Role of cholesterol crystals in atherosclerosis is unmasked by altering tissue preparation methods

Abstract

Standard tissue preparation for light and scanning electron microscopy (SEM) uses ethanol as a dehydrating agent but that can also dissolve cholesterol crystals (CC) leaving behind empty tissue imprints or "clefts". Cholesterol crystals may contribute to plaque rupture by their sharp tips that can tear membranes and trigger inflammation. Therefore, use of ethanol in tissue processing can mask the pathological role of CC. Here we evaluated the amount of cholesterol dissolved from CC with single and complete series of standard graded ethanol concentrations (25-100%) used in tissue preparation. Also, solubility of CC in ethanol at physiological levels was measured. Furthermore, we compared the effect of ethanol on CC in fresh human atherosclerotic plaques to matched segments dehydrated using vacuum (-1 atm, 12h). Tissue crystal density ranging from 0 to +3 was measured semi-quantitatively by SEM. For CC exposed to 25% and 100% ethanol for 10 min each, 0.38% and 95% of CC were dissolved respectively. Also, increase in CC solubility was significant at physiological levels of ethanol (0.16%) compared to water (43.4 ± 18.0 ng/mL vs. 30.9 ± 13.9 ng/mL; p < 0.05). We speculate that this could represent a potential mechanism of cardio-protective effects of alcohol consumption. In atherosclerotic plaques, CC density was lower in ethanol vs. saline treatment (+1.2 vs. +2.8; P < 0.01) with visible dissolving noted by SEM. Ethanol has been used for centuries in tissue preparation for microscopy. Here we demonstrate how current tissue preparation methods greatly alter histological findings with SEM by masking the potential mechanism of plaque rupture.

Cholesterol Crystals and NLRP3 Mediated Inflammation in the Uterine Wall Decidua in Normal and Preeclamptic Pregnancies

“…It must also be determined how different pathological processes in decidua interact since oxidative stress may promote the accumulation of oxLDL and cholesterol crystal formation (16,17). The use of established advanced microscopy methodology for cholesterol imaging learned from atherosclerosis and hepatocytes lipid droplets (47)(48)(49), combined with removal of the crystals by alcohol treatment (50), strongly support that the imaged crystals in decidua are crystalline cholesterol. Still, further verification of the chemical identity of the crystals may be performed by other advanced microscopy techniques, such as coherent anti-Stokes Raman scattering (CARS) imaging.…”

Cholesterol induced heart valve inflammation and injury: efficacy of cholesterol lowering treatment

“…This approach was used because standard methods using ethanol dissolve cholesterol crystals. 16 These tissue segments were then mounted on stubs and gold coated in a sputter coater (EMSCOPE SC500; Emscope, Ashford, UK). The valve surfaces were then examined using a Jeol SEM (model JSM-6300F, Jeol Ltd, Tokyo, Japan).…”

Cholesterol crystal embolization following plaque rupture: a systemic disease with unusual features

“…The incidence of CCE may be expected to increase with a rise in the aging population and the number of vascular procedures performed. The current estimates of incidence are probably greatly underestimated because tissue preparation methods using ethanol dehydration dissolves CCs [ 15 ] . This could greatly skew the data in favor of a lower incidence of CCE when using standard histological methods.…”

“…The external diameter of the arteries plugged with cholesterol emboli vary from 150 µm to 1100 µm in diameter [ 13 ] . Since CCs are readily dissolved when using standard methods with ethanol dehydration for preparing histologic sections, the amount of CCs has been greatly underestimated by light microscopy [ 15, 25, 27 ] . However, by using vacuum dehydration in tissue preparation for scanning electron microscopy or just fresh tissue with fluorescence microscopy, Abela et al were able to demonstrate the vast extent and effect on tissue injury by CCs [ 15, 25­ 27, 33 ] .…”

“…However, the great amount of CCs present in coronary lumen following plaque rupture has been greatly underestimated due to the standard tissue processing methods with ethanol that dissolves CCs ( Fig. 5 ) [ 15, 37 ] . …”