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Effect of sucralfate on gastric permeability in an ex vivo model of stress‐related mucosal disease in dogs

Background Sucralfate is a gastroprotectant with no known systemic effects. The efficacy of sucralfate for prevention and treatment of stress‐related mucosal diseases (SRMD) in dogs is unknown. Hypothesis/Objectives To develop a canine ex vivo model of SRMD and to determine the effect of sucralfate on mucosal barrier function in this model. Animals Gastric antral mucosa was collected immediately postmortem from 29 random‐source apparently healthy dogs euthanized at a local animal control facility. Methods Randomized experimental trial. Sucralfate (100 mg/mL) was applied to ex vivo canine gastric mucosa concurrent with and after acid injury. Barrier function was assessed by measurement of transepithelial electrical resistance (TER) and radiolabeled mannitol flux. Results Application of acidified Ringers solution to the mucosal side of gastric antrum caused a reduction in gastric barrier function, and washout of acidified Ringers solution allowed recovery of barrier function (TER: 34.0 ± 2.8% of control at maximum injury, 71.3 ± 5.5% at recovery, P < .001). Sucralfate application at the time of injury or after injury significantly hastened recovery of barrier function (TER: 118.0 ± 15.2% of control at maximum injury, P < .001 and 111.0 ± 15.5% at recovery, P = .35). Conclusions and Clinical Importance Sucralfate appeared effective at restoring defects in gastric barrier function induced by acid and accelerating repair of tissues subjected to acid in this model, suggesting that sucralfate could have utility for the treatment and prevention of SRMD in dogs.

clinically relevant hemorrhage, and this hemorrhage was associated with a 5-times increased risk of death. 6-8 ICU-hospitalized dogs had a similar rate (7%) of clinically relevant hemorrhage associated with 5fold increased risk of death; this included both dogs with melena (consistent with SRMD) and hematochezia, so the prevalence of SRMD cannot be extrapolated from this study. 9 Forty-nine percent of Alaskan sled dogs develop SRMD. 10 There are no models of the condition relevant to dogs.
In people, stress ulcer prophylaxis (SUP) has become the standard of care for prevention of SRMD in high-risk patient populations.
Human ICUs largely prescribe either proton pump inhibitors (PPIs, 66% of SUP protocols) or histamine 2 receptor antagonists (H 2 RAs, 30% of SUP protocols). 11 Acid suppression with omeprazole leads to proliferation of bacteria in the gastric lumen in both species. 12,13 PPI administration in people carries a 3.5-fold risk for the development of Clostridium difficile-associated diarrhea (CDAD), which is independently associated with longer ICU stays and increased mortality. 11,14 The prevalence of infection by pathogenic Clostridia species in dogs receiving PPI treatment is unknown. Overall, a meta-analysis concluded that the evidence for SUP in human ICU patients is limited and further work is needed to determine the role of various SUP protocols on outcome. 15 A recent review of SRMD in dogs recommended standard use of SUP, particularly PPIs, in critically ill dogs. 16 Just as the efficacy of SUP for critically ill dogs has not been established, the adverse effects of acid suppression in critically ill dogs is unknown. There might be as yet unidentified risk factors for SRMD in dogs, as in people, that would be an indication for SUP.
Sucralfate, an alternative prophylactic treatment for SRMD, is a complex of sucrose and aluminum salts. Sucralfate binds to negatively charged subepithelial proteins exposed during mucosal injury, forming a viscous layer that protects the vascular bed and proliferative zone, allowing for epithelial restitution. 17 Sucralfate absorbs and reduces the activity of pepsin, and is cytoprotective as well as antiapoptotic. 18,19 Sucralfate stimulates mucus synthesis and secretion and bicarbonate secretion. 20,21 Compared with H 2 RAs and PPIs, this drug does not increase bacterial colonization and therefore has a lower likelihood of CDAD and is protective against the development of nosocomial pneumonia in people. 14 It was as effective as H 2 RAs for prevention of overt gastric bleeding events in critically ill people with lower rates of ventilator-associated pneumonia and gastric colonization. 22 Sucralfate does not affect CYP450 enzymes, so there is no effect on the therapeutic effect of concurrently administered medications. No SUP protocol has been examined for treatment or prevention of SRMD in dogs.
The objectives of this study were to develop an ex vivo SRMD model of canine gastric mucosa and to examine the effect of sucralfate on mucosal barrier function. Sucralfate was administered concurrently with acid injury and immediately after the injury with the aim of determining its efficacy both as a protective and reparative drug. The antral and pyloric regions of gastric mucosa were used as they are the most frequent sites for gastric ulceration in dogs. 13 The precise age of the dogs was unknown in most cases, but ranged from $8 months to 10 years-of-age. The dogs were typically mixed breed, ranging in size from $10 to 30 kg. All dogs were euthanized with an overdose of sodium pentobarbital. Immediately after euthanasia, the entire antral-pyloric section of the stomach was

| Ussing chambers
Mucosa was bathed on both the mucosal and serosal sides of the tissue mounted on the chambers with 10 mL of oxygenated Ringer's solution maintained at 378C by water-jacketed reservoirs. As previously described, 24 10 mmol/L glucose was added to the serosal bathing solution, which was balanced with the addition of 10 mmol/L mannitol in the mucosal bathing solution. Treatments were applied to individual tissues after a 30-minute incubation period.  Journal of Veterinary Internal Medicine | 671 additional 25 dogs was used in studies to further evaluate this model and as has been previously described. 25  Ringer's solution was titrated to pH 1.2 with hydrochloric acid. Sucralfate was dissolved in dimethyl sulfoxide (DMSO) at 1 g/mL as it was the maximum dose that was soluble; one milliliter was added to the 10-mL chamber to give a final dose of 100 mg/mL, or a total of 1 g.
DMSO was used as the recommended vehicle by the manufacturer.
One gram was selected as it is the recommended dose in vivo. 26 Sucralfate 1 acidified Ringer's solution was applied to the mucosal aspect of the tissue for 45 minutes. After this injury period, acidified Ringer's solution was removed and replaced with neutral Ringer's solution. When acidified Ringer's was removed, sucralfate was added with the neutral Ringer's solution to maintain a 100 mg/mL concentration throughout this experiment. One Ussing chamber was maintained with neutral Ringer's solution and another treated with acidified Ringer's solution without sucralfate as controls. Each of these treatments was applied to the gastric mucosa of 9 dogs. Tissue from each dog was used in both treatment and control groups. Outcome measures were TER, 3 H-mannitol flux, and histological evaluation.

| Stage 2: Sucralfate after acid injury
Acid injury was induced as previously described. Immediately after acid injury (75 minutes), 1 mL of 1 g/mL of sucralfate (sucrose octasulfate) in DMSO was added to neutral Ringer's solution on the mucosal aspect of the tissue to give a final dose of 100 mg/mL. Controls included uninjured control, acid injury control, uninjured control 1 sucralfate, and injured control 1 vehicle (DMSO). Outcome measures were TER, 3 H-mannitol flux, and histological evaluation. These treatments were applied to mucosa from 8 dogs. Tissue from each dog was used in both treatment and control groups.

| Transepithelial resistance
The spontaneous potential difference (PD) was measured with Ringeragar bridges connected to calomel electrodes, and the PD was shortcircuited through silver-silver chloride electrodes with a voltage clamp that corrected for fluid resistance. TER (XÁcm 2 ) was calculated from the spontaneous PD and short circuit current (Isc). If the spontaneous PD was between 21 and 1 mV, tissues were current clamped at 6 100 lA for 5-seconds and the PD was recorded. The Isc and PD were recorded every 15 minutes for 210 minutes. Data were entered into spreadsheets that calculated TER from Isc and PD using Ohm's law.

| 3 H-mannitol flux
As a second indicator of gastric permeability, flux of 3 H-labeled mannitol across the mucosa was measured. Two-hundred molar of 3 H-radiolabeled mannitol was added to the mucosal reservoir. Samples were taken from both the serosal and mucosal reservoirs after 3 minutes to establish baseline radioactivity. As is standardly performed, 24 two 1-hour mucosalto-serosal fluxes were performed by sampling serosal bathing solutions at 1 and 2 hours after addition of radiolabeled mannitol.

| Histological examination
Gastric mucosal samples were taken for each dog before mounting on Ussing chambers. After 210 minutes, the tissues were collected from each treatment group in Carnoy's fixative for 24 hours and transferred to 70% ethanol. Samples were sectioned at 5 mm, stained with hematoxylin and eosin (H&E), and viewed with a light microscope. Additional sections from each treatment was stained with Alcian blue and periodic acid-Schiff (PAS) stain to stain the gastric mucus layer and viewed with a light microscope.

| Statistical analysis
A 2-way repeated measures ANOVA was used to compare TER data. If two groups were compared, a t-test (parametric data) or rank sum test (nonparametric data) was used. If there were more than two groups, a one-way ANOVA was used to analyze prostanoid and flux data; a Kruskal-Wallis ANOVA on ranks was used to analyze nonparametric prostanoid and flux data. The Tukey's post hoc test was used to detect differences among treatments and time when significance was detected during the initial ANOVA. Histologic data was descriptively reported.
Significance was set at P < .05. Data are represented at means 6 SE.  The effect of sucralfate on uninjured tissue was also examined.
Transepithelial resistance and mannitol flux were not different between control tissue and uninjured control with sucralfate added at 75 minutes (data not shown). In addition, DMSO, the vehicle for sucralfate delivery, was administered after acid injury; acid injury with the addition of DMSO showed similar TER recovery to acid-injured tissues in the absence of DMSO (data not shown).
Histologically, control tissues maintained largely unchanged morphology compared with baseline tissues taken at the time of tissue collection with only occasional apoptotic cells ( Figure 5A,B). However, exposure to HCl induced moderate to marked sloughing of the superficial gastric epithelium with associated thickening of the mucus layer ( Figure 5C). When sucralfate was administered concurrently with acid, little to no tissue sloughing or apoptosis was noted in any samples although the mucus/sucralfate layer was of a similar thickness to the acid-injured control tissue ( Figure 5D).

| Stage 2: Sucralfate after acid injury: Sucralfate enhances recovery when administered after acid injury
To evaluate sucralfate as a treatment for pre-existing alterations in barrier function, sucralfate was applied after the initial acid injury in gastric tissues from n 5 8 dogs. The acid injury was applied as described previously. Immediately after acid injury (at the completion of the 45-minute injury period), sucralfate was applied to the mucosal bathing reservoir to give a concentration of 100 mg/mL in neutral Ringer's solution. In The mechanism by which sucralfate exerted is protective and reparative effects to injured mucosa was not specifically examined in the current study. Histologically, PAS/Alcian blue demonstrated a thick proteoglycan layer in sucralfate treated tissues (data not shown), implying a potential role of sucralfate in increasing mucus production. However, we could not conceive of an accurate method to measure significant differences in the mucus layer. For example, in Figure 3, injury causes an apparent increased thickness of the mucus layer, but the irregular nature of this thickening precluded measuring and statistically analyzing these changes. Instead, we relied on our primary goal of measuring gastric barrier function in treated tissues.
There are several limitations to the use of the Ussing chamber model to study gastric injury and specifically SRMD. It allows only for the study of peracute injury (induced within an hour) because of the viability of canine gastric mucosa ex vivo, so the duration of efficacy of sucralfate could not be examined. Drugs that have a delayed effect on tissues, such as a PPI, would be difficult to investigate without pretreatment of dogs before euthanasia. The Ussing chamber model is not an ideal replication of splanchnic hypoperfusion and does not allow for the interaction of other host factors, such as gastric motility or concurrent medications. However, it has the advantage of allowing for the examination of barrier function in a more comprehensive manner than would be possible with an in vitro or in vivo model. This investigation was completed using tissue from dogs that were previously scheduled for euthanasia and included a variety of breeds and ages. The effect of age, sex, and neuter status did not affect baseline barrier function (data not shown). However, there was a large FIG URE 6 Treatment of acid-injured tissues with sucralfate enhances recovery. A, Acid injury induced a significant decrease in TER compared with uninjured control (1, P 5 .002). When sucralfate was administered after acid injury, TER recovered to a higher level than untreated, injured tissue. Acid-injured tissue treated with sucralfate had a significantly higher TER from 150 minutes until the end of the experiment (*P < .035, n 5 8). B, There is not a significant effect of injury or sucralfate treatment on 3

CONFLICT OF INTEREST DECLARATION
Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
Authors declare no IACUC or other approval was needed.