Brain Research 764 Ž1997. 188–196
Research report
Temporal profiles of the in vitro phosphorylation rate and immunocontent of
glial fibrillary acidic protein Ž GFAP. after kainic acid-induced lesions in area
CA1 of the rat hippocampus: demonstration of a novel phosphoprotein
associated with gliosis
Guido Lenz a , Luis Manozzo a , Simone Gottardo a , Matilde Achaval b , Christianne Salbego a ,
Richard Rodnight a, )
a
Departamento de Bioquımica,
Instituto de Ciencias
Basicas
de Saude,
´
ˆ
´
´ Rua Ramiro Barcelos 2600-Anexo, 90.035.003 Porto Alegre, RS, Brazil
b
Departamento de Ciencias
Morfologicas
Instituto de Biociencias,
UFRGS (Centro), 90.046-900 Porto Alegre, RS, Brazil
ˆ
´
ˆ
Accepted 1 April 1997
Abstract
The in vitro phosphorylation rate and immunocontent of glial fibrillary acidic protein was studied in slices of area CA1 of the rat
hippocampus after stereotaxic injection of 1 nmol of kainic acid. For controls the contralateral hippocampus was injected with saline.
Hippocampal tissue was incubated with w 32 Pxphosphate and analysed by two-dimensional electrophoresis for phosphorylation rate and by
immunoblotting for immunocontent. Both these parameters decreased during the first 4 days after injection and then started to increase at
10 days and continued to increase until at least 84 days. Except for a small excess of phosphorylation rate at 28 days, the relationship
between immunocontent and in vitro phosphorylation rate of glial fibrillary acidic protein remained constant, indicating that the reactive
gliosis was not associated with hypo- or a major hyperphosphorylation of this protein. Histology showed a pronounced loss of CA1
pyramidal cells 1 day after injection. At 28 days after injection the pyramidal cells had disappeared and only a few abnormal neurones
were present. In contrast, immunocytochemistry after 28 days showed a marked increase in astrocytes reacting positive to the antibody in
the strata radiatum and lacunosum moleculare. Besides glial fibrillary acidic protein the expression of several other proteins was
upregulated as a result of the injection of kainic acid. These included phosphovimentin and an unknown phosphoprotein designated pp25
which co-migrated on 2-D gels with a prominent phosphoprotein expressed in primary cultures of astrocytes. Pp25 was expressed in
lesioned tissue more frequently than phosphovimentin and with a time course that started earlier. Of particular interest was the expression
of pp25 in the contralateral saline-injected hippocampus 1 day after injection of kainic acid. It is possible that pp25 will prove to be a
sensitive marker of gliosis. q 1997 Elsevier Science B.V.
Keywords: Glial fibrillary acidic protein; Vimentin; Protein phosphorylation; Kainic acid; Neurotoxicity; Excitatory amino acid; Gliosis; Astrocyte
1. Introduction
Almost any type of injury to the brain results in a
generalized glial reaction known as gliosis, characterized
by hypertrophy and in some cases proliferation, of microglia, macrophages and astroglia and the upregulation of
the expression of the astrocyte marker protein glial fibrillary acidic protein ŽGFAP. and the immature intermediate
filament protein vimentin Žrecently reviewed in Refs.
w5,15,23,30,41,43x.. For the experimental investigation of
this phenomenon, lesions in laboratory animals induced by
excitotoxicity w1,4,14,18,29,32,36x, mechanical trauma
)
Corresponding author. Fax: q55 Ž51. 316-5535.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.
PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 4 5 6 - 3
w28,34,53x, ischaemia w8,29,47x and neurotoxins w45x have
been extensively used. The temporal aspects and precise
cellular nature of the response varies with the kind and
severity of the lesion w15x. In the case of excitotoxic
lesions the initial phases of the glial reaction involve
predominantly microglia and macrophages w1,33x; subsequently astrocytes hypertrophy, and in some situations
proliferate, leading eventually to the formation of a gliotic
scar w14,29x.
One aspect of gliosis that has not received attention
concerns the phosphorylation state of GFAP. This protein,
like other intermediate filament proteins, undergoes multisite cyclic phosphorylation and dephosphorylation, a process which appears to be an important factor in regulating
G. Lenz et al.r Brain Research 764 (1997) 188–196
the dynamic equilibrium between polymerized and depolymerized GFAP Žreviewed in w25x.. Fluorescent labelling
studies have demonstrated that this state of dynamic equilibrium consists of the continuous exchange of a small
pool of soluble subunits with the intact filamentous structure and as such is a recognized feature of the turnover of
intermediate filaments w16,19,37,54x. Phosphorylation of
disassembled subunits inhibits their assembly into filaments w2,25x with subunit exchange in the case of GFAP
being suppressed in proportion to the extent of phosphorylation w38x. Thus changes in the phosphorylation state of
intermediate filament proteins could have profound effects
on the structure of the cytoskeleton, as shown by a study in
which hyperphosphorylation of vimentin induced by the
antitumour drug fostriecin was associated with intermediate filament reorganization w24x. Moreover, protein phosphorylation plays a crucial role in dividing cells, where
evidence from cell cultures has shown that the disassembly
of the cytoskeleton that precedes mitosis in some cells is
regulated by a site-specific increase in the phosphorylation
state of vimentin and GFAP w10,17,35,40x. Whether GFAP
phosphorylation plays a regulatory role in the hypertrophy
of reactive astrocytes is not known. Studies using antisense
mRNA for GFAP and reexpression of the protein w9x have
shown that its presence is necessary for the formation of
stable astrocyte processes. Extension of processes is a
characteristic feature of the lesion-induced hypertrophy of
astrocytes and possibly, as suggested by Weinstein et al.
w55x, phosphorylation of the protein during hypertrophy
could be important for interactions with other cytoskeletal
proteins or cell surface components. Other approaches to
the problem have used primary cultures of astrocytes. In
these preparations agents that affect protein phosphorylation can induce changes in morphology similar to those
occurring in reactive gliosis, but evidence for a causal
relationship is contradictory. For example, differentiation
of primary cultures of astrocytes from flat polygonal cells
to process-bearing cells did not require phosphorylation of
GFAP by cyclic AMP-dependent protein kinase w49x, but
the same morphology change induced by activation of
protein kinase C was prevented by inhibition of this enzyme w22x.
In the present work we compared the in vitro rate of
phosphorylation and immunocontent of GFAP in slices
from the CA1 region at intervals of up to 12 weeks after
direct injection of kainic acid ŽKA. into the hippocampus.
No evidence for hypo- or a major hyperphosphorylation of
GFAP was observed, but the expression of several phosphoproteins of probable astrocytic origin was upregulated.
2. Materials and methods
2.1. Compounds
w 32 PxNa 2 HPO4 was purchased from CNEN ŽSao
˜ Paulo..
Acrylamide, bisacrylamide, KA, polyclonal anti-GFAP and
189
anti-vimentin, monoclonal anti-b-tubulin III, anti-rabbit
IgG, biotinylated anti-goat and anti-mouse IgGs and peroxidase anti-peroxidase complex were obtained from Sigma.
Biotinylated anti-rabbit IgG, streptavidin-biotinylated
horseradish peroxidase complex and reagents for the detection of GFAP and vimentin by chemiluminescence came
from Amersham International.
2.2. Surgery
Male Wistar rats Ž270–320 g. were used. They were
maintained in a ventilated room at constant temperature
with free access to diet and water. All animal procedures
followed NIH guidelines and were approved by the local
authorities. KA Ž0.2 m l of a 5 mM solution neutralized
with NaOH. was injected stereotaxically during 10 min
under deep thiopental anaesthesia with the following coordinates according to Paxinos and Watson w57x: bregma,
y4.3 mm; left lateral, 3.0 mm; depth, 2.6 mm from dura
ŽFig. 1.. The contralateral hippocampus was injected with
0.9% NaCl using the same coordinates.
2.3. Histology and immunocytochemistry
Animals were deeply anaesthetized with thiopental and
perfused through the heart with 100 ml of 0.9% NaCl
followed by 400 ml of 10% formol saline. The brains were
removed and after further fixation, dehydration and embedding coronal sections Ž10 m m. were cut and stained
with haematoxylinreosin.
For immunocytochemistry tissue sections were pre-incubated in 3% H 2 O 2 in 10% methanol to inactivate endogenous peroxidases and then in 3% normal goat serum
for 1 h to block non-specific binding sites. After washing
with phosphate-buffered saline containing 0.4% Triton X100 ŽPBS-Triton. the sections were incubated at 48C for 48
h with polyclonal 1:200 anti-GFAP or 1:400 monoclonal
anti-b-tubulin III, followed by 1:50 anti-rabbit IgG ŽGFAP.
Fig. 1. Diagram based on Paxinos and Watson w57x at y4.3 mm from
bregma showing the location of the needle tip and the area of tissue
analysed. Transverse hippocampal slices were cut across the lesion with a
McIlwain chopper and microslices Ž1 mm diameter. punched out from the
CA1 area as close as possible to the centre of the lesion adjacent to the
site of injection as indicated in the diagram.
190
G. Lenz et al.r Brain Research 764 (1997) 188–196
or 1:200 biotinylated anti-mouse IgG Ž b-tubulin. for 2 h at
room temperature. The sections were then treated for 1.5 h
with 1:500 peroxidase anti-peroxidase ŽSigma. to detect
GFAP or 1:150 streptavidin-biotinylated horseradish peroxidase ŽAmersham. to detect b-tubulin. Between each
step sections were washed with PBS-Triton. Colour was
then developed with diaminobenzidine and H 2 O 2 .
2.4. Preparation and labelling of slices
tin Ž1:400., secondary antibodies Ž1:500 biotinylated antirabbit IgG for GFAP or 1:400 biotinylated anti-goat IgG
for vimentin. and streptavidin-biotinylated horseradish peroxidase complex.
2.7. Quantitation and statistical analysis
Assessment of in vitro phosphorylation rate was made
by densitometric scanning of the autoradiographs as described previously w56x, except that peak areas rather than
Slices were prepared and labelled as described previously w56x. Briefly, rats were killed by decapitation 1, 4, 7,
14, 28 and 84 days after surgery. Hippocampi were dissected on ice within 3 min and transverse slices Ž0.4 mm.
were prepared with a McIlwain chopper. Microslices Ždiameter 1 mm. were then punched out from the CA1 area
adjacent to the site of injection Žsee Fig. 1.. The basic
incubation medium contained ŽmM.: NaCl, 124; KCl, 4;
MgSO4 , 1.2; Na-HEPES ŽpH 7.4., 25; glucose, 12; CaCl 2 ,
1 and was gassed with O 2 . Two microslices obtained from
adjacent slices were preincubated in 100 m l of the basic
medium for 1 h and then with 50 m l of medium containing
60 m Ci of w 32 Pxorthophosphate for 1 h at 308C. The
reaction was stopped with 1 ml of 10% trichloroacetic acid
ŽTCA.. After a minimum of 10 min in ice slices were
washed twice by decantation with 4% TCA to remove
excess radioactivity, briefly with water to remove acid and
then immediately dissolved in a sample solution of 9.2 M
urea, 12.5 mM lysine, 0.2% SDS and 4% Nonidet-P-40.
An aliquot was then taken for determination of protein and
2-mercaptoethanol was added to a final concentration of
2%.
2.5. Two-dimensional electrophoresis
The samples were analysed by non-equilibrium pH
gradient electrophoresis ŽNEPHGE. for the first dimension
and SDS-PAGE slab gels for the second dimension as
described previously w56x. Ten m g of protein from each
sample were applied to first dimension rod gels. After
NEPHGE, a pair of rod gels derived from the saline-injected control sample and the KA sample of the same
animal, were mounted on one second dimension slab gel
Ž9%T or 13%T acrylamide.. Gels were dried and exposed
to X-ray films at y708C with intensifying screens.
2.6. Immunoblotting for GFAP and Õimentin
Gel pieces containing GFAP and vimentin Ž2 = 2 cm.
were electroblotted onto nitrocellulose membranes using a
semi-dry transfer cell ŽTrans-blot RD, BioRad.. Proteins
were immunodetected with polyclonal antibodies and
chemiluminescence using Kodak X-Omat X-ray film essentially as described previously w20x. Briefly, membranes
were blocked overnight with 5% powdered milk and then
treated in sequence with anti-GFAP Ž1:500. or anti-vimen-
Fig. 2. Sections of lesioned hippocam pus stained w ith
haematoxylinreosin. Ž1. Saline-injected, 1 day; Ž2. KA-injected, 1 day;
Ž3. KA-injected, 28 days. p, pyramidal cell layer of CA1; r, str. radiatum;
lm, str. lacunosum moleculare; m, molecular layer of the dentate gyrus; h,
hilus. Note the increased density of cells in the stratum lacunosum
moleculare 1 day post injection; these cells are probably microglia, since
they did not react with an antibody to GFAP Žsee Fig. 3.. At 28 days
neurones in the pyramidal cell layer have disappeared and a marked
increase can be seen in the number of cells shown in Fig. 3 to react with
anti-GFAP. Scale bar s 200 m m.
G. Lenz et al.r Brain Research 764 (1997) 188–196
peak heights of the GFAP spots were measured. Briefly,
on each autoradiograph, representing a saline-injected control sample and a KA sample, the areas of the control
peaks were normalized to 100% and the percentage difference from the KA peaks calculated. To quantify immunodetectable GFAP, the X-ray films obtained from the
chemiluminescence procedure were scanned and analysed
by the same method. Data were plotted as logarithms of
the percentages Žsee Fig. 5.. Significance was calculated
using a paired t-test, values from each gel being treated as
one pair.
191
3. Results
3.1. Histology and immunocytochemistry
To assess the extent of neuronal damage after injection
of KA, sections across the lesion were stained with haematoxylinreosin. One day after injection the pyramidal cell
bodies in the CA1 region were mainly affected ŽFig. 2..
Many of these neurones had pycnotic nuclei, while others
exhibited intensely eosinophilic cytoplasm with strongly
basophilic eccentric nuclei. Only a few neurones with
Fig. 3. Immunostaining for GFAP in the lesioned hippocampus using a polyclonal antibody. Panels 1 and 2: saline-injected, 1 day; panels 3 and 4:
KA-injected, 1 day; panels 5 and 6: KA-injected, 28 days. p, pyramidal cell layer of CA1; r, str. radiatum; lm, str. lacunosum moleculare; m, molecular
layer of the dentate gyrus ŽDG.; h, hilus. Note the great increase in GFAP-positive cells in the strata radiatum and lacunosum moleculare 28 days after
injection of KA. Arrows point to GFAP-positive cells. Scales bars s 200 m m Žin 1, 3 and 5.; s 50 m m Žin 2, 4 and 6.. Panels 2, 4 and 6 correspond to the
squares in 1, 3 and 5.
192
G. Lenz et al.r Brain Research 764 (1997) 188–196
normal appearance were seen. In the stratum lacunosum
moleculare an increase was observed in the number of
small cells with intensely staining nuclei ŽFig. 2.; these
cells are probably microglia since they did not react with
an antibody to GFAP ŽFig. 3..
After 28 days the pyramidal cells in CA1 had largely
disappeared and the remaining neurones in this layer were
shrunken. Neurones with condensed cytoplasm were present in the stratum radiatum. In the strata radiatum and
lacunosum moleculare there was an increase in the number
of cells shown by immunocytochemistry to be mainly
astrocytes Žsee Fig. 3..
Immunocytochemistry of a neurone-specific antibody to
b-tubulin provided further information on the extent of
neuronal cell death. At 4 days after injection only a few
neurones remained in the CA1 area and at 28 days the
lesion had spread to the entire hippocampus Ždata not
shown..
Immunocytochemistry using anti-GFAP at 1 day after
injection showed a slight fall in the number of immunoreactive GFAP-positive cells compared with the saline-injected hippocampus ŽFig. 3.. At 28 days the lesion exhibited numerous GFAP-positive cells in the strata radiatum
and lacunosum moleculare ŽFig. 3..
3.2. Immunocontent of GFAP and phosphorylation rate
The immunocontent and in vitro phosphorylation rate of
GFAP, in relation to the contralateral saline-injected con-
trol, decreased during the first 24 h after injection of KA,
returned to control levels by day 7 and then continued to
increase during the following 11 weeks ŽFigs. 4 and 5.. At
all intervals the relationship between immunocontent and
phosphorylation rate remained constant, except for a small
excess of phosphorylation at day 28 ŽFig. 5.. This excess
was statistically significant on a paired t-test Ž P - 0.05.,
but did not reach significance when the means were compared with a non-paired t-test.
Since it was possible that GFAP phosphorylation also
increased on the contralateral saline-injected hippocampus,
we compared phosphorylation rate in the saline-injected
side of KA-treated rats with the rate in saline-injected
hippocampi from untreated animals. There was a slight
tendency for contralateral GFAP phosphorylation to increase in the treated animals, but no consistent statistically
significant changes were observed Ž n s 6–7, t s 0.42..
Certain other changes in the pattern of protein phosphorylation occurred in the KA-injected hippocampus. Phosphovimentin appeared at day 4 and persisted in the majority of experiments until day 14; subsequently Žday 28. it
was only occasionally seen and it was consistently absent
at day 84 ŽFig. 4A and Table 1.. A similar profile was
exhibited by an unknown low Mr phosphoprotein Ždesignated pp25., except that in this case the protein occurred
more frequently than phosphovimentin and in three out of
11 experiments was present 1 day after lesioning ŽTable 1..
The observation that the appearance of these proteins was
not quite as consistent as the increase in GFAP immuno-
Fig. 4. Autoradiographs of gels prepared from the lesioned area at various intervals after KA injection. A: two-dimensional separations on 9% gels.
Arrowheads point to GFAP; the arrow points to pp25. v, phosphovimentin; 1, synapsin 1; 2, MARCKS. B: autoradiographs of 14% gels. Arrows point to
pp25; horizontal arrowheads indicate putative astrocytic phosphoproteins upregulated in the lesion; p, PEA15 Žan astrocytic protein w3,16x whose
expression was unchanged in the lesion.. Note that the control autoradiographs were derived from normal adult hippocampus and not from the
saline-injected hippocampi. The panel marked ‘culture’ in B was derived from a culture of primary astrocytes.
G. Lenz et al.r Brain Research 764 (1997) 188–196
193
immunocontent and phosphorylation rate increased in the
contralateral saline-injected hippocampus after ipsilateral
KA injection, in comparison with normal untreated hippocampus. However, both phosphovimentin and pp25 appeared in the contralateral hippocampus after injection of
KA: phosphovimentin between days 4 and 14 and pp25
between days 1 and 14 ŽTable 1.. The expression of pp25
on the saline-injected side was more consistent than that of
phosphovimentin and at day 1 this exceeded expression on
the KA-injected side.
Fig. 5. Changes in GFAP phosphorylation rate and immunocontent in the
lesioned area after KA injection expressed as logs of the percentage
increase over the control saline-injected hippocampus. Values significantly different from control by a paired t-test are marked as follows:
)
P - 0.05; ) ) P - 0.01. At 28 days the increase in phosphorylation rate
was only significantly different from the increase in immunocontent on a
paired t-test Ž P - 0.05..
4. Discussion
Taking the in vitro rate of phosphorylation of GFAP as
a rough measure of the phosphorylation state of the protein
in vivo, our results provide no evidence for a major change
in GFAP phosphorylation in the KA-lesioned hippocampus: with the exception of the small excess of phosphorylation rate over immunocontent at 28 days, the increase in
GFAP content after KA injection was accompanied by an
equivalent increase in associated protein kinase activity.
Hypophosphorylation of GFAP post lesion would suggest
that phosphorylation was not necessary for astrocytic hypertrophy; conversely, hyperphosphorylation might have
reflected an increased rate of turnover of the
assemblyrdisassembly cycle or a significant number of
dividing astrocytes. The small excess of phosphorylation at
28 days and the high density of immunoreactive astrocytes
at this interval ŽFig. 3. is consistent with astrocyte hyperplasia. Clear evidence for astrocytic cell division has been
reported in the lesioned hippocampus from double labelling studies with anti-GFAP and thymidine, for example
in the CA3 region consequent on neurodegeneration following an excitotoxic lesion in the amygdala w39x Žsee also
Ref. w31x..
The initial loss of GFAP immunocontent as shown by
reactivity Žsee Table 1. presumably reflected varying degrees of damage.
Pp25 is probably an astrocytic protein since its migration on 2-D gels corresponded exactly to a phosphoprotein
expressed in primary cultures of astrocytes Žafter 7 days.
prepared from neonatal hippocampus ŽFig. 4B.. Furthermore, both in the case of samples from lesioned tissue and
astrocyte cultures the protein tended to focus in two or
three spots and these co-migrated when a mixture of
samples was analysed Ždata not shown.. In addition the
phosphorylation rate of two low Mr phosphoproteins
Žhorizontal arrowheads in Fig. 4B., which were also present on autoradiographs prepared from astrocyte cultures,
was increased in the lesioned tissue. By contrast, the
phosphorylation rate of a protein which is probably the
astrocytic phosphoprotein PEA15 w3,13x was unchanged
after injection of KA ŽFig. 4B, designated ‘p’..
In the absence of an absolute measure of GFAP concentration we were unable to judge the extent to which its
Table 1
Semiquantitative temporal profile of the occurrence of phosphovimentin and pp25 after injection of kainic acid
Days after injection
1
S
Vimentin
n
KArS
pp25
n
KArS
Total N
0
0
4
7
28
84
S
KA
S
KA
S
KA
S
KA
S
KA
0
0
1.0
3
1.8
5
0.9
4
2.5
8
0.8
3
2.5
10
0.9
2
2.6
3
0
0
0
0
1.4
4
0
0
0
0
1.8
1.6
10
14
KA
1.0
3
1.4
7
2.7
2.0
7
1.2
7
3.3
1.7
8
0.8
8
3
1.8
11
1.0
2
0.63
1.5
1.4
2.2
1.4
11
9
10
11
9
7
Spot density on the autoradiographs was assessed visually by two observers on a scale of 0–4, where 0 s protein absent and 4 s protein strongly labelled.
The values cited are the average scores for the saline-injected ŽS. and kainic acid-injected hippocampus ŽKA.. N, total number of films analysed; n,
number of times the protein was present at each time interval. The ratio KArS is a measure of the relative occurrence of the proteins on the two sides of
the brain.
194
G. Lenz et al.r Brain Research 764 (1997) 188–196
immunoblotting ŽFig. 5. has also been observed by quantifying GFAP immunoreactivity histochemically in other
experimental models using the hippocampus – in the CA3
area after intraventricular KA w29x and in CA1 and CA3
areas after ischaemia w8,47x. Immunoblotting may be a
more sensitive method to detect changes in GFAP since
we observed only a small decrease in immunoreactive
GFAP cells by immunocytochemistry 1 day after KA
injection ŽFig. 3.. However, an early loss of GFAP is not a
consistent finding in excitotoxic lesions Že.g., Ref. w39x.
and its occurrence is presumably related to variations in
the experimental model. Interestingly, delayed loss of
GFAP immunoreactivity has been observed in the KA-lesioned thalamus w14x and in animals treated with glucocorticoids w44x or exposed to ammonia w42x. In the present
study the loss was probably partly due to a gliotoxic effect
of KA since astrocytes possess receptors for KA w11,26x,
and partly a consequence of astrocyte swelling induced by
neuronal cell death w41,47x.
The transient expression of the intermediate filament
protein vimentin Žas phosphovimentin, Fig. 4. found in the
present study has been observed by immunocytochemistry
in most models of gliosis w15,29,41,46,51x. Double labelling studies have shown that this expression occurs in
reactive astrocytes that are also GFAP-positive w7,27x. In
the normal adult rodent brain vimentin expression is restricted to specialized glia such as ependymal cells w52x
and the Bergmann glia of the cerebellum w6x and this has
led to the suggestion that vimentin may be a more specific
marker of gliosis than GFAP w41x. Moreover, since vimentin is the main intermediate filament protein in astrocytes during the first 10–12 postnatal days and is then
briefly co-expressed with GFAP before being replaced by
the latter w6,12,48,52x, it has been suggested that
vimentin-positive astrocytes may represent an immature
form about to enter the cell cycle w43x. This cannot be
concluded from our results and indeed other studies have
shown that most reactive astrocytes possess the complex
stellar morphology and other phenotypic features typical of
mature fibrous astrocytes w50x.
A significant finding in the present work was the very
early post-injection expression of the novel phosphoprotein
pp25 ŽTable 1.. This protein co-migrates with a prominent
phosphorylated product in primary cultures of astrocytes
labelled with w 32 Pxphosphate, but its identity is at present
unknown. While we have not excluded the expression of
pp25 in neurones and other cell types it is relevant to note
that unlike GFAP and vimentin Žbefore postnatal day 10.,
the protein was not detected on gels prepared from normal
hippocampal tissue Žsee Fig. 4B. or from other brain
regions Ždata not shown.. This fact and also the fact that it
was expressed earlier and more frequently than phosphovimentin suggests that pp25 could be a more sensitive
marker of KA-induced gliosis than vimentin. It is also
particularly interesting that the initial Ž1 day post injection.
expression of pp25 occurred more frequently in the con-
tralateral saline-injected hippocampus than on the lesioned
side ŽTable 1.. This is a surprising finding and is unlikely
to be related to neuronal cell death since direct injection of
KA into the hippocampus results in only a delayed death
of the majority of neurones on the contralateral hippocampus w32x. As well as pp25, increased expression of phosphovimentin was observed in the contralateral hippocampus, but this response was delayed a few days and occurred less frequently ŽTable 1.. Secondary glial responses
to lesions of the CNS have been frequently described
w21,39x, but to our knowledge the pp25 response is the first
example of a secondary response of a putative astrocytic
protein in an excitotoxic lesion exceeding the response in
the lesioned area and occurring within 1 day of lesioning.
Studies on the nature of pp25 are in progress.
Acknowledgements
Supported by the Brazilian agencies CNPq, FAPERGS,
FINEP and PROPESP and the European Commission
ŽC11) -CT94-0116.
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