Keywords anatomy - muscle - necropsy - pathology
Introduction
The diaphragm (DIA) is the primary muscle of inspiration. Therefore, its uncompromised
function is essential to support the ventilatory and gas exchange demands.[1 ] Many patients with chronic obstructive pulmonary disease (COPD) or emphysema show
high levels of DIA activity both at rest and during exercise, in which the DIA may
experience maximal activation leading to irreversible muscle injury, mechanical failure,
and death.[1 ]
[2 ]
[3 ]
The analysis of frozen muscle biopsies has become a routine method in the evaluation
of muscle structure and function in health and disease.[4 ]
[5 ] However, the analysis of frozen muscle specimens is not widely available in countries
with limited medical facilities.[6 ]
Usually, postmortem studies use formalin-fixed and paraffin-embedded (FFPE) methods
in the health sciences.[7 ]
[8 ] Studies of postmortem bone,[9 ] ligament,[10 ]
[11 ] skin,[12 ] articular cartilage,[13 ] and spinal segments [14 ] show relatively small variations from their live mechanical properties. Although
many changes in skeletal muscle properties have been hypothesized in postmortem tissues,
there is still limited quantitative and qualitative data available.
As the successful evaluation of postmortem muscle integrity in FFPE muscle sections
has rarely been described, the present study aimed to elucidate a reproducible FFPE
method for this type of analysis in postmortem DIA muscle.
Materials and Methods
Autopsy verification is mandatory in Brazil to define the cause of death for most
individuals who die of natural causes. The São Paulo Autopsy Service (Serviço de Verificação
de Óbito [SVO], in the Portuguese acronym) is the major morgue serving the metropolitan
area of São Paulo, Brazil.[8 ] The present study was approved by the ethical committee of the Faculdade de Medicina
of Universidade de São Paulo under the registration number 2.209.383. The division
of pathology from the Instituto do Coração (INCOR) provided the material for the present
study exclusively for the sake of methodological standard in future projects.
Muscle Samples
Specimens from the right lobule[15 ] of the DIA muscle was obtained within 1 hour of sudden death[5 ]
[16 ] from a previously physically healthy 51-year-old man who died of myocardial infarction.
Diaphragm strips (∼ 5 × 5 × 5 mm) were removed at 4 to 6 cm from the central tendon
(midcostal) to avoid the muscle fibers that radiate toward this tendon insert.[1 ]
[15 ] The DIA strips were gently washed in saline solution, placed in a standard histological
cassette (SWINGSETTE™ Tissue Processing/Embedding Cassettes, Histocell Soluções em Anatomia Patológica
Ltda., São Paulo, SP, Brazil) to re-establish the initial length, and immediately
fixed in 10% formalin, frozen at 4°C in a refrigerator, and stored for 24 hours.[5 ]
[16 ] Then, the tissue samples were processed into paraffin-embedded blocks.[6 ]
Histological Staining
Transversal sections of 6 μm thick [15 ] were cut from each paraffin block for histological staining. The sections were stained
with the hematoxylin and eosin (H&E) standard method for the general structure of
the sample.[4 ] The sections were also stained with standard methods [4 ]
[17 ] for: (a) Picrosirius red (collagen fibers); (b) Verhoeff-Van Gieson (elastic fibers);
and (c) Congo red (amyloid deposits).
The qualitative evaluation of the tissue was performed by photographing 40 fields
selected at random for each staining technique. The images were captured using a Zeiss
Microscope (Binocular microscope Axio Lab.A1 with phototube, Carl Zeiss, Thornwood,
NY, USA) with the specific software AxioVision, Version 4.8 (Zeiss, Thornwood, NY,
USA).
Results
The H&E stain positivity indicated a well-preserved muscle ([Fig. 1A ]). The Picrosirius red stain gave superior results under a polarized filter ([Fig. 1B ]), in which different collagen fiber types were observed in the endomysium, in the
perimysium, and in the epimysium. Additionally, we could observe a weak positive stain
for elastic fibers ([Fig. 2A ]), and amyloid ([Fig. 2B ]).
Fig. 1 Cross-sectional images of the diaphragm (DIA) muscle stained with hematoxylin and
eosin (A) or picrosirius red (B) (100x magnification). A) Red arrow: muscle fiber;
yellow arrow: inflammatory process. (B) Picrosirius red stain in polarized microscopy
showing type I collagen fibers (red); intermediate fibers (yellow/orange); and type
III collagen fibers (green).
Fig. 2 Cross-sectional images of the diaphragm (DIA) muscle stained with the Verhoeff-Van
Gieson (A) or Congo red (B) (400x magnification). A) Red arrows: elastic fibers. B)
Congo red stain in polarized microscopy showing amyloid (yellow arrow).
Discussion
With death, a series of postmortem events initiate, including the loss of enzyme activities.[5 ] The reliability of postmortem muscle samples depends on the extent of these cellular
alterations.
Eriksson et al[5 ] demonstrated that muscle samples stored for a maximum of 10 days in controlled temperature
(4°C) are reliable as nitrogen stored samples. Van Ee et al[16 ] had similar results, indicating that lower temperatures may maintain the integrity
of the muscle fibers. We have frozen the samples at 4°C, which maintained its integrity.
Nevertheless, it is reported that muscles stiffen as they enter rigor mortis.[18 ] An experimental study by Fitzgerald[19 ] reported that the elastic compliance decreased 95%, and that the viscous compliance
decreased 98.3% in relation to the living muscle tissue values at ∼ 6.5 hours after
death. Van Ee et al[16 ] reported that a period of 0.5 hours postmortem (prerigor) had only a modest effect
on the mechanical properties of the muscle, while at 48 hours postmortem (postrigor),
the response changed greatly. We have chosen to freeze the samples 1 hour postmortem
to maintain the prerigor integrity.
A pilot study of an autolytic change in rat diaphragms demonstrated that, up to 96
hours postmortem, the only postmortem morphological artifact was a decrease in the
intensity of the H&E staining.[20 ] This artifact was not observed in the present study. The Picrosirius red stain was
successful because it specifically labels collagen molecules without relying on the
recognition of antigens, which may be degraded postmortem or during fixation.[15 ]
[17 ] Although we have observed a decrease in the intensity of the Verhoeff-Van Gieson
staining, Rodrigues et al[21 ] observed that the viscoelastic properties of the DIA decreases with aging, which
may explain our finding. Finally, the Congo red staining was efficient to demonstrate
amyloid deposits, as has been shown by previous studies with FFPE in the nervous system.[22 ]
[23 ]
Recently, Suriyonplengsaeng et al[6 ] successfully developed an immunohistochemistry (IHC) technique, with heat-mediated
antigen retrieval, for FFPE muscle biopsy specimens. This study may encourage more
researches to use IHC on FFPE muscles in autopsy studies as well.
The present study has some weaknesses that need to be acknowledged for proper interpretation.
The main limitation of the present study is the small sample size, which was limited
to one patient. However, the small sample size would not explain our positive findings.
Due to the limited literature, we could not carry out an extensive discussion about
our findings. Nevertheless, we have highlighted in the present study a simple and
valid methodological approach for the analysis of postmortem muscle that could be
improved and discussed in depth in further studies.
Conclusion
In summary, we demonstrate a simple and reproducible technique for FFPE in postmortem
tissue. We suggest that this method could become a valuable tool for the diagnosis
of anatomopathological changes in the DIA muscle.
