B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

Ava i l ab l e on l i ne a t www.sc i enced i r ec t . com

www.e l sev i e r . com/ loca te /b ra i n res

Research Report

Characterisation of the effect of knockout of the amyloidprecursor protein on outcome following mild traumaticbrain injury

Frances Corrigana, b, Robert Vinka, b, Peter C. Blumbergsa, b, Colin L. Mastersc,Roberto Cappaid, 1, Corinna van den Heuvela, b,⁎, 1

aDiscipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide SA, AustraliabCentre for Neurological Diseases, Hanson Institute, SA, AustraliacMental Health Research Institute, Victoria Parkville 3052, AustraliadDepartment of Pathology and Bio21 Molecular Science and BioTechnology Institute, The University of Melbourne, Victoria, 3010, Australia

A R T I C L E I N F O

⁎ Corresponding author at: Discipline of AnatAustralia. Fax: +61 8 82223392.

E-mail address: corinna.vandenheuvel@a1 Co-senior authors.

0006-8993/$ – see front matter © 2012 Elseviedoi:10.1016/j.brainres.2012.02.045

A B S T R A C T

Article history:Accepted 19 February 2012Available online 28 February 2012

The amyloid precursor protein (APP) increases following traumatic brain injury (TBI), althoughthe functional significance of this remains unclear largely because the functions of thesubsequent APP metabolites are so different: Aβ is neurotoxic whilst sAPPα is neuroprotective.To investigate this further, APP wildtype and knockout mice were subjected to mild diffuse TBIand their outcomes compared. APP knockout mice displayed significantly worse cognitive andmotor deficits, as demonstrated by the BarnesMaze and rotarod respectively, than APPwildtypemice. This was associated with a significant increase in hippocampal and cortical cell loss, aswell as axonal injury, in APP knockout mice and an impaired neuroreparative response as indi-cated by diminished GAP-43 immunoreactivity when compared to APP wildtype mice. Thisstudy is the first to demonstrate that endogenousAPP is beneficial followingmild TBI, suggestingthat the upregulation of APP observed following injury is an acute protective response.

© 2012 Elsevier B.V. All rights reserved.

Keywords:Amyloid precursor proteinCaspase-3GAP-43sAPPαSynaptophysinTraumatic brain injury

1. Introduction

Motor vehicle accidents and their sequela, in particular traumaticbrain injury (TBI) will surpass many diseases as the major causeof death and disability by the year 2020, according to the WorldHealth Organization (Hyder et al., 2007). A significant amount ofcell death occurs in the days following the traumatic event dueto the initiationof secondary injury factors suchas inflammation,excitotoxicity andoxidative stress (Gaetz, 2004). Anumberof neu-roprotective and neurotrophic pathways are activated in re-

omy and Pathology, Scho

delaide.edu.au (C. van de

r B.V. All rights reserved.

sponse to this injury in order to facilitate a reparative response.It is thought that the amyloid precursor protein (APP) may be in-volved in these processes as it is acutely upregulated within in-jured neurons and reactive astrocytes following TBI (Chen et al.,2004; Van den Heuvel et al., 1999). Importantly its metabolite,sAPPα, has neuroprotective activity (Goodman and Mattson,1994; Thornton et al., 2006). Indeed this significant post-traumatic upregulation appears to be a normal acute phase re-sponse to neuronal stress (Gentleman et al., 1993) with a similarincrease seen in cells exposed to ischaemia (Nihashi et al., 2001;

ol of Medical Sciences, University of Adelaide, Adelaide, SA 5005,

n Heuvel).

88 B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

Popa-Wagner et al., 1998) and excitotoxicity (Gordon-Krajcer andGajkowska, 2001). The functional significance, however, remainspoorly understood.

APP is a ubiquitously expressed, highly conserved integralmembrane glycoprotein (Mattson, 1997). Only a small proportionof APP remains as a full length protein, withmost rapidly under-going proteolytic processing via two pathways (Herreman et al.,2003). The majority of APP is processed within the non-amyloidogenic pathway via the action of α secretase, to formthe protective α form of APP (sAPPα) (Suh and Checler, 2002),whilst a small proportion is cleaved by β and γ secretases withinthe amyloidogenic pathway to produce toxic Aβ (Koo et al., 1996),themain component of plaques associatedwith Alzheimer's dis-ease. The disparate actions of these APP metabolites highlightswhy the role of APP following TBI remains controversial.

The upregulation of APP seen following TBI has been associat-ed with increased hippocampal cell death in rodents (Murakamiet al., 1998) This was attributed to an increase in the productionof Aβ, which may be facilitated by a transient elevation in β-secretase activity seen post-injury in rodents (Blasko et al.,2004). Indeed co-accumulation of Aβ with APP in swollen axonsand neuronal cell bodies has been observed within days in bothrodent (Stone et al., 2002) and swine models (Chen et al., 2004;Smith et al., 1999) of diffuse TBI. In swine sparse diffuse Aβ pla-ques were also observed up to 6months following injury, thefirst animal model to replicate findings that Aβ plaques arefound in up to 30% of people who die acutely of TBI (Roberts etal., 1991). Data on levels of Aβ within the ventricular cerebrospi-nal fluid following human TBI are contradictory with both an in-crease (Olsson et al., 2004; Raby et al., 1998) and a decrease (Kay etal., 2003) in levels reported. Regardless, a direct link between in-creased levels ofAPP, enhancement ofAβproduction and toxicityhas never been demonstrated (Iwata et al., 2002).

Alternatively, it is thought that theupregulationofAPP follow-ing TBI may represent a protective cellular response, with sAPPαshown to attenuateneuronal cell death induced by excitotoxicity,ischaemia and oxidative stress (Furukawa et al., 1996; Goodmanand Mattson, 1994; Mattson et al., 1993). In addition sAPPα canpromote repair processes, through its well characterised effectson neurite outgrowth (Ohsawa et al., 1997; Qiu et al., 1995; Wangand Ha, 2004), synaptogenesis (Bell et al., 2006) and neurogenesis(Caille et al., 2004). The importance of APP post-injury washighlighted by the finding that knocking out the APP orthologuein Drosophilia, APPL, leads to an increase in mortality comparedto wild-type flies at 1 and 2 weeks following brain trauma in-duced by a needle injury (Leyssen et al., 2005). Therefore theaim of the current study was to assess the importance of endog-enous APP by determiningwhether knockout of APPwouldwors-en functional outcome, increaseneuronal damageand inhibit thereparative response following mild diffuse TBI (mTBI) in mice.

Fig. 1 – NSS for mice, as assessed at 1 h post-injury. Resultsare expressed as medians±interquartile range (n=5 pergroup).

2. Results

2.1. Acute neurological response

Sham-injured APP+/+ and APP−/− mice righted themselvesspontaneously within 120–135 s after removal of anaesthesia.In contrast, the return of the righting reflex (indicative of theduration of unconsciousness) was significantly longer in

injured mice (APP−/− mice, 329±33 s; APP+/+ mice 348±52 s).When assessed for NSS at 1 h following TBI, injured micescored higher than shams, with a median NSS of 3 comparedto 1, although this did not reach significance (Fig. 1). No differ-ences were noted in NSS between APP+/+ and APP−/− mice.According to Tsenter et al. (2008), 1 h post-injury NSS definesthe severity of trauma, with a NSS of less than 5, as seen with-in this study, indicative of a mild TBI.

2.2. Motor outcome

Motor outcome was determined using the rotarod. Results areexpressed as a percentage of each mouse's own pre-injurybaseline level, to compensate for phenotypic affects as APP−/− mice demonstrate forearm weakness (Zheng et al., 1995).Although there is a significant difference between APP−/−and APP+/+ sham animals when the data is expressed asraw scores (Fig. 2A), there is no significant difference whenexpressed as a percentage of each mouse's own baselinelevel (Fig. 2B), allowing a direct comparison between APP−/−and APP+/+ injured mice. This injury model did not producedetectablemotor deficits in APP+/+mice, with thesemice per-forming between 101±5.5% and 103.1±4.2% of their pre-injurytime. In contrast, injured APP−/− mice remained significantlyimpaired on all days of testing post-injury when compared totheir shams (p<0.05) and to the injured APP+/+ mice (p<0.05).Although the APP−/− mice improved over the testing periodfrom 75.2±16.9% of pre-injury level on day 1 to 86.5±11.8%on day 7, they did not return to an uninjured baseline level.

2.3. Cognitive outcome

The cognitive performance of the mice post-injury was mea-sured using the Barnes Maze, which assesses the ability tofind a previously learned escape hole. There were no signifi-cant differences between APP−/− and APP+/+ shams, withboth groups showing improvement in escape latency com-pared to their pre-injury time, APP−/− shams from 38.6±19.1to 22.3±11.2 s and APP+/+ shams from 34.5±17.9 to 16.1±8.9 s(Fig. 3A). Although injured APP+/+ mice were not significantlydifferent to their shams on any day tested post-injury therewas an increase in the escape latency on day 2 following injury,in 44±26.46 s compared to 23.6±12.5 s in APP+/+ shams at thistime point. Injured APP+/+ mice then returned to sham level

Fig. 2 – Motor outcome, as assessed on the rotarod,expressed as raw scores (A) and % pre-injury time (B). APP−/−mice have a small, but significant, impairment in motorperformance following TBI, which was not present in APP+/+mice. Results are expressed as means±SEM. (n=8 per group)(^^^p<0.001 compared to APP+/+ shams, ###p<0.001,##p<0.01, #p<0.05 compared to APP−/− shams, ***p<0.001,**p<0.01, *p<0.05 compared to APP+/+ injured mice).

Fig. 3 – Cognitive outcome as determined by escape latency(A), number of errors (B) and ability to learn a new spatialcontingency (C) on the Barnes Maze. Results are expressed asmeans±SEM (APP+/+ sham, APP−/− sham, APP−/−inj n=8 pergroup; APP+/+ inj n=7). (*p<0.05 compared to injured APP+/+mice, ^p<0.05, ^^p<0.01 compared to APP−/− shams).

89B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

taking 24.0±11.1 and 15.5±13.2 s to locate the escape hole ondays 4 and 6 post-injury compared to 16.1±8.9 and 22±11.5 sin their shams. In contrast the APP−/− mice were significantlyimpaired when compared to injured APP+/+ mice on days 2, 4and 6 post-injury (p<0.05). However, they did improve overthe testing period, with an escape latency of 71.37±43.1 s onday 2 compared to 51.02±28.6 and 41.75±15.4 s on days 4 and 6.

The increase in escape latency in injured APP−/− mice wasassociated with an increase in number of errors with 15, 13 and10 on days 2, 4 and 6 post-injury, compared to 10, 6 and 8 inAPP+/+ mice, with this reaching significance on day 4 post-injury (Fig. 3C). Furthermore, following injury APP−/− mice weresignificantly different to their sham controls on days 2 and 4,with APP−/− shams making 7 and 4 errors respectively. Therewere no significant differences between the APP+/+ and APP−/−shams.

The ability of mice to learn a new location for the escapehole over 3 trials is shown in Fig. 3B. The APP+/+ injuredmice were not different to their shams with performance im-proving steadily over the 3 trials from 73.75 s on trial 1 to31.625 s by trial 3, compared to 83.62 s to 25.37 s in APP+/+shams. On the other hand APP−/− mice were significantly im-paired in their ability to learn the new location, seen as a sig-nificant increase in the amount of time taken on trial 2 whencompared to APP−/− shams (p<0.05) and APP+/+ injured mice

(p<0.05). However, by the third trial there were no differencesbetween the 4 groups.

2.4. Neuronal loss

Vulnerability of cells post-injurywas assessed by evaluating thenumber of healthy neurons within the CA3 region of the hippo-campus (Figs. 4A–F) and the cortex (Figs. 4G–L). Following injurythe CA3 region of the APP+/+ mice looked similar to their shamcontrols, whereas in the APP−/− mice there were increasingnumbers of pyknotic neurons from 3 to 7 days with evidenceof neuronal loss. Although the APP+/+ mice exhibited neuronalloss within the cortex, the damage was greater in APP−/− mice.These observationswere confirmed by a count of the number ofhealthy neurons within the CA3 region (Fig. 4M) and cortex un-derneath the impact site (Fig. 4N), with APP−/− mice exhibitingsignificantly lower numbers than APP+/+ mice in the CA3 re-gion at day 3 post-injury and in the cortex at both 3 and 7 daysafter injury (p<0.05).

Fig. 4 – H&E labelled sections within the CA3 region of the hippocampus (A–F) and cortex directly underneath the impact site (G–L)(scale bar=100μm). Neuronal countswithin the CA3 region of the hippocampus (G) and cortex underneath the impact (H), found thatAPP−/−mice have significantly greater neuronal damage than APP+/+mice in both these regions. (n=5 per group). *p<0.05, **p<0.01compared to injured APP+/+ mice; ^^^^p<0.0001, ^p<0.05 compared to APP−/− shams; ####p<0.0001 compared to APP+/+ shams.

90 B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

Fig. 5 – Assessment of axonal injury utilising NFH (A, C, E, G) and SYP (B, D, F, H) immunoreactivity within the corpus callosum.Scale bar=10 μm. Quantification of the number of NFH positive axonal profiles within the central part of the corpus callosum (I)found that APP−/− mice had significantly greater axonal injury than APP+/+ mice.

91B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

2.5. Axonal injury

Following TBI, accumulation of APP within axons, indicativeof axonal injury was noted within the corpus callosum andbrainstem of wildtype mice with neuronal cell body APP

immunostaining was also noted to be higher following injurywithin the hippocampus than seen in shams (SupplementaryFig. 1). As axonal injury could not be assessed by this methodin APP−/− mice, neurofilament heavy chain (NFH) and synap-tophysin (SYP) immunostaining were utilised to determine

92 B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

the extent of neurofilament compaction and the impairmentin axonal transport respectively (Fig. 5). NFH immunostainingshowed an increase in thickened axons within the centralportion of the corpus callosum in both APP+/+ and APP−/−mice at day 3 following injury, although the numbersappeared greater in APP−/−mice. Similarly, SYP immunoreac-tivity, seen as swellings along the length of the axon,appeared increased in APP−/− mice in comparison to APP+/+mice post-injury. In contrast synaptophysin staining was ab-sent within the white matter in sham animals. Quantificationof the number of NFH immunoreactive profiles within thecentral portion of the corpus callosum found that APP−/−mice had a significant increase following TBI, with 72.8±35.4damaged axons per mm2 compared to 39.9±19.7 in APP+/+mice (p<0.05). Both APP−/− and APP+/+ mice had significantlygreater numbers than their respective shams (p<0.01).

2.6. GAP-43 immunoreactivity following mTBI

Immunoreactivity of GAP-43 within the hippocampus andcortex was used to determine the strength of the reparativeresponse seen post-injury (Figs. 6A–L). GAP-43, is a neural spe-cific membrane associated phosphoprotein which is one com-ponent of injury-induced plasticity as it is associated withpromotion of neuronal sprouting and neurite extensionwhich facilitate synaptic remodelling (Bendotti et al., 1997).The APP+/+ animals had an increase in GAP-43 immunoreac-tivity within the CA1 region of the hippocampus at days 3 and7 following injury and in the cortex at day 7 post-injury, withimmunoreactivity clearly higher than that seen in the APP+/+or APP−/− shams. In contrast GAP-43 immunoreactivity wassimilar to that seen in shams at both time points in APP−/−animals following mTBI. This observation was supported by acount of the number of GAP-43 +ve cells within the cortexand CA1 region of the hippocampus (p<0.05), with a significantincrease in APP+/+ mice at both days 3 and 7. Indeed at day 7APP+/+ mice had 305.8±126.9 GAP-43 +ve cells/mm2 comparedto 93.4±107.34 GAP-43 +ve cells/mm2 in APP−/− mice (Fig. 6M).Similarly in the cortex a significant increase occurred at day 7following TBI (p<0.01) with APP+/+ mice exhibiting 282.5±113.2GAP-43 +ve cells/mm2 compared to 45±36.8 GAP-43 +vecells/mm2 (Fig. 6N).

2.7. Levels of activated caspase-3 following mTBI

Levels of activated caspase-3 were determined within the hip-pocampus (Fig. 7A) and cortex (Fig. 7B) in order to reflect apo-ptotic cell death following injury using an ELISA. The APP−/−mice had significantly higher levels at day 3 post-injury inthe hippocampus (p<0.001) and cortex (p<0.01) when com-pared to APP+/+ mice. At this time point activated caspase-3levels were 3 times higher in the cortex, and 2 times higherin the hippocampus in the APP−/− mice. By day 7, levelsremained elevated in the APP−/− mice, with an absorbancelevel of 0.314±.09 in the hippocampus compared to 0.218±.06

Fig. 6 – GAP-43 immunolabelled sections within the right CA1 rebar=100 μm. A count of the number of GAP-43 +ve cells within tthat levels were higher in APP+/+ than APP−/− mice following in

in the APP+/+ mice, with a similar pattern in the cortex, withAPP−/− mice at 0.224±.05 compared to 0.163±.02 in the APP+/+mice, but this was not significant. There was no significantdifference in activated caspase-3 levels between APP+/+ andAPP−/− shammice.

3. Discussion

This study demonstrates that lack of APP appears to makemice more vulnerable following mTBI, with APP−/− micedemonstrating greater functional deficits, increased vulnera-bility of neurons and an impaired reparative response. Thismodel of mTBI allows the study of the role of endogenousneuroprotective pathways without the complications of largeamounts of tissue loss. Mild TBI is commonly referred to asconcussion, and is defined as traumatically induced disrup-tion of brain function seen as a short loss of consciousness,memory dysfunction or GCS of 13–15 (Shukla and Devi, 2010;Teasdale and Jennett, 1974). Axonal injury is thought to beone of the key pathological features of mTBI, with Blumbergset al. finding evidence of multifocal APP accumulation in axo-nal swellings in the white matter of 5 patients diagnosed withmTBI who died from unrelated causes (Blumbergs et al., 1994).This has been supported by more recent neuroimaging stud-ies of mTBI which describe signal changes that could be attrib-uted to the presence of diffuse axonal injury within whitematter (Bazarian et al., 2007; Inglese et al., 2005; Messe et al.,2010). As such the brief loss of consciousness and evidenceof axonal injury within the corpus callosum and brain stem,coupled with a 1 h NSS of less than 5 seen within this studyare indicative of a mild TBI.

Following injury, APP+/+ mice had significant cortical cellloss under the impact site and a non-significant increase inescape latency on day 2 following injury when compared totheir shams. In contrast APP−/− mice had a significant in-crease in escape latency on the Barnes Maze on days 2–6post-injury, which was associated with an increase in thenumber of errors, which reached significance on day 4. Theyalso displayed significant impairment in learning a new spa-tial contingency when the escape hole wasmoved. In additionAPP−/− mice had a small but significant impairment inrotarod performance in the first week post-injury. These func-tional deficits were correlated with increased neuronal cellloss, an exacerbation of axonal injury, an increase in levelsof active caspase-3 and a decrease in levels of GAP-43 whencompared to APP+/+ mice.

In this study the injury parameters were altered to ensurethat the level of primary injury was the same, as knockoutstrains can display different susceptibility to the impact (Flierlet al., 2009). This was of particular concern due to reported dif-ferences in brain weight of around 10% in APP−/− mice (Ringet al., 2007), although no major neuronal loss within the cortexor hippocampus of adult APP−/− mice has been noted (Hermset al., 2004; Phinney et al., 1999). The acute neurological

gion of the hippocampus (A–F) and right cortex (G–L). Scalehe CA1 region (M) and cortex (N). (n=5 per group), indicatedjury. (*p<0.05, ***p<0.001 compared to APP−/− injured mice).

93B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

Fig. 7 – Levels of activated caspase-3 within the hippocampus(A) and cortex (B), showing a significant increase in APP−/− butnot APP+/+ mice at day 3 post-injury. (n=5 per group)(**p<0.01, ***p<0.001 compared to APP+/+ injured mice).

94 B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

response to injury, as determined by the righting reflex and 1 hNSS score,were the same inAPP+/+ andAPP−/−mice,with boththesemeasures found to be reliable indicators of injury severity(Flierl et al., 2009; Fujimoto et al., 2004; Tsenter et al., 2008).Given the similar immediate response to the injury, it is likelythat the exacerbation of deficits in APP−/−mice is due to an in-crease in the levels of secondary injury.

It should also be noted that the APP−/− phenotype, whichincludes forearmweakness (Zheng et al., 1995) and age relatedcognitive deficits (Dawson et al., 1999) is not the cause of theimpairments observed in these mice following injury.Although there is a difference between APP+/+ and APP−/−shamanimals in termsof raw rotarod scores, only theAPP−/− in-jured animals demonstrated a motor deficit following injury.Furthermore, at this age (10–16 weeks), the APP−/− mice did notdemonstrate cognitive deficits on the Barnes Maze, with nosignificant differences between escape latency in APP+/+ andAPP−/− sham animals.

The exacerbation of functional deficits inAPP−/−mice corre-sponded with increased neuronal damage, elevated levels ofactive-caspase 3 and exacerbated axonal injury in APP−/−mice following injury. Axonal injury was assessed utilisingtwo differentmarkers, in NFH and SYP, to examine impairmentin neurofilament compaction and axonal transport respectivelydue to reports that these can occur in separate populations ofaxons following TBI (Marmarou et al., 2005). Synaptophysin isa component of synaptic vesicles, with its presence within axo-nal swellings thought to indicate impairment in anterogradetransport (Creed et al., 2011). Neurofilament consists of threepolypeptide chains of different weights, namely NF-light, NF-medium and NF-heavy, with both NFM and NFH observed infocal axonal swellings following TBI (Hellewell et al., 2010;

Raghupathi and Margulies, 2002). This is thought to be due todephosphorylation or proteolysis of the side arm domains ofNF, which disrupt the axonal cytoskeleton causing subsequentswelling (Povlishock et al., 1997). In APP−/− mice axonal injuryas detected by both NFH and SYP positive axonal swellingswas greater than that in APP+/+ mice, with quantification ofNFH immunoreactive lengths within the corpus callosum con-firming this observation.

The increased vulnerability of neuronal cell bodies andtheir axons post-injury is most likely due to the lack of neuro-protective sAPPα. Adding sAPPα to cultured neuronal cells in-creases their resistance to excitotoxic, metabolic andoxidative insults (Furukawa et al., 1996; Goodman andMattson, 1994; Mattson et al., 1993), whilst interocerebroven-tricular infusion of sAPPα decreases levels of neuronal deathfollowing TBI (Thornton et al., 2006), and transient global cere-bral ischaemia (Smith-Swintosky et al., 1994).

In addition to increasing the vulnerability of cells, the lackof APP impedes the ability to initiate a reparative response, inthe current study this was reflected by a decrease in theimmunostaining of GAP-43 in the APP−/− mice post-injurywhen compared to APP+/+ mice. GAP-43 is elevated followingTBI (Christman et al., 1997; Emery et al., 2000; Hulsebosch etal., 1998; Thompson et al., 2006), and is associated with the pe-riod of synaptic organisation and axonal organisation(Bendotti et al., 1997). This occurs through its ability to pro-mote neuronal sprouting and neurite extension and to en-hance synaptic remodelling through formation of novelneuronal connections (Bendotti et al., 1997), although this re-sponse may be inhibited with increasing injury severity(Thompson et al., 2006). Previous studies have shown thatcells that are GAP-43 positive are responsible for formationof new axon collaterals following transection of matureaxons in CA3 pyramidal cells (McKinney et al., 1999) and le-sions of the perforant pathway (Lin et al., 1992). The increasein GAP-43 noted in APP+/+ mice is most likely in response toaxonal and dendritic injury. Although no overt cell loss wasnoted within the hippocampus of APP+/+ mice, and onlyminor neuronal injury within the cortex, it is likely that the in-jury would have resulted in ultrastructural changes. Evidentlythese were not sufficient to induce a cognitive deficit detect-able on the Barnes Maze, but could have provided the stimu-lus for the production of GAP-43 noted within this study. Assuch the lack of GAP-43 in APP−/− mice following injury canbe seen as an inhibition of axonal plasticity that would nor-mally occur in response to post-traumatic axonal damage.

The deficits seen in these APP−/− mice can most likely beattributed to lack of sAPPα since its in vitro application causesneurite outgrowth (Araki et al., 1991; Bhasin et al., 1991;Ohsawa et al., 1997; Qiu et al., 1995; Saitoh et al., 1989), whilstraising the levels of sAPPα in vivo increases cortical synapto-genesis (Bell et al., 2006). APP can act synergistically withnerve growth factor (NGF) to enhance its neurotrophic effects(Wallace et al., 1997), with this important for promoting theplasticity associated with recovery following brain injury.

This study suggests that at least immediately following amild injury the beneficial effects of sAPPα, outweigh any po-tentially negative effects of other APP metabolites. This notonly includes Aβ, but also the APP intracellular domain(AICD) and C31, a product of caspase cleavage of the APP

95B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

cytoplasmic domain, which have been suggested to be pro-apoptotic (Kim et al., 2004). Non-transgenic mice are unableto develop the Aβ plaque pathology that is seen in clinicalTBI, with this thought to be due to a three amino acid differ-ence in Aβ sequence (Johnson et al.). Furthermore, rodent Aβis thought to be less toxic, as it does not reduce Cu (II) as effec-tively as human Aβ, which may affect its ability to promoteoxidative stress (Opazo et al., 2000). However, although rodentAβ does not form plaques, it has been shown to form β-sheetfibrils in vitro (Fraser et al., 1992), but not as aggressively ashuman Aβ (Boyd-Kimball et al., 2004). Furthermore, inductionof protein oxidation and lipid peroxidation in primary neu-rons has been demonstrated with rodent Aβ, with this trigger-ing apoptosis and cell death, although at a slower rate thanhuman Aβ (Boyd-Kimball et al., 2004).

Nonetheless, upregulation of soluble Aβ has been demon-strated following TBI in rodents (Blasko et al., 2004; Iwata etal., 2002; Loane et al., 2009; Mannix et al., 2010), as has accumu-lation of Aβwithin damaged axons (Stone et al., 2002). This sug-gests that in rodent models increased Aβ production may stillinfluence secondary injury mechanisms following TBI despitethe lack of plaque formation. Indeed progressive cortical deteri-oration observed in injured adult APOε4 mice, that have re-duced Aβ clearance, was associated with high levels of Aβ inboth cerebral hemispheres at 1 month after injury (Mannix etal., 2010). Furthermore, increasing Aβ clearance without affect-ing APP processing through treatment with the cholesterol ef-flux transporter ABCA1 (ATP-binding cassette A1), improvedfunctional recovery and significantly reduced lesion volume innon-transgenic mice (Loane et al., 2011). However, future stud-ies in specieswith the humanAβ sequencemay provide furtherevidence on the role that endogenous APP plays following TBI.

This study identifies a role for endogenous APP inmaintain-ing cognitive and motor abilities following a mild diffuse TBI inmice by reducing cell death and promoting a reparative re-sponse, with this most likely due to the actions of sAPPα. Longterm recovery after brain injury is known to involve processessuch as neurite outgrowth, synaptic plasticity and neuron re-generation. As such it could be beneficial to either modulatethe proteolytic processing of endogenous APP to enhance levelsof sAPPα post-injury, or to develop an exogenous agent that rep-licates this activity in order to improve functional outcome fol-lowing TBI.

4. Experimental procedure

Generation of APP−/− mice has been described previously(Zheng et al., 1995), with both the APP+/+ and APP−/− miceon the same background strain, C57BLbj×129sv. All studieswere performed within the guidelines established by theNHMRC of Australia and were approved by the Animal EthicsCommittees of the Institute of Medical and Veterinary Sci-ences and the University of Adelaide.

A total of 30APP+/+ (29.2±1.42 g) and 30APP−/− (26.2±2.36 g)male mice were used in this experiment comprising 20 injuredmice and 10 uninjured mice. Injury was induced using a modi-fied version of the Marmarou model of diffuse axonal injury(Marmarou et al., 1994). Ten to sixteen week old mice wereanaesthetizedwith isoflurane and the skull exposed bymidline

incision so that a stainless steel disc (1 mm in diameter, 1 mmthick) could be fixed rigidly to the mouse's skull centrally be-tween the lambda and bregma. After being placed on a foambed (60/80 density/hardness), injury was induced by droppinga 95 g steel weight from 1.2 or 1.3 m, with the APP−/− mice in-jured from the lower height. This injury was found to induce amild diffuse axonal injury, with injured axons seen within thecorpus callosum and brain stem (Supplementary data). Shamoperatedmicewere surgically prepared but not injured. Follow-ing injury, righting reflex, which is measured as the time takenfor an animal to return to a spontaneous upright position fol-lowing injury, was recorded.

4.1. Acute neurological response

At 1 h post-injury a subset of mice (n=5 per group) were ratedon the 10 point Neurological Severity Scale (NSS), a compositebehavioural scale designed to measure the general neurologi-cal state, as described in detail elsewhere (Beni-Adani et al.,2001; Flierl et al., 2009). The NSS recorded at 1 h post-injuryis considered to be a measure of injury severity (Flierl et al.,2009; Tsenter et al., 2008). The mice were assessed for the fol-lowing items: presence of paresis; inability to walk straight;impairment of seeking behaviour; absence of startle reflex; in-ability to exit a 30 cm diameter circle; inability to walk on 3, 2and 1 cm wide beams, and inability to balance on a 0.7 cmwide beam and a 0.5 cm diameter round beam for at least10 s. If a mouse failed to complete one of these items a valueof 1 was added to its NSS score, with a score of 10 thus indicat-ing the greatest impairment.

4.2. Motor outcome

Post-traumatic motor deficits were assessed using a rotarod de-vice (n=8 per group). Briefly, this device consists of a motorisedassembly of 12 rods that are 2mm in diameter, which increasesin speed from 0 to 30 revolutions per minute (rpm) at a rate of3 rpm/10 s. The mice were required to walk on the rods, withthe latency to fall from the rotating bars or to grip the rods andspin two consecutive rotations recorded. APP+/+ mice wereexpected to complete 180 s on the rotarod, whilst APP−/− micewere only expected to complete 120 s due to the forearm weak-ness that is part of their phenotype (27). All mice werepre-trained daily for 5 days, with their best time taken as theirpre-injury baseline level and then assessed for 7 days afterinjury.

4.3. Cognitive outcome

The Barnes maze paradigm exploits the natural inclination ofsmall rodents to seek escape to a darkly lit, sheltered environ-ment (Barnes, 1979) and consists of a white circular platform(diameter 120 cm) elevated 70 cm above the floor, fromwhich the mouse could escape into 1 of 40 holes (5 cm in di-ameter) evenly spaced around the perimeter. The escapehole was connected to an escape box with distinct spatialclues located all around the maze to aid the mice in findingthe correct hole. As such mice have to use a hippocampal de-pendent spatial strategy in order to find the escape hole(Koopmans et al., 2003).

96 B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

During pre-training, animals were initially habituated byplacing them into the escape box and leaving them there for3 min. Oneminute later, the first trial started, with the animalplaced in a start chamber, comprising a plastic box in the cen-tre of the platform. After 10 s the start chamber was removed,a buzzer and a bright light turned on and the mouse was setfree to explore the maze. The trial finished when the mousehad either entered the escape box or 3 min had passed. Ifthe mouse did not find the escape box within 3 min, the ex-perimenter gently guided the animal there. Once the mousehad entered the escape tunnel, the buzzer and light wereturned off and the mouse was allowed to remain for 1 min. Ifa mouse failed to find the escape box they were given a sec-ond trial that day, with a period of approximately 1 h betweentrials. Animals were pre-trained for 5 days prior to injury, withtheir best time taken as their pre-injury baseline level. Assess-ment was conducted on days 2, 4 and 6 post-injury, with es-cape latency (time in seconds) for the mice to find and enterthe escape box with front paws and trunk recorded, as wellas the number of errors, which were counted each time themouse placed its head within a hole which did not containthe escape box manually recorded (APP+/+ sham, APP−/−sham, APP−/− inj n=8; APP+/+ inj n=7).

On day 7 post-injury the escape boxwas switched to a differ-ent, randomly chosen hole to test the ability of mice to learn anew spatial contingency. Micewere allowed three trials, spaced1 h apart, to learn the location of the new holewith their escapelatency recorded as above.

4.4. Histological analysis

Mice were transcardially perfused with 10% formalin on day 3(n=5 per group) or 7 (n=5 per group) post-injury. Serial sec-tions of 5 μm thickness were taken from paraffin embeddedtissue in the region −2.3 mm from the bregma, with this re-gion directly underneath the impact site, and stained withstandard H&E, GAP-43 (1:1000, Novocastra), NFH (Abcam1:10000) or SYP (1:1000, Dako). Following overnight incubationwith either of the specific biotinylated monoclonal antibodies,secondary antibody was applied (1:250 Sigma-Aldrich), fol-lowed by streptavidin peroxidase conjugate (1:1000), withbound antibody then detected with 3,3-diaminobenzidine tet-rahydrochloride (DAB) in the presence of hydrogen peroxideand sections counterstained with haematoxylin. The slideswere incubated with DAB solution for 7 min, with the timenoted for every 15 slides to allow precise determination of in-cubation timewithin a batch to ensure consistency of stainingacross slides. Furthermore, negative controls where the pri-mary antibody was not applied were included.

Sections stained with H&E were used to assess neuronalnumbers within the selectively vulnerable CA3 region of thehippocampus and the cortex directly underneath the impactsite, with the right side analysed (Supplementary Fig. 1A).Four serial sections per mouse were used, with the NDP viewsoftware used to place a rectangle over the CA3 region andcortex to act as a counting frame. The regions within thisframewere sequentially imaged at 40×magnification and dig-itally reconstructed into a montage, to allowmanual countingof the neurons using the cell count software associated withImage J. Only histologically normal appearing neurons with

a clearly defined cell body and nucleus were counted, whilstthose that were only partially seen due to the level of section-ing or were only partially within the counting frame were notincluded, nor were those that appeared pyknotic (dark,shrunken with no visible nucleus). The average number ofneurons was then calculated by totalling the number of neu-rons and dividing this by the calculated area of the countingframe. A similar methodwas used for assessment of the num-ber of GAP-43 positive cells within the cortex underneath theimpact site and the CA1 region (Supplementary Fig. 1B). Twoserial sections per mouse were used, with cells showingclear GAP-43 immunostaining counted, with the final resultexpressed as GAP-43 positive cells/mm2.

To evaluate axonal injury, the number of NFH immunopo-sitive axonal lengths within the central portion of the corpuscallosum was counted. A counting frame was placed overthe area, and the number of injured axons/mm2 determined.

4.5. Caspase-3 ELISA

At either day 3 or 7 post-injury (n=5 per group) mice were re-anaesthetized with isoflurane, decapitated and the brain wasremoved. The hippocampus and the region of cortex directlyunder the impact site were dissected and rapidly frozen in liq-uid nitrogen. Following homogenisation, the amount of proteinwas determined using theBioRad protein assay, with each sam-ple diluted with tris-buffered saline to 400 ng protein per 100 μlof TBS. 100 μl of each sample was loaded in triplicate, and thewells were blocked with 0.2% gelatine before being incubatedwith anti-active caspase 3 (1:1000 BioVision). Secondary anti-rabbit horseradish peroxidise conjugate (1:2000 Rockford) wasadded before the liquid substrate system 3,3′5,5′-tetramethyl-benzidine (Sigma) was used to reveal protein expression. Thereaction was stopped after 100 s with 0.5 M sulfuric acid andlevels of expression determined by reading absorbance at450 nm on an Ascent Multiscan plate reader. To demonstratereproducibility the ELISA was conducted 3 times.

4.6. Statistical analysis

Nonparametric data were analysed with the Kruskal Wallis testfollowed by multiple post-hoc comparisons with Dunn's tests.Parametric data was analysed using a two-way analysis of vari-ance (ANOVA) followed by Bonferonni t tests using GraphpadPrism software. A p value of less than 0.05 was considered signif-icant in all experiments.

Supplementary materials related to this article can befound online at doi:10.1016/j.brainres.2012.02.045

Acknowledgments

We thank Prof. Hui Zheng for providing the APP knockoutmice and Dr Stephen Helps, Dr Emma Thornton, Dr Jenna Zei-bell and Jim Manavis for expert technical assistance. We alsothank the staff within the IMVS animal care facility. Thiswork was funded in part by grants from the Brain Foundationand the Neurological Research Foundation. RC is a NationalHealth and Medical Research Council Senior Research Fellow.

97B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

R E F E R E N C E S

Araki, W., Kitaguchi, N., Tokushima, Y., Ishii, K., Aratake, H.,Shimohama, S., Nakamura, S., Kimura, J., 1991. Trophic effectof beta-amyloid precursor protein on cerebral cortical neuronsin culture. Biochem. Biophys. Res. Commun. 181, 265–271.

Barnes, C.A., 1979. Memory deficits associated with senescence: aneurophysiological and behavioral study in the rat. J. Comp.Physiol. Psychol. 93, 74–104.

Bazarian, J.J., Zhong, J., Blyth, B., Zhu, T., Kavcic, V., Peterson, D.,2007. Diffusion tensor imaging detects clinically importantaxonal damage after mild traumatic brain injury: a pilot study.J. Neurotrauma 24, 1447–1459.

Bell, K.F., Zheng, L., Fahrenholz, F., Cuello, A.C., 2006. ADAM-10over-expression increases cortical synaptogenesis. Neurobiol.Aging 29, 554–565.

Bendotti, C., Baldessari, S., Pende, M., Southgate, T., Guglielmetti, F.,Samanin, R., 1997. Relationship betweenGAP-43 expression in thedentate gyrus and synaptic reorganization of hippocampalmossyfibres in rats treated with kainic acid. Eur. J. Neurosci. 9, 93–101.

Beni-Adani, L., Gozes, I., Cohen, Y., Assaf, Y., Steingart, R.A.,Brenneman, D.E., Eizenberg, O., Trembolver, V., Shohami, E., 2001.A peptide derived from activity-dependent neuroprotectiveprotein (ADNP) ameliorates injury response in closed head injuryin mice. J. Pharmacol. Exp. Ther. 296, 57–63.

Bhasin, R., Van Nostrand, W.E., Saitoh, T., Donets, M.A., Barnes,E.A., Quitschke, W.W., Goldgaber, D., 1991. Expression of activesecreted forms of human amyloid beta-protein precursor byrecombinant baculovirus-infected insect cells. Proc. Natl.Acad. Sci. U. S. A. 88, 10307–10311.

Blasko, I., Beer, R., Bigl, M., Apelt, J., Franz, G., Rudzki, D.,Ransmayr, G., Kampfl, A., Schliebs, R., 2004. Experimentaltraumatic brain injury in rats stimulates the expression,production and activity of Alzheimer's disease beta-secretase(BACE-1). J. Neural Transm. 111, 523–536.

Blumbergs, P.C., Scott, G., Manavis, J., Wainwright, H., Simpson,D.A., McLean, A.J., 1994. Staining of amyloid precursor protein tostudy axonal damage inmild head injury. Lancet 344, 1055–1056.

Boyd-Kimball, D., Sultana, R., Mohmmad-Abdul, H., Butterfield,D.A., 2004. Rodent Abeta(1–42) exhibits oxidative stressproperties similar to those of human Abeta(1–42): Implicationsfor proposed mechanisms of toxicity. J. Alzheimers Dis. 6,515–525.

Caille, I., Allinquant, B., Dupont, E., Bouillot, C., Langer, A., Muller,U., Prochiantz, A., 2004. Soluble form of amyloid precursorprotein regulates proliferation of progenitors in the adultsubventricular zone. Development 131, 2173–2181.

Chen, X.H., Siman, R., Iwata, A., Meaney, D.F., Trojanowski, J.Q.,Smith, D.H., 2004. Long-term accumulation of amyloid-beta,beta-secretase, presenilin-1, and caspase-3 in damaged axonsfollowing brain trauma. Am. J. Pathol. 165, 357–371.

Christman, C.W., Salvant Jr., J.B., Walker, S.A., Povlishock, J.T., 1997.Characterization of a prolonged regenerative attempt bydiffusely injured axons following traumatic brain injury in adultcat: a light and electron microscopic immunocytochemicalstudy. Acta Neuropathol. 94, 329–337.

Creed, J.A., DiLeonardi, A.M., Fox, D.P., Tessler, A.R., Raghupathi,R., 2011. Concussive brain trauma in the mouse results inacute cognitive deficits and sustained impairment of axonalfunction. J. Neurotrauma 28, 547–563.

Dawson, G.R., Seabrook, G.R., Zheng, H., Smith, D.W., Graham, S.,O'Dowd, G., Bowery, B.J., Boyce, S., Trumbauer, M.E., Chen, H.Y.,Van der Ploeg, L.H., Sirinathsinghji, D.J., 1999. Age-relatedcognitive deficits, impaired long-termpotentiation and reductionin synaptic marker density in mice lacking the beta-amyloidprecursor protein. Neuroscience 90, 1–13.

Emery, D.L., Raghupathi, R., Saatman, K.E., Fischer, I., Grady, M.S.,McIntosh, T.K., 2000. Bilateral growth-related protein expression

suggests a transient increase in regenerative potential followingbrain trauma. J. Comp. Neurol. 424, 521–531.

Flierl, M.A., Stahel, P.F., Beauchamp, K.M., Morgan, S.J., Smith,W.R., Shohami, E., 2009. Mouse closed head injury modelinduced by a weight-drop device. Nat. Protoc. 4, 1328–1337.

Fraser, P.E., Nguyen, J.T., Chin, D.T., Kirschner, D.A., 1992. Effectsof sulfate ions on Alzheimer beta/A4 peptide assemblies:implications for amyloid fibril-proteoglycan interactions. J.Neurochem. 59, 1531–1540.

Fujimoto, S.T., Longhi, L., Saatman, K.E., Conte, V., Stocchetti, N.,McIntosh, T.K., 2004. Motor and cognitive function evaluationfollowing experimental traumatic brain injury. Neurosci.Biobehav. Rev. 28, 365–378.

Furukawa, K., Sopher, B.L., Rydel, R.E., Begley, J.G., Pham, D.G.,Martin, G.M., Fox, M., Mattson, M.P., 1996. Increasedactivity-regulating and neuroprotective efficacy ofalpha-secretase-derived secreted amyloid precursor proteinconferred by a C-terminal heparin-binding domain. J.Neurochem. 67, 1882–1896.

Gaetz, M., 2004. The neurophysiology of brain injury. Clin.Neurophysiol. 115, 4–18.

Gentleman, S.M., Graham, D.I., Roberts, G.W., 1993. Molecularpathology of head trauma: altered beta APP metabolism andthe aetiology of Alzheimer's disease. Prog. Brain Res. 96,237–246.

Goodman, Y., Mattson, M.P., 1994. Secreted forms of beta-amyloidprecursor protein protect hippocampal neurons against amyloidbeta-peptide-induced oxidative injury. Exp. Neurol. 128, 1–12.

Gordon-Krajcer, W., Gajkowska, B., 2001. Excitotoxicity-inducedexpression of amyloid precursor protein (beta-APP) in thehippocampus and cortex of rat brain. An electron-microscopyand biochemical study. Folia Neuropathol. 39, 163–173.

Hellewell, S.C., Yan, E.B., Agyapomaa, D.A., Bye, N.,Morganti-Kossmann, M.C., 2010. Post-traumatic hypoxiaexacerbates brain tissue damage: analysis of axonal injury andglial responses. J. Neurotrauma 27, 1997–2010.

Herms, J., Anliker, B., Heber, S., Ring, S., Fuhrmann, M.,Kretzschmar, H., Sisodia, S., Muller, U., 2004. Cortical dysplasiaresembling human type 2 lissencephaly in mice lacking allthree APP family members. EMBO J. 23, 4106–4115.

Herreman, A., Van Gassen, G., Bentahir, M., Nyabi, O., Craessaerts, K.,Mueller, U., Annaert, W., De Strooper, B., 2003. gamma-Secretaseactivity requires the presenilin-dependent trafficking of nicastrinthrough the Golgi apparatus but not its complex glycosylation. J.Cell Sci. 116, 1127–1136.

Hulsebosch, C.E., DeWitt, D.S., Jenkins, L.W., Prough, D.S., 1998.Traumatic brain injury in rats results in increased expressionof Gap-43 that correlates with behavioral recovery. Neurosci.Lett. 255, 83–86.

Hyder, A.A., Wunderlich, C.A., Puvanachandra, P., Gururaj, G.,Kobusingye, O.C., 2007. The impact of traumatic brain injuries:a global perspective. NeuroRehabilitation 22, 341–353.

Inglese, M., Makani, S., Johnson, G., Cohen, B.A., Silver, J.A., Gonen,O., Grossman, R.I., 2005. Diffuse axonal injury in mildtraumatic brain injury: a diffusion tensor imaging study. J.Neurosurg. 103, 298–303.

Iwata, A., Chen, X.H., McIntosh, T.K., Browne, K.D., Smith, D.H., 2002.Long-termaccumulation of amyloid-beta in axons following braintrauma without persistent upregulation of amyloid precursorprotein genes. J. Neuropathol. Exp. Neurol. 61, 1056–1068.

Johnson, V.E., Stewart, W., Smith, D.H., 2010. Traumatic brain in-jury and amyloid-beta pathology: a link to Alzheimer's dis-ease? Nat. Rev, Neurosci.

Kay, A.D., Petzold, A., Kerr, M., Keir, G., Thompson, E., Nicoll, J.A.,2003. Alterations in cerebrospinal fluid apolipoprotein E andamyloid beta-protein after traumatic brain injury. J.Neurotrauma 20, 943–952.

Kim, H.S., Kim, E.M., Kim, N.J., Chang, K.A., Choi, Y., Ahn, K.W.,Lee, J.H., Kim, S., Park, C.H., Suh, Y.H., 2004. Inhibition of

98 B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

histone deacetylation enhances the neurotoxicity induced bythe C-terminal fragments of amyloid precursor protein. J.Neurosci. Res. 75, 117–124.

Koo, E.H., Squazzo, S.L., Selkoe, D.J., Koo, C.H., 1996. Traffickingof cell-surface amyloid beta-protein precursor. I. Secretion,endocytosis and recycling as detected by labeled monoclonalantibody. J. Cell Sci. 109 (Pt 5), 991–998.

Koopmans, G., Blokland, A., van Nieuwenhuijzen, P., Prickaerts, J.,2003. Assessment of spatial learning abilities of mice in a newcircular maze. Physiol. Behav. 79, 683–693.

Leyssen, M., Ayaz, D., Hebert, S.S., Reeve, S., De Strooper, B.,Hassan, B.A., 2005. Amyloid precursor protein promotespost-developmental neurite arborization in the Drosophilabrain. EMBO J. 24, 2944–2955.

Lin, L.H., Bock, S., Carpenter, K., Rose, M., Norden, J.J., 1992.Synthesis and transport of GAP-43 in entorhinal cortexneurons and perforant pathway during lesion-inducedsprouting and reactive synaptogenesis. Brain Res. Mol. BrainRes. 14, 147–153.

Loane, D.J., Pocivavsek, A., Moussa, C.E., Thompson, R., Matsuoka,Y., Faden, A.I., Rebeck, G.W., Burns, M.P., 2009. Amyloidprecursor protein secretases as therapeutic targets fortraumatic brain injury. Nat. Med. 15, 377–379.

Loane, D.J., Washington, P.M., Vardanian, L., Pocivavsek, A., Hoe,H.S., Duff, K.E., Cernak, I., Rebeck, G.W., Faden, A.I., Burns, M.P.,2011. Modulation of ABCA1 by an LXR agonist reducesbeta-amyloid levels and improves outcome after traumaticbrain injury. J. Neurotrauma 28, 225–236.

Mannix, R.C., Zhang, J., Park, J., Zhang, X., Bilal, K., Walker, K.,Tanzi, R.E., Tesco, G., Whalen, M.J., 2010. Age-dependent effectof apolipoprotein E4 on functional outcome after controlledcortical impact in mice. J. Cereb. Blood FlowMetab. 31, 351–361.

Marmarou, A., Foda, M.A., van den Brink, W., Campbell, J., Kita, H.,Demetriadou, K., 1994. A new model of diffuse brain injury inrats. Part I: Pathophysiology and biomechanics. J. Neurosurg.80, 291–300.

Marmarou, C.R., Walker, S.A., Davis, C.L., Povlishock, J.T., 2005.Quantitative analysis of the relationship between intra- axonalneurofilament compaction and impaired axonal transportfollowing diffuse traumatic brain injury. J. Neurotrauma 22,1066–1080.

Mattson, M.P., 1997. Cellular actions of beta-amyloid precursorprotein and its soluble and fibrillogenic derivatives. Physiol.Rev. 77, 1081–1132.

Mattson, M.P., Cheng, B., Culwell, A.R., Esch, F.S., Lieberburg, I.,Rydel, R.E., 1993. Evidence for excitoprotective and intraneuronalcalcium-regulating roles for secreted forms of the beta-amyloidprecursor protein. Neuron 10, 243–254.

McKinney, R.A., Luthi, A., Bandtlow, C.E., Gahwiler, B.H., Thompson,S.M., 1999. Selective glutamate receptor antagonists can induceor prevent axonal sprouting in rat hippocampal slice cultures.Proc. Natl. Acad. Sci. U. S. A. 96, 11631–11636.

Messe, A., Caplain, S., Paradot, G., Garrigue, D., Mineo, J.F., SotoAres, G., Ducreux, D., Vignaud, F., Rozec, G., Desal, H., Pelegrini-Issac, M., Montreuil, M., Benali, H., Lehericy, S., 2010. Diffusiontensor imaging and white matter lesions at the subacute stagein mild traumatic brain injury with persistent neurobehavioralimpairment. Hum. Brain Mapp. 32, 999–1011.

Murakami, N., Yamaki, T., Iwamoto, Y., Sakakibara, T., Kobori, N.,Fushiki, S., Ueda, S., 1998. Experimental brain injury inducesexpression of amyloid precursor protein, whichmay be related toneuronal loss in the hippocampus. J. Neurotrauma 15, 993–1003.

Nihashi, T., Inao, S., Kajita, Y., Kawai, T., Sugimoto, T., Niwa, M.,Kabeya, R., Hata, N., Hayashi, S., Yoshida, J., 2001. Expressionand distribution of beta amyloid precursor protein and betaamyloid peptide in reactive astrocytes after transient middlecerebral artery occlusion. Acta Neurochir. (Wien) 143, 287–295.

Ohsawa, I., Takamura, C., Kohsaka, S., 1997. The amino-terminalregion of amyloid precursor protein is responsible for neurite

outgrowth in rat neocortical explant culture. Biochem. Biophys.Res. Commun. 236, 59–65.

Olsson, A., Csajbok, L., Ost, M., Hoglund, K., Nylen, K., Rosengren,L., Nellgard, B., Blennow, K., 2004. Marked increase ofbeta-amyloid(1–42) and amyloid precursor protein inventricular cerebrospinal fluid after severe traumatic braininjury. J. Neurol. 251, 870–876.

Opazo, C., Ruiz, F.H., Inestrosa, N.C., 2000. Amyloid-beta-peptidereduces copper(II) to copper(I) independent of its aggregationstate. Biol. Res. 33, 125–131.

Phinney, A.L., Calhoun, M.E., Wolfer, D.P., Lipp, H.P., Zheng, H.,Jucker, M., 1999. No hippocampal neuron or synaptic boutonloss in learning-impaired aged beta-amyloid precursorprotein-null mice. Neuroscience 90, 1207–1216.

Popa-Wagner, A., Schroder, E., Walker, L.C., Kessler, C., 1998.beta-Amyloid precursor protein and ss-amyloid peptideimmunoreactivity in the rat brain after middle cerebral arteryocclusion: effect of age. Stroke 29, 2196–2202.

Povlishock, J.T., Marmarou, A., McIntosh, T., Trojanowski, J.Q.,Moroi, J., 1997. Impact acceleration injury in the rat: evidencefor focal axolemmal change and related neurofilamentsidearm alteration. J. Neuropathol. Exp. Neurol. 56, 347–359.

Qiu, W.Q., Ferreira, A., Miller, C., Koo, E.H., Selkoe, D.J., 1995.Cell-surface beta-amyloid precursor protein stimulates neuriteoutgrowth of hippocampal neurons in an isoform-dependentmanner. J. Neurosci. 15, 2157–2167.

Raby, C.A., Morganti-Kossmann, M.C., Kossmann, T., Stahel, P.F.,Watson, M.D., Evans, L.M., Mehta, P.D., Spiegel, K., Kuo, Y.M.,Roher, A.E., Emmerling, M.R., 1998. Traumatic brain injuryincreases beta-amyloid peptide 1–42 in cerebrospinal fluid. J.Neurochem. 71, 2505–2509.

Raghupathi, R., Margulies, S.S., 2002. Traumatic axonal injuryafter closed head injury in the neonatal pig. J. Neurotrauma 19,843–853.

Ring, S., Weyer, S.W., Kilian, S.B., Waldron, E., Pietrzik, C.U.,Filippov, M.A., Herms, J., Buchholz, C., Eckman, C.B., Korte, M.,Wolfer, D.P., Muller, U.C., 2007. The secreted beta-amyloidprecursor protein ectodomain APPs alpha is sufficientto rescue the anatomical, behavioral, andelectrophysiological abnormalities of APP-deficient mice. J.Neurosci. 27, 7817–7826.

Roberts, G.W., Gentleman, S.M., Lynch, A., Graham, D.I., 1991. betaA4 amyloid protein deposition in brain after head trauma.Lancet 338, 1422–1423.

Saitoh, T., Sundsmo, M., Roch, J.M., Kimura, N., Cole, G., Schubert,D., Oltersdorf, T., Schenk, D.B., 1989. Secreted form of amyloidbeta protein precursor is involved in the growth regulation offibroblasts. Cell 58, 615–622.

Shukla, D., Devi, B.I., 2010. Mild traumatic brain injuries in adults.J. Neurosci. Rural Pract. 1, 82–88.

Smith, D.H., Chen, X.H., Nonaka, M., Trojanowski, J.Q., Lee, V.M.,Saatman, K.E., Leoni, M.J., Xu, B.N., Wolf, J.A., Meaney, D.F.,1999. Accumulation of amyloid beta and tau and the formationof neurofilament inclusions following diffuse brain injury inthe pig. J. Neuropathol. Exp. Neurol. 58, 982–992.

Smith-Swintosky, V.L., Pettigrew, L.C., Craddock, S.D., Culwell, A.R.,Rydel, R.E., Mattson, M.P., 1994. Secreted forms of beta-amyloidprecursor protein protect against ischemic brain injury. J.Neurochem. 63, 781–784.

Stone, J.R., Okonkwo, D.O., Singleton, R.H., Mutlu, L.K., Helm, G.A.,Povlishock, J.T., 2002. Caspase-3-mediated cleavage of amyloidprecursor protein and formation of amyloid Beta peptide intraumatic axonal injury. J. Neurotrauma 19, 601–614.

Suh, Y.H., Checler, F., 2002. Amyloid precursor protein, presenilins,and alpha-synuclein: molecular pathogenesis andpharmacological applications in Alzheimer's disease.Pharmacol. Rev. 54, 469–525.

Teasdale, G., Jennett, B., 1974. Assessment of coma and impairedconsciousness. A practical scale. Lancet 2, 81–84.

99B R A I N R E S E A R C H 1 4 5 1 ( 2 0 1 2 ) 8 7 – 9 9

Thompson, S.N., Gibson, T.R., Thompson, B.M., Deng, Y., Hall, E.D.,2006. Relationship of calpain-mediated proteolysis to theexpression of axonal and synaptic plasticity markers followingtraumatic brain injury in mice. Exp. Neurol. 201, 253–265.

Thornton, E., Vink, R., Blumbergs, P.C., Van Den Heuvel, C., 2006.Soluble amyloid precursor protein alpha reduces neuronal injuryand improves functional outcome following diffuse traumaticbrain injury in rats. Brain Res. 1094, 38–46.

Tsenter, J., Beni-Adani, L., Assaf, Y., Alexandrovich, A.G.,Trembovler, V., Shohami, E., 2008. Dynamic changes in therecovery after traumatic brain injury in mice: effect of injuryseverity on T2-weighted MRI abnormalities, and motor andcognitive functions. J. Neurotrauma 25, 324–333.

Van den Heuvel, C., Blumbergs, P.C., Finnie, J.W., Manavis, J., Jones,N.R., Reilly, P.L., Pereira, R.A., 1999. Upregulation of amyloid

precursor protein messenger RNA in response to traumaticbrain injury: an ovine head impact model. Exp. Neurol. 159,441–450.

Wallace, W.C., Akar, C.A., Lyons, W.E., 1997. Amyloid precursorprotein potentiates the neurotrophic activity of NGF. Brain Res.Mol. Brain Res. 52, 201–212.

Wang, Y., Ha, Y., 2004. The X-ray structure of an antiparallel dimerof the human amyloid precursor protein E2 domain.Mol. Cell 15,343–353.

Zheng, H., Jiang, M., Trumbauer, M.E., Sirinathsinghji, D.J.,Hopkins, R., Smith, D.W., Heavens, R.P., Dawson, G.R., Boyce,S., Conner, M.W., Stevens, K.A., Slunt, H.H., Sisoda, S.S., Chen,H.Y., Van der Ploeg, L.H., 1995. beta-Amyloid precursorprotein-deficient mice show reactive gliosis and decreasedlocomotor activity. Cell 81, 525–531.