Download Somatostatin-like immunoreactive primary sensory neurons

Bull Vet Inst Pulawy 58, 651-656, 2014
DOI: 10.2478/bvip-2014-0099
Somatostatin-like immunoreactive primary sensory
neurons supplying the porcine adrenal glands
in physiological conditions
and after adrenalectomy
Sławomir Gonkowski
Department of Clinical Physiology, Faculty of Veterinary Medicine,
Warmia and Mazury University in Olsztyn, 10-957 Olsztyn, Poland,
[email protected]
Received: April 10, 2014
Accepted: September 25, 2014
Abstract
Retrograde neuronal tracing, using fast blue, in combination with a single-labelling immunofluorescence technique, was
applied to determine whether somatostatin (SOM) participates in sensory innervating of the porcine adrenal glands in
physiological conditions and after adrenalectomy. In control animals, SOM–like immunoreactive neurons comprised 7.0 ± 0.7%
of adrenal gland-projecting cells in dorsal root ganglia (DRG) at neuromeres Th6-7 and 6.5 ± 1.2% at neuromeres Th12-14. After
adrenalectomy the percentage of SOM-positive DRG cells considerably increased and attained the level of 44.7 ± 2.5% at
neuromeres Th6-7 and 36.6 ± 1.7% at neuromeres Th12-14. The obtained results demonstrate that SOM is not only
a neuromediator within sensory neurones supplying the porcine adrenal glands, but also suggest the role of this substance during
repairing processes within the nervous system after adrenalectomy.
Keywords: swine, adrenal glands, somatostatin, dorsal root ganglia, adrenalectomy.
Introduction
Somatostatin (SOM) is a tetradecapeptide (14
amino acids) which was isolated for the first time in
1973 from the ovine hypothalamus and was
demonstrated to inhibit growth hormone secretion (6).
This substance is an active factor widely distributed in
the body of all vertebrates and some invertebrate
species, which can be produced by neurons,
neuroendocrine, inflammatory and immune cells (18).
Functions of somatostatin are mediated by five types of
receptors (sst1-5), all of which are members of the
G protein coupled receptor superfamily (8). In spite of
the fact that physiological functions of SOM are
relatively well established, some aspects of its action
remain not clear. One of them is the participation of
somatostatin in sensory innervation of the adrenal
glands.
SOM is an important regulatory peptide in the
central and peripheral nervous system (2).
Nevertheless, it is well known as an active substance
within the autonomic nervous system (11, 15, 24), it
may also take part in the transmission of sensory and
nociceptive information (3, 20). It is known that
primary sensory neurons are located in sensory nuclei
of cranial nerves, as well as in dorsal root ganglia
(DRG). Neurons in the DRG are bipolar cells, which
have perikarya within ganglia and send their endings
centrally to the dorsal horn of the spinal cord and
peripherally to the skin and internal organs. Until now,
both somatostatin and its receptors have been described
in the DRG of various species, including humans (3,
14, 23). The presence of SOM has been mainly
observed in small–cell–diameter neurons of the DRG,
and prevalent types of somatostatin receptors in these
ganglia are two variant forms of sst2 (sst2A and sst2B)
(23).
Nowadays, somatostatin is considered to be
a strong antinociceptive factor (12, 13), which inhibits
the conduction of sensory neurons and may be used as
an analgesic factor during various pathological
processes. On the other hand, some investigations have
shown pro-nociceptive action of intrathecally
administered somatostatin (22).
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S. Gonkowski/Bull Vet Inst Pulawy/58 (2014) 651-656
Antinociceptive actions of somatostatin suggest
that this substance plays important roles during
pathological processes. Moreover, SOM is a wellknown anti-inflammatory agent, which down-regulates
lymphocyte proliferation, reduces immunoglobulin
production, and inhibits the release of proinflammatory cytokines (25). However, up till now,
functions of SOM in the sensory and nociceptive
conducting neurons, especially during pathological
states connected with damage of nervous fibers and
adrenal glands remain unknown. Therefore, the aim of
the present study was the description of SOM–like
immunoreactive (SOM-LI) neurons of the DRG
supplying the porcine adrenal glands under
physiological conditions and after adrenalectomy. It
should be noted that pig becomes the most popular
experimental animal because of considerable
similarities to human in anatomical, physiological, and
pathological characteristics (26).
Material and Methods
The present study was performed on ten immature
sows of the Large White Polish breed (12-18 kg b.w.,
approximately 8 weeks old). The animals were kept in
standard laboratory conditions. All experimental
operations were conducted in compliance with the
instructions of the Local Ethical Committee in Olsztyn.
The pigs were divided into two groups: control
(group C; n = 5) and experimental (group A after
adrenalectomy, n = 5). All animals were pre-treated
with atropine (Polfa, Poland, 0.4 mg/kg, s.c.) and
propionyl promasine (Stresnil, Janssen, Belgium,
0.8 mg/kg, i.m.) 15 min before application of the main
anaesthetic - sodium thiopental (Thiopental; Sandoz,
Austria; 20 mg/kg i.v.), and were subjected to median
laparotomy. The left adrenal gland was injected with
20 μL of 5% aqueous solution of fast blue (FB, Dr.
K. Illing GmbH&KG, Germany; four injections, 5 μL
each) using a Hamilton syringe equipped with 26 gauge
needle. A great attention was paid to avoiding any
contamination of the surrounding tissues with FB due
to the hydrostatic leakage from the injection canal.
After convalescence period of three weeks,
animals of group A were subjected to reoperation,
when, after laparotomy, the adrenalectomy of the left
adrenal gland was performed. One week after the
adrenalectomy, all animals (groups C and A) were reanaesthetised, and then euthanized by an overdose of
sodium thiopental. Afterwards, they were perfused
transcardially with 4% buffered paraformaldehyde
(pH 7.4) prepared ex tempore.
Left dorsal root ganglia from neuromeres Th6-7
and Th12-13 (previous investigations (9) demonstrated
that the major number of sensory neurones supplying
the left porcine adrenal gland are localised in these
segments) were taken from all animals. The ganglia
were rinsed in a 0.1 M buffer solution, pH 7.4, for 72 h
at 4C and then kept at 4C in a buffered 18% sucrose
solution. Finally, the ganglia were cut with a cryostat
(at -22C) into 10 μm serial sections. These sections
were examined under a fluorescence Olympus BX51
microscope equipped with an appropriate filter set to
localise all FB + neurons. The diameter of perikaryon
of each FB – positive cell (in this section, where the
nucleus was clearly visible) was measured by means of
image analysis software (AnalySIS version 3.0, Soft
Imaging System GmbH, Germany). The neurons were
divided into three size –classes: small (diameter up to
30 μm), medium (diameter 31-50 μm), and large
(diameter >51 μm).
All FB - labelled neurons (at least 80 cells per
each experimental animal) were processed for the
routine single labelling immunofluorescence method
(10), using primary SOM antibody (monoclonal rat
antiserum from Chemicon Int. Inc., USA, catalogue
No. MAB354, work dilution: 1:50). After air-drying at
room temperature for 45 min, the sections were
incubated with solution containing 10% of goat serum,
0.1% of bovine serum albumin, 0.01% of NaN 3, Triton
X-100, and thimerozal in PBS for 1 h at room
temperature, then incubated with the SOM antiserum
(overnight; at room temperature), and further incubated
with goat anti-rat FITC conjugated IgG (ICN
Biomedicals, USA, dilution 1:400) (1 h, at room
temperature). Each step of immunolabelling was
followed by rising the sections with PBS (3 × 10 min,
pH 7.4). Standard controls, i.e. pre-absorption of the
primary antibody with appropriate antigen and
omission and replacement of primary antibody by nonimmune serum were performed to test antibody and
specificity of the method. The pre-absorption test was
performed as follows: sections of DRG were incubated
with “working” dilution of anti-somatostatin antibody,
which was pre-absorbed for 18 h at 37C with 20 μg of
human SOM (Hölzel Diagnostika GmbH, Germany).
The labelled sections were analysed using
Olympus fluorescence microscope equipped with epiillumination and appropriate filter sets for FITC and
FB. Relationships between immunohistochemical
staining and FB distribution were examined by
interchanging filters. Finally, the obtained data was
pooled, expressed as means ±SEM and statistically
analysed using GraphPad Prism 5 software (graphPad
Software, USA). The differences were considered
statistically significant at P ≤ 0.05.
Results
SOM-positive neurones supplying the adrenal
glands were observed in DRG of control pigs, as well
as in animals after adrenalectomy (Fig. 1, Table 1).
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Table 1. The percentage of somatostatin – like immunoreactive neurons supplying porcine adrenal glands in dorsal root
ganglia under physiological conditions (group C) and after adrenalectomy (group A)
Neuromeres
Group C
Group A
Th 6-7
7.07 ± 0.7a
44.7 ± 2.5b
Th 12-13
6.5 ± 1.2a
36.6 ± 1.7c
Statistically significant data (P ≤ 0.05) is marked by different letters, and not significant data is marked by the same letter
A
1 FB
1 SOM
2 FB
2 SOM
B
1 FB
1 SOM
2 FB
2 SOM
Fig. 1. Somatostatin – like immunoreactive neurons supplying the adrenal glands (arrows) in
dorsal root ganglia within neuromeres Th6-7 (A) and Th12-13 (B) under physiological
conditions (1) and after adrenalectomy (2). Scale bar 20 μm
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Under physiological conditions, only small
percentage of FB-positive cells revealed the presence
of the SOM. These values amounted to 7.0 ± 0.7% in
segments Th6-7 and 6.5 ± 1.2% in neuromeres
Th12-13, and did not display statistically significant
differences between groups of neuromeres (Table 1).
Moreover, in control animals, the SOM was
visualised in all size – classes of FB-positive neurons
(Table 2), without considerable quantitative differences
between them. The percentage of SOM-LI cells
amounted to 6.4 ± 1.0%, 6.5 ± 0.3%, and 7.7 ± 0.3% in
small, medium, and large FB+ cells, respectively.
After adrenalectomy, the number of FB – positive
neurons immunoreactive to SOM rapidly increased
within both groups of neuromeres. Changes were more
visible in segments Th6-7, where the percentage of
SOM-LI cells containing FB amounted to 44.7 ± 2.5%.
In segments Th12-13 this value was lower and
amounted to 36.6 ± 1.7% (Table 1). In contrast to
control animals, after adrenalectomy, statistically
significant differences in the percentage of SOM-LI
neurons were observed between Th6-7 and Th12-13
segments. In experimental animals, the expression of
SOM increased especially in small and middle-size
cells, where the percentage of SOM-positive neurons
was 59.2 ± 1.2% and 46.1 ± 1.0%, respectively
(Table 2). In large perikarya, these changes were less
visible and were expressed by an increase in the
percentage of SOM-LI cells to 16.7 ± 1.5%. Moreover,
after adrenalectomy statistically significant differences
in the percentage of SOM-positive cells were observed
between particular size-classes of neurons.
Table 2. The percentage of somatostatin – like immunoreactive
neurons supplying porcine adrenal glands in dorsal root ganglia under
physiological conditions (group C) and after adrenalectomy (group
A) in particular size – classes of cells
Diameter of neurons
(μm)
Group C
Group A
<30
6.4 ± 1.0a
59.2 ± 1.2b
31-50
a
6.5 ± 0.3
46.1 ± 1.0c
>51
7.7 ± 0.3a
16.7 ± 1.5d
Statistically significant data (P ≤ 0.05) is marked by different letters,
and not significant data is marked by the same letters
Discussion
The results obtained show that the SOM takes part
in sensory innervating of porcine adrenal glands. Under
physiological conditions, the participation of this
peptide in conducting of sensory stimuli from adrenal
glands seems to be minor, but during pathological
states the significance of the SOM in these processes
clearly increases.
In previous studies, SOM-positive DRG neurons,
as well as somatostatin receptors have been observed in
various species, including humans (3, 14, 15, 24). On
the other hand, SOM has not been reported in DRG
cells supplying the adrenal glands in rodents (14), but it
should be pointed out that data concerning the chemical
coding of sensory neurons supplying these glands is
scant and fragmentary, and limited to typical sensory
neuromediator such as substance P (29).
During the study, the percentage of SOM-positive
neurons supplying the adrenal glands was not high in
control animals. It is in agreement with previous
studies on pig, where the number of SOM-LI neuronal
cells within DRG has fluctuated around 1% (3). On the
other hand, till now somatostatin has been observed
only in small neurons of DRG (16), while in the present
study, various classes of somatostatin-positive cells
were noted, and differences in expression of SOM in
particular size-classes of cells have not been visible
under physiological conditions.
It should be pointed out that at present, not only
the functions of SOM within the adrenal glands, but
also roles of all sensory innervation of these organs are
not known. Some studies suggest that sensory neurons
supplying the adrenal glands, beside conduction of pain
stimuli during pathological processes, may send
information connected with baro- and/or chemoreflexes (19) and, following a change of blood pressure,
affect the secretion of catecholamins by the adrenal
medulla. Additionally, colocalisation of SOM with
substance P, nitric oxide synthase, calcitonin gene
related peptide, and galanin (14) in intra-adrenal
nervous fibres, which partially can be the peripheral
endings of DRG neurons, suggest the similar functions
of the mentioned substances, i.e. effect on secretion of
adrenaline, noradrenaline, and steroid hormones
(17, 28).
The functions of somatostatin in conducting of
sensory and pain stimuli are discussed, but still not
clear. It is known that this substance may act mainly as
an anti-nociceptive factor (12, 13), but some
investigations have demonstrated pro-nociceptive
properties of this peptide (22). Previous studies have
demonstrated that somatostatin and its analogue octreotide (OCT) exhibit anti-nociceptive and antiinflammatory properties and can evoke the analgesic
effects during different types of pathological pain (7,
21). Moreover, previous investigations have also
revealed inhibitory effects of somatostatin on sensory
neurons within dorsal horn of the spinal cord (7).
A dramatic post-adrenalectomy increase in SOMpositive sensory neurons, supplying the porcine adrenal
glands, observed in the present investigation is not
completely in agreement with previous studies. Till
now, this fact was not observed in other species, in
which, in contrast to present results, the reduction of
SOM-positive sensory neurons has been described after
axotomy (27). It is possible that an increase in the
number of SOM-positive sensory neurons in the DRG
after injury of their peripheral endings is characteristic
of the pig. Some investigations concerning this species,
in spite of their fragmentary character, seem to confirm
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the mentioned argument. Namely, post-axotomical
augmentation of SOM-like immunoreactivity has been
described in the porcine autonomic nervous system
(15). Moreover, scanty information concerning SOMpositive sensory neurons in porcine DRG revealed that
the percentage of such cells supplying the urinary
bladder increases under the influence of selected toxins
(4, 5).
An increase in the number of SOM-LI neurons of
dorsal root ganglia during pathological processes,
observed both in previous studies (4, 5, 15) and in the
present study, may arise from the augmentation of
SOM synthesis as an adaptive process within the
nervous system, but it could also indicate an inhibition
of the SOM release from nerve endings. The
augmentation of SOM synthesis, which on the grounds
of well-known anti-nociceptive actions of this peptide
(12, 13) is more probable, can be connected with
altered activity of the sensory neurons under noxious
factors. Nonetheless, these mechanisms are very
difficult to explain. Thus, the changes observed in the
present study may be due to primary (damage of
nervous endings) or secondary (pain) results of
adrenalectomy, and can be a reflection of changes in
the transcriptional, translational, or metabolic level.
The differences in the reaction to adrenalectomy
between neurons within neuromeres Th6-7 and
Th12-14 are also worth to be noted. More intensive
increase in the number of SOM-LI FB-labelled neurons
located at neuromeres Th6-7 may suggest that SOMpositive cells of these segments play a more important
role in the regulation of sensory and/or pain
conductivity from the adrenal glands during
pathological processes. Moreover, neurons in
neuromeres Th6-7 can respond more rapidly to damage
of their peripheral endings, which may indicate their
greater dependence on trophic factors produced by
tissues innervated by them.
At present, sensory functions of SOM after
axotomy, and especially after adrenalectomy, are not
known. They can be only generally elucidated by
repairing processes in destroyed cells. It is possible,
that SOM in the porcine axotomised neurons take
control of the functions of nerve growth factors, which
are necessary for regeneration of destroyed fibres. It is
also known that some growth factors, such as glial-cellline-derived neurotrophic factor (GDNF), which is in
charge of repairing processes, modulate the activity and
induce the release of SOM from both central and
peripheral terminals of adult sensory neurons (16).
Neuroprotective and/or neurodegenerative functions of
SOM are more probable. It is well-known that an injury
to neurons causes an increase in the expression of
neurotransmitters, which promote the regeneration of
injured cells, at least in in vitro experiments (1).
Moreover, some actions of SOM after adrenalectomy
may be due to relatively well-known anti-nociceptive
and anti-inflammatory properties of this peptide (21).
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To sum up, results obtained in the study show that
somatostatin takes part in the sensory innervation of
porcine adrenal glands, and is involved in response to
the damaging factors within these organs. Nevertheless,
the exact roles of somatostatin within sensory nervous
system under physiological conditions and during
pathological processes are not fully explained and
require further investigations.
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