Immunohistochemical Localization of Some Neurotrophic Factors and Their Receptors in the Rat Carotid Body

doi:10.4236/nm.2013.44042

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Neuroscience & Medicine, 2013, 4, 284-289

Published Online December 2013 (http://www.scirp.org/journal/nm)

http://dx.doi.org/10.4236/nm.2013.44042

Open Access NM

Immunohistochemical Localization of Some Neurotrophic

Factors and Their Receptors in the Rat Carotid Body

Dimitrinka Y. Atanasova1,2, Nikolai E. Lazarov1,3*

1Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria; 2Institute of Experimental Morphology, Pathology and

Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria; 3Department of Anatomy and Histology, Medical

University of Sofia, Sofia, Bulgaria.

Email: *[email protected]

Received October 14th, 2013; revised November 10th, 2013; accepted December 5th, 2013

Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original

work is properly cited.

ABSTRACT

The carotid body (CB) is a small neural crest-derived organ that registers oxygen and glucose levels in blood and regu-

lates ventilation. The most abundant cell type in the CB glomeruli is glomus or type I cells, which is enveloped by proc-

esses of sustentacular or type II cells. Growth and neurotrophic factors have been established as signaling molecules

played an important role in the development of the CB. To gain insight whether these signaling molecules are present in

the adult rat CB, we examined the expression and cellular localization of some neurotrophic factors and their corre-

sponding receptors in this organ by immunohistochemistry. The results showed the presence of nerve growth factor

(NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) as well as p75NTR,

tyrosine kinase A receptor (TrkA), tyrosine kinase B receptor (TrkB) and GDNF family receptor alpha1 (GFRα1) in the

adult CB. At the light-microscopical level, the immunoreactivity for NGF and both its low-affinity (p75) and high-af-

finity (TrkA) receptors was detected in the majority of glomus cells and also in a subset of sustentacular cells. BDNF

and its receptors, p75 and TrkB, were observed in the glomus cells, too. Remarkably, the immunohistochemical analysis

revealed that the neuron-like glomus cells, but not the glial-like sustentacular cells, expressed GDNF and GFRα1.

Taken together with prior results, it can be inferred that neurotrophins may be involved in the CB cell differentiation

and survival in adulthood, and may exert a potent glomic protective action as well. It is also presumable that GDNF

production by glomus cells plays a pivotal role in permitting long-term viability of CB grafts, which permits their po-

tential applicability in cell therapy as a promising tool in neurodegenerative disorders.

Keywords: Carotid Body; Immunohistochemistry; Neurotrophin Receptors; Trophic Factors; Rat

1. Introduction

The carotid body (CB) is the main peripheral arterial

chemoreceptor that detects blood levels of O2, CO2/H+

and glucose, and responds to their changes by regulating

breathing [1,2]. It is a small ovoid mass of tissue bilater-

ally located at the bifurcation of the common carotid ar-

tery, just before blood chemicals reach the brain, an or-

gan that is excessively sensitive to oxygen and glucose

deprivation. The CB is composed of two juxtaposed cell

types, neuron-like type I or glomus cells and glial-like

type II, also known as sustentacular cells, which are ag-

gregated in richly vascularized cell clusters, called

glomeruli [3]. Chemical and electrical synapses between

these cells allow for complex sensory processing in the

organ [2]. Furthermore, the principal type cells, i.e., the

glomus cell, contain secretory granules packed with pu-

tative transmitters and are thus considered the chemo-

sensory cells of the organ [1,4], while the type II cells, in

addition to their supporting role, have recently been as-

sumed to be the CB stem cells [5]. The neural crest ori-

gin of the CB suggests that it may require a trophic sup-

port for a continued survival and cell differentiation.

It has been demonstrated that CB chemosensitivity to

hypoxia develops during the early postnatal period. This

functional maturation partially depends on structural and

neurochemical changes, which are promoted by some

neurotransmitters, trophic factors and their receptors,

acting on the CB cell population in autocrine or paracrine

ways [6-8]. Besides, there is a convincing evidence that

neurotrophic factors from CB cells and nerve fibers also

*Corresponding author.

Immunohistochemical Localization of Some Neurotrophic Factors and

Their Receptors in the Rat Carotid Body

285

play an important role in maintaining the structural and

functional specialization of both CB components in

adulthood [9]. Indeed, recent studies have revealed that

glomus cells in the adult rat, of a neural crest origin, ex-

press a number of growth factors, implying their para-

crine-autocrine role in the CB [10,11].

In order to determine the survival requirements of the

parenchymal cells in this organ in adult life, we exam-

ined the immunohistochemical distribution and cellular

localization of some neurotrophic factors and their cor-

responding receptors in the CB of normal adult rats.

2. Materials and Methods

2.1. Animals and Tissue Preparation

The experiments were carried out on male Wistar rats

(300 g body weight). The experiments were conducted in

accordance with the ethical guidelines of the EU Direc-

tive 2010/63/EU for the protection of animals used for

scientific purposes following a standard protocol estab-

lished by the Bioethical Commission of the Biomedical

Research at the Institute of Neurobiology of the Bulgar-

ian Academy of Sciences. All efforts were made to

minimize the number of animals used and their suffering.

The rats were deeply anesthetized with Nembutal (50

mg/kg, i.p., Abbott) and transcardially perfused first with

0.05 M phosphate buffered saline (PBS), followed by 4%

paraformaldehyde in 0.1 M phosphate buffer (PB), pH

7.3. After perfusion, the carotid bifurcations were

quickly removed, both CBs were immediately dissected

out and the specimens were postfixed in the same fixa-

tive overnight at 4˚C. Thereafter, the tissue blocks were

embedded in paraffin and cut into 7 µm thick sections.

2.2. Immunohistochemical Procedure

The sections of CBs were deparaffinized with xylene and

ethanol, and processed for ABC (avidin-biotin-horse-

radish peroxidase complex) immunohistochemistry to

determine the neurotrophin profile of their chemorecep-

tor elements. Briefly, the endogenous peroxidase was

blocked with 1.2% hydrogen peroxide in absolute

methanol, followed by antigen retrieval in 10 mM citrate

buffer (pH 6.0) for up to 30 min in a microwave oven.

After washing in PBS, the sections were preincubated for

60 min at room temperature in 5% normal goat serum to

avoid unspecific staining. Between the separate steps, the

sections were rinsed with cold PBS/Triton X-100. Sub-

sequently, they were incubated in a humid chamber

overnight at 4˚C with the respective primary antibodies

(see Table 1 for suppliers and working dilutions). After

rinsing in PBS, the sections were incubated for 2 h at

room temperature with the appropriate secondary anti-

body, biotinylated goat anti-rabbit IgG (Dianova, Ham-

burg, Germany), goat anti-mouse IgG or rabbit anti-goat

IgG (both from Vector Laboratories, Burlingame, CA,

USA), at a dilution of 1:500, 1:250 or 1:200, respectively.

Following rinsing, the ABC complex (Vectastain Elite

Kit; Vector) was applied. After a color development with

diaminobenzidine as a chromogen for 3 to 5 min, the

sections were dehydrated in graded alcohols, cleared in

xylene, coverslipped with Entellan and air-dried.

2.3. Antisera Specificity Tests

We applied positive and negative controls to test the

specificity of the antibodies used in this study. For im-

munoreaction specificity testing, omission of the specific

primary antibodies by their replacement with PBS or

non-immune serum, at the same dilution as the primary

antiserum, was performed and no specific immunostain-

ing was found under these conditions. The specificities of

the antibodies used in this study have been described in

detail previously [12]. In particular, the primary antisera

Table 1. Primary antibodies used for immunohistochemistry.

Primary antiserum (Antigen) Host species/Type Supplier Cat. № Working dilution

NGF rabbit/polyclonal Santa Cruz sc-548 1:500

BDNF rabbit/polyclonal Santa Cruz sc-546 1:500

NT-3 goat/polyclonal Santa Cruz sc-13380 1:500

GDNF mouse/monoclonal Santa Cruz sc-13147 1:500

GFRα1 rabbit/polyclonal Santa Cruz sc-10716 1:500

p75NTR mouse/monoclonal DAKO M 3507 1:200

TrkA rabbit/polyclonal Santa Cruz sc-118 1:500

TrkB rabbit/polyclonal Santa Cruz sc-8316 1:500

TrkC rabbit/ polyclonal Santa Cruz sc-117 1:500

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Immunohistochemical Localization of Some Neurotrophic Factors and

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286

were preabsorbed for 2 h at room temperature or for 24 h

at 4˚C with the respective synthetic antigen at a concen-

tration of 20 or 200 µg/ml antiserum at its working dilu-

tion and the preabsorbed antisera were substituted for

non-absorbed antisera in the immunohistochemical pro-

cedure. Preabsorbed antibodies failed to stain any tissues

of the CB. The antibodies were further characterized with

tissue from regions known to contain the studied antigens.

Immunolabeled sections of various brain regions at the

level of the trigeminal sensory nuclear complex and the

trigeminal ganglion were used as positive controls for

each of the tested neurotrophic factors.

2.4. Image Processing and Analysis

After immunostaining, the specimens were examined and

photographed with a Nikon research microscope equipped

with a DXM1200c digital camera. The digital images

were saved in a TIF format, and matched for brightness,

contrast and removal of artifacts using Adobe Photoshop

CS3 software (Adobe Systems, Inc., San Jose, CA). The

number of immunopositive cells was determined in a

semiquantitative way for the two given cell populations.

The resultant values provide a relative quantity of the

immunostained glomus and sustentacular cells.

3. Results

3.1. Nerve Growth Factor Family

This small family of neurotrophic factors consists of a

group of neurotrophins, including the nerve growth fac-

tor (NGF), brain-derived neurotrophic factor (BDNF),

neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4, also

known as NT-5). They promote neuronal differentiation,

survival and plasticity by signaling via both a common

low-affinity (pan)neurotrophin receptor p75NTR and high-

affinity transmembrane receptors belonging to the tyro-

sine receptor kinase (Trk) proto-oncogene family. The

latter include TrkA, TrkB and TrkC, and exhibit binding

specificity. TrkA is a specific receptor for the NGF, TrkB

is a receptor for the BDNF and NT-4, while TrkC is a

receptor for NT-3. The number of immunopositive cells

and relative expression levels of the examined neurotro-

phins and their receptors are summarized in Table 2.

Our experiments showed that the majority of the adult

glomus cells and a subset of the sustentacular cells in the

rat CB were NGF immunopositive (Figure 1(A)). The

immunoreactive cells were spread throughout the CB.

The immunostaining in the glomus cells was strong

while that in the sustentacular cells was moderate and

primarily localized in their cytoplasmic processes sur-

rounding the glomus cells. None of the immunopositive

cells showed staining in their nuclei. Further, immunore-

activity for p75 (Figure 1(B)) and TrkA (Figure 1(C))

Table 2. Distributional pattern and relative expression lev-

els of neurotrophins and their receptors in the adult rat

carotid body.

Neurotrophic factors NGF BDNF NT3 GDNF

Glomus cells +++ +++ +++ +++

Sustentacular cells

+ + + −

Neurotrophin

receptors p75NTR TrkA TrkB TrkCGFRα1

Glomus cells +++ +++ +++ +++ +++

Sustentacular cells+ + + − +

(−), no immunopositive cells observed; (+), <50% immunopositive cells;

(++), 50% - 90% immunopositive cells; (+++), >90% immunopositive cells.

Figure 1. Immunohistochemical demonstration of members

of the NGF family and their receptors in the CB of adult

rats. Light photomicrographs showing that the cell clusters

exhibit immunoreactivity for NGF (A) and its low-affinity

p75NTR (B) and high-affinity TrkA (C) receptors. Note that

the immunostaining is observed in the cytoplasm of a large

number of glomus cells and in a subset of sustentacular cells.

The cytoplasmic processes of the latter are also endowed

with TrkA receptor (arrows). (D) Intense BDNF immunore-

activity in numerous glomus cells and scattered sustentacu-

lar cells. Expression of TrkB (E) and TrkC (F) receptor sub-

types is seen in virtually all glomus cells. Scale bar = 50 µm.

was detected in most of the glomus and in some susten-

tacular cells, as well as in nerve fibers within and around

the cell clusters. Similarly, NT-3 (not shown) and BDNF

immunostaining was observed in a lot of glomus cells

and a few sustentacular cells in the CB glomeruli (Figure

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Immunohistochemical Localization of Some Neurotrophic Factors and

Their Receptors in the Rat Carotid Body

287

1(D)). In addition, almost all glomus cells were richly

endowed with TrkB (Figure 1(E)) and TrkC (Figure

1(F)) receptors.

3.2. Glial Cell Line-Derived Neurotrophic Factor

Family

Using antibodies directed against the glial cell line-de-

rived neurotrophic factor (GDNF) and its receptors, we

identified a large number of immunoreactive cells in the

CB, typically aggregated in evenly distributed cell clus-

ters. In particular, virtually all glomus (type I) cells in the

CB were immunopositive for GDNF (Figure 2(A)), with

no notable labeling of the elongated sustentacular (type II)

cells, which surrounded the glomeruli. Accordingly, all

groups of BDNF-immunoreactive cells were also immu-

nostained for the GDNF family receptor alpha-1

(GFRα1), which specifically binds the GDNF (Figure

2(B)). Our attempts to detect RET protein expression by

Figure 2. Immunohistochemical demonstration of GDNF

and its specific receptor in the CB of adult rats. (A) Repre-

sentative photomicrograph indicating the presence of

GDNF-immunoreactive cell groups in the CB glomeruli.

Note that only the glomus cells are immunopositive. (B)

High-resolution micrograph showing the expression of

GFRα1 receptor in the adult CB. A large number of type I

cells exhibit strong immunoreactivity for this receptor sub-

type with similar distributional patterns. (C) No immuno-

reactivity is observed in the section incubated with a pre-

absorbed primary antibody. Scale bars = 50 µm.

immunohistochemistry have failed so far, probably be-

cause its localization is mostly restricted to nerve endings

which are very few in the CB.

Control sections treated with the primary antisera pre-

absorbed with the respective synthetic antigens did not

display any staining (Figure 2(C)).

4. Discussion

The CB is the major arterial oxygen sensor providing

essential afferent input required for the maintenance of

breathing. There is compelling evidence that neurotro-

phic factors such as BDNF and GDNF, working in tan-

dem, are important regulators for the development of

peripheral chemoafferent neurons; the latter provide

hypoxic drive to the brainstem respiratory network [13,

14]. Previous studies have shown that many growth and

trophic factors are present in the glomus type I cells of

the mammalian CB (for a recent review, see [9]). Our

results provide immunohistochemical evidence that the

parenchymal cells in the adult rat CB express certain

neurotrophins belonging to the NGF and GDNF families

as well as their corresponding receptors. In particular,

here we demonstrate the occurrence of NGF, BDNF,

NT-3 and GDNF proteins in the vast majority of glomus

cells and a subset of sustentacular cells which are also

endowed with their cognate receptors p75, TrkA, TrkB

and GFRα1. Such expressional patterns are consistent

with the conclusion that the glomus cells may exert tro-

phic and/or regulatory impact on the adjacent CB cells in

adult life through neurotrophins, which play autocrine

and/or paracrine roles in promoting the survival, growth

and maturation of these cells [11,15].

In addition to their established role in maintaining the

structural and functional specialization of the CB in

adulthood, available evidence strongly suggests that

neurotrophins also promote neuronal precursor cell pro-

liferation and differentiation of distinct neural crest-de-

rived cells (see [16] and references therein). Indeed,

glomus and sustentacular cells, like sympathetic neurons

and chromaffin cells of the adrenal medulla, originate

from the neural crest and phenotypically resemble sym-

pathoadrenal chromaffin cells [17,18]. Our data confirm

prior findings demonstrating that NGF and its high-af-

finity receptor TrkA are preferentially expressed by the

CB glomus cells [10]. However, there is evidence that

the glomus cells do not depend on NGF (as do sympa-

thetic neurons) for survival in vitro and, thus, their

growth requirements are different from other cells of

neural crest origin [19]. In this respect, it has been pro-

posed that NGF interacts cooperatively with the basic

fibroblast growth factor to promote proliferation and sur-

vival of the CB cell population [20]. We have further

shown that NGF and TrkA immunoreactivity also occurs

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Immunohistochemical Localization of Some Neurotrophic Factors and

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288

in the sustentacular cells of the rat CB. It is well known

that NGF is an essential growth factor supporting stem

cell self-renewal outside the nervous system and, thus,

serves as a potential marker for progenitor cells. This

could explain the occurrence of immunoreactivity for this

neurotrophin and its receptors also in the sustentacular

cells which have lately been proposed to be the CB stem

cells giving rise to new glomus cells upon hypoxia [5,

21].

Several recent reports suggest that the expression of

BDNF, GDNF and their receptors in virtually all glomus

cells, which are known to be highly dopaminergic [22],

might account for their potential applicability in neuro-

logical cell therapy [23-26]. Moreover, it has been shown

that nerve fibers innervating the CB and dopaminergic

neurons in the rat petrosal ganglion (PG) also exhibit

BDNF immunoreactivity [27]. Hence, it is plausible to

assume that BDNF and GDNF potently promote the

survival of dopaminergic neurons like CB glomus cells,

and also exert a trophic effect on chemoafferent neurons

of the PG, acting as a target-derived survival factor in

adult life [27,28]. It has also been proposed that BDNF

selectively produced by glomus cells may support local

vascular morphogenesis in the CB [27] whereas GDNF

may act as a protector of catecholaminergic neurons

against toxic damage and oxidative stress [29]. In line

with this, we were able to demonstrate the presence of

GFRα1 in glomus cells of the adult rat CB whose pres-

ence in them was also confirmed by RT-PCR [23]. It is

likely that BDNF acts mainly via its specific receptor in

the adult rat CB.

Unlike the other neurotrophic factors of the NGF fam-

ily, NT-3 and NT-4 have been examined in less detail in

the adult rat CB. In the present study, we demonstrated

the presence of NT-3 and its specific receptor TrkC, but

not of NT-4, in the CB glomeruli. Nonetheless, we were

able to show that TrkB, which is a common receptor for

the BDNF and NT-4, is expressed in almost all glomus

cells. Besides, it is well known that NT-3 binds, though

with less affinity, to other related Trk receptors, TrkA

and TrkB, as well [16,30]. Accordingly, it seems that the

survival of glomus cells also depends on TrkB signaling.

The source of this signaling is, however, still remains

unidentified.

5. Conclusion

In conclusion, it can be inferred that the majority of the

glomus cells and a subset of the sustentacular cells in the

adult rat CB contain high levels of NGF, BDNF and

GDNF and their corresponding receptors. Taken together,

our results suggest that the principal CB cells depend on

certain locally produced trophic factors for their survival

in adult life. It is possible, however, that other neurotro-

phic factors previously reported to be present in the rat

CB [9,11] also play a role as survival factors for the

natural CB cell populations in adulthood. Given that sur-

vival depends on cell-cell interactions, glomus cells may

exert via the entire cocktail of neurotrophins present in

the trophic and/or regulatory control on the adjacent

neuron-like and glial-like cells in the CB, thus implying

their autocrine and paracrine action. Last but not least,

neurotrophins and in particular GDNF might prove to be

good candidates for the maintenance of the long-term

viability of CB grafts, thus permitting the successful de-

velopment of neurological cell replacement therapy.

However, additional experimental and clinical studies are

needed to determine whether this animal model is appli-

cable in humans.

6. Acknowledgements

This work was financially supported by the European

Social Fund and Republic of Bulgaria, Operational

Programme “Development of Human Resources” 2007-

2013 framework (grant BG051PO001-3.3.060048 from

04.10.2012 to DA). The authors wish to thank Professor

Anton Tonchev (Medical University of Varna) for the

generous gift of the primary antibodies, and Dr Angel

Dandov for his critical reading of this manuscript.

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