Intravital imaging of three different microvascular beds in SARS-CoV-2-infected mice.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) enters the respiratory tract, where it infects the alveoli epithelial lining. However, patients have sequelae that extend well beyond the alveoli into the pulmonary vasculature and, perhaps, beyond to the brain and other organs. Because of the dynamic events within blood vessels, histology does not report platelet and neutrophil behavior. Because of the rapid nontranscriptional response of these cells, neither single-cell RNA sequencing nor proteomics report robustly on their critical behaviors. We used intravital microscopy in level-3 containment to examine the pathogenesis of SARS-CoV-2 within 3 organs in mice expressing human angiotensin converting enzyme 2 (ACE-2) ubiquitously (CAG-AC-70) or on epithelium (K18-promoter). Using a neon-green SARS-CoV-2, we observed both the epithelium and endothelium infected in AC70 mice but only the epithelium in K18 mice. There were increased neutrophils in the microcirculation but not in the alveoli of the lungs of AC70 mice. Platelets formed large aggregates in the pulmonary capillaries. Despite only neurons being infected within the brain, profound neutrophil adhesion forming the nidus of large platelet aggregates were observed in the cerebral microcirculation, with many nonperfused microvessels. Neutrophils breached the brain endothelial layer associated with a significant disruption of the blood-brain-barrier. Despite ubiquitous ACE-2 expression, CAG-AC-70 mice had very small increases in blood cytokine, no increase in thrombin, no infected circulating cells, and no liver involvement suggesting limited systemic effects. In summary, our imaging of SARS-CoV-2-infected mice gave direct evidence that there is a significant perturbation locally in the lung and brain microcirculation induced by local viral infection leading to increased local inflammation and thrombosis in these organs.

Negating Tissue Contracture Improves Volume Maintenance and Longevity of In Vivo Engineered Tissues.

BACKGROUND: Engineering large, complex tissues in vivo requires robust vascularization to optimize survival, growth, and function. Previously, the authors used a "chamber" model that promotes intense angiogenesis in vivo as a platform for functional three-dimensional muscle and renal engineering. A silicone membrane used to define the structure and to contain the constructs is successful in the short term. However, over time, generated tissues contract and decrease in size in a manner similar to capsular contracture seen around many commonly used surgical implants. The authors hypothesized that modification of the chamber structure or internal surface would promote tissue adherence and maintain construct volume. METHODS: Three chamber configurations were tested against volume maintenance. Previously studied, smooth silicone surfaces were compared to chambers modified for improved tissue adherence, with multiple transmembrane perforations or lined with a commercially available textured surface. Tissues were allowed to mature long term in a rat model, before analysis. RESULTS: On explantation, average tissue masses were 49, 102, and 122 mg; average volumes were 74, 158 and 176 µl; and average cross-sectional areas were 1.6, 6.7, and 8.7 mm for the smooth, perforated, and textured groups, respectively. Both perforated and textured designs demonstrated significantly greater measures than the smooth-surfaced constructs in all respects. CONCLUSIONS: By modifying the design of chambers supporting vascularized, three-dimensional, in vivo tissue engineering constructs, generated tissue mass, volume, and area can be maintained over a long time course. Successful progress in the scale-up of construct size should follow, leading to improved potential for development of increasingly complex engineered tissues.

Rat epigastric flow-through flap as a modulated arteriovenous fistula: model for the radial artery flow-through flap in distal arterial bypass.

The authors describe a rat flap model that is useful for flow studies. It is an epigastric flow-through flap that mimics the clinical use of a radial artery flow-through (RAFT) flap that has been used as an adjunct to a distal lower extremity arterial bypass graft to improve patency when there is potential high outflow resistance. The hypotheses were that this RAFT flap serves two purposes: 1) it allows additional blood flow through the skin flap and drainage via the vena comitans to increase the blood flow through the bypass graft and help to maintain bypass graft patency; and 2) it acts as a modulating arteriovenous fistula in which the additional flow through the vena comitans of the flow-through flap fluctuates with distal arterial outflow resistance. The rat epigastric flow-through flap model was designed to test these hypotheses. High outflow resistance was induced by sequentially ligating the outflow vessels of the rat femoral artery. Using this model, an increase in blood flow to the skin via the epigastric artery of the flow-through flap was demonstrated as outflow obstruction increased. Then, the patency rates of the flow-through flap bypass were compared to an interpositional arterial graft. The flow-through flap maintained patency while the arterial interposition bypass thrombosed, with near total outflow obstruction induced by serial ligation of the outflow vessels (75 percent patent anastomoses at 1 week for flow-through flap vs. 0 percent for the arterial graft). This flow study demonstrates the inherent ability of the flow-through flap to divert blood flow through the skin capillaries when there is high arterial outflow resistance. The authors believe that a flow-through flap such as the RAFT flap can be an important adjunct to the traditional distal arterial bypass in a subset of patients with high outflow resistance in the recipient artery.