동종 및 이종근육조직과 Adjuvant의 반복주사로 인한 가토근육조직변화에 관한 연구

Abstract

[한글]

Experimental Myositis in Rabbits Produced by Injections of Homologous Muscle Tissue

with Adjuvant

Byung Ho Choi

Department of Pathology, Yonsei University College of Medicine

(Directed by D.S. Kim, M.D.)

Recent interests in so-called auto-immune disease have stimulated many workers in

this field to attempt a production of an experimental disease based on auto-immune

processes in order to elucidate the nature and the mechanisms of various human

disorders.

Hurst was among the first to observe paralysis in rabbits following repeated

subcutaneous or intramuscular injections of saline suspensions of human brain.

Rivers, Sprunt and Berry were able to produce clinical neurologic disease in

monkeys with repeated injections of extracts and saline suspensions of normal

rabbit brain, and they also observed histologic evidence of encephalomyelitis with

demyelination in the affected monkeys. Ferraro and Jervis repeated and confirmed

the work of Rivers and his co-workers. Since that time the mechanism of the

production of these lesions offered a fruitful Held for speculation.

Adjuvants consisting of Mycobacteria in water-in-oil emulsions, when combined

with antigens, have been known to enhance and prolong antibody formation. Employing

these adjuvants, a method became available for the rapid production of disseminated

encephalomyelitis. Experimental allergic encephalorrlyelitis has been used in the

purification of rabies vaccine and in efforts to throw light on the pathogenesis of

multiple sclerosis. Extensive studies have been carried out on the nature and the

mechanism of this disease process thus this has become a highly reproducible

experimental disease, readily induced in a wide variety of animals by injection of

nervous tissue plus mycobacterial adjuvant.

It has also been shown that the central nervous system is not the only organ in

which inflammatory and degenerative changes can be induced by injecting the

homologous tissue combined with adjutants. The technic was used for the production

of aspermatogenesis with homologous testicular tissue, of granulomatous

thyroiditis, of nephrosis, and of isoimmune adrenalitis etc. Employing the adjuvant

technic, an attempt is made to produce an experimental myositis in rabbits

utilizing homologous and heterologous skeletal muscle tissue. A further attempt is

made to correlate the disease so produced to a certain human muscular disorder.

Materials and Methods:

Healthy albino rabbits of a mixed breed, weighing around 2.0 kg. were used.

Antigen was prepared by the following technic. A skeletal muscle was aseptically

removed from normal rabbit thigh and washed several times in sterile isotonic

saline solution. It was cut into small pieces on a freezing microtome. 4.0 gm. of

the clean muscle was ground in a mortar with 8.5 ml. of Bayol F(liquid petrolatum,

U.S.P.) and 1.5 ml. of Arlacel containing 5.0 mg. of killed Mycobacterium

butyricum. Preparation of guinea pig muscle and dog muscle antigens was the same as

that for rabbit muscle.

All animals received five successive injections of a particular antigenic

mixture(0.8 ml. in divided doses) in each of the four footpads simultaneously

between a week interval. The animals were sacrificed around 20 days after the last

immunizing injection. The animals were divided into five groups, The first group

received saline suspensions of a normal rabbit muscle without adjuvant. The second

group received injections of adjutant alone. The third, fourth and the last groups

raceived an antigenic mixture containing adjuvant plus the muscles of rabbit,

guinea pig and dog respectively. The skeletal muscle tissue of different animal

species were used as 10.0 per cent suspensions in isotonic saline solution for skin

testing. For all tests, 0.1 ml. was given intradermally in a previously shaved area

of theflan? Reactions were read at 24 and 48 hours and were observed for several

days thereafter. A complete autopsy was performed on all animals. The gross

features of the organs were carefully recorded and put into 10.0 per cent neutral

formalin. Representative sections from all organs and from both sides of thigh,

calf, paravertebral and bracheal muscles were embedded in paraffin. All sections

were stained with hematoxylin and eosin. Additional sections were stained with

phosphotungstic acid hematoxylin (P.T.A.H.), acid-fast stain, Oil-Red-O for fat,

and Mallory's alan stain when indicated.

Results:

As shown in Table 2, a significant pathologic alteration of a skeletal muscle was

only seen in the group of animals that were injected with an antigenic mixture

composed of rabbit muscle and adjuvant. Five of the animals in this group

minifested the most severe changes characterized by focal and diffuse infiltration

of lymphocytes and histiocytes within the perimysial and endomysial connective

tissues. The cellular exudates also contained a considerable number of

polymorpho-nuclear leukocytes and eosinophiles. Scattered plasma cells were also

seen. The muscle fibers in the viscinity of inflammatory reaction showed varying

degrees of degenerative changes characterized by loss, fragmentation, and

homogenization of myofibrils occasionally leading to a complete loss of the muscle

fiber itself. These changes caused a variation in the size of muscle fibers.

Sarcolemmal nuclei were swollen, hyperplastic and showed occasional central

migration. In other areas, the changes were characterized mainly by focal nodular

infiltration of mononuclear cells and eosinophiles within the connective tissue

particularly around the small veins. In these areas, the muscle fibers appeared to

be intact. Minimal cellular infiltrations in the muscle tissue with a very slight

reactive changes in the muscle fibers were also seen in the group of animals

receiving adjuvant injection alone, and in the animals receiving muscles of

different species with adjuvant, However the changes observed were net of a

specific nature and of significant magnitude. Visceral organs of the animals

receiving adjuvant either alone or with muscle tissue produced hyperplasia of

reticuloendothelial cells and focal collection of lymphocytes in lungs, liver,

kidney and heart.

The result of skin test is summarized in Table 3. Although the muscle tissues of

different species resulted in positive reactions to the respective antigens, but

there was no apparent cross reaction between the animal groups. It was apparent

that there was no notable correlation between the severity of the skin reaction and

of the histopathological changes.

Comment and Conclusion:

The result of the present investigation offers an interesting speculation as to

the nature and the significance of this type of lesion and to the understanding of

a certain human muscle disorder.

In view of the method whereby the disease is produced, it was necessary to make

certain that an infectious agent had not been introduced with the antigenic

mixture. Bacterial cultures of the exudates obtained from grossly visible lesions

did not grow out any organisms. Furthermore intradermal, intraperitoneal or

subcutaneous injections into normal animals of minced muscles freshly taken from

the diseased parts, have failed to transmit the disease. Because of these reasons

the possibility of infection was ruled out.

Another possibility considered was the effects of adjuvant injections. Although

considerably large amount of adjuvant was repeatedly given to a group of animals,

this was not responsible for producing any significant alterations in skeletal

muscle. There were some changes however which could be attributable to the adjuvant

in various visceral organs which corresponded to the descriptions reported by

others.

The conclusion that the skeletal muscle lesions seen in the present investigation

is due to an immunologic process is based on the nature of the technic used to

produce them, on the nature of the histopathological changes and on the

inflammatory character of the lesions in the absence of an infectious agent. With

the evidences in hand, it appears probable that the rabbit skeletal muscle tissue

contains an organ-specific antigen which is capable of producing antibody which

will react with homologous tissue.

Many theories and facts have been presented in the past to explain the precise

pathogenesis how an immunologic reaction of this nature gives rise to the lesion

actually observed. The present investigation was not specifically directed for this

purpose and did not disclose any direct evidences for this explanation. Perhaps, as

suggested by others, increased vascular permeability, and infiltration of

mono-nuclear cells were the initial factor and the damage of the muscle was

secondary. The damage is probably immunologically specific since mononuclear cells

from immunologic states analogous to this situation have been shown to possess a

specific destructive capacity for antigen-containing cells.

The etiology of a group of human muscle disease known as polymyositis has never

been established. The unpredictable clinical course of these diseases and the

varigated pathologic features have led to a conclusion that the generalized

polymyositis in man is not a single disease entity. It is the general tendency to

include polymyositis in the group of "collagen diseases" because of its known

association with scleroderma or disseminated lupus erythematosus. The prominent

pathologic features of polymyositis are degeneration of muscle fibers and presence

of an inflammatory change.

From the morphologic standpoint, the experimental disease presently described

shows many similarities to that of polymyositis. There was also same features

similar to nodular interstitial myositis occurring in cases of "collagen diseases."

It is definitely presumptuous to reach a conclusion that a morphological

similarities would make the two diseases as the same entity. However considering

the fact that there are evidences to suggest a hypersensitive mechanism in a

certain "collagers diseases," and that there are reported effectiveness of

cortisone and A.C.T.H. in the treatment of polymyositis, the proposed idea is not

at all groundless.

It is suggested that perhaps an immunological process may be at play in one of

the pathogenic pathways of polymyositis.

[영문]

Recent interests in so-called auto-immune disease have stimulated many workers in this field to attempt a production of an experimental disease based on auto-immune processes in order to elucidate the nature and the mechanisms of various human

disorders.

Hurst was among the first to observe paralysis in rabbits following repeated subcutaneous or intramuscular injections of saline suspensions of human brain.

Rivers, Sprunt and Berry were able to produce clinical neurologic disease in monkeys with repeated injections of extracts and saline suspensions of normal rabbit brain, and they also observed histologic evidence of encephalomyelitis with demyelination in the affected monkeys. Ferraro and Jervis repeated and confirmed

the work of Rivers and his co-workers. Since that time the mechanism of the production of these lesions offered a fruitful Held for speculation.

Adjuvants consisting of Mycobacteria in water-in-oil emulsions, when combined with antigens, have been known to enhance and prolong antibody formation. Employing these adjuvants, a method became available for the rapid production of disseminated

encephalomyelitis. Experimental allergic encephalorrlyelitis has been used in the purification of rabies vaccine and in efforts to throw light on the pathogenesis of multiple sclerosis. Extensive studies have been carried out on the nature and the mechanism of this disease process thus this has become a highly reproducible

experimental disease, readily induced in a wide variety of animals by injection of nervous tissue plus mycobacterial adjuvant.

It has also been shown that the central nervous system is not the only organ in which inflammatory and degenerative changes can be induced by injecting the homologous tissue combined with adjutants. The technic was used for the production of aspermatogenesis with homologous testicular tissue, of granulomatous thyroiditis, of nephrosis, and of isoimmune adrenalitis etc. Employing the adjuvant technic, an attempt is made to produce an experimental myositis in rabbits utilizing homologous and heterologous skeletal muscle tissue. A further attempt is made to correlate the disease so produced to a certain human muscular disorder.

Materials and Methods:

Healthy albino rabbits of a mixed breed, weighing around 2.0 kg. were used.

Antigen was prepared by the following technic. A skeletal muscle was aseptically removed from normal rabbit thigh and washed several times in sterile isotonic saline solution. It was cut into small pieces on a freezing microtome. 4.0 gm. of the clean muscle was ground in a mortar with 8.5 ml. of Bayol F(liquid petrolatum, U.S.P.) and 1.5 ml. of Arlacel containing 5.0 mg. of killed Mycobacterium butyricum. Preparation of guinea pig muscle and dog muscle antigens was the same as that for rabbit muscle.

All animals received five successive injections of a particular antigenic mixture(0.8 ml. in divided doses) in each of the four footpads simultaneously between a week interval. The animals were sacrificed around 20 days after the last immunizing injection. The animals were divided into five groups, The first group

received saline suspensions of a normal rabbit muscle without adjuvant. The second group received injections of adjutant alone. The third, fourth and the last groups raceived an antigenic mixture containing adjuvant plus the muscles of rabbit,

guinea pig and dog respectively. The skeletal muscle tissue of different animal species were used as 10.0 per cent suspensions in isotonic saline solution for skin testing. For all tests, 0.1 ml. was given intradermally in a previously shaved area of theflan? Reactions were read at 24 and 48 hours and were observed for several days thereafter. A complete autopsy was performed on all animals. The gross features of the organs were carefully recorded and put into 10.0 per cent neutral formalin. Representative sections from all organs and from both sides of thigh, calf, paravertebral and bracheal muscles were embedded in paraffin. All sections were stained with hematoxylin and eosin. Additional sections were stained with phosphotungstic acid hematoxylin (P.T.A.H.), acid-fast stain, Oil-Red-O for fat,

and Mallory's alan stain when indicated.

Results:

As shown in Table 2, a significant pathologic alteration of a skeletal muscle was only seen in the group of animals that were injected with an antigenic mixture composed of rabbit muscle and adjuvant. Five of the animals in this group minifested the most severe changes characterized by focal and diffuse infiltration

of lymphocytes and histiocytes within the perimysial and endomysial connective tissues. The cellular exudates also contained a considerable number of polymorpho-nuclear leukocytes and eosinophiles. Scattered plasma cells were also seen. The muscle fibers in the viscinity of inflammatory reaction showed varying degrees of degenerative changes characterized by loss, fragmentation, and homogenization of myofibrils occasionally leading to a complete loss of the muscle fiber itself. These changes caused a variation in the size of muscle fibers.

Sarcolemmal nuclei were swollen, hyperplastic and showed occasional central migration. In other areas, the changes were characterized mainly by focal nodular infiltration of mononuclear cells and eosinophiles within the connective tissue particularly around the small veins. In these areas, the muscle fibers appeared to be intact. Minimal cellular infiltrations in the muscle tissue with a very slight reactive changes in the muscle fibers were also seen in the group of animals receiving adjuvant injection alone, and in the animals receiving muscles of

different species with adjuvant, However the changes observed were net of a specific nature and of significant magnitude. Visceral organs of the animals receiving adjuvant either alone or with muscle tissue produced hyperplasia of reticuloendothelial cells and focal collection of lymphocytes in lungs, liver,

kidney and heart.

The result of skin test is summarized in Table 3. Although the muscle tissues of different species resulted in positive reactions to the respective antigens, but there was no apparent cross reaction between the animal groups. It was apparent

that there was no notable correlation between the severity of the skin reaction and of the histopathological changes.

Comment and Conclusion:

The result of the present investigation offers an interesting speculation as to the nature and the significance of this type of lesion and to the understanding of a certain human muscle disorder.

In view of the method whereby the disease is produced, it was necessary to make certain that an infectious agent had not been introduced with the antigenic mixture. Bacterial cultures of the exudates obtained from grossly visible lesions did not grow out any organisms. Furthermore intradermal, intraperitoneal or

subcutaneous injections into normal animals of minced muscles freshly taken from the diseased parts, have failed to transmit the disease. Because of these reasons the possibility of infection was ruled out.

Another possibility considered was the effects of adjuvant injections. Although considerably large amount of adjuvant was repeatedly given to a group of animals, this was not responsible for producing any significant alterations in skeletal muscle. There were some changes however which could be attributable to the adjuvant in various visceral organs which corresponded to the descriptions reported by others.

The conclusion that the skeletal muscle lesions seen in the present investigation is due to an immunologic process is based on the nature of the technic used to produce them, on the nature of the histopathological changes and on the inflammatory character of the lesions in the absence of an infectious agent. With the evidences in hand, it appears probable that the rabbit skeletal muscle tissue contains an organ-specific antigen which is capable of producing antibody which will react with homologous tissue.

Many theories and facts have been presented in the past to explain the precise pathogenesis how an immunologic reaction of this nature gives rise to the lesion actually observed. The present investigation was not specifically directed for this

purpose and did not disclose any direct evidences for this explanation. Perhaps, as suggested by others, increased vascular permeability, and infiltration of mono-nuclear cells were the initial factor and the damage of the muscle was secondary. The damage is probably immunologically specific since mononuclear cells from immunologic states analogous to this situation have been shown to possess a specific destructive capacity for antigen-containing cells.

The etiology of a group of human muscle disease known as polymyositis has never been established. The unpredictable clinical course of these diseases and the varigated pathologic features have led to a conclusion that the generalized

polymyositis in man is not a single disease entity. It is the general tendency to include polymyositis in the group of "collagen diseases" because of its known association with scleroderma or disseminated lupus erythematosus. The prominent

pathologic features of polymyositis are degeneration of muscle fibers and presence of an inflammatory change.

From the morphologic standpoint, the experimental disease presently described shows many similarities to that of polymyositis. There was also same features similar to nodular interstitial myositis occurring in cases of "collagen diseases."

It is definitely presumptuous to reach a conclusion that a morphological similarities would make the two diseases as the same entity. However considering the fact that there are evidences to suggest a hypersensitive mechanism in a

certain "collagers diseases," and that there are reported effectiveness of cortisone and A.C.T.H. in the treatment of polymyositis, the proposed idea is not at all groundless.

It is suggested that perhaps an immunological process may be at play in one of the pathogenic pathways of polymyositis.