INTRODUCTION
Acute gastric mucosal lesion (AGML) is one of the most common visceral complications early after severe burns. In patients with thermal injury that involves 30% or were of the total body surface area (TBSA), there was a 14% to 25% incidence of clinically evident gastrointestinal complications and 83.5% of the patients had endoscopic evidence of gastrointestinal disease[1]. Although it was reported recently that, burn-induced stress ulcer occurred less frequently wit h the advances of intensive care supports, AGML still caused a high mortality when complicated with severe bleeding[2], which was recognized as a potentially life- threatening event in such critically ill patients[3,4]. Therefore, such gastrointestinal complications after cutaneous thermal burn remain a problem of great interest and importance.
Several hypotheses have been proposed to explain the mechanism of burn-induced gastric mucosal injury, but no single factor appears to be invariably capable of producing lesions of the gastric mucosa[5]. Traditionally, increased gastric acid production has been long considered as one part of the stress response and the main contributor to the pathogenesis of AGML after severe burns[6]. Consequently, much attention has been paid to acid-neutralizing and/or in hibiting agents in the prevention and treatment of burn-induced gastrointestinal complications. In recent years, however, it is increasingly and widely assumed that tissue ischemia resulting from hypoperfusion is the initial and principal factor, which may trigger re-perfusion injury, for the AGML formation[7]. Meanwhile, the necessity and rationality of AGML prophylaxis by using acid-neu tralizing and/or inhibiting agents have also been challenged[8,9].
Gastric acid secretion is an active metabolic process with energy consumption, which requires sustained and adequate blood supply[8]. It has well been documented that the splanchnic circulation is the first to be reduced in critical illness and the gut is one of the first organs to have the adequacy of its tissue oxygenation compromised in shock[10,11]. We therefore presumed that the gastric acid production in burn shock period might be reduced, which is contrary to what we have thought of before but remains lack of direct evidence.
With this background, the present study is conducted to serially determine the gastric acid production, and the changes of blood flow and energy charge of the gastric mucosa during burn shock period, in order to elucidate the characteristics of gastric acid production in early postburn stage and their mechanisms, as well as to provide useful information for the AGML prophylaxis at clinical settings.
MATERIALS AND METHODS
Animals
Healthy adult Wistar rats of either sex, weighing 220 g ± 30 g, were employed in the study. They were housed in individual metabolic cages in a temperature conditioned room (22 °C-24 °C) with a 12 h light-dark cycle, allowed access to standard rat chow (provided by experimental animal center, Third Military Medical University) and water ad libium, and acc limatized to the surroundings for 7 days prior to the experiments.
Burn injury and resuscitation
Animals were fasted for 12 h before burn injury, and during 48 h postburn period they were allowed water ad libitum. After induction of anesthesia with 1% pentobarbital sodium ( 30 mg/kg, ip ), dorsal hair was shaved, and animals were placed in a wooden template designed to expose 30% of the total body surface area (TBSA), and then immersed in water at 92 °C for 20 seconds, which results in a clearly demarcated full-thickness burn. One hour after burn injury, the animals were resuscitated with 10 mL of warm 0.9% NaCl (normal saline solution, 37 °C) given by intraperitoneal injection. Control animals were similarly anesthetized, shaved and resuscitated but not burned.
The animals burned were randomly divided into five groups for the different measurements and assays that were performed 3, 6, 12, 24 and 48 h postburn ( PBH3, PBH6, PBH12, PBH24 and PBH48).
Measurement of gastric acid production
Three hours prior to each timepoint, animals were anaesthetized, laparotomized, and then the pylorus were ligated. After sacrificed by decapitation at each tim epoint, rats were re-laparotomized to obtain the gastric juice. The pH and volume of each collection were recorded and by using a microtitrator its hydrogen ion concentration was measured by titration with 0.02 mol/L sodium hydroxide to an endpoint indicated by phenolphthalein. The total acidity and total acid output of each gastric juice collection were calculated.
Determination of gastric mucosal blood flow
GMBF was determined as previously described[12]. Radioactive biomicrospheres were prepared with toad red blood cells labeled with 99mTc. At each timepoint, anesthetized animals underwent cannulation of right carotid artery for the injection of radioactive microspheres with PE-50 polyethylene tubing (inside diameter 0.58 mm and outside 0.97 mm). The catheter was care fully advanced into the left ventricle, as confirmed by the ventricular pressure curves monitored with a four-channel physiological recorder. Another catheter for drawing a reference blood sample was introduced into the aorta abdominalis via left femoral artery. The prepared suspension of radioactive microspheres was mixed vigorously for at least 2 min before each injection. Then 0.2 mL of the suspension in an injection syringe (approximately 1.5-2.0 × 105 microspheres) was counted for radioactivity by a γ-scintillation counter before being slowly and uniformly injected into the left ventricle during a 30sec period and the infusion tube was flushed with 0.2 mL heparinized saline solution. The injection syringe was rinsed five times with saline solution into a counting tube for measurement of residual radioactivity in the syringe. Thus net radioactivity injected into the animal was the original minus residual radioactivity. Withdrawal of the reference blood sample, having st arted 20sec before the microsphere injection, was performed by a syringe pump at a constant rate of 0.4 mL/min for 90sec. After withdrawal of the reference blood, animals were killed with an overdose of sodium pentobarbital. The gastric mucosa was sampled, weighed and then counted in γ-scintillation counter. The GMBL was calculated by the following equation and expressed as “mL/min·g tissue”[13]:
Biochemical assays of gastric mucosal energy charge
At each timepoint, the glandular mucosa of stomach was sampled by scraping with razor and stored in liquid nitrogen. On determination, adenine nucleotides were assayed as previously reported with some modifications[14]. Briefly, the sample was powdered in a liquid nitrogen bath and then weighed and homogenized in 20 volume of 10% perchloric acid for deproteinization. The homogenate was centrifuged for 30 min at 12000 × g. The pH of the resulting supernatent was adjusted to 7.0-7.6 with 5 mol K2CO3/L. Then another centrifugation was performed and the supernatent was used to assay for adenine nucleotides by using high-per formance liquid chromatography with a reverse-phase column at a flow rate of 1 mL/min with a buffer of 0.1 mol PBS/L. The ATP, ADP and AMP con centrations in gastric mucosa were then obtained from the eluant fractions. The adenylate energy charge was calculated according to the following equation:
Energy charge = (ATP + 0.5 × ADP) / (ATP + ADP + AMP)
Statistical analysis
Data are expressed as mean ± SE. Experi mental results were analyzed by analysis of variance and t tests for multiple comparisons. P values less than 0.05 were considered to be statistically significant.
DISCUSSION
It has been long considered that certain amount of hydrogenion existing in gastric lumen is the prerequisite for the AGML formation[15,16]. However, the roles of gastric acid in the pathogenesis of AGML have not been fully elucidated so far. In a clinical study, Pruitt et al[6] noted that the mean outputs of total gastric acid in burn patients with normal mucosa, AGML, and AGML with complications (such as bleeding and perforation) were 1.42, 3.32, and 5.37 mmol/h respectively. Lucas et al[17] also found an increase in gastric acid production was positively related the severity of AGML in traumatic patients observed with endoscopy. Interventions designed to decrease gastric acid production, such as vagotomy or administration of aluminum hydroxide or anticholinergic drugs, have all been shown to decrease the incidence of ulcers. In the cace of a burn injury or other severe injury, the presence of acid even at subnormal levels may be sufficient to produce gastrointestinal complications. However, in humans and experimental animals there has not been a consistent association of acute gastric ulcerations after burn injury with hypersecretion of gastric acid[18].
The early phase after severe burn is the critical period for AGML formation. It was reported that the incidence of AMGL within 72 h reached as high as 76%[1,19]. Therefore, investigating the changes of gastric acid production in shock period is both theoretically and practically of importance for the further understanding of the pathogenesis of AGML and, on this basis, improving its treatment and prophylaxis. In present study, we showed that gastric juice volume, total acidity and acid output in burned rats during the early phase of severe burns were significantly decreased compared with unburned controls, with the lowest at 12th h and persisting at lower levels until 48 h postburn. In particular, the total acid outputs at the 6th, 12th, and 24th h in burn animals corresponded only to 1/9, 1/63, and 1/2 of those in control rats. These results indicate that in burn shock period the gastric acid secretion is markedly inhibited.
The production of gastric acid is an active metabolic process with energy consumption, requiring sustained and adequate blood supply[8]. It is well known that during the period of hypovolemic shock such as that induced by burn, blood is preferentially shunted to the “vital” organs, such as the brain and heart, at expense of the splanchnic circulation, causing a sharp reduce of blood flow to the gastrointestinal tract[20]. Therefore, ischemia may be a major factor in the development of AGML after shock and injury. In a report by Horton, blood flow to the small intestine and stomach decreased significantly 5 h postburn, but flow returned to the normal level 24 h after burn only in the small intestine, but not in the stomach[18]. In this murine burn model, we noted that there was a significantly decrease in GMBF and EC with a trend consistent with the serious inhibition of gastric acid production after severe burns. Thus, the decreased acid production might be resulted from ischemia and hypoxia of the gastric mucosa that are metabolically unable to produce normal quantities of acid. Furthermore, while being a causative factor for the AGML formation, gastric acid is also one of the most important barriers against invading pathogenic miero-organisms[21]. Gastric pH < 3.5 is usually bactericidal for most species[22]. In this sense, the inhibition of acid secretion in early postburn period is the manifestations of both local impairment of gastric mucosal function, as well as the dysfunction of host defense mechanism as a whole.
Prophylaxis for stress ulceration continues to be an important part of the management of critically burned patients. However, controversy remains regarding the necessity and rationality of the regimen used[23,24]. Available options at present include antacids, various H2-receptor antagonists, prostaglandins, proton pump inhibitors, and sucralfate, nearly all of which exert their pharmacological actions, to more or less extent, through the acid-neutralizing and/or inhibiting mechanism[25]. In fact, postburn acute mucosal lesions occurred not only in the gastric mucosa, but also in some organs that cannot produce acid such as small and large intestines, and even the gallbladder mucosa[26]. In patients without titratable gastric acid, diffuse erosive gastritis remains occurring within 72 h of burn injury[18]. It was documented in some reports that the incidence of pathological changes in gastric mucosa was not alleviated by using antacids in clinical settings[27], but could be prevented or reduced by satisfactory resuscitation and advanced intensive care support, even in a condition lack of anti acid prophylaxis[28,29]. In burned rats, Skolleborg et al[5] found with postburn fluid resuscitation sufficient to maintain aortic blood presure, gastric mucosal erosions were prevented even when gastric pH was at 1.0. All of these, combined with our results, show in different aspects that gastric acid is not a leading and crucial, but an aggravating factor that functions on the basis of ischemic impairments, for the AGML formation. Whereas, the present AGML prophylaxis mainly by the use of anti-and neutralizing acid drugs is not only making the mucosa more susceptible to acid injury[30], but also resulting in numerous side-effects including breaking the defensive barrier of gastric acid, which may lead to the colonization and translocation of gut organisms and thus increase the risk of nosocomical infections[25,27,28,31]; therefore, taking effective measures to improve splanchnic blood perfusion as early as possible postburn may be more preferable than the mere blockade of gastric acid production for the AGML prevention and treatment.
