We compared the predictive value of a series of behavioral tests, either singly or in combination, as a function of injury severity following CCI in mice. These included the classical or standard Morris water maze (sMWM) and reversal Morris water maze (rMWM) tests, novel object recognition (NOR), passive avoidance (PA), tail suspension (TS), beam walk (BW), and open-field locomotor activity. The goals were to examine the following questions: (1) whether more detailed behavioral assessment than is usually employed is feasible in experimental TBI; (2) to what degree the various behavioral tasks reflect lesion changes and loss of cells in specific anatomical regions; (3) whether the use of multiple behavioral outcomes enhances the ability to discriminate across levels of injury severity; and (4) whether a combined assessment “neuroscore” could be developed that increases predictive value in distinguishing injury severity.
The sMWM, considered to be a test of anterograde spatial learning and memory,36
has been commonly used to examine learning and memory deficits in rodents after TBI.10,37
It was reported that the time to locate the platform in the MWM was TBI severity-dependent in a rat CCI model.15
The rMWM paradigm has been far less studied following TBI.23,38,39
One day after the sMWM probe test, the platform was moved to the opposite quadrant (southwest), and the mice were trained for 4 consecutive days in the rMWM followed by another probe trial. In the rMWM task the animal must develop a new escape strategy to locate a submerged platform no longer placed in the quadrant where the animal had previously found it.39,40
Our data showed that the rMWM paradigm was superior to the sMWM in distinguishing mild from moderate and severe injury in the training phase. In addition, moderately- and severely-injured mice displayed more deficits in spatial memory in the rMWM probe test than the sMWM probe test. As the rMWM evaluates inhibitory learning, in which mice must learn to suppress a previously learned response,41,42
the present results suggest that the extinction of previously acquired memories and the reacquisition process may provide a more sensitive measure than the traditional MWM test for identifying cognitive deficits after experimental TBI. However, despite the fact that the rMWM may be a more sensitive test for discriminating injury-severity-related deficits in cognition, it requires 10 days of testing. This longer duration of testing reduces the ability of the rMWM to identify injury-related changes on a short time scale, which may somewhat diminish its usability in experimental TBI studies.
As the MWM task is particularly sensitive to hippocampal dysfunction,43
neuronal cell losses in sub-regions of the hippocampus were quantified to examine the correlation between MWM and neuronal cell loss. In the present study we found that neuronal cell loss in the CA1, CA2/3, and DG sub-regions of the hippocampus was injury-severity-dependent, and correlated with cognitive dysfunction as demonstrated by both the sMWM and rMWM tests. However, the Spearman rho correlation coefficients between latency of rMWM and sMWM on day 4 and cell loss in these respective hippocampal sub-regions differed: for CA1, this was −0.95 and −0.92, respectively; for CA2/3, the correlations were −0.94 and −0.92; and for DG the correlations were −0.91 and −0.93. These data suggest that the latency on day 4 in the rMWM has a better correlation with CA1 and CA2/3 neuronal cell loss than the sMWM. Similarly, the reversal probe test showed higher correlations to CA2/3 neuronal cell counts compared to the standard probe test (0.97 versus 0.90, respectively), whereas correlations to CA1 (0.94 [reverse probe] versus 0.93 [probe]), and DG (0.92 [reverse probe] versus 0.94 [probe]) neuronal cell counts were similar. These data further suggest that the sMWM and rMWM reflect cell loss to different degrees in different anatomical regions in the hippocampus.
In the present study, we observed that some severely-injured mice were somewhat reluctant to swim, tending at times to float rather than swim; this may in part reflect motor deficits, fatigue, and/or depressive-like behavior in severely-injured mice, as suggested by a trend toward a reduced swim speed in the MWM, significantly decreased activity in the open-field test, and increased immobility time in the TS, respectively. Thus the MWM results in severely-injured animals may not reflect pure cognitive deficits, but may be confounded by motor and affective impairments. Therefore, we also examined the search strategy used by the injured mice to locate the hidden platform.24
This parallels the type of search strategy analysis proposed for the Barnes maze,44,45
which was effectively used previously in this TBI model.12
Sham-injured mice primarily employed a spatial strategy to locate the platform; use of this strategy decreased with injury severity. In contrast, sham-injured mice rarely used a looping strategy to locate the platform, and this strategy was more prominently used with increased injury severity. Moreover, whereas sham animals swam in the center of the maze to find the hidden platform, injured animals spent most of their time swimming around the perimeter of the maze.
The NOR task evaluates non-spatial hippocampal memory,26–28
and is therefore complementary to spatial memory assessment by the MWM test. NOR has been widely used to measure cognitive function after experimental TBI in rodents.46–48
In the present study, mildly-injured mice spent a similar length of time with the novel object as sham-injured mice, whereas moderately- and severely-injured mice spent significantly less time. Correlation coefficients between NOR and CA1, CA2/3, and DG neuronal cell counts, were 0.91, 0.91, and 0.80, respectively.
The hippocampus plays an important role in contextual memory; but PA learning involves both contextual memory and amygdala-dependent emotional memory.49,50
Thus, performance on tests of PA learning decreases as a result of defects in either contextual or emotional memory. Generally, the dorsal hippocampus is specifically involved in memory function, and the ventral hippocampus modulates emotional and affective processes.51
Our histological analysis was focused on the dorsal region of the hippocampus, in the area directly underneath the impact site. The mildly-, moderately-, and severely-injured groups had significantly reduced transfer latency in the PA test compared with the sham-injured group, but this test did not effectively discriminate between injury severity levels. Correlation coefficients between PA and CA1, CA2/3, and DG neuronal cell counts were 0.93, 0.94, and 0.88, respectively.
Stereological methods were used to examine total lesion volume and hippocampal cell loss at 28 days post-injury for each group of mice. Both approaches showed strong correlations between histological changes and injury severity. Conclusions about which sub-regions of the hippocampus are most involved in the mediation of spatial learning have been conflicting. It was reported that dorsal hippocampal lesions have more profound effects than ventral hippocampal lesions.52,53
Morris and colleagues54
showed that intraventricular infusion of amino-phosphonovaleric acid (AP5) impairs hidden-platform MWM performance while blocking long-term potentiation (LPT) in the hippocampal dentate gyrus. Hicks and colleagues55
reported a significant correlation between post-traumatic memory scores using the rMWM after mild lateral fluid percussion and neuronal loss in the hilus of the dentate gyrus. However, another study using genetically-engineered mice showed that blunted LTP in hippocampal CA1 area coincided with impaired MWM learning.56
Our results were more nuanced. We found significant correlations between cognitive dysfunction and neuronal cell loss in the CA1, CA2/3, and DG sub-regions, but these differed as a function of each specific cognitive task ().
The hippocampus is important for tasks such as the MWM, which depends on relating or combining information from multiple sources.57
However, a task that does not have such requirements, such as NOR, may reflect involvement of larger areas, including both the hippocampus and the cortex adjacent to the hippocampus.58
Broadbent and colleagues suggested that larger hippocampal lesions may be needed to impair recognition memory than are needed to impair spatial memory.59
This is consistent with our finding that mildly-injured mice showed cognitive impairments in the MWM, but not in NOR. Few studies have examined the correlation between NOR and neuronal cell loss in hippocampal sub-regions. By measuring differential neuronal activation produced by novel and familiar pictures, it was found that novel pictures produce greater activation in subfield CA1 and less activation in the DG.60
Consistent with these observations, the present study demonstrated that the correlation between NOR and CA1 (r=0.91) and CA2/3 (r=0.91) is greater than that between NOR and DG (r=0.80), suggesting a more critical role for CA1 and CA2/3 in the mediation of novel recognition memory. In addition, we compared cognitive dysfunction assessed by each behavioral test with neuronal cell loss in the CA1, CA2/3, and DG sub-regions of the hippocampus, and determined the Spearman rho correlation coefficient and the p
value for each regression analysis. This analysis demonstrated that the correlation between the hippocampal neuronal number and the NOR and PA tests were less significant than hippocampal neuronal number and the sMWM, rMWM, probe, and reverse probe tests ().
Numerous groups have examined sensorimotor or vestibulomotor changes after rodent TBI.61–65
A previous report from our laboratory showed that moderately-injured mice had significantly more foot-faults than sham-injured mice in the beam-walk test.21
Flierl and colleagues evaluated sensorimotor function and cortical tissue damage after TBI as a function of injury severity in mice,65
but the correlation between sensorimotor function and lesion volume was not studied.35
In the present study, we found that changes in sensorimotor function using the beam-walk test reflected injury severity. Furthermore, in the present experimental conditions, injury-severity-dependent impairments in sensorimotor function were significantly correlated with lesion volumes in the injured cortex (r2
Combinations of behavioral tests have long been used in acute experimental brain and spinal cord injury models to increase sensitivity for discriminating both injury mechanism and the response to therapeutic interventions.66–71
Faden and colleagues developed several versions of a composite score for fluid percussion-induced TBI in the rat,72–74
adapted from prior work in stroke and spinal cord injury. Saatman and colleagues19
developed a composite score that included the MWM test and a motor functional behavioral test. A previous study by Hamm75
used multiple neurobehavioral tasks to evaluate the outcomes of TBI, including reflex suppression (pinna, corneal, and righting reflex), vestibulomotor function (beam balance, beam walking, and rotarod), and cognitive function. Here we sought to build upon and extend these earlier approaches by examining multiple cognitive and motor functional behavioral outcomes. We evaluated and compared various combinations of cognitive, affective, and motor function behavioral tests, to determine if such combined scores could better discriminate injury severity than individual scores. Results from the sMWM probe trial, the rMWM probe trial, the TS test, and the BW test were each converted to an ordinal scale ranging from 0 to 5. Two indices from the MWM were used to calculate CS, because the sMWM measures spatial memory acquisition, whereas the rMWM measures spatial retention memory after memory extinguishing and reacquisition. Further, the TS test represents affective behavior function, while the BW test represents sensorimotor function. Therefore, the CS provides an overall assessment of neurological function in injured mice. These four individual scores were combined, without weighting, to yield a 20-point CS ranging from 0 (severe) to 20 (sham). This CS served to increase differences between the sham- and mildly-injured group from 1–2 points to 4–7 points, enhancing the ability to observe significant differences between these groups, often a problem for studies examining mild or concussive-like insults. Here we found an increased discriminative ability for the CS compared to individual behavioral test scores (), which may allow better delineation between groups for future genetic or pharmacological studies. Notably, in a more recent pharmacological neuroprotection study (manuscript in preparation), we show that the CS provides superior discrimination of functional recovery than individual behavioral tests.
Despite the apparent benefits of using batteries of behavioral tasks to evaluate injury-severity-dependent outcomes after TBI, the present study also revealed some limitations. For example, the severely-injured, but not mildly- or moderately-injured mice, demonstrated less movement in the MWM and reduced locomotor activity in the open-field task. Fatigue, anxiety, depression, or some combination may contribute to reduced swimming ability and locomotor activity in severely-injured mice. To overcome such potential confounding issues, several modifications of the MWM should be considered: (1) reducing the number of daily trials in the test to three; (2) reducing the number of training days to 3; and (3) restricting the mice to a smaller area around the target hidden platform to facilitate locating the platform on the first day of training. In the present study, the severely-injured mice also demonstrated significantly longer times to locate the visible platform, which may have been related to fatigue. The performance of a visible cue test at an earlier time point should be considered to circumvent this issue.
Taken together, the data from this study demonstrate that multiple cognitive, affective, and motor tasks are able to delineate injury severity after CCI in mice. Both cortical lesion volume and neuronal cell loss in sub-regions of the hippocampus were well correlated with sensorimotor and cognitive dysfunction. Further, the composite score developed in the present study was able to better discriminate injury severity levels than traditional individual outcome tests. The CS may therefore be helpful in screening new neuroprotective agents or in investigating secondary injury mechanisms using transgenic mouse models.