPS-induced expression of TNF and IL-1 mRNAs in DRG neurons. LPS-induced expression of TNF and IL-1 mRNAs in DRG occurred exclusively in DRG non-neuronal cells. Thus, the previously reported concept that TNF and IL-1 are synthesized by DRG neurons should be dismissed. To the contrary, the present data indicate that endogenous TNF and IL-1 in DRG are exclusively synthesized by non-neuronal cells implicating that they may act on DRG neurons in a paracrine manner. (5) In contrast to a previous report indicating that IL-1R1 is expressed in all DRG cells, the present study demonstrated that IL-1R1 mRNA is expressed only in a subpopulation of DRG neurons and in some DRG non-neuronal cells as well. IL-1R1 exhibited substantial coincidence with presumed nociceptive neurons expressing VR1, SP or CGRP. The results of the present study suggest that endogenous and exogenous IL-1 may directly activate DRG neurons via IL-1R1 to preferentially modulate nociceptive functions. In addition, IL-1 may act on DRG non-neuronal cells to cause further release of IL-1. (6) For the first time, the functional LPS receptor-TLR4 was demonstrated to be expressed in DRG neuronal and non-neuronal cells at the mRNA level. The neuronal expression of TLR4 was limited to a subset of DRG neurons where it exhibited substantial coincidence with presumed nociceptive neurons expressing VR1, SP or CGRP. The mRNA coding for the LPS receptor accessory protein CD14 was totally absent from DRG neurons of control rats and of rats after systemic LPS. LPS-induced expression of CD14 occurred in DRG non-neuronal cells. The present data indicate that LPS may directly act on primary sensory neurons via TLR4 or indirectly act on primary sensory neurons via TLR4 and CD14. This implies that primary sensory neurons of DRG may detect an infectious state by directly sensing LPS via TLR4. Taken together, this study provides new insights into the cellular and molecular basis of TNF, IL-1 and LPS mediated primary sensory neurotransmission related to pain and neurogenic inflammation. In addition, the present study provides new evidence that the primary sensory neurons of DRG may have an important role as immunosensors to detect and control microbial infection and inflammation

Magnetic Crosstalk Suppression and Probe Miniaturization of Coupled Core Fluxgate Sensors

This paper demonstrates the probe structure optimization of coupled core fluxgate magnetic sensors through finite element analysis. The obtained modelling results have been used to optimize the probe structures from horizontal- to vertical- arrangements for magnetic crosstalk suppression and probe miniaturization. The finite element analysis show that with the same distance between each adjacent fluxgate elements, the magnetic crosstalk is suppressed by 6 times and the volume is reduced by 2 times after the optimization. Furthermore, the miniaturized probes with low magnetic crosstalk have been designed and implemented. The experimental results which showed more than 5 times suppression of magnetic crosstalk verified the simulation results. Therefore, the results provide detailed reference to cope with the contradiction between volume miniaturization and magnetic crosstalk suppression in magnetic sensor-array design

Performance degradation effect countermeasures in residence times difference (RTD) fluxgate magnetic sensors

This paper aims to explore the detection defect of residence times difference (RTD) fluxgate working in low-power mode and present the countermeasures for sensor resolution improvement and linearity enhancement. The main defects are amplitude and symmetry changes induced in the output spikes of fluxgate probe due to the magnetic field. These defects lead to thresholds deviation and asymmetry, then cause severe performance degradation especially on detection resolution and linearity according to the RTD theory. To overcome such effects, the optimized RTD method based on voltage extraction and feedback technology is proposed to implement magnetic field compensation and achieve a zero-field running regime of the RTD fluxgate. In this regard, the sensor linearity is improved by a factor of 38, and the resolution degradation effect is suppressed more than 6 times, verified by the laboratory experiments. The optimized detection method proposed in this paper demonstrated a great potential to achieve lower power consumption and will make the RTD fluxgate more promising technology among bio-magnetic applications

Identification of Genes Related to White and Black Plumage Formation by RNA-Seq from White and Black Feather Bulbs in Ducks

To elucidate the genes involved in the formation of white and black plumage in ducks, RNA from white and black feather bulbs of an F2 population were analyzed using RNA-Seq. A total of 2,642 expressed sequence tags showed significant differential expression between white and black feather bulbs. Among these tags, 186 matched 133 annotated genes that grouped into 94 pathways. A number of genes controlling melanogenesis showed differential expression between the two types of feather bulbs. This differential expression was confirmed by qPCR analysis and demonstrated that Tyr (Tyrosinase) and Tyrp1 (Tyrosinase-related protein-1) were expressed not in W-W (white feather bulb from white dorsal plumage) and W-WB (white feather bulb from white-black dorsal plumage) but in B-B (black feather bulb from black dorsal plumage) and B-WB (black feather bulb from white-black dorsal plumage) feather bulbs. Tyrp2 (Tyrosinase-related protein-2) gene did not show expression in the four types of feather bulbs but expressed in retina. C-kit (The tyrosine kinase receptor) expressed in all of the samples but the relative mRNA expression in B-B or B-WB was approximately 10 fold higher than that in W-W or W-WB. Additionally, only one of the two Mitf isoforms was associated with plumage color determination. Downregulation of c-Kit and Mitf in feather bulbs may be the cause of white plumage in the duck