Andris Kreicbergs

Substance P- and CGRP-immunoreactive nerves in bone

The present study demonstrates the occurrence of substance P (SP)- and calcitonin gene related peptide (CGRP)-immunoreactive nerve fibres in bone, bone marrow, periosteum, synovial membrane and soft tissues adjacent to the bone. The distribution pattern of the two types of nerves was similar, although the CGRP-positive fibres generally were more numerous. Both types of nerves were particularly abundant near the epiphyseal plate, in the bone marrow of patella and epiphyses, and in the periosteum. Many SP- and CGRP-immunoreactive fibres were also observed around blood vessels.

Neuropeptide Y-, tyrosine hydroxylase- and vasoactive intestinal polypeptide-immunoreactive nerves in bone and surrounding tissues

Nerve fibres immunoreactive to neuropeptide Y (NPY), tyrosine hydroxylase (TH) and vasoactive intestinal polypeptide (VIP) were demonstrated in rat bone and adjacent tissues. The distribution of NPY- and TH-positive fibres differed from that of VIP-positive fibres. NPY- and TH-immunoreactive fibres were almost exclusively found close to or within the blood vessel walls, mostly in the vicinity of the epiphyseal plate, but also in the Volkmann canals. VIP-positive fibres were predominantly present in the epiphysis and periosteum and only occasionally around blood vessels. This study demonstrates that bone and surrounding tissues have a supply of both noradrenergic and peptide-containing nerves. The differential distribution of these nerves may reflect specific roles in the local regulation of bone physiology, such as blood flow, bone formation or resorption.

DNA flow analysis of soft tissue tumors.

The cellular DNA content of 81 soft tissue tumors was determined by means of flow cytometry and related to conventional histologic classification of the same tumors. Comparison of histologic and cytometric analysis showed that all 23 benign tumors were diploid (normal DNA content), whereas the malignant group included both diploid and aneuploid (abnormal DNA content) lesions. There appeared to be a relationship between tumor grade and ploidy level in that 92% of Grade II, 28% of Grade III, and 11% of Grade IV lesions were diploid. Cell distribution analysis, feasible in 51 cases, disclosed that diploid lesions had a low proportion of S and G2+M cells and most aneuploid lesions a high proportion, indicating a relationship between ploidy level and proliferative activity. The current study shows that solid mesenchymal tumors may be analyzed by DNA flow cytometry. Regardless of histogenetic type, it appears that benign and low‐grade tumors are diploid and high‐grade tumors, in general, are aneuploid. As to exceptions, DNA analysis may prove to give information beyond that obtained by subjective histologic interpretation. Thus, adequate follow‐up might show that high‐grade lesions with a diploid DNA content are associated with a better prognosis than expected from histologic classification. Cancer 59:128–133, 1987.

Early nerve regeneration after Achilles tendon rupture – a prerequisite for healing? A study in the rat

Nerve regeneration during healing of Achilles tendon rupture in the rat was studied by immunohistochemistry including semiquantitative assessment. Neuronal markers for regenerating and mature fibers, ie., growth associated protein 43 (GAP‐43) and protein gene product 9.5 (PGP 9.5), respectively, were analyzed at different time points (1–16 weeks) post‐rupture. In the paratenon, both the ruptured and intact contralateral tendon (control) consistently exhibited immunoreactivity to the two neuronal markers. However, in the proper tendinous tissue only the ruptured tendon showed immunoreactivity to GAP‐43 and PGP 9.5. This expression was seen already at week 1 post‐rupture to reach a peak at week 6 followed by a successive drop till week 16. Also the occurrence of sensory and autonomic fibers according to immunoreactivity for calcitonin gene‐related peptide (CGRP) and neuropeptide Y (NPY), respectively, was analyzed. CGRP‐positivity was abundantly seen from weeks 2–6 in both perivascular and sprouting free nerve endings in the proper tendon tissue undergoing healing. NPY appeared later, at weeks 6–8 post‐rupture around blood vessels mainly located in the surrounding loose connective tissue. Apart from a role in vasoaction (CGRP, vasodilatory; NPY, vasoconstrictory), both neuropeptides have been implicated in fibroblast and endothelial cell proliferation required for angiogenesis. The present study shows that early healing of ruptured tendons is characterized by an orchestrated, temporal appearance of nerve fibers expressing peptides with different actions. The observed pattern of neuronal regeneration and neuropeptide expression may prove to be important for normal connective tissue healing.

Neuronal plasticity in relation to nociception and healing of rat achilles tendon

Nerve regeneration and the occurrence of three neuropeptides; i.e. substance P (SP), calcitonin gene related peptide (CGRP) and galanin (GAL), were studied during healing of tendon rupture in the rat by semi‐quantitative immunohistochemistry. The neuronal findings were related to nociception as assessed by hindpaw withdrawal latencies at thermal and mechanical tests.

Fixation and demineralization of bone tissue for immunohistochemical staining of neuropeptides

SummaryThe present study in the rat demonstrates the feasibility of applying immunohistochemical staining techniques on bone tissue for studies of substances such as neuropeptides contained in nerve fibers. Two fixation procedures, as well as the influence of demineralization on neuropeptide antigenicity, were studied in bone and for comparison in small intestine.In vivo perfusion with paraformaldehyde and picric acid, followed by demineralization in a solution of either EDTA-cacodylate or buffered EDTA-sucrose, proved to be the most appropriate with respect to preserved substances were tested. In the bone tissue, immunoreactivity was found to four neuropeptides: substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y, and also to the catecolamine-synthesizing enzyme tyrosine hydroxylase. The described method for identifying intraosseal neuropeptides offers a new means of studying skeletal innervation and bioactive substances in bone tissue.

Low risk of recurrence of enchondroma and low-grade chondrosarcoma in extremities. 80 patients followed for 2-25 years.

We analyzed the clinical course in 40 patients with enchondroma and 40 with low-grade chondrosarcoma of the extremities after a median follow-up of 7 years. 13 patients with enchondroma and 2 with chondrosarcoma had only open biopsy and they had no signs of further progression of the lesions. Among 23 patients with enchondroma and 23 with chondrosarcoma who were treated by intralesional curettage, 3 had local recurrences. The 10-year local recurrence rate was 0.04 in the enchondroma group and 0.09 in the chondrosarcoma group. There were no metastases. The results imply that enchondroma and low-grade chondrosarcoma of the extremities should be treated with limited surgery. The morbidity associated with en bloc resection and reconstruction can apparently be obviated without jeopardizing the limb or survival.

The development of autonomic innervation in bone and joints of the rat

The development of autonomic nerves in the hindlimb skeleton, was studied in rats from gestational day (G) 15 to postnatal day (P) 24 by immunoreactivity to neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). Control labelling with antisera to neurofilaments, protein gene-product 9.5 (PGP 9.5), and nerve terminals, synaptophysin (SYN), showed nerve fibres at G15 and nerve terminals at G19 in the perichondrial tissue. From P4, nerve fibres and terminals were observed within the bone organ. Noradrenergic sympathetic nerves, containing NPY, were first discerned at birth, G21, in the perichondrial tissue and within the bone organ at P4. Autonomic cholinergic nerve fibres, indicated by immunoreactivity to VIP, exhibited a similar temporal and regional occurrence. The diaphyseal parts were first supplied with autonomic nerves at P4. The nerve fibres extended into the metaphyses at P6-8 and finally into the epiphyses at P10, concomitant with the first signs of mineralization. Vascular as well as non-vascular nerve fibres were seen. The study shows that developing bone organ is supplied with autonomic nerves from birth, and the the growth of nerves parallels the mineralisation process. Previous studies have demonstrated that NPY potently inhibits parathyroid hormone (PTH) induced effects on osteoblastic bone cells and that VIP is a strong inductor of bone resorption. NPY and VIP also exert vasoregulatory effects. The combined findings suggest an autonomic influence on bone development.

Bone Reinnervation After Fracture: A Study in the Rat

Reinnervation after tibial fracture in the rat was studied by analyzing the occurrence of growth‐associated protein 43 (GAP‐43), a marker for regenerating nerve fibers, and protein gene product 9.5 (PGP‐9.5), a marker for mature nerve fibers, by immunohistochemistry. At 3 days postfracture, GAP‐43‐immunoreactive nerve fibers were first observed in the fracture hematoma and periosteum. At 7 days postfracture, abundant sprouting of GAP‐43‐positive fibers was seen in the callus, hyperplastic periosteum, and edge of fibrocartilage. In the latter region, the nerve fibers were nonvascular, showing dense ramifications and terminal sprouting close to chondroid cells. At 14 days and 21 days postfracture, many GAP‐43‐positive fibers were still sprouting into the fibrocartilage and new woven bone. Fine varicose GAP‐43‐positive fibers also were present in the bone marrow. In contrast to GAP‐43, PGP‐9.5‐positive nerve fibers were observed only occasionally at 3 days postfracture but gradually increased in number from day 14 to 21. Our study shows that intense nerve regeneration occurs in early fracture healing partly unrelated to neovascularization. Considering that neuronal mediators have been shown to participate in local bone formation and resorption, the nerve regeneration observed may prove to be essential for delivery of neuronal mediators required for normal callus formation and/or neovascularization.

Autonomic innervation of tendons, ligaments and joint capsules. A morphologic and quantitative study in the rat

We analyzed the neuronal occurrence of autonomic transmitters; noradrenaline (NA), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), in the Achilles tendon, medial and lateral collateral ligaments and knee joint capsule in the rat — by immunohistochemistry (IHC). In addition, the tissue concentrations of the sympathetic neuropeptide, NPY, and the parasympathetic peptide, VIP, were determined by radioimmunoassay (RIA). IHC demonstrated nerve fibers containing sympathetic vasoconstrictors — NA and NPY‐ and the parasympathetic vasodilator, VIP, in all tissues. NPY‐ and NA‐positive nerve fibers were predominantly observed in larger blood vessels, whereas, nerve fibers immunoreactive to VIP were found in smaller vessels. In many nerve fibers a co‐localization of the transmitters was seen. RIA showed that the concentration of NPY compared to VIP was 15‐times higher in ligaments and twice as high in tendons and capsules. The differences noted may reflect a difference in vulnerability to degenerative conditions. In pathological conditions, dysregulation of autonomic transmitters in hypovascularized tissues subjected to repetitive mechanical load may contribute to tissue hypoxia leading to degeneration and rupture of tendons and ligaments.