The aim of this study was to examine the expression of nicotinamide adenine dinucleotide
phosphate-diaphorase (NADPH-d) activity in human dental pulps and determine
whether there are changes of the activity in chronically inflamed pulp tissue. Nineteen
pulps with clinical diagnosis of chronic pulpitis were collected during endodontic
treatment. The healthy controls were obtained from teeth extracted for orthodontic
therapy. The clinical diagnosis was confirmed by histological analysis. Healthy pulps
showed stratified odontoblasts in peripheral parts, while in central area there was normal
connective tissue. Chronically inflamed pulps showed less expressed stratification
of odontoblasts and infiltration of lymphocytes, polymorphonuclear leukocytes, plasma
cells and mastocytes. NADPH-d granular reactivity was assessed semi quantitatively
under the light microscope by a single observer and scored on an intensity scale from
negative reaction to very strong reaction. In healthy human pulps, NADPH-d activity
was strong to very strong in odontoblastic layer. Endothelial cells and Schwann cells
showed strong NADPH-d reactivity, while the other parts of central area were weakly
positive. Similar distribution of reactivity was expressed also in chronically inflamed
pulp; moderate to strong reaction was observed in stromal area as result of positive reaction
in inflammatory cells and endothelial cells of abundant newly formed capillaries

D. Buković

E. Neziri

I. Miletić

J. Talan-Hranilović

S. Jukić

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Coll. Antropol. 26 (2002) 2: 681–688UDC 616.314.18-002.2:616-074Original scientific paperNicotinamide Adenine DinucleotidePhosphate-Diaphorase (NADPH-d)Histochemistry Detecting NOS inHealthy and Chronically InflamedPulpS. Juki}1, J. Talan-Hranilovi}2, D. Bukovi}3, I. Mileti}1 and E. Neziri41 Department of Dental Pathology, School of Dentistry, University of Zagreb, Zagreb, Croatia2 Department of Pathology »Ljudevit Jurak«, University Hospital »Sestremilosrdnice«, Zagreb, Croatia3 Department of Prosthetic Dentistry, School of Dentistry, University of Zagreb, Zagreb,Croatia4 Private Dental practice, Gare{nica, CroatiaA B S T R A C TThe aim of this study was to examine the expression of nicotinamide adenine dinu-cleotide phosphate-diaphorase (NADPH-d) activity in human dental pulps and deter-mine whether there are changes of the activity in chronically inflamed pulp tissue. Ni-neteen pulps with clinical diagnosis of chronic pulpitis were collected during endodon-tic treatment. The healthy controls were obtained from teeth extracted for orthodontictherapy. The clinical diagnosis was confirmed by histological analysis. Healthy pulpsshowed stratified odontoblasts in peripheral parts, while in central area there was nor-mal connective tissue. Chronically inflamed pulps showed less expressed stratificationof odontoblasts and infiltration of lymphocytes, polymorphonuclear leukocytes, plasmacells and mastocytes. NADPH-d granular reactivity was assessed semi quantitativelyunder the light microscope by a single observer and scored on an intensity scale fromnegative reaction to very strong reaction. In healthy human pulps, NADPH-d activitywas strong to very strong in odontoblastic layer. Endothelial cells and Schwann cellsshowed strong NADPH-d reactivity, while the other parts of central area were weaklypositive. Similar distribution of reactivity was expressed also in chronically inflamedpulp; moderate to strong reaction was observed in stromal area as result of positive re-action in inflammatory cells and endothelial cells of abundant newly formed capillar-ies.681Received for publication October 18, 2002IntroductionThe endogenous production of nitricoxide (NO) plays a role in the regulationof the physiological processes such asblood vessel tone and neurotransmission,as well as in host defence and immuni-ty1,2. The finding of nitric oxide synthaseexpression in human cancers indicates itsrole in pathophysiology of carcinogene-sis3. Nitric oxide is synthesized via oxida-tion of arginine by a family of nitric oxidesynthases (NOS). There are three iso-forms of NOS. Two of them are dependenton elevated intracellular Ca2+ and becau-se of their constitutive appearance aretermed cNOS. These isoforms are NOS1(also called nNOS or ncNOS) which isfound in neurons and NOS3 (eNOS orecNOS) which originated from endothe-lial cells. The NOS2 cloned from macro-phages is independent of elevated intra-cellular Ca2+. Its expression is inducible(iNOS) by cytokines and bacterial lipo-polysaccharide4. However, all NOS areflavoproteins that require nicotinamideadenine dinucleotid phosphate (NADPH)and tetrahydrobiopterin as co-factors5.Nicotinamide adenine dinucleotide phos-phate-diaphorase (NADPH-d) has beenfound to be highly resistant to many pro-tein fixatives and hence visualisation ofNADPH-d activity by histochemical reac-tion of tissue sections has become a viabletechnique for localising NOS activity6,7.In previous research NADPH-d histo-chemistry of dental pulp has been done onrats and cats8. The expression of NADPH-dactivity has been never examined in hu-man pulp tissue yet, neither the changesof its activity in chronically inflamedpulp. In this study we tried to localizeNADPH-d activity in human dental pulpand to find out whether there are changesof the NADPH-d activity in chronicallyinflamed pulp tissue.Material and MethodsNineteen samples of human inflamedpulp tissue were obtained from teeth dur-ing endodontic treatment with clinical di-agnosis of chronic pulpitis. The patientswere between 20 and 45 years old. Ninepulp samples were obtained from femaleand ten from male patients. The pulp tis-sue was extirpated by Hödstrom files(Maillefer, Ballaigues, Switzerland) afteranaesthesia and preparing the access ca-vity. The tissue was removed gently fromthe endodontic file by tweezers and pla-ced in aluminium foil, compressed to eva-cuate the air and frozen in fluid nitrogen.Fifteen healthy controls (seven femaleand eight male) were obtained from teethextracted because of orthodontic reasons.Extracted teeth were grooved by high-speed diamond drill with water coolant toone half of the thickness of dentine alongthe tooth axis. They were split by sharpextraction lever and hammer. The pulptissue was extirpated and processed asdescribed. The samples were frozen incryostat at –25  C, and 8  m-thick sec-tions of the tissue were cut.The incubation media for NADPH-dhistochemistry was used according themethod described by Pearce9. Preparedincubation media was put on tissue cutsmounted on glass slides and left 20 minat 37  C. Then the glasses were washedonce in formol saline, twice in distilledwater and enclosed in gelatine. Also adja-cent sections were stained with haema-toxylin and eosin.All samples were examined under thelight microscope. The reaction of NADPH-dis granular in the cells. Reactivity was as-sessed by a single observer and scored byexamination of five randomly chosen fieldson an intensity scale from 0 to ++++, with0 indicating negative reactivity; + slightreactivity; ++, moderate reactivity; +++strong reactivity; and ++++ very strongreactivity e.g. dense granular reaction. To682S. Juki} et al.: NADPH-d in the Human Pulp, Coll. Antropol. 26 (2002) 2: 681–688enable statistic analysis intensity scalewas transformed to numbers: + – 1, ++ –2, +++ – 3, ++++ – 4. Statistical analysiswas done by descriptive statistic for pop-ulation (p < 0.05).ResultsThe sections of the healthy pulp show-ed peripheral area with more or less con-served palisade arrangement of odonto-blasts with subodontoblastic layer that islacking the cell nucleus. Cell rich zonewas underlying peripheral part. Centralpart consists of fibroblasts, blood vessels,nerves and connective fibbers (Figure 1).Chronically inflamed pulp tissue showedabundance of lymphocytes, polymorpho-nuclear leukocytes, plasma cells and ma-stocytes. Odontoblasts had irregular strati-fication. Mineralizations were often found(Figure 2).683S. Juki} et al.: NADPH-d in the Human Pulp, Coll. Antropol. 26 (2002) 2: 681–688Fig. 1. Healthy pulp (HE, original magnification  40).Fig. 2. Inflamed pulp with disarrangement of odontoblasts, abundanceof capillaries and oedema (HE, original magnification  100).In human healthy pulps, NADPH-dactivity was strong (+++) to very strong(++++) and observed predominantly inodontoblastic cells. Endothelial cells andSchwann cells showed strong reactivity.In the other cells of the stromal area theactivity was slight (+) (Figure 3).Strong (+++) to very strong (++++)NADPH-d activity was obtained in odon-toblasts of chronically inflamed dentalpulp. Strong reactivity (+++) was observ-ed in stromal part of the pulp tissue dueto strong activity expressed in polymor-phonuclear leukocytes and lymphocytes,and plenty of new-born capillaries withNADPH-d positive endothelial cells (Fig-ure 4).684S. Juki} et al.: NADPH-d in the Human Pulp, Coll. Antropol. 26 (2002) 2: 681–688Fig. 3. NADPH-d immunohistochemistry of healthy pulp showing positive odontoblastic and sub-odontoblastic layer, endothelial cells and cells surrounding nerve (original magnification  40).Fig. 4. NADPH-d immunohistochemistry of chronically inflamed pulp(original magnification  100).The difference between healthy andinflamed pulp was statistically signifi-cant only in the central parts of the pulp(Table 1).DiscussionIn this study the NADPH-d activitywas examined in healthy and diseasedhuman dental pulp. We found prominentNADPH-d activity in the odontoblasts,endothelial cells and Schwann cells. Al-though, there is evidence of interspeciesdifferences in the expression of NADPH-d/NOS reactivity, and NADPH-d histoche-mistry is not always coincident withNOS10, the localization of NADPH-d inthe human pulp, found in this study is inaccordance with studies that examinedother mammalian species. The localiza-tion of NADPH-d activity in the dentalpulp of the rat incisor and molar teeth,and cat teeth are well documented in1993 by Kerozoudis et al.8. They demon-strated the activity in odontoblastic layer,subodontoblastic layer and endothelialcells of pulpal blood vessels. In the radi-cular part of incisor pulp, however onlycells in subodontoblastic layer showedNADPH-d activity. This author failed todetect NADPH-d positive nerve fibers incontrast to Lohinai et al.11 who found asmall number of NADPH-d positive nervefibers scattered in the connective tissueof dog’s and cat’s pulp axons occupying aperivascular location.In spite of destruction of odontoblasticprocesses during endodontic treatmentwith Hödstrom files in this study, distinctexpression of NADPH-d could be noticedin the odontoblastic cells. Kerozoudis etal.8 demineralized tooth hard tissue andpreserve cell processes that extend deepin to the dentine and found NADPH-dpositive Tomes’s processes of odontoblastsin rats. Odontoblasts are cells of mesen-chimal origin and they are derived di-rectly from neural crest cells12. They arehighly specialized cells involved in secre-tion of dentine matrix, its mineralization,dentine sensitivity and dentine nutrition.The expression of NADPH-d activity asthe marker of NOS in odontoblasts con-firms that they are unique cells and theirrole in the pulp physiological and patho-logical conditions could be even morecom0plex than it is assumed13. As theyare first cells facing various noxa fromoral environment, the role of NO releas-ing could be their local communicationwith other cells. NO accounts for tonic re-laxation of all types of blood vessels and itact as neurotransmitter in central andperipheral nervous system14. Also NO isproposed to be a second messenger in themanagement of dentine hypersensitivityby the agency of K+ 15. This view is sup-ported by studies that report analgesia inassociation with increased NO produc-tion through a modulation of nociceptiveinput and downregulation of sensitizednociceptors. It is assumed as a conse-685S. Juki} et al.: NADPH-d in the Human Pulp, Coll. Antropol. 26 (2002) 2: 681–688TABLE 1ACTIVITY OF NADPH-D IN HEALTHY AND CHRONICALLY INFLAMED HUMAN PULPHealthypulpChron. in-flamed pulp t F pHealthypulpChron. in-flamed pulpX X N NOdontoblasts 3.33 3.34 –0.04 32 0.97 15 19Endothelial cells 2.87 2.89 –0.11 32 0.91 15 19Schwann cells 3.13 3.58 –1.62 32 0.12 15 19Stromal part 1.27 2.45 –4.93 32 0.00 15 19quence of ability of NO to activate the sol-uble guanylic cyclases16 which produceincrease level of intraneural cGMP17.Endothelial cells of the human pulpshowed marked NADPH-d activity whatis in accordance with study of Lohinai etal11. who found positive NOS/NADPH-dendothelial cells of dog’s and cat’s pulpsand inferior alveolar artery. The fact thata NOS-basal dependent vasodilatationexists in the dental pulp, as in other tis-sues, was demonstrated, also by Lohinaiet al.18. They showed the high responsive-ness of dental pulp vasculature to exoge-nous NO. Therefore, it is presumed thatthe stimulated release of endogenous NOis involved in vasoregulation of dentalpulp.Although, the presence of small num-ber of NADPH-d positive and NOS immu-noreactive axons in the dental pulp ofcats and dogs was reported previously17,our study is the first in the literature thatdescribes occurrence of NADPH-d posi-tive Schwann cells in the pulp. NADPH-dpositive perisynaptic Schwann cells werefound in the frog neuromuscular junc-tions and immunohistochemical labelingof neuronal NOS was detected at themembrane and occasionally in the cyto-plasm of these cells. The author proposedthat NO may act as diffusible glial mes-senger that modulate synaptic activityand synapse formation in the neuromus-cular junction19. According that, the roleof NOS in Schwann cells of the pulp couldbe the modulation of signal transmissionthrough the pulp nerve fibers.Chronically inflamed human pulp ex-hibited NADPH-d activity in polymor-phonuclear leukocytes and lymphocytes.Also endothelial cells of numerous new-born capillaries and disordered odonto-blasts contribute to strong activity ofNADPH-d central parts of the inflamedpulp. Chronic inflammation is more oftenrelated to the carious lesion, and thenumber of inflammatory cells depends onthe progression of the lesion20. Also deepcavity preparation results in acute in-flammation that progress to chronic, gra-nulomatous inflammation. Results of thisstudy are similar to findings of Law etal.21 who examined NADPH-d reactivityand NOS immunoreactivity following toothpreparation in rats. They found increasedintensity of NADPH-d and macrophageNOS reactivity surrounding the inflamedarea of pulp. Also pulp vessels supplyinginflamed area showed increased NADPH-dreactivity21.Nitric oxide has been found to play animportant role in many chronic inflam-matory diseases22,23. Macrophage NOSproduces a large amount of NO with ap-propriate stimulation and induces sub-stantial tissue damage. The exact nature,positive or negative, of NO is still a meterof scientific debate. Okuda et al.24 foundthat experimental allergic encephalomye-litis of mice is characterized by elevationof nitric oxide. The authors suggest thatexcessive NO via NOS played an impor-tant role in eliminating inflammatorycells through apoptotic mechanism. How-ever, researchers have noticed the sup-pressive effect of activated macrophageson proliferate responses of lymphocytesto mitogens or antigens. Also the leuko-cyte cell adhesion to endothelial cells isinhibited by NO25. Therefore, the modu-lating role of NO in regulation of inflam-mation, tissue destruction, and metabo-lite dysfunction has been proposed.The presence of iNOS immunoreac-tivity in radicular cyst which is mostly aconsequence of pathological events in pulp,was examined by Takeichi et al.4. Theimmunoreactive iNOS is present in epi-thelial cells, endothelial cells, fibroblasts,macrophages and polymorph nuclear leu-cocytes. Interesting finding is that theproximity of blood of vessels influencesiNOS production. Unlike PMNL and ma-crophages adjacent to blood vessels whichexhibit positive iNOS production, the dis-686S. Juki} et al.: NADPH-d in the Human Pulp, Coll. Antropol. 26 (2002) 2: 681–688tant cells are iNOS reactivity weak ornegative. This is explained by distribu-tion of different types of T-helper cells,which may or may not synthesize iNOS.The iNOS production could be stimulatedwith bacterial lipopolysaharids (LPS) andinflammatory cytokines, such as inter-leukin-1 , tumor necrosis factor and inter-feron- (INF- ). Close vicinity of iNOS-pro-ducing cells and INF- -producing cells,and appearance of immunoreactivity forboth molecules in some cells indicates theinteraction of iNOS producing cells andINF- -producing cells in radicular cysts.The interaction of the pulp and perira-dicular tissue allow as to compare thisfindings with ours, so we assumed thatthe stimulation of iNOS by bacterial LPSand inflammatory cytokines plays signifi-cant role in pathogenicity of the chronicalpulp inflammation. Takeichi et al.4 pro-poses that NO inhibitors (NG-monome-thyl-L-arginine) could be used throughroot canal as a pharmacological therapyfor periapical lesions, so the addition ofsuch inhibitors in materials that are usedfor the pulp capping could prevent the on-set of inflammation caused by productionof large amounts of NO.These facts, together with further in-vestigation that should carried on aboutthe distinct type of NOS expressed in spe-cific parts of healthy and inflamed pulp,could clarify the exact role of NO in physi-ology and pathophysiology of this tissue.R E F E R E N C E S1. CLANCY, R. M., A. R. AMIN, S. B. ABRAM-SON, Arthitis Rheum., 41 (1998) 1141. — 2. CLAN-CY, R. M., S. B. ABRAMSON, Proc. Soc. Exper. Biol.Med., 210 (1995) 93. — 3. AMBS, S., W. G. MER-RIAM, M. O. OGUNFUSIKA, W. P. BENNETT, N. IS-HIBE, S. P. HUSSAIN, E. E. TZENG, D. A. GELLER,T. R. BILLIAR, C. C. HARRIS, Nature Med., 12(1998) 1371. — 4. TAKEICHI, O., M. HAYASHI, T.TSURUMUACHI, T. TOMITA, H. OGHIHARA, B.OGISO, T. SAITO, Int. End. J., 32 (1999) 124. — 5.WOLF, G., Hist. Histophat., 12. (1997) 251. — 6.HOPE, B. T., G. J. MICHAEL, K. M. KNIGGE, S. R.VINCENT, Proc. Natl. Acad. Sci. USA, 88 (191) 2811.— 7. SCHMIDT, H. H., T. D. WARNER, K. ISHII, H.SHENG, F. MURAD, Science, 255 (1992) 721. — 8.KEREZOUDIS, N. P., L. OLGART, K. FRIED, Histo-chemistry, 100 (1993) 319. — 9. PEARCE, A. E. G.:Histochemistry, theoretical and applied, Vol I. (Saun-ders Co, London, 1968). — 10. WANG, H., H. C. CHRIS-TIAN, J. F. MORRIS, J. Endocrinol., 154 (1997) R 7.— 11. LOHINAI, Z., A. D. SZÉKELY, P. BENEDEK,A. CSILLAG, Neoursci. Lett., 227 (1995) 91. — 12.TEN CATE, A. R., Oral histology. (The C. V. MosbyCo, St. Louis, 1980). — 12. AZINOVI], Z., K. SRDJAK--JORGI], B. LAZI], Coll. Anthropol., 22 Suppl. (1998)25. — 13. RODE, B., Acta Clin. Croat., 39 (2000) 41.— 14. MCCORMACK, K., R. DAVIS, Pain, 68 (1996)5. — 15. LEZ, C., A. PIRKI], D. BUKOVI], R.FURE[, J. DJELMI[, M. IVANI[EVI], B. HODEK,Coll. Anthropol., 26 (2002) 273. — 16. KOESLING,D., E. BÖHME, G. SCHULTZ, FASEB J., 5 (1991)2785. — 17. LOHINAI, Z., I. BALLA, J. MARCZIS, Z.VASS, A. G. B. KOVÁCH, J. Dent. Res., 74 (1995)1501. — 18. DESCARRIES, L., M. CAI, F. ROBI-TAILLE J. Neurocyt. 27 (1998) 829. — 19. IZUMI, T.,I. KOBAYASHI, K. OKAMURA, K. MATSUO, T. KI-YOSHIMA, Y. ISHIBASHI, H. INOUE, H. SAKAI,Arch. Oral Biol., 41 (1996) 627. — 20. LAW, A. S., K.R. BAUMGARDNER, S. T. MELLER, G. F.GEBHART, J. Dent. Res., 78 (1999) 1585. — 21.TAKASHI, T., T. KONDOH, K. KAMEI, H. SEKI, M.FAKUDA, H. NAGAI, H. TAKANO, Y. YAMAZAKI,A. YOKOHAMA, Oral Surg. Oral Med. Oral Pathol.Oral Radiol. Endod., 82 (1996) 505. — 22. BRO-ZOVI], S., V. VU^I]EVI]-BORAS, D. BUKOVI],Coll. Anthropol., 25 (2001) 633. — 23. OKUDA, Y., S.SAKODA, H. FUJIMURA, T. YANAGIHARA, Bio-chem. Biophys. Res. Com., 267 (2000) 826. — 24.TAKEICHI, O., I. SAITO, M. HAYASHI, T. TSURU-MUACHI, T. SAITO, J. Endodon., 24 (1998) 157.S. Juki}Department of Dental Pathology, School of Dentistry, University of Zagreb,Gunduli}eva 5, 10000 Zagreb, Croatia687S. Juki} et al.: NADPH-d in the Human Pulp, Coll. Antropol. 26 (2002) 2: 681–688SINTAZA DU[IKOVA OKSIDA (NOS) U ZDRAVOJ I KRONI^NOUPALJENOJ ZUBNOJ PULPI PRIKAZANA HISTOKEMIJOMNIKOTINAMID ADENIN DINUKLEOTID FOSFAT – DIJAFORAZE(NADPH-d)S A @ E T A KSvrha istra`ivanja je bila ispitati ekspresiju koenzima nikotinamid adenin dinu-kleotid fosfat-dijaforaze (NADPH-d) u ljudskoj pulpi i odrediti postoji li razlika u nje-govoj aktivnosti u kroni~no upaljenoj pulpi. Devetnaest uzoraka je prikupljeno iz zubikoja su bila u postupku endodontskog lije~enja s klini~kom dijagnozom kroni~nog pul-pitisa. Zdravi uzorci su dobiveni iz zubiju ekstrahiranih zbog ortodontskih razloga.Pulpe su ekstirpirane, smrznute i obra|ene histokemijskim postupkomza prikazivanjeNADPH-d. Uzorci su ispitani pod svjetlosnim mikroskopom. U zdravim uzorcima ak-tivnost NADPH-d je jaka do vrlo jaka u odontoblasti~nom u subodontoblasti~nom sloju.Jaka aktivnost je prona|ena u endotelnim stanicama krvnih `ila i stanicama kojeokru`uju `iv~ana vlakna, dok su preostali sredi{njeg dijela pulpe bili jako slabo do sla-bo pozitivni. Sli~na raspodjela aktivnosti je prona|ena u kroni~no upaljenoj pulpi, te jeumjerena do jaka aktivnost, raspr{ene rasprostranjenosti, prona|ena u sredi{njem di-jelu gdje su prevladavale upalne stanice.688S. Juki} et al.: NADPH-d in the Human Pulp, Coll. Antropol. 26 (2002) 2: 681–688

Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase (NADPH-d) Histochemistry Detecting NOS in Healthy and Chronically Inflamed Pulp

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