Masayuki Kitano

Dynamic imaging of pancreatic diseases by contrast enhanced coded phase inversion harmonic ultrasonography

Background: Coded phase inversion harmonic ultrasonography, a newly available sonographic technique, enables visualisation of slow flow in minute vessels in a real time fashion with the use of a sonographic contrast agent containing monosaccharide. Our purpose was to employ this novel technique to observe microvessels in pancreatic tumours. Subjects and methods: Sixty five patients with suspicious pancreatic tumours received contrast enhanced coded phase inversion harmonic ultrasonography, contrast enhanced computed tomography, and endosonography. Final diagnoses based on histological findings were pancreatic ductal carcinomas in 49 patients, inflammatory pseudotumours with chronic pancreatitis in seven, and endocrine tumours in nine. For contrast enhanced coded harmonic ultrasonography, Levovist, a contrast agent, was injected intravenously as a bolus. When the first microbubble signal appeared in the pancreas, images of the ideal scanning plane were displayed in a real time continuous fashion (vessel images). Subsequently, interval delay scanning (perfusion images) was taken to demonstrate parenchymal flow. Tumour vascularity was evaluated by using the two types of imaging. Sensitivities for depicting pancreatic tumours were compared between three examinations. Results: Contrast enhanced ultrasonography demonstrated tumour vessels in 67% of pancreatic ductal carcinomas, although most were relatively hypovascular compared with the surrounding pancreatic tissue. The vascular patterns of tumours obtained by contrast enhanced ultrasonography were closely correlated with those obtained by contrast enhanced computed tomography. Values for sensitivity in depicting pancreatic tumours of 2 cm or less in size were 68% for contrast enhanced computed tomography, 95% for endosonography, and 95% for contrast enhanced ultrasonography. Conclusion: Contrast enhanced coded phase inversion harmonic ultrasonography successfully visualised fine vessels in pancreatic tumours and may play a pivotal role in the depiction and differential diagnosis of pancreatic tumours.

Gastric submucosal microdialysis: a method to study gastrin- and food-evoked mobilization of ECL-cell histamine in conscious rats.

Rat stomach ECL cells are rich in histamine and chromogranin A-derived peptides, such as pancreastatin. Gastrin causes the parietal cells to secrete acid by flooding them with histamine from the ECL cells. In the past, gastric histamine release has been studied using anaesthetized, surgically manipulated animals or isolated gastric mucosa, glands or ECL cells. We monitored gastric histamine mobilization in intact conscious rats by subjecting them to gastric submucosal microdialysis. A microdialysis probe was implanted into the submucosa of the acid-producing part of the stomach (day 1). The rats had access to food and water or were deprived of food (48 h), starting on day 2 after implantation of the probe. On day 4, the rats received food or gastrin (intravenous infusion), and sampling of microdialysate commenced. Samples (flow rate 1.2 microl min(-1)) were collected every 20 or 60 min, and the histamine and pancreastatin concentrations were determined. The serum gastrin concentration was determined in tail vein blood. Exogenous gastrin (4-h infusion) raised microdialysate histamine and pancreastatin dose-dependently. This effect was prevented by gastrin receptor blockade (YM022). Depletion of ECL-cell histamine by alpha-fluoromethylhistidine, an irreversible inhibitor of the histamine-forming enzyme, suppressed the gastrin-evoked release of histamine but not that of pancreastatin. Fasting lowered serum gastrin and microdialysate histamine by 50%, while refeeding raised serum gastrin and microdialysate histamine and pancreastatin 3-fold. We conclude that histamine mobilized by gastrin and food intake derives from ECL cells because: 1) Histamine and pancreastatin were released concomitantly, 2) histamine mobilization following gastrin or food intake was prevented by gastrin receptor blockade, and 3) mobilization of histamine (but not pancreastatin) was abolished by alpha-fluoromethylhistidine. Hence, gastric submucosal microdialysis allows us to monitor the mobilization of ECL-cell histamine in intact conscious rats under various experimental conditions not previously accessible to study. While gastrin receptor blockade lowered post-prandial release of ECL-cell histamine by about 80%, unilateral vagotomy reduced post-prandial mobilization of ECL-cell histamine by about 50%. Hence, both gastrin and vagal excitation contribute to the post-prandial release of ECL-cell histamine.

Anaesthetic agents inhibit gastrin‐stimulated but not basal histamine release from rat stomach ECL cells

By mobilizing histamine in response to gastrin, the ECL cells in the oxyntic mucosa play a key role in the control of the parietal cells and hence of gastric acid secretion. General anaesthesia suppresses basal and gastrin‐ and histamine‐stimulated acid secretion. The present study examines if the effect of anaesthesia on basal and gastrin‐stimulated acid secretion is associated with suppressed ECL‐cell histamine secretion. A microdialysis probe was implanted in the submucosa of the ventral aspect of the acid‐producing part of the stomach (32 rats). Three days later, ECL‐cell histamine mobilization was monitored 2 h before and 4 h after the start of intravenous infusion of gastrin (5 nmol kg−1 h−1). The rats were either conscious or anaesthetized. Four commonly used anaesthetic agents were given 1 h before the start of the experiments by intraperitoneal injection: chloral hydrate (300 mg kg−1), pentobarbitone (40 mg kg−1), urethane (1.5 g kg−1) and a mixture of fluanisone/fentanyl/midazolam (15/0.5/7.5 mg kg−1). In a parallel series of experiments, basal‐ and gastrin‐induced acid secretion was monitored in six conscious and 25 anaesthetized (see above) chronic gastric fistula rats. All anaesthetic agents lowered gastrin‐stimulated acid secretion; also the basal acid output was reduced (fluanisone/fentanyl/midazolam was an exception). Anaesthesia reduced gastrin‐stimulated but not basal histamine release by 55–80%. The reduction in gastrin‐induced acid response (70–95%) was strongly correlated to the reduction in gastrin‐induced histamine mobilization. The correlation is in line with the view that the reduced acid response to gastrin reflects impaired histamine mobilization. Rat stomach ECL cells were purified by counter‐flow elutriation. Gastrin‐evoked histamine mobilization from the isolated ECL cells was determined in the absence or presence of anaesthetic agents in the medium. With the exception of urethane, they inhibited gastrin‐evoked histamine secretion dose‐dependently, indicating a direct effect on the ECL cells. Anaesthetized rats are widely used to study acid secretion and ECL‐cell histamine release. The present results illustrate the short‐comings of such an approach in that a number of anaesthetic agents were found to impair not only acid secretion but also the secretion of ECL‐cell histamine – some acting in a direct manner.

The vagus regulates histamine mobilization from rat stomach ECL cells by controlling their sensitivity to gastrin.

The ECL cells in the oxyntic mucosa secrete histamine in response to gastrin, stimulating parietal cells to produce acid. Do they also operate under nervous control? The present study examines histamine mobilization from rat stomach ECL cells in situ in response to acute vagal excitation and to food or gastrin following vagal or sympathetic denervation. Applying the technique of microdialysis, we monitored the release of histamine by radioimmunoassay. Microdialysis probes were placed in the submucosa on either side of the stomach, 3 days before experiments. The rats were awake during microdialysis except when subjected to electrical vagal stimulation. One‐sided electrical vagal stimulation raised serum gastrin and mobilized gastric histamine. However, gastrin receptor blockade prevented the histamine mobilization, indicating that circulating gastrin accounts for the response. Vagal excitation by hypoglycaemia (insulin) or pylorus ligation did not mobilize either gastrin or histamine. The histamine response to food was almost abolished by gastrin receptor blockade, and it was halved on the denervated side after unilateral subdiaphragmatic vagotomy. While the histamine response to a near‐maximally effective dose of gastrin was unaffected by vagotomy, the response to low gastrin doses was reduced significantly. Abdominal ganglionic sympathectomy failed to affect the histamine response to either food or gastrin. In conclusion, gastrin is responsible for most of the food‐evoked mobilization of ECL‐cell histamine. The histamine response to electrical vagal stimulation reflects the effect of circulating gastrin rather than a direct action of the vagus on the ECL cells. Vagal denervation was accompanied by an impaired histamine response to food intake, probably reflecting the right‐ward shift of the serum gastrin concentration–histamine response curve. The results suggest that the vagus controls the sensitivity of the ECL cells to gastrin.

Long‐lasting cholecystokinin2 receptor blockade after a single subcutaneous injection of YF476 or YM022

Histamine‐forming ECL cells in the rat stomach operate under the control of gastrin. They represent a convenient target for studying cholecystokinin‐B/gastrin (CCK2) receptor antagonists in vivo. We examined the effectiveness and duration of action of two CCK2 antagonists, YM022 and YF476, with respect to their effect on ECL‐cell histidine decarboxylase (HDC) activity in the rat. Oral administration of subcutaneous deposition of YF476 or YM022 reduced the HDC activity. The maximum/near‐maximum dose for both drugs and for both modes of administration was 300 μmol kg−1 (effects measured 24 h after dose). At this dose and time the serum concentration of YF476 was 20–40 nmol l−1. The dose 300 μmol kg−1 was used in all subsequent studies. A single subcutaneous injection of YF476 inhibited the HDC activity for 8 weeks. The circulating concentration of YF476 remained high for the same period of time (15 nmol l−1). Subcutaneous YM022 suppressed the HDC activity for 4 weeks. A single oral dose of YF476 or YM022 inhibited the HDC activity for 2–3 days. Chronic gastric fistula rats were used to study the effect of subcutaneous YF476 on gastrin‐stimulated acid secretion. A single injection of YF476 prevented gastrin from causing an acid response for at least 4 weeks (the longest time studied). We conclude that a single subcutaneous injection of 300 μmol kg−1 YF476 causes blockade of CCK2 receptors in the stomach of the rat for 8 weeks thus providing a convenient method for studies of the consequences of long‐term CCK2 receptor inhibition.

VALUE OF COMPUTED TOMOGRAPHY FOR EVALUATING THE INJECTION SITE IN ENDOSONOGRAPHY-GUIDED CELIAC PLEXUS NEUROLYSIS

Endosonography‐guided celiac plexus neurolysis (EUS‐CPN) safely and effectively relieves pain associated with intra‐abdominal malignancies when the neurolytic is accurately injected. We applied contrast medium to evaluate the ethanol injection sites in patients who received EUS‐CPN due to abdominal pain caused by malignancies. We injected, under the guidance of endoscopic ultrasonography (EUS), ethanol containing 10% contrast medium into the celiac plexus of patients with intra‐abdominal pain due to malignancies. Immediately after the endoscopic therapy, patients underwent computed tomography (CT) to confirm the injection site. Images of distribution of injected solutions were classified into three groups. Injected solution dispersed in unilateral and bilateral anterocrural space was defined as ‘unilateral injection’ or ‘bilateral injection’, respectively. Injected solution located out of the anterocrural space was defined as ‘inappropriate injection’. Pre‐ and postprocedure pain was assessed using a standard analog scale. Before and 2, 4, 8, 12, and 16 weeks after the procedure, pain scores were evaluated. From April 2003 to May 2005, 13 patients were enrolled in this study. Improvement of pain score in the ‘bilateral injection’ and ‘unilateral injection’ groups was significantly superior to the change in the ‘inappropriate injection’ group. Although EUS‐CPN was effective in eight of 13 patients (61.5%), additional EUS‐CPN to the ‘inappropriate injection group’ increased the response rate to 84.6%. Injection of ethanol to the anterocrural space by EUS‐CPN produced adequate pain relief. Immediate examination by CT for confirmation of injection sites after EUS‐CPN would increase the likelihood of induction of pain relief.

α-Fluoromethylhistidine depletes histamine from secreting but not from non-secreting rat stomach ECL cells

Histamine in the oxyntic mucosa of the rat stomach occurs in mast cells (10%) and ECL cells (90%). Unlike the mast cells, the ECL cells operate under the control of gastrin. alpha-Fluoromethylhistidine, an irreversible inhibitor of the histamine-forming enzyme, histidine decarboxylase depletes ECL-cell but not mast-cell histamine. This report shows that the effectiveness by which histidine decarboxylase inhibition depletes ECL-cell histamine depends on the rate of histamine secretion. Rats received alpha-fluoromethylhistidine by continuous subcutaneous infusion for 24 h. Maximally effective doses (>/=3 mg/kg/h) inhibited histidine decarboxylase and reduced oxyntic mucosal histamine in fed rats by 80-90%. In fasted rats, the reduction was 50%. alpha-Fluoromethylhistidine greatly reduced the number of histamine-immunoreactive ECL cells (immunocytochemistry) and of secretory vesicles in the ECL cells (electron microscopy) in fed but not in fasted rats. The half-life of oxyntic mucosal histamine (determined upon histidine decarboxylase inhibition) was 2.6 h in fed rats and 19.4 h in fasted rats. The amount of histamine secreted in response to gastrin (monitored by gastric submucosal microdialysis) was greatly reduced by alpha-fluoromethylhistidine in fed rats but not in fasted rats. ECL cells were isolated from rat stomach by elutriation (80% purity). Their histamine content was determined after culture, with or without alpha-fluoromethylhistidine, in the presence of varying concentrations of gastrin. In a medium containing 10 nM gastrin, ECL cells responded to a maximally effective concentration of alpha-fluoromethylhistidine (0.1 nM) with 80% reduction in histamine content. In the absence of gastrin, ECL cells responded to alpha-fluoromethylhistidine with 45% reduction of histamine; the releasable histamine pool was unaffected. In conclusion, the combination of histidine decarboxylase inhibition and a high rate of histamine secretion will promptly exhaust the ECL-cell histamine pool, while histidine decarboxylase inhibition and a low secretion rate will affect the histamine pool much less.

Clinical significance of vascular assessment by contrast-enhanced harmonic ultrasonography of pancreatic carcinomas.

resolution, and low sensitivity to slow flow, were inevitable.7 Several specific modes for contrast enhancement have recently been developed,7 which sensitively depict the nonlinear signals produced by oscillation or destruction of microbubbles with a good spatial resolution devoid of Doppler-related artifacts and realize so-called contrast harmonic imaging.7 Contrast harmonic imaging visualizes the vascular structure of pancreatic tumors more clearly than does contrastenhanced power Doppler ultrasound. This modality is superior to spiral CT for depiction of pancreatic tumors of 2 cm or less in diameter.19 In addition, contrast harmonic imaging enables the depiction of the margin of some tumors not detected by fundamental B-mode US. Although slight dilatation of the main pancreatic duct on US conducted as screening tests appears to be a risk factor for pancreatic cancer,20 the outline of some tumors with a dilated duct of the pancreas is often unclear on fundamental B-mode US. Subsequent contrast harmonic imaging is capable of demonstrating the outline of the tumors clearly, suggesting that visualization of vascularity may assist in discriminating the tumor from the surrounding tissue in US examination.19 Another crucial role that contrast harmonic imaging plays in the clinical practice of treating pancreatic diseases is the depiction of vessels in 36%–75% of pancreatic ductal carcinomas, which are not generally depicted by contrast-enhanced CT.19,21–26 In this issue of the Journal of Gastroenterology, Numata et al. 26 compared evaluation of the vascularity of pancreatic cancers by contrast-enhanced US to histological findings. Blood flow was depicted in 75% of the pancreatic cancers. Regions rich in vessels indicated inflammation, presence of both carcinoma cells and residual acinar cells, and large vessels. On the other hand, Regions with no vessels corresponded to severe fibrosis, necrosis, and mucin. The relationship between vascularity and histoMost ductal adenocarcinomas of the pancreas are generally accepted as “hypovascular” as compared to the surrounding parenchyma on contrast-enhanced computed tomography (CT).1–5 Contrast-enhanced ultrasonography (US) was first reported with the use of carbon dioxide microbubbles,6 which depicted that 91% of the ductal carcinomas were hypovascular, while 95% of the inflammatory pancreatic masses were isovascular. This ultrasound technique makes differentiation of inflammatory masses from pancreatic ductal carcinomas possible by the presence of intratumoral vessels; however, it requires essentially an angiographic technique that is relatively invasive because carbon dioxide microbubbles should be selectively infused into the celiac artery or superior mesenteric artery. Development of intravenous ultrasound contrast agents have produced the advantage of being able to observe vascularity of hepatic and pancreatic tumors noninvasively by transabdominal ultrasound.7 Levovist (Schering AG, Berlin Germany), a sonography contrast agent that is infused intravenously, has been developed concurrently and is known to be well tolerated with a fairly good safety profile.7 Conventional color and power Doppler sonography enhanced by intravenous application of Levovist allowed us to determine tumor vascularization of pancreatic neuroendocrine tumors.8– 10 The contrast agent significantly improved the characterization of pancreatic neuroendocrine tumors compared with non-enhanced sonography.8–10 There are several reports on the usefulness of contrast-enhanced power Doppler ultrasonography in differential diagnosis of pancreatic carcinomas and chronic focal pancreatitis.11–14 The contrast-enhanced power Doppler technique has also been used in endosonography.15–18 Nevertheless, several limitations in Doppler US studies with Levovist, such as blooming artifacts, poor spatial