The structure and mechanical properties of collecting lymphatic vessels: an investigation using multimodal nonlinear microscopy

Abstract

This study employed nonlinear microscopy on fresh, unstained and unfixed collecting lymphatic vessels to determine the wall structure and its relationships to the mechanical properties of the tissue. Fresh bovine mesenteric collecting lymphatic vessels were mounted in a vessel bath and imaged under different luminal pressures (0-30 cmH(2)O pressure head), and longitudinal tensions. The entire wall thickness was imaged, using two-photon fluorescence to visualize elastin, second harmonic generation to image the collagen, and coherent anti-Stokes Raman scattering to image the cell membrane. The adventitial fat cells were coupled to the wall within the elastin-rich network of fibres. The medial smooth muscle cells were too densely packed to resolve the boundaries of individual cells in en face images, but in tissue sections their appearance was consistent with electron microscopic data. Two distinct populations of collagen fibre were revealed. Large fibre (15-25 microm diameter) bundles were present in the inner media and small fibres (2-5 microm diameter) were distributed throughout the wall. The responses to longitudinal tension and luminal pressure indicated that the larger fibres resist the longitudinal strain and the smaller oppose pressure forces. Individual elastin fibres were of uniform thickness (1-3 microm) and interwove amongst themselves and between the collagen fibres. The network was probably too sparse directly to support mechanical loads and we speculate that its main function is to maintain the organization of collagen bundles during recovery from contraction.

Fig. 1

Apparatus. Top: Schematic diagram of the nonlinear microscope. Below: Lymphatic vessel perfusion rig.

Fig. 3

Three-dimensional reconstructions of TPF image of elastin fibres in relaxed vessel. The collagen autofluorescence has been enhanced to demonstrate the relationship of elastin to the large spiral collagen fibre bundles. (A–C) Relaxed vessel, zero pressure. (D–F) In situ strain, 15 cmH₂O luminal pressure. 25-μm-grid squares.

Fig. 2

Second harmonic generation images of collagen in intact bovine collecting vessel wall at different luminal pressures and longitudinal tensions: tangential images with the long axis of the vessel running from left to right, field of view 240 × 240 μm. The top images show fine collagen fibres in the outer wall and the lower panel shows the coarser fibres in the inner wall of the same vessel. (A,E) Relaxed length, zero pressure. (B,F) In situ length, zero pressure. (C,G) In situ length 15 cmH₂O luminal pressure. (D) In situ length, 30 cmH₂O luminal pressure.

Fig. 5

Principal directions of collagen (□) and elastin (○) fibres as a function of luminal pressure at different depths through the vessel wall. 180° represents the direction of the longitudinal axis of the vessel.

Fig. 4

TPF images of an intact vessel wall at different luminal pressures and longitudinal tensions: Image orientation, conditions and magnification as in Fig. 2A–D. No background subtraction has been performed, enabling thin, intensely fluorescent elastin fibres to be visualized against a background of broad collagen fibre bundles.

Fig. 6

Cells of the vessel wall (CARS, red) with superimposed images of elastin TPF (green) and collagen (SHG, blue). (A) Adventitial fat cells embedded in the outer layers of elastin and collagen. The vessel axis runs from right to left (vessel fixed to better preserve the integrity of the fatty tissue). (B) Luminal surface, showing endothelial layer. Fresh tissue, orientation as in A. (C) Transverse section of the wall, the lumen is on the right.