Somatostatin-like immunoreactivity in primary afferents of the medial articular nerve and colocalization with substance P in the cat.

The proportion of somatostatin-containing dorsal root ganglion cells innervating the knee joint of the cat via the medial articular nerve was determined by using retrograde labeling with fast blue and immunohistochemistry. Immunoreactivity was found in 8.6% of labeled cell bodies. In colchicine-treated ganglia, the proportion increased to 16.8%. Only small and intermediate-sized perikarya showed somatostatin-like immunoreactivity, indicating that this neuropeptide is synthesized predominantly in primary afferent units with unmyelinated sensory axons but may also be present in primary afferents with thinly myelinated sensory fibers. Colchicine treatment had no influence on the cell size distribution. Colocalization of somatostatin with substance P was determined by comparing the proportions of immunopositive dorsal root ganglion cells after incubation with antibodies against substance P or somatostatin or with a mixture of both. Substance P-like immunoreactivity was found in 18.1% (untreated ganglia) and 19.6% (colchicine treated ganglia) of the labeled neurons. After incubation with a mixed antibody solution, 18.2% of joint afferents in untreated and 19.9% of the cells in colchicine-treated ganglia were immunopositive. Comparing this result with the results obtained using somatostatin and substance P antibodies alone, one can calculate that both neuropeptides are colocalized in about 17% of the cat's knee joint afferents. About 3% of the neurons contain only substance P, whereas almost none of the neurons contain only somatostatin. Based on this fact, one can assume that both neuropeptides are coreleased in peripheral tissue as well as in the central nervous system.

Substance P- and calcitonin gene-related peptide immunoreactivity in primary afferent neurons of the cat's knee joint.

The distribution of substance P and calcitonin gene-related peptide was determined in primary afferent neurons of the medial and posterior articular nerve of the cat's knee joint. Perikarya of articular afferents were visualized by retrograde labelling with the fluorescent dye Fast Blue which was applied at the transected end of the peripheral nerves. Substance P was found in about 17% of labelled medial articular afferents and in about 16% of labelled posterior articular afferents, respectively, whereas calcitonin gene-related peptide was present in about 35 and 32% of the medial and posterior articular nerve cells, respectively. Taking into account that these neuropeptides are known to be co-localized, probably not more than one-third of the joint afferents contain substance P and/or calcitonin gene-related peptide. Quantification of cell diameters revealed that substance P was found only in small- or intermediate-sized perikarya (less than 50 microns) indicating that this peptide is predominantly found in unmyelinated neurons. Calcitonin gene-related peptide was present mainly in small- and intermediate- but also in some large-sized neurons (greater than 50 microns) providing evidence that this peptide is found in unmyelinated and to a lesser extent in myelinated neurons. This is consistent with previous studies that show that substance P and calcitonin gene-related peptide are present primarily in unmyelinated and thinly myelinated primary afferents. When the portion of substance P-positive neurons of the medial articular nerve is compared to the number of articular afferents displaying a nociceptive function as determined in earlier electrophysiological studies, it can be calculated that at most 30% of the nociceptive-specific articular afferents contain this neuropeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple method for a specific retrograde labelling of dorsal root and sympathetic ganglion cells innervating the knee joint of the cat.

To examine the biochemical characteristics of primary afferents and sympathetic efferents innervating the normal and inflamed knee joint of the cat, the corresponding perikarya have to be labelled retrogradely. In the present study we used an injection of the fluorescent dye Fast Blue through the intact skin and the patellar ligament into the centre of the knee joint cavity and examined the efficiency of this kind of labelling. The segmental distributions of labelled cell profiles in cryostat sections of dorsal root ganglia (DRG) and paravertebral ganglia of the sympathetic trunk agreed with data obtained previously after applying the dye at the transected end of the two main articular nerves. The soma size distribution of labelled DRG cells was identical to previous results indicating that the perikarya labelled in the present study may be a representative proportion of the knee joint innervation. Axotomy of the medial and posterior articular nerves reduced the number of labelled cell profiles of about 85%. Taken together, these data indicate that an injection of Fast Blue into the knee joint cavity of the cat revealed a highly specific and efficient retrograde labelling of the neuronal cell bodies innervating the knee joint.

NADPH-diaphorase reactivity in articular afferents of a normal and inflamed knee joint in the cat.

The distribution of NADPH-diaphorase was studied in retrogradely labelled dorsal root ganglion cells innervating the knee joint of the cat. A strong staining reaction was found in 7.5 +/- 1.9% (mean +/- S.D. of 9 normal joints and 6393 labelled perikarya) of the articular afferents. An acute inflammation (32 h) significantly increased this proportion to 10.9 +/- 2.2% (mean +/- S.D. of 5 inflamed joints and 3933 labelled perikarya). The diameter distribution of the somata with a positive NADPH-diaphorase reaction ranged from 18 to 46 microns with a maximum at 24-28 microns. These data indicate that a small proportion of knee joint primary afferents may be able to release nitric oxide playing a role in synaptic transmission and in regulatory functions within the peripheral tissue under normal and pathophysiological conditions.

Neurokinin A-like immunoreactivity in articular afferents of the cat.

Dorsal root ganglion cells with axons innervating the cat's knee joint via the medial articular nerve were retrogradely labelled with Fast blue. Neurokinin A-like immunoreactivity was found in 4.5 +/- 1.1% (mean +/- S.D. of 5 nerves and 695 cells) of the articular afferents. Colchicine treatment of the ganglia increased the percentage of immunopositive cells to 8.5 +/- 0.7% (mean +/- S.D. of 6 nerves and 554 cells) after 3-22 h. The diameter distribution of the immunopositive somata ranged from 20 to 50 microns with a maximum at 26-30 microns. Comparing the proportions of neurokinin A-immunopositive cells with those of substance P, it can be calculated on the basis of mRNA encoding that neurokinin A is synthetized in about half of the substance P-containing primary articular afferents.

The effect of a unilateral inflammation at the rat's ankle joint on the expression of preprotachykinin-A mRNA and preprosomatostatin mRNA in dorsal root ganglion cells--a study using non-radioactive in situ hybridization.

In rats with an acute (2 days) and chronic (20 days) unilateral ankle joint inflammation (induced by Freund's complete adjuvant), the proportion of dorsal root ganglion cells containing preprotachykinin-A mRNA or preprosomatostatin mRNA was determined using non-radioactive in situ hybridization. At the acute stage of inflammation, the proportion of neurons containing preprotachykinin-A mRNA was similar to that in control rats. At the chronic stage, the proportion of neurons expressing preprotachykinin-A mRNA was significantly higher on the inflamed side than on the contralateral side. The proportion of dorsal root ganglion cells containing preprosomatostatin mRNA did not change. These data suggest that inflammation influences the synthesis of substance P but not of somatostatin in afferent neurons.

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.

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.

Calcitonin gene-related peptide- and substance P-like immunoreactive fibers in the spermatic nerve and testis of the dog.

In order to determine if calcitonin gene-related peptide (CGRP) and substance P (SP) coexist in peripheral spermatic nerve fibers, we carried out a double-staining immunofluorescence study using confocal microscopy and fluorescence microscopy. CGRP- and SP-like immunoreactivity (LI) coexisted in the spermatic nerve trunk and in the single fibers running along the surface of the testis. The great majority of the SP-containing fibers also held CGRP-LI, although some fibers contained CGRP-LI without SP-LI. These observations are consistent with previous observations on testicular dorsal root ganglion neurons. Additionally, we carried out an immunogold silver staining for CGRP and found CGRP-containing nerve bundles, single nerve fibers and their nerve terminals. Some CGRP-containing nerve terminals were located very superficially in the tunica albuginea (<5 microm from the surface).

Examination of colocalization of calcitonin gene-related peptide- and substance P-like immunoreactivity in the knee joint of the dog.

It is generally assumed that the majority of substance P (SP)-containing afferents are also immunoreactive for calcitonin gene-related peptide (CGRP). In order to determine whether this is also the case in articular afferents where the contents of these peptides are low, we carried out a double labeling study using Fast Blue (FB) as a retrograde tracer injected into the center of the knee joint cavity of the dog together with immunohistochemistry for SP and CGRP. After 7-36 days of survival, dorsal root ganglia (DRGs, L4-S1) were removed. Labeled cells were found mainly (94%) in L5 - 6 DRGs, and SP- and CGRP-like immunoreactivity was found in about 17 and 29% of FB-labeled cells, respectively. The coexistence of SP and CGRP was observed in 10.4% of articular afferents and only 62.7% of SP-positive articular neurons contained CGRP, a much lower ratio than in other afferents of the dog such as testicular afferents. Our data suggest that these peptides are not always released together and that they do not always work together in the joint under normal conditions.

Peripheral modulation of rat knee joint afferent mechanosensitivity by nociceptin/orphanin FQ.

The peripheral effects of nociceptin were examined in normal and acutely inflamed rat knee joints by analyzing single unit recordings from articular primary afferents in response to normal and extreme rotation of the knee. Bolus close intraarterial injection of nociceptin (0.01, 1 and 100 microM) caused a sensitization of normal and inflamed knee joint afferents in response to movements in the normal working range of the joint. When the joint was hyper-rotated, nociceptin had no significant effect on afferent discharge rate in normal knees, however, in inflamed joints the top dose of the neuropeptide caused a decrease in articular mechanosensitivity. These findings suggest that nociceptin seems to be involved in the control of peripheral nociceptive mechanisms, although the behaviour of the peptide is dependent upon the inflammatory status of the tissue.

Release of endogenous histamine in the hypothalamus of anaesthetized cats and conscious, freely moving rabbits.

The hypothalamus of anaesthetized cats and conscious, freely moving rabbits was superfused with CSF through double-walled, push-pull cannulae and the release of endogenous histamine was determined in the superfusates by a radioenzymatic assay. In the posterior hypothalamic area of the anaesthetized cat, the rate of release of endogenous histamine varied rhythmically; phases of high rate of release appeared at 60 min cycles. The release of histamine was increased by electrical stimulation of the superfused area, as well as by hypothalamic superfusion with potassium-rich CSF. In the conscious rabbit, the anterior hypothalamic area and the posterior hypothalamic nucleus were superfused simultaneously. In both regions, the resting release of histamine varied rhythmically at approximately 70 min cycles. Phases of high or low-rate of release in the anterior hypothalamic area coincided with the corresponding phases in the posterior hypothalamic nucleus. The rhythmic release of endogenous histamine in the hypothalamus, as well as the ability of depolarizing stimuli to enhance the release of the amine support the idea that histamine acts as a neurotransmitter in the central nervous system.

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.

Innervation of the dura mater encephali of cat and rat: ultrastructure and calcitonin gene-related peptide-like and substance P-like immunoreactivity.

Ultrastructural, immunocytochemical, and immunoelectron microscopical examinations are reported that describe the morphology of putative sensory nerve endings in the dura mater encephali of the rat and the cat. Morphometrical measurements and reconstructions showed that in the cat the mean diameter of axons, the bare area of axolemma, and the content of mitochondria and vesicles are highly variable in dural nerve endings. Nerve fibers with a high volume density of mitochondria are thought to be sensory, while nerve fibers containing many small vesicles are considered autonomic. There is, however, a broad overlap of mitochondria-rich and vesicle-rich nerve fibers in the dura, so that discrimination between sensory and autonomic endings by these characteristics frequently fails. Whole-mount preparations treated cytochemically for detection of substance P- and calcitonin gene-related peptide-like immunoreactivity in the rat and the cat showed a network of immunopositive nerve fibers in the vicinity of dural blood vessels. Most of these peptidergic and probably sensory nerve fibers were found terminating in the dural connective tissue far from vessels. Calcitonin gene-related peptide-positive nerve fibers were much more abundant than substance P-positive fibers. Immunoelectron microscopic preparations revealed that calcitonin gene-related peptide- and substance P-like immunoreactivity is found in a small proportion of generally thin unmyelinated nerve fibers. These proportions were very similar in the rat and the cat. Summarizing the recent literature, the morphological characteristics of putative sensory nerve fibers in the dura mater are discussed in relation to their possible functional significance for neurogenic inflammation and nociception.

[Localization of the neurokinin 1 receptor in hip joints of patients with painful osteoarthritis]. / Die Lokalisation des Neurokinin 1-Rezeptors im Hüftgelenk von Patienten mit schmerzhafter Osteoarthrose.

INTRODUCTION: Recent studies on osteoarthritis have focused on nociceptive substance P (SP) containing afferent nerve fibres. The effects of SP are known to be mainly mediated by the tachykinin receptor neurokinin 1 (NK1-R). AIM: The aim of the present study was to demonstrate the NK1-R in human joint tissues. METHODS: The hip joint capsule of three patients with painful hip osteoarthritis (Group 1), three patients with femoral neck fracture showing no cartilage destruction (Group 2, controls) and the soft tissue of the fossa acetabuli of Group 1 were resected during hip arthroplasty implantation. The tissue samples were cut into small blocks and immersion-fixed in Zamboni's fixative. The specimens were frozen, cut into 50 microm sections and immunostained using a standard immunohistochemical staining protocol. RESULTS: In Groups 1 and 2 the NK1-receptor was localised in the wall of venous vessels, on Schwann cells of nerve bundles and on nerve fibres. In the osteoarthritis group the staining pattern was similar but the number of NK1-bearing cell structures seemed to be enhanced. CONCLUSIONS: The present study provides the first evidence of NK1-R in the human hip joint. In patients with painful osteoarthritis the density of NK1-R-positive cell structures seemed to be increased. The localisation of the NK1 receptor on different cell types suggests multiple effects of SP in normal and osteoarthritic joints.

[Calcitonin gene-related peptide immunoreactive nerve fibres in the dura mater encephali of the rat: experiments related to the neurogenic inflammation of meningeal structures.]. / Calcitonin gene-related peptide immunreaktive Nervenfasern in der Dura mater encephali der Ratte : Experimente zur neurogenen Entzündung der Hirnhäute.

In this paper morphological and physiological experiments are described that refer to the concept of neurogenic inflammation of meningeal structures as a putative source of migrainous pain and other headaches. The main emphasis of this study carried out on the duramater encephali of the rat was the functional role of calcitonin generelated peptide (CGRP), a vasodilatory neuropeptide of fine afferent nerve fibres. Immunocytochemical preparations showed that the parietal dura mater was densely innervated by CGRP immunoreactive nerve fibres, the distribution and ultrastructure of which were examined by ligh and electron microscopy. The dense innervation around the medial meningeal artery suggested a vasomotor function of these peptidergic fibres. In further experiments the CGRP immune product of the nerve fibres could be diminished by electrical stimulation of the dura mater. Extracellular recordings from trigeminal ganglion cells showed that electrical and mechanical stimulation of large dural vessels activate trigeminal afferents. In a final series of experiments the dural blood flow around branches of the medial meningeal artery was measured with a laser Doppler flowmeter. The blood flow was increased by electrical stimulation of the dura, the size of this effect depending on stimulus strength and frequency. This increase was inhibited in a dose-dependent manner by the competitive CGRP antagonist CGRP(8-37), which shows an involvement of CGRP in the regulation of meningeal blood flow. We conclude that stimulation of trigeminal afferents innervating the dura mater releases CGRP from peptidergic afferent terminals, thereby causing vasodilatation and increasing the meningeal blood flow, an important component of neurogenic inflammation. The preparation decribed will be used for further studies on basic mechanisms of neurogenic inflammation and nociception in meningeal structures.