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INNOVATIVE METHODOLOGY
Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi
Submitted 15 May 2006 ; accepted in final form 14 July 2006
| ABSTRACT |
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10-mm diameter) plastic chambers to the serosa of the abdominal wall of anesthetized CD1 and C57BL mice. The chamber constrained transfer across the area of the chamber base and facilitated mixing, volumetric, and concentration measurements vs. time for mannitol, serum albumin, and osmotic and hydrostatic pressure-driven convection. The mass transfer coefficient of mannitol (MTCM) and of serum albumin (MTCBSA), hydrostatic pressure-driven flux (JP), and osmotic filtration (Josm) were calculated from the time-dependent volume and concentration data. The units of all parameters (µl·min1·cm2) were compared with previously derived parameters from SD rats with a one-way ANOVA. Results indicated small but significant differences in MTCBSA (x102): CD1, 9.72 ± 1.97, n = 6; C57BL, 7.13 ± 1.52, n = 10; rat, 12.5 ± 1.6, n = 17 (P = 0.03). ANOVAs of all other parameters were not significant and confirmed our hypothesis: MTCM (CD1, 3.20 ± 0.38, n = 7; C57BL, 2.34 ± 0.41, n = 6; rat, 2.72 ± 0.23 n = 19), JP (CD1, 0.77 ± 0.15, n = 10; C57BL, 0.33 ± 0.13, n = 15; rat, 0.51 ± 0.16, n = 9), or Josm (CD1, 0.92 ± 0.35, n = 6; C57BL, 0.49 ± 0.35, n = 6; rat 1.72 ± 0.35, n = 6). We conclude that elimination of the variable peritoneal transfer area normalizes calculated transport characteristics and facilitates comparison between species. animal model; peritoneum; transport; mass transfer
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Animal (11, 12) and human studies (2, 3) have demonstrated that only 3040% of the anatomic peritoneum is in contact with a relatively large peritoneal volume (23 liters in humans or 3550 ml in rats); furthermore, with movement or change in body position, the duration of contact with any one surface is variable. While the MTCs of different surfaces are relatively of the same magnitude (8), the true contact area in a "whole-cavity" experiment is typically unknown.
The peritoneal volume used in an animal or human study determines the surface area of contact and the MTAC (2, 11). Different laboratories utilize different volumes in rats and different durations of experiments to describe the transport phenomena (1, 13, 15, 17). In the case of mice, there is similar variation in a wide variation of techniques utilized in these animals (12, 16). Ni and colleagues (16) utilize a very small volume of 2 ml of hypertonic solution (7.5% glucose in an isotonic salt solution). Flessner (11), on the other hand, has used as much as 10 ml in similar-size animals. This wide variation in volumes can lead to significant differences in the contact surface area and estimated MTAC, and it makes the studies from different laboratories difficult to compare. Acute or chronic studies in which the peritoneum is altered may result in increased or decreased surface contact area and produce even more variation in the calculation of transport parameters.
We hypothesize that elimination of the peritoneal contact area as a variable will result in transport rates or parameters, which are of the same order of magnitude. To address this hypothesis, we used transport chambers (9) to fix the contact area in two species of mice to investigate the differences between different species and to compare these to our previous rat studies. Our findings support the hypothesis.
| METHODS |
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FITC-BSA was purchased from Sigma (St. Louis, MO) and was used as delivered. Checks of label purity (9) have demonstrated that there is essentially no separation of the label from the protein.
The chamber solution for studying mannitol transport in the osmotically induced, hypertonic flux from the tissue consisted of 4% mannitol in a Krebs-Ringer-bicarbonate (KRB) solution (see Ref. 7 for details of solution). [14C]mannitol in a concentration of 1 µCi/ml was mixed in this solution. The solution for macromolecular transport was made up of 20 mg of FITC-BSA in 0.5 ml of KRB. For studying hydrostatic pressure-driven convection into the tissue, 5% unlabeled BSA+0.05% Evans blue dye were mixed in KRB with 1 µCi of 125I-labeled IgG per ml (see Ref. 13 for details of this protein and the daily process of removal of free 125I label).
Animals: surgical preparation. CD1 and C57BL mice were purchased from Charles River Laboratories and had weights of 2330 and 2228 g, respectively, at the time of experiment. Sprague-Dawley (SD) rats were purchased from Charles River Laboratories and had weights of 180220 g. A minimum of five animals was utilized in every experimental protocol. Each animal was weighed and anesthetized with 2% isoflurane introduced via a Euthanex Small Animal Anesthesia System (model 2500, Palmer, PA). Rectal temperature was maintained at 3538°C with overhead heating lamps and the Euthanex water-jacketed warming system. A catheter was placed in the carotid artery for sampling and blood pressure monitoring; mean arterial blood pressure was always >70 mmHg and in a similar range to that of SD rats. An internal jugular catheter was placed for infusion of fluids and tracer molecules. Diffusion chambers were constructed of polystyrene centrifuge tubes and were scaled down from our previous rat chambers because of the smaller size of the mouse (8); the internal diameter of the chamber was typically 910 mm. To access the peritoneum, a bloodless, vertical incision was made along the linea alba, which separates the two sides of the abdominal wall and the respective circulations of each side. The animal was then placed on its side, and one side of the abdominal wall was carefully stretched to its original size; abdominal viscera were retracted with warm, moist gauze pads, and the abdominal wall was exposed for chamber placement. The chamber was affixed to the abdominal wall with cyanoacrylate glue. After adhesion, 1 ml of KRB was placed into the chamber for 30 min to allow tissue to recover from the procedure. Warmed KRB was dripped onto gauze pads surrounding the chamber and the remaining abdominal tissues to keep them at physiological temperature. Details of chamber placement and an extensive investigation of effects of the chamber on the tissue can be found elsewhere (5, 8).
All animal procedures were reviewed and approved by the University of Mississippi Institutional Animal Care and Use Committee.
Experimental protocols. Figure 1A displays the method for hydrostatic pressure-driven convection, and Fig. 1B displays the method for large- and small-solute transfer and osmotic ultrafiltration. Methods were analogous to our previous techniques in rats (810).
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To study the transport of serum albumin from the blood into the chamber, the FITC-BSA solution was injected in a volume of
0.3 ml of isotonic KRB intravenously over 23 min. The chamber was filled with a preweighed volume of
0.6 ml of isotonic KRB. The height was <1 cm to prevent any hydrostatic pressure-driven convection into the tissue (13). The volume of the chamber was checked hourly over 3 h. Thirty-microliter samples of the blood were taken hourly. At 3 h, the volume of the chamber fluid was measured, while the fluorescence was determined for each sample of plasma and the chamber solution (Turner Designs TD700, Sunnyvale, CA). At the end of the experiment, the chamber was removed and the chamber area was determined.
To determine the hydrostatic pressure-driven convection into the tissue, a tall chamber of 7-cm height was utilized and filled with the solution of KRB with 5% BSA plus Evans Blue dye and 125I-IgG. The tracer is a volume marker, and its disappearance correlates with the fluid transfer to the tissue (13). The Evans Blue dye, bound to the albumin, provides a visual check on where the convection occurs within the tissue. Fifty-microliter samples of the solution volume at time 0 and then hourly for 180 min were collected to determine the concentration of the tracer, and the solution was removed hourly and weighed to follow the volume change, as a check for leaks at the chamber base. At 180 min, the fluid was removed and the chamber was removed and its area was measured. After death of the animal, the tissue underlying the chamber, including the muscle and skin in the surrounding area, was removed and counted with a gamma counter (Beckman Instrument, Fullerton, CA) to determine total deposition of IgG into the tissue. The clearance of labeled IgG to the tissue was used to calculate the hydrostatic pressure-driven flux into the tissue (see Eq. 5 below).
Calculations.
Statistical calculations were carried out using NCSS 97 (Number Crunching Statistical System, Kaysville, UT). One-way ANOVA was utilized to compare different data sets. Probability of a type I error was set at P
0.05 for significance. The rat data were obtained in the same manner as these experiments or taken from previous publications for comparison (5, 8).
The following equation (similar to Eq. 1) is fitted to the mass transfer data for mannitol or FITC-BSA using the program Scientist (Micromath, Salt Lake City, UT).
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The osmotically induced fluid from the tissue to the chamber (Fluxosm) was calculated as follows
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Vchamber is the change in chamber volume, and
t is time.
Fluid flux from the chamber into the tissue due to hydrostatic pressure (FluxP)-driven convection is calculated as a clearance term for the IgG from the chamber into the underlying tissue as follows
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| RESULTS |
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| DISCUSSION |
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8 ml/h (18). Flessner and colleagues (11) showed in measurements in separate sets of animals close correspondence between the measured surface contact area and the MTAC. Keshaviah and colleagues (14) found a linear relationship between the MTACs for urea, creatinine, and glucose over a range of fill volumes from 0.5 to
3 liters in 10 patients and concluded that this was due to an increasing peritoneal contact area. Chagnac and colleagues (2) studied the solute exchange in 10 patients dialyzed with either 2- or 3-liter dwells. They measured the peritoneal contact area for a 2-liter solution as 0.57 ± 0.03 m2, which corresponded to a MTAC for creatinine of 10.6 ± 0.7 ml/min; the corresponding values for the 3-liter dwell were 0.67 ± 0.04 m2 with a MTAC of 13.6 ± 1.2 ml/min. A comparison of the ratio of the MTACs (0.77) with that of the areas (0.85) provides further clinical support for Eq. 1.
Transport data obtained from whole-cavity experiments in mice depend on the osmolality and volume of the solution and vary with the technique employed. Ni and colleagues (16) injected 2 ml of 7% glucose solution into 30-g mice and obtained
34 µl/min of volume flow into the cavity; the MTAC for urea was calculated to be 28 µl/min. Using the scaling factor of (peritoneal volume)2/3 (4), a 10-ml volume would increase the surface area by a factor of
2.9 and would theoretically (see Eq. 2) raise the rate of mass and fluid transfer by the same amount. The discordance between methods used by different laboratories makes comparisons within a species difficult and between the rat and the mouse almost impossible. The range of MTACurea for rats is 825 ml/h (6, 18), while the value for mice with a 2-ml volume is
1.7 ml/h (16). We hypothesized that the differences between experiments within rodent species and between rodent species (rat vs. mouse) were due to the different surface contact areas. Elimination of this variable might simplify experimental comparison and extend data sets from different rodents.
To address the question of similarity of transport between rodent species, we have utilized a scaled-down version of the transport chamber technique that we developed in rats (8) and have applied this to mice. To validate the comparison between results for the rat with a larger chamber and determinations in mice with the smaller chamber, experiments were repeated in rats with the chambers used in mice. For MTCmannitol and Fluxosm, no differences were noted. This demonstrates that data obtained from the smaller chambers are a valid comparison with that obtained in the larger chambers.
To address our hypothesis that elimination of the peritoneal contact area as a variable will result in transport rates or parameters, which are of the same order of magnitude, we carried out one-way ANOVAs to compare data from both groups of mice and SD rats. Only one of the parameters measured (MTCBSA) was significantly different. Comparisons with a one-way ANOVA between the rat measurements and either the CD1 mice or the C57BL did not reveal any other significant differences in the MTCM, Fluxosm, and FluxP. Because of the differences in Fluxosm between C57BL mice and the SD rats, we combined all of the rat data and compared it to the mice data with a one-way ANOVA; with the increase in the number of data points (n = 15 vs. n = 6 or 9), the difference between C57BL and rats became statistically significant. However, there was not a significant difference for Fluxosm between the CD1 and SD rats. These variations in statistical analyses are most likely due to the nature of invasive animal procedures, which are subject to variations in animal batches, preoperative state of hydration, and surgical technique of different technicians.
The physiological principle of the importance of the surface contact area is not so much dependent on statistics but on the relative magnitude of the measured variables. All the measurements were well within an order of magnitude for each category. These results further demonstrate that once the surface contact area is removed as a variable, all of these species appear to be quite similar in their transport characteristics for the four different types of transport that occur during peritoneal dialysis. While there is variation in these measurements from animal to animal, our hypothesis of these measurements being of the same order of magnitude has been upheld. With these techniques, we can compare and translate results from mice to rats and rats to mice.
| GRANT |
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| ACKNOWLEDGMENTS |
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| FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
| REFERENCES |
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This article has been cited by other articles:
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B. Rippe HOW TO ASSESS TRANSPORT IN ANIMALS? Perit. Dial. Int., February 1, 2009; 29(Supplement_2): S32 - S35. [Abstract] [Full Text] [PDF] |
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M. F. Flessner, K. Credit, K. Henderson, H. M. Vanpelt, R. Potter, Z. He, J. Henegar, and B. Robert Peritoneal Changes after Exposure to Sterile Solutions by Catheter J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2294 - 2302. [Abstract] [Full Text] [PDF] |
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