Materials and methods

Induction of joint inflammation
In 33 female Lewis rats 10 weeks old (Charles River, Sulzfeld, Germany), an inflammation was induced in the right knee joint. In the first step the rats received a subcutaneous injection of 500 μg of antigen [methylated bovine serum albumin (m-BSA); Sigma, Deisenhofen, Germany] in 500 μl of saline and emulsified with 500 μl of Freund's complete adjuvant [supplemented with 2 mg/ml Mycobacterium tuberculosis strain H37RA (Difco, Detroit, MI, USA] and an intraperitoneal injection of 2 × 109 heat-inactivated Bordetella pertussis (Pertussis-Referenzlabor, Krankenhaus Berlin-Friedrichshain, Berlin, Germany). The same immunization procedure was repeated seven days later. After a further 14 days a sterile solution of antigen (m-BSA) (500 μg in 50 μl of saline) was injected into the cavity of the right knee joint (day 0). Eight animals received the same immunization procedure excluding the injection of antigen into the knee joint. The mediolateral diameters of the knee joints were measured at regular intervals by means of a vernier caliper (see Fig. 1a).
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).
Two series of experiments were performed. In the first series (n = 31 rats), we determined the expression of SP-binding and BK-binding sites in the DRGs of lumbar segments L1–L5. The nerves innervating the knee joint are contained in these segments. In the second series of experiments (n = 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
At several time points after the induction of AIA, the behaviour of rats (at least n = 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
Because the fixation of the animals by perfusion impairs the binding of gold-labelled peptides and because the structure of DRGs in cryostat sections is not well maintained, we removed the DRGs after killing the animals and put the neurons into short-term culture. The DRGs of both sides were dissected quickly from segments L1–L5 or from segments C1–C7. The ganglia were incubated at 37°C in 0.28 U/ml collagenase type A (Boehringer, Mannheim, Germany) dissolved in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Eggenstein, Germany) for 100 min. After being washed with phosphate-buffered saline (PBS, 20 mmol, pH 7.4), the ganglia were incubated in PBS containing 25,000 U/ml trypsin (Sigma) for 11 min at 37°C. Then the ganglia were dissociated into single cells by gentle agitation and by trituration through a fire-polished Pasteur pipette. The cells were washed three times in DMEM by centrifugation (500 g, 5 min). The final cell pellet was suspended in Ham's F-12 medium (Gibco BRL) containing 10% heat-inactivated horse serum (Gibco BRL), 100 U/ml penicillin (Gibco BRL), 100 μg/ml streptomycin (Gibco BRL) and 100 ng/ml nerve growth factor (7S, recombinant human; Boehringer). Cells were plated on 13 mm glass coverslips coated with poly-(L-lysine) (200 μg/ml) and kept for 18 h at 37°C in a humidified incubator gassed with 3.5% CO2in 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
SP (Sigma) (1 μmol) or 1 μmol of BK (Bachem, Heidelberg, Germany) was dissolved in 500 μl of HEPES (20 mmol, pH 7.5). This solution was added to 6 nmol of sulpho-N-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
To determine the specificity of the SP–gold or BK–gold complex used in the binding studies, a displacement control was performed. Neurons were incubated in 1 μmol/ml unlabelled SP or 1 μmol/ml unlabelled BK together with 0.3 nmol/ml peptide–gold. The unlabelled peptides should decrease or prevent binding of the gold-labelled peptides. Furthermore, to test whether SP–gold is bound specifically to NK1 receptors, 0.3 nmol/ml SP–gold was incubated in the presence of 1 μmol/ml [Sar9, Met(O2)11]-SP (Bachem), a specific agonist at the NK1 receptor. To examine whether the binding of BK–gold is related to BK receptors, 0.3 nmol/ml BK–gold was incubated in the presence of 1 μmol/ml of D-Arg (Hyp3-Thi5,8-D-Phe7)-BK, a BK analogue specific for the B1 receptor (Bachem), and/or 1 μmol/ml of (Des-Arg10)-Lys-BK, a B2 receptor agonist (Bachem).

Data analysis
At different time points after induction of inflammation, DRG preparations of two to five rats were used to determine SP–gold-binding and BK–gold-binding sites. The DRGs of untreated rats served as the control group. DRGs were also removed from immunized but non-arthritic animals (day 0 group). To analyse the data, from every coverslip 50 or 100 structurally intact neurons were examined with a light microscope (Zeiss; Axiophot, Jena, Germany) coupled to a CCD colour video camera [AVT-BC6(0)] and an image-analysing system (Kontron, Eching, Germany). On each coverslip, neurons were randomly selected; neurons obstructed by other neurons or by tissue were not included. In total 30 500 neurons were analysed. The relative grey value (grey value of the soma/grey value of the substrate background) was determined for each soma. From each individual binding experiment one coverslip was used to perform a displacement control incubation (see above). This allowed us to determine the grey value range of neurons with no SP–gold or no BK–gold binding in each experiment (see Fig. 5, white bars). In all other coverslips of the particular experiment, neurons were considered as positive for peptide-binding sites if they had a relative grey value above that of neurons from the control incubation. For the final analysis, data from the experiments were pooled. Group results are expressed as means and standard deviations. To evaluate the cell size, the cross-sectional area was taken from each selected neuron. For statistical evaluation, four groups of DRGs were formed, namely DRGs of untreated control rats, of the acute phase (1 and 3 days), of the chronic phase (21 and 42 days) and of the intermittent phase (10 days) of AIA. For comparison of the AIA groups with the control group the Mann–Whitney U-test was used, with adjustment of significance values for multiple comparisons; significance was accepted at P <0.05 [41].