Aging in Rodent Brain: Alteration in Astrocyte Population

Neurobiology t~fAging, Vol. 1, pp. 16%173. Printedin the U.S.A.

Aging in Rodent Brain: Alteration in Astrocyte Population B R I A N R. U N S W O R T H A N D L Y N D A H. F L E M I N G 2

D e p a r t m e n t o f Biology, Marquette University, Milwaukee, WI 53233 R e c e i v e d 17 J u n e 1980 UNSWORTH, B. R. AND L. H. FLEMING. Aging in rodent brain: Alteration in astrocyte population. NEUROBIOL. AGING 1(2) 169-173, 1980.--A method for the preparation of bulk isolated glial cells was modified to provide astrocytes suitable for biochemical analysis. Using this method, the astrocyte-enriched fraction from senescent mouse or rat brain stem could not be recovered from the sucrose interface at which 6 month brain stem astroglia accumulated. An alteration in the buoyant density of the senescent glial cells was demonstrated by using continuous diatrizoate gradients. The involvement of the astrocyte cell population in this age-related shift in buoyant density was confirmed using antiserum specific for glial fibrillary acidic protein. Aging rodent brain Diatrizoate gradients

Astrocyte population

Cell separation

EVIDENCE is accumulating to support the concept that the progressive loss of neurons associated with aging [3, 15, 17, 25] is accompanied by a concomitant increase in glial cell number [20,24] and possibly by altered glial cell morphology [8, 13, 14]. Since glial cells constitute the major brain cell population, and are intimately involved with the modulation of neuronal activity [12,23], the role of glial cells during aging is of considerable interest. However, a better understanding of cell function will require biochemical analysis of bulk isolated glial cells. As an initial approach to determine whether the deterioration in brain function with aging is associated with alteration in glial cell function, astrocyte-enriched fractions were prepared from mouse or rat brain stem, telencephalon, and cerebellum. Although several techniques are available for the preparation of bulk-isolated brain cells [10], methods developed for purifying both neuronal and glial cells from the same cell suspension suffer from the limitations imposed by low yield and technical difficulty. To facilitate subsequent analysis of glial cell biochemistry, brain cells were not dissociated by exposure to proteolytic enzymes, as trypsin is known to enter isolated cells [9], and is detrimental to astrocyte membrane receptors [11]. A preliminary account of this work has been presented [7]. METHOD Glial cells were isolated by a modification of the procedure of Sellinger et al. [19]. Mice (CF1 strain) from our aging colony [4] or rats (Fischer 344 or Sprague-Dawley) were decapitated, the brains were rapidly removed to ice-cold 0.32

GFAP immunofluorescence

M sucrose, and dissected into brain regions [22]. The brain region to be analyzed was placed in 7.5% PVP with 10 mM Ca ++ and minced with scissors. (The PVP, or polyvinylpyrrolidone, provided by the Sigma Chemical Co., has a normal molecular weight of 40,000.) The minced tissue was gently homogenized by 5 strokes in a loosely fitting (clearance 0.025 cm) glass-teflon homogenizer (A. H. Thomas & Co.), then sieved by passage through nylon cloth of 75 p~ mesh. The filtrate was made up to approximately 15 ml in volume with Ca++-PVP and layered on a two-step gradient (Fig. 1) consisting of 22 ml of 1.0 M sucrose with 10 mM Ca ÷÷ and 18 mi of 1.75 M sucrose with 10 mM Ca ++. The tube was centrifuged as indicated using a Spinco SW 25.2 rotor and the impure glial cells were recovered from the 1.0 M-1.75 M sucrose interface. (The pelleted neuronal perikarya were discarded.) The cells were washed with 0.32 M sucrose, resuspended in 5 ml of Ca++-PVP, layered on Gradient 2 which consisted of 10 ml 30% (w/v) Ficoll in 0.32 M sucrose, 20 ml of 1.2 M sucrose and 18 ml 1.65 M sucrose and centrifuged. (The Ficoll, obtained from the Sigma Chemical Co., has an average molecular weight of 400,000.) The Ficoll-sucrose gradient was included for its proven ability to resolve glial cells from synaptosomes and mitochondria [5]. The band collecting at the 1.2 M-1.65 M sucrose interface represented glial cells in the 'young' brain preparations. However, few cells banded in this region of the gradient in the "senescent' brain preparations, and in this case, glial cells were recovered from the upper regions of the 1.2 M sucrose. Impure glial cells were washed and resuspended in 15 ml of 0.32 M sucrose and layered on the third gradient which consisted of 22 ml of 1.3 M sucrose and 18 ml of 1.65 M sucrose. Purified glial cells were collected as indicated (Fig. 1).

~This study was supported by a grant from the NIH (N.S.--12334). 2Send reprint requests to present address: Mount Sinai Medical Center, Department of Medicine, Section of Neurology, Milwaukee, WI 53233.

C o p y r i g h t © 1980 A N K H O I n t e r n a t i o n a l Inc.--0197-4580/80/020169-05501.00/0

170

UNSWORTH AND FLEMING Gradient I

Gradient 2

10

/

/

Gradient 3

~--..12MSucrose

20Kx rprn

0"31qM Sucrose

st,,,,

3MSucrole

5Kx rpm 15min

I1U

~JLIAL CELLS

FIG. 1. After homogenization and sieving, the brain cell suspension was subjected to buoyant density gradient centrifugation in a series of discontinuous gradients.

Purified giial cells were suspended in 0.9% NaCI and layered on the top of linear gradients consisting of 25-50% diatrizoate (Renografin-76, E. R. Squibb & Sons, Princeton, New Jersey) which had been diluted with half strength Krebs-Pdnger solution [21]. The gradients were centrifuged at 25,000 rpm for 20 min in a Spinco SW 27 rotor. Buoyant density indicators were provided by 3/16" dia. markers (Small Parts Inc., Miami, Florida). The upper marker was nylon (specific gravity 1.14) and the lower marker was lucite (specific gravity 1.34). Morphological criteria were applied to estimate the cellular composition of the fractions and, hence, to gauge the efficiency of the separation procedure. In addition, since morphological features alone are insufficient to unequivocally identify isolated astrocytes, antiserum to glial fibrillary acidic protein (GFAP was generously provided by Dr. A. Biguami) was used as a marker for astrocytes in the indirect immunofluorescence technique of Antanitus et al. [1].

RESULTS A bulk isolation technique [19] was used to prepare an astrocyte-enriched fraction from gross dissected regions of the 'young' 6 or 12 month rodent brain. When this procedure was applied to senescent 24--30 month old mouse and 24-36 month old rat brain regions, a paucity of cells accumulated at the 1.2-1.65 M sucrose interface of gradient 2 (Fig: 1). Microscopic examination revealed that a band of glial-like particles could be recovered from the upper region of the 1.2 M sucrose layer (Fig. 2). This age-related alteration in the site of accumulation of the cell fraction enriched in astrocytes

was most notable in the brain stem, involved a smaller percentage of the telencephalon glial cell population, and was never observed in suspensions prepared from the senescent cerebellum. To confirm that aging of the brain stem was associated with an apparent alteration in the buoyant density of the astrocyte population, purified cell fractions from 'young' and aged animals were separated on continuous diatrizoate gradients and compared. This approach was based on the reported ability of diatrizoate to distinguish between cells on the basis of size and/or buoyant density [21]. It also eliminates any possible synaptosomal contamination since synaptosomes do not enter diatrizoate gradients of these concentrations [21]. Astroglia prepared from either "young' or senescent animals could be distinguished by the concentration of diatrizoate in which each predominantly banded (Fig. 3a and b). The glial cell fraction prepared from 30 month rat brain stem banded between 27-30% diatrizoate (Fig. 3a). while the equivalent fraction from 6 month rat brain stem banded between 33% and 41% diatrizoate (Fig. 3b). This shift in buoyant density with aging was observed in all preparations from 40 senescent mice and 15 senescent rats. Since the alteration in buoyant density o f astrocyte-enriched fractions was seen in both aged mice and aged rats of two different strains, it would not appear to be an isolated occurrence. Glial cells recovered from the diatrizoate gradients were identified as astrocytes by the indirect sandwich technique, using anti-GFAP serum (Fig. 3c,d,e,f). Although the 'young' and 'old' astrocytes could be distinguished on the basis of buoyant density, they were morphologically indistinguishable by gross examination with phase contrast or immunofluorescence.

ASTROCYTE P O P U L A T I O N DURING AGING

171

FIG. 2. Phase contrast micrographs of glial cells isolated from the brain stem of a, b, 6 month old mouse ( × 1500); c, d, 30 month old mouse ( × 1500final magnification). The cells were collected from gradient 3, Fig. 1.

DISCUSSION The preparation of bulk isolated glial cells from rat and mouse brain has revealed age-related buoyant density changes associated with the astrocyte population in the brain stem. However, a smaller percentage of the astrocytes in the aged telencephalon exhibited the shift in buoyant density. Since it was never seen in aged cerebellar suspensions, this phenomenon may have regional specificity in the aging brain. The specific histochemical localization of GFAP to astrocytes is well established [2,16]. Studies of anti-GFAP crossreactivity have shown that it does not react with neurofibrils [6] or neurofibriUary tangles [6,18], thereby confirming, that it is the astrocyte population that exhibits the age-related change in buoyant density. The age-related change in buoyant density could be caused by an adherence of other brain constituents to the astrocytes. However, a considerable adherence would be

necessary to cause this change. Such an adherence might also be expected to interfere with anti-GFAP binding, thereby causing a change in the distribution of immunofluorescent staining. Since there seems to be no difference in immunofluorescent staining or in the gross morphology of 'old' or 'young' astrocytes, it is unlikely that this is the case. The role of glial cells in aging of the central nervous system has provoked considerable interest, but little is known regarding alteration of glial function during aging. It has been inferred from culture studies, that the cholinergic neuronal enzyme choline acetyltransferase may be expressed by aging glial cells [23], and the excessive increase of glial-specific proteins in senescent rat brains [24] has been interpreted as an aging-related alteration in glial cell metabolism. The preparation of bulk isolated glial cells from rat and mouse brain has revealed age-related buoyant density changes in the brain stem astrocyte population and should stimulate studies to identify functional properties specifically associated with senescence.

172

UNSWORTH AND FLEMIN(i

a w

b

w

FIG. 3. (a) Gliai cells purified from 'senescent' 30 month old rat brain stem. The cells were recovered between 27-30% diatrizoate. This alteration in buoyant density was observed using both mouse and rat senescent brain stem. (b) Glial cells purified from 'young' 6 month old rat brain stem. The cells were recovered between 33-41% diatrizoate and this banding pattern was observed in many preparations from both mouse and rat brain stem. (c,d) 'Young' 6 month old rat glial cells recovered from the diatrizoate gradient and stained with anti-GFAP serum (× 1275 final magnification). (e,f) 'Old' 30 month rat glial cells recovered from the diatrizoate gradient and stained with anti-GFAP serum ( × 1275 final magnification).

ASTROCYTE POPULATION

DURING AGING

173

REFERENCES

I. Antanitus, D. J., B. H. Choi and L. W. Lapham. Immunofluorescence staining of astrocytes in vitro using antiserum to GFA protein. Brain Res. 89: 363-367, 1975. 2. Bignami, A., L. F. Eng, D. Dahl and C. T. Uyeda. Localization of the glial fibrillary acidic protein in astroyctes by immunofluorescence. Brain Res. 43: 42%435, 1972. 3. Brody, H. Cell Counts in Cerebral Cortex and Brainstem. In: Alzheimer's Disease: Senile Dementia and Related Disorders (Aging Vol. 7), edited by R. Katzman, R. D. Terry and K. L. Bick. New York: Raven Press, 1978, pp. 345-351. 4. Caron, P. C. and B. R. Unsworth. Alteration of the activity and molecular form of thymidine kinase during development and aging in the mouse cerebellum. Mech. Ageing Devl. 8: 181-195, 1978. 5. Cotman, C., H. Herschman and D. Taylor. Subcellular fractionation of cultured glial cells. J. Neurobiol. 2: 16%180, 1971. 6. Dahl, D. and A. Bignami. Immunochemical cross-reactivity of normal neurofibrils and aluminum-induced neurofibrillary tangles. lmmunofluorescence study with antineurofilament serum. Expl Neurol. 58: 74--80, 1978. 7. Fleming, L. H. and B. R. Unsworth. Aging in mouse brain: Change in astrocyte population. Soc. Neurosci. Abstr. 4:112, 1978. 8. Geinisman, Y., W. Bondareff and J. T. Dodge. Hypertrophy of astroglial processes in the dentate gyrus of the senescent rat. Am. J. Anat. 153: 537-544, 1978. 9. Guarnieu, M., S. R. Cohen and C. Ginns. Cells isolated from trypsin-treated brain contain trypsin. J. Neurochem. 26: 41-44, 1976. 10. Hamberger, A. and A. Sellstrom. Techniques for separation of neurons and glia and their application to metabolic studies. In: Metabolic Compartmentation in Relation to Structure and Function in Brain, edited by S. Berl and D. Schneider. New York: Plenum Press, 1975, pp. 145-166. 1 I. Henn, F. A., J. Deering and D. Anderson. Receptor studies on isolated astroglial cell fractions prepared with and without trypsin. Neurochem. Res. 5: 45%464, 1980. 12. Kimbelberg, H. K., S. Narumi and R. S. Rourke. Enzymatic and morphological properties of primary rat brain astrocyte cultures, and enzyme development in vivo. Brain Res. 153: 55-77, 1978. 13. Lansfield, P. W., G. Rose, L. Sandles, T. C. Wohlstadter and G. Lynch. Patterns of astroglial hypertrophy and neuronal degeneration in the hippocampus of aged memory-deficient rats. J. Gerontol. 32: 3-12, 1977.

14. Lindsey, J. D., P. W. Lansfield and G. Lynch. Early onset and topographical distribution of hypertrophied astrocytes in hippocampus of aging rats: a quantitative study. J. Gerontol. 34: 661-671, 1979. 15. McGeer, P. L. and E. G. McGeer. Aging and neurotransmitter systems. In: Adv. exp. Med. Biol. Vol. 113, edited by C. E. Finch, D. C. Potter and A. D. Kenney. New York: Plenum Press, 1978, pp. 41-57. 16. Schachner, M., E. T. Hedley-White, D. W. Hsu, G. Shoonmaker and A. Bignami. Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immuno-peroxidase labelling. J. Cell Biol. 75: 67-73, 1977. 17. Scheibel, M. E. and A. B. Scheibel. Structural changes in the aging brain. In: Aging Vol. 1, edited by H. Brody, D. Harmon and J. M. Ordy. New York: Raven Press, 1975, pp. 11-37. 18. Selkoe, D. J., R. K. H. Liem, S. H. Yen and M. L. Shelanski. Biochemical and immunological characterization of neurofilaments in experimental neurofibrillary degeneration induced by aluminum. Brain Res. 163: 235-252, 1979. 19. Sellinger, O. Z., J. M. Azcunta, D. E. Johnson, W. G. Ohlsson and Z. Lodin. Independence of protein synthesis and drug uptake in nerve cell bodies and glial cells isolated by a new technique. Nature, New Biol. 230: 253-256, 1971. 20. Sturrock, R. R. Quantitative morphological changes in neurons and neuroglia in the indusium griseum of aging mice. J. Gerontol. 32: 647-658, 1977. 21. Tamir, H., M. M. Rapport and L. Roizin. Preparation of synaptosomes and vesicles with sodium diatrizoate. J. Neurochem. 23: 943-949, 1974. 22. Unsworth, B. R. and D. R. Hafeman. Tetrodotoxin binding as a marker for functional differentiation of various brain regions during chick and mouse development. J. Neurochem. 24: 261270, 1975. 23. Vernadakis, A., R. Nidess, B. Culver and E. Bragg Arnold. Glial cells: modulators of neuronal environment. Mech. Ageing Devl. 9: 553-566,. 1979. 24. Wintzerith, M., G. Labourdette. J. P. Delaunoy and P. Mandel. Nucleic acids, total and glial protein changes in rat brain with aging. Age 1: 125-129, 1978. 25~ Wisniewski, H. M. and R. D. Terry. Neuropathology of the aging brain. In: Neurobiology o f Aging. edited by R. D. Terry and S. Gershon. New York: Raven Press, 1976, pp. 225-231.