Hydrocortisone and exercise effects on articular cartilage in rats

463

Hydrocortisone and Exercise Effects on Articular Cartilage in Rats Prem P. Gogia, PhD, PT, Marybeth

Brown, PhD, PT, Saud AI-Obaidi,

PhD, PT

ABSTR.4CT. Gogia PP. Brown M, Al-Obaidi S. Hydrocortisone and exercise effects on articular cartilage in rats. .4rch Phys Med Rehabil 1993;74:463-7. l The combined effects of hydrocortisone and running exercise on articular cartilage were assessed in female SpragueDawley rats. Animals were divided into three groups: 12 control (C), 12 who received an injection of 0. I mL hydrocortisone once a week for three weeks (HC), and 12 rats who received three weekly injections of hydrocortisone and ran twice daily for six weeks (HC + run). Previous study revealed that rats that ran on a treadmill for three to 12 months did not have articular cartilage that was different from controls and thus a fourth group of rats, runners only, was not included in this analysis. .4t sacrifice, both knees were examined, photographed, and subsequently decalcified, then sectioned at 6~ and stained. HC t run rats had significantly more surface degeneration on femoral cartilage than HC or C rats. Eight of I2 HC t run rats displayed fibrotic invasion and/or subchondral bone replacement of degenerated articular cartilage, a feature not seen in HC or C rats. Cross-sections from HC t run rats displayed areas of cell death, and loss of matrix staining. Results suggest that, in rats, running esercise combined with intraarticular injections of hydrocortisone is more detrimental to articular cartilage than hydrocortisone or running alone. ‘~8 1993 by the American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and

Rehabilitation

In the past four decades. clinicians have used intraarticular corticosteroid injections for treatment of a variety of .ioint conditions such as osteoarthritis. rheumatoid arthritis.‘.’ acute traumatic arthritis, acute gouty arthritis, and synovitis. lntraarticular injection of corticosteroids have been reported to produce a remarkable antiinflammatorv. analgesic effect in patients with inflammatory signs.J Cor$costeroids offer relief through partial or complete remission of symptoms associated with joint diseases. However, a serious side effect. degenerative joint disease, has been reported following intraarticular injections given at intervals varying from one week to several months.“.5-4 Corticosteroids inhibit chondrocyte production of protein polysaccharides. which are the major constituents of articular cartilage ground substance.‘O Behrens and colleagues” reported a persistent and highly significant reduction in the synthesis of proteins, collagen, and proteoglycans in the articular cartilage of rabbits who received weekly injections of glucocorticoids.‘” They also reported a progressive loss of endoplasmic reticulum, mitochondria, and Golgi apparatus as the number of injections increased. Exercise has been reported to affect articular cartilage

positively bq increasing its thickness. enhancing the infusion of nutrients. and increasing matris synthesis.“‘” However. no report appears in the literature on the interrelationship between intraarticular corticosteroids and exercise. In the past few years. administration of intraarticular corticosteroids to reduce inflammation of an acute or chronic injury in athletes has become a common practice. Athletes typically return to full intensity sport activities within a feu hours to one to two days after receiving intraarticular corticosteroids. One possible outcome is that the deleterious effects of corticosteroids on articular cartilage may be exacerbated by strenuous sports activitier. The purpose ofthis study was to ‘determine the combined etfects of hydrocortisone and running exercise on articular cartilage in rats.

!METHODS Animals Thirty-six mature female Sprague-Hawley rats approximately XOg in weight (range. 183’ to 23g) were obtained from a hreedel” and housed in an appro! ed animal facility where chow and water were freei! available. Temperature (23°C) and hours of light and dark ( 13/l 2) were controlled. Upon receipt. rats were randomlh divided into three groups of 17 rats each. The first group of rats served as controls (C) and during the six-week protocol, C rats were handled daily. weighed weekly, and did not run. The second group also served as controls but were given a total of three in.iections of hydrocortisone acetate” (HC) spaced one week apart. The I2 rats in this group were given injections ofO.1 mL ( I .75mg/kg. a high normal dose) hydrocortisone into both knee joints.

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464

CORTISONE

AND EXERCISE

EFFECTS ON CARTILAGE,

Gogia

and femur were photographed en face through a dissecting microscope at a magnification of 16x. Care was taken while photographing to keep the cartilage surface moist using physiologic saline. After the right knees were photographed. the tibia and femur were placed in 10%)neutral buffered formalin. The left knee was removed intact and also placed in fixative. Both knees were decalcified.” embedded in paraffin, sagittally sectioned through the entire joint at 6~, and stained with hematoxylin and eosin (H&E) for routine morphology. The sections were also stained with alcian blue (pH 2.4) and periodic acid-schiff (PAS) combined to demonstrate the relative distribution of acid glycosaminoglycans (GAGS) and to determine chondrocyte loss, changes in matrix staining characteristics, fibrocystic or subchondral bony intrusion into the articular cartilage, fibrillation and cracking of cartilage. and osteophyte formation. Fig l--India ink technique. Ink adheres to areas of degenerated articular cartilage, X132. Femoral condyle from a cortisone injected control rat (He-only).

Injections were given at the beginning of weeks 1, 2. and 3 of the six-week study period. HC rats also were handled daily and weighed weekly, but did not run. Experimental animals received hydrocortisone injections and ran twice daily for six weeks (HC + run). Hydrocortisone injections were given at the same time (beginning of weeks 1.2. and 3) and dosage as HC rats. HC + run rats were also weighed weekly. A fourth group of rats, runners only, was not included at this time because a previous study (MB, unpublished data) revealed that voluntary running exercise for periods of between three months and one year had no significant deleterious effect on articular cartilage, when comparisons were made with sedentary age-matched controls. Age appears to be the only factor contributing to an increased amount of degenerative change. Therefore, inclusion of a six-week running group was not justified for this study.

Running Protocol HC + run rats ran voluntarily on a motorized treadmill for an hour each morning and afternoon. The initial speed of 12 m/min was increased to a moderate intensity (70% of HR maximum) of 19 m/min by the end of the second week. The 19 m/min speed was maintained for the duration of the study. Running time was increased from 35 minutes on day 1 to 60 minutes by day 5. Thus, HC + run rats ran for one hour twice daily for five of the six weeks of the study.

Data Management Obvious areas of cartilage degeneration (eg, erosion, pitting), as evidenced by India ink adherence, were counted on each tibia1 and femoral condyle. Counts were totaled to derive the number of lesions per tibia1 and femoral condyle for each group of rats. A I X 3 ANOVA was performed to determine significant differences between groups in the number of lesions per tibia and femur. The area of each lesion was obtained by tracing an outline of the lesion from photographs with a Hewlett-Packard digitizer’ programmed to provide area data in pm’. A 1 X 3 ANOVA was used to determine significant differences 0, < .05) in average lesion size for each group of rats. Twenty-four photographs ( 12 tibia, I2 femur) encompassing approximately 60% of the total tibia1 and femoral articular cartilage were taken from H&E stained sections for each animal. The number of chondrocytes per unit area ( 1.07mm’) was counted from a randomly selected segment of each photograph. This 1.07mm’ area was derived because of variations in cartilage thickness. The average number of chondrocytes/unit area was determined for each animal and group. A 1 X 3 ANOVA was used to determine if differences in cell count between groups were significant. Representative data are presented for tibia1 cartilage as femoral cartilage cell data were nearly identical. From the same 12 photographs. tibia1 and femoral cartilage thickness (area) was determined. If significance was achieved for any of the measures, a

Tissue Preparation All rats were killed with an intraperitoneal injection of sodium pentobarbital. The right knee of each rat was opened and the meniscus was removed. A coating of India ink was applied to the surface of the tibia and femur as described by Meachim. “India ink does not adhere to intact cartilage but ink will adhere to areas of cartilage degeneration and will fill holes (fig 1). Next the lndia ink coated tibia

Arch Phys bled Rehabil Vol74, May 1993

Table 1: Mean Animal Body Weight in Grams

Week 0

Week 6

X + SD

X + SD 337 1* 18)

Controls HC only

203 (k12) 1Y8(+12)

HC + Run

196 1*5)

Percentage Change

t17

“+o(k16)

t22.

244 (tl4)

i14

CORTISONE AND EXERCISE EFFECTS ON CARTILAGE, Gogia

465

Table 2: Average Number and Area of Degenerative Lesions \I).

of Rats With I ,cnion3

No. of <‘ondyles With I.csions

Scheffe post-hoc test was used to determine cant differenc~es lay.

where signih-

Total _

Lesions

Average

il,esion TArea/Cnndyle

Ohservcd

cartilage

ti

(pm*)

SD)

Thickness

No differences the three groups.

in cartilage

thickness

were observed

for

RESULTS All cortisone in.jected week period of study to and HC t run animals group (table I ). 411 HC ning protocol. cartilage

rats gained weight during the sixthe extent that weight gain for HC was comparable to that of the C t run animals completed the run-

Breakdown

Some of the C rats displayed small areas of cartilage degeneration. but not to the degree as HC and HC + run animals (table 2). An increase in the number of rat condyles with lesions on tibia and femur was observed for both groups of hydrocortlsone injected rats but signiticance was achieved only for HC + run animals. Significantly more areas of cartilage degeneration and a greater area occupied by degenerative change were seen on the femur of HC + run rats (table I!). There was a trend for the number of tibia1 areas with obvious breakdown to be greater in the injected rats when compared to C animals. Significant ditferences in the area occupied by degenerative lesions on thr tibia were not observed.

Cell Loss Empty lacunac suggesting chondrocyte loss was consistentI> obser\cd in all HC+run rats. Retrospective examination of slides revealed 100% accuracy for two examiners (PPG. MB) in determining which animals. on the basis of ccl1 death. wcrc in the HC+run group. Cell loss was patchy. however. and thus. a trend toward significant differences in cell counts for the HC t run group was observed (table 3).

Table 3: Cartilage Cell Counts per 1.07 pm2 of Tibia1 Cartilage* ______

( ‘ontl,ila tI(’ ~Vll!

tic‘ + ill,, * \o v81l1ti~w.li ditlcwnc,c\

~_..

Rlean

SD

127.x l3l.O l0l.I _~__

31.2 33.3 39.9 ..__--

Histological

Observations

Differential staining of the matrix was a consistent feature observed in both groups of hydrocortisone injected rats but moreso in the HC + run group. Generally, alcian bluePAS staining was less intense in HC t run animals. Areas of cartilage with chondrocyte loss tended tcb display a complete absence of matrix staining. Cystic lesions filled with fibrotic material were found within the articular cartilage only in HC run rats (fig 3). In eight of 12 HC + run rats. subchondral bone or fibrotic material had invaded the lower and middle zones of articular cartilage. Fibrotic cysts were found in articular cartilage from several HC rats; invasion ofsubchondral bone was not seen in any of the HC or C rats.

DISCUSSION Results suggest that running exercise in combination with intraarticular cortisone injections results in damage to femoral articular cartilage in rats. Though a trend toward articular damage was seen in all the injected animals, our results do not strongly support previous investigators reporting significant damage with intraarticular injections.3.5‘“.” HC rats were killed three weehs after their final hydrocortisone injection. Perhaps the three week recovery time for HC rats masked a period of transient damage (es7 mild breakdown, cell loss). Our data do question the advisability of exercising while receiving intraarticular injections of cortisone. It appeared that running resulted in cartilage breakdown as evidenced by the greater number and size of lesions in runners as well as the presence of fibrotic material and subchondral bone where articular cartilage should have been. Exercise may enhance cartilage nutrition and matrix synthesis under normal circumstances.“.‘3 but the positive etiects of exercise were lost for the femur under the conditions of this study. Why rat femoral cartilage was affected to a greater extent than tibia1 is unclear. The detrimental eflects of immobilization and disuse on articular cartilage are well known.“‘.” Though results of this study do not support running exercise after HC injection. joint immobilization or rest are probably not indi-

Arch Phys Med Rehabil VoI74, May 1993

CORTISONE AND EXERCISE EFFECTS ON CARTILAGE, Gogia

466

Fig 2-(A)

Normal articular cartilage. (B) Example of cell loss and fibrotic invasion of articular cartilage from an HC + run rat (H&E, magnification 550X: before reduction).

cated either. Perhaps intermittent active motion or continuous passive motion would be a better alternative for maintaining the viability of articular cartilage after cortisone.‘8 Unquestionably, hydrocortisone acetate caused a decrease in matrix synthesis (acid GAGS) in all HC + run animals and some HC rats as evidenced by the differential staining properties of the matrix with alcian blue-periodic acid schiff. Loss of matrix staining appeared related to chondrocyte death but may also have resulted from depressed matrix synthesis. ‘o.‘9Whether the decrease in matrix was more severe in runners could not be adequately discerned by light microscopy and more definitive biochemical assays need to be done in future studies. In contrast to the findings of others, animals in this study did not lose body weight.” Why our animals did not lose weight is unclear but may relate to dosage of cortisone given, age of the rats. and the administration of local as opposed to systemic injections. Loss ofbody weight was not a confounding variable in this study and results obtained are likely due to HC injection and running exercise.

1 _.

3.

4.

5. 6.

7. 8.

9. IO.

CONCLUSION Six weeks of running exercise combined with three intraarticular cortisone injections spaced one week apart was more deleterious to femoral articular cartilage in rats than hydrocortisone alone. i\cknowledgcment: I hr authors uould like to thank Dr. k’. K. Chithara. St. M~\ry’s Hospital. Tucson. AZ [or slide prc‘paration.

Holden G, Kendall PH. The newer corticosteroids injection. Ann Phys Med 19626: 17X-83.

Arch Phys Med Rehabil Vol74, May 1993

12. 13. 14. 15. 16.

References I.

11.

for local

Murdoch WR. Will G. Methyl prednisolone acetate in intraarticular therapy. Br Med J 1961: I :604-h. Steinberg CLR, Duthie RD, Piva AE. Charcoat-like arthropathy following inta-articular hydrocortisone. JAMA 1962: I8 I: 85 l-4. Hollander JL. Brown EM, Jessar RA. Brown CY. Hydrocortisone and cortisone injected into arthritic joints. comparative cfYects of and use of hydrocortisone as a local arthritic agent. JAMA 1951:147:1629-35. Bentley G. Papain-induced degenerative arthritis ofthc hip in rabbits. J Bone Joint Surg 197 I :53B:324-37. Bentley G. Goodfellow JW. Disorganization of the knee following intra-articular hydrocortisone injections. J Bone Joint Surg I969:5 I B:498-504. Chandler GN, Wright V. Deleterious effect of intra-articular hydrocortisone. Lancet 1458266 l-3. Chandler GN. Wright V. Hartfall SJ. Intra-articular therapy in rheumatoid arthritis. comparison of hydrocortisone tertiary butyl acetate and hydrocortisone acetate. Lancet 1958:2:659-6 I. Sweetnam DR. Mason RM. Murray RO. Steroid arthropathy of the hip. Br Med J 1960: I : 1392-4. Behrens F. Shepard N. Mitchell N. Alteration of rabbit articular cartilage of intra-articular injections of glucocorticoids. J Bone Joint Surg 1975:57A:70-6. Ekholm R. Norback B. On the relationship between articular changes and function. Acta Orthop Scan I95 I :2 I :8 I-98. Lanier RR. EfTects of exercise on the knee joints of inbred mice. Anat Ret 1946:94:3 I l-9. Saaf J. Effect of exercise on articular cartilage. Acta Orthop Stand 1950:2O(Suppl 7):1-83. Meachim G. Light microscopy of India ink preparations of fibrillated cartilage. Ann Rheum Dis 1972:3 1:457-64. Miller WT. Rcstifo RA. Steroid arthropathy. Radiology 1966:86:652-7. Enneking WF. Horow’itz M. The intra-articular effects of immobilization on the human knee. J Bone Joint Surg 1972:54A:973-85.

CORTISONE

AND

EXERCISE

EFFECTS

immobiliration on rabbit articular cartilage. J Anat I97 I ; 10X:397-507. IX. Salter RB. Simmons DF. Malcolm BW, et al. The biological effect of continuous passive motion on the healing of fullthickness defects in articular cartilage. J Bone Joint Surg I 9X0%3A: 1’32-5 I, .IY. Kopta JA. Blosser JA. Elasticity of articular cartilage: effects of intra-articular administration and medial mcniscctom! Clin Orthop I Y69:64:2 l-33. 20. Ishikawa K. Ohira T. Sakata H. Effects of intra-articular in-

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jections of halopredone diac~~atc on the arlic,ular cartilage of rat knees: ;L comparison with mcth!Iprcdnisnl(~ne acetate. Toxic01 4ppl Pharmacol I ‘)X1:75:-1I!-%‘).

17. Sood SC. A study of the effects of experimental

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Errata In the article “Changes in Nerve Conduction and Pi/PCr Ratio During Denervation-Reinnervation of the Gastrocsoleus Muscles of Rats” (Arch Phy.s Med Rehabil 199273: I 155-9) the ATP peaks in figure 2 were mlslabeled. The correct figure is reprinted here. In the article “Surgical Decompression of Impingement in the Weightbearing Shoulder’. (Arch Phys Med Rehabil I YY3;74:3?47) the death dagger should appear next to Robert W. Hussey. MD.

REINNERVATED I8

PARTS ITig

2-j’

PER

,rli

,mst-crush)

MILLION

P NblR spectra obtained in the hind foreleg muscles

(gastrocsoleus) of the rat after sciatic nerve crush. Note an clevation of Pi peak in the totally denervated (two weeks postcrush) muscles and a normalization of the Pi peak in thr winnerbated (eight weeks postcrush) muscles. Arch Phys Med Rehabil Vol74, May 1993