Gross and histomorphological effects of therapeutic ultrasound (1 Watt/Cm2) in exprerimental acute...

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GROSS AND HISTOMORPHOLOGICAL EFFECTS OF THERAPEUTIC ULTRASOUND (1 WATT/CM 2) IN

EXPERIMENTAL ACUTE TRAUMATIC ARTHRITIS IN DONKEYS**

K.I. Singh, PhD]; V.K. Sobti, PhD2; K.S. Roy, PhD 3

the superficial layer (devoid of chondrocytes) and the rest of the cartilage layers were histologically normal.

S U M M A R Y

Acute aseptic arthritis was induced in 8 healthy donkeys aged 3-4 years and weighing 80-100 kg. The animals were divided into two groups (A and B) of four animals each. Group A served as a control where as in group B pulsed ultrasound therapy was applied for 10 minutes @ 1 Watt/Cm 2 from day 2 to 8 after induction of arthritis. The gross changes in the joint capsule, synovial membrane and articular cartilage were quite mild in ultrasound treated animals as compared to controls. Microscopically, the joint capsule showed complete sloughing of the intimal layer and the subintimal layer showed severe inflammatory reaction or even complete necrosis of the fibrous capsule and synovial membrane in untreated animals. The joint capsule of ultrasound treated animals showed an advanced healing stage of the synovial membrane though still some inflammatory reaction was present in the subintimal layer. Synovial membrane of untreated animals showed less of acid mucopolysaccharides material and more of neutral mucopolysaccharides as compared to treated animals. Calcium deposition was not detectable in the joint capsule of the treatment group. Degeneration of articular cartilage was observed microscopically in control animals as marked by fibrillation and desquamation of perichondrial tissue with necrosis of chondrocytes in different layers or even complete sloughing of the articular cartilage. Articular cartilage of ultrasound treated animals had mild changes in

Authors' addresses: 1Department of Veterinary Surgery and Radiology, 2Department of Clinics and Continuing Education; 3Department of Anatomy and Histology, Punjab Agricultural University, Ludhiana-141004, India. **A part of Ph.D. dissertation submitted by first author to Punjab Agricultural University, India.

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

After intra-articular or systemic medication, physiotherapy procedures constitute the second most common form of treatment for different arthropathies in different species of animals. Ultrasound therapy which is non inva- sive has been used widely with a good success rate for treatment of arthritis in dogs, sheep and calves. 17 How- ever, the detailed investigations on the beneficial effects of this form of physiotherapy in the management of equine arthritis are still lacking. The study was undertaken to assess the cellular effects of therapeutic ultrasound (1 Watt/ Cm a) in acute arthritis in donkeys.

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

The present study was designed using 8 clinically healthy donkeys, aged 3-4 years and weighing 80-100 kg. A fortnight before the start of the experiment, all animals were given 2.5 ml of adsorbed tetanus toxoid a intramuscu- larly. Deworming was also done with albendazole b @ 5 mg/kg body weight. During the experimental period, the managemental and feeding practices were similar and constant for all the animals. A day before the start of the experiment, the donkeys were clinically examined along with radiography of the carpal joints and declared fit for the experiment. The left carpal joint area was shaved and painted with povidone-iodine. ~

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

150 JOURNAL OF EQUINE VETERINARY SCIENCE

Figure 1. Intercarpal joint from donkey with induced acute arthritis showing hemorrhagic synovial membrane (SM) and thin eroded (er) hemorrhagic articular carti lage (AC).

In all the animals, the left carpal joint was prepared aseptically. After 2-3 washings, about 1 ml of lignocain hydrochloride e was injected subcutaneously in the depression felt over the intercarpal joint at the dorsomedial aspect of the carpus. This was followed by final preparation of the carpal joint and painting with the povidone-iodine antisep- tic solution. In standing position, the left knee was flexed and the intercarpal joint was entered in the depression on medial to extensor carpi radialis tendon with a disposable 1 inch 20 gauge needle. About 2 ml of synovia was aspirated with a disposable syringe. After collection of synovia, 0.5 ml of turpentine oil mixed with 0.5 ml of gentamycin f was injected into the intercarpal joint through the same needle. The animals were left loose to move around freely on the subsequent days till the end of the study.

All eight animals were randomly divided into two groups (A and B) of four animals each. Group A animals were kept as control. Group B animals were treated with 1Watt/Cm 2 pulsed ultrasound therapy (keeping slow circu- latory movement of the transducer) 48 hours after the induction of acute arthritis. The application time was for l0 minutes daily for 7 successive days.

All the animals of both groups were sacrificed on the 30th day after induction of arthritis. The affected carpus and contralateral carpus were harvested by cutting the proximal and distal bones to the carpus. The joints were de- skinned with a No. 20 scalpel blade. The intercarpal joints of both the affected and normal joints were opened care- fully, avoiding any damage to the articular cartilage. The

aAdsorbed Tetanus Toxoid I.P., Bio Vaccines Pvt. Ltd., 332 Chevella 501503, A.P., India bAnalgon| Wockhardt Veterinary Ltd., Dr. Annie Besant Road, Bombay 40018. CMicroshieid* PVS-S, Johnson and Johnson, NR Jet Enterprises Ltd., 64- 66 Senapati Bapat Marg, Bombay 40016, India. dOil Turpentine I.P., Vivek Pharma, 20, Champsi Bhimji Road, Mazagaon, Bombay 40010, India eLignocain Hydrochloride I.P. Injection, BAIF Laboratories Ltd., Wagholi- 412207, Pune, Maharashtra, India fGentim Vet. Merind Limited, New India Centre, 17 Cooperage Road, Bombay 400039.

Figure 2, Intercarpal joint from donkey with induced acute arthritis and treated with 1 Watt/Cm 2 ultrasound therapy showing hemorrhagic synovial membrane (H) and smooth articular cartilage. joint capsule and the articular cartilage were observed for any gross pathological change.

To study the histopathological and histochemical changes, tissue samples of articular cartilage and joint capsule of the diseased joints as well as contralateral normal joints were harvested. Articular cartilage was col- lected from the base with a fine saw. All the samples were fixed in 10% buffered neutral formalin and in Bouin's fixative. After processing, 5 micron thick sections of col- lected tissues were obtained and stained with:

1. Hematoxylin and Eosin stain for histomorphological studies. 8

2. Alcian blue (AB) / Periodic Acid Schiff (PAS) stain to study acidic and neutral mucopolysaccharides at pH-1. 8

3. Masson's trichrome stain to study fibrous tissue/ collagen. 8

4. Best 's Caramine stain to demonstrate glycogen. 8 5. Vonkossa stain counter stain with Nuclear Fast

Red to demonstrate calcium deposits. 8

RESULTS

Macroscopic Findings In group A, the affected carpal joint showed thickened

periarticular tissue and joint capsule. The synovial membrane had hemorrhagic spots with blood clots present all around its surface. A mild swelling of the periarticular tissue and joint capsule was observed in group B. The synovial membrane of this group was mildly hemorrhagic and showed proliferation at one or two places in one animal of this group. The articular cartilage of the affected joints in group A was reddish black in appearance. The cartilage became thin and at many locations, it was eroded with hemorrhagic spots (Fig. 1). Reddish fibrinous material was also seen adhered to the surface. The articular cartilage of group B was smooth without any erosion, whitish yellow with appreciable shine (Fig. 2). Few focal hemorrhagic

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Figure 3, Section of joint capsule from the donkey with induced acute arthritis showing necrosis of intima, edema (E) and necrosis (N) in the subintimal layer. H & E x70.

Figure 5. Section of articular cartilage of the intercarpal joint from donkey with induced acute arthritis showing marked degenerative changes and complete sloughing off the articular cartilage (arrow). Masson's Trichrome x 70.

Figure 4. Section of joint capsule from the donkey with induced acute arthritis showing necrosis of synovial membrane and joint capsule. H & E x70.

spots were present on the articular surface of the carpal bones and some fibrinous material was sticking to it.

Microscopic Observation of Joint Tissue In group A, an intimal layer of synovial membrane was

completely sloughed off. The subintimal layer showed edema and leukocytic infiltration (Fig.3). There were in- creased numbers of blood vessels and hemorrhagic spots. The deeper layer of the joint capsule was also edematous and connective tissue bundles appeared to be loosened. In one animal, the synovial membrane and part of fibrous capsule showed necrosis. At certain locations even the calcified mass was encountered over tunica intima and in the subintimal layer (Fig.4). The tangential layer of articular cartilage in this group showed marked fibrillation and desquamation of perichondrial tissue (Fig.5). The chondrocytes were necrosed. The intermediate layer showed vacuolation and necrosis of chondrocytes and the lacunae could be seen without chondrocytes. The deeper layer also showed necrosis of chondrocytes and separation from the underlying calcified layer was evident (Fig.6). The osseocartilgenous tissue was a little disorganized. In one

Figure 6. Section of articular cartilage of the intercarpal joint from donkey with induced acute arthritis showing degeneration of articular cartilage and its separation from underlying calcified layer (arrow). H & E x 70.

animal, there was complete sloughing of the articular cartilage leaving the calcified cartilage exposed.

The joint capsule from group B showed loss of the tunica intima at places. The subintimal tissue had necrotic masses along with granulation tissue. Inflammatory cells could be seen around the necrotic mass. Mild hemorrhage was also seen in the subintimal tissue. The fibrous layer was normal with some adipose tissue. However, some tissue sections of this group had normal intimal tissue. The subintimal layer had numbers of mesenchymal cells (Fig.7) and locations where there was hemorrhagic spots and inflammatory reaction. In these tissue sections, the intimal tissue had large numbers of infoldings. The tangential layer of articular cartilage was devoid of chondrocytes whereas an intermediate layer had normal chondrocytes (Fig.8). The radiate layer was almost normal but the number of chondrocytes were less and the ground substance was less eosinophilic. There was a clear cut zone between the intermediate layer and basal radiate layer of hyaline cartilage. The calcified layer of articular cartilage was almost normal.


Figure 7, Section of joint capsule from the donkey with induced acute arthritis and treated with 1 Watt/Cm 2 ultrasound therapy, showing mesenchymal tissue (m) in subintima and infolding (I) of synovial membrane. H & E x 70.

Figure 9, Section of joint capsule from donkey with induced acute arthritis showing loss of alcinophilic material and increase in PAS positive materials. AB/PAS x 70.

Figure 8, Section of articular cartilage of intercarpal joint from donkey with induced acute arthritis and treated with 1 Watt/Cm 2 ultrasound therapy showing tangential layer (T) devoid of chondrocytes. H & E x 70.

Histochemical Changes Synovial membrane from group A animals showed

loss of acid mucopolysaccharides but it had more neutral mucopolysaccharides (Fig. 9). Over all, the reaction to the stain was more poor than normal. In the articular cartilage of this group, the acid mucopolysaccharide material was more in the intermediate layer. It was less in both the tangential and radiate layer. The neutral mucopolysaccharides could not be seen in the matrix of lacunae and chondrocytes.

The joint capsule of group B animals had a low content of acid and neutral mucopolysaccharides particularly in the intirna and subintima (Fig. 10). The distribution of acid and neutral mucopolysaccharides in articular cartilage was comparable to normal.

As compared to normal joint capsule, the intima, subintima and fibrous layer of joint capsule in group A showed weak reaction of glycogen. A loss of glycogen in chondrocytes of articular cartilage was seen. A weak

Figure 10. Section of joint capsule from donkey with induced acute arthritis and treated with 1 Watt/Cm 2 ultrasound therapy showing mixture of weak AB/PAS reaction. AB/PAS x 70.

reaction in the intimal and subintimal layer was also seen in group B but there was a slight increase in the glycogen content of chondrocytes of articular cartilage.

In group A, large amounts of calcium deposit could be seen in the intimal and subintimal tissue and in a few areas of the fibrous capsule (Fig. 11). However, no calcium could be seen in any part of articular cartilage except ossified cartilage which is a normal site of deposition (Fig. 12).

In ultrasound treated animals, the joint capsule and articular cartilage were devoid of any calcium deposit (Fig.13). However one specimen had deposition of calcium on the intima of synovial membrane.

DISCUSSION

On gross examination, the periarticular tissue includ- ing the joint capsule was found to be thickened and the synovial membrane severely hemorrhagic in the control


Figure 11. Section of joint capsule from donkey with induced acute arthritis showing calcium deposition (CA) in intimal and subintimal layers of synovial membrane and focal deposition if fibrous capsule. Vonkossa counter stain with Nuclear Fast Red x 70.

animals. The gross changes in the fibrous joint capsule and synovial membrane were much milder in ultrasound treated animals. As compared to the reddish black extensively eroded thin and hemorrhagic articular cartilage of control animals, the gross appearance of articular cartilage of the treatment group was close to normal though it was whitish with slightly less shine. In equines, acute aseptic arthritis resulted in yellow to yellow brown discoloration, 9 hemorrhage and thickening of the joint capsule. 911 The joint capsule has been reported to be thickened with hemorrhage in acute aseptic arthritis of calves and dogs with the articular cartilage grossly thinned and eroded in calves.7'~ 2,~ 3 The treatment with therapeutic ultrasound in the above study improved the condition of the joint tissue as observed in the present study.

The microscopic changes of the joint capsule of the control animals showed complete sloughing of the intima and subintimal layer evidenced by severe inflammatory reaction or even complete necrosis of the fibrous capsule and synovial membrane. In the same place, calcification of different layers of the joint capsule or calcification of the necrotic mass was also observed reflecting the severity of the inflammatory reaction produced by turpentine oil. The severity of these changes in the joint capsule of ultrasound treated animals was far less than those in the controls, showing advanced healing stages of synovial membrane and fibrous joint capsule though still some inflammatory reaction was present in subintimal tissue. The intimal surface showed large numbers of infoldings.

The synovial membrane of untreated animals showed loss of AB reaction and was more PAS positive, indicating loss of acid mucopolysaccharides. Animals treated with ultrasound therapy showed low acid and neutral mucopolysaccharide contents reaction in the intima and subintima but the fibrous layer seemed to be normal indicating healing of joint capsule was in progress.

Degenerative changes of the articular cartilage was

Figure 12. Section of articular cartilage of intercarpal joint from donkey with induced acute arthritis showing absence of calcium deposition in cartilage except calcified layer (C). Vonkossa counter stain with Nuclear Fast Red x 70.

Figure 13. Section of articular cartilage of intercarpal joint from donkey with induced acute arthritis and treated with lWatt/Cm 2 ultrasound therapy, showing absence of calcium deposition in cartilage except calcified layer (C). Vonkossa counter stain with Nuclear Fast Red x 70.

observed microscopically in the untreated animals. Similar changes in articular cartilage have been reported in various arthritic conditions in horses. 11,1418 Olsson 19 explained that chondromalacia, the earliest sign of degeneration of articular cartilage was due to decreased content of sulfated mucopolysaccharides in the ground substance as demonstrated by loss of alcinophilia and basophilia in the section. Articular cartilage of ultrasound treated animals had very mild changes in the tangential layer and the rest of the cartilage layers were histologically near normal. Thera- peutic ultrasound accelerates not only the resolution of the inflammatory processes by way of its thermal as well as non thermal effects, 2~ but also accelerates the healing process by increasing the rate of growth of replacement tissues at the site of injury, a3 Therapeutic ultrasound has also been demonstrated to be beneficial in the healing of the joint capsule and articular cartilage induced with chemical aseptic arthritis in calves as well as in dogs. 7,1a,13

Alcinophilic reaction was more in the intermediate


layer of articular cartilage of the control group indicating loss of sulfated / acidic mucopolysaccharides (chondroitin sulfate) in tangential as well as radiate layer of the articular cartilage. Acid mucopolysaccharides are responsible for holding collagen fibers together in the cartilage and hence maintain the integrity of the cartilage. 1~ Due to loss of acidic mucopolysaccharides, the cartilage becomes more prone to degenerative changes. 12 A very high degree of degenerative changes were noted in the articular cartilage of the control animals in the present study. Acidic and neutral mucopolysaccharides of articular cartilage of the ultrasound treated group was comparable to normal, showing complete healing of the articular cartilage in this group of animals. This could be possible because of the sup- pressed inflammatory reaction and healing of the joint capsule. In horses, loss of histochemical staining of amor- phous intercellular matrix was attributed to the loss of glycosaminoglycans and the increase in the degenerative changes in the articular cartilage? 1,~4-~8

As compared to the normal joint capsule, the synovial membrane of the animals of both groups showed a weak reaction of glycogen but there was no change in the outer fibrous layer. The decreased glycogen content of synovial membrane indicated poor nutrition to the synovial membrane. A loss of glycogen in the chondrocytes of the articular cartilage was seen in the control group whereas an improvement in the glycogen content of the chondrocytes of articular cartilage was seen in animals treated with ultrasound therapy. This indicates that the healing was progressive in animals receiving ultrasound therapy.

In the control animals, calcium deposition could be seen in various parts of the joint capsule. Subintimal calcium deposits had also been reported in traumatic arthritis in cattlefl 4 and in DJD in dogs. 2s This indicated advanced inflammatory changes in control animals in which necrosed tissue had a tendency to calcify. The joint capsule of ultrasound treated animals showed no calcium deposit reflecting normalization of the joint capsule.

From this study it was concluded that treatment with ultrasound therapy @ lWatt/Cm a resulted in satisfactory healing of joint tissue and the articular cartilage and, hence, it prevented the development of degenerative joint disease as observed in untreated animals.

R E F E R E N C E S

1. Borisov MS: Ultrasonic therapy of experimental aseptic synovitis in sheep: total protein and protein fractions in synovia. Sbornik Nauchnykh Trudor, Moskovskaya Veterinarnaya Akademiya 1972;62:78-79.

2. Chichhanov VP, Irenkov IP: Ultrasonic and cortisone therapy of acute aseptic synovitis in calves: cells in blood and synovia. Sbornik Nauchnykh Trudor, Muskovskaya Veterinarnaya Akademiya 1972;62:80-81.

3. Borisov MS:Total protein and protein fractions in synovial fluid and blood serum of sheep with acute aseptic synovitis: Influence of ultrasonic and hydrocortisone therapy. Trudy

Muskovskoi Veterinarnoi Akademii 1973;65:128-130. 4. Jankovic' -Zagorcic' A: Simultaneous application of DD

current (diadynamic current) and ultrasound in the therapy of lesions of the locomotor apparatus in the dogs. Acta Veterinariya, Yugoslavia 1976;26:195-201.

5. Borisov MS, Entuganov VG, Pechonova NN, Kuznechikova TL: Spontaneous osteoarthritis of the canine stifle joint: clinical findings, synovial fluid cytology and ultrasonic therapy. Sbornik Nauchnykh Trudov Moskovskaya Veterinarnaya Akademiya 1978;100:92-93.

6. Plakhotin MV, Borisov MS, Akopyan VB: Efficacy of ultrasonic therapy and hydrocortisone phonophoresis (for synovitis and arthritis in domestic animals). Veterinariya, Moscow USSR 1980; 10:52-53.

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

8. Luna LG: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed. New York, McGraw Hill Book Co., 1968.

9. 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;43: 51-56.

10. Welch RD, Watkins JP, De Bowes RM, Leipold HW: Effect of intra-articular administration of dimethylsulfoxide on chemically induced synovitis in immature horses. Am J Vet Res 1991 ;52:934-939.

11. Gustafson SB, Trotter GW, Norrdin RW, Wrigley PH, Lamar C: Evaluation of intra-articular administered sodium monoiodoacetate-induced chemical injury to articular cartilage of horses. Am J Vet Res 1992;53:1193-1202.

12. Bhatia R, Sobti VK, Roy KS: Gross and histopathological observations on the effects of therapeutic ultrasound in experimental acute chemical arthritis in calves. J Vet Med A 1992a;39:168-173,

13. Bhatia R, Sobti VK, Roy KS: Gross and histopathological observations on the effects of therapeutic ultrasound in experimental acute traumatic arthritis in calves. Indian J Anim Sc 1992b;62:517-520.

14. Mcllwraith CW, Van Sickle DC: Experimentally induced arthritis of the equine carpus: Histologic and histochemical changes in the articular cartilage. Am J Vet Res 1981 ;42:209-217.

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

16. Gustafson SB, Trotter GW, Wrigley PH, Norrdin RW, Lamar C: Evaluation of intra-articular sodium monoiodoacetate chemical injury to equine articular cartilage. Vet Surgery 1989; 18: 73-74.

17. Ono M, Okada K, Numakunai S, Ohshima K: Pathological studies on arthrosis in horses. Journalofthe FacultyofAgriculture, Iwate University. 1989; 19:93-106.

18. Trotter GW, Yovich JV, Mcllwraith CW, Norrdin RW: Effect of intramuscular polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage. Can J Vet Res 1989;53:224-230.

19. Olsson SE: Degenerative joint disease (osteoarthrosis):A review with special reference to the dog. J Small AnOn Pract. 1971 ;12:333-342.

20. Downer AH: Ultrasound therapy for animals. Mod Vet Pract 1976;57:523-524.

21. Siddiqui HD: Rehabilitation of orthopaedically disabled. Indian Med Gaz 1976;6:89-92.

22. Dyson M, Suckling J: Stimulation of tissue repair by ultrasound: A survey of mechanisms involved. Physiotherapy 1978;64:105-108.

23. Dyson M, Pond JB: The effect of pulsed ultrasound on tissue regeneration. Physiotherapy 1970;56:132-142.

24. Van Pelt RW: Traumatic arthritis in cattle. Am J Vet Res 1968;29:1883-1890.

25. Pedersen NC, Pool R:Caninejointdisease. Vet Clin North Am 1978;8:465-493.


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