| Summary: | Background: Arterial stiffening is an important predictor of cardiovascular risk. In order to identify the pathogenic mechanisms it is necessary to characterise arterial micro-structure and –mechanical properties. Here, we test the hypothesis that chemical fixation (employed in microscopical preparation regimens) may adversely influence arterial structure and stiffness.
Methods: Common carotid arteries (CCA) from young male Wistar rats (225–250g, n=4) were excised. Gross stiffness was determined by wire myography for arterial segments untreated (CON), pre-treated by paraformaldehyde fixation (4%)(FIX) or snap frozen (FRZ). Cryo-sections 5μm thick were prepared from bisected fixed (FIX) or untreated (CON), frozen CCA. Acoustic wave speed (related to tissue elastic modulus) was then characterised by scanning acoustic microscopy (SAM) whilst vessel morphology was quantified from H&E stained sections.
Results: Fixation increased both medial layer thickness (CON 38±3, FIX 55±10 μm, P<0.05) and lamellar spacing (CON 11±4, FIX 15±6 μm, P<0.05) but had no effect on lumen diameter (CON 331±2, FIX 314±29 μm, P>0.05). Fixation significantly increased incremental elastic modulus whilst freezing alone had no effect (CON 0.86±0.15, FIX 1.39±0.10, FRZ 0.99±0.04μm, P<0.05). SAM demonstrated increased stiffness with fixation (CON 1697±21, FIX 1776±32 ms−1, P<0.05) which was pronounced within the inter-lamellar regions (inter-lamellar CON 1629±9, FIX 1678±10 ms−1 P<0.05).
Conclusions: The chemical fixation steps commonly used in microscopical preparation regimens can induce localised changes in the structure and stiffness of arterial compartments. We suggest therefore that cryo-preservation, which preserves the gross-mechanical behaviour of the intact artery, may also maintain the micro-structural and micro-mechanical characteristics of the vessel.
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