Direct measurement of acid permeation into rat oesophagus

Abstract

Background and aims: The early responses of the oesophageal mucosa to acid perfusion may predict subsequent pathology. Mucosal responses to luminal acid may result either from acid permeating through the mucosa or from other unknown transduction mechanisms. In order to better understand the dynamics of acid permeation into the oesophageal mucosa, we measured interstitial pH (pH(int)) of the oesophageal basal epithelial layer, pre-epithelial layer thickness, and blood flow in rats in vivo during luminal acid challenge. A novel confocal microscopic technique was used in vitro to measure pH(int) from defined cellular sites in response to luminal and basolateral acidification.

Methods: 5-(and-6)-Carboxyfluorescein (CF) and carboxy-seminapthorhodofluor-1 (SNARF-1) fluorescence was used to measure pH(int) by conventional and confocal microscopy, respectively, in urethane anaesthetised rats. Pre-epithelial layer thickness was measured optically with carbon particles as markers. Blood flow was measured with laser Doppler flowmetry.

Results: Luminal acidification failed to alter pH(int) in vivo and in vitro, but pH(int) was lowered by modest serosal acidification. Pre-epithelial layer thickness and blood flow increased significantly during luminal surface acid perfusion. Indomethacin had no effect on any acid related response.

Conclusion: In this first dynamic measurement of oesophageal acid permeation and pre-epithelial layer thickness, pH(int) was preserved in spite of high luminal acidity by two complementary techniques. Despite the apparent permeability barrier to acid permeation, oesophageal blood flow and thickness responded to luminal acid perfusion.

Figure 1

In vitro calibration curve of 5-(and-b-) carboxyfluorescein (CF) fluorescence. The curve was generated by loading excised oesophageal mucosa with CF, as described in materials and methods.

Figure 2

Optical measurement of oesophageal pre-epithelial layer thickness using carbon particles. A light rod was used to illuminate the mucosal surface at an acute angle in order to visualise the surface detail of the mucosa. Arrow indicates carbon particles on the luminal pre-epithelial layer surface. In (A), the focal plane is at the pre-epithelial layer surface, at the level of the carbon particles. Note the cluster of carbon particles denoted by the white arrow. (B) The focal plane has been lowered to that of the mucosal surface. Note that the carbon particles cluster is out of focus whereas the mucosal surface is in focus. The Z axis travel of the microscope between the two focal planes is considered to be equivalent to the thickness of the overlying pre-epithelial layer.

Figure 3

Oesophageal permeability to carboxy-seminapthorhodofluor-1 (SNARF-1). Confocal images of excised oesophagus in vitro depicted in the X-Z focal plane, for simultaneous imaging along the lumen to interstitium axis, were used to measure the depth of SNARF-1 permeation. All images were taken at 640 nm and 580 nm emission from the same tissue location at different times. (A) Image taken without SNARF-1. (b) Ten minutes’ exposure to luminally perfused SNARF-1. Note the white staining of the superficial cells but absence of deeper penetration of the dye. (c) Ten minutes’ exposure to luminally and serosally perfused SNARF-1. Note grey fluorescence of the lower pre-epithelial layers, and absence of penetration into the central pre-epithelial layer of the epithelium (black central band). (D) During washout with saline (no fluorescence in perfusate), SNARF-1 was retained in both fluorescent pre-epithelial layers.

Figure 4

X-Y plane confocal images of oesophageal mucosa following perfusion with carboxy-seminapthorhodofluor-1 (SNARF-1). Images were taken from a single area of the mucosa taken at different focal planes along the Z axis. A copy of fig 3C▶ is included to orient the subsequent images. For image (A), the focal plane was at the epithelial surface (stratum corneum); for (B), the stratum spinosum; for (C), the basal cell pre-epithelial layer; and in (D), the subepithelial connective tissue. Note that SNARF-1 fluorescence, which appears white, was taken up by the flattened polygonal surface cells, indicating that these cells are non-viable, and also permeated the interstitial space in the basal cell and subepithelial pre-epithelial layers, as seen by a honeycomb-like pattern in the left potion of (C) (arrow), but did not permeate the stratum spinosum (B).

Figure 5

Interstitial pH (pH_int) of the basal pre-epithelial layer of rat oesophagus measured with carboxy-seminapthorhodofluor-1 (SNARF-1). X-Z images were taken from a single tissue location. Colours in the images correspond to pH (green-acid; red-alkaline). The arrow indicates interstitial fluorescence in the basal pre-epithelial layer from which pH_int was measured. Top: (A) With basolateral and apical perfusates pH=7.4, the pH_int of the basal pre-epithelial layer is alkaline (yellow); (B) acidifying the luminal perfusate to pH 1.5 did not affect pH_int of the basal pre-epithelial layer; (C) acidifying the basal pre-epithelial layer to pH 6.5 acidified the basal pre-epithelial layer, as manifest by the green fluorescence; (D) pH_int of the basal pre-epithelial layer recovered after the pH of the basolateral perfusate was changed to pH 7.4, despite the apical perfusate pH of 1.5. Bottom: (A) time course of pH_int for one of the experiments. AP, pH of apical; BL, pH of basolateral perfusions. (B) Data summary (n=6). **p<0.01 versus perfusion with AP/BL pH 7.4/7.4; †p<0.01 versus perfusion with pH 1.5/7.4.

Figure 6

Microscopic image of oesophageal mucosa demonstrating interstitial extravasation of intravenous 5-(and-6)-carboxyfluorescein (CF). (A) Transillumination demonstrates submucosal blood vessels. (B) Twelve seconds after CF injection intravenously the submucosal vessels fluoresce under 495 nm excitation. The fluorescence appears to be confined to the intravascular space. (C) Nineteen seconds after injection, extravasation is seen in the vicinity of the vessels. (D) Forty eight seconds after injection, the interstitium fluoresces whereas the vessels appear dark, indicating complete extravasation of the circulating dye into the interstitium.

Figure 7

Time course of fluorescence intensity of oesophageal mucosa measured at 530 nm, with excitation alternately at 450 and 495 nm. Note that fluorescence increased and then decreased over time when the tissue was excited at either wavelength. The ratio calculated from the fluorescence measurements was stable, consistent with a stable pH_int. Air injection at 60 minutes lowered the ratio, consistent with the development of interstitial acidosis. CF, 5- (and-6)-carboxyfluorescein.

Figure 8

Effect of perfusion of the luminal surface with acid and with indomethacin intraperitoneally on pH_int of oesophageal mucosa measured in vivo. Note that neither intervention affected pH_int, consistent with failure of acid to permeate from the luminal surface to the vassal pre-epithelial layer, where the dye was present to report pH_int.

Figure 9

Change in the thickness of the pre-epithelial layer overlying the oesophageal mucosa. The pre-epithelial layer was significantly thickened during pH 1 perfusion but was not affected by indomethacin (Indo) intraperitoneally. *p<0.05 versus pH 7 perfusion by ANOVA.

Figure 10

Relative blood flow of oesophageal mucosa. Acid perfusion increased significantly mucosal blood flow but indomethacin (Indo) did not affect this acid induced increase. *p<0.05 versus pH 7 perfusion by ANOVA.