Mahmood Ahmed

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.

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.

Capsaicin effects on substance P and CGRP in rat adjuvant arthritis.

The effects of capsaicin on the sensory neuropeptides substance P and calcitonin gene-related peptide were analyzed in the ankle joints and dorsal root ganglia (L2-L6) of adult female Lewis rats. The study included 23 normal rats and 23 arthritic rats, all injected subcutaneously with capsaicin (total dose 200 mg/kg bw). Another two groups of animals from a previous study, i.e., 23 normal rats and 23 arthritic rats not given capsaicin served as controls. Adjuvant arthritis was induced by inoculation with heat-killed mycobacteria. The morphological distribution of sensory neuropeptides was assessed by immunohistochemistry and the tissue concentrations were determined by radioimmunoassay. In normal rats, capsaicin significantly reduced the concentrations of substance P and calcitonin gene-related peptide in ankle joints (54 and 36%, respectively) as well as dorsal root ganglia (40 and 54%, respectively). In arthritic rats those pretreated with capsaicin had significantly lower concentrations of substance P and calcitonin gene-related peptide in dorsal root ganglia (19 and 42%, respectively) compared to the arthritic controls. In the ankle joints, however, only the SP concentration was reduced (42%). Notably, this was accompanied by a 40% reduction in inflammatory response as assessed by comparing the ankle joint weights of the experimental groups. In general, there was a good correlation between the neuropeptide concentrations in ipsilateral ankle joints and the corresponding dorsal root ganglia as assessed in individual rats. The present study of adjuvant induced arthritis shows that capsaicin administration reduces the otherwise up-regulated levels of sensory neuropeptides in dorsal root ganglia and ankle joints. However, capsaicin at the dose given can only mitigate, not completely prevent the development of joint inflammation. Nonetheless, the findings suggest that antineuronal therapy targeted against specific neurotransmitters may prove useful in inflammatory joint disease.

Sensory neuropeptidergic pattern in tendon, ligament and joint capsule. A study in the rat

The normal occurrence of sensory neuropeptides in tendons, ligaments and joint capsules in the rat was analyzed by immunohistochemistry and radioimmunoassay (RIA). Nerve fibres immunoreactive to substance P (SP), calcitonin gene-related peptide (CGRP), neurokinin A, galanin and somatostatin were identified in the Achilles tendon as well as the collateral ligaments and joint capsule of the knee. The neuropeptidergic fibres were predominantly found in the epiligament and paratenon. However, SP- and CGRP-positive fibres were also seen in the proper ligament and tendon tissues. RIA showed higher concentrations of SP and CGRP in tendons than in ligaments and capsules. The morphological and quantitative data obtained on sensory neuropeptides in normal tendons, ligaments and joint capsules may be used as a reference for tissue analysis in painful and inflammatory conditions of the locomotor apparatus.

Extraction and quantitation of neuropeptides in bone by radioimmunoassay.

The feasibility of extracting and quantifying neuropeptides in bone by radioimmunoassay was investigated in a study including 60 diaphyseal rat femora. Substance P, calcitonin gene-related peptide, neuropeptide Y and vasoactive intestinal polypeptide, previously identified in bone by immunohistochemistry, were extracted from separate homogenates of bone, periosteum and bone marrow in a solution of 4% EDTA and 2 M acetic acid. Measurable amounts of all four neuropeptides in bone, periosteum and bone marrow were obtained by radioimmunoassay in a reproducible manner. The neuropeptide immunoreactivities were characterized by reverse-phase high performance liquid chromatography. Among the four neuropeptides analyzed, neuropeptide Y consistently exhibited the highest concentrations in the different tissues. Overall, cortical bone showed the lowest neuropeptide concentrations. The concentration of vasoactive intestinal polypeptide was higher in periosteum than in bone marrow, whereas that of calcitonin gene-related peptide was uniform in these tissues. The distributional differences observed in bone tissue may be explained by a variety of physiological roles attributed to neuropeptides such as regulation of nociception, vasoactivity, immune function and local bone metabolism. The described methodology offers a new means of investigating a neuropeptidergic involvement in various disorders of the skeleton.

Attenuation of pain and inflammation in adjuvant-induced arthritis by the proteasome inhibitor MG132.

OBJECTIVE In rheumatoid arthritis (RA), pain and joint destruction are initiated and propagated by the production of proinflammatory mediators. Synthesis of these mediators is regulated by the transcription factor NF-kappaB, which is controlled by the ubiquitin proteasome system (UPS). The present study explored the effects of the proteasome inhibitor MG132 on inflammation, pain, joint destruction, and expression of sensory neuropeptides as markers of neuronal response in a rat model of arthritis. METHODS Arthritis was induced in rats by injection of heat-killed Mycobacterium butyricum. Arthritis severity was scored, and nociception was evaluated by mechanical pressure applied to the hind paw. Joint destruction was assessed by radiologic and histologic analyses. NF-kappaB DNA-binding activity was analyzed by electromobility shift assay, and changes in the expression of the p50 NF-kappaB subunit and the proinflammatory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) were detected by immunohistochemistry. RESULTS Arthritic rats treated with MG132 demonstrated a marked reduction in inflammation, pain, and joint destruction. The elevated DNA-binding activity of the NF-kappaB/p50 homodimer and p50, as well as the neuronal expression of SP and CGRP, observed in the ankle joints of arthritic rats were normalized after treatment with MG132. CONCLUSION In arthritic rats, inhibition of proteasome reduced the severity of arthritis and reversed the pain behavior associated with joint inflammation. These effects may be mediated through the inhibition of NF-kappaB activation and may possibly involve the peripheral nervous system. New generations of nontoxic proteasome inhibitors may represent a novel pharmacotherapy for RA.

Type 2 diabetes impairs tendon repair after injury in a rat model

Type 2 diabetes adversely affects the properties of native connective tissue. The underlying mechanisms, however, by which diabetes alters connective tissue metabolism, especially tendon, are poorly defined. The aim of this study was to determine the effect of type 2 diabetes on the mechanical, histological, and molecular properties of the intact and healing Achilles tendon. The right Achilles tendon was transected in 11 male diabetic Goto-Kakizaki (GK) and 10 age- and sex-matched Wistar control rats, while the left Achilles tendon was left intact. At 2 wk postinjury the intact and injured tendons were assessed by biomechanical testing and histology. The gene expression of collagen I and III, biglycan, versican, MMP-13, and MMP-3 was measured by quantitative RT-PCR, and their protein distribution was studied by immunohistochemistry. Intact tendons exhibited only small differences between the groups. In injured tendons, however, a significantly smaller transverse area and lower stiffness was found in diabetic GK compared with Wistar control rats. This correlated with impaired structural organization of collagen fibers and a reduced expression of collagen I and III in the injured tendons of the diabetic GK compared with Wistar control. Moreover, MMP-3 gene expression was downregulated in the injured diabetic GK tendons compared with injured Wistar controls. Our results indicate that in a rat model of diabetes tendon healing is impaired mainly due to altered expression of collagen and MMPs reflecting decreased degradation of matrix proteins and impaired tissue remodeling. Further our data suggest that therapeutic modulation of collagens or MMPs might be targets for new regenerative approaches in operated, injured, or maybe also degenerative tendon diseases in diabetes.

Extraction of neuropeptides from joint tissue for quantitation by radioimmunoassay. A study in the rat

The feasibility of extracting neuropeptides from rat knee joints for quantitation by radioimmunoassay was tested. The investigation, based on 25 adult Lewis rats, focused on substance P, calcitonin gene-related peptide, neuropeptide Y, and vasoactive intestinal polypeptide. The relative recovery of the peptides in different extraction media was assessed Both knee joints including the articulating epiphysis were dissected and cut into small pieces. The series was divided into five subgroups, 10 joints in each, for extraction in five different media: 1) 1 M acetic acid in 4% EDTA, 2) 2 M acetic acid in 4% EDTA, 3) neutral water in 4% EDTA, 4) 2 M acetic acid in 4% EDTA and 95% alcohol, and 5) 2 M acetic acid without EDTA. Measureable concentrations of the four neuropeptides were reproducibly assessed by RIA. Although all extraction media provided measurable concentrations, 2 M acetic acid in 4% EDTA was found to give the highest overall yield of the four neuropeptides analyzed. Reverse-phase HPLC confirmed that the immunoreactivities assessed by RIA corresponded to the four neuropeptides of interest. Experimental and clinical evidence suggest a neurogenic involvement in the pathophysiology of inflammatory joint disease, e.g., rheumatoid arthritis. The extraction procedure described offers a means of determining neuropeptide concentrations in joint tissue under normal and pathologic conditions by RIA.

Alteration in endogenous opioid systems due to chronic inflammatory pain conditions

The influence of chronic arthritic pain on two endogenous opioid peptides, dynorphin B and [Met5]enkephalin-Arg6-Phe7, and multiple opioid receptors in discrete brain, lumbar spinal cord and pituitary pools was investigated. Using radioimmunoassay and receptor binding assay, we examined the changes in regional opioid peptide levels and opioid receptor activity due to chronic inflammation in adjuvant arthritic rats. At 4 weeks post-inoculation, increased levels of immunoreactive dynorphin B and [Met5]enkephalin-Arg6-Phe7 were measured in tissues of arthritic rats compared with controls. No significant changes in mu-, delta- or kappa-opioid receptors were seen after chronic inflammation. Taken together, these results indicate that in chronic arthritis, opioid receptor changes do not follow the peptide alterations of pro-dynorphin and pro-enkephalin systems. Thus, dynamic modification and modulation of nociceptive information takes place during chronic inflammation. This supports the key role of the central nervous system in chronic inflammatory pain conditions.