Physical exercise might influence the risk of oxygen-induced acute neurotoxicity.

OBJECTIVE: Hyperoxia can induce acute neurotoxicity with generalized seizures. Hyperoxia-induced reduction in cerebral blood flow velocity (CBFV) might be protective. It is unclear whether dynamic exercise during hyperoxia can overcome CBFV-reduction and thus possibly increase the risk of neurotoxicity. METHODS: We studied CBFV with both-sided transcranial Doppler with fixed transducer-position and heart rate under increasing hyperoxic conditions in nine professional military oxygen divers. The divers performed dynamic exercise on a bicycle-ergometer in a hyperbaric chamber (ergometries I-III, 21kPa, 100kPa, 150kPa pO2), with continuous blood pressure (ergometries I, II), end-tidal CO2 (PetCO2; ergometry I) being measured. RESULTS: Systolic (CBFVsyst) and diastolic CBFV (CBFVdiast) readings at rest decreased with increasing pO2. During exercise, CBFVsyst and CBFVdiast significantly increased in parallel with increasing pO2, despite reduced flow velocities at rest. ERGOMETRY I: CBFVsyst increased from 65.0 +/- 11.3 cm/second at rest to 80.2 +/- 23.4cm/s during maximum workload (n.s.), diastolic from 14.5 +/- 4.1 cm/second to 15.6 +/- 7.5 cm/s (n.s.). PetCO2 increased from 43.4 +/- 7.8mmHg to 50.0 +/- 7.5mmHg. ERGOMETRY II: CBFVsyst increased from 58.2 +/- 16.5 cm/second to 99.7 +/- 17.0 cm/s (p<0.001), diastolic from 14.0 +/- 10.7 cm/second to 29.4 +/- 11.1 cm/second (p<0.01). ERGOMETRY III: CBFVsyst increased from 54.4 +/-15.0cm/second to 109.4 +/- 22.3cm/s (p<0.001), diastolic from 14.7 +/- 10.4 cm/second to 35.5 +/- 9.3 cm/second (p<0.01). INTERPRETATION: Physical exercise overrules the decrease in CBFV during hyperoxia and leads to even higher CBFV-increases with increasing pO2. A tendency towards CO2 retainment with elevated PetCOz may be causative and thus heighten the risk of oxygen-induced neurotoxicity.

[Study protocol to improve the quality of delirium management in intensive care]. / Protokoll einer Studie zur Qualitätsverbesserung des Delirmanagements auf der Intensivstation.

BACKGROUND: Delirium in cardiac surgery patients is common and is associated with prolonged mechanical ventilation and hospital stay as well as higher mortality. Protocols may improve outcome. In our cardiac surgery intensive care unit (ICU), patients with delirium have not received standardized treatment so far. HYPOTHESIS: In cardiac surgery ICU patients, standardized delirium management will lead after a 4­week introduction, compared to nonstandardized treatment, to a reduction of delirium duration. METHODS: Prospective before/after study to evaluate a quality improvement project for delirium management over 12 weeks including 140 patients. INCLUSION CRITERIA: (a) ≥18 years, (b) consent for research with their data. EXCLUSION CRITERIA: (a) palliative status, (b) present during both the before/after phase, (c) pregnancy, (d) included in a competitive study, or (e) delirium not assessable. The implementation includes the introduction of a protocol with interprofessional training, bedside-teaching, pocket cards, posters, and reminders. The primary outcome is the duration of delirium, assessed four times a day with validated instruments. Secondary outcome measures include delirium incidence, duration of mechanical ventilation, length of stay in ICU and hospital, mortality, nursing/therapeutic interventions, cumulative doses of delirium-related drugs, and complications of delirium for a follow-up of 28 days. Empirical data will be analyzed with descriptive and inferential statistics. OBJECTIVES: The purpose of the study is a reduction of the duration and frequency of delirium in cardiac ICU patients and will provide evidence of the effect size of the introduction of a delirium management.

The centromere-kinetochore complex: a repeat subunit model.

The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species.

Neuroprotection by oxygen in acute transient focal cerebral ischemia is dose dependent and shows superiority of hyperbaric oxygenation.

The neuroprotective effect of oxygen after acute stroke in rats has been shown previously. However, the question of optimal dosing still remains unanswered. Thus, we investigated the use of oxygen at different concentrations by either normobaric oxygenation (NBO) or hyperbaric oxygenation (HBO) at different pressures in a model of transient ischemia/reperfusion in rats. Animals underwent 90 min of middle cerebral artery occlusion (MCAO) followed by 90 min of reperfusion before oxygen treatment. Oxygen was applied either by NBO (100% O(2); 1.0 absolute atmosphere, ATA) or HBO (100% O(2); 1.5, 2.0, 2.5 or 3.0 ATA) for 1 h. Primary endpoints were infarct volume and clinical outcome measured 24 h and 7 days following the MCAO. A statistically significant and long-lasting reduction in infarct volume was seen in the HBO 2.5 ATA and 3.0 ATA groups over a period of 7 days. The reduced infarct volume was accompanied with a statistically significant improvement in clinical outcome in the high-dose oxygen-treated groups. The presented data indicate that oxygen is a highly neuroprotective molecule in transient focal cerebral ischemia in rats, when applied early and at high doses. The effect is dose dependent and shows a superiority of HBO over NBO, when the primary endpoints infarct volume reduction and clinical outcome are analyzed. These data are important for the development of new acute stroke treatment studies in humans.

Alterations in the DNA metabolism of MCa-11 mouse mammary tumor cells grown in vivo and in vitro.

Mouse mammary carcinoma (MCa-11) cells were grown in vitro in exponential, plateau-fed, and starved monolayer cultures or as 100-, 250-, and 500-microns tissue culture spheroids, and in vivo as small (4-mm diameter) and large (12-mm diameter) tumors. In all of these forms, the growth rates of the MCa-11 cells were found to decrease after an initial rapid proliferation of a relatively small number of cells. The DNA distributions of these cells during different rates of growth in vitro and in vivo, as well as the proportion and intensity of labeling of the S-phase cells with [3H]thymidine and [3H]deoxyuridine, were measured by flow and absorption cytometry. We found that significant numbers of MCa-11 cells remained in S phase, even after the growth rates in vivo and in vitro had slowed. However, as growth rates decreased, the intensity and proportion of S-phase cells labeled with exogenous DNA precursors decreased. We conclude that progressive alterations, including possible slowing and cessation, of replicative DNA synthesis occur in S-phase tumor cells as the metabolic constraints on tumor growth are increased.

Thrombectomy vs. Systemic Thrombolysis in Acute Embolic Stroke with High Clot Burden: A Retrospective Analysis.

PURPOSE: The efficacy of i. v. thrombolysis in acute stroke with high clot burden is limited. Successful recanalization is very unlikely if the thrombus length exceeds 7 mm. Thus this retrospective controlled study evaluated the efficacy and safety of neurothrombectomy in the treatment of acute embolic stroke in patients selected by a thrombus length of ≥ 8 mm using the stent retriever Trevo(®) device. MATERIALS AND METHODS: 40 patients with acute occlusion of the anterior intracranial arteries with a thrombus length of ≥ 8 mm were treated with neurothrombectomy. We compared the outcome with a historical cohort of 42 patients with a thrombus length of ≥ 8 mm that received i. v. thrombolysis only. Clinical outcome was assessed by modified Rankin scale in both groups at discharge and on day 90. RESULTS: Patients did not differ in age, mRS on admission, thrombus length or time from symptom onset to i. v. thrombolysis, but the thrombectomy group had higher NIHSS on admission. Successful recanalization was achieved in 33/40 patients (83 %) with neurothrombectomy. 15 patients received i. v. thrombolysis prior to neurothrombectomy. Median mRS at discharge was 3.5 (1.25 - 5) vs. 5 (4 - 6; p < 0.01) and on day 90 3 (1 - 4) vs. 5 (4 - 6; p < 0.01). Symptomatic hemorrhage occurred in 3 vs. 7 patients. 3 vs. 17 patients died within 90 days (thrombectomy vs. control each). There were only a few intervention-related complications. CONCLUSION: Thrombectomy in acute stroke with high clot burden using the Trevo(®) device has a low risk and improved clinical outcome compared to i. v. thrombolysis alone. Treatment selection by a clot length of ≥ 8 mm might be a powerful approach to improve the outcome of mechanical thrombectomy. KEY POINTS: • Clot length of ≥ 8 mm might be a valuable criterion for indicating neurothrombectomy. • Thrombolysis only in high clot burden is associated with poor clinical outcome. • Thrombectomy using the Trevo(®) stent retriever is safe and effective.

Computerized measurement of the DNA content, areas, and autoradiographic grains of the same nuclei: demonstration that lightly (3H)thymidine-labeled bone marrow cells are predominantly in G0/G1 and G2.

A computerized "flying spot" microdensitometer and scanning stage have been combined to measure the cellular DNA content, nuclear areas, and autoradiographic grain areas of the same cells. The slide positions of the Feulgen-stained, (3H)thymidine-labeled cells are mapped with the computerized stage, and nuclear DNA content and areas are then determined by integral absorbance measurements at 588 nm. Following autoradiograph preparation, the cells are relocated and the areas of the autoradiographic grains over each nucleus are measured at a light wavelength (625 nm) and an optical density setting (greater than 0.10) that do not detect the Feulgen stain. The microcomputer calculates the portion of each nucleus covered with autoradiographic grains (grain area proportion, GAP), and it links the GAP value to the DNA content of each nucleus in the computer file for subsequent sorting and analysis. By using this system in a study of mouse bone marrow cells labeled in vivo with (3H)thymidine, we found that all S-phase cells were clearly labeled after 8 or more days of autoradiographic exposure. Prolonged exposures (up to 64 days) led to detection of lightly labeled cells (0.1 less than GAP less than 0.8) with G1/G0 and G2 DNA content.

Determining the origins and the structural aberrations of small marker chromosomes in two cases of 45,X/46,X, + mar by use of chromosome-specific DNA probes.

A 17-year-old girl (S.M.) and a 13-year-old girl (C.L.) both with Ullrich-Turner syndrome (UTS) were found to have 45,X/46,X, + mar mosaicism. The marker chromosomes in both patients were very small in size. In S.M. the marker chromosome was present in 80% of phytohemagglutinin-stimulated lymphocytes, 28% of skin fibroblasts, and 11-20% of gonadal fibroblasts. In C.L., the small marker chromosome was found in 50% of stimulated lymphocytes. S.M. is of normal height, but C.L. is short. Molecular hybridization with a number of Y-specific DNA probes demonstrated their presence in S.M. but absence in C.L. In situ hybridization with Y-specific and X-centromere-specific DNA probes confirmed the Y origin of the marker chromosome in S.M. and the X origin of the minute chromosome in C.L. Biotinylated centromere and telomere probes were also used for in situ hybridization to show the presence of centromeric and telomeric sequences in the Y-marker chromosome, suggesting that the deletion of this marker chromosome is interstitial.

Telomere staining of human chromosomes and the mechanism of radiation-induced dicentric formation.

The majority of models of radiation action developed over the past half century hold that the curvilinear dose responses exhibited by eukaryotic cells to sparsely ionizing radiations result from the interaction of pairs of lesions produced in sensitive targets of the cell. Within this conceptual framework, chromosomal exchange aberrations (e.g., interchanges) are believed to occur through the interaction of damaged sites on both chromosomes participating in the exchange. In contrast, the model proposed by Chadwick and Leenhouts (as well as some other models) suggests that such exchanges arise from initial radiation damage to only one chromosome, which then becomes associated with an undamaged chromosome. A particular aspect of this theory is that asymmetrical exchanges, such as dicentrics, may be formed from the rejoining of a broken end of one chromosome to the telomere of another. By using a DNA probe that specifically hybridizes to the telomeric region of human chromosomes, we were able to test this assertion directly. After scanning more than 200 dicentrics produced in normal human fibroblasts by 6 Gy of 60Co gamma rays, virtually none were found that contained telomeres located between the centromeres of this aberration type. Therefore, since the proposed telomere-break rejoining process, per se, is not necessarily a central element of the Chadwick-Leenhouts model, we suggest the theory be modified to exclude this mechanism.

In vivo cytogenetic effects of oil shale retort process waters.

The induction of cytogenetic effects by oil shale retort process waters from 3 types of pilot plant retorts were examined in murine bone marrow. Each of the process waters induced increased frequencies of structural aberrations in mice treated with 3 daily intraperitoneal injections of the waters. The same treatment had no effect on the frequency of sister chromatid exchanges. Mice given a 1% solution of an above-ground retort water ad libitum for 8 weeks consumed about 1 ml/kg per day of the process water and had a frequency of aberrations comparable to mice given the same dose intraperitoneally for 3 days. Transplacental exposure of C3H mouse embryos indicated that clastogenic compounds in the above-ground retort process water can cross the placenta and induce chromosomal aberrations in embryonic tissues.

Cytogenetic effects of cis-platinum(II)diamminedichloride on human lymphocyte cultures.

Human lymphocytes were treated with the antitumor agent cis-platinum)II)diamminedichloride (PDD) during either the last 24 h or 48 h of incubation. A dose-dependent effect was observed for both inhibition of mitotic activity and increased frequency of metaphases with chromosomal aberrations. The aberrations observed consisted primarily of chromatid breaks. Statistical analysis of 3244 PDD-induced breakpoints demonstrated a significantly nonrandom distribution of breakpoints between chromosomes. The pattern of distribution varied with the type of aberration. Only chromosome number 9 had a significant increase of breakpoints for each type of aberration analyzed. The breakpoints were located predominantly in lightly staining G-gands. Certain individual bands had relatively high frequencies of breakpoints, indicating a specific interaciton occurs between PDD and the DNA of human lymphocytes in vitro.

Labeling of human centromeres using an alphoid DNA consensus sequence: application to the scoring of chromosome aberrations.

Precise identification of centromeres is required for accurate scoring of asymmetrical chromosome aberrations, such as dicentrics. The centromeric regions of all human chromosomes can be labeled by in situ hybridization of a 30 nucleotide oligomer having the sequence of a conserved region of an alphoid DNA consensus sequence. Fluorescent detection of the hybridized probe allows rapid identification of centromeres and accurate scoring of dicentrics, multicentrics, acentric fragments, and the centromeric content of ring chromosomes. This procedure provides a novel approach for scoring these complex chromosome aberrations, particularly damage induced by radiation or radiomimetic agents.

SV40 T antigen induced chromosomal changes reflect a process that is both clastogenic and aneuploidogenic and is ongoing throughout neoplastic progression of human fibroblasts.

In human fibroblasts, the expression of SV40 large T antigen is known to cause a variety of chromosomal aberrations and especially dicentric chromosomes. In some cases, the later aberrations have been reported to be reversible telomeric associations. We report here aberration and chromosome number studies of twenty-nine T antigen positive lineages, studied from their initiation by transfection of T antigen sequences into human diploid fibroblasts, until crisis or immortalization occurred or, in some cases until the lines became tumorigenic in nude mice. The data show that T antigen consistently produced chromosomal instability of both number and structure by an active process that began before transformation indicators were positive and continued throughout neoplastic progression. The most frequently observed aberrations were dicentric chromosomes, which were shown to be true dicentrics by examination by in situ hybridization with telomeric sequences. These data are consistent with the hypothesis that T antigen causes human fibroblasts to become neoplastically transformed by successive rounds of chromosomal mutation and lineage evolution.

Nonrandom distribution of chromosomal aberrations induced by three chemicals.

The G-band locations of 3244 breakpoints induced by cis-platinum (II) diamminedichloride (PDD), 1460 breakpoints induced by cytosine arabinoside (ara-C), and 1257 breakpoints induced by triethylenemelamine (TEM) in human lymphocyte chromosomes were identified. The breakpoints induced by each of these chemicals demonstrated a significantly nonrandom distribution within the human karyotype. The overall pattern of the interarm distribution was dependent upon the chemical used, but certain chromosomes arms exhibited similar responses to all 3 chemicals. Comparison of the frequencies of breakpoints within individual G-bands indicated that (1) certain bands were susceptible to damage induced by all 3 chemicals; (2) certain bands were resistant to damage by all 3 chemicals; (3) certain bands demonstrated variable susceptibility to induced damage dependent upon the chemical agent; and (4) other bands demonstrated near expected frequencies of damage (by length) to all 3 agents.

A combined testing protocol approach for mutagenicity testing.

The antischistosomal agent, hycanthone methanesulfonate (HMS), was employed to illustrate the utility of carrying out several mutagenicity tests in a single concurrent animal experiment. Several commonly used procedures that were successfully integrated into a multiple testing protocol included (1) metaphase analysis in bone marrow, (2) micronucleus test in bone marrow, (3) analysis of the urine for mutagenic constituents, and (4) the host-mediated assay using Salmonella typhimurium. In addition to these animal studies, in vitro mutagenicity testing with and without activation was carried out using S. typhimurium. HMS produced positive, dose--response effects in in vitro tests, metaphase analysis, micronucleus test, and urine analysis, but not in the host-mediated assay. The results of these integrated techniques suggest that such a protocol may be a benefit to those concerned with mutagenicity testing of chemicals.

Hepatotoxic doses of dioxin do not damage mouse bone marrow chromosomes.

We report on a study of the cytogenetic and hepatotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mice of the C57B1/6J (with high-affinity TCDD receptor) or DBA/2J (with low-affinity TCDD receptor) strains were given single intraperitoneal injections of 50, 100 or 150 micrograms of TCDD/kg body weight. At various times (8-48 h) after injection, we examined bone marrow cells for cytogenetic effects by performing structural aberration, sister-chromatid exchange, and micronucleus tests. 1 month after exposure, liver sections were studied for hepatotoxic effects. We found no evidence of chromosome damage by TCDD given in doses that cause liver damage in both strains of mice.

The XRCC2 and XRCC3 repair genes are required for chromosome stability in mammalian cells.

The irs1 and irs1SF hamster cell lines are mutated for the XRCC2 and XRCC3 genes, respectively. Both show heightened sensitivity to ionizing radiation and particularly to the DNA cross-linking chemical mitomycin C (MMC). Frequencies of spontaneous chromosomal aberration have previously been reported to be higher in these two cell lines than in parental, wild-type cell lines. Microcell-mediated chromosome transfer was used to introduce complementing or non-complementing human chromosomes into each cell line. irs1 cells received human chromosome 7 (which contains the human XRCC2 gene) or, as a control, human chromosome 4. irs1SF cells received human chromosome 14 (which contains the XRCC3 gene) or human chromosome 7. For each set of hybrid cell lines, clones carrying the complementing human chromosome recovered MMC resistance to near-wild-type levels, while control clones carrying noncomplementing chromosomes remained sensitive to MMC. Fluorescence in situ hybridization with a human-specific probe revealed that the human chromosome in complemented clones remained intact in almost all cells even after extended passage. However, the human chromosome in noncomplemented clones frequently underwent chromosome rearrangements including breaks, deletions, and translocations. Chromosome aberrations accumulated slowly in the noncomplemented clones over subsequent passages, with some particular deletions and unbalanced translocations persistently transmitted throughout individual subclones. Our results indicate that the XRCC2 and XRCC3 genes, which are now considered members of the RAD51 gene family, play essential roles in maintaining chromosome stability during cell division. This may reflect roles in DNA repair, possibly via homologous recombination.