Animals
All animal care and experimental protocols were approved by Institutional Animal Care and Use Committee of Tajen University, and were in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85–23, revised 1996). Adult male Sprague–Dawley rats weighing 250–300 g (n = 142) were purchased from the Experimental Animal Center of the National Science Council and BioLASCO, Taiwan, Republic of China. They were housed under conditions of constant temperature (23 ± 0.5 °C) and humidity (50 ± 5 %) with a standard 12 h light–dark cycle (08:00–20:00) and unrestricted access to standard laboratory rat chow (Purina) and tap water. All animals were allowed to acclimatize for at least 7 days before experimental manipulations. All efforts were made to reduce the numbers of animals used and to minimize animal suffering at each stage of the experiment.
General animal preparation
The preparatory surgery that included intubation of the trachea and cannulation of the femoral artery and both femoral veins was performed under an induction dose of pentobarbital sodium (50 mg/kg, i.p.) [17, 22]. During recording session, the anesthesia was maintained by continuous intravenous infusion of propofol (Zeneca Pharmaceuticals, Macclesfield, UK) at 20–25 mg/kg/h, which provided satisfactory anesthetic maintenance while preserving the capacity of neural control of cardiovascular functions [23]. Pulsatile and mean SAP (MSAP), as well as heart rate (HR), was continuously displayed on a polygraph (Gould RS3400, Valley View, OH, USA) via a pressure transducer (BD P23XL, Franklin Lakes, NJ, USA). Animals were ventilated mechanically by the use of a small rodent ventilator (Harvard 683, South Natik, MA, USA) to maintain an end-tidal CO2 within 4.0-4.5 %, as monitored by a capnograph (Datex Normocap, Helsinki, Finland). This procedure was conducted to minimize possible confounding cardiovascular changes secondary to respiratory perturbation. The head of the animal was thereafter fixed to a stereotaxic head holder (Kopf 1430, Tujunga, CA, USA), and the rest of the body was placed on a thermostatically controlled pad to maintain rectal temperature of 37 ± 0.5 °C. All data were collected from animals with a steady baseline MSAP above 90 mmHg at the beginning of the recording period.
Recording and power spectral analysis of SAP signals
The waveform signals of SAP recorded from the femoral artery were simultaneously subject to online power spectral analysis as detailed previously [23–25]. The SAP signals were resampled at 256 Hz by an eight-point averaging algorithm, and analyzed was truncated into 16-s (2,048 points) time segments with 50 % overlap. A Hamming window in the time domain was used to decline the leakage effect [24, 26]. Power spectral density of different frequency components of SAP signals was computed using a fast Fourier transform. We were particularly interested in the very low-frequency (0–0.25 Hz) and low-frequency (0.25-0.8 Hz) components of the SAP spectrum. These frequency components of SAP signals were reported to take origin from the RVLM [25] and their power density reflect the prevailing sympathetic neurogenic vasomotor tone [17, 22, 23, 25, 27]. The power densities of these two spectral components were displayed during the experiment, alongside SAP, MSAP and HR, in an online and real-time manner.
Microinjection of test agents into the RVLM
Similar to the procedures described previously [17, 27], microinjection bilaterally of test agents into the functionally identified RVLM sites was performed stereotaxically and sequentially with a glass micropipette (external tip diameter: 50–80 μm) connected to a 0.5-μL Hamilton microsyringe (Reno, NV, USA). The stereotaxic coordinates for the RVLM were 4.5 to 5.0 mm posterior to lambda, 1.8 to 2.1 mm lateral to midline, and 8.0 to 8.5 mm below the dorsal surface of the cerebellum. These coordinates were selected to cover the ventrolateral medulla in which both ER mRNA [19] and protein [20, 28] are distributed, and where functionally identified sympathetic premotor neurons are located [21]. At the beginning of each experiment, the functional location of RVLM neurons on either side was established by monitoring a transient pressor response (15–25 mmHg) after microinjection of L-glutamate (1 nmol, Sigma–Aldrich). Subsequent microinjections of test agents were delivered to the identified pressor loci. The test agents used in this study included 17β-estradiol-3-sulphate sodium (E2β; Sigma–Aldrich, St. Louis, MO, USA); a selective ERα agonist, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1 H-pyrazole (PPT; Tocris Cookson Inc., Bristol, UK); a selective ERβ agonist, diarylpropionitrile (DPN; Tocris Cookson); a nonspecific ER antagonist, ICI 182780 (Tocris Cookson); a selective ERα antagonist, methyl-piperidino-pyrazole (MPP; Tocris Cookson); a selective ERβ antagonist, R,R-tetrahydrochrysene (R,R-THC; Tocris Cookson); a transcription inhibitor, actinomycin D (AMD; Tocris Cookson); a PI3K inhibitor, LY294002 (Calbiochem, San Diego, CA, USA); an Akt inhibitor (Calbiochem); an antisense oligonucleotide (ASON) against the rat ERα or ERβ mRNA (Quality Systems, Taipei, Taiwan) or the scrambled (SCR) ERα or ERβ oligonucleotide (Quality Systems). For ERα mRNA, the ASON sequence was 5’-CATGGTCATGGTCAG-3’ and the SCR sequence was 5’-ATCGTGGATCGTGAC-3’ [29]. For ERβ mRNA, the ASON sequence was 5’-GAATGTCATAGCTGA-3’ and the SCR sequence was 5’-AAGGTTATCGCAAGT-3’ [29]. A total volume of 50 nl was microinjected to each side of RVLM. The dose of test agents and treatment regimen were adopted from our preliminary experiments and previous studies [17, 30] that used the same test agents for the same purpose as in this study. The dose of each antagonist or inhibitor used in this study has been shown in our pilot studies to significantly inhibit cardiovascular responses induced by its specific ligand or enzyme. All test agents were freshly dissolved in artificial cerebrospinal fluid (aCSF) at pH 7.4, except for ICI 182780, AMD and LY294002, which used 5 % dimethyl sulfoxide (DMSO) as the solvent. The composition of aCSF was (in mM): NaCl 117, NaHCO3 25, glucose 11, KCl 4.7, CaCl2 2.5, MgCl2 1.2, and NaH2PO4 1.2. Control experiments showed that these vehicles had no significant effect on baseline MSAP or HR during the 120 min observation period. To avoid confounding effects of drug interactions, each animal received only one treatment of synthetic estrogen, selective ERα, ERβ agonist or vehicle, given alone or in combination with one test agent. Pretreatment with microinjection into the bilateral RVLM of AMD, or ERα or ERβ ASON or SCR, were carried out 1 h or 24 h respectively, prior to DPN application.
Construction and purification of recombinant AdPTEN
To construct adenovirus encoding phosphatase and tensin homologues deleted on chromosome 10 (AdPTEN) or green fluorescence protein (AdGFP), human PTEN complementary DNA or GFP was subcloned into the adenovirus transfer vector pCA13 (Microbix, Toronto, Ontario, Canada) to yield the pCA13-PTEN or pCA13-GFP fusion protein. Subsequently, recombinant adenovirus was generated by cotransfection of pCA13-PTEN or pCA13-GFP with the pJM17 vector (Microbix, Toronto, Ontario, Canada), a plasmid containing the adenoviral genome, into 293 cells [27]. The adenoviruses were purified by CsCl ultracentrifugation and desalted by G-25 gel-filtration chromatography. The titers of the adenoviruses were determined by measuring optical density at 260 nm and plaque-forming assays using 293 cells. AdGFP served as the control viruses [27].
Adenovirus-mediated PTEN gene transfer into the RVLM
The gene transfer of PTEN into the RVLM was performed by microinjection bilaterally into the nucleus of AdGFP or AdPTEN 7 days prior to DPN administration. An adenoviral suspension containing 5 × 108 plaque-forming units (pfu)/100 nl was administered into each injection site over 10–15 min using a glass micropipette [27]. The coordinates used are the same for microinjection of the test agents. Following the microinjection, the muscle layers covering the fourth ventricle were sutured. Body temperature was maintained at 37 °C with heating pads until the animals had recovered from surgery. The rats were allowed to recover in their home cages with free access to food and water. Only animals that showed progressive weight gain after the gene transfer were used in subsequent experiments.
Protein extraction and Western blot analysis
For quantitative analysis of PTEN protein expression in the RVLM, the ventrolateral medulla was rapidly removed and placed on dry ice. Tissues on both sides at the level of RVLM (0.5 to 1.5 mm rostral to the obex) that contained the injection sites were collected, and the same medullary samples thus obtained from 4–6 rats under the same experimental condition were pooled and stored at −80 °C until further analysis. Western blot analysis [27, 31, 32] was carried out on protein extraction from the RVLM for PTEN or β-tubulin. The primary antiserum used included rabbit polyclonal antiserum against PTEN (1:1000; Upstate Biotechnology, Lake Placid, NY, USA) or β-tubulin (1:10000; Sigma–Aldrich). This was followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10000; Jackson Immunoresearch Laboratories, West Grove, PA, USA). Specific antibody–antigen complex was detected using an enhanced chemiluminescence Western blot detection system (Perkin–Elmer Life Sciences, Boston, MA, USA). The amount of detected protein was quantified by Photo-Print Plus software (ETS Vilber-Lourmat, France) and was expressed as fold change relative to basal PTEN protein level. β-tubulin served as an internal control to demonstrate equal loading of the proteins.
Immunohistochemical staining
The procedures of immunohistochemical staining were described previously [33]. At day 7 after the gene transfer, rats were deeply anesthetized with pentobarbital sodium (100 mg/kg, i.p.) and perfused transcardially with warm heparinized saline, followed by ice-cold 4 % paraformaldehyde in 0.1 M PBS (pH 7.4). The brain stem was rapidly removed and post-fixed in the latter solution overnight at 4 °C. 35-μm coronal sections of the rostral medulla oblongata were cut using a cryostat. After pre-absorption in gelatin (0.375 %), normal horse serum (3 %) and triton-X 100 (0.2 %) in PBS, the sections were incubated with a rabbit polyclonal antibody against PTEN (1:1000; Wako). After incubation in biotinylated horse anti-mouse IgG (1:200; Jackson ImmunoResearch), the sections were rinsed in PBS and incubated with AB complexes (Vectastain ABC elite kit, Vector Laboratories, Burlingame, CA). This was followed by incubation with a 3,3’-diaminobenzidine substrate kit f (Vector Laboratories). Sections were rinsed in PBS and dehydrated by passing through graded series of ethanol and xylene. Sections were mounted and observed under a light microscope (BX53, Olympus optical, Tokyo, Japan).
Brain histology
At the conclusion of each experiment, the animal was killed by an overdose of pentobarbital sodium (100 mg/kg, i.p.), and the brain stem was removed from animals and fixed in 30 % sucrose in 10 % formaldehyde–saline solution for at least 72 h. Histological verification of the location of microinjection sites was carried out on frozen 25-μm sections stained with 1 % Neutral red (Sigma–Aldrich).
Statistical analysis
All values are expressed as mean ± SEM. For functional experiments, the time course of the effects of various treatments on MSAP, HR or power density of vasomotor components of SAP spectrum were assessed using two-way analysis of variance (ANOVA) with repeated measures for group difference. For biochemical experiments, differences between treatment groups were assessed using one-way ANOVA. This was followed by the Scheffé multiple-range test for post hoc assessment of individual means. The maximal changes in the hemodynamic parameters were evaluated with Student’s t-test. P < 0.05 was considered statistically significant.
