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KMID : 0378019670100120069
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New Medical Journal
1967 Volume.10 No. 12 p.69 ~ p.86
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Immunohistochemical Studies of Isoimmune Myocarditis in Rabbits
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Abstract
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Recently there are growing evidences that autoimmune mechanisms are involved in the pathogenesis of several diseases. This has stimulated many workers to attempt a production´ of an experimental disease based on autoimmune processes in order to investigate the nature and the mechanisms in human disorders.
Hurst (1932) observed paralysis in the rabbit following repeated subcutaneous or intramuscular -injections of saline suspensions of human brain. Ribers +et al. (1933), and Ferraro and Jervis (1940) experimentally induced neurologic disease in monkeys with repeated injections of extracts and saline .suspension of normal rabbit brain, and also observed encephala -yelitis with demyelination in the affected monkeys.
Cavelti (1947) reported histologic changes similar to those of rheumatic fever in rats and rabbits following repeated injections of saline suspension of homologous heart muscle and skeletal muscle with killed streptococci. Jaffe and Holz (1948) have also produced experimental allergic myocarditis by injections of saline suspension of homologous heart muscle. Laufer et al.(1963) observed experimental granulomatous inflammtion in the heart by modifying the method of Jaffe and Holz. Many other workers also induced experimental myocarditis and detected specific antibody in experimental animals by repeated injections of homologous or heterologous heart homogenates (Grey and Davies, 1951; Kaplan, 1958; Asharson and Dumonde, 1963; Davies et al., 1964).
The present study was an attempt to evaluate 1) the effect of homologous heart muscle homogenate on the heart; 2) the effect of the specific anti-heart serum on the myocardium; 3) the correlation of the lesion to a certain human myocardial disorder.
Materials and Methods:
Albino rabbits, weighing about 2.0 kg. were used. The animals were divided into four groups. The first group received saline alone. The second group received injections of saline and Freund´s complete adjuvant mixture. The third group received a saline suspension of cardiac muscle homogenate without adjuvant. The fourth group received saline suspension of cardiac muscle homogenate plus adjuvant.
Antigen was prepared as follows: After washing the homologous rabbit cardiac muscle with isotonic saline several times, homogenate of-the heart muscle was prepared and then iypophillized. The saline suspension with 4.0 gm. of the lypophillized powder was mixed with 8.0 ml. of bayol F and 1.5 ml. of Tween 80 containing 5.0 mg. of killed mycobacterium butricum.
Anti-rabbit Coomb´s serum was prepared in the goose by subcutaneous injection of purified rabbit gamma globulin in aluminum hydroxide gel biweekly for 4 weeps. The purified anti-rabbit goose gamma globulin (anti-rabbit Coomb´s serum) was prepared and labeled with fluorescine isothiocyanate by the method of Coons et al. (1950) and William et ai. (1960).The experimental animals were given 5 successive injections of antigen into the sole of both hind Meet at weekly intervals. All animals were bleed for the antibody titration, which was carried out by the passive hemagglutination method.
All animals were sacrified and necropsied about two weeks after the last injection of antigen. The organs were grossly examined and a part of each was put into 10% neutral formalin, the rest being :stored at-30C until use.
Staining for F.I.T.C. conjugated antibody; Frozen sections of heart and various other tissues were fixed in 95% ethanol (v/v) in coplin jars for 15 minutes at 37C, in a water bath. Each section was
l rinsed with buffered saline and covered with a drop of F.I.T.C. labeled anti-rabbit goose gamma g1bbulin. The slides were kept in a small moist chamber for 30 rninutes at room temperature to prevent -evaporation. They were washed as before and mounted in buffered glycerol (Reagent glycerol 9 parts, :buffered saline 1 part) and were examined under the fluorescent microscope.
The other part of the heart and other organs were prepared for paraffin sections. All sections were stained with hematoxylin and eosin. Additional stainings included P.T.A.H., Mallory´s azan stain, acid-fast s`tiain, and oil-red-O stain.
Results
1. The results of antibody titration:
In group III the antibody titer showed 1 : 20 and 1 : 40 in six out of 10 animals. In group IV titration showed generally marked increment, some of which reached I : 1280 and I : 640 but certain .animals revealed less than 1 : 160. In group I and II there was no hemagglutination.
2: Histopathological and histochemical findings:
a, Control group (Group I)
Heart: The sections of cardiac muscle showed no particular changes, and also no particular .abnormalities were observed in the other organs.
b. Adjuvant and Saline mixture Group (Group II)
Heart: The cardiac muscle fibers showed no particular changes. However there was minimal :infiltration of small and large mononuclear cells in the interstitial connective tissue. No characteristic -abnonmalities were observed in the other organs or tissues. The lymph nodes of the injected site -showed reactive hyperplasia.
c. Antigenic mixture of rabbit cardiac muscle and saline (Group III).
Heart: The cardiac muscle fibers showed morphological changes, patchy in distridution, mainly in
´ -the ventricular wall. Minimal or moderate infiltration of small and large mononuclear cells and histio-
cytes were frequently found. Edema of the interstitial connective tissue was generally marked by
cleft-fake spaces and fragmentation. The sarcolemmal nuclei were slightly increased in number, and
a
showed variable sized and shaped pyknotic nuclei.
The other organs: The regional popiiteal Iymph-nodes at the injected site showed reactive hyperplasia. .Macrophges and large eosinophilic immature reticular cells were seen in the medulla of of the lymph .nodes. Slight Fibrosis was noted in the liver of No. 22 animal.
d. Injection with an antigen-adjuvant mixture: muscle, adjuvant and saline (Group IV).
The cardiac muscle fibers showed marked morphological changes in all animals. Many areas revealed clumps of macrophages, lymphocytes, land a few plasma cells around degenerated and atrophic muscle :fibers. The caliber of cardiac muscle fibers was very variable, polygonal, round, oval or fused with each .other. The myocardial fibers showed varying degrees of degenerative changes characterized by disappearance, fragmentation, separation, homogenization of myofibrils, hyaline degeneration, swelling and marked atrophy. The obscuring of cross striation was seen. Edema of interstitial connective tissue was generally marked, varying in severity. Sometimes there were dense myofibrils, separated by cleft like spaces, and fragmentation. The sarcolemmal nuclei were slightly increased m number, and varyed. in shape and size.
The other organs: The spleen showed marked morphologic alterations, hyperplasia of germinal center, disappearance of mantle zone, and monocytic proliferation in _ the medulla. The regional lymph. nodes of the injection site showed reactive hyperplasia. The brain, liver, kidney and muscle tissues. were showed no definite changes.
3. Fluorescent antibody in tissues or cells:
As shown in table 4, the fluorescent antibody in cardiac muscle was seen only in group IV.
Fluorescent antibody was found in the ventricular wall of the heart, in the interstitial connective tissue and sarcoplasm. The area where pathological lesions were - severe emitted less fluorescence than the area without or with milder histologic lesions. The fluorescence was not observed in the sarcolemmai nuclei, valves, pericardium, or the walls of small veins. Sometimes, the fluorescence emitted was
mostly along cross striations.
Conclusion
1. Using the mixture of homologous cardiac muscle, adjuvant, and saline as antigen, experimental. myocarditis could be produced.
2. The specific antibody against heart muscle homogenates demonstrated in the serum.
3. ,:It appears that there is no direct relationship between the degree of serum antibody titer and the sverity of histological lesions.
However, in general fluorescene was most evident in group IV, which showed highest titration and severest histologic changes.
It is suggested that immunological processes may play in a part the pathogenesis of myocarditis.
There is no demonstrated fluoroscene which is the specific antibody against heart muscle homogenates in the brain lung liver adrenal glands and skeletal muscle tissues. It is suggested that organ specific processes may play in this experiments.
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