Feasibility and comparison of laparoscopic laser speckle contrast imaging to near-infrared display of indocyanine green in intraoperative tissue blood flow/tissue perfusion in preclinical porcine models.

OBJECTIVE: To determine if laser speckle contrast imaging (LSCI) mitigates variations and subjectivity in the use and interpretation of indocyanine green (ICG) fluorescence in the current visualization paradigm of real-time intraoperative tissue blood flow/perfusion in clinically relevant scenarios. METHODS: De novo laparoscopic imaging form-factor detecting real-time blood flow using LSCI and blood volume by near-infrared fluorescence (NIRF) of ICG was compared to ICG NIRF alone, for dye-less real-time visualization of tissue blood flow/perfusion. Experienced surgeons examined LSCI and ICG in segmentally devascularized intestine, partial gastrectomy, and the renal hilum across six porcine models. Precision and accuracy of identifying demarcating lines of ischemia/perfusion in tissues were determined in blinded subjects with varying levels of surgical experience. RESULTS: Unlike ICG, LSCI perfusion detection was real time (latency < 150 ms: p < 0.01), repeatable and on-demand without fluorophore injection. Operating surgeons (n = 6) precisely and accurately identified concordant demarcating lines in white light, LSCI, and ICG modes immediately. Blinded subjects (n = 21) demonstrated similar spatial-temporal precision and accuracy with all three modes ≤ 2 min after ICG injection, and discordance in ICG mode at ≥ 5 min in devascularized small intestine (p < 0.0001) and in partial gastrectomy (p < 0.0001). CONCLUSIONS: Combining LSCI for near real-time blood flow detection with ICG fluorescence for blood volume detection significantly improves precision and accuracy of perfusion detection in tissue locations over time, in real time, and repeatably on-demand than ICG alone.

A novel assay provides sensitive measurement of physiologically relevant changes in albumin permeability in isolated human and rodent glomeruli.

Increased urinary albumin excretion is a key feature of glomerular disease but has limitations as a measure of glomerular permeability. Here we describe a novel assay to measure the apparent albumin permeability of single capillaries in glomeruli, isolated from perfused kidneys cleared of red blood cells. The rate of decline of the albumin concentration within the capillary lumen was quantified using confocal microscopy and used to calculate apparent permeability. The assay was extensively validated and provided robust, reproducible estimates of glomerular albumin permeability. These values were comparable with previous in vivo data, showing this assay could be applied to human as well as rodent glomeruli. To confirm this, we showed that targeted endothelial glycocalyx disruption resulted in increased glomerular albumin permeability in mice. Furthermore, incubation with plasma from patients with post-transplant recurrence of nephrotic syndrome increased albumin permeability in rat glomeruli compared to remission plasma. Finally, in glomeruli isolated from rats with early diabetes there was a significant increase in albumin permeability and loss of endothelial glycocalyx, both of which were ameliorated by angiopoietin-1. Thus, a glomerular permeability assay, producing physiologically relevant values with sufficient sensitivity to measure changes in glomerular permeability and independent of tubular function, was developed and validated. This assay significantly advances the ability to study biology and disease in rodent and human glomeruli.