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GROWTH, DIFFERENTIATION, AND APOPTOSIS
1Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; and 2Division of Gastroenterology, Departments of Physiology and Clinical Sciences, University of Liverpool, Liverpool, United Kingdom
Submitted 20 July 2006 ; accepted in final form 20 October 2007
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ABSTRACT |
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200 µm2 of gastric surface cells with high laser intensity, causing rapid bleaching of NAD(P)H fluorescence in optically targeted cells. In both Cox-1+/– and Cox-1–/– mice, a similar initial damage area expanded to include bystander epithelial cells over the next 2–5 min, with larger maximal damage noted in Cox-1–/– mice. The maximal damage size seen in Cox-1–/– mice could be reduced by exogenous dimethyl-PGE2. All damaged cells exfoliated, and the underlying epithelium was coincidently repaired over a time interval that was briefer in Cox-1+/– (12 ± 2 min, n = 12) than in Cox-1–/– (24 ± 4 min, n = 14) mice. Directly after damage, pH increased transiently in the juxtamucosal layer (maximal at 3–6 min). A smaller peak pH change was noted in Cox-1–/– mice (
pH = 0.3 ± 0.04) than in Cox-1+/– mice (pH = 0.6 ± 0.2). Recovery to normal surface pH took longer in Cox-1–/– mice (27 ± 5 min) than in Cox-1+/– mice (12 ± 1 min). In conclusion, constitutive loss of Cox-1 leaves the gastric mucosa more prone to damage and slowed repair of microlesions. autofluorescence; photodamage; laser scanning microscopy; two-photon microscopy; reduced nicotinamide adenine dinucleotide; surface pH; cyclooxygenase
The most imperiled component of the gastric mucosa is the layer of epithelial cells directly exposed to the gastric lumen. Injury and repair of this epithelium have been extensively studied in vivo and in vitro (9, 10, 12, 16–18, 25, 26). Animal models are favored for studying gastric repair processes as they can provide responses of physiological significance for understanding similar events in human ulcer disease (26). Gastric injury responses have been studied in mammalian and amphibian (frog) models (11, 15, 27–29). Although these studies of acute damage have helped to resolve early response mechanisms affecting lesion development and repair, they often required the creation of macroscopic and/or pervasive damage to quickly identify regions of injury. Thus, acute damage is often imposed by harsh treatments like pure ethanol, hypertonic saline solution, boiling water, cold probe, or ischemia-reperfusion (15, 17, 18, 29, 33). Conversely, the response to more physiologically relevant or modest stressors [e.g., acetic acid, nonsteroidal anti-inflammatory drugs (NSAIDs), Helicobacter pylori, or stress-inducing procedures like cold restraint] requires longer times to develop small sporadic lesions in which repair processes are difficult to study (19, 27, 28, 34, 37). In models of severe damage, the creation of transmural wounds cross all lines of defense, inflammatory processes are activated, and repair is slow (28). However, superficial injury of the epithelium has been shown to recover more quickly (12, 16–18). Despite the availability of numerous gastric damage models, the field lacks a model in which the early events of damage and repair can be tracked in isolated sites of microscopic, superficial damage.
Much of our knowledge about gastric repair after superficial injury comes from a particularly detailed microscopic examination of the acute response to brief exposure to absolute ethanol (12, 16–18). After virtually the entire surface epithelium had been selectively damaged with this insult, tissue was fixed and examined 30 s to 60 min later. It was observed that the necrotic epithelium rapidly detached, forming a "mucoid cap" over the epithelium composed of mucus, cell debris, plasma, and fibrin. Surviving cells in the gastric pits migrated out to the surface to restitute the epithelium (12). Although the restitution process started within 3 min, it was concluded that epithelial repair could start only after the necrotic cells detached and lifted off the basal lamina (18). Since a higher pH was observed under the mucoid cap (7, 36), it was proposed that one function of the cap was to trap secreted bicarbonate to allow a favorable environment for restitution. Expansion of these findings also included the observation that the exogenous addition of PGE2 reduced the depth of ethanol-induced damage (17).
Prostaglandins confer gastric cytoprotection by preventing cellular necrosis produced by strong and mild irritants (including NSAIDs such as indomethacin) through a mechanism distinct from inhibition of acid secretion (23). In the gastric mucosa, most prostaglandins are generated via the cyclooxygenase-1 (Cox-1) isoform, and only a small part are Cox-2 derived (8, 19, 21, 31–34, 37). Prostaglandins produced by Cox-1 are known to regulate both gastric surface pH and blood flow (3, 4, 31, 34).
Recent developments in in vivo imaging of the gastric mucosa (3–5) have led to the development of a new model of gastric damage that makes possible selective injuries to the surface epithelium of the corpus, real-time observations of expansion and restitution of microscopic lesions, and the ability to simultaneously measure extracellular pH at the gastric surface. Two-photon microscopy imaging is performed using low light levels that are nondestructive to both the fluorophore and cell (38, 39). However, brief exposure to focused higher light intensity can lead to destruction of the light-absorbing molecule (photobleaching), which results in the release of toxic side products that induce cell damage and death. This is the mechanism by which photodynamic therapy can kill cancer cells that have selectively absorbed photosensitizing chemicals (2, 6, 22). Recently, the use of two-photon absorption has been applied to cause focal cell damage (13, 20, 22, 35). Endogenous cytosolic fluorophores, like NAD(P)H, can be visualized by both conventional and two-photon fluorescence imaging, providing insights into cell and tissue bioenergetics (22, 24, 38, 40). Using NAD(P)H as the optical target of laser light to generate a mouse model of superficial gastric damage, we found that resolution of microscopic damage proceeds by a different sequence of events than previously reported and requires, at least in part, Cox-1 activity.
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METHODS |
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3 mo of age. All animals were fed a standard rodent diet and had free access to water. The surgical preparation for these experiments has been previously described (3, 4). Briefly, the stomachs of anesthetized mice (Inactin, Sigma, St. Louis, MO) were exteriorized and everted to expose the gastric mucosa. The mouse was placed supine on the stage of an inverted confocal/two-photon microscope (Zeiss LSM 510 NLO) such that a portion of the exposed mucosa protruded into a perfusion chamber that was warmed to keep the animal's body temperature at 37°C. All experimental procedures were approved by the Animal Care and Use Committee of the University of Cincinnati. Live Tissue Microscopy Time course experiments were performed to image the gastric mucosa of the corpus region of the stomach using a Zeiss C-Apo x40 objective. At each time point, a set of four images was collected as described below, with time points spaced by variable intervals of 30 s to 3 min. The mucosal surface was superfused (0.2 ml/min, KD Scientific push/pull syringe pump) with a pH 3 slightly buffered solution containing 150 mM NaCl, 4 mM Homopipes (Research Organics, Cleveland, OH), and 10 µM Cl-NERF as the pH indicator (Invitrogen) to report extracellular pH at the gastric surface, as previously described (3–5). Cl-NERF was alternately excited at 514 and 458 nm, and confocal fluorescence emission (>530 nm) images were obtained; 514-to-458-nm ratio images were calibrated to pH as previously described (3, 4). To image autofluorescence [putative NAD(P)H], tissue was excited at 710 nm with a femtosecond-pulsed titanium sapphire (Ti-Sa) laser for two-photon excitation (Mai Tai, Spectra-Physics) using 60- to 70-mW light input to the scan head. NAD(P)H fluorescence was collected at 390–485 nm. Confocal images of 710-nm reflectance were collected simultaneously to landmark cellular structures (5).
Two-Photon Microlesions A small rectangular region of the gastric surface (a 150- to 200-µm2 area placed initially over 3–5 cells) was repetitively scanned with 300–340 mW of 710-nm light into the scan head for 5–10 s. The light dose at the surface was calculated as milliWatts per second per micrometer squared, knowing the power of the illuminating light (300–340 mW), the size of an individual pixel (0.102 µm2/pixel, 0.32-µm x 0.32-µm pixel size), and how long each pixel was illuminated to induce photodamage (128–192 µs total when 1.28-µs laser dwell time/pixel was multiplied by 100–150 scan iterations over each individual pixel) (6).
The complete damage-repair time course was followed for an individual lesion, and the process was then repeated one to eight times per animal using separated lesions. All experimental outcomes were evaluated in 8–10 animals, and there was no trend in the outcomes that suggested that tissue responses were different in initial versus later lesions in the same animal (data not shown).
Cox-1–/– Rescue With Dimethyl-PGE2 To ask if exogenous PGE2 rescued the Cox-1–/– phenotype, tissue was first measured during a damage-repair cycle according to our routine protocol, and 0.1 mg/kg 16,16-dimethyl-PGE2 (dmPGE2; Sigma) was then added topically to the surgically exposed gastric mucosal surface for 5 min. Tissue superfusion was then resumed with solution lacking dmPGE2, and a second round of damage and repair was measured in a region of the corpus distant from the site of the first damage. dmPGE2 was solubilized in 95% ethanol and then diluted to a working concentration in saline (final ethanol: <2%).
Image Analysis All image analysis was performed using Metamorph software (Universal Imaging, West Chester, PA). Using ratio images of Cl-NERF fluorescence acquired during the time course of damage and repair, extracellular pH was measured in a square region (16–27 µm2) adjacent to the site of damage and routinely within 20-µm distance from the gastric surface. The distance from surface varied modestly (up to 50 µm when tissue was very mobile) during measurements between images due to the gradual expansion of the wound and detachment of injured cells.
To measure the extent of damage, the area of surface epithelial cells that lost NAD(P)H autofluorescence was measured over time. Since loss of NAD(P)H resulted in a dark region that could be devoid of any material, simultaneously collected confocal reflectance images were used to identify regions retaining cellular material. A binary (inverted) mask of autofluorescence was used to identify regions with viable cells and was compared pixel by pixel with the reflectance image. Multiplying these two images on a pixel-by-pixel basis allowed the definition of damaged cells as the area lacking NAD(P)H but still attached and continuous with the epithelial layer. A second independent assay of cell viability was also used to quantify the damage area. Damaged cells became permeable toward the Cl-NERF dye in the perfusate and could be quantified by measuring the area of intracellular Cl-NERF fluorescence.
Statistics Data are presented as means ± SE, and statistical comparisons were made with an unpaired two-tailed Student's t-test, with a P < 0.05 significance.
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RESULTS |
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Imaging Cellular Autofluorescence Using 710-nm excitation, two-photon fluorescence was observed at 390- to 485-nm emission. Similar to other laboratories who have imaged NAD(P)H (peak 465-nm emission), fluorescence was observed in the cytosol (Fig. 1A) (13, 22, 24). Our working hypothesis is that the fluorophore we observe is NAD(P)H, but for current purposes the important observation is of a cytoplasmic fluorescent molecule that can act as a substrate for two-photon light absorption.
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600-fold compared with settings used for autofluorescence imaging. Completing this high light exposure required 5–10 s and caused a prompt decrease in cytoplasmic autofluorescence in the targeted region (see Supplemental Fig. 1 and Fig. 1).1 In nine experiments, within 5–8 s after repetitive scanning, we observed that autofluorescence in the targeted region decreased to 38 ± 8% of the starting autofluorescence. In contrast, adjacent areas in the same microscope field that were subjected to only low power laser scanning had 103 ± 1% autofluorescence over the same time course and 87 ± 7% of the starting autofluorescence even after 1 h of imaging. In the adjacent areas, there was no evidence of photodamage to tissue. The loss of autofluorescence in the targeted region of the gastric surface was sustained in the targeted region (Fig. 1D). The decreased fluorescence preceded a notable deterioration of cell morphology. When the focal plane was favorable for observation, intracellular changes of morphology were noticed (data not shown) that resembled necrosis (the cell polygonal contour became oval while loosening contact with neighboring cells, vacuolation of the cytoplasm). In some cases, cells in targeted regions also showed spherical cavitation lesions (likely caused by plasma formation from high laser exposure) and increased fluorescence of cell membranes (likely from oxidized membrane lipids from high laser exposure). Injury and Restitution of Surface Epithelium As shown in Fig. 1, the progression of injury could be qualitatively monitored on combined reflectance (red) and autofluorescence (green) images, and the same progression of events occurred for both Cox-1+/– (Fig. 1, A–H) and Cox-1–/– (Fig. 1, I–P) mice. Healthy surface epithelial cells are evident as a yellow overlay, indicating an autofluorescent and reflective cytoplasm (arrow, Fig. 1, A and I). Nuclei are not autofluorescent and were therefore visible in reflectance images only. Immediately after high power laser scanning, the targeted cytoplasm loses autofluorescence but is still visible in reflectance (arrow, Fig. 1, B and J).
Interestingly, we observed that the wound expanded beyond the region directly exposed to light. Over time, adjacent cells lose cytoplasmic autofluorescence and were visible in reflectance only (Fig. 1, C, D, K, and L). Associated with changing volume and shape of deteriorating cells, distances between cells changed as stretching, shrinking, and detaching occurred. Injured cells lacking autofluorescence gradually shed and overlayed the surface, forming a mucoid cap that appeared only in reflectance (double arrow, Fig. 1, E and M).
In contrast to observations after damage to the entire surface epithelium (18), at the time when injured cell detachment completed, the underlying epithelium was left apparently intact with no evident discontinuity in the cell layer by either reflectance or autofluorescence measures (Fig. 1, F–H and N–P). These initial qualitative observations were evaluated quantitatively, as described below.
Measuring the Size of Damage As described in METHODS, we simultaneously collect both autofluorescence and confocal reflectance images of the gastric mucosa at each time point. The autofluorescence image includes all cell types that contained the responsive fluorophore (viable cells), as well as dark regions that contained dead or dying cells, nuclei, and noncellular regions (Fig. 2, A and A '). The reflectance image simply reports all structures (cells, mucus, and debris) that reflected 710-nm light (Fig. 2B). An overlay of autofluorescence and reflectance images illustrating the different elements reported in these two modalities is shown in Fig. 2C. Observations made in Fig. 2 suggest that dead cells can be observed as regions lacking autofluorescence but still being reflective. Regions thus defined can include more than unhealthy cells (e.g., it also includes nuclei and mucus/debris), so identifying dead cells by this protocol also required subjective analysis of images to identify regions that were within the epithelial layer adjacent to the original site of photodamage. In this way, measurement of noncellular (but reflective) areas was excluded. To perform this analysis, a binary mask created from the autofluorescence image (to identify regions either with or without fluorescence; Fig. 2D) was multiplied by the reflectance image (Fig. 2E). The size of damage was measured on the resultant image by drawing a region of interest around the reflective and nonautofluorescent area adjacent to the optically targeted region. The same region was independently verified on the original autofluorescence images and also sporadically on combined reflectance and autofluorescence images.
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233 µm2) and Cox-1+/– mice (186 µm2) were indistinguishable and similar to the area of high intensity light that was scanned across the tissue (150–200 µm2).
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A second independent measure of cell damage was also used to quantify the damage area. As described in METHODS, we simultaneously measured extracellular Cl-NERF fluorescence in the juxtamucosal spaces. This led to the unexpected observation that the damaged cells that lost autofluorescence also became permeable toward the Cl-NERF dye in the perfusate. This provided an independent assay of cell viability compared with loss of autofluorescence. We compared kinetics of Cl-NERF uptake and loss of autofluorescence in cells adjacent to the photodamaged area using images acquired sequentially at intervals of 2 s (Fig. 4). As shown in three different cells, NAD(P)H autofluorescence decreased gradually over 3 min (not instantaneously, as occurs in directly photodamaged cells; Supplemental Fig. 1), and the cellular uptake of Cl-NERF increased over approximately the same time interval. Based on the use of Cl-NERF uptake as an independent measure of cell injury, the area of damage assayed as Cl-NERF-positive cells was compared versus the NAD(P)H/reflectance measure. In 10 experiments, the damaged area reported by intracellular Cl-NERF at 5 min after photodamage was 133 ± 14% of the area of decreased NAD(P)H autofluorescence. The Cl-NERF assay was qualitatively similar but is predicted to lead to overestimation caused by the inclusion of dye-filled pits and the uncertain boundary when the edge of tissue merges with exfoliating cells.
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Time to Exfoliate Injured epithelial cells were gradually pushed from the surface and trapped in the mucoid layer, while the area of damage within the epithelium correspondingly decreased. To quantify the time needed to undergo repair, we selected the time point at which the damage area shrank to the size of the initial damage. Using this time point, the compiled results shown in Table 1 showed that exfoliation and subsequent restitution took significantly longer for Cox-1–/– mice versus Cox-1+/– mice.
Extracellular pH at the Site of Damage It has been shown that alkali egress into the lumen occurs in response to global gastric damage (1, 11, 29, 30). We sought to test if this was also observed in response to microscopic lesions. Figure 5 shows ratio images of extracellular Cl-NERF adjacent to the gastric surface from a representative experiment. A region of increased pH transiently appeared over the site of damage created by the two-photon laser and spread over the surrounding area as the wound progressed. Figure 6 shows representative time courses of gastric surface pH measurements during the response to photodamage in Cox-1+/– (A) and Cox-1–/– (B) mice. Results are compiled in Table 1, which showed that compared with Cox-1+/– mice, the absence of Cox-1 was associated with 1) initial surface pH values that were significantly lower, 2) maximal pH increases that were significantly smaller after damage, and 3) a slower recovery to the starting surface pH after damage.
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DISCUSSION |
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In the three- to five-cell area targeted for high-power laser scanning, endogenous autofluorescence abruptly decreased and stayed low over time. The mechanism of photodamage by two-photon light absorption was not studied in this initial work, where we focused on evaluating the tissue repair response. However, based on studies of cellular mechanisms of photodynamic therapy, cell death is potentially via absorption of light energy by cytosolic and mitochondrial NAD(P)H, consequent disruption of membranes, and cytotoxicity by reactive oxygen species (ROS) (35). In photodynamic therapy, necrosis occurs when membranes are affected first, and apoptosis when mitochondria are affected first (followed by rapid ROS formation) (2, 6, 22). Our variable observation (data not shown) of cavitation vacuoles and induced membrane fluorescence suggest that other effects of high laser light power could also play a role.
Ito and Lacy (12) used a pervasive superficial ethanol-injury model, and some of the stages and elements of expansion and repair of superficial injury that they describe are also present in our study of isolated superficial microlesions. In both models, cells that become compromised (e.g., lose autofluorescence and become permeable to Cl-NERF) will exfoliate, and the remaining viable cells then restore the epithelial layer (12, 16–18). Similar to Ito and Lacy's study (12), we found a mucoid cap also formed over lesions, affecting a small number of cells. However, our model reveals new facets that could not be discovered previously. Initially during injury, cells keep the same position within the surface monolayer, presumably because tight junctions and the basolateral membrane of affected mucous cells remain intact in the damage expansion phase. Damage then expands to surrounding cells that also exfoliate. Exfoliation occurs in very tight time synchrony with restoration of the epithelial layer and somewhat less tightly with restoration of normal surface pH control. Most importantly, repair of the epithelial layer started while only a few injured cells lifted off, not after basal lamina was totally denuded, as in Ito and Lacy's model.
Movement of epithelial cells adjacent to the damaged cells was noticed over time (data not shown), eventually helping to extrude them, but whether these adjacent cells filled the space left by the exfoliated cells or whether cells came from another source, deeper inside glands or from the lamina propria, could not be determined in our analyses because the imaging plane does not image all cells surrounding the damage, and extensive tissue motion compromises analysis of individual cells.
The disruption of the Cox-1 gene slowed down the process of repair/restitution. We chose to compare Cox-1+/– with Cox-1–/– mice so that only one allele was different between animals, because our prior work showed that there is no strong compensatory upregulation of Cox-2 in Cox-1–/– mice and because the physiological response after exogenous prostaglandin (at least in terms of surface pH control) was equivalent among Cox-1+/+, Cox-1+/–, and Cox-1–/– mice (4). We observed that exfoliation took significantly longer for Cox-1–/– tissue compared with Cox-1+/– tissue and that the maximal size of damage was larger in Cox-1–/– mice, although the initial size of damage was similar. Our data support that Cox-1 mediates increased resistance of the mucosa to damage. Cox-1-derived prostaglandins may protect the mucosa by suppressing the release of proulcerogenic factors (nitric oxide, TNF-
, and platelet-activating factor). Among other potential regulatory effects of Cox-1, inhibition of bicarbonate secretion as a consequence of Cox-1 elimination could result in increased gastric damage or inhibited repair (3, 4). When we transiently exposed the gastric mucosa of Cox-1–/– animals to exogenous PGE2, we saw that surface pH values were significantly increased before and after damage and that the maximal damage size was smaller. Results suggest that Cox-1-derived PGE2 is a potential mediator of gastric protection against the early events that lead to damage expansion.
After the acute application of mild irritants to the stomach, Takeuchi and colleagues (14, 31–34) observed that the decrease in acid secretion and increase in gastric mucosal blood flow was attenuated under the administration of indomethacin and concluded that Cox-1 is responsible for maintaining the mucosal integrity after barrier disruption. Alternatively, Wallace and colleagues (8, 37) demonstrated that only modest damage was observed in response to selective inhibition of a single Cox isoform in a model of prolonged NSAID-induced gastric injury, whereas inhibition of both isoforms produced more extensive damage. The basis for these differences among damage models remains speculative, but in all cases (including the microlesion photodamage model) a protective role of Cox-1 was identified. It is important to recognize that the source of the transient pH increase after photodamage is unknown. Results suggest that Cox-1 is likely to regulate the process, at least in part, and that a known activator of gastric bicarbonate secretion (PGE2) is at least competent to alter the surface pH response after injury. At this point, it cannot accurately be determined whether the pH increase is due to a decrease in acid secretion, an increase in bicarbonate secretion/leak, or a combination of both.
In the pervasive superficial damage models, a mucoid cap (containing exfoliated cells) and surface pH increases were observed soon after damage (8, 12, 16–18, 36). For this reason, the mucoid cap was proposed as a structure that acted to hold secreted bicarbonate at the surface (36). However, we observed that the surface pH increase occurs prior to mucoid cap formation (i.e., cell exfoliation). Thus, our results demonstrate that the mucoid cap is not required to generate a region of raised pH over the site of damage. The simplest model consistent with all observations is that the increased pH is a function of the damaged or denuded epithelium and not the presence of an overlying mucoid cap. It remains to be determined whether the loss of prompt epithelial repair that is observed in the absence of Cox-1 has its basis in the loss of surface pH control or another regulatory function of Cox-1.
In summary, our results in a microscopic damage model suggest that the gastric epithelium is capable of rapid repair of the epithelial layer through a process that allows epithelial integrity to be restored concurrent with cell exfoliation. During most of the process of expanding and resolving cell damage, a surge of pH increase is observed over the damage area that does not appear to be a direct consequence of the egress of damaged cells from the epithelial layer.
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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.
1 The online version of this article contains supplemental data. 
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) or Cox-1–/– (
) mice at the indicated time points. The horizontal dashed line identifies the size of initial damage.
