The most striking result of this study was that the hypothesized cellular changes in the gastrointestinal mucosa after experimental induction of GDV were more profound in the jejunum than the fundus. While a decrease in ATP concentration in the fundus of group 3 was observed, this occurred only after 210 min of dilation and volvulus and was not observed in the dogs in group 2 that underwent 120 min of experimentally induced GDV. If the energy charge is considered, group 3 showed an earlier change with a significant decrease at 120 min in both the fundus and the pylorus. The energy charge for group 2 did not change. This apparent decrease in cellular energy availability did not coincide with any changes in mucosal conductance in the fundus. In contrast, there were profound decreases in jejunal ATP concentration in both groups 2 and 3 after rotation of the stomach, and recovery to baseline concentrations after derotation and perfusion in group 2. The changes in overall energy charge mirrored the changes in ATP concentration. This decrease in jejunal ATP concentration and energy charge did parallel an increase in mucosal conductance in these dogs although, interestingly, despite a recovery in ATP concentration and energy charge in group 2, the mucosal conductance continued to increase.
The ATP was measured in these dogs to assess mucosal cell metabolic status. Under aerobic metabolism, degradation of glucose to carbon dioxide and water is the principal source of ATP used for cellular functions, such as active membrane transport and mitochondrial activity. The ATP is replenished under aerobic conditions by oxidative phosphorylation and conversion of ADP within the mitochondria (16
). This is an efficient process with a maximum of 38 molecules of ATP formed for each molecule of glucose degraded. During organ ischemia, anaerobic glycolysis is activated. This is an inefficient system for the synthesis of ATP (17
) and ATP utilization exceeds production, resulting in decreased intracellular ATP concentrations (18
). With oxygen unavailable, lactic acid accumulates in the cell and the glycolytic pathway is blocked. Depletion of intracellular energy (ATP) and subsequent inhibition of ATP-dependent cellular functions, such as sodium and potassium channels, are implicated in the alterations of cell membrane transport that occur during ischemia. These alterations lead to the failure to maintain ionic gradients and membrane structural integrity (19
Thus, it was hypothesized that gastrointestinal mucosal ischemia and subsequent cellular hypoxia induced by GDV would cause a depletion of cellular ATP, which in turn would lead to alterations in cell membrane transport. It was postulated that this cellular change would be reflected by an increase in cell membrane conductance. Total tissue conductance was measured in this study, which indirectly assesses cell membrane permeability and reflects the contribution of both active and passive ion movements (21
). An increase in total tissue conductance may reflect stimulation of active transport (which would require ATP) by some unknown mechanism during GDV or an increase in passive diffusion, which would reflect deterioration of mucosal cell tight-junctions.
How well the ATP concentration, cellular damage, mucosal permeability, and mucosal conductance parallel is unclear. The ATP concentrations and energy charge in tissues have been proposed as markers of the extent of ischemic injury, as well as indicators of organ recovery, but with equivocal results (14
). In addition, tissue concentrations of ATP and its metabolites do not appear to be an indicator of ischemia duration and do not correspond to morphological changes (13
). The measurement of ATP in this study does not quantify the concentration per cell but is an overall representation of mucosal content. It may reflect a high concentration in a few cells or a low concentration in many cells. Energy charge is used to reflect the overall energy status of the tissue, taking into account the contribution of ADP and AMP (14
). It is obvious that some cells in the jejunum were still capable of ATP synthesis, evidenced by the restoration of baseline ATP concentration and energy charge after derotation and reperfusion. This change may reflect a profound increase in the ATP concentration of a few cells, which possibly have undergone an almost rebound effect, particularly in face of the now aerobic synthesis of ATP from abundant ADP and AMP precursors. Lactic acid concentration or creatine phosphate in the mucosa was not measured in this study but may have lent a perspective of the effect of hypoxia on the mucosal cells. Creatine phosphate has been shown to be an indicator of ischemia in muscular tissues (24
). Serum lactic acid concentrations have been measured in clinical cases of GDV and appear to be increased in dogs with gastric necrosis. However, since serum lactic acid was measured, this may reflect an overall metabolic acidotic state rather than be related to the gastric mucosa specifically (25
The increase in jejunal conductance in both groups 2 and 3 during the first 120 min did parallel a decrease in ATP concentration, which might reflect an unknown stimulation of the active transport mechanisms with concurrent consumption of ATP. Conversely, it could reflect cellular ATP depletion due to ischemic conditions and subsequent membrane dysfunction with uncontrolled passive ion transfer due to tight-junction disruption. After reperfusion, during which the ATP concentration returned to normal in group 2, the conductance remained increased above baseline. When combining these results with the microscopic findings, it would suggest that passive transport is affected and it is more likely that the changes in mucosal conductance reflect a decrease in cell membrane function. Additional samples assessed for unidirectional mannitol flux would have been useful to directly assess mucosal permeability and differentiate the conductance findings (26
); however, we were restricted in the number of samples that could be harvested without interfering with the ongoing in vivo experiment and further sampling.
The fact that the jejunal cellular conductance continued to increase in group 2, despite the restoration of ATP concentrations, may suggest cellular effects from other sources beside energy depletion. Damage from oxygen-derived free radicals produced during reperfusion may be involved (24
). Ongoing cell death after reperfusion has been proposed as a factor in studies evaluating equine intestinal ischemia. In a model of venous strangulating obstruction in the equine jejunum, reperfusion after venous occlusion produced similar, although less severe, changes than prolonged venous occlusion (27
). In a study of no-flow ischemia experimentally induced by 720 degrees of volvulus of the ascending colon in ponies, ATP content of the colonic tissue was reduced by 92% after 120 min of ischemia and recovered to 44% of controls after derotation and 120 min of reperfusion (28
).This result and other results in this study led these investigators to also conclude that much of the damage seen during the reperfusion may be a continuation of injury induced during the ischemic period and not specific to reperfusion per se.
Changes in ADP and AMP during this study were variable and did not follow any patterns that could be associated with ATP concentrations. It might be expected that during ischemia, as ATP is broken down, the concentration of ADP and AMP may increase. However, ADP and AMP could also be utilized. It might also be expected, as ATP increases after reperfusion, that ADP and AMP may decrease as they are used to synthesize ATP. Without an obvious pattern of change and without molecular labeling, it was difficult to interpret whether ADP and AMP concentrations reflected the process of synthesis or breakdown. Energy charge was calculated to account for the contribution of ADP and AMP to the overall energy status of the mucosa. Where there were profound changes in the ATP concentration, such as in the jejunum, energy charge mirrored these changes, as expected (14
); however, where changes were less pronounced, the energy charge was more sensitive, as was observed in the fundus results of group 3.
An unexpected finding was the significant increase in ATP concentration seen in the fundic mucosa in group 1. The reason for this is unclear. This may represent a spurious shift to ATP synthesis in these animals, as there was a slight decrease in ADP and AMP concentrations at this time and energy charge was not affected. Since a substantial amount of blood was extravasated after biopsies, red cell breakdown and absorption of intraluminal ATP by the mucosal cells, if possible, could have been an additional source of ATP and may have elevated the ATP concentration of the gastric mucosa. If in fact the increase seen in group 1 should have been expected in groups 2 and 3, then the overall decrease in ATP concentration seen in group 3 is actually more profound than simply the decrease observed from baseline and a decrease equal to this amount, whatever that may be, may have occurred in group 2.
The experimental model used in this study causes per-acute GDV. The decision to maintain intragastric pressure at 30 mmHg, apply 235 degrees of volvulus, and the duration of the ischemic time periods were based upon previous experiments (6
). Our model most closely resembled that of Davidson et al (7
) and hemodynamic, gross, and microscopic changes in our study were consistent with that study. Although changes in ATP content and cellular conductance were not seen in the fundus, overall energy charge changes were seen and assessment of changes in microscopic features and hemodynamic parameters, support that the experimental model was severe. The microscopic changes in the stomach and jejunum showed edema and congestion primarily in the mucosal layer. Thus, although minimal changes in ATP and mucosal conductance were seen in the gastric mucosa, the authors believe this datum is a true reflection of the changes of this model. It is important to remember that this is a per-acute experimental model. How this per-acute model extends to the clinical disease and the full-thickness integrity loss of the fundus that is seen in some dogs is unknown. The authors believe gastric necrosis to be a different, possibly later phenomenon, and may be related to thromboembolic episodes. In many clinical cases, by the time the jejunum is assessed, the stomach has been decompressed and repositioned, and portal and caval compression have been alleviated. Any abnormal gross changes in the jejunum quickly recover after gastric decompression and repositioning and the authors caution that while the jejunum may appear normal in these cases, it is important to realize that mucosal changes have occurred.
Hemodynamic changes in this model were profound with significant decreases in mean arterial blood pressure and increases in portal pressure during gastric dilation and volvulus. The reason for the decrease in mean arterial blood pressure in group 1 is likely related to anesthesia and since the mean arterial blood pressure rarely went below 60 mmHg in these dogs, they did not benefit from the higher intravenous fluid administration rates that were given to groups 2 and 3. The higher fluid administration rates may have accounted for the significant drop in total protein of groups 2 and 3, although the PCV did not appear diluted. A possible explanation would be protein loss into the gastrointestinal lumen.
The significance of the findings of this study relate to the per-acute period and suggest that changes in jejunal mucosal activity is an important event. While the focus of clinical and experimental research is generally on the stomach and implicate it as the major source of pathophysiologic changes in GDV, changes in the jejunum may play an equal or more significant role. The measurement of cellular ATP concentration in mucosa does not give a clear indication of overall cell function and does not directly relate to changes in conductance.