Christensen, Trace A

Abstract Intravital imaging studies have provided insights into the spatial and temporal variations of platelet activation and thrombin generation that occur during hemostasis; however, these studies are generally limited to small vessels due to the practical limitations of imaging in thicker tissues. Recent advances in cleared tissue fluorescence imaging as well as volume electron microscopy (vEM) coupled with machine learning–based image segmentation provide an opportunity for analysis of the 3-dimensional structure of complex tissues. We utilized these technologies to examine hemostatic plugs from murine jugular veins and carotid arteries to investigate the spatial distribution of platelet activation and biochemical responses in these disparate physiologic contexts. Both venous and arterial hemostatic plugs had a heterogeneous structure with regions of sparsely and densely packed platelets. Despite similar injury sizes, arterial hemostatic plugs were at least an order of magnitude larger than venous plugs. The difference in plug size was primarily due to a 19-fold increase in the population of densely packed platelets in the extravascular compartment. Venous plugs displayed significant platelet aggregation extending into the vessel lumen and developed distinctive fibrin and red blood cell–filled cavities. Complementary fluorescence microscopy revealed that platelet activation was spatially heterogeneous in both contexts, with α-granule secretion and phosphatidylserine exposure confined to specific microenvironments, highlighting tightly regulated thrombin activity. Overall, our findings reveal both conserved and distinct mechanisms of hemostatic thrombus formation in different physiologic contexts. They also demonstrate the power of vEM coupled with machine learning–based image segmentation for the quantitative analysis of large imaging data sets from complex tissues.

Key Points Understanding how heme proteins/heme induce AKI and whether NAD + -boosting ameliorates heme protein–mediated AKI is clinically relevant. Heme proteins/heme directly decrease kidney NAD + content and NAD + -boosting is markedly protective in heme protein–mediated AKI. These findings provide new insights in heme protein–mediated AKI and support interest in NAD + -boosting as a therapeutic strategy in AKI. Background Understanding how heme proteins and heme induce AKI is clinically relevant from numerous perspectives, and, in this regard, a widely used model of heme protein–mediated AKI (HP-AKI) involves the glycerol model in mice. Using this model, we have previously demonstrated that NAD + content is decreased in HP-AKI. Because there is significant current interest in NAD + -boosting as a strategy in clinical AKI, we examined the effect of NAD + -boosting in this model. Methods NAD + -boosting was achieved by the administration of nicotinamide mononucleotide (NMN) in the glycerol model of HP-AKI. The effect of NMN, compared with vehicle, was examined in mice with HP-AKI and with sham AKI at day 1 after HP-AKI was induced. Results The administration of NMN preserved kidney NAD + content in HP-AKI, and compared with the administration of vehicle in HP-AKI, NMN improved glomerular filtration markers; reduced histologic injury as assessed by tubular necrosis, dilation, and cast formation and by assessment of dystrophic calcification; reduced expression of kidney injury marker 1, a sensitive marker of AKI; preserved mitochondrial ultrastructure and increased expression of proteins that promote mitochondrial integrity; reduced apoptosis as assessed by terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling staining and genes and protein expression that contribute to apoptosis, while increasing expression of an anti-apoptotic gene; reduced expression of several renal injury-related genes; and mitigated the severity of the senescence phenotype, as assessed by multiple markers. Finally, the administration of myoglobin or heme in vivo diminished kidney NAD + content in mice with intact kidneys. Conclusions These findings demonstrate the remarkable protective effects of NAD + -boosting by NMN in HP-AKI as revealed by a multitude of markers relevant to AKI. The reduction in NAD + content in the kidney in HP-AKI may reflect the direct effects of heme proteins and/or heme. We suggest that these findings support current interest in NAD + -boosting as a therapeutic strategy in clinical AKI.

Diabetic pregnancy increases the offspring’s risk of neonatal and adult cardiovascular disease (CVD). We previously used a rat model (Sprague–Dawley) to show that diabetic pregnancy impairs mitochondrial bioenergetics, dynamics, mitophagy, and quality control in the offspring’s heart, and we hypothesized that mitochondrial dysfunction during early development influences the adult myocardium structure to confer cardiometabolic disease risk with aging. Here, we used 3D serial block face-scanning electron microscopy (SBF-SEM) to analyze perinuclear (PN) and intrafibrillar (IF) mitochondrial networks in the left ventricular sections from control and pregestational diabetes-exposed newborn (NB) rats that were three-week-old and four-month-old. Diabetes-exposed myocardium had 50% fewer PN and 20% fewer IF mitochondria at birth but counts increased more rapidly, resulting in no difference at three weeks and 35% more PN and 49% more IF mitochondria by four months. Despite rising counts, mitochondria volumes remained significantly lower at every developmental timepoint. This shows that diabetic pregnancy causes maldevelopment of the myocardial mitochondrial reticulum which likely contributes to adult CVD.

The exact physiological role of BACE2 in endothelial function of systemic arteries is unknown. Our study shows that thromboxane A2 and prostaglandin F2α are responsible for impairment of endothelium-dependent relaxations in endothelium-specific BACE2-deficient mice. The data support an important role of BACE2 in the control of endothelial arachidonic acid metabolism and vasomotor function.

Our understanding of inner ear hair cell ultrastructure has heretofore relied upon two-dimensional imaging; however, serial block-face scanning electron microscopy (SBFSEM) changes this paradigm allowing for three-dimensional evaluation. We compared inner ear hair cells of the apical cristae in myo7aa −/− null zebrafish, a model of human Usher Syndrome type 1B, to hair cells in wild-type zebrafish by SBFSEM to investigate possible ribbon synapse ultrastructural differences. Previously, it has been shown that compared to wild type, myo7aa −/− zebrafish neuromast hair cells have fewer ribbon synapses yet similar ribbon areas. We expect the recapitulation of these results within the inner ear apical crista hair cells furthering the knowledge of three-dimensional ribbon synapse structure while resolving the feasibility of therapeutically targeting myo7aa −/− mutant ribbons. In this report, we evaluated ribbon synapse number, volume, surface area, and sphericity. Localization of ribbons and their distance from the nearest innervation were also evaluated. We determined that myo7aa −/− mutant ribbon synapses are smaller in volume and surface area; however, all other measurements were not significantly different from wild-type zebrafish. Because the ribbon synapses are nearly indistinguishable between the myo7aa −/− mutant and wild type, it suggests that the ribbons are structurally receptive, supporting that therapeutic intervention may be feasible.

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.