However, evoked signal changes can be used to assess

However, evoked signal changes can be used to assess find FAQ flow changes and are given in percent-changes from baseline [21,22].Somatosensory stimulation was carried out with electrical pulses applied by small needle electrodes inserted under the skin of the right forepaw (PSM Module 676, HSE, March-Hugstetten, Germany). Electric brain activity was recorded monopolarily with an active calomel electrode at 0.5 mm behind the laser probe and an indifferent calomel electrode placed on the nasal bone. Signals were recorded and amplified (BPA Module 675, HSE, March-Hugstetten, Germany) and somatosensory evoked potentials (SEP) were averaged using the Neurodyn acquisition software (HSE, March-Hugstetten, Germany). Evoked potential amplitudes were calculated from the N2-P1-amplitude differences and the latency between the start of stimulation and occurrence of the P1-peak was obtained.

Approximately 60 minutes before the stimulation experiments, isoflurane/N2O anesthesia was discontinued and replaced by intravenous application of ��-chloralose (80 mg/kg; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Supplementary doses of chloralose (30 mg/kg) were given every hour. During chloralose anesthesia, the animals were ventilated with a nitrogen/oxygen mixture of 1/1.Neurovascular coupling measurementSomatosensory activation was carried out by electrical stimulation of the right forepaw with rectangular pulses of 0.3 ms width and a repetition frequency of 2 Hz for 30 seconds. The stimulation current was kept constant at 1.5 mA so that systemic blood pressure changes did not occur [21-23].

Allowing a rest of 30 seconds after each stimulation train, activation-rest cycles were repeated 10 times to increase signal to noise ratio. Flow velocity responses were averaged and relative responses were calculated in relation to the resting phase, setting the resting phase to zero. The evoked flow velocity responses were calculated from the averaged relative flow velocity signals under conditions of stimulation.Clinical chemistryAt the end of the experiments Drug_discovery blood samples were drawn into tubes containing aprotinin (Trasylol, Bayer AG, Leverkusen, Germany), immediately centrifugated and separated, after which plasma was stored at -80��C until analyses. The neuron-specific enolase (NSE) levels were determined using an ELISA (NSE EIA kit; Hoffmann-La Roche, Basel, Switzerland). The S-100B protein was determined with an immunoluminometric assay (Sangtec 100 LIA; Sangtec Medical, Bromma, Sweden) using monoclonal antibodies specific for the beta subunit of the S-100 protein. Cytokine analysis were performed for IL-6, TNF��, interferon (IFN) �� using commercialized rat ELISA sets (BD Bioscience, Heidelberg, Germany).

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