Male AQP4+/+ and AQP4-/- mice, 3 to 4 months old, weighing 25 to 33 g, were kindly provided by Dr. Gang Hu, Jiangsu Key Laboratory of Neurodegeneration, Department of Anatomy, Histology, and Pharmacology of Nanjing Medical University in China. Investigators were blinded to genotype information in all experiments. Mice were kept under environmentally controlled conditions (ambient temperature, 22 ± 2°C; humidity, 40%) on a 12 h light-dark cycle with free access to food and water. All animals were treated according to protocols approved by the Institutional Animal Care and Use Committee of Fudan University.
Autologous whole blood-induced intracerebral hemorrhage
Mice were anesthetized with 10% chloral hydrate (350 mg/kg) and were placed in a stereotaxic frame (Alcott Biotech, Shanghai, China). Through a hole drilled in the skull, a 32-gage needle was implanted into the striatum, 2.0 mm lateral to the midline, 1.0 mm anterior to the coronal suture, and at a depth of 4.0 mm from the surface of the brain. Each mouse was microinjected with 5 μl of autologous whole blood (right striatum) taken from the tail vein over 10 min using a 5 μl microinfusion pump (ALC-IP600, Alcott Biotech). The needle was then pulled out without blood reflux after 5 min and the wound was sutured. Only mice with neurological deficit were regarded as a successful model and approximately 10% of the total number of mice were excluded, owing to absence of neurological deficit or to unexpected death.
This study was divided into nine parts.
Two groups (n =4 each) of animals (AQP4+/+ and AQP4-/- mice) received an intracerebral infusion of 5 μl of autologous whole blood. Physiological values were measured 15 minutes before and 15 and 30 minutes after ICH.
Two groups (n =24 each) of animals (AQP4+/+ and AQP4-/- mice) received an intracerebral infusion of 5 μl of autologous whole blood. Hoechst staining and neurological testing were measured at days 0 (n =6), 1 (n =6), 3 (n =6), and 7 (n =6).
Two groups (n =40 each) of animals (AQP4+/+ and AQP4-/- mice) received an intracerebral infusion of 5 μl of autologous whole blood. Mice were observed daily and cumulative mortality was recorded through day 14 post-hemorrhage.
Two groups (n =48 each) of animals (AQP4+/+ and AQP4-/- mice) received an intracerebral infusion of 5 μl of autologous whole blood. Immunocytochemical studies were conducted with antibodies against neuron-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), and TUNEL, to determine the cell types involved. Measurements were made at days 0 (n =12), 1 (n =12), 3 (n =12), and 7 (n =12).
Two groups (n =24 each) of animals (AQP4+/+ and AQP4-/- mice) received an intracerebral infusion of 5 μl of autologous whole blood. Western blotting was used to detect apoptosis-related protein expression following ICH. Measurements were made at days 0 (n =6), 1 (n =6), 3 (n =6), and 7 (n =6).
Two groups (n =6 each) of animals (AQP4+/+ and AQP4-/- mice) received an intracerebral infusion of 5 μl of autologous whole blood. A cytokine protein assay was used to measure cytokine release in AQP4-re and AQP4+/+ mice 3 days (n =6) after ICH.
Two groups (n =24 each) of animals (AQP4+/+ and AQP4-/- mice) received an intracerebral infusion of 5 μl of autologous whole blood. ELISA was used to verify measured release of cytokines at days 0 (n =6), 1 (n =6), 3 (n =6), and 7 (n =6).
Two groups (n =48 each) of animals (AQP4+/+ and AQP4-/- mice) received an intracerebral infusion of 5 μl of autologous whole blood. After the lateral ventricle was infused with interleukin-1 receptor antagonist (IL-1ra) or tumor necrosis factor binding protein (TNFbp) e, TUNEL staining was measured at days 0 (n =12, 6 mice for each inhibitor), 1 (n =12), 3 (n =12), and 7 (n =12).
Two groups (n =24 each) of animals (AQP4+/+ and AQP4-/- mice) received an intracerebral infusion of 5 μl of autologous whole blood. Western blotting was used to measure IL-1Β and TNF-Α receptor and phosphorylation at days 0 (n =6), 1 (n =6), 3 (n =6), and 7 (n =6).
Mice were observed daily and cumulative mortality was recorded until day 14 post-hemorrhage. Kaplan-Meier survival plots were produced using a log rank test with GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego, CA, USA). Significance was considered for P =0.05 level.
Measurement of physiological variables
Because the blood volume required for analytic assays was large enough to cause hypovolemia, a separate series was performed to define the physiological state of mice subjected to ICH. In two groups of mice (AQP4+/+ and AQP4-/- mice) (n =4 each), the right femoral artery was catheterized for continuous blood pressure monitoring and periodic blood sampling for arterial pH, blood gases (PaO2, PaCO2 ), and glucose (Ciba Corning Diagnostics Corp. East Walpole, MA, USA). Physiological values were measured 15 min before and 15 and 30 min after ICH. These animals thus underwent all acute procedures, including ICH and behavioral testing, and were then killed by an overdose of chloral hydrate anesthesia. All analyses were conducted by an observer blinded to the genotype of the mice.
Neurological deficits were evaluated by an observer blinded to the age and genotype of each mouse, including AQP4+/+ and AQP4-/- mice, at 1, 3, and 7 days after blood injection. Tests included a postural reflex test, which examined upper-body posture while the animal was suspended by the tail, and a forelimb placing test, which examined sensorimotor integration in the forelimb to visual, tactile, and proprioceptive stimuli. Neurological function was graded on a scale of 0 to 12 (normal score, 0; maximal score, 12), as described previously . Mice that did not demonstrate a right upper extremity paresis during ICH were excluded from further study.
Brain tissue processing
Both AQP4+/+ and AQP4-/- mice were anesthetized with intraperitoneal administration of chloral hydrate. The animals were then perfused transcardially through the ascending aorta, with 20 ml of warm (37°C) 0.9% NaCl containing heparin (10000 U/l) followed by 100 ml of a freshly prepared ice-cold solution of 4% formaldehyde in PBS (0.1 M, pH 7.4). Brains were dissected and separated. The brain tissues were fixed at 4°C in the same fixative for 24 h.
For immunofluorescence staining, the following antibodies were used: mouse anti-NeuN (1:500; Millipore, Billerica, MA, USA) and rabbit anti-GFAP antibody (1:100; Millipore). Sections were incubated with primary antibodies at 4°C overnight and then incubated with secondary antibodies (1:200) for 90 minutes. Fluorescein isothiocyanate (FITC) coupled goat anti-rabbit IgG, Cy3 coupled goat anti-rabbit IgG, and Cy3 coupled goat anti-mouse IgG (all from Jackson ImmunoResearch, West Grove, PA, USA) were used as appropriate. Primary antibody omission incubations with either blocking solution or PBS were performed, to test the specificity of immunoreactivity. The TUNEL method was used with an in-situ cell death detection kit (Roche, Mannheim, Germany) to assess for apoptotic cells. In sections colabeled with TUNEL, a Tdt reaction mix was applied after primary antibodies were rinsed off. The Tdt reaction mix contained biotinylated dNTPs, Tdt buffer, and Tdt enzyme (Gibco, Gaithersburg, MD, USA) in ultrapure H2O. After 1 hour (37°C), slides were rinsed in 0.1 M EDTA, pH 8.0 for 2 to 5 minutes, PBS briefly, and then incubated with secondary antibody plus DN-Avidin-FITC (Vector Labs, Burlingame, CA, USA) for 1 h at room temperature. Finally, sections were rinsed in PBS and covered with a cover slip.
For Hoechst staining, sections were stained with Hoechst 33258 (10 minutes), rinsed and cleared in ethanol and xylenes, and covered with a cover slip under Permount (Santa Cruz Biotechnology, Dallas, TX, USA).
Cytokine protein array analysis
Brain tissues were harvested from AQP4+/+ and AQP4-/- mice 3 days after the induction of ICH. Supernatants from brain tissues were analyzed for cytokines using the Mouse Cytokine Array II (RayBiotech, Norcross, GA, USA) according to the manufacturer’s protocol. Briefly, arrayed antibody membranes were incubated with blocking buffer for 30 min at room temperature. Membranes were then probed with 3 ml of conditioned media for 2 h at room temperature. After washing three times with 2 ml of washing buffer, the membranes were incubated with biotin-conjugated anticytokine antibody diluted in blocking buffer for 2 h at room temperature. After the washings, the membranes were developed with an ECL-type system (provided by the kit), exposed to X-ray film (Kodak, Rochester, NY, USA), and processed by autoradiography (Kodak). Autoradiographs of the arrays were scanned to determine the density of the protein array spots and analyzed with a TINA 2.0 program (Raytest, Strasbenhardt, Germany). Relative protein concentrations of different samples were analyzed by comparing densities resulting from subtraction of the blank and normalization for the positive controls.
Treatment with IL-1ra and TNFbp
The IL-1Β inhibitor IL-1ra and the TNF-Α inhibitor TNFbp were used. In the appropriate treatment groups, mice were stereotactically injected with inhibitors into the lateral ventricle (0.9 mm lateral to the midline, 0.1 mm posterior to the coronal suture and at a depth of 3.1 mm from the surface of the brain). Injections of 5 μg IL-1ra (Amgen, Thousand Oaks, CA, USA) or 3 mg/kg TNFbp (Amgen Inc., Boulder, CO, USA) were given 30 minutes after induction of ICH. Mice were then killed 1, 3, or 7 days after induction of ICH, and brains were processed for quantification of TUNEL staining.
Whole brain tissues were homogenized and the samples were loaded onto 4% stacking/12% separating SDS-polyacrylamide gels. The proteins were electrophoretically transferred onto nitrocellulose membranes. After the blocking, the blotting membranes were incubated with specific primary antibodies as follows: rabbit polyclonal against the active cleaved fragment (17 kDa) of caspase-3 (1:1000; Idun Pharmaceuticals, Los Angeles, CA, USA), rabbit polyclonal directed against the active cleaved fragment (20 kDa) of caspase-8 (1:1000, Santa Cruz Biotechnology), rabbit polyclonal directed against the cleaved fragment of caspase-9 (38 kDa) (1:1000, CST, Boston, MA, USA), rabbit polyclonal against Bcl-2, mouse monoclonal against Bax antibodies (1:100, Santa Cruz Biotechnology), rabbit polyclonal against IL-1Β receptor and IL-1Β phosphorylation receptor (1:1000, CST), and rabbit polyclonal against TNF-Α receptor I and TNF-Α receptor I phosphorylation (1:1000, CST) for 2 h at room temperature and then with biotinylated goat anti-rabbit IgG (1: 200, Vector Labs) for 1 h at room temperature. The proteins were visualized using the ABC reagent (Vectastain ABC Kit, Vector Labs) and the ECL detection kit (Amersham Pharmacia Biotech, Baie-D’Urfe, QC, Canada). The intensity of blots was quantified using the Leica Image Processing and Analysis System (Frankfurt, Hesse, Germany). β-actin was used as an internal control.
For cell counting, brain sections of 8 μm thick were cut through the needle entry site, starting with the first appearance of TUNEL-stained or Hoechst-stained cells, extending to the most caudal parts of the hematoma zone, at 1, 3, and 7 days after ICH (n =6 per group). Six brain sections per mouse were selected that showed the greatest difference in TUNEL staining or Hoechst staining from the control group. The total hemispheric areas of each section were traced using an image analyzer. Morphometric analysis involved computer-assisted hand delineation of the area of the striatum. Labeled profiles were counted only if the first recognizable profile of the cell soma came into focus within the counting frame. Each of the coronal sections described was used for analysis. Each group included six mice, and six brain sections from each mouse were counted. The mean number of positive cells per section was recorded.
All data were presented as mean ± standard error of the mean. Statistical analysis was performed with two-tailed Student t tests or one-way analysis of variance (ANOVA) followed by a post-hoc test for group differences at each time point using SPSS 11.0 for Windows (SPSS Inc, Chicago, IL, USA). Differences were considered significant at P <0.05. All analyses were conducted by an observer blinded to the genotype of the mice.