Frequency analysis of catecholamine axonal morphology in human brain

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Journal of the Neurtdogtcal ~'tc~t~ i i~ ( 19t)3~ { I~1 i i:~ 1993 Elsevier Science Publishers B.V All rights r'eselved till?2 51t)X 93 $~f~.ttl)

JNS 04108

Frequency analysis of catecholamine axonal morphology in human brain II. A l z h e i m e r ' s d i s e a s e and h i p p o c a m p a l s y m p a t h e t i c ingrowth Rosemarie M. Booze a Charles F. Mactutus a, Catherine R. Gutman h and James N. Davis b Department of Pharmacology and College of Pharmacy, University of Kentucky Medical Center, Lexington, KY40536-0084, USA, and b Departments of Medicine (Neurology) and Pharmacology, Alzheimer's Disease Research Center, Duke University Medical Center, Durham, NC 27705, USA (Received 15 October, 1992) (Revised, received 13 April, 1993) (Accepted 18 April, 1993)

Key words: Tyrosine hydroxylase; Frontal cortex; Hippocampus; Calcarine cortex; Immunostaining; Noradrenergic

Summary We have examined the various diverse morphologies of catecholamine axons in the brains of patients with Alzheimer's disease. Alzheimer's disease and aged control brain tissue were obtained by a rapid autopsy protocol (mean postmortem delay < 1 h). Tissue blocks from the superior frontal cortex (Brodmann area 9), the hippocampat gyrus, and the calcarine cortex (Brodmann area 17) were processed for identification of catecholamine axons using tyrosine hydroxylase immunocytochemistry. A total of 1275 tyrosine hydroxylase immunoreactive axons were randomly sampled from coded sections and classified into one of six distinct axon-type categories. The axon classification from patients with Alzheimer's disease significantly differed from those of an age-matched control population in the hippocampus. The Alzheimer's disease brains were decreased in the frequency of very long, thin, tyrosine hydroxylase immunoreactive axons (type 1) and had an increased frequency of shorter, tortuous, axons (type 3). These selective quantitative shifts in hippocampal catecholaminergic axon morphology are consistent with the hypothesis that sympathetic noradrenergic axons invade the hippocampus of patients with Alzheimer's disease. Multivariate modeling of the frequency sampling data found that the axon type classification scheme successfully predicted the presence of Alzheimer's disease. In particular, the use of quantitative neuroanatomical measures of the catecholaminergic system in human brain tissue was found to have errorless predictive ability with respect to late onset ( > 75 years) Alzheimer's disease. In summary, the use of quantitative neuroanatomical measures of catecholamine axonal morphologies in Alzheimer's disease brain tissue identified a specific frequency shift which may represent hippocampal sympathetic ingrowth and this unique measure was found to have predictive utility with respect to Alzheimer's disease.

Introduction P o s t m o r t e m examinations of brains from patients with Alzheimer's disease have often found alterations in catecholaminergic neurochemical systems (Ichimiya et al. 1986; Perry et al. 1981; Powers et al. 1988; Cervera et al. 1990). These alterations include loss of catecholamine cell bodies as well as pathological abnormalities in catecholaminergic axons (Bondareff et al. 1982; Marcyniuk et al. 1986; Iverson et al. 1983; Tomlinson et al. 1981; Mann et al. 1980; Chan-Palay and Asan 1989). In particular, pathological abnormalities in catecholamine axons have been reported in the

Correspondence to: Rosemarie M. Booze, Ph.D., Department of Pharmacology, University of Kentucky College of Medicine, MS-305 UKMC, Lexington, KY 40536-0084, USA. Tel.: (606) 233-6507; Fax: (606) 258-1981.

forebrain (Berger et al. 1980) and in the hippocampus (Powers et al. 1988; Torack and Morris 1992) of patients with Alzheimer's disease. Thus, the central catecholaminergic systems appear to be one of several known neurochemical systems which exhibit various pathological alterations in Alzheimer's disease brain tissue. An unexplored source of abnormal catecholamine axons in Alzheimer's disease hippocampus may arise consequent to the well-documented loss of cholinergic neurochemical systems in Alzheimer's disease (Whitehouse et al. 1982; Davies and Maloney 1976). The loss of analogous cholinergic neurons in rodents results in peripheral sympathetic noradrenergic axons sprouting into the hippocampal parenchymal tissue (Crutcher et al. 1981; Loy and Moore 1977; Booze et ai. 1986). Sympathetic ingrowth has been observed in the rat hippocampus after destruction of the medial septal

111 nucleus, nucleus of the diagonal band, or transection of the fimbria/fornix. Substantial evidence has accumulated that sympathetic ingrowth is a specific response to loss of cholinergic innervation originating from the basal forebrain (Crutcher 1987). Given that Alzheimer's disease is associated with loss of basal forebrain cholinergic neurons (Whitehouse et al. 1982; Davies and Maloney 1976; Saper 1988), it is possible that sympathetic ingrowth might occur in the hippocampal formation of patients with Alzheimer's disease. In rats, the presence of hippocampal sympathetic ingrowth is detrimental to the performance of a cognitive spatial memory task (Harrell and Parsons 1988; Harrell et al. 1990; Ayyagari et al. 1991). The presence of abnormal catecholaminergic sympathetic axons in the hippocampus might therefore be anticipated to magnify and exacerbate the cognitive deficit in Alzheimer's disease. In the present experiment we began to explore if a portion of the observed alterations in catecholaminergic axons in Alzheimer's disease brain tissue might represent sympathetic ingrowth. In rodents, hippocampal sympathetic ingrowth axons have a characteristic thick, knobby, appearance that easily distinguishes them from the thin, varicose, axons of locus ceruleus neurons (Booze et al. 1986; Booze and Davis 1987; Crutcher et al. 1981). However, in the human brain, the morphologic appearance of catecholamine axons is unquestionably more diverse than that of the rat (Gaspar et al. 1985; Powers et al. 1988; Booze et al. 1993). We therefore first determined the frequency of the various types of catecholamine axons in brain tissue obtained via rapid autopsy from Alzheimer's and aged control patients. Second, multivariate modeling of the data was performed to test whether the frequency of catecholamine axonal pathology was predictive of the presence of Alzheimer's disease.

Methods

Brain material Human brain tissue was obtained using the rapid autopsy protocol implemented at the Alzheimer's disease research center at Duke University. Alzheimer's brain tissue was obtained from 13 subjects (5 males, 8 females) with a mean age of 80 years (range 57-93). The mean postmortem interval from time of death to placement of the tissue blocks in cold fixative for the rapid autopsy brains was 56 min (range 22-90 min). Independent neuropathological examinations of brain tissue from these subjects, conducted according to established criteria (Khachaturian 1985; Terry et al. 1981), confirmed the established clinical diagnosis of Alzheimer's disease. Tissue from the rapid autopsy control group was drawn from 4 subjects (3 males, 1 female) with a mean age of 68 years (range 47-80).

The postmortem interval from death to fixation was 58 min (range 35-75 min). The pathological diagnoses of these patients included breast cancer, stroke (not involving areas of interest to the current study), and subcortical gliosis. The left hemisphere of each subject was cut into 1-2 cm thick coronal slabs. The appropriate tissue blocks were cut from the slabs and immediately placed in cold 4% buffered paraformaldehyde for a period of 24 h at 4°C. The immersion-fixed tissue blocks were then transferred to phosphate-buffered saline (PBS; 0.1 M NaPO 4 in 0.9% NaCI, pH 7.4), until further processed. Tissue blocks were obtained from the superior frontal cortex (plate 37 in Hausman 1969), at the level of the anterior tip of the caudate nucleus, the depths of the calcarine fissure of the striate visual cortex (plate 45 in Hausman 1969) at the tip of the posterior horn, and the hippocampal gyrus through the middle part of the thalamus at the level of the substantia nigra (plate 45 in Hausman 1969). In addition to these landmarks, cytoarchitectonic criteria of Braak (1984) were applied to confirm the localization of cortical areas. The pineal body from several of the agematched autopsies was also collected to confirm antigenic recognition of sympathetic axons in human tissue.

Immunocytochemical methods The brain tissue was carefully blocked according to standardized landmarks and sectioned using a vibrating microtome (50 p~m thickness; Vibratome, Lancer Instruments). The free-floating sections were washed in a Tris-saline buffer (TBS; 0.1 M Tris-HCl in 0.9% NaCI, pH 7.6). Endogenous peroxidases were neutralized prior to staining by incubation in 1% H 2 O J m e t h a n o l for 30 rain. The tissue sections were placed in 2% normal goat (blocking) serum for a period of 40 rain, and were then directly transferred to a 1 : 1000 dilution of polyclonal anti-tyrosine hydroxylase antisera (Eugene Tech Intl., Allendale, N J). Western blots testing the specificity of this antibody against rat adrenal glands demonstrated a single immunoreactive band for T H at 60 kDa. Free-floating tissue sections were incubated with the primary tyrosine hydroxylase antibody for 16 h at 4°C. Following primary incubation, the tissue sections were washed in PBS and subsequently incubated for 40 min in goat anti-rabbit biotinylated secondary immunoglobulin. A modification of the avidin-biotin complex (ABC kit, Vector Labs, Burlingame, CA) technique was used to localize T H - I R (Hsu et al. 1981; Booze et al. 1988). Following a brief (approx. 5 rain) reaction with 0.05% diaminobenzidine in 0.015% H202, sections were immediately processed for co-localization of amyloid plaques and neurofibrillary tangles using thioflavin-S staining (Schwartz 1972). The free-floating sections were washed in PBS and trans-

I12 ferred into a 1% thioflavin-S solution for 5 min. The tissue sections were differentiated in 70% ethanol and washed in distilled water for 5 rain. The co-stained sections were then mounted on glass microscope slides, air-dried, dehydrated through a series of alcohol/ xylene solutions, and coverslipped with permanent non-fluorescent mounting media (Entellan, EM Science, Germany). Additional control sections were prepared as above, except that they were incubated with normal rabbit serum instead of the TH-antiserum. No immunoreactive axons were observed in these control tissue sections.

Histological methods Tissue sections adjacent to those processed for immunocytochemistry were collected and stained for Nissl substance as well as hematoxylin and eosin. These sections aided in identification of cytoarchitectural landmarks and in neuropathological evaluations of the individual cases. All tyrosine hydroxylase immunoreacted sections were co-stained with thioflavin-S. The TH-IR/thioflavin-S co-stained sections were examined with brightfield optics as well as fluorescence micr,oscopy in order to determine the coexistence of amyloid plaques and neurofibriUary tangles with tyrosine hydroxylase immunoreactive axons. Thioflavin-S staining did not alter tyrosine hydroxylase immunoreactivity or interfere with brightfield microscopy of the sections.

Frequency sampling methods Tissue sections processed for tyrosine hydroxylase immunoreactivity and amyloid staining (thioflavin-S) were randomized and assigned a code number. Two observers naive with regard to information concerning subject, region, and classification examined the coded slides. A reticule grid (100 x 100 squares) was superimposed over the entire tissue section and observed with a low power (10 × ) objective. Squares to be sampled were randomly selected across the vertical and horizontal axes of the entire tissue section. Each square selected was examined for tyrosine hydroxylase axons with a high power (40 × ) objective. Twenty-five axons were sampled from each of 51 tissue sections obtained from 23 subjects. A total of 1275 axons were sampled: 650 from rapid autopsy Alzheimer's brains, 275 from rapid autopsy age-matched control brains, and 350 from delayed autopsy age-matched control brains. Each of the 25 axons/tissue section were classified into one of 6 axon type categories by two independent observers. In this manner, each tissue section was randomly sampled for T H - I R axons, without regard for subregional anatomical distribution. Several commonly employed neuropathological markers were also evaluated to confirm the presence and extent of Alzheimer's disease on the immunoreacted tissue sections. Both amyloid plaques and neu-

rofibrillary tangles were quantified on the coded scc tions using fluorescence microscopy of lhc thioflavin-S stain. The extent of amyloid plaque formation was estimated by counting the number of fluorescent amyloid cores in 5 adjacent, but not overlapping, low-power (10 × objective)microscope fields. The counting fields sequentially spanned all lamina of both the frontal and calcarine cortical regions (Rogers and Morrison 1985). The hippocampal counts were obtained from the molecular layer of fascia dentata (Crain and Burger 1988). In these same areas, the presence of neurofibrillary tangles was recorded as 11= absent or rare tangles, + = moderate number of tangles, and + + = extensive tangles. Whole brain weight and gross pathological information were collected and recorded at autopsy. Information regarding the clinical course of the patients with Alzheimer's disease was obtained from inspection of the patient histories. The first written record of memory impairments, as reported either by the family members or physician, was defined as the onset date for Alzheimer's disease.

Data analysis The axon count data were analyzed using non-parametric chi-square tests, which are appropriate for categorical data (Siegel 1956). Both the Pearson chi-square and the likelihood-ratio chi-square were calculated; the most conservative statistic is reported. Multiple linear regression analyses were used to perform correlations with clinical data (Winer 1972).

Predictive statistical modeling Discriminant function analyses (BMDP Statistical Software 1987) were employed in predictive modeling studies to determine how well the observed shifts in tyrosine hydroxylase axon-type morphology (1) correctly identified individuals with regard to their group membership as well as (2) classify additional individuals using cross-validation. For example, if the changes in hippocampai tyrosine hydroxylase axon type morphology are a strong characteristic of Alzheimer's disease, then this data should be able to successfully predict patient classification. Furthermore, if the shift of axon types which discriminates Alzheimer's patients from rapid autopsy controls is not influenced by any changes in axon type morphology which occur as a function of autopsy delay (Booze et al. 1993), then all delayed autopsy patients should be correctly identified as controls.

Results

Alzheimer's disease markers Tissue from clinically diagnosed and neuropathologically confirmed Alzheimer's disease patients was ex-

113 TABLE 1 MORPHOLOGICAL CHARACTERISTICS OF BRAINS FROM PATIENTS WITH ALZHEIMER'S DISEASE AND AGED CONTROL BRAINS Measure

Alzheimer's Aged control disease Rapid Delayed autopsy autopsy

Brain weight (g) :' Amyloid plaques ~' Superior frontal cortex Dentate gyrus molecular laycr Calcarine cortex Neurofibrilla~ tangles ~ Superior frontal cortex Dentate gyrus Calcarine cortex

1028+42.4

121(/+20.8

1249+24.8

10.3± 1.6

<1

<1

6.6 + 1.3 5.7 + 1.2

<1 0.0

1.3±1.3 <1

+ ++ +

0 0 0

" Values represent mean .± SEM. h Values represent mean ± SEM of the number of amyloid plaques in five adjacent low-power fields (10×). ~ The presence of neurofibrillary tangles was scored: 0 = no or rare tangles. + moderate number of tangles and + + = extensive tangle formation.

a m i n e d . A n a t o m i c a l m e a s u r e s i n c l u d e d b r a i n weight, c o u n t s of a m y l o i d p l a q u e s , a n d a r a t i n g o f the relative n u m b e r of n e u r o f i b r i l l a r y t a n g l e s ( T a b l e 1). A l z h e i m e r ' s d i s e a s e significantly d e c r e a s e d b r a i n weight ( F ( 2 , 2 0 ) = 8.0, P < 0.003). L a r g e n u m b e r s of p l a q u e s of v a r i o u s sizes w e r e f o u n d in all b r a i n regions o f the A l z h e i m e r ' s d i s e a s e subjects ( F ( 2 , 2 0 ) = 21.7, P < 0.001). N e u r o f i b rillary t a n g l e s w e r e o b s e r v e d only in the A l z h e i m e r ' s group. T h e a g e - m a t c h e d c o n t r o l p a t i e n t s w e r e also e x a m i n e d for e v i d e n c e of A l z h e i m e r ' s disease. In n o n e o f the a g e - m a t c h e d c o n t r o l subjects was t h e r e e v i d e n c e of A l z h e i m e r ' s - t y p e p a t h o l o g i c a l a l t e r a t i o n s .

short a n d twisted, with multifocal swellings. T y p e 5 was c h a r a c t e r i z e d by t h i c k - c a l i b e r straight axons which l a c k e d swellings. T y p e 6 were thick c a l i b e r axons with multifocal, bulbous, axonal distentions. A l t h o u g h axons in c a t e g o r i e s 4, 5, a n d 6 w e r e much less f r e q u e n t , they w e r e clearly d i s t i n g u i s h a b l e as u n i q u e classes of axon m o r p h o l o g i e s . N o t m o r e t h a n 1% of all tyrosine hydroxylase axons e n c o u n t e r e d were unclassifiable and 1 - 2 % o c c u r r e d in clusters consisting of several axon types. T h e s e rare axon m o r p h o l o g i e s and axonal clusters were not i n c o r p o r a t e d into the axon count d a t a b a s e . T r a i n e d o b s e r v e r s w e r e readily able to disc r i m i n a t e b e t w e e n the d i f f e r e n t axon m o r p h o l o g i e s a n d place the axons into m o r p h o l o g i c a l c a t e g o r i e s 1 t h r o u g h 6 ( B o o z e et al. 19931.

Sensitit,ity (Alzheimer's disease) A significant d i f f e r e n c e b e t w e e n the A l z h e i m e r ' s a n d c o n t r o l b r a i n tissue was d e t e c t e d in the analysis of axon type f r e q u e n c i e s (X2(51 = 11.8, P < 0.037). T h e c o m p o n e n t s of X 2 e s t i m a t e s i d e n t i f i e d that axon types 1, 3 a n d 4 a c c o u n t e d for the m a j o r i t y of the ~(2 value (40%, 28% a n d 21%). Axon types 2 a n d 5 each cont r i b u t e d less t h a n 2% to shift in axon m o r p h o l o g i e s in A l z h e i m e r ' s disease. T h e i n t e r p r e t a t i o n of these c h a n g e s in A l z h e i m e r ' s d i s e a s e is f u r t h e r clarified if the total p e r c e n t a g e s of the various axon types is c o n s i d e r e d ; the relative f r e q u e n c i e s of axon types 1 t h r o u g h 6 w e r e 19%, 56%, 13%, 3C4, 4%, and 6%, respectively. Thus, d e s p i t e the c o n t r i b u t i o n of axon

Tyrosine hydroxylase axonal morphology T h e tyrosine hydroxylase a n t i b o d y was f o u n d to stain axons of varying d i a m e t e r s in the h u m a n pineal. This finding c o n f i r m e d the positive a n t i g e n i c recognition of n o r a d r e n e r g i c s y m p a t h e t i c axons in h u m a n tissue by the antibody. E x a m i n a t i o n of the tissue sections from frontal cortex, h i p p o c a m p u s , a n d c a l c a r i n e cortex i d e n t i f i e d 6 distinct types o f tyrosine hydroxylase imm u n o r e a c t i v e axons on the basis of m o r p h o l o g y (Fig. 1). T h e s e differing axonal m o r p h o l o g i e s w e r e a r b i t r a r ily d e f i n e d as axon types 1 t h r o u g h 6. T y p e 1 was c h a r a c t e r i z e d by very long (up to 2 m m ) fine, straight, axons that l a c k e d varicosities. T y p e 2 was very fine a n d h a d highly varicose axons, which o c c a s i o n a l l y e x t e n d e d c o n s i d e r a b l e distances. T y p e 3 c o n s i s t e d of short, tortuous, t h i c k - c a l i b e r axons. T h e s e axon types (1, 2, a n d 3) w e r e the most f r e q u e n t m o r p h o l o g i e s a n d c o m p r i s e d the m a j o r i t y of those c a t e g o r i z e d . T y p e 4 axons w e r e

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Fig. 1. Schematic drawings of the six different types of tyrosine hydroxylase immunoreactive axonal morphologies. Type 1 was characterized by very long fine and straight axons that lacked varicosities. Type 2 was very fine with highly varicose axons. Type 3 consisted of short, tortuous, thick-caliber axons. Type 4 axons were short and twisted with multifocal swellings. Type 5 was characterized by thickcaliber straight axons which lacked varicosities. Type 6 were thick caliber axons with multifocal, bulbous, axonal distensions (calibration bar = 50 #m).

114 FRONTAL CORTEX

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mortem d e l a y ( B o o z e e t al. 1993), a c c o u n t e d for a total of only 2 % of the effect of A l z h e i m e r ' s disease on h i p p o c a m p a l t i s s u e ( r e p r e s e n t i n g 4 2 % a n d 3 % o f tile total percentage of fibers identified).

Clinical correlations In order to determine if the axon type changes were related to the clinical course of Alzheimer's disease, we assessed the relationship between the duration and age of onset Alzheimer's disease (memory impairment) with the shifts in axon types 1 and 3. As may be seen in Fig. 3, there was an inverse, although nonsignificant, correlation between the extent of the shift in hip-

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Fig. 2. The relative frequency (%) of the catecholaminergic axon types in AD and aged control brain tissue obtained via rapid autopsy protocol. Regional analyses found significant shifts in the hippocampal axon types 1 and 3 as a function of AD (X2(5) = 14.5, P < 0.01).

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type 4 to the overall chi-square the analytical utility of this axon type is questionable given that it was so rare (3%) in all brain regions. In contrast, the changes in axon types 1 and 3 appeared to identify robust differences in axon types of at least moderate prevalence in Alzheimer's tissue.

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Regional analyses (Fig. 2) found significant shifts in axon type classification which were restricted to the hippocampus (X2(5)= 14.5, P < 0.01); no statistically detectable changes were evident in the frontal cortex (X2(5) = 4.8, P > 0.10) or calcarine cortex (X2(5) = 6.0, P > 0.10). The components of X 2 estimates confirmed, as indicated above, that axon types 1 and 3 accounted for nearly all of the variation attributable to Alzheimer's disease (59% and 32%, respectively). Axon types 1 and 3 represented 19% and 20% of the total percentage of axons identified in the hippoeampus. Alzheimer's brain tissue contained 16% fewer type 1 axons and 12% more frequent type 3 axons, relative to age-matched controls. In contrast, axon types 2 and 5,

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Fig. 3. The relationship between the clinical measures of AD and the shifts in axon types 1 and 3. The top panel illustrates a moderate negative correlation, though nonsignificant, between duration of AD and the extent of shift in hippocampal axon type morphology (r = 0.65, (1,5)= 3.6, P > 0.10). The middle panel illustrates a strong positive correlation between the extent of the shift in hippocampal axon type morphology and the age at onset of memory impairment (r = 0.84, F(1,5)= 11.8, P < 0.019). The bottom panel displays the multiple correlation of duration and age of onset of memory impairment with the extent of the shift in hippocampal axon type morphology (r = 0.95, F(2,4) = 18.6, P < 0.009). The clinical measures of both duration and age of onset of AD provided significant coefficients to this multiple correlation (P < 0.04).

115 pocampal axon type morphology and the duration of memory impairment (r = -0.65, F(1,5) = 3.6, P > 0.10) and a significant positive correlation between the extent of the shift in hippocampal axon type morphology and the age of onset of memory impairment (r = 0.84, F(1,5) = 11.8, P < 0.019). However, when duration and age of onset of memory impairment were considered jointly, there was a striking correlation with the extent of the shift in hippocampal axon type morphology (r = 0.95, F(2,4) = 18.6, P < 0.009) with both duration and age of onset of Alzheimer's disease providing significant contributions to the multiple correlation ( P < 0.04). A similar, but nonsignificant relationship between the shift in axon type morphology within the frontal cortex and the duration and age of onset of Alzheimer's disease was also observed (r = 0.96, F(2,1) = 5.5, P > 0.10). Unfortunately, the 40% fewer sections of frontal cortical tissue available for examination adversely affected the power of the statistical analyses and also called into question the stability of this correlation coefficient. The relationship between axon type shifts in the calcarine cortex and the duration and age of onset of Alzheimer's disease was clearly not significant (r = 0.38, F(2,4) < 1.0, P > 0.10). A significant difference among these three multiple correlations between regional axon type shifts and the duration and age of onset of Alzheimer's disease was also directly confirmed (F(6,9) = 3.86, P < 0.035). Collectively, these findings support the conclusion that only the shift in hippocampal axon type morphology was predictive of clinical course of Alzheimer's disease. The relationship of the clinical course of Alzheimer's disease, as reflected in the measures of age of onset and duration of Alzheimer's disease (memory impairment), to the classic neuropathological marker, amyloid plaques, was also investigated. Average brain plaque counts were used as the dependent variable because we found no significant regional differences in the total number of plaques. The relationship of the plaques to Alzheimer's disease age of onset and duration was strong, but did not attain statistical significance in this study (r = 0.84, F(2,4) = 1.0, P < 0.097).

• ALZHEIMER'S O RAPID AUTOPSYCONTROL DELAYEDAUTOPSYCONTROL (PREDICTED) I

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Patient classification To the extent that the shifts of axon types 1 and 3 are good predictors of Alzheimer's disease, it is anticipated that the Alzheimer's patients will be correctly

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Fig. 4. Patient classification and cross-validation were determined on the basis of the canonical variable representing the quantitative shift in axon types 1 and 3. Group membership was correctly identified for 86% of the patients. The cross-validation sample was 100% correctly identified.

classified relative to rapid autopsy controls. The shift of axon types 1 and 3 accurately predicted the identification of 71% of the Alzheimer's patients and 100% of the rapid autopsy controls (Fig. 4). The two Alzheimer's patients which displayed no evidence of type 1 and 3 axon shifts were appropriately 'misclassified'. It is of note that these two patients were also those which had the earliest age of onset of Alzheimer's disease. Similarly, to the extent that the shifts in axon types 2 and 5 are good predictors of autopsy delay (Booze et al. 1993), one would anticipate the correct classification of rapid autopsy controls relative to delay autopsy controls. The shift of axon types 2 and 5, as previously reported (Booze et al. 1993), correctly identified the origin of 72% of all control brain tissue obtained, 82% of the brain tissue obtained from rapid autopsy controis and 64% of the tissue from delayed autopsy controls (Fig. 5). The brain tissue which was misclassified displayed no regional specificity (3 from frontal cortex, 1 from hippocampus, and 3 from calcarine cortex).

• RAPIDAUTOPSYCONTROL O DELAYEDAUTOPSYCONTROL O ALZHEIMER'S (PREDICTED)

Predictit~e statistical modeling The above results suggested that the classification of catecholaminergic axon type significantly differentiated Alzheimer's patients from an age-matched control population. The classification of patients according to their group membership as well as the results of the crossvalidation sample, as shown in Figs. 4 and 5, confirmed this suggestion.

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CANONICAL VARIABLE Fig. 5. Patient classification and cross-validation were determined on the basis of the canonical variable representing the quantitative shift in axon types 2 and 5. Group membership was correctly predicted for 72% of the control patients. The cross validation sample was 73% correctly identified as having been obtained under rapid autopsy protocol.

Cross-l ~alidation The most critical aspect of classification is how well additional subjects, not employed in the identification of the axon type shifts, are classified by these functions. If the shift of axon types (1 and 3) which discriminates Alzheimer's patients from rapid autopsy controls is not influenced by any changes in axon type morphology which occur as a function of autopsy delay, then the delayed autopsy patients should be correctly identified as controls. The cross-validation sample comprising axon type 1 and 3 counts from delayed autopsy patients was 100% correctly identified as controls (Fig. 4). That no tissue was misclassified provides support for the differentiation of changes noted in axon types 1 and 3 as unique to Alzheimer's disease, and are therefore not attributable to postmortem changes. A similar cross validation was performed with the Alzheimer's patients for the axon type shift (2 and 5) which discriminates rapid autopsy from delayed autopsy controls (Booze et al. 1993). The cross-validation sample consisting of axon types 2 and 5 counts from Alzheimer's patients displayed a distribution quite distinct from the delayed autopsy controls, but quite comparable to the rapid autopsy controls (Fig. 5). Specifically, 73% of the Alzheimer's disease brain tissue samples were correctly identified as being obtained via the rapid autopsy protocol. The brain tissue which was misclassified again displayed no regional specificity (12 from frontal cortex; 3 from hippocampus; 2 from calcarine cortex). Thus, the quantitative axon type profiles not only correctly identified patient classification, but to a large extent, could accurately classify a cross-validation sample. These results suggest that Alzheimer's disease produces a characteristic pattern of tyrosine hydroxylase axon morphologies which is not evident in normal aging processes and is not an artifact of delayed tissue processing. Moreover, the quantitative axon type profiles were found to have errorless predictive ability with respect to late onset ( > 75 years) Alzheimer's disease.

Discussion

We found that the relative proportions of catecholaminergic axon categories are selectively altered in the Alzheimer's disease hippocampus. No significant shifts in catecholamine axons were found in the superior frontal cortex or calcarine cortex of Alzheimer's disease brain tissue. The selective shifts in axon profiles were not due to delays in processing, as tissue obtained via rapid autopsy was compared between the aged control and Alzheimer's groups. Furthermore, the proportional shifts in hippocampal axon morphology were qualitatively and quantitatively different from

those occurring as a consequence ol delayed postmortem tissue fixation (Booze et al. 1993). Thus, selective changes in catecholamine axons occur in Alzheimer's tissue which are independent from those which are a consequence of postmortem delay. Predictive modeling of the hippocampal data found that the shifts in axon categories were successful in discriminating Alzheimer's disease hippocampus from aged control hippocampus. We therefore speculate that the specific shifts in hippocampal axon morphology may represent a form of sympathetic ingrowth in the Alzheimer's disease brain. Sympathetic ingrowth occurs in response to loss of septal cholinergic input to the hippocampus in animal models (Crutcher 1987). In Alzheimer's disease, the extent of hippocampal cholinergic denervation may vary depending on whether the medial septal cholinergic afferent pathway is damaged (Bird et al. 1983). In some patients the septal cholinergic innervation appears to be relatively spared, with extensive damage to the entorhinal cortex and perforant axon pathway (Geddes et al. 1985); however, in other patients the entorhinal and septal inputs to the hippocampus are both damaged in Alzheimer's disease (Hyman et al. 1987). Correlating cholinergic markers in the hippocampus of patients with Alzheimer's disease and axon type profiles would provide further evidence for the presence of sympathetic ingrowth in a subset of Alzheimer's patients. Our results suggest further study of catecholaminergic alterations (and possible sympathetic ingrowth) need to be evaluated relative to the individual pathological changes present in each Alzheimer's disease brain. Given that the extent of sympathetic ingrowth would be expected to be related to the individual patterns of cholinergic pathology, and also to reflect the age-dependent progression of Alzheimer's disease, we did not find putative sympathetic ingrowth axons in patients which had a presumptive age of onset prior to 75 years. These data must, at present, be viewed cautiously as the precise onset of Alzheimer's disease and the duration of Alzheimer's disease are difficult to establish in a retrospective study. Nevertheless, as sympathetic ingrowth appears to be associated with the late onset form of Alzheimer's disease, further examination of sympathetic ingrowth would be most effectively directed at this specific Alzheimer's disease subpopulation. It has been reported that patients with late onset Alzheimer's disease have more profound loss of cholinergic markers in the hippocampus, relative to other regions (Bird et al. 1983). In contrast, patients with early-onset Alzheimer's disease have more severe loss of cholinergic markers in neocortical areas. If more severe loss of hippocampal cholinergic innervation occurs in the late onset form of Alzheimer's disease, then it seems reasonable, and consistent with the present

117

study, that these patients would be most liable to display hippocampal sympathetic ingrowth. It has been previously reported that counts of amyloid plaques (Blessed et al. 1968; Perry et al. 1978) and neurofibrillary tangles (Wilcock and Esiri 1982) correlate with the dementia rating of patients with Alzheimer's disease. Accordingly, the relationships of the traditional neuropathological markers of Alzheimer's disease (Ball 1978), neurofibrillary tangles and amyloid plaques to clinical course of Alzheimer's disease were also investigated. We found the traditional neuropathological measures of Alzheimer's disease - abundant senile plaques and neurofibrillary tangles - to be good markers of the presence of the disease. However, the lack of a significant correlation between the onset/duration of Alzheimer's dementia and plaques suggests, similar to other reports (Katzman et al. 1988), that plaque numbers may not meaningfully relate to the clinical course and severity of Alzheimer's disease. Moreover, our results are similar to those obtained by Mann et al. (1988) in which a small number of Alzheimer's patients were brain biopsled early in their disease and the brains were examined again 3 - 7 years later at autopsy. The clinical progression of Alzheimer's disease did not correlate with the pathological progression of the disease, but plaques were found to be very good markers of the presence of Alzheimer's disease. Whether sympathetic ingrowth occurs in Alzheimer's disease remains an open question due to the lack of selective markers for sympathetic axons in human brain tissue. However, given the changes we have observed in the Alzheimer's hippocampus, it seems reasonable to pursue the sympathetic ingrowth hypothesis in Alzheimer's tissue. The present findings indicate that this sympathetic ingrowth hypothesis could be best studied in the future using hippocampal tissue from patients with late onset Alzheimer's disease ( > 75 years) with clear loss of cholinergic afferents to the hippocampal formation. The importance of investigating this hypothesis lies in the finding of cognitive impairments in animals which have sympathetic ingrowth (Harrcll and Parsons 1988; Harrell et al. 1990; Ayyagari et al. 1991), and the possibility that such a phenomenon might contribute to a n d / o r accelerate the dementia of Alzheimer's disease. Further pursuit of the sympathetic ingrowth model will help fully elucidate the role of both central and peripheral catecholaminergic alterations in the cognitive dysfunction present in Alzheimer's disease. Acknowledgements This work was supported by N I H grant AG1(1747, AG-10836 and the American Federation for Aging Research to R.M.B.; NIH grants AG05128 and NS06233 to J.N.D., and the Veterans Administration. The assistance of Dr. Barbara Crain in the neuropathological evaluations is gratefully acknowledged. We also thank Ms. Gall Cook and Marl Szymanski for their dedicated

work in the rapid autopsy protocol and Carol L. Smith fl)r secretarial assistance.

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