[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.

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.

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.

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.

DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes.

Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere signal at the points of fusion, a clear indication of impaired end-capping. Telomeric fusions were not observed in any of the repair-proficient controls and occurred only rarely in a p53 null mutant. In striking contrast, chromosomal end fusions that retained telomeric sequence were observed in nontransformed DNA-PK(cs)-deficient cells, where they were a major source of chromosomal instability. Metacentric chromosomes created by telomeric fusion became even more abundant in these cells after spontaneous immortalization. Restoration of repair proficiency through transfection with a functional cDNA copy of the human DNA-PK(cs) gene reduced the number of fusions compared with a negative transfection control. Virally transformed cells derived from Ku70 and Ku80 knockout mice also displayed end-to-end fusions. These studies demonstrate that DNA double-strand break repair genes play a dual role in maintaining chromosomal stability in mammalian cells, the known role in repairing incidental DNA damage, as well as a new protective role in telomeric end-capping.

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.

Ordered tandem arrangement of chromosomes in the sperm heads of monotreme mammals.

A very old unanswered question in classical cytology is whether chromosomes are arranged randomly in sperm or whether they occupy specific positions. Even with modern methods of chromosome painting, it is difficult to resolve this question for the very condensed and almost spherical sperm head of most mammals. We have taken advantage of the unusual fibrillar sperm head of monotreme mammals (echidna and platypus) to examine the position of chromosome landmarks in a two-dimensional array. We used fluorescence and radioactive in situ hybridization to telomeric, rDNA, and unique sequences to show that chromosomes are arranged tandemly and in a defined order in the sperm nucleus.

CO-FISH reveals inversions associated with isochromosome formation.

Despite the likely prevalence and documented biological impact of inverted DNA sequences in humans and other species, our ability to detect them on a routine basis is limited. The technique of chromosome orientation fluorescence in situ hybridization (CO-FISH) was used to detect obligate chromosome inversions associated with isochromosome formation in two human cell lines. Simultaneous hybridization of a strand-specific telomeric probe allowed us to deduce the absolute orientation of repetitive DNA sequences associated with the inverted region. These results show that, in principle, CO-FISH could be used to detect virtually any type of inversion, including those likely to escape detection by other methods. Prospective applications of the technique are discussed in relation to its principal limitation, the present availability of suitable single-stranded DNA probes.

On the origin of lateral asymmetry.

Lateral asymmetry refers to unequal fluorescent intensity between adjacent regions of sister chromatids. It has been observed in the centromeric regions of mitotic chromosomes of mouse or human origin when cells are grown in 5-bromo-2'-deoxyuridine (BrdU) for a single round of DNA synthesis. The chromosome-orientation fluorescence in situ hybridization (CO-FISH) technique was used with pseudodiploid mouse cells to show that the regions of asymmetrical brightness coincide with major satellite repetitive DNA, and that the more heavily BrdU-substituted chromatid is the one that fluoresces less brightly. These observations support a 20 year old hypothesis on the origin of lateral asymmetry. Other observations suggest that differential loss of DNA from the heavily substituted chromatid also contributes to lateral asymmetry.

A new method for detecting pericentric inversions using COD-FISH.

A new approach for detecting chromosomal inversions, based on the recently developed technique of chromosome orientation and direction fluorescence in situ hybridization (COD-FISH), is presented. COD-FISH is a strand-specific modification of standard FISH technology which allows the hybridization of single-stranded probes to one, and only one, chromatid of a metaphase chromosome. It can be used to determine the absolute 5'-to-3' direction of DNA target sequences with respect to the short-to-long arm direction of a given chromosome. Since an inversion reverses the orientation of DNA sequences within the inverted region, an inversion becomes detectable as a "switch" in probe signal from one chromatid to the other, when compared to a reference probe outside of the inverted region. Pericentric inversions in chromosomes 1, 8, 10, and X, which had previously been identified by chromosome banding, were analyzed by the COD-FISH technique. The results presented here demonstrate that COD-FISH can be used for the detection of pericentric inversions and that, in some instances, it provides additional information not obtainable by more conventional methods of cytogenetic analysis. Practical limitations of the COD-FISH technique are also discussed.

FISH analysis of the telomere sequences of bulldog ants (Myrmecia: formicidae).

Chromosomes from several species of ants from the genus Myrmecia were hybridized with deoxyoligomer probes of either (T2AG2)7, the putative insect telomere repeat sequence, or (T2AG3)7, the vertebrate telomere repeat sequence. While both sequences hybridized over a range of stringency conditions, (T2AG2)n was clearly the predominant sequence at the termini of the Myrmecia chromosomes. No interstitial sites of either sequence were detected. The genus Myrmecia has a wide range of karyotypes, with chromosome numbers ranging from 2n=2-84. It has been hypothesized that the ancestral karyotype was 2n=4 and karyotype evolution proceeded with an increase in chromosome number. In the absence of detectable interstitial sites of telomere sequence, it is interesting to speculate on the origin of the new telomeres as the chromosome numbers increased.

Direction of DNA sequences within chromatids determined using strand-specific FISH.

The 5' to 3' direction of DNA strands within chromatids of metaphase chromosomes can be determined by using simultaneous hybridization of a single strand of the telomere probe and a single strand of a repetitive sequence to slides pretreated for strand-specific hybridization. The telomere probe identifies the direction of the DNA helical strand remaining in each chromatid of the metaphase chromosomes. The direction of the repetitive sequence is then determined from the direction of the strand to which it hybridizes. This method was used to determine the 5' to 3' direction of three repetitive DNA sequences, each for a different human repeat family.

Identification of trisomy in Macaca fascicularis by fluorescence in situ hybridization with a human chromosome 13 DNA library.

A juvenile macaque monkey with abnormal phenotypic and behavioral features was studied cytogenetically. An additional autosome was found in over 90% of the animal's cultured cells. This chromosome, subsequently identified as number 16 in the macaque karyotype by G-banding, was shown to be mostly homologous with human chromosome 13 using fluorescence in situ hybridization of a human chromosome specific cosmid library. Although the monkey, now deceased, exhibited some abnormal physical and behavioral features, none of the severe clinical characteristics associated with human chromosome 13 trisomy were apparent. We suggest that the incomplete expression of 13-trisomy observed could result if the macaque chromosome were deficient in some of the region(s) of chromosome 13 common to humans affected with the disorder.

Chromosome localization and orientation of the simple sequence repeat of human satellite I DNA.

The predominant chromosomal locations of human satellite I DNA were detected using fluorescent in situ hybridization (FISH). Synthetic deoxyoligonucleotides designed from consensus sequences of the simple sequence repeats of satellite 1 were used as probes. The most abundant satellite I repeat, the -A-B-A-B-A- form, is located at the pericentromeric regions of chromosomes 3, 4, 13, 14, 15, 21, and 22. The less abundant -B-B-B-form was not detected on chromosome 4, but was present at all the other locations. A variation of FISH that allows strand-specific hybridization of single-stranded probes (CO-FISH) determined that the human satellite I sequences are predominantly arranged in head-to-tail fashion along the DNA strand.

Strand-specific FISH reveals orientation of chromosome 18 alphoid DNA.

A method is described for making in situ hybridization strand specific. Through the use of a synthetic DNA probe of a repetitive sequence in the centromeric region of chromosome 18, it is shown that the repeats exist in a head-to-tail tandem array. The method should be useful for studies of the molecular organization and mapping of chromosomes.

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.

Highly conserved repetitive DNA sequences are present at human centromeres.

Highly conserved repetitive DNA sequence clones, largely consisting of (GGAAT)n repeats, have been isolated from a human recombinant repetitive DNA library by high-stringency hybridization with rodent repetitive DNA. This sequence, the predominant repetitive sequence in human satellites II and III, is similar to the essential core DNA of the Saccharomyces cerevisiae centromere, centromere DNA element (CDE) III. In situ hybridization to human telophase and Drosophila polytene chromosomes shows localization of the (GGAAT)n sequence to centromeric regions. Hyperchromicity studies indicate that the (GGAAT)n sequence exhibits unusual hydrogen bonding properties. The purine-rich strand alone has the same thermal stability as the duplex. Hyperchromicity studies of synthetic DNA variants indicate that all sequences with the composition (AATGN)n exhibit this unusual thermal stability. DNA-mobility-shift assays indicate that specific HeLa-cell nuclear proteins recognize this sequence with a relative affinity greater than 10(5). The extreme evolutionary conservation of this DNA sequence, its centromeric location, its unusual hydrogen bonding properties, its high affinity for specific nuclear proteins, and its similarity to functional centromeres isolated from yeast suggest that this sequence may be a component of the functional human centromere.

Interstitial hybridization sites of the (TTAGGG)n telomeric sequence on the chromosomes of some North American hylid frogs.

Interstitial hybridization sites for the (TTAGGG)n telomeric repeat sequence were present in all seven species of hylid frogs examined and in a triploid hybrid between two of the species. Intra- and interspecific differences and similarities in hybridization sites agreed with what is known about the systematics of these species. Chromosome fusions, fissions, and inversions do not appear to have played a role in the evolution of the interstitial sites for the telomeric repeat in the species examined.