All experimental procedures carried out in this study have been approved by the Institutional Animal Care and Use Committee of the Kaohsiung Chang Gung Memorial Hospital (#96008), and were in compliance with the guidelines for animal care and use set forth by this committee.
Adult, male Sprague–Dawley rats (206–248 g; n = 322) were purchased from the Experimental Animal Center of the National Applied Research Laboratories, Taiwan. They were housed in our Association for Assessment and Accreditation of Laboratory Animal Care International-accredited Center for Laboratory Animals under temperature control (24 ± 0.5°C) and 12-h light–dark cycle (lights on during 08:00–20:00). Standard laboratory rat chow and tap water were available ad libitum. Animals were allowed to acclimatize for at least 7 days prior to experimental manipulations.
Under an initial pentobarbital sodium anesthesia (50 mg kg-1, IP), the trachea was intubated and the right femoral artery and both femoral veins were cannulated. Animals received thereafter intravenous infusion of propofol (Zeneca, Macclesfield, UK) at 20 mg kg-1 h-1. This scheme provides satisfactory anesthetic maintenance while preserving the capacity of central cardiovascular regulation . The head of animals was fixed to a stereotaxic headholder (Kopf, Tujunga, CA, USA), and body temperature was maintained at 37°C with a heating pad. Animals were allowed to breathe spontaneously with room air during the recording session.
Experimental endotoxemia model of brain death
An experimental endotoxemia model of brain death  was used, employing intravenous administration of Escherichia coli lipopolysaccharide (LPS; serotype 0111:B4, Sigma-Aldrich, St. Louis, MO, USA) (15 mg˙kg-1) as the insult, with saline serving as the vehicle control in some experiments. Temporal changes in SAP recorded from the femoral artery were routinely followed for 240 min. The SAP signals were also subject simultaneously to on-line and real-time power spectral analysis . We were particularly interested in the LF (0.25-0.8 Hz) component in the SAP spectrum because its power density represents the most crucial link between our animal model and clinical observations from patients who died of systemic inflammatory response syndrome . As we reported previously [15–17], the LF spectral component underwent triphasic changes that composed of a reduction (Phase I), augmentation (Phase II) and disappearance (Phase III) of its power density. Of note is that the last phase resembles that observed in our brain dead patients .
Index for mortality
We assessed mortality by constructing a survival curve over 240 min after intravenous administration of LPS. Animals that succumbed to experimental endotoxemia exhibited a dramatic reduction of loss in the power density of the LF component of SAP signals before death [15–17].
Collection of tissue samples from RVLM
We routinely collected tissue samples from RVLM [15–17] at the peak of each phase of experimental brain death. Medullary tissues collected from anesthetized animals but without treatment served as sham-controls. Tissues from both sides of the ventrolateral medulla, at the level of RVLM (0.5 to 1.5 mm rostral to the obex), were collected by micropunches made with a 1 mm (id) stainless steel bore to cover the anatomical boundaries of RVLM. The concentration of total proteins extracted was determined by the BCA Protein Assay (Pierce, Rockford, IL, USA).
Sample preparation for proteomic and western blot analysis
As in previous studies [13, 18, 19], samples from RVLM were mixed with a tissue protein extraction reagent that contains protease inhibitor and T-PER (Roche, Basel, Switzerland). After being homogenized on ice, the mixture was centrifuged at 10,000 rpm. The supernatant was concentrated by precipitation overnight at −20°C using 10% trichloroacetic acid (TCA) and 0.1% dithiothreitol (DTT), followed by centrifugation at 13,000 rpm at 4°C. The pellet was air-dried after being washed twice with ice-cold acetone, and was reconstituted with a rehydration solution (2% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 0.5% immobilized pH gradient (IPG) buffer, 8 M urea, 15 mM DTT and trace bromophenol blue).
2-Dimensional electrophoresis (2-DE)
The methods for 2-DE in a recent study  were used. In brief, isoelectric focusing (IEF) in the first dimension was carried out with immobiline DryStrip gels (13 cm in length, linear pH gradient 3–10; GE Healthcare, Piscataway, NJ, USA). The gels were rehydrated for 16 h with 300 μL rehydration solution covered by mineral oil, with the strips placed gel-side-down in an IPGphor strip holder (Amersham Pharmacia Biotech, Piscataway, NJ, USA). An IPGphor Isoelectric Focusing System (Amersham Pharmacia Biotech) was used for IEF, which was carried out at 20°C. A three-phase program was used for both analytical and preparative gels. The first phase was set at 500 V for 1 h, the second at 1,000 V for 1 h, and the third at a linear gradient spanning from 8,000 V to 16,000 V for 2 h. After the IEF run, the IPG gel strips were kept at −80°C or prepared directly for second-dimension electrophoresis.
The IPG gel strips were incubated at room temperature prior to second-dimension electrophoresis in a sodium dodecyl sulfate (SDS) equilibration solution (50 mM Tris–HCl, pH 8.8, 6 M urea, 2% SDS, 30% glycerol, and trace bromophenol blue) that contains 1% DTT; to be followed by incubation in SDS equilibration solution that contains 2.5% iodoacetamide. Second dimension run on the gels was carried out at 4°C on running SDS-PAGE gels (16 x 15 cm) without stacking using a Hoefer SE 600 (Amersham Pharmacia Biotech), and the IPG gel strips were embedded on top of the gels with 1% agarose. Electrophoresis was performed at 30 mA/gel for 5 h until bromophenol blue reached the bottom of the gel, after which the 2-D gels were stained with either Coomassie blue or silver nitrate.
The silver-stained 2-D gels were scanned in an ImageScanner II (Amersham Pharmacia Biotech), and the images were processed using Adobe Photoshop and PowerPoint software. Protein spots were checked manually to eliminate poorly detectable spots or artifacts due to gel distortion during silver staining, after which they were quantified and numbered using ImageMaster 2D Platinum (GE Healthcare). For this purpose, the intensity of each detected spot was first determined by ImageMaster, followed by computation of the area at 75% of the spot intensity. The volume of each spot in the 2-D gel was calculated based on these two parameters, and the relative volume of each spot was expressed as a percentage of the total volume of all quantified spots.
In-gel digestion and MALDI-TOF mass spectrometry
As in a recent proteomics study , protein spots excised from the Coomassie blue-stained gels were destained with 0.2 ml acetonitrile and dried in a centrifugal evaporator. After rehydration in 10 mM DTT, alkylated with 100 mM iodoacetamide, the dried gels were digested on ice with digestion buffer made of 0.02 mg/ml trypsin gold (mass spectrometry grade; Promega, Madison, WI, USA) and 50 mM NH4HCO3. After removing excess solution, proteins were further digested for 15 h at 37°C. Peptides extracted with 50% acetonitrile in 5% formic acid were desalted and concentrated using in-tip reversed-phase resin (Zip Tip C18; Millipore, Billerica, MA, USA). Peptide mixtures were applied to the sample target and air-dried after being eluted from the Zip Tip with 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile. After mixing with the matrix (α-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitrile, 0.1% TFA), the sample was analyzed in a MALDI-TOF mass spectrometer system (Voyager DE-PRO, Applied Biosystems, Foster City, CA, USA) [13, 18, 19].
We measured the peptide masses as mono-isotopic masses. Peptide mass fingerprinting was searched against the NCBI database using MASCOT 2.1 (Matrix Science, Boston, MA, USA). Settings in the algorithm included Rattus as taxonomy, trypsin as enzyme, maximum of one missed cleavage site and assuming carbamidomethyl as a fixed modification of cysteine and oxidized methionine as a variable modification. Mass tolerance was set to 100 ppm.
Western blot analysis
Western blot analysis [13, 15–19] was carried out using a rabbit polyclonal antiserum against UCH-L1 (Santa Cruz, Santa Cruz, CA, USA); or a mouse monoclonal antiserum against ubiquitin (Santa Cruz) or β-actin (Chemicon, Temecula, CA, USA). This was followed by incubation with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences, Little Chalfont, Bucks, UK) for UCH-L1; or sheep anti-mouse IgG (Amersham Biosciences) for ubiquitin or β-actin. Specific antibody-antigen complex was detected by an enhanced chemiluminescence Western blot detection system (Santa Cruz). The amount of protein was quantified by the ImageMaster Video Documentation System software (Amersham Biosciences), and was expressed as the ratio relative to β-actin protein.
Isolation of RNA and real-time polymerase chain reaction (PCR)
Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsgad, CA, USA). All RNA isolated was quantified by spectrophotometry and the optical density 260/280 nm ratio was determined. As in our previous studies [13, 16], reverse transcriptase reaction was performed using a SuperScript Preamplification System (Invitrogen) for the first-strand cDNA synthesis. Real-time PCR analysis was performed by amplification of cDNA using a LightCycler® (Roche). PCR reaction for each sample was carried out in duplicate for all the cDNA and for the GAPDH control. Primers were designed using the sequence information of the NCBI database by Roche LightCycler® probe design software 2.0, and oligonucleotides were synthesized by Genemed Biotechnologies (Taipei, Taiwan).
The primer pairs used for amplification of target genes were:
DTT, Dithiothreitol; IEF, Isoelectric focusing; IκB, Inhibitory-κB; IPG, Immobilized pH gradient; LF component, Low-frequency component; LPS, Lipopolysaccharide; NF-κB, Nuclear factor-κB; NOS I, Nitric oxide synthase I; NOS II, Nitric oxide synthase II; RVLM, Rostral ventrolateral medulla; SAP, Systemic arterial blood pressure; SDS, Sodium dodecyl sulfate; TCA, Trichloroacetic acid; TFA, Trifluoroacetic acid; UCHs, Ubiquitin C-terminal hydrolases; UCH-L1, Ubiquitin C-terminal hydrolase isozyme-L1; UPS, Ubiquitin-proteasome system; psma-1, 5’-GTTGTAACCTCGCCGGA-3’ (forward primer) 5’-CTGCATGTGTCTTCGACTT-3’ (reverse primer); ubb, 5’-CGCACCCTCTCTGATTACA-3’ (forward primer) and 5’-CAAAGATGAGCCTCTGCTG-3’ (reverse primer); uch-l1, 5’-AAACGGAGAAGTTGTCCC-3’ (forward primer) and 5’-CGTCCACATTATTGAACAGGATAAA-3’ (reverse primer); gapdh, 5’-CTTCTCTTGTGACAAAGTGGAC-3’ (forward primer) 5’-TTAGCGGGATCTCGCTC-3’ (reverse primer).
Fluorescence signals from the amplified products were quantitatively assessed using the LightCycler® software program (version 3.5; Roche). Second derivative maximum mode was chosen with baseline adjustment set in the arithmetic mode. The relative changes in mRNA expression were determined by the fold-change analysis in which
Note that Ct value is the cycle number at which fluorescence signal crosses the threshold.
We measured proteasome activity with a commercial kit ( Proteasome-Glo 3-Substrate System; Promega, Madison, WI) according to the recommended protocol. Proteins extracted from the ventrolateral medulla were incubated with proteasome-Glo reagent at room temperature for 2 h. For the purpose of the present study, we used the luminogenic substrates provided for the detection of chymotrypsin-like activity, Suc-LLVY-aminoluciferin; trypsin-like activity, Z-LRR-aminoluciferin and caspase-like activity, Z-nLPnLD-aminoluciferin. Proteasome activity was measured with a luminometer (Berthold Technologies, Bad Wildbad, Germany) and expressed as fold changes against sham-controls.
Microinjection of test agents into RVLM
Test agents were microinjected bilaterally and sequentially into RVLM via a glass micropipette connected to a 0.5-μL Hamilton (Reno, NV, USA) microsyringe [15–19]. The coordinates used were: 4.5 to 5 mm posterior to the lambda, 1.8 to 2.1 mm lateral to the midline and 8.1 to 8.4 mm below the dorsal surface of the cerebellum. As a routine, a total volume of 50 nl was delivered to each side of RVLM over 2–3 min to allow for complete diffusion of the test agents to an area approximately 800 x 800 μm within the anatomical boundaries of RVLM . Test agents used included a non-selective proteasome inhibitor , lactacystin (Calbiochem, San Diego, CA, USA); a specific inhibitor of chymotrypsin-like proteasomal activity , proteasome inhibitor II (Calbiochem); a general inhibitor of ubiquitin-recycling , ubiquitin aldehyde (Calbiochem); and a potent, reversible, competitive and active site-directed inhibitor of UCH-L1 , UCH-L1 inhibitor (Calbiochem).The doses were adopted from previous reports that used those test agents for the same purpose as in this study. Lactacystin or ubiquitin aldehyde was dissolved in artificial cerebrospinal fluid, and proteasome inhibitor II or UCH-L1 inhibitor was dissolved in 10% and 40% DMSO respectively. Possible volume effect of microinjection was controlled by injecting the same amount of solvent. All test agents or their vehicles were given 30 min before LPS administration. To avoid the confounding effects of drug interactions, each animal received only one test agent.
All values are expressed as mean ± SEM. Changes in protein expression, real-time PCR products or enzyme activity in RVLM during each phase of experimental brain death was used for statistical analysis. One-way ANOVA was used to assess group means. This was followed by the Scheffé multiple-range test for post hoc assessment of individual means. Mortality rate was assessed by the Fisher exact test. P < 0.05 was considered to be statistically significant.