Methylprednisolone이 골조직에 미치는 영향에 관한 실험적 연구

Abstract

[한글]

Corticosteroid의 광범위한 이용에 따라 이외 장기사용에 외한 각종 부작용을 연구 이해하는 것은 의의있는 일이라고 생각한다. 골조직에 초래되는 corticonteriod의 부작용으로서는 골다공증(osteoporosis)과 무혈성 골괴사(avascular necrosis)가 중요하다. 그 중

무혈성 골괴사에 관하여는 내인성 및 외인성 hypercorticism에 의한 다수외 임상증례가 보고되고, 이를 토대로 한 실험적 연구결과들이 발표되고 있으나 정확한 발생기전에 대하여는 아직도 논란이 많다. 그 기건에 관한 대표적인 설명으로는 골다공증에 따른 이차적인 골절의 결과라는 주장과 혈액의 과응고상태를 일으키기 때문이라는 주장, 그리고 corticosteroid가 지방간을 초래하고 여기서 유래하는 지방색전에 의한다는 학설이 있으나 지금까지의 연구결과로는 어느것도 만족할 만한 이론에 이르지 못하고 있다. 따라서 cortic

osteroid가 골조직에 미치는 영향을 조사하고 톡히 무혈성 골괴사의 기전을 구명코자 본실험을 시행하였다.

실험동물로는 체중 2kg내외의 웅성가토와 200gm내외의 웅성백서 각 36마리씩을 사용하였으며 각 9마리는 대조군으로, 27마리는 corticosteroid 투여군으로 정하였다. corticosteroid투여군에는 methylprednisolone 0.5mg/kg을 매일 1회씩 대퇴부에 근육주사하였으며 대조군에는 동량의 saline을 근육주사하였다. 약물을 2, 4 및 6주간 투여한 후에 실험군 9마리와 대조군 3마리를 도살하였다. 도살직전 혈청 triglyceride와 total cholesterol치를 측정하였고, 전신의 골변화를 보기 위하여 x-선 사진을 촬영한 후 radiodensitometry로 대퇴골, 상박골 및 제1요추의 radiodensity를 측정하였다. 도살직후 대퇴골과 간장및 폐장을 적출하여 골조직과 폐장에서는 일반적 변화를 관찰하기 위한 hematoxylin-eosin염색과 혈관내 지질을 검색키 위한 oil red-0 및 sudan black염색을 시행하였으며, 간장에서는 hematoxylin-eosin과 old red-0염색외에 PAS 및 diastase-PAS염색으로 당원변동을 조사하였다.

실험결과를 요약하면 다음과 같다.

1. Methylprednisolone투여는 가토와 백서의 혈청 triglyceride 및 total cholesterol치를 증가시켰으며 이러한 증가는 가토에서 더욱 현저하였다.

2. 전신 x-선사진의 densitometry상 methylprednisolone은 가토와 백서의 골조직에 다공성변화를 초래하였다.

3. 조직학적 소견상 methylprednisolone투여는 가토의 대투골두 연골하 골조직에 파골세포의 증가와 더불어 골조직의 파괴 및 섬유화를 초래하였다.

4. 지방염색소견상 rnethylprednisolone투여는 가토 대퇴골두의 연골하 모세혈관내에고농도 지질함유를 초래하였다. 백서에서는 이러한 변화를 볼 수 없었으며, 간의 지방변성과 폐희 지방색전소견도 관찰되지 않았다.

5. Methylprednisolone투여는 가토와 백서의 간에 다소의 당원증가를 일으켰다.

이상의 결과를 종합하면 methylprednisolone투여는 가토의 혈청내 지질을 상승시키고대퇴골두 연골하 골조직에서만 모세혈관내에 고농도 지질함유와 함께 골조직의 파괴를 초래하였다.따라서 고지질혈청에 의한 골조직 모세혈관의 혈행장애는 가토에 methylprednisolone을 투여할 때 일어나는 무혈성 골괴사의 한 기전이라고 사료되었다.

An Experimental Study of the Effect of Methylprednisolone on the Bone

Jong Bo Hong

Department of Medical Science The Graduate School, Yonsei University

(Directed by Professor Yoo Bock Lee, M.D.)

Among various side effects of long-term use of corticosteroid, osteoporosis and

avascular necrosis of the femoral head have been implicated as the major

oomplications affecting the bone. However, the exact pathogenic mechanism of

steroid-induced avascular necrosis of the bone remains controversial, in apite of

increasing clinieal and experimental reports on avascular bone necrosis due to

either exogenous or endogenous hypercorticism. Some investigators proposed that

steroid induces osteoporosis and subsequent microfracture which leads to vascular

injury and ischemic bone necrosis, while others have believed that

hypercoagulability following steroid treatment leads to intravascular thrombus

formation which results in ischemic necrosis of bone. Another hypothesia is that

excessive corticosteroids induce fatty liver with subsequent systemic fat

embolisation and eventual aseptic necrosis. However, no one theory seems to satisfy

all inatances of avascular bone necrosis. Therefore, the present study is aimed to

elucidate the mechanism of corticosteroid induced avascular bone necrosis by

studying the effect of corticosteroid on bone.

A total of 72 animals, 30 male rahbits and 36 male rats, were used for the

experiment, and divided into two groups, control and experimental. Control group

comprised 9 animals from each species and was injected normal saline

intramuscularly once every day. Experimental group comprised 27 animals of each

species and was injected methylprednisolone, a short acting glucocorticoid,

intramuacularlr in a dose of 0.5 mg/kg, once every day for 2 to 6 weeks. Animals

were sacrificed after 2, 4 and 6 weeks treatments of methylprednisolone. At the

time of sacrifice, blood samples were obtained to measure serum triglyceride and

total cholesterol concentration. A whole body x-ray was taken from each animal

under anesthesis to determine the degree of osteoporotic change with densitometric

measurement of the femur, humerus and lumbar vertebra on the x-ray film. For the

histelogic examinations, bilateral femoral bones, liver and lungs were removed. All

tissues were fixed in 10% neutral formalin and the femoral bones were decalcified

by the technic of Jones and Sakovitch(1965). Decalcified femoral bones were

bisected longitudinally to include the head portion. One half of the decalcified

femoral bones and portions of the liver and lungs were embedded in paraffin, and

processed through routine histologic technic. About 5 μ thick sections from

paraffin blocks were stained routinely with H-E to examine histologic changes of

the bones, liver and lungs. PAS and diastase-PAS staining were also applied to the

liver to evaluate glycogen content. With the remaining portion of femoral bones,

liver and lungs, frozen sections were prepared and stained with oil red-0 or sudan

black to evaluate fatty changes in the liver, and presence of fat in vascular

spaces in the lunge and bones. Through theae examinations, following results were

obtained.

Results and summary:

1. Excessive administration of methrlprednisolone brought about elevationas of

the serum triglyceride anti total cholesterol levels in both rabbits and rats, but

more pronouced in rabbits.

2. Excesaive adminiatration of methylprednisolone induced generalized

osteoporotic change in both rabbits and rats as determined by radiodensitometric

measurement on whole body x-ray film.

3. Histologic alterations of the bone were observed only in rabbits, and

consisted of demineralization, destruction of bony trabecullae by increased

osteoclastic actlvities and fibrosis in marrow spaces, selectively involving

subchondral area of the femoral head. No such changes were observed in rats.

4. Fat staining demonstrated impaction of stronggly positive sudanophilic

material in subchondral capillaries of the femoral head in rabbits, whereas no such

staining was observed in rats and control animals. No evidence of fatty liver or

fat embolization in the lungs were observed.

5. The liver showed moderate glycogen accumulation in both rabbits and rats.

These results suggest that the compromtsed blood flow particularly at subchondral

capillaries of the femoral head due to hyperlipidemic blood may play an important

role in the development of avascular necrosis following methylprednisolone

administration in rabbits.

[영문]

Among various side effects of long-term use of corticosteroid, osteoporosis and avascular necrosis of the femoral head have been implicated as the major oomplications affecting the bone. However, the exact pathogenic mechanism of steroid-induced avascular necrosis of the bone remains controversial, in apite of

increasing clinieal and experimental reports on avascular bone necrosis due to either exogenous or endogenous hypercorticism. Some investigators proposed that steroid induces osteoporosis and subsequent microfracture which leads to vascular injury and ischemic bone necrosis, while others have believed that hypercoagulability following steroid treatment leads to intravascular thrombus formation which results in ischemic necrosis of bone. Another hypothesia is that excessive corticosteroids induce fatty liver with subsequent systemic fat

embolisation and eventual aseptic necrosis. However, no one theory seems to satisfy all inatances of avascular bone necrosis. Therefore, the present study is aimed to elucidate the mechanism of corticosteroid induced avascular bone necrosis by studying the effect of corticosteroid on bone.

A total of 72 animals, 30 male rahbits and 36 male rats, were used for the experiment, and divided into two groups, control and experimental. Control group comprised 9 animals from each species and was injected normal saline intramuscularly once every day. Experimental group comprised 27 animals of each species and was injected methylprednisolone, a short acting glucocorticoid,

intramuacularlr in a dose of 0.5 mg/kg, once every day for 2 to 6 weeks. Animals were sacrificed after 2, 4 and 6 weeks treatments of methylprednisolone. At the time of sacrifice, blood samples were obtained to measure serum triglyceride and total cholesterol concentration. A whole body x-ray was taken from each animal

under anesthesis to determine the degree of osteoporotic change with densitometric measurement of the femur, humerus and lumbar vertebra on the x-ray film. For the histelogic examinations, bilateral femoral bones, liver and lungs were removed. All tissues were fixed in 10% neutral formalin and the femoral bones were decalcified by the technic of Jones and Sakovitch(1965). Decalcified femoral bones were bisected longitudinally to include the head portion. One half of the decalcified femoral bones and portions of the liver and lungs were embedded in paraffin, and

processed through routine histologic technic. About 5 μ thick sections from paraffin blocks were stained routinely with H-E to examine histologic changes of the bones, liver and lungs. PAS and diastase-PAS staining were also applied to the liver to evaluate glycogen content. With the remaining portion of femoral bones,

liver and lungs, frozen sections were prepared and stained with oil red-0 or sudan black to evaluate fatty changes in the liver, and presence of fat in vascular spaces in the lunge and bones. Through theae examinations, following results were obtained.

Results and summary:

1. Excessive administration of methrlprednisolone brought about elevationas of the serum triglyceride anti total cholesterol levels in both rabbits and rats, but more pronouced in rabbits.

2. Excesaive adminiatration of methylprednisolone induced generalized osteoporotic change in both rabbits and rats as determined by radiodensitometric measurement on whole body x-ray film.

3. Histologic alterations of the bone were observed only in rabbits, and consisted of demineralization, destruction of bony trabecullae by increased osteoclastic actlvities and fibrosis in marrow spaces, selectively involving subchondral area of the femoral head. No such changes were observed in rats.

4. Fat staining demonstrated impaction of stronggly positive sudanophilic material in subchondral capillaries of the femoral head in rabbits, whereas no such staining was observed in rats and control animals. No evidence of fatty liver or fat embolization in the lungs were observed.

5. The liver showed moderate glycogen accumulation in both rabbits and rats.

These results suggest that the compromtsed blood flow particularly at subchondral capillaries of the femoral head due to hyperlipidemic blood may play an important role in the development of avascular necrosis following methylprednisolone

administration in rabbits.