Ultrasonography of the optic nerve sheath to assess intracranial pressure changes after ventriculo-peritoneal shunt surgery in children with hydrocephalus: a prospective observational study.

The optic nerve sheath diameter has been verified by various clinical studies as a non-invasive indicator of intracranial hypertension. The aim of this study was to compare the optic nerve sheath diameter before and immediately after ventriculo-peritoneal shunt surgery in children with hydrocephalus. We analysed transorbital ultrasonographic images recorded after induction of anaesthesia and 30 min after shunt insertion in 34 children, measuring the optic nerve sheath diameters using a linear ultrasound probe. The mean (SD) optic nerve sheath diameters were 5.4 (0.6) mm (right) and 5.3 (0.7) mm (left) before surgery and 4.4 (0.5) mm (right) and 4.5 (0.7) mm (left) after surgery (p < 0.0001 for before and after comparisons for both eyes). The technique allows rapid and non-invasive assessment of intracranial pressure to guide appropriate postoperative management.

A comparison of sedation protocols for gastric endoscopic submucosal dissection: moderate sedation with analgesic supplementation vs analgesia targeted light sedation.

BACKGROUND: Moderate to deep sedation has been recommended during endoscopic submucosal dissection (ESD). However, it is often accompanied by adverse events such as respiratory depression or aspiration pneumonia. This study investigated the respiratory complications and ESD outcomes of two sedation protocols: moderate sedation with analgesic supplementation (MSAS) and analgesia targeted light sedation (ATLS). METHODS: The clinical data of 293 patients who underwent ESD between May and December 2012 were reviewed. During the first 4 months, 155 patients were managed by moderate sedation [Modified Observer Assessment of Alertness/Sedation (MOAA/S) at 2-3] with the MSAS protocol. During the latter period, 138 patients were managed using the ATLS protocol (MOAA/S at 4-5). For both protocols, propofol and remifentanil were infused for sedation and pain control, respectively. RESULTS: The ATLS protocol required less propofol [22.9 (sd 17.3) vs 88.1 (44.0) µg kg(-1) min(-1), P<0.001] and more remifentanil [6.8 (sd 3.1) vs 4.9 (3.0) µg kg(-1) hr(-1), P<0.001] than the MSAS protocol. The desaturation events during the procedure occurred significantly less often (2.2 vs 12.9%, P=0.001) and recovery was significantly faster [19.7 (sd 4.8) vs 27.9 (16.0) min, P<0.001] with the ATLS protocol than with the MSAS protocol. The incidence of aspiration pneumonia with the ATLS protocol was 1.4% compared with 5.2% with the MSAS protocol (P=0.109). There were no differences in outcomes and complications of ESD. CONCLUSION: The ATLS protocol reduced the incidence of desaturation events without affecting ESD performance compared with the MSAS protocol. There was also a trend towards a low incidence of aspiration pneumonia with the ATLS protocol.

The effect of pneumoperitoneum in the steep Trendelenburg position on cerebral oxygenation.

BACKGROUND: daVinci robot-assisted laparoscopic radical prostatectomy (RALP) requires pneumoperitoneum in the steep Trendelenburg position, which results in increased intracranial pressure and cerebral blood flow. The aim of this study was to evaluate the effect of pneumoperitoneum in a 30 degrees Trendelenburg position on cerebral oxygenation using regional cerebral oxygen saturation (rSO2). METHODS: Thirty-two male patients of ASA I and II physical status without previous episodes of cerebral ischemia or hemorrhage undergoing daVinci RALP were enrolled. The rSO2 was continuously monitored with near-infrared spectroscopy (INVOS) 5100) during the study period. Measurements were obtained immediately after anesthesia induction (T0; baseline), 5 min after a 30 degrees Trendelenburg position (T1), 5 min after 15 mmHg pneumoperitoneum in a supine position (T2), 30, 60 and 120 min after the pneumoperitoneum in a Trendelenburg position (T3, T4 and T5, respectively) and after desufflation in a supine position (T6). RESULTS: The change in the left and right rSO2 was statistically significant (Left P=0.004 and Right P=0.023). Both the right and the left rSO2 increased significantly during pneumoperitoneum in a Trendelenburg position (from T3 to T5) and at T6 compared with the baseline value at T0. The partial pressure of carbon dioxide (PaCO2) was increased significantly at T2, T3, T5 and T6 compared with the baseline value at T0. CONCLUSIONS: During daVinci RALP, cerebral oxygenation, as assessed by rSO2, increased slightly, which suggests that the procedure did not induce cerebral ischemia. The PaCO2 should be maintained within the normal limit during pneumoperitoneum in a Trendelenburg position in patients undergoing daVinci RALP because the rSO2 increased in conjunctions with the increase in PaCO2.

Neural substrates, experimental evidences and functional hypothesis of acupuncture mechanisms.

OBJECTIVES: Although acupuncture therapy has demonstrated itself to be effective in several clinical areas, the underlying mechanisms of acupuncture in general and the analgesic effect in particular are, however, still not clearly delineated. We, therefore, have studied acupuncture analgesic effect through fMRI and proposed a hypothesis, based on the obtained result, which will enlighten the central role of the brain in acupuncture therapy. METHODS: The proposed model, termed as a broad sense hypothalamus-pituitary-adrenal (BS-HPA) axis, was based on our observed neuroimaging results. The model incorporates the stress-induced HPA axis model together with neuro-immune interaction including the cholinergic anti-inflammatory model. RESULTS: The obtained results coupled with accumulating evidence suggest that the central nervous system is essential for the processing of these effects via its modulation of the autonomic nervous system, neuroimmune system and hormonal regulation. CONCLUSIONS: Based on our fMRI study, it appears that understanding the effects of acupuncture within a neuroscience-based framework is vital. Further, we have proposed the broad sense-HPA axis hypothesis which incorporates the experimental results.

Drosophila as a model to study human brain degenerative diseases.

Drosophila has been an ideal system in which to identify molecules and define pathways involved in development, in part because of the powerful genetic approaches that are possible. Many of the molecules and pathways important in development in Drosophila are evolutionarily conserved between fly and human. With its highly evolved nervous system, amenability to genetic analysis, and the full genomic sequence available, Drosophila is a valuable tool for investigating and understanding the molecular mechanisms of neurodegenerative diseases.In order to have neurodegenerative Drosophila mutants, I screened EMS treated X chromosomes and P-element inserted 2nd and 3rd chromosomes in Drosophila for reduced life span and neurodegeneration. Twenty-one neurodegenerative mutants including bubblegum, spongecake, and eggroll were isolated and were named by virtue of their brain lesions. Each mutant has distinct pattern of degeneration in specific regions of the brain. Degeneration occurs in lamina and retina region in bubblegum. In spongecake vacuolization can only be seen in the optic lobe, especially in the medulla region. Multilamellated inclusions are wide-spread in the brain of eggroll. It showed not only do the pathologies iin fly brains resemble that of human diseases including Creutzfeldt Jakob disease, Tach-Sachs and Niemann-Pick disease, but the gene involved in the pathological pathway in the bubblegum mutant also functions as in the human adrenoleukodystrophy.

The monitoring of somatosensory evoked potentials and neurologic complications in aneurysm surgery.

Somatosensory evoked potential (SSEP) changes during cerebral aneurysm surgery and their relationship to postoperative neurologic complications have been studied on many occasions. However, it is still a matter of debate whether SSEP monitoring is really helpful in detecting or preventing neurologic complications. We studied 87 patients undergoing aneurysm surgery of the anterior cerebral circulation and SSEPs were monitored in 60 of these patients. All patients were grade 2 by the subarachnoid hemorrhage (SAH) grading system. Median nerve SSEP was monitored for middle cerebral or internal carotid artery aneurysms and posterior tibial nerve SSEP for anterior cerebral artery aneurysms. A decrease in the cortical amplitude of more than 50%, compared with control, was considered significant and interventions were then taken to reverse the SSEP. The pre- and postoperative neurologic deficits of each patient were evaluated immediately before and after surgery. No significant difference was found in the incidence of postoperative neurologic complications in the SSEP monitored (15% [9/60]) and unmonitored patients (22% [6/27]). In the SSEP monitored patients, the amplitudes of SSEPs decreased significantly in 14 patients and 4 of these showed neurologic complications. However, SSEP amplitudes were not significantly changed in 46 patients, and 5 of these showed neurologic complications. Significant changes in the amplitude of SSEP might represent neuronal injury, but the absence of change in the SSEP cannot guarantee patient safety. Our results suggest that SSEP monitoring may be useful for detecting the danger of neuronal injury, but that it does not reduce the incidence of neurologic complications in aneurysm surgery.

Identification of an amino acid defining the distinct properties of murine beta1 and beta3 subunit-containing GABA(A) receptors.

Murine gamma-aminobutyric acid (GABA) type A homomeric receptors made of beta1 subunits are profoundly different, when expressed in Xenopus oocytes, from beta3 homomeric receptors. Application of the intravenous general anesthetic pentobarbital, etomidate, or propofol to beta3 homomeric receptors allows current flow. In contrast, beta1 homomers do not respond to any of these agents. Through construction of chimeric beta1/beta3 receptors, we identified a single amino acid that determines the pharmacological difference between the two beta subunits. When the serine residue present in the wild-type nonresponsive beta1 subunit is replaced by an asparagine found in the same position in the beta3 subunit, the resulting point-mutated beta1S265N forms receptors responsive to intravenous general anesthetics, like the wild-type beta3 subunits. Conversely, after mutation of the wild-type beta3 to beta3N265S, the homomeric receptor loses its ability to respond to these same general anesthetics. Wild-type-to-mutant titration experiments showed that the nonresponsive phenotype is dominant: A single nonresponsive residue within a pentameric receptor is sufficient to render the receptor nonresponsive. In alpha1betax or alpha1betaxgamma2 heteromeric receptors, the same residue manifests as a partial determinant of the degree of potentiation of the GABA-induced current by some general anesthetics. The location of this amino acid at the extracellular end of the second transmembrane segment, its influence in both homomeric and heteromeric receptor function, and its dominant behavior suggest that this residue of the beta subunit is involved in an allosteric modulation of the receptor.

Nondepolarizing neuromuscular blockers inhibit the serotonin-type 3A receptor expressed in Xenopus oocytes.

UNLABELLED: Molecular cloning and sequence comparison indicates a high degree of structural homology between muscle nicotinic acetylcholine (nACh) and serotonin-type 3 (5-HT(3A)) receptors, both members of the direct ligand-gated family of ion channels. Because of the structural similarities and common evolutionary origin of these receptors, neuromuscular blockers (competitive nACh antagonists) may demonstrate pharmacologic cross talk and exhibit attributes of 5-HT(3A) receptor antagonists. We examined six clinically-used neuromuscular blockers for their ability to antagonize currents flowing through the 5-HT(3A) receptors in voltage clamped Xenopus oocytes. The neuromuscular blockers reversibly inhibited the 5-HT(3A) receptor-gated current in the rank order potency of (IC50 mean +/- SEM): d-tubocurarine (0.046 +/- 0.003 microM), atracurium (0.40 +/- 0.03 microM), mivacurium (15.1 +/- 2.93 microM), vecuronium (16.3 +/- 2.24 microM), and rocuronium (19.5 +/- 2.31 microM). Gallamine was essentially inactive as a 5-HT(3A) receptor antagonist with an extrapolated IC50 of 1170 microM. We demonstrate that drugs classically known as competitive nACh receptor antagonists also block 5-HT(3A) receptors. It is likely that certain neuromuscular blockers share pharmacological properties with 5-HT(3A) receptor antagonists, such as a reduction in postoperative nausea and vomiting. With careful drug selection, pharmacological cross talk could potentially be used to minimize polypharmacy and optimize patient management. IMPLICATIONS: Muscle nicotinic acetylcholine and serotonin-type 3A (5-HT(3A)) receptors are similar. Therefore neuromuscular relaxants may block 5-HT(3A) receptors. Our pharmacological study demonstrates that neuromuscular relaxants, as with ondansetron, are 5-HT(3A) receptor antagonists. It is likely that certain neuromuscular relaxants exhibit ondansetron-like clinical properties, such as reduction in postoperative nausea and vomiting.

The infusion rate of mivacurium or atracurium for cesarean section compared with gynecological procedures.

Mivacurium is mainly metabolized by plasma cholinesterase, whereas atracurium is removed by Hofman elimination. The purpose of this study was to compare the infusion rate of atracurium and mivacurium in maintaining surgical relaxation, and to compare their recovery indices between parturients and non-pregnant women. Muscle relaxation was maintained by the continuous infusion of relaxants to retain the first response of train-of-four (TOF) at 5% of control. When mivacurium was used, Bolus-T5 (duration from the end of mivacurium bolus injection to 5% single twitch recovery) was measured. After discontinuing the infusion, the recovery index was measured. The infusion rate of mivacurium, not atracurium, was significantly lower in parturients and Bolus-T5 of parturients was significantly longer than that of non-pregnant women. There was no significant difference in the recovery indices of both relaxants. The authors concluded that the infusion rate of mivacurium in maintaining muscle relaxation in parturients should be reduced compared to the rate in non-pregnant women and measuring Bolus-T5 may be helpful in determining the infusion rate to maintain muscle relaxation.

Preventing neurodegeneration in the Drosophila mutant bubblegum.

The Drosophila melanogaster recessive mutant bubblegum (bgm) exhibits adult neurodegeneration, with marked dilation of photoreceptor axons. The bubblegum mutant shows elevated levels of very long chain fatty acids (VLCFAs), as seen in the human disease adrenoleukodystrophy (ALD). In ALD, the excess can be lowered by dietary treatment with "Lorenzo's oil," a mixture of unsaturated fatty acids. Feeding the fly mutant one of the components, glyceryl trioleate oil, blocked the accumulation of excess VLCFAs as well as development of the pathology. Mutant flies thus provide a potential model system for studying mechanisms of neurodegenerative disease and screening drugs for treatment.

Wolbachia, normally a symbiont of Drosophila, can be virulent, causing degeneration and early death.

Wolbachia, a maternally transmitted microorganism of the Rickettsial family, is known to cause cytoplasmic incompatibility, parthenogenesis, or feminization in various insect species. The bacterium-host relationship is usually symbiotic: incompatibility between infected males and uninfected females can enhance reproductive isolation and evolution, whereas the other mechanisms enhance progeny production. We have discovered a variant Wolbachia carried by Drosophila melanogaster in which this cozy relationship is abrogated. Although quiescent during the fly's development, it begins massive proliferation in the adult, causing widespread degeneration of tissues, including brain, retina, and muscle, culminating in early death. Tetracycline treatment of carrier flies eliminates both the bacteria and the degeneration, restoring normal life-span. The 16s rDNA sequence is over 98% identical to Wolbachia known from other insects. Examination of laboratory strains of D. melanogaster commonly used in genetic experiments reveals that a large proportion actually carry Wolbachia in a nonvirulent form, which might affect their longevity and behavior.

Spongecake and eggroll: two hereditary diseases in Drosophila resemble patterns of human brain degeneration.

Various neuronal degenerative diseases are characterized by late onset, relentless progression, and finally death. Many have a direct genetic basis; others are of still unknown etiological mechanisms [1,2]. The study of human neurodegenerative diseases is complicated by the difficulty of obtaining tissue samples at various stages of progression, especially early in the course of the disease. Since neurodegeneration occurs in many organisms [3-5], model organisms amenable to genetic and molecular techniques, such as the mouse, offer important advantages. Much less laborious and expensive are worms or flies, which have short generation times and can be rapidly screened for mutations. To investigate the use of the fly as a model system for identifying genes related to such diseases, we screened for mutants having reduced lifespan, then examined them for brain degeneration. We describe here two such mutants, each with a different pattern of degeneration as characterized by light and transmission electron microscopy. The brain of the aging spongecake mutant exhibits regionally specific, membrane-bound vacuoles similar to those seen in spongiform degenerations such as Creutzfeldt-Jakob disease [6,7]. The mutant eggroll develops dense, multilamellated structures in the brain, resembling ones found in lipid storage diseases such as Tay-Sachs [8].

Identification of a phosphorylation site for calcium/calmodulindependent protein kinase II in the NR2B subunit of the N-methyl-D-aspartate receptor.

The N-methyl-D-aspartate (NMDA) subtype of excitatory glutamate receptors plays critical roles in embryonic and adult synaptic plasticity in the central nervous system. The receptor is a heteromultimer of core subunits, NR1, and one or more regulatory subunits, NR2A-D. Protein phosphorylation can regulate NMDA receptor function (Lieberman, D. N., and Mody, I. (1994) Nature 369, 235-239; Wang, Y. T., and Salter, M. W. (1994) Nature 369, 233-235; Wang, L. -Y., Orser, B. A., Brautigan, D. L., and MacDonald, J. F. (1994) Nature 369, 230-232). Here we identify a major phosphorylation site on subunit NR2B that is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), an abundant protein kinase located at postsynaptic sites in glutamatergic synapses. For the initial identification of the site, we constructed a recombinant fusion protein containing 334 amino acids of the C terminus of the NR2B subunit and phosphorylated it with CaM kinase II in vitro. By peptide mapping, automated sequencing, and mass spectrometry, we identified the major site of phosphorylation on the fusion protein as Ser-383, corresponding to Ser-1303 of full-length NR2B. The Km for phosphorylation of this site in the fusion protein was approximately 50 nM, much lower than that of other known substrates for CaM kinase II, suggesting that the receptor is a high affinity substrate. We show that serine 1303 in the full-length NR2B and/or the cognate site in NR2A is a major site of phosphorylation of the receptor both in the postsynaptic density fraction and in living hippocampal neurons.

EcoRI variant N199H has enhanced specific activity.

The Asn199 residue of the EcoRI restriction endonuclease has been replaced with other amino acids to investigate whether it mediates nucleotide recognition or catalytic activity. Cassette mutagenesis gave variants of EcoRI: N199D, N199H, N199L, N199R, N199S and N199V. Their relative cleavage rates were found to be in the following order: N199H > EcoRI (wild type; wt) > N199L > N199V > N199S > N199R > N199D. In particular, EcoRI variant N199H showed about a twofold higher specific activity than that of the wt enzyme.

Chemical synthesis and cloning of human beta-endorphin gene in Escherichia coli.

Total synthesis of human beta-endorphin gene has been designed for the expression in bacterial system. Eight individual oligonucleotides corresponding to the beta-endorphin gene were chemically synthesized and joined through the enzyme-catalyzed reaction. The final yield of the 111-nucleotide-long synthetic beta-endorphin gene construct was about 10% of the total oligonucleotide used. The synthetic human beta-endorphin gene was cloned into the bacterium Escherichia coli, using pUC8 vector and shown to have the correct nucleotide sequences as designed.

Comparison of plasma inorganic fluoride concentration with sevoflurane-N2O and enflurane-N2O anesthesia.

Plasma inorganic fluoride concentrations were measured in adult patients without hepatic or renal disease following sevoflurane-N2O anesthesia (n = 7) or enflurane-N2O anesthesia (n = 6). The anesthetic dosage of sevoflurane and enflurane was 6.48 +/- 2.15 %-hours and 6.57 +/- 2.50 %-hours, respectively. The mean peak plasma inorganic fluoride concentration in the sevoflurane group was 19.5 +/- 13.4 mumol/L 1 hour after anesthesia, which decreased to preanesthetic levels 24 hours after anesthesia. In the enflurane group the values were 13.2 +/- 5.8 mumol/L at the end of anesthesia and decreased, but remained, still twice as high as the preanesthetic level 24 hours after anesthesia. The relationship of plasma inorganic fluoride concentration and anesthetic dosage was more pronounced in the sevoflurane group (r = 0.68, slope = 4.2) than in the enflurane group (r = 0.39, slope = 1.2). In conclusion, sevoflurane-N2O anesthesia results in similar subnephrotoxic levels of plasma inorganic fluoride as enflurane-N2O anesthesia, and although the fluoride concentration had a better correlation to anesthetic dosage in the sevoflurane group than in the enflurane group, its excretion was faster in the sevoflurane group than in the enflurane group.

Sigma F, the first compartment-specific transcription factor of B. subtilis, is regulated by an anti-sigma factor that is also a protein kinase.

The establishment of compartment-specific transcription in sporulating cells of B. subtilis is governed at the level of the activity of transcription factor sigma F. Genetic experiments have suggested that SpoIIAA and SpoIIAB, the other products of the sigma F operon, are involved in regulating sigma F activity. This activity is inhibited in the predivisional cell but specifically released from inhibition in the prespore about 1.5 hr after sporulation is induced. We now show that purified SpoIIAB inhibits transcription directed by sigma F in vitro. We note that the amino acid sequence of SpoIIAB shows some similarity to a group of bacterial histidine protein kinases, and we find that SpoIIAB is indeed a protein kinase that phosphorylates SpoIIAA on a serine residue. We suggest that this phosphorylation is responsible for the compartment-specific release of sigma F activity, perhaps through the formation of a tight complex between SpoIIAB and phosphorylated SpoIIAA.

Activity of mutant sigma F proteins truncated near the C terminus.

sigma F, the product of the spoIIAC gene of Bacillus subtilis, is homologous in amino acid sequence throughout most of its length with several other sigma factors of B. subtilis and Escherichia coli. However, 8 residues from the C terminus the homology abruptly breaks down, suggesting that the C-terminal tail of the protein may be dispensable. It is known that an amber mutation at the 11th codon (wild-type glutamine 245) from the C terminus abolishes the function of the sigma factor. We have now placed chain-terminating codons at the ninth codon (wild-type lysine 247), the eighth codon (wild-type valine 248), or the seventh codon (wild-type glutamine 249) from the C terminus. We have tested the resulting mutants for their capacity to sporulate and for their ability to transcribe from a promoter (spoIIIG) that is normally read by RNA polymerase bound to sigma F (E sigma F). The results indicate that a mutant sigma F lacking the terminal 7 residues functions almost normally, which suggests that glutamine 249 is dispensable. By contrast, lysine 247 is crucial for the activity of sigma F: deletion of the 9 C-terminal residues totally inactivates the protein. When the terminal 8 residues were deleted, placing lysine 247 at the C terminus, the transcriptional activity of the factor is reduced by about 80%: we attribute this effect to neutralization of the positive charge of lysine 247 by formation of a salt bridge with the -COO- terminus.

Search for the optimal sequence of the ribosome binding site by random oligonucleotide-directed mutagenesis.

Synthetic DNA duplexes corresponding to the ribosome binding site (RBS) were synthesized through the phosphite method on solid support. The synthetic RBS DNA with partial random sequences was inserted into an appropriate site between the lpp-lac promoter and the beta-galactosidase structural gene in plasmid pMKT2. The level of beta-galactosidase expression was correlated with the color intensity of the recombinant colonies on X-gal plates. The bluest colonies were isolated and characterized with respect to beta-galactosidase enzyme activity and RBS sequence. There was good correlation between color intensity and the level of the enzyme activity, and this provided a reliable phenotypic screening method in the search for the optimal regulatory sequences. Novel RBS sequences obtained here show not only the unique nucleotide distribution, but also strong complemetarity to the 3' end region of 16S rRNA, from which could be deduced a generalized RBS sequence, the position of the SD region, and the 16S rRNA position mediated during translation initiation.