Modulation of Rat Cancer-Induced Bone Pain is Independent of Spinal Microglia Activity.

The dissemination of cancer to bone can cause significant cancer-induced bone pain (CIBP), severely impairing the patient's quality of life. Several rodent models have been developed to explore the nociceptive mechanisms of CIBP, including intratibial inoculation of breast carcinoma cells in syngeneic Sprague Dawley rats. Using this model, we investigated whether resident spinal microglial cells are involved in the transmission and modulation of CIBP, a long-debated disease feature. Immunohistochemical staining of ionizing calcium-binding adaptor molecule 1 (Iba-1) and phosphorylated p38-mitogen-activated protein kinase (P-p38 MAPK) showed no spinal microglial reaction in cancer-bearing rats, independently of disease stage, sex, or carcinoma cell line. As a positive control, significant upregulation of both Iba-1 and P-p38 was observed in a rat model of neuropathic pain. Additionally, intrathecal administration of the microglial inhibitor minocycline did not ameliorate pain-like behaviors in cancer-bearing rats, in contrast to spinal morphine administration. Our results indicate that microglial reaction is not a main player in CIBP, adding to the debate that even within the same models of CIBP, significant variations are seen in disease features considered potential drug targets. We suggest that this heterogeneity may reflect the clinical landscape, underscoring the need for understanding the translational value of CIBP models.

Differential Pain-Related Behaviors and Bone Disease in Immunocompetent Mouse Models of Myeloma.

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.

Von Frey testing revisited: Provision of an online algorithm for improved accuracy of 50% thresholds.

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.

Innervation is higher above Bone Remodeling Surfaces and in Cortical Pores in Human Bone: Lessons from patients with primary hyperparathyroidism.

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.