Investigation into the Effects of Fixation on the Mechanical Properties of Acute Ischemic Stroke Embolus Analogs

Open Access
- Author:
- Hiller, Gretchen
- Graduate Program:
- Biomedical Engineering
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- October 29, 2021
- Committee Members:
- Keefe B Manning, Thesis Advisor/Co-Advisor
Francesco Costanzo, Committee Member
Daniel Hayes, Program Head/Chair
Peter Butler, Committee Member - Keywords:
- acute ischemic stroke
stroke
clot
thrombosis
embolization
blood analog
mechanical
viscoelastic
elastic modulus
histology - Abstract:
- Acute Ischemic Stroke (AIS) is the result of embolic occlusion of a cerebral artery. Currently, there is little understanding why some thromboemboli are successfully removed and others are not. The goal of this study was to characterize mechanical properties, and histological compositions of emboli to better understand why some might be more difficult to remove. This goal was accomplished by developing bovine embolus analogs (EAs) to mimic the properties of human thromboemboli in AIS patients. Since the human clots collected are stored and transported in paraformaldehyde (PFA) phosphate-buffered saline (PBS), this study seeks to characterize the mechanical properties of bovine EAs stored in PFA, and PBS solutions and compare them to control EAs. In addition, this study seeks to determine the histological compositions of bovine EAs. Platelet rich plasma (PRP) and red blood cells (RBCs) blood components were combined in different ratios to form four samples of reconstituted blood with varying hematocrits. Reconstituted samples were sectioned into 24 well plates and stored at body temperature, half of the EAs formed under static conditions and half under dynamic conditions, which entailed the 24 well plates to be placed on a rocker. To determine the mechanical properties both stress-relaxation, and cyclic compression tests were performed. Incremental loads were applied and measured using an Instron. Some samples were than fixed in 4% PFA solution while other samples were stored in 1% PBS solution. Following both fixation in PFA and storage in PFA the samples were again put through the same mechanical tests. Significance was determined by performing a two-tailed t-test with a 95% confidence interval and an alpha value of 0.05. The results showed differences in mechanical properties between unfixed EAs and EAs fixed in 4% PFA. Specifically, the percent of stress relaxation decreased when the EAs were fixed in PFA. In addition, the elastic moduli of the fixed EAs were significantly higher than the unfixed, revealing that PFA causes stiffening. The results also showed that there was no statistical difference in mechanical properties between the control EAs and EAs stored in 1% PBS. The four samples of reconstituted blood with varying hematocrits were put through histology to find the compositions of RBCs, platelets, and fibrin in each EA. The samples were fixed in 4% PFA solution, embedded in paraffin wax, then sectioned and mounted on to glass slides. The glass slides were then dewaxed and stained using the Carstairs’ staining method, which stains RBCS yellow, platelets blue, and fibrin red/pink. The slides were imaged under brightfield microscopy, and the images were processed using ImageJ to determine the percent compositions of RBCs, platelets, and fibrin at the core and periphery of the EAs. Significance was determined by performing a two-tailed t-test with a 95% confidence interval and an alpha value of 0.05. Only EAs formed under static conditions resulted in significantly different compositions between the core and periphery. The results showed that most EAs had a higher composition of platelets along the periphery, higher composition of fibrin at the core, and RBCs were found to be equally dispersed throughout the EAs.