Einfluss der Anästhetika Sevofluran und Propofol auf den nekrotischen und den apoptotischen Zelltod nach inkompletter zerebraler Hemisphärenischämie und Reperfusion in der Ratte über einen Beobachtungszeitraum von 28 Tagen

The long-term effect of sevoflurane and propofol on necrotic and apoptotic cell death after incomplete cerebral ischemia and reperfusion in the rat The present study investigates the effect of the anesthetic agents propofol and sevoflurane on irreversible cell damage (necrosis) and programmed cell death (apoptosis) for a time period of 28 days after incomplete transient cerebral ischemia and reperfusion in the rat. Ninety-six fasted male Sprague-Dawley rats (415±40g) were anesthetized, intubated and ventilated with 2 Vol% isoflurane and N2O/O2 (FiO2=0.33). Catheters were inserted into the right femoral artery and vein as well as in the right jugular vein for drug administration and blood withdrawal. At the end of surgery animals were randomly assigned to one of the following groups: control (n=32): 25 µg/kg/h fentanyl i.v. and N2O/O2 (FiO2=0.33); propofol (n=32): 25 µg/kg/h propofol i.v. and O2/air (FiO2=0.33); sevoflurane (n=32) 2 Vol% sevoflurane in O2/air (FiO2=0.33). Ischemia (45min) was produced by unilateral common carotid artery occlusion plus hemorrhagic hypotension (MAP=40 mmHg). Pericranial temperature (37.5°C), arterial blood gases and pH were maintained constant. Animals were then randomly assigned to a postischemic reperfusion time of 01, 03, 07, or 28 days. At the end of the observation period the animals were deeply anesthetized and killed, the brains were removed, frozen at –70 °C, and sectioned for further evaluation. Hematoxylin-Eosin staining was used to evaluate eosinophilic cell damage and tissue damage in 7 μm sections in the hippocampus, dentate gyrus and surrounding tissues. Immunohistochemistry was used to detect activated caspase-3 as a marker of apoptotic cell death. To distinguish activated caspase-3-positive neurons from other cells, a double staining, using the specific neuronal marker NeuN was used additionally. The results showed that with the exception of one animal in the sevoflurane group there is no tissue damage or eosinophilic cell damage in either the propofol or the sevoflurane treated animals up to 28 days after ischemia. The only eosinophilic tissue damage was present in the control group. About one percent of the hippocampal neurons of all three groups were activated caspase-3-positive, independently of the observation period. Though there was a tendency in both treatment groups to a lower number of activated caspase-3-positive cells. Results of the double staining showed that activated caspase-3, though mainly expressed in neurons, is also expressed in other cells. The present study showed that propofol and sevoflurane both produce a sustained inhibition of eosinophilic cell damage up to 28 days after incomplete cerebral ischemia in rats. Although the amount of activated caspase-3-positive neurons was similar in the three groups there was a tendency towards a lower number in the treatment groups compared to control. This suggests that neuroprotection seen with both, propofol and sevoflurane, involves anti-necrotic mechanisms rather than anti-apoptotic mechanisms. Further investigations will be required in the future to investigate the detailed mechanisms of propofol and sevoflurane to develop a successful treatment of ischemic insults and their consequences.Einfluss der Anästhetika Sevofluran und Propofol auf den nekrotischen und den apoptotischen Zelltod nach inkompletter zerebraler Hemisphärenischämie und Reperfusion in der Ratte über einen Beobachtungszeitraum von 28 Tagen Die hier vorliegende Studie befasst sich mit dem Einfluss der Anästhetika Sevofluran und Propofol auf den nekrotischen und apoptotischen Zelltod nach inkompletter zerebraler Hemisphärenischämie mir Reperfusion in der Ratte über einen Beobachtungszeitraum von 28 Tagen. Zu diesem Zweck wurden 96 männliche Sprague-Dawley-Ratten (415±40g) im Versuch mit Isofluran (2,0 Vol% in O2/N2O mit FiO2=0,33) anästhesiert, intubiert und beatmet. Um die physiologischen Variablen zu überwachen (arterielle Blutdruckmessung und Blutentnahme) und konstant zu halten (Applikation von Medikamenten), wurden in die rechte A. und V. femoralis sowie in die V. jugularis Katheter gelegt und verschiedene Sonden angebracht (perikranielle sowie rektale Temperaturmessung, Messung der Hirndurchblutung). Nach abgeschlossener Präparation wurden die Tiere randomisiert einer der folgenden Gruppen zugewiesen: Kontroll-Gruppe (n=32): Ischämie mit einer Fentanyl/N2O-Narkose, Propofol-Gruppe (n=32): Ischämie unter Propofol-Narkose i.v. (25µg/kg/h), Sevofluran-Gruppe (n=32): Ischämie unter Sevofluran-Narkose (2 Vol%). Mittels eines temporären Verschlusses der A. carotis communis und einer gleichzeitigen hämorrhagischen Hypotension (MAP bei 40 mmHg), induzierte man in den drei Gruppen eine 45-minütige inkomplette zerebrale Ischämie. Nach einer Reperfusionszeit von einem, drei, sieben und 28 Tagen (je n=8) wurden die Tiere in Narkose getötet, die Gehirne entnommen, um sie nach dem Schneiden für die weitere Bearbeitung bei –70° C zu gefrieren. In der HE-Färbung wurde die Anzahl der eosinophil gefärbten Zellen ermittelt und das Ausmaß des ischämischen Schadens bestimmt. Über die immunhistochemische Detektion des Proteins akt. Caspase-3, einem Apoptose-Indikator, wurde in den Gehirnschnitten Apoptose nachgewiesen. Um im Anschluss an diese Einfachfärbung eine Differenzierung der akt. Caspase-3-positiven Zellen in Neurone und andere Zellen zu ermöglichen, wurden die Gehirnpräparate einer Doppelfärbung von akt. Caspase-3 und NeuN, einem ausschließlich neuronalen Strukturprotein, unterzogen. Es zeigte sich in den Ergebnissen der HE-Färbung, dass in den mit Propofol und Sevofluran behandelten Gruppen die Anzahl der eosinophilen Zellen und die Ausprägung des ischämischen Schadens signifikant niedriger war als in der Kontroll-Gruppe. Die Menge der akt. Caspase-3-positiven Zellen ist in allen drei Gruppen gering, was an der Art des gewählten Tiermodells oder an der Art der Untersuchung mit der Immunhistochemie liegen kann. Tendenziell erkennt man eine Reduktion akt. Caspase-3-positiver Zellen in den Gruppen Propofol und Sevofluran im Vergleich zur Kontroll-Gruppe. Die Ergebnisse der Doppelfärbung zeigen, dass neben Neuronen als Hauptexpressionsort akt. Caspase-3 auch andere, nicht neuronale Zellen diesen Apoptose-Indikator herstellen. Auch hier zeigen Gehirnschnitte von mit Propofol bzw. Sevofluran behandelten Tieren tendenziell eine geringere Menge akt. Caspase-3-positiver Neurone. Diese Beobachtungen lassen den Schluss zu, dass die beiden untersuchten Anästhetika Propofol und Sevofluran in vivo eine neuroprotektive Wirkung auf neuronales Gewebe besitzen. Es werden jedoch sicherlich weitere Untersuchungen notwendig sein, um mehr über die Wirkmechanismen dieser beiden Anästhetika in Erfahrung zu bringen und so gezielt ein Konzept für erfolgreiche Therapien ischämischer Ereignisse und ihrer Folgeerscheinungen formulieren zu können

A mobile phone based alarm system for supervising vital parameters in free moving rats

Background: Study protocols involving experimental animals often require the monitoring of different parameters not only in anesthetized, but also in free moving animals. Most animal research involves small rodents, in which continuously monitoring parameters such as temperature and heart rate is very stressful for the awake animals or simply not possible. Aim of the underlying study was to monitor heart rate, temperature and activity and to assess inflammation in the heart, lungs, liver and kidney in the early postoperative phase after experimental cardiopulmonary bypass involving 45 min of deep hypothermic circulatory arrest in rats. Besides continuous monitoring of heart rate, temperature and behavioural activity, the main focus was on avoiding uncontrolled death of an animal in the early postoperative phase in order to harvest relevant organs before autolysis would render them unsuitable for the assessment of inflammation. Findings: We therefore set up a telemetry-based system (Data Science International, DSI™) that continuously monitored the rat’s temperature, heart rate and activity in their cages. The data collection using telemetry was combined with an analysis software (Microsoft excel™), a webmail application (GMX) and a text message-service. Whenever an animal’s heart rate dropped below the pre-defined threshold of 150 beats per minute (bpm), a notification in the form of a text message was automatically sent to the experimenter’s mobile phone. With

Emergency care: then, now, and next.

Five decades ago, hospitals staffed their emergency rooms with rotating community physicians or unsupervised hospital staff. Ambulance service was frequently provided by a local funeral home. Beginning in the late 1960s and accelerating thereafter, emergency care swiftly evolved into its current form. Today, modern emergency departments not only are capable of providing around-the-clock lifesaving care in individual emergencies and disasters. They also conduct timely diagnostic workups, provide access to after-hours acute care, and serve as the "safety net of the safety net" for millions of low-income and uninsured patients. But the field's success has led to a new set of challenges. To overcome them, emergency care must become more integrated, regionalized, prevention oriented, and innovative

Emergency care: then, now, and next.

Five decades ago, hospitals staffed their emergency rooms with rotating community physicians or unsupervised hospital staff. Ambulance service was frequently provided by a local funeral home. Beginning in the late 1960s and accelerating thereafter, emergency care swiftly evolved into its current form. Today, modern emergency departments not only are capable of providing around-the-clock lifesaving care in individual emergencies and disasters. They also conduct timely diagnostic workups, provide access to after-hours acute care, and serve as the "safety net of the safety net" for millions of low-income and uninsured patients. But the field's success has led to a new set of challenges. To overcome them, emergency care must become more integrated, regionalized, prevention oriented, and innovative

Emergency Care: Then, Now, And Next

Five decades ago, hospitals staffed their emergency rooms with rotating community physicians or unsupervised hospital staff. Ambulance service was frequently provided by a local funeral home. Beginning in the late 1960s and accelerating thereafter, emergency care swiftly evolved into its current form. Today, modern emergency departments not only are capable of providing around-the-clock lifesaving care in individual emergencies and disasters. They also conduct timely diagnostic workups, provide access to after-hours acute care, and serve as the "safety net of the safety net" for millions of low-income and uninsured patients. But the field's success has led to a new set of challenges. To overcome them, emergency care must become more integrated, regionalized, prevention oriented, and innovative

Data from: Electromyographic permutation entropy quantifies diaphragmatic denervation and reinnervation

Spontaneous reinnervation after diaphragmatic paralysis due to trauma, surgery, tumors and spinal cord injuries is frequently observed. A possible explanation could be collateral reinnervation, since the diaphragm is commonly double-innervated by the (accessory) phrenic nerve. Permutation entropy (PeEn), a complexity measure for time series, may reflect a functional state of neuromuscular transmission by quantifying the complexity of interactions across neural and muscular networks. In an established rat model, electromyographic signals of the diaphragm after phrenicotomy were analyzed using PeEn quantifying denervation and reinnervation. Thirty-three anesthetized rats were unilaterally phrenicotomized. After 1, 3, 9, 27 and 81 days, diaphragmatic electromyographic PeEn was analyzed in vivo from sternal, mid-costal and crural areas of both hemidiaphragms. After euthanasia of the animals, both hemidiaphragms were dissected for fiber type evaluation. The electromyographic incidence of an accessory phrenic nerve was 76%. At day 1 after phrenicotomy, PeEn (normalized values) was significantly diminished in the sternal (median: 0.69; interquartile range: 0.66–0.75) and mid-costal area (0.68; 0.66–0.72) compared to the non-denervated side (0.84; 0.78–0.90) at threshold p<0.05. In the crural area, innervated by the accessory phrenic nerve, PeEn remained unchanged (0.79; 0.72–0.86). During reinnervation over 81 days, PeEn normalized in the mid-costal area (0.84; 0.77–0.86), whereas it remained reduced in the sternal area (0.77; 0.70–0.81). Fiber type grouping, a histological sign for reinnervation, was found in the mid-costal area in 20% after 27 days and in 80% after 81 days. Collateral reinnervation can restore diaphragm activity after phrenicotomy. Electromyographic PeEn represents a new, distinctive assessment characterizing intramuscular function following denervation and reinnervation

Sevoflurane anesthesia induced changes in the expression of <i>N</i>-methyl-D-aspartate (NMDA) receptor subunits in the hippocampus.

<p>Western blot analysis was used to determine the protein expression levels of various receptor subunits in the hippocampi of anesthetized (sev) and non-anesthetized (sham) mice. Changes in expression levels in the sev group are expressed as relative values normalized to the grey values of the sham group. *P<0.05 reveals an upregulation of the <i>N</i>-methyl-D-aspartate receptor type 1 and 2B subunits (NR1 and NR2B) after sevoflurane anesthesia. No other receptor subunit was significantly altered. Example immunoblots are depicted in the insets. NR2A  =  NMDA receptor type 2A subunit; α₂-GABA_A  = α₂ subunit of γ-aminobutyric acid type A receptor; GluR1, GluR2/3, GluR4  = α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits 1, 2/3, 4; GluR6/7 =  kainate receptor subunit 6/7, β₂-nAch  = β₂ subunit of nicotinic acetylcholine receptor.</p

Long-term potentiation (LTP) in hippocampal brain slices of anesthetized and sham-treated mice was not different.

<p>24 h after sevoflurane anesthesia (sev) or sham treatment (sham), brain slices of the animals were prepared and hippocampal LTP was assessed as elevation of field excitatory postsynaptic potential slopes (fEPSP slopes) after high frequency stimulation (HFS). HFS led to an LTP of fEPSP slopes, which was not significantly different between the two groups. Each symbol represents the averaged fEPSP slopes normalized with respect to the 5 min baseline period before HFS. Insets show fEPSP recordings before and 40 min after HFS.</p

Mice that underwent a sevoflurane anesthesia showed better cognitive performance.

<p>On days one to eight after undergoing a sevoflurane anesthesia (sev) or sham treatment (sham), cognitive performance and behavorial parameters were assessed using the modified hole board test, a task in which the animals are trained to search for food rewards hidden in marked cylinders. <b>A</b>: Time that each animal required for performing the trial plotted against time. <b>B</b>: Number of marked and baited holes, which were not visited at all during one trial (left) and number of non-marked holes which were visited (right) plotted against time. <b>C</b>: Number of times the mouse enters the board plotted against time. <b>D</b>: Number of times the mouse crossed the marked lines per minute plotted against time. Each group consisted of 24 animals. Each symbol represents averaged data from four trials per day. * p<0.05 reveals better cognitive performance (beginning on day three) and better learning (beginning on day two), as well as an attenuated anxiety-related behavior (beginning on day six) in anesthetized mice.</p

Exemplarily fiber type grouping in one rat as a histologic parameter of reinnervation (81 days after phrenicotomy in the mid-costal area).

<p>Exemplarily fiber type grouping in one rat as a histologic parameter of reinnervation (81 days after phrenicotomy in the mid-costal area).</p