Influence of intrinsic noise generated by a thermotesting device on thermal sensory detection and thermal pain detection thresholds.

Various factors can influence thermal perception threshold measurements and contribute significantly to unwanted variability of the tests. To minimize this variability, testing should be performed under strictly controlled conditions. Identifying the factors that increase the variability and eliminating their influence should increase reliability and reproducibility. Currently available thermotesting devices use a water-cooling system that generates a continuous noise of approximately 60 dB. In order to analyze whether this noise could influence the thermal threshold measurements we compared the thresholds obtained with a silent thermotesting device to those obtained with a commercially available device. The subjects were tested with one randomly chosen device on 1 day and with the other device 7 days later. At each session, heat, heat pain, cold, and cold pain thresholds were determined with three measurements. Bland-Altman analysis was used to assess agreement in measurements obtained with different devices and it was shown that the intersubject variability of the thresholds obtained with the two devices was comparable for all four thresholds tested. In contrast, the intrasubject variability of the thresholds for heat, heat pain, and cold pain detection was significantly lower with the silent device. Our results show that thermal sensory thresholds measured with the two devices are comparable. However, our data suggest that, for studies with repeated measurements on the same subjects, a silent thermotesting device may allow detection of smaller differences in the treatment effects and/or may permit the use of a smaller number of tested subjects. Muscle Nerve 40: 257-263, 2009.

Effects of positive end-expiratory pressure on systemic haemodynamics, with special interest to central venous and common iliac venous pressure in liver transplanted patients.

BACKGROUND AND OBJECTIVES: Positive end-expiratory pressure may alter cardiac function and systemic haemodynamics. As transplanted livers may be sensitive to liver congestion, the aim of our study was to evaluate the effect of positive end-expiratory pressure on the cardiovascular system and in particular on central venous and iliac venous pressure in liver transplanted patients. PATIENTS AND METHODS: Seventy-two liver transplant patients were enrolled in this prospective, interventional study. On admission to our Intensive Care Unit all patients were ventilated in a biphasic positive airway pressure mode. Haemodynamic effects of three randomly set levels of end-expiratory pressures (0, 5 and 10 mbar) were studied in the immediate postoperative period in all patients. Mean arterial pressure, central venous pressure, pulmonary capillary wedge pressure, central iliac venous pressure and cardiac index were recorded and analysed at each of the three end-expiratory pressure levels. RESULTS: The values of central- and wedge-pressure significantly increased with increased end-expiratory pressure. Central venous pressure increased by 24% and wedge pressure showed a 6% increase at 10 mbar in comparison to 0 mbar. The values for cardiac index and mean arterial pressure showed no statistically significant difference at 10 mbar as compared to 0 and 5 mbar. The mean pulmonary arterial and common iliac venous pressure were unaffected by different positive end-expiratory pressure levels. CONCLUSIONS: Short-term pressure controlled ventilation with end-expiratory pressure up to 10 mbar does not significantly impair systemic haemodynamics in liver-transplanted patients. Further studies are needed to determine whether these findings could be confirmed with higher pressure levels and/or over a longer period of ventilation time.

[Characteristics of repeated emergency physician use]. / Einsatzcharakteristik wiederholter Notarzteinsätze.

OBJECTIVE: Repeated use of emergency physicians might result in excessive costs and has discouraging effects on emergency department staff. To our knowledge, no data are available on the characteristic of repeated use of emergency physicians (EP) in the German emergency medical services. Accordingly, this retrospective survey focused on emergency cases on-the-scene, which necessitated EP intervention. METHODS: Following data from the local EMS computerised database (Medlinq, EASY, Hamburg, Germany) were retrospectively analysed for the period from January to December of 2002: total number of EP interventions, person who engaged the emergency call, number of repeat EP-users with two to three calls, number of frequent EP-users with more than three calls, their sex, age, NACA. Probably inappropriate repeat users were defined as patients with no apparent underlying distress on-the-scene, lack of preclinical findings which could explain the symptoms initially mentioned, and refuse of hospital referral by the patient. The rate of repeated inappropriate EP use was defined as the ratio of the number of repeated inappropriate use over the total number of EP-engagements in the year 2002. RESULTS: 6064 EP interventions were analysed. The rate of the repeated use was 15.5 % (939/6064) including 3.6 % (218/6064) frequent users. The main medical causes (80 %) of repeated EP use were cardiovascular, neurological, respiratory, and psychiatric. The rate of repeated inappropriate EP use was 0.2 % (11/6064). CONCLUSION: The rate of repeated inappropriate EP use in this middle sized town was low. More than four fifths of all repeated calls were for cardiovascular, respiratory, neurological, and psychiatric distress.

Airway anesthesia alone does not explain attenuation of histamine-induced bronchospasm by local anesthetics: a comparison of lidocaine, ropivacaine, and dyclonine.

BACKGROUND: Lidocaine inhalation attenuates histamine-induced bronchospasm while evoking airway anesthesia. Because this occurs at plasma concentrations much lower than those required for intravenous lidocaine to attenuate bronchial reactivity, this effect is likely related to topical airway anesthesia and presumably independent of the specific local anesthetic used. Therefore, the authors tested the effect of dyclonine, lidocaine, and ropivacaine inhalation on histamine-induced bronchospasm in 15 volunteers with bronchial hyperreactivity. METHODS: Bronchial hyperreactivity was verified by an inhalational histamine challenge. Histamine challenge was repeated after inhalation of dyclonine, lidocaine, ropivacaine, or placebo on 4 different days in a randomized, double-blind fashion. Lung function, bronchial hyperreactivity to histamine, duration of local anesthesia, and lidocaine and ropivacaine plasma concentrations were measured. Statistical analyses were performed with the Friedman and Wilcoxon rank tests. Data are presented as mean +/- SD. RESULTS: The inhaled histamine concentration necessary for a 20% decrease of forced expiratory volume in 1 s (PC20) was 7.0 +/- 5.0 mg/ml at the screening evaluation. Lidocaine and ropivacaine inhalation increased PC20 significantly to 16.1 +/- 12.9 and 16.5 +/- 13.6 mg/ml (P = 0.007), whereas inhalation of dyclonine and saline did not (9.1 +/- 8.4 and 6.1 +/- 5.0 mg/ml, P = 0.7268). Furthermore, in contrast to saline and lidocaine, inhalation of both ropivacaine and dyclonine significantly decreased forced expiratory volume in 1 s from baseline (P = 0.0016 and 0.0018, respectively). The longest lasting and most intense anesthesia developed after dyclonine inhalation (48 +/- 13 vs. 28 +/- 8 [lidocaine] and 25 +/- 4 min [ropivacaine]). CONCLUSION: Both lidocaine and the new amide local anesthetic ropivacaine significantly attenuate histamine-induced bronchospasm. In contrast, dyclonine, despite its longer lasting and more intense local anesthesia, does not alter histamine-evoked bronchoconstriction and irritates the airways. Thus, airway anesthesia alone does not necessarily attenuate bronchial hyperreactivity. Other properties of inhaled local anesthetics may be responsible for attenuation of bronchial hyperreactivity.

S(+)-ketamine increases muscle sympathetic activity and maintains the neural response to hypotensive challenges in humans.

BACKGROUND: S(+)-Ketamine is reported to exert twofold greater analgesic and hypnotic effects but a shorter recovery time in comparison with racemic ketamine, indicating possible differential effects of stereoisomers. However, cardiovascular regulation during S(+)-ketamine anesthesia has not been studied. Muscle sympathetic activity (MSA) may be an indicator of the underlying alterations of sympathetic outflow. Whether S(+)-ketamine decreases MSA in a similar manner as the racemate is not known. Thus, the authors tested the hypothesis that S(+)-ketamine changes MSA and the muscle sympathetic response to a hypotensive challenge. METHODS: Muscle sympathetic activity was recorded by microneurography in the peroneal nerve of six healthy participants before and during anesthesia with S(+)-ketamine (670 microg/kg intravenously followed by 15 microg x kg(-1) x min(-1)). Catecholamine and ketamine plasma concentrations, heart rate, and arterial blood pressure were also determined. MSA responses to a hypotensive challenge were assessed by injection of sodium nitroprusside (2-10 microg/kg) before and during S(+)-ketamine anesthesia. In the final step, increased arterial pressure observed during anesthesia with S(+)-ketamine was adjusted to preanesthetic values by sodium nitroprusside infusion (1-6 microg x kg(-1) x min(-1)). RESULTS: Anesthesia with S(+)-ketamine (ketamine plasma concentration 713 +/- 295 microg/l) significantly increased MSA burst frequency (mean +/- SD; 18 +/- 6 to 35 +/- 11 bursts/min) and burst incidence (32 +/- 10 to 48 +/- 15 bursts/100 heartbeats) and was associated with a doubling of norepinephrine plasma concentration (from 159 +/- 52 to 373 +/- 136 pg/ml) parallel to the increase in MSA. Heart rate and arterial blood pressure also significantly increased. When increased arterial pressure during S(+)-ketamine was decreased to awake values with sodium nitroprusside, MSA increased further (to 53 +/- 24 bursts/min and 60 +/- 20 bursts/100 heartbeats, respectively). The MSA increase in response to the hypotensive challenge was fully maintained during anesthesia with S(+)-ketamine. CONCLUSIONS: S(+)-Ketamine increases efferent sympathetic outflow to muscle. Despite increased MSA and arterial pressure during S(+)-ketamine anesthesia, the increase in MSA in response to arterial hypotension is maintained.

Canine leptomeningeal organ culture: a new experimental model for cerebrovascular beta-amyloidosis.

Cerebral amyloid angiopathy (CAA) is a neuropathological feature of Alzheimer's disease and a common cause of cerebral hemorrhage in the elderly. The pathogenetic mechanisms leading to the deposition of Alzheimer amyloid beta-protein (A beta) in cortical and leptomeningeal vessel walls are unknown. There are no experimental models which reproduce the pathological changes of CAA. In this study, leptomeninges from young and old dogs with pre-existing CAA were cultured in cell culture medium or cerebrospinal fluid and their viability, histological appearance and metabolic activity were analyzed during the culture. In addition, living leptomeninges of old and young dogs were incubated with fluorescein-conjugated A beta and the uptake of A beta was studied by fluorescence microscopy. Leptomeninges from young and old dogs were viable up to 8 weeks in culture. They contain many small- and medium-sized arterioles, the main vessel type affected by CAA. Histology and immunohistochemistry showed excellent preservation of the vessel wall microarchitecture up to 4 weeks in culture. The cultures were metabolically active as shown by the de novo production of beta-amyloid precursor protein. Exogenously added A beta was focally deposited in the vessel walls of old, but not young dogs. In conclusion, the organ culture of canine leptomeninges is easy to perform and appears suitable to investigate the pathogenesis and the progression of CAA.

Cyanide-induced apoptosis and oxidative stress in differentiated PC12 cells.

Terminally differentiated PC12 cells are a useful neuron-like model for studying programmed cell death in response to nerve growth factor (NGF) deprivation. This in vitro model was used to investigate the mechanism by which cyanide-induced histotoxic hypoxia produces neuronal degeneration. Treatment of undifferentiated PC12 cells with 0.1 mM KCN for 24 h did not produce cell death. In contrast, treatment of differentiated PC12 cell cultures with 0.1 mM KCN for 24 h increased cell death by 43% when compared with control cultures, as measured by trypan blue dye exclusion and lactate dehydrogenase release assays. The Ca2+/Mg(2+)-dependent endonuclease inhibitor aurintricarboxylic acid and the transcriptional inhibitor actinomycin D partially attenuated hypoxic toxicity, suggesting roles for endonuclease activation and transcription in this model of neuronal death. Extracted DNA from cyanide-treated neurons demonstrated cleavage into oligonucleosomal fragments on gel electrophoresis. Transmission electron microscopic analysis showed morphological changes consistent with apoptotic cell death, including membrane blebbing and convolution, as well as chromatin condensation and margination to the nuclear membrane. Addition of either ascorbate or catalase to the cultures partially attenuated the loss of cell viability induced by cyanide, and decreased the incidence of apoptotic cells after treatment, based on the in situ detection of DNA strand breaks. The ability of cyanide to elevate intracellular oxidant species was determined by microfluorescence in differentiated PC12 cells loaded with the oxidant-sensitive dye 2',7'-dichlorofluorescin. Exposure of cells to 0.1 mM KCN produced a rapid generation of oxidants that was blocked approximately 50% by ascorbate or catalase. These observations indicate that cyanide induces apoptosis in terminally differentiated, and not undifferentiated, PC12 cells, and that antioxidants significantly reduce the incidence of cyanide-induced apoptosis.

Selective binding of soluble Abeta1-40 and Abeta1-42 to a subset of senile plaques.

Alzheimer's disease is characterized by the progressive accumulation of amyloid-beta protein (Abeta) in senile plaques and cerebral amyloid angiopathy. It is not known whether the plaque growth is a continuous and homogeneous process or whether some plaques have a more rapid evolution. As plaques grow by the deposition of Abeta, we used an in situ binding technique to analyze the deposition of fluorescein-conjugated and biotinylated Abeta1 40 and Abeta1-42 in cryosections of brains from Alzheimer's disease patients. Only a subset of senile plaques but all cerebrovascular Abeta deposits were labeled by both Abeta1-40 and Abeta1-42. Striking differences in binding were observed among adjacent plaques. Quantitative analysis showed that on average 60% of all plaques were labeled with Abeta1-42 and 31% of all plaques were labeled with Abeta1-40 (n=7; P<0.001). Confocal laser scanning microscopy of double-labeled sections revealed that the newly deposited Abeta was only partially co-localized to pre-existing Abeta and apolipoprotein E and was not co-localized to heparan sulfate proteoglycan. Abeta binding was preserved after glycolytic pretreatment with periodic acid. Our results suggest that at a given time point only a subset of active senile plaques accumulate A(beta) and that plaque growth may be conditioned by the presence of other distinct plaque components different from Abeta, apolipoprotein E or heparan sulfate proteoglycan.

Loss of vessel wall viability in cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is a neuropathological feature of Alzeheimer's disease and an important cause of cerebral haemorrhage in the elderly. CAA is characterized by the deposition of Alzheimer amyloid beta protein (A beta) in cerebral and leptomeningeal vessel walls. In order to study the effect of cerebrovascular A beta deposits in vivo, living canine leptomeninges obtained from old dogs affected by CAA were analysed by confocal laser scanning microscopy after immunofluorescence staining for A beta and viability staining with fluorescein diacetate (FDA). Simultaneous detection of the two signals showed a segmental loss of leptomeningeal vessel wall viability at some site of A beta deposition. Many of the non-viable vessels segments were also dilated, suggesting that A beta-induced vascular cell death creates the loci minores resistentiae for the development of cerebral haemorrhage in CAA.

Activation of protein kinase C by trimethyltin: relevance to neurotoxicity.

The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5-20 microM TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24-h period. TMT treatment was accompanied by a low level of PKC down-regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 microM). Phorbol myristate-induced PKC down-regulation or inhibition with chelerythrine provided protection against TMT-induced cytotoxicity. It was concluded that TMT-induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.

Experimental deposition of Alzheimer amyloid beta-protein in canine leptomeningeal vessels.

To study the pathogenesis of cerebral amyloid angiopathy (CAA), organ cultures of canine leptomeninges were incubated with fluorescein-conjugated amyloid beta-protein (FA beta, residues 1-40; 10 nM to 200 microM). Fluorescence microscopy showed focal and dose-dependent FA beta binding to blood vessels affected by CAA at FA beta-concentrations as low as 10 nM. The new A beta deposits appeared to be extracellular and were localized to the middle and outer layers of leptomeningeal arterioles. FA beta partially co-localized with apolipoprotein E (ApoE) as revealed by confocal microscopy, suggesting that A beta in situ binds to ApoE. Young dogs or old dogs without CAA showed no deposition of FA beta. Our results indicate that after initiation of CAA pathology, physiological concentrations of soluble A beta are sufficient to sustain its further deposition and therefore the progression of CAA.

Neuroprotective effects of PKC inhibition against chemical hypoxia.

The effect of potassium cyanide-induced chemical hypoxia on protein kinase C (PKC) translocation and cell injury was studied in differentiated PC12 cells. The cellular distribution of PKC in control cells and cells exposed to 100 microM and 1 mM KCN for 30 min. was visualized by use of an anti-PKC antibody and confocal laser scanning microscope. In control differentiated PC12 cells, PKC was localized perinuclearly, while following 12-phorbol 13-myristate acetate (PMA) or KCN it was translocated to the plasma and organelle membranes. Western blot analysis was used to quantify the translocation. Chemical hypoxia increased the membrane-bound PKC to 210% of control levels, while chelerythrine, a PKC inhibitor, and block of calcium influx into the cells (with calcium channel blocker and calcium-free medium) prevented this effect. Cyanide-induced PKC translocation persisted for at least 120 min. Cell injury was monitored by measuring lactate dehydrogenase (LDH) efflux from the cells 24 hr after addition of cyanide. PKC activation plays a role in hypoxic damage, since PKC down-regulation (by overnight exposure to PMA) or inhibition (with chelerythrine or staurosporine) conferred protection against KCN-induced cytotoxicity. Ca2+ channel blocker nifedipine also protected against chemical hypoxia. None of the pretreatments rendered complete protection against cyanide-induced hypoxia, indicating that PKC-independent mechanism(s) are also activated during chemical hypoxia and contribute to cell injury.

MK-801 prevents cyanide-induced changes of Fos levels in rat brain.

The effect of acute cyanide intoxication on levels of transcriptional regulatory proteins Fos and c-Jun in rat cortex, hippocampus, cerebellum and brain stem was studied. Western blot analysis showed a differential effect of cyanide on Fos levels in the selected brain areas. The most prominent changes were seen 60 min. following ip. injection of KCN in all brain areas except the brain stem, which showed the maximal change 120 min. following cyanide. Fos levels were doubled in cortex and cerebellum and decreased to below 70% of the control levels in hippocampus. Levels of c-Jun were not altered 60 min. following cyanide treatment. Pretreatment with the NMDA receptor antagonist, MK-801, prevented the cyanide-induced changes of Fos. The differential effect of cyanide on Fos levels in different brain areas and the blockade of these changes by MK-801 suggest involvement of multiple neuronal pathways, including the excitatory amino acid (EAA) neurotransmitter system. It is concluded that cyanide alters levels of the transcriptional regulatory protein Fos through activation of the EAA neurotransmitter system and, thus, may affect gene expression in neuronal or glia cells.

Cyanide induces protein kinase C translocation: blockade by NMDA antagonists.

Activation and translocation of protein kinase C (PKC) during KCN-induced histotoxic hypoxia was studied in rat brain slices prepared from cerebellum, hippocampus, and cortex. Treatment with 1-10 mM KCN produced a significant increase in PKC translocation and enzyme activity in the particulate fraction of cerebellar and hippocampal slices. In cortical slices, PKC activity was not affected by cyanide treatment. The membrane-associated PKC activity reached a maximum 30 minutes after incubation with KCN and remained elevated up to 60 minutes in both the hippocampus and cerebellum. Pretreatment with MK-801 and APV, specific NMDA receptor antagonists, blocked the cyanide-stimulated translocation in the hippocampus and cerebellum, whereas CNQX, an AMPA/kainate receptor antagonist, did not alter the response. These results demonstrate that cyanide stimulates PKC activation and translocation from the cytosol to membranes in select brain areas and NMDA receptor activation mediates this process.

Dopaminergic neurotoxicity of cyanide: neurochemical, histological, and behavioral characterization.

Previous reports have shown that dopamine (DA) is depleted in the brains of animals treated with cyanide. To develop a model for studying the mechanisms of cyanide-induced changes in dopaminergic systems, mice were treated with cyanide (KCN, 6 mg/kg, sc) twice a day for 7 days and 16 hr after the last dose neurochemical, histological, or behavioral parameters were evaluated. DA levels in KCN-treated animals decreased in the striatum (41%), hippocampus (30%), and cerebral cortex (13%) as compared to saline-treated controls. In striatal and hippocampal tissues, but not in cerebral cortex, malondialdehyde levels increased 43 and 57%, respectively, as compared to controls, indicating that peroxidation of lipids occurred in these brain areas. Over 30% of the treated mice exhibited decreased locomotor activity and akinesia, which were suppressed by l-DOPA (100 mg/kg, ip). Tyrosine hydroxylase (TH) immunohistochemical examination of brains from cyanide-treated animals showed a reduced number of TH-positive cells in substantia nigra, indicating a loss of dopaminergic neurons. In contrast, acute cyanide (KCN, 6 mg/kg, sc) did not produce significant neurochemical or behavioral changes. Under these treatment conditions, cyanide produces a central dopaminergic toxicity which is characterized by decreased DA levels in select brain areas, impaired locomotor activity, and neuronal damage.