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(Investigative Ophthalmology and Visual Science. 2001;42:3158-3164.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Long-Term Changes in Myosin Heavy Chain Composition after Botulinum Toxin A Injection into Rat Medial Rectus Muscle

Branka Stirn Kranjc1, Janez Sketelj2, Anne D’Albis3 and Ida Erzen4

1 From the University Eye Hospital, Ljubljana, Slovenia; 2 Institute of Pathophysiology and the 4 Institute of Anatomy, Medical Faculty, Ljubljana, Slovenia; and the 3 Laboratoire de Biologie Physicochimique, Université Paris-Sud, Orsay, France.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To study long-term changes of extraocular muscles after botulinum toxin (Botx) A–induced paralysis, with special emphasis on myosin heavy chain (MyHC) isoform pattern in muscle fibers.

METHODS. Botx A (5 IU) was injected into the ocular medial rectus (MR) muscles of adult rats. After 1, 5, and 8 months muscle cross sections were examined immunohistochemically, histochemically, and morphometrically. MyHC content was analyzed by gel electrophoresis.

RESULTS. Paralyzed MR muscles displayed mildly atrophic and hypertrophic muscle fibers and decreased oxidative metabolism, due to decreased succinate dehydrogenase activity. However, muscle morphology was not grossly disturbed. MyHC profile was shifted toward slower isoforms. Electrophoretic analysis showed that the share of MyHCI, and especially of MyHCIIa and MyHCIIx/d, increased several fold, whereas the share of MyHCIIb decreased heavily during the first 5 months. Immunohistochemical analysis generally mirrored the results obtained by electrophoresis. Moreover, specific extraocular MyHC isoform MyHCeom disappeared and could not be detected during the whole experimental period. The portion of MyHCIIb relatively increased 8 months after Botx A injection, although the MyHC profile was still far from normal.

CONCLUSIONS. These long-lasting changes in Botx A–paralyzed ocular MR muscles most probably reflect their inability to regain their unique functional characteristics after new motor end plate formation and recovery of muscle contraction.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
In ophthalmology, botulinum toxin type A (Botx A) is used for the treatment of strabismus and focal dystonias. Botx A is a potent presynaptic neuromuscular blocking agent.1 The presynaptic effect of the toxin induces denervation-like alterations in the motor innervation of skeletal muscle fibers.2 Physiological, histochemical, and ultrastructural changes in muscle fibers have also been observed as a consequence of Botx paralysis.3 4 Its paralytic effect is prolonged, although temporary. The postparalysis recovery of function is probably due to the new motor end plate formation produced by motoneuron axon sprouting in muscle fibers.5

However, important differences have been noted regarding the severity and duration of changes, as well as subsequent recovery, in fast and slow skeletal muscles.3

Morphologic and histochemical features of the extraocular muscles (EOMs) mirror their physiological characteristics, which make EOM the fastest and most fatigue-resistant skeletal muscles.6 EOMs are composed of two layers: the peripheral orbital layer (OL) and the more central global layer (GL). The OL is narrow and consists of smaller muscle fibers (diameter mostly <20 µm), whereas the GL is wider, fills the central portion of the muscle, and contains larger muscle fibers (diameter mostly >20 µm).7 In the GL, three singly innervated fiber (SIF) types are present—fast oxidative, glycolytic, and oxidative glycolytic—expressing either MyHCIIa or MyHCIIb, and many muscle fibers coexpress both isoforms, possibly also MyHCIIx/d and extraocular MyHC (MyHCeom). One multiply innervated fiber (MIF) type in the GL exhibits low oxidative and glycolytic activity, and coexpresses MyHC{alpha}cardiac, MyHCß-slow, and MyHC-slow tonic. In the OL, one SIF type with high oxidative activity is found, which coexpresses MyHCIIa and MyHCIIb and possibly MyHCIIx/d and neonatal MyHC (MyHCneo). The OL MIF type with intermediate oxidative activity coexpresses MyHCI and MyHCIIa and possibly MyHCIIx/d and MyHCneo.8 9 The presence of multiply innervated EOM fibers is unique in mature mammalian skeletal muscles and is thought to play a role in precise rotary eye movements.7 Persistence of immature neonatal protein isoforms into adulthood, such as MyHCneo, and expression of a specific MyHC isoform, MyHCeom, is also specific to these muscles.

Because the EOMs exhibit a unique fiber type composition and function, we presumed a distinctive muscle fiber response after Botx injection.7 9 Earlier findings suggest that the efficacy of Botx treatments in neuromuscular disorders of monkey EOM is dependent not only on the type of disorder but also on the properties of the affected muscle and its motor control.10 11 12 These studies have shown that the Botx paralysis of the EOM does not produce a typical denervation atrophy of all muscle fiber types but, instead, results in selective long-term morphologic alteration of the SIFs in the OL. These fibers have a high mitochondrial content and dense capillary vascular network.

New neuromuscular junction formation in Botx-paralyzed muscles appears to follow similar paths in rats, monkeys, and humans.13 In the present study, we examined the composition of different MyHC isoforms in the rat ocular medial rectus (MR) muscles after neuromuscular paralysis induced by Botx A.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The study was designed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. Female Wistar rats (body weight 220–250 g at the time of surgery) were anesthetized before injection of 5 IU/0.1 ml botulinum A (Oculinum; Smith Kettewell, San Francisco, CA) into the exposed bellies of the left ocular MR muscles. For direct muscle viewing, conjunctival incisions were performed at the medial limbal area. Because no differences were observed between normal nontreated ocular MR muscles and contralateral MR muscles, both served as controls. The rats were painlessly killed by exsanguination under ether anesthesia 1, 5, and 8 months later. At least five ocular MR muscles for each experimental period were collected and frozen in liquid nitrogen. Thereafter, 10-µm-thick serial cross sections (mainly from the muscle belly) and individual proximal and distal muscle cross sections were analyzed morphometrically, histochemically, and immunohistochemically. Isoforms of MyHC isolated from whole-muscle homogenates were analyzed by SDS glycerol gel electrophoresis.

Histochemistry
Histochemical reaction to demonstrate succinate dehydrogenase (SDH) activity in muscle fibers was performed as described.14

Immunohistochemistry
MyHC isoforms in individual muscle fibers were demonstrated by binding of specific monoclonal antibodies against various isoforms of MyHCs: ß slow (BAD5), IIa (SC71), and IIb (BFF3)15 and MyHCneo (Novocastra, Newcastle-upon-Tyne, UK). BF35 antibody15 binding to all these MyHC isoforms, but not to the MyHC IIx/d isoform, was used to check for pure MyHCIIx/d fibers.

Peroxidase-conjugated rabbit anti-mouse IgG (Dakopatts, Glostrup, Denmark) served as the secondary antibody. All antibodies were diluted in phosphate-buffered saline (PBS) with the addition of 0.3% to 0.5% normal bovine serum to prevent unspecific binding. Rat serum was added during application of the secondary antibodies.

Diaminobenzidine tetrahydrochloride (DAB) or 4-chloro-1-naphtol (C1N) were used as chromogens. DAB (0.05%) in 0.2 M acetate buffer (pH 5.2), with 0.01% hydrogen peroxide or 0.05% C1N in 0.05 M Tris-HCl buffer (pH 7.4–7.6; Sigma, St. Louis, MO), with 0.028% hydrogen peroxide, were used. Control sections were incubated without the primary antibody.

SDS Gel Electrophoresis of MyHCs
Myosin was extracted from whole-muscle homogenates of at least five rat ocular MR muscles isolated 1, 5, and 8 months after Botx-induced toxic paralysis. MyHC isoforms were analyzed by electrophoresis in 8% polyacrylamide slab gels in the presence of 0.4% SDS and 30% glycerol. The procedure was performed at 70 V at 4°C for 40 hours.16 Separated MyHC bands were stained with Coomassie blue (Sigma). Relative amounts of different MyHC isoforms were quantified densitometrically.

Morphometrical Analysis and Statistics
Muscle fibers in the global layer (GL) and orbital layer (OL) of rat ocular MR muscles after Botx-induced paralysis and control muscles were analyzed using photomicrographs or video camera–captured images (at least 12,000 muscle fibers from the GL and 4000 fibers from the OL of five muscles for each study period). Using our own computer-assisted methodology,17 we determined the numerical and area percentage of muscle fiber types, their diameter, and diameter distribution. SE was calculated for all parameters, and the t-test was used to establish statistically significant differences (Systat for Windows, ver. 5; SPSS, Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Gross muscle fiber morphology in Botx-paralyzed ocular MR muscles was not evidently disturbed during the course of the experiment (1, 5, and 8 months; Fig. 1 ). Centrally located nuclei were present only in individual muscle fibers in the GL of ocular MR muscles.



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  		Figure 1. SDH activity in control rat ocular MR muscle and in Botx-injected muscles after 1 month, 5 months, and 8 months. Compared with control muscle, SDH activity was reduced in Botx-injected muscles, especially in the orbital muscle layer.

 
In the GL, the changes in the average muscle fiber diameters were not significant during the whole experimental period. The range of muscle fiber diameters greatly increased, however, and some truly hypertrophic fibers were observed, compared with control muscles. Muscle fibers of the OL became significantly smaller after the first month of paralysis (P < 0.05) with the median diameter 14.8 µm versus 16.5 µm in the control OL muscle fibers. The atrophy of the OL muscle fibers was more obvious after 8 months (the median diameter, 12.5 µm; P < 0.01).

Histochemical Analysis of SDH Activity
Paralyzed ocular MR muscle fibers in the OL exhibited lower SDH activity, than muscle fibers in the control muscles during 1, 5, and 8 months of analysis (Fig. 1) . The percentage of the oxidative muscle fibers in the GL was reduced to 16% ± 5% in comparison to 30% ± 2.5% in the control ocular MR muscles (P < 0.05), 1 month after Botx injection and did not change, even after 8 months.

Immunohistochemical Analysis of MyHC
In the GL of paralyzed ocular MR muscles the percentage of MyHCI-positive muscle fibers was higher (P < 0.05) than that in the control muscles (18% ± 5% vs. 10% ± 3%), at 8 months (Fig. 2A) . In both muscle layers a high content of MyHCIIa was detected in small-diameter fibers, and a lower concentration of MyHCIIa in many larger diameter fibers was detected early, after the first month of paralysis (not shown) as well as later on (Figs. 2A 2B) . The reaction to MyHCIIb was feeble in comparison to that in normal MR muscles but was present in many muscle fibers, especially in the GL of the paralyzed muscles during the entire study period of 8 months. We detected no pure MyHCIIx/d-containing fibers using the antibody BF35.



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  		Figure 2. (A) Immunohistochemical analysis showing muscle fibers containing (Aa) MyHCI, (Ab) MyHCIIa, (Ac) MyHCIIb, and (Ad) MyHCneo in the GL of control rat ocular MR muscle and in experimental muscles 5 and 8 months after Botx injection. Fibers 3, 4, and 5 are fast SIF fibers, and 6 are slow, MIF fibers. (B) Immunohistochemical analysis showing muscle fibers containing (Ba) MyHCI, (Bb) MyHCIIa, (Bc) MyHC IIb, and (Bd) MyHCneo in the OL of control rat ocular MR muscle and in experimental muscles, 5 and 8 months after Botx injection. Fibers 1 are SIF fibers and 2 are MIF fibers. Scale bar, 50 µm.

 
In the OL of paralyzed MR muscles, MyHCI-positive fibers were not significantly more numerous than in control muscles. Their variation along the muscle length was comparable with control MR muscles. MyHCneo-containing fibers in the paralyzed muscles were present in the OL, as in the control ocular MR muscles, from 1 to 8 months after Botx injection (Fig. 2) . The results of the immunohistochemical reaction against MyHCneo varied along the muscle length. Toward the distal muscle portion away from the muscle belly they showed some staining in few, predominantly small fibers of the GL, which are fast SIFs and are normally the most oxidative fibers in the GL.

Although most fibers were hybrid fibers, no coexistence of MyHCI with MyHCIIa was detected in the GL of experimental muscles. In contrast to control muscles, colocalization of MyHCI with MyHCneo was found also in the GL. The same fiber types, marked from 1 to 6,9 as described earlier in normal rat MR muscles, were detected also in this study throughout the entire experimental period (Figs. 2A 2B) .

SDS Gel Electrophoresis of MyHC
MyHC isoforms separated by SDS gel electrophoresis are presented in Figures 3 and 4 . The relative amount of MyHCI isoform displayed a slow increase during analysis that was more apparent after 5 and 8 months after Botx injection (P < 0.05). The percentages of MyHCIIx/d and MyHCIIa isoform increased several fold during early stages of the experiment (P < 0.01). Eight months after paralysis, these two MyHC isoforms decreased somewhat but were still higher than in control ocular MR muscles. The percentage of MyHCIIb isoform decreased to approximately one third of normal value during the first 5 months after paralysis (P < 0.001). Thereafter, the share of MyHCIIb increased, but was still significantly lower than in control muscles after 8 months. MyHCeom, which contributed approximately 25% of all MyHC content in normal ocular MR muscles, was not detectable in paralyzed muscles even after 8 months. We could not detect MyHCneo isoform with the method used.



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  		Figure 3. MyHC isoforms, separated by SDS glycerol gel electrophoresis in control rat ocular MR muscles and Botx-injected muscles 1, 5, and 8 months after Botx-induced paralysis.

 


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  		Figure 4. SDS gel electrophoresis of rat ocular MR muscle homogenates (n = 5, for each experimental group). Relative amounts (mean ± SE) of MyHC isoforms (MyHCI, MyHCIIa, MyHCIIx/d, MyHCIIb, MyHCeom) in control rat ocular MR muscles (control), 1 (1 mB), 5 (5 mB), and 8 months (8 mB) after Botx-induced toxic paralysis are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The present study is the first long-term evaluation of the ocular MR muscles after Botx-induced paralysis with special emphasis on the changes in the MyHC isoform profiles in the muscle fibers triggered by temporary paralysis. Recovery of muscle function that follows Botx-induced paralysis after a few weeks is due to preterminal and terminal sprouting of motor axons in nonfunctional motor end plates and the formation of new functional neuromuscular junctions near the former ones. Contractile muscle activity returns after maturation of these junctions in a few weeks.5 13 18 19 20 In the soleus muscle, the initial growth rate of sprouting axons was relatively low (3 µm per day), but after a few weeks rose to 15 µm per day.21 Microscopic examination of paralyzed muscles in our experiments supports the idea that muscle reinnervation occurred as expected. Botx application caused no gross changes in muscle fiber morphology, in accordance with observations in paralyzed human orbicularis oculi muscle.22 Some muscle fibers, especially in the GL, became transiently hypertrophic, which is a typical response of temporarily denervated ocular muscles to reinnervation.23 Centrally located nuclei in paralyzed ocular MR muscles in our rats were very rare, whereas in permanently denervated ocular muscles their presence increased to approximately 20% of all nuclei.24

The most important observation in our study, however, is that in spite of meager morphologic muscle fiber changes after muscle chemical paralysis, the changes in MyHC isoform pattern, observed during the 8 months after Botx paralysis, were profound and long-lasting. Transformations evidently occurred among the existing fiber types1 2 3 4 5 6 that are hybrid fibers already present in normal controls.9 The available antibodies did not permit us to detect new, not-yet-described combinations of MyHC isoforms that would result in additional hybrid fiber types. Electrophoretic analysis of MyHC isoforms isolated from paralyzed ocular MR muscles demonstrated that during the first month after Botx application, the percentages of the MyHCIIa and MyHCIIx/d increased from less than 10% to 30% and 40%, respectively. This happened at the expense of the two fastest MyHC isoforms. The share of the MyHCIIb isoform decreased from approximately 50% to 15%. The fastest MyHCeom, which is specific for the extraocular muscles,25 26 27 and normally contributes approximately 25% to the total MyHC content in the ocular MR muscles (see also Refs. 9 and 27 ) virtually disappeared during the first month of paralysis. No significant change in MyHC profile occurred thereafter during the next 4 months.

At 8 months after Botx application, a shift toward a normal MyHC profile was observed. The shares of MyHCIIa and MyHCIIx/d decreased and that of MyHCIIb increased. The situation, however, was still far from normal. Most notably, the MyHCeom was still below the level of detection. The share of the slow MyHCI increased, and 8 months after Botx application, it was still significantly higher than in normal muscles. In short, the MyHC expression in Botx-paralyzed ocular MR muscles shifted toward slower isoforms, with complete loss of MyHCeom, and did not normalize, even after 8 months. A similar trend was observable by immunohistochemical analysis of muscle fibers in the paralyzed ocular MR muscles, but the differences were not so striking. The percentage of MyHCI-containing fibers in the GL increased during the experimental period, and the intensity of the immunohistochemical staining against MyHCIIb in muscle fibers of both layers was weaker than in normal muscles.

Unfortunately, we were not able to detect MyHCeom immunohistochemically. The reason for relatively less obvious changes observed by immunohistochemical technique in the paralyzed ocular MR muscles may have been that most muscle fibers in normal extraocular muscles are hybrid fibers—that is, they contain two or even three MyHC isoforms.9 Coexistence of MyHCI and MyHCneo within some fibers may lead to the assumption that after Botx injection, new fibers (MyHCI containing) are generated rather than that existing fast fibers are transformed to slow (MIF) fibers, which are the least oxidative and least glycolytic EOM fibers in the GL. However, we assume that the increase of relative amounts of MyHCIIa and MyHCIIx/d is at the expense of MyHCeom and MyHCIIb (Figs. 3 4) , although this was not clearly seen immunohistochemically (Fig. 2) . Again, different shades of gray in the immunohistochemical staining point to the presence of hybrid fibers in paralyzed muscles, which are expected to be even more numerous than in controls.

MyHC expression in muscle fibers is regulated by several factors, such as muscle load or hormonal status, but the neural activation pattern of muscle fibers seems to be most important.28 A phasic, high-frequency, short-train pattern of activation promotes expression of fast MyHC type II. However, experiments with electrical stimulation of denervated muscles have shown that a very special pattern of stimulation is required for maximal expression of the fastest MyHCIIb. Stimulation of a denervated rat fast muscle, in which before denervation the MyHCIIb predominated, with simple short high-frequency trains of impulses did not restore either the normal muscle-shortening velocity or the share of MyHCIIb.29 30 However, a special triplet pattern of only three impulses with a very short first interspike interval normalized the shortening velocity in denervated fast muscles.29 This peculiar pattern mimics dominant features of fast-motor-unit activity.31

It is interesting that the predominant share of the MyHCIIb in fast rat muscles also is not restored, even 6 to 8 months after nerve crush injury followed by immediate nerve regeneration and muscle reinnervation.32 33 It seems as though the new regenerated motor nerve endings are not competent to transmit all the physiological range of impulse patterns or even a temporary denervation causes a long-lasting change in firing pattern of motor units, due to some other cause. Recovery from Botx paralysis also mimics temporary denervation and requires axon growth, establishment of new neuromuscular junctions, and their maturation.

Therefore, we hypothesize that a long-term shift of MyHC content in Botx-paralyzed ocular MR muscles probably reflects long-lasting changes in neural activation of ocular MR muscles after Botx application. This may be due to the inability of newly formed neuromuscular junctions to transmit the complete frequency range of impulses characteristic of normal MR muscles. We are aware of no electrophysiologic study that has examined this problem. However, indirect evidence supports this view. Mechanical properties of the cat superior oblique muscles were studied during reinnervation after trochlear nerve axotomy. Although reinnervation was complete in 4 months, the muscles showed increased twitch time-to-peak—that is, slower contraction—for another 20 months.34 When the recovery of the Botx-paralyzed orbicularis oculi muscle in the monkey was studied, the amplitude of the blink completely recovered in approximately 30 days, but the peak velocity of the blink leveled off at approximately 70% of control and showed no further improvement for another 2 months.19 This indicates that maximal velocity of contraction is depressed for a long period in recovering Botx-paralyzed muscles, which is in accordance with the decreased expression of the fastest MyHCs observed in our experiments. In addition, it has been shown that the {gamma}-motoneuron nerve endings on muscle spindles in ocular muscles are even more rapidly affected by Botx application than the {alpha}-motoneuron ending and that consequent changes in proprioceptive input to oculomotor centers occur.35 Long-term changes in myotatic reflex control of ocular muscle activity after Botx application may, therefore, also affect its pattern of activation during recovery and expression of its MyHCs.

Disappearance of the specific MyHCeom from the ocular MR muscles recovering after Botx paralysis for the whole observation period is the most striking change observed in our experiments. This MyHC isoform is normally present only in the OL of the EOM in the rat, most probably in the SIFs. It is not distributed uniformly along the muscle fibers but is present principally in the central, end plate region of the muscle, whereas the immature (embryonic and neonatal) isoforms are excluded from this region and are present in the remainder of the fibers.36 Observed complete downregulation of this form in paralyzed MR muscles is in line with other signs of specific long-term sensitivity of singly innervated OL fibers to transient Botx-induced paralysis: long-term atrophy, dispersion, and permanently reduced content of mitochondria,10 and a long-lasting decrease of oxidative metabolism revealed by decreased SDH activity (the current study).

These OL SIF fibers seem to play a major role in precise alignment of the eyes and in maintaining tonic eye position.7 Long-term or even irreversible changes in these muscles, produced by Botx-induced temporary paralysis, may represent the foundation of successful Botx-induced correction of strabismus, sometimes with permanent effects after a single injection.37 In a recent review article, the usefulness of Botx treatment for infantile esotropia was emphasized.38 Long-lasting changes that we observed in rat ocular MR muscles after Botx-induced paralysis, especially in their MyHC composition and profile, probably reflect these muscles’ inability to completely regain their specific structural and functional characteristics. As stated in the review, it is conceivable that continued evaluation of the treated children as they mature will reveal unanticipated problems, such as late consecutive exotropia. In view of our results, it seems even more important to continue to observe these patients when cooperation permits.38



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  		Figure . (Continued)

 

   Acknowledgements
 
The authors thank Ana Tomazini, Marko Slak, and Milan Stevanec for technical assistance.


   Footnotes
 
Supported by the Ministry of Science and Technology, Slovenia, and the Centre National De Recherche Scientifique, France.

Submitted for publication February 20, 2001; revised July 30, 2001; accepted August 17, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Branka Stirn Kranjc, University Eye Hospital, Zaloska 29 A, 1000 Ljubljana, Slovenia. branka.stirn{at}guest.arnes.si


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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