ORIGINAL PAPERS

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ORIGINAL PAPERS

ORIGINAL PAPERS
Dent. Med. Probl. 2007, 44, 3, 314–322
ISSN 1644−387X
© Copyright by Silesian Piasts University of Medicine in Wrocław
and Polish Stomatological Association
BARBARA NOWAK1, RAFAŁ OBUCHOWICZ2, WOJCIECH SZCZEPAŃSKI3,
JOANNA ZARZECKA1, WIESŁAW W. PAWLIK2
Importance of Sensory Innervation
in the Leptin Induced Protection of Oral Mucosa
in the Experimental Model
Znaczenie unerwienia czuciowego w ochronie błony śluzowej jamy ustnej
indukowanej przez leptynę w modelu doświadczalnym
1
Department of Conservative Dentistry with Endodontics, Jagiellonian University Medical College, Kraków,
Poland
2
Department of Physiology, Jagiellonian University Medical College, Kraków, Poland
3
Department of Pathology, Jagiellonian University Medical College, Kraków, Poland
Abstract
Background. Ulcerations of an oral mucosa are a common clinical problem. It is reported that in the oral cavity
biologically active leptin receptor OB−R is present on the sensory fibres, known for their protective role in the
mucosa of the GI tract.
Objectives. To evaluate the role of exogenous leptin in to establish healing of ulcerations of the oral mucosa and
mechanisms underlying this action.
Material and Methods. 64 Wistar rats were used. Animals were anesthetized. Oral mucosa ulcerations were pro−
duced (70% acetic acid). After 7 days of healing, ulcer area (UA) was determined and examined histologically.
Microcirculatory blood flow (LDBF) in the ulcer margin was measured. Eight experimental groups were estab−
lished: I – placebo (0.9% saline). II – control (leptin 25 µg/kg s.c.). III – leptin after sensory denervation (capsaicin).
IV – leptin + nitric oxide synthase blocker (L−NNA). V – leptin + L−NNA + nitric oxide donor (SNAP). VI – lep−
tin + CGRP. VII – leptin + CGRP1 receptor blocker (CGRP 8–37). VIII – leptin + L−NNA + CGRP.
Results. Leptin administration markedly decreased UA by 35%, and increased LDBF by 29% in comparison to
placebo group. In sensory denervated animals and after CGRP1 receptor blockade neither protective effects of lep−
tin nor increase in LDBF were observed (UA were markedly increased with concomitant decrease of LDBF in com−
parison to control). Similar effect was observed after blockade of NO synthase, NO donor administration abolished
this effect. After administration of leptin with CGRP significant reduction of UA by 37% was observed with
increase of LDBF by 45% in comparison to the control group. This effect were abolished by administration of NO
synthase blocker.
Conclusions. Leptin accelerates healing of experimentally induced ulcerations of the oral mucosa. This effect
depends on CGRP released from sensory capsaicin sensitive fibres. Observed protective effects are at least in part
due to increased microcirculatory blood flow in the ulcer healing zone what is evoked by NO derived vasorelax−
ation (Dent. Med. Probl. 2007, 44, 3, 314–322).
Key words: oral mucosa, leptin, protection, sensory fibres.
Streszczenie
Wprowadzenie. Owrzodzenia jamy ustnej są częstym problemem klinicznym. Ostatnio wykazano obecność bio−
logicznie aktywnego receptora OB−R dla leptyny na włóknach czuciowych jamy ustnej.
Cel pracy. Wyjaśnienie roli egzogennie podawanej leptyny w gojeniu owrzodzeń jamy ustnej oraz zbadanie me−
chanizmu jej ochronnego działania.
Materiał i metody. Doświadczenia przeprowadzono na znieczulonych 64 szczurach rasy Wistar. Owrzodzenia ja−
my ustnej były wywołane przez podanie 70% kwasu octowego na błonę śluzową policzka szczura. Po 7 dniach
oceniano powierzchnię owrzodzeń (UA) oraz mikrokrążeniowy przepływ krwi w brzegu owrzodzenia (LDBF).
Zwierzęta podzielono na 7 grup doświadczalnych. I grupa – placebo (0,9% NaCl). II grupa – leptyna (25 µg/kg s.c
Sensory Innervation in the Leptin Induced Protection of Oral Mucosa
315
– kontrolna). III grupa odnerwienie sensoryczne – przewlekłe podawanie kapsaicyny. Grupa IV – leptyna + lek bloku−
jący syntazę tlenku azotu (L−NNA). Grupa V – leptyna + LNNA + donor NO (SNAP). Grupa VI – leptyna + CGRP.
Grupa VII – leptyna + lek blokujący receptory CGRP1 – (CGRP8–37). Grupa VIII – leptyna + LNNA + CGRP.
Wyniki. Podanie leptyny znamiennie zmniejszało powierzchnię owrzodzeń o 35%, czemu towarzyszył istotny
wzrost przepływu mikrokrążeniowego w brzegu owrzodzenia o 29% w porównaniu do grupy placebo. U zwierząt
odnerwionych sensorycznie oraz po podaniu leku blokującego receptor CGRP1 nie obserwowano ochronnego dzia−
łania leptyny oraz wzrostu LDBF (w grupach tych UA było znamiennie zwiększone) LDBF zmniejszyło się w po−
równaniu z grupą kontrolną. Podobne działanie obserwowano po zablokowaniu syntazy NO. Podawanie donora
NO niwelowało je. Po podaniu leptyny oraz CGRP obserwowano znamienne zmniejszenie UA o 37% z towarzy−
szącym wzrostem LDBF w brzegu owrzodzenia o 45% w porównaniu do grupy kontrolnej. Działanie było znie−
sione przez wcześniejsze zastosowanie leku blokującego syntazę tlenku azotu.
Wnioski. Leptyna znamiennie przyspiesza gojenie doświadczalnych owrzodzeń błony śluzowej jamy ustnej. Dzia−
łanie to zależy od CGRP uwalnianego z zakończeń kapsaicynowrażliwych neuronów czuciowych. Obserwowane
działanie ochronne koreluje ze zwiększonym ukrwieniem obszaru gojenia i zależą od działania tlenku azotu roz−
szerzającego naczynia (Dent. Med. Probl. 2007, 44, 3, 314–322).
Słowa kluczowe: błona śluzowa jamy ustnej, leptyna, ochrona, włókna czuciowe.
Mucosa of the oral cavity is particularly sus−
ceptible to trauma as it is constantly exposed to
different physical and chemical irritants applied
with food. Beside of challenging physiological
stimuli (associated with food intake), there are
many entities pathological and iatrogenic – ie. dia−
betes, infections, chemotherapy or radiotherapy
severely affecting mucosal integrity.
Mechanisms of resistance of the mucosa of
gastrointestinal tract (GI) to exogenous and
endogenous irritants are well understood due to
investigations upon integrity of the stomach wall
considered as mostly endangered to mechanical,
thermal and especially chemical trauma [1, 2]. It is
widely accepted that nociceptive afferents wide−
spread in the mucosa act not only as sensors of dif−
ferent parameters of luminal content but also play
a role in the maintenance of mucosal resistance.
Large number of studies has confirmed ability and
importance of sensory fibres and their neurohor−
monal mediator CGRP to improve mucosal viabil−
ity, what is primarily associated with increased
blood flow and therefore oxygenation at the site of
injury or healing ulceration [3]. The importance of
neuropeptides released by sensory fibres in the
protection of the mucosa of oral cavity is not so
well know. There is an evidence that buccal and
gingival mucosa is richly innervated by sensory
fibres which originates from trigeminal and spinal
afferent nerves [4, 5]. The common feature of
afferent neurons is ability to release neuromedia−
tors following contact with noxious factors (most−
ly chemical, thermal or mechanical). One of the
effects of an active peptides release is vasodilata−
tion of resistance of microcirculatory vessels that
leads to potentially protective effect of hyperemia
with redness, excessive mucous production and
local warmth of affected area, collectively known
as neurogenic inflammation [6]. This effect sug−
gests to be associated with nitric oxide synthase
activation (NOS) which produces strong vasodila−
tor (NO) currently proposed as the end−mediator
of the protective effect of sensory neurons [7].
Collectively those mechanisms evoked by activa−
tion of local afferent fibres, could potentially
accelerate healing of the oral mucosa injured by
various factors.
Leptin is a molecule like cytokine, primarily
discovered as a hormone involved in the control of
body weight and energy expenditure. This
pleiotropic 167 aminoacid peptide is predominate−
ly secreted by a white adipocytes (there are also
different sources of leptin presented as tissues of
GI and reproductive tract). Mean hormone level in
peripheral blood is 16 pmol/L. This value is body
mass and digestive phase dependent. Biological
effects of leptin are induced via its specific mem−
brane OB−R receptor, a transmembrane moiety
acting through tyrosine kinase. Peripherally local−
ized OB−R receptors have been primarily detected
in gastric, endocrine and exocrine pancreatic and
intestinal tissue [8–11]. Recently the presence of
OB−R receptors was confirmed in salivary glands
and oral mucosa, and in particular on afferent
C fibres which are abundant in the oral cavity
mucosa [12]. Protective influence of leptin in the
oral cavity in the model of experimental ulcers
healing hed been proposed already [13], but the
mechanisms of enhanced healing of mucosal
ulcerations induced in this part of digestive tract
after exposure to exogenous leptin has not been
evaluated yet.
Therefore the aim of current study was to
investigate the role of exogenously administered
leptin in the healing of experimentally induced
ulcerations of the buccal mucosa. We also attempt−
ed to clarify the mechanism of the potential pro−
tective action of the leptin on buccal mucosa with
special interest in the evaluation of the importance
of sensory fibres in this phenomenon.
316
B. NOWAK et al.
Material and Methods
Experimental animals were 64 Wistar rats of
both sex, weighing 270–320 grams. Experimental
procedures conform to guidelines of Animals
Research Committee of Jagiellonian University.
Experimental animals were divided in 8
groups (n = 8). I placebo group – administered
with saline 0,5 ml s.c. II control group – leptin
alone given in the dose of 25 µg/kg s.c., III –
administration of leptin (25 µg/kg s.c.), in sensory
denervated animals (capsaicin in increasing
doses). IV – leptin (25 µg/kg s.c.), + nitric oxide
synthase blocker L−NNA (40 mg/kg i.p.), V – lep−
tin (25 µg/kg s.c.) + L−NNA (40 mg/kg i.p.) +
nitric oxide donor SNAP (25 mmol/kg i.p.), VI –
leptin (25 µg/kg s.c.) + CGRP (20 µg/kg s.c.), VII
– leptin (25 µg/kg s.c.)+ CGRP 8–37 (100 µg/kg
i.p.), VIII – leptin (20 µg/kg s.c.) + L−NNA (40
mg/kg i.p.) + CGRP (20 µg/kg s.c.).
Drugs Used for Experiment
For the current study following drugs were
used: leptin – an agonist of OB−R receptor at the
dose of 25 µg/kg s.c., L−NNA a selective blocker
of nitric oxide synthase – administered at the dose
of 40 mg/kg i.p., nitric oxide donor (SNAP) –
intraperitoneally in the dose of 25 mmol/kg, a neu−
rohormonal mediator Calcitonin Gene Related
Peptide (CGRP) – 20 ug/kg s.c., selective blocker
of CGRP1 receptor – intraperitoneally in dose of
100 µg/kg. Leptin was given three times daily
alone or collectively, with other aforementioned
drugs in the period of ulcer healing (7 days).
Animals were fasted for 24 hours with free
access to water allowed. Rats were anesthetized
with Sodium phentobarbital (20 mg/kg i.p.). In the
group of animals, were importance of sensory
C fibres in the mediation of leptin effects was stud−
ied, ablation of sensory neurons under ether anes−
thesia with use of increasing doses of capsaicin (5,
10, 25 and 50 mg/kg s.c.) was performed, in the 72
hours time period, one week before induction of
buccal ulcerations. Ulcerations of buccal mucosa
were produced with use of plastic mold (4 mm
diameter). Mold was tightly placed to exposed
buccal mucosa. 40 µL of 70% acetic acid was
applied and allowed to remain with the contact
with buccal mucosa for 30 seconds. This proce−
dure evoked immediate necrosis of mucosa in an
area of 10 ± 1 mm2. Necrotic area was gently
rinsed with water. Animals were kept for 7 days in
the cages at room temperature with free access to
food (standard rodent meal) and water. In 6th day
of experiment animals were fasted. After next 24
hours rats were provided with anesthesia (Sodium
pentobarbital 20 mg/kg i.p.), then placed on heat−
ing pad, body temperature was kept at 37oC and
monitored with rectal thermistor with regulator
(FST TR−100). After induction of anaesthesia in
each animal microcirculatory blood flow was
determined in the margin of the ulceration and
adjacent healthy buccal mucosa with Laser
Doppler Flowmeter (Periflux 4001 Master,
Perimed Sweden). Signal (a 420 nm light beam)
was emitted and collected with use of fiberoptic
probe positioned against the surface of the buccal
mucosa. The probe was secured aside of the ani−
mal with use of special dedicated holder designed
to eliminate movement of tip of the probe against
the examined tissue. Spectral Doppler signal
inversion in the close pole was detected and
processed with use of fast Fourier analysis.
Microcirculatory blood flow was expressed in
arbitrary units (PU). Buccal tissue was positioned
on experimental table and holded with metal clips.
After completed measurements animals were sac−
rificed by excessive dose of the anesthetics.
Buccal mucosa with the ulceration produced and
adjacent tissue was photographed. Area of the
ulceration was determined with use of planimetri−
cal 2D analysis of high resolution photograph
(Olympus 20 D digital camera). Buccal mucosa
was removed and processed for histological analy−
sis using standard histologic techniques including:
formalin fixation, dehydration and paraffin
embedding, then cuted in 4−µm sections and
stained with Hematoxillin and Eosin. Specimens
were assessed with use of the optical microscope
(100–200 × magnification) by pathologist, not pro−
vided with information according pharmacological
agents used in different groups. Level of mucosal
tissue injury was expressed with the use of 4 points
scale prepared exclusively for the study. Grade I
– epithelialised mucosa without inflammatory
infiltration. Grade II – epithelial mucosa with
subepithelial inflammatory infiltration. Grade III
– partially healed ulcer with central area of granu−
lation tissue and inflammatory infiltration. Grade IV
– not healed ulcer lined, with granulation tissue
with underlying inflammatory infiltration.
The significance of changes in measured para−
meters was determined using Student’s test for
either paired data with a confidence limit of 0 < 0.05.
Percentage differences in specific parameters were
compared with control, calculated as a mean average
± S.E.M., with n = 8 per each experimental group.
Results
In the placebo group (group I) microcirculato−
ry blood flow was 160 and 210 PU in the healthy
317
buccal mucosa and ulcer margin respectively.
Ulcer damage reached 4 in the own histological
scale. Administration of leptin (group II) evoked
marked increase of LDBF in the buccal mucosa
and the margin of the ulcer by 16 and 29% respec−
tively. Buccal ulcerations were markedly healed in
comparison to placebo group (Fig. 1). Ulcer area
was decreased by 35% reaching point 2 in own
histological scale (Fig. 6, 8).
200
*
group after leptin administration (25 µg/kg s.c.)
grupa po podaniu leptyny
140
120
100
80
sensory denervation (5, 10, 25, 50 mg/kg s.c. Capsaicin/72 h)
odnerwienie sensoryczne
sensory denervation (5, 10, 25, 50 mg/kg s.c. Capsaicin/72 h) + leptin (25 µg/kg s.c.)
odnerwienie sensoryczne + leptyna
*
150
*
100
*
*
n.s.
50
microcirculatory blood flow (PU)
mikrokr¹¿eniowy przep³yw krwi
placebo group (0.9% NaCl s.c.)
grupa placebo
180
160
leptin alone (25 µg/kg s.c.)
sama leptyna
n.s.
0
% change in comparison to placebo group
% zmian w porównaniu z grup¹ placebo
200
% change in comparison to leptin alone
% zmian w porównaniu z sam¹ leptyn¹
*
60
40
20
ulcer area (mm2)
powierzchnia owrzodzeñ
Fig. 2. Changes of microcirculatory blood flow in the
ulcer margin and area of the ulceration after 7 days of
healing in comparison to leptin alone group. Asterisks
indicate significant difference in comparison to group
after leptin alone
Ryc. 2. Zmiany w mikrokrążeniu krwi w porównaniu
z grupą z leptyną. Gwiazdki wskazują na istotną
różnicę w porównaniu z grupą z leptyną
0
ulcer area (mm2)
healing in comparison to placebo group. Asterisks
indicate significant difference in comparison to place−
bo group
Ryc. 1. zmiany mikrokrążeniowego przepływu krwi
w brzegu oraz obszarze owrzodzenia po 7 dniach goje−
nia w porównaniu z grupą placebo. Gwiazdki wskazują
na istotną różnicę w porównaniu z grupą placebo
200
sama leptyna
leptin (25 µg s.c.) + L-NNA (40 mg/kg i.p.)
leptyna + L-NNA
150
leptin (25 µg/kg s.c.) + L-NNA (40 mg/kg i.p.)
+ SNAP ( 25 mmol/kg i.p.)
leptyna + L-NNA + SNAP
*
100
*
50
p<0,05
p<0,05
0
After leptin administration in sensory dener−
vated animals (group III) marked decrease of
microcirculatory blood flow in the buccal mucosa
and ulcer margin by 26 and 35% in comparison
with leptin alone (control group) was observed
(Fig. 2). Ulcer area was increased by 45% in com−
parison with leptin alone and was estimated as 4 in
histological scale (Fig. 6, 10).
Administration of leptin in animals pretreated
with nitric oxide blocker (group IV) evoked a sig−
nificant decrease of microcirculatory blood flow in
the buccal mucosa and ulcer margin by 18 and
24% in comparison with leptin alone group. Ulcer
area was increased by 39% in comparison with
leptin alone and was estimated as 4 in histological
scale (Fig. 6).
In the group where leptin and NO blocker
administration were followed by the treatment
with nitric oxide donor (group V), no marked dif−
ference on the microcirculatory blood flow in the
buccal mucosa and ulcer margin in comparison to
leptin alone group was observed. Differences in
histological picture and area of the ulcerations
ulcer area (mm2)
after leptin alone
were not significant in comparison to leptin alone
group (Fig. 3).
Pretreatment with leptin of the group were
CGRP was administered (group VI) evoked signif−
icant increase of LDBF by 33 and 46% in buccal
mucosa and ulcer margin respectively in compari−
son to leptin alone. Ulcer area was reduced by 37%
in comparison to leptin alone. Histological picture
of the ulcerations were estimated as 1 (Fig. 6, 7).
Blockade of CGRP1 receptors by CGRP 8–37
(group VII) induced marked decrease of LDBF by
26 and 19% in buccal mucosa and ulcer margin
318
B. NOWAK et al.
own scale of histological injury
w³asna skala uszkodzeñ histologicznych
5
sama leptyna
4,5
leptin (25 µg s.c.) + CGRP (20 µg/kg s.c.)
leptyna + CGRP
200
4
leptin (25 µg/kg s.c.) + CGRP 8-37 (100 µg/kg i.p.)
leptyna + CGRP 8-37
*
150
*
3,5
3
2,5
100
*
*
2
1,5
p<0,05
50
p<0,05
1
0,5
0
0
ulcer area (mm2)
after leptin alone
placebo
leptin
alone
sama
leptyna
leptin +
leptin +
capsaicin
L-NNA
leptyna + leptyna +
kapsaicyna
L-NNA
leptin +
L-NNA +
SNAP
leptyna +
L-NNA +
SNAP
leptin
leptin +
leptin +
+ CGRP CGRP 8-37 CGRP +
leptyna + leptyna +
L-NNA
CGRP
CGRP 8-37 leptyna +
CGRP +
L-NNA
Fig. 6. Own scale of histological injury based on the
histological picture of the specimens of injured buccal
mucosa obtained in a different experimental groups
Ryc. 6. Własna skala zmian histopatologicznych. Skala
uwzględnia obrazy histopatologiczne wycinków uszkod−
zonej błony śluzowej policzka w różnych grupach
Discussion
respectively (Fig. 4). Ulcer area was increased by
24% in comparison to leptin alone and reached
3 in the own histological scale (Fig. 6, 7).
Administration of CGRP after blockade of
nitric oxide synthase by L−NNA (group VIII)
evoked significant decrease of microcirculatory
blood flow in the buccal mucosa and ulcer margin
by 36 and 25% in comparison with leptin alone
group (Fig. 5). Ulcer area was increased by 23% in
comparison with leptin alone and was estimated as
4 in histological scale (Fig. 6, 10).
200
sama leptyna
leptin (25 µg s.c.) + L-NNA (40 mg/kg i.p.)
leptyna + L-NNA
150
100
50
leptin (25 µg/kg s.c.) + L-NNA (40 mg/kg i.p.)
+ CGRP (20 µg/kg s.c.)
leptyna + L-NNA + CGRP
*
*
*
*
n.s.
n.s.
0
ulcer area (mm2)
after leptin alone
Ulcer healing is a complex process. Activation
of fibroblasts, keratinocytes and white blood cells
secreting different lymphokines and growth fac−
tors forms a complex network of matrix−cellular
level interactions what finally leads to formation
of granulation tissue in the zone of repair [14]. All
those processes relay on oxygen delivery to the
site of the healing and are strictly dependent on
function of the vascular system, especially from
status of local microcirculation of mucosa at the
site of the injury. Relaxation of arterioles and pre−
capillary sphincters in the terms of tissue damage
maintains hyperemia what plays an important role
in the healing process [15, 16].
In the present study we demonstrate that
exogenously administered leptin induces marked
increase of microcirculatory blood flow in the oral
mucosa (at the ulcer margin – zone of repair) what
was accompanied with significant acceleration of
ulcer healing. Observed correlation between
increased microcirculatory blood flow and elevat−
ed mucosal resistance (increased healing rate of
the injury) has a strong support in the studies per−
formed upon influence of leptin on healing of the
gastric ulcers induced by stress or different nox−
ious factors [17]. The ability of leptin to improve
tissue viability in correlation with locally
increased blood flow was also presented in the
model of acute pancreatitis [18]. In the present
study we focus our attention on importance of sen−
sory fibres in the mediation of protective influence
of leptin. Mucosal functional hyperemia in the GI
tract and oral cavity strongly depends on activa−
319
Fig. 7. Histological specimen of the injured mucosa.
Ulcer healing, grade I. Epithelialised mucosa without
inflammatory infiltration (HE 50 ×)
Ryc. 7. Obraz histopatologiczny uszkodzonej błony
śluzowej. Gojenie owrzodzenia, stopień I.
Nabłonkowanie bez nacieku zapalnego (HE 50 ×)
Ulcer healing grade III. Partially healed ulcer with
central area of granulation tissue and inflammatory
infiltration (HE 100 ×)
śluzowej. Gojenie owrzodzenia, stopień III.
Owrzodzenie częściowo wygojone z tkanką ziarni−
nową i naciekiem zapalnym pośrodkowo (HE 100 ×)
Ulcer healing grade II. Epithelialised mucosa with
subepithelial inflammatory infiltration (HE 200 ×)
śluzowej. Gojenie owrzodzenia, stopień II.
Nabłonkowanie z podnabłonkowym naciekiem zapal−
nym (HE 200 ×)
Ulcer healing grade IV. Not healed ulcer lined with
granulation tissue with underlying inflammatory infil−
tration (HE 100 ×)
śluzowej. Gojenie owrzodzenia, stopień IV. Niewygo−
jone owrzodzenie z tkanką ziarninową i naciekiem za−
palnym (HE 100 ×)
tion of the sensory fibres that mediates local short
reflexes, including vascular [19]. It is confirmed
that biologically active (potentially stimulating)
OB−R leptin receptors are present on afferent sen−
sory fibres which densely innervate gingival and
buccal mucosa. Common feature of this peptide
releasing fibres (hence known as peptydergic) is
a presence of vanilloid VR1 receptor activated by
capsaicin – pungent principle of red pepper [20,
21]. Buccal sensory fibres are mostly from spinal
origin where they predominate – inferior alveolar
nerve consists up to 80% of thin sensory fibres,
which anatomically branches in the tissue, follow−
ing the course of small caliber submucosal blood
vessels. Trigeminal system also leads sensory
fibres but they are in the minority (up to 50%),
among motor and autonomic fibres and innervate
dental pulp, taste buds and mucosa of the palate
mostly [4, 5, 22]. One of the well documented fea−
ture of those slow conducting, thin, unmielinated
fibres is an ability to release CGRP and neu−
rokinins substance P (SP), neurokinin 1 (NK1) and
320
neurokinin 2 (NK2) in the vicinity of submucosal
arterioles and veins [23, 24]. Neurohormons, if
released, act as potent vasodiltatory substances
able to abolish tonic vasoconstrictory effects of
postganglionic sympathetic adrenergic neurons
what induces hyperemia. As the presence of OB−
R receptors on peptydergic neurons had been dis−
covered, we proposed that leptin acting through
biologically active receptors is compatible to acti−
vate sensory fibres in the oral cavity and induce
vasodilation at the site of mucosal damage. To
prove this thesis we partially abolished sensory
C fibres by capsaicin and we observed marked
decrease of mucosal resistance expressed by
reduced healing rate of the ulcers (assessed histo−
logically). Moreover, in the chronically denervat−
ed animals significant decrease of microcirculato−
ry blood flow in the margin of the ulcerations in
the comparison to normal animals with preserved
sensory innervation was observed. This is kept
under observation of authors reporting that protec−
tive effect of leptin was abolished after sensory
fibres ablation in different parts of digestive sys−
tem (especially stomach, intestine and pancreas)
[8, 11, 17, 25, 26].
CGRP is currently proposed to be a mediator
of sensory neurons inducing vasodilatation and
hence protection in the GI tract. No data according
importance of the CGRP in the leptin mediated
protection of the oral mucosa exists, therefore we
performed experiments in order to clarify its role
in the protection of this part of digestive tract.
Blockade of CGRP1 receptor markedly decreased
(observed after leptin alone) protective effect of
leptin on buccal mucosa. Microcirculatory blood
flow in the ulcer margin was markedly diminished
but not abolished in comparison to leptin alone
group. This effect might be due to action of the
other mediators as tachykinins released concomi−
tantly with CGRP from endings of activated
C fibres that is kept under observation made by
authors investigating role of CGRP in the protec−
tion of the other parts of GI tract [3, 24].
Importance of CGRP as an mediator of the protec−
tive action of leptin in the oral mucosa was
stressed by results of the experiments, in cases
where CGRP was administered to leptin pretreated
animals. Marked increase of microcirculatory
blood flow in the ulcer margin was observed. That
was accompanied by histologically proved accel−
eration of the ulcer healing in the comparison to
leptin alone. Those effects supported our former
observations according close correlation between
ulcer healing rate and increase of blood flow in
granulation tissue in the ulcer margin what sug−
gests importance of CGRP as a vasoactive media−
tor in the leptin induced mucosal resistance.
B. NOWAK et al.
The importance of nitric oxide as an end medi−
ator of neuropeptide induced protection in the GI
tract is proved. Especially in the gastric and
intestinal mucosa, this gas molecule was proposed
to be a key factor participating in the healing of
ulcerations. Moreover, this fat soluble, signal par−
ticle is recognized to be responsible for the main−
tenance of the viability of tissues of the digestive
tract [7, 16]. Importance of nitric oxide in the
mediation of neurally mediated, leptin induced
protection of the oral mucosa has not been
explored yet. In our model the blockade of nitric
oxide synthase by the use of modified substrate for
the enzyme (L−nitro arginine), evoked marked
decrease of microcirculatory blood flow in the
margin of the ulcer with subsequent decrease of
healing in comparison to leptin alone group.
Administration of SNAP – an NO donor reversed
effect of the blocker what confirmed importance
of NO in the mediation of the leptin induced buc−
cal mucosa protection. This observations are con−
sistent with recognized experimentally in different
parts of GI tract ability of NO to mediate protec−
tive properties of neuropeptides [27, 28]. It is sug−
gested that observed effects are possibly due to
ability of NO to induce vasodilatation and hence
increase delivery of nutrients to the healing zone.
Those data indicate that vasoactive neuropeptide
(CGRP) collectively with its gas end mediator
(NO) influenes the increase of local capillary
exchange area.
To examine the involvement of NO in CGRP
buccal mucosa protection induced by leptin we
applied CGRP to rats treated with leptin but pread−
ministered with L−NNA. There was no significant
difference in the microcirculatory blood flow and
healing rate of gastric ulcerations in the animals
pretreated by NO synthase blocker alone, and with
the group were CGRP was administered collec−
tively with L−NNA (in both groups leptin was
given). It suggests that action of leptin in the oral
mucosa healing is strictly dependent on nitric
oxide action as final mediator of CGRP induced
vasodilatation. This is currently not recognized, if
protective action of leptin solely depends on
improved nutrients and oxygen delivery due to
increased capillary exchange area, or observed
effects are more coused by the direct stimulation
of the tissue [13]. It could not be excluded that
local circulatory effects in the ulcer margin
observed after leptin administration originate from
the increased angiogenesis at the site of the ulcer
healing with secondary elevation of LDBF.
Significant differences in microcirculatory blood
flow in the healthy oral mucosa between placebo
only and leptin pretreated groups speaks for
importance of the hormone in the control of micro−
circulation, especially vasorelaxation inducing
effect. Phenomenon of stimulated angiogenesis
and increased blood flow induced due to relax−
ation of smooth muscles in resistance vessels wall
are probably simultaneous effects in the margin of
ulcerations.
To conclude we propose that exogenously
administered leptin accelerates healing of acute
321
ulcerations of the oral mucosa. Phenomenon of
increased microcirculatory blood flow in the oral
mucosa accompanying accelerated healing effect
was recognized, what at least in part is due to
increased oxygen delivery and capillary exchange
area. Observed effect is dependent on CGRP which
acts through nitric oxide what leads to decrease of
the tone of precapillary sphincteric vessels.
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Address for correspondence:
Barbara Nowak
Department of Conservative Dentistry with Endodontics
Jagiellonian University Medical College
Montelupich 4
31−155 Kraków
Poland
Received: 6.09.2007
Revised: 25.09.2007
Accepted: 25.09.2007
Praca wpłynęła do Redakcji: 6.09.2007 r.
Po recenzji: 25.09.2007 r.
Zaakceptowano do druku: 25.09.2007 r.

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