1 Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521; and 2 Division of Physiology, Centre for Vascular Biology and Medicine, King's College London, Guy's Campus, London SE1 1UL, United Kingdom
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ABSTRACT |
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A method involving surgical exposure of the colonic mucosa, fluorescent dye addition, and confocal microscopy has been developed for monitoring colonic crypt function in vivo in mice. Na+ concentration in the extracellular pericryptal space of descending colon was measured using a low-affinity Na+-sensitive fluorescent indicator consisting of an Na+-sensitive chromophore (sodium red) and an Na+-insensitive chromophore (Bodipy-fl) immobilized on 200-nm-diameter polystyrene beads. The Na+ indicator beads accumulated in the pericryptal spaces surrounding the colonic crypts after a 1-h exposure of the colonic luminal surface to the bead suspension. Na+ concentration ([Na+]) in the pericryptal space was 491 ± 62 mM (n = 4). After a 70-min exposure to amiloride (0.25 mM), pericryptal [Na+] was reduced to 152 ± 21 mM. Blockage of the crypt lumen with mineral oil droplets reduced pericryptal [Na+] to 204 ± 44 mM. Exposure of the colonic mucosa to FITC-dextran (4.5 kDa) led to rapid accumulation of the dye into the crypt lumen with a half time of 19.8 ± 1.0 s, which was increased to 77.9 ± 6.0 s after amiloride treatment. These results establish an in vivo fluorescence method to measure colonic crypt function and provide direct evidence for accumulation of a hypertonic absorbate in the pericryptal space of descending colon. The pericryptal space represents the first example of a hypertonic extracellular compartment in mammals that is not created by a countercurrent amplification mechanism.
water transport; fluid absorption; colonic crypt
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INTRODUCTION |
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SEVERAL LINES OF EVIDENCE suggest that fecal dehydration in the colon involves active salt accumulation in a pericryptal space, resulting in water movement from the crypt lumen into the pericryptal space and the generation of a cryptal suction force. Previous studies have shown that colonic absorbates are hypertonic (3, 16) and that descending colonic crypts are surrounded by a myofibroblastic pericryptal sheath (9). It is thought that the pericryptal sheath creates a barrier to Na+ movement resulting in a hypertonic compartment. In isolated colonic mucosa, the Na+-sensitive fluorescent dye sodium green accumulates in the pericryptal space in vitro to an extent related to the accumulation of Na+ in the pericryptal space (10, 11, 15). Pericryptal sheath growth in the descending colon is stimulated by angiotensin II and aldosterone during feeding of a low-Na+ diet. This correlates with an enhanced capacity to absorb fluid against a hydraulic resistance in rats fed a low-Na+ diet (11). Additionally, damage to the pericryptal sheath by ionizing radiation makes it leaky and correlates with a reduced capacity to generate hard feces (20).
The purpose of this study was to develop an in vivo method to measure crypt function to test the hypothesis that pericryptal fluid is hypertonic and that the hypertonicity is created by active salt transport from the crypt lumen. A surgical procedure was developed to expose the colonic mucosa to allow for direct visualization of crypts by fluorescence confocal microscopy. A low-affinity ratiometric Na+-sensitive fluorescent indicator, which was described recently in measurements of salt concentration in the airway surface liquid (6), was introduced into the pericryptal space to report the local Na+ concentration. The indicator has been shown to be insensitive to pH and organic solutes (6). Our results provide compelling evidence for a hypertonic pericryptal space that is created by active fluid transport, representing the first example of a hypertonic extracellular compartment that is not created by a countercurrent amplification.
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MATERIALS AND METHODS |
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In vivo colon preparation.
Mice (CD1 strain, 30-40 g) were anesthetized with pentobarbital
sodium (5 mg/kg ip). An abdominal incision was made, and the descending
colon was elevated above the incision without compromise of the
mesenteric blood supply. The colon was ligated with a silk suture
proximal to the section being investigated. With the use of
microscissors, the colon was opened with a 0.5-cm transverse incision
along the antimesenteric border, and the feces were removed. The cut
edge of the colonic mucosa was immobilized using a forked clamp (Fig.
1). The fork holds the tissue, providing
a flat surface for viewing the mucosa by confocal microscopy, and
permits blood flow via the mesenteric vessels. The mucosal surface was
maintained in buffer (120 mM NaCl, 25 mM NaHCO3, 3.3 mM
KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, and 10 mM glucose,
pH 7.4; 296 mosmol/kgH2O) by continuous-drip perfusion. For
Na+-free experiments, the mucosa was washed thoroughly and
replaced in buffer containing 124 mM choline chloride, 5.8 mM KCl, 0.3 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose, and 20 mM HEPES, pH 7.4. The mouse and clamp were fixed rigidly to an aluminum
plate on the stage of an upright confocal fluorescence microscope. Body temperature was maintained using a heating lamp. The surgical preparation takes ~5 min. At the end of each experiment, the mouse was killed by an overdose of pentobarbital sodium. For blood flow experiments, the carotid artery was cannulated and 0.2 ml of 200-nm rhodamine beads was injected. Rhodamine fluorescence was monitored in
time in the colon by rapid image acquisition (every 0.2 s) after
injection. Time of fluorescence appearance was calculated from the
number of captured images before fluorescence. For oxygen experiments,
a Harvard Apparatus ventilator was used to ventilate mice with 100%
oxygen. All protocols were approved by the University of California,
San Francisco, Committee on Animal Research.
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Fluorescent indicators. Fifty micrograms of sodium red (sample provided by Molecular Probes, Eugene, OR; catalog no. 71351) and 30 µg of Bodipy-fl in 2% methanol-water were added to a 1% (vol/vol) suspension of 200-nm-diameter carboxyl latex beads (Polymer Lab, Amherst, MA) suspended in 4 ml of water. After they were shaken at room temperature for 1 h, the beads were centrifuged and repetitively redispersed by brief sonication in distilled water until no free dye was present in the supernatant. The chromophores remained quantitatively immobilized on the beads during measurements and for >2 mo of storage in water at 4°C in the dark. Before experiments, the beads were centrifuged and resuspended by brief sonication in the appropriate buffer, and the crypts were loaded with the dye with 200 µl of a 10% (vol/vol) solution by incubation for 1 h.
For calibration of the Na+ sensitivity of the indicator, beads were added to solutions containing 0-700 mM Na+ (counterion choline). Sodium red and Bodipy-fl image pairs were obtained for each calibration solution. Na+ indicator calibration was also verified by measurement of ratios on the colonic mucosal surface. The fluorescence ratio remained stable forMicroscopy. Fluorescence microscopy was carried out using a Leitz epifluorescence microscope equipped with a Nipkow wheel coaxial-confocal attachment (Technical Instruments, San Francisco, CA). The colonic crypts were viewed with a Nikon CF Plan ×50 ELWD air objective (NA 0.55, working distance 8.7 mm) or Nikon CF Plan ×20 SLWD (NA 0.35, working distance 20.5 mm) air objective. Confocal fluorescence images were acquired using a cooled charge-coupled device camera (model AT200; Photometrics, Tucson, AZ) with a back-thinned 14-bit detector (model TK 512 CB, Tetronix).
Sodium red and Bodipy-fl fluorescence were observed with standard rhodamine and fluorescein filter sets. For z scanning, the microscope fine focus was driven by a microstepper motor (Compumotor, 12,200 steps/revolution, 1,400 steps/200 µm). Motor control and signal detection were controlled by custom-written LabView software.Na+ ratio analysis. Image pairs of sodium red and Bodipy-fl were acquired (exposure time 1 s) for the same field. Background images were obtained under the same conditions but without loading with the dye. Analysis was performed using PMIS software (Photometrics). Ratio images (red-to-green fluorescence) were obtained by pixel-by-pixel division of background-subtracted images. Background values were <10% of signal. Averaged ratios were obtained by integration of red and green fluorescence intensities over specified regions of interest. Two to three crypts were measured from each set of images.
Dextran uptake. For visualization of crypts, the colonic mucosa was exposed to an isotonic solution containing rhodamine-dextran (mol wt 500,000) for 10 min. After the field of view was reduced to one or a few crypts, a solution containing FITC-dextran (mol wt 10,000, 0.2 mg/ml; Molecular Probes) was added at the surface, and time courses and z scans were carried out during visualization with the fluorescein filter set. Time-course data were normalized to the change in fluorescence over the time course. The z-scan data were normalized to fluorescence intensity at the crypt opening and to that at the base of the crypt.
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RESULTS |
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Na+ sensitivity calibration of
ratiometric indicator.
The Na+ indicator calibration was confirmed by recording
images of the colonic surface immediately after addition of the
indicator suspended in solutions of known Na+
concentrations. The fluorescence ratio vs. Na+ calibration
curve showed increasing red-to-green fluorescence ratio with increasing
Na+ concentration (Fig. 2A;
n = 5), in agreement with results obtained for this
indicator applied to the airway surface liquid (6). Indicator fluorescence ratio was sensitive to changes in
Na+ concentration to 700 mM, and the ratio was not
sensitive to pH in the range 6-8 (not shown). The calibration
curve was used to deduce Na+ concentrations in the
pericryptal space in vivo from measured fluorescence intensities.
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Na+ concentration in the pericryptal
space in vivo and effect of amiloride.
The Na+ indicator beads were loaded into the pericryptal
space by addition of 0.1 ml of a bead suspension in Krebs-Ringer
solution onto the colonic mucosa. After the external indicator was
washed off, confocal images indicated greatest fluorescence in the
pericryptal spaces at a depth of 10-40 µm from the surface (Fig.
3). The fluorescence intensity ratio in
the pericryptal space in mouse descending colon of 13.1 ± 0.8 gave a pericryptal Na+ concentration of 491 ± 62 mM
(n = 4 mice). Addition of amiloride (0.25 mM) to the
luminal bathing solution resulted in a slow decrease in fluorescence
ratio to 6.9 ± 0.9, giving a pericryptal Na+
concentration of 152 ± 21 mM (n = 3) after 70 min
(P = 0.005, Student's unpaired 2-tailed
t-test; Fig. 2B). To confirm that pericryptal
hypertonicity is dependent on active Na+ absorption, the
pericryptal Na+ concentration was measured after
replacement of the bathing solution with Na+-free buffer.
The Na+ concentration in the pericryptal space decreased
over an 80-min time course to 207 ± 26 mM (Fig. 2C).
To look at the blood flow, fluorescent beads (200-nm rhodamine beads)
were injected intravenously. Fluorescence was then followed in colonic
capillaries in cut-and-clamped sections and unclamped sections. There
was no significant difference in the time of fluorescence appearance
(1.40 ± 0.12 and 1.47 ± 0.13 s in unclamped and
clamped sections, respectively), showing that microvascular perfusion
is unimpaired in these experiments. To further confirm that the tissue
was viable, pericryptal Na+ concentration was monitored
while the mouse was ventilated with 100% oxygen. The pericryptal
Na+ concentration in the oxygen-ventilated preparation (504 mM) was not significantly different from that in the normal
preparation. Also there was no evidence of large amounts of mucous
production during the experiments.
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Na+ concentration after treatment with mineral (paraffin) oil. Mineral (paraffin) oil droplets have been previously shown to block fluid uptake into the rat colonic crypts (11). A 1% (vol/vol) emulsion of mineral oil prepared by sonication in PBS containing 0.1 mM deoxycholate was layered on the mucosa for 15 min and then removed by washing with Krebs solution. By bright-field microscopy, it was observed that the crypt lumens were loaded with oil droplets. At 40 min after the oil was applied, the pericryptal Na+ concentration was reduced to 204 ± 44 mM (P = 0.017; Fig. 2D). Thus the principal route for Na+ entry into the pericryptal space is via the crypt lumen, since the oil droplets do not affect the surface mucosa (11, 24).
Time course of dextran uptake. To show that the hypertonic Na+ in the pericryptal space is generating an osmotic pressure gradient that results in fluid uptake into the crypt lumens, we measured FITC-dextran uptake into mouse descending colonic crypts in vivo. A larger osmotic gradient is predicted to increase the rate of FITC-dextran uptake (11, 12).
The uptake of FITC-dextran was measured from the time course of fluorescence change at a fixed distance of 30 µm below the crypt opening at the mucosal surface. The time course in Fig. 4 shows a rapid increase in fluorescence intensity with a half time of 19.8 ± 1.0 s. Treatment with amiloride (0.25 mM) increased the half time of dextran uptake to 77.9 ± 6.0 s (P < 0.01; Fig. 4).
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Concentration polarization of dextran along crypt length.
FITC-dextran accumulates in the crypt lumen as a result of
concentration polarization (12). The accumulation results
from convective uptake of dextran by solvent drag in the mass flow stream entering the crypt lumen. This mass flow is produced by the
suction tension within the crypt lumen resulting from the large osmotic
gradient generated across the crypt wall. Dextran accumulates in the
lumen, because its diffusive mobility is much lower than its convective
mobility. The extent of accumulation is a function of the rates of
dextran diffusion within the crypt lumen and permeation across the
crypt wall, as well as the rate of mass flow. The FITC-dextran
concentration profile along the length of the crypt lumens was obtained
by z scans of fluorescence intensity down the crypt. The
profile (Fig. 5) shows accumulation of
dextran 20-80 µm from the crypt opening above the concentration at the crypt lumen. The accumulation of FITC-dextran is abolished after
amiloride treatment. This Na+ pump-dependent concentration
polarization of FITC-dextran in the crypt lumen demonstrates that the
osmotic gradient across the crypt wall is used to generate a mass fluid
flow into the crypt lumens in vivo.
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DISCUSSION |
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Hypertonic pericryptal spaces in vivo.
The principal finding of this study is that murine descending colonic
crypts in vivo can generate and sustain a very large osmotic gradient
as a result of active transport of Na+ from the crypt lumen
into the pericryptal space. Na+ concentration in the
pericryptal space, as reported by Na+ indicator
fluorescence intensity ratios in the pericryptal spaces, was two- to
threefold higher than in the external bathing solution. The ratiometric
Na+ indicator was sensitive to changes in
Na+ concentration at 500 mM.
Dextran uptake into crypt lumen in vivo. The demonstration that FITC-labeled dextran is accumulated into murine crypts in vivo to a higher concentration in the lumen than in the external solution confirms previous work using rat mucosa in vitro (12). Amiloride is a specific inhibitor of Na+ uptake across the apical border mainly by inhibition of the epithelial Na+ conductance channel and also by inhibition of the Na+/H+ exchanger (2). Amiloride has no direct action on water transport. Hence, the only plausible explanation for the threefold slower rate of dextran accumulation into a crypt with amiloride present is that crypt fluid absorption is dependent on an Na+ pump-driven process. Hence, murine crypts in vivo absorb fluid as a consequence of the hypertonicity generated in the pericryptal spaces.
The large sustained osmotic gradient across the cryptal barrier probably requires a very low osmotic water permeability. Fluid absorption rates in murine colonic mucosa in vivo are ~30 µl · cm ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. Zhenjun Diwu (Molecular Probes) for synthesis of sodium red.
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FOOTNOTES |
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* J. R. Thiagarajah and S. Jayaraman contributed equally to this work.
This work was supported by the Wellcome Trust, a Peter Baker Travelling Award to J. Thiagarajah, National Institutes of Health Grants HL-60288, HL-58198, DK-35124, and DK-43840, and grants from the Cystic Fibrosis Foundation.
Address for reprint requests and other correspondence: R. J. Naftalin, Div. of Physiology, Centre for Vascular Biology and Medicine, King's College London, Guy's Campus, London SE1 1UL, UK (E-mail: richard.naftalin{at}kcl.ac.uk).
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.
Received 17 November 2000; accepted in final form 6 August 2001.
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