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431
original article
Arq Bras Endocrinol Metab. 2013;57/6
Decreased astrocytic GFAP
expression in streptozotocin-
induced diabetes after gliotoxic
lesion in the rat brainstem
Expressão astrocitária diminuída de GFAP no diabetes induzido por
estreptozotocina após lesão gliotóxica no tronco encefálico de ratos
Eduardo Fernandes Bondan1,2, Maria de Fátima Monteiro Martins1,2,
Flávio Cesar Viani2
ABSTRACT
Objective:
The aim of this study was to evaluate the effect of diabetic hyperglycemia on as-
trocyte function, estimated by means of glial brillary acidic protein – GFAP – immunohisto-
chemical expression.
Materials and methods:
Adult male rats received a single intravenous
injection of streptozotocin (50 mg/kg) and were submitted 10 days later to a single injection of
10 microlitres 0.1% EB solution or 0.9% saline solution into the cisterna pontis. Ten microliters of
0.1% EB or 0.9% saline solution were also injected in non-diabetic rats. Animals were anesthe-
tized and perfused through the heart 15 and 31 days after EB or saline injection, and brainstem
sections were collected for ultrastructural analysis and GFAP immunohistochemical staining.
Results:
The GFAP brown-stained areas were evaluated by colorimetry using a computerized
image analysis system and the results have shown that diabetes hindered the increase of GFAP
astrocyte expression in the EB-injected group compared to non-diabetic animals. However, dia-
betes did not affect GFAP response in the saline-injected group or in control animals.
Conclu-
sion:
Streptozotocin-induced diabetic condition reduced astrocytic GFAP expression following
gliotoxic injury.
Arq Bras Endocrinol Metab. 2013;57(6):431-6
Keywords
Astrocytes; GFAP; central nervous system; diabetes mellitus; ethidium bromide
RESUMO
Objetivo:
O objetivo deste estudo foi avaliar o efeito da hiperglicemia na função astrocitária,
estimada pela expressão imuno-histoquímica da proteína glial brilar ácida – GFAP.
Materiais
e métodos:
Ratos machos adultos receberam uma injeção intravenosa única de estreptozoto-
cina (50 mg/kg) e foram submetidos, 10 dias após, à injeção de 10 microlitros de solução de BE
0,1% ou de salina 0,9% na cisterna pontina. Dez microlitros de BE 0,1% ou salina 0,9% foram
também injetados em ratos não diabéticos. Os animais foram anestesiados e perfundidos por
via intracardíaca aos 15 e 31 dias pós-injeção de BE ou salina, e amostras de tronco encefálico
foram coletadas para estudo ultraestrutural e análise imuno-histoquímica para a GFAP.
Resul-
tados:
Utilizando um sistema computadorizado de análise de imagens, os resultados das áreas
coradas em marrom pela GFAP, medidas por colorimetria, mostram que o diabetes reduziu o
aumento de expressão dessa proteína no grupo injetado com BE em comparação aos animais
não diabéticos, mas não alterou a resposta no grupo injetado com salina ou nos controles
diabéticos.
Conclusão:
O estado diabético induzido pela estreptozotocina reduziu a expressão
astrocitária de GFAP após dano gliotóxico.
Arq Bras Endocrinol Metab. 2013;57(6):431-6
Descritores
Astrócitos; GFAP; sistema nervoso central; diabetes melito; brometo de etídio
1 Post-Graduate Program in
Environmental and Experimental
Pathology, Universidade
Paulista, São Paulo, SP, Brazil
2 Veterinary Medicine Department,
Universidade Cruzeiro do
Sul, São Paulo, SP, Brazil
Correspondence to:
Eduardo Fernandes Bondan
Rua Caconde, 125/51
01425-011 – São Paulo, SP, Brasil
bondan@uol.com.br
Received on Jan/15/2013
Accepted on Apr/2/2013
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432
Arq Bras Endocrinol Metab. 2013;57/6
INTRODUCTION
It is widely described that ethidium bromide (EB)
injection in the white matter of the central nervous
system (CNS) acts like a gliotoxin, causing local oli-
godendroglial and astrocytic death, with consequent
demyelination (although naked axons remain preser-
ved), blood-brain barrier disruption, and Schwann
cell invasion due to the glia limitans breakdown (1-5).
Surviving astrocytes present vigorous reaction around
the injury site with increased immunorreactivity to
the specic cell marker, glial brillary acidic protein
(GFAP), and reexpression of vimentin (VIM) (5).
Hyperglycemia found in diabetes mellitus is known to
cause well-characterized morphological and functional
changes in peripheral neurons and Schwann cells (6).
Much less is known about the effects of hyperglycemia
on CNS cells, mainly on glia. It is recognized that dia-
betes exacerbates astrocytic (7,8) and neuronal (9,10)
damage induced by ischemia and reperfusion. On the
other hand, insulin treatment prevents diabetes-indu-
ced alterations in astrocyte glutamate uptake and re-
verts the decreased GFAP expression in rats at 4 and
8 weeks of diabetes duration (11). Glial modications
were clearly pointed out in some studies (12,13) using
streptozotocin-diabetic rats after the injection of EB,
with marked delay on macrophagic scavenging activi-
ty of myelin debris, on oligodendrocyte and Schwann
cell remyelination (12), as well as on blood-brain bar-
rier repair (13), although astrocytic response was not
properly investigated and compared between diabetic
and non-diabetic animals. In such context, the aim of
the present investigation was to evaluate the effect of
diabetic hyperglycemia on astrocyte function (estima-
ted by means of GFAP immunohistochemical expres-
sion) in rats injected or not with EB in the brainstem,
serving as normal homeostatic regulators in the neural
microenvironment or as reactive and repairing cells af-
ter injury.
MATERIALS AND METHODS
This experiment was approved by the Ethics Com-
mission of Universidade Paulista (protocol number
002/09). Adult male Wistar rats, 3 to 4 months old,
were used, from which some received, after 12 hours of
fasting, a single injection of streptozotocin (50 mg/kg,
Sigma) in 0.01M citrate buffer (pH 4.5) into the tail
vein. Ten days after that, blood glucose was measured
and animals with levels of 300 mg/dL or more were
considered diabetic. At this time, they were submitted
to a local injection of 10 microlitres of 0.1% EB (group
I) or 0.9% saline (group II) solution into the cisterna
pontis. All rats were anaesthetized with ketamine and
xylazine (5:1; 0.1 ml/100 g) and a burr hole was made
on the right side of the skull, 8 mm rostral to the fron-
toparietal suture.
Injections were performed freehand using a Hamil-
ton Syringe, tted with a 35o angled polished 26-gauge
needle into the cisterna pontis, an enlarged subarach-
noid space below the ventral surface of the pons. Non-
diabetic rats also received 10 microlitres of 0.1% EB
solution (group III) or 0.9% saline solution (group IV).
Diabetic (group V) and non-diabetic rats (group VI)
were also used without receiving any intracisternal in-
jection (control groups).
Body weight and blood glucose levels (Dextrostix,
Ames) were recorded at 3 different times – at the mo-
ment of the streptozotocin injection, 10 days after and
at the time of euthanasia. Water and food were given ad
libitum during the experimental period. All rats were
anaesthetized; some were submitted to intracardiac
perfusion with buffered 10% formaldehyde (for im-
munohistochemical purpose), and some with 4% glu-
taraldehyde in 0.1 M Sorensen phosphate buffer (pH
7.4) (for transmission electron microscopy study) 15
and 31 days after intracisternal injection or not. Thin
slices of the brainstem (pons and mesencephalon) were
collected and post-xed in 0.1% osmium tetroxide,
dehydrated with graded acetones and embedded in
Araldite 502 resin, following transitional stages in ace-
tone. Thick sections were stained with 0.25% alkaline
toluidine blue. Selected areas were trimmed, and thin
sections were stained with 2% uranyl and lead acetate
and viewed in a JEM -1200 EX2 JEOL transmission
electron microscope.
Immunohistochemical protocol was initiated by
the deparafnization of histologic sections in xylene
and re-hydration in alcohol. Endogenous peroxidase
was blocked at room temperature with specic blocker
Dako S2001; the material was then incubated for 30
minutes (PBS-BSA 5%) to block unspecic proteins.
Two washes with PBS were carried out between all
incubations. An enzymatic process of antigenic reacti-
vation was used to demonstrate astrocytes, employing
pronase (S2013, Dako) during 15 minutes, followed
by the application of rabbit anti-cow GFAP antibody
(ZO334, Dako), diluted at 1:1000, for 16 hours. Sec-
GFAP expression in diabetic rats
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433
Arq Bras Endocrinol Metab. 2013;57/6
ondary goat anti-rabbit antibody (E0433, Dako) was
used as the binding antibody diluted at 1:100 for 30
minutes. The material was revealed with DAB (diami-
nobenzidine) for 5 minutes and counterstained with
Harris hematoxylin 1:2.
Astrocytic evaluation was done at the 31st day in
groups I, II, III and IV using a computerized image
analysis system (Image-Pro-Plus 4.5, Media Cybernet-
ics, Silver Spring, USA), measuring by colorimetry the
area stained in brown in a total area of 302,952.5 µm2,
chosen from the lesion edge, where astrocytic reaction
occurred. Negative controls for immunostaining (sec-
tions lacking primary antibody application) were done.
Data were analyzed by t test and statistical signicance
was set at p < 0.05.
RESULTS
The EB-induced lesions were similar to those previou-
sly described in the brainstem of diabetic (12,13) and
non-diabetic rats (2,5). In general terms, they were
characterized by demyelinated areas in the ventral sur-
face of the pons and mesencephalon containing, in the
central region, phagocytic cells, some myelin-derived
membranes in a distended extracellular space, as well
as naked axons. At the periphery, the presence of oli-
godendrocytes and Schwann cells was noted, the latter
occurring in areas of an enlarged extracellular space
devoid of astrocytic prolongments, notably around
blood vessels and in subpial areas. Astrocyte processes
were invariably seen near the incipient, but preponde-
rant, oligodendroglial remyelination (Figure 1A and
B), and Schwann cells also appeared to contribute to
myelin repair. Ultrastructural analysis apparently sho-
wed that astrocytic processes among oligodendrocyte
remyelinated axons were slightly thinner in diabetic
animals compared with non-diabetic ones. Although
oligodendroglia prevailed in the brainstem myelin re-
pair from the 15th to the 31st day, sheaths formed by
Schwann cells in astrocyte-free areas were thicker than
those produced by oligodendrocytes during the same
period. Lymphocytes and inltrating pial cells were
also observed, the rst contacting phagocytic cells and
myelin debris.
All rats submitted to streptozotocin injection pre-
sented hyperglycemia (levels from 300 to 650 mg/
dL) at the 10th day and at perfusion day. During the
experimental period they developed characteristic
polyuria, polydipsia, and weight loss (body weight
data are shown in Table 1). As previously described
(12,13), diabetic rats from group I presented delayed
macrophage activity at the 15th and 31st day after EB
injection, as shown by the nding of huge amounts of
myelin-derived membranes in the extracellular space,
and a lesser extent of remyelination by both oligoden-
drocytes and Schwann cells at the edges of the lesions
in comparison with non-diabetic rats from group III.
A greater proportion of axons persisted without myelin
and remyelinated ones clearly presented thinner my-
elin sheaths.
Figure 1. EB-induced lesions in groups I (A, diabetic) and III (B, non-diabetic) 15 days post-injection. Note the presence of axons in initial oligodendroglial
remyelination (R) among astrocytic processes (a). Note in B some hypertrophic astrocyte prolongments (a) with greater bundles of intermediate laments.
Electron micrographs - A) Bar = 2 µm; B) Bar = 1 µm.
AB
GFAP expression in diabetic rats
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Arq Bras Endocrinol Metab. 2013;57/6
In saline-injected rats from groups II (diabetic)
and IV (non-diabetic), mild lesions circumscribed to
the pons and along the needle track were detected in
just one animal from each group at day 15 post-injec-
tion, probably due to the surgical procedure, and no
difference was noted between them. Ultrastructural
analysis of these two lesions showed a small and focal
expansion of the extracellular space, containing some
loose lamellae and few phagocytic macrophages. No
evidence of primary demyelination or loss of neuroglia
was found.
EB-induced lesions presented increased astrocyte re-
action close to the edges of the injury site, expressed by
the nding of thickened and strongly brown-stained as-
trocytic processes 15 and 31 days post-injection (Figu re
2A and B), although no astrocytes were observed in the
central areas of the lesions. Astrocytic immunoreactivity
following saline injection was very discrete.
In both diabetic and non-diabetic groups, GFAP-
stained areas at 31 days were signicantly greater in EB-
injected rats than in saline-injected or control animals
(Table 2), but were smaller in the diabetic rats (group I,
41669.63 ± 7204.08) in comparison with non-diabetic
AB
Table 1. Body weight of the animals in the experimental groups
Diabetic groups Non-diabetic groups
I-EB injection II-Saline injection V-Control III-EB injection IV-Saline injection VI-Control
Day -10
BW (g)
(16)
271.4 ± 8,7
(16)
268.4 ± 5.9
(16)
276.5 ± 7.7
(16)
258.8 ± 4.9
(16)
265.6 ± 3.7
(16)
278.8 ± 3.4
Day 0
BW (g)
(16)
268.2 ± 6.4
(16)
267.6 ± 3.1
(16)
274.5 ± 3.6
(16)
264.2 ± 4.1
(16)
278.3 ± 2.8
(16)
285.5 ± 2.9
Day 15
BW (g)
(16)
256.3 ± 7.2
(16)
259.1 ± 6.1
(16)
246.3 ± 5.5
(16)
266.6 ± 5.9
(16)
286.3 ± 4.4
(16)
289.1 ± 7.8
Day 31
BW (g)
(8)
238.4 ± 8.8
(8)
246.8 ± 8.2
(8)
239.3 ± 9.3
(8)
317.7 ± 6.1
(8)
328.5 ± 4.5
(8)
325.3 ± 3.2
BW: body weight; Day-10: day of streptozotocin injection in groups I, II and III; Day 0: day of EB administration in groups I and III or day of saline administration in groups II and IV.
Data are presented as means ± standard deviations (SD) for the number of rats given in parenthesis.
Figure 2. GFAP immunohistochemical expression at 31 days in EB-induced lesions from diabetic (A) and non-diabetic rats (B). A and B) Bar = 50 µm.
GFAP expression in diabetic rats
ones (III, 55354.38 ± 5825.37; p = 0.001). As for dia-
betic rats compared with non-diabetic, no difference
was found between control animals (groups V and VI)
and saline-injected rats (groups II and IV), nor for dia-
betic groups II and V and non-diabetic groups IV and
VI, with rats injected with saline solution or not.
DISCUSSION
Astrocytes play a key role in CNS homeostasis, inclu-
ding maintenance of the blood-brain barrier, neuropro-
tection from reactive oxygen species, regulation of neu-
ronal activity and synaptic transmission, energy supply,
as well as control of extracellular pH and ion and neu-
rotransmitter concentrations, among many other func-
tions (14,15).
Intermediate laments of astrocytes are composed
mainly of GFAP, and this protein has become the best
known astrocytic marker (15-17). Intermediate la-
ments are one of the three components of cytoskeleton
and the term “intermediate” reects their thickness
(about 10 nm) which falls between the thickness of the
other two cytoskeletal components – actin laments
Copyright© ABE&M todos os direitos reservados.
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Arq Bras Endocrinol Metab. 2013;57/6
(about 6 nm) and microtubules (about 23 nm). In
contrast to microtubules and actin laments, the com-
position of intermediate laments changes among cell
types, their developmental stages, and functional sta-
tus (16). Astrocyte precursors and immature astrocytes
present principally nestin and vimentin and, as astro-
cytes mature, nestin expression disappears, GFAP be-
comes increasingly expressed, and vimentin decreases
to undetectable levels (15).
Insults to the CNS, such as trauma, ischemia, tu-
mors, neuroinammation, and neurodegenerative
disorders lead to astrocytic activation, also known as
reactive gliosis or astrogliosis, increasing the produc-
tion of intermediate laments. In such conditions, re-
active astrocytes become highly positive for GFAP and
vimentin, also reexpressing the third lament protein,
nestin (14-16). Reexpression of vimentin and strong
astrocytic immunoreactivity to GFAP were clearly seen
by Bondan and cols. (5) following EB injection in the
rat brainstem from the 3rd to the 31st day post-gliotoxic
injection.
This increased GFAP expression around the EB-in-
duced lesions was also conrmed in the present study,
but it was noted in the streptozotocin-diabetic rats that
somehow diabetes hindered the increase in this expres-
sion, although the same was not observed after saline
solution injection. Besides, diabetic rats that did not re-
ceive any intracisternal injection (EB or saline; control
group) had no signicant difference in GFAP expres-
sion from non-diabetic ones, suggesting that such dif-
ference caused by the diabetic status was only detected
when a strong glial response to injury was induced (5).
Meanwhile, decreased astrocyte GFAP expression in
type 1 diabetic rats was also found by other researchers
with no additional harmful condition beyond diabetes
(11,18-22). On the other hand, insulin treatment has
shown to prevent diabetes-induced decreases in astro-
cytic GFAP content (11)
Although astrocytes were not individually counted
in our study, the decrease in GFAP content seen in
diabetic rats apparently reected a decrease in GFAP
expression rather than a decrease in the number of as-
trocytes. This observation is similar to that of Coleman
and cols. (11), and differs from Lechuga-Sancho and
cols. (21), who reported a decrease in rat hypothalamic
astrocyte numbers after 6 weeks of diabetes onset. It is
recognized that astrocyte counts based on quantica-
tions of GFAP-positive cells are not really representa-
tive, as diminution of GFAP immunoreactivity could
lead to undercounting.
While it was initially thought that astrocyte pro-
liferation was a major component of glial scar, it has
been demonstrated repeatedly that there are actually
few astrocytes undergoing cell division during gliosis
(17). To corroborate this afrmation it is important to
notice that no astrocyte in mitotic activity was seen in
our studies, with astrocytic response following gliotoxic
lesions (4,5).
The association of reactive astrocytes with enhanced
GFAP and cellular hypertrophy, coupled with the in vi-
tro observations that mature astrocytes do not represent
a supportive environment for axon growth, has led to
a widespread concept that reactive astrocytes are always
detrimental to regeneration in the CNS. However, it
Table 2. Areas with GFAP staining in µm2 in a total area of 302,952.5 µm2 at 31 days, in rats diabetic or not, injected with EB or not
Animal
Diabetic groups Non-diabetic groups
I-EB injection
(µm2)
II-Saline injection
(µm2)
V-Control
(µm2)
III-EB injection
(µm2)
IV-Saline injection
(µm2)
VI-Control
(µm2)
1 50,241 5,834 9,313 47,281 6,132 6,249
2 60,312 10,105 8,131 39,522 5,041 8,833
3 48,154 4,581 5,121 51,430 4,190 5,214
4 53,826 6,912 4,297 40,265 7,013 5,575
5 57,115 6,134 5,642 34,127 7,264 7,266
6 61,232 7,283 6,715 36,112 6,145 4,127
7 62,841 8,145 4,237 50,717 4,827 4,483
8 49,114 4,905 8,541 33,903 8,549 9,028
Mean 55,354.38a6,737.38c6,499.6c41,669.63b6,145.13c6,346.88c
standard deviation (SD) ±5,825.36 ±1,806.27 ±1,978.4 ±7,204.08 ±1,442.37 ±1,871.01
Distinct letters indicate signicant differences (p < 0.05).
GFAP expression in diabetic rats
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Arq Bras Endocrinol Metab. 2013;57/6
has been proved in several models of brain and spinal
cord injury that not all reactive astrocytes produce non-
permissive molecules for neural regeneration, such as
tenascin and condroitin sulfate family of proteoglycans,
which are inhibitory for neurite outgrowth (17). In op-
position to this proteoglycan up-regulation associated
with regenerative failure in vivo, reactive astrocytes also
produce molecules that can support regeneration, such
as laminin (17). In addition, astrocytes are recognized
to support oligodendrocytes during myelination and
remyelination (15), and this role is particularly impor-
tant after myelin loss due to naturally occurring or ex-
perimentally induced demyelinating processes, such as
the EB gliotoxic model.
The reaction of astrocytes following trauma form-
ing a glial scar surrounding the area of lesion could wall
off the damaged area in an attempt to isolate the injury
site and prevent any further damage to the nearby tis-
sue, which is still viable.
Decreases in GFAP expression are invariably asso-
ciated with detrimental conditions in the CNS (23)
and this was also expected in relation to diabetes. Due
to the complex scenario of CNS repair, the ambiguous
roles of astrocytes in nervous tissue remodeling after
injury, and as diabetes apparently negatively affects as-
trocyte reaction, it is difcult to precisely infer if this
impaired astrocytic response would play a benecial
or a deleterious inuence on the restoration of mor-
phological and functional tissue integrity after neural
damage.
Acknowledgements: this study was supported by São Paulo Rese-
arch Foundation (Fapesp – 2008/58696-2) and National Coun-
sel of Technological and Scientic Development (CNPq).
Disclosure: no potential conict of interest relevant to this article
was reported.
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GFAP expression in diabetic rats
