Mounting evidence from animal studies suggests a role of the nervous system in bone physiology. However, little is known about the nerve fiber localization to human bone compartments and bone surface events. This study reveals the density and distribution of nerves in human bone and the association of nerve profiles to bone remodeling events and vascular structures in iliac crest biopsies isolated from patients diagnosed with primary hyperparathyroidism (PHPT). Bone sections were sequentially double-immunostained for tyrosine hydroxylase (TH), a marker for sympathetic nerves, followed by protein gene product 9.5 (PGP9.5), a pan-neuronal marker, or double-immunostained for either PGP9.5 or TH in combination with CD34, an endothelial marker. In the bone marrow, the nerve profile density was significantly higher above remodeling surfaces as compared to quiescent bone surfaces. Ninety-five percentages of all nerve profiles were associated with vascular structures with the highest association to capillaries and arterioles. Moreover, vasculature with innervation was denser above bone remodeling surfaces. Finally, the nerve profiles density was 5-fold higher in the intracortical pores compared to bone marrow and periosteum. In conclusion, the study shows an anatomical link between innervation and bone remodeling in human bone.

Background In the pain field, it is essential to quantify nociceptive responses. The response to the application of von Frey filaments to the skin measures tactile sensitivity and is a surrogate marker of allodynia in states of peripheral and/or central sensitization. The method is widely used across species within the pain field. However, uncertainties appear to exist regarding the appropriate method for analysing obtained data. Therefore, there is a need for refinement of the calculations for transformation of raw data to quantifiable data. Methods Here, we briefly review the fundamentals behind von Frey testing using the standard up‐down method and the associated statistics and show how different parameters of the statistical equation influence the calculated 50% threshold results. We discuss how to obtain the most accurate estimations in a given experimental setting. Results To enhance accuracy and reproducibility across laboratories, we present an easy to use algorithm that calculates 50% thresholds based on the exact filaments and their interval using math beyond the traditional methods. This tool is available to the everyday user of von Frey filaments and allows the insertion of all imaginable ranges of filaments and is thus applicable to data derived in any species. Conclusion We advocate for the use of this algorithm to minimize inaccuracies and to improve internal and external reproducibility. Significance The von Frey testing procedure is standard for assessing peripheral and central sensitization but is associated with inaccuracies and lack of transparency in the associated math. Here, we describe these problems and present a novel statistical algorithm that calculates the exact thresholds using math beyond the traditional methods. The online platform is transparent, free of charge and easy to use also for the everyday user of von Frey filaments. Application of this resource will ultimately reduce errors due to methodological misinterpretations and increase reproducibility across laboratories.

Selective pharmacological tool compounds are invaluable for understanding the functions of the various ionotropic glutamate receptor subtypes. For the kainate receptors, these compounds are few. Here we have synthesized nine novel quinoxaline-2,3-diones with substitutions in the 7-position to investigate the structure–activity relationship at kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Compound 11 exhibited the highest binding affinity across GluK1–3 while having selectivity toward kainate vs AMPA receptors. Compound 11 potently inhibited glutamate evoked currents at homomeric GluK1 and GluK3 receptors in HEK293 cells with K b values of 65 and 39 nM, respectively. The binding mode of 11 in the ligand binding domain of GluK1 was investigated by X-ray crystallography, revealing that 11 stabilizes the receptor in an open conformation, consistent with its demonstrated antagonism. Furthermore, 11 was tested for analgesic effects in the mouse tail flick test where it significantly increased tail flick latency at doses where 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo­[f]-quinoxaline-7-sulfonamide (NBQX) was ineffective.

Bone pain is a serious and debilitating symptom of multiple myeloma (MM) that impairs the quality of life of patients. The underlying mechanisms of the pain are unknown and understudied, and there is a need for immunocompetent preclinical models of myeloma‐induced bone pain. The aim of this study was to provide the first in‐depth behavioral characterization of an immunocompetent mouse model of MM presenting the clinical disease features: osteolytic bone disease and bone pain. We hypothesized that a widely used syngeneic model of MM, established by systemic inoculation of green fluorescent protein‐tagged myeloma cells (5TGM1‐GFP) in immunocompetent C57Bl/KaLwRijHsd (BKAL) mice, would present pain‐related behaviors. Disease phenotype was confirmed by splenomegaly, high serum paraprotein, and tumor infiltration in the bone marrow of the hind limbs; however, myeloma‐bearing mice did not present pain‐related behaviors or substantial bone disease. Thus, we investigated an alternative model in which 5TGM1‐GFP cells were directly inoculated into the intrafemoral medullary cavity. This localized myeloma model presented the hallmarks of the disease, including high serum paraprotein, tumor growth, and osteolytic bone lesions. Compared with control mice, myeloma‐bearing mice presented myeloma‐induced pain‐related behaviors, a phenotype that was reversed by systemic morphine treatment. Micro‐computed tomography analyses of the myeloma‐inoculated femurs showed bone disease in cortical and trabecular bone. Repeated systemic bisphosphonate treatment induced an amelioration of the nociceptive phenotype, but did not completely reverse it. Furthermore, intrafemorally injected mice presented a profound denervation of the myeloma‐bearing bones, a previously unknown feature of the disease. This study reports the intrafemoral inoculation of 5TGM1‐GFP cells as a robust immunocompetent model of myeloma‐induced bone pain, with consistent bone loss. Moreover, the data suggest that myeloma‐induced bone pain is caused by a combinatorial mechanism including osteolysis and bone marrow denervation. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

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