JOURNAL OF NEUROTRAUMA Volume 9, Number 1, 1992 Mary Ann Liebert, Inc., Publishers
Cognitive Deficits Following Traumatic Brain Injury Produced by Controlled Cortical Impact ROBERT J. AMRITA K.
HAMM,1 C. EDWARD DIXON,2 DAPHNE M. GBADEBO,1 SINGHA,1 LARRY W. JENKINS,3 BRUCE G. LYETH,3 and RONALD L.
HAYES2
ABSTRACT Traumatic brain injury produces significant cognitive deficits in humans. This experiment used a controlled cortical impact model of experimental brain injury to examine the effects of brain injury on spatial learning and memory using the Morris water maze task. Rats (n 8) were injured at a moderate level of cortical impact injury (6 m/sec, 1.5-2.0 mm deformation). Eight additional rats served as a sham-injured control group. Morris water maze performance was assessed on days 11-15 and 30-34 following injury. Results revealed that brain-injured rats exhibited significant deficits (p < 0.05) in maze performance at both testing intervals. Since the Morris water maze task is particularly sensitive to hippocampal dysfunction, the results of the present experiment support the hypothesis that the hippocampus is preferentially vulnerable to damage following traumatic brain injury. These results demonstrate that controlled cortical impact brain injury produces enduring cognitive deficits analogous to those observed after human brain injury. =
INTRODUCTION
Cognitive example,
and prominent sequela of traumatic brain
injury (TBI) (Binder, 1986; al., 1981, 1982, Stuss et al., 1985). For brain-injured patients have difficulty remembering lists of numbers (Brooks, 1972), performing matching-to-sample tasks (McLean et al., 1983), and delayed recall (Stuss et al., 1985). Although cognitive function is impaired following TBI in humans, most animal model studies of TBI have exclusively used measurements of simple reflexes and motor function as their outcome variables. However, a few studies have impairment is A common
Gronwall and
Wrightson, 1974; Levin et al., 1982; Rimel
et
examined memory function following TBI. In a previous study, Lyeth et al. (1990) evaluated memory deficits following fluid percussion brain injury with the radial-arm maze. They found that working memory was impaired for up to 15 days after injury. In the reference memory aspect of the task, no differences were observed between the injured and uninjured animals. Working memory has also been assessed for 7 days after
Departments of 'Psychology, 2Rehabilitative Medicine, College of Virginia, Richmond. Virginia.
and
Medical
11
^Neurosurgery, Virginia Commonwealth University and
HAMM ET AL.
fluid
percussion TBI with a delayed-matching-to-sample procedure (Napper et al., 1989). In this study working memory deficits were observed at the longer delay intervals (>20 sec) for 4 days following TBI. These data suggest that animals exhibit transitory deficits in working memory following fluid percussion TBI. Several observations indicate that those brain regions critical for learning and memory, the hippocampus and cortex, show enhanced susceptibility to damage from traumatic brain injury. Changes in ACh and NMDA receptor binding have been observed following fluid percussion TBI in the dentate gyms and CA1 regions of the hippocampus and in the adjacent neocortex (Oleniak et al., 1988; Miller et al., 1990). Fluid percussion TBI also suppresses long-term potentiation (LTP, which is thought to be an electrophysiological correlate of memory formation) of the hippocampal Schaeffer collateral/CAl system (Miyazaki et al., in press). Finally, fluid percussion TBI results in selective, enhanced vulnerability of hippocampal CAÍ neurons to a subsequent post-TBI ischémie event (Jenkins et al., 1988, 1989). Our laboratory has recently developed a new rodent model of TBI. This model uses a modification of a cortical impact device developed at the General Motors Research Laboratories (Lighthall, 1988). The purpose of the present experiment is to assess cognitive function in rats following controlled cortical impact brain injury using the Morris water maze procedure (Morris, 1981). Morris water maze performance is sensitive to lesions of the hippocampus and cortex (e.g., Morris et al., 1982, 1986a) and to manipulations of cholinergic and glutamatergic function (e.g., Sutherland et al., 1982; Morris et al., 1986b). Thus, the Morris water maze task is sensitive to damage in anatomical regions that are particularly vulnerable to fluid percussion TBI (hippocampus and cortex) and to changes in neurotransmitter systems (ACh and glutamate) that are altered following fluid percussion TBI. Whether the cortical impact model of TBI produces similar changes is not presently known. However, it seems reasonable to assume that similar changes would occur following cortical impact TBI. If this assumption is correct, the Morris water maze, with its sensitivity to brain regions and neurotransmitters that are altered following TBI, would be ideally suited for detecting the effects of TBI. Impairment of maze performance in brain-injured animals would make possible subsequent studies examining the possible functional significance of previously described cortical and hippocampal changes following TBI. METHODS Animals Sixteen male Sprague-Dawley rats* (Hilltop Lab Animals, Inc., Scottsdale, PA) weighing between 300 and 350 g were used. Animals were housed in individual cages with food and water continually available. The animal colony was maintained at a temperature of 20-22°C with a 0600(light)/1800(dark) cycle.
Apparati Cortical impact device. The controlled cortical injury device used by Lighthall (1988) was modified for with rodents. Our injury device consists of a small- (1.975 cm) bore, double-acting, stroke-constrained, pneumatic cylinder with a 5.0 cm stroke. The cylinder is rigidly mounted in a vertical position on a crossbar, which can be precisely adjusted in the vertical axis. The lower rod end has an impactor tip attached (i.e., that part of the shaft that comes into contact with the exposed dura mater). The impactor head was constructed from a 10 mm diameter aluminum rod with a spherical tip. The upper rod end was attached to the transducer core of a linear velocity displacement transducer (LVDT, Shaevitz Model 500 HR). The velocity of the impactor shaft can be controlled by variation of the gas pressure used to power the impactor. Impact velocity is directly use
*The rationale and methods for the use of an animal model in this experiment were reviewed by our Institutional Animal Care and Use Committee. This research follows procedures outlined in the Guide for the Care and Use of Laboratory Animals, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health NIH Publication 85-23, Revised 1985, and Public Health Service Policy on Humane Care and Use ofLaboratory Animals by Awardee Institutions, NIH Guide for Grants and Contracts, Vol. 14, No. 8, June 25, 1985.
12
COGNITIVE DEFICITS FOLLOWING CORTICAL IMPACT measured by the LVDT that produces an analog signal recorded by a PC-based data acquisition system (R. C. Electronics) for analysis of time/displacement parameters of the impactor. Morris water maze. The water maze uses a 180 cm diameter and 60 cm high metal pool. The pool was painted white and filled with water to a depth of 28 cm. The water was made opaque by the addition of approximately 500 ml of nontoxic white latex paint. A platform 10 cm in diameter and 26 cm high (i.e., 2 cm below the water's surface) was used as the hidden goal platform. The pool was located in a 2.5 x 2.5 m room with numerous extra-maze cues (e.g., windows, pipes, bookcase) that remained constant throughout the
experiment.
The animal's movements within the Morris water maze were recorded and analyzed with a video camera, VP112 video scanning unit, and tracking software (HVS Image). This tracking equipment allowed the measurement of latency to reach the hidden goal platform; path length; percentage of time spent in each of 3 equally spaced concentric rings (the first ring. A, included the 60 cm diameter circle in the center of the pool; the second ring, B, included the area from 30 to 60 cm from the center of the pool; the outside ring, C, included the area from 60 cm from the center of the pool to its outside wall; see Fig. 1).
Procedures Production of brain injury. All rats were initially anesthetized with 4% isoflurane with a 2:1 N20/02 mixture in a vented anesthesia chamber. Following endotracheal intubation, rats were mechanically ventilated with a 2% isoflurane and a 2:1 N20/02 mixture. The rats were mounted in the injury device stereotaxic frame in a supine position secured by ear bars and incisor bar. The animal's head was held in a horizontal plane with respect to the interaural line. A midline incision was made, the soft tissues reflected, and a 10 mm diameter craniectomy was made centrally between bregma and lambda. Anesthetic gases were discontinued immediately before injury. Injured rats (n 8) received a cortical impact at 6 m/sec, 1.5-2.0 mm deformation. Sham =
A schematic representation of the Morris water maze. Animals begin a trial from one of the four cardinal labeled S. The Gs represent the four possible hidden goal locations. The pool was divided into three imaginary positions concentric rings (rings A-C) to quantify swimming location of the rats.
FIG. 1.
13
HAMM ET AL.
(n 8) underwent identical surgical procedures but were not injured. Core body temperature was monitored continuously by a rectal thermistor probe and maintained at 37-38°C. After injury, the scalp was sutured closed, and the animal extubated. Morris water maze. Experimenters conducting this phase of the procedure were blind to animal's group membership. To avoid the motor deficits typically observed for several days (typically 2-4 days at the injury level used in this study; Dixon et al., submitted) following brain injury, testing started in day 11 after injury. Rats were given 4 trials per day for 5 consecutive days (days 11-15). For each daily block of four trials, rats were placed in the pool by hand facing the wall. Rats started a trial once from each of the four possible start locations (north, east, south, west; see Fig. 1). The order of starting locations was randomized. The goal platform was positioned 45 cm from the outside wall and was placed in either the northeast, southeast, southwest, or northwest quadrant of the maze (see Fig. 1). The location of the platform was held constant for each animal, but varied between animals, with two animals from each group having the same goal location. Rats were given a maximum of 120 sec to find the hidden platform. If the rat failed to find the platform after 120 sec, it was placed on the platform by the experimenter. All rats were allowed to remain on the platform for 30 sec before being placed in a heated incubator between trials. There was a 4 min intertrial interval. On days 30-34, rats were again tested in the water maze. On these trials the goal platform was moved to a different location from that used on days 11-15. All other aspects of the procedure remained the same. rats
=
Histopathologic Analysis Following postinjury survival of 35 days, animals were anesthetized with sodium pentobarbital (100 mg/kg) and transcardially perfused with 10% buffered formalin. Subsequent to perfusion fixation, each brain was removed and examined for evidence of gross intraparenchymal bleeds and contusions. Brains subjected to light microscopic examination were blocked, dehydrated in ascending concentrations of alcohol and xylene, and infiltrated with paraffin. Each brain was sectioned at 8 p,m and stained with hematoxylin and eosin. Slides from each brain were examined qualitatively with a Nikon Optiphot light microscope by a trained evaluator who
was unaware
of the animal's treatment.
RESULTS Maze
Performance
To ensure that the assessment of cognitive function was not confounded by motor deficits following cortical impact, beam-balance and beam-walking tasks (Dixon et al., 1987) were assessed following injury. By the third day after injury, there were no significant performance differences between the injured and sham animals on these two tasks. In addition, the swimming speed of the rats was measured on the first day of Morris water maze testing. The distance that the rats swam on the 4 trials that comprised day 11 was divided by the total time they spent swimming. The mean swim speed (± SEM) was 22.0 (1.8) for the sham-injured group and 19.3 (1.4) for the injured group. These means were not significantly different. Thus, the injured and sham groups did not differ in their ability to perform the motor requirements of the task used to assess cognitive performance. The mean latency of the four daily trial was calculated for each animal. Figure 2 presents the mean latency of the animals in the injured and sham groups to find the hidden platform on days 11-15 after injury. These data were analyzed by a 2 (group) x 5 (days) split-plot analysis of variance followed by a Duncan Multiple Range test to examine group differences on specific days. These tests revealed that the injured animals took significantly (p < 0.05) longer than sham-injured animals to find the hidden platform on each day. Figure 3 illustrates the search patterns of two representative animals from the two groups on trial 4 of the first day of maze testing (day 11). The percentage of time the animals swam in the outside ring (ring C, see Fig. 1 ) on days 11-15 was analyzed by a 2 (group) x 5 (day) analysis of variance (ANOVA; see Fig. 4). Duncan test indicated that the injured group spent significantly (p < 0.05) more time in the outside ring than the sham-injured group on days 13-15. Analysis of data on days 30-34 revealed that the injured animals took significantly (p < 0.05) longer than sham-injured animals to find the hidden platform on each day, except day 33 (See Fig. 2). The search patterns 14
COGNITIVE DEFICITS FOLLOWING CORTICAL IMPACT
1201
- O*" •
Injured Sham
100 o»
80 H
«
60
es
40H
o
O 20
0
i-r-
11
12
13
14
15
30
3 1
32
33
34
Days FIG. 2.
The
latency of groups to find the goal during the two testing times: days
11-15 and 30-34 after
injury.
are displayed in Figure 5 for a representative animal from each group on trial 25 (the fourth trial on day 32). An ANOVA of the time spent swimming in ring C on days 30-34 indicated that the sham-injured animals swam less in ring C than the injured animals on days 30-34 (see Fig. 5).
Histopathologic Findings With the level of impact employed in this study, there was no gross light microscopic evidence of tissue destruction or underlying histopathologic change either at or remote from the site of impact (see Fig. 6). Furthermore, no overt light microscopic evidence of neuronal loss was found in any brain region examined. However, given the long delay between the injury and the examination of the tissue, coupled with the superficial examination conducted, immediate and more subtle neuropathologic changes could not be discounted.
Maze Search Patterns Sham
FIG. 3.
The
on
Trial 4
Injured
Injury
swimming tracks on the fourth trial (the last trial on day 1 ) of a representative animal from each group. 15
HAMM ET AL. 100
----O-•
90
Injured Sham
80 70
i.kg, ..-I.
60 50 C
40 30 20 10
0
i
11
12
13
14
15
30
31
32
33
34
Days FIG. 4.
The percentage of time the groups spent
swimming in the outside ring (ring C, see Fig. 1) of the maze over test
days. DISCUSSION A number of important findings result from this study. Brain injury produced by controlled cortical impact caused a significant impairment of spatial learning performance in rats that persisted for more than 1 month after injury. These cognitive deficits occurred in the absence of structural damage as assessed by quantitative light microscopic examination. The duration of cognitive dysfunction at this injury level far exceeded any detectable impairments of motor dysfunction. Detailed analysis of maze performance may provide some insight into the anatomical correlates mediating cognitive dysfunction in this model. Controlled cortical impact produced an impairment in maze performance that was still present 1 month after injury. In the rat's life span, a 1 month impairment of cognitive function would be approximately equivalent
Maze Search Pattern
Sham
FIG. 5
A
Injury
on
Trial 25
Injured
sample swimming pattern from an injured and a sham-injured animal on trial 25 (the first trial of day 31). 16
COGNITIVE DEFICITS FOLLOWING CORTICAL IMPACT
FIG. 6.
Coronal section from a representative rat injured with a central cortical impact. Note the normal structural in this middorsal hippocampal section. (Hematoxylin-eosin stain, 20 x magnification.)
integrity and neuronal densities
2 year deficit in a human's life span. The enduring cognitive impairment was confirmed by both differences in latencies to find the hidden platform and in search patterns. The typical search path of a sham-injured animal on days 11-15 (See Fig. 3) shows that the animal swam in the central portion of the maze to find the hidden platform. In contrast, the typical search pattern of an injured animal demonstrated that the animal spent most of its time swimming on the perimeter of the maze, suggesting that the spatial location of the hidden platform animal had not been learned. On days 30-34 (see Fig. 5), the injured animal decreased its swim time around the perimeter of the maze and displayed a random search pattern in the central portion of the maze. Decreasing the time spent swimming in the outside portion of the maze will produce shorter goal latencies than swimming around the perimeter of the maze. However, the injured animal still did not demonstrate that the specific spatial location of the hidden platform in the maze had been learned. An important interpretive consideration in the assessment of animal cognition is the possibility that motor impairment may confound the assessment. In the present experiment, beam balance and beam walking motor deficits associated with cortical impact injury had recovered to preinjury performance levels 8 days before the beginning of maze testing. In addition, the analysis of swim speed revealed no significant difference between the injured and control groups, indicating that the injured and control groups did not differ in their ability to perform the motor requirements of the Morris water maze test. Therefore, these data indicate that motor performance was not a confounding variable in the assessment of cognitive function of the injured rats in the to
a
present study.
The magnitude of cortical impact used in the present study produced cognitive impairment without evidence of gross neuronal destruction. Enduring cognitive deficits have also been observed in the absence of tissue destruction following central fluid percussion injury (Lyeth et al., 1990). Although tissue destruction caused by lesion (Morris et al., 1982) or ischemia (Volpe et al., 1984) is sufficient to produce cognitive impairment, the present and previous findings (Lyeth et al., 1990) in TBI suggest that tissue destruction is not a necessary condition for cognitive impairment. Manipulations of brain cholinergic and glutamatergic function (Sutherland et al., 1982; Morris et al., 1986b) that do not produce neuronal cell death also disrupt Morris water maze performance. Other models of TBI (i.e., fluid percussion) have demonstrated that activation of cholinergic (Lyeth et al., 1988; Robinson et al., 1990) and glutamatergic (Faden et al., 1989; Hayes et al., 1988) systems mediate components of the functional deficits associated with TBI. Thus, the impairment of maze performance observed in the present experiment may be the result of a subtle neurochemical disruption of the brain's normal neurotransmission rather than gross tissue destruction. 17
HAMM ET AL. From the results of the present study it is impossible to determine which areas of the injured brain are responsible for the maze deficits observed following TBI. However, further analysis in light of recent research indicating regionally specific mediation of cognitive function allows one to speculate on possible regional dysfunction following injury. Recent research has shown that spatial navigation ability and specific/episodic memory function are hippocampally mediated while the prefrontal cortex appears to mediate executive functions that involve learning rules necessary to solve a problem (Winocur and Moscovitch, 1990). If one assumes that the ability to navigate to the specific location of the hidden goal platform is governed by the hippocampus and that the learning of nonspatial strategies to help locate the platform is primarily mediated by the prefrontal cortex, the present experiment suggests a differential contribution of the hippocampus and prefrontal cortex to maze performance deficits observed after brain injury. Both the goal latency and swim pattern data indicate that the injured animals do improve their performance over days following TBI. The search pattern data suggest that the improved performance of the injured animals is primarily the result of a change in swimming strategy (i.e., swimming in the center of the maze rather than around the perimeter). While this change in swimming strategy reduces the latency to reach the hidden platform, the search patterns of the injured rats indicate that they do not know the specific spatial location of the goal platform. The performance of the injured rats can be characterized as learning a nonspatial strategy of "do not swim next to the wall." If this analysis is correct, it suggests that the injured animals are capable of learning a task-related rule that requires prefrontal cortex integrity. Injured animals did not learn the spatial navigation strategies that require hippocampal function to find the hidden platform quickly. Thus, the behavioral analysis implies that the primary anatomical source of the impairment of maze performance is the hippocampus rather than the prefrontal cortex. Additional research will be required to confirm our speculations regarding the anatomical substrates and neurotransmitters involved in the impairment of cognitive function following traumatic brain injury. However, this experiment has demonstrated enduring deficits in cognitive function of rats following brain injury similar to the long-lasting cognitive impairment observed in cases of human head injury. Thus, the findings of this study do suggest the potential of the cortical impact model for examining therapeutic treatments and mechanisms mediating the chronic cognitive dysfunction exhibited after traumatic brain injury.
ACKNOWLEDGMENTS The authors wish to express their appreciation to Renee Charles worth, Robert Panter, and Diane Taylor for their excellent technical assistance. The research was supported in part by grants NS12857 (RJH), CCR 303547 (CED), and NS 21458 (RLH).
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COGNITIVE DEFICITS FOLLOWING CORTICAL IMPACT JENKINS, L.W., LYETH, B.C. LEWELT. W., MOSZYNSKI, K., DEWITT, D.S., BALSTER. R.L., MILLER, L.P., CLIFTON, G.L., YOUNG, H.F., and HAYES, R.L. (1988). Combined pre-trauma scopolamine and
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LYETH, B.C. DIXON, CE.. JENKINS, L.W., HAMM, R.J., ALBERICO, A., YOUNG. H.F., STONNINGTON, H.H., and HAYES, R.L. (1988). Effects of scopolamine treatment on long-term behavioral deficits following concussive head
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and rats
Address reprint requests to: Dr. Robert J. Hamm Commonwealth Virginia University Division of Neurosurgery 1101 East Marshall Street Room B2002, Sanger Hall Richmond, VA 23298
20
Cognitive Deficits Following Traumatic Brain Injury Produced by Controlled Cortical Impact ROBERT J. AMRITA K.
HAMM,1 C. EDWARD DIXON,2 DAPHNE M. GBADEBO,1 SINGHA,1 LARRY W. JENKINS,3 BRUCE G. LYETH,3 and RONALD L.
HAYES2
ABSTRACT Traumatic brain injury produces significant cognitive deficits in humans. This experiment used a controlled cortical impact model of experimental brain injury to examine the effects of brain injury on spatial learning and memory using the Morris water maze task. Rats (n 8) were injured at a moderate level of cortical impact injury (6 m/sec, 1.5-2.0 mm deformation). Eight additional rats served as a sham-injured control group. Morris water maze performance was assessed on days 11-15 and 30-34 following injury. Results revealed that brain-injured rats exhibited significant deficits (p < 0.05) in maze performance at both testing intervals. Since the Morris water maze task is particularly sensitive to hippocampal dysfunction, the results of the present experiment support the hypothesis that the hippocampus is preferentially vulnerable to damage following traumatic brain injury. These results demonstrate that controlled cortical impact brain injury produces enduring cognitive deficits analogous to those observed after human brain injury. =
INTRODUCTION
Cognitive example,
and prominent sequela of traumatic brain
injury (TBI) (Binder, 1986; al., 1981, 1982, Stuss et al., 1985). For brain-injured patients have difficulty remembering lists of numbers (Brooks, 1972), performing matching-to-sample tasks (McLean et al., 1983), and delayed recall (Stuss et al., 1985). Although cognitive function is impaired following TBI in humans, most animal model studies of TBI have exclusively used measurements of simple reflexes and motor function as their outcome variables. However, a few studies have impairment is A common
Gronwall and
Wrightson, 1974; Levin et al., 1982; Rimel
et
examined memory function following TBI. In a previous study, Lyeth et al. (1990) evaluated memory deficits following fluid percussion brain injury with the radial-arm maze. They found that working memory was impaired for up to 15 days after injury. In the reference memory aspect of the task, no differences were observed between the injured and uninjured animals. Working memory has also been assessed for 7 days after
Departments of 'Psychology, 2Rehabilitative Medicine, College of Virginia, Richmond. Virginia.
and
Medical
11
^Neurosurgery, Virginia Commonwealth University and
HAMM ET AL.
fluid
percussion TBI with a delayed-matching-to-sample procedure (Napper et al., 1989). In this study working memory deficits were observed at the longer delay intervals (>20 sec) for 4 days following TBI. These data suggest that animals exhibit transitory deficits in working memory following fluid percussion TBI. Several observations indicate that those brain regions critical for learning and memory, the hippocampus and cortex, show enhanced susceptibility to damage from traumatic brain injury. Changes in ACh and NMDA receptor binding have been observed following fluid percussion TBI in the dentate gyms and CA1 regions of the hippocampus and in the adjacent neocortex (Oleniak et al., 1988; Miller et al., 1990). Fluid percussion TBI also suppresses long-term potentiation (LTP, which is thought to be an electrophysiological correlate of memory formation) of the hippocampal Schaeffer collateral/CAl system (Miyazaki et al., in press). Finally, fluid percussion TBI results in selective, enhanced vulnerability of hippocampal CAÍ neurons to a subsequent post-TBI ischémie event (Jenkins et al., 1988, 1989). Our laboratory has recently developed a new rodent model of TBI. This model uses a modification of a cortical impact device developed at the General Motors Research Laboratories (Lighthall, 1988). The purpose of the present experiment is to assess cognitive function in rats following controlled cortical impact brain injury using the Morris water maze procedure (Morris, 1981). Morris water maze performance is sensitive to lesions of the hippocampus and cortex (e.g., Morris et al., 1982, 1986a) and to manipulations of cholinergic and glutamatergic function (e.g., Sutherland et al., 1982; Morris et al., 1986b). Thus, the Morris water maze task is sensitive to damage in anatomical regions that are particularly vulnerable to fluid percussion TBI (hippocampus and cortex) and to changes in neurotransmitter systems (ACh and glutamate) that are altered following fluid percussion TBI. Whether the cortical impact model of TBI produces similar changes is not presently known. However, it seems reasonable to assume that similar changes would occur following cortical impact TBI. If this assumption is correct, the Morris water maze, with its sensitivity to brain regions and neurotransmitters that are altered following TBI, would be ideally suited for detecting the effects of TBI. Impairment of maze performance in brain-injured animals would make possible subsequent studies examining the possible functional significance of previously described cortical and hippocampal changes following TBI. METHODS Animals Sixteen male Sprague-Dawley rats* (Hilltop Lab Animals, Inc., Scottsdale, PA) weighing between 300 and 350 g were used. Animals were housed in individual cages with food and water continually available. The animal colony was maintained at a temperature of 20-22°C with a 0600(light)/1800(dark) cycle.
Apparati Cortical impact device. The controlled cortical injury device used by Lighthall (1988) was modified for with rodents. Our injury device consists of a small- (1.975 cm) bore, double-acting, stroke-constrained, pneumatic cylinder with a 5.0 cm stroke. The cylinder is rigidly mounted in a vertical position on a crossbar, which can be precisely adjusted in the vertical axis. The lower rod end has an impactor tip attached (i.e., that part of the shaft that comes into contact with the exposed dura mater). The impactor head was constructed from a 10 mm diameter aluminum rod with a spherical tip. The upper rod end was attached to the transducer core of a linear velocity displacement transducer (LVDT, Shaevitz Model 500 HR). The velocity of the impactor shaft can be controlled by variation of the gas pressure used to power the impactor. Impact velocity is directly use
*The rationale and methods for the use of an animal model in this experiment were reviewed by our Institutional Animal Care and Use Committee. This research follows procedures outlined in the Guide for the Care and Use of Laboratory Animals, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health NIH Publication 85-23, Revised 1985, and Public Health Service Policy on Humane Care and Use ofLaboratory Animals by Awardee Institutions, NIH Guide for Grants and Contracts, Vol. 14, No. 8, June 25, 1985.
12
COGNITIVE DEFICITS FOLLOWING CORTICAL IMPACT measured by the LVDT that produces an analog signal recorded by a PC-based data acquisition system (R. C. Electronics) for analysis of time/displacement parameters of the impactor. Morris water maze. The water maze uses a 180 cm diameter and 60 cm high metal pool. The pool was painted white and filled with water to a depth of 28 cm. The water was made opaque by the addition of approximately 500 ml of nontoxic white latex paint. A platform 10 cm in diameter and 26 cm high (i.e., 2 cm below the water's surface) was used as the hidden goal platform. The pool was located in a 2.5 x 2.5 m room with numerous extra-maze cues (e.g., windows, pipes, bookcase) that remained constant throughout the
experiment.
The animal's movements within the Morris water maze were recorded and analyzed with a video camera, VP112 video scanning unit, and tracking software (HVS Image). This tracking equipment allowed the measurement of latency to reach the hidden goal platform; path length; percentage of time spent in each of 3 equally spaced concentric rings (the first ring. A, included the 60 cm diameter circle in the center of the pool; the second ring, B, included the area from 30 to 60 cm from the center of the pool; the outside ring, C, included the area from 60 cm from the center of the pool to its outside wall; see Fig. 1).
Procedures Production of brain injury. All rats were initially anesthetized with 4% isoflurane with a 2:1 N20/02 mixture in a vented anesthesia chamber. Following endotracheal intubation, rats were mechanically ventilated with a 2% isoflurane and a 2:1 N20/02 mixture. The rats were mounted in the injury device stereotaxic frame in a supine position secured by ear bars and incisor bar. The animal's head was held in a horizontal plane with respect to the interaural line. A midline incision was made, the soft tissues reflected, and a 10 mm diameter craniectomy was made centrally between bregma and lambda. Anesthetic gases were discontinued immediately before injury. Injured rats (n 8) received a cortical impact at 6 m/sec, 1.5-2.0 mm deformation. Sham =
A schematic representation of the Morris water maze. Animals begin a trial from one of the four cardinal labeled S. The Gs represent the four possible hidden goal locations. The pool was divided into three imaginary positions concentric rings (rings A-C) to quantify swimming location of the rats.
FIG. 1.
13
HAMM ET AL.
(n 8) underwent identical surgical procedures but were not injured. Core body temperature was monitored continuously by a rectal thermistor probe and maintained at 37-38°C. After injury, the scalp was sutured closed, and the animal extubated. Morris water maze. Experimenters conducting this phase of the procedure were blind to animal's group membership. To avoid the motor deficits typically observed for several days (typically 2-4 days at the injury level used in this study; Dixon et al., submitted) following brain injury, testing started in day 11 after injury. Rats were given 4 trials per day for 5 consecutive days (days 11-15). For each daily block of four trials, rats were placed in the pool by hand facing the wall. Rats started a trial once from each of the four possible start locations (north, east, south, west; see Fig. 1). The order of starting locations was randomized. The goal platform was positioned 45 cm from the outside wall and was placed in either the northeast, southeast, southwest, or northwest quadrant of the maze (see Fig. 1). The location of the platform was held constant for each animal, but varied between animals, with two animals from each group having the same goal location. Rats were given a maximum of 120 sec to find the hidden platform. If the rat failed to find the platform after 120 sec, it was placed on the platform by the experimenter. All rats were allowed to remain on the platform for 30 sec before being placed in a heated incubator between trials. There was a 4 min intertrial interval. On days 30-34, rats were again tested in the water maze. On these trials the goal platform was moved to a different location from that used on days 11-15. All other aspects of the procedure remained the same. rats
=
Histopathologic Analysis Following postinjury survival of 35 days, animals were anesthetized with sodium pentobarbital (100 mg/kg) and transcardially perfused with 10% buffered formalin. Subsequent to perfusion fixation, each brain was removed and examined for evidence of gross intraparenchymal bleeds and contusions. Brains subjected to light microscopic examination were blocked, dehydrated in ascending concentrations of alcohol and xylene, and infiltrated with paraffin. Each brain was sectioned at 8 p,m and stained with hematoxylin and eosin. Slides from each brain were examined qualitatively with a Nikon Optiphot light microscope by a trained evaluator who
was unaware
of the animal's treatment.
RESULTS Maze
Performance
To ensure that the assessment of cognitive function was not confounded by motor deficits following cortical impact, beam-balance and beam-walking tasks (Dixon et al., 1987) were assessed following injury. By the third day after injury, there were no significant performance differences between the injured and sham animals on these two tasks. In addition, the swimming speed of the rats was measured on the first day of Morris water maze testing. The distance that the rats swam on the 4 trials that comprised day 11 was divided by the total time they spent swimming. The mean swim speed (± SEM) was 22.0 (1.8) for the sham-injured group and 19.3 (1.4) for the injured group. These means were not significantly different. Thus, the injured and sham groups did not differ in their ability to perform the motor requirements of the task used to assess cognitive performance. The mean latency of the four daily trial was calculated for each animal. Figure 2 presents the mean latency of the animals in the injured and sham groups to find the hidden platform on days 11-15 after injury. These data were analyzed by a 2 (group) x 5 (days) split-plot analysis of variance followed by a Duncan Multiple Range test to examine group differences on specific days. These tests revealed that the injured animals took significantly (p < 0.05) longer than sham-injured animals to find the hidden platform on each day. Figure 3 illustrates the search patterns of two representative animals from the two groups on trial 4 of the first day of maze testing (day 11). The percentage of time the animals swam in the outside ring (ring C, see Fig. 1 ) on days 11-15 was analyzed by a 2 (group) x 5 (day) analysis of variance (ANOVA; see Fig. 4). Duncan test indicated that the injured group spent significantly (p < 0.05) more time in the outside ring than the sham-injured group on days 13-15. Analysis of data on days 30-34 revealed that the injured animals took significantly (p < 0.05) longer than sham-injured animals to find the hidden platform on each day, except day 33 (See Fig. 2). The search patterns 14
COGNITIVE DEFICITS FOLLOWING CORTICAL IMPACT
1201
- O*" •
Injured Sham
100 o»
80 H
«
60
es
40H
o
O 20
0
i-r-
11
12
13
14
15
30
3 1
32
33
34
Days FIG. 2.
The
latency of groups to find the goal during the two testing times: days
11-15 and 30-34 after
injury.
are displayed in Figure 5 for a representative animal from each group on trial 25 (the fourth trial on day 32). An ANOVA of the time spent swimming in ring C on days 30-34 indicated that the sham-injured animals swam less in ring C than the injured animals on days 30-34 (see Fig. 5).
Histopathologic Findings With the level of impact employed in this study, there was no gross light microscopic evidence of tissue destruction or underlying histopathologic change either at or remote from the site of impact (see Fig. 6). Furthermore, no overt light microscopic evidence of neuronal loss was found in any brain region examined. However, given the long delay between the injury and the examination of the tissue, coupled with the superficial examination conducted, immediate and more subtle neuropathologic changes could not be discounted.
Maze Search Patterns Sham
FIG. 3.
The
on
Trial 4
Injured
Injury
swimming tracks on the fourth trial (the last trial on day 1 ) of a representative animal from each group. 15
HAMM ET AL. 100
----O-•
90
Injured Sham
80 70
i.kg, ..-I.
60 50 C
40 30 20 10
0
i
11
12
13
14
15
30
31
32
33
34
Days FIG. 4.
The percentage of time the groups spent
swimming in the outside ring (ring C, see Fig. 1) of the maze over test
days. DISCUSSION A number of important findings result from this study. Brain injury produced by controlled cortical impact caused a significant impairment of spatial learning performance in rats that persisted for more than 1 month after injury. These cognitive deficits occurred in the absence of structural damage as assessed by quantitative light microscopic examination. The duration of cognitive dysfunction at this injury level far exceeded any detectable impairments of motor dysfunction. Detailed analysis of maze performance may provide some insight into the anatomical correlates mediating cognitive dysfunction in this model. Controlled cortical impact produced an impairment in maze performance that was still present 1 month after injury. In the rat's life span, a 1 month impairment of cognitive function would be approximately equivalent
Maze Search Pattern
Sham
FIG. 5
A
Injury
on
Trial 25
Injured
sample swimming pattern from an injured and a sham-injured animal on trial 25 (the first trial of day 31). 16
COGNITIVE DEFICITS FOLLOWING CORTICAL IMPACT
FIG. 6.
Coronal section from a representative rat injured with a central cortical impact. Note the normal structural in this middorsal hippocampal section. (Hematoxylin-eosin stain, 20 x magnification.)
integrity and neuronal densities
2 year deficit in a human's life span. The enduring cognitive impairment was confirmed by both differences in latencies to find the hidden platform and in search patterns. The typical search path of a sham-injured animal on days 11-15 (See Fig. 3) shows that the animal swam in the central portion of the maze to find the hidden platform. In contrast, the typical search pattern of an injured animal demonstrated that the animal spent most of its time swimming on the perimeter of the maze, suggesting that the spatial location of the hidden platform animal had not been learned. On days 30-34 (see Fig. 5), the injured animal decreased its swim time around the perimeter of the maze and displayed a random search pattern in the central portion of the maze. Decreasing the time spent swimming in the outside portion of the maze will produce shorter goal latencies than swimming around the perimeter of the maze. However, the injured animal still did not demonstrate that the specific spatial location of the hidden platform in the maze had been learned. An important interpretive consideration in the assessment of animal cognition is the possibility that motor impairment may confound the assessment. In the present experiment, beam balance and beam walking motor deficits associated with cortical impact injury had recovered to preinjury performance levels 8 days before the beginning of maze testing. In addition, the analysis of swim speed revealed no significant difference between the injured and control groups, indicating that the injured and control groups did not differ in their ability to perform the motor requirements of the Morris water maze test. Therefore, these data indicate that motor performance was not a confounding variable in the assessment of cognitive function of the injured rats in the to
a
present study.
The magnitude of cortical impact used in the present study produced cognitive impairment without evidence of gross neuronal destruction. Enduring cognitive deficits have also been observed in the absence of tissue destruction following central fluid percussion injury (Lyeth et al., 1990). Although tissue destruction caused by lesion (Morris et al., 1982) or ischemia (Volpe et al., 1984) is sufficient to produce cognitive impairment, the present and previous findings (Lyeth et al., 1990) in TBI suggest that tissue destruction is not a necessary condition for cognitive impairment. Manipulations of brain cholinergic and glutamatergic function (Sutherland et al., 1982; Morris et al., 1986b) that do not produce neuronal cell death also disrupt Morris water maze performance. Other models of TBI (i.e., fluid percussion) have demonstrated that activation of cholinergic (Lyeth et al., 1988; Robinson et al., 1990) and glutamatergic (Faden et al., 1989; Hayes et al., 1988) systems mediate components of the functional deficits associated with TBI. Thus, the impairment of maze performance observed in the present experiment may be the result of a subtle neurochemical disruption of the brain's normal neurotransmission rather than gross tissue destruction. 17
HAMM ET AL. From the results of the present study it is impossible to determine which areas of the injured brain are responsible for the maze deficits observed following TBI. However, further analysis in light of recent research indicating regionally specific mediation of cognitive function allows one to speculate on possible regional dysfunction following injury. Recent research has shown that spatial navigation ability and specific/episodic memory function are hippocampally mediated while the prefrontal cortex appears to mediate executive functions that involve learning rules necessary to solve a problem (Winocur and Moscovitch, 1990). If one assumes that the ability to navigate to the specific location of the hidden goal platform is governed by the hippocampus and that the learning of nonspatial strategies to help locate the platform is primarily mediated by the prefrontal cortex, the present experiment suggests a differential contribution of the hippocampus and prefrontal cortex to maze performance deficits observed after brain injury. Both the goal latency and swim pattern data indicate that the injured animals do improve their performance over days following TBI. The search pattern data suggest that the improved performance of the injured animals is primarily the result of a change in swimming strategy (i.e., swimming in the center of the maze rather than around the perimeter). While this change in swimming strategy reduces the latency to reach the hidden platform, the search patterns of the injured rats indicate that they do not know the specific spatial location of the goal platform. The performance of the injured rats can be characterized as learning a nonspatial strategy of "do not swim next to the wall." If this analysis is correct, it suggests that the injured animals are capable of learning a task-related rule that requires prefrontal cortex integrity. Injured animals did not learn the spatial navigation strategies that require hippocampal function to find the hidden platform quickly. Thus, the behavioral analysis implies that the primary anatomical source of the impairment of maze performance is the hippocampus rather than the prefrontal cortex. Additional research will be required to confirm our speculations regarding the anatomical substrates and neurotransmitters involved in the impairment of cognitive function following traumatic brain injury. However, this experiment has demonstrated enduring deficits in cognitive function of rats following brain injury similar to the long-lasting cognitive impairment observed in cases of human head injury. Thus, the findings of this study do suggest the potential of the cortical impact model for examining therapeutic treatments and mechanisms mediating the chronic cognitive dysfunction exhibited after traumatic brain injury.
ACKNOWLEDGMENTS The authors wish to express their appreciation to Renee Charles worth, Robert Panter, and Diane Taylor for their excellent technical assistance. The research was supported in part by grants NS12857 (RJH), CCR 303547 (CED), and NS 21458 (RLH).
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and rats
Address reprint requests to: Dr. Robert J. Hamm Commonwealth Virginia University Division of Neurosurgery 1101 East Marshall Street Room B2002, Sanger Hall Richmond, VA 23298
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