Amyloid β accumulation in axons after traumatic brain injury in humans

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Object. Although plaques composed of amyloid β (Aβ) have been found shortly after traumatic brain injury (TBI) in humans, the source for this Aβ has not been identified. In the present study, the authors explored the potential relationship between Aβ accumulation in damaged axons and associated Aβ plaque formation.

Methods. The authors performed an immunohistochemical analysis of paraffin-embedded sections of brain from 12 patients who died after TBI and from two control patients by using antibodies selective for Aβ peptides, amyloid precursor protein (APP), and neurofilament (NF) proteins. In nine brain-injured patients, extensive colocalizations of Aβ, APP, and NF protein were found in swollen axons. Many of these immunoreactive axonal profiles were present close to Aβ plaques or were surrounded by Aβ staining, which spread out into the tissue. Immunoreactive profiles were not found in the brains of the control patients.

Conclusions. The results of this study indicate that damaged axons can serve as a large reservoir of Aβ, which may contribute to Aβ plaque formation after TBI in humans.

There is considerable evidence that links TBI and AD. Several epidemiological studies have demonstrated that TBI is an epigenetic risk factor for the development of AD.5,11–13,18,21 In addition, plaques composed of Aβ have been found within days after a single incident of TBI in humans, similar to the hallmark pathological characteristics of plaque in cases of AD.6,19 Nonetheless, mechanisms underlying the potential relationship have yet to be elucidated. Using an inertial brain injury model in pigs that produces diffuse axonal injury, large accumulations of Aβ in damaged axons were found to be distributed throughout the brain,23 suggesting a potential source of Aβ for plaque formation. In the present study, the research team questioned whether Aβ accumulation in axons also occurs in brain-injured humans and if this pathological finding is coincident with Aβ plaque formation.

Materials and Methods

Brain Tissue

Brain specimens from 14 patients (eight male and six female patients) were selected from the Glasgow head-injury database for evaluation. Twelve of these specimens were obtained in patients who had suffered fatal head injuries caused by automobile crashes or falls (details are provided in Table 1). These patients survived from 18 hours to 9 days (mean 2.8 days) after injury and their ages at death ranged from 16 to 71 years (mean 43.4 years). Initial immunohistochemical screening of brain sections from these patients, as part of the database protocol, suggested plaquelike Aβ immunoreactivities by using a monoclonal antibody raised to the amino acid sequence Aβ epitope 8–17 (Dako, Cambridge, UK). Brain sections were also obtained from two additional patients, who were deemed controls because they died of septicemic shock or acute tracheobronchitis, with survival times from presentation to death noted as 31 and 71 hours. All brain sections that were examined contained white matter tracts from either the subcortex, deep white matter, or corpus callosum.

TABLE 1

Clinical details of 12 patients with head injuries and two control patients*

Case No.Age (yrs), SexSurvival Post-injuryCause of InjuryTime to PM ExamSkull FractureICHContusionsDAIIBDSide of SwellingrICPOther FindingsCause of Death
116, M48 hrsRTA37 hrsyesSDHyes1modrtyesnoneTBI
257, M19 hrsNK17 hrsyesyesyes1minltyesnoneTBI
360, F24 hrsRTA63 hrsyesSDHyes1minnoneyesnoneTBI
456, F4 daysfall26 hrsyesSDHyes1minnoneyeshepatitisTBI
529, M18 hrsRTA12 hrsyesnoyes0severeltyesMFMI
665, M23 hrsfall54 hrsyesSDHno0modrtyesCLTBI
759, M4 daysfall37 hrsyesEDHyes0minnoneyesFLTBI
859, F5 daysfall14 hrsyesSDHyes0minltyesnoneTBI
971, M19 hrsfall17 hrsyesSDHyes0minnoneyesnoneTBI
1031, F9 daysRTA84 hrsnonoyes3minbilatyesnoneTBI
1117, M37 hrsRTA63 hrsnonoyes2severebilatyesnoneTBI
1233, F5 daysRTA31 hrsyesSDHyes3minnoneyesnoneTBI
1321, F71 hrsSS
1433, M31 hrsATB

ATB = acute tracheobronchitis; CL = cirrhosis of liver; DAI = diffuse traumatic axonal injury severity grades 0–3; EDH = extradural hematoma; FL = fatty liver; IBD = ischemic brain damage; ICH = intracranial hematoma; MF = multiple fractures; MI = multiple injuries; mod = moderate; NK = not known; PM = postmortem; rICP = raised intracranial pressure; RTA = road traffic accident; SDH = subdural hematoma; SS = septicemic shock.

In each case ApoE genotyping was performed on formalin-fixed tissue, as described previously.14,28

Immunohistochemical Analysis

Seventy adjacent tissue sections were used for the immunohistochemical analysis. After dewaxing, the sections were treated with a solution of 3% H2O2 in methanol for 30 minutes to extinguish endogenous peroxidation. The sections were treated with 88% formic acid for 5 minutes for antigen retrieval and then blocked by placing the sections in 5% normal horse serum for 30 minutes. Subsequently, the sections were incubated overnight in blocking serum at 4°C with a series of primary antibodies specific for NF protein, APP, and Aβ (Table 2). Immunoreactivity was visualized using 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories, Carpinteria, CA). These sections were then dehydrated, counterstained with hematoxylin, and mounted. Double labeling was performed with secondary antibodies of fluorescein sheep anti—mouse IgG (Jackson Immuno-Research Laboratories, Inc., West Grove, PA; 1:200) and Texas red goat anti—rabbit IgG (Molecular Probes, Inc., Eugene, OR; 1:200) or with rhodamine-conjugated rabbit anti—goat IgG (Sigma Chemical Co., St. Louis, MO; 1:200).

TABLE 2

Summary of antibodies used for immunohistochemical analysis

AntibodyEpitope Protein/Amino AcidsType*DilutionSupplier
N52NF-HmAb1:400Sigma Chemical Co., 
 St. Louis, MO 
SMI-31NF-HmAb1:1000Sternberger Monoclonals, 
 Inc., Lutherville, 
 MD 
22C11APP/60–100mAb1:5Boehringer, Indianapolis, 
 IN 
6F/3DAβ/8–17mAb1:50Dako, Cambridge, UK 
amyloidAβ/1–17pAb1:100Santa Cruz Biotechnology, 
 1–17 Inc., Santa 
 Cruz, CA 
amyloidAβ/C-20pAb1:100Santa Cruz Biotechnology, 
C-20 Inc. 
2332Aβ/1–17pAb1:4000Dr. V. M. Y. Lee, University 
 of Pennsylvania, 
 Philadelphia, 
PA 
13335Aβ/1–42pAb1:1000Dr. V. M. Y. Lee 
βA27Aβ/1–40mAb1:1000Dr. V. M. Y. Lee 
4G8Aβ/17–24mAb1:1000Dr. V. M. Y. Lee 
BC05Aβ/42(43)mAb1:200Dr. V. M. Y. Lee 

mAb = monoclonal antibody; pAb = polyclonal antibody.

Semiquantitative Analysis

To explore the relationships between the extent of pathological changes and the age of the patient, postinjury survival interval, and ApoE genotype for each case, the number of immunoreactive profiles of axonal bulbs, plaques, neurons, and corpora amylacea were determined in a semiquantitative fashion by applying a ranked scale. This was done by an observer blinded to the circumstances of each case, as has been described previously in detail.23,26 Only immunoreactive profiles that could be clearly identified based on morphological characteristics were counted in each section. These profiles were counted in three to five fields (each field measuring ∼ 1.2 mm2) for each brain section and an average for each profile type was determined per field. A simple scale was used to express the extent of damage in each case in the following manner: +, fewer than five profiles; ++, six to 15 profiles; and +++, more than 15 profiles.

Results
Axonal Pathology

No axonal injury or plaquelike profiles were detected by any stain in the control brains. In brain sections obtained from the 12 brain-injured patients, varying densities of axonal bulbs and axonal swellings were observed following trauma (18 hours—9 days postinjury), with a relatively high density of axonal damage found in 10 patients with brain injuries (Fig. 1A–C, Table 3). These axonal lesions were identified by antibodies targeting NF protein (N52 and SMI-31) and APP (22C11). In nine of these cases, extensive Aβ immunoreactivity was found in the terminal swellings of disconnected axons, which was elucidated by the following antibodies: amyloid 1–17, amyloid C-20, 2332, 13335, and BC05. Double immunostaining demonstrated colocalization of immunoreactivity of Aβ with APP and NF protein in the axonal bulbs (Fig. 2A–L). Although all Aβ-immunoreactive axons colabeled with APP and NF immunoreactivity, many varicose axonal swellings only stained for APP and NF. For many Aβ-immunoreactive axons, we also observed a dissemination of Aβ immunoreactivity into the surrounding tissue. In three head-injured patients, Aβ immunoreactivity was not detected, despite the identification of extensive axonal damage (Table 3).

Fig. 1.
Fig. 1.

Representative photomicrographs of APP, Aβ, and NF protein accumulations in the brain following TBI in patients. The APP and NF protein accumulations in swollen axons are shown as darkly staining immunoreactivity to the antibodies 22C11(A) and N52 (B and C). The Aβ plaque formations are observed in cortical gray matter stained by 6F/3D antibody (D). In the white matter, Aβ plaques are found close to axonal bulbs, which also stain for Aβ, by applying the antibodies 13335 and Aβ17 (arrows in E and F, respectively). Immunoreactivity for NF protein was also occasionally detected in neurons (G and H) and in corpora amylacea (I). Scale bars = 20 µm.

TABLE 3

Comparison of patient age, postinjury survival, and ApoE genotype with pathological findings*

Postinjury Survival (hrs)Axonal BulbsPlaquesNeuronsCorpora Amylacea
Case No.Patient AgeApoE GenotypeAPPNFPAPPNFPAPPNFPAPPNFP 
116483/3+++++++++++++
257194/4+++++++++
360243/4++++++++++++++++++
456943/4+++++++++++++++
529183/4++++++++++++++++++
665234/4+++++++++++
759963/3+++++++++++++++
8591204/4+++++
971194/4++++++
10312162/3++++++++
1117372/3++++++++++++++++
12331203/4++++++
1321713/4
1433313/3

Plaques, axonal bulbs, and corpora amylacea were counted in different fields and average immunoreactive plaques and bulbs were classified as follows: +, less than five deposits; ++, six to 15 deposits; +++, more than 15 deposits. Abbreviation: NFP = NF protein.

Fig. 2.
Fig. 2.

Representative fluorescence photomicrographs demonstrating coaccumulations of APP, Aβ, and NF proteins in the brain following TBI in patients. Double fluorescence immunohistochemical analysis reveals colocalization in axonal bulbs of Aβ (green area in A and G visualized using amyloid 1–17 antibody, and green area in D and red area in J visualized by 13335 antibody) with either APP (red areas in B and E stained by 22C11 antibody) or NF protein (red areas in H and green areas in K stained by N52 antibody). Superimposing the double-stained tissue demonstrates a complete overlap in the immunoreactive axon bulbs (yellow areas in C, F, I and L). Plaques immunoreactive to the Aβ 13335 antibody were found in the white matter (M, large central profile marked by downward pointing arrow), in proximity to axonal bulbs (M, smaller profiles marked by diagonal arrows). Double-staining this section for NF protein with N52 antibody demonstrates coaccumulation of NF protein with Aβ in the axonal bulbs, but no NF staining in the region of the Aβ plaque (N; both superimposed in O). Accumulation of NF protein was also observed in some cortical neurons (P) that colocalized with Aβ (Q; both superimposed in R). Scale bars = 20 µm.

Plaquelike Profiles Immunoreactive for Aβ

In eight specimens obtained in brain-injured patients that demonstrated Aβ immunoreactivity in axons, plaquelike profiles immunoreactive for Aβ were identified in the cortex by using the antibodies 2332, 13335, 4G8, βA27, 6F/3D, and BC05 (Fig. 1D and E, Fig. 2M–O, and Table 3). Some Aβ plaquelike profiles were also found in the white matter close to Aβ-immunoreactive axons. Although swollen axons consistently demonstrated NF protein accumulation, as expected, no accumulation of NF protein was found in plaques labeled for Aβ peptide (Fig. 2M–O).

Accumulation of Aβ and APP in Neurons

In seven specimens obtained in brain-injured patients, immunoreactivity for Aβ and APP was identified in the cytoplasm of neurons throughout the cortex by using the antibodies 2332, 13335, amyloid 1–17, amyloid C-20, and 6F3D (Fig. 1G and H, Fig. 2P–R, and Table 3).

Immunoreactivity for Aβ and NF in the Corpora Amylacea

In nine specimens obtained from brain-injured patients, immunoreactivity for Aβ and NF protein was found in discrete round profiles that appeared to be corpora amylacea. Distinguishing the corpora amylacea is important because they stain with many antibodies and could potentially be confused with axonal swellings (Fig. 1I and Table 3).

Comparisons of Patient Age, Postinjury Survival, and ApoE Genotype With Pathological Findings

No obvious relationship was found between the extent of the damage demonstrated by immunoreactivity in the axonal bulbs, plaques, neurons, and corpora amylacea, and the age, postinjury survival, or ApoE genotype of the brain-injured patients (Table 3). It should be noted, however, that the total number of cases was relatively small for such comparisons. Nonetheless, a consistent association was found; in all cases in which Aβ plaques were identified, Aβ accumulation in damaged axons was also observed.

Discussion

In this report we present the first evidence of extensive Aβ accumulation in the terminal ends of disconnected axons after brain trauma in humans. Many of these immunoreactive axon profiles were found close to Aβ plaques or surrounded by Aβ staining that spread out into the tissue. In addition, Aβ immunoreactivity in damaged axons was consistently found to colocalize with staining for APP and NF protein. On the basis of these data we can infer that damaged axons may represent a key source of Aβ peptides for plaque formation after TBI in humans.

Axonal injury is one of the most important and most common features of diffuse TBI in humans.1,24 It has been well established that damage to axons can lead to impaired axonal transport and massive accumulation of proteins, including APP.4,10,15,16,22,27,29 Although it has long been suspected that this accumulated APP could provide an ample substrate for Aβ production,10,16,22,25 only recently has Aβ been identified in damaged axons after inertial brain trauma in the pig and following head impact in the rat.9,23,27 In both the pig model and the present study, Aβ was found to coaccumulate with APP in swollen axons throughout the injured brain. Nevertheless, Aβ was not found in all damaged axons. Typically, Aβ was identified in axon bulbs (discrete terminal swellings of disconnected axons), but not in elongated varicose axonal swellings that were otherwise visualized by APP staining. Thus, impaired axonal transport alone does not appear to account for the accumulation of Aβ. Rather, axon disconnection may favor processes that lead to the production and/or accumulation of Aβ, such as enhanced proteolysis of APP. Whether these processes are active in all patients with axonal damage remains to be determined. In the present study, brain sections from three patients revealed extensive areas of axonal injury with no Aβ immunoreactivity. Nonetheless, this observation does not rule out the possibility that Aβ could have accumulated in regions that were not examined or that Aβ formation and accumulation may be more delayed in some patients.

It is important to consider that the large pool of axonal Aβ may play a role in further pathogenesis after brain trauma. During lysis or leakage of axonal bulbs, Aβ may be released into surrounding tissue and cerebrospinal fluid. This process may partially account for recent reports in which large increases in Aβ peptides have been described in the cerebrospinal fluid of brain-injured patients.3,17 Conceivably, released axonal Aβ could aggregate in the tissue parenchyma, potentially representing a mechanism of Aβ plaque formation after brain trauma. In the present study, only patients with substantial Aβ accumulation in axons also demonstrated a large number of Aβ plaques. Although relatively few Aβ plaques were found in the pig model of inertial brain injury, those plaques were consistently found close to axonal Aβ accumulations.23 It is not presently clear why humans apparently have an increased susceptibility, compared with pigs, to form Aβ plaques after trauma; however, species-specific amino acid sequencing and processing of APP may partially account for this difference, as has been previously suggested with regard to rodents.16

It is also important to note that our current data corroborate earlier findings of Aβ plaque formation shortly after brain trauma in humans.6,19 There has been some conflict in this area, with one study failing to elucidate Aβ plaques in brain-injured patients.2 Although reasons for this disparity are not clear, differences in immunohistochemical analyses between laboratories may have accounted for variable outcomes. In the present study we used a panel of antibodies specific to Aβ epitopes in combination with antigen retrieval techniques to confirm the presence of Aβ plaques, as well as to identify Aβ accumulations in damaged axons.

Previously, it has been found that patients with AD who have the ApoE ∈4 allele have increased Aβ deposits.20 Individuals with ApoE ∈4 also have a worsened outcome after TBI7,28 and are more likely to have Aβ plaques in their brains shortly following injury.8,14 Because of the limited number of cases, the present data do not demonstrate a relationship between ApoE genotype and Aβ plaques following brain trauma. Nonetheless, in all cases in which Aβ plaques were identified, extensive Aβ accumulation in damaged axons was also found, often close by. This observation supports a potential link between these two posttrauma pathological conditions.

Conclusions

It is recognized that axonal injury is one of the most common and extensive pathological conditions of brain trauma in humans. Accordingly, our current finding of Aβ accumulation in damaged axons reveals a potentially important reservoir of A that can form within days after brain trauma. Release of axonal Aβ may represent a key mechanism leading to further pathogenesis, including Aβ plaque formation. Ultimately, this process may play a role in the link between brain trauma and the development of AD.

Acknowledgments

We thank John Q. Trojanowski and Virginia M. Y. Lee, Department of Laboratory Medicine, University of Pennsylvania, for their generous donation of antibodies and their helpful comments.

References

  • 1.

    Adams JHGraham DIGennarelli TAet al: Diffuse axonal injury in non-missile head injury. J Neurol Neurosurg Psychiatry 54:4814831991J Neurol Neurosurg Psychiatry 54:

  • 2.

    Adle-Biassette HDuyckaerts CWasowicz Met al: Beta AP deposition and head trauma. Neurobiol Aging 17:4154191996Neurobiol Aging 17:

  • 3.

    Emmerling MRMorganti-Kossmann MCKossmann Tet al: Traumatic brain injury elevates the Alzheimer's amyloid peptide A beta 42 in human CSF. A possible role for nerve cell injury. Ann NY Acad Sci 903:1181222000Ann NY Acad Sci 903:

  • 4.

    Gentleman SMNash MJSweeting CJet al: Related beta-amyloid precursor protein (beta APP) as a marker for axonal injury after head injury. Neurosci Lett 160:1391441993Neurosci Lett 160:

  • 5.

    Gottlieb S: Head injury doubles the risk of Alzheimer's disease. Br Med J 321:11002000Gottlieb S: Head injury doubles the risk of Alzheimer's disease. Br Med J 321:

  • 6.

    Graham DIGentleman SMLynch Aet al: Distribution of betaamyloid protein in the brain following severe head injury. Neuropathol Appl Neurobiol 21:27341995Neuropathol Appl Neurobiol 21:

  • 7.

    Friedman GFroom PSazbon Let al: Apolipoprotein E-epsilon4 genotype predicts a poor outcome in survivors of traumatic brain injury. Neurology 52:2442481999Neurology 52:

  • 8.

    Horsburgh KMcCarron MOWhite Fet al: The role of apolipoprotein E in Alzheimer's disease, acute brain injury and cerebrovascular disease: evidence of common mechanisms and utility of animal models. Neurobiol Aging 21:2452552000Neurobiol Aging 21:

  • 9.

    Iwata AChen XHMcIntosh TKet al Long-term accumulation of amyloid-β in axons following brain trauma without persistent upregulation of amyloid precursor protein genes. J Neuropathol Exp Neurol 61:105610682002J Neuropathol Exp Neurol 61:

  • 10.

    Lewen ALi GLNilsson Pet al: Traumatic brain injury in rat produces changes of β-amyloid precursor protein immunoreactivity. Neuroreport 6:3573601995Neuroreport 6:

  • 11.

    Lye TCShores EA: Traumatic brain injury as a risk factor for Alzheimer's disease: a review. Neuropsychol Rev 10:1151292000Neuropsychol Rev 10:

  • 12.

    Mortimer JAvan Duijn CMChandra VHead trauma as a risk factor for Alzheimer's disease: a collaborative re-analysis of case-control studies. EURODEM Risk Factors Research Group. Int J Epidemiol 20 (Suppl 2):S28S351991Int J Epidemiol 20 (Suppl 2):

  • 13.

    Nemetz PNLeibson CNaessens JMet al: Traumatic brain injury and time to onset of Alzheimer's disease: a population-based study. Am J Epidemiol 149:32401999Am J Epidemiol 149:

  • 14.

    Nicoll JARoberts GWGraham DI: Apolipoprotein E epsilon 4 allele is associated with deposition of amyloid beta-protein following head injury. Nat Med 1:1351371995Nat Med 1:

  • 15.

    Pierce JESmith DHTrojanowski JQet al: Enduring cognitive, neurobehavioral and histopathological changes persist for up to one year following severe experimental brain injury in rats. Neuroscience 87:3593691998Neuroscience 87:

  • 16.

    Pierce JETrojanowski JQGraham DIet al: Immunohistochemical characterization of alterations in the distribution of amyloid percursor proteins and β-amyloid peptide after experimental brain injury in the rat. J Neurosci 16:108310901996J Neurosci 16:

  • 17.

    Raby CAMorganti-Kossmann MCKossmann Tet al: Traumatic brain injury increases beta-amyloid peptide 1–42 in cerebrospinal fluid. J Neurochem 71:250525091998J Neurochem 71:

  • 18.

    Rasmusson DXBrandt JMartin DBet al: Head injury as a risk factor in Alzheimer's disease. Brain Inj 9:2132191995Brain Inj 9:

  • 19.

    Roberts GWGentleman SMLynch Aet al: beta A4 amyloid protein deposition in brain after head trauma. Lancet 338:142214231991Lancet 338:

  • 20.

    Schmechel DESaunders AMStrittmatter WJet al: Increased amyloid beta-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease. Proc Natl Acad Sci USA 90:964996531993Proc Natl Acad Sci USA 90:

  • 21.

    Schofield PWTang MMarder Ket al: Alzheimer's disease after remote head injury: an incidence study. J Neurol Neurosurg Psychiatry 62:1191241997J Neurol Neurosurg Psychiatry 62:

  • 22.

    Sherriff FEBridges LRSivaloganathan S: Early detection of axonal injury after human head trauma using immunocytochemistry for beta-amyloid precursor protein. Acta Neuropathol 87:55621994Acta Neuropathol 87:

  • 23.

    Smith DHChen XHNonaka Met al: Accumulation of amyloid beta and tau and the formation of neurofilament inclusions following diffuse brain injury in the pig. J Neuropathol Exp Neurol 58:9829921999J Neuropathol Exp Neurol 58:

  • 24.

    Smith DHMeaney DF: Axonal damage in traumatic brain injury. Neuroscientist 6:4834952000Neuroscientist 6:

  • 25.

    Smith DHNakamura MMcIntosh TKet al: Brain trauma induces massive hippocampal neuron death linked to a surge in beta-amyloid levels in mice overexpressing mutant amyloid precursor protein. Am J Pathol 153:100510101998Am J Pathol 153:

  • 26.

    SmithDHNonakaMMiller Ret al: Immediate coma following inertial brain injury in dependent on axonal damage in the brainstem. J Neurosurg 93:3153222000J Neurosurg 93:

  • 27.

    Stone JROkonkwo DOSingleton RHet al: Caspase-3-mediated cleavage of amyloid precursor protein and formation of amyloid Beta peptide in traumatic axonal injury. J Neurotrauma 19:6016142002J Neurotrauma 19:

  • 28.

    Teasdale GMNicoll JAMurray Get al: Association of apolipoprotein E polymorphism with outcome after head injury. Lancet 350:106910711997Lancet 350:

  • 29.

    Wilkinson AEBridges LRSivaloganathan S: Correlation of survival time with size of axonal swellings in diffuse axonal injury. Acta Neuropathol 98:1972021999Acta Neuropathol 98:

This work was supported by National Institutes of Health Grant Nos. NS08803, NS38104, and AG21527 to Dr. Smith.

Article Information

Address reprint requests to: Douglas H. Smith, M.D., Department of Neurosurgery, University of Pennsylvania, 105c Hayden Hall, 3320 Smith Walk, Philadelphia, Pennsylvania 19104–6316. email: smithdou@mail.med.upenn.edu.

© AANS, except where prohibited by US copyright law."

Headings

Figures

  • View in gallery

    Representative photomicrographs of APP, Aβ, and NF protein accumulations in the brain following TBI in patients. The APP and NF protein accumulations in swollen axons are shown as darkly staining immunoreactivity to the antibodies 22C11(A) and N52 (B and C). The Aβ plaque formations are observed in cortical gray matter stained by 6F/3D antibody (D). In the white matter, Aβ plaques are found close to axonal bulbs, which also stain for Aβ, by applying the antibodies 13335 and Aβ17 (arrows in E and F, respectively). Immunoreactivity for NF protein was also occasionally detected in neurons (G and H) and in corpora amylacea (I). Scale bars = 20 µm.

  • View in gallery

    Representative fluorescence photomicrographs demonstrating coaccumulations of APP, Aβ, and NF proteins in the brain following TBI in patients. Double fluorescence immunohistochemical analysis reveals colocalization in axonal bulbs of Aβ (green area in A and G visualized using amyloid 1–17 antibody, and green area in D and red area in J visualized by 13335 antibody) with either APP (red areas in B and E stained by 22C11 antibody) or NF protein (red areas in H and green areas in K stained by N52 antibody). Superimposing the double-stained tissue demonstrates a complete overlap in the immunoreactive axon bulbs (yellow areas in C, F, I and L). Plaques immunoreactive to the Aβ 13335 antibody were found in the white matter (M, large central profile marked by downward pointing arrow), in proximity to axonal bulbs (M, smaller profiles marked by diagonal arrows). Double-staining this section for NF protein with N52 antibody demonstrates coaccumulation of NF protein with Aβ in the axonal bulbs, but no NF staining in the region of the Aβ plaque (N; both superimposed in O). Accumulation of NF protein was also observed in some cortical neurons (P) that colocalized with Aβ (Q; both superimposed in R). Scale bars = 20 µm.

References

1.

Adams JHGraham DIGennarelli TAet al: Diffuse axonal injury in non-missile head injury. J Neurol Neurosurg Psychiatry 54:4814831991J Neurol Neurosurg Psychiatry 54:

2.

Adle-Biassette HDuyckaerts CWasowicz Met al: Beta AP deposition and head trauma. Neurobiol Aging 17:4154191996Neurobiol Aging 17:

3.

Emmerling MRMorganti-Kossmann MCKossmann Tet al: Traumatic brain injury elevates the Alzheimer's amyloid peptide A beta 42 in human CSF. A possible role for nerve cell injury. Ann NY Acad Sci 903:1181222000Ann NY Acad Sci 903:

4.

Gentleman SMNash MJSweeting CJet al: Related beta-amyloid precursor protein (beta APP) as a marker for axonal injury after head injury. Neurosci Lett 160:1391441993Neurosci Lett 160:

5.

Gottlieb S: Head injury doubles the risk of Alzheimer's disease. Br Med J 321:11002000Gottlieb S: Head injury doubles the risk of Alzheimer's disease. Br Med J 321:

6.

Graham DIGentleman SMLynch Aet al: Distribution of betaamyloid protein in the brain following severe head injury. Neuropathol Appl Neurobiol 21:27341995Neuropathol Appl Neurobiol 21:

7.

Friedman GFroom PSazbon Let al: Apolipoprotein E-epsilon4 genotype predicts a poor outcome in survivors of traumatic brain injury. Neurology 52:2442481999Neurology 52:

8.

Horsburgh KMcCarron MOWhite Fet al: The role of apolipoprotein E in Alzheimer's disease, acute brain injury and cerebrovascular disease: evidence of common mechanisms and utility of animal models. Neurobiol Aging 21:2452552000Neurobiol Aging 21:

9.

Iwata AChen XHMcIntosh TKet al Long-term accumulation of amyloid-β in axons following brain trauma without persistent upregulation of amyloid precursor protein genes. J Neuropathol Exp Neurol 61:105610682002J Neuropathol Exp Neurol 61:

10.

Lewen ALi GLNilsson Pet al: Traumatic brain injury in rat produces changes of β-amyloid precursor protein immunoreactivity. Neuroreport 6:3573601995Neuroreport 6:

11.

Lye TCShores EA: Traumatic brain injury as a risk factor for Alzheimer's disease: a review. Neuropsychol Rev 10:1151292000Neuropsychol Rev 10:

12.

Mortimer JAvan Duijn CMChandra VHead trauma as a risk factor for Alzheimer's disease: a collaborative re-analysis of case-control studies. EURODEM Risk Factors Research Group. Int J Epidemiol 20 (Suppl 2):S28S351991Int J Epidemiol 20 (Suppl 2):

13.

Nemetz PNLeibson CNaessens JMet al: Traumatic brain injury and time to onset of Alzheimer's disease: a population-based study. Am J Epidemiol 149:32401999Am J Epidemiol 149:

14.

Nicoll JARoberts GWGraham DI: Apolipoprotein E epsilon 4 allele is associated with deposition of amyloid beta-protein following head injury. Nat Med 1:1351371995Nat Med 1:

15.

Pierce JESmith DHTrojanowski JQet al: Enduring cognitive, neurobehavioral and histopathological changes persist for up to one year following severe experimental brain injury in rats. Neuroscience 87:3593691998Neuroscience 87:

16.

Pierce JETrojanowski JQGraham DIet al: Immunohistochemical characterization of alterations in the distribution of amyloid percursor proteins and β-amyloid peptide after experimental brain injury in the rat. J Neurosci 16:108310901996J Neurosci 16:

17.

Raby CAMorganti-Kossmann MCKossmann Tet al: Traumatic brain injury increases beta-amyloid peptide 1–42 in cerebrospinal fluid. J Neurochem 71:250525091998J Neurochem 71:

18.

Rasmusson DXBrandt JMartin DBet al: Head injury as a risk factor in Alzheimer's disease. Brain Inj 9:2132191995Brain Inj 9:

19.

Roberts GWGentleman SMLynch Aet al: beta A4 amyloid protein deposition in brain after head trauma. Lancet 338:142214231991Lancet 338:

20.

Schmechel DESaunders AMStrittmatter WJet al: Increased amyloid beta-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease. Proc Natl Acad Sci USA 90:964996531993Proc Natl Acad Sci USA 90:

21.

Schofield PWTang MMarder Ket al: Alzheimer's disease after remote head injury: an incidence study. J Neurol Neurosurg Psychiatry 62:1191241997J Neurol Neurosurg Psychiatry 62:

22.

Sherriff FEBridges LRSivaloganathan S: Early detection of axonal injury after human head trauma using immunocytochemistry for beta-amyloid precursor protein. Acta Neuropathol 87:55621994Acta Neuropathol 87:

23.

Smith DHChen XHNonaka Met al: Accumulation of amyloid beta and tau and the formation of neurofilament inclusions following diffuse brain injury in the pig. J Neuropathol Exp Neurol 58:9829921999J Neuropathol Exp Neurol 58:

24.

Smith DHMeaney DF: Axonal damage in traumatic brain injury. Neuroscientist 6:4834952000Neuroscientist 6:

25.

Smith DHNakamura MMcIntosh TKet al: Brain trauma induces massive hippocampal neuron death linked to a surge in beta-amyloid levels in mice overexpressing mutant amyloid precursor protein. Am J Pathol 153:100510101998Am J Pathol 153:

26.

SmithDHNonakaMMiller Ret al: Immediate coma following inertial brain injury in dependent on axonal damage in the brainstem. J Neurosurg 93:3153222000J Neurosurg 93:

27.

Stone JROkonkwo DOSingleton RHet al: Caspase-3-mediated cleavage of amyloid precursor protein and formation of amyloid Beta peptide in traumatic axonal injury. J Neurotrauma 19:6016142002J Neurotrauma 19:

28.

Teasdale GMNicoll JAMurray Get al: Association of apolipoprotein E polymorphism with outcome after head injury. Lancet 350:106910711997Lancet 350:

29.

Wilkinson AEBridges LRSivaloganathan S: Correlation of survival time with size of axonal swellings in diffuse axonal injury. Acta Neuropathol 98:1972021999Acta Neuropathol 98:

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