Pancreatic sphincter precutting to gain selective access to the common bile duct: a series of 172 patients.

BACKGROUND AND STUDY AIMS: Several precutting techniques have been described in cases of failed access to the common bile duct. We describe our experience with pancreatic sphincter precutting in an upward direction, and report its success rates and complications. PATIENTS AND METHODS: A total of 172 patients underwent a procedure using this technique between January 1989 and December 2001. The technique consisted of a medium-to-large precut along the midline, above the papillary elevation, using either the common channel or the pancreatic duct in the ampulla of Vater as a guide. The septum between the pancreatic duct and the bile duct was removed and separate openings to the pancreatic and bile ducts were created, followed by complete biliary sphincterotomy. RESULTS: Biliary cannulation and sphincterotomy was successful in 163 of the 172 study patients (95 %). Mild complications, which were all managed conservatively, occurred in 17 patients (10 %). This complication rate was significantly higher than our complication rate for standard endoscopic sphincterotomy, which was 0.8 % in 1770 patients ( P < 0.0001). CONCLUSIONS: Pancreatic sphincter precutting is an effective and safe technique for patients in whom selective cannulation of the common bile duct has failed. Further prospective comparative studies of other precutting techniques will better define its clinical value.

Apoptosis in the hydropic cochlea of guinea pigs following immune reaction of the endolymphatic sac: immunohistochemical analysis.

Apoptotic change in the cochlea was studied by immunohistochemistry after the injection of keyhole limpet haemocyanin (KLH) into the right endolymphatic sac of guinea pigs. Apoptosis was examined with the specific antibody to single-stranded DNA (ssDNA). Endolymphatic hydrops became evident in the cochlea 1 day after the injection of KLH (n = 6). Increased ssDNA expression could be detected in the spiral ligament and the stria vascularis. The temporal bones in the control group did not show any ssDNA immunoreactivity (n = 6). Apoptosis is the process of the cell death. Our findings imply that apoptotic changes are involved in endolymphatic hydrops. These phenomena could lead to cochlear dysfunction as seen in endolymphatic hydrops.

Detection of apoptotic change in the lipopolysaccharide (LPS)-treated cochlea of guinea pigs.

This study was undertaken to examine, electrophysiologically and immunohistochemically, the effect of endotoxin on the guinea pig cochlea. A bacterial endotoxin (lipopolysaccharide, LPS, 5 mg/ml, 0.2 ml) was injected into the middle ear trans-tympanically. The electrocochleograms were continuously recorded from before to 48 h after the injection with an electrode inserted into the facial canal. Then, the animals were sacrificed by intracardiac perfusion of a fixative, temporal bones were removed and immunohistochemically stained for single-stranded DNA (ssDNA) and caspase 3 (CPP32). ssDNA was detected at 48 h in the stria vascularis and spiral ligament. CPP32 was observed in the stria vascularis, the spiral ligament and the organ of Corti. The threshold of the compound action potential increased significantly at 48 h in the LPS group. These results suggest that the activation of CPP32 and fragmentation of DNA are involved in the dysfunction of the cochlea observed under inflammatory conditions.

Detection of single-stranded DNA (ssDNA) in the vestibule of guinea pigs after the application of cisplatinum (CDDP).

BACKGROUND: Cisplatinum (CDDP) has a toxic effect on the inner ear. CDDP reacts with DNA and leads to apoptotic cell death. In this study, the presence of single-stranded DNA (ssDNA) was examined immunohistochemically. MATERIALS AND METHODS: CDDP (10 mg/kg b.w.) was injected into the guinea pigs intra-peritoneally. Animals were sacrificed three days after the injection. The temporal bones were fixed via cardiac infusion. The tissues were embedded in paraffin and then used for immunohistochemical examination. RESULTS: That ssDNA was detected in the CDDP treated vestibule. The sensory epithelium, transitional area, dark cells and vestibular ganglion cells exhibited a positive immunoreactivity to ssDNA. CONCLUSIONS: DNA fragmentation is a characteristic feature of apoptosis. Fragmented DNA is detected as ssDNA. Our results indicate that CDDP involves the apoptosis in the inner ear and this is the cause of the ototoxicity of CDDP.

Expression of myeloperoxidase and cochlear dysfunction in the lipopolysaccharide-treated guinea pig.

In this study, the effect of endotoxin on the guinea pig cochlea was examined by electrophysiology and immunohistochemistry. Bacterial lipopolysaccharide (LPS) was injected into the middle ear transtympanically. Electrocochleograms were measured immediately and 48 h after the injection with an electrode inserted into the facial canal. After the electrophysiological measurement, the animals were killed with an intracardiac perfusion of fixative and the temporal bones were removed and processed for immunohistochemistry with anti-myeloperoxidase (MPO) antibody. MPO could be detected after 48 h in the lateral wall and the organ of Corti. After injection of LPS, the threshold of the compound action potential worsened significantly at 48 h in the LPS group. These results suggest that MPO and reactive oxygen species are involved in cochlea dysfunction under inflammatory conditions.

Familial amyloidotic polyneuropathy (ATTR Val30Met) with widespread cerebral amyloid angiopathy and lethal cerebral hemorrhage.

We report an autopsy case of familial amyloidotic polyneuropathy (FAP) with cerebral hemorrhage. A 38-year-old woman with a typical FAP pedigree started developing severe diarrhea and sensori-motor polyneuropathy at the age of 28 years; autonomic nervous system, heart and renal dysfunction manifested themselves in the following years. Genetic analysis revealed a single amino acid substitution at codon 30 of transthyretin (ATTR Val30Met). Ten years after her initial symptoms, the patient died of a sudden convulsive attack and respiratory failure. Autopsy revealed lethal cerebral hemorrhages and uremic lungs. Histochemical and immunohistochemical analyses revealed TTR-derived amyloid protein in every tissue examined, particularly in glomeruli and peripheral vessels. Severe meningo-cerebrovascular amyloidosis was also detected. Because uremia causes oxidative damage to the vascular system and amyloid formation is closely associated with oxidative stress, it is possible that uremic endothelial damage facilitated an unusual cerebral amyloid deposition. In typical FAP (ATTR Val30Met), cerebral amyloid angiopathy does not usually have clinical manifestations. However, cerebral amyloid angiopathy should be considered to explain FAP symptoms when some risk factors such as uremic vascular damage are accompanying features.

Expression of inducible nitric oxide synthase in the cochlea following immune response in the endolymphatic sac of guinea pigs.

Immunohistochemical study for inducible nitric oxide synthase (iNOS or NOS II) in the cochlea of guinea pigs was performed after the injection of keyhole limpet hemocyanin (KLH) into the endolymphatic sac. Morphological changes were observed in the cochlea of all animals after the injection of KLH. Increased iNOS expression was detected in the lateral wall, organ of Corti and ganglion cells. It is known that high levels of nitric oxide can lead to inner ear dysfunction. Our results suggest that iNOS may mediate the inner ear disturbance as seen in endolymphatic hydrops.

Mechanism of endothelin 1 production in the cochlea of rats.

The purpose of this study was to elucidate the mechanism of endothelin 1 (ET1) production in the cochlea of rats. Animals were anesthetized with pentobarbital sodium intraperitoneally and sacrificed by cardiac perfusion with warm saline followed by periodate-lysine-paraformaldehyde. The temporal bones and kidneys were fixed overnight in the same fixative, decalcified with 5% EDTA and embedded in paraffin. Immunohistochemical staining was performed using the labeled streptavidin biotin method to detect the localization of big endothelin 1 (BET1), ET1 and endothelin-converting enzyme 1 (ECE1). Immunoreactivity for BET1, ET1 and ECE1 was localized to the small vessels of the bony labyrinth adjacent to the spiral ligament. Immunohistochemistry for ET1 and BET1 was also localized to the spiral ligament. These results suggest that ET1 may be produced in the small vessels of the bony labyrinth adjacent to the spiral ligament and may play an important role in the cochlear function.

Expression of inducible nitric oxide synthase (iNOS/NOS II) in the hydropic vestibule after injection of keyhole limpet hemocyanin into the endolymphatic sac of guinea pigs.

This study was undertaken to examine the expression of inducible nitric oxide synthase (iNOS / NOS II) in the hydropic vestibule of guinea pigs. Animals were systemically sensitized with 500 microg of keyhole limpet hemocyanin. Two weeks after the first injection, keyhole limpet hemocyanin (100 microg/5 microl) was injected into the endolymphatic sac following the intradural approach, and the next day temporal bones were removed for the immunohistochemical examination. Endolymphatic hydrops was evidenced by the expansion of the Reissner's membrane in the cochlea after direct injection of keyhole limpet hemocyanin into the endolymphatic sac. Inducible nitric oxide synthase expression was increased in the sensory cells, supporting cells and vestibular ganglion cells, while temporal bones, where only phosphate buffered saline was injected, did not show any inducible nitric oxide synthase immunoreactivity. High levels of inducible nitric oxide synthase-catalyzed nitric oxide were detected prior to the development of the inner ear dysfunction. Our results suggest that the occurrence of inducible nitric oxide synthase immunoreactivity parallels the inner ear disturbance as seen in endolymphatic hydrops.

Induction of apoptotic pathway in the vestibule of cisplatin (CDDP)-treated guinea pigs.

BACKGROUND: During the process of apoptosis, double-stranded DNA is broken into single-stranded DNA by the action of caspases and caspase-activated deoxyribonuclease. We immunohistochemically examined the apoptotic changes induced by cisplatin in the vestibule of guinea pigs. MATERIALS AND METHODS: Cisplatin (10 mg/kg b.w.) was intraperitoneally injected into guinea pigs and, 3 days after the injection, the animals were sacrificed by intracardiac perfusion of fixative. The temporal bones were then removed and immunohistochemically stained for caspase-activated deoxyribonuclease or caspase 3. RESULTS: Both caspase-activated deoxyribonuclease and caspase 3 were observed in the dark cell area, transitional area and the sensory epithelium. CONCLUSION: These findings suggest that apoptosis is involved in the vestibular dysfunction of the CDDP-treated patients.

Detection of single-stranded DNA in the hydropic vestibule after the direct injection of antigen into the endolymphatic sac of guinea pigs.

Immunohistochemical study for single-stranded DNA (ssDNA) with vestibule of guinea pigs was performed after the injection of keyhole limpet hemocyanin (KLH) into the right endolymphatic sac. Endolymphatic hydrops became evident by expansion of the Reissner's membrane in the cochlea of all animals 1 day after the injection of KLH. Increased ssDNA expression was detected in the sensory epithelium and transitional area, while temporal bones in the control group did not show any ssDNA immunoreactivities. ssDNA is accompanied with the apoptotic change in the vestibule. Our results suggest that apoptotic changes could be involved in the hydropic vestibule and these phenomena lead inner ear disturbance as seen in endolymphatic hydrops.

Semicircular canal occlusion causes permanent VOR changes.

We measured the guinea pig horizontal vestibulo-ocular reflex (hVOR) to high acceleration impulsive head rotations following a unilateral lateral semicircular canal (LSCC) occlusion. We found a significant hVOR deficit for rotations toward the side of the occluded LSCC and this deficit did not show systematic changes over 3 months. We considered the LSCC nerve was still functional as shown by the normal appearance of the crista of the LSCC ampulla and also electrical stimulation of the LSCC. We conclude that the VOR during angular acceleration in response to high acceleration shows no adaptive plasticity following a unilateral LSCC occlusion.

Passive transfer of experimental autoimmune labyrinthitis.

The aim of the present study was to establish an animal model of autoimmune labyrinthitis using heterologous inner ear antigen (IEAg) and to elucidate whether the experimentally induced labyrinthitis could be passively transferred. Cochlear and vestibular membranous labyrinthine tissues from bovine temporal bones were used as IEAg. Donor mice were inoculated intracutaneously at multiple sites with an emulsion consisting of equal parts of IEAg and complete Freund's adjuvant. After 10 days, mononuclear cells were collected from lymph nodes, spleen and blood of the donor mice and injected intravenously into naive recipient mice. Cellular infiltration was observed in the perilymphatic space of the cochlea of all donor and recipient mice. Endolymphatic hydrops was also observed in 63% of donor and 42% of recipient mice. These findings suggest that the experimentally induced labyrinthitis observed in this animal model was probably due to an autoimmune reaction to the IEAg and was passively transferred by a cell-mediated immune reaction.

Experimental autoimmune labyrinthitis induced by cell-mediated immune reaction.

This study was designed to establish an experimental cell-mediated autoimmune labyrinthitis model in C57BL/6 mice, which could exhibit high reproducibility and be adopted for precise immunological analysis. Inner ear antigen (IEA) was prepared from bovine membranous labyrinth. Following pretreatment with cyclophosphamide (CP) and primary sensitization with IEA/FCA, many inflammatory cells infiltrated transiently into the perilymphatic and endolymphatic regions of the cochlea, vestibule and the endolymphatic sac, but not into the kidney, lung, brain or liver. This reaction occurred from day 7 and peaked on day 12 in all animals, and then rapidly reduced. This reaction occurred in all experimental mice during day 10 to day 12. The control mice showed no cellular reaction in the inner ear. These results suggest that the inner ear may possess cross-species organ-specific antigen and that a cell-mediated immune reaction may play an important role in the induction of autoimmune labyrinthitis.

An in vitro coculture model of transmigrant monocytes and foam cell formation.

To analyze in vitro the migration of monocytes to the subendothelial space, their differentiation into macrophages, and the subsequent formation of foam cells in vitro, we have developed a 2-coculture system with rabbit aortic endothelial cells (AECs), aortic smooth muscle cells (SMCs), and a mixture of matrix proteins on polyethylene filters in chemotaxis chambers. AECs were seeded on a mixture of type I and IV collagen with or without various types of serum lipoproteins (method 1) or on matrix proteins secreted by SMCs (method 2). In these coculture systems, rabbit AECs can maintain a well-preserved monolayer for up to 2 weeks. When human CD14-positive monocytes were added to the upper medium of the system, with monocyte chemotactic protein-1 treatment approximately 60% of the monocytes transmigrated within 24 hours and were retained for up to 7 days, whereas without MCP-1 treatment, <30% of monocytes transmigrated. On day 1, transmigrant monocytes were negative for immunostaining of type I and II macrophage scavenger receptors but by day 3, became positive for scavenger receptors as well as other macrophage markers. When oxidized low density lipoprotein was added to the matrix layer of the method I coculture, on day 4 transmigrant cells exhibited lipid deposit droplets, and by day 7, they had the appearance of typical foam cells. Some of the transmigrant cells recovered in the lower medium on day 7 also appeared to be foam cells, indicating foam cell motility and escape from the coculture layer through the filter. In summary, this coculture system is a useful in vitro tool to dissect the cellular and molecular events that make up the process of foam cell formation.

Intercellular adhesion molecule-1 expression in the inner ear of rats following secondary immune reaction in the endolymphatic sac.

An immunological aetiology for inner ear diseases has long been proposed. The endolymphatic sac (ES) is the only immunoprivileged site in the inner ear with a resident population of immunocompetent cells. By keyhole limpet hemocyanin (KLH) challenge into the ES of systemically pre-immunized guinea pigs, we previously demonstrated an infiltration of inflammatory cells into the perilymphatic space of the cochlea. In order to understand the mechanisms involved in the recruitment of immunocompetent cells into the inner ear, and their relation to the development of endolymphatic hydrops (EH), we investigated the expression and time-kinetics of intercellular adhesion molecule 1 (ICAM-1) in the inner ear of systemically pre-immunized rats after antigen (KLH) challenge into the ES, its relation to cell infiltration in the cochlea and subsequent development of EH. By immunohistochemistry, strong ICAM-1 expression was detected in the spiral ligament, suprastrial region, spiral prominence, spiral modiolar veins, spiral collecting venules, surface membrane of the perilymphatic compartment, perilymphatic space and ES of immunized rats, but not of control rats. ICAM-1 expression was detected at 5-6 h, peaked at 10-15 h, and gradually reduced by 2 weeks. Cell infiltration into the cochlea started at 6-12 h and peaked at day one. By 6 h, 50% of challenged rats developed EH. This figure rose to 70% at 12 h, and then gradually reduced. However, immunoreactivity for KLH (antigen) was only detected in the ES. These results emphasize that the sac is the central immunological organ of the inner ear, and suggest that ICAM-1 may play a pivotal role in the aetiology of immune-mediated inner ear diseases through the recruitment of immunocompetent cells into the inner ear and subsequent development of EH.

Distribution of endothelin-1-like activity in the cochlea of normal guinea pigs.

Distribution of endothelin-1 (ET-1) in the cochlea of normal guinea pigs was determined by immunohistochemistry. ET-1 activity was identified with the mouse anti-human ET-1 IgG1 monoclonal antibody. ET-1 activity was distributed in the modiolus, spiral ligament, stria vascularis, spiral prominence. Reissner's membrane, supporting cells of the organ of Corti and spiral ganglion cells. These findings suggest that ET-1 may be involved in the regulation of fluid volume and ions of the cochlea.

Preliminary study of the role of endothelin-1 in the homeostasis of the inner ear.

Endothelin (ET), originally characterized as a 21-residue vasoconstrictor peptide from endothelial cells, has been reported to act as a local hormonal regulator of pressure, fluid, ions, and neurotransduction. Our previous studies suggested an important role of ET-1 in the inner ear. The present study investigated the time kinetics of ET-1 in the epithelium of the endolymphatic sac (ES) of guinea pigs and its relation to the development of endolymphatic hydrops (EH) following locally mounted secondary immune reaction. In the duration between 12 h and day 1, ET-1-like activity completely disappeared from the epithelium of the ES and was associated with the accumulation of inflammatory cells in the ES and a rapid development of EH. On day 7, ET-1-like activity recovered as a consequence of the decrease of inflammatory cells and reduction of EH. These findings suggest that ET-1 may play an important role as one of the regulators maintaining the fluid balance.

Distribution of endothelin-1-like activity in the vestibule of normal guinea pigs.

Endothelin (ET) has been revealed to be a local hormonal regulator of pressure, fluid, ions and neurotransmitters. In order to investigate the mechanism of homeostasis within the microenvironment of the inner ear, the present study has examined the distribution of ET in the vestibule of normal guinea pigs, by immunohistochemistry using mouse antihuman ET IgG1 monoclonal antibody. ET-like activity was identified in the sensory epithelial cells, supporting cells, dark cells, transitional cells, vestibular membrane, semicircular wall cells and vestibular ganglion cells. These findings suggest that ET may play an important role in maintaining homeostasis of the vestibule.

Fluctuating hearing loss following immune reaction in the endolymphatic sac of guinea pigs.

This study has investigated immune injuries to the inner ear auditory system of guinea pigs. Following secondary antigen challenge to the endolymphatic sac, the mean hearing threshold significantly increased in the early phase from day 1 to day 3 and thereafter recovered. In the early phase, hearing threshold significantly increased simultaneously to the elevation of perilymph antibody levels. The size of hydrops was not the only factor that causes an increase in hearing loss as well as in AP/SP ratio. Scale-out hearing loss was seen in 2 animals with severe degeneration of the stria vascularis as well as the organ of Corti associated with the inflammatory cellular infiltration especially in the perilymphatic space, even in the absence of keyhole limpet hemocyanin antigen in the cochlea. On the other hand, control animals did not suffer hearing loss. These results suggest that an immune reaction in the endolymphatic sac is a possible pathogenic etiology of Ménière's disease or sudden deafness.