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ELSEVIER Regulatory Peptides 55 (1995) 85-102

Capsaicin effects on substance P and CGRP in rat adjuvant arthritis

Mahmood Ahmed a,,, Anders Bjurholm a, Gopala Rao Srinivasan b, Tomas Lundeberg c, Elvar Theodorsson b, Marianne Schultzberg d, Andris Kreicbergs ~

~Department q[' Orthopaedics, Karolinska Hospital, S-171 76 Stockholm, Sweden b Department ~f Clinical Chemistry. Karolinska Hospital, S-171 76 Stockholm, Sweden

~Departrnent of Physiology, Karolinska Institute, S-171 77 Stockholm, Sweden 'l Department of Clinical Neuroscience and Family Medicine. Geriatric Medicine, Novum, KFC, S-141 86 Huddinge, Sweden

Received 25 February 1994; revised version received and accepted 30 September 1994

Abstract

The effects of capsaicin on the sensory neuropeptides substance P and calcitonin gene-related peptide were analyzed in the ankle joints and dorsal root ganglia (L2-L6) of adult female Lewis rats. The study included 23 normal rats and 23 arthritic rats, all injected subcutaneously with capsaicin (total dose 200 mg/kg bw). Another two groups of animals from a previous study, i.e., 23 normal rats and 23 arthritic rats not given capsaicin served as controls. Adjuvant arthritis was induced by inoculation with heat-killed mycobacteria. The morphological distribution of sensory neuropeptides was assessed by immunohistochemistry and the tissue concentrations were determined by radioimmunoassay. In normal rats, capsaicin significantly reduced the concentrations of substance P and calcitonin gene-related peptide in ankle joints (54 and 36~o, respectively) as well as dorsal root ganglia (40 and 54%, respectively). In arthritic rats those pretreated with capsaicin had significantly lower concentrations of substance P and calcitonin gene-related peptide in dorsal root ganglia (19 and 42 %, respectively) compared to the arthritic controls. In the ankle joints, however, only the SP concentration was reduced (42?/0). Notably, this was accompanied by a 40% reduction in inflammatory response as assessed by compar- ing the ankle joint weights of the experimental groups. In general, there was a good correlation between the neuropeptide concentrations in ipsilateral ankle joints and the corresponding dorsal root ganglia as assessed in individual rats. The present study of adjuvant induced arthritis shows that capsaicin administration reduces the otherwise up-regulated lev- cls of sensory neuropeptides in dorsal root ganglia and ankle joints. However, capsaicin at the dose given can only mitigate, not completely prevent the development of joint inflammation. Nonetheless, the findings suggest that antineuronal therapy targeted against specific neurotransmitters may prove useful in inflammatory joint disease.

Kevwords: Ankle joint; Dorsal root ganglia; Mycobacterium; Neuropeptide

* Corresponding author. Fax: +46 8 7295778.

/)167-0115/95/$9.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 4 ) 0 0 0 9 5 - 6

86 M. Ahmed et al. / Regulatory Peptides 55 (1995) 85-102

1. Introduction

Capsaicin, naturally occurring in red pepper, is a well known neurotoxin for sensory neurons. It specifically elicits neurotransmitter release from small sized neurons of dorsal root ganglia and their projections [1-4,7,8,38]. Several studies have shown that capsaicin depletes sensory neuropeptides, i.e., substance P (SP) and calcitonin gene-related peptide (CGRP) from cells in dorsal root ganglia and from terminals in the dorsal horn and the periphery [ 1-6,44]. In dorsal root ganglia, the site of SP and CGRP synthesis, capsalcin specifically depletes small sized cells [ 1,2,6,7]. Capsaicin administration to neonatal rats causes degeneration of small size primary sensory neurons [1,8-11 ], whereas in adult rats it results in a reversible depletion of sensory neuropeptides [12,13]. The effects of capsaicin on dorsal root ganglia are not confined to SP and CGRP. Thus, capsaicin has been reported to deplete somatostatin, vasoactive intestinal polypeptide (VIP) and neurokinin A (NKA) from dorsal root ganglia and dorsal horn [2,14,15]. Notably, SP fibres of sensory origin in autonomic ganglia are also affected by capsaicin [16,17].

Capsaicin administration has been shown to at- tenuate adjuvant arthritis in rats [ 18,19]. In patients with osteoarthrosis of finger joints, topical capsaicin treatment was recently reported to reduce the signs of inflammation [20,21]. These, as well as other clinical and experimental observations, strongly indicate that the nervous system, notably the sensory, has a pathophysiological role in inflammatory joint disease [18,19,22-26]. It has been shown that the expression of sensory neuropeptides are altered in arthritis [24-30]. In a recent study of adjuvant rat arthritis based on immunohistochemistry and radioimmunoassay (RIA) we found a significant increase in SP-like immunoreactivity (LI) and CGRP-LI in ankle joints and the corresponding dorsal root ganglia [31 ]. Although capsaicin has been reported to mitigate the classical signs of arthritis, most likely by an antineuronal mechanism, its effect on sensory

neuropeptides in arthritic joints has not been clari- fied.

In the present study, the effect of capsaicin on sensory neuropeptides in ankle joints and the corresponding dorsal root ganglia was investigated in both normal and arthritic rats by immunohistochemistry and RIA.

2. Materials and methods

The study included 46 female Lewis rats (bw 230- 250 g) treated with capsaicin. The series was divided into 2 experimental groups, 23 rats in each. One group received capsaicin only, whereas the other was also inoculated with heat-killed mycobacteria (My- cobacterium butyricum) to induce arthritis. Another two groups reported previously [ 31 ] served as con- trois; one comprised 23 untreated normal rats, and the other included 23 rats with adjuvant arthritis induced by the mycobacteria. The animals were housed 5/cage at 21 ° C in a 12 h light/dark cycle with water and pellets ad libitum according to the Karo- linska Institute protocol.

Capsaicin (50 mg/kg bw, dissolved in 10~o etha- nol and 10~o Tween-80 in isotonic saline) was injected subcutaneously in all 46 rats (deeply anaesthetized with ether) for four consecutive days (total dose 200 mg/kg). To reduce respiratory symptoms, the animals were injected with theophylline (5 mg/ kg) intraperitoneally (i.p.) prior to each capsalcin injection. 23 of the animals were also inoculated with mycobacteria 1 week after capsaicin administration was completed, i.e., on day 11.

Arthritis was induced by intradermal injection (0.05 ml) of a suspension of heat-killed mycobacteria in paraffin oil (10 mg/ml) into the base of the tall [32]. The 23 normal controls received 0.05 ml paraffin oil via the same route.

Radiography of the ankle joints (not shown) was performed 11 and 29 days post inoculation in 10 arthritic rats and 4 normal controls. The weights of the dissected ankle joints were determined as a mea-

M. Ahmed et al. / Regulatory Peptides 55 (1995) 85-102

Table 1

Comparison of ankle joint weights (mg)

87

Control rats" Arthritic rats a Capsaicin + arthritic rats median: U Q - L Q median: U Q - L Q median: U Q - L Q

Ankle joints, Rt 210:220-210 440:500-420*** 310:360-250'** Ankle joints, Lt 204:220-190 425:500-400*** 285:370-250***

Comparison of ankle joints weights are made between the control, untreated arthritic and capsaicin pretreated arthritic rats. Each value (mg) corresponds to the median and upper-lower quartiles (UQ-LQ), n = 18. Differences are expressed by asterisks according to the level of significance: *** P < 0.001, Rt = right; Lt = left. a The date on control and arthritic rats are taken from Ref. [31 ].

sure of the degree of inflammation to permit comparison between the experimental groups: i.e., control rats, arthritic rats and capsalcin pretreated arthritic rats (Table 1). Histological analysis (hema- toxylin-eosin) of ankle joints from these three groups was also performed to assess inflammatory changes (Fig. 1A-C).

The end-point of the experiment was set at day 29 after the mycobacteria inoculation. Thus, in the experimental group receiving pretreatment with capsalcin for 4 days followed by an interval of 7 days before inoculation with mycobacteria, the whole experimental period covered 40 days, i.e., 4 + 7 + 29 days. For meaningful comparison, the animals given capsalcin only, were also killed 40 days after the first capsaicin injection.

In each experimental group, 18 rats were taken for RIA and 5 rats for immunohistochemistry. In each rat, the dissection comprised both ankle joints and the lumbar dorsal root ganglia (L2-L6). The specimens for RIA were prepared and analyzed sepa- rately with the exception of the dorsal root ganglia. Thus, the latter were pooled as left and right specimens from the L2-L6 levels in each rat, i.e., 5 ganglia in each pool yielding a total of 18 right and 18 left samples from each experimental group. This per- mitted correlative studies of the neuropeptide concentrations in a given ankle joint and the corresponding dorsal root ganglia.

2.1. Radioimmunoassay

The rats were killed by decapitation during ether anaesthesia 29 days post inoculation with mycobacteria. The ankle joint including the tarsal joints was dissected as one specimen, bilaterally. Thus, the specimen consisted of capsular/synovial tissue as well as the distal tibial epiphysis, the whole of talus and calcaneus, and the tarsal bones. The dorsal root ganglia (L2-L6) were excised bilaterally, and pooled as left and right side specimens. Thus, each rat yielded two samples, each consisting of 5 ganglia. All of the dissected tissues were immediately frozen on dry ice and kept at -70°C until neuropeptide extraction.

Each frozen sample was weighed before extraction. The neuropeptides were extracted and quanti- fied as described recently for bone and joint tissue [33,34]. Briefly, the samples were cut into small pieces, boiled for 10 min in 10 volumes of 2 tool/1 acetic acid in 4~ o ethylene diaminotetraacetic acid (EDTA), homogenized in a Polytron (15 s), soni- cated (30 s) and centrifuged 3000 g for 15 min. The supernatants were diluted in 10 ml RIA buffer and kept at -20°C until analysis.

The analysis was performed using the following antisera:

Substance P-like immunoreactivity (SP-LI) was assessed using antiserum SP2 [35] raised in a rab-


bit against conjugated rat SP. The antiserum reacts with SP and SP sulfoxide, but not with other tachy- kinins. High performance liquid chromatography (HPLC) purified ~25I-tyrosine rat SP was used as radioligand and rat SP was used as standard. The detection limit was 3 pmol/1. Intra- and interassay coefficients of variation were 7 and 11 ~o, respectively.

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was analyzed using antiserum CGRP8 raised in a rabbit against conjugated rat C G R P . H P L C purified J25I-histidyl rat C G R P was used as radioligand and rat C G R P as standard. The detection limit of the assay for rat C G R P was 9 pmol/1 and cross-reactivity of the assay to SP, neurokinin A, neurokinin B, neuropeptide K, gastrin, neurotensin, bombesin, neuropeptide Y and calcitonin was less than 0.01~o. Cross-reactivity with human C G R P ~ and/~ was 93 and 24~o, respectively, and with rat C G R P ~ and/~, 100 and 120~/o, respectively. Intra- and interassay coefficients of variation were 8 and 14~o, respectively.

2.2. High performance liquid chromatography

Fig. 1. (A-C) Micrographs ofhematoxylin-eosin stained sections of rat ankle joints showing the synovial membrane of control (A), arthritic (B) and capsaicin pretreated arthritic rats (C). Note the greatly hypertropied synovium in arthritic rats with intense chronic inflammatory cell infilteration (B). These changes are extensive (B) than those in the capsaicin pretreated arthritic rats (C). No signs of inflammation can be seen in the control synovium

In two previous studies [31,33], reverse phase H P L C was applied to extracts of normal knee joints as well as normal and arthritic ankle joints to char- acterize the neuropeptides of interest. Extraction in 2 tool/1 acetic acid in 4~J o EDTA was found to pro- vide optimum yield of both sensory and autonomic neuropeptides. HPLC analysis of the immunoreactive material from joint samples with regard to SP and C G R P consistently resulted in a main peak elut- ing in the position of the corresponding synthetic peptide. Thus, no evidence of multiple immunore- activities was noted for SP and CGRP. As the same extraction procedure and antisera were used in the present study, the HPLC analysis was not repeated.

(A). S = synovium, C = joint cavity, Ta = talus, Ti = distal tibia. The original magnification is 87 × in all micrographs.

M. Ahmed et aL / Regulatory Peptides 55 (1995) 85-102 89

2.3. lmmunohistochemistry

The rats were anaesthetized by i.p. injection of sodium pentobarbitone (60 mg/kg bw). Intra-arterial perfusion with phosphate-buffered saline (PB S) preceded perfusion with the fixative consisiting of 4~o paraformaldehyde in 0.2 mol/1 SOrensen phosphate buffer, pH 7.3, containing 0.2~o picric acid.

The dissection of the ankle joints and dorsal root ganglia was performed as described for the RIA. All samples were immersed in the same fixative for two days at + 4 ° C. The joint specimens containing bone were subjected to demineralization in a 4~o EDTA solution at pH 7.3 for approximately 3 weeks as described previously [36], whereas the dorsal root ganglia did not require this step.

All specimens were rinsed for at least 2 days in 20~o sucrose in 0.1 tool/1 S0rensen phosphate buffer, pH 7.2, containing sodium azide and bacitracin (Sigma, St. Louis). The tissues were cut on a Leitz cryostat to a section thickness of 15 #m. The sections were mounted on chrome alum-gelatin coated slides and processed for an indirect immunofluorescence method as described by Coons [37]. Briefly, the sections were rinsed for 15 min in 0.01 tool/1 PBS, pH 7.3, and then incubated overnight in a humid atmosphere at + 4°C with antiserum to SP, CGRP (dilution 1:200) and Protein Gene Product 9.5 (PGP 9.5) (dilution 1:400).

The SP antiserum was raised in rabbits against the synthetic undecapeptide (Peninsula Laborato- ries Europe Ltd., St. Helens, UK). The CGRP antiserum was raised in rabbits against the synthetic 37 amino acid peptide of rat CGRP (Peninsula Labo- ratories Europe Ltd., St. Helens, UK). The PGP 9.5 antiserum was obtained from Ultraclone Ltd., Cam- bridge, UK. The characteristics of this antiserum have been reported perviously [38].

After incubation with the primary antisera, the sections were rinsed in PBS for 2 × 10 rain, and then incubated with fluorescein isothiocyanate (FITC)- conjugated goat anti-rabbit antibodies (diluted 1:10, Amersham Sweden, Solna) for 30 min at + 37°C. A

final rinse in PBS for 15 min preceded mounting in a mixture ofgiycerol p-phenylenediamine [39]. Con- trois were performed by omitting the primary antisera. Addition of 50 #g/ml peptide to the correspond- hag antiserum prior to application on tissue sections served as another control. A Nikon epifluorescence microscope was used to analyze the sections, and T-Max black and white film (Kodak, Rochester) was used for photography.

2.4. Statistics

The median and upper/lower quartiles were used as measures of central tendency and variation. Data were tested for normality using the Anderson- Darling test [36]. The Mann-Whitney test was used to assess differences between two groups. Tukey multiple comparison test was used to assess differences between the four groups. Spearman's rank coefficient was used to analyze the correlation between groups. P values < 0.05 were considered significant.

3. Results

3.1. Arthritis

In the control group of 23 rats inoculated with mycobacteria, signs of arthritis as reported previously [27], appeared in the ankle joints 9 to 13 days after injection. There was bilateral hind paw swelling, warmth and redness, which persisted until the end of the experiment. There were no radiographical abnormalities on day 11. However, on day 29 there were bony erosions, periosteal thickening and joint space narrowing. In the 23 rats pretreated with capsaicin before inoculation with mycobacteria, there were similar inflammatory signs, although they were less pronounced. The control rats showed no mac- roscopical, histological (Fig. 1A) or radiographical signs of joint inflammation [31]. Comparison of ankle joint weights (left and right sides combined)


showed significantly higher values (109~ increase) in arthritic rats compared to control rats (Table 1). In arthritic rats pretreated with capsaicin, the increase in weight was 44~o compared to control rats. Notably, there was a significant reduction (40~o; P<0.001) in ankle joint weights of capsaicin pretreated arthritic rats compared to that of untreated arthritic rats (Table 1). Histological analysis of ankle joints (hemotoxylin-eosin staining) from arthritic rats showed synovial hypertrophy and chronic inflammatory cell infiltration (Fig. 1B). In arthritic rats pretreated with capsaicin, there were similar inflammatory changes, although they were less pronounced (Fig. 1C).

Table 2a

Tissue concentrations of SP-LI in control and capsaicin treated rats

ControP vs. Capsaicin median: U Q - L Q median: U Q - L Q

Ankle joints, Rt 0.47:0.55-0.31 0.22:0.27-0.18"** Ankle joints, Lt 0.50:0.55-0.38 0.23:0.27-0.18"** DRG-Rt 3.59:4.43-3.15 2.00:2.34-1.45"** DRG-Lt 4.02:4.50-3.72 2.55:2.79-1.95"**

Table 2b

Tissue concentrations of SP-LI in arthritic and capsaicin pretreated arthritic rats

3.2. Radioimmunoassay

The concentrations of CGRP-LI in the different tissues were consistently higher than the SP-LI concentrations. In all experimental groups, the concentrations of SP-LI and CGRP-LI were higher in the dorsal root ganglia than in the ankle joints (Tables 2 and 3).

In normal rats, capsaicin administration significantly lowered the concentrations of SP-LI and CGRP-LI in ankle joints and dorsal root ganglia (Tables 2A and 3A). The mean decrease (left and right sides combined) in SP-LI and CGRP-LI in ankle joints was 54 and 36~o, respectively. In dorsal root ganglia, the corresponding figures were 40 and 54~o.

In arthritic rats, those pretreated with capsaicin had lower concentrations of SP-LI in both ankle joints and dorsal root ganglia than the rats not pretreated with capsaicin. The mean decrease (left and right sides combined) in SP-LI was 42~o in ankle joints and 19~o in dorsal root ganglia (Table 2B). The CGRP-LI concentration was reduced only in the dorsal root ganglia (mean 42~o) (Table 3B).

Comparative analysis of ipsilateral ankle joints and the corresponding dorsal root ganglia in individual animals disclosed consistent correlations with regard to both SP and CGRP concentrations (Table 4A,B).

Arthritic a vs. Capsaicin + arthritic median: U Q - L Q median: U Q - L Q

Ankle joints-Rt 0.80:0.90-0.77 0.48:0.61-0.45"** Ankle joints-Lt 0.81:0.86-0.75 0.46:0.58-0.41"** DRG-Rt 5.80:6.38-5.04 5.13:5.34-4.71"** DRG-Lt 5.48:5.95-5.03 4.15:4.60-3.62"**

The concentrations were obtained from RIA measurements in tissue extracts from indiviual animals as described in Materials and methods. Each value (pmol/g) corresponds to the median and upper-lower quartiles (UQ-LQ), n = 18. Differences are expressed by asterisks according to the level of significance: *** P<0.0001, Rt = right, Lt = left, DRG = dorsal root ganglia, vs. = versus, a The data on arthritic rats are taken from Ref. [31].

3.3. Immunohistochemistry

In the arthritic control rats, as reported previously [ 31 ], there was a clear increase in SP- and CGRP-LI in the synovium of ankle joints (cf. Fig. 2A and B and Fig. 4A and B) and dorsal root ganglia (cf. Fig. 3A and C and Fig. 5A and C).

In capsaicin treated rats, there was a clear reduction in SP and CGRP immunoreactivity in all tissues analyzed. The effect of capsaicin seemed more pronounced for SP than CGRP. The effect was also more obvious in normal than in arthritic rats.

M. Ahmed et al. / Regulatory Peptides 55 (1995) 85-102 91

Table 3a

Tissue concentrations of CGRP-LI in control and capsaicin treated rats

ControP vs. Capsaicin median: U Q - L Q median: U Q - L Q

Ankle joints, Rt 1.34:1.82-1.27 1.09:1.22-0.97"** Ankle joints, Lt 1.80:2.15-1.54 0.91:0.81-1.11"** D R G - R t 23.04:27.94-19.34 10.58:13.15-9.67"** D R G - L t 20.63:27.63-15.61 9.34:12.81-8.95"**

Table 3b

Tissue concentrations of CGRP-LI in arthritic and capsiacin pretreated arthritic rats

Arthritic a vs. Capsalcin + arthritic median: U Q - L Q median: U Q - L Q

Ankle joints, Rt 2.53:2.61-2.12 2.47:2.61-1.90 NS Ankle joints, Lt 2.77:2.81-1.80 2.42:2.81-1.75 NS DRG-Rt 37.53:50.85-24.65 20.02:27.68-12.86"** DRG-Lt 25.35:39.35-29.99 17.34:20.88-11.65"**

The concentrations were obtained from RIA measurements in tissue extracts from indiviual animals as described in Materials and methods. Each value (pmol/g) corresponds to the median and upper-lower quartiles (UQ-L Q) , n = 18. Differences are expressed by asterisks according to the level of significance: *** P < 0.001, Rt = right, Lt = left, D R G = dorsal root ganglia, NS = non-significant, vs. = versus, a The data on arthritic rats are taken from Ref. [31].

SP In normal rats, SP immunoreactive nerve fibres

were almost completely absent in the ankle joint synovium (cf. Fig. 2A and C) and only occasional SP-positive cells were seen in the dorsal root ganglia (cf. Fig. 3A and B) after capsaicin treatment. SP- positive fibres could not be detected in bone, periosteum and bone marrow of capsaicin treated rats.

In arthritic rats pretreated with capsaicin, some SP immunoreactive fibres could still be found in the synovium (cf. Fig. 2B and D), but no SP-positive fibres were seen in bone, periosteum or bone marrow. In the dorsal root ganglia, SP-LI was only found in a few cells (cf. Fig. 3C and D).

CGRP In normal rats, CGRP-LI was clearly reduced but

still discernible in the ankle joint synovium (cf. Fig. 4A and C) and the dorsal root ganglia after capsaicin treatment. The CGRP-positive fibres in the synovium were predominantly non-vascular. A few immunoreactive fibres were also seen in the periosteum, bone and bone marrow. In the dorsal root ganglia, a number of CGRP-positive cells could still be found (cf. Fig. 5A and B).

In arthritic rats, those pretreated with capsaicin showed a clear reduction in CGRP immunoreactive fibres in the ankle joint synovium (cf. Fig. 4B and E). The remaining fibres were both vascular and non- vascular. A substantial number of CGRP-positive fibres were still observed in the periosteum (Fig. 6E), bone (Fig. 4D) and bone marrow despite the capsaicin pretreatment. In the dorsal root ganglia, a large number of CGRP-positive cells could still be seen (cf. Fig. 5C and D).

PGP 9.5 The distribution of PGP 9.5 immunoreactiive fi-

bres in normal as well as arthritic rats pretreated with capsaicin was similar to that of SP- and CGRP-positive fibres. The number of PGP 9.5 immunoreactive fibres in the synovium was clearly reduced by capsaicin in both normal and arthritic rats (cf. Fig. 6A and D, B and C).

4. Discussion

In a previous study of adjuvant induced arthritis in the rat, we observed a significant up-regulation of SP-LI and CGRP-LI in ankle joints and dorsal root ganglia [ 31 ]. In the present study, pretreatment with capsaicin was found to mitigate this upregulation, thus complying with previous reports showing that the neurotoxin capsaicin reduces the expression of sensory neuropeptides, which have been implicated in adjuvant arthritis [18,19]. However, the preven- tive effect ofcapsaicin on adjuvant arthritis was only


Table 4a

Correlations of SP-LI between paired tissues (right and left side) and between ipsilateral ankle joints and dorsal root ganglia

ControP Capsaicin Arthritic a Capsaicin + arthritic

Ankle joints Rt, Lt 0.52* 0.85*** 0.65** 0.45* DRG Rt, Lt 0.47* 0.32 NS 0.59** 0.25 NS Ankle joints Rt, DRG Rt 0.59** 0.50* 0.82*** 0.43* Ankle joints Lt, DRG It 0.49* 0.70*** 0.60** 0.50*

Table 4b

Correlations of CGRP-LI between paired tissues (right and left side) and between ipsilateral ankle joints and dorsal root ganglia

Control Capsaicin Arthritic a Capsaicin + arthritic

Ankle joints Rt, Lt 0.66* 0.57** 0.75*** 0.59** DRG Rt, Lt 0.46* 0.46* 0.73*** 0.19 NS Ankle joints Rt, DRG Lt 0.84*** 0.57** 0.73*** 0.42* Ankle joints Lt, DRG Lt 0.57** 0.48* 0.80*** 0.40*

Values were given as Spearman's rank coefficients. Each represents 18 paired comparisons. The level of significance is represented by asterisks: * P< 0.05, ** P< 0.01, *** P< 0.001, NS = non-significant, Rt = right, Lt = left, DRG = dorsal root ganglia, a The data on normal and arthritic rats are taken from Ref. [31 ].

partial as reflected by persisting inflammatory signs (c.f. [ 4 1 ] ) a n d increased ankle joint weights. In arthritic rats, the capsaicin induced reduction in ankle joint weights and sensory neuropeptide levels was essentially o f the same magnitude.

Capsaicin is known to elicit neurotransmitter release from small sized neurons (type B) of dorsal root ganglia [1-4 ,42] and their projections of un- myelinated C and thinly myelinated A b fibres [1,2,7,8,42]. The majority of these neurons synthe- size both SP and C G R P [43-44] . A substantial proport ion of C G R P , in contrast to SP, is also syn- thesized in large sized cells (type A) [43-47] , from which myelinated fibres arise [48 ]. It has been shown that capsaicin administration to neonatal rats reduces SP-LI in dorsal root ganglia by 7 4 ~ [2,42] and C G R P - L I by 5 0 - 6 0 ~ [4,5]. Thus, the small sized neurons, producing most of the SP found in dorsal root ganglia, are more vulnerable to capsaicin than the large sized neurons mainly producing C G R P . Moreover, it has been reported that unmy-

elinated fibres are more sensitive to capsaicin than myelinated fibres [8-12,42] . Differential effects of capsaicin on various other neuropeptides in dorsal root ganglia have also been described [2].

The present immunohistochemical findings in normal rats would seem to comply with a higher sensitivity to capsaicin of the small type cells than of the large type cells. Thus, there was an almost complete disappearance of SP-LI in the synovium and the dorsal root ganglia after capsaicin treatment, whereas CGRP-posi t ive structures were still observed, conceivably representing projections of large sized cells, known to be resistant to capsaicin. How- ever, our immunohistochemical findings suggesting a difference between the response of SP and C G R P to capsaicin are not entirely in agreement with our R IA results. Firstly, measurable levels of SP were still present in the tissues after capsaicin treatment. Sec- ondly, the capsaicin induced reductions of SP and C G R P in normal ankle joints and dorsal root ganglia were of approximately the same magnitude [ 36 -


Fig. 2.


Fig. 3.

M. Ahmed et al. /Regulatory Peptides 55 (1995) 85-102 95

Fig. 4.

96 M Ahmed et aL / Regulatory Peptides 55 (1995) 85-102

Fig. 5.


Fig. 6.


54~o ]. Presumably , the immunohis tochemica l findings suggesting a difference between SP and C G R P in response to capsa ic in are due to the difference in t issue concent ra t ions . Thus, a reduct ion by half o f SP, which normal ly occurs at very low tissue con- centra t ions , seems to leave too small amounts for detect ion by immunohis tochemis t ry . In contras t , C G R P , which normal ly occurs at high t issue con- centra t ions , is still easily de tec ted by immunohis - tochemis t ry despi te a reduct ion by half.

These observa t ions would seem to reflect the difference in the detec t ion limit of immunohis tochemistry and R I A [49]. Al though immunohis tochemis - try provides valid informat ion about the morphologica l dis t r ibut ion o f neuropept ides , it is not as reliable as R I A in detect ing small amounts . Ad- mittedly, our R I A analysis may entail some non- neuronal SP and C G R P , e.g., in interst i t ial t issue and blood. However , the immunohis tochemica l analysis o f normal t issues demons t r a t ed that the SP and C G R P immunoreact iv i t ies indeed occur red in

neuronal structures. In addit ion, staining with antiserum to the neuronal marke r P G P 9.5 revealed a similarity in appea rance of P G P 9.5 immunoreac t ive nerve fibres and the SP and CG RP-pos i t i ve fibres. F r o m the present s tudy based on bo th immunohis- tochemis t ry and RIA, it appears that capsaic in at the dose employed in adul t rats only par t ly depletes SP and C G R P from sensory neurons, as also descr ibed in other studies [2]. Moreover , there does not seem to be any difference in capsa ic in response between SP and C G R P according to RIA.

The par t ia l effect o f capsaic in observed in normal rats may explain that t rea tment with capsaic in before inoculat ion with mycobac te r ia could not prevent the deve lopment of arthritis, nor an up- regulat ion o f sensory neuropept ides f rom subnormal levels. I t seems tha t the neuropept ide deplet ing effect o f capsa ic in at the given dose is incomplete , reversible and counte rac ted by the induct ion of arthritis. The lat ter caused an up-regulat ion from subnormal levels to levels that seemed to vary according to site

Fig. 2. (A-D) Immunofluorescence micrographs of sections of rat ankle joints after incubation with antiserum to SP. SP immunoreactive nerve fibres are observed in normal (A), arthritic (B) and arthritic rats pretreated with capsalcin (D). Numerous SP-positive nerve fibres are found in the synovium of arthritic rats (B), whereas a smaller number of fibres is seen in normal rats (A) and arthritic rats pretreated with capsaicin (D). No SP-positive fibres can be seen in the synovium of a normal rat pretreated with capsaicin (C). Arrows indicate immunoreactive fibres, s = synovium, b = bone. The original magnification of (A) and (C) is 250 x and that of (B) and (D) is 175 x. Fig. 3. (A-D) Immunofluorescence micrographs of sections of rat dorsal root ganglia after incubation with antiserum to SP. Some SP immunoreactive cells can be seen in the dorsal root ganglion of a normal rat (A), while a larger number of SP-positive cells is observed in the arthritic rat (C). The number of SP-positive cells is smaller in normal and arthritic rats pretreated with capsalcin (B and D, respectively). Arrows indicate SP-positive cells. The original magnification is 175 × in all micrographs. Fig. 4. (A-E) Immunofluorescence micrographs of sections of rat ankle joints after incubation with antiserum to CGRP. CGRP immmunoreactive nerve fibres are observed (arrows) in the synovial membrane of normal (A), arthritic (B), capsaicin pretreated normal (C) and capsaicin pretreated arthritic rats (E). A smaller number of CGRP-positive fibres can be seen in the synovium of capsaicin pretreated normal and arthritic rats (C and E) as compared to the synovium of rats not pretreated with capsalcin (A and B). A few CGRP-positive fibres can still be seen (arrows) in bone (D) and periosteum (Fig. 5E) of the distal tibia of arthritic rats pretreated with capsaicin, s = synovium, b = bone. The original magnification is 87 × in (A, B, D, and E) and 175 x in (C). Fig. 5. (A-D) Immunofluorescence micrographs of sections of rat dorsal root ganglia after incubation with antiserum to CGRP. The number of CGRP immunoreactive cells is larger in normal (A) than in capsaicin pretreated normal rats (C). No clear difference can be seen between the number of CGRP-positive cells in arthritic (B) and capsaicin pretreated arthritic (D) rats. The original magnification is 87 × in all micrographs. Fig. 6. (A-E) Immunofluorescence micrographs of sections of rat ankle joints after incubation with antisera to PGP 9.5 (A-D) and CGRP (E). Pretreatment of normal and arthritic rats with capsaicin resulted in a smaller number of PGP 9.5-positive fibres (arrows) in the synovium, (D and C) as compared to the synovium of normal and arthritic rats not pretreated with capsaicin (A and B). s = synovium, b = bone. The original magnification is 87 x in (A-C and E) and 175 x in (D).

M. Ahmed et al. / Regulatory

and neuropeptide. In ankle joints, the SP concentration reached the normal level, whereas that of CGRP clearly exceeded the normal level (+ 55~o). Conversely, in dorsal root ganglia the SP concentration exceeded the normal level ( + 22 ~o), whereas that of CGRP reached a level below the normal (-14Fo). However, these figures in relation to normal levels should be interpreted with caution, since the actual starting levels are unknown. Thus, they represent the net effect of an initial downregulation by capsalcin and a subsequent upregulation by in- duction of arthritis. The specific mechanisms behind the differential effects of capsaicin on SP-LI and CGRP-LI at different sites in arthritic rats remain unknown. Although the differences were moderate, it may be speculated that arthritis induces expression of sensory neuropeptides in capsaicin resistent neurons, which are not normally of the SP or CGRP phenotype.

An important factor, which most likely influences the levels of SP and CGRP in normal as well as inflamed tissues is nerve growth factor (NGF). Under normal conditions, it is well known that NGF plays a significant role in maintaining the function of sensory and sympathetic neurons [50,51]. In adult rats, it has been shown that NGF treatment induces increased synthesis of SP and CGRP in the dorsal root ganglia [52,53]. In addition, NGF deprivation has been shown to cause a downregulation of SP [54,55]. In a study of adult guinea pigs, capsaicin treatment was found to impair the retrograde transport of NGF to the dorsal root ganglia. This was presumed to cause the decreased synthesis of SP noted [56]. Notably, NGF administration to guinea pigs and rats pretreated with capsaicin was found to reverse the effect of capsalcin. In fact, SP was found to be upregulated in the dorsal root ganglia [56,57]. In arthritis, it has been shown that the concentration of NGF is increased in synovial fluid and synovial membrane [58-60]. In a study on paw inflammation, there was a parallel upregulation of NGF and sensory neuropeptides in the sciatic nerve [61]. In all, these observations show that capsaicin has a

Peptides 55 (1995) 85-102 99

downregulatory effect on NGF, whereas arthritis has an upregulatory effect. Whether the effect of arthritis is stronger than that of capsaicin, as indicated by our results, has yet to be demonstrated.

It seems reasonable to summarize from the present investigation of the capsalcin effect in arthritis that SP and CGRP levels end up approximately inter- mediate between those noted in rats given capsaicin only and those in rats given mycobacteria only. It may prove that a complete depletion of SP and CGRP in adult rats can be achieved by higher doses ofcapsaicin than those employed in the present study (200 mg/kg bw). Whether higher doses of capsaicin would completely prevent the development of arhri- tis has yet to be demonstrated. In a study on adult rats by Gamse and co-workers [2], capsaicin elicited a dose-dependent depletion of SP in dorsal root ganglia (33 Yo by 125 mg/kg and 67 ~/o by 950 mg/kg), although the highest dose did not result in a complete depletion of SP. In the dorsal horn and saphe- nous nerve there was no further depletion by higher doses. In neonatal rats, a lower dose (50 mg/kg bw) of capsaicin decreased SP by 74~o in the dorsal root ganglia [2,42].

The present study also specifically explored the effects of capsaicin on sensory neuropeptides in individual ankle joints and corresponding dorsal root ganglia. Comparative analysis disclosed a consistent correlation between the concentrations of sensory neuropeptides in ipsilateral dorsal root ganglia and ankle joints. However, it remains unknown whether the correlations observed reflect depletion at both anatomical sites or only peripherally.

It may be summarized that capsaicin significantly reduces SP- and CGRP-LI in dorsal root ganglia and ankle joints of normal rats. Pretreatment with capsaicin mitigates, but does not prevent the development of adjuvant arthritis. It is reasonable to as- sume that capsaicin induced reduction of SP and CGRP in inflamed joints can alleviate hyperalgesia and swelling associated with arthritis. However, the remaining SP and CGRP may still contribute to joint inflammation, possibly in conjunction with, or


in addition to, other factors, e.g., from the immune system.

Acknowledgements

This study was supported by grants from the Royal 80 Year Funds of King Gustaf V, Uggla's Founda- tion, Ake Wibergs Foundation, the Karolinska In- stitute Research Funds, Lundbergs Foundation, the Swedish Association Against Rheumatic Disease, the Swedish Medical Research Council (12X-08652- 01, 12X-07464) and the Swedish Society for Medi- cal Research.

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