EUROPEAN JOURNAL OF PHARMACOLOGY24 (1973) 108-112.

NORTH-HOLLANDPUBLISHINGCOMPANY

Short communication CORRELATIONS BETWEEN PATHOLOGICAL CHANGES IN T H E H I N D PAWS O F R A T S WITH ADJUVANT ARTHRITIS AND THEIR RESPONSE TO ANTI-INFLAMMATORY AND ANALGESIC DRUGS R. Duane SOFIA, Heidi B. VASSAR and Susan D. NALEPA
Department of Pharmacology, WallaceLaboratories, Cranbury, New Jersey 08512, U.S.A.

Received 3 September 1973

Accepted 7 September 1973

R.D. SOFIA, H.B. VASSAR and S.D. NALEPA, Correlations between pathological changes in the hind paws of rats with adjuvant arthritis and their response to anti-inflammatory and analgesic drugs, European J. Pharmacol. 24 (1973) 108-112. Pathological alterations associated with adjuvant-induced arthritis in rats, including increased paw volume and temperature and decreased pain threshold, become increasingly severe throughout the course of development of the disease (21 days). Acetyl salicylic acid and indomethacin produced significant reductions in swelling and pyresis observed in both hind paws, with little or no effect on pain threshold. Conversely, d-propoxyphene HC1produced analgesia in both hind paws even though inflammation and increased paw temperature were not significantlv reduced. Adjuvant-induced arthritis .Acetyl salicylic acid d-Propoxyphene HC1 Indomethacin Paw volume Paw temperature Pain threshold Rectal temperature Body weight gain

1. Introduction
Adjuvant-induced polyarthritis in the rat was first described by Pearson (1956). Since that time several investigation into the clinical development (Pearson, 1963; Newbould, 1963; Ward and Cloud, 1966; Weimer and Humelbaugh, 1967; Glenn et al., 1967; and Katz and Piliero, 1969), histopathology (Pearson and Wood, 1963; Burstein and Waksman, 1964; and Glenn and Gray, 1965) and sensitivity of this model to clinically useful anti-arthritic drugs (Newbould, 1963; Graeme et al., 1966; and Winter and Nuss, 1966) have established that adjuvant arthritis is the best available animal model of rheumatoid arthritis. During the development of adjuvant arthritis it has been well documented that paw volume (Pearson, 1956; and Newbould, 1963) and temperature (Walz et al., 1971) significantly increase while body weight

gain is markedly retarded (Newbould, 1963). However, it has not been established to what extent pain threshold in these animals might be altered or if core body temperature is affected by the disease. The purpose of the following investigation was to determine if any correlation(s) existed among the pathological changes mentioned above in adjuvant arthritis and how various non-steroidal anti-inflammatory and analgesic agents might affect these alterations.

2. Materials and methods

Adjuvant arthritis was induced in male SpragueDawley rats (Charles River) weighing 140-180 g at the start of the experiment by the subplantar injection of heat-killed Mycobacterium tuberculosis (Newbould, 1963) into their right hind paws (0.1 ml of a

R.D. Sofia et aL, Pathological changes in adjuvant arthritis

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5 mg/ml suspension in mineral oil). Non-arthritic control animals were sham-injected. A total of 10 rats were used in each experimental group. Appropriate concentrations of acetyl salicylic acid, indomethacin or d-propoxyphene. HC1 were blended into a commercial diet and fed ad libitum. In addition, water was continuously available to the animals. Group mean daily drug dosage was calculated from food consumption and body weight data. The following parameters were measured at weekly intervals (Day 0, 7, 14 and 21) during the course of the experiment: (a) hind paw volume to the level of the lateral malleolus by water displacement; (b) hind paw temperature by means of a Telethermometer (model 43 TD Yellow Springs Instrument Corporation, Yellow Springs, Ohio, U.S.A.) and a 'bango' thermistor probe; (c) rectal temperature using the same Telethermometer, but a regular thermistor probe inserted a constant depth of 3.5 cm; (d) pain threshold in g of pressure using an 'Analgesy-Meter' (Ugo Basile, Milan, Italy); and (e) body weight. On Day 0 these measurements were made just prior to injection of M. tuberculosis. Group mean (-+ S.E.M.) volumes were determined at each test interval and the Student's t-test used to statistically evaluate the data.

3. Results Fig. 1 clearly demonstrates that as the volume of the injected hind paw of arthritic rats increased pain threshold was substantially reduced, while a sharp rise in temperature also occurred. It should also be noted that the elevation in paw volume and decrease in pain threshold were significantly greater at each succeeding testing period. However, paw temperature of the arthritic rats reached peak elevation by Day 14. The paw volume and temperature and pain threshold in sham-injected non-arthritic control rats did not significantly change throughout the course of the experiment. A similar relationship between increases in paw volume and temperature of the contralateral, uninjected (Fig. 1, left) paw of arthritic rats was evident (right panel). These changes, although highly significant, were not of the magnitude exhibited by the injected paw. On the other hand, pain threshold in this paw was not significantly altered at any testing time. Table 1 shows the effect of acetyl salicylic acid, indomethacin and d-propoxyphene. HC1 on these various changes in adjuvant arthritic rats measured on Day 21 of the experiment. The 0.1% concentration of acetyl salicylic acid (82.0 mg/kg/day) in the diet did not significantly alter any of the manifestations of the disease in the injected hind paw. However, the elevations of paw volume and temperature were significantly reduced by 0.2% acetyl salicylic acid in the diet when compared to the arthritic control group, but did not return to the level of the non-arthritic animals. It is interesting that even the high dose of acetyl salicylic acid (163.0 mg/kg/day) had no effect on the lowered pain threshold. On the other hand, indomethacin resulted in a dose-dependent reversal of all pathological changes associated with the uninjected paw. Both concentrations of this drug significantly reduced paw volume while only the highest concentration (0.002%, 1.6 mg/kg/day) significantly reduced paw temperature and brought the pain threshold back to that of the non-arthritic control group. As expected, d-propoxyphene. HC1 had no significant therapeutic effects on the inflammatory and local pyretic reactions of the injected paw, but the reduced pain threshold was significantly reversed. The effect of these drugs on Day 21 volume and

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Fig. 1. Alterations in volume, pain threshold and temperature of injected and uninjected hind paws of non-arthritic ( e - - - e ) and arthritic ( o - - - o ) rats measured at weekly intervals during the development of adjuvant-induced athritis.

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R.D. Sofia et al., Pathological changes in adjuvant arthritis

Table 1 The effect of acetyl salicylic acid, indomethacin and d-propoxyphene.HCl on various pathological changes associated with adjuvantinduced arthritis in rats. Treatment group a [Drug] in diet (%) Actual mean dosage consumed (mg/kg/ day) Injected hind paw Day 21 volume (ml)b Day 21 pain threshold (% of Day0) c A Paw temperature (c)b (Day 21Day 0) Uninjected hind paw Day 21 volume (ml) b Day 21 pain threshold (% of Day0) c A Paw temperature (C) b (Day 21Day 0) Body weight gain (g)b

(Day 21Day 0)

0.1 0.2 Indomethacin 0.001 0.002 d-Propoxyphene. HC1 0.015 0.03

Non-arthritic control Arthritic control Acetyl salicylic acid

82.0 163.0 0.8 1.6 11.6 25.7

2.30 5.43 4.76 3.15 3.85 2.96 5.59 5.61

0.08* 0.24 0.23 0.12" 0.21" 0.18" 0.52 0.43

92* 64 57 65 55 96* 90* 93*

0.1 4.0 3.8 1.8 4.0 1.1 3.8 3.6

0.1" 0.6 0.4 -+0.6* 0.7 0.3* 0.8 0.7

2.31 0.08* 3.84 0.23 3.38 -+0.21 2.56 0.07* 2.93 0.14" 2.28 0.10" 3.76 0.22 3.38 0.30

98 94 105 114 107 107 145" 140"

0.4 0.1" 3.8 0.7 3.8 0.4 2.3 0.4* 2.8 -+ 0.6 1.2 -+ 0.4* 3.8 0.7 3.1 0.7

149 56 69 94 96 106 73 73

3.8* 7.7 4.3 -+ 7.8* 2.8* 8.8* 9.2 6.1

a n = 10 rats per group. b Data expressed as the mean -+ S.E.M. c Mean pain threshold of the group on Day 21 expressed as a % of the mean pain threshold of that group on Day 0. * p < 0.05 when compared with the arthritic control group.

temperature measurements o f the contralateral uninjected hind paw were remarkably similar to those resuits observed on the injected paw. Paw volume was significantly reduced by the 0.2% d r u g - d i e t admixture o f acetyl salicylic acid, while both concentrations o f indomethacin were effective in a dose-related fashion. Moreover, the highest concentration o f indomethacin (0.002%) completely suppressed edema formation. Neither concentration o f d-propoxyphene HC1 significantly affected inflammation in the uninjected paw. Similarly, only the highest concentration o f acetyl salicylic acid and indomethacin had some beneficial local anti-pyretic action in this paw. Pain threshold in the uninjected hind paw o f untreated arthritic rats was not reduced (fig. 1, table 1). Moreover, neither d r u g - d i e t admixture o f acetyl salicylic acid or indomethacin resulted in significant alteration of this parameter. However, both 0.015 and 0.03% d - p r o p o x y p h e n e . HC1 significantly elevated Day 21 pain threshold in the uninjected inflamed hind paw when compared to both the nonarthritic and arthritic control groups. Table 1 also points out that body weight gain in

the arthritic control rats was significantly retarded when compared to the non-arthritic control group. Of the drugs studied only the highest concentration o f acetyl salicylic acid and both doses of indomethacin partially restored body weight gain. Finally, rectal temperature was not significantly changed in any group during the course o f the 21 day experiment.

4. Discussion
Results o f the present investigation point out the fact that some good correlations exist between various pathological changes occurring in adjuvant-induced arthritis in rats. For instance, closely associated with the primary lesion o f the disease, i.e., marked elevation in the volume of the uninjected paw, were an elevation in paw temperature and increased sensitivity to a painful or noxious stimulus. Similar changes, although o f a lesser magnitude, were observed in the secondary lesion site (uninjected paw) with the exception that pain threshold was not signif-

R.D. Sofia et al., Pathological changes in ad/uvant arthritis

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icantly altered. The reason(s) for this observation are not understood. This finding was quite surprising since one might speculate that at least on Day 21, when the elevations in volume and temperature of the uninjected paw were nearly identical to those values obtained on Day 7 in the injected paw, a similar reduction in pain threshold might have occurred. Dose-dependent reductions in paw volume and temperature of both the injected and uninjected hind paws were observed in the acetyl salicylic acid- and indomethacin-treated arthritic groups, thus confirming the work of Walz et al. (1971). Likewise, a partial restoration of body weight gain was also noticed in these groups. On the other hand, only the highest concentration of indomethacin (0.002%, 1.6 mg/kg/ day) used was able to restore pain threshold in the injected paw to the level observed in the non-arthritic control group. It is interesting to note that in those rats treated with the high dose of indomethacin paw volume was essentially the same as the non-arthritic control group. On the other hand, neither acetyl salicylic acid nor indomethacin significantly affected pain threshold of the uninjected hind paw. Neither concentration of d-propoxyphene-HC1 had beneficial effects on paw volume and temperature changes or retardation o f body weight gain associated with the primary or secondary lesions o f adjuvant arthritis. Unlike the correlation between reduction in paw volume and restoration of 'normal' pain threshold in the injected paw of arthritic rats receiving the high concentration of indomethacin, both concentrations of d-propoxyphene .HC1 returned pain threshold to non-arthritic control level even though no reduction in swelling was observed. Moreover, pain threshold in the contralateral uninjected paw was significantly elevated by both doses o f d-propoxyphene HC1. These data support the findings of Winter and Flataker (1965) that analgesics acting through central or peripheral mechanisms could be distinguished by their effect on injected or uninjected paw pain thresholds using the Randall-Selitto paw pressure test. These investigators reported that morphine elevated pain threshold in both paws, whereas drugs such as acetyl salicylic acid and indomethacin increased pain threshold only in the injected paw. It has been clearly demonstrated by the elegant experiments o f Perper et al. (1971) that through manipulation of the adjuvant arthritis test one can

selectively differentiate between effective anti-inflammatory and immunosuppressive agents. Hence, the resuits of the present investigation point out that useful information concerning the potential analgesic activity of a given compound and insight to its mechanism of action might also be determined in the adjuvant arthritis test.

Acknowledgments The authors wish to thank Merck Sharp & Dohme, West Point, Pennsylvania and Eli Lilly and Company, Indianapolis, Indiana for the generous supplies of indomethacin and d-propoxyphene HC1, respectively, used in this investigation. References Burstein, N.A. and B.H. Waksman, 1964, The pathogenesis of adjuvant disease in the rat. I. A histologic study of early lesions in the joihts and skin, Yale J. Biol. Med. 37,177. Glenn, E.M. and J. Gray, 1965, Adjuvant-induced polyarthritis in rats: biologic and histologic background, Amer. J. Vet. Res. 26, 1180. Glenn, E.M., B.J. Bowman, W. Kooyers, T. Koslowske and M.L. Myers, 1967, The pharmacology of 2,3-bis-(p-methoxyphenyl)-indole (indoxole), J. Pharmacol. Exptl. Therap. 155,157. Graeme, M.L., E. Fabry and E.B. Sigg, 1966, Mycobacterial adjuvant periarthritis in rodents and its modification by anti-inflammatory agents, J. Pharmacol. Exptl. Therap. 153,373. Katz, L. and S.J. Piliero, 1969, A study of adjuvant-induced polyarthritis in the rat with special reference to associated immunological phenomena, Ann. N.Y. Acad. Sci. 147, 515. Newbould, B.B., 1963, Chemotherapy of arthritis induced in rats by mycobacterial adjuvant, Brit. J. Pharmacol. 21, 127. Pearson, C.M., 1956, Development of arthritis, periarthritis and periostitis in rats given adjuvants, Proc. Soc. Exptl. Biol. Med. 91, 95. Pearson, C.M., 1963, Experimental joint disease (observations on adjuvant-induced arthritis), J. Chron. Dis. 16,863. Pearson, C.M. and F.D. Wood, 1963, Studies of arthritis and other lesions induced in rats by the injection of mycobacterial adjuvant. VII. Pathologic details of the arthritis and spondylitis, Amer. J. Pathol. 42, 73. Perper, R.J., B. Alvarez, C. Colombo and H. Schroder, 1971, The use of a standardized adjuvant arthritis assay to differentiate between anti-inflammatory and immunosuppressive agents, Proc. Soc. Exptl. Biol. Med. 137,506. Walz, D.T., M.J. DiMartino and A. Misher, 1971, Adjuvantinduced arthritis in rats. II. Drug effects on physiologic,

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biochemical and immunologic parameters, J. Pharmacol. Exptl. Therap. 178,223. Ward, J.R. and R.S. Cloud, 1966, Comparative effect of antirheumatic drugs on adjuvant-induced polyarthritis in rats, J. Pharmacol. Exptl. Therap. 152, 116. Weimer, H.E. and C. Humelbaugh, 1967,,The effects of periodic challenge on the response of q-AP globulin and

other acute-phase reactants of rats serum to tissue injury, Can. J. Physiol. Pharmacol. 45, 241. Winter, CA. and L. Flataker, 1965, Reaction thresholds to pressure in edematous hind paws of rats and responses to analgesic drugs, J. Pharmacol. Exptl. Therap. 150,165. Winter, C.A. and G.W. Nuss, 1966, Treatment of adjuvant arthritis in rats with anti-inflammatory drugs, Arthritis Rheum. 9.394.