Neurobiology of Aging, Vol. 8, pp. 1-6. Copyright ~' Pergamon Journals Ltd., 1987. Printed in the U.S.A. 0197-4580/87 $3.00 + .00

An Immunohistochemical Quantification of Fibrous Astrocytes in the

Aging Human Cerebral Cortex

L. A. H A N S E N , D. M. A R M S T R O N G A N D R. D. T E R R Y 1

D e p a r t m e n t o f Neurosc i ences , Universi ty oj" California, San Diego

R e c e i v e d 17 A p r i l 1986

HANSEN, L. A., D. M. ARMSTRONG AND R. D. TERRY. An immunohistochemical quantification of fibrous astro- cytes in the aging human cerebral cortex. NEUROBIOL AGING 8(1) 1-6, 1987.--1n order to determine whether cortical fibrous astrocytes increase with age, we studied 25 patients ranging in age from 24 to 100 years with no clinical or pathological evidence of dementia or other cerebral disorder. Paraffin sections of mid-frontal cortex were obtained and stained with the avidin-biotin immunolabeling procedure for glial intermediate filament protein. The resulting im- munolabeled fibrous astrocytes were then counted in the molecular and cellular (cortical laminae 2-6) layers. Populations of fibrous astrocytes in both layers varied widely among individuals, and in the molecular layer their numbers were not significantly correlated with advancing age. In the cellular layer, however, despite widely ranging cell counts among individuals within the same decades of life, there was a significant linear increase with age. Our data suggest that the increase occurs or accelerates significantly after age 70, but the case numbers preclude reaching such a conclusion with statistical confidence. However, when the patients are divided into those less than 70 and those older, fibrous astrocytes in the cellular layer are shown to be significantly increased in the latter group compared to the former.

Fibrous astrocyte Aging Neocortex Quantification lmmunohistochemistry

F I B R O U S astrocyte populat ions in the cerebral cor tex are increased in patients with Alzhe imer ' s disease and senile dement ia o f the Alzhe imer type when compared to age- matched controls [ 10,14]. Al though the significance of these findings is not comple te ly unders tood, it is thought that this gliosis might be related to neuronal loss or degenerat ion and disorganized neurite patterns. It is a c o m m o n impression that as t rocytes in the cor tex also increase during normal ag- ing, al though quant i ta t ive measurements of these cell popu- lations have been few [16]. Invest igat ions of fibrous gliosis as an age-related phenomenon in animals have not produced consis tent results [2,3]. In this s tudy, we employed the highly sensit ive avidin-biotin immunolabel ing procedure using antibodies against glial intermediate f i lament protein (GFP) in order to label fibrous as t rocytes in sections of mid- frontal cor tex from patients ranging in age be tween 24 and 100 years. We sought to determine if there is a significant increase in fibrous as t rocytes in the molecular or cellular (cortical laminae 2-6) layers, or both, with normal aging. If such an increase does occur , compar isons can be made be- tween the astrocyt ic gliosis seen in human aging and that reported in exper imental animals. Further , the fibrous gliosis of normal aging can be contras ted with that seen in progres- sive neocort ical degenera t ive disease (i.e., A lzhe imer ' s dis- ease).

METHOD

Twenty-f ive cases were selected with patient ages ranging from 24 to 100 years. The brains were obtained from seven source institutions. Eight cases came from the Jewish H o m e and Hospital for the Aged in N e w York, four each from the hospital of the Albert Einstein College of Medicine and Johns Hopkins Universi ty , three each from the Univers i ty of Vermont and the San Diego Veterans Administrat ion Hospi- tal, two from the Bronx Municipal Hospital , also at Einstein, and one f rom the Queens County Medical Examine r ' s Office. At least three cases from each decade from the fourth to the tenth were included. The subjects had been clinically free of dement ia according to the hospital chart or the attend- ing staff. Among those o v e r 60, any patient with a Blessed score greater than 8 was excluded. The causes of death var- ied, but in no instance was a pat ient ' s demise attr ibutable to a primary neurological disorder. Cardiovascular disease was responsible for seven deaths (cases 9, 13, 14, 20, 21, 22 and 24) and carc inoma for five (cases 7, 10, 11, 16, and 19). Three patients died o f intestinal necrosis or perforation with peritonitis (cases 15, 17, and 18). Pulmonary embolism (case 25), b ronchopneumonia (case 23), and acute asphyxia in a fire (case 3) each accounted for one death. The terminal events in the patients from Johns Hopkins (cases 2, 4, 5 and

1Requests for reprints should be addressed to R. D. Terry, University of California, San Diego, School of Medicine, Department of Neurosciences, M-024, La Jolla, CA 92093.

2 H A N S E N , A R M S T R O N G A N D T E R R Y

FIG. 1. Fibrous astrocytes in the molecular layer. Note positive labeling of perikarya and stellate cytoplasmic processes with antibody to glial intermediate filament protein. Avidin-biotin im- munolabeling preparation, x200.

FIG. 2. In the cellular layer fibrous astrocytes occurred (A) in pairs, (B) in small clusters, and tC) in relation to blood vessels, sending their processes to the vascular walls. Avidin-biotin immunolabeling preparation. × 200.

6) and the Univers i ty of Vermont (cases 1, 8, and 12) are unavailable. However , these cases were screened by neuropathologists at those institutions before being contrib- uted specifically for use as normal controls in aging studies. All brains were examined grossly and microscopical ly and found to be neuropathological ly normal. None of the brains from patients less than 70 contained any neuritic plaques or neurofibril lary tangles. Only a few neuritic plaques and no

neurofibrillary tangles were present in the neocor tex in the patients older than 70, and one or two neurofibril lary tangles at most were seen in the h ippocampus in some of these cases.

The brains had been removed at autopsy in the routine fashion. Pos t -mor tem times varied but never exceeded 24 hours. The brains were fixed in formalin for two weeks. Subsequent ly , tissue blocks were taken from the midfrontal

FIBROUS ASTROCYTES IN AGING 3

Case No. Age

1 24 2 25 3 31 4 33 5 33 6 36 7 41 8 45 9 47

10 54 11 57 12 58 13 65 14 66 15 67 16 69 17 74 18 74 19 78 20 84 21 84 22 85 23 92 24 92 25 100

TABLE 1

MID-FRONTAL CORTEX FIBROUS ASTROCYTE COUNTS

Mean Astrocyte Counts:

Cellular Standard Molecular Sex Layer Deviation Layer

Standard Deviation

M 2.0 0.6 29.8 7.9 M 2.0 2.0 23.5 12.8 M 3.8 2.4 11.6 1.5 M 3.9 2.4 39.1 8.9 M 5.2 2.5 33.3 6.9 M 10.8 7.7 16.8 7.9 M 7.0 5.3 43.3 5.5 M 1.7 1.6 16.5 4.8 M 22.3 8.9 26.0 12.5 M 8.7 5.6 46.2 9.7 F 10.2 5.9 56.5 13.2 M 2.6 1.0 53.3 10.6 F 21.5 9.6 40.3 6.2 M 1.2 1.4 54.6 6.2 M 6.3 3.0 51.3 9.1 M 2.7 1.8 27.0 8.5 F 16.5 9.4 36.2 6.3 M 4.3 3.4 52.0 9.7 M 25.9 3.6 26.1 7.9 F 8.5 5.0 43.5 I0.1 F 9.5 5.9 66.0 12.7 F 33.3 11.5 62.5 8.8 F 13.5 9.2 35.0 12.1 F 38.2 29.6 27.7 9.2 F 16.5 11.8 17.5 6.9

region approximately 10 cm caudal to the frontal pole. The blocks were oriented to ensure that the cortex along the sulcus was cut perpendicular to the surface. The tissue was embedded in paraffin and sectioned at 10 microns.

The anti-astrocyte filament serum utilized in this study was raised in rabbits by immunization to a 49,000 molecular weight polypeptide from a human brain filament preparation. The specificity and characterization of this antibody have been described [4]. Anti-glial intermediate filament protein (GFP) and anti-glial fibrillary acidic protein (GFAP) are widely recognized as providing identical staining patterns and both label fibrous astrocytes.

The paraffin embedded tissue sections were dehydrated through graded alcohols in preparation for the avidin-biotin immunolabeling procedure [8]. This method consists of the following series of steps.

(1) Thirty minute incubation in 0.3% H20~ in 0.1 M Tris- saline.

(2) One hour incubation in 0.1 M Tris containing 3% goat serum and 0.25% Triton, pH 7.4.

(3) Twenty-four hour incubation at 23°C with antiserum to glial intermediate filament protein diluted 1:500 with 1% goat serum in Tris-saline.

(4) Incubation for one hour with biotinylated goat anti- rabbit IgG (Vector Laboratories) diluted 1:200 with Tris- saline containing 1% goat serum.

(5) One hour incubation with ABC complex (Vector Lab- oratories) diluted 1:100 with Tris-saline containing 1% goat serum.

(6) Treatment for 15 minutes with 0.05% solution of 3-3'-diaminobenzidine, 0.01% hydrogen peroxide and nickel chloride in 0.1 M Tris buffer.

The sections were then counterstained with hematoxylin. Method specificity was confirmed by substituting non- immune rabbit serum for the primary antibody and observing no immunoreactivity in the resulting preparations.

It was determined that the difference in optical density between the immunolabeled fibrous astrocytes and the neuropil was insufficient for unedited computerized count- ing. Since the extensive edited procedure would have amounted to essentially manual counting, we decided not to utilize automated analysis.

Counting of fibrous astrocytes was done at a magnifica- tion of 125× with a Leitz microscope. We subdivided the astrocyte counts into molecular and cellular (cortical laminae 2-6) layers since fibrous astrocytes in the former greatly out- number those in the latter, and combined figures obscure any increase in the cellular layer alone. Criteria for including a cell as a fibrous astrocyte were (1) discernable nucleus, (2) positively stained perikaryon, and (3) steilate cytoplasmic processes. Two immunolabeled sections were prepared from the midfrontal cortex tissue block in each case. On each section three areas were identified where the cortical ribbon was truly perpendicular to the white matter. The counted area in each such region was 1.4 mm in length along the pial surface and extended through the full cortical thickness. This procedure resulted in six fibrous astrocyte counts per case from the molecular layer and six per case from the cellular

40

35

30.

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I I

A S T 25 R O C Y T E 20

C O IJ N T 15 S

4 H A N S E N , A R M S T R O N G A N D T E R R Y

2 0 30 aO 50 60 70 80 90 IO0 I I0

A G E

FIG. 3. In the cellular layer fibrous astrocyte counts correlated with age (r=0.551,1)<0.01), but the scatter suggested that the increase occurs predomi- nantly in later life.

21 -30 (N=2)

31 -40 (N=4)

41 -50 (N=3)

51 -60 (N=3)

61 -70 (N=4)

71 -80 (N=3)

81 -90 (N=3)

91 -100 (N=3)

M F A N F I 8 R O U S A S T R O C Y T E C O U N T S

5 10 15 20 25

- - - - • SD=0

SD=3.3

SD=10.7

SD=9.3

Iso=14.o

>D=13.5

FIG. 4. When the cellular layer astrocyte counts are grouped by decades, a tendency tot increas- ing means beyond age seventy is revealed. Sd=standard deviation, N=number of cases per decade.

layer. For purposes of compar ing cases , these figures were then ave raged to p rov ide single f ibrous as t rocy te coun t s pe r case in each of the two layers.

The count ing was done by one o b s e r v e r utilizing a single blind me thod . Fol lowing the compi la t ion of data , l inear re- gress ion analys is was pe r fo rmed by the s t andard stat ist ical me thod to de t e rmine age-re la ted changes in a s t rocy te popu- lat ions. We also divided the cases into two age ca tegor ies ,

those above 70 years and those below. This was done to de t e rmine if mean as t rocy te popula t ions differed signifi- cant ly be tween the two age groups. A two-ta i led S tuden t t - tes t was used for compar ing these data.

R E S U L T S

Fibrous as t rocy te popula t ions in the molecu la r layer ex-

FIBROUS ASTROCYTES IN AGING 5

ceeded, and often dwarfed, those in the cellular layer in nearly every case regardless of age. Their processes in the superficial layer formed a meshwork and could be seen ex- tending to blood vessels and the pial surface (Fig. 1).

As to the cellular layer, fibrous astrocytes were usually more prevalent in the deeper cortical laminae. In cases with high astrocyte counts the positive cells often occurred in clusters but without apparent associated neuronal loss, i.e., the clustering did not represent either microscopic infarction or plaque with glial scarring. Fibrous astrocytes were also prevalent in the vicinity of blood vessels, extending their processes to the vascular walls. Not infrequently, the astro- cytes occurred in closely approximated pairs, but mitoses were never seen (Fig. 2).

The quantitative results of this study are displayed in Table 1. Mean fibrous astrocyte counts for the molecular and cellular layers were determined by averaging the values from each of the six cortical areas counted per case. The cor- reponding standard deviations are also provided. Fibrous as- trocyte counts in both the molecular and cellular layers showed great individual variation even between patients of similar or identical ages. The counts in the molecular layer did not correlate either with those in the cellular or with advancing age. Despite the wide range in cellular layer counts, however, a statistically significant increase with age was present (r=.551, p<0.01, Fig. 3). The distribution suggests that fibrous astrocyte populations are, relative to individual variations, comparatively stable during most of adult life and then increase at about age 70. To explicate this trend, we divided the cases into decades (Fig. 4). Although the tendency for counts to increase beyond age 70 was again apparent, restricted case numbers do not allow statistically valid regression analysis between astrocyte population and age in subgroups of patients below 70 and those above. However, when mean cellular layer counts in these groups are compared, the differences are significant (t=3.18, p<0.01).

DISCUSSION

Studies of age-related changes in glial cell populations in the cerebral cortex of experimental animals have yielded conflicting results. In many cases, the glial cells have not been characterized as oligodendroglia or fibrous or protoplasmic astrocytes. Nevertheless, in the mouse cortex the total number of glia has been shown to increase steadily throughout life from five to 720 days [7]. In rats, one study has shown an increase in the combined astrocyte-oligoden- droglia population in aged animals, possibly as a consistent phenomenon associated with the aging process [2]. How- ever, other investigators found no increase in astrocytes and oligodendroglia in the aging rodent [3]. Hypertrophic astro- cytes, seen with Cajal 's gold chloride stain, have been de- scribed in the hippocampus of aged, memory-deficient rats, associated with significant loss of pyramidal cells [9]. As- trogliosis was not found in other regions of the diencephalon and telencephalon, but a widened molecular layer was noted in these aged animals. Image analysis of GFAP positive as- trocytes in the rat cortex from adolescence to senescence has revealed increased cytoplasmic size and the develop- ment of thicker cell processes in the astrocytes of the aged rats. The total number of astrocytes, however, appeared un- changed with age [1]. This continuous increase in astrocyte cell size, observed from 1 to 30 months of age, may correlate with the development of fibrous astrocytes from pre-existing cortical protoplasmic forms.

In human brains, many observers perceive a mild to mod- erate increase in cortical astrocytes with aging. Most counts of cortical fibrous astrocytes, however, have examined population differences between patients with Alzheimer 's disease or senile dementia of the Alzheimer type and age matched controls [10,14]. One study found greatly increased numbers of fibrous astrocytes in the cellular layer in the SDAT cases but similar populations in the molecular layers of both the demented and control groups [14]. Another group looking at the same question found that fibrous astrocytes were increased in both layers in the AD and SDAT group compared with controls [10].

Another immunohistochemical study of gliosis in the cor- tex of dementia patients and age matched controls also examined the fibrous astrocyte populations in normal aging [16]. Fibrous astrocytes were found to increase with age in the cellular and molecular layers of the superior frontal and parahippocampal gyri [16]. The present study confirms the frontal lobe cellular layer increase, with slightly higher levels of statistical significance (r=0.551, p<0.01 compared to r=0.410, p<0.05). The earlier work also shows about the same degree of variation among individuals of similar ages, with standard deviations approaching or exceeding means when cases are grouped by decades. This variability is often encountered in quantitative evaluations of the human CNS. One analysis has shown instances where, in a single cortical area, normal patients within the same decade of life vary in total glial and neuron counts by a factor of four [6]. Our results differ from those of the earlier study [16] with regard to the molecular layer, since we were unable to demonstrate a correlation between astrocyte counts and age (r=0.318). This discrepancy could be attributable to differing antibody sensitivities, staining and counting methods, or patient popu- lations. Indirect support for an absence of age related fibrous gliosis in the molecular layer is the finding of no correlation between age and molecular layer thickness, expressed either in millimeters or as a percentage of total cortical thickness (Terry, unpublished data).

It should not be inferred from the results of this study that the age related fibrous gliosis seen in the midfrontal cortex necessarily occurs to the same extent throughout the cere- brum. The human brain doesn' t display uniform aging changes, either grossly or microscopically. The frontal cor- tex shows macroscopic shrinkage during aging, whereas the parieto-occipital cortex does not change in size [5]. Micro- scopic changes in neuronal size and density also predomi- nate in the frontal lobe [5]. It is likely that the degree of neocortical fibrous gliosis is proportional to the other aging changes occurring in various cerebral regions.

The increasing numbers of fibrous astrocytes are con- ceivably derived from two sources. The closely approx- imated pairs of cells might imply proliferation of those fi- brous astrocytes which are found in small numbers in the cortex of even quite young individuals. Alternatively, the protoplasmic astrocytes normally resident in the cortex may be capable of giving rise to fibrous astrocytes through mor- phologic transformation. A recent immunohistochemical study, however, concludes that fibrous and protoplasmic as- trocytes are biochemically, antigenically, and devel- opmentally distinct classes of glia, although when dis- sociated from the CNS and grown in culture protoplasmic astrocytes can be induced to make more GFAP and glial filaments [12]. Fibrous astrocytes are familiar and conspicu- ous components in many pathological alterations of the CNS, both those associated with neuronal loss (e.g., de-

6 H A N S E N , A R M S T R O N G A N D T E R R Y

genera t ive and i schemic d iseases) and those wi thou t it (e.g., mult iple sc lerosis p laques and e d e m a t o u s regions ad jacen t to metas tases ) . Despi te the above m e n t i o n e d expe r imen ta l work , this ubiqui ty of f ibrous a s t rocy tes in so m a n y var ied pathologic condi t ions , in the a b s e n c e of any conv inc ing cand ida te for the role of pr imi t ive p r ecu r so r cell, has con- v inced mos t neuropa tho log i s t s tha t cor t ical f ibrous as t ro- cy tes may of ten or iginate t h rough t r ans fo rma t ion of proto- p lasmic forms.

The s t imulus for cel lular layer f ibrous gliosis in aging is still specula t ive . Age-re la ted a l te ra t ions in cort ical n e u r o n s to which the a s t rocy te s may be r e spond ing are unde r con- t inuing inves t igat ion. Data or iginat ing in our l abora to ry [15] and e l sewhere [5] emphas ize neurona l shr inkage , while o thers s t ress neurona l depopu la t ion [6]. E i the r p rocess would predic t co r r e spond ing axonal pa thology. Exper i - menta l work has d e m o n s t r a t e d astroglial r e s p o n s e s ( includ- ing inc reased glial fibril lary acidic prote in) a ssoc ia ted with axonal injury a long f iber t rac ts distal to si tes of actual dam- age [13]. O the r s have also recen t ly s h o w n tha t astroglial cells have the capac i ty to p roduce t rophic and neur i t e -p roduc ing agents in vi t ro and tha t these subs t ances are found in normal

CNS t issue, suggest ing that a s t rocy tes may produce t hem in vivo as they do in vi t ro [11]. F u r t h e r m o r e , adult neu rons f rom the sympa the t i c super ior cervical gangl ion r e spond to ne rve g rowth fac to r ( secre ted by a s t rocy te s in vitro) with neur i t ic ou tg rowth I l l ] . The cel lular layer f ibrous gliosis could the re fore be a morpholog ic mani fes ta t ion of a t rophic r e sponse by the a s t rocy te to axona l degene ra t ion or peri- karyal sh r inkage occur r ing in the aging cor tex. The more se- vere cort ical gliosis seen in pa t ien ts wi th ex tens ive neurona l loss (e.g., A l z h e i m e r ' s d isease) might t hen r ep re sen t an as- troglial r e sponse to inexorable neurona l depopu la t ion and assoc ia ted axonal loss.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge Dr. Shu-Hui Yen who so kindly provided us with the antibody utilized in this study. We also wish to acknowledge Susanne Almstrom for technical assistance and Eleanor Michels for secretarial support. Finally, a number of grants from the National Institutes of Health, AGO5344, AGO5386, 5P50 AGO5131, and from the McKnight Foundation have supported this work.

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