The aim of the present study was to explore the clinical, neuroelectrophysiological and muscular pathological characteristics of chronic progressive external ophthalmoplegia (CPEO) and to improve the understanding of CPEO. Clinical manifestations, neuroelectrophysiology and pathological features of muscle biopsies from 12 patients with CPEO were retrospectively analyzed. The average age of onset for the 12 patients (6 males and 6 females) was 17.2 years. All patients had different degrees of blepharoptosis. A total of 11 patients experienced ocular dyskinesia, but diplopia was rare. Electrophysiological testing in 12 patients revealed abnormal changes in 6 patients, including 4 patients with a myogenic lesion, 1 patient with a neurogenic lesion, and 1 patient with mixed myogenic/neurogenic lesions. Two patients had slow sensory nerve conduction velocity. Muscle biopsies in 12 patients demonstrated ragged-red, irregular and broken fibers in 11 patients through Gomori trichrome and hematoxylin and eosin (H&E) staining, increased lipid levels in some muscle fibers in 4 patients through Οil Ρed O staining and abnormal distribution of type I and II muscle fibers in 3 patients through ATPase staining. Electron microscopy in 5 patients showed an increased number of mitochondria and abnormal mitochondrial aggregation between submucosa and myofibrils in 4 patients. These findings suggest that the possibility of CPEO should be considered if patients present with obvious extraocular muscle paralysis without diplopia. Furthermore, the identification of ragged-red fibers by Gomori trichrome and H&E staining of muscle biopsies from patients is an important basis for the diagnosis of CPEO.
Chronic progressive external ophthalmoplegia (CPEO) is a mitochondrial disease characterized by chronic progressive blepharoptosis, ocular dyskinesia, ptosis, limited of eye movement and bulbar muscle weakness of varied severity (
CPEO is a rare disorder with a low prevalence. In the 20 years between January 1997 and January 2018, only 12 patients with CPEO were encountered among a total of 1,060 muscle biopsies that were performed at the People's Hospital of Jiaozuo City. In the present study, the clinical data of the 12 patients with CEPO were retrospectively analyzed, with the aim of investigating the clinical manifestations, neuroelectrophysiology and muscle pathology of patients with CEPO, while improving the current understanding of CPEO and promoting early diagnosis.
The present study was approved by the ethics committee of the People's Hospital of Jiaozuo City (Jiaozuo, China). All patients have provided written informed consent for publication.
This retrospective study summarized and analyzed the clinical data of 12 patients who had been diagnosed with CPEO from a total of 1,060 muscle biopsies performed in the People's Hospital of Jiaozuo City between January 1997 and January 2018. There were 6 males and 6 females, aged between 9 and 56 years, with an average age of 32 years. The disease duration was 2-25 years, with an average of 13 years. The onset age was 7-35 years, with an average of 17.2 years.
Concentric needle electromyography as well as sensory and motor nerve conduction velocities in all the patients were measured using an Evoked Potential/Electromyography Measuring System (MEB-9200K; Nihon Kohden Corporation). Deltoid, biceps brachii, iliopsoas, gluteus maximus, vastus medialis, tibialis anterior and gastrocnemius muscles were selected for electromyography. Data were recorded and analyzed, including: i) spontaneous potential during the quiet period (occurrence in more than two sites was considered an abnormality); ii) time limit, amplitude and percentage of polyphasic waves of 20 motor unit action potentials during contractions at small forces; and iii) waveform and peak-to-peak amplitude of the recruitment potential during contractions at large forces. Conduction velocities of the median nerve, ulnar nerve, common peroneal nerve, posterior tibial nerve and sural nerve were determined. The ulnar nerve and axillary nerve were selected for repetitive nerve stimulation.
All the patients underwent open biopsy after local anesthesia, including biceps brachii in 8 cases, deltoid in 1 case and quadriceps femoris in 3 cases. A portion of each muscle specimen was frozen in liquid nitrogen, and sliced into 8-µm frozen sections. Sections were subjected to conventional histological, enzyme and histochemical staining, including hematoxylin and eosin (H&E), modified Gomori trichrome, reduced nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), oil red O, periodic acid-Schiff reaction and ATPase (pH 4.2, 4.3, 10.6 and 10.8). Results were visualized using a light microscope (magnifications, x200 and x400).
Frozen sections were first stained with hematoxylin solution for 5-10 min, rinsed under running water for 5 min and stained with eosin solution for 3 min at room temperature. The sections were then dehydrated with an alcohol gradient, cleared in xylene and mounted in neutral gum.
Frozen sections were immersed in hematoxylin solution for 5-10 min at room temperature, rinsed under running water for 5 min, and then immersed in Gomori trichrome solution for 30 min at room temperature. After rinsing with distilled water, the sections were dehydrated with an alcohol gradient, cleared in xylene and mounted in neutral gum.
Frozen sections were incubated with NADH-TR staining solution for 30 min in a thermostat at 37˚C, rinsed with distilled water, air-dried and finally sealed with glycerin gelatin.
Frozen sections were stained in Oil Red O staining solution for 30 min at room temperature. Following rinsing with running water for 5 min, sections were counterstained with hematoxylin solution for 1 min at room temperature, rinsed with running water for 2 min, and sealed with glycerin gelatin.
Frozen sections were fixed in Carnoy's solution for 10 min at room temperature. Following rinsing with running water for 5 min, the sections were immersed in 1% periodic acid solution for 5 min, rinsed with distilled water for 2 min, and then immersed in Schiff solution for 10 min at room temperature. After washing with running water for 5 min, sections were counterstained with hematoxylin solution for 2-3 min at room temperature, and rinsed with running water for 5 min. Then the sections were dehydrated with an alcohol gradient, cleared in xylene and mounted in neutral gum.
Two 8-µm frozen sections per patient were incubated with sodium acetate solution (pH 4.2 and pH 4.3) for 10 min at room temperature. Another two, 8-µm frozen sections were incubated with calcium barbital solution (pH 10.6 and pH 10.8) at 37˚C for 10 min. The four sections were then rinsed with running water for 5 min and incubated with ATP/calcium barbital solution (pH 9.4) at 37˚C for 30 min. They were then incubated in 2% cobalt chloride for 3 min at room temperature, rinsed with distilled water, immersed in 1% ammonium sulfide for 3 min at room temperature, and rinsed with running water for 5 min.
Another portion of each muscle specimen was fixed in 2.5% glutaraldehyde for 24 h at room temperature; washed three times with sodium dimethylarsenate buffer; post-fixed in 1% osmic acid (in sodium dimethylarsenate buffer) for 2 h at room temperature; dehydrated with either an alcohol or acetone series; and then embedded in epoxy resin 618 at 37˚C for 24 h and at 60˚C for 48 h. The specimens were sliced into ultrathin sections (60 nm) using a glass knife. The sections were stained with uranyl acetate at room temperature for 20 min, rinsed thoroughly with water and stained with lead citrate at room temperature for 20 min, following which these sections were dried and observed using a H-7500 transmission electron microscope (magnification, x10,000; Hitachi, Ltd.).
All 12 patients had varying degrees of chronic progressive blepharoptosis. The eyelids covered half of the pupil in 6 patients, two-thirds of the pupil in 4 patients and the whole pupil in 2 patients (
Needle electromyography in 12 patients revealed abnormal changes in 6 patients, including myogenic lesions in 4 patients, a neurogenic lesion in 1 patient and mixed myogenic/neurogenic lesions in 1 patient. A decreased sensory nerve conduction velocity was observed in 2 patients. One of these 2 patients had a decreased sensory conduction velocity of the median nerve and posterior tibial nerve, and the other had a decreased sensory conduction velocity of the sural nerve. Negative symptoms were determined in 12 patients after repetitive stimulation.
H&E staining demonstrated that among the 12 patients, muscle fiber size was normal in 10 patients and a few angular atrophic muscle fibers were visible in 2 patients. In 11 patients, basophilic muscle fibers were irregularly scattered in the sarcoplasm and some fibers were broken (
Transmission electron microscopy showed that among the 5 patients tested, there were increased mitochondria and abnormal mitochondrial aggregation with varied shapes and sizes observed between the subsarcolemma and myofibrils in 4 of the 5 patients (
CPEO is a rare mitochondrial myopathy that can occur at any age, but is commonly found in children and young people (
Neuroelectrophysiological testing is a conventional examination for the diagnosis of neuromuscular diseases. Repetitive nerve stimulation is an effective method for differentiating CPEO from other neuromuscular diseases (
Muscle biopsies are an important method for the diagnosis of mitochondrial myopathy in CPEO. In 1963, Engel and Cunningham (
With the rapid development of molecular tests, many mtDNA fragment losses and point mutations have been identified in patients with CPEO (
It should be noted that the present study has limitations. Firstly, the sample size is small. Since CPEO is a rare disorder, inclusion of more patients with CPEO is very difficult, but the inclusion of more patients in future multi-center studies would be useful to clarify the clinical feature of different types of CPEO. Secondly, a number of possible factors were not analyzed, for example the disease duration and age of the patients could have affected the results and needs to be further investigated. Finally, given the importance of gene detection in the diagnosis of CPEO, further studies are needed to investigate the genetic abnormalities associated with patients with CPEO, to further improve the molecular-pathological diagnosis of CPEO.
In conclusion, the present study suggests that if patients present with obvious extraocular muscle paralysis and do not have symptoms of diplopia, the possibility of CPEO should be considered. Ragged-red fibers identified by Gomori trichrome staining and the H&E staining of muscle biopsies from patients is essential for confirming the diagnosis of CPEO.
Not applicable.
No funding was received.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
HL contributed to the study conception and design. QQQ, HL, QQ, XZ, YZ contributed to the acquisition, analysis and interpretation of data. All authors read and approved the final manuscript.
The present study was approved by the Ethics Committee of the People's Hospital of Jiaozuo City. All patients have given written informed consent.
All patients have given written informed consent for publication.
The authors declare that they have no competing interests.
In a patient with blepharoptosis, the right eyelid covered half of the pupil, with binocular fixation in the central position.
Representative staining images of tissues from patients with chronic progressive external ophthalmoplegia. (A) Hematoxylin and eosin staining of muscle biopsies showed normally sized muscle fibers, abnormal scattered deeply-stained muscle fibers with basophilic sarcoplasm and some broken muscle fibers (magnification, x400). (B) Modified Gomori trichrome staining of muscle biopsies showed a number of typical ragged-red fibers in the muscle tissue (magnification, x400). (C) Nicotinamide adenine dinucleotide tetrazolium reductase staining of muscle biopsies showed that the edges of the muscle fibers were deeply stained blue (magnification, x400). (D) Oil Red O staining of muscle biopsies showed a significant increase in red particles in some of the muscle fibers, suggesting an increase in lipids (magnification, x400). (E) ATPase staining of muscle biopsies showed abnormal distribution of type I and II muscle fibers which appear as fiber-type grouping (magnification, x200). (F) Periodic acid-Schiff reaction staining of muscle biopsies revealed no abnormalities (magnification, x400).
Representative transmission electron microscopy images of patients with chronic progressive external ophthalmoplegia. (A) Massive abnormal subsarcolemma mitochondrial aggregation in a patient, as shown by the yellow arrow. Lead-uranium double staining (magnification, x12,500). (B) Massive abnormal mitochondrial aggregations were observed, with varied shapes and sizes between subsarcolemmas and myofibrils, as demonstrated by the yellow arrow. Lead-uranium double staining (magnification, x12,500)