Elsevier

Neuroscience

Volume 113, Issue 1, 2 August 2002, Pages 155-166
Neuroscience

Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur

https://doi.org/10.1016/S0306-4522(02)00165-3Get rights and content

Abstract

Although skeletal pain plays a major role in reducing the quality of life in patients suffering from osteoarthritis, Paget’s disease, sickle cell anemia and bone cancer, little is known about the mechanisms that generate and maintain this pain. To define the peripheral fibers involved in transmitting and modulating skeletal pain, we used immunohistochemistry with antigen retrieval, confocal microscopy and three-dimensional image reconstruction of the bone to examine the sensory and sympathetic innervation of mineralized bone, bone marrow and periosteum of the normal mouse femur. Thinly myelinated and unmyelinated peptidergic sensory fibers were labeled with antibodies raised against calcitonin gene-related peptide (CGRP) and the unmyelinated, non-peptidergic sensory fibers were labeled with the isolectin B4 (Bandeira simplicifolia). Myelinated sensory fibers were labeled with an antibody raised against 200-kDa neurofilament H (clone RT-97). Sympathetic fibers were labeled with an antibody raised against tyrosine hydroxylase. CGRP, RT-97, and tyrosine hydroxylase immunoreactive fibers, but not isolectin B4 positive fibers, were present throughout the bone marrow, mineralized bone and the periosteum. While the periosteum is the most densely innervated tissue, when the total volume of each tissue is considered, the bone marrow receives the greatest total number of sensory and sympathetic fibers followed by mineralized bone and then periosteum.

Understanding the sensory and sympathetic innervation of bone should provide a better understanding of the mechanisms that drive bone pain and aid in developing therapeutic strategies for treating skeletal pain.

Section snippets

Experimental animals

Experiments were performed on 43 C3H/HeJ mice (36 adult male C3H/HeJ mice for immunohistochemistry, three adult male C3H/HeJ mice for volumetric analysis, and four 7-day-old neonates for testing antibody antigenicity) (Jackson Laboratories, Bar Harbor, ME, USA). All the mice except for the neonates were 6–7 weeks old and weighed 20–25 g. The mice were housed in accordance with National Institutes of Health guidelines and kept in a vivarium maintained at 22°C with a 12-h alternating light–dark

Innervation pattern and distribution of CGRP immunoreactive nerve fibers in the periosteum, mineralized bone and bone marrow

Thinly myelinated or unmyelinated peptidergic sensory fibers were labeled with an antibody raised against CGRP. CGRP immunoreactive (CGRP-ir) nerve fibers were found throughout the periosteum, which is composed mainly of fibrous connective tissue approximately 30–60 μm thick and envelops the entire outer surface of mineralized bone except at the points of ligament and tendon insertion (Marieb and Mallat, 1997). When the periosteum was viewed in a whole mount preparation, CGRP-ir nerve fibers

Sensory and sympathetic innervation of normal bone

In the present report, we show that in addition to CGRP and TH expressing fibers that innervate the mineralized bone, bone marrow, and periosteum, there is also a population of myelinated primary afferent sensory neurons fibers which express RT-97 that innervate the bone. The presence of both CGRP-ir and RT-97-ir fibers suggests that the mineralized bone, the bone marrow and the periosteum receive innervation from both unmyelinated and myelinated sensory neurons, which would presumably include

Acknowledgements

This work was supported by NIH Grants NINDS NS23970, NIDA 11986, NIDCR Training Grant DEO7288 (D.B.M.), DE00270 (M.C.S.), AR43595, a Merit Review from the Veterans Administration and a grant from the Supercomputing Institute for Digital Simulation and Advanced Computation at the University of Minnesota.

References (63)

  • I.A. Silver et al.

    Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts

    Exp. Cell Res.

    (1988)
  • K. Suzuki et al.

    Ascites sarcoma 180, a tumor associated with hypercalcemia, secretes potent bone-resorbing factors including transforming growth factor alpha, interleukin-1 alpha and interleukin-6

    Bone Miner.

    (1994)
  • Z. Tabarowski et al.

    Noradrenergic and peptidergic innervation of the mouse femur bone marrow

    Acta Histochim.

    (1996)
  • Y. Tang et al.

    Dynamic norepinephrine alterations in bone marrow evidence of functional innervation

    J. Neuroimmunol.

    (1999)
  • M. Tominaga et al.

    The cloned capsaicin receptor integrates multiple pain-producing [see comments]

    Neuron

    (1998)
  • M. Ahmed et al.

    Neuropeptide Y, tyrosine hydroxylase and vasoactive intestinal polypeptide-immunoreactive nerve fibers in the vertebral bodies, discs, dura mater, and spinal ligaments of the rat lumbar spine

    Spine

    (1993)
  • Alder, C.-P., 2000. Bone Diseases. Springer-Verlag,...
  • F.J. Alvarez et al.

    Nociceptors for the 21st century

    Curr. Rev. Pain

    (2000)
  • S.E. Asmus et al.

    Developmental changes in the transmitter properties of sympathetic neurons that innervate the periosteum

    J. Neurosci.

    (2000)
  • Bilezikian, J.P., Raisz, L.G., Rodan, G.A., 1995. Principles of Bone Biology. Academic Press,...
  • A. Bjurholm

    Neuroendocrine peptides in bone

    Int. Orthop.

    (1991)
  • M.J. Caterina et al.

    The capsaicin receptor a heat-activated ion channel in the pain [see comments]

    Nature

    (1997)
  • Claus-Peter, A., 2000a. Bone fractures. In: Bone Diseases. Springer-Verlag, Berlin, pp....
  • Claus-Peter, A., 2000b. Bone tumors. In: Bone Diseases. Springer-Verlag, Berlin, pp....
  • Claus-Peter, A., 2000c. Disorders of skeletal developement. In: Bone Diseases. Springer-Verlag, Berlin, pp....
  • Claus-Peter, A., 2000d. Osteoproses and osteopathies. In: Bone Diseases. Springer-Verlag, Berlin, pp....
  • M. Farr et al.

    Inflammation causes a long-term hyperexcitability in the nociceptive sensory neurons of Aplysia

    Learn. Memory

    (1999)
  • Foley, K.M., 1993. Pain assesment and cancer pain syndromes. In: Doyle, G.W.H.a.N.M.D. (Ed.), Oxford Palliative...
  • Goltzman, D.a.R., Rabbani, S.A., 2000. Pathogenesis of osteoblastic metastases. In: Body, J.-J. (Ed.), Tumor Bone...
  • M.H. Goni et al.

    Hypercalcemia of cancer: an update

    Anticancer Res.

    (1993)
  • T.A. Guise et al.

    Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis

    J. Clin. Invest.

    (1996)
  • Cited by (554)

    View all citing articles on Scopus
    View full text