title: Use of multiple fluorophores for evaluating microvascular permeability in control rats and rats with sepsis.
abstract: 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 Blood samples were withdrawn between 5 min and 144 h, and the fluorescence of the labelled proteins was determined.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. TER(PEG-Alb) was significantly lower than TER(albumin) 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.
authors.affiliation: Department of Medicine, University of Toledo College of Medicine, Toledo, OH 43614, USA. ragheb.assaly@utoledo.edu
authors.forename: Ragheb A
authors.initials: RA
authors.lastname: Assaly
authors.forename: Robert H
authors.initials: RH
authors.lastname: Habib
authors.forename: Mustafa
authors.initials: M
authors.lastname: Azizi
authors.forename: Joseph I
authors.initials: JI
authors.lastname: Shapiro
authors.forename: J David
authors.initials: JD
authors.lastname: Dignam
mesh_terms: D000418:Albumins; D000818:Animals; D019559:Capillary Leak Syndrome / Q000175:diagnosis* / Q000209:etiology / Q000503:physiopathology; D002199:Capillary Permeability; D019793:Fluorescein; D005453:Fluorescence; D008833:Microcirculation; D008954:Models, Biological; D011092:Polyethylene Glycols; D051381:Rats; D017207:Rats, Sprague-Dawley; D018805:Sepsis / Q000150:complications* / Q000503:physiopathology
chemical_list: D000418:Albumins; D011092:Polyethylene Glycols; D019793:Fluorescein