Transient cognitive deficits are associated with the reversible accumulation of amyloid precursor protein after mild traumatic brain injury

Neuroscience Letters 409 (2006) 182–186

Transient cognitive deficits are associated with the reversible accumulation of amyloid precursor protein after mild traumatic brain injury Shihong Li a,∗ , Toshihiko Kuroiwa b , Satoru Ishibashi c , Liyuan Sun c , Shu Endo d , Kikuo Ohno a a

Departments of Neurosurgery, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8510, Japan b Pathophysiology, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8510, Japan c Neurology, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8510, Japan d Animal Research Center, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8510, Japan Received 14 June 2006; received in revised form 15 September 2006; accepted 17 September 2006

Abstract Mild traumatic brain injury (MTBI) may frequently cause transient behavioral abnormalities without observable morphological findings. In this study, we investigated neuropathological mechanisms underlying transient cognitive deficits after MTBI. Mongolian gerbils were subjected to experimental MTBI. At various time points after injury, behavioral changes were evaluated by the open-field test and T-maze test, and immunohistochemistry of microtubule-associated protein (MAP2) and amyloid precursor protein (APP) was performed to examine disruptions of the neuronal cytoskeleton and axonal transport, respectively. Transient cognitive deficits were observed after MTBI. Sustained MAP2 loss was found within the cortical impact site, but not the hippocampus. Transient APP accumulation at the same time as transient cognitive deficits occurred in the ipsilateral hemisphere, particularly in the subcortical white matter. These results suggest that the axonal dysfunction indicated by the reversible APP accumulation in the white matter, but not the sustained neuronal cytoskeletal damage reflected by the cortical MAP2 loss confined to the impact site, is responsible for the transient functional deficits after MTBI. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Axonal injury; Microtubule-associated protein2; Mongolian gerbil; Neuronal cytoskeleton

Mild traumatic brain injury (MTBI) represents 70% to 90% of all human traumatic brain injuries (TBI) [6]. Even without observable morphological brain damage, MTBI patients frequently suffer from cognitive deficits, emotional difficulties, and behavioral disturbances [19,25]. The pathological mechanisms underlying these functional impairments and the recovery process are not fully understood. Diffuse axonal injury is one of the most important types of brain damage that can occur after MTBI [1]. Clinical studies have shown that axonal injury may cause significant learning and memory dysfunction [1,10]. Since disruption of axonal transport results in amyloid precursor protein (APP) accumulation, APP has been used as a sensitive marker of axonal injury [10,27]. The disruption of the neuronal cytoskeleton is a prominent pathological finding in acute experimental MTBI, and the degradation or loss of microtubule-associated protein (MAP2) serves

∗

Corresponding author. Tel.: +81 3 5803 5266; fax: +81 3 5803 0140. E-mail address: [email protected] (S. Li).

0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.09.054

as a reliable marker of damage to the neuronal cytoarchitecture [24]. In addition, alterations in MAP2 immunoreactivity appear to be associated with functional recovery after brain injury [5]. Using Mongolian gerbils for studies on cerebral ischemia have proved that this model is reliable and can induce predictable neuropathological and behavioral outcomes in both short-term and long-term investigations [18,16]. Previously in our laboratory, we produced transient or prolonged hyperlocomotion and working memory deficits following experimental TBI induced by mild or moderate lateral fluid percussion injury (LFPI) in Mongolian gerbils [21]. However, the processes mediating the functional recovery in this model are unclear. Here we performed MAP2 and APP immunohistochemistry after mild LFPI in Mongolian gerbils to examine post-injury neuronal cytoskeleton and axon changes, and their potential involvement in the transient behavioral abnormalities after MTBI. Male Mongolian gerbils ranging in age from 22 to 28 weeks and in weight from 65 to 80 g were housed in groups of three or four and maintained on a 14/10-h light/dark cycle with unlimited access to food and water. All animal procedures were approved

S. Li et al. / Neuroscience Letters 409 (2006) 182–186

by the Animal Experiment Committee of Tokyo Medical and Dental University, in compliance with the guidelines for animal experimentation of the National Institute of Heath. Animals were randomly divided into a sham-operated group (SHAM; n = 11) and an MTBI group (MTBI; n = 23). LFPI was induced as previously described [21]. Briefly, each animal was anesthetized with ketamine hydrochloride (50 mg/kg, i.m.), supplemented as necessary. All wounds were infiltrated with 2.0% lidocaine hydrochloride during the surgical preparation and throughout the experiment. When using rats and gerbils for experimental TBI, the application of ketamine hydrochloride in combination with topical injection of 2.0% lidocaine hydrochloride has been proved a reliable anesthetic method with fewer side effects [21,23]. The animals were allowed to breathe spontaneously throughout all surgical procedures. The animal was placed in a stereotaxic frame, and a round craniotomy (3.5 mm in diameter) was made on the right parietal cortex, with center coordinates midway between the bregma and lambda and 2.5 mm lateral to the midline. LFPI of mild severity (0.7–0.9 atm) was induced. SHAM animals received anesthesia and underwent all of the surgical procedures except the delivery of the LFPI. The animals were placed on heating pads to maintain normothermia during the surgical procedure and for 2 h after injury. Animals (MTBI-7 d, n = 8; SHAM-7 d, n = 8) were tested in open-field and T-maze tests before injury and at 6 h, 24 h, and 3, 5, and 7 days after injury. We used the open-field test to evaluate spontaneous locomotor activity. Animals were placed individually in an open field apparatus (85 cm × 85 cm at the bottom) and allowed to start from one of the four corners selected randomly by the experimenter. The total distance moved (cm/10 min) by each animal was analyzed as spontaneous locomotor activity by using a video-tracking system and Smart software (Bio Research Center, Nagoya, Japan). The T-maze spontaneous alternation task has been used to test exploratory behavior and working memory [21,11]. Each animal was allowed to alternate between the left and right goal arms of a T-shaped maze (60 cm [stem] × 25 cm [arm] × 10 cm [width]) throughout a 15trial continuous alternation session. The spontaneous alternation rate (SAR) was calculated as the ratio of the alternating choices to the total number of choices (50%, random choice; 100%, alternation at every trial; 0%, no alternation) [11]. Animals were anesthetized and perfused with 4% paraformaldehyde at 6 h (MTBI-6 h, n = 5; SHAM-6 h, n = 1), 24 h (MTBI-24 h, n = 5; SHAM-24 h, n = 1), Day 3

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(MTBI-3 d, n = 5; SHAM-3 d, n = 1), and Day 7 (MTBI-7 d, n = 8; SHAM-7 d, n = 8; used in Behavioral Evaluations above). The brain was cut into six serial coronal sections every 2.0 mm from the level of the anterior pole of the caudate nucleus to the posterior pole. The sections were embedded in paraffin. Each coronal section was sliced to the thickness of 4 ␮m and processed for the immunohistochemical detection of neuronal cytoskeletal disruption (MAP2, mouse monoclonal antibody, Sigma, St. Louis, MO, USA) and injured axons (APP, mouse monoclonal antibody 22C11, Chemicon, Temecula, CA, USA). Briefly, after blocking with 5% normal horse serum, sections were incubated with primary antibodies (MAP2: 1:1000; APP: 1:500) overnight at 4 ◦ C. After further rinsing, biotinylated secondary antibody was applied. The avidin–biotin complex method (Vector, Burlingame, CA, USA) was used for antibody detection, with 3,3 -diaminobenzidine (DAB) as the chromogen. Following the reaction with DAB, slides were washed, dehydrated, and coverslipped. Omission of primary antibodies served as negative controls. Alterations in MAP2 immunoreactivity were quantified in the ipsilateral cortex and hippocampus. Areas with negative MAP2 immunostaining in six coronal sections were traced on an image of each scanned section with NIH Image software. The total volume of MAP2 loss (indirect lesion volume) of the ipsilateral hemisphere on each section was calculated as a percentage of the volume of the contralateral hemisphere [26]. To assess the APP immunoreactivity, predefined regions rich in axons were selected in the ipsilateral subcortical white matter, and APP immunoreactivity for each animal was evaluated semi-quantitatively [27,15]. Briefly, ipsilateral corpus callosum, external capsule, internal capsule, and caudate putamen were selected as predefined regions for APP score. Each coronal section was then analyzed for APP by light microscopy and a semi-quantitative rating was given: 0 for no APP, 1 for only scattered APP accumulation, 2 for a moderate amount of APP accumulation (no more than 50% of the total axons), and 3 for large amounts of APP accumulation (more than 50% of the total axons). The total APP accumulation score for each animal was the sum of the scores for the six coronal levels. Data are presented as means ± S.E.M. Statistical analyses were performed using two-way analysis of variance (ANOVA). All post-hoc comparisons between groups used Fisher’s PLSD test. A difference was considered statistically significant at P < 0.05.

Fig. 1. Results of behavioral tests. (A) Total distance moved in the open-field test; (B) spontaneous alternation rate in the T-maze test. * P < 0.05.

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Our mild LFPI treatment did not induce apnea or seizures in any animal. The total distance moved by the MTBI group in the open-field test was significantly greater than that by the SHAM animals at 6 h (8241.5 ± 703.7 cm, P < 0.0001), 24 h (6470.0 ± 1005.3 cm, P < 0.0001), and 3 days (4240.3 ± 513.8 cm, P = 0.0491), and returned to sham-treated levels from Day 5 (3706.3 ± 534.0 cm, P = 0.521) (Fig. 1A). The animals tended to choose the left and right arms with equal frequency in the T-maze test before the injury (Fig. 1B). The SAR in the MTBI group was below the random alternation rate (<50%) at 6 and 24 h, recovered to 57.1% ± 3.3% at Day 3, and then remained high until Day 7 (Fig. 1B). In SHAM animals, MAP2 immunolabeling was present in neuronal cell bodies and apical dendrites. In MTBI animals, MAP2 immunoreactivity decreased dramatically within the impact site throughout the entire 7-day post-injury period, with consistent sizes of immunostaining loss. No loss of MAP2 immunostaining was observed in the hippocampus or contralateral cortex of MTBI animals (Fig. 2A–D). Quantitative evaluation revealed no significant time-dependent change in cortical

Fig. 2. Photomicrographs of MAP2 immunostaining (A–D). (A) The cortex of a sham-treated animal; (B) MAP2 loss within the impact site of an MTBI animal at 6 h; (C) the hippocampus of a sham-treated animal; (D) the hippocampus of an MTBI animal at 6 h; (E) quantitative evaluation for MAP2 loss at various times after injury. The volume of the lesions did not change significantly over time. Scale bar = 50 ␮m.

MAP2 loss throughout the post-trauma period (P = 0.756; Fig. 2E). No APP accumulation was found in any SHAM animal. Maximal APP accumulation was observed at 6 and 24 h after injury. Ipsilateral subcortical white matter, especially at the level of the ectorhinal cortex and subiculum, showed the most robust APP accumulation. Areas of the ipsilateral hippocampus and cortex showed increased APP immunoreactivity in a small number of neuronal perikarya. By 3 days after injury, the extent and intensity of APP immunoreactivity had declined, and APPexpressing axons were difficult to identify on Day 7 (Fig. 3A–L). The semi-quantitative analysis for APP immunoreactivity revealed significant difference in APP accumulation in the ipsilateral subcortical white matter over time during the post-trauma period (P < 0.0001; Fig. 3M). The APP accumulation score was at a high level at 6 h (8.8 ± 1.0) and 24 h (7.4 ± 0.7) after injury, then recovered significantly on the third day (4.6 ± 0.8; P < 0.05 versus 6 and 24 h), and furthermore decreased to 2.1 ± 0.4 on the seventh day (P < 0.01 versus 6, 24 h and 3 days). MTBI is the most common type of clinical TBI. Despite its negligible mortality rate, MTBI frequently results in cognitive deficits and behavioral dysfunction [2]. The contribution of neuropathological factors to these functional impairments is controversial, mainly due to the difficulties in identifying neural tissue damage in patients with MTBI. Therefore, experimental studies using appropriate animal models of MTBI are crucial to a better understanding of its pathophysiology. Our Mongolian gerbil MTBI model reproduced transient neurobehavioral abnormalities including the posttraumatic hyperlocomotion and working memory dysfunction seen in patients. These posttraumatic deficits significantly correlate with neural tissue damage after experimental TBI, and the duration of recovery depends on the injury severity [21]. In addition, we found mild histopathological damage, including axonal changes indicated by reversible APP accumulations, which corresponded with the transient behavioral changes after injury. Such an axonal abnormality is potentially important in understanding transient dysfunction after MTBI. Although prolonged cognitive deficits can follow experimental MTBI [28], symptoms after clinical MTBI are usually transient, and only a small percentage of individuals develop long-lasting problems [8]. Better understanding of these transient functional impairments and of their recovery process will lead to better functional prediction and effective MTBI treatments. The gerbil MTBI model mimics some critical components of clinical MTBI—transient cognitive deficits and minor brain damage, and may therefore be a useful experimental tool for testing putative therapeutic strategies for MTBI. Loss or disruption of MAP2 is an early marker for damage of neuronal cytoskeleton following experimental TBI [24]. Since MAP2 plays an important role in neurite extension and neural plasticity [17], its disruption could contribute to the development of cognitive dysfunction after TBI. In this study, we tested this hypothesis in a gerbil MTBI model. MAP2 immunolabeling decreased at the impact site of the ipsilateral cortex within 6 h after injury (Fig. 2B), indicating early microtubule vulnerability following MTBI. The quantitative analysis for MAP2

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Fig. 3. Photomicrographs of APP immunostaining in identical brain regions in the ipsilateral hemisphere (subcortical white matter: A–D; cortex: E–H; hippocampus: I–L), of a sham-treated animal (A, E, I), and an MTBI animal at 6 h (B, F, J), 3 days (C, G, K) and 7 days after injury (D, H, L). (M) semi-quantitative evaluation for APP accumulation at various times after injury. Scale bar = 50 ␮m. * P < 0.05.

immunostaining revealed that the cortical loss of MAP2 was sustained in the impact site through Day 7 without significant change in volume over time (Fig. 2E). The early and sustained cortical loss of MAP2 found in our study corresponds to those of previous animal MTBI studies [24,14], but we observed no overt hippocampal involvement, and others have reported significant MAP2 loss in the ipsilateral hippocampus. The relatively mild injury in our study may account for the absence of MAP2 loss in the hippocampus, since hippocampal vulnerability to experimental MTBI correlates with injury severity [9]. Although we could neither confirm the recovery of MAP2 disruption nor correlate it with the transient cognitive deficits, return of MAP2 immunoreactivity in the contused cortex occurred 21 days after mild weight-drop injury in the rat [20], therefore, future study should be conducted in a chronic time period following TBI in various severities to examine the potential recovery of MAP2

disruption and its correlation with the behavioral deficits probably missed by the present study. APP is transported by fast anterograde axonal transport and serves as a sensitive marker of axonal damage [27]. The interruption of axoplasmic flow due to traumatic or ischemic insult results in the accumulation of APP [27,12]. In addition, axonal damage due to experimental TBI can be either reversible or irreversible depending on the injury severity [4]. We found an acute axonal dysfunction reflected by changes of APP immunoreactivity. The temporal profile of this transient APP accumulation is similar to that in previous studies using APP as a marker for axonal injury following moderate TBI [12,7]. In those studies, transient increases of APP immunoreactivity could result from both recovery of axonal transport and rapid axonal loss due to damage at the origin of the cell body for these axons [4]. However, the latter seems unlikely in our model, since the mild LFPI

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in the current study caused only very subtle and limited cortical neuronal damage, and no hippocampal involvement. Future studies should use electron microscopy to clarify the reversibility of the damage at the ultrastructural level. By applying a semi-quantitative analysis for APP accumulation in the ipsilateral subcortical white matter, we also found that the increased APP immunoreactivity coincided temporally with transient cognitive deficits following MTBI (Fig. 3M). Axonal injury is an almost universal sequela of TBI, including mild cases [1,3]. Previous clinical findings have shown a strong correlation between axonal injury and cognitive dysfunction [1,10]. The vulnerability of the white matter to experimental TBI, as well as the strong correlation between functional deficits and white matter injury, have been demonstrated by our previous work [21]. Taken together, these findings suggest that axonal dysfunction reflected by the reversible APP accumulations, particularly in the subcortical white matter, plays a primary role in the transient cognitive deficits following mild LFPI in gerbils. Although cognitive deficits after MTBI are often described as a psychological disturbance rather than trauma-induced damage to brain tissue, clinical studies have suggested there may be a neuropathological contribution [22,13]. Our study experimentally confirms the neuropathological involvement in transient cognitive deficits following MTBI. We successfully reproduced transient cognitive deficits and minor brain damage by inducing mild LFPI in Mongolian gerbils. The axonal dysfunction reflected by the reversible APP accumulation in the subcortical white matter, instead of the sustained disruption of neuronal cytoskeleton confined to the impact site, may be a primary cause of the transient cognitive deficits following experimental MTBI. Our findings support the speculation from clinical studies that neuropathological factors contribute to functional impairments following MTBI.

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