ARTICLE |
Correspondence to: Steven E. Arnold, Center for Neurobiology and Behavior, U. of Pennsylvania School of Medicine, 142 Clinical Research Bldg., 415 Curie Boulevard, Philadelphia, PA 19104. E-mail: alveus@mail.med.upenn.edu
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Summary |
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Pathological alterations in dendrites and axons (i.e., neuritic pathologies) occur in the normal aging brain as well as in brains from elders with mild cognitive impairment and neurodegenerative dementia. These alterations may correlate with clinical measures of cognitive abilities, but the contribution of neuropil threads (NTs), which constitute 8590% of cortical tau pathology, has not been clear because of the lack of quantitative methodologies. We combined quantitative fractionation and image analysis to devise a strategy for measuring the burden of tau-rich NTs in the entorhinal and perirhinal cortex of brains from elders with and without cognitive impairment, including dementia due to Alzheimer's disease (AD). On the basis of data presented here using this novel strategy, we conclude that this quantitative imaging technique will facilitate efforts to determine the behavioral correlations of neuritic lesions in AD and other brain disorders.
(J Histochem Cytochem 48:16271637, 2000)
Key Words: Alzheimer's disease, neurodegenerative disease, stereology, entorhinal cortex, neuropil threads, neurofibrillary tangles, tau, image analysis
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Introduction |
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Symptoms of neurodegenerative dementias begin with an insidious preclinical phase before the emergence of diagnostic clinical features. This phase may be paralleled by the slow accumulation of the characteristic neuropathological lesions of these diseases, which may precede the clinical expression of dementia by years (
Although earlier proposals for standard neuropathological criteria for the diagnosis of definite AD relied primarily on Aß senile plaques (SPs) (Khachaturian, CERAD), subsequent studies have shown that Aß SPs may be very abundant in the brains of aged individuals who do not exhibit cognitive impairment (
In addition to NFTs, tau-rich neurofibrillary alterations of AD include neuropil threads (NTs) and SP-associated dystrophic neurites (
In addition to the AD brain (
Quantitation of the NT burden is critical in clinicopathological correlative studies of the relative contribution of NTs to measures of the clinical manifestations of neurodegenerative dementias. For this reason, we developed a protocol for the quantitation of NTs in the postmortem brains of elders participating in a large-scale, prospective longitudinal study of cognition in normal and pathological aging. Here we describe a fractionation methodology for sampling brain tissue as well as an image analysis technique that sensitively and reliably quantifies NTs. On the basis of data presented here, we conclude that our protocol enables accurate assessment of the NTs burden in the normal aging brain as well as in the brains of elders with mild cognitive impairment or AD. Moreover, this approach should enable the quantification of normal and pathological neurites labeled by various means in the developing human brain, other brain disorders, and in animal models of these disorders.
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Materials and Methods |
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Subjects
This study included elders with no cognitive impairment (NCI; n = 14), and cognitive impairment (CI), i.e., elders with mild cognitive impairment (n = 9) or dementia due to AD (n = 8; see Table 1). All individuals were participants in the Religious Orders Study, a large, prospective, longitudinal clinicopathological investigation of aging and dementia in Catholic nuns, priests, and brothers. Details of the clinical evaluation procedures have been previously reported (
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Clinical Evaluation
The clinical evaluation was designed to establish the presence or absence of impaired cognition, including dementia, and to determine the etiology of these impairments, with particular attention paid to common conditions, including AD, stroke, Parkinson's disease (PD), and depression. Briefly, a team of investigators led by a neurologist performed annual uniform, structured evaluations of each participant. The medical history included questions about cognitive decline, stroke, PD, head injury, tumor, depression, and other medical problems, as well as medications used by the participant within the previous 14 days of the examination. Neurological examination was performed by trained nurse clinicians and included an assessment of signs of stroke and parkinsonism. Trained neuropsychology technicians administered a battery of cognitive tests chosen to measure a range of cognitive abilities, with emphasis on those affected by aging and AD. These tests included the Mini-Mental State Examination (MMSE) and other tests recommended by CERAD (
Tissue Preparation and Pathological Evaluation
At the time of autopsy, brains were removed and processed as described previously (
Immunohistochemistry
Free-floating cryoprotected sections were washed in 0.1 M phosphate buffer (PB). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in 0.1 M PB for 30 min and then washed in a TrisNaClTriton (TNT; 0.2 M NaCl, 0.5% Triton, 0.1 M Tris) solution. Next, sections were preincubated for 1 hr in 10% horse serum in TNT. Thereafter, tissues were processed by the avidinbiotin method using the Vectastain ABC elite kit (Vector Laboratories; Burlingame, CA). Sections were incubated for 72 hr at 4C with PHF-6 (source: V.M.-Y. Lee), a monoclonal antibody directed against a defined epitope containing phosphorylated T231 residue in PHF-tau (
Immunodetection and Quantitation
Individual coronal sections were chosen from series that contained perirhinal (transentorhinal) and entorhinal cortex at the intermediate level of subfield EI and these regions were delineated at low magnification according to cytoarchitectural criteria (
To analyze the individual images for NT burden, 24-bit color images obtained from the random systematic sampling (4060 images per perirhinal cortex or entorhinal cortex) were converted to 8-bit gray scale images by using conversion algorithms associated with Adobe Photoshop 5.02 (Adobe Systems; Tucson, AZ). Area analysis was performed using the public domain Object-Image 1.62p15 (developed by Norbert Vischer, http://simon.bio.uva.nl/objectimage. html). Overall data reduction yielded a value for the area occupied by the immunopositive zone or the area fraction (burden) defined as the percent fraction of labeled to unstained tissue area.
The analysis algorithm segmented each image along the intensity domain into two fractions, the labeled and the background compartment. To do so reliably, the system arbitrated between two automatic thresholding algorithms, ISODATA (Iterative Self-Organizing Data Analysis) and triangulation (
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The thresholding step separates from the background PHF-tau immunoreactive structures that are a combination of both NFTs and NTs. A size filter then separates out NFTs from this segmented compartment. The resulting binary image can then be edited manually to reduce false-positives and -negatives.
The sensitivity of this measure to the camera settings employed during image acquisition system was handled by strictly adhering to a fixed protocol. All microvideographs were captured using a Cohu 1300 series color CCD camera (Cohu/Electronic Division; San Diego, CA) with the gamma set to 1.0 and the autogain off. The analysis results are a function of illumination intensity as described in the results (lamp setting on the microscope is not a reliable indicator of illumination level). To assure consistent light level from one imaging session to the next, a calibration protocol was employed. Before each imaging session, the Leica DMRBE microscope was calibrated under Köhler illumination using an optical density standard (Kodak Step Tablet #152 3422), 0.19 OD, which was cut and attached to a microscope slide. The standard was imaged using ImageJ (National Institutes of Mental Health, available on the Internet at http://rsb.info.nih.gov/ij/). ImageJ calculates and displays a histogram of the distribution of gray values along the x-axis. The light illumination is adjusted until the mean value of the image histogram equals a predefined value, which in these studies was 85 (scale 0255).
Statistics
Statistical analysis included ANOVA, post-hoc testing, and rank correlations (Spearman's rank correlation). All testing was performed with StatView version 5.01 (SAS Institute, http://www.statview.com).
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Results |
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Image Analysis
We assessed the performance of our image processing approach and its sensitivity to illumination intensity. There was a dramatic effect of light level on analysis results (Fig 2). As light level increased, the measured NT burden decreased. Although the absolute burden is not relevant, it is critical that a consistent protocol is followed when all data of a given study are imaged. To standardize analysis, we selected a level that yielded a gray value of 85 (range 0255) when the 0.19 optical density (OD) calibration standard was imaged.
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By employing a standardized image acquisition protocol, images obtained on different days can be compared. We repeatedly captured three random images from the entorhinal cortex of cases with either low, moderate, or marked NT burden over a period of 6 months. To ensure that the specific location of each image for temporal comparison was identifiable, the exact coordinates of each of the images was determined using the 3-axes motorized stage. Images were then analyzed for NT burden as described above. No substantial difference was detected (Fig 3).
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We examined false-positive/false-negative rates on images using the standard protocol. The images were obtained from cases with a low level of NT burden (n = 1), a moderate NT burden (n = 2), and marked NT burden (n = 1; Table 2). Examples of images from those cases and the segmentation results are shown in Fig 4. To examine the performance rate, the binary images generated by the program were further manually edited. The NT quantification accuracy varied depending on the thread burden, with 3.4% false-negatives when the thread burden was mild, 9.8% when the thread burden was moderate, and 9.7% when the thread burden was marked (Table 2). More importantly, there was a 0.0% false-positive measurement regardless of the thread burden.
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We also determined how the percent contribution of NTs compares to the overall PHF-tau immunoreactivity which includes NFTs. In images of perirhinal and entorhinal cortex from three participants (low, moderate, and marked PHF-tau immunoreactivity), NT burden contributed 91.4 ± 2.5% (n = 6; range 87.393.9%) of the PHF-tau immunoreactivity, and NFTs contributed only 8.6 ± 2.5% (n = 6; range 6.112.7%). Even in a case with a high NFT density (44.6 NFTs/mm2 in the perirhinal cortex and 21.7 NFTs/mm2 in the entorhinal cortex), the NFT contribution of PHF-tau immunoreactivity was <13%.
Sampling Paradigm for NTs and NFTs
For NTs and NFTs, we identified a participant whose brain PHF-tau burden was considered to be moderate with a heterogeneous distribution. Throughout the contour sampled (32 x 106 µm2), we positioned an unbiased counting frame of known area, 40,000 µm2, along a rectangular grid (Table 3) superimposed on the perirhinal and entorhinal cortex by the StereoInvestigator software. The coordinated movement along the x-axis and y-axis was achieved by StereoInvestigator software integration, with stepping motors attached to the microscope stage. For the NT burden measurements, we sampled the entorhinal cortex of a moderate NT burden case with a heterogeneous distribution and a mild NT burden case. Three sets of images were obtained from each case. The moderate thread burden case was examined by using a systematic random sample of 20 images, 40 images, and 60 images (Table 3). A total of 4060 images or 25 35% of the area must be sampled to obtain actual values of thread burden. A mild thread burden case (Table 3) was examined by using a systematic random sample of 20 images, 30 images, 40 images, and 50 images. It is apparent that, regardless of thread burden, attention must be directed towards adequate sampling because the NT burden is negatively correlated with sampling when there is a heterogeneous distribution.
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For NFTs, we determined that a sampling scheme which included at least 50 images (67% of total area) was ample to estimate accurately the NFT density and to simultaneously be efficient. By doubling the number of images taken in a systematic random sample (n = 100; Table 3), the change in NFTs was not significantly different (Student's t-test; t = 0.615, p>0.05). In contrast, by reducing the number of images taken (n = 25; Table 3), our NFT density decreased by almost 30%, which was significantly different (Student's t-test; t = 3.145, p<0.01).
Pathology
Perirhinal cortex (Fig 5A) and entorhinal cortex (Fig 5B) NT burdens (percent area) were highly correlated (Fig 6; r = 0.9 (r2 = 0.83), p<0.0001) and increased significantly in persons with CI compared to NCI participants, as expected (t = -2.82, p<0.0065).
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Discussion |
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We developed systematic automated measurement methods that enabled us to determine the NT burden expressed as area fraction occupied in the perirhinal and entorhinal cortices. Neuropil threads, also called curly fibers or dystrophic neurites, are abnormal neuronal processes that contain PHFs formed by PHF-tau (
The increasing clinical and biological interest in dendrite and axonal damage in neurodegenerative disease, e.g., NTs in AD (-synuclein have revealed extensive networks of dystrophic processes, termed Lewy neurites. These Lewy neurites are seen in the brains of patients with sporadic DLB, PD, and the LB variant of AD (
Accurate estimation of neuropil filament burden requires stereological analyses as much as does evaluation of neuron or synapse number (
Both
The quantification of PHF-tau-positive structures yielded a composite score, defined as a combination of the black pixels that identified both NFTs and NTs. To separate the area contribution of NFTs and NTs, an NIH Image macro was developed to efficiently and accurately delineate and exclude NFTs and produce values that are exclusively defined as NTs. This macro selects (on the basis of contrast) the objects (NFTs) in a x200 magnification (x20 objective) PHF-tau field, filters out objects too small (individual NTs) or too large (masses of NTs) to be NFTs, and then measures the area of each object in the image. The variable "size cut" determines which objects get counted. Observer editing is then performed on each image to shield against false-positives and -negatives. Although additional data analysis is in progress, our results indicate that NT burden contributes heavily, with 91.4 ± 2.5% of the PHF-tau immunoreactivity, and that NFTs contributed only 8.6 ± 2.5%. The necessity for increased accuracy of NTs is being presently evaluated in clinicopathologic studies.
New guidelines for postmortem diagnosis of AD were established in 1997 (
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Acknowledgments |
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Supported by the following NIH/NIA grants: AG15819, AG10124, AG14449, and AG10161.
We are indebted to the altruism and support of the hundreds of Nuns, Priests and Brothers from the following groups participating in the Religious Orders Study: Archdiocesan priests of Chicago, Dubuque, and Milwaukee; Benedictine Monks, Lisle, IL and Collegeville, MN; Benedictine Sisters of Erie, Erie, PA; Capuchins, Appleton, WI; Christian Brothers, Chicago, IL and Memphis, TN; Diocesan priests of Gary, IN; Dominicans, River Forest, IL; Felician Sisters, Chicago, IL; Franciscan Handmaids of Mary, New York, NY; Franciscans, Chicago, IL; Holy Spirit Missionary Sisters, Techny, IL; Maryknolls, Los Altos, CA and Maryknoll, NY; Norbertines, DePere, WI; Oblate Sisters of Providence, Baltimore, MD; Passionists, Chicago, IL; Servites, Chicago, IL; Sinsinawa Dominican Sisters, Chicago, IL and Sinsinawa, WI; Sisters of Charity, BVM, Chicago, IL and Dubuque, IA; Sisters of the Holy Family, New Orleans, LA; Sisters of the Holy Family of Nazareth, Des Plaines, IL; Sisters of Mercy of the Americas, Chicago, IL, Aurora, IL, and Erie, PA; Sisters of St Benedict, St Cloud and St Joseph, MN; Sisters of St Casimir, Chicago, IL; Sisters of St Francis of Mary Immaculate, Joliet, IL; Sisters of St Joseph of LaGrange, LaGrange Park, IL; Society of Divine Words, Techny, IL; Trappists, Gethsemane, KY and Peosta, IA. We are also indebted to the dedication and hard work of Julie Bach, MSW, Study Coordinator.
Received for publication February 28, 2000; accepted June 9, 2000.
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