Animals and Ethics
Adult male Sprague-Dawley rats (260-300 g), were obtained from the Experimental Animal Centre of Central South University, and used for this study according to the Guide for the Care and Use of Laboratory Animals. The animal experiments were approved by the animal ethics committee of Xiangya Hospital Central South University. Animals were housed in a temperature-controlled (24±1°C) room and maintained under diurnal lighting conditions (12-hour light/dark). They had free access to food and water.
Induction of Global Cerebral Ischemia
The animals were anesthetized by intraperitoneal injection of chloral hydrate (350 mg/kg body weight), placed in a supine position on a surgical board, and the extremities were immobilized. Tracheal intubation was orally intubated with a 14-gauge catheter under direct vision and fixed at a distance of 6 cm between the distal end of the catheter and the incisor. Electrocardiogram (ECG) monitoring was performed using limb leads. A 24 G closed-vein indwelling needle was inserted into right femoral vein to establish transfusion passage. A 22 G closed-vein indwelling needle was inserted into the right femoral artery. The three-way cock was connected to the remaining arterial needle. One end was connected to an injector with heparin, and the other end was connected to the physiological monitoring recorder to monitor arterial blood pressure. An esophageal pacing electrode was implanted in the esophagus of the rat approximately 7.5 cm in depth. We induced ventricular fibrillation (VF) with 6 mA alternating current at a frequency of 50 hertz (Hz), and the wave width is 4 ms. Electrical stimulation was performed for 90 seconds. The electrical stimulation parameters were determined according to our pre-test. CA was identified using the following criteria (13): 1) the systolic arterial pressure after electrical stimulation quickly fell to below 25 mm Hg; 2) pulsations in the arterial pressure waveform disappeared; and 3) the electrocardiographic wave displayed on the cardiac monitor indicated VF, pulseless electrical activity (PEA), or asystole after electrical stimulation stopped. After reaching the criteria for CA, the animals were observed with no intervention for 6 minutes. After 6 minutes of CA, cardiopulmonary resuscitation (CPR) was performed. Rats were resuscitated with epinephrine (20 μg/kg intravenous injection), mechanical ventilation (100% fraction of inspiration O2 [FiO2], 60 breathes/minute), and chest compressions (200/minute) performed by the instrument which was made by ourselves. External biphasic defibrillation was performed after 1 minute of CPR if the ECG showed VF. If restoration of spontaneous circulation (ROSC) could not be achieved within 5 minutes, resuscitation efforts were stopped. In successfully resuscitated animals, ventilation rate was adjusted according to the results of blood gas analyses and partial pressure of carbon dioxide (PCO2) in arterial blood was controlled around 40 mm Hg. The oxygen content decreased gradually to 21-30%, balanced with nitrous oxide (N2O). They were monitored with the ECG and hemodynamics for 4 hours. During this period, the rats with weak autonomous respirations were mechanically ventilated. Their respiratory condition was evaluated every 15 minutes to determine whether the mechanical ventilation should be continued. Mechanical ventilation was stopped after 4 hours, all tubes were removed and wounds were sutured. Each rat was fed in a separate cage. Body temperature during each experiment was maintained at 37℃ by an infrared heat lamp regulated by feedback from a rectal temperature probe. The indicators of ROSC included recovery of a supraventricular rhythm and a mean arterial pressure ≥60 mm Hg that was sustained for >10 minutes.
Rats were randomly assigned to the following groups: 1) sham group, 2) CA group, 3) CA + NSC23766 group (NSC), and 4) CA + saline group (Saline). Arterial and venous catheterization, anesthesia and endotracheal intubation were performed in the sham group. An esophageal electrode was implanted in the sham group with a length of 4 cm from the incisor, and then electrical stimulation using the same parameters was performed for 90 seconds to induce generalized twitching but not CA. In the three CA groups, VF was induced for 6 minutes and then standard CPR was performed. Animals in the NSC group were given NSC23766 (Tocris Bioscience, Ellisville, MO, USA), which was dissolved in 10 μl saline and administered bilaterally into the lateral cerebral ventricles of rats 15 minutes before ischemia for a total dose of 50 μg. The dose of Rac1 GTPase inhibitor was determined on the basis of previous studies (5, 10). Rats in Saline group received bilateral saline injections. The survival duration after resuscitation was observed until 9 days. At 24, 72 hours and 9 days after ROSC, the neurological function of rats was scored using the neurological deficit score (NDS) evaluation method (14). Rats were tested for coordination, motor and sensory function (Table 1) (14). NDS was determined by an examiner blinded to the groups. The scores of NDS range from 0 (brain dead) to 80 (normal). Rats with the greatest neurological deficits died or were sacrificed between 6 hours and 9 days after resuscitation.
For hippocampal neuronal survival analysis, the rats that survived for 9 days and non-arrested controls were deeply anesthetized with chloral hydrate and transcardially perfused with 200 ml of normal saline, followed by 300 ml of 4% paraformaldehyde in 0.1 mmol/L phosphate buffered saline at 4°C (pH 7.4). Brains were quickly removed and fixed in 4% paraformaldehyde at 4°C for an additional 24 hours. After fixation, the brains were embedded in paraffin. Serial 5 μm coronal sections were made at the level of anterior hippocampus and nissl staining. The entire length of the hippocampal pyramidal cell layer in the hippocampus was viewed under high power light microscopy (400×). Neurons with rounded cell bodies and clearly visible nucleoli were counted. The number of neuronal surviving was evaluated in CA1 region of pyramidal cells in hippocampus.
Isolation of Mitochondria
After ROSC of 6 hours, rats were deeply anesthetized with chloral hydrate and decapitated. The brains were quickly removed, and the hippocampal CA1 region was dissected on ice. The cytosolic and mitochondrial fractions of the harvested brain tissues were prepared as previously described (15). Each brain tissue sample was homogenized in ice-cold lysis buffer (200 mM mannitol, 80 mM N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid [HEPES]-potassium hydroxide [KOH] [pH 7.4], and the protease inhibitor cocktail) by 35 strokes of gentle pounding in a glass tissue grinder. Homogenates were centrifuged at 750×g for 10 minutes at 4℃. After removing a portion of the supernatant, the remaining supernatant was centrifuged at 8,000×g for 20 minutes at 4℃. The mitochondrial fraction was obtained after washing the resultant pellets three times and resuspending the pellet in lysis buffer. The cytosolic fraction was obtained by centrifuging the supernatants of the 8,000×g centrifugation step at 100,000×g for 1 hour at 4℃.
Rac1 Activation Assay
Assays of Rac1 activation were performed using p21-activated kinase (PAK1)-p21-binding domain (PBD) color agarose beads according to the manufacturer's protocol (Rac1 Activation Assay Kit, Cell Biolabs). Briefly, 400 μg samples were mixed with 40 μl of PAK1-PBD agarose beads and incubated for 1 hour at 4℃. The reaction was terminated by addition of MgCl2. The agarose beads were collected by spinning at 14,000×g for 30 seconds at 4℃ and the supernatants removed. Wash the bead 3 times with Assay Buffer and boil it in sample buffer. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membrane, and detected by immunoblotting using an anti-Rac1-specific antibody (1:1000, Cell Biolabs).
Measurement of ROS
ROS formation was detected with 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA), a fluorescent probe, according to the instruction of ROS assay kit (Beyotime Institute of Biotechnology, China) with slightly improvement. In brief, 10 μl of mitochondria (5-10 μg) was suspended in 170 μl HEPES buffer (120 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 0.1 mM MgCl2, 5.0 mM NaHCO3, 6.0 mM glucose, 1.0 mM CaCl2, 10 mM HEPES, pH 7.4) in 96-well plates; 20 μl of 100 μM DCFH-DA was added to each well, for a final volume of 200 μl. The fluorescence was then measured at 488 nm excitation and 525 nm emissions by a fluorescent plate reader (Genios, TECAN). Increased fluorescence intensity was considered to be indicative of an increase in intracellular ROS.
Western Blotting Analysis
In order to examine the protein levels of antioxidants in the ischemic CA1 region, the animals that survived for 1 days and non-arrested controls were used for western blot analysis, according to previous study. Equal amounts of protein were loaded onto a suitable polyacrylamide gel. After electrophoresis, separated proteins were transferred to nitrocellulose transfer membranes (PVDF; Millipore, Bedford, MA, USA) and blocked in 5% non-fat dry milk prepared in 1X tris buffered saline with tween 20 (TBST), followed by incubation with Trx2 (diluted 1:200, Santa Cruz, CA, USA), Prx3 (diluted 1:3000, Abcam, Cambridge, MA, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000, Cell Signaling Technology, Beverly, MA, USA) overnight at 4℃. Subsequently, the membranes were washed with 1X TBST and the membranes incubated with appropriate secondary antibodies for 2 hours at room temperature. The blots were developed using an enhanced chemiluminescence (ECL) plus detection system (Beyotime Institute of Biotechnology, China) and relative band density measured using the FluorChem FC2 System (NatureGene Corp., Medford, NJ, USA).
Morris Water Maze
To evaluate spatial learning and memory, rats were placed in a circular pool (1.6 m in diameter and 60 cm high). A platform (12 cm) submerged 2.0 cm beneath the surface of water (25±1℃). An automatic tracking system was used to record escape latency and the swimming path. The water maze, which was conceptually divided into four quadrants, was located in a quiet room that was decorated with visual cues. Rats were placed into the maze at different starting positions and received training of searching an unmarked platform composing of 4 trials per day with maximum trial duration of 120 seconds and 30 seconds on platform at the end of trials. In the probe trial performed 24 hours after the last training session, the platform was removed and the memory of platform position was evaluated by putting a rat to opposite quadrant to platform quadrant. The percent of time spent in the target quadrant, which previously contained the platform, and the number of times the rat crossed over the exact location of the former platform, were recorded for each rat. After testing, the rats were sacrificed for Nissl staining.
Data were expressed as means ± standard deviation (SD). Normal distribution for the variable was tested by Kolmogorov-Smirnov test and all variables were normally distributed. Group differences in escape latency in the Morris water maze task were analyzed using a two-way analysis of variance (ANOVA) with repeated measures. The two factors of this analysis were group and training day. Other data were analyzed using a one-way ANOVA with the Student-Newman-Keuls or Dunnett's test (SPSS16.0, Inc., Cary, NC, USA). P<0.05 was considered statistically significant.