Aortic coarctation hypertension induces fibroblast growth factor-2 immunoreactivity in the stimulated nucleus tractus solitarii

ORIGINAL PAPER

Aortic coarctation hypertension induces fibroblast growth factor-2immunoreactivity in the stimulated nucleus tractus solitarii

Debora Rejane Fior-Chadi ÆTatiana Cristina Nogueira Varella ÆJessica Ruivo Maximino Æ Gerson Chadi

Received: 28 February 2007 / Accepted: 16 May 2007 / Published online: 12 June 2007

� Springer Science+Business Media B.V. 2007

Abstract The actions of neurotrophic factors i.e. basic

fibroblast growth factor (bFGF, FGF-2) to neurons are

related not only to neuronal development and maintenance

but also to synaptic plasticity regarding neurotransmission.

We analyzed here the levels of FGF-2 immunoreactivity in

the nucleus tractus solitarii (NTS) of Wistar Kyoto rats in

response to alterations of neuronal activity promoted by the

stimulation of the baroreceptor reflex following an aortic

coarctation-induced-hypertension. The FGF-2 immunore-

activity (IR) was found in the cytoplasm of the neurons and

in the nuclei of the glial cells in the NTS. A large number of

NTS neurons expressed FOS immunoreactivity 4 h after

coarctation, as an indication of neuronal activity. Stereol-

ogical methods showed an increased number of FGF-2

immunoreactive (ir) neuronal profiles (90%) and glial

profiles (149%) in the NTS of the 72 h aortic coarctated

rats. 1-week later, FGF-2 ir neurons were still increased

(54%) but no change was found in the number of FGF-2 ir

glial profiles. The double immunoperoxidase method

revealed that the majority of the FGF-2 ir glial cells was

glial fibrillary acidic protein (GFAP) positive astrocytes.

GFAP immunohistochemistry showed an astroglial reaction

at 72 h time-interval (55%) but not 1 week after stimula-

tion. The number of the cresyl violet positive neurons and

OX42 ir profiles (marker of activated microglia) in the NTS

of coarctated rats were not different from control by 1 week

and 1 month after the surgery, indicating a lack of NTS

injury in this period following coarctation hypertension.

FGF-2 may be an important neurotrophic factor in areas

involved in the control of blood pressure. The increased

FGF-2 IR in the NTS cells following neuronal stimulation

may represent trophic and plastic adaptive responses in this

nucleus in an autocrine/paracrine fashion.

Keywords Fibroblast growth factor-2 �Coarctation hypertension � Nucleus tractus solitarii �Immunohistochemistry � Astrocytes � Neuronal stimulation

Introduction

Basic fibroblast growth factor (bFGF, FGF-2) is a mitogenic

protein with actions on multiple cell types such as fibro-

blasts, nerve cells and glial cells (Baird and Klagsbrun

1991). FGF-2 has also been described as a potent angio-

genic factor (Gospodarowicz 1990; Puumala et al. 1990).

FGF-2 plays a role in the survival and plasticity of neurons

in many brain regions of adult rats (Walicke 1988; Chadi

et al. 1993a).

Much has been learned about the trophic properties of

FGF-2 on neuronal survival from in vitro and in vivo studies,

however the role of this growth factor in response to altera-

tions of neuronal activity has been poorly investigated. It has

been suggested that the expression of FGF-2 in the brain of

adult animals changes in response to an excessive neuronal

excitation as observed following electroconvulsive shock

(Follesa et al. 1994) and after convulsive seizures (Riva et al.

1992) or after physical exercise (Gomez-Pinilla et al. 1997).

Several peptide growth factors have been postulated to be

important in the initiation and progression of vascular growth

D. R. Fior-Chadi � T. C. N. Varella

Department of Physiology, Institute of Biosciences,

University of Sao Paulo, Sao Paulo 05508-900, Brazil

J. R. Maximino � G. Chadi (&)

Department of Neurology, University of Sao Paulo School of

Medicine, Av. Dr. Arnaldo 455, 2nd Floor, Room 2119,

Sao Paulo 01246-903, Brazil

e-mail: [email protected]

123

J Mol Hist (2007) 38:285–294

DOI 10.1007/s10735-007-9101-x

following different types of experimental hypertension

(Saltis et al. 1993; Zettler and Rush 1993; Anwar and

Delafontaine 1994), however little information is available

on the involvement of neurotrophic factors in the central

control blood pressure areas.

The subdiaphragmatic aortic coarctation is an experi-

mental model used to induce a rapid and sustained hyper-

tension in rats (Krieger 1970; Michelini et al. 1992). When

arterial pressure raises there is an increased afferent activity

directed to the nucleus tractus solitarii (NTS) (Mifflin et al.

1988; Langhorst et al. 1992; Li and Dampney 1992; Rogers

et al. 1993; Li and Dampney 1994), the primary site of

integration of the baroreceptor reflex (Ciriello and Calaresu

1981; Spyer 1981; Ross et al. 1985; Chapleau et al. 1995).

The NTS, in turn, outputs to efferent neurons that provide the

parasympathetic (Loewy and Burton 1978; Nosaka et al.

1979) and sympathetic (Soato and Krieger 1974; Cravo et al.

1991; Rogers et al. 1993) innervations of the cardiovascular

system. The aortic coarctation model of induced hyperten-

sion was employed in this study since it promotes a moderate

elevation in the level of arterial pressure leading to neuronal

activation in the NTS without neuronal damage. Stroke and

expression of neurotrophic factors have been observed only

in ageing stroke-prone spontaneously hypertensive rats that

show mean arterial pressure around 240 mmHg (Lin et al.

1997) but not in other models of less severe inducible

hypertension.

In the present work the coarctation hypertension

approach was used to produce a sustained increase of neu-

ronal activity in the NTS, without neuronal damage, in

order to evaluate whether this activation may modify the

FGF-2 immunoreactivity in the NTS cells of the Wistar

Kyoto rats.

Methods

Animal treatment

Specific pathogen-free adult male Wistar Kyoto rats from

the University of Sao Paulo (USP) with body weight (b.w.)

of 250 g were used in the experiments. The animals were

kept under standardized lighting conditions (lights on at

7:00 a.m. and off at 7:00 p.m.), at a constant temperature

of 23�C and with free access to food pellets and tap water.

The rats were submitted to a sham-operation or to a sub-

diaphragmatic aortic coarctation as previously described

(Krieger 1970). Briefly, median laparotomy was performed

under chloral hydrate (7%, Merck, Germany, 350 mg/Kg/

b.w., i.p) anaesthesia and the abdominal aorta was isolated.

A cotton thread was used to constrict the aorta immediately

below the diaphragm and the extent of narrowing was

limited by a hypodermic needle of 0.7 or 0.8 mm in

diameter depending on rat weight. In the sham-operation

the aorta was exposed and isolated in an identical manner

but it was not tied. The animals were sacrificed 72 h

(n = 11), 1 week (n = 8) and 1 month (n = 9) after the

surgery. In the day before killing, the rats from groups of

72 h and 1 week were submitted to arterial pressure

recording. Rats were then anaesthetized with ether (Sigma,

USA) and a tygon catheter filled with heparinized (500 UI/

ml, Liquemine, Roche) saline was placed in the ascending

aorta through the brachial artery. The other end of the

cannula was slipped beneath the skin and exteriorized on

the back of the neck for arterial pressure recording. After a

20 min stabilization period, the arterial pressure was re-

corded during 15 min in the freely moving rats by means of

a transducer connected to a Recorder (Narco Bio-Systems,

USA). Finally, a set of sham-operated (n = 5) and aortic-

coarctated (n = 5) rats were sacrificed 4 h after surgery in

order to evaluate FOS protein expression in the NTS

neuronal cells, as a marker of neuronal activity in this

nucleus. Those animals were not submitted to arterial

pressure recording.

Immunohistochemical procedures

After blood pressure recording the rats were deeply

anaesthetized with chloral hydrate 10% (420 mg/kg, b.w.,

i.p.) and killed by a perfusion through a cannula inserted in

the ascending aorta with 50 ml of isotonic saline at room

temperature followed by 350 ml of fixation fluid (4�C)

during 6 min. The fixative (Zamboni and De Martino 1969)

consisted of 4% (w/v) paraformaldehyde and 0.2% (v/v)

picric acid in 0.1 M phosphate buffer (pH 6.9). The brains

were dissected out and kept in the fixative solution for

90 min. The fixed brains were washed in 10% sucrose

dissolved in 0.1 M phosphate buffered saline (PBS) for

2 days, frozen in dry ice and stored at –70�C freezer.

Coronal adjacent serial 50 lm thick sections were obtained

in a cryostat (Leica, CM 3000, Germany) from the NTS

region, from rostro-caudal level –13.40 mm to –14.30 mm

according to the atlas of Paxinos and Watson (1986). The

sections were sampled systematically during sectioning.

Five series in a rostro-caudal order including every fifth

section were taken to immunohistochemistry.

The immunoreactivities were detected using avidin-

biotin peroxidase technique (Hsu et al. 1981). Floating

sections were washed 2 · 10 min in PBS (0.1 M, pH 7.4)

at room temperature and incubated with normal goat serum

(NGS) 5% for 30 min at room temperature under shaking.

Following, the sections from the series were incubated for

48 h at 4�C under shaking with the rabbit polyclonal FGF-

2 antiserum (Gonzalez et al. 1990) (diluted 1:800), a rabbit

policlonal glial fibrillary acidic protein (GFAP, a marker

for astrocytes, Dakopatts, Denmark, diluted 1:200) or also

286 J Mol Hist (2007) 38:285–294

123

with a mouse monoclonal antibody against the OX42 (a

marker for microglia, Harlan, England, diluted 1:600). The

GFAP and OX42 immunoreactivities were counterstained

with cresyl violet to allow interalia the demonstration of

the nuclei in the astrocytes and microglial profiles as well

as the nucleoli and the Nissl substance in the neuronal

profiles. Sections from rats sacrificed 4 h after surgery

were incubated with a sheep polyclonal anti-FOS antibody

(Affinity, UK) as described above. The antibodies were

diluted in PBS containing 0.5% Triton X-100 (Sigma) and

1% bovine serum albumin (Sigma). The detection of the

antibodies was achieved by the indirect immunoperoxidase

method (ABC) (Hsu et al. 1981) using the avidin-biotin

peroxidase technique as previously described (Chadi et al.

1993a, b). After washing in PBS (3 · 10 min), the sections

were incubated with a biotinylated goat anti-rabbit, or

horse anti-mouse or rabbit anti-goat immunoglobulins

(diluted 1:200, Vector, USA) for 1 h. In a third step the

sections were washed in PBS and incubated with avidin-

biotin peroxidase complex (both diluted 1:100, Vectastain,

Vector) during 45 min. The staining was performed using

0.03% of 3, 3¢ diaminobenzidine tetrahydrochloride (DAB,

Sigma) as a chromogen and 0.05% (v/v) of H2O2 (Sigma)

during 8 min, which gave a brownish colour to the

immunoreaction. For standardization of the immunohisto-

chemical procedure we have used a dilution of the primary

antibody and a concentration of DAB far from saturation

and an incubation time adjusted so that the darkest

elements in the brain sections were below saturation (Zoli

et al. 1990). The FGF-2 antiserum used is a well charac-

terized polyclonal antiserum raised against the n terminal

(residues 1–24) of the synthetic peptide of bovine FGF-2

(1–146) (Gonzalez et al. 1990). This antiserum does not

recognize acidic FGF (FGF-1, cross reactivity less than

1%) (Gonzalez et al. 1990). The OX42 antibody employed

in the present analysis recognizes the complement receptor

type 3 (CR3), an immunologically important surface mol-

ecule of mononuclear phagocytes and it is considered a

well established tool to label resting and activated micro-

glia in the central nervous system (Ling et al. 1990). As

control, sections were incubated overnight at 4�C with the

FGF-2 antiserum pre-incubated with human recombinant

FGF-2 (50 lg/ml, for 24 h at 4�C). To further analyze the

specificity of the immunostainings, sections were incubated

with the solvent of the primary and secondary antibodies or

with the solvent of the avidin-biotin solution and processed

at the same time with the experimental sections.

Double immunolabeling

The two-colour immunoperoxidase method was employed

in a series of sections for the simultaneously detection of

the FGF-2 and the GFAP immunoreactivities. The FGF-2

immunoreactivity was firstly demonstrated as described

above. Following the DAB reaction, the sections were

rinsed several times in PBS and incubated during 48 h in a

humidified chamber with the rabbit polyclonal antiserum

against GFAP, as described above (1:400). After several

rinses in PBS, the sections were incubated with biotiny-

lated goat anti-rabbit immunoglobulins (1:200, Vector) for

1 h at room temperature and with avidin-biotin peroxidase

couplex (both diluted 1:100, Vectastain, Vector) for

45 min at room temperature. The staining was performed

using 4-chloro naphtol 0.05% (Sigma) as a chromogen and

0.05% (v/v) of H2O2 (Sigma) during 10 min. This proce-

dure gave brownish and bluish colours to the FGF-2 and

GFAP immunoreactions, respectively.

Quantitative stereological analysis

The FGF-2, GFAP and OX42 immunoreactive cell profiles

and the neurons stained by cresil violet were quantified by

stereological analysis. The optical fractionator (West and

Gundersen 1990; Chadi et al. 1993a; Janson and Moller

1993) was used to estimate the number of FGF-2, GFAP

and OX42 immunoreactive profiles in the sampled volume

of the NTS, bilaterally.

As described, every fifth section was systematically

sampled (f1 = 5). The series of sections immunostained to

the above described antisera were analyzed using a CAST-

system (Computer Assisted Stereological Toolbox). Briefly:

an Olympus BX50 microscope (Olympus, Denmark) was

interfaced with a computer (IBM 330-P75, USA) and a

colour video camera (JAL 2040, Protec, Japan), both linked

to a colour video monitor (G70, IBM). The GRID software

package (Interactivision, Silkeborg, Denmark) was used to

generate sampling (counting) frames as an overlay image to

the microscopic image on the monitor as well as to control

the motorized X-Y stage (Lang, Huttenberg, FRG). A

microcator (MT12, Heidenhain, FRG) was linked to the

microscope to monitor movements in a vertical (Z) direction.

The border of a square, of an area of 75 lm2, representing the

sampled region to be quantified was outlined on both sides

of the NTS using a 4 · objective. The step rates were entered

(50 lm), after which the program created series of uniformly

sampled fields of vision throughout the entire delimitated

region. For counting the cells, a 100 · oil-immersion

objective with the numerical aperture 1.4 was used.

A counting frame (with a known area, aframe) was created by

the GRID software, and by knowing the step-rates it was

possible to calculate the second sampled fraction (f2):

(X step-length�Y step-length)/aframe). The sampling volume

(disector) in the Z-axis extended 9 lm deep (height of the

disector) after excluding the parts of the section close to the

slide and coverslip (3 lm). Also the total thickness of the

section was measured, giving the third sampling fraction (f3):

J Mol Hist (2007) 38:285–294 287

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height of the section/height of the disector. After having

counted all cells (P

Q–) fulfilling the criteria for being

sampled, the total number of cells (Ntotal) in the sampled

region was estimated: Ntotal =P

Q–�f1�f2�f3The coefficient of error (CE) for the estimated total

number of immunoreactive particles in the sample volume

in the NTS of each animal was calculated (Gundersen and

Jensen 1987; Pakkenberg and Gundersen 1988; West et al.

1991).

The cytoplasmatic and nuclear localization of the FGF-2

immunoreactivity (Chadi et al. 1993b; Cintra et al. 1991)

were taken into account in the discrimination of the neu-

ronal and glial FGF-2 cell profiles (see the results).

Statistical analysis

The statistical analysis was performed using the non-

parametric two-tailed Mann-Whitney U-test (Hollander

and Wolfe 1973). The numbers represent the mean ±

S.E.M.

Results

Blood pressure recording

The subdiaphragmatic aortic coarctation resulted in a sus-

tained moderate hypertensive response observed at 72 h

and 1 week following the surgery. 72-h after the surgery

the arterial pressure was 158 ± 2.4 mmHg in the coarctated

rats (n = 5) and 109 ± 6.9 mmHg in the sham operated rats

(n = 6) (P < 0.05), according to the Mann-Whitney

U-test). 1 week after the surgery the mean arterial pressure

was 156 ± 2.4 mmHg in the coarctated rats (n = 4) and

114 ± 1.9 mmHg in the sham operated rats (n = 4)

(P < 0.05, according to the Mann-Whitney U-test). Arterial

pressure was not recorded after 1 week since it is known

that the arterial pressure is maintained constant after this

period (Giuliano and Brezenoff 1987; also unpublished

observations of our laboratory).

Qualitative analysis of the FGF-2 immunoreactivity

The FGF-2 immunoreactivity was seen in the nucleus of

glial cells and in the cytoplasm of neurons in all extent of

the analyzed regions in the NTS (Fig. 1), area postrema,

dorsal motor nucleus of the vagus and other brain stem

nuclei. The FGF-2 immunoreactivity was also observed in

some fibres within the NTS (Fig. 1).

The aortic coarctation increased the intensity and den-

sity of the FGF-2 immunoreactive cell profiles in the NTS

of the 72 h (Fig. 1) and 1 week operated rats.

Quantitative analysis

The stereological method employing the optical fraction-

ator demonstrated an increased number of the cytoplasmic

FGF-2 immunoreactive neuronal profiles (90 ± 23% of the

control group, n = 5) and nuclear FGF-2 immunoreactive

glial profiles (149 ± 11% of the control group, n = 5) on

both sides of the NTS of the coarctated rats at the 72 h

time-interval (Fig. 2A). 1 week after the coarctation the

Fig. 1 Microphotographs of coronal sections showing the FGF-2

immunoreactivity in the nucleus tractus solitarii (NTS) of the sham-

operated (control) (A, B) and the 72 h aortic coarctated (C, D) rats. Band D represent higher magnification of areas depicted from the

nucleus tractus solitarii of A and C, respectively. The FGF-2

immunoreactivity is present in the cytoplasm of neurons (arrows)

and in the nuclei of glial cells (arrowheads) of the NTS. The FGF-2

immunoreactivity is also observed in some neuronal fibres in the NTS

(open arrow). The aortic coarctation increases the FGF-2 immuno-

reactivity in the neurons and glial cells 72 h after the surgery.

Bars = 100 lm (A, C) and 10 lm (B, D)

288 J Mol Hist (2007) 38:285–294

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number of FGF-2 immunoreactive neuronal profiles was

still increased (54 ± 11% of the control group, n = 4) on

both sides of the NTS (Fig. 2B). By this time no change in

the number of the FGF-2 immunoreactive glial profiles was

observed (Fig. 2B).

The GFAP immunohistochemistry showed an astroglial

reaction in the NTS 72 h after aortic coarctation. The

number of the GFAP immunoreactive profiles increased by

55 ± 5.8% in the NTS of the 72 h coarctated rats compared

to the control rats (n = 5) (Fig. 2). 1 week following the

coarctation hypertension no alteration in the number of

GFAP immunoreactive profiles was observed (Fig. 2). The

GFAP immunoreactive astroglial profiles are showen in

Fig. 3 (A–D).

In order to evaluate whether the hypertensive state has

injuried the NTS neurons we have quantified the OX42

immunoreactive profiles (Fig. 3E), a marker for microglia,

and also the number of neurons in this nucleus after a

period of 1 week and 1 month of coarctation hypertension.

No difference was observed either in the number of neu-

rons (111 ± 11% and 115 ± 18.8%, compared to the

respective control group (100%), 1 week and 1 month,

respectively) and in the OX42 immunoreactive profiles

(117 ± 13% and 118 ± 5% compared to the respective

control group 100%, 1 week and 1 month, respectively).

Co-distribution of FGF-2 and GFAP imunoreactivities

The two-colour immunoperoxidase procedures for simulta-

neous detection of the FGF-2 and GFAP immunoreactivities

revealed that the majority of the nuclear FGF-2 immuno-

reactive cell profiles were GFAP positive astrocytes at the

studied time-intervals (Fig. 3 A–C).

FOS protein immunoreactivity

The NTS of sham-operated rats showed only scattered FOS

positive profiles, however the majority of NTS neuronal

cells from aortic coarctated rats sacrificed 4 h after surgery

labeled for FOS immunoreactivity (Fig. 4).

The control experiments revealed that the sections

incubated with the FGF-2 antibody pre-absorbed with hu-

man recombinant FGF-2 showed no immunoreactivity in

the neurons and glial cells (data not shown). Sections

incubated with the solvent of the primary or secondary

antisera as well as with the solvent of the avidin-biotin

solution showed no reaction (data not shown).

Discussion

FGF-2 is a multifunctional protein which actions address

several types of cells (Baird and Klagsbrun 1991). It has

been demonstrated that this polypeptide induces prolifera-

tion and differentiation of embryonic neurons from multi-

ple central nervous system regions (Gospodarowicz et al.

1976; Walicke 1988). In the adult brain, FGF-2 has been

postulated to participate in the maintenance and survival of

neurons (Kushima et al. 1992). The FGF-2 can also protect

neurons from different types of injury (Gomez-Pinilla et al.

1992; Chadi et al. 1993a; Frim et al. 1993; Nozaki et al.

1993; Koketsu et al. 1994).

Fig. 2 Figure showing the effect of 72 h (A) and 1 week (B)

coarctation hypertension on the FGF-2 immunoreactive (ir) neuronal

and glial cell profiles as well GFAP immunoreactive profiles of the

nucleus tractus solitarii (NTS) of the rat. The bars show the average of

the number of profiles obtained from stereological analysis in both

sides of the NTS of the aortic coarctated rats. The values are

expressed as percent of the control. The dashed line represents the

100% values of the controls for 72 h and 1 week periods. The values

of control and coarctated rats were, respectively for 72 h: 1958 ± 17

and 3750 ± 32 for FGF-2 neuronal profiles, 4325 ± 49 and

10772 ± 22 for FGF-2 glial profiles, and 5290 ± 41 and 8240 ± 5

for GFAP profiles and for 1 week: 4205 ± 7 and 6484 ± 11 for FGF-

2 neuronal profiles, 1142 ± 6 and 1305 ± 5 for FGF-2 glial profiles,

and 5910 ± 15 and 6265 ± 16 for GFAP profiles. *P < 0.01

according to the Mann-Whitney U-test

J Mol Hist (2007) 38:285–294 289

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The present study has demonstrated, using a polyclonal

antibody, the presence of FGF-2 immunoreactivity in the

cytoplasm of neurons, and in the neuronal fibres in the NTS of

the rat. These findings are in agreement with previous obser-

vations which have demonstrated FGF-2 immunoreactivity in

the neurons of several brainstem nuclei using different poly-

clonal antibodies (Grothe et al. 1991; Matsuyama et al. 1992).

Our study has also described FGF-2 immunoreactivity in the

nuclei of astrocytes of the NTS. The present results are

in accordance with previous works which have described,

using monoclonal and polyclonal antisera, the presence of the

FGF-2 immunoreactivity in the nuclei of glial cells widely

distributed in the brain (Cintra et al. 1991; Woodward et al.

1992; Chadi et al. 1994). Previous papers have shown that low

molecular weight forms of the FGF-2 localize mainly in the

cytoplasm of neurons while higher forms are present in the

astrocytes nuclei through out brain and in neuronal nuclei of

few brain regions like pyramidal layer of the hippocampus

(Florkiewicz et al. 1991). Thus the data of the present paper

may refer to the changes of different molecular weight forms

of the molecule in distinct cell population in the NTS.

Few studies have investigated the role of growth factors

in the areas of blood pressure control. The demonstration of

the receptor for neurotrophic factor in the neurons of the

NTS (Mufson et al. 1992) suggests that those molecules

may play actions in the cardiovascular centers.

Different experimental designs have been used to study

the role of neurotrophic factors in the central nervous

Fig. 3 Double

immunoperoxidase procedures

have been used to colocalize

FGF-2 and glial fibrilary acidic

protein (GFAP, marker for

astrocytes) immunoreactivities

in the nucleus tractus solitarii

(NTS) of the 72 h aortic

coarctated rat (A–C). FGF-2

and GFAP immunoreactivities

are identified as a brownish

colour (diaminobenzidine as a

chromogen) and a bluish colour

(4-chloro naphtol as a

chromogen), respectively. B and

C represent higher

magnification of areas depicted

from the NTS showed in A. The

arrows indicate the presence of

FGF-2 immunoreactivity in the

nuclei of astrocytes and the

open arrows show the presence

of FGF-2 immunoreactivity in

the cytoplasm of neurons of the

NTS (B, C). (he panels D and Eshow GFAP immunoreactive

astrocytes (arrows)

counterstained with cresil violet

and OX42 immunoreactive

microglias (arrows) in the NTS

of control rats. Bars = 100 lm

(A), 20 lm (B), 10 lm (C, D,E)

290 J Mol Hist (2007) 38:285–294

123

system. Exogenous administration of growth factors to the

brain (Gomez-Pinilla et al. 1992; Otto and Unsicker 1990;

Chadi et al. 1993a; Koketsu et al. 1994), neuronal lesions

(Finklestein et al. 1988; Park and Mytilineou 1992; Frim

et al. 1993; Chadi et al. 1994) and electrical stimulation

(Follesa et al. 1994) applied to the central neuronal path-

ways are commonly employed.

Lesions to the central nervous system have been described

to induce a strong expression of FGF-2 mRNA and protein in

activated astroglial cells in the area of the injury (Finklestein

et al. 1988; Gomez-Pinilla et al. 1990; Logan et al. 1992;

Chadi et al. 1994). Riva and collaborators (1992) as well as

Follesa and collaborators (1994) described increased levels

of the FGF-2 mRNA following focally-evoked convulsive

seizures. Although an increasing number of studies have

pointed out the role of FGF-2 following cellular lesion, few

works have attempted to investigate the regulation of FGF-2

in response to physiological alterations of neuronal activity.

The present work has employed the coarctation hyper-

tension approach to evaluate whether a physiological

increase of the neuronal activity can modify the FGF-2

immunoreactivity in the NTS of the rat. It is well known

that during 48–72 h following the coarctation, the NTS is

highly stimulated by the intense afferent activity which

determines an increased vagal tone and an intense brady-

cardia at 3–6 h after coarctation (Soato and Krieger 1974;

Michelini et al. 1992).

FOS immunoreactivity has been considered a valuable

tool in the demonstration of activated neurons after stim-

ulus (Erickson and Millhorn 1991). The presence of FOS

positive neurons in the NTS of aortic coarctated rats 4 h

after surgery confirms the activation of that nucleus after

acute hypertension.

Our study has demonstrated an increased FGF-2

immunoreactivity, which may reflect an increased synthe-

sis of the FGF-2 protein, in neurons and astrocytes in the

NTS following the coarctation hypertension. It seems

likely that the changes of the FGF-2 immunoreactivity in

the NTS were probably induced by the increase in the

neuronal activity within this nucleus of the coarctated rats.

Several studies reported that physiological levels of affer-

ent activity are capable of regulating the synthesis of

neurotrophic factors (Isackson 1995). Gall and Isackson

(1989) observed an increased nerve growth factor mRNA

in response to afferent activity following limbic seizures.

Furthermore, experiments involving sub-seizure stimula-

tion (Castren et al. 1993) and physical exercise (Neeper

et al. 1995) have been shown to increase neurotrophins

expression in hippocampus. It was also demonstrated that

light regulates expression of brain derived neurotrophic

factor mRNA in rat visual cortex (Castren et al. 1992).

Moreover, long-term environmental enrichment leads to

regional increases in neurotrophin levels in the rat brain

(Ickes et al. 2000). Expression of neurotrophic factors in

brain activated areas may regulate the necessary synaptic

plasticity phenomena that in turn modulate local synaptic

transmittion (Bramham and Messaoudi 2005). It should be

considered the analysis of the FGF-2 mRNA expression

after aortic coarctation hypertension to confirm the changes

at the protein level, however, one should be aware that the

level and the timing of the FGF-2 gene expression may not

correspond to that found after neuronal lesion (Chadi et al.

1994).

It is possible that the increased neuronal activity induced

by the coarctation hypertension promoted a long lasting

increase in the neuronal FGF-2 immunoreactivity. The

Fig. 4 Microphotographs of coronal sections showing FOS immu-

noreactive neurons in the nucleus tractus solitarii (NTS) of rats 4 h

after aortic coarctation-induced baroreceptor reflex activation (A, B).

B represents a higher magnification of a NTS region showed in A.

Only scatted neuronal cell profiles showed FOS immunoreactivity in

the NTS of sham-operated (control) rats (C). Bars = 100 lm (A, C)

and 20 lm (B)

J Mol Hist (2007) 38:285–294 291

123

neuronal FGF-2 might modulate autocrine trophic and

plastic actions in the NTS neurons. Furthermore, the tran-

sient enhancement of the glial FGF-2 immunoreactivity in

the NTS could be due to the neuronal-glial interaction in

the NTS following the coarctation hypertension in a para-

crine fashion.

In our experiments slight activation of astrocytes was

observed at 72 h time-interval, following the aortic coarcta-

tion, as seen by the increased GFAP immunoreactivity.

Previous studies have demonstrated that a physiological

increased neuronal activity can activate brain astrocytes.

Repeated but kindled seizures applied in one side of the

hippocampus resulted in bilateral increase of the GFAP

mRNA synthesis (Steward et al. 1991). Moreover, short

period of spontaneous physical exercise is sufficient to induce

astroglial activation in rat hippocampus (Gomez-Pinilla et al.

1997). In the present work, it is possible that the coarctation

hypertension has led to a slight and short-lasting activation of

NTS astrocytes to transiently synthesize glial FGF-2 which

may act as a paracrine factor to NTS neurons. It has been

described the role of astroglial FGF-2 in the trophic state of

neurons of several parts of the brain (Engele and Bohn 1991;

Chadi et al. 1994).

The signals triggering the changes in the trophic/plastic

state in the NTS after coarctation hypertension were not

identified. The proto oncogene c-fos was suggested to

participate in the regulation of neurotrophic factor (Morgan

and Curran 1991) and was described to be expressed in the

NTS in response to stimulation of afferent fibres (Erickson

and Millhorn 1991; Rutherfurd et al. 1992) which is in line

with present observation of FOS expression in the NTS

neurons 4 h after aortic coarctation. It should be mentioned

that the majority of NTS neurons of stimulated rats in the

present analysis expressed FOS protein which indicates

that the up-regulation of the FGF-2 protein may have

occurred in those cells.

Recent studies have indicated that the excitatory amino

acid glutamate is the neurotransmitter of the baroreceptor

primary afferent fibres (Guyenet et al. 1987; Pawloski-Dahm

and Gordon 1992). Lawrence and Jarrot (1994) observed

increases of glutamate in microdialysis lisates in the NTS of

phenylephrine-induced hypertensive rats. Moreover, previous

work has demonstrated that low doses of glutamate induce

FGF-2 mRNA in neurons and astrocytes in culture (Pechan

et al. 1993). Thus, increased FGF-2 immunoreactivity

observed in the stimulated NTS may be due to the increased

release of glutamate from the baroreceptor terminals which, in

turn, may have stimulated the FGF-2 synthesis in the NTS

cells, underlining the role of the neurotrophic factor in events

related to neuroplasticity in the central areas of blood pressure

control.

Furthermore, it is known that the glutamate can become

neurotoxic by persistently NMDA receptor activation

(Novelli et al. 1988). In this way the synthesis and local

release of the FGF-2 following coarctation hypertension

might be able to protect the NTS neurons from excitotox-

icity (Cheng et al. 1993; Mattson et al. 1993).

However, it is unlikely that neurotoxicity may have

taken place following coarctation hypertension since we

have not observed any alteration in the OX42 immunore-

activity 1 week and 1 month following the aortic coarcta-

tion. Furthermore, it is postulated that the degree of

degeneration of the injured neurons is related to the

intensity of the microglial reaction (Giulian and Robertson

1990; Giulian 1993). Moreover, we have not found changes

in the number of NTS neurons up to 1 month of aortic

coarctation.

Thus, there is no indication of neuronal lesion of stimu-

lated NTS in the present model and the upregulated FGF-2

may be involved in local neuronal plasticity (Isackson 1995)

and changes in neurotransmitter phenotype (Otto and

Unsicker 1990) may represent a trophic adaptive response in

this nucleus.

In conclusion the presence of the FGF-2 immunoreactivity

in the neurons and astrocytes of the NTS indicates that the

FGF-2 may be an important neurotrophic factor in the areas

involved in blood pressure control. The increased FGF-2

immunoreactivity in the NTS following an increased neu-

ronal activity induced by the coarctation hypertension may

represent a neurotrophic/neuroplastic adaptive response in

this nucleus.

Acknowledgements This work was supported by grants from

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

98/13122-5; 99/01319-1) and Conselho Nacional para o Desen-

volvimento Cientıfico e Tecnologico (CNPq: 521004/97-1; 311797/

2006-7), Brasil. We are grateful to Professor Kjell Fuxe, Karolinska

Institute, Stockholm, Sweden, for valuable discussion and to Dr.

Andrew Baird Whittier Institute, La Jolla, USA for the generous gift

of the FGF-2 antibody. Varella, T.C.N. was a FAPESP undergraduate

trainee fellow (96/9387-8).

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