Frequency dependent changes in mechanosensitivity of rat knee joint afferents after antidromic saphenous nerve stimulation.

The aim of the present study was to examine the effect of electrical saphenous nerve stimulation (14 V, 1-10 Hz) on the mechanosensitivity of rat knee joint afferents. The responses to passive joint rotations at defined torque were recorded from slowly conducting knee joint afferent nerve fibres (0.6-20.0 m/s). After repeated nerve stimulation with 1 Hz, the mechanosensitivity of about 79% of the units was significantly affected. The effects were most prominent at a torque close to the mechanical threshold. In about 46% of the examined nerve fibres a significant increase was obtained, whereas about 33% reduced their mechanosensitivity. The sensitisation was prevented by an application of 5 microM phentolamine, an alpha-adrenergic receptor blocker, together with a neuropeptide Y receptor blocker. An inhibition of N-type Ca(2+) channels by an application of 1 microM omega-conotoxin GVIA caused comparable changes of the mechanosensitivity during the electrical stimulation. Electrical nerve stimulation with higher frequencies resulted in a further reduction of the mean response to joint rotations. After stimulation with 10 Hz, there was a nearly complete loss of mechanosensitivity.In conclusion, antidromic electrical nerve stimulation leads to a frequency dependent transient decrease of the mechanosensitivity. A sensitisation was only obtained at 1 Hz, but this effect may be based on the influence of sympathetic nerve fibres.

Three-dimensional reconstruction of scleral cold thermoreceptors of the cat eye.

Sensory endings that respond to local cooling were identified electrophysiologically in the cat's sclera. Functionally identified scleral thermal fibers were then used to analyze the structural characteristics of cold receptor endings. Four Adelta units sensitive to controlled cooling of their scleral receptive fields were recorded. The receptive areas were mapped, demarcated with pins and examined electron microscopically using extensive three-dimensional reconstructions. The supporting tissue within the receptive areas of cold units consisted of dense collageneous tissue with a small number of blood vessels that were either veins or capillaries. Adelta nerve fibers were found within these tissue blocks presumably corresponding with cold sensitive fibers. Small nerves and single nerve fibers devoid of a perineurium were found in all parts of the tissue, only occasionally passing a blood vessel. The terminal portions showed axonal swellings all along the unmyelinated segment filled with mitochondria, glycogen particles, and some vesicles. About 30% of the terminal axonal membrane is not covered by Schwann cells. In the unmyelinated distal portion, the mitochondrial content ranged from 0.012 to 0.038 microm(3) mitochondrial volume per microm(2) nerve fiber membrane. In comparison with sensory endings in the cat's knee joint, cold receptors in the cat sclera showed many similarities in their three-dimensional structure with polymodal nociceptor endings of the knee joint but contain less mitochondria. This suggests that cold sensory endings do not require specialized cellular processes for the transduction of cold stimuli, as is the case for multimodal transduction and sensitization in the terminal portion of polymodal nociceptors.

Histological demonstration of voltage dependent calcium channels on calcitonin gene-related peptide-immunoreactive nerve fibres in the mouse knee joint.

The afferent (excitability) and efferent functions (release of neuropeptides) of primary afferent nerve fibres are based on Ca(2+)-influx. The aim of the present study was to examine the presence of L- and N-type Ca(2+)-channels at sensory nerve fibres in the mouse knee joint capsule. Specific fluorescent labelled channel blockers and antisera against these channel subtypes were combined with an immunohistochemical staining for calcitonin gene-related peptide (CGRP), a neuropeptide that is widely distributed in primary afferents. There was a nearly complete colocalisation of CGRP immunoreactivity and the binding of omega-conotoxin GVIA (toxin VIA of Conus geographus or BODIPY-verapamil (BODIPY(R) FL verapamil, hydrochloride) demonstrating the presence of N-type and L-type Ca(2+)-channels, respectively. These data were further confirmed by identical results obtained after an immunohistochemical demonstration of the two channel subtypes at the peptidergic nerve fibres.

The neurokinin-1 receptor antagonist RP 67580 reduces the sensitization of primary afferents by substance P in the rat.

The inflammatory mediator substance P (SP) produces a variety of biological effects in several tissues by binding to the tachykinin receptor neurokinin 1 (NK1) and, to a lesser extent, by binding to the neurokinin 2 receptor (NK2). To assess the sensitizing effect of SP on articular afferent fibres the NK1receptor antagonist RP 67580 was applied in normal and acutely inflamed rat knee joints. Altogether 38 fine afferent nerve fibres from the rat knee with conduction velocities of 0.71-13.5 m/s were recorded as single units, during non-noxious and noxious joint rotations. SP, injected i.a. as a bolus close to the knee joint, was able to sensitize 45.5% (10 of 22) of the units recorded from normal joints and 33.3% (five of 15) of afferents from inflamed joints. The following i.a. application of RP 67580 in a range of 20-200 nmol antagonized in a dose-dependent manner the sensitizing effect of SP in a large proportion of slowly conducting articular afferents from normal (66.7%) and inflamed (46.2%) knee joints. Subsequent SP application enhanced the afferent sensitivity further. The electrophysiological results presented here further support the suggestion that the sensitization of afferents by SP in the rat knee joint is mediated mainly by the NK1 receptor, which is probably located on the primary afferents.

Composition of the medial and posterior articular nerves of the mouse knee joint.

The number and the distribution of fiber size in the medial (MAN) and posterior (PAN) articular nerves of the mouse knee joint were studied by electron microscopy. The MAN contained 75 +/- 28 nerve fibers consisting of 63 +/- 24 unmyelinated and 12 +/- 6 myelinated fibers. The PAN was composed of 195 +/- 50 nerve fibers, namely 129 +/- 28 unmyelinated and 66 +/- 24 myelinated fibers. A skewed unimodal distribution of the unmyelinated nerve fiber diameters was seen in both nerves ranging from 0.1 to 1.2 microm with a maximum between 0.3 and 0.6 microm. The myelinated nerve fibers in the MAN ranged from 1 to 8 microm with a peak between 2 and 5 microm. In the PAN, their diameters ranged from 1 to 12 microm with a clearly visible peak at 4-5 microm and a plateau at 8-9 microm that may represent a second maximum. These data show that the knee joint innervation of the mouse is comparable to those of the cat and rat concerning the types of nerve fibers and the composition of the two nerves. However, in relation to the much smaller area of tissue to be innervated the total number of primary afferents is considerable smaller in the mouse.

Neuropeptide Y changes the excitability of fine afferent units in the rat knee joint.

1. The aim of the present study was to examine the effects of the sympathetic co-transmitter Neuropeptide Y on primary afferent nerve fibres of the rat knee joint. The responses to passive joint rotations at defined torque were recorded from 41 slowly conducting afferent nerve fibres (0.9 - 18.8 m s(-1)) innervating the knee joint capsule. 2. About 70% of the joint afferents were significantly affected in their mechanosensitivity by topical application of Neuropeptide Y. Significant effects occurred at a concentration of 10 nM. 3. Decreased mechanosensitivity was observed in about 40% of nerve fibres, whereas 30% of the units increased the mechanosensitivity. In addition, in about 35% of the fibres resting activity was induced or increased. Neither the conduction velocity nor the mechanical threshold of the units correlated with the described effects of Neuropeptide Y. 4. NPY(13--36), a specific Y2-receptor agonist, only modulated the mechanosensitivity, with no effect on the resting activity. The effects on the mechanosensitivity were similar to Neuropeptide Y, i.e. increase and decrease of the response. 5. Studies with the Y1-agonist (Leu(31), Pro(34))-NPY showed that activation of the Y1-receptor predominantly resulted in an enhanced mechanosensitivity and an induction or increase of a resting activity. The opposite effect was observed by application of BIBP 3226 BS, a Y1-receptor antagonist. 6. In conclusion, these data indicate that Neuropeptide Y affects the excitability of sensory nerve fibre endings.

Cortical projection of the rat knee joint innervation and its processing in the somatosensory areas SI and SII.

In recent years the rat knee joint has become an important model for the study of nociception of deep tissues. In contrast to the cortical processing of superficial pain, the knowledge about the processing of deep pain evoked by noxious stimuli in tissues such as tendons, bone, and joint is sparse. To obtain a basis for further functional studies, the projections of the knee joint in the cerebral cortex were determined. Cortical surface potentials evoked by electrical stimulation of the posterior articular nerve were recorded by a platinum ball electrode. Evoked activity was found in the primary somatosensory area SI in an area of about 3 x 3 mm on the contralateral side. Its center was located about 3 mm caudal to the bregma and about 3 mm lateral to the superior sagittal sinus. A small projection in SII was found on the lateral side of the cortex about 6 mm lateral from SI. This area had a size of about 1 x 1 mm, and the amplitudes of the potentials were smaller but had similar latencies to those in SI. An additional projection with small potentials and longer latencies was observed in SI on the ipsilateral side. Cooling of the contralateral SI revealed deprivation of the ipsilateral evoked potentials in SI whereas the potentials in SII remained unchanged. These data indicate that information from the knee joint is processed in parallel in SI and SII on the contralateral side and that there is an additional serial processing in SI on the ipsilateral side.

Responses of fine primary afferent nerve fibres innervating the rat knee joint to defined torque.

In total 23 rat knee joint afferent units with conduction velocities of 1.2-17 m/s were recorded extracellularly during inward and outward joint rotations of defined torque using a newly developed torque-meter. There was not a linear relation between the torque and the obtained angle of the joint. The mechanical thresholds of the units ranged from 10 to 60 mNm. During 10 s of stimulation with 60 mNm they responded with 12-300 action potentials. Two mechanosensitive nerve fibres could not be activated via knee joint rotation at any torque at all. The units could be classified according to their response pattern during stimulation: (1) phasic-tonic response behaviour; (2) constant discharge rate; (3) delayed onset of activity. In conclusion, the novel torque-meter allows a precise characterisation of the mechanical threshold and the corresponding response of fine afferent joint units during knee joint rotation.

Galanin influences the mechanosensitivity of sensory endings in the rat knee joint.

The aim of the present study was to examine the effect of galanin on group III and IV afferent nerve fibres (n = 53) innervating normal and acutely inflamed knee joints in rats. They responded to local mechanical stimulation, movements of the joint and i.a. injections of KCl close to the joint. Single i.a. bolus injections of galanin (0.1 mM, 0.2 mL) caused no direct responses of the units. In normal and acutely inflamed joints, about half of the units did not change the responses to knee joint rotation. A significant reduction of the responses to noxious movements was found in approximately 40% of the units reaching a mean value of 57% in normal joints and 70% in inflamed joints compared with control movements. In approximately 10% the responses increased to 143% in normal joints and 120% in inflamed joints. Injection of a galanin receptor antagonist (M35) doubled the responses to noxious movements in 36% of the units in normal joints and reduced it in 18% to 86% of the control movements, indicating a tonic release and influence on the mechanosensitivity of a proportion of primary afferents by galanin. In conclusion, these data further support the hypothesis that the mechanosensitivity of fine afferent nerve fibres is regulated by a mixture of different substances being released into the innervated tissue. Besides the action of several pro-inflammatory peptides there seems to exist a tonic inhibitory system.

Peripheral application of cyclo-somatostatin, a somatostatin antagonist, increases the mechanosensitivity of rat knee joint afferents.

Recently it has been shown that neuropeptides may be involved in the regulation of the mechanosensitivity of fine afferent nerve fibres. Somatostatin seems to provide a tonic inhibition of the sensitivity of sensory endings in normal tissue. In the present study cyclo-somatostatin, a somatostatin antagonist, was used to test this hypothesis. In a normal joint, 50% (8/16) of the examined primary afferents significantly increased their responses to noxious joint movements after the cyclo-somatostatin application. In an acutely inflamed joint, however, only 9% (1/11) of the nerve fibres significantly increased the response to noxious movements. These data further support the hypothesis that the mechanosensitivity of articular afferents in a normal joint is also regulated by a balance of pro-inflammatory peptides and anti-inflammatory peptides such as somatostatin. In an inflamed joint, pro-inflammatory peptides predominate and together with other mediators, they may be involved in the sensitization of the peripheral nerve fibres.

Distribution of somatostatin receptor subtypes in rat lumbar spinal cord examined with gold-labelled somatostatin and anti-receptor antibodies.

Using gold-labelled somatostatin, somatostatin binding sites were predominantly found in laminae I-III, X and on motorneurones of the rat lumbar spinal cord. A comparison with immunohistochemical staining using antisera against somatostatin receptor sequences revealed that the marked binding in laminae I-III coincided with the presence of somatostatin receptor-like immunoreactivity for the receptor subtypes 1, 2 and 3. Binding sites on motorneurones were only paralleled by an immunoreaction for subtype 3. In lamina X, however, the lack of a positive immunoreaction indicates that in this part other subtypes may be present.

Labelling of peptides with 1.4-nm gold particles to demonstrate their binding sites in the rat spinal cord.

Recently we presented a method to label the neuropeptide substance P with a 1.4-nm gold particle covalently bound at the N-terminus that can be used for demonstrating its binding sites in histological sections. In this study we examined whether the peptides neuropeptide Y, somatostatin, calcitonin gene-related peptide and bradykinin can be labelled in the same way. Polyacrylamide gel electrophoresis revealed a reduction in mobility for peptide-gold conjugates over gold particles alone consistent with peptide binding. In cryostat sections of the rat lumbar spinal cord, the peptides showed a distinct binding pattern in the grey matter corresponding to data of studies using autoradiographic methods. Therefore, we conclude that this simple and fast method can be used for labelling peptides in general to demonstrate their binding sites in histological sections, provided the peptide binds by its C-terminus.

[Histopathological findings in the proprioception of the shoulder joint]. / Histomorphologische Befunde zur Propriozeption am Schultergelenk.

Purpose of this study was the evaluation of distribution and morphology of mechanoreceptors in the glenohumeral joint capsule and rotator cuff in comparison to the coracoacromial ligament by means of specific immunfluorescence microscopy. The complemente joint capsules, rotator cuffs and coracoacromial ligaments of three fresh cadaver shoulder were harvesed. Serial cryostate sections were taken and alternately incubated with antiserum against neurofilament, lamin or myelin of peripheral nerves. The antibody-reaction was visualized with fluorescin lg-G. The nerve endings were photographed and computer-aided 3-dimensional reconstructions were performed. Three types of corpuscular and free nerve endings of different morphology were found in different distributions: whereas the Ruffini corpuscles were much more frequent in the coracoacromial ligament and rotator cuff, Pacini endings were predominantly found in the joint capsule. Generally corpuscular nerve endings were more frequent in the coracoacromial ligament and the rotator cuff than in the antero-inferior capsule and the number of corpuscles increased from medial to lateral within the anterior and inferior parts of the capsule. The dense ligamentous tissue was almost aneural whereas the periarticular fatty or loose connective tissue contained nerve fibres and nerve endings. In view of the results of other experimental and clinical studies the high frequency of Ruffini and Pacini endings in the rotator cuff and coracoacromial ligament suggest, that both are involved in the neurosensory control of glenohumeral stability and subacromial impingement. In contrast our findings in the joint capsule do not clearly prove, that those joint receptors predominantly maintain joint stability.

Plasticity in the expression of bradykinin binding sites in sensory neurons after mechanical nerve injury.

The pro-inflammatory mediator bradykinin plays an important role in hyperalgesia during inflammatory conditions. Here, we used unilateral ligation of the sciatic nerve to investigate whether the expression of bradykinin binding sites in isolated rat dorsal root ganglion neurons is changed following nerve injury. Under control conditions, the percentage of neurons expressing bradykinin binding sites increased from 52% at day 0.8 in culture to 93% at day 1.8 and decreased to 67% at day 3.8. Following nerve ligation either two or 10 days prior to the isolation of the somata, the percentage of neurons from ipsilateral ganglia that expressed bradykinin binding sites was already 87% and 86%, respectively, at day 0.8 in culture; this level was maintained at day 1.8 and decreased slightly at day 3.8. In control neurons, high densities of bradykinin binding sites on individual neurons were observed no sooner than at day 1.8, but already at day 0.8 following nerve ligation, due to a "de novo" expression of B1 receptors and augmentation of B2 receptors. Neurons from the contralateral side responded similarly to ipsilateral neurons after a two day nerve ligation, however, after either a 10 day ligation or a sham operation neurons responded similarly to control neurons. These data are the first evidence that expression of B1 receptors is induced and expression of B2 receptors is enhanced in sensory neurons following nerve ligation. Under pathophysiological conditions, increased expression of subtypes of bradykinin receptors in sensory neurons could contribute to chronic pain conditions.

Nerve growth factor regulates the expression of bradykinin binding sites on adult sensory neurons via the neurotrophin receptor p75.

Neurotrophins mediate specific effects on sensory neurons through tyrosine kinase receptors. Most of these neurons also co-express the neurotrophin receptor p75 (p75NTR), but its function has remained obscure. We now show that nerve growth factor but not brain-derived neurotrophic factor or neurotrophin-3 selectively increases the expression of bradykinin binding sites on cultured dorsal root ganglion neurons from adult mouse via p75NTR. This up-regulation of bradykinin binding sites did not occur in neurons from mice lacking p75NTR or in neurons from wild-type mice treated with p75NTR-blocking antibody, indicating that tyrosine kinase receptors alone are not sufficient to trigger this physiological neuronal response. Thus, the interaction of nerve growth factor with p75NTR is an important factor contributing to chronic pain conditions.

Histomorphological findings on proprioception in the shoulder.

Purpose of this study was the evaluation of distribution and morphology of mechanoreceptors in the glenohumeral joint capsule and rotator cuff in comparison to the coracoacromial ligament by means of specific immunfluorescence microscopy. The complemente joint capsules, rotator cuffs and coracoacromial ligaments of three fresh cadaver shoulder were harvested. Serial cryostate sections were taken and alternately incubated with antiserum against neurofilament, lamin or myelin of peripheral nerves. The antibody-reaction was visualized with fluorescin Ig-G. The nerve endings were photographed and computer-aided 3-dimensional reconstructions were performed. Three types of corpuscular and free nerve endings of different morphology were found in different distributions: whereas the Ruffini corpuscles were much more frequent in the coracoacromial ligament and rotator cuff, Pacini endings were predominantly found in the joint capsule. Generally corpuscular nerve endings were more frequent in the coracoacromial ligament and the rotator cuff than in the antero-inferior capsule and the number of corpuscles increased from medial to lateral within the anterior and inferior parts of the capsule. The dense ligamentous tissue was almost aneural whereas the periarticular fatty or loose connective tissue contained nerve fibres and nerve endings. In view of the results of other experimental and clinical studies the high frequency of Ruffini and Pacini endings in the rotator cuff and coracoacromial ligament suggest, that both are involved in the neurosensory control of glenohumeral stability and subacromial impingement. In contrast our findings in the joint capsule do not clearly proove, that those joint receptors predominantly maintain joint stability.

Quantification of cat's articular afferents containing calcitonin gene-related peptide or substance P innervating normal and acutely inflamed knee joints.

In cats with an acute (32 h) unilateral knee joint inflammation the proportion of calcitonin gene-related peptide-(CGRP) and substance P-(SP) immunoreactive articular afferents, retrogradely labelled by Fast Blue (FB), were determined using immunohistochemistry. The proportion of neurons containing CGRP was significantly higher on the inflamed side (52%) than on the contralateral side (39%) and in controls (42%). However, the proportion of SP-immunoreactive articular perikarya on the inflamed side (26%) did not differ from the contralateral side (24%) and the control cats (22%). These data indicate that acute inflammation induces the synthesis of CGRP but not of SP in joint afferents.

Quantitative examination of calcitonin gene-related peptide immunoreactive nerve fibres in the cat knee joint capsule.

The knee joint of the cat has been used extensively to study the morphology and function of primary afferents in a deep somatic tissue. A proportion of these neurones synthesizes various neuropeptides, with calcitonin gene-related peptide being the most prominent. In the present study we examined the distribution and density of nerve fibers immunoreactive for calcitonin gene-related peptide within the medial articular capsule. The fibres were predominantly located in the superficial layer of the capsule. They formed a dense innervation pattern, mainly accompanying blood vessels. Electron microscopy showed that most fibres were in close proximity to small arteries. The highest innervation density was found in parts of the capsule that were located over the epicondyle of the femur with 21 +/- 12 fibres per mm2 (mean +/- SD). In the tissue over the joint cleft this density was lower, with 11 +/- 6 fibres per mm2. In conclusion, the high innervation density of the knee joint capsule by nerve fibres containing calcitonin gene-related peptide supports the hypothesis of an important regulatory function of this peptide in normal tissue.

Sensitisation of articular afferents in normal and inflamed knee joints by substance P in the rat.

To examine whether substance P (SP) influences the response properties of fine articular afferents in normal and acutely inflamed joints, single units were recorded from the rat knee during normal and noxious joint rotations. Only three of 39 units were activated by a single bolus injection of 0.1 mM SP. However, 35% (7/20) of the nerve fibres from the normal joint and 21% (4/19) of the units from the inflamed joint significantly increased their responses to movements after the SP injection. This was most prominent during noxious movements in normal joints, whereas in inflamed joints increase of responses occurred mainly during normal movements. These data indicate that SP may also be involved in the process of sensitisation of primary afferents during an inflammation.