Reversal of opioid-induced bladder dysfunction by intravenous naloxone and methylnaltrexone.

Peripheral mechanisms may be involved in opioid actions on the urinary bladder. This double-blind study investigated whether opioid inhibition of bladder function is reversed by methylnaltrexone, a peripheral opioid antagonist. Thirteen healthy male volunteers received an intravenous (i.v.) infusion of remifentanil, 0.15 mcg/kg/min, then a single i.v. dose of study medication (methylnaltrexone 0.3 mg/kg, naloxone 0.01 mg/kg, or saline). Urodynamics were measured with indwelling bladder and rectal catheters, and pupil size was assessed with infrared pupillometry. Remifentanil decreased detrusor pressure in 21/25 sessions and caused complete urinary retention in 18/25. Voiding was possible in 7/7, 5/12, and 0/6 sessions after naloxone, methylnaltrexone, and saline, respectively (P=0.0013). Remifentanil caused marked miosis that was reversed by naloxone, but not methylnaltrexone or placebo (P<0.0001). The pupil data confirm that methylnaltrexone did not reverse central opioid effects. Reversal of urinary retention by methylnaltrexone indicates that peripheral mechanisms may play a role in opioid-induced bladder dysfunction.

Genetically modified fibroblasts induce angiogenesis in the rat epigastric island flap.

METHODS: Gene therapy was tested for inducing functional angiogenesis in the superficial rat epigastric island flap to allow earlier pedicle division. Autologous rat fibroblasts were grown, harvested, cultured and retrovirally transfected to produce platelet-derived growth factor AA (PDGF-AA), an angiogenetically active protein. Stable gene expression was monitored by PDGF-AA enzyme-linked immunosorbent assay (ELISA). One hundred and eighty animals were divided into three groups (I-III) and a bilateral flap created in each animal. In all experiments, the right-sided flap was subjected to experimental treatment and the left-sided flap served as control (1ml saline 0.9%). During flap elevation, group I received 5X10(6) GMFB (genetically modified fibroblasts) plus 1 ml Dulbecco's modified Eagle's medium. Group II was treated with 5x10(6) NMFB (non-modified fibroblasts) plus 1 ml medium and group III received 1 ml medium only. The flaps were sutured back and the vascular pedicle was bilaterally ligated and divided in each of ten animals during the following 6 days. After 7 days, the flaps were harvested, the amount of necrosis measured and histologically examined. RESULTS: The GMFB produced up to 560 times more PDGF-AA than the NMFB, measured by ELISA. The GMFB-treated flaps tolerated surgical division of the vascular pedicle significantly earlier than groups II and III. Histologically, fibroblasts persisted in all flaps of groups I and II, without major inflammatory reaction. In all GMFB-treated flaps, massive angiogenesis could be demonstrated. CONCLUSION: By means of retroviral gene transfer, autologous rat fibroblasts can be genetically modified for stable expression of the PDGF-A gene to produce high amounts of PDGF-AA, which is angiogenetically active. After injection into the panniculus carnosus, these cells induce functional angiogenesis to permit earlier division of the vascular pedicle in this flap model.

Metabolic effects of stress mediators on cultured hepatocytes.

Stress mediators play a major role in inducing the hypermetabolic stress state in the liver after major injuries. The majority of studies on the effect of mediators on hepatocytes have focused on single factor effects or on the effect of very complex additives (e. g., serum), and there are no reports which have rigorously identified specific interactions between stress mediators. We used a factorial design experimental approach to evaluate the effects of a four to five day exposure to hormone (glucagon, hydrocortisone, and epinephrine) and cytokine [tumor necrosis factor-alpha (TNF-alpha) interleukin-1beta (IL-1beta) and interleukin-6 (IL-6)] stress mediators on stable cultures of rat hepatocytes. Both individual-factor effects and two factor interactions on the metabolism of urea, glucose, lactate, ketone bodies, albumin, and fibrinogen were evaluated. The cultured hepatocyte model exhibited physiologic responses to the applied stress mediators. While hydrocortisone and epinephrine had no effect, glucagon induced an increase in glucose and urea synthesis. Interleukin-6 increased fibrinogen and decreased albumin production. Furthermore, IL-6 and glucagon caused an increase in the ketone-body ratio (KBR = [acetoacetate]/[beta-hydroxybutyrate]), which is in equilibrium with the intramitochondrial NAD+/NADH. Tumor necrosis factor-alpha and IL-1beta, on the other hand, decreased the KBR. An important two-factor interaction between IL-1beta and IL-6 was identified, namely that IL-1beta effectively negates the positive effect of IL-6 on the KBR when both are present. These results provide further understanding of the effect of stress mediators on hepatic function and metabolism. These effects may have important implications in the pathogenesis of progressive organ dysfunction which often follows prolonged inflammatory states triggered by major injuries.

[Functional angiogenesis induction in epigastric islet flap rat model after genetic modification of fibroblasts]. / Funktionelle Angiogeneseinduktion im epigastrischen Insellappen-modell der Ratte nach genetischer Modifikation von Fibroblasten.

Gene therapy was tested for inducing functional angiogenesis in the superficial rat epigastric island flap to allow earlier pedicle division. Autologous rat fibroblasts were grown, harvested, cultured and retrovirally transfected to produce PDGF-AA, an angiogenetically active protein. Stable gene expression was monitored by PDGF-AA ELISA. 180 animals were divided into 3 groups (I-III) and a bilateral flap created in each animal. In all experiments, the rightsided flap was subjected to experimental treatment and the left-sided flap served as control (1 ml saline 0.9%). During flap elevation, group I received 5 x 10(6) GMFB (genetically modified fibroblasts) plus 1 ml DMEM as medium. Group II was treated with 5 x 10(6) NMFB (non modified fibroblasts) plus 1 ml medium and group III received 1 ml medium alone. The flaps were sutured back and the vascular pedicle was bilaterally ligated and divided in each 10 animals during the following 6 days. 7 days later, the flaps were harvested, the amount of necrosis measured and histologically examined. The GMFB produced up the 560-times more PDGF-AA than the NMFB, measured by ELISA. The GMFB-treated flaps tolerated surgical division of the vascular pedicle significantly earlier than groups II and III. Histologically, fibroblasts persisted in all flaps of groups I and II without major inflammatory reaction. In all GMFB-treated flaps, massive angiogenesis could be demonstrated. By means of retroviral gene transfer autologous rat fibroblasts can be genetically modified for stable expression of the PDGF-A gene to produce high amounts of PDGF-AA, which is angiogenetically active. After injection into the panniculus carnosus, these cells induce functional angiogenesis to permit earlier division of the vascular pedicle in this flap model.

Extracorporeal plasma perfusion of cultured hepatocytes: effect of intermittent perfusion on hepatocyte function and morphology.

The most promising approaches to developing a temporary bioartificial liver support system involve incorporating cultured primary hepatocytes into an extracorporeal perfusion device. As a result, it is important to characterize both the phenotypic response of these cells during extracorporeal perfusion and the critical factors involved in maintaining differentiated cell function over extended periods of perfusion. In this study, hepatocytes cultured in a collagen sandwich configuration were connected to a rat via a hollow fiber plasma separator and perfused with plasma on line. Perfusions were either continuous for 48 hr or intermittent for up to 174 hr with 6 hr per day of extracorporeal plasma perfusion alternating with 18 hr of culture medium perfusion. During perfusion cell morphology was continuously monitored by time-lapse video microscopy. After the procedure, hepatocytes were returned to static culture and function was evaluated by measuring the rates of urea synthesis daily for 7 days. During plasma perfusion all hepatocytes accumulated cytoplasmic lipid droplets in a time dependent manner. Urea synthesis was maintained at initial levels for up to 20 hr of continuous plasma perfusion. However, urea synthesis rates were reduced by 31 and 52% after 30 and 48 hr of continuous plasma exposure, respectively. With intermittent perfusions, as well as with control cells perfused with culture medium only, urea synthesis rates did not decrease for at least 78 hr of total perfusion. There was no difference between the urea synthesis rates after 48 hr of cumulative plasma exposure time between cells subjected to continuous and intermittent plasma perfusion. These results suggest that cultured hepatocytes may be exposed to plasma for at least 20 hr with no significant reduction in liver-specific function. Furthermore, an intermittent plasma perfusion schedule can be used to divide the useful plasma perfusion time over several days with no adverse effects on cell function.

Effects of plasma exposure on cultured hepatocytes: Implications for bioartificial liver support.

In order to examine their potential for use in a bioartificial liver, hepatocytes maintained in a collagen sandwich configuration were cultured for 9 days in heparinized rat plasma. The cells exhibited a progressive accumulation of cytoplasmic lipid droplets which proved to be mainly triglyceride (TG). The rate of TG accumulation correlated with the free fatty acid (FFA) content of the plasma. Removal of FFA and TG from plasma by ether extraction significantly reduced the rate and extent of TG accumulation. A smaller reduction in the rate and extent of TG accumulation was observed when cells were maintained in an oxygen enriched environment. The lipid accumulation suppressed urea synthesis, but clearance of the drug diazepam, although constitutively depressed in plasma, appeared unaffected by the accumulation. The functional and morphological effects of plasma exposure could be fully reversed after at least 6 days of plasma exposure by returning the cells to culture medium.The results indicate that elevated FFA in plasma induces lipid accumulation, which inhibits urea synthesis in cultured hepatocytes. This suggests that estimates of the cell number needed for effective liver support should not be based upon function measurements conducted in culture media. Furthermore, optimization of bioartificial liver support device use may have to be governed by the need to limit the plasma exposure of cultured hepatocytes. However, the highly responsive nature of these cultures and the reversibility of the plasma effects suggest that the collagen sandwich culture system is a promising foundation for the development of an effective bioartificial liver support system. (c) 1996 John Wiley & Sons, Inc.

Effects of hypothermia on the function, membrane integrity, and cytoskeletal structure of hepatocytes.

To increase the storage time of livers for transplantation, a better understanding of hypothermia-induced hepatocyte damage is necessary. To this end, we have characterized the effects of hypothermia on long-term function and cytoskeletal organization of hepatocytes cultured in the collagen sandwich configuration, which maintains the expression of liver-specific functions for several weeks. In these studies, cultured hepatocytes (maintained at 37 degrees C for 7 days) were exposed to 4 degrees C in Leibovitz-15 (L15), University of Wisconsin (UW) solution, or L15 supplemented with 2.5 g% polyethylene glycol (PEG) for various time periods followed by a return to normothermia. When L15 medium was used, the long-term albumin secretion rate of cultured hepatocytes was decreased by 50% after 4 h, and by 95% after 24 h of exposure to 4 degrees C. Amorphous precipitates of F-actin and fragmented short microtubules were also observed after 4 and 12 h of hypothermia, respectively. Similar results were obtained when hepatocytes were stored in UW solution. However, in L15 supplemented with PEG, no significant reduction in long-term albumin secretion rates and intact actin and microtubule morphology was observed even after 24 h of exposure to 4 degrees C. The membrane integrity and long-term albumin secretion of hepatocytes stored in the presence of PEG were decreased to approximately 50% only after 48 h of exposure to 4 degrees C. Thus, PEG may be a useful additive in preservation solutions for hepatocytes in hepatocyte-based liver support systems and for intact tissue as well.

Disturbed esophageal motility after total gastrectomy.

Esophageal manometry with a catheter microtransducer was performed as a functional diagnostic method on 30 patients after total gastrectomy because of gastric cancer (18 men, 12 women with a mean age of 64 +/- 3.7 years). Subsequently their symptoms were recorded. 21 of the patients (70%) complained of reflux discomfort and symptoms of disturbed peristalsis (dysphagia, odynophagia). 29 patients (93%) showed pathological patterns of contraction (repetitive, simultaneous, deformed, multipeak contractions) especially in the distal part of the esophagus. The contractile force was decreased on average by 10 mmHg in that area. The resting pressure of the upper sphincter was also decreased by about 10 mmHg. These results can be explained by an increased postoperative reflux (absence of the lower sphincter) and the changed biomechanics of the esophagus (decreased longitudinal tension) caused by the operation. The results of this study demonstrate the importance of postoperative manometry in total gastrectomized patients.

Desensitization of adenosine A2 receptors in the striatum of the rat following chronic treatment with diazepam.

Following prolonged treatment (7 days) with diazepam (10 mg/kg/day, using ALZET mini-osmotic pumps) in rats, the function of adenosine receptors was assessed in specific structures of the brain, using both agonist ligand binding and adenylate cyclase assays. Binding to A1 receptors was quantified using [3H]N6-[(R)-1-methyl-2-phenylethyl] adenosine, a selective ligand at A1 receptors. Differences in the binding of this ligand and that of [3H]5'-N-ethylcarboxamide adenosine, which binds to both A1 and A2 subtypes of receptors with similar affinities, were used to quantify A2 receptors. Treatment with diazepam failed to alter the binding of [3H]N6-[(R)-1-methyl-2-phenylethyl] adenosine in all areas of the brain studied. However, the binding of A2 receptors and A2 receptor-mediated stimulation of adenylate-cyclase were significantly attenuated in striatal membranes from diazepam-treated rats. Thus, the present study indicated that functional adenosine A2 receptors were desensitized after prolonged treatment with diazepam, since decreased agonist binding to A2 receptors paralleled an attenuation in the stimulation by adenosine of the activity of adenylate cyclase, an effect mediated by the A2 receptor. These results further indicate that the changes in adenosine A2 receptors correlated with significant short-lasting alterations in the sleep-wake cycle during the withdrawal of diazepam. The alterations in sleep-wakefulness did not correlate with the effect of diazepam on benzodiazepine receptors since no changes were observed in the binding of benzodiazepine receptors.