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Stroke & Ischemia

    Neuroprotection against global ischaemia
    • The advantages and disadvantages of the most widely used models are listed.
      • Gerbil-model : Advantages ; Simple operation: only short occlusion of common carotid arteries is needed
        Disadvantages ; Less possibilities for behavioral testing. Variable outcome due to variations in cerebral circulation
      • Rats: two-vessel occlusion-model : Advantages ; One stage surgery: only common carotid arteries are occluded, Occlusion is reversible, Possibility to control respiration by ventilation
        Disadvantages ; Needs induction of hypotension, Needs anaesthesia during occlusion, this may complicate the interpretation of outcome
      • Rats: four-vessel occlusion-model : Advantages ; No anaesthesia needed during ischemic occlusion Well-documented, the most used global ischemia model. Solidly validated
        Disadvantages ; - two-stage operation, highly invasive operation for the closure of vertebral arteries. Procedure only partly reversible: vertebral arteries are permanently closed. Variable outcome within one strain and variation in susceptibility in different strains
    • The hippocampus is the brain structure most severely affected by global ischemia. It is also the region known to play an important role in certain learning and memory processes. Deficits in learning and memory in rats have also been detected after global ischemia and patients having hypoxic/ischemic hippocampal damage are amnesic.
    • Ischemic rats are impaired in tasks requiring working and spatial memory, such as T-maze, radial arm maze, water-maze, circular platform task and delayed non-matching to sample/position (DNMS/P) tasks. Global ischemia also leads to locomotor hyperactivity in an open-field test.
    • Pulsinelli WA, Brierley JB: A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10: 267-272, 1979.
    • Apparatus : Vibratome, two mechanical manipulators, peristaltic pump, thermostatically controlled moist chamber, electric stimulator, amplifier, oscilloscope, software for data storage and analysis.
    • see :

    Neuroprotection against focal ischemia

    • The advantages and disadvantages of the most widely used Rat focal ischemia models are listed.
      • MCA occlusion-model( permanent ) :
        • Advantages ; Selection of the occlusion site allows to some extent the choice of the affected brain area
        • Disadvantages ;
            1) Penetration of drugs to the infarcted area is limited,
            2) No recanalization involved,
            3) Variation in infarct size,
            4) Some models need craniectomy
      • MCA occlusion-model ( transient ) :
        • Advantages ;
            1) Probably the most widely used experimental stroke model,
            2) Possible to assess reperfusion damage following recanalization
            3) Possible to have drug penetration to the occluded area
        • Disadvantages ;
            1) variation in infarct size
      • Photochemically induced focal cerebral thrombosis:
        • Advantages ;
            1) Possibility for precise location and size of infarcted cortical area
            2) Invasive procedures minimal
            3) Ppossibility to study antiplatelet and thrombolytic therapy
        • Disadvantages ;
            1) Eend-arterial occlusion, which is resistant to therapies based on enhancement of collateral perfusion
            2) Differs in some respects from human stroke, e.g. no penumbra
      • Miscellaneous models of cerebral embolism and thrombosis:
        • Disadvantages ; 1) Random and unpredictable location and size of the lesion
    • Rats having focal cerebral ischemia following MCA occlusion have been shown to have deficits in several tests assessing sensorimotor and cognitive functions, which are collated.
      Behavioral testBehavioral taskBehavioral assessment
      Beam-walking testWalking on a beamAbility to maintain balance and hindlimb slips during walking
      Foot-fault testWalking on a gridLimb misplacement asymmetry while moving around a grid
      Rota-rod testWalking on a rotating rodTime of staying on a rod
      Running wheel testRunning in a wheelForelimb slips during running
      Montoya's staircase testReaching and grasping of food pellets Fine movements of forepaws
      Limb-placing testForelimb and hindlimb placementResponse to proprioceptive and tactile stimuli
      Tape testRemoving of sticky tapes from forepawsExtinction, preference for removing of adhesive stimuli
      Sensory inattention testOrientation to sensory (visual, olfactory or tactile) stimuliTendency to orientate and investigate impinging stimuli
      Prehensile traction testPrehensile tractionTime of traction
      Vertical screen testAbility to stay on a vertical screenForelimb and hindlimb muscular strength
      Cylinder testForelimb usage in vertical movementsAsymmetry in forelimb usage
      Passive avoidance testAvoidance of aversive stimulusAssociative memory
      Water-maze testMemorizing the location of a hidden platformSpatial learning


    • Apparatus : Vibratome, two mechanical manipulators, peristaltic pump, thermostatically controlled moist chamber, electric stimulator, amplifier, oscilloscope, software for data storage and analysis.
    • see :

    Behavioral Test of ischemia

    • Water-maze test(I-VI).
      • To assess spatial learning, a modified version of the Morris water-maze task was used. The water-maze pool (Ø 150 cm, depth 74 cm, filled to a height of 52 cm with clear water at temperature 20±2 ºC) was a circular fiber-glass tank, painted black. The pool was divided into four quadrants of equal surface area. The starting locations were called north, south, east, and west, and were located arbitrarily at equal distances on the pool rim. The platform (10 cm diameter, composed of black rubber) was located with its top surface 2.5 cm below the water line in the middle of the quadrant 25 cm from the pool rim. However, in the north-west quadrant (I) the center of the platform was 22 cm from the north-south axis and 20 cm from the pool rim. The swim paths were monitored by a video camera connected to a computer through an image analyzer. If the rat failed to find the hidden platform within 70 s, it was placed on the platform. The animal was allowed to remain on the platform for 10 s and to rest for either 30 s or 1 min. The first, third, and fourth trials of the day were started from one of the points located farthest from the platform. The start point was changed after each trial. Escape latency (time to reach the platform) and path length the animal swam to find the platform were used to assess the acquisition of the water-maze task. Swimming speed (path length/escape latency) was used to assess the motor activity of rats in this task. The shorter the latency to find the platform, the better the memory for its location was considered to be. At the end of the testing period, a probe trial of 70 seconds without the platform was used to assess how well the animals remembered the location of the platform (i.e. by number of passes over the previous platform location). Different searching strategies were also analyzed (percentage of time spent in three equal zones of the pool). A visible platform in a new location was used to determine whether the groups had differences in recognition ability or escape motivation. In experiment I testing was performed for 2 days after each of the three atipamezole/saline treatment periods (on days 11, 12, 20, 21, 29 and 30 after global ischemia induction). The rats had 6 trials on each test day. The platform was changed to a different quadrant each day. After the last two water-maze test days the animals were tested in another room to evaluate their performance in a new environment (days 31 and 32). In experiment II rats were exposed to a water-maze task of eight trials 12 days after global ischemia induction. On the previous day, the animals had been allowed a habituation swim of 90 s without the platform. In experiment III the spatial learning ability of rats was assessed in the water maze on days 22-24 after focal cerebral ischemia. Rats were given five trials from the first through the third test day. There was also an additional probe trial without the platform given on the third day to test how well the animals remembered the location of the hidden platform. On the fourth day (postoperative day 25), the rats were given four trials to find a visible platform placed in the south-east quadrant. In experiment IV the water-maze paradigm was the same as the previous one, except that no visible platform was used.
    • Open-arena test (I).
      • The open-arena test was performed in the same room as the first series of water-maze tests in experiment I. The open-arena test was used for assessing of the exploratory activity on postoperative days 2, 10, 19 and 28. The apparatus was placed on the rim of the water-maze. Each rat was placed in the middle of a black painted square (110 x 110 cm, walls 30 cm), and was monitored for 15 min by a video-camera connected to a computer through an image analyzer (in 3-minute sessions that were interrupted by a 25-second break during which the computer loaded the next program). The computer system registered the distance traveled, and the number of rearings (rearing up on hind legs), number of fecal boli, and time spent grooming were observed by the experimenter.
    • Limb-placing test (III, IV) .
      • This test was a modified version of a test described by De Ryck et al. (De Ryck et al. 1989). The rats were habituated to handling before the induction of ischemia. The limb-placing test was used for assigning ischemic animals to behaviorally equal groups the day after induction of ischemia and the same test was used to assess recovery of rats on postoperative days 2 through 11, 16, and 21 (III) and on days 3, 5, 7, 9, 11, 16, 21 and 32 (IV). This test had seven limb-placing tasks to assess the integration of forelimb and hindlimb responses to tactile and proprioceptive stimulation. The tasks were scored as follows: 2 points, the rat performed normally; 1 point, the rat performed with a delay (2 s) and/or incompletely; and 0 points, the rat did not perform normally. Both sides of the body were tested. In the first task, the rat was suspended 10 cm over a table. Rats normally stretch both of their forelimbs towards the table. In the second task, the rat was positioned towards the table and its forelimbs were placed on the table. Each forelimb was gently pulled down and retrieval and placement were checked. Rats normally replace the limb on the table. The third task was the same as the second except that, by keeping the rat's head upward in a 45º angle, the rat was prohibited from seeing the table or contacting it with its vibrissae. Next, the rats were placed along the table edge to check for lateral placement of the each forelimb (fourth task) and hindlimb (fifth task). In the sixth task, the rat was again positioned towards the table, the hindlimbs just over the table edge. Each hindlimb was pulled down and gently stimulated by pushing it towards the side of the table. In the seventh task, the forelimbs were placed on the edge of the table and the rat was gently pushed from behind toward the edge. Rats normally rats resist the pushing, but injured rats cannot keep their grip and the injured limb slips off the edge.
      • De Ryck M, Van Reempts J, Borgers M, Wauquier A, Janssen PA: Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke 20: 1383-1390, 1989.
    • Beam-walking test.
      • The beam-walking test was used to assess deficits in coordination and integration of motor movement, especially in the hindlimb. The rats were trained to traverse the beam for 3 days before the induction of ischemia and by the end of the training period all rats had learned the task. The animals were tested from days 2 to 7 after ischemia. A beam-walking apparatus consisted of a square beam (2.5 cm wide, 122 cm long, at 42 cm high) connected to a black box (20.5 x 25 cm, 25 cm). A bright light was placed above the start point to motivate the rats to traverse the beam. The performance of the rats was rated as follows: the rat was not able to stay on the beam, 0 points; the rat did not move, but was able to stay on the beam, 1 point; the rat tried to traverse the beam, but fell, 2 points; the rat traversed the beam with more than 50% footslips of the affected hindlimb, 3 points; the rat traversed the beam with more than one footslip, but less than 50%, 4 points; the rat had only one slip of the hindlimb, 5 points; the rat traversed the beam without any slips of the hindlimb, 6 points.
    • Foot-slip test (III, IV)
      • The rats were trained to run in a wheel for 4 days before the induction of ischemia. To assess motor coordination and proprioception, the accuracy of forelimb placement of rats was quantified using running in a wheel in a foot-slip test (on days 2 to 11, 16, and 21 (III) and 2, 3, 5, 7, 9, 11, 16, 21 and 32 (IV) after induction of focal cerebral ischemia). The running wheel (Ø 29 cm, with transparent plastic walls, rungs 2 cm apart) had an adjustable motor and rotated 6 times per minute. Performance was recorded via a camera connected to a video recorder and a monitor. The performance of the rats was assessed by calculating the slip ratio of the affected forelimb (number of slips/number of steps taken) over 2 min.
    • Staircase test (IV)
      • A modified version of the staircase test by Montoya et al. (Montoya et al. 1991) was used for evaluation of the forelimb food pellet reaching and grasping abilities of the rats from different levels of a staircase on days 27 to 32 after induction of ischemia. The test was preceded by 36 h of food deprivation. The PlexiglasTM test apparatus has an elevated central platform with a staircase on both sides. The staircases have six steps, of which the five upper steps were each baited with a chow pellet (45 mg, Campden Instruments Ltd, UK). The rat was placed on the platform and was allowed to collect the pellets during four trials each of five min duration. During each trial, the number of pellets reached but dropped as well as successfully retrieved pellets from both sides were calculated. After each test, the rats were given approximately 15 g of standard food pellets.
      • Montoya CP, Campell Hope LJ, Pemberton KD, Dunnett SB: The ‘staircase test’: a measure of independent forelimb reaching and grasping abilities in rats. J Neurosci Methods 36: 219-228, 1991.