Characterization of patients
The patients in this study suffered from idiopathic Parkinson’s disease and camptocormia and were prospectively included in this study between 2004 and 2010. The diagnosis of Parkinson’s disease was made according to the British Parkinson’s Disease Society Brain Bank criteria [
18]. Camptocormia is defined here as a marked anterior flexion of the thoracolumbar spine of at least 30° (with or without additional laterodeviation), appearing in the standing or walking position and disappearing in the recumbent position, but without any signs of a fixed kyphosis like in osteoporotic kyphosis. Absence of clinically detectable flexor-dystonia is required. All patients were stabilized on optimal anti-Parkinson medication. Informed consent to take part in the study was signed by all patients. The study was approved by the ethical committee of the Medical Faculty of the University Hospital Schleswig-Holstein (D 469/11).
The clinical details of our camptocormia patients were reported by Margraf et al
. [
27]. Briefly, the Parkinson patients showed mean disease duration of 13.5 years. Mean duration of camptocormia was 25 months (5–72 months). A muscle biopsy was offered to all Parkinson patients who had consulted the UK-SH Department of Neurology because of their camptocormia. The muscle biopsies were performed to determine whether the camptocormia could be related to a treatable cause. The most affected part, according to MR imaging of the paraspinal muscles, was selected for the biopsy. All biopsies that were taken in the patient group defined above were used for the present histopathological study to avoid a possible selection bias. Details of the patients are given in Table
1.
Table 1
Details of patients and biopsy findings of Parkinson disease patients
B1 | 1 | 55 | F | 4 | 8 | 800/– | 30 | Lumbar | + | + | + | + | + | + | + | + | 10:1 |
B2 | 2 | 59 | F | 10 | 28 | 700/– | 90 | Th12 | + | + | n.e. | + | 0 | + | + | + | 10:1 |
B3 | 13 | 72 | M | 17 | 30 | 600/– | 80 | Lumbar | + | + | + | + | + | + | + | + | 3:1 |
B4 | 5 | 64 | M | 7 | 32 | 1,400/– | 60 | Thoraco-lumbar | + | + | Ø | + | 0 | + | + | + | 1:1.5 |
B5 | 8 | 68 | F | 22 | 5 | –/DBS | 70 | Th12–L1 | + | + | + | + | 0 | + | + | + | 10:1 |
B6 | 11 | 69 | M | 15 | 12 | 300/– | 60 | L3 | + | + | + | + | + | + | + | + | 20:1 |
B8 | 4 | 62 | M | 10 | 5 | 800/– | 30 | L1 | + | + | + | + | + | + | + | + | 20:1 |
B9 | 10 | 68 | F | 41 | 54 | 500/DBS | 90 | Not specified | + | + | + | + | + | + | + | + | 40:1 |
B10 | 6 | 66 | F | 11 | 36 | 800/– | 45 | Th12–L1 | n.e. | n.e. | + | + | + | + | + | 0 | n.e. |
B11 | 9 | 68 | M | 9 | 72 | 1,350/– | 70 | Cervical | + | + | + | + | + | + | + | 0 | 1:1 |
B12 | 7 | 67 | M | 10 | 17 | 850/– | 45 | Th10–12 | + | + | + | + | 0 | + | + | + | 1:1 |
B13 | 3 | 61 | F | 13 | 17 | 1,200/– | 90 | Th12–L1 | + | + | Ø | + | + | + | + | + | 5:1 |
B14 | – | 70 | M | 6 | 9 | 750/– | 30–40 | Not specified | + | + | + | + | + | + | + | + | 40:1 |
The severity of the clinical symptoms was scored as described by Margraf et al. [
27]. This score encompasses the inclination angle, the circumstances of occurrence, the time of day when the symptoms occur, the degree of back pain and the limitation in daily activities caused by the camptocormia.
Histological investigations of muscle specimens
Standard muscle evaluation was performed using stainings of cryostat sections. These stainings included hematoxylin and eosin (H&E), elastica van-Giesson, periodic acid Schiff reaction (PAS), oil red O (OrO), modified Gomori trichrome, nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR), menadione-linked α-glycerophosphate dehydrogenase (MAG), myoadenylate deaminase (MAD), succinic dehydrogenase (SDH), cytochrome oxidase (COX), combination of COX and SDH, acid phosphatase, non-specific esterase, adenosine triphosphatase (ATPase) at pH values of 4.4, 4.7 and 10.4, which were prepared according to standard procedures [
12].
As indicated in the figures, quantification per power field or arbitrary units were used. For counts per field the mean of 5–15 fields was calculated, depending on the size of the biopsy. Nuclear bags and fiber splittings were counted in medium power fields corresponding to a 200-fold magnification. Ragged red fibers, COX-deficient fibers and whorled fibers were counted in low power fields corresponding to a 100-fold magnification. The fiber type distribution was calculated by the relation of type-1 to type-2 fibers. Some changes were calculated in percent of all fibers. These were the number of fibers showing internal nuclei, the number of fibers showing acid phosphatase activity and the number of fibers showing defects in the NADH reaction. If quantification by counting was not applicable, a five-step rating system corresponding to absent, mild, moderate, severe and extensive, was used. The criteria for the semiquantitative rating were—for caliber variation of myofibers: 0 = uniformity, 1 = mild variation, 2 = moderate variation, 3 = variation showing atrophy or hypertrophic fibers, 4 = variation showing atrophy and hypertrophic fibers; for endomysial fibrosis: 0 = absent, 1 = fibrotic sprout into endomysium, 2 = continuous fine endomysial fibrosis, 3 = continuous moderate endomysial fibrosis, 4 = nearly all fibers were trapped by endomysial collagen, for neurogenic atrophy: 0 = no, 1 = single atrophic fibers of both types, 2 = reticular distribution of some atrophic fibers, 3 = several atrophic fibers or small groups, 4 = large groups of atrophic fibers of both types; for fiber type-2 atrophy: 0 = no, 1 = occasional, 2 = scattered but some, 3 = several, 4 = nearly all type-2 fibers.
To compare the myopathic changes with clinical symptoms, we calculated a score that includes the main aspects of the myopathic changes. These are structural defects, acid phosphatase activity in muscle fibers, endomysial fibrosis, caliber variation, whorled fibers, internal nuclei and fiber splitting. The severity of changes for all included parameters was standardized to a scaling system ranging from 0 to 10. To do so, the semiquantitative values were multiplied by 2.5, the percent values divided by 10. All mean values of counts per power fields were within the range of ten and were taken as they were.
Immunohistochemical stainings
All immunohistochemical staining results reported here were obtained from frozen sections that were fixed for 15 min in ice-cold acetone. With the antibody reactions that were performed without epitope retrieval of the slides we examined inflammatory reactions, lysosomal structures, fiber typing, small vessels, mitochondria and protein deposition. The following antibodies were used: CD3 (rat monoclonal, AbD Serotec), CD8 (mouse monoclonal, Dako), CD20 (mouse monoclonal, clone L26, Dako), KiM1P (mouse monoclonal, gift from the Pathology Department of UK-SH), MHC I (mouse monoclonal, clone W6/32, Dako), cathepsin D (rabbit polyclonal, Dianova), LAMP-1 (mouse monoclonal, clone E5, Santa Cruz), SERCA 1 (mouse monoclonal, clone VE121G9, Thermo Scientific), α-5-laminin (mouse monoclonal, clone 4C7, Chemicon), prohibitin (mouse monoclonal, Abcam), sarcomeric α-actinin (mouse monoclonal, clone EA-53, Sigma), α-B-crystallin (rabbit polyclonal, Oncogene), smooth muscle actin (mouse monoclonal, clone 1A4, Beckman Coulter), dystrophin I, rod region, clone Dy4/6D3, dystrophin II, C-terminal, clone Dy8/6C65, dystrophin III, N-terminal, clone Dy10/12B2 (all three were mouse monoclonals from Novocastra), desmin (mouse monoclonal, clone D33, Dako), filamin (mouse monoclonal, clone FLMN 01, Dianova), myotilin (mouse monoclonal, Novocastra), p62 (mouse monoclonal, BD Bioscience), titin (mouse monoclonal, clone T11, Sigma), triadin (mouse monoclonal, clone GE4.90, Acris-Antibodies) and ZASP (rabbit polyclonal, clone LDB-3, Sigma). For the detection of α-synuclein deposits, antibodies LB509 (mouse monoclonal, Signet) and pSer129-α-Syn (rabbit polyclonal, Abcam) were used. As secondary antibodies, the alkaline phosphatase-coupled goat anti-mouse or goat anti-rabbit from Dako were used and vizualized by neufuchsine. Alternatively, the biotinylated sheep anti-mouse or donkey anti-rabbit from GE Healthcare or rabbit anti-rat from Dako were used, detected by horse radish peroxidise coupled extravidin (Sigma) and vizualized by either DAB or AEC as chromogen.
PET blot
To avoid any influence of a neurodegenerative disease on changes in muscles of our control group, we used antibodies against β-amyloid (clone 6E10, mouse monoclonal, Chemicon) or phosphorylated tau (AT8, mouse monoclonal, Covance) to exclude individuals who showed intracerebral protein aggregate deposits according to CERAD C or Braak and Braak IV to VI and antibodies LB509 (Signet) and pSer129-α-Syn (Abcam) to exclude individuals in whom α-synuclein aggregates were detectable in the brain tissue. For the detection of these protein aggregates, the most sensitive PET blot method was used according to the protocol by Kramer and Schulz-Schaeffer [
23]. For the sensitive detection of α-synuclein aggregates, the autopsy and biopsy muscles were investigated using the regular PET blot protocol [
23] and a postfixed frozen tissue blotting protocol as described by Daus et al. [
7].
Electron microscopic investigation
For electron microscopy, glutaraldehyde-fixed specimens were postfixed with 1% osmium tetroxide (Serva, Heidelberg, Germany) and embedded in araldite (Serva). Ultrathin sections were contrasted with lead citrate (Serva) and uranyl acetate (Serva). Investigations were performed on a Zeiss EM10 transmission electron microscope (Zeiss, Oberkochen, Germany) equipped with a MegaView III imaging system (Olympus Soft Imaging Systems GmbH, Münster, Germany).