Sensory Innervation of Human Bone: An Immunohistochemical Study to Further Understand Bone Pain.

Skeletal diseases and their surgical treatment induce severe pain. The innervation density of bone potentially explains the severe pain reported. Animal studies concluded that sensory myelinated A∂-fibers and unmyelinated C-fibers are mainly responsible for conducting bone pain, and that the innervation density of these nerve fibers was highest in periosteum. However, literature regarding sensory innervation of human bone is scarce. This observational study aimed to quantify sensory nerve fiber density in periosteum, cortical bone, and bone marrow of axial and appendicular human bones using immunohistochemistry and confocal microscopy. Multivariate Poisson regression analysis demonstrated that the total number of sensory and sympathetic nerve fibers was highest in periosteum, followed by bone marrow, and cortical bone for all bones studied. Bone from thoracic vertebral bodies contained most sensory nerve fibers, followed by the upper extremity, lower extremity, and parietal neurocranium. The number of nerve fibers declined with age and did not differ between male and female specimens. Sensory nerve fibers were organized as a branched network throughout the periosteum. The current results provide an explanation for the severe pain accompanying skeletal disease, fracture, or surgery. Further, the results could provide more insight into mechanisms that generate and maintain skeletal pain and might aid in developing new treatment strategies. PERSPECTIVE: This article presents the innervation of human bone and assesses the effect of age, gender, bone compartment and type of bone on innervation density. The presented data provide an explanation for the severity of bone pain arising from skeletal diseases and their surgical treatment.

Identifying spinal afferent (sensory) nerve endings that innervate the marrow cavity and periosteum using anterograde tracing.

While sensory and sympathetic neurons are known to innervate bone, previous studies have found it difficult to unequivocally identify and characterize only those that are of sensory origin. In this study, we have utilized an in vivo anterograde tracing technique to selectively label spinal afferent (sensory) nerve endings that innervate the periosteum and marrow cavity of murine long bones. Unilateral injections of dextran-biotin (anterograde tracer; 20% in saline, 50-100 nl) were made into L3-L5 dorsal root ganglia. After a 10-day recovery period to allow sufficient time for selective anterograde transport of the tracer to nerve terminal endings in bone, the periosteum (whole-mount) and underlying bone were collected, processed to reveal anterograde labeling, and immuno-labeled with antibodies directed against protein gene product (pan-neuronal marker; PGP9.5), tyrosine hydroxylase (sympathetic neuron marker; TH), calcitonin gene-related protein (peptidergic nociceptor marker; CGRP), and/or neurofilament 200 (myelinated axon marker; NF200). Anterograde-labeled nerve endings were dispersed throughout the periosteum and marrow cavity and could be identified in close apposition to blood vessels and at sites distant from them. The periosteum and the marrow cavity were each innervated by myelinated (NF200+) sensory neurons, and unmyelinated (NF200-) sensory neurons that were either peptidergic (CGRP+) or nonpeptidergic (CGRP-). Spinal afferent nerve endings did not express TH, and lacked the cylindrical morphology around blood vessels characteristic of sympathetic innervation. This approach to selective labeling of sensory nerve terminal endings will help to better identify how different sub-populations of sensory neurons, and their peripheral nerve terminal endings, interact with bone.

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.

The transient receptor potential cation channel subfamily V members 1 and 2, P2X purinoceptor 3 and calcitonin gene-related peptide in sensory neurons of the rat trigeminal ganglion, innervating the periosteum, masseter muscle and facial skin.

OBJECTIVE: Distribution of the transient receptor potential cation channel subfamily V members 1 (TRPV1) and 2 (TRPV2), and P2X purinoceptor 3 (P2 × 3) was investigated in rat trigeminal ganglion neurons innervating the periosteum, masseter muscle and facial skin. DESIGN: Double immunofluorescence method for TRPV1 and TRPV2 ion channels or ATP receptor P2 × 3 with calcitonin gene-related peptide (CGRP) was performed on trigeminal ganglion neurons retrogradely labeled from the mandibular periosteum, masseter muscle, or facial skin in 15 male Wistar rats. RESULTS: The cell size of periosteum neurons (mean ± S.D. = 810.7 ± 36.1 µ m2) was smaller than that of masseter muscle neurons (927.0 ± 75.6 µ m2), and larger than that of facial skin neurons (661.3 ± 82.2 µ m2). Periosteum neurons contained TRPV1- (26.7%), TRPV2- (47.1%) and P2 × 3-immunoreactivity (50.1%). Expression of TRPV2-immunoreactivity was more abundant among periosteum neurons than among facial skin neurons (16.1%). Regarding to TRPV1 and P2 × 3 expression, however, there was no significant difference between periosteum neurons and, masseter muscle and facial skin neurons. TRPV1- immunoreactive trigeminal ganglion neurons which innervated the periosteum, masseter muscle and facial skin mostly had small and medium-sized cell bodies, whereas TRPV2- and P2 × 3-immunoreactive trigeminal ganglion neurons innervating those tissues were of various cell body sizes. Approximately 20% of periosteum (19.2%), masseter muscle (19.2%) and facial skin (21.5%) neurons contained both TRPV1- and CGRP-immunoreactivity. Some periosteum neurons also co-expressed CGRP-immunoreactivity with TRPV2- (10.9%) or P2 × 3- immunoreactivity (11.1%). Distributions of perivascular and free nerve fibers containing CGRP and either TRPV1, TRPV2, or P2 × 3 were occasionally very similar in the mandibular periosteum. CONCLUSIONS: The present study indicated that trigeminal ganglion nociceptors innervating the periosteum as well as those innervating the masseter muscle and facial skin have vanilloid, acidic, thermal, mechanical and ATP sensors. In some periosteum neurons, CGRP may act as inflammatory mediator through activation of TRPV1, TRPV2 and P2 × 3.

Biodegradable nanofiber-membrane for sustainable release of lidocaine at the femoral fracture site as a periosteal block: In vitro and in vivo studies in a rabbit model.

The aim of this study was to evaluate the efficacy of a biodegradable, lidocaine-embedded, nanofibrous membrane for the sustainable analgesic release onto fragments of a segmental femoral fracture site. Membranes of three different lidocaine concentrations (10%, 30%, and 50%) were produced via an electrospinning technique. In vitro lidocaine release was assessed by high-performance liquid chromatography. A femoral segmental fracture, with intramedullary Kirschner-wire fixation and polycaprolactone stent enveloping the fracture site, was set-up in a rabbit model for in vivo assessment of post-operative recovery of activity. Eighteen rabbits were randomly assigned to three groups (six rabbits per group): group A comprised of rabbits with femoral fractures and underwent fixation; group B comprised of a comparable fracture model to that of group A with the implantation of lidocaine-loaded nanofibers; and group C, the control group, received only anesthesia. The following variables were measured: change in body weight, food and water intake before and after surgery, and total activity count post-surgery. All membranes eluted effective levels of lidocaine for more than 3 weeks post-surgery. Rabbits in group B showed faster recovery of activity post-operatively, compared with those in group A, which confirmed the pain relief efficacy of the lidocaine-embedded nanofibers. Nanofibers with sustainable lidocaine release have adequate efficacy and durability for pain relief in rabbits with segmental long bone fractures.

The sensory innervation of the calvarial periosteum is nociceptive and contributes to headache-like behavior.

Headaches are thought to result from the activation and sensitization of nociceptors that innervate deep cephalic tissues. A large body of evidence supports the view that some types of headaches originate intracranially, from activation of sensory neurons that innervate the cranial meninges. However, the notion of an extracranial origin of headaches continues to be entertained, although the identity of deep extracranial cephalic tissues that might contribute to headaches remains elusive. Here we employed anatomical, electrophysiological, and behavioral approaches in rats to test the hypothesis that the sensory innervation of the calvarial periosteum is nociceptive. Neural tracing indicated that the calvarial periosteum overlying the frontal and parietal bones is innervated primarily by small and medium-sized neurons in the trigeminal ganglion's ophthalmic division. In vivo single-unit recording in the trigeminal ganglion revealed that calvarial periosteal afferents have slowly conducting axons, are mechanosensitive, and respond to inflammatory mediators, consistent with a nociceptive function. Two distinct neuronal populations were distinguished based on their peripheral axonal trajectory: one that reached the periosteum through extracranial branches of the trigeminal nerve, and another that took an intracranial trajectory, innervating the cranial dura and apparently reaching the periosteum via the calvarial sutures. In behavioral studies, inflammatory stimulation of these afferents promoted periorbital tactile hypersensitivity, a sensory change linked to primary headaches. Activation and sensitization of calvarial periosteal afferents could play a role in mediating primary headaches of extracranial and perhaps also intracranial origin, as well as secondary headaches such as postcraniotomy and posttraumatic headaches. Targeting calvarial periosteal afferents may be effective in ameliorating these headaches.

Innervation of rat and human dura mater and pericranial tissues in the parieto-temporal region by meningeal afferents.

OBJECTIVE: To reinvestigate the innervation pattern of the dura mater of rat and human middle cranial fossa, the morpho-functional substrate of headache generation, and adjacent extracranial tissues with neuronal in vitro tracing. BACKGROUND: This study was initiated by recent structural and functional findings of meningeal afferent fibers which innervate the cranial dura mater and may project to extracranial tissues. METHODS: Anterograde and retrograde neuronal in vitro tracing was made in formaldehyde fixed hemisected rat and human skulls. The fluorescent tracer DiI was applied to proximally cut meningeal nerves in rat and to distal branches of the spinosus nerve in human calvaria lined by dura mater. After several weeks, the dura mater and deep extracranial tissues were examined with fluorescence microscopy. RESULTS: In addition to a network of meningeal nerve fibers, several fiber bundles were observed, leaving the skull through emissary canals and fissures to innervate the pericranial temporal, parietal, and occipital periosteum. Traced fibers were seen spreading into deep layers of the temporal and upper neck muscles. Retrograde neuronal tracing revealed labeled cell bodies exclusively in the mandibular and maxillary division of the rat trigeminal ganglion, and centrally projecting fibers were identified in the spinal trigeminal tract. Electron microscopy of the cross-sected spinosus nerve showed myelinated and unmyelinated axons with similar numbers in human and rat. CONCLUSIONS: We conclude that a proportion of meningeal afferents innervates extracranial tissues like periosteum and pericranial muscles via collaterals projecting through the skull. These afferents may be nociceptive, some may subserve proprioceptive functions. The finding of extracranial projections of meningeal afferents may be important for our understanding of extracranial impacts on headache generation and therapy.

Loss of sympathetic nerve fibers in vital intertrochanteric bone cylinders lateral to osteonecrosis of the femoral head.

OBJECTIVES: Although etiology in osteonecrosis of the femoral head mainly depends on alterations of bone blood flow, vasoregulatory nerve fibers of the sympathetic and sensory nervous system have never been investigated in bone of osteonecrosis patients. This study aimed to demonstrate density of sympathetic and sensory nerve fibers in femoral head and, for comparison, adjacent periosteum, and synovium of the hip joint in patients with osteonecrosis. METHODS: Immunofluorescence staining techniques were applied using specific nerve fiber markers. A total of 10 patients with early femoral head osteonecrosis (ARCO I-II), 10 with late femoral head osteonecrosis (ARCO III-IV), and 10 patients with osteoarthritis of the hip were investigated. RESULTS: In the bone of the femoral head, density of sympathetic nerve fibers was lower in early and late osteonecrosis compared to osteoarthritis. There was a marked preponderance of sympathetic over sensory nerve fibers in bone of osteoarthritis patients, which was opposite in early and late femoral head osteonecrosis. In periosteum, density of sympathetic nerve fibers was similar in all three groups but density of sensory nerve fibers and cellularity were higher in early osteonecrosis compared to the other two groups. DISCUSSION/CONCLUSIONS: Due to the different affinity of norepinephrine for α-adrenoceptors (high affinity) and ß-adrenoceptors (low affinity), the loss of sympathetic nerve fibers relative to sensory nerve fibers in femoral head osteonecrosis might change the femoral head blood flow (towards α-adrenergic vasoconstriction). Higher density of sensory nerve fibers and cellularity in periosteum might indicate an inflammatory response in early osteonecrosis.

Analysis of the sensory innervations of the greater trochanter for improving the treatment of greater trochanteric pain syndrome.

In medical practice, greater trochanteric pain syndrome has an incidence of 5.6 per 1,000 adults per year, and affects up to 25% of patients with knee osteoarthritis and low back pain in industrialized nations. It also occurs as a complication after total hip arthroplasty. Different etiologies of the pain syndrome have been discussed, but an exact cause remains unknown. The purpose of this study was to obtain a better understanding of the sensory innervations of the greater trochanter in attempt to improve the treatment of this syndrome. Therefore, we dissected the gluteal region of seven adult and one fetal formalin fixed cadavers, and both macroscopic and microscopic examination was performed. We found a small sensory nerve supply to the periosteum and bursae of the greater trochanter. This nerve is a branch of the n. femoralis and accompanies the arteria and vena circumflexa femoris medialis and their trochanteric branches to the greater trochanter. This nerve enters the periosteum of the greater trochanter directly caudal to the tendon of the inferior gemellus muscle. This new anatomical information may be helpful in improving therapy, such as interventional denervation of the greater trochanter or anatomically guided injections with corticosteroids and local anesthetics.

Different sympathetic pathways control the metabolism of distinct bone envelopes.

Bone remodeling, the mechanism that modulates bone mass adaptation, is controlled by the sympathetic nervous system through the catecholaminergic pathway. However, resorption in the mandible periosteum envelope is associated with cholinergic Vasoactive Intestinal Peptide (VIP)-positive nerve fibers sensitive to sympathetic neurotoxics, suggesting that different sympathetic pathways may control distinct bone envelopes. In this study, we assessed the role of distinct sympathetic pathways on rat femur and mandible envelopes. To this goal, adult male Wistar rats were chemically sympathectomized or treated with agonists/antagonists of the catecholaminergic and cholinergic pathways; femora and mandibles were sampled. Histomorphometric analysis showed that sympathectomy decreased the number of preosteoclasts and RANKL-expressing osteoblasts in mandible periosteum but had no effect on femur trabecular bone. In contrast, pharmacological stimulation or repression of the catecholaminergic cell receptors impacted the femur trabecular bone and mandible endosteal retromolar zone. VIP treatment of sympathectomized rats rescued the disturbances of the mandible periosteum and alveolar wall whereas the cholinergic pathway had no effect on the catecholaminergic-dependent envelopes. We also found that VIP receptor-1 was weakly expressed in periosteal osteoblasts in the mandible and was increased by VIP treatment, whereas osteoblasts of the retromolar envelope that was innervated only by tyrosine hydroxylase-immunoreactive fibers, constitutively expressed beta-2 adrenergic receptors. These data highlight the complexity of the sympathetic control of bone metabolism. Both the embryological origin of the bone (endochondral for the femur, membranous for the mandibular periosteum and the socket wall) and environmental factors specific to the innervated envelope may influence the phenotype of the sympathetic innervation. We suggest that an origin-dependent imprint of bone cells through osteoblast-nerve interactions determines the type of autonomous system innervating a particular bone envelope.

The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A.

Although skeletal pain is a leading cause of chronic pain and disability, relatively little is known about the specific populations of nerve fibers that innervate the skeleton. Recent studies have reported that therapies blocking nerve growth factor (NGF) or its cognate receptor, tropomyosin receptor kinase A (TrkA) are efficacious in attenuating skeletal pain. A potential factor to consider when assessing the analgesic efficacy of targeting NGF-TrkA signaling in a pain state is the fraction of NGF-responsive TrkA+ nociceptors that innervate the tissue from which the pain is arising, as this innervation and the analgesic efficacy of targeting NGF-TrkA signaling may vary considerably from tissue to tissue. To explore this in the skeleton, tissue slices and whole mount preparations of the normal, adult mouse femur were analyzed using immunohistochemistry and confocal microscopy. Analysis of these preparations revealed that 80% of the unmyelinated/thinly myelinated sensory nerve fibers that express calcitonin gene-related peptide (CGRP) and innervate the periosteum, mineralized bone and bone marrow also express TrkA. Similarly, the majority of myelinated sensory nerve fibers that express neurofilament 200 kDa (NF200) which innervate the periosteum, mineralized bone and bone marrow also co-express TrkA. In the normal femur, the relative density of CGRP+, NF200+ and TrkA+ sensory nerve fibers per unit volume is: periosteum>bone marrow>mineralized bone>cartilage with the respective relative densities being 100:2:0.1:0. The observation that the majority of sensory nerve fibers innervating the skeleton express TrkA+, may in part explain why therapies that block NGF/TrkA pathway are highly efficacious in attenuating skeletal pain.

Structural and cellular features in metaphyseal and diaphyseal periosteum of osteoporotic rats.

Despite the important physiological role of periosteum in the pathogenesis and treatment of osteoporosis, little is known about the structural and cellular characteristics of periosteum in osteoporosis. To study the structural and cellular differences in both diaphyseal and metaphyseal periosteum of osteoporotic rats, samples from the right femur of osteoporotic and normal female Lewis rats were collected and tissue sections were stained with hematoxylin and eosin, antibodies or staining kit against tartrate resistant acid phosphatase (TRAP), alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF), von Willebrand (vWF), tyrosine hydroxylase (TH) and calcitonin gene-related peptide (CGRP). The results showed that the osteoporotic rats had much thicker and more cellular cambial layer of metaphyseal periosteum compared with other periosteal areas and normal rats (P < 0.001). The number of TRAP(+) osteoclasts in bone resorption pits, VEGF(+) cells and the degree of vascularization were found to be greater in the cambial layer of metaphyseal periosteum of osteoporotic rats (P < 0.05), while no significant difference was detected in the number of ALP(+) cells between the two groups. Sympathetic nerve fibers identified by TH staining were predominantly located in the cambial layer of metaphyseal periosteum of osteoporotic rats. No obvious difference in the expression of CGRP between the two groups was found. In conclusion, periosteum may play an important role in the cortical bone resorption in osteoporotic rats and this pathological process may be regulated by the sympathetic nervous system.

A phenotypically restricted set of primary afferent nerve fibers innervate the bone versus skin: therapeutic opportunity for treating skeletal pain.

Although musculoskeletal pain is one of the most common causes of chronic pain and physical disability in both developing and developed countries, relatively little is known about the nerve fibers and mechanisms that drive skeletal pain. Small diameter sensory nerve fibers, most of which are C-fiber nociceptors, can be separated into two broad populations: the peptide-rich and peptide-poor nerve fibers. Peptide-rich nerve fibers express substance P (SP) and calcitonin gene-related peptide (CGRP). In contrast, the peptide-poor nerve fibers bind to isolectin B4 (IB(4)) and express the purinergic receptor P(2)X(3) and Mas-related G protein-coupled receptor member d (Mrgprd). In the present report, we used mice in which the Mrgprd(+) nerve fibers express genetically encoded axonal tracers to determine the peptide-rich and peptide-poor sensory nerve fibers that innervate the glabrous skin of the hindpaw as compared to the bone marrow, mineralized bone and periosteum of the femur. Whereas the skin is richly innervated by CGRP(+), SP(+), P(2)X(3)(+) and Mrgprd(+) sensory nerve fibers, the bone marrow, mineralized bone and periosteum receive a significant innervation by SP(+) and CGRP(+), but not Mrgprd(+) and P(2)X(3)(+) nerve fibers. This lack of redundancy in the populations of C-fibers that innervate the bone may present a unique therapeutic opportunity for targeting skeletal pain as the peptide-rich and peptide-poor sensory nerve fibers generally express a different repertoire of receptors and channels to detect noxious stimuli. Thus, therapies that target the specific types of C-nerve fibers that innervate the bone may be uniquely effective in attenuating skeletal pain as compared to skin pain.

Cutaneous innervation of lower eyelid.

The loss of skin sensation or numbness after lower blepharoplasty is not uncommon. The aim of this study is to elucidate the infraorbital nerve (ION) and zygomaticofacial nerve (ZFN) in detail. Twenty-one hemifaces of 14 fresh Korean adult cadavers were dissected. Infraorbital nerve and ZFN came out of infraorbital foramen and zygomaticofacial foramen. They ran along superficial to the periosteum within and beneath the epimysium of the orbicularis oculi muscle and then through orbicularis oculi muscle perpendicularly and distributed to the skin. The distal branch approached to the lower border of the tarsal plate. Most terminal branches (93.8%) of ION were distributed medial to the lateral canthus. Only a few branches (6.2%) were lateral to the lateral canthus. Most (99.4%) terminal branches of ZFN were distributed lateral to the lateral canthus. Very few (0.6%) branches were medial to the lateral canthus. We conclude that the skin-muscle flap infringes less than the skin flap on the terminal branches of ION and ZFN in exposure of the orbital floor as well as in lower blepharoplasty.

A quantitative analysis of the nerve fibres of the acetabular periosteum of dogs.

There are many techniques for the treatment of hip dysplasia, and novel research is currently being undertaken in the hope of obtaining more efficient and less traumatic techniques. The denervation of the hip joint capsule is a simple and effective technique that allows recovery of the functional activity of the affected limbs in significantly less time than other techniques. This surgical procedure consists of removing the acetabular periosteum, thus eliminating the nerve fibres with consequent analgesia. The aim of this investigation was to quantify the number of nerve fibres present in different regions of the acetabular periosteum. The knowledge of regional differences is potentially valuable for the refining of the denervation technique of the hip joint capsule. Thirty canine acetabular fragments were used to compare the nerve fibre density of the periosteum. The results showed a significant difference between the mean density of nerve fibres at the cranial and dorsal-lateral portion (approximately 75 fibres/mm2) and caudal lateral portion (approximately 60 fibres/mm2) of the acetabulum. Those fibres at the periosteum are almost positioned in a sagittal plane, pointing towards the joint capsule, suggesting the same density in the latter region. These results indicate a new approach to the articular denervation technique, thus obtaining even better results for the treatment of hip dysplasia in dogs.

Post-traumatic orbital reconstruction: anatomical landmarks and the concept of the deep orbit.

Dissection deep within the orbit is a cause for concern to surgeons because of the perceived risks of injuring critical structures such as the contents of the superior orbital fissure and the optic nerve. Although "safe distances" (those distances within which it is considered safe to dissect within the orbit) have been described, these are of limited value if the orbit is severely disrupted or is congenitally shallow. In addition, traumatic defects in the orbital floor, in particular, often extend beyond these distances. Reliable landmarks based on the relations between anatomical structures within the orbit, rather than absolute distances, are described that permit safe dissection within the orbit. We present the concept of the deep orbit and describe its relevance to repair of injuries.

How many types of cholinergic sympathetic neuron are there in the rat stellate ganglion?

Sympathetic cholinergic postganglionic neurons are present in many sympathetic ganglia. Three classes of sympathetic cholinergic neuron have been reported in mammals; sudomotor neurons, vasodilator neurons and neurons innervating the periosteum. We have examined thoracic sympathetic ganglia in rats to determine if any other classes of cholinergic neurons exist. We could identify cholinergic sudomotor neurons and neurons innervating the rib periosteum, but confirmed that cholinergic sympathetic vasodilator neurons are absent in this species. Sudomotor neurons contained vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) and always lacked calbindin. Cholinergic neurons innervating the periosteum contained VIP and sometimes calbindin, but always lacked CGRP. Cholinergic neurons innervating the periosteum were usually surrounded by terminals immunoreactive for CGRP. We conclude that if any undiscovered populations of cholinergic neurons exist in the rat thoracic sympathetic chain, then they are indistinguishable in size, neurochemistry and inputs from sudomotor or cholinergic neurons innervating the periosteum. It may be that the latter two populations account for all cholinergic neurons in the rat thoracic sympathetic chain ganglia.

Sympathetic nervous system does not mediate the load-induced cortical new bone formation.

UNLABELLED: The contribution of the SNS to bone's response to mechanical loading is unclear. Using a noninvasive model of axial loading of the murine tibia, we found that sciatic neurectomy enhances load-induced new cortical bone formation and that pharmacological blockade of the SNS does not affect such responses, indicating that the SNS does not mediate the osteogenic effects of loading in cortical bone. INTRODUCTION: There is increasing evidence that the sympathetic nervous system (SNS) contributes to the regulation of bone mass and may influence remodeling by modulating bones' response to mechanical load-bearing. The aim of this study was to examine the effect of sciatic neurectomy (SN) on the changes in cortical bone formation induced in response to mechanical loading and to investigate whether the SNS is directly involved in such load-induced responses. MATERIALS AND METHODS: Accordingly, load-induced responses were compared in tibias of growing and adult control C57Bl/J6 mice and in mice submitted to unilateral SN; noninvasive axial loading that induced 2,000 microstrain on the tibia lateral midshaft cortex was applied cyclically, 5 or 100 days after surgery, for 7 minutes, 3 days/week for 2 weeks, and mice received calcein on the third and last days of loading. Tibias were processed for histomorphometry, and transverse confocal images from diaphyseal sites were analyzed to quantify new cortical bone formation. Chemical SNS inactivation was achieved by prolonged daily treatment with guanethidine sulfate (GS) or by the introduction of propranolol in drinking water. RESULTS: Our results show that new cortical bone formation is enhanced by loading in all tibial sites examined and that load-induced periosteal and endosteal new bone formation was greater in the SN groups compared with sham-operated controls. This SN-related enhancement in load-induced cortical bone formation in tibias was more pronounced 100 days after neurectomy than after 5 days, suggesting that longer periods of immobilization promote a greater sensitivity to loading. In contrast, the increases in new bone formation induced in response to mechanical loading were similar in mice treated with either GS or propranolol compared with controls, indicating that inactivation of the SNS has no effect on load-induced cortical new bone formation. CONCLUSIONS: This study shows that SN, or the absence of loading function it entails, enhances loading-related new cortical bone formation in the tibia independently of the SNS.

Temporal summation of pain evoked by mechanical stimulation in deep and superficial tissue.

UNLABELLED: Temporal summation of deep tissue pain has been suggested to be facilitated in chronic musculoskeletal pain syndromes. This study aimed to test whether temporal summation of mechanical induced pressure pain is (1) more pronounced at short (1 second) interstimulus intervals (ISIs) compared with long ISI (30 seconds), (2) more potent than summation elicited by pure skin stimulation, and (3) attenuated in women compared with men. Twelve age-matched men and 12 women were included. A computer-controlled pressure stimulator with a probe surface of 1 cm2 was used to give 10 stimulations to the tibialis anterior, tibia periosteum, and the first web of the hand. Sequential stimulation at pressure pain threshold intensity was applied with different ISIs (1, 3, 5, 10, and 30 seconds). The pain intensity was assessed on a visual analog scale (VAS) after each individual stimulus. The VAS scores after the 10th stimulation with 1-second ISI were increased (P < .05) by 418% +/- 77%, 378% +/- 89%, and 234% +/- 66% compared with the first stimulation for tibia, tibialis anterior, and web, respectively. Temporal summation of pain was observed for all ISIs in tibialis anterior and tibia, eg, 30-second ISI evoked a VAS increase of 192% +/- 71 % (tibia) and 117% +/- 42% (tibialis anterior) compared with the first stimulation. The VAS score after the 10th web stimulation was smaller (P < .05) than that of the 10th tibialis anterior or tibia stimulation. A regression analysis between stimulation number and VAS score showed that the pain intensity increased progressively (1) more for 1-second ISIs compared with longer ISIs (P < .01) and (2) faster in deep tissue compared with skin (P < .01). No gender difference was observed. The temporal summation might be related to both central and peripheral mechanisms. PERSPECTIVE: Pain originating in deep tissue influences central pain processing systems more than superficial tissue. This might be of importance in patients with musculoskeletal pain.

Distribution of galanin in bone and joint tissues.

The article describes the distribution of galanin in normal bone and joint tissues. Periosteum, cortical bone, bone marrow, and synovial membrane of normal rats were analyzed. Immunoelectron microscopy (iEM) was used to analyze the distribution of galanin, and radioimmunoassay (RIA) was used to determine its concentration. Immunoelectron microscopy showed that galanin is abundant in nerve fibers and endothelial cells in the periosteum and also in macrophage-like-cells and nerve fibers of the synovial membrane. The concentration of galanin measured by RIA showed the highest concentration in bone marrow, followed by periosteum and cortical bone. This study demonstrates that galanin is present and can be quantified in different compartments of bone and joint tissues and illustrates the possible role of galanin under physiological conditions.