AGE-DEPENDENT MORPHOLOGICAL CHANGES IN dBcAMP- TREATED
DIEGO G. GICOVATE, MARIA I. BERRIA
Departamento de Microbiología, Facultad de Medicina, Universidad
de Buenos Aires
Key words: aging astrocytes, dBcAMP treatment, image
changes in cell morphology are conspicuous features of astrocyte
reaction, we resorted to an histometric approach to evaluate age
influence on such morphological response to activating stimuli. To
this end, first subcultre of rat brain astrocytes at 1, 9 or 21 days
in vitro (DIV) were treated during 2 hs with 1 mM of dBcAMP, a
chemical compound known to induce cell differentiation. Following
treatment, immunoperoxidase labeling of GFAP, specific marker of
astrocyte activation, was carried out. Although total count of GFAP-
positive cell foci was greater in treated samples in all times tested,
when such cell foci were evaluated by image analysis, differences
between perimeter/area ratios of such foci were only statistifically
significant at 1 DIV. It may be concluded that while dBcAMP effect is
maintained despite astrocyte agining, the morphological pattern of
response varies markedly along the observation period.
morfológicos dependientes de la edad en astrocitos tratados con
dBcAMP. Dado que los cambios morfológicos son componente
mensurable de la reacción astrocitaria, se recurrió a un
procedimiento histométrico para evaluar la influencia de la edad
celular en la respuesta a los estimulos diferenciadores.
Asi, subcultivos astrocitarios derivados de encéfalo de rata fueron
tratados a los días 1, 9 y 21 de edad con dBcAMP, un compuesto de
reconocida acción estimulante sobre ese tipo celular.
Consecutivamente a la marcación por inmunoperoxidasa de la proteína
gliofibrilar ácida (GFAP), que es marcador específico del astrocito
activado, por análisis de imagen se determinó el perímetro y área
de los focos celulares positivos. Aunque en todos los tiempos
considerados el número total de focos GFAP-positivos fue mayor en los
cultivos tratados, la relación perímetro/área de dichos focos sólo
significativas al día 1 de desarrollo in vitro. Se concluye que, si
bien la acción diferenciadora se mantuvo pese al envejecimiento
astrocitario, el perfil morfológico de la respuesta celular varió
marcadamente a lo largo del período de observación.
Recibido: 21-VII-1996 Accepted: 3-VII-1996
Postal address: Lic. Diego G. Gicovate, Departamento de
Microbiología, Facultad de Medicina, UBA, Paraguay 2155, 1121 Buenos Aires, Argentina
Activated astrocytes, commonly recognized morphologically by their
enlarged size and numerous cytoplasmic processes, and
immunocytochemically by the massive accumulation of GFAP-positive
filaments, arise following any type of CNS injury, whether physical,
chemical, immunological, viral or bacterial1. The fact that astroglial
responsiveness is maintained in spite of aging, even at times when
cell senescence becomes associated with diminished brain functions, is
still not understood2.
Since astrogliosis phenomena on the whole organism may somehow be
recapitulated by cell cultures affording the advantages of easy
manipulation and feasible control, cultured astrocytes has been
extensively used notwithstanding the limitations of such in vitro
model. In its connection, cell aging has been shown to lead to a
gradual increase in spontaneously differentiated cells with a
concomitant drop in their division potential3. On the other hand, it
is well known that the addition of dibutyryl cyclic AMP (dBcAMP) to
the culture medium of immature astrocytes results in their
differentiation into reactive-like astrocytes4,
since dBcAMP is capable of inhibiting growth and altering morphology
of such cells, thus providing a suitable in vitro counterpart of in
situ response to neural injury5, within the limitations of the
currently employed criteria to define the reactive state6.
With the aim to evaluating age influence on cell response to dBcAMP,
we resorted to astroglial cultures obtained from brains of newborn
Wistar rats as previously described7. After 1, 9 or 21 days in vitro
(DIV) of first subculture in coverslips of Leighton tubes, cell
monolayers were treated with 1 mM dBcAMP in PBS during 2 hs at 37C.
Treated samples, as well as matched controls incubated in PBS alone,
were harvested, washed 3 times with 0.05 M Tris-saline buffer, and
fixed with chilled acid methanol for 20 min at -20C. GFAP labeling was
then carried out by the peroxidase-antiperoxidase (PAP method).
Accordingly, antisera of rabbit anti-GFAP (Sigma Lab, St Louis, MO,
USA) at 1/1600 dilution, goat anti-rabbit (Pell-Freez Biological,
Tustin, CA, USA) at 1/200 and PAP-rabbit (Cappel, Cochranville, PA,
USA) at 1/200 were consecutively employed. Potential sources of
variation in peroxidase development were ruled out by normalized
conditions: 0.03% diaminobenzidine tetrahydrochloride (Fluka Labs,
hauppage, NY, USA) plus 0.02% hydrogen peroxide for 10 min, followed
by washing with tap water. To evaluate morphological changes in
treated cultures vs controls, a suitable software (IMAGE PRO-PLUS 1.1,
Media Cybernetics, Silver Spring, MD, USA) was employed. Hardware
consisted of a black and white CCD video camara coupled to a Zeiss
microscope. Under standardized conditions using a 500/510 nm green
filter, the perimeter and area of 20 randomly selected GFAP- positive
foci were determined for each sample. Recorded values were entered
into an standard spreadsheet in order to calculate means and standard
deviations (SD). Regarding two or more stained astrocytes as a labeled
focus, such foci were counted in randomly selected microscopic fields
at 12 x final magnification. Student t test was used for statistical
analysis taking p < 0.05 as significant. As regards histometric
analysis of each positive focus, since individual cell coutlines could
not clearly be discerned, we resorted to an alternative approach,
taking the ratio between perimeter and area of each selected focus. In
the case of single GFAP-positive cells, we were able to evaluate
growth of astroglial processes by means of a graticule of 13
concentric circles, the firstbeing the most proximal and the 13th the
most distal. This graticule was superimposed on the image of isolated
GFAP-immunolabed cells visualized on the monitor screen. The
perikaryon of each selected stained cell was placed in the center of
the graticule and all the intersections produced by stained processes
with circles were counted. For each sample, the first 15 single
labeled cells to be encountered were measured and data analyzed using
a two-way ANOVA.
In this study, we evaluated effects of astrocyte aging on the
differentiation- inducing properties of dBcAMP, as expressed by the
morphological changes taking place in GFAP-positive cells. Mean total
focus count ± SEM was significantly greater in treated cultures (p
< 0.01) than in controls at 1 DIV (8 ± 0.9 vs 5 ± 0.6), 9 DIV (10
± 0.7 vs 6 ± 0.4) and 21 DIV (5 ± 0.4 vs 3 ± 0.4).
As shown in Table 1, the perimeter/area ratio was only significantly
greater in treated vs control samples (p < 0.05) at 1 DIV, while at
longer times tested,differences failted to reach statistical
significance. The increase in perimeter/area ratio recorded in treated
cultures at 1 DIV, seems due to cell body retractionrather than
changes in focus perimter, since the major changes took place in area
alone. The trend towards significance suggested by values recorded at
21 DIV was disregarded, since by that time controls had shown some
degree of cell degeneration as evidenced by intracytoplasmic
granulation accompanied by an increase in cell detritus.
Although cultures of lower density than employed herein would have
allowed a more accurate delimitation of the cells making up
GFAP-positive foci, it should not be overlooked that cells grown at
high and low densities exhibited distinct morphologies8, and such
culture modality may markedly affect astrocyte properties, among them
cell response to dBcAMP9. In this connection, when single
GFAP-positive cells were evaluated resorting to the graticule and by a
two-way ANOVA (Fig. 1), at all times assayed the single labeled
astrocytes from treated samples invariably displayed a greater number
of cell processes intersecting with concentric graticule circles 2 and
3, since the first was disregarded because on occasion it was occupied
by the cell body itself.
Interestingly enough, cell process branching as depicted by
intersections with circles 4 to 10 was greater in controls at 1 DIV
but not at later times. It should be pointed out that in treated
samples, particularly at 1 DIV, most cell processes intersecting with
circles 2-3 readily reached and even surpassed the outer circle. On
the basis of results achieved, cell aging failed to exert inhibitory
effect on astrocyte response to dBcAMP, as disclosed by the number of
GFAP-positive cell foci, although the perimeter/area ratio of such
foci was significantly greater at 1 DIV alone. As regards single
labeled cells from treated cultures, their morphological pattern was
also significantly different at 1 DIV, since by this time
dBcAMP had induced longer and more numerous cell processes but less
branching. It may be concluded that while dBcAMP effect is maintained
despite astrocyte aging, the morphological pattern of response varies
Although GFAP expression has proven reduced in many long-term cultures
to the extent that their identification becomes difficult10, the
capability of such aged cells to re-enter the division cycle11,
suggests that cultured astrocytes may undergo a transition in a manner
partly resembling reactive astrogliosis in intact brain. Abundant
evidence indicates that, in the aging whole organism, the neuroglial
proliferative ability arises in a previously quiescent population of
cells which may not have divided for prolonged periods of time and
which display few markers of cytodifferentiation [for references see2,
However, there are also examples of astrocyte reaction solely
disclosed by an hypertrophy occurring in absence of cell division14.
At any rate, signals which rigger aged astrocyte activation
remain to be elucidated. In this sense, the in vitro system may
represent a valid tool to discern the stereotypic events involved in
the aging process. Thus, the long-term astroglial culture becomes a
useful substrate to develop procedures intended to speed up, slow down
or arrest a glial reaction which, in the whole organism, may behave as
beneficial or detrimental depending on the timing13.
Acknowledgements: To Dr. Joaquín García-Estrada, a former
fellow at Instituto Cajal, Madrid, who kindly provided the graticule
employed as test grid. This work was partly supported by grants from
CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas)
and University of Buenos Aires.
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Fig. 1. Vertical bars depict the number of intersections of single
GFAP- positive astrocytes with concentric circles (from 1 the most
proximal to 13 the most distal) of the test grid used to evaluate cell
morphology. Each determination corresponds to 15 single labeled cells
to be encountered. DIV: days in vitro.
TABLE 1.-- Morphological changes of GFAP-positive foci in dBcAMP
treated vs untreated astroglial cultures.
Days in Perim./Nucleus ratio Area/Nucleus ratio Perim./Area ratio
vitro control dBcAMP control dBcAMP control dBcAMP
1 51 ± 31 63 ± 66 518 ±
464 275 ± 124* 0.104 ± 0.1 0.204 ± 0.1*
9 57 ± 37 110 ± 304 781 ±
1005 1535 ± 2874 0.122 ± 0.1 0.210 ± 0.4
21 70 ± 53 185 ± 377 749 ±
686 1016 ± 398 0.097 ± 0.1 0.161 ± 0.3
* Significant differences (p < 0.05) between ratios calculated
mean values (± SD).