Progressive changes in T₁, T₂ and left‐ventricular histo‐architecture in the fixed and embedded rat heart

Abstract

Chemical tissue fixation, followed by embedding in either agarose or Fomblin, is common practice in time-intensive MRI studies of ex vivo biological samples, and is required to prevent tissue autolysis and sample motion. However, the combined effect of fixation and sample embedding may alter tissue structure and MRI properties. We investigated the progressive changes in T 1 and T 2 relaxation times, and the arrangement of locally prevailing cardiomyocyte orientation determined using diffusion tensor imaging, in embedded ex vivo rat hearts fixed using Karnovsky's solution (glutaraldehyde-formaldehyde mix). Three embedding media were investigated: (i) standard agarose (n = 3 hearts); (ii) Fomblin (n = 4 hearts); and (iii) iso-osmotic agarose (n = 3 hearts); in the latter, the osmolarity of the fixative and embedding medium was adjusted to 300 mOsm to match more closely that of native tissue. The T 1 relaxation time in the myocardium showed a pronounced decrease over a 48-h period following embedding in Fomblin (−11.3 ± 6.2%; mean ± standard deviation), but was stable in standard agarose-and iso-osmotic agarose-embedded hearts. The mean myocardial T 2 relaxation time increased in all embedded hearts: by 35.1 ± 14.7% with standard agarose embedding, 13.1 ± 5.6% with Fomblin and 13.3 ± 1.4% with iso-osmotic agarose. Deviation in the orientation of the primary eigenvector of the diffusion tensor occurred in all hearts (mean angular changes of 6.6°, 3.2° and 1.9° per voxel after 48 h in agarose-, Fomblin-and iso-osmotic agarose-embedded hearts, respectively), indicative of progressive structural changes in myocardial histo-architecture, in spite of previous exposure to fast-acting tissue fixation. Our results suggest that progressive structural changes occur in chemically fixed myocardium, and that the extent of these changes is modulated by the embedding medium, and by osmotic gradients between the fixative in the tissue and the surrounding medium.

Histo-anatomical structure of the living isolated rat heart in two contraction states assessed by diffusion tensor MRI

“…However, as changes in absolute sheet angles were compared (essentially treating sheets as one population), sheet intersection-angles were not analysed. Also, tissue in contracture was chemically fixed prior to imaging, which alters histo-anatomical structure of rat ventricular tissue (Hales et al., 2011). To the best of our knowledge, this is the first study performing DTI on the same live-perfused heart in two different mechanical states.…”

“…Due to its non-destructive nature, DTI may be used to measure changes in fibre- and sheet-orientation in the same heart, at different stages of the cardiac cycle. This would avoid the impact of histological tissue processing on parameters assessed in-vitro (Hales et al., 2011). However, cardiac and respiratory motion, together with limited spatial resolution, low signal-to-noise ratio, and long scan-times, present major technical hurdles for in-vivo application of cardiac DTI.…”

DT-MRI measurement of myolaminar structure: Accuracy and sensitivity to time post-fixation, b-value and number of directions

“…This would seem to contradict the findings of [15] who reported progressive changes following transferring fixed tissue to an embedding medium, and hypothesized that this was due to osmotic gradients. We did not transfer the heart from fixation medium to an embedding medium and therefore such osmotic gradients would be minimized.…”

“…The aim of this study is to examine the sensitivity of the monoexponential diffusion model to imaging parameters by assessing accuracy against high-spatial resolution MRI (HR-MRI) images of the same heart. It has recently been shown that DT-MRI determined fiber orientation changes with time post embedding in ex vivo fixed non-perfused hearts, although the global architectural organization does not change [15]. We have therefore explored the sensitivity of orientation measurements to time post-fixation for both DT-MRI and HR-MRI.…”

A computational pipeline for quantification of mouse myocardial stiffness parameters

“…A 3D fast spin-echo sequence was used as reported previously [18] with the following imaging parameters: TR = 1 s, number of echoes = 8, TE eff = 44.25 ms, matrix size = 256×256×192, number of averages = 4, field of view 13×13×13 mm 3 (yielding a resolution of 51×51×68μm 3 ), maximum b-value=688 s/mm 2 (including imaging gradients and cross-terms between imaging and diffusion gradients). Eight non-collinear gradients directions were used, arranged according to an optimized scheme based on the electrostatic repulsion principle [19].…”