Use of multiple fluorophores for evaluating microvascular permeability in control rats and rats with sepsis

Capillary leak accompanying systemic inflammatory response conditions is a significant clinical problem. In the present study, we describe and verify a method for studying capillary leak that is based on the injection of proteins that differ significantly in size and have spectrally distinguishable fluorophores. Control (n = 11) and post-CLP (caecal ligation and puncture; n = 14) Sprague-Dawley rats were injected with tracer amounts of albumin and PEG-Alb [albumin covalently linked to methoxy-poly(ethylene glycol)] labelled with fluorescein and Texas Red. Blood samples were withdrawn between 5 min and 144 h, and the fluorescence of the labelled proteins was determined. The relative retention of the PEG-Alb and albumin was assessed via measurement of the TER (transcapillary escape rate; in %/h) and the t(50%) estimate, defined as the time when the actual concentration reached 50% of its baseline. The concentration-time trends for both albumin and PEG-Alb tracers exhibited two-compartmental behaviour and were analysed using bi-exponential modelling. Retention times were significantly greater for PEG-Alb in both control and CLP rats. TERPEG-Alb was significantly lower than TERalbumin for both control (8.1 +/- 5.6 compared with 14.8 +/- 7.1 %/h respectively; P < 0.01) and CLP (14.8 +/- 6.6 compared with 22.5 +/- 7.3 %/h respectively; P < 0.001) rats. The t(50%[PEG-Alb]) was substantially greater than the corresponding t(50%) [albumin] for both control (29.8 +/- 9.8 compared with 7.2 +/- 2.0 h respectively; P < 0.001) and CLP (12.9 +/- 5.6 compared with 5.1 +/- 1.6 h respectively; P < 0.001) rats. The result was similar irrespective of the fluorophore-protein combination, validating the multifluorophore technique. In conclusion, the double-fluorophore approach described in the present study may provide the future basis for a method to quantify capillary leak in disease.