Capsaicin-sensitive sensory nerve fibers contribute to the generation and maintenance of skeletal fracture pain

Abstract

Although skeletal pain can have a marked impact on a patient's functional status and quality of life, relatively little is known about the specific populations of peripheral nerve fibers that drive non-malignant bone pain. In the present report, neonatal male Sprague-Dawley rats were treated with capsaicin or vehicle and femoral fracture was produced when the animals were young adults (15-16 weeks old). Capsaicin treatment, but not vehicle, resulted in a significant (>70%) depletion in the density of calcitonin-gene related peptide positive (CGRP(+)) sensory nerve fibers, but not 200 kDa neurofilament H positive (NF200(+)) sensory nerve fibers in the periosteum. The periosteum is a thin, cellular and fibrous tissue that tightly adheres to the outer surface of all but the articulated surface of bone and appears to play a pivotal role in driving fracture pain. In animals treated with capsaicin, but not vehicle, there was a 50% reduction in the severity, but no change in the time course, of fracture-induced skeletal pain-related behaviors as measured by spontaneous flinching, guarding and weight bearing. These results suggest that both capsaicin-sensitive (primarily CGRP(+) C-fibers) and capsaicin-insensitive (primarily NF200(+) A-delta fibers) sensory nerve fibers participate in driving skeletal fracture pain. Skeletal pain can be a significant impediment to functional recovery following trauma-induced fracture, osteoporosis-induced fracture and orthopedic surgery procedures such as knee and hip replacement. Understanding the specific populations of sensory nerve fibers that need to be targeted to inhibit the generation and maintenance of skeletal pain may allow the development of more specific mechanism-based therapies that can effectively attenuate acute and chronic skeletal pain.

Figure 1

Representative radiographs showing a naïve, pin alone and pin + fracture femur in the male adult Sprague Dawley rat. A stainless steel pin was implanted into the intramedullary space of the femur 21 days prior to fracture in order to provide mechanical stability to allow bone healing. Closed mid-diaphyseal fractures of the left femur were produced in rats using a 3-point impactor device. Radiographic images of femurs from naïve (A), pin (B), and pin + fracture two days post-fracture (C). Scale bar = 4.0 mm. Arrows indicate the fracture site. Brackets indicate the location where the whole mount preparations of the femoral periosteum were removed for immunohistochemical analysis.

Figure 2

A set of diagrams illustrating the anatomical localization of the periosteum of the bone and its innervation by sensory nerve fibers. The periosteum is a thin, cellular and fibrous tissue that is densely innervated by sensory nerve fibers and is tightly adherent to the outer surface of cortical bone (A). For illustrations purposes representative confocal images of the CGRP⁺ nerve fibers (in pink/purple) from periosteal whole preparations were acquired and overlapped on a three dimensional image of the femur obtained by microcomputed tomography (B). High magnification of this illustration shows the degree and pattern of the sensory innervation of the periosteum by CGRP⁺ sensory nerve fibers (C). As mechanical distortion of either the cortical bone and tightly adherent periosteum is highly painful and stabilizing the fractured bone and periosteum significantly attenuates fracture pain, it is thought that activation and sensitization of nociceptors that innervate the periosteum are pivotally involved in driving fracture-induced bone pain.

Figure 3

Neonatal capsaicin treatment results in depletion of CGRP⁺ (primarily unmyelinated) but not of NF200⁺ (primarily myelinated) sensory nerve fibers in the periosteum. Representative confocal images of whole mount preparations of the periosteum that were stained for calcitonin gene related peptide (CGRP) and neurofilament-200 (NF200) sensory nerve fibers obtained from the femurs of non-fractured 18 week-old rats that received neonatal injection of vehicle (A, C) or capsaicin (B, D). CGRP is a neuropeptide found in predominantly unmyelinated (C-fibers) and some thinly myelinated (A-delta) sensory nerve fibers (A, B), whereas NF200 is expressed by myelinated (A-delta and A-beta) primary afferent sensory nerve fibers (C, D). Neonatal capsaicin treatment reduced the number of CGRP⁺ sensory fibers as compared to vehicle treatment (A, B) but did not reduce the number of NF200⁺ sensory fibers (C, D). Confocal images (40µm z-series) were projected from 160 optical sections acquired at 0.25µm intervals. Scale bar A–D: 50 µm.

Figure 4

Depletion of capsaicin sensitive sensory nerve fibers results in attenuation but not the abolition of fracture induced pain behaviors. Following closed fracture of the femur, rats treated with vehicle alone exhibited dramatically increased time spent spontaneously guarding (A), a greater number of spontaneous flinches (B), and reduced weight bearing of the fractured limb (C) as compared to naïve animals or animals that received intramedullary pin placement alone. Animals that received neonatal capsaicin treatment showed a reduction of fracture-induced pain-related behaviors at nearly all time points examined as compared to vehicle treated rats with femoral fracture. Note that the onset and time course of the pain behaviors following fracture were not modified by neonatal capsaicin treatment suggesting that both the C-fibers and A-delta fibers that normally innervate the bone participate in sensing and transmitting bone fracture pain. Histogram showing the effect of capsaicin treatment on the mineralized callus area of the fractured femur as measured by radiographic analysis (D). Although there was a small, but statistically significant , increase in callus area in the capsaicin-treated + fracture vs. the vehicle-treated + fracture animals at days 14 and 28 post-fracture, there was no significant difference in the overall fracture site bridging score at any of the times examined (E). Data are presented as the mean ± standard error of the mean. *p <0.05 fracture + capsaicin-treated rats vs. fracture+vehicle-treated rats.