ABSTRACT
BACKGROUND: Because of the relative lack of understanding of the mechanisms that drive skeletal pain, the purpose of this study was to adapt a previously validated closed femur fracture model to quantitatively evaluate skeletal pain in female and male rats. METHODS: Three-month-old female and male Sprague-Dawley rats were anesthetized, and a stainless steel pin was inserted into the intramedullary space of the left femur. Three weeks later, the rats were reanesthetized, and left femoral diaphyses were fractured using a standardized impactor device. At 1-21 days after fracture, skeletal pain was measured by quantitatively assessing spontaneous guarding, spontaneous flinching, and weight bearing of the fractured hind limb. RESULTS: Females and males showed highly robust pain behaviors that were maximal at day 1 after fracture and returned gradually to normal nonfractured levels at days 14-21 after fracture. The magnitude of fracture pain was not significantly different at most time points between female and male rats. In both females and males, the pain-related behaviors were attenuated by subcutaneous morphine in a dose-dependent manner. CONCLUSIONS: This model may help in developing a mechanism-based understanding of the factors that generate and maintain fracture pain in both females and males and in translating these findings into new therapies for treating fracture pain.
Subject(s)
Bone Diseases/physiopathology , Disease Models, Animal , Femoral Fractures/physiopathology , Pain Measurement/methods , Pain/physiopathology , Animals , Bone Diseases/drug therapy , Female , Femoral Fractures/drug therapy , Male , Morphine/therapeutic use , Pain/drug therapy , Rats , Rats, Sprague-DawleyABSTRACT
Although bone fracture frequently results in significant pain and can lead to increased morbidity and mortality, it is still not clearly understood how sensory neurons are organized to detect fracture pain. In the present report we focused on the periosteum, as this thin tissue is highly innervated and tightly adherent to the outer surface of bone. To define the organization and distribution of the sensory and sympathetic fibers in the mouse femoral periosteum, we used whole-mount preparations, transverse sections, immunofluoresence and laser scanning confocal microscopy. While both the outer fibrous layer and the inner more cellular cambium layer of the periosteum receive an extensive innervation by calcitonin gene-related peptide (CGRP) and 200-kDa neurofilament (NF200) positive sensory fibers as well as tyrosine hydroxylase (TH) positive sympathetic fibers, there is a differential organization of sensory vs. sympathetic fibers within the periosteum. In both layers, the great majority of TH+ fibers are closely associated with CD31+ blood vessels and wind around the larger vessels in a corkscrew pattern. In contrast, the majority of CGRP+ and NF200+ sensory fibers in both layers lack a clear association with CD31+ blood vessels and appear to be organized in a dense net-like meshwork to detect mechanical distortion of periosteum and bone. This organization would explain why stabilization/fixation causes a marked attenuation of movement-evoked fracture pain. Understanding the organization, plasticity and molecular characteristics of sensory and sympathetic nerve fibers innervating the skeleton may permit the development of novel mechanism-based therapies for treating non-malignant skeletal pain.
Subject(s)
Calcitonin Gene-Related Peptide/metabolism , Nerve Fibers/metabolism , Nerve Net/cytology , Periosteum/anatomy & histology , Animals , Bromodeoxyuridine/metabolism , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , Nerve Net/metabolism , Neurofilament Proteins/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Current therapies to treat skeletal fracture pain are extremely limited. Some non-steroidal anti-inflammatory drugs have been shown to inhibit bone healing and opiates induce cognitive dysfunction and respiratory depression which are especially problematic in the elderly suffering from osteoporotic fractures. In the present report, we developed a closed femur fracture pain model in the mouse where skeletal pain behaviors such as flinching and guarding of the fractured limb are reversed by 10mg/kg morphine. Using this model we showed that the administration of a monoclonal antibody against nerve growth factor (anti-NGF) reduced fracture-induced pain-related behaviors by over 50%. Treatment with anti-NGF reduced c-Fos and dynorphin up-regulation in the spinal cord at day 2 post-fracture. However, anti-NGF treatment did not reduce p-ERK and c-Fos expression at 20 and 90 min, respectively, following fracture. This suggests NGF is involved in maintenance but not the acute generation of fracture pain. Anti-NGF therapy did not inhibit bone healing as measured by callus formation, bridging of the fracture site or mechanical strength of the bone. As the anti-NGF antibody does not appreciably cross the blood-brain barrier, the present data suggest that the anti-hyperalgesic action of anti-NGF therapy results from blockade of activation and/or sensitization of the CGRP/trkA positive fibers that normally constitute the majority of sensory fibers that innervate the bone. These results demonstrate that NGF plays a significant role in driving fracture pain and that NGF sequestering therapies may be efficacious in attenuating this pain.
