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Effect of prednisolone on motor end-plate fine structure A morphometric study in hamsters.

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Effect of Prednisolone on Motor
End-Plate Fine Structure: A Morphometric
Study in Hamsters
S. C h o k r o v e r t y , M B B S , M R C P , M. G. Reyes, MD, M. C h o k r o v e r t y , MBBS, a n d R. Kaplan, BS
The fine structure of quadriceps motor end-plates i n hamsters was analyzed quantitatively one, t w o , four, seven, and
thirty-two weeks following intraperitoneal injections of prednisolone. Except for transient increases i n postsynaptic
length and m e m b r a n e profile concentration after prednisolone administration at dosages of 4 m g per kilogram of
body w e i g h t f o r o n e w e e k and 2 m g per kilogram for f o u r weeks, mean values f o r various measurable profiles in t h e
presynaptic and postsynaptic regions showed no significant differences between control and treated animals.
Chokroverty S, Reyes MG, Chokroverty M, et al: Effect of prednisolone o n motor end-plate fine structure: a
morphometric study In hamsters. Ann Neurol 3.358-365, 1978
R e c e n t investigations have demonstrated that myasthenia gravis is an a u t o i m m u n e disease [7, 141 and
that many patients benefit from corticotropin and corticosteroid t r e a t m e n t [ 3 , 91. T h e corticosteroids are
t h o u g h t t o act by their effect of immunosuppression
[ 3 , 91. W h e t h e r corticosteroids also e x e r t a direct
action o n t h e neuromuscular junction has not been
settled. Electrophysiological observations in normal
animals a n d in vitro [ l , 2, 10, 12, 17, 181 have
suggested a direct action of corticotropin and corticosteroid o n neuromuscular transmission, while a r e c e n t
report d e n i e d such a n effect [ 111. T w o experimental
studies i n rabbits [15, 161 s h o w e d m i n o r m o r p h o l o g ical alterations of the neuromuscular junctions a t the
light microscopical level after prolonged administration of corticotropin a n d corticosteroids. The p r e s e n t
study s o u g h t morphological evidence for a direct effect of p r e d n i s o l o n e o n myoneural junctions b y
m o r p h o m e t r i c study of m o t o r end-plate fine structure
i n hamsters.
Materials and Methods
Studies were conducted in 20 male, 90-day-old Syrian golde n hamsters weighing 100 to 120 gm. Ten animals received
daily injections of prednisolone, 2 mg per kilogram of body
weight, intraperitoneally for six days a week. Eight hamsters
received n o injection and served as controls. The remaining
2 hamsters received prednisolone, 4 mg per kilogram, daily
for one week. Pairs of control and experimental animals
were killed by intraperitoneal injections of pentobarbital
sodium at the end of one, two, four, seven, and thirty-two
weeks. The prednisolone-treated hamsters killed 3t the end
From the Neurology Research Laboratory and Neurology Service,
Veterans Administration Hospital, Hines, and the Department of
Pathology, Mount Sinai Hospital Medical Center, Chicago, 1L.
of four weeks did not have paired controls, so t h e mean
values from the controls after two to seven weeks were used
for statistical comparison. The prednisolone-treated animals
received no further corticosteroid between seven and
thirty-two weeks to evaluate possible delayed changes after
the drug was discontinued. The 2 animals receiving prednisolone at a dosage of 4 mg per kilogram were killed at
the end of one week. Immediately after the animals were
killed, portions of the quadrireps femoris muscles containing the motor end-plates were removed.
For morphometric study, Engel’s [ 5 ]technique of locating
motor end-plates for electron microscopy was modified by
substituting the method of Lehrer and Ornstein [13] for
demonstrating esterase activities at the myoneural junction.
The strip of muscle was fixed in cold 3% glutaraldehyde in
Millonig’s buffer at pH 7.4and gently teased into parallel
divisions under a dissecting microscope. One end of each
division was then trimmed with a razor in a straight o r
oblique line for identification, and each division was separated into two strips. One strip of each pair was reacted for
specific and nonspecific esterasc activities according to the
a-naphthyl acetate technique of Lehrer and Ornstein [ 131,
dehydrated in graded alcohol, cleared in propylene oxide,
and infiltrated with araldite. The unreacted strips were osmicatecl, dehydrated, cleared, and similarly infiltrated. The
strips were reviewed under a dissecting microscope t o identify the end-plate zones; the corresponding zone in the
unreacted strip of the pair was demarcated with a razor blacle
and cut out from the rest of the strip with a fine saw. After
the demarcated end-plate zone was trimmed, thick sections
(1.5 to 2 k m ) were cut, stained with toluidine, and examined
at high power under the light microscope to identify 1 o r
more end-plates. The block was further trimmed around the
end-plates, and thin sections were cut on a Porter Blum
Accepted for publication Nov 2, 19’7.
Address reprint requests t o Dr Chokroverty, PO Box 127.Hines,
IL 60141.
358 0364-5134178/0003-0413$01.25 @ 1978 by S. Chokroverty
ultramicrotome and examined using an RCA EMU-3H
electron microscope.
The technique of Engel and Santa [81 was used for
morphometric measurements. All end-plate regions observed in the electron microscope were photographed and
analyzed at a final magnification of 31,000 or 36,700. We
examined 83 end-plates and 279 nerve terminals from controls and 103 end-plates and 333 nerve terminals from the
prednisolone-treated animals by electron microscopy. It
should be noted that more than 1 nerve terminal can be
found in an end-plate. The following were measured: (1)the
presynaptic membrane length (that sector of the axoplasmic
membrane not occupied by Schwann cells), expressed in
microns and determined by a map reader; ( 2 ) the nerve
terminal area, expressed in square microns, measured by a
planimeter; (3) the number of synaptic vesicles per square
micron of nerve terminal area; ( 4 )the postsynaptic area of
the junctional folds and clefts associated with a given nerve
terminal area, measured by a planimeter and expressed in
square microns; ( 5 ) the postsynaptic membrane length, in
microns, associated with a given nerve terminal area, measured by counting the number of junctional fold intersections afcer a grid o f evenly spaced zig-zag lines was placed
over the electron micrograph, as devised by Engel and
Santa [ 8 ] ; and ( 6 ) the membrane profile concentration, or
ratio of postsynaptic membrane length to postsynaptic area.
Student’s 2 test was employed for statistical comparison
using the two-tailed distribution, withp values below 0.05
considered significant.
Results
Qualita title Ob~evt’a
t i a ns
Marked morphological variations were noted in control and prednisolone-treated end-plates, reflecting
both differences in sectional geometry and true variations. The complexity of the junctional folds and
clefts varied. In some regions these appeared normal
(Fig la), in other regions they appeared simple (Fig
Ib), and in still others they seemed to be of intermediate complexity (Fig 2). T h e number and distribution of synaptic vesicles per unit nerve terminal area
also differed from end-plate t o end-plate and from one
region to another (Figs lb, 3). Similar variations were
noted in the nerve terminal area. Some nerve terminals were large (Fig lb), whereas others were small
(Fig 12). IR some regions the nerve terminal appeared
distended, with an excess of neurofilaments (Fig 4 ) ;
others were filled with abundant mitochondria (Fig
It?), while some were devoid of mitochondria (Fig
la). The postsynaptic region appeared denuded of
the nerve terminal in occasional sections. All these
conformational changes were noted in both control
and prednisolone-treated animals. Similar conformationai changes of the normal end-plate have
previously been observed [GI. But the complex postjunctional region was seen slightly more frequently
in the animals receiving prcdnisolone at a dosage of 4
mg per kilogram of body weight for one week and in
the treated ( 2 mg per kilogram) end-plates after four
weeks.
Quantitative Observations
Tables 1 and 2 give morphometric data o n the presynaptic and postsynaptic regions, respectiveiy.
The mean values for nerve terminal length, nerve
terminal area, and number of synaptic vesicles per unit
nerve terminal area were similar ( p > 0.05) between
the control and the prednisolone-treated end-plates
after one, two, four, seven, and thirty-two weeks.
The mean postsynaptic area did not differ significantly ( p > 0.05) between control and treated endplates at the end of each period of study. However, the
mean values for postsynaptic membrane length and
membrane profile concentration in the end-plate regions after one week were larger @ < 0.05) in the
animals receiving prednisolone at a dosage of 4 mg per
kilogram of body weight, but not in those receiving
the drug at 2 m g p e r kilogram. Similarly, postsynaptic
membrane lengrh and membrane profile concentration were increased Cp < 0.05) in the prednisolonecreated end-plates at the end of four weeks. But the
mean values for postsynaptic membrane length and
membrane profile concentration in the prednisolonetreated end-plates at the end of two, seven, and
thirty-two weeks did not differ @ > 0.05) from those
of control end-plates.
Discussion
We observed no significant morphological abnormalities by qualitative and quantitative electron microscopical methods in the presynaptic regions of
hamster motor end-plates after prednisolone treatment for one, two, four, and seven weeks. These
findings provide no morphological counterpart for the
recent electrophysiological observations of direct effects of corticosteroid and corticotropin on rhe presynaptic regions in animal experiments [ I , 2, 10, 12,
17, 181. Wilson and co-workers [ 171 found that prednisolone increased the frequency but decreased the
amplitude of miniature end-plate potentials in rat
phrenic nerve-diaphragm preparations. Prednisolone
and dexamethasone injections given intraperitoneall y
counteracted the presynaptic blocking effect of
hernicholinium in rat phrenic nerve-diaphragm [ 181
and sciatic nerve-cibialis anterior muscle [12] preparations. In denervation experiments in cat soleus
motor nerves, Hall et a1 [ 101 demonstrated preservation of excitability after intramuscular injections of
triamcinolone, indicating a direct effect o n neuronal
excitability. Birnberger et al [27 found a decrease in
the quantum content of end-plate potentials and an
increase in transmission failure rate after addition of
corticotropin in rat phrenic nerve-diaphragm preparation in vitro. Following single massive intravenous
Chokroverry et al: Prednisolone Effect on Motor End-Places
359
F i g I . Prednisolone 12 mglkgl-treated ( a )and control (b) endplates a f e r one week. The smaller newe terminal in ( a ) i s devoid
of mitochondria. The large newe terminal in (b) is associated
with simple junttionalfolcls and t1efi.s(arrows). S1,naptir-t'esi-
360 Annals of Neurology Vol 3 No 4
April 19'8
des i n the netwe terminal Ib) are sparse andare distributed
mostly along the margin. Scale: I p. ( N = nerve terminal;JF =
jwnc-tionalfold; JC =junction deft: my = niyofihvils.)
F i g 2. Motor end-pkute (pre~?itJokone-treuted,four
weeks) with
t u o neme terniincrkJ iN). Note r o m p h lunctronal region (IR) of
f o W i and rlejtJ Stuh. I p.
Chokroverty et al: Prednisolone Effect on Motor End-Plates
361
F i g -3. Control (tzrio weeks) motor end-plate Shoioing portion o j a
large wer1,e terminul ( N )distended with abundant .synaptic. 1,e.ri c h . Scafe: 1 p . t S = Srhzuann cell; my = iti.yofihri/c.,
362
Annals of Neurology
Vol 3
No 4 April 1078
*
Table 1 . Afovphonletrir. Ddta IMran Stundard Errov of Mean) of the Presynaptic Regions
of Control und Predni.rolone-treated Quudriceps Motor End-Plutes in Hamsters after I , 2. 4, 7 , and 32 Week.!
Thirry-two Wecks
Four Wteks
Two Weeks
O n e Wcck
Seven Weeks
Treated
i n o rrearmcnr
Dotermination
Prt-synaprar
mernbmne
icnhTh l f i !
NMW
rurminal
drrd IfiJI
N n of
Conrrol
$97 t
0.22
IN = 92)
2.Vl f
0. 3 5
IN = 9 2 )
-5 I 2 2
"vsKles/fiz
I" n r r Y C
4.23
IN = [jL)
Trrarud.
2 m$ky
5 32 t
0 68
fN = 671
3 95
0 74
IN 6 7 )
- 2 66 ?
4.1 3
iN = 6')
~
Treared,
4 mdkg
4 3'
?
0 51
f N = 351
2 31 t
0.43
i N = 151
05 z
4.20
IN = 35)
Control
Treated,
2 mdkg
4 23 ?
0.36
( N = 64)
IN = 5 3 i
4.86?
3.81 t
0.58
0.36
IN = 64)
IN = 54)
71.26 t
64 5 9 ?
4.46
4.56
IN = 6 1 )
IN = 5 1 )
4.X1
0.41
2
Treated,
Control
2
mdkn
407 f
4.02 t
0.36
0.22
r N = 70)
2 64 t
0.25
IN = 7 2 )
IN = 4 2 )
3 80 f
0.50
IN = 4 2 )
67 60 2
5.7
i N = 3x1
61.08 t
4.65
IN = 5 8 )
Control
5.35 t
0.32
IN = 2 0 )
291
0 42
?
I N = 20)
64.09
7.11
r
(N = 161
Tread,
2 mdkg
4
LO z
0.50
IN = 44)
306 ?
0.41
( N = 45)
68.60 ?
6.OO
(N
= 41)
Control
butween
32 wk)
3.39 f
5.19 5
0.33
IN = 61)
2.38f
0.24
0.29
(N = 6 1 )
65 39 t
9.80
( N = 58)
7
and
(N = (10)
2.55 t
(1.24
( N = 60)
51.34t
1.35
iN = 5 2 )
rcrm,,,<,l
N
=
number o f nerve cerminal regions analyzed.
Chokroverty et al: Prednisolone Effect on Motor End-Plates
363
Table 2. Morphometric Data (Mean ? Standard Error of Mean) of Postsynaptic Regions
of Control and Prednisolone-treated Quadriceps Motor End-Plates in Hamsters after I , 2 , 4, 7, and 32 WeekJ
Thirty-two Weeks
One Week
Two Weeks
Determination
Control
Treared.
2 mdkg
Treated.
4 mdkg
Postsynaptic
area (p’)
5.57 f
0.33
(N = 92)
25.28 e
1.4 5
(N = 92)
7.05 f
0.94
f N = 67)
31.83 2
3.21
( N = 67)
5.57
0.84
(N = 35)
37.34 f
5.60
(N = 35)
p < 0.05’
6.76 2
0.22
( N = 35)
Postsynaptic
membrane
lengrh (pF
Membrane
profile
concentrarion (pipp)
4.66 f
0.1 I
(N = 32)
4.92 I
0.16
( N = 67)
Control
5 60
0.54
?
( N = 64)
26.06 2
2.86
IN = 63)
4.65 ?
0. I 3
( N = 61)
Treared,
2 mdkg
Control
5.40 2
0.57
( N = 54)
23.87 f
2.56
( N = 53)
5.36 f
0.43
( N = 42)
25.67
2.15
( N = 40)
4.30 f
0.17
(N = 53)
p < 0.09
S e w n Weeks
Four Weeks
Treated.
*
2 mgikg
Control
6.66 f
0.73
3.26 c
0.32
( N = 72)
38.27 f
4.64
(N = 72)
p < 0.09
4.77 f
5.16 I
0.15
0. I4
IN = 40)
( N = 72)
p < O.OSb
Treated.
2 mdkg
( N = 20)
3.41 2
0.44
( N = 45)
14.05 -r
1.64
( N = 40)
4.89 c
0.17
(N = 20)
4.43 -r
0 18
( N = 40)
( N = 20)
15.97 c
1.78
Trearpd
(no [rearrnent
Control
3.43 2
0.28
( N = 61)
16.16 z
1.45
( N = 54)
4.95
0.27
2
(N = 54)
berween 7 and
32 wk)
3 32
0.22
?
( N = 60)
14.36 c
0.98
( N = 60)
4 41 c
0 I>
IN = 60)
“The measurements did nor include the intersections of the zig-zag lines with that portion of the postsynaptic membrane bordering the primary
synaptic cleft.
’Significant difference from control value.
N = number of postsynaptic regions analyzed.
doses of methylprednisolone, Baker and co-workers
[ 11 noted direct prejunctional facilitation manifested
by augmentation of the neurogenic posctetanic potentiation in cat soleus nerve-muscle preparation. This
facilitation was preceded by a transient inhibition of
motor nerve terminals.
Prolonged prednisolone treatment also did not alter
the postsynaptic area of hamster motor end-plates.
Similarly, the postsynaptic membrane length and the
ratio of postsynaptic length to postsynaptic area after
prednisolone treatment did not show significant deviations from control values except after four weeks of
prednisolone treatment at 2 mg per kilogram or one
week of treatment at 4 mg per kilogram. The enlarged
postsynaptic membrane length and increased postsynaptic membrane profile concentration after high
doses of prednisolone or prolonged treatment
suggested a dose-related effect. However, the absence
of these changes after two weeks of prednisolone
treatment makes this conclusion improbable. Possibly
a critical period of prednisolone treatment induces
slight temporary and nonspecific changes in postsynaptic membrane length.
We doubt that the inconstant and minor alterations
in postsynaptic length in the presence of normal posrsynaptic area represent an anatomically significant effect of prednisolone on the neuromuscular junctions.
However, the data do not exclude an enzymatic action
not reflected by morphological alterations. This possibility is supported by our recent findings [4]of reduction of acetylcholinesterase in hamster quadriceps
muscles containing end-plates after corticotropin injections. Thus the data may indirectly support the
suggestion that the rherapeutic effects of corticosteroids in myasthenia gravis are not mediated by a
direct action [ l l l on the neuromuscular junction, but
by immunosuppression [ 3 , 91.
364 Annals of Neurology
Vol 3 No 4
April 1978
Supported in part by the Medical Research Service of the Veterans
Administrarion.
Hiro Tonaki assisted in preparing the electron micrographs.
References
1. Baker T, Riker WF, Hall ED: Effects of a single methylprednisolone dose on a facilitatory response of mammalian motor
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2. Birnberger KL, Rude1 R, Struppler A: ACTH and neuromuscuiar transmission: electrophysiological in uitro investigation of
the effects of corticotropin and an A C T H fragment o n
neuromuscular transmission. Ann Neurol 1:270-275, 1977
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corticosteroids in generalized myasthenia gravis: comparative
studies and role in management. Ann NY Acad Sci 274:577595, 1976
4. Chokroverty S, Bernsohn J, Reyes MG, et al: Effect of adrenocorticotropic hormone on muscle acetylcholinesterase
and nonspecific esterase. Acta Neurol Scand 55:226-230,
1977
5 . Engel AG: Locating motor end-plate for electron microscopy.
Mayo Clin Proc 45:450-454, 1970
6. Engel AG, Jerusalem F, Tsujihata M, et al: T h e neuromuscular junction in myopathies: a quantitative ultrastructural
study, in Bradley W G , Gardner-Medwin D, Walton JN (eds):
Recent Advances in Myology. Amsterdam, Excerpta Medica
International Congress Series 3 10:132-143, 1975
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(IgG and C3) at the motor end plate in myasthenia gravis:
ultrastructural and light microscopic localization and electrophysiologic correlations. Mayo Clin Proc 52:267-280,
1977
8 . Engel AG, Santa T: Histometric analysis of the ultrastructure
of the neuromuscular junction in myasthenia gravis and in
myasthenic syndrome. Ann NY Acad Sci 183:46-63, 1971
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10. Hall ED, Baker T,Riker WF: Glucocorticoid preservation of
motor nerve function during early degeneration. Ann Neurol
1:263-269, 1977
11. Hofmann WW: Antimyasthenic action of corticosteroids. Arch
Neurol 34:356-360, 1977
12. Leeuwin RS, Wolters ECMJ: Effect ofcorticosteroids on sciatic
nerve-tibialis anterior muscles of rats treated with
hemicholinium-3: An experimental approach to a possible
mechanism of action of corticosteroids in myasthenia gravis.
Neurology (Minneap) 27:171-177, 1977
13. Lehrer GM, Ornstein L A diazo coupling method for the
electron microscopic localization of cholinesterase. J Biophys
Biochem 6:399-409, 1959
14. Lindstrom JM, Lennon VA, Seybold ME, et al: Experimental
autoimmune myasthenia gravis and myasthenia gravis:
biochemical and immunochemical aspects. Ann N Y Acad Sci
274:254-274, 1976
15. Shapiro MS, NambaT, Grob D: The effect of corticotropin on
the neuromuscular junction. Neurology (Minneap) 18:10181022, 1968
16. Tuncbay TO, Ketel WB, Boshes B: Cortisone effects on
myoneural junction. Neurology (Minneap) 15:3 14-320,1965
17. Wilson RW, Ward MD, Johns TR Corticosteroids: a direct
effect at the neuromuscular junction. Neurology (Minneap)
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18. Wolters ECMJ, Leeuwin RS: Effect of corticosteroids on the
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Chokroverty et al: Prednisolone Effect on M o t o r End-Plates
365
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