The effects of age on maintenance of intense neuromuscular block with rocuronium.

BACKGROUND: Increasing age is associated with a longer duration of action of neuromuscular block. The aim of this study was to determine the influence of ageing on the recovery of the post-tetanic count (PTC) from rocuronium-induced neuromuscular block. METHODS: Twenty-two younger (20-60 years) and 22 older (> 70 years) patients were enrolled in this study. After induction of anaesthesia with fentanyl and propofol, all patients initially received 1 mg/kg rocuronium and neuromuscular block were evaluated by contractions of the adductor pollicis muscle to ulnar nerve train-of-four stimulation using an acceleromyograph. Subsequently, intense rocuronium-induced block was determined every 6 min using the PTC during 1.0-1.5% sevoflurane and remifentanil anaesthesia. When the first response to the PTC stimulus was detected, 0.2 mg/kg rocuronium was additionally administered, and again, spontaneous recovery of neuromuscular function was monitored until the first response to the PTC reappeared. RESULTS: Median values (range) of the times from the administration of 1 mg/kg and 0.2 mg/kg rocuronium until recovery of the first detectable PTC were significantly longer in the older [51.0 (27-100) min, P < 0.0001 and 30.0 (12-66) min, P = 0.0036, respectively] than the younger patients [31.5 (21-45) min and 18.0 (12-36) min, respectively]. CONCLUSION: The times from rocuronium injection to reappearance of the first response to PTC stimulation are approximately twofold longer and more variable in older than younger patients. Hence, the dosing interval of rocuronium should be adjusted using neuromuscular monitoring when maintaining intense neuromuscular block, especially in older patients.

Correlation between cardiac output and reversibility of rocuronium-induced moderate neuromuscular block with sugammadex.

BACKGROUND: The aim of this study was to evaluate the correlation between cardiac output (CO) and reversibility of rocuronium-induced moderate neuromuscular block with sugammadex in elderly patients. METHODS: Fifty elderly (≥ 65 years) patients were enrolled in this study. During 1.0-1.5% end-tidal sevoflurane and remifentanil anaesthesia, contraction of the adductor pollicis muscle in response to ulnar nerve stimulation was acceleromyographically quantified. All patients initially received 1 mg/kg rocuronium followed by 0.2 mg/kg whenever the second twitch T2 of the train-of-four (TOF) response reappeared. CO was measured throughout the study using a FloTrac™/Vigileo™ monitor. After completion of surgery and at the reappearance of T2, the time required for a bolus dose of 2 mg/kg sugammadex to facilitate recovery to a TOF ratio of 0.9 was recorded, and its correlation with CO was analysed. RESULTS: Adequate recovery of neuromuscular block was achieved after sugammadex in all patients. Mean CO at the time of reversal with sugammadex was 5.3 l/min (1.3), and recovery time to a TOF ratio of 0.9 was 173.4 s (54.8). A statistically significant inverse correlation was seen between the time to recovery to a TOF ratio of 0.9 and CO [reversal time (s) = -27.7·CO + 298.7, R(2) = 0.461, P < 0.0001]. CONCLUSIONS: The time to reach a TOF ratio of 0.9 following sugammadex is dependent on CO in elderly patients.

Isolation, purification, and characterization of a collagen-associated serpin, caspin, produced by murine colon adenocarcinoma cells.

A 45-kDa serpin secreted by a murine colon adenocarcinoma cell line, colon26, was isolated, purified, and characterized. It was found to bind specifically to type I collagen with high affinity and to type III collagen with lower affinity. Immunohistochemical studies of murine embryonic tissues showed a specific distribution of this collagen-associated serpin, named caspin, in relation to the formation of bone, cartilage, teeth, and basement membrane. The expression of caspin in high and low lung metastatic subclones of colon26 cell lines was inversely correlated with their metastatic capacity: low lung metastatic cells secreted higher amounts of caspin than their high lung metastatic counterparts. Caspin also demonstrated high homology with human pigment epithelium-derived factor/early population doubling level cDNA-1, which reportedly induces neuronal differentiation of human retinoblastoma cells and is expressed in association with G0 growth arrest. These findings suggest that caspin/pigment epithelium-derived factor/early population doubling level cDNA-1 is a novel factor that might play a crucial role in embryogenesis and tumor metastasis through binding to the extracellular matrix.

Prolonged infection of mouse brain neurons with murine cytomegalovirus after pre- and perinatal infection.

The susceptibility of mice at different developmental stages to a relatively low titer of cell culture-passaged murine cytomegalovirus (MCMV) infection was compared in terms of the urinary excretion of MCMV examined by plaque assay and in terms of the distribution of viral infection, determined by immunohistochemistry, using antibodies specific to the early nuclear antigen of MCMV. Viral infection on day 8.5 of gestation (E8.5) into the conceptus and intraperitoneal infection on day 15.5 of gestation (E15.5), postnatal day 2 (P2), postnatal day 11 (P11), and 30 days after birth (P30), respectively, were performed. Embryonal and perinatal mice were more susceptible to MCMV in terms of urinary excretion of the virus and the presence of viral antigen-positive cells in the brain, lungs, and kidneys. In the embryonal and perinatal infection, the viral antigen-positive cells in the neurons of the cerebral cortex and hippocampus were retained late after birth, even though the positive cells in the lungs and kidneys had disappeared. In the mice infected on E8.5, small clusters of viral antigen-positive cells were detected only in the cortex and hippocampus late after birth, without the urinary excretion of virus. These results suggest that when mice are infected with MCMV at the embryonal and perinatal stages, elimination of the infected neurons is delayed compared with that of the other cells in the lungs and kidneys. These findings provide a model for the analysis of pathogenesis of the subclinical congenital CMV infection that manifested clinically late after birth in humans as brain disorders.

Microphthalmia and cerebral atrophy induced in mouse embryos by infection with murine cytomegalovirus in midgestation.

Microphthalmia and cerebral atrophies were induced in mouse embryos after injection of murine cytomegalovirus (MCMV) into the conceptus at midgestation. The concepti of ICR mice on day 8.5 of gestation were injected with MCMV through the uterine wall, then pregnancies were allowed to continue. On day 15.5 of gestation, microphthalmia was observed in 19.2% of the MCMV-injected embryos (1 x 10(4) plaque-forming units). As the survival rate decreased when pregnancies were allowed to continue further, incidence of microphthalmia decreased, whereas cerebral atrophies, determined by examining the histological sections, were observed in 17.6% of the surviving mouse fetuses on day 18.5 of gestation. Microphthalmia was confirmed by microscopically measuring the eyes on the serial coronal sections. There were two types of microphthalmia: one with marked hypoplastic eye with periglobular mesenchymal proliferation, the other with small eye and lens without the mesenchymal proliferation. Immunohistochemical analysis was performed using antibodies specific to the nuclear antigen of MCMV. Viral antigen-positive cells were widely distributed in the mesenchymes around the oral and nasal cavities and in the mesenchymes around the brain, especially in the endothelial cells of the vessels and the perivascular mesodermal cells. In the eyes, viral antigen-positive cells were observed in mononuclear blood cells in the cavities of the vitreous bodies. These results suggest that the primary target of congenital cytomegalovirus infection may be the mesenchymal cells; then the infection extends to the eyes and brain. In addition, the mesenchymal infection may also disrupt their organogenesis, resulting in microphthalmia and cerebral atrophy. This experimental system may provide a model similar to congenital cytomegalovirus infection in humans.

A spigelian hernia in an infant.

The case of a two-month old Japanese girl with a left side Spigelian hernia that developed during the course of recovery from the surgical treatment of a meningomyelocele and ventriculo-peritoneal shunting (V-P shunting) is reported on. At this time, to our knowledge, 20 cases involving infants and children have been reported on in world-wide medical literature. Of these cases, 4 cases were caused by trauma, 1 by abdominal surgery, and the others had no evident causes. Our patient had a meningomyelocele, and underwent a V-P shunting. However, a cause and effect relationship between the Spigelian hernia and the treatment of the meningomyelocele followed by V-P shunting is inconceivable. We doubt any relationship between the preceding neurosurgery and the development of the hernia. This is the first case of infantile Spigelian hernia reported on in Japan.

Two patients with chromosome 6q terminal deletions with breakpoints at q24.3 and q25.3.

We report on 2 patients with de novo terminal deletion of 6q. The first was a 4-month-old boy whose karyotype was 46,XY,del(6)(q24.3); the second a 2-year-old girl whose karyotype was 46, XX, del(6)(q25.3). The main anomalies in both patients included mental retardation, minor craniofacial and cerebral anomalies, and cardiac defects. The characteristic manifestations were imperforate anus in the first patient, and retinitis proliferans and a triatrial heart in the other. Comparison of clinical findings of our 2 patients with those of 18 previously reported patients with similar phenotypes suggests that terminal deletion of the 6q23 or 6q25 band is critical in producing the main anomalies of del(6q) syndrome.

Susceptibility of mouse embryo to murine cytomegalovirus infection in early and mid-gestation stages.

The susceptibility of mouse embryonic cells to murine cytomegalovirus (MCMV) infection was studied by injecting the virus in the early and mid-gestation stages. For the early stage, blastocysts from BDF1 mice were injected with MCMV or minimal essential medium (MEM) by micromanipulator and returned to the uteri of pseudopregnant ICR mice. On day 11 of gestation, the embryos were examined immunohistochemically, using antibody specific to the early antigen of MCMV, and the placentae were examined by plaque assay. No infection was detected by either method. Furthermore, no infection was detected in MCMV-infected blastocysts that were cultured and examined for infection by immunofluorescence. For mid-gestation embryos, the conceptus was injected with MCMV on day 8.5 of gestation and was subjected to immunohistochemical analysis from day 10.5 to 12.5 of gestation. Viral antigen-positive cells were first observed in the placentae, then antigen-positive cells appeared among the blood cells, endothelial and mesodermal cells of the embryos. On day 12.5 of gestation, clusters of viral antigen-positive cells were sometimes observed in the hearts and livers. Although the incidence was lower, viral antigen-positive cells were also observed in the neuroectoderm and the eyes. These results suggest that MCMV does not infect early embryos and that infection first occurs in the placenta of postimplantation embryos, whence it extends through the blood cells to the endothelial and mesodermal cells of different embryonic regions, eventually extending to the neuroectoderm.

Viral DNA detected by in situ hybridization in the developing mouse brain infected with murine cytomegalovirus.

To investigate the pathogenesis of brain abnormalities caused by congenital cytomegalovirus (CMV) infection, we previously reported experimental murine models of brain damage induced by intraventricular injection of murine CMV (MCMV) at the late stage of gestation. In the present study, viral DNA-positive cells in the damaged brain at different postnatal stages detected by in situ hybridization were compared with viral antigen-positive cells detected by an immunoperoxidase method using a monoclonal antibody against the immediate early antigen. At birth, the number of viral DNA-positive cells almost equalled that of viral antigen-positive cells. Seven to ten days after birth, the number of viral DNA-positive cells in the brain of MCMV-injected mice was one-fifth that of viral antigen-positive cells. Viral DNA-positive cells were more numerous in the hippocampus than in the cortex, and their density was dependent on the presence of viral antigen-positive cells. Dotted reaction products were observed in the nuclei of viral DNA-positive cells. These cells were rarely detected in lesions of later stages such as atrophy of the cortex and hippocampus, or the wall of the cystic lesions. These results suggest that viral DNA-positive cells detected by in situ hybridization are infected cells in which viral DNA replication is occurring actively.

Postnatal porencephaly induced in mouse by murine cytomegalovirus.

Mouse embryos were infected with murine cytomegalovirus (MCMV) by injecting the virus into the cerebral ventricles at the late gestation. After deliveries, offspring were fed by the mothers until 4 weeks. Cystic brain lesions, regarded as porencephaly or paraventricular cysts, were observed in about 20% of the MCMV-injected offspring 3 to 4 weeks after birth. The porencephaly involved the cerebral cortex and the white matter, and sometimes opened to the ventricles, while the paraventricular cysts involved the white matter. The inner surfaces of the cysts were covered with thin monolayer cells. Around the cystic lesions, perivascular cuffings were sometimes observed in the meninges and the basal regions. Viral antigen-positive cells were observed in the cortex and the hippocampus but were hardly observed along the cystic walls. Immunohistochemical double staining using antibodies specific for the viral antigen and specific for factor VIII-related antigen showed that the brain capillary endothelial cells had susceptibility to MCMV infection, and in addition that some neurons in the cortex and the hippocampus had the same susceptibility. These findings suggest that there are at least two ways by which MCMV induce abnormalities in the developing mouse brains; migration of MCMV-infected neurons and affinity to the endothelial cells of the brain vessels to this virus.

Susceptibility of brain cells to murine cytomegalovirus infection in the developing mouse brain.

Mouse embryos were infected with murine cytomegalovirus (MCMV) by injecting the virus into the cerebral ventricles in the late stage of gestation; the brains of the offspring were than analyzed using the histological and immunohistochemical methods. Brains of the offspring, which were injected with relatively high titers of MCMV [1 X 10(4) plaque-forming units (pfu)] on day 13 of gestation exo utero or on day 15 of gestation in utero, showed massiv necrosis of the cerebral cortex with gliomesodermal proliferation around 9 to 10 days after birth. In these brains, viral antigen-positive cells were observed in zonal arrangement in the lesion-free cortex and in the hippocampus. Immunohistochemical double staining showed that some of the viral antigen-positive cells had also reacted with antibody to neuron-specific enolase at the same time, but had hardly reacted with antibodies to brain-type creatine kinase or glial fibrillary acidic protein. Brains of the offspring, which were injected with relatively low titers of virus (1 x 10(3) pfu) on day 15 of gestation, showed zonal arrangement of viral antigen-positive cells mainly in the cerebral cortex and in the hippocampus 7 days after birth, although the numbers of the positive cells were low. Fourteen days after birth, some of these offspring showed atrophy of the cerebral cortex and the hippocampus. These results suggest that some of the neuronal cells in the cerebral cortex and the hippocampus have special susceptibility to MCMV infection.

Induction of S-100b (beta beta) protein in human teratocarcinoma cells.

Human teratocarcinoma NT2/D1 cells undergo differentiation into a variety of cell types, including neurons, treated with retinoic acid. In the present study, the concentrations of alpha S-100 and beta S-100 proteins (alpha and beta subunits of S-100 proteins), and three subunits (alpha, beta and gamma) of enolase in NT2/D1 cells were measured using the sensitive enzyme immunoassay method. The concentration of beta S-100 was markedly increased in the cells after treatment with retinoic acid, whereas the concentration of alpha S-100 was undetectably low, indicating that the S-100b (beta beta) protein was induced by retinoic acid. On the other hand, the concentrations of the three forms of enolase isozymes did not change in the same culture. The induction of S-100b protein was not observed in the NT2/D1 cells after treatment with forskolin, dibutyryl cyclic AMP or cholera toxin. The indirect double-labeled immunofluorescence, using antibodies specific to beta S-100 and monoclonal antibodies specific to neurofilaments, revealed that both the S-100b protein and the neurofilaments were induced in the same subpopulation of cells which underwent neuronal differentiation.

Viral replication in HeLa/fibroblast hybrid cells infected with human cytomegalovirus.

Three human hybrid cell lines were generated by the fusion of D 98OR, a HeLa cell variant, and TIG human diploid fibroblasts. Chromosome numbers of the hybrid cells fell between that of D 98OR cells and the combined chromosome number of the two cell lines, with three marker chromosomes identical to those of D 98OR cells. One hybrid, B-3, produced large amounts of human fibronectin as analyzed by immunohistochemistry. Only human cytomegalovirus (HCMV) infected B-3 cells showed positive fluorescence as detected by human antiserum to HCMV. Further cloning of B-3 by limiting dilution resulted in two cloned hybrids with markedly enhanced virus production as compared with B-3 cells. Treatment of these two clones with phorbol ester further enhanced virus production. These cloned hybrids may provide a tool to analyze host cell factors controlling the transcription and replication of HCMV.

Monoclonal antibodies to guinea-pig cytomegalovirus: an immunoelectron microscopic study.

Three groups of monoclonal antibodies which reacted with cells infected by guinea-pig cytomegalovirus (GPCMV) were prepared. The first group of antibodies immunoprecipitated a 50 000 mol. wt. (50K) polypeptide of GPCMV-infected cells. This polypeptide was identified as part of the nuclear inclusion by immunofluorescence. This nuclear fluorescence was markedly diminished when the infected cells were incubated in the presence of phosphonoacetic acid. By immunoelectron microscopy the antibodies reacted mainly with filamentous structures (26 to 28 nm in diameter) in nuclear inclusions and occasionally stained nucleocapsids. Neither intracytoplasmic nor extracellular virions reacted with the antibodies. Therefore, the 50K protein with which the monoclonal antibodies reacted was nuclear inclusion-specific and a non-structural protein. The second group of antibodies reacted with a 76K polypeptide of the infected cells which was a matrix protein found in both cytoplasmic inclusions and extracellular dense virions. The third group of antibodies mainly reacted with a virion core protein by immunoelectron microscopy.