Quantitative capillary blood flow spatial analysis in skeletal muscle during sepsis
Authors: Mignemi Nicholas A. , Kilchrist Kameron V. , McClatchey Mason A. , Williams Ian Miller , Duvall Craig L. , Wasserman David H. , McGuinness Owen P. 
Published: June 2021

Introduction

Treatment of sepsis accounts for 23 billion healthcare dollars annually, and one in three hospitalized patients are diagnosed with sepsis. While current therapeutic strategies have been effective, sepsis‐induced mortality rates still remain greater than 10% and 40% with severe sepsis. Thus, new therapeutic approaches are needed.

A defining characteristic of sepsis is that it induces hyperglycemia and an insulin resistant state. Skeletal muscle is one of the largest consumers of glucose in the body. Muscle glucose uptake can be controlled by: delivery of glucose through the vasculature, transport of glucose into the muscle, and muscle glucose metabolism. Hyperglycemia and insulin resistance may be due to a defect in one or more of these processes. Our lab and others have shown that inflammatory states cause alterations in vascular flow to the skeletal muscle. Further, changes in vascular flow have been shown to correlate with sepsis‐induced hyperglycemia and insulin resistance. Given that both sepsis and inflammation induce insulin resistance and are associated with impaired muscle blood flow, we hypothesize that protecting the endothelium from sepsis‐induced inflammation will prevent insulin resistance. We will test this hypothesis through the use of mice with a vascular specific knock down of NFκB. However, few tools exist too accurately and reproducibly measure changes in capillary flow with single‐capillary spatial resolution. To being testing the hypothesis we sought to develop and validate a method to measure capillary flow patterns to assess changes in microvascular flow and heterogeneity in the skeletal muscle.

Method

Intravital microscopy was used to track fluorescent beads in mouse gastrocnemius muscle capillaries, and these data were used to build spatial capillary velocity profiles. Briefly, C57/BL6 mice with jugular vein catheters were anesthetized with isoflurane and the gastrocnemius was surgically exposed and positioned on a coverslip with warmed saline. Mice were then injected with 3 μm florescent beads and imaged at 111 frames per second. Resulting videos of binarized fluorescent beads were subjected to a positional tracking algorithm to yield a dataset of position and velocity data for tracked beads. This dataset was used to build capillary flow velocity maps for enhanced spatial visualization of flow data. Data analysis was conducted on both bead tracking data and capillary flow velocity maps using MATLAB. To assess whether the method could detect changes in capillary flow, mice were subjected to saline or lipopolysaccharide (2 mg/kg; LPS).

Results

The average vessel track length was found to be 450 μm and the average vessel flow rate in the gastrocnemius was determined to be 1030 μm/second. To prevent tracking bead vessel selection bias, we averaged the spatial velocities of vessels, and determined the average spatial velocities of capillaries to be 970 μm/second. We calculated the sample size needed to determine a 10–50% change from baseline for all variables (Table 1). LPS decreased muscle capillary flow velocity and increased flow heterogeneity (Figure 1).

Conclusion

We have developed a method to quantitate microvascular flow in the skeletal muscle of mice using flourescent beads. The average flow distribution and velocity of mice was consistent and stable. Additionally, when mice were challenged with LPS, we were able to detect a decrease in capillary flow and an increase in flow heterogeneity within the skeletal muscle.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.