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Experimental Studies |
1 From the Russell H. Morgan Department of Radiology and Radiological Science, Division of Cardiovascular and Interventional Radiology, Johns Hopkins Medical Institutes, Johns Hopkins Hospital, Blalock 545, 600 N Wolfe St, Baltimore, MD 21287. Received May 8, 2006; revision requested July 6; revision received July 26; accepted August 29; final version accepted December 6. Supported by National Institutes of Health grants 1 K08 EB004348-01 and R01 HL61672. Address correspondence to A.A. (e-mail: aarepal{at}jhmi.edu).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONADVANCE IN KNOWLEDGEReferences |
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Materials and Methods: The institutional animal care and use committee approved this study. Adult healthy swine (40–45 kg, n = 8) were tested. GACE was performed by infusing morrhuate sodium selectively into the left gastric artery. Six swine (animals A–F) underwent left GACE by using a dose-escalating regimen of morrhuate sodium, whereas two control swine underwent a sham procedure. Weight and fasting plasma ghrelin levels were compared in swine at baseline and at weeks 1–4. At week 4, stomachs were excised and analyzed. Analysis of the change in ghrelin values and weight was performed with both paired t test and unpaired Student t test.
Results: In control swine (n = 2), there was no significant difference in ghrelin values before (844.8 pg/mL ± 40 [standard deviation]) and after (997 pg/mL ± 93) the procedure (P = .5). Swine that received a low dose of morrhuate sodium (animals A–D) showed a significant increase in serum ghrelin values from 683.7 pg/mL ± 241 to 1555.9 pg/mL ± 312 (P = .002). At a higher dose, the mean baseline ghrelin values decreased from 466 pg/mL to 187 pg/mL ± 162. Weight changes of +1.4% and +8.6% were seen in swine that underwent GACE and control swine, respectively. Histochemical staining showed preservation of overall tissue architecture and parietal cells.
Conclusion: Use of GACE can result in increased or suppressed ghrelin levels.
© RSNA, 2007
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INTRODUCTION |
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In situations such as weight loss, there is a compensatory increase in ghrelin levels that has been identified as a potential cause for failed weight loss attempts during dieting (6). Also, gastric bypass surgery appears to suppress ghrelin secretion by isolating the gastric fundus from ingested nutrients and may explain the long-term effectiveness of this procedure in maintaining weight loss (8,9). On the other hand, increased ghrelin levels have been shown to exert a beneficial hemodynamic effect on the cardiovascular system, with improvement of endothelial function and improved vascular homeostasis (10,11). Therefore, the capability to induce a state of either increased or suppressed systemic ghrelin levels could have important implications for weight loss and the cardiovascular system by controlling energy balance. Thus, the purpose of our study was to prospectively test, in a porcine model, the hypothesis that catheter-directed gastric artery chemical embolization (GACE) can help in the modulation of systemic ghrelin levels.
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MATERIALS AND METHODS |
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Catheter-directed GACE
All GACE procedures were performed by one individual (A.A.), an interventional radiologist with 6 years of experience. Percutaneous access of the right femoral artery was achieved by using ultrasonographic guidance and the Seldinger technique. After the femoral artery was accessed, a 5-F sheath (Cordis, Miami, Fla) was placed in the femoral artery. Angiographic procedures were performed with an interventional angiographic system (Infinix CS-i; Toshiba America Medical Systems, Tustin, Calif). By using standard 5-F angiographic catheters (Omni Sos; Angiodynamics, Queensbury, NY), selective catheterization of the celiac artery was performed from the transfemoral approach. Digital subtraction angiography (DSA) of the celiac and superior mesenteric arteries was performed to delineate vascular anatomy to the gastric fundus, liver, spleen, pancreas, and small bowel (Fig 1a). After identification of the left gastric artery and other potential accessory gastric arteries, vessels to the fundus were superselectively catheterized by using a 3-F microcatheter system (SP3; Boston Scientific, Natick, Mass). Prior to ablation, superselective DSA was repeated through the microcatheters to delineate the fundal vessels (Fig 1b).
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Postablation Protocol and Ghrelin Level Analysis
Swine were placed in standard housing and fed a diet ad libitum. In all swine, fasting serum ghrelin values were obtained at baseline and at weeks 1–4. At each time after the procedure, two blood samples were drawn for ghrelin level determination. Blood samples were drawn for baseline values immediately prior to the procedure and were taken from either an ear vein or a femoral vein by one individual (B.P.B. or T.P.) at each time. After the procedure, all blood samples were drawn in the morning after an overnight fast. Blood was immediately transferred to a glass tube containing disodium ethylenediaminetetraacetic acid (1 mg/mL) and aprotinin (500 U/mL) and centrifuged immediately. Ghrelin levels were measured by using a radioimmunoassay with iodine 125–labeled bioactive ghrelin as the tracer and a rabbit polyclonal antibody (Phoenix Pharmaceuticals, Belmont, Calif). Weights were obtained at the same times as the blood samples by the same two individuals who performed the ghrelin level determinations. At week 4, swine were humanely killed with pentobarbital sodium (100 mg/kg), and the stomachs were surgically excised for histopathologic analysis.
Histopathologic Analysis
Histopathologic analysis was performed and supervised by a board-certified pathologist with 20 years of experience (E.M.). Because of a processing error, histopathologic analysis was performed in only four of six swine. One control swine and one experimental swine were processed incorrectly to get adequate tissue for immunohistochemical analysis. Tissue sections (5 µm thick) were cut from paraffin-embedded blocks on a microtome and mounted from warm water (40°C) onto adhesive microscope slides and allowed to dry overnight at room temperature. Contiguous tissue sections were processed for standard hematoxylin-eosin staining and for immunohistochemical analysis. For immunohistochemical analysis, tissue sections were removed from paraffin blocks in xylene and rehydrated by using serial washes with decreasing concentrations of ethanol (100%–70%). For immunohistochemical detection of ghrelin, rabbit antighrelin (porcine) IgG in a ratio of 1:100 (No. 00101; Phoenix Pharmaceuticals) was used. As a secondary antibody, goat antirabbit 594 in a ratio of 1:1000 (No. A11012; Molecular Probes, Eugene, Ore) was used.
Tissue sections were incubated overnight at 4°C with primary antibodies diluted in 0.1 M phosphate-buffered saline containing 10% normal goat serum and then with the appropriate secondary antibodies for 2 hours at room temperature. Sections were embedded with mounting medium containing 4', 6-diamine-2-phenylindole nuclear counterstain (Vectashield; Vector, Burlingame, Calif). Immunofluorescence analysis was performed by using epifluorescence microscopes (Olympus X51 and IX71; Olympus, Center Valley, Pa) equipped with a digital acquisition system (DP-70; Olympus). Histopathologic analysis was performed to evaluate overall tissue architecture, ulcerations, damage to gastric mucosa, and viability of parietal cells.
Statistical Analysis
Because this study was a dose-escalating feasibility pilot trial, a power calculation was not performed. The primary end point was change in serum ghrelin levels.
Control swine.—A paired t test was used to compare baseline ghrelin levels with the mean ghrelin levels at weeks 1–4. Also, a paired t test was used to compare baseline levels with levels at week 4.
Pre- and postprocedural ghrelin values.—The mean ghrelin values of the four low-dose swine (animals A–D) and the mean ghrelin values of the two control swine, with standard deviations, were plotted at baseline and at weeks 1–4 (Fig 2). A paired t test was used to compare baseline ghrelin values with the mean ghrelin values at weeks 1–4. Finally, a paired t test was used to compare baseline levels with levels at week 4 only.
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Percentage change.—Percentage changes in ghrelin values from baseline levels to levels at weeks 1–4 were calculated for all swine and for the two control swine. A paired t test was used to compare the percentage change from baseline levels to levels at week 4 in the experimental swine with the percentage change between those levels in the control swine. The percentage change between the experimental and the control swine groups was compared by using the unpaired Student t test.
Weight change.—Analysis of the weight change was performed with the paired Student t test.
All statistical analyses were performed with statistical software (GraphPad InStat, version 3.0; GraphPad Software, San Diego, Calif). A difference was considered statistically significant with P < .05 (two-tailed test).
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RESULTS |
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Serum Ghrelin Levels
Control swine.—In the control swine (n = 2), the baseline ghrelin value was 844.8 pg/mL ± 40. After sham embolization with saline, the mean serum ghrelin level was 997 pg/mL ± 93 (P = .51). There was no statistically significant change in ghrelin values from baseline levels to levels at week 4 in control swine (P = .71) (Fig 2).
Pre- and postprocedural ghrelin values.—In swine that received a low dose of morrhuate sodium (n = 4, animals A–D), GACE resulted in a significant increase in serum ghrelin values from a baseline level of 683.7 pg/mL ± 241 to a postprocedural level of 1555.9 pg/mL ± 312 (t = 10.47, df = 3, P = .002). In comparing baseline levels with levels at week 4, in animals A–D, there was a significant increase in ghrelin values (P = .002); in control swine, there was no difference in ghrelin values at baseline and those at week 4 (P = .71). At a higher dose (animal E), the mean baseline ghrelin value decreased from 466 pg/mL ± 162 to 187 pg/mL ± 162. In animal F, the highest dose of 40 mL (2000 mg) was lethal.
Comparison of experimental and control swine.—After the procedure, the mean ghrelin value in experimental swine (animals A–D) was 1555.9 pg/mL ± 312, and the mean ghrelin value in control swine (animals E and F) was 997 pg/mL ± 93. With the unpaired Student t test, a significant difference was observed between the control and experimental groups (t = 3.88, df = 3, P = .008).
Percentage change.—In animals A–D, there was a significant increase of +284% ± 76 at week 4 (t = 4.853, df = 3, P = .02). In control swine, there was no significant percentage change from values at baseline to those at week 4 (105% ± 61.4, P = .7). The mean percentage change in all low-dose experimental swine (animals A–D) was 244.8% ± 34, and the mean percentage change in control swine (animals E and F) was 104% ± 23.4. With the unpaired Student t test, a significant difference was observed between the control and experimental groups (t = 6.8, df = 6, P < .001).
Weight Change
In control swine and swine that underwent GACE, the baseline weight was 85.05 lb ± 6.3 (38.5 kg ± 2.9) and 84.3 lb ± 3.5 (38.2 kg ± 1.6), respectively, and the difference between the two groups was not significant (P = .2). In control swine, the average increase in body weight was +8.6% ± 0.9 (increase of 6.7 lb ± 0.2 [3.0 kg ± 0.1]). In swine that underwent GACE, the average increase in body weight was +1.4% ± 10.9 (increase of 1.3 lb ± 9 [0.6 kg ± 4]). This difference was not statistically significant (P > .05).
Histopathologic Findings
Antighrelin immunohistochemical analysis revealed markedly decreased ghrelin content in the gastric fundus of swine that underwent embolization with morrhuate sodium as compared with control swine (Fig 3). Notably, as demonstrated with hematoxylin-eosin staining, although ghrelin production was reduced after embolization, overall tissue architecture remained well preserved. Systematic sampling of the body and antrum similarly showed no discernible changes after embolization. Hematoxylin-eosin–stained tissue sections revealed intimal thickening of fundal vasculature consistent with the known action of the sclerosant morrhuate sodium. Small microulcers were also noted at the gastroesophageal junction in all animals.
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DISCUSSION |
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Since the identification of the neuropeptide ghrelin, extensive data in both animals and humans have shown its potent orexigenic effects. In rat studies, peripheral or central administration of ghrelin shows very potent short-term increases in food intake and growth hormone secretion (12,13). Mechanisms to antagonize or suppress the effects of ghrelin level on the central nervous system have resulted in dramatic weight loss and change in appetite (14,15). In humans, there is a consistent pattern of ghrelin levels in which the levels increase shortly before and decrease immediately after every meal (7). In situations such as weight loss, a compensatory increase in ghrelin levels may contribute to the difficulty in maintaining body weight. With obese patients, foods fail to suppress systemic ghrelin levels, which could impair postprandial satiety and may initiate overeating (4). Moreover, the ghrelin level appears to have a substantial role in the long-term effect of weight loss in bariatric surgery. In bariatric surgery in which isolation of the gastric fundus from ingested nutrients occurs, ghrelin profiles are reduced by 77% compared with the behavior of these profiles in control patients (8,9,16,17). Furthermore, the normal diurnal pattern is interrupted and the meal-initiated fluctuations are blunted (8,9). On the basis of these findings, achievement of low systemic ghrelin levels has become a potential strategy to control obesity and maintain weight loss.
Anatomically, the left gastric artery, which arises from the celiac axis, provides the dominant blood flow to the fundus of the stomach, the richest source of ghrelin (6). On the basis of the results of our study, it appears that the gastric arteries can be used as a means to target fundal functionality. By using a dose-escalating scale, we were able to directly manipulate systemic ghrelin levels; such manipulation resulted in a threefold increase in these levels. Our data suggest that incomplete ablation (achieved with lower doses) may obviate any feedback inhibition of ghrelin production with resultant overexpression of ghrelin by the remaining viable oxyntic cells. The levels we were able to achieve in our model are similar to levels that could otherwise be achieved by exogenous intravenous administration of ghrelin. These ghrelin levels have an important clinical benefit in left ventricular function in a setting of congestive heart failure and vascular endothelial function in patients with metabolic syndrome (10,11,18,19).
Because of the potent orexigenic effect of ghrelin, this hormone has been a target for the treatment of obesity and weight loss. In our study, use of gastric arteries with a high dose of morrhuate sodium allowed directed ablation of only the gastric fundus, with preservation of the remaining gastric mucosa. Through this highly targeted approach, we were able to achieve a dramatic decrease in systemic ghrelin levels up to –77% from baseline levels at 1 month—a decrease that is similar to the decrease in levels in patients who have undergone bariatric surgery. Also, as shown by using histopathologic analysis, this procedure can be performed with minimal damage to the gastric mucosa. Therefore, this technique allows selective ablation of the ghrelin-producing portion of the stomach, with resultant suppression of systemic ghrelin levels.
Our study had limitations. First, there was a lack of statistical significance in the weight changes in animals that underwent GACE. Because this study was a dose-escalating trial, weight would be expected to vary dependent on the levels of ghrelin present with the administration of each dose of morrhuate sodium. In addition, because animals were fed an ad libitum diet, calibration of food intake and measurements for appetite were not performed. We believe that the combination of all these variables probably contributed to the overall lack of significance of weight change in both groups. Second, complications were seen with this procedure. We noticed small microulcers at the gastroesophageal junction in all animals. These nontarget ablations occurred as a result of the close proximity of all the gastric arteries that supply the fundus and esophagus. In addition, the esophageal arteries, which arise from the left gastric artery, can be difficult to identify because of their size. To minimize nontarget ablations, dedicated high-resolution angiography of the vasculature is necessary to properly depict all the fundal and esophageal branches prior to ablation.
Practical application: On the basis of our findings, there are several potential avenues of further investigation. As shown with findings in our study, use of the gastric arteries to deliver therapeutic agents for local-regional control of ghrelin expression may become a feasible strategy. Various agents targeting ghrelin have been described but are limited in their applicability either because a high dose is required or because it is necessary to deliver these agents directly into the central nervous system (14,15). Because of both the ease and effectiveness of our technique, further refinements could allow delivery of a variety of agents directly to the gastric fundus that would induce overexpression or sustained suppression of ghrelin levels. By implementing such steps, an alternative minimally invasive technique may emerge that would affect energy balance and, potentially, obesity and cardiovascular disease.
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ADVANCE IN KNOWLEDGE |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONADVANCE IN KNOWLEDGEReferences |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: DSA = digital subtraction angiography • GACE = gastric artery chemical embolization
Authors stated no financial relationship to disclose.
Author contributions: Guarantor of integrity of entire study, A.A.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, A.A., T.P.; experimental studies, all authors; statistical analysis, A.A., T.P.; and manuscript editing, A.A., E.M.
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References |
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