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Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry

Article (PDF Available) inActa Neuropathologica 112(4):389-404 · November 2006with177 Reads
DOI: 10.1007/s00401-006-0127-z · Source: PubMed
Assessment of Alzheimer's disease (AD)-related neurofibrillary pathology requires a procedure that permits a sufficient differentiation between initial, intermediate, and late stages. The gradual deposition of a hyperphosphorylated tau protein within select neuronal types in specific nuclei or areas is central to the disease process. The staging of AD-related neurofibrillary pathology originally described in 1991 was performed on unconventionally thick sections (100 mum) using a modern silver technique and reflected the progress of the disease process based chiefly on the topographic expansion of the lesions. To better meet the demands of routine laboratories this procedure is revised here by adapting tissue selection and processing to the needs of paraffin-embedded sections (5-15 mum) and by introducing a robust immunoreaction (AT8) for hyperphosphorylated tau protein that can be processed on an automated basis. It is anticipated that this revised methodological protocol will enable a more uniform application of the staging procedure.
Acta Neuropathol (2006) 112:389–404
DOI 10.1007/s00401-006-0127-z
Staging of Alzheimer disease-associated neuroWbrillary
pathology using paraYn sections and immunocytochemistry
Heiko Braak · Irina AlafuzoV · Thomas Arzberger ·
Hans Kretzschmar · Kelly Del Tredici
Received: 8 June 2006 / Revised: 21 July 2006 / Accepted: 21 July 2006 / Published online: 12 August 2006
© Springer-Verlag 2006
Abstract Assessment of Alzheimer’s disease (AD)-
related neuroWbrillary pathology requires a procedure
that permits a suYcient diVerentiation between initial,
intermediate, and late stages. The gradual deposition
of a hyperphosphorylated tau protein within select
neuronal types in speciWc nuclei or areas is central to
the disease process. The staging of AD-related neuroW-
brillary pathology originally described in 1991 was per-
formed on unconventionally thick sections (100 m)
using a modern silver technique and reXected the pro-
gress of the disease process based chieXy on the topo-
graphic expansion of the lesions. To better meet the
demands of routine laboratories this procedure is
revised here by adapting tissue selection and process-
ing to the needs of paraYn-embedded sections (5–15 m)
and by introducing a robust immunoreaction (AT8) for
hyperphosphorylated tau protein that can be processed
on an automated basis. It is anticipated that this
revised methodological protocol will enable a more
uniform application of the staging procedure.
Keywords Alzheimer’s disease · NeuroWbrillary
changes · Immunocytochemistry ·
Hyperphosphorylated tau protein · Neuropathologic
staging · Pretangles
The development of intraneuronal lesions at selec-
tively vulnerable brain sites is central to the pathologi-
cal process in Alzheimer’s disease (AD) [42, 46, 55, 56,
58, 94]. The lesions consist chieXy of hyperphosphory-
lated tau protein and include pretangle material, neu-
roWbrillary tangles (NFTs) in cell bodies, neuropil
threads (NTs) in neuronal processes, and material in
dystrophic nerve cell processes of neuritic plaques
(NPs) [7, 19, 30].
The AD-related pathological process spans decades
and, during this time, the distribution pattern of the
lesions develops according to a predictable sequence
[8, 21, 89, 93, for a broader discussion of the histopath-
ological diagnosis of AD, see 54, 69, 76]. A staging sys-
tem for the intraneuronal lesions introduced in 1991
diVerentiated initial, intermediate, and late phases of
the disease process in both non-symptomatic and
symptomatic individuals [21, 2428,
39, 40, 43, 44, 63,
71, 75, 7982, 84, 85, 87]. In 1997, this staging system
This study was made possible by funding from the German
Research Council (Deutsche Forschungsgemeinschaft) and
BrainNet Europe II (European Commission LSHM-CT-2004-
503039). This publication reXects only the viewpoint of the
authors, the European Community is not responsible for its use
or contents.
H. Braak (&) · K. Del Tredici
Institute for Clinical Neuroanatomy,
J.W. Goethe University Clinic, Theodor Stern Kai 7,
60590 Frankfurt/Main, Germany
I. AlafuzoV
Institute of Clinical Medicine, Pathology,
Kuopio University and University Hospital,
Harjulantie 1, P.O. Box 1627, 70211 Kuopio, Finland
T. Arzberger · H. Kretzschmar
Institute of Neuropathology, Ludwig Maximilians University,
81377 Munich, Germany
K. Del Tredici
Clinic for Psychiatry and Neurology Winnenden,
71364 Winnenden, Germany
390 Acta Neuropathol (2006) 112:389–404
was incorporated into the NIH-Reagan criteria for the
neuropathological diagnosis of AD [64, 66, 83]. The
Braak system was based upon assessment of two
100 m sections processed according to the silver-
iodate technique proposed by Gallyas [4951, 67, 68,
78]. The Wrst section included the hippocampal forma-
tion at uncal level, the anterior parahippocampal
gyrus, and portions of the adjoining occipito-temporal
gyrus. The second section, taken from the occipital
neocortex, encompassed portions of the striate area,
parastriate area, and peristriate region (Fig. 1). Dis-
tinctive diVerences in the topographical distribution
pattern of the neuroWbrillary lesions enabled the
observer to assign a given autopsy case to one of six
stages [21, 22]. This simple system had the advantage
of being reproducible without having to rely on quan-
titative assessments or knowledge of patient-related
data (age, gender, cognitive status). Furthermore, the
result of the silver reactions in unconventionally thick
sections provided a means of “reading” a given stage
with the unaided eye.
For routine diagnostic purposes, however, such a
system is problematic because it calls for unusually
thick sections cut from blocks embedded in an uncon-
ventional medium [polyethylene glycol (PEG)] [91].
In addition, the method requires that free Xoating sec-
tions be stained by experienced laboratory assistants
using a non-automated silver technique. These fea-
tures drastically limit the feasibility of the original
staging protocol for routine diagnostic use in the
majority of neuropathological laboratories [35]. At the
same time, they account for the fact that the staging
system has found broad acceptance in a research con-
text while having been subjected to numerous modiW-
cations [9, 18, 36, 39, 40, 53, 5962, 6971, 75, 76, 83, 88].
Neuropathologists in routine diagnostic praxis as well
as reference centers that maintain brain banks are
interested in a uniform staging procedure so that the
material submitted by various participating institu-
tions can be used and evaluated according to the same
criteria. Such a staging system must be reproducible,
cost-eVective, and easy.
In recent years, sensitive immunocytochemical
methods have been developed, the application of
which makes it possible to reliably detect not only
incipient neuroWbrillary pathology in mildly involved
brain regions of non-symptomatic individuals but also,
with disease progression, the full extent of the intran-
euronal pathology in the end phase [19, 90]. NeuroW-
brillary changes of the Alzheimer type consist of stable
proteins that are impervious to postmortal delay or
suboptimal Wxation conditions, and immunoreactions
for demonstration of hyperphosphorylated tau protein
can be carried out even on tissue that has been stored
for decades in formaldehyde [1, 72].
Immunoreactions against hyperphosphorylated tau,
however, cannot fully replace the Gallyas silver stain-
ing method (see Technical addendum), inasmuch as
both techniques identify partially diVerent structures.
At the beginning of the intraneuronal changes, a solu-
ble and non-argyrophilic material develops, Wlling the
somata of involved nerve cells as well as dendritic pro-
cesses and axons. Thereafter, the distal dendritic
segments become snarled and develop dilated append-
ages. The soluble “pretangle” material is identiWable in
immunoreactions for hyperphosphorylated tau protein
but remains Gallyas-negative. It is the harbinger of an
argyrophilic, insoluble, and non-biodegradable Wbril-
lary material that emerges after cross-linkage and
aggregation of the soluble pretangle material [7, 19, 96].
The Gallyas-positive neuroWbrillary aggregations grad-
ually Wll the cytoplasm, sometimes inWltrating the prox-
imal dendrites to form a neuroWbrillary tangle, and
appear in dendritic segments as NTs without involving
the axon [19, 45, 95]. Following neuronal death, the
abnormal material remains visible in the tissue as
extraneuronal ghost tangles or tombstone tangles.
With time, ghost tangles are no longer immunoreactive
for hyperphosphorylated tau protein and their argyro-
philia gradually becomes less pronounced [17, 19, 34].
In summary, the pretangle material can only be
detected by immunocytochemistry and this fact has
been taken into account in the revised staging proce-
dure presented here (Fig. 2).
In view of the progress that has been made in the
demonstration of the neuroWbrillary changes of the
Alzheimer type, it seemed expedient to revise the 1991
staging procedure by introducing immunoreactions for
visualization of hyperphosphorylated tau and by adapt-
ing the tissue selection and processing to the demands
of the routine diagnostic laboratory. The goal remains
the same, namely to stage the AD-related neuroWbril-
lary pathology in six stages, as previously, with empha-
sis this time on the plexuses formed of both pretangle
and tangle material, but using paraYn sections immu-
nostained for hyperphosphorylated tau and processed
on an automated basis. To illustrate the advantages
and disadvantages of both methods, required brain
regions with lesions representing AD stages I–VI have
been digitally photographed both in silver- and immu-
nostained 100 m PEG sections and in 7
m paraYn
sections immunostained for hyperphosphorylated tau
protein (AT8–antibody). The revised procedure is
intended to facilitate the uniform application of the
staging procedure, which now can be performed with
greater eYciency than previously.
Acta Neuropathol (2006) 112:389–404 391
392 Acta Neuropathol (2006) 112:389–404
Fixation and macroscopic preparation
Brains obtained at autopsy should be Wxed by immer-
sion in 10% formalin (4% aqueous solution of HCHO)
for one week or longer. Partially remove the meninges
to uncover the rhinal sulcus, collateral sulcus, and calc-
arine Wssure (Fig. 1a–d).
Whereas the original staging procedure requires
evaluation of thick silver-stained sections from two rela-
tively large blocks of cortical tissue, the revised version
uses immunostained paraYn sections microtomed from
three blocks of conventional size that Wt routine tissue
cassettes. Figure 1d shows the cutting lines for removal
of the three blocks and, in addition, those for the classi-
cal view of the hippocampal formation. Alternatively,
the tissue on one side of a cut can be used for conven-
tional paraYn embedding (thin sections), and that on
the other side for PEG embedding (thick sections).
The Wrst block includes anteromedial portions of the
temporal lobe cut at the mid-uncal or amygdala level
(frontal section through the temporal lobe at the level
of the mamillary bodies) encompassing anterior por-
tions of both the parahippocampal gyrus and adjoining
occipito-temporal gyrus. The cutting line runs through
the rhinal sulcus (Fig. 1a, d). The sections from this
block contain central portions of the entorhinal region
and the adjoining transentorhinal region, the latter of
which is concealed in the depths of the rhinal sulcus
[23, 98]. This block is essential for assessment of neuro-
Wbrillary AD stages I–III.
The second block simpliWes assessment of stage IV.
It is obtained from the same slice as the Wrst block and
includes portions of the medial and superior temporal
gyri (Fig. 1a, d). As an alternative to the Wrst two
blocks, the entire slice through the temporal lobe can
be used, provided slides of suYcient size are available.
Reduction of this slice to two blocks is recommended
to avoid exceeding the size of conventional tissue cas-
The third block is removed halfway between the
occipital pole and the junction of the parieto-occipital
sulcus with the calcarine Wssure. The cut is oriented
perpendicular to the calcarine Wssure (Fig. 1c, d).
Again, the size of the block has been reduced to Wt
standard tissue cassettes. Care has to be taken that the
block includes part of the lower bank of the calcarine
Wssure and the adjoining basal occipital gyri encom-
passing portions of the neocortex, i.e., the peristriate
region, parastriate Weld, and a clearly deWnable pri-
mary Weld, the striate area (Brodmann Weld 17 with the
macroscopically identiWable line of Gennari). This
block is indispensable for recognition of the neuroW-
brillary AD stages V and VI.
Mounted paraYn sections of 5–15 m thickness
are de-waxed and re-hydrated.
The monoclonal antibody AT8 (Innogenetics,
Belgium) is one of several commercially available spe-
ciWc antibodies that show robust immunoreactivity for
hyperphosphorylated tau protein, and a recently pub-
lished immunocytochemical trial using this antibody
has yielded reproducible results [1]. AT8 does not
cross-react with normal tau epitopes or require special
pre-treatments, and it is exceptionally reliable in
human autopsy material regardless of the length of the
Wxation time in formaldehyde and/or the condition of
the preserved tissue [16, 19, 57, 77]. When performed
on paraYn sections (5–15 m), AT8-immunoreactions
permit counter-staining for other structures of interest,
1 S
eme s
ng t
on o
e t
s o
required for staging of AD-related neuroWbrillary changes. The
rst block at the far left (a) includes anteromedial portions of the
temporal lobe. It is cut at the mid-uncal or amygdala level (frontal
section at the level of the mamillary bodies) and includes the
parahippocampal and adjoining occipito-temporal gyri (see en-
larged insert below a). The cutting line runs through the rhinal sul-
cus. The second block comes from the same level and includes
part of the medial and superior temporal gyri (a). The third block
at the far right (c) is removed from basal portions of the occipital
lobe. The cut is oriented perpendicular to the calcarine Wssure.
The block includes the neocortex covering the lower bank of the
calcarine Wssure and the adjoining basal occipital gyri. It thus
shows portions of the peristriate region as well as of the parastri-
ate and striate areas (see enlarged insert below c). (b) This block
provides the classical view of the hippocampal formation and is
removed at the level of the lateral geniculate nucleus. It is rou-
tinely dissected for diverse diagnostic purposes of the hippocam-
pal formation. The cutting line runs through the collateral sulcus
(d). The parahippocampal gyrus at this latitude abuts posteriorly
on the lingual gyrus and contains either posterior portions of the
entorhinal and transentorhinal regions or lingual neocortex. Inso-
far as the Wrst temporal block at mid-uncal level is essential for
the evaluation of the transentorhinal and entorhinal regions
(diagnosis of AD stages I–III), the classical hippocampus section
is not absolutely required for staging. The middle drawing in the
second row indicated by a double frame shows the anatomical
landmarks of the entorhinal region seen basally. Note the wart-
like elevations in anterior portions of the parahippocampal gyrus
roughly outlining the extent of the entorhinal allocortex. The low-
er schemata highlight the lamination pattern of the areas that
need to be evaluated for staging purposes. The various allocorti-
cal and neocortical laminae are indicated across the upper mar-
gins. 17, 18, 19 striate area, parastriate area, peristriate region.
Abbreviations: CA1 Wrst sector of the Ammon’s horn, ent entorh-
inal region, parasubic parasubiculum, presubic presubiculum,
temp. neocortex temporal neocortex, tre transentorhinal region
(mesocortex), transentorhin. transentorhinal
Acta Neuropathol (2006) 112:389–404 393
provided that diaminobenzidine is used as a chromo-
gen. Homogeneous immunoreactions can also be
achieved using PEG sections (50–150 m) (Fig. 2). The
sections are incubated for 40 h at 4°C with the AT8
antibody (1:2,000) and thereafter processed for 2 h
with the second biotinylated antibody (anti-mouse
IgG). Reactions are visualized with the ABC-complex
(Vectastain) and 3,3-diaminobenzidine (Sigma).
Prolonged Wxation of brain tissue in a formalde-
hyde solution may cause metachromatic precipitations
394 Acta Neuropathol (2006) 112:389–404
(Buscaino bodies or mucocytes) [73]. Components of
this material partially react with silver methods and
also may interfere with immunoreactions. The precipi-
tations can be removed with pyridine or a tenside solu-
tion [1 unit volume Tween 20 (Merck-Schuchardt
822184) and 9 unit volumes de-ionized water] at 80°C
for 30 min or both. The sections are then rinsed thor-
oughly under running tap water and transferred to
de-ionized water.
Comparison between Gallyas silver- and
AT8-immunostained thick (100 m) sections
Figure 2 is designed to facilitate a direct comparison
between selected cortical areas in 100 m thick PEG-
embedded sections. The Wrst section of each pair has
been silverstained according to a modiWed version of
the technique originally proposed by Gallyas [22, 31,
4951, 67, 68, 78], whereas the second serial section
(i.e., back-to-back sections from the identical tissue
block) underwent staining with the antibody AT8.
Intraneuronal neuroWbrillary tangles are visualized
with equal clarity by both methods (Fig. 2). Further, the
plexuses of argyrophilic NTs are visible not only in the
Gallyas sections but also in the AT8-immunostained sec-
tions—in the latter, however, the plexuses can be seen to
include additional pathologically altered neuronal com-
ponents (dendrites, axons) that contain non-argyrophilic
“pretangle” material (see Fig. 2f, h). Such immuno-
stained plexuses appear much more compact than those
depicted by the silver stain, and their obvious advantage
is that they can be recognized immediately with the
naked eye (see Fig. 3). This applies particularly to the
AT8-immunoreactive plexuses located in the deep corti-
cal layers (see Fig. 2v, x, z, z), whereas in Gallyas sec-
tions such macroscopic recognition is not always
possible (compare Fig. 2v with Fig. 2u).
Nonetheless, the distribution pattern of the immu-
noreactive cortical alterations throughout the various
Welds that are crucial for staging purposes corresponds
to that of the argyrophilic lesions (Fig. 2) and, as such,
it allows the observer to trace the progress of the neu-
roWbrillary pathology in both silverstained (Fig. 2) and
immunostained sections alike (Fig. 3). The greater
emphasis on the abnormal plexuses in AT8-immunore-
active sections, however, facilitates the immediate
diagnostic assessment of the stages, as, for instance, is
readily evident even in the scaled down photographs of
the hemisphere sections shown in Fig. 3. These immu-
nopositive plexuses are still visible macroscopically in
paraYn sections (5–15 m), and it is helpful, initially,
without using the microscope, to view all three slides
against a light background to assign them preliminarily
to a given stage.
The Wnal diagnosis is essentially based on recogni-
tion of the topographical distribution pattern of the
neuroWbrillary pathology and calls for a precise knowl-
edge of which regions in the cerebral cortex, in which
sequence, develop the AD-related neuroWbrillary
lesions. This decision can be made with almost the
same degree of accuracy regardless of whether immu-
nostained or silverstained sections are employed,
although, based on experience, there is a slight ten-
dency to assign a higher stage to the immunostained
slides. The frequency of stage I cases, for example, is
somewhat higher in AT8-immunostained sections
because in the incipient phases of the disease process
AT8-immunopositive nerve cells appear that still lack
argyrophilic material. Thus, it is advisable to perform
the staging procedure using either the Gallyas or AT8
technique but not both methods.
2 C
son o
yas s
ver- an
of cortical neuroWbrillary pathology as seen in adjacent serial
100 m polyethylene glycol-embedded sections. The distribution
pattern of the lesions throughout the various cortical Welds that
are necessary for staging purposes basically corresponds in both
methods. It is possible with either technique to assess the progress
of the neuroWbrillary pathology. In the revised staging procedure,
however, the greater emphasis on the presence of abnormal plex-
uses, which also include non-argyrophilic pretangle material in
AT8-ir sections, facilitates rapid diagnostic assessment of the
stages. a–d stage I: Mild involvement is conWned to the transen-
torhinal region. Note that the plexus of AT8-ir nerve cell pro-
cesses (b and d) is more conspicuous than that of argyrophilic
neuropil threads (a and c). Sections originate from a non-dement-
ed 62-year-old male. e–h stage II: Lesional density increases and
the pathology extends into the entorhinal region. Layer pre-
gradually sinks into a deeper position at the border between en-
torhinal and transentorhinal region (arrowhead). Note the great-
er breadth of the ir-plexus in comparison to silverstained nerve
cell processes (compare f and h with e and g). Immunoreactions
begin to show the deep entorhinal plexus (pri-). The sections
were obtained from a non-demented 78-year-old male. i–n stage
II: The pathology in the outer and inner entorhinal (i, j) and
transentorhinal (k, l) cellular layers worsens, and lesions extend
into the adjoining neocortical association areas of the fusiform
(occipito-temporal) gyrus (m, n). The sections originate from an
85-year-old female. o–t stage IV: The density of the lesions
increases in both the entorhinal region (o, p) and fusiform gyrus
(q–r) with a gradual decrease of the pallid lines (lamina dissecans
in p and outer line of Baillarger in r). The neuroWbrillary pathol-
ogy now extends up to the medial temporal gyrus (s, t). Sections
were taken from an 80-year-old female. u–x stage V: The lesions
extend widely into the occipital lobe and appear in the peristriate
region. Note the presence of a deep plexus in AT8-immunoreac-
tions (v, x). Sections were obtained from a 66-year-old demented
female. y-z stage VI: Lesions are visible even in the parastriate
and striate areas of the occipital neocortex. Note the clear-cut line
in layer V of the striate area (z and z). The sections originate
from a demented 75-year-old male. Scale bar in a applies to all
overviews and that in c to all micrographs of cortical areas
Acta Neuropathol (2006) 112:389–404 395
396 Acta Neuropathol (2006) 112:389–404
The staging system
AD-related neuroWbrillary changes occur at predis-
posed cortical and subcortical sites. The distribution
pattern and developmental sequence of the lesions are
predictable and permit identiWcation of six stages,
which can be subsumed under three more general
units: I–II, III–IV, V–VI [46, 21, 28, 32, 37, 38, 47, 65,
66, 83]. Initial diagnosis as to whether the bulk of the
abnormal tau protein is detectable in the transentorhi-
nal and entorhinal regions (stages I–II), in the limbic
allocortex and adjoining neocortex (stages III–IV), or
in the neocortex, including the secondary and primary
Welds (stages V–VI), simpliWes the subsequent task of
Cases without cortical AD-related neuroWbrillary
pathology The transentorhinal region as well as the
entorhinal region and hippocampal formation remain
devoid of AT8-immunoreactive nerve cells (Fig. 4a, b).
Major characteristics of stages I–II
Stage I: Lesions develop in the transentorhinal region
(Figs. 2a–c, 3a, 4c, d) [23] Subcortical nuclei (i.e.,
locus coeruleus, magnocellular nuclei of the basal fore-
brain) occasionally show the earliest alterations in the
absence of cortical involvement [29]. The transentorhi-
nal region is the Wrst site in the cerebral cortex to
become involved. AT8-immunoreactive (ir) projection
cells contain hyperphosphorylated tau in both the cell
body and all of its neuronal processes (Fig. 4c, d). Late
phases of the stage show abundant AT8-ir neurons that
permit recognition of the descent of the superWcial
entorhinal cellular layer (pre-, i.e., the outer layer-
of the external principal lamina) from its uppermost
position at the entorhinal border to its deepest position
at the transition towards the adjoining temporal neo-
cortex (Figs. 1, 3a) [23]. The entorhinal region proper
remains uninvolved or minimally involved.
Stage II: Lesions extend into the entorhinal region
(Figs. 2e, f, 3b, 4e) From the transentorhinal region,
the lesions encroach upon the entorhinal region, par-
ticularly its superWcial cellular layer, pre-
(Figs. 1, 3b,
4e–g). The deep layer, pri-, gradually becomes visible
(Figs. 2f, 3b, 4e), shows sharply deWned upper and
lower boundaries, and is separated from pre- by the
broad, wedge-shaped lamina dissecans (myelinated
Wber plexus) [23, 98]. AT8-ir pyramidal cells appear in
sectors 1 and 2 of the hippocampal Ammon’s horn
(CA1/CA2) (Fig. 3b). Dilations develop transiently in
apical dendrites that pass through the stratum lacuno-
sum moleculare of CA1 [20]. Scattered NPs appear in
CA1. Fine networks of AT8-ir neurites form in both
the stratum radiatum and stratum oriens.
Major characteristics of stages III–IV
Stage III: Lesions extend into the neocortex of the
fusiform and lingual gyri (Figs. 2i–n, 3c, d, 4h) The
lesions in stage II sites become more severe. The outer
entorhinal cellular layers (pre-, pre-, pre-) and most
3 S
ary pat
polyethylene glycol-embedded hemisphere sections immuno-
stained for hyperphosphorylated tau (AT8, Innogenetics). a stage
: Involvement is slight and all but conWned to the transentorhinal
region (part of the temporal mesocortex), located on the medial
surface of the rhinal sulcus. The section originates from a non-de-
mented 80-year-old female. b stage II: Additional immunoreac-
tivity occurs in layer pre- or layer II of the entorhinal region. The
layer gradually sinks into a deeper position in the transentorhinal
region (arrow). The border between the entorhinal and transen-
torhinal regions is clearly recognizable in these early stages
(arrowhead). Furthermore, the lesions make headway into the
hippocampus (arrow). The section was obtained from a non-de-
mented 80-year-old male. c stage III: The lesions in the hippocam-
pal formation worsen. Entorhinal layers pre- and, additionally,
pri- of the deep layers become strongly involved. Lesions extend
through the transentorhinal region into the adjoining high order
sensory association areas of the temporal neocortex. The lesions
generally do not extend beyond the occipito-temporal gyrus lat-
erally (arrow) and lingual gyrus posteriorly. The section origi-
nates from a 90-year-old female. d stage III: A Xat section through
the entire basal surface of the temporal lobe reveals the severe
involvement of the entorhinal cortex (anterior portions of the
parahippocampal gyrus) at stage III and shows the tendency o
the pathology to extend from there into the adjacent neocortex,
i.e., occipito-temporal gyrus laterally (arrow) and lingual gyrus
posteriorly (arrow). e stage IV: The third and fourth sectors of the
Ammon’s horn and a large portion of the insular cortex (arrow)
become aVected. The involvement of the neocortical high order
sensory association cortex of the temporal lobe now extends up to
the medial temporal gyrus and stops short of the superior tempo-
ral gyrus (arrow). The primary Welds of the neocortex (see trans-
verse gyrus of Heschl) and, to a large extent, also the premotor
and Wrst order sensory association areas of the neocortex remain
intact. This section was taken from an 82-year-old demented fe-
male. f stage V: In addition to the presence of AD-related lesions
in all of the regions involved in stage IV, pathological changes ap-
pear in the superior temporal gyrus and even encroach to a mild
degree upon the premotor and Wrst order sensory association ar-
eas of the neocortex. g stage V: In the occipital lobe, the peristri-
ate region shows varying degrees of aVection, and lesions
occasionally can even be seen in the parastriate area. Stage V sec-
tions were obtained from a 90-year-old female with dementia.
h–i stage VI: Strong immunoreactivity can be detected even in the
Wrst order sensory association areas (e.g., the parastriate area)
and the primary areas of the neocortex (e.g., the striate area) o
the occipital neocortex. Compare the superior temporal gyrus
and transverse gyrus of Heschl at stage V with the same structures
at stage VI. Both stage VI sections originate from a severely de-
mented 70-year-old female Alzheimer patient. Scale bar applies
to all thick sections
Acta Neuropathol (2006) 112:389–404 397
of the molecular layer become Wlled with intermeshing
AT8-ir neurites, whereas the pale lamina dissecans
contains a few radially oriented neurites. The deep
layer pri- is heavily aVected and gradually thins in the
transentorhinal region as it approaches the temporal
neocortex (Figs. 3c, 4h). CA1 appears band-like, and
transient dendritic changes in CA1 reach their culmi-
nation point [20]. CA2 is Wlled with large and strongly
AT8-ir pyramidal cells. A moderate number of mossy
cells with characteristic dendritic excrescences appear
in CA3 and CA4 [97]. The granule cells of the fascia
dentata remain uninvolved. AT8-ir sections showing
the classical view of the hippocampal formation
(Figs. 1b, d) facilitate recognition of lesions in the fas-
cia dentata and Ammon’s horn [92].
From the transentorhinal region, the lesions encroach
upon the neocortex of the fusiform and lingual gyri,
and then diminish markedly beyond this point
(Figs. 3d, 4j). An AT8-ir plexus Wlls the cellular layers
of the temporal neocortex (Figs. 2n, 4l). The outer line
of Baillarger is barely developed and gradually
becomes recognizable only with increasing distance
from the transentorhinal region. A few NPs develop in
the outer layers II–IV.
Stage IV: The disease process progresses more widely
into neocortical association areas (Figs. 2o–t, 3e,
5a) Lesional density increases in sites aVected in
stage III. A few AT8-ir pyramidal cells appear in the
subiculum. The density of the neuritic plexuses of the
entorhinal and transentorhinal regions increases and
causes a corresponding blurring of the lamina dissec-
ans. The deep plexus spans all of the deep layers: pri-,
pri-, and pri-, and from there penetrates widely into
the white substance. This aspect of maximum involve-
ment undergoes little change until the end-phase of
AD. Thus, the pathological features of the entorhinal
and transentorhinal regions must not be taken into
account for further diVerentiation of stages V and VI.
CA1/CA2 are recognizable as dense bands. The vari-
cose dendritic segments vanish from CA1 without
leaving behind any remnants. Large numbers of mossy
cells in CA3 and CA4 become AT8-ir. A few AT8-ir
granule cells appear in the fascia dentata.
In stage IV, the pathology extends broadly into the
mature neocortex. A slight decrease in the immunore-
activity of neocortical NPs can be recognized starting
at the border facing the transentorhinal region. Dense
neuritic plexuses develop up to the middle temporal
convolution (Figs. 2s, t, 4a–c), and a rapid decrease in
the severity of the lesions occurs at the transition to the
superior temporal gyrus (Fig. 3e). The occipital neo-
cortex is unaVected (Fig. 5e, d) or contains blotch-like
local accumulations of AT8-ir pyramidal cells and/or
NPs in the peristriate region (Brodmann area 19).
Major characteristics of stages V–VI:
Stage V: The neocortical pathology extends fanlike in
frontal, superolateral, and occipital directions, and
reaches the peristriate region (Figs. 2u–x, 3f, g, 5
j) From sites involved at stage IV, the lesions appear
in hitherto uninvolved areas and extend widely into the
Wrst temporal convolution (Fig. 3f) as well as into high
order association areas of the frontal, parietal, and
occipital neocortex (peristriate region, Figs. 2v–x, 3g).
Initially, unevenly and loosely distributed NPs appear
in layers II and III, followed by large numbers of AT8-
ir pyramidal cells in layers IIIa, b and V. The lower
border of the outer neuritic plexus in layers II-IIIab
blurs at its transition to the uninvolved layers IIIc and
IV (outer line of Baillarger, Fig. 5g). In stage V, the
deep plexus of layer V is narrow and tends not to
extend into layer VI and the white matter (Fig. 5g, h).
The same pattern (only less pronounced) is seen in sec-
ondary areas of the neocortex, where uneven accumu-
lations of NPs predominate. AVection of layer V is
weak (Fig. 5j). The primary visual Weld (striate area)
contains only isolated signs of the pathology consisting
of NPs (Fig. 5i, j). Isolated AT8-ir neurons also can be
seen in layer IIIab (Fig. 5i, j).
Stage VI: The pathology reaches the secondary and pri-
mary neocortical areas and, in the occipital lobe, extends
into the striate area (Figs. 2y–z, 3h, i) Most areas of the
neocortex show severe aVection and nearly all layers
are Wlled with AT8-ir neurites. As such, the outer line
4 P
rogress o
ary pat
ogy, as seen
paraYn sections immunostained for hyperphosphorylated tau
(AT8, Innogenetics). a, b Control case displaying no AT8-ir
intraneuronal changes. Note that even the transentorhinal region
is devoid of immunoreactivity. c, d stage I: The Wrst AT8-ir pyra-
midal cells often are more easily detected in thick sections than in
paraYn material. Closer inspection of the predilection site (tran-
sentorhinal region in d, framed area in c), however, reveals the
initial lesions. The meshwork of ir-neurites is as yet not well
developed. e–g stage II: Many AT8-ir projection neurons are seen
in the transentorhinal region accompanied by a well-developed
plexus of ir-neurites (f, g). The pathology also extends into the en-
torhinal layers pre- and pri- (arrowheads in e). h–l stage III:
The transentorhinal and entorhinal regions are more severely
involved than in the preceeding stage, and the pathology now ex-
tends into the adjoining temporal neocortex of the occipito-
temporal and lingual gyri (h, j). The middle temporal gyrus re-
mains uninvolved (i). Scale bar in a, c, e, h is also valid for i and
Fig. 5 a, e, f, j, k, o below
398 Acta Neuropathol (2006) 112:389–404
Acta Neuropathol (2006) 112:389–404 399
400 Acta Neuropathol (2006) 112:389–404
of Baillarger—a pallid stripe in stage V—begins to blur
(Fig. 5l). Layer V still appears as a recognizable band
but continues into the neuritic plexus of layer VI. The
underlying white substance contains AT8-ir axons. A
decrease in immunoreactivity of NPs is seen in many
neocortical areas and is most pronounced in the basal
temporal Welds. In the occipital lobe, the pathology
breaches the parastriate and striate areas (Figs. 2y–z,
3i, 5m–o). Large numbers of NPs and AT8-ir nerve
cells appear in layers II and IIIab. Baillarger’s outer
line or the line of Gennari maintains a light appear-
ance, interrupted only by radially oriented AT8-ir neu-
rites. A sharply drawn AT8-ir plexus follows in layer V
(Figs. 2z, z, 5n, o).
By applying the silver technique proposed by Biels-
chowsky [1115] Alzheimer [2, 3] became the Wrst to
describe the NFTs that develop in the course of the dis-
ease that bears his name. This staining technique has
been in use for decades but has been subjected to
numerous modiWcations [10, 33, 36, 48, 52, 74, 99]. In
systematic studies, Gallyas [49, 50] replaced the critical
steps of the Bielschowsky technique by means of more
manageable reactions and developed a reliable method
for selectively demonstrating AD-related neuroWbril-
lary changes. Following its adaptation for use on
100 m thick sections, this method was standardized
for a procedure to stage the development of the corti-
cal AD-related neuroWbrillary pathology that gradually
found international acceptance [64, 66, 83]. Nonethe-
less, one of the unavoidable pitfalls associated with
using silverstaining techniques is that diVerent labora-
tories produce results of widely varying quality [1].
Subsequently, the 1991 staging protocol, too, under-
went a series of permutations, among them the applica-
tion of various types of silver impregnations, the
analysis of cortical sites that are fundamentally less
well suited for the procedure, and the use of tissue sec-
tions, the thickness of which diVered from that origi-
nally proposed [8, 9, 18, 21, 22, 36
, 3941, 53, 54, 5961,
6971, 75, 76, 78, 83]. A radical reduction of the diVer-
ent stages also has been suggested [62].
Since AD is an ongoing and not a static process,
every staging procedure is, de facto, an artiWcial con-
struct. It is the extent of brain involvement rather than
qualitative changes in the neuroWbrillary pathology
that increases with disease progression and, as such,
the concept of six neuropathological stages (and only
six stages) is not entirely amenable to pathological
states of a “transitional” nature that do not fulWll the
criteria for one of the six neuroWbrillary stages
described above.
Here, a revised version of the 1991 staging proce-
dure is presented that can be performed on paraYn
sections of conventional thickness, which have been
immunostained with the AT8 antibody and processed
on an automated basis, thereby fulWlling the demands
of the routine laboratory. The simplicity and unifor-
mity of any staging system is the prerequisite for eVec-
tive comparisons of results among laboratories and for
reliable as well as reproducible classiWcation of a dis-
ease process [35, 86].
Technical addendum
The previous staging protocol relied upon an advanced
but inexpensive silver technique that exploits the physi-
cal development of the nucleation sites and in so doing
permits careful control of the entire staining procedure
[4951]. Insoluble Wbrillary AD-related material can be
visualized virtually in the absence of distracting back-
ground staining [66, 67, 78]. The technique can be
applied to routinely Wxed autopsy material, even when
the material has been stored for decades in formalde-
hyde solutions. It facilitates processing of large numbers
and/or large sections (e.g., hemisphere sections). A
homogeneous staining that permeates the entire thick-
ness of a section is achieved even in 50–150 m sections
[22]. Thin paraYn sections (5–15 m) can also be used
5 P
rogress o
ary pat
ogy, as seen
paraYn sections immunostained for hyperphosphorylated tau
(AT8, Innogenetics). a–e stage IV: The disease process extends
into the high order sensory association neocortex of the temporal
lobe (temp.) and includes the medial temporal gyrus (a–c). The
peristriate region as well as the parastriate Weld and striate area o
the occipital lobe (occ.) still lack the neuroWbrillary pathology
(e, d). f–j stage V: Large numbers of neuritic plaques appear in the
neocortex (g, h). Pathological changes now encroach to a mild de-
gree upon premotor areas and Wrst order sensory association
Welds. In the occipital lobe (j), it is chieXy the peristriate region
(h) that shows varying degrees of aVection, and lesions occasion-
ally even develop in the parastriate area. The striate area remains
uninvolved (i, j). k–o stage VI: Drastic aVection of the neocortex
is seen at stage VI with involvement of almost all areas. Strong
immunoreactivity can be recognized even in premotor areas and
Wrst order sensory association areas (e.g., the parastriate area m),
as well as in primary neocortical areas (e.g., the striate area n).
The borderline between the striate and the parastriate areas is
drawn easily and—owing to the sudden cessation of the line o
Gennari (plexus of myelinated Wbers)—usually can be detected
with the unaided eye. A key feature of stage VI is the involvement
of the striate area (n), characterized by a dense neuritic mesh in
layer V with sharply drawn upper and lower boundaries. Note
that the myelin-rich line of Gennari (layer IVb n) is virtually de-
void of neuroWbrillary pathology. Scale bar in n also applies to g,
h, i, l, m
Acta Neuropathol (2006) 112:389–404 401
and counter-stained for easy identiWcation of cytoarchi-
tectonic units or speciWc nuclei [31]. NeuroWbrillary
changes of the Alzheimer type (NFTs, NTs, NPs)
appear in black and, thus, contrast well against an
almost unstained background. Connective tissue, glial
Wlaments, normal components of the neuronal cytoskel-
eton, Pick bodies, Lewy bodies/neurites, and corpora
amylacea remain unstained. Abnormal tau-protein in
argyrophilic grain disease (AGD), in progressive supra-
nuclear palsy (PSP), corticobasal degeneration (CBD),
and Niemann Pick type C (NPC) can be visualized as
well [46, 78].
The staging procedure originally required section-
ing at a thickness of 100 m. Silverstained or immuno-
stained sections of such thickness are optimal for the
demands of low power (stereo) microscopy and
greatly facilitate recognition of the laminar and areal
distribution pattern of the lesions (see hemisphere
sections in Fig. 3). Sections of this thickness can be
gained from non-embedded brain tissue with the aid
of a vibratome or a freezing microtome. Alterna-
tively, the tissue blocks can be embedded in PEG [91]
and sectioned with a sliding microtome. Application
of PEG (400 and 1,000: Merck-Schuchardt 807 485
and 807 488) is rapid, simple, and causes little shrink-
age [22].
The blocks are transferred from 96% ethanol to
PEG 400 and their surfaces covered with blotting
paper. Blocks are placed on a rotating table and after
having sunk to the bottom (this can take several days),
they are transferred to fresh PEG 400 for an addi-
tional day. Then, transfer to PEG 1000 at 54°C for
1 day. Embed in fresh PEG 1000, mount, and section
at 50–150 m. Transfer sections to 70% ethanol to
remove the embedding medium. Store sections in
formaldehyde solutions. Prior to staining, transfer sec-
tions to de-ionized water. It is important to note that
the Gallyas silver technique displays only highly
aggregated Wbrillary material, whereas the AT8-
immunoreaction also visualizes the non-argyrophilic
material that initially develops within involved neu-
rons (pretangle material).
Acknowledgments The skillful assistance (tissue processing and
staining) of Dr. R.A. Kauppinen (Kuopio), Mr. M. Bouzrou
(Frankfurt/Main), and (illustrations) Ms. I. Szász (Frankfurt/
Main) is gratefully acknowledged.
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  • ... Additional sections from selected blocks were stained with modified Bielschowsky silver stain, and others, with antibodies directed against α-synuclein (1:40, Leica, Buffalo Grove, IL, USA) (including cerebral cortex, cingulate gyrus, hippocampal formation, globus pallidum, putamen, amygdala, subthalamic nucleus, midbrain with substantia nigra, pons with the locus ceruleus, and medulla with the dorsal vagal nucleus), β-amyloid (1:400, Biocare Medical, Concord, CA, USA), and hyperphosphorylated tau (AT8) (1:200, Thermoscientific, Rockford, IL, USA), performed in an automated immunostainer (Ventana, Benchmark Ultra, Tuscon, AZ). Braak and Braak AD staging for neurofibrillary tangles [35,36] and the Consortium to Establish a Registry for AD (CERAD) ratings for neuritic plaques [37] were assigned. A standard 3 × 20 × 25 mm parasagittal, formalin-fixed, tissue block was harvested from the neocerebellum; this block included cortex, white matter and dentate nucleus [25,26]. ...
  • ... In recent years, much attention has focused on the observation that histological deposits of tau appear to develop in a stereotypical spatial and temporal pattern linked to the appearance of disease symptoms [3]. This phenomenon has prompted many to suggest that tau is physically transferred from neuron to neuron along anatomical pathways [4][5][6]. This so-called 'pathogenic spread model' has at least four components: (1) release from donor neurons, (2) aggregation (which could occur before or after step 1), (3) uptake into certain recipient neurons, and (4) induction of aggregation in the recipient cells [7]. ...
  • ... Pathological diagnosis and neuropathological evaluation was performed on formalin-fixed, paraffin-embedded tissue from different brain areas as previously described [23]. Staging of AD pathology was evaluated according to modified assessment of Braak and Alafuzoff [28]. Clinical and pathological diagnosis, sex, age, postmortem delay, brain area, and the Braak scores for NFTs and amyloid load of all analyzed cases (n = 16) are listed in Supplementary Table 1. ...
To assess the feasibility of surgical techniques and to analyze clinical and radiological outcome in different fields of MISS. The main focus is on degenerative spine disease, infection, metastati…" [more]
    Tau lesions (pretangles, neuropil threads, neurofibrillary tangles) that develop in a few types of nerve cells in the brain are essential to the pathogenesis of Alzheimer's disease (AD). The formation of non-argyrophilic pretangles marks the beginning of the pathological process and is of increasing interest because it is temporally closer to the prevailing conditions that induce the... [Show full abstract]
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      Brains of 42 individuals between the ages of 4 and 29 were examined with antibodies (AT8, 4G8) and silver stains for the presence of intraneuronal and extracellular protein aggregates associated with Alzheimer's disease. Thirty-eight of 42 (38/42) cases displayed abnormally phosphorylated tau protein (pretangle material) in nerve cells or in portions of their cellular processes, and 41/42... [Show full abstract]
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