Adult male C57BL/6J mice (6-8 weeks, body weight 25-30 g) were obtained from the SLAC company (Shanghai, China). Mice are kept in cages (four mice per cage), fed with standard laboratory fodder and water and maintained under a 12-12 h light-dark cycle with controlled room temperature (25°C) and humidity (50%). All animal experiments were approved by the Institutional Animal Use and Care Committee at Soochow University and were implemented under the guidelines of Animals Use and Care of the National Institutes of Health (NIH) and the Animal Research: Reporting in Vivo Experiments (ARRIVE).
Intracerebral Hemorrhage and Bilateral Tibial Fracture Model
The mouse ICH model was established as our previously reported [13,20]. In brief, mice were fixed in stereotaxic frame (David Kopf Instruments, Tujunga, California) after anesthetized with 4% chloral hydrate by intraperitoneal injection. Then carried out craniotomy after exposed the skull. Subsequently, 0.5 μL (0.1 μL/min) saline containing 0.05 units of bacterial collagenase (type IV, Sigma, USA) was injected into the left striatum at coordinates 1.0 mm anterior and 2.0 mm lateral to bregma and 3.5 mm below the cortical surface, using a microinjection system (LongerPump, China). The needle was kept for another 5 min after injection to prevent leakage. Needle was inserted into the striatum after craniotomy but the collagenase was not injected in sham group (Sham).
Tibial fracture (TF) surgery was performed according to the literature described previously . Briefly, the bilateral tibia with an intramedullary fixation was fractured after mice anesthetized. The model of ICH accompanied by fracture (MI) was performed fracture surgery immediately after ICH. After the surgery, all mice were placed next to a heater to maintain their normal body temperature and put back to cages when they recovered from anesthesia. All of the above operations were performed under aseptic conditions.
Experimental Design and Reagent
In experiment 1, to explore whether a concomitant bilateral tibial fracture affect the progressions of ICH, the detailed experimental groups were divided as follows: Sham, ICH, TF and MI.
In experiment 2, to determine the effect of IL-13 in the pathophysiological process of MI model, the detailed experimental groups were divided as follows: MI with PBS and MI with IL-13. The detailed experiment design was showed in Fig. 1.
Recombinant mouse IL-13 were obtained from Novoprotein company (Shanghai, China). For MI + IL-13 group, IL-13 (0.2 μg / 2μL per mouse) was injected into the contralateral lateral ventricle (1.0 mm rear and 1.0 mm lateral to bregma and 2.5 mm below the cortical surface) within 5 min after ICH treatment. The needle was kept for another 5 min after injection to prevent leakage.
Mice were deeply euthanized after surgery and ipsilateral striatum was sampled (n = 5 per group). Proteins were extracted by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China) and allowed to lyse for 30 min on ice. Then the brain tissues were lysed by ultrasonic homogenizer (Scientz Biotechnology, China). After that, the homogenate was centrifuged at 12000 revolutions per min for 25 min at 4°C. The protein concentration in the supernatant was measured using NanoDrop 2000 spectrophotometers (Thermo Fisher Scientific, USA). 60 μg proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto hybond-polyvinylidene difluoride (PVDF) membranes. The PVDFs were blocked with 5% bovine serum albumin (BSA), then the blots were probed with respectively primary antibodies overnight at 4 °C. After washing with tris buffered saline-tween (TBST), the PVDFs were incubated with corresponding horseradish peroxidase (HRP) –conjugated secondary antibody for 2 h at room temperature. ECL chemiluminescence system (Clinx Science Instruments, China) were used for immunodetection. Image J (NIH, USA) software was used for the analysis of band density.
Mice were perfused under deep anesthesia with ice-cold PBS whereafter perfusion with 4 % formalin at 24 h after surgeries (n = 5 per group). The brain was removed and soaked in formalin overnight at 4°C and dehydrated with 10-30% sucrose in the next 3 days. Frozen brains which embedded in optimal cutting temperature (OCT, Sakura, Japan) were cut into 10 μm per slice from the front of bleeding area to the end continuously using freezing microtome (Thermo Fisher Scientific, USA). Slices were blocked by 5% BSA for 2 h. Then the slices were incubated with corresponding primary antibodies at 4°C overnight. After phosphate buffer saline-tween (PBST) washed, the slices were incubated with double fluorochrome conjugated antibodies at room temperature for 2 h. Thereafter, the cell nuclei were stained with DAPI (Beyotime Biotechnology, China) for 30 sec. Images were taken with a fluorescence microscope (Nikon, Japan), and the images were processed with Image J software.
The preliminary treatment of the slices as described above (n = 5 per group). After incubated with respectively primary antibodies at 4°C overnight, the slices were hatched with HRP conjugated secondary antibody for 1 h at room temperature. Using dimethylbenzidine (DAB) to coloration, then the slices were washed and nuclei were stained with hematoxylin. After water rinsing and dehydrating with concentration gradient ethanol (75-100%), we used xylene to transparency, then cover glasses and neutral resin were used to seal the slices. Images were taken with an optical microscope (Nikon, Japan)
Propidium Iodide Staining
As previously described , loss of plasma membrane integrity was evaluated by intraperitoneal injection of propidium iodide (PI, 5 mg/mL, Beyotime Biotechnology, China) for 200 μL per mouse 1 h before sacrificed (n = 5 per group). The brains were removed quickly and frozen in liquid nitrogen vapor, then cut into 10 μm frozen slices. Sections were incubated with DAPI for 30 sec to stained cell nuclei. Images were photographed and analyzed by fluorescence microscope. PI positive cells in mice were counted in 200 × fields in the peripheral area of hematoma randomly.
Bleeding Volume Measurement
The frozen sections were prepared as previously described (n = 5 per group). Sections were incubated in prussian blue dye (Sangon Biotech, Shanghai) for 1 h at room temperature after dehydrated with 95% ethanol for 10 min and sections were subjected to washing by PBST. Subsequently, 0.3% hydrogen peroxide-methanol solution were used for 15 min then washing sections. We used DAB to enhance the coloration until the hemorrhagic focus turned brown, then wash the dye away with running water. The process of dehydration, transparency and seal were described above. Finally, the bleeding volumn was analyzed by Image J software.
Brain Water Content Measurement
Mice were sacrificed 24 h after surgery (n = 5 per group). The brains were removed and the cerebellum was isolated first, followed by the separation of the ipsilateral (lesion side) and contralateral hemispheres along the sagittal suture. These three parts of tissue samples were immediately weighed on an electronic analytical balance (Mettler Toledo, Switzerland) to the nearest 0.1 mg and the wet weight were recorded. Then these samples were dried in a drying oven at 160°C for 24 h and weighted again to obtain the dry weight. The percentage of brain water content was: (wet weight - dry weight) / wet weight × 100%.
Corner test was performed to detect sensorimotor and postural asymmetries after injury (n = 7 per group). Mice were allowed to crawl into a 30˚ corner. When mice entered the corner, they stood up against the wall and turned to face the open end. For normal mice, the number of right and left turns would be equal. The lesion side of ICH mice lost the ability to sense stimulation, so they used the contralateral limb more, causing them to move toward the injured side (on the left in this test) more frequently. Test was repeated 10 times for each mouse. The percentage of turning direction was: (left turns / total turns) × 100%.
For the wire grip test, it was aim to evaluate the motor function deficits after injury (n = 7 per group). A metal wire (45 cm long) was suspended 45 cm above a soft platform. The mice’s tails were gently grasped, then slowly released after they catch the metal wire with their forelimbs. The experiment was repeated three times for each mouse, with a 10-minute rest interval. An average value was calculated for each mouse on every day of testing. The scoring criterion was based on previous described . Mice were trained for five consecutive days prior to surgery for both corner test and wire grip test.
The Morris-water maze (MWM) test was performed as previously described . A circular pool with a diameter of 120 cm was filled with water at a depth of 30 cm and the temperature of the water was maintained at about 25°C. Then a circular transparent platform about 8 cm in diameter was placed 1 cm below the surface of the water, about 30 cm from the wall of pool. The pool is divided into four quadrants, with four highly visible cues located on the walls. Mice were trained for five consecutive days (n = 7 per group). Before each training, the mice were subjected to recognize the visible cues on the wall of the pool, then they were gently put in the water and the timing began immediately. Mice were given 90 sec to find the platform. Once they found it, they were allowed to stay for 3 sec, then recorded how long it took. If they could not find the platform within 90 sec, the time was recorded for 90 sec and mice were placed on the platform for 15 sec. Each mouse was trained twice a day with an interval of more than 30 min. At the end of the training, they were placed next to a heater to restore their body temperature. When motor function was restored after surgery, we began to test the spatial memory ability of mice. The escaped latency (the time it took the mice to get on the platform) and crossing number (the number of crossed the original position of the platform after removing it) were used to assess spatial memory ability.
As previously described , tibia collected from different groups were subjected to Micro-CT (micro-computed tomography) scanning (SkyScan 1176, Aartselaar, Belgium). The parameters of X-Ray were set at a current of 500 µA with the voltage of 50 kV and the scanning per layer was 9 µm. Cross sections of fracture site were selected for quantification of bone mineral density (BMD, mg/cc) and bone volume as a proportion of total tissue volume (BV/TV, %).
Data are expressed as the mean ± SEM. Students’ t-test was used for the comparison between two groups, and one-way ANOVA followed by Tukey’s multiple comparisons test was used in which there were more than two groups. Behavioral tests (Corner test and wire-grip test) were analyzed by two-way ANOVA (time and treatment) followed by Tukey’s multiple comparisons test. p < 0.05 was considered statistically significant. All data were processed with GraphPad Prism 7 (GraphPad Software Inc., San Diego, USA).