Jaspersen, Adam

Abstract Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high‐resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.

Introduction: Characterization of acute ischemic stroke thrombi has typically focused on the two dimensional (2D) histological characterization of the thrombus. Little is known about the three dimensional (3D) structure of these occlusive thrombi. Electron Microscopy (EM) can be used to obtain 3D information about the organization of clot components. Individual structures can be segmented and qualitative and quantitative information obtained from the clots. Methods: Following IRB approval from Mayo Clinic, acute ischemic stroke clot samples were collected from 10 patients treated at Mayo Clinic. Samples were immediately fixed in fresh modified Trumps fixative solution. A representative fragment of clot was taken and split longitudinally into two pieces; one piece was sent for 3D-EM analysis and the other for histological processing and analysis. Hematoxylin and eosin stain (H&E) and Martius Scarlett Blue (MSB) histological stains were used for conventional histopathology evaluation and components analysis. Immunohistochemical (IHC) staining using anti-CD42b (Platelets) was applied to confirm the presence of platelets in each clot. Serial block-face scanning electron microscopy was used to generate high-resolution three-dimensional images. Results: The 2D morphological features of the clot were identified using H&E, MSB and anti-CD42b staining. The 3D organization of thrombi varies greatly, both within and between clots depending on the constituents. Red Blood Cell-rich areas are mainly composed of Red Blood Cells packaged tightly together, with a small number of Fibrin strands interwoven between the cells. Fibrin-rich areas are characterized by dense networks of fibrin strands along with platelets and White Blood Cells with some sparse Red Blood Cells present. Platelet-rich areas are composed of large volumes of de-granulated platelets, some fibrin stands and white blood cells and an absence of Red Blood Cells. Conclusions: 3D electron microscopy can provide important information on the organization of components within thrombi. This could provide key information on the age and etiology of the thrombus. In addition, it may help to inform thrombolytic treatment and medical device design.

Background Characterization of acute ischemic stroke (AIS) clots has typically focused on two-dimensional histological analysis of the thrombus. The three-dimensional (3D) architecture and distribution of components within emboli have not been fully investigated. The aim of this study was to examine the composition and microstructure of AIS clots using histology and serial block-face scanning electron microscopy (SBFSEM). Methods As part of the multi-institutional STRIP registry, 10 consecutive AIS emboli were collected from 10 patients treated by mechanical thrombectomy. Histological and immunohistochemical analysis was performed to determine clot composition. SBFSEM was used to assess the ultrastructural organization of the clots and specific features of individual components. Results Quantification of Martius Scarlett Blue stain identified fibrin (44.4%) and red blood cells (RBCs, 32.6%) as the main components. Immunohistochemistry showed a mean platelet and von Willebrand factor content of 23.9% and 11.8%, respectively. The 3D organization of emboli varied greatly depending on the region analyzed. RBC-rich areas were composed mainly of tightly packed RBCs deformed into polyhedrocytes with scant fibrin fibers interwoven between cells. The regions with mixed composition showed thick fibrin fibers along with platelets, white blood cells and RBC clusters. Fibrin-rich areas contained dense fibrin masses with sparse RBC. In three cases, the fibrin formed a grid-like or a sponge-like pattern, likely due to thrombolytic treatment. Segmentation showed that fibrin fibers were thinner and less densely packed in these cases. Conclusions 3D-SEM provides novel and potentially clinically relevant information on clot components and ultrastructure which may help to inform thrombolytic treatment and medical device design.