2.1. Animal surgery
For adult cerebral hypoxia-ischemia (HI), eight-to-twelve week-old male CD1 (Charles River, Wilmington, MA) and Thy1-YFP mice (Jackson Laboratories Stock no. 003782, Bar Harbor, ME;) were challenged by transient cerebral HI, performed as described previously with minor modifications (Adhami et al., 2006
; Shereen et al., 2011
). Briefly, animals were anesthetized by intraperitoneal (IP) injection of avertin, and the right common carotid artery was ligated by two releasable (Mule) knots of 4-O silt suture. After carotid ligation, mice were infused with 7.5% O2 balanced by 92.5% N2 through a face-mask for 60 min. The body tempeture of mice was maintained between 37.5° to 38.5° C by Digi-Sense Benchtop RTD connected with a heating lamp and rectal probe (EW-89000-10; Cole Parmer, Vernon Hills, IL). The knots on the common carotid artery were released at the end of hypoxic stress.
For lipopolysaccharide (LPS)-sensititized neonatal cerebral HI, LPS (0.3 mg/kg, Sigma) was IP-injected to 5-day-old CD1 mice at 72 h before cerebral HI, as previously described with minor modifications (Eklind et al., 2005
). At postnatal day 8, mice were anesthetized by 2% isoflurane and subjected to permanent ligation of the right common carotid artery. Mouse pups recovered for 1.5 h and were then exposed to hypoxia in glass chambers containing 10% oxygen and 90% N2 in a waterbacth kept at 37° C. After hypoxic exposure, mice were returned to dams in the animal care facility. The in-vivo
TTC-labeling procedure was performed at 48 h recovery. These animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and conform to the National Institutes of Health Guide for Care and Use of Laboratory Animals.
2.2. In-vivo TTC-labeling method
To disrupt BBB, mannitol (0.5 M~ 1.4M, Sigma) prepared in PBS at a temperature of 370 C was IP-injected to animals (~0.1 ml/g body weight) for 5 to 180 min as indicated in the text. Mice were anesthetized with avertin and transcardially perfused of PBS followed by 10 ml of 2% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma). At 10 min after transcardial TTC perfusion, the brains of animals were removed and placed into 4% paraformaldehyde. Alternatively, the extracted brains can be incubated in warm phosphate-saline buffer (PBS) for 30 min to enhance TTC-staining. For biochemical analysis, brains were removed after TTC-perfusion and divided into the contralateral (the left cortex) and lesion sides (the right cortex) for protein extraction.
2.3. Quantification of brain infarction
The post-fixed brains after in-vivo
TTC-labeling were sectioned into 0.8 mm thick coronal slices with Vibratome (Stoelting, Wood Dale, IL) and photographed for quantification as previously described (Yang et al., 2009
). Briefly, digital images of 5 slices in each brain were analyzed using the NIH ImageJ 1.4 software. Brain damage was expressed as the ratio of the infarcted area (white area in the right side) to the area of the undamged, contralateral hemisphere.
2.4. Histology and immunohistochemistry
The fixed brains after in-vivo TTC-labeling were transferred into graded 30% sucrose solution and frozen in O.C.T. compounds for sectioning at 50 µm-thickness using a sliding microtome (SM2000R, Leica, Wetzlar, Germany. YFP was visualized with an Olympus epifluorescent microscope (BX-51). For immunohitochemistry, the following antibodies were used: mouse anti-HSP70 (SPA810; Stressgen, Victoria, Canada), rabbit anti-DARPP32 (A31656; Chemicon, Temecula, CA), and mouse anti-microtubule-associated protein 2 (MAP2, Sigma. St. Louis, MO). The immunoreactivity was detected sequentially by biotinylated secondary antibodies, the Vectastain ABC kit (Vector Lab, Burlingame, CA), and the diaminobenzidine tetrahydrochloride (DAB) reaction.
2.5. Cell fractionation and immunoblot analyses
The cortex from fresh brain tissue after in-vivo
TTC-labeling was subjected to protein extraction or mitochondrial-cytosol fractionation, as described previously (Adhami et al., 2006
). For total protein extraction, three part of cortex (contralateral side with red staining and the ipslateral side with or without red staining) were seperated and homogenized in TLB buffer [20 mmol/L Tris, pH 7.4, 137 mmol/L NaCl, 25 mmol/L β-glycerophosphate, 25 mmol/L Na-pyrophosphate, 2 mmol/L EDTA, 1 mmol/L Na3
, 1% Triton X-100, 10% glycerol, 1 mmol/L phenylmethyl sulfonyl fluoride, protease inhibitor cocktail (Sigma)]. For mitochondrial-cytosol fractionation, the tissue was homogenized in cold buffer (20 mmol/L HEPES, pH 7.4, 250 mmol/L sucrose, 10 mmol/L KCl, 1.5 mmol/L MgCl2
, 1mmol/L EDTA, 1 mmol/L EGTA, 0.7% protease inhibitor cocktail, 1 mmol/L Na3
). The extracted protein samples were processed for immunoblotting and visualized by HRP-reactive chemiluminescence reagents (Amersham Biosciences, Arlington Heights, IL). Primary antibodies were rabbit anti-cytochrome c (#4272; Cell Signaling, Danvers, MA), goat anti-AIF (#sc9416, Santa Cruz, Santa Cruz, CA), mouse anti-cytochrome oxidase subunit IV (#A21348; Molecular Probes, Invitrogen, Carlsbad, CA), mouse anti-HSP70, rabbit anti-Bcl-xL (#sc634, Santa Cruz), rabbit anti-caspase-3 (#9662; Cell Signaling) and mouse anti-β-actin (#A544, Sigma).
2.6. Reverse-transcription PCR
The RNA from fresh brain tissues after in-vivo
TTC labeling was extracted using TRIzol reagent (Invitrogen) for RT-PCR analysis, as described previously (Sun et al., 2010
). Brifely, total RNA was processed with the high-capcacity cDNA reverse transcription Kit (Applied BioSystems, Foster City, CA) to transcript cDNAs. The semi-quantitative PCR of mice Hsp70, Caspase-3, Bim-EL, Bcl-xL
cDNAs were detected using the following primers, and the cDNA of a housekeeping gene β-actin was measured in parallel as an internal control: Hsp70
, 5'-AAGCAGACGCAGACCTTCAC-3’ and 5'-AGATGACCTCCTGGCACTTG-3’; Caspase-3
5'-CTATCTGGACAGTAGTTACAAAAT-3’ and 5'-CAGTCAGAGCTCCGGCAGTAG-3’; Bim-EL
, 5'- CTACCAGATCCCCACTTTTC-3’ and 5'-ACCCTCCTTGTGTAAGTTTC-3’; Bcl-xL
, 5'-AGGCAGGCGATGAGTTTGAA-3’ and 5'-TGAAGCGCTCCTGGCCTTTC-3’; Tspo
, 5'-ATGGGGTATGGCTCCTACATAGT-3’ and 5'-CCACTGACAAGCAGAAGATCG-3’; and β-actin
, 5'-GAAGCACTTGCGGTGCACGAT-3’ and 5'-GAAGCACTTGCGGTGCACGAT-3’. Reaction products were separated by electrophoresis on a 2% agarose gel. Bands were visualized using an electrophoresis image analysis system (Eastman Kodak Co., Rochester, NY).
2.7 Matrix metalloproteinase (MMP) zymography
MMP-9 and MMP-2 zymogram was performed, as previously described (Yang et al., 2009
). Briefly, MMP-9 and MMP-2 in brain extracts was pulled-down using the gelatin Sepharose™ 4B beads (GE Healthcare, Buckinghamshire, UK) and separated by electrophoresis in a polyacrylamide gel containing 0.15% gelatin. After electrophoresis, gels were washed twice with 2.5% Triton X-100 and incubated in reaction buffer (50 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, 5 mmol/L CaCl2
) at 37° C overnight. The gel was stained with Coomassie blue and destained to reveal the protease activity. Shown are the inverted image of zymogram gels.
2.8. Statistical Analysis
Quantitative data of infarct area were expressed as means ± SEM and compared between HI plus LPS and HI groups using unpaired t-test. Statistical significance was assumed at p < 0.05.