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Assessment of Neurological Deficits
Neurological scoring test
Neurological deficits were also assessed using a modification of the Bederson neurological scale, as previously described (Kawano et al, 2006). Neurological scores were 0 (normal motor function), 1 (flexion of torso and contralateral forelimb when mouse was lifted by the tail), 2 (circling to the contralateral side when mouse was held by the tail on a flat surface, but normal posture at rest), 3 (leaning to the contralateral side at rest), and 4 (no spontaneous motor activity).
The corner test was used to assess sensory-motor integration (Li et al, 2004; Zhang et al, 2002). Briefly, the mouse was placed between two cardboard pieces. The two boards were gradually moved closer to form an angle of approximately 30° enclosing the mouse from both sides. The anterior parts of the boards were not allowed to touch, such that a small opening toward the front was always present. When the mouse entered into the deep part of the corner, the facial whiskers were touched by the two boards. Then, the mouse stood up on its hindlimbs, and turned back to face the open end. Ten trials were performed for each mouse and the percentage of right turns was calculated (Zhang et al, 2002). In healthy mice, there was a 50% chance that the mice made a right turn (Li et al, 2004; Zhang et al, 2002).
Wire hanging test
The wire hanging test is a simple approach used to measure neuromuscular ability (muscle tone and grip strength) of a rodent by assessing the animal's ability to hang suspended by its forepaws from a ~ 2 cm mm wire 30 cm above a sawdust covered surface for a maximum time of 1 min (Insel, 2001; Ogura et al., 2001). This method likewise evaluates motor coordination by noting the ability of the animal to recruit its hind limbs and tail in order to grip the wire (Freitag et al., 2003). Each mouse was habituated on the day before testing then allowed to descend 5 times on a single session. The time that elapsed until the animal fell was blindly recorded three times and the cutoff time was set at 1 min. The test was repeated 3 times for each animal with a 5 mins rest between trials and the falling latencies from 3 trials were averaged. The test was performed twice for each mouse with results from each mouse being averaged and data analyzed by one-way ANOVA with a Bonferroni post hoc test.
Rotarod, Horizontal Ladder and Other Neurological Tests
The rotarod test measures motor function. There are variations in how it is conducted that makes comparison between studies difficult. Thal, et al., placed rats on the device for 10 seconds . Rotation then started and accelerated to 40 revolutions per minute (rpm) within 90 seconds and then remained constant for 30 more seconds. The trial was repeated 5 minutes later and the trial was stopped if the animal fell off or gripped the rungs and spun for 2 revolutions. No sham animals were included. Another method was performed in the double hemorrhage rat model . The rotation was increased from 4 to 40 rpm over 5 minutes for 3 trials per day for 28 days after SAH, sham surgery or saline injection. SAH was associated with marked, persistent deficits for 28 days.
Silasi and Colbourne did not detect differences in tapered beam walking or horizontal ladder function in rats undergoing sham or endovascular perforation SAH for up to 21 days after SAH .
Plus maze test
The elevated plus maze, a widely used method for assessing anxiety (File, 2001), consists of two closed arms and two open arms (each arm measured 30×5 cm) that are elevated 30 cm above the floor. The arms are arranged in the form of a “plus” sign with a square center platform (5×5 cm) at the intersection of the arms. Mice were acclimated to the testing room for 1 h prior to testing. Each mouse was placed individually onto the central square at the intersection of the four arms, facing an open arm, and allowed to explore the maze for 5 min. A mouse was considered to be in either an open or closed arm when all four paws were inside the arm (Brunner et al., 1999). To reduce lingering olfactory cues, the apparatus was thoroughly wiped with a clean damp cloth between trials. All sessions were videotaped for review. The following behaviors were measured: (1) the number of entries amouse made onto either the closed or open arms from the central square. (2) The percentage of time a mouse spent on the open arms. (3) Stretch-attend posturing (SAP): the number of times a mouse stretched forward and then return to its original position without moving its hind paws (File, 2001). Collected data\ were used to calculate percentage of open arms entries [open arms entries/ (close arm entries+open arms entries)×100], percentage of time on open arms [time on open arms/(time on close arms+time on open arms)×100], closed arm entries and SAPs that occurred within the entire plus maze. Data were analyzed by one-way ANOVA with Bonferroni post hoc test.
Marble burying test
The marble burying procedure, which assesses obsessive compulsive tendencies, was adapted, with minor modifications, from previous studies (Skalisz et al., 2004; Woods- Kettelberger et al., 1997). Briefly, mice were acclimated to the testing room for 1 h prior to test chamber adaptation (Day 1) and prior to testing (Day 2). During adaptation, each animal was individually acclimated for 30 min to the clear polypropylene testing chamber (13 cm×20 cm×30 cm) containing a 5 cm deep layer of sawdust bedding. On day 2, the mice were individually placed in the test chamber, which then contained 24 clear glass marbles (one cm in diameter) placed in six rows of four, on top of a 5 cm deep layer of sawdust. The number of marbles buried in 30 min was recorded and data analyzed using a one-way ANOVA with a Bonferroni post hoc test. In addition, a 1 h test assessment was also conducted for both the gabrb3−/− and gabrb3+/+ mice to assure that any handling induced hyperactivity exhibited by the gabrb3−/− did not directly confound the assessment.
Water Maze Test
The Morris water maze is a circular pool (90 cm in diameter and 45 cm in height) with a featureless inner surface. The pool was filled to a depth of 30 cm with water containing 500 ml of milk (20_1 °C). The tank was placed in a dimly lit, soundproof test room with various visual cues. The pool was conceptually divided into quadrants. A white platform (6 cm in diameter and 29 cm high) was then placed in one of the pool quadrants and submerged 1 cm below the water surface so that it was invisible at water level. The first experimental day was dedicated to swimming training for 60 s in the absence of the platform. During the four subsequent days the mice were given four trials per day with the platform in place. When a mouse located the platform, it was permitted to remain on it for 10 s. If the mouse did not locate the platform within 60 s, it was placed on the platform for 10 s. The animal was taken to its home cage and was allowed to dry up under an infrared lamp after each trial. The time interval between each trial was 30 s. During each trial, the time taken to find the hidden platform (latency) was recorded using a video camera-based Ethovision System (Nodulus, Wageningen, The Netherlands). For October 2009 1711 each training trial, mice were placed in the water facing the pool wall at one of the pool quadrants in a different order each day. One day after the last training trial sessions, mice were subjected to a probe trial session in which the platform was removed from the pool, allowing the mice to swim for 60 s to search for it. A record was kept of the swimming time in the pool quadrant where the platform had previously been placed. Ginsenoside Rh2 (40 mg/kg, p.o.) or tacrine (10 mg/kg, p.o.) as a positive control was given 1 h before the first trial session at every consecutive day. Memory impairment was induced in mice with scopolamine (0.9 mg/kg, i.p.) at 30 min after treatment of the test agent. Control group received 10% Tween 80 solution only.
The Morris water-maze was performed as described previously.22) The experimental apparatus consisted of circular water tank (diameter, 100 cm; height, 35 cm) containing water at 23 °C at a depth of 15 cm and rendered opaque by the addition of powdered milk. A transparent platform was positioned inside the tank such that its top was submerged 2 cm below the water surface. The first training day dedicated to swimming training for 60 s in the absence of the platform. During the four subsequent days the mice were given three trial sessions per day with the platform in place. When a mouse located the platform, it was permitted to remain on it for 10 s. If the mouse did not locate the platform within 120 s, it was placed on the platform for 10 s. In each training trial, the time required to find the hidden platform was recorded. After several trials, the probe trial was conducted after the second injection of Ab 25—35 (at beginning of 6 weeks).
Numerous aspects of learning, memory and neurobehavior can be tested in this apparatus . There are 2 studies employing it after experimental SAH (Table 1)[38,39]. Takata, et al., studied rats undergoing 2 injections of blood or saline into the cisterna magna . Mortality was not reported but would be expected to be high based on prior studies and the massive amount of blood that was injected. Rats were tested for escape latency, swimming speed and swim distance for 16, 60-second trials 29 to 35 days after SAH. The platform was placed in a different quadrant each day and rats were placed randomly in 1 of 4 locations in the maze. If the platform was not found, the rat was placed on the platform for 30 seconds in the first trial or 15 seconds in subsequent trials . The procedure tests learning and short-term memory. SAH was associated with significantly longer escape latency, swim distance and faster swimming speed. Morris water maze testing correlated with neuronal counts in the hippocampus and neocortex.
Silasi and Colbourne compared rats with endovascular perforation SAH to sham-operated animals . They were tested in the Morris water maze from approximately day 21 to 40 after SAH. The procedure was similar to that of Takata, et al., but with 4 trials of 90 seconds per day. SAH rats had longer escape latency and swim distance on days the platform was moved to a new location (every second day). There were Fluoro Jade stained neurons in 4 of 5 SAH rats examined but no other histopathological changes.
After behavioral test, the mice were anesthetized with ethyl ether and perfused transcardially with PBS followed by 4% paraformaldehyde for fixation. The brains were removed and further fixed in 4% paraformaldehyde overnight at 4 °C. The fixed brain was cut into 30mm sections on a sliding microtome, and the section stained with
hematoxylin and eosin (HE).
Beam Balance Test
The beam balance test assesses motor and vestibular function by quantifying the ability to balance on a narrow wooden beam (diameter of 1-2.5 cm) for up to 60 seconds [1,13,14,25,29,40]. Parameters are beam balance time (duration the animal steadily remains on the beam) and beam balance score . Beam balance score is descriptive and examiner-dependent .
Rats with single hemorrhage SAH exhibited significantly increased beam balance score 1 day after SAH compared to their function before SAH and to sham-operated and artificial CSF-injected animals . In later studies, the beam balance test was carried out on a wooden beam with a diameter of 1 cm which may increase the sensitivity compared to the 1.5 cm diameter .
Most studies using the beam balance were done by one laboratory and although the creation of SAH and behavioral assessments were the same, the results varied, suggesting that the sensitivity is relatively low (Table 1). Deficits usually were detected only in the first 1 to 2 days after SAH [14,24,25]. Variable results may be due to several factors including that the score is subjective and descriptive . The severity of SAH caused by cisternal blood injection also is variable . Finally, the beam balance test is not standardized and there is variability in the diameter, length, shape and composition of the beam which may affect the results . Nevertheless, the results consistently show deficits in the first 24 hours after SAH that tend to resolve after that.
Beam Walking Test
The beam walking test is a learned avoidance test. A pre-training session is preceded by a negative reinforcement paradigm in which termination of the adverse stimuli (noise and light) serves as a reinforcement reward. The time taken to traverse the beam and enter a darkened goal box in order to terminate the loud white noise and bright light is measured to assess memory, motivation, attention, somatomotor and locomotor function .
Most [1,13,25,40] but not all  studies document that rats with SAH created by cisterna magna blood injection take a significantly longer time to traverse the beam compared to before SAH and to sham-operated controls for up to 4 days after SAH. In general, the deficit was maximal 1 day after SAH and then gradually improved. All studies were from one laboratory. Since memory, motivation and attention are involved, this test should be more sensitive to brain injury associated with SAH and this does seem to be the case compared to the other tests described above that assess mainly motor functions.