Hay, J. R., Johnson, V. E., Young, A. M.H., Smith, D. H., and Stewart, W.

(2015)
Blood-brain barrier disruption is an early event that may persist for many years
after traumatic brain injury in humans. Journal of Neuropathology and
Experimental Neurology, 74(12), pp. 1147-1157.

There may be differences between this version and the published version. You are
advised to consult the publisher’s version if you wish to cite from it.

http://eprints.gla.ac.uk/122331/

Deposited on: 8 August 2016

Enlighten – Research publications by members of the University of Glasgow

http://eprints.gla.ac.uk
 Blood brain barrier disruption is an early event that may persist for many

years following traumatic brain injury in humans

Jennifer Hay1,2, Victoria E. Johnson3, Adam M. H. Young2, Douglas H. Smith3 & William
Stewart1,2

1. University of Glasgow, Glasgow G12 8QQ, UK

2. Department of Neuropathology, Queen Elizabeth University Hospital, Glasgow G51 4TF,
UK
3. Penn Centre for Brain Injury and Repair and Department of Neurosurgery, Perelman
School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

Correspondence: Dr William Stewart, MBChB, Ph.D, FRCPath.

Department of Neuropathology
Queen Elizabeth University Hospital
1345 Govan Rd
Glasgow G51 4TF, UK.

Email: William.Stewart@glasgow.ac.uk
Tel: +44 (0)141 354 9535

Support
This study was supported by; the National Institutes of Health (NIH) Grants NS038104,
NS056202; the United States Department of Defense Grant No. PT110785; and The Sackler
Institute Endowment Fund, Department of Neurology, Queen Elizabeth University Hospital,
Glasgow

Page 1 of 22
Abstract

Traumatic brain injury (TBI) is a risk factor for dementia, with studies describing a mixed

neurodegenerative pathology in late survivors. However, the mechanisms driving this post-

TBI neurodegeneration remain elusive. Increasingly, blood brain barrier (BBB) disruption is

recognized in a range of neurological disorders, including dementias; although little is known

of the consequences of TBI on the BBB. From the Glasgow TBI Archive autopsy cases of

single, moderate or severe TBI (n=70) were selected to include a range of survivals from

acute (10h to 13days) to long-term (1 to 47years) survival, together with age-matched,

uninjured controls (n=21). Multiple brain regions were examined for fibrinogen (FBG) and

immunoglobulin G (IgG) immunohistochemistry. Following TBI, 40% of patients dying in

the acute phase and 47% of those surviving a year or more from injury showed multi-focal,

abnormal, perivascular and parenchymal FBG and IgG immunostaining localized to grey

matter, with preferential distribution towards the crests of gyri and deep neocortical layers. In

contrast, where present, controls showed only limited, localized immunostaining. These

preliminary data demonstrate evidence of widespread BBB disruption in a proportion of TBI

patients emerging in the acute phase and, intriguingly, persisting in a high proportion of late

survivors.

Key words: Traumatic brain injury, chronic traumatic encephalopathy, blood brain barrier,

fibrinogen.

Page 2 of 22
Introduction

Traumatic brain injury (TBI) represents a major health concern, with 3.5 million cases in the

United States each year (1). In addition to the immediate health impacts of the injury, TBI is

acknowledged as the strongest environmental risk factor for the development of

neurodegenerative disease, typically reported as Alzheimer’s disease (AD)(2-13) in type or,

more recently, recognized as chronic traumatic encephalopathy (CTE)(14-16). Corresponding

to this, autopsy studies in material from patients exposed to either single moderate or severe

or repetitive mild brain injury reveal a complex of neurodegenerative pathologies including

pathologies in tau, amyloid-² and TDP-43, neuronal loss, neuroinflammation and white

matter degradation (13,15-20). However, the mechanisms driving these late, post-TBI

neurodegenerative pathologies remain elusive.

Disruption in blood brain barrier (BBB) integrity is increasingly recognized in a variety of

disorders, including ischemia (21), multiple sclerosis (22) and neurodegeneration (23, 24). In

particular, imaging (25) and animal modelling (23, 26) studies in AD suggest BBB

dysfunction is an early event in the course of disease and may contribute to its pathogenesis,

perhaps as a result of impaired amyloid-² clearance or associated neuroinflammation (23,27-

31). Furthermore, neuropathology studies report complex vascular changes associated with

BBB dysfunction in AD (32-34), with evidence of a correlation between histological evidence

of BBB leakage and Alzheimer-type pathologies in ‘normal’ ageing (35).

Following TBI, acute phase BBB disruption has been demonstrated in several animal models

(36-48) though with conflicting data on the time-course of this disruption some studies

suggesting BBB disruption is short lived, resolving within hours of injury (36-47), while

others suggest a more dynamic, biphasic course following injury, with early phase BBB

disruption at 3-6 hours post injury followed by later, further BBB at 1-3 (38, 48). In support

of this ‘late’ BBB dysregulation after TBI is the observation of focal immunoglobulin G (IgG)
Page 3 of 22
deposition around callosal blood vessels ipsilateral to controlled cortical impact in mice at 3

months survival from injury (49).

Consistent with observations in animal models, evidence of acute BBB permeability

following severe TBI in humans is provided by reports of elevations in CSF to serum albumin

quotient (50-53) and in serum S100² levels (50-53) after injury. Of note, such evidence of

acute BBB disruption following TBI might predict a population of patients with poor long-

term outcome (52). Furthermore, neuroimaging evidence of BBB disruption has been

reported in patients following TBI, even after clinically mild or moderate injury; in some

cases persisting for years at the site of focal injury (contusions) and with greater frequency in

patients with post-traumatic epilepsy (54). Intriguingly, imaging evidence of BBB disruption

has been observed in American football participants, independent of clinical evidence of TBI,

possibly as a result of exposure to ‘sub-concussive’ head impacts(55).

Thus there is evidence from animal models and limited clinical studies of BBB disruption

following TBI. However, the extent and time course of this TBI-associated BBB disruption in

humans remains poorly understood. In particular, evidence of BBB disruption at late time

points post-injury has not been explored. Here, we utilize material from the Glasgow TBI

Archive to characterize the extent, distribution and temporal dynamics of BBB disruption

following closed head injury, with respect to age-matched, uninjured controls.

Materials and Methods

Case Selection and Brain Tissue Preparation

Ethical approval for this study was granted by the West of Scotland Research Ethics Service.

From the Glasgow TBI Archive, cases with a history of single moderate or severe TBI aged

60 years or under at time of death were selected, representing a range of survival times from

injury to include: acute cases with survival times of 10 hours to <14 days (n=27); intermediate
Page 4 of 22
cases with survival times of 14 days to <1 year (n=11); and long-term cases with survival

times from 1 year to 47 years (n=32). Detailed reports from the original diagnostic

autopsywere available for all cases , where necessary supplemented by forensic and clinical

records, and confirmed a clinical history of moderate or severe TBI as defined by Glasgow

Coma Scale at presentation. Age matched, uninjured controls aged 60 years or under at time

of death and with no known history of neurological disease or history of TBI (n=21) were

selected for comparison. Across the cohorts, post-mortem delays were comparable (p>0.05 in

all comparisons across cohorts; Student t-test). Demographics, clinical data and detail on

neuropathology findings at the original autopsy for each cohort are presented in Table 1.

At the time of the original diagnostic autopsy, whole brains were immersion fixed in 10%

formal saline for a minimum of 3 weeks then dissected, sampled following a standardized

block selection protocol and processed to paraffin using standard techniques. Paraffin tissue

blocks from a coronal slice of the cerebral hemispheres at the mid-thalamic level were

identified for analysis to include: the thalamus; corpus callosum with adjacent cingulate and

superior frontal gyri; hippocampus at the level of the lateral geniculate nucleus extending

through the entorhinal cortex, collateral sulcus and fusiform gyrus; and the insular cortex.

From these tissue blocks 8µm sections were prepared for Hematoxylin and eosin staining and

immunohistochemistry.

Hematoxylin and eosin staining

Hematoxylin and eosin (H&E) staining was performed on sections from all tissue blocks.

Slides were deparaffinized in xylene and rehydrated to water followed by immersion for 10

minutes in hematoxylin (Mayer’s, Leica Microsystems, Wetzlar, Germany). After rinsing and

immersion in Scott’s tap water substitute (Leica Microsystems, Wetzlar, Germany), slides

were differentiated in 1% acid alcohol and rinsed. The sections were then immersed in 25%

aqueous eosin Y solution (TCS Biosciences, Buckingham, UK) for 5 minutes, rinsed, then
Page 5 of 22
dehyrated, cleared and coverslipped.

Immunohistochemistry

Following deparaffinization and rehydration, sections were immersed in 3% aqueous H2O2

(15 minutes) to quench endogenous peroxidase activity. Antigen retrieval was performed via

microwave pressure cooker for 8 minutes in preheated 0.1M Tris EDTA buffer. Subsequent

blocking was achieved by applying 50µL of normal horse serum (Vector Labs, Burlingame,

CA, USA) per 5 mL of Optimax buffer (BioGenex, San Ramon, CA, USA) for 30 minutes.

Incubation with the primary antibody was then performed for 20 h at 4°C. Polyclonal rabbit

anti-human antibodies for fibrinogen (FBG) and immunoglobulin G (IgG) (Dako, Carpinteria,

CA, USA) were utilized at dilutions of 1:17,500 and 1:10,000 respectively. A biotinylated

secondary antibody was then applied for 30 minutes followed by avidin biotin complex as per

the manufacturer’s instructions (Vectastain Universal Elite kit, Vector Labs, Burlingame, CA,

USA). Finally, visualization was achieved using the DAB peroxidase substrate kit

(VectorLabs, Burlingame, CA, USA) followed by counterstaining with haematoxylin. Known

positive tissue sections were run in parallel with test sections in all antibody runs in addition

to sections with primary antibody omitted as standard controls for antibody specificity. All

sections were viewed using a Leica DMRB light microscope (Leica Microsystems, Wetzlar,

Germany). In addition, sections were scanned at 20x using a Hamamastu Nanozoomer 2.0-HT

slide scanner, with the images viewed via the SlidePath Digital Image Hub application (Leica

Microsystems, Wetzlar, Germany).

Analysis of Immunohistochemical Findings

All observations were conducted blind to demographic and clinical data by two independent

observers (JH, AY). Anatomically distinct regions were defined for assessment to include the

neocortical grey matter of the cingulate gyrus; cingulate sulcus; superior frontal gyrus;

parahippocampal gyrus; collateral sulcus; fusiform gyrus and insular cortex. A subanalysis of
Page 6 of 22
neocortical grey matter was performed by dividing the cortex into superficial (layers I-II), mid

(layers III-IV) and deep layers (V- VI). In addition, the white matter of the midline and lateral

extent of the corpus callosum and internal capsule; hippocampal sectors CA1-4 and the

thalamus (sub-divided into medial, intermediate and lateral regions) were assessed. Each

anatomical region was reviewed and a semi-quantitative assessment of the frequency and

intensity of immunoreactivity determined as absent (0), sparse (1), moderate (2) or extensive

(3). Representative examples of immunohistochemical findings and the corresponding semi-

quantitative score are shown in Figure 1.

Statistical analysis

All data were analyzed using Minitab (v.16; Minitab Inc.). The Chi-square Test was used to

assess differences in data between and within cohorts.

Results

Fibrinogen Immunoreactivity After TBI

Controls

In uninjured controls, where present, FBG immunoreactivity was observed in a predominantly

perivascular distribution, highlighting small vessels throughout the neuropil. In addition,

occasional, sparse immunoreactive neurons and glia were observed. In the overwhelming

majority of controls (17 of 21), this FBG immunoreactivity was limited (score 0 or 1) (Figure

2a) and localized (Figure 3). In the remaining 4 controls, localized foci of moderate (score 2)

levels of FBG immunoreactivity were observed in single tissue blocks; in two cases localized

to the thalamus, one to the fusiform gyrus and the last to the superior frontal gyrus (Figure 4).

Causes of death in these controls with localized, moderate FBG immunoreactivity were

mixed; one sudden death in epilepsy, one sudden death of cardiac origin, one sepsis with

multi-organ failure and one pneumonia.

Page 7 of 22
Acute TBI survival

In material from patients dying acutely following TBI (10 hours to <14 days survival), in

addition to perivascular FBG immunoreactivity, regions of more confluent, diffuse

immunostaining were also present (Figure 2b). Indeed, in contrast to the typically limited

abnormal FBG immunoreactivity observed in controls, in this acute TBI survival cohort 88%

(p<0.001; ChiSq) of cases showed at least one anatomical region with moderate (score 2) or

extensive (score 3) FBG immunoreactivity. Further, in contrast to the localized pathology in

controls, following TBI increased FBG immunoreactivity often appeared multifocal,

involving 2 or more regions in 11 out of 27 (40%) acute survival cases (Figure 3). Of the

regions examined, moderate or extensive FBG immunoreactivity was detected most

frequently in material from the cingulate and superior frontal gyri (16 cases), followed by

thalamus (12 cases), medial temporal lobe (11 cases) and insular cortex (6 cases) (Figure 4).

Within the neocortical material (cingulate, superior frontal, fusiform and parahippocampal)

there was evidence of preferential anatomical distribution, with moderate or extensive FBG

immunoreactivity more evident in the crests of gyri than in the depths of the adjacent sulci

(Figure 5a) in both cingulate and medial temporal lobe cortical regions. Further, there was

localization of abnormal FBG immunoreactivity towards mid (layers 3 and 4) and deep

(layers 5 and 6) cortical layers rather than superficial (layers 1 and 2) cortical layers (Figure

2b; Figure 5b) in all cortical regions. Notably, only one acute survival case showed focal,

moderate FBG immunoreactivity in the hippocampal formation (sector CA2), with no acute

survival case showing more than localized, limited FBG immunoreactivity in the corpus

callosum, similar to controls. No localization to a particular thalamic sub-region was evident,

neither was the observed abnormal FBG staining localized to focal pathologies of TBI.

Intermediate TBI survival

Similar to material from patients dying acutely following TBI, regions of moderate or

extensive FBG immunoreactivity were present in a greater number of intermediate (>2 weeks
Page 8 of 22
to 1 year post-TBI) TBI survival patients than in controls, with 7 of 11 (64%; p=0.004 ChiSq)

demonstrating at least one anatomical sub-region with moderate or high FBG

immunoreactivity; showing multifocality in 4 of these cases (Figure 3). Again, material from

the region of the cingulate and superior frontal gyri most frequently showed increased staining

(6 cases), followed by medial temporal lobe (4 cases), thalamus (2 cases) and insular cortex (2

cases) (Figure 4). As in acute survival material, there was a predilection for gyral crests over

sulcal depths in the cingulate gyrus and medial temporal lobe and, though the small number of

cases precludes formal statistical assessment, a trend towards preferential staining in mid to

deep rather than superficial neocortical layers in all cortical regions (Figures 5a and 5b).

There was no notable FBG immunoreactivity in the white matter of the corpus callosum, with

only 2 cases showing more than mild staining in the hippocampus.

Long-term TBI survival

Once again, a higher proportion of long-term survival cases showed evidence of increased

FBG immunoreactivity when compared to matched, non-injured controls (Figure 2c), with at

least one anatomical region showing moderate or extensive FBG immunoreactivity in 22 of

32 cases (69%) (p<0.001; ChiSq). Again, multifocality was common, being present in 15 of

the long term survival cases (47%) (Figure 3); this increased staining most frequent in

material from the medial temporal lobe (17 cases), followed by cingulate and superior frontal

gyri and thalamus (14 cases each) and insular cortex (5 cases) (Figure 4). As in acute and

intermediate cases, a preferential distribution to the crests of gyri over depths of sulci was

evident (Figure 5a). Further, within the crests of gyri, FBG immunoreactivity was greater in

the mid and deeper layers than the superficial layers of the cortex. Thus, in the superior

frontal gyrus 56% showed moderate or high FBG immunostaining in the deeper layers

compared to just 12% in the superficial layers (p=0.001; ChiSq) (Figure 5b) this pattern was

reflected in all cortical regions assessed. In the medial temporal lobe 40% showed high FBG

immunostaining in the deep layers compared to 18% in the superficial layers (p=0.1379;
Page 9 of 22
ChiSq). In contrast to earlier survival time points and controls, 23% (p=0.023; ChiSq) of

cases in this long-term survival cohort showed hippocampal FBG immunoreactivity greater

than mild in at least one hippocampal sector, though with no clear preferential distribution to

any particular sector. In keeping with this widespread increased FBG immunoreactivity in

these long-term survival cases, 58% showed increased thalamic staining, versus just 10% of

controls (p=0.001; ChiSq). However, once again, no increase in FBG immunoreactivity was

detected in material from the corpus callosum.

Immunoglobulin G Immunoreactivity After TBI

Controls

Observations in the IgG stained material corroborated the observations in material stained for

FBG (Figure 2d). Thus, at all survival points and in controls the extent and distribution of IgG

immunoreactivity was comparable to FBG immunoreactivity with regards to greater

perivascular and confluent, diffuse staining IgG immunoreactivity in a proportion of TBI

cases at all survivals when compared to controls. Specifically, IgG immunoreactivity in the

control cohort was limited with only 4 of 21 (19%) cases displaying moderate IgG

immunoreactivity (score of 2); in two cases this was localised to the superior frontal gyrus,

one case localised to the thalamus and one to the fusiform gyrus. Again, no multifocality was

observed and causes of death included sudden death in epilepsy (n=2), drug overdose (n=1),

and pneumonia (n=1).

Acute TBI survival

The acute TBI survival cohort demonstrated moderate or high IgG immunoreactivity in at

least one anatomical sub-region in 63% of cases (p<0.005 ChiSq), with multifocality present

in 10 out of 27 (37%). Of the regions examined, moderate or extensive IgG immunoreactivity

was detected most frequently in material from the medial temporal lobe (10 cases), followed

by the cingulate/superior frontal gyri and the thalamus (8 cases) then the insular cortex (2
Page 10 of 22
cases). As with FBG immunoreactivity there was evidence of preferential anatomical

distribution, with moderate or extensive IgG immunoreactivity more evident in the crests of

gyri than in the depths of the adjacent sulci and in the mid to cortical deep layers when

compared to the superficial cortical layers.

Intermediate TBI survival

Following intermediate survival from TBI, regions of moderate or widespread IgG

immunoreactivity were present in a greater number than in controls with 6 of 11 (54%;

p=0.05 ChiSq) demonstrating at least one anatomical sub-region with moderate/high IgG

immunoreactivity, with multifocality present in 45%.

Long-term TBI survival

The long-term survival TBI cohort demonstrated 71% (23 out of 32 cases) of moderate/high

IgG immunoreactivity in at least one anatomical sub-region (p=0.0002; ChiSq). Multifocality

again was common with 14 of the 32 long-term TBI cases (43%) showing immunoreactivity

in 2 or more sub-regions. Increased IgG staining was most frequent in the medial temporal

lobe (13 cases each), followed by cingulate/ superior frontal gyri and thalamus (12 cases) and

insular cortex (3 cases). The pattern and distribution of IgG immunoreactivity echoed the

results in fibrinogen prepared material, with preferential distribution to the crests of gyri over

the depths of sulci in both cingulate gyri and hippocampal gyri, and greater IgG

immunoreactivity observed in the mid and deeper layers versus the superficial neocortical

layers.

Association of BBB disruption with TBI-associated pathologies

Reviewing H&E stained sections from the multiple tissue blocks examined for

immunohistochemical evidence of BBB disruption confirmed anticipated BBB disruption in

association with acute, focal hemorrhagic pathologies, in addition to more widespread and
Page 11 of 22
diffuse BBB disruption independent of focal pathology. Specifically, in material from acute

TBI survivors 21 TBI-associated focal hemorrhagic or contusional lesions were observed

across 13 of the 27 cases, with virtually all (12 of 20 lesions) displaying some degree of

extravascular fibrinogen immunoreactivity in the surrounding parenchyma. However, in the

majority of these cases (9 of 13; 69%) BBB disruption was not confined to the region of focal

pathology, with evidence of widespread, diffuse BBB also present in the remaining tissue

blocks. Notably, in the 14 acute survival TBI cases without focal hemorrhage or contusion, 12

(86%) also displayed high fibrinogen immunoreactivity in at least one region examined.

In material from patients surviving beyond the acute phase of injury evidence of healed

hemorrhages or contusions, with histological features consistent with lesions dating from the

time of the original TBI, were present in 2 of 11 intermediate survival (comprising 6

individual lesions) and 8 of 32 long-term survival cases (comprising 12 distinct lesions).

While not as extensive as observed in relation to focal hemorrhages or contusions in acute

survivors, some evidence of BBB disruption was observed in the immediate locality of all 6

lesions in material from intermediate survivors. In contrast, evidence of focal BBB disruption

in association with healed hemorrhages or contusions was less frequently observed in material

from long-term survivors, with just 6 of 12 lesions associated with abnormal FBG or IgG

immunoreactivity. Of note, both of the intermediate survival cases and all 8 of the long-term

survival cases with BBB disruption in association with focal pathologies also displayed more

widespread, diffuse BBB disruption remote from focal pathology. Further, 6 of 9 intermediate

and 15 of 24 long-term survival cases showing no evidence of focal hemorrhagic or

contusional pathology in the material examined displayed evidence of high FBG

immunoreactivity in at least one region.

In addition to these focal hemorrhagic and contusional pathologies, histological evidence of

diffuse hypoxic/ ischemic injury, microgliosis and/or astroglisosis in varying stages of

Page 12 of 22
evolution was identified in a proportion of TBI cases across all survivals. No correlation

between these TBI-related pathologies and evidence of BBB disruption was observed.

Discussion

Here we demonstrate histological evidence of widespread, diffuse blood-brain barrier (BBB)

disruption in survivors of a single moderate or severe TBI when compared to non-injured

controls. Notably, evidence of increased and widespread extravascular immunoreactivity for

fibrinogen (FBG) and immunoglobulin G (IgG) was observed not only in material from

patients dying in the acute phase post-injury, but also in a high proportion of those surviving

many years after injury. As such, these data raise the possibility that a single moderate or

severe TBI is responsible for immediate and long-lasting alterations in BBB function

following injury, which might contribute to post-TBI neurodegeneration.

Under normal conditions, the plasma proteins fibrinogen (340kDa) and IgG (150kDa) do not

cross the BBB. However, we observed widespread, multifocal, perivascular and parenchymal

FBG and IgG deposition in approximately half of patients dying within the first 2 weeks

following a single moderate or severe TBI; indicating that these proteins had crossed the BBB

in the acute phase following injury. Similar acute BBB leakage has been identified in various

animal models of TBI, as revealed by the presence of either extravasated serum proteins or

intravascularly injected labels in the brain parenchyma (36-44, 46, 56, 57) following injury. In

these models often extensive, acute BBB leakage can be observed in the absence of

hemorrhage, consistent with our observations in this human, autopsy-acquired tissue.

As regards possible structural correlates of this acute BBB disruption, ultrastructural studies

in animal models of TBI have identified a variety of alterations in vascular endothelia in the
Page 13 of 22
acute phase following injury, including the formation of vacuoles, craters and microvilli (47,

57-59) some of which were later identified in human TBI (60, 61). These very rapid changes

in vascular structure and function have been attributed to a range of mechanisms such as

direct perturbation of the vessels by mechanical forces, including the immediate disruption of

vascular endothelial cells. Additional, secondary insults following injury might also be

detrimental to normal endothelial integrity and function, including acute rises in arterial

pressure or intravascular thrombus formation (45, 56, 57). Finally, active physiological

changes have also been described in both animals and humans following TBI, including

increased transendothelial vesicular transport with otherwise intact tight junctions (47, 59, 61,

62) as well as alterations to other components of the BBB, such as early astrocyte disruption

and swelling (45, 58).

Studies suggest that these acute BBB disruptions may confer worse long-term outcome

following TBI clinically (52), although the relative role of this pathology is unknown. Indeed,

acute BBB opening after TBI may simply be one of many common pathological features of a

more severely injured brain, which collectively contribute to poor outcome. Nonetheless,

specific deleterious mechanisms of BBB disruption following TBI may include influx of fluid

together with chemical and protein mediators promoting vasogenic edema, disruption in the

normal pathways to clear toxic metabolites, and a failure to deliver normal metabolites vital

for function. In turn, the temporal course and relative contribution of these various

consequences of BBB disruption may differ with the mode of injury.

Although data from animal models are reported as showing evidence of a biphasic BBB

opening (38, 48), in the present study we identified no clear evidence of defined phases of

BBB disruption across our various survival cohorts, in particular in the acute phase.

Nevertheless, the possibility of distinct phases of BBB disruption following injury cannot be

entirely excluded, with the diverse and heterogeneous nature of post-mortem TBI cases with
Page 14 of 22
varying intercurrent illnesses, survival times and causes of injury conceivably masking such

an observation. Nonetheless, studies specifically directed at understanding the underlying

mechanisms of this BBB dysfunction over time might reveal important potential therapeutic

targets with defined windows of opportunity.

While a degree of acute BBB disruption was anticipated, the number of cases, often with

extensive BBB disruption following long-term survival from TBI was, nonetheless,

surprising. Specifically, extensive, multifocal, extravascular FBG and IgG deposition was

observed in approximately half of those surviving a year or more from injury in our cohort.

Notably, clearance of large soluble proteins from the brain parenchyma occurs via convective

bulk flow of interstitial fluid over the order of hours to days, or more rapidly via reverse

transcytosis, such as occurs with IgG (63). Thus, the observation of soluble plasma proteins in

the brain parenchyma of patients exposed to TBI more than a year prior to death is consistent

with on-going BBB disruption, rather than evidence of protein deposited at the time of injury

and not cleared in the intervening period.

The pattern and distribution of chronic BBB disruption post-TBI was more frequently

localized to the grey matter of the neocortical ribbon and deep grey nuclei, with a preferential

distribution in the former to mid and deeper cortical layers over superficial layers and the

crests of gyri over depths of sulci. This pattern indicates preferential vulnerability in specific

locations within a more global and diffuse process, rather than a heterogeneous multifocal

pathology. Undoubtedly this observation presents challenging and interesting data given the

contrast with current descriptions of aspects of the neuropathology of post-TBI

neurodegeneration and CTE where localization of tau pathologies to the depths of sulci is

reported. However, at present, the pathologies of CTE are provisional, therefore should be

taken as such. More research into these conflicting observations should be carried out in the

future.
Page 15 of 22
Notably our data demonstrate no association with evidence of widespread and diffuse BBB

disruption and the presence of other TBI-associated pathologies. Specifically, BBB disruption

was not localized to areas of focal TBI pathologies, such as contusions or hemorrhages.

Further, there was no correlation between diffuse and widespread BBB disruption and diffuse

ischemic pathology. Nevertheless, the number of cases and necessary heterogeneity in

pathologies in this cohort limit the ability to determine whether specific diffuse primary or

secondary pathologies in the acute phase can account for later patterns of BBB disruption.

However, this will be worthy of exploration using larger post-mortem cohorts and, possibly,

animal models.

In recent years, the chronic clinical and neuropathological sequelae of TBI have been

increasingly reported, with particular attention paid to the association of TBI survival with

increased risk of progressive neurodegenerative disorders, specifically CTE (14-16).

Neuropathological examination of autopsy acquired material from patients with survival

greater than a year from a single moderate or severe TBI reveals an increased frequency of

neurodegeneration-associated pathologies when compared to material from matched controls,

including amyloid-² plaques, neurofibrillary tangles, inflammation and white matter

degradation (13, 17, 18, 20). Notably, the role of the BBB in neurodegeneration is of

increasing interest, with evidence indicating that changes to the brain’s microvasculature may

actively contribute to development of AD pathology (for detailed review see (23)). Various

micro-vascular changes have been reported in AD including changes to vessel structure and

density (33), BBB breakdown and leakage (32, 35), and secretion of potentially neurotoxic

factors from the vascular endothelium (64). While the extravasation of serum proteins such as

fibrinogen has also been shown to correlate with the presence of Alzheimer-type

pathology(35), the cause or effect relationship between alterations in the vasculature and

associated neurodegenerative pathologies is not well defined. However, increasing clinical

Page 16 of 22
and experimental data suggest vascular changes can occur early and may contribute to states

of hypoperfusion promoting neuronal injury (23, 33, 65, 66). Studies to evaluate potential

associations between BBB breakdown and other pathologies associated with

neurodegenerative disease will be an important future consideration.

Perhaps the most widely studied aspect of BBB with regards to AD, is its role in the clearance

and sequestration of amyloid beta to the peripheral circulation (28, 67-69). Interestingly,

individuals with high serum fibrinogen have been shown at greater risk of AD (70, 71).

Fibrinogen was also shown to accelerate inflammation and neurovascular damage in a mouse

mode of AD (72). While the number of cases described in the present study precludes analysis

of multiple co-variants, the interplay and temporal relationship between BBB disruption and

subsequent progressive neurodegenerative pathologies will be an important direction for

future investigation.

Conclusion

Here we present the first preliminary data indicating that after just a single moderate or severe

TBI there is neuropathological evidence of widespread, diffuse, multifocal BBB disruption in

just under one half of patients, even after many years of survival from injury. Given that

vascular dysfunction may be an important contributor to neurodegenerative disorders, acute

and chronic BBB alterations post-TBI will be important to examine in this context.

Page 17 of 22
Table Legend

Table 1. Demographic and clinical data for all groups.

Figure Legends:

Fig. 1 Representative examples of the patterns of FBG immunoreactivity encountered

(a) Section from the superior frontal gyrus of a 47 year old male TBI patient who died 1 year

following a fall. No abnormal FBG immunoreactivity is present (score of 0). (b) Limited,

faint perivascular FBG immunoreactivity (score of 1) from the superior frontal gyrus of a 60

year old male TBI patient who died 16 years after a road traffic accident. (c) More

widespread, moderate perivascular FBG immunoreactivity (score of 2) in the superior frontal

gyrus of a 60 year old male TBI patient who survived 10 hours after a fall. (d) Extensive

perivascular and adjacent parenchymal FBG immunoreactivity (score of 3) in the superior

frontal gyrus of a 50 year old male TBI patient who survived 1 year after an assault. (e)

Extensive perivascular FBG immunoreactivity in thalamic region of a 60 year old male TBI

patient who survived 8 days after a fall. (f) Limited perivascular FBG immunoreactivity in

parahippocampal region of a 56 year old female TBI patient who survived 24 hours after a

road traffic accident. Scale bars = 1mm and apply to all corresponding images.

Page 18 of 22
Fig. 2 Representative images of FBG and IgG immunoreactivity following TBI and in

controls. Absence of FBG immunoreactivity in the superior frontal gyrus of a 46 year old

male with no history of TBI. (b) Extensive FBG immunoreactivity in the superior frontal

gyrus, with preferential distribution of staining to the mid and deep cortical layers, in a 20

year old male TBI patient who survived 2 days following an assault. Extensive FBG (c) and

IgG (d) immunoreactivity in the adjacent sections from the superior frontal gyrus of a 60 year

old male TBI patient who survived 18 years following a fall. Scale bars = 1mm and apply to

all corresponding images.

Fig. 3 Extent of abnormal FBG immunoreactivity following TBI at varying survivals

versus controls. Whilst moderate or extensive FBG immunoreactivity was an uncommon

observation in controls, occurring in just 4 of 21 cases (19%), it was a frequent observation

following TBI at all survival time points assessed, being present in 88%, 62% and 69% of

acute, intermediate and long-term survival cases respectively. Furthermore, in controls

abnormal FBG immunostaining was restricted to single anatomical regions, in contrast to the

often multifocal pathology in material following TBI. (*p<0.005; **p<0.001; Chi-square TBI

cohort v control fibrinogen positivity).

Fig. 4 Regional distribution of FBG immunoreactivity following TBI versus controls. At

all survival intervals and in each region analyzed there was evidence of BBB disruption

following TBI, evidenced by moderate/ extensive FBG immunoreactivity, in a higher

proportion of TBI survivors than matched, non-injured controls in material from the (a)

cingulate/superior frontal gyri, (b) thalamus (c) hippocampus and (d) insular cortex.

(*p<0.05; **p<0.01; ***p<0.005; +p<0.0005; ++p<0.0001; Chi-square TBI cohort v control)

Fig. 5 Neocortical distribution of FBG following TBI. (a) Across all survival time points

there was a clear preferential distribution of abnormal FBG immunoreactivity to the crests of

Page 19 of 22
gyri when compared to the depths of sulci, as illustrated here for the superior frontal gyrus

versus the adjacent cingulate sulcus (*p<0.01; **p<0.001; Chi-square sulcus versus gyrus).

(b) Further, within the neocortical grey there was preferential distribution of abnormal

staining to the mid (layers 3 and 4) and deep (layers 5 and 6) cortical layers when compared

to superficial layers (layers 1 and 2). (+p<0.01; ++p<0.005; Chi-square deep versus

superficial cortical layers).

Page 20 of 22
Table 1.

TBI: Acute TBI: Intermediate TBI: Long-Term Controls

Survival (n=27) Survival (n=11) Survival (n=32) (n=21)
Mean age (Range) 44.4 years 32 years 46.3 years 34.3 years
(9-60) (17-56) (19-60) (14-60)

Males 17 11 31 12
(63%) (100%) (97%) (57.1%)
Mean PM Delay (Range) 56.1 hours 74.7 hours 65.5 hours 71.6 hours
(3-240) (26-192) (9-184.5) (12-144)

Mean Survival Interval (Range) 69.3 hours 72.8 days 7.8 years Not applicable
(6-216) (14-279) (1-47)
Cause of Fall 15 2 15 Not applicable
TBI (56%) (18%) (47%) (No history TBI)
RTA 7 5 5
(26%) (42%) (16%)
Assault 4 3 8
(15%) (25%) (25%)
Unknown 1 1 4
(4%) (8%) (12%)
Cause of Head injury 25(93%) 4 (33%) 0 0
Death Bronchopneumonia 2(7%) 4(33%) 7(22%) 1(4.8%)
ARDS 0 2(17%) 1(3%) 0
Pulmonary 0 1(8%) 0 0
thromboembolism
Heart disease 0 0 6 (19%) 4(16.67%)
Alcohol related 0 0 2(6%) 0
Pyelonephritis 0 0 1(3%) 0
Multi-organ failure 0 0 1(3%) 0
GIT hemorrhage 0 0 1(3%) 0
Polytrauma 0 0 1(3%) 0
Drug overdose 0 0 0 4(16.67%)
SUDEP 7(22%) 8(38.1%)
Pulmonary edema 0 0 1(3%) 0
Septicemia 0 0 0 2(8.3%)
Inhalation of gastric 0 0 0 2(8.3%)
contents
Unknown 0 0 4(12%) 0
TBI Skull fracture 20(74%) 6(54%) 15(47%) 0
Pathologies DAI 11(40%) 10(91%) 6(19%) 0
Brain Swelling 13(48%) 2(18%) 0 0
SDH 20(74%) 4(36%) 13(41%) 0
EDH 0 0 3(9%) 0
SAH 15(56%) 0 1(3%) 0
Key: TBI = traumatic brain injury; SUDEP = sudden unexpected death in epilepsy; GIT = gastrointestinal tract; ARDS
= acute respiratory distress syndrome; RTA = road traffic accident; DAI = Diffuse Axonal Injury; SDH = Subdural
hemorrhage; EDH = Extradural hemorrhage; SAH = Subarachnoid hemorrhage.

Page 21 of 22
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