Monoarticular antigen-induced arthritis leads to pronounced bilateral upregulation of the expression of neurokinin 1 and bradykinin 2 receptors in dorsal root ganglion neurons of rats

Abstract

This study describes the upregulation of neurokinin 1 and bradykinin 2 receptors in dorsal root ganglion (DRG) neurons in the course of antigen-induced arthritis (AIA) in the rat knee. In the acute phase of AIA, which was characterized by pronounced hyperalgesia, there was a substantial bilateral increase in the proportion of lumbar DRG neurons that express neurokinin 1 receptors (activated by substance P) and bradykinin 2 receptors. In the chronic phase the upregulation of bradykinin 2 receptors persisted on the side of inflammation. The increase in the receptor expression is relevant for the generation of acute and chronic inflammatory pain. Ongoing pain and hyperalgesia (enhanced pain response to stimulation of the tissue) are major symptoms of arthritis. Arthritic pain results from the activation and sensitization of primary afferent nociceptive nerve fibres ('pain fibres') supplying the tissue (peripheral sensitization) and from the activation and sensitization of nociceptive neurons in the central nervous system (central sensitization). After sensitization, nociceptive neurons respond more strongly to mechanical and thermal stimulation of the tissue, and their activation threshold is lowered. The activation and sensitization of primary afferent fibres results from the action of inflammatory mediators such as bradykinin (BK), prostaglandins and others on membrane receptors located on these neurons. BK is a potent pain-producing substance that is contained in inflammatory exudates. Up to 50% of the primary afferent nerve fibres have receptors for BK. When primary afferent nerve fibres are activated they can release neuropeptides such as substance P (SP) and calcitonin gene-related peptide from their sensory endings in the tissue. SP contributes to the inflammatory changes in the innervated tissue (neurogenic inflammation), and it might also support the sensitization of nociceptive nerve fibres by binding to neurokinin 1 (NK1) receptors. NK1 receptors are normally expressed on a small proportion of the primary afferent nerve fibres. Because the expression of receptors on the primary afferent neurons is essential for the pain-producing action of inflammatory mediators and neuropeptides, we investigated in the present study whether the expression of BK and NK1 receptors on primary afferent neurons is altered during the acute and chronic phases of an antigen-induced arthritis (AIA). AIA resembles in many aspects the inflammatory process of human rheumatoid arthritis. Because peptide receptors are expressed not only in the terminals of the primary afferent units but also in the cell bodies, we removed dorsal root ganglia (DRGs) of both sides from control rats and from rats with the acute or chronic phase of AIA and determined, after short-term culture of the neurons, the proportion of DRG neurons that expressed the receptors in the different phases of AIA. We also characterized the inflammatory process and the nociceptive behaviour of the rats in the course of AIA. . After immunization, m-BSA was injected into the right knee joint cavity to induce arthritis. The joint swelling was measured at regular intervals. Nociceptive (pain) responses to mechanical stimulation of the injected and the contralateral knee were monitored in the course of AIA. Groups of rats were killed at different time points after the induction of AIA, and inflammation and destruction in the knee joint were graded by histological examination. The DRGs of both sides were dissected from segments L1–L5 and C1–C7 from arthritic rats, from eight immunized rats without arthritis and from ten normal control rats. Excised DRGs were dissociated into single cells which were cultured for 18 h. )-Lys-BK. ). The initial phase of AIA was characterized by strong joint swelling and a predominantly granulocytic infiltration of the synovial membrane and the joint cavity (acute inflammatory changes). In the later phases of AIA (10–84 days after induction of AIA) the joint showed persistent swelling, and signs of chronic arthritic alterations such as infiltration of mononuclear leucocytes, hyperplasia of synovial lining layer (pannus formation) and erosions of cartilage and bone were predominant. The contralateral knee joints appeared normal at all time points. Destruction was observed only in the injected knee but some proteoglycan loss was also noted in the non-injected, contralateral knee. In the acute and initial chronic phases of AIA (1–29 days) the rats showed mechanical hyperalgesia in the inflamed knee (limping, withdrawal response to gentle pressure onto the knee). In the acute phase (up to 9 days) a pain response was also seen when gentle pressure was applied to the contralateral knee. ]–SP in three experiments, showing that SP–gold was bound to NK1 receptors. = 5) of AIA was the binding of BK–gold decreased by the coadministration of BK–gold and the B1 agonist. By contrast, in these experimental groups the binding of BK–gold was suppressed by the coadministration of the B2 agonist. These results show that B2 receptors, but not B1 receptors, were expressed in both normal animals and in animals with AIA. These results show that in AIA in the rat the expression of SP-binding and BK-binding sites in the perikarya of DRGs L1–L5 is markedly upregulated in the course of knee inflammation. Although the inflammation was induced on one side only, the initial changes in the binding sites were found in the lumbar DRGs of both sides. No upregulation of SP-binding or BK-binding sites was observed in the cervical DRGs. The expression of SP-binding sites was upregulated only in the first days of AIA, that is, in the acute phase, in which the pain responses to mechanical stimulation were most pronounced. By contrast, the upregulation of BK-binding sites on the side of AIA persisted for up to 42 days, that is, in the acute and chronic phase of AIA. Only the B2 receptor, not the B1 receptor, was upregulated. The coincidence of the enhanced expression of NK1 and BK receptors on sensory neurons and the pain behaviour suggests that the upregulation of these receptors is relevant for the generation and maintenance of arthritic pain. In the acute phase of AIA, approximately 50% of the lumbar DRG neurons showed an expression of SP-binding sites. Because peptide receptors are transported to the periphery, the marked upregulation of SP-binding receptors probably leads to an enhanced density of receptors in the sensory endings of the primary afferent units. This will permit SP to sensitize more neurons under inflammatory conditions than under normal conditions. However, the expression of NK1 receptors was upregulated only in the acute phase of inflammation, suggesting that SP and NK1 receptors are less important for the generation of hyperalgesia in the chronic phase of AIA. Because BK is one of the most potent algesic compounds, the functional consequence of the upregulation of BK receptors is likely to be of immediate importance for the generation and maintenance of inflammatory pain. The persistence of the upregulation of BK receptors on the side of inflammation suggests that BK receptors should be an interesting target for pain treatment in the acute and chronic phases. Only B2 receptors were identified in normal animals and in rats with AIA. This is surprising because previous pharmacological studies have provided evidence that, during inflammation, B1 receptors can be newly expressed. Receptor upregulation in the acute phase of AIA was bilateral and almost symmetrical. However, hyperalgesia was much more pronounced on the inflamed side. It is most likely that receptors on the contralateral side were not readily activated because in the absence of gross inflammation the local concentration of the ligands BK and SP was probably quite low. We hypothesize that the bilateral changes in receptor expression are generated at least in part by mechanisms involving the nervous system. Symmetrical segmental changes can be produced only by the symmetrical innervation, involving either the sympathetic nervous system or the primary afferent fibres. Under inflammatory conditions, primary afferent fibres can be antidromically activated bilaterally in the entry zone of afferent fibres in the spinal cord, and it was proposed that this antidromic activation might release neuropeptides and thus contribute to neurogenic inflammation. Because both sympathetic efferent fibres and primary afferent nerve fibres can aggravate inflammatory symptoms, it is also conceivable that they are involved in the regulation of receptor expression in primary afferent neurons. A neurogenic mechanism might also have been responsible for the bilateral degradation of articular cartilage in the present study.

Introduction:

Ongoing pain and hyperalgesia (enhanced pain response to stimulation of the tissue) are major symptoms of arthritis. Arthritic pain results from the activation and sensitization of primary afferent nociceptive nerve fibres ('pain fibres') supplying the tissue (peripheral sensitization) and from the activation and sensitization of nociceptive neurons in the central nervous system (central sensitization). After sensitization, nociceptive neurons respond more strongly to mechanical and thermal stimulation of the tissue, and their activation threshold is lowered. The activation and sensitization of primary afferent fibres results from the action of inflammatory mediators such as bradykinin (BK), prostaglandins and others on membrane receptors located on these neurons. BK is a potent pain-producing substance that is contained in inflammatory exudates. Up to 50% of the primary afferent nerve fibres have receptors for BK. When primary afferent nerve fibres are activated they can release neuropeptides such as substance P (SP) and calcitonin gene-related peptide from their sensory endings in the tissue. SP contributes to the inflammatory changes in the innervated tissue (neurogenic inflammation), and it might also support the sensitization of nociceptive nerve fibres by binding to neurokinin 1 (NK1) receptors. NK1 receptors are normally expressed on a small proportion of the primary afferent nerve fibres.

Aims:

Because the expression of receptors on the primary afferent neurons is essential for the pain-producing action of inflammatory mediators and neuropeptides, we investigated in the present study whether the expression of BK and NK1 receptors on primary afferent neurons is altered during the acute and chronic phases of an antigen-induced arthritis (AIA). AIA resembles in many aspects the inflammatory process of human rheumatoid arthritis. Because peptide receptors are expressed not only in the terminals of the primary afferent units but also in the cell bodies, we removed dorsal root ganglia (DRGs) of both sides from control rats and from rats with the acute or chronic phase of AIA and determined, after short-term culture of the neurons, the proportion of DRG neurons that expressed the receptors in the different phases of AIA. We also characterized the inflammatory process and the nociceptive behaviour of the rats in the course of AIA.

Materials and methods:

. After immunization, m-BSA was injected into the right knee joint cavity to induce arthritis. The joint swelling was measured at regular intervals. Nociceptive (pain) responses to mechanical stimulation of the injected and the contralateral knee were monitored in the course of AIA. Groups of rats were killed at different time points after the induction of AIA, and inflammation and destruction in the knee joint were graded by histological examination. The DRGs of both sides were dissected from segments L1–L5 and C1–C7 from arthritic rats, from eight immunized rats without arthritis and from ten normal control rats. Excised DRGs were dissociated into single cells which were cultured for 18 h.

)-Lys-BK.

Results:

). The initial phase of AIA was characterized by strong joint swelling and a predominantly granulocytic infiltration of the synovial membrane and the joint cavity (acute inflammatory changes). In the later phases of AIA (10–84 days after induction of AIA) the joint showed persistent swelling, and signs of chronic arthritic alterations such as infiltration of mononuclear leucocytes, hyperplasia of synovial lining layer (pannus formation) and erosions of cartilage and bone were predominant. The contralateral knee joints appeared normal at all time points. Destruction was observed only in the injected knee but some proteoglycan loss was also noted in the non-injected, contralateral knee. In the acute and initial chronic phases of AIA (1–29 days) the rats showed mechanical hyperalgesia in the inflamed knee (limping, withdrawal response to gentle pressure onto the knee). In the acute phase (up to 9 days) a pain response was also seen when gentle pressure was applied to the contralateral knee.

]–SP in three experiments, showing that SP–gold was bound to NK1 receptors.

= 5) of AIA was the binding of BK–gold decreased by the coadministration of BK–gold and the B1 agonist. By contrast, in these experimental groups the binding of BK–gold was suppressed by the coadministration of the B2 agonist. These results show that B2 receptors, but not B1 receptors, were expressed in both normal animals and in animals with AIA.

Discussion:

These results show that in AIA in the rat the expression of SP-binding and BK-binding sites in the perikarya of DRGs L1–L5 is markedly upregulated in the course of knee inflammation. Although the inflammation was induced on one side only, the initial changes in the binding sites were found in the lumbar DRGs of both sides. No upregulation of SP-binding or BK-binding sites was observed in the cervical DRGs. The expression of SP-binding sites was upregulated only in the first days of AIA, that is, in the acute phase, in which the pain responses to mechanical stimulation were most pronounced. By contrast, the upregulation of BK-binding sites on the side of AIA persisted for up to 42 days, that is, in the acute and chronic phase of AIA. Only the B2 receptor, not the B1 receptor, was upregulated. The coincidence of the enhanced expression of NK1 and BK receptors on sensory neurons and the pain behaviour suggests that the upregulation of these receptors is relevant for the generation and maintenance of arthritic pain.

In the acute phase of AIA, approximately 50% of the lumbar DRG neurons showed an expression of SP-binding sites. Because peptide receptors are transported to the periphery, the marked upregulation of SP-binding receptors probably leads to an enhanced density of receptors in the sensory endings of the primary afferent units. This will permit SP to sensitize more neurons under inflammatory conditions than under normal conditions. However, the expression of NK1 receptors was upregulated only in the acute phase of inflammation, suggesting that SP and NK1 receptors are less important for the generation of hyperalgesia in the chronic phase of AIA.

Because BK is one of the most potent algesic compounds, the functional consequence of the upregulation of BK receptors is likely to be of immediate importance for the generation and maintenance of inflammatory pain. The persistence of the upregulation of BK receptors on the side of inflammation suggests that BK receptors should be an interesting target for pain treatment in the acute and chronic phases. Only B2 receptors were identified in normal animals and in rats with AIA. This is surprising because previous pharmacological studies have provided evidence that, during inflammation, B1 receptors can be newly expressed.

Receptor upregulation in the acute phase of AIA was bilateral and almost symmetrical. However, hyperalgesia was much more pronounced on the inflamed side. It is most likely that receptors on the contralateral side were not readily activated because in the absence of gross inflammation the local concentration of the ligands BK and SP was probably quite low. We hypothesize that the bilateral changes in receptor expression are generated at least in part by mechanisms involving the nervous system. Symmetrical segmental changes can be produced only by the symmetrical innervation, involving either the sympathetic nervous system or the primary afferent fibres. Under inflammatory conditions, primary afferent fibres can be antidromically activated bilaterally in the entry zone of afferent fibres in the spinal cord, and it was proposed that this antidromic activation might release neuropeptides and thus contribute to neurogenic inflammation. Because both sympathetic efferent fibres and primary afferent nerve fibres can aggravate inflammatory symptoms, it is also conceivable that they are involved in the regulation of receptor expression in primary afferent neurons. A neurogenic mechanism might also have been responsible for the bilateral degradation of articular cartilage in the present study.

Introduction

]. Subgroups of primary afferent neurons also express receptors for neuropeptides such as substance P (SP) that are released from primary afferent fibres.

].

]. Thus, under normal conditions, the role of SP is questionable.

]. We therefore removed DRGs of both sides from control rats and from rats with the acute or chronic phase of AIA and determined, after short-term culture of the neurons, the proportion of DRG neurons that expressed the receptors under the different experimental conditions. We also characterized the inflammatory process and the nociceptive behaviour of the rats in the course of AIA.

Induction of joint inflammation

).

At 1, 3, 10, 21, 42 or 84 days after the induction of inflammation in the knee joint, the rats were killed by cervical dislocation during anaesthesia with ether. A total of 10 untreated rats of the same age and sex were used as normal control animals. The immunized rats without arthritis induction were killed 14 days after the second immunization (day 0).

= 20 rats) we again took DRGs from segments L1–L5 to determine the BK receptor subtypes, and we removed DRGs from cervical segments C1–C7 to determine the expression of NK1 and BK receptors in the primary afferent neurons at the cervical level. In addition, a detailed documentation of the behaviour was performed in the second series. All procedures complied with the regulations of the Thuringian Commission for Animal Protection.

Histology and grading of arthritis

After the rats had been killed, both knee joints were removed, skinned, fixed in 4% buffered formalin, decalcified in EDTA, embedded in paraffin, cut into 5 μm frontal sections and stained with haematoxylin–eosin for microscopic examination. Three sections per knee joint were examined in a blind fashion by two independent observers (PKP and RB) with the use of a semiquantitative score (0 = no, 1 = mild, 2 = moderate, 3 = severe alterations). The acute inflammatory reaction was assessed by evaluating the quantity of fibrin exudation and the relative number and density of granulocytes in the synovial membrane and in the joint space. The chronic inflammatory reaction was quantified on the basis of the relative number and density of infiltrating mononuclear leucocytes in the synovial membrane, the degree of synovial hyperplasia, and the extent of fibrosis in the synovial tissues. The histological score is the sum of the three parameters evaluated. A score from 0 to 4 was used to assess the degree of cartilage destruction: 0, no destruction; 1, unequivocal erosions of less than 10% of cartilage and bone cross sections; 2, erosion of 10–25%; 3, erosion of 25–50%; 4, erosion of more than 50% of cartilage and bone cross sections. Additional sections were stained with safranin O to determine the loss of proteoglycan in the cartilage matrix, with the use of the same score as for the evaluation of inflammatory reactions.

Testing of nociceptive behaviour

= 5 rats for each time point) was assessed by using a score. First each rat was placed in a box in which it could move freely. The severity of disturbances of walking was graded: 4, no walking; 3, walking on three legs; 2, limping with the leg with inflammation; 1, limping with the leg with inflamed knee only after pressure on the knee; 0, normal walking. Other parameters (exploratory behaviour, standing on hindlimbs) were also checked and documented. Thereafter the rats were held in the hand by one experimenter and the following tests were performed by the other experimenter: flexion/extension of the left and right knee joint as well as an application of moderate non-painful and strong, lightly painful pressure onto the ankle joints and the knee joints. In all these cases we determined whether the rat showed a nociceptive reaction, namely a withdrawal of the stimulated leg. By using a mechanical device that applied pressure to a small area, we administered pressure to the lateral side of the knee joint and determined the pressure range at which the rat withdrew the leg. Responses to pressure in the range 0–100 g were scored 3 (this stimulus evokes a touch sensation in humans), responses to pressure in the range 100–200 g were scored 2 (this stimulus evokes a pressure sensation), responses to 200–250 g were scored 1 and a lack of response to 250 g was scored 0 (the application of 250 g evokes a weak pain sensation). For analysis, the scores of all animals were added and divided by the number of animals tested in the group.

Preparation of the DRGs

in air. Cells were fed after 14–16h with supplemented Ham's F-12 medium (see above).

Preparation of the SP–gold and BK–gold conjugates

-hydroxysuccinimido Nanogold reagent (BioTrend, Köln, Germany), dissolved in 500 μl of doubly distilled water and incubated for 1 h at room temperature. To separate SP–gold or BK–gold conjugates from unbound SP or BK, membrane centrifugation (Amicon Microcon-10 system) was used. The SP–gold and BK–gold conjugates were dissolved in PBS containing 0.1% bovine serum albumin (BSA), 0.2 mol of sucrose, 4 μg/ml leupeptin and 10 mmol of sodium azide. This solution was aliquoted and stored at –20°C for a maximum of 3 months.

SP–gold and BK–gold binding to cultured neurons

The cells were pre-fixed with 2% paraformaldehyde and 0.05% glutaraldehyde in 0.1 mol of phosphate buffer (pH 7.2) for 30 min. After being washed with PBS (20 mmol, pH 7.4), the cells were pretreated with 50 mmol of glycine in PBS and thereafter with 5% BSA and 0.1% gelatine in PBS for 30min. The cells were then washed with 0.1% acetylated BSA (BSA-C) and incubated overnight with 0.3 nmol/ml SP–gold in PBS or with 0.3 nmol/ml BK–gold containing 0.1% BSA-C, bacitracin (40 μg/ml), leupeptin(4 μg/ml) and chymostatin (2 μg/ml) at 4°C in a moist chamber. After being washed with PBS plus 0.1% BSA-C and thereafter with PBS to remove unbound SP–gold or BK–gold, cells were postfixed with 2% glutaraldehyde in PBS for 10 min. After extensive washing with PBS and doubly distilled water, the gold particles were intensified with silver enhancer (R-Gent, pH 5.5; BioTrend) for 15 min at 22°C. The reaction was stopped by washing in doubly distilled water. The preparations were dehydrated and embedded in DePeX (Fluka, Neu-Ulm, Germany).

Control incubations

)-Lys-BK, a B2 receptor agonist (Bachem).

Data analysis

].

Joint inflammation

depicts the depletion of cartilage proteoglycan (evidenced by the loss of staining with safranin O). Although destruction was observed only in the injected knee, some proteoglycan loss was also noted in the non-injected, contralateral knee.

Nociceptive behaviour (pain response)

). Between 14 and 29 days of AIA, the rats showed a withdrawal response when the injected knee was compressed between two fingers with moderate intensity, whereas strong and painful compression was necessary to evoke a withdrawal response to pressure on the contralateral knee.

Expression of SP–gold- and BK–gold-binding sites in DRGs from control and arthritic rats

) appear dark as a result of the silver staining of the gold particles (arrows). The next sections will illustrate the data analysis and summarize the data on the receptor expression.

Lumbar DRGs

).

) neurons.

the histograms on the left side show the grey density of the neurons from two experimental groups treated only with SP-gold. A proportion of these neurons exhibited grey values of more than 0.16, indicating binding of SP-gold. The histograms on the right side show that neurons with grey values of more than 0.16 were not found when SP-gold was administered together with the specific NK1 receptor agonist. Thus SP-gold specifically labelled NK1 receptors.

Cervical DRGs

= 5), and in 5 rats at 42 days of AIA in the knee.

Lumbar DRGs

).

= 3 rats) of inflammation. At 84 days after the induction of AIA, 51.0 ± 12.7% of the neurons showed an expression of BK–gold-binding sites and this was close to the prearthritic values. In the contralateral DRG of the same animals, however, the proportion of BK–gold-labelled neurons declined in the intermediate phase (day 10) and chronic phase (days 21–84) of AIA and was not significantly different from the control value. Thus the increase in BK–gold-labelled neurons was persistent on the side where the inflammation had been induced, and transient on the opposite side.

). Under all conditions and at all time points, BK–gold binding was found mainly in small and medium-sized neurons. The binding of BK–gold in these experiments was suppressed by the administration of a mixture of both the B1 and B2 agonists (data not shown).

= 5) of AIA was the binding of BK-gold reduced by the coadministration of BK–gold and the B1 agonist. By contrast, in these experimental groups the binding of BK–gold was suppressed by the coadministration of the B2 agonist. These data show that in both normal animals and animals with AIA, B2 receptors but not B1 receptors were expressed.

Cervical DRGs

= 5), and in 5rats at 42 days of AIA in the knee.

Inflammation and nociceptive behaviour

The limping during walking and the withdrawal responses to pressure on the joint show that the rats experienced pain during mechanical stimulation of the inflamed joint (mechanical hyperalgesia). Mechanical hyperalgesia is also a leading symptom of human arthritis, and thus AIA in the rat seems to be a suitable model for studying the mechanisms of arthritic pain. Hyperalgesia was most marked in the acute phase and less severe when the acute phase subsided. Interestingly, later stages of AIA were not obviously painful although the chronic inflammation and joint destruction persisted. Although hyperalgesia was pronounced in the knee with inflammation, signs of weak hyperalgesia were also noted on the contralateral side at 1–9 days of AIA because the mechanical thresholds were lowered. However, the pain response was quite asymmetrical; the main symptoms were on the injected side.

Methodological considerations for the use of DRG

. However, the cell body of DRG neurons is thought to serve as a valid model for the sensory endings, for three reasons:

].

].

].

We therefore believe that the receptor expression in the cell bodies of DRG neurons also represents the receptor expression in the sensory terminals of the DRG neurons.

; this could influence the basal expression of receptors, for example. However, the neurons from different experimental groups were cultured in the same way, and the distribution of the cell sizes was similar in all preparations. Therefore changes in the expression of binding sites are most probably the outcome of pathophysiological changes in the rat during the inflammation.

Expression and upregulation of SP-binding sites during inflammation

]. There is still debate on whether or not the effect of SP on sensory endings is important for the generation of pain (see below).

], we believe that the SP–gold-binding sites represent NK1 receptors.

Because peptide receptors are transported to the periphery, the marked upregulation of SP-binding receptors probably leads to an enhanced density of receptors in the sensory endings of the primary afferent units. This allows SP to sensitize more neurons under inflammatory conditions than under normal conditions. It is therefore likely that the upregulation of NK1 receptors contributes to hyperalgesia during arthritis. Indeed, the upregulation of NK1 receptors coincided with the marked pain responses that were seen in the acute phase of inflammation. However, the final proof of the involvement of NK1 receptors in the arthritic pain will require additional functional experiments. In the chronic phase NK1 receptors seem to be less important.

Expression and upregulation of BK-binding sites during inflammation

]. Although the functional effects of SP on nociceptive afferent neurons are still under discussion (see the last paragraph), there is full agreement that BK causes pain in animals and humans. In the present study, DRGs from AIA rats exhibited more neurons with BK-binding sites. Thus, during AIA more primary afferent neurons can be activated and sensitized by the inflammatory mediator BK, and thus the upregulation of the BK-binding sites is likely to be an important mechanism by which arthritis causes severe pain. Because the increased expression of BK-binding sites persisted for up to 42 days on the side of inflammation, BK-binding sites should be an interesting target for pain treatment in the acute and chronic phases of arthritis.

]. We therefore conclude that the increased expression of BK receptors in AIA is entirely due to an upregulation of the B2 receptor.

Bilateral effects and possible mechanisms of upregulation of receptors

Receptor upregulation in the acute phase of AIA was bilateral and almost symmetrical. However, the hyperalgesia was much more pronounced on the inflamed side. That the pain responses were asymmetrical in spite of a symmetrical receptor upregulation is most probably due to the fact that the receptors on the contralateral side were not readily activated because in the absence of gross inflammation the local concentration of the ligands BK and SP was probably quite low.

] have been observed on the contralateral side. However, during AIA the contralateral knee did not show gross inflammatory alterations; it is therefore questionable whether the enhanced receptor expression in the contralateral DRGs was due to the spreading of the inflammatory disease to the contralateral side.

] has been shown; it is therefore conceivable that efferent activity from the central nervous system to the periphery might also influence the expression of receptors in DRG neurons.

Acknowledgements

R Bräuer and PK Petrow were supported by grants from the German Ministry of Education and Research (FKZ 01ZZ 9602). We thank Dr R Vollandt (Institut für Medizinische Statistik, Informatik und Dokumentation, University of Jena) for performing the statistical evaluation, and Mrs H Kümpel and Mrs H Börner for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Scha 404/9-2).

Figures and Tables

Loss of proteoglycans (staining with safranin O) in the cartilage of the injected knee and the contralateral knee. All values are means and standard deviations.

Proportions of DRG neurons with binding of BK–gold in control animals and in different groups of animals with AIA. Results for DRG neurons from immunized animals without knee injection are shown at 0 days. White bars, contralateral to injected knee; black bars, ipsilateral to injected knee.

the standard deviation was zero at most time points.

and subsequent enhancement with silver. The neurons were cultured for 18 h. The black staining indicates binding of SP–gold or BK–gold (arrows).

= 500, from five cultures) from AIA rats (21 days). White bars, neurons exhibiting grey densities that were in the range of those observed in neurons from control incubations; black bars, neurons exhibiting grey densities that were higher than those observed in the neurons from control incubations.

Size distribution of the cultured neurons from different experimental groups. The white bars show the proportions of neurons with different areas from experiments in which SP–gold-binding sites were determined. The neurons with SP–gold-binding sites are shown by the black bars.

]-SP (1 μmol/ml) together.

= 300, from three cultures) from AIA rats (42 days). White bars, neurons exhibiting grey densities that were in the range of those observed in neurons from control incubations; black bars, neurons exhibiting grey densities that were higher than those observed in the neurons from control incubations.

Size distribution of the cultured neurons from different experimental groups. The white bars show the proportions of neurons with different areas from experiments in which BK–gold-binding sites were determined. The neurons with BK–gold-binding sites are shown by the black bars.

Expression of neurokinin 1 and bradykinin receptors in cervical DRGs ipsilateral to the inflamed knee joint

Values shown are the means and standard deviations.

Expression of bradykinin 1 and bradykinin 2 receptors in DRGs L1–L5

Values shown are the means and standard deviations. Abbreviations: i.l., ipsilateral to injected knee; c.l., contralateral to injected knee.

Keywords

• antigen-induced arthritis
• bradykinin receptor
• dorsal root ganglion neurons
• neurokinin 1 receptor
• pain