Behavioral tests
To evaluate changes in neurological function associated with ischemia, the rats were subjected to a variety of locomotor, sensory, learning and memory tests before and after surgery (Schallert, 2006). All testing are performed from 9-11 AM by the same investigator. Results obtained before surgery were used to define 100% functionality for each animal on each test, and functional recovery was expressed as percent recovery relative to the pre-surgery baseline.
Neurological status
Following surgery, rats are observed for circling behavior if pulled gently by the tail. Rats circling toward the infarcted brain side were ranked as grade 3. Rats that did not circle but fully stretched their forelimbs were graded as 2. Rats that did not circle, but partially stretched their forelimbs were graded as 1. Rats showing no reaction were graded as 0 (Bederson et al., 1986; Ohlsson & Johansson, 1995).
Limb-placement symmetry
To assess the symmetry of limb placement, the rat was gently held by the tail, and forelimb placement on the edge of a table was scored as follows: 3 – forelimbs extended symmetrically; 2 – forelimbs on the left side extended more or less slowly than those on the right side; 1 – limbs on the left side showed minimal movement; and 0 – forelimb on left side did not move at all [10].
Symmetry in the movement of four limbs
Rats are inspected while free-running on a table. Score, 0 – left limbs not used for moving; 1 – left limbs were minimally used; 2 – four limbs used asymmetrically with the left limbs moving slower and to a lesser extent than the right limbs; 3 – all four limbs used to the same extent (Ohlsson & Johansson, 1995).
Body proprioception
The rat is touched lightly with a blunt stick on each side of the body, and the reaction to the stimulus was observed. Scores indicate the following: 3 – rat reacted by turning head and was equally startled by the stimulus on both sides; 2 – rat reacted slowly to stimulus on the left side; and 1 – rat did not respond to the stimulus placed on the left side [Popa-Wagner et al., 2010].
Response to vibrissae touch
A blunt stick was brushed against the vibrissae on each side by an experimenter who remained behind the rats to avoid entering the visual fields. Scores indicate the following: 3 – rat reacted by turning head or was equally startled by the stimulus on both sides; 2 – rat reacted slowly to the stimulus on the left side; and 1 – rat did not respond to stimulation on the left side [10].
Beam-walking test (Rotarod)
The beam-walking, or Rotarod, task assesses fine vestibulomotor function in the MCAO model [Goldstein, 1993 ]. Each rat is tested for its ability to negotiate a rotating (6 rpm) horizontal rod. The time taken for the rat to traverse the rotating cylinder and join a group of rats visible at the finish line was measured. The score assessment was two-fold: (i) time (seconds) required to traverse the rotating cylinder and, (ii) the score as follows: 0, rat falls immediately (onto a soft surface); 1- rat does not walk forward, but stays on the Rotarod; 2 – rat walks, but falls before reaching the goal; 3 – rat traverses the rod successfully, but the limbs are used asymmetrically; 4 – the left hindlimb is used less than 50% of the time taken to traverse the rod; 5 – the rat successfully traverses the rod, but with some difficulties; 6 – no mistakes, symmetric movements.
Inclined plane
We test the ability of each animal to maintain its position at a given angle on an inclined plane [Buchhold et al., 2007]. The relative angle at which the rat could no longer maintain its position was taken as a measure of functional impairment. This test was conducted once before surgery and daily thereafter.
Spontaneous activity
Each rat is placed in front of a large cage with a small entryway. After entering the cage, the number of crossings between two walls was counted within a 30 min test period [10]. This test was conducted only on days 4, 8, 13, 18 und 27 in order to maintain the interest of the rats in exploring the new environment.
Asymmetric sensorimotor deficit: Adhesive Tape Removal Test
We assess the asymmetry of sensorimotor deficit of the forelimbs induced by unilateral MCAO by the adhesive tape removal test. In short, sticky patches were applied on the distal hairless parts of the forelimbs and the removal time from both limbs was measured. Three trials were done separately for each limb and the means of the values were noted. If the animal did not remove the tape within 180 s, the timer was stopped. Results are given as ratio of time needed to remove the adhesive tape from one forelimb divided by the sum of time needed to remove it from both forelimbs (Popa-Wagner et al., 2010).
Spatial working memory: Radial-arm maze
Rats were tested on an eight-arm radial maze that was elevated 60 cm above the floor and had an octagonal central area. One of the eight arms served permanently as a starting compartment, whereas the other seven arms served as goal arms. The starting compartment and each goal arm were separated from the rest of the apparatus by guillotine doors. Each arm was labeled by different black & white cues. Food-deprived rats were placed in the maze and had to remember the “rewarding” arm, i.e. the arm that previously had a food pellet at the end. For each rat the baited arm was kept constant (Olton & Papas, 1979). We assigned a score of 0 for successfully finding the baited arm, and 1 for failure to do so. Each trial consisted of two runs within 20 minutes. Two consecutive successful runs were needed to obtain a score of 0; otherwise the assigned score was 1. Pre-operative training was not conducted for this test. The maze was kept odor-free, and was cleaned during and after testing each animal.
Spatial learning based on positive reinforcement, working and reference memory
was evaluated in the labyrinth food-finding test (maze test). Before the test, the animals were deprived of food but had unlimited access to water. The rats were trained in the maze test during 2 wks for the young rats and 3 wks for the aged rats (which learned more slowly) with food placed in the end-point of a complex route consisting of 3 consecutive T-mazes (Schwarting & Borta, 2005). The food was the reward for finding the way. The animals always were placed in the same area of the maze (start area). Every two days the rats were fed freely after the test for 30 min. We assigned a score of 0 for successfully finding the baited arm. Because the apparatus was made up of 3 T-mazes, the rats could commit 1, 2 or 3 errors in each trial.
Morris Water Maze
The Morris water-maze task was used to assess spatial learning and memory. One week before surgery, aged rats were trained to find a submerged platform in a large (180 cm-diameter) pool filled to within 20 cm of the upper edge with water maintained at 26 °C. The pool was divided into four compass quadrants (north, south, east, and west). Several visual stimuli were placed in each of the four quadrants. For the acquisition of spatial learning, each animal underwent a set of four trials per day for seven days. Before the first trial, the rat was placed on the hidden platform for 30 s by the investigator. Each trial consisted of placing the rat in the water at one of the randomly selected four starting locations around the pool perimeter. Each rat was allowed a maximum of 60 s to find the hidden platform and remain on it for 30s. If a rat failed to find the platform within 60 s, the rat was placed on the platform for 30 s by the investigator. The time and distance required to find the hidden platform during these four acquisition trials were averaged (Tottori et al., 2002). The swim path was recorded by an image analysis system (VideoMot2, TSE, Bad Homburg, Germany) that computed path length and percentage of time spent in each of 4 quadrants. Functional restoration of spatial learning and memory was estimated by weekly testing after MCAO and in total for 8 weeks.
Summary of functional tests after stroke
Rotating Pole
T-Maze
Rotor-Rod
Morris water navigation test
i-Plane
Cylinder Test
Neurological status