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1、Anesthesiology 2005; 102:60615 2005 American Society of Anesthesiologists, Inc. Lippincott Williams Si- monsen Laboratories, Gilroy, CA) were given an intra- peritoneal injection of ketamine (10 mg/kg) and diaze- pam (0.2 mg/kg) and anesthetized with 12% halothane. The rats were decapitated, and the
2、 hippocampi were removed and placed in 4C Geys Balanced Salt Solution (UCSF Cell Culture Facility, San Francisco, CA) contain- ing 50?Madenosine and 0.038 mg/ml ketamine. Next, the hippocampi were transversely sliced (400?m thick) with a tissue slicer (Siskiyou Design Instruments, Grants Pass, OR) a
3、nd stored in the Geys Balanced Salt Solution at 4C for 1 h.24The slices were then transferred onto 25-mm-diameter membrane inserts (Millicell-CM; Milli- pore, Bedford, MA), and put into six-well culture trays with 1.2 ml slice culture medium per well. The sliceculture media for the first 48 h consis
4、ted of 50% Minimal Essential Medium (Eagles with Earles Balanced Salt So- lution; UCSF Cell Culture Facility), 25% Earles balanced salt solution (UCSF Cell Culture Facility), and 25% heat- inactivated horse serum (Hyclone Laboratories, South San Francisco, CA) with 6.5 mg/ml glucose, 50?Maden- osine
5、, and 5 mMKCl; subsequent media lacked adeno- sine. Slices were kept in culture for 714 days before study. Slices were discarded if they showed more thanslight propidium iodide (PI) fluorescence (see section on assessment of cell death) at the beginning of the survival studies.Experiment Design Cult
6、ures were exposed to hypoxia by placing them into a 2-l airtight Billups-Rothenberg Modular Incubator Chamber (Del Mar, CA) through which 95% N25% CO2 gas, preheated to 37C, was passed at 510 l/min. The temperature of the chamber was kept at 37C by both passing preheated gas through the chamber and
7、by plac- ing a heat lamp over the chamber. The temperature inside the chamber was monitored with a thermocouplethermometer. After 10 min of gas flow, the chamber was sealed and placed in a 37C incubator. The partial pres- sure of oxygen was approximately 0.10.2 mmHg, mea- sured with a Clark-type oxy
8、gen electrode. After hypoxia,the culture tray was removed from the chamber, briefly opened to restore oxygenation, and returned to theincubator. For cultures treated with isoflurane, gasflowed through a calibrated vaporizer before entering the Billups-Rothenberg chamber. The hypoxic gas andisofluran
9、e entered the chamber at the same time. In experiments involving inhibitors of MAP kinases or pro- tein kinase B/Akt, the compounds were added to the culture media just before the start of hypoxia, and the media was replaced at the end of the hypoxia. A 10-?M concentration of the MEK1/2 inhibitor U0
10、126 was cho- sen because this is the minimal concentration required to produce inhibition of phosphorylation of MAPK p42/44 in slice cultures12and in addition has been shown to prevent MAPK signaling in cultured neurons.25Assessment of Cell Death Cell viability was assessed 2 and 3 days after hypoxi
11、a with PI (Molecular Probes, Eugene OR), a highly polarfluorescent dye that penetrates damaged plasma mem- branes and binds to DNA. Slice culture media containing 2.3?MPI was added to the wells of the culture trays. After 15 min, the slices were examined with a Nikon Diaphot 200 inverted microscope
12、(Nikon Corporation,Tokyo, Japan), and digital images of fluorescence were taken using a SPOT Jr. digital camera (Diagnostic Instru- ments Inc., Sterling Heights, MI). Excitation light wave- length was 490 nm, and emission was 590 nm. The sensitivity of the camera and intensity of the excitation ligh
13、t was standardized so as to be identical from day today. PI fluorescence was measured in the dentate gyrus, CA1, and CA3 regions of the hippocampal slices. Slices were imaged prior to hypoxia to obtain a PI image assumed to represent 0% cell death. We found that maximum posthypoxia death occurred af
14、ter 2 or 3 days and declined during the next 11 days.1Serial measure-ments of PI fluorescence intensity were made in pre-defined areas (manually outlining CA1, CA3, and dentate separately) for each slice using NIH Image software (free software from the US National Institutes of Health, Wash- ington,
15、 DC). Thus, cell death was followed in the same regions of each slice after hypoxia. After the measure-ment of PI fluorescence on the third day after hypoxia,607MAP KINASES IN ISOFLURANE NEUROPROTECTIONAnesthesiology, V 102, No 3, Mar 2005all of the neurons in the slice were killed with the applicat
16、ion of 100?Mpotassium cyanide and 2 mM sodium iodoacetate to the cultures for 2030 min toproduce a fluorescence signal equal to 100% neurondeath in all regions of interest. Twelve to 24 h later, finalimages of PI fluorescence (equated to 100% cell death) were acquired. The percentage of dead cells at 2 and 3 days after hypoxia were then calculated based on these values, because a linear relation exists between celldeath and PI fluorescence intensity.23,24Measurements of Ca2?i
