AMPA and NMDA glutamate receptors are found in both peptidergic and non-peptidergic primary afferent neurons in the rat

Two distinct classes of nociceptive primary afferents, peptidergic and non-peptidergic, respond similarly to acute noxious stimulation; however the peptidergic afferents are more likely to play a role in inflammatory pain, while the non-peptidergic afferents may be more characteristically involved in neuropathic pain. Using multiple immunofluorescence, we determined the proportions of neurons in the rat L4 dorsal root ganglion (DRG) that co-express AMPA or NMDA glutamate receptors and markers for the peptidergic and non-peptidergic classes of primary afferents, substance P and P2X3, respectively. The fraction of DRG neurons immunostained for the NR1 subunit of the NMDA receptor (40%) was significantly higher than that of DRG neurons immunostained for the GluR2/3 (27%) or the GluR4 (34%) subunits of the AMPA receptor. Of all DRG neurons double-immunostained for glutamate receptor subunits and either marker for peptidergic and non-peptidergic afferents, a significantly larger proportion expressed GluR4 than GluR2/3 or NR1 and in a significantly larger proportion of P2X3- than SP-positive DRG neurons. These observations support the idea that nociceptors, involved primarily in the mediation of neuropathic pain, may be presynaptically modulated by GluR4-containing AMPA receptors

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

In this method we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays

Increased expression of CGRP in sensory afferents of arthritic mice – effect of genetic deletion of the vanilloid receptor TRPV1

The neuropeptide calcitonin gene-related peptide (CGRP), expressed by nociceptive sensory afferents in joints, is an important mediator in the pathogenesis of arthritis. Capsaicin causes neurons in the dorsal root ganglia (DRG) to release CGRP from their central and/or peripheral axons, suggesting a functional link between CGRP and the capsaicin receptor TRPV1. The expression of both TRPV1 and CGRP have been reported to increase in several models of arthritis but the specific involvement of TRPV1-expressing articular afferents that can release CGRP remains unclear. We here wanted to ascertain whether the increase in the number of CGRP-positive primary afferents during arthritis may be affected by genetic deletion of TRPV1. For this, we quantified the expression of CGRP in primary afferent neurons in DRG in wild type mice (WT) vs. TRPV1-KO mice with adjuvant-induced arthritis (AIA), using immunohistochemistry. We found that the fraction of DRG neurons that were immunopositive for CGRP 1) was higher in naïve TRPV1-KO mice than in naïve WT mice, 2) increased progressively 3–21 days after induction of AIA, and 3) this increase was bilateral but significantly greater on the CFA-injected side than on the IFA-injected side in TRPV1-KO mice. The increased expression of CGRP in AIA may reflect a phenotypic switch of primary afferents from non-peptidergic to peptidergic and the larger increase in TRPV1-KO mice may represent a plastic change to compensate for the missing receptor in a major sensory circuit

Influence of the vanilloid receptor TRPV1 on the activation of spinal cord glia in mouse models of pain

Although activation of spinal glia has been implicated in the development of pathological pain, the mechanisms underlying glial activation are not fully understood. One such mechanism may be triggered by reaction to neuroactive substances released from central axons of sensory afferents. The vanilloid receptor TRPV1, a nonselective cation channel in nociceptive sensory afferents, mediates the release of neurotransmitters, such as glutamate and CGRP in the dorsal horn, which can subsequently activate glia. To test the hypothesis that activation of spinal glia is mediated, at least in part, by TRPV1, we studied the expression of markers for microglia (Ionized calcium-binding adapter molecule 1, Iba1) and astrocytes (Glial Fibrillary Acidic Protein, GFAP) in the spinal cord of TRPV1 knockout mice (KO) vs. wild-type mice (WT) in models of acute (intraplantar capsaicin), inflammatory (Adjuvant-Induced Arthritis, AIA), and neuropathic pain (Partial Sciatic Nerve Ligation, PSNL). We found that i) naïve KO mice had denser immunostaining for both Iba1 and GFAP than naïve WT mice, ii) the immunostaining for Iba1 increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 7–14 days after AIA or PSNL, and was significantly greater in WT than in KO mice 3 days after capsaicin, 7–14 days after AIA, and 7 days after PSNL, iii) the immunostaining for GFAP increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 14–21 days after AIA or PSNL, and was significantly greater in WT than in KO mice 14 days after AIA or PSNL. Our results suggest that TRPV1 plays a role in the activation of spinal glia in mice with nociceptive, inflammatory, and neuropathic pain

Effect of genetic deletion of the vanilloid receptor TRPV1 on the expression of Substance P in sensory neurons of mice with adjuvant-induced arthritis

The neuropeptide Substance P (SP), expressed by nociceptive sensory afferents in joints, plays an important role in the pathogenesis of arthritis. Capsaicin causes neurons in the dorsal root ganglia (DRG) to release SP from their central and peripheral axons, suggesting a functional link between SP and the capsaicin receptor, the transient receptor potential vanilloid 1 (TRPV1). The expression of both TRPV1 and SP have been reported to increase in several models of arthritis but the specific involvement of TRPV1-expressing articular afferents that can release SP is not completely understood. We here wanted to ascertain whether the increase in the number of SP-positive primary afferents in arthritis may be affected by genetic deletion of TRPV1. For this, we used immunohistochemistry to quantify the expression of SP in primary afferent neurons in wild type mice (WT) vs. TRPV1-knockout (KO) mice with adjuvant-induced arthritis (AIA). We found that the expression of SP in DRG 1) increased significantly over naïve level in both WT and KO mice 3 weeks after AIA, 2) was significantly higher in KO mice than in WT mice in naïve mice and 2-3 weeks after AIA, 3) was significantly higheron the side of AIA than on the contralateral, vehicle-injected side at all time points in WT mice, but not in KO mice, and 4) increased predominantly in small-size neurons in KO mice and in small- and medium-size neurons in WT mice. Since the size distribution of SP-positive DRG neurons in arthritic TRPV1-KO mice was not significantly different from that in naïve mice, we speculate that the increased expression of SP is unlikely to reflect recruitment of A-fiber primary afferents and that the higher expression of SP in KO mice may represent a plastic change to compensate for the missing receptor in a major sensory circuit

Ethanol and Acetaminophen Synergistically Induce Hepatic Aggregation and TCH346-Insensitive Nuclear Translocation of GAPDH

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signals during cellular stress via several post-translational modifications that change its folding properties, protein-protein interactions and sub-cellular localization. We examined GAPDH properties in acute mouse liver injury due to ethanol and/or acetaminophen (APAP) treatment. Synergistic robust and time-dependent nuclear accumulation and aggregation of GAPDH were observed only in combined, but not individual, ethanol/APAP treatments. The small molecule GAPDH-targeting compound TCH346 partially attenuated liver damage possibly via mitochondrial mechanisms, and independent of nuclear accumulation and aggregation of GAPDH. These findings provide a novel potential mechanism for hepatotoxicity caused by combined alcohol and acetaminophen exposure

Synovial Joints: from Development to Homeostasis

Synovial joint morphogenesis occurs through the condensation of mesenchymal cells into a non-cartilaginous region known as interzone, and the specification of progenitor cells that commit to the articular fate. Although several signaling molecules are expressed by the interzone, the mechanism is poorly understood. For treatments of cartilage injuries, it is critical to discover the presence of joint progenitor cells in adult tissues and their expression gene pattern. Potential stem cells niches have been found in different joint regions, such as the surface zone of articular cartilage, synovium and groove of Ranvier. Inherited joint malformation as well as joint degenerating conditions are often associated with other skeletal defects, and may be seen as the failure of morphogenic factors to establish the correct microenvironment in cartilage and bone. Therefore, exploring how joints form can help us understand how cartilage and bone are damaged and to develop drugs to reactivate this developing mechanism

CC-Chemokine Receptor-2 Expression in Osteoblasts Contributes to Cartilage and Bone Damage during Post-Traumatic Osteoarthritis

In osteoarthritis (OA), bone changes are radiological hallmarks and are considered important for disease progression. The C-C chemokine receptor-2 (CCR2) has been shown to play an important role in bone physiology. In this study, we investigated whether Ccr2 osteoblast-specific inactivation at different times during post-traumatic OA (PTOA) progression improves joint structures, bone parameters, and pain. We used a tamoxifen-inducible Ccr2 inactivation in Collagen1α-expressing cells to obtain osteoblasts lacking Ccr2 (CCR2-Col1αKO). We stimulated PTOA changes in CCR2-Col1αKO and CCR2+/+ mice using the destabilization of the meniscus model (DMM), inducing recombination before or after DMM (early- vs. late-inactivation). Joint damage was evaluated at two, four, eight, and twelve weeks post-DMM using multiple scores: articular-cartilage structure (ACS), Safranin-O, histomorphometry, osteophyte size/maturity, subchondral bone thickness and synovial hyperplasia. Spontaneous and evoked pain were assessed for up to 20 weeks. We found that early osteoblast-Ccr2 inactivation delayed articular cartilage damage and matrix degeneration compared to CCR2+/+, as well as DMM-induced bone thickness. Osteophyte formation and maturation were only minimally affected. Late Collagen1α-Ccr2 deletion led to less evident improvements. Osteoblast-Ccr2 deletion also improved static measures of pain, while evoked pain did not change. Our study demonstrates that Ccr2 expression in osteoblasts contributes to PTOA disease progression and pain by affecting both cartilage and bone tissues

Decoy Receptor CXCR7 Modulates Adrenomedullin-Mediated Cardiac and Lymphatic Vascular Development

Atypical 7-transmembrane receptors, often called decoy receptors, act promiscuously as molecular sinks to regulate ligand bioavailability and consequently temper the signaling of canonical G protein-coupled receptor (GPCR) pathways. Loss of mammalian CXCR7, the most recently described decoy receptor, results in postnatal lethality due to aberrant cardiac development and myocyte hyperplasia. Here, we provide the molecular underpinning for this proliferative phenotype by demonstrating that the dosage and signaling of adrenomedullin (Adm = gene, AM = protein)—a mitogenic peptide-hormone required for normal cardiovascular development—is tightly controlled by CXCR7. To this end, Cxcr7−/− mice exhibit gain-of-function cardiac and lymphatic vascular phenotypes which can be reversed upon genetic depletion of adrenomedullin ligand. In addition to identifying a biological ligand accountable for the phenotypes of Cxcr7−/− mice, these results reveal a previously underappreciated role for decoy receptors as molecular rheostats in controlling the timing and extent of GPCR-mediated cardiac and vascular development

Fetal-derived adrenomedullin mediates the innate immune milieu of the placenta

The remodeling of maternal uterine spiral arteries (SAs) is an essential process for ensuring low-resistance, high-capacitance blood flow to the growing fetus. Failure of SAs to remodel is causally associated with preeclampsia, a common and life-threatening complication of pregnancy that is harmful to both mother and fetus. Here, using both loss-of-function and gain-of-function genetic mouse models, we show that expression of the pregnancy-related peptide adrenomedullin (AM) by fetal trophoblast cells is necessary and sufficient to promote appropriate recruitment and activation of maternal uterine NK (uNK) cells to the placenta and ultimately facilitate remodeling of maternal SAs. Placentas that lacked either AM or its receptor exhibited reduced fetal vessel branching in the labyrinth, failed SA remodeling and reendothelialization, and markedly reduced numbers of maternal uNK cells. In contrast, overexpression of AM caused a reversal of these phenotypes with a concomitant increase in uNK cell content in vivo. Moreover, AM dose-dependently stimulated the secretion of numerous chemokines, cytokines, and MMPs from uNK cells, which in turn induced VSMC apoptosis. These data identify an essential function for fetal-derived factors in the maternal vascular adaptation to pregnancy and underscore the importance of exploring AM as a biomarker and therapeutic agent for preeclampsia