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Global Translational Medicine Elastolysis of porcine aortic adventitia

and the aortas would have been discarded if not acquired cell, was used to uniaxially measure the tensile properties
for this study. of isolated adventitial specimens (n = 30 for each of the
Fifty aortic segments selected from the abdominal five sets of samples). The thickness and width of strips
region were used in this study. They were stored in a (1 × 3 cm) were checked with a digital caliper (Digimax
freezer (−80 C) and thawed when needed. The adventitial Global Inc., Toronto, Canada). Thirty measurements
o
specimens were prepared as previously reported . In for each set of samples, that is, untreated, digested (1 h,
[21]
brief, after removing the extraneous fat and connective 10 U/mL), digested (1 h, 10 U/mL)+irradiated, digested
tissue from the external surface of the vessel, dissection (48 h, 20 U/mL), digested (48 h, 20 U/mL)+irradiated,
was initiated at a corner with a surgical scalpel, and the were carried out along the longitudinal direction at a set
adventitia was then carefully peeled off by hand. gauge distance of 16 mm, and a speed of 10 mm/min.
The values of elastic modulus (Young’s modulus) were
2.2. In vitro elastolysis calculated from the linear region of the recorded stress-
Five sets of 10 adventitial specimens (5 × 3 cm each) were strain plots. The mechanical testing was carried out in its
soaked in phosphate-buffered saline (PBS) for 1 h, and entirety by the same researcher.
then each was placed in a Petri dish (6 cm in diameter). 2.5. Histology
For elastolysis, an elastase solution containing 10 U/mL
was prepared by diluting the original enzyme solution Aortic adventitial tissue specimens, native or treated, were
with an aqueous solution made up of Tris buffer with fixed in 10% neutral buffered formaldehyde solution for
pH 7.8 (100 mM), calcium chloride (1 mM) and sodium 24 h, washed 3 times, and stored in PBS. The specimens
azide (0.02% w/v). Portions (6 mL) of this elastase solution were then placed in plastic tissue processing cassettes


were added to the dishes to cover the samples in two of the and processed for 9 h in the Tissue-Tek VIP 6 Tissue
sets. The dishes were then placed in an orbital shaker and Processor (Sakura Finetek, Torrance, California, USA).
shaken at 160 rpm for 1 h at 37°C. Two sets of 10 specimens The processed samples were embedded in paraffin with the
each were processed. One set was subjected to elastolysis region of interest facing down. Sections (4-μm thick) were
only; the second set was subjected to elastolysis and then cut with a Leica RM2245 microtome (Leica Biosystems,
irradiated. For comparison, a third set of 10 specimens Nussloch, Germany) and collected onto labeled SuperFrost
was subjected to the same protocol without elastolysis Plus slides (Menzel-Gläser, Braunschweig, Germany).
and irradiation. In a separate experiment, the fourth set of The slides were manually stained using a Verhoeff-Van
specimens was subjected to elastolysis in 20 U/mL elastase Gieson stain kit (Australian Biostain, Traralgon, Australia)
for 48 h, and the fifth set to the same protocol followed by following the supplier’s VVG/EVG Protocol and then
irradiation. Following elastolysis, the samples were rinsed coverslipped with the Tissue-Tek automated coverslipper.
3 times in cold water. Before further testing, all specimens The slides were then scanned with a ×40 objective using
were kept in PBS at room temperature. an Olympus VS120 Slide Scanner (Olympus, Sydney,
Australia), and images were acquired using the Olympus
2.3. Irradiation procedure VS-ASW software (v2.9). Data were then visualized using
The rectangular strips of adventitia scheduled for the Olympus OlyVIA™ software (v3.1).
irradiation (5 × 3 cm) were soaked in the RF solution for 2.6. Statistical analysis
30 min at room temperature. Each specimen was then
exposed to UV-A radiation (at a wavelength of 365 nm) The plotted data point for n = 30 were displayed as mean
generated by a UV Curing System OmniCure 1500 values ± standard error of the mean. For the statistical
(Excelitas Technologies Corp., Waltham, Massachusetts, comparison of the values in three groups, the GraphPad®
USA). The localized irradiance on the target was monitored Prism software (Version 6.0) was used to perform the one-
with a radiometer Dymax ACCU-CAL 50 (Dymax Corp., way analysis of variance (ANOVA) in conjunction with
Torrington, Connecticut, USA). The required value for Tukey-Kramer multiple comparisons. For the comparison
the irradiance was obtained by adjusting the distance of two groups of values, a Student’s t-test was used.
between the radiation source and the target. Each side of 3. Results and discussion
the specimen was exposed to an irradiance of 45 mW/cm
2
for 10 min. In the quest for a better understanding of the pathogenesis
of aortic aneurysms, experimental selective enzymolysis
2.4. Mechanical testing of the two major components (elastin and collagen) of the
An Instron Materials Testing System Model #5943 (Instron, vascular wall remained one of the preferred methodologies
Norwood, Massachusetts, USA), equipped with a 50-N load for creating aneurysmal animal models [23-25] and— to a

Volume 2 Issue 2 (2023) 3 https://doi.org/10.36922/gtm.0897