Heat killed multi-serotype Shigella immunogens induced humoral immunity and protection against heterologous challenge in rabbit model.

Recently we have shown the homologous protective efficacy of heat killed multi-serotype Shigella (HKMS) immunogens in a guinea pig colitis model. In our present study, we have advanced our research by immunizing rabbits with a reduced number of oral doses and evaluating the host's adaptive immune responses. The duration of immunogenicity and subsequently protective efficacy was determined against wild type heterologous Shigella strains in a rabbit luminal model. After three successive oral immunizations with HKMS immunogens, serum and lymphocyte supernatant antibody titer against the heterologous shigellae were reciprocally increased and remained at an elevated level up to 180 days. Serogroup and serotype specific O-antigen of lipopolysaccharide and immunogenic proteins of heterologous challenge strains were detected by immunoblot assay. Up-regulation of IL-12p35, IFN-γ and IL-10 mRNA expression was detected in immunized rabbit peripheral blood mononuclear cells (PBMC) after stimulation with HKMS in vitro. HKMS-specific plasma cell response was confirmed by production of a relatively higher level of HKMS-specific IgG in immunized PBMC supernatant compared to control group. Furthermore, the immunized groups of rabbits exhibited complete protection against wild type heterologous shigellae challenge. Thus HKMS immunogens induced humoral and Th1-mediated adaptive immunity and provided complete protection in a rabbit model. These immunogens could be a broad spectrum non-living vaccine candidate for human use in the near future.

Role of Shigella flexneri 2a 34 kDa outer membrane protein in induction of protective immune response.

Emergence of Shigella vaccine is in great need in developing countries. In this paper we have shown that 34 kDa Shigella flexneri 2a outer membrane protein has a role in eliciting immune responses. When injected parentarally this protein gives significant protection against challenge with virulent Shigella flexneri 2a. Macrophages activated with the 34 kDa protein resulted in the dose dependent production of nitric oxide, the highly reactive free radical responsible for killing of invading bacterial pathogen. Also, treatment of murine peritoneal macrophages with the 34 kDa protein showed dose dependent increase in the production of tumor necrosis factor-alpha and interleukin-12. However, there was no dose dependent increase in interleukin-10 production. These data indicated that the 34 kDa outer membrane protein has the ability to modulate the protective immune response against the invading bacterial pathogen, mainly through TH1 mediated pathway. So, the 34 kDa outer mebrane protein can be one of the major components for developing subunit vaccine against shigellosis.

Enterotoxigenicity of mature 45-kilodalton and processed 35-kilodalton forms of hemagglutinin protease purified from a cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain.

Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 microg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 microg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21.

Protective efficacy of oral immunization with heat-killed Shigella flexneri 2a in animal model: study of cross protection, immune response and antigenic recognition.

Oral immunization of rabbits with four doses of 10(11) heat-killed Shigella flexneri 2a showed 100% protection against challenge with virulent S. flexneri 2a. After orally immunizing Guinea pigs with four doses of heat-killed S. flexneri 2a 100% protection could be shown against ocular challenge with the same virulent S. flexneri 2a strain but this conferred no protection against challenge with Shigella dysenteriae type 1. In enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments both whole cell lysate-envelope (WCL-E) fraction and outer membrane proteins (OMPs) were recognized by the antisera. Though protective mechanism in shigellosis is not established with certainty, outer membrane proteins (specially 38, 34, 23 and 20kDa proteins) may be the major antigens in the induction of protective immune responses as indicated by this observation.

Zinc supplementation as adjunct therapy in children with measles accompanied by pneumonia: a double-blind, randomized controlled trial.

BACKGROUND: Zinc deficiency, common in developing countries, is associated with decreased immunocompetence. Zinc supplementation benefits children with acute and persistent diarrhea and prevents pneumonia. Most deaths from vaccine-preventable diseases are from measles and whooping cough; pneumonia is the most common complication of measles and often the proximate cause of related deaths. OBJECTIVE: We evaluated the effect of zinc supplementation on episodes of illness in children with measles accompanied by pneumonia. DESIGN: In a double-blind, randomized controlled trial, children aged 9 mo-15 y who were admitted to the Infectious Diseases Hospital in Calcutta with clinically severe measles accompanied by pneumonia and who had been ill for

Evidence of calcium influx across the plasma membrane depends upon the initial rise of cytosolic calcium with activation of IP(3) in rat enterocytes by heat-stable enterotoxin of Vibrio cholerae non-O1.

In response to heat-stable enterotoxin of Vibrio cholerae non-O1, the initial rise of cytosolic Ca(2+) occurred with activation of IP(3). Chelation of extracellular Ca(2+) with EGTA and suspension of cells in Ca(2+) free buffer both demonstrated the involvement of internal stores in the rise of [Ca(2+)]i. Cells pretreated with dantrolene resulted in decrease of [Ca(2+)]i response which suggested that the rise of intracellular level of Ca(2+) was mostly due to the mobilization from IP(3) sensitive stores. When the cytosolic Ca(2+) was chelated by loading the cells with BAPTA, NAG-ST could not induce Ca(2+) entry to the cell as assessed by Mn(2+) quenching of fura-2 fluorescence which suggested that calcium influx across the plasma membrane depends upon initial rise of this bivalent cation that maintained the sustained phase of [Ca(2+)]i response. Addition of toxin to the fura-2-loaded cells, preincubated with lanthanum chloride, resulted in reduction of [Ca(2+)]i level with a short duration of irregular sustained phase further suggesting that the influx of Ca(2+) across the plasma membrane might be through the calcium channel.

Rise in free intracellular calcium in HeLa cells infected with aggregative Klebsiella pneumoniae strains isolated from cases of diarrhoea.

BACKGROUND & OBJECTIVES: Klebsiella pneumoniae strains occasionally cause diarrhoea in humans. This study was done to determine the involvement of calcium in the pathogenesis of aggregative K. pneumoniae strains. METHODS: A total of nine strains of K. pneumoniae were tested for adherence assay in HeLa cell line. A representative strain CO-1215 was used for [Ca2+]i study using Fura-2 fluorescence. RESULTS: Infection of cultured HeLa cells with aggregative K. pneumoniae strain resulted in five-fold elevation of intracellular level of free calcium ([Ca2+]i) with maximum Ca2+ influx at 3 h after bacterial infection. Chelation of extracellular Ca2+ with [ethylenebis(oxyethylenenitrile)] tetraacetic acid and suspension of cells in Ca2+ free buffer suggested that the rise of Ca2+ in aggregative K. pneumoniae infected HeLa cells was due to influx of Ca2+ from extracellular medium. INTERPRETATION & CONCLUSIONS: This study showed aggregative adherence in HeLa cells and this adherence leads to influx of extracellular Ca2+. The unrestricted passage of calcium ions across cell membranes could cause phosphorylation of proteins involved in ion transport across the membrane, which could result in secretory diarrhoea. Further work is in progress to study the enterotoxicity of these strains in an in vitro rabbit intestinal model.

Escherichia coli heat stable enterotoxin receptors & guanylyl cyclase activity in the intestinal brush border membrane of hamsters & guinea pigs.

BACKGROUND & OBJECTIVES: Although Escherichia coli heat stable enterotoxin (STa) causes diarrhoea in laboratory animals, no studies were done to find out the species specific variation of distribution of the STa receptors in laboratory animals. The present investigation evaluates the density of STa receptors and the guanylyl cyclase (GC) activity in the small intestinal epithelial cells of hamsters and guinea pigs. METHODS: Brush border membrane (BBM) was prepared from the small intestines of hamsters and guinea pigs. Receptor binding assay, GC assay and autoradiography were performed to determine the density of STa receptors, the GC activity and molecular weights of the STa binding proteins respectively. RESULTS: The receptor densities, per mg BBM protein at equilibrium, were found to be 4.1 x 10(9) and 1.5 x 10(12) in hamsters and guinea pigs respectively. The GC activity was found to be lower in STa treated hamster BBM compared to that of guinea pig. Scatchard analysis of the stoichiometric data showed a linear plot, and STa bound with association constants of 0.31 x 10(12) M-1 and 1.04 x 10(12) M-1 in hamsters and guinea pigs respectively. Autoradiographic analysis of the SDS-PAGE, revealed that 125I-STa bound apparently to a 45 kDa membrane protein in hamster and a 115 kDa membrane protein in guinea pig. INTERPRETATION & CONCLUSIONS: It appears that a lower density of STa receptor exists in hamsters compared to that in guinea pigs. STa binds with a single population of STa receptors in each species with different ligand binding affinities. Also, the molecular weights of the STa binding proteins differ in these species. Moreover, the GC activity was found to be lower in hamsters than in guinea pigs.

Comparison of the effects of inhalational anaesthetic agents on sympathetic activity in rabbits.

The effects of inhalational anaesthetic agents on renal sympathetic nerve activity (RSNA) were compared in anaesthetized rabbits. Concentrations of 6% desflurane, 1.2% isoflurane, and 2.4% enflurane increased mean RSNA up to 32, 36 and 44% while higher concentrations, of 12, 2.4 and 3.2% depressed it by 42, 83 and 5%, respectively. For halothane RSNA was unchanged up to 0.8% and decreased by 36% at 1.6% concentration. Nitrous oxide increased RSNA up to 28% at 50% concentration. Maximum reductions in mean arterial pressure (MAP) were 60% for both 2.4% isoflurane and 3.2% enflurane, 50% for 12% desflurane and 1.6% halothane, while 70% nitrous oxide increased MAP by 22%. In conclusion, unlike the entirely depressive effects of halothane, the effects of desflurane, isoflurane and enflurane were biphasic involving excitation at lower concentrations and depression of RSNA and a reduction in MAP at higher concentrations. Nitrous oxide caused increases in both RSNA and MAP.

Vagally mediated sympathoexcitation and central depression by desflurane in rabbits.

The effects of desflurane on renal sympathetic nerve activity (RSNA) were studied in intact or vagotomized anaesthetized rabbits with initial concentrations of 4.5-18%, subsequently equilibrated to end-tidal concentrations from 3%, 6%, 9% and 12% each for 20 min allowing sympathetic activity to stabilize. In intact animals, immediate transient increases in mean sympathetic activity from 27% to 63% were closely related to initial concentrations from 4.5% to 18%. During subsequent equilibration this remained elevated by 25-30% up to 6%, returned to control at 9% and fell by 33% at 12%. Bilateral vagotomy abolished sympathoexcitation apart from small increases in sympathetic activity, for example 14% at 4.5% (P < 0.05). We conclude that increases in inspired desflurane concentrations evoked rapid transient vagally mediated reflex sympathoexcitation with a small extra-vagal contribution. Central depression of sympathetic activity started at 6% and was 33% below baseline at 12%.

The effects of propofol on heart rate, arterial pressure and adelta and C somatosympathetic reflexes in anaesthetized dogs.

The effects of propofol on mean arterial pressure, heart rate and Adelta and C somatosympathetic reflexes, recorded in renal nerves, evoked by repeated individual supramaximal electrical stimuli applied to radial nerves, were observed in anaesthetized, paralysed and artificially ventilated dogs. Propofol was infused at rates from 0.4 to 2.0 mg kg-1 min-1. Mean C and Adelta reflexes were abolished at plasma concentrations (mean, SEM) of 24.3 (3.3) and 29.2 (2.6) microg mL-1 (P < 0.05), respectively, when mean arterial pressure and mean heart rate were reduced by approximately 55% (P < 0.01) and 26% (P > 0.05), respectively. Recovery of Adelta and C reflexes occurred at plasma concentrations of 13.1 (2.3) and 9.9 (1.3) microg mL-1 (P > 0.05), respectively. There was a log- arithmically linearly related fall in mean arterial pressure by 70% up to a plasma concentration approximately 97 microg mL-1 (r 2=0.7) with a 28% reduction in heart rate which was uncorrelated with the plasma concentrations (r 2=0.12). In conclusion, propofol abolished Adelta and C responses at comparable plasma concentrations and caused a major reduction in both mean arterial pressure and heart rate which is consistent with resetting of the baroreflexes. The reduction in mean arterial pressure was logarithmically, linearly correlated with a progressive increase in plasma concentrations without evidence of a ceiling effect.

Effects of propofol and remifentanil on phrenic nerve activity and nociceptive cardiovascular responses in rabbits.

BACKGROUND: The effects of propofol, remifentanil, and their combination on phrenic nerve activity (PNA), resting heart rate (HR), mean arterial pressure (MAP), and nociceptive cardiovascular responses were studied in rabbits. METHODS: Basal anesthesia and constant blood gas tensions were maintained with alpha-chloralose and mechanical ventilation. PNA, HR, MAP, and maximum changes in HR and MAP (deltaHR, deltaMAP) evoked by electrical nerve stimulation of tibial nerves were recorded. The comparative effects were observed for propofol at infusion rates from 0.05 to 3.2 mg x kg(-1) x min(-1) (group I) and remifentanil from 0.0125 to 12.8 microg x kg(-1) x min(-1) alone (group II), and during constant infusions of propofol at rates of 0.1 and 0.8 mg x kg(-1) x min(-1) (groups III and IV, respectively). Finally, the effect of remifentanil on propofol blood levels was observed (group V). RESULTS: The infusion rates for 50% depression (ED50) of PNA, deltaHR, and deltaMAP were 0.41, 1.32, and 1.58 mg x kg-(1) x min(-1) for propofol, and 0.115, 0.125, and 1.090 microg x kg(-1) x min(-1) for remifentanil, respectively. The ratios for the ED50 values of deltaHR and deltaMAP to PNA were 3.2 and 3.9 for propofol, and 1.1 and 9.5 for remifentanil, respectively. Analysis of the expected and observed responses and isobologrms showed that although their combined effects on PNA, resting HR, and MAP, and deltaMAP were synergistic for deltaHR, they were merely additive. Remifentanil had no effect on propofol blood levels. CONCLUSION: PNA was abolished by propofol and remifentanil, alone and in combination, before significant depression of nociceptive pressor responses occurred. Their combined effects on PNA, HR, MAP, and deltaMAP are greater than additive, ie., synergistic. Unlike propofol, remifentanil obtunded pressor responses more than the resting circulation.

Morphological and cytoskeletal changes caused by non-membrane damaging cytotoxin of Vibrio cholerae on int 407 and HeLa cells.

Vibrio cholerae produces a non-membrane damaging cytotoxin (NMDCY), also known as cell rounding factor, which causes rapid rounding of cultured cells like HeLa, CHO and Vero and reportedly elicits enterotoxic activity in the rabbit ileal loop assay. Pursuing the concept that NMDCY might be an accessory factor contributing to the diarrhea caused by V. cholerae, we investigated the effect of NMDCY on Int 407 (intestinal cell line) and HeLa (non-intestinal cell line) cells using light, fluorescent and electron microscopy to gain insight into the cellular response evoked by NMDCY. Binding assays showed that NMDCY has affinity for both Int 407 and HeLa cells. Changes in the internal organelles and cytoskeletal structures of the cell lines were documented indicating changes in the secretory and metabolic function of the toxin-treated cells. Toxin-treated cells visualized under the electron microscope revealed retraction of cell body, formation of blebs on cell surface, changes in mitochondria having dilated and rarefied matrix and an extensively developed Golgi apparatus, endoplasmic reticulum and lysosomes compared to those in normal cells. Immunofluorescence study showed restructuring of microfilament network represented by actin, filamin and vinculin, as also of the microtubular component, tubulin and the intermediate filament, vimentin. Immunogold study further revealed that the toxin is internalized even within the nucleus. Moreover, a rise in the intracellular calcium level of the NMDCY-treated cells leads us to hypothesize that a cascade of events results in the final impairment of the cell machinery.

Specific actions of halothane, isoflurane, and desflurane on sympathetic activity and A delta and C somatosympathetic reflexes recorded in renal nerves in dogs.

BACKGROUND: This was a study of the relative effects on directly recorded sympathetic activity of desflurane, isoflurane, and halothane. METHODS: Renal sympathetic nerve activity (RSNA) was recorded with bipolar electrodes in renal nerves exposed retroperitoneally in anesthetized (alpha-chloralose), paralyzed (succinylcholine), and artificially ventilated dogs. Somatosympathetic responses were evoked by supramaximal electrical stimulation of radial nerves (0.33 Hz, 30 V, 0.5 ms). Spontaneous and evoked activity were rectified, averaged, and integrated to allow quantitative comparison of the effects of 3-12% desflurane, 0.6-2.4% isoflurane, and 0.4-1.6% halothane. RESULTS: Increasing concentrations of isoflurane progressively depressed mean RSNA, Adelta, and C reflexes by 40% (P < 0.01), 50% (P < 0.01) and 70% (P < 0.001) respectively at 2.4% concentration. Halothane depressed both reflexes equally by approximately 60% (P < 0.01) at 1.6% concentration, without significant depression of spontaneous RSNA. Desflurane increased and subsequently decreased RSNA by 37% (P < 0.02) and 65% (P < 0.001) at concentrations of 6% and 12% respectively, and although somatosympathetic reflexes remained unchanged up to 9%, both were depressed equally by 70% (P < 0.01) at 12% concentration. CONCLUSION: After equilibration, lower concentrations of desflurane remained excitatory, but, like isoflurane, higher concentrations depressed RSNA. The effect of halothane on RSNA was insignificant. Isoflurane depressed C more than Adelta somatosympathetic reflexes, which is uncorrelated with lipid solubility because desflurane and halothane, which have the highest and lowest minimum alveolar concentration, respectively, depressed both equally.

Propofol, bradycardia and the Bezold-Jarisch reflex in rabbits.

Propofol may cause profound bradycardia and asystole, which are mediated indirectly via cardiac innervation but could involve direct effects on the sino-atrial (SA) node and the conducting system of the heart. To test the hypothesis that propofol may also activate Bezold-Jarisch reflexes to cause bradycardia, 5-hydroxytryptamine (5-HT), veratridine and propofol were injected into the left ventricle of the heart in both intact and vagotomized rabbits. 5-HT and veratridine produced an acute, rapid, dose-dependent decrease in mean heart rate (delta HR) and a decrease in mean arterial pressure (delta MAP) together with transient but severe depression and abolition of renal sympathetic nerve activity (RSNA). Bilateral vagotomy greatly attenuated these responses; for example, at the highest dose of 5-HT (8 micrograms kg-1), delta HR, delta MAP and duration of abolition of RSNA were reduced by 57% (P < 0.001), 53% (P < 0.05) and 79% (P < 0.05), respectively. In contrast, reductions in delta HR and delta MAP produced by propofol were statistically significant only at very high doses (8 mg kg-1). Propofol depressed but did not abolish RSNA, and bilateral vagotomy had no effect on any of these responses. These results indicate that the cause of acute bradycardia after administration of propofol does not involve the Bezold-Jarisch reflex.

The combined effects of sevoflurane and remifentanil on central respiratory activity and nociceptive cardiovascular responses in anesthetized rabbits.

UNLABELLED: We studied the effects of sevoflurane and remifentanil, alone and in combination, on phrenic nerve activity (PNA), resting heart rate (HR), arterial pressure (MAP), and changes in HR (delta HR) and MAP (delta MAP) evoked by electrical stimulation of tibial nerves in anesthetized rabbits. The 50% effective dose (95% confidence intervals) for the depressant effects of sevoflurane on delta HR, delta MAP, and PNA were 2.3 (1.8%-2.6%), 2.7 (2.3%-2.9%), and 3.4 (3.1%-3.7%), respectively, and for remifentanil were 0.100 (0.050-0.132), 0.850 (0.720-1.450), and 0.090 (0.080-0.145) microgram.kg-1.min-1, which were reduced to 0.046 (0.021-0.065), 0.110 (0.080-0.200), and 0.030 (0.020-0.040) microgram.kg-1.min-1, respectively, by 1% sevoflurane. Depression of evoked cardiovascular responses relative to PNA was greater for sevoflurane and less for remifentanil both alone and in combination with sevoflurane. Sevoflurane acted synergistically with remifentanil on PNA and delta MAP, but not delta HR, for which their combined effect was additive. Coadministration of 1% sevoflurane with the highest infusion rate of remifentanil (1.6 micrograms.kg-1.min-1) used during combined administration reduced resting HR and MAP by 25% (P < 0.05) and 41% (P < 0.05), respectively, which was greater than the predicted reductions of only 14% and 15% if their combined effects had been additive. We conclude that sevoflurane caused a relatively greater depression of nociceptive cardioaccelerator and pressor responses compared with PNA and vice versa for remifentanil. When coadministered, their combined effects on PNA, resting HR, MAP, and delta MAP were synergistic, whereas they were merely additive for delta HR. IMPLICATIONS: Although sevoflurane caused relatively greater depression of nociceptive cardiovascular responses compared with phrenic nerve activity, remifentanil either alone or combined with sevoflurane caused a much greater depression of phrenic nerve activity than cardio-accelerator and pressor responses. This could imply that, during major surgery using anesthesia combining sevoflurane and remifentanil, spontaneous ventilation is not acceptable, and depression of the resting circulation may be much greater than anticipated.

Killed oral Shigella vaccine made from Shigella flexneri 2a protects against challenge in the rabbit model of shigellosis.

The protective efficacy of an orally administered heat-killed virulent strain of Shigella flexneri 2a (5 weekly oral doses) was evaluated in 25 rabbits (14 immunized and 11 non-immunized controls) against challenge with the same strain of Shigella using the rabbit model of shigellosis. All 11 non-immunized rabbits developed bloody diarrhoea following challenge and 6 (54%) died. None of the 14 immunized rabbits developed diarrhoea (all had pellet stools) but 3 (21%) died from causes not associated with diarrhoea. Protection from diarrhoea and dysentery following oral immunization with a killed Shigella species was 100% and highly significant. Death following challenge was 2.5-fold higher in the non-immunized group (p = 0.115) but was not significant. These promising results suggest that further studies should be undertaken to develop a killed oral vaccine against shigellosis.

Binding of Escherichia coli heat-stable toxin and rise of guanylyl cyclase activity in the brush-border membranes of rabbit intestinal epithelial cells.

The study examines the age-related differences in the density of Escherichia coli heat-stable enterotoxin (STa) receptors in the small intestine of rabbits. The number of STa receptors was found to be 1.7 x 10(12) in 14-day old rabbits compared to 2.4 x 10(9) in 14-week old rabbits per milligram brush-border membrane protein. The STa-induced guanylyl cyclase activity in the intestinal brush-border membranes was found to be stimulated by 6.2 folds over the basal enzyme activity in 14-day old rabbits, whereas in the 14-week old rabbits, it was 4 folds over the basal activity. Moreover, the enzyme activity remained lower in the adult rabbits compared to the younger ones. Autoradiographic analysis of sodium dodecyl sulphate polyacrylamide gel electrophoresis showed two STa-binding proteins of apparent molecular weights of 140 and 38 kDa in the intestinal brush-border membranes of rabbits.

Synergistic antinociceptive interaction between sevoflurane and intrathecal fentanyl in dogs.

This study determined the nature of the antinociceptive interaction between sevoflurane and intrathecal fentanyl on somatosympathetic reflexes in anaesthetized dogs. Afferent A delta- and C-fibre-mediated somatosympathetic reflexes, evoked by supramaximal electrical stimulation of tibial nerves, were recorded from renal sympathetic nerves. The effect of fentanyl alone, administered intrathecally (i.t.) in incremental doses from 2 to 64 micrograms, was compared with the effect of the same doses during the administration of 1.5% sevoflurane. The mean ED50s for the depressant effect of fentanyl (i.t.) on A delta and C reflexes were 35.6 micrograms and 14.2 micrograms while 1.5% sevoflurane, when administered alone, depressed them by 15.5% (P < 0.05) and 27.5% (P < 0.01) respectively. During the administration of 1.5% sevoflurane, the mean ED50s of fentanyl (i.t.) for the depression of A delta and C reflexes were reduced by 76% and 75%, to 8.5 micrograms and 3.5 micrograms respectively. The combined antinociceptive effects of sevoflurane and intrathecal fentanyl were not additive but exhibited a high degree of synergistic interaction.

Synergistic interaction between the effects of propofol and midazolam with fentanyl on phrenic nerve activity in rabbits.

BACKGROUND: It has been shown that the depressive effects of both propofol and midazolam on consciousness are synergistic with opioids, but the nature of their interactions on other physiological systems, e.g. respiration, has not been fully investigated. The present study examined the effect of propofol and midazolam alone and in combination with fentanyl on phrenic nerve activity (PNA) and whether such interactions are additive or synergistic. METHODS: PNA was recorded in 27 anaesthetised and artificially ventilated rabbits. In three groups, propofol, fentanyl and midazolam were administered intravenously in incremental doses to construct dose-response curves for the depressant effects of each one on PNA. In another two groups, the effect of pretreatment with either fentanyl 1 microgram.kg-1 i.v. or midazolam 0.05 mg.kg-1 i.v. on the effects of propofol and fentanyl respectively on PNA were studied. RESULTS: Propofol and fentanyl caused a dose-dependent depression of PNA with complete abolition at the highest total doses of 16 mg.kg-1 i.v. and 32 micrograms.kg-1 i.v., respectively. In contrast, midazolam in incremental doses to a total of 0.8 mg.kg-1 reduced mean PNA by 63%, but approximately 12% of PNA remained at a total dose as high as 6.4 mg.kg-1. The mean ED50s, calculated from dose-response curves, were 5.4 mg.kg-1, 3.9 micrograms.kg-1 and 0.4 mg.kg-1 for propofol, fentanyl and midazolam, respectively. Initial doses of either fentanyl 1 microgram.kg-1 i.v. or midazolam 0.05 mg.kg-1 i.v. acted synergistically with subsequent doses of either propofol or fentanyl to abolish PNA at total doses of 8 mg.kg-1 and 8 micrograms.kg-1, respectively. CONCLUSION: Fentanyl has a synergistic interaction with both propofol and midazolam on PNA and hence potentially on respiration.