Immunohistochemical and ultrastructural analysis of sporadic inclusion body myositis: a case series

Early clinical symptoms which should raise suspicion of s-IBM include difficulty in climbing stairs and rising from a sedentary position, aching of thighs and knees and difficulties in using different hand tools [30]. In addition, weakness of the thigh’s quadriceps muscles, increased serum CK level, the presence of RVs and atrophic myofibers in muscle biopsies could help to diagnose patients suffering from s-IBM [9, 31]. There are some reports showing that physical effort may improve skeletal muscle strength and function, which would encourage its implementation immediately after s-IBM has been diagnosed [6, 32]. Since s-IBM is an untreatable disease establishing the final diagnosis is critical for patients in order to avoid an ineffective immunosuppressive therapy with its many side effects [9, 15], and to start physiotherapy [33].

In our study the clinical features, including weakness of upper and lower extremities, difficulty in climbing stairs as well as patients’ ages were typical and similar to other reports [12, 27, 34]. Moreover, similarly to Hermanns et al. [4] and Sakai et al. [17] we observed periodical increases in the levels of CK and LDH enzymes in the studied patients’ serum. In patients from a study conducted in China the average value of CK was 397.38 IU/L, whereas for LDH it was 218.21 IU/L [12]. In our study, the average values of CK and LDH were much higher, 1068 IU/L and 344 IU/L, respectively. As is well known, serum CK and LDH levels increase during skeletal muscle damage and degeneration. Therefore, a muscle biopsy in every case where IBM is suspected is essential for a definitive diagnosis even if there are no features of muscle weakness according to the MRC scale [13, 26, 35, 36].

We found that skeletal muscle biopsies of IBM patients presented typical changes which have been well described by others, including hypertrophy of connective and adipose tissues, focal occurrence of necrotic fibers, RVs, a pattern of inflammatory cell infiltrations or pathological protein aggregates [3, 4, 9, 12, 17, 37]. In most cases we saw myofiber atrophy with subsequent invasion of connective and adipose tissue. The results are in accordance with observations made by others [12, 17, 27, 36, 37]. In a few cases hypertrophic myofibers in IBM patients were also reported [12, 27]. However, muscle fiber hypertrophy seems to be more characteristic of skeletal muscle dystrophies than inflammatory myopathy [38]. We have also demonstrated the presence of necrotic myofibers which could occur in muscles of IBM patients, as was described by Kierdaszuk et al. [37]. The necrotic process is probably caused by the action of perforin and granzymes, enzymes secreted by T lymphocytes [15]. The necrosis of single myofibers is very often associated with muscle fiber regeneration. Indeed, our biopsies found myofibers with centrally located myonuclei which are typical for muscle regeneration. Moreover, in our biopsies we saw moderately intense infiltration of mononuclear cells. To identify the type of inflammatory cell we used an immunohistochemical method, which had been previously suggested by others [17, 37, 39]. Our results indicated that cytotoxic CD8 + T lymphocytes [40] as well as macrophages in infiltrates are present. Current reports suggest that cN1A is a reliable marker for s-IBM, although information on this subject is still insufficient. To our knowledge, only two research teams have presented the diagnostic utility of cN1A antibody on paraffin sections [41, 42]. Our results are the third and so far, they confirm the accumulation of cN1A protein around the vacuoles and myonuclei. Moreover, aggregates of Tau protein, beta-amyloid and apolipoprotein B were detected in the examined fibers. These results are in accordance with some other researchers who suggest that they are typical for most IBM cases [9, 37, 39]. However, Sakai et al. [17] found that in muscle biopsies taken from IBM patients beta-amyloid aggregates were not always present, especially in those patients with granuloma formation inside myofibers. We also found in the sarcoplasm of individual muscle fibers the central or paracentral RVs which are key elements in diagnosing s-IBM in muscles. Here, we presented unique images from the transmission electron microscope showing the real ultrastructure of RVs. To the best of our knowledge, the actual ultrastructure of vacuoles with granules at the margins has not been described. It has been suggested that the formation of RVs could be associated with the process of autophagy or myonuclei degeneration in myofibers. Recently, typical autophagic proteins and nuclear membrane proteins in these vacuoles were seen [10, 13, 30, 43].

In some cases, tubulofilamentous inclusions within myofibers have been seen at the ultrastructural level [3, 4]. In our study such structures were not found, implying that they do not always exist in this disease, which is consistent with diagnostic criteria of definite IBM according to Griggs (1995) [29]. However, as others, we found typical, for IBM, whorled osmiophilic membrane debris, myelinoid bodies and lipid-like inclusion bodies which were predominant in the sarcoplasm. A few authors have reported the presence of some nuclear and cytoplasmic inclusions in IBM muscle biopsies which were located adjacent to the tubulofilamentous structures [4] or between the myofilaments [44]. However, in contrast to others, we did not detect inclusion bodies in the myonuclei [2, 37]. According to other researchers [6], at least three vacuolated fibers should be examined by electron microscopy in order to find inclusions. Interestingly, we noticed abnormalities in the triad ultrastructure, which are characteristic of centronuclear myopathy [45, 46].

As was described above, the morphological features of the disease and detection of typical proteins are not homogenous in each case. We are aware of the main limitation of this work, namely the small number of patients, and the lack of some laboratory tests such as antibodies or magnetic resonance imaging which are not available in our university. Furthermore, they are not routinely used in regular practice. Diagnostic imaging can primarily support the differential diagnosis of inflammatory muscle disease, due to characteristic patterns of muscle involvement [47]. This article summarizes the practical aspects of the s-IBM-diagnosis by using a combination of immunohistochemical and ultrastructural assessment. Current studies on early recognition of IBM are focused on searching for new molecular biomarkers which could be detected in skeletal muscle biopsies [7, 9, 10, 24, 48, 49, 50].