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Alzheimers pathology in human temporal cortex surgically excised after severe brain injury
Milos D. Ikonomovic a,b, Kunihiro Uryu c, Eric E. Abrahamson a, John R. Ciallella a, John Q. Trojanowski c, Virginia M.-Y. Lee c, Robert S. Clark d, Donald W. Marion e, Stephen R. Wisniewski f, Steven T. DeKosky a,b,*
Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA Department of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA c Department of Pathology and Laboratory Medicine, Center for Neurodegenerative Disease Research, Institute on Aging, University of Pennsylvania, Philadelphia, PA 19104, USA d Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA e Department of Neurosurgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA f Epidemiology Data Center, Graduate School of Public Health, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
b a

Received 22 January 2004; revised 20 May 2004; accepted 10 June 2004 Available online 2 September 2004

Abstract Traumatic brain injury (TBI) is a risk factor for the development of Alzheimers disease (AD). This immunohistochemical study determined the extent of AD-related changes in temporal cortex resected from individuals treated surgically for severe TBI. Antisera generated against Ah species (total Ah, Ah1 42, and Ah1 40), the C-terminal of the Ah precursor protein (APP), apolipoprotein E (apoE), and markers of neuron structure and degeneration (tau, ubiquitin, a-, h-, and g-synuclein) were used to examine the extent of Ah plaque deposition and neurodegenerative changes in 18 TBI subjects (ages 18 64 years). Diffuse cortical Ah deposits were observed in one third of subjects (aged 35 62 years) as early as 2 h after injury, with only one (35-year old) individual exhibiting mature, dense-cored plaques. Plaque-like deposits, neurons, glia, and axonal changes were also immunostained with APP and apoE antibodies. In plaque-positive cases, the only statistically significant change in cellular immunostaining was increased neuronal APP ( P = 0.013). There was no significant correlation between the distribution of Ah plaques and markers of neuronal degeneration. Diffuse tau immunostaining was localized to neuronal cell soma, axons or glial cells in a larger subset of individuals. Tau-positive, neurofibrillary tangle (NFT)-like changes were detected in only two subjects, both of more advanced age and who were without Ah deposits. Other neurodegenerative changes, evidenced by ubiquitin- and synuclein-immunoreactive neurons, were abundant in the majority of cases. Our results demonstrate a differential distribution and course of intra- and extra-cellular AD-like changes during the acute phase following severe TBI in humans. Ah plaques and early evidence of neuronal degenerative changes can develop rapidly after TBI, while fully developed NFTs most likely result from more chronic disease- or injury-related processes. These observations lend further support to the hypothesis that head trauma significantly increases the risk of developing pathological and clinical symptoms of AD, and provide insight into the molecular mechanisms that initiate these pathological cascades very early during severe brain injury. D 2004 Elsevier Inc. All rights reserved.
Keywords: Alzheimers disease; Ah plaque; Glial cell

Introduction Traumatic brain injury (TBI) is an environmental risk factor for the development of chronic neurodegenerative
* Corresponding author. Department of Neurology, University of Pittsburgh School of Medicine, 3471 Fifth Avenue, Suite 811, Pittsburgh, PA 15213. Fax: +1-412-692-4526. E-mail address: DeKoskyST@upmc.edu (S.T. DeKosky). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.06.011

disorders, including Alzheimers disease (AD). TBI correlates with accelerated cognitive decline in head-injured, aged subjects (Luukinen et al., 1999), and there is an increased risk for AD dementia in patients who, in adulthood, sustained a severe head injury (Guo et al., 2000; McKenzie et al., 1994; Nemetz et al., 1999; Plassman et al., 2000; Roberts et al., 1991, 1994, 1993; Schofield et al., 1997). One of the detrimental consequences of TBI involves altered processing of amyloid precursor protein (APP), which potentially con-

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tributes to AD-like pathological cascades (DeKosky et al., 1998; McIntosh et al., 1998). In AD, amyloid h peptide (Ah) overproduction and deposition induce inflammatory cytokine infiltration with microglial activation and oxidative stress (Altstiel and Sperber, 1991; Butterfield et al., 2002; Buxbaum et al., 1992; Forloni et al., 1992; Griffin et al., 1989, 1995), the sequelae of which are also involved in trauma-related neurological dysfunction (Rothwell et al., 1997). TBI studies in both animal models (Ciallella et al., 2002; Masumura et al., 2000; Murakami et al., 1998; Pierce et al., 1996; Smith et al., 1999; Uryu et al., 2002) and postmortem human tissue (Gentleman et al., 1993; Graham et al., 1995; McKenzie et al., 1994; Roberts et al., 1991, 1994) demonstrated that increased APP production and/or accumulation can develop after injury. However, evidence of ADlike extracellular Ah deposition has been described only in post-mortem human brain tissue so current knowledge of amyloidogenic APP metabolism and Ah accumulation after human TBI derives almost exclusively from studies of victims of severe, lethal TBI (Roberts et al., 1994). Notably, AD-like neurodegenerative changes as well as intracellular accumulation of neurofibrillary tangle (NFT)-like pathologies were not found in these patients, with exception of subjects with repetitive head injury (Geddes et al., 1999; Schmidt et al., 2001). In the present study, we collected freshly resected human brain tissue from subjects who underwent surgical treatment within hours of TBI and assessed the extent of AD-like extracellular and intracellular changes during this acute post-injury period. We further examined possible associations between these changes and variables such as subjects age, primary lesion characteristics, hypothermia treatment, and functional outcome.

bromide) was used to prevent shivering during hypothermia. All normothermic patients also received neuromuscular paralytic medication for a minimum of 24 h post injury. CT scans taken at the hospital admission were read and classified as described in Marshall et al. (1991). Ah plaque immunostaining from TBI subjects was compared to that in temporal cortex obtained post-mortem from subjects who died of causes not related to head trauma. This group included 10 non-demented subjects (age range 50 75; M/F = 7/3; APOEq4 1/10) and 10 patients with end-stage Alzheimers disease (AD; age range 76 90, M/F = 3/7; APOEq4 6/10) who had been enrolled and followed in the University of Pittsburgh Alzheimers Disease Research Center (ADRC). Neuropathological confirmation of AD diagnosis was made according to established criteria (Anonymous, 1997; Khachaturian, 1985; Mirra et al., 1991) by ADRC Brain Bank neuropathologists. TBI and non-demented control subjects had no concomitant neurological disorders and no history of AD. In addition to control and AD samples, comparison of TBI data was made with surgery samples resected from temporal lobe epilepsy patients. These were processed with antibodies to tau and Ah, to support the specificity of these antibodies in surgery samples. The data on these patients were published previously (Matsuo et al., 1994). Surgical acquisition of tissue specimens Brain specimens were obtained under the approval of the University of Pittsburgh Institutional Review Board for the Clinical Core of the University of Pittsburghs Brain Trauma Research Center and for the University of Pittsburgh ADRC. All head-injured patients underwent decompressive craniectomy, either to relieve intractable cerebral swelling or for removal of severely traumatized brain tissue. Under the Clinical Trauma Center protocol for management of lifethreatening intracranial hypertension, temporal cortex tissue samples were taken in the operating room. Only tissue samples removed for decompression of the brain, and that would normally be discarded, were used; no tissue was removed solely for this study. Written informed consent was obtained from family members for each patient. Equal numbers of subjects (n = 9) contributed tissue samples from the left or right temporal cortex (Table 1). Tissue processing for immunohistochemistry and histology Cortical tissue was immersion-fixed in 2% paraformaldehyde in phosphate buffer (PB) for 4 h at 4jC. Tissue was cryoprotected in graded sucrose and embedded in OCT. Tissue sections were cut at 30 A for free-floating immunohistochemistry (IHC) with antibodies against Ah (10D5, Ah40, Ah42), APP, and apoE (Table 2), as well as for Thioflavin-S (Thio-S, Sigma, St. Louis, MO) and X-34 (Styren et al., 2000) histochemistry. Before IHC, but not histochemistry, tissue was pre-treated with 99% formic acid

Materials and methods Subjects The study examined 18 patients (age range 18 64 years), admitted to the University of Pittsburgh Medical Center (UPMC) between June 1999 and May 2001 for surgical treatment of severe closed head injuries (Glasgow Coma Scale, GCS score < 9). Patients demographic and clinical variables are listed in Table 1. Data on the initial status (GCS; Teasdale and Jennett, 1974) and functional outcome at 3, 6, and 12 months after injury (Glasgow Outcome Scale, GOS; Jennett et al., 1981) were obtained for all TBI patients as described previously (Clark et al., 1999; Marion et al., 1997). Time intervals from the initial head injury to surgical excision of traumatized brain tissue ranged from 2 to 19 h, except for two subjects who were admitted 58 and 74 h after TBI (Table 1). Half of the patients had been randomized to 48 h of hypothermia (Clifton et al., 1993), the other half was normothermic. Subjects in the hypothermic group were maintained at 32 33jC for 48 h, then rewarmed to 37 38jC over 12 h. Systemic neuromuscular paralytic medication (vecuronium

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Table 1 Case demographics, injury/lesion specifics, and functional outcomes Plaque-positive cases (n = 8) Case # With amyloid-beta 1 Age Gender APOE Injury type h post-TBI Side/region GCS Lesion type Lesion side/region Lesion density Lesion volume CT delay (days) H/N treatment GOS 3 months GOS 6 months GOS 12 months 35 M 34 n/a 2 L/T 4 EC L high 27.5 none N 2 2 3 2 60 F 34 Fall 16 R/T 9 IC R/T high 14.7 none N 2 3 3 3 62 F 33 Fall 16 R/T 9 IC R/T high 24.5 none N 4 5 5 4 36 F 34 A 10 R/T 7 IC L/T high 10.5 none N 4 4 1 5 45 M 33 Fall 9.2 R/T 5 IC R/T high 47.3 none H 4 4 4 6 39 M 33 MV 12 R/T 3 EC L high 67.2 none N 1 1 1 No amyloid-beta* 7 24 M 33 AT 2.8 L/T 3 EC L high 86.4 none H 2 2 2 8 21 M 33 n/a 4.4 R/T 3 EC L/F low 21.6 18 d N 2 3 3

Plaque-negative cases (n = 10) Case # Age Gender APOE Injury type Hours post-TBI Tissue side/region GCS Lesion type Lesion side/region Lesion density Lesion volume CT delay (days) H/N treatment GOS 3 months GOS 6 months GOS 12 months 9 37 M 33 n/a 74 L/T 8 n/a n/a n/a n/a none H 2 3 4 10 42 F 33 Fall 2.7 L/T 4 n/a n/a n/a n/a none H 1 1 1 11 59 M 33 Fall 7 L/T 6 IC R/BG high 165.4 none H 1 1 1 12 30 F 34 MC 19 R/T 7 EC R med 49.4 3d H n/a n/a 4 13 64 F 33 n/a 2.2 R/T 4 EC R med 69.42 none N 1 1 1 14 23 M 34 MV 7.5 L/T 3 EC/IC R/F high/high 27.4/20.2 none N 2 2 2 15 51 M 23 HO 3.3 L/T 7 IC L/BG high 18.7 none H 1 1 1 16 40 M 33 Fall 9.3 R/T 7 EC/IC R/F high/med 24.7/23.1 none H 4 4 4 17 n/a M 33 Fall 3.1 L/T 4 EC L high 53.3 16 d N 1 1 1 18 18 F n/a MV 58 L/T 6 EC R low 16.4 21 d H 2 3 2

Abbreviations: A = assault; APOE = apolipoprotein E genotype; AT = all terrain vehicle accident; BG = basal ganglia; d = days; EC = extracerebral; F = frontal cortex; GCS = Glasgow Coma Score; H/N = hypothermia/normothermia; HO = hit with object; IC = intracerebral; L = left; MC = motorcycle accident; MV = motor vehicle accident; NFTs = neurofibrillary tangles; R = right; T = temporal cortex. * Plaque-positive cases marked as no amyloid-beta were positive for APP and/or apoE plaques.

Table 2 Antibodies used for immunohistochemistry Antibody 10D5 Ah1 40 Ah1 42 ApoE Anti-6 17026 PHF-1 NFL 202 303 Ubiquitin Epitope Ah1 16 Ah40 Ah42 apoE APP590 695 pan-tau PS396/pS404 NF-L Syn ahg Syn a (conf.) Ubiquitin Host mouse rabbit rabbit goat rabbit rabbit mouse rabbit mouse mouse mouse Dilution 1:3,000 1:500 1:500 1:10,000 1:10 1:10,000 1:1,000 1:5,000 1:20,000 1:500 1:1,000 Source Elan Pharm. Chemicon Chemicon Genzyme Athena Neurosci. CNDR P. Davies CNDR CNDR CNDR Chemicon

Ah = amyloid h protein; apoE = apolipoprotein E; PHF = paired helical filaments; NFL = neurofilament; Syn = synuclein.

(Sigma) for 5 min. The IHC procedure for human tissue samples has been described in detail (Ikonomovic et al., 1997). Briefly, tissue sections were rinsed between all steps with 0.1% Triton X-100 in 0.1 M PBS. Sections were blocked with normal serum and incubated with primary antibodies for 16 h at 4jC. The antigen antibody reaction was visualized by incubating sections with appropriate affinity-purified, biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) for 1 h at room temperature, followed by an avidin biotin complex (ABC kit, Vector Laboratories), and imidazole acetate buffer (IAB) containing 3,3V-diaminobenzidine (DAB, Sigma), nickel ammonium sulfate, and hydrogen peroxide. Immunostained sections were rinsed in IAB and mounted from PB onto gelatin-coated glass slides, coverslipped, and examined with standard bright field microscopy.

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IHC with markers of neuronal degeneration, including anti-tau, anti-neurofilament (NF) low Mr subunit (NFL), antiubiquitin, and anti-synuclein Syn202 and Syn303 (conformation-dependent) antibodies (Table 2), followed the same staining protocol on 6-A-thick formalin-fixed, paraffinembedded sections from a portion of excised temporal cortex tissue adjacent to that processed in paraformaldehyde for Ah staining. After visualizing immunoreaction with DAB as chromogen, tissue sections were counterstained with hematoxylin. Additional tissue sections were processed as described but without the primary antibody as a control for nonspecific immunostaining. In no instance was immunostaining observed in such control sections. For each antibody, tissue sections from all cases were processed simultaneously, including tissue from control and confirmed AD cases from the ADRC, which served as negative and positive controls, respectively, for Ah plaque and NFT staining (see Fig. 2). Nissl-stained adjacent sections were used to determine tissue cytoarchitecture. Three tissue sections from each case were also stained for aggregated Ah using fluorescent compounds Thio-S and X-34, and examined on an Olympus microscope with a fluorescent attachment. Semi-quantitative estimation of pathological markers In two randomly chosen coded IHC tissue sections from each case, frequencies of Ah, apolipoprotein E (apoE), APP, and of neurodegenerative markers were estimated (Table 3). These semi-quantitative evaluations reflected area density of immunoreactive (IR) plaques or neuronal/glial elements. That amount of surgical tissue was limited, and obtained only in one brain region (temporal cortex), precluded more quantitative analysis of neuropathological changes, and prevented full evaluation of plaques by CERAD criteria that require analysis of multiple brain regions. Two investigators blinded to diagnosis and other clinical findings performed analyses of both plaques and neuronal changes in TBI, controls, AD, and epilepsy samples. In the case of plaques, sections free of deposits were given a score of 0; those showing mild plaque pathology with only focal collections of deposits separated by uninvolved cortex were scored 1; moderate plaque deposition, characterized by diffuse cortical involvement with plaques estimated to cover 30 50% of cortical area were scored as 2; while diffuse cortical involvement with estimated 50% or more of the cortical area covered with plaques was characterized as severe plaque pathology with a score of 3. For semiquantitative assessment of tau-, NFL-, ubiquitin-, and synuclein-IR elements, the absence of immunoreactivity was scored 0, cases with an infrequent number of profiles
Notes to Table 3: * Plaque-positive group > plaque negative group for APP neurons ( P = 0.0133). ** Neurofibrillary tangles.

Table 3 Semi-quantitative analysis of immunohistochemical markers of Alzheimers changes in temporal cortex from TBI cases Case # Plaque-positive cases Plaque-negative cases

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Ab total Plaques Neurons Glia Axons 3 0 0 0 2 1 0 0 3 0 0 0 1 0 0 0 2 0 0 1 2 0 1 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 2 1 2

Ab 1 40 Plaques 2 Neurons 0 Glia 0 Axons 0 Ab 1 42 Plaques 3 Neurons 0 Glia 0 Axons 0 apoE Plaques Neurons Glia Axons

1 0 1 0

0 0 0 0

1 0 1 0

1 0 1 0

1 1 1 0

0 0 0 0

0 1 1 0

0 1 1 0

0 1 1 0

0 0 0 0

0 1 1 0

0 0 0 0

0 1 1 0

0 0 0 0

0 0 0 0

0 0 0 0

0 1 1 0

2 1 1 0

3 0 0 0

1 0 1 0

2 0 1 0

2 1 1 0

0 0 2 0

0 1 2 0

0 1 1 0

0 1 1 0

0 1 1 0

0 0 1 0

0 0 0 0

0 0 1 0

0 0 0 0

0 0 0 0

0 0 0 0

0 2 0 0

3 0 2 0

1 3 3 0

3 1 2 0

1 0 3 0

2 1 3 0

1 0 3 0

0 1 2 0

1 0 3 0

0 1 2 0

0 0 2 0

0 0 2 0

0 1 3 0

0 0 1 0

0 1 3 0

0 0 3 0

0 3 3 0

0 0 3 0

0 1 1 0

APP c-term* Plaques 2 1 Neurons 3 3 Glia 0 0 Axons 3 0

2 3 0 2

0 0 1 0

0 0 0 0

1 3 0 1

1 3 0 0

1 3 0 0

0 1 0 1

0 2 0 0

0 1 1 0

0 2 0 0

0 1 0 0

0 0 1 0

0 0 0 0

0 1 1 0

0 0 1 2

0 3 0 3

Tau Neurons 0 0 0 0 0 0 0 0 0 0 Glia 1 0 0 0 0 0 0 0 00 Axons 0 1 1 1 1 1 1 1 0 0 NFL Neurons Glia Spheroid Axons

1** 0 0 0 0 1

1** 0 1 0 0 1 0 0 0 0 1 1 1 0 1 1 0 1

2 0 1 0

3 0 0 1

3 0 2 2

1 0 0 0

1 0 0 0

2 0 0 0

3 0 1 2

3 0 2 1

2 0 0 1

3 0 0 1

3 0 2 0

2 0 1 0

2 0 0 1

2 0 0 0

3 0 0 2

1 0 0 0

3 0 0 1

1 0 0 1

Ubiquitin Neurons 2 Glia 2 Spheroid 2 Axons 0 Syn202 Neurons Glia Spheroid Axons Syn303 Neurons Glia Spheroid Axons

2 2 0 0

0 0 0 0

3 2 1 0

2 2 0 0

0 0 2 0

2 2 0 0

3 2 1 0

3 3 1 0

3 0 3 0

3 0 2 0

2 1 2 0

2 0 2 0

3 2 2 0

3 2 1 0

3 3 1 0

3 2 2 0

2 2 2 0

0 0 0 0

0 1 0 0

2 2 0 0

1 1 0 0

1 1 0 0

2 1 0 0

2 2 0 0

1 1 0 0

0 1 0 0

1 1 0 0

1 1 0 0

0 0 0 0

1 1 0 0

0 0 0 0

1 1 0 0

2 2 0 0

2 1 0 0

0 1 0 0

0 0 0 0

0 0 0 0

3 2 0 0

2 2 0 0

2 2 0 0

2 2 0 0

2 2 2 0

1 1 0 0

0 0 2 0

2 3 0 0

2 2 0 0

2 2 0 0

1 1 0 0

0 0 0 0

1 2 0 0

1 1 0 0

2 2 0 0

2 2 0 0

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were scored 1, cases with a moderate number of profiles were scored 2, and cases with clustered or widespread profiles were scored 3. Statistical analysis For statistical analysis, TBI cases were divided into two groups based on the presence (plaque-positive) or absence (plaque-negative) of plaque deposits containing Ah and/or related proteins (e.g., APP, apoE). The two groups were compared based on their demographic (age, gender, APOE genotype), clinical (time to surgery, GCS, GOS, hypothermia treatment), and lesion (type, side, volume, density) characteristics using Fishers Exact tests, Wilcoxon tests and two-sample t tests. The two groups were also compared based on their semi-quantitative scores for immunostained cellular elements using Fishers Exact Statistic.

Results AD plaque markers after TBI In eight of the 18 subjects suffering severe TBI, temporal cortex contained AD-like plaque deposits (plaque-positive cases) of Ah and/or related proteins (e.g., APP, apoE). There were no such plaques detectable in the remaining 10 (plaque-negative) TBI subjects, although some of these cases showed immunoreactive pyramidal neurons, axons, and glia (Table 3, Figs. 1D F). Plaque-positive and plaque-negative groups were similar when compared by demographic variables and APOE genotype (percent of APOEq4 allele carriers). The two groups were also not statistically different with respect to clinical variables shown in Table 1, including lesion characteristics analyzed on CT scans, hypothermia treatment, initial severity (GCS), or functional outcome scores on the GOS scale. For each subject, the specific pattern of plaque and cellular staining with antibodies against total Ah, Ah1 40, Ah1 42, apoE, and APP is shown in Table 3 and described below. All AD subjects had numerous cortical plaques detected with all five markers and taupositive NFT (score of 3 for all), consistent with their neuropathological diagnosis. In contrast, only two controls, both of advanced age (z74 years), had scarce, diffuse Ah deposits (score of 1) and no (score of 0) NFT. There was no positive immunostaining with markers of plaques or neuronal changes in surgery samples from epilepsy patients described in the study by Matsuo et al. (1994). Total Ab (10D5-positive) immunostaining Temporal cortex in six of the eight plaque-positive TBI cases contained 10D5-IR Ah plaques (Table 3). These plaques were identified as extracellular deposits of Ah in
Fig. 1. 10D5-immunoreactive plaques and cellular deposits in surgically excised temporal cortex from subjects who sustained severe traumatic brain injury. Compact amyloid beta (Ah) deposits are detected as early as 2 h (A; case #1) after injury and are characterized by an irregular fleecy morphology. Plaques are also present at 16 h after injury (B; case #3), with comparable abundance, and better-defined borders. Ah deposits in TBI are also present as large and dispersed neuropil accumulates (C; case #4). Intracellular Ah deposits are observed in the cytoplasm of cortical pyramidal neurons (D) and in axons (E) of a plaque-negative case (case #18). Sporadic, Ah-positive glial cells are seen in another plaque-negative case (F; case #10). Scale bar 200 A (A C); 100 A (D F).

the neuropil, not associated with vascular elements. There was a considerable inter-subject variability in the abundance, size, and morphology of 10D5-IR plaques. They were most abundant in subjects #1 (35 year old; 2 h postTBI; Tables 1 and 3) and #3 (62 year old; 16 h post-TBI; Tables 1 and 3), and appeared as well-defined circular deposits in the neuropil (Figs. 1A and B). Similar, less frequent deposits were also observed in cases #2, #5, and #6 (Table 3). The distribution of Ah plaques in TBI subjects contrasted with that in our AD cases, which served as positive staining controls and consistently showed abundant 10D5-IR Ah plaques throughout the temporal cortex (not shown). In one Ah plaque-positive TBI subject (#4), 10D5-IR plaques were scarce, and characterized by diffuse and irregular accumulations of Ah (Fig. 1C), similar to those found in two aged control brains (not shown). Two of the plaque-positive TBI subjects (#7 and #8) had no Ah deposits, despite the presence of APP and/or apoE plaques (Table 3). In these cases, we found only an overall increase of 10D5 immunostaining in neuropil (not shown). Plaquepositive and plaque-negative groups were not statistically different regarding the presence of 10D5-IR pyramidal neurons, axons, and glial cells.

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Ab40 and Ab42 immunohistochemistry Immunohistochemical staining for Ah1 40 and Ah1 42 was performed to determine the proportion of Ah species in plaques after TBI. There was a close correspondence in the abundance and morphology of Ah42- and 10D5-IR plaques (Table 3). When comparing the Ah42- and Ah40-IR deposits in TBI cases, Ah40-IR plaques were smaller and less numerous (Figs. 2A and B). Plaques in TBI cases showed a restricted regional organization, whereas in AD cases, they were more abundant and distributed throughout the entire cortical region (Figs. 2E and F). Ah40-IR plaques were present in all but one of the 10D5/Ah42 plaque-positive TBI cases (case #3, Table 3); notably, this case was also characterized by elevated and diffuse Ah40 neuropil immunoreactivity. There were no vascular Ah deposits detected

with Ah40 and Ah42 antibodies or with the general marker of Ah (10D5 immunostaining) in temporal cortex of TBI cases. Neuronal and glial cellular elements were less frequently immunostained (Table 3). Thio-S/X-34 histochemistry Thio-S and X-34 staining of aggregated Ah in plaques was detected in one TBI subject only (case #1, Fig. 2C). NFT and neuropil threads were not histochemically detected in any TBI subjects, including case #1. Every AD case showed abundant thio-S and X-34 fluorescent plaques, NFT, neuropil threads, and vascular elements (Fig. 2G), while no such staining was present in the control cases or the epilepsy surgical samples. Apolipoprotein E immunohistochemistry ApoE was localized to plaques in a subset of TBI cases. All cases with Ah-IR plaques also showed apoE-IR plaques (Table 3). One subject (#8) without Ah-IR plaques had a very small number of apoE- and APP-IR deposits. ApoE-IR deposits were readily detectable as relatively small, compact formations that resembled Ah40-IR plaques in their size and shape, and 10D5-IR or Ah42-IR plaques in frequency and distribution (Table 3, Fig. 2D). As with other plaque markers in TBI cases, apoE-IR plaques were similar, although less numerous, to those in AD cases (Figs. 2D and H). There were no apoE-IR plaques in control subjects. In addition to plaques, apoE immunoreactivity was observed in neurons and glial cells after TBI. Glial apoE immunoreactivity was the most consistent feature of plaque-negative cases. There were no significant differences in apoE-IR cell elements between plaque-positive and plaque-negative groups. C-terminal APP fragments immunohistochemistry The immunohistochemical profile of neuropil deposits was characterized further by examining the distribution of C-terminal APP (APP675 695, anti-6 antibody) fragments. TBI subjects showed a complex pattern of anti-6 immunostaining in neurites, neurons, and glia (Fig. 3). APP-IR plaques were seen in almost every Ah plaque-positive case, as well as in two subjects without Ah plaques (Table 3). These APP deposits appeared as darkly stained aggregations of neuritic elements or as more diffuse neuropil accumulations with poorly defined borders (Fig. 3A). Similar deposits were seen in AD cases, but were absent in controls (not shown). In TBI cases, the frequency of APP-IR plaques was generally low, comparable to that of Ah40-IR plaques. In addition to plaques, the majority (13/18) of TBI cases showed APP-IR neurons, axons, and to lesser extent glia (Table 3). Large pyramidal neurons were particularly darkly immunostained in their soma and apical dendrites, and were often accompanied by numerous APP-IR neuritic processes scattered throughout the neuropil (Fig. 3B). These processes

Fig. 2. Temporal cortex from a subject who sustained severe TBI, (case #1, A D) and an AD patient (E H), immunostained for Ah42 (A, E), Ah40 (B, F), X-34 (C, G), or apoE (D, H). In the temporal cortex of the TBI case, Ah42 plaques (A) are more abundant than Ah40 plaques (B). Frequency of plaques stained with X-34 (C) is closer to that of Ah40, while the distribution of apoE plaques (D) is similar to Ah42 plaques. AD temporal cortex shows a profusion of all plaque types (E H). Not only plaques, but also NFT, neuropil threads, and vascular amyloidosis are abundantly stained in AD cases (G). Scale bar 200 A.

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statistically significant difference in the presence of APPIR neurons when comparing plaque-positive and plaquenegative cases ( P = 0.013); frequencies of APP-IR axons and glia were not statistically different between the two groups. AD neuropathology markers in TBI Cellular pathology was further evaluated by examining the distribution of tau, synuclein, and ubiquitin immunostaining in TBI cases. The majority of TBI subjects showed tau (PHF-1)-IR dystrophic axons in the white matter (Table 3), consistent with diffuse axonal injury (Fig. 4A). In addition, fine tau-IR deposits were dispersed in white matter of some cases, likely the remnants of degenerated axons. Light, diffuse tau immunoreactivity in neuronal somata/ dendrites was detected in only two cases (cases #15, 51 years and #18, 18 years; Table 3). These cases were negative for our plaque markers (Ah, apoE, or APP). In two other plaque-negative cases, pyramidal cell soma showed a unique pattern of tau immunoreactivity that resembled NFTs (cases #11, 59 years and #13, 64 years; Table 3), appearing as dense somatodendritic staining. Consistent with the appearance of NFTs, some of these PHF-IR inclusions were also thio-S-positive (not shown). Normal neuronal cell bodies were NFL-IR in all cases examined, without evidence of pathological alterations at the level of the perikarya. Irregular axonal NFL staining resembling classic diffuse axonal injury was observed in 10 cases, and six cases showed NFL-IR axonal spheroids (data not shown).

Fig. 3. Immunostaining with antibodies against the C-terminus of APP in temporal cortex from subjects who sustained severe traumatic brain injury. A number of APP-immunoreactive plaques, of inconsistent shape and size, are seen in case #1 (A). Intense APP immunostaining is observed in soma and processes of many pyramidal neurons from case #2 (B). Most of these cells appear dysmorphic, as do many APP-immunoreactive corkscrewshaped neurites crossing the neuropil. APP immunostaining is similarly dense in axonal processes within the white matter, seen as bulbous axonal swellings (case #1, C and D). Scale bar 200 A (A, C); 100 A (B, D).

were intensely stained and frequently characterized with a twisted, corkscrew-like morphology. APP immunostaining was also localized to numerous, darkly stained axonal processes and bulbous swellings in the white matter of TBI tissue samples (Figs. 3C and D). This pattern of axonal staining was not observed in control brains. There was a

Fig. 4. Temporal cortex from TBI subjects immunostained using antisera generated against paired helical filaments (A, PHF-1), synuclein (B, Syn202; C, Syn303), or ubiquitin (D), and counterstained with hematoxylin. Positive immunoreaction product is detected in axons (A) and cell bodies (B D). Scale bar 20 A.

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Synuclein Syn202 (which recognizes alpha-, beta-, and gamma-synuclein) immunostaining was observed in cortical neuropil and in the cytoplasm of small clusters of neurons (Fig. 4B). In addition, pericyte and muscle cells in the vascular wall showed intense Syn202 staining (not shown). The Syn303 antibody (which recognizes oxidized alphasynuclein) showed cytoplasmic staining in neurons and some glial cells, while punctuate immunoreaction product was also detected in the neuropil (Fig. 4C). Ubiquitin antibodies also labeled small intracellular elements in neuronal and glial cells (Fig. 4D). Ubiquitin-IR pericytes and other cells, presumably of microglial or macrophage lineage, were detected near blood vessels (not shown). There were no significant differences in the presence of these markers of neuronal degeneration between plaquepositive and plaque negative groups. Further, none of the abnormal neurodegenerative pathologies described above were detected in control tissue samples or in surgical temporal lobe epilepsy samples studied earlier with antibodies to tau and Ah (Matsuo et al., 1994).

Discussion This analysis of surgically resected temporal cortex from survivors of severe TBI reveals that the formation of immunohistochemically detectable extracellular Ah deposits can occur as early as 2 h after injury, demonstrating the extraordinary rapidity with which AD-like pathology can develop after acute neuronal insult. Extracellular deposits of Ah, APP, and apoE were documented in 30% of subjects examined, corroborating and extending several previous post-mortem analyses (Horsburgh et al., 2000; Roberts et al., 1991, 1994). In addition, these biopsy data are not confounded by hard-to-control variables such as agonal state and post-mortem interval, providing compelling evidence that amyloidogenic changes observed after TBI are not due to post-mortem artifacts. We remain, however, confronted with a critical issue, that of whether the observed AD-like pathogenic changes were preexistent (e.g., age- or pre-symptomatic disease-related) or injury-induced. Several compelling lines of evidence argue against age-related amyloidosis being a confounding factor in the interpretation of our results. First, the emergence of Ah plaques in nondemented, non-injured subjects is clearly age dependent, with the incidence of detectable Ah deposition being virtually absent until the sixth decade of life (Braak and Braak, 1997). Second, there are distinct differences in the morphological characteristics of presumed injury-induced plaques described in the current study, the diffuse amyloidosis of aged control subjects, and the extensive Ah pathology characteristic of AD. Third, there is considerable evidence that TBI induces elevated amyloidogenic APP processing in experimental models of TBI (Ciallella et al., 2002; Masumura et al., 2000; Murakami et al., 1998; Pierce et al., 1996; Smith et al., 1999; Uryu et al., 2002).

Ah plaques are virtually absent in populations younger than 50 years (Dayan, 1970; Mackenzie, 1994), and only after the eighth decade of life they are detected in 50% of subjects (Crystal et al., 1988; Delaere et al., 1990; Dickson et al., 1991; Katzman et al., 1988). The extensive nonselective autopsy investigation by Braak and Braak (1997) rarely detected Ah plaques in younger subjects. None of the 58 cases examined in the 31 35 years age range, and only two of the 83 subjects in the 36 40 years age range had some Ah plaques (Braak and Braak, 1997). Furthermore, Ah deposits were detected in only 20 of the 207 people (<10%) in their fifth decade of life (Braak and Braak, 1997). Notably, non-selective studies do not provide participants clinical history, thus one must be cautious in ruling out the potential contribution of older brain injuries and/or disease processes as causative factors in the formation of Ah deposits even in the normal older subjects in the report by Braak and Braak (1997). An additional large-scale human TBI study by Roberts et al. (1994) did not detect Ah deposits in a normal control (non-injured) population under the age of 60. None of the younger plaque-positive subjects in this study had a family history of early-onset AD. Thus, it is highly unlikely that our TBI patients had preexisting plaque pathology that could account for the observations made in the current report. Several important similarities as well as differences exist between Ah plaques in AD and those detected after TBI. AD cases in our study consistently had numerous, compact and hard core Ah deposits, as well as abundant neurofibrillary tangles (NFT) and neuropil threads. In contrast, TBI subjects were virtually devoid of dense-cored plaques, NFT, and neuropil threads. In agreement with this, a high incidence of diffuse plaques after head injury has been reported previously (Clinton et al., 1991). In the present study, only one of our TBI subjects, a 35-year-old male, had detectable dense-cored plaques. Notably, this case was characterized by the most abundant Ah40 deposits. In contrast, one plaque-positive TBI subject (without dense-cored plaques) exhibited abundant Ah42 plaques in the absence of Ah40 deposits. Overall, Ah42 deposits were more abundant than Ah40 deposits in the remaining plaque-positive TBI cases, suggesting that, similar to plaque genesis in AD, Ah42 deposition may precede that of Ah40 after TBI, and the formation of dense-cored plaques might require a substantial accumulation of Ah40 species. The observed preponderance of Ah42 over Ah40 plaques in TBI subjects is in agreement with previous post-mortem analyses of TBI (Gentleman et al., 1997; Horsburgh et al., 2000) and AD (Iwatsubo et al., 1994, 1995). Further studies using very early post-injury brain pathology and animal models are needed to understand the mechanisms underlying such rapid histopathological changes after TBI, including the sequence of deposition and/or contribution of the different Ah species in the formation of more mature AD-like lesions. ApoE was also localized to plaques after TBI. In contrast to the post-mortem study by Horsburgh et al. (2000), we

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found apoE deposits in all of our Ah plaque-positive TBI subjects, as well as in one individual with no detectable Ah deposits. Moreover, apoE and Ah42 plaques were similarly abundant, with one case exhibiting numerous apoE and Ah42 plaques in the absence of Ah40 deposits. Thus, the deposition of apoE could, together with Ah42, represent an initial plaque component or, alternatively, reflect a role for apoE in Ah clearance from the neuropil. It is possible that age-related changes in efficacy of apoE-mediated Ah clearance could contribute to apoE-dependent amyloidogenesis after TBI, as it might occur in AD (Gomez-Isla et al., 1996; McNamara et al., 1998; Schmechel et al., 1993). In support of this notion, the APOEq4 genotype is associated with increased Ah load and risk for developing AD (Mayeux et al., 1993a; McNamara et al., 1998), as well as increased Ah deposition after injury in a mouse model of AD (Hartman et al., 2002) and in human TBI (Horsburgh et al., 2000; Mayeux et al., 1993b, 1995; Nicoll et al., 1995). Although we did not observe such a relationship in our TBI cases, greater numbers of subjects should be examined for this and other possible correlations, considering that a higher prevalence of the APOEq4 allele appears to be linked with Ah deposits even in some aged control subjects (Arai et al., 1999). Our results appear to contradict previous findings by Nakagawa et al. (1999), (2000), who reported that brain trauma can diminish existing Ah plaques in transgenic, APP over-expressing mice. However, severe injury in those studies created a large cavity at the site of impact and analysis. Thus, it is possible that the absence of plaques was related to significant loss of APP-producing neurons due to lesion formation. Another study of transgenic mice reported that head injury causing a minor scar, but not cavity-related neuronal loss, lead to accelerated plaque formation (Uryu et al., 2002). The latter observation is more comparable to our present study, which was of surgical samples that were not the site of the primary injury, and were not affected directly by necrotic lesion and associated neuronal loss. Thus, remaining injured neurons, and axons, are likely source of increased APP and Ah production after TBI. Increased numbers of APP-immunoreactive neuronal cell bodies were the only statistically significant cellular feature distinguishing the plaque-positive TBI group. The dystrophic morphology of intensely APP-labeled neurons indicates an early neurodegenerative process that could result in a surge in Ah production, as demonstrated experimentally in traumatized APP transgenic mice (Smith et al., 1998). Furthermore, the distribution of amyloidogenic APP fragments (c-terminal APP) and Ah in cortical plaques following TBI suggests that plaque-deposited Ah could be derived in part from APP in axonal ends of injured neurons projecting to this brain region. C-terminal fragments of APP are increased in experimental models of TBI (Ciallella et al., 2002; Stone et al., 2000) and in AD brains where they likely represent an initial step in Ah production (Hardy, 1992; Wilson et al., 1999). The observation that increased APP processing and

accumulation of c-terminal APP fragments were detected (albeit to a significantly lesser extent) in plaque-negative cases, and were not present in normal controls, suggests that they might precede Ah plaque formation after TBI. Although our surgically extracted tissue samples were not the sites of primary mechanical injury, they exhibited a variety of neurodegenerative changes, including ubiquitin, synuclein, and PHF tau-IR neuronal and axonal elements, as well as spheroid inclusions. A similar pattern of immunostaining was described previously in the brains of patients with various neurodegenerative diseases associated with alpha-synuclein pathology (Trojanowski and Lee, 2002), and alpha synuclein-IR axonal spheroids were present in cases of TBI (Newell et al., 1999). Furthermore, recurrent TBI in dementia pugilistica is associated with neuropathological PHF-tau profiles (Geddes et al., 1999; Schmidt et al., 2001), indicating that in chronic or repetitive brain injury tau can undergo pathological phosphorylation and neuronal accumulation similar to that observed in AD brains (Lee et al., 2001; Roberts, 1988; Schmidt et al., 2001). Notably, both Ah plaque-positive and -negative TBI cases had tau-, ubiquitin-, and synuclein-positive cell profiles. While evidence of such lesions developing within hours after a single injury is important for our understanding of neuronal vulnerability to TBI, the present study could not establish a clear link between the expression of these markers of neurodegenerative changes and Ah plaques. Subjects who showed NFTlike profiles were relatively older, cautioning against a cause effect relationship between acute TBI and NFT formation. Unlike Ah plaque deposition, intracellular NFT formation may be the result of a slower and more chronic neurodegenerative process. Thus, in contrast to Ah deposition, AD-like NFT changes either require longer evolution, or are not pathological consequences of a single acute TBI. Neither Ah deposition nor neuronal degeneration in temporal cortex in TBI cases correlated significantly with subjects age, severity of brain injury, post-injury interval, or APOE genotype. We cannot exclude the possibility that Ah or other AD-like changes have developed in some other cortical areas that we were not able to examine. Unlike postmortem studies, we were limited in our investigation only to the single brain region (temporal cortex) that was the target of the surgical intervention. However, post-mortem studies indicate that temporal cortex is the area most often affected and most likely to show Ah changes after TBI (Roberts et al., 1994). In conclusion, our study provides new evidence that acute brain injury is frequently associated with AD-like pathology, and that such changes can evolve very rapidly (i.e., within hours). These data and the extensive literature strongly argue that early AD pathology, as a pre-existing condition, contributes little, if at all, to these observations. How dynamic these changes are over the long term, and their possible relationship with functional and pathological outcomes, remains to be determined in greater number of cases. Future studies focusing on additional plaque-associ-

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ated molecules (e.g., acute phase molecules ACT, IL-1h, in addition to apoE) will be critical for a better understanding of regulatory factors underlying Ah deposition and/or clearance from cortical tissue following TBI.

Acknowledgments We thank Yetta I. Wilbur, Barbara A. Isanski, and Daniel Martinez for expert technical assistance. We are grateful to the University of Pittsburgh Alzheimers Disease Research Center (AG05133), the Brain Trauma Research Center (NS30318), and Ava Puccio for brain tissue processing. We also thank Dr. Peter Davies for providing PHF-1 antibodies. Supported by NINDS NS30318, NIA AG05133, NIA AG11542, MH18273.

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