An experimental study in pigs

Selective reduction of bone blood flow by short-term treatment of high-dose methylprednisolone: An experimental study in pigs

Drescher, W

Treatment with corticosteroids is a risk factor for non-traumatic avascular necrosis of the femoral

head, but the pathological mechanism is poorly understood. Short-term treatment with high doses of methylprednisolone is used in severe neurotrauma and after kidney and heart transplantation. We investigated the effect of such treatment on the pattern of perfusion of the femoral head and of bone in general in the pig.

We allocated 15 immature pigs to treatment with high-dose methylprednisolone (20 mg/kg per day intramuscularly for three days, followed by 10 mg/kg intramuscularly for a further 11 days) and 15 to a control group. Perfusion of the systematically subdivided femoral head, proximal femur, acetabulum, humerus, and soft tissues was determined by the microsphere technique. Blood flow in bone was severely reduced in the steroid-treated group. The reduction of flow affected all the segments and the entire epiphysis of the femoral head. No changes in flow were found in non-osseous tissue. Short-term treatment with high-dose methylprednisolone causes reduction of osseous blood flow which may be the pathogenetic factor in the early stage of steroid-induced osteonecrosis.

J Bone Joint Surg [Br] 2001;83-B:274-7.

Received 29 June 1999; Accepted after revision 17 December 1999

Treatment with corticosteroids is a known risk factor for the development of femoral head necrosis (FHN) after kidney and heart transplantation and after neurotrauma1 in both adults and children,2 but the pathological mechanism is poorly understood. Reduction of blood flow in the femoral head has been suggested as a mechanism. In one studey in rabbits blood flow in the femoral head was shown to decrease after 6, 8 and 10 weeks of treatment with steroids,3 and in another investigation multifocal osteonecrosis was shown to be present four weeks after treatment with a bolus of steroid.4 A statistically significant correlation was found between the cumulative dose of methylprednisolone in the first month after heart transplantation and the development of avascular necrosis.5

In patients with FHN, uneven blood flow with a lower flow within the necrotic sequester has been described.6

Our aim was to investigate in pigs the effect of treatment with high-dose methylprednisolone on blood flow in fone in general and on the pattern of regional perfusion of the epiphysis of the femoral head.

Materials and Methods

We used 30 immature Danish landrace pigs of both genders, weighing 46.0 to 50.0 kg with a mean age of 100 days. They were divided randomly into an experimental group (n = 15) undergoing short-term treatment with high– dose steroids and a control group (n = 15).

The experimental group was treated with intramuscular methylprednisolone (Solu-Medrol; Pharmacia & Upjohn, Copenhagen, Denmark) for 14 days. For the first three days, they received 1000 mg/day, followed by 500 mg/day for the next 11 days. On the 14th day, blood flow was measured using radioactive tracer microspheres.

The animals were premeditated with 25 mg of midazolam (Dormicum; Hoffman-La Roche, Basel, Switzerland) and 200 mg of azaperon (Stresnil; Janssen Pharmaceutica, Beerse, Belgium) intramuscularly. Intravenous anaesthesia was induced by 20 mg of etomidate (Hypnomidate; Janssen Pharmaceutica) and after orotracheal intubation maintained by a combination of 30 ml of ketamine (Ketaminol Vet; Veterinaria, Switzerland) (50 mg/ml), 4 ml of pethidinhydrochloride (Petidin Amino; Amino AG, Switzerland), 6 ml of midazolam (5 mg/ml), 6 ml of pancuron (Pavulon; Organon Teknika, Turnhout, Belgium) (2 mg/ml) and 4 ml saline at a rate of 20 ml/hour. The pigs were positioned supine and ventilated on a Servo Ventilator 900 (Siemens-Elema, Sweden) with the hips in the neutral position.

Sheaths (Fast-Cath; Daig Corporation, Minnetonka, USA) were placed in both common carotid arteries (7F) and in one jugular vein (6F). The systolic, diastolic, and mean arterial blood pressures were monitored in a carotid artery by a pressure transducer (Uniflow; Baxter Healthcare Corporation, Santa Ana, California) on a CardioMed CM– 4008 Physiological Trace System (Medi-Stim AS, Oslo, Norway), on which the ECG and rectal temperature were also monitored continuously. Blood-gas analysis was performed every 30 minutes on an ABL 510 blood-gas analyser (Radiometer A/S, Copenhagen, Denmark). Radioactive tracer microspheres (New England Nuclear, Boston, Massachusetts) with a diameter of 15 (mu)m labelled with the isotope ^sup 113^Sn were used to measure regional blood flow.7 Two other types of microsphere, labelled with ^sup 103^Ru and ^sup 141^Ce, were administered afterwards for purposes unrelated to the subject of this paper.

For administration of the microspheres, a pigtail catheter (6.0 F, Cook, Denmark) was advanced into the left ventricle under radiographic control through the sheath in the right carotid artery and another (6.0 F) into the thoracic aorta via the sheath within the left carotid artery. Each vial of microspheres contained 5.0 +/- 10^sup 6^ spheres suspended in 5 ml of 10% Dextran. Before injection the vial was agitated for five minutes on a Whirlmixer (Fisons AG, Loughborough, UK). The spheres were injected through the pigtail catheter into the left ventricle over a period of 30 seconds followed by flushing with 5 ml of heparin-saline at 37 deg C. Reference blood sampling from the aorta was started 30 seconds before injection of the spheres and continued until four minutes after the injection.

For determination of the cardiac output (CO) the total activity in each vial was measured in an Amersham Calibrator (ARC 120, Capintec Inc, New Jersey) before injection. After the experiment, all the remnants were measured and subtracted to obtain the injected dose in megaBecquerel (MBq). A predetermined MBq quantity of each type of microsphere was suspended in tap water and vortexed, and ten aliquots of 1 ml were withdrawn for determination of the counting efficiency (counts per minute/MBq) of the gamma counter used for the isotopes.

Statistical analysis. The normal distribution of the raw data presented as the mean SEM was documented by Q-Q– plotting. Homogeneity of variances was achieved by logo transformation of the raw data. Significance was determined by the paired samples t-test when comparing the left and right hips and by the independent samples t-test when comparing the two experimental groups. p values of less than 0.05 (two-tailed) were considered to be significant.

Results

No significant differences were found between the right and left hip in either of the groups and therefore the mean value for both hips together was taken (Table I).

In the steroid-treated (CS) group there was a general reduction in bone regional blood flow (RBF) (Table I) compared with the control (NCS) group. The RBF in the epiphysis of the femoral head was 38%, in the proximal femoral metaphyseal spongiosa 32%, in the corticalis 31%, and in the acetabular bone 57% of those of the NCS group. For the humeral diaphysis the RBF in the CS group was 32% of that of the NCS animals (Table II). The absolute RBF of the epiphysis of the femoral head was 1.16 +/- 0.16 ml*min^sup -1^ in the NCS group and 0.42 +/- 0.05 ml*min^sup -1^ in the CS animals (p

The specific CO (CO/kg/body-weight) was higher in the NCS animals than in the CS animals (92.3 +/- 9.4 ml*min^sup -1^kg^sup -1^ body-weight v 64.7 +/- 4.8 ml*min^sup -1^ kg^sup -1^ body-weight; p

An overall reduction in the RBF of the epiphysis of the femoral head was found in the CS-treated animals without a tendency to lower blood flow in the craniomedial aspect (Fig. 1). The four craniomedial subregions were combined and the RBF of these and the combined RBF of the remaining subregions of the femoral head were calculated (Table III). No difference was found between these two regions.

Discussion

Osteonecrosis of the femoral head is still common after kidney and heart transplantation5,8 and is regarded as an effect of treatment with corticosteroids.9 In transplantation procedures in children, the incidence of necrosis of the femoral head has been shown to be equal to8 or even higher than that in adults. Necrosis of the femoral head develops predominantly in patients with a high rate of rejection. Such patients frequently receive a pulsed administration of high doses of corticosteroids containing 1g of methylprednisolone daily for three days.5

Our study has shown a pronounced general reduction of blood flow in bone after two weeks of treatment with high– dose methylprednisolone. The RBF in the epiphysis of the femoral head of CS-treated animals was reduced to half the fraction of the CO compared with that of the NCS animals. There was an overall reduction in the RBF of the epiphysis of the femoral head and that of the proximal femur, acetabular bone, and humeral bone. The RBF of the soft tissue of the hip was unchanged, which suggests that the effect of corticosteroids on the RBF of bone is selective.

Decreased arterial inflow or increased venous outflow resistance can reduce intraosseous blood flow. Based on clinical studies of osteonecrosis of varying aetiology including treatment with glucocorticoids, thrombophilia and hypofibrinolysis have been suggested as the common, major cause of osteonecrosis.10 Thrombotic occlusion of the venous outflow of the femoral head originating from thrombophilia or hypofibrinolysis is supposed to cause venous hypertension and reduced arterial perfusion of bone.11 Thrombophilia has recently been discussed as a pathological mechanism in Legg-Perthes’ disease.12-14 The blood supply of the immature epiphysis of the femoral head depends exclusively on extraosseous intracapsular vessels because the growth plate imposes a barrier on the intraosseous blood supply at this age.15 In our study this may make the femoral head more vulnerable to steroid-induced reduction of blood flow.

Wang et al16,17 found that the intrafemoral pressures in steroid-treated rabbits were increased by 2.5 times. Hypertrophy of fat cells was found in the femur and humerus of rabbits treated with high doses of methylprednisolone for five months. Recently, in vitro adipogenesis was induced by steroids in a pluripotent cell line from bone marrow.18 In the rigid intraosseous compartment, growth of fat cells may cause a rise in intraosseous pressure, and thereby compress the thin-walled sinusoids, with a subsequent decrease in bone blood flow.3 We have found, however, decreased blood flow in the cortical bone of the proximal femur which does not contain sinusoids within the vascular bed.19

Degenerative changes in the arteries and arterioles of the capsule of the hip and the femoral head have been found in cadavers of renal transplant patients without clinical hip symptoms. There was thickening of the intima, gross diminution in the number and calibre of the vessels in the arteries of the femoral head and infarcts of subchondral bone.20

The number of stenotic superior retinacular veins was found to be higher in femoral heads obtained at postmortem from steroid-treated patients than in those from patients without steroid therapy.21 In seven out of 30 steroid-treated rabbits, proliferation of foam cells was observed in the intima of the ear veins.22

Bunger et al23 estimated the total and segmental blood flow of the epiphysis of the femoral head in carrageenan induced coxitis in dogs. Histologically, necrosis has been found to be localised particularly in the craniomedial zone of the femoral head. In our study, the epiphysis has been systematically subdivided for the quantitative estimation of blood flow. This subdivision into 24 columns and their separate gamma counting allowed us to investigate the perfusion pattern of the femoral head in detail (Fig. 1). We found no tendency to a lower RBF craniomedially in the CS-treated animals. In patients with non-traumatic necrosis of the femoral head, the blood flow as estimated by laser doppler flowmetry was lower in the necrotic area than in the remaining part of the femoral head.6

We conclude that treatment with high-dose methylprednisolone reduces blood flow in both cortical and cancellous bone selectively in the pig. There appears to be an overall reduction in the RBF in the epiphysis of the femoral head with no tendency to uneven distribution. Reduction of blood flow in bone may be an important pathogenetic factor in the early stages of steroid-induced osteonecrosis.

This study was supported by grants from the Institute for Experimental Clinical Research, Aarhus University, the Danish Rheumatism Association, the Helga- and Peter Kornings Foundation, and the Danish Orthopaedic Society.

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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W. Drescher, T. Schneider, C. Becker, J. Hobolth, W. Ruther, E. S. Hansen, C. Bunger

From the University Hospital Aarhus, Denmark

W. Drescher, MD, Resident

E. S. Hansen, MD, Assistant Professor

J. Hobolth, Medical Student

C. Bunger, MD, DMSc, Professor

Department of Orthopaedics, Aarhus University, 8000 Aarhus C, Denmark.

T. Schneider, MD, PhD, Head of Department

Orthopaedic Hospital, ‘Dreifaltigkeits-Krankenhaus’, 50933 Cologne, Germany.

C. Becker, MD, Resident

Department of Orthopaedics, Heinrich-Heine-University, 40225 Dusseldorf, Germany.

W. Ruther, MD, Professor and Head of the Department

Department of Orthopaedics, University of Hamburg, 20246 Hamburg, Germany.

Correspondence should be sent to Dr W. Drescher.

Copyright British Editorial Society of Bone & Joint Surgery Mar 2001

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