~

Previous studies have described an experimental model of myasthenia gravis (MG) produced by immunizing animals with thymus extracts. In view of the hypothesis that the autoimmune process in MG may be initiated within the thymus itself. we have reexamined this model using presently available methods to evaluate its resemblance to MG. We immunized Lewis rats with extracts of rat thymus in Freund’s adjuvant, as originally described by G. Goldstein. Five procedures were used to test for myas- thenic characteristics: (1) repetitive nerve stimulation; (2) recording of miniature endplate potentials; (3) assays of anti-ACh receptor antibodies in serum; (4) determination of ACh receptors at neuromuscular junctions by [lz51] a-bungarotoxin binding; and (5) evaluation of the histology of the thymus gland. Our results showed that the thymus-immunized animals did not demonstrate abnormalities in any of these parameters. We conclude that immunization of rats with thymus extracts, as described, failed to pro- duce a myasthenia-like condition.

MUSCLE & NERVE 3:293-297 1980

CRITICAL RE EXAM1 N AT1 ON OF THE THYMUS IMMUNIZATION MODEL OF MYASTHENIA GRAVIS

ANNE MURPHY, BA, DANIEL 6. DRACHMAN, MD,

and JOSEPH C. EGGLESTON, MD S. SATYA-MURTI, MD, ALAN PESTRONK, MD,

T h e thymus gland has long been suspected to play an important role in myasthenia gravis (MG). Clinjcal and pathological evidence implicating the thymus includes a high incidence of thyniic hyperplasia (approximately 65%) and neoplasia (approximately 10%) in myasthenic patient^"^,^^ Furthermore, thymectomy ha5 been reported to produce beneficial effects in up to 857; of patients with MG.3,30

In 1966, Goldstein and WhittinghamLo re-

From the Department of Neurology (Ms. Murphy, Drs. Drachman, Satya- Murti, and Pestronk) and the Department of Pathology (Dr. Eggleston), The Johns Hopkins University School of Medicine, Baltimore, MD.

Acknowledgments This work was supported by grants 51301 HD04817, 5P01 NS10920, and a Viets Fellowship from the Myasthenia Gravis Foun- dation The authors would like to acknowledge the expert technical as- sistance of Mr. R Adams. M i C W. Angus, and Miss M S Powell, and efficient help in oreparation of the manuscriot by Mrs C. Barlow and Mrs 3. Kane.

Address reprint requests to Dr. Drachrnat. a! the Depaement of Neurol- ogy, The Johns Hopkins University School of Medicine 1721 East Madi- son St., Baltimore, MD 21205

Received for publication June 27, 1979, revised manuscript accepted for pubiication November 1. 1979.

01 48-639)(/0304/0293 $OO.OO/O 1980 Houghton Mifflin Professional Publishers

portcd that immunization of rodents with extracts of thymus glands produced an animal model with features of hlG. For several reasons, this work and subsequent reports by Goldstein and coauthors 7-11

have been the subject of continuing controversy. First, efforts to reproduce the original results have met with varying success: approximately half the attempts have yielded negative r e s ~ i l t s , ~ ~ ~ ~ ” ~ ~ ~ although others have been confirmatory.’“ Further, electrophysiological methods used for de1ec:ting the myasthenia-like features were open to critic:ism because of mechanically unstable re- cording electrodes and rapid stimulation rates used for repetitive nerve stimulation tests$j

Despite rhese difficulties, the thymus immuni- zation model continues to have certain theoretical attractions. If verifiable, it might help to explain the link between the thymus gland and the neu- romuscular disorder in MG. Moreover, it might provide support for the hypothesis that t.hr mto- immune response in MG originates w i ~ i , , - * the thymus gland.1,1G,34,36 Because of these con- siderations, we thought it appropriate to reexam- ine the thymus immunization model critically, using a battery of presently available methods to cvaluatc its resemblance to human MG.

Thymus Immunization Model of MG MUSCLE & NERVE JuIiAug 1980 293

MATERIALS AND METHODS

Throughout the study, every effort was made to follow the previously described protocol for im- munization7 in detail.

Preparation of Antigen. Thymus glands were re- moved from young, female Lewis rats. After mincing, the glands were homogenized in phos- phate-buffered saline (PBS), pH 7.4, to a con- centration of 20% wt/vol using a motor-driven Teflon pestle. The homogenates were centrifuged at 6,000 or 12,000 g for 10 minutes. The superna- tant was either used immediately or lyophilized and stored at -70°C for later US^'.^,^

Immunization. Either fresh thymus extract or lyophilized material reconstituted to 10 mg/ml was mixed with an equal volume of complete Freund's adjuvant (CFA). In the initial group of 15 female Lewis rats, 0.1 ml of thymic extract was injected into the foot pads, as described by Goldstein and Whittirighani.'o After obtaining negative results in this group of animals, we immunized a second group of nine rats with larger doses consisting of 0.4 or 0.6 nil of thymus extract intradermally, re- peated four or five times at 2-week intervals. Con- trol rats were either untreated (18 rats), or given injections of Ringer's solution (four rats) or CFA (seven rats).

Evaluation of Myasthenic Features. Five different methods were used to test for myasthenic features.

I . Repetitive nenie stimulation. Repetitive nerve stimulation was carried out. in 15 rats immunized with a single injection of thymus extract and in 16 untreated controls. Fourteen to 24 days after im- munization, the rats were anesthetized with chlo- ral hydrate (400 mgikg, intraperitoneally) and strapped to a platform with the right leg firmly taped to a metal rod. One electrode was secured to the skin over the mid-belly of the triceps surae muscle, and the other electrode was fastened over the Achilles tendon with a metal clip. The right sciatic nerve was surgically exposed, and a silver wire stimulator probe was gently hooked around it. The overlying skin was closed around the probe with pins, and the wound was kept moist with mineral oil. Supramaximal stimuli at rates of 10, 30, and 50 Hz were supplied by a Grass S-48 stimulator (Grass Instrument Co., Quincy, MA) through a stimulus isolation unit.. The mus- cle action potentials were amplified by a differen- tial amplifier (DAM-6) (W-P Instruments Co., Hamden, CT), displayed on a Tektronix oscil- loscope (Tektronix Co., Beaverton, OR), and re-

corded on Polaroid@ film (Polaroid Corp., Cam- bridge, MA) for measurement. The percentage decrement or increment in amplitude of the tenth potential as compared to the first. was measured.

2. Measurement of miniuture endplate potentials. Miniature endplate potentials (MEPPs) were mea- sured in the diaphragms of nine immunized test animals, arid seven CFA-injected and four Ringer-injected control rats. Under chloral hy- drate anesthesia, the left hemidiaphragms were quickly removed and pinned at resting length in petri dishes lined with Silastic@ (Dow Corning, Midland, MI). 'rhey were maintained in Trowell's '1.4 medium:? gassed with a mixture of 95% 0, and 5% CO, at 22°C. Intracellular recordings were made with conventional glass microelectrodes of 5-15 M a resistance filled with 3 M KCI. The signal was amplified with a Grass P16 amplifier (Grass Instrument Go.) and displayed on a Tek- tronix storage oscilloscope. Twenty MEPPs were measured in each of 20 fibers per hemidiaphragm. Only f-ocal recordings (rise time, less than 2.0 msec) from fibers with resting membrane potentials of 60 mV or more were measured. The mean MEPP amplitude for each fiber was calculated, and cor- rected to a resting potential of 7.5 IIIV.'~

3. Determinulion of ACh receptors per neuromuscu- lar junction. The number of ACh receptors per neuromuscular junction was determined in the right hemidiaphragms of nine thymus-immunized rats, five CFA-irijectcd rats, and two untreated controls as previously Bundles of parallel fibers were dissected from the hemidia- phragms and incubated at 37°C for 3 to 4 hours in modified Ham's F12 medium2' containing 0.2- 0.3 pg of '251-labeled a-bungarotoxin (a-BuTx) per ml (specific activity = 2-5 x lo4 Cdmol). The fibers were washed thoroughly, fixed in 4% glu- taraldehyde, and the neuromuscular junctions were stained for acetylcholinesterase (AChE). The muscle strips were further dissected into small bundles, and t.he fibers in each bundle (usually 10-100) were counted under a dissecting micro- scope. The radioactivity was measured in a gamma spectrometer. The number of receptor sites per neuromuscular junction was calculated from the amount of bound radioactivity and the number ofjunctions.

4 . Determination of anti-ACh receptor antibody. Anti-ACh receptor antibody in the serum of nine immunized and three CFA-injected controls was determined by a radioimmunoassay similar to those previously d e s ~ r i b e d . ~ , ~ ~ 'This assay mea- sures the amount of antibody that binds to 1251-a- Bu'l'x-labeled ACh receptor obtained from dener-

294 Thymus Immunization Model of MG MUSCLE & NERVE Jul/Aug 1980

Table 1. Repetitive nerve stimulation: change in amplitude of 10th potential a

Stimulation rate No. of

Group animals 10 Hz 30 Hz 50 Hz

Untreated controls 16 +2.15% ? 0.66% +2.55% ? 0.9% -1.38% i 1.34%

Thymus-immunized rats 15 +2.99% ? 1.24% +4.06% 0.96% -0.13% i 1.33%

9 a t a represent the mean percentage change + SEM The percentage change was calculated by [(amplitude of 10th potentral ~ amplitude of Ist pofential)lamplitude of Ist potential] x 700

Table 2. The number of ACh receptors per neuromuscular junction (NMJ) and the MEPP amplitudes in thymus-immunized and control rats

MEPP AChiNMJ ampi itude No of

Animal no Immunization" injections x 107 (mV -c SEM)

Experimental 1 2 3 4 5 6 7 8 9

Cont ro I 1 2 3 4 5 6 7 8 9

10 1 1 12 13

L L L F F F F L L

C FA C FA C FA C FA C FA C FA C FA

Ringer Ringer Ringer Ringer

Untreated Untreated

5 5 5 5 5 5 5 4 4

1 1 1 1 1 1 1 1 1 1 1

~

-

3 1 3 1 1 6 4 8 3 1 3 4 5 3 3 9 3 1

3 6 3 7 4 2 1 9 3 7

~ b

..

-

- -

~

2 4 2 5

0 572 f 0 037 0 574 ? 0 038 0 666 ? 0 054 0 505 ? 0 047 0 598 c 0 045 0 620 2 0 034 0 571 t 0 034 0 526 2 0 031 0 645 t 0 039

0 648 i 0 071 3 54C i 0 051 0 505 i 0 025 0 660 c 0 049 0 810 t 0 022 0 402 -c 0 032 0 436 ? 0 031 0 616 -c 0 043 0 515 -c 0 035 0 562 f 0 044 0 528 ? 0 036

-

__

y = lyophilized thymus, F = fresh thymus, CFA = complete Freund's adjuvant b- = no data available.

vated rat muscles, with a sensitivity of 1.6 x mol/ml.

5. Erialuation of' the hislology vf the thywius gland. The histology of the thymus gland in nine im- munized and three CFA-injected rats was evalu- ated by light microscopy. The thymus glands were dissected from the animals a t the termination of each experiment. They were immediately fixed by immersion in 10% formol saline. The tissue was then embedded in paraffin, and multiple 6-pm- thick sections, stained with herriatoxylin and eosin, were prepared. The slides were coded and exam- ined under the microscope by two of the authors (J.E., D.B.D.) without knowledge of the treatment the animals had received.

RESULTS

The parameters measured gave no iridicatiori of myasthenia-like features in the thymus-immunized rats. The response to repetitive nerve stimulation at 10, 30, and 50 Hz was not significantly different from that in the controls (table 1). In human MG a decremental response of more than 10%-15?; is considered abnormal.12,28 The greatest decrement for any individual test ariinial was 6.3%) at a rate of 50 per second.

MEPP amplitude4 were not significantly differ- ent in the thymus-immunized and control groups of animals (table 2). '['he mean MEPP amplitude for the immunized group was 0.586 k 0.017 (SEM) as compared with 0.566 2 0.034 in the 11 control

Thymus Immunization Model of MG MUSCLE & NERVE JuliAug 1980 295

animals. The lowest amplitude of MEPPs in the thymus-immunized animals was 0.505 mV ? 0.047 mV which is well within the normal range.

No reduction of the junctional ACh receptors was found in the thymus-immunized rats as com- pared to the controls (table 2). The mean number of ACh receptors per neuromuscular junction was 3.49 2 0.36 (SEM) x lo7 in the immunized group, and 3.14 ? 0.32 x lo7 in the controls.

The serum of each thymus-immunized rat was tested 2 to 5 times for anti-ACh receptor anti- body, and the mean titers were below thc detect- able level. By (:oritrast, rats with experimental au- toimmune MG (EAMG) have mean titers of anti- body to rat ACh receptor of approximately 12 x 10-" mol/ml one month after immunization with Torpedo ACh receptor (Noguchi SA, Drachrnan DB, Adarns RN, Hirsch R: Lymphocyte respori- siveness to acetylcholine receptor in rats with exper- imental myasthenia gravis. Ann i\'eiiro/ (in press).

Evaluation of' thymus pathology showed no ab- normalities in the thymus-immunized rats as com- pared to the controls. There was no increase in medullary germinal ccnters, in contrast to the findings of Goldstein and Whittingham."+''

DISCUSSION

According to present concepts, the basic abnor- mality in MG is a reduction of available ACh re- ceptors due to an autoimmune attack? Recently, ,4Ch receptor has been found in extracts of thymic tissue,'^'" and surface ACh receptors have been demonstrated on muscle cells grown from human and rodent thymuses.I6 These findings suggest a possible mechanism by which immunization with thymus gland preparations might produce an ex- perimental model of MG, due to an aut.oimmune response to ACh receptor present in the thymus. Although the present understanding of MG docs not support Goldstein's and Manganaro's8 original idea of a neuromuscular blocking agent released from the thymus glands of immunized animals, the model itself seemed sufficiently interesting to war- rant further investigation.

This study was an attempt to reexamine thymus-immunized rats for evidence of MG. In order to resolve the problem definitively, we have utilized five different parameters to test for the presence of myasthenic features. Two of these methods (determination of ACh receptors at neuromuscular junctions, and assays for anti-ACh receptor antibodies) were not previously available at the time that the model was originally described. Further, the method of repetitive nerve stimula- tion used here provides rigid fixation of the ani-

mal's limb and firm attachment of the stimulating and recording electrodes, thus eliminating objec- tions raised to previous s t ~ i d i e s . ' ~ , ~ ~

The present techniques have previously been used successfully to detect myasthenic characteris- tics in both the passive transfer model of MG",."' and the EAMG mode1,2°-23~29 (Pestronk A, unpub- lished observations). However, in the present study none of the animals immunized with thymic ex- tracts demonstrated any of the features of MG. If the thymus immunization model were to produce MG, the most plausible mechanism would be that the ACh receptor in the thymus might serve as a source of antigen to produce EAMG. In order to explore t.his possibility further, we measured the amount of ACh receptor in the thymic extracts by means of a precipitation assay developed in our laboratory (Angus CW, Powell MS, Drachman DB, in preparation). The results indicated that the ex- tracts contained 0.665 x lo-" mol of ACh recep- tor per g wet weight (mean of three thymuses from 4-week old Lewis rats). This concentration of ACh receptor is somewhat higher than previously re- po~-ted,'~,'~ but the amount injected (0.016 p g per injection) is almost certainly insufficient to elicit an autoimmune response in view of the fact that ho- mologous ACh receptor was

Negative results such as these must always be interpreted with caution, since they do not explain the discrepancy between the findings of different. groups of investigators. Perhaps there was sonie difference in the rats used, although the Lewis strain of rat.s used in all t.he experiments is known to be highly inbred. Perhaps a difference in the thymus glands, a variation in the composition of the adjuvant, or some other undefined factor ac- counts for the differences reported.

At the least, however, we can say that the thymus imniunization model is capricious. It has failed in our hands to produce animals with any of the accepted physiological, biochemical, or inimu- nological changes in myasthenia. Although the ex- perimental model itself seems unworkable, this need not cast doubt on the thymus as the site of origin of the autoimmune response in MG. ln- deed, the hypothesis that the autoimmune re- sponse in human MG may be initiated within the thymus gland itself remains attractive. Some alter- ation in the myoid cells or the immunocompetent lymphocytes of the thymus, perhaps initiated by viral infection, may serve to break tolerance, and thereby trigger an autoimmune response. Such a process would fit well with present concepts of the pathogenesis of MG as an autoimmune disorder directed primarily against ACh receptors.

296 Thymus Immunization Model of MG MUSCLE & NERVE Jul/Aug 1980

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Thymus Immunization Model of MG MUSCLE & NERVE JuliAug 1980 297