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Keywords:

  • Myasthenia gravis;
  • muscle atrophy;
  • corticosteroids;
  • prognosis;
  • morphometry

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Selective atrophy of type II muscle fibres has been long recognized as an enigmatic but consistent feature of myasthenia gravis (MG) muscle; however, the pathophysiology and the mechanism of that change have remained obscure. In the present study, the results of histomorphometric analysis performed on muscle biopsies from 207 thymectomized seropositive MG patients were correlated with clinical features of MG to demonstrate possible pathophysiological associations and potential prognostic impact. The atrophy of type II fibres was verified in 35 cases (16.9%), being more pronounced in fibres of IIB subtype. It was neither significantly associated with the duration and severity of MG nor with the age of the patients. On the other hand, we demonstrated that the atrophy associated with long-term treatment with corticosteroids, and correlated with increasing doses. Thus, we suppose that the atrophy of type II muscle fibres in seropositive MG is steroid induced rather than MG-associated event. Although the MG patients with atrophy of type II fibres did not differ from the remaining MG cases in terms of improvement in the disease during the follow-up period, our analysis provides clear evidence that they presented a significantly slower tendency to reach an asymptomatic state after thymectomy. Therefore, the steroid-induced atrophy of type II fibres in MG muscle might be considered to be an unfavourable prognostic factor.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Myasthenia gravis (MG) is an autoimmune disease characterized by a humoural immune attack upon the acetylcholine receptor (AChR) resulting in the characteristic defect of neuromuscular transmission [1, 2]. Because the diagnosis of MG is based on the typical clinical pattern, neurophysiological and serological investigations, the patients are rarely (if ever) referred for a muscle biopsy [2]. However, the structural changes in MG muscles may be of interest in atypical cases when a neuropathologist deals with the problem of diagnostic alternatives between MG and other myopathies [3]. Although one would not expect major morphological changes in skeletal muscle tissue outside the neuromuscular junction, there were a few single reports in the early second half of the last century indicating an occasional presence of small focal collections of lymphocytes and atrophy of type II muscle fibres [4–6]. These results have been then adopted by major diagnostic textbooks and selective type II fibre atrophy has been thought to be a consistent feature of MG muscle [7, 8]. However, the pathogenesis of these changes has remained obscure. Recently, we have provided evidence that the lymphocytic infiltration in MG muscle is heavily associated with a thymic tumour and that it represents rather a consequence of the ‘spillover’ and infiltration of thymoma-derived mature naive T cells than a true cell-mediated autoimmune inflammation [9]. In the present study, we performed a histomorphometric analysis in a large series of MG muscle biopsies to assess the frequency of the reported selective atrophy of type II muscle fibres. Furthermore, we aimed at correlating the results with clinical features of MG in order to demonstrate possible pathophysiological associations and their potential prognostic impact.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patients

Two hundred seven MG patients, who underwent thymectomy in accordance with the generally agreed therapeutic guidelines [1, 2] (i.e. approximately 20–30% of newly diagnosed MG patients per year) with available follow-up data for at least 18 months after thymectomy in University Hospital Motol, Prague, were retrospectively included in the study. The diagnosis of MG was based on a typical clinical pattern and electromyography (EMG) with decrement at repetitive stimulation and/or increased jitter at single-fibre EMG [2]. The study was restricted to patients presenting increased serum levels of anti-AChR antibodies (seropositive MG). A standard transsternal thymectomy was performed in all the patients (T-3a according to the Myasthenia Gravis Foundation of America (MGFA) thymectomy classification [10]) and the thymus was examined histologically in each case.

Control samples from sternothyroid muscle were taken in 18 patients without MG during thoracic or neck surgery (mean age 43.7 ± 6.2 years).

The study protocol was approved by the Ethics Committee of the University Hospital Motol in Prague, Czech Republic. All patients gave written informed consent.

Evaluation of MG severity and outcome of MG patients

Because the recently recommended Quantitative MG Score for Disease Severity [10] could not be obtained from all patients at the onset of the disease in this retrospective study, the initial severity of MG was graded using a modified Osserman’s scale (0 – no symptoms; 1 – ocular MG; 2 – mild generalized MG without bulbar involvement; 3 – mild generalized MG weakness including bulbar involvement; 4 – moderate generalized MG with moderate bulbar and respiratory muscle weakness; 5 – severe generalized MG), as published elsewhere [11, 12]. The clinical data were collected both at the time of initial diagnosis and shortly preoperatively. A detailed report of the medications for each patient was obtained before the surgery. After thymectomy, all patients were regularly followed (from 19 up to 75 months; mean, 35.0 ± 13.3 months) at intervals of 3 months for the first year post-operatively and then every 6 months onwards each year to the end of follow-up. The post-intervention change in MG status was evaluated in our cohort using the modified stratification outlined in the MGFA recommendations for MG clinical research [10] (complete stable remission (CSR), pharmacological remission (PR), improved with minimal manifestations of MG, unchanged or worse) at every point of follow-up (Table 1). The remission rate was defined as the ratio of the number of asymptomatic patients (i.e. patients in CSR or PR) to the total number of patients whose information concerning the neurological status was available at the follow-up visit. The improvement rate was defined as the ratio of the number of patients, who improved their MG status after thymectomy (i.e. patients in CSR, PR or those who improved with minimal manifestation of MG) to the total number of patients.

Table 1.  Categories defined for the evaluation of the post-intervention status of MG patients after thymectomy
  1. MG, myasthenia gravis.

Complete stable remissionThe patient has had no symptoms or signs of MG for at least 6 months and has received no therapy for MG during that time.
Pharmacological remissionThe same criteria as for complete stable remission except that the patient continues to take some form of therapy for MG.
Improved with minimal manifestationsA substantial decrease in pre-treatment clinical manifestations (the patient has no symptoms of functional limitations from MG but has some weakness on examination of some muscles detectable by careful examination) or a sustained substantial reduction in MG medications
UnchangedNo substantial change in pre-treatment clinical manifestations or reduction in MG medications.
WorseA substantial increase in pre-treatment clinical manifestations or a substantial increase in MG medications.

Muscle biopsy – histology and histochemistry

An excision from the sternothyroid muscle (approximately 10 × 5 × 5 mm in size) was obtained from all the thymectomized MG patients and control patients from the margin of the operational field at the onset of surgery.

Muscle tissues were snap frozen in isopentane (2-methylbutane, Sigma-Aldrich Co., St. Louis, MO, USA) cooled in liquid nitrogen. Cryosections were examined by routine haematoxylin and eosin staining. To distinguish between the different muscle fibre types, sections were tested for myofibrillar adenosine triphosphatase (ATPase) activity using a standard histochemical reaction with pre-incubation at pH values of 4.3, 4.6 and 9.4 (described elsewhere [13]).

Morphometrical analysis

Quantitative morphometry was subsequently performed on three 10-μm serial cryosections from each sample freshly stained for ATPase pH 4.6, where three basic fibre types (I, IIA and IIB) could be identified (Fig. 1). The number of muscle fibres evaluated in each sample ranged from 175 to 568 (mean, 243.7 ± 58.5). Measurements were performed with a computer-based image processing system (QuickPhoto Pro 2.2 Software, Olympus Corp.) interfaced to a light microscope (Olympus BX51, Tokyo, Japan). All fibres in a cross-section devoid of freeze artefact and with fibres orientated perpendicularly to the section were counted for each specimen to obtain the percentage of fibre type prevalence. To quantify the degree of changes in fibre size distribution we used a simple but reliable method devised by Brooke and Engel [14]: The 100× scans were computerized and the ‘lesser diameter’ (the maximum diameter across the lesser aspect of the muscle fibre) of muscle fibres were measured and histograms were plotted for each fibre type separately. The atrophy factor (AF) and hypertrophy factor (HF) for each fibre type was calculated from the histograms as described elsewhere [14] and exemplified in Fig. 2. After considering the morphometric values obtained in the control samples (see below), where more than 95% of muscle fibres in histograms were between 40 and 70 μm and no case reached the AF/HF for any fibre type more than 100, the upper limits for AF and HF were arbitrarily applied similarly as recommended for other muscles [14, 15]: AF were established as 150 and 100 for males and females, respectively. The upper limit for the HF was used 200; however, no case of fibre type hypertrophy was observed in our series and thus it will not be further considered in this report.

image

Figure 1. Frozen section from sternothyroid muscle biopsy of a myasthenia gravis patient tested for myofibrillar adenosine triphosphatase with pre-incubation at pH value of 4.6, where three basic muscle fibre types can be identified: type I – dark fibres, type IIA – pale fibres, type IIB – intermediate staining. Note the atrophy of type II fibres accentuated in the IIB subtype. Bar scale = 100 μm.

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image

Figure 2. Histograms showing the distribution of fibre types I, IIA and IIB in samples of sternothyroid muscle of a control patient (A) and in a case of type II fibre atrophy (B) in sternothyroid muscle of myasthenia gravis patients. The atrophy factor (AF) is counted as follows: The number of fibres with diameter in the range 30 and 40 μm are multiplied by 1, the numbers of those between 20 and 30 μm by 2, the numbers of those from 10 to 20 μm by 3 and the numbers in the group less than 10 μm by 4. Added together, the result is divided by the total number of the appropriate fibres in the histogram and multiplied by 1000. AF for the type IIB fibres in the graph B: (2 × 4 + 3 × 3 + 18 × 2 + 12 × 1)/43 × 1000 = 1511.6. Note: D, diameter; P, prevalence (percentage) of fibres types

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Statistical analysis

The normally distributed continuous data were expressed as means ± S.D. The Student’s t-test and the Fisher’s exact test or chi-square test were used to compare the continuous and categorical variables, respectively. For the outcome analysis, crude cumulative remission rates and improvement rates were calculated and compared between the tested groups at specific points of follow-up period. Further, we determined the Kaplan–Meier estimate of the time to reach an asymptomatic state, defined as time from surgery to first date, when the remission (CSR or PR lasting at least 6 months or to the end of follow-up) was achieved. Univariate analysis was performed with a log-rank test to assess the strength of association between the subgroups of patients and the outcome. Relative risks (hazard ratios) were also computed using univariate Cox proportional hazards regression analysis. The analytical work was performed with SPSS (version 11, SPSS, Inc.) software. Two-tailed probability (P)-values < 0.05 were considered significant. A confidence interval was taken at 95%.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

MG patients profiles

In our series, 207 MG patients (92 males and 115 females, mean age 41.0 ± 14.9 years) with the mean duration of the disease before surgery of 13.0 ± 14.5 months presented before surgery severity grades of MG from 1 to 4 (proportions are given in Table 2). The mean preoperative anti-AChR antibody titre was 7.1 ± 3.3 nmol/l. Thirty-five (16.9%) patients were diagnosed with thymoma. The mean duration of preoperative symptoms was 3.4 ± 1.6 months for the patients with thymoma (mean age 50.7 ± 14.1 years), in contrast to 14.9 ± 15.2 months for those patients without thymoma (mean age 39.1 ± 14.3 years). All patients in the study were long-term treated with cholinesterase inhibitors (pyridostigmine or ambenonium) before the surgery. One hundred eleven (53.6%) patients required long-term therapy with corticosteroids, either with prednisolone (n= 92, 44.4%) or equivalent (methylprednisolone, n= 19, 18.4%). Some of them were additionally treated in combination with azathioprine (n= 38, 18.3%), mycophenolate mofetil (n= 5, 2.4%) or underwent plasmapheresis before surgery (n= 26, 12.6%).

Table 2.  Preoperative evaluation of MG patients and the duration of follow-up after thymectomy. Patients with absence (group A) or presence (group B) of type II muscle fibres atrophy are compared
  Total n= 207 Group A n= 172 Group B n= 35 P-value
  1. Anti-AChR, serum level of the anti-acetylcholine receptor antibodies; G, Osserman’s grade of myasthenia gravis; MG, myasthenia gravis; N.S., not significant; S.D., standard deviation.

Age, years (mean ± S.D.)41.0 ± 14.941.8 ± 15.137.1 ± 13.0N.S.
Duration of MG symptoms, months (mean ± S.D.)13.0 ± 14.514.0 ± 15.016.3 ± 13.7N.S.
Severity of MG (mean ± S.D.)2.45 ± 0.802.45 ± 0.812.45 ± 0.77N.S.
   G 1, n (%)27 (13.0)23 (13.4)4 (11.4)N.S.
   G 2, n (%)73 (35.3)60 (34.9)13 (37.1)N.S.
   G 3, n (%)93 (44.9)77 (44.8)16 (45.7)N.S.
   G 4, n (%)14 (6.8)12 (7.0)2 (5.7)N.S.
   G 5, n (%)0 (0)0 (0)0 (0)N.S.
Anti-AChR (nmol/l), mean ± S.D.7.1 ± 3.37.3 ± 2.97.0 ± 3.3N.S.
Follow-up duration, mean months ± S.D. (range)35.0 ± 13.334.7 ± 13.136.2 ± 15.0N.S.
(19–75)(19–75)(20–68) 

Morphometric analysis

Controls

The mean diameters and prevalence of fibre types I, IIA and IIB obtained in control samples are shown in Fig. 3. The mean values of the AF and HF calculated from histograms were 31.5 ± 26.7 and 42.7 ± 32.1 with range 0–87 and 0–91, respectively.

image

Figure 3. Histograms showing the mean diameter and the fibre type prevalence of type I, IIA and IIB muscle fibres in controls and in the myasthenia gravis patients with absence (A) or presence (B) of type II fibres atrophy.

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Myasthenia gravis patients

The morphometric study followed by the analysis of the histograms confirmed the selective atrophy of type II fibres in 35 cases (16.9%) with AF range of 214 to 1752 (mean 738.1 ± 431.9; median 631). In cases with type II fibres atrophy, the type IIB fibres were more affected than the type IIA fibres (Fig. 3). In all cases, we observed typical checkerboard pattern of muscle fibre type distribution. Only scarce atrophic fibres were angulated in their shape, with most of the atrophic fibres being rounded. No signs of definite neurogenic remodelling of muscle (i.e. fibre type grouping or group atrophy) were noted in our series. In the non-atrophic cases of MG muscle, the mean fibre diameters of fibres type I, IIA and IIB did not significantly differ from controls (Fig. 3). No significant change in size distribution of type I fibres was revealed. The prevalence of fibre types I, IIA and IIB in both the non-atrophic and the atrophic cases did not significantly differ from controls (Fig. 3).

Clinically, all the MG patients with type II fibres atrophy presented at the disease onset, shortly before thymectomy and during the follow-up period pure MG without any obvious additional muscle weakness and/or myopathic changes in EMG or elevation of serum creatine kinase (CK) and myoglobin.

Correlation and outcome analysis

Because the outcome of thymoma MG patients was not shown repeatedly to be different from those without thymoma [16, 17], the thymoma and non-thymoma patients were considered together. The studied MG patients were divided into two groups based on the absence (group A) or presence (group B) of the atrophy of type II muscle fibres.

Preoperative clinical parameters

Patients from both groups did not significantly differ concerning the age, at the onset of the disease, the severity of MG, the anti-AChR antibodies titre, as well as the mean duration of the disease before surgery (Table 2). The type II fibre atrophy has not been identified in MG patients with course of the disease shorter than 6 months.

Thymus pathology

The selective atrophy of type II fibres was significantly associated with non-neoplastic pathology in thymus, because no such case was found in thymoma MG patients of our series. In the non-thymoma group, the prevalence of type II fibre atrophy was not significantly different in patients with atrophic thymus (11 of 51 patients, 21.6%) when compared to those with thymic follicular hyperplasia (24 of 121 patients, 19.8%) (P= 0.839).

Outcome analysis

The outcome of the whole cohort of the MG patients after thymectomy in respect to the preoperative parameters of the disease are given in Table 3. The tendency to improve after thymectomy in patients with less severe MG, shorter duration of the disease before surgery and in younger patients was evident. Concerning the presence of type II fibre atrophy, there was no significant difference in the follow-up duration between the groups A and B (see Table 2). The comparison of the two groups concerning the stratification of the outcome in specific points of the follow-up period is shown in Table 4. As depicted in Fig. 4, MG patients with atrophy of type II fibres showed significantly slower tendency to achieve the asymptomatic state (either CSR or PR). However, the overall rate of improvement did not significantly differ between the two groups, i.e. most patients from both groups improved after thymectomy similarly, but higher proportion of patients with atrophic type II fibres remained presenting minimal MG symptoms requiring treatment. In accordance to that, the Kaplan–Meier estimate analysed by log-rank test revealed, that the subgroups significantly differed in time to achieve the asymptomatic state (P= 0.009) (Fig. 5); the appropriate hazard ratio (CI) computed using univariate Cox regression analysis was 2.0 (1.2–3.4) (P= 0.019).

Table 3.  Stratified neurological outcome of the whole cohort of MG patients after thymectomy at the first year of follow-up according to the severity of MG, age of the patients at the onset of the disease and the duration of the disease before surgery
   N Total 207 CSR 28 PR 37 I-MM 99 U 37 W 6
  1. CSR, complete stable remission; G, Osserman's grade of myasthenia gravis; I-MM, improved with minimal manifestations; MG, myasthenia gravis; PR, pharmacological remission; U, unchanged; W, worse.

Severity of MG
   G1, n (%)278 (29.6)9 (33.3)9 (33.3)1 (3.7)0 (0)
   G2, n (%)7315 (20.5)22 (30.1)26 (35.6)9 (12.3)1 (1.4)
   G3, n (%)935 (5.4)5 (5.4)59 (63.4)21 (22.6)3 (3.2)
   G4, n (%)140 (0)1 (7.1)5 (35.7)6 (42.9)2 (14.3)
Age
   ≤35years, n (%)5511 (20.0)14 (25.5)14 (25.5)15 (27.3)1 (1.8)
   >35years, n (%)15217 (11.2)23 (15.1)85 (55.9)22 (14.5)5 (3.3)
Duration of MG symptoms
   ≤12 months, n (%)9817 (17.3)19 (19.4)39 (39.8)20 (20.4)3 (3.1)
   >12 months, n (%)10911 (10.1)18 (16.5)60 (55.0)17 (15.6)3 (2.8)
Table 4.  Stratified neurological outcome of MG patients after thymectomy at the first, second and third year of follow-up. The remission rates (i.e. the proportion of asymptomatic MG patients) and the improvement rates (i.e. the proportion of MG patients who improved after thymectomy) are also given. Patients with absence (group A) or presence (group B) of type II muscle fibres atrophy are compared. Two-tailed P-value of the Fisher’s exact test is given
  1y 2y3y
  1. MM, minimal manifestations; N.S., not significant; *Significant.

Number of patients, total (A/B)207 (172/35)146 (117/29)65 (53/12)
Complete stable remission, n (%)28 (13.5)28 (26.0)22 (33.8)
   Group A25 (14.5)35 (29.9)21 (39.6)
   Group B3 (8.6)3 (10.3)1 (8.3)
   P-valueN.S. p= 0.034* P= 0.047*
Pharmacological remission, n (%)37 (17.9)29 (19.9)11 (16.9)
   Group A33 (19.2)26 (22.2)10 (18.9)
   Group B4 (11.4)3 (10.3)1 (8.3)
   P-valueN.S.N.S.N.S.
Improved with MM, n (%)99 (47.8)55 (37.7)21 (32.3)
   Group A80 (46.5)39 (33.3)13 (24.5)
   Group B19 (54.3)16 (55.2)8 (66.7)
   P-valueN.S. p= 0.038* p= 0.015*
Unchanged, n (%)37 (17.9)19 (13.0)10 (15.4)
   Group A30 (17.4)14 (12.0)8 (15.1)
   Group B7 (20.0)5 (17.2)2 (16.7)
   P-valueN.S.N.S.N.S.
Worse, n (%)6 (2.9)5 (3.4)1 (1.5)
   Group A4(2.3)3 (2.6)1 (1.9)
   Group B2 (5.7)2 (6.9)0
   P-valueN.S.N.S.N.S.
Remission rate, n (%) 65 (31.4)67 (45.9)33 (50.8)
   Group A58 (33.7)61 (52.1)31 (58.5)
   Group B7 (20.0)6 (20.7)2 (16.7)
   P-value P= 0.020* P= 0.003* P= 0.011*
Improvement rate, n (%) 164 (79.2)122 (83.6)54 (83.1)
   Group A138 (80.2)100 (85.5)44 (83.0)
   Group B26 (74.3)22 (75.9)10 (83.3)
   P-valueN.S.N.S.N.S.
image

Figure 4. Graph showing the progression of remission rate (RR) and improvement rate (IM) during the follow-up period in patients with atrophy of type II fibres (RR+, IM+) in comparison to those without atrophy (RR−, IM−).

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image

Figure 5. The rate of asymptomatic myasthenia gravis patients (i.e. remission rate) over time after thymectomy grouped according to the absence (group A) or presence (group B) of type II fibre atrophy, calculated by the Kaplan–Meier method. P-value of log-rank testing.

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MG treatment before surgery

As indicated in Fig. 6, type II fibre atrophy was not seen in MG patients without corticosteroid treatment and in those treated with corticosteroids with doses 10 mg or less per day (prednisolone or equivalent of methylprednisolone). The prevalence of type II fibres atrophy was significantly increased with increasing dose of long-term treatment with corticosteroids reaching prevalence of almost 73% in MG patients with highest doses. The presence of atrophic fibres prevailed in patients treated with methylprednisolone when compared to those with prednisolone. No statistically significant association was revealed between the prevalence of type II fibres atrophy and the treatment by less frequently used drugs: The type II fibre atrophy was found in none of patients treated with mycophenolate mofetil, in 2 of 38 (5.2%) patients treated with azathioprine and in 3 of 26 (11.5%), who underwent plasmapheresis before surgery.

image

Figure 6. Histogram showing the proportion of myasthenia gravis patients who presented type II muscle fibres atrophy depending on the dose and type of long-term preoperative steroid treatment.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Atrophy of type II muscle fibres in MG muscles was first noted based upon non-quantified observations of single cases or small series of patients [4–6]. In 1969, the pioneering morphometric study of MG muscle by Brooke and Engel demonstrated quantitatively the presence of type II fibres atrophy in more than half of 32 studied MG muscles [18]. However, the mechanism and significance of that change were not considered in those reports. Perhaps due to the low value of muscle biopsy for the diagnosis of MG, this enigmatic fact stood apart from the major field of view of MG research for several decades; however, selective atrophy of a muscle fibre type may become of importance in biopsy differential diagnosis [2, 3]. Our quantitative morphometric analysis confirmed that the atrophy of type II fibres occurs in seropositive MG muscles, but its frequency remains low – it was verified by morphometry in less than 17% of our MG patients. It was neither significantly associated with the duration and severity of MG nor with the age of the patients. On the other hand, we demonstrated that the atrophy associated with long-term treatment with corticosteroids, even in correlation with the increasing dose. Therefore, we suppose that the atrophy of type II muscle fibres in seropositive MG represents a steroid-induced change rather than MG-associated event. It is also supported by the accentuation of atrophy in the IIB subtype of muscle fibres, which is a well-established reaction of muscle tissue to steroid administration known from both experiments [19, 20] and clinical settings as chronic steroid myopathy [21, 22]. Some authors reported also a shift in fibre type prevalence in muscles following corticosteroid treatment with reduction in the percentage of type IIB fibres [19, 23]; on the contrary, other studies [20] have not been able to show such change, as was also the case in our study.

The incidence of chronic steroid myopathy has varied from 7% to 60%[24, 25] in patients receiving corticosteroid treatment for various diseases with marked difference in the effects on muscle depending on the type of steroids used. The most distinct muscle changes were observed in patients who received fluorinated steroids (triamcinilone or dexamethasone), but milder forms of myopathy were detected under treatment with any corticosteroid [26, 27]. In our series, the selective atrophy of type II fibres was more pronounced in patients treated with methylprednisolone when compared to those who received prednisolone, which supports a previous experimental observations [27]. The cumulative dose of steroid has been reported to be significantly related to the development of myopathy [25] and it was also evident in our series. The absence of atrophy of type II fibres in our thymoma MG patients may correspond with the short mean duration of symptoms and therapy before thymectomy.

The mechanisms by which corticosteroids can act at the molecular signalling pathway in muscle atrophy have been largely elucidated in recent years [21, 28]. The stimulation of proteolysis or the inhibition of protein synthesis in muscle atrophy as induced by steroids was shown to occur at least in part due to the activation of the ubiquitin–proteasome pathway, in which there is increased expression of the genes that encode E3 ubiquitin ligases, mainly muscle ring finger1 (MuRF1) and muscle atrophy F-box (MAFbx) also called atrogin-1 [28, 29]. In relation to this, an interesting observation was reported in anti-AChR antibody negative (‘seronegative’) MG associated with antibodies against muscle specific kinase (MuSK) [30], where clinically evident atrophy of muscles especially of the facial and lingual muscles is a major feature [31]. The ability of MuSK antibodies to increase expression of MuRF1 especially in facial muscles [31] was demonstrated, thereby providing possible mechanism for the prominent muscle atrophy in this subtype of MG. In our series of seropositive MG cases, atrophy represented only a microscopic phenomenon. Whether the anti-AChR antibodies could act and contribute to the atrophy the similar way as anti-MuSK antibodies has not been shown. Extraordinarily rare MG cases (sometimes reported as ‘myasthenic neuromyopathy’) present clinically and histologically with accentuated neurogenic atrophy [32, 33]; however, no histological signs of neurogenic atrophy were features in our cases.

Despite the histological pattern of steroid myopathy, no obvious clinical symptoms of an additional myopathy were noted during the course of MG in our patients. The explanation may be provided by the fact that the severity of atrophy observed in our patients was lower when compared with the cases referred to a muscle biopsy due to the advanced symptomatic steroid myopathy, where the AF for type II fibres often reaches levels of more than 2000. We speculate, that the degree of the atrophy of type II fibres in our MG cases did not cross the threshold to be obvious in clinical manifestation or, more likely, minimal symptoms of steroid-induced weakness might have been attributed to the ongoing MG. A disclosure of the asymptomatic steroid-induced myopathic changes in muscle would require repeated EMG testing of MG patients during the course of the MG treatment, which is not in common use.

The clinical profile of MG patients included in our study as well as the results of our outcome analysis are in accordance with recent studies reporting outcome of MG patients after thymectomy in larger series [16, 17, 34, 35]. Although the MG patient with atrophy of type II fibres did not differ from the remaining cases in the sense of improvement of the disease during the follow-up period, the analysis provides clear evidence that they presented (not dramatic but significant) decrease in the tendency to reach an asymptomatic state after thymectomy. Therefore, the atrophy of type II fibres in MG muscle biopsy might be considered an unfavourable prognostic factor.

In conclusion, we have confirmed the previous observations that selective atrophy of type II muscle fibres occurs in MG muscle; however, it is less frequent than previously expected and rather represents the sub-clinical consequence of the chronic steroid administration for MG. Whether such MG patients would profit from a change of therapeutic strategy following muscle biopsy is to be considered.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The study was supported by the Grant of Ministry of Health of the Czech Republic No. IGA MZCR NR/8924-3 and by the Research Project No. VZ FNM 00064203.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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