Reagents and antibodies
RNAlater and HotStarTaq® Master Mix Kit were purchased from Qiagen (Valencia, CA, USA). TRizol Reagent was purchased from Thermo Fisher Scientific (Waltham, MA, USA). 5 X All-In-One RT MasterMix (with AccuRT Genomic DNA Removal Kit) was purchased from Applied Biological Materials Inc. (Richmond, BC, Canada). RIPA buffer, BCA Protein Assay kit, Chemiluminescent Substrate Detection kit and HRP-linked goat anti mouse IgG were purchased from Boster (Wuhan, China). Protease inhibitor cocktail and phosphatase inhibitors were purchased from Roche (Basel, Basel City, Switzerland). Rabbit antibodies against VGluT1, EAAT2, EAAT3, tyrosine hydroxylase (TH), Glial fibrillary acidic protein (GFAP), and mouse antibody against VGluT3 were all purchased from Abcam (Cambridge, UK). Rabbit antibodies against VGluT2, EAAT1, EAAT3, β-actin and mouse antibody against GFAP, Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 555 goat anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Diaminobenzidine, isoflurane, sodium pentobarbital and mouse anti-TH antibody were ordered from Sigma (St Louis, MO, USA). Polink-2 plus® Polymer HRP Detection System was obtained from ZSGB-BIO (Beijing, China).
Animals and Cyclic intermittent hypoxia exposure
Male Sprague-Dawley rats were obtained from Beijing Vital River Laboratory Animal Technology Co, Ltd. (Beijing, China), aged 8 weeks and weighed 240-250 g at entry into the protocol. Rats were housed under room temperature and standard humidity (50 ± 5%) with a 12 hrs day/night circle with laboratory chow and water ad libitum. All procedures performed in this study were in accordance with national animal research regulations, and all animal experimental protocols were approved by the Institutional Animal Ethics Committee at the First Afflicted Hospital of Xinxiang Medical University.
Cyclic intermittent hypoxia exposure and control procedures were similar with previously report procedures . In brief, rats in their home cages were placed inside the Oxycycler Model A84XOV (Biospherix, Redfield, NY, USA) hypoxia system and exposed to CIH or room air. O2 fraction (FIO2) in each chamber was monitored and regulated by two timer-controlled valves. The chamber was flushed with 100% N2 to inspired FIO2 nadir of 10% for 3 min. The FIO2 gradually returned to 21% over the remainder of each cycle. The exposure cyclic was repeated every 6 min for 8 hrs/day from 8:30 to 16:30 for 14 consecutive days during rat sleeping hours. For the control group, the control rats underwent the same exposure, but the chamber was flushed with compressed air. After completion of CIH exposure, all animals were assigned to the following studies.
Human carotid body
Human surgical specimens were obtained from The First Affiliated Hospital of Xinxiang Medical University with consent from patients (granting approval number: 2016008). Human carotid body specimen was obtained from a patient with left carotid body paraganglioma and human cerebral cortex tissue was obtained from a patient with craniocerebral trauma. The protocol related to human subjects was conducted in accordance with the declaration of Helsinki, and approved by the Ethics Committee of The First Affiliated Hospital of Xinxiang Medical University.
Rat carotid body harvest
After anesthesia was achieved by inhalation of 2% isoflurane, the rat was decapitated and the carotid bifurcations were rapidly removed and placed in 95% O2-5% CO2 statured ice-cold PBS. The CBs were dissected and then immediately soaked in RNAlater and stored at -80°C until analyzed. The approximate time from euthanasia to removal of the CB to placement in RNAlater was about 4 min.
RNA extraction and RT-PCR
Total RNA was extracted from human CB specimen using Trizol-reagent. For the reverse transcription (RT), 500 ng of total RNA reversely transcribed into cDNA after gDNA removal using 5 X All-In-One RT MasterMix (with AccuRT Genomic DNA Removal Kit), in accordance to the manufacturer's protocol. Instead of RT MasterMix, DEPC-H2O was used in reverse transcription reaction to obtain a negative cDNA control. The mRNA expression level was detected by PCR in a Veriti® 96-well Thermal cycler (Applied Biosystems, Foster City, CA, USA) using HotStarTaq® Master Mix Kit. According to the manufacturer’s protocol, 10 μl of HotStarTaq Master Mix, 1μl of gene-specific primer pairs and 2 μl of cDNA samples were mixed together to produce a final volume of 20 μl. The PCR reactive conditions were 95°C for 10 min, followed by 40 cycles at 94°C for 50 sec, then annealing temperature for 50 sec and 72°C for 1 min, then ending at 72°C for 10 min. An equal volume of the negative cDNA control was used in the PCR as negative PCR control. The PCR product was loaded into 1.2% agarose gel. A total of 3 technical replicates were conducted. All primers were exon spanning and designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/). Details of all primers are listed in table 1.
Table 1. Sequence of human primers used in RT-PCR experiment
VGluT: vesicular glutamate transporter, EAAT: excitatory amino acid transporter, Tm: temperature, F: forward primer, R: reverse primer.
Protein extraction and western blot analysis
Protein was extracted from 16 CBs pooled from eight Control or eight CIH rats and human CB specimen. Tissues were homogenized with RIPA buffer containing phosphatase inhibitors and protease inhibitor cocktail. The supernatant was collected after centrifuged at 12,000 g/min for 10 min at 4°C and protein concentration was detected by BCA protein assay kit. 35 mg of protein was loaded into 8% SDS-PAGE gel, and then transferred to PVDF membrane (Millipore, Darmstadt, HE, Germany) at 200 mA for 2 h. After blocking with 5% skim milk, the membrane was probed with primary antibodies including: rabbit anti-VGluT1 (1:1000), anti-VGluT2 (1:1000), anti-EAAT1 (1:1000), anti-EAAT2 (1:1000), anti-EAAT3 (1:6000), anti-EAAT3 (1:1000), β-actin (1:1000) and mouse anti-VGluT3 (1:1000) antibodies. The membrane was then washed with TBS-T and incubated with second HRP-linked goat anti rabbit (1:8000) or HRP-linked goat anti mouse (1:5000) at room temperature for 1 h. After incubation, the membranes were washed three times with TBS-T and immersed in Chemiluminescent Substrate Detection kit and detected by AmershamTM Imager 600 system (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), in accordance to the manufacturer’s instructions. A total of 3 technical replicates were conducted.
Rats were fixed with 4% neutral buffered formalin and the CBs were removed. The paraffin-embedded CB sections prepared by Shandon™ Finesse™ 325 Microtomes (Thermo Fisher Scientific, Waltham, USA) at 3 μm thickness were used. The sections were deparaffinized by being heated at 45°C for 1 h and cleared through xylene solution, then rehydrated through washes in decreasing grades of ethanol (100%, 95%, 80%, and 60%) for 3 min each. After washing with PBS, the slides were incubated in citrate buffer (pH 6.0) for 15 min at 100°C to retrieve antigen. Then, sections were immersed to 3% H2O2 to block endogenous peroxidase activity. After being blocked with 10% goat serum, sections were incubated overnight at 4°C with following primary antibodies including: rabbit anti-EAAT2 (1:200), rabbit anti-EAAT3 (1:50) and mouse anti-VGluT3 (1:100). After washing three times in PBS containing 0.2% Triton X-100, sections were incubated with Polink-2 plus® Polymer HRP Detection System, in accordance with the manufacturer’s instruction. Finally, sections were washed, and the antibody-antigen complex was visualized by incubating the sections with 0.01% H2O2 and 0.05% diaminobenzidine to yield a reddish-brown crystalline product. Negative staining control was prepared by omitting the primary antibody. The staining was examined through the Nikon H600L microscope and photographed with Nikon digital camera DS-Fi1c (Nikon, Tokyo, Japan).
Double immunofluorescence staining
CB paraffin sections prepared as above were exposed to a mixture of two primary antibodies as follows: rabbit anti-EAAT2 (1:50) with mouse anti-GFAP (1:200); rabbit anti-EAAT2 (1:50) with mouse anti-TH (1:2000); rabbit anti-EAAT3 (1:50) with mouse anti-TH (1:2000); rabbit anti- EAAT3 (1:50) with mouse anti-GFAP (1:200); mouse anti-VGluT3 (1:100) with rabbit anti-TH (1:100); mouse anti-VGluT3 (1:100) with rabbit anti-GFAP (1:100). After washing, the slides were incubated with a mixture of Alexa Fluro 488 goat anti-rabbit IgG (1:400) and Alexa Fluro 555 goat anti-mouse IgG (1:400). Negative staining control was prepared by omitting the primary antibody. The staining was examined with the Axio Observer A1 microscope and photographed with the AxioCam MRc5 camera (Carl Zeiss, Gottingen, Germany).
The data were presented as means ± S.D. Statistical evaluation was conducted by unpaired Student’s t-test. P < 0.05 was considered significant.