Lee, Donghee
(Department of Mechanical Engineering, Kookmin University)
,
Kim, Jeong Chul
(Institute of Medical and Biological Engineering, Medical Research Center, Seoul National University)
,
Shin, Eunkyoung
(Clinical Research Institute, Seoul National University Hospital)
,
Ju, Kyung Don
(Clinical Research Institute, Seoul National University Hospital)
,
Oh, Kook-Hwan
(Division of Nephrology, Department of Internal Medicine, Seoul National University Hospital)
,
Kim, Hee Chan
(Institute of Medical and Biological Engineering, Medical Research Center, Seoul National University)
,
Kang, Eungtaek
(Department of Internal Medicine, Chung-Ang University Hospital)
,
Kim, Jung Kyung
(Department of Mechanical Engineering, Kookmin University)
Fluorescence recovery after photobleaching (FRAP) is a well-established method commonly used to measure the diffusion of fluorescent solutes and biomolecules in living cells or tissues. Here a fiber-optic-based FRAP (f-FRAP) system was developed, and validated using macromolecules in water and agaro...
Fluorescence recovery after photobleaching (FRAP) is a well-established method commonly used to measure the diffusion of fluorescent solutes and biomolecules in living cells or tissues. Here a fiber-optic-based FRAP (f-FRAP) system was developed, and validated using macromolecules in water and agarose gels of different concentrations. We applied f-FRAP to measure the site-specific diffusion of fluorescein (NaFluo) in peritoneal membranes (PMs) on the liver, cecum, and kidney of a living rat during peritoneal dialysis. Diffusion of fluorescein in PM varied in a time-dependent manner according to the type of organ ($D_{PM\;on\;Liver}/D_{NaFluo}=0.199{\pm}0.085$, $D_{PM\;on\;Cecum}/D_{NaFluo}=0.292{\pm}0.151$, $D_{PM\;on\;Kidney}/D_{NaFluo}=0.218{\pm}0.110$). The proposed method allows direct quantitative measurement of the three-dimensional diffusion in local PM in vivo, which was previously inaccessible by peritoneal function test methods such as peritoneal equilibration test (PET) and standardized PM assessment (SPA). f-FRAP is promising for local and dynamic assessments of peritoneal pathophysiology and the mass transport properties of PMs, presumed to be affected by variation of tissue structures over different organs and functional changes of the PM with years of peritoneal dialysis.
Fluorescence recovery after photobleaching (FRAP) is a well-established method commonly used to measure the diffusion of fluorescent solutes and biomolecules in living cells or tissues. Here a fiber-optic-based FRAP (f-FRAP) system was developed, and validated using macromolecules in water and agarose gels of different concentrations. We applied f-FRAP to measure the site-specific diffusion of fluorescein (NaFluo) in peritoneal membranes (PMs) on the liver, cecum, and kidney of a living rat during peritoneal dialysis. Diffusion of fluorescein in PM varied in a time-dependent manner according to the type of organ ($D_{PM\;on\;Liver}/D_{NaFluo}=0.199{\pm}0.085$, $D_{PM\;on\;Cecum}/D_{NaFluo}=0.292{\pm}0.151$, $D_{PM\;on\;Kidney}/D_{NaFluo}=0.218{\pm}0.110$). The proposed method allows direct quantitative measurement of the three-dimensional diffusion in local PM in vivo, which was previously inaccessible by peritoneal function test methods such as peritoneal equilibration test (PET) and standardized PM assessment (SPA). f-FRAP is promising for local and dynamic assessments of peritoneal pathophysiology and the mass transport properties of PMs, presumed to be affected by variation of tissue structures over different organs and functional changes of the PM with years of peritoneal dialysis.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
가설 설정
(a) Concentrations of NaFluo and glucose during PET. (b) Concentration profiles of solutes with time in logarithmic scale.
The concentrations of both NaFluo and fluorescein dextrans were 4 mg/ml. We hypothesized that the pore size of the agarose gel would affect the diffusion of molecules. The concentration of agarose was controlled to make different pore sizes of agarose gel.
제안 방법
5% gel has been reported to be 450 nm [16]. All measured diffusion coefficients were normalized with respect to the diffusion coefficient of NaFluo in solution using the same optical-fiber tip to correct the geometrical variation of the illumination volume at the tip of the fiber.
In this study we developed a fiber-optic-based FRAP (f-FRAP) apparatus to measure site-specific and time-dependent diffusion of fluorescent solutes in the PM of a living rat, which is not accessible by conventional testing methods. In contrast to existing optical techniques with limited penetration depth of light, light can be delivered through a flexible optical fiber with a micron-sized tip to the deep, confined parts in living mice for FRAP measurements, as demonstrated in previous studies [11, 12].
In this study we developed the f-FRAP system to measure the transmembrane diffusion of solutes at the PM of the rat. We validated the device using in vitro experiments, and it showed reliable performance in the measurement of diffusion coefficients.
This work was supported by the National Research Foundation (NRF) grant funded by the Ministry of Education (NRF-2009-0075194, NRF-2012S1A2A1A01029148), and the Human Resources Development Program (20134010200580) of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) funded by the Ministry of Trade, Industry and Energy, Republic of Korea. The authors thank Kyunghoon Kim and Seongjun Kim for their assistance in measuring the 3D illumination beam profile.
In contrast to existing optical techniques with limited penetration depth of light, light can be delivered through a flexible optical fiber with a micron-sized tip to the deep, confined parts in living mice for FRAP measurements, as demonstrated in previous studies [11, 12]. The proposed f-FRAP method was validated using in vitro tests with solutions and agarose gels mixed with fluorescein or fluorescein dextrans, and applied to the measurement of dynamic diffusion rates of solutes in the PMs on the liver, cecum, and kidney of rats during PD.
Both diffusion and ultrafiltration in the PM can be measured using an optical-fiber tip with axis perpendicular to the flow direction, combined with fluorescent makers with large molecular weights. The proposed method can be used to assess morphological and functional effects in the PM with respect to PD solutions or drugs, in combination with PET and SPA. Future work with fluorescent glucose analogues can provide more precise insight into peritoneal transport, specifically, in an animal model of peritonitis and peritoneal sclerosis.
To determine the feasibility of the proposed technique for investigating tissue structure of the PM, relative diffusion coefficients were also measured using different concentrations (0.5, 2.5, and 4.5%) of agarose gels mixed with NaFluo or fluorescein dextrans (3 and 10 kDa). The concentrations of both NaFluo and fluorescein dextrans were 4 mg/ml.
1(d), which shows the actual excitation field in our photobleaching experiments. To validate the proposed f-FRAP system, relative diffusion coefficients (D/D0) were measured using solutions of fluorescein sodium salt (NaFluo; 376 Da, excitation peak = 460 nm, emission peak = 515 nm, F6377; Sigma-Aldrich, USA) and fluorescein dextrans (3, 10, and 70 kDa, excitation peak = 494 nm, emission peak = 521 nm; Invitrogen, USA) to investigate the effects of the molecular weights of molecules on relative diffusion coefficients. The Stokes-Einstein equation was used to compare the theoretical and experimental values of D/D0 for fluorescein dextran solutions:
In this study we developed the f-FRAP system to measure the transmembrane diffusion of solutes at the PM of the rat. We validated the device using in vitro experiments, and it showed reliable performance in the measurement of diffusion coefficients. We applied the f-FRAP to direct measurement of mass transport in the PMs of rats.
We found that the diffusion characteristics of the PM vary according to both the positions of the PM and time. A future study will examine solute transport characteristics in normal and abnormal PMs. Both diffusion and ultrafiltration in the PM can be measured using an optical-fiber tip with axis perpendicular to the flow direction, combined with fluorescent makers with large molecular weights.
참고문헌 (28)
B. Rippe, "A three-pore model of peritoneal transport," Peritoneal Dialysis International 13 (Suppl 2), S35-S38 (1993).
E. Goffin, "Peritoneal membrane structural and functional changes during peritoneal dialysis," Semin. Dial. 21, 258-265 (2008).
W. Van Biesen, A. Van Der Tol, N. Veys, N. Lameire, and R. Vanholder, "Evaluation of the peritoneal membrane function by three letter word acronyms: PET, PDC, SPA, PD-Adequest, POL: What to do?," Contrib. Nephrol. 150, 37-41 (2006).
M. M. Pannekeet, A. L. Imholz, D. G. Struijk, G. C. Koomen, M. J. Langedijk, N. Schouten, R. de Waart, J. Hiralall, and R. T. Krediet, "The standard peritoneal permeability analysis: a tool for the assessment of peritoneal permeability characteristics in CAPD patients," Kidney Int. 48, 866-875 (1995).
D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, "Mobility measurement by analysis of fluorescence photobleaching recovery kinetics," Biophys. J. 16, 1055-1069 (1976).
J. D. Bryers and F. Drummond, "Local macromolecule diffusion coefficients in structurally non-uniform bacterial biofilms using fluorescence recovery after photobleaching (frap)," Biotechnol. Bioeng. 60, 462-473 (1998).
H. A. Leddy and F. Guilak, "Site-specific moleculars diffusion in articular cartilage measured using fluorescence recovery after photobleaching," Ann. Biomed. Eng. 31, 753-760 (2003).
M. C. Papadopoulos, J. K. Kim, and A. S. Verkman, "Extracellular space diffusion in central nervous system: Anisotropic diffusion measured by elliptical surface photobleaching," Biophys. J. 89, 3660-3668 (2005).
K. H. Lee, S. J. Shin, C. B. Kim, J. K. Kim, Y. W. Cho, B. G. Chung, and S. H. Lee, "Microfluidic synthesis of pure chitosan microfibers for bio-artificial liver chip," Lab. Chip. 10, 1328-1334 (2010).
J. R. Thiagarajah, J. K. Kim, M. Magzoub, and A. S. Verkman, "Slowed diffusion in tumors revealed by microfiberoptic epifluorescence photobleaching," Nat. Meth. 3, 275-280 (2006).
Z. Zador, M. Magzoub, S. Jin, G. T. Manley, M. C. Papadopoulos, and A. S. Verkman, "Microfiberoptic fluorescence photobleaching reveals size-dependent macromolecule diffusion in extracellular space deep in brain," FASEB J. 22, 870-879 (2008).
T. J. Feder, I. Brust-Mascher, J. P. Slattery, B. Baird, and W. W. Webb, "Constrained diffusion or immobile fraction on cell surfaces: A new interpretation," Biophys. J. 70, 2767-2773 (1996).
J. Yguerabide, J. A. Schmidt, and E. E. Yguerabide, "Lateral mobility in membranes as detected by fluorescence recovery after photobleaching," Biophys. J. 40, 69-75 (1982).
B. Rippe and D. Venturoli, "Fluid loss from the peritoneal cavity by back-filtration through the small pores of the three-pore model," Kidney Int. 73, 985-986 (2008).
S. J. Davies, J. Bryan, L. Phillips, and G. I. Russell, "Longitudinal changes in peritoneal kinetics: the effects of peritoneal dialysis and peritonitis," Nephrology Dialysis Transplantation 11, 498-506 (1996).
J. Burkart and J. M. Henrich, "Problems with solute clearance and ultrafiltration in continuous peritoneal dialysis," UpToDate (2013).
W. Smit, N. Schouten, N. van den Berg, M. J. Langedijk, D. G. Struijk, and R. T. Krediet, "Analysis of the prevalence and causes of ultrafiltration failure during long-term peritoneal dialysis: a cross-sectional study," Peritoneal Dialysis International 24, 562-70 (2004).
S. J. Davies, E. A. Brown, N. E. Frandsen, A. S. Rodrigues, A. Rodriguez-Carmona, A. Vychytil, E. Macnamara, A. Ekstrand, A. Tranaeus, and J. C. Filho, "Longitudinal membrane function in functionally anuric patients treated with APD: data from EAPOS on the effects of glucose and icodextrin prescription," Kidney Int. 67, 1609-1615 (2005).
M. F. Flessner and R. L. Dedrick, "Role of the liver in small-solute transport during perit Flessner oneal dialysis," J. Am. Soc. Nephrol. 5, 116-120 (1994).
L. Gotloib, A. Shustak, P. Bar-Sella, and V. Eiali, "Heterogeneous density and ultrastructure of rabbit's peritoneal microvasculature," Int. J. Artif. Organs. 7, 123-125 (1984).
C. Ronco, "The "nearest capillary" hypothesis: A novel approach to peritoneal transport physiology," Perit. Dial. Int. 16, 121-125 (1996).
T. Casalini, M. Salvalaglio, G. Perale, M. Masi, and C. Cavallotti, "Diffusion and aggregation of sodium fluorescein in aqueous solutions," J. Phys. Chem. B. 115, 12896-12904 (2011).
※ AI-Helper는 부적절한 답변을 할 수 있습니다.