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 Sensory Innervation in the Leptin Induced Protection of Oral Mucosa 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 microcirculatory blood flow (PU) mikrokr¹¿eniowy przep³yw krwi ulcer area (mm2) powierzchnia owrzodzeñ Fig. 1. Changes of microcirculatory blood flow in the ulcer margin and area of the ulceration after 7 days of 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 % change in comparison to leptin alone % zmian w porównaniu z sam¹ leptyn¹ 200 leptin alone (25 µg/kg s.c.) 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 microcirculatory blood flow (PU) mikrokr¹¿eniowy przep³yw krwi ulcer area (mm2) powierzchnia owrzodzeñ Fig. 3. 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. 3. Zmiany w mikrokrążeniu krwi w porównaniu z grupą z leptyną. Gwiazdki wskazują na istotną różnicę w porównaniu z grupą z leptyną 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 % change in comparison to leptin alone % zmian w porównaniu z sam¹ leptyn¹ 5 leptin alone (25 µg/kg s.c.) 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 microcirculatory blood flow (PU) mikrokr¹¿eniowy przep³yw krwi ulcer area (mm2) powierzchnia owrzodzeñ Fig. 4. 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. 4. Zmiany w mikrokrążeniu krwi w porównaniu z grupą z leptyną. Gwiazdki wskazują na istotną różnicę w porównaniu z grupą z leptyną 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). % change in comparison to leptin alone % zmian w porównaniu z sam¹ leptyn¹ 200 leptin alone (25 µg/kg s.c.) 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 microcirculatory blood flow (PU) mikrokr¹¿eniowy przep³yw krwi ulcer area (mm2) powierzchnia owrzodzeñ Fig. 5. 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. 5. Zmiany w mikrokrążeniu krwi w porównaniu z grupą z leptyną. Gwiazdki wskazują na istotną różnicę w porównaniu z grupą z leptyną 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− Sensory Innervation in the Leptin Induced Protection of Oral Mucosa 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 ×) Fig. 9. Histological specimen of the injured mucosa. Ulcer healing grade III. Partially healed ulcer with central area of granulation tissue and inflammatory infiltration (HE 100 ×) Ryc. 9. Obraz histopatologiczny uszkodzonej błony śluzowej. Gojenie owrzodzenia, stopień III. Owrzodzenie częściowo wygojone z tkanką ziarni− nową i naciekiem zapalnym pośrodkowo (HE 100 ×) Fig. 8. Histological specimen of the injured mucosa. Ulcer healing grade II. Epithelialised mucosa with subepithelial inflammatory infiltration (HE 200 ×) Ryc. 8. Obraz histopatologiczny uszkodzonej błony śluzowej. Gojenie owrzodzenia, stopień II. Nabłonkowanie z podnabłonkowym naciekiem zapal− nym (HE 200 ×) Fig. 10. Histological specimen of the injured mucosa. Ulcer healing grade IV. Not healed ulcer lined with granulation tissue with underlying inflammatory infil− tration (HE 100 ×) Ryc. 10. Obraz histopatologiczny uszkodzonej błony 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− Sensory Innervation in the Leptin Induced Protection of Oral Mucosa 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. 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L.: Intrinsic regulation of CGRP release by dental pulp sympathetic fibres. J. Dent. Res. 2003, 82, 398–401. [23] FRISTAD I., VANDEVSKA−RADUNOVIC V., FJELD K., WIMALAWANSA S. J., HALS KVINNSLAND I.: NK1, NK2, NK3 and CGRP1 receptors identified in oral soft tissues, and in bone and dental hard tissue cells. Cell Tissue Res. 2003, 311, 383–391. 322 B. NOWAK et al. [24] HOLZER P.: Local effector function of capsaicin−sensitive sensory nerve endings: involvement of tachykinins, cal− citonin gene related peptide and other neuropeptides. Neuroscience 1988, 24, 739–768. [25] KONTUREK P. C., KONTUREK S. J., BRZOZOWSKI T., JAWOREK J., HAHN E. G.: Role of leptin in the stomach and the pancreas. J. Physiol. (Paris) 2001, 95, 345–354. [26] HACIOGLU A., ALGIN C., PASAOGLU O., KANBAK G.: Protective effect of leptin against ischemia/reperfusion injury in the rat small intestine. BMC Gastroenterol. 2005, 21, 37–42. [27] KAWASAKI H.: Regulation of vascular function by perivascular calcitonin−gene−related peptide−containing nerves. Jpn. J. Pharmacol. 2002, 88, 39–43. [28] PAWLIK W. W., OBUCHOWICZ R., BIERNAT J., SENDUR R., JAWOREK J.: Role of calcitonin gene related peptide in the modulation of intestinal circulatory, metabolic, and myoelectric activity during ischemia/reperfusion. J. Physiol. Pharmacol. 2000, 51, 933–942. 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.