Capillary Network Morphometry of Pig Soleus Muscle Significantly Changes in 24 Hours After Death

Abstract

Capillary network characteristics are invaluable for diagnostics of muscle diseases. Biopsy material is limited in size and mostly not accessible for intensive research. Therefore, especially in human tissue, studies are performed on autopsy material. To approach the problem whether it is reliable to deduce hypotheses from autopsy material to explain physiological and pathological processes, we studied capillarity in pig soleus muscle 1 and 24 hr after death. Capillaries and muscle fibers were immunofluorescently marked, and images were acquired with a confocal microscope. Characteristics of the capillary network were estimated by image analysis methods using several plugins of the Ellipse program. Twenty-four hours after death, the measured characteristics of the capillary network differ by up to 50% when compared with samples excised 1 hr after death. Muscle fiber diameter, the measured capillary length, and tortuosity were reduced, and capillary network became more anisotropic. The main postmortem change that affects capillaries is evidently geometric deformation of muscle tissue. In conclusion, when comparing results from biopsy samples with those from autopsy samples, the effect of postmortem changes on the measured parameters must be carefully considered.

Horizontal deformation of skeletal muscle thick sections visualised by confocal microscopy

“…To minimise the tissue deformation effects when studying skeletal muscles in thick sections, the procedures that minimise factors contributing to tissue section deformation should be employed. The tissue samples should be harvested as soon as possible after animal sacrifice to reduce the postmortem changes of muscle tissue 8 . As quick temperature changes of frozen samples can exaggerate shrinkage, the temperature of medium where sections are put after cutting and fixative should be kept cold on ice 9,10 .…”

“…In skeletal muscle research, 3D analysis is especially important for studying changes in microvasculature, since the capillary network is a complex interwoven 3D structure which cannot be reliably estimated by stereological methods in 2D 4–6 . One of the well‐established methods for visualising tissue structure and cellular or subcellular particles in 3D is fluorescence confocal and multiphoton microscopy of thick tissue sections 4,7,8 . However, thick tissue sections are prone to artefacts, especially section deformations that may significantly influence some stereological and morphometric results like muscle fibre diameter and capillary length, but not dimensionless parameters like object number and Euler‐Poincaré characteristics 1 …”

Skeletal Muscle Microvascular Dysfunction in Obesity-Related Insulin Resistance: Pathophysiological Mechanisms and Therapeutic Perspectives

“…Additionally, application of various stimuli including local ischaemia, temperature changes, and vasoactive agents such as acetylcholine, adenosine, serotonin, bradykinin, and sodium nitroprusside, can be used to study microvascular response. Histological assessment of skeletal muscle microvasculature is conventionally accomplished by two-dimensional (2D) analyses of tissue cross-sections [52,53], although recently, a three-dimensional (3D) analytic technique that overcomes the usual technical biases and inconsistencies associated with the traditional 2D approach has been proposed [54][55][56].…”