Ikeda-Matsuo et al. 10.1073/pnas.0604400103. |
Supporting Figure 7
Supporting Table 1
Supporting Figure 8
Supporting Table 2
Supporting Materials and Methods
Fig. 7. Expression of mPGES-1 in sham-operated (SHAM) animals, MCA-occluded (MCAO) animals, or animals without surgery and ischemia. (A) Immunostaining for mPGES-1 in coronal brain slices of SHAM animals killed 24 h after surgery and animals without surgery and ischemia (no-ischemia, no-surgery; NINS). Representative data from four animals are presented. (B) Immunostaining for mPGES-1 in the postischemic brain using nonabsorbed mPGES-1 antibody () or preabsorbed mPGES-1 antibody by blocking peptide (+). (Scale bar, 5 mm.) (CF) Western blot analysis of the expression of mPGES-1 (C and E) and COX-2 (D and F) in the hippocampus (C and D) and striatum (E and F) of the ipsilateral (i) or contralateral (c) hemisphere 24 h after ischemia (MCAO) or SHAM. The left (l) or right (r) hemispheres of animals without surgery and ischemia (NINS) were used as controls. Quantitated data from immunoblotting with mPGES-1 or COX-2 antibody were scaled to a percentage of the maximal response (ipsilateral cortex 24 h after ischemia). n = 4 animals per group.
Fig. 8. Changes of cerebral blood flow (CBF) and anatomy of cerebral arterial vasculature. (A) Changes of CBF before (b), during (0-120), and immediately (r0) or 30 min (r30) after ischemia. The data are expressed as a percentage of the preocclusion value (n = 7 animals per group). (B) Anatomy of cerebral arterial vasculature in mPGES-1 KO (/) and WT (+/+) mice. There were no anatomical differences in the circle of Willis or in the origins of the cerebral arteries between the genotypes. Arrows indicate the middle cerebral artery. (Scale bar, 5 mm.)
Table 1. Physiological parameters of mPGES-1 KO (−/−) and WT (+/+) mice
mPGES-1 | +/+ | −/− |
Preocclusion (n = 4) | ||
pH | 7.40 ± 0.03 | 7.39 ± 0.02 |
pCO2, mmHg | 29.43 ± 3.72 | 34.60 ± 3.38 |
pO2, mmHg | 94.95 ± 1.76 | 90.88 ± 2.83 |
MAP, mmHg | 94.83 ± 2.29 | 88.05 ± 4.43 |
90 min-occlusion (n = 5) | ||
pH | 7.38 ± 0.02 | 7.36 ± 0.01 |
pCO2, mmHg | 38.68 ± 2.70 | 34.18 ± 0.71 |
pO2, mmHg | 96.50 ± 3.33 | 98.08 ± 3.58 |
MAP, mmHg | 91.70 ± 4.98 | 94.20 ± 8.60 |
Postreperfusion (n = 4) | ||
pH | 7.33 ± 0.01 | 7.31 ± 0.02 |
pCO2, mmHg | 34.93 ± 1.25 | 38.03 ± 2.43 |
pO2, mmHg | 91.53 ± 2.74 | 91.68 ± 1.79 |
MAP, mmHg | 84.40 ± 3.56 | 80.87 ± 4.46 |
There were no significant differences between genotypes in the values of pH, pO2, pCO2 , and mean arterial pressure (MAP) measured before, during (90 min after occlusion), and after (30 min after reperfusion) ischemia.
Table 2. Gender differences in infarct volume, edema, and behavioral dysfunctions
Ischemic symptoms | Male +/+ | Female +/+ | Male −/− | Female −/− |
Infarct volume | ||||
Whole brain, % | 12.84 ± 1.42 (10) | 14.9 ± 1.42 (8) | 4.60 ± 0.93 (7)** | 5.44 ± 1.15 (8)** |
Cortex, % | 9.47 ± 1.15 (9) | 8.57 ± 0.87 (7) | 2.88 ± 0.71 (8)** | 3.22 ± 0.87 (8) |
Edema rate, % | 12.83 ± 1.95 (10) | 15.52 ± 1.64 (6) | 7.58 ± 1.18 (7) | 7.34 ± 1.37 (9)* |
Neurological score§ | 1.84 ± 0.14 (19) | 1.86 ± 0.34 (7) | 1.06 ± 0.11 (16)** | 0.83 ± 0.17 (6)* |
Motor activity | ||||
SHAM | 46.86 ± 4.76 (7) | 51.75 ± 2.50 (4) | 46.40 ± 7.75 (5) | 41.60 ± 8.25 (5) |
MCAO | 10.58 ± 2.00 (19) | 7.43 ± 1.90 (7) | 30.93 ± 3.79 (15)** | 29.83 ± 9.10 (6)* |
Number of animals investigated is shown in parentheses. There were no significant gender differences. *, P < 0.05, and **, P < 0.01 vs. +/+ of each gender.
The volume of infarcted brain tissue 24 h after ischemia was estimated and expressed as a percentage of the corrected tissue volume in the mPGES-1 KO (−/−) and WT (+/+) mice.
The corrected edema percentage 24 h after ischemia.§
The neurological score was measured 24 h after ischemia. Scoring was as follows: 0, no deficit; 1, flexion of the torso and contralateral forelimb when lifted by the tail; 2, contralateral forelimb weakness upon application of pressure to the side of the body; 3, circling to the affected side; and 4, no spontaneous locomotor activity. The number of times the mice crossed the lines over a period of 1 min was measured by using MCA-occluded (MCAO) or sham-operated (SHAM) mice.Supporting Materials and Methods
Middle Cerebral Artery Occlusion (MCAO). The occlusion of the middle cerebral artery (MCA) was achieved by inserting a nylon monofilament (4-0 for rats and 6-0 for mice; with a heat-blunted tip coated with silicon) thread through the proximal external carotid artery into the internal carotid artery and up to the MCA (18 mm for rats and 9 mm for mice from the internal carotid/pterygopalatine artery bifurcation). The occlusion of the MCA was maintained for 2 h, followed by reperfusion. If flexion of the torso and contralateral forelimb were not observed when the animal was lifted by the tail, if abnormal barrel rolling was observed, or if cerebral hemorrhage was observed, the animal was removed from further study.
Intracerebroventricular (i.c.v.) Injection.
One-microliter solutions of either prostaglandin E2 (PGE2) (1.25 or 2.5 ng/ml) or the vehicle (0.25% dimithylsulfoxide in artificial cerebrospinal fluid; 127 mM NaCl/1.6 mM KCl/1.24 mM KH2PO4/1.3 mM MgSO4/2.4 mM CaCl2/26 mM NaHCO3/10 mM glucose) were infused stereotaxically into the bilateral cerebroventricle (1.5 mm lateral, 0.7 mm caudal to the bregma and 2.0 mm from the surface with the skull; total PGE2 0, 2.5, or 5 ng) just before the MCAO.Staining of Brain Slice.
Animals were anesthetized and perfused transcardially with saline, followed by 4% paraformaldehyde (PFA) in PBS. The brains were removed, postfixed overnight in 4% PFA and 4% sucrose in PBS, and then cryoprotected in 10%, 15%, and 20% sucrose in PBS for 1 day each. The brains were then frozen, and coronal sections (20 mm) were cut by using a cryostat. Primary antibodies were mPGES-1 (1:250 dilution; Cayman Chemical, Ann Arbor, MI), cyclooxygenase-2 (COX-2) (1:250; Santa Cruz Biotechnology), CD-11b (1:50; Serotec), Neu-N (1:2,000; Chemicon), GFAP (1:2,000; Sigma-Aldrich), von Willebrand factor (VWF) (1:25; DAKO), and caspase-3 (1:100; Cell Signaling Technology, Beverly, MA). Staining was performed according to protocol of the avidinbiotin complex kit (Vector Laboratories). The antigen peptide for mPGES-1 (Cayman Chemical) was used for an absorbance control experiment.Western Blotting.
Dissected brain tissues were lysed by homogenization in 10 mM Hepes-buffered solution (pH 7.4) containing 5 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 1 mg/ml pepstatin, and 50% (vol/vol) glycerol, followed by sonication and centrifugation at 15,000 × g for 10 min at 4°C. A total of 50 mg of proteins of each sample was separated by SDS/PAGE and then transferred to Immobilon-P membranes (Millipore) as described (1). After blocking with 5% skim milk in Tris-buffered saline-Tween 20 (TBS-T) overnight, the membranes were incubated with the first antibodies against mPGES-1 (1:500 dilution), mPGES-2 (1:1,000; Cayman Chemical), cPGES (1:250, Cayman Chemical), COX-1 (1:250, Cayman Chemical), COX-2 (1:1,000), and caspase-3 (1:500) in TBS-T for 1.5 h. After washing with TBS-T, horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) were added at a 1:10,000 dilution in TBS-T and incubated for 1 h. After washing with TBS-T, the protein bands were visualized with enhanced chemiluminescence (ECL) Western blot detection reagents (Amersham Pharmacia Biosciences).Northern Blotting.
Rats were killed 6 h after ischemia, and their brains were dissected as described above. Total RNA was extracted with TRIzol reagent (Life Technologies), and approximately equal amounts (»15 mg) of the total RNAs were applied to 1.2 M formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore). The resulting blots were hybridized with the cDNA probes for mPGES-1, COX-2, and GAPDH that had been labeled with [32P]dCTP by a Megaprime DNA labeling system (Amersham Pharmacia Biosciences). After washing the membrane, the blots were visualized by autoradiography with X-Omat AR films (Eastman Kodak).Quantification of Infarct Volume.
The animals were killed 24 h after reperfusion. Brains were sectioned coronally into five 2-mm sections at the location from which the distance to the prefrontal was 1, 3, 5, and 7 mm, respectively, and incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC) in saline for 10 min at 37°C. The TTC-unstained white area of the whole brain, cerebral cortex, or striatum and the whole brain of each posterior face of the slice were measured by using SCION IMAGE software (Scion, Frederick, MD) and numerically integrated across the thickness of the slice to obtain an estimate of the infarct volume and whole brain volume, respectively. The volumes from all slices were summed to calculate the total infarct volume and were expressed as percentage of the whole brain volume [infarct volume of whole brain, cortex, or striatum = (infarct volume of whole brain, cortex, or striatum/volume of whole brain) ´ 100]. The degree of edema was calculated as follows [edema percent = (volume of postischemic hemisphere volume of contralateral hemisphere)/volume of contralateral hemisphere ´ 100].1. Ikeda-Matsuo, Y., Ikegaya, Y., Matsuki, N., Uematsu, S., Akira, S. & Sasaki, Y. (2005) J. Neurochem. 94, 15461558.