Transient local presence of nerve fibers at onset of secondary ossification in the rat knee joint

In view of recent evidence that nerves may be involved in bone formation, the present study examines the local occurrence of axons at the onset of secondary ossification center formation in the knee region of developing rats. Radiographic and histological examination showed that secondary ossification center formation commenced at day 10. At day 15 the epiphyseal ossification had reached a relatively mature state. As seen by light microscopy, cartilage canals first appeared at day 5, reaching the epiphyseal center by day 9. Axons exhibiting a neurofilament-like immunoreactivity emerged from the perichondrial plexa into the cartilage canals. Many calcitonin gene-related peptide (CGRP)-immunoreactive and substance P (SP)-immunoreactive axons were found in the canals, as well as in the perichondrium. Axons with tyrosine hydroxylase-like immunoreactivity were not found in the canals, but such fibers occurred in relation to blood vessels at other sites. The canal-related axons disappeared between days 13 and 15, and the canals themselves did not persist beyond bone formation. As seen in the electron microscope, an individual canal contained 3–10 unmyelinated Schwann cell-enclosed axons with diameters of 0.1–2.0 m. These observations show that putative sensory unmyelinated axons with CGRP-and SP-like immunoreactivity are transiently present during initiation of bone formation in developing epiphyses. Whether there is a causal relation between transient innervation and osteogenesis remains to be determined.     

Distribution of nerve endings and sensory neuropeptides in rat synovium, meniscus and bone.

The nervous system collects information from the outer world by specific senses and from the interior milieu by somatic senses. This information is processed and stored in memory and affects various bodily functions through the efferent arm of the nervous system. The efferent chemical neuropeptide message is transported intra-axonally to the site of action, which imparts site-specificity to the peripheral, paracrine neuropeptide effects. In the present study, immunohistochemistry using the immunoperoxidase method with nickel amplification was applied to visualize the topographical distribution of articular nerve fibres and nerve endings using the markers PGP 9.5 and synaptophysin, respectively. Furthermore, to get a comprehensive idea of the sensory innervation of the articular and para-articular tissue, antisera to calcitonin gene-related peptide (CGRP) and substance P were employed. Samples were collected after fixation by perfusion followed by immersion in fixative and decalcification by a special method, which also allows studies of the bone innervation. PGP 9.5- and synaptophysin-immunoreactive type IVa and IVb nerve fibres and endings were found in the synovial lining and sublining tissue and in the vascularized peripheral parts of the menisci. Furthermore, periosteum, bone marrow and the epiphyseal growth plates were also innervated, whereas innervation of the diaphyseal and metaphyseal bone was more sparse. PGP 9.5- and synaptophysin-immunoreactive nerves were also characterized by their CRGP, and to some extent, substance P content. Because of their distribution, the peripheral peptide-containing type IVa and IVb nerve fibres and nerve endings are in a position to participate in the pathogenesis of arthritis, including aspects of nociception, tissue remodelling and neurogenic inflammation.     

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

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.     

Ontogeny of sensory nerves in the developing skeleton

Background: Previous studies have shown that the neuropeptide calcitonin gene-related peptide (CGRP) have an influence on osteoclastic bone resorption and that CGRP and substance P (SP), both wellknown markers for sensory neurons, behave as growth factors. Materials and Methods: The ontogeny of the sensory nerves in the hindlimb skeleton of the rat was studied from gestational day (GD) 15 to neonatal day (ND) 24 by immunohistochemistry. Neurofilaments and nerve terminals were labelled with protein gene-product 9.5 (PGP 9.5) and synaptophycin (SYN) respectively. Results: PGP 9.5 appeared at GD 15 and SYN at GD 19, both in the perichondrial tissue of the long bones. One week later, at ND 4 nerve fibre, immunoreactive to PGP 9.5 and SYN were observed within the bone organ. Sensory nerves, indicated by CGRP and SP, were first discerned at GD 18–19 in the periosteal tissue of the diaphyseal and metaphseal regions and in the bone organ at ND 4. Approximately at ND 6, vascular as well as non-vascular nerves extended into the metaphyses and at ND 8 into the epiphyses, concomitant with the first signs of mineralization. Conclusions: The study shows that a functional sensory nerve supply of the developing bone organ occurs immediatly prior to partus, apparently parallel with an increasing mineralization. The combined findings may indicate a sensory influence on developmental processes in the skeleton. © 1995 Wiley-Liss, Inc.     

Distribution of Matrix Proteins in Perichondrium and Periosteum During the Incorporation of Meckel's Cartilage into Ossifying Mandible in Midterm Human Fetuses: An Immunohistochemical Study

Immunohistochemical localization of versican and tenascin‐C were performed; the periosteum of ossifying mandible and the perichondrium of Meckel's cartilage, of vertebral cartilage, and of mandibular condylar cartilage were examined in midterm human fetuses. Versican immunoreactivity was restricted and evident only in perichondrium of Meckel's cartilage and vertebral cartilage; conversely, tenascin‐C immunoreactivity was only evident in periosteum. Therefore, versican and tenascin‐C can be used as molecular markers for human fetal perichondrium and fetal periosteum, respectively. Meckel's cartilage underwent endochondral ossification when it was incorporated into the ossifying mandible at the deciduous lateral incisor region. Versican immunoreactivity in the perichondrium gradually became weak toward the anterior primary bone marrow. Tenascin‐C immunoreactivity in the primary bone marrow was also weak, but tenascin‐C positive areas did not overlap with versican‐positive areas; therefore, degradation of the perichondrium probably progressed slowly. Meanwhile, versican‐positive perichondrium and tenascin‐C‐positive periosteum around the bone collar in vertebral cartilage were clearly discriminated. Therefore, the degradation of Meckel's cartilage perichondrium during endochondral ossification occurred at a different rate than did degradation of vertebral cartilage perichondrium. Additionally, the perichondrium of mandibular condylar cartilage showed tenascin‐C immunoreactivity, but not versican immunoreactivity. That perichondrium of mandibular condylar cartilage has immunoreactivity characteristic of other periosteum tissues may indicate that this cartilage is actually distinct from primary cartilage and representative of secondary cartilage. Anat Rec, 297:1208–1217, 2014. © 2014 Wiley Periodicals, Inc.     

Crocodilian bone-tendon and bone-ligament interfaces

We investigated bone-tendon (27 sites) and bone-ligament (12 sites) interfaces in six pairs of crocodile limbs and girdles under light microscopy. These crocodilian interfaces often included a direct, unmediated insertion in which the tendon or ligament fibers inserted directly into the bone itself without fibrocartilaginous mediation. This was quite different from the usual direct insertion known in mammals and lizards. Fibrocartilaginous tissue at the bone-tendon interface is generally believed to protect tendon fibers against shear stress. Other types of insertions were found in the crocodilian epiphyses, namely, hyaline cartilage and pseudofibrocartilaginous insertions. Notably, a thick periosteum/perichondrium and subchondral layer was involved at both interfaces. The thick periosteum/perichondrium seemed to form along the epiphyseal hyaline cartilage and might function in replacement of fibrocartilaginous tissues. Crocodilian thick periosteum/perichondrium would be expected to reinforce the limb and girdle bones--especially their epiphyses, in which secondary centers of ossification are absent. The subchondral layer--a kind of fibrocartilaginous tissue--seemed to play the role of the growth plate in compensating for the absence of secondary centers of ossification. Therefore, we hypothesized that the crocodile-specific bone-tendon interfaces were the result of these specializations of bone development and growth. In crocodiles, the disadvantages of the single ossification center are effectively compensated for by specialized morphologies, including these interfaces. Specialized bone growth provides the crocodile with the largest body size of the recent reptiles and an extremely fast method of locomotion.     

Organization of a unique net-like meshwork of CGRP+ sensory fibers in the mouse periosteum: implications for the generation and maintenance of bone fracture pain

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.     

Structure, formation and role of cartilage canals in the developing bone

In the long bones, endochondral bone formation proceeds via the development of a diaphyseal primary ossification centre (POC) and an epiphyseal secondary ossification centre (SOC). The growth plate, the essential structure for longitudinal bone growth, is located between these two sites of ossification. Basically, endochondral bone development depends upon neovascularization, and the early generation of vascularized cartilage canals is an initial event, clearly preceding the formation of the SOC. These canals form a discrete network within the cartilaginous epiphysis giving rise to the formation of the marrow space followed by the establishment of the SOC. These processes require excavation of the provisional cartilaginous matrix which is eventually replaced by permanent bone matrix. In this review, we discuss the formation of the cartilage canals and the importance of their cells in the ossification process. Special attention is paid to the enzymes required in disintegration of the cartilaginous matrix which, in turn, will allow for the invasion of new vessels. Furthermore, we show that the mesenchymal cells of the cartilage canals express bone-relevant proteins and transform into osteocytes. We conclude that the canals are essential for normal epiphyseal bone development, the establishment of the growth plate and ultimately longitudinal growth of the bones.     

Expression of Semaphorin-3A and its receptors in endochondral ossification: potential role in skeletal development and innervation.

Bone tissue is densely innervated, and there is increasing evidence for a neural control of bone metabolism. Semaphorin-3A is a very important regulator of neuronal targeting in the peripheral nervous system as well as in angiogenesis, and knockout of the Semaphorin-3A gene induces abnormal bone and cartilage development. We analyzed the spatial and temporal expression patterns of Semaphorin-3A signaling molecules during endochondral ossification, in parallel with the establishment of innervation. We show that osteoblasts and chondrocytes differentiated in vitro express most members of the Semaphorin-3A signaling system (Semaphorin-3A, Neuropilin-1, and Plexins-A1 and -A2). In vitro, osteoclasts express most receptor chains but not the ligand. In situ, these molecules are all expressed in the periosteum and by resting, prehypertrophic and hypertrophic chondrocytes in ossification centers before the onset of neurovascular invasion. They are detected later in osteoblasts and also osteoclasts, with differences in intensity and regional distribution. Semaphorin-3A and Neuropilin-1 are also expressed in the bone marrow. Plexin-A3 is not expressed by bone cell lineages in vitro. It is detected early in the periosteum and hypertrophic chondrocytes. After the onset of ossification, this chain is restricted to a network of cell processes in close vicinity to the cells lining the trabeculae, similar to the pattern observed for neural markers at the same stages. After birth, while the density of innervation decreases, Plexin-A3 is strongly expressed by blood vessels on the ossification front. In conclusion, Semaphorin-3A signaling is present in bone and seems to precede or coincide at the temporal but also spatial level with the invasion of bone by blood vessels and nerve fibers. Expression patterns suggest Plexin-A3/Neuropilin-1 as a candidate receptor in target cells for the regulation of bone innervation by Semaphorin-3A.     

Occurrence, distribution and ontogeny of CGRP immunoreactivity in the rat lower respiratory tract: effect of capsaicin treatment and surgical denervations

The occurrence and distribution of calcitonin gene-related peptide (CGRP) immunoreactivity in the rat respiratory tract were investigated by means of immunocytochemistry and radioimmunoassay using antibodies raised in rabbits to synthetic rat CGRP. Substantial amounts of CGRP immunoreactivity (range 5-37 pmol/g) were detected in all parts of the respiratory tract, the highest being in the stem bronchus. Gel filtration chromatography of extractable CGRP immunoreactivity revealed one single peak, eluting at the position of synthetic rat CGRP. CGRP immunoreactivity was localized both in mucosal endocrine cells and nerve fibres from the larynx down to the peripheral lung. CGRP-immunoreactive endocrine cells were found singly in trachea and stem bronchi and in groups in intrapulmonary airways. They appeared at a late stage of gestation (17 days), reached a maximum number near term and decreased after birth to maintain a population similar to that of the adult animals by postnatal day 21. Similarly, CGRP-immunoreactive nerve fibres were first identified by day 18 of the gestation period and reached the adult distribution by postnatal day 21. CGRP-immunoreactive nerve fibres were localized among smooth muscle, seromucous glands, beneath and within the epithelium of the airways and around blood vessels. CGRP was also found in sensory ganglia and in motor end plates of the larynx musculature. Neonatal pretreatment with capsaicin caused a marked reduction in CGRP immunoreactivity of nerve fibres in the respiratory tracts as well as a less marked decrease in the population of CGRP-containing endocrine cells of the lung. No change was seen in motor end plates immunostaining. Vagal ligation experiments revealed that CGRP-immunoreactive nerve fibres travelling in the vagus originate mainly from neurons located in the jugular ganglion. Infranodosal right vagal ligation induced a marked loss in CGRP-immunoreactive nerves of the trachea, and of the ipsilateral stem bronchus, but no changes were observed in peripheral lung. By contrast infranodosal left side vagal ligation caused a decrease in CGRP-immunoreactive nerves of the ipsilateral lung and bronchus without affecting the peptide content in the trachea. Left vagal ligation also induced a marked increase in both the intensity of staining and number of CGRP-immunoreactive endocrine cells in the lung. We conclude that CGRP immunoreactivity is localized in both nerve fibres and endocrine cells and is associated principally with the afferent (sensory) innervation of the respiratory tract.(ABSTRACT TRUNCATED AT 400 WORDS)     

An Immunohistochemical Study of Matrix Components in Early‐Stage Vascular Canals Within Mandibular Condylar Cartilage in Midterm Human Fetuses

Matrix components of vascular canals (VCs) in human fetal mandibular condylar cartilage (15–16 weeks of gestation) were analyzed by immunohistochemistry. Prevascular canals (PVCs), consisting of spindle‐shaped cells without capillary invasion, were observed within the cartilage. Intense immunoreactivity for collagen type I, weak immunoreactivity for aggrecan and tenascin‐C, weak hyaluronan (HA) staining, and abundant argyrophilic fibers in PVCs indicated that they contain noncartilaginous fibrous connective tissues that was different from those in the perichondrium/periosteum. These structural and immunohistochemical features of PVCs are different from those of previously reported cartilage canals of the long bone. Capillaries entered the VCs from the periosteum and ascended through VCs. Following capillary invasion, loose connective tissue had formed in the lower part of VCs, and immunoreactivity for collagen types I and III, tenascin‐C, and HA staining was evident in the matrix of loose connective tissue. No chondroclasts or osteogenic cells were seen at the front of capillary invasion, although small, mononuclear tartrate‐resistant acid phosphatase (TRAP)‐positive cells were present. Meanwhile, TRAP‐positive, multinucleated chondroclasts and flattened, osteoblast‐like cells were observed in the loose connective tissue at the lower part of VCs. These results may indicate slow progress of endochondral ossification in human fetal mandibular condyle. Further, unique matrix components in PVCs/VCs, which were different from those in cartilage canals in long bone, may reflect the difference of speed of endochondral ossification in cartilage canals and human fetal mandibular condyles. Anat Rec, 298:1560–1571, 2015. © 2015 Wiley Periodicals, Inc.     

Distribution and coexistence of neuropeptides in nerve fibres in the temporomandibular joint of late gestation fetal sheep

The density and distribution of nerve fibres immunoreactive to antisera for PGP 9.5 (general neuronal marker), calcitonin gene related peptide (CGRP) and substance P (SP) (markers for sensory neurons), as well as neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and tyrosine hydroxylase (TH) (markers for autonomic fibres), were examined in the temporomandibular joint (TMJ) of late gestation fetal sheep. This work formed part of a project investigating the influence of age and osteoarthritis on the innervation of the TMJ, and was undertaken to determine whether the innervation of the joint at 140 d gestation (17 d before birth) differed from that in the mature adult. Immunofluorescence microscopy was applied to serial sections of the capsule, disc and synovial membrane of 10 joints from 5 fetuses and image analysis was used for the quantitative assessment. The capsule, synovial membrane and the disc contained fibres immunoreactive (IR) to antisera for PGP 9.5, SP and CGRP. NPY-IR fibres were only visible in the loose connective tissue of the capsule. No VIP- or TH-IR nerve fibres were detected in the fetal TMJ. There was no statistically detectable difference between the density of nerve fibres immunoreactive to CGRP or PGP9.5 antisera in the capsule or disc. Substance P-immunoreactivity (IR) was relatively weak in all samples examined. Scattered branches of CGRP-IR fibres were found deep in the disc proper. The lack of receptor endings, other than free nerve endings in the TMJ of the late fetal sheep, might be a reflection of the functional and anatomical immaturity of the TMJ, as reflected in the immature, gross and microscopic appearance of the disc, the inferior joint compartment and articular surface of the condyle at this stage. These results demonstrate that the capsule, synovial membrane and disc in the TMJ of fetal sheep at 140 d gestation age are innervated with sensory fibres, while autonomic fibres are located in the capsule only. The findings also support the view that the disc is innervated at an early stage of life but at a later stage the density of innervation in the central part of the disc regresses and the innervation remains only peripherally in the adult TMJ disc. Further work is required to determine (1) at what stage sympathetic fibres innervate the disc and the synovium, and (2) when the mechanoreceptive nerve endings develop.     

The postnatal development of the insertions of the medial collateral ligament in the rat knee

Bone soft tissue remodelling at the femoral and tibial insertions of the medial collateral ligament (MCL) of the rat knee was monitored at regular intervals from birth to 120 days of age in 40 Sprague Dawley rats. At birth the femoral insertion originated from the perichondrium of the epiphysis. By day 8 the perichondrium within the insertion had turned into fibrocartilage. Secondary ossification of the femoral epiphysis had progressed in the region near to the insertion site by day 15. The epiphyseal cartilage was entirely replaced by bone by day 40 except for the fibrocartilage within the insertion. After that stage, no qualitative change in zonal insertion characteristics was observed, but only increase in size and decrease in cellularity. At birth, the tibial ligament inserted onto the thin cortical bone of the metaphysis via periosteum. At day 8, osteoclasts started to resorb the thin cortical bone at the ligament insertion, thus forming a metaphyseal depression between days 10 and 20. From days 20 to 120, the insertion remained qualitatively unchanged, showing three zones, the ligament, periosteum, and metaphyseal trabecular bone. The deep periosteal layer showed osteoclastic activity in the proximal part and osteoblastic activity in the distal part. The migration mechanism of the ligament insertion during growth seems to be caused by this growth-related osteoclastic resorption of the proximal metaphyseal bone and by simultaneous osteogenic activity, which successively cements the distal part of the ligament to bone. The persistence of the periosteal layer and the metaphyseal depression for up to 120 days may be regarded as a sign of continuing growth in this animal model. This is the first investigation showing that the formation of the metaphyseal depression is a purely postnatal event, and suggests that this process might be initiated by the change in mode of growth and joint biomechanics after birth, enabling ligament development and migration in a growing and increasingly loaded weight-bearing joint. The mainly resorptive process, which takes place during development of the tibial MCL insertion, may account for the tensile failure of this ligament that commonly occurs at this site during growth. The pronounced morphological differences between the chondral femoral and the periosteal tibial attachment of the adult MCL are apparently caused by the different postnatal developmental processes at epiphyses and metaphyses.Presented in part at the meeting of the Orthopaedic Research Society, Orlando, Florida, 13–16 February, 1995     

Calcitonin gene-related peptide-immunoreactive nerve fibers in mandibular periosteum of rat: evidence for primary afferent origin

Peptidergic neurons may play a role in the local regulation of bone mineralization. The neuropeptide vasoactive intestinal peptide (VIP) increases bone resorption in vitro, while calcitonin gene-related peptide (CGRP) has been shown to inhibit bone resorption in vitro. We have previously reported that sympathetic nerves with VIP-immunoreactivity innervate bone and periosteum. In the present study we sought to determine if CGRP fibers, like VIP fibers, exist in periosteum and what their origin might be. In whole-mount preparations of mandibular periosteum from rat, CGRP- and VIP-immunoreactive (IR) nerve fibers were present as networks within the periosteum. In preparations using two-color immunofluorescence, most CGRP-IR fibers were also immunoreactive for substance P (SP). In rats in which the subperiosteal space subjacent to the mandibular molars was injected with Fast blue or Fluoro-gold, retrogradely labeled cells were seen in ipsilateral trigeminal ganglia, superior cervical ganglia, and nodose ganglia. Individual cells labeled with both CGRP immunoreactivity and retrograde tracer were seen only in the mandibular portion of the trigeminal ganglion. These data suggest that CGRP-IR nerve fibers in periosteum may be of primary afferent origin. Given the reported effects of CGRP on bone mineralization, the present results suggest that primary afferent nerves containing CGRP and SP, as well as sympathetic nerves containing VIP, may play a role in focal bone remodeling.     

Atrial and ventricular rat coronary arteries are differently supplied by noradrenergic, cholinergic and nitrergic, but not sensory nerve fibres

The present immunohistochemical study set out to determine the extent of perivascular innervation in the rat heart, using markers for noradrenergic sympathetic fibres (tyrosine hydroxylase = TH), cholinergic parasympathetic fibres (vesicular acetylcholine transporter = VAChT), nitrergic fibres (neuronal NO synthase = nNOS), and peptidergic sensory fibres (calcitonin gene-related peptide = CGRP). For each of these antigens, the vascular innervation density was assessed separately in the atria, the basal and the apical parts of the ventricles, and was correlated to the inner vascular diameter. The four major findings are: (1) Each of these neurochemically defined populations shows an individual distribution pattern significantly different from the others with respect to correlation with vascular diameter and occurrence along atrial versus ventricular vessels. (2) Among autonomic efferent axons, nNOS-containing fibres are far less numerous than cholinergic and noradrenergic fibres. (3) Autonomic efferent axons (noradrenergic, cholinergic, nitrergic) are much more abundant around atrial than ventricular vessels, whereas perivascular CGRP-immunoreactive sensory nerve fibres are equally distributed in the various parts of the heart. (4) Noradrenergic and cholinergic axons preferentially innervate small-diameter vessels (negative linear correlation between index of innervation and vascular diameter), whereas the supply with CGRP-immunoreactive sensory nerve fibres does not change with vascular diameter. Collectively, the present study shows individual distribution patterns for each of the neurochemically defined populations of perivascular axons along the atrial and ventricular coronary arteries, indicating a highly differentiated nervous regulation of atrial versus ventricular, and large-diameter versus resistance vessels.     

Expression of neuropeptides and growth-associated protein 43 (GAP-43) in cutaneous and mucosal nerve structures of the adult rat lower lip after mental nerve section.

The reinnervation of the adult rat lower lip has been investigated after unilateral section of the mental nerve. Rats were sacrificed at 4, 7, 9, 14, 30, and 90 days after the operation. A further group of animals with section of the mental nerve and block of the alveolar nerve regeneration, was sacrificed at 14 days. Specimens were processed for immunocytochemistry with antibodies against PGP 9.5, GAP-43 or neuropeptides (CGRP, SP and VIP). Four days after nerve section, axonal degeneration seems evident in the mental nerve branches and inside skin and mucosa. GAP-43 immunoreactivity is intense in the mental nerve 7 days after nerve section and it reaches its maximal expression and distribution in peripheral nerve fibres at 14 days. At 30 days, the decline in its expression is associated with the increase of PGP9.5-, SP-, and CGRP immunopositivity. VIP is observed only in perivascular fibres at all times observed. Present results suggest that, after sensory denervation of the rat lip, nerve fibres in skin and mucosa remain at lower density than normal. The different time courses in the expression of neuropeptides and GAP-43 suggest a possible early involvement of GAP-43 in peripheral nerve regeneration.     

Absence of cartilage canals in the long bone extremities of four species of skeletally immature marsupials

The cartilaginous epiphyses and physes from the bone extremities of four species of skeletally immature marsupials were studied. The microscopic and ultrastructural features of the marsupial tissues were compared with similar samples from a neonatal lamb and a 1-day-old chick. Chondrocyte differentiation and endochondral ossification appeared similar in physes from the marsupials, foetal lamb, and 1-day-old chick. However, unlike the lamb and chick, which both contained cartilage canals, there were no cartilage canals in the epiphyseal or physeal cartilage from the marsupials. Many of the epiphyseal chondrocytes from the marsupial specimens contained large lipid droplets. It is suggested that the lipids in marsupial chondrocytes may be utilized in metabolic pathways. Despite hypertrophy of chondrocytes, there were no epiphyseal ossification centers in the femoral heads of the marsupial specimens; this was possibly due to the absence of cartilage canals, which are considered a source of osteoproginator cells. This study indicates that physeal and epiphyseal cartilage in marsupials is viable and functions in an avascular environment; this may be due to unique metabolic properties of the chondrocytes.     

The effect of intra-articular capsaicin on nerve fibres within the synovium of the rat knee joint

The aim of this study was to establish the effects of intra-articular capsaicin (pelargonic acid vallinylamide) on synovial innervation of the rat knee. Rats were sacrificed 1, 2, 4 and 7 days after intra-articular injection of capsaicin and joint tissues stained with either conventional haematoxylin and eosin (H and E) or with specific antibodies to the calcitonin gene-related peptide (CGRP), substance P (both of which are markers for primary afferent fibres), the C-flanking peptide of neuropeptide Y (CPON) (localised in postganglionic sympathetic fibres), or protein gene product 9.5 (a pan-neuronal marker). At lower concentrations (0.1% and 0.25%), capsaicin produced no change in peptide staining pattern or histological appearance. At 0.5% capsaicin, there was complete loss of nerve fibres showing positive staining for CGRP and substance P at all time points. Staining for CPON and protein gene product 9.5 was still present, but decreased, 1 and 2 days after treatment and virtually absent at 4 and 7 days. These findings provide evidence for partially selective denervation induced by 0.5% capsaicin, in contrast to 1% capsaicin which abolished staining for all peptide markers, indicating a total ablation of nerve fibres. A consistent but unexpected finding was the presence of a severe inflammatory response in joints treated with 0.5% and 1% capsaicin. An influx of polymorphonuclear leucocytes was found to occur within 4 h of injection, with progressive appearance of mononuclear cells after this time. We conclude that it is difficult to specifically deplete sensory nerve fibres from the synovium by means of local capsaicin injection. Although selective loss of staining for sensory nerve fibres could be achieved by injection of 0.5% capsaicin, there was progressive non-specific loss of post-ganglionic autonomic fibres which may be related to the severe inflammatory response provoked by the higher doses of capsaicin.     

Changes in nociceptive sensory innervation in the epidermis of the rat lower lip skin in a model of neuropathic pain

The epidermis is innervated by fine nerve endings that are important in the perception of nociceptive stimuli. However, their role in neuropathic pain is controversial. In this paper, changes in the innervation patterns of epidermal sensory afferent fibres in the rat lower lip have been studied following bilateral chronic constriction injury (CCI) of the mental nerve-a purely sensory branch of the trigeminal nerve. Sections of the lower lip were processed for immunocytochemistry using antibodies against Protein Gene Product (PGP) 9.5 and Calcitonin Gene-Related Peptide (CGRP) to identify the non-peptidergic and the peptidergic populations of nociceptive small diameter primary sensory afferent fibres. Peptidergic fibres co-localised both markers and the non-peptidergic fibres only stained for PGP 9.5 and not for CGRP. We quantified the total fibre length per 6000 microm(2) in the epidermis at several time points following CCI. Our data indicate that both fibre populations were significantly decreased at 2 weeks post-CCI, followed by fibre re-growth at levels above those seen in sham-operated animals at 4 weeks; however, this increase was only statistically significant for the non-peptidergic population. At 8 weeks post-CCI, the fibre lengths of both populations did not differ significantly from shams. This transient hyper-innervation of the epidermis by one subpopulation of nociceptive fibres coincided with the occurrence of spontaneous pain or dysesthetic sensations which we detected in a previous study in the same animal model. Therefore, we speculate that this transient hyper-innervation of the epidermis following injury could play a role in nociception in these animals.     

Noradrenergic and cholinergic innervation of the bone marrow

Bone marrow is supplied by sensory and autonomic innervation. Although it is well established that hematopoiesis is regulated by cytokines and cell-to-cell contacts, the role played by neuromediators on the proliferation, differentiation and release of hematopoietic cells is still controversial. We studied the innervation of rat femur bone marrow by means of fluorescence histochemistry and immunohistochemistry. Glyoxylic acid-induced fluorescence was used to demonstrate catecholaminergic nerve fibers. The immunoperoxidase method with nickel amplification was applied to detect the distribution of nerve fibers using antibodies against the general neuronal marker PGP 9.5 (neuron-specific cytoplasmic protein), while the cholinacetyltransferase immunoreactivity was studied by immunohistochemistry. Our results show the presence of an extensive network of innervation in the rat bone marrow, providing a morphological basis for the neural modulation of hemopoiesis.