The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Virginia (Charlottesville, VA, USA). All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publications number 80-23) revised in 2011. All efforts were made to minimize the number of animals used and their suffering.
Animal groups and surgery
Eight-week-old male CD-1 adult mice (N=22) weighing 28-32 g from Charles River Laboratories International Inc. (Wilmington, MA, USA) were housed under 12-hour light and 12-hour dark cycle with free access to chow and water. These mice were randomly subjected to either left common carotid artery occlusion (LCCAO) surgery (N=13) or sham surgery (N=9). For anesthesia induction, mice were exposed to 5% isoflurane for 3-5 minutes. Anesthesia was maintained by 2.0% isoflurane. Midline cervical incision was then performed, and the left common carotid artery was carefully isolated from the arterial sheath to avoid damage to vagus nerve. The artery was then doubly ligated with 4-0 nylon suture. Sham surgery was performed by exposing the left common carotid artery without carotid ligation. During anesthesia, anal temperature was strictly maintained at 37±0.2 ℃ by warming blanket and lamps. The inhaled and exhaled gases were also monitored to maintain normal end-tidal CO2 concentrations.
Two weeks after surgery, mice were subjected to Barnes maze as we previously described (12). To test their spatial learning and memory, mice were first placed in the middle of a circular platform with 20 equally spaced holes (SD Instruments, San Diego, CA). One of these holes was connected to a dark chamber called target box. Aversive noise (85 dB) and bright light (200 W) shed on the platform were used to encourage mice to find the target box. They had a spatial acquisition phase that lasted for 4 days with 3 minutes per trial, 2 trials per day and 15 minutes between each trial. Animals then went through the reference memory phase to test the short-term retention on day 5 and long-term retention on day 12. No test or handling was performed from day 5 to day 12. The latency to find the target box during each trial was recorded with the assistance of ANY-Maze video tracking system (SD Instruments).
One day after Barnes maze test, mice were subjected to fear conditioning test as we previously described (12). Each mouse was placed into a test chamber wiped with 70% alcohol and exposed to 3 tone-foot shock pairings (tone: 2000 Hz, 85 dB, 30 seconds; foot shock: 1 mA, 2 seconds) with an intertrial interval of 1 minute in a relatively dark room. The mouse was removed from this test chamber 30 seconds after the conditioning stimuli. The animal was placed back to the same chamber without the tone and shock 24 hours later for 8 minutes. The animal was placed 2 hours later into another test chamber that had different context and smell from the first test chamber in a relatively light room. This second chamber was wiped with 1% acetic acid. Freezing was recorded for 3 minutes without the tone stimulus. The tone was then turned on for 3 cycles, each cycle for 30 seconds followed by 1-minute inter-cycle interval (4.5 minutes in total). Animal behavior in these two chambers was video recorded. The freezing behavior in the 8 minutes in the first chamber (context-related) and 4.5 minutes in the second chamber (tone-related) was scored in an 8 seconds interval by an observer who was blind to the group assignment. Animals in all groups were sacrificed immediately after fear conditioning test to harvest the brain for the following histo-pathological examinations.
Histo-morphology was assessed after HE staining of brain sections as described before (13). Animals were deeply anesthetized with 5% isoflurane. They were then transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.4). The mice were decapitated, and the brains were then immersed in 4% paraformaldehyde for 24 hours, processed for paraffin embedding, and sectioned into 5 μm thick sections on a rotary microtome. Coronal sections consisting of the dorsal hippocampus and corpus callosum were selected and used in HE staining. The paraffin-embedded sections were dewaxed by baking at 55-65 ℃ for 90 minutes, cleaned with xylene two times with each for 5 minutes, and rehydrated with 100% ethanol twice with each for 5 minutes and 70% ethanol once for 5 minutes. After 5-minute washes in distilled water, the sections were stained in hematoxylin for 2 minutes and then washed two times in running water for 1 minute. The sections were dipped in acid alcohol solution and then washed again using running water for 1 minute. After rinsing each section in ammonia alcohol for 5 seconds, 10 dips were performed in Eosin-Y solution for 30-45 seconds to counterstain the sections. Following two additional rinses in 100% ethanol with each for 1 minute, the sections were dehydrated in graded alcohols, cleared in xylene and cover-slipped with Permount. Six coronal brain sections were imaged per animal in the brain area from Bregma -1.06 to Bregma -2.70 based on the mouse brain stereotaxic coordinates.
Neuronal and axonal degeneration were examined by using a modified amino-cupric sliver staining (14). Briefly, mice were deeply anesthetized with 5% isoflurane, and then perfused transcardially with 4% paraformaldehyde in 0.1 M cacodylate buffer. The brain was removed and stored in the same fixative solution for 24 hours at 4 ℃. The brain was embedded in 0.5% gelatin solution and cut into 50 μm thick sections by vibrotome. The brain sections were washed five times with distilled water for 5 minutes each in a staining tray, and then were transferred into cupric-silver solution in a dish. This dish was wrapped and placed on a hot plate at 40 ℃ for 1 hour and then left in the hood at room temperature overnight. On the next day, the staining tray was shaken in a dish with 100% acetone for 30 to 90 seconds and then was quick transferred to a dish with silver diamine incubation solution. The incubation lasted for 35 minutes. The sections were then transferred to the reducing agent solution for 5 minutes, and then transferred to ferricyanide bleaching solution for another 10 minutes with continuous gentle agitation. When dark straw color appeared on the slice, the sections were washed in distilled water two times with each for 5 minutes and then were stabilized in 2% sodium thiosulfate solution for 1 minute with light agitation. After differentiation and stabilization, the sections were rinsed again in distilled water for 2 to 5 minutes, and then mounted on gelatin-treated glass slides. The slices were then rinsed with distilled water and dehydrated in graded ethanol solution. After cleaning in pure xylene, the slices were cover-slipped with Permount before the xylene dried out. Four coronal brain sections were imaged per animal in the brain area from Bregma -1.06 to Bregma -2.70 based on the mouse brain stereotaxic coordinates.
Results are presented as means±S.D. Data from the training sessions of Barnes maze were analyzed by two-way repeated measures analysis of variance followed by Tukey's test. The other data were analyzed by one-way analysis of variance followed by Tukey's test or by Student's t test as appropriate. Differences were considered significant at a P<0.05. All statistical analyses were performed with SigmaStat (Systat Software, Inc., Point Richmond, CA, USA).