Online Supplement
CIRCULATIONAHA/2004/475913; VERSION: 3
Methods
Animals
Male adult Sprague-Dawley rats (185 to 215 g, n = 143) purchased from the Experimental Animal Center, National Science Council, Taiwan were used. All experimental procedures were carried out in compliance with the guidelines of our institutional animal care committee.
General Preparation
In experiments that evaluated cardiovascular functions, preparatory surgery was carried out under an induction dose of pentobarbital sodium (50 mg/kg, IP). Rats received thereafter continuous intravenous infusion of propofol (30 mg‧kg-1‧h-1),1,2,3,4 which provided satisfactory anesthetic maintenance while preserving the capacity of central cardiovascular regulation.4 Pulsatile and mean systemic arterial pressure (MSAP), as well as heart rate (HR), were recorded on a polygraph (Gould RS3400).1,2,3 Animals were mechanically ventilated to maintain end-tidal CO2 to be within 4 to 5%, as monitored by a capnograph (Datex Normocap).
Evaluation of Sympathetic Vasomotor Tone
The SAP signals recorded were simultaneously subject to on-line power spectral analysis.1,2,,3,4 We were particularly interested in the very low-frequency (00.25 Hz) and low-frequency (0.250.8 Hz) components in the SAP spectrum. Our laboratory demonstrated previously5 that these spectral components of SAP signals take origin from the RVLM, and their power density reflects the prevailing sympathetic neurogenic vasomotor tone.1,2,3,4
Induction of Experimental Endotoxemia
LPS (20 mg/kg, serotype 0111:B4; Sigma) was administered intravenously to induce endotoxemia.1,6 Injection of the same amount of saline served as vehicle and volume control. Temporal changes in MSAP, HR or power density of vasomotor components of the SAP signals were routinely followed for 6 hours.
Microinjection of Oligonucleotide or Decoy DNA into the RVLM
Microinjection bilaterally of oligonucleotide using glass micropipette, at a volume of 100 nL per side, was carried out stereotaxically and sequentially into functionally identified RVLM sites as detailed previously.2,3,6 Each animal received a single injection of an antisense oligonucleotide that targets against the coding region (nt 61-78) of the mouse heat-inducible hsp70 gene (Genemed Biotechnologies), a sense or a scrambled hsp70 oligonucleotide (Genemed Biotechnologies),7 or the kB decoy or scrambled DNA.8 The dose and treatment regimen were adopted from previous reports7,8 that used the oligonucleotide or decoy DNA for the same purpose as in the present study. Microinjection of aCSF served as the vehicle and volume control.
Heat Shock Induction
Under pentobarbital anesthesia, rats were placed on a temperature-controlled electric heating pad set at 45°C. Hyperthermic HS was induced by maintaining the core temperature of animals at 42 ± 0.5°C for 15 minutes,7,9 as monitored by a thermistor probe placed in the colon. Animals were thereafter allowed to recover at room temperature for the time-interval stipulated for each experiment. Normothermic (NT) controls were similarly anesthetized but received no HS induction. Sham-controls were anesthetized without receiving further experimental treatments.
Isolation of Total RNA and Reverse Transcription-Polymerase Chain Reaction
At the conclusion of some physiological experiments, the brain was rapidly removed and placed on dry ice. Isolation and extraction of total RNA from the ventrolateral part of medulla oblongata, at the level of RVLM (0.5 to 2.5 mm rostral to the obex), and reverse transcription-polymerase chain reaction (RT-PCR) analysis of iNOS, nNOS or GAPDH mRNA were carried out.1,10,11 The amount of mRNA products for iNOS or nNOS was analyzed by the ImageMaster VDS analysis software (Amersham Pharmacia Biotech), and was expressed as the ratio to GAPDH mRNA product, which served as the internal control.
Protein Extraction and Western Blot Analysis
At the conclusion of some physiological experiments, the brain was rapidly removed and placed on dry ice. Tissues on both sides of the ventrolateral part of the medulla oblongata, at the level of RVLM, were collected by micropunches made with a stainless steel bore (1 mm i.d.) and frozen in liquid nitrogen. 7 Medullary samples thus obtained from 4-6 rats under the same experimental condition were stored at -80°C and were pooled to provide sufficient tissue for analysis. Western blot analysis was carried out using a mouse monoclonal antiserum against the inducible form of HSP70 (StressGen) or IKKa (Calbiochem), or a rabbit polyclonal antiserum against IkBa (Calbiochem), iNOS (Santa Cruz) or p65 subunit of NF-kB (Santa Cruz). This was followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson) for HSP70, IKKa or goat anti-rabbit IgG (Jackson) for IkBa, iNOS or NF-kB. Specific antibody-antigen complex was detected by an enhanced chemiluminescence Western blot detection system (NENTM, Life Science Products). The amount of protein product was quantified by the ImageMaster Video Documentation System (Amersham Pharmacia Biotech), and was expressed as the ratio to b-tubulin protein.
Nuclear Protein Extraction and Electrophoretic Mobility Shift Assay
Nuclear protein extracts were prepared from the ventrolateral medulla that included RVLM. Medullary tissues obtained from 5 to 6 rats were homogenized, centrifuged and pellets were collected to prepare purified nuclei. Nuclear protein was extracted by modifying the procedures reported by Hutchison et al.12 and was stored at -85°C until further use. Protein concentrations were determined by the Bradford assay.
Electrophoretic mobility shift assay (EMSA) for activated NF-kB was performed following the procedures reported by Yeh et al.8 Double-stranded synthetic oligonucleotide probe for NF-kB was end labeled using [g-32P]ATP and T4 polynucleotide kinase (Promega). DNA and protein complexes were resolved on 4% Tris-glycine nondenaturing polyacrylamide gels. After electrophoresis, gels were dried and exposed to photographic film. For supershift studies, a polyclonal antiserum against NF-kB p65, p50 or c-Rel (Santa Cruz) was added to the DNA binding reaction cocktail and incubated for 30 minutes. Binding of the antiserum to the appropriate transcription factor was indicated by a supershift in the EMSA.
IKK Assay
An immune complex kinase assay was performed to detect IKK activity according to the procedures reported by Huang et al.13 Equal amounts of protein extracts were immunoprecipitated with IKKa antiserum together with protein G-agarose beads. IKK kinase activity was determined in a kinase buffer containing 1 mg of GST-IkBa (1-317) (Santa Cruz), 25 mM ATP and 3 mCi of [g-32P]ATP. The kinase reaction was terminated by the addition of 5x SDS-PAGE sample buffer, and phosphorylated IkBa was resolved by SDS-PAGE gel electrophoresis and visualized by autoradiography. The other half of the immunoprecipitates was subject to SDS-PAGE and immunoblotting with anti-IKKa antiserum to verify equal loading of the kinase.
Histology
The brain stem of some animals was removed at the end of the physiological experiment and fixed in 30% sucrose in 10% formaldehyde-saline solution for at least 72 hours. Frozen 25-mm sections of the medulla oblongata were stained with 1% neural red for histological verification of the location of microinjection sites. 1% Evans blue was added to the microinjection solution to facilitate this process.
Statistical Analysis
All values are expressed as mean ± SE. One-way or 2-way ANOVA with repeated measures was used, as appropriate, to assess group means. This was followed by the Scheffé multiple range test for post hoc assessment of individual means. A value of P < 0.05 was considered to be statistically significant.
References
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2. Chan SHH, Wang LL, Ou CC, et al. Contribution of peroxynitrite to fatal cardiovascular depression induced by overproduction of nitric oxide in rostral ventrolateral medulla of the rat. Neuropharmacology. 2002;43:889898.
3. Chan SHH, Wang LL, Wang SH, et al. Differential cardiovascular responses to blockade of nNOS or iNOS in rostral ventrolateral medulla of the rat. Br J Pharmacol. 2001;133:606-614.
4. Yang CH, Shyr MH, Kuo TBJ, et al. Effects of propofol on nociceptive response and power spectra of electroencephalographic and systemic arterial pressure signals in the rat: correlation with plasma concentration. J Pharmacol Exp Ther. 1995;275:15681574.
5. Kuo TBJ, Yang CHH, Chan SHH. Selective activation of vasomotor component of SAP spectrum by nucleus reticularis ventrolateralis in rats. Am J Physiol. 1997;272:H458H492.
6. Chuang YC, Chan JYH, Chang AYW, et al. Neuroprotective effects of coenzyme Q10 at rostral ventrolateral medulla against fatality during experimental endotoxemia in the rat. Shock. 2003;19:427-432.
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9. Li PL, Chao YM, Chan SHH, et al. Potentiation of baroreceptor reflex response by heat shock protein 70 in nucleus tractus solitarii confers cardiovascular protection during heatstroke. Circulation. 2001;103:2114-2119.
10. Feinstein DL, Galea E, Aquino DA, et al. Heat shock protein 70 suppresses astroglial-inducible nitric oxide synthase expression by decreasing NFkB activation. J Biol Chem. 1996;271:17724-17732.
11. Hauser GJ, Dayao EK, Wasserloos K, et al. HSP induction inhibits iNOS mRNA expression and attenuates hypotension in endotoxin-challenged rats. Am J Physiol. 1996;271:H2529-H2535.
12. Hutchison KA, Dittmar KD, Czar MJ, et al. Proof that hsp70 is required for assembly of the glucocorticoid receptor into a heterocomplex with hsp90. J Biol Chem. 1994;269:5043-5049.
13. Huang KC, Chen CW, Chen JC, et al. HMG-CoA reductase inhibitors inhibit inducible nitric oxide synthase gene expression in macrophages. J Biomed Sci. 2003;10:396-405.
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