(Stroke. 1995;26:2338-2346.)
© 1995 American Heart Association, Inc.
Articles |
Nonocclusive Common Carotid Artery Thrombosis in the Rat Results in Reversible Sensorimotor and Cognitive Behavioral Deficits
From the Cerebral Vascular Disease Research Center, Department of Neurology, University of Miami School of Medicine, and Department of Psychology (E.J.G.), University of Miami, Coral Gables, Fla.
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Background and Purpose Microemboli released during transient ischemic attack, stroke, and cardiac surgery are thought to cause a variety of functional deficits in humans. The purpose of this study was to characterize the type and extent of neurobehavioral deficits present after photochemically induced common carotid artery thrombosis (CCAT), a thromboembolic model of stroke in the rat that results in a platelet emboli shower.
Methods Thirty-two male Wistar rats were assigned to four groups. Groups 1 (n=8) and 3 (n=8) were long-term (6-week survival) and short-term (2-week survival) experimental groups subjected to right CCAT with the use of the photochemical technique. Groups 2 (n=8) and 4 (n=8) served as sham-operated controls for each experimental group. A battery of behavioral tests was applied daily beginning 24 hours after thrombosis; this consisted of elicited forelimb placing, postural reflex, beam balance, beam walking, and open field activity. Cognitive testing with a water maze task was performed on post-CCAT days 30 to 33 for groups 1 and 2 and on post-CCAT day 2 for groups 3 and 4. Ten-micrometer coronal brain sections were stained with hematoxylin and eosin, and infarct location and frequency were determined.
Results Significant sensorimotor deficits were observed, which recovered within 2 weeks after CCAT. The data that follow are derived by combining the two experimental groups and comparing these with the two sham groups. The following tests showed significant effects after CCAT: contralateral elicited forelimb placing, ipsilateral elicited forelimb placing, beam balance, and beam walking score. Cognitive dysfunction was seen acutely (group 3 animals) at 2 days after CCAT; Morris water maze length and latency to target were significantly greater in the experimental group. No deficits were seen in postural reflex, open field activity, or delayed cognitive testing. Histopathological assessment revealed small infarcts in 11 of 16 thrombosed rats. However, a strong relationship between neurobehavioral deficits and infarct location was not consistently demonstrated.
Conclusions CCAT produces consistent sensorimotor and cognitive behavioral deficits that recover within 2 weeks of injury. Behavioral outcome was not necessarily associated with overt histopathological damage, suggesting that reversible injury mechanisms, both vascular and neuronal, may be partly responsible for the temporary loss of function. These data strengthen the role of CCAT as a clinically relevant model of thromboembolic stroke.
Key Words: behavior, animal • cerebral ischemia • cognition • stroke • rats
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Introduction |
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The release of microemboli into the cerebral circulation is a common event after vascular thrombosis, cardiac arrest, and cardiac surgery.1 2 3 The deposition of platelet aggregates in the microvasculature often results in transient ischemic attacks and acute stroke and can cause subsequent sensory, motor, and cognitive deficits.2 Correlations have been made in cardiopulmonary bypass patients indicating that the degree of functional defects is proportional to the quantity of emboli recorded on transcranial Doppler flow studies.3
Experimental models of embolic stroke commonly use the injection of microspheres or autologous clots into a major vessel to mimic thromboembolic events.4 5 Although these models may have some utility in assessing basic ischemic pathological mechanisms and consequences, they are not based on active thrombotic processes, which have been shown experimentally to precipitate an array of microvascular and hemodynamic changes occurring after capture of cerebral emboli.6
Photochemically induced nonocclusive common carotid artery thrombosis (CCAT) is a rodent model of unilateral carotid stenosis with subsequent platelet embolization.7 Hemodynamic, microvascular, and blood-brain barrier abnormalities have been observed in this model.8 9 10 11 Blood-borne factors possibly released from the forming carotid thrombus and from subsequent platelet emboli have been detected in the downstream circulation.12 13 Histopathology after CCAT shows the presence of small, focal infarcts and ischemic cells most commonly located in the ipsilateral cortex.7 9 Occasionally, platelet emboli and structural damage are also noted in the ipsilateral hippocampus, striatum, thalamus, and the contralateral cortex.7 10
The purpose of this investigation was to characterize the acute and chronic behavioral deficits seen after CCAT and to define a battery of standard tests that are sensitive to changes in functional outcome in this model. These tests will provide a measure in the screening of therapeutic strategies in the future. Particular interest was given to examining deficits typically seen clinically after thromboembolic stroke.
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Materials and Methods |
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The experiments were performed on 32 male Wistar rats weighing between 250 and 350 g obtained from Charles River Breeders. The rats were housed individually under 12-hour light/dark cycles and were given food and water ad libitum. Rats were randomly separated into four groups of 8 animals. Groups 1 and 3 were long-term (6-week survival) and short-term (2-week survival) experimental groups, respectively, and groups 2 and 4 served as sham-operated controls (photosensitive dye injection; no laser irradiation) for each time point.
Surgery
Rats were deprived of food 12 hours before surgery. CCAT
was produced with the use of the photochemical
technique.7 11 12 Briefly, the animals were
anesthetized with halothane, intubated, and artificially
ventilated with a rodent respirator. Rats were maintained on a mixture
of 70% nitrous oxide, 30% oxygen, and 1% halothane. Jugular venous
and tail arterial catheters were inserted for the
administration of drugs and the measurement of blood gases and blood
pressure. Muscle relaxation was achieved by the injection of
pancuronium bromide. Under a Zeiss operating microscope, the right
common carotid artery was exposed by blunt dissection and retraction of
the surrounding musculature. The vagus nerve was carefully separated
from the common carotid artery, and the artery was freely suspended in
a saline bath.
A tunable argon dye laser (wavelength, 562 nm; peak power, 325 mW) was focused onto the right common carotid artery for 10 minutes. Simultaneously, the photosensitizing dye rose bengal (15 mg/mL in 0.9% saline) was injected intravenously into the circulation over a period of 90 seconds. This irradiation procedure has been shown to produce 50% to 75% stenosis.13 After the irradiation, catheters were removed, wounds were sutured, and the animals were allowed to recover and return to their home cages. Body temperature was monitored throughout the procedure with a rectal probe and was maintained between 36°C and 37°C with a heating pad.
Behavioral Protocol
Each animal was handled for 3 days before testing and was
trained as needed for each task. The animals were tested daily
beginning 24 hours after surgery unless otherwise noted by an
experimenter blinded to the experimental procedure. Since the injury
after CCAT may be widespread and typically includes damage to the
cortex, striatum, and hippocampus, a battery of behavioral tests was
chosen to evaluate functional outcome. Each behavioral test is
described in detail below.
Elicited Forelimb Placing
Forelimb placing to three separate stimuli (visual, tactile, and
proprioceptive) was measured to assess sensorimotor
integration.14 Visual placing is accomplished by cupping
the rat in the experimenter's hands while allowing the forelimbs to
dangle freely. The rat is tilted and lowered toward a tabletop. A
normal rat will reach toward the table with both forelimbs extended.
Visual placing is also assessed by moving the rat toward the table in a
sideways direction for each side. Tactile placing is evaluated by
tilting the animal's head upward at a 45° angle to the table while
allowing one forelimb to dangle freely. The rat is brought toward the
table's edge, and the forelimb is lightly touched; this is performed
for the dorsal and the lateral surfaces of each forelimb. Normal rats
will immediately place the forelimb on the table. Similarly,
proprioceptive placing is accomplished by forcefully contacting the
dorsal surface of each forelimb with the table edge. For each test, a
score of 0 was given for normal, immediate placing. A score of 1 was
given if the placing was delayed or incomplete. A score of 2 indicated
absent placing. A maximum score of 10 was possible for each
forelimb derived from the sum of the five scores (0 to 2) from dorsal
and lateral tactile stimulation, dorsal proprioceptive
stimulation, and forward and sideways visual stimulation.
Postural Reflex
This test is sensitive to damage in the cortex and
striatum.15 The rat was suspended by the tail 1 m above a
table top. He was slowly lowered toward the table, and his posturing
was observed. A normal rat will extend both forelimbs toward the table.
This behavior received a score of 0. A score of 1 was given if the rat
flexed one or both forelimbs. This animal is then given the lateral
push test. This involves placing the rat on a sheet of
plastic-coated paper and applying lateral pressure behind the
shoulders in the left and right directions. If the rat was unable to
resist the force equally in both directions, he received a score of
2.
Beam Balance
Beam balance is sensitive to motor cortical
insults.16 17 This task is used to assess gross
vestibulomotor function by requiring a rat to balance steadily on a
narrow beam. The test involves three 60-second training trials 24 hours
before surgery to acquire baseline data. The apparatus
consists of a 3/4-inch-wide beam, 10 inches in length, suspended 1
ft above a table top. The rat is positioned on the beam and must
maintain steady posture with all limbs on top of the beam for 60
seconds. The animal's performance was rated with the scale of
Clifton and colleagues,18 which ranges from 1 to 6, with a
score of 1 being normal and a score of 6 indicating that the animal was
unable to support itself on the beam.
Beam Walking
This is a test of sensorimotor integration specifically
examining hindlimb function. The testing apparatus and
rating procedures were adapted from Feeney and
colleagues.16 A 1-inch-wide beam, 4 ft in length, is
suspended 3 ft above the floor in a dimly lit room. At the far end of
the beam is a darkened goal box with a narrow entryway. At equal
distances along the beam, four 3-inch metal screws are positioned,
angling away from the beam's center. A white noise generator and
bright light source at the start of the beam motivate the animal to
traverse the beam and enter the goal box. Once inside the goal box, the
stimuli are terminated. The rat's latency to reach the goal box (in
seconds) and hindlimb performance as he traversed the beam
(based on a 1 to 7 rating scale) are recorded. A score of 7
indicates normal beam walking with less than 2 foot slips, and a score
of 1 indicates that the rat is unable to traverse the beam in less than
80 seconds. Each rat is trained for 3 days before surgery to acquire
the task and to achieve normal performance (a score of 7) on
three consecutive trials. Three baseline trials are collected 24 hours
before surgery, and three testing trials are recorded daily
thereafter. Mean values of latency and score for each day were
computed.
Open Field Activity Assay
Elevated open field activity is characteristic of hippocampally
damaged animals19 20 21 22 and has been observed in animals with
striatal/cortical damage as well.23 Rats are placed in a
novel environment (3x3 ft) with 3-inch-square grid markings on the
base. The number of times a rat crosses a grid line is recorded for
a 5-minute period. The animal was tested once before surgery and on
alternate days thereafter.
Morris Water Maze
This is a task that measures learning and memory in an aqueous
environment. Briefly, the rat was placed in a 4-ft-diameter water
tank that is visually separated into four quadrants. In the center of
one quadrant, the goal quadrant, a platform is hidden 1 inch below the
waterline. The rat was placed in the tank at one of four start
locations (north, south, east, west) and swam for up to 60 seconds
until it found the platform. Once the animal located the platform, it
remained on it for 20 seconds. In the event that an animal did not
locate the platform, the observer positioned the animal on the platform
at the end of the 60-second swim period. During a 2- to 4-minute
intertrial interval, the animals were kept warm by infrared heating
lamps. The temperature of the water maze was 20±0.5°C. The latency
and path length of the rat were measured by a video tracking system
interfaced to a computer.
Rats from groups 1 and 2 (6 weeks; experimental and sham, respectively) were tested on a simple place task with a hidden platform at 5 weeks after CCAT for 3 days and with a visible platform for 1 day. In these animals no pretraining was given. Rats from groups 3 and 4 (2 weeks; experimental and sham, respectively) were trained with a hidden platform for 2 days before CCAT and were tested again 2 days after CCAT. Each day of testing consisted of eight randomized trials. In addition, at the end of the third day of testing for groups 1 and 2 and at the end of day 2 after CCAT for groups 3 and 4, a probe trial was given in which the platform was removed from the tank. This is designed to test whether the animal has a learned bias to navigate toward the goal quadrant or if the animal has located the hidden platform using a different approach. The percentage of time spent in the goal quadrant was recorded for each rat for a 60-second period.
Histopathology
At designated time periods (2 or 6 weeks), animals were
perfusion-fixed and their brains removed for histopathological
analysis. Briefly, rats were deeply anesthetized with
halothane for 10 minutes and perfused transcardially with
physiological saline for 2 minutes and then FAM (a
mixture of 40% formaldehyde, glacial acetic acid, and methanol; 1:1:8
by volume; 20 minutes) at a pressure of 120 mm Hg. Heads were removed
and stored in chilled fixative overnight. Brains were extracted and
embedded in paraffin, and 10-µm sections were then prepared at
200-µm intervals for staining with hematoxylin and eosin. Semiserial
sections were examined under low power (x10), and camera lucida
drawings were made. From these, infarct location and quantity were
tabulated.
Statistical Analysis
Data from behavioral tests were analyzed with
two-way ANOVAs with treatment group and postsurgical testing day as
factors. Fisher's tests were used to evaluate statistically
significant post hoc comparisons. One-way ANOVA was performed on
physiological variables.
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Results |
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Physiological variables, as shown in Table 1
, were within normal ranges and did not differ
significantly between experimental and sham groups.
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Behavioral Outcome
Long-term (6-week) survival animals (groups 1 and 2; n=7 each)
were compared, and the CCAT animals were found to recover within a
2-week period to presurgical baseline levels in all sensorimotor tests.
For this reason, additional animals (groups 3 and 4; n=8 each) were
allowed to survive for 2 weeks only. Behavioral data from groups 1 and
3 (experimental) and groups 2 and 4 (sham) were compared and did not
differ within each condition; therefore, they were analyzed
together for tests common to all groups. Two animals, one animal each
from groups 1 and 2, were excluded from the final analysis for
technical reasons.
Elicited Forelimb Placing
In all of the experimental animals, mild to moderate elicited
forelimb placing deficits were present. These were observed in the
contralateral forelimb of all CCAT rats and in the ipsilateral forelimb
of 50% of CCAT rats. Statistically significant effects were seen in
the contralateral forelimb when CCAT animals were compared with sham
animals beginning 24 hours after thrombosis. These effects recovered
within 14 days. Analyses of contralateral placing indicated
significant effects of group (F1,27=26.8,
P<.001), test day (F13,312=10,
P<.001), and a group-by-day interaction
(F13,312=8.75, P<.001). Follow-up
comparisons indicated that CCAT and sham animals differed on each of
the first 10 postsurgical days (Fig 1
left).
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Analyses of ipsilateral placing indicated significant
effects of group (F1,27=6.20, P<.02),
test day (F13,351=2.78, P<.001), and a
group-by-day interaction (F13,351=2.54,
P<.003). Fisher's follow-up comparisons indicated that
CCAT and sham animals differed on each of postsurgical testing days 1
to 7 (Fig 1
right). In both cases, data from sham
animals are not always visible since a score of 0 indicates normal
placing, and most sham animals were unaffected by the surgery.
Postural Reflex
No postural reflex abnormalities were observed in CCAT or sham
animals. All animals exhibited normal forelimb extension.
Beam Balance
Mild beam balance abnormalities were detected in CCAT animals
compared with sham animals. Analysis of the beam balance data
revealed a significant group-by-day interaction
(F13,351=2.86, P<.001) as well as an effect of
days (F13,351=3.27, P<.001). Follow-up
comparisons indicated significant main effects of group only on day 6
(P<.015). Beam balance data are displayed in Fig 2.
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Beam Walking
Beam walking latencies were normalized with respect to the
animal's presurgical baseline level. Beam walking latency from CCAT
animals was not statistically different from controls, although there
was a trend for CCAT animals to exhibit longer latencies in traversing
the beam. The beam walking score of CCAT animals was significantly
albeit modestly impaired compared with sham animals for 2 weeks after
CCAT. Analysis of the beam walk score revealed significant
effects of treatment group (F1,27=8.49,
P<.008). Post hoc comparisons indicated that the scores of
CCAT and sham animals differed on each of the 14 postsurgical testing
days, with the exception of days 2 and 11. Beam walking score data are
presented in Fig 3. A hindlimb deficit was
indicated by any score below 7.
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Open Field Activity Assay
No differences were seen in activity levels between CCAT and sham
animals after thrombosis.
Morris Water Maze
Distance and latency-to-target data were normalized to
presurgical levels. At 5 weeks after thrombosis, CCAT animals performed
as well as sham animals on the hidden platform learning set task,
visible platform test, and probe trial, as indicated by comparable
latencies and lengths to target.
Acute water maze testing at 2 days after CCAT showed that retention and
learning in CCAT animals were significantly impaired compared with sham
animals. Latency (P<.01) and length (P<.001) to
target were prolonged in CCAT rats. Fig 4 left shows
post-CCAT latency minus pre-CCAT baseline latency for experimental and
control animals. Fig 4 right is a similar bar graph of
length to target. The data show that CCAT rats performed worse after
surgery, with a higher average latency and length to target than
presurgical levels and compared with sham animals. The
performance of sham animals improved, as indicated by a
negative length and latency value. A strong trend (P=.058)
existed for CCAT animals to spend less time in the goal quadrant during
the probe trial than CCAT animals, although this did not reach
statistical significance as defined in the other tasks
(P<.05).
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A postsurgical motor defect that may have resulted in the rats' inability to perform in the water maze task was ruled out by comparing swim speeds before and after CCAT in those animals tested acutely and by comparing swim speeds of sham to experimental animals in those tested chronically (where no presurgery testing was performed). These did not significantly differ for any of the groups (data not shown).
In summary, nonocclusive CCAT in the rat produces consistent, mild to moderate sensory, fine and gross motor, and acute cognitive neurobehavioral deficits. The sensorimotor deficits are transient and recover within 2 weeks. CCAT, by the photochemical technique, does not result in long-term learning and memory deficits, postural reflex abnormalities, or open field hyperactivity.
Histopathology
Histopathological changes were observed in 11 of 16 CCAT animals
but in no sham animals. As shown in Table 2, the
majority of small embolic infarcts were seen in the ipsilateral cortex
and occasionally in the ipsilateral hippocampus; no damage was observed
in the brain stem or thalamus. The number of brain regions exhibiting
neuronal damage varied between animals; as few as one infarct was
documented in one animal, while another rat presented with a
total of 14. In general, the pattern of injury was not different
between short- and long-term survival animals, although the number
of observable infarcts was considerably less at 6 weeks than at 2
weeks.
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Fig 5 summarizes the histopathological characteristics
of the lesions 2 or 6 weeks after CCAT. At 2 weeks after CCAT (Fig 5A through 5C), infarcts were generally well demarcated
and contained reactive astrocytes and macrophages. In some
cases damage was more subtle, and selective neuronal necrosis was
detected (Fig 5C). At 6 weeks after CCAT, infarcts
appeared shrunken and at times difficult to visualize at low power (Fig 5D). Within the hippocampus, multiple sites of injury
were occasionally seen. In some cases damage to the
pyramidal cell layer as well as the dentate gyrus was
observed (Fig 5E and 5F).
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Brain/Behavior Relationships
As noted previously, there existed considerable variability in the
histopathology observed after CCAT. On the other hand, the behavioral
data appeared surprisingly consistent. Since strong
relationships could not be determined by evaluating the overall outcome
of the groups, we decided to examine specific animals for
behavioral/histopathological correlations. The animals chosen were
those in which extremes in neurobehavioral outcome or pathology were
observed. Animal numbers refer to those in Table 2.
The most significant histopathology in group 1, 5 infarcts at 6 weeks after thrombosis, was seen in animal 4. Although the damage affected the forelimb cortex and hippocampus, the sensorimotor outcome in this animal was not noticeably different from others in the group, and the water maze function at 5 weeks was not affected.
Animal 6 in group 1 (not included in table) did not have any observable damage on hematoxylin and eosin staining. The rat possessed moderate contralateral placing deficits persisting for 7 days and mild beam balance and beam walking abnormalities that recovered in 4 and 2 days, respectively. As in animal 4, no cognitive deficits were observed at 5 weeks. This animal is an example of the presence of transient sensorimotor dysfunction in the absence of overt histopathology.
Interestingly, animal 1 in group 3 (2-week survival), with multiple infarcts in the cortex, hippocampus, and striatum, had the most robust and prolonged forelimb placing deficits of all animals tested, with recovery only at day 14. In this same animal, beam balance and beam walk abnormalities were mild, but recovery was again not complete until day 13. Water maze deficits were present, as they were in all animals of group 2, but these did not deviate greatly from the mean values.
Animal 6 in group 3 had only three observable injury areas: two in the cortex and one in the hippocampus. This animal had sensorimotor deficits (beam and placing) very close to the reported means, but somewhat surprisingly, the water maze performance was the worst of 8 animals tested (latency to target, 32.97 seconds; length to target, 1452.84 cm).
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Discussion |
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The data from this study demonstrate that photochemically induced nonocclusive CCAT leads to mild neurobehavioral deficits that normalize over a 14-day period. Sensorimotor deficits were seen in all thrombosed rats; in all those tested acutely, cognitive deficits were present. While the most robust neurobehavioral changes were associated with the thrombosed hemisphere, significant abnormalities were also detected within the contralateral hemisphere. The description of the neurobehavioral correlates of CCAT is important because a major goal of stroke therapy is to prevent the neurological consequences of stroke and to restore brain function after injury. Our analysis indicates that a battery of diverse behavioral tasks is useful to fully characterize the functional status of the rat after CCAT. This model of thromboembolic stroke should be advantageous in the study of injury mechanisms underlying neurobehavioral impairments and in the evaluation of pharmacological therapeutic interventions.
Previous studies of photochemically induced thrombosis have demonstrated that CCAT leads to subsequent platelet embolization to the brain.7 10 24 Ultrastructural studies have detected platelet emboli within pial and parenchymal arterioles immediately after CCAT,9 25 while autoradiographic images of indium-labeled platelets have demonstrated widespread platelet accumulation throughout the thrombosed hemisphere as early as 15 minutes after thrombosis.10 24 Thus, occlusive platelet emboli originating at the site of the injured carotid artery have been implicated in the histopathological changes associated with this thrombotic model. In this regard, regional cerebral blood flow studies using [14C]iodoantipyrine have demonstrated multiple ischemic foci 30 minutes after CCAT.8 Thus, some of the neurobehavioral consequences of CCAT reported in the present study would be expected to have resulted from structural damage induced by occlusive platelet emboli.
In the present study left (contralateral) elicited forelimb placing deficits were observed in all thrombosed rats. Histopathological assessment of these rats showed that 10 of the 16 thrombosed rats demonstrated damage within the right cortical forelimb region. In a previous indium-labeled platelet study, CCAT was shown to lead to platelet accumulation commonly within the vascular boundary zones between the anterior and middle cerebral artery territories.10 Importantly, this boundary zone partially overlaps the deep layers of the cortical forelimb area in the rat.26 It has been demonstrated that these deep cortical layers, particularly layer V, are critical in forelimb placing reactions.14 It is also known from clinical studies that boundary zone (ie, watershed) areas are a common site of cerebral infarction.27 28 Thus, although this thromboembolic model results in a shower of platelet emboli, the cortical forelimb region appears to be selectively capable of sequestering emboli and thus is vulnerable to CCAT injury.
Recent experimental studies, including autoradiographic studies after photochemically induced CCAT, have shown that platelet accumulation is largely transient and not necessarily associated with overt histopathological damage.10 29 For example, in baboons, evidence for transient platelet accumulation after the intracarotid infusion of 111In-labeled platelets has been reported.29 Of particular interest to the present discussion was the finding that focal neurological function was temporarily impaired after the intracarotid infusion of labeled platelets, as shown by the ipsilateral loss of somatosensory evoked potentials.29 However, neuropathological examination of these animals failed to show detectable thrombotic occlusion or parenchymal damage. In clinical studies in which transcranial Doppler techniques are used to assess possible vascular perturbations during cardiopulmonary bypass surgery or in patients at risk for stroke, a high incidence of embolization has been reported.30 31 32 33 34 Interestingly, these patients can present with neurological symptoms that are not associated with obvious structural damage.
While histopathological correlates to the forelimb deficits were seen in the present study, similar structural-functional relationships were not seen in regard to other neurobehavioral tasks. For example, although cognitive dysfunction was seen to varying degrees in all thrombosed rats tested at 2 days after CCAT, overt hippocampal injury was documented in only 4 of 16 rats. This apparent lack of a hippocampal histopathological correlate to abnormal water maze performance has previously been shown in various models of cerebral ischemia and head trauma.35 36 For example, after fluid percussion brain injury in rats, which results in prolonged cognitive dysfunction, histopathological damage to the hippocampus is not necessary for the presence of water maze deficits.35
Although water maze performance is classically related to the hippocampus, other structures, including the cortex and striatum, may also be involved.37 38 It is therefore possible that damage to other brain regions or white matter tracts may have participated in the cognitive disturbances seen after CCAT. Lesions encompassing mainly the frontal and parietal cortices have been shown to produce a deficit in a water maze task.37 38 Interruption of the cortico-hippocampal circuitry due to local changes in blood flow39 and overexcitation of these inputs with concomitant excitatory neurotransmitter release35 have also been associated with water maze performance deficits. Regardless of the exact mechanisms underlying this dysfunction, the present data indicate that CCAT leads to learning and retention deficits in the rat that have clinical correlates.3
Acute microvascular abnormalities induced by platelet embolization or by the release of blood-borne factors from the carotid thrombus might also have participated in some of the behavioral deficits described in this study. For example, widespread blood-brain barrier dysfunction and elevated plasma serotonin levels are early consequences of CCAT.9 12 13 Since an intact blood-brain barrier is necessary for normal neuronal function,40 transient failure of the blood-brain barrier might allow blood-borne neuroactive substances to gain access to extravascular pools and thereby affect neuronal function. In addition, there also exists the possibility that CCAT induced sublethal neuronal injury that was not detectable by light microscopic analysis. Ultrastructural studies will therefore be necessary to determine whether fine structural abnormalities within selective neuronal populations correlate with any of the behavioral consequences of CCAT.
While the majority of neurobehavioral deficits were associated with the right thrombosed hemisphere, ipsilateral forelimb placing deficits were also detected in 50% of the thrombosed rats. With the presence of significant group differences between thrombosed and control rats, this indicates that unilateral carotid thrombosis can influence the function of the contralateral hemisphere. Interestingly, in a previous autoradiographic study a low frequency of indium-labeled platelets was identified in the contralateral cerebral cortex 15 minutes after CCAT and was commonly seen within the boundary zone of the anterior and middle cerebral artery territories.10 In the present study small infarcts within the contralateral cortex were identified in only 3 of 16 rats. Thus, platelet embolization to the contralateral hemisphere may be partly responsible for the ipsilateral forelimb placing deficits seen in our study.
By 14 days after CCAT, the neurobehavioral abnormalities had disappeared. The transient nature of these abnormalities is consistent with the observation that in a rat model of microsphere-induced embolic stroke, sensorimotor abnormalities also were significantly attenuated by 14 days.41 The observation of reversible behavioral abnormalities after brain injury is common to many rodent models.36 42 43 44 For example, after middle cerebral artery occlusion in normotensive rats, neurobehavioral deficits including reflex and sensorimotor function deficits recover to preoperative levels by day 30 after ischemia in rats with large cortical infarcts.36 Significant recovery occurs in most stroke patients during the initial months after injury.45 46 Potential recovery mechanisms after brain injury include plasticity and circuit reorganization as well as the normalization of function in remote regions not structurally damaged (ie, diaschisis).47 Even though it appears that CCAT does not truly duplicate the rapid recovery of neurological deficits by which transient ischemic attack is characterized, CCAT may ultimately represent a clinically relevant model of mild thromboembolic stroke that can be used to investigate this important research area.
To our knowledge, a thromboembolic stroke model has not previously been described with respect to functional outcome with a variety of tasks such as we have employed. As a result of the variable nature of such injury models, it is generally assumed that inconsistent morphology and unpredictable dysfunction will occur. Although the patterns of embolic infarction after CCAT were indeed diffuse and varied considerably across animals, we were able to demonstrate a consistent series of deficits that normalized within 2 weeks. While in some animals neurobehavioral deficits were correlated with structural abnormalities, this was not necessarily true in all cases. With the combination of neurobehavioral and histopathological outcome measures, CCAT may be valuable in the screening of therapeutic strategies relating to the treatment of thromboembolic stroke.
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Acknowledgments |
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This study was supported by US Public Health Service grants NS-27127, NS-23244, and NS-05820. The authors wish to thank Susan Kraydieh and Marcilia Halley for expert technical assistance. Nancy Alexis is a member of the Neuroscience Program at the University of Miami.
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Footnotes |
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Reprint requests to Nancy E. Alexis, MS, Department of Neurology (D4-5), University of Miami School of Medicine, PO Box 016960, Miami, FL 33101.
Received May 4, 1995; revision received August 17, 1995; accepted September 13, 1995.
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