Assessment of the Intestinal Barrier with Five Different Permeability Tests in Healthy C57BL/6J and BALB/cJ Mice.

BACKGROUND: Intestinal permeability is thought to be of major relevance for digestive and nutrition-related diseases, and therefore has been studied in numerous mouse models of disease. However, it is unclear which tools are the preferable ones, and how normal values should be defined. AIMS: To compare different in vivo permeability tests in healthy mice of commonly used genetic backgrounds. METHODS: We assessed the intestinal barrier in male and female C57BL/6J and BALB/cJ mice of different ages, using four orally administered permeability markers, FITC-dextran 4000 (FITC-D4000) and ovalbumin (OVA) measured in plasma, and polyethylene glycol (PEG) and lactulose/mannitol (Lac/Man) measured in urine, and by assessing lipopolysaccharide (LPS) in portal vein plasma. RESULTS: After gavage, FITC-D4000, OVA, Lac/Man, and PEG400, but not PEG4000, were detectable in plasma or urine. Female mice tended to have a higher permeability according to the FITC-D4000, OVA, and PEG400 tests, but the Lac/Man ratio was higher in males. No significant differences between the two mouse strains of young and old mice were observed except for mannitol recovery, which was higher in BALB/cJ mice compared to C57BL/6J mice (p < 0.05). Virtually no LPS was detected in healthy mice. For all markers, normal values have been defined based on 5th-95th percentile ranges of our data. CONCLUSION: Selected oral permeability tests, such as FITC-D4000, OVA, PEG400, and Lac/Man, as well as LPS measurements in portal vein plasma, could be suitable for the evaluation of the intestinal barrier in mice, if used in a standardized way.

Simultaneous assessment of glomerular filtration and barrier function in live zebrafish.

The zebrafish pronephros is a well-established model to study glomerular development, structure, and function. A few methods have been described to evaluate glomerular barrier function in zebrafish larvae so far. However, there is a need to assess glomerular filtration as well. In the present study, we extended the available methods by simultaneously measuring the intravascular clearances of Alexa fluor 647-conjugated 10-kDa dextran and FITC-conjugated 500-kDa dextran as indicators of glomerular filtration and barrier function, respectively. After intravascular injection of the dextrans, mean fluorescence intensities of both dextrans were measured in the cardinal vein of living zebrafish (4 days postfertilization) by confocal microscopy over time. We demonstrated that injected 10-kDa dextran was rapidly cleared from the circulation, became visible in the lumen of the pronephric tubule, quickly accumulated in tubular cells, and was detectably excreted at the cloaca. In contrast, 500-kDa dextran could not be visualized in the tubule at any time point. To check whether alterations in glomerular function can be quantified by our method, we injected morpholino oligonucleotides (MOs) against zebrafish nonmuscle myosin heavy chain IIA (zMyh9) or apolipoprotein L1 (zApol1). While glomerular filtration was reduced in zebrafish nonmuscle myosin heavy chain IIA MO-injected larvae, glomerular barrier function remained intact. In contrast, in zebrafish apolipoprotein L1 MO-injected larvae, glomerular barrier function was compromised as 500-kDa dextran disappeared from the circulation and became visible in tubular cells. In summary, we present a novel method that allows to simultaneously assess glomerular filtration and barrier function in live zebrafish.

A method for assessment of blood volume parameters in pregnant sheep using fluorescein-labelled dextran.

The assessment of blood volume parameters in clinical and research settings has been limited by methods that involve radioactivity, complex assays or are unreliable. We aimed to design a method for measuring blood volume parameters that was non-radioactive, simple, cheap and reliable. We have used a commercially available fluorescein-labelled 250kDa dextran, a large inert molecule, and have measured dilution of this through the intravascular space of pregnant ewes. From this estimation of plasma volume and measured hematocrit, we have calculated blood volume and red cell volume. The blood volume results are 6% lower than those obtained using radiolabelled red cells, but there is no significant difference in red cell volume between methods. The coefficient of variation for repeated measurements of plasma volume measurements is 3.8%. This is a simple, reliable, cheap and non-radioactive method for estimating blood volume parameters in pregnant sheep, and may prove useful in other settings.

An assessment of the potential use of anionic dextrans as a plasma substitute.

Several problems exist when dextrans are used as plasma substitutes. High molecular weight dextrans can cause red cell aggregation and increased blood viscosity. Low molecular weight dextrans, although shown to improve circulation and promote flow, are removed rather rapidly from the circulation due to high premeation rates across capillary walls. In the present study, a small anionic charge is introduced onto the dextran to make it electrostatically negative. Since capillary walls have been shown to retain negatively charged solutes in preference to neutral solutes, the anionic dextran should retain its effectiveness for longer periods of time compared to similar sized neutral dextran. Studies were done on eight unanaesthetized dogs to compare the relative disappearance rates of dextran and anionic dextran (carboxymethyl dextran) from the circulation. It was shown that anionic dextrans do remain in the circulation over a longer period of time compared to neutral dextrans.