The cholinergic system in joint health and osteoarthritis: a narrative-review.

Osteoarthritis (OA) poses a major health and economic burden worldwide due to an increasing number of patients and the unavailability of disease-modifying drugs. In this review, the latest understanding of the involvement of the cholinergic system in joint homeostasis and OA will be outlined. First of all, the current evidence on the presence of the cholinergic system in the normal and OA joint will be described. Cholinergic innervation as well as the non-neuronal cholinergic system are detected. In a variety of inflammatory diseases, the classic cholinergic anti-inflammatory pathway lately received a lot of attention as via this pathway cholinergic agonists can reduce inflammation. The role of this cholinergic anti-inflammatory pathway in the context of OA will be discussed. Activation of this pathway improved the progression of the disease. Secondly, chondrocyte hypertrophy plays a pivotal role in osteophyte formation and OA development; the impact of the cholinergic system on hypertrophic chondroblasts and endochondral ossification will be evaluated. Cholinergic stimulation increased chondrocyte proliferation, delayed chondrocyte differentiation and caused early mineralisation. Moreover, acetylcholinesterase and butyrylcholinesterase affect the endochondral ossification via an acetylcholine-independent pathway. Thirdly, subchondral bone is critical for cartilage homeostasis and metabolism; the cholinergic system in subchondral bone homeostasis and disorders will be explored. An increase in osteoblast proliferation and osteoclast apoptosis is observed. Lastly, current therapeutic strategies for OA are limited to symptom relief; here the impact of smoking on disease progression and the potential of acetylcholinesterase inhibitors as candidate disease-modifying drug for OA will be discussed.

Synovial nerve fiber density decreases with naturally-occurring osteoarthritis in horses.

OBJECTIVE: To measure the nerve fiber density in synovial membranes from healthy and OA equine joints and to investigate the relationship between synovial innervation and OA severity, synovial vascularity and synovitis. DESIGN: Twenty-five equine metacarpophalangeal joints were collected post-mortem. The joints were dissected and the macroscopic lesions of the articular cartilage were scored. Synovial membrane specimens (n = 50) were harvested, fixed, sectioned and scored histologically. Immunohistochemical staining and immunofluorescence with S-100 protein, that identifies nerve fibers, and ⍺-actin, that stains vascular smooth muscle, were also performed on site-matched specimens and the relationships between these tissues was interrogated. RESULTS: The nerve fiber density was higher in the superficial layer (≤200 µm) of the synovium when compared to the deeper layer in control equine joints (mean difference (95% C.I.): 0.054% (0.018%, 0.11%)). In osteoarthritic joints, synovial innervation decreased in the superficial layer with increasing macroscopic OA score (ß (SEM), 95% C.I.: -0.0061 (0.00021), -0.0011, -0.00017). The blood vessel density was also higher in the superficial layer of the synovium compared to the deep layer in the control (mean difference (95% C.I.): 1.1% (0.36%, 2.3%)) and OA (mean difference (95% C.I.): 0.60% (0.22%, 1.2%)) equine joints. Moreover, considering all synovial specimens, higher nerve fiber density in the deep layer positively correlated with blood vessel density (ß (SEM), 95% C.I.: 0.11 (0.036), 0.035, 0.18). CONCLUSION: The reduction in nerve fiber density with advanced cartilage degeneration suggests that peripheral neuropathy is associated with equine OA. Whether this link is associated with neuropathic pain, requires further investigation.

Histopathological analysis of the posterior cruciate ligament in primary osteoarthritis.

BACKGROUND: According to some authors, the indication of an arthroplasty maintaining the posterior cruciate ligament (PCL) demands adequate structural preservation and proprioceptive function of this ligament. The nervous fibers contained in the synovial neurovascular bundle (NVB) around the PCL are involved in proprioception. A study evaluating the grade of PCL and NVB degeneration by using clinical, radiological, and perioperative parameters in knee arthritis patients, in theory, could help surgeons in the decision of preserving or not preserving the PCL in a particular patient. QUESTIONS: (1) Can the degree of the PCL collagen fibers degeneration be predicted by clinical, radiographic, and perioperative parameters in knee arthritis patients? (2) Is the NVB histological degeneration status predictable using clinical, radiographic, and perioperative parameters in the same subset of patients? (3) Is there a correlation between the degree of the PCL collagen fibers degeneration and NVB status in knee arthritis patients? METHODS: Eighty-nine PCLs (85 patients) obtained from total knee replacement surgery were studied. The histologic degeneration of PCL collagen fibers and the NVB status (preserved, degenerated, not detected) were evaluated. These histological degeneration patterns were correlated with clinical and radiographic parameters and with anterior cruciate ligament (ACL) status. RESULTS: A small prevalence of preserved NVB was related to Grades IV and V of Ahlbäck's classification, ACL absence, and severe PCL degeneration. The clinical and radiological parameters studied were not able to predict the grade of histological degeneration of the PCL. CONCLUSIONS: Ahlbäck's classification and ACL status provided useful information about NVB integrity. LEVEL OF EVIDENCE: Basic Science Level IV.

Cervical spine meniscoids: an update on their morphological characteristics and potential clinical significance.

PURPOSE: Cervical spine meniscoids are intra-articular folds of synovial membrane that have been theorised to have potential clinical significance in neck pain. Recent anatomical and clinical research has re-visited the pathoanatomical capacity of these structures. The purpose of this review is to discuss cervical spine meniscoid morphology in light of recently published work, to provide an update on the plausible relevance of these structures to clinical practice. METHODS: Narrative review critically discussing basic science and clinical research regarding cervical spine meniscoids, with focus upon implications for clinical practice. RESULTS: Basic science research indicates that cervical spine meniscoids can be innervated and appear to vary in morphology in the presence of articular degeneration. In a clinical population, associations have been observed between cervical spine meniscoid morphology and presence of cervical spine symptoms. CONCLUSIONS: Recent studies regarding cervical spine meniscoid morphology provide further evidence of pathoanatomical capacity of these structures. Further research is required, however, in clinical populations to empirically investigate specific theorised mechanisms of cervical spine meniscoid involvement in neck pain.

The innervation of synovium of human osteoarthritic joints in comparison with normal rat and sheep synovium.

OBJECTIVE: To study whether osteoarthritis (OA) in the knee is associated with a change of the innervation pattern in the synovial layer. DESIGN: In synovial tissue from the normal knee joint of rat and sheep we studied the presence of vessels and of nerve fibres using transmission electron microscopy and immunohistochemistry. Synovial material was also obtained from patients who underwent total knee replacement surgery. This material was examined for inflammatory changes, and the presence of vessels and nerve fibres was assessed. RESULTS: The synovium in the parapatellar region of the normal knee joint of rat and sheep exhibited a dense capillary and neuronal network. It was entered by calcitonin gene-related peptide containing sensory fibres and tyrosine hydroxylase-positive sympathetic nerve fibres. Synovial material from patients with knee OA exhibited different degrees of inflammation. Synovial material without inflammation exhibited a similar vascular and neuronal network as the normal knee joint from rat and sheep. However, in synovium with inflammatory changes we found a significant decrease of nerve fibres in depth ranges close to the synovial lining layer depending on the degree of inflammation whereas deeper regions were less affected. CONCLUSIONS: Inflammatory changes in the synovium of OA joints are associated with a massive destruction of the capillary and neuronal network which is present in normal synovium. Due to the disappearance of the sensory fibres it is unlikely that OA pain is initiated directly in the synovium. The loss of normally innervated vascularisation may have multiple consequences for the physiological functions of the synovium.

Sensory innervation and inflammatory cytokines in hypertrophic synovia associated with pain transmission in osteoarthritis of the hip: a case-control study.

OBJECTIVE: To clarify the sensory innervation and inflammatory cytokines in hypertrophic synovia associated with pain transmission in OA of the hip. METHODS: A piece of the synovium was extracted during reconstruction surgery in 50 patients with OA of the hip as an inflammatory synovium and in 12 patients with femoral neck fracture as a normal synovium. Each sample was immersed in fixative solution, sectioned on a cryostat, and then processed for immunohistochemistry using antibodies as follows: neuron-specific class III ß-tubulin (TuJ-1) as a general marker for nerve fibres, calcitonin gene-related peptide (CGRP) for sensory nerve fibres, nuclear factor κB (NF-κB) for the protein complex controlling the transcription of DNA in cellular responses to painful stimuli, and TNF-α for cytokines involved in acute inflammation. The number of immunopositive cells and fibres were counted using a fluorescence microscope. RESULTS: In the inflammatory synovium of OA of the hip, TuJ-1 was positive in 46% (23 hips). Of those positive for TuJ-1, 78% (18 hips) were also positive for CGRP, but 22% (5 hips) were negative for CGRP. NF-κB was positive in 68% (34 hips). Of those positive for NF-κB, 76% (26 hips) were also positive for TNF-α, but 24% (8 hips) were negative for TNF-α. In normal synovia, all four substances were negative. CONCLUSION: We suggest sensory innervation and inflammatory cytokines in hypertrophic synovia are associated with nociception in OA of the hip. TRIAL REGISTRATION: University Hospital Medical Information Network, www.umin.ac.jp, UMIN000001335.

Increased density of sympathetic nerve fibers in metabolically activated fat tissue surrounding human synovium and mouse lymph nodes in arthritis.

OBJECTIVE: To investigate the density of sympathetic nerve fibers in and the metabolic activation of fat tissue surrounding human synovium in rheumatoid arthritis (RA)/osteoarthritis (OA) and in the draining lymph nodes of arthritic and normal mice. METHODS: Using immunofluorescence and immunohistochemistry, the density of sympathetic nerve fibers and the presence of nerve repellent factors were investigated. The metabolic activation of fat tissue was estimated by the occurrence of small-vacuole adipocytes, expression of ß3-adrenoceptors, and adipose tissue weight. RESULTS: The density of sympathetic nerve fibers was markedly increased in fat tissue surrounding RA synovium compared with that in fat tissue surrounding OA synovium. In adipose tissue adjacent to draining lymph nodes, the density of sympathetic nerve fibers was higher in arthritic mice compared with normal mice. In human synovium and mouse draining lymph nodes, the 2 sympathetic nerve repellent factors, semaphorin 3C and semaphorin 3F, were highly expressed. In arthritic compared with normal mice, the fat tissue around lymph nodes was markedly lighter, adipocytes had more fragmented lipid droplets, and fat tissue demonstrated high expression of ß3-adrenoceptors. CONCLUSION: This study demonstrated an increased density of sympathetic nerve fibers in metabolically activated fat tissue surrounding human RA synovium and the draining lymph nodes of arthritic mice. Because sympathetic neurotransmitters stimulate lipolysis, the repulsion of sympathetic nerve fibers from inflamed regions and their increased occurrence in fat tissue probably represent an adaptive program to support the proinflammatory process by releasing energy-rich substrates.

Catecholamine-producing cells in the synovial tissue during arthritis: modulation of sympathetic neurotransmitters as new therapeutic target.

BACKGROUND: The proinflammatory and anti-inflammatory role of the sympathetic nervous system in early and late inflammation is an unresolved paradox. A drastic loss of sympathetic nerve fibres in the synovial tissue of patients with rheumatoid arthritis (RA) has previously been demonstrated. The presence of tyrosine hydroxylase (TH)-positive cells in RA and osteoarthritis (OA) has been determined, but the role of these cells in inflammation is still unclear. OBJECTIVE: To characterise TH-positive cells in inflamed RA and OA synovial tissue and to study their role in inflammation. METHODS: Synovial samples were obtained from 32 patients with OA and 19 patients with RA and from 10 control patients. Synovial tissue samples were used for immunofluorescence staining. Synovial cells were isolated by tissue digestion and immediately used for cell culture. For in vivo experiments, collagen type-II arthritis in DBA/1J mice was induced. RESULTS: TH+ cells were present only in inflamed tissue and not in controls. Catecholamine-storing vesicles and vesicular monoamine transporter 2 (VMAT2) were identified in the synovial tissue. Experimental increase of cytoplasmic catecholamines by VMAT2 blockade strongly reduced tumour necrosis factor (TNF) independently of canonical extracellular ß-adrenergic signalling. In addition, VMAT2 blockade increased cyclic AMP (cAMP) and cAMP responsive element binding protein, responsible for TNF inhibition. In vivo, appearance of VMAT2 positive cells was confirmed. VMAT2 blockade ameliorated inflammation also in vivo. CONCLUSIONS: This study demonstrates that local catecholamine-producing cells start to replace sympathetic nerve fibres around the onset of disease, and modulation of locally produced catecholamines has strong anti-inflammatory effects in vivo and in vitro.

Galantamine in rheumatoid arthritis: A cross talk of parasympathetic and sympathetic system regulates synovium-derived microRNAs and related pathogenic pathways.

The acetylcholinesterase inhibitor, galantamine, has shown therapeutic effect in rat model of rheumatoid arthritis. Hence, the current study aims at determining the mode of action of galantamine by examining different synovium-derived microRNAs (miRs) and their related pathogenic pathways. The study also focuses on how parasympathetic and sympathetic pathways in the synovial tissue could affect the mode of action and anti-arthritic effect of galantamine. Chemical sympathectomy was initiated in 12 adjuvant arthritic rats by exposure to 6-hydroxydopamine (6-OHDA; 2 × 50 mg/kg) on day 9 after adjuvant injection and again (2 × 100 mg/kg) one week later. Six rats were treated with galantamine (2.5 mg/kg/day) to explore the influence of sympathetic impairment on galantamine effect. Another twelve additional adjuvant arthritic rats were exposed to the selective α7 nicotinic acetylcholine receptor blocker methylcaconitine citrate (MLA; 5.6 mg/kg/day), 15 min before galantamine treatment. As control, six adjuvant arthritic rats were treated with galantamine alone. Treatment proceeded for 5 days, from day 14 till day 18 post-adjuvant injection. Different miRs and their related pathogenic pathways were examined. Tyrosine hydroxylase (TH) expression was also measured in joint tissue. Galantamine affected the expression of the different miRs and their related parameters. Both, 6-OHDA and MLA, interrupted the anti-inflammatory/anti-arthritic effect of galantamine to different extent. Additionally, TH expression in the synovium was affected by galantamine, suggesting a novel pathogenic target in the treatment of rheumatoid arthritis.

The pattern of distribution of PGP 9.5 and TNF-alpha immunoreactive sensory nerve fibers in the labrum and synovium of the human hip joint.

To date, there has been no report clarifying the existence of sensory nerve fibers as the origin of the hip joint pain of osteoarthritis. We examined the existence of sensory nerve fibers in osteoarthritis (OA), osteonecrosis of the femoral head (ONFH), and femoral neck fracture of the human hip joint. Ten labra of 10 human hip joints were harvested during a total hip arthroplasty. Each labrum was separated into 12 sections and we used three sections for analysis, which included 2 weight-bearing and 1 non-weight-bearing portion. Protein gene product 9.5 (PGP 9.5) immunoreactive sensory nerve fibers were found in the labrum and synovium harvested from the weight-bearing portion in the OA group. Some of these sensory nerve fibers were also positive for tumour necrosis factor alpha (TNF). The PGP 9.5 immunoreactive sensory nerve fibers existed in the labrum tissue and inflammatory TNF positive cells were observed in the hyperplastic synovium. On the other hand, we could not demonstrate PGP 9.5 or TNF immunoreactive sensory nerve fibers and cells in any of the ONFH group or the non-weight-bearing portion in the OA group. These data suggest that the pain of ONFH and OA of the hip joint have different pathogenetic mechanisms and that the invasion of sensory nerve fibers containing TNF may be involved in the pathogenesis of pain in the human hip joint affected by OA.

Intraneural ganglia: a clinical problem deserving a mechanistic explanation and model.

Intraneural ganglion cysts have been considered a curiosity for 2 centuries. Based on a unifying articular (synovial) theory, recent evidence has provided a logical explanation for their formation and propagation. The fundamental principle is that of a joint origin and a capsular defect through which synovial fluid escapes following the articular branch, typically into the parent nerve. A stereotypical, reproducible appearance has been characterized that suggests a shared pathogenesis. In the present report the authors will provide a mechanistic explanation that can then be mathematically tested using a preliminary model created by finite element analysis.

Adherence of intraneural ganglia of the upper extremity to the principles of the unifying articular (synovial) theory.

OBJECT: Intraneural ganglia are nonneoplastic mucinous cysts contained within the epineurium of peripheral nerves. Their pathogenesis has been controversial. Historically, the majority of authors have favored de novo formation (degenerative theory). Because of their rarity, intraneural ganglia affecting the upper limb have been misunderstood. This study was designed to critically analyze the literature and to test the hypothesis that intraneural ganglia of the upper limb act analogously to those in the lower limb, being derived from an articular source (synovial theory). METHODS: Two patients with digital intraneural cysts were included in the study. An extensive literature review of intraneural ganglia of the upper limb was undertaken to provide the historical basis for the study. RESULTS: In both cases, the digital intraneural ganglia were demonstrated to have joint connections; the one patient in whom an articular branch was not appreciated initially had evidence on postoperative MR images of persistence of intraneural cyst after simple decompression was performed. Eighty-six cases of intraneural lesions were identified in varied locations of the upper limb: the most common sites were the ulnar nerve at the elbow and wrist, occurring 38 and 22 times, respectively. Joint connections were present in only 20% of the cases published by other groups. CONCLUSIONS: The authors believe that the fundamental principles of the unifying articular (synovial) theory (that is, articular branch connections, cyst fluid following a path of least resistance, and the role of pressure fluxes) previously described to explain intraneural ganglia in the lower limb apply to those cases in the upper limb. In their opinion, the joint connection is often not identified because of the cysts' rarity, radiologists' and surgeons' inexperience, and the difficulty visualizing and demonstrating it because of the small size of the cysts. Furthermore, they believe that recurrence (subclinical or clinical) is not only underreported but also predictable after simple decompression that fails to address the articular branch. In contrast, intraneural recurrence can be eliminated with disconnection of the articular branch.

Lower density of synovial nerve fibres positive for calcitonin gene-related peptide relative to substance P in rheumatoid arthritis but not in osteoarthritis.

OBJECTIVES: Sensory nerve fibres (NFs) contain two major neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP). The pro-inflammatory role of SP is known, while CGRP has anti-inflammatory activities by inhibiting T helper type 1 cytokines, TNF secretion and leucocyte proliferation. We demonstrated the increase of SP-positive NFs in RA as compared with OA. This study investigated the density of CGRP-positive NFs relative to SP-positive NFs or sympathetic NFs in synovial tissue of patients with RA and OA. METHODS: By immunofluorescent staining of synovial tissue of 25 patients with RA and 35 patients with OA, NFs positive for CGRP, SP and tyrosine hydroxylase (sympathetic NFs) were quantified. RESULTS: Density of CGRP-positive NFs was higher in OA than in RA, and density of SP-positive NFs tended to be higher in RA. In RA patients, comparison of CGRP-positive and SP-positive NFs in the same synovial tissue demonstrated less CGRP-positive than SP-positive NFs. The ratio of CGRP-positive NFs to SP-positive NFs was lower in RA as compared with OA. In OA, but not in RA, density of CGRP-positive NFs positively correlated with density of sympathetic NFs, which is much lower in RA patients. CONCLUSION: The preponderance of SP-positive NFs over CGRP-positive NFs or sympathetic NFs most probably supports the pro-inflammatory process in patients with RA. The reasons for the loss of CGRP in sensory NFs are not known.

Localization of SP- and CGRP-immunopositive nerve fibers in the hip joint of patients with painful osteoarthritis and of patients with painless failed total hip arthroplasties.

Using immunohistochemical methods we determined the presence of SP- and CGRP-immunopositive nerve fibers in the hip joint of patients with femoral neck fracture (controls, group 1), painful osteoarthritis (group 2), and painless failed total hip arthroplasties (group 3). Immunoreactive nerve fibers were found in the soft tissue of the fossa acetabuli as well as in the subintimal part of the synovial layer in the hip joint capsule of groups 1 and 2. In the capsule of controls the innervation density had a median of 5.7fibers/cm(2) for CGRP-ir and 3.2fibers/cm(2) for SP-ir afferents. In the osteoarthritic group, the density significantly increased to a median of 15.6fibers/cm(2) for CGRP-ir and 8.2fibers/cm(2) for SP-ir neurons (p=0.05). Patients with failed hip arthroplasties completely lacked these neuropeptide containing afferents. Innervation density in the fossa acetabuli of osteoarthritc patients showed a median of 14.1fibers/cm(2) for CGRP-ir and 5.9fibers/cm(2) for SP-ir afferents. From these data we assume that the hip joint capsule and the soft tissue of the fossa acetabuli are important triggers of nociception. This is supported by the fact, that patients with loosened total hip arthroplasties, where we failed to detect SP- and CGRP-immunoreactive fibers, did not feel pain. The upregulation of SP- and CGRP-positive neurons in response to arthritic stages suggests a mechanism involving neuropeptides in the maintenance of a painful degenerative joint disease and in mediating noxious stimuli from the periphery. Furthermore, these findings help to explain clinical observations, such as effectiveness of local therapy to control hip pain with intraarticular injection, synovectomy and denervation procedures.

Predominance of synovial sensory nerve fibers in arthrofibrosis following total knee arthroplasty compared to osteoarthritis of the knee.

BACKGROUND: So far, there exists no golden standard for the treatment of arthrofibrosis (AF) following total knee arthroplasty (TKA). Although pain is a hallmark of AF, nociceptive nerve fibers have never been investigated in affected joint tissue. METHODS: A total of 24 patients with osteoarthritis (OA) of the knee (n = 12) and post-TKA AF of the knee (n = 12) were included. Along evaluation of typical clinical signs and symptoms by using the Knee Society Clinical Rating System (KSS), the Knee Injury and Osteoarthritis Outcome Score (KOOS), and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC index), the innervation of joint tissue was studied by semiquantitative immunofluorescence of nerve fibers. RESULTS: Patients with AF compared to OA had a lower KSS and lower KOOS. In all compartments (anterior, medial, and lateral recesses), the density of synovial sympathetic nerve fibers was significantly higher in OA compared to AF, which was also true for the density of sensory nerve fibers in the medial and lateral recesses. In synovial tissue of the anterior recess of patients with AF compared to OA, the density of nociceptive sensory nerve fibers was significantly higher relative to sympathetic nerve fibers. This was similarly observed in the neighboring infrapatellar fat pad of the knee. CONCLUSIONS: Similar as in many painful musculoskeletal diseases, this study indicates that patients with arthrofibrosis of the knee after TKA demonstrate a preponderance of profibrotic sensory nerve fibers over antifibrotic sympathetic nerve fibers. This could serve as a starting point for AF therapy with specific antifibrotic pain medication or regional anesthetic techniques.

Anti-inflammatory cooperativity of corticosteroids and norepinephrine in rheumatoid arthritis synovial tissue in vivo and in vitro.

Corticosteroids (CS) and norepinephrine (NE) support each other's biological effects. Thus, deficiency of cortisol and reduced synovial sympathetic innervation (SSI) may be proinflammatory in rheumatoid arthritis (RA). This study tested the anti-inflammatory cooperativity of CS and NE in human RA synovial tissue. In an in vivo study, 32 patients with RA (with prior CS therapy/without SSI: n=7; without prior CS therapy/with SSI: 6; with prior CS therapy/with SSI: 19) were investigated for synovial inflammation. In an in vitro study with synoviocytes from RA and OA patients, the separate and combined effects of cortisol and NE were studied. In the in vivo study, patients with prior CS therapy/with SSI showed lower secretion of synovial IL-8 than the other groups, lower synovial density of T cells and macrophages, and lower overall inflammation. In the in vitro study, a cooperative suppressive effect of NE (10(-6) M to 10(-8) M) and cortisol (10(-6) M and 10(-7) M) on secretion of IL-8 and TNF from primary early culture mixed RA synoviocytes was observed. This cooperative effect was not observed in OA synoviocytes. In the same RA and OA patients, the cooperative effect was lost in 3rd passage synovial fibroblasts. This study demonstrates the cooperativity of cortisol and NE for inhibition of proinflammatory mediators produced in the synovial tissue of RA patients. These results underscore that coupling of an efficient secretion of systemic cortisol together with local production of NE is important in order to lower synovial inflammation.

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.

Tachykinin regulation of basal synovial blood flow.

1. Experiments were performed to investigate the role of endogenously released tachykinins in the regulation of blood flow to the rat knee joint. Synovial perfusion was assessed by laser Doppler perfusion imaging, which permitted spatial measurement of relative changes in perfusion from control (pre drug administration), expressed as the percentage change. Most experiments were performed on the exposed medial aspect of the knee joint capsule. 2. Neither the selective tachykinin NK1 receptor antagonist, FK888, nor the selective tachykinin NK2 receptor antagonist, SR48968, significantly influenced synovial blood flow at doses of 10(-12), 10(-10) and 10(-8) mol. However, topical co-administration of these agents produced significant dose-dependent reductions in basal synovial perfusion of 6.3 +/- 4.6 and 12.0 +/- 3.4 and 19.9 +/- 2.6%, respectively; n = 29. The non-selective tachykinin NK1/NK2 receptor antagonist, FK224, also produced significant (at 10(-10) and 10(-8) mol), but less potent, reductions in perfusion of 5.3 +/- 4.0, 8.4 +/- 2.2 and 5.9 +/- 2.8%, respectively; n = 25. 3. Topical administration of the alpha 1-, alpha 2-adrenoceptor antagonist phenoxybenzamine elicited a 31.3 +/- 6.2% increase in blood flow which was substantially reduced to 10.4 +/- 3.8% by co-administration of the FK888 and SR48968 (both at 10(-8) mol; n = 8-13), suggesting that normally there is sympathetic vasoconstrictor "tone' which is opposed by the vasodilator action of endogenous tachykinins. 4. One week after surgical interruption of the nerve supply to the knee joint, co-administration of FK888 and SR48968 (both at 10(-8) mol) now produced slight vasodilatation (6.7 +/- 4.6%; n = 9) which did not differ significantly from vehicle treatment. Depletion of tachykinins from sensory nerve fibres by systemic capsaicin administration also resulted in abolition of the vasoconstrictor effect of FK888 and SR48968 (both at 10(-8) mol), with these agents only producing a slight vasodilatation (2.5 +/- 5.3%; n = 6). 5. By use of a near infra-red laser source it was possible to image knee joint perfusion transcutaneously, the overlying skin being left intact. In this more physiological situation, close intra-arterial injection of the combination of FK888 and SR48968 (both at 10(-8) mol) again elicited vasoconstriction (48.8 +/- 16.2% reduction in blood flow; n = 4). 6. These results indicate that endogenous tachykinins may be continuously released from sensory fibers innervating the joint. Basal release of tachykinins could therefore be an important physiological influence opposing sympathetic vasoconstrictor tone.

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.

The distribution of peptide-containing nerves in the synovia of the cat knee joint.

Synovial capsule in cats is extensively innervated by a network with axonal diameter ranging from 0.6-3 microns according to its position and neuropeptide content. Nerve markers such as Neuron Specific Enolase (NSE) and Neurofilament triplet protein (NF) could be observed only when the axonal fibre attained a critical diameter of over the 3 microns limit. The relatively thick fibres (1-3 microns) show positive immunoreactivity for Substance P (SP), 5-hydroxytryptamine (5-HT), and Vasoactive Intestinal Peptide (VIP), and seldom coreact with NSE and NF, whereas, the thinnest fibres (0.6-0.8 microns) characterized to contain either Methionine or Leucine Enkephalin (M-Enk, L-Enk) did not coreact positively with axonal markers. We found that different anesthetics may effect variably the immunoreactivity of some neuropeptides (SP, L-Enk, 5-HT) while others (VIP, M-Enk) remained unaffected. Based on our data and the few reported ones in the pertinent literature, it is judged that urethane is the anesthetic of choice in experimental studies of neuropeptides. Our findings of isolated positive immunoreactive cell bodies to enkephalin in synovia might suggest the presence of intrinsic relay system, where the enkephalin acts as suppressor of SP and VIP release from the sunovium nerve terminals. Such a local inter-relationship between different neuropeptide systems might have a practical role on the understanding of the pathogenesis of different arthritic processes as well as therapeutic strategy in the future.