Reviewed

CLINICAL, RADIOLOGICAL, HEMATOLOGICAL AND SYNOVIAL FLUID OBSERVATIONS ON THE EFFECT OF AN ANTIARTHRITIC DRUG (ART) IN

EXPERIMENTAL ASEPTIC ACUTE ARTHRITIS IN EQUINES

K.I. Singh, PhDt; V.K. Sobti, PhD2; A.K. Srivastava, PhD 3

inflammation and aided in normalization of various biochemical and cytological parameters both of synovia and plasma.

S U M M A R Y

Aseptic acute arthritis of the intercarpal joint was induced with 0.5 mt of turpentine oil in eight clinically healthy donkeys, aged three to four years and weighing 80- 100 kg. Group A (four animals) served as control whereas in Group B, an antiarthritic drug (ART) was administered orally @ 20 grams from days 2 to 26. As compared to controls, there was an early return to normal stance and weight bearing in the treatment group. The degree of severity of lameness persisted for a longer time in the control group as compared to treated animals. Synovial total leukocytic count progressively increased in the control group whereas the antiarthritic drug treatment caused a decrease in the synovial total leukocytic count. Synovial acid phosphatase activity gradually decreased in the antiarthritic drug treated animals. Synovial aspartate aminotransferase activity was near normal on the 30th day in animals given the antiarthritic drug. Plasma total protein, alkaline phosphatase and aspartate aminotransferase did not show any significant variation in either group. Synovial and plasma lactic dehydrogenase also did not show any significant variation in the treatment group. Overall, the administration of an antiarthritic drug caused reduction in

Authors' addresses: 1AsSistant Professor, Department of Surgery and Radiology, Centre of Advanced Studies; 2professor and Head, Department of Surgery and Radiology, Centre of Advanced Studies; aprofessor and Head, Department of Pharmacology rand Toxicology, Punjab Agricultural University, College of Veterinary Science, Ludhiana - 141004, India. Note: This manuscript is part of a Ph.D. Dissertation submitted by the first author to Punjab Agricultural University.

I N T R O D U C T I O N

Accidental injuries to the joint may lead to acute arthritis and lameness in equines. Any delay in treatment can lead to secondary degenerative joint disease (DJD) which many times becomes a permanent cause of disability. To treat this condition systemic, as well as intra-articular, medication of various drugs like corticosteroids, hyaluronic acid, polysulfated glycosaminoglycans, etc., have been attempted with varying results.l,2,3,4.5 An antiarthritic drug (ART) which is a herbal preparation containing Allium sativum, Cyperus rotundus and Zingiber officinalis has been acclaimed to have beneficial effects in arthritis in donkeys. 6,7 The present study was conducted to investigate the detailed effects of this drug in acute arthritis in equines.

M A T E R I A L S A N D M E T H O D S

Eight clinically healthy donkeys, aged three to four years and weighing 80-100 kg were given 2.5 ml of adsorbed tetanus toxoid a intramuscularly fifteen days before the start of the experiment. Deworming was also done with albendazole b at 5 mg/kg body weight. The management and feeding conditions were kept similar for all the animals.

Pilot trials were conducted on four additional donkeys to standardize the dose of turpentine oil c for induction of acute arthritis in the intercarpal joint and to establish the

Volume 19, Number 8, 1999 511

protocol of recording various observations/parameters. A dose of 0.5 ml of turpentine oil mixed with 0.5 ml of gentamicin sulfate (containing 20 mg of gentamicin) intra-articularly proved optimum to induce acute arthritis in donkeys.

In all the animals the left carpal joint was prepared aseptically. About 1.0 ml of lignocain d was injected subcutaneously in the depression felt over the intercarpal joint at the dorsomedial aspect of the carpus. In standing position, the left knee was flexed and the intercarpal joint was entered using a disposable 1.0 inch, 20 gauge needle. About 2.0 ml of synovia was aspirated. Thereafter, 0.5 ml of turpentine oil mixed with 0.5 ml of gentamicin was injected into the intercarpal joint through the same needle. The animals were allowed to move freely on the subsequent days till the end of the study.

After induction of acute arthritis, the eight donkeys were randomly divided into two groups A and B of four animals each. Group A served as the control in which no treatment was given. The animals of Group B were given 20 gms of ART e daily for 25 days, starting 48 hours after the induction of arthritis. The drug was mixed in a sugar base and given orally. As per recommendations of the manufacturers, 15 ml of Livol f was given orally seven days before the start of ART administration.

The parameters studied were: (a) Clinical Observations: The rectal temperature,

respiration and pulse rate and joint circumference (mid carpus) were recorded at days 0 (before induction of arthritis), 1 to 4, 9, 15, 20, 25 and 30 (before sacrificing the animal). The rest of the clinical observations (lameness, warmth of the affected carpal joint, pain on flexion, flexion angle (~ swelling of the carpus, general activity, standing posture and feed intake) were recorded daily from day 0 to 15 and then on days 20, 25 and 30. Lameness was recorded on walk and was graded as ++++ (no weight bearing on the affected limb), +++ (slight weight bearing on affected limb), ++ (moderate weight bearing), + (mild lameness) and - (normal walk), and scores of 4, 3, 2, 1 and 0 were awarded, respectively.

Warmth of the affected carpal surface was judged by placing the back side of the hand on the skin of the affected carpal surface and was graded as warm (+++), moderately warm (++), mildly warm (+) and normal temperature, as in the contra lateral limb, and scores of 3, 2, 1 and 0 were given, respectively.

To judge the pain on flexion, the affected carpal joint was flexed and the pain felt by the animal was classified as very severe (++++), severe (+++), moderate (++), mild (+) and nil (-). The scoring rates of 4, 3, 2, 1 and 0 were awarded, respectively. Flexion angle (~ was estimated by keeping the radius ulna as the base of the angle and flexing the leg at the carpal.

Swelling of the carpus was judged grossly and was rated as severe (+++), moderate (++), mild (+) and near

normal (-) with a scoring number of 3, 2, 1 and 0 was given, respectively.

(b) Blood and Plasma Analysis: Ten ml of venous blood was collected from the jugular vein in heparanized (1:1000 heparing used) glass vials on days 0 (before induction of arthritis), 2, 9, 20 and 30. It was checked for hemoglobin (Hb), total leukocyte count (TLC) and differential leukocyte count (DLC). The plasma was separated and stored in deep freeze at minus 20~ The biochemical parameters estimated were total protein (gin/ 100 ml of plasma); alkaline phosphatase and acid pho- sphatase (nmol phenol produced/min/ml of plasma); aspartate aminotransferase and alanine aminotransferase (nmol of pyruvate formed/min/ml of plasma) and lactic dehydrogenase (n mol/min/ml) by methods as described by Wooton. 8

(c) Synovial Analysis: The synovial fluid was collected aseptically in the heparinized glass vials. The schedule of sampling was the same as for the blood collection. It was examined for its appearance, viscosity (by placing a synovial drop on the thumb and then touching it with the index finger. Separating the fingers produced a string which was measured before breaking), clot or precipitate formation, TLC and DLC. The biochemical parameters estimated were the same as those of plasma.

(d) Radiography: Plain radiographs of the affected carpus in anterioposterior, lateral and lateral flexed views were taken on day 0 (before induction of arthritis), 20 and 30.

(e) Microbial Assay: To check the accidental microbial contamination of the joint, a drop of synovia was cultured in nutrient broth and blood agar at various intervals till the end of the experiment.

Statistical Analysis: Rectal temperature, respiration, pulse rate and joint circumference, hematological and biochemical parameters of blood and plasma and synovial cytological and biochemical parameters were subjected to analysis of variance (ANOVA) followed by a critical difference (CD) test. 9 A probability level of P< 0.05 was considered as statistically significant.

R E S U L T S A N D D I S C U S S I O N

All the synovial samples cultured in nutrient broth and on blood agar showed no microbial growth in either group indicating that the arthritis induced was of an aseptic nature in all the animals of both the groups.

The drug (ART) used for the treatment of acute arthritis was well tolerated by the animals as none of them showed any untoward clinical symptom like diarrhea, vomition or colic. The animals showed no resistance in swallowing the drug as it was palatable to them and there was no loss of drug during administration.

Turpentine oil, being an irritant in nature, caused acute

512 JOURNAL OF EQUINE VETERINARY SCIENCE

aseptic arthritis which closely simulated clinical traumatic arthritis as also reported by Bhatia. 1~ The joints appeared swollen three to four hours after injection of turpentine oil indicating severe inflammation of the joint. All the animals showed reduced feed intake till day 4, followed by normal intake tilt the end of the study.

The rectal temperature rose significantly (p < 0.05) for the first three days after induction of arthritis in both the groups (Table 1). The initial rise of rectal temperature could be due to acute inflammatory change in the joint. The respiration rate in ART treated animals did not show any significant variation at various intervals. However, in the control group it decreased significantly (p < 0.05) after day 3 as compared to the base value. On days 20 and 25, the respiration rate was near normal but on day 30 it again decreased significantly (p < 0.05). An initial significant (p < 0.05) rise in pulse rate after induction of acute arthritis was observed in animals of both the groups (Table 1). The pain caused by severe inflammation of the joint tissue might have contributed towards the increased pulse rate. However, the pulse rate was near normal after day 9 in ART treated animals but it remained significantly high (p < 0.05) even on day 30 in the control group animals.

After induction of arthritis, joint circumference

increased significantly (p < 0.05) from the base value at various recording intervals in both the groups and the increase was maximum on day 3 followed by gradual decrease on subsequent recordings till the end of the experiment (Table 1).

Due to severe inflammation, all the animals kept the leg flexed and the toe was just touching the ground after induction of acute arthritis. All the animals of both the groups did not use the affected leg during walking for the first two to three days due to severe soft tissue inflammatory changes. They were provided with comfortable soft bedding and were given assistance while getting up. The control group animals with severe pain were given a bolus of analgin as per the need to provide temporary relief. In the control group animals, the carpal swelling was severe on day 3 followed by moderate swelling till day 13 after induction of the acute arthritis. Thereafter it became mild till the last recording on day 30. In drug treated animals the carpus swelling was moderate from days 1-13 after induction of arthritis and thereafter milder swelling till day 20. The carpus appeared to be normal on days 25 and 30. The carpus swelling observations were confirmed by joint circumference recordings (Table 1). The control group animals showed severe lameness on the affected leg from

Table 1. SEM + of rectal tempera tu re (~ respirat ion rate (per min), pu lse rate (per min) and jo int c i rcumference (cm) at var ious record ing intervals in Groups A and B.

Day 0 1 2 3 4 9 15 20 25 30

Group

Rectal Temperature (~

A 97.35 100.85 a 100.55 a 99.30 a 97.00 96.55 bi 97.15 bcd 96.30 bcdj 96.85 bcd 98.50 bcd

_+1.56 +0,81 _+0.90 _+0.67 _+0.42 +0.59 _+0.78 _+0.41 _+0.82 _+0.68 B 98.55 101.30 a 101.20 a 100.90 a 99.80 99.00 bcd 99.10 bcd 97.55 bcde 97.65 bcde 97.83 bcde

_+1.28 -+1.02 +0.73 _+0.88 _+0.77 -+0.76 +_0.87 -+0.84 _+0.70 _+0.94

Respiration Rate (per min)

A 12.5 10.50 10.50 9.50 a 8.25 a 7.50 ab 8.00 a 10.00 10.00 8.50 a

-+1.71 _+0.50 -+1.26 _+0.96 -+0.25 +0.50 -+0.82 _+1.41 _+0.82 _+0.50

B 7.50 10.50 10.50 9.25 9.25 8.00 10.50 9.00 10.00 8.25

�9 +0.50 _+2.22 -+1.26 _+1.60 +_1.60 _+0.82 _+0.96 +0.71 +_1.15 _+1.31

Pulse Rate (per min)

A 35.00 57.75 a 73.00 ab 60.00 ac 46.00 bcd 42.00 39.00 bcd 39.50 bcd 44.00 bcd 50.00 ac

_+2.38 _+7.12 -+9.38 _+4.32 _+2.58 _+2.58 _+1.91 _+4.03 -+2.31 _+5.03

B 45.00 79.00 a 76.00 a 65.00 ab 59.50 abc 66.00 abc 52.00 bcd 52.00 bcd 50.00 bcd 49.00 bcd

+_6,61 +_3.00 _+9.80 -+8.06 _+5.44 _+11.83 _+7.66 +_7.83 +_9.31 _+6.61

Joint Circumference (cm) A 18.83 21.65 a 22.25 a 22.75 ab 22.60 ab 21.63 aed 20.80 acde 20.88 acde 20.80 acde 20.43 acde

_+.48 _+0.53 _+0.43 _+0.43 _+0.45 _+0,47 _+0.68 _+0.72 -+0.64 -+1.04

B 19.67 22.!3 a 22.80 a 23.27 ab 23.23 ab 22.20 abc 21.53 ade 21.47 ade 20.93 ade 20.67 ade

_+0,44 +_0.32 _+0.35 -+0.62 +0.64 -+0.82 -+0.97 _+0.98 _+0.72 _+0.60 a=Significant from day 0 b=Significant from day 1 c=Significant from day 2 d=Significant from day 3 e=Significant from day 4 h=Significant from day 20 i=Significant from day 30

Volume 19, Number 8, 1999 513

Table 2 . Mean + SEM of hemato log ica l parameters at var ious recording intervals in Groups A and B.

Day 0 2 9 20 30

Group

Hemoglobin (gm%)

A 9.28 9.00 8.95 7,30 8.53

+_0.49 _+0.76 +_0,40 _+0.37 -+0,42

B 8.73 9.05 7.83 9.38 7.90

+_0.26 +_0.84 +_.0.75 +_1.07 +0.06

Blood TLC (mm 3)

A 8150 11975 13300 8750 10200

+_1699 _+1280 +_1899 +676 +1347 A

B 9250 11850 12375 11000 8775

_+1594 +-87 ,+1449 ,+1243 _+1193 B

Neutrophils (%)

A 49 66 58 53 49

,+5 ,+2 +_3 ,+5 +_6

B 57 64 67 55 51 A

+_2 _+4 ,+6 +_5 ,+4

Lymphocytes(%) B

A 48 32 40 45 48

+_4 ,+2 ,+4 ,+5 ,+5

B 41 35 32 45 c 48 bc

+_2 -+5 ,+5 +_5 +_4

Monocytes(%)

A 2 1 2 2 2

+0 _+0 -+1 -+1 _+0

B 1 1 1 0 1

�9 +0 _+0 _+o +0 _+0

Eosinophils (%) A

A 1 2 1 1 2

+-1 +-1 +0 +0 -+1 B

B 1 2 1 1 1

+_0 +_1 +0 +_1 +_1

b=Significant from day 2; c=Significant from day 9,

days 4-9 and moderate lameness from days 10-15 and were mildly lame on days 20 to 30. The animals given ART showed an appreciable decrease in lameness (severe lameness on days 4 and 5 and moderately lame from days 6 to 11) as compared to control group animals and were seen walking normally on day 25. It showed that the drug ART helped in early resolution of the inflammation of the joint and its surrounding tissue.

The degree of pain on flexion increased with the passage of time after induction of arthritis in the control group animals and it was severely painful from day 8 onward till the end of the experiment. Administration of ART showed a gradual decrease in pain on flexion and it was very mild at the end of the experiment, further confirming its anti-inflammatory properties. The joint capsule is well innervated with proprioceptive pain end organs t~ andjoint reaction is associated with the release of prostaglandins resulting in pain and swelling} z Rose la reported that pain on flexion was one of the most consistent

Table 3 , Mean + SEM of p lasma biochemical parameters at var ious recording intervals in Groups A and B.

Day 0 2 9 20 30 Group

A

B

Total Proteins (gms/100 ml of plasma)

10.00 10.95 10.35 10.15 10.50

+-I.20 _+0.43 +-0.73 +_0,81 +1.18

14.98 11,60 9.80 11.88 12.10

+_2.79 _+1.14 +-0.47 +_1.05 +-0.60

Plasma alkaline phosphatase

(n mol phenol produced/min/ml of plasma)

131.83 68.61 119.28 72.77 142.45

+-27.69 + _ 1 2 . 7 0 +_34.35 +_7.26 +_24.95

93.13 106.32 125.11 116.27 92.93

+_13.36 + _ 1 4 . 3 9 + _ 2 7 . 5 4 • +_16.50

Plasma acid phosphatase

(n mol phenol produced/min/ml of plasma)

3.63 2.51 6.69 3.62 14.79 abcd

+-0.88 +-0.90 +3.68 +0.32 +_1.58

3.88 4.01 14.81 ab 12.27 ab 5.04 cd

+_1.01 +1.43 +_1.22 _+2.81 +_0.53

Plasma aspartate aminotransferase

(n mol of pyruvate formed/min/ml of plasma)

104.99 96.05 108.37 91.30 122.09

+_10.59 +_8.69 +_.9.34 +-7.75 +-16.87

97.28 93.77 78.40 89.08 109.16

+10.70 +12.41 +-11.47 +_3,93 +-6.19

Plasma alanine aminotransferase

(n mol pyruvate formed/min/ml of plasma)

31.90 32.46 33.46 21.31 a 21.09 a

+1.69 +_3.25 _+.5.87 _+5.04 _+2.02

28.36 32.66 16.95 18.51 14.81

+-3.13 +-3.19 +-7.30 +-4.97 +_2.65

Plasma LDH (n mol/min/ml)

109.09 278.31 a 319.12 a 275,00 356.23 a

+39.32 - + 1 0 . 9 1 +-12.30 -+43.10 +-7.21

135.67 181.76 207.68 174.56 235.58

+-27.88 -+43.79 + _ 5 3 . 6 7 +_44.31 +_47.69 a=Significant from base value b=Significant from day 2 c=Significant from day 9 d=Significant from day 20

clinical findings in acute arthritis of equines. The synovia collected on day 2 increased in volume

significantly (p < 0.05 ) as shown in Table 4 and changed from light straw color to dirty red and had flakes in both the groups. The change in color and the presence of flakes was suggestive of severe inflammation of the synovial membrane in both the groups. On subsequent collections, it decreased significantly (p < 0.05) in volume and was homogeneously red/sanguinous in color in both groups except for one animal of Group B in which its color turned towards normal. The decrease in synoviai volume in the later phase was indicative of reduced inflammation and the synovial membrane was in the reparative phase.

514 J O U R N A L OF E Q U I N E V E T E R I N A R Y S C I E N C E

Table 4. M e a n + S E M of synov ia l v o l u m e (ml), synov ia l v iscos i ty (cm) and synov ia l cy to log i ca l p a r a m e t e r s a t var ious record ing in te rva ls in Groups A and B.

Day 0 2 9 20 30

Group

Synovial Volume (ml)

A 1.17 1.98 a 0.33 ab 0.35 ab 0.63 b

-+0.63 -+0.10 -+0.11 -+0.10 -+0.26

B 1.28 2.15 a 0.48 ab 0.43 ab 0.31 ab

-+0.19 -+0.29 -+0.05 +0.06 -+0.06

Synovial Viscosity (cm)

A 1.20 0.35 a NIL NIL NIL

�9 +0.12 _+O.24

B 1.00 0.33 a NIL NIL NIL

�9 +0.00 -+0.33

Synovial TLC (mm 3)

A 2325 7225 a 30033 a 42200 ab 5422 c

-+289 -+1348 -+13517 _+14212 _+2326

B 2713 9362 a 4066 b 6213 a 3833

�9 +603 -+1033 +1257 _+1180 +2485

Synovial Neutrophils (%)

A 53 63 a 49 b 49 b 52

�9 +2 -+4 _+3 _+3 _+ 10

B 48 53 50 52 58 a

�9 +1 _+4 -+3 -+2 -+2

Synovial Lymphocytes (%)

A 47 36 50 51 47

+_2 _+4 -+3 -+3 -+5

B 51 47 49 48 41 a

-+1 _+3 _+3 _+1 -+2

Synovial Monocytes (%)

A 0 1 0 0 0

�9 +0 _+0 -+0 -+O -+0

B 0 0 O 0 1 �9 +0 -+0 -+0 +_0 +0

Synovial Eosinophils (%) A 1 1 1 0 3

�9 +0 _+0 _+0 _+0 _+0 B 1 1 1 1 1

�9 +0 -+0 -+0 -+0 -+0 aSignificant from base value b=Significant from day 2 c=Significant from day 9

The synovial viscosity decreased significantly (p < 0.05) on day 2 after induction of arthritis in all the animals of both the groups and was nil on subsequent intervals indicating either decreased polymerization of hyaluronic acid or increased alkaline phosphatase activity or decreased synovial mucin. 14,15,16 In dogs and horses, pathological or inflammatory conditions of the joint resulted in decreased viscosity.t6,17,~8

Synovial TLC increased progressively in the control animals and was significantly (p < 0.05) high from base till day 20 (Table 4). In the treatment group, it also increased significantly (p < 0.05) on day 2 (before the start of the therapy) but decreased subsequently as the treatment started

Table 5 . M e a n + S E M of synov ia l b i ochemica l p a r a m e t e r s a t va r i ous record ing in te rva ls in Groups A and B.

Day 0 2 9 20 30 Group

A

B

A

B

A

B

A

B

A

B

A

B

Synovial Total Proteins (gm/100 ml of synovia)

4.15 9.60 a 12.73 a 9.90 a 8.27 a +0.78 _+1.09 _+1.39 _+1.15 -+0.58

3.40 10.45 a 4.80 b 9.20 ac ND +0.73 -+1.27 -+1.39 -+0.81

Synovial alkaline phosphatase (n mol of phenol produced/min/ml of synovia)

41.68 246.41 a 331.46 ab 226.10 ac 238.37 a -+2.53 -+5.62 -+15.77 -+37.26 -+68.62 48.28 179.83 a 359.57 ab 276.16abc 168.68 acd �9 +8.84 _+26.97 -+34.21 -+12.53 -+35.92

Synovial acid phosphatase (n mol of phenol produced/min/ml of synovia) 4.15 31.91 a 29.87 a 28.21 a 16.51

�9 +1.30 -+5.39 -+4.65 _+7.45 -+8.42 2.84 32.16 a 24.87 a 14.73 b 11.43 bc

�9 +0.67 _+5.92 _+7.51 -+8.97 -+3.28 Synovial aspartate aminotransferase

(n mol of pyruvate formed/min/ml of synovia) 99.02 177.60 a 63.46 ab 115.10 bc 183.89 ac �9 +5.53 _+9.58 _+10.86 -+12.32 _+30.20

100.94 182.99 a 135.84 b 135.28 113.99 b �9 +8.83 -+7.26 -+9.82 -+44.33 -+26.99

Synovial alanine aminotransferase (n mol of pyruvate formed/min/mt of synovia)

107.82 323.72 a 726.75 ab 573.01 a 260.12 acd +23.48 +61.05 +27.42 +87.17 -+30.96 120.95 321.76 a 348.54 a 372.21 a 169.09 +13.94 +59.61 _+96.44 _+107.94 -+30.62

Synovial LDH (n mol/min/ml)

272.76 378.18 a 282.52 b 346.55 ac 314.17 b �9 +40.37 _+10.43 _+34.67 -+17.99 _+14.40 241.13 362.92 223.15 333.09 265.68 �9 +41.69 -+22.07 -+9.73 _+65.43 _+17.41

"=Significant from base value b=Significant from day 2 c=Significant from day 9 d=Significant from day 20 ND=Not determined

and was near normal at the end of the study. A quantitative change in leukocytes present in the synovial fluid provides an indication of the inflammation of the synovial membrane.19 The decreased value of TLC in the treatment group as compared to the control group was suggestive of the and-inflammatory effect of ART.

Blood TLC increased moderately in both the groups but at the end of the study it was near the base value in ART treated animals (Table 2). Blood differential cell counts showed insignificant variations at most of the observation periods and were comparable to the synovial differential count. A slight rise in blood TLC was indicative of mild systemic reaction to induced arthritis in the present study.

Synovial total proteins increased significantly (p < 0.05) on day 2 after induction of acute arthritis in all the animals of both the groups but remained significantly (p < 0.05)

Volume 19, Number 8, 1999 515

Figure 1. Thirty-day radiograph [anteroposterior (AP), lateral (L) and lateral flexed (LF) views] of donkey with induced acute arthritis showing increased soft tissue density (arrow) around the carpal joint.

increased at all the subsequent intervals in the control animals only (Table 5). In Group B, synovial total protein decreased on day 9 and was near the base value but again rose significantly (p < 0.05) on day 20. With increasing joint inflammation, the total synovial proteins tend to approach those of plasma. 2~ The increased total proteins in synovial fluid have been attributed to increased permeability of synovial membrane in equines and human beings.13,22,23,24,25 However, administration of ART caused a decrease in synovial total proteins as compared to the control group animals.

Synovial alkaline phosphatase (ALP) activity increased significantly (p < 0.05) at various recording intervals in both the groups (Table 5). However, the magnitude of increase was less in the treatment group as compared to controls. The increased alkaline phosphatase values of synovial fluid indicated inflammatory changes in the joint. The increased values of synovial ALP in turn might have also helped in reduction of synovial viscosity.IS However, plasma alkaline phosphatase did not show significant variations at various recording intervals in both the groups (Table 3).There was an early return of synovial acid phosphatase (ACP) activity to normal in ART treated animals as compared to that of controls of the present study (Table 5). Plasma ACP remained significantly high (p < 0.05) in the control animals on day 30 (Table 3). Synovial aspartate aminotranseferase (AAT) activity increased significantly (p < 0.05) two days (before the start of therapy) after induction of arthritis in both the groups and at the last observations in the control group (Table 5). Its value decreased gradually in ART treated animals and was near normal on day 30. Plasma AAT did not show any significant variation at various recording intervals (Table 3). Synovial alanine aminotranseferase (ALT) also increased at various recording intervals in both the groups (Table 5). Its activity decreased at the last observation in the treatment group. Plasma ALT activity was found decreased on day 20 and 30 in the control group, However, no significant

Figure 2. Thirty-day radiograph [anteroposterior (AP), lateral (L) and lateral flexed (LF) views] of donkey with induced acute arthritis showing decreased soft tissue density (arrow) around the carpal joint.

change was observed in the treatment group (Table 3). Synovial lactic dehydrogenase (LDH) activity found statistically increased (p < 0.05) on days 2 and 20 (Table 5) and plasma LDH up to day 30 (Table 3) in the control group animals. Synovial and plasma LDH values of ART treated animals did not show any significant variation. Overall results of this study indicate that synovial enzymic activity was comparatively less in the treatment group indicating less inflammatory changes in drug treated animals as compared to control group animals. The increased enzyme activity in the joint fluid may result from one of the several mechanisms which include the release of enzymes from leukocytes, from necrotic or inflamed synovial tissue and production and release of an increased amount of enzymes by altered synovial tissueY A positive correlation observed between the leukocytes in the field and the enzyme levels is considered as evidence for the release of enzymes from the leukocytes. A loss of semipermeability of the synovial membrane has also been correlated with the significant elevation of synovial enzymes like AAT, ALP and LDH in acute joint disease of horses. 25

The radiographic findings in the present study were visually compared. The radiographs showed increased soft tissue density after induction of arthritis in both the groups. It decreased appreciably in Group B as compared to controls on day 30 indicating resolution soft tissue inflammation with ART treatment, However, no bone density or new bone formation was observed till the end of the experiment in both the groups (Figs. 1 and 2). Increased soft tissue density was also observed in acute aseptic arthritis in horses. 533

In conclusion, the oral administration of ART was very easy, simple and convenient as compared to other cumbersome invasive techniques like intra-articular injection of various agents. ART treatment helped in decreasing periarticular swelling and arthralgia as evident by the early return to normal stance, decreased lameness andpain on flexion. Various synovial and blood biochemical

516 JOURNAL OF EQUINE VETERINARY SCIENCE

and cyto logica l parameters re turned to normal at the end of

the study jus t i fy ing its an t i - in f lammatory effects in acute

arthritis in equines .

F O O T N O T E S

aAdsorbed Tetanus Toxoid I.R, Bio Vaccines Pvt. Ltd., 332, Chevella 501503, A.P., India. bAnalgon| Wockhardt Veterinary Ltd., Dr. Annie Besant Road, Bombay-400018, India. cOil Turpentine I.P., Vivek Pharma, 20, Champsi Bhimji Road, Mazagaon, Bombay 400010, India. dLignocain Hydrochlodde I.P. Injection, BAIF Laboratories Ltd., Wagholi-412207, Pune, Maharashtra, India. eART (Antiarthritic drug), Indian Herbs Research and Supply Co. Pvt. Ltd., P. Box No.5, Shardanagar, Saharnpur-247001, U.R, India. fLivol, P.F.S, concentrate, Indian Herbs Research and Supply Co., 5-B, Veerasandra, Industrial Area, Bangalore-562158, India gHeparin Sodium Extrapure, S.d. Fine Chem. Ltd., Boisar-401501, India.

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21. Stashak TD: Adam's Lameness in Horses (4th ed) Philadelphia: Lea and Febiger, 1987;339-447.

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R E F E R E N C E S

1. Rydell NW, Butler J, Balazas EA: Hyaluronic acid in synovial fluid. IV. Effect of intraarticular injection of hyaluronic acid on the clinical symptoms of arthritis in track horses. Acta Vet Scand 1970; 11:139-155.

2. Auer JA, Fackelman GE, Gingerich DA, Fetter AW: Effect of hyaluronic acid in naturally occurring and experimentally induced osteoarthritis. Am J Vet Res 1980;41:568-574.

3. Yovich JV, Trotter GW, Mcllwraith CW, Norrdin RW: Effect of polysulphated glycosaminoglycan on chemical and physical defects in equine articular cartilage. Am J Vet Res 1987;48:1407-1414.

4. Collins EA: Use of polysulphated glycosaminoglycan in equine lameness. Vet Rec 1989;124:89-90.

5. Bowman KF, Purohit RC, Ganjam VK, Pechman RD Jr, Vaughan JT: Thermographic evaluation of corticosteroid efficacy in amphotericin B induced arthritis in ponies. Am J Vet Res 1983;44:51-56.

6. Singh KI, Sobti VK, Roy KS: Gross and histomorphological effects of antiarthritic drug in experimental acute arthritis in the donkey. Indian JAnim Sci 1997; 67:376-380.

7. Labana Amar Iqbal Singh, Sobti VK, Massarat Khan, Saigal RP: Histopathological studies on the synovial membrane of donkey with induced chronic arthritis following ultrasound therapy and antiarthritic drug treatment. Indian JAnim Sci 1998;68:1135- 1138.

8. Wooton IDP: Microanalysis in Medical Biochemistry(4th ed.) London: J & A Churchill Ltd., 1964.

9. Snedecor GW, Cochran WG: Statistical Methods (6th ed) Calcutta, India: Oxford and IBH Publishing Co., 1967.

10. Bhatia R: Studies on ultrasound therapy for induced traumatic arthritis in canines and bovines. MVSc Thesis submitted to Punjab Agricultural University, India, 1990.

11. Jubb KVF, Kennedy PC, Palmer N: Pathology of domestic animals (3rd ed) Vol 1. NY: Academic Press, 1985;91-98.

12. Dick WC, Proven C: Adrenergic control of canine synovial perfusion in experimentally induced osteoarthritis and synovitis. Res Vet Sci 1970;11:587-590.

13. Rose R J: The diagnosis and treatment of arthritis in horses. NZ Vet J 1983;31:13-15.

14. Van PeltRW: Propertiesofequinesynovialfluid. JAm Vet Med Assoc 1962; 141:1051-1061.

15. Van Pelt RW: Effect of alkaline phosphatase in vitro on the relative viscosity and mucin precipitate quality of bovine synovial fluid. Am J Vet Res 1971 ;32:1937-1943.

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