Experiments were performed according to a protocol approved by the Yonsei University Animal care and Use committee in accordance with NIH guidelines.
Male Hsp70 transgenic mice (Hsp70 Tg) and wild-type (Wt) littermates originally created by Dillmann et al (University of California, San Diego; Maber et al, 1995) were used.
2. Transient Middle Cerebral Artery Occlusion Model (tMCAO)
Ischemia was induced using the tMCAO model. Mice were anesthetized by face mask using 3% to 4% isofluorane followed by 1.5% to 2% for maintenance.
Physiological parameters including body temperature, blood pressure, and arterial blood gases were monitored and maintained in the normal range. A 6-0 nylon monofilament suture was inserted into the common carotid artery to occlude the ostium of middle cerebral artery. After 2h, the suture was inserted of middle cerebral artery. After 2h, the suture was retracted to allow reperfusion. Twenty-four hours later, the animals were euthanized in a CO2 chamber and then perfused transcardially with heparinized saline. Brains were harvested for different assays.
3. Primary astrocyte culture
Primary cortical astrocytes were cultured from 1 to 3 day old postnatal ICR
mice and maintainedin Minimum essential medium (MEM, Gibco, USA) containing 10% fetal bovine serum and 10% equine serum (Hyclone, USA).
Briefly, hemispheres of new bone ICR mice were removed aseptically from the skulls, freed of the meanings.
4. Heat Pretreatment
Astrocyte cultures were washed three times with balanced salt solution (BSS5.5) containing 5.5mM glucose, 116mM NaCl, 1.8mM CaCl2, 0.8mM MgSO4, 5.4mM KCl, 1mM NaH2PO4, 14.7mM NaHCO3, and HEPSE at pH7.4. The culture medium was then exchange with BSS5.5 and incubation continued at 43℃ for 30
min.
5. Oxygen and Glucose Deprivation (OGD)
Cultures of either astrocyte were deprived of glucose and oxygen by changing the culture medium to balanced salt solution containing no glucose (BSS0.0) and kept in an oxygen-free chamber at 37℃ for 6h. Cultures were then transferred to a 37℃ incubator with 5% CO2 and reperfused with glucose at a concentration of 5.5mmol/L (BSS5.5) at normoxia for 24h. All experiments were performed in triplicate.
6. Cresyl violet stain
After animals were perfused, brains were sunk in 20% sucrose, frozen, and cut into 20㎛ sections on a cryostat. Brain sections from four consecutive coronal levels of brain were stained with cresyl violet.
7. Hoeschst-PI nuclear staining
The cell death was evaluated by staining of non-viable cell with propidium iodide (Sigma, USA), and live cell with Hoechst 33258 dye (Sigma, USA).
Staining with the fluorescent dyes propidium iodide and Hoechst 33258 allows discrimination of apoptotic cells on the basis of nuclear morphology and evaluation of membrane integrity. Hoeschst dye was added to the culture medium to a final concentration of 2-5㎍/㎖ and the cultured cells were kept at 37℃ for 30min. Propidium iodide solution was the added (final concentration 2-5㎍/㎖) just before observation in a Olympus microscope equipped for epifluorescence with UV filter block.
8. Two-dimensional electrophoresis
Protein sample preparation : Cultured cell pellets were washed twice in ice-cold PBS (GIBCO, USA) and lysed in sample buffer composed with 7M urea, 2M Thiourea containing 4%(w/v) 3-[(3-cholamidopropy)dimethyammonio]-1-propanesulfonate (CHAPS,) 1%(w/v) dithiothreitol (DTT) and 2%(w/v)
pharmalyte and 1mM benzamidine. Proteins were extracted for one hour at room temperature with vortexing. After centrifugation at 15,000rpm for one hour at 15℃, insoluble material was discarded and soluble fraction was used for two-dimensional gel electrophoresis. Protein loading was normalized by Bradford assay.
A. 2D PAGE
IPG dry strips were equilibrated for 12-16h with 7M urea, thiourea containing 2% 3-[(3-cholam-idopropy) dimethyammonio]-1-propanesulfonate (CHAPS,), 1% dithiothreitol (DTT), 1% pharmalyte and respectively loaded with 200㎍
of sample. Isoelectric focusing (IEF) was performed at 20℃ using a Multiphor II electrophoresis unit and EPS 3500 XL power supply (Amersham Biosciences, Sweden) following manufacturer’s instruction. For IEF, the voltage was linearly increased from 150 to 3,500V during 3h for sample entry followed by constant 3,500V, with focusing complete after 96kVh. Prior to the second dimension, strips were incubated for 10 minutes in equilibration buffer (50mM Tris-Cl, pH6.8 containing 6M urea, 2% SDS and 30% glycerol), first with 1%
DTT and second with 2.5% iodoacetamide. Equilibrated strips were inserted onto SDS-PAGE gels (20-24cm, 10-16%). SDS-PAGE was performed using Hoefer DALT 2D system (Amersham Biosciences, Sweden) following manufacture’s instruction. 2D gels were run at 20℃ for 1.7kVh. And then 2D
gels were silver stained as described by Oakley et al. but fixing and sensitization step with glutaraldehyde was omitted.
B. Image analysis
Quantitative analysis of digitized images was carried out using the PDQuest software (version 7.0, BioRad) according to the protocols provided by the manufacturer. Quantity of each spot was normalized by total valid spot intensity. Protein spot were selected for the significant expression variation deviated over two fold in its expression level compared with control or normal sample.
9. MALDI-TOF analysis
Enzymatic digestion of protein in-gel spot were enzymatically digested in-gel in a manner similar to that previously described by Shevchenko et al. and using modified porcine typsin. Gel pieces were washed with 50% acetonitrile to remove SDS, salt and stain, dried to remove solvent and then rehydrated with trypsin(8-10㎍/㎕) and incubated 8-10h at 37℃. The proteolytic reaction was terminated by addition of 5㎕ 0.5% trifluoroacetic acid. Typtic peptides were recovered by combining the aqueous phase from several extractions of gel pieces with 50%
aqueous acetonitrile. After concentration the peptide mixture was desalted using C18ZipTips(Millipore, USA), and peptides eluted in 1-5㎕ of acetonitrile. An aliquot of this solution was mixed with an equal volume of a saturated solution of
cyano-4-hydroxycinnamic acid in 50% aqueous acetonitirle, and l㎕ of mixture spotted onto a target plate, and then protein analysis were performed using a Ettan MALDI-TOF (Amersham Biosciences, Sweeden).
10. Western blot analysis
Proteins were isolated from mouse brain and astrocyte and lysed in solubilizing buffer (1x PBS, 1% nonidet P-40. 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitors-PMSF, aprotinin and sodium orthovandate). Equal amounts of protein extracts were separated by 10% SDS-PAGE and transferred to PVDR membrane (Millipore, USA). The membrane was blocked with 5% nonfat milk in TBS containing 0.05% and reacted with antibody mouse anti-Hsp70 (1:1000, Stressgen, USA), rabbit anti-NF-κB (1:1000, Calbiochem, Germany), mouse anti-phospho-IκB (1:1000, Santa Cruz, USA), rabbit anti-Phospho-p38 MAPK (1:1000, Cell Signaling, USA), rabbit anti-Phospho-STAT-1 (1:1000, Cell Signaling, USA), rabbit anti-Phospho-SAPK/JNK (1:1000, Cell Signaling, USA), rabbit anti-c-Fos (1:1000, Calbiochem, UK) and rabbit anti-Phospho-c-Jun (1:1000, Cell Signaling, USA). The membrane was then incubated with the secondary antibody and thoroughly washed. Immunoreactive bands were visualized with a SuperSignal (Thermo, USA).
11. Co-immunoprecipitation
This was performed by following a protocol from Stressgen Biotechnologies (Catalog and Technical Reference Guide) with minor modifications. Whole brain tissue and astrocyte cell lysates were pre-cleared by adding 50㎖ Protein A/G PLUS-Agarose (Santa Cruz, USA), 2㎎ of tissue lysate in 1mL of complete RIPA buffer. Precleared lysates (200㎖) were then incubated with 2.5㎎ of mouse monoclonal Anti-Hsp70 antibody (Stressgen, USA) or an IgG isotype control (2.5㎎ normal mouse IgG, sc-2025; Santa Cruz, USA) at 4℃ overnight. The Protein A/G PLUS-Agarose was then collected and the supernatant was aspirated off by microcentrifuging the mixture for 2min at 4,300g. After washing all reactions five times, samples were boiled for 5min and then microcentrifuged briefly to pellet Protein A/G PLUS-Agarose. Twenty microliters of the supernatants were used for Western blot analysis. It reacted with antibody mouse anti-Hsp70 (1:1000, Stressgen, USA), rabbit anti-NF-κB (1:1000, Calbiochem, Germany), mouse anti-IκB (1:1000, Santa Cruz, USA), rabbit anti-p38 MAPK (1:1000, Cell Signaling, USA), rabbit anti-Phospho-STAT-1 (1:1000, Cell Signaling, USA), rabbit anti-SAPK/JNK (1:1000, Cell Signaling, USA), rabbit anti-c-Fos (1:1000, Calbiochem, UK) and rabbit anti-c-Jun (1:1000, Cell Signaling, USA).
12. Phosphorylation ELISA assay
Cell extracts were subjected to the Immunoassay Kit (Biosource, USA) according to the manufacturer’s specifications. Add 100㎕ of the standards and samples to the appropriate microtiter wells. Tap gently on side of plate to thoroughly mix and incubated for 2h at room temperature. Pipette 100㎕ of detection antibody solution into each well except the chromogen blanks and incubate for 1h at room temperature. Add 100㎕ anti-rabbit IgG-HRP working solution to each wells and incubate for 30min at room temperature. Add 100㎕ of stabilized chromogen to each well and incubate for 30min at room temperature and in the dark. The O.D. values at 450nm can only be read after the stop solution has been added to each well.
13. Electrophoretic-Mobility Shift Assay (EMSA)
Nuclear extracts were subjected to the EMSA “Gel Shift” Kit (Panomics, CA) according to the manufacturer’s specifications. This assay enables the simultaneous detection and semiquantitative comparison of the DNA-binding activity of NF-κB AGTTGAGGGGACTTTCCCAGGC-3’), AP-1 (5’-GCCTTGATGACTCAGCCGGAA) and STAT-1 (5’-CATGTTATGCATATTC CTGTA AGTG-3’) from nuclear extracts in the mouse brain and primary cultured astroctye. Briefly, biotin-labeled DNA-binding oligonucleotides were incubated with 10㎎ of nuclear extract at 15℃ for 30min to allow the formation of
NF-κB/DNA, AP-1/DNA and STAT-1/DNA complexes. This complexes were separated from the free probes by 6% non-denaturing gel electrophoresis in 0.5x TBE at 120V for 15min. The probes in the complexes were then extracted, ethanol precipitated and hybridized to the EMSA “Gel Shift” Kit array. Detection of signals was obtained using the SuperSignal (Thermo, USA).
14. RT-PCR Reaction
Total RNA was isolated and purified with Trizol Reagent (Invitrogen, USA) according to the protocol recommended by the manufacturer. RNA was quantified by measuring the absorbance at 260nm, and the ratio was 1.8 or higher. cDNA synthesis of mRNA was carried out by reverse transcription (RT). Normalization of the samples was accomplished using the reverse transcription-polymerase chain reaction (RT-PCR). PCR amplification for MMP-9, IL-1β, TNF- α and GAPDH were performed at 94℃ for 30 sec, at 53℃ for 30sec and at 72℃ for 30sce for 35 cycles. The sequences of the specific primers were as follows: sense, 5'-AAATGTGGGTGTACACAGGC-3' and antisense 5’ TTCACCTCATTTT GGAAACT-3' for MMP-9; sense, 5’-CTCCATTGAGCTTTGTACAAGC-3’
and antisense, 5’-GGGGTTGACCATGTAGTCGT-3’ for IL-1β; sense, 5’-TCAGCCTCTTCTCATTCCTGC-3’ and antisense, 5’-TTGGTGGTTTGCTACG ACGTG-3’ for TNF-α; sense, 5’-ACCACAGTCCATGCCATCAC-3’ and antisense, 5’-TCCACCACCC TGTTGCTGTA-3’ for GAPDH. The PCR products were separated by electrophoresis in 1.0% agarose gels with ethidium bromide.