Interaction of the photobactericides methylene blue and toluidine blue with a fluorophore in Pseudomonas aeruginosa cells.

BACKGROUND AND OBJECTIVES: The difference in photobactericidal efficacy between methylene blue (MB) and toluidine blue (TB) may be explained by their involvement with proteins, lipopolysaccharides (LPS), and siderophores and siderophore-receptor protein complexes on the bacterial outer membrane. This study aims to determine if this is the case by using the fluorescence given off by a pseudomonal siderophore named pyoverdin. STUDY DESIGN/MATERIALS AND METHODS: Confocal laser scanning microscopy was used to observe the fluorescence of Pseudomonas aeruginosa cells excited at 488 nm in the presence of increasing dye concentrations. RESULTS: Cellular fluorescence at 522 nm progressively decreased with increasing dye concentrations. The Stern-Volmer constants for cellular fluorescence quenching with the dyes were compared to the association constants for dyes complexed with LPS. The quenching of cellular fluorescence was associated with the formation of a ground-state complex between the dyes and pyoverdin-FpvA protein system. MB readily complexed with this system, whereas TB complexed more strongly with LPS. CONCLUSION: The different affinities of the dyes for both pyoverdin-protein and LPS will affect the contributions of the dyes' interactions with these biopolymers to the overall bacterial photodamage.

Comparison of covered versus uncovered wire mesh stents in the canine biliary tract.

Self-expanding wire mesh stents have been developed for endoscopic placement across malignant biliary strictures, but tumor ingrowth may limit the usefulness of open mesh stents. We reasoned that coating the wire mesh might prevent tumor ingrowth. Tissue response to covered and uncovered stents was compared in dogs. Stents were surgically placed in the bile ducts of 22 mongrel dogs through the sphincter of Oddi. Either a silicone-covered stent or an uncovered stent was inserted. Liver function test values remained normal throughout a 1- or 3-month study. Necropsy revealed that all ducts were unobstructed. Bile duct histologic examination revealed mild-to-moderate cellular infiltration in all animals. Mucosal hyperplasia was more marked in the animals with uncovered stents and the bare wires became deeply embedded in bile duct epithelium, whereas the wires of covered stents did not. We conclude that covered stents are well tolerated by the canine bile duct. These results suggest that such stents may be removable, making self-expanding metal stents an appropriate treatment for both benign and malignant biliary strictures.

The effect of bipolar electrosurgical coagulation waveform on a rat uterine model of fallopian tube sterilization.

Laparoscopic bipolar electrosurgical sterilization has been employed for nearly two decades. This bipolar procedure was intended to reduce the incidence of unintended complications, such as bowel burns. However, compared to the monopolar procedure, it produced a higher failure rate. This study examines the role of the low power-high voltage coagulation waveform on the effectiveness of electrosurgical desiccation on the rat uterine model of the fallopian tube. Desiccations were performed with both Kleppinger and Storz forceps using generator settings from 2 to 6. Desiccations performed to the visual endpoint (blanch, swell, and collapse) required from 23.8 to 30.0 joules, although procedures performed at a generator setting of 2 required long application times, an average 15.4 sec (Storz) and 28.2 (Kleppinger). All bipolar procedures led to a loss of tissue at the desiccation site at 12 weeks postsurgery. Therefore, the electrosurgical coagulation waveform proved to be effective in producing adequate in vivo tissue destruction even at very low generator settings.

Bipolar electrosurgical sphincterotomy.

We tested a newly developed bipolar sphincterotome. Monopolar and bipolar sphincterotomes were employed to cut small bowel smoothly and hemostatically. The bipolar instrument required 17.1 watts while the monopolar required 29.2 watts; these values are significantly different at p = 0.005. Bipolar sphincterotomies were performed via open surgical access on eight dogs. The animals were allowed to recover and were followed for 6 weeks. There was no evidence of stenosis or common duct dilation at autopsy, and serum alkaline phosphatase and bilirubin were within normal limits. Both sphincterotomes were used to cut tissue in vitro and histological examination displayed no evidence of thermal injury at the return electrode site of the bipolar sphincterotome. The lower power levels required by the bipolar sphincterotome may decrease procedure complications.

The interaction between electrosurgical generators, endoscopic electrodes, and tissue.

Endoscopic monopolar and bipolar devices were compared during cutting and coagulation. It was observed on appropriate animal models that the initial resistance (impedance) values recorded correspond to the normal tissue impedance at the electrode. The subsequent impedance values increase 25 to 50 ohms for the coagulator which relates to tissue desiccation and for the cutting electrodes the impedance increases greater than 1000 ohms during the arcing process. At similar power settings, typical monopolar generators produce maximum power at 300 to 500 ohms while typical bipolar generators produce maximum power at 25 to 100 ohms. With impedances greater than 1000 ohms, monopolar generators are capable of higher power output than are bipolar generators. Since cutting is a high impedance process, bipolar cutting electrodes do not perform as intended with typical bipolar generators. Therefore, bipolar cutting electrodes should be employed with a monopolar generator or a generator designed specifically for their use.

In vivo evaluation of monopolar versus bipolar electrosurgical polypectomy snares.

The energy required and tissue damage in bipolar and monopolar polypectomy snares were compared in a canine model. The bipolar snare required an average of 34 joules of energy, whereas the monopolar snare required 228 joules to cut the same diameter of gastric mucosa tended into a polypoid structure (p = 0.0005). The reduced energy delivered to the tissue from the bipolar procedure resulted in only 32% average depth of damage to the underlying gastric wall, whereas the monopolar procedure caused an average 69% (p = 0.001). Surgically created polyps required 247 joules and 69 joules for corresponding monopolar and bipolar polypectomy (p = 0.001). The decreased energy required and the correspondingly reduced damage caused to the underlying bowel wall by the bipolar snare should reduce the incidence of perforation and post-polypectomy syndrome. The bipolar snare completes a local circuit about the snare, eliminating the return electrode and, consequently, the possibility of any return electrode burns. The bipolar snare thus provides an added safety margin during polypectomy.

Spontaneous passage of glass beads from the canine gallbladder: facilitation by sphincterotomy.

To investigate the mechanism by which ablation of the sphincter of Oddi prevents gallstone formation, we assessed passage of glass beads out of the gallbladders of dogs with sphincterotomy and sham sphincterotomy. One month after bead implantation, dogs with an intact sphincter passed 52%, 26%, 22%, 10%, 0%, and 0% of beads with diameters of 2, 3, 4, 5, 6, and 8 mm, respectively. For the same respective bead diameters, dogs with a sphincterotomy passed 90%, 90%, 88%, 75%, 75%, and 42% of beads (p less than 0.05 for all bead diameters). No beads were in the common bile duct of any dog. In separate dogs studied by cholescintigraphy, sphincterotomy significantly increased gallbladder ejection fraction from 0.46 to 0.76 (p less than 0.01). In addition, sphincterotomy significantly lowered resting gallbladder volume from 24.4 to 15.8 ml (p less than 0.025) and lowered cholecystokinin-stimulated gallbladder volume from 13.3 to 5.9 ml (p less than 0.025). These data indicate that even with an intact sphincter, small solids can pass from the gallbladder and into the duodenum. Sphincterotomy facilitates passage of solids, apparently by general improvement in gallbladder emptying. Facilitated passage of crystals, microliths, or small stones seems the most likely explanation for prevention of gallstone formation by sphincterotomy.

Effect of bombesin and gastrin-releasing peptide on canine sphincter of Oddi.

This study reports the effects of bombesin and gastrin-releasing peptide (GRP) on the canine sphincter of Oddi using a method that allows repeated cannulation of the biliary sphincter in the unanesthetized animal through a Thomas cannula placed opposite the biliary papilla. Immediately after intravenous administration of bombesin or GRP, phasic sphincter contractions disappeared, basal sphincter pressures fell, and common bile duct pressures rose. Because bombesin releases cholecystokinin (CCK) and CCK resulted in a similar pattern to that of bombesin, the bombesin effect on the sphincter of Oddi may have been secondary to CCK's effect on the sphincter. To test if the bombesin effect on the sphincter of Oddi was due to the release of CCK, we blocked CCK release by administration of somatostatin, having first established that somatostatin blocked endogenous CCK release in our animal model by use of an intraduodenal infusion of lipid. Exogenous administration of bombesin failed to alter sphincter of Oddi or common bile duct pressures in dogs treated with somatostatin. Somatostatin did not, however, block CCK's effect on gallbladder contraction, since exogenous administration of CCK after somatostatin injection resulted in the pressure changes in the biliary tree typical of CCK-induced gallbladder contraction. Thus bombesin administration appears to result in sphincter relaxation and gallbladder contraction by the release of endogenous CCK rather than by a direct effect. The increase in common bile duct pressures was due to gallbladder contraction, since this rise in pressure was abolished by cholecystectomy. The peptide effect on sphincter contraction and basal sphincter pressure were unaffected by cholecystectomy.

Diurnal rhythm of HMG CoA reductase activity in canine intestine is independent of luminal contents.

Activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) measured in isolated segments of canine intestinal mucosa showed a distinct diurnal rhythm. Total activity changed over a twofold range with a peak occurring during midday, shortly after feeding. Since the isolated segments had no contact with luminal contents, the rhythm was not directly related to food components or bile salts. Humoral or neural influences must mediate the rhythm. The diurnal rhythm persisted for at least 3-5 mo, but was lost by 10 mo following formation of the isolated segment, possibly because of mucosal involution.

Lysolecithin-lipid interactions in disruption of the canine gastric mucosal barrier.

Lysolecithin has been implicated in the pathogenesis of gastric ulcer and reflux gastritis. By use of canine Heidenhain pouches, we examined effects of lysolecithin on the gastric mucosal barrier both at neutral (PO4 buffer, pH 7) and acidic (0.15 M HCl) pH with respect to interaction of lysolecithin with membrane lipids. At both pH values, 2 mM lysolecithin significantly increased forward diffusion of Na+ and backdiffusion of H+. Both solutions also caused significant efflux of membrane phospholipid and cholesterol. Saturation of neutral and acidic lysolecithin solutions with either lecithin or cholesterol significantly diminished or completely prevented disruption of the barrier to H+ backdiffusion and Na+ forward diffusion. In agreement with other reports, we also demonstrated formation of soluble lysolecithin-lecithin mixed micelles but formation of insoluble lysolecithin-cholesterol complexes. Finally, by use of [3H]polyethylene glycol and [14C]lysolecithin, we demonstrated more rapid mucosal uptake of lysolecithin at acidic than at neutral pH for any given experimental condition. Specifically, mean mucosal uptake of lysolecithin from acidic solutions saturated with lecithin (which did not disrupt the gastric mucosal barrier) was greater than uptake from neutral solutions without added lipid (which did disrupt the barrier). These studies suggest that lysolecithin-lipid interactions play an important role in lysolecithin-induced injury to the gastric mucosa. The most important of these interactions appears to occur in luminal lysolecithin micelles rather than within mucosal membranes.

Peptidergic regulation of feeding in the dog (Canis familiaris).

Although the incidence of obesity in the domesticated dog is high, few studies have investigated the regulation of food intake in this species. In the present study we investigated the response of the dog to a number of putative satiety agents including cholecystokinin (CCK), bombesin, calcitonin and naloxone. CCK significantly suppressed food intake during a scheduled fifteen minute meal in intact dogs and in dogs receiving total subdiaphragmatic vagotomies. Emesis occurred following injection of higher doses of CCK in most dogs. Bombesin and calcitonin reduced intake in both normal and vagotomized dogs, although higher doses of calcitonin were needed to significantly suppress feeding in vagotomized dogs compared with intact animals. Naloxone reduced feeding by as much as 60% in intact and vagotomized animals. Glucagon suppressed feeding in intact dogs, but not in vagotomized animals. Somatostatin and pancreatic polypeptide did not alter food intake. Thus the domesticated dog responds somewhat differently to some neuropeptides compared with the laboratory rat stressing the importance of examining the regulation of food intake across species.

Demodulated low frequency currents from electrosurgical procedures.

Low frequency currents produced incidental to electrosurgical procedures are usually only a minor threat unless they are coupled directly into the heart through a catheter or a thoracic surgical procedure. However, should the low frequency blocking capacitors in the electrosurgical generator fail in the conducting state, extremely dangerous low frequency currents of many milliamperes may flow into the patient. Even with properly functioning capacitors, low frequencies up to several milliamperes may be produced by some generators. The results of this study show that the coagulation mode of operation produces greater amounts of low frequency currents than the "pure cutting" mode at the same power setting. These results also demonstrate that the higher the generator power output, the higher the magnitude of the low frequency currents.

A miniature current probe for measuring electrosurgical currents.

The measurement of high-frequency electrosurgical currents at the surgical site is complicated by a generator output circuit isolated from ground, as well as radiation and displacement current losses along connecting wires. An inductive probe can accurately measure the actual electrosurgical current at the active electrode.

Inhibition of gallstone formation by sphincterotomy in the prairie dog: reversal by atropine.

Sphincterotomy has previously been shown to decrease gallbladder volume and gallbladder bile stasis while inhibiting gallstone formation in the prairie dog. We tested the possibility that atropine administration might reverse the effects of sphincterotomy on gallbladder mechanical function and gallbladder bile stasis and thereby reverse the inhibition of gallstone formation seen after sphincterotomy. Sixteen prairie dogs underwent sphincterotomy and were placed on a high cholesterol diet known to consistently induce gallstone formation. After 6 wk, in addition to the lithogenic diet, 8 animals were administered atropine. A control group of 8 animals was continued on the lithogenic diet. At death, animals that had undergone sphincterotomy and received atropine had a larger mean gallbladder volume (2.7 +/- 0.4 ml) than sphincterotomized control animals (0.66 +/- 0.2 ml, p less than 0.005), more total lipid stored in the gallbladder (176.1 +/- 33.9 mumol, sphincterotomy and atropine vs. 42.8 +/- 17.3 mumol, sphincterotomy, p less than 0.005), and a larger fraction of administered radiolabeled bile salt stored in the gallbladder (51.5 /- 7.3%, sphincterotomy and atropine vs. 24.5 +/- 10.7 sphincterotomy, p less than 0.05). The ratio of the fraction of [3H]cholic acid in the gallbladder (given 1 day before death) to the fraction of [14C]cholic acid in the gallbladder (given 4 days earlier) was lower for animals that underwent sphincterotomy and received atropine (0.63 +/- 0.04) than for sphincterotomized control animals (1.0 +/- 0.15, p less than 0.05), indicating a return of gallbladder bile stasis in the atropine-treated group. Confirming our previous work, only 1 of 8 animals that underwent sphincterotomy had gallstones. In contrast, 8 of 8 atropine-treated animals formed stones (p less than 0.005). Gallbladder bile of both groups were equally supersaturated with cholesterol. These findings provide direct support for the role of gallbladder bile stasis in the pathogenesis of cholesterol gallstone formation in this model.

Bile acid and bile salt disrupt gastric mucosal barrier in the dog by different mechanisms.

The present study was undertaken to assess the mechanism by which protonated taurocholic acid disrupts the gastric mucosal barrier. By the criterion of lecithin solubilization, the critical micellar concentration of taurocholic acid (pH 1) was 4.5 mM, as opposed to 3.0 mM for sodium taurocholate (pH 7). In canine Heidenhain pouches, taurocholic acid significantly increased net forward diffusion of Na+ and backdiffusion of H+ at concentrations of 9, 4.5, and 3.5 mM, indicating that micelle formation was not required for disruption of the gastric mucosal barrier by this bile acid. Saturation of the 9 mM taurocholic acid solution with lecithin (and cholesterol) did not prevent disruption of the gastric mucosal barrier. At 9 mM, taurocholic acid was absorbed from the pouches at a mean rate of 1,150 +/- 115 nmol/min in contrast to an absorption rate of 225 +/- 10 nmol/min for sodium taurocholate at the same concentration. These findings indicate that, unlike ionized bile salts, disruption of the gastric mucosal barrier by taurocholic acid is mediated largely by uptake of bile acid by the gastric mucosa rather than dissolution of mucosal membrane lipids.

A canine model of gastric and colonic polyps.

Polyps were produced in the canine stomach and colon by a surgical technique. In a total of 18 dogs 26 polyps were produced in the stomach or colon. Twenty of the 26 polyps were viable when endoscopically and microscopically examined. Polyps of approximately 1 cm in diameter and 3 to 4 cm in length can be produced without sloughing. The gross and microscopic characteristics of the canine polyp are quite similar to the human condition. Obviously the adenomatous tissue of the polyp head was not reproduced, but this area is not generally cut during polypectomy. The surgical produced polyps appear to be useful as a model to study the effects of electrosurgical cutting and for training in the technique of polypectomy.