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 Blood samples were withdrawn between 5 min and 144 h, and the fluorescence of the labelled proteins was determined.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 t50% 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 t50%[PEG–Alb] was substantially greater than the corresponding t50%[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.
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January 2008
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Research Article|
December 11 2007
Use of multiple fluorophores for evaluating microvascular permeability in control rats and rats with sepsis
Ragheb A. Assaly;
*Department of Medicine, University of Toledo College of Medicine, Toledo, OH 43614, U.S.A.
Correspondence: Dr Ragheb A. Assaly (email ragheb.assaly@utoledo.edu).
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Robert H. Habib;
Robert H. Habib
†Saint Vincent Mercy Medical Center, Toledo, OH 43608, U.S.A.
‡Department of Surgery, University of Toledo College of Medicine, Toledo, OH 43614, U.S.A.
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Mustafa Azizi;
Mustafa Azizi
*Department of Medicine, University of Toledo College of Medicine, Toledo, OH 43614, U.S.A.
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Joseph I. Shapiro;
Joseph I. Shapiro
*Department of Medicine, University of Toledo College of Medicine, Toledo, OH 43614, U.S.A.
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J. David Dignam
J. David Dignam
§Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, OH 43614, U.S.A.
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Publisher: Portland Press Ltd
Received:
June 28 2007
Revision Received:
August 22 2007
Accepted:
August 29 2007
Accepted Manuscript online:
August 29 2007
Online ISSN: 1470-8736
Print ISSN: 0143-5221
© The Authors Journal compilation © 2008 Biochemical Society
2008
Clin Sci (Lond) (2008) 114 (2): 123–130.
Article history
Received:
June 28 2007
Revision Received:
August 22 2007
Accepted:
August 29 2007
Accepted Manuscript online:
August 29 2007
Citation
Ragheb A. Assaly, Robert H. Habib, Mustafa Azizi, Joseph I. Shapiro, J. David Dignam; Use of multiple fluorophores for evaluating microvascular permeability in control rats and rats with sepsis. Clin Sci (Lond) 1 January 2008; 114 (2): 123–130. doi: https://doi.org/10.1042/CS20070219
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